Swertiamarin supplementation prevents obesity-related chronic inflammation and insulin resistance in mice fed a high-fat diet.

Obesity is characterized by low-grade chronic inflammation, which underlies insulin resistance and non-alcoholic fatty liver disease (NAFLD). Swertiamarin is a secoiridoid glycoside that has been reported to ameliorate diabetes and NAFLD in animal models. However, the effects of swertiamarin on obesity-related inflammation and insulin resistance have not been fully elucidated. Thus, this study investigated the effects of swertiamarin on inflammation and insulin resistance in high-fat diet (HFD)-induced obese mice. C57BL/6 mice were fed a HFD or HFD containing swertiamarin for 8 weeks. Obesity-induced insulin resistance and inflammation were assessed in the epididymal white adipose tissue (eWAT) and livers of the mice. Swertiamarin attenuated HFD-induced weight gain, glucose intolerance, oxidative stress, and insulin resistance, and enhanced insulin signalling in mice. Compared to HFD-fed mice, the swertiamarin-treated mice exhibited increased lipolysis and reduced adipocyte hypertrophy and macrophage infiltration in eWAT. Moreover, swertiamarin alleviated HFD-mediated hepatic steatosis and inflammation by suppressing activation of the p38 MAPK and NF-κB pathways within the eWAT and liver of obese mice. In conclusion, supplementation with swertiamarin attenuated weight gain and hepatic steatosis, and alleviated obesity-associated inflammation and insulin resistance, in obese mice.

Simultaneous determination of multiple components in rat plasma and pharmacokinetic studies at a pharmacodynamic dose of Xian-Ling-Gu-Bao capsule by UPLC-MS/MS.

Xian-Ling-Gu-Bao capsule (XLGB) is an effective traditional Chinese medicine prescription (TCMP) that is used for the prevention and treatment of osteoporosis in China. A rapid, simple, efficient and stable method based on UPLC-MS/MS technology was developed for simultaneous determination of multiple components of XLGB in rat plasma. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). For twenty-one selected quantitative prototypes, all calibration curves showed favourable linearity (r>0.9932) in linear ranges. The lower limits of quantification (LLOQs) were 2 ng/mL for psoralen (PL), 2.5 ng/mL for asperosaponin VI (AS), 1 ng/mL for isopsoralen (IPS) and sweroside (SW), 0.5 ng/mL for magnoflorine (MA), bavachinin (BVN), tanshinone IIA (TA), timosaponin BII (TBII) and icaritin (ICT), 0.1 ng/mL for epimedin B (EB) and epimedin C (EC), 0.05 ng/mL for icariin (IC), isobavachalcone (IBC), psoralidin (PD), bavachin (BV), bavachalcone (BC), epimedin A (EA) and isobavachin (IBV), 0.02 ng/mL for neobavaisoflavone (NEO) and icariside I (ICI) and 0.01 ng/mL for icariside II (ICII). The intra-day and inter-day (low, medium, high) precision (relative standard deviation) for all analytes was less than 8.63%, and the accuracies (as relative error) were in the range of -12.45% to 8.91%. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The validated method was successfully applied to the pharmacokinetics (PK) studies of the twenty-one prototypes at pharmacodynamic doses (0.3 and 1 g/kg/day). In addition, dynamic profiles of 28 metabolites (phase II conjugates: 23 glucuronide conjugates, 2 sulfate conjugates and 3 glucuronide or sulfate conjugates) were also monitored by their area/IS area-time curves. As a result, coumarins, prenylated flavonoids from Psoraleae Fructus, alkaloids and prenylated flavonol glycosides from Epimedii Herba, and iridoid glycosides, triterpenoid saponins from Dipsaci Asperoidis Radix were considered to be the key effective substances of XLGB due to their high exposure and appropriate pharmacokinetic features. This is the first report to reveal pharmacodynamic ingredients by a reversed pharmacodynamic (PD) - pharmacokinetics (PK) study.

Gentiopicrin exerts anti-rheumatic effect in human fibroblast-like synoviocytes via inhibition of p38MAPK/NF-κB pathway.

