CN117298178A - 一种黄酮提取物在制备预防或治疗酒精性肝损伤中的用途 - Google Patents
一种黄酮提取物在制备预防或治疗酒精性肝损伤中的用途 Download PDFInfo
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Abstract
本发明提供一种黄酮提取物在制备预防或治疗酒精性肝损伤药物中的用途。本发明通过动物实验对黄酮提取物治疗酒精性肝损伤的活性进行验证，发现本发明所述的黄酮提取物具有显著的酒精性肝损伤修复效果。
Description
技术领域
本发明涉及医药领域，具体涉及一种黄酮提取物在制备预防或治疗酒精性肝损伤药物中的用途。
背景技术
酒精性肝损伤，即酒精性肝病（alcoholic liver disease，ALD），是长期大量饮酒后酒精对肝脏产生毒性损伤并导致相关健康问题的疾病，在世界范围内ALD发病率呈逐年上升趋势，因此备受关注。临床上将ALD分为酒精性脂肪肝（alcoholic fatty liver，AFL）、酒精性肝炎（alcoholic hepatitis，AH）、酒精性肝纤维化(alcoholic liver fibrosis，ALF)和酒精性肝硬化（alcoholic cirrhosis，AC）。
威灵仙为毛茛科植物威灵仙Clematis chinensis Osbeck、棉团铁线莲Clematishexapetala Pall.或东北铁线莲Clematis manshurica Rupr.的干燥根和根茎，具有祛风湿、通经络之功效，临床上常用于治疗风湿痹痛、肢体麻木、筋脉拘挛与屈伸不利等症。但是传统中药疗法中，突出的不足之处是有效物质含量不高，且植物本身可能含有损伤肝肾的物质，由于制备方法粗犷没有去除，增加了药物不良反应的发生率。目前对威灵仙的研究，大部分集中于皂苷部位，黄酮类应用于解酒护肝的常见化合物为二氢杨梅素，二氢杨梅素即藤茶的主要活性成分，民间就有喝藤茶来清热解毒、抗炎、护肝的应用历史。目前对威灵仙黄酮类的活性研究较少，且未见威灵仙黄酮类应用于酒精性肝损伤的报道。
发明内容
本发明要解决的技术问题是提供了威灵仙总黄酮提取物在制备预防或治疗酒精性肝损伤或解酒保肝的药物中的用途，同时提供了所述威灵仙总黄酮提取物的制备方法，其总黄酮含量在70%以上，且纯化过程简单环保。
本发明威灵仙总黄酮提取物可以通过以下技术方案制备：
步骤1：取威灵仙药材，粉碎，用70％乙醇水溶液回流提取，收集提取液并浓缩得到浸膏；
步骤2：将浸膏用大孔树脂柱层析进行分离纯化，先后依次用水，20%，60%和95%的乙醇水溶液进行洗脱；
步骤3：收集60％乙醇洗脱液，减压浓缩至干得威灵仙总黄酮粗提取物。
优选的，威灵仙总黄酮提取物的制备方法还包括步骤4：用100℃沸水搅拌溶解步骤3所得威灵仙总黄酮粗提取物，趁热快速过滤后，滤液静置冷却至室温，再次过滤，所得固体即为威灵仙总黄酮精制提取物。
更优选的，步骤1中威灵仙药材与70%乙醇水溶液的重量比为1：8，提取温度为60℃，提取时间为40分钟，提取2次。
更优选的，步骤2中大孔树脂柱层析为LX20和D101混合树脂，混合体积比为LX20：D101=2：1。
更优选的，步骤4中所述沸水与E1重量比为30：1，搅拌时间为15分钟。
进一步地，所述药物还包括药学上可接受的载体。
进一步地，所述药物添加常规辅料，按照常规工艺制成临床上可接受的片剂、胶囊剂、口服液剂、散剂、滴丸剂、颗粒剂。
进一步地，所述酒精性肝损伤包括：酒精性脂肪肝、酒精性肝纤维化、酒精性肝炎、酒精性肝硬化。
