Identification of three triterpenoids in almond hulls

Article abstract of DOI:10.1021/jf9908289

Three triterpenoids, betulinic acid, oleanolic acid, and ursolic acid, were isolated as their methyl esters (treatment with diazomethane) from diethyl ether extracts of almond hulls (Nonpareil variety) using flash chromatography and preparative high-performance liquid chromatography. The triterpenoids, which comprised ~1% of the hulls, were characterized using chromatographic and spectroscopic methods. These studies demonstrate that almond hulls are a rich source of these triterpenoids, which have reported anti-inflammatory, anti-HIV, and anti-cancer activities.

Full text of DOI:10.1021/jf9908289

J. Agric. Food Chem. 2000, 48, 3437−3439
3437
Id en tifica tion of Th r ee Tr iter p en oid s in Alm on d Hu lls
Gary Takeoka,* Lan Dao, Roy Teranishi, Rosalind Wong, Stephan Flessa, Leslie Harden, and
Richard Edwards
Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture,
800 Buchanan Street, Albany, California 94710
Three triterpenoids, betulinic acid, oleanolic acid, and ursolic acid, were isolated as their methyl
esters (treatment with diazomethane) from diethyl ether extracts of almond hulls (Nonpareil variety)
using flash chromatography and preparative high-performance liquid chromatography. The triter-
penoids, which comprised ∼1% of the hulls, were characterized using chromatographic and
spectroscopic methods. These studies demonstrate that almond hulls are a rich source of these
triterpenoids, which have reported anti-inflammatory, anti-HIV, and anti-cancer activities.
Keyw or d s: Betulinic acid; oleanolic acid; ursolic acid
INTRODUCTION
to act as a selective inhibitor of human melanoma in
cell culture and animal models that function by induc-
tion of apoptosis (Pisha et al., 1995). Preclinical develop-
ment is currently being conducted to explore the poten-
tial use of betulinic acid for the treatment or prevention
of human melanoma. In our continuing search for novel
phytonutrients in agricultural products, we examined
the chemical composition of almond hulls to characterize
the unknown triterpenoid(s) and other constituents.
Almonds [Prunus dulcis (Mill.) D.A. Webb] are a
major California commodity, generating $858 million in
gross sales in 1995. The annual production of almond
hulls is currently in excess of 600000 tons (1.2 billion
pounds), with this product mainly used as livestock feed.
Previous studies by Buttery et al. (1980) had revealed
that direct extraction of almond hulls with diethyl ether
gave a white solid (1-1.5% of the hulls) having mass
and infrared spectra quite similar to those of oleanolic
acid. Triterpenoids such as oleanolic acid and ursolic
acid occur especially in the waxy coatings of leaves and
on fruits such as apple and pear and may serve as insect
antifeedants and antimicrobial agents (Harborne, 1998).
Although triterpenoids have had rather limited medici-
nal use, recent studies indicate their great potential as
drugs (Mahato et al., 1992). Betulinic acid has been
shown to exhibit significant anti-HIV activity, inhibiting
HIV replication in H9 lymphocytes with an EC50 value
(concentration of the test sample that was able to
suppress HIV replication by 50%) of 1.4 mM, whereas
its IC50 (concentration of test sample that was toxic to
50% of the mock-infected cells) for inhibiting uninfected
H9 cell growth was 13 mM [therapeutic index (TI ) 9.3);
defined as toxicity (IC50) divided by anti-HIV activity
(EC50)] (Fujioka et al., 1994). Similarly, oleanolic acid
was found to inhibit HIV-1 replication in acutely
infected H9 cells with an EC50 value of 1.7 mg/mL while
inhibiting H9 cell growth with an IC50 value of 21.8 mg/
mL (Kashiwada et al., 1998). These same researchers
(Kashiwada et al., 1998) also reported the anti-HIV
activity of ursolic acid, which had an EC50 value of 2.0
mg/mL while exhibiting slight toxicity (IC50 ) 6.5 mg/
mL, TI ) 3.3). Ursolic acid has also shown significant
cytotoxicity in the lymphocytic leukemia cells P-388
(ED50 ) 3.15 mg/mL) and L-1210 (ED50 ) 4.00 mg/mL)
as well as the human lung carcinoma cell A-549 (ED50
) 4.00 mg/mL) (Lee et al., 1988). Oleanolic acid has been
proposed as an anti-inflammatory and antiarthritic
agent (Singh et al., 1994). Betulinic acid has been shown
EXPERIMENTAL PROCEDURES
Ma ter ia ls. Almond hulls were supplied by the Northern
Merced Hulling Association (Ballico, CA). Oleanolic acid was
isolated from olives, and ursolic acid was isolated from apple
peel. Betulinic acid methyl ester was obtained from Indofine
Chemical Co., Inc. (Somerville, NJ ). HPLC grade methanol was
supplied by Fisher Scientific (Fair Lawn, NJ ).
Extr a ction . Dried almond hulls were ground in a Wiley
mill to pass a 6.4 mm screen. The ground hulls were extracted
with freshly distilled diethyl ether [containing ∼0.001% Ethyl
antioxidant 330 (1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-
hydroxybenzyl)benzene)] in a Soxhlet extractor.
Meth yla tion . A portion of the white solid obtained from
the diethyl ether extraction (1.97 g) was treated with CH2N2
in diethyl ether; the methylation products were further puri-
fied by flash chromatography and by HPLC. Oleanolic acid
(0.31 g) and ursolic acid (0.10 g) were treated with CH2N2 in
diethyl ether and purified by HPLC. CH2N2 was generated
according to the procedure described by Black (1983).
F la sh Ch r om a togr a p h y. A portion of the methylated
extract (1.21 g) was separated by flash chromatography on a
glass column (33 cm × 2.3 cm i.d.; bed height ) 23 cm) packed
with silica gel (Merck grade 9385, 230-400 mesh). Using
pentane/diethyl ether mixtures, the following fractions were
obtained: fraction 1 (pentane; 150 mL), fraction 2 (95:5 v/v;
150 mL), fraction 3 (70:30 v/v; 150 mL), fraction 4 (50:50 v/v;
150 mL), fraction 5 (30:70 v/v; 150 mL), and fraction 6 (diethyl
ether; 150 mL).
An a lytica l HP LC-MS. An HP 1100 liquid chromatograph
equipped with a manual injector (Rheodyne model 1725) fitted
with a 20 mL sample loop and an HP 1100 diode array detector
(DAD) was coupled with an HP 1100 mass selective detector
(MSD). Atmospheric pressure chemical ionization (APCI) was
utilized with the following mass spectrometer operating condi-
tions: gas temperature, 350 °C, with a nitrogen flow rate of
4.2 L/min; nebulizer pressure, 60 psi; vaporizer temperature,
* Author to whom correspondence should be addressed
[telephone (510) 559-5668; fax (510) 559-5958; e-mail
grt@pw.usda.gov].
10.1021/jf9908289 This article not subject to U.S. Copyright. Published 2000 by the American Chemical Society
Published on Web 07/08/2000

