Development of a liquid-liquid microextraction GC-MS method for simultaneous determination and pharmacokinetic analysis of β- elemene in rat plasma after administration of citronella grass extract / 药学学报

We established a simple and sensitive GC-MS method for the determination of β-elemene in rat plasma and measured the pharmacokinetics of citronella grass extract in rats. Plasma samples were pretreated using liquid-liquid microextraction: 100 μL of plasma sample (containing naphthalene as the internal standard) was extracted with 50 μL of n-hexane. The determination was performed on DB-5ms column (30 m×0.25 mm, 0.25 μm). The initial column temperature was 60 ℃ and raised to 160 ℃ at a rate of 50 ℃·min-1, maintained for 3 min, and finally increased to 260 ℃ for 3 min. Helium was the carrier gas and the flow rate was 0.15 mL·min-1. The injection volume was 2 μL. EI and selected monitored ions pattern were used for ion scanning with m/z 128 (naphthalene) and m/z 93 (β-elemene). Citronella grass extract was administered to rats by intragastric administration and intravenous administration (containing β-elemene 55 mg·kg-1), and plasma was collected and prepared using an automated blood collection system. The linear range of β-elemene in plasma was 1.0-250 ng·mL-1 (r = 0.997), the limit of quantification was 1.0 ng·mL-1, the accuracy was -4.47% - -0.85%, the extraction recovery was between 56.02%-66.89%, and no obvious matrix effect (94.28%-108.63%) was found. The main pharmacokinetic parameters of β-elemene were AUC0-t (23.56 ± 4.40) ng·mL-1, tmax (1.67 ± 0.58) h, Cmax (7.36 ± 0.69) ng·mL-1, MRT0-t (2.76 ± 0.27) h, t1/2z (2.73 ± 1.36) h, Vz (7.39 ± 3.18) L·kg-1, CLz (1.95 ± 0.51) L·h-1·kg-1, and the absolute bioavailability was about 8.78%. The method is simple, accurate, and sensitive, and is suitable for the pharmacokinetic analysis of β-elemene in citronella grass extract in rats. All animal studies were implemented according to protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

Research progress on culture technologies,chemical components,and pharmacological activities of Saussurea involucrata cells / 中国中药杂志

Saussurea involucrata,a traditional Chinese medicinal material,is effective in the treatment of rheumatoid arthritis with cold-dampness blockage syndrome,cold pain in lower abdomen,and menstrual irregularities. However,due to the specific habitat,low natural reproduction rate,slow growth,and overexploitation,it is at the high risk of extinction. S. involucrata cells can be obtained through callus culture,suspension culture,and hairy root culture. This study highlighted the influences of reactor type,culture system,precursor,elicitor type, and light wavelength on the suspension culture of S. involucrate cells. The chemical components of S. involucrata cells mainly include phenylpropanoids,flavonoids,lignans,and steroids,among which phenylpropanoids are the most abundant. S. involucrata cells have multiple pharmacological activities of anti-inflammation,analgesia,activating blood and resolving stasis,immunoregulation,increasing bone density,lowering blood lipids,anti-hypoxia,anti-exercise fatigue,anti-radiation,anti-obesity,and anti-oxidation. Moreover,it has the potential of treating aplastic anemia. This study reviews the cell culture technologies,chemical components,and pharmacological activities of S. involucrata cells,laying a basis for the further research,development,and utilization.

Comparison of Related Mechanism in Rats with Ulcerative Colitis After Treatment of Three Regiments / 中国实验方剂学杂志

Objective:This study was designed to compare inflammatory response, water carriage and gut brain axis in rats with ulcerative colitis (UC) after treatment of three regiments, Huangqintang (HQT), Sishenwan (SSW), and Tongxie Yaofang(TXYF). Method:After approved by Institute of Chinese Materia Medica Ethics Committees in China Academy of Chinese Medical Sciences, UC in rats was induced by using a compound method (trinitrobenzenesulfonic acid plus ethanol). Rats were randomly divided into control, disease, positive control salazosulfapyridine (SASP, 0.5 g·kg-1), HQT (20 g·kg-1), SSW(26 g·kg-1), and TXYF group(22 g·kg-1). After 5 days of treatment, colonic tissues and the blood were taken for various assays. Damage of colonic tissues was detected by hematoxylin-eosin staining (HE). The distribution of Vasoactine intrestinal (VIP), 5-hydroxytrytamine (5-HT), P-substance (SP) in the blood and serum were detected by enzymelinked immunosorbent assay (ELISA) and immunohistochemistry (IHC), the levels of aquaporin3 (AQP3) and Aquaporin4 (AQP4) in the serum were detected by Western blot, the mRNA expression of Extracellular regulated protein kinases 1 (Erk1) and p38 mitogen-activated protein kinase (p38 MAPK) in the serum were detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). Result:The brain-gut peptide results showed that compared with the normal group, the content of 5-HT and VIP in model group were significantly decreased (P<0.01), the content of SP were decreased, but there was no significant statistical difference, compared with the disease group, the content of 5-HT in SASP and TXYF group were clearly increased (P<0.05), the increment of VIP and SP in SASP, HQT, TXYF group were significant (P<0.05). Compared with the normal group, the content of AQP3 in model group were significantly increased(P<0.01), the content of AQP4 were clearly decreased(P<0.01), compared with the disease group, the content of AQP4 in SASP and HQT group were clearly increased (P<0.05), whereas the levels of AQP3 in HQT group were most significant reduced (P<0.01). Compared with the disease group, the expression of Erk1 and p38 were clearly reduced (P<0.01), with the most significant reduce being the expression in HQT group. Conclusion:Three regiments all have therapeutic effects on UC, manifested by improvements of the signs and mental status of UC rats. However, in terms of gut-brain axis disturbance improvement, the therapeutic effect of TXYF was superior than HQT and SSW, whereas in terms of inflammatory response suppression and water carriage accomodation, the therapeutic effect of HQT was superior than SSW and TXYF.

