美洲大蠊生物学特性及药用价值研究
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摘要
美洲大蠊(Periplaneta americana)是世界上分布最广、最为常见的室内卫生害虫之一,它能携带和传播多种疾病,也能引起变态反应性疾病;在中药中可用作消炎、镇痛、治疗创伤和治疗心血管疾病的重要成分。为了深入了解它的生物学特性和药用价值,并为这一天然药物资源的发掘和综合利用提供全面、系统的实验依据;我们以其作为研究对象,在前人工作的基础上对其进行了比较系统的研究,内容包括生物学特性研究、药用有效成分的提取与分析、提取物的药效学研究、以及应用美洲大蠊成虫免疫兔血清筛选美洲大蠊若虫cDNA文库,并对发现的新基因进行了生物信息学分析。
一.美洲大蠊生物学特性研究:
通过室内自然条件下人工饲养美洲大蠊,全面系统地观察记录了它的生活史和正常生存条件要求,掌握了它们的交配、生殖产卵、孵化、生长、蜕皮和羽化等生命活动过程,以及它的食性、食量、生活环境、活动特点等行为特性,系统描述了其形态特征,统计分析了其产卵量、孵化率、食量的季节性变化,为对其进行综合防治和规模化人工养殖提供、补充了详细、系统的实验资料。
二.美洲大蠊药用有效成份的提取与分离:
通过改进提取工艺,探索出了一种新的美洲大蠊成虫药用有效成份提取方法。首先用PBS抽提得到美洲大蠊成虫粗提物;用索氏提取器提取油脂,随后用硫酸铵盐析法得到总蛋白,多糖由乙醇沉淀所得,剩下的为可溶性小分子物质。经分析发现,美洲大蠊成虫中油脂含量为4.97%,油脂中含油酸、亚油酸和占很大比例的长链烷烃;多糖的含量低,仅0.49%。
三.美洲大蠊提取物的药效学研究:
首先以美洲大蠊成虫粗提物为试药,观察不同药物剂量对二甲苯致小鼠耳廓肿胀、小鼠棉球性肉芽组织增生、鸡蛋清致小鼠足跖肿胀、小鼠醋酸性扭体反应、小鼠热板法的镇痛作用、大鼠应激性胃溃疡、小鼠醋酸烧灼性胃溃疡、小鼠无水乙醇胃粘膜损伤等的抑制作用。实验结果表明,美洲大蠊成虫提取物具有明显且较全面的抗炎、镇痛作用,与对照组相比具有统计学意义。组织病理学观察进一步证实了美洲大蠊提取物的抗炎、消肿作用。同时建立小鼠肝脏肉芽肿模型,注射美洲大蠊的几种提取成分以观察是否有减轻免疫反应或抑制日本血吸虫病的作用。
四.美洲大蠊免疫兔血清筛选美洲大蠊若虫cDNA文库:
应用美洲大蠊成虫免疫兔血清,筛选美洲大蠊若虫cDNA文库,从获得阳性克隆中挑选4个进行测序,发现其中两个序列与已知基因无显著同源性,确认为新基因,分别命名为Parcxpwxxq01、Parcxpwxxq02,并成功登录GenBank,登陆号分别为DQ522316、DQ522315。该研究利用现代分子生物学技术筛选出了具有药用价值的新基因。
五.新基因编码蛋白质的生物信息学分析:
运用现代生物信息学分析方法,利用有关程序和软件,对获得的新基因进行了编码蛋白质的结构和功能预测,包括物理化学性质、二级结构与折叠类型、亲水性参数和柔曲性参数预测、以及特定功能位点分析等。
总结
本文围绕医学昆虫美洲大蠊的药用价值开展了一系列研究工作。在人工饲养条件下,进行了室内繁殖、发育及其生物学特性和行为学特性的细致观察,成功地开发出了新的提取方法,并对其进行药效学试验。另外,我们成功制备了美洲大蠊免疫血清并对美洲大蠊若虫cDNA文库进行了筛选,发现了2个新基因。上述结果为美洲大蠊药用价值的进一步开发提供了具有重要参考价值的实验依据。
The medical insect Periplaneta Americana(cockroach) is one of the interior insects,who is widely dispersed,common and closely related to humanity.As a medicinal materials which was earliest recorded in the Qin Dynasty,and all Chinese material medicine are collected.So,it had a long history in China.In this paper,we used P.Americana as the subject of experiment,on the basis of experience summed up by predcessors, arounding its medicinal value,we carried out a series systematic research, in order to provide scientific basis for unearthing,exploiting and comprehensive utilizing of this medical insect P.Americana
1.Studies on biological characteristics of P.Americana
During 2005～2006,we carried out systematic investigations on domestic reproduction,development,biological characteristics and ethological features of P.Americana.The biological characteristis including reproduction of adults,laying eggs,hatching,development of nymphae,moulting,emergence and the ethological features including negative phototaxis,aggregation,food habit at different developmental stages were observed.Blogical parameters such as duration for eggs, duration for nymphae,hatching rate,capacity for eating.The life cycle was detailed described.The results can provide the experimental data for artificial feeding and reproduction of medicinal insect P.Americana in large-scale.
