Suppression of lung inflammation by the ethanol extract of Chung-Sang and the possible role of Nrf2.

BACKGROUND: Asian traditional herbal remedies are typically a concoction of a major and several complementary herbs. While balancing out any adverse effect of the major herb, the complementary herbs could dilute the efficacy of the major herb, resulting in a suboptimal therapeutic effect of an herbal remedy. Here, we formulated Chung-Sang (CS) by collating five major herbs, which are used against inflammatory diseases, and tested whether an experimental formula composed of only major herbs is effective in suppressing inflammation without significant side effects. METHODS: The 50% ethanol extract of CS (eCS) was fingerprinted by HPLC. Cytotoxicity to RAW 264.7 cells was determined by an MTT assay and a flow cytometer. Nuclear NF-κB and Nrf2 were analyzed by western blot. Ubiquitinated Nrf2 was similarly analyzed following immunoprecipitation of Nrf2. Acute lung inflammation and sepsis were induced in C57BL/6 mice. The effects of eCS on lung disease were measured by HE staining of lung sections, a differential cell counting of bronchoalveolar lavage fluid, a myeloperoxidase (MPO) assay, a real-time qPCR, and Kaplan-Meier survival of mice. RESULTS: eCS neither elicited cytotoxicity nor reactive oxygen species. While not suppressing NF-κB, eCS activated Nrf2, reduced the ubiquitination of Nrf2, and consequently induced the expression of Nrf2-dependent genes. In an acute lung inflammation mouse model, an intratracheal (i.t.) eCS suppressed neutrophil infiltration, the expression of inflammatory cytokine genes, and MPO activity. In a sepsis mouse model, a single i.t. eCS was sufficient to significantly decrease mouse mortality. CONCLUSIONS: eCS could suppress severe lung inflammation in mice. This effect seemed to associate with eCS activating Nrf2. Our findings suggest that herbal remedies consisting of only major herbs are worth considering.

Eclipta prostrata promotes the induction of anagen, sustains the anagen phase through regulation of FGF-7 and FGF-5.

CONTEXT: Eclipta prostrata L. (Asteraceae) (EP) has been widely used for the treatment of skin disease in Asian traditional medicine. OBJECTIVE: This study investigates the potency of EP in promoting hair growth in vivo and in vitro. MATERIALS AND METHODS: C57BL/6N mice were divided into four groups (n = 4) as follows: control (topical treatment of normal saline), topical 3% minoxidil to the dorsal skin of mice for 14 days, and low (1 mg/day) and high (10 mg/day) doses of EP orally administered once a day for 14 days. Dorsal hairs of C57BL/6N mice were depilated to synchronize anagen induction. Hair growth activity was evaluated by gross and microscopic observations. Sections of dorsal skin were stained with haematoxylin and eosin. We also treated the various concentrations of EP (5, 10 and 50 µg/mL) for 24 h on the human dermal papilla cells (HDPs) and examined the effects of EP on the expression of FGF-7 and mTOR signalling. RESULTS: EP enhanced the induction of anagen in the dorsal skin of mice, characterized by the appearance of inner root sheath along with hair shaft, the emergence of hair shaft through the epidermis. EP increased the expression of FGF-7, while decreased the level of FGF-5 in C57/BL6 mice. EP also increased the expression of FGF-7, activated the mTOR signalling in HDPs. DISCUSSION AND CONCLUSIONS: These results suggest that EP has a potency to enhance the growth of hair follicle, promoting hair growth through regulation of FGF-7 and FGF-5.

Douchi (fermented Glycine max Merr.) alleviates atopic dermatitis-like skin lesions in NC/Nga mice by regulation of PKC and IL-4.