To investigate the effect of gentiopicrin on the expressions of inflammatory factors in human fibroblast-like synoviocytes (HFLS) and the underlying mechanism. Human fibroblast-like synoviocytes (HFLS) were cultured in vitro at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % fetal bovine serum (FBS) in a humidified incubator containing 5 % CO2. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-tetrazolium bromide (MTT) assay, while real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the expressions of interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) mRNAs. The expressions of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) were determined using Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IL-1ß and IL-6 in cell lysate. Treatment with 5-25 µM gentiopicrin did not significantly affect the number of viable cells, when compared with control group (p > 0.05). However, at 50 and 100 µM gentiopicrin, the number of viable cells were significantly increased, relative to control group (p < 0.05). Results of qRT-PCR showed that the expression levels of IL-1ß and IL-6 mRNAs were significantly higher in TNF-α group than in control group (p < 0.05). However, treatment with gentiopicrin significantly and dose-dependently decreased their expression levels compared with TNF-α group (p < 0.05). Western blotting results showed that the expressions of p-p38MAPK and NF-κB-p65 proteins were significantly upregulated in TNF-α group, when compared with control group (p < 0.05). However, treatment with gentiopicrin significantly and dose-dependently down-regulated the expression of these proteins compared with TNF-α group (p < 0.05). The levels of IL-1ß and IL-6 in cell lysate were significantly higher in TNF-α group than in control group (p < 0.05). However, treatment with gentiopicrin, and p38MAPK/NF-κB pathway inhibitors (SB203580 and BAY11-7082) significantly reduced the levels of these inflammatory factors compared with TNF-α group (p < 0.05).  Gentiopicrin has therapeutic potential for Rheumatoid arthritis (RA  ) through a mechanism involving the inhibition of p38MAPK/NF-κB pathway.

Sweroside Alleviated LPS-Induced Inflammation via SIRT1 Mediating NF-κB and FOXO1 Signaling Pathways in RAW264.7 Cells.

Pterocephalus hookeri was used as a traditional Chinese medicine for the treatment of rheumatoid arthritis. Sweroside was a main iridoid isolated from P. hookeri. The present study aimed to investigate the anti-inflammatory effect mechanism of sweroside. In RAW264.7 cells induced by lipopolysaccharide (LPS), the abnormal proliferation, the NO content increase, and the downregulated Sirtuin1 (SIRT1) expression were observed. Sweroside could alleviate the inflammation by inhibiting cell proliferation through arresting the cell cycle at the G0/G1 phase, by suppressing pro-inflammatory cytokines and by promoting anti-inflammatory cytokines in LPS-induced RAW264.7 cells. Further mechanism research indicated that sweroside could activate the SIRT1, then suppress the nuclear factor-kappa B (NF-κB) and promote the Forkhead transcription factor O1 (FOXO1) signaling pathways. The present study indicated that sweroside may be the main anti-inflammatory constituent of P. hookeri and a promising candidate for anti-inflammation therapy.

The therapeutic effect of aucubin-supplemented hyaluronic acid on interleukin-1beta-stimulated human articular chondrocytes.

BACKGROUND: Injection of exogenous hyaluronic acid (HA) into the joint capsule improves symptoms of early stage osteoarthritis (OA). However, reactive oxygen species degrade HA into small oligosaccharides that can elicit pro-inflammatory responses. Likewise, disturbance of the antioxidant enzyme system and generation of oxidative stress by pro-inflammatory cytokines worsen knee OA. Accordingly, we proposed the use of aucubin, an antioxidant and anti-inflammatory compound, as a versatile adjuvant to HA for treating OA. METHODS: Primary human chondrocytes were cultured in media supplemented with aucubin in a series of concentrations (0, 0.01, 0.1, 1, and 10 µg/ml) to study dose-dependent toxicity. We then evaluated the therapeutic effects of HA (100 µg/ml) supplemented with aucubin (10 µg/ml) on interleukin-1 beta (IL-1ß, 10 ng/ml)-stimulated chondrocytes. RESULTS: The use of aucubin did not change cell viability or alter lactate dehydrogenase release to normal chondrocytes. Although the proliferation and sulfated glycosaminoglycan production were not affected, aucubin partially restored the hypertrophic transformation of chondrocytes. Relative to treatment with HA or aucubin alone, real-time PCR revealed that aucubin-supplemented HA down-regulated the mRNA levels of tumor necrosis factor-alpha (TNF-α), corrected collagen type 1 and aggrecan, and up-regulated tissue inhibitor of metalloproteinase 1. Moreover, ELISA testing also showed a reduced TNF-α production. Although superoxide dismutases activity was still distributed, aucubin restored total antioxidant capacity of IL-1ß-stimulated chondrocytes. Western blotting further showed that aucubin inhibited cyclooxygenase-2 and regulated the nuclear factor (erythroid-derived 2)-like 2 pathway. CONCLUSION: Aucubin can enhance the anti-catabolic and anti-inflammatory effects of HA on OA chondrocytes.

