Studies On The Active Compounds Against Penicilum. Italicum From Propolis And Its Antifungal Action

Propolis is a resinous material collected by bees from plant buds and exudates, which is employed for resisting pathogenic microorganism, construction and repair of the honeycomb and sterilization of surroundings. There was a substantial evidence indicating that propolis had broad antimicrobial activity and was named as "natural antibiotics". At the same time, propolis is a complex mixture of chemical constituents including flavonoids, phenolic acids and esters, terpenes, fatty acids and amino acid etc. More than 300 kinds of compounds were identified from propolis of different origin. It indicated propolis was a desirable material for screening out natural antimicrobial. In current study, Penicillum italicum （P.italicum） , an important pathogenic fungi which lead to blue mold during the citrus storage as the indicator., bioassay was used to separate the active compound ; the action mechanism of active compounds against P.italicum was studied; Inhibitory effect of propolis ethyl acetate extract（PEAE） against citrus blue mold was evaluated. The main results as follow:1. The extracted solvent and origin of propolis affected the chemical constituent and antifungal activity against P. italicum. Propolis ethyl acetate extract（PEAE） and propolis ethanol extract （PEE） demonstrated the most powerful activities, followed by propolis chloroform extract, while propolis petroleum ether extract and propolis water extract showed weak acivities, and the inhibitory activities were consistant with the contents of flavonoids or phenols in the corresponding extracts. Propolis from Hebei Baoding was of the highest contents of flavonoids or phenols and strongest inhibitory effect on the P. italicum; followed by Hebei Yongnian propolis; Propolis from Hubei Dangyang was last one.2. The antifungal fractions from PEE and PEAE were separated by organic solvent extract, thin-layer chromatography （TLC） or column chromatography combined with bioassay and they had the similar chemical constitutes assayed by HPLC. The color reaction and UV behaviour for flavonoids were determined to identify the substance in the active fraction .The results showed its color became blue when meeting with NaHBO₃; Its characteristic wavelength was 292 nm in methol and would be shifed 38nm to the longer wave in MeONa or NaAc. This main flavonoids in active fraction were of the typical characteristic of 5,7-dihydroxyflavanone. Using the HPLC-PDA-ESI-MS method, it was deduced that the main compounds in the active fraction were pinobanksin, pinocembrine, chrysin and galangin; and pinocembrine had the highest relative content in the four compounds.3. Purified pinocembrine, chrysin and galangin were attained by further separating active fraction through sephadex LH-20 chromatography, which purity for HPLC were 87.13%, 97.00%, 94.55% respectively. Among the three compounds, pinocembrine had the strongest inhibitory effect against P.italicum, followed by chrysin, and galangin was the last one; Their inhibition percents against P.italicum were 70.28%, 34.12%,12.5% separately at the concentration of 200 mg/L.4. Effect of pinocembrine on the P.italicum were evaluated by growth rate method . Pinocembine appeared obviously inhibitory effect on the P.italicum and had the line relaxtion between the concentration and inhibition rate. The regression equation of toxicity was y = 1.77 lnx+1.54, （ r =0.98283, P =0.01717）. EC₅₀=89.06 mg/L and EC₉₀ =469.78 mg/L. At the same time, pinocembrime were still of the stable antifungal activity at at the temeperature range of 35～100℃and pH4～7.5. Pinocembrine treatment damaged the cell structure of P.italicum during spore germination and mycelium culture. Microscopic structure of P.italicum hyphas treated with pinocembrine became swollen and abnormal, the protoplasm concentrated and the cell wall degraded, which indicated that hyphas deem to death. The results of ultrastructure indicated evident plasmolysis, intracellular components were extravasation. At the same time, activity of chitinase andβ-1, 3-glucanase were higher than contolled after treated with pinocembrine. Pinocembrine also destroyed the integrality of cell membrane and the relative leakage rate was increasing greatly after treated with pinocembrine.6. Indexes on respiration metabolism and material metabolism were measured , the results indicated mycelium treated with pinocembine had the lower respiratory rate , ATP, ADP , AMP and protein contents . After culturing 8h and 24h, protein in the treated mycelium were only up to 62.29% and 42.72% compared with controlled. While the reduced sugar content in treated mycelium was higher than controlled mycelium at 24 h. It suggested that pinocembrine treatment blocked energy metabolism of mycelium through inhibited respiration; At the same time , Cell wall degrading enzyme in the mycelium accelerated the degradation of chitin and dextran. therefore , it led to the accumulation of reduced sugar and decreasing of protein, mycelium became deathward.7. Compared with control, PEAE treatment decreased the disease incidence of blue mold and green mold , and delayed the decline of color , total soluble solids（TSS） ,titratable acidity（TA） ,vitamin C level and eatable quality of citrus fruits during storage at 26°C. Therefore, PEAE could effectively controlled postharvest diseases of citrus fruit during storage.8.Expressive time of propolis extract on the induced resistance against citrus blue mold and influence of propolis extract on the activities of defensive related enzymes were investigated. The results showed that at 48 h after treatment with propolis extract, citrus fruit exhibited the strongest resistance against blue mold. Propolis extract treatment maintained higher level of the total phenolic contents, enhanced activities of phenylalanine ammonia-lyase, polyphenol oxidase and peroxidase, and stimulated chitinase in citrus fruit peel. Therefore, propolis treatment strengthened the defense system of citrus fruits and reduced disease incidence of citrus blue mold, and appeared great potential on postharvest disease control of citrus fruit.