龟甲、水蛭的品种与质量研究

  本文关键词：龟甲、水蛭的品种与质量研究，由笔耕文化传播整理发布。

        本文从原动物鉴定,DNA条形码研究,有效成分或指标成分的含量测定、指纹图谱等几个方面讨论水蛭、龟甲的质量,具体研究分以下几个方面：研究目的：本课题针对动物类中药质量标准存在的问题,以常用动物药水蛭、龟甲为研究对象,初步拟定以下研究目标：通过将DNA条形码、常规鉴定方法与指纹图谱相结合,完善水蛭、龟甲真伪优劣鉴定方法,为建立一套客观化、规范化、符合中医药特色的动物类中药质量评价方法和质量标准打下基础。研究方法：本实验采用以下实验方法：一、常规鉴定：包括原动物鉴定、性状鉴定、显微鉴定、TLC薄层色谱鉴定等。根据结果,初步判断其品种。二、DNA条形码研究方法：包括试剂盒DNA提取法,运用Taq酶或TaqMIX酶进行PCR扩增；测序后得到DNA条形码。最后通过DNA条形码进行变异位点、遗传N-J树等分析,对水蛭、龟甲进行客观的品种鉴定。通过常规鉴定和DNA条形码,找出适合快速、客观、准确鉴定水蛭、龟甲真伪的方法。三、有效成分和指标成分含量测定：根据文献报道,初步确定次黄嘌呤及L-羟脯氨酸为标准物质,考察水蛭药材的次黄嘌呤及龟甲药材的胶原蛋白含量。另根据2010版《中国药典》,考察水蛭的凝血酶效价。判断药材质量。四、指纹图谱的比对：运用高效液相测定法,参照文献所述的方法,并自己加以改良,确定高效液相指纹图谱的建立方法。绘制蚂蟥药材的高效液相指纹图谱,并对其进行比对和研究。研究结果：性状鉴定：药材鉴别龟甲为上甲、下甲盾片上的纹理,水蛭为颜色、形态大小等。TLC结果为龟甲的正品和混伪品均可在相同的位置与标准品(胆固醇)和龟甲标准药材形成相似的点；水蛭的正品和混伪品均可在相同的位置与水蛭标准药材形成相似的点。DNA条形码：龟甲方面,研究所用的23个样品的基于COI基因的DNA条形码长度范围为651-706bp。药材龟甲样品条形码长度为569bp。GC含量范围为40.8-45.0%。乌龟与其混伪品的在序列长度及GC含量上均有差异,乌龟及其混伪品有238个变异位点。遗传N-J树表明龟甲的正品乌龟聚集在一起,各种混伪品聚集在一起,且支持率接近100。水蛭方面,研究所用的12个样品的DNA条形码长度范围为649-675bp。GC含量范围为29.8%-35.5%,蚂蟥、水蛭、柳叶蚂蟥与其混伪品的条形码长度及GC含量上均有差异。蚂蟥、水蛭、柳叶蚂蟥及其混伪品有303个变异位点。遗传N-J树表明水蛭的正品来源聚集在一起,各种混伪品聚集在一起,且支持率近100含量测定：浸出物含量：龟甲0.75%-8.08%,正品合格率66.7%,伪品比正品含量低；水蛭3.7%-16.68%,正品合格率25%,伪品比正品含量低。水蛭凝血酶效价2.9u-633.2u,不同品种、不同炮制方法的效价差距很大。次黄嘌呤含量测定的标准曲线的回归方程为y=143.8x+0.573, R2=0.999,含量从0.044-1.958mg/g。龟甲的胶原蛋白含量标准曲线回归方程为y=30189x+147, R2=0.999,含量从2.32-16.68%,不同品种、炮制与否对含量影响很大。指纹图谱：用建立的色谱条件,测定得到的蚂蟥HPLC指纹图谱,共有10个共有峰,其中可对次黄嘌呤、黄嘌呤色谱峰进行指认。共有峰相对峰面积RSD值为24.47%-77.7%,10个不同产地的水蛭样品指纹图谱相似度为0.669-0.994。研究结论：运用《中国药典》2010版方法进行常规鉴定,可以区别龟甲的原动物。但是水蛭的原动物、龟甲水蛭的药材、饮片等通过性状、显微、薄层鉴定则较为困难,需要更加快速、准确的方法进行龟甲、水蛭的品种鉴定。基于此,笔者研究出龟甲、水蛭原动物及药材的DNA提取、PCR扩增方法,得到DNA条形码,根据实验结果分析可以得出以下结论：由于龟甲、水蛭的正品与混唯品有较多的变异位点及遗传N-J树正品与混伪品可以明显的区分开,因此基于COI序列的DNA条形码技术可以很好地鉴定龟甲、水蛭及其混伪品,为该药材的快速、准确鉴定品种提供了新的方法。但是,由于DNA条形码只能对龟甲、水蛭的真伪做出鉴定,无法判别其质量,因此需要结合含量测定,才能科学、准确的评价龟甲、水蛭的质量。关于水蛭、龟甲的质量,笔者采用有效成分或指标成分含量测定的方法判断其质量。运用《药典》方法,通过水蛭、龟甲的浸出物含量进行测定,粗略评价水蛭、龟甲质量发现,由于水蛭加工方法的影响,市场占有量最大的蚂蟥浸出物含量差异很大,且炮制成饮片后,龟甲、水蛭浸出物含量降低。通过对水蛭凝血酶效价的测量,初步判断水蛭抗凝血能力的强弱,发现水蛭的抗凝血能力与其品种有很大的关系,吸血水蛭品种抗凝血能力强,不吸血水蛭品种抗疑血能力差。通过对水蛭主流指标成分次黄嘌呤含量及龟甲有效成分胶原蛋白含量测定,得到以下结论：水蛭方面,从测量结果中可以看出,正品中,蚂蟥的次黄嘌呤含量最高,水蛭最低。这与凝血酶效价的结果相反。由于水蛭的抗凝血成分还没有确定,因此,只能通过测定该成分从一方面评价水蛭的质量,但是对于市场上的主流品种蚂蟥来说,该指标对质量评价有一定的意义。龟甲方面,龟甲的正品来源乌龟胶原蛋白的含量最高,黄喉拟水龟较乌龟含量稍低,常见混伪品红耳侧线龟的含量较低。经过煮、刮等炮制后,龟甲的胶原蛋白含量会大幅度降低,这与浸出物含量相吻合。因此,从含量测定的角度来讲,生龟甲有效成分含量高于炙龟甲。本研究以蚂蟥为例,对不同产地的10份样品进行指纹图谱的研究,不同产地的蚂蟥药材的指纹图谱相似性较高,说明不同产地蚂蟥含有的化学成分大致相当。但是共有峰的峰面积RSD值很大,说明这样本蚂蟥的共有成分含量差异很大,这应与养殖方法养殖环境、炮制有关。综上所述,本实验研究通过单一常规鉴定评价水蛭、龟甲质量的弊端,探索性的将DNA条形码与水蛭、龟甲传统四大鉴定相结合,综合判定水蛭、龟甲的质量。最后,为建立动物药材的综合质量判定标准,及为2015版药典提供实验基础。本研究创新点为：1、本项目探索从龟甲、水蛭及其混伪品的原动物、药材中提取DNA的方法,第一次从龟甲、水蛭的药材中成功获得可测序的DNA,并首次完整、系统分析龟甲、水蛭正品与混伪品DNA条形码特征(包括变异位点、遗传N-J树等情况)。2、首次利用2010版药典及文献,对龟甲、水蛭进行系统品种和质量研究。3、本课题根据指纹图谱建立的一系列要求,以蚂蟥为例,首次引入黄嘌呤为标准品,与黄嘌呤组成混标,系统的完成整个蚂蟥指纹图谱的建立工作。

