CN114886906A - 黄酮糖苷组合物在制备预防或治疗脂代谢紊乱性疾病的药物中的应用 - Google Patents
黄酮糖苷组合物在制备预防或治疗脂代谢紊乱性疾病的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种黄酮糖苷组合物的新用途,所述黄酮糖苷组合物包括柳穿鱼叶苷和蒙花苷。通过肥胖小鼠的高胆固醇血症和肝脏脂肪变性模型,经实验验证,本发明的黄酮糖苷组合物能有效降低西方饮食诱导的肥胖小鼠体脂率和血清总胆固醇水平,改善肝脏脂肪变性,可作为活性成分应用于降高胆固醇血脂或改善肝脏组织脂肪变的药物和/或开发成有助于维持血脂健康水平的保健品和膳食补充剂。
Description
技术领域
本发明属于中药、天然药物和保健品技术领域,具体涉及一种黄酮糖苷组合物在制备预防或治疗脂代谢紊乱性疾病的药物中的应用。
背景技术
随着人类社会的发展,营养过剩使肥胖成为一大危害全球人类健康的重要因素。肥胖的发生往往伴随着多种糖脂代谢紊乱性疾病,包括高脂血症、非酒精性脂肪肝、动脉粥样硬化性心血管疾病等。高脂血症一般包括高胆固醇血症、高甘油三酯血症或混合型的血脂异常。糖脂代谢紊乱性疾病发病隐匿,机制较为复杂,且病程较长,往往进展到后期才被人所重视。流行病学证据显示,控制饮食和积极锻炼是防治糖脂代谢紊乱性疾病行之有效的方法。然而,面对好食肥甘、久坐少动的现代生活常态,有效的药物干预也不可或缺。高脂血症的治疗药物主要包括降脂类药物、抗氧化应激损伤类药物等。这些药物主要是通过减轻肥胖、降低血清总胆固醇和总甘油三酯、维持体内脂代谢稳态平衡等方式来改善和逆转病理进程。
近年来,中药或天然提取物,尤其是药食同源性植物提取物,以其副作用小的优势成为糖脂代谢紊乱性疾病预防和治疗药物研究中的热点。其中,许多植物类黄酮提取物以其具有的抗氧化、抑菌、抗炎、护肝等生物活性,被广泛地深入研究和开发。因此,基于饮食诱导的脂代谢紊乱动物模型,从食药两用资源中去寻找和发现具有防治高脂血症、脂质异位沉积的天然药物(植物药),以及具有维持血脂健康水平的保健品和膳食补充剂,有着重要的社会意义和应用价值。
大蓟Cirsium japonicum DC.,菊科蓟属植物,别名大刺儿菜、大刺盖、山萝卜、刺萝卜等,以其干燥地上部分入药,收载于中国药典I部(2020版),也是国家批准的保健食品原料(卫法监发[2002]51号)。本申请人前期主要聚焦于研究其化学成分,如CN202110800921.6公开了“采用大孔树脂串联动态轴向压缩柱分离纯化大蓟总苷的方法”。但是,迄今,尚未发现关于本发明所述的黄酮糖苷组合物在预防或治疗高胆固醇血症、非酒精性脂肪肝,以及在维持血脂健康水平的保健品和膳食补充剂方面的应用。
发明内容
本发明的首要目的在于提供一种黄酮糖苷组合物在制备预防或治疗脂代谢紊乱性疾病的药物中的应用。
本发明是通过以下技术方案实现:
一种黄酮糖苷组合物在制备预防或治疗脂代谢紊乱性疾病的药物中的应用,所述黄酮糖苷组合物包括柳穿鱼叶苷和蒙花苷。
本发明所述的黄酮糖苷组合物可参考CN202110800921.6从药食同源的大蓟中提取制备得到,优选的,基于黄酮糖苷组合物的总质量,柳穿鱼叶苷和蒙花苷的质量和占90%以上,其中,柳穿鱼叶苷占80~90%,蒙花苷占10~15%。
本发明所述的脂代谢紊乱性疾病包括高胆固醇血症或非酒精性脂肪肝。
