Discovery, isolation and structural identification of alkaloid glycosides in six traditional Chinese medicine such as Coptis chinensis / 中国中药杂志

Alkaloids are important active ingredients occurring in many traditional Chinese medicines, and alkaloid glycosides are one of their existence forms. The introduction of saccharide units improves the water solubility of alkaloid glycosides thus presenting better biological activity.Because of the low content in plants, alkaloid glycosides have been not comprehensively studied. In this study, ultrahigh performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry(UPLC-QTOF-MS/MS) was employed to identify and analyze the alkaloid glycosides in Coptis chinensis, Phellodendron chinense, Menispermum dauricum, Sinomenium acutum, Tinospora sagittata and Stephania tetrandra. The results showed that except Tinospora sagittata, the other five herbal medicines contained alkaloid glycosides. Furthermore, the alkaloid glycosides in each herbal medicine were identified based on UV absorption spectra, quasimolecular ion peaks in MS, fragment ions information in the MS/MS, and previous literature reports. A total of 42 alkaloid glycosides were identified. More alkaloid glycosides were identified in C. chinensis and Menispermum dauricum, and eleven in C. chinensis were potential new compounds. Furthermore, the alkaloid glycosides in the water extract of C. chinensis were coarsely se-parated by macroporous adsorption resin, purified by column chromatography with D151 cation exchange resin, ODS and MCI, combined with semi-preparative high performance liquid chromatography. Two new alkaloid glycosides were obtained, and their structures were identified by mass spectrometry and NMR data as(S)-7-hydroxy-1-(p-hydroxybenzyl)-2,2-N,N-dimethyl-1,2,3,4-tetrahydroisoquinoline-6-O-β-D-glucopyranoside and(S)-N-methyltetrahydropalmatubine-9-O-β-D-glucopyranoside, respectively. This study is of great significance for enriching the information about the chemical composition and the in-depth development of C. chinensis. Meanwhile, it can provide a reference for rapid identification and isolation of alkaloid glycosides from other Chinese herbal medicines.

Progress in studies and control strategies for diseases of Coptis chinensis / 中国中药杂志

Coptis chinensis is one of bulk traditional herbal medicines in China. In recent years, the occurrence of various diseases has caused great yield loss and quality reduction of C. chinensis, which has become an important threat of herbal medicine industry. Here we reviewed the symptoms, pathogens, epidemiology and control methods of 6 common diseases of C. chinensis including root rot, southern blight, violet root rot, leaf spot, powdery mildew, and anthracnose. This review aims at providing guidance for the disease diagnostic, pathogen identification, and control strategies of the diseases on C. chinensis, and facilitate the growth of traditional medicine industry.

Variation in physicochemical properties and bacterial community structure in rhizosphere soil of Coptis chinensis tow cropping modes / 中国中药杂志

The natural forest and artificial shed are the main cropping modes of Coptis chinensis. This study is aimed to reveal the rhizosphere soil bacterial community structure difference between under tow C. chinensis cropping modes-natural forest and artificial shed, and to assist us to completely understand soil quality condition,and provide theoretical guidance for soil improvement and C. chinensis planting. The rhizosphere soil samples of 1-5-year-old C. chinensis under tow cropping modes-natural forest and artificial shed were collected. Illumina high-throughput sequencing technology was used to analyze the alpha diversity, community composition, community structure of soil bacteria under the tow cropping modes,and the effects of soil nutriment indices on soil bacterial community structure. Through the analysis of species number, Shannon, Chao1 index and ACE index of bacterial community, it was found that the bacterial diversity of 1-year-old C. chinensis soil under natural forest cropping mode was significantly lower than that under artificial shed cropping mode, and the diversity of bacterial communities in soil of 2-5-years old C. chinensis were not significant different between two cropping modes. A total of 53 phyla,60 classes,140 orders and 266 families were detected in the rhizosphere soil of C. chinensis under the cropping modes of natural forest, respectively. The rhizosphere soil of C. chinensis under the cropping modes of artificial shed included 54 phyla,65 classes,140 orders and 264 families, respectively. Under the two cropping modes, the top 10 dominant species of bacterial community abundance are the same, they are Proteobacteria, Acidobacteria, Actinobacteria,Bacteroidetes, Planctomycetes, Chloroflexi, Verrucomicrobia, Gemmatimonadetes, Firmicutes and Cyanobacteria, but there are differences in the abundance sequence. The top 10 dominant species of bacterial community abundance accounted for 74.36% to 74.30% of the total bacteria, and 3.15% to 3.92% of the bacteria are unclassified. The results of Metastat analysis showed that the abundance of Gemmatimonadetes in the rhizosphere soil of C. chinensis under the cropping modes the artificial shed was significantly higher than that under the natural forest cropping mode(P<0.05). MRPP analysis of community structure differences showed that under tow cropping modes, there were significant differences in the bacterial community structure of 1-4-year-old soil bacteria, among which the difference between 1-year-old soil samples was the largest. With the increase of cropping years, the difference gradually decreases, and there is no significant difference in the bacterial community structure between 5-year-old soil samples. RDA analysis and correlation analysis of bacterial community structure and soil physical and chemical properties showed that the order of environmental factors on the rhizosphere soil bacteria of Coptis chinensis was: pH>available P> total P> total K>bulk density>total N>available N>organic matter. The results are helpful to understand the soil health of C. chinensis and provide scientific basis and theoretical guidance for soil improvement and C. chinensis planting.