3438 J. Agric. Food Chem., Vol. 48, No. 8, 2000
Takeoka et al.
315 °C; capillary voltage, 2500 V; and fragmentor, 60 V. The
column was a 250 mm × 4.6 mm i.d. Luna C18(2) (particle
size ) 5 mm; Phenomenex, Torrance, CA) equipped with a
Supelguard LC-18 guard column (2 cm × 2.1 mm i.d.; Supelco,
Bellefonte, PA). Methanol was used as the mobile phase at a
flow rate of 0.7 mL/min. The instrument was controlled and
the data were processed by an HP ChemStation (rev. A.06.01
[403]).
P r ep a r a tive HP LC. The preparative HPLC system con-
sisted of Gilson model 305 and 306 pumps, a Gilson 806
manometric module, a Gilson 811C dynamic mixer (Gilson
Medical Electronics, Inc., Middleton, WI), a manual injector
(Rheodyne model 1725) fitted with a 900 µL sample loop
(PEEK tubing), and an HP 1047A refractive index detector. A
C18 reversed phase Rainin Dynamax preparative column (250
mm × 21.4 mm i.d., 8 mm, 100 Å; Varian Associates, Walnut
Creek, CA) was directly coupled to a guard column (50 mm ×
21.4 mm i.d.) containing the same packing material. The
mobile phase was 100% methanol. The flow rate was 4.4 mL/
min. The three triterpenoids contained in fraction 4 were
isolated, and their purity was checked by analytical LC-MS.
When necessary, the compounds were rechromatographed
using a mobile phase of 98:2 v/v methanol/water. Oleanolic
acid (obtained from olives) and ursolic acid (obtained from
apple peel) were methylated (diazomethane) and subsequently
purified using the above HPLC conditions.
Ca p illa r y GC. An HP 5890 series II gas chromatograph
equipped with a flame ionization detector (FID) was used. A
DB-1HT fused silica capillary column (30 m × 0.25 mm i.d.;
df ) 0.1 mm) was also used. The linear velocity of helium
carrier gas was 34.4 cm/s (150 °C). The oven temperature was
programmed from 150 °C (4 min isothermal) to 290 °C at 2
°C/min. The injector and detector temperatures were 250 and
310 °C, respectively.
F igu r e 1. Chemical structures of methylated almond hull
triterpenoids.
Ta ble 1. 13C NMR Da ta of Alm on d Hu ll Tr iter p en oid s
(All Mea su r ed in CDCl3)
C
methyl betulinate methyl oleanolatea methyl ursolateb
Ma ss Sp ectr om etr y. The spectra were obtained via DCI
using a VG70/70 mass spectrometer. The ionization energy was
70 eV, and the EI source temperature was 205 °C. Methyl
betulinate had the following mass spectrum: 470 (M+, 19), 452
(32), 411(15), 410 (19), 262 (38), 220 (20), 207 (36), 189 (100).
Methyl oleanolate had the following mass spectrum: 470 (M+,
5), 452 (4), 411 (3), 410 (3), 262 (70), 203 (100), 189 (28). Methyl
ursolate had the following mass spectrum: 470 (M+, 6), 452
(8), 411 (5), 410 (5), 262 (69), 203 (100), 189 (34).
NMR Sp ectr oscop y. NMR spectra were obtained at 298
K from samples in CDCl3 with TMS as an internal standard
on a Bruker model ARX400 spectrometer at a frequency of
100.62 MHz for carbon and 400.13 MHz for proton. One- and
two-dimensional experiments were run for both nuclei. The
