Melanogenesis-inhibitory activity and cancer chemopreventive effect of glucosylcucurbic acid from shea (vitellaria paradoxa) kernels

Article abstract of DOI:10.1002/cbdv.201400424

Two jasmonate derivatives, glucosylcucurbic acid (1) and methyl glucosylcucurbate (2), were isolated from the MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels. These and their deglucosylated derivatives, cucurbic acid (3) and methyl cucurbate (4), were evaluated for their melanogenesis-inhibitory and cancer chemopreventive potencies. Compounds 1, 3, and 4 exhibited potent melanogenesis-inhibitory activities in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells. Western-blot analysis revealed that compounds 1 and 3 reduced the protein levels of MITF (=microphthalmia-associated transcription factor), tyrosinase, TRP-1 (=tyrosine-related protein 1), and TRP-2 mostly in a concentration-dependent manner. In addition, compound 1 exhibited inhibitory effects against Epstein￡Barr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, against TPA-induced inflammation in mice, and against skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter.

Full text of DOI:10.1002/cbdv.201400424

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Melanogenesis-Inhibitory Activity and Cancer Chemopreventive Effect of
Glucosylcucurbic Acid from Shea (Vitellaria paradoxa) Kernels
by Jie Zhanga), Masahiro Kuritaa), Kodai Ebinaa), Motohiko Ukiyaa), Harukuni Tokudab),
Ken Yasukawac), Eliot T. Mastersd), Naoto Shimizue), Momoko Akihisaf), Feng Fengg), and
Toshihiro Akihisa*a)h)
a) College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo
101-8308, Japan
b) Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640,
Japan
c) School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan
d) World Agroforestry Centre (ICRAF), Nelson Marlborough Institute of Technology (NMIT), Nelson
7010, New Zealand
e) Application Center, Agilent Technologies Japan, Ltd., 9-1 Takakura-cho, Hachioji-shi, Tokyo
192-0033, Japan
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) Department of Natural Medicine Chemistry, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, P. R. China
h) Akihisa Medical Clinic, 1086-3 Kamo, Sanda-shi, Hyogo 669-1311, Japan
(fax: þ81-79-567-1980; e-mail: akihisa_toshihiro@yahoo.co.jp)
Two jasmonate derivatives, glucosylcucurbic acid (1) and methyl glucosylcucurbate (2), were
isolated from the MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels. These and
their deglucosylated derivatives, cucurbic acid (3) and methyl cucurbate (4), were evaluated for their
melanogenesis-inhibitory and cancer chemopreventive potencies. Compounds 1, 3, and 4 exhibited
potent melanogenesis-inhibitory activities in a-melanocyte-stimulating hormone (a-MSH)-stimulated
B16 melanoma cells. Western-blot analysis revealed that compounds 1 and 3 reduced the protein levels of
MITF (¼ microphthalmia-associated transcription factor), tyrosinase, TRP-1 (¼tyrosine-related protein
1), and TRP-2 mostly in a concentration-dependent manner. In addition, compound 1 exhibited
inhibitory effects against EpsteinÀBarr virus early antigen (EBV-EA) activation induced with 12-O-
tetradecanoylphorbol-13-acetate (TPA ) in Raji cells, against TPA-induced inflammation in mice, and
against skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-
dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter.
Introduction. – The Shea butter tree (Vitellaria paradoxa C.F. Gaertner; synonyms
Butyrospermum paradoxum (C. F. Gaertn.) Hepper, Butyrospermum parkii (G.
Don) Kotschy; belonging to family Sapotaceae) is indigenous to the savanna belt
extending across sub-Saharan Africa north of the equator, ranging from Senegal in the
west to Ethiopia and Uganda in the east [1–4]. 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 the seed kernel, consisting of an olein and a stearin fraction, along
with non-saponifiable (non-lipid) compounds. Fractionated shea stearin is used
ꢀ 2015 Verlag Helvetica Chimica Acta AG, Zürich

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primarily as a cocoa-butter substitute or extender in chocolate manufacture [4]. These
applications are due to properties imparted by the structures of its component
triacylglycerols. In addition, shea butter is increasingly 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 [5]. To characterize and quantify
the constituents of shea butter among widely dispersed Vitellaria paradoxa populations,
we have recently determined the content and composition of 36 shea kernel samples
from seven sub-Saharan countries, with a focus on fatty acids, triacylglycerols,
triterpene alcohol fractions from the NSL, and triterpene ester composition of the
kernel lipids (hexane extracts) [6][7]. In addition, we have 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 [8].
