Phytochemistry 108 (2014) 157–170

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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 School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-855, Japan 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 b c

a r t i c l e

i n f o

Article history: Received 25 April 2014 Accepted 3 September 2014 Available online 21 October 2014 Keywords: Shea butter Vitellaria paradoxa Sapotaceae Shea kernel Oleanane-type triterpene glycoside Antioxidant activity Anti-inflammatory activity Melanogenesis-inhibitory activity Epstein–Barr virus early antigen (EBV-EA) Cytotoxic activity

a b s t r a c t 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 cells induced by a-melanocyte-stimulating hormone (a-MSH); generation of 1,1-diphenyl-2-picrylhydrazyl (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 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 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 ⇑ 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: [email protected] (T. Akihisa). http://dx.doi.org/10.1016/j.phytochem.2014.09.017 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

the seed kernel, consisting of an olein fraction and a stearin fraction along with non-saponifiable (non-lipid) compounds. Fractionated 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 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 (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

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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 pharmacological and cosmeceutical potentials of the constituents of defatted shea kernel were of interest, since there seems to be little industrial 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.

2. Results and discussion 2.1. Melanogenesis-inhibitory, antioxidant, EBV-EA-inductioninhibitory, 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 soluble hydrophilic components. The MeOH extract was fractionated into EtOAc-, n-BuOH-, and H2O-soluble fractions. Extracted fractions were evaluated for melanogenesis-inhibitory and cytotoxic activities in a-melanocyte-stimulated hormone (a-MSH)-stimulated 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 13acetate (TPA) in Raji cells, anti-inflammatory activity against TPAinduced inflammation in mice, and cytotoxic activity against four human cancer cell lines by means of a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, respectively. As compiled in Table 1, the MeOH extract, and the EtOAcand BuOH-soluble fractions exhibited potent melanogenesis-inhibitory activities (28.2–58.0% melanin content) at 100 lg ml1 concentration, which were more potent than that of the reference arbutin (4-hydroxyphenyl b-D-glucopyranoside; 87.1% melanin content at 100 lg ml1), but with some cytotoxicities (37.9– 63.4% cell viability). The MeOH extract and the three fractions exhibited potent DPPH free radical-scavenging activities (IC50 6.8–24.3 lg ml1) similar to, though slightly less inhibitory than, that of the reference a-tocopherol (IC50 5.6 lg ml1; Table 1). Upon evaluation of the inhibitory effects against TPA (20 ng)-induced EBV-EA activation in Raji cells, the MeOH extract and the EtOAcsoluble fraction exhibited potent inhibitory effects (6.9% and 5.3% induction of EBV-EA at 100 lg ml1 concentration, respectively), while both the MeOH extract and the H2O-soluble fraction showed inhibitory activity (70% and 81% inhibition at 1.0 mg ml1 concen-

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 lg ml1, 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 lg ml1). 2.2. Isolation, identification, and structure elucidation All three fractions from the MeOH extract were subjected to successive 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 compounds, 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 as tieghemelin A (3), arginine C (5), and 3-O-b-D-glucopyranosyl 16a-hydroxyprotobassic acid (7) (Gosse et al., 2002), butyroside D (4) and 3-O-b-D-glucuronopyranosyl protobassic acid (10) (Li et al., 1994), 3-O-b-D-glucuronopyranosyl 16a-hydroxyprotobassic acid (6) (Gosse et al., 2002; Li et al., 1994), Mi-glycoside I (11) and 3O-b-D-glucopyranosyl bassic acid (15) (Nigam et al., 1992), protobassic acid (12) (Nigam et al., 1992; Sahu, 1996; Toyota et al., 1990), bassic acid (16) (Sahu, 1996; Toyota et al., 1990), spinasterol 3-O-bD-glucopyranoside (17) and 22-dihydrospinasterol 3-O-b-D-glucopyranoside (18) (as the tetraacetate derivatives, 17a and 18a, respectively) (Furuya et al., 1990; Kojima et al., 1990), (2S,4S)-2O-(b-D-glucopyranosyl)pentane-2,4-diol (19) and (2R,4S)-2-O-(bD-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 ()-epicatechin (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 optical 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 spectroscopic 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.

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 (%)

Control (100% DMSO) MeOH extract EtOAc-soluble fraction BuOH-soluble fraction H2O-soluble fraction Arbutinc a-Tocopherolc a

DPPH Free-radical-scavenging activity, IC50 (lg ml1)b

Cell viability (%)

10 lg ml1

100 lg ml1

10 lg ml1

100 lg ml1

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 5.6 ± 0.1

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 ll ml1. b Values of fourfold experiments. Concentration of DMSO in the sample solution was 5 ll/ml. c Reference compounds.

