CN110123859B - 具有防治肝损伤的菊叶提取物及其应用 - Google Patents
具有防治肝损伤的菊叶提取物及其应用 Download PDFInfo
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- CN110123859B CN110123859B CN201910563890.XA CN201910563890A CN110123859B CN 110123859 B CN110123859 B CN 110123859B CN 201910563890 A CN201910563890 A CN 201910563890A CN 110123859 B CN110123859 B CN 110123859B
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Abstract
本发明公开了具有防治肝损伤的菊叶提取物及其应用,该发明主要由菊叶的乙醇提取物经过有机溶剂萃取脱脂、大孔吸附树脂和中压色谱分离凝胶MCI富集精制而成,通过大量试验筛选制备得到,无毒副作用、健康安全,实验结果表明本发明提取物能够改善D‑氨基半乳糖所致化学性肝损伤及对乙酰氨基酚所致药源性肝损伤,显著降低血清中转氨酶水平和脂质过氧化损伤,增加机体谷胱甘肽含量,从而具有肝损伤保护作用。本发明提供的制备方法简单、便捷,自动化程度高,能很好地保留原料中的有效成分,无毒副作用,可以用于制备防治肝损伤的药物及保健品。
Description
技术领域
本发明涉及一种天然药食同源植物传统非药用部位的综合开发利用,特别是涉及一种菊叶提取物及其在制备防治肝损伤药物及保健品中的应用。
背景技术
肝脏作为全身最大的实质性器官和主要的解毒器官,担负人体的重要生理功能,如合成肝糖原和凝血因子、异物代谢、激素代谢、储存脂溶性维生素、产生胆汁等。由于肝脏承担众多生理功能及其充沛的血流量,肝脏同时容易受到内、外各种致病因素的侵袭而受到损失。根据病因的不同,肝损伤主要包括病毒性肝炎(乙肝、丙肝等)、酒精性脂肪肝、非酒精性脂肪肝、化学性肝损伤、药源性肝损伤等。目前,虽然有逍遥丸、丹栀逍遥片、疏肝解郁胶囊等养肝护肝的中成药,但柴胡、何首乌、雷公藤等中药已经被明确报道具有肝毒性,服用含有肝毒性的中药的中成药养肝护肝可能进一步引发中药药源性肝损伤。
菊花始载于《神农本草经》,列为上品,“久服利血气,轻身延年”,历来作为具有清肝明目功效的代表性药食同源品种。菊叶为菊科植物菊(Chrysanthemum morifoliumRamat.)的叶子,据《本草纲目》记载菊“茎叶根实并同……其苗可蔬,叶可啜,花可饵,根实可药,囊之可枕,酿之可饮”可知,菊叶自古既是食品又是药品,对机体安全性较高,适合开发为保健食品和功能食品。据调研,在菊花采收过程中,大量依然苍翠的菊叶被废弃,其中富含大量的木犀草素、香叶木素等黄酮类成分及绿原酸、二咖啡酰基奎宁酸等酚酸类成分,研究表明该类黄酮类和酚酸类成分具有显著的肝损伤保护作用。
肝损伤的机制之一是组织过氧化损伤,D-氨基半乳糖(D-GalN)是肝细胞磷酸尿嘧啶核苷干扰剂,其能够与肝细胞内的尿苷二磷酸(UDP)结合形成UDP-半乳糖胺复合物,使尿苷三磷酸(UTP)耗竭,从而使尿苷类化合物无法环化,最终导致肝细胞坏死。此外,GalN可引起肝细胞内钙-镁比例失调,Ca2+内流增加,导致胞内Ca2+增加、Mg2+减少。胞内Ca2+增加可抑制线粒体功能,激活磷脂酶,分解膜磷脂,破坏溶酶体膜,使蛋白水解酶释放,加速氧自由基的产生和脂质过氧化反应,进一步加剧肝细胞损伤。作为常用的非甾体类解热镇痛药,对乙酰氨基酚(acetaminophen,APAP)诱导的肝损伤是药源性肝损伤的代表,也是筛选保肝药物的常用实验模型。APAP在肝细胞内经CYP450酶系代谢转化生成高活性的中间代谢产物N-乙酰-对-苯醌亚胺(NAPQI),可通过坏死和凋亡的方式造成肝细胞的死亡,进而活化人体自然杀伤细胞、中性粒细胞,使炎性因子过表达,从而激活固有免疫系统;此外,APAP在肝内代谢过程中产生自由基可引起肝细胞膜脂质过氧化,并可通过破坏钙稳态而产生细胞毒性,导致肝损伤,尤以肝脏小叶中央型坏死为常见。
