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Bioassay-guided isolation and identification of cytotoxic compounds from Bolbostemma paniculatum
Journal of Ethnopharmacology 169 (2015) 18–23
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Bioassay-guided isolation and identification of cytotoxic compounds from Bolbostemma paniculatum Yun Tang a,b, Wei Li a, Jiaqing Cao b,c, Wei Li b,c, Yuqing Zhao b,c,n a
Key Laboratory of Natural Active Pharmaceutical Constituents of Jiangxi Province, Yichun University, Yichun 336000, PR China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China c Key Laboratory of Structure-Based Drug Design & Discovery (Shenyang Pharmaceutical University), Ministry of Education, Shenyang 110016, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 January 2015 Received in revised form 25 March 2015 Accepted 3 April 2015 Available online 13 April 2015
Ethnopharmacological relevance: Bolbostemma paniculatum (Maxim.) Franquet (B. paniculatum), also named “Tu-bei-mu” in Chinese folk medicines, has been described in application for the treatment of tumors, warts, inflammation and toxication in traditional Chinese medicinal books. The major constituents in B. paniculatum are triterpenoid saponins, which have been proved to possess dramatically cytotoxic activity and antivirus activity. The aim of this study is to isolate and identify the active triterpenoid saponin from the bulb of B. paniculatum by a bioassay-guided method. Materials and methods: Four cucurbitacine triterpenoid sapogenins and 11 triterpenoid saponins were isolated from the active EtOAc and n-BuOH extract of B. paniculatum by using bioassay-guided screening. Their structures were elucidated based on the spectroscopic methods and compared with published data. Cytotoxic activities of isolated compounds were determined by MTT assay. Results: Four cucurbitacine triterpenoid sapogenins, isocucurbitacin B(1), 23,24-dihydroisocucurbitacin B(2), cucurbitacin E(3), 23,24-dihydrocucurbitacin E(4), and 11 triterpenoid saponins, tubeimosideI(5), tubeimoside III(6), tubeimoside V(7), dexylosyltubeimoside III(8), lobatoside C(9), tubeimoside A(10), tumeimoside B(11), lobatoside A(12), tubeimoside C(13), tubeimoside IV(14), 7β,18,20,26-tetrahydroxy(20S)-dammar-24E-en-3-O-α-L-(4-acetyl)arabinopyranosyl-(1-2)-β-D-glucopyranoside(15) were isolated from the active EtOAc and n-BuOH extracts. Of them, compounds 2, 4, 9 and 12 were firstly isolated from the Bolbostemma genus. MTT assay revealed that compounds 1, 3 and 4 had significantly activities against HeLa and HT-29 human cancer cells with IC50 values ranging from 0.93 to 9.73 μM. It is worth mentioning that compound 4's activities against the two cell lines are 12- and 8-fold that of the positive control drug (5-Fu). Whereas, the cyclic bisdesmosides 5–9 exerted significantly activities on BGC-823, HeLa, HT-29 and MCF-7 cancer cells with IC50 values ranging from 1.30 to 15.64 μM. And 6's activities against the four cell lines are 6-, 3-, 10- and 16-fold that of 5-Fu and 8's activities against the four cell lines are 5-, 3-, 14- and 9-fold that of 5-Fu. Conclusion: The cytotoxic activity of the bulbs of B. paniculatum is mainly ascribable to cucurbitacine triterpenoid sapogenins (1–4) and the cyclic bisdesmosides (5–9). The cyclic bisdesmosides are the main anti-cancer active compounds of B. paniculatum. The above results provide scientific evidence to support, to some extent, the ethnomedicinal use of B. paniculatum as anticancer remedies in traditional Chinese medicine. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Bolbostemma paniculatum Triterpenoid Tubeimoside Cyclic bisdesmosides Cytotoxicity
1. Introduction Abbreviations: B. paniculatum, Bolbostemma paniculatum (Maxim.) Franquet; EtOAc, ethyl acetate; n-BuOH, n-butanol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylterazolium bromide; IC50, half maximal inhibitory concentration; 5-Fu, fluorouracil; ODS, octadecylsilyl; OD, optical density; SAR, structure–activity relationship; NMR, nuclear magnetic resonance; TLC, thin layer chromatography; HPLC, high performance liquid chromatography; CC, column chromatography; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide; Rha, rhamnose; Ara, arabinose; Glc, glucose; Xyl, xylose n Corresponding author. E-mail address: [email protected] (Y. Zhao). http://dx.doi.org/10.1016/j.jep.2015.04.003 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Cancer is harmful to human health and it has become the leading cause of death in the last 50 years (Ullah and Aatif, 2009). In past decades, searching new anticancer agent from animals and plants source has drawn increasingly attention of researchers. Bolbostemma paniculatum, mainly distributed in Shaanxi, Shanxi, Henan and Shandong Province of China, is one of the Chinese folk medicines recorded in Supplement to the Compendium of Materia Medica, and it was used in the Qing Dynasty (Zhao, 1983). It has been described as a
Y. Tang et al. / Journal of Ethnopharmacology 169 (2015) 18–23
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folk remedy for the treatment of tumors, warts, and detoxication for thousands of years (Committee of Pharmacopeia, 2010). Moreover, B. paniculatum has also been applied in the treatment of cancer for a long time because of its abundant triterpinoid saponins (Sindambiwe et al., 1998). Triterpenoids with cytotoxic activity against HeLa (cervical cancer) (Xu et al., 2011), A549 (lung carcinoma) (Zhang et al., 2011), BGC-823 (gastric carcinoma) (Zhang et al., 2013), CNE-2Z (nasopharyngeal carcinoma) (Ma et al., 2008), HepG2 (liver cancer) (Yin et al., 2011), SKOV-3 (ovarian cancer) (Chen et al., 2012), EC109 (esophageal cancer) (Xu et al., 2013), JEG-3(choriocarcinoma) (Huang et al., 2011), A2780 (ovarian cancer) (Liu et al., 2011), and HL-60 (promyelocytic leukemia) (Yu et al., 1996) cell lines had been reported to be the major constituents of B. paniculatum. In addition, a few of cucurbitacins with cytotoxicity had also been isolated and identified from the bulbs of B. paniculatum (Cassady and Suffness, 1980). Based on above knowledge, searching for newly cytotoxic compounds from B. paniculatum is desirable. In current study, a systematic chemical investigation was carried out on the bulbs of B. paniculatum. The cytotoxic activities were tested against several human cancer cell lines including BGC-823 gastric cancer cell line, Hela cervical carcinoma cell line, HT-29 colon cancer cell line and MCF-7 breast cancer cell line. A bioassay-guided screening method was conducted to separate pure compound (s) responsible for their activities. The chemical structures of isolated compounds were established with the aid of extensive NMR spectroscopic, massspectral analyses and published data.
0:1) as eluant to provide 14 fractions A–N based on TLC analysis. Fraction I was subjected to silica gel column chromatography (CC) eluted with CH2Cl2:CH3OH:H2O (the lower phase) (7:2:1, 7:3:1) to obtain 10 sub-fractions (I-1-1, I-1-2, I-1-3,…I-1-10). Fraction I-8 was applied to ODS silica gel CC eluted with 50% MeOH–H2O to afford 15 (25 mg) and eluted with 70% MeOH–H2O to afford 13 (25 mg). Fraction J was chromatographed on silica gel eluted with CH2Cl2– MeOH–H2O (the lower phase) (7:2.5:1) to collect 11 sub-fractions (J-11, J-1-2, J-1-3…J-1-11). Fraction J-7 was applied to ODS silica gel CC eluted with MeOH–H2O (3:10; 1:2, 7:10, 9:10) to afford 12 (20.0 mg) and 11 (28.0 mg) as pure compounds, and 10 with few impurities. Compound 10 (25.0 mg) was purified by preparative HPLC using 75% MeOH–H2O. Fraction J-8 was applied to ODS silica gel CC eluted with MeOH–H2O (3:10; 1:2, 7:10, 9:10) to afford 14 (30.0 mg). Fraction K was chromatographed on silica gel eluted with CH2Cl2–MeOH–H2O (the lower phase) (7: 3: 1) to collect 7 sub-fractions (K-1-7). Fraction K6 was applied to ODS silica gel CC eluted with MeOH–H2O (3:10; 1:2, 7:10, 9:10) to collect 4 fractions (K-6-1, K-6-2, K-6-3, K-6-4). Fraction K6-3 was applied to preparative HPLC using 67% MeOH–H2O to afford 5 (20 mg) and 6 (29 mg) as pure compounds. Fraction L was applied to ODS silica gel CC eluted with MeOH–H2O (3:10; 1:2, 7:10, 9:10) to collect 2 sub-fractions (L-1, L-2). Fraction L-1 was further purified by using recrystallization to afford 3 (15 mg) and 4 (18 mg) as pure compounds. Fraction L-2 was applied to preparative HPLC using 50% MeOH–H2O to afford 1 (20 mg) and 2 (25 mg) as pure compounds. Once again, the isolated compounds 5–15 were tested for cytotoxic activity to identify the bioactive compound(s).
