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volume 33
Phytochemicals in
Human Health
Protection, Nutrition,
and Plant Defense
RECENT ADVANCES IN PHYTOCHEMISTRY
Proceedings of the Phytochemical Society of North America
General Editor: John T. Romeo, University of South Florida, Tampa, Florida
A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new
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recent advances in phytochemistry
volume 33
Phytochemicals in
Human Health
Protection, Nutrition,
and Plant Defense
Edited by
John T. Romeo
University of South Florida
Tampa, Florida
Cover:
Flax seed. The richest natural source 01 the plant lignan, secoisolariciresinol diglycoside
(SOG), translormed to cancer-protecting mammalian lignans by intestinal bacteria.
(Courtesy 01 Lilian U. Thompson)
Intsia bijuga. Heartwood. Cross section 01 vesicles, one 01 which (lighter color) is lull 01
pentahydroxy Ilavonoid, robinetin. Oarker adjacent vessel contains a mixture 01 other
Ilavonoids and tri- and tetra-hydroxy stilbenes. Compounds are assumed to lunction in
decay resistance (Courtesy 01 W. E. Hillis).
Proceedings 01 the 38th Annual Meeting 01 the Phytochemical Society 01 North America
on Phytochemicals in Human Health Protection, Nutrition, and Plant Oelense, held July
26-31,1998, in Pullman, Washington
ISBN 978-1-4613-7123-6
© 1999Springer Science+Business Media New York
Originally published by Kluwer Academic / Plenum Publishers in 1999
Softcover reprint of the hardcaver Ist editian 1999
Since 1994, the Phytochemical Society of North America has devoted its
annual symposia to topics with biological perspectives. Our last four volumes
have dealt with medicinal plants (1994), plant/insect interactions (1995), food
phytochemicals (1996), and plant/microbe interactions (1997), respectively.
The Symposium held in Pullman, Washington, July 26-31, 1998 brought
many aspects of these previous symposia once again to the forefront. This
time, however, there was greater emphasis on the potential applications of
phytochemistry to the diverse topics of human health and nutrition and plant
defense. As we learned about innovative uses of molecular biology as it is being
applied to these topics, we were reminded once again of the biochemical
foundation on which these advances rest. On the occasion of the 75 th birthday of
G.H. Neal Towers, which we were privileged to celebrate, a perspective of
where we began and how far we have advanced was made patently real for those
in attendance.
The papers assembled in this volume were presented during the Sympo-
sium. Roughly grouped under three broad topics, they include: I. Drug Discov-
ery and Pathway Engineering toward New MedicinallNutriceutical Targets
(papers by Cragg, Croteau, Thompson, Costa, McLaughlin, Dixon, and Matern),
2. Roles for Polyphenols-Biosynthesis and Applications (Gross, Hillis, Haslam,
and Ferreira), 3. New Chemical Prospects and Plant Defense (Asakawa, Selmar,
Houghton, and Mizutani).
It is estimated that 80% of the world's population relies on traditional med-
icines for primary health care. For the rest of the world, about 114 of all pre-
scriptions contain plant extracts or active principles derived from higher plants.
The role of the U.S. National Cancer Institute in the development of new drugs
is reviewed by Cragg. A road map through this agency is provided along with
current protocols and collaborative procedures. Plant natural products continue
to be major players in cancer treatment, accounting for up to 62% of commer-
cially available drugs. Successes and prospects are discussed. Currently, approx-
imately 1,000 Pacific yew trees must be harvested to produced a kilogram oftaxol,
used for treating breast, ovarian, and other cancers. Treatment for ovarian cancer
Vll
viii PREFACE
alone would consume 90,000 trees annually. Walker and Croteau have focused
their work on elucidating the biosynthesis of taxol and its related taxoids. They
use a sequential approach, which examines the enzymes catalyzing each trans-
formation, one which involves both in vivo and in vitro studies and molecular
genetics. Ultimately, this information will be utilized to engineer biological
systems to improve yields of taxoids.
The anticancer effects of lignans, particularly effective against mammary,
colon, skin, and prostate cancers, are discussed by Thompson. Diets high in flax,
the richest source, contain plant lignans that are acted upon by bacterial flora in
the colon of humans to produce enterodiol and enterolactone, the putative pro-
tective agents. Epidemiological and animal studies indicate that both hormone-
related (weak or antiestrogenic effects) and non-hormone-related (antioxidant,
antiangiogenic) mechanisms may be responsible. Costa and colleagues demon-
strate that the major enzymatic steps for synthesizing these protective lignans
are now understood. There is potential for using gene transfer and biotechnolog-
ical manipulation of regulatory enzymes to enhance levels of the beneficial
compounds.
McLaughlin and Chang update their studies of Annonaceous acetogenins.
Since 1993, by using relatively simple bioassays, over 200 bioactive compounds
have been described and evaluated. Structural/activity studies have identified
. compounds having both antitumor and pesticidal effects. Acetogenins inhibit
mitochondrial electron transport systems, and, as a consequence, they are espe-
cially toxic to multiple drug resistant tumor cells and pesticide resistant insects
that possess ATP-dependent xenobiotic efflux systems. In another paper which
links plant defense and human health protection, Dixon and colleagues show how
molecular biology can answer definitively such questions as the efficacy of phy-
toalexins. By cloning and manipulating genes, not only can plant resistance to
disease be improved, but added human health benefits are likely. Antiestrogens,
which are chemopreventive for breast cancer, and soy, which is correlated with
decreased prostrate cancer, may also have hormonelike activity in plants. Matern
also emphasizes structure/activity relationships in his discussion of the medici-
nal potential and biosynthesis of coumarins, compounds isolated from many plant
and microbial sources. Coumarin roles as anti-HIV inhibitors and as treatment
for skin disorders are among those singled out. He details recent developments
in biosynthesis and localization, and points out that cladistic analysis of DNA
sequences should lead to classification of enzymes, and mutational studies to the
identification of domains responsible for catalytic specificities. Biotechnological
generation of plants with modified coumarin biosynthesis and future medical
applications are probable.
The importance of phenolic compounds and their potential for human bet-
terment was apparent in several papers. The widely accepted view that tannins
function as insect feeding deterrents was both reinforced and challenged by
Gross. The diversity of tannins, and our recognition of new emerging roles for
PREFACE ix
them, such as viral inhibitors, which enable some insects to thrive by feeding on
phenolic containing plants, forces us to rethink long-held ideas concerning their
functions as feeding deterrents. Similarly, we realize their vast potential for
medical applications. Extensive enzyme studies have both identified metabolic
intermediates and have provided tools for the elucidation of biochemical reac-
tion mechanisms. There is a need for cellular localization studies, information
on transport vehicles, knowledge of final deposition sites, and application of
molecular biology for insight into the unique structures of tannin-synthesizing
enzymes. Hillis examines an often neglected source of natural products, the heart-
wood, a major feature of which is increased formation of secondary metabolites,
especially phenolics. He examines the anatomy of heart wood and its associated
tissues, and also the mechanisms by which compounds are selectively con-
centrated there. Potential fruit of such studies could lead toward more efficient
syntheses of complex natural substances.
The health-promoting effects of tea, fruit juices, and red wine are at least
partially attributable to polymeric proanthocyanidins. The chemical work of
Ferreira and colleagues demonstrates how conformation analysis is leading to an
understanding of the complexation of these compounds with other biomolecules,
and thus, a better understanding of their salutary effects. Haslam and colleagues
discuss astringency and polyphenollprotein interactions. Structure/activity rela-
tionships, the role of water, and hydrophobic effects and interactions are
described. While admitting that structural problems still remain to be solved, they
call for increasing emphasis on studies ranging from metabolism through ecology,
to practical and applied problems.
A group of papers focus more directly, but not exclusively, on plant defense.
Asakawa writes on the phytochemistry ofbryophytes. An imposing array of active
terpenoids and aromatic compounds are present in Liverworts with about 80% of
the sesqui- and diterpenoids being enantiomers of those found in higher plants.
Only about 5% of bryophytes have been studied chemically. Many are distin-
guished by characteristic odors and taste, pungency and bitterness, antimicrobial
and antifungal activity, insect antifeedant activity, nematocidal and piscicidal
activity, and plant growth inhibitory activity. Increasing medical applications are
being discovered, ranging from cytotoxic and anti-HIV activity to possible
chemoprevention of osteoporosis and allergy. Similarly, interest in the genus Bud-
dleja is relatively recent. A number of uses of extract of this plant, which include
wound healing, treatment for liver and bronchial complaints, and antifungal,
antibacterial, analgesic, and sedative effects are known. Houghton and Mensah
show that an understanding ofthe role ofthe plant's contained iridoids, f1avonoids,
and phenylethanoids is emerging. Selmar summarizes the biology of cyanogenic
glucosides and related nutritional problems. The idea that high concentrations
in certain plants function as repellents is generally accepted, but the role of low
concentrations, widespread in most food plants, is less understood. A putative
function as a metabolic mediator involved in signaling and influencing plant
x PREFACE
John T. Romeo
University of South Florida
CONTENTS
15. Plant Ecochemicals from the Viewpoint of Plant Defense ........... 393
Junya Mizutani
Introduction 2
Anticancer Agents Derived from Natural Resources: The NCI Role ...... . 3
Current Status of the NCI Natural Products Drug Discovery Program ... . 7
Drug Discovery ............................................. . 7
Preclinical Development ...................................... . 9
Clinical Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10
Natural Products Drug Development: The Supply Issue ................ 10
Paclitaxel (Taxol®) ........................................... 13
Potential Anti-HIY Agent, Michellamine B ......................... 15
Collaboration in Drug Discovery and Development: The NCI Role . . . . . .. 17
Screening Agreement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 18
Collaboration in Preclinical Development: Rapid Access to Intervention
Development (RAID) ....................................... 18
INTRODUCTION
Over the ages, humans have relied on nature for their basic needs for the pro-
duction offoodstutTs, shelters, clothing, means of transportation, fertilizers, flavors
and fragrances, and not least, medicines. Plants have formed the basis of sophisti-
cated traditional medicine systems that have been in existence for thousands of
years in countries such as China l and India. 2 These plant-basedsystems continue
to play an essential role in health care, and it has been estimated by the World Health
Organization that approximately 80% of the world's inhabitants rely mainly on tra-
ditional medicines for their primary health care. 3 Plant products also play an impor-
tant role in the health care systems of the remaining 20% of the population mainly
residing in developed countries. Analysis of data on prescriptions dispensed from
community pharmacies in the United States from 1959 to 1980 indicates that about
25% contained plant extracts or active principles derived from higher plants, and
at least 119 chemical substances, derived from 90 plant species, can be considered
as important drugs currently in use in one or more countries. 3 Of these 119 drugs,
74% were discovered as a result of chemical studies directed at the isolation ofthe
active substances from plants used in traditional medicine. Well-known examples
of plant-derived medicinal agents include: the antimalarial drug quinine, obtained
from the bark of Cinchona officinalis; the analgesics, codeine and morphine from
Papaver somniferum; the antihypertensive reserpine from Rauwolfia serpentina;
and the cardiac glycoside, digoxin, from Digitalis purpurea. 4
While marine organisms do not have a history of use in traditional medi-
cine, the ancient Phoenicians employed a chemical secretion from marine mol-
luscs to produce purple dyes for woolen cloth, and seaweeds have long been used
to fertilize the soil. The world's oceans, covering more than 70% of the earth's
NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT 3
The United States National Cancer Institute (NCI) was established in 1937,
its mission being "to provide for, foster, and aid in coordinating research related
to cancer." In 1955, NCI set up the Cancer Chemotherapy National Service Center
(CCNSC) to coordinate a national voluntary cooperative cancer chemotherapy
program, involving the procurement of drugs, screening, preclinical studies, and
clinical evaluation of new agents. By 1958, the initial service nature of the orga-
nization had evolved into a drug research and development program with input
from academic sources and substantial participation of the pharmaceutical indus-
try. The responsibility for drug discovery and preclinical development at NCI now
rests with the Developmental Therapeutics Program (DTP), a major component
of the Division of Cancer Treatment and Diagnosis (DCTD). Thus, NCI has, for
the past forty years, provided a resource for the preclinical screening of com-
pounds and materials submitted by grantees, contractors, pharmaceutical and
chemical companies, and other scientists and institutions, public and private,
worldwide, and has played a major role in the discovery and development of many
of the available commercial and investigational anticancer agents. During this
period, more than 400,000 chemicals, both synthetic and natural, have been
screened for antitumor activity.
Initially, most of the materials screened were pure compounds of synthetic
origin, but the program also recognized that natural products were an excellent
source of chemicals with a wide variety of biological activities. During the early
4 G. M. CRAGG et at.
years of the CCNSC, the screening of natural products was concerned mainly with
the testing of microbial fermentation products, and, prior to 1960, only about 1,500
plant extracts were screened for antitumor activity. Plants have a long history of
use in the treatment of cancer, 7 although many of the claims for the efficacy of such
treatment should be viewed with some skepticism because cancer, as a specific
disease entity, is likely to be poorly defined in terms of folklore and traditional
medicine. 8 Earlier work on the isolation of active antitumor agents from Podophyl-
lum peltatum L., the Mayapple, found throughout the eastern U.S. and used by early
American cultures for the treatment of skin lesions and warts, and the discovery
and development of vinblastine and vincristine, from the rosy periwinkle, Catha-
ranthus roseus (L.) G. Don, used in the treatment of childhood leukemia and other
cancers, however, provided convincing evidence that plants could be sources of a
variety of novel potential cancer chemotherapeutic agents (Fig. 1).8 Epipodophyl-
lotoxin, isolated as the active antitumor agent from various species of Podophyl-
lum, was semisynthetic ally converted into the clinically active agents, etoposide
and teniposide. Thus, the decision was made to explore plants more extensively as
sources of agents with antitumor activity, and, in 1960, an interagency agreement
was established with the United States Department of Agriculture (USDA)
for the collection of plants for screening in the CCNSC program. A small
number of animal extracts, mainly of marine origin, also were tested beginning in
1960, but by the end of 1968, only 1,000 animal extracts had been screened.
The pace of investigation of marine invertebrates accelerated in the 1970s, and by
1982, over 16,000 extracts had been screened. In contrast, however, from 1960 to
1982, over 180,000 microbial fermentation products and over 114,000 plant-
derived extracts were tested for in vivo antitumor activity, mainly using the LI210
and P388 mouse leukemia models. Extracts showing significant activity were
subjected to bioassay-guided fractionation, and the isolated active agents were
submitted for secondary testing against panels comprising four to eight animal
tumor models and human tumor xenografts. 9 Those agents showing significant
activity in the secondary panel were assigned priorities for preclinical and
clinical development.
Of the 92 anticancer drugs commercially available prior to 1983 in the
United States and approved worldwide between 1983 and 1994, approximately
62% can be related to natural origin. 6 While the majority of these drugs were
discovered outside the NCI program, the NCI did playa significant role in the
development of many of them. Two plant-derived agents, paclitaxel (taxol®)
and camptothecin, were discovered through the NCI program, and, while camp-
tothecin failed as a clinical candidate in the 1970s, its derivatives, topotecan,
9-amino camptothecin, and irenotecan (CPT-II), are currently showing clinical
efficacy against a variety of cancer disease types (Fig. I ).10 Other plant-derived
drugs in clinical trials are homoharringtonine, isolated from the small Chinese
evergreen tree, Cephalotaxus harringtonia var. Drupacea (Sieb. & Zucc.)
Koidzumi, and 4-ipomeanol, a pneumotoxic furan derivative produced by
NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT 5
A'
R-+~ o
HO
0
0
HO
Camptothecin : R' = R2 = R3 = H
Etoposide: R = CH3;
Topotecan: R'
R2 = H ; R3 = CH 3CH2
= OH; R2 = CH2N(CH3b; R3 = H
Teniposide : R = (A
OH
N
N
H
""""/
o
II
~
CH'CO OOH
o 0
II II
Ph-C-NH-CH-CH-C-O
I
Ph
I
OH
.H
HO . ~, 0
: H :
PhCO OCCH 3
II II
o 0
<
~Oll_
elL"
o
Ipomeanol
Harringtonine: R = I - ~
C02 Ma
OH HO O
Homoharringtonine: R = ~_
C02 Me
sweet potatoes (Ipomoea hatatas) infected with the fungus, Fusarium solani
(Fig. 2). Homoharringtonine has shown activity against various leukemias and
is in Phase III clinical trials, while ipomeanol is in early clinical trials for
treatment of patients with lung cancer. I I A number of plant-derived agents were
entered into clinical trials by the NCI, but the trials were terminated due to lack
of efficacy or unacceptable toxicity. I I Among these agents were acronycine,
bruceantin, maytansine, and thalicarpine, all of which could serve as cytotoxins
for linking to monoclonal antibodies or other "carrier" molecules targeted to
specific tumors.
Many of the commercial drugs of microbial origin, such as actinomycin D,
doxorubicin (adriamycin), and mitomycin C, were discovered by research groups
associated with the pharmaceutical industry, and this trend continues, generally
in close collaboration with the NCI in the developmental phases. Much of the
drug discovery effort in the marine area, however, has been supported by the NCI
through contract or grant mechanisms. While no marine-organism-derived agent
has yet been approved for commercial development, several agents, including
bryostatin I and dolastatin lO (Fig. 4), are in clinical trials; 12 bryostatin I is
showing some promising activity in trials against melanoma. 13
Most drugs currently available for cancer therapy are effective predominantly
against rapidly proliferating tumors, such as leukemias and lymphomas, but, with
some notable exceptions, such as paclitaxel, show little useful activity against the
slow-growing adult solid tumors, such as lung, colon, prostatic, pancreatic, and
brain tumors. In the early 1980s, the NCI program was discontinued because it was
perceived that few novel active leads were being isolated from natural sources. Of
NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT 7
particular concern was the failure to yield agents possessing activity against the
solid tumor disease-types. This apparent failure might, however, be attributed more
to the nature of the primary screens being used at the time, rather than to a
deficiency of nature. Continued use of the primary P388 mouse leukemia screen
appeared to be detecting only previously identified active compounds or chemical
structure types having little or no activity against solid tumors. In retrospect, these
results might be attributed to the use of a single disease-specific model as the
primary screen that filtered out those agents with potential specificity against
tumors other than mouse leukemia or closely related human diseases.
In an attempt to overcome this deficiency, NCI developed an alternative,
disease-oriented, preclinical anticancer drug discovery strategy aimed at the dis-
covery of new agents for disease-specific clinical trials in relevant cancer patient
populations. 14
Drug Discovery
During 1985-1990, the NCI developed a new in vitro primary screen based
upon a diverse panel of human tumor cell lines. 14 The screen currently comprises
sixty cell lines derived from nine cancer types, and organized into subpanels
representing leukemia, lung, colon, central nervous system, melanoma, ovarian,
renal, prostate and breast. In late 1998, a preliminary prescreen comprising three
cell lines will be introduced, and all materials will be tested in the prescreen.
Those materials showing significant activity in one or more of the three lines will
be advanced to the sixty cell line screen for further evaluation.
With the development of the new in vitro screening strategy, the NCI once
again turned to nature as a potential source of novel anticancer agents, and a new
natural products acquisition program was implemented in 1986. Contracts for the
cultivation and extraction of fungi and cyanobacteria and for the collection of
marine invertebrates and terrestrial plants were initiated in 1986, and with the
exception of fungi and cyanobacteria, these programs continue to operate. Marine
organism collections originally focused in the Caribbean and Australasia, but have
now expanded to the Central and Southern Pacific and to the Indian Ocean (off
East and Southern Africa) through a contract with the Coral Reef Research Foun-
dation, which is based in Palau in Micronesia. Terrestrial plant collections have
been carried out in over 25 countries in tropical and subtropical regions world-
wide through contracts with the Missouri Botanical Garden (Africa and Mada-
gascar), the New York Botanical Garden (Central and South America), and the
University of Illinois at Chicago (Southeast Asia), and have been expanded to the
continental United States through a contract with the Morton Arboretum.
In carrying out these collections, the NCI contractors work closely with
8 G. M. CRAGG et a/.
STAGES:
Bulk
Synthesis
Preliminary
Tox& Ph arm
OVERALL MANAGEMENT:
CANCER OR AIDS OPERATING COMMITIEE
Figure 3. The NCI decision network process.
ing of extracts was discontinued, and alternative assays involving the use of target
enzymes are now being used.
Preclinical Development
Those agents showing significant in vivo activity are presented to the NCI
Division of Cancer Treatment and Diagnosis (DCTD) Decision Network Com-
mittee (DNC), and, if approved by the DNC, the agent is entered into preclinical
and clinical development. The Decision Network Process divides the preclinical
drug development process into stages designated as DNIIA, DNIIB, and DNIII
as described below (Fig. 3).
• An adequate supply of natural product is procured to permit preclini-
cal and clinical development (discussed in detail below).
• Formulation studies are performed to develop a suitable vehicle to sol-
ubilize the drug for administration to patients, generally by intravenous
injection or infusion in the case of cancer. The low solubility of many
natural products in water poses considerable problems, but these can
be overcome by use of co-solvents or emulsifying agents (surfactants)
such as Cremophore EL (polyoxyethylated castor oil).
• Pharmacological evaluation determines the best route and schedule of
administration to achieve optimal activity of the drug in animal models,
the half-lives and bioavailability of the drug in blood and plasma, the
10 G. M. CRAGG et al.
rates of clearance and the routes of excretion, and the identity and rates
of formation of possible metabolites.
• In the final preclinical step, toxicological studies are performed to
determine the type and degree of major toxicities. in rodent and dog
models. These studies help to establish the safe starting doses for
administration to human patients in clinical trials.
Clinical Development
The critical first step in the development of any natural product drug is the
procurement of an adequate supply to meet the requirements for preclinical and
clinical investigation. While total synthesis may be considered as a potential
route for bulk production of the active agent, it is worth noting that the structures
of most bioactive natural products are complex, and bench-scale syntheses
often are not readily adapted to large-scale economic production. Isolation from
the natural source, therefore, often provides the most economically viable method
of production. It also should be noted that, of the established plant-derived
commercial anticancer drugs, vinblastine and vincristine are still produced by
isolation from Catharanthus rose us grown in various regions worldwide, while
etoposide and teniposide are semisynthetically produced from natural precursors
isolated from Podophyllum emodii harvested in India and Pakistan (Fig. I).
The problems associated with the large-scale production of paclitaxel also
have been resolved through semisynthesis from natural precursors, such as
baccatin III and IO-desacetylbaccatin Ill, isolated from the needles of various
Taxus species. IS
NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT II
Bryostatin-1
NSC 339555
HO
Flavopiridol UCN-01
NSC 649890 NSC 638850
KRN5500
NSC 650426
Dolastatin 10
NSC 376128
Figure 4. Some natural product-derived anticancer agents in development by the NCI.
12 G. M. CRAGG et al.
H,C
H,C
CH,
CH,
Calanolide A
Michellamine B
CH,
Prostratin
Conocurvone
The initial raw material collection sample (0.3-1.0 kg) generally will yield
enough extract (10-40 g) to permit isolation of the pure, active constituent in
sufficient milligram quantity for complete structural elucidation. Subsequent
secondary testing and preclinical development, however, might require gram or
even kilogram quantities, depending on the degree of activity and toxicity of the
active agent.
In order to obtain sufficient quantities of an active agent for early pre-
clinical development, recollections of 5-50kg of the raw material, preferably
from the original collection location, might be necessary. Should the preclinical
studies justify development ofthe agent towards clinical trials, considerably larger
amounts of material would be required. The performance of large recollections
necessitates surveys of the distribution and abundance of source organisms as
NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT 13
well as determination of the variation of drug content in the various parts in the
case of plants, and the fluctuation of content with time and season of harvesting.
In addition, the potential for mass cultivation or aquaculture of the source organ-
ism needs to be assessed. If problems are encountered due to scarcity of the wild
source or inability to adapt it to cultivation, a search for alternative sources is
necessary. Other species of the same genus, or closely related genera, can be ana-
lyzed for drug content, and techniques, such as tissue culture, can be investigated.
Paclitaxel (Taxol®)
Probably the most significant drug discovered and developed through the
NCI natural products program is the plant-derived agent, paclitaxel (Fig. I). Pacli-
taxel was isolated through an NCI contract with Drs. Monroe Wall and Mansukh
Wani of Research Triangle Institute from the bark of the Pacific Yew tree (Taxus
brevifalia) in 1969 from samples collected by the USDA as part of the early
exploratory plant screening program. Like many other potential anticancer agents
at that time, paclitaxel only showed moderate activity against the then current
mouse leukemia models and was not considered of particular interest. It was only
the observation of its activity in new test systems (the 816 melanoma and several
human tumor xenografts) developed in the mid- to late 1970s that revived inter-
est. This interest was further heightened by the discovery of its unique mecha-
nism of action by Dr. Susan Horwitz of Albert Einstein School of Medicine;
paclitaxel polymerizes tubulin and stabilizes microtubules, thereby inhibiting
mitosis and cell division. '9
These observations promoted the development ofpaclitaxel which advanced
through preclinical studies (e.g. animal toxicology) to initiation of Phase I clinical
trials in 1983. The early trials were fraught with serious problems of toxicity, par-
ticularly allergic reactions including anaphylaxis, which brought it close to being
dropped from clinical studies. The toxicity was traced back to the poor solubility
of taxol in aqueous systems which required the use of high concentrations of
the emulsifying agent, Cremophore EL (a castor oil derivative), in the preparation
of a suitable vehicle for parenteral administration; Cremophore EL is known
to elicit hypersensitivity reactions. These problems were alleviated by the use
ofionger infusion times (e.g. 24 hours every 14-21 days) and premedication with
anti-allergy drugs.
Due to the slow progress of paclitaxel through Phase I clinical trials and
doubts about its clinical efficacy, only sufficient drug for a moderate number of
trials was isolated in bulk (several kilograms) from the Pacific Yew bark. This
later became a problem when important activity was found in Phase II trials in
ovarian cancer in 1987 and interest in the drug greatly intensified. The observa-
tion of approximately 30% response rates in trials with patients having refractory
ovarian cancer resulted in a tremendous demand for the drug. 20 The yield ofpacli-
taxel isolated was about 1 gram per 301bs of bark, and the average Pacific Yew
14 G. M. CRAGG et al.
tree (about 100 years in age) yielded about 20lbs of bark (equivalent to 1.5 trees
per gram). Given that about 12,000 women were dying annually in the U.S. from
advanced ovarian cancer, and that the usual treatment required about 2 grams of
paclitaxel per patient, 24,000 grams were needed, amounting to the destruction
of 36,000 trees. Meanwhile, significant activity also was observed in the treat-
ment of patients with metastatic breast cancer (40,000 deaths per year), and
responses were being observed also in patients with other forms of advanced
malignancy, including lung cancer, malignant melanoma, and lymphomas.
A detailed analysis of the paclitaxel supply crisis and its eventual solution
has been published, 18 so only a brief review is presented here. The initial source
was the bark of the Pacific Yew, Taxus brevi/alia, an understory tree growing in
the forests of the Pacific northwest from northern California into British Colum-
bia. The taxol supply needs for preclinical and early clinical studies were met
easily by bark collections in Oregon between 1976 and 1985, ranging in size from
2,000 pounds to 15,000 pounds. Later observations of responses in the treatment
of patients with a variety of solid tumors, including malignant melanoma and
ovarian cancer, led to an escalation in demand for drug, resulting in several 60,000
pound-collections between 1987 and 1989. These collections raised concerns
about their impact on the continued existence of the tree, but inventories con-
ducted by the USDA Forest Service and Bureau of Land Management and funded
by Bristol-Myers Squibb (BMS) determined that the tree was abundant (estimates
of>100 million trees) on government land. Over 1.6 million pounds of bark were
harvested under strictly controlled conditions in each of the years 1991 and 1992
by Hauser Northwest, a subsidiary of Hauser Chemical Research (HCR) under
contract to BMS. These collections resulted in the production of hundreds of
kilograms of paclitaxel by HCR.
Both NCI and BMS realized that alternative sources of paclitaxel would
need to be developed to permit its eventual marketing as a clinical drug, and NCI
organized workshops in 1990 and 1992 to promote research into various aspects
of paclitaxel. Analytical surveys of the needles of a number of Taxus species
collected from several countries, including Canada, Georgia, Mexico, Russia,
Ukraine, and the United States were performed, and the content of paclitaxel and
key baccatin precursors in various Taxus cultivars was determined. Though the
paclitaxel content ofthe needles generally was lower than that of the bark, needles
of several species and cultivars were found to be relatively good sources of bac-
catin precursors. The pioneering studies, by the French research team of Greene,
Poitier and coworkers, of the semisynthetic conversion of 10-desacetylbaccatin
III, isolated from the needles of T baccata, to paclitaxel,21 and the subsequent
development of more efficient conversion processes,22 led to the large-scale pro-
duction of paclitaxel and related compounds, such as taxotere,23 from renewable
needle resources, and the solution of the supply problem. Significant advances
also have also been made in the production of paclitaxel through plant tissue
culture using technology developed by the company Phyton Catalytic, working
NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT 15
with BMS,24 while there also have been promising developments in the isolation
from microbial sources. 25
these observations and the in vitro activity against an impressive range of HIV-l
and HIV-2 strains, there were serious disadvantages which precluded advance-
ment of michellamine B to clinical trials. The difference between the toxic dose
level and the anticipated level required for effective antiviral activity was small,
indicative of a narrow therapeutic index. Further toxicology studies in primates
confirmed the very narrow therapeutic index, and indicated potential neurologi-
cal toxicity. Based on these observations, NeI discontinued further studies aimed
at clinical development.
Despite this decision, it is possible that the pharmacological and toxico-
logical profiles can be improved through analogue synthesis. Such studies could
require substantial quantities of the natural product, or the successful synthetic
studies of Bringmann and his group could provide a satisfactory solution. 28 The
isolation of the novel antimalarial compounds, the korupensamines (Fig. 6), from
10'
OH OCH3 OH OCH3
g'
CH3
HO CH3
OH CH3
9
korupensamine A korupensamine B
g' g'
CH3 CH3
HO CH3 HO
11
OH
korupensamine C korupensamine D
As noted above, much of the NCI drug discovery and development effort
has been, and continues to be, carried out through collaborations with research
organizations and the pharmaceutical industry worldwide. Many of the naturally
derived anticancer agents were developed through such efforts. Thus, the discovery
and preclinical development of etoposide and teniposide, semisynthetic derivatives
of the natural product epipodophyllotoxin, were performed by Sandoz investiga-
tors, and the NCI played a substantial role in the clinical development.
Though paclitaxel (taxol®) was discovered by Wall and Wani with NCI IS
contract support, the key to solving the supply problem was the semisynthetic con-
version ofbaccatin III derivatives to paclitaxel (and taxane analogs) pioneered by
the French group led by Poitier,21 followed by the development of alternative con-
version methods by the Holton group,22 supported by the NCI and Bristol-Myers
Squibb. IS The semisynthetic analog, taxotere (docetaxel), produced through a col-
laborative agreement between the Centre National de la Recherche Scientifique
(CNRS) and Rhone-Poulenc Rorer, after undergoing extensive clinical evaluation
in Europe and North America under auspices of organizations, such as the Euro-
pean Organization for Research and Treatment of Cancer (EORTC), and the Cana-
dian and U.S. National Cancer Institutes, 23 is now in clinical use in Europe and
North America. Indeed, there is close collaboration between the EORTC, the
United Kingdom Cancer Research Campaign (CRC), and the NCI in the preclini-
cal and clinical development of many anticancer agents, such as bryostatin I, dolas-
tatin 10, aphidicolin glycinate, rhizoxin, pancratistatin, and phyllanthoside.
Drugs, such as bleomycin, aclacinomycin, and deoxyspergualin, were dis-
covered by the Umezawa group at the Institute of Microbial Chemistry in Japan
and developed in collaboration with the NCI; a number of the agents currently
in early clinical development at the NCI, such as UCN-OI, and quinocarmycin
and spicamycin analogs, are the result of collaborations between Japanese com-
panies, such as Kyowa Hakko Kogyo, Fujisawa Pharmaceutical Co. Ltd, and Kirin
Brewery Ltd, and the NCL I2
The DTP of the NCI thus complements the efforts of the pharmaceutical
industry and other research organizations through taking positive leads, which
industry might consider too uncertain to sponsor, and conducting the "high risk"
research necessary to determine their potential utility as anticancer drugs. In pro-
moting drug discovery and development, the DTP/NCI has formulated various
mechanisms for establishing collaborations with research groups worldwide.
18 G. M. CRAGG et ai.
Screening Agreement
In the late 1970s and early 1980s, many significant discoveries were made
in such fields as biochemistry, molecular biology, embryology, and carcinogene-
NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT 19
sis that had the potential for the development of new strategies and agents for
cancer treatment; most investigators, however, were working only in their own
areas of expertise without the benefit of close liaison with experts in the many
disciplines required to discover and develop new therapies and strategies. In
response to the need to coordinate these research efforts, the NCI initiated the
NCDDG Program in the early 1980s with the goal of bringing together scientists
from academia, industry, and government, in the form of consortia, in a focused
effort aimed at the discovery of new drugs. 30 The inclusion of an industrial com-
ponent in almost all consortia has had positive effects in helping to orient the aca-
demic component( s) towards drug development, and maintaining a focus on the
final outcomes of drug discovery in terms of clinical trials and marketable prod-
ucts, as well as contributing high quality scientists and resources to the Program.
Involvement of NCI Staff has enabled the NCI to contribute its considerable
resources and expertise in cancer drug development, including extensive com-
puterized databases and repositories of compounds tested over more than 40
years, primary and secondary screening systems, and all the resources necessary
for preclinical development of agents meeting selection criteria of the NCI Deci-
sion Network Committee or RAID program. The consortia, headed by a Princi-
pal Investigator, submit proposals based on independent ideas, rather than in
response to specific topics proposed by the NCI, thereby permitting the widest
scope and the greatest degree of innovative science, and encouraging diversity in
the discovery of new drugs and therapeutic approaches.
The National Cooperative Natural Product Drug Discovery Group
(NCNPDDG) Program is one of four such programs, the other three being
directed at studies of Mechanisms of Action, Specific Diseases (e.g. lung and
colon cancer), and Preclinical Model Development. Since 1989, twelve NCN-
PDDGs have been awarded encompassing the study of all natural sources, includ-
ing plants, marine bacteria and invertebrates, microalgae, cyanophytes, and
dinoflagellates, and using a variety of assays, such as molecular targets, mecha-
nism-based assays, cell lines and in vivo systems.
oped policies for the distribution of extracts from the NPR to qualified organiza-
tions, subject to the signing of a legally-binding Material Transfer Agreement
(MTA) (see DTP WWW Homepage below).
To be considered for access to the NPR, organizations are required to
submit short proposals outlining the nature of their screening systems and demon-
strating the capability to process active extracts and develop any isolated active
agents towards clinical trials and commercial production. Approved organizations
have to enter into an MTA with DCTD, with one of the key terms being the
requirement that the recipient organization negotiate suitable terms of collabora-
tion and compensation with the source country(ies) of any extract(s) shown to
exhibit significant activity in the organization's screens. While the current poli-
cies only apply to the testing of extracts in screens pertaining to activity against
cancer, AIDS, and related opportunistic infections, as well as diseases of concern
to developing countries (e.g. malaria, parasitic diseases), the extension to testing
against all human diseases currently is being considered.
The NCI DTP offers access to a considerable body of data and background
information through its WWW Homepage:http://dtp.nci.nih.gov/
Publicly available data include results from the human tumor cell line
screen and AIDS antiviral drug screen, the expression of molecular targets in
cell lines, and 2D and 3D structural information. Background information is
available on the drug screen and the behavior of "standard agents," NCI investi-
gational drugs, analysis of screening data by COMPARE,14 the AIDS antiviral
drug screen, and the 3D database. Data and information are only available on
so-called "open compounds" which are not subject to the terms of confidential
submission.
In providing screening data on extracts, they are identified by code numbers
only; details of the origin of the extracts, such as source organism taxonomy and
location of collection, may only be obtained by individuals or organizations pre-
pared to sign agreements binding them to terms of confidentiality and require-
ments regarding collaboration with, and compensation of, source countries. Such
requirements are in line with the NCI commitments to the source countries
through its LOC and the MTA.
tion (NSF) and components of the National Institutes of Health (NIH), including
the NCI, the National Institute of Allergy and Infectious Diseases (NIAID), the
National Heart, Lung, and Blood Institute (NHLBI), and the National Institute of
Mental Health (NIMH). The goals of the Program are research into drug discov-
ery from natural sources, linked to the identification, inventory, and conservation
of biodiversity, a primary concern of the NSF, and economic development in
developing countries. 30 All these goals are linked to the provision of suitable
training and infrastructure building.
Five awards, four involving countries in Central and South America and one
involving the West African countries of Cameroon and Nigeria, were awarded in
1993 and 1994, and are administered through the NIH Fogarty International
Center. A significant challenge in the development of the ICBGs was the estab-
lishment of principles related to intellectual property rights and the protection of
the rights of the participating source (developing) countries, including commu-
nities and indigenous peoples. While it was possible to develop guidelines for use
in negotiating contracts and agreements, no single set of contractual terms could
apply to all participants, and awardees have developed unique mechanisms and
agreements to suit the particular circumstances of the organizations and countries
involved. 35 As integrated conservation and development projects, the long term
evaluation of this Program will depend on how successful the projects are in
demonstrating the economic value of biodiversity in providing new pharmaceu-
ticals and sustainable natural products-based industries for the participating
developing countries. The Program was recently recompeted. Five new awards,
involving countries in Central and South America (Argentina, Chile, Mexico,
Panama, and Suriname), Africa (Cameroon, Madagascar, and Nigeria), and
Southeast Asia (Vietnam) were awarded in September, 1998.
lakes. Valuable products and information are certain to result from the cloning
and understanding of the novel genes which will be discovered through these
processes.
The report concludes that "these new microorganisms provide a vast
untapped reservoir of genetic and metabolic diversity, the harvesting and study
of which will have far-reaching, positive effects for society in areas such as
enhanced food production, medicine (e.g. antibiotic discovery), bioremediation
of waste materials, and agriculture. 37"
Combinatorial Biosynthesis
CONCLUSION
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Medica, Vols I and 2, Singapore, World Scientific Publishing.
2. KAPOOR, L.D. 1990. CRC Handbook of Ayurvedic Medicinal Plants. Boca Raton,
Florida, CRC Press.
3. FARNSWORTH, N.R., AKERELE, 0., BINGEL, A.S., SOEJARTO, D.O., GUO, Z. 1985.
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4. KINGHORN A.D. 1994. The discovery of drugs from higher plants. In: The Discovery
of Natural Products with Therapeutic Potential (VP Gullo, ed.), Butterworth-Heinemann,
Boston, pp. 81-108.
5. MCCONNEL, 0., LONGLEY, R.E., KOEHN, F.E. 1994. The discovery of marine natural
products with therapeutic potential. In: The Discovery of Natural Products with Thera-
peutic Potential (VP Gullo, ed.), Butterworth-Heinemann, Boston, pp. /09-174.
NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT 27
6. CRAGG, G.M., NEWMAN, OJ., SNADER, K.M. 1997. Natural products in drug dis-
covery and development. 1. Nat. Prod. 60: 52-60.
7. HARTWELL 1.L. 1982. Plants Used Against Cancer. Quarterman, Lawrence, MA.
8. CRAGG, G.M., BOYD, M.R., CARDELLINA II, 1.H., NEWMAN, OJ., SNADER, K.M.,
MCCLOUD, T.G. 1994. Ethnobotany and drug discovery: The experience of the US
National Cancer Institute. In: Ethnobotany and the Search for New Drugs. Ciba Foun-
dation Symposium 185, (OJ Chadwick, J Marsh, eds.), Wiley & Sons, Chichester, U.K.,
pp. 178-196.
9. DRISCOLL, 1.S. 1984. The preclinical new drug research program of the National Cancer
Institute. Cancer Treat. Rep. 68: 63-76.
10. POTMEISEL, M., PINEDO, H. 1995. Camptothecins: New Anticancer Agents. Boca
Raton, Florida, CRC Press.
II. CRAGG, G.M., BOYD, M.R., CARDELLINA 1I, 1.H., GREVER, M.R., SCHEPARTZ,
S.A., SNADER, K.M., SUFFNESS, M. 1993. Role of plants in the National Cancer Insti-
tute drug discovery and development program. In: Human Medicinal Agents from Plants.
Am Chern Soc Symposium Series 534, (AD Kinghorn, MF Balandrin, eds.), Amer. Chern.
Soc., Washington, DC, pp. 80-95.
12. CHRISTIAN, M.e., PLUDA, 1.M., HO, T.e., ARBUCK, S.G., MURGO, AJ.,
SAUSVILLE, E.A. 1997. Promising new agents under development by the Division of
Cancer Treatment, Diagnosis, and Centers of the National Cancer Institute. Scm. Oncol.
24: 219-240.
13. PHILIP, PA., REA, D., THAVASU, P., CARMICHEL, 1., STUART, N.S.A., ROCKETT,
H., TALBOT, D.e., GANESAN, T., PETTIT, G.R., BALK WILL, E, HARRIS, A.L. 1993.
Phase I study ofbryostatin I: Assessment of interleukin 6 and tumor necrosis factor alpha
induction in vivo. 1. Natl. Cancer Inst. 85: 1812-1818.
14. BOYD, M.R., PAULL, K.D. 1995. Some practical considerations and applications of the
National Cancer Institute in vitro anticancer drug discovery screen. Drug Dev. Res. 34:
91-109.
IS. MAYS, T.D., MAZAN, K.D., CRAGG, G.M., BOYD, M.R. 1997. "Triangular privity"-a
working paradigm for the equitable sharing of benefits from biodiversity research
and development. In: Global Genetic Resources: Access, Ownership, and Intellectual
Property Rights, (KE Hoagland and AY Rossman, eds.), Association of Systematics
Collections, Washington DC, pp. 279-298.
16. BOYD, M.R. 1988. Strategies for the identification of new agents for the treatment of
AIDS: A national program to facilitate the discovery and preclinical development of new
candidates for clinical evaluation. In: "AIDS: Etiology, Diagnosis, Treatment and Pre-
vention" (VT. DeVita, S. Hellman and S.A. Rosenberg, cds.), 1.B. Lippincott, Philadel-
phia, pp. 305-319.
17. WEISLOW, O.S., KISER, R., FINE, D.L, BADER, 1., SHOEMAKER, R.H., BOYD, M.R.
1989. New soluble-formazan assay for HIV-I cytopathic effects: Application to high-flux
screening of synthetic and natural products for AIDS-antiviral activity. 1. Natl. Cancer
Inst. 81: 577-586.
18. CRAGG, G.M., SCHEPARTZ, S.A., SUFFNESS, M., GREVER, M.R. 1993. The taxol
supply crisis. New NCI policies for handling the large-scale production of novel natural
product anticancer and anti-HIV agents. 1. Nat. Prod. 56: 1657-1668.
19. HORWITZ, S.B. 1992. Mechanism of action of taxol. Trends Pharmacol. Sci. 13:
134-136.
20. MCQUIRE, WP, ROWINSKY, EX, ROSENSHEIM, N.B., GRUMBINO, Ee.,
CETTINGER, D.S., ARMSTRONG, D.K., DONEHOWER, R.e. 1989. Taxol: A unique
antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms.
Ann. Intern. Med. III: 273-279.
28 G. M. CRAGG et al.
35. ROSENTHAL,1. 1997. OECD Proceedings: Investing in Biological Diversity. The Cairns
Conference, Australia, 25-28 March, 1996. OECD Publications, Paris, pp. 253-273.
36. BALANDRIN, M.E, KINGHORN, A.D., FARNSWORTH, N.R. 1993. Plant-derived
natural products in drug discovery and development. An overview. In: Human Medicinal
Agents from Plants (AD Kinghorn and MF Balandrin, eds.) Am. Chern. Soc. Symposium
Series 534, Amer. Chern. Soc., Washington, DC, pp. 2-12.
37. YOUNG, P. 1997. Major microbial diversity initiative recommended. ASM News. 63:
417-421.
38. SAUSVILLE, E.A. 1997. Targeted toxins. In: Encyclopedia of Cancer, Vo!' III. Acade-
mic Press, Inc., pp. 1703-1714.
39. MELTON, R.G., SHERWOOD, R.E 1996. Antibody-enzyme conjugates for cancer
therapy. 1. Nat!. Cancer Inst. 88: 153-165.
40. MCDANIEL, R., EBERT-KHOSLA, S., HOPWOOD, D.A., KHOSLA, C. 1995. Ratio-
nal design of aromatic polyketide natural products by recombinant assembly of enzymatic
units. Nature. 375: 549-554.
41. HORAN, A.C. 1994. Actinomycetes: A continuos source of novel natural products. In:
The Discovery of Natural Products with Therapeutic Potential (VP Gullo, ed.), Butter-
worth-Heinemann, Boston, pp. 3-30.
Chapter Two
TAXOL BIOSYNTHESIS
A Review of Some Determinant Steps
Introduction .................................................. 32
Alternative Sources of Taxol Supply ............................. 32
Semi-synthesis ............................................ 32
Total Synthesis ......... '. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 34
Biological Sources. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 34
Elucidating the Biosynthetic Pathway ......................... , . . .. 35
Formation of the Universal Diterpene Precursor:
Geranylgeranyl Diphosphate ................................. 35
Taxadiene Synthase: The First Committed Enzyme of
Taxol Biosynthesis ......................................... 36
Taxoids Are Biosynthesized by a Mevalonate-Independent
Pathway .................................................. 37
Hydroxylation of the Taxadiene Nucleus ......................... 37
Subsequent Hydroxylation Steps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 38
Acylation Reactions ........... , ................................ 38
Taxadienyl Acetate as the Third Specific Intermediate in
Taxol Biosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 38
Conversion of IO-Deacetylbaccatin III to Baccatin III . . . . . . . . . . . . .. 40
C4/C20 Epoxidation and Oxetane Ring Formation. . . . . . . . . . . . . . . . . .. 40
Oxidation at C9 ................. , ....... , ................... 41
C 13 Side-Chain Formation and Assembly ,........................ 42
Origin of Side-Chain N-Benzoate Function ........................ 44
Conclusion ........................... , . . . . . . . . . . . . . . . . . . . . . . .. 45
Phytochemicals in Human Health Protection, Nutrition, and Plant Defense, edited by Romeo.
Kluwer Academic I Plenum Publishers, New York, 1999.
31
32 K. WALKER AND R. CROTEAU
INTRODUCTION
Taxol l (Fig. I) is one of the structurally more complex members ofthe taxoid
family characterized by the tricyclic diterpene taxane ring system. Taxol was first
purified from Pacific Yew (Taxus brevi/alia) bark in 1970 2 after extracts from this
material showed cytostatic activity against cancer cells. 3 Classical antineoplastic
drugs (e.g., vinblastine, colchicine, podophyllotoxin, maytansine, and others) act
by inhibiting polymerization of micro tubules. However, in 1979, Taxol was found
to exhibit a novel cytostatic mechanism that involves the promotion oftubulin poly-
merization and stablization of assembled microtubules with consequent blocking
of normal mitotic spindle development and cell division. 4 Discodermolides 5 and
epothilones6 are other natural products with this unusual mode of action. Taxol, as
a new, alternative cell spindle poison, was ultimately promoted to clinical testing.
