新型抗蝰蛇毒血清对不同蝰蛇毒作用的实验研究

新型抗蝰蛇毒血清是采用国产圆斑蝰蛇毒与缅甸蝰蛇毒按一定比例免疫马匹后 ,从马血浆中精制而成。作者通过免疫电泳及动物实验 ,观察新型抗蝰蛇毒血清对国产圆斑蝰蛇毒与缅甸蝰蛇毒的免疫沉淀反应、体外中和作用、体内保护作用等方面的作用。结果 :急性毒性实验 :国产圆斑蝰蛇毒和缅甸蝰蛇毒的 LD50 (腹腔注射 )分别为 (0 .30 6± 0 .0 0 3) mg/ kg、 (0 .2 90± 0 .0 0 3) mgkg。体外中和实验表明新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒均有中和作用 ,其中和抗体效价依次为 2 750μg/ ml (约 450 LD50 )、 2 4 90μg/ml(约 430 L…     

抗蝰蛇毒IgY灌胃对蝰蛇伤小鼠保护作用的研究

目的 探讨抗蝰蛇蛇毒鸡卵黄抗体(immunoglobulin yolk,IgY)经腹腔注射或灌胃对蝰蛇伤小鼠保护作用,为其口服制剂的应用奠定理论基础.方法 用蝰蛇原毒单一抗原免疫母鸡,收集第12天以后的鸡蛋,用水稀释法提取抗蝰蛇毒IgY;间接法ELISA检测IgY的效价和灌胃小鼠体内IgY的吸收时间;以胃排空率实验确定蛇伤前灌胃IgY的最佳时间,以腹腔注射和不同浓度IgY灌胃实验分别检验IgY对蝰蛇伤小鼠的保护作用.结果 初次免疫后第12天开始收集抗体,水稀释法提取的IgY效价为1∶6400;不同浓度IgY灌胃后,2.5～3.5 h胃排空率均达61.8%以上,血浆中抗体效价亦相应达高峰;腹腔注射和IgY灌胃实验对蛇伤小鼠的保护作用明显,小鼠的存活时间与阴性IgY组小鼠相比差异显著(P＜0.05);同等效应下,灌胃IgY的有效剂量大约为腹腔注射的10～15倍.结论 抗蝰蛇蛇毒IgY经腹腔注射和灌胃实验均能有效保护蝰蛇伤小鼠.     

葛根总黄酮提取工艺的研究

目的:筛选提取葛根总黄酮的最佳工艺条件。方法:采用单因素和正交试验对提取葛根总黄酮的工艺条件进行优化;利用紫外可见分光光度计测量葛根总黄酮的含量;利用高效液相色谱仪测量葛根素的含量;分别以葛根素和葛根总黄酮的含量为指标进行最优工艺的评价。结果:最佳工艺条件是以9倍量的30%乙醇提取3次,每次1h。结论:该提取工艺简单,成本低,适合于生产应用。     

中西医结合治疗蝰蛇咬伤75例临床体会

目的总结中西医结合抢救治疗蝰蛇咬伤的方法。方法采用抗蝮蛇毒血清和抗五步蛇毒血清配伍代替抗蝰蛇毒血清以及中西医结合综合抢救治疗蝰蛇咬伤患者75例。结果 75例患者中,治愈58例,好转13例,无效3例,死亡1例。有效率94.67%。结论在缺乏抗蝰蛇毒血清的情况下,抗蝮蛇毒血清和抗五步蛇毒血清配伍使用以及中西医结合综合抢救治疗蝰蛇咬伤能提高抢救成功率。     

6种晋产野生葛根中葛根素、大豆苷和总黄酮含量比较

采用高效液相色谱法对山西省5地6处野生葛根中主要有效成分葛根素、大豆苷及总黄酮含量进行了分析,并对HPLC法的测定条件(流动相和洗脱程序)进行了优化。结果表明,6种晋产野生葛根中主要有效成分含量差异显著,葛根素的含量为1.46%~4.48%,大豆苷的含量为0.11%~0.62%,总黄酮的含量为11.19%~26.14%。葛根素、总黄酮含量与纬度高低呈正相关。晋南绛县么里镇小北山与夏县泗交镇太宽河自然保护区的野生葛根中葛根素含量低于《中国药典》规定的2.4%,而晋北灵丘县的野生葛根中葛根素含量则高达4.48%。     

