六月青有效萃取物筛选与护肝作用及其作用机制的实验研究
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摘要
众所周知,肝脏疾病尤其是病毒性肝炎,对人类健康危害极大。目前,虽然在病毒性肝炎预防和治疗研究方面取得了巨大成绩,但可用于临床治疗的抗肝炎病毒药物非常有限,它们的治疗效果也不理想。因此,研制新型抗HBV药仍是面临的主要课题。
六月青,是抗HBV复方——复方六月雪的主要组成药物之一。本课题组对复方六月雪做了大量体内、体外的实验研究,现已证实:在体内,复方六月雪对CCl_4、AP和D-GalN所致急性化学性肝损伤有明显的保护作用,能明显地降低ALT、AST活性,提高肝脏细胞色素P450及细胞色素b5的含量;对于感染鸭乙型肝炎病毒(DHBV)的广西麻鸭,复方六月雪能降低其血清ALT、AST的活性和DHBsAg、DHBeAg、DHBV-DNA的含量。在体外,采用直接加药法和血清药理学法观察复方六月雪对HepG2.2.15细胞的作用,结果对HepG2.2.15细胞分泌HBsAg、HBeAg和HBV-DNA的合成复方六月雪表现出较强的抑制作用,且抑制作用有明显的量效和时效反应关系。也就是说,复方六月雪体内及体外均有显著的抗HBV作用。而关于六月青的研究文献中,除了本课题组对其植物鉴定学等研究文献外,目前尚未见到其他尤其是化学成分、药理作用方面的文献报道。基于对复方六月雪的实验研究结果,本文对六月青展开系统的实验研究:先对六月青化学成分进行定性实验,然后用固-液萃取法将其分成不同萃取物,之后通过体内的抗肝损伤作用、对免疫功能的影响、体外细胞毒性作用、对HBV的抑制等实验筛选具有活性作用的萃取物,并对有效萃取物保肝作用机制进行实验研究。
【目的】通过体内、体外实验筛选六月青萃取物中的有效部位,并对有效部位体外抗HBV作用、体内抗化学性急性肝损伤及作用机制进行探讨研究。
【方法】1、六月青化学成分定性分析试验与萃取物分离:采用传统的化学成分定性分析方法进行分析,用极性由小到大的溶剂石油醚、氯仿、乙酸乙酯、水饱和正丁醇、60%乙醇依次进行萃取分离,对60%乙醇萃取物进行化学成分定性检识、紫外扫描和HPLC定性分析。2、60%乙醇萃取物对HepG2.2.15细胞DNA的抑制作用:对其体外抗HBV作用进行研究,采用FQ-PCR法检测不同浓度60%乙醇萃取物作用后HepG2.2.15细胞内DNA的含量。3、60%乙醇萃取物对急性肝损伤大鼠的影响:采用CCl_4、D-GalN致大鼠急性肝损伤,以肝脏重量指数、血清ALT、AST、ALP、Alb、Glo、TP、T.Bil、G-GT、LDH及肝组织病理切片为指标,观察60%乙醇萃取物对急性肝损伤大鼠的保护作用。4、60%乙醇萃取物对急性肝损伤小鼠的影响:以CCl_4、AP、D-GalN作为急性肝损伤诱导剂,引起小鼠急性肝损伤,观察肝脏重量指数,胸腺重量指数,脾脏重量指数,血清ALT、AST、ALP、Alb、T-AOC,肝组织中SOD、MDA、TP、NO、GSH、GSH-Px及肝组织病理的变化,探讨60%乙醇萃取物对急性肝损伤的保护作用及作用机制。5六月青各萃取物体内试验:以四氯化碳所致急性肝损伤和环磷酰胺所致免疫功能低下小鼠为模型,观察六月青各萃取物对急性肝损伤小鼠的肝脏指数、血清ALT和AST活性的影响,对免疫功能低下小鼠胸腺和脾脏重量、血清溶菌酶和TNF-α的影响。6、六月青各萃取物体外对HepG2.2.15细胞的HBsAg、HBeAg的抑制作用:采用WST-8法检测各萃取物的细胞毒性,测定细胞存活率,计算TC_(50);ELISA法测定各萃取物直接作用细胞后培养上清中HBsAg、HBeAg的含量,计算各萃取物的抗原抑制百分率、半数抑制浓度(IC_(50))及治疗指数(TI)。7、六月青水、醇总提取物抗急性肝损伤的实验:以四氯化碳诱导小鼠急性肝损伤,检测血清ALT、AST活性。
