1份生产用中药蛇粗毒的DNA分子鉴定

目的 利用PCR技术鉴定干燥蛇粗毒的来源物种,并对基因组DNA的提取效果及提取过程中需注意的若干问题进行分析和讨论,同时还对若干针对不同长度目的 基因扩增引物的使用效果进行验证.方法 从1份已保存了7年的生产用干燥蛇粗毒中提取总DNA,并以之为模板进行PCR扩增、测序和序列分析.结果 使用该方法可从蛇粗毒中提取得到微量的总DNA,并成功扩增得到0.45～1.18 kb不同长度范围的目的 片段;测序结果提交NCBI,并与CenBank中的同源序列进行BLAST比对.结论 该蛇粗毒样品来自眼镜蛇,且极可能由多个不同的亚种或单倍型所组成.该技术有可能推广用于对动物粗毒的来源进行准确和直接地鉴定.     

炮山甲及其混伪品的DNA微型条形码鉴定研究

目的:建立基于DNA微型条形码的炮山甲及其混伪品的分子鉴定技术。方法:采用改良的DNA提取方法提取中华穿山甲与其易混物种炮制甲片的DNA,利用线粒体COⅠ、16S rRNA和12S rRNA基因的标准及微型条形码引物进行PCR。双向测序后进行BLAST和序列位点分析,再计算种内和种间K2P遗传距离,并构建NJ系统树。结果:16S rRNA(L2513/H2714)和12S rRNA(L1085/H1259)引物对在所有甲片DNA中均能扩增成功,所获序列与Genbank序列的BLAST比对相似度大于98%。中华穿山甲与其易混物种的种间遗传距离远大于其种内遗传距离,而且各物种在NJ树中表现出单系性,其中16S rRNA微型条形码能将穿山甲属聚在一起。结论:基于16S rRNA的微型条形码技术可有效鉴别炮山甲及其混伪品。     

哈氏蜈蚣线粒体基因组全序列分析

[目的]获取哈氏蜈蚣线粒体基因组全序列。[方法]应用差速离心以及滚环扩增的方法得到纯度较高的线粒体DNA,采用Illumina HiSeq X Ten技术对扩增得到的哈氏蜈蚣线粒体DNA进行测序,采用从头组装和mapping近缘种的方法拼接组装线粒体基因组,应用MITOS在线注释及与近缘种进行本地注释等方法注释线粒体基因组。[结果]获得14 538 bp哈氏蜈蚣线粒体基因组全序列。其共有20个tRNA及13个蛋白基因,2个rRNA(16S rRNA、12S rRNA)。[结论]获取哈氏蜈蚣线粒体基因组全序列,并可用于药用蜈蚣的资源调查与保护研究。     

二株具有抗菌活性红树林土壤放线菌的分离与鉴定

目的 了解广西北海红树林土壤放线菌的抗菌活性.方法 采用海水配制高氏培养基分离广西北海红树林土壤中的放线菌.提取二株典型放线菌发酵液进行抗菌实验,同时提取其总DNA,用放线菌通用引物对16S rDNA进行PCR扩增,对获得的扩增结果进行DNA序列测定.结果 提取到的二株典型放线菌发酵液具有抗菌活性,同时将二株典型放线菌菌株的16S rDNA,经测序后对测序结果进行比对显示,所获的BH200951和BH200954菌株与链霉菌属形态学特征、生理生化特征和基于16S rDNA (16S rRNA)基因序列的系统发育分析,BH200951被鉴定为链霉菌属的有效发表种链霉菌的菌株Streptomyces aurantiogriseus AY999773的变种,BH200954初步被鉴定为链霉菌属的有效发表种仙台链霉菌.结论 分离来自广西北海红树林土壤中的二菌株其发酵产物有很强的抗菌活性,具有进行新药开发利用的潜力.     

