【作者】 马美湖；

【导师】 谭济才；

【作者基本信息】 湖南农业大学 ， 茶学， 2005， 博士

【摘要】 本研究旨在:①进一步探讨茶多酚与茶氨酸的功能,组成以茶多酚、茶氨酸为主体的强效复合剂,开展抗菌抑菌、增强机体抵抗力、抗瘤抑瘤以及抗（降）血脂的实验研究;②通过实验研究,为茶氨酸、茶多酚的生理功能科学应用提供有效的基础数据;③利用其抗菌抑菌和增强动物机体免疫力的功能,为开发安全无公害纯天然植物饲料添加剂,推进无公害畜产品生产,提供科学的基础依据;④为开发以茶多酚和茶氨酸为主,具有增强免疫力和抵抗力、抗瘤抑瘤、抗（降）血脂的药品、功能性食品提供实验依据;⑤为寻求天然植物成分复合剂抗“SARS”、“禽流感”等人畜共患的病毒、细菌性流行病提供实验依据。 首先利用7种常见、易得、具有类似功能的植物提取物成分,进行茶多酚、茶氨酸功能增效物的筛选L₈（2⁷）的体外抗菌实验。结果显示,鱼腥草素、牛至油同茶多酚、茶氨酸配合抗菌抑菌增效效果较好,形成了茶多酚复合剂。茶多酚复合剂分别对Colibacillus、Ssalmonella、Staphylococcus aureus抑菌效果及有效浓度L₉（3⁴）配合的实验,得出对Colibacillus、Salmonella和Staphylococcus aureus最低抑菌浓度（MIC）分别为1.25、0.625 0.625mg·ml^-1,最低杀菌浓度（MBC）为2.5、1.25、1.25mg·ml^-1;对Colibacillus、Salmonella、Staphylococcus aureus作用时间与抑菌率（IBR）的关系发现均随时间和浓度的增加提高,并发现了试物同细菌作用的僵持期（4～8h）;小鼠体内抑菌实验表明,在感染Colibacillus和Staphylococcus aureus之前18h灌喂茶多酚复合剂,死亡率（72h）分别为40%、30%,感染后1h灌喂死亡率（72h）分别为70%、60%,同对照组比差异极显著（p<0.01）和显著（p<0.05）。茶多酚复合剂有很好的体内抗菌效果。 采用0、50、100、150mg·kg^-1d^-1BW^-1茶多酚复合剂灌喂,增强小鼠免疫功能机理的实验,设阴性对照组（C₀）和低（C₁）、中（C₂）、高（C₃）4个剂量组,每组10～15只小鼠,预饲7d,实验期30～45d。末次灌喂24h后分别测定小鼠细胞免疫功能、体液免疫功能、非特异性免疫等指标。结果表明:茶多酚复合剂明显刺激脾淋巴细胞增殖和转化,各组的0D差值均高于C₀组,有显著或极显著性差异更多还原

【Abstract】 The research was focused on the further studies of the function of tea-polyphenols、 theane, r-glutamylethylamide, and the experimental studies of antibiosis and bacteriostasis、 improving the resistance of organism、 anticancer and reducing the blood fat, based on the compound of tea-polyphenols, theane,r-glutamylethylamide, which mainly consists of the two substances. Through the experiment, the basic effective data for the scientifical application of the physiological function of tea-polyphenols and theane,r-glutamylethylamide were provided; the scientific basic thereunder was offered for the safety and non-hazard natural plant feed additive and the production of non-hazard animal product by the utilization of the function of its antibiosis and increasing the physiological immunity; the experimental gist was given for the medicine and functional health protection food which can enhance immunity and resistance , resist cancer and tumor、 reducing blood fat; the scientifical foundation was provided for searching some natural plant composition which can resist the virus of some epidemic such as"SARS"、 "bird flu" etc..The compound consisting mainly of Tea-polyphenols and theane,r-glutamylethylamide has been gotten by the L₈ （2⁷） vitro experiment for the filtration of synergy of Tea-polyphenols、 theane,r-glutamylethylamide using the effect of seven kinds of materials with similar function extracted from plant to Colibacillus、 Ssalmonella、 Staphylococcus aureus （namely TP compound）. Then develop the L₉ （3⁴） experiments of the effect of antibiosis TP compound to Colibacillus、Ssalmonella、Staphylococcus aureus respectively and the valuable concentration, and measure the minimum immunity concentration （MIC）, minimum sterilization concentration （MBC）; in order to assure the animal intestine experiments successful, the experiment of the pre-test of dosage of and the effect of resisting by TP compound Colibacillus、 Staphylococcus aureus in rat intestine were adopted. The result is: Houttuyninum , Origanum Vulgare L ,Tea-polyphenols, theane,r-glutamylethylamide cooperating together can enhance the effect of antibiosis . MIC of the compound to Colibacillus, Salmonella and Staphylococcus aureu is 1.25 > 0.625 0.625 mg ? ml"1, MBCis2.5 , 1.25 , 1.25mg?mrS The relation of acting time of TP compound to Colibacillus > Salmonella, Staphylococcus aureus and IBR is increasing by the increasing of time and concentration. 18h before infected by Colibacillus and Staphylococcus aureus, the rats were fed by TP compound （preventing group）, the death rate （ 72h ） was 40 % , 30% respectively , 70%, 60% respectively fed infected for lh （treating group）, but 100% in physiological saline group. Preventing group,treating group compared to comparison group ,the differentia is very notable （p<0.01） and notable （p<0.05）? TP compound had good effect on intestine antibiosis.The experiments of increasing the immunity function of rats were adopted ,using 0, 50, 100, 150 mg-kg’Id’1BW^ITTP compound to feed, respectively arrange the negative comparison group（Co） and low （Q） , mid （C2） , high （C3） 4groups, every group has 10 15 rats, fed for 7d, experimental period 30⁴5d, feeding TP compound 0.4 ml-one’Id’1? 