Screening Studies On Fungistasis Of Plant Extracts And Isolation Of Active Substance

Thirty-three species of plant including 24 families collected from north-east and south-east in Guangxi province in China were extracted with methanol. Bioactivity of these extracts against Fusarium oxysporum f. sp. Niveum, Pestalotia calabae Westond, Thielaviopsis paradoxa (de Seynes) V. Hohnel, Glorosprium musarum(Berk & Curt) Arx, Alternaria kikuchiana Takana, Mycosphaerella sentina(Fr.) Schroeter and Sphaceloma fawcettii Jenkins were measured by mycelium growth rate method at concentration of 10g/L after treated 72h. The results showed that the extract of Acorus tatarinowii Schott has a broad spectrum of antimicrobial, Test for the 7 species of plant fungal pathogens have 100% of the antibacterial activity. The extract's inhibition from Mahonia bealei (Fort.) Carr. to Pestalotia calabae Westond is 94.78%. Most plant extracts had strong inhibition to Alternaria kikuchiana Takana, the inhibitions of Eucalyptus tereticornis Smith, Camellia oleifera Abel., Schima Superba, Flueggea suffruticosa (Pall.) Baill and Rhus chinensis Mill(root) were 100%, the antifungal activity of Eucalyptus robusta Smith and Toxicodendron succedaneum (L.)O.Kuntze were also more than 90%, the extract of Schima Superba is the most effective to Alternaria kikuchiana (Fr) Keissl and it's antimicrobial activitie is 100% with concention of 2g/L.The inhibitions of Piper puberulum (Benth) Maxim to Mycosphaerella sentina(Fr.) Schroeter and Atropa belladonna L.(fruit) to Sphaceloma fawcettii Jenkins were more than 90%. 18 species plant extracts from Eucalyptus tereticornis Smith, Camellia oleifera Abel., Eucalyptus citriodora Hook.f., Buddleja asiatica Lour., Cocculus orbiculatus (Linn.) DC., Atropa belladonna Linn., Dalbergia hupeana Hance, Rhus chinensis Mill.(root), Holboellia fargesii Reaub., Flueggea suffruticosa (Pall.) Baill, Evodia lepta (Spreng.) Merr., Mahonia bealei (Fort.) Carr., Schefflera minutistellata Merr. ex Li, Evodia fargesii Dode, Photinia prunifolia (Hook. et Arn.) Lindl., Tacca chantrieri Andre, Cassytha filiformis Linn., Piper wallichii (Miq.) Hand.-Mazz. have inhibition to hypha. Their inhibition ratio are higer than 60% separately. But Croton lachnocarpus Benth., Strophanthus divaricatus(Lour.) Hook. et . Arn., Lygodium japonicum-(Thunb.) Sw., Rhus chinensis Mill.(leaf), Gelsemium elegans(Gardn. et Champ.) Benth., Tacca chantrieri Andre and Actinidia chinensis Planch. have promotion to hypha.In order to further compare the antifungal activity of plant extract, toxicty test of 13 higher activity plant extracts were further determined, the results indicated that the toxicty of Acorus tatarinowii Schott to 7 species of pathogens are more than 1.0g/L. The toxity of Schima Superba to Alternaria kikuchiana (Fr) Keissl is most distinct, it's EC50 of methonel extract is only 0.0532 g/L. The EC50 valus of 72h of Camellia oleifera Abel again Thielaviopsis paradoxa (de Seynes) V. Hohnel and Alternaria kikuchiana Takana are respectively 0.8374g/L and 0.6956g/L. Buddleja asiatica Lour. to Thielaviopsis paradoxa (de Seynes) V. Hohnel and Atropa belladonna L. to Glorosprium musarum(Berk & Curt)Arx are also distinct, which EC50 are 0.7320g/L and 0.9012g/L relatively.Based on above results, Schima Superba and Acorus tatarinowii Schott. were selected for further investigation of their active ingredients. The methanol crude extract from Schima Superba was preliminarily partitioned into three parties by solid-liquid extraction with petroleum ether, trichloromethane, n-butanol. The n-butanol fraction was found to be the most effective part in inhibiting mycelial growth of Alternaria kikuchiana Takana. Which fungistatic rate was 99.35% with concention of 1g/L after treated 72h. The methanol crude extract from Acorus tatarinowii Schott. was preliminarily partitioned into four parties by liquid -liquid extraction with petroleum ether, ethylacetate, n-butanol and water. The activity of petroleum ether extract against 7 species of pathogens was much higher than that of the others, suggesting that the active ingredients be mainly existed in the part of petroleum ether extract. That's inhibitive rate are 100% with concention of 5g/L.Further separation of n-butanol estract of Schima Superba was conducted with silica gel column chromatography. Bioactivity was measured with Mycelium growth rate method. The results indicated that HM1-147~153 was the most effective against Alternaria kikuchiana Takana at a concention of 0.2g/L. Inhibitory effects of 24h, 48h, 72h were 86.67%, 80.40%and 81.42% respectively. Compounds of of HM1-147~153 were conducted with polyamide column chromatography. HM2-2~3 and HM2-1 have good inhibition to mycelial growth at concention of 0.1g/L. Their antifungal rate of 72h were 89.57% and 77.73%. They also could inhibite the mycelial growth of Thielaviopsis paradoxa (de Seynes) V. Hohnel. Their inhibition rate were 71.43% and 67.57%.Compounds of petroleum ether extrac of Acorus tatarinowii Schott. were isolated by silica gel column chromatography. Scp1-5~6 and Scp1-7~15 were tested to be the most effective part in inhibiting mycelial growth of 7 species of pathogens. Their activity were much higher than that of others and their antibacterial ratio are 100% with concention of 2g/L after 72h treated.Further separation and Purification of Scp1-5~15 by silica gel column chromatography and solvent recrystallization. Bioactivity was measured by means of mycelium growth rate and spore germination. Scp2-9~14 has high inhibition rate to 7 species of pathogens. Further Purification of Scp2-9~14. Result from TLC test with 4 different developing solvent system and were coloured by vapor of iodine, ultraviolet, sulfuric acid and identified by spectrum analysis of 1H-NMR, 13C-NMR, MS and in comparison with data reported in previous references. The main active compound was 1,2-dimetoxy-4-(2-propenyl)benzene (DPB2). Bioassay results revealed that EC50 values of 72h of DPB2 to Thielaviopsis paradoxa (de Seynes) V. Hohnel, Pestalotia mangiferae P.Henn, Fusarium oxysporum f. sp. Niveum, Alternaria kikuchiana Takana, Glorosprium musarum (Berk & Curt)Arx, Sphaceloma fawcettii Jenkins, Mycosphaerella sentina(Fr.) Schroeter were 0.1021g/L, 0.0997g/L, 0.0805g/L, 0.1742g/L, 0.1503g/L, 0.1853g/L and 0.1924 g/L.