【作者】 陈光亮；

【导师】 徐叔云； 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2005， 博士

【摘要】 第一部分 总皂苷的提取及含量测定 目的 从牛膝、萆薢中分别提取总皂苷,测定提取物中总皂苷含量,了解提取物用于药效学试验的可行性。 方法 牛膝切片采用水提、石油醚脱脂、醇提、饱和正丁醇萃取、过氧化铝柱提取总皂苷,采用紫外—可见分光光度法,以齐墩果酸为对照品测定牛膝提取物中总皂苷的含量。萆薢切片乙醇提取、石油醚脱脂、饱和正丁醇萃取、加丙酮沉淀提取总皂苷,采用紫外—可见分光光度法,以薯蓣皂苷元为对照品测定测定萆薢提取物中总皂苷的含量。 结果 齐墩果酸在2.77 μg～13.85 μg范围内线性关系良好,平均加样回收率为97.0%,牛膝提取物中总皂苷含量为80.%。薯蓣皂苷元在5.12 μg～25.6 μg范围内线性关系良好,样品平均加样回收率为97.5%。萆薢提取物中总皂苷含量为53.1%。 结论 上述提取总皂苷方法可行,稳定,总成分含量能满足药效学试验的要求。 第二部分 萆薢总皂苷+牛膝总皂苷的组方合理性研究 目的 从降低血尿酸水平和抗炎作用两方面探讨牛膝总皂苷和萆薢总皂苷的组方合理性及剂量配比。 方法 采用尿酸型小鼠高尿酸血症模型和酵母膏致小鼠高尿酸血症模型以及小鼠耳肿胀模型和微晶型尿酸钠致大鼠急性痛风性关节炎模型模型。按照Codrug软件,得优化组方。 结果 在尿酸型小鼠高尿酸血症模型和酵母膏致小鼠高尿酸血症模型,萆薢总皂苷组、牛膝总皂苷+萆薢总皂苷组和阳性对照组小鼠血清尿酸水平显著降低（P<0.01）,牛膝总皂苷480、240、120 mg.kg^-1组小鼠血清尿酸水平与模型组比较无显著性差异。萆薢总皂苷组与牛膝总皂苷+萆薢总皂苷组相比小鼠血清尿酸水平也无明显差异。牛膝总皂苷、萆薢总皂苷对小鼠耳肿胀均有显著的抑制作用,牛膝总皂苷+萆薢总皂苷组耳肿胀抑制率均强于单药组。更多还原

【Abstract】 Part 1 Extracting of total saponin and research on their qualityAim To extract the total saponin of Dioscorea and Achyranthes and research their quality. Methods The total saponin of Achyranthes bidentata (TSA) was extracted by water, alcohol, n-butanol.With oleanolic acid as control, the content of total saponin was determinated by UV. Total saponin of Dioscorea (TSD) was extracted by alcohol, n-butanol. With diosgenin as control, the content of total saponin was determinated by UV.Results The linear relationship of oleanolic acid was better between 2.77μg~13.85μg, average recovery rate was 97% and the content of total saponin of Achyranthes was 80%. The linear relationship of diosgenin was better between 5.12μg~25.6μg, average recovery rate was 97.5% and the content of total saponin was Dioscorea 53.1%.Conclusion The methods was well done to extract total saponin and stable. The ingrements comformed to the rules of animal experiments.Part 2 Study on the rationality of TSD plus TSAAim To study the rationality and the dose of TSD and TSA according to the decrease ofserum uric acid level and anti-inflammatory of TSD and TSA.Methods Mice hyperuricemic models were made by uric acid ip or yeast extract paste ig. Inthese models the effect of TSD, TDA on uric acid level was observed. And mice ear swellingwas induced by dimethylbenzene, rat feet swelling was induced by MSU. In these modelsanti-inflammatory effect of TSD, TSA was measured. According to Codrug software, thegrading-up combination prescription was obtained.Results In the mice hyperuricemic models, the serum uric acid level of TSD group, TSDplus TSA group and positive control group was significantly reduced compared with themodel group(P < 0. 01) .The serum uric acid level of TSA group has no difference from that of the model group. The serum uric acid level of TSD group has no difference from that TSD plus TSA group yet. Both of TSD and TSA could markedly inhibit the mice ear swelling. Moreover the inhibitory of the group of TSD plus TSA was more stronger than the group of TSD or TSA. In the mice ear swelling model, TSA120 mg-kg’1 plus TSD 240mg. kg"1 could reach the maximal inhibitory effect; in the rat feet swelling model TSA 60 mg-kg"1 plus TSD 120 mg. kg"1 could reach the maximal inhibitory effect.Conclusion TSD could reduce the mice serum uric acid level, but TSA could not. Both TSD and TSA had anti-inflammatory effect. TSD plus TSA had more anti-inflammatory effect. In these models the best proportion of TSA and TSD was 1:2.Part 3 Establish animal hyperuricemia model and studies on the effects and mechanisms of TSD A on animal experimental hyperuricemiaAim To establish animal hyperuricemia model and studies on the effects and mechanisms of TSDA on animal experimental hyperuricemia.Methods (l)Mouse was given different dose of uric acid by intraperitoneal injection(ip), the serum uric acid level was detected after 1 h later. (2) Yeast extract paste 7. 