Biological evaluation and molecular docking of baicalin and scutellarin as Helicobacter pylori urease inhibitors.

ETHNOPHARMACOLOGICAL RELEVANCE: Baicalin and scutellarin are the principal bioactive components of Scutellaria baicalensis Georgi which has extensively been incorporated into heat-clearing and detoxification formulas for the treatment of Helicobacter pylori-related gastrointestinal disorders in traditional Chinese medicine. However, the mechanism of action remained to be defined. AIM OF THE STUDY: To explore the inhibitory effect, kinetics and mechanism of Helicobacter pylori urease (the vital pathogenetic factor for Helicobacter pylori infection) inhibition by baicalin and scutellarin, for their therapeutic potential. MATERIALS AND METHODS: The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of baicalin and scutellarin was characterized with IC50 values, compared to acetohydroxamic acid (AHA), a well known Helicobacter pylori urease inhibitor. Lineweaver-Burk and Dixon plots for the Helicobacter pylori urease inhibition of baicalin and scutellarin was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni(2+) binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode. Moreover, cytotoxicity experiment using Gastric Epithelial Cells (GES-1) was evaluated. RESULTS: Baicalin and scutellarin effectively suppressed Helicobacter pylori urease in dose-dependent and time-independent manner with IC50 of 0.82±0.07 mM and 0.47±0.04 mM, respectively, compared to AHA (IC50=0.14±0.05 mM). Structure-activity relationship disclosed 4'-hydroxyl gave flavones an advantage to binding with Helicobacter pylori urease. Kinetic analysis revealed that the types of inhibition were non-competitive and reversible with inhibition constant Ki of 0.14±0.01 mM and 0.18±0.02 mM for baicalin and scutellarin, respectively. The mechanism of urease inhibition was considered to be blockage of the SH groups of Helicobacter pylori urease, since thiol reagents (L,D-dithiothreitol, L-cysteine and glutathione) abolished the inhibitory action and competitive active site Ni(2+) binding inhibitors (boric acid and sodium fluoride) carried invalid effect. Molecular docking study further supported the structure-activity analysis and indicated that baicalin and scutellarin interacted with the key residues Cys321 located on the mobile flap through S-H·π interaction, but did not interact with active site Ni(2+). Moreover, Baicalin (at 0.59-1.05 mM concentrations) and scutellarin (at 0.23-0.71 mM concentrations) did not exhibit significant cytotoxicity to GES-1. CONCLUSIONS: Baicalin and scutellarin were non-competitive inhibitors targeting sulfhydryl groups especially Cys321 around the active site of Helicobacter pylori urease, representing potential to be good candidate for future research as urease inhibitor for treatment of Helicobacter pylori infection. Furthermore, our work gave additional scientific support to the use of Scutellaria baicalensis in traditional Chinese medicine (TCM) to treat gastrointestinal disorders.

Pogostone suppresses proinflammatory mediator production and protects against endotoxic shock in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Pogostemon cablin (Blanco) Benth is a well-known medicinal herb commonly used in many Asian countries for inflammatory diseases. Pogostone (PO), a natural product isolated from Pogostemon cablin, is known to exert various pharmacological activities. This study aimed to investigate the anti-inflammatory property of PO, to elucidate its mechanism of action, and to evaluate its potential acute toxicity. MATERIALS AND METHODS: The in vitro anti-inflammatory activity of PO was assessed using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The protein and mRNA levels of proinflammatory mediators were measured with ELISA and RT-PCR, respectively. Proteins of the NF-κB and MAPK family were determined by Western blot to investigate the underlying molecular mechanisms. The in vivo anti-inflammatory activity of PO was tested using LPS-induced endotoxic shock in mice. In addition, the median lethal dose (LD50) of PO in mice was tested in an acute toxicity test. RESULTS: In vitro, PO significantly inhibited the protein and mRNA expression of proinflammatory mediators including TNF-α, IL-6, IL-1ß, NO, and PGE2. The action mechanism of the anti-inflammatory activity of PO was partly dependent on inhibition of the activation of NF-κB and the phosphorylation of JNK and p38 MAPK. In vivo, PO was able to significantly reduce the mortality induced by LPS in mice. Furthermore, PO could markedly suppress the production of the proinflammatory mediators in serum, and attenuate liver and lung injury. The action mechanisms of PO during endotoxic shock may be attributed to down-regulation of the mRNA expression of inflammatory mediators in multiple organs via inhibition of the activation of NF-κB and the phosphorylation of p38 MAPK. Moreover, the LD50 of PO in mice was about 163mg/kg with intravenous administration, which was about 8-fold higher than the dose used in the animal experiment. CONCLUSIONS: Our findings regarding the anti-inflammatory effect of PO and the underlying molecular mechanisms help justify the use of Pogostemon cablin in Chinese medicine for the treatment of inflammatory diseases. More importantly, the results also render PO a promising anti-inflammatory agent worthy of further development into a pharmaceutical drug for the treatment of septic shock.

