CN110585250B - 长白落叶松树根总黄酮在抑制尿酸升高和预防治疗痛风疾病中的应用 - Google Patents
长白落叶松树根总黄酮在抑制尿酸升高和预防治疗痛风疾病中的应用 Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
Abstract
本发明涉及长白落叶松(Lanix olgensis Henry)树根总黄酮在抑制尿酸升高和预防治疗痛风方面的应用。本发明长白落叶松树根总黄酮属于纯天然产物，可以直接取材长白落叶松树根、也可以为木材加工后的副产物经过提取纯化制备得到，经研究证明长白落叶松树根中含有丰富的黄酮类物质，并具有抑制尿酸升高作用，可制备预防治疗痛风疾病的药物及保健食品。
Description
技术领域
本发明涉及长白落叶松树根总黄酮的提取制备方法，经药理实验证明具有新的医药用途。具体的说是长白落叶松树根总黄酮具有抑制尿酸升高的作用，具有预防和治疗痛风疾病的作用，属于中药领域。
背景技术
痛风是有疼痛的致残性急性发作特征的炎症性关节炎，它是由高尿酸血症和关节周围的尿酸盐晶体沉积引起的症状。高尿酸血症是痛风的主要病因。研究表明，高尿酸血症与尿酸盐转运蛋白的表达和功能改变有关。血清尿酸水平长期增加或患有高尿酸血症是痛风形成的主要病理条件。
流行病学调查数据表明，近年来，中国人群中高尿酸血症的发病率也呈直线上升且年轻化趋势，从1996-2006年，我国的高尿酸血症发病率约增加了10倍，高尿酸血症已成为继高血压、高血脂、高血糖之后的临床“第四高”。据保守估计，目前我国高尿酸血症患者人数已达1.2亿，约占人口总数的9.0％。高尿酸血症因无明显临床症状而不被人们重视。但事实上，长期高尿酸血症对人体的主要影响之一是尿酸盐结晶沉积在关节及肾脏而引起的痛风。高尿酸血症是引起痛风的重要生化基础，约5％～12％的高尿酸血症患者会发展成为痛风。痛风不仅可以侵犯骨和关节，还容易累及肾脏和心血管系统。更为重要的是，国内外大量流行病学研究表明，高尿酸血症与代谢综合征的许多成分如肥胖、高血压、血脂异常、高血糖症、冠心病及胰岛素抵抗等密切相关，并已成为代谢综合征的一部分，其与肥胖、高脂血症、高血压、糖尿病、动脉粥样硬化等疾病呈显著正相关。与高血压、胰岛素抵抗，肥胖、高脂血症、糖耐量异常等同等协同作用，加重动脉硬化，提高心脑血管疾病的发生率。由此可见，高尿酸血症已成为威胁人类健康的重要疾病。
高尿酸血症长期以来几乎完全依赖别嘌呤醇，但临床上有相当比例的高尿酸血症患者接受别嘌呤醇治疗不能达到血清尿酸浓度的推荐控制目标。此外，尽管别嘌呤醇的安全性较好，但其在肾功能受损患者中的副反应发生率及严重程度均有提高。较新的降尿酸药物如非布司他和培格洛替酶，临床应用中也有一定的副反应，如肝酶水平升高、皮疹、腹泻、头痛以及恶心、挫伤/瘀斑、鼻咽炎、便秘和呕吐等。因此，寻找新的安全、有效的降尿酸功能性成分具有重要的现实意义和应用价值。
长白落叶松(Lanix olgensis Henry)是松科落叶松属的落叶乔木，是我国东北、内蒙古大兴安岭林区的主要森林组成树种，其面积约占大兴安岭林地面积的70％。长白落叶松树根中含有大量的原花青素、黄酮等化合物，具有抗氧化、抗癌、降低胆固醇、保护心血管等功效，是很有利用价值的天然产物。