本发明通过对菊叶活性成分进行提取、富集和精制,将其应用于制备防治肝损伤的药物及保健品开发中,既充分发掘菊的资源价值、延伸菊产业链、实现菊资源的综合利用和产业绿色发展,同时减少菊花采收过程中大量菊叶浪费。但是,如何对菊叶进行系统研究,尤其是将其应用于防治肝损伤的药物和保健品开发,目前并无相关研究。因此,对菊叶进行分离、富集和纯化,制备其防治肝损伤的活性成分,具有重要的临床意义。
发明内容
发明目的:本发明的目的在于提供一种具有防治肝损伤的菊叶提取物,其能够对化学性肝损伤和药源性肝损伤在内的各种肝损伤具有良好的防治作用;本发明的另一目的是提供上述菊叶活性提取物的制备方法和应用。
技术方案:为了实现以上目的,本发明所述的具有防治肝损伤的菊叶提取物的制备方法,其包括以下步骤:
一种具有防治肝损伤的菊叶活性提取物,它由下述制备方法得到:
(1)取药用菊花叶,去除杂质、干燥、粉碎,投入提取罐中,加入5~20倍量的体积分数50%~95%乙醇,加热回流提取1~3次,过滤、合并提取液,减压回收乙醇至无醇味后,得菊叶乙醇提取物浸膏,备用;
(2)取步骤(1)得到的乙醇提取物浸膏,加入10~20倍体积量的水混悬均匀,加入5~20倍有机试剂萃取1~3次脱除叶绿素和蜡质,取萃取后余液,备用;
(3)取步骤(2)得到的萃取后余液,上聚酰胺柱色谱分离,先用水洗脱,再用体积分数为20%~95%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位粗提物;
(4)取步骤(3)得到的菊叶活性部位粗提物,上中压色谱分离凝胶柱分离,先用水洗脱,再用体积分数为10%~85%乙醇洗脱,并收集体积分数为10%~85%乙醇洗脱液,浓缩,经减压干燥得到菊叶活性提取物。
作为优选方案,所述的具有防治肝损伤的菊叶提取物,步骤(1)中菊叶干燥方式为为阴干、晒干、40-60℃热风干燥、微波干燥、冷冻干燥,特别优选45℃热风干燥。
作为优选方案,所述的具有防治肝损伤的菊叶提取物,步骤(2)中有机试剂为正己烷、环己烷、二氯甲烷、石油醚,特别优选二氯甲烷。
作为优选方案,所述的具有防治肝损伤的菊叶提取物,步骤(3)中聚酰胺粒径为60-400目,特别优选60-80目。
作为优选方案,所述的具有防治肝损伤的菊叶提取物,步骤(4)中的中压色谱分离凝胶柱(MCI-GEL)型号为CA、CDR、CHP、CK、CQH、CQP、SCA,特别优选CHP。
作为优选方案,以上所述的菊叶提取物,其特征在于,步骤(2)中所述步骤为:取步骤(1)得到的菊叶醇提浸膏,加入15倍量水混悬均匀后,依次加入10倍、8倍、6倍量的二氯甲烷萃取,取萃取后余液,备用。二氯甲烷萃取3次基本能够出去菊叶中大部分的叶绿素、蜡质、脂肪酸等脂溶性杂志,从而能够与黄酮类、酚酸类活性成分分离。
作为优选方案,以上所述的菊叶提取物,其特征在于,步骤(3)中聚酰胺树脂的粒径为60-80目,富集步骤为:先用水洗脱除去水溶性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位粗提物。通过聚酰胺填料中的酰胺键的氢键作用,可以将菊叶中的酚酸类、黄酮苷类活性成分富集。
作为优选方案,以上所述的菊叶提取物,其特征在于,步骤(4)中MCI树脂型号为反相CHP型,精制步骤为:先用体积分数10%的乙醇洗脱除去大极性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位提取物。反相型MCI与聚酰胺的串联使用结合反相色谱的疏水相互作用和聚酰胺的氢键吸附作用,使得活性提取物纯度更高。10%的乙醇洗脱可以进一步去除水溶性单、寡糖、氨基酸等,40%的乙醇洗脱可进一步纯化酚酸类、黄酮苷类活性成分,将其与保留在柱子上的脂溶性成分进一步分离。