Fe3+络合萃取法从野葛根中分离葛根素

利用Fe3 + 能够和葛根素生成可溶性络合物的性质建立了一种从中药野葛根中萃取葛根素的新型分离方法。以甲醇冷浸从野葛根中提取葛根总黄酮 ,将其进行水解、中和 ,再给水解葛根总黄酮中加入FeCl3 使葛根素与Fe3 + 络合溶解 ,过滤除去其它不溶性物质 ,用盐酸解聚Fe3 + 葛根素络合物 ,则得葛根素粗品 ,将其重结晶可得葛根素。同时 ,利用分光光度法确定了Fe3 + 葛根素络合物解聚的最佳酸度。利用TLC标准品对照、IR和分光光度法对产品进行了定性和定量分析。该方法从葛根中提取葛根素收率为 1 2 % ,纯度为 96 5 % ,具有操作简便、工艺流程简单 ,容易实现工业化的优点。     

葛根抗腹泻药效组分实验研究

目的:验证葛根抗腹泻的药效组分,为葛根的质量评价提供科学数据。方法:在经方葛根芩连汤的基础上分析葛根抗腹泻的药效组分,采用组合药理实验辅助验证药效组分。结果:抗腹泻相关实验中葛根水煎液中、高剂量组、葛根药效组分组、葛根芩连汤组与阴性对照组比较均有显著性差异(P0.01)。结论:葛根素-大豆苷-大豆苷元-3'-羟基葛根素是葛根抗腹泻的药效组分。     

广西圆斑蝰蛇毒凝血因子X激活物在实验鼠体内的促凝血作用研究

目的研究国产圆斑蝰蛇(Vipera russellii)毒中凝血因子X激活物(RVV-X)在实验动物体内的促凝血作用。方法对从广西圆斑蝰蛇毒中分离纯化出的具有止血作用的RVV-X单一有效成分进行了实验鼠促凝血作用的研究。主要测定的指标包括出血时间(BT)、全血凝固时间(CT)、血浆复钙时间(RT)、激活部分凝血激酶时间(APTT)、纤维蛋白原含量(FIB)、优球蛋白溶解时间(ELT)以及血小板数量(PLT)。结果该药物的促凝血作用很强,注射很小的剂量(0.000313u/kg)的RVV-X就可以显著地缩短小鼠出血时间,对大鼠也表现出明显的促凝血作用。结论从广西圆斑蝰蛇毒中分离纯化出的RVV-X在极低的剂量下就可以发挥其凝血、止血作用,安全范围很大。本试验为其将来开发研制成为一种全新的止血药提供了一些基础资料。     

圆斑蝰蛇咬伤中毒35例临床分析

目的分析圆斑蝰蛇咬伤中毒的临床特点,总结圆斑蝰蛇咬伤中毒合理有效的治疗方法。方法对2006年6月~2017年12月广西中医药大学第一附属医院与广西医科大学第一附属医院收治确诊为圆斑蝰蛇咬伤中毒患者的临床资料进行回顾性分析。结果 (1)一般情况:本组35例患者中,男21例(60.00%),女14例(40.00%),男女比例为3∶2;年龄8~63岁,平均(44.73±5.84)岁。(2)临床特征与转归:35例患者均出现凝血障碍的症状,其中并发非典型DIC 8例(22.85%),并发急性肾功能衰竭8例(22.85%),并发内脏出血10例(28.57%),并发脑出血5例(14.29%)。全部患者均治愈,治愈率100%。(3)应用抗蝮蛇毒血清治疗情况:应用抗蝮蛇毒血清治疗(应用抗蝮蛇毒血清组)17例,未用抗蝮蛇毒血清治疗(未应用抗蝮蛇毒血清组)18例,两组的疗效和并发症比较差异无统计学意义(P0.05)。结论 (1)圆斑蝰蛇咬伤中毒以青壮年男性多见,咬伤部位多见于下肢,好发于夏秋两季。(2)圆斑蝰蛇咬伤中毒出现凝血功能障碍、非典型DIC综合征表现,可能是消耗性凝血功能障碍导致。(3)应用抗蝮蛇毒血清治疗圆斑蝰蛇咬伤中毒是否有效还需更多的循证医学资料支持。     

