CN112274557A - 冬凌草叶水提物粉末,其制备,抗动脉血栓作用及应用 - Google Patents
冬凌草叶水提物粉末,其制备,抗动脉血栓作用及应用 Download PDFInfo
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Abstract
本发明公开了冬凌草叶水提物粉末,其制备方法以及在治疗动脉血栓和静脉血栓中的应用。通过本发明所述的方法制备得到的冬凌草叶水提物粉末含25种奎尼酸类化合物和11种槲皮素类化合物。实验证明,本发明的冬凌草叶水提物粉末具有优秀的抗动脉血栓和抗静脉血栓作用。因而提出本发明为治疗动脉血栓和静脉血栓提供了有效的技术手段。
Description
技术领域
本发明涉及一种制备冬凌草叶水提物粉末的方法,涉及由该方法制备的冬凌草叶水提物粉末在制备抗动脉血栓和抗静脉血栓药物中的应用。本发明属于生物医药领域。
背景技术
动脉栓塞已成为目前发病率高和死亡率高的疾病之一。动脉血栓形成对暂时性脑缺血发作,急性冠状动脉综合症,心肌梗塞和心房颤动负责。在房颤中有18％-47％病人患有冠状动脉疾病,在伴随冠状动脉疾病的房颤患者中有大约20％接受经皮冠状动脉介入治疗。动脉血栓形成还对人工心脏瓣膜,动静脉瘘和其他手术后的动脉血栓及不稳定型心绞痛负责。例如肝移植手术之后,患者面临肝脏动脉血栓风险。此外,抗磷脂综合症患者也面临动脉血栓风险。虽然肿瘤与静脉血栓的关联比与动脉血栓的关联广泛,但是对特殊恶性肿瘤及肿瘤治疗中动脉血栓包括外周动脉血栓发病的认识正在日益加深。动脉插管及缺血性中风则使得儿童动脉血栓病例日益增加。十多年前就开始警惕可卡因滥用导致的动脉血栓风险。因为病因不同,所以在传统观念下静脉血栓和动脉血栓被看作两种不同的疾病。最近的流行病学研究表明,静脉血栓和动脉血栓之间的关联性难以割断。这种状况可以归结于它们的风险因子相互重叠。这样一来,动脉血栓的预防和治疗便越来越受到重视。
直接口服抗凝是动脉血栓临床治疗的唯一策略。尽管口服抗凝剂对动脉血栓的疗效确切,但是都有出血副作用。例如在有效的口服剂量下,阿司匹林可诱发消化道出血或颅内出血。这种风险大幅度限制了患者的受益面。临床需要疗效可与阿司匹林媲美,又没有阿司匹林样消化道出血或颅内出血风险的药物。针对这个临床需求,国内外研究人员付出了大量心血。可是,一直取得没有实质性进展。
冬凌草是唇形科香茶菜属植物碎米桠Rabdosiarubescens(Hemsl.)Hara的全草。冬凌草为多年生草本植物,广泛分布于我国黄河长江流域,主产于河南济源太行山和王屋山地区。因具有清热解毒,清咽利喉和消肿止痛功能,民间一直将冬凌草全草煮水饮用。在过去的30多年里,冬凌草的化学成分及药理作用研究主要集中在叶和杆的脂溶性部分, 代表性成分为冬凌草甲素和冬凌草乙素。迄今没有任何与冬凌草叶的水提物的化学成分及治疗作用相关的研究见诸文献。
发明人长期从事冬凌草叶水提物粉末的化学成分及治疗作用研究。发明人对冬凌草叶水提物粉末的化学组分进行质谱分析,发现了36种未见报道的组分,主要包括桂皮酰奎尼酸和多取代槲皮素。虽然报道过冬凌草叶水提物粉末单一组分的生物活性,但是全组分联合的生物活性并不知情。通过反复研究发明人发现,这28种组分可治疗动脉血栓。与冬凌草的已知发明相比,本发明的冬凌草叶水提物粉末可治疗动脉血栓和抗静脉血栓的发明达到了一个全新高度。根据这些认识,发明人提出了本发明。
发明内容
本发明要解决的技术问题是提供一种制备冬凌草叶水提物粉末的方法,以及由该方法制得的冬凌草叶水提物粉末。实验证明,由本发明制备的冬凌草叶水提物粉末有抗动脉血栓和抗静脉血栓作用。为了达到所述目的,本发明采用了以下六个技术手段。
第一个技术手段是提出了本发明的一种冬凌草叶水提物粉末,所述冬凌草叶水提物粉末含25种奎尼酸类化合物及11种槲皮素类化合物,所述25种奎尼酸类化合物为 3-O-[E-2-(3-羧基-1-丙烯羰氧)-3,5-二甲酰氧-4-(3-羧基-2-丙烯酰氧)-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(6-羟基-1-己烯羰氧)-3-(4-羟基-1-丁烯羰氧)-4-(3-羧基-1-丙烯羰氧)-5-甲氧-6-(5-羧基-1-戊烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(3-羧基丙羰氧)-3,5-二甲氧-4-(3-羧基丙酰氧)-6-(6-羧基己羰氧)桂皮酰]奎尼酸,3-O-[E-2-(6-羟基己羰氧)-3,5-二甲酰氧-4-(3-羟基丙羰氧)-6-(6-羧基己羰氧)桂皮酰]奎尼酸,3-O-[E-2-(7-羧基-1,3-庚二烯羰氧)-3,4-二(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸, 