Limonoids from Azadirachta indica var. siamensis extracts and their cytotoxic and melanogenesis-inhibitory activities.

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Limonoids from Azadirachta indica var. siamensis Extracts and Their Cytotoxic and Melanogenesis-Inhibitory Activities by Aranya Manosroi a ), Worapong Kitdamrongtham a ), Kenta Ishii b ), Takuro Shinozaki b ), Yosuke Tachi b ), Mio Takagi b ), Kodai Ebina b ), Jie Zhang b ), Jiradej Manosroi* a ), Rima Akihisa c ), and Toshihiro Akihisa* b ) c ) a ) Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand (phone: þ 66-53-944338; fax: þ 66-53-894169; e-mail: [email protected]) b ) College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan c ) Akihisa Medical Clinic, 1086-3 Kamo, Sanda-shi, Hyogo 669-1311, Japan (fax: þ 81-79-5671980; e-mail: [email protected])

Six new limonoids, 7-benzoyl-17-epinimbocinol (5), 3-acetyl-7-tigloylnimbidinin (8), 1-isovaleroyl1-detigloylsalanninolide (15), 2,3-dihydro-3a-methoxynimbolide (16), deacetyl-20,21-epoxy-20,22-dihydro-21-deoxyisonimbinolide (26), and deacetyl-20,21,22,23-tetrahydro-20,22-dihydroxy-21,23-dimethoxynimbin (27), along with 28 known limonoids, 1 – 4, 6, 7, 9 – 14, 17 – 25, and 28 – 34, and two known flavonoids, 35 and 36, have been isolated from the extracts of bark, leaves, roots, and seeds of Azadirachta indica A. Juss. var. siamensis Valeton (Siamese neem tree; Meliaceae). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. All of these compounds were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines. Eleven compounds, 1, 2, 4 – 7, 13, 16, 17, 29, and 30, exhibited potent cytotoxicities against one or more cell lines with IC50 values in the range of 0.1 – 9.3 mm. Compound 16 induced apoptotic cell death in AZ521 cells upon evaluation of the apoptosis-inducing activity by flow cytometric analysis. Western blot analysis on AZ521 cells revealed that compound 16 activated caspases-3, -8, and -9, while increasing the ratio of Bax/Bcl-2. This suggested that 16 induced apoptosis via both mitochondrial and death receptor pathways in AZ521. In addition, upon evaluation of all compounds against the melanogenesis in B16 melanoma cells induced with amelanocyte-stimulating hormone (a-MSH), 20 limonoids, i.e., 1 – 3, 6, 9 – 11, 18, 19, 21 – 29, 32, and 34, and two flavonoids, 35 and 36, exhibited melanogenesis-inhibitory activities, with no, or almost no, toxicities to the cells at lower and/or higher concentrations, which were more potent than the reference arbutin, a known melanogenesis inhibitor. Western blot analysis showed that nimbin (18) reduced the protein levels of microphtalmia-associated transcription factor (MITF), tyrosinase, tyrosine-related protein 1 (TRP-1), and TRP-2 mostly in a concentration-dependent manner, indicating that 18 inhibits melanogenesis 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.

Introduction. – Siamese neem tree (Azadirachta indica A. Juss. var. siamensis Valeton), one of the two varieties of neem tree (Azadirachta indica A. Juss.) of the family Meliaceae, occurs throughout Southeast Asia including Laos, Myanmar, Cambodia, and Thailand. Phenotype characteristics of Siamese neem tree have reportedly include less branching, longer and thicker leaflets, larger and denser inflorescence, and larger fruits [1]. Several parts of A. indica var. siamensis are used for various medicinal purposes. The flowers were traditionally used as a bitter tonic, febrifuge, and to treat 2014 Verlag Helvetica Chimica Acta AG, Zrich

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nasal polyposis, while the stem bark was used for the treatment of diarrhea and amoebic dysentery. The fruit was used as an anthelmintic, and for treatment of hemorrhoids and kidney complaints. The root bark was used as an antipyretic, emetic, and for the treatment of skin diseases. Leaves were used as a bitter tonic, to stimulate gastric secretion, and to treat malaria fever [2]. There are very few publications concerning the chemical constituents and biological activities of A. indica var. siamensis. The flower extract induced decrease of the activity of phase-I enzymes cytochrome P450, aniline hydroxylase (ANH), and aminopyrone-N-demethylase (AMD), while increasing the activity of phase-II enzyme glutathione-S-transferase (GST) in rats [3]. Water extracts from leaves, stem, stem bark, and branches exhibit moderate toxicities against the oriental fruit fly (Dacus dorsalis Hel.) [4]. Nimbolide, a limonoid from A. indica var. siamensis, showed antimalarial activity against Plasmodium falciparum in culture [5]. In addition, strong antioxidant activities of leaf, fruit, and stem bark extracts from A. indica var. siamensis have been reported [6]. In the course of a search for potential bioactive compounds from Meliaceae plants, a detailed investigation on the limonoid constituents of A. indica (neem tree) seed extracts was undertaken, and this revealed that some limonoids exhibit potent inhibitory activities against melanogenesis in B16 melanoma cells, against 12-Otetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, and against

508


TPA-induced EpsteinBarr virus early antigen (EBV-EA) activation [7] [8], as well as cytotoxic and apoptosis-inducing activities [9]. Further, we have reported that some limonoids from the fruits of Melia azedarach (chinaberry tree) exhibited potent cytotoxic activities against HL60, A549, AZ521, and SK-BR-3 human cancer cell lines [10], and apoptosis-inducing activities against HL60 and AZ521 cell lines [11]. In our continuing study on the constituents of Meliaceae plants, the flower extract of A. indica var. siamensis has been demonstrated to contain various flavonoids and limonoids, and some of these compounds have been shown to exhibit potent melanogenesis-inhibitory and cytotoxic activities [12]. Our continuing study on the bark, leaf, root, and seed extracts of A. indica var. siamensis led us to the isolation of 34 limonoids, 1 – 34, and two flavonoids, 35 and 36, including six new limonoids, 5, 8, 15, 16, 26, and 27. Herein, we describe the structure elucidation of these compounds and evaluation of their cytotoxic activities against four human cancer cell lines, as well as the inhibitory activities against the melanogenesis in a-melanocyte-stimulating hormone (a-MSH)-stimulated B16 melanoma cells. Results and Discussions. – Cytotoxic and Melanogenesis-Inhibitory Activities of the Extracts of A. indica var. siamensis. Dried and powdered bark, leaves, and roots of A. indica var. siamensis were extracted with MeOH, while dried and powdered seeds of the plant were extracted with hexane and MeOH successively. The MeOH extracts of the bark and roots were fractionated into hexane-, AcOEt-, BuOH-, and H2O-soluble fractions, while the MeOH extracts of leaves and seeds were fractionated into hexane-, AcOEt-, and H2O-soluble fractions, and AcOEt-, BuOH-, and H2O-soluble fractions, respectively. All extracts and fractions were evaluated for their cytotoxic activities against four human cancer cell lines, HL60 (leukemia), A549 (lung), AZ521 (stomach), and SK-BR-3 (breast), by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay, and the results are collected in Table 1. Among the extracts evaluated, the MeOH extract of the roots was cytotoxic especially against HL60 (IC50 7.3 mg/ml) and AZ521 (IC50 2.6 mg/ml) cells, the MeOH extract of the leaves against HL60 (IC50 7.9 mg/ml) cells, and the hexane extract of the seeds against SK-BR-3 cells (IC50 8.7 mg/ml). On the other hand, the MeOH extracts of the bark and seeds showed no cytotoxicity against all cell lines, and moderate cytotoxicities against three of the four cell lines tested, respectively. The hexane-soluble fraction of the root MeOH extract exhibited the most potent cytotoxicities (IC50 0.8 – 2.3 mg/ml) against all cell lines tested which were more potent than the reference chemotherapeutic drug, cisplatin (Table 1), and the AcOEt-soluble fraction of the seed MeOH extract also showed potent cytotoxicities (IC50 7.0 – 8.8 mg/ml) against all cancer cell lines tested. All extracts and fractions of A. indica var. siamensis were then evaluated for their melanogenesis-inhibitory and cytotoxic activities in a-MSH-stimulated B16 melanoma cells (Table 1). To assess the risk/benefit ratio of each extract or fraction, 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 extract or fraction and concentration (Table 1). A test sample with smaller A/C ratio would be a lower-risk skin-whitening agent. The MeOH extracts of the bark and leaves and the hexane extract of the seeds exhibited small A/C ratios, i.e., 0.61 – 0.82, with

96.5 2.9

87.4 1.1 107.2 1.4 87.4 0.7 106.5 5.9 108.4 5.2

43.6 0.9 5.6 0.1 33.7 4.5 88.3 2.1 93.9 8.2

80.9 4.2 96.7 5.0 79.5 3.5 105.1 3.6

80.1 4.2 56.1 1.7 57.1 1.9 99.8 7.0 100.5 6.1

100.0 3.1

10 mg/ml

87.1 2.8

0.3 0.3 68.7 5.3 0.1 0.3 96.0 7.1 107.1 9.7

8.9 0.4 3.1 0.4 22.4 1.9 41.3 3.2 93.3 4.3

43.4 2.5 29.5 1.9 9.8 2.0 54.9 4.1

26.4 1.8 16.5 0.9 21.9 1.3 97.9 1.5 94.6 7.1

100.0 3.1

100 mg/ml

Cell viability [%]

1.02

0.61 1.03 0.69 0.74 0.80

0.06 0.25 0.11 0.86 1.09

0.82 0.52 1.28 0.77

0.70 1.36 1.13 0.67 0.77

10 mg/ml

0.79

84.0 0.17 > 100 0.79 0.77

0.22 0.32 0.15 0.64 0.96

0.31 0.17 1.06 0.87

1.34 3.46 0.05 0.16 0.35

100 mg/ml

A/C Ratio c )

a ) IC50 values based on triplicate five points. Cells were treated with the test samples (1 10 4 – 1 10 6 g/ml) for 48 h, and cell viability was analyzed by the MTT assay. Each value represents the mean S.D. b ) 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 ¼ 3). Concentration of DMSO in the sample solution was 2 ml/ml. c ) A/C Ratio: activity/cytotoxicity ratio, which was obtained by dividing the melanin content [%] by the cell viability [%] at each concentration. d ) Reference compounds.

