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Cytotoxic and Melanogenesis-Inhibitory Activities of Limonoids from the Leaves of Azadirachta indica (Neem) by Mio Takagi a ), Yosuke Tachi a ), Jie Zhang a ), Takuro Shinozaki a ), Kenta Ishii a ), Takashi Kikuchi a ), Motohiko Ukiya a ), Norihiro Banno b ), Harukuni Tokuda c ), and Toshihiro Akihisa* a ) d ) a
) College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan b ) Ichimaru Pharcos Company Ltd., 318-1 Asagi, Motosu-shi, Gifu 501-0475, Japan c ) Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan d ) Akihisa Medical Clinic, 1086-3 Kamo, Sanda-shi, Hyogo 669-1311, Japan (fax: þ 81-79-5671980; e-mail: [email protected])
Seventeen limonoids (tetranortriterpenoids), 1 – 17, including three new compounds, i.e., 17defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide (14), 17-defurano-17-(2x-2,5-dihydro-2hydroxy-5-oxofuran-3-yl)-28-deoxonimbolide (15), and 17-defurano-17-(5x-2,5-dihydro-5-hydroxy-2oxofuran-3-yl)-2’,3’-dehydrosalannol (17), were isolated from an EtOH extract of the leaf of neem (Azadirachta indica). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines, seven compounds, i.e., 1 – 3, 12, 13, 15, and 16, exhibited potent cytotoxicities with IC50 values in the range of 0.1 – 9.9 mm against one or more cell lines. Among these compounds, cytotoxicity of nimonol (1; IC50 2.8 mm) against HL60 cells was demonstrated to be mainly due to the induction of apoptosis by flow cytometry. Western blot analysis suggested that compound 1 induced apoptosis via both the mitochondrial and death receptor-mediated pathways in HL60 cells. In addition, when compounds 1 – 17 were evaluated for their inhibitory activities against melanogenesis in B16 melanoma cells, induced with a-melanocyte-stimulating hormone (a-MSH), seven compounds, 1, 2, 4 – 6, 15, and 16, exhibited inhibitory activities with 31 – 94% reduction of melanin content at 10 mm concentration with no or low toxicity to the cells (82 – 112% of cell viability at 10 mm). All 17 compounds were further evaluated for their inhibitory effects against the EpsteinBarr virus early antigen (EBV-EA) activation induced by 12O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells.
Introduction. – Plants of the Meliaceae family have been well documented for their ability to metabolize structurally diverse and biologically significant limonoids and triterpenoids [1] [2]. In the course of a search for potential bioactive compounds from Meliaceae plants, a detailed investigation on the limonoid constituents of the seed extracts of Azadirachta indica A. Juss. (neem tree) was carried out, and this revealed that some limonoids exhibit potent inhibitory activities against melanogenesis in B16 melanoma cells, against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, and against TPA-induced EpsteinBarr virus early antigen (EBV-EA) activation [3] [4], as well as cytotoxic activities against HL60, A549, AZ521, SK-BR-3, and CRL1579 human cancer cell lines, and apoptosis-inducing activity against HL60 cell line [5]. Further, we have reported that some limonoids from the fruits of Melia 2014 Verlag Helvetica Chimica Acta AG, Zrich
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azedarach exhibited potent cytotoxic activities against HL60, A549, AZ521, and SKBR-3 human cancer cell lines [6], and apoptosis-inducing activities against HL60 and AZ521 cell lines [7]. In a continuing study on the limonoid constituents of Meliaceae plants, the leaf extract of A. indica was investigated, resulting in the isolation of 17 limonoids, 1 – 17, including three new compounds, 14, 15, and 17 (see below). Herein, we describes the structure elucidation of these compounds and evaluation of their cytotoxic activities against four human cancer cell lines, melanogenesis-inhibitory activities in amelanocyte-stimulating hormone (a-MSH)-stimulated B16 melanoma cells, and inhibitory effects on the induction of EBV-EA activation induced with TPA in Raji cells. Extract of neem leaves has been therapeutically used as folk medicine to control leprosy, eye problem, epistaxis, intestinal worms, anorexia, biliousness, and skin ulcers [8] [9], and it has been reported to be nontoxic and non-mutagenic [10], and to possess a wide range of biological prosperities such as immunomodulatory, anti-inflammatory, antihyperglycaemic, antiulcer, antimalarial, antifungal, antibacterial, antiviral, antioxidant, antimutagenic, chemopreventive, and anticarcinogenic activities [8 – 15]. Results and Discussions. – Cytotoxic, Melanogenesis-Inhibitory, and EBV-EA Induction-Inhibitory Activities of A. indica Leaf Extracts. Dried and powdered leaves of A. indica were extracted with EtOH, and the extract was passed through activated charcoal column in order to remove chlorophyll. The almost chlorophyll-free fraction was partitioned into AcOEt- and H2O-soluble fractions. The EtOH extract and the two fractions were evaluated for their cytotoxic activities against a human leukemia cell line, HL60, by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, melanogenesis-inhibitory activities in a-MSH-stimulated B16 melanoma cell line, and for inhibitory effects on EpsteinBarr virus early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. As compiled in Table 1, while the AcOEt-soluble fraction exhibited cytotoxicity (IC50 65.1 mg/ml) against HL60 cell line, and melanogenesis-inhibitory activity (9.1% melanin content with 39.7% cell viability at 100 mg/ml), the EtOH extract and the H2O-soluble fraction were inactive against both HL60 (IC50 > 100 mg/ml) and a-MSHstimulated B16 melanoma cell lines ( > 100% melanin content with 95% cell viability at 100 mg/ml). In addition, the AcOEt-soluble fraction exhibited the most potent inhibitory effect against TPA (20 ng/32 pmol)-induced EBV-EA activation in Raji cells (9.0% induction of EBV-EA at 100 weight ratio/20 ng TPA concentration) among the others. The AcOEt-soluble fraction was further investigated in this study. Isolation, Identification, and Structure Elucidation of Compounds. The AcOEtsoluble fraction was subjected to column chromatography (CC) over SiO2 and ODS, and reversed-phase preparative high-performance liquid chromatography (HPLC) which led to the isolation of seventeen compounds, 1 – 17, of which 14, 15, and 17 were new compounds. The fourteen known compounds were identified as nimonol (1) [16], isonimocinolide (2) [17], nimonolactone (3) [18], munronolide (4) [19], 6-deacetylnimbin (5) [20], 6-homodeacetylnimbin (6) [21], 6-deacetylnimbinolactone (7) [16], 6deacetylnimbinolide (8) [22], 6-deacetylisonimbonolide (9) [22], nimbinene (10) [23], 6-deacetylnimbinene (11) [23], nimbolide (12) [24], 28-deoxonimbolide (13) [24] [25],
Cell viability [%]
100.0 3.1 95.2 5.1 39.7 1.5 94.6 3.5 87.1 2.8
Melanin content [%]
100.0 4.2 107.5 0.4 9.1 0.6 122.7 5.8 68.9 2.3
IC50 [mg/ml] b )
> 100 65.1 2.4 > 100 1.3 0.3
Melanogenesis-inhibitory and cytotoxic activities c ) at 100 mg/ml in B16 cells
Cytotoxic activities in HL60 cells a )
11.6 1.3 (60) 9.0 1.3 (60) 14.6 1.5 (50)
100
52.7 1.5 51.5 1.3 56.9 1.5
10
Drug concentration e )
100 1.3 100 1.4 100 1.1
1
Percentage EBV-EA induction d )
a ) Cells were treated with test samples (1 10 4 1 10 6 g/ml) for 48 h, and cell viability was analyzed by MTT assay. b ) The IC50 value is the concentration of compound required to inhibit the growth of the cells by 50%. This was obtained on the basis of triplicate assay results. c ) Melanin content [%] and cell viability [%] were determined based on the absorbances at 405, and 570 (test wavelength) – 630 (reference wavelength) nm, resp., by comparison with those for DMSO (100%). Each value represents the mean S.D. of three determinations. Concentration of DMSO in the sample solution was 2 ml/ml. d ) Values represent percentages relative to the positive control value, TPA (32 pmol, 20 ng) representing 100%. e ) Concentrations in terms of weight ratio/20 ng TPA. Values in parentheses are viability percentages of Raji cells. f ) Reference compounds.
Control (100% DMSO ) EtOH extract AcOEt-Soluble fraction H2O-Soluble fraction Cisplatin f ) Arbutin f )
Extract or fraction
Table 1. Cytotoxic Activities in HL60 Cells, Melanogenesis-Inhibitory and Cytotoxic Activities in B16 Mouse Melanoma Cells, and Percentage of EpsteinBarr Early Antigen ( EBV-EA ) Induction of Azadirachta indica Leaf Extracts CHEMISTRY & BIODIVERSITY – Vol. 11 (2014) 453
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and 2’,3’-dehydrosalannol (16) [26] by comparison of their spectral data with those in the literature. Of these known compounds, while ten limonoids, 1, 2, 5, 8 – 13, and 16, have already been found in A. indica, limonoid 4 has so far been found only in another Meliaceae plant, Munronia henryi [19]. On the other hand, whereas limonoids 3 [18], 6 [21], and 7 [18] have previously been obtained by structural modifications of natural limonoids 1, 12, and 5, respectively, this study seems to be the first instance of their isolation from a natural source. The structures of three new compounds, 14, 15, and 17, were elucidated on the basis of spectroscopic analysis and comparison with literature data as described below. Compound 14 displayed a [M þ Na] þ ion peak in its positive-ion mode HR-ESI mass spectrum at m/z 491.2019 (C27H32NaO þ7 ; calc. 491.2040), indicating the molecular formula C27H32O7. The UV spectrum showed absorption maximum at 233 nm indicating an a,b-unsaturated ketone system, while the IR spectrum showed the absorption bands of g-lactone (1750 cm 1), ester C¼O (1729 cm 1), and conjugated cyclohexenone (1676 cm 1) functions. The 13C- and 1H-NMR data (Table 2) of 14 were analogous to those of compound 13 [24] [25], although the furan-3-yl ring signals for 13 were lacking for 14. The presence of an a,b-unsaturated-g-lactone ring instead of the furan-3-yl ring at C(17) was deduced from the 1H-NMR (d(H) 4.77 (dd, J ¼ 1.4, 3.2; CH2(23)) and 7.21 (dd, J ¼ 1.8, 3.2; HC(22))), and 13C-NMR signals (d(C) 135.2 (C(20)), 174.2 (C(21)), 144.9 (C(22)), and 70.4 (C(23))) [6] [27]. The HMBC correlations (Table 2) between HC(17) and C(20), C(21), and C(22) indicated that the b-substituted-g-lactone ring was located at C(17). Hence, the structure of 14 was assigned as 17-defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide. The NOE correlations between HbC(16) (d(H) 2.08), HC(17) (d(H) 3.58), and Me(18) (d(H) 1.75) (Fig. 1) supported the a-orientation of the g-lactone ring at C(17). Compound 15 exhibited a [M þ Na] þ ion peak in its positive-ion mode HR-ESI mass spectrum at m/z 507.2028 (C27H32NaO þ8 ; calc. 507.1994), consistent with the
Fig. 1. Major NOE correlations ( $ ) for compounds 14 and 17. Drawings correspond to energyminimized conformation of compounds. Calculation was performed using CAChe Conformation Search with the MM2 force field (CaChe version 6.01; Fujitsu Co., Tokyo, Japan).