Bushen­Yizhi formula ameliorates cognition deficits and attenuates oxidative stress­related neuronal apoptosis in scopolamine­induced senescence in mice.

Bushen­Yizhi formula (BSYZ), a traditional Chinese medicine formula consisting of six herbs has been reported to possess a neuroprotective effect. The present study aimed to investigate the effects of BSYZ on learning and memory abilities, as well as oxidative stress and neuronal apoptosis in the hippocampus of scopolamine (SCOP)­induced senescence in mice, in order to reveal whether BSYZ is a potential therapeutic agent for Alzheimer's disease (AD). A high­performance liquid chromatography (HPLC) fingerprint was applied to provide a chemical profile of BSYZ. Extracts of BSYZ were orally administered to mice with SCOP­induced memory impairment for two weeks. The learning and memory abilities were determined by the Morris water maze test. The oxidant stress­related indices, such as activity of superoxide dismutase (SOD) and levels of glutathione (GSH) and malondialdehyde (MDA) were examined in hippocampus of SCOP­treated mice. The cell death ratio was assessed by TUNEL staining, while apoptotic­related proteins including Bcl­2 and Bax were determined by immuno-fluorescent staining and western blot analysis. Caspase­3 was determined by western blot analysis. Consequently, a chromatographic condition, which was conducted at 35˚C with a flow rate of 0.8 ml/min on the Gemini C18 column with mobile phase of acetonitrile and water­phosphoric acid (100:0.1, v/v), was established to yield common fingerprint chromatography under 203 nm with a similarity index of 0.986 within 10 batches of BSYZ samples. BSYZ at a dose of 2.92 g/kg significantly improved the cognitive ability, restored the abnormal activity of SOD and increased the levels of MDA and GSH induced by SCOP. Moreover, the neural apoptosis in the hippocampus of SCOP­treated mice was reversed by BSYZ by regulating the expression of Bcl­2, Bax and caspase­3. The results demonstrated that BSYZ had neuroprotective effects in SCOP­induced senescence in mice by ameliorating oxidative stress and neuronal apoptosis in the brain, supporting its potential in AD treatment.

Inactivation of jack bean urease by scutellarin: elucidation of inhibitory efficacy, kinetics and mechanism.

In the present study, the inactivation effect of scutellarin (SL) on jack bean urease was investigated to elucidate the inhibitory potency, kinetics and mechanism of inhibition. It was revealed that SL acted as a concentration- and time-dependent inactivator of urease characteristic of slow-binding inhibition with an IC50 of 1.35±0.15 mM. The rapid formation of the initial SL-urease complex with an inhibition constant of Ki=5.37×10(-2) mM was followed by a slow isomerization into the final complex with the overall inhibition constant of Ki*=3.49×10(-3) mM. High effectiveness of thiol protectors, such as L-cysteine (L-cys), 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) significantly slowed down the rate of inactivation, indicating the strategic role of the active site sulfhydryl group in the blocking process. While the insignificant protection by boric acid and fluoride from the inactivation further confirmed that the active site cysteine should be obligatory for urease inhibition, which was also rationalized by the molecular docking study. The inhibition of SL on urease proved to be reversible since SL-blocked urease could be reactivated by DTT application and multidilution. The results obtained indicated that urease inactivation resulted from the reaction between SL and the sulfhydryl group.

