【作者】 傅永红；

【导师】 李红玉；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 目的:对单味藏药甘青乌头提取物以及复方十八味诃子利尿丸进行了抑菌作用研究;深入考察了兔耳草提取物体内外抗HSV-2病毒活性及其机制;初步检测了甘青乌头生物碱类提取物对肿瘤细胞增殖的抑制作用。通过实验筛选出具有抗菌、抗病毒以及抑制肿瘤细胞增殖的活性部位。方法:本研究采用有机溶剂提取法得到甘青乌头和兔耳草全草的提取物。采用琼脂扩散法和微量稀释法以抑菌圈和最小抑菌浓度为指标对单味甘青乌头乙醇总提取物、挥发油以及生物碱类提取物和藏药复方十八味诃子利尿丸进行了体外抑菌活性研究。采用MTT法结合CPE法测定兔耳草乙醇总提取物和总皂苷对Vero细胞的毒性,以ACV为阳性对照采用空斑减少法结合定量荧光PCR法考查了兔耳草乙醇总提取物和总皂苷体外抗HSV-2病毒活性及其机制。通过建立HSV-2病毒小鼠脑炎模型和生殖器模型对兔耳草乙醇总提取物和总皂苷体内抗病毒活性进行了初步探讨。采用SRB法检测了甘青乌头生物碱类提取物对肝癌细胞HepG-2和肺癌细胞lewis增殖的抑制作用。结果:抗菌活性研究显示:单味藏药甘青乌头乙醇总提取物、挥发油以及生物碱类提取物对金黄色葡萄球菌、耐药表皮葡萄球菌、枯草芽孢杆菌、蜡状芽孢杆菌、粪肠球菌、大肠埃希菌、铜绿假单孢杆菌和白色念珠菌均形成了直径范围为9～19 mm的抑菌圈,提取物对各种菌种的MIC范围为0.125～25.00 mg/ml。藏药复方十八味诃子利尿丸对实验菌种具有抑制作用,形成直径范围为8～19 mm大小的抑菌圈,对各种菌的MIC范围为3.125～50 mg╱ml。抗病毒活性研究:兔耳草乙醇总提取物、总皂苷和阳性对照ACV对Vero细胞的半数中毒浓度TC₅₀分别为0.2103 mg/ml、0.1127 mg/ml和11.02 mg/ml,对病毒直接杀灭的半数有效浓度EC₅₀分别为0.08825 mg/ml、0.03255 mg/ml和0.2529 mg╱ml,治疗指数TI分别为2.4、3.5和44.3。抑制病毒侵入细胞作用的半数有效浓度EC₅₀分别为0.1478 mg/ml、0.09333mg/ml和0.2105mg/ml,治疗指数TI分别为1.4、1.2和52.4。兔耳草乙醇总提取物、总皂苷对小鼠灌胃的LD₅₀分别为1.27 g/kg和0.5g/kg。0.125 g/kg剂量组明显延长了小鼠的平均存活时间,总皂苷在该剂量组对小鼠的保护率为10%。生殖器感染小鼠给药一周后,小鼠生殖器疱疹病变出现好转,两周后小鼠基本康复。抑制肿瘤细胞增殖活性研究:甘青乌头脂溶性生物碱和水溶性生物碱对HepG-2细胞和Lewis细胞均具有明显的抑制作用,抑制作用随药物浓度的增加和作用时间的延长而增强。结论:抗菌活性研究:单味藏药甘青乌头各种提取物对实验菌种抑制作用:挥发油成分＞水溶性生物碱＞脂溶性生物碱＞乙醇总体提取物。各种提取物对革兰阳性（G+）菌的抑制效应比革兰阴性（G-）菌显著一些。对耐药菌和真菌效果最好,尤其是生物碱类该特性非常突出。藏药复方十八味诃子利尿丸对革兰阳性（G+）菌比对革兰阴性（G-）菌的抑制作用强,对白色念珠菌抑制效果最好。抗病毒活性研究:对HSV-2的直接杀灭和病毒侵入细胞的阻断作用是兔耳草乙醇总提取物和总皂苷体外抗病毒的主要作用途径。提取物高剂量治疗组对HSV-2病毒引起的小鼠脑炎和生殖器感染有一定的治疗作用。抑制肿瘤细胞增殖活性研究:甘青乌头脂溶性生物碱和水溶性生物碱对HepG-2细胞和Lewis细胞均具有明显的抑制作用且抑制作用随药物浓度的增加和作用时间的延长而增强,其中水溶性生物碱比脂溶性生物碱的抑制作用更加明显。更多还原

【Abstract】 Objective:In order to screen the active extracts of antibacterial,antivirus and antitumor effects in tibetan medicine,the antibacterial activity of A.tanguticum extracts and the shiba wei hezi liniao wan was studied,and the anti-HSV-2 virus activity and mechanisms of L.brevituba extracts was studied in vitro and in vivo.The activity of A.tanguticum alkaloid extracts on the proliferation of tumor cells was also studied initially.Method:The extracts of A.tanguticum and L.brevituba were made by the organic solvent method.The antibacterial activity of A.tanguticum ethanol extracts,Volatile oil and alkaloids and the prescription shiba wei hezi liniao wan was studied in vitro by using the agar disc diffusion method and a broth microdilution method.The cytotoxicity of L.brevituba ethanol extract and saponins to Vero was assayed by MTT and CPE.As ACV was used as positive reference drug, the anti-HSV-2 virus activity and mechanisms was studied in vitro by using CPE,plaque reduction assay and a real-time PCR.Mice encephalitis and genital herps model were established by infected with HSV-2 to evaluate the anti-HSV-2 virus activity in vivo preliminary.The activity of A.tanguticum alkaloid extracts on the proliferation of tumor cells was studied initially by using SRB.Result:The antibacterial