【作者】 丁红秀；

【导师】 高荫榆；

【作者基本信息】 南昌大学 ， 食品科学， 2007， 博士

【摘要】 毛竹（Phylbstachys pubescens）又名“楠竹”、“孟宗竹”,我国毛竹种植总面积达300多万hm²,年产近700000万株,产量占我国所有竹种的70%以上,是我国经济价值最大的竹种。按照每株产2公斤鲜竹叶计算,每年可产毛竹叶1400000公斤,数量惊人。毛竹叶中含有大量的对人体有益的活性物质,目前毛竹叶的利用在国内几为空白,一般作为废弃物抛弃,造成林业资源的巨大浪费。本研究可丰富天然产物资源库,为相关资源开发利用提供具有学术参考价值的文献。利用酶法中试综合提取的毛竹叶粗多糖（CPMBL）为原料,系统地研究毛竹叶多糖的分离纯化、构成及功能活性等。通过组合膜（微滤膜、超滤膜、纳滤膜）对CPMBL进行分离及DEAE—纤维素柱层析纯化;采用平板法进行抗、抑菌活性研究;以活性氧清除率、DPPH法、TBA法等研究PMBL体外抗氧化能力;通过动物实验研究PMBL的体内抗氧化、抗疲劳、降血糖及脂类代谢调节等方面的生理功能。研究结果与分析:1、CPMBL的分离、纯化及构成研究对CPMBL进行分离纯化,获取PMBL纯品进一步研究。实验结果:组合膜分离得到超滤多糖（分子量>6000D）和纳滤多糖（分子量2000～6000D）,纯度分别为37.37%和51.76%;UV检测无明显的蛋白质和核酸特征吸收峰;DEAE-纤维素柱层析分离得到七个洗脱组分;选择其中洗脱峰面积较大的三个,经HPLC测定为高纯度多糖均一组分,相对分子量分别为:20378D、14367D、5932D;定性实验显示三者均含硫酸基,为无蛋白缀合的非淀粉酸性多糖;IC分析其单糖组成,PMBL主要由鼠李糖、阿拉伯糖、甘露糖、半乳糖、葡萄糖、果糖六种单糖等构成;IR分析表明,PMBL是以β—D—吡喃糖为主要糖环结构多糖类,其分子内存在果呋喃糖骨架结构。结果表明:以上方法可行,组合膜具有较好的分离、纯化效果。2、PMBL的抗、抑菌研究以CPMBL和精制PMBL作对比实验,选择革兰氏阳性菌（G⁺）和革兰氏阴性菌（G^-）及真菌为试验菌,考察PMBL的抗、抑菌效果。实验结果:CPMBL液对细菌表现出显著的广谱抗、抑菌效果,对真菌无作用;PMBL无明显的抗菌活性。结果表明:PMBL没有抗、抑菌作用。3、PMBL的体外抗氧化研究通过研究PMBL对活性氧自由基清除率、DPPH清除作用、抗脂质过氧化及抗H₂O₂诱导红细胞溶血作用等,考察PMBL体外抗氧化活性。实验结果:PMBL对O₂^-和·OH均具有较强清除作用,清除能力均优于Vc和芦丁;对DPPH的清除效果良好,IC₅₀为36.08mg/L,弱于Vc和芦丁;对亚油酸脂质过氧化有一定抑制作用;对H₂O₂诱导红细胞溶血有显著抑制作用,浓度为30mg/l时对H₂O₂所诱导的红细胞溶血抑制率为38.119%,强于同浓度Vc和芦丁。结果表明:PMBL体外抗氧化作用主要体现为对活性氧自由基具有显著清除作用,而对有机自由基包括DPPH和脂质过氧化反应中产生的过氧化自由基清除能力相对较弱;体外抗氧化能力存在明显量效关系。4、PMBL对老龄小鼠体内抗氧化研究以正常喂养的老龄小鼠作对照,灌胃PMBL考察其体内抗氧化活性。实验结果:PMBL可以显著增加血清及肝、肾组织等蛋白质含量;降低血清及组织MDA含量;提高小鼠的血清及心、肝、肾组织SOD和GSH-px活力;提高老龄小鼠体内总抗氧化能力（TAOC）;血清、心、肝、肾组织TAOC/MDA比值均显著高于正常对照组。结果表明:PMBL可促进血清及组织蛋白质合成,延缓动物组织及细胞结构及功能衰退;能减少体内MDA生成,提高体内抗氧化系统酶活性,较好地维持老龄小鼠体内抗氧化/氧化平衡状态。5、PMBL的抗疲劳作用研究以正常喂养小鼠组作对照,不同剂量PMBL灌胃,考察其抗疲劳活性。实验结果:PMBL能延长小白鼠负重游泳时间,降低了运动后血尿素氮（BUN）的增量,提高了血清乳酸脱氢酶（LDH）活力,显著增加肌糖元和肝糖元储备量,提高抗疲劳能力。结果表明:PMBL具有缓解体力疲劳作用。6、PMBL的降血糖作用研究通过四氧嘧啶法诱发建立小鼠糖尿病模型,考察PMBL降血糖活性;并研究其对正常小鼠血糖值的影响。实验结果:PMBL可显著降低由四氧嘧啶致糖尿病小白鼠血糖值,缓减糖尿病病鼠消瘦、体重减轻及多饮多食症状;对正常小白鼠的血糖值、体重、摄食量等无不良影响。结果表明:PMBL具有显著的降血糖活性;对正常小白鼠血糖无不良影响。7、PMBL对脂类代谢调节作用考察PMBL对小鼠脂类代谢调节作用:更多还原

【Abstract】 As the highest economically valuable bamboo species,Moso bamboo（Phylbstachys pubescens）is widely planted in china,with an annual production of about 7 billion roots.Despite the considerable presence of various beneficial active components,the leaves of Moso bamboo were generally discarded without effective utilization,which cause a huge waste of forestry resource.This research can enrich the resource reservoir of natural products and offer valuable reference to the development of activated component In this research,we firstly extracted the active crude polysaccharide from Moso bamboo leaves（CPMBL）using enzymic methods.And the isolation and purification technology was systematically studied,as well as the chemical composition and the biological activities of PMBL.CPMBL was isolated by combined membrane technology and purified by DEAE-cellulose column chromatography;The bacteriostatic and antimicrobial effects of PMBL were observed by plate way.The antioxidant effects in vitro of PMBL were invested by studying the scavenging capacities of ROS,1,1-diphenyl 2-picrylhydrazyl（DPPH） and the antioxidant activities on lipids（TBA）,etal.Finally,we investigated several biological functions of PMBL by animal experiment,including bacteriostasis,anti-oxidation,anti-fatigation and decreasing blood sugar and blood fat,respectively.Research results and analysis1.Isolation,purification and composition analysis of PMBLCPMBL was purified and refined PMBL was obtained,further research showed that:two different components were obtained by combined membrane