Mechanism of Astragali Radix-Coptis Rhizoma pair in treating type 2 diabetes mellitus based on network pharmacology / 中国中药杂志

This study aims to explore the main mechanism of Astragali Radix-Coptis Rhizoma pair(hereinafter referred to as the pair) in the treatment of type 2 diabetes mellitus(T2 DM) based on network pharmacology and animal experiment. The main Chinese medicine compound prescriptions for T2 DM were retrieved from CNKI database and the medicinals with high frequency among these prescriptions were screened. The active components in the above medicinals were searched from TCMSP, TCMID, and previous research, targets of the components from SwissTargetPrediction and SEA, and targets for the treatment of T2 DM from DISGENET, TTD, and DrugBank. Thereby, the medicinal-component-disease-target network was constructed with Cytoscape. The targets were input in String database to yield the related proteins and the protein-protein interaction(PPI) network was constructed by Cytoscape. The biological functions of proteins in the PPI network were analyzed by Cluego. Then, high-fat high-sugar diet and 30 mg·kg~(-1) streptozotocin(STZ, intraperitoneal injection, once) were employed to induce T2 DM in rats and the T2 DM rats were classified into the control group, model group, positive drug(metformin) group, and pair group. After one month of administration, the changes of blood glucose and blood lipids [triglyceride(TG), cholesterol(CHO), low density lipoprotein(LDL), high density lipoprotein(HDL)] were detected with biochemical methods and pathological changes of islet and collagen deposition in pancreatic tissue by HE staining and Masson staining, respectively. The result showed that pair can be used for T2 DM treatment. ras-related C3 botulinum toxin substrate 1(RAC1), paraoxonase 1(PON1), beta-galactoside alpha 2,6-sialyltransferase 1(ST6 GAL1), insulin receptor(INSR), sex hormone-binding globulin(SHBG), ileal sodium/bile acid cotransporter(SLC10 A2), endothelin-1 receptor A(EDNRA), peroxisome proliferator-activated receptor A(PPARA), endothelin receptor B(EDNRB), and 5-hydroxytryptamine receptor 2 A(HTR2 A) were the targets of the pair for the treatment of T2 DM. The main biological functions of the pair were regulating the metabolism of blood glucose and li-pids and protecting the cardiovascular system. The fasting blood glucose, and serum TG, CHO, and LDL were higher(P<0.01) and the HDL was lower(P<0.05) in the model group than in the control group on the 7 th, 14 th, and 28 th days. The fas-ting blood glucose and the serum TG, CHO, and LDL decreased(P<0.05) and the serum HDL increased(P<0.05) in the metformin group and the pair group as compared with those in the model group on the 14 th and 28 th days. There were no significant differences in blood glucose, TG, CHO, LDL, and HDL between the metformin group and the pair group. Rats in the model group demonstrated damaged structures of islets and pancreas, obviously increased deposition of collagen in islets and pancreas, and blurred cell boundaries. Metformin and the pair significantly alleviated the damaged structures and collagen deposition. The pair can effectively regulate the disorders of blood glucose and lipid metabolism in T2 DM and protect the structure and functions of pancreas and islets by controlling cardiovascular system, which is worthy of clinical application and can be used for drug development.

