In the Laboratory

Investigation of Secondary Metabolites in Plants

A General Protocol for Undergraduate Research in Natural Products
Jonathan Cannon, Du Li, Steven G. Wood, Noel L. Owen,* Alexandra Gromova, and Vladislav Lutsky
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602; *noel_owen@byu.edu

Secondary metabolites are compounds found in higher

plants that are not involved in the major metabolic pathways.
These compounds are however, believed to play important
roles in plants. For example, some of them are toxic and help
in defending the plant from predators; others have odors that
attract insects and thus facilitate pollination. Many of these
compounds have important medicinal properties and are useful
drugs. In fact, a large percentage of all known medicines have
their origins in natural products found in plants.
A number of pharmaceutical companies and universities
have research groups that investigate the bioactivity and
potential medicinal uses of plant secondary metabolites. We
have been engaged in such research over the course of the
past ten years or so and much of the work in our laboratory
has been done by undergraduate students. Plant secondary
metabolite research cuts across many scientific disciplines such
as botany, microbiology, organic chemistry, biochemistry, and
other chemical sciences. Consequently such research provides
a unique opportunity to expose students to working in an
interdisciplinary environment. In this paper we (i) outline
typical experimental procedures that can be used to extract
and isolate individual chemical constituents from a plant, (ii)
suggest some simple procedures to test for selected bioactivity,
and (iii) explain how the molecular structures of the natural
products may be determined using spectroscopic techniques.
We use the Siberian plant Astragalus danicus as our example.
The work done on this plant will illustrate the multitude and
complexity of the chemical components found in plants. The
general scheme and approaches outlined in this paper may be
adapted for undergraduates at most colleges and universities.
The Plant Source
We have found that plants having a history of medicinal
use by indigenous peoples to treat various illnesses are the most
interesting candidates for study. However, any convenient
plant material such as commercial herbal products would be
suitable for this exercise. Considerable work has already been
done on many common medicinal plants. Therefore, it is very
valuable (imperative for serious research work) to check the
literature on the chosen plant in order to avoid duplication
of effort (1).
To obtain a sufficient amount of a purified secondary
metabolite for total structure determination, approximately
0.52 kg of dried plant material is required. For some plants,
the season in which they are collected governs to a large degree
the nature and quantities of the metabolites present in the stems
or leaves. The best method of collecting the plant material is

Permanent address: Irkutsk Institute of Chemistry, Siberian Branch

of the RAS, ul. Favorskogo, 664033 Irkutsk, Russian Federation.

1234

freezing in the field using solid CO2 or liquid N2. The frozen
plant material may then be stored frozen or subsequently
freeze-dried. Alternatively, the plants may be carefully dried
in the shade (drying in the sun may result in photodegradation of the constituents). Before extraction, the plant
material should be macerated if fresh or frozen, or ground
up if dried. If the sample is small or the plant material is
fresh or frozen, a pestle and mortar is sufficient; otherwise, a
blender, mechanical grinder, or mill works well.
Initial Extraction Scheme
We have worked out a scheme to yield three extracts of
differing polarity. A small-scale initial extraction is sufficient
to provide enough extract for bio-testing. The dried, ground
plant material (5 g) is covered with two volumes of methylene
chloride and stirred for 20 min. The solvent is filtered off,
the extraction is repeated, and the collected solvent is taken
to dryness.
If the plant material is fresh or frozen, the initial extraction
is carried out with methylene chloride/methanol (50/50) and
the subsequent extractions are carried out as described.
This process is repeated utilizing methanol as the solvent.
The methanol extracts are added to the residue from methylene
chloride extractions and the resulting solution is extracted
three times with equal volumes of hexane (addition of a small
amount of water may be necessary to facilitate phase separation).
The phases are separated and pooled. After removal of the
solvents, the hexane and methanol phases yield nonpolar and
polar organic extracts, respectively. Since in most instances
any native use of these plants for medicinal purposes involves
aqueous extraction or poultices, a hot water (90 C) extract may
be prepared from the extracted plant material or, if preferred,
from a new sample, resulting in a third (aqueous) extract.
There may be instances when the indigenous ethnobotanical
procedure involves acidic or basic conditions, but in the
majority of cases neutral water can be used.
The Isolation and the Purification Scheme
A larger-scale extraction of the plant material based on
the nature of the initial extract demonstrating the bioactivity is
initiated. An appropriate extraction solvent is chosen and a
500-g plant sample is extracted three times with two volumes
of solvent. The solvent is removed (a rotary evaporator is useful
here) and the resulting crude extract is tested to confirm the
bioactivity. The crude extract is analyzed by TLC and on the
basis of the result, a silica gel column is often run to
subfractionate the crude extract. The subfractions are then
tested for bioactivity and the active subfraction is chosen for
further purification. The active compounds are usually obtained
in pure form following additional chromatographic steps. If