黄酮类化合物也是长白落叶松树根提取物中非常重要的一类化合物，对植物的生长、发育、开花、结果及防菌防病等方面有着十分重要的作用。而且黄酮类化合物生理活性多种多样，具有心血管系统活性、抗菌及抗病毒活性、抗肿瘤活性、抗氧化自由基活性、抗炎镇痛活性及保肝活性和抗疲劳作用，此外还有降压、降血脂、抗衰老、提高机体免疫力等药理活性。另外，随着食品工业的发展与消费观念的改变，天然活性成分的保健食品成为现代人追逐的目标，其中黄酮类化合物以纯天然、高活性、见效快、作用广泛等特点日益受到人们的关注。黄酮类化合物的提取方法主要有溶剂法、微波提取法、超声波提取法、超临界流体萃取法、酶法提取、双水相提取法、半仿生提取法、膜分离法、热压流体萃取法、高压液相提取法等。
本发明可进一步扩大长白落叶松树根的开发和利用，具有良好的社会效益和可观的经济效益。
本发明完成前，还未有长白落叶松树根总黄酮的制备方法及用于抑制尿酸升高的报道。
发明内容
发明的目的之一在于提供长白落叶松树根总黄酮的新用途，具体是一种长白落叶松树根总黄酮在控制尿酸升高中的应用，更具体地是长白落叶松树根总黄酮在预防和治疗高尿酸血症和痛风的药物及保健品中的应用。
本发明目的之二是落叶松树根总黄酮是从松科植物长白落叶松 (Lanixolgensis Henry)的树根中经过提取、纯化得到，总黄酮含量在85％以上。
本发明突出贡献在于：从天然资源长白落叶松树根中富集总黄酮，得到落叶松树根总黄酮，具有抑制尿酸升高的作用。提供一种新药用资源及新医药用途，这是本发明最突出的贡献及最显著的创新性。
本发明首次采用了水浸泡除去皂苷、水溶性多糖等杂质成分，然后利用含氧化钙的乙醇提取纯化，提取液不经过浓缩，直接经过大孔树脂柱吸附纯化，解决了乙醇提取液浓缩后具有大量析出物，不利于在树脂柱上吸附纯化的问题。本发明先用水浸泡，除去大量水溶性杂质，然后用含氧化钙的乙醇提取，除去一些酚酸类及鞣质类成分，提取液通过树脂柱除掉脂溶性杂质成分，也是本发明的创新点之一。
本发明的另一目的是提供一种从长白落叶松树根中提取总黄酮的制备方法，其特征在于：将长白落叶松树根粗粉用35-50℃温水浸提1-3次，每次8-12小时，滤过，收取浸提后的落叶松树根粗粉，用 4-8倍量含1-3％氧化钙的60-90％乙醇提取1-3次，滤过，合并滤液，用乙醇体积1-2％的活性炭脱色，过滤，得乙醇提取液；取乙醇提取液，通过预先用40-80％乙醇处理好的AB-8大孔树脂吸附树脂，再用 1-3倍柱体积的40-80％乙醇洗脱，收集流出液及洗脱液，回收乙醇，滤过，滤饼水洗，干燥，得长白落叶松树根总黄酮。
进一步优选：将落叶松树根粗粉用40℃温水浸提3次，每次12 小时，滤过，收取浸提后的落叶松树根粗粉，用6倍量含2％氧化钙的60％乙醇提取3次，滤过，收集乙醇提取液，用乙醇体积2％的活性炭脱色，过滤，得乙醇提取液；取乙醇提取液，通过预先用60％乙醇处理好的AB-8大孔树脂吸附树脂，再用1倍柱体积的60％乙醇洗脱，收集流出液及洗脱液，回收乙醇，滤过，滤饼水洗，干燥，得长白落叶松树根总黄酮。
由于本发明首次公开了长白落叶松树根总黄酮具有抑制尿酸升高的作用，因此，将长白落叶松树根总黄酮单独或与其它活性组份或辅料配合制成药剂，只要是该药剂具有抑制尿酸升高的作用，均属于本发明的保护范围。本发明的组合物在制成任何一种剂型时，均具有抑制尿酸升高的作用，均在本发明的保护范围。
