Rhizoma Coptidis Alkaloids Research Articles

Palmatine ameliorates N-methyl-N'-nitrosoguanidine-induced chronic atrophic gastritis through the STAT1/CXCL10 axis.

Chronic atrophic gastritis (CAG) is a prevalent preneoplastic condition of the stomach. Palmatine (PAL), an isoquinoline alkaloid isolated from Rhizoma Coptidis (RC), has significant anti-inflammatory properties and is often used to treat gastrointestinal disorders. However, the mechanism of PAL on CAG remains unclear. In this study, N-methyl-N'-nitrosoguanidine (MNNG) was used to induce CAG inflammatory disease models invivo and invitro. The efficacy of five alkaloids in RC and the dose-dependent effects of the most effective PAL in CAG mice were evaluated in two animal experiments. RNA-seq and western blot revealed that PAL significantly improved IL-17, TNF, and NF-kappa B inflammation-related signaling pathways. Further hub gene prediction and experimental validation revealed that PAL modulated the STAT1/CXCL10 axis, thereby exerting attenuation of CAG through the regulation of IL-17, TNF-α, and p-p65 expression. In conclusion, PAL was proposed to mitigate MNNG-induced CAG, potentially through the inhibition of oxidative stress and inflammatory responses via the STAT1/CXCL10 axis. This approach is an effective complement to the use of PAL in the treatment of CAG.

Epiberberine: a potential rumen microbial urease inhibitor to reduce ammonia release screened by targeting UreG

Rumen microbial urease inhibitors have been proposed for regulating nitrogen emission and improving nitrogen utilization efficiency in ruminant livestock industry. However, studies on plant-derived natural inhibitors of rumen microbial urease are limited. Urease accessory protein UreG, plays a crucial role in facilitating urease maturation, is a new target for design of urease inhibitor. The objective of this study was to select the potential effective inhibitor of rumen microbial urease from major protoberberine alkaloids in Rhizoma Coptidis by targeting UreG. Our results showed that berberine chloride and epiberberine exerted superior inhibition potential than other alkaloids based on GTPase activity study of UreG. Berberine chloride inhibition of UreG was mixed type, while inhibition kinetics type of epiberberine was uncompetitive. Furthermore, epiberberine was found to be more effective than berberine chloride in inhibiting the combination of nickel towards UreG and inducing changes in the second structure of UreG. Molecular modeling provided the rational structural basis for the higher inhibition potential of epiberberine, amino acid residues in G1 motif and G3 motif of UreG formed interactions with D ring of berberine chloride, while interacted with A ring and D ring of epiberberine. We further demonstrated the efficacy of epiberberine in the ruminal microbial fermentation with low ammonia release and urea degradation. In conclusion, our study clearly indicates that epiberberine is a promising candidate as a safe and effective inhibitor of rumen microbial urease and provides an optimal strategy and suitable feed additive for regulating nitrogen excretion in ruminants in the future.Key points• Epiberberine is the most effective inhibitor of rumen urease from Rhizoma Coptidis.• Urease accessory protein UreG is an effective target for design of urease inhibitor.• Epiberberine may be used as natural feed additive to reducing NH3 release in ruminants.

Integrating transcriptomics and network pharmacology to reveal the mechanisms of total Rhizoma Coptidis alkaloids against nonalcoholic steatohepatitis

