Isolation,Structure Identification And Antifungal Activity Of Components From Tupistra Chinensis And Application

Postharvest fruits are vulnerable to fungal infection in the process of storage and transportation,due to mechanical damage or resistance reduced in the process of ripening,resulting in rot,deterioration,and loss of edible value,and causing a huge waste of resources and economic losses.It is of great significance for the development of agricultural economy of China to strengthen the preservation of fruits and reduce the decay of fruits.In recent years,natural resource preservatives have attracted extensive attention in the field of fruit storage due to their low toxicity and environmental pollution.Plant resources are renewable and rich in China.The application of plant extracts in preservation is correspond with the concept of national green development,and has important economic value and practical significance.In this paper,isolation,structure identification,antifungal activity and mechanism of components from T.chinensis rhizomes were studied,the effects of extract of T.chinensis as antifungal agent on postharvest storage quality of Ponkan were studied.The main research contents and results are as follows:（1）Five kinds of plants:rhizome of T.chinensis,the leaves of Artemisia argyi,Xanthium sibiricm,Eucalyptus tereticornis and Pyrola calliantha as raw materials,the inhibitory effects of95%ethanol crude extracts on six kinds of fruit saprolegnoid fungi such as Penicillium digitatum,Rhizopus stolonifera and Botrytis cinerea were determined by mycelial growth rate method.Through comprehensive comparison,it was found that the 95%ethanol extract of T.chinensis had good antifungal effects.The crude 95%ethanol extract was dispersed with water,and then extracted with petroleum ether,ethyl acetate and n-butanol in turn,concentrated and dried to obtain petroleum ether extracts,ethyl acetate extracts and n-butanol extracts,and their activities were determined.The results showed that ethyl acetate extracts had good antifungal activity.（2）The ethyl acetate extracts from rhizomes of T.chinensis were separated by various chromatographic separation methods（silica gel column chromatography,rapid preparative chromatography,PTLC and PHPLC,etc.）and identified by MS,IR,NMR(¹H NMR,¹³C NMR,HSQC,¹H-¹H COSY,HMBC and NOESY).Among the 27 compounds isolated,20 compounds were steroidal saponins including 13 new compounds,which were 2β-acetoxy-1α,3α,4α,5α,7α-pentahydroxy-spirost-25（27）-en-6-one（1）,（25S）-2β-acetoxy-1α,3α,4α,5α,7α-pentahydroxyspirostan-6-one（2）,3β-acetoxy-1β,2β,4β,5β,7β-pentahydroxy-spirost-25（27）-en-6-one（3）,（25S）-2α-acetoxy-1β,3β,4β,5β,7β-pentahydroxyspirostan-6-one（4）,（25S）-4β-acetoxy-1β,2β,3β,5β,7β-pentahydroxyspirostan-6-one（5）,Spirost-（25S）-1α-acetoxy-2β,3β,4β,5α,6α,7β-hexaol（6）,（25S）-1β-O-acetyl-2β-hydroxyspirostan-3-O-β-D-glucopyranoside（7）,1β,5β-dihydroxyspirostan-25（27）-en-3-O-acetyl-β-D-glucopyranoside（8）,（25R）-1β-hydroxyspirostan-5（6）-en-3-O-acetyl-β-D-glucopyranoside（9）/（25S）-1β-hydroxyspirostan-5（6）-en-3-O-acetyl-β-D-glucopyranoside（10）,（25R）-1α-O-acetyl-5（6）-en-spirostan-3-O-β-D-glucopyranoside（11）/（25S）-1α-O-acetyl-5（6）-en-spirostan-3-O-β-D-glucopyranoside（12）,（25R）-1β,17α-dihydroxy-5β-spirostan-3-O-β-D-glucopyranoside（13）,and seven known compounds were:1β,2β,3β,4β,5β,7α-hexahydroxyspirost-25（27）-en-6-one（16）,（25S）-1β,2β,3β,4β,5β,7α-hexahydroxyspirost-6-one（17）,3-epi-neoruscogenin-3-O-β-D-glucopyranoside（18）,Isoepiruscogenin-3-O-β-D-glucopyranoside（19）,Epiruscogenin-3-O-β-D-glucopyranoside（20）,5β-25（27）-en-1β-hydroxyspirost-3-O-β-D-glucopyranoside（21）,tokorogenin-1-O-β-D-glucopyranoside（22）;Five compounds were cardiosteroids,including two new compounds,14（15）,20（22）-dien-5β-card-3-O-acetyl-β-D-glucopyranosyl-β-D-fucopyranose（14）,5（6）,20（22）-dien-card-3-O-acetyl-β-D-glucopyranosyl-β-D-fucopyranose（15）,and three known compounds,3α-acetoxy-5β-card-14（15）-enolide（23）,3α-hydroxy-5β-card-14（15）,20（22）-dienolide（24）,3α-acetoxy-14α-hydroxy-5β-card-20（22）-enolide（25）;and two compounds:Tupichinol A（26）and Tupichinol B（27）were flavanols.（3）The antifungal activities of the compounds isolated from T.chinensis were studied.The results showed that steroidal saponins（compounds 7,8,9/10,11/12,18,19/20,and 21）had strong antifungal activities against both tested fungal strains,and their IC₅₀ values（molar concentration）were lower than those of the two positive controls（sodium benzoate and potassium sorbate）.The activities of steroidal saponins（compounds 1,2,16,and 17）and cardiac steroids（compounds 14,23,24,and 25）were lower.According to the preliminary analysis of the relationship between structure and activity,the antifungal activity of spirostanol saponins with methyl（R or S）at 25 position of spirostanol aglycone is higher than that of vinyl(Δ^25（27）).（4）The possible antifungal mechanisms of steroidal saponins compounds（19/20）were studied by observing mycelial morphology and measuring cell membrane permeability and ergosterol content of P.digitatum and R.stolonifera.The results showed that steroidal saponins has a great influence on mycelial morphology:the outer wall of mycelia treated with steroidal saponins was rough,thin,wrinkled and curved,the permeability of cell membrane was increased,and the content of ergosterol was decreased,which indicated that the mechanism of inhibiting fungi might be to destroy the cell membrane of fungi.（5）The effect of 95%ethanol crude extracts and ethyl acetate extracts of T.chinensis incorporated into rosin resin coating on postharvest storage quality of Ponkan was studied.The results showed that adding 95%ethanol extracts and ethyl acetate extracts could effectively reduce the decay rate of Ponkan,delay the increase of malondialdehyde content and the decrease of VC content,and maintain the freshness of the fruit.It can be seen that the extracts have good preservative effect on Ponkan,and is expected to be applied in the coating preservative of citrus.