为实现以上技术方案，长白落叶松树根总黄酮含量通过以下方法测定：
对照品溶液的制备精密称取芦丁对照品10mg，置100mL量瓶中，用甲醇溶解，并稀释至刻度，摇匀，精密量取5mL置10mL量瓶中，用甲醇稀释至刻度，摇匀，即得。
供试品溶液的制备取长白落叶松树根总黄酮粉末，精密称定 50mg，至50mL容量瓶中，加甲醇适量，超声处理20min，取出，放置室温，加甲醇定容至刻度，摇匀，取2mL至25mL容量瓶中，加甲醇定容至刻度即得。
测定法分别取对照品溶液、供试品溶液、随行空白。照分光光度法(《中国药典》2010年版一部附录V B)，在波长340nm处分别测定吸收度，计算。
长白落叶松树根总黄酮(简称总黄酮)具有预防和治疗高尿酸血症的作用，这些药理作用，通过以下药效学试验例得到证实。
实验原料：本药效学中用到的长白落叶松树根总黄酮为本发明人提供。按照实例1制备而成。本药效学试验中用到的别嘌呤醇为广西彼迪药业有限公司生产(批号为：20180301)。
1、急性毒性实验研究
选用小白鼠20只，18～20g，雌雄各半。以动物能耐受的最大给药浓度(0.5g/mL)、最大给药容积(0.4mL/10g体重)一日内灌胃给药1次，连续观察7天内有无毒性反应及动物死亡。测得结果是：小鼠7天内动物均无一死亡，在此剂量下未观察到明显急性毒性反应。其小鼠最大给药量为10g药粉/kg体重/d。按70kg体重计算，该给药剂量约为临床日拟用量100倍。
2、长白落叶松树根总黄酮对小鼠高尿酸血症(氧嗪酸钾盐法)的影响
取昆明种小鼠60只。雌雄各半。随机分为6组(正常对照组，模型组，总黄酮高、中、低剂量组，别嘌醇组)。按组给药5天，每天1 次。末次给药1小时后，除空白组外，其余各组动物腹腔注射氧嗪酸钾盐300mg/kg，造成小鼠高尿酸血症。注射1h后，各组动物摘眼球取血，2500r/min离心5min，取血清测血尿酸值，进行组间差异性比较。结果见表1。
表1长白落叶松树根总黄酮对小鼠高尿酸血症的影响(n＝10，X±S)
注：△与空白组比较P<0.01，**与模型组比较P<0.01。
试验结果表明，长白落叶松树根总黄酮各剂量组对小鼠(氧嗪酸钾盐)所致高尿酸血症模型血尿酸有良好的抑制作用。
3、长白落叶松树根总黄酮对小鼠血尿酸(黄嘌呤法)的影响
取60只雄性小鼠，随机分为6组(正常对照组，模型组，总黄酮高、中、低剂量组，别嘌醇组)，每组10只。每日均按0.1mL·l0g灌胃。6d后，除正常对照组外，其他5组小鼠分别腹腔注射10％黄嘌呤 0.1mL·l0g，30min后取血测血尿酸值。
表2长白落叶松树根总黄酮对小鼠血尿酸的影响(n＝10，X±S)
注：△与空白组比较：P<0.01，**与模型组比较P<0.01。
模型组血尿酸含量显著高于正常对照组，差异有显著意义 (P<0.01)，用药组与模型组比较血尿酸含量差异非常显著，均低于模型组(P<0.0l)。进一步说明长白落叶松树根总黄酮降低血尿酸含量的机制与别嘌呤醇一样，是减少尿酸生成，减轻其对组织的损害，从而有效控制痛风发作。
从以上的毒性及药效学实验可知，长白落叶松树根总黄酮具有抑制尿酸生成的作用，从而可以治疗和预防痛风，长期服用没有副作用，并且可以制成口服制剂，服用方便。