Potential mechanism of Huangqin decoction for the treatment of ulcerative colitis based on network pharmacology / 药学学报

To study the mechanism of Huangqin decoction (HQT) in the treatment of ulcerative colitis (UC) by using network pharmacology, chemical components and targets related to the four herbs of Chinese meteria medical in HQT were searched through the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) to construct the interaction network diagram of the target point of the compounds. The UC-related targets were screened through OMIM, TTD, and GeneCard databases. The compound-target network was constructed using Cytoseape_v3.7.1 software; based on the STRING database, a target interaction network for HQT for UC was constructed, and the core target of HQT for UC was selected based on topological parameters. GO (gene ontology) biological process enrichment analysis and KEGG (KEGG pathway analysis) pathway annotation analysis were performed on the disease and drug intersection targets using the R package clusterprofile version 3.12.0 in Bioconductor. The HQT compound-UC target network contains 128 compounds and corresponding targets 141. The core targets are AKTI, IL6, PTGS2, IL10, IL1β and so on. GO functional enrichment analysis yielded 151 GO terms, and KEGG pathway enrichment screening resulted in 33 associations with UC, mainly involving PI3K-AKT signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway, Toll-like receptor signaling pathway and so on. The synergetic effect of HQT with multi-components and multi-pathway was confirmed by network pharmacology, and the main possible mechanism of HQT in treating UC was predicted, which lay a foundation for the identification of effective components, the mechanism of action, and clinical application.

The effects of Huangqin Tang on oxidative stress and ulcerative colitis in rats through the Nrf2 signal pathway / 药学学报

This study aimed to investigate the effect of Huangqin Tang (HQT) on oxidative stress associated with ulcerative colitis in rats, and to explore its antioxidant mechanism. After approved by Institute of Chinese Materia Medica Ethics Committees in China Academy of Chinese Medical Sciences, the rats were given 2,4,6-trinitrobenzenesulfonic (TNBS)/ethanol mixture to induce the ulcerative colitis (UC), and were randomly divided into normal group, model group, the salazosulfapyridine (SASP) group, and high, middle or low dose (20, 10, 5 g·kg-1) of HQT groups. After 5 days of treatment, the activity of catalase (CAT) from micrococcus lysodeikticus, glutathione peroxidase (GSH-px), myeloperoxidase (MPO), superoxide dismutase (SOD) were detected by biochemical assays. The levels of lipid peroxide (LPO) were detected by ELISA. The positive protein expression of nuclear factor erythroid-2-related factor 2 (Nrf2) were detected by immunohistochemistry method and the downstream antioxidant enzymes of Nrf2 were determined by Western blot analyses. The levels of SOD, CAT and GSH-px activities in the normal group were significantly higher than the model group, while the serum MPO activity in the model group was obviously increased (P<0.05 or P<0.01). Compared with the model group, there was a significant difference in the activity of CAT in the high and middle dose groups of HQT (P<0.05 or P<0.01), the activity of GSH-px in the high, middle and low dose groups of HQT were apparently higher than the model group (P<0.05); The serum levels of LPO in the model group were significantly higher than those in the normal group (P<0.05), while the up-regulating effects on LPO were reversed by the high and middle dose groups of HQT (P<0.05). The expression of Nrf2 in the high-dose group of HQT and SASP group was statistically significant (P<0.05). The results of Western blot showed that compared with the model group, each of the HQT and SASP group could increase the heme oxygenase (HO-1) and NAD[P]H: quinone oxidoreductase 1 (NQO-1) expression in a dose-dependence manner. HQT has significant anti-oxidative stress and obviously improves the signs, mental status and defecation of UC rats. The mechanism of action for HQT maybe related to activate the Nrf2 pathway and increase the expression of Ⅱ phase metabolic enzymes such as HO-1 and NQO-1, reduce the content of LPO and MPO in serum and enhance the activity of SOD, CAT and GSH-px.

