Evaluation of drug interactions of Saposhnikoviae Radix and its major components with astragaloside IV and paeoniflorin using in vitro and in vivo experiments.

Saposhnikoviae Radix (SR) may enhance the pharmacodynamics of Huangqi Chifeng Tang (HQCFT) in the treatment of cerebral infarction according to our previous research, but the underlying mechanism is unknown. Herein, an in vivo pharmacokinetic assay in rats and in vitro MDCK-MDR1 cell assays were used to investigate the possible mechanism of SR, its main components, and its interactions with Astragali Radix (AR) and Paeoniae Radix (PR). An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC‒MS/MS)-based analytical method for quantifying astragaloside IV (ASIV) and paeoniflorin (PAE) in microdialysis and transport samples was developed. The pharmacokinetic parameters of SR were determined using noncompartmental analyses CCK-8 assays were used to detect the cytotoxicity of ASIV, PAE, cimifugin (CIM), prim-o-glucosylcimifugin (POG) and their combinations. Moreover, drug transport was studied using MDCK-MDR1 cells. Western blotting was performed to measure the protein expression levels of P-GP and MRP1. Claudin-5, ZO-1, and F-actin expression was determined via immunohistochemical staining of MDCK-MDR1 cells. harmacokinetic studies revealed that, compared with those of Huangqi Chifeng Tang-Saposhnikoviae Radix (HQCFT-SR), the Tmax of ASIV increased by 11.11 %, and the MRT0-t and Tmax of PAE increased by 11.19 % and 20 %, respectively, in the HQCFT group. Transport studies revealed that when ASIV was coincubated with 28 µM CIM or POG, the apparent permeability coefficient (Papp) increased by 71.52 % and 50.33 %, respectively. Coincubation of PAE with 120 µM CIM or POG increased the Papp by 87.62 % and 60.95 %, respectively. Moreover, CIM and POG significantly downregulated P-gp and MRP1 (P < 0.05), inhibited the expression of Claudin-5, ZO-1, and F-actin (P < 0.05), and affected intercellular tight junctions (TJs). In conclusion, our study successfully established a selective, sensitive and reproducible UPLC‒MS/MS analytical method to detect drug‒drug interactions between SR, AR and PR in vivo and in vitro, which is beneficial for enhancing the therapeutic efficacies of AR and PR. Moreover, this study provides a theoretical basis for further research on the use of SR as a drug carrier.

Prim-O-glucosycimifugin attenuates liver injury in septic mice by inhibiting NLRP3 inflammasome/caspase-1 signaling cascades in macrophages.

BACKGROUND: Liver dysfunction and liver failure are serious complications of sepsis, directly leading to septic progression and death. Now, there is no specific therapeutics available for sepsis-related liver dysfunction. Prim-O-glucosylcimifugin (POG), a chromone richest in the roots of Saposhnikovia divaricata (Turcz.) Schischk, is usually used to treat headache, rheumatoid arthritis and tetanus. While, the underlying mechanisms of POG against sepsis-induced liver damage and dysfunction are still not clear. PURPOSE: To study the anti-sepsis effect of POG, and its pharmacological mechanism to protect liver injury by weakening the function of macrophages in septic livers through inhibiting NOD-like receptor protein 3 (NLRP3) inflammasome pathway. METHOD: In vivo experiments, septic mouse model was induced by cecal ligation and puncture (CLP), and then the mortality was detected, liver inflammatory damages and plasma biomarkers of liver injury were evaluated by histopathological staining and biochemical assays, respectively. In vitro experiments, mouse primary peritoneal macrophages were treated with lipopolysaccharide (LPS) and ATP, and then the activated-inflammasomes, macrophage migration and polarization were detected by ASC immunofluorescence staining, transwell and flow cytometry assays, respectively. NLRP3 inflammasome components NLRP3, caspase-1, IL-1ß and IL-18 protein expressions were detected using western blot assays, and the contents of IL-1ß and IL-18 were measured by ELISA assays. RESULTS: POG treatment significantly decreased the mortality, liver inflammatory damages, hepatocyte apoptosis and plasma biomarkers of liver injury in CLP-challenged male WT mice, which were comparable to those in ibuprofen (a putative anti-inflammatory drug)-supplemented septic male WT mice and septic NLRP3 deficient-male mice. POG supplementation significantly suppressed NLRP3 inflammasome activation in septic liver tissues and cultured macrophages, by significantly reducing NLRP3, cleaved-caspase-1, IL-1ß and IL-18 levels, the activated-inflammasome ASC specks, and macrophage infiltration and migration, as well as M1-like polarization, but significantly increasing M2-like polarization. These findings were similar to the pharmacological effects of ibuprofen, NLRP3 deficiency, and a special NLRP3 inhibitor, MCC950. CONCLUSION: POG protected against sepsis by inhibiting NLRP3 inflammasome-mediated macrophage activation in septic liver and attenuating liver inflammatory injury, indicating that it may be a potential anti-sepsis drug candidate.