isolation,including ultrafiltrationing polysaccharide solution（UPMBL,M_W>6000D）and nanofiltration polysaccharide solution（NMBL,2000D<M_W<6000D）,Its polysaccharide purity is 37.37%and 51.76%respectively.In UV detection proved that there is no obvious characteristic absorption peaks for either protein or nucleic acid.Seven pure PMBL obtained from UPMBL and NPMBL by DEAE-cellulose column chromatography.with three relatively higher production among them,Its molecular weight and purity were assayed by HPLC.Result show that they can be proved to be composed with high purity polysaccharides,and its molecular weight were UPMBL_W（20378D）、UPMBL₁（14367D）、NPMBL_W（5932D）respectively.which are mainly rhamnose,arabinose,mannose,galactose,glucose and fructose as well.Analysis of infrared spectrum showed that,there is little difference in sugar residue composition between UPMBL_W and NPMBL_W.Experimental results proved that above methods are feasible to isolate and purify the CPMBL in order to get pure PMBL production.2.The antimicrobial activity effect of PMBLThe bacteriostatic and antimicrobial effect of CPMBL and PMBL on gram-positive bacteria（G⁺）and gram-negative bacteria（G^-）were observed.The Mowings are the results: CPMBL has strong bacteriostatic and antimicrobial effects,but PMBL has no obvious antimicrobial activity; Result show that CPMBL has broad - spectrum antimicrobial.and it hasn’t effects on fungi. PMBL hasn’t bacteriostatic and antimicrobial effects3、The antioxidant effect in vitro of PMBLThe antioxidant effects in vitro of PMBL were invested by studying the scavenging capacities of ROS,1,1-diphenyl 2-picrylhydrazyl（DPPH）and the antioxidant activities on lipids using thiobarbituric acid（TBA）method.The followings are the results:the scavenging effect of PMBL on ROS is good,on O₂^<sup>IC₅₀=5.98mg/Land·OH(IC₅₀=5.55mg/L)are stronger than Vc’s(IC₅₀=6.59mg/L)and rutin’s(IC₅₀=17.77mg/L.on DPPH free radical,the scavenging effect of PMBL(IC₅₀=36.08mg/L)is good,but weaker than Vc’s(IC₅₀=21.05mg/L)and rutin’s (IC₅₀=15.88mg).PMBL has some antioxidant activities on linoleic acidThe erythrocyte hemolytic test results show that,PMBL has obvious inhibition to erythrocyte hemolysis caused by H₂O₂,the inhibition rate of 30mg/l PMBL is 38.119%,stronger than Vc’s and rutin’s.and With the concentration increasing,the inhibition is more obvious,which shows that there are obvious dose-effect relationship for PMBL to erythrocyte hemolytic caused by H₂O₂. The results showed that:The antioxidant effects in vitro of PMBL embody mainly that it has significant scavenging effect on oxygen free radical,and has more weak scavenging effect on organic free radical including DPPH,lipid peroxidation et.al;The antioxidant ability in vitro of PMBL has a dose-effect relationship obviously.4.The antioxidant effect of PMBL in vivo on aged miceCompared with normal aged mice,the antioxidant effect in vivo of PMBL on aged mice was studied.The followings are the results:PMBL can significantly increase contents of protein in the serum,liver & kidney,decrease the contents of MDA in the serum and the organs, increase activity of SOD and GSH-Px in the serum,heart,liver,kidney and the total antioxidant competency.The ratio of TAOC/MDA shows that,TAOC/MDA of PMBL group in the serum, heart,liver、kidney is obvious higher than the normal group.The results show that,PMBL can promote portein synthesis in serum and organs,mitigate recession of the tissue and cytoarchitecture,improve the function,balance the