名贵中药冬虫夏草品种及蛋白组分研究

【作者】 任艳；

【导师】 卢先明； 万德光；

【作者基本信息】 成都中医药大学 ， 中药学， 2013， 博士

【摘要】 冬虫夏草是一味来源独特、疗效显著的名贵中药,在我国具有悠久的药用历史,具有“补肾益肺、止血化痰”的功效,主要用于治疗久咳虚喘、阳痿遗精、劳嗽咯血、腰膝酸软等肺肾疾病。自18世纪（1785年）,英国真菌学家Dickson首次将其带回西方,冬虫夏草这味神奇的中药从此走出国门,走向世界,也打开了冬虫夏草现代研究的大门。现代药学研究证明,冬虫夏草富含多种化学成分,其补益作用主要表现在对机体免疫调节方面有着独特疗效。随着人们对亚健康越来越重视,冬虫夏草这味性味温和的天然补益药市场需求日益增大,而其年产量有限,人工培育等替代品研究尚不成熟。其价格昂贵,逐年升高,市场出现了大量的掺伪品、混淆品,并且流通渠道较为混乱、不规范。而《中国药典》对冬虫夏草品种定义存在值得商榷的地方,对其质量控制标准仅以在虫生真菌内广泛存在的腺苷作为指标性成分,难以有效地、有针对性的鉴别真伪并进行质量评价。蛋白类成分是冬虫夏草化学成分的重要组成部分,蛋白质本身就是品种遗传信启、在细胞水平的表达,并且通常具有良好的生物活性,蛋白组学是目前国内外研究热点,特别是其在差异性蛋白表达、功能性蛋白筛选方面具有独特的优势,而一直以来对冬虫夏草蛋白成分的研究较为滞后,相关报道极少。鉴于此,本课题对冬虫夏草开展了以下几方面的研究工作：1、正品冬虫夏草品种研究通过对冬虫夏草菌体、虫体的基原品种考证,阐述了冬虫夏草菌的分类学研究进展。根据分子系统学研究最新进展,在2007年,冬虫夏草的有性型及全型已归类为子囊菌纲、线形虫草科、线性虫草属Ophiocordyceps sinensis （Berk.）G. H. Sung, J.M.Sung, Hywel-Jones&Spatafora.,简写为Ophiocordyceps sinensis （Berk.） G.H. Sung et al.,建议药典对菌的基原物种进行更新。通过对冬虫夏草虫体基原品种考证发现,目前冬虫夏草虫体品种为59种,大多数学者公认冬虫夏草菌的寄主昆虫集中在Hepialidae蝠蛾属Hepialus田首次对中药冬虫夏草的命名进行系统的研究,对历版中国药典及相关文献进行分析,表明“冬虫夏草”作为药材中文名比较客观、合适,但专属性不强；中药冬虫夏草应与冬虫夏草菌区别,其药材的拉丁名不应直接等同于冬虫夏草菌的属名,建议结合虫体部位与冬虫夏草菌最新学名进行药材命名：Ophiocordyceps Hepialus。2、冬虫夏草蛋白质2-DE方法优化与特征性蛋白的鉴定在课题组前期研究的基础上,本论文进一步对适用于冬虫夏草蛋白2-DE方法进行考察,得到了最佳方法条件：（1）上样量600μg（2）被动水化方式（3）搭建盐桥进行深度除盐；重现性研究结果显示该方法对同一产地同一批次样品的匹配率达到89.5%,表明该方法稳定可靠。首次对冬虫夏草2-DE过程中蛋白含量测定方法进行研究,确定了Bradford法是作为冬虫夏草可溶性蛋白检测的最适方法。