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J. Zhang et al. / Phytochemistry 108 (2014) 157–170 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. Extract or fraction

Percentage EBV-EA inductiona Drug conentrationb (lg ml1) 100

MeOH extract EtOAc-soluble fraction BuOH-soluble fraction H2O-soluble fraction Indomethacine Cisplatine a b c d e

6.9 ± 0.4 5.3 ± 0.5 14 ± 0.6 13.0 ± 0.6

(60) (60) (50) (50)

Inhibition of inflammation

Cytotoxicity, IC50 (lg ml1)d HL-60 A549

AZ521

SK-BR-3

10

1

I.R. (%)c

(Leukemia)

(Lung)

(Stomach)

(Breast)

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

1.3 ± 0.3

5.5 ± 0.6

2.9 ± 0.2

5.6 ± 0.2

Values represent relative percentages to the positive control value. TPA (32 pmol, 20 ng) = 100%. Concentrations in terms of weight ratio 20 ng1 TPA. Values in parentheses are viability percentage of Raji cells. Percent inhibitory ratio (I.R.) at 1.0 mg ear1. IC50 Value was obtained on the basis of triplicate assay results. Reference compounds.

The HRESIMS of compound 1 displayed a sodiated molecular ion at m/z 997.4599 [M+Na]+ consistent with a molecular formula of C47H74O21. The HRESIMS2 experiment of the [M+Na]+ gave fragments 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 hexuronic acid). In the 1H NMR spectrum (Table 3) of the aglycone moiety 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 aglycone moiety of 1 were in accord with those of 16a-hydroxyprotobassic acid (13) (Nigam et al., 1992; Eskander et al., 2006; SànchezMedina 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 aglycon), dH 5.00 [H-1 of a-rhamnopyranosyl (a-Rhap) group] and dC 76.0 [C-2 of a-arabinopyranosyl (a-Arap) group], and dH 5.73 (H1 of a-Arap) and dC 177.0 (C-28 of the aglycon), which suggested the substitution patterns of the aglycone by the sugar moieties assigned as shown in Fig. 1. The coupling constant data (JH–1,H– 2 = 3.7 Hz; JH–2,H–3 = 5.0 Hz) for a-Arap indicated that it was present in the 1C4 conformation, as observed in similar triterpenoid saponins (Eskander et al., 2005, 2006; Sánchez-Medina et al., 2009). Upon acid hydrolysis, compound 1 afforded D-glucuronic acid, L-rhamnose, and L-arabinose, which were identified by GLC 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, the structure of 1 was assigned as 3b-[(b-D-glucuronopyranosyl)oxy]-2b,6b,16a,23-tetrahydroxyolean-12-en-28-oic acid O-a-L-rhamnopyranosyl-(1?2)-a-L-arabinopyranosyl ester (paradoxoside A). Compound 2 exhibited a [M+Na]+ ion peak at m/z 1129.5011 in the HRESIMS, corresponding to a molecular formula of C52H82O25. The 1H and 13C NMR spectra (Table 3) of 2 were almost superimposable with those of 1 except that the former showed additional signals of a b-xylopyranosyl (b-Xylp) moiety. Lower-field glycosylation shifts (+9.5 ppm) (Matsumoto et al., 1990) of the C-4 signal of 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 a-Rhap 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 D-xylose as the sugar units, and aglycone, 13 (Nigam et al., 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-

olean-12-en-28-oic acid O-b-D-xylopyranosyl-(1?4)-a-L-rhamnopyranosyl-(1?2)-a-L-arabinopyranosyl ester (paradoxoside B). The HRESIMS of compound 8 exhibited a quasi-molecular ion at m/z 695.3998 [M+H]+, consistent with a molecular formula of C37H58O12. The MS2 of the [M+H]+ gave a fragment at m/z 469.3309 [(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 8 exhibited six tertiary methyl groups, a primary 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 Dglucuronic acid as the sugar and 12 as the aglycone. The above evidence, 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 experiments of 8 confirmed that this possesses the structure 3b-[(b-Dmethyl glucuronopyranosyl)oxy]-2b,6b,16a,23-tetrahydroxyolean-12-en-28-oic acid (paradoxoside C). Compound 9 exhibited a [M+Na]+ peak at m/z 851.4427 in the HRESIMS, corresponding to a molecular formula of C42H68O16. 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 glucose moieties. Upon acid hydrolysis, 9 furnished 12 and D-glucose, demonstrating that 9 possesses 12 as the aglycone moiety with two D-glucosyl units as the sugar moiety. HMBC experiments 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 aglycone). Hence, structure 9 was established as 3b-[(b-D-glucopyranosyl-(1?3)-b-D-glucopyranosyl)oxy]-2b,6b,16a,23-tetrahydroxyolean-12-en-28-oic acid (paradoxoside D). 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,

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J. Zhang et al. / Phytochemistry 108 (2014) 157–170 29 30

