中草药  2015, Vol. 46 Issue (24): 3623-3629
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夏枯草的化学成分及其三萜成分的抗肿瘤活性研究
柏玉冰1,3, 李春2, 周亚敏1,3, 皮胜玲1,3, 夏伯候1,3, 李亚梅1,3, 林丽美1,3 , 廖端芳1,3    
1. 湖南中医药大学, 湖南 长沙 410208;
2. 中国中医科学院中药研究所, 北京 100700;
3. 湖南省中药不良成分快速检测及脱除工程技术研究中心, 湖南 长沙 410208
摘要: 目的 研究夏枯草Prunella vulgaris 果穗的化学成分及其抗乳腺癌细胞活性。方法 运用硅胶、ODS、Sephadex LH-20等色谱方法进行分离纯化,根据理化性质和波谱数据并参考文献鉴定化合物的结构;通过MTT法,对化合物体外抗乳腺癌细胞MCF-7、MDA-MB-231的活性进行筛选。结果 从夏枯草果穗中分离得到14个化合物,分别鉴定为寡肽(1)、5α,8α-过氧麦角-6,22-二烯-3β-醇(2)、β-香树素(3)、白桦脂酸(4)、3-羟基-11-烯-11,12-脱氢-28,13-乌苏酸内酯(5)、大戟醇(6)、2α,3α,24-三羟基-12-烯-28-齐墩果酸(7)、candelabrone 12-methyl ether(8)、cyclopentaneacetic acid(9)、2α-羟基熊果酸(10)、α-菠菜甾醇(11)、齐墩果酸(12)、熊果酸(13)、β-谷甾醇(14)。结论 化合物25689均为首次从该属植物中分离得到;化合物1013对乳腺癌细胞MCF-7、MDA-MB-231及正常乳腺细胞MCF-10A均具有明显抑制作用;化合物4对乳腺癌细胞MCF-7、MDA-MB-231具有明显抑制作用,而对正常乳腺细胞MCF-10A抑制不明显,能选择性地抑制肿瘤细胞。
关键词: 夏枯草     三萜     3-羟基-11-烯-11,12-脱氢-28,13-乌苏酸内酯     大戟醇     白桦脂酸     乳腺癌细胞    
Chemical constituents of triterpenoids from Prunella vulgaris and their antitumor activities
BAI Yu-bing1,3, LI Chun2, ZHOU Ya-min1,3, PI Sheng-ling1,3, XIA Bo-hou1,3, LI Ya-mei1,3, LIN Li-mei1,3, LIAO Duan-fang1,3    
1. Hunan University of Chinese Medicine, Changsha 410208, China;
2. Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;
3. Hunan Engineering Center for Rapid Test and Removal of Toxic and Harmful Substances in Chinese Medicine, Changsha 410208, China
Abstract: Objective To study the chemical constituents from Prunella vulgaris and their antitumor activities. Methods Silica gel, reverse-phase octadecylsilyl (ODS), Sephadex LH-20 chromatographic methods, and HPLC were applied to isolating and purifying compounds. MS and NMR spectroscopic methods were used to determine their structures. Furthermore, the cytotoxicity of these chemical components for MCF-7, MDA-MB-231, and MCF-10A cell lines was measured by MTT method. Results Forteen compounds were isolated from the fruits of P. vulgaris and their structures were identified as: autantiamide acetate (1), 5α,8α-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol (2), β-amyrin (3), betulic acid (4), 3-hydroxy-11-en-11,12-dehydrogenation-28,13-oic acid lactone (5), eburicol (6), 2α,3α,24-trihydroxyolean-12-en-28-oic acid (7), candelabrone 12-methyl ether (8), cyclopentaneacetic acid (9), 2α,3β-dihydroxyursa-12-en-28-oic acid (10), α-spinasterol (11), oleanolic acid (12), ursolic acid (13), and β-sitosterol (14). Conclusion Compounds 2, 5, 6, 8, and 9 are isolated from the genus of Prunella L. for the first time. The results of cytotoxic assay indicate that compounds 10 and 13 can obviously inhibit the activity of MCF-7, MDA-MB-231, and the normal cell lines MCF-10A. Compound 4 can selectly inhibit the activity of MCF-7 and MDA-MB-231 while show no effect on the normal cell lines MCF-10A.
Key words: Prunella vulgaris L.     triterpenoids     3-hydroxy-11-en-11,12-dehydrogenation-28,13-oic acid lactone     eburicol     betulic acid     breast cancer cell    