本发明将上述黄酮糖苷组合物进行了生物活性评价研究,采用西方饮食诱导的C57BL6小鼠肥胖性肝脂肪变模型,通过测定体脂率、血清总胆固醇水平和ALT酶活性,以及肝组织病理学评价等指标,证实此黄酮糖苷组合物能有效降低模型小鼠的体脂率和血清总胆固醇水平,改善肝脏组织脂肪变,极具开发价值。
本发明以黄酮糖苷组合物为活性成分,加上药学上可接受的载体,通过本领域常规手段制备成预防或治疗脂代谢紊乱性疾病(饮食诱导的高胆固醇血症和肝脏脂肪变性)的药物剂型,适宜的剂型可以为片剂、胶囊剂、粉剂、颗粒剂、丸剂、散剂等。
本发明还提供了上述的黄酮糖苷组合物在制备维持血脂健康水平的保健品或膳食补充剂中的应用。
本发明与现有技术相比,具有如下有益效果:
本发明提供了一种黄酮糖苷组合物的新用途,通过肥胖小鼠的高胆固醇血症和肝脏脂肪变性模型,经实验验证,本发明的黄酮糖苷组合物能有效降低西方饮食诱导的肥胖小鼠体脂率和血清总胆固醇水平,改善肝脏脂肪变性,可作为活性成分应用于降高胆固醇血脂或改善肝脏组织脂肪变的药物和/或开发成有助于维持血脂健康水平的保健品和膳食补充剂。
附图说明
图1为实施例1中黄酮糖苷组合物的体外抗氧化活性和抗炎活性测试结果;
图2为实施例2的给药方案流程图;
图3为实施例2中黄酮糖苷组合物对西方饮食饲养小鼠的体脂率、血清胆固醇和肝脏ALT水平的影响;
图4为实施例2中黄酮糖苷组合物对西方饮食饲养小鼠的皮下脂肪组织形态学的影响图;
图5为实施例2中黄酮糖苷组合物对西方饮食饲养小鼠的肝组织病理学变化图;
图6为实施例3的给药方案流程图;
图7为实施例3中黄酮糖苷组合物苷对西方饮食饲养小鼠的体脂率、血清胆固醇和肝脏ALT水平的影响;
图8为实施例3中黄酮糖苷组合物对西方饮食饲养小鼠的皮下脂肪组织形态学的影响图;
图9为实施例3中黄酮糖苷组合物对西方饮食饲养小鼠的肝组织病理学变化图;
图10为实施例3中黄酮糖苷组合物对西方饮食饲养小鼠的血清代谢组学分析结果;
图11为实施例2和3中黄酮糖苷组合物与HMGCR和CYP7A1的分子对接结果。
具体实施方式
下面通过具体实施方式来进一步说明本发明,以下实施例为本发明具体的实施方式,但本发明的实施方式并不受下述实施例的限制。
本发明实施例所采用的黄酮糖苷组合物参照CN202110800921.6制备得到,其中基于黄酮糖苷组合物的总质量,柳穿鱼叶苷和蒙花苷的质量和占90%以上,其中,柳穿鱼叶苷占80~90%,蒙花苷占10~15%。
实施例1:黄酮糖苷组合物的体外抗氧化活性和抗炎活性
1.抗氧化活性
本发明所述黄酮糖苷组合物的抗氧化活性以其ORAC值表示。所用试剂以浓度为10mM的PBS(pH 7.4)为溶剂配制,现配现用。Trolox和样品分别加纯水或DMSO溶解,制备成母液,再用PBS梯度稀释成不同浓度的溶液。向黑色非透明平底96孔板中加入不同浓度的Trolox溶液(作阳性对照)、样品溶液或PBS(作空白对照)25 μL,再加浓度为10 nM的荧光素钠溶液150 μL,37 ℃下密封静置孵育30 min,迅速加入240 mM的AAPH溶液25 μL后,测定荧光强度,设定程序为激发波长485 nm、发射波长520 nm,每90 s循环测定1次,至荧光强度衰退到稳定为止。设置3个复孔。分别以Trolox和样品的相对荧光强度(relativefluorescence intensity)为纵坐标、时间(time)为横坐标作相对荧光强度衰退曲线图,ORAC值以Trolox当量表示,单位为μmol TE/g。
测定结果如图1A和图1 B所示,本发明所述黄酮糖苷组合物的ORAC值为4207 ±137 μmol TE/g。
