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中药植物美洲商陆抗菌活性及作用机理研究

Research on Antifungal Activities of Chinese Traditional Medicine Phytolacca Amercana Roxb and Its Action Mechanism

【作者】 杨帮

【导师】 赵志模; 丁伟;

【作者基本信息】 西南农业大学 , 农药学, 2005, 硕士

【摘要】 美洲商陆作为一种传统中药在中国已经有上百年的历史。自上世纪50年代以来,关于美洲商陆的研究报道逐渐引起国内外学者的关注,对其在医药、食品、工业和农业上的应用研究也随之发展起来。医药方面的研究表明该植物具有调节免疫、利尿、抗菌消炎、抗病毒、抗肿瘤、杀寄生虫等作用。然而关于美洲商陆在农药方面的研究报道很少。 本项研究在本人导师对23种中药植物初步筛选的基础上,选择商陆属植物美洲美洲商陆做为研究对象,从农药学的角度研究美洲商陆提取物的抑菌活性,明确了抑菌活性成分在其植株不同部位和不同极性溶剂中的分布情况,并对抑菌活性成分进行了提取分离及初步的抑菌活性研究。现将主要结果摘要如下: 1.美洲商陆各部位溶剂提取物的抑菌活性研究 1.1 美洲商陆抑菌活性成分在植株中分布的部位 将美洲商陆植株分为根、茎、叶和果四部分,研究了该植物各部分系列溶剂顺序提取物对四种农业病原真菌的抑制作用,结果证实美洲商陆植株不同部位提取物对这四种病原真菌均有一定的抑制作用,且抑菌谱基本相同,即对柑橘绿霉和小麦纹枯病菌的抑制作用较强。不同部位提取物对同一种病原菌活性也存在一定差异,叶、根和果甲醇提取物在相同浓度下的抑菌活性明显高于茎。这可能是因为茎中的活性成分含量较低,或是其他杂质含量较高所致。 1.2 美洲商陆抑菌活性成分最佳提取溶剂 测定了美洲商陆植株各部分系列溶剂顺序提取物对四种病原菌的抑菌活性,结果表明,茎、叶、根和果四部分甲醇提取物的抑菌活性远远高于其他三种溶剂提取物,这表明美洲商陆的抑菌活性成分难溶于石油醚、苯、乙醚等低极性溶剂而易溶于低级醇。因此,甲醇或乙醇是美洲商陆植株各部分抑菌活性成分的最适提取溶剂。 1.3 美洲商陆提取物对不同病原菌生长的抑菌效果 采用生长速率法,测定了美洲商陆系列溶剂顺序提取物对玉米小斑、棉花枯萎、柑橘绿霉和小麦纹枯病菌四种农业病原真菌的抑制作用,结果表明美洲商陆提取物对这四种病原真菌均有一定的抑制作用,其中对柑橘绿霉和小麦纹枯病菌的抑制作用较强,尤其是柑橘绿霉病菌。因此选择柑橘绿霉病菌作为活性追踪供试菌。 1.4 抑菌活性成分性质的初步研究 由美洲商陆抑菌活性成分主要存在于甲醇顺序提取物,可以推断出美洲商陆抑菌活性成分是一种或几种强极性化合物。根据文献上关于美洲商陆医药抑菌活性成分的研究,排