夏枯草Prunellae Spica为唇形科(Labiatae)夏枯草属Prunella L. 植物夏枯草Prunella vulgaris L. 的干燥果穗,味辛、苦,性寒,入肝、胆经。具有清火、明目、散结、消肿的功能,用于目赤肿痛、目珠夜育、头痛眩晕、瘰疬、瘿瘤、乳痈肿痛[1]。研究表明,夏枯草中主要含有三萜及其皂苷、酚酸、甾醇及其苷、黄酮及其苷、有机酸、挥发油及糖类等成分[2, 3]。药效关系研究表明,夏枯草中抗病毒活性的主要贡献成分为鞣质和多糖[4, 5],抗炎活性成分为迷迭香酸[6],抗氧化活性成分为酚酸和多糖[7, 8],而其抗肿瘤主要贡献成分为三萜类,如齐墩果酸可以明显抑制肺腺癌SPC-A-1细胞的生长[9];熊果酸对A549、SK-OV-3、SK-MEL-2和HCT15细胞均有温和的细胞毒性作用[10];3α,19α,24-三羟基-12-烯-28-乌苏酸和3β,16α,24-三羟基-12-烯-28-齐墩果酸对人肺癌细胞A549瘤株有明显的抑制作用[11];2α,3α-三羟基-12-烯-28-乌苏酸还可诱导人急性白血病Jurkat T细胞的凋亡[12]。早在《神农本草经》中就有记载“夏枯草,味苦、辛,主寒热瘰疬,头创,破症,散结···”,现代研究表明,夏枯草对于乳腺癌的治疗也有效[13]。但是,目前其抗乳腺癌研究方面的药效物质基础研究尚不够明确。因此,本课题组对其进一步的物质基础研究和选择抗肿瘤主体成分三萜类进行抗乳腺癌细胞活性筛选研究。本实验从夏枯草果穗中分离得到了14个化合物,分别鉴定为寡肽(autantiamide acetate,1)、5α,8α-过氧麦角-6,22-二烯-3β-醇 [5α,8α-epidioxy-(22E,24R)-ergosta-6,22- dien-3β-ol,2]、β-香树素(β-amyrin,3)、白桦脂酸(betulic acid,4)、3-羟基-11-烯-11,12-脱氢-28,13-乌苏酸内酯(3-hydroxy-11-en-11,12-dehydrogenation- 28,13-oic acid lactone,5)、大戟醇(eburicol,6)、2α,3α,24-三羟基-12-烯-28-齐墩果酸(2α,3α,24- trihydroxyolean-12-en-28-oic acid,7)、candelabrone 12-methyl ether(8)、cyclopentaneacetic acid(9)、2α-羟基熊果酸(2α,3β-dihydroxyursa-12-en-28-oic acid,10)、α-菠菜甾醇(α-spinasterol,11)、齐墩果酸(oleanolic acid,12)、熊果酸(ursolic acid,13)、β-谷甾醇(14)。其中化合物2、5689为首次从该属植物中分离得到。并通过MTT法,对三萜类化合物3457101213进行体外抗乳腺癌细胞的活性进行筛选。化合物1013能明显抑制MCF-7、MDA-MB-231、MCF-10A细胞增殖;化合物4能明显抑制乳腺癌细胞MCF-7、MDA-MB-231的增殖,而对MCF-10A抑制不明显,能选择性地抑制肿瘤细胞。

1 仪器与材料

GT-2127QT型超声仪(昆山市超声仪器有限公司),RE-52A型旋转蒸发仪(上海亚荣生化仪器厂),Bruker AV-400型核磁共振光谱仪(德国Bruker公司),Agilent 1100 Series LC/MSD Trap质谱仪(美国Agilent公司),X-4型显微熔点仪(杭州科博仪器有限公司),凝胶Sephadex LH-20(美国Pharmacia公司),薄层色谱硅胶预涂板(青岛海洋化工厂),ODS(50 mm,YMC,日本东京Merck公司),柱色谱硅胶(80~100、200~300、300~400目,青岛海洋化工厂),溶剂均为分析纯或化学纯。CJ-1F型医用净化工作台(苏州冯氏实验动物设备有限公司),台式低速离心机(湖南凯达实业发展有限公司),DMIL型倒置显微镜(Leica),3111型CO2培养箱(Thermo),MR-96A酶标仪(深圳迈瑞生物医疗电子股份有限公司)。

