Patchouli alcohol attenuates the cognitive deficits in a transgenic mouse model of Alzheimer's disease via modulating neuropathology and gut microbiota through suppressing C/EBPß/AEP pathway.

BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by progressive cognitive dysfunctions and behavioral impairments. Patchouli alcohol (PA), isolated from Pogostemonis Herba, exhibits multiple pharmacological properties, including neuroprotective effects. This study aimed to investigate the therapeutic effects of PA against AD using the TgCRND8 transgenic AD mouse model, and to explore the underlying mechanisms targeting CCAAT/enhancer-binding protein ß/asparagine endopeptidase (C/EBPß/AEP) signaling pathway. METHODS: After genotyping to confirm the transgenicity, drug treatments were administered intragastrically once daily to 3-month-old TgCRND8 mice for 4 consecutive months. Several behavioral tests were applied to assess different aspects of neurological functions. Then the brain and colon tissues were harvested for in-depth mechanistic studies. To further verify whether PA exerts anti-AD effects via modulating C/EBPß/AEP signaling pathway in TgCRND8 mice, adeno-associated virus (AAV) vectors encoding CEBP/ß were bilaterally injected into the hippocampal CA1 region in TgCRND8 mice to overexpress C/EBPß. Additionally, the fecal microbiota transplantation (FMT) experiment was performed to verify the potential role of gut microbiota on the anti-AD effects of PA. RESULTS: Our results showed that PA treatment significantly improved activities of daily living (ADL), ameliorated the anxiety-related behavioral deficits and cognitive impairments in TgCRND8 mice. PA modulated the amyloid precursor protein (APP) processing. PA also markedly reduced the levels of beta-amyloid (Aß) 40 and Aß42, suppressed Aß plaque burdens, inhibited tau protein hyperphosphorylation at several sites and relieved neuroinflammation in the brains of TgCRND8 mice. Moreover, PA restored gut dysbiosis and inhibited the activation of the C/EBPß/AEP signaling pathway in the brain and colon tissues of TgCRND8 mice. Interestingly, PA strikingly alleviated the AD-like pathologies induced by the overexpression of C/EBPß in TgCRND8 mice. Additionally, the FMT of fecal microbiota from the PA-treated TgCRND8 mice significantly alleviated the cognitive impairments and AD-like pathologies in the germ-free TgCRND8 mice. CONCLUSION: All these findings amply demonstrated that PA could ameliorate the cognitive deficits in TgCRND8 mice via suppressing Aß plaques deposition, hyperphosphorylation of tau protein, neuroinflammation and gut dysbiosis through inhibiting the activation of C/EBPß/AEP pathway, suggesting that PA is a promising naturally occurring chemical worthy of further development into the pharmaceutical treatment of AD.

Traditional Patchouli essential oil modulates the host's immune responses and gut microbiota and exhibits potent anti-cancer effects in ApcMin /+ mice.

Patchouli Essential Oil (PEO) has been used as a scent for various healing purposes since the ancient Egyptian period. The primary source of the oil is Pogostemon cablin (PC), a medicinal plant for treating gastrointestinal symptoms. However, the pharmacological function has not been addressed. Here, we report the cancer prevention and gut microbiota (GM) modulating property of PEO and its derivatives patchouli alcohol (PA) and pogostone (PO) in the ApcMin /+ colorectal cancer mice model. We found that PEO, PA, and PO significantly reduced the tumor burden. At the same time, it strengthened the epithelial barrier, evidenced by substantially increasing the number of the goblet and Paneth cells and upregulation of tight junction and adhesion molecules. In addition, PEO, PA, and PO shifted M1 to M2 macrophage phenotypes and remodeled the inflammatory milieu of ApcMin /+ mice. We also found suppression of CD4+CD25+ and stimulation CD4+ CD8+ cells in the spleen, blood, mesenteric lymph nodes (MLNs), and Peyer's patches (PPs) of the treated mice. The composition of the gut microbiome of the drug-treated mice was distinct from the control mice. The drugs stimulated the short-chain fatty acids (SCFAs)-producers and the key SCFA-sensing receptors (GPR41, GPR43, and GPR109a). The activation of SCFAs/GPSs also triggered the alterations of PPAR-γ, PYY, and HSDCs signaling mediators in the treated mice. Our work showed that PEO and its derivatives exert potent anti-cancer effects by modulating gut microbiota and improving the intestinal microenvironment of the ApcMmin /+ mice.

