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Identification of compounds with cytotoxic activity from Millettia dorwardi Coll. Et. Hemsl
Phytochemistry Letters 25 (2018) 60–64
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Identification of compounds with cytotoxic activity from Millettia dorwardi Coll. Et. Hemsl
T
Kai Chenb, Huan Tanga, Li Zhenga, Lun Wangb, Linlin Xuea, Ai-hua Penga, Ming-hai Tanga, ⁎ Chaofeng Longc, Xiaoxin Chenc, Hao-yu Yea, , Li-juan Chena a
Lab of Natural Product Drugs, Cancer Center, West China Medical School, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China School of Chemical Engineering, Sichuan University, Chengdu 610041, China c Guangdong Zhongsheng Pharmaceutical Co Ltd, Dongguan 440100, Guangdong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Millettia dorwardi Coll. Et Cytotoxicity Separation Lignan derivatives
Two new lignan derivatives and nine known compounds were isolated from the EtOAc and n-BuOH extracts of Millettia dorwardi Coll. Et. Hemsl. The structures of the compounds were determined by 1D and 2D NMR, CD and HRMS. Then, cytotoxic effects of these compounds were evaluated against five cancer cell lines (HepG2, HCT116, MCF7, Raji and KG-1). Results showed the isolated compounds have no obvious cytotoxic activity, but millepurpan (5) showed moderate growth inhibitory activity against Raij and KG-1 cells, with IC50 values of 36.13 and 30.11 μM, respectively. Further activity evaluation revealed that millepurpan (5) could induce G1 arrest and apoptosis in KG-1 cells. The lignan skeleton was isolated from Millettia genus for the first time and all compounds were firstly reported from Millettia dorwardi Coll. Et.
1. Introduction
2. Results and discussion
The genus Millettia (Leguminosae) is represented by over 200 species and is distributed in tropical Africa, Asia, and Australasia (Thulin, 1983). Modern pharmacological researches have demonstrated that parts of Millettia species were found to have various biological activities such as antitumor (Ngamga et al., 2007), cardiovascular function (Huang et al., 2008), anti-estrogen (Chihiro et al., 2006), antioxidant (Yu et al., 2007), insecticidal (Shin et al., 1942), piscicidal (Singhal et al., 1982) activities due to the presence of flavonoids and its derivatives. In our previous study of Millettia pachycarpa Benth and M. dielsiana Harms, various anti-tumor and anti-inflammatory constituents were discovered and researched (Li et al., 2012; Ye et al., 2012; Wu et al., 2013; Ye et al., 2014). Millettia dorwardi Coll. et Hemsl is one of 50 wild species of Millettia distributed in China, which grows in Yunnan and Guizhou provinces. Our previous phytochemical investigation of nhexane part of ethanol extract from this species led to the isolation of new isoflavone glycoside and eleven known compounds (Chen et al., 2015). In our ongoing research for the discovery of new bioactive constituents from Millettia dorwardi Coll. et Hemsl, two new lignan derivatives were isolated from the vine stems, together with nine known compounds. Herein, we report the isolation and structure determination of these compounds, as well as their cytotoxic activities against human cancer cell lines.
Eleven compounds were isolated from the ethyl acetate (EA) and nBuOH fraction of the 95% ethanol extract of the powdered stems of Millettia dorwardi Coll. et Hemsl. Their structures were identified by spectroscopic analysis including 1D and 2D NMR and HRMS data. In addition to the two new lignan derivatives, nine known compounds were identified as (6S,7R,8R)-7a-[(β-D-glucopyranosyl)oxy]lyoniresinol (3) (Wangteeraprasert and Likhitwitayawuid, 2009), dibenz[b,f] oxepin-1,6diol, 8-methoxy-7-methyl-pacharin (4) (Anjaneyulu et al., 1984), millepurpan (5) (Chihiro et al., 2000), medicarpin (6) (Higgins, 1972), dibutyl phthalate (7) (Tong et al., 2013), adenosine (8) (Reist et al., 1968), uridine (9) (Jones et al., 1970), 2,6,2′,6′-tetramethoxy-4,4′-bis (2,3epoxy-1-hydroxypropyl) biphenyl (10) (Phrutivorapongkul et al., 2003), afromosin glucoside (11) (Eade et al., 1978). The above known compounds were determined and confirmed by comparing their MS, NMR data, and optical rotation with those of literature (Fig. 1). Compound 1 was obtained as brown oil. Its molecular formula was established as C39H48O16 by HRESIMS at m/z 795.2839 ([M + Na]+), suggesting the presence of 16 ° of unsaturations. The 1H and 13C NMR data of compound 1 closely resembled those of 3 (Wangteeraprasert and Likhitwitayawuid, 2009), indicating that 1 also contained the aglycon lyoniresinol connected to a glucose moiety through the C (7a) to C (1”) ether linkage. However, several 13C NMR spectral differences
⁎
Corresponding author. E-mail address: [email protected] (H.-y. Ye).
https://doi.org/10.1016/j.phytol.2018.03.004 Received 14 November 2017; Received in revised form 14 February 2018; Accepted 1 March 2018 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of isolated compounds (1–11) from the vine stems of Millettia dorwardi Coll. Et. Hemsl.
Fig. 2. Key HMBC, COSY and NOESY corrections of compounds 1 and 2.
studies. It is known that for this class of lignans and their glucosides, the sign of the couplets at 287 and 273 nm reflects the orientation of the aryl substituent at C (8) (Ohashi et al., 1994; Sakakibara et al., 1974). In this study, compound 1 showed positive and negative peaks at 287 and 273 nm, respectively (Fig. 3), and was therefore assigned the (6S,7R,8R)-absolute configuration, consistent with the earlier report (Yang et al., 2005). Hence, compound 1 was identified as (6S,7R,8R)6a-[10-propenoic acid, 11-(4”',5”'-dimethoxyphenyl)]-7a-[(β-glucopyranosyl)oxy]lyoniresinol. Compound 2 was obtained as a pale yellow oil. Based on HRESIMS analysis, its molecular formula is C40H50O17. Its 1H and 13C NMR datum (Table 1) were very similar to those for compound 1, with the only difference being due to the replacement of the C-3′′′ hydrogen proton in
between these two compounds were noticed. An obvious structure of caffeic acid was found from the 13C NMR spectrum, a trans double bond (δC 146.48 and 116.41), a carbonyl connected with oxygen (δC 169.20), an aromatic hydrocarbons (δC 128.77, 125.20, 112.96, 152.85, 150.77 and 111.47). The presence of signals for two additional methoxy groups (δC 56.60 and 56.44) both in the 1H and 13C NMR spectra of 1 suggested the presence of two methoxy groups on caffeic acid moiety. Additionally, the structure of caffeic acid could only connect to C-6a (δC 68.56). The NOESY cross peaks obtained for H-C (6), H-C (7), and H-C (8) indicated that the relative configurations at C (6), C (7), and C (8) of compound 1 were identical with those of compound 2, which was consistence with 3 (Fig. 2) (Wangteeraprasert and Likhitwitayawuid, 2009). Conclusive evidence came from the circular dichroism (CD) 61
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Fig. 3. CD curves of compounds 1 (—) and 2 (- - -).
