Active Ingredients and Mechanism of Action of Rhizoma Coptidis against Type 2 Diabetes Based on Network-Pharmacology and Bioinformatics

A pharmacological network of"component/target/pathway"for Rhizoma coptidis against type 2 diabetes(T2D)was established by network-pharmacology,and the active components of Rhizoma coptidis and its mechanism were explored.A literature-based and database study of the components of Rhizoma coptidis was carried out and screened by ADME paramcters.The targets of Rhizoma coptidis were predicted by the ligand similarity method.Related pathways were analyzed with databases,and software was used to construct a "component/target path" network.The mechanism was further confirmed by GEO database with R software.A total of 12 active components were screened from Rhizoma coptidis,involving 57 targets including MAPKI,STAT3,INSR,and 38 signaling pathways were associated with T2D.Related signaling pathways included essential pathways for T2D such as insulin resistance,and pathways that had indirect effect on T2D.It was suggested that Rhizoma coptidis may exert its effects against T2D through multi-component,multi-target,and multi-pathway forms.

Effects of Cinnamon Granules on Pharmacokinetics of Berberine in Rhizoma Coptidis Granules in Healthy Male Volunteers

The effects of Cinnamon granules on pharmacokinetics of berberine in Rhizoma Coptidis granules in healthy male volunteers,and the compatibility mechanism of Jiao-Tai-Wan（JTW） composed of Rhizoma Coptidis granules and Cinnamon granules were investigated.The concentration of berberine in plasma of healthy male volunteers was determined directly by high performance liquid chromatogra-phy（HPLC） after an oral administration of Rhizoma Coptidis granules alone or combined with Cinnamon granules（JTW）.The plasma concentration-time curves of berberine were plotted.The data were analyzed with Drug and Statistics（DAS） 2.0 pharmacokinetic program（Chinese Pharmacology Society） to obtain the main pharmacokinetic parameters.The results showed that the plasma concentration-time curve of berberine was described by a two-compartment model.The Cmax,Tmax,t1/2 and CLz/F of berberine in Rhizoma Coptidis granules were 360.883 μg/L,2.0 h,3.882 h,119.320 L.h-1.kg-1 respectively,and those of berberine in JTW were 396.124 μg/L,1.5 h,4.727 h,57.709 L.h-1.kg-1 respectively.It was suggested that Rhizoma Coptidis granules combined with Cinnamon granules could increase the plasma concentration of berberine,promote berberine absorption and lengthen the detention time of berberine in healthy male volunteers.

Rapid and simultaneous determination of moisture and berberine content in Coptidis Rhizoma and Phellodendri Chinensis Cortex by near-infrared spectroscopy and chemometrics

Coptidis Rhizoma(Chinese:Huanglian)and Phellodendri Chinensis Cortex(Chinese:Huangbo)are widely used Traditional Chinese Medicine,and often used in combination because of their similar pharmacological effects in clinical practice.However,the quality control methods of the two drugs are different and complicated,which is time consuming and laborious in practical application.In this paper,rapid and simultaneous determination of moisture and berberine in Coptidis Rhizoma(CR)and Phellodendri Chinensis Cortex(PC)was realized by near-infrared spectroscopy(NIRs)combined with global models.Competitive adaptive reweighted sampling(CARS)and successive projection algorithm(SPA)method were applied for variable selection.Principal component analysis(PCA)and partial least squares regression method(PLSR)were applied for qualitative and quantitative analysis,respectively.The characteristic variables of berberine showed similarity and consistency in distribution,providing basis for the global models.For moisture content,the global model had relative standard error of prediction set(RSEP)value of 3.04%and 2.53%for CR and PC,respectively.For berberine content,the global model had RSEP value of 5.41%and 3.97%for CR and PC,respectively.These results indicated the global models based on CARS-PLS method achieved satisfactory prediction for moisture and berberine content,improving the determination e±ciency.Furthermore,the greater range and larger number of samples enhanced the reliance of the global model.The NIRs combined with global models could be a powerful tool for quality control of CR and PC.