【结果】1、化学成分定性分析结显示:六月青中含有皂苷、强心苷类、内酯类、香豆素类、酚类、有机酸、氨基酸、多肽、蛋白质、糖和苷类、鞣质类成分。薄层(TLC)对比实验表明不含靛蓝和靛玉红,用不同极性溶剂将六月青醇总提物进行萃取分离,得到石油醚、氯仿、乙酸乙酯、水饱和正丁醇、60%乙醇部位共五个萃取物。60%乙醇萃取物化学成分定性分析结果提示:60%乙醇萃取物在209nm处有强吸收,可能含有皂苷类、强心苷、香豆素类、内酯类、植物甾醇、三萜类化学成分。2、60%乙醇萃取物能明显抑制HepG2.2.15细胞内HBV-DNA的复制,降低HBV-DNA的拷贝数,升高循环数(CT值)。3、对于CCl_4、D-GalN所致急性肝损伤的大鼠,60%乙醇萃取物能减轻肝脏重量,降低血清ALT、AST、ALP、G-GT、LDH活性,提高Alb、Glo、TP含量,减少T.Bil含量。4、对于CCl_4、AP、D-GalN所致急性肝损伤小鼠,60%乙醇萃取物能减轻肝脏重量,增加胸腺重量,降低血清ALT、AST、ALP活性,提高Alb含量和T-AOC活性,提高肝组织中SOD、GSH、GSH-Px活性,降低肝组织中MDA含量和NO的活性。5、在六月青各萃取物中,石油醚部位、氯仿部位的高剂量和低剂量组及60%乙醇部位高剂量组的肝指数均明显低于CCl_4模型组,石油醚部位、氯仿部位及60%乙醇部位的高剂量和低剂量组均能显著地降低ALT活性,氯仿部位、60%乙醇部位高剂量组能明显降低AST活性,其他部位对AST、ALT活性没有明显影响。对于免疫抑制小鼠,五个萃取物均能增加胸腺重量和提高血清溶菌酶含量,对脾脏重量均无显著性影响,其中乙酸乙酯部位、水饱和正丁醇部位、60%EtOH部位还能提高血清TNF-α含量。6、六月青各萃取物体外对HepG2.2.15细胞的影响:①氯仿部位、乙酸乙酯部位、水饱和正丁醇部位均有一定的细胞毒性,石油醚部位、60%乙醇部位对细胞的抑制随浓度增加而增强,TC_(50)分别为1.036mg/ml、7.818mg/ml,②细胞培养上清中HBsAg、HBeAg检测结果显示:石油醚部位、60%乙醇部位对HepG2.2.15细胞分泌HBsAg和HBeAg有较强的抑制作用(TI均大于2),60%乙醇部位高效低毒。7、LYQ水、醇总提取物的最大耐受量(MTD)分别为153.0g生药/kg体重、369.0g生药/kg体重,分别相当于临床用药量的46、110.8倍。对于四氯化碳导致的ALT、AST活性升高,六月青水总提取物高、中剂量和醇总提取物高剂量能显著降低ALT、AST的活性(P＜0.01),六月青水总提取物低剂量能显著降低ALT活性,但不能显著降低AST活性,醇总提物中、低剂量对ALT、AST活性的降低作用不显著。
【结论】1、六月青含有皂苷、内酯类、香豆素类、内酯类、酚类等多种化学成分,其水、醇总提取物对急性肝损伤有保护作用。2、用固-液萃取法将其分成石油醚、氯仿、乙酸乙酯、水饱和正丁醇、60%乙醇部位共五个萃取物,其中60%乙醇部位在209nm处有强吸收,可能含有皂苷类、香豆素类、内酯类、植物甾醇、三萜类化学成分。药效学研究表明,在体外有显著的抗HBV作用,能显著抑制HBV-DNA的复制和HepG2.2.15HBsAg和HBeAg的表达;在体内有增强免疫功能作用,提高血清溶菌酶和TNF-α含量;对急性化学性肝损伤保护作用显著,降低血清中ALT、AST、ALP活性,提高血清Alb含量和T-AOC活性;可提高肝组织中SOD、GSH、GSH-Px活性,降低肝组织中MDA含量和NO的活性,从而增强机体氧化防御体系的功能,其作用机制可能与抑制脂质过氧化反应有关。3、此外,石油醚部位有一定的增强免疫功能和保护肝脏作用,能明显抑制HepG2.2.15细胞分泌HBsAg和HBeAg。4、氯仿、乙酸乙酯、水饱和正丁醇部位有增强免疫功能的作用,对HepG2.2.15细胞有一定的毒性,其中氯仿部位对急性化学性损伤的肝脏有保护作用。
Studies on the screening of active extracts of Liuyueqing and their hepatoprotective effect and action mechanism
As we all know,hepatic disease,especially viral liver hepatitis,is the most harmful disease to human health.Recently,there are many achievements on the prevention and treatment of viral liver diseases.But the drugs which are used to treat the viral liver hepatitis are few clinically now.And the curative effect of these medicines is not what we wanted.Therefore,it is an important issue for us to develop new drugs to inhibit HBV.
