绞股蓝有效部位对SAMP8小鼠抗老年性痴呆作用及其机制研究
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摘要
目的：
研究绞股蓝有效部位对快速性老化痴呆小鼠(SAMP8小鼠)抗老年性痴呆(AD)作用及其机制。对中药绞股蓝提取分离出的部位进行体外抗老年性痴呆筛选,确定其有效部位后,进行体内实验,探究绞股蓝对SAMP8小鼠的抗AD作用及机制,为绞股蓝抗老年性痴呆作用提供实验信息及科学依据。
方法：
1.绞股蓝的提取、分离：将干燥的绞股蓝全草制成粗粉,称取25.0kg,分别用95%、70%、50%乙醇浸渍提取3次,每次浸泡4-5d,合并提取液,减压回收乙醇。得乙醇总提取物后用水溶解成混悬液,依次用石油醚、乙酸乙酯、正丁醇萃取,回收溶剂。
2.(1)对中药绞股蓝有效部位进行体外筛选：采用MTT法,体外直接给药测定绞股蓝有效部位对NG-108细胞增殖的影响,用Aβ25-35片段损伤NG-108细胞,造成体外AD模型,测定绞股蓝有效部位对小鼠NG-108细胞的增殖作用,通过两种方式,在药效试验跟踪下,从中筛选出药效较强的有效部位。
(2)体外抗老年性痴呆的作用研究：采用MTT法,直接加药和含药血清检测绞股蓝有效部位(正丁醇部位)对Aβ25-35片段造成NG-108细胞的损伤的体外AD模型的增殖作用：用Hoechst33258染色法、流式细胞法观察正丁醇部位对Aβ25-35造成NG-108细胞损伤作用的影响,采用Elisa法检测半胱天冬氨酸蛋白酶-3(caspase-3)的表达水平。
3.体内抗老年性痴呆作用研究：
采用健康正常老化小鼠(SAMR1小鼠)10只和快速性老化痴呆模型小鼠模型(SAMP8小鼠)50只,随机把SAMP8小鼠分为SAMP8模型组、阳性对照组(石杉碱甲0.15mg/kg);绞股蓝正丁醇部位高剂量组(500mg/kg);绞股蓝正丁醇部位中剂量组(250mg/kg);绞股蓝正丁醇部位低剂量组(125mg/kg);每组10只小鼠。石杉碱甲组和各给药组按以上剂量给药,SAMR1组给予等容积的双蒸水,各组均灌胃给药(ig),给药容量均为10mL/kg,每天给药1次,连续给药8周。末次给药后24h,用Morris水迷宫方法以逃避潜伏期、寻求次数和平台停留时间为指标对小鼠记忆行为进行检测,观察中药绞股蓝正丁醇部位对小鼠行为学的影响。采用ELISA法测定SOD、MDA、 GSH、Aβ、细胞色素C的含量。采用免疫组化法检测APP、p75NTR、pJNK及凋亡蛋白Bax、抗凋亡蛋白Bcl-2和半胱天冬氨酸蛋白酶-3(caspase-3)的表达水平；采用RT-PCR法对SAMP8大脑中APP、BACE-1、p75NTR、pJNKmRNA进行半定量分析；用Western Blot法测定P53的表达。
4.急性毒性试验(最大耐受量Maximaltolerance dose, MTD测定)：取昆明种小鼠20只,体重18-22g,雌雄各半,禁食不禁水12小时后,给予绞股蓝正丁醇部位,以最大浓度和最大体积,灌胃给药(ig)。给药后连续14天观察动物的毒性症状及死亡情况,每间隔7天称重1次。计算MTD的倍数,小鼠体重增加百分率。(小鼠MTD倍数=每只小鼠耐受药量/小鼠平均体重×成人平均体重(60kg)/成人每日用量)。
结果：
1.实验得到乙醇总提取物9kg。用水溶解成混悬液,依次用石油醚、乙酸乙酯、正丁醇萃取,回收溶剂,得石油醚部位286.1g；乙酸乙酯部位524.0g；正丁醇部位974.2g；水部位1303.0g。
2.(1)绞股蓝提取物体外筛选：绞股蓝提取物对体外NG-108细胞增殖的影响：
①绞股蓝石油醚部位和正丁醇部位在5～40μg/mL浓度下对NG-108细胞均有明显的增殖作用(P<0.05或P<0.01)。绞股蓝水提部位在5～40μg/mL浓度对NG-108细胞无增殖作用,而在80μg/mL浓度下对NG-108细胞显示有增殖作用(P<0.05)。乙酸乙酯部位对NG-108细胞有增殖趋势,但与空白组比较无显著性意义。
②Aβ25-35片段造成NG-108细胞损伤后的对细胞增殖作用的影响：绞股蓝正丁醇部位在5～20μg/mL浓度下对NG-108细胞均有明显的增殖作用(P<0.05)。绞股蓝石油醚部位和水提部位在5μg/mL浓度对NG-108细胞有增殖作用(P<0.05)。而乙酸乙酯部位对NG-108细胞有增殖趋势,但与25μMMol/L Aβ25-35损伤组比较无显著性差异,筛选出绞股蓝正丁醇部位为有效部位。
(2)股蓝正丁醇部位体外抗老年性痴呆(AD)作用机制研究：
①绞股蓝正丁醇部位对体外Aβ25-35损伤的NG-108细胞增殖的影响：绞股蓝正丁醇部位在5、10μg/mL浓度下对Aβ25-35损伤的NG-108细胞均有比较明显的增殖作用(P<0.01),在20、40μg/mL浓度对Aβ25-35损伤的NG-108细胞有增殖趋势,但与Aβ25-35损伤组比较无显著性意义。
②绞股蓝正丁醇部位含药血清对体外Aβ25-35损伤的NG-108细胞增殖的影响：绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞体外生长有促进增殖作用,10%绞股蓝正丁醇部位含药血清与10%空白含药血清比较,有显著性意义(P<0.05)；20%绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞有增殖趋势,但与20%空白含药血清比较,无显著性意义。
③Hoechst33258染色：直接给药法表明,绞股蓝正丁醇部位在5、10μg/mL浓度下对Aβ25-35损伤NG-108细胞的凋亡有明显的抑制的作用(P<0.05),20μg/mL浓度下对Aβ25-35损伤NG-108细胞也有一定的抑制作用,但与Aβ25-35损伤组比较无显著性意义；含药血清法表明,绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞凋亡有抑制的作用,10%绞股蓝正丁醇部位含药血清与10%空白含药血清比较,有显著性意义(P<0.05)；20%绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞有增殖趋势,但与20%空白含药血清比较,无显著性意义。
④流式细胞法：直接接给药法表明,绞股蓝正丁醇部位在5、10μg/mL浓度下对Aβ25-35损伤NG-108细胞的凋亡有明显的抑制的作用(P<0.05),20μg/mL浓度下对AP25-35损伤NG-108细胞也有一定的抑制作用,但与Aβ25-35损伤组比较无显著性意义。含药血清法表明,绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞凋亡有抑制的作用,10%绞股蓝正丁醇部位含药血清与10%空白含药血清比较,有显著性意义(P<0.05)；20%绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞有增殖趋势,但与20%空白含药血清比较,无显著性意义。
3.绞股蓝正丁醇部位对快速性老化痴呆模型SAMP8小鼠作用的影响
(1) Morris水迷宫学习能力测试：游泳能力测定结果显示,SAMP8模型组和SAMR1组及各给药组之间比较没有显著性差异(P>0.05)。定位航行实验结果显示：①潜伏期：SAMP8模型组的逃避潜伏期与其他各组比明显延长,其他组(除了高剂量组)与SAMP8模型组对比有非常显著性差异(P<0.01),②寻求次数：SAMP8模型组平均寻求次数均少于其他各给药组,其他组(除了高剂量组)与SAMP8模型组对比有显著性的差异(P<0.05)；③平台停留时间：SAMP8模型组平均停留时间比其他给组短,其他组(除了高剂量组)与对比SAMP8模型组在统计学上无显著差异外,都有显著性的差异(P<0.05)。
(2)绞股蓝正丁醇部位对老年性痴呆小鼠体内自由基代谢的影响：②SOD: SAMR1组、石杉碱甲组、中剂量组和低剂量与SAMP8模型组相比有非常显著性差异(P<0.O1)。②GSH-PX:与SAMP8模型组相比,SAMR1组、石杉碱甲组有非常显著性差异(P<0.01), SAMP8模型组与低剂量组相比有显著性差异(P<0.05)。③MDA: SAMR1组、石杉碱甲组和低剂量与SAMP8模型组相比有非常显著性差异(P<0.01),中剂量组与SAMP8模型组相比有显著性差异(P<0.05)。