Thus, we were interested in the evaluation of pharmacological and cosmeceutical
potentials of the constituents of defatted shea kernel, since there seems to be little
industrial utilization of defatted shea kernel (residue), other than as fuel. In our recent
study, we have isolated several oleanane-type triterpene acids and their glycosides,
phenolic compounds, sugars, and other polar constituents from the defatted shea
kernel, and have evaluated their bioactivity [9]. Our continuing study on the
constituents of defatted shea kernel has led to the isolation of two jasmonate
derivatives, glucosylcucurbic acid (1) and methyl glucosylcucurbate (2). Herein, we
report the evaluation of melanogenesis-inhibitory and cancer chemopreventive
potencies of compounds 1 and 2, and their deglucosylated derivatives, cucurbic acid
(3) and methyl cucurbate (4), respectively.
Compounds 1–4 have so far been isolated from the seeds of Cucurbita pepo
(Cucurbitaceae) [10][11]. Occurrence of compound 2 in Boronia megastigma
(Rutaceae) [12], of 3 in Phyllunthus urinaria (Euphorbiaceae) [13], Secare cereal
(Poaceae) [14], Anemia phyllitidis (Anemiaceae) [14], and Botryodiplodia theobromae
(Ascomycota fungus) [15], and of 4 in P. urinaria [13] and B. megastigma [16] also have
been reported previously. These compounds possess a cyclopentanoid C12-acid
structure similar to jasmonic acid and its analogs which are widely distributed in the
plant kindgom and have been found as plant regulators [17]. Compounds 1 [10], 2 [10],
3 [10][18][19], and 4 [10][20][21] also have been reported to possess plant-growth
regulatory activity.
Results and Discussion. – Isolation and Identification. The defatted residue of shea
kernel (collected in Nigeria) [6] was extracted with MeOH to yield a MeOH extract
which was suspended in H2O, and partitioned successively with AcOEt and BuOH to
yield AcOEt-, BuOH-, and H2O-soluble fractions sequentially [9]. The BuOH-soluble

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549
fraction was subjected to column chromatography (CC) on Diaion HP-20, octadecyl
silica gel (ODS), and subsequently on SiO2 which eventually yielded compounds 1 and
2. Compounds 1 and 2 were identified as glucosylcucurbic acid (3-O-b-d-glucopyr-
anosylcucurbic acid) and methyl glucosylcucurbate (methyl 3-O-b-d-glucopyranosyl-
cucurbate), respectively, on the basis of spectroscopic and chemical methods, and
1
comparison of their MS and H-NMR data with those in the literature [11]. This
study seems to be the first for the isolation of compounds 1 and 2 from a Sapotaceae
plant.
Acid Hydrolysis of 1 and 2, and Preparation of Methyl Cucurbate (4). Acid
hydrolysis of both 1 and 2 with aqueous CF3COOH solution yielded cucurbic acid (3)
[14][19] as an aglycon. The sugar moiety liberated from the hydrolysis of both 1 and 2
was identified as d-glucose by GLC analysis of the (trimethylsilyl)-thiazolidine
derivative [22]. Treatment of 3 with (trimethylsilyl)diazomethane (¼diazomethyltri-
methylsilane) as described in [23] afforded methyl cucurbate (4) [14][19][21][24].
Melanogenesis-Inhibitory Activity. Compounds 1–4 were evaluated for melano-
genesis inhibition in a-melanocyte-stimulating hormone (a-MSH)-stimulated B16
melanoma cells (Table 1). The cytotoxic activities of these compounds against B16
melanoma cells were also determined by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay. To assess the risk/benefit ratio for
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 concentration (Table 1).
Compound with smaller A/C ratio would be a lower-risk skin-whitening agent. Among
the compounds tested, compound 3 exhibited potent inhibitory effects (46.3–75.2%
melanin content) with no, or almost no, toxicity to the cells (80.6–102.2% cell viability)
at all concentrations tested (10, 30, and 100 mm), and their A/C ratios were in the range
of 0.57–0.73 at these concentrations. On the other hand, compound 1 at a higher
concentration (61.0% melanin content, 84.9% cell viability, and A/C ratio of 0.72 at
100 mm), and compound 4 at lower concentrations (66.2 and 59.3% melanin contents,
105.6 and 102.2% cell viabilities, and A/C ratios of 0.63 and 0.58, resp., at 10 and 30 mm),
exhibited inhibitory activities with no or low cytotoxicity. The inhibitory activities on
melanogenesis of these compounds were superior to a known standard melanogenesis-
inhibitor, arbutin (4-hydroxyphenyl b-d-glucopyranoside; 92.7, 91.0, and 71.5%
melanin contents at 10, 30, and 100 mm, resp.), which has been recognized as a useful
depigmentation compound for skin whitening in the cosmetic industry [25], and hence
compounds 1, 3, and 4 may be valuable as potential skin-whitening agents.
