【作者】 胡明辉；

【导师】 赵昱；

【作者基本信息】 浙江大学 ， 生药学， 2007， 硕士

【摘要】 第一章黄酮木脂素水飞蓟宾类和黄酮醇类衍生物的抗氧化活性研究活性氧（Reactive oxygen species,ROS）包括超氧阴离子（·O₂^-）和羟自由基（OH·）等,正常情况下,ROS有生理功能,如杀菌,但当其产生量超出了机体的清除能力时,就会产生氧压,损伤细胞成分,在许多疾病中发挥重要作用。ROS引起的损伤与多种疾病的发生发展有关,如神经系统疾病（脑卒中、阿尔茨海默病、帕金森病等）、心血管疾病、动脉粥样硬化、高血压、自身免疫性疾病、肿瘤、病毒（AIDS、病毒性肝炎等）和衰老等。氧自由基也可作为细胞内信息转导信号引发细胞浆内核转录因子（如nuclearfactorκB,NF-κB）的激活,后者进入核内与炎症、免疫相关的基因启动子结合,引起核内有关的基因转录增加,是很多急性、慢性炎症及免疫失调等疾病发生发展的分子生物学基础。因此研究和开发新的抗氧化剂（天然产物或合成的天然产物衍生物）来防治与自由基相关的疾病有重要的意义。黄酮木脂素（Flavonolignans）是一类以黄酮和苯丙素类衍生物缩合而成的黄酮类化合物。该类化合物主要的天然产物有水飞蓟宾,异水飞蓟宾,水飞蓟亭,水飞蓟宁,2,3-脱氢水飞蓟宾,Sinaiticin等,大量的研究表明,水飞蓟宾等黄酮木脂素类化合物具有很多的药理活性,抗氧化、保肝护肝、抗肿瘤、抗炎、抗心血管疾病、抗糖尿病和抗药源性肾毒性等作用。黄酮木脂素类化合物的众多生物作用,证实其本身是较为理想的抗肿瘤,抗病毒性和化学性肝损伤,抗炎症和其他疾病的一类母体化合物。对该类化合物的深入研究正吸引着众多科学工作者更大的兴趣。二氢黄酮醇（dihydroflavonols）是指C环2,3位为单键,3位有一羟基取代的黄酮类化合物。许多该类化合物如黄杉素（taxifolin）,落新妇苷（astilbin）、二氢杨梅素（dihydromyricetin,ampelopsin）等具有多种生物活性。此类化合物具有抗氧化、保肝、抗肿瘤、抗病毒、抗炎、抗菌、抗高血压和高血脂等药理活性。研究这两类天然产物的新衍生物的抗氧化活性,可进一步了解黄酮木脂素类和黄酮醇类化合物的抗氧化机理,为开发新的抗氧化剂提供实验依据。目的:采用不同的体外抗氧化模型,从不同层次考察黄酮木脂素水飞蓟宾类和黄酮醇类新衍生物的抗氧化活性和构效关系,为进一步开发有效的抗氧化剂提供药理依据。方法:采用自由基清除模型,保护由双氧水诱导的神经细胞损伤模型和抗脂质过氧化模型,从抑制自由基产生、清除自由基及抗氧化这三个不同层次从体外考察本实验室合成的两类天然产物衍生物的抗氧化活性和研究它们的构效关系。结果:水飞蓟宾类衍生物中化合物2有清除·O₂^-作用,其IC₅₀=2.65×10^-5mol/L,活性略强于阳性对照(IC₅₀=3.81×10^-5mol/L)。二氢黄酮醇类衍生物中化合物11,12,13,16,21显示了一定的O₂^-清除活性,IC₅₀分别为33.7±0.87,18.78±0.84,35.2±1.12,30.05±0.65和36.32±1.55μg/mL,但活性低于阳性对照的IC₅₀值11.6±0.55μg/mL。化合物9-13,21有清除DPPH的作用,IC₅₀分别为11.73±0.46,32.82±1.21,6.64±0.72,13.50±0.75,15.50±0.56和2.65±0.45μg/mL,阳性对照的IC₅₀值为1.58±0.46μg/mL。化合物10,11,13,21有抗脂质过氧化作用,IC₅₀分别为20.52±0.65,11.55±0.82,3.52±0.25,18.74±0.47μg/mL,阳性对照的IC₅₀值为8.63±0.53μg/mL。化合物21还能显著地保护PC12细胞不受H₂O₂损伤的作用。结论:化合物21有抑制·O₂^-产生、清除自由基、抗脂质过氧化和保护神经元细胞的作用,有进一步研究的意义;化合物11,12,13,16,21有清除·O₂^-作用,化合物9-13,21有清除DPPH作用,化合物10,11,13,21有抗脂质过氧化作用。第二章美洲大蠊的抗肿瘤活性研究美洲大蠊这一蜚蠊属昆虫在我国有着广泛的分布,虽然它被认为是病菌的携带者和食物的破坏者,但它也有可用的地方。在民间,美洲大蠊作为药用动物已有悠久的历史,最早记载于《神农本草经》,常将它用来治疗烫伤、灼伤,以及小儿疳疾。我们从体内和体外两个不同层面研究美洲大蠊提取物的抗肿瘤活性,为开发新的抗肿瘤中药制剂提供一定的实验依据。目的:采用体外培养的肿瘤细胞株CNE、Hela、K562、PC3、P388 D1和体内S180肿瘤移植模型,分别测定美洲大蠊粗提物样品的体外、体内抗肿瘤活性,为下一步的研究奠定实验基础。方法:以肿瘤细胞株为靶细胞,用MTT法测定样品抑制肿瘤细胞增殖的作用,用以判断样品的体外抗肿瘤活性;采用S180肿瘤移植模型,计算样品的体内抑瘤率,用以判断样品的体内抗肿瘤活性。结果:体外细胞毒实验,在147个粗提物样品中有将近50个样品对CNE等肿瘤细胞有抑制活性,其中RL-A0711-（17-20）等样品对K562的抑制活性很高,其IC₅₀值小于10μg/mL。体内小鼠S180肉瘤移植实验,样品SLY-1的高、低剂量组对S180肉瘤均有抑制作用,抑制率分别为36.23%和50.72%,脾脏指数分别为6.84和5.75,胸腺指数分别为1.93和2.25。样品SLY-2只有高剂量组有抑制作用,抑制率为39.13%,脾脏指数为6.84,胸腺指数为2.13。结论:美洲大蠊提取物在体内体外均对肿瘤细胞有抑制作用,其抑瘤机理可能与提高小鼠的机体免疫功能有关。更多还原

【Abstract】 Chapter I Study the antioxidant properties of novel silybin and dihydroflavonol derivativesThe reduction of molecular oxygen leads to the formation of reactive oxygen species （ROS） including superoxide anion radicals （·O₂^-） and hydroxyl （OH·） radicals. Generally, the ROS has some physiological function such as bacterial ingestion. However, when the imbalance between cellular production of free radicals and the ability of cells to defend against them occurs, the ROS cause oxidative damage to cell components, and may play an important role in various pathological conditions. This damage may contritute to a variety of diseases in nervous system, such as stroke （a disease in acute central nervous system injury）, Parkinson’s disease and Alzheimer’s disease （AD）. It also results in other pathological processes, including cardiac disease, atherosclerosis, hypertension, autoimmune rheumatic disease, cancer, viral disease （such as AIDS） and aging. ROS, as the signal of message transduction intra-cellular, it also can activate nuclear factorκB （NF-κB）, which binds to the promoter, related to inflammation and immunity, and increases the genetic transcription in nuclear. The occurrence and development of many diseases, such as actue, chronic inflammation, and immune imbalance and so on, may be attributed to this molecular biology base. Therefore there is considerable interest in the discovery and development of efficient synthetic or natural antioxidants to protect and therapy various diseases related to free radicals.Flavonolignan is a kind of flavones, which is coupled by catechol flavones and phenylpropanoids. The representative compounds of flavonolignans are silybin, isosilybin, silichristin, silidianin and 2,3-dehydrosilybin, which were isolated from silymarin. A lot of researches indicated that flavonolignans have many bioactivies such as antioxidant, hepatoprotective, anti-tumor, anti-inflammation, anti-cardiovascular diseases, anti-diabetes and anti-renal toxicity from medicinal resource. Flavonolignan, as a kind of lead compounds, possessing several of bioactivities, attracts many researchers to study their structure-activity relationship （SAR） and to develop new derivatives.Dihydroflavonols, belonging to flavonoids, in which 2,3-position is a single bond and 3-position is substituted by hydroxyl, are an important class of secondary plant metabolites. Many dihydroflavonols such as taxifolin, astilbin, ampelopsin show excellent activities. Dihydroflavonols exhibit a wide spectrum of pharmacological properties, including antioxidative, hepato-protective, antineoplastic, anti-virus, anti-inflammation, anti-fungi, anti-hypertension, and anti-hyperlipemia activities.The present study would be benefit for understanding the mechanisms of antioxidative activity of flavonolignans, such as silybin derivatives and dihydroflavonols and further optimize their application.Subject: We elucidate the antioxidant properties of these new derivatives of flavonolignans and dihydroflavonols and determine their structure-activity relationship as atitioxidants by using various experimental models. And this may be contributed to discover and develop new antioxidants.Methods: We established inhibition of xanthine oxidase, scavenging free radicals, protection of PC12 cells-insulted by H₂O₂ and inhibition of lipid peroxidation models to assay the antioxidant properties of new derivatives of flavonolignans and dihydroflavonols from different levels, and to determine their SARs.Results: Compound 2, one of silybin derivatives, demonstrated excellent antioxidant effect on scavenging superoxide anion free radicals, the IC₅₀ of which was 2.65×10^-5 mol/L, while the IC₅₀ of quercetin （referfence compound） was 3.81×10^-5 