Study On Antifungal Activities And Active Ingredients Of Clerodendrum Inerme (L.) Gaertn

Sixteen species of mangrove plants, belonging to thirteen families, were collected from Fangchenggang city in Guangxi province for screening of antifungal activities. According to the result, Clerodendrum inerme (L.) Gaertn., which had significant inhibition against spores of three pathogens, was selected to be further investigated for active ingredients. The active ingredients were isolated and identified from Clerodendrum inerme.The inhibitory activities of the methanol extracts from sixteen species of mangrove plants against spores of Thielaviopsis paradoxa, Fusarium oxysporum f. sp. niveum and Sphaceloma fawcettii were determined with the method of conidia germination. The results showed that the extracts of Clerodendrum inerme and Myoporum bontioides had significant inhibition against spores of the three pathogens at 10g/L, with the inhibition rates being above 77.29%. The extracts of Aegiceras corniculatum, Excoecaria agallocha, Lumnitzera racemosa and Heritiera littoralis had significant inhibition against spores of Thielaviopsis paradoxa, but low inhibitory activities against spores of the other two pathogens. The extracts of other plants had low inhibitory activities against spores of the three pathogens.In order to further compare the inhibitory activities of the plant extracts, toxicity of five plant extracts to spores of Thielaviopsis paradoxa was determined. The extracts of Lumnitzera racemosa, Aegiceras corniculatum, Clerodendrum inerme, Excoecaria agallocha, Myoporum bontioides had significant inhibition against spores of Thielaviopsis paradoxa, with the EC50 values of 0.3852g/L,0.5714g/L,3.1910g/L,3.4384g/L,4.8205g/L, respectively. The extracts of Clerodendrum inerme and Myoporum bontioides had significant inhibition against spores of Fusarium oxysporum f. sp. niveum and Sphaceloma fawcettii. The EC50 values of extracts from Clerodendrum inerme and Myoporum bontioides to spores of Fusarium oxysporum f. sp. niveum were 4.2549g/L and 2.9947g/L, and that of extracts from Clerodendrum inerme and Myoporum bontioides against spores of Sphaceloma fawcettii. were 4.9397g/L and 4.2949g/L.According to above results, Clerodendrum inerme was selected for further investigation. The inhibitory activities of extracts from different parts of Clerodendrum inerme against seven pathogens were measured in laboratory with growth rate method. The results showed that the extracts from leaf had the highest inhibition against seven pathogens. The extracts from leaf of Clerodendrum inerme had significant inhibition against Thielaviopsis paradoxa, Alternaria kikuchiana, Mycosphaerella sentina, Sphaceloma fawcettii and Glorosprium musarum with the inhibition rates being above 60%.The methanol extract from leaf of Clerodendrum inerme was preliminarily separated into 4 parts by liquid-liquid extraction. The petroleum ether fraction had high inhibition against Thielaviopsis paradoxa, Alternaria kikuchiana, Mycosphaerella sentina, Sphaceloma fawcettii, Glorosprium musarum, with the inhibition rates of 96.47%, 96.70%, 62.84%, 70.52% and 70.26%, respectively. The ethyl acetate fraction had high inhibition against the above five pathogens, with the inhibition rates of 94.13%, 96.57%, 61.37%, 63.70% and 58.43%. The n-butanol fraction and the water fraction had low inhibitory activities. The results showed that the petroleum ether fraction and the ethyl acetate fraction were the most effective portions.Further separation of the petroleum ether fraction was conducted with silica gel column chromatography. Bioactivity was measured with growth rate method. The results indicated that KLS had the highest inhibition against the five pathogens. Further separation of the ethyl acetate fraction was conducted with silica gel column chromatography, and KLS was isolated from it. Further separation of KLS was conducted with ODS-A column chromatography, and two compounds were isolated. Results from TLC test with different solvent system and EI-MS and NMR showed that two compounds were kaurenoid diterpenes.Toxicity of KLS-46 to the five pathogens was measured with growth rate method. The results indicated that the EC50 values of KLS-46 to Thielaviopsis paradoxa, Alternaria kikuchiana, Mycosphaerella sentina, Sphaceloma fawcettii, Glorosprium musarum were 1.0993×10-2g/L, 9.7912×10-3g/L, 7.8840×10-2g/L, 6.9873×10-2g/L and 4.1717×10-2g/L, respectively. KLS-46 had similar toxicity to Alternaria kikuchiana with carbendazim, but lower toxicity to the other four pathogens than carbendazim. The inhibitory activities of KLS-46 against spores of Thielaviopsis paradoxa, Fusarium oxysporum f. sp. niveum and Sphaceloma fawcettii were determined with the method of conidia germination. The results showed that the inhibition rates were 98.31%, 100.00% and 98.15%. KLS-46 had significant inhibition against spores of all three pathogens.Toxicity of KLS-54 to the five pathogens was measured. The results indicated that the EC50 values of KLS-54 to Alternaria kikuchiana, Thielaviopsis paradoxa, Mycosphaerella sentina, Sphaceloma fawcettii, Glorosprium musarum were 9.7753×10-3g/L, 9.2637×10-3g/L, 5.1065×10-2g/L, 9.7486×10-2g/L and 4.7521×10-2g/L, respectively. KLS-54 had similar toxicity to Alternaria kikuchiana with carbendazim, but lower toxicity to the other four pathogens than carbendazim. The inhibitory activities of KLS-54 against spores of Thielaviopsis paradoxa, Fusarium oxysporum f. sp. niveum and Sphaceloma fawcettii were determined. The results showed that the inhibition rates of KLS-54 were 99.83%, 100.00%and 99.33%. KLS-54 had significant inhibition against spores of all three pathogens.