Acetylenic spiroketal enol ethers from Artemisia rupestris and their synergistic antibacterial effects on methicillin-resistant Staphylococcus aureus.

Synergistic bioassay-guided isolation of the extracts of Artemisia rupestris L, which belongs to the family Asteraceae, afforded two acetylenic spiroketal enol ethers, namely rupesdiynes A (1) and B (2). Their structures were determined based on spectroscopic analysis and experimental and calculated ECD investigations. The two compounds exhibited synergistic activity and were able to reduce the minimum inhibitory concentration (MIC) of oxacillin four-fold, with a fractional inhibitory concentration index (FICI) of 0.5 in combination with oxacillin against the oxacillin-resistant EMRSA-16. Biofilm formation inhibitory and Ethidium bromide (EtBr) efflux assay were further employed to verify the possible mechanism of the synergistic antibacterial effect. Additionally, molecular docking studies were conducted to investigate the binding affinities of the two compounds with penicillin-binding protein 2a (PBP2a) of EMRSA-16. Taken together, rupesdiynes A (1) and rupesdiyne B (2) showed moderate synergistic activity against EMRSA-16 with oxacillin via inhibiting biofilm formation and efflux pump activity, respectively.

Natural Product BO-1 as an Inner Responsive Molecule Inhibits Antimicrobial-Resistant Staphylococcus aureus via Synergism.

Multidrug-resistant Staphylococcus aureus, a Gram-positive bacterium that causes several difficult-to-treat human infections, is a considerable threat to global healthcare. We hypothesize that there exist inner responsive molecules (IRMs) which can function synergistically with antibiotics to restore the sensitivity of resistant bacteria to existing antibiotics without inducing new antibiotic resistance. An investigation of the extracts of the Chinese medicinal herb Piper betle L. led to the isolation of six benzoate esters, BO-1-BO-6. Among these, BO-1 as a distinct IRM displayed considerable synergism by potentiating antibacterial activity against five antibiotic-resistant S. aureus strains. Mechanistic studies demonstrated that BO-1 acted as a suppressing drug resistance IRM via inhibiting efflux activity. A combination of BO-1 with ciprofloxacin significantly inhibited resistance to this antibiotic and reversed its resistance in the S. aureus strain. Furthermore, BO-1 effectively enhanced the activity of ciprofloxacin against the efflux fluoroquinolone-resistant S. aureus strain SA1199B that caused infection in two animal models and significantly decreased the inflammatory factors IL-6 and C-reactive protein of the infected mice, thereby showing the practice utility of this approach.

Synergistic anticancer effect of flavonoids from Sophora alopecuroides with Sorafenib against hepatocellular carcinoma.

Sorafenib (SF), a multi-kinase inhibitor, is the first FDA-approved systemic chemotherapy drug for advanced hepatocellular carcinoma (HCC). However, its clinical application is limited by severe toxicity and side effects associated with high applied doses. Sophora alopecuroides L. is traditionally used as Chinese herbal medicine for treating gastrointestinal diseases, bacillary dysentery, viral hepatitis, and other diseases, and exerts an important role in anti-tumor. Hence, we investigated the synergistic actions of seventeen flavonoids from this herb combined with SF against HCC cell lines and their primary mechanism. In the experiment, most compounds were found to prominently enhance the inhibitory effects of SF on HCC cells than their alone treatment. Among them, three compounds leachianone A (1), sophoraflavanone G (3), and trifolirhizin (17) exhibited significantly synergistic anticancer activities against MHCC97H cells at low concentration with IC50 of SF reduced by 5.8-fold, 3.6-fold, and 3.5-fold corresponding their CI values of 0.49, 0.66, and 0.46 respectively. Importantly, compounds 3 or 17 combined with SF could synergistically induce MHCC97H cells apoptosis via the endogenously mitochondrial-mediated apoptotic pathway, involving higher Bax/Bcl-2 expressions with the activation of caspase-9 and -3, and arrest the cell cycle in G1 phases. Strikingly, this synergistic effect was also closely related to the co-suppression of ERK and AKT signaling pathways. Furthermore, compound 3 significantly enhanced the suppression of SF on tumor growth in the HepG2 xenograft model, with a 79.3% inhibition ratio at high concentration, without systemic toxicity, compared to either agent alone. These results demonstrate that the combination treatment of flavonoid 3 and SF at low doses exert synergistic anticancer effects on HCC cells in vitro and in vivo.