Liuyueqing is one of main herbs of compound liuyuexue(CLYX)which can inhibit HBV.Our team has done many experimental researches on CLYX.It has been proved that CLYX can protect markedly against the acute chemical liver injury induced by CCl_4,AP and D-GalN,and can significantly decrease the activities of ALT and AST and increase the contents of cytochrome P450 and cytochrome b5.CLYX also can decrease the activities of ALT and AST as well as the contents of DHBsAg,DHBeAg and DHBV-DNA in serum of Guangxi brown spot duckling infected by DHBV.In vitro,through directly adding CLYX and the serum with CLYX into the medium of HepG2.2.15 cell,the effect of CLYX on HepG2.2.15 cell was observed.CLYX can obviously inhibit the secretion of HBsAg and HBeAg and the synthesis of HBV-DNA,and this inhibition is dose-and time-depedent. That is to say,CLYX has the inhibitive effects on HBV not only in vitro but also in vivo. In the references of researching on Liuyueqing,except for pharmacognosical report of Liuyueqing done in our laboratory,there is no other reports,especially on chemical composition and pharmacology.Based on the results of the experimental research on CLYX,this research will carry on the systemic experiment for Liuyueqing:Firstly,the constituent of Liuyueqing will qualitatively be analyzed.Secondly,the different extracts will be gotten by solid-liquid isolation method.Thirdly,the active extracts of liver protective effect will be screened by these methods,including the studies on the hepatoprotection of the active extracts against chemical liver injury,the effect on immune function in vivo,the inhibitive effect of HBV,and the toxicity of cell in vitro.The mechanism of liver protective effect of the active extracts will be studied by the experiment.
【Objective】To screen the active extracts from five Liuyueqing(LYQ)extracts in vivo and in vitro,and to study the effects of these extracts on HBV in vitro and their protective effect and possible mechanism against chemical acute liver injury.
【Methods】1.The traditional qualitative analysis method of chemical constituent was used to analyze LYQ extracts.The isolation of LYQ constituents:The several extracts were isolated from LYQ by traditional methods with different solvent,which were petroliem, chloroform,ethyl acetate,n-butyl alcohol,and 60%alchohol.60%alchohol portion was analyzed by qualitative analysis of chemical constituent,UV scanning and high performance liquid chromatography(HPLC).2.The inhibitory effect of 60%alcoholic portion on HBV-DNA in HepG2.2.15 cell:Different concentrations of 60%alcoholic portion were added into culture fluid respectively,then the quantity of HBV-DNA in cell was detected by FQ-PCR subsequently.3.The effect of 60%alcoholic portion on acute liver injury in rats: The models of CCl_4-and D-Gal-induced acute live injuries in rat were used in the study on the protective effect of 60%alcoholic portion.The liver weight index,ALT,AST ALP,Alb, Glo,TP,T.Bil,G-GT and LDH in serum were detected.The hepatic pathological changes were examined.4.The effect of 60%alcoholic portion on acute liver injury in mice:The CCl_4-,AP-and D-GalN-induced acute liver injuries were used to study the protective effect and possible mechanisms of 60%alcoholic portion in mice.The indexes of liver,thymus gland and spleen were detected.The activities of ALT,AST,ALP and T-AOC,and Alb content in serum were determined.The activities of SOD,NO,GSH and GSH-Px as well as the levels of MDA in hepatic tissue were also measured.The hepatic pathological changes were also examined.5.The concentration of tumor necrosis factorα(TNF-α),the activity of serum lysozyme,and the indexes of thymus gland and spleen were determined in immunosuppressed mice induced by cyclophosphamide.6.The effect of LYQ extracts on HepG2.2.15 cell in vitro:The toxicity of LYQ extracts on HepG-2.2.15 cell and cell viability were evaluated by WST-8 method and TC_(50)was calculated.HBsAg and HBeAg in cell culture medium were detected by ELISA method.The inhibition ratio of antigen,IC50 and TI were calculated.7.To measure MTDs of water extract and alcohol extract of LYQ. The protective effect of water extract and alcohol extract of LYQ against acute liver injury: The activities of serum ALT and AST,and the liver weight index were detected in mice with acute liver injury induced by carbon tetrachloride(CCl_4)after the treatment with water extract and alcohol extract of LYQ.
