Screening development for anti-hemotoxic activity of Thai herbs against eastern Russell’s viper Daboia Siamensis (Smith, 1917) venom

Abstract

The eastern Russell’s viper, Daboia siamensis, is one of the important venomous snakes in Thailand. Its venom possesses hematotoxin causing pathological alterations to circulatory and renal systems. Although antivenom serum is used for standard medical treatment, its cost per dose, ineffectiveness for some symptoms, and potential to develop allergic reactions in patients has called attention to an alternative remedy including the medicinal herb. To find effective herbs, appropriate screening assays are needed. This study aims to develop in vitro and in vivo screening assays and use for screening Thai herbs with anti-hematotoxic activity against D. siamensis venom. For in vitro assay, five assays corresponding to 5 biological activities of venom were developed. Three assays for phospholipase, coagulation, and fibrinogenolytic activities have been successfully developed and effectively showed a marked difference between positive and negative controls. Two assays for proteolytic and hemagglutination activities were proven to be not suitable for the screening because of the low activity in D. siamensis venom. For in vivo assay, an alternative chick embryo assay for hematotoxic activities of D. siamensis venom was developed. The microanatomy of embryonic vasculature was verified as suitable markers for the hematotoxic effect of snake venom. Subsequently, 10 herbal extracts were screened with the validated in vitro assays. The results showed that Areca catechu L. is the most effective herb with 45.93% inhibition against the venom. Various concentration of A. catechu extract was used to neutralize D. siamensis venom at LD50 concentration (6.35 µg/µL), and the mixture applied to the chick embryo. It was found that A. catechu extract reduced the mortality of chick embryo in a dose dependent manner. The median inhibitory dose (ID50) of A. catechu against D. siamensis venom in chick embryo during 4-hour exposure was calculated at 4.42 µg/µL. With the strong anti-hemotoxic effect and the low toxicity of A. catechu (LD50 of 445.16 µg/µL), this herb should be further developed into the herbal remedy for snakebit treatment.