98.7 9.7

68.9 2.3

5.6 0.2

Cisplatin d ) Arbutin d )

2.9 0.2

25.2 1.6 11.5 1.0 28.8 5.6 75.7 7.2 82.6 7.9

Seeds Hexane extract 19.8 8.6 38.0 2.5 12.4 1.4 8.7 1.4 53.2 7.8 MeOH Extract 45.5 1.0 > 100 65.0 8.1 71.6 3.4 110.7 2.8 AcOEt-Soluble fraction 7.5 0.1 7.2 2.3 8.8 0.7 7.0 0.8 60.4 3.9 BuOH-Soluble fraction > 100 > 100 > 100 > 100 78.4 7.2 H2O-Soluble fraction > 100 > 100 > 100 > 100 87.2 8.4 5.5 0.6

2.0 0.4 1.0 0.4 3.4 0.6 26.6 0.5 89.6 3.4

Roots MeOH Extract 7.3 0.5 15.5 0.4 2.6 0.5 25.1 3.4 2.6 0.5 Hexane-soluble fraction 0.9 0.1 1.0 0.3 0.8 0.4 2.3 0.2 1.4 0.7 AcOEt-Soluble fraction 8.3 0.7 > 100 9.5 1.4 29.9 0.7 3.8 1.6 BuOH-Soluble fraction > 100 > 100 > 100 > 100 75.6 2.9 H2O-Soluble fraction > 100 > 100 > 100 > 100 102.2 0.7

1.3 0.3

13.3 2.1 5.1 2.3 10.4 1.2 47.9 3.2

> 100 > 100 > 100 > 100

> 100 > 100 66.5 4.8 81.1 0.6 47.3 7.9 50.4 9.2 34.4 4.2 > 100 101.6 5.2 > 100 > 100 81.3 6.4

Leaves MeOH Extract 7.9 3.0 Hexane-soluble fraction 50.9 8.7 AcOEt-Soluble fraction 70.4 4.1 H2O-Soluble fraction > 100

56.0 3.1 76.1 4.0 64.8 6.2 66.5 7.0 77.7 7.3

Bark MeOH Extract > 100 > 100 > 100 > 100 Hexane-soluble fraction 13.9 3.1 27.5 5.3 36.2 6.5 22.4 5.6 AcOEt-Soluble fraction 11.7 2.3 > 100 86.7 4.4 > 100 BuOH-Soluble fraction 84.9 1.5 > 100 95.6 8.9 > 100 76.4 1.2 > 100 87.9 6.2 > 100 H2O-Soluble fraction 35.3 5.5 57.1 2.5 1.2 2.4 16.1 1.5 32.9 2.8

100.0 4.2

Melanin content [%] 100 mg/ml

SK-BR-3 ( Breast) 10 mg/ml

AZ521 ( Stomach)

Melanogenesis-inhibitory activity and cytotoxicity b ), and A/C ratio c )

100.0 4.2

HL60 A549 ( Leukemia) ( Lung)

Cytotoxicity, IC50 [mg/ml] a )

Control (100% DMSO )

Extract or fraction

Table 1. Cytotoxicities in Human Cancer Cells, and Melanogenesis-Inhibitory Activities and Cytotoxicities in B16 Mouse Melanoma Cell Line of Azadirachta indica var. siamensis Extracts

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53.2 – 66.5% melanin content without significant inhibition of cell proliferation (80.1 – 87.4% cell viability) at 10 mg/ml, which were more potent than those of known melanogenesis inhibitor and useful depigmentation compound for skin whitening in the cosmetic industry [13], namely, arbutin (4-hydroxyphenyl b-d-glucopyranoside; A/C ratio 1.02 with 98.7% melanin content and 96.5% cell viability at 10 mg/ml). In addition, the BuOH- and H2O-soluble fractions of the bark extract, the hexane- and H2O-soluble fractions of the leaf extract, the BuOH-soluble fraction of the root extract, and the AcOEt-, BuOH-, and H2O-soluble fractions of the seed extract exhibited small A/C ratios, i.e., 0.52 – 0.86, with 50.4 – 81.3% melanin content, and with no or low cytotoxicities (87.4 – 108.4% cell viability) at 10 mg/ml. Among these, the BuOH- and H2O-soluble fractions of the bark extract showed very small A/C ratios, i.e., 0.16 and 0.35, respectively, with potent melanogenesis-inhibitory activities (16.1 and 32.9% melanin content, resp.), and with almost no cytotoxicity (97.9 and 94.6% cell viability, resp.) at 100 mg/ml, suggesting that these fractions may be candidates as potential skinwhitening agents. The MeOH extract of the root, and its hexane- and AcOEt-soluble fractions also exhibited very small A/C ratios at 10 and 100 mg/ml, i.e., 0.06 – 0.25 and 0.15 – 0.32, respectively, with significant melanogenesis-inhibitory activities (1.4 – 3.8 and 1.0 – 3.4% melanin contents at 10 and 100 mg/ml, resp.), but most of the inhibitory activities might be due to their cytotoxicities (5.6 – 43.6 and 3.1 – 22.4% cell viabilities at 10 and 100 mg/ml, resp.). The AcOEt-soluble fraction of the bark extract, the hexane- and AcOEt-soluble fractions of the leaf and root MeOH extracts, and the hexane extract and the AcOEtsoluble fraction of the MeOH extract of seeds were further investigated for their constituents in this study. Investigation of the other fractions is still undergoing. Isolation, Identification, and Structure Elucidation of Compounds. i) Bark Extract. The AcOEt-soluble fraction of the A. indica var. siamensis bark MeOH extract was subjected to column chromatography (CC) over SiO2 and octadecyl silica gel (ODS), and reversed-phase (RP) preparative high-performance liquid chromatography (HPLC), which enabled the isolation of 13 limonoids, 8 – 10, 17 – 20, 23, 24, 28, and 32 – 34, and two flavonoids, 35 and 36, of which compound 8 was a new compound. The 14 known compounds were identified as 3-acetyl-7-tigloylvilasinin lactone (9) [14], salannin (10) [15], nimbolide (17) [16], nimbin (18) [17], 6-deacetylnimbin (19) [17], nimbandiol (20) [18], isonimbinolide (23) [19], 6-deacetylisonimbinolide (24) [20], 3acetyl-11-methoxy-1-tigloylazadirachtinin (28) [21], nimbisonol (32) [22], nimbione (33) [19], nimbionone (34) [19], (þ)-catechin (35) [23] [24], and ()-epicatechin (36) [23] [24] by comparison of their spectral data with those in the literature. ii) Leaf Extract. The hexane extract of A. indica var. siamensis leaves was passed through Diaion HP-20 column to remove chlorophylls. The almost chlorophyll-free eluate was subjected to CC (SiO2 and ODS) and preparative HPLC to yield two limonoids, 25 and 26, of which 26 was a new compound. The known compound 25 was identified as 6-deacetylnimbinolactone [25] by spectral comparison with literature. The AcOEt-soluble fraction of the MeOH extract was subjected to CC (SiO2 ) and preparative HPLC which afforded four limonoids, 22 – 24 and 27, of which 27 was a new compound. Among the three known compounds, 22 was identified as 6-deacetylnimbinolide [20] by spectral comparison with literature, while the identification of 23 and 24 was described above.


511

iii) Root Extract. The hexane- and AcOEt-soluble fractions of the MeOH extract of A. indica var. siamensis roots were submitted to CC (SiO2 and ODS) and preparative HPLC to give seven compounds 11 – 13, 15, 16, 18, and 20, and four compounds 14, 19, 21, and 31, respectively, of which 15 and 16 were new compounds. Among the nine known compounds, 18 – 20 have been isolated also from the bark extract as described above, and the six other known compounds were identified as 3-deacetylsalannin (11) [17], salannol (12) [26], salannol 3-acetate (13) [26], 1-benzoyl-1-decinnamoylohchinin acetate (14) [27], 6-acetylnimbandiol (21) [18], and nimbidiol (31) [28] by comparison of their spectral data with those in the literature. iv) Seed Extract. Three compounds, 2 – 4, from the hexane extract, and six compounds, 1, 5 – 7, 29, and 30, from the AcOEt-soluble fraction of the MeOH extract of A. indica var. siamensis seeds were isolated by CC (SiO2 and ODS) and preparative HPLC. Among these compounds, 5 was a new compound, and the other eight known compounds were identified as nimonol (1) [29], 23-deoxyazadironolide (2) [7], azadiradione (3) [30], 7-benzoylnimbocinol (4; 7-deacetyl-7-benzoylazadiradione) [31], epoxyazadiradione (6) [30], 7-deacetyl-7-benzoylepoxyazadiradione (7) [31], gedunin (29) [32], and 7-deacetyl-7-benzoylgedunin (30) [31] by comparison of their spectral data with those in the literature. The structures of six new compounds, 5, 8, 15, 16, 26, and 27, were elucidated on the basis of spectroscopic analysis and comparison with literature as described below. Compound 5 gave a [M þ Na] þ ion peak in the positive-ion-mode HR-ESI-MS at m/z 535.2450 (C33H36NaO þ5 ; calc. 535.2460), consistent with the molecular formula C33H36O5 . The UV spectrum showed an absorption maximum at 231 nm, evidencing the presence of an a,b-unsaturated ketone system, while the IR spectrum displayed the absorption bands of ester C¼O (1728 cm 1), conjugated cyclopentenone (1716 cm 1), conjugated cyclohexenone (1672 cm 1), and furanyl (875 cm 1) functions. The 1H- and 13 C-NMR (Tables 2 and 3, resp.) data of 5 and 17-epiazadiradione [33] differed only slightly from each other. The signals for the AcO group were missing, but there were additional signals corresponding to a benzoyl (Bz) group. The HMB cross-correlation (Fig. 1) for HC(7) (d(H) 5.64) with C(7’) (d(C) 165.2), and the NOE correlation (Fig. 1) between the 1H-signals of HC(7) and Me(30) (d(H) 1.36) of 5 indicated that the Bz group was located at C(7) in an a-orientation. Further NOE correlation between the 1H-signals of HC(17) (d(H) 3.28) and Me(18) (d(H) 1.48) of 5 supported that the furanyl ring at C(17) was b-oriented. Hence, the structure of 5 was assigned as 7-benzoyl-17-epinimbocinol ( ¼ 7-deacetyl-7-benzoyl-17-epiazadiradione). Compound 8 gave a [M þ Na] þ ion peak in the positive-ion-mode HR-ESI-MS at m/z 589.2797 (C33H42NaO þ8 ; calc. 589.2777), consistent with the molecular formula C33H42O8 . The IR spectrum of 8 indicated the presence of OH (3340 cm 1), ester C¼O (1725 cm 1), C¼O (1710 cm 1), and furan (875 cm 1) functions. The 1H- and 13C-NMR (Tables 2 and 3, resp.) data of 8 indicated the presence of four tertiary Me groups(d(H) 0.96, 1.02, 1.14, and 1.19), one Ac Me group (d(H) 2.06), one tigloyl group (d(H) 1.78 (Me(4’)), 1.86 (Me(5’)), and 6.89 (HC(3’))) [14], one CH2O group (d(H) 3.31 and 3.53 (2d, J ¼ 7.8); d(C) 77.9), three CHO groups (d(H) 3.55, 5.11, and 5.75), one vinyl CH group (d(H) 5.68; d(C) 123.6), one C¼O moiety (d(C) 213.7), and a b-substituted furan ring (d(H) 6.56, 7.32, and 7.33; d(C) 112.5, 124.6, 140.9, and 142.3) [34], suggesting that 8 possesses a nimbidinin- (12-oxovilasinin-) type skeletal structure [34 –

4.95 (dd, J ¼ 2.3, 3.7)

5.11 (t, J ¼ 2.7)

2.33 (dd, J ¼ 6.0, 15.6, Ha ), 2.84 (dd, J ¼ 6.0, 15.6, Hb )

2.06 (dt, J ¼ 4.0, 16.0, Ha ), 1.83 (ddt, J ¼ 4.0, 12.8, 16.8, Hb ) 1.46 – 1.55 (m, 2 H ) 5.96 (s)

11

12

15

0.96 (s)

1.25 (s)

1.26 (s)

19

0.91 (s)

1.69 (d, J ¼ 1.4)

1.83 (d, J ¼ 1.8)

1.48 (s)

18

1.02 (s)

3.66 (br. d, J ¼ 8.7)

3.51 (br. d, J ¼ 7.8)

3.42 (br. d, J ¼ 6.4)

3.28 (s)

17

1.21 (s)

1.77 (d, J ¼ 1.8)

2.94 (br. d, J ¼ 7.7)

2.34 (dd, J ¼ 6.4, 12.6, Ha ), 2.00 (dt, J ¼ 6.4, 12.6, Hb )

2.10 (dt, J ¼ 8.2, 12.1, Ha ), 2.23 (dd, J ¼ 6.6, 12.1, Hb )

2.38 (t, J ¼ 5.5, Ha ), 2.13 (t, J ¼ 5.7, Hb )

5.57 (tt-like, J ¼ 1.5, 4.6) 5.43 (tt, J ¼ 1.8, 6.4)

2.32 – 2.43 (m, 2 H )