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molecular formula C27H32O8 . The UV absorption observed at 231 nm suggested the presence of an a,b-unsaturated ketone system. The IR spectrum of 15 indicated the presence of OH (3458 cm 1), g-lactone (1752 cm 1), ester C¼O (1735 cm 1), and conjugated cyclohexenone (1680 cm 1) functions. The 13C- and 1H-NMR spectra
18 19 20 21 22 23
17
12 13 14 15 16
10 11
6 7 8 9
3 4 5
1 2
Position
152.3 1, 4, 5, 28, 29 42.1 49.0 1, 3, 4,6, 7, 9, 28, 29 72.4 7 85.6 5, 6, 8, 9 50.8 41.4 1, 12, 14
46.3 32.6 8, 9, 10, 12 8, 9, 10, 12 174.0 133.1 148.5 5.35 (tq, J ¼ 1.8, 6.4) 87.4 13, 14 2.18 (dd, J ¼ 6.9, 12.4, Ha ) 40.1 13, 14, 15, 20 2.08 (dt, J ¼ 8.7, 12.4, Hb ) 14, 15, 17, 20 3.58 (br. d, J ¼ 9.2) 48.7 13, 14, 15, 16, 20, 21, 22 1.75 (d, J ¼ 1.8) 13.2 13, 14, 17 1.17 (s) 14.6 1, 5, 9, 10 135.2 174.2 7.21 (dd, J ¼ 1.8, 3.2) 144.9 17, 20, 21, 23 4.77 (2 H, dd, J ¼ 1.4, 3.2) 70.4 20, 21, 22 2.37 (dd, J ¼ 5.2, 16.3) 3.26 (dd, J ¼ 5.7, 16.3)
2.55 (t, J ¼ 5.5)
4.13 (dd, J ¼ 3.7, 12.8) 4.24 (d, J ¼ 3.7)
2.77 (d, J ¼ 12.4)
7.06 (d, J ¼ 9.6)
202.5 130.2 4, 10 152.4 1, 4, 5,28 42.0 49.0 1, 3, 4, 6, 7, 9, 10, 28, 29 72.3 5, 7 85.5 5, 6, 7, 9, 14 50.9 41.5 8, 10, 11, 12, 19, 30 46.2 32.9 8, 9, 10, 12
202.3 129.9 4, 10
d(C ) HMBC (H!C)
174.2 131.0 149.7 5.62 (br. t, J ¼ 7.7) 88.3 13 2.38 (dt, J ¼ 7.0, 12.9, Ha ) 39.0 13, 14, 15, 20 2.18 (dd, J ¼ 8.7, 12.9, Hb ) 15, 17, 20 3.73 (br. d, J ¼ 9.0) 53.1 13, 15, 16, 20, 21, 22 1.73 (d, J ¼ 1.8) 13.0 13, 14, 17 1.18 (s) 14.2 1, 5, 9, 10 168.8 5.76 (s) 97.3 23 5.93 (s) 120.2 17, 20, 21, 23 170.1
3.17 (dd, J ¼ 6.4, 16.5) 3.25 (dd, J ¼ 3.9, 16.5)
2.51 (dd, J ¼ 3.7, 6.4)
4.11 (dd, J ¼ 3.7, 12.8) 4.24 (d, J ¼ 3.7)
2.71 (d, J ¼ 12.4)
7.08 (d, J ¼ 10.2)
5.89 (d, J ¼ 10.1)
d( H)
5.88 (d, J ¼ 10.1)
15 d( H )
14 d(C ) HMBC (H!C)
71.1 1’, 3, 5, 10 30.7 3, 4, 10 4, 10 70.8 1, 5 44.1 38.9 4, 6,7,9, 10, 19, 28, 29 72.2 7 86.2 5, 6, 8, 9, 30 48.1 39.0 8, 10, 11, 12, 19, 30 40.6 29.9 8, 9, 10, 12 8, 9, 10, 12 174.7 132.4 147.8 87.3 13 40.2 15, 17, 20 13, 14, 15,20 48.7 13, 14, 15,16, 20, 21, 22 13.3 13, 14, 17 15.2 1, 5, 9, 10 137.1 171.4 141.6 17, 20, 21, 23 96.8 20, 21
5.05 (t, J ¼ 3.2) 1.99 (dt, J ¼ 3.2, 9.2, Ha ) 2.31 – 2.28 (m, Hb ) 3.88 (t, J ¼ 3.0)
6.80 (br. s) 5.95 (d, J ¼ 10.5)
1.82 (d, J ¼ 1.8) 0.91 (s)
5.37 – 5.44 (m) 2.10 – 2.15 (m, Hb ) 2.33 – 2.38 (m, Ha ) 3.48 (br. d, J ¼ 7.3)
2.14 – 2.20 (m) 2.38 – 2.43 (m)
2.23 – 2.40 (m)
3.98 (dd, J ¼ 3.2, 12.4) 4.26 (d, J ¼ 3.2)
2.58 (d, J ¼ 12.6)
d(C ) HMBC (H!C)
d( H )
17
Table 2. 1H- and 13C- (400 and 100 MHz, resp.), and HMBC NMR Data of Compounds 14, 15, and 17 (recorded in CDCl3 ; d in ppm, J in Hz)
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28 20.7 17.3 51.8
79.5
4, 5, 6, 29 3, 4, 29 3, 4, 28 7, 8, 9, 14 12
3.75 (br. d, J ¼ 7.3, Ha ) 3.81 (d, J ¼ 7.3, Hb ) 1.35 (s) 1.29 (s) 3.77 (s)
d( H )
d( H)
d(C ) HMBC (H!C)
15
14
3.72 (br. d, J ¼ 7.3, Ha ) 3.81 (d, J ¼ 7.3, Hb ) 29 1.35 (s) 30 1.31 (s) COOMe 3.67 (s) 1’ 2’ 3’ 4’ 5’
Position
Table 2 (cont.)
20.6 17.1 52.5
79.4
3, 4, 5, 6, 29 3, 4, 5, 6, 29 3, 4, 5, 28 7, 8, 9, 14 12
d(C ) HMBC (H!C)
2.11 (d, J ¼ 0.9) 1.97 (d, J ¼ 0.9)
5.72 (br. s)
4.12 (br. d, J ¼ 7.3, Ha ) 3.64 (d, J ¼ 7.3, Hb ) 1.13 (s) 1.29 (s) 3.45 (s)
d( H )
17
77.9 5, 6, 29 5, 6, 29 19.5 3, 4, 5, 28 16.2 7, 8, 9, 14 52.5 12 164.4 114.6 1’, 4’, 5’ 160.1 20.3 2’, 3’, 5’ 27.4 2’, 3’, 4’
d(C ) HMBC (H!C)
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(Table 2) of 15 were closely similar to those of 14, although the a,b-unsaturated-glactone ring signals for 14 were lacking for 15. Instead of the g-lactone ring, the presence of a 21-hydroxybut-20(22)-ene-21,23-g-lactone ring at C(17) was deduced from the 1H-NMR (d(H) 5.76 (s; HC(21)) and 5.93 (s; HC(22)), and 13C-NMR signals (d(C) 168.8 (C(20)), 97.3 (C(21)), 120.2 (C(22)), and 170.1 (C(23))) [6] [17] [22] [28]. The HMBC cross-correlations (Table 2) of HC(17) (d(H) 3.73) with C(20), C(21), and C(22), and of HC(22) (d(H) 5.93) with C(17), C(20), C(21), and C(23), and the NOE correlations between HbC(16) (d(H) 2.18), HC(17) (d(H) 3.73), and Me(18) (d(H) 1.73) of 15 evidenced that the b-substituted-g-lactone ring was located at C(17) with an a-orientation. Thus, the structure of 15 was elucidated as 17defurano-17-(2x-2,5-dihydro-2-hydroxy-5-oxofuran-3-yl)-28-deoxonimbolide. The configuration at C(21) of 15 remained undetermined. The molecular formula of compound 17 was determined as C32H42O10 on the basis of its positive-ion mode HR-ESI mass spectrum (m/z 609.2677 ([M þ Na] þ , C32H42NaO þ10 ; calc. 609.2675). The UV spectrum displayed an absorption maximum at 220 nm consistent with an a,b-unsaturated ketone system, while the IR spectrum indicated the presence of OH (3418 cm 1), g-lactone (1746 cm 1), and ester C¼O (1730 cm 1) functions. The 13C- and 1H-NMR data (Table 2) of 17 were analogous to those of compound 16 [26], although the furan-3-yl ring signals for 16 were lacking in the spectra of 17. The presence of a g-hydroxy-a,b-unsaturated-g-lactone ring instead of a furan-3-yl ring at C(17) was deduced from the 1H-NMR (d(H) 5.95 (d, J ¼ 10.5; HC(23)) and 6.80 (br. s; HC(22))), and 13C-NMR signals (d(C) 137.1 (C(20)), 171.4 (C(21)), 141.6 (C(22)), and 96.8 (C(23))) [6] [17] [22]. The HMBC correlations (Table 2) of HC(17) with C(20), C(21), and C(22) indicated the presence of bsubstituted-g-lactone ring. Hence, the structure of 17 was assigned as 17-defurano-17(5x-2,5-dihydro-5-hydroxy-2-oxofuran-3-yl)-2’,3’-dehydrosalannol. The NOE correlations (Fig. 1) between HbC(16) (d(H) 2.10 – 2.15), HC(17) (d(H) 3.48), and Me(18) (d(H) 1.82) indicated that the g-lactone ring at C(17) was a-oriented. The configuration at C(23) of 17 remained undetermined. Cytotoxic Activities. The cytotoxic activities of compounds 1 – 17, 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 as compiled in Table 3. All compounds, except 4 and 7, exhibited cytotoxicities against one or more cancer cell lines tested with IC50 values in the range of 0.1 – 97.9 mm. In particular, the cytotoxic activities of compounds 1 – 3, 12, 13, 15, and 16 against HL60 (IC50 0.1 – 9.4 mm), of compounds 2 and 13 against A549 (IC50 7.6 and 9.3 mm, resp.), of compounds 1, 12, 13, and 15 against AZ521 (IC50 0.8 – 9.9 mm), and of compounds 1, 12, and 13 against Sk-BR-3 (IC50 1.7 – 7.8 mm) were observed to be 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). Among the active compounds, potent cytotoxicities of compounds 12 [2] [25] [28 – 30] and 13 [2] [25] [30] against a wide variety of human cancer cell lines have previously been reported, and it has been stated that compound 12 is the principal cytotoxic principle of neem leaf extract [31]. In addition, compound 12 has been investigated for the mechanisms of apoptosis-inducing activities against human leukemia (U937) [29], colon (HCT-119 and HT-29) [32], and hepatocarcinoma (HepG2) [33] cell lines.
4.2 1.1
18.4 1.9
9.5 0.5
49.6 2.5 > 100
9.9 0.5
0.8 0.03 2.4 0.4 72.2 4.1
46.1 6.2 78.0 4.6 > 100 76.0 3.9 > 100 > 100 64.0 5.7
> 100
6.5 0.8 13.0 4.2 17.0 0.3
AZ521 ( Stomach)
18.8 0.6
22.8 1.7 > 100
24.9 2.9
> 100 1.7 0.1 83.4 2.4
47.1 3.9 67.4 3.4 > 100 > 100 > 100 > 100 91.6 3.8
> 100
6.7 1.0 7.8 0.7 19.4 2.1
SK-BR-3 ( Breast)
a ) Cells were treated with compounds (1 10 4 1 10 6 m) for 48 h, and cell viability was evaluated by the MTT assay. b ) The IC50 values based on triplicate five points. c ) Data reproduced from [5]. d ) Reference compound.
Cisplatin d )
> 100 > 100
37.8 5.1
2.1 0.1 9.4 1.2 41.6 5.8
> 100 9.3 1.0 > 100
> 100 97.9 7.8 > 100 > 100 > 100 81.3 5.4 > 100
> 100
19.9 1.1 7.6 2.7 46.8 2.5
A549 ( Lung)
0.1 0.01 2.7 0.2 18.5 2.5
11.6 2.3 21.2 3.4 > 100 28.8 2.1 75.8 3.9 17.2 2.1 25.6 3.7
Nimbin-type limonoids 5 6-Deacetylnimbin c ) 6 6-Homodeacetylnimbin 7 6-Deacetylnimbinolactone 8 6-Deacetylnimbinolide 9 6-Deacetylisonimbinolide 10 Nimbinene 11 6-Deacetylnimbinene
Salannin-type limonoids 12 Nimbolide 13 28-Deoxonimbolide c ) 14 17-Defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide 15 17-Defurano-17-(2x-2,5-dihydro-2-hydroxy-5-oxofuran3-yl)-28-deoxonimbolide 16 2’,3’-Dehydrosalannol 17 17-Defurano-17-(5x-2,5-dihydro-5-hydroxy-2-oxofuran3-yl)-2’,3’-dehydrosalannol
> 100
2.8 0.1 5.9 1.1 7.9 1.0
HL60 ( Leukemia)
Vilasanin-type limonoid 4 Munronolide
Azadirone-type limonoids 1 Nimonol 2 Isonimocinolide 3 Nimonolactone
Compound
Table 3. Cytotoxic Activities ( IC50 S.D. [mm]) of the Compounds Isolated from Azadirachta indica Leaf Extract against Four Human Cancer Cells a ) b )
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On the basis of the results in Table 3, it can be concluded that azadirone-, nimbin-, and salannin-type limonoids possess in general potent or moderate cytotoxic activities and, those of azadirone- and salannin-types are more active than those of nimbin-type. Apoptosis-Inducing Activity of Compound 1. Compound 1, which exhibited potent cytotoxic activity against HL60 cells (IC50 2.8 mm), was evaluated for its apoptosisinducing activity using HL60 cells. HL60 Cells were incubated with the 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 [34]. Annexin V is a Ca-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, PI double positive), or necrotic (annexin V negative, PI positive) cell death. The ratio of early apoptotic cells (Fig. 2, lower right) increased after treatment with 1 in HL60 cells for 24 h (from 3.2 to 22.6%) and 48 h (36.5%), and that of late apoptotic cells (upper right) increased after 48 h (from 4.8 to 45.8%). These results demonstrated that most of the cytotoxicity of compound 1 against HL60 cells is due to inducing apoptotic cell death.