BACKGROUND: Douchi (fermented Glycine max Merr.) is produced from fermented soybeans, which is widely used in traditional herbal medicine. In this study, we investigated whether Douchi attenuates protein kinase C (PKC) and interleukin (IL)-4 response and cutaneous inflammation in Atopic dermatitis (AD)-like NC/Nga mice. METHODS: To induce AD-like skin lesions, D. farinae antigen was applied to the dorsal skin of 3-week-old NC/Nga mice. After inducing AD, Douchi extract was administered 20 mg/kg daily for 3 weeks to the Douchi-treated mice group. We identified the changes of skin barrier and Th2 differentiation through PKC and IL-4 by immunohistochemistry. RESULTS: Douchi treatment of NC/Nga mice significantly reduced clinical scores (p < 0.01) and histological features. The levels of PKC and IL-4 were significantly reduced in the Douchi-treated group (p < 0.01). The reduction of IL-4 and PKC led to decrease of inflammatory factors such as substance P, inducible nitric oxide synthase (iNOS) and Matrix metallopeptidase 9 (MMP-9) (all p < 0.01). Douchi also down-regulated Th1 markers (IL-12, TNF-α) as well as Th2 markers (IL-4, p-IκB) (p < 0.01). CONCLUSION: Douchi alleviates AD-like skin lesions through suppressing of PKC and IL-4. These results also lead to diminish levels of substance P, iNOS and MMP-9 in skin lesions. Therefore, Douchi may have potential applications for the prevention and treatment of AD.

Anti-inflammatory and antipruritic effects of luteolin from Perilla (P. frutescens L.) leaves.

Perilla (Perilla frutescens L.) leaves have shown therapeutic efficacy in the treatment of inflammatory disorders, allergies, bronchial asthma, and systemic damage due to free radicals. In the present study we analyzed the active constituents in perilla leaves using high-performance liquid chromatography (HPLC) and isolated luteolin, a polyphenolic flavonoid. We investigated the anti-inflammatory and antipruritic properties of luteolin. Luteolin inhibited the secretion of inflammatory cytokines such as interleukin-1ß (IL-1 ß) and tumor necrosis factor-α (TNF-α) from human mast cells (HMC-1) stimulated with phorbol myristate acetate plus calcium ionophore A23187 in a dose-dependent manner. Luteolin also significantly reduced the histamine release from rat peritoneal mast cells stimulated by compound 48/80, a potent histamine liberator. Furthermore, the administration of luteolin markedly inhibited the scratching behavior and vascular permeability induced by pruritogens, such as compound 48/80 or serotonin, in ICR mice. These results suggested that luteolin has potential as a therapeutic agent against inflammation and itch-related skin diseases.

Anti-Inflammatory Effects of Weigela subsessilis Callus Extract via Suppression of MAPK and NF-κB Signaling.

Weigela subsessilis is used in folk medicine to treat pain and allergic syndromes in Korea. However, the antibacterial and anti-inflammatory activities of W. subsessilis callus extract remain unexplored. In this study, we aimed to evaluate the W. subsessilis callus of pharmacological activity. Therefore, we first established in vitro calluses of W.subsessilis via plant tissue culture methods. We then evaluated the antioxidant and anti-inflammatory effects of W. subsessilis callus extract in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells. The W. subsessilis callus extract showed antioxidant and anti-inflammatory effects. These effects were regulated via suppression of mitogen-activated protein kinase signaling through LPS-induced translocation of nuclear factor kappa B (NF-κB) p65 from the cytoplasm to the nucleus. W. subsessilis callus extract also showed antibacterial and anti-inflammatory activities in Propionibacterium acnes-treated HaCaT keratinocyte cells. These results indicate that W. subsessilis callus extract has antioxidant, antibacterial and anti-inflammatory activities, suggesting its possible application in the treatment of inflammatory disorders.

Galuteolin, identified in the extract of thymus quinquecostatus flowers, is involved in inhibiting melanin biosynthesis in B16/F10 melanoma cells.

To enhance the skin whitening effect, tyrosinase activity and melanin biosynthesis needs to be suppressed in the skin. To achieve this goal, we examined the extract of Thymus quinquecostatus flowers, and identified a functional ingredient, galuteolin. Galuteolin effectively inhibited melanin biosynthesis in B16/F10 cells, partially suppressing tyrosinase activity. Therefore, this study suggests that galuteolin can be used as a cosmetic ingredient for skin whitening.

