CN107693663A - 延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用 - Google Patents
延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用 Download PDFInfo
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Abstract
本发明公开了延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用。所制备的延龄草甾体皂苷类有效成分本身是偏诺皂苷元、偏诺皂苷元‑3‑O‑α‑L‑吡喃鼠李糖基(1→2)‑β‑D‑葡萄糖苷、偏诺皂苷元‑3‑O‑α‑L‑吡喃鼠李糖基(1→4)‑[α‑L‑吡喃鼠李糖基(1→2)]‑β‑D‑葡萄糖苷和偏诺皂苷元‑3‑O‑α‑L‑吡喃鼠李糖基(1→4)‑α‑L‑吡喃鼠李糖基(1→4)‑[α‑L‑吡喃鼠李糖基(1→2)]‑β‑D‑葡萄糖苷中的一种或多种,因拥有共同的偏诺皂苷元‑3‑O‑α‑L‑吡喃鼠李糖基(1→2)‑β‑D‑葡萄糖苷等结构单元,而在人体中具有类似的治疗功效,主要用于治疗由心脑血管系统疾病形成的脑出血或脑缺血再灌注损伤及其脑血管病后遗症疾病的临床药物应用。
Description
技术领域
本发明涉及延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用,属于中医药制备技术领域。
背景技术
延龄草为百合科延龄草属植物延龄草Trillium tschonoskii Maxim.,俗名头顶一颗珠、芋儿七、狮儿七等,是传统的名贵中药,有延年益寿的功效。主治头晕目眩、失眠、跌打损伤、外伤出血、神经衰弱、高血压病、脑震荡后遗症等疾病,是恩施土家族著名民间药之一。目前国内外对延龄草属植物的药理活性研究表明延龄草有较强的抗炎、免疫调节、改善大鼠学习记忆功能和抗衰老等作用。发明人采用脑缺血再灌注损伤大鼠实验模型,从神经功能评分、脑指数、脑梗死体积等方面研究证实延龄草甾体皂苷类有效成分偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷具有抗脑缺血再灌注损伤的显著药理疗效。同时,根据心脑血管系统疾病形成的脑血管病后遗的临床表现与病因病机原理,构建长期脑缺血或多次梗塞脑缺血形成的血管性痴呆或阿尔默茨海默病模型,即利用腹腔注射半乳糖促老化与反复结扎双侧颈总动脉致脑低灌注缺血及鼠脑室一次性注射凝聚态Aβ蛋白制作异质性及多因性阿尔茨海默病(Heterogeneitys/Multi-factors' Alzheimer's Disease,H/MAD)模型,应用H/MAD模型证实偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷具有改善脑血管病后遗的显著药理疗效。上述研究还没有报道,我们通过系统的体内外实验研究,首先发现延龄草甾体皂苷类有效成分对脑缺血再灌注损伤及其脑血管病后遗症疾病具有良好的治疗作用。
发明内容
本发明目的是提供延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用。
本发明采用了下述技术方案。
延龄草甾体皂苷类有效成分的制备方法如下:
(一)制备延龄草药材或重楼药材甾体皂苷类提取物:将延龄草药材或重楼药材粉碎成粗粉,用0-100%乙醇浸泡0.5-2小时,4-12倍量溶剂加热回流提取2-4次,每次1-3小时,过滤,合并滤液。上述滤液与45-80℃减压浓缩至0.5-1.5g生药材/ml,0-4℃冷藏过夜,析胶,过滤,滤液再用正丁醇萃取回收得浸膏稀释上大孔吸附树脂或滤液直接上大孔吸附树脂AB-8型或D-101型或HPD-400或各种型号大孔吸附树脂富集,依次用乙醇水由0-50%洗脱,再用乙醇水由50-95%洗脱得到各洗脱部位,主要收集乙醇水60-85%的洗脱部位,各洗脱部位在45-80℃减压回收溶剂至乙醇适量浓度,用冷冻干燥法或喷雾干燥进行干燥,收集冻干粉或喷雾干燥粉,即得延龄草药材或重楼药材甾体皂苷类提取物。
(二)制备延龄草甾体皂苷类有效成分:将所得延龄草或重楼药材提取物反复上硅胶柱,上样后依次用三氯甲烷:甲醇或二氯甲烷:甲醇或三氯甲烷:甲醇:水或二氯甲烷:甲醇:水梯度洗脱,主要收集三氯甲烷(或二氯甲烷):甲醇:水为65:35:1或60:30:1的洗脱流份,以上述甾体皂苷类有效成分中偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的4种单体作为对照品TLC或HPLC检测,合并相应流份。上述流份于45-80℃减压浓缩蒸干,用适量蒸馏水混悬,上反相硅胶ODS-C18柱色谱或SephadexLH-20柱色谱或制备高效液相色谱技术分离纯化,依次用乙醇水(或乙腈水)由0-50%洗脱,再用乙醇水50-95%(或乙腈水)洗脱,洗脱液用冷冻干燥法或喷雾干燥进行干燥,收集冻干粉或喷雾干燥粉,用10-95%乙醇结晶并重结晶,即得制备延龄草甾体皂苷类有效成分。
前述方法制备的延龄草甾体皂苷类偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷中的一种或多种有效成分可用于制备脑缺血再灌注损伤及其脑血管病后遗症药物。所述药物包括口服液、胶囊、片剂、泡腾片、粉针剂、水针剂、注射剂、雾化剂、微乳剂、凝胶剂、纳米制剂或各种已知剂型制剂或各种可接受剂型制剂,所述药物还包括各种单方制剂和复方制剂。