Ethnopharmacological relevanceNon-alcoholic steatohepatitis (NASH) has emerged as a major cause of cirrhosis and hepatocellular carcinoma, posing a significant threat to public health. Rhizoma Coptidis, a traditional Chinese medicinal herb has been shown to have significant curative effects on liver diseases. Total Rhizoma Coptidis Alkaloids (TRCA) is a primarily alkaloid mixture extracted from Rhizoma Coptidis, and its constituents are widely accepted to have hepatoprotective effects. Aim of the studyThis work aimed to investigate the efficacy and potential mechanisms of TRCA in ameliorating NASH through both in vitro experiments and in vivo mouse models. Materials and methodsThe study employed a mice model induced by a high-fat diet (HFD) to evaluate the effectiveness and pharmacological mechanisms of TRCA in alleviating NASH. Transcriptomic sequencing and network pharmacology were used to explore the possible targets and mechanisms of TRCA to ameliorate NASH. Further validation was performed in free fatty acid (FFA)-induced human hepatocytes (LO2) and human hepatocellular carcinoma cells (HepG2). ResultsTRCA effectively ameliorated the main features of NASH such as lipid accumulation, hepatitis and hepatic fibrosis in the liver tissue of mice induced by HFD, as well as improved glucose tolerance and insulin resistance in mice. Combined with transcriptomic and network pharmacological analyses, 68 core targets associated with the improvement of NASH by TRCA were obtained. According to the KEGG results, the core targets were significantly enriched in the PI3K-AKT signaling pathway whereas TRCA ameliorated the aberrant down-regulation of the PI3K-AKT signaling pathway induced by HFD. Furthermore, the five highest-ranked genes were obtained by PPI network analysis. Moreover, our findings suggest that TRCA may impede the progression of HFD-induced NASH by regulating the expression of PPARG, MMP9, ALB, CCL2, and EGFR. ConclusionsTRCA can ameliorate HFD-induced liver injury by modulating aberrant downregulation of the PI3K-AKT signaling pathway. Key proteins such as PPARG, MMP9, ALB, CCL2, and EGFR may be critical targets for TRCA to ameliorate NASH. This finding supports using Rhizoma Coptidis, a well-known herbal medicine, as a potential therapeutic agent for NASH.

Epiberberine inhibits Helicobacter pylori and reduces host apoptosis and inflammatory damage by down-regulating urease expression

Ethnopharmacological relevanceWith dramatically increasing antibiotic resistance in Helicobacter pylori (H. pylori), it is urgent to find alternative therapeutic agents. Rhizoma Coptidis is a traditional Chinese medicine for gastrointestinal diseases and shows excellent anti-H. pylori effect. Epiberberine (EPI), as one of the major alkaloids of Rhizoma Coptidis, has been reported to have urease-inhibiting activity, but its scavenging effect on H. pylori and the potential mechanism remain unclear. Aim of the studyTo investigate the inhibitory effect of EPI on H. pylori and explore its multi-action on Helicobacter pylori urease (HPU). Materials and methodsUsing minimum inhibitory concentration (MIC) assay, minimum bactericidal concentration (MBC) assay, growth inhibition kinetics assay, bacterial resistance development, transmission electron microscope (TEM) assay, and animal experiments to investigate the inhibitory effect of EPI on H. pylori in vitro and in vivo. Using the Berthelot method, molecular docking and thermal displacement experiments to verify that EPI inhibits urease activity by interacting with HPU. Using transcriptome data, Real-Time PCR (RT-PCR) experiments to investigate the alterations in the expression of urease subunit ureB gene after EPI treatment. Using MTT cell viability assay, Hoechst 33342 staining method, JC-1 reagent detection method, western blot experiments, and Griess method to investigate the anti-apoptosis and anti-inflammation actions of EPI on gastric epithelial cells (GES-1) induced by HPU. ResultsIn vitro experiments proved that EPI has significant anti-H. pylori activity without drug resistance, induces H. pylori fragmentation and apoptosis. In vivo experiments showed that EPI has a certain clearance effect of H. pylori, and can reduce gastric inflammation caused by H. pylori infection. Transcriptome data, RT-PCR experiments, and other experiments demonstrate that EPI has a triple effect: (1) inhibiting the expression of HPU subunits ureB, (2) directly inhibiting urease activity by interacting with HPU, and (3) inhibiting HPU-induced apoptosis and inflammation in GES-1. ConclusionsEPI is an excellent anti-H. pylori agent and reduces host apoptosis and inflammation by inhibiting the activity of urease and down-regulating the expression of ureB.

