Chinese Journal of Natural Chinese Journal of Natural Medicines 2012, 10(3): 0222−0225 Medicines

doi: 10.3724/SP.J.1009.2012.00222

A new prenylated chalcone from the seeds of pachycarpa

SU Xue-Hui, LI Cong-Ying, ZHONG Yu-Jiao, YUAN Zhi-Peng, LI Yan-Fang*, LIANG Bing

Department of Pharmaceutics & Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, Available online 20 May 2012

[ABSTRACT] AIM: To investigate the chemical constituents from the seeds of Millettia pachycarpa. METHODS: The compounds were isolated and purified by repeated column chromatography. Their structures were elucidated on the basis of spectroscopic analyses, and their cytotoxic activities against human tumor cell lines (7860, A549, A2780, Hela, K562) and murine tumor cell lines (LL/2, B16) were evaluated by MTT assay. RESULTS: Four were obtained and their structures were identified as 3-hydroxy-4-methoxylonchocarpin (1), 4-methoxylonchocarpin (2), isobavachromene (3), and dorspoinsettifolin (4). CONCLUSION: Compound 1 is a new prenylated chalcone, and compounds 2–4 were isolated from this for the first time. Compounds 1–4 selec- tively showed moderate inhibition against one or more of the tested cell lines. [KEY WORDS] Millettia pachycarpa; Flavonoids; Cytotoxic activities [CLC Number] R284; R965 [Document code] A [Article ID] 1672-3651(2012)03-0222-04

investigation of bioactive compounds from this has led 1 Introduction to the isolation of a new chalcone, 3-hydroxy-4-methoxy- The genus Millettia (Leguminosae) includes about 200 lonchocarpin (1) and three known compounds (2–4), whose distributed in the tropical and subtropical regions of structural elucidation and cytotoxicity are reported herein. [1] the world, with about 46 species in many parts of China . 2 Results and Discussion Great interest has been generated in this genus because of the abundance of their isoflavonoids [2]. Millettia pachycarpa 2.1 Chemistry Benth (Houguojixueteng in Chinese), is mainly distributed in Extensive chromatographic purification of the extract of Sichuan, Yunnan, Guangdong, Guangxi, and Guizhou prov- the seeds of Millettia pachycarpa led to the isolation of a new inces in China and has been used as an anthelminthic, a chalcone, (1), along with three known compounds, including medication capable of causing the evacuation of parasitic 4-methoxylonchocarpin (2) [7] isobavachromene (3) [17] and [3] intestinal worms . Previous phytochemical studies on this dorspoinsettifolin (4) [18], which were readily identified by plant have led to the identification of flavonoids, , comparison of spectroscopic and mass-spectrometric data [4-11] and terpenoids . Some of these substances exhibited an- with those reported in the literature. [12-13] tiestrogenic activities . Compound 1, obtained as yellow needles, showed the [M In continuation of our study of cytotoxic secondary me- + H]+ at m/z 353.138 6 in the HR-ESI-MS, which corre- tabolites of this plant, we previously reported preparative sponds to C21H21O5 (calcd. 353.138 9). It was clear from the isolation and purification of three rotenoids and one isofla- NMR spectra that 1 was a chalcone. The IR absorption at 1 vone (barbigerone) by high-speed counter-current chroma- 650 cm-l indicated that 1 was a 2'-hydroxychalcone. The tography (HSCCC) [14]. In addition, barbigerone induces highly deshielded proton signal at δ 13.78 and the chemical apoptosis in murine lung-cancer cells via the mitochondrial shift of the carbonyl function at δ 191.9 were noted as further apoptotic pathway by bioactivity investigation [15-16]. Further evidence for the chelated hydroxyl and conjugated carbonyl

moieties, respectively. 1H NMR of 1 (Table 1) revealed nine

[Received on] 10-Oct.-2011 proton resonance signals from δ 5–8, two of which were as- [*Corresponding author] LI Yan-Fang: Associate Prof.: Tel/Fax: signable to the pyran group (see below). Two of the proton 86-28-85405221; E-mail: [email protected] These authors have no any conflict of interest to declare. signals form an AB system at δ 7.42 and 7.80 (d, J = 15.2 Hz),

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Table 1 1H (400 MHz) and 13C NMR (100 MHz) data for the large coupling constant indicating the trans geometry of a compound 1 in CDCl3 (J in Hz) double bond. Thus an ABX system at δ 7.27 (d, J = 2.0 Hz),