实施例
本发明是通过如下的实验施例得以实现(证实)，但不仅限于本实例。
实例1长白落叶松树根总黄酮的制备方法
将长白落叶松树根粗粉20kg，用40℃温水浸提3次，每次12小时，滤过，收取浸提后的长白落叶松树根粗粉，用6倍量含2％氧化钙的60％乙醇提取3次，滤过，收集乙醇提取液，用乙醇体积2％的活性炭脱色，过滤，得乙醇提取液；取乙醇提取液，通过预先用60％乙醇处理好的AB-8大孔树脂吸附树脂，再用1倍柱体积的60％乙醇洗脱，收集流出液及洗脱液，回收乙醇，滤过，滤饼水洗，干燥，得落叶松树根总黄酮1232g，总黄酮含量94.6％。
实施例2(胶囊剂)
处方：长白落叶松树根总黄酮：200g
淀粉：40g
羧甲基淀粉钠：10g
3％羟兵甲基纤维素水溶液适量
长白落叶松树根总黄酮、淀粉、羧甲基淀粉钠，混合均匀，用 3％羟丙甲基纤维素水溶液制粒，干燥，整粒，装入0号胶囊，制成 1000粒，即得。每粒含落叶松树根总黄酮200mg。每次2粒。每日2 次。
实施例3(片剂)
处方：长白落叶松树根总黄酮：200g
乳糖：40g
羧甲基淀粉钠：7.5g
3％羟丙甲基纤维素水溶液适量
滑石粉：2.5g
长白落叶松树根总黄酮、乳糖、羧甲基淀粉钠，混合均匀，用 3％羟丙甲基纤维素水溶液制粒，干燥，整粒，加入滑石粉混匀，压片，制成1000片，即得。每片含落叶松树根总黄酮200mg。每次2 片。每日3次。
实施例4(颗粒剂)
处方：长白落叶松树根总黄酮：200g
糖粉：300g
长白落叶松树根总黄酮、糖粉，混合均匀，用适量乙醇制粒，干燥、整粒、分装，制成500袋，即得。每次1袋。每日2次。
实施例5(滴丸剂)
处方：长白落叶松树根总黄酮：20g
聚乙二醇6000：25g
聚乙二醇4000：15g
称取25g聚乙二醇6000，10g聚乙二醇4000在水浴上熔化，加入长白落叶松树根总黄酮15g，搅拌均匀，倾入保温管中，调节恒温装置，使药液在80-90℃下滴入冷却过的液体石蜡(12-14℃)中，滴完后，将药丸倾入滤纸上吸干石蜡油，制成1000粒，即得。每次20粒。每日 2次。
Claims (3)
1.一种抑制尿酸升高及预防治疗痛风的长白落叶松(Lanix olgensis Henry)树根总黄酮，其特征在于，长白落叶松树根总黄酮是由以下方法提取纯化：
(1)将长白落叶松树根粗粉用35-50℃温水浸提1-3次，每次8-12小时，滤过，收取浸提后的长白落叶松树根粗粉，用4-8倍量含1-3％氧化钙的60-90％乙醇提取1-3次，滤过，合并滤液，用乙醇体积1-2％的活性炭脱色，过滤，得乙醇提取液；
(2)取乙醇提取液，通过预先用40-80％乙醇处理好的AB-8大孔树脂吸附树脂，再用1-3倍柱体积的40-80％乙醇洗脱，收集流出液及洗脱液，回收乙醇，滤过，滤饼水洗，干燥，得长白落叶松树根总黄酮。
2.根据权利要求1所述的一种抑制尿酸升高及预防治疗痛风的长白落叶松树根总黄酮，根据药剂学要求，制成片剂、胶囊剂、颗粒剂、粉剂、口服液中的任何一种口服制剂。
3.根据权利要求1所述的一种抑制尿酸升高及预防治疗痛风的长白落叶松树根总黄酮在制备抑制尿酸升高及预防治疗痛风的药品中的应用。
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