2.抗炎活性
本发明所述黄酮糖苷组合物的抗炎化活性以其对环氧合酶-2(COX-2)的抑制效果来评价,具体操作按照COX-2抑制剂测定试剂盒使用说明执行。
测定结果发现50μg/mL的此组合物的抑制率为64.97%。此组合物和阳性药塞来昔布的剂量效应如图1C所示,相应的IC50值分别为32 μg/mL和144 nM。
由上述的体外实验证实:本发明所述黄酮糖苷组合物具有很好的抗氧化和抗炎活性。
实施例2:黄酮糖苷组合物预防给药降低饮食诱导肥胖小鼠体脂率和血清总胆固醇水平,改善肝脏脂肪变性
1、实验药品与试剂
小鼠血清总胆固醇(TC)和谷丙转氨酶(ALT)测定试剂盒购自上海荣盛生物药业有限公司。黄酮糖苷组合物(批号20211010,柳穿鱼叶苷占85.61%,蒙花苷占10.55%)。
2、实验动物
健康雄性C57BL/6J小鼠48只,5周龄,体重16-18g,SPF级,购自广东省实验动物中心,许可证号:SCXK(粤)2018-0002,合格证号:No. 44007200093110。动物饲养在广东药科大学实验动物中心SPF级动物房。动物饲养温度:20~25 ℃,湿度:40%-70 %,12 h : 12 h昼夜间断照明;自由进食饮水。本实验获得了广东药科大学实验动物伦理委员会批准,所有操作符合《广东药科大学实验动物管理条例》。
实验流程如图2所示。所有动物在适应性喂养1周后,根据小鼠体重分为对照组(Cont,n=8)、西方饮食模型组(WD,n=10)、预防给药高剂量组(Y-H,n=10)、预防给药低剂量组(Y-L,n=10)和阿托伐他汀钙组(A-tin,n=10)。对照组给予维持饲料和饮用水,其余各组给予高脂饲料(西方饮食模型饲料TP26304,南通特洛菲饲料科技有限公司)和果葡糖浆饮水(用果葡糖浆F55,加灭菌水稀释至果糖含量约为3.75%);但预防给药组同时灌胃相应剂量的组合物(低剂量为80mg/kg体重,高剂量为160mg/kg体重),阳性药组灌胃阿托伐他汀钙(立普妥,10mg/kg体重)。
3、实验方法
实验结束前两天,用活体动物体成份分析仪(Minispec LF90Ⅱ,德国Bruker公司)测定小鼠身体成分组成,其中包括脂肪质量(Fat mass,g)和瘦肉体重(Lean mass,g)。体脂率=脂肪质量/体重×100%,瘦体重指数=瘦体质量/体重×100%。
实验结束时,将小鼠麻醉,摘眼球取血,室温凝血后,离心,取血清,用商品试剂盒测定小鼠血清总胆固醇水平。
同时取小鼠肝脏和腹股沟皮下脂肪,将其中一部分用4%多聚甲醛保存用于组织切片,另一部分液氮速冻后-80℃冷冻。
取相同部位的肝组织,经常规4%多聚甲醛中固定,石蜡包埋,切片,H&E染色,光镜下观察。血清总胆固醇(TC),谷丙转氨酶(ALT)水平均使用商品试剂盒,根据说明书进行测定。
所有数据均采用平均值±标准差(`X± SD) 表示。采用SPSS 21.0软件进行统计分析,比较两组间的差异采用独立样本t检验分析,以p<0.05 表示两组间差异有统计学意义。
4、实验结果
研究发现,如图3所示,与Cont组相比,WD组小鼠体重和体脂率增加明显,在高剂量黄酮糖苷组合物的干预下,Y-H组小鼠体脂率有显著下降,瘦肉比重显著上升。说明此组合物有改善体脂率的作用。