三斑海马及其混伪品的DNA条形码分子鉴定研究

目的 建立三斑海马Hippocampus trimaculatus的COI、16 S rRNA和ATP6的条形码序列数据库,应用DNA条形码技术从分子水平快速准确鉴定三斑海马和其他正伪品海马,探讨海马属药材鉴定的新方法。方法 提取三斑海马药材的基因组DNA,PCR扩增COI、16 S rRNA和ATP6的序列并进行双向测序,所得序列采用软件Codon Code Aligner V4.2对测序峰图进行校对拼接,应用ClustalX软件进行序列比对,MAGA5.0软件计算三斑海马的种内种间遗传距离(Kimura2-Parameter,K2P),构建邻接树(Neighbor-joingtree,NJTree)聚类分析不同品种海马药材的鉴定结果。结果 获得的三斑海马线粒体COI、16 S rRNA、ATP6序列长度分别649、572、603～605 bp,其中3个条形码序列的种内变异位点碱基数分别为8、4和15,种内的变异率较小,COI、16 S r RNA、ATP6序列的平均种内K2P遗传距离0.002、0.001和0.006,均远小于三斑海马的种间K2P距离;NJ树结果显示三斑海马与其他海马均可明显区分,具有良好的单系性。结论 COI、16 S r RNA、ATP6序列作为条形码均可以鉴定三斑海马及其他混伪品海马药材,为动物类药材及其混伪品和近源物种的分子鉴定提供依据,为对保障海马临床用药安全提供了新的技术手段。     

《中国药典》中蕲蛇饮片分子鉴定方法的研究和探讨

目的：运用多种分子鉴定方法,从技术要求和标准编写方面对蕲蛇饮片现行质量标准提出建议。方法：本研究选取蕲蛇所在蝰科Viperidae蝮亚科Crotalinae的7个近缘物种和9种常见的蕲蛇伪品物种作为研究对象,采用DNA 条形码技术扩增16S rRNA序列,运用电泳检视法,根据扩增条带的有无评价模板DNA的质量;16S rRNA序列扩增产物通过双向测序,运用BioEdit软件拼接,双端对齐后剪切比对,利用MEGA 5.1软件构建邻接（NJ）系统发育树;采用蕲蛇饮片现行标准方法进行同步鉴别。结果：16S rRNA序列的电泳检视结果可有效评价蕲蛇样品的模板DNA质量,避免由于模板DNA质量不佳导致的假阴性结果的可能性;基于16S rRNA条形码序列建立的NJ树可以鉴别蕲蛇饮片及其混淆品;蕲蛇饮片现行标准方法专属性良好。结论：DNA条形码方法、现行标准方法以及NJ树三者相互结合,从不同角度对蕲蛇饮片进行真伪鉴别,有效弥补蕲蛇饮片现行标准方法的不足,为其他中药品种的分子鉴定技术的质量标准制定提供了参考。     

甘南野生狭叶红景天与四裂红景天的rRNA基因间隔区PCR产物电泳图谱的初步研究

目的:分析甘肃省甘南地区野生狭叶红景天与四裂红景天中提取的DNA中rRNA基因内转录间隔区PCR扩增产物的电泳图谱,为不同品种鉴别和品质评价从分子水平提供依据.方法:提取2种红景天中药材的核基因组DNA,利用合成的特异性PCR引物对所提取的DNA中rRNA基因内转录间隔序列进行nPCR扩增,扩增产物进行琼脂糖凝胶电泳以得到电泳图谱并进行分析.结果:琼脂糖凝胶电泳图谱显示不同种红景天的rRNA基因内转录间隔区ITS1序列长度均在340 bp左右,ITS2序列长度均在410 bp左右,且具有明显的可比性.已具备足够的遗传信息量进行碱基序列分析.结论:不同DNA中rRNA基因内转录间隔区PCR扩增产物可作为从分子水平进行鉴别的标记之一.     

家种及野生秦艽、麻花秦艽的rRNA基因间隔区PCR产物电泳图谱的初步研究

目的：分析甘肃不同地区家种及野生中药材秦艽Gentiana macrophylla、麻花秦艽G.straminea中提取的DNA中rRNA基因内转录间隔区PCR扩增产物的电泳图谱，为秦艽、麻花秦艽等不同品种鉴别和品质评价从分子水平提供依据。方法：提取中药材家种及野生秦艽、麻花秦艽的核基因组DNA，利用合成的特异性PC及引物对所提取的DNA中rRNA基因内转录间隔序列进行nPCR扩增，扩增产物行琼脂糖凝胶电泳以得到电泳图谱并进行分析。结果：琼脂糖凝胶电泳图谱显示不同秦艽DNA中rRNA基因内转录间隔区长度均在360bp左右，已具备足够的遗传信息量进行碱基序列分析。结论：不同秦艽DNA中rRNA基因内转录间隔区PCR扩增产物可作为从分子水平进行鉴别的标记之一。     