2. Materials and methods
2.3. Chemical elucidation of compounds 1–15
2.1. Plant material
The structures of compound 1–15 were elucidated by spectroscopic methods and the 1H and 13C NMR spectral data were compared with earlier reports (Zheng et al., 2007; Shi et al., 1994; Nobuo et al., 2001; Kong et al., 1986; Kasai et al., 1988; Cheng et al., 2006; Tang et al., 2005, 2014; Ma et al., 2006; Fujioka et al., 1989; Liu et al., 2004). Compound 1: White needle crystals (EtOAc), 1H NMR (300 MHz, CDCl3): δ 0.81(3H, s), 0.97(3H, s), 1.18(3H, s), 1.26(3H, s), 1.33(3H, s), 1.41(3H, s), 1.54(3H, s), 1.56(3H, s), 2.00(3H, s), 5.94 (1H, d, J¼5.7 Hz), 6.43(1H, d, J¼15.6 Hz), 7.05(1H, d, J¼15.6 Hz). 13C NMR (75 MHz, CDCl3): δ 38.8(C-1), 210.6(C-2), 80.2(C-3), 46.7(C-4), 138.1(C-5), 121.9 (C-6), 23.9(C-7), 42.7(C-8), 48.3(C-9), 36.2(C-10), 211.8(C-11), 48.5(C12), 50.6(C-13), 47.9(C-14), 45.3(C-15), 71.2(C-16), 58.0(C-17), 19.8(C18), 20.0(C-19), 78.1(C-20), 23.7(C-21), 202.4(C-22), 120.2(C-23), 152.0 (C-24), 79.3(C-25), 25.9(C-26), 26.4(C-27), 20.9(C-28), 24.1(C-29), 18.8 (C-30), 170.2(C-31), 21.9(C-32). Compound 2: White needle crystals (EtOAc), 1H NMR (600 MHz, CDCl3): 0.81(3H, s), 0.96(3H, s), 1.18(3H, s), 1.27(3H, s), 1.33(3H, s), 1.40 (3H, s), 1.43(3H, s), 1.45(3H, s), 1.96(3H, s), 5.94 (1H, d, J¼6.0 Hz). 13C NMR(150 MHz, CDCl3): δ 38.8(C-1), 210.6(C-2), 80.2(C-3), 46.7(C-4), 138.2(C-5), 121.9(C-6), 23.8(C-7), 42.7(C-8), 48.4(C-9), 36.3(C-10), 211.8 (C-11), 48.6(C-12), 48.2(C-13), 50.6(C-14), 45.5(C-15), 70.9(C-16), 57.7 (C-17), 20.0(C-18), 18.7(C-19), 78.9(C-20), 24.4(C-21), 213.9(C-22), 30.7 (C-23), 34.7(C-24), 81.3(C-25), 26.1(C-26), 25.8(C-27), 21.0(C-28), 24.1 (C-29), 19.8(C-30), 170.4(C-31), 22.4(C-32). Compound 3: White needle crystals (EtOAc), 1H NMR (600 MHz, CDCl3): δ 1.00(3H, s), 1.03(3H, s), 1.25(3 H, s), 1.36(3H, s), 1.39(3H, s), 1.44(3 H, s), 1.54(3H, s), 1.57(3H, s), 2.01(3H, s), 5.77 (1H, br.s), 5.96(1H, br.s), 6.46(1H, d, J¼ 15.6 Hz), 7.06(1H, d, J¼15.6 Hz). 13C NMR (150 MHz, CDCl3): δ 114.8(C-1), 144.6(C-2), 198.7(C-3), 47.5(C-4), 136.8(C-5), 120.8(C-6), 23.6(C-7), 41.6(C-8), 48.9(C-9), 34.7(C-10), 212.9(C-11), 48.9(C-12), 50.7(C-13), 48.1(C-14), 45.6(C-15), 71.3(C-16), 58.2(C-17), 19.9(C-18), 20.1(C-19), 78.2(C-20), 24.0(C-21), 202.5(C-22), 120.4(C-23), 152.0(C-24), 79.3(C-25), 25.9(C-26), 26.5(C-27), 20.2(C28), 28.0(C-29), 18.4(C-30), 170.3(C-31), 21.9(C-32). Compound 4: White needle crystals (EtOAc), 1H NMR(600 MHz, CDCl3): δ 0.99(3H, s), 1.04(3H, s), 1.25(3H, s), 1.36(3H, s), 1.40(3H,
The bulbs of B. paniculatum (Maxim.) Franquet (Cucurbitaceae) were collected in Shaanxi province of People's Republic of China. A voucher specimen of this herb (No. 2010085) was identified by Prof. Jincai Lu of Shenyang Pharmaceutical University. The voucher specimen was deposited in our lab. 2.2. Plant extraction and purification under bioassay-guided screening The bulbs of B. paniculatum (50.0 kg) were extracted with 75% EtOH (80 L 3) under refluxing for 2 h. The filtrate obtained was concentrated under reduced pressure to yield a dark residue (15 kg). Part of the crude ethanolic extract (8 kg) was suspended in water (28 L) and successively extracted with petroleum ether, EtOAC and nbutanol sequentially (3 28 L) to afford dried petroleum ether-, EtOAc-, n-BuOH- and H2O-soluble extracts. Each of these extracts was tested for its cytotoxic activity. The EtOAc extract showed moderate cytotoxicity against the BGC-823, HeLa, HT-29 and MCF-7 cell lines, the n-BuOH extract were clearly the most active against the BGC-823, HeLa, HT-29 and MCF-7 cell lines. Further fractionation of EtOAc extract (200 g) with liquid chromatography (silica gel, eluted with mixture of petroleum ether/acetone from 40:1 to 2:1) afforded four major fractions A–D based on TLC analysis. Fraction B was further purified with radial chromatography (silica gel, eluted with mixture of petroleum ether/EtOAc from 20:1 to 0:1) to afford two sub-fractions (FB-1, FB-2). FB-1 was purified by sephadex LH-20 and then applied to preparative HPLC using 54% MeOH–H2O to afford 2(8 mg), 3 (8.5 mg) and 4 (8 mg) as pure compounds. Fraction B was further purified by using recrystallization to afford 1 (20 mg) as pure compound. Once again, the isolated compounds 1–4 were tested for cytotoxic activity to identify the bioactive compound(s). The n-BuOH-soluble extract (500 g) was then chromatographied over silica gel column using a gradient of CH2Cl2:CH3OH (from 20:1 to
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s), 1.42(3H, s), 1.44(3H, s), 1.46(3H, s), 1.97(3H, s), 5.77 (1H, br.s), 5. 96(1H, d, J ¼2.4 Hz). 13C NMR(150 MHz, CDCl3): δ 114.8(C-1), 144.6 (C-2), 198.7(C-3), 47.5(C-4), 136.8(C-5), 120.8(C-6), 23.6(C-7), 41.6 (C-8), 48.4(C-9), 34.7(C-10), 212.8(C-11), 48.8(C-12), 48.9(C-13), 50.7(C-14), 45.7(C-15), 71.0(C-16), 57.8(C-17), 19.8(C-18), 18.2(C19), 78.9(C-20), 24.5(C-21), 213.9(C-22), 30.7(C-23), 34.8(C-24), 81.3(C-25), 26.2(C-26), 25.9(C-27), 20.1(C-28), 27.9(C-29), 20.2(C30), 170.4(C-31), 22.4(C-32). Compound 5: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.92(3H, s), 0.92(3H, s), 1.16(3H, s), 1.25(3H, s), 1.53(3H, s), 1.58(3H, s), 1.64(3H, s), 2.07(3H, s), 5.62(1H, br. s), 4.99(1H, br. s), 5.06 (1H, br. s), 5.41(1H, br. s), 5.57(1H, br. s), 6.11(1H, m). 13C NMR (150 MHz, C5D5N): δ 44.0(C-1), 69.4(C-2), 82.9(C-3), 43.1(C-4), 48.5(C5), 18.2(C-6), 33.1(C-7), 40.0(C-8), 47.3(C-9), 37.2(C-10), 23.9(C-11), 123.3(C-12), 144.2(C-13), 41.8(C-14), 29.1(C-15), 22.7(C-16), 47.0 (C-17), 41.3(C-18), 45.9(C-19), 30.8(C-20), 33.9(C-21), 32.2(C-22), 64.4 (C-23), 15.6(C-24), 17.6(C-25), 17.6(C-26), 25.9(C-27), 176.0(C-28), 33.1 (C-29), 23.6(C-30), 171.2(C-10 ), 46.2(C-20 ), 70.1(C-30 ), 48.0(C-40 ), 171.4 (C-50 ), 26.4(C-60 ), 103.1(GlcC-1), 79.6(Glc-C-2), 78.9(GlcC-3), 71.4(GlcC-4), 78.5(GlcC-5), 62.4(GlcC-6), 104.4(AraC-1), 73.7(AraC-2), 72.5(AraC-3), 72.4 (AraC-4), 64.5(AraC-5), 94.0(Ara'C-1), 74.7(Ara'C-2), 70.9(Ara'C-3), 67.4 (Ara'C-4), 64.3(Ara'C-5), 100.5(RhaC-1), 72.2(RhaC-2), 78.1(RhaC-3), 73.1 (RhaC-4), 68.0(RhaC-5), 18.3(RhaC-6), 106.7(XylC-1), 74.5(XylC-2), 78.3 (XylC-3), 70.6(XylC-4), 66.9(XylC-5). Compound 6: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.99(3H, s), 1.05(3H, s), 1.39(3H, s), 1.47(3H, d, J¼ 6.0 Hz), 1.50(3H, s), 1.71(3H, s), 1.89(3H, s), 1.97(3H, s), 5.66(1H, br. s), 5.12(1H, d, J¼7.2 Hz), 5.16(1H, d, J¼ 7.2 Hz), 5.33(1H, d, J¼ 7.8 Hz), 5.95(1H, d, J¼ 7.8Hz), 6.33(1H, br. s). 13C NMR(150 MHz, C5D5N): δ 44.1(C-1), 70.0 (C-2), 82.9(C-3), 42.6(C-4), 47.7(C-5), 18.7(C-6), 33.2(C-7), 40.3(C-8), 47.7(C-9), 36.8(C-10), 24.0(C-11), 122.9(C-12), 144.6(C-13), 42.1(C-14), 37.0(C-15), 73.4(C-16), 49.3(C-17), 40.9(C-18), 46.5(C-19), 30.8(C-20), 36.0(C-21), 32.4(C-22), 64.3(C-23), 15.3(C-24), 17.5(C-25), 17.8(C-26), 27.3(C-27), 175.7(C-28), 33.7(C-29), 24.3(C-30), 171.3(C-10 ), 47.2(C-20 ), 70.1(C-30 ), 47.0(C-40 ), 171.5(C-50 ), 25.9(C-60 ), 103.4(GlcC-1), 83.8(GlcC-2), 78.0(GlcC-3), 70.9(GlcC-4), 77.3(GlcC-5), 62.4(GlcC-6), 105.5(Glc'C-1), 77.1 (Glc'C-2), 77.8(Glc'C-3), 70.9(Glc'C-4), 75.7(Glc'C-5), 64.8(Glc'C-6), 94.5 (AraC-1), 76.7(AraC-2), 74.8(AraC-3), 69.5(AraC-4), 67.4(AraC-5), 102.4 (RhaC-1), 72.5(RhaC-2), 78.2(RhaC-3), 73.5(RhaC-4), 68.0(RhaC-5), 18.3 (RhaC-6), 106.3(XylC-1), 74.7(XylC-2), 78.0(XylC-3), 70.9(XylC-4), 67.0 (XylC-5). Compound 7: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.89(3H, s), 0.92(3H, s), 1.34(3H, s), 1.38(3H, s), 1.45(3H, s), 1.52(3H, d, J¼ 6.0 Hz), 1.64(3H, s), 1.86(3H, s), 5.48(1H, br. s), 5.08(1H, br. s), 5.14(1H, d, J¼7.2 Hz), 5.32(1H, d, J¼7.2 Hz), 5.91(1H, d, J¼6.6 Hz), 6.44(1H, br. s). 13C NMR(150 MHz, C5D5N): δ 44.1(C-1), 69.7(C-2), 82.9(C-3), 42.4(C-4), 47.8(C-5), 18.5(C-6), 33.8(C-7), 40.2(C-8), 48.8 (C-9), 37.0(C-10), 22.7(C-11), 123.0(C-12), 144.2(C-13), 42.4(C-14), 29.3 (C-15), 24.1(C-16), 47.4(C-17), 41.5(C-18), 46.3(C-19), 30.8(C-20), 34.1 (C-21), 32.4(C-22), 64.2(C-23), 15.2(C-24), 17.5(C-25), 17.8(C-26), 26.3 (C-27), 176.5(C-28), 33.1(C-29), 23.6(C-30), 171.9(C-10 ), 46.4(C-20 ), 70.1 (C-30 ), 46.7(C-40 ), 171.4(C-50 ), 26.4(C-60 ), 102.9(GlcC-1), 84.2(GlcC-2), 78.3 (GlcC-3), 69.7(GlcC-4), 78.0(GlcC-5), 62.5(GlcC-6), 105.6(Glc'C-1), 77.1(Glc'C2), 77.5(Glc'C-3), 70.8(Glc'C-4), 75.7(Glc'C-5), 64.5(Glc'C-6), 94.6(AraC-1), 76.0(AraC-2), 75.1(AraC-3), 71.0(AraC-4), 67.5(AraC-5), 102.4(RhaC-1), 72.6 (RhaC-2), 78.3(RhaC-3), 73.6(RhaC-4), 67.9(RhaC-5), 18.3(RhaC-6), 106.6 (XylC-1), 74.9(XylC-2), 77.9(XylC-3), 70.98(XylC-4), 67.1(XylC-5). Compound 8: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.98(3H, s), 1.03(3H, s), 1.22(3H, s), 1.37(3H, s), 1.45(3H, d, J¼ 6Hz), 1.61(3H, s), 1.70(3H, s), 1.94(3H, s), 5.65(1H, br. s), 5.09(1H, d, J¼ 7.8 Hz), 5.36(1H, d,J¼7.8 Hz), 5.96(1H, d, J¼7.8 Hz), 6.42(1H, br. s). 13 C NMR(150 MHz, C5D5N): δ 44.2(C-1), 70.0(C-2), 83.2(C-3), 42.4(C4), 47.7(C-5), 18.4(C-6), 33.2(C-7), 40.3(C-8), 47.2(C-9), 37.0(C-10), 24.0 (C-11), 122.9(C-12), 144.7(C-13), 42.1(C-14), 36.9(C-15), 73.5(C-16), 49.2 (C-17), 40.9(C-18), 46.6(C-19), 30.8(C-20), 36.0(C-21), 32.5(C-22), 64.5 (C-23), 15.2(C-24), 17.4(C-25), 17.6(C-26), 27.4(C-27), 175.7(C-28), 33.7