The Mechanisms of Yu Ping Feng San in Tracking the Cisplatin-Resistance by Regulating ATP-Binding Cassette Transporter and Glutathione S-Transferase in Lung Cancer Cells.

Cisplatin is one of the first line anti-cancer drugs prescribed for treatment of solid tumors; however, the chemotherapeutic drug resistance is still a major obstacle of cisplatin in treating cancers. Yu Ping Feng San (YPFS), a well-known ancient Chinese herbal combination formula consisting of Astragali Radix, Atractylodis Macrocephalae Rhizoma and Saposhnikoviae Radix, is prescribed as a herbal decoction to treat immune disorders in clinic. To understand the fast-onset action of YPFS as an anti-cancer drug to fight against the drug resistance of cisplatin, we provided detailed analyses of intracellular cisplatin accumulation, cell viability, and expressions and activities of ATP-binding cassette transporters and glutathione S-transferases (GSTs) in YPFS-treated lung cancer cell lines. In cultured A549 or its cisplatin-resistance A549/DDP cells, application of YPFS increased accumulation of intracellular cisplatin, resulting in lower cell viability. In parallel, the activities and expressions of ATP-binding cassette transporters and GSTs were down-regulated in the presence of YPFS. The expression of p65 subunit of NF-κB complex was reduced by treating the cultures with YPFS, leading to a high ratio of Bax/Bcl-2, i.e. increasing the rate of cell death. Prim-O-glucosylcimifugin, one of the abundant ingredients in YPFS, modulated the activity of GSTs, and then elevated cisplatin accumulation, resulting in increased cell apoptosis. The present result supports the notion of YPFS in reversing drug resistance of cisplatin in lung cancer cells by elevating of intracellular cisplatin, and the underlying mechanism may be down regulating the activities and expressions of ATP-binding cassette transporters and GSTs.

Simultaneous determination of calycosin, prim-O-glucosylcimifugin, and paeoniflorin in rat plasma by HPLC-MS/MS: application in the pharmacokinetic analysis of HQCF.

OBJECTIVE: This study aimed to develop and validate a high-performance liquid chromatography-tandem mass spectrometry method to simultaneously determine three bioactive components of the Huangqi Chifeng decoction (HQCF) in rat plasma. METHODS: Taxol was used as an internal standard in the developed method. Chromatographic separation was performed on a C18 column using a gradient elution with 0.1% formic acid in acetonitrile (v/v) and 0.1% formic acid in water (v/v) as the mobile phases at a flow rate of 0.4 mL·minute-1. All compounds were monitored via selected reaction monitoring with an electrospray ionization source. RESULTS: The lower limits of quantification of paeoniflorin, calycosin, and prim-O-glucosylcimifugin were 15.0, 0.75, and 0.75 ng·mL-1, respectively. The calibration curves indicated optimal linearity (r > 0.99) across the concentration ranges. The specificity, precision, accuracy, recovery, matrix effect, and stability of the method were validated. This method was successfully applied in a pharmacokinetics study of the three compounds in rat plasma. CONCLUSION: The pharmacokinetics results provide insights into the mechanisms of HQCF in vivo and its future clinical application.

