Anti-inflammatory effect, plasma effective components and therapeutic targets of Huanglian Jiedu Decoction on ulcerative colitis mice / 中国中药杂志

This paper was to investigate the effect of Huanglian Jiedu Decoction(HLJD) on ulcerative colitis(UC) in mice, and determine the effective components in plasma, and virtually screen its therapeutic target, and predict its mechanism. Sixty Balb/c mice were randomly divided into blank group, model group, mesalazine treatment group(0.3 g·kg~(-1)), and HLJD treatment groups(24.66, 12.33, 6.17 g·kg~(-1)). Excepted for the blank group, all the mice in HLJD and mesalazine treatment groups were gavage administration. All mice freely drank 2.5% DSS solution for seven days to induce UC. The disease activity index(DAI) was detected each day. At the end of the experiment, HE staining was used to observe the pathological changes in colon. The content of IL-1β, IL-6 and TNF-α in colon were determined by ELISA. The effective components in plasma were determined by UPLC-Q-TOF-MS. The reverse docking in PharmMapper was used to screen the component targets. The disease targets of UC were collected by searching TTD, OMIM and GeneCards databases. The intersection of the component targets and disease targets was selected as the therapeutic targets. Then the therapeutic targets were imported into the STRING for GO and KEGG enrichment analysis. Discovery Studio was used to simulate the docking between the components and the targets. RESULTS:: showed that the DAI in the model group increased significantly(P<0.05), and the number of inflammatory cells and infiltration degree increased significantly compared with the blank group. The DAI in HLJD treatment group was significantly reduced(P<0.05), and the number and infiltration degree of inflammatory cells were reduced compared with the model group. The ELISA results showed that the levels of IL-1β, IL-6 and TNF-α were increased significantly in the model group(P<0.01) compared with the blank group, and significantly down regulated in the HLJD treatment group(P<0.05) compared with the model group. After UPLC-Q-TOF-MS analyse, ten components were identified. The network pharmacology analysis showed that the action targets were significantly enriched in 129 of biological processes, such as response to organic substance, chemical and oxygen-containing compound, etc., as well as 16 of signal pathways, such as IL-17, TNF and hepatitis B signal pathways, were enriched too. The results of molecular docking showed that limonin, palmatine and berberine could bind to CASP3 and MMP9 by hydrogen bond. In conclusion, HLJD could alleviate the colonic mucosal inflammatory infiltration and mucosal damage in UC mice. The mechanism may be related to the anti-inflammatory effect on UC mice by reducing the levels of IL-1β, IL-6 and TNF-α in colon through limonin, palmatine and berberine regulating IL-17 signal pathway and TNF signal pathway via CASP3 and MMP9 meditated.

Effect of Palmatine Binding to MYC G-quadruplex on Proliferation and Apoptosis of Colon Cancer Cells and Molecular Mechanism / 中国实验方剂学杂志

Objective:To explore whether palmatine interferes with the proliferation and apoptosis of colon cancer HCT116 cells by binding to G-quadruplex in the promoter region of MYC proto-oncogene and its possible molecular mechanism. Method:Fluorescence spectrum was used to analyze the binding ability of palmatine to MYC G-quadruplex. Circular dichroism analysis was conducted to confirm the effect of palmatine on the configuration of MYC G-quadruplex， followed by the prediction of their binding mode based on molecular docking and the localization analysis of palmatine in HCT116 cells under a fluorescence microscope. The effects of palmatine on MYC gene transcription and MYC protein expression were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot， respectively. The effects of palmatine on the viability and apoptosis of HCT116 cells were further assayed by thiazolyl blue tetrazolium bromide （MTT） assay and flow cytometry. Result:As revealed by fluorescence spectrum and molecular docking analysis， palmatine might bind to G-quadruplex of MYC gene through stacking. Circular dichroism analysis showed that palmatine could maintain the parallel configuration of MYC<italic> </italic>G-quadruplex. It was discovered in fluorescence imaging that palmatine was distributed in the nucleus and bond to G-quadruplex of MYC gene. In addition， palmatine inhibited MYC gene transcription， MYC protein expression， as well as the viability of HCT116 cells， and promoted the apoptosis of HCT116 cells. Conclusion:Palmatine is able to bind to MYC G-quadruplex to further inhibit the expression of MYC gene and protein expression， which may be one of the molecular mechanisms of palmatine in suppressing the proliferation of colon cancer HCT116 cells and facilitating their apoptosis.