化合物3457101213(实验室自制),阳性对照品顺铂(批号130922,齐鲁制药有限公司),高糖DMEM培养基(HyClone,NXJ0709),胎牛血清(HyClone,NXC0582),噻唑蓝(Sigma,MKBH7489V),二甲基亚砜(国药集团化学试剂有限公司,批号20120705),胰蛋白酶(北京鼎国昌盛生物技术有限公司,批号04D10151)。夏枯草植物样本于2013年5月采于安徽夏枯草GAP基地,经湖南中医药大学刘塔斯教授和龚力民讲师共同鉴定为唇形科夏枯草属植物夏枯草Prunella vulgaris L.。

人乳腺癌细胞MCF-7、MDA-MB-231购自于中国科学院上海细胞库,人正常乳腺细胞MCF-10A购自上海复祥生物科技有限公司。

2 提取与分离

夏枯草果穗90 kg粉碎后经甲醇超声提取3次,每次40 min,合并提取液,减压浓缩得总浸膏2.13 kg,甲醇溶解,经硅胶柱色谱,依次用石油醚、醋酸乙酯、甲醇洗脱,减压浓缩得各部分浸膏。

取醋酸乙酯部分(880 g),经硅胶柱色谱(200~300目),用洗脱剂石油醚-醋酸乙酯(10∶1、5∶1、2∶1)梯度洗脱,得馏份Fr. 1~7。Fr. 2经硅胶柱色谱(200~300目)分离,石油醚-醋酸乙酯9∶1洗脱,得12个馏份,馏份3~5重结晶得化合物3(830 mg),馏份9~11重结晶得化合物6(27 mg);Fr. 4经硅胶柱色谱(200~300目)分离,石油醚-醋酸乙酯7∶1洗脱,得27个馏份,馏份11~15减压浓缩后得化合物11(2.56 g),馏份19~22减压浓缩后得化合物4(520 mg);Fr. 5经硅胶柱色谱(200~300目)分离,经二氯甲烷-丙酮100∶1洗脱,得152个馏份,馏份22~28析出晶体14(57 mg),馏份55~71析出晶体12(1.62 g),馏份80~102经硅胶柱色谱(200~300目)分离,石油醚-醋酸乙酯3∶1,得到化合物5(236 mg),馏份112~120析出化合物13(1.73 g),馏份137~140经Sephadex LH-20纯化得到化合物8(7 mg)。Fr. 6经硅胶柱色谱及ODS柱色谱分离,得55个馏份,馏份22~27得化合物2(375 mg),馏份35~40得化合物1(211 mg)。

取甲醇部位73 g,经硅胶柱色谱(200~300目)分离,用石油醚-醋酸乙酯(3∶1、1∶1、0∶1)梯度洗脱,得到组分Fr. 1~6。Fr. 3经硅胶及ODS柱色谱分离,得78个馏分,馏分23~31得化合物10(76 mg),馏分35~44得化合物9(10 mg);Fr. 5经硅胶柱色谱分离,二氯甲烷-甲醇40∶1洗脱,得到化合物7(360 mg)。

3 结构鉴定

化合物1:白色粉末(甲醇),紫外254 nm下呈暗斑。1H-NMR (400 MHz, CDCl3) δ: 4.77 (1H, d, J = 5.6 Hz, H-2), 3.21 (1H, dd, J = 13.6, 5.6 Hz, H-3a), 3.05 (1H, dd, J = 13.6, 5.6 Hz, H-3b), 7.25 (5H, m, H-5, 9), 7.72 (2H, d, J = 8.4 Hz, H-3′, 7′), 7.44 (2H, t, J = 7.6 Hz, H-4′, 6′), 7.53 (1H, t, J = 7.4 Hz, H-5′), 4.35 (1H, m, H-1″), 2.75 (2H, m, H-2″), 7.07 (2H, m, H-4″, 8″), 7.15 (2H, m, H-5″, 7″), 3.82 (1H, dd, J = 11.4, 4.2 Hz, H-9″), 3.93 (1H, dd, J = 11.4, 5.0 Hz, H-9″), 2.02 (3H, s, -COCH3), 5.99 (1H, d, J = 8.4 Hz, α-NH), 6.76 (1H, d, J = 6.4 Hz, β-NH);13C-NMR (100 MHz, CDCl3) δ: 170.2 (C-1), 55.0 (C-2), 38.4 (C-3), 136.6 (C-4), 128.5 (C-5, 9), 129.3 (C-6, 8), 127.1 (C-7), 167.1 (C-1′), 133.6 (C-2′), 128.6 (C-3′, 7′), 127.0 (C-4′, 6′), 131.9 (C-5′), 49.4 (C-1″), 37.4 (C-2″), 136.7 (C-3″), 128.7 (C-4″, 8″), 129.1 (C-5″, 7″), 126.7 (C-6″), 64.5 (C-9″), 170.7 (-CO-), 20.8 (-CH3)。以上数据与文献报道一致[14],故鉴定化合物1为寡肽。