Position δ H δ C δ 7.13 (1H, dd, J = 8.4, 2.0 Hz) and δ 6.87 (1H, d, J = 8.4 Hz) 1 128.5 s located in ring B could all be observed. The remaining two proton signals forming an AX system at δ 6.38 and δ 7.72 2 7.27 (d, J = 2.0) 113.0 d were assigned to H-5′ and H-6′, respectively, because of the 3 145.9 s downfield chemical shift of the latter. 4 148.8 s The signals that could be assigned to the 2, 2-dimethylp- 5 7.13 (dd, J = 8.4, 2.0 ) 110.5 d yran group were as follows: δ 1.47 and δ 1.47 (both 3H s, 6 6.87 (d, J = 8.4) 122.9 d two of CH3), δ 5.59 (d, J = 10.0 Hz) and 6.75 (d, J = 10.0 Hz). α 7.80 (d, J = 15.2) 118.4 d The proton spectrum of 1 also showed a methoxyl group at δ β 7.42 (d, J = 15.2) 144.2 d 3.94. The foregoing data are consistent with the structure of 1. 1 13 β′ 191.9 s The H and C NMR spectroscopic data of 1 were very close to those of 3-methoxy-4-hydroxylonchocarpin, reported from 1′ 109.4 s Cube´ Resin Insecticide [19]. The placement of the methoxyl 2′ 160.9 s group at position 4 rather than position 3 was based on corre- 3′ 114.1 s lation of H-5 at δ 6.87 with methoxyl proton δ 3.94 in 4′ 159.7 s NOESY spectrum. The 13C NMR signals (Table 1) were fully 5′ 6.38 (d, J = 8.8) 108.2 d assigned on the basis of DEPT spectra, HSQC and HMBC 6′ 7.72 (d, J = 8.8) 130.8 d experiments. 4″ 6.75 (d, J = 10.0) 115.9 d 2.2 Biological studies Four compounds 1–4 were tested for their cytotoxicities 5″ 5.59 (d, J = 10.0) 128.0 d in vitro against human tumor cell lines (7860, A549, A2780, 6″ 77.8 s Hela, K562) and murine tumor cell lines (LL/2, B16) by the 7″ 1.47 (s) 28.4 q standard MTT with cisplatin as positive control. As shown in 8″ 1.47 (s) 28.4 q Table 2, compound 1 demonstrated potent inhibition against OMe 3.94 (s) 58.0 q K562 and moderate cytotoxicity against A2780, B16, and OH-6′ 13.78 (s) LL/2 cancer cell lines, while 2 exhibited weak toxicity against K562, Hela and LL/2 cancer cell lines. Compound 3 OH-3 5.69 (s) showed moderate cytotoxicity against

Fig. 1 Structure of compounds 1–4 and selected HMBC, NOESY correlations of 1

Table 2 Cytotoxic activities of compounds 1–4 (n = 3, x ± s )

–1 Compounds IC50 /(μg ·mL ) 7860 A549 A2780 B16 Hela K562 LL/2 1 > 40 n.t. 21.4 ± 0.5 24.5 ± 0.3 > 40 2.4 ± 0.6 13.8 ± 0.9 2 > 40 n.t. > 40 > 40 23.4 ± 0.8 18.9 ± 0.4 21.7 ± 0.3 3 20.4 ± 0.3 27.1 ± 0.1 18.2 ± 0.2 21.0 ± 0.7 17.5 ± 0.3 15.4 ± 0.4 19.9 ± 0.4 4 > 40 n.t. > 40 > 40 > 40 22.7 ± 0.5 23.6 ± 0.5 a) Cisplatin 6.7 ± 1.1 9.5 ± 0.5 10.2 ± 1.3 5.2 ± 0.7 4.8 ± 0.7 4.5 ± 0.2 0.54 ± 0.3 a) Positive control n. t.: not tested

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all tested cell lines. Compound 4 also inhibited the prolifera- UV (MeOH): 247 (3.96), 281 (3.99) nm. IR (KBr) υ 3 450 tion of cell lines K562 and LL/2. (br s, OH), 1 608 (C=O), 1 583, 1 510, 1 487, 1 286, 1 236, 1 120, 975, 800 cm–1; 1H and 13C NMR see Table 1. 3 Experimental + + HR-ESI-MS m/z 353.138 6 [M + H] (C22H25O4 , calcd. 3.1 General experimental procedures 353.138 9).