Taxol and its semi-synthetic congener, TaxotEre (Fig. I ),7 have attracted con-
siderable interest during the last ten years for their use in chemotherapy. 8 Currently,
investigations on dose-dense scheduling have provided both shorter treatment time
and palliative therapy for advanced disease in higher risk patients undergoing
taxane chemotherapy.9 These two taxanes, either alone or in combination with cis-
platin, or anthracyclines, doxorubicin and epirubicin, 10. I I have proven to be
effective in the treatment of ovarian, breast, head, neck, and lung cancers. 10.12.13
The increased utilization ofTaxol, and structurally-related taxoids, in cancer
chemotherapy has precluded obtaining this compound exclusively from the origi-
nallimited source, the bark of the Pacific yew (Taxus brevi/alia). Only 100~ 170
mg Taxol can be isolated from -I kg bark from a mature tree (-0.02% yield).14.15
Roughly 1,000 trees are needed to produce I kg Taxol, and a full ten course treat-
ment requires about 3 g Taxo!. Taxol for the treatment of ovarian cancer alone would
consume -90,000 trees annually,15 placing an exorbitant long term demand on this
limited resource; therefore, alternative sources of supply are being sought.
:i...,
::c
tTl
farnesyl and isopentenyl geranylgeranyl (/J.
diphosphates diphosphate Vi
MM/'
G. / ' T "'-B:8
~.B BH~
----+~''''.. 5~ ,
~
04
1 H
H 20 H 20 H H 20
~'-M'--
verticillene taxa-4(5), 11 (l2)-diene
Rl0 OOH
~ ----+
~ I"'" ~
R20"'" 1 ~ H~ 0
~"'~OH ----+
OH OBz OAe
----+
H 20
over taxadiene synthase activity, as well as the coincidence of the time course
curves for both enzymes, suggests that the prenyltransferase is not rate-limiting in
induced Taxol production.
The two enzymes described above (OOPP synthase and taxadiene synthase)
are both translated as preproteins bearing N-terminal targeting sequences that
direct them to the plastids for processing to mature forms. It is now generally
accepted that the monoterpenes, diterpenes, and tetraterpenes of higher plants
are biosynthesized exclusively in plastids via isopentenyl diphosphate 54 .55 derived
from the mevalonate-independent pathway.56,57 Thus, it was entirely consistent
when Zenk and co-workers58 demonstrated that the taxoid, taxuyunnanine C,
derived from [U-\3C]- or [1-\3C]glucose in T chinesis cell cultures, yielded a label-
ing pattern (evaluated by NMR spectroscopy) that was inconsistent with the meval-
onate pathway but that bore salient features reminiscent ofthe alternative pathway
for isoprenoid biosynthesis,56 Particularly compelling was the observation oflong
range 13C_13C coupling in taxuyannine C, which proved that a contiguously-labeled
C 3 precursor (precluded by the classical mevalonate pathway) undergoes intramol-
ecular rearrangement during the formation ofthe precursor isoprene unit (Fig. 2).58
In T chinesis cell cultures, [1 ,2- 13 C]-acetate was incorporated only into the acetate
side-chains, not the tricyclic terpenoid core.
(Fig. 1).59 This microsomal hydroxylase fulfilled all of the expected requirements
of a cytochrome P450 mixed function monooxygenase (heme thiolate protein),
including blue light (450 nm) reversal of CO inhibition. 59 Whether the mechanism
for this hydroxylation involves an epoxide intermediate or a regiospecific radical
rebound-oxygen insertion at C5 directly is not yet known. The rearranged double
bond and the stereochemistry of this first oxygenated intermediate constrain the
mechanistic possibilities for subsequent elaboration at this site of the oxetane
moiety ofTaxol. 50
Acylation Reactions
:i....,
~ ~
----=--=.
---+
A~---+ ::r:
11"'" ; '" II""': "', ....'9 t'I'I
:: ~ :: ?/ [/J
1 H 4 OH 1 H 4 OAe OAe
~ H 20 H 20 20
en
HO HO
B
""'OH
---+ "''''OH
---+
20 20
~
'-0
40 K. WALKER AND R. CROTEAU
R~00"o7~"o~o
20 20 20 AcO 20
4(5)-ene 4(20)-ene-5a-ol 4(20)-epoxy-5a-ol oxetane ring
Figure 4. Proposed progression ofbiosynthetic transformations at C4, C5, and C20 oftaxoids
leading to the oxetane moiety.
Oxidation at C9
a)
x = H or acetvl
b)
c)
Figure 5. Proposed mechanism for oxetane ring formation (see text for details).
~""
I """ C0 2 H_ _ _....
~
Oi""" ----1~. 04o,H
""
I
C0 2 H
E-cinnamic acid Z-cinnamic acid Z-cinnamic acid epoxide
~H2 + AcO
~C02H
t::JH 2 0 .?'
~-phenylalanine ~O""""" o
. C~U 6H OH
~H2 ~ N-debenzoyltaxol
l~oO
2
~C02H
~I
phenylisoserine
o NH 0
crY~'
o
OH
Taxol
Figure 6. Summary of C 13 side-chain formation and assembly. The phenylpropanoid moiety
of the side-chain is derived from phenylalanine by initial rearrangement to ~-phenylalanine. The
cinnamate derivatives are not incorporated into taxol in vivo. A, phenylalanine ammonia lyase;
B, phenylalanine aminomutase; C, baccatin 111-13-0-phenylisoserinyl transferase; D, N-
debenzoyltaxol-N-benzoyl transferase.
the reaction, the mixture was diluted with carrier (S)-phenylalanine and (R,s)-I3-
phenylalanine, and these metabolites were derivatized to the corresponding N-
benzoyl methyl esters which were extensively purified to indicate a minimum
of 0.17% conversion to l3-phenylalanine. The configuration of the enzymatically
formed product was established as (3R)-I3-phenylalanine by similar means,
but involved conversion to the (I S)-camphanoate methyl ester and co-
chromatography with the authentic diastereomic derivative. Hence, this mutase
44 K. WALKER AND R. CROTEAU
H~
.f
tl; :,yNH2 phenylalanine aminomutase
~
~ S ... , Ho
// C02H
phenylalanine p-phenylalanine
(and presumably the C2-benzoate ofTaxol) originates, in part, from each phenyl-
propanoid. 72 •75 In higher plants, benzoate has been shown to derive from cinnamic
acid, most likely via 3-hydroxy-3-phenyl propionate; subsequent reactions yield
benzoyl CoA and acetyl COA. 76 Since in T. brevifalia, cinnamic acid is not incor-
porated into Taxol, the conversion ofphenylisoserine and (X- and ~-phenylalanine
to the benzoate moiety must occur by an alternate route.
CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
I. Paclitaxel is the generic name for Taxol which is a registered trademark of Bristol-
Myers Squibb. Because of the greater familiarity with the word "Taxol", wc use it
throughout.
2. WANI, M.e., TAYLOR, H.L., WALL, M.E., COGGON, P., MCPHAIL, A.T. 1971. Plant
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3. WALL, M.E., WAN!, M.e. 1995. Paclitaxcl: From discovery to clinic. In: Taxane
46 K. WALKER AND R. CROTEAU
Anticancer Agents: Basic Science and Current Status. ACS Symposium Series 583 (G. I.
Georg, T.T. Chen, I. Ojima, D.M. Vyas., eds.) Washington, DC, pp. 18-30.
4. SCHIFF, P.B., FANT, 1., HORWITZ, S.B. 1979. Promotion of microtubule assembly in
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5. KOWALSKI, R.1., GIANNAKAKOU, P., GUNASEKERA, S.P., LONGLEY, R.E., DAY,
B. w., HAMEL, E. 1997. The microtubule-stabilizing agent discodermolide competitively
inhibits the binding of pac litaxel (Taxol) to tubulin polymers, enhances tubulin nucleation
reactions more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant
cells. Mol. Pharmacol. 52:613-622.
6. NICOLAOU, K.C., ROSCHANGAR, E, VOURLOUMIS, D. 1998. Chemical biology of
epothilones. Angew. Chern. Int. Ed. Eng. 37:2015-2045.
7. Taxotere is a registered trademark of Rhone-Poulenc Rorer; the generic name for this
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throughout.
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TAXOL BIOSYNTHESIS 47
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TAXOL BIOSYNTHESIS 49
to determine the steric course of key reaction steps in the biosynthesis of Taxol,
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31 :3883-3887 (and references therein).
Chapter Three
Lilian U. Thompson
Introduction .. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 51
Sources of Mammalian Lignan Precursors . . . . . . . . . . . . . . . . . . . . . . . . . .. 53
Anticancer Effects of Lignans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 54
Mammary Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 54
Colon Cancer ............................................... 56
Skin and Prostate Cancers ..................................... 57
Mechanism of Lignan Effect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 58
Application in Cancer Prevention and Treatment. . . . . . . . . . . . . . . . . . . . .. 60
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 61
INTRODUCTION
Epidemiological studies suggest that diets rich in whole grains, fruits, and
vegetables are associated with a lower risk of cancer. I Although the low fat and
high fiber content of such diets appear to contribute to their health benefits, many
studies suggest that their non-nutritive components or phytochemicals also
contribute to anticancer effects. The lignans are thought to be one of these
phytochemicals.
Lignans are phenolic compounds formed by the union of two cinnamic acid
residues and, thus, have a dibenzylbutane skeleton structure. 2 What has been sug-
gested to be cancer protective, however, are the mammalian Iignans enterodiol
Phytochemicals in Human Health Protection, Nutrition. and Plant Defense, edited by Romeo.
Kluwer Academic / Plenum Publishers, New York, 1999.
51
52 L. U. THOMPSON
(ED) and enterolactone (EL) which are produced when plant lignans are acted
upon by the bacterial flora in the colon of humans or animalsY ED is produced
from the plant lignan secoisolariciresinol diglycoside (SDO) and EL from
matairesinol (Fig. I), although there may be other plant lignan precursors that
have not yet been identified. ED may be converted to EL, but not the reverse,
under the colonic conditions. The mammalian lignans differ from plant lignans
in that mammalian lignans have the hydroxyl groups in the meta position while
plant lignans have the oxygenated substituents primarily in the para position
(Fig. I). Once mammalian lignans are produced in the colon, they undergo
enterohepatic circulation i.e. they are absorbed, transported to the liver, and
secreted in bile. 5.6 A portion reaches the kidney and eventually is excreted in the
urine. Thus, urinary excretion of ED and EL has been used as an index of plant
lignan intake.
OH
o
H
H CHi)H
CHi)H oxidation
~
colonic bacteria
OH
OH
Enterodiol (ED) Enterolactone (EL)
Figure 1. Metabolism of plant lignans to mammalian lignans.
ROLE OF LIGNANS IN CARCINOGENESIS 53
The mammalian lignans are produced in the human colon; thus, an in vitro
fermentation method using human fecal inoculum to simulate human colonic
HO~
OH
1713-Estradiol
Figure 2. Chemical structure of estradiol and tamoxifen.
54 L. U. THOMPSON
fermentation was used to screen 66 common plant foods for their precursors. 13
This showed that precursors are widely distributed in oil seeds, whole grains,
legumes, fruits, vegetables, and seaweeds, but flaxseed, also known as linseed,
contains the highest concentration with values 75-800 times higher than the other
foods (Table 1). The result was in agreement with an early observation that urinary
lignan excretion in rats fed flaxseed was over 100 times higher than the other ten
grains that were tested. 3 This finding was further confirmed in a recent direct
analysis of the precursors SDO and matairesinol in many plant foods. 14 .15
Although the lignan concentration differs with variety, planting location and year
of harvest,16 flaxseed remains the richest source of the mammalian lignan pre-
cursors, particularly the SDO. Human feeding studies showed a 5 to 195 fold
increase in urinary lignan excretion upon diet supplementation with 10 to 50 g of
flaxseed per day.13.17.20 All these findings suggest that flaxseed is a good' food
model to test the anticancer effects of lignans.
Mammary Cancer
are considered early markers of cancer risk since highly proliferating cells are
more susceptible to carcinogen damage,22 while nuclear aberrations are lethal
events that have been associated with carcinogen exposure and susceptibility of
the cells to carcinogen.23 Both cell proliferation and nuclear aberration were sig-
nificantly reduced by the flaxseed diets. 21 The urinary lignan excretion increased
with flaxseed dose and was negatively related to the number of nuclear aberra-
tions in the mammary epithelial cells. This suggests the potential of lignans in
flaxseed to reduce the number of tumors if provided prior to carcinogen expo-
sure. Indeed, when the experiment was repeated with 5% flaxseed fed only at the
pre-initiation stage and with tumor as the endpoint (i.e. up to 20 weeks after
DMBA treatment), a reduction in the number of tumors, but not in tumor size,
was observed compared with the control. 24 In contrast, when provided only after
carcinogen exposure (early promotion stage), 5% flaxseed significantly reduced
the tumor volume by over 50%, but not the tumor number. 24
To establish that the anticancer effect of flaxseed was due to the mammalian
lignans produced from SDG, the SDG was isolated from flaxseed 25 and tested for
its effect at the early promotion stage. 26 SDG, fed at a level equivalent to that con-
sumed in the 5% flaxseed diet (i.e. 1.5mg/day), resulted in 47% fewer tumors
than the control. It did not reduce significantly the tumor incidence and volume.
The results were in contrast to that seen with flaxseed,24 and are perhaps related
to the fact that in the flaxseed study, the basal diet (control) was fed for 4 weeks
prior to carcinogen injection, while in the SDG study, the basal diet was fed for
only one week. Nonetheless, this provided the first direct evidence that a mam-
malian lignan precursor can influence carcinogenesis although the nature of the
effect depended on the experimental design.
Flaxseed (supplemented at a level of 2.5% and 5%) and the SDG and
flaxseed oil equivalent to the amount in 5% flaxseed diet were also tested for their
effect on tumor development when fed to rats starting 13 weeks after carcinogen
treatment i.e. when the tumor size was about I cm in diameter. 27 After seven weeks
of treatment, all treatment groups had established tumor volumes that were more
than 50% smaller than the initial volume, while the tumor volumes in the control
group did not significantly change (Table 2). The fact that both the SDG and oil
groups had tumor sizes which did not differ significantly from that of the 5%
flaxseed group indicate that both components contribute to the effect seen with
flaxseed. However, the number and volume of new tumors that appeared during
the seven week treatment were the lowest in the SDG group, suggesting a stronger
cancer protective effect of the SDG than the oil when provided at a late promo-
tion stage of carcinogenesis. Considering all tumors (i.e. established and new),
the volumes in animals fed the 2.5% flaxseed, 5% flaxseed, and SDG equivalent
to that in 5% flaxseed level, were significantly lower than those fed the control
or flaxseed oil diet. A significant negative relationship was observed between the
urinary mammalian lignan excretion and the established tumor size at the end of
the study, providing further evidence that mammalian lignans are effective in
56 L. U. THOMPSON
Table 2. Tumor characteristics of rats fed the high fat basal diet (BD) alone or
supplemented with secoisolariciresinol diglycoside (SDG, 2,OOOnmollday), flaxseed
oil (OIL, 1.82% w/w) or flaxseed (2.5% or 5% , w/w)*
Established
tumor
volume, em3
New tumor Total tumor New tumor
Dietary group Initial Final volume, em 3 volume, em 3 number
BD 0.741 0.635 0.213 0.321 2.4
SDG 0.668 0.304 0.052 0.115 1.2
OIL 0.644 0.299 0.187 0.223 2.7
2.5% F 0.700 0.187 0.094 0.126 1.8
5.0% 0.569 0.149 0.128 0.135 2.4
*All values are geometric means, except the new tumor number.
Summarized from reference,"
reducing established tumor growth. The flaxseed oil does not have lignans; thus,
its inhibitory effect on established tumor growth may be related to the metabolic
effect of its alpha linolenic acid which is present in high concentration (57% of
total fatty acids).
Colon Cancer
The mammalian lignans are produced from plant precursors in the colon;34
therefore, this is a body site where they may exert their primary effects. To deter-
mine the role of lignans on colon carcinogenesis, rats were treated with the colon
carcinogen azoxymethane and then fed diets supplemented with either 5% or 10%
flaxseed or defatted flaxseed for 4 weeks.2x Upon sacrifice, the number of aber-
rant crypts (AC) or AC per focus (ACF) in their colons was determined. ACF are
thought to be valid putative preneoplastic markers of colon carcinogenesis. 29
Compared with the control, a significantly lower number of AC (41-53%) and
ACF (48-57%) were observed with the flaxseed diets, indicating a protective
effect on colon cancer. However, the urinary lignan excretion or the flaxseed levels
did not have a linear relationship with the number of AC or ACF suggesting that
there is a dose limit to the protective effect of the lignans. The effect of flaxseed
also did not differ significantly from that of defatted flaxseed, indicating that the
effect was not due to the oil content.
To further establish the role of the SDG, in a second study, groups of
azoxymethane-treated rats were fed either the basal diet (control) or the basal diet
supplemented with a lower level of flaxseed (2.5% or 5%) or defatted flaxseed,
or SDO equivalent to that in the 5% flaxseed (i.e. 1.5 mg/day).30 The defatted
flaxseed diets had the same lignan content as the flaxseed diets but lacked the
flaxseed oil, so the role of the flaxseed oil could be further differentiated from
ROLE OF LIGNANS IN CARCINOGENESIS 57
that of the SDG. After a longer feeding time of 100 days, a lower number of ACF
and AC per ACF was again observed in all treatment groups compared with the
control. The control group already had four microadenomas and two polyps, while
the treatment groups did not have any, indicating that the control group was pro-
gressing faster towards tumor formation than those fed either the flaxseed or the
SDG. The observed effect was thought to be unrelated to the fiber content of
flaxseed since no significant differences were observed among diets in pH and
cecal short chain fatty acids, the products of fiber fermentation. On the other hand,
a significant negative relationship was observed between the urinary lignan excre-
tion and the number of AC per ACE Together with the fact that SDG caused an
effect which did not differ significantly from that of the 5% flaxseed, this rela-
tionship further suggests that the mammalian lignans derived from the SDG in
flaxseed have colon cancer protective effect.
Although the above studies did not have tumor formation as the endpoint,
a subsequent in vitro study showed that the cell proliferation of four human colon
tumor cell lines (HCT-15, LS 174T, Caco-2, and T-84) can be inhibited by the
mammalian lignans, with EL being more effective than ED. 31 Cell proliferation
was inhibited by 55-88% at 100 uM ED or EL concentration, a level that is high
but is achievable in the colon with the intake of up to 25-50g flaxseed. 31 The
effect was not estrogen-related since estradiol has no effect on the cell prolifera-
tion of these tumor cells.
While this result may be suggestive of a Iignan effect on prostate cancer, it is not
conclusive.
Many mechanisms have been suggested for the effect of lignans on car-
cinogenesis although they are based primarily on results derived from in vitro
studies. 34- 47 Lignans have weak estrogenic/antiestrogenic as well as non-hormone
related properties which are summarized in Table 3. 35-47 At high concentrations
and in the presence of estradiol, EL and ED appear to have anti estrogenic and
antiproliferative effects on estrogen receptor positive breast cancer cells, while at
low concentrations, they are more estrogenic. 35 -39 They bind to human and rat
alpha-fetoprotein, a plasma carcino-embryonic protein that regulates the growth
of proliferative and tumor cells. 40 This is important since competition with estro-
gen for binding to alpha fetoprotein may help modulate the proliferative-
enhancing activity of the estrogen on the target tissue. They have been shown to
bind to nuclear type II estrogen binding site,41 a component of the genome that
regulates estrogen-stimulated growth. They inhibit the activities of enzymes
involved in sex hormone synthesis, such as aromatase, 5-alpha reductase, and 17-
beta hydroxy steroid dehydrogenase. 42 -44 Aromatase is a cytochrome P-450
enzyme that catalyzes the formation of estrogen from androstenedione; thus, it
can lower the amount of available estrogen that may increase breast tumor growth.
The 5-alpha reductase is needed in the conversion of testosterone to the main
prostatic androgen, 5 alpha dihydrotestosterone; thus, its inhibition may have
implications in prostate cancer development. The l7-beta hydroxy steroid dehy-
two 30-g slices of flaxseed-containing bread will provide about 7.4 umol of
lignans. 2o This amount is higher than the estimated lignan intake of nonvegetari-
ans (4.6umol/day) with a plant food intake on the upper end of current diet rec-
ommendations (i.e. 8 servings of whole grains and 8 servings of fruit and/or
vegetables) and is the same as the lignan intake of vegetarians (7.4umol/day) who
may have a higher intake of plant foods (i.e. 9 servings of whole grains, 10 serv-
ings of fruits and/or vegetables, 2 servings of legumes, and one serving of
oilseeds). Thus, an individual may be able to double or triple their intake of
tignans with the addition of some flaxseed containing breads in their diet.
CONCLUSION
Epidemiological studies suggest that lignans may have some cancer pro-
tective effects. Animal studies have shown that flaxseed, the richest source of
mammalian lignan precursor, can (a) reduce the mammary tumor incidence,
number, and size depending on the time of exposure, (b) reduce the development
and growth of colon tumors, and (c) reduce the tumor size and incidence oflung
metastasis of melanoma cells. The SDG, ED, and EL produced effects similar to
flaxseed, confirming that mammalian lignans derived from SDG have anticancer
effects. In vitro studies and limited in vivo studies suggest that both hormone
related (i.e. weak estrogenic/antiestrogenic) and non-hormone related (i.e. antiox-
idant, anti angiogenic ) mechanisms may be responsible for this effect. Although
bioavailability studies suggest some similarities in the metabolism of lignans in
vitro, and in rats and humans, the anticancer effects of lignans have yet to be
demonstrated in prospective studies in humans. If confirmed to be cancer pro-
tective without adverse effects, increased tignan consumption not only from
whole grains, legumes, fruits, and vegetables but also from a small amount of
flaxseed or flaxseed-supplemented products may be recommended.
ACKNOWLEDGMENT
REFERENCES
18. LAMPE, 1.W, MARTINI, M.C., KURZER, M.S., ADLERCREUTZ, H., SLAVIN, 1.1..
1994. Urinary lignan and isoflavonoid excretion in premenopausal women consuming
flaxseed powder. Am. 1. Clin. Nutr. 60: 122~128.
19. CUNNANE, S.c., HAMADEH, MJ., LIEDE, A.C., THOMPSON, loU, WOLEVER,
T.M., JENKINS, DJ. 1995. Nutritional attributes of traditional flaxseed in healthy young
adults. Am. 1. Clin. Nutr. 61: 62--68.
20. NESBITT, P.D., THOMPSON, loU 1997. Lignans in homemade and commercial
products containing flaxseed. Nutr. Cancer 29: 222~227.
21. SERRAINO, M., THOMPSON, loU 1991. The effect of flaxseed supplementation on
early risk markers for mammary carcinogenesis. Cancer Lett. 60: 135~142.
22. RUSSO, 1., RUSSO, I.H. 1978. DNA labelling index and structure of the rat mammary
gland as determinants of its susceptibility to carcinogenesis. 1. Natl. Cancer Inst. 61:
1451~1459.
23. SHARKEY, M., BRUCE, R. 1986. Quantitation of nuclear aberrations as a screen for
agents damaging to mammary epithelium. Carcinogenesis 7: 1991 ~ 1995.
24. SERRAINO, M., THOMPSON, loU 1992. The effect of flaxseed supplementation on the
initiation and promotional stages of mammary tumorigenesis. Nutr. Cancer 17: 153~ 159.
25. RICKARD, S.E., ORCHESON, 1..1., SEIDL, M.M., LUYENGI, 1.., FONG, H.H.,
THOMPSON, loU 1996. Dose-dependent production of mammalian lignans in rats and
in vitro from the purified precursor secoisolariciresinol diglycoside in flaxseed. 1. Nutr.
126: 2012~2019.
26. THOMPSON, loU, SEIDL, M., RICKARD, S., ORCHESON, 1.., FONG, H. 1996.
Antitumorigenic effect of a mammalian lignan precursor from flaxseed. Nutr. Cancer 26:
159~165.
27. THOMPSON, loU, RICKARD, S.E., ORCHESON, 1..1., SEIDL, M.M. 1996. Flaxseed
and its lignan and oil components reduce mammary tumor growth at a late stage of car-
cinogenesis. Carcinogenesis 17: 1373~1376.
28. SERRAINO, M., THOMPSON, loU 1992. Flaxseed supplementation and early markers
of colon carcinogenesis. Cancer Lett. 63: 159~165.
29. BIRD, R.P. 1995. Role of aberrant crypt foci in understanding the pathogenesis of colon
cancer. Cancer Lett. 93: 55-71.
30. JENAB, M., THOMPSON, L.u. 1996. The influence of flaxseed and lignans on colon
carcinogenesis and beta-glucuronidase activity. Carcinogenesis 17: 1343~ 1348.
31. SUNG, M.-K., LAUTENS, M., THOMPSON, L.U 1998. Mammalian lignans inhibit the
growth of estrogen-independent human colon tumor cells. Anticancer Res. 18:
1405-1408.
32. YAN, 1.., YEE, 1.A., LI, D., MCGUIRE, M.H., THOMPSON, L.U 1998. Dietary flaxseed
supplementation and experimental metastasis of melanoma cells in mice. Cancer Lett.
124: 181~186.
33. LANDSTROM, M., ZHANG, 1.X., HALLMANS, G., AMAN, P., BERGH, A.,
DAMBER, 1.E., MAZUR, W, WAHALA, K., ADLERCREUTZ, H. 1998. Inhibitory
effects of soy and rye diets on the development of Dunning R3327 prostate adenocarci-
noma in rats. Prostate 36: 15 I ~ 161.
34. ADLERCREUTZ, H., MAZUR, W 1997. Phytoestrogens and western diseases. Ann.
Med. 29: 95-120.
35. MOUSAVI, Y, ADLERCREUTZ, H. 1992. Enterolactone and estradiol inhibit each
other's proliferative effect on MCF-7 breast cancer cells in culture. 1. Steroid Biochem.
Mol. BioI. 41: 615~619.
36. WANG, c., KURZER, M.S. 1997. Phytoestrogen concentration determines effects on
DNA synthesis in human breast cancer cells. Nutr. Cancer 28: 236-247.
37. WELSHONS, WV, MURPHY, C.S., KOCH, R., CALAF, G., JORDAN, Vc. 1987.
64 L. U. THOMPSON
Introduction ................................................... 67
Dietary Lignans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 68
Cancer-Preventing Lignans in Plants ............................... 70
Lignan Precursors and Biosynthetic Pathways. . . . . . . . . . . . . . . . . . . . . . .. 76
Laccases .................................................... 76
Dirigent Proteins ....................... '. . . . . . . . . . . . . . . . . . . . .. 76
PinoresinollLariciresinol Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 80
Secoisolariciresinol Dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 81
Secoisolariciresinol Glucosyltransferase .......................... 82
Enhanced Levels of Cancer-Preventing Lignans in Plants: Towards
Enginnering Their Metabolic Pathways. . . . . . . . . . . . . . . . . . . . .. 82
INTRODUCTION
Phytochemicals in Human Health Protection. Nutrition, and Plant Detense, edited by Romeo.
Kluwer Academic / Plenum Publishers, New York. 1999.
67
68 M. A. COSTA et al.
DIETARY LIGNANS
»
z
HO MeO o
OH OGle
HO~8'0 ~
OH 1.# 8 OGle ::t:
HO m
:;>;:I
o (')
P"I e5'"0
~ OH [J)
OH
Enterodiol 4 Enterolaetone 5 Secoisolariciresinol diglucoside 6
0-
'-0
Figure 1. Various 8-8' linked lignans and the "mammalian" lignans, enterodiol 4 and enterolactone 5.
70 M. A. COSTA et al.
testinal tract of the plant lignans, secoisolariciresinol (2) and matairesinol (3). In
contrast, those population groups that have a positive correlation between
high grain intake and urinary enterolactone (5) excretion appear to be more pro-
tected, e.g. young vegetarians. In this respect, Table 18 shows various dietary
groups and the presumed health risks associated with low consumption of daily
levels of grain, as revealed by differences in enterolactone excretion. Note also
that a high lignan excretion has been observed in subjects with a low risk of colon
cancer.9
Such differences initially were thought to be due to either the amount or
type of dietary fiber ingested, but comparisons of populations consuming Western
vs. vegetarian diets established similar levels of total dietary fiber intake. Other
studies showed that increased fiber levels in the diet did not have distinct health
benefits,1O suggesting that additional factors, associated with plant food items in
the diet, had a beneficial effect. 11.12
Eventually, two classes of plant-derived phenolics, the "mammalian"
lignans and the isoflavonoids, were attributed roles as cancer-preventive sub-
stances. Both classes of these metabolites are diphenolic compounds having
certain structural similarities to the natural and synthetic estrogens and antie-
strogens,13 and the term "phytoestrogen" was subsequently coined. In addition to
cancer prevention, dietary phytoestrogens also are believed to play a role in
limiting the effects of menopause, osteoporosis, and heart disease. Indeed, the
importance of lignans in medicine, human health, and nutrition has since become
well recognized. lo
The role of the "mammalian" lignans, enterodiol (4) and enterolactone (5),
in helping prevent carcinogenesis has been summarized in detail in numerous
contributions,14.15 and the reader is referred to Chapter 3 by Thompson in this
CANCER-PREVENTING LIGNANS IN CEREAL GRAINS AND OTHER CROPS 71
volume. They are formed from secoisolariciresinol (2) and matairesinol (3),
through metabolism by facultative gastrointestinal microflora,16 and differ from
plant lignans in that they have phenolic hydroxyl groups only in the meta posi-
tion of the aromatic rings (Fig. 2). Possible pathways for the conversion of 2 and
3 into 4 and 5, respectively, have been described elsewhere; 17 mammalian lignans
were first detected in the urine of female rats and humans. 18,19
The presence of mammalian lignans in humans can influence sex hormone
metabolism and various biological activities, including intracellular enzyme
levels, protein biosynthesis, growth factor action, and malignant cell proliferation
and angiogenesis. These properties have been interpreted as providing the basis
MeO MeO
HO HO
OH OH
Secoisolariciresinol 2 Matairesinol 3
MeO MeO
OH
"'",,/OH
OMe
HO HO
OH
OH
._---------------..,.
OH
Enterodiol 4 Enterolactone 5
for cancer growth inhibition and prevention of cancer initiation. 14 Proposed mech-
anisms on how mammalian lignans (so-called phytoestrogens) may exert their
effects include that of decreasing free estrogen concentrations resulting from
stimulation of sex hormone binding globulin synthesis in the liver. \3 However,
mammalian lignans are also effective in preventing the initiation and growth of
animal tumors,15 and it has been shown that synthetic enterolactone (5) is cyto-
toxic to human lymphoid cells. IS Although the precise physiological and phar-
macological effects of the dietary lignans are not yet established, a positive
correlation has been identified between diet and reduction in onset of "Western
diseases", including breast, prostate, and colon cancers.
The dietary lignans, secoisolariciresinol, matairesinol, and secoisolari-
ciresinol diglucoside (SOG, 6) are present in grains, various seeds, berries,
fruits, and vegetables (Tables 2-5) as shown. The main known source is
flaxseed, which contains SOG in amounts 75 to 800 times higher than in other
foods (Table 5).15,10,2\ Additionally, legumes such as peanuts, alfalfa sprouts,
kudzu leaf, and soybeans, also contain secoisolariciresinol (see Table 3).22
Other data have also been presented ls .23 indicating the in vitro formation of
"mammalian" lignans following digestion of human dietary food items (Table 4).
Indeed, the comparison of foods in Table 4 serves as a possible indicator of the
large differences in amounts of "mammalian" lignans produced from different
foods in the diet, and presumably reflects the levels of lignans present in the plant
materials consumed. Of special interest is the high level produced when flaxseed
is used as substrate. 23 On the other hand, the values present in mekuba seaweed
are unusual, given that the phenylpropanoid pathway is presumed to be absent (to
any great extent) in algae. 24 Whether the mammalian lignans are exclusively due
to secoisolariciresinol and matairesinol has also been questionable, given that
Adlercreutz l4 noted that, in rats fed rye (Secale cereale) bran, more mammalian
lignans were excreted than the lignans deemed present. Another important aspect
is the effect that different levels and types of fiber in the diet may have on lignan
metabolism, since this may alter the populations of bacterial species present in
the intestine. Indeed, Reddy et at. 25 summarized the effects of diverse dietary
habits on activities and levels of the gut microflora, revealing differences in con-
centrations of anaerobes and aerobes and in associated metabolism of fecal bile
acids.
In addition to 2 and 3, other lignans have important pharmacological
roles (Table 2, Fig. 3).2 These include pinoresinol (1) from Forsythia in term edia ,
a blood platelet activating factor, and sesamin (7) and sesamolinol (8), antioxi-
dants from sesame (Sesamum indicum) seeds, whose intake reduces the choles-
terol levels in humans. 2 Indeed, the mechanism by which sesamin exerts
its antioxidant effects has been studied using rats fed experimental diets: admin-
istration of sesamin resulted in an increase in tocopherol levels/ 6 where the
role of sesamin on tocopherol levels appears to involve competition with y-
tocopherol oxidation. Akimoto et al.27 also discuss its multiple biological
functions, which include interference with linoleic acid metabolism, hypocholes-
terolemic action, enhancement of hepatic detoxication of chemicals and
alcohol, protective effect on hypertension, cardiovascular hypertrophy, and chem-
ically induced mammary tumors. Moreover, sesamin is a hydroxyl radical
scavenger and a potent inhibitor of NADPH-dependent microsomal lipid
peroxidation.
Interestingly, a high tea (both black and green) intake is also negatively
correlated with the occurrence of coronary heart disease. Mazur et al. 28 have
reported relatively high levels of secoisolariciresinol and matairesinol in tea as
being (partly) responsible for its antimutagenic, anticarcinogenic, and antioxida-
tive properties. Other lignans of biological importance include podophyllotoxin
(9) from the mayapple (Podophyllum peltatum) rhizomes which, as its semi-
synthetic etoposide (10) and teniposide (11) derivatives, is among only a few
plant compounds effcctive for cancer treatment. 6•29 Another is gomisin A (12),
isolated from Schizandra chinensis fruits, which has the ability to protect the
CANCER-PREVENTING LIGNANS IN CEREAL GRAINS AND OTHER CROPS 75
(+)-Sesamin 7 (+)-SesamolinoI8
OH
t~.8,0
O~
~ 0
MoOVOM, OMe
(~)-Podophyllotoxin 9 Teniposide II
HO
HO
MeO OH
(+)-Gomisin A 12 Nordihydroguaiaretic acid 13
Laccases
Dirigent Proteins
OH ~OH ~OH ~
tTl
Pinoresinoll :;0
"t:I
le- oxidant 0"""~ OMe lariciresinol
Dirigent protein
[:g
reductase tr'~
)3 r> OM'
NADPH
3 ~""" 0 ~"""HO
. OMe
t"'"
OH HOY HOY
I
OMe OMe
Coniferyl alcohol 14 (+)-Pinoresinol la (+)-Lariciresinol 19a r:/l
~
Z
("J
Pinoresinoll tTl
lariciresinol I NADPH [:g
reductase >-
t"'"
MeO MeO ~
OH Z
o r:/l
(-)-Secoisolariciresinol OH
HO HO
dehydrogenase ~
NAD(Pt ~
::r:
tTl
::tI
("J
OH OH
(-)-Matairesinol 3a (-)-Secoisolariciresinol 2a ~
r:/l
A OH
HO
j
OMe (±)-Dehydrodiconiferyl
alcohols ISallSb
OH
OH
M~~O"
OMe
I e- oxidation 8.8'
HO
OH o
(and other OMe (±)-Pinoresinols tall b
Coniferyl alcohol 14 resonance hybrids)
8-0-4'
H&HOg ~OH
O~
""" OMe
I
# OMe
OH erythrolthreo (±)-Guaiacylglycerol
8-0-4' coniferyl alcohol ethers 16a/16b
B
OH
le- oxidation
Dirigent protein
OH
Coniferyl alcohol 14 Putative enzyme-bound intermediate (+)-Pinoresinolla
suggesting that the native protein may be a trimer.38 This 78 kDa protein, which
does not have any measurable (oxidative) catalytic capacity on its own, can, in
the presence ofa one-electron oxidant, align the coniferyl alcohol radicals formed
in such a way that stereoselective coupling occurs. The term dirigent protein
(Latin, dirigere: to guide or align) was introduced to describe the proposed unique
function of this 78 kDa protein. 38 Note, however, that stereoselective coupling is
only observed when E-coniferyl alcohol is used as a substrate, but not with either
E-p-coumaryl (17) or E-sinapyl (18) alcohols.
OH OH
MeO OMe
OH OH
p- Coumaryl alcohol 17 Sinapyl alcohol 18
To further study the specificity of the coupling reaction in vitro, two dis-
tinct but very similar cDNAs, psdFi 1 and psdFi2, encoding the dirigent protein,
were obtained40 by screening a cDNA library made from F. intermedia young
stems. 39 This was done by using a -370-base pair peR product generated from
primers designed from the amino terminus and several internal amino-acid
microsequences of the dirigent protein. The gene sequence encodes a protein of
18 kDa and the protein is preceded by a signal peptide. It contains four potential
N-glycosylation sites and four potential phosphorylation sites. It has no introns
and shows no homology to any other proteins of known function. This cDNA was
cloned into a baculovirus expression vector system and was expressed in
Spodoptera Jrugiperda Sf9 insect cells. SDS-PAGE revealed that the insect
culture produced recombinant dirigent protein consisting of - four bands ranging
in size from 22 to 27kDa (Fig. 6, lane 4), vs. the single band of 27kDa for the
native protein (Fig. 6, lane 2). When subjected to an enzymatic deglycosylation
procedure, only a single band of -18 kDa was present (Fig. 6, lane 5), which cor-
responds with the calculated molecular weight based on the amino acid sequence.
In vitro assays confirmed that only the (+)-enantiomer of pinoresinol (la) was
being formed when the recombinant dirigent protein was incubated with
E-coniferyl alcohol (14) and laccase as shown (Fig. 7).40 In addition, the dirigent
protein was also cloned into an inducible promoter vector and was expressed
in Schneider 2 (S2) Drosophila melanogaster cells. This convenient system
produces the dirigent protein' in stable transformed insect cells.
80 M. A. COSTA et al.
A 2 3 4 5
kDa
94.0-
67.0 -
43.0 -
30.0 -
20.1 -
Figure 6. A. SDS-PAGE and B. western blot of native
Forsythia intermedia (lanes I and 2) and recombinant
(Spodopterafbaculovirus expressed, lanes 3 to 5) diri-
B
2 3 4 5 gent protein. Lanes I (lOOllg) and 3 (25Ilg): crude total
30.0 - cellular protein extracts. Lanes 2 and 4 (OAllg, each):
purified proteins. Lane 5 contains OAllg of the degly-
20.1 - cosylated form of the recombinant protein: u
In flaxseed, it was shown that the opposite enantiomeric form of pin ore sino I
is formed: when flaxseed cell-free extracts are incubated in presence of E-[3H]-
coniferyl alcohol, only (-)-pinoresinol (1 b) is generated. 17 A flaxseed cDNA
library has been constructed and it is being screened to obtain the corresponding
dirigent protein gene. How both the dirigent protein genes and the correspond-
ing proteins from Forsythia and Linum differ in engendering the formation of (+)-
and (-)-pinoresinol (la and Ib), respectively, is of great interest.
PinoresinollLariciresinol Reductases
200 (+ )-pinoresinol ] a
a0-
:s
o
:~ 100
g
o
:g Figure 7. Chiral HPLC analy-
0::
sis of [9,9'-3HJ-pinoresinol 1
obtained following incubation
with lac case and recombinant
dirigent protein; as can be
o 10 20 30 seen essentially only the (+)-
Elution volume (ml) antipode la is formed.
CANCER-PREVENTING LIGNANS IN CEREAL GRAINS AND OTHER CROPS 81
Secoisolariciresinol Dehydrogenase
OH OH OH
(-)-Secoisolariciresinol 2a Lactol20 (-)-Matairesinol 3a
(20) compound was also detected (Fig. 8) with the recombinant protein, but not
with the corresponding native enzyme.
Secoisolariciresinol Glucosyltransferase
This protein catalyses the final step in SDO formation in flax (L. usitatis-
simum) seed, with maximum secoisolariciresinol glucosyltransferase activity
being observed at week two of flax seed development; it appears to be localized
in the seed coat (1.D. Ford, Washington State University, unpublished results). A
partially purified putative secoisolariciresinol diglucosyl transferase, when incu-
bated in the presence of UDP[ 1-3H]glucose, converts (±)-secoisolariciresinols
(2a/2b) into (+)-[3H]secoisolariciresinol diglucoside (6) as shown by HPLC
analysis and liquid scintillation counting. Further confirmation of the product was
obtained by IH NMR analysis of the acetylated derivative of the enzymatic
product. This glucosyltransferase, capable of converting secoisolariciresinol (2)
into SDO is being purified from flax seeds.
With essentially all of the genes encoding the biochemical pathway from
coniferyl alcohol to secoisolariciresinol and matairesinol in hand, the opportunity
now exists to biotechnologically enhance existing levels in lignan-producing
plants (e.g. flax) or to introduce (regulatory genes or perhaps even the entire
pathway) into those which do not normally biosynthesize the lignans to any con-
siderable extent (e.g. rice, rye and wheat). As shown in Tables 3 and 4, lignans
differentially accumulate in foods, such as wheat, flax, sesame, fruits, and veg-
etables. Accordingly, manipulation of regulatory enzymatic steps would appear
to be a useful strategy to enhance/increase levels of these beneficial compounds.
CANCER-PREVENTING LIGNANS IN CEREAL GRAINS AND OTHER CROPS 83
ACKNOWLEDGMENTS
REFERENCES
SEIDL, M., CHEUNG, F. 1996. Anticancer effects of flaxseed Iignans. In: Natural
Antioxidants and Food Quality in Atherosclerosis and Cancer Prevention. (Kumpulainen,
J.T., Salonen, J.K., eds), The Royal Society of Chemistry, Cambridge, England, pp
356-364.
16. BORRIELLO, S.P, SETCHELL, K.D.R., AXELSON, M., LAWSON, A.M. 1985.
Production and metabolism of lignans by the human faecal flora. l Appl. Bacteriol.
58: 37-43.
17. FORD, J.D., DAVIN, L.B., LEWIS, N.G. 1999. Lignans and health: Cancer chemopre-
venti on and biotechnological opportunities with plant lignans. In: Plant Polyphenols 2:
Chemistry and Biology. (Gross, G.G., Hemingway, R.W, Yoshida, T., eds), Plenum Press,
New York, pp ( •• ).
18. SETCHELL, K.D.R., LAWSON, A.M., MITCHELL, F.L., ADLERCREUTZ, H., KIRK,
D.N., AXELSON, M. 1980. Lignans in man and in animal species. Nature 287: 740-742.
19. STITCH, S.R., TOUMBA, J.K., GROEN, M.B., FUNKE, C.w., LEEMHUIS, 1., VINK,
1., WOODS, G.F. 1980. Excretion, isolation and structure of a new phenolic constituent
of female urine. Nature 287: 738-740.
20. NESBITT, P.D., THOMPSON, L.u. 1997. Lignans in homemade and commercial
products containing flaxseed. Nutr. Cancer 29: 222-227.
21. ADLERCREUTZ, H., MAZUR, W 1997. Phyto-oestrogens and Western diseases. Anal.
Med. 29: 95-120.
22. MAZUR, WM., DUKE, lA., W AHALA, K., RASKU, S., ADLERCREUTZ, H. 1998.
Isoflavonoid and lignan in legumes: Nutritional and health aspects in humans. Nutr.
Biochem. 9: 193-200.
23. THOMPSON, L.u., ROBB, P, SERRAINO, M., CHEUNG, F. 1991. Mammalian lignan
production from various foods. Nutr. Cancer 16: 43-52.
24. lEWIS, N.G., DAVIN, L.B., SARKANEN, S. 1999. The Nature and function of lignins.
In: Comprehensive Natural Products Chemistry. Vol. 3. (Barton, Sir D.H.R., Nakanishi,
K., Meth-Cohn, 0., eds), Elsevier, London, pp 618-739.
25. REDDY, B.S., COHEN, L.A., McCOY, G.D., HILL, P., WEISBURGER, 1.H., WYNDER,
E.L. 1989. Nutrition and its relation to cancer. Adv. Cancer Res. 32: 327-345.
26. KAMAL-ELDIN, A., PETTERSSON, D., APPELQVIST, L.-A. 1996. The in vivo antiox-
idant properties of sesame lignans. In: Natural Antioxidants and Food Quality in Ather-
osclerosis and Cancer Prevention. (Kumpulainen, J.T., Salonen, J.K., eds), The Royal
Society of Chemistry, Cambridge, England, pp 230-235.
27. AKIMOTO, K., ASAMI, S., TANAKA, T., SHIMIZU, S., SUGANO, M., YAMADA, H.
1996. Antioxidant activity of sesamin on NADP-dependent lipid peroxidation in liver
microsomcs. In: Natural Antioxidants and Food Quality in Atherosclerosis and Cancer
Prevention. (Kumpulainen, J.T., Salonen, 1.K., cds), The Royal Society of Chemistry,
Cambridge, England, pp 241-246.
28. MAZUR, WM., W AHALA, K., RASKU, S., SALALlA, A., HASE, T., ADLER-
CREUTZ, H. 1998. Lignan and isoflavonoid concentrations in tea and coffee. Br. 1. Nutr.
79: 37-45.
29. GOEl, H.C., PRASAD, H.C., SHARMA, A. 1998. Antitumor and radioprotective action
of Podophyllum hexandrum. Ind. 1. Exp. BioI. 36: 583-587.
30. KO, K.M., IP, S.P., POON, M.K.T., WU, S.S., CHE, c.T., NG, K.H., KONG, Yc. 1995.
Effect of a lignan-enriched fructus schisandrae extract on hepatic glutathione status in
rats: protection against carbon tetrachloride toxicity. Planta Medica 61: 134-137.
31. IP, S.P, MAK, D.H.F., LI, pc., POON, M.K.T., KO, K.M. 1996. Effect of a lignan-
enriched extract of Schisandra chinensis on aflatoxin BI and cadmium chloride-induced
hepatotoxicity in rats. Pharmacology and Toxicology 78: 413-416.
32. YAMADA, S., MURAWAKI, Y, KAWASAKI, H. 1993. Preventive effect of gomisin A,
86 M. A. COSTA et al.
50. HILDER, VA., GATEHOUSE, A.M.R. 1990. Transforming plants as a means of crop
protection against insects. Outlook on Agriculture 19: 179-183.
51. YOUNG, R. 1995. Improved tomato products: Utilising modern biotechnology. Food
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52. KRAMER, M.G., REDENBAUGH, K. 1994. Commercialization of a tomato with an
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53. SHINTANI, D., DELLAPENNA, D. 1998. Elevating the vitamin E content of plants
through metabolic engineering. Science 282: 2098-2100.
54. BROWER, V 1998. Nutraceuticals: Poised for a healthy slice of the healthcare market?
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Chapter Five
Introduction ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 89
Methodology .................................................. 91
Recent Progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 93
Non-Acetogenin Compounds ................................... 93
New Annonaceous Acetogenin Compounds ........................ 100
Biological Studies with the Annonaceous Acetogenins ............... 120
Summary ..................................................... 124
INTRODUCTION
89
90 1. L. McLAUGHLIN AND C.-J. CHANG
natural, environmentally friendly pesticides will gross nearly one billion dollars
in 1998. Thus, the discovery of phytochemicals as new antitumor and pesticidal
agents is justifiable and should be fundable.
