Flavonoid Profile of the Genista tridentata L., a Species Used Traditionally to Treat Inflammatory Processes.

Ethnopharmacological surveys on Portuguese flora reveal that Genista tridentata L. is a shrub used in traditional medicine for the treatment of various inflammation-related health problems, although scientific support of its benefits is still necessary. In order to establish the anti-inflammatory potential of G. tridentata and support its traditional use, ethanolic extracts of three sections of the plant (root, stem, and leaves) were subjected to in vitro evaluation of anti-inflammatory activity using lipopolysaccharide (LPS)-stimulates macrophages as an inflammation model. Simultaneously, we also aimed to establish the extracts' flavonoids profile. The ethanolic extracts, obtained by Soxhlet extraction, profile of the three sections confirmed their richness in flavonoids, being three prenylated flavonoids isolated and characterized in the root, including a new natural compound, the 3-methoxymundulin. The extracts from the three plant sections showed strong antioxidant activity at the cellular level and significantly inhibit the LPS-triggered NO production by downregulating Nos2 gene transcription and consequently iNOS expression. Additionally, root and stem extracts also decreased the LPS-induced transcription of the pro-inflammatory genes Il1b, Il6, and Ptgs2. Thus, the results support the anti-inflammatory properties attributed to G. tridentate preparations. Relevantly, the roots of the shrub, plant part not used, is an unexplored source of compounds with pharmacological and nutraceutical value.

Chemical constituents and biological activities of Albizia myriophylla wood.

CONTEXT: Albizia myriophylla Benth (Leguminosae) is a medicinal plant widely used in Thailand and other Asian countries as a folk medicine remedy for many ailments. OBJECTIVE: The objective of this study is to investigate the chemical compositions, antibacterial activity, and cytotoxicity of A. myriophylla wood. MATERIALS AND METHODS: The structure identification of the isolated compounds was established using spectroscopic methods. In vitro antibacterial activity against Streptococcus mutans, Staphylococcus aureus, and Bacillus cereus and the cytotoxicity against KB cells of extracts and compounds from A. myriophylla were performed using broth microdilution and resazurin microplate assays, respectively. The lupinifolin content in A. myriophylla extracts was quantified by high-performance liquid chromatography. RESULTS: A rare flavan-3,4-diol (1) together with eight known compounds (2-9) were isolated from the wood of A. myriophylla. Compounds 4-9 exhibited anti-S. mutans activity, of which lupinifolin (5) was the most potent with an MIC value of 0.98 µg/mL, followed by its dihydroxy derivative 4 with an MIC value of 62.5 µg/mL. Compounds 4 and 5 also displayed marked antibacterial activity against B. cereus and S. aureus (MIC value 15.63-125 µg/mL) and showed strong cytotoxic activity against KB cells (IC50 value 4.95-12.55 µg/mL). The lupinifolin contents in ethanol extracts from two different collections of this plant originating from central and southern Thailand were 93.85 and 0.04 mg/g, respectively. CONCLUSION: This is the first report of compounds 1-4 from A. myriophylla. Compounds 4 and 5 showed potent antibacterial and cytotoxic activities compared with other isolates. The anti-S. mutans activity of A. myriophylla extracts seems to be related to the lupinifolin content.

Antibiofilm Formation Activity of Lupinifolin Against Methicillin-Resistant Staphylococcus aureus.