Based on appearance and index components to study on standards of commodity classes of Saposhnikovia Radix / 中草药

Objective: A new table of standards of commodity classes of Saposhnikovia Radix is formed through literature market and local area survey. To explore the scientific nature and practicability of new standards of commodity classes of Saposhnikovia Radix, according to the appearance and index components related to quality of Saposhnikovia Radix, through statistical analysis and comparison of characteristic spectrum. Methods: Samples of different specifications and grades were collected from Northeast China, Inner Mongolia, Hebei, and the other main production areas, mainstream medicine market. Measuring samples appearance, extractum, characteristic spectrum of four chromones and analyzing data and discussing the relationship between standards of commodity classes and the appearance and index components to provide datas and theoretical support for the new table. Results: According to appearance character, the standards of commodity classes of Saposhnikovia Radix divided into three specifications of wild, imitates wild, cultivated were science based. At the same time, it is suggested to increase the content of the cimifugin as the index to distinguish between wild and cultivated Saposhnikovia Radix. Conclusion: There were significant differences in appearance and index components between different standards of Saposhnikovia Radix. Whether different classes of Saposhnikovia Radix only depend on size remains to be discussed.

Prim-O-glucosylcimifugin enhances the antitumour effect of PD-1 inhibition by targeting myeloid-derived suppressor cells.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that play an important role in immune evasion, PD-1/PD-L1 inhibitor tolerance and tumour progression. Therefore, MDSCs are potential targets for cancer immunotherapy. In this study, we screened an effective polymorphonuclear MDSC (PMN-MDSC) inhibitor from the Traditional Chinese Medicine Library and evaluated its synergistic antitumour effects with PD-1 inhibitor. METHODS: In the present study, we found that PMN-MDSCs accumulate heavily in the spleen and bone marrow of melanoma (B16-F10) tumour-bearing mice. Then, we determined the top 10 key proteins in the upregulated KEGG pathways of PMN-MDSCs in tumour-bearing mice through proteomics and Cytoscape analysis. The key proteins were then used as targets for the screening of PMN-MDSC inhibitors from the traditional Chinese Medicine Library (20000 compounds) through molecular docking and weight calculation of the docking score. Finally, the inhibitory effect of the inhibitor was verified through proteomics and metabolomics analysis in vitro and melanoma (B16-F10) and triple-negative breast cancer (4 T1) mouse tumour models in vivo. RESULTS: Traditional Chinese medicine saposhnikovia root extract Prim-O-glucosylcimifugin (POG) could bind well to the target proteins and inhibit the proliferation, metabolism and immunosuppressive ability of PMN-MDSCs by inhibiting arginine metabolism and the tricarboxylic acid cycle (TCA cycle). POG could also increase CD8 T-lymphocyte infiltration in the tumours and enhance the antitumour effect of PD-1 inhibitor in B16-F10 and 4 T1 mouse tumour models. CONCLUSIONS: POG was successfully screened from the traditional Chinese Medicine library as a PMN-MDSC inhibitor. POG exhibited a good synergistic antitumour effect with PD-1 inhibitor. This study provided a potential option for enhancing the efficacy of PD-1 inhibitors in clinical applications.

The Effect of Prim-O-Glucosylcimifugin on Tryptase-Induced Intestinal Barrier Dysfunction in Caco-2 Cells.

The intestinal barrier dysfunction is a critical pathological change in irritable bowel syndrome (IBS). The objective of this study was to evaluate the effect of Prim-O-glucosylcimifugin (POG) on intestinal barrier dysfunction and reveal possible molecular mechanisms. Human colon adenocarcinoma cell line (Caco-2) cell monolayers induced by tryptase (TRYP) were used to establish an intestinal barrier dysfunction model. Caco-2 cell monolayers from both functional and dysfunctional samples were treated with POG (30, 60 and 120 µg/mL) for 2, 8, 24, 36, 48 and 72 h. The Caco-2 cell monolayers were assessed by measurement of trans-epithelial electrical resistance (TEER) and percentage of fluorescein permeation (PFP). The expression of Protease Activated Receptor 2 (PAR-2) and myosin light chain kinase (MLCK) mRNA was analyzed by RT-PCR and the level of Zonula Occludens-1 (ZO-1) protein expression was determined by Western blot. In addition, the impact of POG on the distribution of the tight juction protein of Occludin was performed by immunofluorescence. Our results showed that POG elevated the TEER and decreased the PFP of the functional Caco-2 cell monolayers, as well as the dysfunctional Caco-2 cell monolayers. Furthermore, POG inhibited the expression of PAR-2 mRNA and MLCK mRNA and increased the level of ZO-1 protein expression in dysfunctional Caco-2 cells. The distribution of the Occludin proteins was ameliorated simultaneously. This study demonstrates that POG can enhance the intestinal barrier function of Caco-2 cell monolayers by inhibiting the expression of PAR-2 and MLCK and up-regulating the expression of ZO-1 protein, and ameliorated the distribution of Occludin protein.