(C-29), 24.4(C-30), 171.6(C-10 ), 47.1(C-20 ), 70.6(C-30 ), 46.3(C-40 ), 171.9 (C-50 ), 26.4(C-60 ), 103.1(GlcC-1), 84.9(GlcC-2), 78.4(GlcC-3), 70.7(GlcC-4), 78.2(GlcC-5), 62.5(GlcC-6), 105.8(Glc'C-1), 77.0(Glc'C-2), 77.6(Glc'C-3), 70.7 (Glc'C-4), 75.7(Glc'C-5), 64.2(Glc'C-6), 94.8(AraC-1), 76.4(AraC-2), 72.5 (AraC-3), 69.9(AraC-4), 67.7(AraC-5), 102.5(RhaC-1), 72.4(RhaC-2), 73.4 (RhaC-3), 75.8(RhaC-4), 68.0(RhaC-5), 18.3(RhaC-6). Compound 9: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.92(3H, s), 0.92(3H, s), 1.09(3H, s), 1.24(3H, s), 1.50(3H, s), 1.54(3H, d, J ¼6 Hz), 1.59(3H, s), 1.90(3H, s), 5.45(1H, br. s), 5.03 (1H, d, J ¼7.2 Hz), 5.56(1H, d, J¼ 7.2Hz), 5.92(1H, t, J ¼9.6 Hz), 6.18 (1H, d, J¼ 4.2 Hz). 13C NMR(150 MHz, C5D5N): δ 44.3(C-1), 68.9(C2), 84.0(C-3), 43.5(C-4), 47.1(C-5), 18.8(C-6), 33.1(C-7), 40.1(C-8), 48.5(C-9), 37.3(C-10), 23.9(C-11), 123.2(C-12), 144.1(C-13), 42.0(C14), 28.9(C-15), 22.9(C-16), 46.9(C-17), 41.5(C-18), 46.0(C-19), 30.8 (C-20), 34.0(C-21), 32.3(C-22), 65.8(C-23), 15.9(C-24), 17.8(C-25), 17.6(C-26), 26.3(C-27), 176.2(C-28), 33.1(C-29), 23.7(C-30), 171.4(C10 ), 47.1(C-20 ), 70.0(C-30 ), 48.0(C-40 ), 171.4(C-50 ), 26.4(C-60 ), 103.5 (GlcC-1), 80.0(Glc-C-2), 78.9(GlcC-3), 71.4(GlcC-4), 78.3(GlcC-5), 62.5 (GlcC-6), 104.7(AraC-1), 73.7(AraC-2), 72.5(AraC-3), 72.5(AraC-4), 64.4 (AraC-5), 94.3(Ara'C-1), 74.9(Ara'C-2), 71.0(Ara'C-3), 67.5(Ara'C-4), 64.7 (Ara'C-5), 100.5(RhaC-1), 72.4(RhaC-2), 70.3(RhaC-3), 75.4(RhaC-4), 67.9(RhaC-5), 18.4(RhaC-6). Compound 10: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 5.38 (1H, t, J¼3.0 Hz), 1.46(3H, s), 1.58(3H, s), 0.99(3H, s), 1.15(3H, s), 0.82(3H, s), 0.87(3H, s), 5.13 (1H, d, J¼7.8 Hz), 5.35 (1H, d, J¼7.8 Hz). 13C NMR(150 MHz, C5D5N): δ 43.5(C-1), 70.1(C-2), 82.3(C3), 42.5(C-4), 47.8(C-5), 17.7(C-6), 32.5(C-7), 39.6(C-8), 47.3(C-9), 36.6 (C-10), 23.5(C-11), 122.9(C-12), 142.4(C-13), 47.6(C-14), 46.7(C-15), 213.3(C-16), 46.7(C-17), 44.6(C-18), 46.5(C-19), 30.8(C-20), 34.5(C-21), 21.0(C-22), 65.3(C-23), 14.6(C-24), 16.9(C-25), 17.5(C-26), 26.9(C-27), 33.2(C-29), 23.3(C-30), 102.9(GlcC-1), 83.4(GlcC-2), 78.0(GlcC-3), 71.1 (GlcC-4), 77.8(GlcC-5), 62.3(GlcC-6), 105.6(Glc'C-1), 76.6(Glc'C-1), 77.8 (Glc'C-1), 70.8(Glc'C-1), 78.2(Glc'C-1), 62.2(Glc'C-1). Compound 11: White powder (Methanol), 1H NMR (600 MHz, C5D5N): δ 1.48(3H, s), 1.58(3H, s), 0.86(3H, s), 1.02(3H, s), 0.95(3H, s), 0.88(3H, s), 5.18 (1H, d, J¼7.8 Hz), 5.39 (1H, d, J¼ 7.8 Hz). 13C NMR (150 MHz, C5D5N): δ 43.6(C-1), 70.4(C-2), 82.7(C-3), 42.8(C-4), 48.3(C5), 18.1(C-6), 33.0(C-7), 38.9(C-8), 48.0(C-9), 37.2(C-10), 23.7(C-11), 118.1(C-12), 142.7(C-13), 43.0(C-14), 42.9(C-15), 23.9(C-16), 49.7(C-17), 37.1(C-18), 38.7(C-19), 30.7(C-20), 44.2(C-21), 213.9(C-22), 65.6(C-23), 14.7(C-24), 17.1(C-25), 16.9(C-26), 25.7(C-27), 33.4(C-29), 24.8(C-30), 103.2(GlcC-1), 83.7(GlcC-2), 78.3(GlcC-3), 71.4(GlcC-4), 78.1(GlcC-5), 62.6 (GlcC-6), 105.9(Glc'C-1), 76.9(Glc'C-2), 78.1(Glc'C-3), 71.1(Glc'C-4), 78.5 (Glc'C-5), 62.5(Glc'C-6). Compound 12: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.93(3H, s), 1.00(3H, s), 1.09(3H, s), 1.28(3H, s), 1.42(3H, s), 1.58(3H, s), 5.50(1H, br. s), 5.16(1H, d, J¼ 7.2 Hz), 5.18(1H, d, J¼ 7.8 Hz). 13C NMR(150 MHz, C5D5N): δ 44.1(C-1), 70.7(C-2), 82.5 (C-3), 42.8(C-4), 47.7(C-5), 17.9(C-6), 33.3(C-7), 39.9(C-8), 48.6(C9), 37.0(C-10), 23.7(C-11), 122.8(C-12), 144.9(C-13), 42.3(C-14), 28.3 (C-15), 24.0(C-16), 46.7(C-17), 42.0(C-18), 46.4(C-19), 31.0(C-20), 34.2(C-21), 33.0(C-22), 64.6(C-23), 14.7(C-24), 17.2(C-25), 17.5(C26), 26.3(C-27), 180.2(C-28), 33.2(C-29), 23.8(C-30), 103.7(GlcC-1), 83.6(GlcC-2), 78.1(GlcC-3), 71.3(GlcC-4), 78.0(GlcC-5), 62.5(GlcC-6), 106.6(AraC-1), 73.9(AraC-2), 74.4(AraC-3), 69.3(AraC-4), 67.3(AraC-5). Compound 13: White needle crystals (Methanol), 1H NMR (600 MHz, C5D5N): δ 1.01(3H, s), 1.48(3H, s), 1.66(3H, s), 1.70(3H, s), 1.34(3H, s), 1.14(3H, s), 4.97 (1H, d, J¼7.2 Hz), 5.19 (1H, d, J¼ 6.6 Hz). 13C NMR (150 MHz, C5D5N): δ 39.6(C-1), 27.1(C-2), 88.8(C-3), 39.7(C-4), 54.8(C5), 30.1(C-6), 78.2(C-7), 49.3(C-8), 52.1(C-9), 37.5(C-10), 22.9(C-11), 28.3(C-12), 44.6(C-13), 50.3(C-14), 36.6(C-15), 25.9(C-16), 49.5(C-17), 61.7(C-18), 16.9(C-19), 74.3(C-20), 26.5(C-21), 41.6(C-22), 23.4(C-23), 126.2(C-24), 130.8(C-25), 17.7(C-26), 25.9(C-27), 27.9(C-28), 16.7(C-29), 16.9(C-30), 105.2(GlcC-1), 83.9(GlcC-2), 78.3(GlcC-3), 71.7(GlcC-4), 78.2 (GlcC-5), 62.9(GlcC-6), 106.7(AraC-1), 73.8(AraC-2), 74.4(AraC-3), 69.2 (AraC-4), 67.1(AraC-5).
Y. Tang et al. / Journal of Ethnopharmacology 169 (2015) 18–23
Compound 14: White needle crystals (Methanol), 1H NMR (600 MHz, C5D5N): δ 1.02(3H, s), 1.15(3H, s), 1.28(3H, s), 1.34(3H, s), 1.48(3H, s), 1.88(3H, s), δ 5.85(1H, t, J ¼7.2 Hz), 4.97(1H, d, J ¼7.8Hz), 5.19(1H, d, J ¼6.6 Hz). 13C NMR(150 MHz,C5D5N): δ 39.6 (C-1), 27.1(C-2), 88.7(C-3), 39.7(C-4), 54.8(C-5), 30.1(C-6), 78.2(C7), 49.3(C-8), 52.1(C-9), 37.5(C-10), 23.0(C-11), 28.3(C-12), 44.6(C13), 50.3(C-14), 36.6(C-15), 25.8(C-16), 49.5(C-17), 61.7(C-18), 16.9 (C-19), 74.3(C-20), 26.5(C-21), 41.4(C-22), 23.0(C-23), 125.6(C-24), 136.1(C-25), 68.2(C-26), 14.0(C-27), 27.9(C-28), 16.7(C-29), 17.0(C30), 105.2(GlcC-1), 83.9(Glc-C-2), 78.2(GlcC-3), 71.7(GlcC-4), 78.1 (GlcC-5), 62.9(GlcC-6), 106.7(AraC-1), 73.8(AraC-2), 74.3(AraC-3), 69.2 (AraC-4), 67.1(AraC-5). Compound 15: White powder (Methanol), 1H –NMR(600 MHz, C5D5N): δ 1.03(3H, s), 1.14(3H, s), 1.28(3H, s), 1.34(3H, s), 1.49(3H, s), 1.88(3H, s), 2.02(3H, s), 5.85(1H, t, J ¼7.2 Hz), 4.95(1H, d, J ¼7.8 Hz), 5.23(1H, d, J¼ 7.2 Hz). 13C NMR(150 MHz, C5D5N): δ 39.5(C-1), 27.0(C-2), 88.8(C-3), 39.7(C-4), 54.7(C-5), 30.1(C-6), 78.2 (C-7), 49.4(C-8), 52.1(C-9), 37.4(C-10), 23.0(C-11), 28.2(C-12), 44.6 (C-13), 50.3(C-14), 36.6(C-15), 25.8(C-16), 49.3(C-17), 61.6(C-18), 16.9(C-19), 74.3(C-20), 26.5(C-21), 41.4(C-22), 22.9(C-23), 125.5(C24), 136.1(C-25), 68.1(C-26), 14.0(C-27), 27.9(C-28), 16.6(C-29), 16.9 (C-30), 170.8( CQO), 21.1(CH3), 105.1(GlcC-1), 83.1(Glc-C-2), 78.2 (GlcC-3), 71.7(GlC-4), 78.3(GlcC-5), 62.8(GlcC-6), 106.4(AraC-1), 74.2 (AraC-2), 72.1(AraC-3), 72.3(AraC-4), 64.5(AraC-5).
2.4. Cytotoxic activity 2.4.1. Cell culture BGC-823, HeLa, and MCF-7 cells were grown in RPMI 1640 supplemented with 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES buffer, 1 mM sodium pyruvate and 2 Mm L-glutamine. HT-29 cells were cultured in DMEM supplemented with 4.5 g/L of glucose. All cell culture media contained 10% FBS and 1% penicillin/streptomycin unless otherwise specified. All cell lines were incubated at 37 1C in a humidified atmosphere containing 5% CO2 (Ivanova et al., 2011). 2.4.2. MTT assay The effects of test compounds on human cancer cell growth, expressed as the percentage of cell survival, were determined using the MTT assay (Ivanova et al., 2011). The cells were grown in 96-well plates at 1 104 cells per well and exposed to the test compounds. After incubation for 48 h, 10 μL of the MTT solution (5 mg/mL; Sigma; St. Louis, MO, USA) were added into each well. The plates were incubated for 2–4 h at 37 1C. The supernatant was then removed and the formazan crystals were dissolved with 100 μL of DMSO. The absorbance at 490 nm was recorded using an OPTI max microplate reader. The cell survival percentages were calculated by dividing the mean OD of compound-containing wells by that of DMSO-control wells. Three independent experiments were accomplished to determine the IC50.