Fig. 2. Detection of compound-1-induced early and late apoptotic cells by annexin VPI double staining in HL60 cells. The cells were cultured with 30 mm 1 for 24 and 48 h.
Caspases are known to mediate the apoptotic pathway [35] [36]. To clarify the mechanism by which compound 1 induces apoptotic cell death, activation of caspases-3, -8, and -9 was evaluated by Western blot analysis. After treatment of HL60 cells with 1 (30 mm), the levels of procaspases-3, -8, and -9 diminished and cleaved caspases-3, -8, and -9 were detected, almost in a time-dependent manner (Fig. 3). These results suggest that compound 1 induced apoptotic cell death via both the mitochondrial and the death-receptor mediated pathways. Next, we investigated the effect of compound 1 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 [37]. Expression of these
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Fig. 3. Western blot analysis of HL60 cells treated with compound 1. Western blot analysis of caspases-3, -8, and -9, and of Bcl-2 and Bax in HL60 cells with compound 1 (30 mm) for 8 and 24 h. The results are from one representative experiment among three runs, which showed similar patterns to one another.
proteins was examined by Western blot analysis. Treatment of HL60 cells with 1 (30 mm) decreased slightly the level of Bcl-2 and increased the level of Bax in a time-dependent manner (Fig. 3). The Bax/Bcl-2 ratio is one of the indices of the intrinsic mechanism of apoptosis in mitochondria [38]. Since compound 1 increased this ratio in HL60, it seems that compound 1-induced apoptosis involved Bax/Bcl-2 signal transduction. Compound 1 is, therefore, suggested to induce apoptosis in HL60 cells by involving the mitochondrial signal transduction pathway. Melanogenesis-Inhibitory Activity. All 17 compounds were evaluated for melanogenesis inhibition in a-MSH-stimulated B16 melanoma cells (Table 4). The cytotoxic activities of these compounds against B16 melanoma cells were also determined by means of MTT 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. A compound with a smaller A/C ratio would be a lowerrisk skin-whitening agent. Among the compounds tested, eleven compounds, i.e., 1, 2, 3 – 7, 10, and 15 – 7, exhibited inhibitory activities (6.1 – 89.2% melanin content) with no or almost no toxicity to the cells (81.7 – 148.2% cell viability) at 10 mm, and their A/C ratios were in the range of 0.07 – 0.86 at that concentration (Table 4). In addition, four compounds, i.e., 6, 10, 16, and 17, were further proved to be lower-risk melanogenesis inhibitors by exhibiting small A/C ratios, i.e., 0.37 – 0.47, with higher cell viabilities, i.e., 72.8 – 142.7%, at 30 mm. These compounds would be more potent melanogenesis inhibitors at lower
57.2 2.0 31.4 1.5 60.3 1.4 88.0 0.8 83.8 5.7 105.4 5.4 78.6 0.0 81.4 1.0 32.6 4.5 5.4 0.5 9.9 0.6 6.1 1.3 43.2 1.6 89.2 5.9 92.7 4.6
Vilasanin-type limonoid 4
Nimbin-type limonoids 5 6 7 8 9 10 11
Salannin-type limonoids 12 13 14 15 16 17
Arbutin d )
71.5 1.3
3.9 3.4 1.5 0.6 8.0 0.8 1.6 0.3 4.5 0.3 17.2 2.4
2.4 0.9 5.9 0.6 125.2 3.4 6.3 0.5 123.8 2.7 8.1 0.7 25.5 1.2
4.9 0.5
2.4 0.5 1.9 0.4 3.6 2.2
102.3 1.5
52.5 2.3 30.5 1.9 24.4 0.8 85.4 5.9 100.6 3.4 148.2 1.4
106.9 3.6 103.9 4.1 101.8 7.0 98.3 1.8 95.3 6.0 101.5 5.9 73.5 6.6
81.7 4.6
111.8 6.3 115.5 4.7 75.3 3.2
101.0 6.4
24.1 6.1 0.4 0.1 53.4 1.9 18.9 0.5 72.8 5.5 142.7 9.7
28.4 2.9 76.5 4.4 101.7 8.5 49.9 8.1 98.7 6.6 88.8 4.1 27.4 2.7
40.4 3.0
59.1 5.2 57.4 3.6 15.2 1.7
100.0 0.7
81.6 6.3
1.1 2.6 0.7 0.2 17.9 2.9 0.4 0.2 29.7 0.5 57.3 3.3
2.3 1.4 18.6 1.4 105.2 8.1 4.7 0.1 103.4 5.7 43.3 0.4 13.1 0.9
4.4 0.3
5.1 0.5 6.1 1.3 0.4 0.2
100.0 0.7
100 mm
A/C Ratio
0.91
0.62 0.18 0.41 0.07 0.43 0.60
0.29 0.58 0.86 0.85 1.11 0.77 1.11
0.70
0.62 0.56 1.82
10 mm
0.90
0.18 0.50 0.53 0.17 0.37 0.46
0.43 0.39 1.01 1.02 1.04 0.57 2.11
0.42
0.30 0.28 4.98
30 mm
0.88
3.55 2.14 0.45 4.00 0.15 0.30
1.04 0.31 1.19 1.34 1.20 0.19 1.95
1.11
0.47 0.31 9.00
100 mm
a ) Melanin content [%] and cell viability [%] were determined based on the absorbances at 405, and 570 (test wavelength) – 630 (reference wavelength) nm, resp., 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-to-cytotoxicity ratio, which was obtained by dividing the melanin content [%] by the cell viability [%] at each concentration. d ) Reference compound.
91.0 4.2
4.4 4.7 0.2 0.6 28.1 2.2 3.2 1.2 27.0 0.9 65.0 8.3
12.3 0.6 30.2 1.0 103.1 0.5 50.9 2.6 102.7 6.8 50.8 1.9 57.8 5.4
17.0 1.0
17.5 0.9 16.3 0.9 75.7 5.9
100.0 0.7
69.3 2.8 65.1 3.9 136.8 2.4
100.0 3.1
100.0 3.1
100.0 3.1
Control (100% DMSO)
Azadirone-type limonoids 1 2 3
30 mm
Cell viability [%] 10 mm
30 mm
10 mm
100 mm
Melanin content [%]
Compound
Table 4. Melanogenesis-Inhibitory and Cytotoxic Activities a ) b ), and A/C Ratios c ) of Compounds Isolated from the Leaf Extract of Azadirachta indica in B16 Mouse Melanoma Cell Line
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concentrations than the reference arbutin (4-hydroxyphenyl b-d-glucopyranoside; 92.7 and 91.0% melanin contents; 102.3 and 101.0% cell viabilities; and A/C ratios 0.91 and 0.90, at 10 and 30 mm, resp.), a known melanogenesis inhibitor and useful depigmentation compound for skin whitening in the cosmetic industry [39]. All compounds, except 7 and 9, exhibited potent melanogenesis-inhibitory activities (1.5 – 25.5% melanin contents) at 100 mm, however, with high cytotoxicities at that concentration (0.4 – 57.3% cell viabilities). In contrast to the melanogenesis inhibitory nature for fifteen compounds, 1 – 6, 8, and 10 – 17, two compounds, i.e., 7 (88.0, 103.1, and 125.2% melanin contents at 10, 30, and 100 mm, resp.) and 9 (105.4, 102.7, and 123.8% melanin contents at 10, 30, and 100 mm, resp.) exhibited induction of melanogenesis in a concentration-dependent manner without exhibiting cytotoxicity (Table 4). This seems to be the first observation of the induction of melanogenesis by limonoids, although several aromatic compounds, e.g., rosmarinic acid [40], 2,3,5,4’-tetrahydroxystilbene-2-O-b-d-glucoside [41], diethylstilbestrol [42], and hesperetin [43], have previously been reported to possess this feature. Inhibitory Effects on EBV-EA Induction. The inhibitory effects on the induction of EBV-EA induced by TPA were examined as a preliminary evaluation of anti-tumorpromoting activity. In Table 5, the inhibitory effects of compounds 1 – 17 against TPA (32 pmol)-induced EBV-EA activation in Raji cells are collected. Even at a concentration of 32 nm (mole ratio of compound to TPA 1000 : 1), high viability (60 – 70%) of Raji cells was observed, indicating the low cytotoxicity of the tested compounds. All compounds tested exhibited inhibitory effects (IC50 413 – 493 mole ratio/32 pmol TPA), which were more potent than, or almost comparable with, that of the reference compound, retinoic acid (IC50 482 mole ratio/32 pmol TPA), one of the retinoids that has been studied as a cancer-chemoprevention agent for various organ site cancers [44]. Since inhibitory effects against EBV-EA induction have been demonstrated to correlate with those against tumor promotion in vivo [45], all 17 compounds may be potential inhibitors of tumor promotion. Compound 12 has recently been shown to exhibit chemopreventive activity against 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis [46]. Conclusions. – This study has established that the AcOEt-soluble fraction of the EtOH extract of Azadirachta indica (neem) leaves exhibits cytotoxic activity against human HL60 cells, melanogenesis-inhibitory activity in a-MSH-stimulated B16 melanoma cells, and inhibitory effects against TPA-induced EBV-EA activation in Raji cells. Seventeen limonoids, 1 – 17, including three new compounds, 14, 15, and 17, have been isolated from the AcOEt-soluble fraction. Evaluation of these compounds established that nimonol (1), isonimocinolide (2), nimonolactone (3), nimbolide (12), 28-deoxonimbolide (13), and 2’,3’-dehydrosalannol (16) exhibit potent cytotoxic activities against HL60 and/or other human cancer cell lines tested, and the cell death of HL60 cells by compound 1 is mainly due to apoptosis. Western blot analysis indicated that compound 1 induced apoptosis via both the mitochondrial and the death receptormediated pathways. It appears that three limonoids, 1, 13, and 16, as well as 12 [31], are highly responsible for the cytotoxicity of the AcOEt-soluble fraction of the A. indica leaf extract, because these compounds constitute the major limonoids of the fraction.
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Table 5. Inhibitory Effects of Compounds Isolated from Neem Leaf Extracts and Reference Compound on the Induction of EpsteinBarr Virus Early Antigen ( EBV-EA ). Compound
Percentage EBV-EA induction a )
IC50 c ) (mole ratio/ 32 pmol TPA )
Drug concentration b ) 1000
500
100
10
Azadirone-type limonoids 1 11.9 0.7 (60) 2 9.3 0.7 (60) 3 9.1 0.7 (70)
49.7 1.5 48.2 1.6 46.2 1.5
73.9 2.5 72.0 2.5 71.9 2.5
100 0.3 100 0.5 100 0.3
470 470 460
Vilasanin-type limonoid 4 7.9 0.6 (60)
48.5 1.4
71.6 2.4
100 0.4
480
Nimbin-type limonoids 4.7 0.3 5 d) 6 3.5 0.4 7 13.8 0.6 8 6.3 0.4 9 12.3 0.7 10 15.3 0.8 11 8.1 0.5
(70) (60) (60) (60) (60) (60) (60)
46.2 1.5 43.7 1.5 51.1 1.8 46.1 1.3 50.0 1.4 52.8 1.5 47.0 1.4
88.8 2.1 71.6 2.2 80.1 2.0 71.3 2.3 80.1 2.0 82.7 2.1 76.1 2.4
100 0.4 98.8 0.5 100 0.4 100 0.3 100 0.5 100 0.5 100 0.3
489 430 493 468 487 487 460
Salannin-type limonoids 12 9.2 0.6 (60) 13 e ) 10.5 0.7 (60) 14 8.0 0.4 (60) 15 7.3 0.3 (60) 16 2.1 0.3 (60) 17 1.7 0.2 (60)
47.9 1.1 49.2 1.4 48.9 1.3 47.8 1.3 43.9 1.2 41.7 1.4
73.0 2.4 76.6 2.3 73.7 2.4 72.6 2.6 71.1 2.5 70.1 2.6
100 0.3 100 0.4 100 0.3 100 0.3 98.0 0.5 97.2 0.4
462 469 481 475 423 413
Retinoic acid f )
49.3 1.6
76.3 2.1
100 0.2
482
21.6 0.9 (60)
a
) Values represent the percentages relative to the positive control, with TPA (32 pmol, 20 ng) representing 100% induction. Values in parentheses are viability percentages of Raji cells. Data are expressed as mean S.D. (n ¼ 3). b ) Concentration in terms of mole ratio/32 pmol TPA. c ) The IC50 value represents the mole ratio of compounds, relative to TPA, required to inhibit 50% of the positive control activated with 32 pmol TPA. d ) Data taken from [3]. e ) Data taken from [4]. f ) Reference compound.