TLC: silica gel GF254 (Qingdao Marine Chemical Fac- 4-Methoxylonchocarpin (2) Yellow needles, mp 1 tory, China); Column chromatography (CC): silica gel (SiO2; 149–152 °C. H NMR (400 MHz, CDCl3): 13.79 (1H, br s, 200–300 and 100–200 mesh, Qingdao Marine Chemical -OH), 7.85 (1H, d, J = 15.2 Hz, H-α), 7.72 (1H, d, J = 9.6 Hz, Factory, China); mp: Kofler melting point apparatus (uncor- H-2΄), 7.61 (2H, d, J = 8.4 Hz, H-2), 7.44 (1H, d, J = 15.2 Hz, rected); IR Spectra: Nicolet FT-IR 200 SXV spectrometer; in H-β), 6.94 (2H, d, J = 8.8 Hz, H-3), 6.75 (1H, d, J = 10 Hz, cm-1; 1H-, and 13C NMR (DEPT) and 2D-NMR spectra: Var- H-4΄΄), 6.38 (1H, d, J = 9.2 Hz, H-3΄), 5.59 (1H, d, J = 10 Hz, H-3΄΄), 3.86 (3H, s, -OCH ), 1.47 (6H, s, CH × 2); 13C NMR ian INOVA 400/54 spectrometer; δ in ppm rel. to Me4Si as 3 3 internal standard, J in Hz. HR-ESI-MS: quadrupole time of + DEPT (100 MHz, CDCl3): 191.9 (s, C=O), 161.8 (s, C-4΄), flight (Q-TOF) premier spectrometer coupled with an ESI 160.9 (s, C-2΄), 159.9 (s, C-4), 144.1 (d, C-β), 130.5 (d, C-6΄), source (Micromass, Simonsway, Manchester, UK). MCI gel 130.3 (d, C-2, 6), 128.1(d, C-2΄΄), 127.5 (s, C-1), 117.8 (d, CHP 20P (Mistubishi Kasei Industry Co. Ltd., Japan) and C-α), 115.9 (d, C-11΄΄), 114.4 (d, C-3, 5), 114.1 (s, C-1΄), Sephadex LH-20 (Merck, Germany) were used. 109.4 (s, C-3΄), 108.2 (d, C-5΄), 77.7 (s, C-3΄΄), 55.4 (q, 3.2 Plant material OCH3), 28.3 (q, CH3 × 2); HR-ESI-MS m/z 337.181 0 [M + + + The seeds of Millettia pachycarpa Benth (Thunb.) Bl. H] (C22H25O3 , calcd. 337.180 3). were purchased at Huashen Co. Ltd., China, in August 2006 Isobavachromene (3) Yellow needles, mp 199–202 °C. 1 and identified by Dr. LI Yan-Fang. A voucher specimen (No. H NMR (400 MHz, DMSO-d6): 14.1 (1H, br s, 6΄-OH), 10.1 P00013) of the plant has been deposited at the Department of (1H, s, 4-OH), 8.19 (1H, d, J = 9.2 Hz, ), 7.80 (4H, d, J = 7.6 Pharmaceutics & Bioengineering, Sichuan University, Hz,), 6.85 (2H, d, J = 8.8 Hz, H-3, 5), 6.64 (1H, d, J =10 Hz, Chengdu, China. H-4΄΄), 6.43 (1H, d, J = 8.8 Hz, H-3΄), 5.78 (1H, d, J =10 Hz, 13 3.3 Extraction and isolation H-3΄΄), 1.43 (6H, s, CH3 × 2); C NMR + DEPT (50.0 MHz, The seeds of M. pachycarpa (5.0 kg) were powdered and DMSO-d6): 192.3 (s, C=O), 160.6 (s, C-4΄), 160.2 (s, C-6΄), extracted three times at reflux with 95% EtOH (each time for 159.2 (s, C-4), 145.2 (d, C-β), 132.1 (d, C-2΄), 131.6 (d, 3 hours). After concentration in vacuum, the extract was sus- C-2,6), 128.9 (d, C-3΄΄), 125.8 (s, C-1), 117.2 (d, C-α), 116.0 (d, C-3,5), 115.1 (d, C-4΄΄), 113.9 (s, C-5΄), 108.6 (s, C-1΄), pended in water and partitioned with petroleum ether and 108.1 (d, C-3΄), 28.3 (q, CH × 2); HR-ESI-MS m/z 321.113 5 EtOAc of equal volume five times successively. Both the 3 [M – H]– (C H O , calcd. 