一种具有防治肝损伤的菊叶活性提取物,它由下述制备方法得到:
(1)取新鲜菊叶,去除杂质,45℃热风干燥后适当粉碎,投入提取罐中,依次加入药材重量20倍、15倍、10倍体积量的95%乙醇,加热回流提取3次,依次为2小时、1.5小时、1小时。过滤、合并三次提取液,减压回收乙醇至无醇味后,得菊叶醇提浸膏,备用;
(2)取步骤(1)得到的菊叶醇提浸膏,加入15倍量水混悬均匀后,依次加入10倍、8倍、6倍量的二氯甲烷萃取,取萃取后余液,备用;
(3)取步骤(2)得到的菊叶萃取后余液,上粒径为60-80目的聚酰胺柱,先用水洗脱除去水溶性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位粗提物;
(4)取步骤(3)得到的菊叶活性部位粗提物,上中压色谱分离凝胶柱MCI-CHP树脂柱,先用体积分数10%的乙醇洗脱除去大极性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位提取物。
本发明对菊叶提取物中各原料所含成分进行分析:以绿原酸为对照品,采用福林-酚法,在760nm处测定吸光度,测得所制备的菊茎叶活性部位中总酚酸的含量为49.26%以上;以芦丁为对照品,采用硝酸铝-亚硝酸钠比色法,应用紫外-可见分光光度在510nm处测定吸光度,采用该方法可知所制备的菊叶活性部位中总黄酮的含量为31.21%以上。由此可知,制备得到的菊叶活性部位中总黄酮类和总酚酸类成分含量之和大于等于80.47%,有效成分含量高。
本发明所述的具有防治肝损伤的菊叶提取物在制备保肝药物及保健品中的应用。
本发明所述的具有防治肝损伤的菊叶提取物在制备防治化学性肝损伤药物及保健品中的应用。
本发明所述的具有防治肝损伤的菊叶提取物在制备防治药源性肝损伤药物及保健品中的应用。
本发明所述的具有防治肝损伤的菊叶提取物与药学上可接受的载体制备成药品、保健食品或功能食品。
一种药物制剂,其本发明所述的防治肝损伤的菊叶活性提取物与药学上可接受的载体制成颗粒剂、片剂、丸剂、散剂、口服液等。
有益效果:本发明提供的防治肝损伤的菊叶活性提取物在菊叶传统应用基础上进行新的用途开发,和现有技术相比具有以下优点:
(1)本发明主要以药用菊花采收过程中产生的废弃物菊叶为原料,制备其活性提取物,开发其新用途,实现变废为宝,可延伸菊产业链,可实现菊资源的综合利用和产业的提质增效,具有很好的经济效应、社会效益、生态效益和环境效益,符合国家的可持续发展战略。
(2)实验结果表明,本发明提供的菊叶活性提取物可以防治化学性肝损伤、药源性肝损伤等诸多因素引起的肝细胞受损,能够显著改善上述疾病的各种症状及临床指标。对D-氨基半乳糖所致小鼠化学性肝损伤,菊叶活性提取物能够降低模型组小鼠肝组织MDA、增加GSH,同时降低血清中AST和ALT,通过提高模型组小鼠抗氧化能力以对抗化学性肝损伤。在对乙酰氨基酚致小鼠药源性肝损伤中,菊叶活性提取物能够减少肝组织中MDA和GSH含量,同时降低血清中AST和ALT,通过纠正紊乱的生化代谢从而防治对乙酰氨基酚导致的药源性肝损伤。
(3)本发明通过比较优选出最佳的制备工艺自动化程度高,可操作性强,工艺路线简单,精制的菊叶活性成分纯度高,杂质少,活性更显著,无毒副作用;制备工艺能源消耗少,环境友好。
具体实施方式
下面结合具体实施例进一步阐明本发明,应理解这些实施例仅用于说明本发明而不用于限制本发明的范围,在阅读了本发明之后,本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
本发明所述的药材和乙醇用量均指得是重量体积比。乙醇均指的是体积分数。
实施例1
1、一种具有防治肝损伤的菊叶活性提取物的制备方法,其包括以下步骤:
(1)取新鲜菊叶,阴干,适当粉碎,置于提取罐中,加入药材重量15倍体积量的95%乙醇,加热回流提取3次,每次1小时,过滤、合并提取液,减压回收乙醇至无醇味后,得菊叶醇提浸膏,备用;