【Results】1.There were saponin,cardiac glycosides,lactone,coumarin,phenols, organic acid,amino acid,polypeptide,protein,carbonhydrates and glycoside in the liuyueqing.The result of thin-layer chromatography(TLC)contrast showed that there were not indigo and indirubin in the extract of LYQ.Different solvent were used to separate the alcohol extract of LYQ into five extracts,which were petroliem portion,chloroform portion, ethyl acetate portion,n-butyl alcohol portion,and 60%alchohol portion.The results of qualitative analysis on 60%alchohol portion showed that there was a strong absorption on 209nm wavelength and there were saponin,cardiac glycosides,lactone,coumarin and triterpenoids in it.2.60%alchohol portion could inhibit the replication of HBA-DNA in HepG2.2.15 cell and decrease the copies of HBV-DNA and could increase circulation.3. In CCl_4-and D-Gal-induced acute liver injuries of rat,60%alchohol portion could lower the weight of enlarged liver and could decrease the activities of ALT,AST,ALP,G-GT and LDH in serum as well as the content of T.Bil,and improve the content of Alb and Glo and TP.4.In acute liver injuries of mice induced by CCl_4 and AP and D-GalN,the weight of enlarged liver was decreased by the treatment with 60%alchohol portion,which could increase the weight of thymus gland.60%alchohol portion could decrease the activities of ALT and.AST and ALP and could increase the content of Alb and activities of T-AOC.60% alchohol portion also increased the activities of SOD,GSH,GSH-Px in hepatic tissue and decreased the content of MDA and NO.All results indicated that 60%alchohol portion could protect markedly against the acute liver injury.5.In five extracts of LYQ,the high and low dosage of petroliem and chloroform portion could lowered the enlarged liver index as well as the high dosage of 60%alchohol portion in CCl_4-induced acute liver injury in mice,and the activities of ALT and AST in serum were decreased in acute liver injuried mice induced by CCl_4.The high and low dosage of petroliem and chloroform and 60% alchohol portion could obviously decrease ALT activity,and AST activity was significantly decreased by the high dosage of chloroform and 60%alchohol portion.The rest of portion had no effect on activities of ALT and AST.In cyclophosphamide immunosuppressed mice, five portions could improve the weight of thymus gland and the activities of lysozyme,but they could not improve the weight of spleen.The level of TNF-αwas obviously improved by ethyl acetate and n-butyl alcohol and 60%alchohol portion.6.The effect of extracts of LYQ on HepG2.2.15 cell in vitro:①Chloroform and ethyl acetate and n-butyl alcohol portion showed some toxicity,but the inhibition effect of 60%alchohol portion on cell was increased with the increase in concentration.②The results of measuring HBsAg and HBeAg in cell culture medium showed that petroliem and 60%alchohol portion could inhibit the expressions of HBsAg and HBeAg in HepG2.2.15 cell(TI＞2)and 60%alchohol portion was active extract.7.The MTDs of aqueous and alcohol extracts of LYQ were respectively 153.0g and 369.0g crude materials/kg body weight,which were 46 and 110.8 times of clinical dose.The high and low dosage of aqueous extract of LYQ and the high dosage of alcohol extract of LYQ could significantly reduce the activities of ALT and AST in serum.The low dosage of aqueous extract of LYQ could obviously decrease the activities of ALT,but could not decrease the activities of AST.The middle and low dosage of alcohol extract of LYQ could not decrease the activities of ALT and AST.
【Conclusion】1.There are many kinds of chemical constituents in LYQ,such as saponin, lactone,coumarin,phenols,amino acid and so on,whose water extract and alcohol extract have hepatoprotective efffcts against acute liver injury.2.Five extracts,which are petroliem,chloroform,ethyl acetate,n-butyl alcohol,and 60%alchohol portions are gotten by solid-liquid isolation method.60%alchohol portion has a strong absorption on 209nm wavelength and possibly contains saponin,lactone,coumarin and triterpenoids.HBV is inhibited by 60%alchohol portion in vitro,which can inhibit the replication of HBV-DNA and the expressions of HBsAg and HBeAg in HepG2.2.15 cell.In vivo,it can enhance immunity function,and obviously increase the contents of lysozyme and TNF-αin serum.It can also protect markedly against the acute chemical liver injury,which decreases the activities of ALT,AST and ALP in serum and the content of MDA and NO in hepatic tissue, and increases the content of Alb and activities of T-AOC in serum and the activities of SOD, GSH,GSH-Px in hepatic tissue.Its possible mechanisms of hepatoprotection are related with the inhibition of lipid peroxidation.3.In addition,petroliem portion can enhance immunity function,protect liver and inhibit the expressions of HBsAg and HBeAg in HepG2.2.15 cell.4.Chloroform,ethyl acetate,n-butyl alcohol portions have the effects of enhancing immunity function and are toxic to HepG2.2.15 cell.Chloroform portion can also protect against the acute liver injury.