24h after the latest feed the index,such as the transfer of lymphocyte, the immunity function of cell and body fluid , the function of monokaryon macrophage,the bioactivity of NK cell and so on, had been measured .the result is : The OD difference value that the transfer ability of lymphocyte induced by ConA of comparison group was lower than other groups. It was a very notable（P<0.05） between C2 and Co, notable（P<0.01） between C3 and Co.TP compound has strong effect that it can stimulate the increasing and transfer of lymphocyte ; according to the later abnormal experiment induced by DNFB, the left and right ear difference weight of the grouping rats all were bigger than that of group Co, among it, the differentia is very notable in group C2 and group C0（P<0.05）,notable （PO.Ol）in group C3 and group .having the effect of accelerating abnormal reaction greatly; the cell number of the rat antibody in group Co were also lower than other groups, group Ci（50mg ? kg-1 ? d-1 ? BW-1） compared with group Co was notable（P<0.05）, group C2 （100 mg ? kg-1 ? d-1 ? BW-1） compared with group C3 （150 mg ? kg-1 ? d-1 ? BW-1） and group Co was very notable （PO.01）, having function of the enlargement of the cell promoted by IgM antibody, so it is very important to enhance the physiological immunity; the rat blood serum hemolysin （mainly include IgG, IgM）were measured, its volume average of group Co was notable（P<0.05）compared with group Ci and very notable （PO.01） compared with group C3 .it indicated that TP compound can increase the rat blood serum hemolysin, namely IgG, IgM, especially IgM; the experiments that the swallow index of the rat groups Co was higher than group were notable（P<0.05）, indicating the material tested have the affect of increasing the function of rat chest> spleen and the monokaryon macrophage; through the experiments of CRBC, the swallow index of monokaryon macrophage to CBRC was above group Co （PO.05）, indicating it can increase the function of normal immunity apparatus such as chest , spleen and the rat normal immunity; the experiment of the bioactivity of NK cell indicated that TP compound had notable effect to this cell （PO.05 or PO.01）, the compound can effectively protect the cytolemma of immunity cell against less damage .control the effusion of LDH, enhance the bioactivity of NK cell; the rat blood serum lysozyme was measured that the group Co was notable or very notable compared with other groups （P<0. 05） （P<0. 01） .The experiment of anticancer effect of TP compound was used to measure the resistance ratio of vitro anticancer, treated for 48h, the anticancer ratio of low, mid, high concentration group was respectively up to 35.92%, 42.88%, 53.85%, notable （PO.05 or PO.01）, treated for 72h, the anticancer ratio of low, mid, high concentration group was respectively up to 41.29%, 46.58%, 61.63%, all had relation with concentration; rat physiological experiments indicated that TP compound had the ability to reduce the weight of tumor greatly （PO.01）, the ratio of anticancer was up to 26%, very notable（P0.01 ）<>using the observation of HE coloration slice up, the tumor cell of tested group appeared different extent shape changing, royal purple colored in cell nucleus, peachblow colored in cytoplasm, and cell arrange tightly , show the character of apoptosis of tumor cell; carried the DNA gel electrophoresis of tumor cell, the tested group appear the trapezium band clearly and important character of apoptosis of tumor celloThe study on anti-Sigo cancer mechanism of TP compound indicated that the S180G1 cell increased from 51.1%（0 u g ?ml-l ）to 80.5%（150 u g ?ml-l ）by TP compound, the proportion of S and G2M cell was reduced, so was index of cell apart （PI）, cell reproduction would weaken generally. Followed by the concentration of TP compound increasing, the ratio of apoptosis of S180 tumor cell increased induced by TP compound from5.9% （0 u g ? ml-l） to 48.2% （150 u g *ml-l）; especially the concentration of 50 u g *ml-l, up to very notable （PO.01）; the expressof SI80 tumor cell PTEN protein was raised greatly by TP compound, respectively increased from 35.61+0.48 （Oug-ml-1） to 