Antibacterial activity of phytochemicals isolated from Atractylodes japonica against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has been emerging worldwide as one of the most important problems in communities and hospitals. Therefore, new agents are needed to treat acute oral infections from MRSA. In this study, antibacterial compounds from the roots of Atractylodes japonica (A. japonica) were isolated and characterized. The compounds were isolated from the root extracts using HPLC-piloted activity-guided fractionations. Four A. japonica compounds were isolated and identified as atractylenolide III (1), atractylenolide I (2), diacetylatractylodiol [(6E,12E)-tetradeca-6,12-diene-8,10-diyne-1,3-diol diacetate, TDEYA, 3). and (6E,12E)-tetradecadiene-8,10-diyne-1,3-diol (TDEA, 4), which was obtained by hydrolysis of TDEYA. The minimum inhibitory concentrations (MICs) was determined in the setting of clinical MRSA isolates. Compound 4 showed anti-MRSA activity with a MIC value of 4-32 µg/mL. The overall results provide promising baseline information for the potential use of the extract of A. japonica as well as some of the isolated compounds in the treatment of bacterial infections.

Anti-Inflammatory Activities of an Extract of In Vitro Grown Adventitious Shoots of Toona sinensis in LPS-Treated RAW264.7 and Propionibacterium acnes-Treated HaCaT Cells.

Toona sinensis has been traditionally used to treat dysentery, enteritis, flatulence, and itchiness. However, the existence of anti-inflammatory effects of T. sinensis on Propionibacterium acnes-induced skin disease is unknown. In vitro cultures of plant cells and tissues produced under controlled conditions offer a continuous production platform for plant natural products including pigments and anti-inflammatory agents. In this study, we determine the anti-inflammatory activities of an extract of in vitro grown adventitious shoots of T. sinensis on P. acnes, the etiologic agent of skin inflammation. The extract of T. sinensis showed antioxidant and anti-inflammatory activity in LPS-treated RAW264.7 cells. It also had antibacterial activity and anti-inflammatory effects on P. acnes-treated HaCaT cells. In addition, these effects were regulated by suppression of the mitogen-activated protein kinase (MAPK) pathways. These results suggesting the potential application of adventitious shoots of T. sinensis grown with an in vitro proliferation system as a medicine for treating P. acnes-induced inflammatory skin disease.

The methanol extract of Guettarda speciosa Linn. Ameliorates acute lung injury in mice.

BACKGROUND: Guettarda speciosa is mainly found in tropical areas in Asia. Although G. speciosa is traditionally used to treat some of the inflammatory disorders, the experimental evidence supporting the anti-inflammatory effect of G. speciosa is limited. Here, we sought to obtain evidence that G. speciosa has anti-inflammatory activity using an acute lung injury (ALI) mouse model and to explore possible underlying mechanisms for the activity. METHODS: The methanol extract of G. speciosa Linn. (MGS) was fingerprinted by HPLC. Cytotoxicity was determined by MTT and flow cytometer. As for an ALI mouse model, C57BL/6 mice received an intratracheal (i.t.) injection of lipopolysaccharide (LPS). The effects of MGS on lung inflammation in the ALI mice were assessed by differential cell counting and FACS of inflammatory cells and hematoxylin and eosin staining of lung tissue. Proteins were analyzed by immunoprecipitation and immunoblotting, and gene expression was by real-time qPCR. Neutrophil elastase activity was measured by ELISA. RESULTS: MGS did not cause metabolic disarray or produce reactive oxygen species that could induce cytotoxicity. Similar to ALI patients, C57BL/6 mice that received an i.t. LPS developed a high level of neutrophils, increased pro-inflammatory cytokines, and inflicted tissue damage in the lung, which was suppressed by i.t. MGS administered at 2 h after LPS. Mechanistically, MGS activated Nrf2, which was related to MGS interrupting the ubiquitin-dependent degradation of Nrf2. MGS suppressed the nuclear localization of NF-κB induced by LPS, suggesting the inhibition of NF-κB activity. Furthermore, MGS inhibited the enzymatic activity of neutrophil elastase. CONCLUSION: MGS could suppress lung inflammation in an ALI mouse model, the effect of which could be attributed to multiple mechanisms, including the activation of Nrf2 and the suppression of NF-κB and neutrophil elastase enzymatic activity by MGS.

Synergistic effects of the combination of 20-hydroxyecdysone with ampicillin and gentamicin against methicillin-resistant Staphylococcus aureus.