    The content of this paper includes the identification of the original a nimal, DNA bar coding, effective components or index component contents det ermination, fingerprint of H1RUDO and TESTUDINIS CAKAPAX ET PLASTRUM, which can evaluation these two herbs quality.Research purposes:this topic aims at traditional identification cannot describe the truth and quality of animal medicinal materials quickly, obje ctively and correctly, choosing HIRUDO and TESTEDIMS CARAPAX ET PLASTKTIM a s the instance, Making out the following research goal-Through the combination of DNA bar code, traditional identification and fingerprint, improve the identification method of HIRUDO and TESTUDINIS CA RAPAX ET PLASTRUM, which can basis for establishment a new, objective, obje ctive, digitized, standardized, quality standard of ani-mal medicinal mater ialsResearch methods:this topic used the following research methods:First, the traditional identification:including animal identificatio-n, character identification, microscopic identification and TLC iden-tificati on of animal medicinal materials, According to the results, preliminary jud ges its strainssecond, DNA barcode:DNA was extracted by kit method, and use univers-a1primer and Taq enzyme or TaqMIX enzyme to amplification, after se-quencin g, DNA bar code is obtained. Through, analysis of mutation si-tes and DNA b ar code genetic N-J tree, the leech and turtle’s strain is identified objec tively.Third, determination of effective or index components:according to t-h e reports, use hypoxanthine and L-hydroxyproline as standard subst-ance, d etermination hypoxanthine of HIRUDO and collagen of TliSTUDINI-S CARAPAX ET PLASTRUM. According to the2010edition of "Pharmaeopoe-ia of China", study the potency of thrombin of HIRUDO.Fourth, fingerprint:using HPLC determination method, according the rep orts to establish HPLC fingerprint. HPLC fingerprint drawing HIRUDO herbs, and compare and Study on its.The conclusion of the study:Using the traditional identification, ca-n make difference between the TESTUDINIS CARAPAX ET PLASTRUM’s origi-nal ani mal. But through the character, microscopic identification an-d TLC, It’s h ard to identification the a-nimal of HIRUDO and materia-ls. For these reaso ns, the author researches the DNA extraction, PCR amplification of leech, t urtles, and their materials, finally obtain-ed DNA bar code successfully, a ccording to the analyze of DNA bar co-des, the following conclusions can be drawn:the TESTUDINIS CARAPAX ET PLASTRUM, HIRUDO and its adulterants has a great many of variable sites. The