(+/-)-Borneol Reverses Mitoxantrone Resistance against P-Glycoprotein.

P-Glycoprotein (Pgp) is a main factor contributing to multidrug resistance and the consequent failure of chemotherapy. Overcoming Pgp efflux is a strategy to improve the efficacy of drugs. (+)-Borneol (BNL1) and (-)-borneol (BNL2) interfere and inhibit Pgp, and thus, the accumulation of drugs increases in cells. However, it is not clear yet how they play the inhibitory effect against Pgp. In this work, the effect and molecular mechanism of borneol enantiomers in reversing mitoxantrone (MTO) resistance against Pgp were explored by in vitro and in silico approaches. Chemosensitizing potential tests showed that BNLs could enhance the efficacy of MTO in MES-SA/MX2 cells, and BNL2 exhibited a stronger potential. The protein expression of Pgp was decreased to some extent by the administration of BNLs. Molecular docking revealed that BNLs could reduce the binding affinity between MTO and Pgp. The results were consistent with the chemosensitizing potential test and were supported by molecular dynamics (MD) simulations. Molecular docking also suggested that BNLs preferred to bind in the drug-binding pocket rather than the nucleotide-binding domain of inward-facing Pgp. The occupied space of BNLs had an evident distance from that of MTO, which was further verified by the conformational analysis after MD simulations. The decomposition of binding free energies revealed the key amino acid residues (GLN195, SER196, TRP232, PHE343, SER344, GLY346, and GLN347) for BNLs to reverse MTO resistance. The results provide an insight into the mechanism through which BNLs reduce the MTO resistance against inward-facing Pgp in the drug-binding pocket through noncompetitive inhibition.

Isorhynchophylline exerts antidepressant-like effects in mice via modulating neuroinflammation and neurotrophins: involvement of the PI3K/Akt/GSK-3ß signaling pathway.

Isorhynchophylline (IRN), an oxindole alkaloid isolated from Uncaria rhynchophylla, elicited distinct antidepressant-like activity in mice. The present study aimed to investigate the antidepressant-like effects of IRN in chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors in mice and to illustrate its possible mechanisms of action. The mice were subjected to CUMS for 6 wk and administered with IRN (20 or 40 mg/kg) daily by oral gavage for 3 wk. The PI3K/protein kinase B (Akt) inhibitor and glycogen synthase kinase-3ß (GSK-3ß) inhibitors were used to determine the involvement of the PI3K/Akt/GSK-3ß pathway in the antidepressant-like effects of IRN in the mice. The results showed that CUMS caused depression-like behaviors in the mice, such as behavioral despair by the forced swim test (FST) and anhedonia by the sucrose preference test. In addition, CUMS could significantly reduce the levels of nerve growth factor and brain-derived neurotrophic factor but markedly increase the release of TNF-α and IL-6 in the hippocampus and cerebral cortex of the mice. Western blotting analysis showed that CUMS markedly suppressed the levels of phosphorylated GSK-3ß (Ser9) and phosphorylated Akt (Ser473) but significantly enhanced the translocation of NF-κB p65 from cytosol to nuclei in the hippocampus and cerebral cortex of the mice. CUMS could also significantly increase the NF-κB binding activity in the hippocampus and cerebral cortex of the mice, whereas IRN treatment could significantly reverse the behavioral and biochemical changes induced by CUMS in the mice. Moreover, the antidepressant-like effect of IRN was completely abolished by the PI3K/Akt inhibitor. Combination treatment with IRN and GSK-3ß inhibitors in the mice exerted a synergistic anti-immobility action in the FST. The results of mechanistic investigations indicated that the antidepressant-like action of IRN was mediated, at least in part, by enhancing neurotrophins and attenuating neuroinflammation via modulating the PI3K/Akt/GSK-3ß pathway.-Xian, Y.-F., Ip, S.-P., Li, H.-Q., Qu, C., Su, Z.-R., Chen, J.-N., Lin, Z.-X. Isorhynchophylline exerts antidepressant-like effects in mice via modulating neuroinflammation and neurotrophins: involvement of the PI3K/Akt/GSK-3ß signaling pathway.

