Triterpene glycosides and other polar constituents of shea (Vitellaria paradoxa) kernels and their bioactivities

Article abstract of DOI:10.1016/j.phytochem.2014.09.017

The MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels was investigated for its constituents, and fifteen oleanane-type triterpene acids and glycosides, two steroid glucosides, two pentane-2,4-diol glucosides, seven phenolic compounds

Full text of DOI:10.1016/j.phytochem.2014.09.017

Phytochemistry 108 (2014) 157–170
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triterpene glycosides and other polar constituents of shea
(Vitellaria paradoxa) kernels and their bioactivities
Jie Zhang a, Masahiro Kurita a, Takuro Shinozaki a, Motohiko Ukiya a, Ken Yasukawa b, Naoto Shimizu c,
Harukuni Tokuda d, Eliot T. Masters e, Momoko Akihisa f, Toshihiro Akihisa a,g,
⇑
a College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan
b School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-855, Japan
c Application Center, Agilent Technologies Japan Ltd., 9-1 Takakura-cho, Hachioji-shi, Tokyo 192-0033, Japan
d Graduate School of Medical Science, Kanazawa University, 13-1 Takara-maschi, Kanazawa 920-8640, Japan
e World Agroforestry Centre (ICRAF), Nelson Marlborough Institute of Technology (NMIT), Nelson 7010, New Zealand
f Department of Endocrinology and Metabolism, Medical Hospital of Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
g Akihisa Medical Clinic, 1086-3 Kamo, Sanda-shi, Hyogo 669-1311, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels was investigated for its
constituents, and fifteen oleanane-type triterpene acids and glycosides, two steroid glucosides, two
pentane-2,4-diol glucosides, seven phenolic compounds, and three sugars, were isolated. The structures
of five triterpene glycosides were elucidated on the basis of spectroscopic and chemical methods. Upon
evaluation of the bioactivity of the isolated compounds, it was found that some or most of the compounds
have potent or moderate inhibitory activities against the following: melanogenesis in B16 melanoma
Received 25 April 2014
Accepted 3 September 2014
Available online 21 October 2014
Keywords:
Shea butter
Vitellaria paradoxa
cells induced by
a-melanocyte-stimulating hormone (a-MSH); generation of 1,1-diphenyl-2-pic-
rylhydrazyl (DPPH) radicals, against Epstein–Barr virus early antigen (EBV-EA) activation induced by
12-O-teradecanoylphorbol 13-acetate (TPA) in Raji cells; t TPA-induced inflammation in mice, and
proliferation of one or more of HL-60, A549, AZ521, and SK-BR-3 human cancer cell lines, respectively.
Western blot analysis established that paradoxoside E inhibits melanogenesis by regulation of expression
of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1
(TRP-1) and TRP-2. In addition, tieghemelin A was demonstrated to exhibit cytotoxic activity against
Sapotaceae
Shea kernel
Oleanane-type triterpene glycoside
Antioxidant activity
Anti-inflammatory activity
Melanogenesis-inhibitory activity
Epstein–Barr virus early antigen (EBV-EA)
Cytotoxic activity
A549 cells (IC50 13.5 lM) mainly due to induction of apoptosis by flow cytometry. The extract of defatted
shea kernels and its constituents may be, therefore, valuable as potential antioxidant, anti-inflammatory,
skin-whitening, chemopreventive, and anticancer agents.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
the seed kernel, consisting of an olein fraction and a stearin frac-
tion along with non-saponifiable (non-lipid) compounds. Fraction-
The shea tree [Vitellaria paradoxa C.F. Gaertner; synonyms
Butyrospermum paradoxum (C.F. Gaertn.) Hepper, Butyrospermum
parkii (G. Don) Kotschy; belonging to the Sapotaceae family] is
indigenous to the savanna belt extending across sub-Saharan
Africa north of the equator, ranging from Mali in the west to
Ethiopia and Uganda in the east (di Vincenzo et al., 2005;
Maranz et al., 2004a, 2004b; Masters et al., 2004). The fruit of
the tree is edible and nutritious, while the most widely valued
product of shea tree is shea butter, the edible fat extracted from
ated shea stearin is used primarily as a cocoa butter substitute or
extender in chocolate manufacture (Masters et al., 2004). These
applications are due to properties imparted by the structures of
its component triacylglycerols. In addition, shea butter is increas-
ingly popular as component of skin care products and cosmetic
product formulations, in part due to the unusually high level of
non-saponifiable lipid (NSL) constituents in the fat (Alander,
2004). In order to characterize and quantify the constituents of
shea butter among widely dispersed V. paradoxa populations, the
contents and compositions of triterpene alcohol fractions from
the NSL, and fatty acid, triacylglycerol, and triterpene ester
compositions of the kernel lipids (hexane extracts) from 36 shea
kernel samples from seven sub-Saharan countries were recently
determined (Akihisa et al., 2010c, 2011). In addition, it was
⇑
Corresponding author at: College of Science and Technology, Nihon University,
1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan. Tel.: +81 79 567
0020; fax: +81 79 567 1980.
E-mail address: akihisa_toshihiro@yahoo.co.jp (T. Akihisa).
http://dx.doi.org/10.1016/j.phytochem.2014.09.017
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

158
J. Zhang et al. / Phytochemistry 108 (2014) 157–170
demonstrated that cinnamyl and acetyl triterpene esters isolated
from the kernel fat could be valuable as anti-inflammatory agents
and chemopreventive agents in chemical carcinogenesis (Akihisa
et al., 2010b). From such perspectives, the evaluation of pharmaco-
logical and cosmeceutical potentials of the constituents of defatted
shea kernel were of interest, since there seems to be little indus-
trial utilization of defatted shea kernel (residue), other than as fuel.
Herein, the isolation of oleanane-type triterpene acids and their
glycosides, phenolic compounds, and other polar constituents from
the defatted shea kernel, and the evaluation of bioactivity of the
isolated compounds, are described.
tration, respectively) against TPA (1.0 lg)-induced inflammation in
mice (Table 2). On the other hand, the MeOH extract and the
EtOAc-soluble fraction exhibited moderate cytotoxic activity
against HL-60 (leukemia) cell line (IC50 76.6 and 69.5 l ,
g mlꢀ1
respectively), and the BuOH-soluble fraction exhibited moderate
cytotoxicity against all of the HL-60, A549 (lung), AZ521 (stomach),
and SK-BR-3 (breast) cell lines tested (IC50 43.2–88.0 l
g mlꢀ1).
2.2. Isolation, identification, and structure elucidation
All three fractions from the MeOH extract were subjected to suc-
cessive column chromatography (CC) on Diaion HP-20, silica gel
(SiO2), octadecyl silica gel (ODS), and Sephadex LH-20 columns,
and to reversed-phase HPLC which led to the isolation of five com-
pounds, 17 and 18 (as the tetraacetate derivatives; 17a and 18a,
respectively), 23, 26, and 27, from the EtOAc-soluble fraction, 20
compounds, 1–3, 6–12, 14–16, 19–22, 24, 25, and 28, from the
BuOH-soluble fraction, and four compounds, 4, 5, 29, and 30, from
the H2O-soluble fraction. Among these, five compounds, 1, 2, 8, 9,
and 14, were new, and the 21 known compounds were identified
2. Results and discussion
2.1. Melanogenesis-inhibitory, antioxidant, EBV-EA-induction-
inhibitory, anti-inflammatory, and cytotoxic activities of defatted shea
kernel extracts
Dried and pulverized shea kernels were treated with hexane to
remove the lipid fraction (Akihisa et al., 2010c), and the defatted
kernels were then treated with MeOH in order to isolate the solu-
ble hydrophilic components. The MeOH extract was fractionated
into EtOAc-, n-BuOH-, and H2O-soluble fractions. Extracted frac-
tions were evaluated for melanogenesis-inhibitory and cytotoxic
as tieghemelin A (3), arginine C (5), and 3-O-b-
16 -hydroxyprotobassic acid (7) (Gosse et al., 2002), butyroside D
(4) and 3-O-b- -glucuronopyranosyl protobassic acid (10) (Li
et al., 1994), 3-O-b- -glucuronopyranosyl 16 -hydroxyprotobassic
acid (6) (Gosse et al., 2002; Li et al., 1994), Mi-glycoside I (11) and 3-
O-b- -glucopyranosyl bassic acid (15) (Nigam et al., 1992), protob-
D-glucopyranosyl
a
D
D
a
activities in
a-melanocyte-stimulated hormone (a-MSH)-stimu-
lated B16 melanoma cells, 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical-scavenging activity, inhibitory effects on Epstein–Barr virus
early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol 13-
acetate (TPA) in Raji cells, anti-inflammatory activity against TPA-
induced inflammation in mice, and cytotoxic activity against four
human cancer cell lines by means of a 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, respec-
tively. As compiled in Table 1, the MeOH extract, and the EtOAc-
and BuOH-soluble fractions exhibited potent melanogenesis-inhib-
D
assic acid (12) (Nigam et al., 1992; Sahu, 1996; Toyota et al., 1990),
bassic acid (16) (Sahu, 1996; Toyota et al., 1990), spinasterol 3-O-b-
D-glucopyranoside (17) and 22-dihydrospinasterol 3-O-b-D-gluco-
pyranoside (18) (as the tetraacetate derivatives, 17a and 18a,
respectively) (Furuya et al., 1990; Kojima et al., 1990), (2S,4S)-2-
O-(b-
D-glucopyranosyl)pentane-2,4-diol (19) and (2R,4S)-2-O-(b-
D-glucopyranosyl)pentane-2,4-diol (20) (Hybelbauerová et al.,
2009; Kaneko et al., 1998), isotachioside (22) (Inoshiri et al.,
1987), gallic acid (23) (Dini, 2011), (+)-catechin (24) and (ꢀ)-epicat-
echin (25) (Seto et al., 1997), quercetin (26) (Atta et al., 2011), rutin
(27) (Savage et al., 2011), and proto-quercitol (28) (Machado and
Lopes, 2005; Wacharasindhu et al., 2009), also known as quercitol,
by comparison of MS, 1H NMR, and 13C NMR spectroscopic and opti-
cal rotation data with corresponding literature data (Fig. 1). On the
other hand, three known compounds, arbutin (21), sucrose (29),
and maltose (30), were identified by comparison of their spectro-
scopic signatures against those of reference standards.
The structures of the five new compounds were elucidated on
the basis of spectroscopic data by comparison with literature as
described below, and their proposed structures were supported
by analysis of the DEPT, 1H–1H COSY, HMQC, HMBC, and NOSEY
data.
itory activities (28.2–58.0% melanin content) at 100
centration, which were more potent than that of the reference
arbutin (4-hydroxyphenyl b- -glucopyranoside; 87.1% melanin
content at 100
g mlꢀ1), but with some cytotoxicities (37.9–
l
g mlꢀ1 con-
D
l
63.4% cell viability). The MeOH extract and the three fractions
exhibited potent DPPH free radical-scavenging activities (IC50
6.8–24.3
that of the reference
l
g mlꢀ1) similar to, though slightly less inhibitory than,
a
-tocopherol (IC50 5.6 l
g mlꢀ1; Table 1). Upon
evaluation of the inhibitory effects against TPA (20 ng)-induced
EBV-EA activation in Raji cells, the MeOH extract and the EtOAc-
soluble fraction exhibited potent inhibitory effects (6.9% and 5.3%
induction of EBV-EA at 100 l
g mlꢀ1 concentration, respectively),
while both the MeOH extract and the H2O-soluble fraction showed
inhibitory activity (70% and 81% inhibition at 1.0 mg mlꢀ1 concen-
Table 1
Melanogenesis-inhibitory activities and cytotoxicities in B16 mouse melanoma cell line, and DPPH free-radical- scavenging activities of defatted shea kernel extract.
Extract or fraction
Melanogenesis-inhibitory activity and cytotoxicitya
Melanin content (%) Cell viability (%)
10 10
g mlꢀ1 g mlꢀ1
DPPH Free-radical-scavenging
b
activity, IC50 (l )
g mlꢀ1
l
100
l
g mlꢀ1
l
100 l
g mlꢀ1
Control (100% DMSO)
MeOH extract
100.0 4.2
92.0 6.7
99.2 1.4
100.5 4.6
101.6 4.6
98.7 9.7
100.0 4.2
28.2 5.9
58.0 3.9
48.4 8.7
114.9 6.0
68.9 2.3
100.0 3.1
101.2 3.0
105.0 3.7
101.8 4.6
108.9 6.5
96.5 2.9
100.0 3.1
51.8 8.2
63.4 3.5
37.9 2.3
90.4 4.6
87.1 2.8
6.8 0.8
24.3 0.7
13.2 1.4
6.8 1.5
EtOAc-soluble fraction
BuOH-soluble fraction
H2O-soluble fraction
Arbutinc
a
-Tocopherolc
5.6 0.1
a
Melanin content and cell viability were determined based on the absorbances at 405 , and 570 (test wavelength) – 630 (reference wavelength) nm, respectively, by
comparison with those for DMSO (100%). Each value represents the mean S.D. (n = 3). Concentration of DMSO in the sample solution was 2
l mlꢀ1
l
.
b
Values of fourfold experiments. Concentration of DMSO in the sample solution was 5
Reference compounds.
ll/ml.
c