genetic N-J tree shows that genuine and adulterants can be separated from the clear zone, so the DNA bar code tech nology can be identify the TESTUDINIS CARAPAX ET PLASTRUM, HIRUDO and its ad ultera-nts. This paper provides a new method for fast, accurate identificat,^ion of species of the medicinal materials. However, because the DNA bar co de only can do TESTUDINIS CARAPAX ET PLASTRUM, HIRUDO authentic-ity identif ication, its quality cannot distinguish. Therefore requir-es a combination of content determination, can be scientific, accura-te evaluation of TESTUD IMS CARAPAX ET PLASTRUM, HIRUDO quality.Using the "Pharmacopoeia" method, the extractives content of HIRUDO, TE STUDIN1S CARAPAX ET PLASTRUM were determined, the result shows that due to the effect of HIRUDO processing method, Whitmania pigra,Whitm-an, the large st amount of market share, has a greatly of difference of extract content.A nd the Chinese herbal pieces has low contents. Th-rough the measurement of the HIRUDO potency of thrombin, make a prel-iminary judgment of HIRUDO anti coagulant ability, it shows that ther-e is a great relationship between-coa gulation HIRUDO and varieties, varieties of blood-sucking HIRUDO anticoagul ant ability, not blood an-ticoagulant HIRUDO species ability.Through the determination of hypoxanthine, index components of HIRUDO, contents and collagen, effective components TESTUDINIS CARAPAX ET PL-ASTRUM. content, obtained the following conclusions:HIRUDO, from the measurement r esults can be seen, genuine of hypoxanthine content Whi-tmania pigra,Whitma n is highest, the lowest is Hirudo nipponica, Wh-itman. It’s in contrast to the results with thrombin. The anticoagul-ant components of HIRUDO has not been determined, therefore, the det-ermination of the composition of HIRUD0is the one side the quality of evaluation, market, mainstream varieties of HIRUDO, the index has c-ertain significance to the quality evaluation. TES TUDINIS CARAPAX ET PLASTRUM, the collagen content in genuine source is the highest, Mau-remys mutica has slightly lower content, common adulterants tr achemy-s scripta is lowest, the collagen content of Chinese herbal pieces o-f TESTUDINIS CARAPAX ET PLASTRUM is greatly reduced. Therefore, from the p erspective of content determination, the content of effective components wi th TESTUDINIS CARAPAX ET PLASTRUM is higher than its Chinese herbal pieces. In this study,HIREDO as example, research of10samples of different o rigin of the fingerprints, fingerprint shows the different origin of high imilarity, chemical composition of different origin contain roughly the sam e. But RSD of the share peak area is