2.64 (t, J ¼ 4.6)

4.02 (d, J ¼ 3.2)

3.90 (dt, J ¼ 3.2, 11.3)

3.30 (d, J ¼ 11.3)

6.40 (d, J ¼ 10.1)

5.84 (d, J ¼ 10.1)

27

1.20 (s)

1.77 (d, J ¼ 1.8)

2.83 (br. d, J ¼ 7.3)

1.80 (dd, J ¼ 8.4, 16.9, Ha ), 2.58 (dd, J ¼ 6.4, 11.9, Hb )

5.57 (br. t, J ¼ 6.7)

2.15 (dd, J ¼ 3.7, 16.5), 2.24 (dd, J ¼ 4.1, 2.86 (dd, J ¼ 5.5, 16.5) 16.9, Ha ), 2.87 (dd, J ¼ 4.1, 16.9, Hb )

2.67 (dd, J ¼ 3.7, 5.3)

4.01 (d, J ¼ 3.2)

3.91(dd, J ¼ 3.2, 11.6)

3.36 (d, J ¼ 11.6)

6.42 (d, J ¼ 10.1)

5.86 (d, J ¼ 10.1)

26

5.46 (tt, J ¼ 2.3, 7.8)

2.07 – 2.15 (m), 2.40 (dd, J ¼ 4.0, 5.6)

4.22 (d, J ¼ 3.7)

16

5.68 (t, J ¼ 2.5)

2.52 (dd, J ¼ 5.5, 17.9, Ha ), 2.27 – 2.35 (m, Hb )

2.72 (t, J ¼ 6.0)

2.64 (dd, J ¼ 7.4, 16.0) 3.44 (dd, J ¼ 5.5, 7.3) 2.51 (br. d, J ¼ 10.1)

9

4.23 (d, J ¼ 3.2)

5.75 (d, J ¼ 3.0)

5.64 (t, J ¼ 2.1)

7

4.52 (dd, J ¼ 3.7, 12.6)

3.95 (dd, J ¼ 3.2, 12.6)

4.23 (d, J ¼ 12.6)

2.03 – 2.17 (m, 2 H )

6

3.36 (d, J ¼ 12.6)

2.64 (d, J ¼ 12.6)

3.81 (dd, J ¼ 2.3, 3.2)

2.58 (dd, J ¼ 2.5, 16.3, Ha ), 2.86 (dd, J ¼ 3.2, 16.3, Hb )

16

2.52 (d, J ¼ 12.6)

2.32 (dd, J ¼ 3.2, 11.9)

5

3

2.20 – 2.30 (m, Ha ), 2.02 – 2.10 (m, Hb )

2.03 (dd, J ¼ 2.8, 15.1, Ha ), 2.34 (dt, J ¼ 3.2, 15.1, Hb )

5.91 (d, J ¼ 10.1)

2

4.86 (t, J ¼ 2.8)

3.55 (t, J ¼ 2.8)

7.18 (d, J ¼ 10.1)

1

15

8

5

Position

Table 2. 1H-NMR Data (400 MHz, CDCl3 ) for Six Compounds from Azadirachta indica var. siamensis Extracts

512 CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)

3.66 (br. d, J ¼ 7.8, Ha ), 1.47 (s) 3.60 (d, J ¼ 7.8, Hb )

3.31 (d, J ¼ 7.8, Ha ), 3.53 (d, J ¼ 7.8, Hb )

1.02 (s)

28

2.09 – 2.16 (m)

6.89 (qq, J ¼ 1.4, 6.9) 1.78 (dd, J ¼ 1.2, 6.9) 1.86 (d, J ¼ 1.2)

7.41 (t, J ¼ 8.2) 7.56 (tt, J ¼ 1.4, 8.2) 7.41 (t, J ¼ 8.2) 7.94 (dd, J ¼ 1.4, 8.2)

3’

4’ 5’

6’

4.90 (s)

27

1.59 (s)

3.45 (s) 3.70 (s)

28-MeO

3.79 (s)

23-MeO 3.71 (s)

1.59 (s) 1.29 (s)

3.50 (s)

3.75 (s)

1.28 (s)

4.86 (d, J ¼ 3.7)

2.74 (dd, J ¼ 0.9, 19.0), 3.74 (br. d, J ¼ 5.6) 2.96 (d, J ¼ 19.0)

5.63 (s)

26

21-MeO

3.54 (s)

3.48 (s)

12-MeO

2.09 (s) 3.40 (s)

2.06 (s)

0.98 (d, J ¼ 6.9) 0.97 (d, J ¼ 6.9)

1.33 (s)

7.33 (t, J ¼ 1.8)

6.34 (dd-like, J ¼ 0.9, 1.7)

3-MeO

3-AcO

2.07 – 2.15 (m), 2.23 – 2.32 (m)

7.94 (dd, J ¼ 1.4, 8.2)

1.20 (s) 1.30 (s)

2’

1.14 (s)

1.10 (s) 1.36 (s)

29

30

1.19 (s)

6.00 (br. d, J ¼ 10.5)

7.32 (br. s)

7.35 (t, J ¼ 2.6)

23

6.90 (t, J ¼ 1.4)

6.56 (d, J ¼ 1.6)

6.05 (d, J ¼ 2.0)

22

7.26 (br. d, J ¼ 0.9)

7.33 (t, J ¼ 1.6)

16

7.17 (s)

15

21

8

5

Position

Table 2 (cont.)


514

Table 3.


C-NMR Data (100 MHz, CDCl3 ) for Six Compounds from Azadirachta indica var. siamensis Extracts

Position

5

8

15

16

26

27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 1’ 2’ 3’ 4’ 5’ 6’ 7’ 3-AcO

156.8 126.1 204.1 44.2 46.8 23.9 74.3 45.1 38.4 40.2 15.8 31.9 47.9 194.7 123.9 207.6 59.5 26.0 19.1 121.3 140.4 110.1 143.2 27.1 21.4 26.5 129.7 129.5 128.6 133.5 128.6 129.5 165.2

71.5 30.1 73.5 42.3 40.1 72.7 73.7 44.3 36.3 39.9 34.0 213.7 61.3 153.7 123.6 33.9 42.8 19.1 15.5 124.6 142.3 112.5 140.9 77.9 18.7 25.6 166.5 128.5 137.1 14.5 12.2

70.4 28.7 71.2 42.7 40.1 72.5 85.9 48.3 39.0 40.2 30.0 175.0 132.9 147.7 87.4 40.2 48.7 13.3 15.5 137.4 171.4 141.9 96.9 77.8 19.5 16.4 172.3 43.4 25.1 22.7 22.5

208.8 39.0 80.9 46.9 42.8 72.5 82.9 49.7 40.7 49.4 33.0 173.0 135.8 145.2 88.4 41.2 12.9 14.6 126.7 138.9 110.5 143.0 175.4 16.2 17.2

202.1 126.2 148.0 47.5 43.3 65.9 87.4 47.8 38.9 47.5 34.1 174.2 130.1 150.7 86.0 37.1 53.4 17.3 16.2 63.7 82.2 34.3 173.3 175.4 17.1 17.3

202.4 126.2 148.3 47.5 43.6 66.1 86.7 47.0 39.3 47.8 34.3 175.4 130.0 151.1 86.9 37.0 61.2 14.1 16.2 78.9 105.0 79.9 108.2 175.4 17.2 17.5

169.0, 20.9

170.5, 21.2 53.0

52.2 56.3 56.5 53.0

3-MeO 12-MeO 21-MeO 23-MeO 28-MeO

52.7

58.1 51.8

52.0

36] bearing with Ac and tigloyl groups at C(3) and C(7), respectively. The HMB crosscorrelations (Fig. 1) of HC(3) (d(H) 5.11) with AcOC(3) (d(C) 169.0), of HC(7) (d(H) 5.75) with C(1’) (d(C) 166.5), and of HC(9) (d(H) 3.44) and CH2(11) (d(H) 2.52 and 2.27 – 2.35), and of Me(18) (d(H) 1.02) with C(12) (d(C) 213.7), and the NOE correlations between the 1H-signals of HC(3), Me(29) (d(H) 1.19), Me(19) (d(H) 0.96), Me(30) (d(H) 1.14), and HC(7), and of Me(30) and HC(17) (d(H) 3.42) of 8


515

Fig. 1. Key HMB (H ! C) and NOE (H H) correlations for compounds 5, 8, 15, 16, 26, and 27. Drawings correspond to energy-minimized conformation of compounds. Calculation was performed using CAChe Conformation Search with the MM2 force field (CaChe version 6.01; Fujitsu Co., Tokyo, Japan).

supported the proposed structure. Thus, the structure of 8 was assigned as 3-acetyl-7tigloylnimbidinin ( ¼ 3-acetyl-12-oxo-7-tigloylvilasinin). The molecular formula of compound 15 was determined as C34H46O11 on the basis of its positive-ion-mode HR-ESI-MS (m/z 631.3143 ([M þ H] þ , C34H47O þ11 ; calc. 631.3118)). The UV absorption at 216 nm suggested the presence of an a,b-unsaturated ketone system, while the IR spectrum displayed the absorption bands for OH (3426 cm 1), g-lactone (1761 cm 1), and ester C¼O (1734 cm 1) functions. The 1Hand 13C-NMR (Tables 2 and 3, resp.) spectroscopic data of 15 were analogous to those of 3-acetylsalannol [37], with the b-furanyl ring signals for 3-acetylsalannol lacking.