Oxyberberine, a novel gut microbiota-mediated metabolite of berberine, possesses superior anti-colitis effect: Impact on intestinal epithelial barrier, gut microbiota profile and TLR4-MyD88-NF-κB pathway.

Berberine (BBR), a naturally-occurring isoquinoline alkaloid isolated from several Chinese herbal medicines, has been widely used for the treatment of dysentery and colitis. However, its blood concentration was less than 1 %, and intestinal microflora-mediated metabolites of BBR were considered to be the important material basis for the bioactivities of BBR. Here, we investigated the anti-colitis activity and potential mechanism of oxyberberine (OBB), a novel gut microbiota metabolite of BBR, in DSS-induced colitis mice. Balb/C mice treated with 3 % DSS in drinking water to induce acute colitis were orally administrated with OBB once daily for 8 days. Clinical symptoms were analyzed, and biological samples were collected for microscopic, immune-inflammation, intestinal barrier function, and gut microbiota analysis. Results showed that OBB significantly attenuated DSS-induced clinical manifestations, colon shortening and histological injury in the mice with colitis, which achieved similar therapeutic effect to azathioprine (AZA) and was superior to BBR. Furthermore, OBB remarkably ameliorated colonic inflammatory response and intestinal epithelial barrier dysfunction. OBB appreciably inhibited TLR4-MyD88-NF-κB signaling pathway through down-regulating the protein expressions of TLR4 and MyD88, inhibiting the phosphorylation of IκBα, and the translocation of NF-κB p65 from cytoplasm to nucleus. Moreover, OBB markedly modulated the gut dysbiosis induced by DSS and restored the dysbacteria to normal level. Taken together, the result for the first time revealed that OBB effectively improved DSS-induced experimental colitis, at least partly through maintaining the colonic integrity, inhibiting inflammation response, and modulating gut microflora profile.

Patchouli Oil Attenuates High Fat Diet-induced Non-alcoholic Hepatic Steatosis.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. Nevertheless, no first-line therapy exists. Hepatic steatosis is the earliest stage of NAFLD, which is characterized by an accumulation of hepatic lipids. Patchouli oil (PO), which is isolated from the well-known Chinese herb named Pogostemon cablin (Blanco) Benth. (Lamiaceae), inhibits hepatic lipid accumulation effectively. However, its potential ability for the treatment of NAFLD had not been reported before. Thus, the objective of this study was to investigate the effectiveness of PO against hepatic steatosis and its underlying mechanisms. We used a high fat diet (HFD)-induced hepatic steatosis model of rats to estimate the effect of PO against NAFLD. Hematoxylin-eosin and oil red O staining were used to analyze the hepatic histopathological changes. ELISA, RT-qPCR, and Western blotting analysis were applied to evaluate the parameters for hepatic steatosis. Our results showed that PO significantly attenuated the lipid profiles and the serum enzymes, evidenced by quantitative and histopathological analyses. It also markedly down-regulated the expression of sterol regulatory element-binding protein 1 (SREPB-1c) with its downstream factors in de novo lipogenesis. And, likewise, in lipid export by very low-density lipoproteins (VLDL), related molecules were dramatically improved. Furthermore, PO observably normalized the aberrant peroxisome proliferator-activated receptor α (PPAR-α) signal in fatty acids oxidation. In conclusion, PO exerted a preventing effect against HFD-induced steatosis and might be due to decrease de novo lipogenesis, promote export of lipids, as well as owing to improve fatty acids oxidation.