This study has further established that some limonoid constituents of the AcOEtsoluble fraction of A. indica leaf extract, i.e., 6-homodeacetylnimbin (6), nimbinene (10), 16, and 17-defurano-17-(5x-2,5-dihydro-5-hydroxy-2-oxofuran-3-yl)-2’,3’-dehydrosalannol (17), exhibit melanogenesis-inhibitory activities while showing no, or almost no, cytotoxicity at lower concentrations. These compounds may be, therefore, valuable as potential skin-whitening agents. In addition, on the basis of the results of in vitro EBV-EA induction, compounds 1 – 17 may be useful as agents that inhibit chemical carcinogenesis. This work has thus provided a further evidence of the importance of A. indica leaf extract and its limonoid constituents as potential anticancer, melanogenesis-inhibitory, and chemopreventive agents.
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Experimental Part General. Column chromatography (CC): activated charcoal (20 – 150 mesh; Nacalai Tesque, Inc., Kyoto, Japan), silica gel (SiO2 , 230 – 400 mesh; Merck & Co. Inc., D-Darmstadt), and ODS (Chromatorex-ODS, 100 – 200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan). Reversed-phase (RP) 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 MeCN/H2O (flow rate 3.0 ml/min) 3 : 2 (system I) or 9 : 11 (system II), or with MeCN/H2O/AcOH 50 : 50 : 0.1 (flow rate, 2.0 ml/min) (system III) or MeOH/H2O 7 : 3 (flow rate, 3.0 ml/min; system IV), or on a Capcell Pak AQ 5 mm column (Shiseido Co., Ltd., Tokyo, Japan) with MeCN/H2O 3 : 2 (flow rate, 3.0 ml/min; system V) or 2 : 3 (flow rate, 2.0 ml/ min; system VI). Optical rotations: JASCO P-1020 digital polarimeter. UV Spectra: JASCO V-630Bio spectrophotometer; lmax (log e) in nm. IR Spectra: JASCO FTIR-300E spectrometer; ˜n in cm 1. 1D- and 2D-NMR spectra: JEOL ECX-400 (1H, 400 MHz; 13C, 100 MHz) spectrometer at r.t.; CDCl3 soln.; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Agilent 1100 LC/MSD TOF (time-of-flight) system (ionization mode, pos.; 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). Chemicals and Materials. Leaves of Azadirachta indica were collected at Andhra Pradesh, India, in August, 2009. The plant was identified by Mr. Sriram Gangadhar (Ichimaru Pharcos Co. Ltd., Gifu, Japan). A voucher specimen (BI091201-2019) is deposited with the Research Laboratory, Ichimaru Pharcos Co. Ltd. The chemicals were purchased as follows: fetal bovine serum (FBS), RPMI-1640 medium, antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin), and non-essential amino acid (NEAA) from Invitrogen Co. (Carlsbad, CA, USA); Eagles minimal essential medium (MEM), Dulbeccos modified Eagles medium (DMEM), arbutin (4-hydroxyphenyl b-d-glucopyranoside), kojic acid, a-MSH, and MTT from Sigma-Aldrich Japan Co. (Tokyo, Japan); and rh annexin V/FITC kit (Bender MedSystems) from Cosmo Bio. Co. Ltd. (Tokyo, Japan). Primary antibodies of anti-MITF, antityrosinase, anti-TRP-1, and anti-TRP-2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-b-actin from Cell Signaling Technology (Beverly, MA, USA). All other chemicals and reagents were of anal. grade. Extraction and Isolation. The dried and powdered leaves of A. indica (3 kg) were extracted with EtOH by maceration at r.t. (38 l; 1 week, 1 ), and the extract was passed through membrane filter (0.45 mm), which, after evaporation of the solvent in vacuo, yielded the EtOH extract (126 g). To remove chlorophylls, a portion of the extract (96 g) was subjected to CC on activated charcoal (230 g) and eluted with MeOH/AcOEt (1 : 0 to 0:l). The almost chlorophyll-free MeOH eluate (48.0 g) was partitioned between AcOEt and H2O to yield the AcOEt- (35.0 g) and H2O-soluble fractions. The AcOEt-soluble fraction was applied to CC (SiO2 (700 g); hexane/AcOEt 4 : 1 to 0 : 1) to yield eleven fractions, Frs. A – K. Fr. D (2087 mg) from the eluate of hexane/AcOEt 3 : 2 was subjected to CC (ODS (60 g); MeOH/H2O 1 : 1 to 1 : 0) to yield six fractions, Frs. D1 – D6. Prep. HPLC of Frs. D3 (218 mg; system I) and D4 (249 mg; system V) afforded compounds 11 (tR 16.8 min; 36.7 mg), 10 (tR 22.8 min; 8.0 mg), and 1 (tR 32.8 min; 60.9 mg), and compound 3 (tR 14.4 min; 3.7 mg), resp. Fr. E (1532 mg) eluted with hexane/AcOEt 3 : 2 was applied to CC (SiO2 (30 g); hexane/AcOEt 1 : 1 to 0 : 1) to give four fractions, Frs. E1 – E4. Prep. HPLC (system II) of Fr. E2 (232 mg) yielded compound 2 (tR 32.8 min; 11.9 mg). Further CC (ODS (10 g); MeOH/H2O 3 : 7 to 1 : 0) and subsequent prep. HPLC (system II) of Fr. E3 (296 mg) afforded compound 7 (tR 63.0 min; 5.0 mg). A portion (70 mg) of Fr. F (1695) from the eluate of hexane/AcOEt 1 : 1 was subjected to prep. HPLC (system IV) to give compound 6 (tR 18.4 min; 2.3 mg). Fr. G (2482 mg) eluted with hexane/AcOEt 1 : 1 was subjected to CC (SiO2 (90 g); hexane/ AcOEt 7 : 3 to 0 : 1) to yield six fractions, Frs. G1 – G6. Prep. HPLC of Frs. G2 (573 mg; system V) and G5 (72 mg; system VI) afforded compounds 5 (tR 22.4 min; 171.1 mg) and 12 (tR 24.4 min; 778.9 mg), and compounds 9 (tR 22.8 min; 10.4 mg) and 8 (tR 24.8 min; 20.1 mg), resp. Fr. H (4049 mg) from the eluate of hexane/AcOEt 1 : 1 was further separated into eleven fractions, Frs. H1 – H11, by CC (SiO2 (70 g); CHCl3/MeOH 1 : 0 to 9 : 1). Prep. HPLC (system III) of Frs. H5 (163 mg) and H7 (95 mg) yielded compounds 15 (tR 15.2 min; 3.3 mg) and 17 (tR 21.6 min; 4.7 mg), and compound 4 (tR 28.0 min; 7.7 mg), resp. Fr. I (2305 mg) eluted with hexane/AcOEt 2 : 3 to 1 : 4 was subjected to CC (SiO2 (100 g); hexane/
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AcOEt 7 : 3 to 0 : 1) to give eight fractions, Frs. I1 – I8. Prep. HPLC of Frs. I2 (267 mg; system I) and I3 (679 mg; system V) furnished compounds 14 (tR 11.6 min; 5.8 mg) and 13 (tR 15.6 min; 56.6 mg), and compound 16 (tR 24.8 min; 41.3 mg), resp. The m.p., specific rotations, UV, IR, and HR-ESI-MS data of three new compounds, 14, 15, and 17, are given below. Their 1H- and 13C-NMR data are compiled in Table 2. 17-Defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide ( ¼ Methyl (2aR,5aR,6S,6aR, 8R,9aR,10aS,10bR,10cS)-8-(2,5-Dihydro-2-oxo-3-furanyl)-2a,5a,6,6a,8,9,9a,10a,10b,10c-decahydro2a,5a,6a,7-tetramethyl-5-oxo-2H,5H-cyclopenta[d]naphtho[2,3-b:1,8-bc]difuran-6-acetate; 14). Fine needles. M.p. 155 – 1588 (MeOH). [a] 25 D ¼ þ 54.5 (c ¼ 1.43, EtOH). UV (MeOH): 233 (3.90) (a,bunsaturated ketone). IR (KBr): 1750 (g-lactone), 1731 (ester C¼O), 1676 (conjugated cyclohexenone). NMR Spectra: see Table 2. HR-ESI-MS: 491.2019 ([M þ Na] þ , C27H32NaO þ7 ; calc. 491.2040). 17-Defurano-17-(5x-2,5-dihydro-5-hydroxy-2-oxofuran-3-yl)-28-deoxonimbolide ( ¼ Methyl (2aR, 5aR,6S,6aR,8R,9aR,10aS,10bR,10cS)-8-[(3x)-2,5-Dihydro-2-hydroxy-furan-3-yl]-2a,5a,6,6a,8,9,9a,10a, 10b,10c-decahydro-2a,5a,6a,7-tetramethyl-5-oxo-2H,5H-cyclopenta[d]naphtho[2,3-b:1,8-bc]difuran-6acetate; 15). Fine needles. M.p. 152 – 1558 (MeOH). [a] 20 D ¼ þ 107.1 (c ¼ 0.82, EtOH). UV (MeOH): 231 (4.01) (a,b-unsaturated ketone). IR (KBr): 3348 (OH), 1752 (g-lactone), 1735 (ester C¼O), 1680 (conjugated cyclohexenone). NMR Spectra : see Table 2. HR-ESI-MS : 507.2028 ( [M þ Na] þ , C27H32NaO þ8 ; calc. 507.1994). 17-Defurano-17-(5x-2,5-dihydroxy-5-hydroxy-2-oxofuran-3-yl)-2’,3’-dehydrosalannol ( ¼ Methyl (2aR,3R,5S,5aR,6R,6aR,8R,9aR,10aS,10bR,10cR)-8-[(5x)-2,5-Dihydro-5-hydroxy-2-oxo-furan-3-yl]2a,4,5,5a,6,6a,8,9,9a,10a,10b,10c-dodecahydro-3-hydroxy-2a,5a,6a,7-tetramethyl-5-[(3-methyl-1-oxo-2buten-1-yl)oxy]-2H,3H-cyclopenta[d]naphtho[2,3-b:1,8-bc]difuran-6-acetate; 17). Fine needles. M.p. 125 – 1288 (MeOH). [a] 25 D ¼ þ 80.7 (c ¼ 1.25, EtOH). UV (MeOH): 220 (3.96) nm (a,b-unsaturated ketone). IR (KBr): 3418 (OH), 1746 (g-lactone), 1730 (ester C¼O), 1713. NMR Spectra: see Table 2. HR-ESI-MS: 609.2677 ([M þ Na] þ , C32H42NaO þ10 ; calc. 609.2675). Cell Lines and Culture Conditions. Cell lines B16 4A5 (mouse melanoma), HL60 (leukemia), AZ521 (stomach), A549 (lung), and SK-BR-3 (breast) were obtained from Riken Cell Bank (Tsukuba, Ibaraki, Japan). Three cell lines, B16 4A5, HL60, and SK-BR-3, were grown in RPMI-1640 medium, while AZ521 and A549 cell lines were grown in DMEM and in 90% DMEM þ 10% MEM þ 0.1 mM NEAA, resp. 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 [47 – 49]. Determination of Cell Proliferation. Cell proliferation was assessed using the MTT based colorimetric assay as described in [47 – 49]. Assay of Melanin Content. Melanogenesis-inhibition assay in a-MSH-stimulated B16 melanoma cells was performed as described in [48]. Annexin VPropidium Iodide (PI) Double Staining. Annexin V-PI double staining was performed as described in [50] with HL60 (1 105 cells). Western Blotting. Western blot analysis was performed with HL60 (1 105 cells) as described in [50] [51]. REFERENCES [1] L. Zhao, C.-H. Huo, L.-R. Shen, Y. Yang, Q. Zhang, Q.-W. Shi, Chem. Biodiversity 2010, 7, 839. [2] Q.-G. Tan, X.-D. Luo, Chem. Rev. 2011, 111, 7437. [3] T. Akihisa, T. Noto, A. Takahashi, Y. Fujita, N. Banno, H. Tokuda, K. Koike, T. Suzuki, K. Yasukawa, Y. Kimura, J. Oleo Sci. 2009, 58, 581. [4] T. Akihisa, A. Takahashi, T. Kikuchi, M. Takagi, K. Watanabe, M. Fukatsu, Y. Fujita, N. Banno, H. Tokuda, K. Yasukawa, J. Oleo Sci. 2011, 60, 53. [5] T. Kikuchi, K. Ishii, T. Noto, A. Takahashi, K. Tabata, T. Suzuki, T. Akihisa, J. Nat. Prod. 2011, 74, 866. [6] T. Akihisa, X. Pan, Y. Nakamura, T. Kikuchi, N. Takahashi, M. Matsumoto, E. Ogihara, M. Fukatsu, K. Koike, H. Tokuda, Phytochemistry 2013, 89, 59.