The emergence of methicillin-resistant of Staphylococcus aureus (MRSA) has led to an urgent need for the discovery and development of new antibacterial agents. As part of an ongoing investigation into the antibacterial properties of the natural products, 20-Hydroxyecdysone (20E), isolated from the roots of Achyranthes japonica Nakai, was found to be active methicillin-resistant Staphylococcus aureus (MRSA), either alone or in combination with ampicillin (AM) or gentamicin (GM), vis checkerboard assay. This study investigated the antibacterial activity of 20E, which exhibited poor antibacterial activity (MIC=250-500 microg/mL) against all the bacterial strains tested. The combined activity of ampicillin (AM), gentamicin (GE) plus 20E against MRSA resulted in fractional inhibitory concentrations (FICs) ranging from 4.00 to 0.031 microg/mL, respectively. Meanwhile, the FIC index ranged from 0.16-4.50, indicating a marked synergistic relationship between AM, GE and 20E against MRSA with enterotoxin gene. Time-kill assays also showed a decrease remarkably between the combination and the more active compound. Therefore, this study demonstrated that AM, GE, and 20E can act synergistically in inhibiting MRSA in vitro.

Inhibition of LPS-induced iNOS, COX-2 and inflammatory mediator expression by paeonol through the MAPKs inactivation in RAW 264.7 cells.

We evaluated the in vivo anti-inflammatory and analgesic activities of orally administered paeonol in mice, and also investigated the anti-inflammatory activity of paeonol in a cell line. Paeonol significantly reduced the edema induced by arachidonic acid in rats. The analgesic effects were assayed using 2 different models, i.e., by acetic acid-induced writhing response and by formalin induced licking and biting time. Moreover, we examined the effects of paeonol on the release of inflammatory mediators such as NO, PGE(2) and IL-6. Our results demonstrated that paeonol inhibited LPS induced expression of NO, PGE(2) and IL-6. Paeonol prevented LPS induced iNOS, COX-2 and ERK activation. Therefore, paeonol appears to have potential as a treatment for inflammatory disease and analgesic.

The medicinal mushroom Auricularia auricula-judae (Bull.) extract has antioxidant activity and promotes procollagen biosynthesis in HaCaT cells.

In this study, Auricularia auricula-judae (Bull.) extract (AAE) had potent antioxidant activity in vitro and promoted the biosynthesis of procollagen, a precursor of collagen in HaCaT cells. In addition, the expression of HAS-3 (hyaluronic acid synthase), which is a moisturizing factor, was increased in HaCaT cells in response to AAE. Therefore, this work suggests that AAE has the potential to exhibit antioxidant activity and promote procollagen biosynthesis in HaCaT cells.

Fermentation of Blackberry with L. plantarum JBMI F5 Enhance the Protection Effect on UVB-Mediated Photoaging in Human Foreskin Fibroblast and Hairless Mice through Regulation of MAPK/NF-κB Signaling.

Chronic and extensive exposure of ultraviolet (UV)-irradiation causes human skin sunburn, inflammation, or photoaging, which is associated with downregulated collagen synthesis. This study investigated the effects of fermented blackberry (Rubus fruticosus B., FBB) by Lactobacillus plantarum JBMI F5 (LP) on UVB-induced photoaging in human foreskin fibroblast (Hs68) as well as in SKH-1 hairless mice. FBB pretreatment inhibited UVB-mediated type-1 procollagen degradation, matrix metalloproteinase (MMP)-1 and MMP-2 protein expression, and suppressed nuclear factor-κB (NF-κB) activation as well as mitogen-activated protein kinase (MAPK) phosphorylation in Hs68. In addition, FBB administration diminished the wrinkle formation in dorsal skin and epidermal thickening in UVB-irradiated hairless mice. Moreover, UVB-induced Type-1 procollagen reduction and antioxidant enzyme inactivation were reversed by FBB administration. These results suggest that FBB may have antiphotoaging effects on UVB-induced wrinkle formation by maintaining the extracellular matrix density in the dermis, which occurs via regulation of reactive oxygen species and related MAPK and NF-κB signaling. Therefore, FBB can be a potential candidate for protecting skin aging against UV irradiation.

Continentalic acid from Aralia continentalis induces growth inhibition and apoptosis in HepG2 cells.

