Reprinted from LIPIDS, November, 1969, Vol. 4, No.
6, Pages: 450-453
New Sources of 9-D-Hydroxy-cis-12-octadecenoic Acid 1
R. G. POWELL, R. KLEIMAN and C. R. SMITH, JR.,
Northern Regional Research Laboratory, 2 Peoria, Illinois 61604
ABSTRACT
9-D -Hydroxy-cis-12-octadecenoic acid
has been isolated from 3 seed oils of the
family Apocynaceae: Holarrhena anti-
dysenterica (73%), Nerium oleander
(II %) and Nerium indicum (8%). The
known occurrence of this acid was
previously limited to the genus
Strophanthus (9-15%). A mixture of
unusual tetra-acid glycerides was isolated
from N. oleander oil by thin layer chro-
matography. Pancreatic lipase hydrolysis
of the glyceride mixture showed that
9 -D-acetoxy-cis-12-octadecenoic acid is
esterified exclusively at an Cl'-glycerol
position and that normal fatty acids
occupy the remaining 2 glycerol posi-
tions. A portion of the hydroxy acid in
N. indicum oil was also acetylated, how-
ever. no acetate was found in either H.
antidysenterica or Strophanthus hispidus
oils.
INTRODUCTION
Gunstone (1) first reported the occurrence
of 9-hydroxy-cis-12-octadecenoic acid in
Strophanthus sarmentosus seed oil and later
showed that it probably is of general occur-
rence (9-15%) in seed oils of the genus
Strophanthus, family Apocynaceae (2). The
structure has been confirmed by synthesis (3)
a nd the naturally occurring "Strophanthus
acid" has been assigned the D configuration (4).
The Strophanthus acid may also be obtained by
partial diimide reduction of naturally occ~rring
9 -D -h y d r 0 xy t ra m-I O,cis-I 2-octadecad!enOlC
acid (5,6).
Strophanthus oils are convenient sources of
min or amounts of 9-D -hydroxy-cis-12-octa-
decenoic acid. however. the low hydroxy acid
content and u'navailability of the oils suggested
a search for additional sources among the
closely related genera Holarrhena and Nerium.
In tltis paper we report the characterization
of 9-D-hydroxy-cis-12-octadecenoic acid from 3
new sources. One, Holarrhena antidysenterica
(Roth) Wall ex. DC. seed oil, contains this acid
as the major component (73%). We also report
the characterization of an unusual tetra-acid
! Presented at the AOCS Meeting, San Francisco,
April, 1969.
2No. Utiliz. Res. Dev. Div., ARS, USDA.
[I]
gl V cerid e mix t ure isolated from Nerium
;l~ander L. seed oil.
PROCEDURES AND DATA
Infrared (IR) spectra were determined on a
Perkin-Elmer 137 instrument as liquid films or
as I % solutions in carbon tetrachloride or car-
bon disulfide. Nuclear magnetic resonance
(NMR) spectra were recorded on Varian A-60
or HA-IOO spectrometers with deuteriochloro-
form solutions containing 1% tetramethylsilane
as an internal standard (unless otherwise indi-
ca ted). Optical rotatory dispersion (ORO)
measurements were made on a Cary Model 60
recording spectropolarimeter at 26° in absolute
methanol and in 0.5 dm cells. Melting points
were determined with a Fisher-Johns melting
point apparatus and are uncorrected ..
Thin layer chromatography (TLC) was per-
formed on plates coated with 0.25 mm or 1.0
mm layers of Silica Gel G or 20% silver nitrate-
impregnated Silica Gel G with 20% or 30%
ether in hexane as the solvent. Bonc ac!d-
impregnated plates were used for analysis of
lipolysis products. Spots on analytical plates
were visualized by charring with sulfuric acid-
chromic acid. After preparative plates were
sprayed with dichlorofluorescein, bands were
visualized under ultraviolet light.
Gas liquid chromatography (GLC) of methyl
esters was conducted as described by Miwa et
al. (7). Triglycerides were analyzed on an F &M
Model 5750 instrument equipped with a 24 X
1/8 in. metal column packed with 3% OV-l.
The instrument was linearly temperature-pro-
grammed at 4 deg/min from 200-400 C.
Oxidative cleavages were according to the
method of von Rudloff (8). Iodine values were
determined by the AOCS method (9). Pan-
creatic lipase hydrolyses, silylations and subse-
quent GLC of silylated lipolysis products were
as described by Tallent and Kleiman (10).
Isolation and Purification of Hydroxy Acids
Seeds were ground and extracted overnight
in a Soxhlet apparatus with petroleum ether
(30-60 C); solvent was removed from the oils
on a rotary evaporator. Both Nerium oleander
and Naium indicum oils exhibited bands in the
IR at 1235 cm-! and 1020 cm-! (acetate)
which were not present in spectra of H. anti-
dvsenterica or Strophanthus hispidus oils.
. Methyl esters were prepared by refluxing the
oils for 2 hr with 5% hydrochloric acid in
 9-HYOROXY-CIS-OCTAOECENOIC ACID