2.Collection and analyses for the medicinal effective component parts derived from adults of P.Americana
The traditional methods of collection medicinal effective component parts have been improved.Firstly,the crude extraction was obtained from Periplaneta Americana adult.Followly,the lipid was obtained by Soxhlet's apparatus;and the total protein was got by ammonium sulfate salt fractionation;the polysaccharides was extracted by ethanol precipitation;the remains was the soluble low molecular weight substance.The content of lipid and the polysaccharides in P.Americana adult was 4.97%,0.47%,respectively.
3.Study on pharmacology of extracts of P.Americana
The result of acute toxicity test showed,extracts of P.Americana were nontoxic.Inhibitive effects of extracts of P.Americana were investigated in different inflammatory models,for instance, dimethylbenzene induced acute inflammatory swelling ears of mice, cotton induced-granuloma tissue proliferous inflammation of mice,egg white induced rat paw edema,acetic acid induced writhing reponse of mice,stress gastric ulcer of mice,acetic acid gastric ulcer of mice, alcohol gastric ulcer of mice.The results showed higher dosage extracts of adult P.Americana played important roles in subsidence of swelling, decreasing pain and diminish.There was obviously statistical difference (P＜0.01 or 0.05=,as compared with control group.The results proved that extracts of P.Americana is indeed worthy of further development for potential medicinal value.At the same time we established the model of liver granuloma,the extraction of P.Americana was injected and the effect of relieving immune response was observed.
4.Screening nymphal P.Americana cDNA library with anti P. Americana
Two positive clone nucleotide sequences which showed no homology with any other known nucleotide sequences were obtained by screening nymphal P.Americana cDNA library with anti-adult P. Americana serum.The two new encoding genes were submitted to GenBank and named as Parcxpwxxq01,Parcxpwxxq02,with the Accession Numbers were DQ522315 and DQ522316.This study provides us a new approach to screen the medicinal genes via molecular biological techniques.
5.Bioinformatical forecast of the structures and biological functions of amino acid sequence deduced by new encoding genes
Applying the softwares provided by the National Center for Biotechnology Information,we analysised the proteins which were codinged by the nucleotide sequences,their structures and functions, including physical characterics,average molecular weights, hydrophobicities,average flexibilities,α-helixes,β-turns and etc.had been applied.
In summary,a series systematic researches arounding the medicinal value of medicinal insect P.Americana were carried out in this paper. Domestic reproduction,development,biological characteristics and ethological features of P.Americanawere comprehensively and systematically investigated under artificial feeding condition.The new genes were found by screening cDNA library with anti- P. Americanawere sera.
The results above provide important valuable experimental data for advanced researches of P.Americana for its medicinal.
一.美洲大蠊生物学特性研究:
通过室内自然条件下人工饲养美洲大蠊,全面系统地观察记录了它的生活史和正常生存条件要求,掌握了它们的交配、生殖产卵、孵化、生长、蜕皮和羽化等生命活动过程,以及它的食性、食量、生活环境、活动特点等行为特性,系统描述了其形态特征,统计分析了其产卵量、孵化率、食量的季节性变化,为对其进行综合防治和规模化人工养殖提供、补充了详细、系统的实验资料。
二.美洲大蠊药用有效成份的提取与分离:
通过改进提取工艺,探索出了一种新的美洲大蠊成虫药用有效成份提取方法。首先用PBS抽提得到美洲大蠊成虫粗提物;用索氏提取器提取油脂,随后用硫酸铵盐析法得到总蛋白,多糖由乙醇沉淀所得,剩下的为可溶性小分子物质。经分析发现,美洲大蠊成虫中油脂含量为4.97%,油脂中含油酸、亚油酸和占很大比例的长链烷烃;多糖的含量低,仅0.49%。
三.美洲大蠊提取物的药效学研究:
首先以美洲大蠊成虫粗提物为试药,观察不同药物剂量对二甲苯致小鼠耳廓肿胀、小鼠棉球性肉芽组织增生、鸡蛋清致小鼠足跖肿胀、小鼠醋酸性扭体反应、小鼠热板法的镇痛作用、大鼠应激性胃溃疡、小鼠醋酸烧灼性胃溃疡、小鼠无水乙醇胃粘膜损伤等的抑制作用。实验结果表明,美洲大蠊成虫提取物具有明显且较全面的抗炎、镇痛作用,与对照组相比具有统计学意义。组织病理学观察进一步证实了美洲大蠊提取物的抗炎、消肿作用。同时建立小鼠肝脏肉芽肿模型,注射美洲大蠊的几种提取成分以观察是否有减轻免疫反应或抑制日本血吸虫病的作用。
四.美洲大蠊免疫兔血清筛选美洲大蠊若虫cDNA文库:
应用美洲大蠊成虫免疫兔血清,筛选美洲大蠊若虫cDNA文库,从获得阳性克隆中挑选4个进行测序,发现其中两个序列与已知基因无显著同源性,确认为新基因,分别命名为Parcxpwxxq01、Parcxpwxxq02,并成功登录GenBank,登陆号分别为DQ522316、DQ522315。该研究利用现代分子生物学技术筛选出了具有药用价值的新基因。
五.新基因编码蛋白质的生物信息学分析:
运用现代生物信息学分析方法,利用有关程序和软件,对获得的新基因进行了编码蛋白质的结构和功能预测,包括物理化学性质、二级结构与折叠类型、亲水性参数和柔曲性参数预测、以及特定功能位点分析等。
总结
本文围绕医学昆虫美洲大蠊的药用价值开展了一系列研究工作。在人工饲养条件下,进行了室内繁殖、发育及其生物学特性和行为学特性的细致观察,成功地开发出了新的提取方法,并对其进行药效学试验。另外,我们成功制备了美洲大蠊免疫血清并对美洲大蠊若虫cDNA文库进行了筛选,发现了2个新基因。上述结果为美洲大蠊药用价值的进一步开发提供了具有重要参考价值的实验依据。