(3)绞股蓝正丁醇部位对老年痴呆小鼠Aβ、APP和BACE-1的影响：
①对于血清中Aβ含量的表达,SAMR1组、石杉碱甲组、低剂量组和中剂量与SAMP8模型组相比有非常显著性差异(P<0.01),高剂量与SAMP8模型组相比有显著性差异(P<0.05)。对于脑内Aβ含量的表达,SAMR1组、石杉碱甲组与SAMP8模型组相比有非常显著性差异(P<0.01),低剂量组、中剂量与SAMP8模型组相比有显著性差异(P<0.05)。②绞股蓝正丁醇部位对小鼠脑组织内APP蛋白免疫阳性神经元的影响：APP免疫阳性神经元主要分布于大脑顶叶皮层,海马CA1、CA3和血管内壁等部位。APP免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组与SAMP8模型比较,有非常显著的差异(P<0.01),低剂量组、中剂量与SAMP8模型组比较亦有显著性差异(P<0.05)。
②绞股蓝正丁醇部位对BACE-1、APPmRNA表达的影响：对于BACE-1, SAMR1组与SAMP8模型组相比有非常显著性差异(P<0.01),石杉碱甲组和低剂量与SAMP8模型组相比有显著性差异(P<0.05),对于APPmRNA, SAMR1组与SAMP8模型组相比有非常显著性差异(P<0.01),石杉碱甲组、中剂量组和低剂量与SAMP8模型组相比有显著性差异(P<0.05)。
(4)绞股蓝正丁醇部位对老年性痴呆小鼠Aβ介导p75NTR凋亡通路的影响：
①绞股蓝正丁醇部位对细胞色素C含量的影响：SAMR1组、石杉碱甲组、低剂量和中剂量组与SAMP8模型组相比有非常显著性差异(P<0.01)。
②绞股蓝正丁醇部位对小鼠脑组织内p75NTR、pJNK、Bax、Bcl-2和caspase-3免疫阳性神经元的影响。(1)p75NTR免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组和低剂量组与SAMP8模型组比较,有非常显著性意义(P<0.01),绞股蓝正丁醇中剂量组与模型组比较,有显著性差异(P<0.05),高剂量与SAMP8模型对照组相比,无显著性差异；(2)pJNK免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组、低剂量和中剂量组与SAMP8模型比较,有非常显著的差异(P<0.01),绞股蓝正丁醇高剂量组与模型组比较,无显著性差异。(3)Bax免疫阳性神经元个数各组比较：SAMR1、石杉碱甲组和绞股蓝正丁醇高、中、低剂量组与SAMP8模型组比较,有非常显著性意义(P<0.01)；(4)Bcl-2免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组和绞股蓝正丁醇的低剂量与模型组比较,有非常显著性意义(P<0.01)；绞股蓝正丁醇中剂量与SAMP8模型组比较,有显著性差异(P<0.05)；(5)caspase-3免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组、低剂量组和中剂量组与SAMP8模型组比较,有非常显著性意义(P<0.01),绞股蓝正丁醇高剂量组与SAMP8模型组比较,有显著性差异(P<0.05)。
③绞股蓝正丁醇部位对p75NTR、pJNKmRNA表达的影响：对于p75NTRmRNA, SAMR1组、石杉碱甲组与SAMP8模型组相比有非常显著性差异(P<0.01),中剂量组和低剂量与SAMP8模型组相比有显著性差异(P<0.05),对于pJNKmRNA, SAMR1组、石杉碱甲组和低剂量与SAMP8模型组相比有非常显著性差异(P<0.01)。
④用、Western Blot法测定P53的表达：SAMR1组、石杉碱甲组、低剂量和中剂量组与SAMP8模型组相比有非常显著性差异(P<0.01),高剂量组与SAMP8模型组相比有显著性差异(P<0.05)。
4.急性毒性试验(MTD):结果表明,给药后14天内小鼠活动,外观、行为、摄食、大小便、呼吸等未见异常,未见毒性反应及死亡发生。给药后小鼠生长发育正常,体重增长正常。绞股蓝正丁醇部位对小鼠经口的MTD为120g药材/kg,相当于成人(60kg体重)日用量(30g药材)的240倍。限于药物浓度及动物的给药容量,无法测定LD50。绞股蓝正丁醇部位毒性反应剂量大于120g(原生药)/kg(体重)。
结论：
1.绞股蓝正丁醇部位在体外对NG-108细胞有较好的增殖作用；对由Aβ25-35片段引起NG-108细胞的损害也有较好的保护作用,Hoechst33258染色及流式细胞法研究显示对Aβ25-35损伤的NG-108细胞凋亡有较好的抑制的作用。其保护机制可能是抑制半胱天冬氨酸蛋白酶-3(caspase-3)的水平有关。
2.绞股蓝正丁醇部位有较好的抗老年性痴呆的作用,其作用的机制可能与改善小鼠的学习、记忆能力；提高小鼠的抗氧化能力；抑制APP、BACE-1蛋白水平,使Aβ生成减少；也有可能与减少Aβ与p75NTR结合,抑制JNK通路,使pJNK含量减少,抑制了p53蛋白,增强下游的Bcl-2/Bax的表达,减少细胞色素C的释放,使半胱天冬蛋白酶-3(caspase-3)的释放减少有关。
3.急性毒性试验,绞股蓝正丁醇部位对小鼠经口的MTD为120g药材/kg,相当于成人日用量的240倍,表明毒性低。
Objective
To study anti-Alzheimer's disease (AD) effect and its mechanism of effective parts of Gynostemma pentaphylla on rapid aging dementia mice (SAMP8mice). Four parts of Gynostemma pentaphylla were isolated to screen senile dementia in vitro, identified the effective constituents of senile dementia, providing experimental evidence for treating senile dementia effect of Gynostemma pentaphyllum.
Methods
1. Extraction and separation of Gynostemma pentaphylla:Making dry Gynostemma pentaphylla into coarse powder, using25.0kg, impregnated with95%,70%,50%of ethanol extraction, respectively3times, soaked4～5d, and then combined extracts, the ethanol was recoveried. dissolved total Ethanol extracts into water suspensions, followed to extract petroleum ether, ethyl acetate, n-butyl alcohol extraction, and then recoveried the solvent.
2. Extraction of effective part of Gynostemma pentaphylla screening in vitro:using MTT method, to study the effect of extract of effective parts of Gynostemma pentaphylla on proliferation of mouse NG-108cells in vitro by directly giving drug, to study the effect of extract of effective parts of Gynostemma pentaphylla on proliferation of mouse NG-108cells in vitro injuried by Aβ25-35fragment.