化合物2:无色针晶(石油醚-丙酮),1%香草醛浓硫酸溶液显色呈墨绿色。1H-NMR (400 MHz, CDCl3) δ: 3.97 (1H, m, H-3), 6.25 (1H, d, J = 8.2 Hz, H-6), 6.51 (1H, d, J = 8.0 Hz, H-7), 0.81 (3H, s, H-18), 0.88 (3H, s, H-19), 0.99 (3H, d, J = 6.8 Hz, H-21), 5.14 (1H, dd, J = 15.6, 8.0 Hz, H-22), 5.22 (1H, dd, J = 15.2, 7.2 Hz, H-23), 0.81 (3H, d, J = 6.4 Hz, H-26), 0.83 (3H, d, J = 6.4 Hz, H-27), 0.90 (3H, d, J = 6.8 Hz, H-28);13C-NMR (100 MHz, CDCl3) δ: 34.6 (C-1), 30.1 (C-2), 66.4 (C-3), 37.0 (C-4), 82.1 (C-5), 135.4 (C-6), 130.7 (C-7), 79.4 (C-8), 51.0 (C-9), 36.9 (C-10), 23.4 (C-11), 39.3 (C-12), 44.5 (C-13), 51.6 (C-14), 20.6 (C-15), 28.6 (C-16), 56.1 (C-17), 12.8 (C-18), 18.2 (C-19), 39.7 (C-20), 20.9 (C-21), 135.2 (C-22), 132.3 (C-23), 42.7 (C-24), 33.0 (C-25), 19.6 (C-26), 19.9 (C-27), 17.5 (C-28)。上述数据与文献报道一致[15],故鉴定化合物2为5α,8α-过氧麦角-6,22-二烯-3β-醇。

化合物3:白色针状结晶(醋酸乙酯),1%香草醛浓硫酸溶液显色呈红色。1H-NMR (400 MHz, CDCl3) δ: 5.18 (1H, t, J = 3.2 Hz, H-12), 3.22 (1H, dd, J = 10.8, 4.8 Hz, H-3), 1.13 (3H, s, H-27), 1.00 (3H, s, H-23), 0.98 (3H, s, H-25), 0.94 (3H, s, H-24), 0.87 (3H, s, H-29), 0.87 (3H, s, H-30), 0.83 (3H, s, H-28), 0.79 (3H, s, H-26);13C-NMR (100 MHz, CDCl3) δ: 38.6 (C-1), 27.2 (C-2), 79.0 (C-3), 39.8 (C-4), 55.2 (C-5), 18.4 (C-6), 32.6 (C-7), 38.8 (C-8), 47.6 (C-9), 36.9 (C-10), 23.5 (C-11), 121.7 (C-12), 145.2 (C-13), 41.7 (C-14), 26.1 (C-15), 26.9 (C-16), 32.5 (C-17), 47.2 (C-18), 46.8 (C-19), 31.1 (C-20), 34.7 (C-21), 37.1 (C-22), 28.1 (C-23), 15.5 (C-24), 15.6 (C-25), 16.8 (C-26), 26.0 (C-27), 28.4 (C-28), 33.3 (C-29), 23.7 (C-30)。上述数据与文献报道基本一致[16],故鉴定化合物3为β-香树素。

化合物4:白色果冻状结晶(氯仿),1%香草醛浓硫酸溶液显色呈红色。ESI-MS m/z: 455 [M-H], 457 [M+H]+1H-NMR (400 MHz, DMSO-d6) δ: 4.69 (1H, s, H-29), 4.56 (1H, s, H-29), 3.16 (1H, d, J = 5.2 Hz, H-3), 1.64 (3H, s, 30-Me), 0.93, 0.87, 0.87, 0.76, 0.65 (3H×5, s, 27, 25, 26, 24, 23-Me);13C-NMR (100 MHz, DMSO-d6) δ: 38.5 (C-1), 27.4 (C-2), 77.0 (C-3), 38.8 (C-4), 55.2 (C-5), 18.2 (C-6), 34.2 (C-7), 40.5 (C-8), 50.2 (C-9), 36.6 (C-10), 20.7 (C-11), 25.6 (C-12), 37.9 (C-13), 42.3 (C-14), 29.5 (C-15), 32.0 (C-16), 55.7 (C-17), 46.9 (C-18), 48.8 (C-19), 150.6 (C-20), 30.4 (C-21), 37.0 (C-22), 28.4 (C-23), 16.0 (C-24), 16.2 (C-25), 16.1 (C-26), 14.7 (C-27), 177.5 (C-28), 109.9 (C-29), 19.2 (C-30)。以上数据与文献报道基本一致[17],故鉴定化合物4为白桦脂酸。

