| 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. |
PDF Full Text Request