CN103191140A - 大黄素-8-O-β-D-葡萄糖苷的抗肿瘤活性及其新用途 - Google Patents
大黄素-8-O-β-D-葡萄糖苷的抗肿瘤活性及其新用途 Download PDFInfo
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Abstract
本发明涉及大黄素-8-O-β-D-葡萄糖苷的抗肿瘤活性及其在制备肿瘤治疗和预防药物或食品中的新用途。在一定浓度范围内，大黄素-8-O-β-D-葡萄糖苷可以选择性地杀伤人肝癌细胞、结直肠癌细胞等多种癌细胞，而不影响人体正常细胞的活力和功能。因此可被用于制备抗肿瘤药物和预防肿瘤发生的药物或食品。
Description
发明领域
本发明涉及大黄素-8-O-β-D-葡萄糖苷的抗肿瘤活性及其在医药和食品领域的新用途。
背景技术
大黄素-8-O-β-D-葡萄糖苷主要分布于蓼科何首乌(Polygonum multiflorum Thunb.)、虎杖(Polygonum cuspidatum Sieb.et Zucc.)、掌叶大黄(Rheum palamatum L.)、豆科决明(Cassia obtusifolia L.)、狭叶番泻(Cassia angustifolia Vahl)、唇形科碎米桠(Rabdosia rubescens Hara)、葡萄科白蔹(Ampelopsis japonica Makino)等多科属药用植物中(王文静，等.云南中医学院学报，2005，28(1)：10-12；陈志强，栾颖，中国药房，2009，20(9)：688-689；田国芳，等.中国中药杂志，2010，35(18)：2437-2439；唐力英，等.中国实验方剂学杂志，2009，15(7)：35-37；邬秋萍，等.中药材，2007，30(10)：1250-1252；吕芳，徐筱杰.中药材，2008，31(9)：1340-1343；赫军，等.沈阳药科大学学报，2009，26(3)：188-190)。过去报道其有一定促智作用，对脑中的真性乙酰胆碱酯酶具有良好的可逆性抑制作用，对β淀粉样蛋白造成的海马神经元损伤具有一定的保护作用，并能显著改善由东莨菪碱导致的小鼠学习记忆功能障碍，显著提高正常小鼠学习记忆获得能力。急性毒性实验表明口服大黄素-8-O-β-D-葡萄糖苷具有较高的安全性(陈万生等，专利号99119950226；中草药，2001，32(1)：39-41)。我们研究发现化合物大黄素-8-O-β-D-葡萄糖苷具有良好的抗肿瘤作用，特别是对人肝癌、结直肠癌等，在一定浓度范围内，对肿瘤细胞具杀伤作用而不影响人正常体细胞的活力和功能。
发明内容
本发明内容涉及化合物大黄素-8-O-β-D-葡萄糖苷的抗肿瘤活性及其在制备癌症治疗和预防药物或食品中的新用途。
本发明所指的抗肿瘤活性为抗肝癌、结直肠癌等多种肿瘤的活性。大黄素-8-O-β-D-葡萄糖苷对多种人体肿瘤细胞，包括肝癌和结直肠癌细胞，具有选择性的杀伤作用。该化合物可剂量依赖性地将人肝癌细胞阻滞在G2-M期，并诱导其发生凋亡，但是不影响人体正常细胞的活力和功能。因此，大黄素-8-O-β-D-葡萄糖苷具有高效抗肿瘤且低毒等特点。据此，大黄素-8-O-β-D-葡萄糖苷可被用于制备抗肿瘤药物和预防肿瘤发生的药物或食品。
附图说明
图1是大黄素-8-O-β-D-葡萄糖苷作用于人肝癌HepG2细胞及人正常肝实质L02细胞0～72h的抑制率图。