进一步的,上述延龄草甾体皂苷类有效成分还包括偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷中的一种或多种的有效成分皂苷元的第21位和第27位的转化羟甲基的羟基或17位羟基或所有糖基的醇羟基被乙酰基、脂肪链烷烃基、芳香族取代基、酰胺基中的任意一种取代,或者是水解后会产生偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷或偏诺皂苷元的物质。
所涉及的脑缺血再灌注损伤是由心肌缺血、脑动脉粥样硬化、微栓塞、血流动力学改变、血液成分的改变、颈内动脉系统短暂性脑缺血发作、椎-基底动脉系统短暂性脑缺血发作、脂肪栓塞、脑血管痉挛形成脑缺血疾病中的任意一种。
所涉及脑血管病后遗症,主要是由心脑血管系统疾病形成的,主要由短暂性脑缺血发作、血栓形成性脑梗死、脑栓塞、腔隙性脑梗死、高血压脑病、慢性起病的缺血性脑病形成的脑血管病后遗症,特别是对长期脑缺血或多次梗塞脑缺血形成的血管性痴呆或阿尔默茨海默病。
化学研究表明,中国的延龄草属和重楼属药材中主要含有甾体皂苷类、倍半萜类、苯丙素苷类和酚酸类成分为主,其中延龄草属和重楼属中甾体皂苷类成分类型基本一致,特别是延龄草属和重楼属药材都主要含有甾体皂苷类有效成分偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷。因此,延龄草甾体皂苷类有效成分的药材来源主要为百合科延龄草属植物延龄草Trillium tschonoskiiMaxim.、吉林延龄草Trillium kamtschaticum Pall.ex Pursh.(或白花延龄草Trillium camschatcense Ker Gawl.)和西藏延龄草Trillium govanianum Wall.ex Royle.的根及根茎或果实或全草。或延龄草甾体皂苷类有效成分的药材来源也可以为百合科重楼属植物云南重楼(滇重楼)Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz.、七叶一枝花(华重楼)Paris polyphylla Smith var. chinensis (Franch.)Hara.、狭叶重楼Paris polyphylla var. stenophylla Franch.、巴山重楼Paris bashanensis F. T. Wang &Tang.、凌云重楼Paris cronquistii (Takht.) H. Li.、金线重楼Paris delavayi Franch.、海南重楼Paris dunniana H. Lév.、球药隔重楼Paris fargesii Franch.、长柱重楼Paris forrestii (Takht.) H.Li.、禄劝花叶重楼Paris luquanensis H. Li.、毛重楼Paris mairei H. Lév.、花叶重楼Paris marmorata Stearn.、四叶重楼Paris quadrifolia L.、皱叶重楼Paris rugosa H. Li & Kurita.、黑籽重楼Paris thibetica Franch.、北重楼Paris verticillata M. Bieb.、南重楼Paris vietnamensis (Takht.) H. Li. 的根茎或全草。
本发明的优点是:提供了延龄草药材或重楼药材中甾体皂苷类有效成分的提取与制备方法,所得延龄草甾体皂苷类偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷中的一种或多种有效成分可用于制备抗脑出血或抗脑缺血再灌注损伤及其脑血管病后遗症药物的口服液、胶囊、片剂、泡腾片、粉针剂、水针剂、注射剂、雾化剂、微乳剂、凝胶剂、纳米制剂或各种已知剂型制剂或各种可接受剂型制剂的原料药物。
延龄草药材或重楼药材中甾体皂苷类有效成分偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷对由心脑血管系统疾病形成的脑缺血及其脑血管病后遗症药物具有良好的治疗作用和较强的生物活性,可能开发一类新药,具有较好市场前景。
延龄草药材或重楼药材中甾体皂苷类有效成分偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷对由短暂性脑缺血发作、血栓形成性脑梗死、脑栓塞、腔隙性脑梗死、高血压脑病、慢性起病的缺血性脑病形成的脑血管病后遗症具有显著疗效,特别是对长期脑缺血或多次梗塞脑缺血导致的血管性痴呆或阿尔默茨海默病具有显著药理疗效。
附图说明
图1为各给药组对脑缺血再灌注损伤模型大鼠神经功能缺损评分的影响;
图2为各给药组对脑缺血再灌注损伤模型大鼠脑指数的影响;
图3为各给药组对脑缺血再灌注损伤模型大鼠脑梗死体积的影响;
图4为各给药组的脑缺血再灌注损伤模型大鼠的脑缺血图;
图5为偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷的分子结构式;
图6为偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的分子结构式;
图7为偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的分子结构式。
具体实施方式
下面结合实施例对本发明作进一步描述:
实施例1