Palmatine ameliorates high fat diet induced impaired glucose tolerance

BACKGROUND: The impaired glucose tolerance (IGT) is a representative prediabetes characterized by defective glucose homeostasis, and palmatine (PAL) is a natural isoquinoline alkaloid with multiple pharmacological effects. Our study aims to investigate the therapeutic effect of PAL on the impaired glucose tolerance. METHODS: Male Sprague-Dawley rats were used to establish an IGT model with high fat diet (HFD). Oral glucose tolerance test (OGTT) and further biochemical analysis were conducted to determine the effect of PAL on glucose intolerance in vivo. Molecular details were clarified in a cellular model of IGT induced by Palmitate (PA) on INS-1 cells. RESULTS: Our study demonstrated a relief of IGT with improved insulin resistance in HFD induced rats after PAL treatment. Besides, promoted pancreas islets function was validated with significantly increased ß cell mass after the treatment of PAL. We further found out that PAL could alleviate the ß cell apoptosis that accounts for ß cell mass loss in IGT model. Moreover, MAPK signaling was investigated in vivo and vitro with the discovery that PAL regulated the MAPK signaling by restricting the ERK and JNK cascades. The insulin secretion assay indicated that PAL significantly promoted the defective insulin secretion in PA-induced INS-1 cells via JNK rather than ERK signaling. Furthermore, PAL treatment was determined to significantly suppress ß cell apoptosis in PA-induced cells. We thus thought that PAL promoted the PA-induced impaired insulin release by inhibiting the ß; cell apoptosis and JNK signaling in vitro. CONCLUSION: In summary, PAL ameliorates HFD-induced IGT with novel mechanisms.

Isolation and identification of chemical constituents from Disporum cantoniense (III) / 中草药

Objective: To study the chemical constituents from Disporum cantoniense. Methods: Separation was carried out by ion exchange chromatography, medium pressure MCI column chromatography, ODS column chromatography, gel column chromatography, preparative and semi preparative liquid chromatography; The structures of the compounds were identified by modern spectral techniques such as mass spectrometry and nuclear magnetic resonance. Results: Eleven compounds were isolated and identified as 2’-β-D-glucopyranosyloxybenzyl-6-α-L-(4’-O-acetyl)-rhamnopyranosyloxy-2-hydroxy-3-methoxybenzoate (1), 4’,7- dihydroxyflavone (2), palmatine (3), marmesinin (4), 4’-O-β-D-glucosyl-5-O-methylvisamminol (5), nodakenetin (6), 2-aminopyridine (7), tenuifoliside A (8), neosakuranin (9), 2″-O-rhamnosylicariside II (10) and baohuoside I (11). Conclusion: Compound 1 is a new compound named disporumoside, compounds 2-11 are isolated from the genus of Disporum for the first time.

Changes of alkaloids in active constituents before and after preparation of Chinese medicine Phellodendron amurense / 国际中医中药杂志

Objective@#To investigate the changes of alkaloids in Chinese medicine Phellodendron amurense.@*Methods@#High performance liquid chromatographywas used to detect the content of berberine, corkaline, jatrorrhizine, palmatinebefore and after processing in different processed products of Chinese medicine Phellodendron amurense. The column was Ecosil C18 column (250 mm × 4.6 mm, 2.5 μm), and the mobile phase was acetonitrile: 0.2% phosphoric acid buffer solution (55:45)(0.4 g of sodium lauryl sulfate per 100 ml of 0.2% phosphoric acid aqueous solution), detection wavelength was 265 nm, column temperature was 35 ℃, flow rate was 1.0 ml/min, and injection volume was 3.0 μl.@*Results@#The best processing temperature of brine-dried cork, anthrax, and wine-running cork was below 160 ℃. Themass fraction of total alkaloid was 4.512 7%, 2.789 4% and 4.047 1% at 100 ℃. Among them, the wine-dried cork and salt water-filled cork products and raw products had a high mass indicating that the auxiliary materials for increasing the dissolution rate of alkaloids were salt and wine. Compared with the raw products, the content of berberine and palmatine in salt water-running cork and wine-running cork was obvious. The content of berberine and palmatine in anthrax was significantly decreased, and the content of corkaline and jatrorrhizine did not change significantly.@*Conclusions@#The changes of alkaloids before and after in different processing of Cortex Phellodendron have showed some differences. The different processing and temperature have a direct influence on the changes of its composition, which can changes the chemical properties of Chinese medicine Phellodendron amurense before and after processing.