(2)取步骤(1)得到的乙醇提取物浸膏,加入10倍体积量的水混悬均匀,加入10倍体积的正己烷萃取1次,取萃取后余液备用;
(3)取步骤(2)得到的菊叶萃取后余液,上粒径为80-100目的聚酰胺柱,先用去离子水洗,并将洗脱液进行动态吸附,至无样品流分;依次用10%乙醇、70%乙醇进行梯度洗脱。收集70%乙醇洗脱液,减压浓缩并干燥,得到菊叶活性部位粗提物;
(4)取步骤(3)得到的菊叶活性部位粗提物,上中压色谱分离凝胶柱MCI-SCA柱,先用体积分数10%的乙醇洗脱除去大极性成分,再用体积分数为50%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位提取物。
本发明对菊叶提取物中各原料所含成分进行分析:以绿原酸为对照品,采用福林-酚法,在760nm处测定吸光度,测得所制备的菊茎叶活性部位中总酚酸的含量为49.26%;以芦丁为对照品,采用硝酸铝-亚硝酸钠比色法,应用紫外-可见分光光度在510nm处测定吸光度,采用该方法可知所制备的菊叶活性部位中总黄酮的含量为31.21%。由此可知,制备得到的菊叶活性部位中总黄酮类和总酚酸类成分含量之和为80.47%。
实施例2
1、一种具有防治肝损伤的菊叶活性提取物的制备方法,其包括以下步骤:
(1)取新鲜菊叶,晒干,揉碎,投入提取罐中,依次加入药材重量20倍、15倍、10倍体积量的95%乙醇,加热回流提取3次,每次1小时,过滤、合并提取液,减压回收乙醇至无醇味后,得菊叶醇提浸膏,备用;
(2)取步骤(1)得到的乙醇提取物浸膏,加入15倍体积量的水混悬均匀,依次加入20倍、15倍、10倍体积的环己烷萃取3次,取萃取后余液备用;
(3)取步骤(2)得到的菊叶萃取后余液,上粒径为100-200目的聚酰胺柱,先用去离子水洗,并将洗脱液进行动态吸附,至无样品流分;依次用10%乙醇、50%乙醇、90%乙醇进行梯度洗脱。收集50%乙醇洗脱液,减压浓缩并干燥,得到菊叶活性部位粗提物;
(4)取步骤(3)得到的菊叶活性部位粗提物,上中压色谱分离凝胶柱MCI-CK柱,先用体积分数10%的乙醇洗脱除去大极性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位提取物。
本发明对菊叶提取物中各原料所含成分进行分析:以绿原酸为对照品,采用福林-酚法,在760nm处测定吸光度,测得所制备的菊茎叶活性部位中总酚酸的含量为49.98%;以芦丁为对照品,采用硝酸铝-亚硝酸钠比色法,应用紫外-可见分光光度在510nm处测定吸光度,采用该方法可知所制备的菊叶活性部位中总黄酮的含量为31.67%。由此可知,制备得到的菊叶活性部位中总黄酮类和总酚酸类成分含量之和为81.65%。
实施例3
1、一种具有防治肝损伤的菊叶活性提取物的制备方法,其包括以下步骤:
(1)取新鲜菊叶,去除杂质,45℃热风干燥后适当粉碎,投入提取罐中,依次加入药材重量20倍、15倍、10倍体积量的95%乙醇,加热回流提取3次,依次为2小时、1.5小时、1小时。过滤、合并三次提取液,减压回收乙醇至无醇味后,得菊叶醇提浸膏,备用;
(2)取步骤(1)得到的菊叶醇提浸膏,加入15倍量水混悬均匀后,依次加入10倍、8倍、6倍量的二氯甲烷萃取,取萃取后余液,备用;
(3)取步骤(2)得到的菊叶萃取后余液,上粒径为60-80目的聚酰胺柱,先用水洗脱除去水溶性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位粗提物;
(4)取步骤(3)得到的菊叶活性部位粗提物,上中压色谱分离凝胶柱MCI-CHP树脂柱,先用体积分数10%的乙醇洗脱除去大极性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位提取物。