一种延龄草甾体皂苷类提取物及其中有效成分偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的制备方法:将延龄草药材粉碎成粗粉,用70%乙醇浸泡1小时,10倍量溶剂加热回流提取3次,每次2小时,过滤,合并滤液。上述滤液与65℃减压浓缩至1.0g生药材/ml,0-4℃冷藏过夜,析胶,过滤,滤液再用正丁醇等有机溶剂萃取,回收有机溶剂,得浸膏加水混悬,上AB-8型号大孔吸附树脂富集,依次用水、30%乙醇、60%乙醇和85%乙醇洗脱,得到各洗脱部位,用冷冻干燥法进行干燥,收集冻干粉,即得。其中85%的洗脱部位以甾体皂苷类成分为主,甾体皂苷总含量大于95%,即得延龄草甾体皂苷类提取物。将上述延龄草甾体皂苷类提取物既85%乙醇的洗脱部位的浸膏加入等量的100-200目硅胶拌匀,50℃减压挥干溶剂,研匀,干法上样,上常压硅胶柱(200-300目,硅胶量为浸膏的30倍),上样后依次用三氯甲烷:甲醇:水梯度洗脱,主要收集三氯甲烷:甲醇:水为65:35:1的流份,以上述延龄草甾体皂苷类有效成分中偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的3种单体作为对照品TLC或HPLC检测,合并相应流份。上述流份于65℃减压浓缩蒸干,用适量蒸馏水混悬,上反相硅胶ODS-C18柱色谱,依次用乙醇水由0-50%洗脱,再用乙醇水50-95%洗脱,主要收集60%-80%洗脱液用冷冻干燥法进行干燥,收集冻干粉,用10-95%乙醇结晶并重结晶,即得。采用上述方法制备延龄草甾体皂苷类有效成分中偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷的得率为1.5-2.6g/kg,经HPLC检测纯度为99.0%;偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的得率为0.8-1.9g/ kg,经HPLC检测纯度为98.6%;偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的得率依次分别为的得率为0.3-0.9g/ kg,经HPLC检测纯度为98.2%;色谱条件 UItimateXB-C18色谱柱(4.6mm×150mm,3μm),流动相为乙腈-水(10%-100%梯度洗脱),检测波长为203nm,流速为1.0ml/min,柱温30℃,进样量20μl。
实施例2
一种延龄草甾体皂苷类提取物及其中有效成分偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的制备方法:将延龄草药材粉碎成粗粉,用60%乙醇浸泡2.0小时,12倍量溶剂加热回流提取3次,每次2小时,过滤,合并滤液。上述滤液与70℃减压浓缩至1g生药材/ml,0-4℃冷藏过夜,析胶,过滤,滤液上D101型号大孔吸附树脂富集,依次用水、30%乙醇、60%乙醇和85%乙醇洗脱,得到各洗脱部位,用喷雾干燥法进行干燥,收集喷雾干粉,即得。其中 85%的洗脱部位以甾体皂苷类成分为主,甾体皂苷总含量大于92%,即得延龄草甾体皂苷类提取物。将上述延龄草甾体皂苷类提取物既85%乙醇的洗脱部位的浸膏加入等量的100-200目硅胶拌匀,50℃减压挥干溶剂,研匀,干法上样,上常压硅胶柱(200-300目,硅胶量为浸膏的25倍),上样后依次用三氯甲烷:甲醇:水梯度洗脱,主要收集三氯甲烷:甲醇:水为60:30:1的洗脱流份,以上述延龄草甾体皂苷类有效成分中偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的3种单体作为对照品TLC或HPLC检测,合并相应流份。上述流份于65℃减压浓缩蒸干,用适量蒸馏水混悬,上SephadexLH-20柱色谱,依次用乙醇水由0-50%洗脱,再用乙醇水50-95%洗脱,洗脱液用喷雾干燥进行干燥,收集喷雾干燥粉,用10-95%乙醇结晶并重结晶,即得。采用上述方法制备延龄草甾体皂苷类有效成分中偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷的得率为1.5-2.6g/kg,经HPLC检测纯度为98.8%;偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的得率为0.8-1.9g/kg,经HPLC检测纯度为98.5%;偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的得率依次分别为的得率为0.3-0.9g/kg,经HPLC检测纯度为97.8%;色谱条件 UItimateXB-C18色谱柱(4.6mm×150mm,3μm),流动相为乙腈-水(10%-100%梯度洗脱),检测波长为203nm,流速为1.0ml/min,柱温30℃,进样量20μl。
实施例3
一种延龄草甾体皂苷类提取物及其中有效成分偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的制备方法:将延龄草药材粉碎成粗粉,用50%乙醇浸泡1.0小时,8倍量溶剂加热回流提取3次,每次2小时,过滤,合并滤液。上述滤液与70℃减压浓缩至1g生药材/ml,0-4℃冷藏过夜,析胶,过滤,滤液上HPD400型号大孔吸附树脂富集,依次用水、30%乙醇、50%乙醇和85%乙醇洗脱,得到各洗脱部位,用喷雾干燥法进行干燥,收集喷雾干粉,即得。其中 85%的洗脱部位以甾体皂苷类成分为主,甾体皂苷总含量大于90%,即得延龄草甾体皂苷类提取物。将上述延龄草甾体皂苷类提取物既85%乙醇的洗脱部位的浸膏加入等量的100-200目硅胶拌匀,50℃减压挥干溶剂,研匀,干法上样,上常压硅胶柱(200-300目,硅胶量为浸膏的20倍),上样后依次用三氯甲烷:甲醇:水梯度洗脱,主要收集三氯甲烷:甲醇:水为65:35:1的洗脱流份,以上述延龄草甾体皂苷类有效成分中偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的3种单体作为对照品TLC或HPLC检测,合并相应流份。上述流份于65℃减压浓缩蒸干,用制备高效液相色谱技术分离纯化,用乙醇水50-95%洗脱,洗脱液经适当浓缩用喷雾干燥进行干燥,收集喷雾干燥粉,用10-95%乙醇结晶并重结晶,即得。采用上述方法制备延龄草甾体皂苷类有效成分中偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷的得率为1.5-2.6g/kg,经HPLC检测纯度为99.3%;偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的得率为0.8-1.9g/kg,经HPLC检测纯度为99.1%;偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的得率依次分别为的得率为0.3-0.9g/kg,经HPLC检测纯度为98.8%;色谱条件 UItimateXB-C18色谱柱(4.6mm×150mm,3μm),流动相为乙腈-水(10%-100%梯度洗脱),检测波长为203nm,流速为1.0ml/min,柱温30℃,进样量20μl。
