永瓣藤化学成分的研究

永瓣藤化学成分的研究陈玲，谢平，戚薇，曾菊珍（药学系中药化学教研室）关键词永瓣藤，化学成分，三萜，蒜盘子甾酮永瓣藤（ＭｏｎｉｍｏｐｅｔａｌｎｍＣｈｉｎｅｎｓｅＲｅ－ｈｄ）系卫矛科永瓣藤属植物［１］，为我国特有的珍稀二类保护植物［２］，分布于江西东北部...     

Effects of β-escin and saponin on the transverse-tubular system and sarcoplasmic reticulum membranes of rat and toad skeletal muscle

 Mechanically skinned skeletal muscle fibres from rat and toad were exposed to the permeabilizing agents β-escin and saponin. The effects of these agents on the sealed transverse tubular system (t-system) and sarcoplasmic reticulum (SR) were examined by looking at changes in the magnitude of the force responses to t-system depolarization, the time course of the fluorescence of fura-2 trapped in the sealed t-system, and changes in the magnitude of caffeine-induced contractures following SR loading with Ca²⁺ under defined conditions. In the presence of 2 μg ml^–1β-escin and saponin, the response to t-system depolarization was not completely abolished, decreasing to a plateau, and a large proportion of fura-2 remained in the sealed t-system. At 10 μg ml^–1, both agents abolished the ability of both rat and toad preparations to respond to t-system depolarization after 3 min of exposure, but a significant amount of fura-2 remained in sealed t-tubules even after exposure to 100 μg ml^–1β-escin and saponin for 10 min. β-Escin took longer than saponin to reduce the t-system depolarizations and fura-2 content of the sealed t-system to a similar level. The ability of the SR to load Ca²⁺ was reduced to a lower level after treatment with β-escin than saponin. This direct effect on the SR occurred at much lower concentrations for rat (2 μg ml^–1β-escin and 10 μg ml^–1 saponin) than toad (10 μg ml^–1β-escin and 150 μg ml^–1 saponin). The reverse order in sensitivities to β-escin and saponin of t-system and SR membranes indicates that the mechanisms of action of β-escin and saponin are different in the two types of membrane. In conclusion, this study shows that: (1) β-escin has a milder action on the surface membrane than saponin; (2) β-escin is a more potent modifier of SR function; (3) simple permeabilization of membranes is not sufficient to explain the effects of β-escin and saponin on muscle membranes; and (4) the t-system network within muscle fibres is not a homogeneous compartment. Received: 18 November 1998 / Received after revision: 6 January 1999 / Accepted: 7 January 1999     

Calmodulin loss in vascular smooth muscle following Triton X-100 or saponin skinning

The calmodulin (CaM) content of intact and chemically skinned strips of rat caudal artery was measured using a¹²⁵I-CaM radioimmunoassay. The total CaM measured following homogenization of arterial tissue with EGTA and EGTA/Triton X-100 was 2.58 mol/kg wet tissue. Based on a smooth muscle volume of 40%, this value corresponds to a cellular CaM concentration of 6.5 M. Approximately 97% of total CaM was soluble and approximately 3% was EGTA-nonextractable. Permeabilization of the plasmalemma with 0.15 mg/ml saponin or 0.5% Triton X-100 caused significant detergent-dependent loss of CaM. At the end of a 1 h skinning period, tissues exposed to saponin lost 30% of total CaM. By comparison, tissues skinned under the same conditions with Triton X-100 lost 50%. During a subsequent 4h exposure to relaxing solution, total tissue CaM continued to decline. The exponential loss over the 5h period was described by a first order model having diffusible and nondiffusible CaM components. The diffusible CaM component of saponin skinned tissue (59%) was significantly less than the diffusible component of those skinned with Triton X-100 (88%); however, the rate coefficients for CaM diffusion (0.78 h^–1 and 0.91 h^–1, respectively) did not statistically differ. The nondiffusible component of CaM was significantly larger in saponin treated strips (42%) than in Triton X-100 permeabilized tissue (12%). Arterial strips skinned with Triton X-100, which were subsequently exposed to relaxing solution for up to 22 h, lost significantly more CaM than those retained in Triton X-100 skinning solution for a comparable duration These studies demonstrate the diffusion of CaM from detergent skinned arterial strips and characterize the time course of that loss.     