J. Zhang et al. / Phytochemistry 108 (2014) 157–170
159
Table 2
Percentage of Epstein–Barr virus early antigen (EBV-EA) induction, inhibition of inflammation, and cytotoxicities in human cancer cells of defatted shea kernel extract.
d
Extract or fraction
Percentage EBV-EA inductiona
Inhibition of inflammation
Cytotoxicity, IC50 (l )
g mlꢀ1
Drug conentrationb
(l
g mlꢀ1
)
HL-60
A549
AZ521
SK-BR-3
(Breast)
100
10
1
I.R. (%)c
(Leukemia)
(Lung)
(Stomach)
MeOH extract
6.9 0.4 (60)
5.3 0.5 (60)
14 0.6 (50)
13.0 0.6 (50)
58.6 2.2
51.4 2.1
64.6 2.4
62.9 2.4
100.0 0.5
98.6 0.7
100.0 0.4
100.0 0.5
70 3.7
47 3.6
53 2.5
81 1.5
96 3.3
76.6 6.2
69.5 3.6
64.6 2.4
>100
>100
>100
43.2 1.8
>100
97.3 1.7
>100
60.3 5.9
>100
>100
>100
88.0 3.1
93.8 5.3
EtOAc-soluble fraction
BuOH-soluble fraction
H2O-soluble fraction
Indomethacine
Cisplatine
1.3 0.3
5.5 0.6
2.9 0.2
5.6 0.2
a
b
c
Values represent relative percentages to the positive control value. TPA (32 pmol, 20 ng) = 100%.
Concentrations in terms of weight ratio 20 ngꢀ1 TPA. Values in parentheses are viability percentage of Raji cells.
Percent inhibitory ratio (I.R.) at 1.0 mg earꢀ1
IC50 Value was obtained on the basis of triplicate assay results.
Reference compounds.
.
d
e
The HRESIMS of compound 1 displayed a sodiated molecular
olean-12-en-28-oic acid O-b-
D-xylopyranosyl-(1?4)-a-L-rhamno-
ion at m/z 997.4599 [M+Na]+ consistent with a molecular formula
of C47H74O21. The HRESIMS2 experiment of the [M+Na]+ gave frag-
ments at m/z 821.4281 [(M+Na) – 176]+ (loss of a hexuronic acid),
m/z 719.3605 [(M+Na) – 132 – 146]+ (loss of a diglycosidic chain
comprising one pentose and one deoxyhexose), and m/z
543.3285 [(M+Na) – 132 – 146 – 176]+ (sequential loss of a hexu-
ronic acid). In the 1H NMR spectrum (Table 3) of the aglycone moi-
ety of 1, six tertiary methyl groups, a primary hydroxy methylene,
four secondary oxymethines, and an olefinic methine were
observed, and the 13C NMR spectroscopic data (Table 3) of the agly-
pyranosyl-(1?2)- -arabinopyranosyl ester (paradoxoside B).
a-L
The HRESIMS of compound 8 exhibited a quasi-molecular ion at
m/z 695.3998 [M+H]+, consistent with a molecular formula of
C
37H58O12. The MS2 of the [M+H]+ gave a fragment at m/z
469.3309 [(M+H) – (175 + Me) – 2 ꢁ 18]+ (losses of a methyl hexo-
suronate moiety and 2H2O). The 1H NMR spectrum (Table 4) of the
aglycone moiety of 8 exhibited six tertiary methyl groups, a pri-
mary hydroxy methylene, three secondary oxymethines, and an
olefinic methine, and the 13C NMR spectroscopic data (Table 4) of
the aglycone moiety of 8 were in accord with those of protobassic
acid (12) (Li et al., 1994; Sahu, 1996). Acid hydrolysis of 8 gave D-
glucuronic acid as the sugar and 12 as the aglycone. The above evi-
dence, coupled with the cross-correlations between dH 4.53 [H-1 of
6-O-methyl-b-glucopyranosiduronic acid (b-MeGlcAp) group] and
dC 83.6 (C-3 of the aglycon), and dH 3.77 (MeO of b-MeGlcAp)
and dC 171.3 (C-6 of b-MeGlcAp) observed in the HMBC experi-
cone moiety of 1 were in accord with those of 16a-hydroxyprotob-
assic acid (13) (Nigam et al., 1992; Eskander et al., 2006; Sànchez-
Medina et al., 2009; Tapondjou et al., 2011). In the HMBC spectrum
of 1, long-range correlations were observed between dH 4.50 [H-1
of b-glucopyranosyl (b-GlcAp) group] and dC 83.5 (C-3 of the agly-
con), dH 5.00 [H-1 of
76.0 [C-2 of -arabinopyranosyl (
1 of -Arap) and dC 177.0 (C-28 of the aglycon), which suggested
a-rhamnopyranosyl (a-Rhap) group] and dC
a
a-Arap) group], and dH 5.73 (H-
ments of 8 confirmed that this possesses the structure 3b-[(b-D-
methyl glucuronopyranosyl)oxy]-2b,6b,16a,23-tetrahydroxyole-
a
the substitution patterns of the aglycone by the sugar moieties
assigned as shown in Fig. 1. The coupling constant data (JH–1,H–
an-12-en-28-oic acid (paradoxoside C).
Compound 9 exhibited a [M+Na]+ peak at m/z 851.4427 in the
2 = 3.7 Hz; JH–2,H–3 = 5.0 Hz) for
a
-Arap indicated that it was pres-
HRESIMS, corresponding to a molecular formula of C42H68O16.
ent in the 1C4 conformation, as observed in similar triterpenoid
saponins (Eskander et al., 2005, 2006; Sánchez-Medina et al.,
The MS2 of the [M+Na]+ gave fragment at m/z 689.3777 [(M+Na)
– 162]+ (loss of a terminal hexose moiety). The 1H and 13C NMR
spectroscopic data (Table 4) of the aglycone moiety of 9 were
essentially the same as those of 8, and the 1H NMR spectrum also
showed two anomeric proton signals [dH 4.51 (1H, d, J = 7.8 Hz) and
4.58 (1H, d, J = 7.8 Hz)], along with other resonances due to two
2009). Upon acid hydrolysis, compound 1 afforded
D-glucuronic
acid, -rhamnose, and -arabinose, which were identified by GLC
L
L
analysis of the trimethylsilyl thiazolidine derivatives (Section 4.5),
in addition to compound 13 (Nigam et al., 1992; Eskander et al.,
2006; Sànchez-Medina et al., 2009; Tapondjou et al., 2011). Hence,
glucose moieties. Upon acid hydrolysis, 9 furnished 12 and
cose, demonstrating that 9 possesses 12 as the aglycone moiety
with two -glucosyl units as the sugar moiety. HMBC experiments
D-glu-
the structure of
ranosyl)oxy]-2b,6b,16
O- -rhamnopyranosyl-(1?2)-
doxoside A).
Compound 2 exhibited a [M+Na]+ ion peak at m/z 1129.5011 in
1
was assigned as 3b-[(b-
,23-tetrahydroxyolean-12-en-28-oic acid
-arabinopyranosyl ester (para-
D-glucuronopy-
a
D
a-
L
a
-
L
showed cross-correlations between dH 4.58 [H-1 of the outer
b-glucopyranose (b-Glcp) group] and dC 88.0 (C-3 of the inner
b-Glcp), and dH 4.51 (H-1 of the inner b-Glcp) and dC 83.7 (C-3 of
the HRESIMS, corresponding to a molecular formula of C52H82O25
.
the aglycone). Hence, structure 9 was established as 3b-[(b-
copyranosyl-(1?3)-b- -glucopyranosyl)oxy]-2b,6b,16 ,23-tetra-
hydroxyolean-12-en-28-oic acid (paradoxoside D).
D-glu-
The 1H and 13C NMR spectra (Table 3) of 2 were almost superim-
posable with those of 1 except that the former showed additional
signals of a b-xylopyranosyl (b-Xylp) moiety. Lower-field glycosyl-
ation shifts (+9.5 ppm) (Matsumoto et al., 1990) of the C-4 signal of
D
a
Compound 14 exhibited a [M+H]+ ion at m/z 677.3892 in the
HRESIMS, compatible with the molecular formula C37H56O11. The
MS2 of the [M+H]+ afforded a fragment at m/z 451.3218 ([(M+H)
– (175 + Me) – 2 ꢁ 18]+ (losses of a methyl hexosuronate moiety
and 2H2O). The 1H NMR spectrum (Table 4) of the aglycone moiety
of 14 exhibited six tertiary methyl groups, a primary hydroxy
methylene, two secondary oxymethines, and two olefinic
methines, and the 13C NMR spectroscopic data (Table 4) of the
aglycone moiety of 8 were in accord with those of bassic acid
(16) (Toyota et al., 1990). Acid hydrolysis of 14 gave D-glucuronic
acid as the sugar and 16 as the aglycone. The above evidence,
a-Rhap (dC 83.2) of 2, along with the long-range correlation
between dH 4.53 (H-1 of b-Xylp) and the C-4 resonance of -Rhap
a
in the HMBC spectrum of 2 suggested the substitution patterns
of the aglycone by the sugar moieties as shown in Fig. 1. Acid
hydrolysis of 2 gave
D-glucuronic acid, L-arabinose, L-rhamnose,
and -xylose as the sugar units, and aglycone, 13 (Nigam et al.,
D
1992; Eskander et al., 2006; Sànchez-Medina et al., 2009;
Tapondjou et al., 2011). Hence, the structure of 2 was established
as 3b-[(b-D-glucuronopyranosyl)oxy]-2b,6b,16a,23-tetrahydroxy-

