Therapeutic effect of oxyberberine on obese non-alcoholic fatty liver disease rats.

BACKGROUND: Berberine (BBR) has been widely used to treat non-alcoholic fatty liver disease (NAFLD). The metabolites of BBR were believed to contribute significantly to its pharmacological effects. Oxyberberine (OBB), a gut microbiota-mediated oxidative metabolite of BBR, has been firstly identified in our recent work. PURPOSE: Here, we aimed to comparatively investigate the anti-NAFLD properties of OBB and BBR. METHODS: The anti-NAFLD effect was evaluated in high-fat diet-induced obese NAFLD rats with biochemical/ELISA tests and histological staining. The related gene and protein expressions were detected by qRT-PCR and Western blotting respectively. Molecular docking and dynamic simulation were also performed to provide further insight. RESULTS: Results indicated OBB remarkably and dose-dependently attenuated the clinical manifestations of NAFLD, which (100 mg/kg) achieved similar therapeutic effect to metformin (300 mg/kg) and was superior to BBR of the same dose. OBB significantly inhibited aberrant phosphorylation of IRS-1 and up-regulated the downstream protein expression and phosphorylation (PI3K, p-Akt/Akt and p-GSK-3ß/GSK-3ß) to improve hepatic insulin signal transduction. Meanwhile, OBB treatment remarkably alleviated inflammation via down-regulating the mRNA expression of MCP-1, Cd68, Nos2, Cd11c, while enhancing Arg1 mRNA expression in white adipose tissue. Moreover, OBB exhibited closer affinity with AMPK in silicon and superior hyperphosphorylation of AMPK in vivo, leading to increased ACC mRNA expression in liver and UCP-1 protein expression in adipose tissue. CONCLUSION: Taken together, compared with BBR, OBB was more capable of maintaining lipid homeostasis between liver and WAT via attenuating hepatic insulin pathway and adipocyte inflammation, which was associated with its property of superior AMPK activator.

Comparison of anti-inflammatory effects of berberine, and its natural oxidative and reduced derivatives from Rhizoma Coptidis in vitro and in vivo.

BACKGROUND: Berberine (BBR) is the most abundant and major active constituent of Rhizoma Coptidis (RC), which has been widely used to treat inflammatory diseases in traditional oriental medicine. Despite BBR has been found to exhibit pronounced anti-inflammatory effect, the anti-inflammatory activities of its natural derivatives were sparsely dissected out. PURPOSE: To comparatively investigate the anti-inflammatory potential of BBR, and its natural oxoderivative (oxyberberine, OBB) and reduced derivative (dihydroberberine, DHBB) in vitro and in vivo, and delineate the possible underlying mechanism. METHODS: LC-MS/MS was used to identify the natural derivatives of BBR in RC. The potential anti-inflammatory properties of BBR and its natural derivatives were comparatively evaluated in vitro by lipopolysaccharide (LPS)-induced RAW264.7 macrophages cells, and in vivo via three typical acute inflammation murine models. Some important inflammation-related molecules were analyzed by ELISA, qRT-PCR and Western blotting. RESULTS: LC-MS/MS led to the identification of BBR, OBB and DHBB in RC ethyl acetate extract. The in vitro assay indicated that BBR, OBB and DHBB (1.25, 2.5 and 5 µM) pretreatment significantly decreased the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), prostaglandinE2 (PGE2) and nitricoxide (NO), and inhibited the mRNA expressions of cyclooxygenase-2 (COX-2) and inducible nitricoxide synthase (iNOS) in a dose-dependent manner, with relative efficiency of OBB > BBR > DHBB. Furthermore, OBB, BBR and DHBB remarkably inhibited the phosphorylation of nuclear factor-κB (NF-κB) p65 and inhibitory kappa Bα (IκBα). In vivo, BBR (20 mg/kg) and OBB (5, 10, and 20 mg/kg) pretreatment significantly ameliorated the xylene-induced ear edema, carrageenan-stimulated paw edema, and acetic acid-elicited vascular permeability in mice in a dose-dependent manner, with OBB exhibiting superior anti-inflammatory effect at the same dose (20 mg/kg). Histopathological analysis indicated that OBB and BBR could markedly attenuate the inflammatory deterioration and decrease the cellular infiltration in paw tissues. Additionally, the carrageenan-induced increases in TNF-α, IL-6, IL-1ß, PGE2 and NO productions, and COX-2 and iNOS mRNA expressions were effectually and concentration-dependently suppressed by OBB and BBR pretreatment. CONCLUSION: The anti-inflammatory activity of BBR and its natural derivatives was in the order of OBB > BBR > DHBB. OBB was for the first time found to be endowed with pronounced anti-inflammatory property, which was probably associated with suppressing the activation of NF-κB signaling pathway, and the subsequent gene expressions and productions of pro-inflammatory mediators. The results might contribute to illuminating the pharmacodynamic underpinnings of RC and provide evidence for developing OBB as a safe and promising natural lead compound in inflammation treatment.