本发明技术方案，具有如下优点：
本发明首次提出威灵仙总黄酮提取物在制备预防或治疗酒精性肝损伤或解酒保肝的药物中的用途。对实验大鼠酒精灌胃构建酒精大鼠模型，发现威灵仙总黄酮提取物对于酒精性肝损伤具有较好的修复效果。
同时本发明提供了威灵仙总黄酮提取物的制备方法，工艺简单易行，仅使用水和乙醇作为溶剂，安全，环保，最后使用沸水溶解冷却结晶的方式进一步提高了总黄酮含量，降低了纯化的成本。
具体实施方式
实施例1威灵仙总黄酮提取物制备及含量测定。
威灵仙总黄酮提取物的制备（黄酮粗提取物及黄酮精制提取物的制备）
取威灵仙药材5kg，粉碎，用40kg的70％乙醇水溶液60℃回流提取，提取2次，每次提取40分钟，合并两次提取液，减压浓缩为浸膏；用大孔树脂柱层析对浸膏进行分离纯化，层析柱直径为8cm，柱体积为3L，大孔树脂为LX20和D101混合树脂（混合体积比为LX20：D101=2：1），先后依次用水，20%，60%和95%的乙醇水溶液进行洗脱，每个梯度洗脱3BV，收集60％乙醇洗脱液，减压浓缩至干得威灵仙总黄酮粗提取物285g，取150g威灵仙总黄酮粗提取物用4.5L的100℃沸水搅拌15分钟（水保持微沸状态）溶解黄酮粗提取物，趁热迅速抽滤，所得滤液静置冷却至室温，再次抽滤，所得固体即为威灵仙总黄酮精制提取物78g。
总黄酮含量测定
仪器：岛津2700紫外可见分光光度计
标准曲线制备：精密吸取0.2mg/mL芦丁对照品溶液1，2，3，4，5，6mL，分别置于50mL容量瓶中。加入95%乙醇至总体积为15mL，依次加入10%硝酸铝溶液1mL，0.98%醋酸钾溶液1mL，摇匀，加水至刻度，静置一小时，于415nm处，以30%乙醇水溶液为空白，测定吸光度，以50mL中芦丁质量为横坐标，吸光度为纵坐标，绘制标准曲线。
试样制备：称取0.500g威灵仙总黄酮提取物，加入95%乙醇水溶液30mL，于65℃水浴中加热溶解后，用95%乙醇水溶液定容至50mL容量瓶得待测液，精密吸取待测液1.0mL，置于50mL容量瓶中，加入95%乙醇至总体积为15mL，依次加入10%硝酸铝溶液1mL，0.98%醋酸钾溶液1mL，摇匀，加水至刻度，静置一小时。空白试液：精密吸取待测液1.0mL，至于50mL容量瓶中，加95%乙醇至总体积为15mL，加水至刻度，摇匀。
以空白试液做参比，在波长415nm处测定试料溶液得吸光度，根据标准曲线求出试料溶液中得黄酮类化合物含量，并由此计算出实施例1中威灵仙总黄酮粗提取物中黄酮含量为73%，威灵仙总黄酮精制提取物中黄酮含量为87.5%。
实施例2威灵仙总黄酮提取物对酒精性肝损伤小鼠的作用。
1.主要试剂
受试物组：实施例1制备的威灵仙总黄酮粗提取物及威灵仙总黄酮精制提取物；对照组：二氢杨梅素。
2.药物配制
药物溶液配置：采用质量百分数为0.5%的羧甲基纤维素钠（CMC-Na）的水溶液分别将威灵仙黄酮粗提取物及威灵仙黄酮精制提取物，二氢杨梅素配制成药物浓度为50mg/mL的受试药物溶液。
3. 动物分组、造模及给药实验动物及饲养
8周龄SPF级雄性SD大鼠50只，体重180~220g，适应性喂养7天后，按体重随机分成5组，每组10只，分别为空白对照组，酒精模型组，威灵仙黄酮粗提取物组，威灵仙黄酮精制提取物组，二氢杨梅素对照组。其中空白对照组每天上午给予生理盐水，前2周生理盐水剂量8mL/kg，第3-6周，生理盐水剂量为12mL/kg；剩余3组为造模小鼠，每天上午给予50%酒精，前2周给酒精剂量8mL/kg，第3-6周给酒精剂量为12mL/kg；