Advances in separation technology (chromatography) and in structural elu-
cidation methods (spectral methods and x-ray crystallography) have simplified
the isolation and characterization of new phytochemicals. Bioassays remain
the most insurmountable roadblock in our quest for new bioactive substances.
Examples of such are: the complex panel of 60 human tumor cell lines at the
National Cancer Institute (NCI, Frederick, Maryland); the robotic, mechanism-
based, screening methods employed by the U.S. pharmaceutical industry; the use
of human tumor xenografts in athymic mice; and the costly panels of indicator
pests maintained by industrial pesticide researchers.
For the independent phytochemical investigator, establishment of cell
culture laboratories requires a full-time commitment of manpower and special-
ized equipment that consumes one's entire research budget. Here at Purdue Uni-
versity we have been aided by a Cell Culture Laboratory funded through the
Purdue Cancer Research Center with NCI support; yet the fees for these services
(up to $120.00/sample in six cell lines) are exorbitant, and we can afford only
confirmatory cell culture tests for our isolated compounds. Similarily, a battery
of mechanism-based assays is too expensive to create and maintain, and we, at
best, can only operate a few of these (protein kinase C, protein-tyrosine kinase)
in our new anticancer drug discovery efforts.2.3 Similarily, the cost of sterile
quarters for maintenance and testing of athymic mice is prohibitive to most
academicians. In pesticide research, the growth and propagation of indicator
pests is perhaps less troublesome and burdensome, but it still consumes funds for
specialized facilities, pest containment, and maintenance manpower. We have
concluded that our best investment of research funds is not on complicated
biological methodology, but on the chemical side of this work.
Thus, our laboratory has sought, developed, and employed general, uncom-
plicated, and affordable bioassay alternatives. Four simple, yet effective, bench-
top bioassays have given us the advantage of detecting both novel and known
phytochemicals with a broad range of structural as well as biological diversity.
These bioassays are: (l) lethality to the larvae of brine shrimp (Artemia salina)
(a rapid, general, bioassay for antitumor and pesticidal compounds), (2) the inhi-
bition of crown gall tumors, induced by plasmid transfer and expression from
Agrobacterium tumefaciens, on discs of potato (Solanum tuberosum) tubers (an
antitumor bioassay), (3) the inhibition or stimulation of frond proliferation of
duckweed (Lemna minor) (an assay for plant growth stimulants and inhibitors),
and (4) lethality to the larvae of yellow fever mosquitoes (Aedes aegyptii) (a test
for pesticides).
Since 1984, over 320 diverse bioactive plant components have been
isolated and characterized in our laboratory by using these methods both for
screening the extracts and for directing the fractionations. Three previous
BIOASSAYS AND ISOLATION OF NEW AGENTS 91
reviews summarize our progress until 1993. 4- 6 This paper updates our progress to
1997.
METHODOLOGY
The materials and methods required for the brine shrimp and potato disc
bioassays, respectively, are described in our original papers/'s and the methods
are updated in later reports including a validation studyY In a recent article, all
of the currently used methods for all four bioassays are described with the back-
grounds, complete references, and statistical considerations. 'o These bioassays
have now become popular. Analyses of literature citations show that the original
brine shrimp test (BST) paper has been quoted hundreds of times and is the most
useful of these methods. 7 These methodologies have been taught by us in over 20
international workshops, and an effort has been made to bring the bioassays
directly to the people where most of the uninvestigated plant species of the world
are located (SE Asia, Africa, Central America, and South America). To conserve
space, only the materials and methods of the BST are presented herein (Table 1,
Fig. 1). Recent papers describe these procedures in Spanish and Chinese and
extend the reading audience. 11,12
In our laboratory, each researcher conducts his/her own brine shrimp bioas-
says on his/her own bench. This simple methodology promotes self-reliance and
rapid turn around (24 hours), and these advantages are important for this type of
work. All known antitumor agents (except prodrugs) are detected by the brine
shrimp and potato disc tests, and all known pesticides, tested so far, are detected
Compound or Extract : O. 5 ml
(20 mg) + 2 ml s o l v c n t - - - - - - - - - - - - - - - + - - -......~ 0.5 ml
1------;- 0.5 ml
Procedures
1. Prepare sea water according to directions on box (38 g sea salt per liter of water), filter.
2. Put sea water in small tank, add shrimp eggs to one side of the divided tank, and cover
this side. The lamp above the other side will attract the hatched shrimp.
3. Allow two days for the shrimp to hatch and mature as nauplii (in warmer climates,
hatching may take place sooner, but the authors use 48-72hr nauplii to be consistent).
4. Prepare vials for testing; for each fraction, test initially at 1,000, 100, 10 Jlglml; prepare
three vials at each concentration for a total of nine vials; weigh 20 mg of sample and add
2 ml of solvent (20 mgl2 ml); from this solution transfer 500, 50, or 5 JlI to vials
corresponding to 1,000, 100, or 10 Jlg/ml, respectively. Evaporate solvent under nitrogen
and then put under high vacuum for about 30 min; volatile solvents will evaporate
overnight. Alternatively, materials may be dissolved in DMSO (dimethylsulfoxide), and up
to 50 JlI may be added per 5 ml of brine before DMSO toxicity will affect the results. An
alternative dilution procedure is illustrated in Fig. I; this procedure avoids the need for
100 JlI and 10 JlI syringes.
5. After two days (when the shrimp larvae are ready), add about 4 ml of sea water to each
vial, count 10 shrimp per vial (30 shrimp per dilution), and adjust the volume with sea
water to 5 mllvial. Place the vials, uncovered, under the lamp. Be sure that vials are not
overheated by the lamp.
6. Twenty-four hours later count and record the number of survivors.
7. Analyze the data with the Finney computer program for probit analysis to determine LC 50
values and 95% confidence intervals. A copy of this program for IBM PCs is available
from J.L. McLaughlin; a $10.00 donation is requested to cover costs, postage, and
handling.
8. Additional dilutions at less than 10 Jlg/ml may be needed to determine the LC so values for
potent materials; also, intermediate concentrations, e.g., at 750, 500, and 250 Jlg/ml can be
prepared and tested to narrow the confidence intervals. By starting with 2mglml (step 4
above) dilutions at 100, 10, and I Jlg/ml are easily prepared for more potent materials.
by the brine shrimp and mosquito larvae tests. Thus, false negatives are not a
major problem. In screening randomly collected plants (over 3,500 species, so
far), extracts of about 5% have shown activity at LC 50 values <500ppm in
the BST. Those with LC 50 values <200 ppm are worthy of confirmatory testing in
the potato disc test and then in cell culture in our search for new antitumor
compounds.
In the past four year period (1993-1997), 98 papers from our laboratory
describing over 200 compounds and their biological effects have been published
BIOASSAYS AND ISOLATION OF NEW AGENTS 93
dealing with this research. Thus, a good level of productivity is possible with a
small group of dedicated scientists using bioassays that really work. Combinato-
rial chemists, molecular biologists, and marine chemists now all compete with
phytochemists for research funds. Consequently, work on higher plants in the
U.S., in the future, will have to be conducted with some unusual flair to give it a
competitive edge. Plant research funds are increasingly difficult to obtain.
Researchers in the countries where unique uninvestigated plant species are abun-
dant are especially urged to collect, screen, and fractionate their available plant
species. New compounds are out there waiting to be found and applied to the
needs of society, and action must be taken soon before the dwindling plant species
of these regions are made extinct.
RECENT PROGRESS
Using the above methods, since 1993, our laboratory has found 207 bioac-
tive compounds which are briefly described and biologically evaluated in this
review. Confirmatory cytotoxicity tests have been made in the panel of human
tumor cell lines at the Cell Culture Laboratory, Purdue Cancer Center. These cell
lines were carefully selected to cover the major types of problematic human solid
tumors. They are A-549 (lung), MCF-7 and MCF-71 Adr (breast), HT-29 (colon),
A-498 (renal), PC-3 (prostate), and PACA-2 (pancreatic). Resistance ratios
(MCF-7 vs. MCF-7/adr, which is resistant to adriamycin) help to select com-
pounds and extracts which are effective against multiple drug resistant (MDR)
tumors. ED50 values are presented in Ilg/ml (ppm). Our seven day MTT assay is
based on mitochondrial reduction of the tetrazoliurn to a blue formazan dye. 13 At
Eli Lilly and Company, bullatacin (61) gave an ED50 value of 1O-14 1lg/m l against
human leukemia (CRF-LEM) cells, and, at Abbott Laboratories, the adjacent bis-
tetrahydrofuran (THF) Annonaceous acetogenins (51, 61, 70) all gave cytotoxic-
ity ED50 values showing potencies at least three orders of magnitude greater than
adriamycin; thus, we have confidence in, and independent confirmation of, the
high levels of cytotoxic potencies presented herein. Cytotoxic ED50 values <41lg1
ml are considered significant. 14 Adriamycin is always run as a positive control.
Most of our recent work has been focused on the potent antitumor and pes-
ticidal acetogenins from the Annonaceae. However, several non-acetogenin com-
pounds are quite promising and will be discussed first followed by the
presentation of our new actogenins and a discussion of various biological studies.
Non-Acetogenin Compounds
that it selectively inhibits, at the substrate binding site, the Syk PTK that medi-
ates the degranulation of antigen-stimulated RBL-2H3 rat tumor mast cells.l7
Thus, this compound and/or its analogs may find future application in the
prevention or treatment of allergies as well as tumors.
2. Knema glomerata Merrill (Myristicaceae): Work on the stem bark ofthis
Philippine species led to the isolation of one new phenylalkylphenol, kneglom-
eratanol (1), and two new acetophenones, kneglomeratanones A (2) and B (3),
together with seven known phenolic compounds (4-10) and three known fla-
vanoids. All of these compounds showed moderate to significant toxicities to three
human tumor cell lines, inhibited the growth of the potato disc crown gall tumors
(PO), and were lethal in the brine shrimp test (BST).18 Since these compounds
do not show selective cytotoxicities, they are not being studied further.
3. Melia volkensii Gurke (Meliaceae): The root bark of this folkloric
medicine from Kenya has yielded ten cytotoxic triterpenes: meliavolin (11),
meliavolkin (12), and melkianin A (13);19 meliavolen (14), melianione (15), 3-
episapelin A (16), and nimbolin B (17)/° meliavolkenin (18);21 and meli-
avolkensins A and B (19, 20).22 Seven of these (11, 12, 14, 15, 18-20) are new
compounds. X-ray crystallography of 13 and the acetate of 11, Mosher esters,
derivative preparations, and spectral analyses (especially NOESY) were used to
elucidate the structures. All of these compounds (especially 17) showed interest-
ing selectivities for the colon cell line (HT-29). Work is continuing with this
species.
OH
o
l' : 2" R,
HO~' (CH2)6~-
2' 9' \0' 0 1"
oj'
HO~'
2 6 2' 0
(CH ,)n ~ CH 3
3 5
4
OH
R,
, '"""~: R'O¥(CH')~
~ R2
27
o Ul o
12
if'.
; - 21
,01 A
·0
5' 7
I o'~9 AcO'"
3
6'
8t
~'7 OH
18
.
'1 '
cr
~ E 24 26
(t
17 20 "OH
0 3
"O A 4'?'" 0\\"
~'r' 2' l' 0" S' 17' 29 28
5' ~ I 7' '"
13
2~h
20
",,\.~
E
24
23
26
14, R ~ H
96 1. L. McLAUGHLIN AND C.-1. CHANG
16
H 9H
HO HO~c:>VV27
;~~23 l'oH
-".:. 26
1
17
H
r=':
14:"" 13"""'" ...... ,,~o
' 0 3 A
Acd" "OCOC= CHCH 3 4(
'7 (
'1' 0\"
I 5';:'-" I 7' 29
CH3 6'
17 18
0 26
4'r
5'
(f
:;:,-..
1'1'3
0'"
2. ~H
HI"
21
OCH 3
2 'I 27
~OCH3
o ,).('H
OH OH
OH
16
Cmpd
23 2.98 3.21
I 3.01
I 2.69
I 1.17 4.65 1.79
rotenone 7.46xI0·'
o o
OAe OAe
16
1711 .. ,1~8
7 9 "H q,
lC-~_O:r 6' ~c-o OR,
"3' >- I
N 26
27 28
H,C
RI R2 R
Ae 0
0,
;e-cH, -Q-
V_ ~
-~
OH
29 33 OCHl
30 Ac 34 H
ctC_CH,-Q
/ -
31 H 0
/c~
32 H 0
-~
I
Cmpd BST A·549 MCF·7 HT·29 A·498 PC·3
I I2.16 I3.71 I3.04 I2.05
PACA·2
26 2.00
I2.31
27 >30 >30 >30 24.99 >30 >30
31
,2
2.85xlO
'\
2.30 3.83xlO
-\
<10
-, -\
1.92
1.I0xl0 2.44xl0
-2 -2
34 5.41xlO 30.9 7.41 1.97x1O 4.56 3.55
adriamycin -3 -\ -, -, , ,
4.16xlO 3.76xlO 3.90xlO 1.87x1O 6.37xlO 1.63xl0
BIOASSAYS AND ISOLATION OF NEW AGENTS 99
hope to reisolate more of31 and test it in vivo against A-498 xenografts in athymic
mice.
6. Serenoa repens (Bart.) Small (Palmae): This is the saw palmetto of
Florida and the Carolinas the berries of which are extracted and sold popularly
as a nutritional supplement to shrink benign prostatic hypertrophy. BST directed
fractionation led to the isolation of l-monoacylglycerides, l-monolauricin (45)
and l-monomyristin (46).28 These compounds are unusual because mammalian
systems produce 2-monoacylglycerides, and, if phosphorylated at C-3 by kinases,
45 and 46 would form powerful surfactants, as are formed from phospholipids
by snake venom lipases; 45 and 46 were only moderately cytotoxic with selec-
tivity for PACA-2 (pancreas) and A-498 (lung). A use patent has been filed with
commercial interest coming from the herb trade industry. Additional bioactive
lipoid compounds are present in trace amounts.
R=
45,
o
II
R= -c~
46,
adriamycin
47 48 49 50
Cmpds PACA·2
47 19.2
48 53% 15 3.48 4.30 2.92 1.82 3.86xI0·' 2.12
49 55% 2.84 4.4IxlO·' 3.30x 10" 6.18xI0·' 1.18x 10" 2.79xlO·'
50 >100 1.98 2.73 3.09 3.10 1.63 1.99
adriamycin .- 4.99xI0·' 2.53xI0·' 3.5IxlO·2 4.28xlO·' 2.76xlO·' 1.33x10·'
samples collected on the same day from 670 trees growing in the same planta-
tion in Maryland; bioactivities vary as much as 900 times from one tree to the
next, showing unexpected germplasm variability.
We have now isolated 43 additional bioactive compounds from the seeds
and bark of A. triloba. These include 18 adjacent bis-THF acetogenins which can
be divided into three types: the asimicin type [asimicin (51), asimin (52), asimi-
cacin (53), asiminecin (54), asiminocin (55), asimilobin (56), parviflorin (57), cis-
and trans-asimicinones (58, 59), and lO-hydroxyasimicin (60)]; the bullatacin
type [bullatacin (61), bullatin (62), squamocin (63), motrilin (64), bullanin (65),
cis-and trans-bullatacinones (66, 67), and bullatetrocin (68)]; and the trilobacin
type [trilobacin (69), trilobin (70), asitribin (71), cis-and trans-trilobacinones (72,
73), and 10-hydroxytrilobacin (74)].44-54 Also included are 16 mono-THF com-
pounds [annonacin, annonacin A, cis-and trans-annonacin A-ones, gigantetrocin
A, cis-and trans-gigantetrocin-A-ones, cis-and trans-isoannonacins, murisolin
(75), 16, 19-cis-murisolin (76), murisolin A (77), cis-and trans-murisolinones (78,
79), and asiminenins A and B (80, 81 ).5253.55 Only one nonadjacent bis-THF ace-
togenin, trilobalicin (82), has been found. 54 The complicated absolute stereo-
chemistries of most of these acetogenins have been determined by IH NMR
analyses of Mosher esters/" sometimes aided by the formation of formaldehyde
acetal derivatives. 57 Some are extremely potent, e.g. 55, 65, and 70 are nonselec-
tively cytotoxic at ED50 values <1 0-12 Ilg/m l. The addition of the fourth hydroxyl
decreases potencies as in 60, 68, and 74.53 HPLC has permitted the difficult
separation of the 2,4-cis and trans ketolactones whose activities are usually
comparable. 4R Compound 56 has its adjacent bis-THF ring system located at C-
10 to C-17 and has only one flanking hydroxyl group at C-18. Compound 57 is
a C-35 acetogenin, and its hydroxylated bis-THF ring system starts at C-13
instead of at C-15. Some of the absolute stereochemistries are not shown here,
but they are available in the published papers and in our latest review paper. 58
~
~&a37
3 35 37
2 R, R,
0
'0
~o
0
0
Rs R3 X Y
A B C D E RI R2 R3 R4 RS R6
I I
51 threo lrans Ihreo trans {hreo X OH H H H H
".:!"~' ".",
32~[CH2),
0"
56 57
~H
31 =
32
34
OH o
68
~In\li
~~~..~
, ~
~
3
o4-o 0 L
35 37
OH OH y
A B C D R
..ns
erythro ./
\
OH
0
22
32
OH 0
OH OH
82
51 2.56x10
·2
8.43x10 -. 8.52x 10 <10
·12
52 ·3 _9 -, ·12
4.6x10 7.99x10 9.57xlO <10
.J ·9 ·12 ·11
53 5.7x10 3.58x10 <10 <10
.J ·9 ·12
54 4.9x10 3.29x10 2.74x10 <10
.J.:! ·12 ·12
55 4.9x 10'3 3.lx10 2.9x10 <10
56 1.06
-, 2.14 1.47
.,
3.01x10 6.30xlO 2.26xlO I.04x10
57 -2 -12
1.72
.,
8.8x 10 <10 5.49x10
58
., ·7 ·7
2.2
I.08x10 I.lxlO 2.0xlO
59 ·7 ·7 .,
2.9xlO <10 <10 1.5 x 10
63
·9 -, ·It
9.7x10 2.94x10 1.52xl0 3.79x10
·12 ·14 -6
64 1.I0xl0 8.52x10 I.96x10 4.46xI0
.) ·14 ·12
65 6.Ox 10 3.llxl0 3.22x10 4.77xI0
69
.J .J .,
8.7x 10 8.02x I 0 3.39x1O <10
70 9.7x10
.J
5.71x 10 2.95x10 2.20x 10
..
71 ·2 -4 ·5
1.69 1.30
-,
2.35x10 2.25x10 1.24x 10 7.04x10 L25x10
75
., ., 3.15 -, ·9
2.36
.J
2.07x 10 5.90x10 6.58x I 0 I.09xlO 1.50xl0
77 2.07xI0" 5.90xlO
. 3.15 6.58xlO
.,
1.09xlO
., 2.36 1.50xlO
.,
78 12.3 1.48xlO
.,
7.93xlO
., 7.54xlO
., 3.44 1.48 1.07xlO
.,
79 18.2
., ., 1.16 1.23
., .,
2.76xlO 2.90xlO 2.14xlO 5.90xlO
80 4.73x10
.,
2.85xlO
.,
2.39xlO
.,
8.12xlO
.,
6.26xlO
., 1.66 8.58xlO
.
81 5.82x10
., 3.22x10
. 3.61xlO
. 6.94xW-' 5.72xlO
., 3.66xlO
.,
6.34xlO
.7
threo trans
trans
Cmpd y
83 OH H o 6
84 OH H o
85 OH H o 6
86 H OH 2
S
R "
~ R
~~O
x
~ 0
OH OH 0 y
R Rl R2 b A S C
''''''', OH -~.~
_M, "
22 21 ~: .. 2 16
~ ~~' . .
34 (CH,l8 Ill: 11 '4 0 9 b 1 a
OH OH a
92,93 96
32 ............... (CH2)'0~(CH2h~(CH2)5
:
OH :OH m 4
I
0
..l35
0
1
97
trans
98
~ 3 35 37
~
04'_ 0
o
y
BIOASSAYS AND ISOLATION OF NEW AGENTS ]07
Cmpd A B m n R
99 saId. trans X
trans
OH
0 0
OR, 0 threo
Cmpd RI R2 Cmpd R m
104 a H 95 0 II
105 a acetonide 96 a 13
-4 -6
101 0.11 1.54x I 0 1.47xl0 3.04xlO· 1
_3 -3
102 0.87 3.64x10 7.llxl0 7.26xI0- 3
-3 -3
103 0.77 <10 6.31x10 <10
-2 -2
104 9.33 1.08xl0 <10 1.58
-3
105 <10 3.47x10 9.32x I 0
-3 -2
106 <10 5.72xI0 2.6
107 0.84
_3 ., -,
7.86x10 7.47x10 3.46x10
108 0.98 -3 -, -2
4.x10 5.6x 10 4.7x10
108 J. L. McLAUGHLIN AND C.-J. CHANG
·1 ·1
109 31.86 2.89x10 1.22 1.18 7.50x10 5.98x10
·1 ·1
8.99x10
adriamycin 5.20xlO
·2
5.75x10
-1
6.41x10
·2
3.42x10
-2
1.21x10
-, -2
2.55x10
bis-THF acetogenins lacking a flanking hydroxyl on the lactone side, are selec-
tively ca. 1,000 times the cytotoxic potency of adriamycin, and show unusually
good activity against A-498 (renal); we hope to test 128 in athymic mice against
A-498 tumor xenografts.
34 34
34
35 ····37 35
CH 3(CHz)a I D CH 3(CH;z),
3. 2 1 3.
D
118 119
"
" OH 6H
37
37
120 121
trans
34
OH
R,
A n R, R2
125 threo OH OH
126 erythro 5 OH OH
127 H H
128 7 H H
110 J. L. McLAUGHLIN AND c.-J. CHANG
117 3.4xlO
., 6. 17xlO·5 5. 18xlO·[ 1.20 2.76 2.06 <10-8
123
.[ -I 2.72 1.44 2.29xlO· 4 3.62xlO-I 2.53xlO·4
1.3 x 10 4.23xlO
.[ .;,
124 7.6xlO 5.55xlO 3.19 1.98 3.39xlO·2 1.35xlO·' 5.69xlO·'
·1
125 4.2xlO 1.04xlO 1.78 1.42 5.40x10· 1 1.65xlO·' 1.41xlO·6
OR,
0
130 R=Et Rl=H
Two highly unusual acetogenins isolated from this species are giganin (96),
the first acetogenin to have neither THF nor epoxide rings,82 and goniocin (131),
the first tris-THF ring acetogenin; the cytotoxic potency of 131 approached that
of adriamycin.83 Gonionenin (132) was isolated, and the C-21122 double bonds
in 132 and in gigantetronenin were oxidized with m-chloroperbenzoic acid to give
epoxides which were then cyclized using perchloric acid to give pairs of adjacent
bis-THF [cyclogonionenin (133) and cyclogonionen C (134)] and nonadjacent
bis-THF [gigantecin (135) and C-18/21-cis-gigantecin (136)] acetogenins. The
resulting bis-THF compounds (133-136) showed enhanced bioactivities, with
135 and 118 being over a billion times more potent than adriamycin against
certain cell lines. 84 Similarly, goniodenin (137) was found with a cis-C-2l/22
double bond and was converted to a pair of tris-THF acetogenins; cyclogonio-
den ins T (138) and C (139), which showed generally enhanced cytotoxicities,
especially for the pancreatic (PACA-2) cellline. 85 Gigantransenins A-C (140-142)
are C-37 mono-THF acetogenins each with an unprecedented trans double
bond; their cytotoxic potencies were close to those of adriamycin.80 Cis-
gigantrionenin (143) and 4-acetyl gigantetrocin A (144) both have unusual
features with 144 showing good selectivity.87 The absolute configurations of
gigantetrocin A, goniothalamicin, and several other acetogenins from this and
other species were determined through formaldehyde acetal derivatives and
Mosher ester methodology.57
Recently, 4-deoxyannomontacin (145), with a mixture of cis-and trans-
annonmontacinones (146, 147),88 and 4-deoxygigantecin (148), with a mixture of
cis- and trans-gigantecinones (149, 150),89 have been isolated; compounds 145
and 148 are less cytotoxic, respectively, than annomontacin (151) and gigantecin
(152), but 145 shows good selective potencies for A-549 (lung) and MCF-7
(breast). These new compounds were equivalent or superior to rotenone in the
YFM test. Compound 145 is abundant and is proposed for in vivo testing against
A-549 (lung) and MCF-7 (breast) tumor xenografts.
c<s
37
22 ~ 21
OH OH 35
2
1 36
1°
10
o
131 132
threo
A I trans
37
threo~
I
threo
/(CH 2) , , \ OH
34 22 21 0 1817 0 14 13 10
OH OH
threo
Ihreo lrans ~ Irans 37
H 3 '36
22 21 \, . I 0
CH,(CH 2h, 18 17'01413 '0 10 4
1
OH o
trans trans
threo threo 37 threo
\ I I
35 OH
35
OH '36
23 21 I 0
23 I
CH 3 (CH 2)& r 18- 17 0-14 13 10 2 CH 3 (CH 2)g 10
22 1 1
OH OH 0 0
15.-
143 144
OH
34
-Cmpd YFM
131
132 21.7
.J ., -4
1.34xl0 4.54xlO 1.12xl0
133 0.19
.J .J .,
1.41xl0 1.19xlO 1.54xl0
134 2.71
.J ., .,
1.07xl0 1.79xl0 4.08xlO
135
., ·12 -4 ·12
3.44xlO <10 1.0xlO <10
·1 ·12 ·12 ·12
136 2.49x10 <10 <10 <10
138 35.3 -4 ., ., ., -4 ·8
6.37x10 7.63x 10 4.67x10 1.67x 10 9.38x10 <10
BIOASSAYS AND ISOLATION OF NEW AGENTS 113
139 40.1
., 2.57 1.07
., .,
4.27x I 0 3.64x10 5.21x10 3.83x 10
140 3.6
., ·2
1.5 1.5
., .,
1.6xlO 1.0xlO 1.8xlO I.7x 10
141 5.8
., -2
1.4 1.6 -, 1.5
2.lxlO 2.1xlO 7.lx10
142 4.2
-, 2.2x 10-2 1.3 1.5 1.5 1.1
1.8xlO
143 2.52 -1 -, -6 -1 -, .,
5.99x 10 2.68x 10 6.94x10 1.39x 10 I.IIxlO 1.15xl0
145 I.3x 10-' 6.45x10-' 5.77xI0-' 1.41x10-' 1.50x10-' 1.73x10-' 1.00xI0-' 1.30
146,147 3.lxI0-' 2.60 3.21 2.55x 10-' 1.44 1.01 6.78xI0-' 1.00
148 4.0xlO- 1 -, 1.0 1.43x10-' 3.28x I 0-' 1.50x 10-' 3.93xlO-' 0.68
I.3lxlO
rotenone IT
mono-THF compounds with the flanking hydroxyl on the right one carbon away
from the ring), and (2,4-cis-and trans)-annomuricin D-ones (176, 177) (which
possess an erythro vicinal diol between the mono-THF and the keto lactone
rings. 99 No bis-THF acetogenins, so far, have been found in guanabana, but some
of these mono-THF compounds show interesting cytotoxic potencies and selec-
tivities, e.g. 153 against HT-29 (colon) cells.
ri QH
A
C
-
R--
m Cmpd
~
n A B C 0
~~
Cmpd RI R2 I~HI Y
1
R R trans S 4 OH
159
160 R R trans OH
-----
35 Y 35
33 33
OH OH~ OH OH
I 0
10 I 0
2 1 2 1
0 0
Cmpd W X Cmpd W Y
/'
./(CH2h~ 2 21
0'"
18
/"(CH2)5~1~O
17 (CH )
4 2
1
0
32
/(CH2h1~~'
?~o
20 190'16
/.,
15 11 R
~A
(CH2)S',
2 33
34 35
0
34 20H OH 11 25 0 OH OH 0
~
32 (CHZ)s
Cmpd
~
~- R RI
170 H H H OH OH OH OH
171 H OH OH H H OH OH
-------------------
BIOASSAYS AND ISOLATION OF NEW AGENTS 115
172
173 H
R2
OH
J R3
H
-r--- 14-
OH
174 OH H OH H
176,177
------
Cmpd PACA-2
153
~ 1
156 2.3 28
~8
160 4.9 47
., ·1
1.8
I.7x 10 2.3x 10
·1
161 0.63 3.3x10 >1.0 >1.0
1 ·1
162 0.69
~
163 0.70
~ , ~ 1
1.56
7.55x10 1.23xl0
1 1
164 0.56 3.34x 10
~
1.03xl0
~
1.66
1 1
165 0.61 3.08x10
~
2.28x 10
~
1.54
166 0.60
., 1.48
9.09x10 6.45x I0
1 1
168,169 0.11 1.74xl0
~
5.70xlO
~
>1.0
.\ .\
171 34 1.08 6.95 2.30 4.92 1.26x1O 4.13xlO
172 34
.\
2.26
.\
2.36
., .\
3.4xlO 7.8xlO 1.95xl0 7.7xlO
.\ .\ ·2
173 8.9 I.7lxlO 17.93 1.63 6.07x10 1.14 3.58xlO
174 11.2
., 3.56 1.64 -\ -\
2.74x10 3.79x10 2.12xlO 162.x10
175 13.8
., 2.97 1.24
.\
2.06x10 2.58x10 2.28x10 4.28x10
176,177 4.8
-, -\ ., -\
1.32 <10
-2
<10 6.llxlO <10 1.22xl0
-------- -----~----
orythro
\ A
~
./ (CH2l.~ (CH2l '" (CH2l. ~_ 2 37
:w I 2 2
R3 OH o , 0 0
Cmpd [--R-Il~
----Ri R3 A
178 OH H H cis
179 OH H H trans
180 H OH H cis
181 H OH H trans
182 H H OH cis
183 H H OH trans
7°
/hreo
erythro
Ifans I trans ~6 37
• , 16/ ~(CH) 4R. 35
", 29 . 0
ISR 0
OR
Cmpd-- Rl R2
184,185 H H OH H
186,187 H OH H H
188,189 OH H H H
200,201 H H H OH
A
4R~37
(CH 2)5--...-""-U ~y g
o
37
34
A B A B
193 cis (12S) threo (24R) 195 trans (12R) threo (24R)
194 cis (l2S) erythro (24S) 196 trans (l2R) erythro (24S)
118 1. L. McLAUGHLIN AND c.-1. CHANG
Cmpd
197
R
H
--r R1
OH
R2
H
R3
198 H H OH H
199 H H H OH
----~---- ----_._------
.2 -l !4 .2
178,179 7.00xlO 8.52xlO 1.86xl0 6.93xlO
180,181
., ., ., 1.51
4.43x10 1.30xlO 2.15x10
182,183 7.53xlO
.,
2.40x10
.,
1.35xl0
. 1.57xl0
.,
184,185
., ., ., ·12
4.05x10 1.66xl0 1.05xlO <10
186,187 1.58xl0
.,
3.29x10
.11
7.63xlO
. 1.09xlO
·12
188,189
., ., ., <10
·12
4.67x10 1.25xl0 1.61xl0
190 7.0xlO
., 1.79xl0
.,
1.00xlO
.,
5.23xlO
.,
191,192
., ., ., .,
1.37xlO 3.37xlO 1.07xl0 2.29x10
193 5.60x10
.,
1.51xlO
.,
8.53x10
.,
5.81xl0
.,
8.23xlO
.,
6.81xlO
. 1.25xl0
·IS
194
., ·11
>1.0 >1.0
., ., ·14
8.19x10 1.88xl0 3.48x10 2.15xl0 2.81x10
195
., ·11 ., ., ., 4.39xlO
., 8.90xl0
·IS
4.33x10 2.15xlO 2.lOxl0 3.67xlO 6.39xlO
197
., ., ., 1.48
., 1.62xlO-2 <10
.,
8.00x10 <10 <10 <10
198
., -8 ., 1.21
., ., <10.,
5.72xlO <10 <10 <10 3.16xlO
199
., ., ., 1.17
., ., <10.,
6.55x10 <10 <10 <10 4.33xlO
200 7.98x10
.,
4.38x10
. 4.46xl0
.,
6.90xl0
.. 4.35 2.33 1.02xl0
.,
201
., ., 1.71 1.45
., 9.04
.,
7.98xlO 3.18x10 1.00xlO 1.19xlO
-.- ... ~~-------------- -- ------~~---
BIOASSAYS AND ISOLATION OF NEW AGENTS 119
from India, contained some errors in the reported structures; I09 we reassigned the
relative stereochemistry of bullatalicin (196) and the bullatalicinones and identi-
fied the erroneous structures as bullatalicin, bullatanocin, and squamostatin A. 110
The bark has been an excellent source of bullatacin (61) for biological evalua-
tions; while purifying some bullatacin by HPLC, a shoulder peak was isolated
and identified as squamotacin (202), a bullatacin analogue in which the hydrox-
ylated bis-THF ring system starts at C-13 instead of C-15; molvizarin (203), a
C-35 analog of bullatacin (61), was also isolated; 202 and 203 showed remark-
able cytotoxic selectivity for the prostate (PC-3) cell line, with 202 showing a
potency more than 100 million times that of adriamycin; data for bullatacin (61),
which is more generally cytotoxic, are given below for comparative purposes. III
A patent has been filed, and 202 should be a major target compound for
synthesis.
Four new mono-THF acetogenins, cis- and trans-mosinones A (203, 204),
mosin B (205), and mosin C (206), along with annoreticuin (207), all possess a
carbonyl group at C-9; all of these compounds show selectivity against the pan-
creatic cell line (PACA-2), with potencies 10 to 100 times that of adriamycin.1I2
A patent has been filed, and 206 is proposed for in vivo tests against PACA-2
xenografts.
CH,
OH OH
m n
202
203
51
203
., ., >1
.; ., ·8 .;
5.26x10 6.30x10 7.32x10 7.09xlO 4.47x10 7.66x10
51
.; ·6 ., >1
., ·9 ·9
1.59xl0 2.44x10 5.9xlO 4.85xlO <10 <10
R3=~ B
~
R,-
~(E)
\ 20 0
I 15 2
9
2
0
OH OH 0 0
R.=~ d~37
R2 =
"0 0
Cmpd. A B C D E N C-1S/C-20
adriamycin 2.57xlO- 1 5.27x 10- 3 1.99x 10- 1 2.00xI0-' 1.02xlO-' 3.2IxI0-' 1.79xlO-'
Hollingworth et at. 117 concluded that bullatacin (61) is the most potent among the
several chemically diverse types of complex I ETS inhibitors known. In our lab-
oratory, Landolt et al. ll8 showed several structure-activity relationships (SARs)
among 20 structurally diverse acetogenins in the inhibition of oxygen uptake by
rat liver mitochondria, and some are more potent than rotenone in this subcellu-
lar system. Mitochondrial testing of 14 additional acetogenins found trilobacin
(69) and asiminacin (54) to be more potent than bullatacin (61).119 Similar SAR
conclusions were obvious when we tested 44 acetogenins in the yellow fever mos-
quito larvae (YFM) assay, with bullatacin (61) and trilobacin (69) giving the best
potencies (LC so values 0.10 and 0.67ppm, respectively; rotenone gave 1.2ppm);
compounds with YFM LC so values of <1.0ppm are considered as new lead
candidates for pesticide development. 12o Summaries of this mechanism and
these SARs have been published in our recent reviews on the Annonaceous
acetogenins. 41.58, 121
A second mode of action helps to explain the selectivity of the acetogenins
in inhibiting tumor vs. normal cells; an ubiquinone-linked NADH oxidase is acti-
vated in the plasma membranes of tumor cells, and this enzymatic activity is
potently inhibited by bullatacin (61) and other acetogenins. 122 The net effect of
this action and the action on the ETS results in intracellular ATP depletion, and
apoptosis (programmed cell death) is a consequence of the build up of radical
oxygen.123 Indeed, we have recently demonstrated that bullatacin (61) induces
apoptosis in WEHI 231 B cells, a murine B cell line that is at the immature stage
of B cell maturation (Geahlen and McLaughlin, unpublished).
The cell inhibition activities of several Annonaceous acetogenins, covering
the three major structural classes ofbis-adjacent, bis-nonadjacent, and single THF
ring compounds and their respective ketolactone rearrangement products, were
tested blind at Wayne State University in an in vitro disc diffusion assay against
three murine (P388, P03, and M17/Adr) and two human (H8 and HI2S) cancer-
ous cell lines, as well as a non-cancerous rat GI epithelial cell line (l18). The
results demonstrated a dose-dependent inhibition of cancerous cell growth, while
non-cancerous cell growth was not inhibited by the same dosages, All of the ace-
togenins, irrespective of their various structural types, inhibited the growth of
adriamycin resistant tumor cells and non-resistant tumor cells at the same levels
of potency. These results showed that the Annonaceous acetogenins are an
extremely potent class of compounds and their inhibition of cell growth can be
effective for drug resistant cancer cells, while exhibiting only minimal toxicity to
normal non-cancerous cells,124
Resistance mechanisms which require an ATP-dependent transporter would
be obvious targets for the useful application of these compounds as antitumor
agents, antimalarials, pesticides, or in any of the several systems in which such
ATP-dependent efflux is a significant factor, An important earlier observation
from the Ncr cell panel was that adriamycin resistant cell lines are almost always
more susceptible to the acetogenins than the respective wild type cells, e,g. MCF-
122 J. L. McLAUGHLIN AND c.-J. CHANG
710gIO GIso -5.86 molar and MCF-7/Adr 10gIO GI so -6.49 molar for bullatacin (61);
the acetogenins also routinely give more impressive selectivity and potency data
in the panel than almost all of the clinically used antitumor drugs. In the
COMPARE program,125 against the NCI bank of 175 "standard agents" for bul-
latacin (61) at all three levels (GI so , TGI so, and LC so ), there was nothing with a
correlation coefficient above 0.7, indicating no close mechanistic relatives. Con-
cerning bullatacin (61) vs. all of the compounds tested to that time, most of the
close matches were with related acetogenins (which we had provided earlier). The
NCI concluded that, " ... the mechanism of action has definite uniqueness."
We decided to confirm, in our own laboratory, the effects of the acetogenins
against multi-drug resistant (MDR) human tumor cells in vitro. Bullatacin (61)
was effectively cytotoxic to MCF-7/Adr (MDR) breast cells, while it was more
cytostatic to the parental MCF-7/wt (wild type) cel1. 126 This assay system was
extended to include 14 of our structurally diverse acetogenins; bullatacin-indexes
illustrated the MDR phenomenon (bullatacin 1.0; adriamycin 258.5; vincristine
141.3; vinblastine 18.8) and suggested several SARs: I) the bis-adjacent THF
acetogenins with three hydroxyls and the bullatacin-type stereochemistry (threo-
trans-threo-trans-erythro) from C-15 to C-24 are generally potent; 2) a spacing
of 13 carbon atoms between the I5-hydroxyl and the y-Iactone seems to be
optimum; and 3) some of the mono-THF compounds, e.g. gigantetrocin A are
potent. 127 Thus, we believe that this is good rational evidence to suggest in vivo
studies with athymic mice bearing xenografts of MDR human tumors; we have
applied for a patent protecting the use of acetogenins in treating MDR tumors.
A logical extension of this work was into the pesticide field. At Purdue's
Entomology Department, we compared six Annonaceous acetogenins (two
each from the mono-THF, nonadjacent bis-THF, and adjacent bis-THF classes)
with five commercial insecticides representing the amidohydrazone, carbamate,
organophosphate, and pyrethroid classes. Dietary toxicities were determined to
insecticide-resistant and insecticide-susceptible strains of Blattella germaniae
(the German cockroach), 2nd and 5th instars. The speed of kill (LTso) values per-
mitted ranking of all 11 compounds, and parviftorin (57) was ranked second with
cypermethrin (a synthetic pyrethroid) having a slight edge. Resistance ratios were
calculated and were, as predicted, lower for the acetogenins than for the synthetic
compounds. Only hydramethylnon (an inhibitor of ATP production at ETS
complex III) gave comparable resistance ratios. Some of the acetogenins were
even more effective against the resistant vs. the nonresistant roaches. Thus, the
acetogenins show great promise in treating insecticide-resistant insects in a new
type of baited "Roach Motel".67b
A glance at the cytotoxicity data tabulated above for our new acetogenins
reveals some remarkable potencies and selectivities for certain tumor cell types.
For example, squamotacin (202), the ring shifted bis-THF analog of bullatacin
(61), is selective for the prostate (PC-3) cell line, with a potency of over 100
million times that of adriamycin; the nonadjacent bis-THF bullatanocinlbullatal-
BIOASSAYS AND ISOLATION OF NEW AGENTS 123
icin series (193-196) and the sylvaticin series (114-117) are extremely potent
(some LCso's at <IO-8Ilg/ml) for the pancreatic (PACA-2) cell line; the unique
tris-THF, cyclogoniodenin T (138), and the 9-carbonyl mono-THF compounds
(203-207) also showed selectivity for PACA-2; the mono-THF, longicin (87), was
surprisingly potent to PACA-2 and the lung (A-549) cell lines; and the asimicin
series (51-55) is extremely potent (LCso's < IO-12Ilg/ml) against the colon (HT-
29) cell line. We predict that specific acetogenins may well become useful in the
therapy of specific tumor types as well as their MDR descendants, and patents
for some of these specific applications have been submitted.
Limited in vivo antitumor tests of the Annonaceous acetogenins have given
additional encouraging results. Uvaricin originally showed in vivo activity against
3PS (P388) (murine lymphocytic leukemia) (157% TIC at I Amg/kg), and rolli-
nones (147% T/C at I Amg/kg) and asimicin (51) (124% T/C at 25.0 Ilg/kg) were
similarly active, with 51 showing fifty times the potency of the other twO. 128 We
reported the activity ofbullatacin (61) and (2,4-cis and trans)-bullatacinones (66,
67) against U2l0 (murine leukemia) in normal mice and in inhibiting tumor
xenografts of A2780 (human ovarian carcinoma) in athymic mice. 107 Bullatacin
(61), effective at only 50.0Ilg/kg, was over 300 times more potent than taxol
against U2l0, and 61, again at 0.05mg/kg, and bullatalicin (196), at I mg/kg
were nearly as effective as cis-platinum, while causing much less weight loss.
Previously unpublished results, obtained by the late G. Grindey at the Eli Lilly
Co., showed similar effectiveness of61 (67% tumor growth inhibition at 50 Ilg/kg)
against X-5563 plasma cell myeloma grafts in normal mice. A favorable ratio of
cytotoxic EDso values vs. LDso values in mice suggested that bullatacin (61)
should be less toxic in vivo than currently used drugs such as cis-platinum. 129 The
difficulties involved in securing additional in vivo studies, at either the NCI
or within the pharmaceutical industry, have been the major road block in the
development of these compounds as new, clinically useful, antitumor agents.
Ames test results (Sitek Research Laboratories, unpublished results) on
F005 from paw paw were negative in 9 out of 10 tests and only slightly positive
(2.5% above background reversions) on one histidine mutant of Salmonella
typhimurium after enzyme activation of the extract. This negative result demon-
strates that the acetogenins are not mutagenic, and these results were to be
expected because the acetogenins, unlike most other antitumor agents, do not
exert their primary effects by interfering with DNA; they inhibit ATP production.
In other unpublished results (Asta Laboratories), bullatacin (61) was 3PS
active (131 % TIC at 50llg/kg) in mice and emetic at 1851lg/kg in pigs; this latter
result demonstrated that the acetogenins likely explain the former use of Eli Lilly's
fluid extract of paw paw seeds as an emetic preparation. 130 Thus, emesis is a def-
inite safety factor should someone ingest excessive amounts of these materials
either intentionally or unintentionally. Antiemetic drugs, as are administered with
cis-platinum, may likely be needed for antitumor treatment with the acetogenins,
and/or emesis can be used as a convenient signal that overdosage has occurred.
124 1. L. McLAUGHLIN AND c.-1. CHANG
SUMMARY
Four simple (bench-top) bioassays are serving us well for the detection and
fractionation monitoring of new plant antitumor and pesticidal agents. These are:
(1) lethality to the larvae of brine shrimp (Artemia salina); (2) the inhibition of
crown gall tumors, induced by plasmid transfer and expression from Agrobac-
terium tumefaciens, on discs of potato (Solanum tuberosum) tubers; (3) the inhi-
bition or stimulation of frond proliferation of duckweed (Lemna minor); and (4)
lethality to the larvae of yellow fever mosquitoes (Aedes aegyptii). Since 1984,
over 320 chemically diverse bioactive plant components have been isolated and
characterized in our laboratory by using these methods. Recently, bioactive com-
pounds from the Meliaceae, Lauraceae, Euphorbiaceae, Laminaceae, and other
plant families have been isolated, but our most exciting leads have been with
the potent acetogenins from the Annonaceae; these compounds are powerful
inhibitors of mitochondrial electron transport systems and of the NADH oxidase
that is prevalent in the plasma membranes of tumorous cells. The consequence is
ATP depletion, and this is especially toxic to multiple drug resistant tumor cells
and pesticide resistant insects that possess ATP-dependent xenobiotic efflux
systems. Structural activity relationship studies (in mitochondrial preparations
and against mosquito larvae) help to define the optimum structural features. This
paper has presented the chemistry and biological testing results of 207 plant
components recently isolated using the simple bioassays described folIowed by
cytotoxicity testing in a panel of six human tumor cell lines.
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125. PAULL, K.D., SHOEMAKER, R.H., HODES, L., MONKS, A., SCUDIERO, D.A.,
RABIN STEIN, L., PLOWMAN, 1., BOYD, M.R. 1989. Display and analysis ofpattems
of differential activity of drugs against human tumor cell lines: Development of mean
graph and COMPARE algorithm. 1. Nat. Can. Inst. 8 I: 10898-1092.
126. OBERLIES, N.H., CROY, Y.L., HARRISON, M.H., MCLAUGHLIN, 1.L. 1997. The
Annonaceous acetogenin bullatacin is cytotoxic against multidrug resistant human
mammary adenocarcinoma cells. Cancer Lett. 115:173-179.
127. OBERLIES, N.H., CHANG, C.J., McLAUGHLIN, 1.L. 1997. Structure-activity rela-
tionships of diverse Annonaceous acetogenins against multi drug resistant human mnam-
mary adenocarcinoma (MCF-7/adr) cells. 1. Med. Chern. 40:2102-2106.
128. RUPPRECHT, 1.K., HUI, Y-H., MCLAUGHLIN, 1.L. 1990. Annonaceous acetogenins:
A review. 1. Nat. Prod. 53:237-278.
129. HOLSCHNEIDER, C.H., JOHNSON, M.T., KNOX, R.M., REZAL, A., RYAN, W.J.,
MONTZ, E.1. 1994. Bullatacin in vivo and in vitro experience in an ovarian cancer model.
Cancer Chern. Pharm. 34: 166- I 70.
130. Anonymous. 1898. Lilly's Handbook of Pharmacy and Therapeutics, 13th edition, Eli
Lilly and Company, Indianapolis, p. 89.
131. FANG, X.-P., RIESER, M.1., GU, Z.-M., LZENG, MCLAUGHLIN, 1.L. 1993. Annona-
ceous acetogenins: An updated review. Phytochem. Anal. 4:27-48 and 49-67.
132. ALALI, F.Q., LIU, x.-X., MCLAUGHLIN, 1.L. 1999. Annonaceous acetogenins: Recent
progress. J. Nat. Prod. 61: (in press).