Mechanism of Melanogenesis Inhibition by Compounds 1 and 3. Tyrosinase, and
tyrosinase-related protein-1 (TRP-1) and TRP-2 are enzymes responsible for the
synthesis of melanin [26]. Regulations of the transcription and activity of these
melanogenic enzymes are effective for depigmentation [27]. Tyrosinase, a rate-limiting
enzyme, catalyzes the hydroxylation of l-tyrosine to l-3,4-dihydroxyphenylalanine (l-
DOPA), and the oxidation of l-DOPA to l-DOPA quinone [28]. TRP-2 functions as
DOPAchrome tautomerase, and TRP-1 catalyzes oxidation of 5,6-dihydroxy-1H-
indole-2-carboxylic acid (DHICA) [28]. Transcription for expression of these enzymes
is regulated by microphthalmia-associated transcription factor (MITF) [29]. To clarify
the mechanism involved in the melanogenesis inhibition by compounds 1 and 3, the

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Fig. 1. Effect on expression of tyrosinase, MITF, TRP-1, and TRP-2 in a-MSH-stimulated B16 melanoma
cells treated with compounds 1 and 3. The results are from one representative experiment among three
runs which displayed similar patterns to one another.
potent melanogenesis inhibitors found in this study, the protein levels of MITF,
tyrosinase, TRP-1, and TRP-2 were evaluated in B16 melanoma cells treated with
compounds 1 and 3 by Western blot analysis. Treatment of B16 melanoma cells with
compounds 1 and 3 reduced protein levels of all of MITF, tyrosinase, TRP-1, and TRP-
2, mostly in a concentration-dependent manner, although the level of expression of
tyrosinase at 30 and 100 mm was inverted for compound 1 (Fig. 1). These results
suggested that compounds 1 and 3 inhibit melanogenesis in the a-MSH-stimulated B16
melanoma cells due to, at least in part, inhibition of the expression of MITF, followed
by a decrease in the expression of tyrosinase, TRP-1, and TRP-2.
Inhibitory Effects on EpsteinÀBarr Virus Early Antigen (EBV-EA) Induction. The
inhibitory effects on the induction of EBV-EA induced by 12-O-tetradecanoylphorbol-
13-acetate (TPA ) were further examined as a preliminary evaluation of antitumor-
promoting activities of compounds 1–4. The results are collected in Table 2, together
with the corresponding data for b-carotene, a vitamin A precursor studied widely in
cancer-chemoprevention animal models, and for retinoic acid, one of the retinoids that
has been studied as a cancer-chemoprevention agent for various organ site cancers
[30]. 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. Among the compounds tested, compound 1 showed an inhibitory effect
with an IC50 value (concentration for 50% inhibition with respect to the positive
control) of 414 molar ratio/32 pmol TPA, which was almost comparable with that of b-
carotene (IC50 397 molar ratio/32 pmol TPA). Whereas the other three compounds, 2–
4, exhibited lower inhibitory effects (IC50 425–448 molar ratio/32 pmol TPA) than b-
carotene; these compounds displayed greater inhibitory action than retinoic acid (IC50
482 molar ratio/32 pmol TPA). Since inhibitory effects against EBV-EA induction
have been demonstrated to correlate with those against tumor promotion in vivo
[31][32], compound 1 may be a potential inhibitor of tumor promotion.