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3. Results and discussion All isolated compounds were identified following analysis of their physicochemical and spectroscopic data and by comparison with published data. Thus, 15 compounds isolated from B. paniculatum bulb were identified as isocucurbitacin B(1) (Zheng et al., 2007; Shi et al., 1994), 23,24-dihydroisocucurbitacin B(2) ( Shi et al., 1994), cucurbitacin E(3) (Nobuo et al., 2001), 23,24-dihydrocucur-bitacinE(4) (Shi et al., 1994), tubeimoside I(5) (Kong et al., 1986), tubeimoside III(6) (Kasai et al., 1988), tubeimoside V(7) (Cheng et al., 2006; Tang et al., 2005), dexylosyltubeimoside III(8) (Ma et al., 2006), lobatoside C(9) (Fujioka et al., 1989), tubeimoside A(10), tumeimoside B(11) (Tang et al., 2014), lobatoside A(12) (Fujioka et al., 1989), tubeimoside C(13) (Tang et al., 2014), tubeimoside IV(14) (Liu et al., 2004), 7β,18,20,26tetrahydroxy-(20S)-dammar-24E-en-3-O-α-L-(4-acetyl)arabinopyranosyl-(1-2)-β-D-glucopyranoside(15) (Liu et al., 2004). This is the first report of the isolation of 23,24-dihydroisocucurbitacin B(2), 23,24-dihydrocucur-bitacinE(4) and lobatoside C(9) from B. paniculatum. Cytotoxicity screening of B. paniculatum extracts and isolated compounds are summarized in Tables 1 and 2. As shown in Tables 1 and 2, EtOAC extract and n-butanol extracts of B. paniculatum displayed cytotoxic activity against tested cells. The EtOAC extract had moderately activity with IC50 values of 50.38, 22.81, 28.09, and 43.54 μM against BCG-823, Hela, HT-29 and MCF-7, respectively. Among the compounds isolated from the EtOAC extract, compounds 1–4 exhibited activities with IC50 values range from 0.93 to 34.61 μM against Hela and HT-29, while compounds 1–4 were inactive with IC50 values 4100 μM against BCG-823 and MCF-7 (See Table 2).
Table 2 The cytotoxicity of compounds against cells tested (the unit for IC50 was μM). Sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 5-Fua
IC50 values BCG-823
Hela
HT-29
MCF-7
4 100 4 100 4 100 4 100 14.78 7 2.30 2.747 0.31 12.617 1.05 3.42 7 0.33 9.137 0.38 36.747 1.73 4 100 4 100 4 100 4 100 4 100 18.107 0.73
7.217 0.42 34.617 1.70 4.92 7 0.39 0.93 7 0.05 15.26 7 1.25 3.377 0.42 9.677 0.57 3.16 70.27 10.22 7 0.53 18.117 1.39 4100 4100 4100 4100 4100 11.617 0.52
9.737 0.57 23.117 1.13 7.30 7 0.33 2.63 70.19 13.20 7 0.67 2.167 0.23 2.08 70.17 1.577 0.11 6.617 0.45 9.69 70.42 4100 4100 4100 4100 4100 23.08 70.82
4100 4100 4100 4100 15.647 0.81 1.30 7 0.15 13.32 7 1.26 2.337 0.15 10.82 7 0.47 60.17 74.26 4100 4100 4100 4100 4100 21.337 1.18
Results are expressed as mean 7 SD. a
5-Fu (fluorouracil) as a positive control.
Table 1 The cytotoxicity of extracts against cells tested (the unit for IC50 was μM). Sample
EtOH extract Petroleum ether extract EtOAC extract n-BuOH extract Water extract Results are expressed as mean7 SD.
IC50 values BCG-823
Hela
HT-29
MCF-7
40.36 7 2.50 4 100 50.38 7 3.53 14.017 2.52 4 100
84.767 6.15 4 100 22.81 7 0.92 20.45 7 1.21 4 100
21.38 7 0.86 4100 28.09 7 1.97 5.05 7 0.27 4100
30.82 7 2.14 4100 43.54 7 3.88 10.54 70.56 4100
22
Y. Tang et al. / Journal of Ethnopharmacology 169 (2015) 18–23
Compound 4 was the most active compound with IC50 values of 0.93 and 2.63 μM against Hela and HT-29 cell lines. Compound 4's activities against the two cell lines are 12- and 8-fold that of the positive control drug (5-Fu). The activity against BCG-823 and MCF-7 observed for EtOAc extract was stronger than that of observed for 1–4, suggesting that the activity could either be attributable to compounds acting synergisticall or to other (nonpurified) phytochemical(s). Additionally, the n-butanol extract had stronger activity against BCG-823, Hela, HT29 and MCF-7 than those of EtOAC extract with IC50 values of 14.01, 20.45, 5.05 and 10.54 μM, respectively. Among the compounds isolated from n-butanol extract, compounds 5–9 showed significantly activities with IC50 values range from 1.30 to 15.64 μM, while compounds 12, 14 and 15 had no activity against the four tested cells (IC50 4100 μM). It is worth mention that 6's activities against the four cell lines are 6-, 3-, 10 and 16-fold that of 5-Fu and 8's activities against the four cell lines are 5-, 3-, 14 and 9-fold that of 5-Fu. Structurally, compounds 5–9 belongs to cyclic bisdesmosides with two oligosaccharides flanked on the positions of C-3 and C-28 of a pentacyclic triterpene and bridged with 3-hydroxy-3-methyl glutarate. Consequently, the macrocyclic structure of 5–9 plays an important role in their potential cytotoxicity. Furthermore, comparing the structures and IC50 values of 6 and 8 with 5, 7 and 9 (See Table 2), it can be concluded that 6 and 8 with an additional hydroxyl group attached to C-16 demonstrated high cytotoxicity, suggesting that the hydroxyl group of C-16 enhanced the cytotoxic activity. The result is consistent well with our previous finding (Li et al., 2012). It is reported that when cyclic bisdesmosides converted to simple triterpene bisdesmosides, the cytotoxicity against Hela cell line disappeared. This result suggested that the macrocyclic structure played an important role in the demonstration of potent cytotoxicity. Furthermore, the terminal sugar of the glycosyl moiety bonded to C-3 might affect the cytotoxicity (Fujioka et al., 1996). The structures of lobatoside B and lobatoside C are quite similar, except for the terminal sugar of the glycosyl moiety bonded to C-3. In the literature, lobatoside B, with GI50 values of 1.15, 1.20 and 1.23 against SW-620, SK-MEL-5 and UO-31 cell lines, the terminal sugar is a hexose, while lobatoside C, with GI50 values of 8.91, 4.17 and 6.03 against the three cell lines, the terminal sugar is an arabinose. This observation suggested that the terminal hexose in the C-3 glycosyl moiety might be important to the potency of these compounds (Fujioka et al., 1996). The structure–activity relationship (SAR) of these compounds correlated well with our previous studies of cyclic bisdesmosides on cytotoxic activity (Li et al., 2012). The key SAR of cyclic bisdesmosides can be seen in Fig. 1. Regarding to other compounds (10–15) isolated from B. paniculatum n-butanol extract, tubeimoside A (10) had moderate cytotoxic activity against BCG-823, Hela, HT-29 and MCF-7 with IC50 values of 36.74, 18.11, 9.69 and 60.17, whilst tubeimoside B (11) and the others (12–15) were inactive (IC50 4100 μM). To the best of our knowledge,
Fig. 1. The key SAR of cyclic bisdesmosides.
this is the first report on EtOAC extract of B. paniculatum along with the isolated compound 10, 11, 13 and 15 screening for cytotoxic activity.
4. Conclusion In this study, EtOAC extract of B. paniculatum and isolated pure compounds (1–4) exhibited higher cytotoxic activities against Hela and HT-29 than those of crude extract. The pure compounds (5–10) isolated from B. paniculatum n-butanol extract exhibited higher cytotoxic activities than the crude extract. This behavior might be due to synergism between compounds present in the extract. In China, B. paniculatum has been described as a folk remedy for the treatment of tumors, warts, and detoxication and it has also been applied in the treatment of cancer. The triterpinoid saponins are considered to be the main active constituents. Base on this study, we can conclude that the cytotoxic activity of B. paniculatum is mainly ascribable to the cyclic bisdesmosides with IC50 values range from 1.30 to 15.64 μM. These results provide some scientific evidence to support, to some extent, the ethno-medicinal use of B. paniculatum as traditional anticancer remedies. The cytotoxicity assay used in this study could only provide preliminary data to help identify constituents with potential anticancer properties. Anticancer drugs with low side effects, apoptosisinducing and targeting specificity to the cancer cells are the drugs of choice. Considering the potential of the cyclic bisdesmosides as anticancer agents, a further study on the mechanism of cell death is needed to provide more convincing evidence.