Molecular identification and alkaloids content of endangered Chinese medicinal plant Coptis chinensis var. brevisepala / 中国中药杂志

According to the records of Chinese materia medica,Coptis chinensis var. brevisepala is an authentic Chinese medicinal plant highly recommended by ancient physicians since its rhizome is like a string of beads and has a good medicinal value. However,its medicinal components and values remain to be studied as it is endangered because of overexploitation. Therefore,this study aims to quantitatively determine its effective components based on UPLC-QTOF-MS,and to compare the contents of isoquinoline alkaloids in C.chinensis var. brevisepala with those in other Coptis species. Meanwhile,molecular methods accurately identified 12 batches of C. chinensis var. brevisepala,9 batches of C. chinensis,4 batches of C. deltoidea,and 1 batch of C. teeta. Gradient elution was performed with Waters CORTECS C18 column( 4. 6 mm× 150 mm,2. 7 μm) and the mobile phase acetonitrile-water with 0. 4% formic acid. Mass spectrometry was conducted in ESI positive mode. The quantitative results showed that 8 main alkaloids had a good linear relationship within the concentration range( R~2>0. 996),with the recovery rate of 95. 18%-105. 0% and the RSD of 0. 28%-3. 7%. Compared with that of other Coptis species,the rhizome of C. chinensis var. brevisepala had the highest contents of berberine and columbamine. The total content of the 8 alkaloids in C. chinensis var. brevisepala was similar to that in C. chinensis but higher than that of the other two species. PCA was performed to compare the alkaloids among the 4 species. Besides,the 8 alkaloids were evaluated in different parts of C. chinensis var. brevisepala. The results indicate that this method is reliable and efficient and can provide a reference for the quality research.

Research progress on medicinal resources of Coptis and its isoquinoline alkaloids / 中国中药杂志

Coptidis Rhizoma is a common Chinese medicinal in clinical practice,with the effects of clearing heat,drying dampness,purging fire,and removing toxin. All the medicinal plants of Coptis can be used for clinical treatment,but some species are endangered due to resource destruction and difficulty in planting. The dominant medicinal components in Coptidis Rhizoma are isoquinoline alkaloids. There are various methods for the analysis and detection of alkaloids,such as LC-MS,HPLC,and TLC,among which LC-MS is the most widely applied. Different plants of Coptis vary in the kind and content of alkaloids. C. chinensis,C. deltoidea,C. teeta,C. chinensis var. brevisepala,C. omeiensis,C. quinquefolia,and C. quinquesecta mainly contain berberine,palmatine,coptisine,jatrorrhizine,and columbamine,five effective alkaloid components. Plant isoquinoline alkaloids( PIAs) have strong pharmacological activity but are difficult to prepare. The application of synthetic biology of PIAs will be helpful for the clinical application of PIAs. This paper reviews the research progress on biological resources of Coptis species and structures of alkaloids as well as analysis methods and synthetic biology for isoquinoline alkaloids in the medicinal plants of Coptis in recent years,which will facilitate the protection of Coptis medicinal resources and the application and development of alkaloids.

Structure-activity relationship of coptis alkaloid bitterness inhibited by mPEG-PLLA / 中国中药杂志

