Cailan Li

Epiberberine, a natural protoberberine alkaloid, inhibits urease of Helicobacter pylori and jack bean: Susceptibility and mechanism

Abstract In our previous study, Rhizoma Coptidis extract was found to exert more potent inhibitory effect than its major component berberine towards urease from Helicobacter pylori (HPU) and jack bean (JBU). In continuation of our work, the present study was designed to further comparatively investigate the urease inhibitory activities of five major protoberberine alkaloids in Rhizoma Coptidis, namely berberine, palmatine, coptisine, epiberberine, jateorhizine to identify the bioactive constituent, and illuminate the potential mechanism of action. Results indicated that the five protoberberine alkaloids acted as concentration‐dependent inactivators of urease with IC50 values ranging between 3.0 and 5087 &mgr;M for HPU and 2.3–> 10,000 &mgr;M for JBU, respectively. Notably, epiberberine (EB) was found to be the most potent inhibitor against both ureases with IC50 values of 3.0 ± 0.01 &mgr;M for HPU and 2.3 ± 0.01 &mgr;M for JBU, which was more effective than the standard urease inhibitor, acetohydroxamic acid (83 ± 0.01 &mgr;M for HPU and 22 ± 0.01 &mgr;M for JBU, respectively). Further kinetic analysis revealed that the type of EB inhibition against HPU was slow‐binding and uncompetitive, with Ki of 10.6 ± 0.01 &mgr;M, while slow‐binding and competitive against JBU with Ki of 4.6 ± 0.01 &mgr;M. Addition of thiol reagents, such as l‐cysteine, glutathione and dithiothreitol, significantly abolished the inhibition, while Ni2+ competitive inhibitors, boric acid and sodium fluoride, synergetically inhibited urease with EB, indicating the obligatory role of the active site sulfhydryl group for the inhibition. In addition, binding of EB with the urease proved to be reversible, as about 65% and 90% enzymatic activity of HPU and JBU, respectively, could be restored by dithiothreitol application. These findings highlighted the potential role of Rhizoma Coptidis protoberberine alkaloids, especially EB, as a lead urease inhibitor in the treatment of diseases associated with ureolytic bacteria. Thus, EB had good potential for further development into a promising therapeutic approach for the treatment of urease‐related diseases. Graphical Abstract Figure. No Caption available.

Comparison of Helicobacter pylori Urease Inhibition by Rhizoma Coptidis, Cortex Phellodendri and Berberine: Mechanisms of Interaction with the Sulfhydryl Group

Rhizoma Coptidis, Cortex Phellodendri, and berberine were reported to inhibit Helicobacter pylori. However, the underlying mechanism remained elusive. Urease plays a vital role in H. pylori colonization and virulence. In this work, aqueous extracts of Rhizoma Coptidis, Cortex Phellodendri of different origins, and purified berberine were investigated against H. pylori urease and jack bean urease to elucidate the inhibitory capacity, kinetics, and mechanism. Results showed that berberine was the major chemical component in Rhizoma Coptidis and Cortex Phellodendri, and the content of berberine in Rhizoma Coptidis was higher than in Cortex Phellodendri. The IC50 values of Rhizoma Coptidis were significantly lower than those Cortex Phellodendri and purified berberine, of which Coptis chinensis was shown to be the most active concentration- and time-dependent urease inhibitor. The Lineweaver-Burk plot analysis indicated that the inhibition pattern of C. chinensis against urease was noncompetitive for both H. pylori urease and jack bean urease. Thiol protectors (L-cysteine, glutathione, and dithiothreithol) significantly protected urease from the loss of enzymatic activity, while fluoride and boric acid showed weaker protection, indicating the active-site sulfhydryl group was possibly responsible for its inhibition. Furthermore, the urease inhibition proved to be reversible since C. chinensis-blocked urease could be reactivated by glutathione. The results suggested that the anti-urease activity of Rhizoma Coptidis was superior to that of Cortex Phellodendri and berberine, which was believed to be more likely to correlate to the content of total alkaloids rather than berberine monomer. The concentration- and time-dependent, reversible, and noncompetitive inhibition against urease by C. chinensis might be attributed to its interaction with the sulfhydryl group of the active site of urease.

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 activity of coptisine free base in mice through inhibition of NF-κB and MAPK signaling pathways