Palmatine ameliorated murine colitis by suppressing tryptophan metabolism and regulating gut microbiota.

Inflammatory bowel disease (IBD), majorly include Crohn's disease (CD) and ulcerative colitis (UC), is chronic and relapsing inflammatory disorders of the gastrointestinal tract, which treatment options remain limited. Here we examined the therapeutic effects of an isoquinoline alkaloid, Palmatine (Pal), on mice experimental colitis induced by dextran sulfate sodium (DSS) and explored underlying mechanisms. Colitis was induced in BALB/c mice by administering 3% DSS in drinking water for 7 days. Pal (50 and 100 mg kg-1) and the positive drug Sulfasalazine (SASP, 200 mg kg-1) were orally administered for 7 days. Disease activity index (DAI) was evaluated on day 8, and colonic tissues were collected for biochemistry analysis. The fecal microbiota was characterized by high-throughput Illumina MiSeq sequencing. And plasma metabolic changes were detected by UPLC-MS. Our results showed that Pal treatment significantly reduced DAI scores and ameliorated colonic injury in mice with DSS-induced colitis. Mucosal integrity was improved and cell apoptosis was inhibited. Moreover, gut microbiota analysis showed that mice received Pal-treatment have higher relative abundance of Bacteroidetes and Firmicutes, but reduced amount of Proteobacteria. Moreover, Pal not only suppressed tryptophan catabolism in plasma, but also decreased the protein expression of indoleamine 2,3-dioxygenase 1 (IDO-1, the rate-limiting enzyme of tryptophan catabolism) in colon tissue. This is consolidated by molecular docking, which suggested that Pal is a potent IDO-1 inhibitor. Taken together, our findings demonstrate that Pal ameliorated DSS-induced colitis by mitigating colonic injury, preventing gut microbiota dysbiosis, and regulating tryptophan catabolism, which indicated that Pal has great therapeutic potential for colitis.

Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process.

In this study, we examined the anti-Helicobactor pylori effects of the main protoberberine-type alkaloids in Rhizoma Coptidis. Coptisine exerted varying antibacterial and bactericidal effects against three standard H. pylori strains and eleven clinical isolates, including four drug-resistant strains, with minimum inhibitory concentrations ranging from 25 to 50 µg/mL and minimal bactericidal concentrations ranging from 37.5 to 125 µg/mL. Coptisine's anti-H. pylori effects derived from specific inhibition of urease in vivo. In vitro, coptisine inactivated urease in a concentration-dependent manner through slow-binding inhibition and involved binding to the urease active site sulfhydryl group. Coptisine inhibition of H. pylori urease (HPU) was mixed type, while inhibition of jack bean urease was non-competitive. Importantly, coptisine also inhibited HPU by binding to its nickel metallocentre. Besides, coptisine interfered with urease maturation by inhibiting activity of prototypical urease accessory protein UreG and formation of UreG dimers and by promoting dissociation of nickel from UreG dimers. These findings demonstrate that coptisine inhibits urease activity by targeting its active site and inhibiting its maturation, thereby effectively inhibiting H. pylori. Coptisine may thus be an effective anti-H. pylori agent.

Cleavable Multifunctional Targeting Mixed Micelles with Sequential pH-Triggered TAT Peptide Activation for Improved Antihepatocellular Carcinoma Efficacy.