HepG2, MCF7, HCT116, KG-1 and Raji with 5-fluorouracil as the positive control for HepG2 and HCT116, taxol for MCF7, cytarabine for KG-1, and vinblastine for Raji cell (Table 2). Compounds 1-3, 6-11 showed weak inhibitory activity with the inhibitory rate ranging from 0.7 to 62.5% at the concentration of 100 μM. Compound 4 exhibited moderate effects against four cancer cell lines, HepG2, HCT116, Raji and KG-1, with IC50 values of 52.03, 68.89, 40.17 and 61.22 μM, respectively. Compound 5 showed the best cytotoxic activity among the tested compounds, with IC50 value of 38.07, 46.85, 36.13 and 30.11 μM, respectively. To test whether compound 5 induced KG-1 cells apoptosis, we further measured the apoptosis of KG-1 cells by Annexin V/PI staining with flow cytometry. The result proved that the proportions of Annexin V-positive apoptotic cells were increased significantly in KG-1 cells when it was treated with 20 and 40 μM compound 5 for 24 h (Fig. 4A). Moreover, the impact of compound 5 on cell cycle progression was also investigated. As shown in Fig. 4B, G1 population of compound 5-treated KG-1 cells was increased during 24 h posttreatment, suggesting that compound 5 treatment caused accumulation of a G1 population in KG-1 cells.
Table 1 1 H- and 13C NMR date of compounds 1, 2 and 3 (400 MHz, CD3OD). Position
1
2 δC
δH 1′ 2′ 3′ 4′ 5′ 6′ 1 2 3 4 5 6 7 8 6a 7a 9 10 11 1” 2” 3” 4” 5” 6” 1”' 2”' 3” 4”' 5”' 6”' OCH3 OCH3 OCH3 OCH3 OCH3
6.41 (s)
6.41 (s)
6.60 (s) 2H 2.7 (m) 2.02 (m) 2.23(m) 4.38 (d, 5.2) 4.35(m), 4.14(m) 3.92 (m), 3.53 (m) 6.33 7.55 4.26 3.35 3.24 3.24 3.33 3.70 3.86
(d, 16) (d, 16) (d, 7.2) (m) (m) (m) (m) (m), (m)
7.12 (d, 8) 6.96 (d, 8)
7.19 (s) 3.36 (s) 6H 3.78 (s) 3.87 (s) 3.86 (s) 3.85 (s)
139 107 149 134.7 149 107 147.6 139.1 148.8 107.9 33.5 37.9 46.7 42.6 68.6 71.8 169.2 116.4 146.5 104.6 78.1 78 75.2 71.6 62.8 128.8 125.2 113 152.9 150.8 111.5 60.3 56.9 56.6 56.6 56.4
3
δH
δC
6.41 (s)
6.41 (s)
6.60 (s) 2H 2.70 (m) 2.02 (m) 2.21(m) 4.38 (d, 5.2) 4.35 (m), 4.14(m) 3.92 (m), 3.53 (m) 6.42 7.56 4.26 3.35 3.24 3.24 3.33 3.70 3.86
(d, 16) (d, 16) (d, 7.2) (m) (m) (m) (m) (m), (m)
6.90 (s)
6.90 (s) 3.37 (s) 3.85 (s) 6H 3.79 (s) 6H 3.87 (s) 3.82 (s)
139 107 149 134.7 149 107 147.6 139.1 148 107.9 33.5 37.9 46.7 42.6 68.7 71.7 169 118.2 146.4 104.6 78.2 77.9 75.2 71.6 62.8 131.6 106.8 154.8 141.3 154.8 106.8 60.3 61.2 56.9 56.8 56.6
δH
δC
6.42 (s)
6.42 (s)
6.60 (s) 2H 2.26 (m) 1.69 (m) 2.04 (m) 4.42 (d, 6.0) 3.60 (m), 3.51(m) 3.88 (m), 3.45 (m)
138.9 106.9 149 134.5 149 106.9 147.6 139.4 148.6 107.8 33.9 40.6 46.7 42.8 66.2 71.5
3. Experimental 3.1. General experimental procedures
4.28 3.30 3.28 3.23 3.20 3.80 3.65
(d, 7.6) (m) (m) (m) (m) (m), (m)
3.85 (s) 6H 3.74 (s) 3.34 (s)
104.9 78.3 78 75.2 71.7 62.9
Column chromatography was carried out on silica gel 60 (0.2–0.5 mm, 0.040–0.063 mm, Merck, Darmstadt, German), MCI gel CHP 20P (Adsorbent Resins, 75–150 μm, Mitsubishi Chemical, Co. Ltd., Japan) and Sephadex LH-20 (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Medium-pressure liquid chromatography (MPLC) (Büchi, Flawil, Switzerland) was performed with a C-605 pump, a C-615 pump manager, a C-635 UV detector, a C-660 fraction collector and a Sepacore Record 1.0 workstation. HPLC was performed on a Waters Alliance 2695 separations module (Empower pro software) connected to a Waters 2996 photodiode array detection system (190–800 nm) using a Sunfire C18 column (150 mm × 4.6 mm id, 5 μm; Waters, Milford, MA, USA). The MS analyses were performed with a QTOF Premier Mass Spectrometer coupled with an ESI source (Micromass, Simonsway, Manchester, UK). NMR spectroscopy was performed on a Bruker Avance 400 NMR (Bruker Biospin, Rheinstetten, Germany). The circular dichroism spectra was performed on JASCO J1500 (Japan).