Coptisine inhibits Helicobacter pylori and reduces the expression of CagA to alleviate host inflammation in vitro and in vivo

Ethnopharmacological relevanceHelicobacter pylori (H. pylori) is a major pathogen colonized in the human stomach and is implicated in gastritis, peptic ulcer, and gastric carcinoma. Antibiotics are useful for eradicating H. pylori but failed for drug resistance, making it urgent to develop effective and safe drugs. Rhizoma Coptidis was reported as one of the most effective Chinese medicines to treat H. pylori-related gastrointestinal diseases, while the precise antimicrobial mechanism remains unclear. Thus, it is of great significance to study the antimicrobial ingredients and corresponding mechanisms of Rhizoma Coptidis. Aim of the studyTo search for the most effective alkaloid against H. pylori in Rhizoma Coptidis and illustrate the probable mechanisms. Materials and methodsFive main alkaloids in Rhizoma Coptidis were isolated. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were tested to determine the most effective one. Bacterial growth experiments, Annexin V-FITC/PI staining, TUNEL staining, and transmission electron microscopy (TEM) were performed to further study the anti-H. pylori activity of coptisine (Cop). The in vivo effect of Cop on H. pylori eradication rate and H. pylori-induced inflammation was investigated in mice. Transcriptomics was used to understand the underlying mechanism of eradicating H. pylori and reducing host inflammation. Western blot, RT-PCR, and ELISA experiments were utilized and confirmed that cagA was one of the targets of Cop. ResultsAccording to the MIC and MBC, Cop was the most effective alkaloid against H. pylori, especially with no drug resistance developed. In vitro experiments showed that Cop inhibited H. pylori by inducing DNA fragmentation, phosphatidylserine exposure, and membrane damage. Cop (150 mg/kg/day) effectively eradicated H. pylori in mice and reduced the levels of IL-2 and IL-6 to relieve gastric inflammation. Transcriptomic analysis revealed that virulence factor cagA was one of the hub genes associated with the inflammation-improving effect of Cop. That is, Cop could decrease the expression of CagA and subsequently reduce the translocation of CagA to gastric epithelial cells, thereby improving the morphology of hummingbird-like phenotype induced by CagA and alleviating inflammation. ConclusionsCop is the most effective alkaloid in Rhizoma Coptidis and might act through multiple mechanisms for H. pylori eradication along with reducing the expression of CagA to alleviate inflammation.

Characterization of the anti-AChE potential and alkaloids in Rhizoma Coptidis from different Coptis species combined with spectrum-effect relationship and molecular docking.

Coptis species are the main source of Rhizoma Coptidis (RC) drugs, which have always been used to treat Alzheimer's disease in the clinical experience of ancient China. However, many species of this genus have been largely underutilized until now. With this fact, this research has been designed to investigate for the first time the anti-acetylcholinesterase (AChE) property of different extracts for RC drugs from four Coptis species (C. chinensis, C. deltoidea, C. teeta and C. omeiensis) and to quantify the main alkaloids. Petroleum ether, ethyl acetate and n-butanol fractions of RC drugs were sequentially collected using an accelerated solvent extraction technique. Spectrum-effect relationship and molecular docking were applied to analyse the relationships between alkaloids and AChE inhibitory activity. The N-butanol extract was proven to be the main active fraction, and C. teeta may be the best source of RC drugs for Alzheimer's disease treatment, with significantly lower IC 20, IC 50 and IC 80 values for AChE inhibition. The UPLC/QqQ-MS quantitative analysis showed that the accumulations of 10 alkaloids in RC drugs from different sources greatly varied. Three data processing methods (Random forest, Boruta and Pearson correlation) comprehensively analysed the spectrum-effect relationship and revealed that columbamine, berberine and palmatine were the most important AChE inhibitors that could be used as quality markers to select RC drugs for Alzheimer's disease treatment. In addition, the dominant compounds were successfully docked against AChE to verify the binding affinity and interactions with the active site. The present study can contribute to the reasonable development and utilization of RC drugs from different sources, especially to provide certain evidence for their application in the treatment of Alzheimer's disease.