Qualitative and Quantitative Analysis for the Quality Control of Rhizoma Coptidis by HPLC-DAD and HPLC-ESI-MS

For quality control purpose, an approach of fingerprinting and simultaneous quantification of five major bioactive constituents of Rhizoma Coptidis was established via a high-performance liquid chromatograph coupled with a photodiode array UV detector（HPLC-DAD） and an electrospray ionization mass spectrometer（HPLC-ESI/MS） The compounds were identified on the basis of the comparison of their mass spectra with literature data and those of standard samples and quantified by the HPLC-DAD method. Baseline separation was achieved on an XTerra C18 column（5 μm, 250 mm×4.6 mm i. d.） with linear gradient elution of formate buffer（consisting of 0.5% formic acid, adjusted to pH=4.5 with ammonia） and acetonitrile（consisting of 0.2% formic acid and 0.2% triethylamine）. The me- thod was validated for linearity（r^2〉0.9995）, repeatability（RSD〈3.1%）, intra- and inter-day precision（RSD〈1.8%） with recovery（99.9%-105.1%）, limits of detection（0.15-0.35 μg/mL）, and limits of quantification（0.53-0.82 μg/mL）. The similarities of 32 batches of Rhizoma Coptidis and their classification according to their manufacturers were based on the retention time and peak areas of the characteristic compounds. The five compounds were selected for quality assessment ofRhizoma coptidis via partial least squares analysis（PLS）.

Network pharmacology-based analysis of effective components and mechanism of Rhizoma coptidis in treating diabetes

Objective:To identify the active ingredients,potential targets,and mechanism of Rhizoma coptidis by bioinformatics method,and to explore the hypoglycemic effect of Rhizoma coptidis by in vitro experiments.Methods:The chemical components of Rhizoma coptidis were collected through database search,and oral bioavailability and drug-likeness were used for preliminary screening.The targets of Rhizoma coptidis and diabetes-related targets were collected by database retrieval and reverse docking techniques,and the biological process of cross-set proteins was analyzed.The inhibitory effects of Rhizoma coptidis onα-glucosidase,α-amylase activity,and advanced glycation end products(AGEs)were determined via in vitro experiments.In addition,the effects of Rhizoma coptidis on preadipocyte differentiation,absorption of glucose by adipocytes,and the level of intracellular triglyceride were investigated using the adipocyte differentiation model.Results:There were 11 potentially active ingredients in Rhizoma coptidis.IL-6,caspase-3,epidermal growth factor receptor(EGFR),MYC,and estrogen receptor 1 were considered as the key genes.The bioinformatics analysis showed that Rhizoma coptidis played an antidiabetic role mainly via biological processes and signaling pathways including hormone receptor activity,glutathione binding,steroid binding,etc.In vitro experiments showed that the extract of Rhizoma coptidis inhibited the activities ofα-glucosidase andα-amylase,and the generation of AGEs;meanwhile,the extract promoted the absorption of glucose by adipocytes.In addition,the extract of Rhizoma coptidis decreased triglyceride level.Conclusions:Our network pharmacology and in vitro experiments demonstrate the anti-diabetic effects and possible underlying mechanisms of Rhizoma coptidis extract.

Associated Bacteriostasis Research of Coptidis Rhizoma-Folium Isatidis and Coptidis Rhizoma-Flos Poprli against Escherichia coli O2

[Objective] The paper was to explore the combined inhibitory effect of rhizoma coptidis-folium isatidis and rhizoma coptidis-flos poprli against Escherichia coli O2.[Method] Contrast test of single and associated bacteriostasis against known serotype E. coli O2 was conducted using microcheckerboard method.[Result] The MIC of rhizoma coptidis, folium isatidis and flos poprli were 1/8 extracting liquid, 1/8 extracting liquid and 1/2 extracting liquid, respectively. When combined with folium isatidis or flos poprli, the MIC of rhizoma coptidis was 1/8 extracting liq-uid or 1/16 extracting liquid compared with single use. When combined with rhizoma coptidis, the MIC of folium isatidis and flos poprli were 1/8 extracting liquid and 1/16 extracting liquid.[Conclusion] When rhizoma coptidis was combined with folium isatidis or flos poprli, the FIC values were 2 and 0.625, performing independent action and additive effect, respectively.