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Table 2. Inhibitory Effects on the Induction of EpsteinÀBarr Virus Early Antigen (EBV-EA) of
Compounds 1–4 and Reference Compounds
Compound
Percentage EBV-EA inductiona)
IC50b) [molar ratio/
32 pmol TPA]
1000c)
500
100
10
1
2
3
4
9.3Æ0.5 (70)
10.0Æ0.7 (70)
7.9Æ0.4 (60)
9.5Æ0.5 (60)
8.6Æ0.5 (70)
15.3Æ1.1 (70)
42.3Æ1.3
44.3Æ1.4
47.8Æ1.6
49.7Æ1.5
34.2Æ1.0
49.3Æ1.4
70.3Æ2.5
71.6 Æ 2.3
71.8 Æ 2.3
75.2Æ2.2
82.1Æ2.0
76.3Æ2.3
96.7Æ0.6
414
425
438
448
397
482
98.9Æ0.6
98.6Æ0.7
100.0Æ0.5
100.0Æ0.3
100.0Æ0.4
b-Carotened)
Retinoic acidd)
a) Values represent the percentages relative to the positive control, with TPA (32 pmol, 20 ng)
representing 100% induction at four different concentrations in terms of molar ratio/32 pmol TPA. Data
are exressed as meanÆS.D. (n ¼ 3). b) IC50 Values represent the molar ratios of compounds, relative to
TPA, required to inhibit 50% of the positive control activated with 32 pmol TPA. c) Values in
parentheses are viability percentages of Raji cells. d) Reference compounds.
Cytotoxic and Anti-Inflammatory Activities. Upon evaluation of cytotoxic activities
of compounds 1–4 against four human cancer cell lines, HL60 (leukemia), AZ521
(stomach), A549 (lung), and SK-BR-3 (breast), only compound 4 exhibited weak
activities against HL60 and AZ521 cells (IC50 values of 74.4 and 50.0 mm, resp.)
(Table 3). In addition, upon evaluation of anti-inflammatory activity of compounds 1
and 2 against TPA-induced inflammation in mice, compound 1 exhibited anti-
inflammatory activity with the 50% inhibitory dose (ID50) of 1.11 mmol/ear, which was
mostly comparable with that of indomethacin, a commercially available anti-
inflammatory drug (ID50 0.91 mmol/ear). This inhibitory activity has been demon-
strated to be closely parallel with that of the inhibition of 7,12-dimethylbenz[a]an-
thracane (DMBA)-TPA papilloma formation in the mouse-skin model [33]. Hence,
compound 1 might be expected to possess high antitumor-promoting activity in the
same animal model.
Table 3. Cytotoxic Activities and Inhibition of Inflammation in Mice of Compounds 1–4
Compound
Cytotoxicity (IC50 ÆS.D. [mm])a)
Inhibition of inflammation
b
HL60
A549
AZ521
SK-BR-3
ID50
)
95% CIc)
(Leukemia) (Lung)
(Stomach) (Breast)
[mmol/ear]
1
2
3
4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.11
1.97
0.94–1.31
1.62–2.40
74.4Æ0.6
50.0Æ0.8 >100
Cisplatind)
4.2Æ1.1
18.4Æ1.9 9.5Æ0.5
18.8Æ0.6
Indomethacind)
0.91
0.76–1.09
a) Cells were treated with compounds (1Â10À4 –1Â10À6 m) for 48 h, and cell viability was analyzed by
the MTT assay. IC50 Values based on triplicate five points. b) ID50, 50% Inhibitory dose. c) 95%
Confidence intervals. d) Reference compounds.

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Two-Stage Carcinogenesis. On the basis of the results of the in vitro and in vivo
assays described above, we evaluated subsequently the inhibitory effect of compound 1
in a tumor model in mouse skin, and the inhibitory effect was compared with that of b-
carotene. The incidence [%] of papilloma-bearing mice and the average numbers of
papillomas per mouse in the two-stage carcinogenesis test in mouse skin using DMBA
as an initiator and TPA as a promoter are presented in Fig. 2,a and b, respectively. The
incidence of the papilloma-bearing mice was high and 100% at ten-weeks promotion in
Group I (the positive control). Per mouse at 10 and 20 weeks of the promotion, 3.7 and
8.6 papillomas, respectively, were found. The formation of papillomas in the mouse skin
was delayed, and the mean numbers of papillomas per mouse were reduced by the
treatment with compound 1 (Group II). Thus, in Group II, treated with compound 1,
the percentage ratios of papilloma-bearing mice were 30% at 10 weeks, and 100% at
20 weeks, and the mean papillomas per mouse were 1.2 at 10 weeks, and 5.9 at 20 weeks.
The inhibitory effect of compound 1 was found to be almost comparable with that of
reference b-carotone (Group III; papilloma bearers: 33 and 100% at 10 and 20 weeks,
resp.; papillomas/mouse: 1.5 and 6.0 at 10 and 20 weeks, resp.).
Fig. 2. Inhibitory effect of compound 1 and reference b-carotene on mouse skin carcinogenesis induced by
DMBA and TPA. a) Percentage of mice with papillomas; b) average number of papillomas per mouse.