[Nasal resorption of prim-O-glucosylcimifugin and 5-O-methylvisammioside in rats].

In this experiment, rat nasal mucosa absorption characteristics of prim-O-glucosylcimifugin and 5-O-methylvisammioside were studied to provide a basis for drug delivery of Toutongning nasal spray. The nasal mucosa absorption test in rats was conducted with in situ nasal perfusion method after pH 6 buffer solution was used to prepare high, medium and low concentrations of prim-O-glucosylcimifugin, 5-O-methylvisammioside mixed solution as liquid circulation in nasal cavity. Then the concentrations of the circulating liquid compositions to be measured were determined by HPLC, and the absorption rates of prim-O-glucosylcimifugin and 5-O-methylvisammioside under different pH conditions were also investigated. According to the results, the absorption rate constant was (0.588±0.041)×10⁻³, (0.547±0.023)×10⁻³, (0.592±0.063)×10⁻³ min⁻¹ for prim-O-glucosylcimifugin high, middle and low concentrations, and (0.438±0.041)×10⁻³, (0.407±0.023)×10⁻³, and (0.412±0.063)×10⁻³ min⁻¹ for 5-O-methylvisammioside high, middle and low concentrations. There was no significant difference among high, middle and low concentration groups, and the absorption under pH 6 was better than that under other pH conditions. Therefore, we can get the conclusion that the main active ingredient of Toutongning nasal sprays can be absorbed through the nasal mucosa, and it is feasible to make nasal spray; in addition, pH 6 of nasal spray is scientific and reasonable.

Prim-O-glucosylcimifugin Attenuates Lipopolysaccharideinduced Inflammatory Response in RAW 264.7 Macrophages.

BACKGROUND: Radix Saposhnikoviae (RS) exerts anti-inflammatory, analgesic, antipyretic, antioxidation effects and has been used in traditional Chinese medicine to treat common colds, headache, and rheumatoid arthritis. Prim-O-glucosylcimifugin (POG) is the highest content chromone and one of the major active constituents in RS. OBJECTIVE: The study was aimed to explore the anti-inflammation effects of POG in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. MATERIALS AND METHODS: Cell viability was detected by Cell Counting Kit-8 assay. Production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 was assessed by enzyme-linked immunosorbent assay. Real-time polymerase chain reaction and Western blot were performed to analyze mRNA and protein levels, respectively. RESULTS: During the whole experiment, 15, 50, and 100 µg/mL of POG had no cytotoxicity on RAW 264.7 cells. POG dose-dependently inhibited the production of NO, TNF-α, IL-1ß, and IL-6 that were induced by LPS. POG treatment downregulated the mRNA and protein expression inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) in LPS-activated RAW 264.7 macrophages in a concentration-dependent manner. Furthermore, LPS-induced JAK2/STAT3 activation was prevented in RAW 264.7 macrophages by POG treatment. STAT3 overexpression significantly reversed the effects of POG on LPS-activated RAW 264.7 macrophages. CONCLUSION: These results demonstrate that POG exerts anti-inflammatory effects through the inhibition of iNOS and COX-2 expression by inhibiting the phosphorylation of JAK2/STAT3. SUMMARY: POG exerts anti-inflammatory effects in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 expression by inhibiting JAK2/STAT3 signaling. Abbreviations used: LPS: Lipopolyssacharide; NO: Nitric oxide; TNF-α: Tumor necrosis factor-α; IL: Interleukin; RS: Radix Saposhnikoviae; POG: Prim-O-glucosylcimifugin; iNOS: Inducible NO synthase; COX2: Cyclooxygenase; FBS: Fetal bovine serum; DMSO: Dimethylsulfoxide; CCK-8: Cell Counting Kit; RIPA: Radio immunoprecipitation assay buffer; ECL: Enhanced chemiluminescence; SD: Standard deviation; ELISA: Enzyme-Linked immunosorbent assay.