60.2 56.9 56.6
compound 1 with a methoxy group in compound 2. This difference was supported by the HMBC correlations from the 2′′′/6′′′ hydrogen proton (δH 6.90, s) to C-15 (δC 146.42), C-4′′′ (δC 141.31) and C-6′′′ (δC 106.81). The absolute configuration of 2 was determined from CD and NOESY date, which was in accordance with 1. (Fig. 2) Thus, compound 2 was identified as (6S,7R,8R)-6a-[10-propenoic acid, 11-(3”',4”',5”'trimethoxyphenyl)]-7a- [(β-glucopyranosyl)oxy]lyoniresinol. These compounds were evaluated for the cytotoxic effects on
3.2. Plant material Vine stems (wet weight 5 kg) of Millettia dorwardi Coll. et Hemsl (Leguminosae) were collected near Manbian village, Mengla county, Yunnan province, P.R. China at an altitude ranging from 800 to 1500 m above mean sea level [coordinates (WGS84): N 21°51′1.97”; E 62
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Table 2 Cytotoxic activity of compounds from Millettia dorwardi Coll. et Hemsl. IC50 (μM) HepG2 4 5 6 7 8 other Taxol 5-fluorouracil cytarabine vinblastine
52.08 ± 38.07 ± 83.22 ± > 100 > 100 > 100 – 37.61 ± – –
HCT116 1.32 0.71 0.82
2.26
68.89 ± 46.85 ± 72.33 ± 54.22 ± > 100
1.21 2.33 0.99 1.52
– 6.20 ± 0.28 – –
Raji
KG-1
MCF7
40.17 ± 0.54 36.13 ± 0.78 70.23 ± 1.05 > 100 > 100
61.22 30.11 61.23 55.33 64.23
– – – 0.09 ± 0.18
– – 0.12 ± 0.02 –
± ± ± ± ±
1.24 1.30 0.86 0.31 1.05
> 100 > 100 > 100 > 100 > 100 0.05 ± 0.01 – – –
compound 7 (20.3 mg). Fr.3–4 (3.4 g) were combined and chromatographed on a silica gel column (200–300 mesh) eluting with PE-EA (40:1 to 1:1, v/v) to yield Fr.s.1–Fr.s.6. Fr.s.2 (213 mg) and Fr.s.4 (172 mg) were further purified by a reverse phase semi-preparative HPLC to yield compounds 4 (6.8 mg), 5 (5.4 mg), 6 (8.2 mg) (MeOHH2O, 75:25, 70:30 and 65:35, v/v, respectively). Fr.9 (5.2 g) was purified by MCI (75–150 μm) eluting with MeOH-water (2:1, 1:1, 1:2, 1:0, v/v) to yield Fr.9.1–9.4. Fr.9.2 (502 mg) was separated by gel permeation chromatography on Sephadex LH-20 (2.5 × 120 cm) with MeOH-water (1:1, v/v) as eluent to afford compounds 1 (7.3 mg), 2 (6.5 mg) and 3 (13.2 mg). Frs. 9.3-9.4 (1.2 g) were combined and purified by semi-preparative HPLC with methanol and water (60:40, v/v) as the eluent, yielding compounds 10 (12.4 mg) and 11 (9.5 mg). Part of n-BuOH extract was subjected to chromatography over MCI eluting with MeOH-water (2:1, 1:1, 1:2, 1:0, v/v) to yield Fr.B.1–4. Fr.B.3 was separated by gel permeation chromatography on Sephadex LH-20 (2.5 × 120 cm) with MeOH-water (1:1, v/v) as eluent to yield Fr.B.3.1–6. Fr.B.3.3 and Fr.B.3.4 were purified by a reverse phase preparative HPLC (MeOH-H2O, 30:70 and 25:75, v/v, respectively), yielding compounds 8 (6.7 mg) and 9 (15.9 mg).
101°15′29.30”]. It was identified by Prof. Kaijiao Jiang, Institute of Materia Medica, Chinese Academy of Medical Science. A voucher specimen (SKLB-201109) was deposited in State Key Laboratory of Biotherapy, Sichuan University. 3.3. Extraction and isolation The air-dried and finely ground stems (3 kg) of Millettia dorwardi Coll. et Hemsl were extracted with 95% ethanol (10 L) at room temperature for three times (10 days each). After concentration in vacuo, the crude (100.6 g) was suspended in H2O (5 L) and then extracted with ethyl acetate (EA) (3 × 5 L) and butyl alcohol (n-BuOH) (3 × 5 L) to obtain an EA extract (46 g) and an n-BuOH extract (25 g). Part of EA extraction was subjected to silica gel column (100–200 mesh) eluting with a petroleum ether (PE)-EA gradient solvent system (1:0 to 0:1, v/ v) and nine fractions were obtained (Fr.1–9) based on their TLC profiles. Fr.1 (2.3 g) was chromatographed on a silica gel column (200–300 mesh) eluting with PE-EA (100:1 to 10:1, v/v) to yield Fr.1.1–1.7. Fr.1.2 (259 mg) was further purified using preparative silica gel column (5–10 μm) with PE-EA (50:1, v/v) as eluent to give
Fig. 4. KG-1 (5 × 105 cells per well) cells were seeded on 6-well plates for 24 h and then incubated with indicated concentrations of compound 5 for 24 h. (A) Cells were stained with Annexin V-FITC/PI before apoptosis analysis by flow cytometry. (B) Cells were stained with PI before cycle analysis by flow cytometry.
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Acknowledgments
The structures of the known compounds were elucidated using 1D and 2D NMR spectra, and MS, and compared with published data.
This project was supported by the National Natural Science Foundation of China (U1402222 and 81402493) and Guangdong Innovative Research Team Program (2011Y073).
3.4. Chemical characterization 3.4.1. Compound 1 Pale brown oily; [α ]20 D = −48.7 (c = 0.04, MeOH)], UV (MeOH) λmax (log ε): 211.5 (4.52), 286.0 (3.83) (nm). HREIMS: m/z 795.2839 ([M + Na]+) (calcd. for C39H48O16, 795.2840); 1H and 13C NMR (see Table 1).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.phytol.2018.03.004. References
3.4.2. Compound 2 Pale yellow oily; [α ]20 D = −49.5 (c = 0.04, MeOH)], UV (MeOH) λmax(log ε): 217.4 (4.43), 290.1 (3.82) (nm). HREIMS: m/z 825.3010 ([M + Na]+) (calcd. for C40H50O17, 825.3004); 1H and 13C NMR (see Table 1).