通过研究了不同产地冬虫夏草蛋白表达图谱的差异性、共同性,找出了特征性蛋白点12个,并对其中的5个进行了初步的结构鉴定,结构鉴定结果为：乙酰乳酸合成酶小亚基类、转录修复偶联因子类、假设蛋白Mnod₇043类、FMN依赖的a-羟基酸脱氢酶类、铁硫氧化还原酶类蛋白类。首次对不同产地冬虫夏草进行多样性分析（差异性蛋白、共有蛋白）,结果表明不同产地正品冬虫夏草蛋白种类差异较大,结合前面品种研究,认为冬虫夏草蛋白的多样性与不同产地优势冬虫夏草菌寄主昆虫的种类有关。这为后续通过冬虫夏草特征性蛋白的对不同冬虫夏草产地溯源奠定了基础：同时提出通过寻找不同来源正品中药的共有蛋白来建立合适的特征性蛋白质表,对多基原中药以及复合物中药质量评价研究提供一条参考思路。3、首次对冬虫夏草中免疫活性蛋白进行研究利用酶联免疫法测定药物对巨噬细胞分泌TNF-a、IL-1、IL-12因子的影响,对冬虫夏草蛋白组分的免疫活性进行研究,优化了体外免疫活性检测的方法：分离得到小鼠腹腔巨噬细胞,培养3h细胞贴壁后给药,LPS（作为后期实验的阳性对照）给药终浓度为10μg·ml-1,给药9h后终止反应,弃掉营养培养基,更换双无培养基,细胞分泌36h后离心,取出上清液即得分别含有TNF-α、IL-1、 IL-12因子的待测样品。试验应在小鼠腹腔巨噬细胞提取纯化后72h内完成。首次对冬虫夏草蛋白组分的稳定性进行了研究,筛选出适合的蛋白组分保存条件：缓冲液PB（pH=7.2,0.45mmol·L-1DTT）作为冬虫夏草蛋白保存液,保存温度为-20℃,保存时间10d。首次通过结合免疫活性跟踪,对冬虫夏草蛋白进行超滤分离,得到4个分子量段,超滤效果较好。免疫活性检测结果表明,冬虫夏草蛋白总提物以及各分子量段蛋白组分对巨噬细胞免疫系统总体表现出多方面的双向调节,包括（1）功能性的双向调节,即不同分子量段对正常巨噬细胞具有一定促进、抑制作用或作用不明显：对免疫状态低下的细胞（β-巯基乙醇造模损伤）,能显著的促进免疫因子的分泌接近正常水平或具有免疫促进作用的的趋势；对免疫状态过度旺盛的细胞（LPS刺激）,能显著的抑制免疫因子的分泌接近正常水平或具有免疫抑制作用的趋势；（2）剂量性双向调节,不同分子量段蛋白样品对免疫因子的调节,没有呈显著的线性关系,但总体上、在一定浓度范围内表现为高浓度促进免疫、低浓度抑制免疫的双向调节作用（3）不同分子量趋势性双向调节,虽然不同分子量段蛋白组分对不同免疫因子表现为不同的免疫调节作用,总体上分析发现,大分子量蛋白组分主要表现为免疫促进作用,随着分子量的降低,免疫抑制作用越明显。通过凝胶层析法对冬虫夏草蛋白组分进行了初步分离,得到了表现为免疫抑制作用、活性较强、重现性好、蛋白种类较为单一、分子量在14.4-20.1kDa的蛋白组分III,为后期活性蛋白的高通量筛选提供依据。通过对冬虫夏草特征性蛋白与活性蛋白的研究,即从鉴别成分与有效成分的角度,对冬虫夏草的质量评价的研究进行了探索,为后期指标性蛋白或蛋白组分寻找奠定基础,为冬虫夏草质量评价从蛋白组分的角度进行补充完善提供科学依据。更多还原

