This study was designed to investigate the effect of Huangqin Tang (HQT) on TLR4/Myd88 pathway and the downstream cytokines in rats with ulcerative colitis (UC) to explore its underlying mechanisms of action. The model of UC rats with cell immunoreactivity was made using a compound method (trinitrobenzene sulfonic acid plus ethanol). Rats were randomly divided into the control group, the model group, the salazosulfapyridine (SASP) group, high, medium and low dose(20, 10, 5 g·kg-1) of HQT groups. After a three-day treatment, production of NO in serum was detected by Griess assay, the levels of interleukin (IL)-4, IL-10, IL-17 and prostaglandin E2（PGE2） in serum were detected by ELISA. After a five-day treatment, the positive protein expressions of COX-2 and iNOS in the colon tissue were determined by ICH method, the protein expressions of TLR4 and MyD88 in colon tissue were determined by Western blot. Compared with the control group, the levels of NO, IL-17, PGE2, the protein expressions of TLR4, MyD88 and the protein positive expressions of COX-2, iNOS were apparently higher in the model group. Compared with model group, the above indexes were significantly improved in the SASP and high-dose HQT groups (P < 0.05). These results show that HQT has a definite effect on UC in rats. Its mechanisms of action may be achieved by inhibiting the activity of TLR4/MyD88/NF-κB signal pathway and down-regulation of NO, IL-17 and PGE2 production.

The quality control processes for herbal medicines have been problematic. Flavonoids are the major active components of Huangqin Tang (HQT, a traditional Chinese medicine formula). In this study, we used a combinative method approach consisting of chromatographic fingerprinting (high performance liquid chromatography; HPLC), quantitative methods and a pharmacodynamic evaluation model to analyze the flavonoids of HQT obtained from different sources. Ten batches of HQT were analyzed by the HPLC fingerprinting method and 26 common peaks were detected, of which 23 peaks corresponded with the chemical profile of HQT. In addition, 11 major compounds were identified by LC–MS analysis (liquid chromatography–tandem mass spectrometer; LC–MSn) and quantified by the HPLC quantitative method approach. The studied 10 batches of HQT were found to be homogeneous in their composition with a similarity between 0.990 and 1.000. The distribution of the 11 identified compounds was found to be very similar among the batches. Only slight pharmacodynamic differences were detected between the different batches, confirming the homogeneity of HQT. The results of this study prove that the combination of chromatographic fingerprinting and quantitative analysis can be readily used for comprehensive quality control of herbal medicines.

To investigate the effect of huangqin tang on expression of cytokines and NF-κB p65 in rats with ulcerative colitis (UC), and to probe into its underlying mechanisms of action. The mode of UC rats with cell immunoreactivity was made using compound method (trinitrobenzene sulfonic acid and ethanol). Rats were randomly divided into control group, model group, SASP group and high dose, middle dose and low dose of huangqin tang group. The food intake, body weight and microscopic damage of rats in each group were evaluated after being treated for five days. The blood and colon tissue were also collected. Production of NO was detected by Griess assay, the expression levels of IL-6, TNF-α, PGE2 were detected by ELISA. ICH method was undertaken to determine the expression of NF-κB p65 protein in colon tissue. The food intake and body weight of model group rats were lower than that of control group. The expression levels of NO, IL-6, TNF-α, PGE2 in serum and NF-κB p65 protein of colon tissue in model group were higher than that of control group. The above indexes were ameliorated in high and middle dose of huangqin tang groups. But there was no significant difference with SASP group. NF-κB p65 may be involved in the pathogenesis of UC, and huangqin tang can inhibit the relative activity of NF-κB p65, and decrease the expression levels of NO, IL-6, TNF-α and PGE2.