Acknowledgment This work was financially supported by E&T modern center for Natural Products of Liaoning Province of China (No. 2008402021) and the National Natural Science Foundation of China, China (NO. 81273389). We are also grateful to the Analytical Center of Shenyang Pharmaceutical University for identification of the measurements of NMR, IR, HR-TOF-MS. References Cassady, J.M., Suffness, M., 1980. Anticancer agents based on natural products models. Academic Press, New York, pp. 201–269. Cheng, G., Zhang, Y., Zhang, X., Tang, H.F., Cao, W.D., Gao, D.K., Wang, X.L., 2006. Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, functions by inducing apoptosis in human glioblastoma U87MG cells. Bioorg. Med. Chem. Lett. 16, 4575–4580. Chen, W.J., Yu, C., Yang, Z., He, J.L., Yin, J., Liu, H.Z., Liu, H.T., Wang, Y.X., 2012. Tubeimoside I induces G2/M phase arrest and apoptosis in SKOV-3 cells through increase of intracellular Ca2 þ and caspase-dependent signaling pathways. Int. J. Oncol. 40, 535–543. Committee of Pharmacopeia, 2010. Chinese Pharmacopoeia. Vol. 1. Chinese Medicine Science and Technology Press, China p. 155. Fujioka, T., Iwamoto, M., Iwase, Y., Hachiyama, S., Okabe, H., Yamauchi, T., Mihashi, K., 1989. Studies on the constituents of Actinostemma lobatum Maxim.IV. Structures of lobatosides C, D and H, the dicrotalic acid esters of bayogenin bisdesmosides isolated from the herb. Chem. Pharm. Bull. 37, 1770–1775. Fujioka, T., Kashiwada, Y., Okabe, H., Mihashi, K., Lee, K.H., 1996. Antitumor agents 171. cytotoxicities of lobatosides B, C, D, and E, cyclic bisdesmosides isolated from Actinostemma lobatum Maxim. Bioorg. Med. Chem. Lett. 6, 2807–2810. Huang, P., Yu, C., Liu, X.Q., Ding, Y.B., Wang, Y.X., He, J.L., 2011. Cytotoxicity of tubeimoside I in human choriocarcinoma JEG-3 cells by induction of cytochromec release and apoptosis via the mitochondrial-related signaling pathway. Int. J. Mol. Med. 28, 579–587. Ivanova, A., Mikhova, B., Batsalova, T., Dzhambazov, B., Kostova, I., 2011. New furostanol saponins from Smilax aspera L. and their in vitro cytotoxicity. Fitoterapia 82, 282–287. Kasai, R., Miyakoshi, M., Nie, R.L., Zhou, J., Matsumoto, K., Morita, T., Nishi, M., Miyahara, K., Tanaka, O., 1988. Saponins from Bolbostemma Paniculatum: cyclic bisdesmosides, tubeimoside II and III. Phytochemistry 27, 1439–1446.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Bioassay-guided isolation and identification of cytotoxic compounds from Bolbostemma paniculatum Yun Tang a,b, Wei Li a, Jiaqing Cao b,c, Wei Li b,c, Yuqing Zhao b,c,n a
Key Laboratory of Natural Active Pharmaceutical Constituents of Jiangxi Province, Yichun University, Yichun 336000, PR China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China c Key Laboratory of Structure-Based Drug Design & Discovery (Shenyang Pharmaceutical University), Ministry of Education, Shenyang 110016, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 January 2015 Received in revised form 25 March 2015 Accepted 3 April 2015 Available online 13 April 2015
Ethnopharmacological relevance: Bolbostemma paniculatum (Maxim.) Franquet (B. paniculatum), also named “Tu-bei-mu” in Chinese folk medicines, has been described in application for the treatment of tumors, warts, inflammation and toxication in traditional Chinese medicinal books. The major constituents in B. paniculatum are triterpenoid saponins, which have been proved to possess dramatically cytotoxic activity and antivirus activity. The aim of this study is to isolate and identify the active triterpenoid saponin from the bulb of B. paniculatum by a bioassay-guided method. Materials and methods: Four cucurbitacine triterpenoid sapogenins and 11 triterpenoid saponins were isolated from the active EtOAc and n-BuOH extract of B. paniculatum by using bioassay-guided screening. Their structures were elucidated based on the spectroscopic methods and compared with published data. Cytotoxic activities of isolated compounds were determined by MTT assay. Results: Four cucurbitacine triterpenoid sapogenins, isocucurbitacin B(1), 23,24-dihydroisocucurbitacin B(2), cucurbitacin E(3), 23,24-dihydrocucurbitacin E(4), and 11 triterpenoid saponins, tubeimosideI(5), tubeimoside III(6), tubeimoside V(7), dexylosyltubeimoside III(8), lobatoside C(9), tubeimoside A(10), tumeimoside B(11), lobatoside A(12), tubeimoside C(13), tubeimoside IV(14), 7β,18,20,26-tetrahydroxy(20S)-dammar-24E-en-3-O-α-L-(4-acetyl)arabinopyranosyl-(1-2)-β-D-glucopyranoside(15) were isolated from the active EtOAc and n-BuOH extracts. Of them, compounds 2, 4, 9 and 12 were firstly isolated from the Bolbostemma genus. MTT assay revealed that compounds 1, 3 and 4 had significantly activities against HeLa and HT-29 human cancer cells with IC50 values ranging from 0.93 to 9.73 μM. It is worth mentioning that compound 4's activities against the two cell lines are 12- and 8-fold that of the positive control drug (5-Fu). Whereas, the cyclic bisdesmosides 5–9 exerted significantly activities on BGC-823, HeLa, HT-29 and MCF-7 cancer cells with IC50 values ranging from 1.30 to 15.64 μM. And 6's activities against the four cell lines are 6-, 3-, 10- and 16-fold that of 5-Fu and 8's activities against the four cell lines are 5-, 3-, 14- and 9-fold that of 5-Fu. Conclusion: The cytotoxic activity of the bulbs of B. paniculatum is mainly ascribable to cucurbitacine triterpenoid sapogenins (1–4) and the cyclic bisdesmosides (5–9). The cyclic bisdesmosides are the main anti-cancer active compounds of B. paniculatum. The above results provide scientific evidence to support, to some extent, the ethnomedicinal use of B. paniculatum as anticancer remedies in traditional Chinese medicine. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Bolbostemma paniculatum Triterpenoid Tubeimoside Cyclic bisdesmosides Cytotoxicity
1. Introduction Abbreviations: B. paniculatum, Bolbostemma paniculatum (Maxim.) Franquet; EtOAc, ethyl acetate; n-BuOH, n-butanol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylterazolium bromide; IC50, half maximal inhibitory concentration; 5-Fu, fluorouracil; ODS, octadecylsilyl; OD, optical density; SAR, structure–activity relationship; NMR, nuclear magnetic resonance; TLC, thin layer chromatography; HPLC, high performance liquid chromatography; CC, column chromatography; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide; Rha, rhamnose; Ara, arabinose; Glc, glucose; Xyl, xylose n Corresponding author. E-mail address: [email protected] (Y. Zhao). http://dx.doi.org/10.1016/j.jep.2015.04.003 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Cancer is harmful to human health and it has become the leading cause of death in the last 50 years (Ullah and Aatif, 2009). In past decades, searching new anticancer agent from animals and plants source has drawn increasingly attention of researchers. Bolbostemma paniculatum, mainly distributed in Shaanxi, Shanxi, Henan and Shandong Province of China, is one of the Chinese folk medicines recorded in Supplement to the Compendium of Materia Medica, and it was used in the Qing Dynasty (Zhao, 1983). It has been described as a
Y. Tang et al. / Journal of Ethnopharmacology 169 (2015) 18–23
19
folk remedy for the treatment of tumors, warts, and detoxication for thousands of years (Committee of Pharmacopeia, 2010). Moreover, B. paniculatum has also been applied in the treatment of cancer for a long time because of its abundant triterpinoid saponins (Sindambiwe et al., 1998). Triterpenoids with cytotoxic activity against HeLa (cervical cancer) (Xu et al., 2011), A549 (lung carcinoma) (Zhang et al., 2011), BGC-823 (gastric carcinoma) (Zhang et al., 2013), CNE-2Z (nasopharyngeal carcinoma) (Ma et al., 2008), HepG2 (liver cancer) (Yin et al., 2011), SKOV-3 (ovarian cancer) (Chen et al., 2012), EC109 (esophageal cancer) (Xu et al., 2013), JEG-3(choriocarcinoma) (Huang et al., 2011), A2780 (ovarian cancer) (Liu et al., 2011), and HL-60 (promyelocytic leukemia) (Yu et al., 1996) cell lines had been reported to be the major constituents of B. paniculatum. In addition, a few of cucurbitacins with cytotoxicity had also been isolated and identified from the bulbs of B. paniculatum (Cassady and Suffness, 1980). Based on above knowledge, searching for newly cytotoxic compounds from B. paniculatum is desirable. In current study, a systematic chemical investigation was carried out on the bulbs of B. paniculatum. The cytotoxic activities were tested against several human cancer cell lines including BGC-823 gastric cancer cell line, Hela cervical carcinoma cell line, HT-29 colon cancer cell line and MCF-7 breast cancer cell line. A bioassay-guided screening method was conducted to separate pure compound (s) responsible for their activities. The chemical structures of isolated compounds were established with the aid of extensive NMR spectroscopic, massspectral analyses and published data.
0:1) as eluant to provide 14 fractions A–N based on TLC analysis. Fraction I was subjected to silica gel column chromatography (CC) eluted with CH2Cl2:CH3OH:H2O (the lower phase) (7:2:1, 7:3:1) to obtain 10 sub-fractions (I-1-1, I-1-2, I-1-3,…I-1-10). Fraction I-8 was applied to ODS silica gel CC eluted with 50% MeOH–H2O to afford 15 (25 mg) and eluted with 70% MeOH–H2O to afford 13 (25 mg). Fraction J was chromatographed on silica gel eluted with CH2Cl2– MeOH–H2O (the lower phase) (7:2.5:1) to collect 11 sub-fractions (J-11, J-1-2, J-1-3…J-1-11). Fraction J-7 was applied to ODS silica gel CC eluted with MeOH–H2O (3:10; 1:2, 7:10, 9:10) to afford 12 (20.0 mg) and 11 (28.0 mg) as pure compounds, and 10 with few impurities. Compound 10 (25.0 mg) was purified by preparative HPLC using 75% MeOH–H2O. Fraction J-8 was applied to ODS silica gel CC eluted with MeOH–H2O (3:10; 1:2, 7:10, 9:10) to afford 14 (30.0 mg). Fraction K was chromatographed on silica gel eluted with CH2Cl2–MeOH–H2O (the lower phase) (7: 3: 1) to collect 7 sub-fractions (K-1-7). Fraction K6 was applied to ODS silica gel CC eluted with MeOH–H2O (3:10; 1:2, 7:10, 9:10) to collect 4 fractions (K-6-1, K-6-2, K-6-3, K-6-4). Fraction K6-3 was applied to preparative HPLC using 67% MeOH–H2O to afford 5 (20 mg) and 6 (29 mg) as pure compounds. Fraction L was applied to ODS silica gel CC eluted with MeOH–H2O (3:10; 1:2, 7:10, 9:10) to collect 2 sub-fractions (L-1, L-2). Fraction L-1 was further purified by using recrystallization to afford 3 (15 mg) and 4 (18 mg) as pure compounds. Fraction L-2 was applied to preparative HPLC using 50% MeOH–H2O to afford 1 (20 mg) and 2 (25 mg) as pure compounds. Once again, the isolated compounds 5–15 were tested for cytotoxic activity to identify the bioactive compound(s).
