人参蛋白活性研究

【作者】 李红艳；

【导师】 赵大庆；

【作者基本信息】 长春中医药大学 ， 中药学， 2010， 博士

【摘要】 人参(Panax ginseng C. A. Meyer.)是我国传统名贵中草药,具有极高的药用价值和广泛的药理作用。本论文主要通过现代分离纯化技术、分析鉴定技术及多水平活性检测技术对人参中研究较少但活性多样的生物大分子-蛋白及多肽进行生物特性研究,为优化人参的炮制加工工艺,揭示人参的临床物质基础,开发创新药物、保健食品等系列产品提供科学依据及技术支持。1、采用PAGE电泳胶内酶活染色方法,对园参(鲜参、生晒参)、山参及林下参中氧化还原酶、水解酶、转移酶、裂解酶、异构酶等5种酶系共22种酶进行活性研究。结果表明,四种参中共有13种酶具有清晰酶活性谱带,其中,鲜参11种,生晒参8种,山参9种,林下参7种。过氧化物酶(POD)、过氧化氢酶(CAT)、苹果酸脱氢酶(MDH)、超氧化物歧化酶(SOD)、酯酶(EST)、酸性磷酸酶(ACP)、淀粉酶(AMY)7种酶为四种参共有,乳酸脱氢酶(LDH)、细胞色素氧化酶(CYT)、谷草转氨酶(GOT)3种酶仅为鲜参所有,磷酸葡萄糖变位酶(PGM)和葡萄糖磷酸异构酶(GPI)仅为山参所有,磷酸葡萄糖脱氢酶(PGD)为生晒参和鲜参共有。四种参酶活性的差别,提示人参因栽培方式、炮制方法等的不同,存在一定质量差异,这为人参的品种选择及临床药用起到一定的借鉴作用。2、选取园参、山参及林下参中活性均较高的7种酶即POD、CAT、SOD、MDH(氧化还原酶类)和EST、ACP、AMY(水解酶类),针对不同产地(10个产地)、不同部位(芦头、主根、须根)及不同年限(4年、5年)的园参(鲜参),进行酶活力测定。结果表明,不同产地及不同部位人参各种酶活力均有较大差异;不同年限人参各种酶活力虽有一定差异,但差别不大。人参不同产地间的酶活力差异,说明人参因栽培地区不同,存在一定的质量差异,这为人参的规范化种植提供一定科学依据;人参不同部位酶活力的差异,说明各种酶在参体内的分布不同,同时说明芦头、主根及须根均具有不可忽视的作用,这为人参的整体入药及临床筛选奠定了一定的理论基础;4年及5年生人参酶活力差别不大,说明人参采收年限4年和5年均可。3、采用中性缓冲液浸提、硫酸铵分级沉淀、等电点沉淀、SP离子交换柱层析、DEAE离子交换柱层析、G-75凝胶过滤柱层析、高效凝胶过滤色谱(HPGFC)及基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)等方法,对园参(鲜参)中活力较高的SOD进行分离纯化,最终得到分子量为31 kDa的人参SOD二聚体。经改良的邻苯三酚自氧化法测定,其酶活力为9480.43 U/mg,纯化倍数为308.51倍。敏感性试验表明,该酶对H2O2、CHCl3-EtOH、Urea、NaN3等变性剂较敏感;稳定性试验表明,该酶70℃以下具有很好的热稳定性,pH4-9范围内具有很好的pH稳定性;紫外-可见光谱测定,该酶最大吸收峰在278 nm。人参SOD的纯化与性质研究,为人参抗衰老的临床药用研究起到一定的指导作用。4、采用中性缓冲液浸提、中空纤维膜过滤(截留分子量10 kDa及2 kDa)、pH 5.0缓冲液透析、Sephadex G-75凝胶柱层析等方法,对园参(生晒参)中具有明显促细胞增殖作用的蛋白进行分离纯化,并采用SDS-PAGE、HPGFC、MALDI-TOF-MS等技术对其进行分子量测定及纯度鉴定,最终得到分子量为8000 Da的多肽(人参生长肽)。将该肽作用于小鼠胚胎成纤维细胞(3T3)、人胚肺二倍体细胞(2BS)及人喉癌细胞(Hep-2),并将其细胞活性与其他人参蛋白活性进行比较,结果表明,该肽具有明显的促进3T3细胞增殖作用,提示人参在维持组织结构和参与损伤修复以及预防或治疗心脏疾病等方面具有不可忽视的作用。5、采用SPF级ICR雄性小鼠进行人参蛋白毒理学检验。通过测定体重及脏器重量等进行不良反应试验,结果表明,人参蛋白对雄性幼年小鼠体重及脏器无明显影响,但具一定的肝脏刺激性,并有促进雄性器官生长的作用;通过经口最大剂量给药,进行急性毒性试验,结果表明,最大给药剂量下各受试小鼠活动正常,无中毒症状,说明人参蛋白安全、无毒。人参蛋白的安全性实验,为人参蛋白的产品开发提供了科学依据,为人参蛋白的功能学研究奠定了实验基础。6、采用昆明种清洁级小鼠进行人参蛋白功能学检验。(1)通过二硝基氟苯(DNFB)致小鼠迟发性超敏(DTH)反应及小鼠碳廓清试验,进行人参蛋白增强免疫力功能检验,结果表明,小鼠耳肿胀度及吞噬指数明显增加,说明人参蛋白具有强免疫功能的作用。(2)通过耐常压缺氧及常温游泳试验,进行人参蛋白提高缺氧耐受力及缓解体力疲劳功能检验,结果表明,小鼠耐缺氧及游泳时间明显延长,说明人参蛋白具有增强耐缺氧及抗疲劳功能的作用。(3)通过60Co-γ射线辐射小鼠试验,进行人参蛋白对辐射危害的辅助保护功能检验,结果表明,小鼠外周血白细胞数明显增加,骨髓细胞微核数明显减少,说明人参蛋白具有增强抗辐射功能的作用;通过角叉菜胶致小鼠非特异性急性炎症反应试验,进行人参蛋白缓解炎症功能检验,结果表明,小鼠足趾肿胀度无明显降低,说明人参蛋白不具有明显的抗炎作用。(4)通过醋酸致小鼠扭体试验及热板致小鼠舔足试验,进行人参蛋白缓解疼痛功能检验,结果表明,小鼠出现扭体时间明显延长,舔足次数明显减少,说明人参蛋白具有一定的镇痛作用。(5)通过测定辐射小鼠及正常小鼠灌服人参蛋白后SOD活性及丙二醛(MDA)含量,进行人参蛋白抗氧化功能检验,结果表明,SOD活性升高、丙二醛(MDA)含量降低,说明人参蛋白具有提高抗氧化功能的作用。综上所述,本论文主要针对人参中的大分子成分—蛋白质进行研究。通过对不同来源人参进行同工酶活力比较,为人参的种质资源鉴定提供酶学依据;通过对人参蛋白进行毒理学与功能学研究,为人参蛋白类药物的开发及应用提供药理学依据。另外,本论文首次比较系统的从人参中分离得到SOD及生长肽纯品,并分别进行理化性质与活性研究,为人参中具有生物活性蛋白的纯化与鉴定奠定了理论与实验基础。更多还原