anhydrous methanol. Esters were recovered by ...'" <r. >0

~ N N ".; ei
ether extraction and were analyzed by GLC. Oil is
yield:>, iodine values and analyses of the methyl
esters by GLC are recorded in Table 1.
Concentrates of the hydroxy esters were ob- >,
tained by preparative TLC. The concentrates ~ '"
2 - ~
,...,
>0 r-:t"- ,...,o
were saponified by refluxing for 1 hr with 1N
potassium hydroxide in ethanol, and unsaponi-
U
-l
v
>,
"'0
>,-
::
" t"-
0
-
fiables were removed by extracting dilute ,:;;;

.s'"
aqueous solutions of the soaps with ether. After en
acidification, the free hydroxy acids were
'~"
,...,
"":
,..., N
-
"- -
recovered by ether extraction. The acids were 0 0
esterified with diazomethane, and the resulting '"'""
esters were purified by preparative TLC. Overall 2
yields were near 80% in each instance (based on .5
" r-:<r. .d-.'"
N
GLC of the total esters). ~
~ "- t"- t"-
<t N

~
Characterization of Hydroxy Acids
'"'"
Hydroxy acid methyl esters from the 4 oils >,
r-:t"- -<i" Nc: :xi0
(H. antidysenterica, N. oleander, N. illdicum, ,...,
"
...E'"
N N
and S. hispidus) were indistinguishable by IR ::g
and NMR. The spectra were consistent with a 0 0

''"" :~=
"'0
C 1 8 methyl ester having 1 hydroxyl and 1 cis
double bond. GLC of the esters gave single CIl
0
r-: r-:
<t
>0
<r.
-
:xi
peaks, > 98%, for each and retention character- '"" 0 " N

istics of all 4 esters were identical. The esters

'"
<.>
g"'"
~
>, U
showed single spots which were not differen- <.>
0
tiated by TLC on either ordinary or silver- OO
~ i
nitrate impregnated plates. J "'0
Samples of hydroxy ester from each of the 4 :0 ~
«: <.>
oils were cleaved oxidatively and methyl esters -;'" o
of the products were prepared by refluxing ""' CIl

with acidic methanol. The IR spectrum of each ~

oxidation product had a strong band at 1773 .8

cm- l characteristic of a 'Y-lactone, and GLC 8
::;
showed methyl hexanoate and a fragment cor- .:
responding to a C l2 'Y-lactone-methyl ester (5). E- ~
.::
~"
Thus, the hydroxy acids from all 4 sources are ~
C l8 acids having hydroxyl at C9 and a cis ~
double bond at C1 2. ORD curves were also ob- :;
tained for each of the 4 methyl ester samples .£
(Table II).
Hydrogenation of the hydroxy ester from H.
anti dysenterica, with palladium-charcoal in
hexane, gave methyl 9-hydroxyoctadecanoate. t""-~-oo