Wuji Wan (WJW), containing Rhizoma Coptidis (Huanglian in Chinese, HL), Frutus Evodiae Rutaecarpae (Wuzhuyu, WZY) and Radix Paeoniae Alba (Baishao, BS), is a classical traditional Chinese medical formula employed in treating intestinal disorders. Berberine (BBR) and palmatine (PMT) are the major active alkaloids in HL and have analgesic and anti-microbial effects. A sensitive, specific and validated ultra-performance liquid chromatography–tandem mass spectrometric method was developed to investigate the pharmacokinetic profiles of BBR and PMT in rat plasma and in situ intestinal perfusion solution. In comparison with the pharmacokinetic parameters of BBR and PMT, t 1/2, C max, T max, AUC, CL and MRT after intragastric (i.g.) administration with HL extract alone, those remarkably changed after i.g. administration with WJW formulas 1 and 2 (herb proportions are 12:2:3 and 12:1:12). Particularly, the oral bioavailability of PMT in WJW formula 1 was significantly increased. In rat intestinal perfusion experiments, the apparent permeability coefficient value of PMT was (1.45 ± 0.72) × 10−5 cm/s when perfusion with HL was performed, and the value was significantly increased to (3.92 ± 0.52) × 10−5 cm/s on perfusion with WJW formula 1. These results indicate that the pharmacokinetic parameters and absorption of BBR and PMT are affected by the other herbs or ingredients from WJW formulas.

Background Stellera chamaejasme L , a traditional Chinese herb, has long been used for treatment of various tumors in the Chinese population. In our previous study, we paid an attention to the cytotoxic and proapoptotic effects of Stellera chamaejasme L extracts (ESC, ESC-1 and ESC-2, the latter two were isolated from ESC) on 4 various tumor cells (NCI-H157, NCI-H460, BEL-7402 and SK-HEP-1) in vitro . ESCs showed significantly inhibitory effects on the 4 tumor cells. ESC-2 had the strongest inhibitory effect and the broadest sensitive cell spectrum. ESC-2 and ESC acted in a similar way against tumor cells, which suggested anti-tumor active fraction of ESC might exist in ESC-2. Here, we further observe the inhibitory and proapoptotic effects of Stellera chamaejasme L extracts in vivo . Methods In this study, we used hollow fiber tumor model to evaluate the inhibitory and proapoptotic effects of Stellera chamaejasme L extracts. Apoptotic rates of the cancer cells retrieved from the hollow fibers were measured with flow cytometric analysis, caspase 3, 8, 9 enzyme activities were detected by colorimetric assay, Fas, Fas-L, TNF-R1 and TNF-α expression were determined with elisa assay and radioimmunoassay respectively. Results The results showed that ESC, ESC-2 all had inhibitory effects on 4 tumor cells. According to the effect strength, dose and antitumor spectrum, the order of antitumor effects of ESCs was: ESC-2 > ESC > ESC-1. NCI-H460 cells were the most sensitive to ESCs. ESC, ESC-2 increased greatly the apoptotic rate and caspase 3, 8 enzyme activities in NCI-H460. ESCs had no significant effects on expression of Fas, Fas-L protein, but TNF-α/TNFR1 protein expression in NCI-H460 cells changed significantly after ESC and ESC-2 treatment. Conclusion ESC-2 had the similar antitumor effect to that of ESC in vivo and further confirmed that ESC-2 may be the main antitumor active fraction of ESC, which was consistent with our previous results in vitro .

The pharmacodynamic (PD) and pharmacokinetic (PK) properties of Huangqin Tang (HQT) were investigated in yeast-induced febrile rats. Blood sample and rectal temperature data of the rats were collected at different times after single oral administration of HQT at 20 g x kg(-1). The plasma concentrations of paeoniflorin, baicalin, wogonoside, baicalein, wogonin, oroxylin A, glycyrrhizic acid and glycyrrhetinic acid were quantified by a sensitive liquid chromatography-tandem mass spectrometric (LC-MS) method. The blood concentrations of PGE2, 1L-1β and TNF-α were detected by radioimmunoassay (RIA). All pharmacokinetic parameters were processed by non-compartmental analysis using WinNonlin software. The potential relationship between the mean concentration of eight constituents and the antifebrile efficacy was investigated by calculating Pearson correlation coefficients. It was found that HQT had significant antifebrile efficacy in yeast-induced febrile rats, but had no effect to normal rats. The antifebrile effect of HQT can be attributed to the inhibition of PGE2, 1L-1β and TNF-α. The constituents (baicalin, wogonoside, baicalein, wogonin, oroxylin A, glycyrrhizic acid and glycyrrhetinic acid) in febrile rats had delayed absorption and elimination, a longer residence time in the body, and higher C(max) and AUC than those in normal rats. Febrile condition could affect the pharmacokinetic behaviour of HQT in vivo; the flavonoids with the same backbone showed the similar fate in the body; baicalein and wogonin had a strong positive correlation (R > 0.66, P ≤ 0.02) with the antifebrile efficacy determined. Together, these constituents demonstrated different pharmacokinetic properties in the febrile body.