2. Materials and methods
2.3. Chemical elucidation of compounds 1–15
2.1. Plant material
The structures of compound 1–15 were elucidated by spectroscopic methods and the 1H and 13C NMR spectral data were compared with earlier reports (Zheng et al., 2007; Shi et al., 1994; Nobuo et al., 2001; Kong et al., 1986; Kasai et al., 1988; Cheng et al., 2006; Tang et al., 2005, 2014; Ma et al., 2006; Fujioka et al., 1989; Liu et al., 2004). Compound 1: White needle crystals (EtOAc), 1H NMR (300 MHz, CDCl3): δ 0.81(3H, s), 0.97(3H, s), 1.18(3H, s), 1.26(3H, s), 1.33(3H, s), 1.41(3H, s), 1.54(3H, s), 1.56(3H, s), 2.00(3H, s), 5.94 (1H, d, J¼5.7 Hz), 6.43(1H, d, J¼15.6 Hz), 7.05(1H, d, J¼15.6 Hz). 13C NMR (75 MHz, CDCl3): δ 38.8(C-1), 210.6(C-2), 80.2(C-3), 46.7(C-4), 138.1(C-5), 121.9 (C-6), 23.9(C-7), 42.7(C-8), 48.3(C-9), 36.2(C-10), 211.8(C-11), 48.5(C12), 50.6(C-13), 47.9(C-14), 45.3(C-15), 71.2(C-16), 58.0(C-17), 19.8(C18), 20.0(C-19), 78.1(C-20), 23.7(C-21), 202.4(C-22), 120.2(C-23), 152.0 (C-24), 79.3(C-25), 25.9(C-26), 26.4(C-27), 20.9(C-28), 24.1(C-29), 18.8 (C-30), 170.2(C-31), 21.9(C-32). Compound 2: White needle crystals (EtOAc), 1H NMR (600 MHz, CDCl3): 0.81(3H, s), 0.96(3H, s), 1.18(3H, s), 1.27(3H, s), 1.33(3H, s), 1.40 (3H, s), 1.43(3H, s), 1.45(3H, s), 1.96(3H, s), 5.94 (1H, d, J¼6.0 Hz). 13C NMR(150 MHz, CDCl3): δ 38.8(C-1), 210.6(C-2), 80.2(C-3), 46.7(C-4), 138.2(C-5), 121.9(C-6), 23.8(C-7), 42.7(C-8), 48.4(C-9), 36.3(C-10), 211.8 (C-11), 48.6(C-12), 48.2(C-13), 50.6(C-14), 45.5(C-15), 70.9(C-16), 57.7 (C-17), 20.0(C-18), 18.7(C-19), 78.9(C-20), 24.4(C-21), 213.9(C-22), 30.7 (C-23), 34.7(C-24), 81.3(C-25), 26.1(C-26), 25.8(C-27), 21.0(C-28), 24.1 (C-29), 19.8(C-30), 170.4(C-31), 22.4(C-32). Compound 3: White needle crystals (EtOAc), 1H NMR (600 MHz, CDCl3): δ 1.00(3H, s), 1.03(3H, s), 1.25(3 H, s), 1.36(3H, s), 1.39(3H, s), 1.44(3 H, s), 1.54(3H, s), 1.57(3H, s), 2.01(3H, s), 5.77 (1H, br.s), 5.96(1H, br.s), 6.46(1H, d, J¼ 15.6 Hz), 7.06(1H, d, J¼15.6 Hz). 13C NMR (150 MHz, CDCl3): δ 114.8(C-1), 144.6(C-2), 198.7(C-3), 47.5(C-4), 136.8(C-5), 120.8(C-6), 23.6(C-7), 41.6(C-8), 48.9(C-9), 34.7(C-10), 212.9(C-11), 48.9(C-12), 50.7(C-13), 48.1(C-14), 45.6(C-15), 71.3(C-16), 58.2(C-17), 19.9(C-18), 20.1(C-19), 78.2(C-20), 24.0(C-21), 202.5(C-22), 120.4(C-23), 152.0(C-24), 79.3(C-25), 25.9(C-26), 26.5(C-27), 20.2(C28), 28.0(C-29), 18.4(C-30), 170.3(C-31), 21.9(C-32). Compound 4: White needle crystals (EtOAc), 1H NMR(600 MHz, CDCl3): δ 0.99(3H, s), 1.04(3H, s), 1.25(3H, s), 1.36(3H, s), 1.40(3H,
The bulbs of B. paniculatum (Maxim.) Franquet (Cucurbitaceae) were collected in Shaanxi province of People's Republic of China. A voucher specimen of this herb (No. 2010085) was identified by Prof. Jincai Lu of Shenyang Pharmaceutical University. The voucher specimen was deposited in our lab. 2.2. Plant extraction and purification under bioassay-guided screening The bulbs of B. paniculatum (50.0 kg) were extracted with 75% EtOH (80 L 3) under refluxing for 2 h. The filtrate obtained was concentrated under reduced pressure to yield a dark residue (15 kg). Part of the crude ethanolic extract (8 kg) was suspended in water (28 L) and successively extracted with petroleum ether, EtOAC and nbutanol sequentially (3 28 L) to afford dried petroleum ether-, EtOAc-, n-BuOH- and H2O-soluble extracts. Each of these extracts was tested for its cytotoxic activity. The EtOAc extract showed moderate cytotoxicity against the BGC-823, HeLa, HT-29 and MCF-7 cell lines, the n-BuOH extract were clearly the most active against the BGC-823, HeLa, HT-29 and MCF-7 cell lines. Further fractionation of EtOAc extract (200 g) with liquid chromatography (silica gel, eluted with mixture of petroleum ether/acetone from 40:1 to 2:1) afforded four major fractions A–D based on TLC analysis. Fraction B was further purified with radial chromatography (silica gel, eluted with mixture of petroleum ether/EtOAc from 20:1 to 0:1) to afford two sub-fractions (FB-1, FB-2). FB-1 was purified by sephadex LH-20 and then applied to preparative HPLC using 54% MeOH–H2O to afford 2(8 mg), 3 (8.5 mg) and 4 (8 mg) as pure compounds. Fraction B was further purified by using recrystallization to afford 1 (20 mg) as pure compound. Once again, the isolated compounds 1–4 were tested for cytotoxic activity to identify the bioactive compound(s). The n-BuOH-soluble extract (500 g) was then chromatographied over silica gel column using a gradient of CH2Cl2:CH3OH (from 20:1 to
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s), 1.42(3H, s), 1.44(3H, s), 1.46(3H, s), 1.97(3H, s), 5.77 (1H, br.s), 5. 96(1H, d, J ¼2.4 Hz). 13C NMR(150 MHz, CDCl3): δ 114.8(C-1), 144.6 (C-2), 198.7(C-3), 47.5(C-4), 136.8(C-5), 120.8(C-6), 23.6(C-7), 41.6 (C-8), 48.4(C-9), 34.7(C-10), 212.8(C-11), 48.8(C-12), 48.9(C-13), 50.7(C-14), 45.7(C-15), 71.0(C-16), 57.8(C-17), 19.8(C-18), 18.2(C19), 78.9(C-20), 24.5(C-21), 213.9(C-22), 30.7(C-23), 34.8(C-24), 81.3(C-25), 26.2(C-26), 25.9(C-27), 20.1(C-28), 27.9(C-29), 20.2(C30), 170.4(C-31), 22.4(C-32). Compound 5: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.92(3H, s), 0.92(3H, s), 1.16(3H, s), 1.25(3H, s), 1.53(3H, s), 1.58(3H, s), 1.64(3H, s), 2.07(3H, s), 5.62(1H, br. s), 4.99(1H, br. s), 5.06 (1H, br. s), 5.41(1H, br. s), 5.57(1H, br. s), 6.11(1H, m). 13C NMR (150 MHz, C5D5N): δ 44.0(C-1), 69.4(C-2), 82.9(C-3), 43.1(C-4), 48.5(C5), 18.2(C-6), 33.1(C-7), 40.0(C-8), 47.3(C-9), 37.2(C-10), 23.9(C-11), 123.3(C-12), 144.2(C-13), 41.8(C-14), 29.1(C-15), 22.7(C-16), 47.0 (C-17), 41.3(C-18), 45.9(C-19), 30.8(C-20), 33.9(C-21), 32.2(C-22), 64.4 (C-23), 15.6(C-24), 17.6(C-25), 17.6(C-26), 25.9(C-27), 176.0(C-28), 33.1 (C-29), 23.6(C-30), 171.2(C-10 ), 46.2(C-20 ), 70.1(C-30 ), 48.0(C-40 ), 171.4 (C-50 ), 26.4(C-60 ), 103.1(GlcC-1), 79.6(Glc-C-2), 78.9(GlcC-3), 71.4(GlcC-4), 78.5(GlcC-5), 62.4(GlcC-6), 104.4(AraC-1), 73.7(AraC-2), 72.5(AraC-3), 72.4 (AraC-4), 64.5(AraC-5), 94.0(Ara'C-1), 74.7(Ara'C-2), 70.9(Ara'C-3), 67.4 (Ara'C-4), 64.3(Ara'C-5), 100.5(RhaC-1), 72.2(RhaC-2), 78.1(RhaC-3), 73.1 (RhaC-4), 68.0(RhaC-5), 18.3(RhaC-6), 106.7(XylC-1), 74.5(XylC-2), 78.3 (XylC-3), 70.6(XylC-4), 66.9(XylC-5). Compound 6: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.99(3H, s), 1.05(3H, s), 1.39(3H, s), 1.47(3H, d, J¼ 6.0 Hz), 1.50(3H, s), 1.71(3H, s), 1.89(3H, s), 1.97(3H, s), 5.66(1H, br. s), 5.12(1H, d, J¼7.2 Hz), 5.16(1H, d, J¼ 7.2 Hz), 5.33(1H, d, J¼ 7.8 Hz), 5.95(1H, d, J¼ 7.8Hz), 6.33(1H, br. s). 13C NMR(150 MHz, C5D5N): δ 44.1(C-1), 70.0 (C-2), 82.9(C-3), 42.6(C-4), 47.7(C-5), 18.7(C-6), 33.2(C-7), 40.3(C-8), 47.7(C-9), 36.8(C-10), 24.0(C-11), 122.9(C-12), 144.6(C-13), 42.1(C-14), 37.0(C-15), 73.4(C-16), 49.3(C-17), 40.9(C-18), 46.5(C-19), 30.8(C-20), 36.0(C-21), 32.4(C-22), 64.3(C-23), 15.3(C-24), 17.5(C-25), 17.8(C-26), 27.3(C-27), 175.7(C-28), 33.7(C-29), 24.3(C-30), 171.3(C-10 ), 47.2(C-20 ), 70.1(C-30 ), 47.0(C-40 ), 171.5(C-50 ), 25.9(C-60 ), 103.4(GlcC-1), 83.8(GlcC-2), 78.0(GlcC-3), 70.9(GlcC-4), 77.3(GlcC-5), 62.4(GlcC-6), 105.5(Glc'C-1), 77.1 (Glc'C-2), 77.8(Glc'C-3), 70.9(Glc'C-4), 75.7(Glc'C-5), 64.8(Glc'C-6), 94.5 (AraC-1), 76.7(AraC-2), 74.8(AraC-3), 69.5(AraC-4), 67.4(AraC-5), 102.4 (RhaC-1), 72.5(RhaC-2), 78.2(RhaC-3), 73.5(RhaC-4), 68.0(RhaC-5), 18.3 (RhaC-6), 106.3(XylC-1), 74.7(XylC-2), 78.0(XylC-3), 70.9(XylC-4), 67.0 (XylC-5). Compound 7: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.89(3H, s), 0.92(3H, s), 1.34(3H, s), 1.38(3H, s), 1.45(3H, s), 1.52(3H, d, J¼ 6.0 Hz), 1.64(3H, s), 1.86(3H, s), 5.48(1H, br. s), 5.08(1H, br. s), 5.14(1H, d, J¼7.2 Hz), 5.32(1H, d, J¼7.2 Hz), 5.91(1H, d, J¼6.6 Hz), 6.44(1H, br. s). 13C NMR(150 MHz, C5D5N): δ 44.1(C-1), 69.7(C-2), 82.9(C-3), 42.4(C-4), 47.8(C-5), 18.5(C-6), 33.8(C-7), 40.2(C-8), 48.8 (C-9), 37.0(C-10), 22.7(C-11), 123.0(C-12), 144.2(C-13), 42.4(C-14), 29.3 (C-15), 24.1(C-16), 47.4(C-17), 41.5(C-18), 46.3(C-19), 30.8(C-20), 34.1 (C-21), 32.4(C-22), 64.2(C-23), 15.2(C-24), 17.5(C-25), 17.8(C-26), 26.3 (C-27), 176.5(C-28), 33.1(C-29), 23.6(C-30), 171.9(C-10 ), 46.4(C-20 ), 70.1 (C-30 ), 46.7(C-40 ), 171.4(C-50 ), 26.4(C-60 ), 102.9(GlcC-1), 84.2(GlcC-2), 78.3 (GlcC-3), 69.7(GlcC-4), 78.0(GlcC-5), 62.5(GlcC-6), 105.6(Glc'C-1), 77.1(Glc'C2), 77.5(Glc'C-3), 70.8(Glc'C-4), 75.7(Glc'C-5), 64.5(Glc'C-6), 94.6(AraC-1), 76.0(AraC-2), 75.1(AraC-3), 71.0(AraC-4), 67.5(AraC-5), 102.4(RhaC-1), 72.6 (RhaC-2), 78.3(RhaC-3), 73.6(RhaC-4), 67.9(RhaC-5), 18.3(RhaC-6), 106.6 (XylC-1), 74.9(XylC-2), 77.9(XylC-3), 70.98(XylC-4), 67.1(XylC-5). Compound 8: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.98(3H, s), 1.03(3H, s), 1.22(3H, s), 1.37(3H, s), 1.45(3H, d, J¼ 6Hz), 1.61(3H, s), 1.70(3H, s), 1.94(3H, s), 5.65(1H, br. s), 5.09(1H, d, J¼ 7.8 Hz), 5.36(1H, d,J¼7.8 Hz), 5.96(1H, d, J¼7.8 Hz), 6.42(1H, br. s). 13 C NMR(150 MHz, C5D5N): δ 44.2(C-1), 70.0(C-2), 83.2(C-3), 42.4(C4), 47.7(C-5), 18.4(C-6), 33.2(C-7), 40.3(C-8), 47.2(C-9), 37.0(C-10), 24.0 (C-11), 122.9(C-12), 144.7(C-13), 42.1(C-14), 36.9(C-15), 73.5(C-16), 49.2 (C-17), 40.9(C-18), 46.6(C-19), 30.8(C-20), 36.0(C-21), 32.5(C-22), 64.5 (C-23), 15.2(C-24), 17.4(C-25), 17.6(C-26), 27.4(C-27), 175.7(C-28), 33.7