半夏和滴水珠抗五步蛇毒中毒作用的实验研究

目的研究滴水珠与半夏生药及其提取物抗五步蛇毒的作用。方法将ICR小鼠随机分为半夏生药组、半夏醇提物组、滴水珠生药组、滴水珠醇提物组、空白对照组、阳性对照组及模型组,各组小鼠均灌胃给药7天,并观察各给药组小鼠的体重变化情况;在末次给药1h后于小鼠腹腔注射五步蛇毒,观察各组小鼠的中毒表现,并统计各组小鼠的死亡率,比较各组小鼠的肝脏指数、脾脏指数及胸腺指数,并对各组小鼠血浆纤维蛋白原（FIB）、血浆凝血酶原时间（PT）、凝血酶时间（TT）、活化部分凝血酶时间（APTT）以及血小板（PLT）、红细胞（RBC）和白细胞（WBC）计数进行比较。结果半夏和滴水珠醇提物、半夏和滴水珠生药均可降低五步蛇毒中毒小鼠的死亡率,对五步蛇毒引起的小鼠PT、TT、APTT上升和FIB下降具有显著的抑制作用,与模型组相比P〈0.05;并可影响五步蛇毒中毒小鼠外周血血小板（PLT）、红细胞（RBC）和白细胞（WBC）计数,与模型组相比P〈0.05。但滴水珠和半夏生药可引起小鼠体重下降,并影响肝脏、胸腺和脾脏指数,与空白对照组相比P〈0.05。结论半夏和滴水珠醇提物及半夏和滴水珠生药均具有一定的抗五步蛇毒作用,乙醇提取能降低半夏和滴水珠的毒性。     

Properties of nerve growth factor from the venom of Bothrops atrox.

A glycoprotein fraction islated in poor yield (approx. 0.04%) from the venom of Bothrops atrox contained nerve growth factor. The material had biological activity, stability, the property of anomalous adsorption onto surfaces, apparent molecular weight and sub-unit structure that were similar to those for nerve growth factor that had been previously purified from the venom of Vipera russelli. Antiserum raised against nerve growth factor from Vipera russelli showed definite cross-reactivity with either the material from Bothrops atrox or a nerve growth factor-containing fraction from the venom of Ancistrodon piscivorus piscivorus, while antiserum against nerve growth factor from mouse had little effect on any of these preparations.     

Toxicities, LD50 prediction and in vivo neutralisation of some elapid and viperid venoms.

Toxicity levels of elapid (Naja naja and Naja oxiana) viperid (Vipera lebetina and Vipera russelli) venoms for mice and rat for intraperitoneal intravenous and intramuscular routes have been determined. The data have been analysed using a mathematical expression to calculate lethal venom concentrations in human snake bite cases. Further, in vivo neutralisation of snake venom potency (after experimental injection) using high voltage-low current electric shock treatment has been attempted. This treatment postponed the death further by 60-90 min in mice in case of elapid envenomation. In case of viperid envenomation such a postponement of death time was not noticed. The death postponement induced by the shock treatment probably refers to structural impairments that occur at molecular level in venom components and their consequent altered interactions with the target tissue or system.     

A comparative study of the biological properties of venoms from snakes of the genus Vipera (true adders)

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms of 13 taxa of Vipera were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate the presence of certain common characteristics among the venoms, particularly if V. russelli is excluded from the comparison. The results also support the recently proposed reassignment of V. russelli to a separate genus. 3. The data show that information on venom biological properties can be used for differentiation of venoms of many species of Vipera. Particularly useful for this purpose are the protease, phosphodiesterase, phospholipase A and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.     

Cross-reactivities of polyclonal antibodies against factor V activating enzyme, a serine proteinase from Vipera lebetina (snake) venom

Antibodies to the factor V activating enzyme from Vipera lebetina venom were produced by immunizing a rabbit with chromatographically purified factor V activating enzyme probes. The antibodies cross-reacted with different protein fractions in 23 snake venoms (ten viperid, eight crotalid, and five elapid venoms) as demonstrated by western immunoblotting. In the venom of Vipera russelli the antibodies recognized only one protein band which probably belonged to factor V activating enzyme.     

蛇伤凉血合剂治疗蝰蛇咬伤的疗效观察

目的观察蛇伤凉血合剂对蝰蛇咬伤的临床疗效。方法将62例病人分为治疗组和对照组,两组均给予常规治疗,治疗组加服蛇伤凉血合剂。观察比较两组预后,局部组织坏死发生率,肢体功能受限发生率,肾损害发生率和血液透析率,住院时间等指标。结果两组病人的临床疗效、肿胀消退时间、疼痛缓解时间、局部坏死发生率、致残率、肾损害发生率和血液透析率、住院时间均有显著性差异（P〈0.05）。结论蛇伤凉血合剂对蝰蛇咬伤有显著疗效。     

Purification and amino-acid sequence of a nerve growth factor from the venom of Vipera russelli russelli.

Nerve growth factor (NGF) was purified from the venom of Vipera russelli russelli by Sephadex G-50 gel filtration, S-Sepharose column chromatography and Blue-Sepharose CL-6B column chromatography. The purified NGF was found to be a glycoprotein, whose apparent molecular mass was estimated to be about 17.5 kDa by SDS-PAGE. The amino-acid sequence was determined by a combination of conventional methods. The V. r. russelli NGF was composed of 117 amino-acid residues with one residue, Asn-21, being N-linked glycosylated and the molecular mass of its protein portion was calculated to be 13,280 Da.     

