| Preliminary studies in the laboratory confirmed that the extracts of the Indocalamus tessellatus(I.tessellatus)had antifungal activity against Alternaria alternate(A.alternate),but the antifungal mechanism was not clear.In this paper,the I.tessellatus leaves were used as experimental material,and the tobacco A.alternate was used as test strain.The antifungal components from I.tessellatus leaves were isolated and identified.Then,the antifungal mechanism of the antifungal components was explored preliminarily.The crude extracts of I.tessellatus leaves were obtained by flash extraction technique.The extraction condition was as follows: I.tessellatus leaves powders which particle size were less than 0.425 mm,were mixed with ethanol,glacial acetic acid and pure water at the ratio of 3:5:3:12(g:m L:m L:m L),the mixture was flash-extracted(flash extraction conditions: immersion for 40 min,voltage 120 V,and 40 s),negative-pressure filtration,the extraction was taken twice.The extracts were combined,concentrated and dried.Then the extracts were dissolved in ethanol,negative-pressure filtration,and the ethanol part was concentrated and dried as the crude extracts.The antifungal activity of the crude extracts from I.tessellatus leaves was determined by mycelium growth rate method and spore germination method.The results showed that the EC50 of the crude extracts to mycelium growth of tobacco A.alternate was 3.08 mg/m L,and the EC50 of the crude extracts to spore germination of tobacco A.alternate was 3.74 mg/m L.Three organic reagents(petroleum ether,ethyl acetate and n-butanol)were used to extract and separate the crude extracts.The antifungal activity of each extract phase was evaluated by mycelium growth rate method.The results showed that the ethyl acetate phase had the highest inhibitory activity to the mycelium growth of tobacco A.alternate,and the inhibitory rate was 54.39% at 3.67 mg/m L.The toxicity test showed that the EC50 of ethyl acetate extracts of I.tessellatus leaves to mycelium growth of tobacco A.alternate was 3.67 mg/m L,and to spore germination was 1.47 mg/m L.Separation and purification of ethyl acetate phase from the crude extracts of I.tessellatus leaves was carried out by Silica gel column chromatography,eluted by changing the polarity of the elution.Finally,the components(Fr.1.2 and Fr.1.3)which showed the best antifungal activity were obtained.The analysis of the components was taken by high performance liquid chromatography-mass spectrometry(LC-MS).The results showed that the Fr.1.2 mainly contained isoswertiajaponin,orientin,or 5,2',4'-trihydroxy-6,7,5'-trimethoxyflavone,and the Fr.1.3 mainly contained tricin-7-O-?-D-glucoside,tricin,diosmetin,or 7-hydroxy-5,3,4'-trimethoxyflavone.The ethyl acetate phase extract from the crude extracts of I.tessellatus leaves was used to study the antifungal mechanism on tobacco A.alternate.The results showed that the antifungal components of the I.tessellatus leaves could destroy the membrane structure of the mycelium of tobacco A.alternate,the electrolyte and nucleic acid in the mycelia outflowed and achieved the balance between internal and external under the condition of 4 mg/m L for 30 hours of treatment.During the mycelia growth of tobacco A.alternate,the antifungal components could inhibit the synthesis of glucan,ergosterol and soluble protein in the mycelia.Compared to the control,the characteristic absorption peak amplitude of each functional group decreased significantly,ergosterol content decreased by 22.34%,and the inhibition rate of soluble protein synthesis was 12.70%.The antifungal components of I.tessellatus leaves could promote sporulation of A.alternate,but the germination rate of spores decreased.Scanning electron microscopy showed that the surface of spores in the experimental group was bulging,the spores were more vulnerable to injury.The target of antifungal constituents components of the I.tessellatus leaves may be in the cell membranes or some metabolic pathways. |
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