Objective To investigate the effects of Saussurea involucrate by cell culture on increasing the calcium content and bone density after 90 days feed in ovariectomized rats. Methods Osteoporosis model rats were induced by ovariectomy, treated with 16.7、33.3、100 mg/(kg?d)cell culture of Saussurea involucrate for 90 days. Body weight was observed, calcium content was measured through atomic absorption method and the bone density was detected by bone sonometers. Results cell culture of Saussurea involucrate(16.7、33.3、100 mg/kg)can increase the calcium content[(380.1 ± 1.9)mg/g、(370.7 ± 1.1)mg/g、(363.5 ± 2.4)mg/g] significantly(P<0.01)than the model group; increase distal femoral bone mineral density[(0.041 ± 0.017)g/cm2、(0.042±0.023)g/cm2、(0.040±0.008)g/cm2] significantly(P<0.01 or 0.05) than the model group(0.022±0.014)g/cm2; and also increase the middle femoral bone mineral density, the low-dose group (0.305±0.030)g/cm2 significantly (P<0.05) than the model group(0.259±0.061)g/cm2. Conclusion The cell culture of Saussurea involucrate has the effect of increasing the bone density.

To elucidate the compatibility principle of Yupingfeng powder, a cocktail probe substrates approaches were developed. The enzymatic activity of cytochrome P450 from rat liver microsome was evaluated after being interfered with 7 prescriptions of Yupingfeng powder, which was designed according to the decomposed recipes design of traditional Chinese medicine. In vitro test, rat liver microsome incubation system was utilized to detect the 50% inhibitory concentrations of Yupingfeng powder with the decomposed recipes to cytochrome P450 (CYP1A2, CYP2A6 ,CYP2C9, CYP2C19, CYP3A4) enzyme. The CYPs IC50 value of Yupingfeng powder with different compatibility were greater than crude drug 1.0 g x L(-1), which indicated that all Yupingfeng powder prescriptions had no significant inhibitory activity to cytochrome P450. For CYP1A2, CYP2C19, CYP3A4, the IC50 value of Yupingfeng powder with the decomposed recipes had a tendency to increase, compared with the major impact factor from Yupingfeng powder. For CYPs, the detected IC50 of Yupingfeng powder with the decomposed recipes tended to decrease, compared with the linearly predicted value. From the point of view of the impact of drugs on the metabolic activity, the compatibility of Yupingfeng powder has certain advantages and reasonable.

The current study aims to investigate the pharmacokinetic properties of Huangqin Tang on different oral doses. An LC-MS method for simultaneous determination of flavonoids and terpenoids in rat plasma was developed and validated. Plasma samples were treated with hydrochloric acid (containing 1% ascorbic acid), precipitated with acetonitrile, separated on a Zorbax SB-C18 column, detected by single quadruple mass spectrometry with an electrospray ionization interface, and quantified using selected ion monitoring mode. All pharmacokinetic parameters were processed by non-compartmental analysis using WinNonlin software. The results of specificity, linearity, intra-day and inter-day precisions, accuracy, and stability for LC-MS assay were suitable for the quantification of paeoniflorin, baicalin, wogonoside, baicalein, wogonin, oroxylin A, glycyrrhizic acid and glycyrrhetinic acid in rat plasma. The concentration-time profiles of baicalin, wogonoside, baicalein, wogonin, oroxylin A and glycyrrhizic acid showed double-peak phenomenon after Huangqin Tang was orally administered at 40 g x kg(-1) dose; all eight constituents in rat plasma showed good dose-exposure relationship within the dosage of 10-40 g x kg(-1); although plasma concentrations were different, the flavonoids with the same backbone showed the similar fate in the body with the corresponding dosage. In conclusion, the LC-MS assay was successfully applied for the pharmacokinetic study of multi-constituents of Huangqin Tang with different doses. Additionally, these constituents demonstrated good pharmacokinetic properties in the body.