19 18

12

HO RO

11 26 9

25 2 3

1

10 5

4

24 23

6

H

OH

1 2 3 4 5 6 7 8 9 10 11 12 13

R GlcA GlcA GlcA GlcA Glc GlcA Glc MeGlcA Glc-(1 3)-GlcGlcA Glc H H

H

20 17

13 H 14 16 15

8

21 22 28

COOR"

RO

OH R' OH OH OH OH OH OH OH H H H H H OH

OH Rha-(1 Xyl-(1 Rha-(1 Api-(1 Rha-(1 H H H H H H H H

2)-Ara4)-Rha-(1 3)-Xyl-(1 3)-Xyl-(1 3)-Xyl-(1

2)-Ara2)-Rha-(1 2)-Ara2)-Rha-(1 2)-Ara2)-Rha-(1 2)-Ara-

H GlcO

19 R = Me, R' = H 20 R = H, R' = Me

O

OH

23 OH

HO

HO

O

O OR OH O 26 R = H 27 R = Rha-(1 6)-Glc

OH OH

24 HO

25

O HO

4

5

OH HO Api

OH

ROOC HO HO

OH

OH

OH

28

OH OH

OH

OH OH

17 18 Δ22

OH

O

O OH

GlcA R = H MeGlcA R = Me

23

OH

HO

21 R = H 22 R = OMe

OH

HO HO

H

H

GlcO

R R'

22

H

R

GlcO

OH

14 R = MeGlcA 15 R = Glc 16 R = H

R"

OH

HO

COOH

H

27

7

H

HO

R'

Me HO HO

OH O2 3

Ara

O

OH 1

OH

4

HO HO

5 3

O 2

Glc

1

OH

O

HO HO

Rha OH

6

OH Xyl

Fig. 1. Structures of compounds 1–28.

coupled with the cross-correlations between dH 5.35 (H-1 of bMeGlcAp) 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 structure 3b-[(b-D-methyl glucuronopyranosyl)oxy]-2b,6b,23-trihydroxyoleana-5,12-dien-28-oic acid (paradoxoside E). This study has thus established that the defatted shea kernel extract contains various triterpene glycosides of oleanolic acid derivatives, i.e., basic acid, protobassic acid, and 16a-hydroxyprotobassic acid, as aglycones, with sugar chains at the C-3 and C-28 positions. It is worth noting that these kinds of triterpene glycosides have been frequently encountered in various species and

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 isolation 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). While (2R,4S)-2-O-(b-D-glucopyranosyl)pentane-2,4-diol (20) has so far been isolated from the fruits of Crescentia cujete (Bignoniaceae) (Kaneko et al., 1998) and from the stems of Vaccinium myritillus (Ericaceae) (Hybelbauerová et al., 2009), its (2S,4S)-isomer (19) has so far been known only as a synthetic compound

161

J. Zhang et al. / Phytochemistry 108 (2014) 157–170 Table 3 H (400 MHz) and

1

Position

Aglycon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Sugar moiety 3-O-b-GlcA 1 2 3 4 5 6 28-O-a-Ara 1 2 3 4 5 2Ara-O-a-Rha 1 2 3 4 5 6 4Rha-O-b-Xyl 1 2 3 4 5

13

C (100 MHz) NMR Spectroscopic data of compounds 1 and 2 isolated from the defatted shea kernel extract. 1 (CD3OD)

2 (CD3OD)

dH (J in Hz)

dC

dH (J in Hz)

dC

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) 1.65 (dd, 5.5, 13.7) 2.04 (m), 2.14 (m) 5.43 (br. t, 3.7)

1.40 (dd, 3.7, 14.7), 1.79 (m) 4.48 (m) 3.11 (dd, 3.7, 14.2) 1.07 (m), 2.27 (t, 13.7) 1.15 1.79 3.41 1.31 1.63 1.07 1.33

(m), 1.89 (m) (m), 1.92 (m) (br. d, 10.1), 3.73 (br. d, 10.1) (s) (s) (s) (s)

0.89 (s) 0.99 (s)

1.31 (br. s) 4.48 (m) 1.56 (br. d, 13.3), 1.81 (br. d, 13.3) 1.65 (dd, 5.5, 13.7) 2.03 (m), 2.14 (m) 5.41 (br. t, 3.2)

1.43 (br. d, 16.9), 1.80 (m) 4.49 (m) 3.07 (dd, 3.7, 14.0) 1.06 (m), 2.27 (t, 13.3) 1.14 1.76 3.43 1.31 1.62 1.05 1.34

(m), 1.90 (m) (m), 1.91 (m) (br. d, 10.1), 3.71 (br. d, 10.1) (s) (s) (s) (s)

0.88 (s) 0.97 (s)

4.50 3.35 3.39 3.42 3.61

(d, 7.8) (m) (br. t, 9.6) (m) (d, 10.1)

104.6 75.0 77.9 73.7 76.0 177.0

4.51 3.35 3.33 3.48 3.60

(d, 7.8) (m) (m) (m) (d, 10.1)