3. Effect of inhibition on AD:using the method of MTT, directly giving and serum detection of Gynostemma pentaphyllum n-butanol extracts to study the mechanism which made model on NG-108cells by A β25-35fragment, through Hoechst33258staining, flow cytometry assay to obsereve NG-108cells apoptosis, Elisa method was detected to the caspase-3expression level.
4. Anti-Alzheimer's disease (AD) effect and its mechanism of effective parts of Gynostemma pentaphy11a on rapid aging dementia mice (SAMP8mice): To observe the effect of Gynostemma pentaphy11a n-butanol extracts on mice behavior. The Morris water maze method was used to seek times, escape latency, platform quadrant than as index to detect the memory behavior of mice. Elisa method was used to determin the content of SOD, MDA, GSH, Aβ and cytochrome C. Immunohistochemistry was used to detect the APP, p75NTR, pJNK, Bax, Bcl-2and cysteine aspartic proteinase-3(caspase-3) expression, Semi-Quantitative analysis was performed on APP, BACE-1, p75NTR and pJNKmRNA in the brain by RT-PCR method. Western blot method was used to detect the expression of p53protein.
5. Acute toxicity test:The n-butanol extract of Gynostemma pentaphyllum, was givien to mice to determin the maximum tolerance dose of mice.
Results
1. Screening in vitro:the effect of Gynostemma pentaphyllum extraction on the proliferation of NG-108cells in vitro:petroleum ether and n-butanol extract of Gynostemma pentaphyllum in5～40μg/mL concentrations on the proliferation of NG-108cells were compared with noramal group, it had significant difference (P<0.05or P<0.01). Water extraction of Gynostemma pentaphyllum at5～40μg/mL concentration on NG-108cells were compared with normal group, it had no significant, but in80μg/mL concentration on NG-108cells, compared with noramal group, it had significant difference (P <0.05). Ethyl acetate had the trend of proliferation on NG-108cells, but compared with the normal group, it had no significant difference.
2. Anti Alzheimer's disease mechanism of Gynostemma pentaphyllum n-butanol extraction in vitro:①Hoechst33258dyeing:directly to drug method indicated that, when Gynostemma pentaphyllum extraction were in5and10μg/mL concentration which injuried by A025-35on NG-108cell of apoptosis compared with the control group, it had significantly difference (P<0.05),20μg/mL concentration which injuried by Aβ25-35on NG-108cell of apoptosis compared with the control group, it had no significant difference. Animal serological methods indicated that, if compared with10%blank serum,10%animal serological had significantly difference(P<0.05). Compared with20%blank serum,20%animal serological had no significantly difference.②Flow cytometry assay:directly to drug method indicated that, when Gynostemma pentaphyllum extraction were in5and10μg/mL concentration which injuried oy Aβ25-35on NG-108cell of apoptosis compared with the control group, it had significant difference (P<0.05),20μg/mL concentration which injuried by Aβ25-35on NG-108cell of apoptosis compared with the control group, it had no significant difference. Animal serological methods indicated that,10%animal serological was compared with10%blank serum, it had significantly difference (P<0.05). Compared with20%blank serum,20%animal serological had no significantly difference.