516


The presence of a g-hydroxy-a,b-unsaturated g-lactone ring instead of a b-furanyl ring at C(17) was deduced from the 1H-NMR (d(H) 6.00 (br. d, J ¼ 10.5; HC(23)) and 6.90 (t, J ¼ 1.4; HC(22))) and 13C-NMR (d(C) 137.4 (C(20)), 171.4 (C(21)), 141.9 (C(22)), and 96.9 (C(23))) signals [10] [20] [38]. The HMB correlations (Fig. 1) of HC(17) and C(20) indicated the presence of b-substituted g-lactone ring at C(17). In addition, the NOE correlations (Fig. 1) between Me(30) (d(H) 1.30), HC(17) (d(H) 3.51), and Me(18) (d(H) 1.83) supported that the g-lactone ring at C(17) was a-oriented. Taking into account these and the close similarity of the 1H-NMR spectroscopic data with those of salanninolide [39], the structure of 15 was assigned as 1-detigloyl-1isovaleloylsalanninolide ( ¼ 17-defurano-17-(4-hydroxybut-2-en-4-olide-2-yl)-3-acetylsalannol). The configuration at C(23) of 15 remained undetermined. The molecular formula of 16 was determined as C28H34O8 , based on its negativeion-mode HR-ESI-MS (m/z 497.2170 ([M H] , C28H33O 8 ; calc. 497.2175)). The IR spectrum of 16 indicated the presence of g-lactone (1789 cm 1), ester C¼O (1748 cm 1), C¼O (1710 cm 1), and furan (874 cm 1) functions. The 1H- and 13C-NMR (Tables 2 and 3, resp.) data of 16 were very close to those of 2,3-dihydronimbolide [40], with additional signals of a secondary MeO group (d(H) 3.40 (s, 3 H), 3.81 (dd, J ¼ 2.3, 3.2, 1 H); d(C) 58.1 (q), 80.9 (d)) for 16. The HMB cross-correlations (Fig. 1) of HC(3) (d(H) 3.81) with C(1) (d(C) 208.8), C(4) (d(C) 46.9), and MeOC(3) (d(C) 58.1), and the NOE correlations (Fig. 1) between the 1H-signals of HC(3), Me(19) (d(H) 1.25), and Me(29) (d(H) 1.47) of 16 revealed that the MeO group was located at C(3) with an a-orientation. Thus, the structure of 16 was elucidated as 2,3-dihydro-3a-methoxynimbolide. The molecular formula of compound 26 was determined as C28H34O10 on the basis of its positive-ion-mode HR-ESI-MS (m/z 531.2238 ([M þ H] þ , C28H35O þ10 ; calc. 531.2230)). The UV spectrum showed an absorption maximum at 215 nm consistent with an a,b-unsaturated ketone system, while the IR spectrum displayed the absorption bands of OH (3451 cm 1), g-lactone (1806 cm 1), ester C¼O (1732 cm 1), and conjugated cyclohexenone (1683 cm 1) functions. The 1H- and 13C-NMR (Tables 2 and 3, resp.) data of 26 were similar to those of 6-deacetylnimbin [16], with the bsubstituted furanyl ring signals for 6-deacetylnimbin lacking for 26. Instead of the bfuranyl ring, the presence of a 3,4-epoxybutan-4-olide-3-yl (b-substituted b,g-epoxy-gbutyrolactone) ring at C(17) was deduced from the 1H-NMR (d(H) 5.63 (s, HC(21)), and 2.74 (dd, J ¼ 0.9, 19.0) and 2.96 (d, J ¼ 19.0) (CH2(22))) and 13C-NMR (d(C) 63.7 (C(20)), 82.2 (C(21)), 34.3 (C(22)), and 173.3 (C(23))) signals in the spectra of 26 [41]. The HMB cross-correlations (Fig. 1) of HC(17) (d(H) 2.94) with C(20), C(21), and C(22), and of HC(21) (d(H) 5.63) with C(23), along with the NOE correlations (Fig. 1) between the 1H-signals of HC(17) and Me(30) (d(H) 1.28) of 26 supported that the b-substituted-g-lactone ring was located at C(17) in an a-orientation. Thus, the structure of 26 was assigned as deacetyl-20,21-epoxy-20,22-dihydro-21-deoxyisonimbinolide ( ¼ 17-defurano-17-(3,4-epoxybutan-4-olide-3-yl)deacetylnimbin). The configurations at C(20) and C(21) of 26 remained undetermined. Compound 27 gave a [M þ Na] þ ion peak in the positive-ion-mode HR-ESI-MS at m/z 617.2585 (C39H42NaO þ12 ; calc. 617.2573), consistent with the molecular formula C39H42O12 . The UV spectrum showed an absorption maximum at 214 nm evidencing the presence of an a,b-unsaturated ketone system, while the IR spectrum displayed the


517

absorption bands of OH (3450 cm 1), ester C¼O (1730 cm 1), and conjugated cyclohexenone (1683 cm 1) functions. The 1H- and 13C-NMR (Tables 2 and 3, resp.) data of 27 were closely related to those of 6-deacetylnimbin [17], with the b-substituted furanyl ring signals for 6-deacetylnimbin lacking for 27. This left a C6H11O5 unit for the side chain, and the 13C-NMR spectrum showed, apart from the signals of the tetracyclic skeleton and its substituents, signals of a CHO group (d(C) 79.9), quaternary C-atom (78.9 (CO)), two acetal C-atoms (d(C) 105.0 and 108.2 (OCO)), and two MeO C-atoms (d(C) 56.3 and 56.5). These features, along with the calculation of the C¼C bond equivalents, suggested that the side chain is a cyclic diacetal. The HMB correlations of CH2(16) (d(H) 1.80 and 2.58) and HC(17) (d(H) 2.83) with the quaternary C-atom signal (d(C) 78.9) (Fig. 1) allowed the assignment of this signal to C(20). Further 13C,1H long-range couplings of HC(21) (d(H) 4.90 (s)) with C(20) and MeOC(21) (d(C) 56.3), and of HC(23) (d(H) 4.86 (d, J ¼ 3.7)) with C(21) (d(C) 105.0), C(22) (d(C) 79.9), and MeOC(23) (d(C) 56.5) observed in the HMBC spectrum, together with the 1H,1H couplings between the HC(22) (d(H) 3.74 (br. d, J ¼ 5.6)) and HC(23) signals observed in the 1H,1H-COSY spectrum of 27 enabled us to formulate 3,4-dihydroxy-2,5-dimethoxytetrahydrofuran ring structure for the side chain. The a-configuration of the side chain at C(17) could be deduced from the NOE correlation between HC(17) and Me(30) observed in the NOESY spectrum of 27. Hence, the structure of 27 was elucidated as deacetyl-20,21,22,23-tetrahydro-20,22dihydroxy-21,23-dimethoxynimbin ( ¼ 17-defurano-17-(2,3-dihydroxy-1,4-dimethoxy1,4-epoxybutan-2-yl)deacetylnimbin). The configurations at C(20), C(21), C(22), and C(23) remained undetermined. Compound 27 is a rare limonoid possessing a 3,4dihydroxy-2,5-dimethoxytetrahydrofuran ring, a fully O-substituted furanyl moiety, as side chain, and only two compounds of this type has so far been isolated from natural sources, i.e., entangosin from Entandrophragma angolense (Meliaceae) [42] and 7-Oacetyl-7-O-debenzoyl-22-hydroxy-21-methoxylimocinin from the flower extract of A. indica var. siamensis [12]. Structural Diversity of Limonoids and Flavonoids in the Bark, Leaf, Root, and Seed Extracts of Azadiracta indica var. siamensis. Although it is highly possible that far more types of limonoids and flavonoids occur, this study enabled isolation and characterization of 36 compounds including ring intact (azadirone and vilasanin types), i.e., 1 – 9, ring C-seco-limonoids (salannin, nimbin, and azadirachtin types), i.e., 10 – 28, ring Dseco-limonoids, i.e., 29 and 30, and degraded limonoids, i.e., 31 – 34, and flavonoids i.e., 35 and 36, from the extracts of bark, leaf, root, and seed parts of A. indica var. siamensis, as compiled in Table 4. Whereas the bark extract contained a wide variety of limonoids (vilasanin, salannin, nimbin, and azadirachtin types of limonoids, and degraded limonoids) along with flavonoids, only a few types of limonoids were detected in the leaf and seed extracts, i.e., nimbin-type limonoids in the leaf extract, and azadironetype and ring-D-seco-limonoids in the seed extract. Predominance of nimbin-type limonoids in the bark and root extracts, i.e., 19 in the bark extract, and 18 in the root extract, may have some behavioral or physiological effects, particularly against insects; in the plant parts [43]. Our recent study has established that the flower extract of A. indica var. siamensis contains various prenylated flavonoids in addition to limonoids [12]. All of the known limonoids and flavonoids, except compound 14, identified in this study in the A. indica var. siamensis extracts, have been detected also in the various

518


Table 4. Yield of Limonoids and Flavonoids from the Bark, Leaf, Root, and Seed Materials of Azadiracta indica var. siamensis No.

Compound name

Yield [mg] Bark (2.0 kg) a )

Ring intact limonoids Azadirone type 1 Nimonol 2 23-Deoxyazadironolide 3 Azadiradione 4 7-Benzoylinimbocinol 5 7-Benzoyl-17-epilnimbocinol 6 Epoxyazadiradione 7 7-Deacetyl-7-benzoyl-14,15-epoxyzadizadione Vilasanin type 8 3-Acetyl-7-tigloylnimbidinin 9 3-Acetyl-7-tigloylvilasinin lactone Ring C-seco limonoids Salannin type 10 Salannin 11 3-Deacetylsalannin 12 Salannol 13 Salannol 3-acetate 14 1-Benzoyl-1-decinnamoylohchinin acetate 15 1-Isovaleroyl-1-detigloylsalanninolide 16 2,3-Dihydro-3a-methoxynimbolide 17 Nimbolide Nimbin type 18 Nimbin 19 6-Deacetylnimbin 20 Nimbandiol 21 6-Acetylnimbandiol 22 6-Deacetylnimbinolide 23 Isonimbinolide 24 6-Deacetylisonimbinolide 25 6-Deacetylnimbinolactone 26 Deacetyl-20,21-epoxy-20,22-dihydro-21-deoxyisonimbinolide 27 Deacetyl-20,21,22,23-tetrahydro-20,22dihydroxy-21,23-dimethoxynimbin Azadirachtin type 28 3-Acetyl-11-methoxy-1-tigloylazadirachtinin

Leaf (500 g) a )

Root (2.0 kg) a )

29.1 10.3 1.9 29.0 10.8 14.8 29.9 5.8 7.9

7.1 3.7 2.0 32.8 1.8 2.4 10.4 1.5 31.9 138.2 2.0

9.4 2.5

413.0 3.4 8.0 3.5 1.8 1.5 1.8 3.2 3.8 3.9

12.1

Ring D-seco limonoids 29 Gedunin 30 7-Deacetyl-7-benzoylgedunin Degraded limonoids 31 Nimbidiol 32 Nimbisonol 33 Nimbione 34 Nimbionone

Seed (720 g) a )

9.1 6.8 73.0 7.4 11.1 5.9


519

Table 4 (cont.) No.

Compound name

Yield [mg] Bark (2.0 kg) a )

Flavonoids 35 (þ)-Catechin 36 ()-Epicatechin a

Leaf (500 g) a )

Root (2.0 kg) a )

Seed (720 g) a )

7.3 6.4

) Weight of dried plant materials.

parts of A. indica (neem tree). Thus, compounds 23 [19], 28 [21], 29 [32], 32 [22], 33 [19], 34 [19], 35 [24], and 36 [24] in the bark; compounds 1 [29] [44], 3 [38], 6 [38], 9 [45], 17 [16] [44] [46], 19, 22, 24, and 25 [44], and 29 [38] in the leaves; compound 31 [28] in the root bark; compounds 2 [7], 3 [7] [8], 4 [31], 6 [7], 7 [31], 9 [7] [14], 10 [8], 11 [8] [17], 12 and 13 [26], 18 and 19 [7] [8] [17], 20 [18], 21 [7] [8] [18], 29 [7], and 30 [7] [31] in the seeds; compound 17 [40] in the stem; and 22 and 24 [20] in the twig of A. indica. Presence of 23, 28, and 32 – 36 in the bark; of 22, 24, and 25 in the leaves; of 31 in the roots; and of 2 – 4, 6, 7, 29, and 30 in the seeds of A. indica var. siamensis is consistent with the results from A. indica. This study seems to be the first for the isolation of compound 14 from a natural source, since this compound has been reported only as the oxidation product of a natural limonoid, ohchinal [27]. Cytotoxic Activity. The cytotoxic activities of compounds 1 – 36, and the reference chemotherapeutic drug, cisplatin, were evaluated against the human cancer cell lines HL60 (leukemia), A549 (lung), AZ521 (stomach), and SK-BR-3 (breast) by the MTT assay, and the results are compiled in Table 5. Although seven compounds, 9, 12, 23, 25, 32, 35, and 36, were inactive against all cell lines tested, the other 29 compounds exhibited cytotoxicities against one or more cancer cell lines with IC50 values in the range of 0.1 – 94.4 mm. In particular, the cytotoxic activities of 1, 2, 4 – 7, 16, 17, 29, and 30 against HL60 (IC50 0.1 – 9.3 mm), of 5 and 13 against A549 (IC50 6.3 and 5.0 mm, resp.), of 1, 4, 5, 16, and 17 against AZ521 (IC50 0.8 – 6.5 mm), and of 1, 5, 7, 16, and 29 against SK-BR-3 (IC50 4.0 – 8.7 mm) superior to or almost comparable with those of the reference cisplatin (HL60: IC50 4.2 mm ; A549: IC50 18.4 mm ; AZ521: IC50 9.5 mm ; and SK-BR-3: IC50 18.8 mm). Based on the results compiled in Tables 1, 4, and 5, it is highly possible that compound 17 is one of the cytotoxic principles for the AcOEt-soluble fraction of the bark MeOH extract, as well as compounds 13 and 16 for the hexanesoluble fraction of the root MeOH extract, and compounds 1, 5 – 7, 29, and 30 for the AcOEt-soluble fraction of the seed MeOH extract, because these compounds are potent cytotoxic constituents of the relevant fractions. In accordance with our recent findings on the limonoid constituents of A. indica leaf extract [44], azadirone-type limonoids exhibited in general more potent cytotoxicities than the other types of limonoids, although compound 17 of salannin type exhibited the highest cytotoxicity against HL60 cells. Compound 17 has previously been reported to exhibit potent cytotoxicities against a wide variety of human cancer cell lines [40] [46 – 49]. In addition, the mechanisms of apoptosis-inducing activities of 17 against human leukemia (U937) [48], colon (HCT-119 and HT-29) [50], and hepatocarcinoma (HepG2) [51] cell lines, as well as of 1 against HL60 cell line [44] have been investigated.

