We consistently swabbed the seed culture of the tested pathogen on an agar plate. Then, we separately impregnated sterilized blank paper discs with different crude extract fractions and placed them on the agar plate. We incubated the plates at 37ºC for 16 h. We noted the antibacterial activity by measuring the diameter of the inhibition zone. We used gentamicin (10 µg/disc) as positive control and kept DMSO (< 1%) as a negative control. We ensured that all the experiments had technical triplicates and we performed them twice to render two biological replicates.

We used a broth micro-dilution method to evaluate the minimum inhibitory concentration (MIC) values of crude extracts using Clinical & Laboratory Standards Institute (CLSI) procedures. Essentially, we added each extract (5 μl) into the wells of a 96 well plate comprising 105 CFU/ml bacterial cells. We incubated the 96 well plates at 37 °C for 16 h. We kept the final concentrations ranging from 250 to 2000 µg/ml. In each test, we included three controls comprising, gentamicin 10 µg/ml (as positive), DMSO < 1% final concentration (as solvent) and bacterial inoculum (as negative). We have taken the MIC value as the lowest concentration of the tested extract showing inhibitory effect against the pathogens, recorded via the Microplate reader (TECAN, Infinite-M200-PRO). We confirmed all tests, having technical triplicates, twice. We observed promising results for both the fractions of ethyl acetate and acetone extracts with which we carried out all chromatographic analyses.

In the present study, we performed all the tests in triplicates and expressed the data obtained as the mean ± standard deviation (S.D). We determined the P values using the student’s T-test, two-tailed distribution, (*) is P ≤ 0.05. These have been reflected in Tables Tables11 and and22 and Figure S2.

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