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Cytotoxic and Melanogenesis-Inhibitory Activities of Limonoids from the Leaves of Azadirachta indica (Neem) by Mio Takagi a ), Yosuke Tachi a ), Jie Zhang a ), Takuro Shinozaki a ), Kenta Ishii a ), Takashi Kikuchi a ), Motohiko Ukiya a ), Norihiro Banno b ), Harukuni Tokuda c ), and Toshihiro Akihisa* a ) d ) a
) College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan b ) Ichimaru Pharcos Company Ltd., 318-1 Asagi, Motosu-shi, Gifu 501-0475, Japan c ) Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan d ) Akihisa Medical Clinic, 1086-3 Kamo, Sanda-shi, Hyogo 669-1311, Japan (fax: þ 81-79-5671980; e-mail: [email protected])
Seventeen limonoids (tetranortriterpenoids), 1 – 17, including three new compounds, i.e., 17defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide (14), 17-defurano-17-(2x-2,5-dihydro-2hydroxy-5-oxofuran-3-yl)-28-deoxonimbolide (15), and 17-defurano-17-(5x-2,5-dihydro-5-hydroxy-2oxofuran-3-yl)-2’,3’-dehydrosalannol (17), were isolated from an EtOH extract of the leaf of neem (Azadirachta indica). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines, seven compounds, i.e., 1 – 3, 12, 13, 15, and 16, exhibited potent cytotoxicities with IC50 values in the range of 0.1 – 9.9 mm against one or more cell lines. Among these compounds, cytotoxicity of nimonol (1; IC50 2.8 mm) against HL60 cells was demonstrated to be mainly due to the induction of apoptosis by flow cytometry. Western blot analysis suggested that compound 1 induced apoptosis via both the mitochondrial and death receptor-mediated pathways in HL60 cells. In addition, when compounds 1 – 17 were evaluated for their inhibitory activities against melanogenesis in B16 melanoma cells, induced with a-melanocyte-stimulating hormone (a-MSH), seven compounds, 1, 2, 4 – 6, 15, and 16, exhibited inhibitory activities with 31 – 94% reduction of melanin content at 10 mm concentration with no or low toxicity to the cells (82 – 112% of cell viability at 10 mm). All 17 compounds were further evaluated for their inhibitory effects against the EpsteinBarr virus early antigen (EBV-EA) activation induced by 12O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells.
Introduction. – Plants of the Meliaceae family have been well documented for their ability to metabolize structurally diverse and biologically significant limonoids and triterpenoids [1] [2]. In the course of a search for potential bioactive compounds from Meliaceae plants, a detailed investigation on the limonoid constituents of the seed extracts of Azadirachta indica A. Juss. (neem tree) was carried out, and this revealed that some limonoids exhibit potent inhibitory activities against melanogenesis in B16 melanoma cells, against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, and against TPA-induced EpsteinBarr virus early antigen (EBV-EA) activation [3] [4], as well as cytotoxic activities against HL60, A549, AZ521, SK-BR-3, and CRL1579 human cancer cell lines, and apoptosis-inducing activity against HL60 cell line [5]. Further, we have reported that some limonoids from the fruits of Melia 2014 Verlag Helvetica Chimica Acta AG, Zrich
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azedarach exhibited potent cytotoxic activities against HL60, A549, AZ521, and SKBR-3 human cancer cell lines [6], and apoptosis-inducing activities against HL60 and AZ521 cell lines [7]. In a continuing study on the limonoid constituents of Meliaceae plants, the leaf extract of A. indica was investigated, resulting in the isolation of 17 limonoids, 1 – 17, including three new compounds, 14, 15, and 17 (see below). Herein, we describes the structure elucidation of these compounds and evaluation of their cytotoxic activities against four human cancer cell lines, melanogenesis-inhibitory activities in amelanocyte-stimulating hormone (a-MSH)-stimulated B16 melanoma cells, and inhibitory effects on the induction of EBV-EA activation induced with TPA in Raji cells. Extract of neem leaves has been therapeutically used as folk medicine to control leprosy, eye problem, epistaxis, intestinal worms, anorexia, biliousness, and skin ulcers [8] [9], and it has been reported to be nontoxic and non-mutagenic [10], and to possess a wide range of biological prosperities such as immunomodulatory, anti-inflammatory, antihyperglycaemic, antiulcer, antimalarial, antifungal, antibacterial, antiviral, antioxidant, antimutagenic, chemopreventive, and anticarcinogenic activities [8 – 15]. Results and Discussions. – Cytotoxic, Melanogenesis-Inhibitory, and EBV-EA Induction-Inhibitory Activities of A. indica Leaf Extracts. Dried and powdered leaves of A. indica were extracted with EtOH, and the extract was passed through activated charcoal column in order to remove chlorophyll. The almost chlorophyll-free fraction was partitioned into AcOEt- and H2O-soluble fractions. The EtOH extract and the two fractions were evaluated for their cytotoxic activities against a human leukemia cell line, HL60, by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, melanogenesis-inhibitory activities in a-MSH-stimulated B16 melanoma cell line, and for inhibitory effects on EpsteinBarr virus early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. As compiled in Table 1, while the AcOEt-soluble fraction exhibited cytotoxicity (IC50 65.1 mg/ml) against HL60 cell line, and melanogenesis-inhibitory activity (9.1% melanin content with 39.7% cell viability at 100 mg/ml), the EtOH extract and the H2O-soluble fraction were inactive against both HL60 (IC50 > 100 mg/ml) and a-MSHstimulated B16 melanoma cell lines ( > 100% melanin content with 95% cell viability at 100 mg/ml). In addition, the AcOEt-soluble fraction exhibited the most potent inhibitory effect against TPA (20 ng/32 pmol)-induced EBV-EA activation in Raji cells (9.0% induction of EBV-EA at 100 weight ratio/20 ng TPA concentration) among the others. The AcOEt-soluble fraction was further investigated in this study. Isolation, Identification, and Structure Elucidation of Compounds. The AcOEtsoluble fraction was subjected to column chromatography (CC) over SiO2 and ODS, and reversed-phase preparative high-performance liquid chromatography (HPLC) which led to the isolation of seventeen compounds, 1 – 17, of which 14, 15, and 17 were new compounds. The fourteen known compounds were identified as nimonol (1) [16], isonimocinolide (2) [17], nimonolactone (3) [18], munronolide (4) [19], 6-deacetylnimbin (5) [20], 6-homodeacetylnimbin (6) [21], 6-deacetylnimbinolactone (7) [16], 6deacetylnimbinolide (8) [22], 6-deacetylisonimbonolide (9) [22], nimbinene (10) [23], 6-deacetylnimbinene (11) [23], nimbolide (12) [24], 28-deoxonimbolide (13) [24] [25],
Cell viability [%]
100.0 3.1 95.2 5.1 39.7 1.5 94.6 3.5 87.1 2.8
Melanin content [%]
100.0 4.2 107.5 0.4 9.1 0.6 122.7 5.8 68.9 2.3
IC50 [mg/ml] b )
> 100 65.1 2.4 > 100 1.3 0.3
Melanogenesis-inhibitory and cytotoxic activities c ) at 100 mg/ml in B16 cells
Cytotoxic activities in HL60 cells a )
11.6 1.3 (60) 9.0 1.3 (60) 14.6 1.5 (50)
100
52.7 1.5 51.5 1.3 56.9 1.5
10
Drug concentration e )
100 1.3 100 1.4 100 1.1
1
Percentage EBV-EA induction d )
a ) Cells were treated with test samples (1 10 4 1 10 6 g/ml) for 48 h, and cell viability was analyzed by MTT assay. b ) The IC50 value is the concentration of compound required to inhibit the growth of the cells by 50%. This was obtained on the basis of triplicate assay results. c ) Melanin content [%] and cell viability [%] were determined based on the absorbances at 405, and 570 (test wavelength) – 630 (reference wavelength) nm, resp., by comparison with those for DMSO (100%). Each value represents the mean S.D. of three determinations. Concentration of DMSO in the sample solution was 2 ml/ml. d ) Values represent percentages relative to the positive control value, TPA (32 pmol, 20 ng) representing 100%. e ) Concentrations in terms of weight ratio/20 ng TPA. Values in parentheses are viability percentages of Raji cells. f ) Reference compounds.
Control (100% DMSO ) EtOH extract AcOEt-Soluble fraction H2O-Soluble fraction Cisplatin f ) Arbutin f )
Extract or fraction
Table 1. Cytotoxic Activities in HL60 Cells, Melanogenesis-Inhibitory and Cytotoxic Activities in B16 Mouse Melanoma Cells, and Percentage of EpsteinBarr Early Antigen ( EBV-EA ) Induction of Azadirachta indica Leaf Extracts CHEMISTRY & BIODIVERSITY – Vol. 11 (2014) 453
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and 2’,3’-dehydrosalannol (16) [26] by comparison of their spectral data with those in the literature. Of these known compounds, while ten limonoids, 1, 2, 5, 8 – 13, and 16, have already been found in A. indica, limonoid 4 has so far been found only in another Meliaceae plant, Munronia henryi [19]. On the other hand, whereas limonoids 3 [18], 6 [21], and 7 [18] have previously been obtained by structural modifications of natural limonoids 1, 12, and 5, respectively, this study seems to be the first instance of their isolation from a natural source. The structures of three new compounds, 14, 15, and 17, were elucidated on the basis of spectroscopic analysis and comparison with literature data as described below. Compound 14 displayed a [M þ Na] þ ion peak in its positive-ion mode HR-ESI mass spectrum at m/z 491.2019 (C27H32NaO þ7 ; calc. 491.2040), indicating the molecular formula C27H32O7. The UV spectrum showed absorption maximum at 233 nm indicating an a,b-unsaturated ketone system, while the IR spectrum showed the absorption bands of g-lactone (1750 cm 1), ester C¼O (1729 cm 1), and conjugated cyclohexenone (1676 cm 1) functions. The 13C- and 1H-NMR data (Table 2) of 14 were analogous to those of compound 13 [24] [25], although the furan-3-yl ring signals for 13 were lacking for 14. The presence of an a,b-unsaturated-g-lactone ring instead of the furan-3-yl ring at C(17) was deduced from the 1H-NMR (d(H) 4.77 (dd, J ¼ 1.4, 3.2; CH2(23)) and 7.21 (dd, J ¼ 1.8, 3.2; HC(22))), and 13C-NMR signals (d(C) 135.2 (C(20)), 174.2 (C(21)), 144.9 (C(22)), and 70.4 (C(23))) [6] [27]. The HMBC correlations (Table 2) between HC(17) and C(20), C(21), and C(22) indicated that the b-substituted-g-lactone ring was located at C(17). Hence, the structure of 14 was assigned as 17-defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide. The NOE correlations between HbC(16) (d(H) 2.08), HC(17) (d(H) 3.58), and Me(18) (d(H) 1.75) (Fig. 1) supported the a-orientation of the g-lactone ring at C(17). Compound 15 exhibited a [M þ Na] þ ion peak in its positive-ion mode HR-ESI mass spectrum at m/z 507.2028 (C27H32NaO þ8 ; calc. 507.1994), consistent with the
Fig. 1. Major NOE correlations ( $ ) for compounds 14 and 17. Drawings correspond to energyminimized conformation of compounds. Calculation was performed using CAChe Conformation Search with the MM2 force field (CaChe version 6.01; Fujitsu Co., Tokyo, Japan).