520


Table 5. Cytotoxic Activities of Compounds Isolated from the Extracts of Azadirachta indica var. siamensis a ) Compound

1 c) 2 3 d) 4 d) 5 6 d) 7 d) 8 9 10 d ) 11 12 d ) 13 14 15 16 17 18 19 e ) 20 21 22 23 24 25 26 27 28 29 d ) 30 d ) 31 32 33 34 35 36 Cisplatin f )

Cytotoxicity, IC50 [mm] b ) HL60 ( Leukemia)

A549 ( Lung)

AZ521 ( Stomach)

SK-BR-3 ( Breast)

2.8 0.1 5.2 0.2 14.7 0.6 5.3 0.6 2.8 1.2 9.3 2.1 3.1 0.9 12.3 2.1 > 100 67.1 4.1 23.5 8.5 > 100 71.9 5.8 12.4 2.6 21.7 0.9 5.0 0.7 0.1 0.01 92.4 4.7 18.4 3.0 > 100 67.3 2.3 28.8 2.1 > 100 49.7 0.2 > 100 24.2 1.9 58.6 5.5 > 100 5.9 1.8 2.9 1.1 40.8 3.1 > 100 63.2 2.4 66.3 3.9 > 100 > 100 4.2 1.1

19.9 1.1 21.9 2.0 93.5 2.4 29.1 0.5 6.3 0.5 42.8 3.7 28.2 1.4 20.9 2.4 > 100 > 100 > 100 > 100 5.0 2.1 61.6 3.8 > 100 12.8 0.4 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 63.9 0.8 51.5 3.6 > 100 > 100 > 100 > 100 > 100 > 100 18.4 1.9

6.5 0.8 94.4 0.4 69.5 0.7 0.9 1.2 3.8 0.3 22.0 1.8 12.1 2.4 21.8 1.6 > 100 55.4 6.1 42.1 4.2 > 100 40.1 2.8 59.8 3.5 > 100 2.6 0.2 0.8 0.03 53.0 1.8 22.4 1.0 62.4 6.4 > 100 76.0 3.9 > 100 > 100 > 100 > 100 > 100 > 100 16.9 1.8 30.7 2.5 21.2 1.9 > 100 > 100 > 100 > 100 > 100 9.5 0.5

6.7 1.0 23.4 1.8 15.9 2.6 21.1 3.6 8.7 0.7 12.3 3.6 4.0 1.1 55.0 1.8 > 100 88.1 1.2 30.6 5.7 > 100 18.1 2.3 82.7 0.5 > 100 8.1 1.2 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 8.3 1.1 > 100 53.8 3.1 > 100 > 100 > 100 > 100 > 100 18.8 0.6

a ) Cells were treated with compounds (1 10 4 – 1 10 6 m) for 48 h, and cell viability was analyzed by the MTT assay. b ) IC50 values based on triplicate five points. Each value represents the mean S.D. c ) Data taken from [44]. d ) Part of data taken from [9]. e ) Data taken from [12]. f ) Reference compound.

Apoptosis-Inducing Activity of Compound 16. Compound 16, which exhibited a potent cytotoxic activity against AZ521 cells (IC50 2.6 mm), was evaluated for its apoptosis-inducing activity using AZ521 cells. AZ521 Cells were incubated with the


521

test compound for 24 and 48 h, and then the cells were analyzed by means of flow cytometry with annexin Vpropidium 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 [52]. 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 used to differentiate between early apoptotic (annexin V positive, PI negative), late apoptotic (annexin V and PI double positive), or necrotic (annexin V negative, PI positive) cell death. The ratio of early apoptotic cells (Fig. 2, lower right) was increased after treatment of AZ521 cells with 16 for 24 h (from 0.6 to 13.2%) and 48 h (to 15.4%), and that of late apoptotic cells (upper right) was increased after 48 h (from 0.7 to 15.0%). These results demonstrated that the cytotoxicity of 16 against AZ521 cells is, at least to some extent, due to induction of apoptotic cell death. Caspases are known to mediate the apoptotic pathway [53] [54]. To clarify the mechanism by which compound 16 induces apoptotic cell death, activations of caspases-3, -8, and -9 were evaluated by Western blot analysis. After treatment of AZ521 cells with 16 (20 mm), the levels of procaspases-3, -8, and -9 diminished, and cleaved caspase-3 was detected, almost in a time-dependent manner (Fig. 3). These results suggest that 16 induced apoptotic cell death via both the mitochondrial and the death receptor-mediated pathways. Then, we investigated the effect of 16 on Bax and Bcl-2. The proapoptotic proteins Bax and Bid, and the antiapoptotic mitochondrial protein Bcl-2 are important regulators of cytochrome c release from mitochondria [55]. Expression of these proteins was examined by Western blot analysis. Treatment of AZ521 cells with compound 16 (20 mm) decreased the level of Bcl-2 in a time-dependent manner, while the level of Bax was almost unchanged (Fig. 3). The Bax/Bcl-2 ratio is one of the indices of the intrinsic mechanism of apoptosis in mitochondria [56]. Since 16 increased this ratio in AZ521, it seems that 16-induced apoptosis involved Bax/Bcl-2 signal transduction. Compound 16, therefore, supported the induction of apoptosis in AZ521 cells by involving the mitochondrial signal transduction pathway.

Fig. 2. Detection of compound 16-induced early and late apoptotic cells by annexin VPI double staining in AZ521 cells. The cells were cultured with compound 16 (20 mm) for 24 and 48 h. Each value is the mean of three experiments.

522


Fig. 3. Western blot analysis of AZ521 cells treated with compound 16. Western blot analysis of caspases-3, -8, and -9, and of Bcl-2 and Bax in AZ521 cells with compound 16 (20 mm) for 24 and 48 h.

Melanogenesis-Inhibitory Activity. All 36 compounds were evaluated for melanogenesis inhibition in a-MSH-stimulated B16 melanoma cells (Table 6). The cytotoxic activities of these compounds against B16 melanoma cells were also determined by means of MTT assay. Among the compounds tested, 15 compounds, i.e., 1 – 3, 6, 9 – 12, 18, 23, 27 – 29, and 34 – 36, exhibited inhibitory activities (28.5 – 83.9% melanin content) with no, or almost no, toxicity to the cells (84.9 – 110.6% cell viability) at 10 mm, and their A/C ratios were in the range of 0.31 – 0.88 at that concentration. Among these 15 compounds, seven, i.e., 10, 18, 23, 27, 28, 35, and 36, in addition to compounds 22 and 24, were further proved to be lower-risk melanogenesis inhibitors by exhibiting 25.5 – 72.1% melanin contents with higher cell viabilities, i.e., 93.2 – 103.2%, at 30 mm (A/C ratios 0.26 – 0.74). In addition, even at a higher concentration (100 mm), six compounds, 19, 21, 24, 25, 32, and 36, exhibited higher cell viability, i.e., 83.3 – 97.9%, with potent melanogenesis inhibitions (22.9 – 57.5% melanin content; A/C ratios 0.26 – 0.69). Thus, 20 limonoids, i.e., 1 – 3, 6, 10 – 12, 18, 19, 21 – 29, 32, and 34, and two flavonoids, 35 and 36, isolated in this study were proved to be lower-risk melanogenesis inhibitors by exhibiting small A/C ratios at lower and/or higher concentrations which were more potent than the reference arbutin (92.7, 91.0, and 71.5% melanin contents; 102.3, 101.0, and 81.6% cell viabilities; and A/C ratios 0.91, 0.90, and 0.88, at 10, 30, and 100 mm, resp.). These compounds might be, at least in part, responsible for the melanogenesis-inhibitory activities of the fractions from which they have been isolated (Tables 1 and 4). While two flavonoids, 35 and 36, have been revealed to be potent melanogenesis inhibitors in this study in a-MSH-stimulated B16 melanoma cells, compound 35 did not significantly decrease the melanin content of B16 melanoma cells without the presence of stimulator [57] [58]. Compound 36 has been reported to suppress melanogenesis in human malignant melanoma cells (HMV-II) stimulated with TPA [59]. Mechanism of Melanogenesis Inhibition by Compound 18. Tyrosinase, and tyrosinase-related protein-1 (TRP-1) and TRP-2 are enzymes responsible for the synthesis of melanin [60]. Regulations of the transcription and activity of these melanogenic

Control (100% DMSO ) 1 d) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 d ) 18 19 e ) 20 21 22 23 24 d ) 25 26 27 28 29

Compound

100.0 3.1 0.1 0.2 0.2 0.9 0.8 0.5 0.5 0.3 3.5 0.5 0.9 1.1 1.7 0.2 1.9 0.2 0.8 1.6 26.2 7.8 8.4 2.1 4.8 1.9 0.1 0.2 3.8 1.8 14.5 6.0 0.9 0.4 3.9 3.4 9.9 0.7 49.6 5.1 1.0 0.4 22.9 1.2 9.0 2.2 5.6 1.5 3.6 0.7 36.6 2.0 25.1 1.5 15.0 0.9 4.1 0.4 0.1 0.2

100.0 0.7 108.5 2.3 110.6 8.0 106.2 3.0 0.8 0.1 2.0 0.3 95.8 7.9 0.8 0.1 73.7 3.0 89.6 5.1 100.2 3.0 92.8 7.5 59.5 4.5 46.7 2.1 60.5 1.2 103.9 4.4 69.9 1.4 52.5 2.3 103.2 2.9 110.4 0.3 55.5 3 102.2 4.2 98.7 3.1 105.3 4.0 104.0 1.6 107.3 4.9 107.2 8.7 98.8 3.4 109.3 1.9 84.9 3.7

100.0 3.1 9.8 2.0 11.8 0.7 6.3 0.3 0.1 0.7 1.0 0.7 4.7 1.0 0.8 0.4 2.2 0.8 13.4 1.7 49.6 1.8 23.7 2.3 20.3 1.4 6.4 0.6 35.8 1.5 83.8 4.2 6.8 0.7 4.4 4.7 56.3 5.0 96.5 2.9 0.4 1.7 101.1 2.0 69.5 7.4 25.5 1.8 51.0 2.9 88.4 4.3 111.7 3.3 36.4 0.7 40.7 0.6 7.2 0.9

100.0 3.1 78.0 4.4 67.9 5.8 33.3 4.0 0.1 0.3 1.0 0.7 31.3 2.7 0.2 0.4 30.3 1.7 47.0 4.0 44.5 0.5 45.5 2.0 37.7 1.2 18.4 1.1 33.3 1.2 96.6 8.4 20.5 0.9 32.6 4.5 65.3 7.6 98.4 6.7 2.2 9.0 103.1 5.6 103.1 9.9 72.5 2.2 96.3 5.6 88.5 9.8 101.3 7.7 61.6 5.7 75.4 1.9 28.5 3.3