Identification, isolation and purification of neurotoxic phospholipases A2 from Vipera russelli venom using polyclonal antibodies.

All the neurotoxic phospholipases A2 present in whole Vipera russelli venom were precipitated selectively from other non-neurotoxic phospholipases A2 and non-phospholipases A2 fractions using antibodies (anti PL-V Ig) raised against one of the purified neurotoxic phospholipases A2 (VRV PL-V). These neurotoxins were identified and isolated in their homogeneous form by chromatographic and electrophoretic methods. The present report of selective isolation and purification of all the neurotoxic phospholipases A2 of V. russelli venom is first of its kind.     

Analysis of Vipera russelli venom using polyclonal antibodies prepared against its purified toxic phospholipase A2 VRV PL-V.

Vipera russelli venom induces predominantly neurotoxic, myotoxic necrotic and hemorrhagic symptoms in experimental animals and has several hydrolytic enzyme activities. In this study, V. russelli venom is characterized both as a PLA2 and as a toxin. Anti PL-V Ig (antibodies to a toxic phospholipase A2 VRV PL-V of V. russelli venom) nullifies the toxicity of whole V. russelli venom to a great extent. The neurotoxic symptoms vanish completely in the presence of anti PL-V Ig. The cross reacting components of whole V. russelli venom were removed by precipitating them from whole venom by the addition of anti PL-V Ig. The non-cross reacting components present in the supernatant were checked for toxicity. There was a significant reduction in toxicity. The LD50 value of the supernatant had increased from 4.1 mg/kg body weight to 11.7 mg/kg body weight and it showed about 34% of the total venom phospholipase A2 activity. It had edema forming, hemorrhagic and hemolytic activity but failed to induce neurotoxic, anticoagulant and myotoxic effects.     

Purification and characterization of an organ specific haemorrhagic toxin from Vipera russelli russelli (Russell's viper) venom

A haemorrhagic toxin (VRR-12) from Vipera russelli russelli (Russell's viper) venom has been purified by ion-exchange chromatography on CM-Sephadex C-50 followed by size-exclusion HPLC to electrophoretically homogeneous state. It is a 12 kDa single polypeptide having 1 mole of Zn+2 ion. This toxin induces intense intestinal haemorrhage and to a lesser extent skeletal muscle haemorrhage in mice. It does not show detectable proteolytic and esterolytic activity with selected substrates under specified conditions, haemolytic and phospholipase activity. When VRR-12, preincubated with bivalent antiserum against Saw-scaled and Russell's viper venom or EDTA was injected, haemorrhagic activity was not reduced, on the other hand preincubation with phenylmethyl sulphonyl fluoride reduced the activity markedly. Biodistribution studies with 125I VRR-12 show that haemorrhagic manifestation by this toxin is not a direct function of the fraction of the totally administered toxin distributed to that tissue.     

Enzymatic activity and inhibition of the neurotoxic complex vipoxin from the venom of Vipera ammodytes meridionalis

Vipoxin from the venom of Vipera ammodytes meridionalis is an unique neurotoxic complex between a toxic phospholipase A2 and a highly homologous non-toxic protein inhibitor. It is an example of evolution of a catalytic and toxic function into inhibitory and non-toxic one. The activity of the V. ammodytes meridionalis toxin is 1.7 times higher than that of the closely related (92% sequence identity) neurotoxic complex RV4/RV7 from the venom of Vipera russelli formosensis The enhanced enzymatic activity of vipoxin is attributed to limited structural changes, in particular to the substitutions G54R and Q78K in the PLA2 subunit of the complex and to the T54R substitution in the inhibitor. Oleyloxyethylphosphocholine, aristolochic acid and vitamin E suppressed the enzymatic activity of vipoxin and its isolated PLA2 subunit. These compounds influence inflammatory processes in which PLA2 is implicated. The peptide Lys-Ala-Ile-Tyr-Ser, which is an integral part of the PLA2 components of the two neurotoxic complexes from V. ammodytes meridionalis and V. russelli formosensis (sequence 70-74) activated vipoxin increasing its PLA2 activity by 23%. This is in contrast to the inhibitory effect of the respective pentapeptides with 70-74 sequences on other group II PLA2s. Surprisingly, the same peptide inhibited 46% of the V. russelli formosensis PLA2 activity. The limited changes in the structure of the two highly homologous neurotoxins lead to considerable differences in their interaction with native peptides.