实施例4
本发明以延龄草药材或重楼药材中获得的甾体皂苷类有效成分偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖为原料药物,对由心脑血管系统疾病形成的脑缺血再灌注损伤的大鼠模型进行药效学研究,证实上述甾体皂苷类有效成分对由心肌缺血、脑动脉粥样硬化、微栓塞、血流动力学改变、血液成分的改变、颈内动脉系统短暂性脑缺血发作、椎-基底动脉系统短暂性脑缺血发作、脂肪栓塞、脑血管痉挛等形成的脑缺血再灌注损伤疾病具有显著药理疗效,具有开发抗脑缺血一类新药的巨大潜力。
1 材料与方法
1.1 实验动物 SPF级健康SD雄性大鼠120只,体重240~280 g,购自湖南斯莱克景达实验动物有限公司,动物许可证编号为SCXK(湘)2016-0002。
1.2 药品与试剂 偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷即重楼皂苷Ⅵ(简称Y2T)、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Y3T)、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖即重楼皂苷Ⅶ(Y4T)、偏诺皂苷元(Y苷元)均由本实验室自行制备(纯度均大于98.8%);尼莫地平片购于亚宝药业集团股份有限公司(批号:170107);脑心通胶囊购于陕西步长制药有限公司(批号20170320)。
1.3 脑缺血再灌注模型的建立 参考Zea longa的方法,采用大脑中动脉阻塞法制备局灶性脑缺血再灌注模型。大鼠称重,腹腔注射10% 水合氯醛(0.35m L/100 g),麻醉后固定。常规消毒手术视野区皮肤,纵向切开颈中部皮肤2 cm,钝性分离皮下组织与肌肉,暴露右侧颈总动脉CCA,小心剪开包裹颈总动脉及迷走神经的鞘膜,并继续分离右颈内动脉ICA及右颈外动脉ECA。在CCA 近心端、远心端分别穿线,近心端系紧,结扎颈总动脉CCA,远心端系一松结备用。分离ECA、ICA,并在ECA穿线结扎,用动脉夹夹闭ICA。距颈总动脉分叉处约4 mm剪一“V”型小口,插入栓线,用备用的丝线打一正结,松开ICA 的动脉夹,然后调整线栓角度,使线栓弧度水平向外,轻轻将线栓送入颅内,轻缓推进直至感觉到阻力,插入深度在18.0~20.0 mm,即完成了大脑中动脉阻塞所致大脑局灶性脑缺血的制备。分层缝合肌肉与皮肤(部分栓线留体外)。于2 h 后轻轻地向外拔出栓线1cm左右至略感阻力即可进行再灌注4 h,剪断皮肤外露的栓线。手术后保温,常规饲养。假损伤组不插入线栓,其余同模型组。
1.4 动物分组及给药 SD大鼠随机分为12组:假损伤组,模型组,尼莫地平组(10mg/kg),脑心通组(2010 mg/kg),Y2T中剂量组(60 mg/kg),Y2T高剂量组(90 mg/kg),Y3T中剂量组(60 mg/kg),Y3T高剂量组(90 mg/kg),Y4T中剂量组(60 mg/kg),Y4T高剂量组(90mg/kg),Y苷元中剂量组(37 mg/kg),Y苷元高剂量组(54 mg/kg),连续灌胃给药8天,第8天灌胃1 h后,制备缺血再灌注模型。假损伤组和模型组给予等体积的生理盐水。
1.5 评价指标
1.5.1 一般情况观察 实验过程中每天观察并记录饮食、饮水情况及精神状态。
1.5.2神经功能评分 参照Zea Longa 5分制评分标准对大鼠神经功能进行盲评:0分,没有神经功能缺损;1分,对侧前爪不能完全伸展,为对侧前肢内收、屈曲;2分,行走时大鼠向对侧转圈、划圈,将大鼠置于地面时,推动患侧肩向对侧移动时,阻力明显降低;3分,行走时大鼠身体向左侧(瘫痪侧)倾倒;4分,意识丧失,不能自发行走;5分,死亡。评分在1~3者,确定大鼠造模成功,纳入模型组。
1.5.3 大鼠脑指数的测定 脑缺血再灌注评分后,大鼠麻醉,取出全脑,生理盐水冲洗,去除嗅球、小脑及脑干,滤纸吸干水分,称重。脑指数=脑湿重(g)/体质量(100 g),可反映血脑屏障结构损伤的情况。
1.5.4 大鼠脑梗死体积的测定 将脑组织称重后,立即放置-20 ℃冰箱中快速冷冻20 min,用手术刀片由前向后行冠状位切片,共5片,每片约 2 mm厚,置 2% 2,3,5- 三苯基氯化四氮唑(TTC)溶液中, 37 ℃避光染色30 min,15 min后翻面。经染色正常脑组织呈玫瑰红色,而梗塞区呈苍白色,界限清晰。染色后再用4%多聚甲醛固定30 min,拍照,用图像处理软件计算脑梗灶体积的百分比。
1.6 统计方法 实验结果以±s表示,采用SAS软件统计分析。采用单因素方差分析,组间两两比较,采用最小显著差法检验。
2 结果
2.1 一般情况 各给药组灌胃期间,大鼠精神状态良好,饮食睡眠正常,体质量与其余组大鼠无明显差异性。
2.2 各给药组对脑缺血再灌注损伤模型大鼠神经功能缺损症状评分的影响
假损伤组没有神经功能缺损;模型组神经功能缺损明显;尼莫地平组、脑心通组、Y2T中剂量组、Y3T中(高)剂量组神经功能缺损评分均较模型组显著降低,差异显著(P<0.01);而Y2T高剂量组、Y4T高剂量组、Y苷元中剂量组神经功能缺损评分较模型组也明显降低(P<0.05)。见表1和图1。