Global gene expression analysis in liver of db/db mice treated with catalpol.

Catalpol, a major bioactive component from Rehmannia glutinosa, which has been used to treat diabetes. The present study was designed to elucidate the anti-diabetic effect and mechanism of action for catalpol in db/db mice. The db/db mice were randomly divided into six groups (10/group) according to their blood glucose levels: db/db control, metformin (positive control), and four dose levels of catalpol treatment (25, 50, 100, and 200 mg·kg-1), and 10 db/m mice were used as the normal control. All the groups were administered orally for 8 weeks. The levels of fasting blood glucose (FBG), random blood glucose (RBG), glucose tolerance, insulin tolerance, and glycated serum protein (GSP) and the globe gene expression in liver tissues were analyzed. Our results showed that catalpol treatment obviously reduced water intake and food intake in a dose-dependent manner. Catalpol treatment also remarkably reduce fasting blood glucose (FBG) and random blood glucose (RBG) in a dose-dependent manner. The RBG-lowering effect of catalpol was better than that of metformin. Furthermore, catalpol significantly improved glucose tolerance and insulin tolerance via increasing insulin sensitivity. Catalpol treatment significantly decreased GSP level. The comparisons of gene expression in liver tissues among normal control mice, db/db mice and catalpol treated mice (200 and 100 mg·kg-1) indicated that there were significant increases in the expressions of 287 genes, whichwere mainly involved in lipid metabolism, response to stress, energy metabolism, and cellular processes, and significant decreases in the expressions of 520 genes, which were mainly involved in cell growth, death, immune system, and response to stress. Four genes expressed differentially were linked to glucose metabolism or insulin signaling pathways, including Irs1 (insulin receptor substrate 1), Idh2 (isocitrate dehydrogenase 2 (NADP+), mitochondrial), G6pd2 (glucose-6-phosphate dehydrogenase 2), and SOCS3 (suppressor of cytokine signaling 3). In conclusion, catalpol ecerted significant hypoglycemic effect and remarkable therapeutic effect in db/db mice via modulating various gene expressions.

Gentiopicroside protects neurons from astrocyte-mediated inflammatory injuries by inhibition of nuclear factor-κB and mitogen-activated protein kinase signaling pathways.

The effects of gentiopicroside (Gent), an active component derived from the traditional Chinese medicine Gentiana macrophylla, on lipopolysaccharide-induced astrocyte activation and subsequent neuronal damage were investigated. Gent significantly inhibited the release of tumor necrosis factor-α, interleukin-1ß, nitric oxide, and prostaglandin E, as well as expressions of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-induced primary astrocytes. Furthermore, Gent relieved neurotoxicity from astrocyte-mediated inflammatory injury. Mechanism studies indicated that Gent significantly suppressed nuclear factor-κB nuclear translocation and down-regulated c-Jun-N-terminal kinase/stress-activated protein kinase mitogen-activated protein kinase phosphorylation levels with little influence on elevated p-p38 levels. Taken together, our findings suggested Gent could prevent the neurotoxicity related to astrocyte-mediated inflammatory injury by inhibition of nuclear factor-κB and mitogen-activated protein kinase signaling pathways. The study also indicated that neuronal injury could be prevented by promptly modulating inflammatory responses of astrocytes.

Gentiopicroside ameliorates bleomycin-induced pulmonary fibrosis in mice via inhibiting inflammatory and fibrotic process.