Main alkaloids of Rhizoma Coptidis improved palmitic acidinduced insulin resistance in HepG2 cells via AMPK and MAPK signaling pathway

Rhizoma Coptidis,a traditional Chinese herbal medicine,has been used for treating diabetes for thousands of years.However,the molecular basis for this action has not been elucidated.In the present study,the effects of seven main alkaloids of Rhizoma Coptidis on glycometabolism were investigated and the related molecular mechanism of five active compounds on insulin resistant(IR)cell model was explored for the first time.Results showed that berberine,palmatine,epiberberine,columbamine and groenlandicine enhanced glucose consumption in the palmitic acid(PA)-induced IR-HepG2 cells,indicating that these compounds could improve IR.In addition,we found that among these active alkaloids,berberine,columbamine,epiberberine and groenlandicine could inhibit the activation of ERK and p38 pathway,while berberine,columbamine,palmatine and epiberberine could activate AMPK pathway.Moreover,palmatine and columbamine regulated the mRNA expression of GLUT2 to ameliorate IR via activating AMPK and inactivating p38 MAPK signal pathway.To sum up,berberine,columbamine,palmatine,epiberberine and groenlandicine might be the active reagents,which contribute to the glucose lowering effects of Rhizoma Coptidis.

Experimental Study of Emilia Sonchifolia Combined with Coptidis Rhizoma for the Prevention and Treatment of Oral Ulcer

Objective: This paper aims to investigate the therapeutic effect of the combination of Emilia Sonchifolia and Coptidis Rhizoma on oral ulcer rats. Methods: 36 SD rats of half male and half female were kept for 7 days, and 6 rats among them were selected as normal group by random sampling method, and the rest rats were randomly divided into model group, positive control group, Emilia Sonchifolia group, Coptidis Rhizoma group and combined group after the establishment of oral ulcer model. The normal group and model group were given blank film, the positive control group was given Guilin Watermelon Frost, and the Emilia Sonchifolia group, Coptidis Rhizoma group and combined group were given the corresponding oral film, which was administered to the ulcer for 7 days continuously, 2 times per day. The healing of oral ulcer was assessed at the end of the last day of administration, and the ulcer area was calculated on the 1st, 3rd, 5th and 7th days after successful modeling, and the serum levels of IL-2 and TNF-α in rats were detected by Emilia Sonchifolia. Results: The grading of ulcer healing in the positive control group and the combined group was better than the other groups, and the difference was statistically significant when compared with the model group (p Coptidis Rhizoma group, Emilia Sonchifolia group, combined group and positive control group was smaller than that in the model group, and the difference was statistically significant (p Emilia Sonchifolia group, Coptidis Rhizoma group and combined group were all effective in reducing the area of oral ulcer, among which the combined group was more effective. The level of pro-inflammatory factor TNF-α was reduced and the level of anti-inflammatory factor IL-2 was increased in the Emilia Sonchifolia group, Coptidis Rhizoma group and combined group, and the difference was statistically significant (p Conclusion: The combination of Coptidis Rhizoma and Emilia Sonchifolia is effective in the treatment of oral ulcer in rats, and the effect of the combination is better than that of the drug alone.

A review on pharmacological effects of Rhizoma Coptidis(Huang lian)

Huang Lian(Rhizoma coptidis, RC), which has effects on clearing damp-heat, quenching fie, and counteracting poison, is a herb frequently used in many traditional formulas. At present, there are many researches on the pharmacological effects of RC. In this review, we summarize the research progress on pharmacological effects of RC from four main aspects, in order to guide clinical application and explore the prospects of the application of RC.