Study on Quality Standards for Shufeng Zhike Extract / 中国中医药信息杂志

Objective To establish the quality standards for Shufeng Zhike Extract;To improve the controll ability of the extract and ensure medicine efficacy. Methods To establish a HPLC method for simultaneous differentiation of Paeoniae Radix Alba, Saposhnikoviae Radix, Polygoni Cuspidati Rhizomaet Radix, Glycyrrhizae Radix et Rhizoma, Belamcandae Rhizoma and Bupleurum in Shufeng Zhike Extract. Phenomenex Kintex C18 column (4.6 mm×100 mm, 2.6 μm) was used. Acetonitrile-0.05%phosphoric acid was as the mobile phase in gradient elution (0–25 min, 10%acetonitrile; 25–26 min, 10%→14% acetonitrile; 26–35 min, 14% acetonitrile; 35–36 min, 14%→34% acetonitrile;36–55 min, 34%acetonitrile) at flow rate of 0.8 mL/min, and column temperature was 40 ℃. The detection wavelength was 250 nm. To establish a HPLC method for simultaneous determination of Paeoniflorin and Prim-O-glucosylcimifugin in Shufeng Zhike Extract, Phenomenex Luna C18 column (4.6 mm×150 mm, 5 μm) was used; acetonitrile-0.05%phosphoric acid (12:88) was as the mobile phase at flow rate of 1 mL/min; column temperature was 40 ℃; the detection wavelength was 250 nm. Results The chromatographic peak separation with HPLC method for simultaneous differentiate of Paeoniae Radix Alba, Saposhnikoviae Radix, Polygoni Cuspidati Rhizoma et Radix, Glycyrrhizae Radix et Rhizoma, Belamcandae Rhizoma and Bupleurumin in Shufeng Zhike Extract was clear and negative control had no interference. The linear range of Paeoniflorin was 0.035 93–2.514 8 μg (r=0.999 5), and the average recovery was 100.54%(n=6). The linear range of Prim-O-glucosylcimifugin was 0.006 7–0.67 μg (r=0.999 5), and the average recovery was 100.39%(n=6). Conclusion The established quality standards are simple, reliable, and accurate. It can rapidly identificate Paeoniae Radix Alba, Saposhnikoviae Radix, Polygoni Cuspidati Rhizomaet Radix, Glycyrrhizae Radix et Rhizoma, Belamcandae Rhizoma and Bupleurum, and suitable for rapid determination of Paeoniflorin and Prim-O-glucosylcimifugin, which can provide the basis for the quality inspection of Shufeng Zhike Extract.

HPLC fingerprint and determination of multi-components in Chitong Xiaoyanling Granules / 中草药

Objective To establish an HPLC fingerprint and to determine six compounds in Chitong Xiaoyanling Granules (CXG) for reference of the effective quality control. Methods The analysis was carried out on an analytical column Dikma Luster ODS (250 mm × 4.6 mm, 5 μm) with gradient elution by methanol (A)-0.1% phosphoric acid solution (B) (0-15 min, 20%-30% A; 15-30 min, 30% A; 30-40 min, 30%-60% A; 40-55 min, 60% A), at the detection wavelengths of 254, 283, 274, and 300 nm and a flow rate of 1.0 mL/min. The column temperature was 30 ℃. Similarity evaluation software was used to evaluate the fingerprint of 10 batches of CXG, and the six marker components were quantified. Results The common mode of the fingerprint was set up with 18 common peaks, and six of them were identified by comparison with the reference. The similar degrees of 10 batches of samples were over 0.9, they were prim-O-glucosylcimifugin, 4'-O-β-D-glucosyl-5-O-methylvisamminol, pulegone, hesperidin, paeonol, and isoimperatorin. The linear ranges were 0.013-0.505 mg/mL (r = 0.999 8), 0.052-2.097 mg/mL (r = 0.999 2), 0.019-0.772 mg/mL (r = 0.998 9), 0.025-1.003 mg/mL (r = 0.999 1), 0.006-0.251 mg/mL (r = 0.999 5), and 0.014-0.576 mg/mL (r = 0.999 4) for prim- O-glucosylcimifugin, 4'-O-β-D-glucosyl-5-O-methylvisamminol, pulegone, hesperidin, paeonol, and isoimperatorin, respectively. The contents of prim-O-glucosylcimifugin, 4'-O-β-D-glucosyl-5-O-methylvisamminol, pulegone, hesperidin, paeonol, and isoimperatorin were 7.267-7.333, 4.260-4.522, 2.033-2.093, 12.234-12.771, 19.023-19.334, and 11.152-11.291 mg/g in 10 batches of samples, respectively. Conclusion The established method has high sensitivity and specificity, and can be used for the quality control of CXG.