Neolignans from Piper betle Have Synergistic Activity against Antibiotic-Resistant Staphylococcus aureus.

A phytochemical investigation of an extract of the leaves of Piper betle, guided by a synergistic antibacterial screen, led to the isolation and structural elucidation of 10 new neolignans, Pibeneolignan A-J (1-10), together with 11 known compounds. The structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic data, single-crystal X-ray diffraction analysis, and experimental and calculated ECD investigations. Compounds 1 and 2 are new naturally occurring neolignan skeletons, based on the cyclohept-2-ene-1,4-dione framework. We propose that these natural products are biosynthetically formed from bicyclic [3.2.1] neolignans by oxidative cleavage and ring opening at C-1' and C-2'. Among these compounds, 9, 13, 15, and 16, in combination with norfloxacin against an effluxing S. aureus strain (SA1199B), exhibited significant synergistic activity with fractional inhibitory concentration indices (FICIs) of 0.078, 0.156, 0.125, and 0.25, respectively. Bacterial growth curves, ethidium bromide (EtBr) efflux, and qRt-PCR were further employed to verify their synergistic antibacterial mechanism. Furthermore, computational molecular modeling suggested the binding of compounds 14-17 and 19 to the active site of the modeled structure of the NorA efflux pump, which is the main efflux pump in SA1199B.

Flavonoids from Artemisia rupestris and their synergistic antibacterial effects on drug-resistant Staphylococcus aureus.

This study seeks to discover flavonoids from a traditional Chinese herb, Artemisia rupestris L., with synergistic antibacterial effects against multidrug-resistant Staphylococcus aureus. Five flavonoids, artemetin (1), chrysosplenetin (2), pachypodol (3), penduletin (4) and chrysoeriol (5) were obtained by various column chromatographic methods. Their chemical structures were determined on the basis of comprehensive spectroscopic analysis and comparison with literature data. Three of the compounds (2, 4 and 5) exhibited synergistic activity when combined with norfloxacin against SA1199B, an effluxing fluoroquinolone-resistant strain. The fractional inhibitory concentration indices (FICIs) of 2, 4 and 5 in combination with norfloxacin were 0.375, 0.079 and 0.266 respectively, suggesting synergy. Compound 5 also showed synergistic effects against EMRSA-15 and EMRSA-16 when combined with ciprofloxacin and oxacillin exhibiting FICIs of 0.024 and 0.375 respectively. Real time ethidium bromide (EtBr) efflux assay, qRT-PCR and molecular docking were employed to explore the mechanisms of the synergistic effects.

Synergism of sophoraflavanone G with norfloxacin against effluxing antibiotic-resistant Staphylococcus aureus.

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), remains a challenge in hospital and community settings. The design and discovery of new compounds to deal with resistant bacteria has become one of the most important areas of anti-infective research today. The aim of this study was to address the problem of MRSA by searching for synergistic natural antibacterial products from traditional Chinese herbs that are not substrates for the efflux mechanisms of MRSA and that overcome bacterial drug resistance by other, as yet undescribed, mechanisms. In vitro synergistic activity was determined using the standard chequerboard method, and mechanistic studies were performed by an ethidium bromide efflux assay. Using in vivo experiments, the efficacies of different concentrations of the combinations were compared in a murine model of pyaemia. The natural product sophoraflavanone G showed specific synergistic antibacterial effects both in vitro and in vivo and may serve as a template for agents with antibiotic-potentiating activity for use against infections caused by S. aureus, including MRSA.