5—30 g ? kg"1 was given to mice by ig once daily for 7,14,21,28 consecutive days, and detected the level of uric acid in serum.(3)Mouse and rat hyperuricemic models were made by orally administering yeast extract paste once per day (30g.kg’], 20g.kg"’, respectively) for 7 days; another mouse hyperuricemia model was generated by intraperitoneal injection once with uric acid 250 mg.kg"1 or potassium oxonate 300 mg-kg"1. (4)The concentration of uric acid in serum or urine was detected by the phosphotungstic acid method, and the activity of xanthine oxidase (XOD) was assayed by a test kit.Results (1) Uric acid 125, 250, 500, 1000 mg.kg1 ip could significantly increase the level of uric acid in mouse serum. The level of uric acid in mouse serum was attained peak at 10min and the hyperuricemia could lasted over 4 hours after uric acid 250mg.kg"Igiven. (2)The serum uric acid level in mice were (271. 8±53. 2), (215. 4+31. 5), (195. 9 + 56. 0), (142.1 ±30. 7) umol ? L"1 in 30 g - kg"1 group; and (226. 8±40. 7), (148. 67±30. 4), (176. 9+27.0), (119.3±27. 4) umol . L"1 in 15 g . kg’1 group after administration 1, 2, 3, 4 weeks; In 7. 5 g ? kg’1 group the serum uric acid level was (117. 0+29.0) umol ? L"1 after 1 week. However the serum uric acid of the control group was (108.1 ± 13.9) umol ? L"1. (3)In the mouse hyperuricemia model induced by uric acid, the serum uric acid of the normal group, model group m,TSD plus TSA group (400 ^ 120n 40 mg. kg"1), benzbromarone group (20 mg. kg"1) and allopurinol group (40 mg. kg"1) were 114. 3+21. 4, 229.6+46.9, 160.4 + 32.0, 162.5 + 27.5, 25. 4 ±35. 5, 126.9 ±19.8, 189. 6 ± 31. 6 u mol. L1 respectively.(4) In the mouse hyperuricemia model induced by yeast extract paste, the serum uric acid level were 118.6±29.2, 244.7±47.2, 161.7±34.6, 144.8±31.0, 175.6± 23.9, 81.8±20. 7, 73.0±19.4umol.L~1 respectively. (5) In the mouse hyperuricemia model induced by potassium oxonate, the serum uric acid level were 98. 2+29.9,179. 7± 39.0,131. 7 ± 20.0,140.8 ± 29.2,160. 6 ± 28. 2,88. 7 ± 21.2,117. 3 ± 32.3 u mol. L"1 respectively. (6) In the rat hyperuricemia model induced by yeast extract paste, the serum uric acid level were 152.6+27.9, 282.2±65. 2, 195. 3±66.3, 190. 7±35.8, 243.8+51.1, 178.6 ±40. 7, 121.3 ±57.3 umol.L"1 respectively; the serum XOD activity were 33. 6±3. 2, 52. 4±4. 4, 42. 3±5. 9, 44. 9±5. 1, 44. 6±6. 7, 48. 8±7. 0, 10.8+2. 2 U. I/1 respectively; 24 h total uric acid excretion, were 23. 5 ±4.82, 43.1 +9.18, 41. 4 ±8.53, 35.3±6. 7, 36. 7±8. 06, 43.8±12. 6, 19. 2±4. 9 mmol respectively.Conclusion (1) Uric acid given by ip could form mice hyperuricemia model, the dosage of 250 mg .kg"1 was better. (2) Yeast extract paste (15~30) g ? kg"1 given by ig could form mice hyperuricemia model, and the hyperuricemia could last 1 ~2 weeks.(3) TSDA possesses a potent anti-hyperuricemic effect on hyperuricemic animals, and the mechanism may be relevant in accelerating the excretion and decreasing the production of uric acid. Part 4 Eeffects and mechanisms of TSDA on gouty arthritis ofrabbit and ratAim To observe the preventive and therapeutic effects and mechanisms of TSD on animal gout arthritis.Methods Rat gouty arthritis model and rabbit gouty arthritis model were established by MSU intraarticular injection, the joint swell degree, inflammatory factor and cytokine in the joint fluid or leachate were detected, moreover histopathologic examination was done, rat feet swelling was induced by carrageenan, mice ear swelling was induced by dimethylbenzene, the mice writhing responewas induced by