160
J. Zhang et al. / Phytochemistry 108 (2014) 157–170
29
30
20
19
18
21
22
12
17
28
11
9
26
13
14
25
H
COOH
H
COOR"
R'
16
HO
RO
HO
RO
15
1
4
8
7
10 H
2
3
H
5
27
6
H
23
OH
24
OH
OH
14 R = MeGlcA
15 R = Glc
16 R = H
R' R"
OH Rha-(1 2)-Ara-
OH Xyl-(1 4)-Rha-(1 2)-Ara-
R
1 GlcA
2 GlcA
3 GlcA
4 GlcA
5 Glc
6 GlcA
7 Glc
OH Rha-(1 3)-Xyl-(1 2)-Rha-(1 2)-Ara-
OH Api-(1 3)-Xyl-(1 2)-Rha-(1 2)-Ara-
OH Rha-(1 3)-Xyl-(1 2)-Rha-(1 2)-Ara-
OH H
22
H
23
OH H
8 MeGlcA
9 Glc-(1 3)-Glc- H
10 GlcA
11 Glc
12 H
H
H
H
H
H
H
H
H
H
H
H
H
OH
17
GlcO
18 Δ22
H
13 H
O
OH
R
OH
OH
GlcO
GlcO
R
R'
HO
OH
OH
19 R = Me, R' = H
21 R = H
20 R = H, R' = Me
22 R = OMe
23
OH
OH
OH
OH
HO
OH
OH
HO
O
O
O
HO
O
OR
OH
OH
OH
OH
OH
OH
24
25
26 R = H
27 R = Rha-(1 6)-Glc
OH
OH
HO
OH
OH
O
6
4
1
HO
HO
5
O2
O
2 OH
HO
HO
4
5
3
1
HO
3
HO
OH
OH
Ara
28
Api
Glc
ROOC
HO
HO
O
O
HO
HO
Me
HO
O
OH
GlcA R = H
OH
HO
OH
Xyl
Rha
MeGlcA R = Me
Fig. 1. Structures of compounds 1–28.
coupled with the cross-correlations between dH 5.35 (H-1 of b-
MeGlcAp) and dC 81.5 (C-3 of the aglycon), and dH 3.68 (MeO of
b-MeGlcAp) and dC 170.7 (C-6 of b-MeGlcAp) observed in the
HMBC experiments of 14 confirmed that this possesses the struc-
ture 3b-[(b-D-methyl glucuronopyranosyl)oxy]-2b,6b,23-trihydr-
oxyoleana-5,12-dien-28-oic acid (paradoxoside E).
plant parts of the family Sapotaceae (Eskander et al., 2005, 2006;
Gosse et al., 2002; Lavaund et al., 1996; Li et al., 1994; Nigam
et al., 1992; Sahu, 1996; Sànchez-Medina et al., 2009; Tapondjou
et al., 2011). This study seems to be the first, however, for the iso-
lation of compound 10 from a natural source, since this compound
has been reported only as the acid hydrolysis product of butyroside
C, isolated from Madhuca butyracea (Sapotaceae) (Li et al., 1994).
This study has thus established that the defatted shea kernel
extract contains various triterpene glycosides of oleanolic acid
While (2R,4S)-2-O-(b-D-glucopyranosyl)pentane-2,4-diol (20) has
derivatives, i.e., basic acid, protobassic acid, and 16
a
-hydroxypro-
so far been isolated from the fruits of Crescentia cujete (Bignonia-
ceae) (Kaneko et al., 1998) and from the stems of Vaccinium
myritillus (Ericaceae) (Hybelbauerová et al., 2009), its (2S,4S)-iso-
mer (19) has so far been known only as a synthetic compound
tobassic acid, as aglycones, with sugar chains at the C-3 and C-28
positions. It is worth noting that these kinds of triterpene glyco-
sides have been frequently encountered in various species and

J. Zhang et al. / Phytochemistry 108 (2014) 157–170
161
Table 3
1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic data of compounds 1 and 2 isolated from the defatted shea kernel extract.
Position
1 (CD3OD)
dH (J in Hz)
2 (CD3OD)
dH (J in Hz)
dC
dC
Aglycon
1
2
3
4
5
6
7
8
1.19 (br. d, 13.7), 2.10 (br. d, 13.7)
4.34 (br. s)
3.59 (m)
46.8
71.0
83.5
44.0
49.0
68.6
41.2
40.0
48.7
37.2
24.6
124.1
143.9
43.3
36.2
74.5
50.4
42.1
47.5
31.3
36.2
31.6
65.2
16.2
19.2
19.0
27.4
177.0
33.3
25.3
1.18 (br. d, 12.8), 2.09 (br. d, 12.8)
4.31 (br. s)
3.59 (d, 4.6)
46.7
71.4
83.6
44.0
48.9
68.6
41.3
39.9
48.7
37.1
24.5
124.1
143.8
43.3
36.3
74.5
50.3
42.2
47.6
31.3
36.4
31.8
65.1
16.2
19.2
19.0
27.3
177.0
33.4
25.1
1.29 (br. s)
4.47 (br. s)
1.57 (br. d, 14.2), 1.81 (dd, 5.0, 14.2)
1.31 (br. s)
4.48 (m)
1.56 (br. d, 13.3), 1.81 (br. d, 13.3)
9
1.65 (dd, 5.5, 13.7)
1.65 (dd, 5.5, 13.7)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.04 (m), 2.14 (m)
5.43 (br. t, 3.7)
2.03 (m), 2.14 (m)
5.41 (br. t, 3.2)
1.40 (dd, 3.7, 14.7), 1.79 (m)
4.48 (m)
1.43 (br. d, 16.9), 1.80 (m)
4.49 (m)
3.11 (dd, 3.7, 14.2)
1.07 (m), 2.27 (t, 13.7)
3.07 (dd, 3.7, 14.0)
1.06 (m), 2.27 (t, 13.3)
1.15 (m), 1.89 (m)
1.79 (m), 1.92 (m)
3.41 (br. d, 10.1), 3.73 (br. d, 10.1)
1.31 (s)
1.63 (s)
1.07 (s)
1.33 (s)
1.14 (m), 1.90 (m)
1.76 (m), 1.91 (m)
3.43 (br. d, 10.1), 3.71 (br. d, 10.1)
1.31 (s)
1.62 (s)
1.05 (s)
1.34 (s)
0.89 (s)
0.99 (s)
0.88 (s)
0.97 (s)
Sugar moiety
3-O-b-GlcA
1
2
3
4
5
6
4.50 (d, 7.8)
3.35 (m)
3.39 (br. t, 9.6)
3.42 (m)
104.6
75.0
77.9
73.7
76.0
4.51 (d, 7.8)
3.35 (m)
3.33 (m)
3.48 (m)
3.60 (d, 10.1)
105.1
74.9
78.1
71.0
75.9
177.0
3.61 (d, 10.1)
177.0
28-O-a-Ara
1
2
3
4
5
5.73 (d, 3.7)
3.79 (dd, 3.7, 5.0)
3.90 (m)
3.86 (m)
93.7
76.0
70.5
66.5
63.1
5.59 (d, 4.1)
3.81 (dd, 4.1, 5.5)
3.86 (m)
3.82 (m)
94.1
75.3
71.9
67.4
64.3
3.50 (dd, 3.7, 10.9), 3.93 (dd, 8.5, 10.9)
3.52 (dd, 2.8, 11.5), 3.91 (dd, 7.3, 11.5)
2Ara-O-
a-Rha
1
2
3
4
5
6
5.00 (d, 1.4)
3.83 (dd, 1.4, 3.2)
3.64 (dd, 3.2, 9.6)
3.38 (m)
3.68 (m)
1.27 (d, 6.0)
101.7
72.3
72.1
73.7
70.7
18.0
5.12 (br. s)
3.86 (m)
3.86 (m)
3.59 (br. t, 9.2)
3.74 (m)
1.30 (d, 6.0)
101.3
72.3
72.1
83.2
68.9
18.1
4Rha-O-b-Xyl
1
2
3
4
5
4.53 (d, 7.8)
3.34 (m)
3.24 (br. t, 8.2)
3.48 (m)
106.5
74.9
77.7
71.0
67.2
3.20 (dd, 10.0, 11.5), 3.86 (dd, 5.0, 11.5)
(Hybelbauerová et al., 2009), and this study seems to be the first
instance of its isolation from a natural source. It cannot be
excluded that compounds 8 and 14, which are the methyl esters
of the glucuronopyranosyl group in each compound, are artefacts
formed from the corresponding demethyl compounds during the
extraction and isolation procedures – although the latter com-
pounds have not been concomitantly isolated in this study. This
study has proved that the BuOH-soluble fraction of the defatted
shea kernel extract contains proto-quercitol (27) in appreciable
amount (Section 4.4). Whereas 28 is a very abundant deoxy-inosi-
tol in Quercus sp. (Fagaceae) (Rodríguez-Sánchez et al., 2010), this
compound has so far been reported only in Mimsops elengi as a
sapotaceous constituent (Misra and Mitra, 1967). Occurrence of
four phenolic compounds, 23–26, along with other phenolics, has
previously been reported in shea kernels (Maranz et al., 2003).
2.3. Melanogenesis-inhibitory activity
Compounds 1–12 and 14–28 (as the tetraacetate derivatives,
17a and 18a, as for 17 and 18, respectively) were evaluated for
melanogenesis inhibition in
a-MSH (melanocyte-stimulating
hormone)-stimulated B16 melanoma cells (Table 5). The cytotoxic