mol/L. Compounds 11, 12, 13, 16, 21 showed activities of scavenging·O₂^-, and IC₅₀ values were 33.7±0.87, 18.78±0.84, 35.2±1.12, 30.05±0.65 and 36.32±1.55μg/mL, respectively, a little weaker than control (IC₅₀=11.6±0.55μg/mL). Compounds 9-13, 21 exhibited anti-DPPH radicals activity, the values of IC₅₀ were 11.73±0.46, 32.82±1.21, 6.64±0.72, 13.50±0.75 and 15.50±0.56, 2.65±0.45μg/mL, respectively, while the IC₅₀ of control was 1.58±0.46μg/mL. Compounds 10, 11, 13, 21 had excellent effect of inhibition of lipid peroxidation. IC₅₀ values were 20.52±0.65, 11.55±0.82, 3.52±0.25, 18.74±0.47μg/mL, respectively, and the IC₅₀ of control was 8.63±0.53μg/mL. Compound 21 could markedly protect PC12 cells, insulted by H₂O₂.Conclusion: All of the data presented here demonstrated that compound 21 has antioxidant properties of scavenging superoxide anion and DPPH free radicals, protecting PC12 from injuring by H₂O₂ and inhibiting lipid peroxidation. Therefore, it is valuable to further study this compound. Compounds11, 12, 13, 16, 21 can scavenging superoxide anion, compounds 9-13, and 21 can capture DPPH radicals and compounds 10, 11, 13,21 can blocking chain reaction by inhibiting lipid peroxidation. Periplaneta americana is widely distributed in China. Although it was well known as an active carrier of pathogenic organisms, as well as for damaging stored products and being aesthetically unappealing and offensive . It was employed as folk medicines for the treatment of toxic disorders and Children’s malnutrition since ancient China. The earliest record could be found in ’ShenNong Bencao Pharmacy’, which was more than two thousands years ago. In this study, the anti-tumor effects of extracts from Periplaneta americana was evaluated with both in vitro and in vivo models.Subject: To evaluate the anti-tumor activities of Periplaneta Americana extracts.Methods: To evaluate the cytotocicities of Periplaneta Americana extracts, such as the human nasopharyngeal carcinoma CNE、carcinoma cervicis Hela、human chronic granulocytic leukemia K562、prostatic carcinoma PC3、gmouse macrophae P388 D1 in vitro and to evaluate the anti-tumor effectivities in vivo by transplanted mouse sarcoma S180 model.Results: In vitro, about 50 in 147 samples more or less inhibited the proliferation of CNE, Hela, P388 D1, K562 and PC3 cell lines, and a few of samples, such as RL-A0711-（17-20） significantly inhibited the proliferation of K562, with the IC₅₀ value < 10μg/mL. In vivo, SLY-1 sample of low dose （500 mg/kg） and SLY-1 of high dose （1500 mg/kg） both inhibited the growth of S180 cell, the inhibitive rate were 36.23% and 50.72%, and the spleen index were 6.84 and 5.74, the thymus index were 1.93 and 2.25. Only SLY-2 sample of high dose （1500 mg/kg） inhibited the growth of S180 cell, the inhibitive rate were 39.13%, and the spleen index were 6.84, the thymus index were 2.13.Conclusion: These results indicated that PAE possesses potent anti-tumor activity on the models that we have employed. Its anti-tumor mechanism are probably associated with the enhancing of immunity.更多还原