Anjaneyulu, A.S.R., Reddy, A.V.R., Reddy, D.S.K., Ward, R.S., 1984. Pacharin: a new dibenzo (2, 3-6, 7) oxepin derivative from Bauhinia racemosa lamk. Tetrahedron 40, 4245–4252. Chen, K., Tang, H., Wu, B., Li, S.C., Peng, A., Ye, H., 2015. Phytochemical investigation of Millettia dorwardi coll. et hemsl. Biochem. Syst. Ecol. 60, 24–27. Chihiro, I., Masataka, I., Tan, H.T.W., Tokuda, H., Mou, X.Y., 2000. Anti-tumor-promoting effects of isoflavonoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis. Cancer Lett. 152, 187–192. Chihiro, I., Masataka, I., Minako, K., 2006. Isoflavonoids with antiestrogenic activity from Millettia pachycarpa. J. Nat. Prod. 69, 138–141. Eade, R.A., McDonald, F.J., Pham, H., 1978. C-Glycosylflavonoids. II, the synthesis of 7, 4'-Di-O-methylpuerarin (8-c-(-D-Glucopyranosyl-7, 4'-dimethoxyisoflavone). Aust. J. Chem. 31, 2699–2706. Higgins, V.J., 1972. Role of the phytoalexin medicarpin in three leaf spot diseases of alfalfa. Physiol. Mol. Plant Pathol. 2, 289–300. Huang, R., Zhang, X., Jiang, W., Jiao, Y., 2008. The protection of jade lang umbrella flavonoids on myocardial ischemia-reperfusion injury. J. Chin. Hosp. Pharm. 28, 870–873. Jones, A.J., Grant, D.M., Winkley, M.W., 1970. Carbon-13 magnetic resonance: XVII. Pyrimidine and purine nucleosides. J. Am. Chem. Soc. 92, 4079–4087. Li, X., Wang, X., Ye, H., Peng, A., Chen, L., 2012. An isoflavone, inhibits tumor angiogenesis and human non-small-cell lung cancer xenografts growth through VEGFR2 signaling pathways. Cancer Chemoth. Pharm. 70, 425–437. Ngamga, D., Free, S.N.Y.F., Tane, P., Fomum, Z.T., 2007. Millaurine A, new guanlidine alkaloid from seeds of Millettia laurentii. Fitoterapia 78, 276–277. Ohashi, K., Watanabe, H., Okumura, Y., Uji, T., Kitagawa, I., 1994. Indonesian medicinal plants: XII. four isomeric lignan-Glucosides from the bark of Aegle marmelos (Rutaceae). Chem. Pharm. Bull. 42, 1924–1926. Phrutivorapongkul, A., Lipipun, V., Ruangrungsi, N., Kirtikara, K., Nishikawa, K., Maruyama, S., Watanabe, T., Ishikawa, T., 2003. Studies on the chemical constituents of stem bark of Millettia leucantha: isolation of new chalcones with cytotoxic, antiherpes simplex virus and anti-inflammatory activities. Chem. Pharm. Bull. 51, 187–190. Reist, E.J., Calkins, D.F., Goodman, L., 1968. The synthesis of adenine nucleosides of 3deoxy-3-C-hydroxymethyl-D-erythrofuranose and 2-deoxy-2-C-hydroxymethyl-D-erythrofuranose. J. Am. Chem. Soc. 90, 3852–3857. Sakakibara, J., Ina, H., Yasue, M., 1974. Studies on the constituents of Tripetaleia paniculata Sieb. et Zucc. IV. On the constituents of the wood. (2). structure of lyoniside. Yakugaku Zasshi 94, 1377–1383. Shin, F.C., Sping, L., Yee, S.C., 1942. Insecticidal action of millettia pachycarpa benth. J. Econ. Entomol. 35, 80–82. Singhal, A.K., Sharma, R.P., Baruah, J.N., Govindan, S.V., Herz, W., 1982. Rotenoids from roots of Millettia pachycarpa. Phytochemistry 21, 949–951. Thulin, M., 1983. Opera Bot. 68, 71. Tong, H., Gao, X., Sheng, Z., Li, Q., Li, S., Li, N., Liu, J., 2013. Galactopoietic activity of dibutyl phthalate isolated from Vaccaria segetalis. J. Northeast. Agr. Univ. (English Edition) 20, 28–33. Wangteeraprasert, R., Likhitwitayawuid, K., 2009. Lignans and a sesquiterpene glucoside from Carissa carandas stem. Helv. Chim. Acta 92, 1217–1223. Wu, W., Ye, H., Wan, L., Han, X., Wang, G., Hu, J., 2013. Millepachine, a novel chalcone, induces G2/M arrest by inhibiting CDK1 activity and causing apoptosis via ROSmitochondrial apoptotic pathway in human hepatocarcinoma cells in vitro and in vivo. Carcinogenesis 34, 1636–1643. Yang, Y.L., Chang, F.R., Wu, Y.C., 2005. Squadinorlignoside, a novel 7, 9'-Dinorlignan from the stems of Annona squamosa, helv. Chim. Acta 88, 2731–2737. Ye, H., Fu, A., Wu, W., Li, Y., Wang, G., Tang, M., Li, S., He, S., 2012. Cytotoxic and apoptotic effects of constituents from Millettia pachycarpa Benth. Fitoterapia 83, 1402–1408. Ye, H., Wu, W., Liu, Z., Xie, C., Tang, M., Li, S., Yang, J., Tang, H., 2014. Bioactivityguided isolation of anti-inflammation flavonoids from the stems of Millettia dielsiana Harms. Fitoterapia 95, 154–159. Yu, Y.Y., Zhang, L.H., Cao, S.W., 2007. Investigation on free radical scavenging activity of flavonoid extracts from Millettla nitida Benth. var. hirsutissima. Nat. Prod. Res. Dev. 19, 741–744.
3.5. In vitro determination of cytotoxic activities The isolated constituents were evaluated for their cytotoxicity against hepatocellular carcinoma (HepG2), breast cancer cell line (MCF7), colon cancer (HCT116), acute myelogenous leukemia cell line (KG-1) and lymphoma cell line (Raji). HepG2, MCF7 and HCT116 cell lines were cultured in DMEM medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) in a 5% CO2 humidified atmosphere at 37 °C. KG-1 and Raji cell lines were cultured in RPMI-1640 medium, with addition of the same concentrations of FBS, penicillin and streptomycin as above. Briefly, 5 × 103 cells/well (100 μL) were seeded in 96-well microplates and cultured at 37 °C in a 5% CO2 humidified atmosphere for 24 h. Then, the cells were treated with fresh culture medium containing various concentrations of tested compounds and incubated at 37 °C under a humidified atmosphere of 95% CO2 for another 48 h. After that, the cells (HepG2, MCF7 and HCT116) were added with a 5 mg/mL MTT solution (20 μL) and incubated for 4 h at 37 °C. Then, the supernatant was removed and formazan crystals were dissolved in DMSO (150 μL). The cells (KG-1 and Raji) were added with a 5 mg/mL MTT solution (20 μL) and incubated for 4 h at 37 °C. Afterwards, the cells were added with a 200 mg/mL sodium dodecyl sulfate (SDS) solution (50 μL) and incubated for 12 h at 37 °C. The absorbance of each well was measured at 570 nm by using a microplate reader (SpectraMax M5, Molecular Devices). The IC50 values (concentration that inhibits 50% of cell growth) of tested compounds were calculated using the prism software. Six-well culture plates were used for cell culture. After incubated for 24 h, various concentrations of compound 5 was added to cells for another 24 h. Then cells were collected and then subjected to the Annexin V/PI Apoptosis Detection kit (Invitrogen) for staining according to the manufacturer’s instructions. The stained cells were analyzed by flow cytometry in one hour after staining. For cell cycle analysis, 6-well culture plates were used for cell culture. After incubated for 24 h, various concentrations of compound 5 was added to cells for another 24 h. The alive cells were collected and washed with PBS for two times, then fixed with 75% (v/v) pre-cold ethanol at 4 °C for 12 h. Cells were washed with PBS for three times and then stained with PI staining buffer (PI, 50 mg/mL) for 20 min. The cell cycle were then subjected to flow cytometry to analyze cell cycle. Approximately 30,000 cells were evaluated for each sample. Conflict of interest The authors declare that they have no conflicts of interest.