very large, so the common ingredient c ontent of samples difference is very big, it should be concerned with the c ultivation, breeding environment, processing method.To sum up, through the research the shortcomings of traditional evaluat ion of HIRUDO, TESTUDINIS CARAPAX ET PLASTRUM quality, explore the DNA bar code and combine four traditional identifications. Make a judgment of HIRED0, TESTUDINIS CARAPAX ET PLASTRUM quality comprehensively. Finally, this pr-oject makes a experimental basis of comprehensive quality of animal medicin e Pharmacopoeia of China2015edition.The research innovations:1, the project is in the traditional Chinese medicine under the guid-an ce of the theory, the molecular identification of medicinal mater-ials base, combined with the traditional character identification, microscopic identi fication, physical and chemical identification and content determination.2, for the first time on the TESTUDINIS CARAPAX ET PLASTRUM, HIRUDO, va riety, quality of original investigation and research system, HIRUDO, TESTU DINIS CARAPAX ET PLASTRUM quality evaluation.3, according to a series of requirements to establish the fingerprin-t of the HIRUDO, for example, establish the work accomplished the f-irst whol e HIRUDO fingerprint system.

龟甲、水蛭的品种与质量研究

中文摘要5-8Abstract8-10前言11-12第一部分 文献综述12-29    1 水蛭、龟甲的本草考证12-16        1.1 水蛭名考12-13        1.2 龟甲名考13-16    2 水蛭化学成分与鉴定的研究进展16-20        2.1 水蛭的药材鉴定与质量评价16-17        2.2 水蛭化学成分研究进展17-19        2.3 总结19-20    3 龟甲药材鉴定与化学成分研究进展20-23        3.1 龟甲的药材鉴定与质量评价研究进展20-21        3.2 龟甲化学成分研究进展21-22        3.3 总结22-23    4 动物DNA条形码研究进展与其在中药领域的应用23-27        4.1 动物DNA条形码的理论基础及操作步骤23-24        4.2 动物DNA条形码的研究进展24-25        4.3 动物DNA条形码技术在中药动物药领域内的应用25-27    5 中药动物药HPLC指纹图谱的研究现状及展望27-29        5.1 中药动物药HPLC指纹图谱的研究现状27-28        5.2 中药动物药HPLC指纹图谱的展望28-29第二部分 实验研究29-94    实验技术路线29-30    1. 龟甲、水蛭样品收集及品种鉴定30-49        1.1 龟甲、水蛭样品收集30-36        1.2 龟甲、水蛭及其混伪品原动物鉴定36-38        1.3 龟甲、水蛭及其混伪品药材性状鉴定38-40        1.4 龟甲、水蛭药材的粉末显微鉴定40-43        1.5 龟甲、水蛭的薄层色谱鉴别43-47        1.6 总结与讨论47-49    2. 龟甲、水蛭及其混伪品DNA条形码研究49-66        2.1 仪器、试剂及样品49-51        2.2 方法摸索51-54        2.3 DNA提取及PCR扩增条件确定54-55        2.4 实验结果55-60        2.5 结果分析60-65        2.6 总结与讨论65-66    3. 龟甲、水蛭浸出物含量测定66-71        3.1 仪器、试剂与样品66-67        3.2 实验方法67-68        3.3 实验结果68-69        3.4 总结与讨论69-71    4.龟甲、水蛭的有效成分或指标成分含量测定71-88        4.1 水蛭凝血酶效价评价71-75        4.2 水蛭次黄嘌呤含量75-81        4.3 龟甲胶原蛋白含量81-88    5. 蚂蟥HPLC指纹图谱研究88-94        5.1 仪器、试剂及样品88-89        5.2 色谱条件89        5.3 方法学考察89-90        5.4 实验结果90-92        5.5 总结与讨论92-94第三部分 参考文献94-100第四部分 附图100-107致谢107-108个人简历108

  本文关键词：龟甲、水蛭的品种与质量研究，，由笔耕文化传播整理发布。