The present study was designed to evaluate antibacterial and cytotoxic evaluation of different extracts of Astragalus membranaceus root against common poultry pathogens. Sequential extraction with hexane, ethanol, chloroform and aqueous solvents was prepared and antibacterial activity was evaluated by using agar well diffusion. Minimum inhibitory concentration (MIC) of plant extracts was evaluated by micro broth dilution test. The extracts exhibiting antimicrobial activity were further evaluated for cytotoxicity by using MTT assay on Vero cell line. Cell culture media was prepared and cell lines were propagated, monolayer was formed. This monolayer was exposed to plant extract dilutions. After 24-48 hours, MTT dye was introduced and cell survival percentage was calculated. Statistical analysis was conducted with Statistic Package for Social Sciences (SPSS for windows version 16, SPSS inc, Chicago, IL, USA). Results of antibacterial activity and MTT assay were compared using DMR posthoc test. Growth of Clostridium perfringens, Escherichia coli, Haemophilus species, Salmonella enterica and Staphylococcus aureus inhibited by all extracts of Astragalus except aqueous extract which shows no zones of inhibition against C. perfringes. MIC values were higher for aqueous extract against all selected bacteria and lowest for chloroform against E. coli, S. enterica and Staph. aureus (208.3ug/ml, 156.25ug/ml, 78.125ug/ml respectively) for hexane against Haemophilus species (833.3ug/ml) and for all three extracts against C.perfringes (1250ug/ml). Hexane, chloroform and ethanol extracts were appeared to be safe at all concentrations except ≥ 2000μg/ml, ≥1000μg/ml and ≥3000μg/ml respectively while aqueous extracts showed cytotoxicity at concentrations ≥625μg/ml. Astragalus membranaceus
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showed antibacterial activity against all selected pathogens. Chloroform and hexane extracts showed greater antibacterial activity than ethanol and aqueous. Cytotoxicity values for chloroform extract are safer than rest of three extracts. Astragalus membranaceus may be used to design traditional medicines for the development of therapeutic agent which will be more safe, effective and economical.