化合物5:白色粉末(甲醇),1%香草醛浓硫酸溶液显色呈红色。ESI-MS m/z: 931 [2M+Na]+1H-NMR (400 MHz, CDCl3) δ: 5.96 (1H, d, J = 10.0 Hz, H-12), 5.30 (1H, dd, J = 10.4, 2.4 Hz, H-11), 3.22 (1H, dd, J = 11.0, 5.0 Hz, H-3), 2.13 (1H, d, J = 5.6, H-18), 1.16 (3H, s, 27-Me), 1.05 (3H, s, 26-Me), 0.99 (3H, s, 25-Me), 0.99 (3H, d, J = 6.0 Hz, 29-Me), 0.93 (3H, d, J = 5.6 Hz, 30-Me), 0.91 (3H, s, 23-Me), 0.78 (3H, s, 24-Me);13C-NMR (100 MHz, CDCl3) δ: 38.2 (C-1), 27.0 (C-2), 78.8 (C-3), 38.9 (C-4), 54.7 (C-5), 17.7 (C-6), 31.2 (C-7), 41.9 (C-8), 53.0 (C-9), 36.3 (C-10), 128.8 (C-11), 133.5 (C-12), 89.7 (C-13), 41.6 (C-14), 25.5 (C-15), 22.8 (C-16), 45.1 (C-17), 60.5 (C-18), 40.2 (C-19), 38.1 (C-20), 30.8 (C-21), 31.3 (C-22), 27.7 (C-23), 14.9 (C-24), 19.1 (C-25), 18.9 (C-26), 16.1 (C-27), 180.0 (C-28), 17.9 (C-29), 17.8 (C-30)。以上数据与文献报道基本一致[18],故鉴定化合物5为3-羟基-11-烯-11,12-脱氢-28,13-乌苏酸内酯。

化合物6:白色粉末(醋酸乙酯),1%香草醛浓硫酸溶液显色呈红色。1H-NMR (400 MHz, CDCl3) δ: 4.72 (1H, brs, H-28a), 4.66 (1H, brs, H-28b), 3.10 (1H, m, H-3), 1.03 (3H, d, J = 7.2 Hz, H-26), 1.02 (3H, d, J = 7.2 Hz, H-27), 1.01 (3H, s, H-29), 0.97 (3H, s, H-19), 0.93 (3H, d, J = 6.4 Hz, H-21), 0.89 (6H, s, H-30, 31), 0.71 (3H, s, H-18);13C-NMR (100 MHz, CDCl3) δ: 35.0 (C-1), 27.3 (C-2), 76.5 (C-3), 39.2 (C-4), 50.4 (C-5), 17.9 (C-6), 28.2 (C-7), 133.2 (C-8), 134.6 (C-9), 36.5 (C-10), 20.7 (C-11), 25.5 (C-12), 31.1 (C-13), 47.0 (C-14), 31.0 (C-15), 30.7 (C-16), 50.4 (C-17), 15.7 (C-18), 18.3 (C-19), 35.0 (C-20), 18.7 (C-21), 33.8 (C-22), 31.2 (C-23), 156.9 (C-24), 31.3 (C-25), 21.8 (C-26), 22.0 (C-27), 105.9 (C-28), 28.1 (C-29), 15.0 (C-30), 24.4 (C-31)。以上数据与文献报道基本一致[19],故鉴定化合物6为大戟醇。