acetic acid, the mice pain induced by warm bath or hot plate, and rat granuloma induced by cotton ball subcutaneously embedding. In these models the swelling degree, pain response latency, granuloma weight and so on were detected respectively.Results hi the rat model, the joint swelling was very obviouse 6 h later and the swelling degree reached the peak at 24h or 48h.The level of NO> TNF-ou PGE2. IL-lp\ IL-6 and IL-8 in the model group was significantly increased and histopathologic examination showed that the synovial cells were seriously injured in the model group, there are many polycyte, histoleucocyte and urete crystal in the joint and soft tissue. TSDA 200, 60mg.kg’1 could significantly inhibit joint swelling of rat and significantly improve the histopathologic changes. TSDA 200mg.kg"1 could improve the gaits and significantly inhibit the increase of NO. TNF- a and PGE2 content as well as the content of IL-6.In the rabbit model, the rabbit joint of the model group appeared red, swelling and higher skin temperature; the fluid increased; WBC in the blood and fluid rose significantly in the model group. The level of NCK TNF-ou PGE2> IL-lp\ IL-6 and IL-8 in the model group was significantly increased and histopathologic examination showed that the synovial cells and soft tissue were seriously injured in the model group. TSDA 100 mg.kg"1 could significantly reduce the fluid in the joint and 100, 30, 10 mg. kg"1 could significantly reduce WBC in the joint fluid. 100, 30 mg.kg"1 inhibited the content of IL-lfi. IL-6 and IL-8.In the model induced by carrageenan, the swellling degree of TSDA 200,60mg.kg" groups was significantly decreased compared with the model group. In the model of cottonball granuloma, the weight of granuloma groups were significantly reduced. In the modelsinduced by dimethylbenzen and writhing respone was induced by acetic acid, TSDA400,120,40 mg.kg’1 could significantly reduce mouse ear swelling and frequency of writhingrespone. TSDA 400 mg.kg"! could prolong pain response latency.Conclusion TSDA possessed antigout arthritis effects, its mechanism may be related toinhibit the product of cytokines.Part 5 Effects of TSDA on cytokine secrection and their gene expression of J774 cells and human peripheral blood mononuclear cellsAim To investigate the effects of TSDA on cytokine secrection and their gene expression of J774 cells and human peripheral blood mononuclear cells (PBMC) in vitro. The effect of TSDA on neutrophil chemotaxis was also observed.Methods Isolated total RNA from different groups of J774 cells and PBMC, TNF-a mRNA, IL-lpmRNA, IL-8mRNA expression with LPS stimulation were investigated by RT-PCR technique; ELIS A technique measured TNF-a, IL-6 contents of J774 cells, neutrophil chemoattractant made with zymosan was observed.Results The results from RT-PCR showed that TSDA could inhibit EL-1 pmRNA,IL-8mRNA expression, the inhibitory rates on IL-lp*mRNA expression in the groups of 100, 1000 mg.L"1 were about 27.2±7.8%> 32.5±10.8% (P<0.01,n=4) respectively; the inhibitory rates on IL-8mRNA expression in the groups of 100,1000 mg.L"1 were about 19.1±7.2%^ 24.0±6.2% (P<0.05,n=4) respectively. In addition, TSDA 10, 100, 1000 mg-L^could inhibit TNFa and IL-6 secretion of J774 cells, the inhibitory rates on TNFa were 32.8%, 56.6%, 85.6% and on IL-6 were 33.4%, 61.8%, 86.1%. And TSDA 1000 mg.L"1 could inhibit TNFamRNA expression, the inhibitory rate was 15.0±6.0%. What’s more TSDA 100, 1000 mg.L"1 could inhibit neutrophil chemotaxis induced by zymosan.Conclusion TSDA could inhibit the synthesis of TNF-a , IL-6 and expression of IL-ipmRNA, IL-8mRNA,TNF-amRNA. Inhibition inflammatory cytokines production更多还原