表1 各给药组对脑缺血再灌注损伤模型大鼠神经功能缺损评分的影响(±s)
注:与模型组比较,* p﹤0.05,** p﹤0.01;n=10。
2.3 各给药组对脑缺血再灌注损伤模型大鼠脑指数的影响
模型组大鼠脑指数明显升高,与假损伤组比较有显著性差异(P<0.01);脑心通组、Y2T中剂量组、Y4T中(高)剂量组较模型组大鼠脑指数明显降低,差异显著(P<0.01);Y3T中(高)剂量组较模型组大鼠脑指数也明显降低(P<0.05);而其余各组较模型组大鼠脑指数改善的不明显。见表2和图2。
表2 各给药组对脑缺血再灌注损伤模型大鼠脑指数的影响(±s)
注:与模型组比较,* p﹤0.05,** p﹤0.01;n=10。
2.4 各给药组对脑缺血再灌注损伤模型大鼠脑梗死体积的影响
TTC染色结果显示(正常脑组织呈玫瑰红色,而梗塞组织呈苍白色,界限清晰),假损伤组未见梗死灶,模型组出现明显梗死灶。尼莫地平组、Y2T中(高)剂量组、Y4T中(高)剂量组、Y苷元高剂量组较模型组梗死灶体积缩小,差异显著(P<0.01);而脑心通组、Y3T中(高)剂量组、Y苷元中剂量组较模型组梗死灶体积也明显缩小(P<0.05)。见表3和图3、4。
表3 各给药组对脑缺血再灌注损伤模型大鼠脑梗死体积的影响(±s)
注:与模型组比较,* p﹤0.05,** p﹤0.01;n=10。
实施例5
本发明以延龄草药材或重楼药材中具有抗脑缺血再灌注损伤的甾体皂苷类有效成分偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖为原料药物,根据心脑血管系统疾病(主要为短暂性脑缺血发作、血栓形成性脑梗死、脑栓塞、腔隙性脑梗死、高血压脑病、慢性起病的缺血性脑病形成的脑血管病后遗症)形成的脑血管病后遗的临床表现与病因病机原理,构建长期脑缺血或多次梗塞脑缺血形成的血管性痴呆或阿尔默茨海默病模型,即利用腹腔注射半乳糖促老化与反复结扎双侧颈总动脉致脑低灌注脑缺血及鼠脑室一次性注射凝聚态Aβ蛋白制作异质性及多因性阿尔茨海默病(Heterogeneitys/Multi-factors' Alzheimer's Disease,H/MAD)模型,应用H/MAD模型证实延龄草甾体皂苷类有效成分偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷具有改善脑血管病后遗的显著药理疗效,具有开发抗脑缺血一类新药的巨大潜力。
1.药品与试剂 偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷(Y2T)、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Y3T)、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Y4T)均为白色粉末,由笔者实验室自行分离得到,质量分数98%。盐酸多奈哌齐(卫材药业有限公司,国药准字 H20050978,批号131246A);D-半乳糖(Sigma公司,批号Lot# 060M00631V);Aβ25-35(Sigma公司,批号Lot# 053M4804V);T-AOC、TChE、CAT、SOD、MDA和GSH-PX检测试剂盒(南京建成生物工程研究所,批号20150512)。
2.动物的分组、造模及给药 将大鼠随机分为9组,每组12只:空白组、H/MAD模型组、盐酸多奈哌齐组、Y2T高剂量组、Y2T中剂量组、Y3T高剂量组、Y3T中剂量组、Y4T高剂量组、Y4T中剂量组。空白组不予处理;模型组与给药组大鼠每天腹腔注射D-半乳糖(50 mg·kg-1),造成亚急性衰老,4 W后先进行双侧颈总动脉反复结扎间段性的阻断血流,造成脑缺血性再灌注损伤病灶。手术完成后,将大鼠喂养3天,取未死亡的状态良好的大鼠再脑内注射凝聚态Aβ25-35。模型组于脑内注射手术后第3天开始每日灌胃2%聚山梨酯水溶液1次;盐酸多奈哌齐组按1 mg·kg-1每日灌胃盐酸多奈哌齐的聚山梨酯水溶液1次;Y2T高(中)剂量组、Y3T高(中)剂量组、Y4T(中)剂量组分别按34.38 mg·kg-1、17.19 mg·kg-1、20.51 mg·kg-1、10.25 mg·kg-1、15.19 mg·kg-1、7.59 mg·kg-1灌胃Y2T、Y3T、Y4T的聚山梨酯水溶液,每天1次,给药6天。
3. Morris水迷宫实验 与空白组比较,模型组大鼠潜伏期延长、过台次减少,在第四象限逗留时间短,差异显著(P<0.01);与模型组比较,给药盐酸多奈哌齐组、Y2T和Y3T各组能缩短潜伏期、增加过台次数,在第四象限逗留时间延长(P<0.05或P<0.01),说明盐酸多奈哌齐、延龄草甾体皂苷Y2T和Y3T各给药组治疗效果明显,而延龄草甾体皂苷Y4T治疗效果不明显。结果见表4。
表4 延龄草甾体皂苷对H/MAD模型大鼠空间探索试验结果(±s,n=10)
与空白组比较,# p﹤0.01;与模型组比较,* p﹤0.05,** p﹤0.01。
4.延龄草甾体皂苷对H/MAD模型大鼠血清中T-AOC、TChE和CAT含量的影响
与空白组比较,模型组大鼠血清中T-AOC、CAT含量明显降低,TChE活力增强,差异显著(P<0.01);与模型组比较,延龄草甾体皂苷Y4T使T-AOC的活力恢复的不明显,差异不显著,能提高CAT含量,降低TChE含量(P<0.05);给药盐酸多奈哌齐组、延龄草甾体皂苷Y2T和Y3T各组能提高T-AOC、CAT含量,降低TChE活力,与模型组相比具有显著差异(P<0.05或P<0.01),说明盐酸多奈哌齐、延龄草甾体皂苷Y2T和Y3T各给药组治疗效果明显,能使H/MAD模型大鼠脑内T-AOC的活力恢复,抗氧化酶CAT含量上升,降低TChE含量,增强清除自由基的能力,拮抗氧化反应,同时防止神经递质的减少,起到防治AD的作用。结果见表5。
