Phenolic secondary metabolites from Acorus calamus (Acorales: Acoraceae) rhizomes: the feeding deterrents for Spodoptera litura (Lepidoptera: Noctuidae).

Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is one of the most destructive pests of various crops cultivated in Thailand. Spodoptera litura larvae, at early stages, attack the leaves and feed on every part of infested crops in later stages. Acorus calamus essential oil contains toxic asarones, which are generalistic cytotoxic compounds. However, the present study is the first attempt to look at safer metabolites from the rhizomes that could deter insect feeding. The objective was to use such compounds as safer residues on crops that would prevent the feeding of herbivorous lepidopterans. Accordingly, phenolic metabolites were isolated and evaluated to establish the feeding deterrence against polyphagous S. litura larvae. Methanol extract of A. calamus, chrysin, and 4-hydroxy acetophenone compounds were the most effective feeding deterrents with FD50 of 87.18, 10.33, and 70.77 µg/cm2, respectively, after 4 h of feeding on treated kale leaves in a no-choice leaf disc assay. Chrysin also reduced carboxylesterase activities (1.37-fold), whereas A. calamus methanol extract reduced glutathione-S-transferase activities (1.44-fold). Some larvae were also seen dead if they consumed the treated kale leaves. Feeding deterrent activity in the methanol extract of A. calamus was due to chrysin and 4-hydroxy acetophenone. The large-scale utilization of such compounds could help develop feeding deterrent strategies in the integrated pest management of lepidopterans.

Gymnema inodorum (Lour.) Decne. Extract Alleviates Oxidative Stress and Inflammatory Mediators Produced by RAW264.7 Macrophages.

Gymnema inodorum (Lour.) Decne. (G. inodorum) is widely used in Northern Thai cuisine as local vegetables and commercial herb tea products. In the present study, G. inodorum extract (GIE) was evaluated for its antioxidant and anti-inflammatory effects in LPS plus IFN-γ-induced RAW264.7 cells. Major compounds in GIE were evaluated using GC-MS and found 16 volatile compounds presenting in the extract. GIE exhibited antioxidant activity by scavenging the intracellular reactive oxygen species (ROS) production and increasing superoxide dismutase 2 (SOD2) mRNA expression in LPS plus IFN-γ-induced RAW264.7 cells. GIE showed anti-inflammatory activity through suppressing nitric oxide (NO), proinflammatory cytokine production interleukin 6 (IL-6) and also downregulation of the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and IL-6 mRNA levels in LPS plus IFN-γ-induced RAW264.7 cells. Mechanism studies showed that GIE suppressed the NF-κB p65 nuclear translocation and slightly decreased the phosphorylation of NF-κB p65 (p-NF-κB p65) protein. Our studies applied the synchrotron radiation-based FTIR microspectroscopy (SR-FTIR), supported by multivariate analysis, to identify the FTIR spectral changes based on macromolecule alterations occurring in RAW264.7 cells. SR-FTIR results demonstrated that the presence of LPS plus IFN-γ in RAW264.7 cells associated with the increase of amide I/amide II ratio (contributing to the alteration of secondary protein structure) and lipid content, whereas glycogen and other carbohydrate content were decreased. These findings lead us to believe that GIE may prevent oxidative damage by scavenging intracellular ROS production and activating the antioxidant gene, SOD2, expression. Therefore, it is possible that the antioxidant properties of GIE could modulate the inflammation process by regulating the ROS levels, which lead to the suppression of proinflammatory cytokines and genes. Therefore, GIE could be developed into a novel antioxidant and anti-inflammatory agent to treat and prevent diseases related to oxidative stress and inflammation.

Intracellular ROS Scavenging and Anti-Inflammatory Activities of Oroxylum indicum Kurz (L.) Extract in LPS plus IFN-γ-Activated RAW264.7 Macrophages.

Oroxylum indicum (L.) Kurz has been used as plant-based food and herbal medicine in many Asian countries. The aim of the present study was to examine the antioxidant and anti-inflammatory activities of O. indicum extract (O. indicum) in RAW264.7 cells activated by LPS plus IFN-γ. The phytochemical compounds in O. indicum were identified by GC-MS and LC-MS/MS. Five flavonoids (luteolin, apigenin, baicalein, oroxylin A, and quercetin) and 27 volatile compounds were found in O. indicum. O. indicum presented antioxidant activities, including reducing ability by FRAP assay and free radical scavenging activity by DPPH assay. Moreover, O. indicum also suppressed LPS plus IFN-γ-activated reactive oxygen species generation in RAW264.7 macrophages. It possessed the potent anti-inflammatory action through suppressing nitric oxide (NO) and IL-6 secretion, possibly due to its ability to scavenge intracellular ROS. The synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectroscopy results showed the alteration of signal intensity and integrated areas relating to lipid and protein of the activated RAW264.7 macrophages compared to unactivated cells. This is the first report of an application of the SR-FTIR technique to evaluate biomolecular changes in activated RAW264.7 cells. Our results indicate that O. indicum may be used as a potential source of nutraceutical for the development of health food supplement or a novel anti-inflammatory herbal medicine.