Summary of Professor Tong Xiaolin's clinical experience of applying Rhizoma Coptidis(Huang Lian)

With the advantages of less toxicity and side effects, traditional Chinese medicine(TCM) is showing a bright future with a good clinical practice. Chinese herbal medicine, which usually contains various active ingredients, could provide multiple therapeutic effects. Professor Tong Xiaolin has accumulated abundant experience during many years of clinical practice and applies Rhizoma Coptidis(RC) well with the treatment of diabetes and its complications. Rhizoma coptidis is a classical natural herb which is used frequently by professor Tong Xiaolin. In this review, we summarize Tong Xiaolin's clinical experience of applying Rhizoma Coptidis(Huang Lian) in the treatment of disease from two aspects as well as introducing some traditional Chinese medical theory and clinical experience, with a prospect of guiding clinician to use RC more suitably and reasonably.

黄连黄柏协同抗痛风作用研究

[目的]研究黄连和黄柏的协同抗痛风作用。[方法]将80只小鼠随机平分为8组,即空白对照组、模型组、别嘌呤醇组、黄连组、黄柏组、黄连黄柏低、中、高剂量组,以小鼠血清尿酸水平和肝脏黄嘌呤氧化酶活性为指标,评价黄连和黄柏联用抗痛风作用。[结果]黄连和黄柏联用可显著降低高尿酸血症小鼠血清尿酸水平(P<0.05),可显著抑制肝脏黄嘌呤氧化酶活性(P<0.05)。同剂量时,其抗痛风作用强于二者单独使用。[结论]黄连和黄柏具有协同抗痛风作用。

火焰原子吸收光谱法测定黄连中金属元素的含量

[目的]利用原子吸收光谱法测定黄连中金属元素的含量。[方法]使用HNO3～HClO4体系消解样品,并采用火焰原子吸收光谱法测定6种不同来源的黄连中的Mg、Fe、Mn、Cu和Zn等5种金属元素的含量。[结果]黄连中含有丰富的金属元素,其中Mg的含量最高,Fe、Mn和Zn含量相差不大,Cu的含量最低;加标回收率在100.10%～107.24%,相对标准偏差在0.19%～3.69%(n=6)。[结论]该方法快速、简便、灵敏,结果准确可靠,可用于黄连的质量控制及资源开发。

川产道地药材三叶黄连HPLC指纹图谱研究

[目的]建立川产道地药材三叶黄连的指纹图谱检测标准。[方法]以盐酸小檗碱为参照物,采用依利特SinoChrom ODS-BP(250mm×4.6 mm,5μm)色谱柱,以乙腈-浓度0.1%乙酸和10 mmol乙酸铵溶液为流动相梯度洗脱,流速为1.0 ml/min,检测波长为345nm。[结果]药材有14个共有峰,共有峰的参数符合"中药注射剂指纹图谱技术要求"的有关规定。[结论]该方法准确、重复性好,建立的指纹图谱检测标准,可作为川产道地药材三叶黄连质量评价与品种鉴别的重要依据之一。

黄连原色浸制标本制作方法的研究

[目的]研究黄连原色浸制标本的制作方法.[方法]以不同生长年限的黄连为对象,摸索1～5年生黄连原色浸制标本的制作方法.[结果]植株经溶液B（10 g/L醋酸铜＋50 ml/L冰醋酸）浸煮10 ～ 30 min,再保存于特定保存液（40 ml/L亚硫酸＋10 ml/L甘油）6个月后仍保持原有色泽,形态逼真,立体感强.[结论]制备过程剔除了甲醛等常规处理的有毒试剂,绿色、高效、安全.应进一步针对不同的药用植物制定特定的标本制作方法,尽可能扩大适用范围,满足标本展示及研究的需要.