【Abstract】 Ginseng(Panax ginseng C. A. Meyer.) is a traditional Chinese herbal medicines and it has high medicinal value and wide range of pharmacological effects. In this study, we conducted experiments on macromolecules of which had varied activities while not being further studied. The biological activity of these macromolecules-namely ginseng proteins and peptides, had been tested via modern separation and purification technology, analysis and identification technology and detection of activity in vitro and in vivo technology in order to provide scientific evidences and technical support for optimizing the process of preparing ginseng, exposing the clinical material base of ginseng and developing a series of ginseng products including innovative drug and health foods etc.1、Using in-gel activity staining method of PAGE electrophoresis to study the activity of 22 kinds of isozyme among 5 different enzyme systems consisting of oxidoreductase system, hydrolase system, transferase system, catenase system and isomerase system in wild ginseng, ginseng in forest and cultivated ginseng. Results show clear isoenzymes bands of 13 kinds, which participate in 11 kinds of fresh ginseng, 8 kinds of dry ginseng, 9 kinds of wild ginseng, 8 kinds of ginseng in forest. The peroxydase(POD), catalase(CAT), malic dehydrogenase (MDH),Superoxide dismutase (SOD), Esterase (EST), acid phosphatase (ACP) and Amylase (AMY) in all ginsengs; lactate dehydrogenase (LDH), cytochrome oxidase (CYT) and glutamic oxaloacetic transaminase (GOT) only in fresh ginseng; phosphoglucomutase (PGM) and glucose-6-phosphate isomerase (GPI) only in wild ginseng; 6-phosphogluconate dehydrogenase (PGD) in fresh and dry ginseng. Differences in enzyme activity of four kinds of ginseng suggest that there are some quality differences in ginseng because of differences of cultivation methods, processing methods, which play a certain reference of the selection of species of ginseng and clinical application.2、Using colorimetric technology of activity detection to test the activity of POD, CAT, SOD, MDH(oxidoreductase system) and EST, ACP, AMY (hydrolase system) which of them appeared to be high in activity in ginseng. Then to make comparison on enzyme activity of ginseng from different sources(10 sources in Jilin province ), with different parts (rhizome、taproot and fibrous root) and of different ages (4 years and 5 years) , respectively. It turned out that ginseng from different sources and with different parts did show a great difference in enzyme activity among each group. And ginseng of different ages had but not so much difference in enzyme ability. Differences in enzyme activity of ginseng from different origins suggest that there are some quality differences because of cultivation regional difference, which provide certain scientific reference for ginseng standardized planting; Differences of the enzyme activity in ginseng different parts show that various enzymes in the body of the distribution are different ,and the role of reed head, main root and fibrous roots can not be ignored, which has laid a theoretical foundation for medicine and clinical screening of the ginsengs; Differences of enzyme activity in 4 and 5 years old ginseng, show that ginseng can be harvested in 4 years or 5years.3、Using method of neutral buffer extraction and ammonium sulfate fractionation to extract crude enzyme from ginseng and then using isoelectric point precipitation, SP ion-exchange chromatography, DEAE ion-exchange chromatography, G-75 gel filtration column chromatography, high performance gel filtration chromatography(HPGFC) and matrix-asisted laser desorption ionization/time of flight mass spectrometry(MALDI-TOF-MS) to further isolate and purify to get a homodimer of SOD with molecule weight of 31 kDa. The activity of the enzyme tested by improved Pyrogallol autoxidation method appeared to be 9480.43 U/mg, inferring that the purification factor was 308.51. The enzyme was sensitive to detergent as H2O2、CHCl3-EtOH、Urea、NaN3 etc and exhibited high thermo stability (70℃) over the pH range from 4.0 to 9.0. Its maximum absorption wavelength was 278 nm and it was sensitive to hydrogen