六月青,是抗HBV复方——复方六月雪的主要组成药物之一。本课题组对复方六月雪做了大量体内、体外的实验研究,现已证实:在体内,复方六月雪对CCl_4、AP和D-GalN所致急性化学性肝损伤有明显的保护作用,能明显地降低ALT、AST活性,提高肝脏细胞色素P450及细胞色素b5的含量;对于感染鸭乙型肝炎病毒(DHBV)的广西麻鸭,复方六月雪能降低其血清ALT、AST的活性和DHBsAg、DHBeAg、DHBV-DNA的含量。在体外,采用直接加药法和血清药理学法观察复方六月雪对HepG2.2.15细胞的作用,结果对HepG2.2.15细胞分泌HBsAg、HBeAg和HBV-DNA的合成复方六月雪表现出较强的抑制作用,且抑制作用有明显的量效和时效反应关系。也就是说,复方六月雪体内及体外均有显著的抗HBV作用。而关于六月青的研究文献中,除了本课题组对其植物鉴定学等研究文献外,目前尚未见到其他尤其是化学成分、药理作用方面的文献报道。基于对复方六月雪的实验研究结果,本文对六月青展开系统的实验研究:先对六月青化学成分进行定性实验,然后用固-液萃取法将其分成不同萃取物,之后通过体内的抗肝损伤作用、对免疫功能的影响、体外细胞毒性作用、对HBV的抑制等实验筛选具有活性作用的萃取物,并对有效萃取物保肝作用机制进行实验研究。
【目的】通过体内、体外实验筛选六月青萃取物中的有效部位,并对有效部位体外抗HBV作用、体内抗化学性急性肝损伤及作用机制进行探讨研究。
【方法】1、六月青化学成分定性分析试验与萃取物分离:采用传统的化学成分定性分析方法进行分析,用极性由小到大的溶剂石油醚、氯仿、乙酸乙酯、水饱和正丁醇、60%乙醇依次进行萃取分离,对60%乙醇萃取物进行化学成分定性检识、紫外扫描和HPLC定性分析。2、60%乙醇萃取物对HepG2.2.15细胞DNA的抑制作用:对其体外抗HBV作用进行研究,采用FQ-PCR法检测不同浓度60%乙醇萃取物作用后HepG2.2.15细胞内DNA的含量。3、60%乙醇萃取物对急性肝损伤大鼠的影响:采用CCl_4、D-GalN致大鼠急性肝损伤,以肝脏重量指数、血清ALT、AST、ALP、Alb、Glo、TP、T.Bil、G-GT、LDH及肝组织病理切片为指标,观察60%乙醇萃取物对急性肝损伤大鼠的保护作用。4、60%乙醇萃取物对急性肝损伤小鼠的影响:以CCl_4、AP、D-GalN作为急性肝损伤诱导剂,引起小鼠急性肝损伤,观察肝脏重量指数,胸腺重量指数,脾脏重量指数,血清ALT、AST、ALP、Alb、T-AOC,肝组织中SOD、MDA、TP、NO、GSH、GSH-Px及肝组织病理的变化,探讨60%乙醇萃取物对急性肝损伤的保护作用及作用机制。5六月青各萃取物体内试验:以四氯化碳所致急性肝损伤和环磷酰胺所致免疫功能低下小鼠为模型,观察六月青各萃取物对急性肝损伤小鼠的肝脏指数、血清ALT和AST活性的影响,对免疫功能低下小鼠胸腺和脾脏重量、血清溶菌酶和TNF-α的影响。6、六月青各萃取物体外对HepG2.2.15细胞的HBsAg、HBeAg的抑制作用:采用WST-8法检测各萃取物的细胞毒性,测定细胞存活率,计算TC_(50);ELISA法测定各萃取物直接作用细胞后培养上清中HBsAg、HBeAg的含量,计算各萃取物的抗原抑制百分率、半数抑制浓度(IC_(50))及治疗指数(TI)。7、六月青水、醇总提取物抗急性肝损伤的实验:以四氯化碳诱导小鼠急性肝损伤,检测血清ALT、AST活性。
【结果】1、化学成分定性分析结显示:六月青中含有皂苷、强心苷类、内酯类、香豆素类、酚类、有机酸、氨基酸、多肽、蛋白质、糖和苷类、鞣质类成分。薄层(TLC)对比实验表明不含靛蓝和靛玉红,用不同极性溶剂将六月青醇总提物进行萃取分离,得到石油醚、氯仿、乙酸乙酯、水饱和正丁醇、60%乙醇部位共五个萃取物。60%乙醇萃取物化学成分定性分析结果提示:60%乙醇萃取物在209nm处有强吸收,可能含有皂苷类、强心苷、香豆素类、内酯类、植物甾醇、三萜类化学成分。2、60%乙醇萃取物能明显抑制HepG2.2.15细胞内HBV-DNA的复制,降低HBV-DNA的拷贝数,升高循环数(CT值)。3、对于CCl_4、D-GalN所致急性肝损伤的大鼠,60%乙醇萃取物能减轻肝脏重量,降低血清ALT、AST、ALP、G-GT、LDH活性,提高Alb、Glo、TP含量,减少T.Bil含量。