【作者】 张为民；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2010， 博士

【摘要】 筛选具有抗病毒和抑菌活性的中草药不仅为开发新型天然药物提供理论依据,而且对中草药资源的开发和利用具有重要的意义。本研究对采自陕西省太白山地区7种中草药各提取部位的体外抑菌作用进行初步筛选,发现太白蓼(Polygonum taipaishanense Kung)和朱砂七(Polygonum cillinerve (Nakai) Ohwl)提取物的体外抑菌活性明显,然后进行了太白蓼和朱砂七抗病毒和抑菌活性部位的筛选、有效成分分离及鉴定,获得以下结果:1.朱砂七乙酸乙酯部位(PCE)、太白蓼乙酸乙酯部位(PTKE)、老鹳草正丁醇部位(GWN)及老鹳草的乙酸乙酯部位(GWE)均有较强的抑菌活性,且具有较宽的抗菌谱。2. PTKE对新城疫病毒(NDV)在鸡胚成纤维细胞(CEF)上增殖的抑制率为32.49%,PTKEB对NDV在CEF上有直接杀灭、抗吸附和抑制增殖作用(抑制率分别为90.52%、65.23%和61.21%),并且与剂量呈正相关性。鸡胚试验证实不同浓度PTKEB药物和病毒混合组对NDV在鸡胚内增殖具有显著的抑制作用(P<0.01)。体外抑菌试验表明,PTKE对大肠埃希菌、金黄色葡萄球菌、沙门菌、巴氏杆菌和无乳链球菌的MBC分别为100 mg/mL、25 mg/mL、50 mg/mL、25 mg/mL和50 mg/mL;分离物PTKEA、PTKEB和PTKEC对金黄色葡萄球菌、沙门菌和无乳链球菌有一定的抑制作用,但对大肠埃希菌和巴氏杆菌没有抑制作用。3. PTKE对蓖麻油和番泻叶所致的小鼠腹泻均有极显著的抑制作用(P<0.01);对乙酸所致家兔肠黏膜毛细血管通透性增高、腹腔注射醋酸所致小鼠腹腔毛细血管通透性增高及皮内注射二甲苯致皮肤毛细血管通透性增高分别具有极显著(P<0.01)和显著(P<0.05)抑制作用;在雏鸡饮水中添加不同浓度PTKE,各试验组雏鸡胸腺指数、脾脏指数和法氏囊指数与对照组相比差异显著或极显著(P<0.05或P<0.01),免疫器官的组织结构发育状况也明显优于对照组;PTKEB浓度为7.81μg/mL时,其对脾脏、胸腺、法氏囊和外周血淋巴细胞的体外增殖作用极显著高于细胞对照组(P<0.01);PTKE对雏鸡的生长有一定的促进作用,但其降低料肉比和增加饲料转化率的趋向不明显。4.朱砂七氯仿部位(PCC)、大黄素(EM)和大黄素甲醚(PH)对NDV在CEF上增殖的抑制作用不明显;朱砂七乙酸乙酯部位(PCE)及进一步分离物PCEA对NDV在CEF上的抑制率为39.12%和72.33%;PCEA可抑制NDV在鸡胚内的增殖,其预防组、药物和病毒混合组与病毒对照组的血凝效价均差异显著(P<0.05),但治疗组与病毒对照组差异不显著。PCE对9株临床分离菌的MBC均小于100 mg/mL,PCC对9株菌也有一定抑制作用;EM对鸡源沙门菌,猪源链球菌,奶牛源无乳链球菌、表皮葡萄球菌和金黄色葡萄球菌的MBC分别为5 mg/mL、2.5 mg/mL、2.5 mg/mL、2.5 mg/mL和1.25 mg/mL;PCEA对鸡源沙门菌,猪源链球菌,奶牛源无乳链球菌、表皮葡萄球菌和金黄色葡萄球菌的MBC分别为24 mg/mL、24 mg/mL、12 mg/mL、6 mg/mL和6 mg/mL。5. EM、PH、PCEA、PCEB和PTKEB对猪传染性胃肠炎病毒(TGEV)均具有一定的直接杀灭作用(抑制率均大于71.28%);PCEA、PCEB和PTKEB可抑制TGEV在ST细胞内增殖,三者对TGEV的生物合成的抑制率分别为82.65%、85.74%和54.04%;EM、PCEA、PCEB和PTKEB对TGEV感染细胞具有一定的吸附保护作用,其抑制率分别为44.85%、69.07%、83.08%和48.28%;不同浓度的PCEA、PCEB和PTKEB对TGEV在ST细胞生物合成的抑制作用随药物剂量的增加而增强。6.经系统预试、柱色谱分离及液相-质谱分析等方法对太白蓼和朱砂七抗病毒和抑菌有效部位的化学成分进行鉴定,结果从PTKEB部位检出儿茶素类和3′,4′,5,7-四甲氧基黄酮,从PCC和PCE检测出大黄素、大黄素甲醚、大黄素-8-β-D-葡萄糖甙等蒽醌类、白藜芦醇和白藜芦醇苷等二苯乙烯类,这些物质为其抗病毒和抑菌作用的主要活性成分。更多还原

【Abstract】 Searching antiviral and antibacterial active components from Chinese herbal medicine will be very helpful not only in providing theoretical evidence for developing new drug, but also in proper exploitation and utilization to resources of Chinese herbal medicine. This study mainly involved in 7 Chinese herbal medicines which belongs to Taibai Moutain area, Shaanxi Province at the basis of in vitro bacteriostasis tests. Selections on antiviral and antibacterial active fractions of Polygonum taipaishanense Kung and Polygonum cillinerve (Nakai) Ohwl in vitro and in vivo, separation and assessment of effective ingredients and other aspects were studied. The results were as following.1. All the Polygonum cillinerve ethyl fluoroacetate fraction (PCE), Polygonum taipaishanense ethyl fluoroacetate fraction (PTKE), Geranium wilfordii n-butanol fraction (GWN) and its ethyl fluoroacetate fraction (GWE) owned better virucidal activity with extensive antibacterial spectrum.2. The inhibition ratio of PTKE on proliferation of NDV in CEF are 32.49%. PTKEB had effect of directly elimination, anti- absorption and inhibiting proliferation on NDV in CEF (inhibition rate were 90.52%, 65.23% and 61.21%, respectively), there are obvious dosage-effect relationship between PTKEB and anti-NDV inhibitory rate. PTKEB can inhibit the proliferation of NDV in chicken embryo with extremely significant difference between herbal toxin mixed group and virus control(P<0.01). PTKE can inhibit E. coli, S. aureus, Salmonella, Pasteurella and S. agalactiae