Effect of extract and components of Berberis wilsonae on improving ability of learning memory and anti-oxidant in mice / 中草药

Objective To determine the content of total alkaloids and their components in roots, stems, and leaves of Berberis wilsonae, and investigate the effects of their extracts and components on the improvement of learning and memory ability and anti-oxidant activity of mice. Methods In this study, the total alkaloid content of roots, stems, leaves, and roots, stem extracts and components of B. wilsonae was determined by acid dye colorimetry. The HPLC method and conditions were established for determination jateorhizine, palmatine, and berberine. The memory impairment mice model was established with scopolamine, and the root and stem extracts and components of B. wilsonae were ig administrated for 30 d. Jumping platform test and water maze test were used to detect the effect of roots and stem extracts and components on learning and memory, and the levels of MDA and T-SOD in serum was determinated by kit method. Results The results showed that the latency of the mice in step-down test was significantly prolonged (P < 0.01), and the reduction of the number of errors was also significant (P < 0.01). To a certain extent, the place navigation time in water maze was shortened (P < 0.01), and the number of errors was reduced significantly (P < 0.05). Extracts and components can significantly increase the level of T-SOD (P < 0.05, 0.01) in mice, and reduce the level of MDA significantly (P < 0.05, 0.01). Conclusion The extracts and components of B. wilsonae had a certain effect on the improvement of learning and memory ability of mice, and the anti-oxidant capacity in vivo was also improved.

HPLC fingerprint and multicomponents determination of Corydalis Decumbentis Rhizoma / 中草药

Objective: To establish the HPLC fingerprint and multicomponents determination of Corydalis Decumbentis Rhizoma, and provide a scientific basis for the improvement of its quality standards. Methods The separation was performed on a chromatographic Diamonsil C18 column (250 mm × 4.6 mm, 5 μm), with acetonitrile-0.2% acetic acid (adjusting pH to 6.0 with triethylamine) as the mobile phase for gradient elution. Volume flow rate was 1.0 mL/min. Column temperature was 35 ℃. Injection was 10 μL and the detection wavelength was 280 nm. The fingerprints of 15 batches of Corydalis Decumbentis Rhizoma were established and evaluated by the similarity evaluation system of TCM (version 2012A), which were divided into two categories by clustering analysis and principal component analysis. Meanwhile, the content of protopine, palmatine, bicuculline and tetrahydropalmatine was determinated. Results:The fingerprint of Corydalis Decumbentis Rhizoma was established. There were 10 common peaks in the fingerprint. Protopine, palmatine, bicuculline and dl-tetrahydropalmatine were separated with good linearity relationships (r > 0.999 9). The average recovery rates of the investigated compounds were 97.12%, 100.09%, 98.53%, and 99.71%, respectively. Conclusion:HPLC fingerprint combined with multicomponents determination can provide the reference for the quality evaluation of Corydalis Decumbentis Rhizoma.

Effect of different decocting methods on active ingredients in Banxia Xiexin Decoction / 中草药