Angelica sinensis Supercritical Fluid CO2 Extract Attenuates D-Galactose-Induced Liver and Kidney Impairment in Mice by Suppressing Oxidative Stress and Inflammation.

Angelica sinensis (AS, Danggui in Chinese) is an important herbal component of various traditional formulae for the management of asthenia and its tonic effects. Although AS has been shown to ameliorate cognitive damage and nerve toxicity in D-galactose (D-gal)-elicited senescent mice brain, its effects on liver and kidney injury have not yet been explored. In this work, mice were subjected to hypodermic injection with D-gal (200 mg/kg) and orally gavaged with AS (20, 40, or 80 mg/kg) once a day for 8 successive weeks. Results revealed that AS significantly improved liver and kidney function as assessed by organ index and functional parameters. In addition, AS pretreatment effectively ameliorated the histological deterioration. AS attenuated the MDA level and markedly enhanced the activities and gene expressions of antioxidative enzymes, namely Cu, Zn-SOD, CAT, and GPx. Furthermore, AS markedly inhibited the D-gal-mediated increment of expressions of inflammatory cytokines iNOS, COX-2, IκBα, p-IκBα, and p65 and promoted the IκBα expression level in both hepatic and renal tissues. In sum, AS pretreatment could effectively guard the liver and kidney of mice from D-gal-induced injury, and the underlying mechanism was deemed to be intimately related to attenuating oxidative response and inflammatory stress.

Efficacy and indication optimization of Chinese medicine (Tiao-Chang Ke-Min granules) for diarrhea-predominant irritable bowel syndrome: study protocol for a randomized controlled trial.

BACKGROUND: Irritable bowel syndrome (IBS) is a chronic, recurring condition, prevalent in the general population. Current medication treatments usually leave patients undertreated. Nowadays, Chinese medicine (CM) is being considered as a promising treatment approach for IBS. However, due to methodological limitations, there is no strong evidence to support CM. Although IBS relapses are common, the relapse assessment has always been neglected in CM study designs. Meanwhile, in clinical practice and studies, it has been found that certain CM formulas can only benefit certain kinds of patients. Discovering what population and illness characteristics likely respond to outcomes may help improve the effectiveness of CM. The aims of this study are to evaluate the efficacy and safety of Tiao-Chang Ke-Min (TCKM) granules for IBS, especially in reducing IBS symptoms' relapse, by a high-quality randomized controlled trial and then to optimize the indication of the TCKM granules. METHODS/DESIGN: This is a parallel-group, randomized, double-blind, placebo-controlled trial embedded with outcome predictive factors. Eligible patients with diarrhea-predominant IBS will be randomized into either a TCKM granule group or a placebo group. Patients from both groups will receive health education. The treatment duration is 4 weeks and the follow-up is 12 weeks. The primary outcome is global improvement measured with adequate relief (AR). The second outcome measures include time until relief, time until first relapse, total relapse times, long-term effectiveness, individual symptoms, IBS-Symptom Severity Score (IBS-SSS), IBS-Quality of Life Questionnaire (IBS-QOL), and Hospital Anxiety and Depression Scale (HADS). Predictive factors associated with patient and illness characteristics have been widely collected. These factors will be embedded in this trial for further identification. DISCUSSION: This trial may provide high-quality evidence on the efficacy and safety of TCKM granules for IBS and a more accurate indication. Importantly, this trial will provide a new research method for improving the therapeutic effects of CM for clinicians and researchers. To address IBS relapse assessment, a series of special definitions of relapse incidents has been made for this trial. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ID: ChiCTR-IOR-17010600 . Registered on 9 February 2017.