Chapter Six
133
134 R. A. DIXON et al.
INTRODUCTION
derivatives are almost ubiquitous among terrestrial plants, the isoflavonoids are
restricted primarily to the Leguminosae, although they occur rarely in other
families such as the Apocynaceae, Pinaceae, Compositae, and Moraceae. 5•6
The nearly 900 naturally occuring isoflavonoid aglycones can be divided into
9 major classes based on differences in their basic carbon skeletons. 5 Members of
these various classes, some of which will be described in more detail later in this
article, are shown in Figure I. The isoflavones and pterocarpans are the most abun-
dant isoflavonoids, with 334 and 152 different structures, respectively, having been
described as of September 1994. This enormous diversity is caused by the various
substituents, for example methoxyl, prenyl, methylenedioxy, that can occur on
many different positions of the A and Brings.
The limited taxonomic distribution of the isoflavonoids is directly related to
the occurrence of the enzyme isoflavone synthase, which catalyzes the aryl migra-
tion reaction leading to the formation of an isoflavone from a flavanone (see below).
It is probable that this enzyme has evolved independently in the Leguminosae and
the other diverse taxa in which isoflavonoids are occassionally found.
HO%HO~I
I I ....- ~ H HO
~ ~ ~I
o ~ I CH 30 ~ OCH 3
OCH 3
OH
Daidzein (isoflavone) (-)Sativan (isoflavan) (-)Medicarpin (pterocarpan)
HO%I
~ ~ o
° ~I OH o >
Coumestrol (coumestan) (+ )Pisatin (pterocarpan)
HO HO
HO%II
~ ;:?
OH ° ~ I OCH 3
Genistein Formononetin
Biochanin A
oo~ H°'COu
~ 1 ;:?
~
I
OH
OH Equol
Estradiol 17-13
Tamoxifen
and increases estrogen receptor and plasma prolactin levels in mice,24 although it
is 15,000 times less potent than estradiol for binding to murine mammary estro-
gen receptors. 24 Thus, its major biological activity is probably as an estrogen
agonist, although its concentration in the "Western" diet is probably too low
to cause physiological effects. However, in humans eating a soy-rich diet,
isoflavonoids may be present in the urine at very high levels. For example, human
adults given a daily diet containing 40 g of soy protein secreted 5.3 mg of equol
per day, compared with only 2-27 Jlg of estrone glucuronide, the principal urinary
estrogen that is released during the follicular phase of the menstrual cycle. 25 This
is a 100-fold increase in urinary equol above that observed in adults who consume
little soy products in their diet.
The observation that phytoestrogens can be assayed by activation of an
estrogen-regulated transcription system in yeast cells 26 provides a method for
rapid screening of these compounds. This approach is now being applied to
extracts from plants, such as Artemesia,27 used in herbal medicine for treatment
of conditions such as menstrual abnormalities. It is likely that several species will
contain bioactive flavonoids and isoflavonoids.
An interesting, but yet unresolved, question is whether plants themselves
contain estrogen receptor-like proteins, and, if so, whether phytoestrogens may
function hormonally in plants. Such a view would have held little weight ten
years ago, but the recent demonstration of the functions of the brassinosteroids in
plant growth and development give credence to the possibility that new
classes of plant regulators may await discovery. In this regard, it is interesting that
dioecious species, such as the Osage orange (Madura pomifera), contain
estrogen-receptor binding compounds, the levels and seasonal variations of which
are consistent with a possible role in sex determination. 26 Although the exact nature
of these compounds has yet to be determined, it is interesting that M. pomifera, a
member of the Moraceae, contains at least three prenylated isoflavonoids. 6
3X ~-SCOA
CH
I 2
COO-
+
CoAS
/3'<:::
I
g
OH
3CoASH
c~~
t
CoAS
t
Malonyl CoA 0 ~ 0 o o o
4-Co"m",o," Co. 'C~
NADPH 1 CHS • ''''"'.~'''
YY
HOyyOH , , ( ) - o : H S
OH 0
-= CoAS
0 o OH 0
I
HO~OH
fj ~ OH
:::,..
I I -
o
2' ,4,4'-Trihydroxychalcone
(isoliquiritigenin)
Figure 3. Reactions catalyzed by chalcone synthase in the presence and absence of chalcone
reductase.
MOLECULAR CONTROLS FOR ISOFLAVONOID BIOSYNTHESIS 141
of the CHS gene family are constitutively expressed in roots and root nodules,
but not in the aerial parts of the plant. However, these family members are
expressed in leaves, at the onset of the isoflavonoid phytoalexin defense response,
following exposure to pathogens or elicitors. 47 The CHS proteins encoded by the
different gene family members are generally very similar in primary sequence,47
and it is not known if they possess different kinetic properties or are differentially
localized in the cell.
Many isoflavonoids lack the 5-hydroxyl group (6'-hydroxyl, chalcone num-
bering), and are thus derived from 2',4,4'-trihydroxychalcone rather than from
naringenin chalcone. The 5-deoxy isoflavonoids are particularly prevalent in
legume roots, and the pterocarpan phytoalexins are invariably of this class. 13C_
Labelling studies indicated that the 5-hydroxyl group is lost prior to the cycliza-
tion of the A-ring of the chalcone,52 presumably at the polyketide stage. Crude
extracts from elicited cell cultures of Glycyrrhiza echinata were shown to produce
2',4,4' -trihydroxychalcone and its corresponding flavanone liquiritigenin, in addi-
tion to naringenin chalcone, from malonyl CoA and 4-coumaroyl CoA in the pres-
ence of high concentrations of NADPH. 53 The chalcone was produced first and
then converted to the flavanone by chalcone isomerase present in the preparation.
The activity was described as 6'-deoxychalcone synthase, and was demonstrated
also in G. echinata protoplasts. 53
Purified soybean CHS was shown to require the presence of a separate
protein, given the trivial name of "chalcone reductase" (CHR), for NADPH-
dependent formation of (iso )liquiritigenin. 54 The reductase was purified to appar-
ent homogeneity and was shown to be a monomer of Mr 34,000, that catalyzed
the transfer of the pro-R-hydrogen of [4-3H]NADPH to the polyketide bound to
CHS, with resultant loss of the hydroxyl function as water (Fig. 3). The enzyme
exhibited approximately 90% maximal activity at a molar ratio (CHS : reductase)
of 2: I, and could co-act with CHS from parsley, a species that does not synthe-
size 6'-deoxychalcone derivatives. 54 This latter point suggests that the multiple
forms of CHS found in legumes are unlikely to be involved differentially in the
formation of 6'-deoxy- and 6'-hydroxy-chalcones, although this remains to be
proven experimentally.
CHR is encoded by a small gene family in soybean 55 and alfalfa,56-59 and
has also recently been cloned from Pueraria lobata and Glycyrrhiza echinata;59.6o
the gene does not appear to be present in species such as carrot and parsley that
do not accumulate 5-deoxy isoftavonoids. 55 The enzyme possesses a leucine
zipper domain, but it is not yet known if this is involved in interactions with CHS.
Although the enzyme is a polyketide reductase, it does not share significant
sequence identity to the reductases of fatty acid synthesis; rather, it is related to
a mammalian aldose reductase and to 2,5-diketo-D-gluconic acid reductase from
Corynebacterium. 55
It is still not clear why co-action of CHR with CHS results in no more than
142 R. A. DIXON et al.
100 A
50
~ 25
.:;
U
a:I
Q)
>
~
<i.i 100
a:
75
Figure 4. Relative transcrip-
50 tion rate kinetics of (A)
CHS (00) and (8) CHR (00)
in relation to PAL (0) in
25 elicitor-treated alfalfa cell sus-
pension cultures. Values from
nuclear transcript run-on
analyses are normalized to
3.0 100% maximum level. Data
Hours post elicitation from ref 59.
BeanchslS
-72
Figure 5. Diagramatic representations of the bean chs15 and alfalfa chr7 promoters. SB =
silencer box. Boxes marked G' contain single base mismatches from the chslS G-box core
sequence (CACGTG). See text for details.
likely, but not yet proven, that these elements bind transcription factors responsi-
ble for the co-ordinated transcriptional activation of CHS and CHR.
Isoftavone Synthase
HOWl ' b
H
V
I OH
HO
HO
~ NADPH
• OH
OH Genistein
(-)(25) Naringenin 2,5,7,4'- Tetrahydroxy-
isoflavanone
shifted by two mass units, whereas there was no corresponding shift in the mol-
ecular ion of the isoflavone, consistent with the subsequent dehydration reaction.
The dehydration reaction appears to be catalyzed by an activity present predom-
inantly in the cytoplasmic supernatant. The 2-hydroxyisoflavanone can also
spontaneously convert to the isoflavone. The enzyme is stereoselective, and (2R)-
naringenin is not a substrate.
The model for the reaction pathway of "isoflavone synthase" (Fig. 6)65 there-
fore involves P450-catalyzed hydroxylation coupled to aryl migration, a reaction
with mechanistic similarities to the well-described proton migration mechanism
of some P450 reactions. Similarities between the mechanism ofIFS and other reac-
tions, such as ring condensation of ent-7-hydroxy-kaurenoic acid to GA I2 aldehyde
in gibberellin biosynthesis, and sterol demethylation, have been discussed. 65
To date, there have been no reports on the purification to homogeneity, or
the molecular cloning, of either of the two enzymes of the IFS complex. The
flavanone 2-hydroxylase cytochrome P450 from Pueraria has been solubilized
with Triton X-IOO, and partially purified by ion exchange chromatography; the
enzymatic reaction could be reconstituted by addition of NADPH cytochrome
P450 reductase. 66 The 2-hydroxyisoflavanone dehydratase has been purified from
elicitor-treated P. lobata cells, and is a soluble monomeric enzyme of subunit Mr
38,000. 60 •67 It is not yet clear whether this enzyme physically associates with the
P450 hydroxylase catalyzing the aryl migration.
The low abundance and extreme lability of the isoflavone synthase
cytochrome P450 make purification by classical procedures a daunting task. The
approach taken in our lab to cloning IFS has been to isolate cDNAs encoding
cytochrome P450s by a PeR-based strategy similar to that reported recently for
Arabidopsis P450s. 68 This is made possible by the existence of small motifs with
significant sequence homology in all known P450s. By using this strategy, we
have isolated 6 novel P450s from an alfalfa cell suspension library, and have
shown that 5 of these are elicitor-inducible (C.L. Steele and R.A. Dixon, unpub-
lished results). After suitable modifications to optimize expression, full length
cDNAs have been expressed in E. coli and in insect cell cultures, and functional
analysis is underway.
COO-
+3 \:OSCOA
malonyl-CoA
phenylalanine p-coumaroyl-CoA
CHS+CHR ~
H0yYHIr<}-OH
4,2',4'-trihydroxychalcone ~ '=-'
CHI ~ 0
oH
7,4'-{jihydroxyflavanone H 0 W - O -
IFS ~°
daidzeinHO~) r-\. . H
~
I°
7-IOMT/
CH,0'O?-o- /' .'·IOMT
,.hydm><ylo~oo,,'o HO~o,,",
IFR ~ 0 HO
(3A,....."''' HO~OC"'
PTS~O HO
HO
(6a, 11 aR)-medicarpin
or (-)-medicarpin
Four internal peptide sequences were obtained from the purified protein,
one of which had high (72%) sequence identity to a region of a catechol-O-
methyltransferase from barley. All 4 internal peptides respectively had about
55% amino acid sequence identity to 4 regions of 6a-hydroxymaackiain 3-0-
methyl transferase from Pisum sativum,77 but had no sequence identity to the
alfalfa COMT or chalcone 2'-0-methyltransferase (ChaIOMT) genes previously
cloned in our laboratory. Sequence information from three internal tryptic pep-
tides was used to design oligonucleotide primers to facilitate cloning of a full-
length IOMT cDNA. At least two different IOMT genes are present in the genome
of tetraploid alfalfa, whereas a single gene is present in chickpea and cowpea. 78
Sequences hybridizing to alfalfa IOMT were not present in the genomes of
tobacco and potato. The alfalfa IOMT8 cDNA was functionally expressed in E.
coli, and the properties of the enzyme shown to be essentially identical to those
of the enzyme purified from cell cultures. 78 Strong induction ofIOMT transcripts
preceded the accumulation of medicarpin in elicitor-treated cell cultures.
We believe that the enzyme with isoflavone 7-0MT activity in vitro methy-
lates the 4'-position in vivo. The unexpected precursor feeding results in alfalfa
can be explained if the OMT is in a "metabolic compartment" or "channel," and
its association with the enzymes producing its substrate or removing its product
could account for the different regiospecificities observed in vivo and in vitro.
The isoflavone synthase and 2' -hydroxylase are both microsomal cytochrome
P450s, with which the 4' -OMT could be physically associated. Thus, only
daidzein formed in situ by microsomal isoflavone synthase, but not exogenously
supplied daidzein, might act as substrate for the OMT. Such close associations
might be necessary if the methyltransferase could only act on the 4'-position
during the aryl migration of the B-ring, the pK of the A-ring 7-hydroxyl making
it the preferred site for methylation in a non-associated in vitro system. We have
expressed the alfalfa IOMT in the yeast two-hybrid system as a fusion to the gal4
DNA binding domain, and used this to screen an elicited alfalfa culture cDNA
library fused to the gal4 transactivation domain for expressed proteins interact-
ing with the methyltransferase. This approach failed to reveal any alfalfa proteins
capable of interacting with the methyltransfrease in yeast cells (IT. Reddy and
R.A. Dixon, unpublished results).
It is now possible to test the methyltransferase channel hypothesis by mol-
ecular genetic strategies. We have recently generated a large number of indepen-
dent alfalfa transformants harboring the IOMT cDNA sequence in both sense and
antisense orientations, under control of the constitutive cauliflower mosaic virus
35S promoter (X.Z. He and R.A. Dixon, unpublished results). Some plants con-
taining sense constructs express high levels of IOMT transcripts in the leaves, an
organ in which IOMT is not normally expressed. Cell suspension cultures have
been initiated from these plants to test for the impact of the transgene expression
on isoflavone metabolite levels. Reduction of medicarpin formation in antisense
148 R. A. DIXON et al.
start site fused to the CHS15 H-box region in an in vitro transcription system (Q.
Zhu, F. McAlister, R.A. Dixon and C. Lamb, unpublished results). Studies are in
progress to confirm the nuclear localization of KAP2 following elicitation, by
using immunocytochemical and fluorescent gene-fusion approaches. The phos-
phorylation status of KAP2 does not affect its DNA binding activity,84 but may
impact its ability to interact with KAPI and possibly other transcription factors.
It may also affect nuclear transport of KAP2.
Screening a cDNA expression library with the region of the CHS15 pro-
moter containing both the H-box I and the G-box led to the cloning of a DNA
binding protein, G/HBFl, that binds both the G-box and H-box (in the context
of the CHS15 promoter).92 G/HBF I is a member of the basic leucine zipper family
of transcription factors, and its closest homo logs in plants are a member of the
opaque family of factors that control zein gene expression in maize, and the
CPRF2 factor that binds the G-box in the parsley CHS promoter. 93
Western blot analysis revealed that elicitation or infection result in the rapid
appearance of a phosphorylated form of GIHBF -1. 92 Use of E. coli expressed
G/HBFl as a substrate in in vitro kinase assays showed that this is associated with
the rapid induction of the protein kinase activity that phosphorylates G/HBF-1.92
To isolate the protein kinase involved in signal transduction to G/HBF -I, we cloned
G/HBF-l as a fusion to the gal4 DNA binding domain as a bait in the yeast two-
hybrid system, and used this to screen a soybean cell culture cDNA library (from
elicited and unelicited cultures) fused to the gal4 transactivation domain for
expressed proteins interacting with G/HBF-1. This screen resulted in the
identification of a calmodulin domain protein kinase (CDPK) that specifically
interacts with G/HBFl (P. Canovas, J.T. Reddy, C. Lamb and R.A. Dixon, unpub-
lished results). CDPKs are calcium dependent, and elicitor-mediated CHS tran-
scription also requires calcium. The two-hybrid screen also identified a protein
phosphatase that might be involved in G/HBF -1 regulation. Baculovirus-expressed
G/HBF-l kinase phosphorylates G/HBF-l in vitro (P. Canovas, S. Hedrick, R.A.
Dixon and C. Lamb, unpublished results). Studies are now in progress to address
functionally the role of G/HBF-l kinase by transgenic approaches.
Transcription of the downstream isoflavone reductase gene is slightly
delayed compared to that of PAL or CHS genes in elicited alfalfa cell suspen-
sions, consistent with its responding to different transcriptional regulators. 48 This
idea is confirmed by the results of in vivo (functional) and in vitro analyses of
the alfalfa isoflavone reductase promoter (S. Yin, B. Miao and N.L Paiva, per-
sonal communication), which appears to be regulated by transcription factor(s)
recognizing sequences not involved in CHS regulation. Availability of alfalfa
CHR and isoflavone O-methyltransferase gene promoters, coupled with genetic
approaches in model legumes, will now enable us to address whether common
or disparate transcriptional regulators are involved in coordinating the transcrip-
tional activation of the isoflavonoid pathway.
MOLECULAR CONTROLS FOR ISOFLAVONOID BIOSYNTHESIS 151
The above studies have identified a protein kinase cascade tightly linked to
the transcriptional activation of genes involved in the elicitor/infection-induced
synthesis of isoftavonoid phytoalexins. However, the induced hypersensitive
defense response in plant cells is a multi component one, involving rapid pro-
duction of active oxygen species (the oxidative burst), induction of anti-oxidative
enzymes, metabolic changes including cell wall reinforcement and phytoalexin
accumulation, production of various pathogenesis-related proteins, and genera-
tion of long distance signals for activation of systemic defense responses. 1,83,94,95
Signal transduction pathways must operate intracellularly (from elicitor reception
to gene activation) and intercellularly, the latter at both short distances (to signal
metabolic changes to the periphery of the hypersensitive lesion), and long dis-
tances (for activation of systemic responses). Understanding the complexities of
these various signal pathways and at what levels they diverge or converge is a
major unresolved problem in molecular plant pathology. We have used a some-
what simplistic pharmacological approach to attempt to dissect signaling path-
ways in a model soybean cell culture system that exhibits a strong oxidative burst,
anti oxidative defenses, and isoftavonoid accumulation in an avirulence gene
dependent manner.
Exposure of suspension cultures of soybean cv Williams 82 to Pseudomonas
syringae pv glycinea (Psg) harboring the avrA avirulence gene, which is recog-
nized by the Rpt resistance gene in Williams 82, results in an oxidative burst that
generates maximum concentrations of hydrogen peroxide approximately 4 to 6
hours after exposure to the bacteria. 96 A burst with similar kinetics is observed in
response to yeast elicitor. However, the large oxidative burst is not observed if the
soybean cells are exposed to Psg harboring the avrC avirulence gene, the corre-
sponding resistance gene for which is absent from cv Williams 82. Likewise, accu-
mulation of the isoftavonoid phytoalexin glyceollin is observed in cultures exposed
to Psg: avrA or yeast elicitor, but not to Psg: avrc. 97
The oxidative burst in cells exposed to Psg: avrA is inhibited by the protein
kinase inhibitor K252a, and the burst can be induced, in the absence of bacteria,
by the protein phosphatase inhibitor cantharidin, indicating the operation of
a finely poised protein phosphorylation/dephosporylation cascade controlling the
activity of the membrane associated NADPH oxidase responsible for active
oxygen generation. 96 The oxidase, and subsequent production of hydrogen
peroxide, is inhibited by the suicide inhibitor diphenylene iodonium (DPI).
Hydrogen peroxide generated in the oxidative b,urst acts as a diffusible
signal for induction of genes encoding anti oxidative enzymes such as glutathione
S-transferase,98 but very high concentrations of hydrogen peroxide only weakly
152 R. A. DIXON et al.
~~[(:)l
[(:-)l~~do kinase cascade
~
Phosphorylation
of GIHBF-l and
other transcription
factors
Salicylic .
acid PhenYlalamne)
t PAL
L - Cinnamic 'lcid enzymes
L 1 Downstream phenylpropanoid/
isoflavonoid pathway enzymes
I Isoflavonoid phytoalexins I
Figure 8. Scheme for signal transduction pathways for induction of the oxidative burst and
isoflavonoid phytoalexin accumulation in soybean cells. See text for details.
induce genes that encode phytoalexin biosynthetic enzymes such as PAL and
CHS. Treatment with DPI completely abolishes glyceollin accumulation
following exposure of soybean cells to Psg: avrA or yeast elicitor, suggesting that
hydrogen peroxide may act as a signal for phytoalexin induction. 97
Several serine protease inhibitors, including phenylmethylsulfonyl fluoride
(PM SF) and diisopropylfluorophosphate (DFP), although themselves inactive
as inducers of the oxidative burst and phytoalexin production, greatly potentiate
these responses if added along with Psg: avrA or yeast elicitor, and potentiation of
the oxidative burst by PMSF or DFP is completely inhibited by K252a or
DPI, suggesting that the potentiated burst operates through the kinase-mediated
pathway leading to stimulation of NADPH oxidase. 97 Interestingly, PMSF or
DFP treatment also results in a strong oxidative burst in soybean cells exposed to
Psg: avrC, and this is again blocked by DP!. However, this burst does not result in
glyceollin accumulation. Thus, generation of hydrogen peroxide is necessary, but
not in itself sufficient, for induction of isoflavonoid phytoalexin accumulation.
MOLECULAR CONTROLS FOR ISOFLAVONOID BIOSYNTHESIS 153
Clearly, a signal for isoflavonoid induction is generated in the Psg: avrA interac-
tion that is not generated in response to Psg:avrC, even when a synergistic inter-
action between Psg: avrC and PMSF or DFP leads to a strong oxidative burst. A
scheme summarizing potential signal pathways for the oxidative burst and
isoflavonoid phytoalexin induction is shown in Figure 8.
Treatment of cell suspension cultures of soybean, alfalfa, or tobacco with
the macromolecular elicitor cryptogein (a 10.3 kDa peptide from Phytophthora
cryptogea) results in the release of low molecular mass (500-2,OOODa) endoge-
nous elicitors that can pass through a dialysis membrane into the surrounding
culture medium. 99 The active compounds induce PAL and IFR transcripts and
enzymatic activity, accumulation of glyceollin, and an oxidative burst in soybean
cells. Release of the factors from soybean cells is inhibited by the RNA synthe-
sis inhibitor actinomycin D, but not by DP!. The activity of the factors released
from tobacco cells in response to cryptogein, or from alfalfa cells in response
to yeast elicitor, is unaffected by pre-treatment with sodium periodate or pro-
teinase K. In contrast, treatment of the factor(s) from soybean cells with pro-
teinase K or trypsin destroys its ability to induce glyceollin, suggesting that it
may be a small peptide. 99 The diffusible elicitors have now been extensively
purified, but structural characterization has been hampered by their extremely low
abundance, in spite of their high biological activity. This potency suggests that
they may indeed be important signal molecules for the intercellular transmission
of the elicitation response.
CONCLUSIONS
Several important advances have been made during the last few years in
our understanding of the molecular biology of isoflavonoid biosynthesis and its
control, and of the important effects of dietary isoflavonoids on human health.
The challenge for the future will be to devise strategies for manipulating the levels
of isoflavonoid compounds in transgenic plants, not only in legumes in which
these compounds occur naturally, but also in other plant species including major
food crops. The aims of these studies will be to increase either natural plant
disease resistance or dietary intake of health-promoting phytochemicals.
ACKNOWLEDGMENTS
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Chapter Seven
Ulrich Matern
INTRODUCTION
161
162 U. MATERN
7 - Methylesculin
Umbelliferone R1 =OH; R2 =H
Esculetin R1 = R2 =OH
Robustic acid
OMe
O~
Phellopterin
the road to detailed molecular and regulatory studies, and significant progress
has been accomplished recently. Furthermore, it has long been known that
some coumarins show remarkable bioactivities, e.g. the anticoagulant effect of
4-hydroxycoumarins or the anti proliferative and phototoxic action of linear
furanocoumarins as well as the inhibitory action on 5-lipoxygenases. 22 - 24 More
recently, new and surprisingly diverse activities have been ascribed to other
coumarins, some of which have received public attention and may serve as lead
structures in drug development. A few such examples will be considered before
briefly reviewing some recent developments in the research of biosynthesis.
( + ) - Calanolide A
)?:~
~~n~
~ 0
(:t
••'
110
,.............. ~
11
12
OH
Figure 2. Structures and bioactivities of calanolides. The anti-HIV-I activity (EC so ) and the
cell toxicity (lC so ) are indicated.
as well as the spatial orientation of the two hydroxy groups in conjunction with
the two pyrone-carbonyls are essential components of the pharmacophor. 32 .33 The
integration of the viral DNA copy into the host genome proceeds in three steps,
beginning with the recession of the 3' end followed by cleavage of the host DNA,
strand insertion into the host, and finally sealing of the nicks by DNA ligase.
Retroviral integrases contain three conserved acidic residues (Asp, Asp, Glu),
which are critical for the nuclease activity; these residues are assumed to facili-
tate the coordination with divalent metal ions and the phosphodiester backbone
during the 3' recession of the viral DNA copy. Accordingly, it was proposed that
the oxygens of the 4-hydroxycoumarin rings become coordinated to the essential
metal ions and thereby disrupt their binding to the enzyme or enzyme-DNA
complex. 32 .33 Taken together, coumarins are now being employed to inhibit all
three pivotal enzyme activities of HIV-I replication, the reverse transcriptase, the
protease, as well as the integrase.
MEDICINAL POTENTIAL AND BIOSYNTHESIS OF PLANT COUMARINS 165
Figure 3. Synthetic coumarin inhibitors of HI V-I integrase. Modification of the tetrameric (left)
to the dimeric 4-hydroxycoumarin (right) considerably reduced the inhibitory efficiency (lC,o).
BIOSYNTHESIS OF COUMARINS
Simple Coumarins
6
eOOH eOOH eOOH
ONH'
OH
/ ~
8° 8°
lfi
-...:::
OH
OH
OH
\ /
ro # 0
Coumarin
0 -HO CO .& 0
Umbelliferone
0
reviewed recently, and the enzymes have been allocated almost exclusively to the
plastids. 49 Unfortunately, the cyclization of cinnamic or 4-coumaric acid to
coumarin and umbelliferone, respectively (Fig. 4), has not been conclusively
elaborated in vitro, and it remains to be established whether the acids or the cor-
responding CoA-esters are the physiological substrates of cyclization.</5o.51 Fur-
thermore, it is unclear whether the cyclization proceeds in two steps, i.e.
artha-hydroxylation followed by lactonization, or in one step as was proposed,
for example, via a spiro-intermediate (Fig. 4 ).cf 50.51 Preliminary evidence in the
literature pointed to the plastids as the site of coumarin cyclization, and this is
supported by recent data on the prenylation of umbelliferone. 51}-52
168 U. MATERN
0"-
'-D
170 U. MATERN
Psoralen
o 0 -<
Z
....,
Umbelliferone ~ ::c:
m
[/).
ti'i
~~ o..,..,
Angelicin
HO b o 0 o ""r<
~ ~....,
I n
o
c:
3:::
Osthenol (-)-Columbianetin >-
;:>;;I
Figure 6. Sequence of reactions converting umbelliferone to linear (top) or angular (bottom) furanocoumarins.
Z
[/).
....,
172 U. MATERN
induction of the specific enzyme activities revealed that each step was catalyzed
by a different enzyme entity.5.8 It is likely that angular furanocoumarins are formed
in a similar way from osthenol (Fig. 6), but these reactions have not yet been
observed in vitro despite the fact that Ammi majus is capable of accumulating the
angular type of coumarins in its fruits at least. 68
The conversion of demethylsuberosin to (+)-marmesin had been proposed
to involve the epoxidation of the side-chain double bond and formation of the
corresponding diol. 3 However, although in principle P450 monooxygenases are
capable of inserting an "oxen" into 0Iefins,69.7o no such intermediate was observed
in vitro during the marmesin synthase reaction. It is more likely, therefore, that
the 7-hydroxyl group of demethylsuberosin favors the one step cyclization to
(+)-marmesin by delocalizing the side-chain double bond electrons. Model
mechanisms proposed for the interaction of the catalytic P450 oxo-derivative
(Fig. 7) with aliphatic double bonds support such a direct cyclization. 71 It should
be noted in this context that linear dihydropyrano- or pyronocoumarins such as
graveolone had been considered to originate from demethylsuberosin by a dif-
ferent cyclization reaction;3.72 preliminary evidence obtained with microsomes
from elicited parsley cells suggested, however, that graveolone is possibly formed
from (+)-marmesin. 5.8
The formation of psoralen from (+)-marmesin is a unique reaction in plant
secondary metabolism, since stoichiometric amounts of acetone must be
released. 3.5 The reaction had been proposed to be driven by the generation of the
carbenium ion of (+)-marmesin at C-3' followed by a 1,3-elimination step.3 This
hypothetical mechanism did not require the hydroxylation at C-3' and implied
a 2',3'-anti-elimination reaction. The identification of psoralen synthase as a
cytochrome P450-dependent monooxygenase in microsomes from elicited Ammi
majus or parsley cells refuted the proposal and pointed to an oxidative mecha-
nism involving iron-oxygen radical species. 5.8.67 After binding of the substrate,
P450 monooxygenases are reduced by cytochrome P450 reductase in the pres-
ence of molecular oxygen to yield both the reactive peroxyiron ([Felll-O-OH] and
oxoiron ([Fe'V-On enzyme species (Fig. 7). Accordingly, a sequential mechanism
appeared possible analogous to the side-chain dealkylation of cholesterol via
pregnenolone to the il '6 _steroid;69.71 this mechanism might involve the oxidative
functionalization of the isopropyloxy substituent in (+)-marmesin and removal of
one carbon by the reactive [Fe'V -0]" porphyryl radical cation (Fig. 7), leaving an
acetyl-derivative, followed by deacetylation through peroxyhemiketal formation
with the strong nucleophile [Felll-O-O-] (Fig. 7).69.71
In cooperation with the group of Boland, we revisited the mechanism of
the psoralen synthase reaction with stereospecifically deuterated substrates. 5.73
These studies revealed the elimination of the hydrogen in syn-orientation to the
isopropyloxy-substituent (Fig. 8). Furthermore, labeled acetone released from the
incubation was trapped with pentafluorobenzylhydroxylamine, clearly indicating
that the isopropyloxy side-chain was removed in one step. The microsomal
MEDICINAL POTENTIAL AND BIOSYNTHESIS OF PLANT COUMARINS 173
S(Cys) ~(Cys)
N~N N :
,;=-! N
/., 'Felll~1 RH e /.. .:::Fe ll
----/
N~I"
-N
N~'"'" "
-N
OH
2
RH
[Fe" l- 0 - OH]
[Fe 1v_ or
Figure 7. Schematic activation of cytochrome P450 yielding the ferric-hydroperoxy species
and the iron-oxo radical. The low-spin iron in the resting enzyme is converting to high-spin iron
upon substrate (RH) binding.
174 U. MATERN
HO~'"
.' <:Co
D,•••
0
H
I ~
bOO
~ + [Fe IV - 0)-
Fe IV 00 +
00
Felli +
~ OH
+
Felli + HDO
+
y
o
Figure 8. Schematic conversion of (+)-[3'-'H,jmarmesin to psoralen and acetone by syn-
elimination. The iron-oxo radical [Felv-Or abstracts the deuterium from carbon 3'. The reactive
intermediate loses the side-chain isopropyl radical by disproportionation, and the isopropyl
radical recombines with the 'HO radical in an oxygen-rebound process.
MEDICINAL POTENTIAL AND BIOSYNTHESIS OF PLANT COUMARINS 175
psoralen synthase accepted also the synthetic pseudosubstrate in which the iso-
propyloxy side-chain was replaced by an acetyl residue (Fig. 9). However, the
diol corresponding to (+ )-marmesin (iso-I,2-propylene residue), considered as
an intermediate in the hypothetical two step reaction, was not accepted; 73 this
diol is known as a natural plant metabolite (prandiolV Taken together, the
[Fe IV -Or porphyryl radical cation at the active site of psoralen synthase attacks
the Hs at C-3'of (+)-marmesin (Fig. 8). Homolytic abstraction of this hydrogen
yields a benzylic radical which then undergoes p-cleavage to psoralen and an
isopropyloxy radical. The latter radical reacts in an oxygen rebound process with
the adjacent porphyryl-Fe1V-OH center to acetone hydrate (Fig. 8).73 Synthetic
iron or manganese tetraphenylporphyryl complexes activated with iodosobenzene
also showed some psoralen synthase activity, but lacked the stereospecificity of
the microsomal enzyme catayzing both syn and anti eliminations. This empha-
sizes the impact of the spatial enzyme structure on the selectivity of the reaction
as was also demonstrated recently for camphor hydroxylase. 74
[Feill-O-OHJ
o
I
II
H3C - C- OH
Felli + HOO
Psoralen
CONCLUSIONS
-..)
-..)
178 U. MATERN
OH
cCCL000
Psoralen
•
Bergaptol
0
J OH
.
0 0
OH OH
Xanthotoxol 5,8-Dihydroxypsoralen
J
J
0
CH 3 0
Xanthotoxin
J
J
eGo
OH CH 3 0
..
0 I 0 I # 0 0
CHaO CHaO
5-Hydroxyxanthotoxin Isopimpinellin
Figure II. Pattern of reactions that may lead from psoralen to isopimpinellin.
MEDICINAL POTENTIAL AND BIOSYNTHESIS OF PLANT COUMARINS 179
ACKNOWLEDGMENTS
The work cited from our laboratory was supported by the Deutsche Forschungs-
gemeinschaft and the Fonds der Chemischen Industrie. Critical reading of the
manuscript by M. Petersen, Marburg, is gratefully acknowledged.
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MEDICINAL POTENTIAL AND BIOSYNTHESIS OF PLANT COUMARINS 183
Georg G. Gross
Universitat Ulm
Abteilung Allgemeine Botanik
D-89069 Ulm, Germany
INTRODUCTION
185
186 G.G.GROSS
o
eOOH
HO¥OH
OH
1,2,3,4,6-Penta-O-galloyl-B-D-glucose (2)
OH OH
OH OH
HO
HO
OH
OH
meta-Digallic acid (5) Ellagic acid (4)
Hexahydroxydiphenic
acid (3)
which C-C linkages are formed between adjacent galloyl residues of pentagal-
loylglucose to yield (R) or (S)-3,4,5,3',4',5'-hexahydroxydiphenoyl (HHDP, 3)
groups, followed by the subsequent formation of dimeric and oligomeric deriva-
tives that are connected via C-C or C-O-C bonds between the galloyl residues.
After the eventual hydrolytical release of these HHDP residues, the free diphenic
acid spontaneously rearranges to the extremely insoluble dilactone, e\lagic acid
(4), which became name-giving for this group of natural products. Gallotannins,
in contrast, originate by a quite different mechanism, i.e. by esterification of
further galloyl units to the pentagalloylglucose (2) core to yield digalloyl residues
which are attached via so-called meta-depside bonds (5). Substitution degrees of
as much as 10-12 galloyl residues have been reported for such gallotannins from
various sources. 2- 5 It should be noted that evidence, based on NMR spectroscopy,
has been presented that gallotannins are mixtures of meta- and para-depsides in
nature. 3- S As this view still awaits supporting experiments, only the traditional
meta-bonds, known from the literature for decades, are used in this chapter for
the illustration of gallotannin structures. To differentiate unequivocally these
polygalloylglucoses (gallotannins sensu strictu) from their one to five-fold sub-
stituted precursors (often referred to as "simple galloylglucose esters"), the term
"complex gallotannins" is used in this chapter.
Innumerable structures of hydrolyzable tannins and related compounds
bave been reported from many laboratories for decades, and this solid chemical
basis has prompted studies on the biochemistry of these complex molecules. This
question has been investigated in the author's laboratory mainly by extensive
enzyme studies, i.e. by a technique having the advantage that it not only allows
the unequivocal identification of metabolic intermediates but also provides oth-
erwise inaccessible information about "activated" intermediates as an indispens-
able tool for the elucidation of biochemical reaction mechanisms. Consequently,
the insights into the pathways described in this chapter have been obtained almost
exclusively by this laborious but also highly evidential method.
gallotannins; and finally (v) the oxidative processes leading from pentagalloyl-
glucose to monomeric and oligomeric ellagitannins. The results obtained from
enzyme studies are reported in this section.
(b)
oA, 6H
5-Dehydroshikimic
(c')
0"
YOH
OH
Protocatechuic
(b, c') ))H
HOYOH
OH
Gallic acid (1)
acid (9) acid (10)
I
(c)
mung bean seedlings II and leaves from Pelargonium 12 suggested the sequence
5-dehydroshikimic (9) ~ protocatechuic (10) ~ gallic acid (1) (route c' in
Fig. 2), but these preliminary results were never corroborated by detailed
investigations.
Ambiguous results regarding the two alternatives were obtained after
feeding experiments with carboxyl-labeled shikimic acid (the biosynthetic route
to gallic acid via C 6C3 compounds involves loss of the radioactive carboxyl group,
while it is retained in the direct aromatization) which suggested the existence of
two routes to gallic acid, the preferential one depending on the plant species and
its developmental status. 13- 16 Experiments with the herbicides L-AOPP (L-2-
aminooxy-3-phenylpropionic acid, an inhibitor of the deamination of L-
phenylalanine to cinnamic acid 6) and glyphosate (N-(phosphonomethyl)glycine,
a phosphoenolpyruvate analog that blocks the activity of 5-enolpyruvylshikimate
dehydrogenase) supported the direct aromatization of shikimate or shikimate
precursors to gallic acid and related phenolics. '7 . '8
Summarizing the conflicting evidence, it appears presently most plausible
to regard the direct aromatization of 5-dehydroshikimic acid (9) at least as a
significant, if not the predominant, route to gallic acid. Strong evidence support-
ing this conclusion has recently been obtained by feeding [13C]glucose to cultures
of the fungus Phycomyces blakesleeanus and to leaves of the dicotyledonous tree
Rhus typhina, followed by determination of isotope distributions of isolated gallic
acid and aromatic amino acids. '9 Interpretation of the resulting isotopomer pat-
terns by a retrobiosynthetic approach showed that gallic acid was derived in both
species from an early intermediate of the shikimate pathway, most probably 5-
dehydroshikimate (9). Notably, the carboxyl group of gallic acid was found to
originate from a C6C I intermediate of the shikimate pathway and not from the
side-chain of a C6C3 metabolite, thus ruling out this latter route as a major
pathway. It was concluded that dehydrogenation of 5-dehydroshikimate (9)
in both the fungus and the plant was the predominant pathway to gallic acid
(Fig. 2, route c) but the alternative route c' (Fig. 2) via protocatechuic acid (10)
could not be excluded on the basis of the available data.
Formation of ~-GlucogalIin
HO* ~
eOOH
OH
I
OH
• \
'\
UDP
b
H~O'e ~
HO
OH
0
OH II
o
~
I1-Glucogallin (11)
OH
I
OH
OH
soon recognized that this thioester was not involved in the biosynthesis of 13-
glucogallin.25 It was found instead that a glucosyltransferase from oak leaves
catalyzed the efficient esterification of free gallic acid with UDP-glucose as the
energy-rich component, yielding f3-glucogallin and related I-O-acyl-f3-D-
glucoses (Fig. 3).26-2R Numerous analogous enzymes catalyzing the formation of
phenolic I-O-acylglucoses were isolated from various plant sources in the mean-
time, thus providing evidence that UDP-glucose functions as the general activated
donor in the esterification of glucose with phenolic acids.
The main pathway. It was apparent from early investigations on the biosyn-
thesis of gallotannins that crude extracts from young oak leaves produced digal-
loylglucose and trigalloylglucose in assay mixtures which contained f3-glucogallin
(11) as sole substrate. Again, as in the preceding formation of f3-glucogallin, no
requirement for galloyl-CoA was observed in these reactions. This surprising
result could be interpreted only with the assumption that f3-glucogallin exerted
an unexpected dual role, evidently acting not only as acceptor substrate but also
as the acyl donor required for such a transformation. 25 Considering the then
known compararatively low group-transfer potential ~Go' of acylglucoses (e.g.
glucose-I-phosphate, ca 21 kJ mol-I; glucose-6-phosphate, ca 10.5 to 12.5 kJ
mol-I ),29 the existence of such an enzyme activity was surprising. This question
was solved meanwhile by the finding that the related cinnamoy I ester, 1-0-
sinapoyl-f3-D-glucose, has an unexpectedly high ~Go' of 35.7kJmol-I.30 i.e. a
value that is comparable to well-known data for acyl-CoA thioesters (ca. 36 kJ
mol-I). It is reasonable to assume that the ~Go' of f3-glucogallin (11) is on the
same order of magnitude.
Subsequent investigations led to the isolation of an acyl transferase from oak
leaves that catalyzed the "disproportionation" of f3-glucogallin (11) by which the
galloyl moiety of the donor was transferred to the glucose-6-0H of the acceptor,
yielding 1,6-di-0-galloyl-f3-D-glucose (12) and free glucose as a deacylated
by-product (Fig. 4V I Two structurally closely related esters, 1-0-
BIOSYNTHESIS, BIODEGRADATION, AND HYDROLYZABLE TANNINS 191
H
° OH
71
O,;:;-c ~ OH oH
Lr tc
I1-Glucogallin (11) OH I1-Glucogallin (11)
",~O, ~O" oI ~
\. . \. .
HO~O,~ '\ HO~O,C 1 h OH ~
ft OH OH Glucose HO OH II Glucose
o o
H OH
° OH OH
71
Lr h71
0, ~ OH OH
I1-Glucogallin (11)
O~j ~ 0" Lo"
'?o
tc ~ oH
1 h OH
\. .
HO~O,C '\
Glucose
HO 0
/
II
0
"O~o"JCloo
/
o /
0 II
0
~
:r:
tTl
[JJ
po
1:0
H §
° OH
tTl
a
0.;::.
o
ix
T
~ I
OH
~
Ci
r..-Glucogallin (11) ?:i
HO:y1 g,o~O\ OH
"-- :"\-
tr: ~
HO~ p~O I ~
Glucose
OH *O=:/ p 'ft h OH Ci
:r:
7 O=CQ- 0 -<
~ I ~
HO OH
r;_ OH ~
OH HO OH
~
N
>-
1:0
l'
1,2,3,4,6-Penta-O-galloyl-B-D-glucose (2) tTl
~
Figure 4. The biosynthetic pathway from l3-glucogallin to pentagalloylglucose. ~
z
[JJ
'D
Y>
194 G. G. GROSS
HO~OH 0 16=0H OH
HO~O,
OH
o ~ I +
HO
OH
'c
II
OH HO~
o
I1-Glucogallin (11) ['4CjGlucose
jr OH
OH
HO~O\
H~O'C ~ I
HO 0 :;/ OH
OH 16=
HO~OH + OH
OH II
o
Glucose ['4CjI1-Glucogallin
Figure 5. Enzymatic galloyl-exchange between ~-glucogallin and glucose. Bold lines symbol-
ize the presence of an appropriate label (e.g. 14e) as prerequisite for the detection and
quantification of this reaction.
ttl
5
[/J
i
-l
::c
tr1
[/J
:0
ttl
5
vtr1
OH OH OH
OH OH OH Cl
r r r
l l l s::
o"c ~ OH v
0" ~ OH OH
OH
0"
'?
n ~ OH OH
OH
I
OO
n ~
+ 1,6-Digalloylglucose
'?o "'" HO
nh h
o "'" + ~ ~
h OH HO~O\ o'c I h OH
HO OH HO~II HO OH OH
HO~o,cg I ~
o 0 v
o=~" ~
1,6-Di-O-galloyl-f1.D-glucose (12) 6-0-Galloylglucose
v
13
HO OH
t<
N
;J>
1,2,6-Tri-O-galloyl-f1-D-glucose (13) ttl
r'
tr1
Figure 6. ~-Glucogallin "independent" enzymatic "disproportionation" of I ,6-digalloylglucose to 1,2,6-trigalloylglucose. ~
~
z
[/J
U>
""
196 G. G. GROSS
+ UDP-Glucose
-UDP
"~~ OH \I
o
~ OH II
o
+ (15), - Glc
\I
HO~\
Ac~~'c
o
II
0
~
1-0-lsobutyroyl-B-D-glucose (17) 1,3-Di-O-isobutyroyl-B-D-glucose
1. (15), - G"
+ (15), - Glc
1,2,3,4-Tetra-O-isobutyroyl-B-D-glucose 1,2,3-Tri-O-isobutyroyl-B-D-glucose
o 2 3
Incubation time (h)
~
OH
O=C ~
rt/
cU OH
:y*~.~ HO h OH
~'"
H HO OH
1 ,2,3,4,6-Penta-O-galloyl-B-D-glucose (2)
OH H
7, ~ OH
""c
0 ... :::,... OH
7,°
Lr
OH ~O"":::"" OH H
O
?i
h h/ ~ OH H 7 0II '-':: OH
' 0 II ,=,
HO :::,... <> 0
Y4 OH
O=C~ _ 0
",:QtX(,. '&~
, "'" ~ Ii o. . . c~
h OH
h-OH
HO OH H
HO OH HO OH*., o
H HO OH
HO ~
OH
OH
4= o
a
4-0-Digalloyl-1,2,3,6-tetra-O- ~
[/J
[/J
2-0-Digalloyl-1,3,4,6-tetra-O- galloyl-B-D-glucose (16)
galloyl-B-D-glucose (18)
IJj
25
[/J
~
::r:
tTl
[/J
.2ii
IJj
,
, OH
~ 25
71 otTl
O""~::-... OH Cl
o
lrr: LOH
-:? oII 6 ~ OH
~
71
HO~~'o HO II
~ ~C/°=y1 c'o~o.....c I II h OH ~
U OH
HO *O~~::-"'H ~
h .~
~
HO
o ~H *o~~ =Q-fio~ ~o o
1-& ::r:
o 0 OH >-<:
HO OH 7 o
~~*O~O
HO OH
.
7 I OH
h OH HO I
* es
HO::-'" OH
~" HO :::-.. OH ~
N
HOYOH OH H >-
IJj
OH r
tTl
2,3-Di-O-digalloyl-1,4,6-tri-O- 2,4-Di-O-digalloyl-1,3,6-tri-O- ~
galloyl-B-D-glucose (19) galloyl-B-D-glucose (17) ~
Z
[/J
Figure 9. Acylation of pentagalloylglucose (2) to hexagalloylglucoses (16, 18) and heptagalloylglucoses (17, 19) catalyzed by galloyl-
transferases A and B (cf. text) from leaves of Rhus typhina. ~-Glucogallin (11) serves as general galloyl donor in these transformations.
Main reactions are symbolized by bold arrows, minor reactions by thin arrows; the dashed arrow symbolizes trace reactivity.
N
o
OH OH
OH OH
0" :,... 1
tv
0" :,... 1 o
HO
o
II
C
6
a OH
OH HO
o
II
C
6
a OH
OH
tv
1 0 0 '" OH 1 0 ",OH
HO:"" 0 ~c""'O 1
o. . . c,.",-::. HO:"" 1
Y C'~
OHA II
tc OH
YC'~ 0 ~c/o o. . . c
tc h-
OH o=c ~ OH
HO~OH o=h-o: + Il-Glucogallin (11) I'" -
OH HO)--\H - Glc
O"c/ O
A
* : OH Q-OH
HO OH
O~ :,... 1
HO
o
II
'<:c
6
a OH
OH
1 0 ",OH
HO
c,~
:,...