Journal of Chemical Education Vol. 78 No. 9 September 2001 JChemEd.chem.wisc.edu

In the Laboratory

the compounds are in the right polarity range, silica gel

chromatography works satisfactorily. Both preparative and
analytical HPLC, if available, are extremely useful to separate
mixtures and determine the purity of the isolated compounds.
The Bio-testing Procedures
We have found the use of relatively simple and straightforward cytotoxicity and antimicrobial assays to be effective in
identifying and confirming the biological activity throughout
the extract fractionation and purification procedures. Cytotoxicity testing is carried out using standard 96-well microtiter
plates seeded (ca. 5 105 cells) with HeLa cells (epithelial
carcinoma, cervix, human, ATTC CCL2). The plates are incubated for at least 4 h and the plant extracts are tested in
triplicate at initial concentrations of 200, 100, and 50 mg/mL.
After a 36-h incubation, the results are determined using sulphorhodamine B dye to stain protein as described by Skehan et
al. (2). This method involves fixing the viable cells to the
plate with a 0.1% perchloric acid solution, washing several
times with water, and staining the cells with a solution of
the dye in 1% acetic acid (4 mg/mL). Excess dye is removed
with subsequent washings of 1% acetic acid and the absorbance
of the dye at 540 nm (directly proportional to the number
of viable cells) is determined by taking up the bound dye in Tris
buffer (10 mM, pH 7.4). The percent viability is determined
relative to untreated control wells.
Antimicrobial testing is done using a gram-positive organism, Staphylococcus aureus (ATCC 6538P), a gram-negative
organism, Pseudomonas aeruginosa (ATTC 27853), and a
yeast, Candida albicans (ATCC 90028). These tests are also
run in 96-well microtiter plates as described by Donaldson
(3). The plant extract samples are added in triplicate to plates
containing appropriate growth media (water and methanol
extracts 200 to 0.1 g/mL, hexane extracts 500 to 0.5 g/mL)
and an inoculum of the microorganism is added. Growth inhibition is determined by comparing the optical density (600
nm) of the plate read directly after inoculation to the optical
density read after a 24-h incubation.

spectroscopy. The former is the method of choice if goodquality crystals can be obtained and if one has access to the
appropriate equipment. 1H and 13C 1-D and 2-D NMR
experiments are usually sufficient to obtain a structure of a small
molecule (under 1000 MW). A quantitative 13C experiment
provides the total number of carbons in a compound. These
data, in conjunction with high-resolution mass spectral data,
are usually sufficient to provide the molecular formula of the
unknown compound. The majority of natural products extracted
from plants are composed only of carbon, hydrogen, and oxygen, with the exception of some classes of nitrogen-containing
compounds such as alkaloids. A NMR DEPT experiment
identifies all quaternary carbon atoms as well as those connected
to one, two, or three hydrogen atoms. The 2-D NMR experiments, COSY and HETCOR, show the connectivity of adjacent hydrogen atoms and hydrogen atoms to carbon atoms,
respectively. Occasionally, more sophisticated experiments
such as NOESY, HMBC, or INADEQUATE are necessary for
complete structure determination. However, these techniques
are often only available in laboratories that have well-trained
spectrometer operators.
A routine infrared or ultraviolet spectrum will always
provide useful information concerning the presence or absence
of specific functional groups in the compound under investigation, provided enough sample is available.
An Example
Plants of the Astragalus genus (the largest genus of the
Fabaceae family) are found worldwide. Astragalus danicus
grows wild in eastern Siberia (Russia) and has been used for

