Preparation, Purification And Identification Of Methanol Extract Of Cynanchum Paniculatum And Neutralization Efficacy Of Deinagkistrodon Acutus Venom

Snake venom is a complex mixture with diversely biological activities and pharmacological activities, mainly consisting of proteins, enzymes, peptides, small molecules and other components, of which the protein(enzyme, peptide) accounts for more than 90%. Venom from the wound into the tissues and organs of victims during and after biting, its various components individually or cooperatively exercise different biological function, ultimately leading to the disorder at the physiological and biochemical activities of the victims. Most victims appear such as flaccid paralysis,bleeding and clotting disorders, edema, muscle necrosis and local tissue ulceration and other symptoms, while a small number of victims will appear ptosis, shortness of breath and other symptoms.At present, anti-snake venom serum is certainly the most effective direct treatment to snakebites. However, the antiserum has many deficiencies. There are various side effects of antiserum such as anaphylaxis, serum sickness, and some bite complications.The disadvantage of antiserum is time consuming, low yielding, expensive and difficult to save. Therefore, we hope to find new complementary therapies to treat snakebite.Snakebite occur at home and abroad(such as Asia, Africa and South America),commonly used herbs first to aid snakebite victims. People will aim at the active substances in these herbs, especially those that are naturally inhibitors and pharmacodynamics of the active compounds.In this study, we used nine kinds of common medical herbs in Chinese medicines(Eclipta prostrata, Curcuma longa L., Houttuynia cordata T., Andrographis paniculata N., Aristolochia fordiana H., Paris polyphylla, Lysimachia paridiformis,lemon B., Cynanchum paniculatum) to do comparative study on the activity of snake venom. Experiments show that Cynanchum paniculatum has the best anti-venom activity. Then we selected Cynanchum paniculatum as the focus study, carried out extracts from Cynanchum paniculatum, then isolated and purified the active constituents from the extracts, finally designed in vivo and in vitro experiments about the activeconstituents to neutralize D. acutus venom.Firstly, Cynanchum paniculatum was extracted with 75% methanol, obtained the methanol extracts and experimental determination of the activity, the results show that:(1) Cynanchum paniculatum methanol extracts exhibited no hemolysis and no damage to mice.(2) The methanol crude extract could neutralize the activities of phospholipases A2, plasmin, hyaluronic acid enzyme with dose-effect relationship.(3) SDS-PAGE experiment further demonstrated Cynanchum paniculatum methanol extracts may neutralize fibrinolytic enzyme activity, with dose-effect relationship.(4) Cynanchum paniculatum methanol extracts could alleviate symptoms of edema on feet and bleeding in the back of the mice, the treatment effect was dose-dependent.Secondly, the Cynanchum paniculatum crude methanol extract was extracted by organic solvents(petroleum ether, chloroform, ethyl acetate, n-butanol) and sephadex LH-20 gel chromatography, obtained three major peaks C1, C2 and C3, and then after fibrinolytic activity experment test, the results showed that only peak C2 with the strongest neutralizing effect, but almost no effect on C1 and C3.Finally, the peak C2 was further isolated by RP-HPLC, and then many peaks were obtained, among which three major peaks P1, P2 and P3 were chosen. After fibrinolytic activity tests, the results showed that only peak P2 with the strongest neutralizing effect,but P1 and P3 peaks without any effects. Peak P2 was identified as paeonolide by mass spectrometry and NMR. The paeonolide could completely neutralized the activity of the hyaluronic acid enzyme. Meanwhile, animal experiments showed that the symptoms of bleeding and edema caused by venom were reduced.In summary, the present study was the first to obtain one anti-D. Acutus venom active consitituent from Cynanchum paniculatum and identified it as paeonolide, which reveals the substance basis that Cynanchum paniculatum was an effective herb to treat snakebite by D. acutus.