3. Anti-Alzheimer's disease effect and its mechanism of n-butanol extraction of Gynostemma pentaphylla on rapid aging dementia mice (SAMP8mice):(1)Morris water maze learning ability test:swimming ability test results showed that SAMR1and the administration group were compared with SAMP8model group, it had no significant difference (P>0.05). Positioning navigation experiment results displayed:①Incubation period:Compared with SAMP8model group, SAMR1and the administration groups had very significant difference (except high dose group)(P<0.01).②Sought number:SAMP8model group average sought number were less than other groups who were administrated, if SAMR1and the administration group were compared with SAMP8model group, it had significant difference (except high dose group)(P<0.05).③Platforms stay, the average stay length of SAMP8model group was shorter than other groups, SAMR1and the administration groups were compared with SAMP8model group, they had significant difference (except high dose group)(P<0.05).
(2)N-butanol extraction of Gynostemma pentaphylla on senile dementia effects of free radical metabolism in mice:①SOD:Compared with the SAMP8model group, SAMR1group, huperzine group and low-dose group had a very significant difference (P<0.01).②GSH-PX:Compared with SAMP8model group, huperzine group had very significant difference (P<0.01), low-dose group and medium-dose group had significant differences (P<0.05).③MDA:Compared with the SAMP8model group, SAMR1group and low-dose group had a very significant difference (P<0.01), medium-dose group had significant differences (P<0.05).
(3)①-butanol extraction of Gynostemma pentaphylla on senile dementia effects of Aβ、APP and BACE-1:For the expression of Aβ content in serum compared with SAMP8model group, SAMR1group, huperzine group and all of drug groups had a very significant differences (P<0.01). Expression of Aβ content in the brain, compared with SAMP8model group, SAMR1group, huperzine group had very significant differences (P<0.01), medium-dose groups and low-dose group had significant differences (P<0.05).②Tissue effect of immunohisto-chemical of n-butanol extraction of Gynostemma pentaphylla on APP proteins in mouse brain:APP-immunoreactive neurons were mainly located in the parietal cortex of the brain, hippocampal CA1, CA3and parts of the inner wall of blood vessels. APP number of immunopositive neurons in the comparison group: compared with SAMP8model group, SAMR1and huperzine group had very significant differences (P<0.01), the low dose group and medium-dose group had significant differences (P<0.05).③Expression effect of n-butanol extraction of Gynostemma pentaphylla on BACE-1and APPmRNA:for BACE-1mRNA, compared with SAMP8model group, SAMR1group had very significantly differences(P<0.01), huperzine group and low-dose group had significant differences (P<0.05). For APPmRNA, compared with SAMP8group, SAMR1group had very significantly differences (P<0.01), huperzine group, medium-dose group and low-dose group had significant differences (P<0.05).
(4)N-butanol extraction of Gynostemma pentaphylla on Aβ in Alzheimer's mice which mediated p75NTR to induce apoptosis:①effects of n-butanol fraction on cytochrome c content:compared with SAMP8model group, SAMR1group, huperzine group, low-dose group and the medium-dose group had very significant difference (P<0.01).②Tissue effect in immunohistochemical of n-butanol extraction of Gynostemma pentaphylla on p75NTR, pJNK, Bax, Bcl-2and the caspase-3. p75NTR number of immunopositive neurons in the comparison group:compared with SAMP8model group, SAMR1, huperzine group and low-dose group had very significant differences (P<0.01), the medium-dose group had significant differences (P<0.05). pJNK number of immunopositive neurons in the comparison group:SAMR1, huperzine group, low-dose group and medium-dose group compared with SAMP8group, they had very significant differences (P<0.01), but high-dose group was compared with SAMP8model group, it had no significant differences. Bax number of immunopositive neurons in the comparison group:compared with SAMP8group, huperzine group and all of drug groups had very significant differences(P<0.01),Bcl-2number of immunopositive neurons in the comparison group:compared with SAMP8model group, SAMR1, huperzine group and low-dose group had very significant differences (P<0.01), but medium-dose group had no significant differences.(5)caspase-3number of immunopositive neurons in the comparison group: compared with SAMP8model group, SAMR1and huperzine group, medium-dose group and low-dose group had very significant differences (P<0.01), but high-dose group had significant differences(P<0.05).③Expression effect of n-butanol extraction of Gynostemma pentaphylla on p75NTR and pJNKmRNA:for p75NTRmRNA, compared with SAMP8group, SAMR1group and huperzine group had very significantly differences (P<0.01), medium-dose group and low-dose group had significant differences (P<0.05). For pJNKmRNA, compared with SAMP8model group, SAMR1group had very significantly differences (P<0.01), huperzine group and the low-dose group had significant differences (P<0.05).
(4) Expression effect of n-butanol extraction of Gynostemma pentaphylla on P53:compared with SAMP8model group, SAMR1group, huperzine group, the medium-dose group and low-dose group had very significantly differences (P<0.01), the high-dose group had significant differences (P<0.05).
(5)Acute toxicity test (MTD):The results showed that the activities, appearance, behavior, feeding of mice within14days after administration were normal. Urine, breath, no toxic reactions and death occurred. The mice growth and weight gain after administration was also normal. n-butanol fraction of Gynostemma pentaphyllum on mice by oral administration was120g/kg herbs, and it was equivalent to adults (60kg weight)daily (30g drug)240times.
Conclusion
(1)N-butanol extraction of Gynostemma pentaphylla in vitro can induce NG-108cells to increase proliferation, it also can protect NG-108cells which injuried by Aβ25-35fragment. Hoechst33258staining and flow cytometry assay can decrese injuried by Aβ25-35, inhibit apoptosis in NG-108cells. The protection mechanism may be related to inhibit the cysteamine aspartic acid protease-3(caspase-3) level.
(2) N-butanol extraction of Gynostemma pentaphylla can anti-old sexual dementia of role, its mechanism may be related to improve learning, and memory capacity of mice. Improve the anti-oxidation capacity of mice. Inhibition APP, and BACE-1protein level, reduce Aβgenerated, also can reduce the combination of Aβand p75NTR, inhibit JNK pathway, reduced pJNK content, inhibit p53protein and increase downstream of BCL-2/Bax of expression, release the content of cytochrome C and reduce caspase-3content.
(3)Acute toxicity testing show that n-butanol fraction of gynostemma pentaphyllum on mice by oral administration is120g/kg120g herbs, equivalent to240times times the adult daily amount, indicate it has low toxicity.