Aerial parts
of the plant

#1
H2O
extract

The Structure Determination

After a pure chemical component is obtained, one of the
first steps in ascertaining its structure is the determination
of its molecular mass. If a high-resolution mass spectrometer
(HRMS) is available, then a precise determination of the mass
of the parent ion will often indicate the most likely molecular
formulas. For many pure natural products, especially glycosides
and related compounds, fast atom bombardment (FAB) is
the mass spectrometric method of choice for analysis, but for
more volatile compounds electron impact (EI) and chemical
ionization (CI) can give useful data. An analysis of the mass
spectral fragmentation pattern will, in many instances suggest
the molecular formulas of the main breakdown fragments,
which is helpful in determining the total structure. Tandem
techniques such as GCMS or HPLCMS can facilitate the
simultaneous separation and the identification of compounds
of interest. Electrospray MS techniques are useful for compounds
with molar masses above about 1000.
The most useful techniques for structure determination
of natural products are, however, X-ray diffraction and NMR

H 2O
MeOH

#2
MeOH
extract

CH 2Cl2

#3
CH2Cl2
extract

#6
Waxes, fats,
lipids, etc.

#4
CHCl3
extract

#5
H2O
soluble

#7
H2O
residue

SIO2 chromatography
(hexane/acetone)

1-BuOH/H2O
partition

#8
1-BuOH
soluble
SIO2 chromatography
(CHCl3/MeOH/H2O)
prep HPLC
(MeOH/H2O)

prep HPLC

#9
Fatty acid
fraction
linolenic
acid (1)

CHCl3/H2O
partition

#10
D-3-Omethyl-chiroinositol (2)

#11
Saponin fraction
monoside (3)
biosides (4, 5)
triosides (6, 7)

Figure 1. Extraction scheme for Astragalus danicus.

JChemEd.chem.wisc.edu Vol. 78 No. 9 September 2001 Journal of Chemical Education

1235

In the Laboratory
Table 1. Data for Compounds Isolated from Astragalus danicus
Compound
No. Name

Formula

Mol. Ion Exptl (HRMS)

Calcd

Lit

Exptl

Value

Ref

Linolenic acid

C18H30O2

[MH]

278.224

278.225

11

D-3-O-Methyl-chiro-inositol

C7H14O6

[M+H]+

195.086

195.087

186187

188

10

Soyasapogenol 3--D-glucuronide C36H58O9

[MH]

633.399

633.400

242244

3a

Soyasapogenol B

458.375

458.376

252253

258260

C30H50O3

[M]

Cloversaponin IV (bioside)

C41H66O13

[MH]

765.442

765.443

220222

Azukisaponin II (bioside)

C42H68O14

[MH]

795.454

795.453

283285

285287 11

Astragaloside VIII (trioside)

C47H76O17

[MH]

911.499

911.500

223225

223224 12

Hespidacin (trioside)

C48H78O18

[MH]

941.511

941.511

240242

245247 13

centuries by healers and native peoples of that area to cure

headaches, to treat post-menstrual symptoms and fatigue, to
increase stamina, etc. The above-ground parts of the plant
were collected during the summer flowering season and airdried. The crushed plant material (ca. 1 kg) was extracted by
a method similar to that outlined above, but the methylene
chloride and methanol extracts were kept separate (see Fig. 1).
A sample of each of the three extracts was tested for bioactivity
against HeLa cells (human cervical cancer), Candida albicans
(fungus), Pseudomonas aeruginosa (gram-negative bacterium),
and Staphylococcus aureus (gram-positive bacterium). The
MeOH and the CH2Cl2 extracts were cytotoxic to HeLa cells
and inhibited the growth of S. aureus.
The chloroform-soluble portion (#4, Fig. 1) of the methanol extract was combined with methylene chloride extract
(#3) on the basis of both TLC and the S. aureus bioassay
results. This combined material (#6) was subjected to SiO2
column chromatography and then preparative HPLC [C-18,
5 m, column; solvents: MeCN/H2O (45:55 to 100:0, v/v)]
providing a fraction that inhibited bacterial growth by 50% at
a concentration of 30 g/mL. An IR spectrum of this fraction
showed a strong carbonyl group absorption at 1712 cm1,
indicative of a COOH group. The major component of this
fraction was easily identified from the 13C NMR spectrum
of this fraction as linolenic acid (1) (4) (Table 1). A subsequent
literature search (57) confirmed that the bioactivity of this
fraction (both antibacterial and cytotoxicity) could be completely accounted for by this polyunsaturated fatty acid. A
second, non-biologically-active compound was also isolated
from fraction #6. This compound was identified on the basis
of its IR, MS, and 1H and 13C NMR spectra as D-3-O-methylchiro-inositol (2).
The genus Astragalus contains a class of plant secondary
metabolites known as saponins, which have a terpenoid
framework with one or more sugar groups attached. Although
these compounds were not active in our bioassays, we were
interested in comparing the structures of the saponins present
in A. danicus with those found in other Astragalus species. A
butanol/water separation technique (Fig. 1) effective in isolating such compounds was employed on the CHCl3-extracted
aqueous potion of the initial MeOH extract. The saponincontaining butanol extract (#11) was further separated by
column chromatography on silica gel (CHCl3/MeOH/H2O
solvent system) and produced three fractions. Five saponins
(Fig. 2) were subsequently isolated from these fractions. The first
fraction contained a single component, 3. After preparative
1236