同样,血生化分析结果表明,与Cont组相比,WD组小鼠小鼠血清胆固醇水平明显增加,说明西方饮食诱导的脂代谢异常;同时,ALT明显上升,说明肝功能受损。而在此黄酮糖苷组合物的干预下,Y-H组小鼠血清胆固醇水平和血清ALT相比模型组有显著降低且具有统计学意义(p<0.05)(图3),提示其对饮食诱导肥胖小鼠的肝脏有保护作用,且能有效降低血清胆固醇水平。
在预防性干预实验过程中,实施例中的WD组和Y-H,Y-L组小鼠,通过对肝脏组织切片的H&E染色观察(图4)、脂肪组织的H&E染色(图5)的病理学观察,发现此组合物可减轻小鼠肝脏脂肪变性,改善肝脏脂质蓄积。
实施例3:黄酮糖苷组合物治疗给药有效降低饮食诱导肥胖小鼠体脂率,改善肝组织脂肪变性,降低血清总胆固醇水平,其作用机制可能与抑制CYP7A1和HMGCR有关
1、实验药品与试剂
小鼠血清总胆固醇(TC)和谷丙转氨酶(ALT)测定试剂盒购自上海荣盛生物药业有限公司。黄酮糖苷组合物(批号20211011,柳穿鱼叶苷占85.90%,蒙花苷占10.02%)。
2、实验动物
健康雄性C57BL/6J小鼠48只,5周龄,体重16-18g,SPF级,购自广东省实验动物中心,许可证号:SCXK(粤)2018-0002,合格证号:No. 44007200093110。动物饲养在广东药科大学实验动物中心SPF级动物房。动物饲养温度:20~25 ℃,湿度:40%-70 %,12 h : 12 h昼夜间断照明;自由进食饮水。本实验获得了广东药科大学实验动物伦理委员会批准,所有操作符合《广东药科大学实验动物管理条例》。
所有动物适应性喂养1周后,根据小鼠体重、分为正常对照组(Cont,n=8)和西方饮食组(WD,n=40)。正常对照组给予维持饲料和饮用水,西方饮食组给予西方饮食模型饲料TP26304(南通特洛菲饲料科技有限公司)和果葡糖浆饮水(用果葡糖浆F55,加灭菌水稀释至果糖含量约为3.75%)。在10周后,依据体重和TC等指标再将西方饮食组分成WD组(WD,n=10)和治疗给药高剂量组(Z-H,n=10)、治疗给药低剂量组(Z-L,n=10)和阿托伐他汀钙组(A-tin,n=10)。WD组继续给予高脂饲料和果葡糖浆饮水,治疗给药组给予高脂饲料和果葡糖浆饮水,同时灌胃给予相应剂量的黄酮糖苷组合物(低剂量100mg/kg体重,高剂量200mg/kg体重),阳性药组给灌胃阿托伐他汀钙(立普妥,10mg/kg体重),具体给药方案如图6所示。
3、实验方法
实验结束前两天,用活体动物体成份分析仪(Minispec LF90Ⅱ,德国Bruker公司)测定小鼠身体成分组成,其中包括脂肪质量(Fat mass,g)及瘦体重(Lean mass,g)。体脂率=脂肪重量/体重×100%,瘦体重指数=瘦体重量/体重×100%。
实验结束时,将小鼠麻醉,摘眼球取血,自然凝血后,离心,取血清,分装,用商品试剂盒测定小鼠血清总胆固醇水平。
同时取小鼠肝脏、腹股沟皮下脂肪,将其中一部分用4%多聚甲醛保存用于组织切片,其余部分-80℃冰箱冻存。
取相同部位的肝组织,经常规4%多聚甲醛中固定,石蜡包埋,切片,H&E染色,光镜下观察。血清TC和ALT水平等均使用商品试剂盒,根据说明书进行测定。
采用基于GC-MS的血清代谢组学分析和生物信息学分析,探究黄酮糖苷小分子组合物的代谢调控通路。
所有数据均采用平均值±标准差(`X± SD) 表示。采用SPSS 21.0软件进行统计分析,比较两组间的差异采用独立样本t检验分析,以p<0.05 表示两组间差异有统计学意义。
4、实验结果