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molecular formula C27H32O8 . The UV absorption observed at 231 nm suggested the presence of an a,b-unsaturated ketone system. The IR spectrum of 15 indicated the presence of OH (3458 cm 1), g-lactone (1752 cm 1), ester C¼O (1735 cm 1), and conjugated cyclohexenone (1680 cm 1) functions. The 13C- and 1H-NMR spectra
18 19 20 21 22 23
17
12 13 14 15 16
10 11
6 7 8 9
3 4 5
1 2
Position
152.3 1, 4, 5, 28, 29 42.1 49.0 1, 3, 4,6, 7, 9, 28, 29 72.4 7 85.6 5, 6, 8, 9 50.8 41.4 1, 12, 14
46.3 32.6 8, 9, 10, 12 8, 9, 10, 12 174.0 133.1 148.5 5.35 (tq, J ¼ 1.8, 6.4) 87.4 13, 14 2.18 (dd, J ¼ 6.9, 12.4, Ha ) 40.1 13, 14, 15, 20 2.08 (dt, J ¼ 8.7, 12.4, Hb ) 14, 15, 17, 20 3.58 (br. d, J ¼ 9.2) 48.7 13, 14, 15, 16, 20, 21, 22 1.75 (d, J ¼ 1.8) 13.2 13, 14, 17 1.17 (s) 14.6 1, 5, 9, 10 135.2 174.2 7.21 (dd, J ¼ 1.8, 3.2) 144.9 17, 20, 21, 23 4.77 (2 H, dd, J ¼ 1.4, 3.2) 70.4 20, 21, 22 2.37 (dd, J ¼ 5.2, 16.3) 3.26 (dd, J ¼ 5.7, 16.3)
2.55 (t, J ¼ 5.5)
4.13 (dd, J ¼ 3.7, 12.8) 4.24 (d, J ¼ 3.7)
2.77 (d, J ¼ 12.4)
7.06 (d, J ¼ 9.6)
202.5 130.2 4, 10 152.4 1, 4, 5,28 42.0 49.0 1, 3, 4, 6, 7, 9, 10, 28, 29 72.3 5, 7 85.5 5, 6, 7, 9, 14 50.9 41.5 8, 10, 11, 12, 19, 30 46.2 32.9 8, 9, 10, 12
202.3 129.9 4, 10
d(C ) HMBC (H!C)
174.2 131.0 149.7 5.62 (br. t, J ¼ 7.7) 88.3 13 2.38 (dt, J ¼ 7.0, 12.9, Ha ) 39.0 13, 14, 15, 20 2.18 (dd, J ¼ 8.7, 12.9, Hb ) 15, 17, 20 3.73 (br. d, J ¼ 9.0) 53.1 13, 15, 16, 20, 21, 22 1.73 (d, J ¼ 1.8) 13.0 13, 14, 17 1.18 (s) 14.2 1, 5, 9, 10 168.8 5.76 (s) 97.3 23 5.93 (s) 120.2 17, 20, 21, 23 170.1
3.17 (dd, J ¼ 6.4, 16.5) 3.25 (dd, J ¼ 3.9, 16.5)
2.51 (dd, J ¼ 3.7, 6.4)
4.11 (dd, J ¼ 3.7, 12.8) 4.24 (d, J ¼ 3.7)
2.71 (d, J ¼ 12.4)
7.08 (d, J ¼ 10.2)
5.89 (d, J ¼ 10.1)
d( H)
5.88 (d, J ¼ 10.1)
15 d( H )
14 d(C ) HMBC (H!C)
71.1 1’, 3, 5, 10 30.7 3, 4, 10 4, 10 70.8 1, 5 44.1 38.9 4, 6,7,9, 10, 19, 28, 29 72.2 7 86.2 5, 6, 8, 9, 30 48.1 39.0 8, 10, 11, 12, 19, 30 40.6 29.9 8, 9, 10, 12 8, 9, 10, 12 174.7 132.4 147.8 87.3 13 40.2 15, 17, 20 13, 14, 15,20 48.7 13, 14, 15,16, 20, 21, 22 13.3 13, 14, 17 15.2 1, 5, 9, 10 137.1 171.4 141.6 17, 20, 21, 23 96.8 20, 21
5.05 (t, J ¼ 3.2) 1.99 (dt, J ¼ 3.2, 9.2, Ha ) 2.31 – 2.28 (m, Hb ) 3.88 (t, J ¼ 3.0)
6.80 (br. s) 5.95 (d, J ¼ 10.5)
1.82 (d, J ¼ 1.8) 0.91 (s)
5.37 – 5.44 (m) 2.10 – 2.15 (m, Hb ) 2.33 – 2.38 (m, Ha ) 3.48 (br. d, J ¼ 7.3)
2.14 – 2.20 (m) 2.38 – 2.43 (m)
2.23 – 2.40 (m)
3.98 (dd, J ¼ 3.2, 12.4) 4.26 (d, J ¼ 3.2)
2.58 (d, J ¼ 12.6)
d(C ) HMBC (H!C)
d( H )
17
Table 2. 1H- and 13C- (400 and 100 MHz, resp.), and HMBC NMR Data of Compounds 14, 15, and 17 (recorded in CDCl3 ; d in ppm, J in Hz)
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28 20.7 17.3 51.8
79.5
4, 5, 6, 29 3, 4, 29 3, 4, 28 7, 8, 9, 14 12
3.75 (br. d, J ¼ 7.3, Ha ) 3.81 (d, J ¼ 7.3, Hb ) 1.35 (s) 1.29 (s) 3.77 (s)
d( H )
d( H)
d(C ) HMBC (H!C)
15
14
3.72 (br. d, J ¼ 7.3, Ha ) 3.81 (d, J ¼ 7.3, Hb ) 29 1.35 (s) 30 1.31 (s) COOMe 3.67 (s) 1’ 2’ 3’ 4’ 5’
Position
Table 2 (cont.)
20.6 17.1 52.5
79.4
3, 4, 5, 6, 29 3, 4, 5, 6, 29 3, 4, 5, 28 7, 8, 9, 14 12
d(C ) HMBC (H!C)
2.11 (d, J ¼ 0.9) 1.97 (d, J ¼ 0.9)
5.72 (br. s)
4.12 (br. d, J ¼ 7.3, Ha ) 3.64 (d, J ¼ 7.3, Hb ) 1.13 (s) 1.29 (s) 3.45 (s)
d( H )
17
77.9 5, 6, 29 5, 6, 29 19.5 3, 4, 5, 28 16.2 7, 8, 9, 14 52.5 12 164.4 114.6 1’, 4’, 5’ 160.1 20.3 2’, 3’, 5’ 27.4 2’, 3’, 4’
d(C ) HMBC (H!C)
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(Table 2) of 15 were closely similar to those of 14, although the a,b-unsaturated-glactone ring signals for 14 were lacking for 15. Instead of the g-lactone ring, the presence of a 21-hydroxybut-20(22)-ene-21,23-g-lactone ring at C(17) was deduced from the 1H-NMR (d(H) 5.76 (s; HC(21)) and 5.93 (s; HC(22)), and 13C-NMR signals (d(C) 168.8 (C(20)), 97.3 (C(21)), 120.2 (C(22)), and 170.1 (C(23))) [6] [17] [22] [28]. The HMBC cross-correlations (Table 2) of HC(17) (d(H) 3.73) with C(20), C(21), and C(22), and of HC(22) (d(H) 5.93) with C(17), C(20), C(21), and C(23), and the NOE correlations between HbC(16) (d(H) 2.18), HC(17) (d(H) 3.73), and Me(18) (d(H) 1.73) of 15 evidenced that the b-substituted-g-lactone ring was located at C(17) with an a-orientation. Thus, the structure of 15 was elucidated as 17defurano-17-(2x-2,5-dihydro-2-hydroxy-5-oxofuran-3-yl)-28-deoxonimbolide. The configuration at C(21) of 15 remained undetermined. The molecular formula of compound 17 was determined as C32H42O10 on the basis of its positive-ion mode HR-ESI mass spectrum (m/z 609.2677 ([M þ Na] þ , C32H42NaO þ10 ; calc. 609.2675). The UV spectrum displayed an absorption maximum at 220 nm consistent with an a,b-unsaturated ketone system, while the IR spectrum indicated the presence of OH (3418 cm 1), g-lactone (1746 cm 1), and ester C¼O (1730 cm 1) functions. The 13C- and 1H-NMR data (Table 2) of 17 were analogous to those of compound 16 [26], although the furan-3-yl ring signals for 16 were lacking in the spectra of 17. The presence of a g-hydroxy-a,b-unsaturated-g-lactone ring instead of a furan-3-yl ring at C(17) was deduced from the 1H-NMR (d(H) 5.95 (d, J ¼ 10.5; HC(23)) and 6.80 (br. s; HC(22))), and 13C-NMR signals (d(C) 137.1 (C(20)), 171.4 (C(21)), 141.6 (C(22)), and 96.8 (C(23))) [6] [17] [22]. The HMBC correlations (Table 2) of HC(17) with C(20), C(21), and C(22) indicated the presence of bsubstituted-g-lactone ring. Hence, the structure of 17 was assigned as 17-defurano-17(5x-2,5-dihydro-5-hydroxy-2-oxofuran-3-yl)-2’,3’-dehydrosalannol. The NOE correlations (Fig. 1) between HbC(16) (d(H) 2.10 – 2.15), HC(17) (d(H) 3.48), and Me(18) (d(H) 1.82) indicated that the g-lactone ring at C(17) was a-oriented. The configuration at C(23) of 17 remained undetermined. Cytotoxic Activities. The cytotoxic activities of compounds 1 – 17, 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 as compiled in Table 3. All compounds, except 4 and 7, exhibited cytotoxicities against one or more cancer cell lines tested with IC50 values in the range of 0.1 – 97.9 mm. In particular, the cytotoxic activities of compounds 1 – 3, 12, 13, 15, and 16 against HL60 (IC50 0.1 – 9.4 mm), of compounds 2 and 13 against A549 (IC50 7.6 and 9.3 mm, resp.), of compounds 1, 12, 13, and 15 against AZ521 (IC50 0.8 – 9.9 mm), and of compounds 1, 12, and 13 against Sk-BR-3 (IC50 1.7 – 7.8 mm) were observed to be 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). Among the active compounds, potent cytotoxicities of compounds 12 [2] [25] [28 – 30] and 13 [2] [25] [30] against a wide variety of human cancer cell lines have previously been reported, and it has been stated that compound 12 is the principal cytotoxic principle of neem leaf extract [31]. In addition, compound 12 has been investigated for the mechanisms of apoptosis-inducing activities against human leukemia (U937) [29], colon (HCT-119 and HT-29) [32], and hepatocarcinoma (HepG2) [33] cell lines.
4.2 1.1
18.4 1.9
9.5 0.5
49.6 2.5 > 100
9.9 0.5
0.8 0.03 2.4 0.4 72.2 4.1
46.1 6.2 78.0 4.6 > 100 76.0 3.9 > 100 > 100 64.0 5.7
> 100
6.5 0.8 13.0 4.2 17.0 0.3
AZ521 ( Stomach)
18.8 0.6
22.8 1.7 > 100
24.9 2.9
> 100 1.7 0.1 83.4 2.4
47.1 3.9 67.4 3.4 > 100 > 100 > 100 > 100 91.6 3.8
> 100
6.7 1.0 7.8 0.7 19.4 2.1
SK-BR-3 ( Breast)
a ) Cells were treated with compounds (1 10 4 1 10 6 m) for 48 h, and cell viability was evaluated by the MTT assay. b ) The IC50 values based on triplicate five points. c ) Data reproduced from [5]. d ) Reference compound.
Cisplatin d )
> 100 > 100
37.8 5.1
2.1 0.1 9.4 1.2 41.6 5.8
> 100 9.3 1.0 > 100
> 100 97.9 7.8 > 100 > 100 > 100 81.3 5.4 > 100
> 100
19.9 1.1 7.6 2.7 46.8 2.5
A549 ( Lung)
0.1 0.01 2.7 0.2 18.5 2.5
11.6 2.3 21.2 3.4 > 100 28.8 2.1 75.8 3.9 17.2 2.1 25.6 3.7
Nimbin-type limonoids 5 6-Deacetylnimbin c ) 6 6-Homodeacetylnimbin 7 6-Deacetylnimbinolactone 8 6-Deacetylnimbinolide 9 6-Deacetylisonimbinolide 10 Nimbinene 11 6-Deacetylnimbinene
Salannin-type limonoids 12 Nimbolide 13 28-Deoxonimbolide c ) 14 17-Defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide 15 17-Defurano-17-(2x-2,5-dihydro-2-hydroxy-5-oxofuran3-yl)-28-deoxonimbolide 16 2’,3’-Dehydrosalannol 17 17-Defurano-17-(5x-2,5-dihydro-5-hydroxy-2-oxofuran3-yl)-2’,3’-dehydrosalannol
> 100
2.8 0.1 5.9 1.1 7.9 1.0
HL60 ( Leukemia)
Vilasanin-type limonoid 4 Munronolide
Azadirone-type limonoids 1 Nimonol 2 Isonimocinolide 3 Nimonolactone
Compound
Table 3. Cytotoxic Activities ( IC50 S.D. [mm]) of the Compounds Isolated from Azadirachta indica Leaf Extract against Four Human Cancer Cells a ) b )
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On the basis of the results in Table 3, it can be concluded that azadirone-, nimbin-, and salannin-type limonoids possess in general potent or moderate cytotoxic activities and, those of azadirone- and salannin-types are more active than those of nimbin-type. Apoptosis-Inducing Activity of Compound 1. Compound 1, which exhibited potent cytotoxic activity against HL60 cells (IC50 2.8 mm), was evaluated for its apoptosisinducing activity using HL60 cells. HL60 Cells were incubated with the 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 [34]. Annexin V is a Ca-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, PI double positive), or necrotic (annexin V negative, PI positive) cell death. The ratio of early apoptotic cells (Fig. 2, lower right) increased after treatment with 1 in HL60 cells for 24 h (from 3.2 to 22.6%) and 48 h (36.5%), and that of late apoptotic cells (upper right) increased after 48 h (from 4.8 to 45.8%). These results demonstrated that most of the cytotoxicity of compound 1 against HL60 cells is due to inducing apoptotic cell death.