141.79+3.01 （150u g ? ml-1）, up to notable level （PO.05, PO.01）, strongly reducing the snippet extent of DNA worked by H2O2（P<0.01）, resisting the reproduction of S^o tumor cell, making the cell stay at Gl/S point, and inducing apoptosis of cell; the effect of resistance express was good according to the Si go gene, such as c-myc, Bcl-2, P53> c-jun> c-fos etc.. induced by different concentration TP compound to H2O2 （0.4mmol ? L-l） . Designing five groups healthy male SD rat, fed for 42d and 70d, carry the experiment of anti-blood fat effect of TP compound, liver index （LI） -. total cholesterin （TC） and three glyceride（TG） were measured respectively and the devision and colority of liver, the result was: between the high fat model group （the second group）and high fat tested group（the third group）,the differentia of LI was very notable （PO.01）, between the second group and the third group, the differentia was notable （PO.05 ） by measuring the TC% TG and FFA . through the observation of liver tissue, in normal comparison group（the first group） the liver was red, tight tissue, side-sharp, surface smooth, and appearance tumefied -> side-round > sunking if pressed, snuff color and white point on surface in the second group.and the third group the soak status of liver fat alleviated, through the micro-observation of the liver tissue, it was integrity of liver leves , polygon of the liver cell, radiation around the centre, sinus hepaticus clear, hepatic cord orderly, vein among sinus hepaticus clear in the first group; and in the second group the construct of sinus hepaticus was destroyed, diffusible denaturalization of liver cell, liver cell nucleus was pressed to aside by fatty drop. The centre vein well invaded by inflammation cell around, sinus hepaticus open; in the third group the fatty liver cell reduced, hepatic cord arranged tightly, the region joined by vessel clear, so was with the liver leaves .The study on rat physiological mechanism affected by TP compound indicated that LI, TC> TG in blood serum ■> high density lipoprotein- cholesterin （HDL-C） > low density lipoprotein -cholesterin (LDL-CX very low density lipoprotein cholesterin（VIDL-C）high fat model group（the second group） compared to normal comparison group （the first group）, the differentia was notable or very notable （P < 0.05. P < 0.01）, high fat tested group （the third group） compared to the first group ,the differentia was not notable （P>0.05）, the second group compared to the third group, the differentia was notable or very notable （P<0.05, P<0.01）; three types of enzyme ofembodying the function of liver metabolism in blood serum and liver damage were measured, Glutamic oxaloacetic transaminase （GOT）between the second group and the third group-. Alkaline phosphataes（ ALP） the differentia was notable or very notable （P<0.05, P<0.01）, although the differentia of Glutamic pyruvic transaminase （GPT） was not notable （P > 0.05）, but the enzyme was lower clearly in the third group ; the second group was highest on the index of atherosclerosis （AS）, coronary heart disease（R-CHD）than the other group, among 1 > 2,3 group ,the differentia was notable or very notable （P < 0.05, P < 0.01）; the second group also was highest on free fatty acid （FFA） in rat liver, propylene glycol (MDAX TC, TG and VIDL-C, among 1, 2, 3group, the differentia was notable or very notable （P < 0.05, P < 0.01）; the mechanism of cell molecule also was measured, the result was :if the concentration of TP compound was 150-100mg ? L-l,the nucleus shift ratio of NF- k B p65 was under Omg ? L-l （PO.05）; if the concentration of TP compound was ranged in 50-100mg ? L-l, the index of positive area of TGF- 3 lmRNA hybridization of the cell in all groups was under Omg ? L-l （PO.05）, if the concentration of TP compound was ranged in 100 150mg ? L-l, the differentia of the effect to cholesterin in THP-1 cell was up to be notable （PO.05） or very notable （PO.Ol）o all results measured of above indicated that TP compound can promote the metabolism and transfer of cholesterin and the, accelerate the metabolism and transfer of liver fat , speed the oxidative decomposition of FFA, reduce the liver cell demage from FFA and provent the injury of liver.更多还原