Nasal resorption of prim-O-glucosylcimifugin and 5-O-methylvisammioside in rats / 中国中药杂志

In this experiment, rat nasal mucosa absorption characteristics of prim-O-glucosylcimifugin and 5-O-methylvisammioside were studied to provide a basis for drug delivery of Toutongning nasal spray. The nasal mucosa absorption test in rats was conducted with in situ nasal perfusion method after pH 6 buffer solution was used to prepare high, medium and low concentrations of prim-O-glucosylcimifugin, 5-O-methylvisammioside mixed solution as liquid circulation in nasal cavity. Then the concentrations of the circulating liquid compositions to be measured were determined by HPLC, and the absorption rates of prim-O-glucosylcimifugin and 5-O-methylvisammioside under different pH conditions were also investigated. According to the results, the absorption rate constant was (0.588±0.041)×10⁻³, (0.547±0.023)×10⁻³, (0.592±0.063)×10⁻³ min⁻¹ for prim-O-glucosylcimifugin high, middle and low concentrations, and (0.438±0.041)×10⁻³, (0.407±0.023)×10⁻³, and (0.412±0.063)×10⁻³ min⁻¹ for 5-O-methylvisammioside high, middle and low concentrations. There was no significant difference among high, middle and low concentration groups, and the absorption under pH 6 was better than that under other pH conditions. Therefore, we can get the conclusion that the main active ingredient of Toutongning nasal sprays can be absorbed through the nasal mucosa, and it is feasible to make nasal spray; in addition, pH 6 of nasal spray is scientific and reasonable.

Simultaneous Determination of Gastrodin, Prim-O-glucosylcimifugin, 5-O-Methylvisammioside, Imperatorin and Isoimperatorin in Yuzhen Powder by HPLC / 中国药师

Objective: To establish an HPLC method for the determination of gastrodin, prim-O-glucosylcimifugin, 5-O-methylvisammioside, imperatorin and isoimperatorin in Yuzhen powder.Methods: A Phenomenex Gemini C18 column(250 mm×4.6 mm, 5μm) was used and the column temperature was 25℃.The flow rate was 1.0 ml·min-1 and the mobile phase consisted of methanol-water with gradient elution.The detection wavelength was 230 nm and the sample size was 5 μl.Results: The linear range of gastrodin was 2.68-214.0 μg·ml-1(r=0.999 9), and the average recovery was 100.2%(RSD=0.9%, n=6);the linear range of prim-O-glucosylcimifugin was 5.22-418.0 μg·ml-1(r=0.999 7), and the average recovery was 99.9%(RSD=0.9%, n=6);the linear range of 5-O-methylvisammioside was 4.57-365.8 μg·ml-1(r=0.999 5), and the average recovery was 99.7%(RSD=1.0%, n=6);the linear range of imperatorin was 5.22-417.2 μg·ml-1(r=0.999 6), and the average recovery was 99.0%(RSD=0.9%, n=6);the linear range of isoimperatorin was 5.29-423.2 μg·ml-1(r=0.999 8), and the average recovery was 100.2%(RSD=0.8%, n=6).Conclusion: The method is simple and accurate, and the contents of the five ingredients can be determined simultaneously.The method can be used for the quality control of Yuzhen powder.

Gu-Ben-Fang-Xiao decoction attenuates sustained airway inflammation by suppressing ER stress response in a murine asthma remission model of respiratory syncytial virus infection.