3-O-[E-2-(7-羧基-1-庚烯羰氧)-3-(6-羟基-1-己烯羰氧)-4-(3-羧基-1-丙烯羰氧)-5-甲酰氧 -6-(6-羧基-1-己烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(4-羟基丁羰氧)-3,4-二(3-羧基丙羰氧)-5- 甲酰氧-6-(6-羧基-1-己烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(5-羧基-1-戊烯羰氧)-3-羧基乙酰氧-4,6-二(3-羧基丙烯酰氧)-5-甲酰氧桂皮酰]奎尼酸,3-O-[E-2,3-二(3-羧基-1-丙烯羰氧)-4-(3-羧基丙烯酰氧)-5-羧基乙酰氧-6-(4-羧基-1-丁烯羰氧)桂皮酰]奎尼酸, 3-O-[E-2,3,4-三(4-羟基丁羰氧)-5-甲酰氧-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸, 3-O-[E-2-(4-羟基-1,3-丁二烯羰氧)-3,4-二(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(6-羧基-1,3,5-己三烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(4-羟基-1,3-丁二烯羰氧)-3-羧乙酰氧-4-(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(4-羧基-1,3-丁二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2,3-二(3-羧基丙羰氧)-4-(3-羧基丙酰氧)-5-甲酰氧-6-(6-羧基-1-己烯羰氧)桂皮酰]奎尼酸,3-O-[E-2,3-二(3-羧基丙羰氧)-4-(3-羟基丙酰氧)-5-羟甲氧-6-(5-羧基戊羰氧)桂皮酰]奎尼酸,3-O-[E-2,6-二(5- 羧基-1-戊烯羰氧)-3-(3-羧基丙羰氧)-4-羧基乙酰氧-5-甲氧桂皮酰]奎尼酸,3-O-[E-2-(8-羧基-1-辛烯羰氧)-3-(3-羧基丙羰氧)-4-(7-羧基-1-庚烯羰氧)-5-羧基乙酰氧-6-(4-羧基-1-丁烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(9-羟基-1-壬烯羰氧)-3-(4-羟基丁羰氧)-4-(8-羟基-1-辛烯羰氧)-5-甲氧-6-(4-羧基-1-丁烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(7-羧基-1,3,5-庚三烯羰氧)-3,4- 二(5-羧基-1,3-戊二烯羰氧)-5-羧基乙酰氧-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸, 3-O-[E-2-(4-羟基-1-丁烯羰氧)-3-(4-羧基-1-丁烯羰氧)-4-(3-羧基丙烯酰氧)-5-羧基乙酰氧 -6-(4-羧基-1,3-丁二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(7-羧基-1,3,5-庚三烯羰氧)-3-(8-羟基 -1,3,5,7-辛四烯羰氧)-4,5-二(4-羧基-1,3-丁二烯酰氧)-6-(6-羧基-1,3,5-己三烯羰氧)桂皮酰] 奎尼酸,3-O-[E-2-(4-羟基丁羰氧)-3,4-二(3-羧基丙羰氧)-5-(3-羟基丙酰氧)-6-(4-羧基丁羰氧)桂皮酰]奎尼酸,3-O-[E-2,6-二(5-羧基-1-戊烯羰氧)-3-(3-羧基丙羰氧)-4-(3-羧基丙酰氧)-5-羟基桂皮酰]奎尼酸,3-O-[E-2,5-二(7-羧基-1,3-庚二烯羰氧)-3-(6-羟基-1,3-己二烯羰氧)-4-(4-羧基-1,3-丁二烯羰氧)-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(5-羟基-1-戊烯羰氧)-3,4-二(4-羧基-1-丁烯羰氧)-5-羧基乙酰氧-6-(4-羧基-1,3-丁二烯羰氧)桂皮酰]奎尼酸和3-O-[E-2,5-二羧基乙酰氧-3-(10-羧基-1,3,5,7-癸四烯羰氧)-4-(8-羧基-1,3,5-辛三烯羰氧)-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸；所述11种槲皮素类化合物为3-O- 