本发明对菊叶提取物中各原料所含成分进行分析:以绿原酸为对照品,采用福林-酚法,在760nm处测定吸光度,测得所制备的菊茎叶活性部位中总酚酸的含量为50.27%;以芦丁为对照品,采用硝酸铝-亚硝酸钠比色法,应用紫外-可见分光光度在510nm处测定吸光度,采用该方法可知所制备的菊叶活性部位中总黄酮的含量为35.94%。由此可知,制备得到的菊叶活性部位中总黄酮类和总酚酸类成分含量之和为86.21%。
实施例4
1、一种具有防治肝损伤的菊叶活性提取物的制备方法,其包括以下步骤:
(1)取新鲜菊叶,去除杂质,微波干燥后揉碎,投入提取罐中,依次加入药材重量20倍、10倍体积量的95%乙醇,加热回流提取2次,依次为3小时。过滤、合并两次提取液,减压回收乙醇至无醇味后,得菊叶醇提浸膏,备用;
(2)取步骤(1)得到的菊叶醇提浸膏,加入10倍量水混悬均匀后,依次加入10倍、8倍、6倍量的石油醚萃取,取萃取后余液,备用;
(3)取步骤(2)得到的菊叶萃取后余液,上粒径为200-400目的聚酰胺柱,先用水洗脱除去水溶性成分,再用体积分数为70%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位粗提物;
(4)取步骤(3)得到的菊叶活性部位粗提物,上MCI-CQH疏水树脂柱,先用体积分数10%的乙醇洗脱除去大极性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位提取物。
本发明对菊叶提取物中各原料所含成分进行分析:以绿原酸为对照品,采用福林-酚法,在760nm处测定吸光度,测得所制备的菊茎叶活性部位中总酚酸的含量为47.91%;以芦丁为对照品,采用硝酸铝-亚硝酸钠比色法,应用紫外-可见分光光度在510nm处测定吸光度,采用该方法可知所制备的菊叶活性部位中总黄酮的含量为33.55%。由此可知,制备得到的菊叶活性部位中总黄酮类和总酚酸类成分含量之和为81.46%。
实施例5
1、一种具有防治肝损伤的菊叶活性提取物的制备方法,其包括以下步骤:
(1)取新鲜菊叶,去除杂质,晒干后揉碎,投入提取罐中,依次加入药材重量20倍、10倍、5倍体积量的95%乙醇,加热回流提取3次,依次为1.5小时。过滤、合并两次提取液,减压回收乙醇至无醇味后,得菊叶醇提浸膏,备用;
(2)取步骤(1)得到的菊叶醇提浸膏,加入10倍量水混悬均匀后,依次加入10倍、5倍量的石油醚萃取,取萃取后余液,备用;
(3)取步骤(2)得到的菊叶萃取后余液,上粒径为60-80目的聚酰胺柱,先用水洗脱除去水溶性成分,再用体积分数为70%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位粗提物;
(4)取步骤(3)得到的菊叶活性部位粗提物,上中压色谱分离凝胶柱MCI-CDR离子交换树脂柱,先用体积分数10%的乙醇洗脱除去大极性成分,再用体积分数为50%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位提取物。
本发明对菊叶提取物中各原料所含成分进行分析:以绿原酸为对照品,采用福林-酚法,在760nm处测定吸光度,测得所制备的菊茎叶活性部位中总酚酸的含量为49.62%%;以芦丁为对照品,采用硝酸铝-亚硝酸钠比色法,应用紫外-可见分光光度在510nm处测定吸光度,采用该方法可知所制备的菊叶活性部位中总黄酮的含量为32.51%。由此可知,制备得到的菊叶活性部位中总黄酮类和总酚酸类成分含量之和为82.13%。
实施例6 D-氨基半乳糖致小鼠化学性肝损伤药效实验
1、药品
D-氨基半乳糖(D-GalN)、羧甲基纤维素钠(CMC-Na纯度≥99%),购自阿拉丁生物科技有限公司。D-GalN临用前用无菌生理盐水配制50mg/ml浓度的溶液,CMC-Na用无菌水配制0.5%的溶液。
2、试剂
谷丙转氨酶(ALT)、谷草转氨酶(AST)、丙二醛(MDA)、微量还原型谷胱甘肽(GSH)及蛋白质测试盒(BCA)购于南京建成科技有限公司。
3、实验动物