Although tumor-targeting nanovehicles for hepatocellular carcinoma (HCC) chemotherapy have attracted great research and clinic interest, the poor cancer penetration, inefficient cellular uptake, and slow intracellular drug release greatly compromise their therapeutic outcomes. In this work, a multifunctional mixed micellar system, consisting of glycyrrhetinic acid (GA) for specific liver-targeting, trans-activator of transcription (TAT) peptide for potent cell penetration, and pH-sensitive poly(ß-amino ester) polymers for acidic-triggered drug release, was developed to provide HCC-targeting delivery and pH-triggered release of doxorubicin (DOX). These micelles were hypothesized to efficaciously accumulate in HCC site by the guide of GA ligands, enter into cancer cells facilitated by the activated TAT peptide on the micellar surface, and finally rapidly release DOX in cytoplasm. To demonstrate this design, DOX was initially loaded in micelles modified with both GA and TAT (DOX/GA@TAT-M) with high drug loading efficiency and pH-sensitive drug release profiles. The HCC-targeting cellular uptake and synergetic anticancer efficacy were tested, indicating DOX/GA@TAT-M could be specifically and effectively internalized into HCC cells by the effect of GA targeting and TAT penetrating with enhanced cytotoxicity. In addition, the prolonged circulation time and enhanced accumulation in tumor facilitated its potent tumor growth inhibition activity in vivo. These results demonstrated that the cleavable multifunctional mixed micelles with tumor targeting, controlled TAT peptide activation, and sequential pH-sensitive drug release could be an efficient strategy for HCC treatment.

Patchouli alcohol ameliorates dextran sodium sulfate-induced experimental colitis and suppresses tryptophan catabolism.

Despite the increased morbidity of ulcerative colitis (UC) in recent years, available treatments remain unsatisfactory. Pogostemon cablin has been widely applied to treat a variety of gastrointestinal disorders in clinic for centuries, in which patchouli alcohol (PA, C15H26O) has been identified as the major active component. This study attempted to determine the bioactivity of PA on dextran sulfate sodium (DSS)-induced mice colitis and clarify the mechanism of action. Acute colitis was induced in mice by 3% DSS for 7 days. The mice were then given PA (10, 20 and 40mg/kg) or sulfasalazine (SASP, 200mg/kg) as positive control via oral administration for 7 days. At the end of study, animals were sacrificed and samples were collected for pathological and other analysis. In addition, a metabolite profiling and a targeted metabolite analysis, based on the Ultra-Performance Liquid Chromatography coupled with mass spectrometry (UPLC-MS) approach, were performed to characterize the metabolic changes in plasma. The results revealed that PA significantly reduced the disease activity index (DAI) and ameliorated the colonic injury of DSS mice. The levels of colonic MPO and cytokines involving TNF-α, IFN-γ, IL-1ß, IL-6, IL-4 and IL-10 also declined. Furthermore, PA improved the intestinal epithelial barrier by enhancing the level of colonic expression of the tight junction (TJ) proteins, for instance ZO-1, ZO-2, claudin-1 and occludin, and by elevating the levels of mucin-1 and mucin-2 mRNA. The study also demonstrated that PA inhibited the DSS-induced cell death signaling by modulating the apoptosis related Bax and Bcl-2 proteins and down-regulating the necroptosis related RIP3 and MLKL proteins. By comparison, up-regulation of IDO-1 and TPH-1 protein expression in DSS group was suppressed by PA, which was in line with the declined levels of kynurenine (Kyn) and 5-hydroxytryptophan (5-HTP) in plasma. The therapeutic effect of PA was evidently reduced when Kyn was given to mice. In summary, the study successfully demonstrated that PA ameliorated DSS-induced mice acute colitis by suppressing inflammation, maintaining the integrity of intestinal epithelial barrier, inhibiting cell death signaling, and suppressing tryptophan catabolism. The results provided valuable information and guidance for using PA in treatment of UC.

Patchoulene Epoxide Isolated from Patchouli Oil Suppresses Acute Inflammation through Inhibition of NF-κB and Downregulation of COX-2/iNOS.