100.0 0.7 39.3 1.3 66.3 1.0 41.1 2.0 0.6 0.1 1.6 0.4 53.6 7.9 0.6 0.3 5.2 0.1 67.4 1.3 92.5 5.0 48.2 3.0 34.6 2.9 22.4 2.0 57.1 2.4 94.7 1.3 33.1 2.6 24.1 6.1 100.0 3.1 113.9 2.3 34.5 2.3 103.7 6.1 107.2 3.4 98.0 3.1 105.1 2.8 100.5 4.1 124.0 2.4 95.5 0.7 102.3 2.0 47.2 1.4

30 mm

10 mm

30 mm

10 mm

100 mm

Cell viability [%]

Melanin content [%]

100.0 0.7 1.3 0.0 1.0 0.1 1.9 0.1 0.8 0.1 1.7 0.4 1.9 0.2 0.6 0.1 0.3 2.9 2.4 0.5 65.2 5.6 25.8 1.9 6.4 3.0 0.3 0.2 12.8 5.6 29.9 4.3 1.1 0.2 1.1 2.6 49.9 9.9 97.9 0.4 0.8 0.7 86.5 2.6 47.2 0.7 32.3 0.9 25.1 1.4 90.6 4.5 83.5 7.3 54.3 6.3 53.9 4.3 1.1 0.1

100 mm

0.72 0.61 0.31 0.13 0.50 0.33 0.25 0.41 0.52 0.44 0.49 0.63 0.39 0.55 0.93 0.29 0.62 0.63 0.89 0.04 1.01 1.04 0.69 0.93 0.82 0.94 0.62 0.69 0.34

10 mm

A/C Ratio

0.25 0.18 0.15 0.17 0.63 0.09 1.33 0.42 0.20 0.54 0.49 0.59 0.29 0.63 0.88 0.21 0.18 0.56 0.85 0.01 0.97 0.65 0.26 0.48 0.88 0.90 0.38 0.40 0.15

30 mm

0.08 0.20 0.42 0.63 2.06 0.47 2.83 6.33 0.33 0.40 0.33 0.75 0.33 0.30 0.48 0.82 3.55 0.20 0.51 1.25 0.26 0.19 0.17 0.14 0.40 0.30 0.28 0.08 0.09

100 mm

Table 6. Melanogenesis-Inhibitory Activities and Cytotoxicities a ) b ), and A/C Ratios c ) of Compounds Isolated from Azadirachta indica var. siamensis Flower Extract in B16 Mouse Melanoma Cell Line


4.9 1.3 0.7 0.2 57.5 9.5 5.7 0.7 10.8 1.4 22.6 1.4 50.7 5.0 71.5 1.3

33.3 0.6 24.4 1.0 97.9 2.6 111.0 9.3 91.5 6.0 95.6 2.8 104.9 7.6 102.3 1.5

0.9 0.8 4.1 0.5 98.9 4.6 52.7 4.4 42.5 1.6 51.9 6.9 72.1 3.2 91.0 4.2

7.0 0.6 8.8 0.5 106.2 4.0 87.6 6.2 69.0 7.4 83.9 8.9 80.9 6.2 92.7 4.6

2.0 0.2 17.6 0.1 88.3 1.9 71.4 2.5 62.6 3.1 93.2 4.3 97.2 8.2 101.0 6.4

30 mm

10 mm

30 mm

10 mm

100 mm

Cell viability [%]

Melanin content [%]

2.0 0.5 18.0 1.1 83.3 4.8 15.9 1.2 35.2 3.3 72.6 2.4 95.8 4.0 81.6 6.3

100 mm

0.21 0.36 1.08 0.79 0.75 0.88 0.77 0.91

10 mm

A/C Ratio

0.45 0.23 1.12 0.74 0.68 0.56 0.74 0.90

30 mm

2.45 0.04 0.69 0.36 0.31 0.31 0.53 0.88

100 mm

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 ¼ 3). Concentration of DMSO in the sample solution was 2 ml/ml. c ) A/C Ratio: activity/cytotoxicity ratio, which was obtained by dividing the melanin content [%] by the cell viability [%] at each concentration. d ) Data taken from [44]. e ) Data taken from [12]. f ) Reference compound.

30 31 32 33 34 35 36 Arbutin f )

Compound

Table 6 (cont.)

524 CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)


525

Fig. 4. Effects on expression of MITF, tyrosinase, TRP-1, and TRP-2 in a-MSH-stimulated B16 melanoma cells treated with compound 18. Relative amount of expressed protein is shown at each immunoblotting band.

enzymes are effective for depigmentation [61]. Tyrosinase, a rate-limiting enzyme, catalyzes the hydroxylation of l-tyrosine to l-(3,4-dihydroxyphenyl)alanine (lDOPA), and the oxidation of l-DOPA to l-DOPA quinone [62]. TRP-2 functions as DOPAchrome tautomerase, and TRP-1 catalyzes oxidation of 5,6-dihydroxy-1Hindole-2-carboxylic acid (DHICA) [62]. Transcription for expression of these enzymes is regulated by microphthalmia-associated transcription factor (MITF) [63]. To clarify the mechanism involved in the melanogenesis inhibition by compound 18, which turned out to be the most abundant limonoid constituent of the root extract and one of the potent melanogenesis inhibitors found in this study, the protein levels of tyrosinase, TRP1, TRP-2, and MITF were evaluated in B16 melanoma cells treated with 18 by Western blot analysis. Treatment of B16 melanoma cells with 18 reduced protein levels of MITF, tyrosinase, TRP-1, and TRP-2 proteins, mostly in a concentration-dependent manner, although the level of expression of tyrosinase at 10 and 30 mm was inverted (Fig. 4). These results suggested that 18 exhibits melanogenesis inhibitory activity in the a-MSHstimulated 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. Conclusions. – This study has established that some of the MeOH and hexane extracts, and the fractions obtained therefrom of the bark, leaves, roots, and seeds of Azadirachta indica var. siamensis (Siamese neem) exhibit potent cytotoxic activities against HL60, A549, A521, and SK-BR-3 human cancer cell lines, and potent melanogenesis-inhibitory activity in a-MSH-stimulated B16 melanoma cells with almost no cytotoxicity to the cells. Investigation on the AcOEt-soluble fraction of the bark extract, the hexane- and AcOEt-soluble fractions of the leaf and root MeOH extracts, and the hexane extract and the AcOEt-soluble fraction of the MeOH extract

526


of seeds has led to the isolation of 34 limonoids, 1 – 34, and two flavonoids, 35 and 36, of which six, i.e., 7-benzoyl-17-epinimbocinol (5), 3-acetyl-7-tigloylnimbidinin (8), 1isovaleroyl-1-detigloylsalanninolide (15), 2,3-dihydro-3a-methoxynimbolide (16), deacetyl-20,21-epoxy-20,22-dihydro-21-deoxyisonimbinolide (26), and deacetyl-20,21, 22,23-tetrahydro-20,22-dihydroxy-21,23-dimethoxynimbin (27), were new compounds. Evaluation of cytotoxic activity against four human cancer cell lines for these compounds established that eleven compounds, 1, 2, 4 – 7, 13, 16, and 17, 29, and 30, exhibit potent cytotoxicities against one or more cell lines, and the cell death of AZ521 cells by treatment with compound 16 is, at least in part, due to apoptosis induced via both the mitochondrial and the death receptor-mediated pathways. This study has further established that 20 limonoids, i.e., 1 – 3, 6, 10 – 12, 18, 19, 21 – 29, 32, and 34, and two flavonoids, 35 and 36, are to be considered lower-risk melanogenesis inhibitors by exhibiting small A/C ratios (activity-to-cytotoxicity ratios) at lower and/or higher concentrations which were more potent than the reference arbutin. Among these compounds, nimbin (18) has been revealed to exert its melanogenesis inhibition, at least in part, by inhibiting expression of MITF, tyrosinase, TRP-1, and TRP-2. This work has thus provided further examples of the importance of the bark, leaf, root, and seed extracts of A. indica var. siamensis, and their limonoid and flavonoid constituents as potential anticancer and skin-whitening agents. Experimental Part General. Column chromatography (CC): Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan), silica gel (SiO2 , 230 – 400 mesh; Merck), and octadecyl silica gel (ODS; Chromatorex-ODS, 100 – 200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan). Prep. HPLC (with refractive-index detector): ODS columns (25 cm 10 mm i.d.) at 258 on a Pegasil ODS-II 5 mm column (Senshu Scientific Co., Ltd., Tokyo, Japan) with MeOH/H2O (system I), MeOH/H2O/AcOH (system II), MeOH/H2O/HCOOH (system III), MeCN/H2O (system IV), MeCN/H2O/AcOH (system V), or with MeCN/H2O/HCOOH (system VI), or on a Capcell pak AQ 5 mm column (Shiseido Co., Ltd., Tokyo, Japan) with MeCN/H2O (system VII), MeCN/H2O/AcOH (system VIII), or with MeCN/H2O/HCOOH (system IX) as mobile phase. Optical rotations: JASCO P-1020 digital polarimeter. UV Spectra: JASCO V-630Bio spectrophotometer; lmax (log e) in nm. IR Spectra: JASCO FTIR-300E spectrometer; ñ in cm 1. 1D- and 2D-NMR spectra: JEOL ECX-400 (1H: 400 and 13C: 100 MHz) spectrometer at r.t.; CDCl3 or (D6 )acetone soln.; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Agilent 1100 LC/MSD TOF (time-offlight) system (cap. voltage, 3000 V; fragmentor voltage, 225 V); m/z. Microplate reader: Sunrise-Basic (Tecan Japan Co., Ltd., Kawasaki, Japan). Flow cytometer: Cell Lab QuantaTM SC (Beckman Coulter K. K., Tokyo, Japan). Materials and Chemicals. The bark, fruits (pulps and seeds), leaves, and roots of Azadirachta indica A. Juss var. siamensis Valeton were collected from Lampang, Thailand, from March to April in 2011 (bark, leaves, and roots), and from October to November in 2011 (fruits). The voucher specimens of the plant materials were authenticated by Mr. J. F. Maxwell, Chiang Mai University (CMU) Botanical Herbarium, and deposited with the Natural Product Research Development Center (NPRDC), Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand (Voucher specimen No. MANOSROI#0046). The chemicals were purchased as follows: fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin) from Invitrogen Co. (Carlsbad, CA, USA); Dulbeccos modified Eagles medium (DMEM), MTT, arbutin, a-MSH, and MTT from SigmaAldrich Japan Co. (Tokyo, Japan); and cisplatin from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals and reagents were of anal. grade. Extraction and Isolation. i) Bark. The dried and powdered bark of A. indica var. siamensis (2.0 kg) was extracted with MeOH by maceration at r.t. (10 l; 1 week, 3 ) to yield a MeOH extract (125 g). The


