本文關(guān)鍵詞：龜甲、水蛭的品種與質(zhì)量研究，由筆耕文化傳播整理發(fā)布。

        本文從原動(dòng)物鑒定,DNA條形碼研究,有效成分或指標(biāo)成分的含量測定、指紋圖譜等幾個(gè)方面討論水蛭、龜甲的質(zhì)量,具體研究分以下幾個(gè)方面：研究目的：本課題針對動(dòng)物類中藥質(zhì)量標(biāo)準(zhǔn)存在的問題,以常用動(dòng)物藥水蛭、龜甲為研究對象,初步擬定以下研究目標(biāo)：通過將DNA條形碼、常規(guī)鑒定方法與指紋圖譜相結(jié)合,完善水蛭、龜甲真?zhèn)蝺?yōu)劣鑒定方法,為建立一套客觀化、規(guī)范化、符合中醫(yī)藥特色的動(dòng)物類中藥質(zhì)量評價(jià)方法和質(zhì)量標(biāo)準(zhǔn)打下基礎(chǔ)。研究方法：本實(shí)驗(yàn)采用以下實(shí)驗(yàn)方法：一、常規(guī)鑒定：包括原動(dòng)物鑒定、性狀鑒定、顯微鑒定、TLC薄層色譜鑒定等。根據(jù)結(jié)果,初步判斷其品種。二、DNA條形碼研究方法：包括試劑盒DNA提取法,運(yùn)用Taq酶或TaqMIX酶進(jìn)行PCR擴(kuò)增；測序后得到DNA條形碼。最后通過DNA條形碼進(jìn)行變異位點(diǎn)、遺傳N-J樹等分析,對水蛭、龜甲進(jìn)行客觀的品種鑒定。通過常規(guī)鑒定和DNA條形碼,找出適合快速、客觀、準(zhǔn)確鑒定水蛭、龜甲真?zhèn)蔚姆椒āＨ�、有效成分和指�?biāo)成分含量測定：根據(jù)文獻(xiàn)報(bào)道,初步確定次黃嘌呤及L-羥脯氨酸為標(biāo)準(zhǔn)物質(zhì),考察水蛭藥材的次黃嘌呤及龜甲藥材的膠原蛋白含量。另根據(jù)2010版《中國藥典》,考察水蛭的凝血酶效價(jià)。判斷藥材質(zhì)量。四、指紋圖譜的比對：運(yùn)用高效液相測定法,參照文獻(xiàn)所述的方法,并自己加以改良,確定高效液相指紋圖譜的建立方法。繪制螞蟥藥材的高效液相指紋圖譜,并對其進(jìn)行比對和研究。研究結(jié)果：性狀鑒定：藥材鑒別龜甲為上甲、下甲盾片上的紋理,水蛭為顏色、形態(tài)大小等。TLC結(jié)果為龜甲的正品和混偽品均可在相同的位置與標(biāo)準(zhǔn)品(膽固醇)和龜甲標(biāo)準(zhǔn)藥材形成相似的點(diǎn)；水蛭的正品和混偽品均可在相同的位置與水蛭標(biāo)準(zhǔn)藥材形成相似的點(diǎn)。DNA條形碼：龜甲方面,研究所用的23個(gè)樣品的基于COI基因的DNA條形碼長度范圍為651-706bp。藥材龜甲樣品條形碼長度為569bp。GC含量范圍為40.8-45.0%。烏龜與其混偽品的在序列長度及GC含量上均有差異,烏龜及其混偽品有238個(gè)變異位點(diǎn)。遺傳N-J樹表明龜甲的正品烏龜聚集在一起,各種混偽品聚集在一起,且支持率接近100。水蛭方面,研究所用的12個(gè)樣品的DNA條形碼長度范圍為649-675bp。GC含量范圍為29.8%-35.5%,螞蟥、水蛭、柳葉螞蟥與其混偽品的條形碼長度及GC含量上均有差異。螞蟥、水蛭、柳葉螞蟥及其混偽品有303個(gè)變異位點(diǎn)。遺傳N-J樹表明水蛭的正品來源聚集在一起,各種混偽品聚集在一起,且支持率近100含量測定：浸出物含量：龜甲0.75%-8.08%,正品合格率66.7%,偽品比正品含量低；水蛭3.7%-16.68%,正品合格率25%,偽品比正品含量低。水蛭凝血酶效價(jià)2.9u-633.2u,不同品種、不同炮制方法的效價(jià)差距很大。次黃嘌呤含量測定的標(biāo)準(zhǔn)曲線的回歸方程為y=143.8x+0.573, R2=0.999,含量從0.044-1.958mg/g。龜甲的膠原蛋白含量標(biāo)準(zhǔn)曲線回歸方程為y=30189x+147, R2=0.999,含量從2.32-16.68%,不同品種、炮制與否對含量影響很大。指紋圖譜：用建立的色譜條件,測定得到的螞蟥HPLC指紋圖譜,共有10個(gè)共有峰,其中可對次黃嘌呤、黃嘌呤色譜峰進(jìn)行指認(rèn)。