火焰原子吸收法测定恩施本地黄连中Mn·Cu和Pb的含量

[目的]利用火焰原子吸收法测定湖北恩施地区黄连中的Mn、Cu和Pb含量。[方法]采用硝酸-高氯酸体系消解黄连样品,用火焰原子吸收光谱法对样品中的Cu、Mn和Pb的含量进行测定。[结果]恩施本地黄连中Mn、Cu的含量丰富,分别为1 084.5和20.05μg/g,但重金属元素Pb未检出;方法的加标回收率为105.5%和99.1%。[结论]该方法快速、准确、简便,可用于黄连资源的开发与利用。

Huanglian decoction suppresses the growth of hepatocellular carcinoma cells by reducing CCNB1 expression

BACKGROUND Hepatocellular carcinoma(HCC)is one of the most prevalent cancers in human populations worldwide.Huanglian decoction is one of the most important Chinese medicine formulas,with the potential to treat cancer.AIM To investigate the role and mechanism of Huanglian decoction on HCC cells.METHODS To identify differentially expressed genes(DEGs),we downloaded gene expression profile data from The Cancer Genome Atlas Liver Hepatocellular Carcinoma and Gene Expression Omnibus(GSE45436)databases.We obtained phytochemicals of the four herbs of Huanglian decoction from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.We also established a regulatory network of DEGs and drug target genes and subsequently analyzed key genes using bioinformatics approaches.Furthermore,we conducted in vitro experiments to explore the effect of Huanglian decoction and to verify the predictions.In particular,the CCNB1 gene was knocked down to verify the primary target of this decoction.Through the identification of the expression levels of key proteins,we determined the primary mechanism of Huanglian decoction in HCC.RESULTS Based on the results of the network pharmacological analysis,we revealed 5 bioactive compounds in Huanglian decoction that act on HCC.In addition,a protein-protein interaction network analysis of the target genes of these five compounds as well as expression and prognosis analyses were performed in tumors.CCNB1 was confirmed to be the primary gene that may be highly expressed in tumors and was significantly associated with a worse prognosis.We also noted that CCNB1 may serve as an independent prognostic indicator in HCC.Moreover,in vitro experiments demonstrated that Huanglian decoction significantly inhibited the growth,migration,and invasiveness of HCC cells and induced cell apoptosis and G2/M phase arrest.Further analysis showed that the decoction may inhibit the growth of HCC cells by downregulating the CCNB1 expression level.After Huanglian decoction treatment,the expression levels of Bax,caspase 3,caspase 9,p21 and p53 in HCC cells were increased,while the expression of CDK1 and CCNB1 was significantly decreased.The p53 signaling pathway was also found to play an important role in this process.CONCLUSION Huanglian decoction has a significant inhibitory effect on HCC cells.CCNB1 is a potential therapeutic target in HCC.Further analysis showed that Huanglian decoction can inhibit HCC cell growth by downregulating the expression of CCNB1 to activate the p53 signaling pathway.

In vitro studies on the multi-target anti-Alzheimer activities of berberine-like alkaloids from Coptidis Rhizoma

Coptidis Rhizoma, a well known heat-clearing Chinese Materia Medica, exhibited multi-target anti-Alzheimer effectsin vitro through inhibition of AI342 aggregation, ACHE, and BACE1. Six berberine-like alkaloids were found to be the majoractive constituents of Coptidis Rhizoma by HPLC analysis along with activity assays. A standard combination of the six majoralkaloids was demonstrated to fully reproduce the in vitro effects of Coptidis Rhizoma. Molecular docking studies revealed thatberberine-like alkaloids could directly bind at the active sites of AI342, ACHE, and BACE1 through hydrophobic interactions,aromatic stacking, and electrostatic interactions.