105.1 74.9 78.1 71.0 75.9 177.0

5.73 3.79 3.90 3.86 3.50

(d, 3.7) (dd, 3.7, 5.0) (m) (m) (dd, 3.7, 10.9), 3.93 (dd, 8.5, 10.9)

93.7 76.0 70.5 66.5 63.1

5.59 3.81 3.86 3.82 3.52

(d, 4.1) (dd, 4.1, 5.5) (m) (m) (dd, 2.8, 11.5), 3.91 (dd, 7.3, 11.5)

94.1 75.3 71.9 67.4 64.3

5.00 3.83 3.64 3.38 3.68 1.27

(d, 1.4) (dd, 1.4, 3.2) (dd, 3.2, 9.6) (m) (m) (d, 6.0)

101.7 72.3 72.1 73.7 70.7 18.0

5.12 3.86 3.86 3.59 3.74 1.30

(br. s) (m) (m) (br. t, 9.2) (m) (d, 6.0)

101.3 72.3 72.1 83.2 68.9 18.1

4.53 3.34 3.24 3.48 3.20

(d, 7.8) (m) (br. t, 8.2) (m) (dd, 10.0, 11.5), 3.86 (dd, 5.0, 11.5)

106.5 74.9 77.7 71.0 67.2

(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 compounds 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-inositol 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 Table 4 H (400 MHz) and

1

Position

Aglycon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

J. Zhang et al. / Phytochemistry 108 (2014) 157–170

13

C (100 MHz) NMR Spectroscopic data of compounds 8, 9, and 14 isolated from the defatted shea kernel extract. 8 (CD3OD)

14 (C5D5N)

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

1.15 2.01 4.24 3.59

(br. d, 14.2) (dd, 1.8, 14.2) (br. q, 2.8) (d, 3.7)

46.5

1.17 2.03 4.34 3.59

(br. d, 14.2) (dd, 1.8, 14.2) (br. q, 2.8) (d, 3.7)

46.5

1.42 2.14 4.85 4.47

46.5

1.32 4.46 1.50 1.78

(br. s) (br. s) (br. d, 14.2) (dd, 3.2, 14.2)

1.32 4.47 1.50 1.78

(br. s) (br. s) (br. d, 14.2) (dd, 3.2, 14.2)

1.60 (m) 1.95 (dt, 4.6, 17.9) 2.11 (ddd, 2.3, 11.5, 17.9) 5.30 (br. t, 3.7)

1.07 1.84 1.60 1.99

(br. d, 17.4) (br. t, 12.8) (br. d, 16.0) (m)

2.87 (dd, 3.2, 13.7) 1.14 (br. d, 10.5) 1.69 (t, 13.7) 1.20 1.38 1.53 1.76 3.43 3.70 1.31 1.61 1.10 1.13

(br. d, 13.0) (dt, 3.2, 13.7) (br. d, 14.2) (m) (d, 11.5) (d, 11.5) (s) (s) (s) (s)

0.91 (s) 0.95 (s)

Sugar moiety 3-O-b-MeGlcA or 3-O-b-Glc 1 4.53 (d, 7.8) 2 3.38 (t, 7.8) 3 3.42 (t, 8.7) 4 3.54 (t, 8.7) 5 3.90 (d, 9.6) 6 MeO-6 3Glc-O-b-Glc 1 2 3 4 5 6

9 (CD3OD)

3.77 (s)

71.6 83.6 44.0 48.8 68.3 41.0 39.6 49.5 37.0 24.5 123.8 144.4 43.5 28.6 24.0 48.4 42.7 47.1 31.5 34.9 33.6 65.0 16.2 19.1 18.7 26.6 181.7 33.7 24.0

105.5 74.8 77.3 73.0 76.4 171.3

1.60 (m) 1.96 (dt, 4.6, 17.9) 2.13 (ddd, 2.3, 11.5, 17.9) 5.30 (br. t, 3.6)

1.08 1.84 1.61 1.98

(br. d, 17.4) (br. t, 12.4) (br. d, 16.9) (m)

2.87 (br. d, 13.7) 1.16 (br. d, 10.5) 1.70 (t, 13.7) 1.20 1.38 1.53 1.76 3.42 3.73 1.31 1.62 1.10 1.14

(br. d, 13.7) (dt, 3.2, 13.7) (br. d, 14.2) (m) (d, 11.0) (d, 11.0) (s) (s) (s) (s)

0.91 (s) 0.95 (s)

4.51 3.54 3.55 3.51 3.32 3.73 3.82

(d, 7.8) (t, 7.8) (t, 9.2) (t, 7.8) (m) (dd, 3.7, 11.9) (dd, 2.3, 11.9)

71.3 83.7 44.0 48.9 68.4 41.1 39.6 49.6 37.1 24.6 123.9 144.5 43.5 28.7 24.0 48.4 42.8 47.2 31.6 34.9 33.6 65.3 16.3 19.2 18.7 26.6 181.8 33.8 24.1