Pulmonary fibrosis (PF) is a chronic and ultimately fatal interstitial lung disease of various causes. The advent of nintedanib and pirfenidone provides treatment options for PF patients for the first time. However, the adverse effects of the two drugs such as gastrointestinal disorders and hepatic dysfunction often lead to treatment discontinuation. Gentiopicroside (GPS) is a natural secoiridoid glycoside from gentian species of medicinal plants, and has a variety of pharmacological activities, including hepatoprotective and cholagogic, anti-inflammatory, antinociceptive, and smooth muscle relaxing activities. The present study aimed to investigate the therapeutical effects of GPS on bleomycin (BLM)-induced PF in mice. Severe lung inflammation and fibrosis were observed in BLM-treated mice. GPS significantly ameliorated inflammatory and fibrotic responses in lungs of PF mice which were confirmed by histopathological examinations including light microscopy and transmission electron microscopy. Additionally, GPS significantly decreased the levels of inflammatory cytokines including TNF-α and IL-1ß in bronchoalveolar lavage fluid and reduced the content of hydroxyproline in lungs of PF mice. Furthermore, GPS significantly downregulated the expression of TGF-ß1 and CTGF in lungs of PF mice. In vitro, GPS inhibited epithelial-mesenchymal transition of A549 cells stimulated by TGF-ß1, in a dose-dependent manner. Our findings suggest that GPS has the potential as an ideal drug candidate for PF, as it has both anti-inflammatory and anti-fibrotic effects. Alveolar epithelial cells and TGF-ß1 may be the main target cells and molecule of GPS on BLM-induced PF, respectively.

The anti-hyperplasia, anti-oxidative and anti-inflammatory properties of Qing Ye Dan and swertiamarin in testosterone-induced benign prostatic hyperplasia in rats.

Qing Ye Dan (QYD) is the whole plant of Swertia mileensis and used in Chinese folk medicine for the treatment of prostatitis, benign prostatic hyperplasia (BPH) and so on. This study was to investigate the effects of QYD and its main component swertiamarin on BPH induced by testosterone in rats. The prostatic expressions of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (ßFGF) and proliferating cell nuclear antigen (PCNA) were detected by immunohistochemistry assay. Prostatic levels of oxidative stress and inflammatory-related factors were also analyzed. Additionally, the prostatic expressions of androgen receptor (AR), estrogen receptor (ER)-α, ER-ß, hypoxia-inducible factor (HIF)-1α, B-cell CLL/lymphoma (Bcl)-2 and Bcl-2-associated X protein (Bax) were measured by western blot. The epithelial-mesenchymal transition (EMT) associated factors were evaluated by quantitative RT-PCR. It showed that QYD and swertiamarin ameliorated the testosterone-induced prostatic hyperplasia and collagen deposition, attenuated the over-expressions of HIF-1α, VEGF, EGF, ßFGF, PCNA, AR and ER-α, reduced the ratio of Bcl-2/Bax, enhanced the expression of ER-ß, inhibited the oxidative stress and local inflammation, as well as relieved prostatic EMT. It suggested that QYD and swertiamarin had prostatic protective potential against BPH.

[Effects of different preparation technologies on concentrations of puerarin and catalpol in plasma and brain of rats after oral administration].

To compare the effects of different preparation technologies on the concentrations of puerarin and catalpol in plasma and brain of rats after oral administration, in order to lay an experimental basis for developing new oral Zige preparations. The nanocrystal, self-microemulsions (tween-80 and Cremophor RH-40 as emulsifiers) and inclusion complex of HP-ß-CD containing puerarin and catalpol were prepared. The concentrations of puerarin and catalpol in plasma and brain of rats after oral administration were determined by HPLC-MS/MS method. The pharmacokinetic parameters and brain target index were compared. The results showed that preparation technologies had different influences on the concentrations of puerarin and catalpol in plasma and brain. The self-microemulsion (tween-80) could significantly increase the oral absorption of puerarin than other technologies(P<0.05), and inclusion complex could remarkably increase the oral absorption of catalpol than nanocrystal(P<0.01). For puerarin, the brain targeting index of inclusion complex was the highest (P<0.05); but for catalpol, the brain targeting index of inclusion complex and self-microemulsions were both higher than nanocrystal (P<0.05). The self-microemulsion(tween-80) had the highest AUCbrain of puerarin than other groups (P<0.01); the inclusion complex had the highest AUCbrain for catalpol, but there was no significant difference compared with self-microemulsions. In conclusion, the self-microemulsion (tween-80) technology could increase the amount of puerarin and catalpol in brain, and was expected to be used in new oral Zige preparations.

Regulatory Effect of Catalpol on Th1/Th2 cells in Mice with Bone Loss Induced by Estrogen Deficiency.