4、对于CCl_4、AP、D-GalN所致急性肝损伤小鼠,60%乙醇萃取物能减轻肝脏重量,增加胸腺重量,降低血清ALT、AST、ALP活性,提高Alb含量和T-AOC活性,提高肝组织中SOD、GSH、GSH-Px活性,降低肝组织中MDA含量和NO的活性。5、在六月青各萃取物中,石油醚部位、氯仿部位的高剂量和低剂量组及60%乙醇部位高剂量组的肝指数均明显低于CCl_4模型组,石油醚部位、氯仿部位及60%乙醇部位的高剂量和低剂量组均能显著地降低ALT活性,氯仿部位、60%乙醇部位高剂量组能明显降低AST活性,其他部位对AST、ALT活性没有明显影响。对于免疫抑制小鼠,五个萃取物均能增加胸腺重量和提高血清溶菌酶含量,对脾脏重量均无显著性影响,其中乙酸乙酯部位、水饱和正丁醇部位、60%EtOH部位还能提高血清TNF-α含量。6、六月青各萃取物体外对HepG2.2.15细胞的影响:①氯仿部位、乙酸乙酯部位、水饱和正丁醇部位均有一定的细胞毒性,石油醚部位、60%乙醇部位对细胞的抑制随浓度增加而增强,TC_(50)分别为1.036mg/ml、7.818mg/ml,②细胞培养上清中HBsAg、HBeAg检测结果显示:石油醚部位、60%乙醇部位对HepG2.2.15细胞分泌HBsAg和HBeAg有较强的抑制作用(TI均大于2),60%乙醇部位高效低毒。7、LYQ水、醇总提取物的最大耐受量(MTD)分别为153.0g生药/kg体重、369.0g生药/kg体重,分别相当于临床用药量的46、110.8倍。对于四氯化碳导致的ALT、AST活性升高,六月青水总提取物高、中剂量和醇总提取物高剂量能显著降低ALT、AST的活性(P＜0.01),六月青水总提取物低剂量能显著降低ALT活性,但不能显著降低AST活性,醇总提物中、低剂量对ALT、AST活性的降低作用不显著。
【结论】1、六月青含有皂苷、内酯类、香豆素类、内酯类、酚类等多种化学成分,其水、醇总提取物对急性肝损伤有保护作用。2、用固-液萃取法将其分成石油醚、氯仿、乙酸乙酯、水饱和正丁醇、60%乙醇部位共五个萃取物,其中60%乙醇部位在209nm处有强吸收,可能含有皂苷类、香豆素类、内酯类、植物甾醇、三萜类化学成分。药效学研究表明,在体外有显著的抗HBV作用,能显著抑制HBV-DNA的复制和HepG2.2.15HBsAg和HBeAg的表达;在体内有增强免疫功能作用,提高血清溶菌酶和TNF-α含量;对急性化学性肝损伤保护作用显著,降低血清中ALT、AST、ALP活性,提高血清Alb含量和T-AOC活性;可提高肝组织中SOD、GSH、GSH-Px活性,降低肝组织中MDA含量和NO的活性,从而增强机体氧化防御体系的功能,其作用机制可能与抑制脂质过氧化反应有关。3、此外,石油醚部位有一定的增强免疫功能和保护肝脏作用,能明显抑制HepG2.2.15细胞分泌HBsAg和HBeAg。4、氯仿、乙酸乙酯、水饱和正丁醇部位有增强免疫功能的作用,对HepG2.2.15细胞有一定的毒性,其中氯仿部位对急性化学性损伤的肝脏有保护作用。
Studies on the screening of active extracts of Liuyueqing and their hepatoprotective effect and action mechanism
As we all know,hepatic disease,especially viral liver hepatitis,is the most harmful disease to human health.Recently,there are many achievements on the prevention and treatment of viral liver diseases.But the drugs which are used to treat the viral liver hepatitis are few clinically now.And the curative effect of these medicines is not what we wanted.Therefore,it is an important issue for us to develop new drugs to inhibit HBV.