共有峰相對峰面積RSD值為24.47%-77.7%,10個(gè)不同產(chǎn)地的水蛭樣品指紋圖譜相似度為0.669-0.994。研究結(jié)論：運(yùn)用《中國藥典》2010版方法進(jìn)行常規(guī)鑒定,可以區(qū)別龜甲的原動(dòng)物。但是水蛭的原動(dòng)物、龜甲水蛭的藥材、飲片等通過性狀、顯微、薄層鑒定則較為困難,需要更加快速、準(zhǔn)確的方法進(jìn)行龜甲、水蛭的品種鑒定�；诖�,筆者研究出龜甲、水蛭原動(dòng)物及藥材的DNA提取、PCR擴(kuò)增方法,得到DNA條形碼,根據(jù)實(shí)驗(yàn)結(jié)果分析可以得出以下結(jié)論：由于龜甲、水蛭的正品與混唯品有較多的變異位點(diǎn)及遺傳N-J樹正品與混偽品可以明顯的區(qū)分開,因此基于COI序列的DNA條形碼技術(shù)可以很好地鑒定龜甲、水蛭及其混偽品,為該藥材的快速、準(zhǔn)確鑒定品種提供了新的方法。但是,由于DNA條形碼只能對龜甲、水蛭的真?zhèn)巫龀鲨b定,無法判別其質(zhì)量,因此需要結(jié)合含量測定,才能科學(xué)、準(zhǔn)確的評價(jià)龜甲、水蛭的質(zhì)量。關(guān)于水蛭、龜甲的質(zhì)量,筆者采用有效成分或指標(biāo)成分含量測定的方法判斷其質(zhì)量。運(yùn)用《藥典》方法,通過水蛭、龜甲的浸出物含量進(jìn)行測定,粗略評價(jià)水蛭、龜甲質(zhì)量發(fā)現(xiàn),由于水蛭加工方法的影響,市場占有量最大的螞蟥浸出物含量差異很大,且炮制成飲片后,龜甲、水蛭浸出物含量降低。通過對水蛭凝血酶效價(jià)的測量,初步判斷水蛭抗凝血能力的強(qiáng)弱,發(fā)現(xiàn)水蛭的抗凝血能力與其品種有很大的關(guān)系,吸血水蛭品種抗凝血能力強(qiáng),不吸血水蛭品種抗疑血能力差。通過對水蛭主流指標(biāo)成分次黃嘌呤含量及龜甲有效成分膠原蛋白含量測定,得到以下結(jié)論：水蛭方面,從測量結(jié)果中可以看出,正品中,螞蟥的次黃嘌呤含量最高,水蛭最低。這與凝血酶效價(jià)的結(jié)果相反。由于水蛭的抗凝血成分還沒有確定,因此,只能通過測定該成分從一方面評價(jià)水蛭的質(zhì)量,但是對于市場上的主流品種螞蟥來說,該指標(biāo)對質(zhì)量評價(jià)有一定的意義。龜甲方面,龜甲的正品來源烏龜膠原蛋白的含量最高,黃喉擬水龜較烏龜含量稍低,常見混偽品紅耳側(cè)線龜?shù)暮枯^低。經(jīng)過煮、刮等炮制后,龜甲的膠原蛋白含量會大幅度降低,這與浸出物含量相吻合。因此,從含量測定的角度來講,生龜甲有效成分含量高于炙龜甲。本研究以螞蟥為例,對不同產(chǎn)地的10份樣品進(jìn)行指紋圖譜的研究,不同產(chǎn)地的螞蟥藥材的指紋圖譜相似性較高,說明不同產(chǎn)地螞蟥含有的化學(xué)成分大致相當(dāng)。但是共有峰的峰面積RSD值很大,說明這樣本螞蟥的共有成分含量差異很大,這應(yīng)與養(yǎng)殖方法養(yǎng)殖環(huán)境、炮制有關(guān)。綜上所述,本實(shí)驗(yàn)研究通過單一常規(guī)鑒定評價(jià)水蛭、龜甲質(zhì)量的弊端,探索性的將DNA條形碼與水蛭、龜甲傳統(tǒng)四大鑒定相結(jié)合,綜合判定水蛭、龜甲的質(zhì)量。最后,為建立動(dòng)物藥材的綜合質(zhì)量判定標(biāo)準(zhǔn),及為2015版藥典提供實(shí)驗(yàn)基礎(chǔ)。本研究創(chuàng)新點(diǎn)為：1、本項(xiàng)目探索從龜甲、水蛭及其混偽品的原動(dòng)物、藥材中提取DNA的方法,第一次從龜甲、水蛭的藥材中成功獲得可測序的DNA,并首次完整、系統(tǒng)分析龜甲、水蛭正品與混偽品DNA條形碼特征(包括變異位點(diǎn)、遺傳N-J樹等情況)。2、首次利用2010版藥典及文獻(xiàn),對龜甲、水蛭進(jìn)行系統(tǒng)品種和質(zhì)量研究。3、本課題根據(jù)指紋圖譜建立的一系列要求,以螞蟥為例,首次引入黃嘌呤為標(biāo)準(zhǔn)品,與黃嘌呤組成混標(biāo),系統(tǒng)的完成整個(gè)螞蟥指紋圖譜的建立工作。