Objective To explore the influence on anti-inflammatory efficacy and bitter cold properties of Coptis processed by Evodia juice.Methods Auricle swelling model induced by croton oil in mice was used in the anti-inflammatory efficacy experiment.The mice were assigned randomly into distilled control group,dexamethasone group,crude drug group,3 processed drug groups that Euodia juice which plant origin was Euodia rutaecarpa(Juss.) var.bodinaieri(Dode) Huang(SMYHL),Evodia rutaecarpa(Juss.) Benth and Euodia rutaecarpa(Juss.) Benth var.officinalis(Dode) Huang,respectively.The crude drug group and the 3 processed drugs groups were orally administrated one time and the dosages were all 6 g/kg.The control group was orallly administrated the same volume water.Dexamethasone group was intraperitoneally injected dexamethasone 30 g/kg.The swelling degree of each group was observed.In the bitter cold properties experiment,rats were assigned randomly into control group,crude drug group and 3 processed drug groups.The crude drug group and the 3 processed drug groups were orally administrated for 14 days and the dosages were all 3 g/kg.The control group was orally administrated the same volume water.The body weight,capacity for eating and drinking,body temperature,heart rate and blood viscosity of rats were measured.Meanwhile,TSH,IL-2,IL-8 in serum and plasma were determined by radioimmunoassay.Results The degree of mouse auricle swelling was reduced by the crude and processed drugs.Fourteen days after medication,the weight,capacity of eating and drinking of rats were declined to different degrees in crude drug group and in processed drugs group as compared with control group.The blood viscosity was increased and IL-2 was decreased in SMYHL group as compared with control group(P <0.05).TSH was decreased in crude drug group as compared with control group(P <0.05).Conclusion There is no obvious change in anti-inflammatory function of Coptis before and after processed.The cold properties of Coptis can be reduced after processed by Evodia Juice.

Ethnopharmacological relevance: Yu Ping Feng San (YPFS, in Chinese: Jade Windscreen Powder), a well-known traditional Chinese medicine, is commonly used to cure the diseases of respiratory systems and immune systems.Aim of the study: A selective and sensitive high-performance liquid chromatography coupled with mass spectrometry method (HPLC-MS) was developed and validated for simultaneous quantification of cycloastragenol, formononetin, calycosin, 4'-O-beta-glucopyranosyl-5-O-methylvisamminol (GMV) and cimifugin in rat plasma after oral administration of Yu Ping Feng San decoction.Materials and methods: Plasma samples were extracted via solid-phase extraction (SPE), separated on a Zorbax SB-C18 column, detected by single quadruple mass spectrometry with an electrospray ionization interface, and quantified using selected ion monitoring mode. The current SPE-HPLC-MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability. The method was applied to a comparative pharmacokinetic study after administration of Yu Ping Feng San to rats at different doses (10, 20 and 40 g/kg).Results: The calibration curves were linear over the ranges 0.50-50 ng/mL and 17.36-1736 ng/mL. Intra- and inter-day precisions (relative standard deviations) were from 0.45% to 10.95%, and accuracy (relative recovery) from 95% to 115%. The extraction recoveries were greater than 88.42% for all analytes. Dose-dependence was shown for some constituents in the drug concentration-time profiles. Among all the active ingredients detected, cimifugin had the highest blood concentration (881-1510 ng/mL), and cycloastragenol had the longest retention time in the rat body (15.06-20.44 h).Conclusion: This analytical method is a selective, sensitive, precise, accurate, and reliable assay for simultaneous determination of cycloastragenol, calycosin, formononetin, GMV, and cimifugin in rat plasma. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

OBJECTIVE:To observe the impact on absorption of berberine and palmatine in different compatibilities of Wuji pill by the perfused rat intestine-liver preparation.METHOD:Use L9 (3(4)) orthogonal design table, establish the perfused rat intestine-liver preparation, the twelve Wuji pill compatibilities duodenal administrated, collect the perfusate at different times points for LC-MS detection, calculate the absorbed score, Ka.RESULT:Evodiae Fructus and the absorption score, Ka of berberine and palmatine are inverse correlated. The most superior portion which promote the absorption is Coptidis Rhizoma-Evodiae Fructus-Paeoniae Radix Alba 3:1:3.CONCLUSION:Evodiae Fructus suppressed the absorption of berberine and palmatine. With the different portion the absorption also have big different.