Melting Point/C

Mass Data ( m /z )

HPLC (C-18; 5 m, column 21.4 250 mm; solvents

MeCN/H2O 45:55, v/v) fractions two and three yielded compounds 4 and 5, and 6 and 7, respectively.
We will consider in detail the structural elucidation of
two of these saponins, 3 and 4, to illustrate methodology and
briefly comment on the elucidation of the remaining three.
The MS data indicated that molecular mass of 4 was greater
than that of 3 by 132 mass units. The proton and carbon
NMR spectra of both compounds were consistent with the
suspected saponin structure: elements of both the cyclic
hydrocarbon terpenoid ring structure and the characteristic
patterns of the oxygenated carbon atoms of sugar groups were
clearly discernible.
Many plant secondary metabolites have attached sugar
groups. The removal of these groups often aids in the determination of both the genin (nonsugar portion) and the individual sugar structures. To this end, acid hydrolysis (3.5%
H2SO4; dioxane/water 1:1, 80 C for 6 h) of compound 4
resulted in the isolation of two components from the nonaqueous part of the hydrolysate by silica gel chromatography.
The first of these (3a) was found to have the molecular

3a
3
4
5
6
7

Monoside
Bioside
Bioside
Trioside
Trioside

R1 (on
sugar unit)

H
S1
S2
S2
S3
S3

H
CH2OH
H
CH2OH

20
22
12

OH

17

1
3
4

RO

CH2OH

S1

S2

COOH
O
OH

OH
OH

OH

R1

OH
OH

OH

Xyl
or
Glc

-D-glucopyranosiduronic acid
-D-xylopyranosil
-D-glucopyranosil
-L-rhamnopyranosil

R1
OH

O
O

Xyl
or
Glc

OH
O

OH

O
CH3
OH OH

Figure 2. Triterpenoid saponins of Astragalus danicus.

Journal of Chemical Education Vol. 78 No. 9 September 2001 JChemEd.chem.wisc.edu

Glc A

OH

Glc A

Glc A:
Xyl:
Glc:
Rha:

O
OH

Glc A

OH

S3
COOH

COOH

Rha

In the Laboratory

formula C30H50O3 from analysis of the HRMS. This formula

corresponds to a degree of unsaturation of 6, indicating a
structure with 6 double bonds or rings. The 1H and 13C NMR
spectra of this compound showed the presence of one trisubstituted double bond ( 5.41; 1H), 7 methyl groups, and
1 primary and 2 secondary hydroxyl groups. These data were
consistent with a pentacyclic triterpenoid with one double
bond. The location and the relative configuration of the three
oxygen-containing substituents (at positions 3, 4, and 22
on the triterpenoid ring system) were determined by further
inspection of NMR chemical shift and coupling-constant
values (Fig. 2). A search of the literature revealed that compound
3a is the known compound soyasapogenol B (8), previously
isolated from soybean.
The second component (a white amorphous powder)
isolated from the hydrolysate of 4 was identified as compound
3 by comparison of their mass spectra (C36H58O9), NMR
spectra, and TLC traces. The NMR spectra of 3 indicated that
the genin portion of this compound has the same structure
as 3a. However, the spectra showed that compounds 3 and
3a differ by the presence of six additional carbon atoms in 3
and a concomitant increase of 176 in mass number. Infrared
and NMR data also indicated the presence in 3 of a COOH
group (at 1716 cm1 and 175.3 ppm, respectively). These
features indicated that compound 3 is the monoside of genin
3a, glycosylated by glucuronic acid. The place of attachment
of the glucuronic acid at C-3 of the genin was determined
by a long-range NMR coupling experiment (HMBC), and
coupling constant data confirmed that the conformation of
this sugar derivative is -D-glucuronic acid. Hence, the hydrolysis
of compound 4 produced both compound 3 and the genin 3a.
Compounds 3 and 4 differed by 132 mass units,
corresponding to a pentose sugar unit. The presence of five
additional carbon atoms in the 13C NMR spectrum of 4
confirmed this, and the pentose moiety was identified as xylose
from its 13C chemical shift values. Analysis of the long-range
NMR coupling constants showed that the xylose was attached
to C-2 of the glucopyranosiduronic acid of compound 3.
The three other saponin compounds (5, 6, and 7) isolated
from the butanol extract of the plant were all based on the
same genin, 3a. Compound 5 contained two sugar groups
and compounds 6 and 7 each contained three sugars. The
structures of all the saponins are shown in Figure 2. Table 1
summarizes the physical properties of all the compounds isolated from A. danicus. None of these saponins were active in
our bioassays, although similar oleanene glucuronides have
shown hepatoprotective activity (9).
Conclusions
The example cited above is among the more difficult cases
encountered, but it illustrates the approach and techniques involved in the isolation and structural elucidation of secondary
plant metabolites. The student involved in this work was well
supervised, but other less involved projects have been successfully completed by students with minimal outside help.

