IN VITRO ANTIBACTERIAL EVALUATION OF CRUDE EXTRACT AND SOLVENT FRACTIONS OF CLERODENDRUM MYRICOIDES AND KALANCHOE DENSIFLORA AGAINST SELECTED BACTERIAL ISOLATES CAUSING BOVINE MASTITIS.

Abstract

Background: Mastitis is a bacterial disease with estimated annual costs of 35 billion USD in the global dairy industry. Although antibiotics are the remedies in mastitis treatment, their effectiveness is lost because of the presence of resistant microbes; so, finding a new remedial option by evaluating the antimicrobial activities of medicinal plants is extremely interesting. Ethiopian plants have potential therapeutic value, yet are confined to traditional practices, where thorough studies reporting in vitro and in vivo antimicrobial properties are rare. Thus, this study aims to evaluate in vitro antibacterial effects of crude extract and solvent fractions of C. myricoides and K. densiflora against S. aureus and E. coli cause bovine mastitis. Methods: An experimental study was conducted on the fresh roots of both plants collected under the guidance of traditional healers. Sample was cleaned, air-dried, and then subjected to cold maceration via 80% methanol with successive fractionation in hexane, chloroform, absolute methanol, and water in order of polarity, and tested for phytochemical ingredients. In vitro antibacterial activity was evaluated via agar well/disc diffusion, broth macrodilution, and time‒kill kinetics assays. The obtained data were analyzed by SPSS version 20 software at the 95% confidence level, and a P value < 0.05 was considered statistically significant. Results: The maximum yield (40.6%) was obtained from crude extracts of C. myricoides, whereas the minimum yield (23.8%) was gained from its hexane fraction. Phytochemical analysis revealed the presence of all the tested secondary metabolites in the crude extracts of both plants. Among all the tested extracts, the crude extract from C. myricoides was the most effective, producing the highest ZOI (24.7 and 22 mm) for S. aureus and E. coli, respectively. The plant had a strongest MIC value of 3.9 mg/mL, with the corresponding MBC value of 7.8 mg/mL in S. aureus. The hexane fraction of K. densiflora had the lowest antibacterial effect, with a ZOI of 6.3 mm in S. aureus. The results from solvent fractions revealed the strongest effects in the methanolic extracts, followed by the subsequent activities shown by fractionates of water, methanol, chloroform, and hexane for both plants. C. myricoides at 4× MIC showed concentration-dependent killing kinetics within the first 2 hrs of incubation in S. aureus. K. densiflora exhibited only bacteriostatic in E. coli. Conclusion: These findings suggest the promising activity of the selected plants, upholding their standardized use as alternative remedies. Thus, further studies on essential bioactive, combination effects, and in vivo tests are warranted.