化合物7:白色粉末(甲醇),1%香草醛浓硫酸溶液显色呈蓝色。ESI-MS m/z: 487 [M-H]1H-NMR (400 MHz, DMSO-d6) δ: 5.17 (1H, brs, H-12), 4.23 (1H, m, H-2), 3.91 (1H, d, J = 3.6 Hz, H-3), 3.71 (1H, brs, H-24b), 3.54 (1H, brs, H-24a), 2.74 (1H, dd, J = 3.6, 13.2 Hz, H-18), 1.09 (3H, s, H-27), 0.94 (3H, s, H-23), 0.88 (6H, s, H-30, 25), 0.85 (3H, s, H-29), 0.68 (3H, s, H-26);13C-NMR (100 MHz, DMSO-d6) δ: 41.6 (C-1), 64.9 (C-2), 72.8 (C-3), 41.6 (C-4), 48.5 (C-5), 18.2 (C-6), 33.0 (C-7), 39.2 (C-8), 47.3 (C-9), 37.9 (C-10), 23.4 (C-11), 121.7 (C-12), 144.1 (C-13), 42.0 (C-14), 27.3 (C-15), 22.8 (C-16), 45.9 (C-17), 41.0 (C-18), 45.7 (C-19), 30.7 (C-20), 33.6 (C-21), 32.3 (C-22), 22.0 (C-23), 64.0 (C-24), 17.0 (C-25), 16.7 (C-26), 25.9 (C-27), 178.8 (C-28), 33.1 (C-29), 23.6 (C-30)。以上数据与文献报道基本一致[20],故鉴定化合物7为2α,3α,24-三羟基-12-烯-28-齐墩果酸。

化合物8:黄色针状晶体(甲醇),紫外254 nm下呈暗斑,365 nm下呈亮黄色荧光,1%香草醛浓硫酸溶液显色呈黄色。1H-NMR (400 MHz, CDCl3) δ: 1.99 (1H, dt, J = 14.4, 8.6 Hz, H-1α), 3.37 (1H, dt, J = 14.4, 6.8 Hz, H-1β), 2.62 (2H, m, H-2), 2.41 (1H, dd, J = 14.4, 2.8 Hz, H-5α), 2.55 (1H, dd, J = 16.4, 3.0 Hz, H-6α), 2.74 (1H, dd, J = 16.4, 14.8 Hz, H-6β), 3.32 (1H, m, H-15), 1.40 (3H, d, J = 7.2 Hz, H-16), 1.39 (3H, d, J = 7.2 Hz, H-17), 1.17 (3H, s, H-18), 1.17 (3H, s, H-19), 1.44 (3H, s, H-20), 3.81 (3H, s, OMe), 13.13 (1H, s, 14-OH), 5.72 (1H, s, 11-OH);13C-NMR (100 MHz, CDCl3) δ: 36.2 (C-1), 34.5 (C-2), 215.7 (C-3), 46.9 (C-4), 48.9 (C-5), 35.5 (C-6), 204.3 (C-7), 112.0 (C-8), 138.9 (C-9), 38.9 (C-10), 133.3 (C-11), 152.4 (C-12), 126.8 (C-13), 158.1 (C-14), 26.1 (C-15), 20.3 (C-16), 20.3 (C-17), 27.0 (C-18), 20.9 (C-19), 17.5 (C-20), 62.2 (-OCH3)。以上数据与文献对照基本一致[21],故鉴定化合物8为candelabrone 12-methyl ether。

化合物9:黄色油状物,1%香草醛浓硫酸溶液显色呈橘红色。ESI-MS m/z: 225 [M-H], 227 [M+H]+1H-NMR (400 MHz, CD3OD) δ: 5.46 (1H, m, H-3′), 5.44 (1H, m, H-2′), 3.55 (2H, t, J = 6.8 Hz, H-5′a), 2.59 (1H, dd, J = 14.4, 4.0 Hz, H-2″b), 2.37 (2H, t, J = 6.0 Hz, H-1′), 2.31 (2H, m, H-4′), 2.30 (1H, m, H-1), 2.24 (1H, m, H-5b), 2.08 (1H, m, H-4a), 1.99 (1H, m, H-2), 1.55 (1H, m, H-5a);13C-NMR (100 MHz, CD3OD) δ: 40.7 (C-1), 56.3 (C-2), 223.6 (C-3), 39.6 (C-4), 29.2 (C-5), 27.1 (C-1′), 129.5 (C-2′), 130.1 (C-3′), 32.6 (C-4′), 63.4 (C-5′), 181.3 (C-1″), 40.8 (C-2″)。以上数据与文献报道基本一致[22],故鉴定化合物9为cyclopentaneacetic acid。