葡萄糖苷-2’-甲酰氧-3’-(3-羧基丙烯酰氧)-4’-羧基乙酰氧-5’-(3-羧基-1-丙烯羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(10-羧基-1,3-癸二烯羰氧)-4’-(5-羧基 -2-戊烯羰氧)-5’-(3-羧基-1-丙烯羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(6-羧基-1-己烯羰氧)-4’-(6-羟基-2-己烯羰氧)-5’-(3-羧基-1-丙烯羰氧)-6’-(5-羟基 -1-戊烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(7-羟基-1,3,5-庚三烯羰氧)-4’-(4-羧基-2- 丁烯羰氧)-5’-(3-羧基-1-丙烯羰氧)-6’-(7-羧基-1,3-庚二烯羰氧)槲皮素,3-O-葡萄糖苷-2’- 甲酰氧-3’-(8-羧基-1,3,5-辛三烯羰氧)-4’-(5-羧基-1-戊烯羰氧)-5’-(5-羧基-1,3-戊二烯羰氧)-6’-(6-羟基-1,3,5-己三烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(4-羧基丁羰氧)-4’-(3-羧基丙羰氧)-5’-(4-羟基丁羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素,3-O-葡萄糖苷 -2’-甲酰氧-3’,6’-二(7-羟基-1-庚烯羰氧)-4’-(4-羧基-1-丁烯羰氧)-5’-(3-羧基-1-丙烯羰氧) 槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(7-羧基-1,3,5-庚三烯羰氧)-4’,5’,6’-三(5-羧基-1,3-丁二烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(7-羧基-1,3,5-庚三烯羰氧)-4’,5’-二(5-羧基 -1,3-戊二烯羰氧)-6’-(7-羧基-1,3-庚二烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(4-羧基丁羰氧)-4’-(3-羧基丙羰氧)-5’-(4-羟基丁羰氧)-6’-(5-羟基戊羰氧)槲皮素和3-O-葡萄糖苷 -2’-甲氧-3’-(7-羟基-1-庚烯羰氧)-4’,5’-二(5-羟基-1-戊烯羰氧)-6’-(8-羟基-1,3-辛二烯羰氧) 槲皮素。
第二个技术手段是提出了制备所述冬凌草叶水提物粉末方法。取冬凌草叶用自来水洗净,在蒸馏水中于50℃-100℃加热0.5-4小时并于200-400rpm搅拌,冷却至室温,过滤,滤饼用蒸馏水洗3次,合并的滤液减压浓缩,得到的粉末即为冬凌草叶水提物粉末。
第三个技术手段是提供冬凌草叶水提物粉末的质谱总离子流图谱。
第四个技术手段是提供与冬凌草叶水提物粉末的质谱总离子流谱中的峰对应的36种组分的化学结构。
第五个技术手段是评价冬凌草叶水提物粉末的抗动脉血栓活性。
第六个技术手段是评价冬凌草叶水提物粉末的抗静脉血栓活性。
附图说明
图1冬凌草叶水提物粉末的UPLC-质谱总离子流图谱。
图2冬凌草叶水提物粉末中的3-O-[E-2-(3-羧基-1-丙烯羰氧)-3,5-二甲酰氧-4-(3-羧基 -2-丙烯酰氧)-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸及裂解产物。
图3冬凌草叶水提物粉末中的3-O-[E-2-(6-羟基-1-己烯羰氧)-3-(4-羟基-1-丁烯羰氧)-4-(3-羧基-1-丙烯羰氧)-5-甲氧-6-(5-羧基-1-戊烯羰氧)桂皮酰]奎尼酸及裂解产物。
图4冬凌草叶水提物粉末中的3-O-[E-2-(3-羧基丙羰氧)-3,5-二甲氧-4-(3-羧基丙酰氧)-6-(6-羧基己羰氧)桂皮酰]奎尼酸及裂解产物。
图5冬凌草叶水提物粉末中的3-O-[E-2-(6-羟基己羰氧)-3,5-二甲酰氧-4-(3-羟基丙羰氧)-6-(6-羧基己羰氧)桂皮酰]奎尼酸及裂解产物.