162
J. Zhang et al. / Phytochemistry 108 (2014) 157–170
Table 4
1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic data of compounds 8, 9, and 14 isolated from the defatted shea kernel extract.
Position
8 (CD3OD)
dH (J in Hz)
9 (CD3OD)
dH (J in Hz)
14 (C5D5N)
dH (J in Hz)
dC
dC
dC
Aglycon
1
1.15 (br. d, 14.2)
2.01 (dd, 1.8, 14.2)
4.24 (br. q, 2.8)
3.59 (d, 3.7)
46.5
1.17 (br. d, 14.2)
2.03 (dd, 1.8, 14.2)
4.34 (br. q, 2.8)
3.59 (d, 3.7)
46.5
1.42 (dd, 3.7, 14.9)
2.14 (dd, 3.7, 14.2)
4.85 (dd, 3.7, 7.3)
4.47 (d, 3.2)
46.5
2
3
4
5
6
7
71.6
83.6
44.0
48.8
68.3
41.0
71.3
83.7
44.0
48.9
68.4
41.1
70.5
81.5
46.7
147.7
120.8
33.1
1.32 (br. s)
4.46 (br. s)
1.50 (br. d, 14.2)
1.78 (dd, 3.2, 14.2)
1.32 (br. s)
4.47 (br. s)
1.50 (br. d, 14.2)
1.78 (dd, 3.2, 14.2)
5.98 (dd, 3.2, 5.0)
2.48 (dd, 5.0, 18.8)
1.76 (dd, 3.2, 18.8)
8
9
10
11
39.6
49.5
37.0
24.5
39.6
49.6
37.1
24.6
38.4
45.9
37.2
24.0
1.60 (m)
1.60 (m)
1.97 (dd, 5.5, 11.0)
1.95 (dt, 4.6, 17.9)
2.11 (ddd, 2.3, 11.5, 17.9)
5.30 (br. t, 3.7)
1.96 (dt, 4.6, 17.9)
2.13 (ddd, 2.3, 11.5, 17.9)
5.30 (br. t, 3.6)
2.04 (m)
2.16 (m)
5.60 (br. t, 3.7)
12
13
14
15
123.8
144.4
43.5
123.9
144.5
43.5
123.0
145.0
43.0
1.07 (br. d, 17.4)
1.84 (br. t, 12.8)
1.60 (br. d, 16.0)
1.99 (m)
28.6
1.08 (br. d, 17.4)
1.84 (br. t, 12.4)
1.61 (br. d, 16.9)
1.98 (m)
28.7
1.26 (m)
2.15 (m)
2.11 (m)
2.19 (m)
27.6
23.5
16
24.0
24.0
17
18
19
48.4
42.7
47.1
48.4
42.8
47.2
46.7
42.4
45.8
2.87 (dd, 3.2, 13.7)
1.14 (br. d, 10.5)
1.69 (t, 13.7)
2.87 (br. d, 13.7)
1.16 (br. d, 10.5)
1.70 (t, 13.7)
3.33 (dd, 3.7, 14.2)
1.80 (br. t, 13.7)
1.29 (br. t, 13.7)
20
21
31.5
34.9
31.6
34.9
30.9
34.2
1.20 (br. d, 13.0)
1.38 (dt, 3.2, 13.7)
1.53 (br. d, 14.2)
1.76 (m)
3.43 (d, 11.5)
3.70 (d, 11.5)
1.31 (s)
1.61 (s)
1.10 (s)
1.13 (s)
1.20 (br. d, 13.7)
1.38 (dt, 3.2, 13.7)
1.53 (br. d, 14.2)
1.76 (m)
3.42 (d, 11.0)
3.73 (d, 11.0)
1.31 (s)
1.62 (s)
1.10 (s)
1.14 (s)
1.20 (m)
1.44 (m)
1.76 (m)
2.07 (m)
4.03 (d, 10.5)
4.52 (d, 10.5)
1.70 (s)
1.71 (s)
1.18 (s)
1.24 (s)
22
23
33.6
65.0
33.6
65.3
33.0
65.5
24
25
26
27
28
29
30
16.2
19.1
18.7
26.6
181.7
33.7
24.0
16.3
19.2
18.7
26.6
181.8
33.8
24.1
23.4
23.7
21.1
26.2
170.7
33.3
23.7
0.91 (s)
0.95 (s)
0.91 (s)
0.95 (s)
0.94 (s)
1.01 (s)
Sugar moiety
3-O-b-MeGlcA or 3-O-b-Glc
1
2
3
4
5
6
4.53 (d, 7.8)
105.5
74.8
77.3
73.0
76.4
171.3
4.51 (d, 7.8)
3.54 (t, 7.8)
3.55 (t, 9.2)
3.51 (t, 7.8)
3.32 (m)
3.73 (dd, 3.7, 11.9)
3.82 (dd, 2.3, 11.9)
104.8
74.6
88.0
69.5
77.2
62.1
5.35 (d, 7.8)
4.04 (t, 9.2)
4.21 (t, 9.2)
4.42 (t, 9.2)
4.53 (d, 9.2)
106.4
75.1
77.6
73.2
77.0
3.38 (t, 7.8)
3.42 (t, 8.7)
3.54 (t, 8.7)
3.90 (d, 9.6)
170.7
MeO-6
3.77 (s)
52.9
3.68 (s)
52.0
3Glc-O-b-Glc
1
2
3
4
5
6
4.58 (d, 7.8)
3.32 (m)
3.40 (t, 8.7)
3.30 (m)
3.32 (m)
3.65 (dd, 5.6, 11.9)
3.91 (dd, 1.8, 11.9)
105.2
75.4
77.7
71.5
78.1
62.6
activities of these compounds against B16 melanoma cells were
also determined by means of MTT (thiazolyl blue tetrazolium bro-
mide) assay. To assess the risk/benefit ratio of each compound, the
relative activities vs. toxicities were calculated by dividing the
melanin content (%), by the cell viability (%), and expressed as an
activity-to-cytotoxicity ratio (A/C ratio) for each compound and
phenolic compounds, 21, 22, and 24–27, and one cyclitol, 28, tested
in this study were proved to be lower-risk melanogenesis inhibi-
tors (22.6–92.1% melanin content, and 72.6–113.9% cell viability)
by exhibiting small A/C ratios (0.31–0.91) at lower and/or higher
concentrations. Among these compounds, 21 is known to be a use-
ful depigmentation compound for skin whitening in the cosmetic
industry (Lim et al., 2009). As far as the oleanolic acid derivatives
tested are concerned, the monodesmosides glycosylated at C-3,
i.e., compounds 6–11, 14, and 15, proved to be more potent
melanogenesis inhibitors than the bisdesmosides glycosylated at
concentration (10, 30, and 100
lM). A compound with smaller A/
C
ratio would be lower-risk skin-whitening agent
a
(Kitdamrongtham et al., 2014). Nine oleanolic acid derivatives,
6–12, 14, and 15, two pentane-2,4-diol glucosides, 19 and 20, six