According to the GC-MS analysis, compositional variation was observed between samples of patchouli oil, of which an unknown compound identified as patchoulene epoxide (PAO) was found only in the long-stored oil, whose biological activity still remains unknown. Therefore, the present study aimed to evaluate the potential anti-inflammatory activity with three in vivo inflammatory models: xylene-induced ear edema, acetic acid-induced vascular permeability, and carrageenan-induced paw edema. Further investigation into its underlying mechanism on carrageenan-induced paw edema was conducted. Results demonstrated that PAO significantly inhibited the ear edema induced by xylene, lowered vascular permeability induced by acetic acid and decreased the paw edema induced by carrageenan. Moreover, PAO markedly decreased levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and nitric oxide (NO), but increased levels of interleukin-4 (IL-4) and interleukin-10 (IL-10). PAO was also shown to significantly downregulate the protein and mRNA expressions of cyclooxygenase-2 (COX-2) and inducible nitric-oxide synthase (iNOS). Western blot analysis revealed that PAO remarkably inhibited p50 and p65 translocation from the cytosol to the nucleus by suppressing IKKß and IκBα phosphorylation. In conclusion, PAO exhibited potent anti-inflammatory activity probably by suppressing the activation of iNOS, COX-2 and NF-κB signaling pathways.

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.

Synthesis of pogostone by one-step.

Pogostone, isolated from Pogostemon cablin, has many biological activities such as potential antibacterial, anticandida, and antifungal. Traditional extraction leads to low output of PO about 17.6 mg/kg from Herba Pogostemonis. The previous literature had reported a synthetic study and the yield had reached 4.48% with strictly controlled reaction conditions. The two methods above cannot meet the large demand of PO; we report a new synthesis method. 4-hydroxy-6-methyl-2-pyrone (1) was added in toluene, with the existence of acylation catalyst 4-dimethylaminopyridine (DMAP), 4-methylvaleric acid (2), and condensing agent dicyclohexylcarbodiimide (DCC), PO was synthesized after the combination of 3-carbon of (1) with 1-OH of (2) in the acylation reaction. The purity had reached 98%, determined by HPLC. The structure was confirmed by spectroscopic methods including infrared, electron ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. PO was totally synthesized in one step including cyclization, with total yield of 27.2%.

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.

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.

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.

Apigenin mediated protection of OGD-evoked neuron-like injury in differentiated PC12 cells.

Ischemic stroke has been confirmed to cause neuronal injury due to its insufficient supply of glucose and oxygen to brain tissue. Previous research has shown that oxidative stress, a result of excessive accumulation of reactive oxygen species (ROS), relates to pathophysiology of ischemic stroke, and causes oxidative damage to biomolecules, eventually leading to programmed cell death. Meanwhile, apigenin has been shown to exhibit antioxidant, anti-inflammatory, anti-cancer properties and neuroprotective action. Hence, this study was to investigate the potential mechanisms underlying the neural protection of apigenin on oxygen and glucose deprivation/reperfusion (OGD/R) induced neuronal injury in differentiated PC12 cells. Cells were pretreated with apigenin for 6 h, and then subjected to OGD for 12 h followed by reperfusion for 24 h. The results showed that OGD/R significantly decreased cell viability, mitochondrial membrane potential, mRNA levels of antioxidant and detoxifying enzymes and Nrf2 protein expression, while elevated the release of LDH, cell apoptosis, intracellular ROS level, P53 protein expression and upregulated its downstream genes in PC12 cells. However, apigenin effectively inhibited these undesirable changes induced by OGD/R. Our findings demonstrate that this compound attenuates OGD/R induced neuronal injury mainly by virtue of its anti-apoptosis and antioxidative properties via affecting the expression of Nrf2 and P53, and their downstream target gene transcription.