ICR小鼠,雄性,体重21.0±1.0g,7w,购于南京市江宁区青龙山动物繁育场,SPF级,实验动物合格证编号:NO.201826493,动物生产许可证号:SCXK(苏)2017-0001;饲养环境为屏障环境系统,温度20~25℃,相对湿度40~70%,换气次数10~20次/小时,每日12h/12h交替照明。
4、实验方法
4.1动物分组
小鼠适应饲养一周后,随机分组,分别为空白组、模型组,各给药组。给药组给予本发明实施例1~5制备得到的菊叶活性提取物。均采取灌胃给药方式,空白对照组和模型组同时给予同体积CMC-Na溶液,各给药组按折合生药量1.2g/Kg体重剂量灌胃给药,每天一次,连续一周。
4.2操作步骤
末次给药所有动物禁食12~16小时,末次给药1h后,除空白组腹腔注射无菌生理盐水外,其余各组小鼠腹腔注射给予500mg/kg的D-GalN。造模后24h各组迅速摘眼球采血,静置30min,3000rpm离心10min取血清,保存于-80℃备用。小鼠处死后,迅速摘取肝脏,置于预冷的生理盐水中反复冲洗,用滤纸拭干。4℃对肝脏进行组织匀浆,3000rpm离心10min取血清,保存于-80℃备用。按照各测试盒操作方法测定肝组织中MDA、GSH和血清中ALT、AST水平。
5.实验结果
由表1可见,与正常组相比,模型组小鼠肝脏组织中MDA显著升高、GSH显著下降,血清中AST和ALT活性显著上升,表明:模型组小鼠肝组织中发生明显的脂质过氧化损伤,消耗了大量谷胱甘肽对抗氧化损伤和解毒;同时肝细胞受损导致胞内的AST和ALT释放至血清中。相比模型组,各给药组肝组织MDA下降、GSH增加,血清中AST和ALT下降,表明各给药组能够提高模型组小鼠抗氧化能力,同时保护受损的肝细胞,以对抗化学性肝损伤。其中,实施例3对GSH的增强作用和AST的降低作用最为显著,同时对MDA和ALT也具有显著的改善作用,综合考虑其对D-氨基半乳糖致小鼠化学性肝损伤肝组织的防治效果最佳。
表1 D-氨基半乳糖致小鼠化学性肝损伤生化检测
组别 | MDA(nmol/mgpr) | GSH(nmol/mgpr) | AST(U/L) | ALT(U/L) |
空白组 | 0.49±0.05 | 7.73±0.62 | 3.74±0.29 | 3.82±0.32 |
模型组 | 0.98±0.08* | 2.21±0.17* | 18.43±1.48** | 14.43±1.37** |
实施例1 | 0.56±0.04# | 8.46±0.79## | 11.61±1.06# | 6.50±0.57# |
实施例2 | 0.60±0.06# | 6.60±0.58# | 13.67±1.29# | 3.45±0.36## |
实施例3 | 0.50±0.05# | 15.57±1.46## | 10.07±1.17# | 3.40±0.42## |
实施例4 | 0.64±0.05 | 7.74±0.62## | 16.85±1.52 | 13.77±1.32 |
实施例5 | 0.53±0.04# | 4.48±0.35 | 17.61±1.62 | 6.59±0.68# |
与空白对照组比较,*p<0.05,**p<0.01。与模型组比较,#p<0.05,##p<0.01。
实施例7对乙酰氨基酚(APAP)致小鼠药源性肝损伤药效实验
1、样品
对乙酰氨基酚(APAP),购自阿拉丁生物科技有限公司,临用前用无菌生理盐水配置一定浓度的溶液。
2、实验动物
同实施例6。
3、实验方法
3.1动物分组
动物分组及给药剂量同实施例6。
3.2操作步骤
末次给药所有动物禁食12~16小时,末次给药30min后,除对照组灌胃0.5%CMC-Na外,其余各组小鼠以200mg/kg剂量灌胃APAP。造模后14h各组迅速摘眼球采血,静置30min,3000rpm离心10min取血清,保存于-80℃备用。小鼠处死后,迅速摘取肝脏,置于预冷的生理盐水中反复冲洗,用滤纸拭干。4℃对肝脏进行组织匀浆,3000rpm离心10min取血清,保存于-80℃备用。按照各测试盒操作方法测定肝组织中MDA、GSH和血清中ALT、AST水平。