图6冬凌草叶水提物粉末中的3-O-[E-2-(7-羧基-1,3-庚二烯羰氧)-3,4-二(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸及裂解产物。
图7冬凌草叶水提物粉末中的3-O-[E-2-(7-羧基-1-庚烯羰氧)-3-(6-羟基-1-己烯羰氧)-4-(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(6-羧基-1-己烯羰氧)桂皮酰]奎尼酸及裂解产物。
图8冬凌草叶水提物粉末中的3-O-[E-2-(4-羟基丁羰氧)-3,4-二(3-羧基丙羰氧)-5-甲酰氧-6-(6-羧基-1-己烯羰氧)桂皮酰]奎尼酸及裂解产物。
图9冬凌草叶水提物粉末中的3-O-[E-2-(5-羧基-1-戊烯羰氧)-3-羧基乙酰氧-4,6-二(3- 羧基丙烯酰氧)-5-甲酰氧桂皮酰]奎尼酸及裂解产物。
图10冬凌草叶水提物粉末中的3-O-[E-2,3-二(3-羧基-1-丙烯羰氧)-4-(3-羧基丙烯酰氧)-5-羧基乙酰氧-6-(4-羧基-1-丁烯羰氧)桂皮酰]奎尼酸及裂解产物。
图11冬凌草叶水提物粉末中的3-O-[E-2,3,4-三(4-羟基丁羰氧)-5-甲酰氧-6-(6-羧基 -1,3-己二烯羰氧)桂皮酰]奎尼酸及裂解产物。
图12冬凌草叶水提物粉末中的3-O-[E-2-(4-羟基-1,3-丁二烯羰氧)-3,4-二(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(6-羧基-1,3,5-己三烯羰氧)桂皮酰]奎尼酸及裂解产物。
图13冬凌草叶水提物粉末中的3-O-[E-2-(4-羟基-1,3-丁二烯羰氧)-3-羧乙酰氧-4-(3- 羧基-1-丙烯羰氧)-5-甲酰氧-6-(4-羧基-1,3-丁二烯羰氧)桂皮酰]奎尼酸及裂解产物。
图14冬凌草叶水提物粉末中的3-O-[E-2,3-二(3-羧基丙羰氧)-4-(3-羧基丙酰氧)-5-甲酰氧-6-(6-羧基-1-己烯羰氧)桂皮酰]奎尼酸及裂解产物。
图15冬凌草叶水提物粉末中的3-O-[E-2,3-二(3-羧基丙羰氧)-4-(3-羟基丙酰氧)-5-羟甲氧-6-(5-羧基戊羰氧)桂皮酰]奎尼酸及裂解产物。
图16冬凌草叶水提物粉末中的3-O-[E-2,6-二(5-羧基-1-戊烯羰氧)-3-(3-羧基丙羰氧)-4-羧基乙酰氧-5-甲氧桂皮酰]奎尼酸及裂解产物。
图17冬凌草叶水提物粉末中的3-O-[E-2-(8-羧基-1-辛烯羰氧)-3-(3-羧基丙羰氧)-4-(7- 羧基-1-庚烯羰氧)-5-羧基乙酰氧-6-(4-羧基-1-丁烯羰氧)桂皮酰]奎尼酸及裂解产物。
图18冬凌草叶水提物粉末中的3-O-[E-2-(9-羟基-1-壬烯羰氧)-3-(4-羟基丁羰氧)-4-(8- 羟基-1-辛烯羰氧)-5-甲氧-6-(4-羧基-1-丁烯羰氧)桂皮酰]奎尼酸及裂解产物。
图19冬凌草叶水提物粉末中的3-O-[E-2-(7-羧基-1,3,5-庚三烯羰氧)-3,4-二(5-羧基 -1,3-戊二烯羰氧)-5-羧基乙酰氧-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸及裂解产物。
图20冬凌草叶水提物粉末中的3-O-[E-2-(4-羟基-1-丁烯羰氧)-3-(4-羧基-1-丁烯羰氧)-4-(3-羧基丙烯酰氧)-5-羧基乙酰氧-6-(4-羧基-1,3-丁二烯羰氧)桂皮酰]奎尼酸及裂解产物。
图21冬凌草叶水提物粉末中的3-O-[E-2-(7-羧基-1,3,5-庚三烯羰氧)-3-(8-羟基 -1,3,5,7-辛四烯羰氧)-4,5-二(4-羧基-1,3-丁二烯酰氧)-6-(6-羧基-1,3,5-己三烯羰氧)桂皮酰] 奎尼酸及裂解产物。
图22冬凌草叶水提物粉末中的3-O-[E-2-(4-羟基丁羰氧)-3,4-二(3-羧基丙羰氧)-5-(3- 羟基丙酰氧)-6-(4-羧基丁羰氧)桂皮酰]奎尼酸及裂解产物。
图23冬凌草叶水提物粉末中的3-O-[E-2,6-二(5-羧基-1-戊烯羰氧)-3-(3-羧基丙羰氧)-4-(3-羧基丙酰氧)-5-羟基桂皮酰]奎尼酸及裂解产物。
图24冬凌草叶水提物粉末中的3-O-[E-2,5-二(7-羧基-1,3-庚二烯羰氧)-3-(6-羟基-1,3- 己二烯羰氧)-4-(4-羧基-1,3-丁二烯羰氧)-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸及裂解产物。
图25冬凌草叶水提物粉末中的3-O-[E-2-(5-羟基-1-戊烯羰氧)-3,4-二(4-羧基-1-丁烯羰氧)-5-羧基乙酰氧-6-(4-羧基-1,3-丁二烯羰氧)桂皮酰]奎尼酸及裂解产物。
图26冬凌草叶水提物粉末中的3-O-[E-2,5-二羧基乙酰氧-3-(10-羧基-1,3,5,7-癸四烯羰氧)-4-(8-羧基-1,3,5-辛三烯羰氧)-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸及裂解产物。