研究绞股蓝有效部位对快速性老化痴呆小鼠(SAMP8小鼠)抗老年性痴呆(AD)作用及其机制。对中药绞股蓝提取分离出的部位进行体外抗老年性痴呆筛选,确定其有效部位后,进行体内实验,探究绞股蓝对SAMP8小鼠的抗AD作用及机制,为绞股蓝抗老年性痴呆作用提供实验信息及科学依据。
方法：
1.绞股蓝的提取、分离：将干燥的绞股蓝全草制成粗粉,称取25.0kg,分别用95%、70%、50%乙醇浸渍提取3次,每次浸泡4-5d,合并提取液,减压回收乙醇。得乙醇总提取物后用水溶解成混悬液,依次用石油醚、乙酸乙酯、正丁醇萃取,回收溶剂。
2.(1)对中药绞股蓝有效部位进行体外筛选：采用MTT法,体外直接给药测定绞股蓝有效部位对NG-108细胞增殖的影响,用Aβ25-35片段损伤NG-108细胞,造成体外AD模型,测定绞股蓝有效部位对小鼠NG-108细胞的增殖作用,通过两种方式,在药效试验跟踪下,从中筛选出药效较强的有效部位。
(2)体外抗老年性痴呆的作用研究：采用MTT法,直接加药和含药血清检测绞股蓝有效部位(正丁醇部位)对Aβ25-35片段造成NG-108细胞的损伤的体外AD模型的增殖作用：用Hoechst33258染色法、流式细胞法观察正丁醇部位对Aβ25-35造成NG-108细胞损伤作用的影响,采用Elisa法检测半胱天冬氨酸蛋白酶-3(caspase-3)的表达水平。
3.体内抗老年性痴呆作用研究：
采用健康正常老化小鼠(SAMR1小鼠)10只和快速性老化痴呆模型小鼠模型(SAMP8小鼠)50只,随机把SAMP8小鼠分为SAMP8模型组、阳性对照组(石杉碱甲0.15mg/kg);绞股蓝正丁醇部位高剂量组(500mg/kg);绞股蓝正丁醇部位中剂量组(250mg/kg);绞股蓝正丁醇部位低剂量组(125mg/kg);每组10只小鼠。石杉碱甲组和各给药组按以上剂量给药,SAMR1组给予等容积的双蒸水,各组均灌胃给药(ig),给药容量均为10mL/kg,每天给药1次,连续给药8周。末次给药后24h,用Morris水迷宫方法以逃避潜伏期、寻求次数和平台停留时间为指标对小鼠记忆行为进行检测,观察中药绞股蓝正丁醇部位对小鼠行为学的影响。采用ELISA法测定SOD、MDA、 GSH、Aβ、细胞色素C的含量。采用免疫组化法检测APP、p75NTR、pJNK及凋亡蛋白Bax、抗凋亡蛋白Bcl-2和半胱天冬氨酸蛋白酶-3(caspase-3)的表达水平；采用RT-PCR法对SAMP8大脑中APP、BACE-1、p75NTR、pJNKmRNA进行半定量分析；用Western Blot法测定P53的表达。
4.急性毒性试验(最大耐受量Maximaltolerance dose, MTD测定)：取昆明种小鼠20只,体重18-22g,雌雄各半,禁食不禁水12小时后,给予绞股蓝正丁醇部位,以最大浓度和最大体积,灌胃给药(ig)。给药后连续14天观察动物的毒性症状及死亡情况,每间隔7天称重1次。计算MTD的倍数,小鼠体重增加百分率。(小鼠MTD倍数=每只小鼠耐受药量/小鼠平均体重×成人平均体重(60kg)/成人每日用量)。
结果：
1.实验得到乙醇总提取物9kg。用水溶解成混悬液,依次用石油醚、乙酸乙酯、正丁醇萃取,回收溶剂,得石油醚部位286.1g；乙酸乙酯部位524.0g；正丁醇部位974.2g；水部位1303.0g。
2.(1)绞股蓝提取物体外筛选：绞股蓝提取物对体外NG-108细胞增殖的影响：
①绞股蓝石油醚部位和正丁醇部位在5～40μg/mL浓度下对NG-108细胞均有明显的增殖作用(P<0.05或P<0.01)。绞股蓝水提部位在5～40μg/mL浓度对NG-108细胞无增殖作用,而在80μg/mL浓度下对NG-108细胞显示有增殖作用(P<0.05)。乙酸乙酯部位对NG-108细胞有增殖趋势,但与空白组比较无显著性意义。
②Aβ25-35片段造成NG-108细胞损伤后的对细胞增殖作用的影响：绞股蓝正丁醇部位在5～20μg/mL浓度下对NG-108细胞均有明显的增殖作用(P<0.05)。绞股蓝石油醚部位和水提部位在5μg/mL浓度对NG-108细胞有增殖作用(P<0.05)。而乙酸乙酯部位对NG-108细胞有增殖趋势,但与25μMMol/L Aβ25-35损伤组比较无显著性差异,筛选出绞股蓝正丁醇部位为有效部位。
(2)股蓝正丁醇部位体外抗老年性痴呆(AD)作用机制研究：
①绞股蓝正丁醇部位对体外Aβ25-35损伤的NG-108细胞增殖的影响：绞股蓝正丁醇部位在5、10μg/mL浓度下对Aβ25-35损伤的NG-108细胞均有比较明显的增殖作用(P<0.01),在20、40μg/mL浓度对Aβ25-35损伤的NG-108细胞有增殖趋势,但与Aβ25-35损伤组比较无显著性意义。
②绞股蓝正丁醇部位含药血清对体外Aβ25-35损伤的NG-108细胞增殖的影响：绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞体外生长有促进增殖作用,10%绞股蓝正丁醇部位含药血清与10%空白含药血清比较,有显著性意义(P<0.05)；20%绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞有增殖趋势,但与20%空白含药血清比较,无显著性意义。
③Hoechst33258染色：直接给药法表明,绞股蓝正丁醇部位在5、10μg/mL浓度下对Aβ25-35损伤NG-108细胞的凋亡有明显的抑制的作用(P<0.05),20μg/mL浓度下对Aβ25-35损伤NG-108细胞也有一定的抑制作用,但与Aβ25-35损伤组比较无显著性意义；含药血清法表明,绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞凋亡有抑制的作用,10%绞股蓝正丁醇部位含药血清与10%空白含药血清比较,有显著性意义(P<0.05)；20%绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞有增殖趋势,但与20%空白含药血清比较,无显著性意义。
④流式细胞法：直接接给药法表明,绞股蓝正丁醇部位在5、10μg/mL浓度下对Aβ25-35损伤NG-108细胞的凋亡有明显的抑制的作用(P<0.05),20μg/mL浓度下对AP25-35损伤NG-108细胞也有一定的抑制作用,但与Aβ25-35损伤组比较无显著性意义。含药血清法表明,绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞凋亡有抑制的作用,10%绞股蓝正丁醇部位含药血清与10%空白含药血清比较,有显著性意义(P<0.05)；20%绞股蓝正丁醇部位含药血清对Aβ25-35损伤的NG-108细胞有增殖趋势,但与20%空白含药血清比较,无显著性意义。
3.绞股蓝正丁醇部位对快速性老化痴呆模型SAMP8小鼠作用的影响
(1) Morris水迷宫学习能力测试：游泳能力测定结果显示,SAMP8模型组和SAMR1组及各给药组之间比较没有显著性差异(P>0.05)。定位航行实验结果显示：①潜伏期：SAMP8模型组的逃避潜伏期与其他各组比明显延长,其他组(除了高剂量组)与SAMP8模型组对比有非常显著性差异(P<0.01),②寻求次数：SAMP8模型组平均寻求次数均少于其他各给药组,其他组(除了高剂量组)与SAMP8模型组对比有显著性的差异(P<0.05)；③平台停留时间：SAMP8模型组平均停留时间比其他给组短,其他组(除了高剂量组)与对比SAMP8模型组在统计学上无显著差异外,都有显著性的差异(P<0.05)。
(2)绞股蓝正丁醇部位对老年性痴呆小鼠体内自由基代谢的影响：②SOD: SAMR1组、石杉碱甲组、中剂量组和低剂量与SAMP8模型组相比有非常显著性差异(P<0.O1)。②GSH-PX:与SAMP8模型组相比,SAMR1组、石杉碱甲组有非常显著性差异(P<0.01), SAMP8模型组与低剂量组相比有显著性差异(P<0.05)。③MDA: SAMR1组、石杉碱甲组和低剂量与SAMP8模型组相比有非常显著性差异(P<0.01),中剂量组与SAMP8模型组相比有显著性差异(P<0.05)。