图2是不同浓度大黄素-8-O-β-D-葡萄糖苷诱导HepG2细胞凋亡图。
图3是大黄素-8-O-β-D-葡萄糖苷对人肝癌HepG2细胞周期的影响图。
图4是大黄素-8-O-β-D-葡萄糖苷作用人结直肠癌HT-29细胞72h的抑制率图。
图5是大黄素-8-O-β-D-葡萄糖苷作用大鼠心肌H9c2细胞48h的抑制率图。
具体实施方式
下面通过具体实施例来对本发明作进一步的说明，但这些实施例只是为了说明，而不是对本发明的限制。
实施例
1.大黄素-8-O-β-D-葡萄糖苷对人肝癌HepG2细胞及人正常肝实质L02细胞活力的影响
采用MTT比色法比较大黄素-8-O-β-D-葡萄糖苷对人肝癌HepG2细胞和人正常肝实质 L02细胞的细胞毒性。两种细胞分别以1600个/孔的密度接种于96孔板，孵育12～24h，设50μg/mL、100μg/mL、200μg/mL、300μg/mL四个给药组，并设空白溶剂对照组(0.1％DMSO)和去甲斑蝥素(NCTD，10μg/mL)阳性对照组，每组8个复孔。分别测0h、24h、48h、72h四个时相各孔细胞的OD值，并做抑制率直方图。抑制率＝(1-加药组OD值/对照组OD值)×100％，结果见图1。
图1不同浓度大黄素-8-O-β-D-葡萄糖苷作用于人肝癌HepG2细胞及人正常肝实质L02细胞0～72h的抑制率图：
A.不同浓度大黄素-8-O-β-D-葡萄糖苷对人肝癌HepG2细胞的抑制率图
B.不同浓度大黄素-8-O-βD-葡萄糖苷对人正常肝实质L02细胞的抑制率图
结论：由图1比较可知，在50～200μg/mL范围内，大黄素-8-O-β-D-葡萄糖苷对肝癌HepG2细胞的生长抑制作用呈现明显的时间-剂量依赖性，而对人正常肝实质L02细胞活力无显著影响。
2.大黄素-8-O-β-D-葡萄糖苷诱导人肝癌HepG2细胞凋亡
本实验采用Annexin V-FITC和PI双染细胞，流式细胞术检测细胞凋亡率。与上述MTT实验相同密度的肝癌HepG2细胞接种于直径10cm平皿中，孵育12～24h，除溶剂空白对照组外分别加50μg/mL、100μg/mL、200μg/mL的大黄素-8-O-β-D-葡萄糖苷作用细胞72h，收集全细胞，离心弃上清，磷酸盐缓冲液洗2遍，加Binding buffer重悬细胞，加10μLAnnexin V-FITC染液避光反应15min，加5μLPI染液混匀后上机检测，结果见图2。
图2不同浓度大黄素-8-O-β-D-葡萄糖苷诱导HepG2细胞凋亡
A大黄素-8-O-β-D-葡萄糖苷50μg/mL组 B大黄素-8-O-β-D葡萄糖苷100μg/mL组C大黄素-8-O-β-D葡萄糖苷200μg/mL组 D空白对照组
各组凋亡率整理见表1：
表1大黄素-8-O-β-D-葡萄糖苷作用人肝癌HepG2细胞72h凋亡率
结论：由图2和表1数据可知，50、100、200μg/mL3个浓度的大黄素-8-O-β-D-葡萄糖苷诱导肝癌HepG2细胞的凋亡率都比空白对照组明显增高，凋亡率随大黄素-8-O-β-D-葡萄糖苷浓度的增加而增加，有明显的剂量依赖性，其中200μg/mL组细胞凋亡率达到33.4％。
3.大黄素-8-O-β-D-葡萄糖苷对人肝癌HepG2细胞周期的影响