peroxide. The studies of purification and characterization of ginseng, play a guiding role for clinical medicinal research of ginseng antiaging.4、Ginseng protein which was extracted by neutral pH buffer and hollow fiber membrane filtration method had been isolated and purified via acidic buffer(pH 5.0) dialysis、hollow fiber membrane ultrafiltration(10 kDa and 2 kDa) and sephadex G-75 gel-filtration chromatography. Then conducting the determination of molecule weight and identification of purification on SDS-PAGE、HPGFC and MALDI-TOF-MS, resulting in a polypeptide(ginseng growth peptide) with molecule weight of 8000Da. Comparing the functional activity on mouse embryonic fibroblasts cells (3T3)、human embryo lung diploid cells (2BS) and human laryngeal carcinoma cells (Hep-2) of this polypeptide with other ginseng proteins (proteinⅠ,Ⅱ, andⅢ), leading to the result that this polypeptide did best in promoting the growth of normal cells. The purification of ginseng growth peptide and significantly promoting the proliferation of 3T3 cells, suggest that the role of ginseng in maintaining the organizational structure and participation in damage repair and prevent or treat heart disease can not be ignored.5、To conduct toxicology tests of ginseng protein on ICR male mouse of SPF level. The results of adverse reaction test suggested that though having certain stimulating effects on liver, ginseng protein either had no obvious influences on the weight and visceral organs of juvenile male mice or promote the growth of Male genitals. Furthermore acute toxicity test showed that mice of maximum dose acted like normal mice without poisoning, indicating that ginseng protein was safe and non-toxicity. The safety experiments of ginseng protein provided a scientific basis for product development of ginseng protein, and layed an experimental foundation of the functional studies of ginseng protein.6、To conduct function tests of ginseng protein on Kunming mice of cleaning level. The results of dinitrofluorobenzene (DNFB) induced delayed-type hypersensitivity (DTH) in mice and mice carbon clearance experiment suggested that the ear swelling degree and phagocytic index had largely increased, informing of the immune enhancing function of ginseng protein; Swimming test at room temperature and hypoxia tolerance test showed that ginseng protein had an ability in enhancing hypoxia function and alleviating physical fatigue because of the prolongation of swimming and hypoxia tolerant time. The radiation of 60Co-γray on mice greatly increased the number of peripheral blood leucocyte and sharply decreased the number of bone marrow micronucleus in mice, informing of anti-radiation function of ginseng. The experiment of carrageenan-induced non-specific acute inflammation in mice showed decreasing trend in mouse toe swelling degree with no statistical significance, indicating that ginseng protein had no obvious anti-inflammatory effects. Acetic acid-induced writhing test and pain threshold measuring test by hot-plate in mice showed that the writhing time of mice had been greatly prolonged and the number of paw-licking had decreased, indicating that ginseng protein had certain effect on easing pain. In addition, our studies also found that after being fed with ginseng protein, normal mice and the radiated ones showed higher SOD activity on the contrary to lower MDA concentration, proving that ginseng protein had the ability in promoting antioxidant function.In summary, The research focuses on bioactive macromolecules of ginseng protein. According to compare with the activity of isozyme from different sources of ginsengs, provides enzymology basis for germplasm resource identification of ginseng. According to function and toxicology testing of ginseng protein, provide a pharmacological basis for the development and applications of ginseng protein drugs. In addition, this research systematically isolated and purified SOD and growth peptide from ginseng, and carried out physical and chemical properties and active research respectively, which layed a theoretical and experimental foundation for purification and identification of the biological activity protein of ginseng.更多还原