ß-Patchoulene from patchouli oil protects against LPS-induced acute lung injury via suppressing NF-κB and activating Nrf2 pathways.

ß-Patchoulene (ß-PAE), a tricyclic sesquiterpene isolated from the essential oil of the leaves and stems of Pogostemon cablin (Blanco) Benth., has been reported to have potent anti-inflammatory activity. The aim of this study was to evaluate the potential protective effect of ß-PAE on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and to illuminate the underlying mechanisms. ALI was induced by intracheal instillation of LPS into lung, and dexamethasone (DEX) was used as a positive control. Results indicated that pretreatment with ß-PAE significantly decreased the mortality rate of mice and lung W/D weight ratio, ameliorated lung pathological changes as compared to model group. Meanwhile, ß-PAE pretreatment markedly inhibited the increase of TNF-α, IL-6 and IL-1ß secretions in the bronchoalveolar lavage fluid, and prevented LPS-induced elevations of MPO activity and MDA level in the lung. Additionally, ß-PAE pretreatment significantly elevated miR-146a expression and suppressed the LPS-induced activation of NF-κB and expression of its mediated genes (TNF-α, IL-6 and IL-1ß). ß-PAE was also observed to markedly upregulate the Nrf2 and HO-1 expression and activate the antioxidant genes (NQO-1, GCLC and HO-1). Taken together, ß-PAE possessed protective effect against LPS-induced ALI, which might be associated with its differential regulation of NF-κB and Nrf2 activities and up-regulation of expression of miR-146a. The results rendered ß-PAE a promising anti-inflammatory agent worthy of further development into a pharmaceutical drug for the treatment of ALI.

Induction of defense-related enzymes in patchouli inoculated with virulent Ralstonia solanacearum

Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.

The immune-regulating effect of Xiao'er Qixingcha in constipated mice induced by high-heat and high-protein diet.

BACKGROUND: Xiao'er Qixingcha (EXQ) has been extensively applied to relieve dyspepsia and constipation in children for hundreds of years in China. However, the therapeutic mechanism underlying its efficacy remained to be defined. The present study aimed to clarify the possible laxative and immune-regulating effects of EXQ on two models of experimental constipation in mice, which mimicked the pediatric constipation caused by high-heat and high-protein diet (HHPD). METHODS: The two models of constipated mice were induced by HHPD or HHPD + atropine respectively. To investigate the laxative and immune-regulating activities of EXQ, animals were treated with three doses of EXQ (0.75, 1.5 and 3 g/kg) for 7 consecutive days. The fecal output parameters (number and weight), weight of intestinal content and, the thymus and spleen indexes were measured. The levels of sIgA, IL-10, TNF-α and LPS in colon and serum were determined by ELISA. Furthermore, the pathological changes of colon tissue were examined after routine H&E staining. RESULTS: Both HHPD and HHPD + atropine treatments obviously inhibited the fecal output and reduced the colonic sIgA, prominently increased the levels of IL-10 and TNF-α in colonic tissue and elevated the contents of LPS in serum and colonic tissues. In contrast, oral administration of EXQ significantly improved the feces characters and dose-dependently decreased the intestinal changes in both models. In HHPD model test, EXQ efficaciously boosted the sIgA level in a dose-dependent manner, significantly elicited decreases in TNF-α and IL-10 levels, and evidently decreased the spleen and thymus indexes. In HHPD + atropine model test, EXQ treatment reversed the pathological changes by not only dramatically decreasing the spleen index and the levels of LPS and IL-10, but also markedly elevating the thymus index. Furthermore, microscopic observation revealed that EXQ treatment maintained the integrity of colonic mucosa, and protected the colonic tissues from inflammation in the both models. CONCLUSIONS: EXQ exhibited prominent laxative activity and effectively protected the colonic mucosal barrier in two models of constipated mice, of which the mechanism might be closely associated with its propulsive and immune-regulating properties. The current results not only validated the rationale for the clinical application of EXQ in pediatric constipation related symptoms, but also threw new light on the immune-inflammatory responses accompanied with chronic constipation pathology.

