【作者】 黄海军；

【导师】 周迎春；

【作者基本信息】 南方医科大学 ， 中西医结合内科， 2008， 硕士

【摘要】 研究背景轮状病毒(Rotavirus,RV)是世界范围内引起婴幼儿严重腹泻的最主要原因,并成为严重的公共卫生问题,特别是在发展中国家。在世界各国,90%以上的婴幼儿在3岁之前均受到了RV的感染,在5岁以下腹泻住院患儿中,RV感染病例占20%～70%,5岁以上的儿童几乎全都感染过RV。人的一生可多次重复RV感染,但有症状感染主要发生在3岁以内的婴幼儿。全球每年有近60万儿童死于RV感染性腹泻,主要是在不发达国家。尽管发达国家因此而死亡的较少,但为此各国都要承担高额的医疗费用。以美国为例,用于治疗RV腹泻的费用每年高达4.54亿美元。国内研究表明在腹泻住院儿童中,40%以上是RV感染所致。我国轮状病毒死亡数估计每年至少达4万人,成为公共卫生资源的主要负担。目前,病毒性腹泻尚无特异的抗病毒药物,而中医药呈现出独特的优势。番石榴叶对轮状病毒性腹泻疗效确切,能够显著的改善腹泻等症状。体外实验亦证实番石榴叶能提高MA-104细胞对轮状病毒的耐受,降低其毒力,破坏轮状病毒颗粒的外壳和内核。发现不同的成分针对引起腹泻的不同机制,多途径发挥止泻作用。如挥发油具有镇痛、抗炎性能;槲皮素能抑制肠道收缩,缓解肠道痉挛,还可减少肠道水分和电解质的分泌。本实验主要研究番石榴叶的有效成分皂苷和挥发油体外直接抗轮状病毒的作用。目的利用体外培养及MTT法检测番石榴叶中皂苷、挥发油的体外抗轮状病毒作用,明确两者在轮状病毒感染的预防和治疗作用。方法1、将不同浓度的皂苷作用于MA-104细胞,MTT法检测细胞存活率,求出其半数细胞毒性浓度(median toxic concentration,TC50)后,予TC50以下的不同浓度的皂苷对轮状病毒感染细胞前后分别进行干预,检测其对病毒感染细胞存活的影响,以明确皂苷对轮状病毒感染的预防和治疗作用;2、将不同浓度的挥发油作用于MA-104细胞,MTT法检测细胞存活率,求出其TC50后,予TC50以下的不同浓度的挥发油对轮状病毒感染细胞前后分别进行干预,检测其对病毒感染细胞存活的影响,以明确挥发油对轮状病毒感染的预防和治疗作用。结果1、细胞存活率和皂苷浓度间关系经Probit回归分析得到皂苷TC50为112.24μg/ml。之后以100μg/ml、75μg/ml、50μg/ml、25μg/ml、1μg/ml预先作用细胞,再以RV感染细胞,或先以RV感染MA104细胞,再以上述不同浓度皂苷作用细胞,继续培养48h后,皂苷100μg/ml、75μg/ml时细胞病变较其它各组均较轻;MTT法检测发现皂苷用于抗RV时,其预防和治疗作用最高抑制率分别为62.12%和77.98%,半数抑制浓度(50%inhibition contraction,IC50)分别为63.83μg/ml和33.19μg/ml,治疗指数分别1.76和3.38。并且在TC50范围以下,不论治疗或预防作用,随着药物浓度的增加,病毒抑制率明显升高,呈量效关系。2、细胞存活率和挥发油浓度间关系经Probit回归分析得到挥发油的TC50为4.09μl/ml。实验结果表明,在挥发油预防作用时用4μl/ml、2μl/ml、1μl/ml、0.5μl/ml、0.25μl/ml分别预先处理细胞后,再以RV感染细胞,培养48小时后,其中挥发油4μl/ml组细胞病变明显较其它各组轻。MTT法检测发现挥发油用于预防轮状病毒感染随着药物浓度的增加,RV的抑制率增加,表现出量效关系,其IC50为2.72μl/ml,治疗指数为1.50。在挥发油治疗作用时,先用RV感染细胞后,再用不同浓度药物干预,发现挥发油同样表现出很好的抗病毒作用,其对RV的抑制率高达86.55%,IC50为1.14μl/ml,治疗指数为3.59。在药物浓度和病毒抑制率上同样表现出量效关系。结论番石榴叶中有效成分皂苷、挥发油具有良好的体外抗轮状病毒的作用。更多还原

