Bioassay-guided identification of Chlorogenic Acid as an anti-snake venom constituent of Ficus racemosa L.: An in vitro and in silico study

Bioassay-guided identification of Chlorogenic Acid as an anti-snake venom constituent of Ficus racemosa L.: An in vitro and in silico study

Abstract

Ethnopharmacological relevance

The inherent limitations of the current anti-snake venom serum therapy have prompted the search for alternative treatment strategies for snakebite envenomation, a neglected tropical disease of the world. Several plant-derived bioactive compounds were reported to possess anti-ophidic properties.

Aim of the study

To evaluate the snake venom toxin neutralizing properties of traditional medicinal plants from Barpeta district of Assam, India.

Materials and methods

Extracts from 19 traditional medicinal plants collected from Barpeta district of Assam, India were screened for anti-snake venom properties. Selected anti-ophidic extract underwent bioassay-guided fractionation using preparative thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). HR-LCMS analysis was employed to identify the major phytocompounds in the bioactive fraction. Molecular docking studies followed by in vitro validations were performed with targeted proteins from Indian “Big Four” snake venoms.

Results

Among all the tested extracts, the aqueous extract of Ficus racemosa bark (AEFRB) demonstrated 100 % neutralization of some of the enzymatic activities of Naja naja and Daboia russelii venom. The HR-LCMS analysis tentatively identified 19 major phytocompounds in the bioactive fraction AEFRB-P3. Among those, Chlorogenic acid (CGA) showed promising binding affinities within the catalytic site of Daboxin P, a PLA2 isolated from Daboia russelii venom. CGA demonstrated in vitro neutralization of the enzymatic activity of Daboxin P with an IC50 of 28.248 ± 1.104 μM. Additionally, CGA demonstrated dose-dependent neutralization of PLA2, anti/pro-coagulant, proteolytic, and indirect haemolytic activities of Indian “Big Four” snake venoms under in vitro conditions.

Conclusion

This is the first mechanistic in vitro characterization of the anti-ophidic properties of F. racemosa and identification of CGA as the active constituent. These findings require further in vivo validation, pharmacokinetic evaluation, safety assessment, and synergistic formulation to translate mechanistic insights into clinically viable therapeutic interventions for management of snakebite envenomation.

Thakuria, J., Saha, D., Chakraborty, R., Mukhopadhyay, R., & Doley, R. (2026). Bioassay-guided identification of Chlorogenic Acid as an anti-snake venom constituent of Ficus racemosa L.: An in vitro and in silico study. Journal of Ethnopharmacology, 358, 121021. https://doi.org/10.1016/j.jep.2025.121021