Tumor formation in all mice was initiated with DMBA (390 nmol) and promoted with TPA (1.7 nmol)
*
twice weekly, starting one week after initiation. Filled circles ( ) represent the untreated control group
~
*
(TPA alone; Group I); open circles ( ) refers to TPAþ1 (85 nmol; Group II); open triangles ( ) refer
to TPAþb-carotene (85 nmol; Group III). After 20 weeks of promotion, a significant difference in the
number of papillomas per mouse between the groups treated with compound 1 and b-carotene, and the
control group was evident (p<0.01, using the Studentꢁs t-test). The numbers (standard deviations in
parentheses) of papillomas per mouse for each group were 8.6 (1.4), 5.9 (0.5), and 6.0 (1.8) for groups I,
II, and III, respectively.
Conclusions. – This work has established that the MeOH extract of defatted shea
(V. paradoxa) kernels contains two jasmonate derivatives, glucosylcucurbic acid (1) and
methyl glucosylcucurbate (2), in addition to triterpene glycosides, phenolics, sugars,
and other polar constituents as has been demonstrated in our recent study [9]. This
seems to be the first isolation of compounds 1 and 2 from a Sapotaceae plant. Upon
evaluation of compounds 1 and 2, and their deglucosylated derivatives, cucurbic acid

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(3) and methyl cucurbate (4), for the melanogenesis-inhibitory activity in a-MSH-
stimulated B16 melanoma cells, compounds 1, 3, and 4 exhibited potent inhibitory
activities, suggesting that these compounds may be valuable as potential skin-whitening
agents. In addition, from the results of the in vitro EBV-EA induction, in vivo TPA-
induced inflammation, and in vivo two-stage carcinogenesis tests, compound 1 may be
useful as agent that inhibit chemical carcinogenesis. The mechanisms by which
compound 1 suppresses the tumor promotion may be due to one or more of the
following effects: i) enhancement of detoxification of carcinogenesis, ii) inhibition of
cytochrome P450-mediated activation of carcinogenesis, iii) ꢂentrapmentꢁ of carcino-
gens and prevention of their interaction with DNA, iv) disruption of the cell cycle and/
or induce apoptosis, v) modulation of nuclear receptors, and vi) suppressing gene
expression by DNA methylation [34]. Compound 1 did not exhibit cytotoxicity against
the cancer cells tested, indicating that this compound did not affect the signaling
pathways involving RAS (rat sarcoma) proteins, since RAS proteins, their regulators,
and the downstream enzymes are activated in many cancer cells by a variety of
mechanisms, including oncogenic mutations of RAS genes [35]. This study seems to be
the first demonstration of anti-melanogenesis activity by compounds 1, 3, and 4, and
antitumor-promoting activity by compound 1.
Experimental Part
General. Column chromatography (CC): Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan),
silica gel (SiO2, 230–400 mesh; Merck & Co., Inc., DE-Darmstadt), and octadecyl silica gel (ODS;
Chromatorex-ODS, 100–200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan). TLC: Silica gel 60 F254
aluminium sheets (Merck); eluent: CHCl3/MeOH/AcOH/H2O 15 :8 :3 :2. GLC: Shimadzu GC-2014
instrument on a DB-17 fused silica cap. column (Agilent Technologies, Inc., Santa Clara, CA, USA;
30 mÂ0.32 mm i.d.; column temp., 2008; injection and detector temp., 2708; He flow rate, 0.4 ml/min;
split ratio, 1:75). Optical rotations: JASCO P-1020 digital polarimeter. 1D- and 2D-NMR spectra: JEOL
ECX-400 (1H: 400 and 13C: 100 MHz) spectrometer at r.t.; CD3OD or C5D5N solns.; d in ppm rel. to
Me4Si as internal standard, J in Hz. HR-ESI-MS: Agilent 1100 LC/MSD TOF (time-of-flight) system
(ionization mode; positive; cap. voltage, 3000 V; fragmentor voltage, 225 V). Microplate reader: Sunrise-
Basic (Tecan Japan Co., Ltd., Kawasaki, Japan).