【Abstract】 BACKGROUNDRotavirus infection remains the most common cause of severe, dehydrating gastroenteritis among children worldwide. Almost every child in the world, in both developed and developing countries, will be infected with rotavirus in the first 5 years of life. Globally, approximately 600,000 children die every year from rotavirus gastroenteritis, with the vast majority of these deaths occurring in the poorest countries. In developed nations, rotavirus infection rarely results in death but remains the most common cause of hospitalizations for acute gastroenteritis in children and leads to major medical and societal costs. In the United States ,the costs on therapy of rotavirus diarrhea up to 454 million US dollars. Researches from china also show that more than 40 percent of diarrhea in hospitalized children, is caused by rotavirus. China’s estimated annual rotavirus deaths of at least 40,000 people and become the main burden of public health resources. At present, there are no specific anti-rotavirus drugs, and TCM has unique advantages.In our long-term clinical practice, we found that using guava leaf as the main drug, supplemented with other medicine, has the exact effect on rotavirus diarrhea, can significantly improve symptoms such as diarrhea. In our previous studies ,we found that guava leaf can enhance the tolerance of MA-104 cells on the rotavirus, reduce the virus’ toxicity, and damage rotavirus particle shell and core. We also found that different ingredients from the guava leaf can treat the rotavirus diarrhea by different mechanisms. Such as volatile oil has analgesic, anti-inflammatory properties; Quercetin can inhibit intestinal contraction, ease intestinal cramps, reduce intestinal secretion of water and electrolytes. In the experiments, we extracted essential oil and saponins from the guava leaf, and studied their anti-rotavirus effect in vitro.OBJECTIVETo evaluate the anti-rotavirus effect of the saponin and volatile oil from Psidium guajava leaves using MTT assay, to conform their roles on prevention and therapy of rotavirus infection.METHODSExtract volatile oil and saponins from Guava leaf.1、Using different concentrations of saponin interfered the MA-104 cells, we get the median cytotoxic concentration(TC50) by MTT assay, Then added the components of saponin of different concentrations which are below the median toxic concen - tration(TC50) to interfere the rotavirus in infecting the MA104 cells. and using the MTT assay to evaluate this components’ effect on the MA104 cell viability before and after its infected by rotavirus ,and determine their anti-rotavirus effects in vitro.2、Using different concentrations of volatile oil interfered the MA-104 cells, we get the median cytotoxic concentration(TC50) by MTT assay, Then added the components of volatile oil of different concentrations which are below the median toxic concentration(TC50) to interfere the rotavirus in infecting the MA104 cells. and using the MTT assay to evaluate this components’ effect on the MA104 cell viability before and after its infected by rotavirus ,and determine their anti-rotavirus effects in vitro. RESULT1、By Probit regression analysis we find the TC50 of saponin on MA-104 cells is112.24ug/ml.then we added saponin of different concentrations which are below the median toxic concentration to interfere the rotavirus in infecting the MA104 cells. 48 hours latter, we found the cytopathic effect in groups of 100μg/ml and 75μg/ml are slighter than other groups either used for prevention or therapy;Through MTT assay analysis, we found the maximum inhibit ratio are 62.12% and 77.98%, the median inhibition contraction are 63.83μg/ml and 33.19μg/ml, and the therapeutic index (TI) are 1.76 and 3.38,when the saponin used for prevention and therapy respectively. Also, The inhibit ratio of rotavirus increase with the raise of concentration of the components, no matter we add it before or after the virus infected the cells.2、Through Probit regression analysis we find the TC50 of volatile oil on MA-104 cells is 4.09μl/ml in this experiment . In our experiment we found the cytopathic effect in group of 4μl/ml is slighter than other groups either used for prevention or therapy ;Through MTT assay analysis, we found the maximum inhibit ratio are 60.04 % and 86.55 %, the median inhibition contraction are 2.72μl/ml and 1.14μl/ml, and the therapeutic index are 1.50 and 3.59, when the volatile oil used for prevention and therapy respectively. Also, The inhibit ratio of rotavirus increase with the raise of concentration of the components, no matter we add it before or after the virus infected the cells.CONCLUSIONThe saponin and volatile oil from Psidium guajava leaves have nice and exact anti-rotavirus effect in vitro.更多还原