Table 5
Melanogenesis-inhibitory activities and cytotoxicities in B16 mouse melanoma cell line of compounds isolated from defatted shea kernel extract.a,b
.
Compound
Melanin content (%)
10
100.0 3.1
Cell viability (%)
10
A/C Ratio
10
lM
30
l
M
100
l
M
l
M
30
l
M
100
l
M
l
M
30
lM
100 lM
Controlc
100.0 3.1
100.0 3.1
100.0 0.7
100.0 0.7
100.0 0.7
Triterpene acid and triterpene glycoside
1
2
3
4
5
6
7
8
115.1 2.3
94.2 2.0
91.2 8.4
96.2 3.1
105.3 4.3
84.7 3.1
86.6 4.5
86.5 3.7
89.2 8.0
86.2 2.2
83.9 4.0
98.3 1.8
101.8 2.3
97.5 2.0
64.6 3.0
104.0 5.1
86.8 0.4
90.9 5.6
103.8 2.7
95.6 5.9
70.1 1.9
77.2 2.8
78.1 4.1
83.9 5.2
81.7 4.5
73.8 1.8
78.7 4.2
80.3 6.5
79.5 0.4
23.6 3.7
78.9 7.4
49.7 5.0
84.8 2.4
22.1 2.4
13.4 2.9
47.7 6.3
52.8 1.7
79.6 4.5
75.2 7.0
81.0 1.9
70.7 3.6
21.6 1.6
42.0 4.9
50.2 6.5
10.4 0.8
90.4 6.0
90.2 2.8
94.6 4.5
98.4 5.4
112.4 4.5
93.4 0.1
100.2 0.9
109.8 5.3
110.2 4.2
109.8 5.3
103.2 3.2
105.4 2.4
104.7 4.0
98.6 1.1
81.8 8.0
85.8 4.0
87.9 4.9
83.1 4.8
90.0 5.5
92.7 5.6
84.9 1.6
96.7 0.5
110.6 4.1
113.9 6.0
110.6 4.0
100.5 3.6
104.6 1.6
99.7 3.1
95.9 4.0
56.0 6.0
74.8 2.6
67.8 3.6
67.6 4.5
45.6 7.5
37.3 3.0
85.0 1.3
90.0 1.6
113.8 6.3
112.1 5.8
109.0 4.0
101.1 7.6
69.6 2.4
90.5 3.8
88.2 1.7
29.4 2.2
1.27
1.04
0.96
0.98
0.94
0.91
0.86
0.79
0.81
0.79
0.81
0.93
0.97
0.99
0.79
1.21
0.99
1.09
1.15
1.03
0.83
0.80
0.71
0.74
0.74
0.73
0.75
0.81
0.83
0.42
1.05
0.73
1.25
0.48
0.36
0.56
0.59
0.70
0.67
0.74
0.70
0.31
0.46
0.57
0.35
9
10
11
12
14
15
16
Steroid glucoside
17ad
96.3 0.6
100.9 9.6
96.3 3.2
97.8 2.6
96.6 3.1
84.1 4.3
96.3 2.4
108.5 4.2
92.7 3.9
103.3 2.5
91.0 5.2
101.1 1.9
1.00
0.93
1.04
0.95
1.06
0.83
18ad
Pentane-2,4-diol glucoside
19
20
84.7 1.5
74.3 2.7
72.1 5.6
55.7 3.3
67.4 7.5
42.5 4.0
96.7 5.1
102.4 3.5
92.9 0.7
108.8 4.1
90.5 4.4
107.3 1.7
0.88
0.73
0.78
0.51
0.74
0.40
Phenolic compound
21
22
92.1 1.2
100.5 1.5
92.2 1.8
83.9 8.9
80.9 6.2
53.4 7.1
80.3 4.4
91.7 2.1
89.1 2.0
85.5 7.1
51.9 6.9
72.1 3.2
40.3 1.9
64.9 4.1
72.2 3.7
75.6 2.5
42.7 9.9
22.6 1.4
50.7 5.0
20.2 1.5
48.3 1.4
101.9 6.9
96.1 0.4
95.8 3.6
95.6 2.8
104.9 7.6
104.6 8.2
109.2 5.8
99.9 3.0
99.3 4.4
93.4 1.0
93.2 4.3
97.2 8.2
75.1 3.1
104.3 3.4
82.1 8.1
106.2 1.7
61.6 4.5
72.6 2.4
95.3 4.0
56.9 1.8
98.3 2.9
0.90
1.05
0.96
0.88
0.77
0.51
0.74
0.92
0.90
0.92
0.56
0.74
0.54
0.62
0.88
0.71
0.69
0.31
0.53
0.36
0.49
23e
24f
25f
26
27
Cyclitol
28
95.7 2.6
91.0 4.1
95.4 1.9
107.3 3.5
107.1 5.7
107.7 1.3
0.89
0.85
0.89
a
Melanin content and cell viability were determined at three different compound concentrations based on the absorbances at 405 and 570 (test wavelength) – 630 (reference wavelength) nm, respectively, by comparison with
those for DMSO (100%).
b
Each value represents the mean S.D. (n = 3). Concentration of DMSO in the sample solution was 2 l .
l mlꢀ1
100% DMSO.
17a = Tetraacetate derivative of 17; 18a = Tetraacetate derivative of 18.
Data taken from Manosroi et al., 2013.
c
d
e
f
Data taken from Manosroi et al., 2014.

164
J. Zhang et al. / Phytochemistry 108 (2014) 157–170
C-3 and C-28, i.e., compounds 1–5. Compounds 26 and 27, and
compounds 6–12, 14, 15, 19–22, 24, and 25, might be, at least in
part, responsible for the melanogenesis-inhibitory activities of
the EtOAc- and BuOH-soluble fractions, respectively (Table 1).
2.4. Mechanism of melanogenesis inhibition by compound 14
Tyrosinase, and tyrosinase-related protein-1 (TRP-1) and TRP-2
are enzymes responsible for the synthesis of melanin (Costin and
Hearing, 2007). Regulation of the transcription and activity of these
melanogenic enzymes are effective for depigmentation (Briqanti
et al., 2003). Tyrosinase, a rate-limiting enzyme, catalyzes the
hydroxylation of
L-tyrosine to l-(3,4-dihydroxyphenyl)alanine
(L-DOPA), and the oxidation of L-DOPA to L-DOPA quinone (Levy
et al., 2006). TRP-2 functions as DOPAchrome tautomerase, and
TRP-1 catalyzes oxidation of 5,6-dihydroxy-1H-indole-2-carbox-
ylic acid (DHICA) (Kobayashi et al., 1994). Transcription for expres-
sion of these enzymes is regulated by microphthalmia-associated
transcription factor (MITF) (Levy et al., 2006). To clarify the mech-
anism involved in the melanogenesis inhibition by compound 14,
which exhibited potent melanogenesis inhibitory activity (42.0%
Fig. 2. Effects on the expression of MITF, tyrosinase, TRP-1, and TRP-2 in
stimulated B16 melanoma cells treated with compound 14.
a-MSH-
Table 6
DPPH Free-radical-scavenging activities and inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) of compounds isolated from defatted shea kernel
extract.
M)a
Percentage EBV-EA inductionb
IC50
d
Compound
DPPH Free-radical-scavenging activity, IC50
(l
1000c
500c
100c
10c
Triterpene acid and triterpene glycoside
1
2
3
4
5
6
7
8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9.8 0.5
10.1 0.6
13.6 0.7
13.2 0.6
14.9 .0.7
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
50.4 1.6
48.0 1.4
52.3 1.6
53.1 1.5
54.9 1.5
42.1 1.6
40.6 1.3
45.6 1.6
48.0 1.4
44.9 1.5
43.8 1.5
35.4 1.4
51.6 1.8
49.3 1.5
39.6 1.3
77.3 2.4
77.6 2.8
81.6 2.1
82.0 2.3
83.7 2.0
72.0 2.4
70.3 2.6
76.8 2.7
77.6 2.8
75.1 2.7
73.8 2.6
63.2 2.5
78.8 2.3
77.9 2.5
67.3 2.5
100.0 0.4
100.0 0.3
100.0 0.2
100.0 0.3
100.0 0.2
94.1 0.5
91.6 0.6
97.6 0.5
100.0 0.3
96.7 0.6
95.3 0.5
91.3 0.5
99.6 0.5
98.9 0.5
92.0 0.6
455
456
470
460
479
348
335
368
410
360
353
330
380
371
339
0
0
0
0.4
0.3
0.4
9
6.5 0.6
10
11
12
14
15
16
0
0
0
0.3
0.3
0.2
1.2 0.3
0
0
0.2
0.3
Steroid glucoside
17ae
18ae
>100
>100
9.1 0.6
8.5 0.4
(70)
(70)
47.1 1.5
46.3 1.6
77.7 2.3
76.6 2.5
100.0 0.3
100.0 0.4
457
450
Pentane-2,4-diol glucoside
19
20
>100
>100
10.7 0.6
10.0 0.5
(60)
(60)
47.0 1.6
46.1 1.5
73.2 2.6
72.0 2.4
100.0 0.4
100.0 0.3
459
453
Phenolic compound
21
22
23f
24
25
26
27
>100
12.1 0.7
9.6 0.6
6.4 0.5
2.4 0.3
3.6 0.2
1.9 0.5
10.1 0.8
(60)
(60)
(70)
(60)
(60)
(70)
(70)
49.8 1.4
46.3 1.4
48.1 1.1
39.5 0.2
41.7 0.3
28.6 1.3
48.5 1.4
75.9 2.5
72.6 2.5
74.8 2.3
71.6 0.3
73.2 0.5
64.2 2.4
73.1 2.4
100.0 0.4
100.0 0.5
100.0 0.3
98.0 0.5
98.6 0.2
91.7 0.7
100.0 0.4
456
439
473
352
381
293
451
46.4 6.1
10.9 2.5
7.1 3.2
5.8 2.3
12.9 3.4
6.0 0.3
Cyclitol
28
>100
19.8 0.8
8.6 0.5
(70)
(70)
59.3 1.5
34.2 1.0
89.4 2.1
82.1 2.0
100.0 0.3
100.0 0.3
563
397
Reference compound
a-Tocopherol
13.0 2.3
b-Carotene
a
Each sample was measured in triplicate. IC50 values were determined by the method of probit-graphic interpolation of six concentration levels.
Values represent the relative percentage to the positive control, with TPA (32 pmol, 20 ng) representing 100% induction at four different concentrations in terms of molar
b
ratio 32 pmolꢀ1 TPA. Data are expressed as mean S.D. (n = 3).
c
Values in parentheses are viability percentages of Raji cells.
IC50 represents the molar ratio of compound, relative to TPA, required to inhibit 50% of the positive control activated with 32 pmol TPA.
17a = Tetraacetate derivative of 17; 18a = Tetraacetate derivative of 18.
Data taken from Manosroi et al., 2013.
d
e
f