Fig. 2. Detection of compound-1-induced early and late apoptotic cells by annexin VPI double staining in HL60 cells. The cells were cultured with 30 mm 1 for 24 and 48 h.
Caspases are known to mediate the apoptotic pathway [35] [36]. To clarify the mechanism by which compound 1 induces apoptotic cell death, activation of caspases-3, -8, and -9 was evaluated by Western blot analysis. After treatment of HL60 cells with 1 (30 mm), the levels of procaspases-3, -8, and -9 diminished and cleaved caspases-3, -8, and -9 were detected, almost in a time-dependent manner (Fig. 3). These results suggest that compound 1 induced apoptotic cell death via both the mitochondrial and the death-receptor mediated pathways. Next, we investigated the effect of compound 1 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 [37]. Expression of these
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Fig. 3. Western blot analysis of HL60 cells treated with compound 1. Western blot analysis of caspases-3, -8, and -9, and of Bcl-2 and Bax in HL60 cells with compound 1 (30 mm) for 8 and 24 h. The results are from one representative experiment among three runs, which showed similar patterns to one another.
proteins was examined by Western blot analysis. Treatment of HL60 cells with 1 (30 mm) decreased slightly the level of Bcl-2 and increased the level of Bax in a time-dependent manner (Fig. 3). The Bax/Bcl-2 ratio is one of the indices of the intrinsic mechanism of apoptosis in mitochondria [38]. Since compound 1 increased this ratio in HL60, it seems that compound 1-induced apoptosis involved Bax/Bcl-2 signal transduction. Compound 1 is, therefore, suggested to induce apoptosis in HL60 cells by involving the mitochondrial signal transduction pathway. Melanogenesis-Inhibitory Activity. All 17 compounds were evaluated for melanogenesis inhibition in a-MSH-stimulated B16 melanoma cells (Table 4). The cytotoxic activities of these compounds against B16 melanoma cells were also determined by means of MTT 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. A compound with a smaller A/C ratio would be a lowerrisk skin-whitening agent. Among the compounds tested, eleven compounds, i.e., 1, 2, 3 – 7, 10, and 15 – 7, exhibited inhibitory activities (6.1 – 89.2% melanin content) with no or almost no toxicity to the cells (81.7 – 148.2% cell viability) at 10 mm, and their A/C ratios were in the range of 0.07 – 0.86 at that concentration (Table 4). In addition, four compounds, i.e., 6, 10, 16, and 17, were further proved to be lower-risk melanogenesis inhibitors by exhibiting small A/C ratios, i.e., 0.37 – 0.47, with higher cell viabilities, i.e., 72.8 – 142.7%, at 30 mm. These compounds would be more potent melanogenesis inhibitors at lower
57.2 2.0 31.4 1.5 60.3 1.4 88.0 0.8 83.8 5.7 105.4 5.4 78.6 0.0 81.4 1.0 32.6 4.5 5.4 0.5 9.9 0.6 6.1 1.3 43.2 1.6 89.2 5.9 92.7 4.6
Vilasanin-type limonoid 4
Nimbin-type limonoids 5 6 7 8 9 10 11
Salannin-type limonoids 12 13 14 15 16 17
Arbutin d )
71.5 1.3
3.9 3.4 1.5 0.6 8.0 0.8 1.6 0.3 4.5 0.3 17.2 2.4
2.4 0.9 5.9 0.6 125.2 3.4 6.3 0.5 123.8 2.7 8.1 0.7 25.5 1.2
4.9 0.5
2.4 0.5 1.9 0.4 3.6 2.2
102.3 1.5
52.5 2.3 30.5 1.9 24.4 0.8 85.4 5.9 100.6 3.4 148.2 1.4
106.9 3.6 103.9 4.1 101.8 7.0 98.3 1.8 95.3 6.0 101.5 5.9 73.5 6.6
81.7 4.6
111.8 6.3 115.5 4.7 75.3 3.2
101.0 6.4
24.1 6.1 0.4 0.1 53.4 1.9 18.9 0.5 72.8 5.5 142.7 9.7
28.4 2.9 76.5 4.4 101.7 8.5 49.9 8.1 98.7 6.6 88.8 4.1 27.4 2.7
40.4 3.0
59.1 5.2 57.4 3.6 15.2 1.7
100.0 0.7
81.6 6.3
1.1 2.6 0.7 0.2 17.9 2.9 0.4 0.2 29.7 0.5 57.3 3.3
2.3 1.4 18.6 1.4 105.2 8.1 4.7 0.1 103.4 5.7 43.3 0.4 13.1 0.9
4.4 0.3
5.1 0.5 6.1 1.3 0.4 0.2
100.0 0.7
100 mm
A/C Ratio
0.91
0.62 0.18 0.41 0.07 0.43 0.60
0.29 0.58 0.86 0.85 1.11 0.77 1.11
0.70
0.62 0.56 1.82
10 mm
0.90
0.18 0.50 0.53 0.17 0.37 0.46
0.43 0.39 1.01 1.02 1.04 0.57 2.11
0.42
0.30 0.28 4.98
30 mm
0.88
3.55 2.14 0.45 4.00 0.15 0.30
1.04 0.31 1.19 1.34 1.20 0.19 1.95
1.11
0.47 0.31 9.00
100 mm
a ) Melanin content [%] and cell viability [%] were determined based on the absorbances at 405, and 570 (test wavelength) – 630 (reference wavelength) nm, resp., 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-to-cytotoxicity ratio, which was obtained by dividing the melanin content [%] by the cell viability [%] at each concentration. d ) Reference compound.
91.0 4.2
4.4 4.7 0.2 0.6 28.1 2.2 3.2 1.2 27.0 0.9 65.0 8.3
12.3 0.6 30.2 1.0 103.1 0.5 50.9 2.6 102.7 6.8 50.8 1.9 57.8 5.4
17.0 1.0
17.5 0.9 16.3 0.9 75.7 5.9
100.0 0.7
69.3 2.8 65.1 3.9 136.8 2.4
100.0 3.1
100.0 3.1
100.0 3.1
Control (100% DMSO)
Azadirone-type limonoids 1 2 3
30 mm
Cell viability [%] 10 mm
30 mm
10 mm
100 mm
Melanin content [%]
Compound
Table 4. Melanogenesis-Inhibitory and Cytotoxic Activities a ) b ), and A/C Ratios c ) of Compounds Isolated from the Leaf Extract of Azadirachta indica in B16 Mouse Melanoma Cell Line
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concentrations than the reference arbutin (4-hydroxyphenyl b-d-glucopyranoside; 92.7 and 91.0% melanin contents; 102.3 and 101.0% cell viabilities; and A/C ratios 0.91 and 0.90, at 10 and 30 mm, resp.), a known melanogenesis inhibitor and useful depigmentation compound for skin whitening in the cosmetic industry [39]. All compounds, except 7 and 9, exhibited potent melanogenesis-inhibitory activities (1.5 – 25.5% melanin contents) at 100 mm, however, with high cytotoxicities at that concentration (0.4 – 57.3% cell viabilities). In contrast to the melanogenesis inhibitory nature for fifteen compounds, 1 – 6, 8, and 10 – 17, two compounds, i.e., 7 (88.0, 103.1, and 125.2% melanin contents at 10, 30, and 100 mm, resp.) and 9 (105.4, 102.7, and 123.8% melanin contents at 10, 30, and 100 mm, resp.) exhibited induction of melanogenesis in a concentration-dependent manner without exhibiting cytotoxicity (Table 4). This seems to be the first observation of the induction of melanogenesis by limonoids, although several aromatic compounds, e.g., rosmarinic acid [40], 2,3,5,4’-tetrahydroxystilbene-2-O-b-d-glucoside [41], diethylstilbestrol [42], and hesperetin [43], have previously been reported to possess this feature. Inhibitory Effects on EBV-EA Induction. The inhibitory effects on the induction of EBV-EA induced by TPA were examined as a preliminary evaluation of anti-tumorpromoting activity. In Table 5, the inhibitory effects of compounds 1 – 17 against TPA (32 pmol)-induced EBV-EA activation in Raji cells are collected. Even at a concentration of 32 nm (mole ratio of compound to TPA 1000 : 1), high viability (60 – 70%) of Raji cells was observed, indicating the low cytotoxicity of the tested compounds. All compounds tested exhibited inhibitory effects (IC50 413 – 493 mole ratio/32 pmol TPA), which were more potent than, or almost comparable with, that of the reference compound, retinoic acid (IC50 482 mole ratio/32 pmol TPA), one of the retinoids that has been studied as a cancer-chemoprevention agent for various organ site cancers [44]. Since inhibitory effects against EBV-EA induction have been demonstrated to correlate with those against tumor promotion in vivo [45], all 17 compounds may be potential inhibitors of tumor promotion. Compound 12 has recently been shown to exhibit chemopreventive activity against 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis [46]. Conclusions. – This study has established that the AcOEt-soluble fraction of the EtOH extract of Azadirachta indica (neem) leaves exhibits cytotoxic activity against human HL60 cells, melanogenesis-inhibitory activity in a-MSH-stimulated B16 melanoma cells, and inhibitory effects against TPA-induced EBV-EA activation in Raji cells. Seventeen limonoids, 1 – 17, including three new compounds, 14, 15, and 17, have been isolated from the AcOEt-soluble fraction. Evaluation of these compounds established that nimonol (1), isonimocinolide (2), nimonolactone (3), nimbolide (12), 28-deoxonimbolide (13), and 2’,3’-dehydrosalannol (16) exhibit potent cytotoxic activities against HL60 and/or other human cancer cell lines tested, and the cell death of HL60 cells by compound 1 is mainly due to apoptosis. Western blot analysis indicated that compound 1 induced apoptosis via both the mitochondrial and the death receptormediated pathways. It appears that three limonoids, 1, 13, and 16, as well as 12 [31], are highly responsible for the cytotoxicity of the AcOEt-soluble fraction of the A. indica leaf extract, because these compounds constitute the major limonoids of the fraction.
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Table 5. Inhibitory Effects of Compounds Isolated from Neem Leaf Extracts and Reference Compound on the Induction of EpsteinBarr Virus Early Antigen ( EBV-EA ). Compound
Percentage EBV-EA induction a )
IC50 c ) (mole ratio/ 32 pmol TPA )
Drug concentration b ) 1000
500
100
10
Azadirone-type limonoids 1 11.9 0.7 (60) 2 9.3 0.7 (60) 3 9.1 0.7 (70)
49.7 1.5 48.2 1.6 46.2 1.5
73.9 2.5 72.0 2.5 71.9 2.5
100 0.3 100 0.5 100 0.3
470 470 460
Vilasanin-type limonoid 4 7.9 0.6 (60)
48.5 1.4
71.6 2.4
100 0.4
480
Nimbin-type limonoids 4.7 0.3 5 d) 6 3.5 0.4 7 13.8 0.6 8 6.3 0.4 9 12.3 0.7 10 15.3 0.8 11 8.1 0.5
(70) (60) (60) (60) (60) (60) (60)
46.2 1.5 43.7 1.5 51.1 1.8 46.1 1.3 50.0 1.4 52.8 1.5 47.0 1.4
88.8 2.1 71.6 2.2 80.1 2.0 71.3 2.3 80.1 2.0 82.7 2.1 76.1 2.4
100 0.4 98.8 0.5 100 0.4 100 0.3 100 0.5 100 0.5 100 0.3
489 430 493 468 487 487 460
Salannin-type limonoids 12 9.2 0.6 (60) 13 e ) 10.5 0.7 (60) 14 8.0 0.4 (60) 15 7.3 0.3 (60) 16 2.1 0.3 (60) 17 1.7 0.2 (60)
47.9 1.1 49.2 1.4 48.9 1.3 47.8 1.3 43.9 1.2 41.7 1.4
73.0 2.4 76.6 2.3 73.7 2.4 72.6 2.6 71.1 2.5 70.1 2.6
100 0.3 100 0.4 100 0.3 100 0.3 98.0 0.5 97.2 0.4
462 469 481 475 423 413
Retinoic acid f )
49.3 1.6
76.3 2.1
100 0.2
482
21.6 0.9 (60)
a
) Values represent the percentages relative to the positive control, with TPA (32 pmol, 20 ng) representing 100% induction. Values in parentheses are viability percentages of Raji cells. Data are expressed as mean S.D. (n ¼ 3). b ) Concentration in terms of mole ratio/32 pmol TPA. c ) The IC50 value represents the mole ratio of compounds, relative to TPA, required to inhibit 50% of the positive control activated with 32 pmol TPA. d ) Data taken from [3]. e ) Data taken from [4]. f ) Reference compound.