Cytotoxicity effects of Rhizoma Coptidis on L929 murine fibroblast cells

Rhizoma Coptidis （RC）, a widely used traditional Chinese medicine, is commonly believed to be non-toxic. However, little is known about its cytotoxicity and relevant mechanisms at cellular and genetic levels. The present study aimed to explore the cytotoxicity of RC and its possible mechanisms related to cell cycle arrest, DNA damage and reactive oxygen species （ROS） level in L929 murine fibroblast cells. The cells were cultured in vitro and treated with different RC concentrations for 24 h. Cell viability was determined by CCK-8 method, morphological changes were observed with an inverted microscope, cell cycle and ROS level were examined by flow cytometry, and DNA damages were detected by comet assay. Our results showed that cell viability was significantly decreased in a dose-dependent manner when the RC concentration was higher than 1 mg/mL. ARC concentration above 1 mg/mL altered the morphology of L929 cells. Both cells at G2/M phase and the ROS level increased in the 2 mg/mL group. Each DNA damage indicator score increased in the groups with the RC concentration of above 0.05 mg/mL. Taken together, our study suggested that RC at a high dosage exhibited cytotoxicity on L929 cells, which was likely to be the consequences of cell cycle arrest, DNA damage and accumulation of intracellular ROS.

Dynamic monitoring of the cytotoxic effects of protoberberine alkaloids from Rhizoma Coptidis on HepG2 cells using the xCELLigence system

AIM: To investigate the cytotoxic effects of the six protoberberine alkaloids(PAs) from Rhizoma Coptidis on HepG2 cells. METHOD: A systematic screening was conducted to investigate the dynamic response of HepG2 cells to the PAs using the impedance-based xCELLigence system. Cisplatin was selected as the positive control. The real time, concentration-response curves and the 50% inhibitory concentrations(IC50) were acquired to evaluate the anticancer activity of the PAs. RESULTS: All of the six PAs inhibited cell growth and induce death in HepG2 cells in a time- and concentration-dependent manner. The IC50 values of cisplatin, berberine, columbamine, coptisine, epiberberine, jatrorrhizine, and palmatine were 5.13, 42.33, 226.54, 36.90, 302.72, 383.54, and 456.96 μg·mL-1, respectively. The results obtained using the xCELLigence system corresponded well with those of the conventional methods. CONCLUSION: The xCELLigence system is a reliable and efficient tool for real-time screening of the cytotoxic effect of compounds in cell-based in vitro assays. Coptisine and berberine, with methylenedioxy group at C2 and C3 on the phenyl ring showed stronger effect.than the other four PAs. However, compared with cisplatin, the six PAs didn't show obvious cytotoxic effect on HepG2 cells.These results provided some useful data for the evaluation of the anticancer compounds, and the clinical application of traditional Chinese medicine.

Study of the anti-MRSA activity of Rhizoma coptidis by chemical fingerprinting and broth microdilution methods

AIM： Methicillin-resistant Staphylococcus aureus （MRSA） is a pathogenic bacterium that causes both hospital- and community-acquired infections, and for which single-drug treatments are becoming less efficient. Rhizoma coptidis has been used for more than two thousand years in China to treat diarrhea, fever, and jaundice. In this study, the anti-MRSA activity of Rhizoma coptidis is examined and its effective components sought. METHODS： The mecA and norA genes were determined by PCR amplification and sequencing. Drug susceptibility of Staphylococcus aureus ATCC43300 was performed using the VITEK2 compact system. The chemical fingerprint of Rhizoma coptidis was investigated using HPLC and preparative liquid chromatography, and the anti-MRSA activity was determined using an improved broth microdilution method. RESULTS： The drug susceptibility test revealed that the penicillin-binding protein phenotype of the strain changed in comparison to penicillin-sensitive Staphylococcus aureus. Ten batches of Rhizoma coptidis showed anti-MRSA activity on the norA-negative Staphylococcus aureus strain, as well as the strain that contained a norA gene. The spectrum-effect relationship revealed that the berberine alkaloids were the effective components, within which berberine, coptisine, palmatine, epiberberine,, and jatrorrhizine were the major components. CONCLUSION： This study lays a foundation for in vivo studies of Rhizoma eoptidis and for the development of multi-component drugs.