(C-29), 24.4(C-30), 171.6(C-10 ), 47.1(C-20 ), 70.6(C-30 ), 46.3(C-40 ), 171.9 (C-50 ), 26.4(C-60 ), 103.1(GlcC-1), 84.9(GlcC-2), 78.4(GlcC-3), 70.7(GlcC-4), 78.2(GlcC-5), 62.5(GlcC-6), 105.8(Glc'C-1), 77.0(Glc'C-2), 77.6(Glc'C-3), 70.7 (Glc'C-4), 75.7(Glc'C-5), 64.2(Glc'C-6), 94.8(AraC-1), 76.4(AraC-2), 72.5 (AraC-3), 69.9(AraC-4), 67.7(AraC-5), 102.5(RhaC-1), 72.4(RhaC-2), 73.4 (RhaC-3), 75.8(RhaC-4), 68.0(RhaC-5), 18.3(RhaC-6). Compound 9: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.92(3H, s), 0.92(3H, s), 1.09(3H, s), 1.24(3H, s), 1.50(3H, s), 1.54(3H, d, J ¼6 Hz), 1.59(3H, s), 1.90(3H, s), 5.45(1H, br. s), 5.03 (1H, d, J ¼7.2 Hz), 5.56(1H, d, J¼ 7.2Hz), 5.92(1H, t, J ¼9.6 Hz), 6.18 (1H, d, J¼ 4.2 Hz). 13C NMR(150 MHz, C5D5N): δ 44.3(C-1), 68.9(C2), 84.0(C-3), 43.5(C-4), 47.1(C-5), 18.8(C-6), 33.1(C-7), 40.1(C-8), 48.5(C-9), 37.3(C-10), 23.9(C-11), 123.2(C-12), 144.1(C-13), 42.0(C14), 28.9(C-15), 22.9(C-16), 46.9(C-17), 41.5(C-18), 46.0(C-19), 30.8 (C-20), 34.0(C-21), 32.3(C-22), 65.8(C-23), 15.9(C-24), 17.8(C-25), 17.6(C-26), 26.3(C-27), 176.2(C-28), 33.1(C-29), 23.7(C-30), 171.4(C10 ), 47.1(C-20 ), 70.0(C-30 ), 48.0(C-40 ), 171.4(C-50 ), 26.4(C-60 ), 103.5 (GlcC-1), 80.0(Glc-C-2), 78.9(GlcC-3), 71.4(GlcC-4), 78.3(GlcC-5), 62.5 (GlcC-6), 104.7(AraC-1), 73.7(AraC-2), 72.5(AraC-3), 72.5(AraC-4), 64.4 (AraC-5), 94.3(Ara'C-1), 74.9(Ara'C-2), 71.0(Ara'C-3), 67.5(Ara'C-4), 64.7 (Ara'C-5), 100.5(RhaC-1), 72.4(RhaC-2), 70.3(RhaC-3), 75.4(RhaC-4), 67.9(RhaC-5), 18.4(RhaC-6). Compound 10: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 5.38 (1H, t, J¼3.0 Hz), 1.46(3H, s), 1.58(3H, s), 0.99(3H, s), 1.15(3H, s), 0.82(3H, s), 0.87(3H, s), 5.13 (1H, d, J¼7.8 Hz), 5.35 (1H, d, J¼7.8 Hz). 13C NMR(150 MHz, C5D5N): δ 43.5(C-1), 70.1(C-2), 82.3(C3), 42.5(C-4), 47.8(C-5), 17.7(C-6), 32.5(C-7), 39.6(C-8), 47.3(C-9), 36.6 (C-10), 23.5(C-11), 122.9(C-12), 142.4(C-13), 47.6(C-14), 46.7(C-15), 213.3(C-16), 46.7(C-17), 44.6(C-18), 46.5(C-19), 30.8(C-20), 34.5(C-21), 21.0(C-22), 65.3(C-23), 14.6(C-24), 16.9(C-25), 17.5(C-26), 26.9(C-27), 33.2(C-29), 23.3(C-30), 102.9(GlcC-1), 83.4(GlcC-2), 78.0(GlcC-3), 71.1 (GlcC-4), 77.8(GlcC-5), 62.3(GlcC-6), 105.6(Glc'C-1), 76.6(Glc'C-1), 77.8 (Glc'C-1), 70.8(Glc'C-1), 78.2(Glc'C-1), 62.2(Glc'C-1). Compound 11: White powder (Methanol), 1H NMR (600 MHz, C5D5N): δ 1.48(3H, s), 1.58(3H, s), 0.86(3H, s), 1.02(3H, s), 0.95(3H, s), 0.88(3H, s), 5.18 (1H, d, J¼7.8 Hz), 5.39 (1H, d, J¼ 7.8 Hz). 13C NMR (150 MHz, C5D5N): δ 43.6(C-1), 70.4(C-2), 82.7(C-3), 42.8(C-4), 48.3(C5), 18.1(C-6), 33.0(C-7), 38.9(C-8), 48.0(C-9), 37.2(C-10), 23.7(C-11), 118.1(C-12), 142.7(C-13), 43.0(C-14), 42.9(C-15), 23.9(C-16), 49.7(C-17), 37.1(C-18), 38.7(C-19), 30.7(C-20), 44.2(C-21), 213.9(C-22), 65.6(C-23), 14.7(C-24), 17.1(C-25), 16.9(C-26), 25.7(C-27), 33.4(C-29), 24.8(C-30), 103.2(GlcC-1), 83.7(GlcC-2), 78.3(GlcC-3), 71.4(GlcC-4), 78.1(GlcC-5), 62.6 (GlcC-6), 105.9(Glc'C-1), 76.9(Glc'C-2), 78.1(Glc'C-3), 71.1(Glc'C-4), 78.5 (Glc'C-5), 62.5(Glc'C-6). Compound 12: White powder (Methanol), 1H NMR(600 MHz, C5D5N): δ 0.93(3H, s), 1.00(3H, s), 1.09(3H, s), 1.28(3H, s), 1.42(3H, s), 1.58(3H, s), 5.50(1H, br. s), 5.16(1H, d, J¼ 7.2 Hz), 5.18(1H, d, J¼ 7.8 Hz). 13C NMR(150 MHz, C5D5N): δ 44.1(C-1), 70.7(C-2), 82.5 (C-3), 42.8(C-4), 47.7(C-5), 17.9(C-6), 33.3(C-7), 39.9(C-8), 48.6(C9), 37.0(C-10), 23.7(C-11), 122.8(C-12), 144.9(C-13), 42.3(C-14), 28.3 (C-15), 24.0(C-16), 46.7(C-17), 42.0(C-18), 46.4(C-19), 31.0(C-20), 34.2(C-21), 33.0(C-22), 64.6(C-23), 14.7(C-24), 17.2(C-25), 17.5(C26), 26.3(C-27), 180.2(C-28), 33.2(C-29), 23.8(C-30), 103.7(GlcC-1), 83.6(GlcC-2), 78.1(GlcC-3), 71.3(GlcC-4), 78.0(GlcC-5), 62.5(GlcC-6), 106.6(AraC-1), 73.9(AraC-2), 74.4(AraC-3), 69.3(AraC-4), 67.3(AraC-5). Compound 13: White needle crystals (Methanol), 1H NMR (600 MHz, C5D5N): δ 1.01(3H, s), 1.48(3H, s), 1.66(3H, s), 1.70(3H, s), 1.34(3H, s), 1.14(3H, s), 4.97 (1H, d, J¼7.2 Hz), 5.19 (1H, d, J¼ 6.6 Hz). 13C NMR (150 MHz, C5D5N): δ 39.6(C-1), 27.1(C-2), 88.8(C-3), 39.7(C-4), 54.8(C5), 30.1(C-6), 78.2(C-7), 49.3(C-8), 52.1(C-9), 37.5(C-10), 22.9(C-11), 28.3(C-12), 44.6(C-13), 50.3(C-14), 36.6(C-15), 25.9(C-16), 49.5(C-17), 61.7(C-18), 16.9(C-19), 74.3(C-20), 26.5(C-21), 41.6(C-22), 23.4(C-23), 126.2(C-24), 130.8(C-25), 17.7(C-26), 25.9(C-27), 27.9(C-28), 16.7(C-29), 16.9(C-30), 105.2(GlcC-1), 83.9(GlcC-2), 78.3(GlcC-3), 71.7(GlcC-4), 78.2 (GlcC-5), 62.9(GlcC-6), 106.7(AraC-1), 73.8(AraC-2), 74.4(AraC-3), 69.2 (AraC-4), 67.1(AraC-5).
Y. Tang et al. / Journal of Ethnopharmacology 169 (2015) 18–23
Compound 14: White needle crystals (Methanol), 1H NMR (600 MHz, C5D5N): δ 1.02(3H, s), 1.15(3H, s), 1.28(3H, s), 1.34(3H, s), 1.48(3H, s), 1.88(3H, s), δ 5.85(1H, t, J ¼7.2 Hz), 4.97(1H, d, J ¼7.8Hz), 5.19(1H, d, J ¼6.6 Hz). 13C NMR(150 MHz,C5D5N): δ 39.6 (C-1), 27.1(C-2), 88.7(C-3), 39.7(C-4), 54.8(C-5), 30.1(C-6), 78.2(C7), 49.3(C-8), 52.1(C-9), 37.5(C-10), 23.0(C-11), 28.3(C-12), 44.6(C13), 50.3(C-14), 36.6(C-15), 25.8(C-16), 49.5(C-17), 61.7(C-18), 16.9 (C-19), 74.3(C-20), 26.5(C-21), 41.4(C-22), 23.0(C-23), 125.6(C-24), 136.1(C-25), 68.2(C-26), 14.0(C-27), 27.9(C-28), 16.7(C-29), 17.0(C30), 105.2(GlcC-1), 83.9(Glc-C-2), 78.2(GlcC-3), 71.7(GlcC-4), 78.1 (GlcC-5), 62.9(GlcC-6), 106.7(AraC-1), 73.8(AraC-2), 74.3(AraC-3), 69.2 (AraC-4), 67.1(AraC-5). Compound 15: White powder (Methanol), 1H –NMR(600 MHz, C5D5N): δ 1.03(3H, s), 1.14(3H, s), 1.28(3H, s), 1.34(3H, s), 1.49(3H, s), 1.88(3H, s), 2.02(3H, s), 5.85(1H, t, J ¼7.2 Hz), 4.95(1H, d, J ¼7.8 Hz), 5.23(1H, d, J¼ 7.2 Hz). 13C NMR(150 MHz, C5D5N): δ 39.5(C-1), 27.0(C-2), 88.8(C-3), 39.7(C-4), 54.7(C-5), 30.1(C-6), 78.2 (C-7), 49.4(C-8), 52.1(C-9), 37.4(C-10), 23.0(C-11), 28.2(C-12), 44.6 (C-13), 50.3(C-14), 36.6(C-15), 25.8(C-16), 49.3(C-17), 61.6(C-18), 16.9(C-19), 74.3(C-20), 26.5(C-21), 41.4(C-22), 22.9(C-23), 125.5(C24), 136.1(C-25), 68.1(C-26), 14.0(C-27), 27.9(C-28), 16.6(C-29), 16.9 (C-30), 170.8( CQO), 21.1(CH3), 105.1(GlcC-1), 83.1(Glc-C-2), 78.2 (GlcC-3), 71.7(GlC-4), 78.3(GlcC-5), 62.8(GlcC-6), 106.4(AraC-1), 74.2 (AraC-2), 72.1(AraC-3), 72.3(AraC-4), 64.5(AraC-5).
2.4. Cytotoxic activity 2.4.1. Cell culture BGC-823, HeLa, and MCF-7 cells were grown in RPMI 1640 supplemented with 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES buffer, 1 mM sodium pyruvate and 2 Mm L-glutamine. HT-29 cells were cultured in DMEM supplemented with 4.5 g/L of glucose. All cell culture media contained 10% FBS and 1% penicillin/streptomycin unless otherwise specified. All cell lines were incubated at 37 1C in a humidified atmosphere containing 5% CO2 (Ivanova et al., 2011). 2.4.2. MTT assay The effects of test compounds on human cancer cell growth, expressed as the percentage of cell survival, were determined using the MTT assay (Ivanova et al., 2011). The cells were grown in 96-well plates at 1 104 cells per well and exposed to the test compounds. After incubation for 48 h, 10 μL of the MTT solution (5 mg/mL; Sigma; St. Louis, MO, USA) were added into each well. The plates were incubated for 2–4 h at 37 1C. The supernatant was then removed and the formazan crystals were dissolved with 100 μL of DMSO. The absorbance at 490 nm was recorded using an OPTI max microplate reader. The cell survival percentages were calculated by dividing the mean OD of compound-containing wells by that of DMSO-control wells. Three independent experiments were accomplished to determine the IC50.