The medical insect Periplaneta Americana(cockroach) is one of the interior insects,who is widely dispersed,common and closely related to humanity.As a medicinal materials which was earliest recorded in the Qin Dynasty,and all Chinese material medicine are collected.So,it had a long history in China.In this paper,we used P.Americana as the subject of experiment,on the basis of experience summed up by predcessors, arounding its medicinal value,we carried out a series systematic research, in order to provide scientific basis for unearthing,exploiting and comprehensive utilizing of this medical insect P.Americana
1.Studies on biological characteristics of P.Americana
During 2005～2006,we carried out systematic investigations on domestic reproduction,development,biological characteristics and ethological features of P.Americana.The biological characteristis including reproduction of adults,laying eggs,hatching,development of nymphae,moulting,emergence and the ethological features including negative phototaxis,aggregation,food habit at different developmental stages were observed.Blogical parameters such as duration for eggs, duration for nymphae,hatching rate,capacity for eating.The life cycle was detailed described.The results can provide the experimental data for artificial feeding and reproduction of medicinal insect P.Americana in large-scale.
2.Collection and analyses for the medicinal effective component parts derived from adults of P.Americana
The traditional methods of collection medicinal effective component parts have been improved.Firstly,the crude extraction was obtained from Periplaneta Americana adult.Followly,the lipid was obtained by Soxhlet's apparatus;and the total protein was got by ammonium sulfate salt fractionation;the polysaccharides was extracted by ethanol precipitation;the remains was the soluble low molecular weight substance.The content of lipid and the polysaccharides in P.Americana adult was 4.97%,0.47%,respectively.
3.Study on pharmacology of extracts of P.Americana
The result of acute toxicity test showed,extracts of P.Americana were nontoxic.Inhibitive effects of extracts of P.Americana were investigated in different inflammatory models,for instance, dimethylbenzene induced acute inflammatory swelling ears of mice, cotton induced-granuloma tissue proliferous inflammation of mice,egg white induced rat paw edema,acetic acid induced writhing reponse of mice,stress gastric ulcer of mice,acetic acid gastric ulcer of mice, alcohol gastric ulcer of mice.The results showed higher dosage extracts of adult P.Americana played important roles in subsidence of swelling, decreasing pain and diminish.There was obviously statistical difference (P＜0.01 or 0.05=,as compared with control group.The results proved that extracts of P.Americana is indeed worthy of further development for potential medicinal value.At the same time we established the model of liver granuloma,the extraction of P.Americana was injected and the effect of relieving immune response was observed.
4.Screening nymphal P.Americana cDNA library with anti P. Americana
Two positive clone nucleotide sequences which showed no homology with any other known nucleotide sequences were obtained by screening nymphal P.Americana cDNA library with anti-adult P. Americana serum.The two new encoding genes were submitted to GenBank and named as Parcxpwxxq01,Parcxpwxxq02,with the Accession Numbers were DQ522315 and DQ522316.This study provides us a new approach to screen the medicinal genes via molecular biological techniques.
5.Bioinformatical forecast of the structures and biological functions of amino acid sequence deduced by new encoding genes
Applying the softwares provided by the National Center for Biotechnology Information,we analysised the proteins which were codinged by the nucleotide sequences,their structures and functions, including physical characterics,average molecular weights, hydrophobicities,average flexibilities,α-helixes,β-turns and etc.had been applied.
In summary,a series systematic researches arounding the medicinal value of medicinal insect P.Americana were carried out in this paper. Domestic reproduction,development,biological characteristics and ethological features of P.Americanawere comprehensively and systematically investigated under artificial feeding condition.The new genes were found by screening cDNA library with anti- P. Americanawere sera.
The results above provide important valuable experimental data for advanced researches of P.Americana for its medicinal.
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