Inhibition of bitterness is a significant measure to improve the compliance and clinical efficacy of traditional Chinese medicine(TCM) decoction. According to the characteristics of TCM decoction, such as high dispersion of bitterness components, multi-component bitterness superposition and strong instantaneous stimulation, the research group put forward a new strategy to inhibit bitterness in the early stage based on the self-assembly characteristics of amphiphilic substances in aqueous solution, in order to reduce the distribution of bitterness components in real solution and achieve the purpose of bitter-masking. It was found that the bitter-masking effect of amphiphilic substances was different on the bitter compounds of various structures. Therefore, it was speculated that there might be a certain relationship between the bitter inhibition effect and the substrate structure. In this paper, the interaction between mPEG-PLLA and five bitter alkaloids(bamatine, jatrorrhizine, berberine, epiberberine and coptisine) in Coptidis Rhizoma was studied to explore the effect of substrate structure on the inhibition of bitterness. The sensory test of volunteers was used to determine the bitter-masking effect of mPEG-PLLA on the decoction of Coptidis Rhizoma and its main bitter alkaloids. The molecular docking and molecular force field were applied to locate the bitter groups and the bitter-masking parts. The relationship between the bitter strength and the structure was analyzed by the surface electrostatic potential of the bitter alkaloids, and the correlation between the bitter-masking effect and the structural parameters of the bitter components was explored by factor analysis, so as to clarify the structure-activity relationship of mPEG-PLLA in masking the bitterness of coptis alkaloids. It was found that mPEG-PLLA had significant taste masking effect on the decoction of Coptidis Rhizoma and five alkaloids. The masking effect was obviously related to the structure of different alkaloids: the effect increased with the increase of the number of hydrogen donors, rotatable bonds, molecular weight, and hydrophobicity, and decreased with the increase of surface electrostatic potential, electrophilicity and binding energy with bitter receptors. In this study, the influence of alkaloid structure of Coptidis Rhizoma on the butter-masking effect of mPEG-PLLA was preliminarily elucidated, providing a scientific basis for better exerting the bitter-masking effect of amphiphilic block copolymers.

Identification of Coptis chinensis root rot disease pathogenic Fusarium spp. fungi / 中国中药杂志

Root rot disease is vital disease of Coptis chinensis, it has bursted in most producing area in recent years, and has caused severe damage. To identify the pathogenic fungi, Fusarium spp. fungi were isolated from rot root, of which the pathogenic fungi were screened with inoculation on C. chinensis root and plant, and identified with molecular and morphological method. The 20 Fusarium spp. fungi were obtained, of which 5 displayed high pathogenicity. It was deduced that F. oxysporum, F. solani and F. tricinctum were the pathogen, possibly pioneer pathogen of C. chinensis root rot disease. Among which F. oxysporum was dominant and deserved to pay more attention. High temperature and high humidity can increase pathogenicity of Fusarium spp. So the global climate warming may lead to temperature rising of C. chinensis producing area and favor the pathogen fungi, which may be one of the main factors leading to bursting of C. chinensis root rot disease. To control the root rot, beside developing and using pesticide, producing base should be moved to a high altitude area.

Biotransformation and enzymatic mechanism of protoberberine alkaloids / 中国中药杂志

Protoberberine alkaloids belong to the quaternary ammonium isoquinoline alkaloids, and are the main active ingredients in traditional Chinese herbal medicines, like Coptis chinensis. They have been widely used to treat such diseases as gastroenteritis, intestinal infections, and conjunctivitis. Studies have shown that structural modification of the protoberberine alkaloids could produce derivative compounds with new pharmacological effects and biological activities, but the transformation mechanism is not clear yet. This article mainly summarizes the researches on the biotransformation and structure modification of protoberberine alkaloids mainly based on berberine, so as to provide background basis and new ideas for studies relating to the mechanism of protoberberine alkaloids and the pharmacological activity and application of new compounds.

Variation in fungal community structures in rhizosphere soil of Coptis chinensis with cropping mode under natural forest and artificial shed / 中国中药杂志

