肽图结合质谱筛选名贵中药阿胶的特异性肽段与鉴定研究

【作者】 叶茂；

【导师】 国锦琳；

【作者基本信息】 成都中医药大学 ， 生药学， 2016， 硕士

【摘要】 阿胶(Asini Corii Colla)为马科动物驴(Equus asinus L.)的干燥皮或鲜皮经煎煮、浓缩制成的固体胶。始载于《神农本草经》。陶弘景曰：“出东阿,故名阿胶”。阿胶性平,味甘,归肺、肝、肾三经,具有养阴补血,润燥止血的疗效,用于血虚萎黄,眩晕心悸,肌痿无力,心烦不眠,虚风内动,肺燥咳嗽,劳嗽咯血,吐血尿血,便血崩漏,妊娠胎漏。目前,对阿胶进行质量评价多根据外观、色泽、质地、气味和氨基酸含量等评价阿胶真伪优劣。当下由于阿胶需求量巨大,其它动物皮加工品层出不穷,“掺伪现象严重,由于其主要成分胶原蛋白氨基酸组成类似,现有方法难以有效鉴别。2015版《中国药典》采用高相液相色谱-质谱联用仪建立胰酶酶解后阿胶的鉴别方案,能初步有效解决阿胶掺伪的问题,但仍存方法标准化等问题建立稳定、有效的鉴别和质量控制方法非常必要。本课题组主要开展了以下工作：1.阿胶质量评价研究进展阿胶其质量评价主要根据阿胶理化性质,通过运动粘度法、薄层色谱法,HPLC指纹图谱法,光谱法、电泳法等方法进行鉴定和质量评价,2015版药典采用质谱方法进行鉴别为最新方法。2.阿胶胶原蛋白前处理及酶解条件研究通过查询NCBI信息库,课题组驴、牛、羊、马的胶原蛋白氨基酸序列,利用DNAMAN 6.0软件对不同种属来源的皮Ⅰ型胶原蛋白α1链氨基酸序列进行同源比对发现存在95%以上相似性。再利用ExPASy PeptideMass软件系统进行胰酶酶切,比对酶切结果显示驴皮酶解产物中存在7段特异性肽段,质荷比为3647.7804、2140.0417、2073.0107、1792.9340、1752.9027、1100.4966、657.3202。可考虑进一步验证后作为其鉴别的特征肽段。通过加热法溶解、二氯甲烷-环己烷萃取系统及超滤法纯化等处理阿胶样品,在验证2015版《中国药典》酶解的方法的同时,优化酶解方案。最终优化酶解条件为：温度38℃,pH范围7.5～8.0,酶解时间12h以上,加酶比4：1～1：1(Trypsin 250 u/mg),从而建立阿胶胶原蛋白生物酶解方法。3.阿胶特异性肽片段筛选与鉴定研究通过质谱法找出存在差异的驴皮中特异性肽段质荷比为536.7653、765.9070、834.9236、714.3693做解卷积计算等到特异性肽段分子量并证实4组离子都为双电荷。而根据前期生物信息学模拟酶切结果比对,有1组离子接近理论情况下驴皮Ⅰ型胶原蛋白alpha-1酶切肽段,建议与另外3个分子量同时作为鉴定依据做不同胶类来源胶原蛋白酶解产物验证,择优选取特异性分子量。初步建立阿胶特异性肽片段高效液相色谱-质谱联用仪鉴别方法为：以C18色谱柱(2.1mm×100mm,2.7 μ m)分离；以乙腈为流动相A,以0.1%甲酸溶液为流动相B,按0-5 min,5%乙腈；5-8 min,5%-10%乙腈；8-32 min,10%-20%乙腈；32～38min,20%-95%乙腈进行梯度洗脱。流速0.30 mL/min；柱温30℃；进样量1.0 μ L。采用正离子(ESI+)模式进行检测,质荷比(m/z)为536.7653(双电荷)、765.9070(双电荷)、834.9236(双电荷)作为检测离子。对提取的供试品离子流色谱中,应同时呈现与对照药材色谱保留时间一致且峰度显著的色谱峰。4.阿胶特异性肽片段质量评价验证经验证,提取离子m/z 536.7653、m/z765.9070、m/z 834.9236能有效鉴别收集到的11组阿胶样品,并能与2015版《中国药典》中所提到的提取离子m/z539.8相同的鉴别能力,根据m/z 539.8作为指标鉴定1号、2号、5号为优品阿胶。课题组初步提出的特异性阿胶鉴别方案对于收集到的11组阿胶样品能进行有效的鉴别。11组阿胶样品中,仅3家企业生产的阿胶样品符合标准,其余8家企业生产的阿胶都有不同程度掺伪掺假现象。更多还原