Coptisine is one of the main constituents of Coptis chinensis which has been widely used for the remedy of inflammatory disorders. Although the biological activities of coptisine have been well known, the pharmacological properties of its free base have seldomly been elucidated thus far. The aim of this study was to investigate the potential anti-inflammatory properties of coptisine free base (CFB, 8-hydroxy-7,8-dihydrocoptisine) on three animal models, namely xylene-induced ear edema, acetic acid-induced vascular permeability and carrageenan-induced paw edema. The results exhibited that CFB exerted a dose-dependent suppression on ear edema induced by xylene, significantly mitigated the aggravation of vascular permeability caused by acetic acid and paw edema induced by carrageenan. Additionally, CFB significantly suppressed the productions of interleukin-1β (IL-1β), interleukin-6 (IL-6), prostaglandinE2 (PGE2) and tumor necrosis factor (TNF-α) in the drug-treated groups as compared with the vehicle group after treatment with carrageenan. Signaling events of nuclear factor-κB (NF-κB) translocation, such as p-IKKα, p-IKKβ, p-IκBα and p65 (nucleus) were significantly inactivated, while inhibitor of nuclear factor κBα (IκBα) and p65 (cytosolic) were markedly up-regulated by CFB. Furthermore, CFB also significantly suppressed the mitogen-activated protein kinase (MAPK) pathway by blocking the phosphorylation of p-p38 (phospho-p38 mitogen-activated protein kinases) and p-JNK (phospho-c-jun N-terminal kinase) but not p-ERK (phospho-extracellular signal-regulated kinase). Hence, CFB efficiently prevented inflammation, at least partially, via inhibition of NF-κB and MAPK pathways. These findings provided a pioneering pharmacological basis for the anti-inflammatory effect of CFB and suggested CFB might be a potential candidate for the therapy of inflammatory disorders.

Protective effect of coptisine free base on indomethacin-induced gastric ulcers in rats: Characterization of potential molecular mechanisms

Aims: The aim of this study was to comparatively investigate the potential gastroprotective effect and underlying mechanisms of coptisine free base (CFB, 8‐hydroxy‐7, 8‐dihydrocoptisine), berberine and lansoprazole against indomethacin‐induced gastric ulcer in rats. Materials and methods: CFB (10, 20 and 40 mg/kg), berberine (20 mg/kg) and lansoprazole (30 mg/kg) were orally administrated to rats prior to indometacin ingestion, and gastric lesions were evaluated macroscopically and histologically, and further analyzed by ELISA, qRT‐PCR and Western blot. Key findings: CFB exerted comparable or superior gastroprotective effect to berberine in protecting against indomethacin‐induced gastric injury. CFB pretreatment significantly enhanced the levels of superoxide dismutase (SOD) and glutathione (GSH), and markedly decreased the malonaldehyde (MDA) content. CFB administration effectively suppressed the levels of myeloperoxidase (MPO), interleukin‐1&bgr; (IL‐1&bgr;), tumor necrosis factor‐&agr; (TNF‐&agr;) and angiotensin II (Ang II). Besides, CFB substantially up‐regulated the mRNA expressions of cyclooxygenase‐1 (COX‐1) and cyclooxygenase‐2 (COX‐2), and promoted gastric mucosal prostaglandin E2 level (PGE2). Furthermore, CFB pretreatment remarkably increased the translocation of nuclear factor erythroid 2‐related factor 2 (Nrf2) from cytosol into the nucleus, and the expression of heme oxygenase‐1 (HO‐1), while significantly decreased the expression of mitogen activated protein Kinase Kinase 6 (MKK6) and translocation of p38 mitogen‐activated protein kinase (p38 MAPK). Significance: This was the first investigation reporting the anti‐ulcer effect of protoberberine alkaloid free base on in vivo rodent model. The gastroprotective mechanism of CFB might involve favorable regulation of antioxidant and anti‐inflammatory status mediated, at least partially, by the Nrf2 signaling pathway and p38 MAPK translocation.

Chongcao-Shencha Attenuates Liver and Kidney Injury through Attenuating Oxidative Stress and Inflammatory Response in D-Galactose-Treated Mice

The Chongcao-Shencha (CCSC), a Chinese herbal compound formula, has been widely used as food material and medicine for enhancing physical strength. The present study investigated the possible effect of CCSC in alleviating the liver and kidney injury in D-galactose- (D-gal-) treated mice and the underlying mechanism. Mice were given a subcutaneous injection of D-gal (200 mg/kg) and orally administered CCSC (200, 400, and 800 mg/kg) daily for 8 weeks. Results indicated that CCSC increased the depressed body weight and organ index induced by D-gal, ameliorated the histological deterioration, and decreased the levels of ALT, AST, BUN, and CRE as compared with D-gal group. Furthermore, CCSC not only elevated the activities of antioxidant enzymes SOD, CAT, and GPx but also upregulated the mRNA expression of SOD1, CAT, and GPx1, while decreasing the MDA level in D-gal-treated mice. Results of western blotting analysis showed that CCSC significantly inhibited the upregulation of expression of nuclear factor kappa B (NF-κB) p65, p-p65, p-IκBα, COX2, and iNOS and inhibited the downregulation of IκBα protein expression caused by D-gal. This study demonstrated that CCSC could attenuate the liver and kidney injury in D-gal-treated mice, and the mechanism might be associated with attenuating oxidative stress and inflammatory response.