321.112 7). petroleum ether and EtOAc solution were evaporated to pro- 20 17 4 Dorspoinsettifolin (4) White needles, mp 158–160 °C. vide 43.5 g petroleum ether extract and 137 g EtOAc extract. 1H NMR (400 MHz, CDCl ): 7.74 (1H, d, J = 8.8 Hz, H-5), The petroleum ether extract (43.5 g) was chromatogra- 3 7.42 (2H, d, J = 8.4 Hz, H-2΄, 6΄), 6.95 (2H, d, J = 8.8 Hz, phed on silica gel (200 g, 350 mm × 60 mm) with light pe- H-3΄, 5΄), 6.62 (1H, d, J = 10 Hz, H-11), 6.49 (1H, d, J = 8.4 troleum-EtOAc mixture of increasing polarity (19 : 1, 9 : 1, 7 : Hz, H-6), 5.56 (1H, d, J = 10 Hz, H-12), 5.4 (1H, dd, J = 2.4, 1, 4 : 1, 2 : 1, 1 : 1, 1 : 2, 1 : 4, 0 : 1) (38 frs of 200 mL). A 13.2 Hz, H-2), 3.84 (3H, s,-OCH ), 3.01 (1H, dd, J = 13.2, total 38 fractions (ca. 200 mL each) were collected. Fractions 3 16.8 Hz, H-3α), 2.80 (1H,dd, J = 13.2, 2.8 Hz, H-3b), 1.44 4–6 provide 2 (578 mg). Fractions 7–10 yielded 4 (157 mg). (3H, s, -CH ), 1.46 (3H, s, -CH ); 13C NMR+DEPT (100.0 Fractions 18–20 were rechromatographed over MCI gel 3 3 MHz, CDCl3): 190.8 (s, C-O), 159.8 (s, C-7), 159.6 (s, C-4΄), column (300 mm × 35 mm) and eluted with acetone/H O [65 : 2 157.7 (s, C-9), 131.0 (s, C-1΄), 128.8 (d, C-5), 127.7 (d, C-2΄), 35, 70 : 30, 75 : 25 and 80 : 20 (V/V), 100 mL each] to afford 127.5 (d, C-6΄), 115.9 (d, C-11), 114.7 (d, C-10), 114.4 (d, 3 (82 mg). Fractions 21–23 was rechromatographed over C-3΄), 111.1 (d, C-6), 109.4 (s, C-8), 79.5 (s, C-2), 77.5 (s, MCI gel column (300 mm × 35 mm) and eluted with ace- C-13), 55.3 (q, OCH3), 44.4 (s, C-3), 28.4 (q, C-14), 28.1 (q, tone/H2O [60 : 40, 65 : 35, 70 : 30, and 75 : 25 (V/V), 100 mL + + C-15); HR-ESI-MS m/z 337.144 3 [M + H] (C21H21O4 , each] to afford 62 subfractions. Subfractions 31–40, was calcd. 337.144 0). further subjected to CC over Sephadex LH-20 (700 mm × 20 mm) eluted with CH2Cl2-MeOH (1 : 1 ) to give 1 (13 mg). 3.4 Biological assay Acknowledgements [20] This was performed as in literature . Financial support by the ChengDa Scholarships of China 4 Identification for SU Xue-Hui and NMR measurements afforded by the Centre of Testing & Analysis (Sichuan University) are grate- 3-Hydroxy-4-methoxylonchocarpin (1) Yellow solid, fully acknowledged.

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厚果鸡血藤种子中的一个新异戊烯基查耳酮

苏雪会, 李聪颖, 钟玉蛟, 袁志鹏, 李延芳*, 梁 冰

四川大学化学工程学院制药与生物工程系, 成都 610065

【摘 要】 目的：研究厚果鸡血藤种子的化学成分。方法：采用硅胶、MCI 等多种层析柱分离手段．运用 NMR 和 MS 等 波谱技术鉴定化合物的结构。结果：从厚果鸡血藤种子中分离并鉴定了 4 个黄酮类化合物：3-hydroxy-4-methoxylonchocarpin (1), 4-methoxylonchocarpin (2), isobavachromene (3), dorspoinsettifolin (4)。结论：化合物 1 为新的查耳酮, 化合物 2–4 为首次从该属植 物中分离得到; 化合物 1–4 对测试的部分细胞株显示了中等的抑制活性。 【关键词】 厚果鸡血藤; 黄酮; 细胞毒活性

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