化合物10:白色粉末(甲醇),1%香草醛浓硫酸溶液显色呈蓝色。1H-NMR (400 MHz, DMSO-d6) δ: 4.72 (1H, brs, H-28a), 4.66 (1H, brs, H-28b), 1.04 (3H, d, J = 11.6 Hz, H-18), 1.02 (3H, s, 27-Me), 1.01 (3H, m, 23, 25, 30-Me), 1.00 (3H, d, J = 6.0 Hz, 29-Me), 0.97 (3H, s, 24-Me), 0.93 (3H, s, 26-Me), 0.87 (3H, s, 26-Me), 0.87 (3H, s, 26-Me), 0.71 (3H, s, 26-Me);13C-NMR (100 MHz, DMSO-d6) δ: 47.3 (C-1), 67.4 (C-2), 82.5 (C-3), 38, 7 (C-4), 55.0 (C-5), 18.3 (C-6), 32.9 (C-7), 38.7 (C-8), 47.2 (C-9), 37.8 (C-10), 23.2 (C-11), 124.8 (C-12), 138.5 (C-13), 42.0 (C-14), 27.8 (C-15), 24.1 (C-16), 47.1 (C-17), 52.6 (C-18), 38.8 (C-19), 38.7 (C-20), 30.5 (C-21), 36.6 (C-22), 29.1 (C-23), 17.2 (C-24), 16.7 (C-25), 17.5 (C-26), 23.5 (C-27), 178.5 (C-28), 17.3 (C-29), 21.4 (C-30)。以上数据与文献报道基本一致[19],故鉴定化合物10为2α-羟基熊果酸。

化合物11:白色针晶(石油醚-醋酸乙酯),1%香草醛浓硫酸溶液显色呈紫红色。ESI-MS m/z: 435 [M+Na]+1H-NMR (400 MHz, CDCl3) δ: 5.15 (2H, dd, J = 14.8, 8.8 Hz, H-22, 23), 5.02 (1H, dd, J = 14.8, 8.6 Hz, H-7), 3.60 (1H, m, H-3), 1.55 (1H, s, OH), 1.02 (3H, d, J = 6.8 Hz, H-21), 0.85 (1H, d, J = 5.6 Hz, H-29), 0.82~0.80 (6H, m, H-26, 27), 0.80 (3H, s, H-19), 0.56 (3H, s, H-18);13C-NMR (100 MHz, CDCl3) δ: 37.1 (C-1), 31.4 (C-2), 71.0 (C-3), 37.9 (C-4), 40.2 (C-5), 29.6 (C-6), 117.4 (C-7), 139.5 (C-8), 49.4 (C-9), 34.2 (C-10), 21.5 (C-11), 39.4 (C-12), 43.2 (C-13), 55.0 (C-14), 23.0 (C-15), 28.5 (C-16), 55.9 (C-17), 12.0 (C-18), 13.0 (C-19), 40.8 (C-20), 21.3 (C-21), 138.1 (C-22), 129.4 (C-23), 51.2 (C-24), 31.8 (C-25), 19.0 (C-26), 21.0 (C-27), 25.4 (C-28), 12.2 (C-29)。以上数据与文献报道基本一致[23],故鉴定化合物11为α-菠甾醇。

化合物12:白色针状结晶(氯仿),1%香草醛浓硫酸溶液显色呈紫红色。ESI-MS m/z: 455 [M-H], 457 [M+H]+1H-NMR (400 MHz, CHCl3) δ: 5.28 (1H, brs, H-12), 3.22 (1H, dd, J = 11.0, 5.5 Hz, H-3), 2.82 (1H, dd, J = 13.6, 4.0 Hz, H-18), 1.13 (3H, s, 27-Me), 0.98 (3H, s, 23-Me), 0.93, 0.91, 0.90, 0.77, 0.75 (3H×5, s, 29, 25, 26, 24-Me);13C-NMR (100 MHz, CHCl3) δ: 38.4 (C-1), 27.1 (C-2), 79.0 (C-3), 38.7 (C-4), 55.2 (C-5), 18.3 (C-6), 32.4 (C-7), 39.3 (C-8), 47.6 (C-9), 37.1 (C-10), 23.4 (C-11), 122.6 (C-12), 143.6 (C-13), 41.6 (C-14), 27.8 (C-15), 23.6 (C-16), 46.5 (C-17), 40.9 (C-18), 45.9 (C-19), 30.7 (C-20), 33.8 (C-21), 32.6 (C-22), 28.1 (C-23), 15.5 (C-24), 15.3 (C-25), 17.1 (C-26), 25.9 (C-27), 183.4 (C-28), 22.9 (C-29), 33.0 (C-30)。以上数据与文献报道基本一致[24],故鉴定化合物12为齐墩果酸。