    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.

龜甲、水蛭的品種與質(zhì)量研究

中文摘要5-8Abstract8-10前言11-12第一部分 文獻(xiàn)綜述12-29    1 水蛭、龜甲的本草考證12-16        1.1 水蛭名考12-13        1.2 龜甲名考13-16    2 水蛭化學(xué)成分與鑒定的研究進(jìn)展16-20        2.1 水蛭的藥材鑒定與質(zhì)量評價(jià)16-17        2.2 水蛭化學(xué)成分研究進(jìn)展17-19        2.3 總結(jié)19-20    3 龜甲藥材鑒定與化學(xué)成分研究進(jìn)展20-23        3.1 龜甲的藥材鑒定與質(zhì)量評價(jià)研究進(jìn)展20-21        3.2 龜甲化學(xué)成分研究進(jìn)展21-22        3.3 總結(jié)22-23    4 動(dòng)物DNA條形碼研究進(jìn)展與其在中藥領(lǐng)域的應(yīng)用23-27        4.1 動(dòng)物DNA條形碼的理論基礎(chǔ)及操作步驟23-24        4.2 動(dòng)物DNA條形碼的研究進(jìn)展24-25        4.3 動(dòng)物DNA條形碼技術(shù)在中藥動(dòng)物藥領(lǐng)域內(nèi)的應(yīng)用25-27    5 中藥動(dòng)物藥HPLC指紋圖譜的研究現(xiàn)狀及展望27-29        5.1 中藥動(dòng)物藥HPLC指紋圖譜的研究現(xiàn)狀27-28        5.2 中藥動(dòng)物藥HPLC指紋圖譜的展望28-29第二部分 實(shí)驗(yàn)研究29-94    實(shí)驗(yàn)技術(shù)路線29-30    1. 龜甲、水蛭樣品收集及品種鑒定30-49        1.1 龜甲、水蛭樣品收集30-36        1.2 龜甲、水蛭及其混偽品原動(dòng)物鑒定36-38        1.3 龜甲、水蛭及其混偽品藥材性狀鑒定38-40        1.4 龜甲、水蛭藥材的粉末顯微鑒定40-43        1.5 龜甲、水蛭的薄層色譜鑒別43-47        1.6 總結(jié)與討論47-49    2. 龜甲、水蛭及其混偽品DNA條形碼研究49-66        2.1 儀器、試劑及樣品49-51        2.2 方法摸索51-54        2.3 DNA提取及PCR擴(kuò)增條件確定54-55        2.4 實(shí)驗(yàn)結(jié)果55-60        2.5 結(jié)果分析60-65        2.6 總結(jié)與討論65-66    3. 龜甲、水蛭浸出物含量測定66-71        3.1 儀器、試劑與樣品66-67        3.2 實(shí)驗(yàn)方法67-68        3.3 實(shí)驗(yàn)結(jié)果68-69        3.4 總結(jié)與討論69-71    4.龜甲、水蛭的有效成分或指標(biāo)成分含量測定71-88        4.1 水蛭凝血酶效價(jià)評價(jià)71-75        4.2 水蛭次黃嘌呤含量75-81        4.3 龜甲膠原蛋白含量81-88    5. 螞蟥HPLC指紋圖譜研究88-94        5.1 儀器、試劑及樣品88-89        5.2 色譜條件89        5.3 方法學(xué)考察89-90        5.4 實(shí)驗(yàn)結(jié)果90-92        5.5 總結(jié)與討論92-94第三部分 參考文獻(xiàn)94-100第四部分 附圖100-107致謝107-108個(gè)人簡歷108

  本文關(guān)鍵詞：龜甲、水蛭的品種與質(zhì)量研究，，由筆耕文化傳播整理發(fā)布。