Liuyueqing is one of main herbs of compound liuyuexue(CLYX)which can inhibit HBV.Our team has done many experimental researches on CLYX.It has been proved that CLYX can protect markedly against the acute chemical liver injury induced by CCl_4,AP and D-GalN,and can significantly decrease the activities of ALT and AST and increase the contents of cytochrome P450 and cytochrome b5.CLYX also can decrease the activities of ALT and AST as well as the contents of DHBsAg,DHBeAg and DHBV-DNA in serum of Guangxi brown spot duckling infected by DHBV.In vitro,through directly adding CLYX and the serum with CLYX into the medium of HepG2.2.15 cell,the effect of CLYX on HepG2.2.15 cell was observed.CLYX can obviously inhibit the secretion of HBsAg and HBeAg and the synthesis of HBV-DNA,and this inhibition is dose-and time-depedent. That is to say,CLYX has the inhibitive effects on HBV not only in vitro but also in vivo. In the references of researching on Liuyueqing,except for pharmacognosical report of Liuyueqing done in our laboratory,there is no other reports,especially on chemical composition and pharmacology.Based on the results of the experimental research on CLYX,this research will carry on the systemic experiment for Liuyueqing:Firstly,the constituent of Liuyueqing will qualitatively be analyzed.Secondly,the different extracts will be gotten by solid-liquid isolation method.Thirdly,the active extracts of liver protective effect will be screened by these methods,including the studies on the hepatoprotection of the active extracts against chemical liver injury,the effect on immune function in vivo,the inhibitive effect of HBV,and the toxicity of cell in vitro.The mechanism of liver protective effect of the active extracts will be studied by the experiment.
【Objective】To screen the active extracts from five Liuyueqing(LYQ)extracts in vivo and in vitro,and to study the effects of these extracts on HBV in vitro and their protective effect and possible mechanism against chemical acute liver injury.
【Methods】1.The traditional qualitative analysis method of chemical constituent was used to analyze LYQ extracts.The isolation of LYQ constituents:The several extracts were isolated from LYQ by traditional methods with different solvent,which were petroliem, chloroform,ethyl acetate,n-butyl alcohol,and 60%alchohol.60%alchohol portion was analyzed by qualitative analysis of chemical constituent,UV scanning and high performance liquid chromatography(HPLC).2.The inhibitory effect of 60%alcoholic portion on HBV-DNA in HepG2.2.15 cell:Different concentrations of 60%alcoholic portion were added into culture fluid respectively,then the quantity of HBV-DNA in cell was detected by FQ-PCR subsequently.3.The effect of 60%alcoholic portion on acute liver injury in rats: The models of CCl_4-and D-Gal-induced acute live injuries in rat were used in the study on the protective effect of 60%alcoholic portion.The liver weight index,ALT,AST ALP,Alb, Glo,TP,T.Bil,G-GT and LDH in serum were detected.The hepatic pathological changes were examined.4.The effect of 60%alcoholic portion on acute liver injury in mice:The CCl_4-,AP-and D-GalN-induced acute liver injuries were used to study the protective effect and possible mechanisms of 60%alcoholic portion in mice.The indexes of liver,thymus gland and spleen were detected.The activities of ALT,AST,ALP and T-AOC,and Alb content in serum were determined.The activities of SOD,NO,GSH and GSH-Px as well as the levels of MDA in hepatic tissue were also measured.The hepatic pathological changes were also examined.5.The concentration of tumor necrosis factorα(TNF-α),the activity of serum lysozyme,and the indexes of thymus gland and spleen were determined in immunosuppressed mice induced by cyclophosphamide.6.The effect of LYQ extracts on HepG2.2.15 cell in vitro:The toxicity of LYQ extracts on HepG-2.2.15 cell and cell viability were evaluated by WST-8 method and TC_(50)was calculated.HBsAg and HBeAg in cell culture medium were detected by ELISA method.The inhibition ratio of antigen,IC50 and TI were calculated.7.To measure MTDs of water extract and alcohol extract of LYQ. The protective effect of water extract and alcohol extract of LYQ against acute liver injury: The activities of serum ALT and AST,and the liver weight index were detected in mice with acute liver injury induced by carbon tetrachloride(CCl_4)after the treatment with water extract and alcohol extract of LYQ.