number of attached protons for 13C signals was determined
from DEPT90 and DEPT135 assays.
1
2
3
4
5
6
7
8
CH2
CH2
CH
C
CH
CH2
CH2
C
CH
C
38.77 CH2
27.45 CH2
79.00 CH
38.48 CH2
27.24 CH2
79.05 CH
38.67
27.26
79.03
38.77
55.27
18.34
33.01
39.53
47.60
37.00
23.32
125.60
138.17
42.02
28.06
24.26
48.12
52.92
39.08
38.90
30.68
36.66
28.16
15.45
15.63
16.93
23.63
178.05
16.93
21.18
51.43
38.89
C
38.78
C
55.41 CH
18.33 CH2
34.38 CH2
55.28 CH
18.37 CH2
32.72 CH2
40.72
50.60 CH
37.24
C
39.32
47.68 CH
37.08
C
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
C
C
CH2
CH2
CH
20.92 CH2
25.57 CH
23.44 CH2
122.40 CH
38.31
42.43
C
C
143.82
41.69
C
C
C
CH2
CH2
C
CH
CH
30.66 CH2
32.21 CH2
27.74 CH2
23.12 CH2
56.60
C
46.76
C
47.00 CH
49.53 CH2
41.35 CH
45.93 CH
30.71 CH
33.90 CH2
32.42 CH2
28.13 CH3
15.58 CH3
15.32 CH3
16.87 CH3
25.96 CH3
178.29 CdO
33.12 CH3
23.66 CH3
51.52
RESULTS AND DISCUSSION
CH
150.58
C
Soxhlet extraction of almond hulls with diethyl ether
gave a white solid (yield ) 3.04 ( 0.14%). A portion of
the solid (1020 mg) was methylated with diazomethane
prior to fractionation by flash chromatography. Fraction
4 (29.4%, 300 mg) displayed three peaks by capillary
gas chromatography that were subsequently identified
as methyl oleanolate (15.6%, IDB-1 ) 3419), methyl
betulinate (40.9%, IDB-1 ) 3433), and methyl ursolate
(43.5%, IDB-1 ) 3473) (Figure 1). This mixture was
separated by preparative HPLC to yield three purified
triterpenoids, which all had apparent molecular weights
of 470 as evidenced by APCI mass spectrometry. In
contrast to the retention order observed by capillary GC,
the compounds had the following retention order (in
order of increasing retention time) by reversed-phase
(C18) HPLC: methyl betulinate, methyl oleanolate, and
methyl ursolate. The three triterpenoids were identified
by comparison of the Kovats indices, EI mass spectra,
and 1H and 13C NMR spectra (Table 1) with those of
CH2
CH2
CH3
CH3
CH3
CH3
CH3
CdO
CH2
CH3
29.71 CH2
36.99 CH2
28.01 CH3
15.37 CH3
15.98 CH3
16.13 CH3
14.74 CH3
176.67 CdO
109.58 CH3
19.40 CH3
51.25
CO2Me
a
Data are in agreement with that of Seo et al. (1981) with the
exception of the value for C-17, where we suspect there was a
typographical error in the paper. Data are in accordance with
b
that of Seo et al. (1981).
authentic reference standards. The mass spectrum of
methyl betulinate was similar to that reported by
Budzikiewicz et al. (1963), whereas the mass spectra of
methyl oleanolate and methyl ursolate closely matched

Identification of Triterpenoids in Almond Hulls
J. Agric. Food Chem., Vol. 48, No. 8, 2000 3439
Kashiwada, Y.; Wang, H.-K.; Nagao, T.; Kitanaka, S.; Yasuda,
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triterpenoids together comprised ∼1% of the hulls, thus
revealing almond hulls as a rich source of these promis-
ing anti-inflammatory, anti-HIV, and anticancer agents.
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Received for review J uly 26, 1999. Accepted May 21, 2000.
J F9908289