This study has further established that some limonoid constituents of the AcOEtsoluble fraction of A. indica leaf extract, i.e., 6-homodeacetylnimbin (6), nimbinene (10), 16, and 17-defurano-17-(5x-2,5-dihydro-5-hydroxy-2-oxofuran-3-yl)-2’,3’-dehydrosalannol (17), exhibit melanogenesis-inhibitory activities while showing no, or almost no, cytotoxicity at lower concentrations. These compounds may be, therefore, valuable as potential skin-whitening agents. In addition, on the basis of the results of in vitro EBV-EA induction, compounds 1 – 17 may be useful as agents that inhibit chemical carcinogenesis. This work has thus provided a further evidence of the importance of A. indica leaf extract and its limonoid constituents as potential anticancer, melanogenesis-inhibitory, and chemopreventive agents.
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Experimental Part General. Column chromatography (CC): activated charcoal (20 – 150 mesh; Nacalai Tesque, Inc., Kyoto, Japan), silica gel (SiO2 , 230 – 400 mesh; Merck & Co. Inc., D-Darmstadt), and ODS (Chromatorex-ODS, 100 – 200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan). Reversed-phase (RP) 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 MeCN/H2O (flow rate 3.0 ml/min) 3 : 2 (system I) or 9 : 11 (system II), or with MeCN/H2O/AcOH 50 : 50 : 0.1 (flow rate, 2.0 ml/min) (system III) or MeOH/H2O 7 : 3 (flow rate, 3.0 ml/min; system IV), or on a Capcell Pak AQ 5 mm column (Shiseido Co., Ltd., Tokyo, Japan) with MeCN/H2O 3 : 2 (flow rate, 3.0 ml/min; system V) or 2 : 3 (flow rate, 2.0 ml/ min; system VI). Optical rotations: JASCO P-1020 digital polarimeter. UV Spectra: JASCO V-630Bio spectrophotometer; lmax (log e) in nm. IR Spectra: JASCO FTIR-300E spectrometer; ˜n in cm 1. 1D- and 2D-NMR spectra: JEOL ECX-400 (1H, 400 MHz; 13C, 100 MHz) spectrometer at r.t.; CDCl3 soln.; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Agilent 1100 LC/MSD TOF (time-of-flight) system (ionization mode, pos.; 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). Chemicals and Materials. Leaves of Azadirachta indica were collected at Andhra Pradesh, India, in August, 2009. The plant was identified by Mr. Sriram Gangadhar (Ichimaru Pharcos Co. Ltd., Gifu, Japan). A voucher specimen (BI091201-2019) is deposited with the Research Laboratory, Ichimaru Pharcos Co. Ltd. The chemicals were purchased as follows: fetal bovine serum (FBS), RPMI-1640 medium, antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin), and non-essential amino acid (NEAA) from Invitrogen Co. (Carlsbad, CA, USA); Eagles minimal essential medium (MEM), Dulbeccos modified Eagles medium (DMEM), arbutin (4-hydroxyphenyl b-d-glucopyranoside), kojic acid, a-MSH, and MTT from Sigma-Aldrich Japan Co. (Tokyo, Japan); and rh annexin V/FITC kit (Bender MedSystems) from Cosmo Bio. Co. Ltd. (Tokyo, Japan). Primary antibodies of anti-MITF, antityrosinase, anti-TRP-1, and anti-TRP-2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-b-actin from Cell Signaling Technology (Beverly, MA, USA). All other chemicals and reagents were of anal. grade. Extraction and Isolation. The dried and powdered leaves of A. indica (3 kg) were extracted with EtOH by maceration at r.t. (38 l; 1 week, 1 ), and the extract was passed through membrane filter (0.45 mm), which, after evaporation of the solvent in vacuo, yielded the EtOH extract (126 g). To remove chlorophylls, a portion of the extract (96 g) was subjected to CC on activated charcoal (230 g) and eluted with MeOH/AcOEt (1 : 0 to 0:l). The almost chlorophyll-free MeOH eluate (48.0 g) was partitioned between AcOEt and H2O to yield the AcOEt- (35.0 g) and H2O-soluble fractions. The AcOEt-soluble fraction was applied to CC (SiO2 (700 g); hexane/AcOEt 4 : 1 to 0 : 1) to yield eleven fractions, Frs. A – K. Fr. D (2087 mg) from the eluate of hexane/AcOEt 3 : 2 was subjected to CC (ODS (60 g); MeOH/H2O 1 : 1 to 1 : 0) to yield six fractions, Frs. D1 – D6. Prep. HPLC of Frs. D3 (218 mg; system I) and D4 (249 mg; system V) afforded compounds 11 (tR 16.8 min; 36.7 mg), 10 (tR 22.8 min; 8.0 mg), and 1 (tR 32.8 min; 60.9 mg), and compound 3 (tR 14.4 min; 3.7 mg), resp. Fr. E (1532 mg) eluted with hexane/AcOEt 3 : 2 was applied to CC (SiO2 (30 g); hexane/AcOEt 1 : 1 to 0 : 1) to give four fractions, Frs. E1 – E4. Prep. HPLC (system II) of Fr. E2 (232 mg) yielded compound 2 (tR 32.8 min; 11.9 mg). Further CC (ODS (10 g); MeOH/H2O 3 : 7 to 1 : 0) and subsequent prep. HPLC (system II) of Fr. E3 (296 mg) afforded compound 7 (tR 63.0 min; 5.0 mg). A portion (70 mg) of Fr. F (1695) from the eluate of hexane/AcOEt 1 : 1 was subjected to prep. HPLC (system IV) to give compound 6 (tR 18.4 min; 2.3 mg). Fr. G (2482 mg) eluted with hexane/AcOEt 1 : 1 was subjected to CC (SiO2 (90 g); hexane/ AcOEt 7 : 3 to 0 : 1) to yield six fractions, Frs. G1 – G6. Prep. HPLC of Frs. G2 (573 mg; system V) and G5 (72 mg; system VI) afforded compounds 5 (tR 22.4 min; 171.1 mg) and 12 (tR 24.4 min; 778.9 mg), and compounds 9 (tR 22.8 min; 10.4 mg) and 8 (tR 24.8 min; 20.1 mg), resp. Fr. H (4049 mg) from the eluate of hexane/AcOEt 1 : 1 was further separated into eleven fractions, Frs. H1 – H11, by CC (SiO2 (70 g); CHCl3/MeOH 1 : 0 to 9 : 1). Prep. HPLC (system III) of Frs. H5 (163 mg) and H7 (95 mg) yielded compounds 15 (tR 15.2 min; 3.3 mg) and 17 (tR 21.6 min; 4.7 mg), and compound 4 (tR 28.0 min; 7.7 mg), resp. Fr. I (2305 mg) eluted with hexane/AcOEt 2 : 3 to 1 : 4 was subjected to CC (SiO2 (100 g); hexane/
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AcOEt 7 : 3 to 0 : 1) to give eight fractions, Frs. I1 – I8. Prep. HPLC of Frs. I2 (267 mg; system I) and I3 (679 mg; system V) furnished compounds 14 (tR 11.6 min; 5.8 mg) and 13 (tR 15.6 min; 56.6 mg), and compound 16 (tR 24.8 min; 41.3 mg), resp. The m.p., specific rotations, UV, IR, and HR-ESI-MS data of three new compounds, 14, 15, and 17, are given below. Their 1H- and 13C-NMR data are compiled in Table 2. 17-Defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide ( ¼ Methyl (2aR,5aR,6S,6aR, 8R,9aR,10aS,10bR,10cS)-8-(2,5-Dihydro-2-oxo-3-furanyl)-2a,5a,6,6a,8,9,9a,10a,10b,10c-decahydro2a,5a,6a,7-tetramethyl-5-oxo-2H,5H-cyclopenta[d]naphtho[2,3-b:1,8-bc]difuran-6-acetate; 14). Fine needles. M.p. 155 – 1588 (MeOH). [a] 25 D ¼ þ 54.5 (c ¼ 1.43, EtOH). UV (MeOH): 233 (3.90) (a,bunsaturated ketone). IR (KBr): 1750 (g-lactone), 1731 (ester C¼O), 1676 (conjugated cyclohexenone). NMR Spectra: see Table 2. HR-ESI-MS: 491.2019 ([M þ Na] þ , C27H32NaO þ7 ; calc. 491.2040). 17-Defurano-17-(5x-2,5-dihydro-5-hydroxy-2-oxofuran-3-yl)-28-deoxonimbolide ( ¼ Methyl (2aR, 5aR,6S,6aR,8R,9aR,10aS,10bR,10cS)-8-[(3x)-2,5-Dihydro-2-hydroxy-furan-3-yl]-2a,5a,6,6a,8,9,9a,10a, 10b,10c-decahydro-2a,5a,6a,7-tetramethyl-5-oxo-2H,5H-cyclopenta[d]naphtho[2,3-b:1,8-bc]difuran-6acetate; 15). Fine needles. M.p. 152 – 1558 (MeOH). [a] 20 D ¼ þ 107.1 (c ¼ 0.82, EtOH). UV (MeOH): 231 (4.01) (a,b-unsaturated ketone). IR (KBr): 3348 (OH), 1752 (g-lactone), 1735 (ester C¼O), 1680 (conjugated cyclohexenone). NMR Spectra : see Table 2. HR-ESI-MS : 507.2028 ( [M þ Na] þ , C27H32NaO þ8 ; calc. 507.1994). 17-Defurano-17-(5x-2,5-dihydroxy-5-hydroxy-2-oxofuran-3-yl)-2’,3’-dehydrosalannol ( ¼ Methyl (2aR,3R,5S,5aR,6R,6aR,8R,9aR,10aS,10bR,10cR)-8-[(5x)-2,5-Dihydro-5-hydroxy-2-oxo-furan-3-yl]2a,4,5,5a,6,6a,8,9,9a,10a,10b,10c-dodecahydro-3-hydroxy-2a,5a,6a,7-tetramethyl-5-[(3-methyl-1-oxo-2buten-1-yl)oxy]-2H,3H-cyclopenta[d]naphtho[2,3-b:1,8-bc]difuran-6-acetate; 17). Fine needles. M.p. 125 – 1288 (MeOH). [a] 25 D ¼ þ 80.7 (c ¼ 1.25, EtOH). UV (MeOH): 220 (3.96) nm (a,b-unsaturated ketone). IR (KBr): 3418 (OH), 1746 (g-lactone), 1730 (ester C¼O), 1713. NMR Spectra: see Table 2. HR-ESI-MS: 609.2677 ([M þ Na] þ , C32H42NaO þ10 ; calc. 609.2675). Cell Lines and Culture Conditions. Cell lines B16 4A5 (mouse melanoma), HL60 (leukemia), AZ521 (stomach), A549 (lung), and SK-BR-3 (breast) were obtained from Riken Cell Bank (Tsukuba, Ibaraki, Japan). Three cell lines, B16 4A5, HL60, and SK-BR-3, were grown in RPMI-1640 medium, while AZ521 and A549 cell lines were grown in DMEM and in 90% DMEM þ 10% MEM þ 0.1 mM NEAA, resp. 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 [47 – 49]. Determination of Cell Proliferation. Cell proliferation was assessed using the MTT based colorimetric assay as described in [47 – 49]. Assay of Melanin Content. Melanogenesis-inhibition assay in a-MSH-stimulated B16 melanoma cells was performed as described in [48]. Annexin VPropidium Iodide (PI) Double Staining. Annexin V-PI double staining was performed as described in [50] with HL60 (1 105 cells). Western Blotting. Western blot analysis was performed with HL60 (1 105 cells) as described in [50] [51]. REFERENCES [1] L. Zhao, C.-H. Huo, L.-R. Shen, Y. Yang, Q. Zhang, Q.-W. Shi, Chem. Biodiversity 2010, 7, 839. [2] Q.-G. Tan, X.-D. Luo, Chem. Rev. 2011, 111, 7437. [3] T. Akihisa, T. Noto, A. Takahashi, Y. Fujita, N. Banno, H. Tokuda, K. Koike, T. Suzuki, K. Yasukawa, Y. Kimura, J. Oleo Sci. 2009, 58, 581. [4] T. Akihisa, A. Takahashi, T. Kikuchi, M. Takagi, K. Watanabe, M. Fukatsu, Y. Fujita, N. Banno, H. Tokuda, K. Yasukawa, J. Oleo Sci. 2011, 60, 53. [5] T. Kikuchi, K. Ishii, T. Noto, A. Takahashi, K. Tabata, T. Suzuki, T. Akihisa, J. Nat. Prod. 2011, 74, 866. [6] T. Akihisa, X. Pan, Y. Nakamura, T. Kikuchi, N. Takahashi, M. Matsumoto, E. Ogihara, M. Fukatsu, K. Koike, H. Tokuda, Phytochemistry 2013, 89, 59.
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