activity of tibetan medicine:the antibacterial activity against Staphylococcus aureus（ATCC 25923）,Staphylococcus epidermidis,Enterococcus faecalis,Escherichia coli （ATCC 25922）,Bacillus subtilis,Bacillus cereus,Pseudomonas aeruginosa（ATCC2785）, Candida albicans of the A.tanguticum ethanol extracts,Volatile oil and alkaloids was studied. The inhibition zones were in the range of 9～19 mm.The MIC was in the range of 0.125～25.00 mg/ml.The antibacterial activity against tested strains of the prescription shiba wei hezi liniao wan was studied.The inhibition zones was in the range of 8～19 mm and the MIC was in the range of 3.125～50 mg/ml.The anti-HSV-2 virus activity of L.brevituba:the TC₅₀of L.brevituba ethanol extract, saponins and ACV were 0.2103 mg/ml,0.1127 mg/ml and 11.02 mg/ml in vero,respectively.The EC₅₀of virucidal activityof extracts of L.brevitub against HSV-2 were 0.08825 mg/ml,0.03255 mg/ml and 0.2529 mg/ml,and TI were 2.4,3.5 and 44.3.For effect of extracts of L.brevitub on HSV-2 attachment to Vero cells,The EC₅₀were 0.1478 mg/ml,0.09333 mg/ml and 0.2105 mg/ml,and TI were 1.4,1.2 and 52.4.The LD₅₀of L.brevituba ethanol extract and saponins were 1.27 g/kg and 0.5 g/kg respectively.Dose 0.125 g/kg prolonged mean survival times.The protected rate of dose 0.125 g/kg for saponins was 10%.Genital infection in mice a week after the administration,genital herpes lesions in mice were improved,and the mice recovered in two weeks.The antitumor activity of A.tanguticum fat-soluble alkaloids and water-soluble alkaloids:the extracts inhibit HepG-2 and Lewis significantly.The effect of extracts enhanced by increasing drug concentration and extensing the time.Conlusion:The antibacterial activity of tibetan medicine:the result showed that A.tanguticum ethanol extracts,Volatile oil and alkaloids effect were Volatile＞water-soluble alkaloids＞fat-soluble alkaloids＞ethanol extracts.The effects of various extracts on gram-positive（G+）were more significant than that on gram-negative（G-）.The effects of various extracts on methicillin-resistant bacteria and fungi were the best,particularly in the alkaloids.Shibawei Heziliniao Wan had more obvious antibacterial activity to gram-positive（G+）than gram-negative（G-）.The effect on Candida albicans was more obviously.The anti-HSV-2 virus activity of L.brevituba:directly killing HSV-2 and preventing HSV-2 attaching to vero cells were the main ways of anti-viral role for L.brevituba ethanol extract and saponins in vitro.High dose of extracts had a certain therapeutic effect in mice infected with the brain and genitalia.The antitumor activity of A.tanguticum fat-soluble alkaloids and water-soluble alkaloids:the extracts inhibited HepG-2 and Lewis significantly and the effect of extracts enhanced by increasing drug concentration and extensing the time.Water-soluble Alkaloids had more obvious effect than fat-soluble.更多还原