YO~c"""O O'c 1
tc fi
1
HO :,... 0, ..... 0
OH*C/
* HO
:,...1
OH
3-0-Trigalloyl-1 ,2,4,6-tetra-0-
OH galloyl-r..-D-glucose (21) o
o
Figure 10. Transformation of 1,2,3,4,6-pentagalloylglucose (2) to 3-0-digalloyl-I,2,4,6-tetra-0-galloyl-~-D-glucose (20) and 3-0- Cl
trigalloyl-l,2,4,6-tetra-O-galloyl-~-D-glucose (21) by galloyitransferase C from leaves of Rhus typhina (see text for details). 23
Vl
Vl
BIOSYNTHESIS, BIODEGRADATION, AND HYDROLYZABLE TANNINS 203
Formation of Ellagitannins
DEGRADATION OF GALLOYLGLUCOSES
2[H]
j-
Ho~OHIR
HO ~ c .......... O-CH, OH
OOH
o ~o\ I
HO r ~_o o~o,-c /0. OH
0 0 II
HO~ I \ / 0
OH co OC
HOOOOH
HO HO HO OH
o
CI
Casuariclin (23)
CI
Figure 11. Proposed steps in the oxidation of 1,2,3,4,6-penta-O-galloyl-f3-D-glucose (4C\ configuration) to monomeric
(5
C/l
C/l
ellagitannins by 4-6 and 2-3 coupling.
BIOSYNTHESIS, BIODEGRADATION, AND HYDROLYZABLE TANNINS 205
LOCALIZATION OF GALLOTANNINS
The recent trends in this field have been eloquently described by Okuda et
al. 87 "The remarkable change in the definition of tannins, due to recent advances
in the chemistry of natural polyphenolic compounds, has been accompanied
by the changes in our recognition of the significance of their existence and
utilization, from that for leathering material to that for health effects."
ACKNOWLEDGMENTS
I thank the many colleagues and recent or previous coworkers that con-
tributed to the results reported in this chapter. Generous financial support from
the Deutsche Forschungsgemeinschaft (Bonn), the Fonds der Chemischen Indus-
trie (Frankfurt/M.) and from research grants of the University of Ulm is
gratefully acknowledged.
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14. SAIJO, R. 1983. Pathway of gallic acid biosynthesis and its esterification with catechins
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27. GROSS, G.G. 1983. Partial purification and properties of UDP-glucose: vanillate 1-0-
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33. DAHL BENDER, B., STRACK, D. 1984. Enzymatic synthesis of 1,2-disinapoylglucose
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82. SATOH, K., NAGAI, E, USHIYAMA, K., YASUDA, I., SETO, T, KANO, I. 1997. Inhi-
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of both moutan cortex and Paeoniae radix. Biochem. Pharmacol. 53: 611-614.
83. NAKASHIMA, H., ICHIYAMA, K., HIRAYAMA, E, UCHINO, K., ITO, M., SAITOH,
T., UEKI, M., YAMAMOTO, N., OGAWARA, H. 1996. Sulfated pentagalloyl glucose
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84. GOTO, H., SHIMADA, Y, AKECHI, Y, KOHTA, K., HATTORI, M., TERASAWA, K.
1996. Endothelium-dependent vasodilator effect of extract prepared from the roots of
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85. CHUNG, K.-T, STEVENS, S.E. jr, LIN, W.-E, WEI, c.1. 1993. Growth inhibition of
selected food-borne bacteria by tannic acid, propyl gallate and related compounds. Lett.
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86. HUNTER, M.D., SCHULTZ, J.c. 1993. Induced plant defenses breached? Phytochemi-
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87. OKUDA, T., YOSHIDA, T, HATANO, T 1992. Polyphenols from asian plants. Structural
diversity and antitumor and antiviral activities, pp. 160-183. in Phenolic Compounds in
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(M. Huang, C. Ho, and C. Lee, eds.), ACS Symposium Ser. Vol. 507, Washington, DC,
1992.
Chapter Nine
WE. Hillis
INTRODUCTION
215
216 W. E. HILLIS
On the other hand, the presence of extractives can also affect pulping and
lower the yield, color, and quality of pulp for papermaking, as well as interfering
with the recovery of pulping chemicals. 2 Some extractives in heartwood can
degrade wood coatings, lower adhesion qualities in bonded products (as with
some eucalypts), and cause corrosion. Others have found important use as
perfumes, dyestuffs, and medicines. In the future, the increasing volume of wood
from short-rotation, fast-grown plantations will contain lesser amounts of
heartwood and its extractives.
Recently it has been found 3.4 that some cells and splits in heartwood contain
pure or high concentrations of complex compounds. These can be biosynthesised
in heartwood from carbohydrates (or lipids to a lesser extent) without formation
of appreciable amounts of waste products. When the complex biological mecha-
nisms of these conversions are ascertained, it should be possible to produce the
complex compounds with the exact properties required in high yield and with
little pollution from an inexhaustible resource.
The phenomenon of heartwood initiation and formation, which occurs in
some trees, is complex and involves various processes in the living tree, some of
which may vary from species to species. The living ray and axial parenchyma
playa basic role in the internally controlled heartwood formation. The efficiency
and age of the sapwood parenchyma before activity increases can be influenced
by environmental conditions, growth rate, and various properties of the tree. A
holistic understanding of the physiological, anatomical, cellular, and biochemi-
cal conditions involved in heartwood formation is required before the phenome-
non can be defined. This has been attempted in three earlier studies. (Bamber
and Fukazawa,s Hillis,6 and Higuchi.7) The present contribution summarizes and
updates the first two, the primary objective being to understand the formation of
extractives resulting from heartwood formation. The latter devotes consideration
to the biochemistry and the enzymological changes in wood.
CAMBIAL REGION
The structural elements of wood are formed in the cambial region, located
between the previously formed bark and the wood of stems and branches. Cambial
cells have a limited life of 4-33 days, and consist of very thin walls containing
mostly water, numerous Jiving organelles, nutrients, sugars, etc. inside a plas-
malemma with embedded enzymes. Most cells divide and expand in about 30
days to become wood, but injuries in the growing season result in exudates with
compositions markedly different from the sapwood or heartwood extractives in
the same tree. 6 Processes controlling growth vary as cells go through stages
of division and enlargement and maturation, until death occurs when maturation
is complete.
THE FORMATION OF HEARTWOOD AND ITS EXTRACTIVES 217
The secondary layer of these small, short, thin-walled cells may be absent. Ray
tissues in angiosperm trees have a more complicated structure in comparison with
coniferous trees. Ray and axial parenchyma are usually of similar size, but in
Pseudosindora palustris the size of apotracheal parenchyma cells (located axially
but away from vessels) is much larger than those of the ray parenchyma. II There
may be storage parenchyma (both horizontal and axial) containing reserve mate-
rials such as starch or fats or specialized cells contiguous with vessels. The
efficiency of parenchyma in heartwood formation may differ. Polyphenols can be
present only in the ray parenchyma of some species, whereas in other species they
are present also in the axial parenchyma. The ray parenchyma cells play an impor-
tant role in heartwood formation, whereas axial parenchyma appears to have dif-
ferent fm:ctions. The specialized cells have numerous large pits between them
and the vessels. They contain living cytoplasm which seems to be involved espe-
cially in carbohydrate metabolism and short-distance transport. 12 Lignification of
the ray parenchyma in the genus Pinus occurs in the transition zone during
heartwood formation. 5.13
Morphological 14.15 and chemical studies6 have revealed differences in func-
tion and physiological condition between various ray parenchyma cells. Starch is
stored mainly in the axial parenchyma which can be arranged in characteristic
patterns. Administration of 14C-labelled phenylalanine to disks of Zelkova serrata
showed that radioactivity was more conspicuous in the ray parenchyma cells than
in the axial parenchyma cells, particularly in the transition zone where most of
the radioactivity was located. 16 This will be discussed later. The dimensions of
ray tissue vary in different plants from one cell wide (uniseriate) to several cells
wide (multiseriate) and from one to twenty cells high. The behavior of the nuclei
in these cells, within a segment of the ray, as well as across the sapwood (and
transition zone) can differ. The cells can occupy 8-40% of the volume of
angiosperms and about 16% of gymnosperms.
Vessels or pores are found only in the axial direction of hardwoods and with
a diameter larger than other cells. Their size differs between and within species,
e.g. in the latter, vessels in the earlywood have larger diameters than those in the
latewood. They are in long lengths of 1-3 meters, thin-walled, and interconnected
through various types of pits, and may be separate or arranged in groups (as
ring-or diffuse-porous) with characteristic patterns. They can be blocked with
tyloses or extractives.
The balloon-shaped tyloses extrude from the secondary wall of living
ray cells through pits into vessels if the pit size exceeds 10 /lm. If the pit size
is smaller, extractives tend to be extruded from the ray cells into vessels. 17
Tyloses are thin-walled and may be lignified. They are regularly produced in
connection with wounding and appear in the living wood bordering dead
wood, either near injuries involving loss of moisture or characteristically at the
heartwood periphery of several species. They develop before heartwood
extractives.
THE FORMATION OF HEARTWOOD AND ITS EXTRACTIVES 219
Resin canals in gymnosperms have a larger diameter than the other cells.
They develop in both vertical and horizontal directions to form an anastomozing
network. In some genera such as Pinus, the canals are lined with thin-walled
epithelial cells which function for several seasons and can produce resinous mate-
rials under high pressure (up to 10 atmospheres). The epithelial cells in resin
canals in the genus Pinus are lignified during the heartwood transition. 5
Due to the fragile nature of the cambial region and its small volume even
in its most active phase, it is difficult to assign the location of its extractives.
During the short periods of rapid metabolism, the cambial region in some euca-
lypts may be separated to allow its extractives to be examined. The main recog-
nizable compounds are (+) catechin, (-) epicatechin, possibly (-) epigallocatechin
gallate, shikimic acid, and 5 derivates of galloyl- and hexahydroxy-diphenic acid
linked to gluco-pyranose, synthesized by the shikimic acid and other pathways. 18
If the cambial region of eucalypts is injured during its most active period, a
fluid of red-brown kino is formed, sometimes in large amounts. Kinos contain
largely proanthocyanidins in various stages of polymerization with distinctive
monomeric polyphenols in some species, such as C-glucosides of flavonoids and
the lignan eudesmin (1) in Eucalyptus hemiphloia. I9 •2o Usually, kino is contained
in the wood in thin veins (1.5-5 mm) of up to 2 meters in length or in pockets
(when the parenchyma bridges are broken) which may contain large amounts.
Often, kino from eucalypts or resin from pines is exuded onto the surface of the
tree from damaged cambial regions and probably serves as defense mechanisms
for those regions.
1: Eudesmin 2: Matairesinol
CH07£H°0
3
HO
1
""
h
H
I""
h
OCH3
OH
3: Hydroxymatairesinol 4: a-Conidendrin
220 W. E. HILLIS
SAPWOOD
Sapwood is the outer annulus of tree stems, branches, and roots. In the
living tree, the fibrous elements die soon after their development has been com-
pleted. On the other hand, many of the ray and axial parenchyma cells remain
alive for a considerable time in the sapwood which has been defined as "the
portion of the wood that, in the living tree, contains living cells and reserve mate-
rials (e.g. starch)".22 The area of sapwood before it is transformed into heartwood
varies considerably among families, genera, and species. Heartwood may never
form, and living cells and starch have been found in the inner tissues of Acer
saccharum, Nothofagus cunninghamii, Alstonia scholaris, etc. (M.M. Chattaway,
pers. com.) over 100 years old. Ring-porous angiosperms usually have narrow
sapwoods and a small number of sapwood rings, whereas the width and ring
number in diffuse-porous woods can be greater and more variable.23 In some
species, all the ray parenchyma live throughout the sapwood. In other species,
they begin to die from the middle sapwood, and in another group, the rays begin
to die from the cambium. lo
The number of growth rings in the sapwood can be characteristic of some
species after the tree has grown out of the juvenile stage, and it remains more or
less constant throughout the life of the tree. The commencement of heartwood
formation varies considerably between genera and species: it is about 3-4 years
with Robinia species, about 5-7 years (1.5-2.5 cm width) with mature eucalypts,
3-12 years with Douglas-fir, about 14 years with P radiata, and 25-70 years with
P sylvestris. 23 There appears to be a strong genetic influence, with family differ-
ences, in the age of the tree when heartwood is formed.6.24.25 The number of heart-
wood rings appears to be essentially unrelated to tree growth in the post-juvenile
growth phase in P radiata. 26 The number of rings is often more consistent than
the width and cross-sectional area which can be influenced by location in the tree,
environmental conditions and site quality,26-29 growth rate/O,31 height in stem, age
of tree, and size of crown. 6 ,32-39 A highly significant relationship has been found
between sapwood area and foliar surface area of four eucalypts. 4o
The sapwood width of Cryptomeria japonica appears to be more dominant
than the number of growth rings in controlling heartwood formation. 41 P
canariensis, having a more narrow sapwood than predicted by age and growth,
tends to display a higher percentage of axial parenchyma in the inner xylem. 42
THE FORMATION OF HEARTWOOD AND ITS EXTRACTIVES 221
The intense resin soaking found in some heartwoods is explained by the high
proportion of living cells in the xylem and their ability to accumulate large
quantities of reserve starch. 42
Trees probably require a particular amount of sapwood, as shown, for
example, by the strong relationship between rate of growth and sapwood width. 43
Also, it has been concluded that heartwood is probably formed when the burden
of maintaining all the sapwood exceeds the capacity of the area or mass of foliage
and increasing respiration costs39 to supply the needs of sap stream conduction
and food storage. 5,44 In a study of heartwood formation, Gerrish 35 used the
"specific leaf burden" (SLB), defined as the ratio of the unit volume ofrespiring
tissue to the unit mass ofleaf. He considered that in the diffuse-porous, evergreen
Metrosideros polymorpha, heartwood formation is controlled by whole-tree ener-
getics rather than by the movement and behavior of the sapstream. Formation of
heartwood may be initiated when the SLB reaches a threshold value such that
all the sapwood in the tree can no longer be maintained. It is possible with
M. polymorpha stands that heartwood initiation is linked to canopy closure.
With the increasing use of plantations, fast-growing species, and shorter
times between harvests, the amount of sapwood in proportion to heartwood in
marketable timber and pulpwood is also increasing. This will affect the use of
wood in various applications. It has been proposed that the principle determin-
ing the amount of sapwood is the provision for the storage of food reserves such
as starch. 5 The patterns of the radial decrease of starch have a strong relationship
with the sapwood width, and trees with a narrow-sapwood and an abrupt decrease
in starch at the heartwood boundary have deep-colored heartwood with distinct
boundaries; other species show a gradual decrease of starch across the
sapwood. 10,23,45 Seasonal changes are observed only in the amount of free fatty
acids in the sapwood of Pinus sylvestris, with the levels being greater at the
beginning and end of the growing season. 46 Changes in vacuole content (where
polyphenols accumulate) also appear in two patterns; one shows no vacuoles in
the outer sapwood but the number gradually increases towards the sapwood/heart-
wood boundary. The other shows large amounts of vacuoles from the outer to the
inner sapwood. 47 Species with a large number of vacuoles in the outer sapwood
have narrow sapwoods. 49 The radial changes of vacuolar content vary according
to species but generally decrease in size with age and disintegrate in the transi-
tion zone and with the death of ray cells. 48 Relationships between width and age
of sapwood with other features have been summarized. 6
The parenchyma cells are the main living cells and they can respond to
injury. In some cases, the fibers in sapwood can retain their cytoplasm and starch. 5
Nobuchi et al. 48 grouped 20 coniferous species according to the survival curves
of sapwood ray cells. All cells in one type, such as that containing Pinus densiflora
and Thuja orientalis, survive from the cambium to the transition zone between
sapwood and heartwood. In another type, such as that containing Pseudotsuga
japonica and Cryptomeria japonica, parts of the ray parenchyma cells die in the
222 W. E. HILLIS
inner sapwood. In the third type, part of the ray parenchyma begins to die in the
outer sapwood, as with Picea abies and Larix leptolepis, and these species do not
show a sharp boundary between sapwood and heartwood. Several angiosperm
trees show an abrupt decrease of living ray parenchyma at the heartwood periph-
ery.48 There is only a weak direct relationship between radial changes in the
proportion of living ray cells and heartwood formation. 48
Sapwood contains low levels of extractives, as in the case of Quercus
Spp49-51 and Thuja plicata. In some species the composition of sapwood extrac-
tives can differ from those in heartwood. In gymnosperms, sapwood has a rela-
tively high free water content (in the lumen) of about 150% compared with 55%
in the heartwood. In Cryptomeria japonica, the radial distribution patterns of
methanol soluble extractives closely resemble those of moisture content. 41 On the
other hand, the sapwood of angiosperms usually contains about 83% free water
compared with an average of 81 % in the heartwood for a number of species. 6
However, the moisture content of the heartwood of some angiosperms may be
higher than that of the sapwood, as with some Eucalyptus, Quercus, Juglans, etc.
species. It is noteworthy there is no change in moisture content at the periphery
of teak heartwood. 52 On this basis, it is not always easy to support the claim that
loss of water is the first step in heartwood formation.
Saranpaa and H6ll 45 considered that the cell wall hemicelluloses of Pinus
sylvestris were partly hydrolyzed during heartwood formation and may be
involved. The polyphenols are formed first in the shikimate and phenylpropanoid
routes, with flavonoids from an additional route of the plant aromatic pathway. 55
Data indicate that the shikimate pathway in the plastids provides amino acids for
protein synthesis, and the cytoplasm provides, in addition, phenylalanine for the
phenylpropanoid and flavonoid segments. The chalcone synthase leading to
flavonoid synthesis is located on the endoplasmic reticulum membrane. 55
In pines, an abrupt change in the composition of extractives occurred in the
narrow transition zone where the triglyceride content decreased rapidly and fatty
acids increased. There was little change in the composition of the resin acids
across a cross-section, although the proportion of them increased more substan-
tially in the heartwood than in the sapwood. The extractives in the earlywood
tissues of Pieea abies had a somewhat higher content of fatty acids than the
corresponding latewood tissues and the degree of saturation of these fatty acids
appeared to be higher. 6
Triacylglycerols and fatty acids are the major reserve lipids of Robinia.
pseudoaeaeia, and they have an energy content that is much lower than that of
starch. 56 Only a small portion is used for heartwood formation. 57 The lipid classes
contain a variety of fatty acids with chain lengths of 14 to 24 carbon atoms and
up to 3 double bonds. At the sapwood/heartwood boundary, the amount of satu-
rated fatty acids begins to decline towards the center of the trunk with a shift
towards a higher degree of un saturation. Steryl esters of palmitic and stearic acids
are the most dominant, and these and other esters reach maximum value on the
heartwood side of the transition zone after which lower quantities are present. 57
The amount of free sterols increases markedly in the same location of the heart-
wood. The six sterols change in proportion across the stem with cholesterol reach-
ing its maximum at the outermost heartwood. In Teetona grandis, the lipid content
increases towards the transition zone with a simultaneous decrease in the amount
of starch. Datta and Kumar 8 suggested that lipids, together with sugars from the
hydrolyzed starch, give rise directly to extractives in the transition zone.
Changes in metabolism towards synthesis of extractives are accompanied
by activity of corresponding enzymes. It has been suggested that apart from
lipases, several other acylester hydrolases (e.g. phospholipases) catalyse lipolytic
reactions in the transition zone,56 and the enzyme turnover is most rapid at the
sapwood-heartwood boundary. Relatively high lipase activities have been found
at the heartwood periphery of R. pseudoaeaeia.
TRANSITION ZONE
J.
increased respiration and biological activity in this zone could expel free water
from the living cells preceding the formation of extractives.
OH
Ho-o--?;O-OH ,"'"
R
.ij
".
Ot-P H
5: Agathal'esinol R~H 7: Sugiresinol RI~R2~OH; R3~R4~R5~H 9: (+}-Ferruginol
6: Sequirin R9)H 8: Hydroxysugiresinol RI~R2~R49)H; R3~R5~H
The transition zone has a sharp boundary with both sapwood and heart-
wood or injured wood, and follows the same outline. The number of annual rings
and width of the transition zone have constant values at the different heights of
C. japonica and other species. 70 Different colored layers can be seen in the
transition zone adjacent to the heartwood of Taxus baccata and Pinus radiata
and some other species. The width of the transition zone can vary throughout the
year. 6 While it has been recorded in a number of angiosperms, it is less obvious
than in gymnosperms.
The percentage of living cells decreases gradually across the transition zone
to zero at the outer heartwood boundary of Cryptomeria japonica. IO ,41,48 Two
marked cytological changes have been observed in parenchyma cells in the tran-
sition zone of this species. The first is at the boundary between the sapwood and
the transition zone, where the cells show conspicuous vacuolarization (polyphe-
nols accumulate) and symptoms of senescence. The second change is seen at the
boundary between the transition zone and the heartwood and indicates involve-
ment with heartwood and extractives formation. 10 The disintegration of the nuclei
in the ray parenchyma cells and the disintegration of the vacuoles begins in the
transition zone after the decrease of the moisture content in the sapwood. This
disorganization of cell compartments can result in an altered71 or intensified72
metabolism to produce polyphenols, particularly in the inner transition zone and
outer heartwood when the protoplasmic membrane disintegrates to release extrac-
tives and cytosolic enzymes such as oxidases. Chattaway's concept72 has been sup-
ported by enzyme assay and oxygen uptake,73,74 nitrogen contents/5 and tracer
experiments. 16,65 The metabolic activity is enhanced at certain times of the vege-
tative period. 7o,76-78 The ray parenchyma cells are considered to be more impor-
tant than axial cells for heartwood formation in C. japonica. 41 Biological
factors as well as non-biological factors can change the nature of the secondary
metabolites after the death of all ray parenchyma cells.
It has been shown that the incorporation of radioactive compounds into
wood extractives in the transition zone in both gymnosperms and angiosperms is
quite high. 53 ,54,76.79,8o It was Harris 81 who first drew attention to the formation of
226 WE. HILLIS
polyphenols in the inner layers of the transition zone in Pinus radiata during the
flush of growth. They became incorporated in heartwood in early summer. In
Eucalyptus polyanthemos, the outer layers of the zone formed a methylellagic
acid glucoside, whereas the inner layers formed proanthocyanidin polymers as
well (WE. Hillis, unpublished). The degree of flavonoid formation in Prunus
yedoensis was much larger in the transition zone than in the inner sapwood. With
Cryptomeria japonica,82 one compound was present in greatest amounts in the
transition zone, whereas the other two major polyphenols were more abundant in
the heartwood. A marked change in the resin composition occurred in the transi-
tion zone of spruce and pine when the triglyceride content decreased and the fatty
acids increased. 6
Greatly increased respiratory activity has been observed at the
sapwood/heartwood boundary in Robinia pseudoacacia. 83 There are several indi-
cations of increased activity of enzymes between sapwood and heartwood. Lipase
is localized in the transition zone of Indian hardwoods,84,85 triacylglycerols are
hydrolyzed in the transition zone in Pinus sylvestris, and significant increases
occur in the amount and type of fatty acid composition at this time,86 Concen-
trations of phenol oxidizing enzymes have been observed at both the outer and
inner peripheries of the transition zone of some eucalypts, This increase could
be due to de novo synthesis and/or introduction of an activator. A number of
co-enzymes of key enzymes have been found to increase near the heartwood
boundary. Two phenol-oxidizing enzymes have been found in fresh P. radiata
heartwood,87 In several species, increased peroxidase and polyphenolic oxidase
activity have been found in the layers adjacent to the heartwood or the transition
zone where they were concentrated in the torus of the pits. Greatest activity occurs
during the winter and dormant seasons. 88 ,89 These oxidase enzymes would be
responsible for the oxidation and polymerization of major portions of polyphe-
nolic extractives. Other enzymes have been reported such as: succinate dehydro-
genase (in Acacia nilotica,84 indicating a zone of high respiration and possibly
associated with mitochondrial involvement in the formation of phenolics);58
adenosine triphosphatase (implicated in cellular transport, membrane permeabil-
ity and high-energy consuming processes in cellular metabolism and which dis-
appears in heartwood); acid phosphatase (concerned with degradation and
transport of carbohydrate); lipase; maltase; aldolase; and amylase, 58 The presence
of glucose-6-phosphatase indicates the operation of the hexose monophosphate
shunt. Its activity in Tectona grandis has been detected in the inner sapwood and
transition zone only when active conversion of metabolic substances is taking
place to form complex heartwood extractives, 58 Peroxidase activity in the inner
sapwood and transition zone peaks during the late growing season, to early
dormant season, and in a period before the maximum emanation of ethylene.
Peroxidase activity decreases in the transition zone of Shorea robusta,90 An abrupt
rise and decline in the activities of succinate dehydrogenase and acid phosphatase
occurs at the heartwood periphery of Azadirachta indica,91
THE FORMATION OF HEARTWOOD AND ITS EXTRACTIVES 227
HEARTWOOD FORMATION
The annual rhythm of activities is similar in all species. Thus, annual ring
formation commences in the spring and ceases towards the end of summer. Heart-
wood formation is initiated in warm temperate zones during the decline of
cambial activity or dormant periods. Before the end of summer, the heartwood
region has expanded to about 70% of the annual ring in Robinia pseudoacacia,
with completion of the formation in winter. Similar results have been observed
with Larix leptolepis, Pinus spp., and Cryptomeria japonica. 95 •96
Translocated carbohydrates and reserve substances provide raw materials
and/or the energy for heartwood formation. Heartwood is typically formed after
changes in reserve substances, and, in the autumn and winter seasons, no con-
spicuous changes are observed. 94 The outer heartwood of Robinia pseudo acacia
appears to contain living parenchyma cells in late summer. 94 ,95 Living cells in the
outer heartwood gradually form heartwood extractives in the autumn and winter
seasons and then die. 95 .96
The extracellular stimulus for heartwood formation is not entirely a process
dependent on age or sapwood volume. Heartwood extractives formation may
happen at different times in neighboring parenchyma cells. Abrupt enlargements
of the heartwood periphery can appear over short radial distances and are part
of the major heartwood region. Also, there are concentric and symmetrical dark
rings in heartwood. The heartwood of a few tropical species Microberlinia
brazzavilliensis (trade name zebrano), Berlinia, Diospyros, and Dalbergia spp.
(see als0 97.98 ) contain regularly spaced, very dark colored, axially long stripes. In
zebrano, they are narrow (l--4mm) and alternate between equally narrow, paral-
lel, light colored stripes which occupy one growth ring. In Thuja plicata 99 and
other species,6 the heartwood contains concentric bands of light colored wood in
a target ring pattern. There is apparently some factor that eliminates the ability
of the sapwood available at the time to convert into heartwood without damag-
ing the cambium's ability to form new sapwood (lA.F. Gardner, pers. com.). Frost
could be a cause in temperate regions 99 but is not likely to be so for tropical and
semi-tropical species or in some stands subject to weather extremes. Perhaps, a
substance is translocated from the cambium to the inner sapwood where it accu-
mulates and reaches a threshold level at the transition zone to initiate heartwood
formation. 43 This compound could be of greater importance for initiating extrac-
tives formation than the maintenance of a particular sapwood volume. Com-
pounds such as water-soluble vitamins 75 or ethylene precursors could induce or
activate genes encoding new enzyme systems.
If an injury to a tree is of sufficient severity to destroy a part of the cambium
and interrupt the files of ray parenchyma with the cambial zone, then the sapwood
situated behind that tissue will not be transformed to heartwood. The affected
region is eventually overgrown by normal cells, which later are transformed to
heartwood, leaving an isolated area of "included sapwood." On the other hand,
injury to the tree can accelerate the formation of heartwood in the neighboring
region. IOO When Abies grandis trees were infested by the balsam woolly aphid
230 W. E. HILLIS
(Adelges piceae), the percentage of heartwood increased, both in terms of age and
area, with the amount formed varying with different intensities of attack. Neither
the nature nor the amount of phenolic extractives was affected. Aphids are known
to change the anatomy and function of the ray parenchyma cells. 101
Heartwood color is often much darker than the surrounding sapwood. Trees
with narrow sapwoods have darker colored heartwoods,47 apparently due to the
greater availability of primary metabolites at the boundary. In Robinia pseudoa-
cacia, the earlywood exhibits the dark color of the heartwood, whereas the late-
wood shows the light color of the' sapwood. 56 The heartwood of some angiosperms
can have irregular bands of different color intensity and width. 98 Since the
amounts of reserve metabolites depend on climatic and environmental factors,
the deposition of heartwood extractives varies from one year to another. 102 Heart-
wood is free from storage starch, most sugars except mannose, xylose, arabinose,
and fats. 45.103 ,104 The small amounts of galactose and arabinose in the heartwood
of Pinus sylvestris might have originated from the breakdown of hemicelluloses
during heartwood formation. 45 ,103
Occasionally, the individual cells of a file of ray parenchyma cells in heart-
wood have different appearances, indicating the presence of secondary metabo-
lites of different composition. Also, in cross-section, the volume of non-structural
material in the vessels can exceed that of the adjacent ray parenchyma cells, indi-
cating a continuous extrusion from the rays of extractives and precursors during
the period of intense metabolic activity.
The process of heartwood formation is accompanied by several physiolog-
ical and cytological changes. Following the examination of several species,14,7I,105
morphology and dimensions of the nuclei in ray parenchyma cells were used to
define the vitality or activity in the transformation of sapwood to heartwood.
Other workers have obtained similar results with other species. 6 Large and oval
cell nuclei (indicative of active tissue) become radially elongated, and the degree
of slenderness decreases with increasing distance from the cambium and then
shows a rapid increase of cell activity in the transition zone (in support of Chat-
taway's theory).72 There is a tendency to form bigger and longer cell nuclei and
to increase the ratio of nucleoli volume to nuclei volume in the transition zone,
with the volumes being largest in particular trees with dark heartwoods. In R.
pseudoacacia and P japonica in spring to mid-summer, the ray parenchyma cells
containing nuclei coincide with the boundary of the sapwood and transition zone,
where one-half of the rays are alive. After this time, ray cells containing nuclei
can sometimes be found in the outer heartwood during late summer. IO Heartwood
formation is considered to occur in a rather short period at the boundary between
the transition zone and heartwood. 10 Recently, Magel et al. 94.106 showed that both
the ray and axial parenchyma in the inner sapwood of R. pseudoacacia are rich
in cell organelles and appear to play an important role during physiological
changes in this region. Cell death in the transition zone starts with vacuolariza-
tion, followed by a decrease in the number of cell organelles, accompanied by
THE FORMATION OF HEARTWOOD AND ITS EXTRACTIVES 231
EXTRACTIVES
different parenchyma and other cells and the sequence of genes and enzymes
which produce these extractives.
As previously mentioned, the term "extractives" used here covers a large
number of organic compounds of different classes which can be extracted from
wood or bark with polar and non-polar solvents 116 and which are the end-
products of metabolism. Some carbohydrates and their derivatives can be removed
with water. Whereas terpenoids can be completely removed, polyphenolic and aro-
matic-based compounds (found in most species) may enter the cell wall and
combine with cell wall components. They may be polymerized or oxidized and
require a weak alkali extraction. In a few species, due to the nature of their initial
extractives, such as ebony, polymerization and other changes can result in materi-
als insoluble in solvents. The term "extraneous material" should be confined to
silica, calcium carbonate, and inorganic based compounds. Extractives are prod-
ucts resulting from enhanced metabolism at the transition zone or heartwood
periphery due to cytological changes in the ray and axial parenchyma. 16,53
Extractives convey a wide range of useful properties to wood, particularly
if or when they enter the cell wall. They have properties such as resistance to
fungi and bacteria as well as water repellency to improve durability, increased
insect resistance, increased crushing strength of wood, low fiber saturation point,
and chromogenic properties to improve appearance. On the other hand, their pres-
ence can affect pulping and lower the yield, color, and quality of pulp for paper-
making, as well as affect recovery processes. They can degrade wood coatings,
lower adhesion qualities in bonded products, cause corrosion, and result in
dermatitis in some people.
The amount of methanol-soluble compounds of the same type in heartwood
increases at an increasing rate from pith to outer heartwood in different species,
and the rate in the inner heartwood may be IOW. 6,118 It is frequently shown by color
intensity that the maximum values decrease from the lower parts to crown height,
probably due to the cell age of the ray parenchyma41 and the availability of
primary metabolites. Slower grown trees of the same species generally contain
larger amounts of extractives. 6 It has been proposed that the amount of polyphe-
nols in heartwood is related to the amount of carbohydrate reaching the periph-
ery.6 The effect of growth rate is exemplified by the arabinogalactan in the lumen
of heartwood tracheids of Larix occidentalis. In an old (300 years) slow grown
tree, the arabinogalactan content in some rings was 24.5%, whereas in a young
fast-grown tree it reached only 7.68%.119
Often, the amount of extractives at the heartwood periphery is in the vicin-
ity of 10%, although the amount may reach 30-35% in Eucalyptus marginata.
The amount of starch in the sapwood is less than this amount, and, consequently,
extra translocated carbohydrate is required for extractives synthesis. Detailed
studies have shown that, in some species, the amount and constituents of extrac-
tives reach their maximum 2-3 rings inside the heartwood periphery,41.82,12o
THE FORMATION OF HEARTWOOD AND ITS EXTRACTIVES 233
possibly due to aqueous solutions diffusing into the heartwood. 41 ,58 It may also be
due to the slow conversion of precursors to end-product.
In some cases, the lower amounts of extractives in inner heartwood is due
to progressive non-enzymatic insolubilization as the wood ages within the
trunk. 121 The extent of insolubilization is probably related to the ease with which
the compounds are oxidized in air when they copolymerize with different cell-
wall polymers. 122 Also, the inner heartwood of Quercus spp. can become darker
than the outer due to hydrolysis and polymerization of the ellagitannins present. 51
It has been claimed the total amount of extractives is the same throughout the
heartwood. 51 The heartwoods of a number of angiosperms become increasingly
acidic as the trees grow and become more mature, mainly due to acetic acid
released from the acetyl groups of hemicelluloses. Values of pH 2.6 or less have
been found in the interior of some eucalypts. Unstable proanthocyanidins
can form red compounds under these conditions over a period of time and then
oxidize to brick-red to brown insoluble polymers121 which have been observed
in red-brown colored heartwoods. Under acidic conditions, ellagitannins can
be slowly hydrolyzed, and polymerized as in Quercus petraea and Castanea
sativa. 49 ,5o After a sharp increase at the heartwood periphery, the amount of ellag-
itannins decreases but the amount of free ellagic and gallic acids increases as the
heartwood ages, 6
Extractives in the cell wall can enter from cell lumens during heartwood
formation. Another possible mechanism is that precursors of polymerized
polyphenols enter or diffuse into the compound middle lamellae, or intercellular
space between the cells, from the pits of the parenchyma cells. From that
location they infiltrate the walls of the fibers via the cell wall capillaries of
the S I and the less permeable, thicker S2 layers of the secondary waiL 123-125
About one-half of the total polyphenols in redwood and incense cedar heartwoods
is estimated to be in the cell wall, and these are not as easily extracted as the
remainder, 126
The amount of extractives in sapwood is small and relatively uniform from
cambium to outer heartwood. At the cambial region of Tsuga spp, (hemlock),
a mixture of phenolic depsides based on shikimic, quinic, ferulic, and caffeic
acids is formed along with glucosides of lignans specific for western or eastern
hemlock 127 Catechin, epicatechin, and proanthocyanidin dimers are generated in
the parenchyma of the sapwood. In the transition zone, the lignans matairesinol
(2), hydroxy-matairesinol (3), a-conidendrin (4), and traces of related phenols
are formed along with a small proportion of a lignin-like materiaL These are
deposited in the parenchyma and bordered pits during heartwood formation, 127
Conifers show a pronounced decrease in fatty acid esters and an increase in free
fatty acids of different composition when the sapwood changes to heartwood,6
While neutral lipids dominate in sapwood of Pinus spp" diterpene resin acids,
mono- and sesqui-terpene volatile oils, and fatty acids are the main extractives
234 W. E. HILLIS
in heartwood. 128 The sapwood extractives of Schinopsis spp. are mainly gallic acid
esters and CIS compounds, with the latter being mainly in the inner sapwood. At
the heartwood periphery, the gallic acid content decreases whereas polymers of
CIS compounds increase sharply.12s
When '4C-phenylalanine was administered to Zelkova serrata, it was found
converted into small amounts of polyphenols that were present in sapwood, mainly
keyakinol (10) and taxifolin 6-C-glucoside (11). In the parenchyma of the transi-
tion zone, the phenylalanine was converted into larger amounts ofkeyakinol (10),
aromadendrin 6-C-glucoside (12) and taxifolin 6-C-glucoside (11), and in the
heartwood into, kaempferol 6-C-glucoside (13), keyakinin (14), and quercetin 6-
C-glucoside (15). This suggests that C-glucosylation occurs at the stage offorma-
tion of the flavanonols in the transition zone, and later, at the heartwood boundary,
an enzyme catalyses their oxidative dehydrogenation to the flavonol derivatives. 16
The transformation of sapwood into heartwood in Douglas-fir alters the composi-
tion of phenolic extractives in three different ways: phenolic glycosides present in
sapwood are absent in heartwood; some polyphenols absent or in small amounts
in the sapwood are biosynthesized at the transition zone; some polyphenols are oxi-
dized to complex polymers which may become partially insolubilized. 121
OH
2
R 10: Keyakinol (Rl=OMe, R2=H)
11: Taxifolin 6-C-Glc (Rl=R2=OH)
12: Aromadendrin 6-C-Glc (R I=OH, R2=H)
OH
13: KaempferoI6-C-Glc (Rl=OH, R2=H)
2
R 14: Keyakinin (p.l=OMe, R2=H)
15:Quercetin 6-C-Glc (R I=R 2=OH)
In conifers, the resin canals are lined with epithelial cells which produce
an oleoresin that, in the sapwood of healthy trees, can achieve a pressure of several
atmospheres. The composition of the oleoresin found in pockets differs from the
resin in sapwood and heartwood.
Heartwood is a "conservative" taxonomic tissue, and its extractives are
characteristic of the family, genus, and even species to which the tree belongs.
Natural products can be used in chemotaxonomy to assist identification and
improve botanical classification. There are two categories. First, the phyletic char-
acters acquired in previous times from unknown causes have the greater taxo-
nomic value. Second, the physiological and biological characters acquired as
adaptions to ecological conditions have taxonomic value mostly limited to the
species leve!. For example, the heartwoods of the Cupressaceae are the sole source
of tropolones such as ~-thujaplicin (16), and the Pinaceae contain a number of
monocyclic terpenoids (17-20). Different classes of polyphenols are characteris-
tic of Prunus, Acacia, Nothofagus, and Eucalyptus genera. 6 However, a few
species have biochemical varieties in which the heartwood components are
significantly different from those normally found in the species. 129
q:H 0
2 2 2 2
16: (3-Thujaplicin 17: Terpinolene 18: (1-Terpinene 19:(3-Terpinene 20:y- Terpinene
two types of parenchyma cells contain different major components. The axial
parenchyma cells of Cryptomeria japonica form significantly more red-colored
materials than do ray parenchyma cells during heartwood formation. 130 The latter
contribute more to the dark color of heartwood color. The parenchyma materials
often are deposited around cross-field pittings and around bordered pit pairs on the
tracheid-lumen side, particularly in latewood. 129 The axial parenchyma cells are
also significantly larger than those ofthe ray parenchyma in Pseudosindora palus-
tris. The phenolic fractions of the ethyl acetate soluble extractives are larger than
the acidic and neutral fractions in the axial parenchyma. The same phenols and the
neutral fraction (mainly sterols) are more abundant in the axial than in the ray
parenchyma. 131
The outermost colored growth ring of the heartwood of Robinia pseudoa-
cacia contains the highest concentration of dihydrorobinetin (21) in winter,
whereas in late summer its concentration is highest in the next inward growth
ring. A hydroxycinnamic acid derivative shows a similar distribution. In contrast
to dihydrorobinetin, robinetin accumulates in significant amounts in the transi-
tion zone. 65 In another study,56 phospholipides in R. pseudoacacia decreased
significantly in the transition zone towards the heartwood boundary so that only
trace amounts were present in the heartwood. Steryl esters were highest at the
heartwood periphery, but free sterols reached their maximum value with increas-
ing depth within the trunk, while relative frequencies of individual free sterols
changed. Phospholipase enzymes in this species also showed a maximal activity
at the heartwood periphery. 56
The heartwood ofCryptomeriajaponica contains the conioids (norlignans)
agatharesinol (5), sequirin (6), sugiresinol (7), hydroxysugiresinol (8), and
ferruginol (9). The radial position of the maxima of each ofthese phenols changes
depending on the height in the tree. 41 In a later study, the abrupt formation of
heterogeneous droplets which appeared in the mainly living ray parenchyma of the
transition zone was considered to be related to the heartwood phenols and their
precursors. These substances migrated from the ray parenchyma to the dead tra-
cheids in the transition zone in the first phase of biosynthesis of heartwood phenols.
Then, in the second phase at the boundary between transition zone and heartwood,
the chemical composition of the oil-like droplets changed from lipid-like materi-
als to those with a phenolic nature. The latter are probably precursors ofthe heart-
wood phenols. In the third stage, changes to the mostly conioid components
increased at different rates inside the heartwood, possibly due to some ray
parenchyma still living in the heartwood or to other biological changes. 107
Juglans nigra,6 but not in Chamaecyparis obtusa I35 ), high amounts of ethylene
have been detected in the transition zone or adjacent tissues in the late dormant
and winter periods. The quantity of ethylene (and phenolic extractives) produced
per unit weight of wood by the living axial and horizontal parenchyma adjacent
to the heartwood periphery is influenced more by individually activated than
total parenchyma cells and, in some cases, by the level of oxygen. 133 When fresh
blocks of P radiata sapwood were stored in a container continuously ventilated
with humidified air containing ethylene, polyphenols were formed but in a
composition resembling that of injured wood. 64
The situation is shown more clearly by the transition zone surrounding a
zone of infection. The wound reaction appears to be most rapid during the
growing season, and more ethylene emanates from this zone than that adjacent
to the heartwood of Pinus radiata or from mechanical wounds. 63 After the ovipos-
itor of Sirex noctilio penetrates the bark of P radiata, it forms two tunnels, one
for its eggs and the other for the fungus Amylostereum oreolalum to feed the
hatched larvae. 63 The sapwood cells are ruptured in the process. Dominant trees
produce much greater amounts of ethylene in response to attack by Sirex noctilio
and lesser amounts by injury than do suppressed trees. 64 Three weeks later,
pinosylvins (22,23) are present in considerable quantities in the lesions. 63 .64
Further cytological and biochemical studies on extractives formation are needed
giving particular attention to each part of the transition zone.
OH
OH
H0Y-O~
::7 I -
HO :::,...
OH
OR
as darker material adjacent to the sapwood boundary. Because of their large size
these polymerized materials are unable to penetrate the cell walls of the fibers in
contrast to the extractives of normal heartwood.
Considerable differences in the amounts of storage carbohydrates
and adenine nucleotides have been observed between different trunks of Fagus syl-
vatica. 104 However, with increasing depth in the trunks, these carbohydrates
decrease in amount up to the boundary of the discolored zone which does
not contain them. The pattern of radial distribution of adenine nucleotides, which
act as energy donors, was similar to the carbohydrates, particularly with the
decrease inside the discolored wood. This pattern indicates the synthesis of com-
pounds takes place at the sapwood-discolored wood boundary after a short phase
of enhanced metabolism, in a similar manner to heartwood extractives. 104
Discolored wood resulting from injury is part of a sequence which can lead
eventually-but not always-to decay. According to the vigor of the tree, the
severity of the wound, and the aggressiveness of the microorganisms, the changes
may stop at a particular stage. Dark sapwood discolorations may with subsequent
healing and diameter growth become incorporated into heartwood with darker
colors. 147
When invading microorganisms spread, they do so vertically through com-
partmentalized tissues. While the margins of the compartments remain intact, the
portion of the column distal to the wounds-the reaction zone-can still contain
pioneer microorganisms. In some cases, these compartments appear as dark bands
in sapwood and heartwood.
Dark irregular streaks characterize some species such as Jug/ans nigra,
Tectona gra n dis, and Acacia melanoxylon. Less common are the narrow axial
bands or streaks of varying color and intensity which are found in a regular or
characteristic arrangement in sapwood or usually heartwood. These streaks can
be localized in tangential directions, such as in Diospyros species,148 or be more
extensive as concentric groups. Most notable are the large number of narrow dark
brown to black streaks in a creamy yellow heartwood of Microberlinia brazzav-
illiensis and the red axial streaks in a brown heartwood of Berlinia Spp.98 Whether
the highly selective formation of specific compounds by the physiologically active
parenchyma is due to inherent genetic cellular differences or to different stimuli
of the parenchyma at the heartwood periphery is unknown.148.149
Stains may also form at the sapwoodlheartwood boundary, and their cause
is uncertain. Soil conditions or root injuries may provide the initial stimuli for
eventual stain formation. 147 The stains contain larger amounts of colored mater-
ial than heartwood, and in Quercus spp., they originate in the ray parenchyma and
eventually become incorporated in the heartwood. Stains can also represent the
early stages of attack by certain species of fungi on the non-living cells of nor-
mally colored, completely formed heartwood, and result in dark discolorations,
such as those caused by Stereum frustulatum, and Fistulina hepatica on oak,
THE FORMATION OF HEARTWOOD AND ITS EXTRACTIVES 241
Eucalyptus marginata, and other species. 6.'47 Such tissue has lower durability than
heartwood.
Target ring patterns of pale color have been observed in Thuja plicata,
Pseudotsuga menziesii, and other species6 and in moon rings of a few consecu-
tive growth rings in Quercus Spp.150 These paler colored rings contain less
extractives than the adjacent heartwood, although the ellagic acid content in
Quercus spp. is higher,'51 and have been attributed to an earlier, cold period when
heartwood formation was interrupted.