Protective Effects of Li-Fei-Xiao-Yan Prescription on Lipopolysaccharide-Induced Acute Lung Injury via Inhibition of Oxidative Stress and the TLR4/NF-κB Pathway.

Li-Fei-Xiao-Yan prescription (LFXY) has been clinically used in China to treat inflammatory and infectious diseases including inflammatory lung diseases. The present study was aimed at evaluating the potential therapeutic effects and potential mechanisms of LFXY in a murine model of lipopolysaccharide- (LPS-) induced acute lung injury (ALI). In this study, the mice were orally pretreated with LFXY or dexamethasone (positive drug) before the intratracheal instillation of LPS. Our data indicated that pretreatment with LFXY enhanced the survival rate of ALI mice, reversed pulmonary edema and permeability, improved LPS-induced lung histopathology impairment, suppressed the excessive inflammatory responses via decreasing the expression of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) and chemokine (MIP-2) and inhibiting inflammatory cells migration, and repressed oxidative stress through the inhibition of MPO and MDA contents and the upregulation of antioxidants (SOD and GSH) activities. Mechanistically, treatment with LFXY significantly prevented LPS-induced TLR4 expression and NF-κB (p65) phosphorylation. Overall, the present study suggests that LFXY protected mice from acute lung injury induced by LPS via inhibition of TLR4/NF-κB p65 activation and upregulation of antioxidative enzymes and it may be a potential preventive and therapeutic agent for ALI in the clinical setting.

Inhibition of Helicobacter pylori and Its Associated Urease by Palmatine: Investigation on the Potential Mechanism.

In this paper, we evaluated the anti-Helicobacter pylori activity and the possible inhibitory effect on its associated urease by Palmatine (Pal) from Coptis chinensis, and explored the potential underlying mechanism. Results indicated that Pal exerted inhibitory effect on four tested H. pylori strains (ATCC 43504, NCTC 26695, SS1 and ICDC 111001) by the agar dilution test with minimum inhibitory concentration (MIC) values ranging from 100 to 200 µg/mL under neutral environment (pH 7.4), and from 75 to 100 µg/mL under acidic conditions (pH 5.3), respectively. Pal was observed to significantly inhibit both H. pylori urease (HPU) and jack bean urease (JBU) in a dose-dependent manner, with IC50 values of 0.53 ± 0.01 mM and 0.03 ± 0.00 mM, respectively, as compared with acetohydroxamic acid, a well-known urease inhibitor (0.07 ± 0.01 mM for HPU and 0.02 ± 0.00 mM for JBU, respectively). Kinetic analyses showed that the type of urease inhibition by Pal was noncompetitive for both HPU and JBU. Higher effectiveness of thiol protectors against urease inhibition than the competitive Ni2+ binding inhibitors was observed, indicating the essential role of the active-site sulfhydryl group in the urease inhibition by Pal. DTT reactivation assay indicated that the inhibition on the two ureases was reversible, further supporting that sulfhydryl group should be obligatory for urease inhibition by Pal. Furthermore, molecular docking study indicated that Pal interacted with the important sulfhydryl groups and inhibited the active enzymatic conformation through N-H ∙ π interaction, but did not interact with the active site Ni2+. Taken together, Pal was an effective inhibitor of H. pylori and its urease targeting the sulfhydryl groups, representing a promising candidate as novel urease inhibitor. This investigation also gave additional scientific support to the use of C. chinensis to treat H. pylori-related gastrointestinal diseases in traditional Chinese medicine. Pal might be a potentially beneficial therapy for gastritis and peptic ulcers induced by H. pylori infection and other urease-related diseases.