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Identification of compounds with cytotoxic activity from Millettia dorwardi Coll. Et. Hemsl
T
Kai Chenb, Huan Tanga, Li Zhenga, Lun Wangb, Linlin Xuea, Ai-hua Penga, Ming-hai Tanga, ⁎ Chaofeng Longc, Xiaoxin Chenc, Hao-yu Yea, , Li-juan Chena a
Lab of Natural Product Drugs, Cancer Center, West China Medical School, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China School of Chemical Engineering, Sichuan University, Chengdu 610041, China c Guangdong Zhongsheng Pharmaceutical Co Ltd, Dongguan 440100, Guangdong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Millettia dorwardi Coll. Et Cytotoxicity Separation Lignan derivatives
Two new lignan derivatives and nine known compounds were isolated from the EtOAc and n-BuOH extracts of Millettia dorwardi Coll. Et. Hemsl. The structures of the compounds were determined by 1D and 2D NMR, CD and HRMS. Then, cytotoxic effects of these compounds were evaluated against five cancer cell lines (HepG2, HCT116, MCF7, Raji and KG-1). Results showed the isolated compounds have no obvious cytotoxic activity, but millepurpan (5) showed moderate growth inhibitory activity against Raij and KG-1 cells, with IC50 values of 36.13 and 30.11 μM, respectively. Further activity evaluation revealed that millepurpan (5) could induce G1 arrest and apoptosis in KG-1 cells. The lignan skeleton was isolated from Millettia genus for the first time and all compounds were firstly reported from Millettia dorwardi Coll. Et.
1. Introduction
2. Results and discussion
The genus Millettia (Leguminosae) is represented by over 200 species and is distributed in tropical Africa, Asia, and Australasia (Thulin, 1983). Modern pharmacological researches have demonstrated that parts of Millettia species were found to have various biological activities such as antitumor (Ngamga et al., 2007), cardiovascular function (Huang et al., 2008), anti-estrogen (Chihiro et al., 2006), antioxidant (Yu et al., 2007), insecticidal (Shin et al., 1942), piscicidal (Singhal et al., 1982) activities due to the presence of flavonoids and its derivatives. In our previous study of Millettia pachycarpa Benth and M. dielsiana Harms, various anti-tumor and anti-inflammatory constituents were discovered and researched (Li et al., 2012; Ye et al., 2012; Wu et al., 2013; Ye et al., 2014). Millettia dorwardi Coll. et Hemsl is one of 50 wild species of Millettia distributed in China, which grows in Yunnan and Guizhou provinces. Our previous phytochemical investigation of nhexane part of ethanol extract from this species led to the isolation of new isoflavone glycoside and eleven known compounds (Chen et al., 2015). In our ongoing research for the discovery of new bioactive constituents from Millettia dorwardi Coll. et Hemsl, two new lignan derivatives were isolated from the vine stems, together with nine known compounds. Herein, we report the isolation and structure determination of these compounds, as well as their cytotoxic activities against human cancer cell lines.
Eleven compounds were isolated from the ethyl acetate (EA) and nBuOH fraction of the 95% ethanol extract of the powdered stems of Millettia dorwardi Coll. et Hemsl. Their structures were identified by spectroscopic analysis including 1D and 2D NMR and HRMS data. In addition to the two new lignan derivatives, nine known compounds were identified as (6S,7R,8R)-7a-[(β-D-glucopyranosyl)oxy]lyoniresinol (3) (Wangteeraprasert and Likhitwitayawuid, 2009), dibenz[b,f] oxepin-1,6diol, 8-methoxy-7-methyl-pacharin (4) (Anjaneyulu et al., 1984), millepurpan (5) (Chihiro et al., 2000), medicarpin (6) (Higgins, 1972), dibutyl phthalate (7) (Tong et al., 2013), adenosine (8) (Reist et al., 1968), uridine (9) (Jones et al., 1970), 2,6,2′,6′-tetramethoxy-4,4′-bis (2,3epoxy-1-hydroxypropyl) biphenyl (10) (Phrutivorapongkul et al., 2003), afromosin glucoside (11) (Eade et al., 1978). The above known compounds were determined and confirmed by comparing their MS, NMR data, and optical rotation with those of literature (Fig. 1). Compound 1 was obtained as brown oil. Its molecular formula was established as C39H48O16 by HRESIMS at m/z 795.2839 ([M + Na]+), suggesting the presence of 16 ° of unsaturations. The 1H and 13C NMR data of compound 1 closely resembled those of 3 (Wangteeraprasert and Likhitwitayawuid, 2009), indicating that 1 also contained the aglycon lyoniresinol connected to a glucose moiety through the C (7a) to C (1”) ether linkage. However, several 13C NMR spectral differences
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Corresponding author. E-mail address: [email protected] (H.-y. Ye).
https://doi.org/10.1016/j.phytol.2018.03.004 Received 14 November 2017; Received in revised form 14 February 2018; Accepted 1 March 2018 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of isolated compounds (1–11) from the vine stems of Millettia dorwardi Coll. Et. Hemsl.
Fig. 2. Key HMBC, COSY and NOESY corrections of compounds 1 and 2.
studies. It is known that for this class of lignans and their glucosides, the sign of the couplets at 287 and 273 nm reflects the orientation of the aryl substituent at C (8) (Ohashi et al., 1994; Sakakibara et al., 1974). In this study, compound 1 showed positive and negative peaks at 287 and 273 nm, respectively (Fig. 3), and was therefore assigned the (6S,7R,8R)-absolute configuration, consistent with the earlier report (Yang et al., 2005). Hence, compound 1 was identified as (6S,7R,8R)6a-[10-propenoic acid, 11-(4”',5”'-dimethoxyphenyl)]-7a-[(β-glucopyranosyl)oxy]lyoniresinol. Compound 2 was obtained as a pale yellow oil. Based on HRESIMS analysis, its molecular formula is C40H50O17. Its 1H and 13C NMR datum (Table 1) were very similar to those for compound 1, with the only difference being due to the replacement of the C-3′′′ hydrogen proton in
between these two compounds were noticed. An obvious structure of caffeic acid was found from the 13C NMR spectrum, a trans double bond (δC 146.48 and 116.41), a carbonyl connected with oxygen (δC 169.20), an aromatic hydrocarbons (δC 128.77, 125.20, 112.96, 152.85, 150.77 and 111.47). The presence of signals for two additional methoxy groups (δC 56.60 and 56.44) both in the 1H and 13C NMR spectra of 1 suggested the presence of two methoxy groups on caffeic acid moiety. Additionally, the structure of caffeic acid could only connect to C-6a (δC 68.56). The NOESY cross peaks obtained for H-C (6), H-C (7), and H-C (8) indicated that the relative configurations at C (6), C (7), and C (8) of compound 1 were identical with those of compound 2, which was consistence with 3 (Fig. 2) (Wangteeraprasert and Likhitwitayawuid, 2009). Conclusive evidence came from the circular dichroism (CD) 61
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Fig. 3. CD curves of compounds 1 (—) and 2 (- - -).