【Results】1.There were saponin,cardiac glycosides,lactone,coumarin,phenols, organic acid,amino acid,polypeptide,protein,carbonhydrates and glycoside in the liuyueqing.The result of thin-layer chromatography(TLC)contrast showed that there were not indigo and indirubin in the extract of LYQ.Different solvent were used to separate the alcohol extract of LYQ into five extracts,which were petroliem portion,chloroform portion, ethyl acetate portion,n-butyl alcohol portion,and 60%alchohol portion.The results of qualitative analysis on 60%alchohol portion showed that there was a strong absorption on 209nm wavelength and there were saponin,cardiac glycosides,lactone,coumarin and triterpenoids in it.2.60%alchohol portion could inhibit the replication of HBA-DNA in HepG2.2.15 cell and decrease the copies of HBV-DNA and could increase circulation.3. In CCl_4-and D-Gal-induced acute liver injuries of rat,60%alchohol portion could lower the weight of enlarged liver and could decrease the activities of ALT,AST,ALP,G-GT and LDH in serum as well as the content of T.Bil,and improve the content of Alb and Glo and TP.4.In acute liver injuries of mice induced by CCl_4 and AP and D-GalN,the weight of enlarged liver was decreased by the treatment with 60%alchohol portion,which could increase the weight of thymus gland.60%alchohol portion could decrease the activities of ALT and.AST and ALP and could increase the content of Alb and activities of T-AOC.60% alchohol portion also increased the activities of SOD,GSH,GSH-Px in hepatic tissue and decreased the content of MDA and NO.All results indicated that 60%alchohol portion could protect markedly against the acute liver injury.5.In five extracts of LYQ,the high and low dosage of petroliem and chloroform portion could lowered the enlarged liver index as well as the high dosage of 60%alchohol portion in CCl_4-induced acute liver injury in mice,and the activities of ALT and AST in serum were decreased in acute liver injuried mice induced by CCl_4.The high and low dosage of petroliem and chloroform and 60% alchohol portion could obviously decrease ALT activity,and AST activity was significantly decreased by the high dosage of chloroform and 60%alchohol portion.The rest of portion had no effect on activities of ALT and AST.In cyclophosphamide immunosuppressed mice, five portions could improve the weight of thymus gland and the activities of lysozyme,but they could not improve the weight of spleen.The level of TNF-αwas obviously improved by ethyl acetate and n-butyl alcohol and 60%alchohol portion.6.The effect of extracts of LYQ on HepG2.2.15 cell in vitro:①Chloroform and ethyl acetate and n-butyl alcohol portion showed some toxicity,but the inhibition effect of 60%alchohol portion on cell was increased with the increase in concentration.②The results of measuring HBsAg and HBeAg in cell culture medium showed that petroliem and 60%alchohol portion could inhibit the expressions of HBsAg and HBeAg in HepG2.2.15 cell(TI＞2)and 60%alchohol portion was active extract.7.The MTDs of aqueous and alcohol extracts of LYQ were respectively 153.0g and 369.0g crude materials/kg body weight,which were 46 and 110.8 times of clinical dose.The high and low dosage of aqueous extract of LYQ and the high dosage of alcohol extract of LYQ could significantly reduce the activities of ALT and AST in serum.The low dosage of aqueous extract of LYQ could obviously decrease the activities of ALT,but could not decrease the activities of AST.The middle and low dosage of alcohol extract of LYQ could not decrease the activities of ALT and AST.
【Conclusion】1.There are many kinds of chemical constituents in LYQ,such as saponin, lactone,coumarin,phenols,amino acid and so on,whose water extract and alcohol extract have hepatoprotective efffcts against acute liver injury.2.Five extracts,which are petroliem,chloroform,ethyl acetate,n-butyl alcohol,and 60%alchohol portions are gotten by solid-liquid isolation method.60%alchohol portion has a strong absorption on 209nm wavelength and possibly contains saponin,lactone,coumarin and triterpenoids.HBV is inhibited by 60%alchohol portion in vitro,which can inhibit the replication of HBV-DNA and the expressions of HBsAg and HBeAg in HepG2.2.15 cell.In vivo,it can enhance immunity function,and obviously increase the contents of lysozyme and TNF-αin serum.It can also protect markedly against the acute chemical liver injury,which decreases the activities of ALT,AST and ALP in serum and the content of MDA and NO in hepatic tissue, and increases the content of Alb and activities of T-AOC in serum and the activities of SOD, GSH,GSH-Px in hepatic tissue.Its possible mechanisms of hepatoprotection are related with the inhibition of lipid peroxidation.3.In addition,petroliem portion can enhance immunity function,protect liver and inhibit the expressions of HBsAg and HBeAg in HepG2.2.15 cell.4.Chloroform,ethyl acetate,n-butyl alcohol portions have the effects of enhancing immunity function and are toxic to HepG2.2.15 cell.Chloroform portion can also protect against the acute liver injury.
引文
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