J. Zhang et al. / Phytochemistry 108 (2014) 157–170
165
Table 7
Inhibition of inflammation in mice and cytotoxic activities of compounds isolated from defatted shea kernel extract.
e
Compound
Inhibition of inflammation
Cytotoxicity, IC50 S.D. (l
M)d,
a
ID50
(l
mol earꢀ1
)
95% CIb
I.R.c (%)
HL-60
A549
AZ521
SK-BR-3
(Breast)
(Leukemia)
(Lung)
(Stomach)
Triterpene acid and triterpene glycoside
1
2
3
4
5
6
7
8
0.18
0.17
0.18
0.12
0.19
0.15–0.21
0.13–0.24
0.14–0.24
0.10–0.13
0.13–0.29
>100
>100
>100
>100
13.5 1.0
19.1 1.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
48.5 3.7
>100
>100
17.9 0.8
77.8 2.0
10.9 1.3
>100
>100
>100
>100
>100
>100
98.2 3.4
>100
>100
86.4 3.6
>100
>100
30.1 0.6
>100
31.4 0.9
>100
>100
>100
>100
>100
>100
19.4 3.2
82.0 5.9
23.0 2.5
15.4 1.8
80.7 0.5
>100
>100
>100
>100
>100
0.07
0.13
0.16
0.13
0.02
0.06–0.08
0.10–0.19
0.13–0.20
0.11–0.15
0.20–0.29
9
10
11
12
14
15
16
72.7 4.3
>100
32.0 1.6
29.7 0.8
7.6 0.1
>100
>100
0.05
0.38
0.03–0.06
0.30–0.47
Phenolic compound
21
22
23
26
27
29
2.5
31 3.9
>100
>100
13.9 7.9
23.3 1.6
>100
>100
>100
>100
>100
>100
>100
>100
29.1 5.5
>100
>100
>100
>100
54.7 7.5
>100
>100
0.94
0.91
0.72–1.24
0.78–1.06
Reference compound
Indomethacin
Cisplatin
0.91
0.76–1.09
96 3.3
4.2 1.1
18.4 1.9
9.5 0.5
18.8 0.6
a
b
c
ID50: 50% Inhibitory dose.
95% Confidence intervals.
Percent inhibitory ratio at 1.0 mg/ear. Each value represents the mean S.D. (n = 5).
d
e
Cells were treated with compounds (1 ꢁ 10ꢀ4 to 1 ꢁ 10ꢀ6 M) for 48 h, and cell viability was analyzed by the MTT assay. IC50 Values based on triplicate five points.
Compounds 17a, 18a, 19, 20, 24, 25, and 28 exhibited IC50 values of >100 lM in all cell lines used.
melanin content) with small A/C ratio (0.46) at 100
lM, the protein
2.6. Inhibitory effects on EBV-EA induction
levels of tyrosinase, TRP-1, TRP-2, and MITF were evaluated in B16
melanoma cells treated with 14 by Western blot analysis. Treatment
of B16 melanoma cells with 14 reduced protein levels of MITF, tyros-
inase, TRP-1, and TRP-2 proteins, mostly in a concentration-depen-
dent manner (Fig. 2). These results suggested that 14 exhibits
melanogenesis inhibitory activity on a a-MSH stimulated B16 mela-
noma cells by, at least in part, inhibiting the expression of MITF, fol-
lowed by decreasing the expression of tyrosinase, TRP-1, and TRP-2.
The inhibitory effects of compounds 1–12 and 14–28 (as the tet-
raacetate derivatives, 17a and 18a, as for 17 and 18, respectively)
against TPA (32 pmol)-induced EBV-EA activation in Raji cells,
together with comparable data for b-carotene, a vitamin A precur-
sor studied widely in cancer chemoprevention animal models, are
compiled in Table 6. Even at a concentration of 32 nmol (molar ratio
of compound to TPA 1000:1), high viability (60–70%) of Raji cells
was observed indicating low cytotoxicity of all compounds. All
compounds showed inhibitory effects with IC50 values (concentra-
tion for 50% inhibition with respect to the positive control) of 293–
563 M ratio 32 pmolꢀ1 TPA. Among these, nine oleanolic acid deriv-
atives, 6–8, 10–12, and 14–16, and three flavonoids without a gly-
cosyl group, 24–26, exhibited potent inhibitory effects (293–380 M
ratio 32 pmolꢀ1 TPA) which were higher than that of b-carotene
(397 M ratio 32 pmolꢀ1 TPA). The bisdesmosides glycosylated at
C-3 and C-28, i.e., 1–5, and the monodesmoside gycosylated at
C-3 with a diglycosyl unit, i.e., 9, of oleanolic acid derivatives
exhibited lower inhibitory effects of EBV induction (IC50 values of
455–479 M ratio 32 pmolꢀ1 TPA) than the monodesmosides glycos-
ylated at C-3 with a monoglycosyl unit, i.e., 6–8, 10, 11, 14, and 15,
and those without a glycosyl group, i.e., 12 and 16. Since the inhib-
itory effects against EBV-EA induction have been demonstrated to
correlate with those against tumor promotion in vivo (Akihisa
et al., 2003; Manosroi et al., 2013), compounds 6–8, 10–12, 14–
16, and 24–26 may be potential inhibitors of tumor promotion.
2.5. DPPH-Radical-scavenging activities
The DPPH-radical-scavenging activities of compounds 1–12 and
14–28 (as the tetraacetate derivatives, 17a and 18a, as for 17 and
18, respectively) are provided in Table 6. Among the compounds
tested, six phenolic compounds, 22–27, exhibited free-radical-
scavenging activities (IC50 5.8–46.4
pounds 23–27 showed strong radical-scavenging activities (IC50
5.8–12.9 M) which were more potent than, or almost equivalent
to, the reference -tocopherol (IC50 13.0 M). This can be
lM), among which, com-
l
a
l
explained by the presence of more phenolic OH groups leading to
higher radical-scavenging activity due to the increase of the H-rad-
ical-donating activity (Bouchet et al., 1998; Manosroi et al., 2010;
Natella et al., 1999). Compounds 23, 26, and 27, and compounds
22, 24, and 25, are considered to be, at least in part, responsible
for the DPPH free-radical-scavenging activities of the EtOAc- and
BuOH-soluble fractions, respectively (Table 1). While no active
ingredient has been isolated from the H2O-soluble fraction in this
study, the presence of various phenolic compounds such as
compounds 21–27 in this fraction is highly probable (Maranz
et al., 2003; Manosroi et al., 2010; Kim et al., 2011), which might
be responsible for the potent DPPH-radical-scavenging activity of
the H2O-soluble fraction (Table 1).
2.7. Anti-inflammatory activity against TPA-induced inflammation in
mice
Ten glycosylated and two unglycosylated oleanolic acid
derivatives, i.e., 1–5, 8–11, and 15, and 13 and 16, respectively,

166
J. Zhang et al. / Phytochemistry 108 (2014) 157–170
and four phenolic compounds, 21, 23, 26, and 27, were evaluated
for their anti-inflammatory activities against TPA-induced inflam-
mation in mice in comparison to indomethacin, a commercially
available anti-inflammatory drug (Table 7). All oleanolic acid
derivatives tested exhibited marked anti-inflammatory activities
A549 (lung), AZ521 (stomach), and SK-BR-3 (breast) by the MTT
assay as compiled in Table 7. While 11 compounds, 3–7, 12, 14–
16, 23, and 26, exhibited potent or moderate cytotoxicities against
one or more cell lines with IC50 values in the range of 7.6–82.0
the other 16 compounds were inactive against all cell lines tested
(IC50 > 100 M). In particular, the cytotoxic activities of 3 and 4
against A549 cell line (IC50 13.5 and 19.1 M, respectively) and
12 against HL-60 cell line (IC50 7.6 M) were more potent than,
or almost comparable with, those of the reference cisplatin [IC50
18.4 M (A549), 4.2 M (HL-60)]. Based on the results compiled
lM,
with the 50% inhibitory doses (ID50) of 0.02–0.38
they were more potent than reference indomethacin (ID50
0.91
mol earꢀ1). Compounds 26 and 27 exhibited anti-inflamma-
tory activities (ID50 0.94 and 0.91
mol earꢀ1, respectively) almost
l
mol earꢀ1, i.e.,
l
l
l
l
l
equivalent to that of reference indomethacin. The anti-inflamma-
tory activity of these compounds has been demonstrated to be
closely parallel with that of the inhibition of DMBA-TPA papilloma
formation in the mouse-skin model (Yasukawa et al., 1996), and
hence the oleanolic acid derivatives tested in this study might be
expected to possess high antitumor-promoting effect in the same
animal model. The high anti-inflammatory activities of various
types of triterpene acids (Akihisa et al., 2007; Banno et al., 2004,
2006; Manosroi et al., 2013) and oleanane-type triterpene glyco-
sides (Manosroi et al., 2013; Ukiya et al., 2006, 2007) have also
been observed in our previous studies.
l
l
in Tables 1 and 7, it is highly possible that two phenolic com-
pounds, 23 and 27, for the EtOAc-soluble fraction, seven oleanolic
acid derivatives, 3, 6, 7, 12, and 14–16, for the BuOH-soluble
fraction, and one oleanolic acid derivative, 5, for the H2O-soluble
fraction are responsible for the cytotoxicities of the fractions,
because these compounds are cytotoxic constituents of the
relevant fractions. With respect to the oleanolic acid derivatives
tested, highly glycosylated bisdesmosides (i.e., 3–5), exhibited, in
general, more potent cytotoxic activities than those with less gly-
cosylated, i.e., 1, 2, 6–11, 14, and 15.
2.8. Cytotoxic activity
2.9. Apoptosis-inducing activity of compound 3
The cytotoxic activities of compounds 1–12 and 14–28 (as the
tetraacetate derivatives, 17a and 18a, as for 17 and 18, respec-
tively), and the reference chemotherapeutic drug, cisplatin, were
evaluated against the human cancer cell lines HL-60 (leukemia),
Compound 3, which exhibited potent cytotoxic activities
against A549 cells (IC50 13.5
lM) was evaluated for its apoptosis-
inducing activity using A549 cells. A549 cells were incubated with
3 (10
lM) for 24 and 48 h, and the cells were subsequently ana-
Compound 3
24
48 (h)
0.7%
4.8%
5.0%
30.8%
13.4%
83.0%
11.6%
50.8%
Negaꢀvecontrol
24
48 (h)
1.4%
1.9%
1.7%
2.0%
2.8%
93.9%
2.8%
93.5%
Annexin V-FITC
Fig. 3. Compound 3 induced apoptosis against A549 cells. A549 cells were cultured with compound 3 (10
lM) for 24 h and 48 h.