Comparison of intestinal bacteria and inflammatory cytokine expression in rats with ulcerative colitis after treatment of three regiments / 药学学报

This study was designed to compare intestinal bacteria and inflammatory cytokine expression in rats with ulcerative colitis (UC) after treatment of three regiments, Huang-qin-tang (HQT), Si-shen-wan (SSW), and Tong-xie-yao-fang (TXYF). After approved by Institute of Chinese Materia Medica Ethics Committees in China Academy of Chinese Medical Sciences, UC in rats was induced by using a compound method (trinitrobenzenesulfonic acid plus ethanol). Rats were randomly divided into control, disease, positive control salazosulfapyridine (SASP, 0.5 g·kg-1), HQT (20 g·kg-1), SSW (26 g·kg-1), and TXYF groups (22 g·kg-1). After 7 days of treatment, colonic tissues and the blood were taken for various assays. Damage of colonic tissues was detected by H&E staining. The levels of tumor necrosis factor-ɑ (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8) and prostaglandin E2 (PGE2) in the serum were detected by the enzyme linked immunosorbent assay (ELISA). Total DNA was extracted from stool samples for analyses of 16SMiseqPE300V3-4 segment using high-throughput sequencing. The inflammatory cytokine results showed that compared with the disease group, the content of IL-6, PGE2, TNF-α in SASP group were decreased (P<0.05), with the most significant decrease being the level of IL-8 (P<0.01), whereas the levels of IL-6, IL-8 and TNF-α in HQT group were reduced (P<0.05) and PGE2 content was clearly reduced (P<0.01). The contents of four cytokines in SSW group were decreased, but there was no statistical difference. While the levels of IL-6 and TNF-α in TXYF group were reduced, and the reductions of IL-8 and PGE2 were significant (P<0.05). The results after sequencing showed that microbiome species richness SSW group > HQT group > TXYF group; the similarity between samples TXYF group > SSW group > HQT group; the species of HQT and TXYF group have greater difference when compared to the disease group. The content of beneficial bacteria in the intestine of HQT group > SSW group > TXYF group. Three regiments all have therapeutic effects on UC, manifested by improvements of the signs and mental status of UC rats. However, in terms of inhibition of inflammatory factors IL-6, IL-8, PGE2 and TNF-α, and regulation of intestinal microbiome, the therapeutic effect of HQT was superior than SSW and TXYF.

Simultaneous determination and quantitation of anthraquinones, lignans and flavonoids in Xiao-Cheng-Qi Tang, Hou-Po-San-Wu Tang and Hou-Po-Da-Huang Tang by HPLC / 药学学报

A simple and selective HPLC method for simultaneous determination and quantification of anthraquinones, lignans and flavonoids in Xiao-Cheng-Qi Tang (XCQT), Hou-Po-San-Wu Tang (HPSWT) and Hou-Po-Da-Huang Tang (HPDHT) was developed and validated. An Agilent Zorbax SB-C 18 (4.6 mm x 250 mm, 5 µm) column with the mobile phase of acetonitrile and 0.5% acetic acid aqueous solution in gradient elution mode was used. The flow rate was 1.0 mL · min(-1) at 30 °C, and injection volume was 10 µL. The detection wavelength was set at 254 nm and 294 nm simultaneously for the quantitative analysis. The current HPLC assay was validated for linearity, intra-day and inter-day precisions, accuracy, recovery and stability. The method was applied to the content comparison of the gallic acid, cinnamic acid, sennoside A, sennoside B, rhein, emodin, aloe-emodin, chrysophanol, physcion, magnolol, honokiol, narirutin, naringin, hesperidin, neohesperidin, hesperetin, naringenin and nobiletin in XCQT, HPSWT and HPDHT. The good linear equations of eighteen constituents were obtained within the investigated ranges (r > 0.998). The recovery of the method was 94.28%-99.89% and the precision was less than 5%. The sample was stable within 16 h. There were some differences between the contents of anthraquinones, lignans and flavonoids in analogous formulae about XCQT. XCQT contained the greatest abundance of anthraquinones and flavonoid, HPSWT contained the greatest abundance lignans. In conclusion, the methods are simple, low-cost, precise, accurate and reliable for the determination of eighteen constituents in analogous formulae about XCQT, and these results provide methodological support for its quality control.

Effect of huangqin tang on the regulatory NF-κB p65 signal pathway in rats with ulcerative colitis / 药学学报

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.

Pharmacokinetics and pharmacodynamics of huangqin tang in febrile rats / 药学学报

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.

LC-MS quantification and pharmacokinetics of the multi-constituents of Huangqin Tang in rat plasma after different single oral doses / 药学学报

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.

Compatibility principle research of Yupingfeng powder based on cocktail probe substrates approaches / 中国中药杂志

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.