Anti-inflammatory effects of Brucea javanica oil emulsion by suppressing NF-κB activation on dextran sulfate sodium-induced ulcerative colitis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Brucea javanica is an important traditional medicinal herb used for the treatment of dysentery, malaria, inflammation and cancer in southeast Asia for many years. However, the anti-inflammatory mechanism of Brucea javanica in the treatment of dysentery (also known as ulcerative colitis, UC) has not been fully illuminated. Brucea javanica oil emulsion (BJOE) is the major active and most common application form of Brucea javanica oil (BJO), which has a variety of pharmacological activities. The aim of this study was to investigate the potential anti-inflammatory effect of BJOE and possible mechanism of action on dextran sulfate sodium (DSS)-induced UC in mice. MATERIALS AND METHODS: The components of BJOE were determined by gas chromatography-mass spectrometry (GC-MS). Balb/C mice with dextran sulfate sodium (DSS, 30mg/mL) induced colitis were treated with BJOE (0.5, 1 and 2g/kg) and two positive drugs (sulfasalazine, SASP, 200mg/kg; and azathioprine, AZA, 13mg/kg) once daily by gavage for 7 days. Mice in normal control group and DSS group were orally given the same volume of distilled water and soybean lecithin suspension (0.15g/kg) respectively. The effects of BJOE on DSS-induced UC were assessed by determination of body weight loss, disease activity index (DAI), colon length, histological analysis, as well as levels of pro-inflammatory cytokines. The mRNA expression of MPO, iNOS and COX-2 in colon tissues was detected by qRT-PCR. In addition, NF-κB p65, p-p65 and IκB-α, p-IκBα protein expression levels in colon tissues were investigated using Western blotting. RESULTS: The major components of BJOE were found to be oleic acid (62.68%) and linoleic acid (19.53%) as detected by GC-MS. Our results indicated that BJOE, SASP and AZA showed beneficial effect on DSS-induced colitis in mice, and significantly reduced the body weight loss and DAI, restored the colon length, repaired colonic pathological variations, decreased histological scores, and decreased the levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-8, IL-17 and IFN-γ) as compared with the DSS group. In addition, the mRNA expression of MPO, iNOS and COX-2 induced by DSS treatment was remarkably inhibited by BJOE, SASP or AZA treatments. Furthermore, when compared with DSS-treated mice, the activation of NF-κB was significantly inhibited by AZA and BJOE treatment. CONCLUSIONS: Our study shows that BJOE possessed appreciable anti-inflammatory effect against murine experimental UC induced by DSS. The protective mechanism of BJOE may involve inhibition of NF-κB signal transduction pathways and subsequent down-regulation of inflammatory mediators. These findings suggest that BJOE might be an efficacious and promising therapeutic approach for the treatment of UC. Our investigation might also provide experimental evidence for the traditional application of Brucea javanica in the treatment of dysentery and might add new dimension to the clinical indications for BJOE.

Gartanin Protects Neurons against Glutamate-Induced Cell Death in HT22 Cells: Independence of Nrf-2 but Involvement of HO-1 and AMPK.