Chemicals and Materials. The shea kernel sample was collected and identified by one of the authors
(E. T. M.) on behalf of the World Agroforestry Centre (ICRAF), an international research institute
constituted under the Consultative Group for International Agricultural Research (GGIAR), in a
parallel research activity to project CFC/FIGOOF/23 ꢂImproving Product Quality and Market Access for
Shea Butter Originating from Sub-Saharan Africaꢁ (ProKaritØ) [36]. Near the geographic center of a
regional sampling mission undertaken from Senegal to South Sudan during the 2006 shea season (May
through July), the specific sample described in this study was obtained as fresh fruit collected from
beneath the crown of a healthy mature shea tree at a site in central Nigeria (longitude E 7827’9’’, latitude
N 9840’53’’, elevation 365 m) [6]. The chemicals were purchased as follows: TPA from ChemSyn
Laboratories (Lenexa, KS, USA); the EBV cell-culture reagents and butanoic acid from Nacalai Tesque,
Inc. (Kyoto, Japan); fetal bovine serum (FBS), RPMI-1640 medium, non-essential amino acid (NEAA),
and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin) from Invitrogen Co. (Carlsbad, CA,
USA); DMBA, indomethacin, Dulbeccoꢁs modified Eagleꢁs medium (DMEM), Eagleꢁs minimal
essential medium (MEM), arbutin, a-MSH, and MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide) from SigmaÀAldrich 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-glucose and TMS-HT kit
(hexamethyldisilazane–trimethylchlorosilane (HMDSÀTMCS) in anh. pyridine) from Wako Pure

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Chemical Industries, Ltd. (Osaka, Japan); and (all-E)-retinoic acid, d-glucose, and l-cysteine methyl
ester hydrochloride from Kanto Chemical Co., Inc. (Tokyo, Japan). All other chemicals and reagents
were of anal. grade.
Isolation of Compounds 1 and 2. The defatted residue (1,900 g) of shea kernel (sample No. 9,
collected in Nigeria) [6] was extracted with MeOH (reflux, 3 h, 3Â ) to yield a MeOH extract (450 g),
which was suspended in H2O, and partitioned successively with AcOEt and BuOH to yield AcOEt-,
BuOH-, and H2O-soluble fractions (69.0, 134.0, and 191.0 g, resp.) sequentially [9]. The BuOH-soluble
fraction (130 g) was subjected to CC (Diaion HP-20 (1000 g); MeOH/H2O, 0 :1!1:0) to give nine
fractions, B1–B9. A portion (26.0 g) of Fr. B7 (27.1 g), eluted with MeOH/H2O 7:3, was subjected to CC
(ODS (700 g); MeOH/H2O, 0 :1!1:0) to afford nine fractions, B7-1–B7-9. Further CC of Fr. B7-5
(3.4 g) (SiO2 (100 g); CHCl3/MeOH, 1:0!13 :7) yielded compounds 2 (9.6 mg; TLC: Rf 0.63) and 1
(50.0 mg; Rf 0.46), from the eluates of CHCl3/MeOH 1:0 and 1:1, resp.
Identification of Compounds 1 and 2. Compounds 1 and 2 were identified as glucosylcucurbic acid
(¼ 3-O-b-d-glucopyranosylcucurbic acid; CAS Reg. No: 62082-23-9) and methyl glucosylcucurbate
(¼ methyl 3-O-b-d-glucopyranosylcucurbate; CAS Reg. No: 62071-04-9), resp., on the basis of the
comparison of their MS and 1H-NMR data with those in the literature [11].
Acid Hydrolysis of 1 and 2, and Preparation of Cucurbic Acid (3) and Methyl Cucurbate (4). Acid
hydrolysis of 1 with aq. CF3COOH soln., as described in [9][37], yielded 3 ({(1R,2S,3S)-3-hydroxy-2-
[(2Z)-pent-2-en-1-yl]cyclopentyl}acetic acid) [14][19] as the aglycon. The sugar moiety liberated showed
one TLC spot with an Rf value of 0.50, compatible with that of authentic glucose. The sugar moiety was
then treated with l-cycteine methyl ester hydrochloride and subsequently with trimethylsilylation
reagent [22]. The GLC of the mixture afforded a peak with a tR value of 16.9 min, which was identical to
that of authentic d-glucose. Authentic l-glucose eluted with tR 18.7 min under the same GLC condition.
By the same acid hydrolysis and derivatization procedures as described above, compound 2 afforded 3 as
an aglycon, and d-glucose as a sugar. These results supported the identification of compounds 1 and 2.
Treatment of 3 with TMS-diazomethane as described in [23] afforded 4 (methyl {(1R,2S,3S)-3-hydroxy-2-
[(2Z)-pent-2-en-1-yl]cyclopentyl}acetate) [14][19][21][24].