in vitro (their MBC were 100, 25, 50, 25 and 50 mg/mL respectively), and its isolates-PTKEA, PTKEB and PTKEC had inhibitory effect on S. aureus, Salmonella and S. agalactiae, but no antibacterial effect on E. coli and Pasteurella.3. PTKE could inhibit not only the diarrhea of mouse caused by Castor Oil and Senna Leaf extreme markedly (P<0.01), but also the heightening of capillary permeability located in rabbit intestine mucosa caused by Acetic Acid (P<0.01). In addition, it also could extremely inhibit heightening of capillary permeability located in abdominal cavity and in skin caused by acetic acid injected intraperitoneally and xylene injected intradermally respectively(P<0.05). The experimental groups were shown significant difference or the most significant difference compared with the contro(lP<0.05 or P<0.01)when added PTKE at different concentration to drinking water. The developmental state of histological structure of immune organs in experimental group were also better than it in control group. There were most significant difference on lymphocyte proliferation of spleen, thymus, bursa of Fabricius and peripheral blood between the PTKEB group at concenteation of 7.81μg/mL and the contro(lP<0.01). PTKE could promote the growth of chicken, but no evident trends could be seen both in reducing the rate of forage to meat and in increasing the transformation efficiency of forage.4. No obvious inhibitory effect could be observed in chloroform extract fraction of Polygonum cillinerve (PCC), emodin (EM) and physcione (PH) on proliferation of NDV in CEF. The inhibitory rate of PCE and its isolates PCEA on proliferation of NDV in CEF were 39.12% and 72.33%, respectively. PCEA can also inhibit the proliferation of NDV in chicken embryo with significant difference in hemagglutination titer between prevention group or herbal toxicity mixed group and virus control group(P<0.05), but no difference could be seen between treated group and virus control. All the minimal bactericidal concentration (MBC) of PCE to 9 bacteria were lower than 100 mg/mL. PCC had some virucidal effects on these bacteria. The MBC of Emodin on Salmonella (chicken), S.suis, S. agalactiae (cow), S.epidermidis (cow) and S. aureus (cow) were 5, 2.5, 2.5 , 2.5 and 1.25 mg/mL, respectively. The MBC of PCEA to Salmonella (chicken), S.suis, S. agalactiae (cow), S. epidermidis (cow), and S. aureus (cow) were 24, 24, 12, 6 and 6 mg/mL, respectively.5. EM, PH, PCEA, PCEB and PTKEB could eliminate TGEV directly, and their inhibitory rate were all more than 71.28 %. PCEA. PCEB and PTKEB could prevent the intracellular duplication of TGEV in ST cell, their inhibitory rate on biosynthesis of TGEV were 82.65 %, 85.74 % and 54.04 %, respectively. EM, PCEA, PCEB and PTKEB had adsorbing protection to infected cell by TGEV (their inhibitory rate were 44.85%, 69.07%, 83.08% and 48.28%, respectively). The inhibitory effect of PCEA, PCEB and PTKEB at different concentration on biosynthesis of TGEV in ST cell enhanced as increase of dosage.6. According to analysis by system pretesting, chromatographic separation and LC-MS,we detected Catechin and 3′,4′,5,7-Tetramethoxyflavone from PTKEB, while Anthraquinone including Emodin, Physcion, Emodin-8-β-D-glucopyranoside and Stilbenes containing Resveratrol and Piceid from PCC and PCE. These compounds were main active components related to antivirus and antibacteria.更多还原