The synergy and mode of action of quercetin plus amoxicillin against amoxicillin-resistant Staphylococcus epidermidis.

BACKGROUND: Staphylococcus epidermidis is one of the most multiple resistances to antibiotics in the recent years. Therefore, practically-prescribed antibiotics in the treatment of these strains are not effective. Plant-derived antibacterial is one of the most interesting sources of new therapeutics. The present study was to investigate antibacterial, synergy and modes of action of quercetin and amoxicillin against amoxicillin-resistant Staphylococcus epidermidis (ARSE). METHODS: The MICs, checkerboard assay, viability curves, cytoplasmic membrane (CM) permeability, enzyme assay, transmission electron microscopy, confocal microscopy and FT-IR microspectroscopy measurement was performed. RESULTS: The MICs of amoxicillin, penicillin, quercetin and kaempferol against all ARSE strains were 16, 200, 256-384 and >1024 µg/mL respectively. Synergistic effects were exhibited on amoxicillin plus quercetin and penicillin plus kaempferol against these strains at FIC index 0.50 and <0.38 respectively. The synergistic activity of quercetin plus amoxicillin was confirmed by the viable count. This combination increased CM permeability, caused marked morphological, peptidoglycan and cytoplasmic membrane damage, increased protein amide I and II, but decreased fatty acid in bacterial cells. The quercetin had an inhibitory activity against ß-lactamase. CONCLUSIONS: So, these findings are the first report that quercetin has the synergistic effect with amoxicillin against ARSE via four modes of actions, inhibit peptidoglycan synthesis and ß-lactamases activity, increase CM permeability and protein amide I and II but decrease fatty acid in bacterial cells. Of course, this flavonol has the dominant potential to develop a brand-new collateral phytochemical agent plus amoxicillin to treat ARSE. Future work should focus on the bioavailability, efficacy and toxicity in animal and human studies, as well as, the synergistic effect on blood and tissue should be evaluated and achieved.

Synergism and the mechanism of action of the combination of α-mangostin isolated from Garcinia mangostana L. and oxacillin against an oxacillin-resistant Staphylococcus saprophyticus.

BACKGROUND: Globally, staphylococci have developed resistance to many antibiotics. New approaches to chemotherapy are needed and one such approach could be to use plant derived actives with conventional antibiotics in a synergestic way. The purpose of this study was to isolate α-mangostin from the mangosteen (Garcinia mangostana L.; GML) and investigate antibacterial activity and mechanisms of action when used singly and when combined with oxacillin against oxacillin-resistant Staphylococcus saprophyticus (ORSS) strains. The isolated α-mangostin was confirmed by HPLC chromatogram and NMR spectroscopy. The minimum inhibitory concentration (MIC), checkerboard and killing curve were determined. The modes of action of these compounds were also investigated by enzyme assay, transmission electron microscopy (TEM), confocal microscopic images, and cytoplasmic membrane (CM) permeabilization studies. RESULTS: The MICs of isolated α-mangostin and oxacillin against these strains were 8 and 128 µg/ml, respectively. Checkerboard assays showed the synergistic activity of isolated α-mangostin (2 µg/ml) plus oxacillin (16 µg/ml) at a fractional inhibitory concentration index (FICI) of 0.37. The kill curve assay confirmed that the viability of oxacillin-resistant Staphylococcus saprophyticus DMST 27055 (ORSS-27055) was dramatically reduced after exposure to isolated α-mangostin (2 µg/ml) plus oxacillin (16 µg/ml). Enzyme assays demonstrated that isolated α-mangostin had an inhibitory activity against ß-lactamase in a dose-dependent manner. TEM results clearly showed that these ORSS-27055 cells treated with this combination caused peptidoglycan and cytoplasmic membrane damage, irregular cell shapes and average cell areas were significantly larger than the control. Clearly, confocal microscopic images confirmed that this combination caused considerable peptidoglycan damage and DNA leakage. In addition, the CM permeability of ORSS-27055 was also increased by this combination of actives. CONCLUSIONS: These findings provide evidence that isolated α-mangostin alone has not only some activity but also shows the synergistic activity with oxacillin against ORSS-27055. The chromone and isoprenyl structures could play a significant role in its action. This synergistic activity may involve three mechanisms of action. Firstly, potential effects of cytoplasmic membrane disruption and increases permeability. Secondly, inhibit ß-lactamase activity. Finally, also damage to the peptidoglycan structure. We proposes the potential to develop a novel adjunct phytopharmaceutical to oxacillin for the treatment of ORSS. Future studies require clinical trials to establish if the synergy reported can be translated to animals and humans.