Anti-Candida albicans activity and pharmacokinetics of pogostone isolated from Pogostemonis Herba.

The present work was designed to evaluate the in vitro and in vivo anti-Candida activity of pogostone (PO), a natural product isolated from Pogostemon cablin (Blanco) Benth. PO showed potent in vitro activity against clinical Candida spp. isolates tested in this study. PO and the reference drug voriconazole (VRC) were equally effective against all the fluconazole-resistant Candida albicans strains, with MIC ranging from 3.1 µg/ml to 50 µg/ml. Besides, PO was fungicidal against all Candida isolates with MFC ranging from 50 µg/ml to 400 µg/ml. By contrast, VRC was fungistatic as it failed to elicit a fungicidal effect against the Candida spp. isolates at the highest tested concentration (400 µg/ml). Furthermore, oral and topical PO administration effectively reduced the fungal load in vagina of vulvovaginal candidiasis mouse models. Topical PO administration (1.0-4.0 mg/kg) demonstrated higher activity against the vulvovaginal candidiasis than VRC (4.0 mg/kg). The pharmacokinetics and safety profile of PO were also investigated. The pharmacokinetics assay revealed that PO was easily absorbed after oral administration in mice, which might account for its in vivo anti-Candida effect. The acute toxicity test showed that the median lethal dose of PO in mice was 355 mg/kg, which was much higher than the daily dose used for the therapeutic experiments. This study demonstrated the potential of PO as a promising candidate for the treatment of Candida infections, particularly for vulvovaginal candidiasis.

[Scenario analysis of integrated model of nutrients in the Miyun Reservoir and its watershed].

Supported by the integrated model of nutrients for the Miyun Reservoir in part I, effects of different control measures were studied on the water quality of the reservoir. Four scenarios were assumed and analyzed. Results of the base case scenario showed that TN concentration of the Miyun Reservoir had highly exceeded the environmental quality standard for surface water, and TP was relatively better. Furthermore, there were many regions that chlorophyll-a concentration exceeded 10 microg/L in the reservoir, and centralized in the reservoir area of Chaohe River. Scenario 1, 2 and 3 investigated effects of different pollution control measures on the water quality of the reservoir. Results showed that the control of nutrient input loads could improve the water quality greatly, especially control of TP loadings would limit algae growth effectively, and regions that chlorophyll-a concentration exceeded 10 microg/L even disappeared. The results indicated that some control measures, such as changing farming style, part treatment on stockbreeding pollution and reducing point source pollutant loadings were very effective and essential to decrease the eutrophic level of the reservoir.

[Integrated model of nutrients for the Miyun Reservoir and its watershed].

In recent years, the Miyun Reservoir faces severe problems of water quality and quantity due to continuous drought. In order to simulate and predict the eutrophic status of the Miyun Reservoir and its watershed, an integrated model was developed based on GIS and RS technology, which was composed of non-point source (NPS) simulation model, aquatic ecological-hydrodynamic model, and river water quality model. Ecological-hydrodynamic model was developed through coupling water quality analysis simulation program (WASP) with environmental fluid dynamics Code (EFDC). SWAT was selected as NPS simulation model, which could also perform water quality simulation in the river. Then the integrated model was calibrated using Markov Chain Monte Carlo method and verified using observed data. Results indicated that observed water quality data laid around the mode curves of simulation distribution, and which also dropped into the confidence interval on 80 percent credibility of water quality simulation distribution. In conclusion, the results show that the integrated model can meet the need of application.