Oxidative stress mediates the pathogenesis of neurodegenerative disorders. Gartanin, a natural xanthone of mangosteen, possesses multipharmacological activities. Herein, the neuroprotection capacity of gartanin against glutamate-induced damage in HT22 cells and its possible mechanism(s) were investigated for the first time. Glutamate resulted in cell death in a dose-dependent manner and supplementation of 1-10 µM gartanin prevented the detrimental effects of glutamate on cell survival. Additional investigations on the underlying mechanisms suggested that gartanin could effectively reduce glutamate-induced intracellular ROS generation and mitochondrial depolarization. We further found that gartanin induced HO-1 expression independent of nuclear factor erythroid-derived 2-like 2 (Nrf2). Subsequent studies revealed that the inhibitory effects of gartanin on glutamate-induced apoptosis were partially blocked by small interfering RNA-mediated knockdown of HO-1. Finally, the protein expression of phosphorylation of AMP-activated protein kinase (AMPK) and its downstream signal molecules, Sirtuin activator (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), increased after gartanin treatment. Taken together, these findings suggest gartanin is a potential neuroprotective agent against glutamate-induced oxidative injury partially through increasing Nrf-2-independed HO-1 and AMPK/SIRT1/PGC-1α signaling pathways.

Evaluation of safety of modified-Danggui Buxue Tang in rodents:immunological, toxicity and hormonal aspects.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Astragali (RA), Radix Angelicae Sinensis (RAS) and Folium Epimedii (FE) are three of the extensively applied herbs among traditional Chinese medicines for gynecological disorders and osteoporosis. A derivative herbal formula-RRF, consisting of the three medicines with a weight ratio of 5:1:5, is derived from a famous Chinese herbal formula-Danggui Buxue Tang (DBT). RRF has shown noteworthy perimenopause ameliorating effect in both ovariectomized rats and natural aging female rats, which might represent a promising candidate for the treatment of perimenopausal disorders. The aim of this study was to evaluate its immunological potential, chronic toxicity and reproductive effects by 26-week repeated daily administration in female rats, in order to optimize its safe use. MATERIALS AND METHODS: The effect of RRF on immunological function was studied by macrophage phagocytosis, immune organ index, serum immunoglobulin level as well as delayed type hypersensitivity (DTH) in mice. For toxicity assessment, acute toxicity study was performed according to fixed dose procedure with a single oral administration of RRF to mice. In the oral chronic toxicity, 120 female rats were administrated RRF orally in 0, 1100, 4400, or 8800mg/kg/day doses for 26 weeks. Clinical signs, mortality, body weights, feed consumption, haemato-biochemical parameters, organ weights, histopathology and reproductive hormone profiles were examined at the end of the 13- and 26-week dosing period, as well as after the 4-week recovery period. RESULTS: Oral administration of RRF at three doses (282, 564 and 1128mg/kg) significantly increased the indices of phagocytosis K, as compared with prednisone acetate (PR) group (p<0.05 or 0.01). Exposure of RRF dose-dependently boosted circulating serum IgM level (all p<0.01) in response to CRBC in PR-induced mice. Furthermore, RRF treatment elicited a significant increment (all p<0.01) in DNFB-induced DTH response and the immune organ indices in a dose-dependent manner in mice, in parellel to DNFB-induced group. In the single dose acute toxicity and repeated dose 90-day chronic toxicity investigations, no toxic signs/mortality were observed. RRF treatment did not cause any toxicologically significant changes in clinical signs, food consumption, body weight, relative organ weight, hematological parameters, clinical chemistry, gross pathology and histopathology between treatment and control groups. No treatment related gross/histopathological lesions were observed and no target organ was identified. Long-term repeated administration of RRF exerted a significant promotion on serum level of steroid hormone estradiol, progesterone and testosterone release, along with decrease of circulating pituitary follicle stimulating hormone, luteinizing hormone, and prolactin levels in female rats. The No Observed Adverse Effect Level (NOAEL) of RRF was determined to be over 8800mg/kg/day for elderly female rats, a dose that was equivalent to 50 times of human dose. CONCLUSION: The present investigation demonstrated that RRF possessed appreciable immunopotentiating activity and had a relatively wide margin of safety. Long-term treatment of RRF exhibited estrogenic properties, and retarded certain age-associated degenerations. RRF might have the potential for further development as a safe and effective alternative/complementary to conventional medication in relieving perimenopausal symptoms.