图27冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’-(3-羧基丙烯酰氧)-4’-羧基乙酰氧-5’-(3-羧基-1-丙烯羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素及裂解产物。
图28冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’-(10-羧基-1,3-癸二烯羰氧)-4’-(5-羧基-2-戊烯羰氧)-5’-(3-羧基-1-丙烯羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素及裂解产物。
图29冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’-(6-羧基-1-己烯羰氧)-4’-(6-羟基-2-己烯羰氧)-5’-(3-羧基-1-丙烯羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素及裂解产物。
图30冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’-(7-羟基-1,3,5-庚三烯羰氧)-4’-(4-羧基-2-丁烯羰氧)-5’-(3-羧基-1-丙烯羰氧)-6’-(7-羧基-1,3-庚二烯羰氧)槲皮素及裂解产物。
图31冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’-(8-羧基-1,3,5-辛三烯羰氧)-4’-(5-羧基-1-戊烯羰氧)-5’-(5-羧基-1,3-戊二烯羰氧)-6’-(6-羟基-1,3,5-己三烯羰氧)槲皮素及裂解产物。
图32冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’-(4-羧基丁羰氧)-4’-(3-羧基丙羰氧)-5’-(4-羟基丁羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素及裂解产物。
图33冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’,6’-二(7-羟基-1-庚烯羰氧)-4’-(4-羧基-1-丁烯羰氧)-5’-(3-羧基-1-丙烯羰氧)槲皮素及裂解产物。
图34冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’-(7-羧基-1,3,5-庚三烯羰氧)-4’,5’,6’-三(5-羧基-1,3-丁二烯羰氧)槲皮素及裂解产物。
图35冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’-(7-羧基-1,3,5-庚三烯羰氧)-4’,5’-二(5-羧基-1,3-戊二烯羰氧)-6’-(7-羧基-1,3-庚二烯羰氧)槲皮素及裂解产物。
图36冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲酰氧-3’-(4-羧基丁羰氧)-4’-(3-羧基丙羰氧)-5’-(4-羟基丁羰氧)-6’-(5-羟基戊羰氧)槲皮素及裂解产物。
图37冬凌草叶水提物粉末中的3-O-葡萄糖苷-2’-甲氧-3’-(7-羟基-1-庚烯羰氧)-4’,5’- 二(5-羟基-1-戊烯羰氧)-6’-(8-羟基-1,3-辛二烯羰氧)槲皮素及裂解产物。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1制备冬凌草叶水提物粉末
干冬凌草叶用自来水洗洗净,取300g洗净的叶在700-1200mL蒸馏水中于50℃-100℃加热0.5-4小时,同时搅拌(200-400rpm)。冷却至室温,过滤,滤液减压浓缩得到90g冬凌草叶水提物粉末。或者新鲜的冬凌草叶用自来水洗洗净,取1350g洗净的叶在1500-2500mL蒸馏水中于50℃-100℃加热0.5-4小时,同时搅拌(200-400rpm)。冷却至室温,过滤,滤液减压浓缩得到90g冬凌草叶水提物粉末。
实施例2测定冬凌草叶水提物粉末的色谱和质谱离子流谱
2-1.样品溶液的制备(10mg/mL)
称取26.7mg冬凌草叶水提物粉末,用2.67mL超纯水中将粉末溶解。得到的溶液经超声震荡1分钟,之后再于13000r/min离心10分钟。取上清液,过0.22μm滤膜,置于样品瓶中供色谱和质谱测定用。
2-2色谱条件
色谱柱:Waters,Acquity
HSS T3柱(2.1×100mm i.d.,1.7μm)；进样体积:2μL； PDA检测器:190-400nm；流动相:水(0.1％甲酸),乙腈；采用这种流动相并按表1的梯度洗色谱柱。
表1流动相梯度表
2-3.测定色谱图
按照上面的色谱测定条件测定并记录冬凌草叶水提物粉末的UPLC色谱图(见说明书附图1)。