The protective effects of the supercritical-carbon dioxide fluid extract of Chrysanthemum indicum against lipopolysaccharide-induced acute lung injury in mice via modulating Toll-like receptor 4 signaling pathway.

The supercritical-carbon dioxide fluid extract of Chrysanthemum indicum Linné. (CFE) has been demonstrated to be effective in suppressing inflammation. The aim of this study is to investigate the preventive action and underlying mechanisms of CFE on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. ALI was induced by intratracheal instillation of LPS into lung, and dexamethasone was used as a positive control. Results revealed that pretreatment with CFE abated LPS-induced lung histopathologic changes, reduced the wet/dry ratio and proinflammatory cytokines productions (TNF-α, IL-1ß, and IL-6), inhibited inflammatory cells migrations and protein leakages, suppressed the levels of MPO and MDA, and upregulated the abilities of antioxidative enzymes (SOD, CAT, and GPx). Furthermore, the pretreatment with CFE downregulated the activations of NF-κB and the expressions of TLR4/MyD88. These results suggested that CFE exerted potential protective effects against LPS-induced ALI in mice and was a potential therapeutic drug for ALI. Its mechanisms were at least partially associated with the modulations of TLR4 signaling pathways.

Characterisation of the metabolism of pogostone in vitro and in vivo using liquid chromatography with mass spectrometry.

INTRODUCTION: Pogostone possesses potent anti-bacterial and anti-fungal activities and has been used for the quality control of essential oil of Pogostemon cablin. Pogostone is easily absorbed after oral administration but its metabolism in mammals remains elusive. OBJECTIVE: To investigate the metabolic profile of pogostone in vitro and in vivo. METHODS: High-performance liquid chromatography coupled with mass spectrometry (LC­MS) techniques were employed. Orbitrap MS and ion trap tandem mass spectrometry (MS/MS) were utilised to analyse the metabolism of pogostone by virtue of the high sensitivity and high selectivity in the measurement. In vitro experiment was carried out using rat liver microsomes while the in vivo study was conducted on rats, which were orally administered with pogostone (80 mg/kg). RESULTS: In total, three mono-hydroxylated, one di-hydroxylated, one mono-oxygenated, one di-oxygenated metabolite, one hydrolysis and one hydroxy conjugated metabolites were found. In addition hydroxylation was demonstrated to be a major metabolic pathway of pogostone. CONCLUSION: LC­MS was demonstrated to be a powerful tool for the metabolite identification of pogostone. The tentative identification of metabolites provides an insight for the metabolic clues of pogostone.

Enhancing hepatoprotective action: oxyberberine amorphous solid dispersion system targeting TLR4.

Oxyberberine (OBB) is a significant natural compound, with excellent hepatoprotective properties. However, the poor water solubility of OBB hinders its release and absorption thus resulting in low bioavailability. To overcome these drawbacks of OBB, amorphous spray-dried powders (ASDs) of OBB were formulated. The dissolution, characterizations, and pharmacokinetics of OBB-ASDs formulation were investigated, and its hepatoprotective action was disquisitive in the D-GalN/LPS-induced acute liver injury (ALI) mouse model. The characterizations of OBB-ASDs indicated that the crystalline form of OBB active pharmaceutical ingredients (API) was changed into an amorphous form in OBB-ASDs. More importantly, OBB-ASDs showed a higher bioavailability than OBB API. In addition, OBB-ASDs treatment restored abnormal histopathological changes, improved liver functions, and relieved hepatic inflammatory mediators and oxidative stress in ALI mice. The spray drying techniques produced an amorphous form of OBB, which could significantly enhance the bioavailability and exhibit excellent hepatoprotective effects, indicating that the OBB-ASDs can exhibit further potential in hepatoprotective drug delivery systems. Our results provide guidance for improving the bioavailability and pharmacological activities of other compounds, especially insoluble natural compounds. Meanwhile, the successful development of OBB-ASDs could shed new light on the research process of poorly soluble medicine.