In this study, we investigated the effects of continentalic acid (CA, (-)-pimara-8(14), 15-diene-19-oic acid), a diterpenic acid, isolated from Aralia continentalis, on the proliferation and apoptosis induction of HepG2 cells. In 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, the inhibitory effect became gradually stronger with the passage of time, 24, 48 and 72 h after treatment with CA, and the most significant effect was observed at 72 h. CA treatment for 72 h induced DNA fragmentation in a dose-dependent manner. Furthermore, flow cytometric analysis of HepG2 cells exposed to CA showed that apoptotic cells increased in a dose-dependent manner. The induction of apoptosis in HepG2 cells by CA was mediated through the activation of caspase-3, Bak, and Bax, and then through the cleavage of peroxisome proliferator-activated receptor (PARP) and the down-regulation of Bcl-2. These results demonstrate that CA efficiently induces apoptosis and is a good candidate for further evaluation as an effective chemotherapeutic agent.

Apoptosis-inducing effect of akebia saponin D from the roots of Dipsacus asper Wall in U937 cells.

Methanol extracts of the root of Dipsacus asper Wall (Dipsacaceae) were found to exhibit apoptosis-inducing activities in U937 (human monocyte-like histiocytic) cells. Investigation of the active n-BuOH fraction led to the isolation of akebia saponin D (ASD). Structure was established by spectroscopic methods. Treatment of U937 cells with ASD induced apoptosis in a dose dependent manner. ASD exerted strong cytotoxicity against human and murine leukemia cells. It is significantly increased the subG1 cell population and expression of p53 and Bax gene. And also ASD enhanced NO production from RAW264.7 macrophage cells. Taken together, these results strongly indicate that ASD may exert apoptosis-inducing activity via induction of apoptosis through activation chiefly via the nitric oxide and apoptosis-related p53 and Bax gene expression. These data provide scientific evidence that Dipsacus asper Wall can be useful as a chemopreventive agent.

Anti-hyperglycemic effects and signaling mechanism of Perilla frutescens sprout extract.

BACKGROUND/OBJECTIVES: Perilla frutescens (L.) Britton var. (PF) sprout is a plant of the labiate family. We have previously reported the protective effects of PF sprout extract on cytokine-induced ß-cell damage. However, the mechanism of action of the PF sprout extract in type 2 diabetes (T2DM) has not been investigated. The present study was designed to study the effects of PF sprout extract and signaling mechanisms in the T2DM mice model using C57BL/KsJ-db/db (db/db) mice. MATERIALS/METHODS: Male db/db mice were orally administered PF sprout extract (100, 300, and 1,000 mg/kg of body weight) or rosiglitazone (RGZ, positive drug, 1 mg/kg of body weight) for 4 weeks. Signaling mechanisms were analyzed using liver tissues and HepG2 cells. RESULTS: The PF sprout extract (300 and 1,000 mg/kg) significantly reduced the fasting blood glucose, serum insulin, triglyceride and total cholesterol levels in db/db mice. PF sprout extract also significantly improved glucose intolerance and insulin sensitivity, decreased hepatic gluconeogenic protein expression, and ameliorated histological alterations of the pancreas and liver. Levels of phosphorylated AMP-activated protein kinase (AMPK) protein expression also increased in the liver after treatment with the extract. In addition, an increase in the phosphorylation of AMPK and decrease in the phosphoenolpyruvate carboxykinase and glucose 6-phosphatase proteins in HepG2 cells were also observed. CONCLUSIONS: Our results sugges that PF sprout displays beneficial effects in the prevention and treatment of type 2 diabetes via modulation of the AMPK pathway and inhibition of gluconeogenesis in the liver.

Anti-inflammatory activity of the decoction of Forsythia suspensa (Thunb.) Vahl is related to Nrf2 and A20.