人参皂甙Rg1、Rb1和维生素E对氧化损伤的牛RPE细胞p53表达的影响

目的：检验氧化损伤的牛RPE细胞是否存在p53基因表达的变化和人参皂甙Rg1、Rb1和维生素E对其的影响,以期进一步研究RPE细胞氧化损害的机理.方法：建立牛RPE细胞氧化损伤模型,原位杂交技术测定RPE细胞p53mRNA的表达,免疫组化技术测定p53蛋白表达.结果：p53蛋白表达阳性的牛RPE细胞的细胞核染成黄色,与次黄嘌吟／黄嘌吟氧化酶（HX／XO）组p53蛋白阳性的细胞数相比较,正常对照组,Rg1（0.1mg.L-1）组及VitE（10mg.L-1）组存在显著性差异（P＜0.05）,而与Rb1（10mg.L-1）组无显著性差异.原位杂交法测定P53mRNA表达,牛RPE细胞P53mRNA表达阳性可在细胞浆内见到位于细胞周边部的蓝紫色絮状物,分别与HX／XO组阳性的细胞相比较,正常对照组,Rg1组及VitE组存在显著性差异（P＜0.05）,而与Rb1组无显著性差异.结论：氧化损伤可导致基因P53mRNA及其蛋白在RPE细胞内的表达明显增加.人参皂甙Rg1和VitE对抗氧自由基对RPE细胞损伤的机制可能是通过清除氧自由基,影响凋亡基困p53的表达,从而有效地抑制氧自由基对RPE细胞的损伤.     

激光共聚焦显微镜比较观察甾体皂苷Trilliuo、Lc253、Lc239对培养心肌细胞内[Ca2+]i的作用

目的：薯蓣皂苷元的皂苷为许多抗心血管中药中的主要活性成分，具有重要研究价值。本研究以经人工合成获得的薯蓣皂苷元皂苷同系物甾体皂苷Trilliuo(署蓣皂苷元葡萄糖苷、Lc253[薯蓣皂苷鼠李糖(1→2)葡萄糖苷]、Lc239(薯蓣皂苷元[鼠李糖(1→2)，葡萄糖(1→3)]葡萄糖苷)为研究对象，通过考察它们对培养心肌细胞钙离子释放的影响，分析其构效关系。方法：利用激光共聚焦显微镜测定加入fluo3—AM培养的心肌细胞中的钙离子变化情况。结果：Lc253能明显增强心肌细胞节律性和呈浓度依赖性增加心肌细胞静息钙离子水平，加入CaCl2 10mmol/L or KCl 60 mmol/L后钙离子荧光峰强度仍维持在较高水平；而Trilliuo and Lc239在10^—5mmol/L浓度时，对心肌细胞静息钙离子水平作用不明显，在高浓度胞外Ca^2 或K^ 情况下，抑制心肌细胞内钙离子升高；其作用特点与verapamil相似，具有钙离子通道阻断作用。结论：研究结果显示三种甾体皂苷对细胞内钙离子浓度有所差异，结构中连接糖的数目与调孔钙内流的活性有重要的相关性。这些皂苷类化合物对钙离子通道的活性有潜在的开发价值。     

加味酸枣仁胶囊的研制

目的:优选加味酸枣仁胶囊的制备工艺.方法:以酸枣仁皂苷A含量为指标,考察提取溶媒、加溶媒量、提取时间三因素对提取效果的影响;采用固定圆锥底法考察粉体的流动性;选择测定酸枣仁皂苷A的含量作为控制本品的质量标准.结果:酸枣仁最佳提取工艺即用70%乙醇提取2次,第1次2 h,第2次1 h,用乙醇量均为8倍药材量;粉末水分控制在3.01%流动性好,休止角平均值为28.2°;该胶囊含酸枣仁皂苷A不得低于11.00mg/粒.结论:上述实验结果可为加味酸枣仁胶囊的制备提供实验依据.     

An experimental vaccine composed of two adjuvants gives protection against Mycoplasma bovis in calves

Mycoplasma bovis is a major pathogen affecting cattle causing bronchopneumonia, mastitis, and other disorders. Only autogenous vaccines made specifically for individual farms are available in parts of Europe and the United States. A novel experimental vaccine composed of a field M. bovis isolate combined with saponin and Emulsigen^® adjuvants was evaluated. Eighteen 3–4 week old calves were placed in three equal groups: vaccinated (Vac), positive control (PC) and negative control (NC). The Vac calves were subcutaneously injected with 8 ml of the vaccine; the PC and NC calves received phosphate buffered saline (PBS). Three weeks later the Vac and PC calves were challenged with a virulent M. bovis strain, the NC group received PBS. Throughout the study clinical observations, microbiology and immunological tests were carried out. Three weeks post challenge two calves from each group were euthanased for necropsy and histopathological examination. The vaccine effectively stimulated the humoral immune response, with high titres of anti-M. bovis specific antibodies and total Ig concentration. This vaccine also intensified the IgA response. A clinically protective effect of the vaccine was demonstrated as it also reduced the gross pathological lung lesions and nasal shedding of M. bovis.     