OBJECTIVE:To in vitro compare the induction of extracts of Stellera chamaejasme ESC, ESC-1 and ESC-2 on NCI-H157 cell apoptotic.METHOD:The apoptosis rate was inspected by flow cytometry; caspase-3, 8, 9 activities was measured by spectrophotometry. Fas, Fas-L, TNF-alpha, Trail-R, Cyto-C, Smac/diablo protein expressions of apoptosis pathway was observed by Elisa method.RESULT:Compared with the control group, ESC, ESC-1, ESC-2 can significantly improve the apoptosis rate of NCI-H157 cell. ESC significantly improved cells caspase-3, 8 activity, ESC-2 can significantly improve the activity of caspase-3, 8, 9. ESC, ESC-1, ESC-2 significantly increased Fas expression and ESC significantly increased Fas/Fas-L ratio. ESC, ESC-1, ESC-2 significantly increased TNF-alpha protein expression. ESC-1 significantly lowered TRAIL-R expression. ESC, ESC-1, ESC-2 had no significant effect on Cyto-C. ESC-1, ESC-2 significantly reduced Smac protein expression.CONCLUSION:The apoptotic effect induced by ESCs may be related to the regulation of death receptor pathway proteins. Induction mechanisms of ESCs were so complicated that it may have a two-way regulatory effect. Its induction in apoptosis is a result from comprehensive regulation and control.

Objective:To study the effect of compatibility with Fructus Evodiae on intestinal absorption of alkaloids in Rhizoma Coptidis and its mechanism in rats.Method: In situ single-pass perfused rat intestinal model was used and the concentrations of berberine and palmatine were determined by HPLC.The drug absorption rate constant(Ka) and apparent absorption coefficient(Papp) were tested to evaluate the effect of compatibility with verapamil or Fructus Evodiae on their absorption.Result: Rhizoma Coptidis extractive and Fructus Evodiae extractive(6∶12) can significantly enhance Ka and Papp of berberine and palmatine.Two alkaloids in Rhizoma Coptidis and Fructus Evodiae extractive(6∶12) promoted their intestine absorption.When the compatible proportion was 6∶1,the absorption of berberine decreased,and the absorption of palmatine increased.Verapamil had a promoting effect on the absorption of berberine and palmatine.Conclusion: Fructus Evodiae extractive can promote the intestine absorption of berberine and palmatine,and the mechanism may be related to its inhibition of P-glycoprotein activity.

Objective:To investigate the recovering effect of Qizheng plaster on the bone defect in rat model.Method: The rat model of bone defect was established.Qizheng plaster was percutaneously given with doses of 2,1,0.5 g · kg-1 for 4 weeks.During the period,the effect was investigated by X-ray examination,serum calcium(Ca),phosphorus(P) and alkaline phosphate(ALP) were measured,and biomechanical parameters were determined,and histopathologic examination was carried out.Result: In 2,1,0.5 g · kg-1 groups,the X-ray examination showed that of bone mineral density(BMD) in the bone defect parts was increased within 7-28 days.The gray value in the treated groups were higher than that of the model group,and had obvious difference on the 14th day(P0.01).There were no obvious changes in the serum ALP,Ca and P.Histopathologic examination showed that the generation rate of bony trabeculae and bony spicules in the 2,1,0.5 g · kg-1 groups were faster than that in the model group within 7-14 days.Conclusion: Qizheng plaster has recovering effect for the bone defect in rat model.