Objective To investigate the effects of the three methods of decocting with deslag, decocting without deslag, and double decocting on the content of nine ingredients baicalin, baicalein, ginsenoside Re, ginsenoside Rb1, monoammonium glycyrrhizinate hydrate, liquiritin, 6-gingerol, berberine hydrochloride, palmatine hydrochloride, and total flavonoids in Banxia Xiexin Decoction (BXD). Methods Nine index components were determined by HPLC. The HPLC analysis was performed on Welch Ultimate XB-C18 column (250 mm × 4.6 mm, 5 μm) with mobile phase of acetonitrile-0.1% phosphate aqueous solution for gradient elution; And carried out at column temperature of 28 ℃, volume flow of 0.9 mL/min, and detection wavelength of 203, 252, 280, and 355 nm. The total flavonoids were determined by colorimetry. Results Nine kinds of ingredients and total flavonoids could be detected in three different decoctions. In the method of decocting with deslag, baicalin, baicalein, ginsenoside Rb1, monoammonium glycyrrhizinate hydrate, and liquiritin increased by 10.01%, 12.88%, 29.09%, 16.75%, and 15.02%, respectively, compared with decocting without deslag; It decreased by 5.54%, 4.15%, 14.49%, 7.85%, and 9.18%, respectively compared with double decocting; Ginsenoside Re, 6-gingerol, berberine hydrochloride, and palmatine hydrochloride increased by 37.90%, 3.78%, 5.33%, and 5.99% compared with decocting without deslag, respectively; compared to the double decocting methods, it increased by 1.07%, 11.57%, 3.41%, and 1.93%. The total flavonoids increased 22.61% higher than decocting without deslag and 6.54% higher than double decocting. Conclusion: The results can effectively reflect the quality difference of different decocting methods. Among the three methods of decoction, the method of decocting without deslag has significantly improved the dissolution of the active ingredients of each component in the decoction, and improve the clinical efficacy of BXD to a certain extent. It provides a good experimental basis for the decocting without deslag method used in Zhang Zhongjing’s Treatise on Febrile Diseases.

RP-HPLC simultaneous determination of five isoquinoline alkaloid in Erhuang Xiaoyan tablets / 重庆医学

Objective To establish a method for determining five isoquinoline alkaloids (including berberine,jatrorrhizine,jatrorrhizind,coptisine and palmatine) in Erhuang Xiaoyan tablets simultaneously.Methods Determination by reversed phase high performance liquid chromatography (RP-HPLC).Column:YMC-Pack ODS-AM (4.6 mm× 250.0 mm,5 μm);mobile phase:elution with 0.1％ (v/v) formic acid aqueous solution-acetonitrile mobile phase gradient;flow rate:0.4 mL/min;injection volume:10 μL;detection wavelength:345 nm;column temperature:27 ℃.Results Five of isoquinoline alkaloids in Erhuang Xiaoyan tablets were separated perfectly.The good linear relationships were obtained in the following ranges,1.526 4-5.342 4μg (r=0.999 9) for berberine,0.403 2-1.411 2 μg (r=0.999 6) for palmatine,0.309 7-1.083 9 μg (r=0.999 8) for coptisine,0.111 2-0.389 2 μg (r=0.999 8) for jatrorrhizine and 0.162 2-0.567 7 μg (r=0.999 6) for epiberberine.The recoveries were 98.69％,98.66％,98.67％,98.67％ and 98.67％,respectively.Conclusion The method possesses high feasibility,strong specificity,high accuracy and good reproducibility,which can be used for the quality control of Erhuang Xiaoyan tablets.

Biliary excretion characteristics of berberine, palmatine and jateorhizine in sandwich-cultured rat hepatocytes / 中国药理学通报

Aim To study the biliary excretion characteristics of berberine,palmatine and jateorhizine in rat hepatocytes.Methods Berberine,palmatine and jateorhizine were incubated with the sandwich-cultured rat hepatocytes (SCRH) in standard Ca2+ buffer or Ca2+ free buffer.The accumulation of the three compounds under different conditions were measured by UPLC-MS/MS.The biliary excretion index and biliary clearance were calculated,and the effect of P-gp or Mrp2 inhibitor on the transport of three compounds was also investigated.Results While the incubation time increased,the accumulation of the three compounds also increased.There were obvious differences in accumulation of berberine,palmatine and jateorhizine in incubations treated with standard buffer and calcium-free buffer.The P-gp inhibitors ciclosporin A and verapamil could inhibit the biliary excretion of berberine,palmatine and jateorhizine.However,the Mrp2 inhibitors MK571 and probenecid had no effect on biliary excretion of the three compounds.Conclusions The biliary excretion of berberine,palmatine and jateorhizine is mainly through an active process.They are all the P-gp substrates other than Mrp2 substrates.