HepG2, MCF7, HCT116, KG-1 and Raji with 5-fluorouracil as the positive control for HepG2 and HCT116, taxol for MCF7, cytarabine for KG-1, and vinblastine for Raji cell (Table 2). Compounds 1-3, 6-11 showed weak inhibitory activity with the inhibitory rate ranging from 0.7 to 62.5% at the concentration of 100 μM. Compound 4 exhibited moderate effects against four cancer cell lines, HepG2, HCT116, Raji and KG-1, with IC50 values of 52.03, 68.89, 40.17 and 61.22 μM, respectively. Compound 5 showed the best cytotoxic activity among the tested compounds, with IC50 value of 38.07, 46.85, 36.13 and 30.11 μM, respectively. To test whether compound 5 induced KG-1 cells apoptosis, we further measured the apoptosis of KG-1 cells by Annexin V/PI staining with flow cytometry. The result proved that the proportions of Annexin V-positive apoptotic cells were increased significantly in KG-1 cells when it was treated with 20 and 40 μM compound 5 for 24 h (Fig. 4A). Moreover, the impact of compound 5 on cell cycle progression was also investigated. As shown in Fig. 4B, G1 population of compound 5-treated KG-1 cells was increased during 24 h posttreatment, suggesting that compound 5 treatment caused accumulation of a G1 population in KG-1 cells.
Table 1 1 H- and 13C NMR date of compounds 1, 2 and 3 (400 MHz, CD3OD). Position
1
2 δC
δH 1′ 2′ 3′ 4′ 5′ 6′ 1 2 3 4 5 6 7 8 6a 7a 9 10 11 1” 2” 3” 4” 5” 6” 1”' 2”' 3” 4”' 5”' 6”' OCH3 OCH3 OCH3 OCH3 OCH3
6.41 (s)
6.41 (s)
6.60 (s) 2H 2.7 (m) 2.02 (m) 2.23(m) 4.38 (d, 5.2) 4.35(m), 4.14(m) 3.92 (m), 3.53 (m) 6.33 7.55 4.26 3.35 3.24 3.24 3.33 3.70 3.86
(d, 16) (d, 16) (d, 7.2) (m) (m) (m) (m) (m), (m)
7.12 (d, 8) 6.96 (d, 8)
7.19 (s) 3.36 (s) 6H 3.78 (s) 3.87 (s) 3.86 (s) 3.85 (s)
139 107 149 134.7 149 107 147.6 139.1 148.8 107.9 33.5 37.9 46.7 42.6 68.6 71.8 169.2 116.4 146.5 104.6 78.1 78 75.2 71.6 62.8 128.8 125.2 113 152.9 150.8 111.5 60.3 56.9 56.6 56.6 56.4
3
δH
δC
6.41 (s)
6.41 (s)
6.60 (s) 2H 2.70 (m) 2.02 (m) 2.21(m) 4.38 (d, 5.2) 4.35 (m), 4.14(m) 3.92 (m), 3.53 (m) 6.42 7.56 4.26 3.35 3.24 3.24 3.33 3.70 3.86
(d, 16) (d, 16) (d, 7.2) (m) (m) (m) (m) (m), (m)
6.90 (s)
6.90 (s) 3.37 (s) 3.85 (s) 6H 3.79 (s) 6H 3.87 (s) 3.82 (s)
139 107 149 134.7 149 107 147.6 139.1 148 107.9 33.5 37.9 46.7 42.6 68.7 71.7 169 118.2 146.4 104.6 78.2 77.9 75.2 71.6 62.8 131.6 106.8 154.8 141.3 154.8 106.8 60.3 61.2 56.9 56.8 56.6
δH
δC
6.42 (s)
6.42 (s)
6.60 (s) 2H 2.26 (m) 1.69 (m) 2.04 (m) 4.42 (d, 6.0) 3.60 (m), 3.51(m) 3.88 (m), 3.45 (m)
138.9 106.9 149 134.5 149 106.9 147.6 139.4 148.6 107.8 33.9 40.6 46.7 42.8 66.2 71.5
3. Experimental 3.1. General experimental procedures
4.28 3.30 3.28 3.23 3.20 3.80 3.65
(d, 7.6) (m) (m) (m) (m) (m), (m)
3.85 (s) 6H 3.74 (s) 3.34 (s)
104.9 78.3 78 75.2 71.7 62.9
Column chromatography was carried out on silica gel 60 (0.2–0.5 mm, 0.040–0.063 mm, Merck, Darmstadt, German), MCI gel CHP 20P (Adsorbent Resins, 75–150 μm, Mitsubishi Chemical, Co. Ltd., Japan) and Sephadex LH-20 (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Medium-pressure liquid chromatography (MPLC) (Büchi, Flawil, Switzerland) was performed with a C-605 pump, a C-615 pump manager, a C-635 UV detector, a C-660 fraction collector and a Sepacore Record 1.0 workstation. HPLC was performed on a Waters Alliance 2695 separations module (Empower pro software) connected to a Waters 2996 photodiode array detection system (190–800 nm) using a Sunfire C18 column (150 mm × 4.6 mm id, 5 μm; Waters, Milford, MA, USA). The MS analyses were performed with a QTOF Premier Mass Spectrometer coupled with an ESI source (Micromass, Simonsway, Manchester, UK). NMR spectroscopy was performed on a Bruker Avance 400 NMR (Bruker Biospin, Rheinstetten, Germany). The circular dichroism spectra was performed on JASCO J1500 (Japan).