Jatrorrhizine from Rhizoma Coptidis exerts an anti-obesity effect in db/db mice

Ethnopharmacological relevanceObesity is closely related to diabetes. Jatrorrhizine (JAT) from Rhizoma Coptidis (RC) can reduce blood glucose and lipid levels. However, the molecular mechanisms for JAT's anti-obesity effect are still not clear. Aim of the studyTo explore the effect of JAT in the treatment of obesity and the underlying molecular mechanisms. Materials and methodsdb/db mice were used as a typical obese animal model in current study. The anti-obesity effects of five alkaloids from RC were compared by feeding the mice for 8 weeks with a dosage of 105 mg/kg while the dose-dependent study (35 mg/kg and 105 mg/kg) of JAT on obese mice was conducted in another 8-week-long animal experiment. Meanwhile, RNA-seq analysis, in vitro experiments, and western blotting were utilized to predict and confirm the potential pathway that JAT participated in improving obesity. ResultsThe experimental results demonstrated that five RC alkaloids caused different degrees of weight loss in db/db obese mice. Among them, JAT showed the best effect. It could significantly reduce the body weight, blood lipid levels, and epididymal fat weight of db/db mice. H&E and Oil red O staining results showed that it could also dramatically improve liver lipid metabolism. The results from RNA-seq suggested that JAT had significantly altered 207 DEGs for the treatment of obesity, among which IRS1 changed the most. Next, GO and KEGG analysis enriched four major lipid metabolism-related pathways: biosynthesis of unsaturated fatty acids, PI3K-AKT signaling pathway, metabolic pathways, and fatty acid elongation. Finally, in vitro experiments and western blotting proved that JAT regulated the expression of IRS1/PI3K/AKT pathway-related proteins in a dose-dependent manner to address obesity. ConclusionsIn conclusion, JAT from RC has an effect on treating obesity, and its anti-obesity effect may be exerted via the IRS1/PI3K/AKT signaling pathway.

Biological Response Profiling Reveals the Functional Differences of Main Alkaloids in Rhizoma Coptidis.

Rhizoma Coptidis (RC) is a widely used traditional Chinese medicine. Although modern research has found that some alkaloids from RC are the pharmacologically active constituents, the differences in their biological effects are not completely clear. This study analyzed the differences in the typical alkaloids in RC at a systematic level and provided comprehensive information on the pharmaceutical mechanisms of the different alkaloids. The ethanol RC extract (RCE) was characterized using HPLC assay. HepG2, 3T3-L1, and RAW264.7 cells were used to detect the cytotoxicity of alkaloids. Transcriptome analyses were performed to elucidate the cellular pathways affected by RCE and alkaloids. HPLC analysis revealed that the typical alkaloids of RCE were berberine, coptisine, and palmatine. Coptisine and berberine displayed a stronger inhibitory effect on cell proliferation than palmatine. The overlapping ratios of differentially expressed genes between RCE and berberine, coptisine, and palmatine were 70.8%, 52.6%, and 42.1%, respectively. Pathway clustering analysis indicated that berberine and coptisine possessed a certain similarity to RCE, and both compounds affected the cell cycle pathway; moreover, some pathways were uniquely enriched by berberine or coptisine. Berberine and coptisine had different regulatory effects on genes involved in lipid metabolism. These results provide comprehensive information on the pharmaceutical mechanisms of the different RC alkaloids and insights into their better combinatory use for the treatment of diseases.

Systematic Study of Crucial Transcription Factors of Coptidis rhizoma Alkaloids against Cerebral Ischemia-Reperfusion Injury.

Coptidis rhizoma alkaloids (CRAs), extracted from Coptidis rhizoma, have been indicated to play important neuroprotective roles, but the mechanism underlying has not been determined, especially from the perspective of transcription factors (TFs). In this study, crucial TFs involved in the protective activity of CRA were revealed based on RNA-Seq technology, proteomics, and network pharmacological analysis of the effects of CRA on middle cerebral artery occlusion-mediated cerebral ischemia-reperfusion (I/R) injury. Importantly, CRA significantly reduced the infarction rate and neurological deficiency score. Moreover, CRA significantly decreased the levels of TNF-α, MCP-1, and IL-1β. In addition, seven TFs, including Ncor1, Smad1, Bhlhe41, Stat3, Sp100, Satb2, and Lrpprc, were found to be crucial TFs, and five of these TFs were associated with inflammation. Furthermore, eight compounds in CRA were associated with the identified TFs through network pharmacological analysis. The alteration of Lrpprc and Sabt2 was further confirmed by measuring their downstream genes, including Pigg, Hhatl, Wdr77, Mpped1, Arpp21, Ppfia3, Rims1, and Cacna2d1 by reverse transcriptase polymerase chain reaction. Thus, these seven TFs may be important targets in CRA-mediated protection against I/R injury. This research provides a new view of the protective effect of CRA against cerebral I/R injury and reveals new therapeutic targets for treating cerebral ischemia.