J. Zhang et al. / Phytochemistry 108 (2014) 157–170
167
lyzed by means of flow cytometry with annexin V–propidium
iodide (PI) double staining. Exposure of the membrane phospho-
lipid phosphatidylserine to the external cellular environment is
one of the earliest markers of apoptotic cell death (Martin et al.,
1995). Annexin V is a calcium-dependent phospholipid-binding
protein with high affinity for phosphatidylserine expressed on
the cell surface. PI does not enter whole cells with intact mem-
branes, and was thus used to differentiate between early apoptotic
(annexin V positive, PI negative), late apoptotic (annexin V, PI dou-
ble positive), or necrotic (annexin V negative, PI positive) cell
death. The ratio of early apoptotic cells (lower right) was increased
after treatment with 3 in A549 cells for 24 h (11.6% vs. 2.8% of neg-
ative control) and 48 h (13.4% vs. 2.8% of negative control), and that
of late apoptotic cells (upper right) was increased after 48 h (30.8%
vs. 2.0% of negative control) (Fig. 3). These results demonstrated
that most of the cytotoxic activity of compound 3 against A549
cells is due to inducing apoptotic cell death.
voltage: 150 V]. GLC: Shimadzu GC-2014 instrument on a DB-17
fused silica glass capillary column (Agilent Technologies, Inc., Santa
Clara, CA, USA; 30 m ꢁ 0.32 mm i.d.; column temp., 200 °C; injec-
tion and detector temp., 270 °C; He flow rate, 0.4 ml minꢀ1; split
ratio, 1:75). SiO2 (Silica gel 60, 230–400 mesh; Merck), Diaion
HP-20 (Mitsubishi Chemical Co., Tokyo, Japan), and ODS (Chromat-
orex-ODS, 100–200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan)
were used for column chromatography (CC). TLC was performed
on Silica gel 60 F254 aluminum sheets (Merck), eluent: CHCl3–
MeOH–AcOH–H2O (15:8:3:2). Reversed-phase preparative HPLC
(with refractive index detector) was carried out on ODS columns
(25 cm ꢁ 10 mm i.d.) at 25 °C and a flow rate of 2.0 ml minꢀ1 of
mobile phase; on a Pegasil ODS SP100 column (Senshu Scientific
Co., Ltd., Tokyo, Japan) with MeCN–H2O–AcOH [30:70:0.2 (HPLC
system I); 28:72:0.2 (HPLC system II); or 100:0:0.2 (HPLC system
III)] or with MeOH–H2O–AcOH [78:22:0.2 (HPLC system IV); or
on a Capcell pak C18 column (Shiseido Co., Ltd., Tokyo, Japan) with
MeCN–H2O–AcOH [28:72:0.2 (HPLC system V)] or with
MeOH–H2O–AcOH [48:52:0.2 (HPLC system VI); 20:80:0.2 (HPLC
system VII); or 2:98:0.2 (HPLC system VIII)].
3. Conclusions
This study has established that the MeOH extract of defatted
shea kernel contains oleanane-type triterpene acids and
glycosides, along with other polar compounds including steroid
glucosides, pentane-2,4-diol glucosides, and other phenolic com-
pounds as active principles, which exhibit melanogenesis-inhibi-
4.2. Chemicals and materials
The shea nut sample used in this study was collected and iden-
tified by one of the authors (E.T.M.) during the 2006 shea season
(May through July) from a healthy mature tree at a site (longitude
E 7°207900, latitude N 9°4005300, elevation 365 m) in central Nigeria
(Akihisa et al., 2010c). The chemicals were purchased as follows:
TPA from ChemSyn Laboratories (Lenexa, KS, USA), EBV cell-culture
reagents and butanoic acid from Nacalai Tesque, Inc. (Kyoto,
Japan), fetal bovine serum (FBS), RPMI-1640 medium, antibiotics
(100 units mlꢀ1 penicillin and 100 mg mlꢀ1 streptomycin), and
non-essential amino acid (NEAA) from Invitrogen Co. (Carlsbad,
CA, USA), DMBA, indomethacin, Dulbecco’s modified Eagle’s med-
ium (D-MEM), Eagle’s minimal essential medium (MEM), arbutin,
tory activity in
a-MSH-stimulated B16 melanoma cells, DPPH
free-radical-scavenging activity, inhibitory effects against TPA-
induced EBV-EA activation in Raji cells, anti-inflammatory activity
against TPA-induced inflammation in mice, and cytotoxic activity
against human HL-60 cells. Among the compounds isolated, 18
compounds, 6–12, 14, 15, 19–22, and 24–28, for the inhibition of
melanogenesis, six phenolic compounds, 22–27, for the DPPH rad-
ical-scavenging activity, 12 compounds 6–8, 10–12, 14–16, and
24–26, for the anti-tumor promoting activity, 12 compounds, 1–
5, 8–11, 15, and 16, for the anti-inflammatory activity, and 11 com-
pounds, 3–7, 12, 14–16, 23, and 26, for the cytotoxic activity
against human cancer cell lines, have been demonstrated to be
the relevant active principles of the extract. While shea butter from
the kernel is the most valued product of shea tree (Alander, 2004;
Masters et al., 2004), this study has, thus, demonstrated that the
extract of defatted shea kernel and its constituents may also be
valuable as potential antioxidants, anti-inflammatory agents,
chemopreventive agents, skin-whitening agents, and as anticancer
agents.
DL-a-tocopherol, a-MSH, and MTT from Sigma–Aldrich Japan Co.
(Tokyo, Japan), primary antibodies of anti-MITF, anti-tyrosinase,
anti-TRP-1, and anti-TRP-2 from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA, USA), anti-b-actin from Cell Signaling Technology
(Beverly, MA, USA),
L-arabinose, L-glucose, D-glucuronic acid,
maltose, sucrose, cisplatin, b-carotene, and TMS-HT kit [hexameth-
yldisilazane–trimethylchlorosilane (HMDS–TMCS) in anhydrous
pyridine] from Wako Pure Chemical Industries, Ltd. (Osaka, Japan),
D-arabinose,
L
-rhamnose, and
D-xylosem from Tokyo Chemical
4. Experimental
Industry Co., Ltd. (Tokyo, Japan),
D
-glucose and -cysteine methyl
L
ester hydrochloride from Kanto Chemical Co., Inc. (Tokyo, Japan),
and recombinant human (rh) Annexin V/FITC kit (Bender
MedSystems) from Cosmo Bio Co., Ltd. (Tokyo, Japan). All other
chemicals and reagents were of analytical grade.
4.1. General
Melting points were determined on a Yanagimoto micro melt-
ing point apparatus and are uncorrected. Optical rotations were
measured on a JASCO P-2200 polarimeter in EtOH at 25 °C. UV
spectra, on a JASCO V-630Bio spectrophotometer, and IR spectra,
using a JASCO FTIR-300 E spectrometer, were recorded in EtOH
and KBr disks, respectively. NMR spectra were acquired with a JEOL
ECX-400 (1H, 400 MHz; 13C, 100 MHz) spectrometer in CD3OD,
C5D5N, or in D2O. Chemical shift (d) values are given in ppm with
TMS as internal standard, and coupling constants (J) in Hz. HRE-
SIMS were obtained on an Agilent 1100 LC/MSD TOF (time-of-
flight) system [ionization mode: positive; nebulizing gas (N2)
pressure: 35 psig; drying gas (N2): flow, 12 1 minꢀ1; temp:
325 °C; capillary voltage: 3000 V; fragmentor voltage: 225 V] and
on an Agilent 6530 LC/QTOF system [ionization mode: positive;
nebulizing gas (N2) pressure: 50 psig; drying gas (N2): flow, 10
1 minꢀ1; temp: 350 °C; capillary voltage: 3000 V; fragmentor
4.3. Cell lines and culture condition
B16 4A5 (mouse melanoma) cell line and four human cancer
cell lines, HL-60 (human leukemia), AZ521 (stomach), A549 (lung),
and SK-BR-3 (breast), were obtained from Riken Cell Bank (Ibaraki,
Japan). Cell lines HL-60 and SK-BR-3 were grown in RPMI 1640
medium, while B16 and A549 cell lines, and AZ521 cell line were
grown in D-MEM and in 90% D-MEM + 10% MEM + 0.1 mM NEAA,
respectively. The medium was supplemented with 10% FBS and
antibiotics. Cells were incubated at 37 °C in a 5% CO2 humidified
incubator. The cells were cultured as described in the literature
(Akihisa et al., 2010a; Kikuchi et al., 2011; Tabata et al., 2005).