Andrographolide sodium bisulphite-induced inactivation of urease: inhibitory potency, kinetics and mechanism.

BACKGROUND: The inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C. METHODS: The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ 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. RESULTS: The IC50 of ASB against JBU and HPU was 3.28±0.13 mM and 3.17±0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki=2.86×10(-3) mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki*=1.33×10(-4) mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap. CONCLUSIONS: ASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.

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.

Selective antibacterial activity of patchouli alcohol against Helicobacter pylori based on inhibition of urease.

The aim of this study is to evaluate the antibacterial activity and urease inhibitory effects of patchouli alcohol (PA), the bioactive ingredient isolated from Pogostemonis Herba, which has been widely used for the treatment of gastrointestinal disorders. The activities of PA against selected bacteria and fungi were determined by agar dilution method. It was demonstrated that PA exhibited selective antibacterial activity against Helicobacter pylori, without influencing the major normal gastrointestinal bacteria. Noticeably, the antibacterial activity of PA was superior to that of amoxicillin, with minimal inhibition concentration value of 78 µg/mL. On the other hand, PA inhibited ureases from H.pylori and jack bean in concentration-dependent fashion with IC50 values of 2.67 ± 0.79 mM and 2.99 ± 0.41 mM, respectively. Lineweaver-Burk plots indicated that the type of inhibition was non-competitive against H.pylori urease whereas uncompetitive against jack bean urease. Reactivation of PA-inactivated urease assay showed DL-dithiothreitol, the thiol reagent, synergistically inactivated urease with PA instead of enzymatic activity recovery. In conclusion, the selective H.pylori antibacterial activity along with urease inhibitory potential of PA could make it a possible drug candidate for the treatment of H.pylori infection.

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.

Gastroprotective effect and mechanism of patchouli alcohol against ethanol, indomethacin and stress-induced ulcer in rats.

Pogostemonis Herba is an important Chinese medicine widely used in the treatment of gastrointestinal dysfunction. Patchouli alcohol (PA), a tricyclic sesquiterpene, is the major active constituent of Pogostemonis Herba. This study aimed to investigate the possible anti-ulcerogenic potential of PA and the underlying mechanism against ethanol, indomethacin and water immersion restraint-induced gastric ulcers in rats. Gross and histological gastric lesions, biochemical and immunological parameters were taken into consideration. The gastric mucus content and the antisecretory activity were analyzed through pylorus ligature model in rats. Results indicated that oral administration with PA significantly reduced the ulcer areas induced by ethanol, indomethacin and water immersion restraint. PA pretreatment significantly promoted gastric prostaglandin E2 (PGE2) and non-protein sulfhydryl group (NP-SH) levels, upregulated the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) mRNA expression, and considerably boosted the gastric blood flow (GBF) and gastric mucus production in comparison with vehicle. In addition, PA modulated the levels of interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α). The levels of glutathione (GSH), catalase (CAT) and malonaldehyde (MDA) were also restored by PA. However, the gastric secretion parameters (pH, volume of gastric juice and pepsin) did not show any significant alteration. These findings suggest that PA exhibited significant gastroprotective effects against gastric ulceration. The underlying mechanisms might involve the stimulation of COX-mediated PGE2, improvement of antioxidant and anti-inflammatory status, preservation of GBF and NP-SH, as well as boost of gastric mucus production.

Insecticidal activity of pogostone against Spodoptera litura and Spodoptera exigua (Lepidoptera: Noctuidae).