(3)绞股蓝正丁醇部位对老年痴呆小鼠Aβ、APP和BACE-1的影响：
①对于血清中Aβ含量的表达,SAMR1组、石杉碱甲组、低剂量组和中剂量与SAMP8模型组相比有非常显著性差异(P<0.01),高剂量与SAMP8模型组相比有显著性差异(P<0.05)。对于脑内Aβ含量的表达,SAMR1组、石杉碱甲组与SAMP8模型组相比有非常显著性差异(P<0.01),低剂量组、中剂量与SAMP8模型组相比有显著性差异(P<0.05)。②绞股蓝正丁醇部位对小鼠脑组织内APP蛋白免疫阳性神经元的影响：APP免疫阳性神经元主要分布于大脑顶叶皮层,海马CA1、CA3和血管内壁等部位。APP免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组与SAMP8模型比较,有非常显著的差异(P<0.01),低剂量组、中剂量与SAMP8模型组比较亦有显著性差异(P<0.05)。
②绞股蓝正丁醇部位对BACE-1、APPmRNA表达的影响：对于BACE-1, SAMR1组与SAMP8模型组相比有非常显著性差异(P<0.01),石杉碱甲组和低剂量与SAMP8模型组相比有显著性差异(P<0.05),对于APPmRNA, SAMR1组与SAMP8模型组相比有非常显著性差异(P<0.01),石杉碱甲组、中剂量组和低剂量与SAMP8模型组相比有显著性差异(P<0.05)。
(4)绞股蓝正丁醇部位对老年性痴呆小鼠Aβ介导p75NTR凋亡通路的影响：
①绞股蓝正丁醇部位对细胞色素C含量的影响：SAMR1组、石杉碱甲组、低剂量和中剂量组与SAMP8模型组相比有非常显著性差异(P<0.01)。
②绞股蓝正丁醇部位对小鼠脑组织内p75NTR、pJNK、Bax、Bcl-2和caspase-3免疫阳性神经元的影响。(1)p75NTR免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组和低剂量组与SAMP8模型组比较,有非常显著性意义(P<0.01),绞股蓝正丁醇中剂量组与模型组比较,有显著性差异(P<0.05),高剂量与SAMP8模型对照组相比,无显著性差异；(2)pJNK免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组、低剂量和中剂量组与SAMP8模型比较,有非常显著的差异(P<0.01),绞股蓝正丁醇高剂量组与模型组比较,无显著性差异。(3)Bax免疫阳性神经元个数各组比较：SAMR1、石杉碱甲组和绞股蓝正丁醇高、中、低剂量组与SAMP8模型组比较,有非常显著性意义(P<0.01)；(4)Bcl-2免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组和绞股蓝正丁醇的低剂量与模型组比较,有非常显著性意义(P<0.01)；绞股蓝正丁醇中剂量与SAMP8模型组比较,有显著性差异(P<0.05)；(5)caspase-3免疫阳性神经元个数各组比较：SAMR1组、石杉碱甲组、低剂量组和中剂量组与SAMP8模型组比较,有非常显著性意义(P<0.01),绞股蓝正丁醇高剂量组与SAMP8模型组比较,有显著性差异(P<0.05)。
③绞股蓝正丁醇部位对p75NTR、pJNKmRNA表达的影响：对于p75NTRmRNA, SAMR1组、石杉碱甲组与SAMP8模型组相比有非常显著性差异(P<0.01),中剂量组和低剂量与SAMP8模型组相比有显著性差异(P<0.05),对于pJNKmRNA, SAMR1组、石杉碱甲组和低剂量与SAMP8模型组相比有非常显著性差异(P<0.01)。
④用、Western Blot法测定P53的表达：SAMR1组、石杉碱甲组、低剂量和中剂量组与SAMP8模型组相比有非常显著性差异(P<0.01),高剂量组与SAMP8模型组相比有显著性差异(P<0.05)。
4.急性毒性试验(MTD):结果表明,给药后14天内小鼠活动,外观、行为、摄食、大小便、呼吸等未见异常,未见毒性反应及死亡发生。给药后小鼠生长发育正常,体重增长正常。绞股蓝正丁醇部位对小鼠经口的MTD为120g药材/kg,相当于成人(60kg体重)日用量(30g药材)的240倍。限于药物浓度及动物的给药容量,无法测定LD50。绞股蓝正丁醇部位毒性反应剂量大于120g(原生药)/kg(体重)。
结论：
1.绞股蓝正丁醇部位在体外对NG-108细胞有较好的增殖作用；对由Aβ25-35片段引起NG-108细胞的损害也有较好的保护作用,Hoechst33258染色及流式细胞法研究显示对Aβ25-35损伤的NG-108细胞凋亡有较好的抑制的作用。其保护机制可能是抑制半胱天冬氨酸蛋白酶-3(caspase-3)的水平有关。
2.绞股蓝正丁醇部位有较好的抗老年性痴呆的作用,其作用的机制可能与改善小鼠的学习、记忆能力；提高小鼠的抗氧化能力；抑制APP、BACE-1蛋白水平,使Aβ生成减少；也有可能与减少Aβ与p75NTR结合,抑制JNK通路,使pJNK含量减少,抑制了p53蛋白,增强下游的Bcl-2/Bax的表达,减少细胞色素C的释放,使半胱天冬蛋白酶-3(caspase-3)的释放减少有关。
3.急性毒性试验,绞股蓝正丁醇部位对小鼠经口的MTD为120g药材/kg,相当于成人日用量的240倍,表明毒性低。
Objective
To study anti-Alzheimer's disease (AD) effect and its mechanism of effective parts of Gynostemma pentaphylla on rapid aging dementia mice (SAMP8mice). Four parts of Gynostemma pentaphylla were isolated to screen senile dementia in vitro, identified the effective constituents of senile dementia, providing experimental evidence for treating senile dementia effect of Gynostemma pentaphyllum.
Methods
1. Extraction and separation of Gynostemma pentaphylla:Making dry Gynostemma pentaphylla into coarse powder, using25.0kg, impregnated with95%,70%,50%of ethanol extraction, respectively3times, soaked4～5d, and then combined extracts, the ethanol was recoveried. dissolved total Ethanol extracts into water suspensions, followed to extract petroleum ether, ethyl acetate, n-butyl alcohol extraction, and then recoveried the solvent.
2. Extraction of effective part of Gynostemma pentaphylla screening in vitro:using MTT method, to study the effect of extract of effective parts of Gynostemma pentaphylla on proliferation of mouse NG-108cells in vitro by directly giving drug, to study the effect of extract of effective parts of Gynostemma pentaphylla on proliferation of mouse NG-108cells in vitro injuried by Aβ25-35fragment.