168
J. Zhang et al. / Phytochemistry 108 (2014) 157–170
4.4. Extraction and isolation
(4.0 mg). Occurrence of three phenolic compounds, 26, 27, and
28, which have been detected in the EtOAc-soluble fraction as
described above, seems likely in the BuOH-soluble fraction as well
– although their occurrence in this fraction was not confirmed in
this study.
Whole nuts were oven-dried at 60 °C over 72 h and decorticat-
ed. Kernels were crushed into powder first. The pulverized sample
was weighed (3705 g), and extracted with hexane (under condi-
tions of reflux, 3 h, 3ꢁ) which gave an extract (1737 g) (Akihisa
et al., 2010c). The defatted residue was then extracted with MeOH
(under conditions of reflux, 3 h, 3ꢁ) to yield a MeOH extract
(450 g) which was suspended in H2O, and partitioned successively
with EtOAc and BuOH to yield EtOAc- (69 g), BuOH- (134 g), and
H2O- (191 g) soluble fractions sequentially.
EtOAc-soluble fraction. A portion of the EtOAc fraction (60.0 g)
was subjected to SiO2 CC (800 g). Step gradient elution was
conducted with hexane–EtOAc (1:0 ? 0:1) and EtOAc–MeOH
(1:0 ? 7:3) to give 14 fractions, A1–A14. Fraction A9 (200 mg),
from the EtOAc eluate, was crystallized from MeOH to yield a
crystalline material (45 mg) which was then acetylated in acetic
anhydride/pyridine. HPLC (system III) of the resulting acetate
yielded compounds 18a (the tetraacetate derivative of 18;
1.7 mg, tR 17.0 min) and 17a (the tetraacetate derivative of 17;
2.0 mg, tR 24.0 min). A portion (403 mg) of the fraction A10
(2.26 g), from the EtOAc eluate , was subjected to HPLC (system
VI) giving compounds 26 (7.2 mg, tR 33.0 min) and 27 (6.6 mg, tR
36.0 min). A portion (650 mg) of the fraction A11 (6.1 g), from
the EtOAc–MeOH (19:1) eluate was passed through a SiO2 CC
[20 g; hexane–EtOAc (7:3 ? 0:1)] to give a fraction (100 mg) from
which was obtained compound 23 (61.9 mg) by crystallization
from MeOH.
H2O-soluble fraction. A portion (90 g) of the H2O-soluble fraction
was subjected to Sephadex LH-20 CC (150 g), eluted with
MeOH–H2O (0:1 ? 1:1) which yielded seven fractions, H1–H7.
The fractions H2 (2.1 g) and H3 (53.9 g), both from the eluates of
H2O, were crystallized from MeOH–H2O (1:1) yielding 30 (1.6 g)
and 29 (1.4 g), respectively. Fraction H4 (20.4 g), from the eluate
of H2O, was subjected to ODS CC (96 g); {MeOH–H2O
(0:1 ? 1:1)] to yield eight fractions, H4–1–H4–8. Fraction H4–7
(724 mg), upon further silica gelCC [25 g; CHCl3–MeOH
(1:0 ? 13:7)], gave a fraction (105 mg) from which were isolated
compounds 5 (16.2 mg, tR 36.0 min) and 4 (20.0 mg, tR 48.0 min)
by HPLC (system II).
4.4.1. Paradoxoside A (1)
White amorphous powder from MeOH, mp 225–228 °C; aD25
ꢀ38.8 (c 0.41, EtOH); UV (EtOH) kmax nm: 264, 394; IR (KBr): mmax
cmꢀ1: 3436 (OH), 2930, 1632 (C@O), 1384, 1073, 1040; For 1H
NMR and 13C NMR spectroscopic data, see Table 3; HRESIMS m/z:
997.4599 [M+Na]+ (calcd for C47H74O21Na, 997.4615); HRESIMS2
m/z: 821.4281 [(M+Na)
–
GlcA]+ (calcd for C41H66O15Na,
821.4299), 719.3605 [(M+Na) – Ara – Rha]+ (calcd for C36H56O13Na,
719.3618), 543.3285 [(M+Na) – Ara – Rha – GlcA]+ (calcd for
C
30H48O7Na, 543.3297), 499.3384 [(M+Na) – Ara – Rha – GlcA –
BuOH-soluble fraction. The BuOH-soluble fraction (130 g) was
subjected to CC [Diaion HP-20 (1000 g); step-gradient elution with
MeOH–H2O (0:1 ? 1:0)] to give nine fractions, B1–B9. Fraction B2
(29.6 g), from the H2O eluate, was crystallized from MeOH–H2O
(1:1) to yield compound 28 (3.9 g). Fraction B3 (4.6 g), from the
H2O eluate, was passed through an ODS CC [120 g; MeOH–H2O
(0:1 ? 7:3)] to afford eight fractions, B3–1–B3–8. Crystallization
of fraction B3–2 (1.7 g) from MeOH yielded compound 21
(137.6 mg). Fraction B3–7 (140 mg) was subjected to silica gel col-
umn chromatography, CC [10 g; CHCl3–MeOH (19:1 ? 0:1)] to give
a fraction (10 mg) from which were isolated compounds 24
(1.3 mg, tR 11.0 min) and 25 (3.9 mg, tR 12.0 min) by HPLC (system
V). Fraction B4 (4.8 g), from the MeOH–H2O (1:9) eluate, was
applied to a SiO2 column [150 g; CHCl3–MeOH (1:0 ? 7:3)] to yield
nine fractions, B4–1–B4–9. Fraction B4–5 (424 mg), upon CC on an
ODS column [MeOH–H2O (0:1 ? 3:17)], yielded a fraction (36 mg)
from which was obtained compound 22 (3.0 mg, tR 15.0 min) and a
fraction (5.0 mg, tR 17.0 min) by HPLC (system VII). Further HPLC
(system VIII) of the latter fraction yielded compounds 20 (0.8 mg,
tR 57.0 min) and 19 (1.4 mg, tR 58.5 min). A portion (26.0 g) of frac-
tion B7 (27.1 g), from theMeOH–H2O (7:3) eluate, was subjected to
ODS CC [700 g; MeOH–H2O (0:1 ? 1:0)] to afford nine fractions,
B7–1–B7–9. Further silica gel CC [100 g; CHCl3–MeOH
(1:0 ? 13:7)] of fraction B7–5 (3.4 g) yielded nine fractions, B7–
5a–B7–5i. HPLC (system I) of fraction B7–5 h (431 mg) gave com-
pounds 1 (12.5 mg, tR 122.0 min), 2 (14.9 mg, tR 130.0 min), and
3 (17.1 mg, tR 140.0 min). Fraction B8 (20.0 g), from the MeOH–
H2O (9:1) eluate was subjected to ODS CC [200 g; MeOH–H2O
(0:1 ? 1:0) to afford six fractions, B8–1–B8–6. Silica gel CC [CHCl3–
MeOH (1:0 ? 0:1)] of a portion (1.4 g) of the fraction B8–4 (8.8 g)
gave eight fractions, B8–4a–B8–4 h. HPLC (system IV) of fraction
B8–4b (50 mg) yielded compounds 12 (18.6 mg, tR 41.0 min) and
14 (4.0 mg, tR 43.9 min). Fraction B8–4f (155 mg), upon repeated
silica gel CC [CHCl3–MeOH (1:0 ? 1:1)], eventually afforded com-
pounds 6 (1.2 mg), 7 (1.0 mg), 8 (25.0 mg), 9 (35.5 mg), 10
(12.3 mg), and 11 (24.7 mg). An EtOAc-soluble portion (26 mg) of
fraction B8–5 (766 mg) was passed through a silica gel column
[hexane–EtOAc (1:0 ? 3:2)] which afforded compound 16
CO2]+ (calcd for C29H48NaO5, 499.3399).
4.4.2. Paradoxoside B (2)
White amorphous powder from MeOH, mp 217–220 °C; aD25
ꢀ35.3 (c 0.35, EtOH); UV (EtOH) kmax nm: 264, 441; IR (KBr): mmax
cmꢀ1: 3436 (OH), 2930, 1638 (C@O), 1385, 1074, 1040; For 1H
NMR and 13C NMR spectroscopic data, see Table 3; HRESIMS m/z:
1129.5011 [M+Na]+ (calcd for C52H82O25Na, 1129.5037); HRESIMS2
m/z: 953.4702 [(M+Na) – GlcA]+ (calcd for C46H74O19Na, 953.4721),
719.3598 [(M+Na) – Xyl – Rha – Ara]+ (calcd for C36H56O13Na,
719.3618), 543.3272 [(M+Na) – Xyl – Rha – Ara – GlcA]+ (calcd
for C30H48O7Na, 543.3297).
4.4.3. Paradoxoside C (8)
White amorphous powder from MeOH, mp 235–238 °C; aD25
+13.5 (c 1.15, EtOH); UV (EtOH) kmax nm: 245, 250, 256; IR (KBr):
mmax cmꢀ1: 3436 (OH), 2930, 1638 (C@O), 1385, 1073, 1040; For
1H NMR and 13C NMR spectroscopic data, see Table 4; HRESIMS
m/z: 695.3998 [M+H]+ (calcd for C37H59O12, 695.4006); HRESIMS2
m/z: 469.3309 [(M+H) – MeGlcA – 2H2O]+ (calcd for C30H45O4,
469.3318).
4.4.4. Paradoxoside D (9)
White amorphous powder from MeOH, mp 241–244 °C; aD25
+20.0 (c 0.87, EtOH); UV (EtOH) kmax nm: 244, 250, 255; IR (KBr):
mmax cmꢀ1: 3410 (OH), 2930, 1700 (C@O), 1384, 1070, 1037; For
1H NMR and 13C NMR spectroscopic data, see Table 4; HRESIMS
m/z: 851.4427 [M+Na]+ (calcd for C42H68O16Na, 851.4405); HRE-
SIMS2 m/z: 689.3777 [(M+Na) – Glc]+ (calcd for C36H58O11Na,
689.3876), 645.3912 [(M+Na) – Glc – CO2]+ (calcd for C35H58O9Na,
645.3978).
4.4.5. Paradoxoside E (14)
White amorphous powder from MeOH, mp 237–240 °C; aD25
+15.5 (c 0.20, EtOH); UV (EtOH) kmax nm: 272; IR (KBr): mmax
cmꢀ1: 3435 (OH), 2930, 1689 (C@O), 1385, 1070, 1037; For 1H
NMR and 13C NMR spectroscopic data, see Table 4; HRESIMS m/z:
677.3892 [M+H]+ (calcd for C37H57O11, 677.3901); HRESIMS2 m/z:

J. Zhang et al. / Phytochemistry 108 (2014) 157–170
169
451.3218 [(M+H)
451.3212).
– MeGlcA –
2H2O]+ (calcd for C30H43O3,
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4.5. Acid hydrolysis of compounds 1, 2, 8, 9, and 14
A solution of each compound (3–5 mg) in H2O (2.0 ml) and 2 M
aq. CF3COOH (11.0 ml) was heated under conditions of reflux in a
water bath for 2 h (Tapondjou et al., 2011). The mixture was then
diluted in H2O (10 ml) and treated with EtOAc (3 ꢁ 3 ml). The com-
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MeOH to remove acid and analyzed by TLC in comparison with
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Appendix A. Supplementary data
Kikuchi, T., Uchiyama, E., Ukiya, M., Tabata, K., Kimura, Y., Suzuki, T., Akihisa, T.,
2011. Cytotoxic and apoptosis-inducing activities of triterpene acids from Poria
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Kim, I.-S., Yang, M., Lee, O.-H., Kang, S.-N., 2011. The antioxidant activity and the
bioactive compound content of Stevia rebaudiana water extract. LWT – Food Sci.
Technol. 44, 1328–1332.
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the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
09.017.
Kitdamrongtham, W., Ishii, K., Ebina, K., Zhang, J., Ukiya, M., Koike, K., Akazawa, H.,
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flowers of Azadirachta indica var. siamensis, and their melanogenesis-inhibitory
and cytotoxic activities. Chem. Biodiversity 11, 73–84.
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Brewington, T., Solano, F., García-Borrón, J.C., Hearing, V.J., 1994. Tyrosinase
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