<b>Background and Objective:</b> Biofilm formation activity of Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is one of the crucial factors rendering this pathogenic bacterium difficult to be eradicated. It has been reported that lupinifolin, is a major phytochemical agent isolated from <i>Derris reticulata</i> Craib. stem possesses antibacterial activity against MRSA. This study aimed to investigate the effects of lupinifolin and its combinations with some antibacterial drugs, including ampicillin, cloxacillin or vancomycin, on the biofilm formation activity of MRSA. <b>Materials and Methods:</b> The crystal violet biofilm formation assay was performed to evaluate the biofilm formation activity. <b>Results:</b> Lupinifolin produced a significant inhibitory activity against MRSA biofilm formation with the median inhibitory concentration (IC₅₀) of 7.96±3.05 µg mL¹ (n = 6) at 24 hrs incubation. Lupinifolin at the concentrations of sub-MICs (1, 2, 4 and 8 µg mL¹) combined with the antibacterial drugs at their sub-MICs also exhibited substantial antibiofilm formation activities. The maximal antibiofilm activity was found with the combination of lupinifolin (8 µg mL¹) and vancomycin (1 µg mL¹) by the percentage inhibition of 102.39±0.89 (n = 8). The antibiofilm formation activities of the combinations between lupinifolin and the antibacterial drugs at various concentrations tested were also significantly higher than those of lupinifolin alone. <b>Conclusion:</b> These results indicated that lupinifolin can potentially be developed as an antibacterial enhancer for the management of biofilm-associated bacterial infections caused by MRSA, in which the current pharmacological treatment is still limited.

Synergistic Activity of Lupinifolin in Combinations with Antibiotics Against Staphylococcus aureus.

<b>Background and Objective:</b> Antibacterial resistance is one of the top global public health problems. The use of natural substances, which can enhance the antibacterial activity of currently used medications, is a promising alternative to oppose antibacterial resistance. The pharmacological activities of lupinifolin, a prenylated flavanone isolated from stems of <i>Derris reticulata</i> Craib., against growth and biofilm formation of <i>Streptococcus mutans</i> and <i>Staphylococcus aureus</i> have been previously documented. Nonetheless, interactions between lupinifolin and other antibacterial agents have not been determined. This study aimed to investigate the effects of lupinifolin in combinations with some antibacterial agents, specifically ampicillin, cloxacillin or vancomycin, against <i>S. mutans</i>, Methicillin-Sensitive <i>S. aureus</i> (MSSA) and Methicillin-Resistant <i>S. aureus</i> (MRSA). <b>Materials and Methods:</b> The checkerboard assay was performed to determine the antibacterial activity of lupinifolin plus the testing antibacterial agents. The Fractional Inhibitory Concentration Index (FICI) was calculated to indicate the interaction between lupinifolin and the antibacterial agent tested. <b>Results:</b> Lupinifolin exerted the synergistic activity when using in combination with ampicillin or cloxacillin against MSSA with the FICIs of <u><</u>0.5. The potential synergistic effect was also observed with lupinifolin plus ampicillin or cloxacillin against MRSA. However, the combination of lupinifolin plus vancomycin resulted in no interaction against MRSA. The combined effects of lupinifolin and ampicillin or cloxacillin against <i>S. mutans</i> were somewhat ambiguous with the borderline values of FICI of 0.5156 and 0.5625, respectively. <b>Conclusion:</b> Lupinifolin potentially plays a role as an antibacterial intensifier against some pathogenic gram-positive bacteria, particularly MSSA and MRSA. Nonetheless, further experiments are required to explain the precise mechanism of synergy.

Mechanism of Action and Biofilm Inhibitory Activity of Lupinifolin Against Multidrug-Resistant Enterococcal Clinical Isolates.

The treatment of enterococcal infections is becoming more difficult because of multidrug resistance (MDR). Lupinifolin, a prenylated flavonoid isolated from Albizia myriophylla Benth., showed a potent antimicrobial activity against enterococci. The aim of this study was to investigate antibacterial activity and action of lupinifolin against MDR enterococcal clinical isolates. Antibacterial properties of lupinifolin against 21 MDR isolates were assessed using broth microdilution method and time-kill assay. To study mode of action of lupinifolin on the isolates, propidium iodide intensity, salt tolerance assay, and electron microscopic analyses were performed. Antibiofilm formation activity of lupinifolin was conducted using crytal violet assay. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration values of lupinifolin against the isolates ranged between 0.5 and 2.0 µg/mL and between 2 and 16 µg/mL, respectively. Lupinifolin at 2MIC and 4MIC inhibited the bacterial growth >2 log colony-forming units (CFU)/mL at 2 hr incubation by time-kill analysis. The compound increased membrane permeability and caused loss of salt tolerance. SEM and TEM micrographs revealed pronounced morphological and ultrastructural changes in the treated bacteria. Crystal violet staining showed the antibiofilm-producing activity of lupinifolin against four MDR enterococci. This study suggested that lupinifolin is an essential antimicrobial agent that could be useful for the treatment of MDR enterococcal infections.