Content Determination of 3 Components in Li'ermian Capsule by HPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of 3 components in Li'ermian capsule,including berberine hydrochloride,palmatine hydrochloride and cinnamaldehyde.METHODS:HPLC method was adopted.The determination was performed on Agilent Zorbax SB-C18 with mobile phase consisted of acetonitrile-0.05 mol/L potassium dihydrogen phosphate solution (23 ∶ 77,V/V) at the flow rate of 1.0 mL/min.The detection wavelengths were set at 345 nm (0-20 min,berberine hydrochloride,palmatine hydrochloride) and 290 nm (＞20-30 min,cinnamaldehyde),and column temperature was 30 ℃.The sample size was 10 μL.RESULTS:The linear ranges of berberine hydrochloride,palmatine hydrochloride and cinnamaldehyde were 0.036 80-0.736 0,0.016 76-0.335 2,0.004 140-0.082 80 μg (r≥0.999 5).The limits of detection were 0.110 4,0.050 3,0.124 2 ng,and the limits of quantitation were 0.368 0,0.167 6,0.414 0 ng,respectively.RSDs of precision,stability (12 h) and reproducibility tests were all lower than 2.0％ (n=6).The recoveries were 96.16％-99.73％ (RSD=0.99％-1.21％,n=6).CONCLUSIONS:Established method is simple,reproducible and suitable for simultaneous determination of 3 components such as berberine hydrochloride in Li'ermian capsule.

Simultaneous Determination of 7 Components in Siwei Jianghuang Decoction Powder by HPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of gallic acid,magnoflorine,ellagic acid,jatrorrhizine hydrochloride,palmatine hydrochloride,berberine hydrochloride and curcumin in Siwei jianghuang decoction powder.METHODS:HPLC method was adopted.The determination was performed on Capcell Pak C18-MG Ⅱ column with mobile phase consisted of acetonitrile-0.1％ phosphoric acid (gradient elution) at the flow rate of 0.8 mL/min.The detection wavelengths were 270 nm (0-60 min,gallic acid,magnoflorine,ellagic acid,jatrorrhizine hydrochloride,palmatine hydrochloride,berberine hydrochloride) and 428 nm (60-70 min,curcumin).The column temperature was set at 30 ℃,and sample size was 10 μL.RESULTS:The linear ranges of gallic acid,magnoflorine,ellagic acid,jatrorrhizine hydrochloride,palmatine hydrochloride,berberine hydrochloride and curcumin were 0.249 6-1.497 6,0.284 0-1.704 0,0.075 6-0.453 6,0.015 9-0.095 9,0.023 6-0.141 6,0.098 2-0.589 0 and 0.060 4-0.362 4 μtg (r≥0.999 8).The limits of detection were 6.24,4.73,7.56,2.36,3.20,6.54,6.04 ng,and the limits of quantitation were 17.47,16.08,20.86,7.31,10.24,19.62,19.32 ng,respectively.RSDs of precision,stability (12 h),reproducibility tests were lower than 2.0％ (n=6).The recoveries were 95.45％-103.47％ (RSD=0.86％-1.98％,n=9).CONCLUSIONS:Established method is simple,accurate,reliable and suitable for simultaneous determination of 7 components such as gallic acid in Siwei jianghuang decoction powder.

Research on interaction between Coptidis Rhizoma extracts and intestinal bacteria / 中草药

Objective To investigate the interaction between Coptidis Rhizoma extracts and intestinal bacteria. Methods The metabolites of Coptidis Rhizoma extracts by human gut microbiota were identified by UPLC-Q-TOF/MS. Furthermore, the effects of Coptidis Rhizoma extracts on the growth of Bifiobactderia, Lactobacilli, Enterobacter, and Enterococcus were evaluated by the colorimetric method. Results Alkaloids in Coptidis Rhizoma were mainly degraded by reduction and demethoxy, among which berberine and coptisine were converted to reductive products and palmatine was metabolized to a demethoxyl product. Additionally, pathogens such as Enterobacter and Enterococcus were obviously inhibited and probiotics such as Lactobacilli and Bifiobactderia were notably promoted by Coptidis Rhizoma extracts. Conclusion Gut microflora can metabolize alkaloids from Coptidis Rhizoma by multiple ways, and Coptidis Rhizoma extracts can improve the intestinal microecology.