60.2 56.9 56.6
compound 1 with a methoxy group in compound 2. This difference was supported by the HMBC correlations from the 2′′′/6′′′ hydrogen proton (δH 6.90, s) to C-15 (δC 146.42), C-4′′′ (δC 141.31) and C-6′′′ (δC 106.81). The absolute configuration of 2 was determined from CD and NOESY date, which was in accordance with 1. (Fig. 2) Thus, compound 2 was identified as (6S,7R,8R)-6a-[10-propenoic acid, 11-(3”',4”',5”'trimethoxyphenyl)]-7a- [(β-glucopyranosyl)oxy]lyoniresinol. These compounds were evaluated for the cytotoxic effects on
3.2. Plant material Vine stems (wet weight 5 kg) of Millettia dorwardi Coll. et Hemsl (Leguminosae) were collected near Manbian village, Mengla county, Yunnan province, P.R. China at an altitude ranging from 800 to 1500 m above mean sea level [coordinates (WGS84): N 21°51′1.97”; E 62
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Table 2 Cytotoxic activity of compounds from Millettia dorwardi Coll. et Hemsl. IC50 (μM) HepG2 4 5 6 7 8 other Taxol 5-fluorouracil cytarabine vinblastine
52.08 ± 38.07 ± 83.22 ± > 100 > 100 > 100 – 37.61 ± – –
HCT116 1.32 0.71 0.82
2.26
68.89 ± 46.85 ± 72.33 ± 54.22 ± > 100
1.21 2.33 0.99 1.52
– 6.20 ± 0.28 – –
Raji
KG-1
MCF7
40.17 ± 0.54 36.13 ± 0.78 70.23 ± 1.05 > 100 > 100
61.22 30.11 61.23 55.33 64.23
– – – 0.09 ± 0.18
– – 0.12 ± 0.02 –
± ± ± ± ±
1.24 1.30 0.86 0.31 1.05
> 100 > 100 > 100 > 100 > 100 0.05 ± 0.01 – – –
compound 7 (20.3 mg). Fr.3–4 (3.4 g) were combined and chromatographed on a silica gel column (200–300 mesh) eluting with PE-EA (40:1 to 1:1, v/v) to yield Fr.s.1–Fr.s.6. Fr.s.2 (213 mg) and Fr.s.4 (172 mg) were further purified by a reverse phase semi-preparative HPLC to yield compounds 4 (6.8 mg), 5 (5.4 mg), 6 (8.2 mg) (MeOHH2O, 75:25, 70:30 and 65:35, v/v, respectively). Fr.9 (5.2 g) was purified by MCI (75–150 μm) eluting with MeOH-water (2:1, 1:1, 1:2, 1:0, v/v) to yield Fr.9.1–9.4. Fr.9.2 (502 mg) was separated by gel permeation chromatography on Sephadex LH-20 (2.5 × 120 cm) with MeOH-water (1:1, v/v) as eluent to afford compounds 1 (7.3 mg), 2 (6.5 mg) and 3 (13.2 mg). Frs. 9.3-9.4 (1.2 g) were combined and purified by semi-preparative HPLC with methanol and water (60:40, v/v) as the eluent, yielding compounds 10 (12.4 mg) and 11 (9.5 mg). Part of n-BuOH extract was subjected to chromatography over MCI eluting with MeOH-water (2:1, 1:1, 1:2, 1:0, v/v) to yield Fr.B.1–4. Fr.B.3 was separated by gel permeation chromatography on Sephadex LH-20 (2.5 × 120 cm) with MeOH-water (1:1, v/v) as eluent to yield Fr.B.3.1–6. Fr.B.3.3 and Fr.B.3.4 were purified by a reverse phase preparative HPLC (MeOH-H2O, 30:70 and 25:75, v/v, respectively), yielding compounds 8 (6.7 mg) and 9 (15.9 mg).
101°15′29.30”]. It was identified by Prof. Kaijiao Jiang, Institute of Materia Medica, Chinese Academy of Medical Science. A voucher specimen (SKLB-201109) was deposited in State Key Laboratory of Biotherapy, Sichuan University. 3.3. Extraction and isolation The air-dried and finely ground stems (3 kg) of Millettia dorwardi Coll. et Hemsl were extracted with 95% ethanol (10 L) at room temperature for three times (10 days each). After concentration in vacuo, the crude (100.6 g) was suspended in H2O (5 L) and then extracted with ethyl acetate (EA) (3 × 5 L) and butyl alcohol (n-BuOH) (3 × 5 L) to obtain an EA extract (46 g) and an n-BuOH extract (25 g). Part of EA extraction was subjected to silica gel column (100–200 mesh) eluting with a petroleum ether (PE)-EA gradient solvent system (1:0 to 0:1, v/ v) and nine fractions were obtained (Fr.1–9) based on their TLC profiles. Fr.1 (2.3 g) was chromatographed on a silica gel column (200–300 mesh) eluting with PE-EA (100:1 to 10:1, v/v) to yield Fr.1.1–1.7. Fr.1.2 (259 mg) was further purified using preparative silica gel column (5–10 μm) with PE-EA (50:1, v/v) as eluent to give
Fig. 4. KG-1 (5 × 105 cells per well) cells were seeded on 6-well plates for 24 h and then incubated with indicated concentrations of compound 5 for 24 h. (A) Cells were stained with Annexin V-FITC/PI before apoptosis analysis by flow cytometry. (B) Cells were stained with PI before cycle analysis by flow cytometry.
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Acknowledgments
The structures of the known compounds were elucidated using 1D and 2D NMR spectra, and MS, and compared with published data.
This project was supported by the National Natural Science Foundation of China (U1402222 and 81402493) and Guangdong Innovative Research Team Program (2011Y073).
3.4. Chemical characterization 3.4.1. Compound 1 Pale brown oily; [α ]20 D = −48.7 (c = 0.04, MeOH)], UV (MeOH) λmax (log ε): 211.5 (4.52), 286.0 (3.83) (nm). HREIMS: m/z 795.2839 ([M + Na]+) (calcd. for C39H48O16, 795.2840); 1H and 13C NMR (see Table 1).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.phytol.2018.03.004. References
3.4.2. Compound 2 Pale yellow oily; [α ]20 D = −49.5 (c = 0.04, MeOH)], UV (MeOH) λmax(log ε): 217.4 (4.43), 290.1 (3.82) (nm). HREIMS: m/z 825.3010 ([M + Na]+) (calcd. for C40H50O17, 825.3004); 1H and 13C NMR (see Table 1).