Since the NMR spectra of compounds 1 and 2 had remained not fully assigned, their 1H- and
1
13C-NMR data, which were fully assigned by 13C-DEPT, H,1H-COSY, HMQC, HMBC, and NOESY
experiments, are given below, along with their HR-ESI-MS and specific-rotation data.
Glucosylcucurbic Acid (¼{(1R,2S,3S)-3-(b-d-Glucopyranosyloxy)-2-[(2Z)-pent-2-en-1-yl]cyclo-
pentyl}acetic Acid; 1). [a]2D0 ¼ À14.4 (c¼1.92, EtOH); (lit. [11]: [a]D ¼ À23.4 (c¼4.5, MeOH)).1H-NMR
(400 MHz, CD3OD): 0.97 (t, J¼7.3, Me(5’)); 1.34 (dt, J¼2.8, 10.1, HaÀC(5)); 1.75 (dddd, J¼2.8, 7.3, 9.2,
14.2, HaÀC(4)); 1.85 (ddd, J¼7.3, 11.0, 14.2, HaÀC(1’)); 1.93 (ddd, J¼4.1, 7.3, 9.2, HbÀC(5)); 2.00–2.09
(m, HbÀC(1’)); 2.06 (q-like, J¼7.3, CH2(4’)); 2.07–2.15 (m, HbÀC(4)); 2.13 (ddt, J¼2.3, 5.9, 12.4,
HÀC(2)); 2.24 (dd, J¼9.2, 15.6, HaÀC(1’’)); 2.40 (dd, J¼6.9, 15.6, HbÀC(1’’)); 2.61 (ddd-like, J¼2.3, 6.9,
15.6, HÀC(1)); 3.13 (t, J¼8.6, HÀC(2’’’)); 3.20 (ddd, J¼2.3, 5.5, 9.6, HÀC(5’’’)); 3.29 (t, J¼8.8, HÀC(4’’’));
3.33 (t, J¼8.9, HÀC(3’’’)); 3.67 (dd, J¼5.5, 11.9, HaÀC(6’’’)); 3.83 (dd, J¼2.3, 11.9, HbÀC(6’’’)); 4.08 (dt,
J¼2.8, 6.4, HÀC(3)); 4.25 (d, J¼7.8, HÀC(1’’’)); 5.36 (dt, J¼7.3, 11.0, HÀC(2’)); 5.45 (dt, J¼7.3, 11.0,
HÀC(3’)). 13C-NMR (100 MHz, CD3OD): 14.6 (C(5)); 21.6 (C(4’)); 25.3 (C(1’)); 29.7 (C(5)); 31.8 (C(4));
36.2 (C(1’’)); 38.1 (C(1)); 48.9 (C(2)); 62.6 (C(6’’’)); 71.6 (C(4’’’)); 75.0 (C(2’’’)); 77.7 (C(5’’’)); 78.1
(C(3’’’)); 85.3 (C(3)); 103.2 (C(1’’’)); 128.4 (C(2’)); 133.9 (C(3’)); 177.5 (C(2’’)). HR-ESI-MS (pos.):
397.1833 ([MþNa]þ , C18H30NaOþ8 ; calc. 397.1838).
Methyl Glucosylcucurbate (¼ Methyl {(1R,2S,3S)-3-(b-d-Glucopyranosyloxy)-2-[(2Z)-pent-2-en-1-
1
yl]cyclopentyl}acete; 2). [a]2D0 ¼ þ29.3 (c¼0.80, EtOH). H-NMR (400 MHz, CD3OD): 0.97 (t, J¼7.3,
Me(5’)); 1.33 (dt, J¼2.8, 10.1, HaÀC(5)); 1.75 (dddd, J¼2.8, 7.3, 9.2, 14.2, HaÀC(4)); 1.77–1.89 (m,
HaÀC(1’)); 1.92 (ddd, J¼4.1, 7.3, 9.2, HbÀC(5)); 1.98–2.10 (m, HbÀC(1’)); 2.05 (q-like, J¼7.3, CH2(4’));
1.98–2.14 (m, HÀC(2)); 2.04–2.15 (m, HbÀC(4)); 2.29 (dd, J¼9.2, 15.6, HaÀC(1’’)); 2.43 (dd, J¼6.9, 15.6,
HbÀC(1’’)); 2.61 (ddd-like, J¼2.3, 7.3, 16.0, HÀC(1)); 3.13 (t, J¼8.8, HÀC(2’’’)); 3.20 (ddd, J¼2.3, 5.5, 9.6,
HÀC(5’’’)); 3.29 (t, J¼8.8, HÀC(4’’’)); 3.33 (t, J¼8.8, HÀC(3’’’)); 3.65 (s, 2’’-MeO), 3.66 (dd, J¼5.5, 11.9,
HaÀC(6’’’)); 3.83 (dd, J¼2.3, 11.9, HbÀC(6’’’)); 4.08 (dt, J¼2.8, 6.4, HÀC(3)); 4.24 (d, J¼7.8, HÀC(1’’’));
5.35 (dt, J¼7.3, 11.0, HÀC(2’)); 5.44 (dt, J¼7.3, 11.0, HÀC(3’)). 13C-NMR (100 MHz, CD3OD): 14.6
(C(5)); 21.6 (C(4’)); 25.3 (C(1’)); 29.6 (C(5)); 31.8 (C(4)); 36.0 (C(1’’)); 38.1 (C(1)); 48.9 (C(2)); 52.0 (2’’-

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
MeO), 62.7 (C(6’’’)); 71.6 (C(4’’’)); 75.0 (C(2’’’)); 77.8 (C(5’’’)); 78.1 (C(3’’’)); 85.3 (C(3)); 103.2 (C(1’’’));
128.4 (C(2’)); 133.9 (C(3’)); 175.6 (C(2’’)). HR-ESI-MS (pos.): 411.1999 ([MþNaþ, C19H32NaO8þ ; calc.
411.1995).
Cell Lines and Culture Conditions. B16 4A5 (mouse melanoma) cell line and four human cancer cell