Pharmacokinetics of Five Alkaloids and their Metabolites in Normal and Diabetic Rats after Oral Administration of Rhizoma coptidis.

Rhizoma coptidis has been clinically used for a long time for the treatment of various diseases in China, such as hypertension, diabetes, and inflammation. Previous studies have shown that alkaloid components of Rhizoma coptidis extract could be extensively metabolized and the metabolites were also considered to be the therapeutic material basis. However, until now, pharmacokinetic studies of the in vivo metabolites have not been revealed yet. The aim of the present study was to characterize the pharmacokinetics and excretions of five main alkaloids (berberine, jatrorrhizine, palmatine, epiberberine, and coptisine) and their seven metabolites (berberrubine, demethyleneberberine, jatrorrhizine-3-O-β-D-glucuronide, thalifendine-10-O-β-D-glucuronide, berberrubine-9-O-β-D-glucuronide, demethyleneberberine-2-O-sulfate, and demethyleneberberine-2-O-β-D-glucuronide) in rats after oral administration of Rhizoma coptidis extract. Meanwhile, comparative pharmacokinetics and excretions of these analytes in diabetic model rats were also investigated, since Rhizoma coptidis is widely used for the treatment of diabetes. Our results showed that the in vivo existing forms of alkaloid components were phase II metabolites, highlighting the glucuronidation metabolic pathway. In diabetic model rats, the utilization of Rhizoma coptidis alkaloids was significantly increased and the biotransformation of berberine into berberrubine was significantly inhibited.

An integrated network pharmacology and transcriptomic method to explore the mechanism of the total Rhizoma Coptidis alkaloids in improving diabetic nephropathy

Ethnopharmacological relevanceRhizoma Coptidis (RC) is a traditional Chinese medicine (TCM) used for treating diabetes (Xiao Ke Zheng), which is firstly recorded in Shennong Bencao Jing. Modern pharmacological studies have confirmed that RC has beneficial effects on diabetes and its complications. Alkaloids are the main active pharmacological component of RC. However, the effect and molecular mechanism of total Rhizoma Coptidis alkaloids (TRCA) in improving diabetic nephropathy (DN) are still unclear. Aim of the studyTo verify the effect of TRCA in the treatment of DN and clarify the molecular mechanism by combining network pharmacology and transcriptomic. Materials and methodsEight-week-old db/db mice were orally administered with normal saline, 100 mg/kg TRCA, and 100 mg/kg berberine (BBR) for 8 weeks. Serum, urine, and kidney samples were collected to measure biological indicators and observe renal pathological changes. Then, the molecular mechanism of TRCA improving DN was predicted by the network pharmacology. Briefly, the main active alkaloids components of TRCA and their targets were collected from the database, as well as the potential targets of DN. Using the Cytoscape software to visualize the interactive network diagram of “ingredient-target”. The GO and KEGG pathways enrichment analysis of the core targets were executed by Metascape. Furthermore, RNA-seq was used to get whole transcriptomes from the kidneys of db/m mice, db/db mice, and db/db mice treated with TRCA. The key differentially expressed genes (DEGs) were gathered to conduct the GO and KEGG pathways enrichment analysis. Finally, the potential pathways were validated by western blotting. ResultsThe administration of BBR or TRCA for 8 weeks significantly reduced the fasting blood glucose (FBG) and body weight of db/db mice, and improved their renal function and lipid disorders. According to H&E, PAS, and Masson staining, both the BBR and TRCA could alleviate renal damage and fibrosis. The Venn diagram had shown that seven alkaloids ingredients collected from TRCA regulated 85 common targets merged in the TRCA and DN. The results of RNA-seq indicated that there are 121 potential targets for TRCA treatment on DN. Intriguingly, both the AGE-RAGE signaling pathway and the PI3k-Akt signaling pathway were included in the KEGG pathways enrichment results of network pharmacology and RNA-seq. Moreover, we verified that TRCA down-regulated the expression of related proteins in the AGEs-RAGE-TGFβ/Smad2 and PI3K-Akt pathways in the kidney tissues. ConclusionsIn summary, the renal protection of TRCA on DN may be related to activation of the AGEs-RAGE-TGFβ/Smad2 and PI3K-Akt signaling pathways.