After preparative TLC and recrystallization ~~I.CiM

M~('.ll""'l

from hexane, the saturated ester melted at

51-52 C and had a plain negative ORD curve
(Table II). Known methyl 9-o-hydroxyocta-
decanoate gives a similar melting point,
51.5-52.8 C, and a nearly superimposable plain
negative ORD curve (11).
A 156 mg portion of methyl 9-hydroxy-cis-
12-octadecenoate, from Holarrhena oil, was
acetylated by allowing to stand overnight in a
mixture of pyridine-acetic anhydride (1: I).
Column chromatography on 2 g of neutral
alumina (hexane) yielded 176 mg of product.
The product gave IR and NMR spectra which
[2 ]
 R. G. POWELL, R. KLEIMAN AND C. R. SMITH, JR.
TABLE II
Optical Rotatory Dispersion of Hydroxy Esters
Ester and source
Methyl 9-0-hydroxy-cis-12-octadecenoate
Holarrhena antidysenterica
Nerium oleander
Nerium indicum
Strophan thus lzispidus
Strophanthus kombe a
Methyl 9-0-hydroxyoctadecanoate
Hydrogenated Holan-hena ester
Hydrogenated methyl dimorphecolate a
aSpecific rotations of these materials were obtained by Applewhite et aJ. (11) and are
listed here for comparison.
were consistent with those expected for methyl
9-acetoxY-cis-12-octadecenoate. GLC and TLC
showed the product to be homogeneous.
Glyceride Characterization
An unknown component was observed upon
TLC analysis of N. oleander oil, which had an
Rf slightly lower than normal triglycerides.
GLC of the oil indicated that acetotriglycerides
and triterpene acetates were absent because no
significant components with carbon numbers
less than C52 were observed. A significant peak
was eluted as Cs 6, however, even though only
2% C20 esters were found in the ester analysis.
The presence of glycerides containing an
acetylated hydroxy acid constituent was indi-
cated by a conspicuous 1235 cm- 1 (acetate)
band in the IR; only weak hydroxyl absorption
was present.
IR of the unusual component, isolated by
preparative TLC, showed a strong 1235 cm- 1
(acetate) band and no hydroxyl. A sharp singlet
was present in the NMR spectrum at 7 8.1
(CC1 4 solution) characteristic of an acetoxy
group. A multiplet was evident at 75.3, due to
a single proton, and a similar multiplet was
noted in the NMR spectrum of methyl
9-acetoxy-12-octadecenoate. This signal was
assigned to the proton on a carbon bearing an
acetoxy group (C 9 ). A multiplet, equivalent to
4 protons, was present at 7 5.9, indicative of
protons on the outer carbons of a glycerol
moiety. Except for the signals at 7 8.1 and 7
5.3, the spectrum was otherwise consistent with
that of a glyceride. This unusual component,
when analyzed by GLC, gave peaks having car-
bon numbers of C S2 , C S4 and CS6 '
After the unusual glyceride mixture was sub-
jected to pancreatic lipase hydrolysis, a product
was isolated that migrated between normal free
acids and diglycerides on TLC. This product
had an IR band at 1705 cm- l , associated with
Specific rotation in degrees
Concentration,
[a] 589 [a1500 [a]400 g/100 ml
-0.83 -1.18 -2.00 2.88
-1.76 -2.30 -3.20 1.13
-1.92 -2.34 -3.40 0.95
-1.87 -2.48 -4.12 2.67
-1.17 -1.76 -2.54 1.02
-0.12 -0.16 -0.30 2.29
-0.17 -0.23 -0.37 1.49
free carboxylic acid, but also had bands at 1724
cm- 1 and 1235 cm- l , characteristic of an
acetoxy derivative. Treatment with diazo-
methane gave the methyl ester which was iden-
tical with known methyl 9-acetoxY-cis-12-octa-
decenoate, as demonstrated by IR, NMR, GLC
and TLC.
A portion of lipolysate of the unusual
glyceride mixture was silylated, and GLC
showed no monoglycerides with acetoxy func-
tional groups. Similar treatment of a lipolysate
of the original oil showed that 5% of the mono-
glycerides contained acyl groups with hydroxy
or acetoxy functions, or both. A portion of the
whole oil lipolysate was treated with diazo-
methane and subsequent GLC demonstrated
that 12% acetoxy and 2% hydroxy methyl
esters were present in the mixture. Thus,
assuming no acyl migration during lipolysis, all
the acetoxyacyl and a portion of the hydroxy-
acyl groups are esterified on the outer glycerol
carbons.
DISCUSSION
The hydroxy acids of H. antidysenterica, N.
oleander, N. indicum and S. hispidus are clearly
identical, and experimental data are consistent
with only one structure: 9-0-hydroxy-cis-12-