【Abstract】 As a kind of Chinese traditional medicine, Phytolacca amercana Roxb has been used for hundreds of years in China. Reports about Phytolacca amercana Roxb began to draw much attention of researchers home and aboard since 1950s. As a result, researches on application of this plant in such field as medicine, food, industry and agriculture had greatly increased. Pharmacological studies showed that Phytolacca amercana Roxb had many bioactivities such as diuresis, improving immunity, antimicrobial, antivirus, diminishing inflammation and so on. However, there has been few reports about researches on applying this plant in pesticide science so far.In this paper, we studied the antifungal activity of abstracts from Phytolacca amercana Roxb, determined the distribution of antifungal components in different parts of this plant and different resolves, and further carried out studies on isolation and antifungal activity of these components. The main results are as follows:l.Studies on antifungal activity of abstracts from different parts of Phytolacca amercana Roxb1.1 Distribution of antifungal components in Phytolacca amercana Roxbwe studied the fungistasis of sequence abstracts from different parts of Phytolacca amercana Roxb on 4 fungi, the results showed that abstracts from different parts of this plant all had certain antifungal activity on the 4 fungi, and had similar antifungal spectrum. There were some differences in antifungal activity of abstracts from different parts to the same fungi. Fungistasis of methyl abstracts from leaf, root and fruit is stronger than that from stem at the same concentration. The reason might be that content of antifungal components in stem is lower than that in other parts, or that impure in stem is higher than that in other parts.1.2 Proper solvent for extracting antifungal components from Phytolacca amercana Roxbwe studied the fungistasis of sequence abstracts from different parts of Phytolacca amercana Roxb on 4 fungi, the results showed that the fungistasis of Methyl alcohol extracts is much stronger than that of other extracts. Which indicated that the solubility of antifungal components in this plant was low in Petroleum oil, Benzene and Ether and high in methanol. In conclusion, methanol or ethanol is proper solvent for extracting antifungal components from Phytolacca amercana Roxb,1.3 The fungistasis of abstracts from Phytolacca amercana Roxb on 4 fungiThe fungistasis of abstracts from Phytolacca amercana Roxb were tested with isolate hypha of 4 fungi (Helminthosporium maydis, Fusarium oxysporium, Penicillium digitatum, Rhizotonia cerealis). The results showed that the fungistasis of extracts on Penicillium digitatum and Rhizotonia cerealis were much stronger, and fungistasis on Penicillium digitatum is the strongest among the 4 fungi.1.4 Preliminary analysis of antifungal components in Phytolacca amercana RoxbThe fact that antifungal components mainly existed in Methyl alcohol sequence extracts showed that antifungal components in Phytolacca amercana Roxb is one or a series of chemicals with high polarity. Based on reports about active components in this plant, we assumed that antifungal components obtained in our experiment belonged to saponins or antimicrobial proteins. Referring to reported methods used to isolate saponins, we isolated saponins from Methyl alcohol sequence extracts. Results of bioassay showed that antifungal activity and spectrum of saponins obtained were consistent with that of Methyl alcohol sequence extracts. It was suggested that the antifungal components in Methyl alcohol sequence extracts belonged to saponins of Phytolacca amercana Roxb. So, it is meaningful to further isolate antifungal components from saponins.2.Researches on methods for isolating saponins from root of Phytolacca amercana RoxbReferring to reported methods we established a set of methods for isolating saponins from root of Phytolacca amercana Roxb based on repeated experiments. After dried plant powder was degreased with series of solvents, and then extracted with Methanol, and finally extracted with n-butyl, the total crude saponins were mainly in the n-butyl partition. The total crude saponins were subjected to Macro-reticular absorption resin in stead of silica gel chromatography to primarily give active components on the basis of shorter time and lower usage of toxic solvents. The active components mentioned above was further isolated by silica gel chromatography to give antifungal components(2F3). Results of TCL showed that 2F3 is consistent of two compounds whose R_f value are so close that we can hardly separate them with our facilities. So 2F3 was used as specimen in the following bioassay.3.Study on antifungal activity of 2F3Using carbendazim as standard fungicide, we tested the fungistasis of 2F3 on Penicilliumdigitatum and Penicillium italicum Wehmer both in substrate and on orange fruits in laboratory.The results showed that the fungistasis of 2F3 and carbendazim on Penicillium digitatum and Penicillium italicum Wehmer were good. Which EC50 were 80.41mg/l and 62.13mg/l to Penicillium digitatum and 130.80mg/l and 55.96mg/l to carbendazim respectively. Fungistasis of carbendazim was stronger than that of 2F3.Results of 15-day controlling test after inoculation showed that 2F3 could effectively control Penicillium digitatum and Penicillium italicum Wehmer on inoculated orange fruits. But the control effect of 2F3 at different concentration decreased with increase of days after treatment. Control effect of 2F3 at 400, 800, 1600 times dilution were 95%, 91%, 88% respectively 15 days after treatment. Control effect carbendazim at 1000 times dilution was better than that of 2F3 at 400 times dilution.Results of 2-month storage test at normal temperature showed that 2F3 could control Penicillium digitatum and Penicillium italicum Wehmer on stored orange fruits. But the control effect of 2F3 at different concentration decreased with the increase of days after treatment. Control effect of 2F3 at 400, 800,1600 times dilution were 80.3%, 76.2%, 71.1% respectively after 1-month storage. Which decreased to 76.0%, 73.5%, 67.6% respectively after 2-month storage. Control effect of carbendazim at 1000 times dilution was 87.6% after 1-month storage and 81.2% after 2-month storage.4 Preliminary studies on antifungal mechanism of 2F3 on Penicillium digitatumConsidering the limits to experimental condition and time, we only studied inhibitory activity of 2F3 to spore and hypha of Penicillium digitatum from the aspect of morphology.The result of spore germination test showed that inhibitory activity of 2F3 to spore germination of Penicillium digitatum is week, the inhibition rate of 2F3 from 100 to 1600 times were all below 60% 12h after treatment. On the other hand, inhibitory activity of 2F3 decreased with the increase of time after treatment, inhibition rate of 2F3 at 100 times dilution were 55%, 49%, 40% when treated after 27% 12h, 24h, 48h, 72h respectively.Results of observation with microscope showed that hypha of Penicillium digitatum did not suffer broken cell wall, bioplasm leaking in PDA substrate with 2F3 at different concentration. Which indicated that 2F3 had no lethality to hypha of Penicillium digitatum, but could obviously inhibit growth of hypha. Under the effect of 2F3, hypha of Penicillium digitatum became misshapen and swelled, space between hypha branches decreased, and growth of hypha slowed down. Which is the immediate reason why growth of Penicillium digitatum was inhibited.

  • 【分类号】R285
  • 【被引频次】7
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