This study is aimed to reveal the rhizosphere soil fungal community structure difference of Coptis chinensis cropping between natural forest and artificial shed modes, and provide theoretical guidance for soil improvement and C. chinensis planting. The rhizosphere soil samples of 1-5-year-old C. chinensis under natural forest and artificial shed modes were collected. Illumina high-throughput sequencing technology was used to analyze the community structure and diversity of soil fungi under the tow cropping modes,and the effects of soil nutriment indices on soil fungal community structure. The results suggested that the abundance and diversity of fungal communities in soil of 2-5-year-old C. chinensis were not significant different in both two cropping modes, but it was significantly higher than that in the 1-year-old C. chinensis. Comparing soil samples from the same year-old C. chinensis under the two cropping modes, it was found that there was no significant difference in the abundance and diversity of fungal communities. The fungal community of the rhizosphere soil was different in composition and abundance between tow cropping modes, and between different planting years. The 17 phyla,59 classes and 155 orders,and 17 phyla,59 classes and 157 orders were detected in the rhizosphere soil of C. chinensis under the cropping modes of natural forest and the artificial shed, respectively. Ascomycota, Basidiomycota and Mortierellomycota were dominant phyla in rhizosphere soil, and the average abundance of the 3 phyla accounted for 74.36% and 74.30% of the total fungi. The results of analysis of similarities showed that there were significant differences in the fungal community structure of 1-year-old and 2-year-old C. chinensis soil fungi, and there was no significant difference in the community structure of 3-5-year-old samples. Under the natural forest cropping mode, there were significant differences among the samples of different years. Under the artificial shed cropping, there were significant differences in fungal community structure between 1-year-old and 3-5-year-old C. chinensis soil, and between 2-year-old and 3-5-year-old C. chinensis soil. The results of canonical correlation analysis showed that soil pH and soil organic matter content were the main factors affecting the soil fungal community structure. Soil organic matter content was positively correlated with Basidiomycota and Cryptomycota, pH was negatively correlated with Basidiomycota and C. ryptomycota. The planting of C. chinensis has promoted the diversity and abundance of rhizosphere fungal community significantly. For the same year-old C. chinensis soil, abundance of fungal community was no significant difference between two cropping modes. There are significant differences in the rhizosphere soil fungal community structure between tow cropping modes in the first two years of planting. Through the interaction between the rhizosphere and the soil and the continuous selection of the rhizosphere to the fungal community, the fungal community structure tended to be the same between the two cropping modes in rhizosphere soil of 3-5-year old C. chinensis. The soil pH and orga-nic matter content were the main factors affecting the change of fungal community structure.

Study on quality of standard decoction of Coptidis Rhizoma based on traditional decoction process / 中国中药杂志

In order to determine the quality evaluation method for standard decoction of Coptidis Rhizoma,15 batches of standard decoction of Coptidis Rhizoma were prepared by using standardized process. Parameters such as traits,p H value,indicative component content,fingerprint similarity,composition transfer rate and dry extract rate were selected as the indexes for quality evaluation. Similarity evaluation and cluster analysis were performed for HPLC fingerprint of standard decoction,and mathematical model was used to study the correlation between dry extract rate,berberine content,berberine transfer rate in standard decoction and berberine content in decoction pieces. The results showed that the similarity of fingerprints was greater than 0. 99 for these 15 batches of standard decoctions of Coptidis Rhizoma. In cluster analysis,the standard decoctions of Coptidis Rhizoma from 4 producing areas were classified into 3 categories,consistent with the content determination results,indicating that there were quality differences among different producing areas.R2 in three linear regression mathematical models established was all greater than 0. 9,with significant difference. The validation of three batches of data showed that the models had good accuracy. Therefore,this model can be used to predict the quality of standard decoction prepared from different Coptidis Rhizoma pieces. In the standard decoction process established in this study,the integrity of the traditional process was greatly preserved,and the established quality evaluation method could be used to comprehensively examine the quality of the standard decoction,which can provide a demonstration for the related research of water extraction preparation containing Coptidis Rhizoma pieces.

Diversity of culturable fungi isolated from Coptis chinensis rot root in Shizhu city of Chongqing / 中国中药杂志

Root rot disease restricts the Coptis chinensis industry in Shizhu of Sichuan province. To disclose fungi composition and pathogen in rot root,so as to prevent and treat the root rot disease,the C. chinensis rot root of 5 years from 4 areas in Shizhu were collected in 3 seasons respectively. The fungi were isolated and molecularly and morphological identified,followed with population statistics. 437 fungi were isolated,belonging to 5 subphylum,11 classes,16 orders,22 families and 28 genus respectively. There are great difference among the fungi compositions of different area,year and sampling season,while there was no obvious variation rule. Ilyonectria sp.,Pythium sp.,Phoma sp,Trichoderma sp.are dominant genus,while Pythium sp.,Ilyonectria sp.,Phoma sp.,Fusarium sp. may contain root rot pathogen. Antagonistic bacteria may be screened from the strains of Trichoderma sp. isolated.