527

extract was suspended in H2O and partitioned with hexane yielding a hexane-soluble fraction (8.3 g). The H2O layer was then partitioned with AcOEt and BuOH successively furnishing AcOEt- , BuOH-, and H2O- soluble fractions (33.5,14.9, and 65.8 g, resp.). The AcOEt-soluble fraction was applied to CC (SiO2 (500 g); hexane/AcOEt 9 : 1 to 1 : 9) to yield ten fractions, Frs. 1 – 10. Fr. 3 (734 mg) from the eluate of hexane/AcOEt 7 : 3 was subjected to CC (SiO2 (18 g); CHCl3/MeOH 99 : 1 to 97 : 3) to yield a fraction (53 mg) from the eluate of CHCl3/MeOH 97 : 3. Prep. HPLC (system VIII; ratio, 60 : 40 : 0.1, flow rate, 2.0 ml/min) of the fraction yielded compound 22 (retention time (tR ) 14.2 min; 2.0 mg). Fr. 6 (1987 mg) eluted with hexane/AcOEt 1 : 1 was subjected to CC (SiO2 (150 g); hexane/AcOEt 4 : 1 to 0 : 1) to give four fractions, Frs. 6a – 6d. CC (ODS (30 g); MeOH/H2O 2 : 3 to 3 : 2) of Fr. 6b (963 mg), eluted with hexane/AcOEt 7 : 3, afforded four fractions, Frs. 6b-1 – 6b-4. Prep. HPLC (system V) of Fr. 6b-2 (353 mg; 58 : 42 : 0.1, flow rate, 3.0 ml/min) and Fr. 6b-3 (155 mg; 55 : 45 : 0.1; flow rate, 3.0 ml/min) afforded 33 (tR 8.0 min; 11.1 mg), 19 (tR 21.6 min; 138.2 mg), and 17 (tR 26.8 min; 1.5 mg), and compounds 32 (tR 7.2 min; 7.4 mg), 34 (tR 8.4 min; 5.9 mg), and 18 (tR 32.0 min; 31.9 mg), resp. Fr. 7 (1685 mg), eluted with hexane/ AcOEt 2 : 3, upon CC (SiO2 (80 g); hexane/AcOEt 1 : 0 to 1 : 1), gave eight fractions, Frs. 7a – 7h. Fr. 7e (548 mg) eluted with hexane/AcOEt 7 : 3 was further subjected to CC (ODS (20 g); MeCN/H2O 2 : 3 to 4 : 2) to give a fraction (148 mg) from which compounds 8 (tR 22.0 min; 5.8 mg) and 10 (tR 23.5 min; 7.1 mg) were isolated by prep. HPLC (system VIII; ratio 62 : 38 : 0.1; flow rate, 3.0 ml/min). Prep. HPLC (system V; ratio, 58 : 42 : 0.1; flow rate, 3.0 ml/min) of Fr. 7f (320 mg), eluted with hexane/AcOEt 7 : 3, yielded compounds 28 (tR 14.4 min; 12.1 mg) and 9 (tR 16.0 min; 7.9 mg). Fr. 9 (3993 mg) from the eluate of hexane/AcOEt 1 : 4 was subjected to CC (SiO2 (200 g); hexane/AcOEt 2 : 3 to 0 : 1) to yield seven fractions, Frs. 9a – 9g. Prep. HPLC (system II; 35 : 65 : 0.5; flow rate, 3.0 ml/min) of Fr. 9b (104 mg), eluted with hexane/AcOEt 2 : 3, gave compounds 35 (tR 7.2 min; 7.3 mg) and 36 (tR 11.2 min; 6.4 mg). Fr. 9c (1339 mg) eluted with hexane/AcOEt 1 : 4, was submitted to CC (ODS (60 g); MeOH/H2O 1 : 9 to 4 : 1) to yield nine fractions, Frs. 9c-1 – 9c-9. Prep. HPLC (system V) of Fr. 9c-5 (112 mg; 40 : 60 : 0.1; flow rate, 3.0 ml/min) and Fr. 9c-6 (80 mg; ratio, 43 : 57 : 0.1; flow rate, 3.0 ml/min) yielded compounds 24 (tR 20.4 min; 2.5 mg) and 23 (tR 35.2 min; 9.4 mg), resp. ii) Leaves. The dried and powdered leaves of A. indica var. siamensis (500 g) were extracted with MeOH by maceration at r.t. (3 l; 3 d, 4 ) to yield a MeOH extract (101 g). The extract was suspended in H2O and partitioned with hexane to yield a hexane-soluble fraction (8.2 g). The H2O layer was then partitioned with AcOEt to give AcOEt- and H2O-soluble fractions (14.6 and 73.6 g, resp.). To remove chlorophylls, the hexane extract was subjected to CC (Diaion HP-20 (150 g)) and eluted with MeOH (1 l) and acetone successively [29]. The almost chlorophyll-free MeOH eluate (5.4 g) was applied to CC (SiO2 (150 g); hexane/AcOEt 9 : 1 to 0 : 1) to yield 13 fractions, Frs. H1 – H13. Fr. H10 (241 mg) from the eluate of hexane/AcOEt 13 : 7 was subjected to CC (SiO2 (6 g); CHCl3/AcOEt 49 : 1 to 17 : 3) to afford a fraction (16 mg) from the eluate of CHCl3/AcOEt 19 : 1. Prep. HPLC (system I; ratio, 7 : 3; flow rate, 2.0 ml/min) of the fraction yielded compound 26 (tR 10.4 min; 3.8 mg). Fr. H13 (601 mg) from the eluate of hexane/AcOEt 0 : 1 was purified by CC (ODS (12 g); MeOH/H2O 1 : 1 to 1 : 0) to give a fraction (425 mg) from the eluate of MeOH/H2O 3 : 2, which was further submitted to CC (SiO2 (6 g); CHCl3/ acetone 19 : 1 to 7 : 3) to yield five fractions, Frs. H13-1 – H13-5. Prep. HPLC (system IV; ratio, 7 : 11; flow rate, 2.0 ml/min) of Fr. H13-2 (15 mg) afforded compound 25 (tR 31.6 min; 3.2 mg). The AcOEt-soluble fraction was applied to CC (SiO2 (388 g); hexane/AcOEt 1 : 0 to 0 : 1) to yield ten fractions, Frs. A1 – A10. Fr. A9 (419 mg) from the eluate of hexane/AcOEt 0 : 1 was subjected to CC (SiO2 (12 g); CHCl3/MeOH 1 : 0 to 0 : 1) to give eight fractions, Frs. A9-1 – A9-8. Prep. HPLC (system IV; ratio, 7 : 11; flow rate, 2.0 ml/ min) of Fr. A9-4 (79 mg) furnished compound 27 (tR 58.4 min; 3.9 mg). Fr. A9 – 5 (126 mg) was separated by CC (SiO2 (6 g); hexane/acetone 1 : 0 to 0 : 1) to afford seven fractions, Frs. A9-5a – A9-5g. Prep. HPLC of Fr. A9-5b (12 mg; system IV; ratio, 2 : 3; flow rate, 2.0 ml/min) and Fr. A9-5c (10 mg; system VII; ratio, 1 : 1; flow rate, 2.0 ml/min) gave compounds 22 (tR 35.2 min; 1.8 mg) and 23 (tR 56.0 min; 1.5 mg), and 24 (tR 60.8 min; 1.8 mg), resp. iii) Roots. The dried and powdered roots of A. indica var. siamensis (2.0 kg) were extracted with MeOH by maceration at r.t. (3 l; 1 week, 3 ) to yield a MeOH extract (92 g). The extract was suspended in H2O and partitioned with hexane yielding a hexane-soluble fraction (21.3 g). The H2O layer was then partitioned with AcOEt and BuOH successively which afforded AcOEt-, BuOH-, and H2O-soluble fractions (15.2, 8.3, and 33.5 g, resp.). The hexane-soluble fraction was applied to CC (SiO2 (690 g);

528


hexane/AcOEt 1 : 0 to 1 : 9) to yield ten fractions, Frs. H1 – H10. Fr. H6 (908 mg) from the eluate of hexane/AcOEt 1 : 1 was subjected to CC (ODS (44 g); MeCN/H2O 2 : 3 to 1 : 0) to give compound 18 (413.0 mg) from the eluate of MeCN/H2O 2 : 3. Fr. H8 (433 mg) eluted with hexane/AcOEt 2 : 3 was subjected to CC (ODS (24 g); MeOH/H2O 2 : 3 to 1 : 0) to afford a fraction (212 mg) from the eluate of MeOH/H2O 2 : 1. Prep. HPLC (system VI; ratio, 65 : 35 : 0.5; flow rate, 2.0 ml/min) of this fraction afforded compound 13 (tR 51.2 min; 32.8 mg). Fr. H9 (215 mg) from the eluate of hexane/AcOEt 1 : 4 was subjected to CC (SiO2 (12 g); CHCl3/acetone 1 : 0 to 9 : 1) to give four fractions, Frs. H9-1 – H9-4. Prep. HPLC of Fr. H9-1 (28 mg; system VI; ratio, 65 : 35 : 0.5; flow rate, 2.0 ml/min) and Fr. H9-2 (7 mg; system VI; ratio, 40 : 60 : 0.5; flow rate, 3.0 ml/min) yielded compounds 16 (tR 20.8 min; 10.4 mg) and 12 (tR 29.6 min; 2.0 mg), and 20 (tR 41.6 min; 8.0 mg), resp. Fr. H10 (197 mg) from the eluate of hexane/AcOEt 1 : 4 was subjected to CC (SiO2 (10 g); CHCl3/acetone 1 : 0 to 0 : 1) to give six fractions, Frs. H10-1 – H10-6. Prep. HPLC of Fr. H10-1 (46 mg; system VI; ratio, 40 : 60 : 0.5; flow rate, 3.0 ml/min) and Fr. H10-2 (19 mg; system VI; ratio, 45 : 55 : 0.5; flow rate, 2.0 ml/min) yielded compounds 15 (tR 61.6 min; 2.4 mg) and 11 (tR 41.6 min; 3.7 mg), resp. The AcOEt-soluble fraction was applied to CC (SiO2 (500 g); hexane/ AcOEt 4 : 1 to 0 : 1) to furnish six fractions, Frs. A1 – A6. Fr. A4 (723 mg) from the eluate of hexane/ AcOEt 2 : 3 was subjected to CC (SiO2 (22 g); hexane/AcOEt 4 : 1 to 1 : 4) to yield four fractions, Frs. A41 – A4-4. Upon further CC (ODS (8 g); MeOH/H2O 2 : 3 to 3 : 2), Fr. A4 – 1 (169 mg) gave compound 31 (73.0 mg). Prep. HPLC (system II; ratio, 65 : 35 : 0.1; flow rate, 2.0 ml/min) of Fr. A4 – 4 (61 mg) provided compound 19 (tR 41.6 min; 3.4 mg). Fr. A6 (1134 mg) eluted with hexane/AcOEt 0 : 1 was subjected to CC (ODS (50 g); MeOH/H2O 2 : 3 to 1 : 0) to give eight fractions, Frs. A6-1 – A6-8. CC (SiO2 (8 g); CHCl3/acetone 1 : 0 to 49 : 3) of Fr. A6-5 (258 mg) afforded a fraction (60 mg) from which compound 21 (tR 41.6 min; 3.5 mg) was isolated by prep. HPLC (system II; ratio, 65 : 35 : 0.1; flow rate, 2.0 ml/min). Further, CC (SiO2 (8 g); CHCl3/acetone 4 : 1 to 3 : 1) of Fr. A6-6 (167 mg) gave a fraction (30 mg). Prep. HPLC (system II; ratio, 70 : 30 : 0.1; flow rate, 2.0 ml/min) of the fraction yielded compound 14 (tR 54.4 min; 1.8 mg). iv) Seeds. The dried and powdered seeds of A. indica var. siamensis (720 g) were extracted with hexane and MeOH successively under reflux (3 l; 2 h, 3 each) to give hexane (19 g) and MeOH (224 g) extracts, resp. The hexane extract was applied to CC (SiO2 (450 g); hexane/AcOEt 1 : 0 to 0 : 1) to yield nine fractions, Frs. H1 – H9. Fr. H5 (1212 mg) from the eluate of hexane/AcOEt 3 : 2 was subjected to CC (SiO2 (60 g); hexane/AcOEt 13 : 7 to 1 : 1) to give seven fractions, Frs. H5-1 – H5-7. Fr. H5-3 (284 mg) was further subjected to CC (ODS (12 g); MeOH/H2O 1 : 1 to 7 : 3) to give 14 fractions, Frs. H5-3a – H5-3n. Prep. HPLC (system I; ratio, 7 : 3; flow rate, 2.0 ml/min) of Fr. H5-3j (20 mg) gave compound 3 (tR 32.8 min; 1.9 mg). Fr. H5-3l and Fr. H5-3n contained one compound each, i.e., compounds 2 (10.3 mg) and 4 (29.0 mg), resp. The MeOH extract was suspended in H2O and partitioned with AcOEt and BuOH successively to yield AcOEt-, BuOH-, and H2O- soluble fractions (77.0, 44.9, and 97.1 g, resp.). The AcOEt-soluble fraction was submittted to CC (SiO2 (1.2 kg); hexane/AcOEt 9 : 1 to 0 : 1) to yield seven fractions, Frs. A1 – A7. Fr. A2 (14.1 g) from the eluate of hexane/AcOEt 7 : 3 was subjected to further CC (SiO2 (520 g); hexane/AcOEt 1 : 0 to 0 : 1) to afford eleven fractions, Frs. A2-1 – A2-11. Fr. A2-7 (5127 mg) and Fr. A2-8 (1121 mg), upon repeated CC (SiO2 ) under the similar conditions as above, yielded fractions A2 – 7a (533 mg) and A2 – 8a (377 mg), resp. CC (ODS (26 g); MeOH/H2O 3 : 2 to 17 : 3) of Fr. A2-7a gave ten fractions, Frs. A2-7a-1 – A2-7a-10. Prep. HPLC of Fr. A2-7a-6 (187 mg; system II; ratio 65 : 35 : 0.1; flow rate, 2.0 ml/min) and Fr. A2-7a-7 (150 mg; system II; ratio, 75 : 25 : 0.1; flow rate, 3.0 ml/min) yielded compound 6 (tR 41.6 min; 9.6 mg), and 7 (tR 41.6 min; 29.9 mg) and 1 (tR 41.6 min; 29.1 mg), resp. On the other hand, CC (ODS (16 g); MeOH/H2O 7 : 3 to 6 : 4) of Fr. A2-8a gave eight fractions, Frs. A2-8a-1 – A2-8a-8. Prep. HPLC of Fr. A2-8a-6 (18 mg) and Fr. A2-8a-7 (23 mg; system II; ratio, 80 : 20 : 0.1; flow rate, 3.0 ml/min), and Fr. A2-8a-8 (21 mg; system II; ratio, 75 : 25 : 0.1; flow rate, 3.0 ml/min) yielded 29 (tR 50.0 min; 9.1 mg), 5 (tR 12.7 min; 10.9 mg), and 30 (tR 24.0 min; 6.8 mg), resp. Data of the New Compounds. 7-O-Benzoyl-17-epinimbocinol ( ¼ (5a,7a,13a,17b)-17-(Furan-3-yl)4,4,8-trimethyl-3,16-dioxoandrosta-1,14-dien-7-yl Benzoate; 5). Amorphous solid. [a] 25 D ¼ 42.7 (c ¼ 0.81, EtOH). UV (EtOH): 231 (4.16). IR (KBr): 1728 (ester C¼O), 1716 (conj. cyclopentenone), 1672 (conj. cyclohexenone), 875 (furan). 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS (pos.): 535.2450 ([M þ Na] þ , C33H36NaO þ5 ; calc. 535.2460).