Wounds are prevented from becoming extensively colonized by pathogens
by two general types of responses: cytological and chemical. When living cells
gradually die under conditions suitable for cellular metabolism, starch, if present,
disappears, and antimicrobial compounds are formed in "reaction zones" to
inhibit decay organisms. 92 •l37 Fungal infection can result in the formation, in
wound or discolored wood, of uncharacteristic compounds, different from those
normally formed in the heartwood of that species. Normal heartwoods of Prunus
spp. contain flavonoids and proanthocyanidins, 15 I but P. domestica affected by
Stereum (now Chondrostereum) purpureum contains patches of the coumarin
scopoletin (24).153 P.jamasakura affected by Trametes (syn. Coriolus) versicolor,
contains large amounts of the lignan cyclo-olivil (25).154 P. yedoensis, affected by
Taphrina wiesneri,155 contains fluorescent gentisic acid (2,5 dihydroxy benzoic
acid) glycosides and other compounds. Stained wood, resulting from colonization
of C. purpureum in Malus pumila, contains at its boundary with sapwood the
biaryl compound aucuparin (26) and a pentacyclic triterpene (27).156 The same
pathogen formed A-pyrufuran (28) in Pyrus communi. 157 When the highly durable
heartwood in a living Thuja plicata is infected by a fungus, Sporothrix sp., a novel
lactone thujin (29) is produced from the thujaplicins e.g. 16. 158 Also, the discol-
ored heartwood in this species, possessing distinct, but irregular transverse
boundaries, can contain fungi including Cylindrocephalum sp. This fungus
reduces the amount of the toxic thujaplicins and lignans but does not affect the
structural integrity of the wood. '59
The amount and composition of terpenoids can also change markedly upon
fungal infestation or insect attack. The ratio of the components in the group of
sesquiterpenes resulting from infection of Ulmus glabra with Ceratocystis ulmi
was different from that when infected with Corio Ius versicolor, and also from
that infected with Chondrestereum purpureum. 160 When the sapwood of Pinus
contorta was attacked by Dendroctonus ponderosae and its associated microor-
ganisms, the amounts of total terpenes and particularly J3-phellandrene (30) were
much higher than those found in normal heartwood. 161
With Liriodendron tulip!era,162 the sapwood contained small amounts of a
single non-phenolic alkaloid-glaucine (31). This compound was the major com-
ponent of the heartwood extractives, which contained substantial amounts of five
other non-phenolic alkaloids, two phenolic alkaloids, two lignans, and one simple
242 W. E. HILLIS
CH30~OH OH
HO "- I I-oH
::::,.. OCH3
OH
phenol. On the other hand, the discolored sapwood (probably originating from
fire) contained nine non-phenolic aporphine alkaloids, eight phenolic aporphine
alkaloids, and two lignans. The composition of these extractives differed from
those in the heartwood in the three discolored zones of the one cross-section. In
two of these zones, the amount was higher than in the heartwood, with glaucine
being the most abundant compound and varying five_fold. 163 In later studies, the
extraordinarily high concentration of glaucine in the discolored sapwood formed
in response to injury to the living tree and its high antifungal activity suggested
that injury induces the tree to stimulate the biosynthesis of glaucine thus
protecting itself against attack of microorganisms that invade the stem after
wounding. 163 This in turn causes deficiency in the O-methyltransferase system of
the tree, resulting in the formation of phenolic tetraoxygenated aporphine
alkaloids. 163 These antifungal alkaloids are probably metabolized in tum to
nonantifungal but antibiotic alkaloid pigments by the fungi.
THE FORMATION OF HEARTWOOD AND ITS EXTRACTIVES 243
HO
OH
HO yh
,;7
~I
I
R
h
oH
o OH
separations between cells and occur in short lengths from the pith. They have no
obvious connection with living tissues, and the deposits, which appear to be formed
in situ, continue to fill them when they enlarge with continued tree growth. 4
Almost pure or high concentrations of the lignans gmelinol (35) in shakes
of Gmelina leichhardtii and cyclo-olivil (25) in Olea cunninghamii, the aromatic
diterpene acid podocarpic acid (36) in shakes of several Dacrydium spp. and
Podocarpus spp, the methyl thujate (37) in Thuja plicata, and camphor (38) in
Dryobalanops aromatica and D. lanceolata have been reported. 6 Three different-
shaped deposits occurred close together in a heart check (split) in the pith region
of Tsuga heterophylla, and each deposit largely contained one of the lignans a-
conidendrin (4) or matairesinol (2) or hydroxymatairesinol (3).166 Other shakes in
T heterophylla contained a-conidendrin 166 or matairesinol. 166 Shake deposits in
T mertensiana contained matairesinol 167 as did those in Abies spp. and Pseudot-
suga menziesiii. 168
Deposits in the shakes of lntsia bijuga contained crystals of pure robinetin.
"'()' C""O
'OCH 3
SUMMARY
Phenolic and similar compounds are present in small amounts. The change from
sapwood to symmetrically-shaped heartwood is due to increased metabolism of
parenchyma cells at the transition zone or heartwood periphery after which all
cells are dead. The changes occur with the passive involvement of other cells.
Increased amounts of ethylene also are formed in some species in response to
insect injury of living sapwood.
A major feature of heartwood is the increased formation of extractives, or
secondary metabolites, or non-structural materials, of a complex composition
which is different from that of the sapwood. The amount and type of the com-
pounds is characteristic of the species, but the amount can be influenced by envi-
ronmental conditions. The formation of natural products probably arose during
evolution as a defense mechanism.
Heartwood extractives are formed at the periphery from starch stored in the
sapwood and from translocated carbohydrate from the cambium. After synthesis,
the parenchyma cells die, and the different cell compartments disintegrate, allow-
ing further changes to the compounds, such as non-specific oxidation and poly-
merization. Diffusion into the cell wall or entry into the lumens of neighboring
cells, notably the entry into vessels in angiosperms, occurs.
Various types of irregularly-shaped discolored wood (false heartwood) are
apparently a form of localized containment system resulting from injury. The
extractives are more highly polymerized or oxidized than those of heartwood
extractives and are less likely to diffuse into the cell wall. Heartwood, discolored
woods, and sapwood affected by microbial growth and other injuries can contain
compounds different in composition from those normally present in heartwood.
A particular fungus can result in different extractives in different species.
In a few species, components of extractives can be formed in a high degree
of purity in vessels of angiosperms, tracheids of gymnosperms, and even in shakes.
Why or how these vessels and tracheids accumulate these complex natural prod-
ucts, that are present in much smaller amounts, if at all, in neighboring tissues, is
not yet know. Understanding the mechanisms for these highly selective formations
could lead to processes to biosynthesize complex compounds without by-products
from simple substrates. These substrates could be provided by simple carbohy-
drates, such as those translocated from the leaves, and available in unlimited quan-
tities. Essential additions would be necessary to support specific enzyme systems.
Synthesis by enzyme systems, using simple inexhaustible resources, and requiring
minimum energy, would provide a more economical way to produce complex
natural substances than those now available.
ACKNOWLEDGMENTS
Dr. G. A Kile, ChiefCSIRO Forestry and Forest Products, provided facilities and
Ms. Heather Forster prepared several drafts of this chapter. I am grateful to Dr.
lA.F. Gardner, Dr. A.F.A. Wallis, and Mr. 1 Ilic for reviewing the manuscript.
246 WE. HILLIS
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THE FORMATION OF HEARTWOOD AND ITS EXTRACTIVES 253
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Chapter Ten
INTRODUCTION
255
256 D. FERREIRA et al.
The limited number of options that are available for the synthesis of proan-
thocyanidin oligomers has been comprehensively reviewed 10- 13 and need not be
discussed here. In principle, a chain extender fIavanyl unit with a good leaving
group (typically OH or SCH2Ph) at C(4) is converted into a strongly electrophilic
center (typically C(4) carbocation or A-ring quinone methide) which is then
trapped by the chain terminating nucleophilic fIavanyl unit, typically a fIavan-3-
01 moiety, to give the regiomeric (4~6)- and (4~8)-bifiavanoids. These then
serve as precursors to trifiavanoids via coupling with the electrophile at the A-
or D-rings, and eventually to tetraflavanoids and higher oligomers (Fig. I).
In the profisetinidin (RI = H), i.e. 5-deoxy (A-ring) series of compounds, the
bifiavanoids have a preference for D-ring coupling in the transformation to the
trimers, while the procyanidin-type bifiavanoids (RI = OH), i.e. 5-oxy (A-ring),
exhibit a remarkable propensity for coupling at C(8) (A-ring). For chain termi-
nating moieties possessing B-rings with nucleophilicity comparable to that of the
A-ring, the former rings often compete favorably as nucleophiles in the process
of interflavanyl bond formation. 13 ,14
A recent application of this protocol to the synthesis of a unique series of
di- and trimeric profisetinidins, and a novel utilization of 4-benzylsulfanylfIavan-
3-01s in the controlled synthesis of di- and trimeric procyanidins under neutral
conditions and some related transformations are now discussed.
6.&OH
HO
('~OH
OH
I
H@forLG=OH
HO~ for LG = OH or SCH2Ph
OH ~OH
~ 0yyol'I"'~OH
HOW
0 l II'···~OH
~
@
OH ~OH
R' R'
C-4 carbocation A-ring quinone methide
~OH ~OH
HO
"""~OH """~OH
OH
nucleophilic flavan-3-ol chain
terminating unit
~OH
HO
""'~OH
~
IE ~OH
I""'~OH
Q OH
OH
(4 - 6)-biflavanoids OH
OH
(4 - 8)-biflavanoids
Tetraflavanoids, etc. _Repetition Triflavanoids via coupling at A- or D-ring with
C-4 carbocation or A-ring quinone methide
stituents of wattle and quebracho tannins (see ref. 15 for appropriate references).
Naturally occurring oligomers exhibit predominantly 2,3-trans relative stereo-
chemistry and possess either 2R,3S or 2S,3R absolute configurations. 10.' ,
Profisetinidins exhibiting 2,3-cis relative configuration of the chain extender
moieties are extremely rare and are hitherto restricted to two tentative (4~6)
bis-fisetinidols from Colophospermum mopane,'6 a promelacacinidin from
Acacia melanoxylon,17 and some proteracacinidins's from A. galpinii's and A.
caffra.'9
The methanol extract of the bark of Pithecellobium dulce (Roxb.) Benth
(Guamuchil, Madras thorn) afforded a series of mono-, di- and trimeric pro-
fisetinidins exhibiting both 2,3-trans and 2,3-cis relative configurations of the
constituent fisetinidol moieties,'5 thus offering the opportunity to rigorously cor-
roborate the structures of the 2,3-cis analogues via synthesis. Such an approach
was additionally motivated by the inability to unequivocally differentiate between
2,3-cis-3,4-trans- and 2,3-cis-3,4-cis-configurations of ABC-units in e.g. com-
pound 4 on the basis of 'H NMR coupling constants.
Thus, separate treatment of epifisetinidol-4~-01 (1) with catechin (2)
and epicatechin (3) under mild acidic conditions afforded the epifisetinidol-
(4~~6)- and (4~~8)-catechins (6) and (4), epifisetinidol-(4~~6)- and (4~~8)
epicatechins (7 and 5), respectively (Fig. 2), both couplings proceeding highly
stereoselectively.20 'H NMR data of the permethylaryl ethers in conjunction with
the circular dichroic properties,z'-23 based on the aromatic quadrant rule,z4 then
permitted unambiguous structure definition of compounds 4-7 and hence of the
natural products 4 and 5. Acid-catalyzed condensation of epifisetinidol-4~-01 (1),
with either catechin (2) or epicatechin (3) in a I : 6 molar ratio, stereoselectively
afforded both the dimeric profisetinidins 4 or 5 and the angular trimeric pro-
fisetinidins 8 or 9. The appropriate derivatizations gave the permethylaryl ether
triacetates with 'H NMR and CD data identical to those of the same derivatives
of the natural products.
The elegance of this simple biomimetic approach to the synthesis of proan-
thocyanidin oligomers was next demonstrated during synthesis of the "mixed"
profisetinidin trimers 10 and 11, i.e. analogues possessing different chain exten-
der units. Trimer 10 with its fisetinidol ABC- and epifisetinidol GHI-unit was
formed by acid-catalyzed reaction of the fisetinidol-(4a~8)-catechin biftavanoid
(12)25 and epifisetinidol-4~-01 (1). The remaining triftavanoid (11) with its epi-
catechin DEF-unit was similarly synthesized using the epifisetinidol-(4~~6)
epicatechin (7) (vide supra) in the acid-catalyzed condensation with fisetinidol-
4a-ol (13). Comparison of the 'H NMR and CD data of the pennethylaryl
ether triacetates of trimers 10 and 11 with those of the same derivatives of
the natural products, again provided unequivocal structural proof for the latter
compounds.
RECENT ADVANCES IN THE CHEMISTRY OF PROANTHOCYANIDINS 259
H0'(l(0i:: ex
'" OH I
OH
HO yyvi ",,·: : ,. .
~OH
0 ex
~ OH
OH
~'OH
OH
OH
2 - ,
3 -
~OH
HO
"""~OH
HO
OH
~OH
"""~OH
OH
, ~
OH IE
~ OH
,
4 -
OH
5 -
6 -
Figure 2. Synthesis of dime ric profisetinidins with epifisetinidol chain extender units.
~OH
o"" .. ~OH
II"
OH ~OH
HOJ: "" .. ~OH
~J.., "I
OH
OH
8 ~ =,
9 ~ = ~OH
HO "",.~OH
OH ~OH
HOJ:
""'~OH
0""" OH
OH
10 ~ =,
11 ~ =-=
HO
~I~'l'"
~
0
""".::::.... r8:IB
0H
OH
~OH
~. OH &OH HOYY°'iI""~
~
HO '8 0 E I OH
""'.::::....
~
DI
::::.... F OH = OH
OH QH
OH 12 13
RECENT ADVANCES IN THE CHEMISTRY OF PROANTHOCYANIDINS 261
(JC 0H (JC 0H
HO ",,,,1
HOycxo
,,"" ~ 1 OH
" W O ,I' ,.. OH 1
1
~ OH
~ ""'OH OH
OH SCH2Ph
2 ~-
14
3 ~-
DMTSF or AgBF 4
R
HO
"" •.
(JC
~1
0H
OH
15 ~ " "R= H
16 ~ " ! ,R = 413-epicatechin
17~,,=,R=H
Figure 3. Synthesis of procyanidin B-1 15 and B-2 16 using Lewis acid activation of 4~
benzy1su1fany1epicatechin 14.
catechin (3) in THF was treated with AgBF4 at the above molar ratios, procyani-
din B-2 (17) was formed in 37% yield, without evidence of the formation of
regiomeric dimers or of higher oligomers. This protocol, thus, compares favor-
ably with the classical acid-catalyzed condensation of catechin-4a-ol and cate-
chin (2),20.26 which gave a mixture of procyanidins B-3 (20) and B-6, the trimeric
procyanidin C(2) (21) (ef Fig. 4) and its 4,6-regioisomer, and the presumed all-
trans-(4-t8)-linked tetraflavanoid analogue (10: 1 : 12: 1 : 3) (45% overall yield).
The scope of the thiophilic Lewis acid mediated interflavanyl bond forma-
tion was extended using 4-benzylsulfanylcatechin (18) (4: 1 mixture of 4~- and
4a_epimers),2o.26 representing the (2R,3S)-2,3-trans-flavan-3-01 extender unit of
the procyanidins, as source of the flavan-3-01 C(4) electrophilic moiety (Fig. 4).
Separate treatment of a mixture of the epimeric 4-benzylsulfanylcatechins (18)
and catechin (2) and epicatechin (3) in THF with AgBF4 afforded procyanidin
B-3 (20) (35%) and B-4 (19) (51%), respectively. The preferences for the for-
mation of 4~- and 4a-interflavanyl bonds by using the epicatechin- and catechin-
4-thiobenzyl ethers (14 and 18), respectively, and for the (4-t8)-interflavanyl
linkages were anticipated,2o.26 i.e. the thiobenzyl ethers are converted by Lewis
acids into relatively stable intermediates permitting both the regioselective attack
of the nucleophile via C(8), where the HOMO displays maximum amplitude, and
the stereoselectivity by approach from the sterically least hindered side.
Finally, procyanidin B-3 (20) and the 4-benzylsulfanylcatechin epimeric
mixture (18) were coupled by using AgBF 4 in THF to give the trimeric pro-
cyanidin (21) (26%) as the only isolable product (Fig. 4). The sequences towards
the procyanidins depicted in Figures 3 and 4 from using AgBF 4 as the thiophilic
Lewis acid, no doubt, offer advantages as far as control over the level of oligomer-
ization, reversibility, and "scattering" of the interflavanyl bond(s) are concerned
in comparison with the formation of these products under conditions previously
developed (see ref. 13 and references cited therein).
(X 0H (X
7"1
0H
Ho):CXo
"" .. ::::,... OH
HOWO"",.::::,... I OH
I :::::,...
1
OH
::::,... OH
H
OH SCH2Ph
2
18 ~ == i and ~ (41)
3
AgBF4
(X
W
0H
HO 0
I "" . : : :,. .
1
OH
:::::,... ~ OH (X0H
(X OH ';
yyvi""':::::"" 0 I
W"
0H HO
OH
HO 0 ·1
~OH
OH OH
~H ~
W
OH ( X IOH 19
HO ' 0
::::,...I "" .. ::::,... OH
~ OH (X0H
I
OH
HO
yyv i """:: :""
'
' 0
OH
~OH
OH
21
Figure 4. Synthesis of procyanidin B-3 20, B-4 19 and C-2 21 using Lewis acid activation of
the 4-benzylsulfanylcatechin epimers 18.
These conditions,20 which have been applied universally for the formation of
C(Sp3)~C(Sp2) interflavanyl linkages, would be less applicable to the formation
of the ether bonds in compounds 22-25. We, thus, selected to activate the elec-
trophilic attributes of one of the flavan-3,4-diol methyl ethers, e.g. 26, via for-
mation of the 4-chloroflavan-3-ol derivative 27, in order to permit the formation
of the crucial ether bond at a near neutral pH value.
264 D. FERREIRA et al.
°H (£rI 0H OH
HO~O2,,"" ~B HO
A I c 3 3
~ 4 ""'OH
QH ~ 'OX"'O~ OH
° ~ 0,1'1
'. 4
"I
.................
OH
HO.",3 _ 0
OH
Q
OH
HO
22 - !
23 - OH
24
H
H0'6?°H
I
° "",·aO
~ 'Cl"'O~
~ I 0 ""
OH
'. OH
HO"'" _ 0
25
OMe OMe
I I
MoO'Oo:" '"
OMe (r OMe (r
MeOty~
I ° : :". ~
4 OAc = "OAc
QAc
OAc
0:OC"
~II"" ° I ""'"
30
N
b OMe O\1e
OMe ~
Me Meo~o~II,.0
MeO i) ~
:- OH
29 OH
"
ii) Ac 20/pyridine
OMe
OMe I
(r
Me0Yy0'lII," ~
VY""OH C1
27
MeO
OM< (J
~0'-lil' ~ I
:Y
aM,
i) l,JU,. :- OH
()~\lk
M,O
OH
JJ
i)
ii) Ac 20/pyridine OH
/
31
ii) Ac 20/pyridine
(rOMe OMe
M"'~::: I
MeO
O~", ".
A
ox: O
..........
lOMe
OMe
t "cf¥"O~
2> D I
OMe
()
34
1:e ~ OMe 32
Miscellaneous
35
(X 0H
'(j()", : : ,. .
HO 0 " I
(X
OH
: :,. (X
0H
I OH
1
HO 0"
HOyYO,l""'::::"" OH yyv'l""::::"" OH
~
1
~OH OH
OH
(X
W
0H
HO 0, I
OH
::::,...1
= OH OH
HO~OI"'(XH
~OH
OH
38
268 D. FERREIRA et al.
B-Type Proanthocyanidins
Rl
cix°
RI
~OR'
6'~
I B H
R20 2II"::::"",
" OR2
HO 0 B I
~.::::,... OH
OR2 A I C
::::,...
=4 OH
OH 11""&OR2
OH
~OH
OR2
HO =
12 I - , RI =R2 =H
~
39 I ' R' = OH, R2=H I E
,,
# OH
40 I - , RI = H, R2 = Me
OH
41 I - , Rl =R2=H
42 l - , Rl = H, R2 = Me 43 RI =H
44 RI=OH
RECENT ADVANCES IN THE CHEMISTRY OF PROANTHOCYANIDINS 269
RIO 0 ..
(E(IE
0Rl
~
"":::".. ORI
D I F
:::".. OH
RI
12/40 ~ 2 RI =H
OH
47 RI =Me
45 RI =OH HO~~O.
-::r OH-::r OH
46 RI =OMe
: :". :J ~ I
:::".. OH
~:J
1 48
(S(
oMe
BI 1
-::r oow
Me ", ".:::".. OMe
47 +50
A I c
:::".. OH
RI
53 Rl =H
54 Rl =D
49 RI = R2 = H
50 RI = Me, R2=H
51 RI = H, R2 = D
52 RI = Me, R2=D
Figure 6. Proposed route to the cleavage of the interflavanyl bond and of the C-ring in
profisetinidins e.g. 12 and permethylaryl ether 40.
sumably results from the relative lability of this bond, imposing a high degree of
SN 1 character to the processes of protonation and delivery of hydride ion.
The mechanism for cleavage of the interfiavanyl bond in the profisetinidin
biflavanoids (Fig. 6) was corroborated using sodium cyanotrideuterioborohydride
[Na(CN)BD31 in TFA. Under these conditions the fisetinidol-(4a~8)-catechin
(12) was converted into catechin (2) (26%) and the (2R)-1,3-dideuterio-1,3-
diarylpropan-2-ol (51) (25%), while the permethylaryl ether (40) and the
fisetinidol-( 4~~8)-catechin hepta-O-methyl ether (42) both gave tetra-O-
methylcatechin (47) (12, 32% resp.), the dideuterio-l ,3-diarylpropan-2-ol tri-O-
methyl ether (52) (14, 16% resp.), and the 4~-deuteriofisetinidol derivative (54)
(12, 14% resp.). Formation of the deuteriated 1,3-diarylpropan-2-0Is (51 and 52)
RECENT ADVANCES IN THE CHEMISTRY OF PROANTHOCYANIDINS 271
A-Type Proanthocyanidins
The double interflavanyl linkage in A-type proanthocyanidins introduces a
high degree of conformational stability which culminates in high-quality and
unequivocal NMR spectra, conspicuously free of the effects of dynamic rotational
isomerism at the dimeric level. Compounds of this class are readily recognizable
from the characteristic AB-doublet CJ3 .4 = 3-4Hz) of C-ring protons in the het-
erocyclic region of their lH NMR spectra, 54 and may possess either (20.,40.)- or
(2~,4~)-double interflavanyl bonds. Two fundamental structural problems, i.e.
establishment of the mode of linkage of the C- to the D-ring, and assignment of
272 D. FERREIRA et al.
RIO RIO
15 RI =H 20 RI =H
55 RI =Me 56 RI =Me
~OMe
MeO
"""~OMe MeO
~OMe
"""~OMe
OH
OMe OMe
57 58
OMe
((
Meo
v ' O Q 0 I' "" ~ I OMe
I
~ ""'OH
D
59
60
RECENT ADVANCES IN THE CHEMISTRY OF PROANTHOCYANIDINS 273
the absolute configuration at the stereocentres of the F-ring, have limited progress
in this field. These and related problems have hitherto been approached via exotic
spectroscopic methods 55 - 58 which prompted us to search for a simpler and general
chemical method that is based upon the reductive cleavage of the acetal func-
tionality of A-type proanthocyanidins. The potential to address these problems by
reduction of either of the c-o acetal bonds was demonstrated 59 for the known
procyanidins A-I (61) and A-2 (62), available from the skins of mature peanuts
(Arachus hypogea),60 by using Na(CN)BH3 in TFA. The readily accessible hepta-
O-methyl ethers (63 and 64) were selected as model compounds with a view to
using the O-substituents of the D-ring as probes for anticipated much simplified
1H NMR studies.
Separate treatment of the hepta-O-methylprocyanidins A-I (63) and A-2
(64) with Na(CN)BH3 in TFA for 1.5 h at O°C (Fig. 7) gave conversion to mix-
tures comprising the starting materials, and, as anticipated from cleavage "a", the
tetrahydropyrano[2,3-f]chromene derivatives (65) (5.2%) and (66) (7%). The
envisaged B-type procyanidin biflavanoids (67 and 68) from the "b" pathway were
not obtained, but instead, the respective monomeric units, i.e. tetra-O-methyl-ent-
catechin (69) (4%) and tri-O-methylcatechin (70) (3.4%) from the A-I derivative
(63), and tetra-O-methyl-ent-catechin (69) (3%) and tri-O-methylepicatechin (71)
(l.3%) from the A-2 derivative (64) were isolated.
Both the carbon-oxygen bonds of the acetal functionality in the procyani-
din A-I (63) and A-2 (64) derivatives are, thus, susceptible to reductive cleavage
under acidic conditions. This process is presumably triggered by the random pro-
tonation of the acetal oxygens and concomitant delivery of the equivalent of
hydride ion at the antibonding (cr*) orbitals of the carbon-oxygen bonds in a pre-
dominant SN2 manner. Such a transfer of hydride ion apparently occurs from
a complex between the reducing agent and the axial C(3) (C-ring) oxygen lone
pair, the proximity of the boron-hydrogen bonds to the backside of the acetal
carbon being a prerequisite for reduction of either one of the acetal bonds. Reduc-
tion, thus, leads to "inversion" of configuration at C(2)(C) of both B-type pro-
cyanidin intermediates (67 and 68), and of the tetrahydropyrano[2,3-f]chromene
derivatives (65 and 66). The chemistry and the unequivocal structure elucida-
tion, including assessment of absolute configuration at all the stereocentres of the
latter class of compounds, are well understood, 61-63 and facilitated confirmation
of the absolute stereochemistry ofring F in the natural product derivatives 63 and
64.
Biflavanoids (67 and 68) are prone to facile cleavage of their interflavanyl
bonds via protonation of the electron-rich phloroglucinol D_ring 49 .5o and attack of
hydride ion at C(4)(C)46 to give the ent-catechin derivative (69) from the ABC-
unit and respectively, the epicatechin and catechin derivatives (70 and 71) from
the DEF-moieties. The "liberation" of the latter two chain terminating flavan-3-
01 units unambiguously defines the D-ring oxygen that is involved in the acetal
274 D. FERREIRA et al.
RIO
ORI 63,641 ..
cleavage a
65 ~ =~
661 = ~
61
62 I:=
63 ~
,RI=H
, Rl=H
,Rl=Me
64
!= ,RI=Me
MeO
67 ~ = I
68 ~=I
MeO
OMe
70 ~ =~
69 71 ~=l
Figure 7. Cleavage of the acetal functionality of proanthocyanidin A-I and A-2 permethylaryl
ethers 63 and 64 with Na(CN)BH3 in TFA.
RECENT ADVANCES IN THE CHEMISTRY OF PROANTHOCYANIDINS 275
Dihydroflavonols
Owing to the ease of the reductive transformation, dihydroftavonol ~
ftavan-3,4-diol,20.64 the dihydroftavonols (four diastereomers for each hydroxyla-
tion pattern) are key compounds as precursors to the electrophilic ftavanyl chain
extender units in the semisynthetic approach to proanthocyanidin oligomers. Only
a limited number of dihydroftavonols with 2,3-trans configuration are available
commercially or from natural sources. Analogues possessing 2,3-cis configura-
tion are exceptionally rare and definitely not available for preparative applica-
tions, which clearly demonstrates the need for a synthetic protocol giving access
to the full range of dihydroftavonol diastereomers with phenolic oxygenation
patterns approximating those of the natural products.
Virtually all the synthetic efforts to synthesize enantiomerically enriched
dihydroftavonols had hitherto focussed on the Julia asymmetric epoxidation of
chalcones 65 -67 and the subsequent transformation of the chalcone epoxides into
dihydroftavonols. The literature covering these developments up to 1990 was
recently comprehensively reviewed. 68 Our own efforts in this regard focussed
mainly on chalcones exhibiting the hydroxylation patterns of naturally occurring
dihydroftavonols,69-71 in contrast to other approaches selecting chalcones with the
minimum number of, or which are devoid of phenolic oxygenation. 72- 76
Epoxidation of the chalcone methyl ethers (72-76) with hydrogen perox-
ide in the triphase system, aq. NaOH/poly-L or D-alanine/CCI4,65-67 gave the
(-)-trans-(77a-81a) (aR,/3S)70 and (+)-trans-epoxides (77b-81b) (as,/3R),70
respectively, in high yields (79-99%) and fair enantiomeric excess (50-85%)
(Fig. 8).77.78 Initial attempts towards cyclization of the epoxides to the corre-
276 D. FERREIRA et al.
- (i)
~~" 0 ",H ""<::::: R5
RIAJl:3 I
# R4
72 Rl = R3 = R5 = H, R' = MOM, R4 = OMe 77 Rl = R3 = R5 = H, R2 = MOM, R4 = OMe
73 Rl =R4 = OMe, R' = MOM, R3 =R5 = H 78 Rl = R4 = OMe, R2 = MOM, R3 = R5 = H
74 Rl =R4=R5 = OMe, R2 = MOM, R3 =H 79 Rl =R4 = R5 = OMe, R2 = MOM, Rl =H
75 Rl = R3 = R4 = OMe, R' = MOM, R5 = H 80 Rl =R3 =R4 = OMe, R2 = MOM, R5 = H
76 Rl = R' = R4 = R5 = OMe, R' = MOM 81 Rl = R3 =R4 = R5 = OMe, R2 = MOM
(XI
W
Rl 0 R4
I I""'~ R5
~ OH
R3 0
87 Rl = R3 =R5 =H, R4=OMe
-
88 Rl =R4= OMe,R3 =R5 =H
89 Rl = R4 = R5 = OMe, R3 = H (iii)
90 Rl = R3 = R4 = OMe, R5 = H +
91 Rl = R3 = R4 = R5 = OMe
,,(XI
W
Rl 0 R4
82 Rl =R3 =R5 =H, R4=OMe
I ""~ R5 83 Rl = R4 = OMe, R3 = R5 = H
84 Rl = R4 = R5 = OMe, R3 = H
~ OH 85 Rl = R3 = R4 = OMe, R5 = H
R3 0 86 Rl = RJ = R4 = R5 = OMe
92 Rl=RJ=R5=H,R4=OMe
93 Rl = R4 = OMe, R3 = R5 = H
94 Rl = R4 = R5 = OMe, R3 = H
95 Rl = R3 = R4 = OMe, R5 = H 77-96a = configuration shown
96 Rl = Rl = R4 = R5 = OMe b = enantiomer
MOM=CH20Me
Flavan-3-ols
The flavan-3-0Is are the most common chain terminating flavanyl units in
naturally occurring proanthocyanidin oligomers.1.3 All four possible diastere-
omers of catechin (3,5,7,3',4'-pentahydroxylation) were encountered, hence
stressing the need for synthetic access, especially in view of the fact that only
(+)-catechin and (-)-epicatechin are readily available among this largest group of
naturally occurring C6 .C 3 .C 6 -metabolites. In order to address the issue of stereo-
control at C(2) and C(3) of the flavan-3-01 molecular framework, we designed a
278 D. FERREIRA et al.
l3-attack
OMOM
(IR,2R)-102b-I06b
i OMOM
a-attack
97-101
RI,R2,R3,R4=H,OMe
(1 S,2S)-102a-I06a
NMR analysis of procyanidin B-1 (15) and B-3 (20) permitted full assign-
ment of the proton and carbon resonances for both the more extended 117 and
compact 118 conformers in the free phenolic form. In organic solvents, the more
extended rotamer 117 of procyanidin B-1 (15) is preferred over the more compact
rotamer (10: 7), but in water, the more compact rotamer dominates (10: 2). When
procyanidin B-3 (20) is dissolved in organic solvents, the more compact rotamer
is slightly preferred (8 : 10). With water as solvent, only trace proportions of the
more extender rotamer are detected. In this solvent, rotational conformation
exchange is detected despite the observation of two distinct and sharp sets of
signals for each rotamer. The heterocyclic ring of the ABC unit exists in an
approximate half-chair conformation in each rotamer for both procyanidin 8-1
(15) and B-3 (20). Coupling constants of the heterocyclic ring of the DEF moiety
in both 15 and 20 indicate substantial axial orientation of the E-ring (see 119 and
120 for E- and A-conformers of the DEF unit of 20). Lineshape analysis of 3-
H(F) indicated that the "abnormal" coupling constants of the F-ring were indica-
tive of a comparatively high-energy skewed-boat conformation for 15 and
between a half-chair and a skewed-boat conformation for 20 rather than to E- ~
A-conformational exchange, which has hitherto been used to explain the smaller
than anticipated coupling constants.
RECENT ADVANCES IN THE CHEMISTRY OF PROANTHOCYANIDINS 281
OH
~
BI
O 1"-
HOW ~ OH
A 1 c 3
~ - OH g
9'O H
OH ~ E 1
HO~'8
9 0 1"- ~ OH
DI F
~ OH
OH
OH
OH
OH
Fl
~
+OH
OH
119 : E-conformer 120 : A-conformer
OH
121
e('7
NH3 ,N~
H l8: 'f-H
H+OC CO-HN H
OH
H.............
B I CO
I 0
CU2
HO
o ,1"" ~ OH N--\-H
H J H
OH
~OH
HO
"I"'~OH
OH
122
CONCLUSION
This review clearly demonstrates that considerable progress has been made
to gain insight into the complex factors that govern the chemistry of the proan-
thocyanidin oligomers. The section on the stereoselective synthesis of
dihydroftavonols and ftavan-3-0Is introduces a fresh breeze into this much
neglected area of proanthocyanidin chemistry, indicating that introduction of chi-
rality at C(2) and C(3) is indeed possible via utilization of the myriad of modern
synthetic methods that are available to researchers in this field. It may be antici-
pated that the rapid advances that have been made in conformational analysis of
these compounds will continue and contribute towards understanding of the intri-
cate principles governing the complexation of proanthocyanidins with other bio-
RECENT ADVANCES IN THE CHEMISTRY OF PROANTHOCYANIDINS 283
molecules. These advances may eventually also contribute towards a better under-
standing of the claimed health-promoting properties of this important group of
natural occurring polyphenols.
ACKNOWLEDGMENTS
We are grateful for the enthusiastic support of our co-workers, E.V Brandt,
B.C.B. Bezuidenhoudt, P.S. van Heerden, R.J.J. Nel, and P.l Steynberg. Financial
support by the Foundation for Research Development, Pretoria and the "Sentrale
Navorsingsfonds" of this University, is gratefully acknowledged.
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7. FRANKEL, E.N., WATERHOUSE, A.L., TEISSEDRE, P.L. 1995. Principal phenolic
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12. FERREIRA, 0., STEYNBERG, J.P., BURGER, IF.W, BEZUIDENHOUDT, B.e.B.
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284 D. FERREIRA et al.
13. FERREIRA, D., STEYNBERG, J.P., ROUX, D.G., BRANDT, E.Y. 1992. Diversity of
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15. STEYNBERG, P.I, STEYNBERG, IP., BRANDT, E.Y., FERREIRA, D., HEMINGWAY,
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18. MALAN, E., SIREEPARSAD, A. 1995. The structure and synthesis of the first dimeric
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21. BOTHA, J.J., FERREIRA, D., ROUX, D.G. 1981. Synthesis of condensed tannins. Part
1. Stereoselective and stereospecific synthesis of optically pure 4-arylfiavan-3-ols, and
assessment of their absolute stereochemistry at C-4 by means of circular dichroism.
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22. VAN DER WESTHUIZEN, 1.H., FERREIRA, D., ROUX, D.G. 1981. Synthesis of
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Trans. I: 1220-1226.
23. BARRETT, M.W, KLYNE, W, SCOPES, P.M., FLETCHER, A.C., PORTER, L.1.,
HASLAM, E. 1979. Plant proanthocyanidins. Part 6. Chiroptical studies. Part 95.
Circular dichroism ofprocyanidins. J. Chern. Soc., Perkin Trans. I: 2375-2377.
24. DE ANGELIS, G.G., WILDMAN, We. 1979. Circular dichroism studies-I. A quad-
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Tetrahedron 25: 5099-5112.
25. YOUNG, D.A., CRONJE, A., BOTES, A.L., FERREIRA, D., ROUX, D.G. 1985.
Synthesis of condensed tannins. Part 14. Bifiavanoid profisetinidins as synthons. The
acid-induced "phlobaphene" reaction. I Chern. Soc., Perkin Trans. I: 2521-2527.
26. DELCOUR, lA., FERREIRA, D., ROUX, D.G. 1983. Synthesis of condensed tannins.
Part 9. The condensation sequence ofleucocyanidin with (+)-catechin and with the resul-
tant procyanidins. 1 Chern. Soc., Perkin Trans. 1: 1711-1717.
27. TROST, B.M., MURAYAMA, E. 1981. Dimethyl(methylthio)sulfonium fluoroborate.
A chemoselective initiator for thionium induced cyclizations. 1 Am. Chern. Soc. 103:
6529-6530.
28. TROST, B.M., SATO, T. 1985. Dimethyl(methylthio)sulfonium tetrafiuoroborate initiated
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719-721.
29. BARRETT, A.G.M., BEZUIDENHOUDT, B.C.B., HOWELL, A.R., LEE, A.C.,
RUSSEL, M.A. 1989. Redox glycosidation via thionoester intermediates. J. Org. Chern.
54: 2275-2277.
30. STEYNBERG, P.1., NEL, R.J.J., VAN RENSBURG, H., BEZUIDENHOUDT, B.C.B.,
RECENT ADVANCES IN THE CHEMISTRY OF PROANTHOCYANIDINS 285
University of Sheffield
Sheffield, u.K., S3 7HF
2Department of Molecular Biology and Biotechnology
Krebs Institute, University of Sheffield
Sheffield, u.K., S 10 2TN
INTRODUCTION
289
290 E. HASLAM et a/.
defense in plants; foodstuffs, nutrition and beverages; fruit and floral pigmentation;
natural glues, varnishes, and exoskeletons; and the influence of diet and the appli-
cation of herbal medicines in the treatment of certain pathological conditions. 2
I 1
[ Protein ( H20)c : Polyphenol ( H20 )d]
[ soluble complexes I
/
New Products
\
[ Protein: Polyphenol] (H 2 0)m
[ insoluble complexes I
STRUCTURE-ACTIVITY RELATIONSHIPS:
POLYPHENOLS
HO
HO
Rugosin D (1)
HO OH
HO~OHO~OH
~o~JS~
0t, O~O~
HO¥OH YOH
OH OH
!3-1,2,3,4,6-Penta-O-galloyl-D-glucose (2)
,1..........
1 ",-' "'"
HO
~
Ol ~,O -"""
~
.& OH
""
"
(XI
OH
OH HO~O ..(.... OH
galloyl esters. etc. ~OH
OH
flavan-3-o1s
Figure 2. Principal aromatic sites in natural polyphenols for the complexation with proteins. '1
High resolution NMR studiesS- lO generally confirm what has always been
assumed, namely that the aromatic nuclei of polyphenols provide the principal
sites for association with proteins. The data obtained from equilibrium dialysis
studies of a range of simple phenols (resorcinol, catechol, pyrogallol) and BSA
gave Scatchard plots whose analysis showed that the affinity of both catechol and
pyrogallol for BSA was three orders of magnitude greater than that of resorci-
no1. 11 Interestingly, resorcinol, at 20 D e, has twice the solubility in water of cate-
chol and pyrogallo1. This observation also gives quantitative support to earlier
ideas that the o-dihydroxy- and o-trihydroxy- aromatic nuclei of natural polyphe-
nols represent the principal sites for complexation with proteins (Fig. 2).
Molar enthalpies of transfer (L1H 0 .tr ) may be analogously determined by
microcalorimetric methods. 6 Plots of (L1H0 .tr ) versus (T L1S 0 .tr ), for a range of
natural galloyl and hexahydroxydiphenoyl esters complexing with BSA, gave
linear plots (slope -1) indicative of enthalpy-entropy compensation. This pro-
portionality indicates that particular features of protein-polyphenol interactions
also may be examined, with suitable caution, by reference to molar enthalpies of
transfer (L1H0 ,tr). Under conditions in which the polyphenol: BSA complex was
precipitated, it was observed that there was little or no heat change on precipita-
tion. This favors the proposition (and often the assumption) that the structures
of the polyphenol: protein complexes are similar in the solid state to those in
solution.
Bradykinin (3)
H~
~--
>
HO
HO~ HO
NH2
H'+A~
N N
1 H
HO f;i
HO~::>
HO
peptide I
1 19
~ • • • • ~ • • • ~~® • • ~ • • ~~
.... ..•
QGP P P QGGP QQRPPQ PGNQ
peptide II 1
• • ~~® • • ~ • • ~~
~ ~ ~
22
Ali ppm
0.2
0.1
• • ~~® • • ~ • • ~~~ • • • • ~ • • • ~
1 n
Figure 4. Peptides I and II. Plot of the mean change in chemical shift (~8) in H20 / d 6 DMSO
(9; I v/v) on going from the free peptide II (22 amino acids) to a I; 5.6 molar ratio of peptide
and 13-1,2,3,4,6-pentagalloyl-D-glucose (2). Chemical shift changes are averaged over all
protons in each residue except for those (e.g. NH) which are exchangeable (e.g. prolyl groups
and arginyl group each have 7 protons).
* *** ** *** * **
• • LlLl®• • Ll.·LlLlLl • • • • Ll • •
1
eLl 22
Figure S. Intermolecular nOes observed in NOESY and ROESY spectra of the complexes
between peptide II and aromatic protons of the 6-galloyl ester group of ~-l ,2,3,4,6-
pentagalloyl-D-glucose (2) such as to suggest that intermolecular contact (*) occured primar-
ily between the galloyl ester groups and specific residues in the peptide chain.
These experiments supported the data obtained earlier with larger protein
molecules, i.e. a clear dependence in the association upon molecular size, the
number and arrangement of phenolic groups, and water solubility of the polyphe-
no!. They also strongly suggested that the principal binding sites on these pep-
tides are the prolyl residues themselves together with the preceding (N-terminal)
amino acid side-chain. Furthermore, measurements in the complexation studies
with both (-)-epigallocatechin-3-0-gallate (4) and 13-1,2,3,4,6-penta-O-
galloyl-D-glucose (2) of significant entropy changes [+80 - 90j deg- I M- 1 and
255 j deg- I M- 1 respectively] strongly indicate that the processes are hydrophobi-
cally driven (water structure breaking) ones.
OH
~OH
H0I(Y0'l,"'VOH
~"'O
OH O~OH
~OH
OH
(-)-epigallocatechin-3-0-gallate ( 4 )
It was concluded that the principal binding sites on the two peptides are
the apolar methylene and methine groups on the prolyl residues themselves. The
interaction was visualized as a hydrophobically driven association between a
galloyl ring and the exposed hydrophobic surface of the pyrrolidine ring, prefer-
entially that face containing the a-proton. Hydrogen bonding of one or two phe-
nolic groups to the tertiary amide carbonyl group on the adjacent peptide linkage
was presumed to be a secondary interaction helping to stabilize the complex, (Fig.
6). Thus, proline-rich peptides and proteins which have an open, random-coil type
of conformation have a high affinity for polyphenols not only as a result of their
extended structures, but also by virtue of the prolyl groups themselves which, in
a figurative sense, provide 'sticky patches" on the protein for the phenolic nuclei
of the polyphenolic substrate.
ASTRINGENCY AND POLYPHENOL PROTEIN INTERACTIONS 299
(a)
\
..
I
I
I
,
,,,
I
precipitation
I
hydrophobic suface layer
polyphenol
,
\
\
,,
\
\
\
, \
, I ;
\ \
(b) \
\
,48,0
\ 0, ..
, ·-0 ~
Figure 7. Polyphenol-protein precipitation processes: (a)-low initial protein concentrations;
(b }--high initial protein concentrations.
( a) (b) (c)
polyphenol I protein
o simple phenol 0
Figure 8. Protein precipitation: the role of simple phenols and their contribution to a hydropho-
bic surface coating-(a)-polyphenols alone; (b}-polyphenols plus simple phenols; (c)-
simple phenols alone.
In ascertaining the proportions of tannin in astringent infusions, great care must be taken to
prevent the presence of any excess of gelatine for when this excess exists, I have found that a
small portion of the solid compound formed is re-dissolved.
WATER
the association processes, the reorganization of the various solvent shells provides
an important driving force for the interactions to take place (Fig. 1). Water is by
far the most common liquid in our environment. Its structure can be represented
by a somewhat distorted tetrahedron with the oxygen atom at the center; the
protons are directed towards two of the vertices, and the lobes of electron
density-the so-called electron lone pairs ("rabbits ears" or "water wings")-are
directed towards the other two. Because water was probably indispensable for the
genesis of life and because of its unique properties, in a pure form and as a
solvent, the structure of water (and ices) has been the subject of intense work
since the last century. The literature is voluminous. However, agreement on a
comprehensive theory of the molecular structure of bulk water has proved elusive.
From the point of view of intermolecular hydrogen bonding, the individual water
molecule is well placed in having both double donor (protons) and double accep-
tor (lone pairs) hydrogen bond functionality based about one central oxygen atom.
It is accepted that the structure and properties of bulk water derive from exten-
sive hydrogen bonding interactions between individual water molecules. It is
the nature and arrangement of these interactions which are still the subject of
debate.
The dual hydrogen bonding functionality of water molecules is seen most
clearly in the crystal structure of ordinary hexagonal ice (Fig. 9) in which each
water molecule has four nearest neighbors to which it is hydrogen bonded. These
four hydrogen bonds are spatially arranged with local tetrahedral symmetry; that
is the oxygen atoms of the neighboring water molecules occupy the vertices of a
regular tetrahedron surrounding the oxygen atom of the central molecule.
This propensity of water to enter into extensive hydrogen bonding networks
extends to the various clathrate hydrates which it forms. As in ice, the hydrogen
bonded water molecules are four coordinate in a distorted tetrahedral arrange-
ment. They are "ice-like" but differ in having large internal cages. 26 Owing to the
steric requirements imposed by the inclusion of other molecules as guests within
these voids, their hydrogen bond networks are organized differently from that of
hexagonal ice.
The melting of ice to produce ordinary liquid water clearly entails funda-
mental changes in the way in which water molecules are arranged relative to one
another. Essentially two models have been developed. Frank's "jlickering cluster"
theory 27.28 invokes the concept of clusters of three or four coordinate water mol-
ecules with a mean life-time of about a nano second. A second type of modeI/9
of which a number of variations have been proposed, is the uniform continuum
model, orignally suggested by Bernal and Fowler. Essential to this second model
is the assumption that all of the hydrogen bonds in liquid water remain intact.
However, they may be stretched, bent, and distorted to an extent that varies with
temperature and pressure; the existence of "broken" hydrogen bonds is not specif-
ically recognized. In the immediate vicinity of a given water molecule, the
arrangement of the quasi-equilibrium positions resembles that in ice, but further
ASTRINGENCY AND POLYPHENOL PROTEIN INTERACTIONS 303
H
I ,
H"";;O"'H HI
H ,
_" / O"-H-.O. H
• 9 ---H-O.. H,' ··H..... I
, ·H ..... / O-H---·O.
H O---H-O HI' " .
I : ....... H...,'
H-'O... H ··o:...-H
H H, I '
/ O"-H-O H
····0 ---H-O. ," "'H I
Figure 9, The water molecule. I
H
"H /H
..... O.. --H-O
..... O-H- .. O
HI' ".
Part-structure of hexagonal ice.
i ........ H '
Hydrogen bonds are shown as , ........ ~H
dotted lines; covalent bonds as full
I
lines. H
away from this center the arrangement and connectivity may be significantly dif-
ferent. In particular, it is likely that the O-H .. ·.... O hydrogen bonds will be bent
and stretched to varying degrees.
of origins, e.g. that tannins were bound by modified collagen and synthetic nylon
polymers such as polycaprolactam, the latter containing the peptide linkage as
the "only reactive grouping. ,,22,31 However, it has long been recognized that the
strong charge solvating property of water and its own intrinsic hydrogen bonding
ability mean that the possibility of obtaining large binding energies from these
forces alone is improbable. 32
Evidence that might be interpreted as showing that hydrophobic interac-
tions are important in the formation of tannin-protein complexes had been noted
earlier, (for example the complexes could be dissociated by detergents and organic
solvents). Hoff and his colleagues/ 3 however, were the first to draw serious atten-
tion to the involvement of hydrophobic groups in the formation and stabilization
of tannin-protein complexes, The hydrophobic nature of tannins was, thus,
demonstrated by their adsorption on uncharged polystyrene resin, Complex for-
mation between condensed tannins and gelatin or poly-L-proline was enhanced
with increasing ionic strength and temperature as predicted for hydrophobic inter-
actions. According to Jencks, "Hydrophobic effects" are probably the single most
important factor providing the driving force for non-covalent intermolecular inter-
actions in aqueous media. 32 They may broadly be defined as an interaction of the
molecules with each other which is stronger than the interaction of the separate
molecules each with water. No mechanism is implied by this definition.