lines, HL60 (human leukemia), AZ521 (stomach), A549 (lung), and SK-BR-3 (breast), were obtained
from Riken Cell Bank (Ibaraki, Japan). Cell lines HL60 and SK-BR-3 were grown in RPMI 1640
medium, while B16 and A549 cell lines, and AZ521 cell line were grown in DMEM and in 90% DMEM
þ10% MEM þ0.1 mm NEAA, resp. The medium was supplemented with 10% FBS and antibiotics.
Cells were incubated at 378 in a 5% CO2 humidified incubator. The cells were cultured as described in
[23][38][39].
Assay of Melanin Content. Melanogenesis inhibition assay in the a-MSH-stimulated B16 melanoma
cells was performed as described in [39].
Western Blot Analysis. Western blot analysis was performed according to the method reported in
[40][41] with a slight modification. Briefly, B16 melanoma cells (1Â105 cells) were exposed to the test
compound (30 and 100 mm), supplemented with a-MSH (0.1 mm) for 48 h. Cells were collected and lysed.
Lysates of total protein were separated by 15% sodium dodecyl sulfate (SDS)Àpolyacrylamide gels and
transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were
incubated with anti-MITF, anti-tyrosinase, anti-TRP-1, anti-TRP-2, and anti-b-actin primary antibodies
at 48 overnight. The blots were then detected with enhanced chemiluminescence (ECL) plus Western
blotting detection system (GE Healthcare, Chalfont St Giles, UK).
In vitro EBV-EA Activation. For the protocol for this in vitro assay, refer to [8][22][42].
Cytotoxicity Assay. Cytotoxic activity was assessed using the MTT-based colorimetric assay as
described in [23][38][39].
Assay of TPA-Induced Ear-Edema Inflammation in Mice. For the protocol for this in vivo assay, refer
to [8][43][44].
In vivo Two-stage Carcinogenesis Assay on Mouse-Skin Papillomas. The experiment, conducted at
Kyoto Prefectural University of Medicine, was performed in accordance with the guidelines of the
Committee for Animal Research, Kyoto Prefectural University of Medicine. Each group of specific
pathogen-free ICE mice obtained from Japan SLC, Inc. (Hamamatsu, Japan) was composed of 15 mice
housed five per cage and given H2O ad libitum. The back of each mouse was shaved with surgical clippers,
and the mouse was treated topically with DMBA (100 mg, 390 nmol) in acetone (0.1 ml) for the initiation
treatment. One week after the initiation, papilloma formation was promoted by the application of TPA
(1 mg, 1.7 nmol) in acetone (0.1 ml) on the skin twice a week for 20 weeks. Group I received the TPA
treatment alone, and Groups II and III received a topical application of test samples (85 nmol) in acetone
(0.1 ml) 1 h before each TPA treatment. The incidence and numbers of papillomas were observed and
detected weekly for 20 weeks; only typical papillomas larger than ca. 1 mm in diameter were counted.
For the protocol for this in vivo assay, cf. [44][45].
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Received November 21, 2014