Metabolic profile of alkaloids in Rhizoma Coptidis in rat plasma, urine and feces after oral administration using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Rhizoma Coptidis (RC) has been used to treat diabetes, pertussis, bacillary dysentery, sore throat, eczema, and aphtha for thousands of years. Alkaloids are the major components in RC, and its curative effect is achieved by oral administration. However, information on its composition in vivo is weak. In this study, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/QTOF-MS) was used to analyze the major active components and their metabolites in rat plasma, urine and feces after oral administration of RC extract. A total of 96 compounds including 8 prototype compounds and 88 metabolites were identified, and hydroxylation, reduction, demethylenation, demethylation, dehydrogenation, sulfation, glucuronidation and methylation were the major metabolic pathways. This study analyzed metabolic processes of the major active components in RC in vivo, which provided important information for its active composition and in vivo mechanism research. Meanwhile, metabolic profile studies on representative compounds provided valuable reference materials to elucidate the full-scale metabolites of RC.

Composition of Ophiopogon Polysaccharide, Notoginseng Total Saponins and Rhizoma Coptidis Alkaloids Inhibits the Myocardial Apoptosis on Diabetic Atherosclerosis Rabbit.

To investigate the effects of Composition of Ophiopogon polysaccharide, Notoginseng total saponins and Rhizoma Coptidis alkaloids (CONR) on myocardial apoptosis of diabetic atherosclerosis (DA) rabbits METHODS: Sixty male New Zealand white rabbits were randomly divided into 6 groups [control group, model group, CONR high-dose group (450 mg/kg), CONR medium-dose group (150 mg/kg), CONR low-dose group (50 mg/kg), and simvastatin group] by using a completely random method, 10 in each group. DA model was established by intravenously injected alloxan combined with high-fat diet and abdominal aortic balloon injury. After mediation for 10 weeks, fasting blood glucose (FBG), glycosylated hemoglobin (GHB), glycosylated serum protein (GSP), fructoseamine (FRA), aldose reductase (AR), advanced glycation end products (AGEs) in serum were measured by enzyme linked immunosorbent assay (ELISA) method; the expression of receptor of AGEs (RAGE) in myocardial tissue were observed by immunohistochemical method; and p-Jun N-terminal kinase (p-JNK), caspase-3, B-cell lymphoma-2 (bcl-2) protein expression in myocardial tissue were measured by Western blotting. The myocardial apoptosis was detected by TdT-mediated dUTPnick-end labeling (TUNEL) method, and apoptosis index (AI) was calculated. Compared with the control group, serum FBG, GHB, GSP, FRA, AR, AGEs and the expression of myocardium RAGE, p-JNK, caspase-3 proteins, as well as apoptosis index (AI) were significantly increased and bcl-2 protein was significantly decreased in the model group (P<0.01). Compared with the model group, the levels of serum FBG, GHB, GSP, FRA and AR showed a significant decline in CONR high- and medium-dose groups (P<0.01). FBG and GHB showed a significant decline in CONR low-dose group (P<0.01). Compared with the model group, the expression of serum AGEs and myocardium RAGE, p-JNK and caspase-3 protein as well as AI were significantly decreased and bcl-2 protein was significantly up-regulated in all treatment groups (P<0.01); high-dose CONR had the most significant effect on abovementioned indices compared with other treatment groups (P<0.01). Middle-dose CONR had better effect on serum AGEs compared with the low-dose group (P<0.01); middle-dose CONR and simvastatin groups had better effect on the expression of caspase-3, bcl-2 protein, myocardium apoptosis compared with the CONR low-dose group (P<0.01). CONR may effectively inhibit myocardial apoptosis on DA rabbits by intervening AGEs-RAGE and JNK, caspase-3, and bcl-2 protein expressions.