本实验采用细胞PI染色、流式细胞仪检测的方法测定大黄素-8-O-β-D-葡萄糖苷对肝癌HepG2细胞周期的影响。与MTT实验相同密度的肝癌HepG2细胞接种于直径10cm的平皿 中，孵育12～24h，加大黄素-8-O-β-D-葡萄糖苷至终浓度为200μg/mL，作用72h，另设溶剂空白对照组。收集全细胞，磷酸盐缓冲液洗2遍，70％冰乙醇4℃固定保存过夜。1000r/min离心5min弃去固定液，用磷酸盐缓冲液洗涤2次。用500μL PBS重悬细胞，加入RNaseA使终浓度为20μg/mL，37℃水浴，45min。加入PI染液使终浓度为50μg/mL，4℃避光染色60min。流式细胞仪检测，结果见图3。
图3大黄素-8-O-β-D-葡萄糖苷对人肝癌HepG2细胞周期的影响
A空白对照
B200μg/mL大黄素-8-O-β-D葡萄糖苷作用72h
结论：由图3，空白对照组G2-M期细胞的比例为11.70％；大黄素-8-O-β-D葡萄糖苷组G2-M期的细胞比例为19.19％，说明大黄素-8-O-β-D葡萄糖苷可以一定程度诱导人肝癌HepG2细胞发生G2-M期阻滞，影响其正常生长。
4.大黄素-8-O-β-D-葡萄糖苷对人结直肠癌HT-29细胞活力的影响
采用MTT比色法检测大黄素-8-O-β-D-葡萄糖苷对人结直肠癌HT-29细胞的细胞毒性。以1600个/孔的密度接种于96孔板，孵育12～24h，药物设50μg/mL、100μg/mL、150μg/mL、200μg/mL、300μg/mL四组，设空白对照组和去甲斑蝥素(NCTD，10μg/mL)阳性对照组，每组8个复孔。测定72h各孔细胞的OD值做大黄素-8-O-β-D-葡萄糖苷抑制率直方图。抑制率＝(1-加药组OD值/对照组OD值)×100％，结果见图4。
图4大黄素-8-O-β-D-葡萄糖苷作用人结直肠癌HT-29细胞72h的抑制率图
结论：由图4可知，在50～300μg/mL剂量范围内，大黄素-8-O-β-D-葡萄糖苷对人结直肠癌HT-29细胞的杀伤作用有明显的剂量依赖性，对癌细胞的生长抑制作用随浓度的升高而增强。
5.大黄素-8-O-β-D-葡萄糖苷对大鼠心肌细胞活力的影响
采用MTT比色法检测大黄素-8-O-β-D-葡萄糖苷对大鼠心肌H9c2细胞的细胞毒性作用。以1600个/孔的密度接种于96孔板，孵育12～24h，大黄素-8-O-β-D-葡萄糖苷设50μg/mL、100μg/mL、150μg/mL、200μg/mL、250μg/mL组，设空白对照组和去甲斑蝥素(NCTD，10μg/mL)阳性对照组，每组8个复孔。测定48h各孔细胞的OD值做抑制率直方图。抑制率＝(1-加药组OD值/对照组OD值)×100％，见图5。
图5大黄素-8-O-β-D-葡萄糖苷作用大鼠心肌H9c2细胞48h的抑制率图
结论：由图5可知，大黄素-8-O-β-D葡萄糖苷在50～250μg/mL浓度范围内对大鼠心肌H9c2细胞的生长毒性作用较小，不影响细胞的正常生长。
Claims (3)
1.大黄素-8-O-β-D-葡萄糖苷的抗肿瘤活性及其在制备肿瘤治疗和预防药物或食品中的新用途。
2.根据权利要求1所述的大黄素-8-O-β-D-葡萄糖苷的抗肿瘤活性，其特征在于所述抗肿瘤活性为抗肝癌和结肠癌活性。
3.根据权利要求1和2所述的大黄素-8-O-β-D-葡萄糖苷的抗肿瘤活性，其特征在于所述抗肿瘤活性为抗肝癌活性。
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