4、实验结果
由表2可见,与正常组相比,模型组小鼠肝脏组织中MDA和GSH显著升高,血清中AST和ALT活性显著上升,表明:模型组小鼠肝细胞受损,同时肝组织中发生明显的脂质过氧化损伤,导致谷胱甘肽代偿性增加以对抗氧化损伤。相比模型组,各给药组肝组织中MDA、GSH以及血清中AST、ALT均有不同程度的下降,其中实施例3对MDA、AST和ALT的下降作用最显著。
表2 对乙酰氨基酚致小鼠药源性肝损伤生化检测
组别 | MDA(nmol/mgpr) | GSH(nmol/mgpr) | AST(U/L) | ALT(U/L) |
空白组 | 0.74±0.06 | 6.87±1.67 | 7.18±0.64 | 3.82±0.32 |
模型组 | 1.45±0.11* | 10.35±5.03* | 22.13±2.06** | 15.86±1.55** |
实施例1 | 0.80±0.07# | 6.75±2.13# | 10.52±1.01## | 7.95±0.81# |
实施例2 | 0.92±0.08 | 8.68±1.50 | 12.33±1.12## | 13.68±1.13 |
实施例3 | 0.58±0.05## | 4.01±1.03## | 10.05±1.08## | 5.23±0.43## |
实施例4 | 0.90±0.08 | 8.41±2.99 | 15.83±1.60# | 5.86±0.52## |
实施例5 | 0.91±0.07 | 5.79±1.04# | 10.14±0.99## | 6.59±0.58## |
与空白对照组比较,*p<0.05,**p<0.01。与模型组比较,#p<0.05,##p<0.01。
以上实验结果表明,本发明提供的菊叶活性提取物可以保护多种原因导致的肝损伤,尤其是化学性肝损伤和药源性肝损伤,从而应用于防治肝损伤药物及保健品开发。对D-氨基半乳糖所致小鼠化学性肝损伤,菊叶活性提取物能够降低模型组小鼠肝组织MDA、增加GSH,同时降低血清中AST和ALT,通过提高模型组小鼠抗氧化能力以对抗化学性肝损伤。在对乙酰氨基酚致小鼠药源性肝损伤中,菊叶活性提取物能够减少肝组织中MDA和GSH含量,同时降低血清中AST和ALT,通过纠正紊乱的生化代谢从而防治对乙酰氨基酚导致的药源性肝损伤。
本发明提供的制备方法工艺路线简单,自动化程度高,活性成分纯度高,杂质少,无毒副作用和不良反应。通过不同的工艺筛选可知,按实施例3制备方法提取得到的酚酸类和黄酮类活性成分纯度最高,且对化学性肝损伤和药源性肝损伤综合防治效果均最佳。取得了很好的预料不到的技术效果。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (1)
1.具有防治肝损伤的菊叶提取物在制备防治化学性肝损伤或药源性肝损伤的药物中的应用;
所述的菊叶活性提取物是通过以下方法制备得到:
(1)取新鲜菊叶,去除杂质,45 ℃ 热风干燥后适当粉碎,投入提取罐中,依次加入药材重量20倍、15倍、10倍体积量的95%乙醇,加热回流提取3次,依次为2小时、1.5小时、1小时,过滤、合并三次提取液,减压回收乙醇至无醇味后,得菊叶醇提浸膏,备用;
(2)取步骤(1)得到的菊叶醇提浸膏,加入15倍量水混悬均匀后,依次加入10倍、8倍、6倍量的二氯甲烷萃取,取萃取后余液,备用;
(3)取步骤(2)得到的菊叶萃取后余液,上粒径为60-80目的聚酰胺柱,先用水洗脱除去水溶性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位粗提物;
(4)取步骤(3)得到的菊叶活性部位粗提物,上中压色谱分离凝胶柱MCI-CHP树脂柱,先用体积分数10%的乙醇洗脱除去大极性成分,再用体积分数为40%的乙醇洗脱,洗脱液减压浓缩,得到菊叶活性部位提取物。
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