Sodium dodecyl sulfate sensitized switchable solvent liquid-phase microextraction for the preconcentration of protoberberine alkaloids in Rhizoma coptidis.

A sodium dodecyl sulfate sensitized switchable solvent liquid-phase microextraction method was developed and applied to the preconcentration of active alkaloids in Rhizoma coptidis followed by high performance liquid chromatography determination. Before extraction, nonionic triethylamine was converted to its cationic form in the presence of carbon dioxide. Then, the ionic solvent carrying target analytes was once more reverted to its nonionic form by adding sodium hydroxide, as well as phase separation and analytes enrichment were realized simultaneously. Several parameters affecting the approach, such as concentration of sodium dodecyl sulfate, extraction solvent volume, sodium hydroxide concentration, sample phase pH, injection solvent type, and extraction time, were investigated and optimized. The possible microextraction mechanism of double micelle supramolecular inclusion was explored. Under the optimum conditions, the enrichment factors of four protoberberine alkaloids were from 101.8 to 152.0. The linear ranges (with r2 ≥0.990) were 0.032-4.23, 0.031-4.33, 0.0026-10.04, and 0.0013-4.13μg/mL for epiberberine, coptisine, palmatine, and berberine, respectively. The detection limits were in the range of 0.16-0.32ng/mL. Satisfactory accuracies (recoveries 98.8-104.6%) and precisions (RSDs 1.9-10.9%) were also obtained. The results showed that the approach is rapid, effective, eco-friendly, and easy-to-handle for the enrichment and detection of active alkaloids in Rhizoma coptidis.

Metabolism of Rhizoma coptidis in Human Urine by Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry.

Rhizoma coptidis extract and its alkaloids were reported to exhibit various pharmacological activities. However, pharmacokinetics investigations indicated that the plasma concentrations of the alkaloids were too low to explain their systemic therapeutic actions. Thus, the metabolic profile of Rhizoma coptidis in humans is yet to be fully investigated and the present study aimed to investigate the metabolic profile of Rhizoma coptidis in human urine after oral administration of Rhizoma coptidis extract. In this study, the metabolism of Rhizoma coptidis at a clinical dose (5g/60kg/day) was investigated using ultra-high-performance liquid chromatography coupled with high-resolution LTQ-Orbitrap mass spectrometry. Totally, 30 constituents including 7 prototypes, 5 sulfation metabolites and 18 glucuronide conjugates were elucidated and identified on the basis of the characteristics of their high-resolution precursor ions, product ions, and chromatographic retention times in human urine. Among the 7 prototypes, 3 prototypes (M20, M26 and M28) were identified definitely by comparing with standards. Based on the metabolites detected in human urine, a possible metabolic pathway of Rhizoma coptidis in vivo was proposed. The results demonstrated that the metabolic fate of Rhizoma coptidis mainly involved sulfation and glucuronidation in human urine and the glucuronide conjugate M14 (berberrubinen-9-O-glucuronide) might be a pharmacokinetic marker for Rhizoma coptidis alkaloids in humans. This study will be helpful to comprehensively understand the metabolic process of Rhizoma coptidis and how Rhizoma coptidis shows its pharmacological effects in humans.

The Effect of Ginger Juice Processing on the Chemical Profiles of Rhizoma coptidis.