Water is a notoriously poor solvent for apolar compounds, such as hydro-
carbons and the noble gases, at moderate temperatures and pressures, This reluc-
tance to dissolve in water has been popularly attributed to the hydrophobicity of
these substances. For an apolar compound to dissolve in water, it must intrude
into a liquid that is characterized by an extended network of hydrogen bonds and
has a high cohesive energy. Many rationalizations have focused upon the large
losses of entropy which accompany the dissolution of non-polar solutes, such as
a noble gas or a hydrocarbon, in water. Frank and Evans 28 first sought to ratio-
nalize the unusual thermodynamic properties of non-polar solutes in water by
postulating a particular ordering of water molecules (structure making) around
the solute. They described the process as "iceberg" formation:
when a rare-gas or a non-polar molecule dissolves in water at room temperature, it modifies the
water structure in the direction of greater 'crystallinity '-the water, so to speak, builds a micro-
scopic iceberg around it.
He (Kauzman) suggested that the water molecules" anarchic distaste for the orderly regimen-
tation imposed upon them by the hydrophobic side chains of the protein forces these side chains
to shy away from water and to congregate in the centre of the protein.
(a)
alanyl
HN
Me,
reo I
(b)
(c)
I
I Me
M'~l,""l
OC NH
Figure 10. Schematic representation of the hydrophobic effect resulting from the congregation
of two amino acid side chains (alanyl & leucyl) of a protein (a to b), after Nemethy and
Scheraga. 37 ,38 There is a reduction in the number of nearest neighbor water molecules; water
molecules are "released" to the bulk medium (c) and this is one of the driving forces for the
interaction.
derived from Quercus and Castanea species (Fig. II). The molecule of p-
1,2,3,4,6-penta-O-galloyl-D-glucopyranose has, in its most favored conforma-
tion, the shape of a circular disc. Molecular models clearly reveal that the
periphery of the molecule is hydrophilic, by virtue of the presence of the pheno-
lic groups, whilst the upper and lower faces of the molecule (disc-shape) are, in
contast, largely hydrophobic in character. p-I,2,3,4,6-Penta-O-galloyl-D-glucose
is amorphous and has a limited solubility in water (-1.0 mM at 20°C). Solutions
of higher concentration, obtained by heating to 50-60°C, readily gel on cooling
to ambient temperature. Such gels probably arise from the ability of the polyphe-
nol to form extensive three dimensional lattices. These would result from verti-
cal stacking of molecules (rather as a pile of coins) with their hydrophobic faces
brought in juxtaposition to minimize water contacts and from intermolecular
ASTRINGENCY AND POLY PHENOL PROTEIN INTERACTIONS 307
OH
Figure 11. ~-1,2,3,4,6-Penta-O-gal1oyl
D-glucose: Castalagin and Vescalagin: a C-l , a-OH , Castalagin ( 5 )
structural comparison. C-l, J3-0H, Vescalagin (6)
"hydrophobic effects" are the major driving forces favoring phenol (and hence
polyphenol) complexation with proteins, they do not answer the question why
such dramatic differences should exist. These comparative properties are certainly
not ones that would have been readily predicted a priori. Thus, although both
vescalagin and castalagin have formally just 6 hydrogen atoms less than ~-
1,2,3,4,6-penta-O-galloyl-D-glucose, all three molecules have a similar molecu-
lar mass and possess 5 aromatic nuclei and 15 phenolic hydroxyl groups. At the
moment, one can only speculate as to the nature of the solvation shell around
polyphenol molecules such as ~-1,2,3,4,6-penta-O-galloyl-D-glucose, castalagin,
and vescalagin. Presumably there will be significant clusters (a cloud) of water
molecules in the vicinity of the three phenolic groups of each galloyl ester TImc-
tion. The distribution of water molecules around the remainder of each aromatic
ring is, in present circumstances, far more difficult to define.
ASTRINGENCY
The objective evaluation of the taste and flavor of foods and beverages still
depends largely upon sensory perception. A fundamental understanding of the
physiology and chemistry underlying various aspects of taste is still lacking. This
is certainly the case with respect to that quality generally referred to as astrin-
gency which influences the acceptability of many fruits and fruit products, such
as fruit juices and wines. Astringency results from a loss of lubrication in the
mouth. It is normally recognized as a feeling of extreme dryness and constric-
tion, roughness or puckeriness of the palate which takes a significant time to
develop. It is diffuse and not confined to a particular region of the palate. The
word astringent is derived from the Latin ad (to) and stringere (bind); thus astrin-
gency is properly defined as a binding reaction, a sensation of touch. Indeed,
astringents in medicine are recognized as substances that bind to and precipitate
proteins. Typical astringents include the salts of multivalent cations (AI, Cr, Zn,
Pb, Ca, B), dehydrating agents such as alcohol and dimethyl ketone, mineral acids
and natural polyphenols (vegetable tannins). A mucous membrane covers all the
exposed surfaces of the mouth which are moistened by the secretions from the
salivary glands. According to Bate-Smith,42 the primary process whereby astrin-
gency develops is via precipitation of proteins and mucopolysaccharides in the
mucous secretions. Accepting this view (which is still broadly assumed), an
understanding of the mechanism of the astringent response focuses attention auto-
matically upon (i) the molecular basis of the action of salivary proteins which
give rise to lubrication in the mouth and (ii) polyphenols and their interactions
with salivary proteins which result in the loss of lubrication.
In terms of taste, natural polyphenols have two distinctive characteristics-
astringency and bitterness. Distinguishing between these qualities is an important
facet in the training of members of taste panels in the food industry. The data
ASTRINGENCY AND POLYPHENOL PROTEIN INTERACTIONS 309
shown in Table 4 were measured by an experienced taster from the food indus-
try (Clapperton, Williamson and Haslam, unpublished data). Increasing aliquots
of each polyphenol were taken and a "quasi-binding" curve established for each
sample. From these curves the minimum threshold concentration for the percep-
tion of astringency in the palate was determined.
Even the most cursory examination of the data in Table 4 indicates a broad
correlation of the astringent response of a particular polyphenol with the picture
presented earlier of the relationship between polyphenolic character and capac-
ity to bind to protein (e.g. molecular size, the number and disposition of pheno-
lic nuclei, conformational flexibility and water solubility). Such a correlation
strongly suggests that, in the case of polyphenols, it is (as originally suggested
by Bate-Smith) their binding to salivary proteins which fundamentally underlies
the development of astringency in the mouth.
Saliva is produced by the major salivary glands which empty their secre-
tions into the oral cavity. The macromolecules in saliva consist almost exclusively
of proteins (-1.0-3.Smg./ml.), and amino acid analyses of human salivary pro-
teins have demonstrated the presence of an unusually large amount of proline
(-16-33% of total amino acids). From the work in severallaboratories,43 it is now
clear that saliva contains a unique group of proteins-proline-rich proteins or
PRPs. They can be sub-divided into acidic (APRP), basic (BPRP), and glycosy-
lated (GPRP) proteins. In human parotid saliva, these account for 17%,23%, and
30%, respectively of the total protein. The acidic proline-rich phosphoproteins
have important biological functions related to providing a protective environment
for teeth and may modulate the adhesion of bacteria to oral surfaces. The bio-
logical function(s) of the glycosylated and basic proline-rich proteins are less
clearly defined but most probably include the binding of oral bacteria and
masticatory-lubricating properties. Salivary proline-rich proteins have a repeti-
310 E. HASLAM et al.
tive primary structure particularly rich in the amino acids proline, asparagine, glu-
tamine, and glycine. The reason for the large number of isoforms of PRPs is not
entirely clear; some smaller APRPs are thought to be derived by post-translational
cleavage of the larger APRPs, and one or more basic PRPs may similarly arise
by proteolysis of acidic PRPs.
The primary structures of three closely related ISO-residue and three 106-
residue human APRPs, considered to be derived by post-translational cleavage of
the larger APRPs, have been defined. 44 The amino acid sequence of the phos-
w
AOO
•
o~
OH
)IN''
OH
)IN''
H~o
H 0 H 0
• P,prolyl ~ N, asparaginyl ~ Q, glutaminyl
1
00~~00~®~00~.~~~00
.•• ..
•• O@O~O~0000~ ••• ~·
.••••
·~~O~.O~·0.~~00-»
»» •• ~
~ ~~ ~~~~ ~
·0.~O • • ~~··O ••••~.
•. .••• .. .••
0.~·
.~
~·~O.~
®.~
•• ~~··O.0 ••0·0
• • • ~~ •• O~~ • • • • • •
150
~ ®.~
~ A,I,L,V • G
Figure 12. Diagrammatic representation of the amino acid sequence of the human acidic PRP:
phosphoprotein PIF -so The break shown in the amino acid sequence betweeen position 51
and 52 [»»>1 illustrates the amphipathic nature of the molecule, with a strongly acidic N-
terminus (-50 amino acids) and the proline-rich C-terminus (-100 amino acids).
ASTRINGENCY AND POLYPHENOL PROTEIN INTERACTIONS 311
phoprotein PIF-s (Fig. 12) has a strongly hydrophilic "head" segment from the
N-terminus to position 51 [»»>] containing two phosphoserines and fourteen
amino acids with a carboxyl side chain (aspartic, glutamic). Conversely, the
remainder of the amino acid sequence to the C-terminus is rich in the amino
acids proline, glycine, asparagine, and glutamine and, in comparison to the N-
terminus, is relatively apolar and hydrophobic. The protein may be likened to a
detergent molecule with a charged, hydrophilic "head" and a non-polar "tail."
Kauffman et al. 45 similarly reported the isolation of eleven basic proline-
rich proteins from the parotid gland of a single individual. All contain segments
with identical or similar amino acid sequences. Protein IB-7 (59 amino acids)
contains two contiguous 21 residue segments (1-22 and 22-42) that are virtually
identical. These segments are followed by a 12 amino acid sequence which is
identical to residues 2-13 and 23-34 (Fig. 12). The contiguous repeat pattern
1-54 recurs in other human parotid basic proline-rich proteins. Such molecules
may similarly be thought of as amphipathic, but in this case the strongly
hydrophilic side chains (lysine and arginine) are not grouped but distributed
throughout the amino acid sequence (Fig. 13).
Similar precise structural information and hence relative molecular masses
of the GPRPs are not yet available. However, human BPRPs and GPRPs are
encoded by a series of 4 genes; each gene gives rise to a precursor protein which
through proteolytic cleavage gives rise to secreted BPRPs and GPRPs of varying
sizes. The amino acid sequences of some GPRPs are known from genetic studies.
They are similar to those of BPRPs and contain a recognition sequence for N-
glycosylation at asparagine. This similarity suggests that these molecules also are
amphipathic, but, until more is known concerning the distribution of the
hydrophilic sugars on the proline-rich amino acid sequence, direct comparison
with either the structures of the BPRPs or APRPs is not possible.
Pre-eminent amongst the proteins that bind polyphenols most strongly are
the salivary pRPS.12 Basic PRPs complex with and precipitate polyphenols, and
.•••••.
1
0.~
.••• ..
O •• ·0.~ •••• ~ •• ~~.~
.•••• .••• ~.~
·0.~
~
~.
~
.0 .p
O~<90<9
59
~ Q.N
o K,R
Figure 13. Diagrammatic representation of the amino acid sequence of the human basic PRP:
187.
312 E. HASLAM et al.
recent observations suggest that both acidic PRPs and glycosylated PRPs are fully
capable of forming soluble complexes with tannins (cf Fig. 1) but ones which
are not readily precipitated by virtue of the strongly solubilizing groups present
elsewhere on the peptides. Complexation is presumed to occur selectively (as with
the BPRPs) in the relatively hydrophobic proline-rich regions of the peptides.
The suggestion that the role of these proteins is as a "first line of defense"
against the detrimental effects of polyphe no Is in the diet of herbivores is a natural
and attractive one. 46 Based on his own and other's work, Bate-Smith42 first suc-
cinctly stated the presumed role of vegetable tannins in plant defense:
From the biological point of view the importance of tannins in plants lies in their effectiveness
as repellants to predators, whether animal or microbial.
triboJogicaJJayer
poJyphenoJs
( c ) gJycosyJated PRPs
,gJycosyJ residues
"
Figure 14. Polyphenol: PRPs interactions; Lubrication and Astringency. (a)-PRP molecules
in solution: random coil conformations giving rise to a tribological layer in the palate;
(b )-collapse of the tribological layer and release of water giving rise to astringency; (c)-
glycosylated PRPs.
and there would be a time delay before the necessary intermolecular collisions
occur but also because of the very nature of polyphenol/protein interactions them-
selves.
The principles which underlie molecular recognition phenomena may be
analyzed not only in terms of the composition, structure, and conformation of
the substrates taking part in the complexation reactions but also in terms of three
idealized concepts. 48 "Die-mould" (jig-saw) matching is essentially static with an
exact fit of donor and acceptor molecules together. "Key-IocR' matching is time
dependent, since the key (donor) invariably has to be maneuvered into the lock
(acceptor) to achieve the correct fit. Finally "hand-in-glove" matching of donor
and acceptor molecules is both time dependent and dynamic. Both donor and
acceptor molecules are mobile and flexible and may assume a variety of subtly
different shapes as complexation proceeds. In such situations, it is important
that both substrates are able to sample a variety of different relative orientations
with respect to each other such that ultimately the maximum number of
strong contacts are made between donor and acceptor species. Such associative
reactions frequently exhibit strong cooperative effects. Present evidence, sum-
marized above, suggests that where polyphenol-protein complexation is at its
most effective it is largely of the "hand-in-glove" type where polyphenol and
protein both possess considerable conformational mobility and structures which
permit the possibility of bringing about multi-dentate complexation. If this view
is accepted, this would also predict a delayed time dependent astringent response
in the palate.
Finally, brief comment should be made on the role of low molecular mass
phenolic compounds, such as (-)-epicatechin, (+)-catechin, (-)-epigallocatechin-
3-0-gallate, and chlorogenic acid, in the development of astringency in the palate.
The evidence from taste panels shows that the threshold values for the percep-
tion of these compounds as astringents in the palate is relatively high, cf Table
4. Work such as that of Englehardt et al. 49 suggests nevertheless that where the
concentration of these compounds in a liquor is high then the liquor is generally
perceived as mildly astringent. A priori it seems unlikely that the explanation is
as suggested for polyphenols in Figure 14, namely direct cross-linking of the
PRPs. The binding of simple phenols to proteins is weak (cf Table 3) and the
principal (if not sole) binding site(s) appears to be the o-dihydroxy, o-trihydroxy
"B'phenolic nuclei. The participation of such substrates in direct cross-linking
reactions seems, for steric reasons, also to be doubtful. Accordingly, any expla-
nation must center upon their relatively high concentrations in the liquor(s) and
the hypothesis that under such conditions the equilibrium (Fig. I) between phe-
nolic compound and PRP is driven towards the intermolecular complex. Coating
of the surface (either wholly or partially) of the PRP would lead to a relatively
hydrophobic phenolic layer. In turn, this would lead to aggregation and to the
release of solvent (water) to the bulk medium. It is interesting to note in this
context that McManus et al. 11 observed that simple phenols, such as pyrogallol,
may indeed induce precipitation of globular proteins from solution so long as the
ASTRINGENCY AND POLYPHENOL PROTEIN INTERACTIONS 315
POSTSCRIPT
Commenting almost thirty years ago on the state of the art of natural
product chemistry one of its most distinguished practitioners, the late T.A.
Geissman,5o wrote bravely, but percipiently:
Increasingly attention will be paid to the role of organic compounds in plants and less attention
to what they are. Certainly structures are important but the determination of structure in itself
is ceasing to be of much interest or importance, and often turns out to be an exercise in the
manipulative and interpretive skill of the investigator. ... The future of phytochemistry is to
use the chemical information as the starting point for inquiry into questions that lie in the realm
of biology.
The story of the chemistry of plant polyphenols as it has unfolded over the
past three decades amply illustrates the truth of Geissman's shrewd observations.
For, whilst structural problems still remain to be solved, attention has turned
increasingly to the questions which are posed by the presence of these complex
metabolites in plant materials. These range from those of metabolism, through
ecology to the practical and applied problems which ensue in food science and
medicine.
To plagiarize the poet John Donne it is certainly a truism to say that in
nature "no molecule is an Island. entire of itself" Many of the intrinsic proper-
ties of plant polyphenols are related to their propensity to associate with other
molecular species. Most notable are those which occur with proteins, and under-
standing the molecular mechanisms involved remains a major scientific challenge
for the 21 st century. The study of the molecular basis of polyphenol-protein inter-
actions described above is one such contribution. It is not only of intrinsic impor-
tance to the wider questions of molecular recognition but it is also of considerable
practical significance to the foodstuffs and beverages industry where the modifi-
cation and control of the astringent response is often a much sought after objec-
tive. Knowledge in this area is similarly important in the increasing use of plant
materials containing polyphenols as sources of anti-oxidants, and as herbal med-
icines. In such cases, the palate, with its associated PRPs, may indeed be seen as
a potential barrier which such potential medicines must first surmount before they
may be absorbed into the plasma.
REFERENCES
1. HASLAM, E. 1997. Vegetable tannage: Where do the tannins go? J.Soc.Leather Tech and
Chemists 81: 45-51.
316 E. HASLAM et al.
43. BENNICK, A. (1982). Salivary proline rich proteins. MoI.Cell.Biochem. 45: 83-99.
44. HAY, D.1., BENNICK, A., SCHLESINGER, D.H., MINAGUCHI, K.,
MADAPALLITAM, G., SCHLUCKEBIER, S.K. 1988. The primary structures of six
human salivary acidic proline-rich proteins (PRP-I, PRP-2, PRP-3, PRP-4, PIF-s and
PIF-f). Biochem.1. 255: 15-21.
45. KAUFFMAN, D.L., BENNICK, A., BLUM, M., KELLER, P.1. 1991. Basic proline-rich
proteins from human parotid saliva: Relationships of covalent structures of ten proteins
from a single individual. Biochemistry 30: 351-3355.
46. MCARTHUR, c., SANSON, G.D., BEAL, A.M. 1995. Salivary proline-rich proteins
in mammals: Roles in oral homeostasis and counteracting dietary tannin. J.Chem.Ecol.
21: 663-691.
47. VAN HOLDE, K.E.1971. Physical Biochemistry, Prentice Hall: New Jersey.
48. WILLIAMS, R.J.P. 1988. Lecture, Biochemical Society, Sheffield, April 15 th •
49. ENGLEHARDT, U.H., KUHR, S., DING, Z. 1992. Influence ofcatechins and theaflavins
on the astringent taste of black tea brews. Z. Lebensm. Unters.Forsch. 195: 108-IlI.
50. GEISSMAN, T.A. 1972. In: Recent Advances in Phytochemistry (VC.Runeckles and
J.E.Watkin, eds.), volume 4, p. 303.
Chapter Twelve
PHYTOCHEMISTRY OF BRYOPHYTES
Biologically Active Terpenoids and Aromatic Compounds
from Liverworts
Yoshinori Asakawa
Phytochemicals in Human Health Protection, Nutrition, and Plant Defense, edited by Romeo.
Kluwer Academic I Plenum Publishers, New York, 1999.
319
320 YASAKAWA
INTRODUCTION
BIOLOGICAL ACTIVITY
Characteristic Scents
Liverworts emit volatile terpenoids or simple aromatic compounds when
crushed. These are responsible for intense sweet-woody, intense turpentine, sweet-
PHYTOCHEMISTRY OF BRYOPHYTES 321
Hepaticae:
Conocephalum conicum Antimicrobial, antifungal, antipyretic, antidotal activity; used to
cure cuts, burns, scalds, fractures, swollen tissue, poisonous
snake bites, and gallstones
Frullania tamarisci Antiseptic activity
Marchantia polymorpha Antipyretic, antihepatic, antidotal, diuretic activity; used to cure
cuts, fractures, poisonous snake bites, burns, scalds, and
open wounds
Reboulia hemisphaerica For blotches, hemostasis, external wounds, and bruises
Continued
86. 3,5-Dihydroxy-2-geranylbibenzyl 96. Mastigophorene D
87. 2-Carboxy-3,4-dihydroxy-5-(3-methyl- 97. Plagiochin A
2-butenyl)bibenzyl 98. d- Tubocurarine
88. Labda-12,14-dien-7,8-diol 99. 7',8'-Dehydromarchantin A
89. Isoriccardin C 100. Isomarchantin C
90. Riccardin C 10 1. II-Epiporelladiolide
91. Lunularic acid 102. I I, I 3-Dehydroporelladiolide
92. Isopolygodial 103. Porellaolide
93. Isosacculatal 104. Porelladiolide
94. Mastigophorene A 105. 3a,4a-Epoxyporelladiolide
95. Mastigophorene B
Some genera of the Hepaticae produce intense pungent and bitter sub-
stances which exhibit interesting biological activities described in subsequent sec-
tions. Most North American liverworts contain unpleasant substances, some of
which taste like immature green pea seeds or pepper.24 Porella vernicosa complex
(P arboris-vitae, P fauriei, P gracillima, P obtusata subsp. macroloba, P roellii
and P vernicosa) contain very pungent substances, and Jamesoniella autumnalis
contains an intense bitter principle whose taste resembles that of the leaf of lilac
and Swertia japonica or the root of Gentiana scabra var. orientalis. The strong
hot taste of Porella vernicosa complex is due to (-)-polygodial (9a).6
We reported that two eudesmanolides, diplophyllolide (10), and ent-
7a-hydroxydiplophyll0lide (11), a germacranolide, tulipinolide (12), two 2,3-
secoaromadendrane-type sesquiterpene hemiacetals, plagiochiline A (13), and
plagiochiline I (14), and a sacculatane-type diterpene dialdehyde, sacculatal (15),
possessing an intense pungent taste, had been isolated from some Chiloscyphus,
Wiesnerella, Plagiochila, Pellia, and Trichocoleopsis species, respectively. 6
Further fractionation of the ether extract of Pellia endiviifolia resulted in the iso-
lation of a new pungent 1~-hydroxysacculatal (16), together with several saccu-
latane-type diterpenoids. 25 The hot taste of Pallavicinia levieri lO and Riccardia
robata var. yakushimensis 26 is due to sacculatal (15). Polygodial and sacculatal
have been obtained from cell suspension cultures from each Iiverwort.27.28 Porella
acutifolia subsp. tosana is a pungent stem-leafy liverwort. Its taste is due to the
presence of hydroperoxysesquiterpene lactones, 1a- (17), and 1~-hydroperoxy-
4a,5~-epoxygermacra-IO (l4),II(l3)-dien-12,18a-0Iides (18).29 When one
chews a whole plant of the stem-leafy liverworts, Plagiochila asplenioides, Pfru-
ticosa, P ovalifolia, and P yokogurensis which contain plagiochiline A (13), one
slowly feels a potent hot taste. It is suggested that 13 might be converted into a
pungent unsaturated dialdehyde by human saliva. Enzymatic treatment of 13 with
XH -
H9
~(5)
Y yP (6) (7)
(8)
amylase in phosphate buffer or with human saliva produces two strong pungent
2,3-secoaromadendrane-type aldehydes, plagiochilal B (19) whose partial
structure is similar to that of the pungent drimane-type sesquiterpene dialdehyde,
polygodial (9a), and furanoplagiochilal (20).30
Most of the species belonging to the Lophoziaceae produce surprisingly
intense bitter substances. Gymnocolea inflata is persistently bitter and induces
vomiting when one chews a few leaves for several seconds. The earlier review
mentioned that this is due to gymnocolin A (21).6 Jungermannia infusca has an
intense bitter taste. This is due to the presence of the infuscasides A-E (22-26).31
The bitterness of Anastrepta orcadensis, Barbilophozia lycopodioides, and
Scapania undulata are attributable to anastreptin A (27),18,32 barbilycopodin
(28),18,32 and scapanin A (29),33 respectively.
Q:Y
CHO CHO CHO
WHO
,;'I HO
~HO
"
"
; :"'r ~.
(9a) (9b) (ge)
yq
::::,...
R
'0,
~
"DAC
R CHO
~HO
(15) R=H
LA (17) (18)
(16) R=OH
OH~ r--
(19) (20)
?
Jik-oAC
W"""
J--:;i
(23) (24)
(25)
y;:C
~
' OA~
'~" :.
a
":. 0
"
(27) (28) (29)
(30a)
ct2t (30b) °
(31) R=H
(35) R=C02 H
(38) R=C02 K
11
Q
~I
H
C 17 H33
(34)
13
(32) R=H
(36) R=C02 H
(39) R=C0 2K
15
11
Figure 4. Allergy inducing sesquiterpene lac tones and long chain alkyl phenols.
PHYTOCHEMISTRY OF BRYOPHYTES 329
MeO H
H H
(57) (58)
Figure 5. Sesquiterpenoids and cyclic and acyclic bis-bibenzyls possessing various biological
activities.
PHYTOCHEMISTRY OF BRYOPHYTES 331
(59)
(60)
(61)
Me
~
(62)
o Me (63)
MeO ~ I
:::::,.. h
(64)
Figure 5. Continued.
332 Y.ASAKAWA
Y., unpublished results). Marchantin A (51) also shows cytotoxicity (TIC 117)
against P-388. 7 Blasia pusilla produces bis(bibenzyl) dimers, pusilatin A-D
(58-t;1). Pusilatins B (59) and C (60) possess DNA polymerase ~ inhibitory activ-
ity (lC50 13.0 and 5.l6IlM), moderate cytotoxicity against KB cell (ED5o 13.1 and
13.0Ilg/ml) and weak HIV-RT inhibitory activity.37 Trichocolea species produce
prenyl ethers, tomentellin (62), demethyltomentellin (63), and trichocolein (64).38
Compound 62 is the major cytotoxic component of T mollissima, active against
BSC cells at 151lg/disk.38 Compound 63 isolated from T tomentella also showed
the same activity.38
also have antifeedant activity against larvae of Japanese Pieris species. 7 A series
of natural drimanes and related synthetic compounds was tested for antifeedant
activity against aphids. 39 Polygodial (9a) from the Porella vernicosa complex and
warburganal (9c) from the African tree Warburgia ugandensis were the most
active substances. Natural (-)-polygodial (9a) and the synthetic (+)-enantiomer
(9b) showed similar levels of activity as aphid antifeedants. (-)-Polygodial killed
mosquito larvae at a concentration of 40 ppm and had mosquito repellent activ-
ity which is stronger than the commercially available DEET. Plagiochilide (65)
isolated from Plagiochila species killed Nilaparvata lugens (Delphacidae) at
lOOllg/ml (Asakawa, Y., unpublished results).
activity at ID50 1.85 )lg/ml. 7 Perrottetins A (75) and D (76) from Radula perrat-
tetii and prenyl bibenzyls (77-81), also from Radula species, riccardin A (47)
from Riccardia multifida, and marchantins D (55) and E (56) from Marchantia
species had calmodulin inhibitory activity (IDso 2.0-95.0 )lg/ml). 7 The simple
bibenzyls (82-85) from Radula and Frullania species also showed weak calmod-
ulin inhibitory activity (IDso 100 )lg/ml) as did the labdane-type diterpene diol,
labda-12, 14-dien-7 ,8-diol (88) (ID50 82 )lg/ml) isolated from Parella perrotte-
tiana. 7 Perrottetin A (75), prenylbibenzyls (76, 84, 86, 87), marchantins D (55)
and E (56), and riccardin A (47) also inhibited 5-lipoxygenase (76-4% at 10-6
mol).7 The following phenolic compounds showed significant cyc\ooxygenase
inhibitory activity: marchantin A (51) (lC 50 46.4 )lM), marchantin B (54) (55.9),
marchantin E (56) (58.0), paleatin B (57) (45.2), perrottetin D (76) (26.2), radu-
lanin H (81) (39.7), isoriccardin C (89) (50.8), and riccardin C (90) (53.5).40
H
~W
I "
"""
..........
b
~
0",
".,
"''''
".
". .
H (69) (70)
~
(71) (72)
0(7~:~ HO
(74)
Figure 6. Sesqui- and diterpenoids, and bibenzyls possessing superoxide anion radical release
inhibitory activity.
PHYTOCHEMISTRY OF BRYOPHYTES 335
Lunularic acid (91), which is found in almost all liverworts as a minor com-
ponent, has anti-hyaluronidase activity (IC 5o 0.13 nM). This activity is stronger
than that of tranilast (N-3',4' -dimethoxycinnamoylanthranilic acid) which is an
anti-allergenic agent developed in Japan for oral administration. Lunularic acid
(91) has been obtained from hydrangenol-~-glucoside via hydrangenol in good
yield. 4l Perrottetin E (49) exhibited inhibitory activity fWAor thrombin (lC 50
I 811M) which is associated with blood coagulation. 42
The strongest piscicides are the pungent (-)-polygodial (9a) from Porella
vernicosa complex and sacculatal (15) from Pellia endiviifolia, Pallavicinia
levieri, Riccardia robata var. yakushimensis, and Trichocoleopsis sacculata.
Killie-fish (Oryzia latipes) is killed within 2 hr by 0.4 ppm solution of9a and 15. 6,7
Sacculatal (15) and 1~-hydroxysacculatal (16) also kill killie-fish within 20 min
at I ppm?5 Killie-fish is also killed within 2 hr by a 0.4 ppm solution of synthetic
pungent (+)-polygodial (9b). Hence, piscicidal activity is not affected by the
chirality of polygodial. Polygodial is also toxic to fresh water bitterlings, which
are killed within 3 min by a 0.4 ppm solution. 7 On the other hand, isopolygodial
(92) from cultured cells of Porella vernicosa and the higher plant Polygonum
hydropiper and isosacculatal (93) from Pellia, Riccardia, and Trichocoleopsis
species show neither piscicidal nor molluscicidal activity even at 1O,000ppm.7
Almost all crude extracts from liverworts which contain bitter or pungent
substances show phytotoxic activity. (-)-Polygodial (9a) inhibits the germination
and root elongation of rice in husk at 100ppm. At a concentration less than
25 ppm, it dramatically promotes root elongation ofrice. 6A3
OH OH OH
(77)
OH OR'
OH
OH
OH
(88)
OH
HO
OH
(91)
HO
(90)
(92)
(94)
(96)
(99)
CONCLUSION
(100)
~
, -..;:
~
R ~
0 0 0
(101 ) (102) (103)
o
(104) (105)
Figure 10. Cyclic bis-bibenzyl, and guaiane-type sesquiterpene lactones possessing cathepsins
Band L inhibitory activity.
340 Y.ASAKAWA
ACKNOWLEDGMENTS
REFERENCES
343
344 P. J. HOUGHTON AND A. Y. MENSAH
which have occurred since then, many of which have shown that the speculated
chemical basis for the reputed activities is in fact correct.
ETHNOPHARMACOLOGY OF BUDDLEJA
Bronchial Complaints
Diuretic Activity
The leaves of some Buddleja species formerly were official in the pharma-
copeias of several Central and South American countries because of their diuretic
activity. Diuresis is achieved by oral administration of a decoction of the leaves,
and occasionally the roots, of the species in question. The effect was ascribed to
the flavonoid and iridoid content of the leaves, but no further work has been
reported to justify or contradict this hypothesis.
Table 1. Ethnopharmacological uses of Buddleja species]
Species Geographical area
WOUND HEALING and related conditions
B. americana L. Mexico
B. asiatica Lour. Nepal, Philippines
B. cordata H.B.K. Mexico
B. davidii Franchet China (Shaanxi Province)
B. diffusa Ruiz et Pav. Peru, Ecuador
B. globosa Lam. Chile
B. incana Ruiz et Pav. Peru, Ecuador
B. officinalis Maxim. China
B. salviifalia Lam. South Africa
B. sessiliflora Kunth Mexico
LIVER AILMENTS
B. americana L. roots Mexico, Guatemala
B. cordata ~-[.B.K. roots Mexico
B. globosa Lam. leaves Chile
B. officinalis Maxim. China
BRONCHIAL CONDITIONS
B. albiflora Hems!. leaves, flowers China
B. cambara Arechav. Brazil
B. curviflora Hook et Am. leaves, flowers China
B. diffusa Ruiz et Pav. Peru, Ecuador
B. globosa Lam. leaves Chile, Bolivia
B. incana Ruiz et Pav. leaves Peru, Ecuador
B. madagascarensis Lam. leaves Madagascar, Uruguay
B. salviifolia Lam. South Africa
B. saligna Willd. South Africa
B. sessiliflora Kunth Mexico
ANTIRHEUMATIC EFFECTS
B. americana L. Mexico, Guatemala
B. diffusa Ruiz et Pav. Peru, Ecuador
B. humboldtiana Roemer & Schultes leaves Texas, USA
B. incana Ruiz et Pav. Peru, Ecuador
B. salviifolia Lam. South Africa
B. sessiliflora Kunth Mexico
DIURETIC EFFECTS
B. americana L. leaves, roots Mexico,Guatemala
B. diffusa Ruiz et Pav. Peru, Ecuador
B. humboldtiana Roemer & Schultes leaves Texas, USA
B. sessiliflora Kunth Mexico
ANALGESIC EFFECTS
B. americana L. leaves, root, bark poultice Mexico, Guatemala,
B. hrasiliensis Jacqu. leaves, roots Brazil
B. diffusa Ruiz et Pav. Bolivia
B. incana Ruiz et Pav. Peru, Ecuador
B. sessiliflora Kunth Mexico
SEDATIVE/TRANQUILIZING EFFECTS
B. americana L. root. Mexico, Guatemala,
B. madagascarensis Lam. leaves Madagascar
B. quinquenaria Cham. et Schltr. Brazil
B. sessiliflora Kunth Mexico
BIOLOGICALLY ACTIVE COMPOUNDS FROM BUDDLEJA SPECIES 347
Sedative/Tranquilizing Effects
R50
R4 R3
Compound R1 R2 R3 R4 Rs
1 Linarin H OCH 3 H H Rutinose
2 Kaempferol H OH OH H H
3 Quercetin OH OH OH H H
4 Acacetin H OCH 3 H H H
5 Luteolin OH OH H H H
6 Luteolin 7-0- OH OH H H Glucose
glucoside
7 6-0H luteolin OH OH H OH H
8 Apigenin-7 -0- H OH H H Glucose
glucoside
9 Rutin H OH O-rutinose H H
10 Scutallerein 7-0- H OH H OH Glucose
glucoside
11 Pectolinarigenin H OCH 3 H OCH 3 H
12 Salvigenin H OCH 3 H OCH 3 CH 3
13 Buddlenoid A 13'0 H OH OH H Cinnamoyl-6-
glucose
14 Buddlenoid B 14'u OCH 3 OH OH H Cinnamoyl-6-
glucose
Flavonoids
Pbenyletbanoids
A compound showing a blue fluorescence under UV light 365 nm had been
noted in the early screening procedures for flavonoids carried out by Bate-Smith in
1962. 9 This compound was shown to be orobanchin (15) and, subsequently, several
other related compounds have been isolated from Buddleja species (Figure 2).4
BIOLOGICALLY ACTIVE COMPOUNDS FROM BUDDLEJA SPECIES 349
HOy~
I
O~
n~O
~R
~
0 ____O,
00
HO ~~O OH I
CH 3 0 ~ OH
HO
HO OH
R R'
15 OH OH Orobanchin
16 H OH Verbascoside
(Acteoside)
CH 2 0H
17 H
H~~O,- Echinacoside
OH
R R' R"
18 Ac H H
19 H Ac H
20 H H Ac
CH20R"
R'O~O ?' OH
o OH ~
c~
HO '" OR
HO
OH
R R' R"
0
21 CH 3
~
H
HO
0
CHaO~
/" 1 '"
22 H H
HO '"
H H
HO~
23 :1 '"
HO
0
CH30~
24 H /'" 1 '"
H
HO '"
0
CH30~ H
25 CH3 /"'1 '"
0
HO '"
CH30~
/"'1 '"
HO '"
26 CH3 H
0
HO~ H
27 CH3 :1 '"
HO
0
CH30~
28 CH3 /"1 '" arabinose
HO '"
0
CH30~
29 /"1 '" apiose
CH3 HO '"
0
30 H HO~ arabinose
:1 '"
HO
Figure 2. Continued.
BIOLOGICALLY ACTIVE COMPOUNDS FROM BUDDLEJA SPECIES 351
Terpenoids
Iridoids. The iridoids of Buddleja belong to the C-9 catalpol and aucubin
group rather than the C-IO type such as loganin. The latter is found characteris-
tically in the Loganiaceae, and the occurrence of the C-9 compounds gives
chemotaxonomic support to the contention that Buddleja should form part of a
separate family rather than be included in the Loganiaceae. Aucubin (31) from
the leaves of B. globosa was the first iridoid to be isolated, and several similar
compounds have been reported since then. 3 The catalpol type of iridoid (34),
where an epoxide group replaces the C6-C7 double bond found in the aucubin
(31) analogues, also occurs, and compounds with ester-linked phenylpropanoid
residues are known for both of these types as well as for the related compound
ajugolY lridoids so far reported (apart from those with a lignan moiety) are
shown in Figure 3.
More recently, a group of compounds consisting of iridoids linked to a
lignan moiety have been isolated from the polar fractions ofthe roots of B. davidii.
(see Fig. 7).
RO H
R~
~
CH2~H O"L~~H
R
0
HO~OHOH
~~ 2
R
0"
HO OH OH
3
H Aucubin
31 34 H Catalpol
~"'
¢
OH
p-Methoxycinnamoyl
3
32 aucubin 5 35 Catalposide
o 0, )
o 0"
33 HO~
"" '
I
0~CCH3 ~'
CH 3 -·.0
o
36 p-Methoxycinnamoyl
catalpol 8
9
Buddlejoside A 2
o 0,
RO~
HO
)o! O~O 3 0"
CH20H
HO/ OH OH
R
37
Vanillylajugol
38 Feruloylajugol
\
CHao~OH
39 HO OH
CHa
Buddlejoside A1 9
o
c~m
R"O
R'O
0
OR 0
'-"::::
0
CH,~O~
HO OH OH
R R' R"
40 H H C~O~
41 CHaCO H C~O~ Buddlejoside A3
42 H CH3CO CH30~
Buddlejoside ~
43 H CH30~ H Buddlejoside ~
47 H H CHaO~ Buddlejoside As
CH3
C~9Y
50 H CH3CO CH30 ~ /, Buddlejoside A12.
CH30
51 H CH3CO CH30~ Buddlejoside A13
HO
Figure 3. Continued.
52 H Buddlejoside A14
53 H Buddlejoside A15
Figure 3. Continued.
CH, Y
+H- CH,
CH 2
R 57 Buddledin 0
54 OH Buddledin A
55 H Buddledin B
56 OOCCH 3 Buddledin C
58 Buddledin E
~
\CH3
." H CH3
CHC/i!.CH3
?'" 0 o
-:~
CH 3 ...~
H R
CH 3 CH 3 CH 3
R R
59 H 61 H Cycloclorenone
60 OH 62 OH 1- Hydroxycycloclorenone
63 Buddlejone
64 Anhydrobuddlejone 65 Anhydrobuddlejol
66 Maytenone
Figure 4. Continued.
R R'
67 OH H Saikosaponin A
68 OH Rhamnose (1- 4) glucose Buddlejasaponin 1
69 OH Xylose (1- 4) glucose Buddlejasaponin 2
70 OH Xylose Buddlejasaponin 3
71 OH Glucose Buddlejasaponin 4
73 Mimengoside B
Lignans
Lignans and neolignans from B. davidii stem, first isolated in 1984, are
somewhat unusual, as some of them are trimeric congeners (see Fig. 6) and
most lignans are dimeric in nature. 19 Several compounds have also been described
where a lignan unit is combined with an iridoid, but in these cases, the iridoid
component is ajugol rather than aucubin or catalpol (see Fig. 7).
OCH 3
OH
CHO
HO
74 Balanophonin 75 Syringaresinol
0 OCH 3
HO OCH 3
CH 2 0H
R
76 CHO Buddlenol A
77 CH 2 0H Buddlenol B
OCH 3
R'
OH
OH
0
OCH 3
CH 3 0
0
0
OCH 3
CH 2 0H
R R'
78 H OCHa Buddlenol C
79 OCHa OCHa Buddlenol D
80 H H Buddlenol E
R OCH 3 H
81 OCHa Buddlenol F
OH
HO
HO
{p 0H
H
~
o
CH3 O~O~H
H~HOH
82
HO
HO
{p 0H
H
~
o
CH 3 O~O~OH
OH H~HOH
84
Figure 7. Lignan-iridoid congeners isolated from Buddleja species.
360 P. 1. HOUGHTON AND A. Y. MENSAH
OH OH
n n
85 20 87 20
86 22 88 22
nant antibacterial compound when tested against Staph. aureus and E. coli. 24 More
recently, a related compound, angoroside A (30) has also been reported from the
same species and been shown to be active against Staph. aureus. 25
wound healing. Catechol f1avonoids such as quercetin (3) are particularly effec-
tive in this context due to chelation with iron, and are important in oxidation
processes and the production of the oxygen free radicals which cause tissue
damage.
Recent studies in our laboratories also have shown that the aqueous extract
of B. globosa leaf displays considerable antioxidant activity. A variety of con-
stituents of this extract were tested as single compounds (Table 3). It can be seen
that both the phenylethanoids such as verbascoside (16) and the f1avonoids such
as linarin (1) display a significant effect.
Liver-Protectant Properties
verbascoside (16) and echinacoside (17), while the iridoids had no activity unless
they contained a phenylpropanoid residue (Table 4).
Antifungal Activity
Table 5. Antifungal activity (MIC;llg/mL) of compounds isolated from B. globosa stembark CHCb extract
ORGANISMS*
COMPOUND AN CA EF PN SB SC SD TI TR
Total extract >1,000 >1,000 7.8 >1,000 >1,000 >1,000 >1,000 7.8 7.8
Buddledin A 54 >1,000 >1,000 12.0 >1,000 >1,000 >1,000 >1,000 12.0 12.0
Budd1edin B 55 >1,000 >1,000 12.0 >1,000 >1,000 >1,000 >1,000 12.0 12.0
Buddlejone 63 >1,000 >1,000 750 >1,000 >1,000 >1,000 >1,000 750 750
Anhydrobuddlejone 64 >1,000 >1,000 750 >1,000 >1,000 >1,000 >1,000 750 750
Maytenone 66 >1,000 >1,000 375 >1,000 >1,000 >1,000 >1,000 750 750
Miconazole (positive control) 1.2 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 ;c
~
'Key to organisms. ::c:
AN Aspergillus niger ATCC 16404. SC Saccharomyces cerivisae ATCC 10234. o
CA Candida albicans ATCC 1023l. SO Scytalidium dimidiatum EL 936. c:
EF Epidermophytonjloccosum SJH. TI Trichophyton interdigitale EL 5171.
PN Penicillium notatum ATCC 11654. TR Trichophyton rubrum EL 5095.
SB Scrophulariopsis brevicaulis EL 3839. ~
~
?>
:<
~
~
rn
~
BIOLOGICALLY ACTIVE COMPOUNDS FROM BUDDLEJA SPECIES 365
species showed that it possessed a dose-related activity against all of them, and
a 0.5% solution of the oil in Tween 80 was comparable in activity to a 2% resor-
cinol solution. 33
Analgesic Properties
In vivo tests using mice and rats have justified the ethnopharmacological
uses of the Central American species of Buddleja as analgesics. 34 The leaves of
B. cordata and also its principal flavonoid, linarin (1), both showed effects in mice
which indicated suppression of pain. A dose of lOOmg/kg of linarin (1) had a
similar effect to 3 mg/kg morphine sulphate for heat-induced pain and a greater
effect than 100mg/kg of acetylsalicylic acid. However, 100mg/kg extract had
much the same effect as the same dose of linarin (1), and this indicates that the
activity could not be due solely to linarin. This was emphasised even more in a
test for acetic acid-induced abdominal writhing where the ED50 of the extract
(22mg/kg) was lower than that of linarin (89.0mg/kg).34
Antirheumatic Uses
Rheumatism is an inflammatory condition and so the anti-inflammatory
activities of Buddleja constituents mentioned above could contribute to the relief
of rheumatic pain. The in vivo reduction of carrageenan-induced rat paw edema
displayed by the extract of B. cordata leaves and linarin (1) is an indication that
anti-rheumatic activity might exist. 29
Other Activities
Eye Treatment. The flowers of B. officinalis, known as "Mi Mueng Ha",
have enjoyed a considerable reputation in Chinese traditional medicine as a treat-
ment for sore eyes and improving the clearness of the eye. 3 The flowers contain
flavonoids, notably linarin (1), and the triterpenoid saikosaponins known as
mimengosides A and B (72,73) as well as the phenylethanoid acteoside. 8,17 The
triterpenoids may well have antiinflammatory activity (see above) which would
alleviate soreness, but a more interesting finding is the inhibitory activity of con-
stituents of the flowers of B. officinalis on aldose reductase when tested in vitro. 35
Aldose reductase is involved in cataract formation as one of the complications of
diabetes and its inhibition would indicate some lessening in the risk of this occur-
ring. A 70% methanolic extract of the flowers showed high activity and, when the
constituents were isolated and tested, four of the flavonoids showed significant
effects at low concentrations (Table 6).
5. CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
I. BRUMMITT, R.K. 1992. Vascular Plant Families and Genera. Royal Botanic Gardens
Kew, p. 510.
2. HUTCHINSON, 1. 1959. The Families of Flowering Plants vol. I. Clarendon Press,
Oxford, p. 373.
3. HOUGHTON, PJ. 1984. Ethnopharmacology of some Buddleja species. 1. Ethnophar-
macology 11: 293~308.
4. YAMAMOTO, A., NITTA, S., MIYASE, 1., UENO, A., WU, L-1. 1993. Phenylethanoid
and lignan-iridoid complex glycosides from roots of Buddleja davidii. Phytochemistry
32: 421---425.
5. HOUGHTON, P1., HIKINO, H. 1989. Antihepatotoxic activity of extracts and con-
stituents of Buddleja species. Planta Medica 55:123~126.
6. MIYASE, 1., AKAHORI, C, KOHSAKA, H., UENO, A. 1991. Acylated iridoid glyco-
sides from Buddleja japonica Hemsl. Chern. Pharm. Bull. 39:2944~2951.
7. YU, H. 1933. Chemical study of Buddleia variabilis. Bull. Soc. Chim. BioI. 15: 482---497.
8. TSENG, K.F., CHANG, S. 1953. Constituents of Buddleja officinalis. Acta Pharmaco-
logica Sinica I: 84~85.
9. BATE-SMITH, E.C 1962. The phenolic constituents of plants and their taxonomic
significance. 1. Linn. Soc. (Bot) 58: 95~173.
10. YOSHIDA, 1., NOBUHARA, 1., UCHIDA, M., OKUDA, 1. 1978. Studies on the con-
stituents of Buddleja species I. Chern. Pharm. Bull. 26: 2535~2542.
II. YOSHIDA, 1., NOBUHARA, 1., FUJII, N., OKUDA, 1. 1978. Studies on the constituents
of Buddleja species II. Chern. Pharm. Bull. 26: 2543~2549.
12. DEVIVAR, A.R., NIETO, D.A., GAVINO, R., PEREZ, A.L. 1995. Isocapnell-9-en-8-one
and 6-alpha-hydroxyisocapnell-9-en-8-one, sesquiterpenes from Buddleia species. Phy-
tochemistry 40: 167~ 170.