Research for dynamic changes of growth and alkaloids of Coptis chinensis / 中国中药杂志

With Coptis chinensis in high-yielding soil as the object,the growth regularity of plant and dynamic change of alkaloid content was studied. The plant growth model of C. chinensis was constructed. The plant height equation was =3.030 9+0.732 6-0.009 6²,the number of leaves equation was =111.882 6-2 234.881 7/+15 218.960 8/²-31 740.960 8/³,the leaf area equation was =-217.136 1+30.552 2-0.359 0²,the roots talk biomass equation was =-2.748 8+0.210 3+0.006 4²,the number of rootstalk equation was =-1.246 0+0.192 6+0.000 8²,the fibrous root biomass equation was =-4.973 5+0.589 4 -0.002 6². The results indicated that the number of leaves and leaf area were increasing continuously after seedling transplanting,the leaf area of 3-year-old C. chinensis reached a maximum value of 425.83 cm²/plant,after declining.The number of leave of 5-year-old C. chinensis reached a maximum value of 70.91. With the increasing of years of growth, the number of rootstalk and rootstalk biomass of C. chinensis was increasing continuously. The biomass growth of 3-year-old and 4-year-old rootstalk was the fastest in the whole development stage of C. chinensis,the annual increase of more than 300%. The change curve of rootstalk number, rootstalk biomass and fibrous root biomass in the whole growth stage was a s-type.The dry matter partition of leafwas the highest in 1-year-old C. chinensis, and then gradually decreased,the change trend of dry matter partition of rootstalk was just the opposite, the dry matter partition of fibrous root increases with the increase of the growing year, reaching the maximum value in 3-year-old, then gradually lower trend. The root-shootratio of 1-year-old C. chinensis was the smallest, then gradually increases, the growth center gradually shifted to the roots from stems and leaves, The weight of underground part of 3-year-old C. chinensis exceeded the aboveground part, the 5-year-old C. chinensis root-shoot ratio reached the maximum value of 1.91:1.With the increasing of years of growth, the contents of coptisine, berberine, epiberberine and palmatine in rootstalk was increasing continuously. The jatrorrhizine content in 2-year-old C. chinensis was significantly lower than that in other years, the content was no significant change after that. The columbamine content reached a maximum value in 3-year-old C. chinensis,then the decreased gradually. The content of magnoflorine gradually increased and reached maximum value in 5-year-old C. chinensis.

Combination of berberine and ciprofloxacin reduces multi-resistant Salmonella strain biofilm formation by depressing mRNA expressions of luxS, rpoE, and ompR

Bacterial biofilms have been demonstrated to be closely related to clinical infections and contribute to drug resistance. Berberine, which is the main component of Coptis chinensis, has been reported to have efficient antibacterial activity. This study aimed to investigate the potential effect of a combination of berberine with ciprofloxacin (CIP) to inhibit Salmonella biofilm formation and its effect on expressions of related genes (rpoE, luxS, and ompR). The fractional inhibitory concentration (FIC) index of the combination of berberine with CIP is 0.75 showing a synergistic antibacterial effect. The biofilm's adhesion rate and growth curve showed that the multi-resistant Salmonella strain had the potential to form a biofilm relative to that of strain CVCC528, and the antibiofilm effects were in a dose-dependent manner. Biofilm microstructures were rarely observed at 1/2 × MIC/FIC concentrations (MIC, minimal inhibition concentration), and the combination had a stronger antibiofilm effect than each of the antimicrobial agents used alone at 1/4 × FIC concentration. LuxS, rpoE, and ompR mRNA expressions were significantly repressed (p < 0.01) at 1/2 × MIC/FIC concentrations, and the berberine and CIP combination repressed mRNA expressions more strongly at the 1/4 × FIC concentration. The results indicate that the combination of berberine and CIP has a synergistic effect and is effective in inhibiting Salmonella biofilm formation via repression of luxS, rpoE, and ompR mRNA expressions.