应用rRNA基因间隔区碱基测序对中药(大黄)进行鉴定

目的:对大黄种子中提取的DNA中rRNA基因内转录间隔区进行PCR扩增,对于PCR扩增,并对PCR扩增产物进行碱基序列测定,以期从分子水平建立对中草药的鉴定标准。方法:常规提取当归、大黄种子DNA,利用合成的特异性引物对其DNA中rRNA基因内转录间隔区进行套式PCR扩增,将扩增产物以四色荧光标记的双脱氧末端终止循环法进行碱基序列测定。结果:经琼脂糖凝胶电泳证实大黄种子中rRNA基因内转录间隔区PRC基因内转录间隔区的完整碱基序列。RRNA基因内转录间隔区的碱基序列可作为分子水平鉴定植物中药材的又一有效途径。     

显齿蛇葡萄查耳酮合成酶基因cDNA克隆及蛋白质序列分析

目的 对显齿蛇葡萄Ampelopsis grossedentata查耳酮合成酶(CHS)基因进行克隆及序列分析.方法 根据已经克隆的植物基因的保守序列设计一对引物,以显齿蛇葡萄总RNA为模板,采用RT-PCR的方法扩增CHS基因序列并连接到pMD 18-T Simple载体上,阳性克隆经PCR检测后进行测序.结果 得到一段1 173 bp的序列,序列分析表明,该片段编码390个氨基酸,与其他高等植物CHS基因氨基酸序列同源性在67.9％以上.结论 首次从显齿蛇葡萄中克隆了CHS基因,为有效利用该基因奠定了基础.     

Neutralizing properties of Musa paradisiaca L. (Musaceae) juice on phospholipase A2, myotoxic, hemorrhagic and lethal activities of crotalidae venoms

The use of plants as medicine has been referred to since ancient peoples, perhaps as early as Neanderthal man. Plants are a source of many biologically active products and nowadays they are of great interest to the pharmaceutical industry. The study of how people of different culture use plants in particular ways has led to the discovery of important new medicines. In this work, we verify the possible activity of Musa paradisiaca L. (Musaceae) against the toxicity of snake venoms. Musa paradisiaca, an important source of food in the world, has also been reported to be popularly used as an anti-venom. Interaction of Musa paradisiaca extract (MsE) with snake venom proteins has been examined in this study. Phospholipase A2 (PLA2), myotoxic and hemorrhagic activities, including lethality in mice, induced by crotalidae venoms were significantly inhibited when different amounts of MsE were mixed with these venoms before assays. On the other hand, mice that received MsE and venoms without previous mixture or by separated routes were not protected against venom toxicity. Partial chemical characterization of MsE showed the presence of polyphenols and tannins and they are known to non-specifically inactivate proteins. We suggest that these compounds can be responsible for the in vitro inhibition of the toxic effects of snake venoms. In conclusion, according to our results, using mice as experimental model, MsE does not show protection against the toxic effects of snake venoms in vivo, but if was very effective when the experiments were done in vitro.     

Anticoagulant and antifibrinogenolytic properties of the aqueous extract from Bauhinia forficata against snake venoms

The aqueous extract from aerial parts of Bauhinia forficata was able to neutralize the clotting activity induced by Bothrops and Crotalus crude venoms. The clotting time, upon human plasma, induced by B. moojeni venom was significantly prolonged. Clotting and fibrinogenolytic activities induced by isolated thrombin-like enzyme from Bothrops jararacussu were totally inhibited after incubation at different ratios. The extract was not able to neutralize the hemorrhagic activity induced by an Bothrops venoms, but it efficiently inhibited the edema induced by Crotalus durissus terrificus venom and isolated PLA2s. In addition, it did not inhibited the phospholipase A2 activity of Bothrops snake venoms. Interaction studies between Bauhinia forficata extract and snake venoms, when analyzed by SDS-PAGE, did not reveal any apparent degradation of the venom proteins. This extract is a promising source of natural inhibitors of serine-proteases involved in blood clotting disturbances induced by snake venoms.     