倒卵叶五加总皂甙对大鼠心肌缺血再灌注损伤后c-fos mRNA表达的影响

目的 :探讨倒卵叶五加总皂甙 (SAOH)对大鼠心肌缺血再灌注后c fosmRNA表达的影响。方法 :采用结扎左冠状动脉前降支 30min ,再灌注复制大鼠心肌缺血再灌注损伤 (IRI)的模型 ,治疗组结扎前 10min注射SAOH ,检测心肌再灌注 4 0min时各组心肌组织乳酸脱氢酶 (LDH)、磷酸肌酸激酶 (CK)、丙二醛 (MDA)的含量和血浆超氧化物歧化酶 (SOD)的活性 ,并用原位杂交的方法观测左室心肌c fosmRNA的表达。结果 :再灌注 4 0min时 ,SAOH 5 0mg/kg组和 10 0mg/kg组心肌LDH、CK、MDA含量分别为 :(1790 .0± 31.4 )kIU/ g蛋白、(182 .0± 6 .8)kIU/ g蛋白、(3.6 9± 0 .11) μmol/ g蛋白和 (10 16 .0± 4 2 .6 )kIU/ g蛋白、(16 8.0± 4 .3)kIU/ g蛋白、(4.37± 0 .17) μmol/ g蛋白 ,显著低于IRI组 :(196 7.0± 6 4 .3)kIU/ g蛋白、(2 0 6 .0± 3.6 )kIU/ g蛋白、(5 .77±0 .2 0 ) μmol/ g蛋白 (均 P <0 .0 1) ;SAOH 5 0mg/kg组和 10 0mg/kg组SOD活性为 (11.70± 0 .2 6 ) μIU/L、(13.0 0± 0 .19) μIU/L ,较IRI组SOD(0 .30± 0 .2 5 ) μIU/L明显升高 (均 P <0 .0 1) ;而SAOH 5 0mg/kg组和 10 0mg/kg组心肌组织c fosmRNA的光密度值 (OD)分别为 0 .12± 0 .0 3、0 .12± 0 .0 3,较IRI组OD值 0 .2 1± 0 .0 3明显减小(均P     

New and safe formulation for scorpion immunotherapy: Comparative study between saponin and FCA adjuvants associated to attenuated venom

Envenoming by scorpion is a major health problem in Maghreb regions as well as in several regions of the world. Immunotherapy is the only effective treatment for scorpion stings. The immune sera are obtained from hyper-immunized animals with a formulation of venom associated to Freund’s Complete Adjuvant (FCA). This formulation seems to protect against several alterations in immunized animals leading to worsening of their health due to added toxicity of native venom and FCA adjuvant. This study aims to provide a more efficient and non-toxic alternative to this formulation. Two formulations of saponin or FCA associated to irradiated venom of Androctonus australis hector (Aah) were used to compare their safety and their efficiency to better enhance the antibody titers against toxic antigens.Both of these formulations were used in immunization schedule of three months. Blood samples were collected every week, cell count, myeloperoxydase (MPO) and eosinophil peroxidase (EPO) activities and specific antibody titers were evaluated. Four months after the last immunization, rabbits were challenged with increased doses of native Aah venom.Results showed that immunization with saponin formulation induced lower inflammatory cell activation as well as reduced MPO and EPO activities compared to that using FCA. The formulation of irradiated venom with saponin seems also to be more efficient in the activation of lymphocytes resulting in higher titers of specific IgG. The immunoprotective effect evaluation showed that the formulation using saponin seems to protected animals until 3 LD50 of native venom compared to that using FCA which protected only until 2 LD50. These results indicate that saponin formulation with irradiated antigen could be more efficient and safe immunizing preparation for the production of sera against scorpion envenomation.     

倒卵叶五加总皂甙对大鼠心肌顿抑的作用

目的　探讨倒卵叶五加总皂甙 (SAOH)对大鼠缺血心肌再灌注后心肌顿抑的作用。方法　采用结扎左冠状动脉前降支 30 m in,再灌注复制大鼠心肌缺血 /再灌注损伤 (IRI)的模型 ,结扎前 10 min注射 SAOH,采用左心室插管 ,动态监测左心室心功能 ,并测定心肌再灌注 40 m in时心肌组织三磷腺苷酶 (ATPase)的活性和血浆一氧化氮(NO)。结果　 IRI组、SAOH1 组 (5 0 mg/ kg)及 SAOH2 组 (10 0 m g/ kg)在缺血及再灌注过程中左心室收缩压和室内压上升或下降最大速率 (L VSP和± d P/ dtmax)均呈进行性下降 ,但 SAOH组下降趋势较缓。 SAOH组在整个再灌注过程中 ,L VSP和± d P/ dtmax均较 IRI组明显提高 ,甚至 SAOH2 组的 +d P/ dtmax值在再灌注即刻、再灌注 10 m in时即高于假手术组 (P均 <0 .0 1) ,再灌注 2 0 m in、30 min时与假手术组无明显差异。再灌注 40 min时 ,SAOH1 组、SAOH2 组较 IRI组心肌肌膜 Na+- K+- ATPase、Ca2 +- ATPase、Mg2 +- ATPase活性和血浆 NO水平明显升高 (P均<0 .0 1)。结论　 SAOH对心肌顿抑有明显的改善作用 ,其机制之一可能与其提高心肌组织 ATPase活性和血浆NO含量 ,减少 Ca2 +超载有关。