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 β-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.

The MTT antibacterial assay is an important method in natural antibacterial component discovery. Researchers can use the MTT antibacterial assay to quickly and efficiently evaluate the antibacterial activity of natural compounds. The aim of this research was to find a method to reduce background colour and bacterial cell interference when using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to study the antibacterial effect of phytochemicals. This study used NZ ‘Hass’ ripe avocado seed extract as an example to develop a silica gel column chromatography method that could combine with the MTT assay to remove the background colour and bacterial cells before the formazan measuring stage. This method is particularly suitable for MTT-based antibacterial inhibition studies when the tested phytochemical extracts have an interfering colour.

Introduction: A rapid procedure was developed for the targeted isolation and assessment of antibacterial compounds from plant‐based materials. The effectiveness of this method was demonstrated using Feijoa sellowiana fruit peels.Objective: The objectives of this study are as follows: develop an efficient procedure utilizing direct thin‐layer chromatography (TLC)‐bioautography to facilitate the targeting, identification, and purification of antibacterial compounds from plant extracts and delineate a method based on TLC‐bioautography to determine the minimum effective dose (MED), alongside a colorimetric broth microdilution aided by high‐performance liquid chromatography (HPLC) for evaluating the isolated active compounds.Methodology: Active compounds were targeted using TLC‐bioautography against Staphylococcus aureus, and the identification was achieved through liquid chromatography–mass spectrometry (LC–MS) combined with Compound Discoverer. Purification was carried out using a customized separation method. The structure was confirmed using nuclear magnetic resonance (NMR) spectroscopy. The MED, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were determined by two enhanced antibacterial assays.Results: The main antibacterial compound identified was flavone. A TLC‐bioautography‐based antibacterial assay and a colorimetric broth microdilution assisted by HPLC were described as the enhanced antibacterial assay protocols. The MED, MIC, and MBC of flavone against S. aureus were found to be 4.2–5.2 μg/cm², 225–275 μg/mL, and 550–650 μg/mL, respectively. Similarly, the MED, MIC, and MBC against Escherichia coli were determined to be 5.2–6.1 μg/cm², 325–375 μg/mL, and 375–425 μg/mL, respectively.Conclusion: This study proposed an enhanced bioassay‐guided separation technique for the isolation of antibacterial compounds from plants, along with two improved methods for assessing the antibacterial efficacy of insoluble or colored compounds.

Traditional Chinese medicine (TCM) serves as a significant adjunct to chemical treatment for chronic diseases. For instance, the administration of Baitouweng decoction (BTWD) has proven effective in the treatment of ulcerative colitis. However, the limited understanding of its pharmacokinetics (PK) has impeded its widespread use. Chinese Bama miniature pigs possess anatomical and physiological similarities to the human body, making them a valuable model for investigating PK properties. Consequently, the identification of PK properties in Bama miniature pigs can provide valuable insights for guiding the clinical application of BTWD in humans. To facilitate this research, a rapid and sensitive UPLC-MS/MS method has been developed for the simultaneous quantification of eleven active ingredients of BTWD in plasma. Chromatographic separation was conducted using an Acquity UPLC HSS T3 C₁₈ column and a gradient mobile phase comprising acetonitrile and water (containing 0.1% acetic acid). The methodology was validated in accordance with the FDA Bioanalytical Method Validation Guidance for Industry. The lower limit of quantitation fell within the range of 0.60–2.01 ng/mL. Pharmacokinetic studies indicated that coptisine chloride, berberine, columbamine, phellodendrine, and obacunone exhibited low , while fraxetin, esculin, fraxin, and pulchinenoside were rapidly absorbed and eliminated from the plasma. These findings have implications for the development of effective components in BTWD and the adjustment of clinical dosage regimens.