Rhizoma coptidis (RC) has been used as an herbal medicine in China for over one thousand years, and it was subjected to specific processing before use as materia medica. Processing is a pharmaceutical technique that aims to enhance the efficacy and/or reduce the toxicity of crude drugs according to traditional Chinese medicine theory. In this study, the chemical profiles of RC, ginger juice processed RC (GRC), and water processed RC (WRC) was determined to reveal the mechanism of processing of RC. UPLC-QTOF-MS analysis of methanol extract of RC, GRC, and WRC has been conducted to investigate the effect of processing on the composition of RC. HPLC-PDA was used to determine the variance of total alkaloids and seven alkaloids of RC during the processing. The volatiles of RC, GRC and ginger juice were separated by distillation, the change of volatiles content was recorded and analyzed, and the qualitative analysis of the volatiles was carried out using GC-MS. The microstructures of RC, GRC and WRC were observed using a light microscope. Results showed that ginger juice/water processing had limited influence on the composition of RC’s methanol extract, but significant influence on the content of some alkaloids in RC. Ginger juice processing significantly increased (p < 0.05) the volatiles content of RC and changed the volatiles composition obviously. Processing also had an influence on the microstructure of RC. This research comprehensively revealed the mechanism of ginger juice processing of RC.

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.

Metabolic profile of Rhizoma coptidis in human plasma determined using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

Rhizoma coptidis extract and its alkaloids show various pharmacological activities, but its metabolic profile in human plasma has not been thoroughly investigated. In the present research, the metabolism of Rhizoma coptidis at a clinical dose (5g/60kg/day) was systematically analyzed to determine its biotransformation processes in human plasma. In this research, the metabolites of Rhizoma coptidis in human plasma after oral administration of Rhizoma coptidis extract at a clinical dose were investigated using ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution LTQ-Orbitrap mass spectrometry. The structural elucidation of the constituents was confirmed by comparing their retention times (tR ) and MSn fragments with those of standards and literature reports. In total, two prototypes and twelve metabolites were detected in human plasma. The two prototypes were confidently identified using reference standards. Of the compounds detected, M7 (berberrubinen-9-O-glucuronide) was the most abundant based on its peak area, which indicates that this compound might be a pharmacokinetic marker for Rhizoma coptidis alkaloids in humans. Based on the metabolites detected in human plasma, a possible metabolic pathway for Rhizoma coptidis in vivo was proposed. The results indicated that the alkaloids in Rhizoma coptidis were extensively biotransformed in vivo mainly via conjugation with glucuronic acid (GluA) or sulfuric acid (SulA) to form phase II metabolites, and the GluA metabolites are likely the dominant form in human plasma. To the best of our knowledge, this is the first in vivo evaluation of the metabolic profile of the whole Rhizoma coptidis extract in human plasma, which is essential for determining the chemicals responsible for the pharmacological activities of Rhizoma coptidis in vivo. Moreover, it would be beneficial for us to further systematically study the pharmacokinetic behavior of Rhizoma coptidis in humans.

Anti-hyperlipidemic effects of Rhizoma Coptidis alkaloids are achieved through modulation of the enterohepatic circulation of bile acids and cross-talk between the gut microbiota and the liver

Molecular and microbiome studies were employed to uncover the mechanisms underlying the cholesterollowering effects of RC alkaloids. Hyperlipidemic mice were treated with RC alkaloids for 35days. After treatment, bile acid levels in serum, liver and feces were investigated. Key targets involved in enterohepatic circulation of bile acids were evaluated. 16S rDNA analysis was used to study the change of gut microbiota in mice. The up-regulated CYP7A1 and BSEP in mouse liver accelerated the synthesis and transportation of bile acids. Bile acids were reabsorbed due to the increased ASBT in mouse ileum. The down-regulated claudin-1 and occludin in ileum contributes to enlarged fetal excretion of bile acids. RC alkaloids treatment restored the disrupted gut microflora in hyperlipidemic mice. These findings present new evidence supporting a key role for RC alkaloids as a regulator of liver–gut axis in hyperlipidemic mice.

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

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 5087μM for HPU and 2.3–>10,000μM for JBU, respectively. Notably, epiberberine (EB) was found to be the most potent inhibitor against both ureases with IC50 values of 3.0±0.01μM for HPU and 2.3±0.01μM for JBU, which was more effective than the standard urease inhibitor, acetohydroxamic acid (83±0.01μM for HPU and 22±0.01μM 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±0.01μM, while slow-binding and competitive against JBU with Ki of 4.6±0.01μM. 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.