Preparation of palmatine-loaded flexible nano-liposomes films based on Bletilla striata polysaccharides / 中草药

Objective To prepare palmatine-loaded flexible nano-liposomes (PFL) films with Bletilla striata polysaccharide (BSP) as membrane material, and evaluate its pharmacy related performance in order to lay the foundation for further application. Methods The PFL was prepared by injection method and the films of PFL based on Bletilla striata polysaccharide (PFL-BPF) was prepared by homogenate coating method. The PFL-BPF was characterized and evaluated by electron microscopy, differential scanning calorimeter (DSC), and in vitro transmucosal membrane experiment. Results The PFL and BSP had good compatibility and easy to film with BSP as membrane material. The appearance of PFL-BPF obtained was smooth, non-bubble, flexible, and suitable stiffness; PFL-BPF had good biological adhesion. The time of scouring the film agent from mucous membrane with normal saline was (130 ± 7) min. At 0.5 h, the dose of PFL-BPF promoting palmatine (PA) infiltration to mucosa was 32.41 μg/g. It was 3.17 times higher than those of PA solution based on BSP (PL-BPF) and 1.9 times for PA common liposomes based on BSP (BLP + PA-BPF) (t-test, P < 0.05); At 2.5 h, it was 2.67 times and 1.89 times higher than those of PL-BPF and BLP + PA-BPF, respectively. It showed that PFL-BPF could significantly promote the water-soluble drug PA through mucosa membrane and release it slowly. The results of DSC showed that the possible mechanism for promoting the absorption of PA through mucosa membrane was that the flexible liposomes disturbed the mucosal epithelial cells and carried the drug into the mucosal tissue. Conclusion The PFL-BPF had the advantages of good film-forming property, lasting adhesive attraction, strong scour resistance, simple and feasible preparation process, and could promote drug permeation into mucosa obviously. Therefore, the flexible nano-liposomes film is a good drug carrier for the transmucosal drug delivery applications and has a wide application prospect.

The effects of essential oils of traditional Chinese medicine on the pharmaceutical properties of flexible nano-liposomes loaded with palmatine chloride / 药学学报

Flexible liposomes are an excellent drug delivery nanocarrier, however, the leakage of drugs from liposomes has become common technical obstacle in the industry and also hindered its further application seriously. It is very urgent and necessary to avoid or reduce the leakage of drugs from liposomes. In this work, five kinds of essential oils such as Folium Artemisiae Argyi oil (FA), Folium Eucalypti oil (FE), Arabian Jasmine oil (AJ), Syzygium Aromaticum oil (SA) and Fructus Forsythiae oil (FF) were encapsulated in the lipid bilayer of palmatine chloride (PC) loaded flexible nano-liposomes (PFL), then the optimal essential oil and its dosage level were determined by the external leakage curve of PC. The female Japanese white rabbits were used to evaluate the vaginal irritancy potential of liposomes samples. The pharmaceutical properties such as encapsulation efficiency, particle size, zeta potential, deformability and structure of liposomes samples were evaluated. In order to investigate the permeability of liposomes samples to deliver PC across skin and mucous membrane in vitro, the side-by-side diffusion cells were used. The results showed that the leakage of hydrosoluble PC from PFL was reduced at different degrees by the essential oils in the lipid bilayer of PFL, however, the reduction in leakage degree was obviously higher for FA than thoses of FE, AJ, SA and FF (P < 0.05), and the highest reduction in leakage degree was obtained when the FA and lipid mass ratio was 1:6. The encapsulation efficiency, particle size, zeta potential and deformability of PFL were not significantly changed after FA was encapsulated in the lipid bilayer of the PFL (P > 0.05), so did the lamellar structure of PFL. In addition, the transdermal and transmucosal permeability of PC were also enhanced obviously by encapsulating FA in the lipid bilayer of PFL, and there was no vaginal/vulvar irritation observed in the rabbits. In summary, the drug leakage was reduced by encapsulating suitable essential oil (such as FA) in the lipid bilayer of flexible liposomes, and the vaginal mucosa permeability were improved for the drug. These results provide a novel technique in the improvement of flexible nano-liposomes for drug delivery.