Anjaneyulu, A.S.R., Reddy, A.V.R., Reddy, D.S.K., Ward, R.S., 1984. Pacharin: a new dibenzo (2, 3-6, 7) oxepin derivative from Bauhinia racemosa lamk. Tetrahedron 40, 4245–4252. Chen, K., Tang, H., Wu, B., Li, S.C., Peng, A., Ye, H., 2015. Phytochemical investigation of Millettia dorwardi coll. et hemsl. Biochem. Syst. Ecol. 60, 24–27. Chihiro, I., Masataka, I., Tan, H.T.W., Tokuda, H., Mou, X.Y., 2000. Anti-tumor-promoting effects of isoflavonoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis. Cancer Lett. 152, 187–192. Chihiro, I., Masataka, I., Minako, K., 2006. Isoflavonoids with antiestrogenic activity from Millettia pachycarpa. J. Nat. Prod. 69, 138–141. Eade, R.A., McDonald, F.J., Pham, H., 1978. C-Glycosylflavonoids. II, the synthesis of 7, 4'-Di-O-methylpuerarin (8-c-(-D-Glucopyranosyl-7, 4'-dimethoxyisoflavone). Aust. J. Chem. 31, 2699–2706. Higgins, V.J., 1972. Role of the phytoalexin medicarpin in three leaf spot diseases of alfalfa. Physiol. Mol. Plant Pathol. 2, 289–300. Huang, R., Zhang, X., Jiang, W., Jiao, Y., 2008. The protection of jade lang umbrella flavonoids on myocardial ischemia-reperfusion injury. J. Chin. Hosp. Pharm. 28, 870–873. Jones, A.J., Grant, D.M., Winkley, M.W., 1970. Carbon-13 magnetic resonance: XVII. Pyrimidine and purine nucleosides. J. Am. Chem. Soc. 92, 4079–4087. Li, X., Wang, X., Ye, H., Peng, A., Chen, L., 2012. An isoflavone, inhibits tumor angiogenesis and human non-small-cell lung cancer xenografts growth through VEGFR2 signaling pathways. Cancer Chemoth. Pharm. 70, 425–437. Ngamga, D., Free, S.N.Y.F., Tane, P., Fomum, Z.T., 2007. Millaurine A, new guanlidine alkaloid from seeds of Millettia laurentii. Fitoterapia 78, 276–277. Ohashi, K., Watanabe, H., Okumura, Y., Uji, T., Kitagawa, I., 1994. Indonesian medicinal plants: XII. four isomeric lignan-Glucosides from the bark of Aegle marmelos (Rutaceae). Chem. Pharm. Bull. 42, 1924–1926. Phrutivorapongkul, A., Lipipun, V., Ruangrungsi, N., Kirtikara, K., Nishikawa, K., Maruyama, S., Watanabe, T., Ishikawa, T., 2003. Studies on the chemical constituents of stem bark of Millettia leucantha: isolation of new chalcones with cytotoxic, antiherpes simplex virus and anti-inflammatory activities. Chem. Pharm. Bull. 51, 187–190. Reist, E.J., Calkins, D.F., Goodman, L., 1968. The synthesis of adenine nucleosides of 3deoxy-3-C-hydroxymethyl-D-erythrofuranose and 2-deoxy-2-C-hydroxymethyl-D-erythrofuranose. J. Am. Chem. Soc. 90, 3852–3857. Sakakibara, J., Ina, H., Yasue, M., 1974. Studies on the constituents of Tripetaleia paniculata Sieb. et Zucc. IV. On the constituents of the wood. (2). structure of lyoniside. Yakugaku Zasshi 94, 1377–1383. Shin, F.C., Sping, L., Yee, S.C., 1942. Insecticidal action of millettia pachycarpa benth. J. Econ. Entomol. 35, 80–82. Singhal, A.K., Sharma, R.P., Baruah, J.N., Govindan, S.V., Herz, W., 1982. Rotenoids from roots of Millettia pachycarpa. Phytochemistry 21, 949–951. Thulin, M., 1983. Opera Bot. 68, 71. Tong, H., Gao, X., Sheng, Z., Li, Q., Li, S., Li, N., Liu, J., 2013. Galactopoietic activity of dibutyl phthalate isolated from Vaccaria segetalis. J. Northeast. Agr. Univ. (English Edition) 20, 28–33. Wangteeraprasert, R., Likhitwitayawuid, K., 2009. Lignans and a sesquiterpene glucoside from Carissa carandas stem. Helv. Chim. Acta 92, 1217–1223. Wu, W., Ye, H., Wan, L., Han, X., Wang, G., Hu, J., 2013. Millepachine, a novel chalcone, induces G2/M arrest by inhibiting CDK1 activity and causing apoptosis via ROSmitochondrial apoptotic pathway in human hepatocarcinoma cells in vitro and in vivo. Carcinogenesis 34, 1636–1643. Yang, Y.L., Chang, F.R., Wu, Y.C., 2005. Squadinorlignoside, a novel 7, 9'-Dinorlignan from the stems of Annona squamosa, helv. Chim. Acta 88, 2731–2737. Ye, H., Fu, A., Wu, W., Li, Y., Wang, G., Tang, M., Li, S., He, S., 2012. Cytotoxic and apoptotic effects of constituents from Millettia pachycarpa Benth. Fitoterapia 83, 1402–1408. Ye, H., Wu, W., Liu, Z., Xie, C., Tang, M., Li, S., Yang, J., Tang, H., 2014. Bioactivityguided isolation of anti-inflammation flavonoids from the stems of Millettia dielsiana Harms. Fitoterapia 95, 154–159. Yu, Y.Y., Zhang, L.H., Cao, S.W., 2007. Investigation on free radical scavenging activity of flavonoid extracts from Millettla nitida Benth. var. hirsutissima. Nat. Prod. Res. Dev. 19, 741–744.
3.5. In vitro determination of cytotoxic activities The isolated constituents were evaluated for their cytotoxicity against hepatocellular carcinoma (HepG2), breast cancer cell line (MCF7), colon cancer (HCT116), acute myelogenous leukemia cell line (KG-1) and lymphoma cell line (Raji). HepG2, MCF7 and HCT116 cell lines were cultured in DMEM medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) in a 5% CO2 humidified atmosphere at 37 °C. KG-1 and Raji cell lines were cultured in RPMI-1640 medium, with addition of the same concentrations of FBS, penicillin and streptomycin as above. Briefly, 5 × 103 cells/well (100 μL) were seeded in 96-well microplates and cultured at 37 °C in a 5% CO2 humidified atmosphere for 24 h. Then, the cells were treated with fresh culture medium containing various concentrations of tested compounds and incubated at 37 °C under a humidified atmosphere of 95% CO2 for another 48 h. After that, the cells (HepG2, MCF7 and HCT116) were added with a 5 mg/mL MTT solution (20 μL) and incubated for 4 h at 37 °C. Then, the supernatant was removed and formazan crystals were dissolved in DMSO (150 μL). The cells (KG-1 and Raji) were added with a 5 mg/mL MTT solution (20 μL) and incubated for 4 h at 37 °C. Afterwards, the cells were added with a 200 mg/mL sodium dodecyl sulfate (SDS) solution (50 μL) and incubated for 12 h at 37 °C. The absorbance of each well was measured at 570 nm by using a microplate reader (SpectraMax M5, Molecular Devices). The IC50 values (concentration that inhibits 50% of cell growth) of tested compounds were calculated using the prism software. Six-well culture plates were used for cell culture. After incubated for 24 h, various concentrations of compound 5 was added to cells for another 24 h. Then cells were collected and then subjected to the Annexin V/PI Apoptosis Detection kit (Invitrogen) for staining according to the manufacturer’s instructions. The stained cells were analyzed by flow cytometry in one hour after staining. For cell cycle analysis, 6-well culture plates were used for cell culture. After incubated for 24 h, various concentrations of compound 5 was added to cells for another 24 h. The alive cells were collected and washed with PBS for two times, then fixed with 75% (v/v) pre-cold ethanol at 4 °C for 12 h. Cells were washed with PBS for three times and then stained with PI staining buffer (PI, 50 mg/mL) for 20 min. The cell cycle were then subjected to flow cytometry to analyze cell cycle. Approximately 30,000 cells were evaluated for each sample. Conflict of interest The authors declare that they have no conflicts of interest.
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