3. Effect of inhibition on AD:using the method of MTT, directly giving and serum detection of Gynostemma pentaphyllum n-butanol extracts to study the mechanism which made model on NG-108cells by A β25-35fragment, through Hoechst33258staining, flow cytometry assay to obsereve NG-108cells apoptosis, Elisa method was detected to the caspase-3expression level.
4. Anti-Alzheimer's disease (AD) effect and its mechanism of effective parts of Gynostemma pentaphy11a on rapid aging dementia mice (SAMP8mice): To observe the effect of Gynostemma pentaphy11a n-butanol extracts on mice behavior. The Morris water maze method was used to seek times, escape latency, platform quadrant than as index to detect the memory behavior of mice. Elisa method was used to determin the content of SOD, MDA, GSH, Aβ and cytochrome C. Immunohistochemistry was used to detect the APP, p75NTR, pJNK, Bax, Bcl-2and cysteine aspartic proteinase-3(caspase-3) expression, Semi-Quantitative analysis was performed on APP, BACE-1, p75NTR and pJNKmRNA in the brain by RT-PCR method. Western blot method was used to detect the expression of p53protein.
5. Acute toxicity test:The n-butanol extract of Gynostemma pentaphyllum, was givien to mice to determin the maximum tolerance dose of mice.
Results
1. Screening in vitro:the effect of Gynostemma pentaphyllum extraction on the proliferation of NG-108cells in vitro:petroleum ether and n-butanol extract of Gynostemma pentaphyllum in5～40μg/mL concentrations on the proliferation of NG-108cells were compared with noramal group, it had significant difference (P<0.05or P<0.01). Water extraction of Gynostemma pentaphyllum at5～40μg/mL concentration on NG-108cells were compared with normal group, it had no significant, but in80μg/mL concentration on NG-108cells, compared with noramal group, it had significant difference (P <0.05). Ethyl acetate had the trend of proliferation on NG-108cells, but compared with the normal group, it had no significant difference.
2. Anti Alzheimer's disease mechanism of Gynostemma pentaphyllum n-butanol extraction in vitro:①Hoechst33258dyeing:directly to drug method indicated that, when Gynostemma pentaphyllum extraction were in5and10μg/mL concentration which injuried by A025-35on NG-108cell of apoptosis compared with the control group, it had significantly difference (P<0.05),20μg/mL concentration which injuried by Aβ25-35on NG-108cell of apoptosis compared with the control group, it had no significant difference. Animal serological methods indicated that, if compared with10%blank serum,10%animal serological had significantly difference(P<0.05). Compared with20%blank serum,20%animal serological had no significantly difference.②Flow cytometry assay:directly to drug method indicated that, when Gynostemma pentaphyllum extraction were in5and10μg/mL concentration which injuried oy Aβ25-35on NG-108cell of apoptosis compared with the control group, it had significant difference (P<0.05),20μg/mL concentration which injuried by Aβ25-35on NG-108cell of apoptosis compared with the control group, it had no significant difference. Animal serological methods indicated that,10%animal serological was compared with10%blank serum, it had significantly difference (P<0.05). Compared with20%blank serum,20%animal serological had no significantly difference.
