HPLC Purification Of Antimicrobial Peptides From Northeast Forest Frogs And The Study Of Anti-Influenza Virus Effect In Vitro

The skin bioactive peptides (BAPs) have many families and lots Of functions, such as anti bacteria, anti fugin, anti tumor and so on. It reported that AMPs have inactivated properties to virus, and do no harm for host cells. For the unique characters of amphibian skin peptides, it is hoped to be the important way of exploring new antibiotic. Cell culture is one of filtrations of antivirus drug, and its characteristics of sensitivity, differential, fast observed, better repetition and less drugs make it a mul-target filtration.Method: The experiment animal is northeast forest frog (Rana dybowskii), we use electrical stimulation to distill the skin secretion, and the secretion purified by Sephadex G-100,50,25. HPLC purification system has been established, and the liquid phase, folw rate has been tested. SAH hydrolase was use to filtrate dybowskii. The candidate components will be study on the cell. In the MDCK cell culture experiment in vitro, antiviral activity against human-influenza virus was estimated in terms of cytopathic effect, Viral titre yields and OD values of MTT colorimetric method to determine the respective effects of anti-penetration. activity against virus. The anti-virus mechanism of the effective anti-influenza virus was studied in three ways: adding medicine to the culture MDCK cell before adding virus, adding virus to the cultured MDCK cell before adding medicine, and adding medicine and virus to the cultured MDCK cell after they mixed for a while.The results were as follows. (1) a mixture of V (acetonitrile): V (methanol): V (water)=5:5:90 is used as liquid phase and the flow rate is 0.5ml·min^-1, UV detection wavelength is performed at 216 nm and 280 nm. Secretion purified by Sephadex G-100,50,25 and HPLC, we gained dybowskiiⅠ～Ⅺat last. (2) dybowskiⅠ,Ⅲ,Ⅳ,Ⅵ,Ⅶcan restrain the SAH hydrolase. (3) In the MDCK cell culture experiment in vitro, antiviral activity against humaninfluenza virus was estimated in terms of cytopathic effect. In the groups of adding medicine before virus, dybowskiiⅢ,Ⅳ,Ⅵ,Ⅶhad shown anti-penetration acticity, and the anti-virus rates are 97.7%,91.7%,70.7% and 78.2% respectively. DybowskiiⅣprovided good protection for cell and good effect in restraining virus propagation. The anti-virus rate was 82.8%, but lower than anti-penetration activity. The results of the three different ways indicated that dybowskiiⅢand dybowskiiⅣprovided good anti-penetration activity, dybowskiiⅣalso showed better effect in restrainning virus propagation, and had good protection for cell. All these samples showed no directly destroy virus activity.Conelutions: (1) We have established HPLC system to purify the Northeast forest frog's skin secretion and we have gained the BAPs dybowskiiⅠ～Ⅺ. (2) dybowskiⅠ,Ⅲ,Ⅳ,Ⅵ,Ⅶcan restrain the SAH hydrolase. (3) We used 3 ways to add drugs in MDCK cell culture, and they provide new method and means to study-the anti-virus activities of Northeast forest frog's skin secretion.There are planty of amphibians' resource in China, especially the Northeast forest frogs in Heilongjiang provience. We found the ant-virus BAPs from the skin secretion in the Northeast forest frog, and it could become the experimental referece for the production of a new speciall ant-virus drug.