Pharmacokinetics and oral bioavailability of palmatine and jatrorrhizine in Huangteng in rats / 中国中药杂志

In this study, the total alkaloids of Huangteng were given to the rats by the methods of intragastric administration and tail vein. After the concentration changes of palmatine and jatrorrhizine in the plasma of rats were determined by RP-HPLC, pharmacokinetic parameters and oral bioavailability were calculated by 3P97 software. After the rats were pre-treated with total alkaloid 60 mg•kg⁻¹ by the methods of intragastric administration and tail vein, the main pharmacokinetic parameters were determined as follows： in the intragastric administration group, the Cmax of palmatine and jatrorrhizine were (0.91±0.06), (0.70±0.08) mg•L⁻¹; tmax of palmatine and jatrorrhizine were (35.24±0.83), (47.76±1.24) min; t1/2 of palmatine and jatrorrhizine were (187.03±1.53), (105.64±16.99) min, AUC of palmatine and jatrorrhizine were (280.30±18.69), (144.36±1.06) mg•min•L⁻¹; in the intravenous injection group, the t1/2 of palmatine and jatrorrhizine were (172.18±12.38), (147.26±1.82) min; AUC of palmatine and jatrorrhizine were (2 553.14±214.91), (328.83±10.81) mg•min•L⁻¹. The oral bioavailability of palmatine was 10.98% and jatrorrhizine was 43.90%.

Discrimination of proliferation inhibiting ingredients in Corydalis soxicola on rat hepatic stellate cell-T6 based on composition-activity relationship / 中草药

Objective: To establish the composition-activity relationship (CAR) model based on study of chemical composition and relating proliferation inhibitory rate of Corydalis soxicola and recognize the anti-hepatic fibrosis active compounds of C. soxicola. Methods: Nine orthogonal C. soxicola extract were analyzed by HPLC, and 21 characteristic peaks were profiled. Anti-hepatic fibrosis activity was investigated by MTT assays on HSC-T6, and the potential active components were identified by scores plot and variable importance in projection (VIP) values by means of orthogonal partial least squares (OPLS) analysis, and the activities of identified components were verified by MTT and flow cytometry. LDH kit was used to detect the effect of dehydrocavidine, palmatine, and berberine on LDH activity. Results: The results showed that six peaks including peaks 13, 14, 16, 18, 19, and 20 were significantly related to anti-hepatic fibrosis activity among nine orthogonal extract from C. soxicola. Peaks 18, 19, and 20 were characterized as dehydrocavidine, palmatine, and berberine, respectively. MTT assay showed that dehydrocavidine, palmatine, and berberine with various concentration significantly inhibited the proliferation of HSC-T6 cells. It was also found in flow cytometry that the apoptotic rates of dehydrocavidine, palmatine, and berberine (0.10 mg/mL) on HSC-T6 were 42.12%, 42.22%, and 36.73%, respectively, which were obviously higher than that of control (1.69%, P < 0.01). No obvious cytotoxic effect was found when the final concentration of dehydrocavidine, palmatine, and berberine were less than 0.15, 0.10, and 0.10 mg/mL, respectively. Conclusion: In this study, it was for the first time found that dehydrocavidine, palmatine, and berberine in C. soxicola extract can effectively inhibit the proliferation and induce apoptosis of HSC-T6 based on composition-activity relationship, and there was no obvious cytotoxic effect of the effective concentration, which showed that they may be the active components with potential anti-hepatic fibrosis effect and have a certain security in the application in C. soxicola. At the same time, it also suggests that the research ideas based on CAR can provide an effective method for the identification of active components in natural plants.