造模2周后，第3周到第6周，每天下午给予治疗药物，给药剂量10mL/kg，其中空白对照组和酒精模型组给予10mL/kg生理盐水，威灵仙黄酮粗提取物组，威灵仙黄酮精制提取物组，二氢杨梅素对照组分别给予10mL/kg对应的受试药物溶液，具体给药方式采用灌胃给药，上午和下午的灌胃时间间隔约6h。
4.指标检测和方法
末次灌胃后禁食不禁水12小时，称重后异氟烷麻醉，腹主动脉取血，分离全血，全自动生化分析仪（AU480）进行检测血清谷草转氨酶（AST）、丙氨酸转氨酶（ALT）、甘油三酯（TG）、总胆固醇（TC）；10%肝组织匀浆测定丙二醛(MDA)。取血结束后，迅速解剖分离肝脏，冷生理盐水漂洗，滤纸拭干后称重，计算肝脏系数。
5.实验结果
各组SD大鼠生化指标及脏器系数如表1所示，由表1可知，酒精模型组大鼠各项指标和空白组相比均显著升高，说明造模成功，而和模型组相比，威灵仙总黄酮粗提取物组各项指标均得到显著改善，效果与对照组二氢杨梅素效果接近，且威灵仙总黄酮精制提取物组改善效果更佳。
表1 SD大鼠生化指标和脏器系数
| 组别 | AST | ALT | TG | TC | MDA | 肝脏系数 |
| 空白对照组 | 102.7±22.8 | 52.2±13.5 | 0.53±0.21 | 1.96±0.33 | 1.69±0.34 | 3.42±0.25 |
| 酒精模型组 | 183.3±35.6 | 100.3±17.2 | 1.24±0.28 | 3.67±0.52 | 2.86±0.47 | 4.28±0.29 |
| 威灵仙总黄酮粗提取物组 | 147.8±33.3** | 71.4±15.8** | 0.71±0.19*** | 2.78±0.46** | 2.32±0.39** | 3.89±0.28* |
| 威灵仙总黄酮精制提取物组 | 129.2±28.7*** | 55.5±16.6*** | 0.66±0.20*** | 2.51±0.42*** | 2.17±0.28*** | 3.61±0.31** |
| 二氢杨梅素对照组 | 143.5±37.0** | 65.3±12.5*** | 0.70±0.16*** | 2.96±0.35** | 2.46±0.31** | 3.78±0.22* |
注：
*表示与模型对照组相比，P<0.05
**表示与模型对照组相比，P<0.01
***表示与模型对照组相比，P<0.001。
Claims (5)
1.一种威灵仙总黄酮提取物在制备预防或治疗酒精性肝损伤或解酒保肝的药物中的用途，其特征在于，所述威灵仙总黄酮提取物中黄酮含量大于70%。
2.根据权利要求1所述的用途，其特征在于，所述威灵仙总黄酮提取物可通过以下步骤制备：
步骤1：取威灵仙药材，粉碎，用70％乙醇水溶液回流提取，收集提取液并浓缩得到浸膏；
步骤2：浸膏用大孔树脂柱层析进行分离纯化，先后依次用水，20%，60%和95%的乙醇水溶液进行洗脱；
步骤3：收集60％乙醇洗脱液，减压浓缩至干得威灵仙总黄酮粗提取物。
3.根据权利要求2所述的用途，其特征在于，所述威灵仙总黄酮提取物的制备还包括步骤4：用100℃沸水搅拌溶解步骤3所得威灵仙总黄酮粗提取物，趁热快速过滤后，滤液静置冷却至室温，再次过滤，所得固体即为威灵仙总黄酮精制提取物。
4.根据权利要求3所述的用途，其特征在于，步骤4中所述沸水与威灵仙总黄酮粗提取物重量比为30：1，搅拌时间为15分钟。
5.根据权利要求1-3任一所述的用途，其特征在于，所述酒精性肝损伤包括：酒精性脂肪肝、酒精性肝纤维化、酒精性肝炎、酒精性肝硬化。
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