ETHNOPHARMACOLOGICAL RELEVANCE: In recent years, asthma has increased dramatically in prevalence with a considerable economic burden all over the world. Long-term remission should be regarded as the promising and meaningful therapeutic goal in asthma management. However, the precise definition criteria and rational therapies for asthma remission have not been well-established. In academia, there is a consensus that even in those who develop asymptomatic remission of asthma, persistent airway inflammation is ubiquitous. Gubenfangxiao decoction (GBFXD) has been widely used in treating asthma remission stage for decades in the Jiangsu Province Hospital of Chinese Medicine, China. We previously demonstrated that GBFXD could downregulate the asthma susceptibility gene ORMDL3, a trigger of Endoplasmic reticulum (ER) stress and unfolded protein response (UPR). AIM THIS STUDY: To investigate the involvement of ER stress and UPR in the anti-inflammatory effects of GBFXD in Respiratory Syncytial Virus (RSV)-OVA-induced asthma remission mice. MATERIALS AND METHODS: Mice were orally administered GBFXD at three doses for 30 days after an RSV-OVA challenge. The levels of inflammation mediators in serum were measured using a Luminex assay and the amount of IFN-γ in lung homogenates was detected using ELISA. The splenic CD4+ and CD8+ T lymphocytes were counted using flow cytometric analysis. The mRNA and protein levels of asthma susceptibility gene ORMDL3, ER stress markers (BIP, CHOP), and three canonical UPR branches (PERK-eIF2a-ATF4, IRE1α-XBP1/IRE1α-JNK-AP1 and ATF6-SERCA2b signal pathways) were detected using real-time RT-PCR and western blot. RESULTS: Histopathological analysis showed that the model group mice still exhibited a sustained airway inflammation even after suspending the OVA-challenge and RSV infections for 30 days. H&E staining results indicated that GBFXD could attenuate sustained airway inflammation. Decreased serum CXCL1 level and increased IFN-γ level in lung homogenate were observed after GBFXD treatment. Reductions in the number of splenic CD4+/CD8+ T lymphocytes were found after DEX treatment. We further confirmed the previous finding that GBFXD could downregulate the expression of ORMDL3. As a result of suppressed UPR, decreased ER stress markers and inhibited UPR branches (PERK and IRE1α signal pathway) were also observed through the significant reduction of signature mRNA and protein expressions after GBFXD treatment. CONCLUSION: GBFXD can significantly attenuate RSV-OVA-induced persistent airway inflammation in murine asthma remission model. These effects may be mediated, at least partially, by inhibiting the activation of ER stress responses.

Antinociceptive Effects of Prim-O-Glucosylcimifugin in Inflammatory Nociception via Reducing Spinal COX-2.

We measured anti-nociceptive activity of prim-o-glucosylcimifugin (POG), a molecule from Saposhnikovia divaricate (Turcz) Schischk. Anti-nociceptive or anti-inflammatory effects of POG on a formalin-induced tonic nociceptive response and a complete Freund's adjuvant (CFA) inoculation-induced rat arthritis pain model were studied. Single subcutaneous injections of POG produced potent anti-nociception in both models that was comparable to indomethacin analgesia. Anti-nociceptive activity of POG was dose-dependent, maximally reducing pain 56.6% with an ED50 of 1.6 mg. Rats given POG over time did not develop tolerance. POG also time-dependently reduced serum TNFα, IL-1ß and IL-6 in arthritic rats and both POG and indomethacin reduced spinal prostaglandin E2 (PGE2). Like indomethacin which inhibits cyclooxygenase-2 (COX-2) activity, POG dose-dependently decreased spinal COX-2 content in arthritic rats. Additionally, POG, and its metabolite cimifugin, downregulated COX-2 expression in vitro. Thus, POG produced potent anti-nociception by downregulating spinal COX-2 expression.

Metabolism studies on prim-O-glucosylcimifugin and cimifugin in human liver microsomes by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Prim-O-glucosylcimifugin (PGCN) and cimifugin (CN) are major constituents of Radix Saposhnikoviae that have antipyretic, analgesic and anti-inflammatory pharmacological activities. However, there were few reports with respect to the metabolism of PGCN and CN in vitro. In this paper, we describe a strategy using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) for fast analysis of the metabolic profile of PGCN and CN in human liver microsomes. In total, five phase I metabolites of PGCN, seven phase I metabolites and two phase II metabolites of CN were identified in the incubation of human liver microsomes. The results revealed that the main phase I metabolic pathways of PGCN were hydroxylation and hydrolysis reactions. The phase I metabolic pathways of CN were found to be hydroxylation, demethylation and dehydrogenation. Meanwhile, the results indicated that O-glucuronidation was the major metabolic pathway of CN in phase II metabolism. The specific UDP-glucuronosyltransferase (UGT) enzymes responsible for CN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A9, UGT2B4 and UGT2B7 might play major roles in the glucuronidation of CN. Overall, this study may be useful for the investigation of metabolic mechanism of PGCN and CN, and it can provide reference and evidence for further pharmacodynamic experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Antinociceptive Effects of Prim-O-Glucosylcimifugin in Inflammatory Nociception via Reducing Spinal COX-2