104.8 74.6 88.0 69.5 77.2 62.1

52.9

(dd, 3.7, 14.9) (dd, 3.7, 14.2) (dd, 3.7, 7.3) (d, 3.2)

5.98 (dd, 3.2, 5.0) 2.48 (dd, 5.0, 18.8) 1.76 (dd, 3.2, 18.8) 1.97 (dd, 5.5, 11.0) 2.04 (m) 2.16 (m) 5.60 (br. t, 3.7)

1.26 2.15 2.11 2.19

(m) (m) (m) (m)

3.33 (dd, 3.7, 14.2) 1.80 (br. t, 13.7) 1.29 (br. t, 13.7) 1.20 1.44 1.76 2.07 4.03 4.52 1.70 1.71 1.18 1.24

(m) (m) (m) (m) (d, 10.5) (d, 10.5) (s) (s) (s) (s)

0.94 (s) 1.01 (s)

5.35 4.04 4.21 4.42 4.53

(d, 7.8) (t, 9.2) (t, 9.2) (t, 9.2) (d, 9.2)

3.68 (s) 4.58 3.32 3.40 3.30 3.32 3.65 3.91

(d, 7.8) (m) (t, 8.7) (m) (m) (dd, 5.6, 11.9) (dd, 1.8, 11.9)

activities of these compounds against B16 melanoma cells were also determined by means of MTT (thiazolyl blue tetrazolium bromide) 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 concentration (10, 30, and 100 lM). A compound with smaller A/ C ratio would be a lower-risk skin-whitening agent (Kitdamrongtham et al., 2014). Nine oleanolic acid derivatives, 6–12, 14, and 15, two pentane-2,4-diol glucosides, 19 and 20, six

70.5 81.5 46.7 147.7 120.8 33.1 38.4 45.9 37.2 24.0 123.0 145.0 43.0 27.6 23.5 46.7 42.4 45.8 30.9 34.2 33.0 65.5 23.4 23.7 21.1 26.2 170.7 33.3 23.7

106.4 75.1 77.6 73.2 77.0 170.7 52.0

105.2 75.4 77.7 71.5 78.1 62.6

phenolic compounds, 21, 22, and 24–27, and one cyclitol, 28, tested in this study were proved to be lower-risk melanogenesis inhibitors (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 useful 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

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 (%)

Cell viability (%)

A/C Ratio

30 lM

100 lM

10 lM

30 lM

100 lM

100.0 ± 3.1

100.0 ± 3.1

100.0 ± 3.1

100.0 ± 0.7

100.0 ± 0.7

100.0 ± 0.7

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

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

Pentane-2,4-diol glucoside 19 84.7 ± 1.5 20 74.3 ± 2.7

72.1 ± 5.6 55.7 ± 3.3

67.4 ± 7.5 42.5 ± 4.0

Phenolic compound 21 22 23e 24f 25f 26 27

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

95.7 ± 2.6

91.0 ± 4.1

Controlc

Triterpene acid and triterpene glycoside 1 115.1 ± 2.3 2 94.2 ± 2.0 3 91.2 ± 8.4 4 96.2 ± 3.1 5 105.3 ± 4.3 6 84.7 ± 3.1 7 86.6 ± 4.5 8 86.5 ± 3.7 9 89.2 ± 8.0 10 86.2 ± 2.2 11 83.9 ± 4.0 12 98.3 ± 1.8 14 101.8 ± 2.3 15 97.5 ± 2.0 16 64.6 ± 3.0 Steroid glucoside 17ad 18ad

Cyclitol 28

10 lM

30 lM

100 lM

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

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

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

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

95.4 ± 1.9

107.3 ± 3.5

107.1 ± 5.7

107.7 ± 1.3

0.89

0.85

0.89

J. Zhang et al. / Phytochemistry 108 (2014) 157–170

10 lM

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 ll ml1. c 100% DMSO. d 17a = Tetraacetate derivative of 17; 18a = Tetraacetate derivative of 18. e Data taken from Manosroi et al., 2013. f Data taken from Manosroi et al., 2014.