Many positive attributes are associated with a project

such as this one. Some of these are summarized below.
1. Discovering what chemicals are found in medicinally
important plants is an exciting adventure to most
students, and their enthusiasm for this kind of project
contributes significantly to their success.
2. The project involves a multidisciplinary approach, and
students benefit from exposure to a wide range of
analytical and bioorganic techniques.
3. This kind of project allows students to carry out more
sophisticated spectral studies in a context that they find
interesting and encouraging to independent learning.
4. Students are introduced to the discovery processes for
new and potentially important medicines.

Acknowledgments
We wish to thank David M. Grant of the University of
Utah for his encouragement and financial support of this work,
and also Brigham Young University for financial assistance.
Literature Cited
1. Duke, J. A. CRC Handbook of Medicinal Herbs; CRC Press:
Boca Raton, FL, 1985; Handbook of Biologically Active
Phytochemicals and Their Activities; CRC Press: Boca Raton,
FL, 1992. Society for Medicinal Plant Research; http://
www.uni-duesseldorf.de/WWW/GA/inhalt.htm. Agricultural
Research Service. Dr. Dukes Phytochemical and Ethnobotanical Databases; http://www.ars-grin.gov/duke/ (accessed May
2001).
2. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenny, S.; Boyd, M. R.
J. Natl. Cancer Institute 1990, 82, 11071112.
3. Donaldson, J. R. A New Approach: Comparing Ethnobotany
and Chemical Ecology Approach in the Search for Medicinal
Plants; Masters Thesis, Department of Botany, Brigham Young
University April 2000.
4. Beilstein 1961, Suppl. Series 3, Vol. 2, p 1508.
5. Raychhowdhury, M. K.; Goswami, R.; Chakrabarti, P. J. Appl.
Bacteriol. 1985, 59, 183.
6. Sagar, P. S.; Das, U. N.; Koratkar, R.; Ramesh, G.; Padma, M.;
Kumar G. S. Cancer Lett. 1992, 63, 189.
7. Ramesh, G.; Das, U. N. Cancer Lett. 1998, 123, 207.
8. Kitagawa, I.; Yoshikawa, M.; Yosioka, I. Chem. Pharm. Bull.
1976, 24, 121.
9. Kinjo, J.; Aoki, K.; Okawa, M.; Shii, Y.; Hirakawa, T.; Nohara, T.;
Nakajima, Y.; Yamazaki, T.; Hosono, T.; Someya, M.; Niiho, Y.;
Kurashige, T. Chem. Pharm. Bull. 1999, 47, 708.
10. Beilstein 1967, Suppl. Series 3, Vol. 6, p 6927.
11. Fukunaga T.; Nishiya, K.; Takeya, T. Chem. Pharm. Bull. 1987,
35, 1610.
12. Kitagawa, I.; Wang, H. K.; Yoshikawa, M. Chem. Pharm. Bull.
1983, 31, 716.
13. Mahato, S. B. Phytochemistry 1991, 30, 3389.

JChemEd.chem.wisc.edu Vol. 78 No. 9 September 2001 Journal of Chemical Education

1237