表5 延龄草甾体皂苷对H/MAD模型大鼠血清中T-AOC、TChE和CAT含量的影响(±s,n=10)
与空白组比较,# p﹤0.01;与模型组比较,* p﹤0.05,** p﹤0.01。
5.延龄草甾体皂苷对H/MAD模型大鼠血清中SOD、MDA和GSH-PX含量的影响
与空白组比较,模型组大鼠SOD、GSH-PX明显降低(P<0.01),而MDA水平显著升高(P<0.01);与模型组比较,盐酸多奈哌齐组、延龄草甾体皂苷Y2T和Y3T各组能使MDA水平明显下降(P<0.01),而SOD、GSH-PX明显上升(P<0.01),说明给药盐酸多奈哌齐、延龄草甾体皂苷Y2T和Y3T治疗有效;与模型组比较,延龄草甾体皂苷Y4T使MDA水平下降的不明显,差异不显著,但可以提高SOD、GSH-PX的活力(P<0.05)。结果见表6。
表6 延龄草甾体皂苷对H/MAD模型大鼠血清中SOD、MDA、GSH-PX含量的影响(±s,n=10)
与空白组比较,# p﹤0.01;与模型组比较,* p﹤0.05,** p﹤0.01。
本实验通过测定H/MAD模型大鼠血清中T-AOC、TChE、CAT、SOD、MDA和GSH-PX的含量,来了解机体受氧自由基的损伤及机体清除自由基能力的强弱,为其作用机制的研究提供依据。结果显示,造模后,大鼠脑组织内自由基产生增加,发生了明显的脂质过氧化反应,偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷(Y2T)、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Y3T)(尤其是各中剂量组)能使H/MAD模型大鼠行为学活动能力有所恢复,能使血清中T-AOC的活力恢复,抗氧化酶CAT含量上升,能明显提高SOD、GSH-PX的活力水平(P<0.05或P<0.01),增强机体清除自由基的能力;能降低TChE的含量,防止神经递质的减少,同时又能降低MDA的含量(P<0.05或P<0.01)以减少机体受自由基损伤的程度,表现出良好的抗氧化损伤作用,而偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Y4T)的治疗作用不明显。
实施例6
中国延龄草属或重楼属药材中甾体皂苷类化合物偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷即重楼皂苷Ⅵ(Y2T)、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Y3T)和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷即重楼皂苷Ⅶ(Y4T)的波谱数据信息与解析。
1.偏诺皂苷元-3-O-α-L-吡喃鼠李糖基-(1→2)-β-D-葡萄糖苷即重楼皂苷Ⅵ(pennogenin-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside),无色针晶(甲醇),分子式为C39H62O13,ESI-MS[M+H]+峰m/z:739.4245。Liebermann-Burchard反应和Molish反应均呈阳性为甾体皂苷。1H-NMR(DMSO-d6,400 MHz) δ: 0.73(3H,s,CH3-18),0.74(3H,d,J=6.0 Hz,CH3-27),0.78(3H,d, J=6.8 Hz,CH3-21),0.95(3H,brs,CH3-19),1.08(3H,d,J=6.0 Hz,Rha-CH3-
6″),5.34 (1H, d,J=4.4 Hz,H-6),5.03 (1H,d,J=0.8 Hz,Rha-H-1″),4.93(1H,d, J=6.0 Hz,Glc-H-1′);13C-NMR (DMSO-d6, 100 MHz)δ:36.9(C-1),30.8(C-2),77.7(C-3),37.6(C-4), 140.3(C-5),121.3(C-6),31.5(C-7),31.2(C-8),51.9 (C-9),36.3(C-10),22.4(C-11),31.4(C-12),43.6(C-13),49.5(C-14),29.7(C-15),88.9(C-16),88.3(C-17),16.6(C-18),17.7(C-19),44.3(C-20),17.1(C-21),108.7(C-22),31.6(C-23),28.0(C-24),29.0(C-25),65.8(C-26),9.3(C-27),98.1(C-1′),76.5(C-2′),76.3(C-3′),71.9(C-4′),76.0(C-5′),60.9(C-6′),100.0(C-1″),70.6(C-2″),70.5(C-3″),70.2(C-4″),67.9(C-5"),19.0(C-6″)。 分子结构式见图5。
2.偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(pennogenin-3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]
-β-D-glucopyranoside),白色针晶(甲醇),mp259-261℃,分子式为 C45H71O17,ESI-MS[M+H]+ 峰m/z:885.4827。Liebermann-Burchard反应和Molish反应均呈阳性为甾体皂苷。1H-NMR(DMSO-d6,400 MHz)δ: 0.73(3H,s,CH3-18),0.74(3H,d,