Anti-snake venom properties of Schizolobium parahyba (Caesalpinoideae) aqueous leaves extract

Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothrops pauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA(2)) and CB (PLA(2) from crotoxin complex). Phospholipase A(2), coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by B. pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of B. pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after B. pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases.     

Antihemorrhagic, antinucleolytic and other antiophidian properties of the aqueous extract from Pentaclethra macroloba

Several Brazilian plants have been utilized in folk medicine as active agents against various effects induced by snake venoms. The inhabitants of the Amazon region use, among others, the macerated bark of a plant popularly named "Pracaxi" (Pentaclethra macroloba Willd) to combat these effects. We report now the antihemorrhagic properties against snake venoms of the aqueous extract of Pentaclethra macroloba (EPema). EPema exhibited full inhibition of hemorrhagic and nucleolytic activities induced by several snake venoms. Additionally, partial inhibition of myotoxic, lethal, phospholipase and edema activities of snake venoms and its isolated PLA(2)s by EPema is reported. In vivo tests showed that EPema is able to totally inhibit a Bothrops jararacussu metalloprotease (BjussuMP-I) induced hemorrhage, suggesting interaction of the extract compounds with this high molecular weight protein. The extract did induce neither hemorrhage nor death in mice when administered alone by i.m. route. When administered separately by i.m. route, the extract did not induce death in mice at 12.5--300 mg/kg doses. Other assays demonstrated that EPema was unable to inhibit fibrinogenolytic and coagulant activities of Bothrops atrox venom. Although the mechanism of action of EPema is still unknown, the finding that no visible change was detected in the electrophoretic pattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. The search for new inhibitors of venom metalloproteases and DNAases are a relevant task. Investigation of snake venom inhibitors can provide useful tools for the elucidation of the action mechanisms of purified toxins. Furthermore, these inhibitors can be used as molecular models for development of new therapeutical agents in the treatment of ophidian accidents.     

Antiophidian properties of the aqueous extract of Mikania glomerata

Aqueous extracts, prepared from dried or fresh roots, stems or leaves of Mikania glomerata, a plant found in Mata Atlantica in Southeastern Brazil, were able to efficiently neutralize different toxic, pharmacological, and enzymatic effects induced by venoms from Bothrops and Crotalus snakes. Phospholipase A(2) activity and the edema induced by Crotalus durissus terrificus venom were inhibited around 100 and approximately 40%, respectively, although this inhibition was only partial for Bothrops venoms. The hemorrhagic activity of Bothrops venoms (Bothrops altenatus, Bothrops moojeni, Bothrops neuwiedi, and Bothrops jararacussu) was significantly inhibited by this vegetal species, while the clotting activity of Crotalus durissus terrificus, Bothrops jararacussu, and Bothrops neuwiedi venoms was totally inhibited. Although, the mechanism of action of Mikania glomerata extract is still unknown, the finding that no visible change was detected in the electrophoretic pattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. Since the extract of Mikania glomerata significantly inhibited the studied snake venoms, it may be used as an alternative treatment to serumtherapy and, in addition, as a rich source of potential inhibitors of PLA(2)s, metalloproteases and serineproteases, enzymes involved in several physiopathological human and animal diseases.     

The protective effect of Mucuna pruriens seeds against snake venom poisoning

Ethnopharmacological relevance

The seed, leaf and root of Mucuna pruriens have been used in traditional medicine for treatments of various diseases. In Nigeria, the seed is used as oral prophylactics for snakebite.

Aim of the study

To study the protective effects of Mucuna pruriens seed extract against the lethalities of various snake venoms.

Materials and methods

Rats were pre-treated with Mucuna pruriens seed extract and challenged with various snake venoms. The effectiveness of anti-Mucuna pruriens (anti-MPE) antibody to neutralize the lethalities of snake venoms was investigated by in vitro neutralization.