octadecenoic acid. The presence of this acid in
S. hispidus oil was demonstrated previously
(12). Assignment of the 0 configuration was
possible as these 4 esters and known methyl
9-0 -hydroxy-cis-12-octadecenoate (11) all gave
plain negative ORD curves that were nearly
superimposable. Confirmation of this assign-
ment was obtained by hydrogenating the
Holarrhena ester; the product gave an ORD
curve essentially the same as that for known
methyl 9-D-hydroxyoctadecanoate (4,11).
Gunstone (1) found that methyl 9-hydroxy-
l2-octadecenoate gives abnormally high iodine
[3]
 9-HYDROXY-CIS-OCT ADECENOIC ACID
values, apparently because of involvement of
the reagent with the hydroxyl group. Similarly,
the iodine value determined for Holarrhena oil
(Table I) is considerably higher than that calcu-
lated from the fatty acid composition. More
satisfactory results may be obtained after
acetylation (1) and the Nerium oils do not
show this discrepancy.
Since pancreatic lipase (EC 3.1.1.3) displays
a strong specificity for the 1 and 3 (or Q and Q')
positions of triglycerides (13), lipolysis with
this enzyme has been widely used to determine
the intraglyceride distribution of various acyl
groups. In Strophanthus oils 9-hydroxy-12-
octadecenoic acid shows strong preference for
the 2 (or (3) glycerol position, and there is no
evidence for triglycerides with more than 3 acyl
groups (14). Since lipolysis of the mixture of
tetra-acid glycerides from N. oleander gave no
monoglycerides with acetoxy functional
gr 0 ups, evidently 9-acetoxy-12-octadecenoic
acid occurs exclusively at I (or both) of the Q
glyceroi positions. However, 9-hydroxy-12-
octadecenoic acid is also present, as shown by
lipolysis of the whole oil, and the major portion
of this acid occurs at the (3 glycerol position.
The tetra-acid glycerides of N. oleander, con-
taining an acetylated monohydroxy acid, are
apparently unique although similar tetra-acid
(I5) and penta-acid (16) glycerides having
acetylated di- or trihydroxy acids are also
known.
The Holarrhena alkaloids are considered
possible starting materials for industrial prepa-
ration of steroid hormones, including the
adrenocortical hormone aldosterone (17). Oils
from other species of Holarrhena may prove
equally rich in 9-hydroxy-12-octadecenoic acid,
and moderate quantities of the oils could
become available as by-products of the pharm-
aceutical industry. At present, H. anti-
dysenterica seed oil is, by far, the richest
known source of 9-D-hydroxy-cis-12-octa-
decenoic acid.
[4 ]
ACKNOWLEDGMENTS
Preliminary screening results and G LC analyses by
F. R. Earle, J. W. Hagemann and R. W. Miller; NMR
spectra by L. W. Tjarks; Holarrhena and Nerium seeds
were furnished by Q. Jones, USDA, Beltsville, Md ..
and the Strophanthus seeds by F. D. Gunstone, Uni-
versity of St. Andrews, Scotland.
REFERENCES
J. Gunstone, F. D.. J. Chern. Soc., 1274-1278
(1952).
2. Gunstone, F. D.. and L. J. Morris. J. Sci. Food
Agr. 10:522-526 (1959).
3. Kennedy, J., A. Lewis, N. J. McCorkindale and R.
A. Raphael, J. Chern. Soc., 4945-4948 (1961).
4. Schroepfer, G. J., Jr., and K. Bloch, J. BioI.
Chern. 240:54-63 (1965).
5. Powell, R. G .. C. R. Smith, Jr. and I. A. Wolff, J.
Org. Chern. 32:1442-1446 (1967).
6. Badami, R. C., and L. J. Morris, JAOCS
42:1119-1121 (1965).
7. Miwa, T. K., K. L. Mikolajczak, F. R. Earle and I.
A. Wolff, Anal. Chern. 32: 1739-1 742 (1960).
8. Rudloff, E. von, Can. J. Chern. 34:1413-1418
(1956).
9. American Oil Chemists' Society. "Official and
Tentative Methods of Analysis," Chicago, 1957.
10. Tallent, W. H., and R. Kleiman, J. Lipid Res.
9: 146-148 (1968).
1 J. Applewhite, T. H., R. G. Binder and W. Gaffield,
J. Org. Chern. 32: 1173-11 78 (1967).
12. Gunstone, F. D., 1. Sci. Food Agr. 4:129-132
(1953).
13. Entressangles, B., H. Sari and P. Desnuelle, Bio-
chim. Biophys. Acta 125:597-600 (1966).
14. Gunstone, F. D., and M. I. Qureshi, J. Sci. Food
Agr. 19:386-388 (1968).
IS. Mikolajczak, K. L., C. R. Smith, Jr. and I. A.
Wolff, Lipids 3:215-220 (1968).
16. Mikolajczak, K. L., and C. R. Smith, Jr., Biochim.
Biophys. Acta 152:244-254 (1968).
17. Cerny, V., and F. Sorm, "The Alkaloids," Vol. 9,
Edited by R. H. F. Manske, Academic Press, New
York, 1967, p. 305.
[Received April 3, 1969]