state of antioxidation/oxidation.5.antifatigue effects of PMBLCompared with normal mice,antifatigue eifects of PMBL was studied by intragastric infusion different dose of PMBL.The followings are the results:PMBL could extend swimming time,decrease the content of blood urea nitrogen,increase the activity of Lactic dehydrogenases,obviously increase reservation of liver glycogen and muscle glycogen.The results show that PMBL has physical fatigue alleviating function6、The hypoglycemic effect of PMBLMice were injected alloxan to cause diabetic model,and the hypoglycemic effect of PMBL on experimental diabetic mice and the influence on the serum glucose of normal mice were observed respectively.The following are the results:PMBL could remarkably decrease the serum glucose,relate weight lose and water intake of diatetic mice,but has no harmful effects on the serum glucose of normal mice.The results show that PMBL has hypoglycemic effect significantly on diatetic mice,and has no harmful effects on normal mice.7、Regulation of PMBL on lipid metabolism（1）Preventive effects of PMBL on hyperlipidemia and fatty liver were evaluated by ig. different dosages PMBL to the mice fed with high fat diet,the experiment lasted for 4 weeks. The followings are the results:PMBL has obvious inhibition to weight gain of the mice caused by high fat feeding,and good relief function to growth disorder caused by liver damage.PMBL can remarkably inhibit the rising of blood lipid,effectively decrease TC,TG、LDL-C in the serum,increase HDL-C in the serum.All dose of PMBL can reduce the liver cell damage, decrease ALT activity in the serum and the content of liver crude fat,inhibit the liver index and hepatomegaly,and the effect is batter than positive control（PC,Xuezhikang）.The results show that PMBL can effectively relief and prevent the formation of hyperlipidemia,fatty liver and liver damages.（2）The lipid-reducing and liver-benefiting effects of PMBL on the hyperlipidemia,fatty liver and liver-damaging mice were studied.The experiment lasted for 2 weeks.The followings are the results.Different dose PMBL all can remarkably decrease TCHO,TG、LDL-C contents in the serum,and improved HDL-C contents in the serum appreciably;Reduce the content of liver crude fat（the highest reduction rate are 58%）,the liver function and liver cell morphometry in PMBL group are superior to HL group significantly,and similar to NG.The study conformed that,All the dose PMBL can remarkably decrease the content of liver crude fat,ALT activity and liver index in the with hyperlipidemia,fatty liver and liver damages.The results show that PMBL has remarkable antilipidemic effect,and can effectively improve the liver function on the mice with hyperlipidemia and fatty liver.liver damage.The research results show that,the application of membrane separation technology is simple and feasible,which can provides a reference to the study of other plant polysaccharides.PMBL has activity of antifatigue,antioxidative,hypoglycemic and antilipidemic in certain degree, which shows that non-traditional medicinal plant polysaccharides has the same biological activities with medicinal plant polysaccharides,non-traditional medicinal edible resource can be used to product polysaccharides to improve the deficient state of excessive exploitation of Chinese Herbal Drugs Resources,and helpful to improve the added value of moso bamboo leaves, promote the development of forestry economy provide new methods and models for related resources更多还原