Correlation between integrated pharmacokinetics and pharmacodynamics of bile processed Rhizoma Coptidis in febrile rats / 药学学报

This study was designed to validate the correlation between integrated pharmacokinetic and therapeutic effects of alkaloids using bile processed Rhizoma Coptidis (BRC). Rats were divided into three groups: normal, disease model, model+BRC. Rats were induced to have an excessive heat syndrome. Rectal temperatures were collected at 0, 3, 6 and 9 h after single oral administration of the drugs. The plasma concentrations of three alkaloids were quantified at different times by UPLC-MS/MS after the administration of BRC. An approach of self-defined weighting coefficiency was created to the holistic pharmacokinetic profiles of alkaloids in BRC. The classified and integrated synthetic concentrations were obtained, and then the pharmacokinetic parameters of alkaloids were calculated from non-compartmental model analysis. The potential relationship between the integrated mean concentration of alkaloids and the antifebrile efficacy was investigated. The holistic t(max) of alkaloids was 1.11 h, the antifebrile effect of BRC at 3 h was improved over the model group. Double peaking appeared in the integrated blood concentration-time curve, the second t(max) of alkaloids was 4.82 h. The antifebrile effects of BRC at 3-6 h were significant, and the antifebrile effects at 6-9 h was decreased significantly. Dynamic variation of alkaloids of BRC in the body exhibited the similarity to the pattern of its antifebrile effect.

Establishment and reliability evaluation of the design space for HPLC analysis of six alkaloids in Coptis chinensis (Huanglian) using Bayesian approach / 中国天然药物

Coptis chinensis (Huanglian) is a commonly used traditional Chinese medicine (TCM) herb and alkaloids are the most important chemical constituents in it. In the present study, an isocratic reverse phase high performance liquid chromatography (RP-HPLC) method allowing the separation of six alkaloids in Huanglian was for the first time developed under the quality by design (QbD) principles. First, five chromatographic parameters were identified to construct a Plackett-Burman experimental design. The critical resolution, analysis time, and peak width were responses modeled by multivariate linear regression. The results showed that the percentage of acetonitrile, concentration of sodium dodecyl sulfate, and concentration of potassium phosphate monobasic were statistically significant parameters (P < 0.05). Then, the Box-Behnken experimental design was applied to further evaluate the interactions between the three parameters on selected responses. Full quadratic models were built and used to establish the analytical design space. Moreover, the reliability of design space was estimated by the Bayesian posterior predictive distribution. The optimal separation was predicted at 40% acetonitrile, 1.7 g·mL(-1) of sodium dodecyl sulfate and 0.03 mol·mL(-1) of potassium phosphate monobasic. Finally, the accuracy profile methodology was used to validate the established HPLC method. The results demonstrated that the QbD concept could be efficiently used to develop a robust RP-HPLC analytical method for Huanglian.

DW2007 Ameliorates Colitis and Rheumatoid Arthritis in Mice by Correcting Th17/Treg Imbalance and Inhibiting NF-κB Activation

In the previous study, the rhizome mixture of Anemarrhena asphodeloides and Coptis chinensis (DW2007), improved TNBS-, oxazolone-, or DSS-induced colitis in mice by regulating macrophage activation. Therefore, to understand the effect of DW2007 on the T cell differentiation involved in the adaptive immunity, we measured its effect on both Th17 and Treg cell differentiation in splenocytes, in the lamina propria of mice with DSS-induced colitis (DIC), and in the spleens of mice with collagen-induced arthritis (CIA). Results showed that DW2007 potently inhibited the differentiation of splenocytes into Th17 cells, but increased Treg cell differentiation in vitro. In the colon of wild type and TLR4−/− mice with DIC, DW2007 potently suppressed DSS-induced colon shortening and myeloperoxidase activity. DW2007 also suppressed collagen-induced paw thickening, clinical index, and myeloperoxidase activity in CIA mice. Overall, DW2007 potently suppressed Th17 cell differentiation in mice with CIA and DIC, but increased Treg cell differentiation. Moreover, DW2007 strongly inhibited the expression of TNF-α and IL-1β, as well as the activation of NF-κB. Based on these findings, DW2007 may ameliorate inflammatory diseases by regulating the innate immunity via the inhibition of macrophage activation and the adaptive immunity via the correction of disturbed Th17/Treg cells.

Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensis

Background Berberine acts primarily as an important active ingredient in Coptis chinensis and has been traditionally applied in clinical treatment. Nevertheless, little information has been released about C. chinensis, as far as functional genes and biosynthetic pathway of berberine are concerned. Here, we isolated a novel (S)-canadine synthase gene (designated as CAS-1) from C. chinensis by using RT-PCR and RACE techniques. Results Bioinformatics analysis showed that the cDNA is 1942 bp in length with a complete open reading frame (ORF) of 1476 bp, and the ORF encodes a polypeptide of 491 amino acids with a calculated molecular mass of 55.29 kDa and a pI value of 8.92. Real-time quantitative PCR analysis showed that CcCAS-1 was constitutively expressed in leaf, petiole and rhizome tissues, especially in the leaves of C. chinensis. However, the results of berberine content in different tissues by high-performance liquid chromatography with photodiode array detection (HPLC-DAD) method showed that the leaves and the petiole tissues have similar content of berberine. Conclusions We found that the berberine content in the rhizome was seven times (more or less) than that in the leaves and the petioles. In addition, the full length coding sequence of CcCAS-1 was inserted into pET-32a and was successfully expressed in Escherichia coli, laying a solid foundation for protein purification, activity assay and multi-clonal antibody preparation. Together, our data suggest that CcCAS-1 is a novel heme-thiolate enzyme essential for berberine biosynthesis in C. chinensis.

Effects of alkaloids from Coptidis Rhizoma on mouse peritoneal macrophages in vitro / 中国中药杂志

This work was mainly studied the effects of the four alkaloids from Coptidis Rhizoma on the mouse peritoneal macrophages in vitro and preliminarily discussed the regulating mechanisms. The effect of alkaloids from Coptidis Rhizoma on the vitality of macrophages was measured by the MTT assay. The effect of alkaloids on the phagocytosis of macrophages was determined by neutral red trial and respiratory burst activity was tested by NBT. The expressions of respiratory-burst-associated genes influenced by alkaloids were detected by qRT-PCR. The conformation change of membrane protein in macrophages by the impact of alkaloids was studied by fluorospectro-photometer. Results showed that the four alkaloids from Coptidis Rhizoma could increase the phagocytosis of macrophages in different level and berberine had the best effect. Berberine, coptisine and palmatine had up-regulation effects on respiratory burst activity of mouse peritoneal macrophages stimulated by PMA and regulatory activity on the mRNA expression of PKC, p40phox or p47phox, whereas the epiberberine had no significant influence on respiratory burst. Moreover, alkaloids from Coptidis Rhizoma could change the conformation of membrane protein and the berberine showed the strongest activity. The results suggested that the four alkaloids from Coptidis Rhizoma might activate macrophages through changing the conformation of membrane protein of macrophages and then enhanced the phagocytosis and respiratory burst activity of macrophages. Furthermore, the regulatory mechanism of alkaloids on the respiratory burst activity of macrophages may be also related to the expression level of PKC, p40phox and p47phox.

Pharmacokinetic and pharmacodynamic characteristics of berberine and jateorhizine in Coptidis Rhizoma powder and their monomeric compounds in type 2 diabetic rats / 中国中药杂志

This article focused on a comparative analysis on the pharmacokinetic and pharmacodynamic characteristics of berberine (BER) and jateorhizine(JAT) in Coptidis Rhizoma powder (HL-P) and their monomeric compounds (BER + JAT, BJ) in type 2 diabetic (T2D) rats to explore the beneficial. effect of HL-P in the treatment of T2D. The T2D rats were treated with HL-P, BER, JAT and BJ, respectively for 63 d. The pharmacokinetic parameters, dynamic changes in blood glucose level and blood lipid values were measured. The results showed that, compared with other corresponding group, t(max), T(½ka) of BER and JAT in HL-P group were reduced, while C(max), AUC(inf), AUC(last), V(L)/F were significantly increased; compared with model group, blood glucose levels were decreased significantly in HL-P group since the 18th day, while those in BER or BJ group were reduced since the 36th day, however, blood glucose levels showed no obvious changes in JAT group; compared with model group, FFA values in all treatment group were decreased significantly. Moreover, TG, HDL and LDL value in HL-P group, LDL value in BER group and HDL value in BJ group were improved significantly. The above results showed that Coptidis Rhizoma powder showed excellent pharmacokinetic characteristics and excellent activity of lowering blood glucose and lipid. It provided a scientific basis for oral application of Coptidis Rhizoma powder in the treatment of T2D.