J=6.0 Hz,CH3-27),0.78(3H,d, J=6.8 Hz,CH3-21),0.95(3H,brs,CH3-19),1.08(3H,
d,J=6.0 Hz,Rha-CH3-6″),1.10(3H,d,J=6.0 Hz,Rha-CH3-6″′),5.33 (1H, d,J=4.4Hz,H-6),5.02 (1H,brs,Rha-H-1″), 4.92(1H,d, J=6.4 Hz,Glc-H-1′),4.70(1H, d, J=2.0 Hz,Rha-H-1″′);13C-NMR (DMSO-d6, 100 MHz)δ:36.8(C-1),30.8(C-2),76.9(C-3),37.6(C-4),140.2(C-5),121.3(C-6),31.5(C-7),31.2(C-8),51.9(C-9),36.3(C-10),22.4(C-11),31.4(C-12),43.6(C-13),49.5(C-14),29.7(C-15),88.9(C-16),88.3(C-17),16.6(C-18),17.8(C-19),44.3(C-20),17.1(C-21),108.7(C-22),31.6(C-23),28.0(C-24),29.0(C-25),65.8(C-26),9.3(C-27),98.1(Glc-C-1′),76.2(C-2′),76.1 (C-3′),71.9(C-4′),75.2(C-5′),60.0(C-6′),100.3(Rha-C-1″),70.7(C-2″),70.5(C-3″),70.4(C-4″),67.9(C-5″),19.0(C-6″),100.5(Rha-C-1″′),71.9(C-2″′),70.5(C-3″′),76.7(C-4″′),68.6(C-5″′),17.7(C-6″′)。分子结构式见图6。
3.偏诺皂苷元-3-O-α-L-吡喃鼠李糖基-(1→4)-O-α-L-吡喃鼠李糖基(1→4)-[O-α-L-吡喃鼠李糖基-(1→2)]-O-β-D-吡喃葡萄糖苷即重楼皂苷Ⅶ(pennogenin-3-O-α-L-
rhamnopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→4)-O-[α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside),白色针晶(甲醇),mp259-261℃,C51H80O21 ,ESI-MS [M+Na]+ 峰m/z: 1053.5272。Liebermann-Burchard反应和Molish反应均呈阳性为甾体皂苷。1H-NMR(DMSO-d6,400 MHz)δ: 0.73(3H,s,CH3-18),0.74(3H,d,J=6.0Hz,CH3-27),0.78(3H,d, J=6.8 Hz,CH3-21),0.95(3H,brs,CH3-19),1.08(3H,d,J=6.0 Hz,Rha-CH3-6″),1.10(3H,d,J=6.0 Hz,Rha-CH3-6″′),1.12(3H,d,J=6.0 Hz,Rha-CH3-6″′′),5.32 (1H, d,J=4.0 Hz,H-6),5.06 (1H,brs,Rha-H-1″″), 5.02 (1H,brs,Rha-H-1″),4.86(1H,d, J=4.0 Hz,Glc-H-1′),4.67(1H, d, J=1.2 Hz,Rha-H-1″′);13C-NMR (DMSO-d6, 100 MHz)δ:36.9(C-1),30.8(C-2),77.8(C-3),37.6(C-4),140.2(C-5),121.3(C-6),31.5(C-7),31.2(C-8),51.9(C-9),36.3(C-10),22.4(C-11),31.4(C-12),43.6(C-13),49.5(C-14),29.7(C-15),88.9(C-16),88.3(C-17),16.6(C-18),17.8(C-19),44.3(C-20),17.1(C-21),108.7(C-22),31.6(C-23),28.0(C-24),29.0(C-25),66.7(C-26),9.3(C-27),98.2(Glc-C-1′),76.2(C-2′),76.0 (C-3′),71.9(C-4′),75.3(C-5′),65.8(C-6′),100.0(Rha-C-1″),70.7(C-2″),70.6(C-3″),70.4(C-4″),67.9(C-5″),19.0(C-6″),100.3(Rha-C-1″′),71.9(C-2″′),70.6(C-3″′),77.0(C-4″′),68.9(C-5″′),17.7(C-6″′) ,101.0(Rha-C-1″′′),71.3(C-2″′′),71.3(C-3″′′),76.0(C-4″′′),69.7(C-5″′′),18.1(C-6″′′)。分子结构式见图7。
Claims (7)
1.延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用,其特征在于,延龄草甾体皂苷类有效成分中偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷中的一种或多种有效成分用于治疗由心脑血管系统疾病形成的脑出血或脑缺血再灌注损伤及其脑血管病后遗症疾病。
2.根据权利要求1中所述延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用,其特征在于,所述的延龄草甾体皂苷类有效成分还包括偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷中的一种或多种有效成分的皂苷元的第21位和第27位的转化羟甲基的羟基或17位羟基或所有糖基的醇羟基被乙酰基、脂肪链烷烃基、芳香族取代基、酰胺基中的任意一种取代,或者是水解后会产生偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷或偏诺皂苷元的物质。