【Abstract】 Asini Corii Colla(E’jiao)is prepared from the skin of Equus asinus by decoction and concentration and thus becomes the glue.It is load in the beginning of 《Sheng Nong’s herbal classic》.Tao hongjing said:"From DongE,it is called E’jiao."E’jiao behaves flat, tastes sweet, act on lung, liver, kidney, nourishing Yin and enrich the blood,it is used for blood deficiency chlorosis, dizziness palpitations, muscle impotent helpless and upset sleepless,wind moving restlessly,cough of phlegm-heat,fatigue syndromes haemoptysis, vomiting blood or bloody urine, metrorrhagia and metrostaxis, abortion.At present, the quality evalution of Asini Corii Colla mainly depends on the appearance, color, texture, smell and amino acids to evaluate the authenticity.Now because of the great demand of Asini Corii Colla, other animal skins processed products emerge in endlessly, adulteration is serious. Because of its main ingredients are similar in collagen amino acid composition, existing methods are difficult to identify effectively.Chinese pharmacopoeia 2015 uses HPLC-MS to establish an identification of trypsin digested Asini Corii Colla,it can effectively solve the problem of adulteration of Asini Corii Colla, preliminary but still exsists standardization problems, so stable and effective identification and quality control method is very necessary. So the main work is as follows:1.Review on the Quality Evaluation of Asini Corii CollaQuality Evaluation of Asini Corii Colla mainly based on physical and chemical properties.Using kinematic viscosity method, thin layer chromatography, HPLC fingerprint method, spectral method, electrophoresis method to identify and evaluate its quality.Chinese pharmacopoeia 2015 using mass spectrometry,as the latest methods,to identify the Asini Corii Colla.2.Pretreatment conditions of collagen enzyme solution of Asini Corii CollaBy querying the NCBI databases, we found the amino acid sequence of collagen of donkeys, cattle, sheep.By using DNAMAN 6.0 to BLAST the collagen alpha-1 type I chain,we found more than 95% similarity.Then use ExPASy PeptideMass software system for Trypsin enzymolysis,enzymolysis results show that there are 7 specific peptides in Asini Corii Colla, mass-to-charge ratio are 3647.7804,2140.0417,2073.0107,1792.9340, 1752.9027,1100.4966,1752.9027.we can consider them as the specific peptides to identify Asini Corii Colla after verification.Dissolved by heating method and extraction system is methylene chloride-hexane and ultrafiltration purification processing samples of Asini Corii Colla.To verify the method of Chinese pharmacopoeia 2015, meanwhile,optimize the enzyme solution.The optimized enzymolysis conditions is:temperature 38 ℃, pH range of 7.5～8.0, enzymolysis time more than 12 h, ratio of object and enzyme is 4:1-1:1 (250 u/mg Trypsin), thus collagen enzymolysis method is established.3.Select the specific peptides of Asini Corii Colla and establish the standard of identificationUsing mass spectrometry to find specific peptides of Asini Corii Colla, the mass-to-charge ratio is 536.7653,765.9070,834.9236,714.3693, and calculate the convolution, it’s confirmed that they are all double charge.According to the results of the previous bioinformatics simulation enzymlysis,1 ions is close to it and the other three molecular weight can be added to the standard of identification until they were validated.Establishment of the specific peptides evaluation method:Separate with C18 chromatographic column (2.1 mm* 100 mm,2.7microns);acetonitrile is mobile phase A, 0.1% formic acid solution is mobile phase B,Elution condition is 0-5 min,5% acetonitrile;5-8 min,5%～10% acetonitrile;8-32 min,10%～20% acetonitrile;32-38 min, 20%～95% acetonitrile.The flow rate of 0.30 mL/min;Column temperature 30 ℃. Sample quantity 1.0μL.Anion (ESI+) model is adopted to improve the detection. Use mass-to-charge ratio (m/z) 536.7653 (double charge),765.9070 (double charge),834.9236 (double charge) to test for the ions of samples, chromatographic retention time should be at the same time to present consistent and kurtosis larger chromatographic peaks.4. Verification of quality evaluation of the specific peptidesExtract ions m/z 536.7653,765.9070,834.9236 can effectively identify the collected 11 Asini Corii Colla samples, and has the same identification ability which Chinese pharmacopoeia 2015 mentioned in the extract ion of m/z539.8, according to the m/z 539.8 as target identification No.1,2,5 are not fake. The preliminary authentication scheme can effectively identify the collected 11 Asini Corii Colla samples.11 groups of samples, only 3 companies’ production meet the criterion, the other 8 companies’ production have different degree of adulteration.更多还原