163

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

Fig. 2. Effects on the expression of MITF, tyrosinase, TRP-1, and TRP-2 in a-MSHstimulated B16 melanoma cells treated with 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-carboxylic acid (DHICA) (Kobayashi et al., 1994). Transcription for expression of these enzymes is regulated by microphthalmia-associated transcription factor (MITF) (Levy et al., 2006). To clarify the mechanism involved in the melanogenesis inhibition by compound 14, which exhibited potent melanogenesis inhibitory activity (42.0%

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. Compound

DPPH Free-radical-scavenging activity, IC50 (lM)a

Percentage EBV-EA inductionb 1000

Triterpene acid and triterpene glycoside 1 >100 2 >100 3 >100 4 >100 5 >100 6 >100 7 >100 8 >100 9 >100 10 >100 11 >100 12 >100 14 >100 15 >100 16 >100 Steroid glucoside 17ae 18ae Pentane-2,4-diol glucoside 19 20 Phenolic compound 21 22 23f 24 25 26 27 Cyclitol 28

c

IC50d c

c

c

500

100

10

9.8 ± 0.5 10.1 ± 0.6 13.6 ± 0.7 13.2 ± 0.6 14.9 ± .0.7 0 ± 0.4 0 ± 0.3 0 ± 0.4 6.5 ± 0.6 0 ± 0.3 0 ± 0.3 0 ± 0.2 1.2 ± 0.3 0 ± 0.2 0 ± 0.3

(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

>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

>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

>100 46.4 ± 6.1 10.9 ± 2.5 7.1 ± 3.2 5.8 ± 2.3 12.9 ± 3.4 6.0 ± 0.3

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

>100

19.8 ± 0.8

(70)

59.3 ± 1.5

89.4 ± 2.1

100.0 ± 0.3

563

8.6 ± 0.5

(70)

34.2 ± 1.0

82.1 ± 2.0

100.0 ± 0.3

397

Reference compound

a-Tocopherol b-Carotene a

13.0 ± 2.3

Each sample was measured in triplicate. IC50 values were determined by the method of probit-graphic interpolation of six concentration levels. b 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 ratio 32 pmol1 TPA. Data are expressed as mean ± S.D. (n = 3). c Values in parentheses are viability percentages of Raji cells. d IC50 represents the molar ratio of compound, relative to TPA, required to inhibit 50% of the positive control activated with 32 pmol TPA. e 17a = Tetraacetate derivative of 17; 18a = Tetraacetate derivative of 18. f Data taken from Manosroi et al., 2013.

165

J. Zhang et al. / Phytochemistry 108 (2014) 157–170 Table 7 Inhibition of inflammation in mice and cytotoxic activities of compounds isolated from defatted shea kernel extract. Compound

ID50a

1

(lmol ear

)

Triterpene acid and triterpene glycoside 1 0.18 2 0.17 3 0.18 4 0.12 5 0.19 6 7 8 0.07 9 0.13 10 0.16 11 0.13 12 0.02 14 15 0.05 16 0.38 Phenolic compound 21 22 23 26 27 Reference compound Indomethacin Cisplatin a b c d e

Cytotoxicity, IC50 ± S.D. (lM)d,

Inhibition of inflammation 95% CI

b

c

I.R. (%)

0.15–0.21 0.13–0.24 0.14–0.24 0.10–0.13 0.13–0.29

0.06–0.08 0.10–0.19 0.13–0.20 0.11–0.15 0.20–0.29 0.03–0.06 0.30–0.47 29± 2.5 31 ± 3.9

0.94 0.91

0.72–1.24 0.78–1.06

0.91

0.76–1.09

e

HL-60

A549

AZ521

SK-BR-3

(Leukemia)

(Lung)

(Stomach)

(Breast)

>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 7.6 ± 0.1 >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 72.7 ± 4.3 >100 32.0 ± 1.6 29.7 ± 0.8

>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

4.2 ± 1.1

18.4 ± 1.9

9.5 ± 0.5

18.8 ± 0.6

96 ± 3.3

ID50: 50% Inhibitory dose. 95% Confidence intervals. Percent inhibitory ratio at 1.0 mg/ear. Each value represents the mean ± S.D. (n = 5). Cells were treated with compounds (1  104 to 1  106 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 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, tyrosinase, TRP-1, and TRP-2 proteins, mostly in a concentration-dependent manner (Fig. 2). These results suggested that 14 exhibits melanogenesis inhibitory activity on a a-MSH stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase, TRP-1, and TRP-2. 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-radicalscavenging activities (IC50 5.8–46.4 lM), among which, compounds 23–27 showed strong radical-scavenging activities (IC50 5.8–12.9 lM) which were more potent than, or almost equivalent to, the reference a-tocopherol (IC50 13.0 lM). This can be explained by the presence of more phenolic OH groups leading to higher radical-scavenging activity due to the increase of the H-radical-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).