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

Abstract &bgr;‐Patchoulene (&bgr;‐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 &bgr;‐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 &bgr;‐PAE significantly decreased the mortality rate of mice and lung W/D weight ratio, ameliorated lung pathological changes as compared to model group. Meanwhile, &bgr;‐PAE pretreatment markedly inhibited the increase of TNF‐&agr;, IL‐6 and IL‐1&bgr; secretions in the bronchoalveolar lavage fluid, and prevented LPS‐induced elevations of MPO activity and MDA level in the lung. Additionally, &bgr;‐PAE pretreatment significantly elevated miR‐146a expression and suppressed the LPS‐induced activation of NF‐&kgr;B and expression of its mediated genes (TNF‐&agr;, IL‐6 and IL‐1&bgr;). &bgr;‐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, &bgr;‐PAE possessed protective effect against LPS‐induced ALI, which might be associated with its differential regulation of NF‐&kgr;B and Nrf2 activities and up‐regulation of expression of miR‐146a. The results rendered &bgr;‐PAE a promising anti‐inflammatory agent worthy of further development into a pharmaceutical drug for the treatment of ALI. Highlights&bgr;‐PAE significantly decreased the mortality rate of mice and lung W/D weight ratio, ameliorated lung pathological changes.&bgr;‐PAE pretreatment markedly inhibited the increase of TNF‐&agr;, IL‐6 and IL‐1&bgr; secretions in the bronchoalveolar lavage fluid.&bgr;‐PAE prevented LPS‐induced elevations of MPO activity and MDA level in the lung.ß‐PAE possessed protective effect against LPS‐induced ALI by inhibiting NF‐&kgr;B and activating Nrf2 pathways.

Protective Effect of Pogostone on 2,4,6-Trinitrobenzenesulfonic Acid-Induced Experimental Colitis via Inhibition of T Helper Cell

Inflammatory bowel disease (IBD) is a chronic immune-related disease mainly caused by the disequilibrium of T helper (Th) cell paradigm? Pogostone (PO) is one of the major chemical constituents of Pogostemon cablin (Blanco) Benth. The present study aims to investigate the potential benefit of PO against IBD in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis model. PO treatment by enema significantly brought down the disease activity index (DAI) of the TNBS-challenged rats, which was manifested by the ameliorated inflammatory features including ulceration, adhesion, and edema. Hematoxylin-eosin (HE) staining and immunohistochemistry analysis showed that PO effectively relived colon damage by restoring epithelium, and more importantly, by inhibiting the infiltration of pro-inflammatory Th1 and Th17 cells in the colon. Additionally, PO inhibited the activity of myeloperoxidase and secretion of inflammatory cytokines including IFN-γ, IL-12p70, IL-17A, and IL-10. Together with our previous findings, the present data indicated that the anti-IBD effect of PO probably related to its direct inhibition on Th cell proliferation and suppression of the cytokines secretion. These results highlighted the potential of PO as a promising candidate to relieve IBD.

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-1β (IL-1β), 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-1β, 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.

Therapeutic efficacy and safety of Kang-ai injection combined with platinum-based doublet chemotherapy in advanced NSCLC: A meta-analysis

Aims: Kang‐ai injection (KA) is a famous Chinese patent medicine authorized by China Food and Drug Administration, which is widely used to treat advanced non‐small cell lung cancer (NSCLC) in China. This meta‐analysis is aimed to evaluate the therapeutic efficacy and safety of KA on advanced NSCLC. Methods: Seven databases were examined for related studies until January 15, 2018. Odds ratio (OR) was used to evaluate tumor response, Karnofsky Performance Scale (KPS) improvement and adverse reactions, and mean difference (MD) was used to estimate immune functions. Key findings: Thirty randomized controlled trials involving 1956 patients with advanced NSCLC were included. The results showed that compared with the platinum‐based doublet chemotherapy (PBDC) alone, KA combined with PBDC could significantly enhance tumor response (OR = 1.69, 95% CI [1.40, 2.04], P < 0.00001), KPS improvement (OR = 3.01, 95% CI [2.36, 3.84], P < 0.00001) and immune functions including the percentages of CD3+ (MD = 8.90, 95% CI [3.06, 14.73], P = 0.003), CD4+ (MD = 9.43, 95% CI [6.32, 12.53], P < 0.00001) and NK (MD = 4.81, 95% CI [1.95, 7.68], P = 0.001) and the ratio of CD4+/CD8+ (MD = 0.29, 95% CI [0.04, 0.53], P = 0.02). Moreover, KA combined with PBDC markedly decreased the incidences of adverse reactions including gastrointestinal reaction (OR = 0.38, 95% CI [0.30, 0.47], P < 0.00001), myelosuppression (OR = 0.32, 95% CI [0.23, 0.45], P < 0.00001) and hair loss (OR = 0.53, 95% CI [0.36, 0.76], P < 0.00001). However, there was no significant difference between the combination treatment group and the control group in the percentage of CD8+ (MD = −2.93, 95% CI [−6.68, 0.82], P = 0.13). Significance: Despite the small sample size and study limitations, the results of this meta‐analysis indicated that the combination therapy of KA and PBDC (especially NP regimen) might be a beneficial therapeutic method for advanced NSCLC patients.