研究发现,在治疗性干预实验过程中,如图7所示,与Cont组相比,WD组组小鼠体重和体脂率增加明显,在高剂量黄酮糖苷组合物的干预下,Z-H组小鼠体脂率有显著下降,瘦肉比重显著上升。说明此组合物有改善体脂率的作用。
血生化分析结果表明,与Cont组相比,WD组小鼠血清胆固醇水平较正常对照组明显增加,说明西方饮食摄入导致脂代谢异常;同时,ALT明显上升,说明肝功能受损。而在此黄酮糖苷小分子组合物的干预下,Z-H组小鼠血清胆固醇水平和血清ALT相比模型组有显著降低且具有统计学意义(p<0.05)(图7),提示其对饮食诱导肥胖小鼠的肝脏损伤有保护作用,且能有效降低血清胆固醇水平。
同样,通过对WD组和Z-H组小鼠的肝脏(图8)和脂肪组织(图9)的病理切片观察,发现组合物可显著减轻肝脏脂肪变性。代谢组学分析结果(图10)和分子对接分析结果发现作用机制可能与其抑制CYP7A1(图11A)和HMGCR(图11B)活性,调控初级胆汁酸生物合成和胆汁分泌通路等有关。
综上,本发明通过肥胖小鼠的高胆固醇血症和肝脏脂肪变性模型,证实了无论在预防性干预,还是在治疗性干预的情形下,本发明所述的黄酮苷组合物均能有效降低饮食诱导模型小鼠体脂率和血清总胆固醇水平,改善肝脏组织脂肪变性。因此,本发明所述的黄酮苷组合物可作为活性成分应用于降高胆固醇血脂或改善肝脏组织脂肪变的药物,以及开发成有助于维持血脂健康水平的保健品和膳食补充剂。
Claims (6)
1.一种黄酮糖苷组合物在制备预防或治疗脂代谢紊乱性疾病的药物中的应用,所述黄酮糖苷组合物包括柳穿鱼叶苷和蒙花苷。
2.一种黄酮糖苷组合物在制备维持血脂健康水平的保健品或膳食补充剂中的应用,所述黄酮糖苷组合物包括柳穿鱼叶苷和蒙花苷。
3.根据权利要求1或2所述的应用,其特征在于,基于黄酮糖苷组合物的总质量,柳穿鱼叶苷和蒙花苷的质量和占90%以上,其中,柳穿鱼叶苷占80~90%,蒙花苷占10~15%。
4.根据权利要求1所述的应用,其特征在于,所述脂代谢紊乱性疾病包括高胆固醇血症或非酒精性脂肪肝。
5.根据权利要求1所述的应用,其特征在于,所述药物是以黄酮糖苷组合物为主要活性成分及药学上可接受的载体。
6.根据权利要求1所述的应用,其特征在于,所述药物的剂型为片剂、胶囊剂、粉剂、颗粒剂、丸剂或散剂。
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CN101112407A (zh) * | 2007-08-03 | 2008-01-30 | 安徽医科大学 | 野菊花乙醇提取物在制备防治酒精性肝病的药物中的应用 |
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CN112516152A (zh) * | 2020-11-18 | 2021-03-19 | 广东药科大学 | 大蓟苷在制备治疗糖脂代谢病药物中的应用及其组合物 |
CN113440547A (zh) * | 2021-07-15 | 2021-09-28 | 广东药科大学 | 采用大孔树脂串联动态轴向压缩柱分离纯化大蓟总苷的方法 |
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CN112516152A (zh) * | 2020-11-18 | 2021-03-19 | 广东药科大学 | 大蓟苷在制备治疗糖脂代谢病药物中的应用及其组合物 |
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