TLC-bioautography directed isolation of antibacterial compounds from active fractionation of Ferula ferulioides.

A novel optimised isolation method, TLC-bioautography, was evaluated and utilised in this research. Antibacterial compounds which were isolated from the dichloromethane extract of Ferula ferulioides (Steud.) Korovin were detected by means of the method. Their structures were elucidated by extensive spectral and chemical methods. Their antibacterial activities against drug-resistant Staphylococcus aureus (S. aureus) strains were evaluated with broth microdilution method, and the results proved that TLC-bioautography was an effective and highly efficient way to screen natural compounds from plant extracts against drug-resistant strains.

Isovalerylshikonin, a new resistance-modifying agent from Arnebia euchroma, supresses antimicrobial resistance of drug-resistant Staphylococcus aureus.

Antimicrobial resistance is the greatest threat to the treatment of bacterial infectious diseases. The development of resistance-modifying agents (RMAs) represents a promising strategy to mitigate the spread of bacterial antimicrobial resistance. In this study, a natural product, isovalerylshikonin (IVS), was isolated from Arnebia euchroma, a traditional Chinese medicine herb, that exhibited marginal antibacterial activity against drug-resistant Staphylococcus aureus RN4220, with a minimum inhibitory concentration (MIC) of 16 mg/L. In addition, a synergistic effect between IVS and streptomycin (STM) was detected by the microdilution antimicrobial chequerboard assay, with a reduction in the MIC of STM by up to 16-fold against strain RN4220. A bacterial ethidium bromide efflux assay and reverse transcription PCR were performed to investigate the synergistic mechanism. IVS significantly inhibited bacterial efflux and expression of msrA mRNA in vitro. A murine peritonitis/sepsis model was employed to test the in vivo synergistic activity of IVS and STM. IVS synergistically decreased bacterial counts with STM in peritoneal, spleen and liver tissue and increased mouse survival with STM in 7 days. The acute toxicity of IVS was tested and the 50% lethal dose (LD50) of IVS with a single exposure was 2.584 g/kg in mice. Overall, IVS, a low-toxicity RMA, exhibited synergistic antibacterial activities in vitro and in vivo against drug-resistant S. aureus. The effects were mediated by suppression of msrA mRNA expression and reduced bacterial efflux. In addition, these data support that IVS is a potential RMA against microbial resistance caused by the MsrA efflux pump.

High-Speed Counter-Current Chromatography with an Online Storage Technique for the Preparative Isolation and Purification of Dihydroflavonoids from Sophora alopecuroides L.

INTRODUCTION: High-speed counter-current chromatography (HSCCC) is an efficient and non-absorption separation technique, but limitations still exist in simultaneous isolation of complex structures of natural products. Moreover, particular methods are various for different kinds of natural products. OBJECTIVE: A novel HSCCC strategy combined with an online storage recycling elution (OSR-CCC) technique was developed for the quick separation of naturally occurring dihydroflavonoids from the extract of the herb Sophora alopecuroides L. METHODOLOGY: In the separation procedure, a storage loop and two six-port valves were connected to a HSCCC system. Effluent A was subjected to an online storage loop and then to recycling separation three times after effluent B was collected in head-to-tail mode. After completion of the recycling separation of effluent A, the elution was switched to tail-to-head mode to collect effluent C. A biphasic solvent system of n-hexane/ethyl acetate/methanol/water (9:6:6:8, v/v/v/v) was used as the separation solvent during the whole elution. RESULTS: Six constituents were isolated simultaneously from the extract (200 mg) of S. alopecuroides by running HSCCC non-stop, and their purities were higher than 95.0%. Their structures were determined as the pterocarpan glycoside sophoratonkin (1) (10.0 mg) and five dihydroflavonoids, alopecurone F (2) (5.4 mg), lehmannin (3) (11.0 mg), alopecurone A (4) (35.0 mg), sophoraflavanone G (5) (21.0 mg), alopecurone B (6) (31.0 mg). CONCLUSION: This recycling HSCCC method combined with an online storage technique could be a rapid, effective and simple approach to isolate stilbene-dihydroflavonoids from herbs of the Sophora genus simultaneously. Copyright © 2017 John Wiley & Sons, Ltd.