化合物13:白色粉末(氯仿),1%香草醛浓硫酸溶液显色呈红色。ESI-MS m/z: 455 [M-H], 457 [M+H]+1H-NMR (400 MHz, DMSO-d6) δ: 5.13 (1H, brs, H-12), 3.00 (1H, m, H-3), 2.11 (1H, d, J = 11.6 Hz, H-18), 1.04 (3H, s, H-27), 0.90 (3H, s, H-29, 23), 0.87 (3H, s, H-24), 0.81 (3H, s, H-30), 0.75 (3H, s, H-26), 0.68 (3H, s, H-25);13C-NMR (100 MHz, DMSO-d6) δ: 38.5 (C-1), 24.1 (C-2), 77.1 (C-3), 36.8 (C-4), 55.0 (C-5), 18.3 (C-6), 33.0 (C-7), 38.8 (C-8), 47.1 (C-9), 36.8 (C-10), 21.3 (C-11), 124.8 (C-12), 138.4 (C-13), 41.9 (C-14), 27.2 (C-15), 23.1 (C-16), 47.3 (C-17), 52.6 (C-18), 38.7 (C-19), 38.6 (C-20), 30.4 (C-21), 36.6 (C-22), 28.5 (C-23), 16.3 (C-24), 15.5 (C-25), 17.2 (C-26), 23.5 (C-27), 178.5 (C-28), 17.2 (C-29), 21.0 (C-30)。以上数据与文献报道基本一致[25],故鉴定化合物13为熊果酸。

化合物14:白色针晶(氯仿),mp 140~141 ℃。薄层色谱展开后以1%香草醛浓硫酸显紫红色,与β-谷甾醇对照品共薄层显相同斑点,混合熔点不下降,确认化合物14为β-谷甾醇。

4 活性筛选

MCF-7、MDA-MB-231、MCF-10A细胞株在37 ℃、5% CO2的饱和湿度环境下,含10%胎牛血清的高糖DMEM完全培养基中培养、传代。接种时用0.25%胰蛋白酶消化液消化,加细胞培养基吹打成均匀细胞悬液,将细胞制成2×104/mL的细胞悬液,每孔100 μL接种于96孔板中。称取一定量的样品,用DMSO完全培养基配制成100 mmol/L的母液,用DMEM配制成200 mmol/L的最高浓度工作液,然后依次配制成60、20、6、2、0.6 μmol/L的工作液。顺铂用DMEM完全培养基配制成浓度为50、15、5、1.5、0.75、0.15 μg/mL的工作液。待细胞贴壁后每孔加入含不同浓度样品或顺铂工作液100 μL,每个浓度设3个复孔。分别于加药后72 h后,每孔加MTT溶液(5 mg/mL)20 μL,4 h后将上清液吸出,每孔加DMSO 150 μL,震荡待结晶完全溶解,酶标仪492 nm测定吸光度(A)值。重复实验3次,按公式计算各组的生长抑制率(IR,IR=1-A药物组/A对照组)。用SPSS 20.0软件计算IC50值。结果见表 1

表 1 三萜化合物对乳腺癌细胞及正常乳腺细胞增殖抑制作用 Table 1 Inhibition of tested triterpenoids on proliferation of MCF-7, MDA-MB-231, and MCF-10A cells
5 结果及讨论

本实验从夏枯草中分离得到14个化合物,其中化合物25689为首次从夏枯草中分离,同时也是首次从该属植物中分离,丰富了其化学成分。并且本实验分离得到的化合物中主要为三萜,且又首次分离得到3-羟基-11-烯-11,12-脱氢-28,13-乌苏酸内酯和大戟醇2个三萜,进一步佐证三萜为夏枯草中主要成分。

活性结果显示,化合物1013能明显抑制MCF-7、MDA-MB-231、MCF-10A细胞增殖;化合物4对乳腺癌细胞MCF-7、MDA-MB-231显示出明显抑制活性,而对MCF-10A抑制不明显,能选择性地抑制肿瘤细胞。中医上,夏枯草是一味软坚散结的传统中药,临床上也广泛应用于乳腺增生、乳腺癌等。而现代药理研究表明,夏枯草三萜是抗肿瘤的主要成分。因此,对夏枯草中分离得到的主体成分三萜进行的抗乳腺癌的活性研究结果可能从物质基础上为揭示其发挥上述临床作用和药理活性提供依据。同时,本研究为进一步研究夏枯草的物质基础奠定了基础。

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