Results

In rats, MPE pre-treatment conferred effective protection against lethality of Naja sputatrix venom and moderate protection against Calloselasma rhodostoma venom. Indirect ELISA and immunoblotting studies showed that there were extensive cross-reactions between anti-MPE IgG and venoms from many different genera of poisonous snakes, suggesting the involvement of immunological neutralization in the protective effect of MPE pre-treatment against snake venom poisoning. In vitro neutralization experiments showed that the anti-MPE antibodies effectively neutralized the lethalities of Asiatic cobra (Naja) venoms, but were not very effective against other venoms tested.

Conclusions

The anti-MPE antibodies could be used in the antiserum therapy of Asiatic cobra (Naja) bites.     

高特异性PCR方法鉴别乌梢蛇及其混淆品

 目的建立一种简便、准确的乌梢蛇药材DNA分子标记鉴别方法。方法根据乌梢蛇及10种常见混淆品线粒体12SrRNA基因序列,设计一对专用于乌梢蛇的鉴别引物HWL-1和HWH-1,用不同的复性温度PCR扩增,确立特异性反应条件,并利用此方法鉴别从市场上购买的各种乌梢蛇药材。结果PCR结果是在65℃复性温度下,正品乌梢蛇得到约320 bp的扩增带,而混淆品在同样的条件下无扩增带。为检验该方法的准确性,对市售乌梢蛇炮制品随机选取13块进行PCR鉴别验证,其中正品9块,混淆品4块,检出率达100%。结论所设计的鉴别引物对正品乌梢蛇有高度的特异性。PCR方法稳定性高,同种不同个体间的种内差异对鉴定结果无影响。本方法简单、准确、快速、灵敏度高、重现性好。     

Effect of Eryngium creticum on the haemolytic activities of snake and scorpion venoms

Aqueous and ethanol extracts of the fresh and dried leaves and roots of Eryngium creticum were tested for their inhibition against snake and scorpion venoms. The fresh leaf extract gave a higher percentage inhibition of the haemolytic activity of the scorpion venom Leiurus quinquesteiartus compared with the dried leaf extract. Extracts of both fresh and dried roots gave 100% inhibition of the snake and scorpion venoms. However, ethanol extracts of the leaves and roots enhanced RBC haemolysis rather than inhibiting venom activities on red blood cells. © 1997 John Wiley & Sons, Ltd.     

7种蛇毒小肽对临床分离耐（多）药结核分枝杆菌的体外活性研究

 目的检测7种从蛇毒分离的小肽是否对临床分离的耐药性结核分枝杆菌菌株具有活性。方法放射性方法检测蛇毒小肽对结核分枝杆菌的最小抑制浓度，细菌存活计数确证放射性方法的结果。结果7种蛇毒小肽对耐药性结核分枝杆菌菌株都有活性。其MIC值分别为(μg·mL_-1)：Opiophagus hannah 5.4，Naja atra 8.6，Bungarus fasciatus 6.4，Trimeresurus stejnegri 12.6，Protobothrops mucrosquamatus 11.8，Protobothrops jerdonii 7，Agksistrodon halys 4.2。结论这些结果是首次报道，为进一步设计和开发新来源的抗结核病新药提供了依据。     

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??OBJECTIVE To develop Zaocys dhumnades DNA extraction and detection kit, evaluate the specificity, stability and repeatability of the kit, and investigate the quality of commercial black tip snake products using the kit. METHODS Using the modern DNA fingerprint technology, the pharmacopoeia Zaocys dhumnades DNA detection method was optimized and improved, and Zaocys dhumnades DNA extraction and detection reagent was developed to obtain the tip snake PCR detection kit. The performance parameters of the kit were evaluated. The kit was used on 18 commercial black tip snake products randomly sampled from Beijing, Tianjin, Changchun and Jilin City.The results were compared with those got by using pharmacopoeia method of DNA test. RESULTS Zaocys dhumnades DNA testing kit was still valid after repeated freezing and thawing for 5, 10 and 20 times. Among the 18 commercial black tip snake samples 14 were quality goods, and four were adulterants. Repeated experiments of specificity, stability and repeatability displayed exactly the same results . The test result of the kit method was consistent with the pharmacopoeia method. And the kit method could fulfil the DNA testing task in ordinary laboratories. CONCLUSION Zaocys dhumnades DNA testing kit can identify tip snake products conveniently, accurately and effectively, which can be applied in the rapid detection of Zaocys dhumnades DNA.