3. Anti-Alzheimer's disease effect and its mechanism of n-butanol extraction of Gynostemma pentaphylla on rapid aging dementia mice (SAMP8mice):(1)Morris water maze learning ability test:swimming ability test results showed that SAMR1and the administration group were compared with SAMP8model group, it had no significant difference (P>0.05). Positioning navigation experiment results displayed:①Incubation period:Compared with SAMP8model group, SAMR1and the administration groups had very significant difference (except high dose group)(P<0.01).②Sought number:SAMP8model group average sought number were less than other groups who were administrated, if SAMR1and the administration group were compared with SAMP8model group, it had significant difference (except high dose group)(P<0.05).③Platforms stay, the average stay length of SAMP8model group was shorter than other groups, SAMR1and the administration groups were compared with SAMP8model group, they had significant difference (except high dose group)(P<0.05).
(2)N-butanol extraction of Gynostemma pentaphylla on senile dementia effects of free radical metabolism in mice:①SOD:Compared with the SAMP8model group, SAMR1group, huperzine group and low-dose group had a very significant difference (P<0.01).②GSH-PX:Compared with SAMP8model group, huperzine group had very significant difference (P<0.01), low-dose group and medium-dose group had significant differences (P<0.05).③MDA:Compared with the SAMP8model group, SAMR1group and low-dose group had a very significant difference (P<0.01), medium-dose group had significant differences (P<0.05).
(3)①-butanol extraction of Gynostemma pentaphylla on senile dementia effects of Aβ、APP and BACE-1:For the expression of Aβ content in serum compared with SAMP8model group, SAMR1group, huperzine group and all of drug groups had a very significant differences (P<0.01). Expression of Aβ content in the brain, compared with SAMP8model group, SAMR1group, huperzine group had very significant differences (P<0.01), medium-dose groups and low-dose group had significant differences (P<0.05).②Tissue effect of immunohisto-chemical of n-butanol extraction of Gynostemma pentaphylla on APP proteins in mouse brain:APP-immunoreactive neurons were mainly located in the parietal cortex of the brain, hippocampal CA1, CA3and parts of the inner wall of blood vessels. APP number of immunopositive neurons in the comparison group: compared with SAMP8model group, SAMR1and huperzine group had very significant differences (P<0.01), the low dose group and medium-dose group had significant differences (P<0.05).③Expression effect of n-butanol extraction of Gynostemma pentaphylla on BACE-1and APPmRNA:for BACE-1mRNA, compared with SAMP8model group, SAMR1group had very significantly differences(P<0.01), huperzine group and low-dose group had significant differences (P<0.05). For APPmRNA, compared with SAMP8group, SAMR1group had very significantly differences (P<0.01), huperzine group, medium-dose group and low-dose group had significant differences (P<0.05).
(4)N-butanol extraction of Gynostemma pentaphylla on Aβ in Alzheimer's mice which mediated p75NTR to induce apoptosis:①effects of n-butanol fraction on cytochrome c content:compared with SAMP8model group, SAMR1group, huperzine group, low-dose group and the medium-dose group had very significant difference (P<0.01).②Tissue effect in immunohistochemical of n-butanol extraction of Gynostemma pentaphylla on p75NTR, pJNK, Bax, Bcl-2and the caspase-3. p75NTR number of immunopositive neurons in the comparison group:compared with SAMP8model group, SAMR1, huperzine group and low-dose group had very significant differences (P<0.01), the medium-dose group had significant differences (P<0.05). pJNK number of immunopositive neurons in the comparison group:SAMR1, huperzine group, low-dose group and medium-dose group compared with SAMP8group, they had very significant differences (P<0.01), but high-dose group was compared with SAMP8model group, it had no significant differences. Bax number of immunopositive neurons in the comparison group:compared with SAMP8group, huperzine group and all of drug groups had very significant differences(P<0.01),Bcl-2number of immunopositive neurons in the comparison group:compared with SAMP8model group, SAMR1, huperzine group and low-dose group had very significant differences (P<0.01), but medium-dose group had no significant differences.(5)caspase-3number of immunopositive neurons in the comparison group: compared with SAMP8model group, SAMR1and huperzine group, medium-dose group and low-dose group had very significant differences (P<0.01), but high-dose group had significant differences(P<0.05).③Expression effect of n-butanol extraction of Gynostemma pentaphylla on p75NTR and pJNKmRNA:for p75NTRmRNA, compared with SAMP8group, SAMR1group and huperzine group had very significantly differences (P<0.01), medium-dose group and low-dose group had significant differences (P<0.05). For pJNKmRNA, compared with SAMP8model group, SAMR1group had very significantly differences (P<0.01), huperzine group and the low-dose group had significant differences (P<0.05).
(4) Expression effect of n-butanol extraction of Gynostemma pentaphylla on P53:compared with SAMP8model group, SAMR1group, huperzine group, the medium-dose group and low-dose group had very significantly differences (P<0.01), the high-dose group had significant differences (P<0.05).
(5)Acute toxicity test (MTD):The results showed that the activities, appearance, behavior, feeding of mice within14days after administration were normal. Urine, breath, no toxic reactions and death occurred. The mice growth and weight gain after administration was also normal. n-butanol fraction of Gynostemma pentaphyllum on mice by oral administration was120g/kg herbs, and it was equivalent to adults (60kg weight)daily (30g drug)240times.
Conclusion
(1)N-butanol extraction of Gynostemma pentaphylla in vitro can induce NG-108cells to increase proliferation, it also can protect NG-108cells which injuried by Aβ25-35fragment. Hoechst33258staining and flow cytometry assay can decrese injuried by Aβ25-35, inhibit apoptosis in NG-108cells. The protection mechanism may be related to inhibit the cysteamine aspartic acid protease-3(caspase-3) level.
(2) N-butanol extraction of Gynostemma pentaphylla can anti-old sexual dementia of role, its mechanism may be related to improve learning, and memory capacity of mice. Improve the anti-oxidation capacity of mice. Inhibition APP, and BACE-1protein level, reduce Aβgenerated, also can reduce the combination of Aβand p75NTR, inhibit JNK pathway, reduced pJNK content, inhibit p53protein and increase downstream of BCL-2/Bax of expression, release the content of cytochrome C and reduce caspase-3content.
(3)Acute toxicity testing show that n-butanol fraction of gynostemma pentaphyllum on mice by oral administration is120g/kg120g herbs, equivalent to240times times the adult daily amount, indicate it has low toxicity.
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