Flavonoids from Sophora moorcroftiana and their synergistic antibacterial effects on MRSA.

Synergy is now a widely recognized approach that has direct applicability for new pharmaceuticals. The ethanolic extract of the aerial parts of the herb Sophora moorcroftiana showed significant antibacterial activity against drug-resistant Staphylococcus aureus, and its minimum inhibitory concentration (MIC) was 8 µg/mL. In a phytochemical study of the extract, five flavonoids were obtained. However, the isolates exhibited antibacterial activity in the range of 32-128 µg/mL, which was weaker than the extract. In combination with antibiotics, the antibacterially inactive compound genistein (1) and diosmetin (4) showed significant synergistic activity against drug-resistant S. aureus. In combination with norfloxacin, genistein (1) reduced the MIC to 16 µg/mL and showed synergy against strain SA1199B with a fractional inhibitory concentration index (FICI) of 0.38. With the antibiotics norfloxacin, streptomycin and ciprofloxacin, diosmetin (4) showed synergy against SA1199B, RN4220 and EMRSA-15, with FICI values of 0.38, 0.38 and 0.09, respectively. In an efflux experiment to elucidate a plausible mechanism for the observed synergy, genistein showed marginal inhibition of the NorA efflux protein.

[Effects of soil texture and water content on the mineralization of soil organic carbon in paddy soils].

To understand how soil texture and water content affect the mineralization of organic C in paddy soil, 3 selected soils (sandy loam, clay loam, and silty clay) were incubated (25 degrees C) with 14 C-labelled rice straw (1.0 g x kg(-1)) at water content varied from 45% to 105% of water holding capacity (WHC). Data indicated that, in the sandy loam and clay loam, the mineralization rate of 14 C-labelled rice straw reached the maximum at 75% WHC, as 53% and 58% of the straw C mineralized in the incubation period of 160 d, whereas in the silty clay, it increased gradually (from 41.8% to 49.0%) as water content increased up to 105% WHC. For all of the three soils, the mineralization rate of soil native organic C reached the maximum at 75% WHC, with 5.8% of the organic C mineralized in the same period for the sandy loam, and 8.0% and 4.8% for the clay loam and silty clay, respectively. As water content increased further, the mineralization rate of native organic C in the three soils significantly declined. The mineralization rate of added rice straw and native organic C in all the three soils, was well fitted with a conic curve. These results suggest that water-logging can decrease the mineralization of organic C in paddy soils.

[Effects of temperature on organic carbon mineralization in paddy soils with different clay content].

An incubation test with three kinds of paddy soil (sandy loam, clay loam, and silty clay soils) in subtropical region was conducted at 10, 15, 20, 25 and 30 degrees C to examine the response of the mineralization of soil organic carbon (SOC) to temperature change. The results showed that during the period of 160 d incubation, the accumulative mineralized amount of SOC in sandy loam, clay loam, and silty clay soils at 30 degrees C was 3.5, 5.2 and 4.7 times as much as that at 10 degrees C, respectively. The mineralization rate was lower and relatively stable at lower temperatures (< or = 20 C), but was higher at the beginning of incubation and decreased and became stable as the time prolonged at higher temperatures (> or = 25 degrees C). During incubation, the temperature coefficient (Q10) of SOC mineralization in test soils fluctuated, with an average Q10 in sandy loam, clay loam, and silty clay soils being 1.92, 2.37 and 2.32, respectively. There was a positive exponential correlation between SOC mineralization constant k and temperature (P < 0.01), and the response of SOC mineralization to temperature change was in the order of clay loam soil > silty clay soil > sandy loam soil.