2-4.测定离子流谱和质谱的条件
电喷雾离子化模式为正(PI)及负(NI)模式。离子模式参数:毛细管电压为1000V,去溶剂气流速为800L/h,温度450℃,源温度为120℃,锥孔气流速为50L/h,喷雾气压为6bar, 碎裂电压为20-45V,取样锥电压为6V,采集模式为MSE continuum分辨率模式,带电粒子的质量数与电荷数之比(m/z)数据采集范围为100-1500,低能量通道Trap碎裂电压为6V, 高能量通道Trap碎裂电压选择梯度电压为20-60V,选取LE(亮氨酸脑啡肽)为质量锁采集 m/z,范围为100-1500。
2-5.记录离子流谱和质谱
按照上面的条件测定并记录冬凌草叶水提物粉末的离子流谱(见说明书附图1)。
实施例3指定冬凌草叶水提物粉末中36种组分的结构
将实施例2的UPLC色谱与质谱联接,测定冬凌草叶水提物粉末的UPLC-质谱。质谱条件是电喷雾离子化的正离子和负离子两种模式。离子模式参数:毛细管电压为1000 V,去溶剂气流速为800L/h,温度450℃,源温度为120℃,锥孔气流速为50L/h,喷雾气压为6bar,碎裂电压为20-45V,取样锥电压为6V,采集模式为MSE continuum分辨率模式,带电粒子的质量数与电荷数之比(m/z)数据采集范围为100-1500,低能量通道Trap碎裂电压为6V,高能量通道Trap碎裂电压选择梯度电压为20-60V,选取LE(亮氨酸脑啡肽) 为质量锁采集m/z,范围为100-1500。在45分钟内分出36个独立峰。根据质谱裂解规律, 这些峰(按照总离子流图谱从左到右的峰顺序)的结构指定见表2。
表2测得的总离子流图谱中峰对应组分保留时间，负离子质量数，结构及名称
实施例4评价冬凌草叶水提物粉末的抗动脉血栓作用
1)将聚乙烯管拉成一端为斜口的细管,定长为10.0cm,分别为右颈静脉(管径较粗) 及左颈动脉(管径较细)插管；中段聚乙烯管定长为8.0cm,血栓线压在颈动脉插管方向, 插管前需在管中充满肝素。
2)将体重200±20g雄性大鼠在手术前适应环境并禁食一天。随机分为5‰CMC-Na(0.2mL/100g,10只大鼠)组,阿司匹林(167μmol/kg,10只大鼠)组,冬凌草叶水提物粉末(50mg/kg,10只大鼠)组。按照规定剂量给大鼠口服药物。给药30min后,大鼠腹腔注射20％乌拉坦溶液麻醉(7mL/kg),2min之后开始手术。手术中将大鼠仰卧位于固定板上,剪开颈部皮肤,分离右颈总动脉及左颈静脉,血管下压线,结扎远心端,在静脉靠远心端处剪一小口,将插管插入静脉端,注射肝素,而后取下注射肝素的注射器,系线固定,再用动脉夹夹住动脉近心端,在靠近远心端方向剪一小口,将动脉端结扎,系线固定后松开动脉夹,建立体外循环旁路。循环15分钟后先剪断静脉端观察血液循环是否正常,若正常从动脉端取出血栓线,在纸上沾干浮血后称重,以血栓重表示活性,数据列入表3。表中的血栓重量表明,在167μmol/kg口服剂量下阿司匹林有效地抑制大鼠患动脉血栓症,在50mg/kg口服剂量下冬凌草叶水提物粉末也有效抑制大鼠患动脉血栓症。也就是说,冬凌草叶水提物粉末有优秀的抗动脉血栓活性。
表3冬凌草叶水提物粉末对大鼠动脉血栓的影响
a)与5‰CMC-Na比p<0.05；n＝10
实施例5评价冬凌草叶水提物粉末的抗静脉血栓作用
实验动物与方法
SD品系雄性大鼠(250±20g),购自北京维通利华实验动物技术有限公司,用于制作大鼠下腔静脉结扎模型。
实验分组和剂量
SD雄性大鼠在实验前适应25-28℃的环境并禁食一天。之后,随机分为阳性对照华法林组,口服剂量为0.82μmol/kg/天；阴性对照生理盐水组,口服剂量为10mL/kg/天；冬凌草叶水提物粉末组,剂量为12.5mg/kg/天。连续口服7天。
评价方法
最后一次给药30min后大鼠腹腔注射20％乌拉坦溶液使麻醉。在手术板上将麻醉大鼠仰卧位固定,备皮和消毒。然后,沿大鼠腹白线打开腹腔。开腹切口为下至凝固腺上至露出肝脏一角。将小肠等器官从腹腔内移出并用浸润过生理盐水的纱布包裹。钝性分离血管周围结缔组织,暴露下腔静脉及分支。在肾静脉下方将腹主动脉和下腔静脉剥离开, 然后用生理盐水浸湿的缝合线在下腔静脉与左肾静脉交汇处将下腔静脉结扎。将从腹腔内移出并用浸润过生理盐水的纱布包裹的肠等器官按解剖位置移回腹腔内,用缝合线逐层缝合腹腔。由于华法林治疗的大鼠随时可死亡,所以30min之后便打开腹腔,逐个将分支结扎,从下腔静脉与左肾静脉的交汇处的结扎处取出2cm下腔静脉,再从下腔静脉中取出血栓称重,数据见表4。
表3冬凌草叶水提物粉末对静脉血栓的影响
| 静脉血栓大鼠的治疗剂 | 剂量 | 静脉血栓湿重:均值±SDmg |
| 生理盐水(n＝8) | 10mL/kg/天 | 22.2±7.0 |
| 华法林(n＝7) | 0.82μmol/kg/天 | 8.7±1.8 |
| 冬凌草叶水提物粉末(n＝7) | 12.5mg/kg/天 | 11.4±2.6<sup>a</sup> |
a)与生理盐水比P<0.01.