We measured anti-nociceptive activity of prim-o-glucosylcimifugin (POG), a molecule from Saposhnikovia divaricate (Turcz) Schischk. Anti-nociceptive or anti-inflammatory effects of POG on a formalin-induced tonic nociceptive response and a complete Freund's adjuvant (CFA) inoculation-induced rat arthritis pain model were studied. Single subcutaneous injections of POG produced potent anti-nociception in both models that was comparable to indomethacin analgesia. Anti-nociceptive activity of POG was dose-dependent, maximally reducing pain 56.6% with an ED₅₀ of 1.6 mg. Rats given POG over time did not develop tolerance. POG also time-dependently reduced serum TNFα, IL-1β and IL-6 in arthritic rats and both POG and indomethacin reduced spinal prostaglandin E2 (PGE₂). Like indomethacin which inhibits cyclooxygenase-2 (COX-2) activity, POG dose-dependently decreased spinal COX-2 content in arthritic rats. Additionally, POG, and its metabolite cimifugin, downregulated COX-2 expression in vitro. Thus, POG produced potent anti-nociception by downregulating spinal COX-2 expression.

Simultaneous Determination of Asarinin, Prim-O-glucosylcimifugin and 5-O-Methylvisammioside in Xinqin Granules by HPLC / 中国药师

Objective:To simultaneously determine the contents of asarinin, prim-O-glucosylcimifugin and 5-O-methylvisammio-side in Xinqin granules. Methods:An HPLC method was used. The determination was performed on a ZORBAX Eclipse XDB-C18 col-umn(150 mm ×4.6mm,5 μm) with the mobile phase consisting of menthol (A)-water (B) with gradient elution. The flow rate was 1. 0 ml·min-1 . The column temperature was 30℃. The detection wavelength was set at 254 nm from 0 to 30 min and 287nm from 30 to 55 min. The injection volume was 10μl. Results:The linear range of prim-O-glucosylcimifugin, asarinin and 5-O-methylvisammio-side was 10.210-163.400 μg·ml-1(r=0.999 7),10.160-162.600 μg·ml-1(r=0.999 8) and 5.015-80.240 μg·ml-1(r=0. 999 8), respectively. The average recovery was 100. 30%(RSD=1. 6%, n=6),101. 53%(RSD=1. 1%,n=6) and 101. 12%(RSD=1. 2%, n=6), respectively. Conclusion: The method is simple and accurate, which can be used in the quality control of Xinqin granules.

Simultaneous determination of seven compounds in Fangzhi nasopharyngeal granules by HPLC / 中国药学杂志

OBJECTIVE: To develop a method for simultaneous determination of gentiopicroside, prim-O-glucosylcimifugin, calycosin-7-O-β-D-glucoside, paeonol, magnolin, imperatorin and isoimperatorin in Fangzhi nasopharyngeal granules by HPLC.

Determination of Prim-O-glucosylcimifugin in Biyan Tablets by HPLC / 中药新药与临床药理

Objective To establish the determination of prim-O-glucosylcimifugin in Biyan Tablets by HPLC.Methods The HPLC determination was carried out on Diamonsil C18 column(4.6 mm? 250 mm,5 ? m).The mobile phase consisted of methanol-acetonirile-H2O(18 :12 :70) with a flow rate of 1.0 mL/min,and the ultraviolet detection wavelength was set at 254 nm.Results Prim-O-glucosylcimifugin showed a good linearity in the range of 0.1888～ 1.534 ? g with the peak area score,the coefficient was 0.9997,and the average recovery was 101.74 %(RSD=2.97 %).Conclusion The method is accurate and reliable,and can be used for the quality control of Biyan Tablets.