BACKGROUND: Essential oil of Pogostemon cablin (Blanco) Benth. has been reported to exhibit strong insecticidal activities, but few studies have focused on the insecticidal activity of its main individual constituent, pogostone (PO). The goal of this research was to investigate the insecticidal activity of PO against two harmful noctuid insects, Spodoptera litura (Fabricius) and Spodoptera exigua (Hübner). RESULTS: In a no-choice assay, PO exhibited strong antifeedant activity against S. litura and S. exigua. PO showed pronounced larvicidal activities, including oral toxicity (LC50 986.88 mg L(-1) and 545.61 mg L(-1) respectively) and contact toxicity (LC50 1041.42 mg L(-1) and 519.48 mg L(-1) respectively) against these two noctuid insects. Additionally, PO treatment significantly increased the larval and pupal developmental period. Furthermore, PO showed moderate ovicidal activities and influenced the emergence and deformity of the moth. However, PO failed to exert a potent effect on adult development. These tested parameters proved to be dose dependent for both insect species. CONCLUSION: PO possesses strong insecticidal activities, especially antifeedant, larvicidal, growth inhibitory and pupicidal activities, against S. litura and S. exigua. PO may partly account for the insecticidal activity of patchouli oil and may be a promising candidate for the control of agricultural insects.

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.

Gastroprotective and anti-Helicobacter pylori potential of herbal formula HZJW: safety and efficacy assessment.

BACKGROUND: A traditional Chinese Medicine (TCM) formula, HZJW, has been applied in clinics in China for gastrointestinal disorders. However, the therapeutic mechanism underlying its efficacy and safety remained to be defined. The present investigation was undertaken to evaluate the formula HZJW for its gastroprotective potential, possible effect on Helicobacter pylori along with safety to justify its anti-ulcer action and safe clinical application. METHODS: The gastroduodenal cytoprotective potential was evaluated in rodent experimental models (HCl/Ethanol and NSAID-induced ulcer protocols). The anti-H. pylori property was assessed by agar dilution assay in vitro and analysis in vivo including rapid urease test, immunogold test and histopathology. For toxicity assessment, acute toxicity study was performed according to fixed dose procedure with a single oral administration of HZJW to mice. In the oral chronic toxicity, rats (80 males, 80 females) were administrated HZJW orally in 0, 1000, 2500, or 5000 mg/kg/day doses for 26 weeks (n = 40/group of each sex). Clinical signs, mortality, body weights, feed consumption, ophthalmology, hematology, serum biochemistry, gross findings, organ weights and histopathology were examined at the end of the 13- and 26-week dosing period, as well as after the 4-week recovery period. RESULTS: In the HCl/Ethanol-induced ulcer model, it was observed that oral administration with HZJW (260, 520 and 1040 mg/kg) and ranitidine (250 mg/kg) significantly reduced the ulcerative lesion index (116.70 ± 36.4, 102.20 ± 18.20, 84.10 ± 12.1 and 73.70 ± 16.70) in a dose-dependent manner, respectively, with respect to control group (134.10 ± 31.69). Significant inhibition was also observed in ulcerative index from aspirin-induced ulcer model, with decreases of 35.40 ± 5.93, 31.30 ± 8.08, 26.80 ± 8.27and 20.40 ± 6.93 for the groups treated with HZJW and ranitidine, in parallel to controls (41.60 ± 10.80). On the other hand, treatment with HZJW efficaciously eradicated H. pylori in infected mice in rapid urease test (RUT) and immunogold antibody assay, as further confirmed by reduction of H. pylori presence in histopathological analysis. In the in vitro assay, MICs for HZJW and amoxicillin (positive control) were 125 and 0.12 µg/mL respectively. The LD50 of HZJW was over 18.0 g/kg for mice. No drug-induced abnormalities were found as clinical signs, body weight, food consumption, hematology, blood biochemistry, ophthalmology and histopathology results across three doses. No target organ was identified. The No Observed Adverse Effect Level (NOAEL) of HZJW was determined to be 5,000 mg/kg/day for both sexes, a dose that was equivalent to 50 times of human dose. CONCLUSIONS: These results suggested the efficacy and safety of HZJW in healing peptic ulcer and combating H. pylori, which corroborated their conventional indications and contributed to their antiulcer pharmacological validation, lending more credence to its clinical application for the traditional treatment of stomach complaints symptomatic of peptic ulcer disease (PUD). HZJW might have the potential for further development as a safe and effective alternative/complementary to conventional medication in treating gastrointestinal (GI) disorders.