在治疗过程中口服0.82μmol/kg/天华法林的大鼠5只因全身性出血死亡,存活的7只大鼠在最后测定静脉血栓湿重时也发现全身性出血副反应；口服生理盐水的8只大鼠及口服12.5mg/kg/天冬凌草叶水提物粉末的7只大鼠在测定静脉血栓湿重时均未见出血副反应。也就是说,冬凌草叶水提物粉末无出血副反应。
为了进一步支持冬凌草叶水提物粉末无出血副反应，本发明在静脉血栓大鼠上测定了它们的尾出血时间。具体操作是,最后一次给药30min后,将鼠尾尖剪去2mm并开始计时。每15s用滤纸将血轻轻拭去,直至滤纸无任何血迹,经历的时间为出血时间。表4 的数据说明,0.82μmol/kg/天华法林显著性地延长大鼠出血时间。冬凌草叶水提物粉末对大鼠尾出血时间无影响。
表4鲜柚子皮水提物粉末对静脉血栓大鼠尾出血时间的影响
| 静脉血栓大鼠的治疗剂 | 剂量 | 尾出血时间:均值±SDs |
| 生理盐水(n＝8) | 10mL/kg/天 | 153.6±43.9 |
| 华法林(n＝7) | 0.82μmol/kg/天 | 346.3±71.9<sup>a</sup> |
| 冬凌草叶水提物粉末(n＝7) | 6mg/kg/天 | 198.3±38.9<sup>b</sup> |
a)与生理盐水比P<0.01；b)与生理盐水比P>0.05,与华法林比P<0.01
综合从上面2个模型获得的数据,本发明的结论是口服剂量为12.5mg/kg/天的冬凌草叶水提物粉末可有效地抑制静脉血栓,不影响静脉血栓大鼠的出血时间,无出血副作用。这两方面都比口服剂量为0.82μmol/kg/天的华法林优秀,冬凌草叶水提物粉末有优秀的抗静脉血栓活性。
Claims (6)
1.一种冬凌草叶水提物粉末,其特征在于，所述冬凌草叶水提物粉末中含有25种奎尼酸类化合物；其中，所述的25种奎尼酸类化合物为3-O-[E-2-(3-羧基-1-丙烯羰氧)-3,5-二甲酰氧-4-(3-羧基-2-丙烯酰氧)-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(6-羟基-1-己烯羰氧)-3-(4-羟基-1-丁烯羰氧)-4-(3-羧基-1-丙烯羰氧)-5-甲氧-6-(5-羧基-1-戊烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(3-羧基丙羰氧)-3,5-二甲氧-4-(3-羧基丙酰氧)-6-(6-羧基己羰氧)桂皮酰]奎尼酸,3-O-[E-2-(6-羟基己羰氧)-3,5-二甲酰氧-4-(3-羟基丙羰氧)-6-(6-羧基己羰氧)桂皮酰]奎尼酸,3-O-[E-2-(7-羧基-1,3-庚二烯羰氧)-3,4-二(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(7-羧基-1-庚烯羰氧)-3-(6-羟基-1-己烯羰氧)-4-(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(6-羧基-1-己烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(4-羟基丁羰氧)-3,4-二(3-羧基丙羰氧)-5-甲酰氧-6-(6-羧基-1-己烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(5-羧基-1-戊烯羰氧)-3-羧基乙酰氧-4,6-二(3-羧基丙烯酰氧)-5-甲酰氧桂皮酰]奎尼酸,3-O-[E-2,3-二(3-羧基-1-丙烯羰氧)-4-(3-羧基丙烯酰氧)-5-羧基乙酰氧-6-(4-羧基-1-丁烯羰氧)桂皮酰]奎尼酸,3-O-[E-2,3,4-三(4-羟基丁羰氧)-5-甲酰氧-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(4-羟基-1,3-丁二烯羰氧)-3,4-二(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(6-羧基-1,3,5-己三烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(4-羟基-1,3-丁二烯羰氧)-3-羧乙酰氧-4-(3-羧基-1-丙烯羰氧)-5-甲酰氧-6-(4-羧基-1,3-丁二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2,3-二(3-羧基丙羰氧)-4-(3-羧基丙酰氧)-5-甲酰氧-6-(6-羧基-1-己烯羰氧)桂皮酰]奎尼酸,3-O-[E-2,3-二(3-羧基丙羰氧)-4-(3-羟基丙酰氧)-5-羟甲氧-6-(5-羧基戊羰氧)桂皮酰]奎尼酸,3-O-[E-2,6-二(5-羧基-1-戊烯羰氧)-3-(3-羧基丙羰氧)-4-羧基乙酰氧-5-甲氧桂皮酰]奎尼酸,3-O-[E-2-(8-羧基-1-辛烯羰氧)-3-(3-羧基丙羰氧)-4-(7-羧基-1-庚烯羰氧)-5-羧基乙酰氧-6-(4-羧基-1-丁烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(9-羟基-1-壬烯羰氧)-3-(4-羟基丁羰氧)-4-(8-羟基-1-辛烯羰氧)-5-甲氧-6-(4-羧基-1-丁烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