ETHNOPHARMACOLOGICAL RELEVANCE: The water extract of Forsythiae Fructus (WFF) is an herbal remedy that is prescribed to treat various inflammatory diseases in traditional Chinese medicine. Although the anti-inflammatory activity of WFF has been reported, the underlying mechanisms for the activity remain unclear. Here, we examined whether the anti-inflammatory activity of WFF is associated with Nrf2, an anti-inflammatory factor, and A20, an ubiquitin-regulator protein that inhibits signaling cascades of endotoxin or cytokines. MATERIALS AND METHODS: The water extract of Forsythia suspensa (Thunb.) Vahl was prepared and fingerprinted by HPLC. Cytotoxicity and intracellular ROS induced by WFF were determined by MTT and FACS analyses, respectively. Nuclear and cytoplasmic proteins were analyzed by immunoblot. Expression of mRNA was analyzed by a semi-quantitative RT-PCR. Expression of proteins or genes was quantitated by Image J. RESULTS: WFF activated Nrf2, inducing the expression of Nrf2-dependent genes, such as HO-1, NQO1, and GCLC in RAW 264.7 cells. On the other hand, WFF suppressed NF-κB induced by LPS or TNF-α, which was coincided with the expression of A20. Conversely, WFF failed to suppress NF-κB when A20 expression was silenced by siRNA. CONCLUSION: WFF activated Nrf2 and expressed A20. Given that Nrf2 suppresses inflammation and A20 broadly disrupts inflammatory signaling cascades, our results suggest that the anti-inflammatory activity of WFF is attributable to Nrf2 and A20.

Suppression of lung inflammation by the methanol extract of Spilanthes acmella Murray is related to differential regulation of NF-κB and Nrf2.

ETHNOPHARMACOLOGICAL RELEVANCE: Although Spilanthes acmella has been used to relieve inflammation, fever, pain, or infection in traditional Asian medicine, experimental evidence supporting these functions is scarce. Here, we examined an anti-inflammatory function and a possible underlying mechanism of S. acmella Murray (SAM). MATERIALS AND METHOD: The methanol extract of SAM was fingerprinted by HPLC. C57BL/6 mice were administered with a single intratracheal (i.t.) LPS and 2 h later with a single i.t. SAM. The effect of SAM on lung inflammation was assessed by histology, semi-quantitative RT-PCR, and MPO assay of lung tissue. The effects of SAM on a pro-inflammatory factor NF-κB and an anti-inflammatory factor Nrf2 were analyzed by immunoblotting of nuclear proteins and by semi-quantitative RT-PCR analysis of mRNA of the genes governed by these transcription factors. V5-Nrf2 was precipitated by an anti-V5 antibody and the ubiquitinated V5-Nrf2 was revealed by immunoblotting of HA-tagged ubiquitin. RESULTS: The i.t. SAM robustly diminished a neutrophilic lung inflammation induced by i.t. LPS treatment of mice. In RAW 264.7 cells, SAM suppressed the nuclear localization of NF-κB and the expression of NF-κB-dependent cytokine genes. SAM increased the level of Nrf2 in the nucleus and the expression of Nrf2-dependent genes while suppressing ubiquitination of Nrf2. CONCLUSION: Our results suggest that SAM can suppress a neutrophilic inflammation in mouse lungs, which is associated with suppressed NF-κB and activated Nrf2. Our results provide experimental evidence supporting the anti-inflammatory function of S. acmella.

A new cytotoxic prenylated chalcone from Sophora flavescens.

We have isolated a new prenylated chalcone from the roots of Sophora flavescens (Leguminosae). We determined that structure of this compound is 7,9,2',4'-tetrahydroxy-8-isopentenyl-5-methoxychalcone (1) on the basis of spectroscopic analysis (1D and 2D NMR data). Compound 1 exhibited potent cytotoxicity against human acute promyelocytic (HL60), mouse lymphocytic (L1210) and human histiocytic (U937) leukemia cells.

Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens.

This study investigated the antibacterial activities of sophoraflavanone G from Sophora flavescens in combination with two antimicrobial agents against oral bacteria. The combined effect of sophoraflavanone G and the antimicrobial agents was evaluated using the checkerboard method to obtain a fractional inhibitory concentration (FIC) index. The sophoraflavanone G+ampicillin (AM) combination was found to have a synergistic effect against S. mutans, S. sanguinis, S. sobrinus, S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis, whereas the sophoraflavanone G+gentamicin (GM) combination had a synergistic effect against S. sanguinis, S. criceti, S. anginosus, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis. Neither combination exhibited any antagonistic interactions (FIC index >4). In particular, the MICs/MBCs for all the bacteria were reduced to one-half - one-sixteenth as a result of the drug combinations. A synergistic interaction was also confirmed by time-kill studies for nine bacteria where the checkerboard suggested synergy. Thus, a strong bactericidal effect was exerted through the drug combinations, plus in vitro data suggested that sophoraflavanone G combined with other antibiotics may be microbiologically beneficial rather than antagonistic.