3.根据权利要求1中所述延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用,其特征在于,所涉及的脑缺血再灌注损伤是由心肌缺血、脑动脉粥样硬化、微栓塞、血流动力学改变、血液成分的改变、颈内动脉系统短暂性脑缺血发作、椎-基底动脉系统短暂性脑缺血发作、脂肪栓塞、脑血管痉挛形成脑缺血疾病中的任意一种。
4.根据权利要求1中所述延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用,其特征在于,所涉及脑血管病后遗症,主要是由心脑血管系统疾病形成的,主要由短暂性脑缺血发作、血栓形成性脑梗死、脑栓塞、腔隙性脑梗死、高血压脑病、慢性起病的缺血性脑病形成的脑血管病后遗症,特别是对长期脑缺血或多次梗塞脑缺血形成的血管性痴呆或阿尔默茨海默病。
5.根据权利要求1中所述延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用,其特征在于,所述药物包括口服液、胶囊、片剂、泡腾片、粉针剂、水针剂、注射剂、雾化剂、微乳剂、凝胶剂、纳米制剂或各种已知剂型制剂或各种可接受剂型制剂,所述药物还包括各种单方制剂和复方制剂。
6.根据权利要求1中所述延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用,其特征在于,所述的延龄草甾体皂苷类偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷中的一种或多种有效成分的制备方法如下:
(一)制备延龄草药材或重楼药材甾体皂苷类提取物:将延龄草药材或重楼药材粉碎成粗粉,用0-100%乙醇浸泡0.5-2小时,4-12倍量溶剂加热回流提取2-4次,每次1-3小时,过滤,合并滤液;上述滤液与45-80℃减压浓缩至0.5-1.5g生药材/ml,0-4℃冷藏过夜,析胶,过滤,滤液直接上大孔吸附树脂AB-8型或D-101型或HPD-400或各种型号大孔吸附树脂富集,依次用乙醇水由0-50%洗脱,再用乙醇水由50-95%洗脱得到各洗脱部位,主要收集乙醇水60-85%的洗脱部位,各洗脱部位在45-80℃减压回收溶剂至乙醇适量浓度,用冷冻干燥法或喷雾干燥进行干燥,收集冻干粉或喷雾干燥粉,即得延龄草药材或重楼药材甾体皂苷类提取物;
(二)制备延龄草甾体皂苷类有效成分:将所得延龄草或重楼药材提取物反复上硅胶柱,上样后依次用三氯甲烷:甲醇或二氯甲烷:甲醇或三氯甲烷:甲醇:水或二氯甲烷:甲醇:水梯度洗脱,主要收集三氯甲烷(或二氯甲烷):甲醇:水为65:35:1或60:30:1的洗脱流份,以上述甾体皂苷类有效成分中偏诺皂苷元、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-葡萄糖苷、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷的4种单体作为对照品TLC或HPLC检测,合并相应流份;上述流份于45-80℃减压浓缩蒸干,用适量蒸馏水混悬,上反相硅胶ODS-C18柱色谱或SephadexLH-20柱色谱或制备高效液相色谱技术分离纯化,依次用乙醇水(或乙腈水)由0-50%洗脱,再用乙醇水50-95%(或乙腈水)洗脱,洗脱液用冷冻干燥法或喷雾干燥进行干燥,收集冻干粉或喷雾干燥粉,用10-95%乙醇结晶并重结晶,即得制备延龄草甾体皂苷类有效成分。
7.根据权利要求6中所述延龄草甾体皂苷类有效成分在制备抗脑缺血再灌注损伤及其脑血管病后遗症药物中的应用,其特征在于,所述的延龄草药材来源主要为百合科延龄草属植物延龄草Trillium tschonoskii Maxim.、吉林延龄草Trillium kamtschaticum Pall.ex Pursh.(或白花延龄草Trillium camschatcense Ker Gawl.)和西藏延龄草Trillium govanianum Wall.ex Royle.的根及根茎或果实或全草;或延龄草药材来源也可以为百合科重楼属植物云南重楼(滇重楼)Paris polyphylla Smith var. yunnanensis(Franch.) Hand.-Mazz.、七叶一枝花(华重楼)Paris polyphylla Smith var. chinensis(Franch.) Hara.、狭叶重楼Paris polyphylla var. stenophylla Franch.、巴山重楼Paris bashanensis F. T. Wang &Tang.、凌云重楼Paris cronquistii (Takht.) H.Li.、金线重楼Paris delavayi Franch.、海南重楼Paris dunniana H. Lév.、球药隔重楼Paris fargesii Franch.、长柱重楼Paris forrestii (Takht.) H.Li.、禄劝花叶重楼Paris luquanensis H. Li.、毛重楼Paris mairei H. Lév.、花叶重楼Paris marmorata Stearn.、四叶重楼Paris quadrifolia L.、皱叶重楼Paris rugosa H. Li & Kurita.、黑籽重楼Paris thibetica Franch.、北重楼Paris verticillata M. Bieb.、南重楼Paris vietnamensis (Takht.) H. Li. 的根茎或全草。
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