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.

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.

Protective Effect of SFE-CO2 of Ligusticum chuanxiong Hort Against d-Galactose-Induced Injury in the Mouse Liver and Kidney.

This study was designed to explore how supercritical fluid CO2 extract of Ligusticum chuanxiong Hort (CX) protects mouse liver and kidney from d-galactose-induced injury. The antioxidant capacity of CX was confirmed both in vitro and in vivo. The d-galactose-induced malondialdehyde increase was attenuated by CX, as well as the increase in aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine level. In addition, the activities of antioxidant enzymes were markedly renewed, and the gene expressions of these enzymes were upregulated in CX groups. The results of histological analysis suggested that CX could effectively attenuate the d-galactose-induced structure damage. Furthermore, results of Western blotting analysis showed that CX significantly inhibited the upregulation of nuclear factor protein expression caused by d-galactose. In conclusion, CX could attenuate the liver and kidney injury in d-galactose-treated mice, and the mechanism might be associated with attenuating oxidative stress and inflammatory response.

Polydatin attenuates d-galactose-induced liver and brain damage through its anti-oxidative, anti-inflammatory and anti-apoptotic effects in mice.

Accumulating evidence has shown that chronic injection of d-galactose (d-gal) can mimic natural aging, with accompanying liver and brain injury. Oxidative stress and apoptosis play a vital role in the aging process. In this study, the antioxidant ability of polydatin (PD) was investigated using four established in vitro systems. An in vivo study was also conducted to investigate the possible protective effect of PD on d-gal-induced liver and brain damage. The results showed that PD had remarkable in vitro free radical scavenging activity on 2,2-diphenyl-1-picryl-hydrazyl (DPPH˙), 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS+˙) radical ions, and hydroxyl and superoxide anions. Results in vivo indicated that, in a group treated with d-gal plus PD, PD remarkably decreased the depression of body weight and organ indexes, reduced the levels of the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and alleviated alterations in liver and brain histopathology. PD also significantly decreased the level of MDA and elevated SOD, GSH-Px, CAT activity and T-AOC levels in the liver and brain. In addition, the levels of inflammatory mediators, such as TNF-α, IL-1ß and IL-6 in serum were markedly reduced after PD treatment. Western blotting results revealed that PD treatment noticeably attenuated the d-gal-induced elevation of Bcl-2/Bax ratio and caspase-3 protein expression in liver and brain. Overall, our findings indicate that PD treatment could effectively attenuate d-gal-induced liver and brain damage, and the mechanism might be associated with decreasing the oxidative stress, inflammation and apoptosis caused by d-gal. PD holds good potential for further development into a promising pharmaceutical candidate for the treatment of age-associated diseases.

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.

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.

Usnic acid protects LPS-induced acute lung injury in mice through attenuating inflammatory responses and oxidative stress.

Usnic acid is a dibenzofuran derivative found in several lichen species, which has been shown to possess several activities, including antiviral, antibiotic, antitumoral, antipyretic, analgesic, antioxidative and anti-inflammatory activities. However, there were few reports on the effects of usnic acid on LPS-induced acute lung injury (ALI). The aim of our study was to explore the effect and possible mechanism of usnic acid on LPS-induced lung injury. In the present study, we found that pretreatment with usnic acid significantly improved survival rate, pulmonary edema. In the meantime, protein content and the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) significantly decreased, and the levels of MPO, MDA, and H2O2 in lung tissue were markedly suppressed after treatment with usnic acid. Meanwhile, the activities of SOD and GSH in lung tissue significantly increased after treatment with usnic acid. Additionally, to evaluate the anti-inflammatory activity of usnic acid, the expression of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and anti-inflammatory cytokine IL-10, and chemokines interleukin-8 (IL-8) and macrophage inflammatory protein-2 (MIP-2) in BALF were studied. The results in the present study indicated that usnic acid attenuated the expression of TNF-α, IL-6, IL-8 and MIP-2. Meanwhile, the improved level of IL-10 in BALF was observed. In conclusion, these data showed that the protective effect of usnic acid on LPS-induced ALI in mice might relate to the suppression of excessive inflammatory responses and oxidative stress in lung tissue. Thus, it was suggested that usnic acid might be a potential therapeutic agent for ALI.