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3. Results and discussion All isolated compounds were identified following analysis of their physicochemical and spectroscopic data and by comparison with published data. Thus, 15 compounds isolated from B. paniculatum bulb were identified as isocucurbitacin B(1) (Zheng et al., 2007; Shi et al., 1994), 23,24-dihydroisocucurbitacin B(2) ( Shi et al., 1994), cucurbitacin E(3) (Nobuo et al., 2001), 23,24-dihydrocucur-bitacinE(4) (Shi et al., 1994), tubeimoside I(5) (Kong et al., 1986), tubeimoside III(6) (Kasai et al., 1988), tubeimoside V(7) (Cheng et al., 2006; Tang et al., 2005), dexylosyltubeimoside III(8) (Ma et al., 2006), lobatoside C(9) (Fujioka et al., 1989), tubeimoside A(10), tumeimoside B(11) (Tang et al., 2014), lobatoside A(12) (Fujioka et al., 1989), tubeimoside C(13) (Tang et al., 2014), tubeimoside IV(14) (Liu et al., 2004), 7β,18,20,26tetrahydroxy-(20S)-dammar-24E-en-3-O-α-L-(4-acetyl)arabinopyranosyl-(1-2)-β-D-glucopyranoside(15) (Liu et al., 2004). This is the first report of the isolation of 23,24-dihydroisocucurbitacin B(2), 23,24-dihydrocucur-bitacinE(4) and lobatoside C(9) from B. paniculatum. Cytotoxicity screening of B. paniculatum extracts and isolated compounds are summarized in Tables 1 and 2. As shown in Tables 1 and 2, EtOAC extract and n-butanol extracts of B. paniculatum displayed cytotoxic activity against tested cells. The EtOAC extract had moderately activity with IC50 values of 50.38, 22.81, 28.09, and 43.54 μM against BCG-823, Hela, HT-29 and MCF-7, respectively. Among the compounds isolated from the EtOAC extract, compounds 1–4 exhibited activities with IC50 values range from 0.93 to 34.61 μM against Hela and HT-29, while compounds 1–4 were inactive with IC50 values 4100 μM against BCG-823 and MCF-7 (See Table 2).
Table 2 The cytotoxicity of compounds against cells tested (the unit for IC50 was μM). Sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 5-Fua
IC50 values BCG-823
Hela
HT-29
MCF-7
4 100 4 100 4 100 4 100 14.78 7 2.30 2.747 0.31 12.617 1.05 3.42 7 0.33 9.137 0.38 36.747 1.73 4 100 4 100 4 100 4 100 4 100 18.107 0.73
7.217 0.42 34.617 1.70 4.92 7 0.39 0.93 7 0.05 15.26 7 1.25 3.377 0.42 9.677 0.57 3.16 70.27 10.22 7 0.53 18.117 1.39 4100 4100 4100 4100 4100 11.617 0.52
9.737 0.57 23.117 1.13 7.30 7 0.33 2.63 70.19 13.20 7 0.67 2.167 0.23 2.08 70.17 1.577 0.11 6.617 0.45 9.69 70.42 4100 4100 4100 4100 4100 23.08 70.82
4100 4100 4100 4100 15.647 0.81 1.30 7 0.15 13.32 7 1.26 2.337 0.15 10.82 7 0.47 60.17 74.26 4100 4100 4100 4100 4100 21.337 1.18
Results are expressed as mean 7 SD. a
5-Fu (fluorouracil) as a positive control.
Table 1 The cytotoxicity of extracts against cells tested (the unit for IC50 was μM). Sample
EtOH extract Petroleum ether extract EtOAC extract n-BuOH extract Water extract Results are expressed as mean7 SD.
IC50 values BCG-823
Hela
HT-29
MCF-7
40.36 7 2.50 4 100 50.38 7 3.53 14.017 2.52 4 100
84.767 6.15 4 100 22.81 7 0.92 20.45 7 1.21 4 100
21.38 7 0.86 4100 28.09 7 1.97 5.05 7 0.27 4100
30.82 7 2.14 4100 43.54 7 3.88 10.54 70.56 4100
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Compound 4 was the most active compound with IC50 values of 0.93 and 2.63 μM against Hela and HT-29 cell lines. Compound 4's activities against the two cell lines are 12- and 8-fold that of the positive control drug (5-Fu). The activity against BCG-823 and MCF-7 observed for EtOAc extract was stronger than that of observed for 1–4, suggesting that the activity could either be attributable to compounds acting synergisticall or to other (nonpurified) phytochemical(s). Additionally, the n-butanol extract had stronger activity against BCG-823, Hela, HT29 and MCF-7 than those of EtOAC extract with IC50 values of 14.01, 20.45, 5.05 and 10.54 μM, respectively. Among the compounds isolated from n-butanol extract, compounds 5–9 showed significantly activities with IC50 values range from 1.30 to 15.64 μM, while compounds 12, 14 and 15 had no activity against the four tested cells (IC50 4100 μM). It is worth mention that 6's activities against the four cell lines are 6-, 3-, 10 and 16-fold that of 5-Fu and 8's activities against the four cell lines are 5-, 3-, 14 and 9-fold that of 5-Fu. Structurally, compounds 5–9 belongs to cyclic bisdesmosides with two oligosaccharides flanked on the positions of C-3 and C-28 of a pentacyclic triterpene and bridged with 3-hydroxy-3-methyl glutarate. Consequently, the macrocyclic structure of 5–9 plays an important role in their potential cytotoxicity. Furthermore, comparing the structures and IC50 values of 6 and 8 with 5, 7 and 9 (See Table 2), it can be concluded that 6 and 8 with an additional hydroxyl group attached to C-16 demonstrated high cytotoxicity, suggesting that the hydroxyl group of C-16 enhanced the cytotoxic activity. The result is consistent well with our previous finding (Li et al., 2012). It is reported that when cyclic bisdesmosides converted to simple triterpene bisdesmosides, the cytotoxicity against Hela cell line disappeared. This result suggested that the macrocyclic structure played an important role in the demonstration of potent cytotoxicity. Furthermore, the terminal sugar of the glycosyl moiety bonded to C-3 might affect the cytotoxicity (Fujioka et al., 1996). The structures of lobatoside B and lobatoside C are quite similar, except for the terminal sugar of the glycosyl moiety bonded to C-3. In the literature, lobatoside B, with GI50 values of 1.15, 1.20 and 1.23 against SW-620, SK-MEL-5 and UO-31 cell lines, the terminal sugar is a hexose, while lobatoside C, with GI50 values of 8.91, 4.17 and 6.03 against the three cell lines, the terminal sugar is an arabinose. This observation suggested that the terminal hexose in the C-3 glycosyl moiety might be important to the potency of these compounds (Fujioka et al., 1996). The structure–activity relationship (SAR) of these compounds correlated well with our previous studies of cyclic bisdesmosides on cytotoxic activity (Li et al., 2012). The key SAR of cyclic bisdesmosides can be seen in Fig. 1. Regarding to other compounds (10–15) isolated from B. paniculatum n-butanol extract, tubeimoside A (10) had moderate cytotoxic activity against BCG-823, Hela, HT-29 and MCF-7 with IC50 values of 36.74, 18.11, 9.69 and 60.17, whilst tubeimoside B (11) and the others (12–15) were inactive (IC50 4100 μM). To the best of our knowledge,
Fig. 1. The key SAR of cyclic bisdesmosides.
this is the first report on EtOAC extract of B. paniculatum along with the isolated compound 10, 11, 13 and 15 screening for cytotoxic activity.
4. Conclusion In this study, EtOAC extract of B. paniculatum and isolated pure compounds (1–4) exhibited higher cytotoxic activities against Hela and HT-29 than those of crude extract. The pure compounds (5–10) isolated from B. paniculatum n-butanol extract exhibited higher cytotoxic activities than the crude extract. This behavior might be due to synergism between compounds present in the extract. In China, B. paniculatum has been described as a folk remedy for the treatment of tumors, warts, and detoxication and it has also been applied in the treatment of cancer. The triterpinoid saponins are considered to be the main active constituents. Base on this study, we can conclude that the cytotoxic activity of B. paniculatum is mainly ascribable to the cyclic bisdesmosides with IC50 values range from 1.30 to 15.64 μM. These results provide some scientific evidence to support, to some extent, the ethno-medicinal use of B. paniculatum as traditional anticancer remedies. The cytotoxicity assay used in this study could only provide preliminary data to help identify constituents with potential anticancer properties. Anticancer drugs with low side effects, apoptosisinducing and targeting specificity to the cancer cells are the drugs of choice. Considering the potential of the cyclic bisdesmosides as anticancer agents, a further study on the mechanism of cell death is needed to provide more convincing evidence.
Acknowledgment This work was financially supported by E&T modern center for Natural Products of Liaoning Province of China (No. 2008402021) and the National Natural Science Foundation of China, China (NO. 81273389). We are also grateful to the Analytical Center of Shenyang Pharmaceutical University for identification of the measurements of NMR, IR, HR-TOF-MS. References Cassady, J.M., Suffness, M., 1980. Anticancer agents based on natural products models. Academic Press, New York, pp. 201–269. Cheng, G., Zhang, Y., Zhang, X., Tang, H.F., Cao, W.D., Gao, D.K., Wang, X.L., 2006. Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, functions by inducing apoptosis in human glioblastoma U87MG cells. Bioorg. Med. Chem. Lett. 16, 4575–4580. Chen, W.J., Yu, C., Yang, Z., He, J.L., Yin, J., Liu, H.Z., Liu, H.T., Wang, Y.X., 2012. Tubeimoside I induces G2/M phase arrest and apoptosis in SKOV-3 cells through increase of intracellular Ca2 þ and caspase-dependent signaling pathways. Int. J. Oncol. 40, 535–543. Committee of Pharmacopeia, 2010. Chinese Pharmacopoeia. Vol. 1. Chinese Medicine Science and Technology Press, China p. 155. Fujioka, T., Iwamoto, M., Iwase, Y., Hachiyama, S., Okabe, H., Yamauchi, T., Mihashi, K., 1989. Studies on the constituents of Actinostemma lobatum Maxim.IV. Structures of lobatosides C, D and H, the dicrotalic acid esters of bayogenin bisdesmosides isolated from the herb. Chem. Pharm. Bull. 37, 1770–1775. Fujioka, T., Kashiwada, Y., Okabe, H., Mihashi, K., Lee, K.H., 1996. Antitumor agents 171. cytotoxicities of lobatosides B, C, D, and E, cyclic bisdesmosides isolated from Actinostemma lobatum Maxim. Bioorg. Med. Chem. Lett. 6, 2807–2810. Huang, P., Yu, C., Liu, X.Q., Ding, Y.B., Wang, Y.X., He, J.L., 2011. Cytotoxicity of tubeimoside I in human choriocarcinoma JEG-3 cells by induction of cytochromec release and apoptosis via the mitochondrial-related signaling pathway. Int. J. Mol. Med. 28, 579–587. Ivanova, A., Mikhova, B., Batsalova, T., Dzhambazov, B., Kostova, I., 2011. New furostanol saponins from Smilax aspera L. and their in vitro cytotoxicity. Fitoterapia 82, 282–287. Kasai, R., Miyakoshi, M., Nie, R.L., Zhou, J., Matsumoto, K., Morita, T., Nishi, M., Miyahara, K., Tanaka, O., 1988. Saponins from Bolbostemma Paniculatum: cyclic bisdesmosides, tubeimoside II and III. Phytochemistry 27, 1439–1446.
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