13. DEVIVAR, A.R., NIETO, D.A., GAVINO, R., PEREZ, A.L. 1996. Isocapnell-9-en-8-one
and 6-alpha-hydroxyisocapnell-9-en-8-one, sesquiterpenes from Buddleia species. Phy-
tochemistry 42: 1709.
14. HOUGHTON, P.l., WOLDEMARIAM, TZ., CANDAU, M., BARNARDO, A.,
KHEN-ALAFUN, O. AND LI SHANGXIAO 1996 Buddlejone, a diterpene from Bud-
dleja albiflora. Phytochemistry 42: 485---488.
15. HOUGHTON, PJ., MENSAH, A.Y., L1AO, Y.H. 1998 Novel diterpenoids from B.
globosa and B. yunnanensis. Phytochemistry (submitted).
16. YAMAMOTO, A., MIYASE, 1., UENO, A., MAEDA, 1. 1991. Buddleja saponins I-IV,
4 new oleanane-triterpene saponins from the aerial parts of Buddleja japonica Hemsl.
Chern. Pharm. Bull. 39: 2764~2766.
17. DING, N., YAHARA, S., NOHARA, 1. 1992. Structure of mimengoside-A and
368 P. I HOUGHTON AND A. Y. MENSAH
CYANIDE IN FOODS
Biology of Cyanogenic Glucosides and Related Nutritional
Problems
Dirk Selmar
INTRODUCTION
Plants that are able to liberate significant amounts of HCN are referred to
as cyanogenic. The main source of the cyanide is the so-called cyanogenic glu-
cosides. These compounds consist of a-hydroxynitriles, also called cyanohydrins,
Phytochemicals in Human Health Protection. Nutrition. and Plant Defense, edited by Romeo.
Kluwer Academic I Plenum Publishers, New York, 1999.
369
370 D. SELMAR
which are stabilized by a sugar. Nearly 3,000 plant species have been reported to
be cyanogenic. 1.2 The HCN liberated from cyanogenic plants is thought to be an
important ecological factor, e.g. in plant defence against herbivores, and must be
clearly distinguished from low levels ofHCN production during ethylene biosyn-
thesis in intact plants. 3 The amounts ofHCN produced during ethylene synthesis
are several magnitudes lower than those concentrations resulting from cyano-
genesis due to tissue disruption.
In addition to several reviews dealing with general aspects of cyanogene-
sis and cyanogenic glycosides,I.4-6 other more specialized ones have been
published. Among these are reviews on the occurrence and distribution of
cyanogenic glycosides/·8 structures,2,7 structural deterrnination,9 function, 10, II
biosynthesis,2,12,13 and occurrence and toxicity in foodstuffs, 14-16 This chapter sum-
marizes briefly the recent progress in the research on cyanogenic glucosides, with
special emphasis on their significance in plant derived foodstuffs and related
nutritional aspects,
It is generally known that cyanide or HCN is toxic, as it inhibits numerous
metabolic processes, The acute toxicity of HCN and cyanide, respectively, is a
consequence of their affinity for various heavy metals, such as iron or copper,
with which they form cyano complexes. Most important is the effect on
cytochromes that results in an efficient inhibition of respiration. In addition,
numerous other metabolic processes are affected (for review see ref, 17). The
lethal dose of cyanide for humans is considered to be about 1 mg per kg body
weight. 18 They are widespread, present in most foodplants, but fortunately their
concentration often is low.
CYANOGENESIS
B - glucosidase
glucose
cyanogenic hydroxynitrile
glucoside
<
(cyanohydrin)
hydroxynitrile
lyase
hydroxynitrile HeN
Figure 1. Cyanogenesis in plants.
differ. In plants like A/ocasia or Trig/ochin, they are highly specific for the
cyanogenic glucoside triglochinin that occurs in these plants. 24 .25 In contrast, the
cyanogenic ~-glucosidases of Sorghum or of Prunus have moderate specificity;
in addition to hydrolyzing the cyanogenic glucosides present in the plants,
they are able to hydrolyze various other glucosides. 26 •27 In flax, cassava, and
Hevea, the related cyanogenic ~-glucosidases are nonspecific and are capable of
hydrolyzing a large variety of different glucosides?8-30 Despite their observed dif-
ferences in substrate specificity, all ~-glucosidases share one important feature:
they do not hydrolyze cyanogenic glycosides that contain two sugar moieties. 22
The inability to hydrolyze cyanogenic diglucosides is of importance with regard
to their translocation (see below).
0, HO~ NH2
_ _ _ _ _ _ _ _ _ _ tyrosine
__~
......
, NADPH - - - - - - - - - - - - - - ------
cytochrom P 450 (
, yr \,
NADP+
, ~COOH
2
~COOH+C02 ~H,
NH O
,,HO ~ I
6H > ,~ HO ~ /
iN,, HN.N~'
O· , ,
NADPH NADP+
HO OH~20 OH,
(E)-p-hydroxyphenyl-
, N-hydroxytyrosine _ _ _ _ _ _ _ _ _ _ _ ~~-~h~d:o~~r~i~e _ _ _ _ _ _ _ ~c~a~o~i~e _ ,;'
,
, cytochrom P 450 ox ,,
HO
D'J
I~ /
N
HO
(Z)-p-hydroxyphenyl-acetaldoxime
G'ucosy'- Glucose
, O2 dO -
transferase -:P'
~ "'-- ~ CN
I
,~ CN ~ ~
~:N I
HO ::::::,... NADPH NADP+ , HoN HO ~ o
p-hydroxyphenyl- , Vi
p-hydroxymandelonitrile dhurrin t11
, acetonitrile , r
a::
;J>
;>::l
Figure 2. Biosynthesis of cyanogenic glucosides. Biochemical reactions involved in dhurrin biosynthesis as outlined by reference 2.
CYANIDE IN FOODS 373
P450 ox> converts this aldoxime to the hydroxynitrile, which then is glucosylated
by a UDP-glucose dependent glucosyltransferase (For review see ref. 2).
Presently, such comprehensive knowledge, including the sequences of the
cytochromes involved, is available only for the Sorghum system. Nevertheless,
due to strong homologies in the properties of the biosynthetic apparatus of other
cyanogenic plants, learned by numerous labeling experiments with 14C-amino
acids, we know that nearly all cyanogenic glucosides are synthesized by an anal-
ogous mechanism. 2 .4 Astonishingly, only six amino acids are used as precursors.
Various structures and their amino acid precursors are listed in Table I. Valine
and isoleucine are the precursors of simple aliphatic cyanogens, such as Iinamarin
or lotaustralin. Branched cyanogenic glucosides, such as heterodendrin, are
derived from leucine. Aromatic cyanogens, like prunasin or dhurrin, are synthe-
sized either from phenylalanine or tyrosine, depending on their hydroxylation. In
addition to these five protein amino acids, cyclopentenyl glycine is a nonprotein
amino acid that serves as precursors for cyanogenic glucosides. A series of
cyclopentenyl glycine derived compounds, such as deidaclin, can be detected in
various members of the Passifloraceae. 6.34
From these basic structures, various derivatives are known, in which either
the aglycone is modified by further hydroxylation, or in which additional sugar
molecules are attached, e.g. amygdalin which corresponds to a prunasin gluco-
side. Overall, about 60 different structures all derived from these six basic struc-
tures, are known (see refs. I, 2, 7).
TRANSLOCATION
valine linamarin
isoleucine (R)-lotaustralin
(S)-epilotaustralin
taraktophyllin =
4-(S)-hydroxy-deidaclin
taraktophyllin-6' -rhamnoside
gynocardin =
cyclopentenylglycine (R )-deidaclin 4-(S)-5-(R)-tetraphyllin A
(S)-tetraphyllin A
@ ~
O-glucose
holocalin = m- hydroxyprunasin
prunasin-6'-malonate vicianin =
prunasin-6' -arabinoside
phenylalanine (R)-prunasin
(S)-sambunigrin
t:::J
H CN proteacin = p-glycosyloxy-dhurrin
''''
C* dhurrin-6'-glucoside nandinin =
'O-glucose 4' -caffeoyl-p-glycosy loxy-
mandelonitrile
HO
tyrosine (S)-dhurrin
(R)-taxiphyllin
CYANIDE IN FOODS 375
sequential
CH3, /
CN diglucosidase
/C,
CH3 O-glucose
I
O-Glucose glucose
simultaneous
diglucosidase
CH3 , /
CN
/C,
CH3 O-glu~ose
O-glucose gentiobiose
transport metabolite. In the second case, the two glucose moieties are split off as
gentiobiose, producing hydroxynitriles-which also occurs in post mortem
cyanogenesis-but now under controlled conditions. The HeN produced quanti-
tatively can be refixed by p-cyanoalanine synthase. This enzyme catalyzes the
reaction of cyanide and cysteine. 37 The resulting p-cyanoalanine is hydrolyzed to
asparagine. 38 In this manner, the nitrogen present in cyanogenic glucosides is
incorporated into asparagine and becomes available for the general nitrogen pool.
Simultaneous cleavage of cyanogenic glucosides results in their metabolization
(for details see ref. 4).
To summerize: In source-tissues, cyanogenic (mono)glucosides are mobi-
lized by glucosylation and translocated as diglucosides into sink-tissue where they
are hydrolyzed. Their metabolic fate is determined by the mode of hydrolysis:
Sequential cleavage leads to a simple translocation, whereas simultaneous clevage
initiates metabolization to non-cyanogenic compounds. Depending on the actual
ratio of activities of simultaneous to sequential diglucosidase, the one or the other
process is favored (Fig. 4).
FUNCTIONS
Ecological Significance
Imetabolization I Itranslocation I
source-
organs
enzymes, they are able to liberate relatively large amounts ofHCN when injured;
these plants are classified as highly or strongly cyanogenic. In contrast, other
plants that synthesize cyanogenic glucosides contain much smaller concen-
trations, often less than 10 nmol per g fresh weight. These are called weakly
cyanogenic plants.
The HCN liberated upon tissue damage-at least in strongly cyanogenic
plants-respresents an important ecological factor by repelling potential herbi-
vores (see refs 39, 40). Studies on the protective role of cyanogenesis also have
been performed (see refs 10, 11). These investigations have shown in general that
it is not the presence of cyanogenic glucosides but the ability of plants to liber-
ate HCN rapidly that represents the protective function. Surprisingly, in some
cases, the carbonyl compound, for instance, benzaldehyde, also acts as a repel-
lent. 41 If th~ protection is due to an effective cyanogenesis, the presence of the
corresponding hydrolytic enzymes is essential for the ecological function. Both,
the l3-glucosidases, which initialize and enable cyanogenesis, and the hydroxyni-
trile lyase are involved in the defensive strategy. The relatively unstable hydrox-
ynitriles may dissociate spontaneously; nevertheless, effective cyanogenesis
requires these lyases which may accelerate HCN-liberation up to 20-fold. 42
Evolutionary Aspects
The ability of plants to synthesize cyanogenic glucosides corresponds to an
ancient character. This can be deduced from their widespread occurrence through-
out the plant kingdom, as well as from the strong similarities in biosynthetic path-
CYANIDE IN FOODS 377
Sorghum bico/or
HNL
from
Prunus serotina
Hevea brasiliensis Prunus amygdalus
dehydrogenases
'1/
"alP-hydrolase fold"
'"p1 / a p-motif"
(catalytic triad) (nucleotid binding domain)
a catalytic triad (Fig. 5). The sequence comparison of the hydroxynitrile lyases
from Prunus and Linum points in a similar direction. These hydroxynitrile lyases
reveal only slight homologies to each other. The lyase from Prunus seems to be
homologous to flavin-dependent dehydrogenases, but the lyase from flax is more
homologous to Zn-containing alcohol dehydrogenases. 5o (Fig. 5) Both groups of
dehydrogenases have their origin in a common putative ancestor, sharing a
nucleotide binding sequence and a ~a~-motif. From these data, it can be deduced
that hydroxynitrile lyases have developed convergently at least four times. 50
As mentioned, the ability to synthesize cyanogenic glucosides is an ancient
character. This means that in the course of evolution, plants existed that were
able to synthesize cyanogenic glucosides but which lacked hydroxynitrile lyases
and, thus, the ability to carry out efficient cyanogenesis. As the repellent effect
for potential herbivores depends on rapid cyanogenesis, the significance of
cyanogenic glucosides in these ancient plants must have been due to different
selective pressures.
for example, the spouting of various plants that normally depends on vernaliza-
tion, can also be induced by application of cyanide. 62
Small amounts of HCN also are produced in the course of ethylene pro-
duction (Fig. 6). The biogenetic precursor of this gaseous plant hormone is 1-
aminocyc1opropane-l-carboxylic acid (ACC). ACC is oxidized by a specific
oxidase to produce equimolar quantities of ethylene, HCN, and CO 2 •3.63 Applica-
tion of exogenous ethylene to plants induces endogenous ethylene production.
Thus, exogenous ethylene application also leads to the liberation of small amounts
1-aminocyclopropane
1-carboxylic acid ethylene
(ACC)
General Aspects
Since cyanogenic glucosides are essentially ubiquitous, and since different
amounts are present in different plants and plant parts, quite different amounts of
cyanide are released upon tissue disruption. In general, during food preparation,
plant tissue is disrupted to different degrees. On the one hand, plant parts may be
consumed as they are, for instance, as fresh vegetables, fruits, and salad greens.
On the other hand, other parts are dried, cooked, ground, or mashed. Because of
these differences in tissue disruption, there are correspondingly strong differences
in the initiation of post-mortem processes such as cyanogenesis.
Depending on the degree of disruption, almost the entire amount of
cyanogenic glucosides originally present in the intact plant may still be present
in some foods, whereas, in others, nearly all of the cyanogenic glucosides are
cleaved, and only the corresponding degradation products are detectable. In sub-
sequent processing steps, the degree of decomposition and evaporation of the
volatile hydroxynitriles and gaseous HCN, respectively, depends on the process-
ing method, especially on the amount of heating. In order to provide solid and
comparable information on the quantity of these compounds in different prod-
ucts, in the following paragraphs, the term HCN-potential is used, which includes
the concentrations of all cyanogens, including the uncleaved cyanogenic gluco-
sides, the hydroxynitriles, free HCN, and cyanide. The data will be given in both
mol and in g of HCN equivalents that would be liberated if all the cyanogenic
compounds were completely dissociated to HCN.
CYANIDE IN FOODS 381
Irhodanese I
CN" + S-SOl" ~ SCN" + SOl" Figure 7. Detoxification of cyanide by
l
excretion
rhodanese. In mammalian liver cells, in
contrast to the artificial substrate thiosulfate,
which is used in standard enzymatic assays,
via urine various sui fane sulfur anions are used. 65
become critical, and about 60mg HCN per day is lethal. Concentrations
of cyanide in foods derived from weakly cyanogenic plants, all of which are in
the Ilg-range, are no problem for our detoxification mechanism and for our
health.
The limiting factor in the detoxification of cyanide is not the rhodanese
reaction, but sulphur availability and excretion of the resulting thiocyanate. 66
Consequently, people who consume cyanide rich foods, e.g., up to twenty mg
per day, have enhanced levels of thiocyanate in the blood which can create
severe health problems (see below). The small amounts of cyanide present in
foods derived from weakly cyanogenic plants do not significantly enhance the
thiocyante level in our blood and, thus, do not cause the dissorders described
below.
Only in special cases does the small amount of cyanide in foods derived
from weakly or medium cyanogenic plants create toxicological problems. Such
an example is the preparation of whisky and other alcoholic beverages. As men-
tioned, leaves of barley contain relatively high concentrations of cyanogenic
glucosides, mostly derived from leucine. Consequently, in the malt and in the
mash, cyanogens and their degradation products also are present. In the course
of the distillation procedure, resultant HCN reacts with ethanol to yield toxic
carbamates. 67 •68
mainly due to the interference of SCN with iodine metabolism. Thus, long term
exposure to significant concentrations of HCN causes or aggravates iodine
deficiency disorders, which are expressed mainly as goiter and cretinism. 73
In addition, various investigations have suggested that the consumption of
cassava products with high residual cyanide content causes neurological and par-
alytic disorders, for instance, konzo and tropical ataxic neuropathy.74.75 In these
cases, however, the correct pathogenic mechanism is unknown, and it is unclear
whether there is a link to cyanide metabolism. There are also some indications
that long-term consumption of increased cyanide concentration in foodstuffs may
induce diabetes. 76 But apart from investigations in Nigeria, in which a correla-
tion between frequent consumption of improperly processed cassava products and
the frequency of aquired diabetes has been observed, no other data are available.
Manihot escu/enta
leaves
(source)
Itranslocation I
transport
via phloem
[ accumulation 1
sequential linamarin
diglucoSid~
tuber
linustatin h-y-d-ro-Iy-s-is'l
rl .
(sink) . asparagine
simultaneo~ acetone / ' "
diglucosidase cyanohydrin I metabolization I
Manihot esculenta
Ibiosynthesis)
~
leaves
(source)
Itranslocation I
transport
"aspired
via phloem metabolism"
sequential
di9lucOSida~
linamarin Iaccumulation I
tuber linustatin Ihydrolysis)
(sink)
simultane~ acetone ' "
diglucosidase cyanohydrin
olization of the imported cyanogens does not appear to play any role in cassava.
Our goal for producing modified cassava plants is outlined in Figure 8b. Tuber
specific suppression of the sequential diglucosidase and concomitant over-
expression of the simultaneous diglucosidase should change the metabolic
fate of linustatin imported into the tubers, causing it to be metabolized into
non-cyanogenic compounds. The other enzymes required for this process, ~
cyanoalanine synthase and cyanoalanine hydrolase, are present naturally in
cassava tubers of the original varieties. 87 In such plants, the leaves should contain
the same amount of cyanogenic glucosides as the original varieties, but in the
root and tubers, the cyanogenic potential should be reduced significantly. Whether
these target plants indeed will have the desired properties, or whether-for
instance due to the initiation of supplementary synthesis of cyanogenic gluco-
sides in the tubers-the cyanogenic potential will again be high, will only be
clarified by experiments.
CYANIDE IN FOODS 387
CONCLUSIONS
ACKNOWLEDGMENTS
I wish to thank Dr. Dave S. Seigler (University of Illinois, Urbana) for crit-
ical reading the manuscript and helpful suggestions related to the sientific content
as well as for linguistic improvements.
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61. TANAKA, 0., CLELAND, c., BEN-TAL, Y. 1983. Effect of ferricyanide, ferrocyanide
and KCN on growth and flowering on the short-day plant Lemna paucicostata. Plant and
Cell Physiology 24(4): 705-711.
62. GASSNER, G., Heuer, W 1927. Praktische Anleitung zum Friihtreiben von Pflanzen
mittels Blausiiure. Verlagsbuchhandlung Paul Parey, Berlin.
63. YANG, S.F., HOFFMANN, N.E. 1984. Ethylene biosynthesis and its regulation in higher
plants. Ann. Rev. P!. Physio!. 35: 155-189.
64. LEHMANN, G., ZINSMEISTER, H.D., ERB, N., NEUNHOEFFER, O. 1979. Uber den
Blausiiuregehalt von Getreiden und Getreideprodukten Z. Erniihrungswiss. 18: 16-22.
65. WESTLEY, J. 1981. Cyanide and sulfane sulfur. pp. 61-76 in Cyanide in Biology
(B. Vennesland, E.E. Conn, C.1. Knowles, J. Westley, F. Wissing, eds.), Academic Press,
London.
66. ROSLING, H. 1994. Measuring effects in humans of dietary cyanide exposure from
cassava. Acta Horticulture 375: 271-283.
67. MACKENZIE, WM., CLYNE, A.H., MACDONALD, L.S. 1990. Ethyl carbamate for-
mation in grain-based spirits: Part II. The identification and determination of cyanide
related species involved in ethyl carbamate formation in scotch whisky. J. Inst. Brewing
96(4): 223-232.
68. COOK, R., MCGAIG, N., MCMILLAN, J.M.B., LUMSDEN, WB. 1990. Ethyl caramate
formation in grain-based spirits: Part III. The primary source. J. lnst. Brewing 96(4):
233-244.
69. BAUDOIN, J.P., BARTHELEMY, J.P., NDUNGO, V 1991. Variability of cyanide con-
tents in the primary and secondary genepools of the lima bean, Phaseolous lunatus 1.
Plant Genetic Resources Newsletter 85: 5-9.
70. SCHlLCHER, H., WILKENS-SAUTER, M. 1986. Quantitative Bestimmung cyanogener
Glycoside in Linum usitatissimum mit Hilfe der HPLC. Fette, Seifen, Anstriche 8:
287-290.
392 D. SELMAR
71. DUFOUR, D.L. 1994. Cassava in Amazonia: Lessons in utilization and safety from native
people. Acta Horticulture 375: 175-182.
72. ESSERS, S. 1995. Removal of cyanogens from cassava roots. CIP-DATA Koninklijke
Bibliothetheek, Den Haag, 131 p.
73. DELANGE, F., EKPECHI, L.O., ROSLING, H. 1994. Cassava cyanogenesis and iodine
deficiency disorders. Acta Horticulture 375: 289-293.
74. TYLLESKAR 1994. The association between cassava and the paralytic disease konzo.
Acta Horticulture 375: 331-339.
75. SPENCER, P 1994. Human consumption of plant material with neuro-toxic potential.
Acta Horticulture 375: 341-348.
76. AKANJI, A.O. 1994. Cassava intake and the risk of diabetes in humans. Acta Horticul-
ture 375: 349-359.
77. SUDARESAN, S., NAMBISAN, B., ESWARI AMMA, e.S. 1987. Bitterness in cassava
in relation to cyanogen content. Indian 1. Agric. Sci. B57: 37-40.
78. BOKANGA, M. 1994. Distribution of cyanogenic potential in cassava germplasm. Acta
Horticulture 375: 117-123.
79. NYE, M.M. 1991. The mis-measure of manioc (Manihot esculenta). Econ. Bot. 45(1):
47-57.
80. PEREIRA, 1.F., SEIGLER, D.S., SPLITTSTOESSER, WE. 1981. Cyanogenesis in sweet
and bitter cultivars of cassava. Hortscience 16: 776-777.
81. KING, N.L.R., BRADBURY, 1.H. 1995. Bitterness of cassava: Identification of a new
apiosyl glucoside and other compounds that affect its bitter-taste. 1. Sci. Food Agric.
68(2): 223-230.
82. DO, L., BOKANGA, M., M0LLER, B.L., HALKER, B.A. 1995. The biosynthesis of
cyanogenic glucosides in roots of cassava. Phytochemistry 39(2): 323-326.
83. BEDIAKO, M.K.B., TAPPER, B.A., PRITCHARD, G.G. 1981. Metabolism, synthetic
site, and translocation of cyanogenic glycosides in cassava. pp. 143-148 in Tropical Root
Crops: Research strategies for the 1980s (E.R. Terry, K.A. Oduro, F. Caveness, eds.),
International Development Research Centre, Ottawa, Canada.
84. MAKAME, M., AKORODA, M.O., HAHM, S.K. 1987. Effects of reciprocal stem grafts
on translocation in cassava. 1. Agric. Sci. Camb. 109: 605-608.
85. DEBRUIJN, G.H. 1973. The cyanogenic character of cassava (Manihot esculenta) pp.
43-48 in Chronic Cassava Toxicity (B. Nestel, R. MacIntyre, eds.), International Devel-
opment Research Centre, Ottawa, Canada.
86. SELMAR, D. 1994. Translocation of cyanogenic glucosides in cassava. Acta Horticul-
ture 375: 61-67.
87. NARTEY, F. 1969. Studies on cassava, Manihot usitatissimum, II. Plant Physiol. 22:
1085-1096.
Chapter Fifteen
Junya Mizutani
Phytochemicals in Human Health Protection, Nutrition, and Plant Defense, edited by Romeo.
Kluwer Academic I Plenum Publishers, New York, 1999.
394 1. MIZUTANI
INTRODUCTION
Chloranthus japonicus
W<0 ~o ~
H
Shizukanolide A
:::-
Shizukanolide B
WQ
Lindenene
y:t(o
Yr< H~
9'" :::- 9'"
0
I #
~ y::q ~ 1#
0
. :::-
9'" ~"
HO\\'" ~
OH
Shizukolidol Isofuranodiene
W<0 ¢eto ~o
Shizuka-aeoradienol
~ :::-
~ H ~ H
l ~
"
OAe
Chloranthalaetone C
"OH
Shizukanolide C
'OAe
Shizukanolide D
Subsequently, from the aerial parts, glechomanolid, and from the leaves,
furanodienone, shizukafuranol, and shizukolidol (Fig. I) were isolated and
identified. 10.1 I Isofuranodiene, shizukaacoradienol, chloranthalactone C, shizu-
kanolide C, and shizukanolide D also were isolated from the roots and identified
(Fig. 1).10-13 The root of C japoniclls was known as Chinese drug. The antimicro-
bial activity of these materials was investigated. Shizuka-nolide A, glechomano-
lid, and isofuranodiene showed little activity against microbes. In contrast,
shizukanolide B (8,9-dehydroshizukanolide) showed antifungal activity and
caused remarkable growth inhibition offungi of the genera Mucor and Rhizopus. IO
Anti-fungal activities of chloranthalactone C and shizukanolide C against Mucor
griseo-cyanus were compared with that of shizukanolide B. Despite, the presence
of an a~,y8-unsaturated lactone moiety in the molecules of chloranthalactone C
and shizukanolide C, they were found to be substantially inactive. 12
Furanogermacranes such as isofuranodiene are considered to be key inter-
mediates of other furanosesquiterpenes. Glechomanolid also may be a biosyn-
396 1. MIZUTANI
()I~
'Y)ppl
Farnesyl pyrophosphate
)I'
)I'
lsofuranodiene Glechomanolid
...................
Shizukanolide A
Lindenene
Wt°
Shizukanolide B
OH
~C~M'
OH
----II.~ Compound X
Shizukaol A
~o
Shizukanolide B
Hand 1, consisting of two lindenane units, were isolated from the roots of C.
japonicus. 1S Their structures were elucidated principally by ID- and 2D-NMR
methods (Fig. 4).
Furthermore, a novel sesquiterpene trimer, trishizukaol A, consisting of
three lindenane units, was isolated from the roots of C. japonicus. The structure
was determined by the abovementioned methods. A structurally related sesqui-
terpene dimer, designated as shizukaol J, was also found in the same plant. 16
Although a number of lindenane dimers have been isolated from the chlorantha-
ceous plants, this is the first example of the isolation of a trimeric lindenane
sesquiterpene. Shizukaol J was determined to be a homo-dimer of compound X.
The plausible biogenetic relationships of trishizukaol A, shizukaol J, and com-
pound X are shown in Fig. S. Subtraction of a proton from C-13 of the compound
X (iindenatriene) and successive double bond migration results in a carbanion at
C-lS which then attacks C-ll' of another molecule of compound X to give the
dimer shizukaol 1. Finally, a Diels-Alder type cycloaddition between the termi-
nal methylene of shizukaol J and the C-lS"/C-4"/C-S"/C-6" diene of the third
molecule of compound X completes the structure of the trimer trishizukaol A
(Fig. S). So far, more than 10 lindenane dimers containing compound X as a struc-
tural unit have been isolated from Chloranthus species. 14-16,19-21 All attempts to
isolate monomeric compound X, however, have failed. Structural variations and
biogenetic correlations of such lindenane dimers suggest that these compounds
are synthesized in plant cells.
Chloranthus serratus
OH OH
SllIzukaol A
" fO
OH
0 0
HO HO
O~O
~ ~O
o~r{l0
·'UOH
0 o 0
Shizukaol C
Shizukaol B Shimkaol G
OH OH OH
C02Mc
OH
'~o-r
o
0
0
HO
00
o
~ ;0 0
0
·'UOH
Figure 4. Structures of lindenane dimer shizukaols A-I isolated from Chloranthus species and
plausible biosynthetic correlations.
Compound X Compound X
(Lindenatriene) (Lindenatriene)
Compound X
Trishizukaol A
Figure 5. Plausible biosynthetic route for the formation oftrishizukaol A and shizukaol J from
compound X (lindenatriene).
Acoragermacrone Acolamone
M o
Zederone
H~O OH
Neoacolamone 7a-Hydroxyneoacolamone
Shizukanolide E
Shizukanolide F
~ 1?
opp
'
,
Famesyl pyrophosphate
O(y
Acoragermacrone
isofuranodiene
•
C(?i
1?
I J
0
~~o
~
o
Furanodienone 'OAc
Acolamone Chloranthalactone C
Zederone Shizukanolide C
Neoacolamone
9?Y
7a-Hydroxyneoacolamone
OH
15' Shizukaol A
OH
/
Compound Y
C0 2 Me
Shizukaol C
OH O~
0 / 011\
C0 2Me
HOo~ofO
o 0
Shizukaol B
Oil Oil
Compound X
(Lindenatriene)
HO
Oil
Cycloshizukaol A
Compound X
(Lindenatriepe)
Figure 9. Cycloshizukaol A isolated from the roots of Chloranthus serratus, possibly derived
biogenetically from two molecules of compound X (Iindenatriene) through a [6 + 6] cycload-
dition reaction.
The methanol extract of the roots and rhizome of Carex .redia Nees var.
miyabei (Franchet) T. Koyama (Cyperaceae) shows strong antibacterial activity
against Staphylococcus aureus. We isolated four 3,5,4'-trihydroxystilbene (resver-
atrol) o!igomers, one dimer, one trimer, and two tetramers. 24 Their structures were
determined from spectroscopic evidence and biogenetic consideration. The dimer
was identified to be viniferin, already identified as a phytoalexin of grapevine
404 J. MIZUTANI
leaves. 25 The trimer and teramers were new compounds and were named
miyabenols C (for the trimer), and A and B (for the tetramers). The stereochem-
istry of miyabenols A and B was later determined, 26 and their structures are shown
(Fig. 10).
OH
OH
OH
OH
Miyabenol A Miyabenol B
HO OH
OH
Miyabenol C Kobophenol A
OH
OH
Hopeaphenol
Kobophenol B
Figure 10. Structures of oligostilbenes (trimer and tetramers) isolated from Carex Species.
PLANT ECOCHEMICALS FROM THE VIEWPOINT OF PLANT DEFENSE 405
Carex kobomugi
An antimicrobial novel tetrastilbene named kobophenol A has been isolated
from the roots and rhizome of Carex kobomugi Ohwi (Cyperaceae), together with
previously known oligostilbenes, miyabenol C and E-viniferin, as a major active
principle. 27 ,28 The structure of kobophenol A has been determined on the basis of
spectroscopic and chemical properties (Fig. 10).
Carex pumila
A novel antimicrobial tetrastilbene constituted of two E-viniferin units
was isolated from the subterranean parts of C. pumila Thunb. (Cyperaceae).29 Its
complex polycyclic structure was elucidated by precise analyses and was named
kobophenol B. The relative stereochemistry of kobophenol B was assigned from
the results of NOESY experiments. Kobophenol B is expected to be biosynthe-
sized from two molecules of (-)-E-viniferin, which has been found in the same
plant, via four succesive electron oxidative coupling reactions. Therefore, the
absolute configuration of kobophenol B can be deduced as depicted in Figure 10.
E-Viniferin was first isolated as a phytoalexin of grapevine (Vilis vinifera,
Vitaceae) leaves. 25 We isolated (-)-E-viniferin and related oligostilbenes (Fig. II)
from Carex species. 24.27 However, no stereochemical assignment for E-vineferin
has been made except that its corresponding 7b,8b-dihydro-erivative, gnetin F
(2b),30 has been shown to have a 7aS, 8aS configuration from CD results (Fig.
11). An ether-soluble phenolic fraction obtained from the methanol extract of
the roots and rhizome of C. pumila was chromatographed on a silica gel column
and subsequent silica gel preparative TLC yielded three oligostilbenes, (-)-E-
viniferin, and miyabenols C and A, which were identified by comparing with
authentic samples. 3 !
The relative configuration of (-)-E-viniferin (la) was next determined by
chemical methods. Permethylation of la with dimethyl sulfate gave a pentamethyl
ether derivative (Sa). Dehydrogenation of Sa with DDQ readily afforded a ben-
zofuran derivative (6). Catalytic hydrogenation of 6 gave a cis-dihydrobenzofu-
ran (7). Cis-trans isomerization at the 7a,8a-positions of7 was smoothly catalyzed
by aluminum chloride to yield a corresponding trans isomer (8). The IH-NMR
spectrum of7b,8b-dihydro-derivative (8a), directly obtained from Sa by catalytic
hydrogenation, was identical with that of 8, but apparently different from that
of 7. Therefore, the relative configuration of la was determined to be trans. The
CD spectrum ofa 7b,8b-dihydro-derivative (2a) of (-)-E-viniferin (la) showed a
completely opposite pattern to that of gnetin F (2b). The absolute configuration
of (-)-E-viniferin (la) from C. pumila was thus concluded to be 7aR,8aR
(Fig. 11).
Interestingly, we found that wild grapevine (Vilis coignetiae) produced (+)-
E-viniferin by Cu2+ induction. 31 Therefore, e-viniferin produced by grapevine is
406 1. MIZUTANI
RO
RO
OR OR
HO
o HO
•
(-)-E- Viniferin
Resveratrol radical I OH
Resveratrol radical II
OH
OH
HO ...
Reveratrol radical II
Miyabenol C
HO
E-Viniferin radical
o
HO
"Resveratrol radical I
HO
Miyabenol C radical
OH
Kobophenol A
hammer. After keeping damaged leaves in 71 of water at 25°C for 24hr, the water
diffusates were extracted with ethyl acetate (21). Column chromatography of the
neutral fraction (0.8 g) on Wako-gel C-200 gave colorless needles (119 mg) as the
anti-microbial principle, named rugosal A (Fig. 13). The structure of rugosal A
was deduced by chemical and spectroscopic (UV, IR, MS, and NMR) methods to
be a novel carotane with an a,p-unsaturated aldehyde group, an endo-peroxide
bridge, and an allyl alcohol partial structure. The corresponding carboxylic acid,
named rugosic acid A, was isolated from the uninjured leaves, but rugosic acid
A did not exhibit antifungal activity.
Since rugosal A has an allylic alcohol partial structure, the exciton chiral-
ity method 36 was expected to be applicable to determined the absolute configura-
tion, after being converted into the corresponding benzoate. Rugosal A benzoate,
however, could not be obtained, and a small amount of an hemiacetal benzoate
(derivative I) was produced (Fig. 13). Therefore, derivative I was produced via a
hemiacetal by heating rugosal A in pyridine followed by the treatment of benzoic
anhydride in triethylamine. Since derivative I is stereochemically equivalent to
rugosal A benzoate, the exciton chirality method is applicable. To exclude an
effect of the aldehyde chromophore on the CD spectrum, derivative I was reduced
with an excess amount of NaBH4 to yield the corresponding alcohol (derivative
15
14
CHO COzH
12
13 Rugosa! A
Rugosa! A hemiaceta!
f ~
Rugosic acid A
~a
CHO CHzOH
Figure] 3. Structures of rugosal A and rugosic acid A, and chemical conversion of rugosal A
to determine its absolute configuration. a, benzoic anhydride/triethylamine; b, NaBH4; c, pyri-
dine/heating. .
410 1. MIZUTANI
Other Sesquiterpenes
13
14
HO
I
2 on'I:, 2'
?"
3 6' ~ OR
4 5'
OH
Apigenin
Figure 14. Structures of isolated compounds from Rosa rugosa leaves and their derivatives.
412 J. MIZUTANI
Figure 15. Hypothetical biosynthetic relation between bisabolanoids and carotanoids of Rosa
rugosa.
larvae. The crude exudate and pure rugosal A were tested for their antifeedant
activity against neonate tobacco cutworm larvae. 48 Both the trichome exudate and
rugosal A retarded feeding of the larvae, and the activity of the crude exudate
was attributed mainly to rugosal A. It is well known that other higher plants (e.g.
Solanaceae and Compositae) also possess glandular trichomes which exude
defensive compounds.
In addition, some other compounds, minor carotanes, non-carotane
(bisabolane and acorane) sesquiterpenes, and 2-phenoxychromones were also
identified as constituents of the exudate. Since it has been suggested that biosyn-
thesis of the carotane sesquiterpenes is highly active in the tip cells of the glan-
dular trichomes, precursor sesquiterpene hydrocarbons were expected to be
present in the exudate. In the investigation ofless polar fractions, some sesquiter-
pene hydrocarbons were detected, and three major sesquiterpene hydrocarbons
[acora-3(4), 7(15)-diene, carota-4(5), II (12)-diene (isodaucene) and carota-I,4-
diene], and two minor [daucene and acora-3(4),7(8)-diene] were identified. 49 The
presence of these carotadienes, which are possibly precursors of the correspond-
ing C-14-oxygenated carotanoids, suggests that the formation of the carotane
skeleton, followed by the regio-specific oxigenation at C-14, occur in the biosyn-
thesis of R. rugosa carotanoids. The structures of these sesquiterpene hydrocar-
bons are also shown in Figure 14.
Lettucenin A
13
CHO
5'
R 1= OMe, R2= Me: lrilin A
RI= OMe, R2= H: lrilin B
Lettucenin A
R 1= R2= H: lrilin C
HO
HO
1'# 2' R2 R
I 3' OH 0
R= H: Apigenin
R= OMe: Hispidulin
6'" 4' RJ
Q
~
yY
H0yY0'('\~ #
OH
OH 0
5,7,2'-Trihydroxyflavanone
Figure 16. Structures of compounds inducibly produced by Taraxacum officinale and Iris
pseudacorus leaves treated with cupric chloride.
PLANT ECOCHEMICALS FROM THE VIEWPOINT OF PLANT DEFENSE 415
Isoftavonoids
Flavonoids
CONCLUSION
lindenane sesquiterpenes and the trimer are produced in Chloranthus species. The
biological meaning of plants synthesizing such sophisticated compounds has not
been made clear, but it is interesting from the viewpoint of phytochemistry.
Carex species (Cyperaceae) produce characteristic oligostilbenes as
defense allelochemicals. High concentrations of tetrastilbenes suggest they play
important roles in the defense mechanisms of the plants. Tetrastilbenes in Carex
species are biosynthesized from resveratrol (3,5,4'-trihydroxystilbene, monomer)
via (-)-E-viniferin (dimer) and miyabenol C (trimer) by means of successive elec-
tron oxidative coupling reactions. The last step, a coupling reaction of miyabenol
C radical with a resveratrol radical followed or not followed by the addition
of water, gives kobophenol A or miyabenol A, respectively. Interestingly, major
components are different from each other among the three Carex species. A
breakthrough in determining the structure of hopeaphenol (tetramer having C2
symmetry) from an isotopomeric point of view has been done by using NMR
spectroscopy.
A wild rose, Rosa rugosa (Rosaceae), produces a novel carotane sesquiter-
pene, rugosal A, with antifungal and antifeedant activities. Carota-I,4-dien-14-
aI, precursor of rugosal A is biosynthesized in the gandular trichomes of R. rugosa
leaves, and then the precursor is converted into rugosal A by autoxidation.
Taraxacum officinale (Compositae) leaves are induced to produce an anti-
fungal guaianolide, lettucenin A. When dandelion leaf is infected with a patho-
genic fungus, lettucenin A is produced around the necrotic spots in sufficient
amounts to inhibit the growth of pathogens. Iris pseudacorus (Iridaceae) leaves
also can be induced to produce several isoftavonoids and ftavonoids as defense
chemicals.
ACKNOWLEDGMENTS
This work was carried out in the Faculty of Agriculture, Hokkaido Univer-
sity in collaboration with colleagues, research associates, and many graduate and
undergraduate students. Their names are indicated in references. Some of works
have financially supported by Japan Science and Technology Corporation (JST).
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INDEX
421
422 INDEX
Lignans, 51, 53--61, 67--68, 73, 76, 219, 233, Matairesinol, 52, 54, 68-74, 76--77, 81-82,
235,241-242,244,347,351,354, 219,233,244
357-358 Maura pomifera, 239
biosynthetic pathways, 68, 76--83 Mayapple, 72, 74; see also Podophyllum
mammalian, 51-55, 57, 60--61, 68-73, 83 Maytenone, 355, 364
mechanisms, 58-59 Medicago truncatula, 148
metabolism, 74 Medicarpin, 135-136, 146-147
plant, 52-53 Medicinal plants, 320, 345, 347
Lignification, 218, 227 Mekuba seaweed, 73
Lignins, 207, 217-219 Melanoma, 13
Linamarase,377 Melia volkensii, 94
Linamarin, 374, 383, 385-386 Meliaceae, 94, 124
Linarin, 348, 361-365 Mesuaferrea, 237
Lindename dimers, see Sesquiterpenoids 8-Methoxypsoralen, 165
Lindenanolides, 399, 403, 416 Methyl thujate, 244
Lindenine, 394, 396 7-0-Methyldihydrokaempferol, 414, 416
Unum, 80, 377-378; see also Flax Methylesculin, 162, 165
L. usitatissimum, 82, 377 O-Methyltransferase, 146, 150, 166, 168, 176,
Linustatin, 385-386 242
Lipids, 216, 222-223, 237 Metrosideros polymorpha, 221
Lipochitooligosaccharide signal molecules, Mevalonate biosynthetic pathway, 37, 170,
136 224
5-Lipoxygenase inhibitory activity, 163, 165, Michellamine B, 12, 14-16
320, 333-334, 336, 361 Microberlinia brazzavilliensis, 229, 240
Uriodendron tulipfera, 241 Mimengosides, 351, 356, 365-366
Liver disease treatment, 345-346; see also Mixtures, 60, 100, I 16
Liver protectant Miyabeno1s, 404--405, 407--408, 417
Liver protectant, 74 Molecular mechanisms, 315
Liverworts, 319-322, 325, 328--329, 332, 335, Molecular recognition, 290, 315
337, 339 Molluscicidal activity, 335, 366
biological activity of, 320, 325, 329, Monoacylglycerides, 99
332-335, 337 Mononetin, 146
bitterness, 320, 325-327 Monoterpenoids, 37, 233, 235, 320, 324,
odor, 321-322 339
pungenc~ 320, 325-327, 332 Moraceae, 135, 138
Loganiaceae, 344, 351 Musci, 320, 338
Lotaustralin, 373 Muscle relaxing activity, 320, 335, 337
Lung cancer, 5, 32 Myristicaceae, 94
Lunularic acid, 334
Luteolin, 348, 361, 365 NADH oxidase, 121, 124, 151-153
Luteolin-7 -O-glucoside, 348, 361 Naphthalenes, 320
Lymphomas, 6, 13 Naringenin, 143
Naringenin chalcone, 140-141
Malus pumila, 241 Nematocidal, 320, 332-333
Mammalian Iignans, 51-55, 57, 60-61, 68-73, Nematode defense, 363
83 Neurite sprouting activity, 320, 335
Manihot esculenta, 377, 379, 383, 385-386; Nicotine, 337
see also Cassava Nod genes, 134, 136
Marchantia, 328-329, 332-334 Nordihydroguaiaretic acid, 75
Marchantins, 329, 332, 335, 337 Norlignans, 236
Marmesin, 170-172, 174-177 Nothofagus cunninghamii, 239
428 INDEX
Oak, 190--191, 196,207,240; see also Phenylethanoids, 347-350, 360, 362, 365
Quercus Phenylalanine aminomutase, 43-44
I-Octen-3-ol,321 Phenylalanine ammonia lyase, 42-43, 145, 149,
Olea cunninghamii, 244 152-153,227-228
Oleoresin, 234-235 Phenylpropanoids,68, 134, 166,223,347,351,
Oligopeptides, 281, 294 362
Oligosaccharides, 377 pathway, 74-76, 166
Oligostilbenes, 403-405, 407-408, 417 Phorbol,97
Orobanchin, 348-349 Phospholipases, 223, 236
Osthenol, 169-172 Photosensitizing, 165
Ovarian cancer, 13-14,32 Phototoxic, 163
Oxidative process inhibition, 359 Phthalides, 320
Phytoalexins, 134-136, 140--141,144, 148,
P388 leukemia, 6, 100 150--152, 168,238,403,405,413
Pacific Yew tree, 13, 14, 32; see also Taxus Phytoestrogens, 53, 60, 70--71,83, 137-138
brevifolia Phytotoxic, 335
Paclitaxel,4--6, 10, 13-14, 17; see also Taxol Picea abies, 222-223, 238
Palmae,·97 Picea mariana, 224
Parsley, 141, 172, 176 Pinaceae, 100, 135, 235
Parvifloracin, 106 Pinoresinol, 68--69, 74, 76-78, 80--81
Passifloraceae, 373 Pinoresinolliariciresinol reductase, 76, 80--81,
Pathogen defense, 232, 239, 242, 363; see also 83
Antipathogen Pinosylvins,237
Pathogen inhibition, 415, 417 Pinus, 218-220,224,228-229,233,237-238
Pathogenesis-related proteins, 151 P. banksiana, 228
Paw paw, 100, 104, 123; see also Asimina P. canariensis, 220
triloba P. contorta, 241
Pea, 135; see also Pisum sativa P. densifiora, 221
Pellia, 325, 329, 333, 335 P. japonica, 230
Pentagalloylglucose, 186-188, 190-191, P. radiata, 220, 225-228, 236--237, 239
193-194,196--197,199-204, P. resinosa, 217
206-208,291-292,294,296-298, P. strobus, 238
305-309 P. sylvestris, 220--221, 223, 226, 230-231
Pentosephosphate pathway, 227 P. taeda, 237
Perrottetins, 329, 334, 337 Pisatin, 135-136
Persea americana, 96 Piscicidal, 320, 335, 351
Pesticidal, 89-90, 93, 100 Pisum sativum, 147
Pesticide, 97, 106, 121-122 Pithecellobium dulce, 258
Phaseolus vulgaris, 73, 140; see also Bean Plagiochila, 322, 325, 329, 332, 335
I3-Phellandrene,241-242 Plagiochilal B, 326, 333, 335
Phellopterin, 162, 165 Plagiochilines, 326, 332-333, 335
Phenol oxidizing enzymes, 226--227 plagiochiline A, 325, 329, 335
Phenolics, 51, 187, 189,208,226,236,242, Plant defense, 134, 151,215,232,290,312,
245,255,275,291,296-300,303, 376,393-394,407,416-417; see also
306,308,314-315,338,354,360, Anti-herbivore; Herbivore defense;
415-416; see also Polyphenols Pesticidal
alkyl, 320, 328 Plant growth inhibitory activity, 320, 335
diphenolic compounds, 70 Plant lignans, 52-53
fatty acid esters, 354, 359 Podocarpid acid, 244
Phenolic O-methyltransferases, 144 Podocarpus, 244
Phenoxychromones, 412 Podophyllotoxin, 32, 72, 74-75
INDEX 429
Tyrosinase inhibitory activity, 362, 366 Weakly cyanogenic plants, 376, 378-382, 384,
387
Ulmus glabra, 241 Wheat, 73, 82-83
Umbelliferone, 161-162, 167-169, 171, 176 Wound healing, 345-346, 359-362
Uniform continuum model, 302 Wounding, 237-238
Urinogenital disinfectant, 347
Xanthotoxin, 178
Vasodilatory effects, 208 Xanthotoxol, 176, 178
Verbascoside, 349, 360-363
Vescalagin, 291-292, 305, 307-309 Yellow fever mosquito (YFM), 90, 97, 99, III,
Vinblastine, 4-5, 10, 32, 122 121, 124
Vincristine, 4-5, 10, 122
Viniferins, 403, 405-408, 417 Zelkova serrata, 218, 234