Objective:To investigate the actions of promoting blood circulation for removing blood stasis by Guanxin Cataplasm and provide the experimental basis for clinical applications of the cataplasm.Method: The rat blood stasis syndrome model was induced by subcutaneous injection of adrenalin with ice-bath,the indexes of hemorheology including fibrinogen,whole blood viscosity,plasma viscosity,packed cell volume and platelet aggregation.The acute rat myocardial ischemia model was established by sublingual vein injection of 0.5 U·kg-1 posterior pituitary,and the electrocardiogram ECGs were recorded by polygraph to determine the incidence of ventricular tachycardia(VT) and ventricular fibrillation(VF),the mean duration and onset time of VT plus VF.Result: Compared with model group,high dosage group of guanxin cataplasm decreased plasma viscosity from(1.101±0.064) mPa·s to(1.039±0.025) mPa·s,platelet aggregation from(54.92±6.53)% to(49.05±4.15)%,and whole blood viscosity under the shear rate of 200 s-1from(3.81±0.31) mPa·s to(3.53±0.27) mPa·s;meanwhile the delayed duration of VT plus VF from(1 133.75±298.71)s to(881.20±115.48)s and onset time of VT plus VF from(20.90±7.74)s up to(36.08±16.91)s with reduced incidence of VT plus VF from 75% to 37.5% were observed.Small and middle dosage group led to a reduction in whole blood viscosity under 10 s-1 from(7.14±0.88) mPa·s to(6.38±0.69) mPa·s and that under 200 s-1 from(3.81±0.31) mPa·s to(3.48±0.15) mPa·s shear rate,and in the incidence decreased from 75% to 37.5% and duration decreased from(1 133.75±298.71) s to(816.43±243.46) s for VT plus VF respectively(P<0.05,P<0.01).Conclusion:Guanxin cataplasm can obviously improve indexes of hemorheology and afford a protection against occurring of VT plus VF,which may contribute to its function of promoting blood circulation for removing blood stasis.

To illustrate the compability rule of Jinlingizi powder, by investigating the effects of Jinlingzi Powder with different compatibility on the enzymatic activity of cytochrome P1 A2 (CYP1A2) from rat liver microsome. The different compability of Jinlingizi powder is designed, based on the orthogonal array L9 (3(4)). In vitro test, rat liver microsomes incubation system is applied to detect the 50% inhibitory concentraton of Jinlingzi powder with different compatibility to cytochrome P1A2 (CYP1A2) enzyme. In vivo experiments, rats is treated orally with the different compability of Jinlingizi powder for 5 days, then be injected with probe drug phenacetin. The biosample from liver tissue is obtained by microdialysis probe, then analysisd by HPLC. The concentration-time data are modulated by software WinNonlin. IC50 data show no significant inhibitory activty to cytochrome P1 A2. Acetaminophen and phenacetin PK parameters indicate that the different compability of Jinlingizi powder can modulate the CYP 1A2 mediated metabolism, which is associate with the compatibility of Jinlingzi powder.

Objective: To explain the compatibility mechanism of Wuji pill in view of absorption and liver uptake.Method: Establish the perfused rat intestine-liver preparation,After the eighteen Wuji Pill compatibilities duodenal administrated,collect the perfusate from portal vein and hepatic vein at different times points(0、1、5、10、15、30、60、90、120min) for LC/MS detection.Calculate the AUC,liver uptake rate,conduct the range analysis and variance analysis.Result: For the absorption of representative components,Evodia was negatively correlated with the chief ingredients of Coptidis Rhizoma and Paeoniae Radix Alba,Paeoniae Radix Alba was positively correlated with the chief ingredients of Coptidis Rhizoma but negatively correlated with the chief ingredients of Evodia;For the liver uptake of representative components,Evodia was positively correlated with the chief ingredients of Coptidis Rhizoma,Coptidis Rhizoma and Paeoniae Radix Alba are negatively correlated with each other.Also received the optimal compatibility for absorption and liver uptake.Conclusion: For the absorption of representative components,Evodia restrain the chief ingredients of Coptidis Rhizoma and Paeoniae Radix Alba,Paeoniae Radix Alba promote the chief ingredients of Coptidis Rhizoma but restrain the chief ingredients of Evodia.For the liver uptake,Evodia promote the chief ingredients of Coptidis Rhizoma,Coptidis Rhizoma and are restrained mutually.