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MAIN CONCLUSION In vitro antimicrobial activity testing of Bassia longifolia methanolic bark extract shows antibacterial activity against gram-positive Staphylococcus aureus strains, both methicillin sensitive S. aureus (MSSA) and methicillin resistant S. aureus (MRSA), as well as anti-biofilm activity on those bacterial strains. The analytical investigation of this extract reveals that present polyphenols consist mainly of tannins, known for their antimicrobial potential. INTRODUCTION The bark of Bassia longifolia Koenig (=Madhuca longifolia Koenig, Sapotaceae) is known to be applied externally as paste in the treatment of cuts and wounds and, as such, represents a part of the phytomedicine in Nepal [1, 2]. This experiments were undertaken to explore the in vitro antimicrobial activity of the methanolic bark extract and its phytochemical composition. After a primary antimicrobial screening on several types of microbes, additional anti-biofilm activity testing was performed on S. aureus experimental models, both methicillin sensitive (MSSA) and methicillin resistant (MRSA) strains. MATERIALS & METHODS Total polyphenolic and tannin contents of methanolic bark extract were determined and expressed as catechin [3]. Standard laboratory microbial strains including S. aureus ATCC 6538, Escherichia coli ATCC 10536, Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404, as well as clinical methicillin resistant S. aureus MFBF 10679 (MRSA), from stock-cultures of the Collection of Microorganisms (MFBF) of the Department of Microbiology, Faculty of Pharmacy and Biochemistry University of Zagreb were used. The Agar well diffusion assay was performed following European Pharmacopoeia guidelines (c = 20.00 mg mL–1 ) [1]. Minimal inhibitory concentrations (MICs) were investigated following EUCAST guidelines [2]. MIC was defined as the lowest concentration of extract that allows no more than 20% growth of microbes in comparison with the extract-free control. The effect on biofilm formation of MSSA and MRSA was evaluated with a crystal violet assay [3]. RESULTS & DISCUSSION The analytical investigation of the methanolic bark extract reveals that the present polyphenols consist mainly of tannins. A total polyphenolic content of 43.1% ± 1.4% was determined and tannins represent the majority with 42.2% ± 2.0%, both expressed as catechin. In vitro antimicrobial activity testing of this extract shows antibacterial activity against gram-positive S. aureus strains, both methicillin sensitive S. aureus ATCC 6538 (MSSA) and methicillin resistant S. aureus MFBF 10679 (MRSA). Measured zones of growth inhibition (ZI) in the Agar well diffusion assay for MSSA and MRSA were 16 ± 1 mm and 15 ± 0 mm, respectively. Minimal inhibitory concentration (MIC) in the Serial microdilution broth assay showed to be 208.17 ± 90.36 µg mL-1 for both strains. No antimicrobial activity was observed for other tested microbial strains in given experimental conditions (E. coli ATCC 10536, C. albicans ATCC 10231 and A. brasiliensis ATCC 16404). The anti-biofilm activity of B. longifolia methanolic extract on biofilm formation of MSSA and MRSA was expressed Polyphenols Communications 2021 224 P2.8 as minimal biofilm-forming inhibition concentration (MBFIC). MBFIC50 and MBFIC90, calculated from linear regression of the log10 of extract concentration vs. % biofilm reduction, represent the lowest extract dilutions at which biofilm formation was inhibited by 50% and 90%, compared to the untreated control. The values for MSSA are as follows, MBFIC50 = 1.38 µg mL-1 and MBFIC90 = 1044.59 µg mL-1 , while values for MRSA showed to be higher, MBFIC50 = 2.52 µg mL1 and MBFIC90 = 1138.43 µg mL-1 . Obtained antimicrobial activity data in this study are generally in agreement with tannins being well known for their antimicrobial activity. Shown in vitro antimicrobial activity of B. longifolia methanolic extracts gives impetus for further in depth antimicrobial and other bioactivity testing in the future, emphasizing in particular resistant S. aureus strains. REFERENCES [1] Council of Europe 2005. European Pharmacopoeia 188–191. [2] ESCMID, EUCAST E.Dis 5.1. 2003. Clinical Microbiology and Infection 9: 1–7. [3] Vlainić, J. ; Kosalec, I. ; Pavić, K. ; Hadjipavlou-Litina, D. ; Pontiki, E, ; Zorc, B. 2018. Journal of Enzyme Inhibition and Medicinal Chemistry 33: 376–82. |