Simultaneous determination of six constituents in Jiaotai Pills by HPLC / 中成药

AIM To establish an HPLC method for the simultaneous content determination of six constituents in Jiaotai Pills (Coptidis Rhizoma and Cinnamomi Cortex).METHODS The analysis of 30％ methanol of this drug was performed on a 30 ℃ Agilent ZORBAX SB-C1s column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of acetonitrile-KH2PO4flowing at 0.8 mL/min in a gradient elution manner,and the detection wavelength was set at 276 nm.RESULTS Epiberberine,jatrorrhizine hydrochloride,coptisine hydrochloride,palmatine chloride,berberine hydrochloride and cinnamaldehyde showed good linear relationships within the ranges of 0.64-41.24 μg/mL (R2 =0.999 9),0.65-43.76 μg/mL (R2 =1.000 0),0.82-52.65 μg/mL (R2 =0.999 9),0.79-50.70 μg/mL (R2 =0.999 9),3.08-197.20 μg/mL (R2=0.999 8) and 0.65-41.65 μg/mL (R2 =0.999 9),whose average recoveries were 98.06％,102.76％,99.27％,99.75％,96.74％ and 101.33％ with the RSDs of 0.56％,0.54％,0.39％,0.55％,0.48％ and 2.14％,respectively.CONCLUSION This accurate,sensitive,stable and reproducible method can be used for the quality control of Jiaotai Pills.

Uptake Characterizations of Berberine, Palmatine and Jateorhizine in Rat Primary Hepatocytes / 中国药学杂志

OBJECTIVE: To evaluate the uptake characterizations of berberine, palmatine and jateorhizine through hepatic uptake model with rat primary hepatocytes. METHODS: Rat hepatocytes were isolated by Seglen's two-step method and cultured in suspension. The activity of transporters was assessed using rosuvastatin which is known as the substrate of the transporters. The tested drugs were incubated with rat hepatocytes at 4 or 37℃ for various periods of time and the drugs were determined using UPLC-MS/MS method to calculate the uptake amounts. The effect of cultural time and temperature on the hepatic uptake of these compounds were assessed. The kinetic parameters such as Km, Vmax and CLactive/CLuptake were calculated from concentration dependent uptake experiments. The effects of positive inhibitors on the uptake of berberine, palmatine and jateorhizine were also surveyed by pre-incubation of the inhibitors with the rat hepatocytes, followed by co-incubation with berberine, palmatine and jateorhizine. RESULTS: The hepatic uptake of berberine, palmatine and jateorhizine increased along with the increase of concentration at 37℃ while the change of hepatic uptake was very little at 4℃. The value of CLactive/CLuptake was 85.26%, 74.90% and 57.74% for berberine, palmatine and jateorhizine. Ibuprofen, digoxin, glycyrrhizin and prazosin which were the known inhibitors of transporters could remarkably reduce the hepatic uptake of berberine and palmatine, respectively. Glycyrrhizin and prazosin could reduce the uptake of jateorhizine. CONCLUSION: The transport of berberine, palmatine and jateorhizine into hepatocytes is mainly through an active process. Berberine and palmatine are the substrates of Oatp1a1, Oatp1a4, Oatp1b2 and Oct1, and the uptake of jateorhizine might be associated with Oatp1b2 and Oct1.

Simultaneous Determination of Five Components in Ermiao Pills by UHPLC with Dual-Wavelength / 中国药学杂志

OBJECTIVE: To develop an ultra-high performance liquid chromatography(UHPLC) method for simultaneous determination of berberine hydrochloride, phellodendrine hydrochloride, palmatine hydrochloride, magnoflorine, and rhizoma atractylodis in Ermiao pills. METHODS: The UHPLC analyses were performed on a Waters BEH C18 column (2.1 mm×100 mm, 1.7 μm)eluted with acetonitrile and 0.1% phosphoric acid(each 100 mL containing 0.1 g sodium dodecyl sulfate). The flow rate was 0.3 mL·min-1, the detection wavelength was set at 280 and 340 nm, and the column temperature was set at 30℃. RESULTS: The calibration curves of the five compositions had good linear relationship in the ranges of the tested concentrations. The precision, stability and repeatability complied with the requirements of methodology. The recoveries were between 96.8%-99.3% respectively. The RSDs were below 2.8%. CONCLUSION: The developed method is simple, sensitive, rapid and accurate. This work provides helpful information for the comprehensive quality evaluation of Ermiao pills.