(7-羧基-1,3,5-庚三烯羰氧)-3,4-二(5-羧基-1,3-戊二烯羰氧)-5-羧基乙酰氧-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(4-羟基-1-丁烯羰氧)-3-(4-羧基-1-丁烯羰氧)-4-(3-羧基丙烯酰氧)-5-羧基乙酰氧-6-(4-羧基-1,3-丁二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(7-羧基-1,3,5-庚三烯羰氧)-3-(8-羟基-1,3,5,7-辛四烯羰氧)-4,5-二(4-羧基-1,3-丁二烯酰氧)-6-(6-羧基-1,3,5-己三烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(4-羟基丁羰氧)-3,4-二(3-羧基丙羰氧)-5-(3-羟基丙酰氧)-6-(4-羧基丁羰氧)桂皮酰]奎尼酸,3-O-[E-2,6-二(5-羧基-1-戊烯羰氧)-3-(3-羧基丙羰氧)-4-(3-羧基丙酰氧)-5-羟基桂皮酰]奎尼酸,3-O-[E-2,5-二(7-羧基-1,3-庚二烯羰氧)-3-(6-羟基-1,3-己二烯羰氧)-4-(4-羧基-1,3-丁二烯羰氧)-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸,3-O-[E-2-(5-羟基-1-戊烯羰氧)-3,4-二(4-羧基-1-丁烯羰氧)-5-羧基乙酰氧-6-(4-羧基-1,3-丁二烯羰氧)桂皮酰]奎尼酸和3-O-[E-2,5-二羧基乙酰氧-3-(10-羧基-1,3,5,7-癸四烯羰氧)-4-(8-羧基-1,3,5-辛三烯羰氧)-6-(6-羧基-1,3-己二烯羰氧)桂皮酰]奎尼酸。
2.如权利要求1所述的冬凌草叶水提物粉末,其特征在于，所述的冬凌草叶水提物粉末中还含有11种槲皮素类化合物；其中，所述的11种槲皮素类化合物为3-O-葡萄糖苷-2’-甲酰氧-3’-(3-羧基丙烯酰氧)-4’-羧基乙酰氧-5’-(3-羧基-1-丙烯羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(10-羧基-1,3-癸二烯羰氧)-4’-(5-羧基-2-戊烯羰氧)-5’-(3-羧基-1-丙烯羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(6-羧基-1-己烯羰氧)-4’-(6-羟基-2-己烯羰氧)-5’-(3-羧基-1-丙烯羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(7-羟基-1,3,5-庚三烯羰氧)-4’-(4-羧基-2-丁烯羰氧)-5’-(3-羧基-1-丙烯羰氧)-6’-(7-羧基-1,3-庚二烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(8-羧基-1,3,5-辛三烯羰氧)-4’-(5-羧基-1-戊烯羰氧)-5’-(5-羧基-1,3-戊二烯羰氧)-6’-(6-羟基-1,3,5-己三烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(4-羧基丁羰氧)-4’-(3-羧基丙羰氧)-5’-(4-羟基丁羰氧)-6’-(5-羟基-1-戊烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’,6’-二(7-羟基-1-庚烯羰氧)-4’-(4-羧基-1-丁烯羰氧)-5’-(3-羧基-1-丙烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(7-羧基-1,3,5-庚三烯羰氧)-4’,5’,6’-三(5-羧基-1,3-丁二烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(7-羧基-1,3,5-庚三烯羰氧)-4’,5’-二(5-羧基-1,3-戊二烯羰氧)-6’-(7-羧基-1,3-庚二烯羰氧)槲皮素,3-O-葡萄糖苷-2’-甲酰氧-3’-(4-羧基丁羰氧)-4’-(3-羧基丙羰氧)-5’-(4-羟基丁羰氧)-6’-(5-羟基戊羰氧)槲皮素和3-O-葡萄糖苷-2’-甲氧-3’-(7-羟基-1-庚烯羰氧)-4’,5’-二(5-羟基-1-戊烯羰氧)-6’-(8-羟基-1,3-辛二烯羰氧)槲皮素。
3.一种制备权利要求1或2所述的冬凌草茎水提物粉末的方法,其特征在于，通过以下方法制备得到：
将冬凌草叶用水洗净,在蒸馏水中加热浸泡并缓慢搅拌,过滤,滤液减压浓缩,得到的粉末即为冬凌草叶水提物粉末。
4.如权利要求3所述的制备方法,其中冬凌草叶用自来水洗净,优选地在蒸馏水中于50℃-100℃加热0.5-4小时并于200-400rpm/min搅拌,冷却至室温,过滤,滤饼用蒸馏水洗3次,合并的滤液减压浓缩,得到的粉末即为冬凌草叶水提物粉末。
5.权利要求1或2所述的冬凌草叶水提物粉末在制备抗动脉血栓药物中的应用。
6.权利要求1或2所述的冬凌草叶水提物粉末在制备抗静脉血栓药物中的应用。
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