Lupinifolin from Albizia myriophylla wood: A study on its antibacterial mechanisms against cariogenic Streptococcus mutans.

OBJECTIVE: To determine the anti-Streptococcus mutans mechanisms of action of lupinifolin from Albizia myriophylla Benth. (Fabaceae) wood and provide scientific evidence to support the traditional use of the plant against dental caries. METHODS: The minimum inhibitory concentration (MIC) was evaluated using the broth micro-dilution method. The effects of lupinifolin on bactericidal activity, bacterial cell walls, and membranes were investigated by time-kill, lysis, and leakage assays, respectively. Electron microscopy was utilized to observe any cell morphological changes caused by the compound. Localization of lupinifolin in S. mutans was detected using the thin layer chromatography technique. RESULTS: The MIC range of lupinifolin against S. mutans (n=6) was 2-4 µg/ml. This compound displayed bactericidal effects on S. mutans ATCC 25175 by 90-99.9% killing at 4MIC-16MIC after 8-24 hours. Lupinifolin-treated cells demonstrated no lysis. However, significant cytoplasmic leakage through the bacterial membrane was observed after treatment with lupinifolin at 4MIC-16MIC. As revealed by ultrastructural analysis, lupinifolin produced some changes in bacterial cell walls and membranes. Moreover, the compound was observed in the cytoplasmic fraction of the lupinifolin-treated cells. These results suggest that lupinifolin can enter the cell of bacteria but does not accumulate in the cell envelope and subsequently disrupts the integrity of the cytoplasmic membrane, leading to cell death. CONCLUSION: The scientific evidence from this study offers valuable insights into the potential role of lupinifolin in pharmaceutical and antibiotic applications and supports the therapeutic effects of A. myriophylla, which has traditionally been used as an alternative treatment for dental caries.

Lupinifolin from Derris reticulata possesses bactericidal activity on Staphylococcus aureus by disrupting bacterial cell membrane.

In this study, lupinifolin, a prenylated flavonoid, was isolated from Derris reticulata stem, identified by NMR spectra and confirmed with mass spectrometry. Lupinifolin was freshly prepared by solubilizing in 0.1 N NaOH and immediately diluted in Müller-Hinton broth for antibacterial testing. The data showed that Gram-positive bacteria were more susceptible to lupinifolin than Gram-negative bacteria. Of four strains of Gram-positive bacteria tested, Staphylococcus aureus was the most susceptible. Using the two-fold microdilution method, it was found that lupinifolin possessed antimicrobial activity against S. aureus with minimum inhibitory concentration and minimum bactericidal concentration of 8 and 16 µg/ml, respectively, which is less potent than ampicillin. However, from the time-effect relationship, it was shown that lupinifolin had faster onset than ampicillin. The faster onset of lupinifolin was confirmed by scanning electron microscopy. To investigate the mechanism of action of lupinifolin, transmission electron microscopy (TEM) was performed to observe the ultrastructure of S. aureus. The TEM images showed that lupinifolin ruptured the bacterial cell membrane and cell wall. Due to its fast onset, it is suggested that the action of lupinifolin is likely to be the direct disruption of the cell membrane. This hypothesis was substantiated by the data from flow cytometry using DiOC2 as an indicator. The result showed that the red/green ratio which indicated bacterial membrane integrity was significantly decreased, similar to the known protonophore carbonyl cyanide 3-chlorophenylhydrazone. It is concluded that lupinifolin inhibits the growth of S. aureus by damaging the bacterial cytoplasmic membrane.