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The increase in bacterial resistance and the decline in the effectiveness of antimicrobial agents are challenging issues for the control of infectious diseases. Traditional Chinese herbal plants are potential sources of new or alternative medicine. Here, we identified antimicrobial components and action modes of the methanol-phase extract from an edible herb Potentilla kleiniana Wight et Arn, which had a 68.18% inhibition rate against 22 species of common pathogenic bacteria. The extract was purified using preparative high-performance liquid chromatography (Prep-HPLC), and three separated fragments (Fragments 1-3) were obtained. Fragment 1 significantly elevated cell surface hydrophobicity and membrane permeability but reduced membrane fluidity, disrupting the cell integrity of the Gram-negative and Gram-positive pathogens tested (p < 0.05). Sixty-six compounds in Fragment 1 were identified using Ultra-HPLC and mass spectrometry (UHPLC-MS). The identified oxymorphone (6.29%) and rutin (6.29%) were predominant in Fragment 1. Multiple cellular metabolic pathways were altered by Fragment 1, such as the repressed ABC transporters, protein translation, and energy supply in two representative Gram-negative and Gram-positive strains (p < 0.05). Overall, this study demonstrates that Fragment 1 from P. kleiniana Wight et Arn is a promising candidate for antibacterial medicine and food preservatives.

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ETHANOPHARMACOLOGICAL RELEVANCE: Potentilla kleiniana Wight et Arn is a wide-spread wild plant in the mountainous areas in southern China. The whole herb has been used as a traditional herbal medicine to treat fever, arthritis, malaria, insect and snake bites, hepatitis, and traumatic injury. In vitro studies have reported the antibacterial activity use of the plant in traditional medicinal systems. AIM OF THE STUDY: The aim of this study was to investigate the inhibitory activity of total flavonoid from Potentilla kleiniana Wight et Arn (TFP) on methicillin-resistant Staphylococcus aureus (MRSA) in planktonic state and biofilm state. MATERIALS AND METHODS: Antibacterial activities of TFP on planktonic MRSA were determined by agar diffusion method, microtiter plate assay and time-kill curve assay. Electrical conductivity, membrane permeability, membrane potential and autoaggregation were analyzed to study TFP effects on planktonic MRSA growth. Crystal violet (CV) staining and confocal laser scanning microscopy (CLSM) were analyzed to study TFP effects on aggregation and maturation of MRSA biofilm. After TFP treatment, extracellular polymeric substances (EPS) production were examined. Morphological changes in planktonic and MRSA biofilm following TFP treatment were determined with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Moreover, α-Toxin protein expression and adhesion-related gene expression were also determined. RESULTS: The minimum inhibitory concentration (MIC) of TFP against MRSA was 20 µg/mL. The agar diffusion method and time-kill curve assay results indicated that TFP inhibited planktonic MRSA growth. TFP treatment significantly inhibited planktonic MRSA growth by inhibiting autoaggregation, α-hemolysin activity, α-Toxin protein expression, but increasing electrolyte leakage, membrane permeability and membrane potential and impacting cell structure. Moreover, TFP treatment significantly inhibited aggregation and maturation on MRSA biofilm by decreasing surface hydrophobicity, EPS production and adhesion-related gene expression. CONCLUSION: The results of this trial provide scientific experimental data on the traditional use of Potentilla Kleiniana Wight et Arn for traumatic injury treatment and further demonstrate the potential of TFP to be developed as a novel anti-biofilm drug.

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INTRODUCTION: Potentilla kleiniana Wight et Arn is widely used as a herbal medicine to treat type 2 diabetes. However, detailed information about its active compounds is lacking. OBJECTIVE: To develop an efficient method for the rapid screening and separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn. METHODOLOGY: Potential α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn were rapidly screened out through ultrafiltration high-performance liquid chromatography mass spectrometry (HPLC-MS), and then followed by a target-guided high-speed counter-current chromatography (HSCCC) separation using two-phase solvent systems composed of n-hexane/ethyl acetate/methanol/water (1:10:1:10, v/v/v/v and 1:10:5:6, v/v/v/v), and adopting increasing flow-rate from 1.5 to 3.0 mL/min after 200 min. Their structures were identified by ultraviolet (UV), MS, proton nuclear magnetic resonance (1 H-NMR) and carbon-13 (13 C)-NMR, and their α-glucosidase inhibitory activities were assessed by in vitro assay. RESULTS: Five α-glucosidase inhibitors including gallic acid (25.7 mg, 98.2%, 1), brevifolincarboxylic acid (9.86 mg, 95.3%, 2), ethyl evifolincarboxylate (13.26 mg, 97.6%, 3), 3,3'-di-O-methylellagic acid-4'-O-ß-d-glucopyranoside (16.26 mg, 95.1%, 4), and 3,3'-di-O-methylellagic acid (10.54 mg, 96.8%, 5) were successfully purified from 250 mg n-butanol extract in a single run. Compounds 1, 2, 4 and 5 exhibited stronger α-glucosidase inhibitory activities[half maximal inhibition concentration (IC50 ) values at 173.41 ± 6.35, 323.46 ± 8.08, 44.63 ± 2.50, and 20.73 ± 2.56 µM, respectively] than acarbose (IC50 value at 332.12 ± 5.52 µM, reference compound). CONCLUSIONS: Notably, compounds 2-5 were reported in the Potentilla kleiniana Wight et Arn for the first time. The results indicated that the proposed method could be applied for the rapid screening and preparative separation of α-glucosidase inhibitors from a complex matrix.

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