TLC-Bioautography-Guided Isolation and Assessment of Antibacterial Compounds from Manuka (Leptospermum scoparium) Leaf and Branch Extracts.

A rapid procedure for the targeted isolation of antibacterial compounds from Manuka (Leptospermum scoparium) leaf and branch extracts was described in this paper. Antibacterial compounds from three different Manuka samples collected from New Zealand and China were compared. The active compounds were targeted by TLC-bioautography against S. aureus and were identified by HR-ESI-MS, and -MS/MS analysis in conjunction with Compound Discoverer 3.3. The major antibacterial component, grandiflorone, was identified, along with 20 ß-triketones, flavonoids, and phloroglucinol derivatives. To verify the software identification, grandiflorone underwent purification via column chromatography, and its structure was elucidated through NMR analysis, ultimately confirming its identity as grandiflorone. This study successfully demonstrated that the leaves and branches remaining after Manuka essential oil distillation serve as excellent source for extracting grandiflorone. Additionally, we proposed an improved TLC-bioautography protocol for evaluating the antibacterial efficacy on solid surfaces, which is suitable for both S. aureus and E. coli. The minimum effective dose (MED) of grandiflorone was observed to be 0.29-0.59 µg/cm2 against S. aureus and 2.34-4.68 µg/cm2 against E. coli, respectively. Furthermore, the synthetic plant growth retardant, paclobutrazol, was isolated from the samples obtained in China. It is hypothesized that this compound may disrupt the synthesis pathway of triketones, consequently diminishing the antibacterial efficacy of Chinese Manuka extract in comparison to that of New Zealand.

An improved MTT colorimetric method for rapid viable bacteria counting.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay has been employed in the analysis of bacterial growth. In comparison to experiments conducted on mammalian cells, the MTT bacterial assay encounters a greater number of interfering factors and obstacles that impact the accuracy of results. In this study, we have elucidated an improved MTT assay protocol and put forth an equation that establishes a correlation between colony-forming units (CFU) and the amount of formazan converted by the bacteria, drawing upon the fundamental principle of the MTT assay. This equation is represented as CFU=kF. Furthermore, we have explicated a methodology to determine the scale factor "k" by employing S. aureus and E. coli as illustrative examples. The findings indicate that S. aureus and E. coli reduce MTT by a cyclic process, from which the optimal reduction time at room temperature was determined to be approximately 30 mins. Furthermore, individual E. coli exhibits an MTT reduction capacity approximately four times greater than that of S. aureus. HPLC analysis proves to be the most accurate method for mitigating interferences during the dissolution and quantification of formazan. Additionally, this study has identified a new constraint related to the narrow linear range (0-125 µg/mL) of formazan concentration-absorbance and has presented strategies to circumvent this limitation.