2.6. Inhibitory effects on EBV-EA induction The inhibitory effects of compounds 1–12 and 14–28 (as the tetraacetate 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 precursor 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 (concentration for 50% inhibition with respect to the positive control) of 293– 563 M ratio 32 pmol1 TPA. Among these, nine oleanolic acid derivatives, 6–8, 10–12, and 14–16, and three flavonoids without a glycosyl group, 24–26, exhibited potent inhibitory effects (293–380 M ratio 32 pmol1 TPA) which were higher than that of b-carotene (397 M ratio 32 pmol1 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 pmol1 TPA) than the monodesmosides glycosylated 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 inhibitory 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. 2.7. 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,

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and four phenolic compounds, 21, 23, 26, and 27, were evaluated for their anti-inflammatory activities against TPA-induced inflammation in mice in comparison to indomethacin, a commercially available anti-inflammatory drug (Table 7). All oleanolic acid derivatives tested exhibited marked anti-inflammatory activities with the 50% inhibitory doses (ID50) of 0.02–0.38 lmol ear1, i.e., they were more potent than reference indomethacin (ID50 0.91 lmol ear1). Compounds 26 and 27 exhibited anti-inflammatory activities (ID50 0.94 and 0.91 lmol ear1, respectively) almost equivalent to that of reference indomethacin. The anti-inflammatory 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 glycosides (Manosroi et al., 2013; Ukiya et al., 2006, 2007) have also been observed in our previous studies.

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 lM, the other 16 compounds were inactive against all cell lines tested (IC50 > 100 lM). In particular, the cytotoxic activities of 3 and 4 against A549 cell line (IC50 13.5 and 19.1 lM, respectively) and 12 against HL-60 cell line (IC50 7.6 lM) were more potent than, or almost comparable with, those of the reference cisplatin [IC50 18.4 lM (A549), 4.2 lM (HL-60)]. Based on the results compiled in Tables 1 and 7, it is highly possible that two phenolic compounds, 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 glycosylated, 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, respectively), 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 apoptosisinducing 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%

83.0%

11.6%

50.8%

13.4%

PI

Negave control 24

48 (h)

1.4%

1.9%

1.7%

2.0%

93.9%

2.8%

93.5%

2.8%

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.

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lyzed by means of flow cytometry with annexin V–propidium iodide (PI) double staining. Exposure of the membrane phospholipid 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 membranes, and was thus used to differentiate between early apoptotic (annexin V positive, PI negative), late apoptotic (annexin V, PI double 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 negative 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. 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 compounds as active principles, which exhibit melanogenesis-inhibitory activity in a-MSH-stimulated B16 melanoma cells, DPPH free-radical-scavenging activity, inhibitory effects against TPAinduced 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 radical-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 compounds, 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. 4. Experimental 4.1. General Melting points were determined on a Yanagimoto micro melting 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. HRESIMS were obtained on an Agilent 1100 LC/MSD TOF (time-offlight) system [ionization mode: positive; nebulizing gas (N2) pressure: 35 psig; drying gas (N2): flow, 12 1 min1; 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 min1; temp: 350 °C; capillary voltage: 3000 V; fragmentor

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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; injection and detector temp., 270 °C; He flow rate, 0.4 ml min1; split ratio, 1:75). SiO2 (Silica gel 60, 230–400 mesh; Merck), Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan), and ODS (Chromatorex-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 min1 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)].

4.2. Chemicals and materials The shea nut sample used in this study was collected and identified 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°20 7900 , latitude N 9°400 5300 , 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 ml1 penicillin and 100 mg ml1 streptomycin), and non-essential amino acid (NEAA) from Invitrogen Co. (Carlsbad, CA, USA), DMBA, indomethacin, Dulbecco’s modified Eagle’s medium (D-MEM), Eagle’s minimal essential medium (MEM), arbutin, 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 [hexamethyldisilazane–trimethylchlorosilane (HMDS–TMCS) in anhydrous pyridine] from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), D-arabinose, L-rhamnose, and D-xylosem from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), D-glucose and L-cysteine methyl 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.

4.3. 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).

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4.4. Extraction and isolation Whole nuts were oven-dried at 60 °C over 72 h and decorticated. Kernels were crushed into powder first. The pulverized sample was weighed (3705 g), and extracted with hexane (under conditions of reflux, 3 h, 3) which gave an extract (1737 g) (Akihisa et al., 2010c). The defatted residue was then extracted with MeOH (under conditions of reflux, 3 h, 3) 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. 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 column 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 fraction 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 compounds 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 compounds 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

(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. 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; a25 D 38.8 (c 0.41, EtOH); UV (EtOH) kmax nm: 264, 394; IR (KBr): mmax cm1: 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 C30H48O7Na, 543.3297), 499.3384 [(M+Na) – Ara – Rha – GlcA – CO2]+ (calcd for C29H48NaO5, 499.3399). 4.4.2. Paradoxoside B (2) White amorphous powder from MeOH, mp 217–220 °C; a25 D 35.3 (c 0.35, EtOH); UV (EtOH) kmax nm: 264, 441; IR (KBr): mmax 1 1 cm : 3436 (OH), 2930, 1638 (C@O), 1385, 1074, 1040; For H 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; a25 D +13.5 (c 1.15, EtOH); UV (EtOH) kmax nm: 245, 250, 256; IR (KBr): mmax cm1: 3436 (OH), 2930, 1638 (C@O), 1385, 1073, 1040; For 1 H 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; a25 D +20.0 (c 0.87, EtOH); UV (EtOH) kmax nm: 244, 250, 255; IR (KBr): mmax cm1: 3410 (OH), 2930, 1700 (C@O), 1384, 1070, 1037; For 1 H NMR and 13C NMR spectroscopic data, see Table 4; HRESIMS m/z: 851.4427 [M+Na]+ (calcd for C42H68O16Na, 851.4405); HRESIMS2 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; a25 D +15.5 (c 0.20, EtOH); UV (EtOH) kmax nm: 272; IR (KBr): mmax cm1: 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:

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451.3218 [(M+H) – MeGlcA – 2H2O]+ (calcd for C30H43O3, 451.3212). 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 combined EtOAc layers thus extracted were washed with H2O and evaporated to dryness to afford the corresponding aglycone. The aqueous residue was concentrated to dryness by repeatedly adding MeOH to remove acid and analyzed by TLC in comparison with sugar standards. The absolute configuration of sugar residues was determined by GLC analysis of their chiral trimethylsilyl thiazolidine derivatives (Hara et al., 1987). The details of the acid hydrolysis of compounds 1, 2, 8, 9, and 14 are described in Supplementary data (S1). 4.6. Biological evaluation The protocols of assay of melanin content, in vitro EBV-EA activation experiment, determination of DPPH free-radical-scavenging activity, assay of TPA-induced ear-edema inflammation in mice, cytotoxicity assay, Annexin V–propidium iodide (PI) double staining, and Western blot analysis (Akazawa et al., 2006; Akihisa et al., 2006, 2007, 2010a; Kikuchi et al., 2007, 2011; Tabata et al., 2005) were described in Supplementary data (S2). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 09.017. References Akazawa, H., Akihisa, T., Taguchi, Y., Banno, N., Yoneima, R., Yasukawa, K., 2006. Melanogenesis inhibitory and free radical scavenging activities of diarylheptanoids and other phenolic compounds from the bark of Acer nikoense. Biol. Pharm. Bull. 29, 1970–1972. Akihisa, T., Yasukawa, K., Tokuda, H., 2003. Potentially cancer chemopreventive and anti-inflammatory terpenoids from natural sources. In: Atta-ur-Rahman (Ed.), Studies in Natural Products Chemistry. Vol. 29: Bioactive Natural Products (Part J). Elsevier Science, Amsterdam, pp. 73–126. Akihisa, T., Taguchi, Y., Yasukawa, K., Tokuda, H., Akazawa, H., Suzuki, T., Kimura, Y., 2006. Acerogenin M, a cyclic diarylheptanoid, and other phenolic compounds from Acer nikoense and their anti-inflammatory and anti-tumor-promoting effects. Chem. Pharm. Bull. 54, 735–739. Akihisa, T., Nakamura, Y., Tagata, M., Tokuda, H., Yasukawa, K., Uchiyama, E., Suzuki, T., Kimura, Y., 2007. Anti-inflammatory and anti-tumor-promoting effects of triterpene acids and sterols from the fungus Ganoderma lucidum. Chem. Biodiversity 4, 224–231. Akihisa, T., Seino, K., Kaneko, E., Watanabe, K., Tochizawa, S., Fukatsu, M., Banno, H., Metori, K., Kimura, Y., 2010a. Melanogenesis inhibitory activities of iridoid-, hemiterpene-, and fatty acid-glycosides from the fruits of Morinda citrifolia (noni). J. Oleo Sci. 59, 49–57. Akihisa, T., Kojima, N., Kikuchi, T., Yasukawa, K., Tokuda, H., Masters, E.T., Manosroi, A., Manosroi, J., 2010b. Anti-inflammatory and chemopreventive effects of triterpene cinnamates and acetates from shea fat. J. Oleo Sci. 59, 273–280. Akihisa, T., Kojima, N., Katoh, N., Ichimura, Y., Suzuki, H., Fukatsu, M., Maranz, S., Masters, E.T., 2010c. Triterpene alcohol and fatty acid composition of shea nuts from seven African countries. J. Oleo Sci. 59, 351–360. Akihisa, T., Kojima, N., Katoh, N., Kikuchi, T., Fukatsu, M., Shimizu, N., Masters, E.T., 2011. Triacylglycerol and triterpene ester composition of shea nuts from seven African countries. J. Oleo Sci. 60, 385–391. Alander, J., 2004. Shea butter – a multifunctional ingredient for food and cosmetics. Lipid Technol. 16, 202–205. Atta, E.M., Hashem, A.I., Ahmed, A.M., Elqosy, S.M., Jaspars, M., El-Sharkaw, E.R., 2011. Phytochemical studies on Diplotaxis harra growing in Sinai. Eur. J. Chem. 2, 535–538. Banno, N., Akihisa, T., Tokuda, H., Yasukawa, K., Higashihara, H., Ukiya, M., Watanabe, K., Kimura, Y., Hasegawa, J., Nishino, H., 2004. Triterpene acid from

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