【Abstract】 Cordyceps is a precious traditional Chinese medicine with significant effect and unique sources.It has a long medicinal history in China with the effect of Tonifying kidney and lung, stoping bleeding phlegm,mainly used in the treatment of chronic cough asthma, impotence, spermatorrhea,Labor cough hemoptysis, lumbar debility, the lung or kidney disease. the18th century （1785）, when British mycologists Dickson brought Cordyceps back to western countries for the first time, this traditional medicine with magic began to be known by the whole world, which also opened the door of modern research about Cordyceps. The modern pharmacology studies have proved that Cordyceps sinensis is rich in a variety of chemical ingredients. Now, its tonic effect mainly clinically manifested of immune regulation with unique effect.With more and more attention to the sub-health, Cordyceps as the tonic has an increasing market deman for its mild nature.But, its annual production is limited,and alternatives such as its artificial cultivation research is still in its crucial stage. Its expensive price, increased year by year. A lot of adulteration products, adulterants, have appeared in the market,and its distribution channels is more chaos, not-standard. The definitions of Cordyceps sinensis varieties in The Chinese Pharmacopoeia are open to question. The quality standards of the Chinese Pharmacopoeia. The index components of Cordyceps in The Chinese Pharmacopoeia is the adenosine class composition that is entomogenous fungi widespread, which is difficult to effectively targeted authenticity and quality evaluation.Protein ingredient is an important part of the chemical composition in Cordyceps.Protein itself is the variety expression of genetic information, and usually has a strong biological activity, proteomics research is the focus of world, especially in differences in protein expression and functional protein screening, which has unique advantages,while the study of Cordyceps protein components has been lagging behind, few research. In view of this, the subject of Cordyceps carried out the research work of the following aspects: 1. Species research of CordycepsResearch base of the original varieties of Cordyceps bacteria, parasites, describes the progress of the study of the taxonomy of Cordyceps sinensis. According to the latest progress of molecular studies in2007. the Teleomorph and Maxi has been classfied as Ascomycetes.Linear Cordyceps Branch,Linear Cordyceps Ophiocordyceps sinensis （Berk.） G. H. Sung, J.M.Sung, Hywel-Jones&Spatafora Abbreviated as Ophiocordyceps sinensis （Berk.） G. H. Sung et al. Propose Pharmacopoeia update the base of the original species of Cordyceps. Through the research of theVarieties of the Cordyceps parasites origin,we found that Cordyceps parasite species has59species, while most scholars have recognized that the host insects of Cordyceps is a Hepialus in Hepialidae Hepialus.More systematic study of Chinese medicine Cordyceps naming and analysis of Calendar Chinese Pharmacopoeia and related literature show that "dongchongxiacao" as a medicinal Chinese name is more objective, appropriate, but not specific.Chinese medicine Cordyceps should be different from Cordyceps fungus, Latin name should not be directly equivalent to the generic name of the Cordyceps fungus.Propose naming medicine material via Combination of the insect body parts and latest scientific name of Cordyceps fungus:Ophiocordyceps Hepialus.2.2-DE method optimization and the identification of the characteristic protein of Cordycepson the basis of Task Force preliminary study, This paper further applicable to Cordyceps protein2-DE method, obtained the best method:（1）.the sample volume of600mg;（2）.passive hydration;（3）.build salt bridge to further demineralization.Reproducibility study results showed that the method of the same batch of samples of the same origin match rate reached89.5%, indicating that the method is reliable。Studies on the determination of protein content in Cordyceps2-DE process showed the Bradford method is optimal as the detection method of Cordyceps soluble protein.Through the research of the difference of different origin cordyceps protein expression profiles and intercommunity,12proteins characteristic point was found, and five of them in the structure of the preliminary appraisal.The structure identified: Acetolactate synthase small subunit of class,Transcription repair coupling factor class,Hypothetical proteins Mnod₇043class,FMN-dependent alpha-hydroxy acid dehydrogenases,Iron-sulfur oxidoreductase protein.Based on analyzing the diversity of different origin cordyceps （Differences in protein, total protein）., results showed that different regions authentic Cordyceps protein species had difference,combining with the variety in front of the study. Speculated that the diversity of Cordeceps protein and different locality advantages cordyceps fungus host insect species..This laid the foundation for the follow-through the Cordyceps characteristic protein on different Cordyceps Origin Traceability. At the same time put forward by finding common proteins of different sources of authentic traditional Chinese medicine, to evaluate the quality of traditional Chinese medicine, which can be a reference for quality evaluation of the traditional Chinese medicine with many origins as well as complex.3. Immunologically active protein of CordycepsBy enzyme-linked immunosorbent assay （ELISA） determined the influence of drugs on macrophage secretion of TNF-a, IL-1, IL-12factor. Research on Cordyceps protein component of the immune activity to optimize the in vitro immune activity detection method:Isolate mouse peritoneal macrophages, Incubate for3h cells adherent, Dosing of LPS （as a positive control for the post-experiment） dosing a final concentration of10μg/ml,Terminate reaction after dosing9h, discard nutrient medium, replace of Antibiotic-free medium.Test should be in mouse peritoneal macrophages,completed within72h after extraction and purificationConducted a study on the stability of the protein component, screened a suitable protein component storage conditions:Buffer PB （pH=7.2,0.45mmol/L DTT） as the Cordyceps protein preservation solution,-20℃for10dThrough a combination of immune activity tracking, ultrafiltration separation Cordyceps protein, got four molecular weight segment.ultrafiltration has better effect.Immune activity test results showed that the total protein extract of Cordyceps and the molecular weight of the protein components had a wide range of bidirectional regulation to immune system macrophages.（1）.Functional bidirectional adjustment,Which was to that protein of different molecular weight on normal macrophages had certain promotion, inhibition, or effect was not obvious;To the low immune status of the cells （3-mercaptoethanol modeling injury）, it can significantly promoted the secretion of immune factors to near normal levels or enhance immune function;To the over-exuberant immune status of the cells （LPS stimulation）, it can significantly inhibited the secretion of immune factors to near normal levels or had a immunosuppressive trend.（2）. Doses of bidirectional regulation. Samples of different molecular weight protein regulation of immune factors, there was not a significant linear relationship. But on the whole, within a certain concentration,there was a two-way regulation in which high concentration promoted immunization,low concentration had an immunosuppression,（3）.Different molecular weight type of bidirectional adjustment.Although the protein components of different molecular weight immunomodulatory effect of different immune factors appeared to be different, on the whole, analysis found, mainly for immunologic enhancement of molecular weight protein components, with lower molecular weight, immunosuppression more obvious.Preliminary separation of protein components of Cordyceps by gel chromatography. Got protein Ⅲ characterized by immune inhibition, activity strong, good reproducibility, types of relatively single, in14.4～20.1kDa protein molecular weight. Which provided the basis for later high-throughput screening of active protein.The research on Cordyceps characteristic protein and active protein, Namely, from the point of the identification of ingredients and active ingredients, explored research on of Cordyceps quality evaluation.which laid the foundation for the later seeking indicators protein or protein components and provided the scientific basis for Cordyceps quality evaluation from proteomics research更多还原