529

3-Acetyl-7-tigloylnimbidinin ( ¼ (1S,3R,3aR,5aR,6S,6aR,9S,9aS,11aR,11bR,11cR)-3-(Acetyloxy)-9(furan-3-yl)-1,2,3,3a,4,5a,6,6a,8,9,9a,10,11,11a,11b,11c-hexadecahydro-1-hydroxy-3a,6a,9a,11b-tetramethyl-10-oxocyclopenta[7,8]phenanthro[10,1-bc]furan-6-yl (2E)-2-Methylbut-2-enoate; 8). Amorphous solid. [a] 20 D ¼ 18.2 (c ¼ 0.30, EtOH). UV (EtOH): 210 (3.81). IR (KBr): 3340 (OH), 1725 (ester C¼O), 1710 (C¼O), 872 (furan). 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS (pos.): 589.2797 ([M þ Na] þ , C33H42NaO þ8 ; calc. 589.2777). 1-Isovaleroyl-1-detigloylsalanninolide ( ¼ (2aR,3R,5S,5aR,6R,6aR,8R,9aR,10aS,10bR,10cR)-3(Acetyloxy)-2a,4,5,5a,6,6a,8,9,9a,10a,10b,10c-dodecahydro-8-(5-hydroxy-2-oxo-2,5-dihydrofuran-3-yl)6-(2-methoxy-2-oxoethyl)-2a,5a,6a,7-tetramethyl-2H,3H-cyclopenta[d]naphtho[2,3-b:1,8-b’c’]difuran-5yl 3-Methylbutanoate; 15). Fine needles (MeOH). M.p. 238 – 2418. [a] 20 D ¼ þ 70.8 (c ¼ 1.36, EtOH). UV (MeOH): 216 (4.03). IR (KBr): 3426 (OH), 1761 (g-lactone), 1734 (ester C¼O). 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS (pos.): 631.3143 ([M þ H] þ , C34H47O þ11 ; calc. 631.3118). 2,3-Dihydro-3a-methoxynimbolide ( ¼ Methyl [(2aS,3R,5aR,6S,6aR,8R,9aR,10aS,10bR,10cR)-8(Furan-3-yl)-2a,4,5,5a,6,6a,8,9,9a,10a,10b,10c-dodecahydro-3-methoxy-2a,5a,6a,7-tetramethyl-2,5-dioxo2H,3H-cyclopenta[d]naphtho[2,3-b:1,8-b’c’]difuran-6-yl]acetate; 16). Fine needles (MeOH). M.p. 198 – 2018. [a] 20 D ¼ þ 78.9 (c ¼ 1.05, EtOH). UV (MeOH): 220 (3.80). IR (KBr): 1789 (g-lactone), 1748 (ester C¼O), 1710 (C¼O), 874 (furan). 1H- and 13C-NMR: see Tables 2 and 3, resp. 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS (neg.): 497.2170 ([M H] , C28H33O 8 ; calc. 497.2175). Deacetyl-20,21-epoxy-20,22-dihydro-21-deoxyisonimbinolide ( ¼ Methyl (2R,3aR,4aS,5R,5aR,6R, 9aR,10S,10aR)-3,3a,4a,5,5a,6,9,9a,10,10a-Decahydro-5-hydroxy-10-(2-methoxy-2-oxoethyl)-1,6,9a,10atetramethyl-9-oxo-2-( 3-oxo-2,6-dioxabicyclo[3.1.0]hex-5-yl)-2H-cyclopenta[b]naphtho[2,3-d]furan-6carboxylate; 26). Fine needles (MeOH). M.p. 137 – 1408. [a] 20 D ¼ þ 55.6 (c ¼ 0.43, EtOH). UV (EtOH): 215 (3.75). IR (KBr): 3451 (OH), 1806 (g-lactone), 1732 (ester C¼O), 1683 (conj. cyclohexenone). 1 H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS (pos.): 531.2238 ([M þ H] þ , C28H35O þ10 ; calc. 531.2230). Deacetyl-20,21,22,23-tetrahydro-20,22-dihydroxy-21,23-dimethoxynimbin ( ¼ Methyl (2R,3aR,4aS, 5R,5aR,6R,9aR,10S,10aR)-3,3a,4a,5,5a,6,9,9a,10,10a-Decahydro-5-hydroxy-6-(methoxycarbonyl)1,6,9a,10a-tetramethyl-9-oxo-2-(tetrahydro-3,4-dihydroxy-2,5-dimethoxyfuran-3-yl)-2H-cyclopenta[b]naphtho[2,3-d]furan-10-acetate; 27). Fine needles (MeOH). M.p. 121 – 1248. [a] 20 D ¼ þ 10.1 (c ¼ 0.60, EtOH). UV (EtOH): 214 (3.96). IR (KBr): 3450 (OH), 1730 (ester C¼O), 1683 (conj. cyclohexenone). HR-ESI-MS (pos.): 617.2585 ([M þ Na] þ , C39H42NaO12 þ ; calc. 617.2573). Cell Lines and Culture Conditions. Cell lines HL60 (leukemia), AZ521 (stomach), A549 (lung), SKBR-3 (breast), and B16 4A5 (mouse melanoma) were obtained from Riken Cell Bank (Tsukuba, Ibaraki, Japan). Cell lines were grown in the following media: HL60 and SK-BR-3 in RPMI-1640 medium, AZ521 and B16 4A5 in DMEM medium, and A549 in 90% DMEM þ 10% MEM (minimum essential medium) þ 0.1 mm NEAA (non-essential amino aciols). The medium was supplemented with 10% FBS and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells were incubated at 378 in a 5% CO2 humidified incubator. The cells were cultured as described in [9] [64 – 66]. Determination of Cell Proliferation. Cell proliferation was assessed using the MTT based colorimetric assay as described in [9] [64 – 66]. Assay of Melanin Content. Melanogenesis-inhibition assay in a-MSH-stimulated B16 melanoma cells was performed as described in [65]. Annexin VPropidium Iodide (PI) Double Staining. Annexin VPI double staining was performed as described in [9] [66 – 68] with AZ521 (1 105 cells). Western Blotting. Western blot analysis was performed according to the previous method with AZ521 cells (1 105 cells) [67] [68] or with B16 melanoma cells (1 105 cells) [69]. REFERENCES [1] K. Sombatsiri, K. Ermel, H. Schmutterer, The neem tree Azadirachta indica A. Juss. and other meliaceous plants, Ed. H. Schmutterer, VCH Verlagsgesellschaft, Weinheim, 1995, p. 589. [2] T. Clayton, P. Soralump, W. Chaukul, R. Temsiririrkkul, Medicinal Plants in Thailand, Vol. 1, Amarin Printing, Bangkok, 1996, p. 44.

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Identification and antifeedant activities of limonoids from Azadirachta indica.

Cytotoxic and melanogenesis-inhibitory activities of limonoids from the leaves of Azadirachta indica (Neem).

Three new limonoids from Azadirachta indica.

Chemical composition, leishmanicidal and cytotoxic activities of the essential oils from Mangifera indica L. var. Rosa and Espada.

Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids from Neem (Azadirachta indica) as Anti-Diabetic Agents.

Three new and other limonoids from the hexane extract of Melia azedarach fruits and their cytotoxic activities.

Limonoids and Triterpenoids from Dysoxylum mollissimum var. glaberrimum.

Analysis of anti-bacterial and anti oxidative activity of Azadirachta indica bark using various solvents extracts.

Investigation of total phenolic content and antioxidant activities of Azadirachta indica roots.

Chemopreventive and Anticancer Activities of Allium victorialis var. platyphyllum Extracts.

Therapeutics Role of Azadirachta indica (Neem) and Their Active Constituents in Diseases Prevention and Treatment.

Studies on the acaricidal mechanism of the active components from neem (Azadirachta indica) oil against Sarcoptes scabiei var. cuniculi.

Bioactive limonoids from the leaves of Azaridachta indica (Neem).

Comparison of cytotoxic activities of extracts from Selaginella species.

Immunomodulatory effects of neem (Azadirachta indica) oil.

On the toxicology of Azadirachta indica leaves.

Two new alkaloids from Portulaca oleracea and their cytotoxic activities.

Limonoids from the leaves of Soymida febrifuga and their insect antifeedant activities.

Azadirachta indica attenuates cisplatin-induced nephrotoxicity and oxidative stress.

Cardiovascular effects of Azadirachta indica extract.

Antimicrobial, antibiofilm and cytotoxic activities of Hakea sericea Schrader extracts.

Antioxidant and cytotoxic activities investigation of tomato seed extracts.

Evaluation of cytotoxic and anti-inflammatory activities of extracts and lectins from Moringa oleifera seeds.

Chemical composition, biological and cytotoxic activities of plant extracts and compounds isolated from Ferula lutea.