PROBLEM: Estradiol (E2 ) deficiency can cause bone loss and the skew of Th1/Th2 cells. However, the correlation between the Th1/Th2 cells and the bone loss induced by estrogen deficiency remains unclear. Our aim was to investigate the role of Th1/Th2 in bone loss induced by estrogen deficiency and elucidated the therapeutical effect of catalpol in this condition. METHOD OF STUDY: Young, sham-operated (Sham), ovariectomized (Ovx), and naturally aged mice, treated with catalpol at different doses or control vehicle, were used in this study as indicated in each experiment. ELISA assay, dual-energy X-ray absorptiometry, and flow cytometry were used to analyze E2 , C-terminal telopeptides of type I collagen (CTx-I), bone mineral density (BMD), and Th1/Th2 subsets, respectively. The mRNA and protein expressions of specific transcription factors for Th1/Th2 cells (T-bet and GATA-3) were analyzed using real-time quantitative PCR and Western blot, respectively. RESULTS: Bone mineral density and E2 levels positively correlated with the proportion of Th2 subset while negatively correlated with that of Th1 subset and the ratio of Th1/Th2. Catalpol alleviated bone loss effectively by regulating Th1/Th2 polarization. Catalpol promoted the expression of Th2-specific transcription factors while inhibited that associated with Th1. CONCLUSION: Th1/Th2 skew is involved in bone loss induced by estrogen deficiency. Catalpol alleviates bone loss effectively by regulating Th1/Th2 paradigm.

Determination and pharmacokinetic study of gentiopicroside, geniposide, baicalin, and swertiamarin in Chinese herbal formulae after oral administration in rats by LC-MS/MS.

A sensitive and efficient liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of gentiopicroside, geniposide, baicalin, and swertiamarin in rat plasma. To avoid the stress caused by restraint or anesthesia, a freely moving rat model was used to investigate the pharmacokinetics of herbal medicine after the administration of a traditional Chinese herbal prescription of Long-Dan-Xie-Gan-Tang (10 g/kg, p.o.). Analytes were separated by a C18 column with a gradient system of methanol-water containing 1 mM ammonium acetate with 0.1% formic acid. The linear ranges were 10-500 ng/mL for gentiopicroside, geniposide, and baicalin, and 5-250 ng/mL for swertiamarin in biological samples. The intra- and inter-day precision (relative standard deviation) ranged from 0.9% to 11.4% and 0.3% to 14.4%, respectively. The accuracy (relative error) was from -6.3% to 10.1% at all quality control levels. The analytical system provided adequate matrix effect and recovery with good precision and accuracy. The pharmacokinetic data demonstrated that the area under concentration-time curve (AUC) values of gentiopicroside, geniposide, baicalin, and swertiamarin were 1417 ± 83.8, 302 ± 25.8, 753 ± 86.2, and 2.5 ± 0.1 min µg/mL. The pharmacokinetic profiles provide constructive information for the dosage regimen of herbal medicine and also contribute to elucidate the absorption mechanism in herbal applications and pharmacological experiments.

Characterization of metabolites of sweroside in rat urine using ultra-high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry and NMR spectroscopy.

Sweroside, a major active iridoid in Swertia pseudochinensis Hara, is recognized as an effective agent in the treatment of liver injury. Based on previous reports, the relatively short half-life (64 min) and poor bioavailability (approximately 0.31%) in rats suggested that not only sweroside itself but also its metabolites could be responsible for the observed hepato-protective effect. However, few studies have been carried out on the metabolism of sweroside. Therefore, the present study aimed at identifying the metabolites of sweroside in rat urine after a single oral dose (100 mg/kg). With ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), the metabolic profile revealed 11 metabolites in rat urine, including phase I, phase II and aglycone-related products. The chemical structures of metabolites were proposed based on accurate mass measurements of protonated or deprotonated molecules and their fragmentation patterns. Our findings showed that the aglycone of sweroside (M05) and its glucuronide conjugate (M06) were principal circulating metabolites in rats. While several other metabolic transformations, occurring via reduction, N-heterocyclization and N-acetylation after deglycosylation, were also observed. Two metabolites (M05 and M06) were isolated from the rat urine for structural elucidation and identifcation of reaction sites. Both M05 and M06 were characterized by (1)H, (13)C and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. UHPLC/Q-TOF-MS analysis has provided an important analytical platform to gather metabolic profile of sweroside.

Analgesic activity of catalpol in rodent models of neuropathic pain, and its spinal mechanism.

Neuropathic pain is a major health issue that represents considerable social and economic burden worldwidely. In this study, we investigated the potential of catalpol, an iridoid glucoside of Rehmannia glutinosa Steud, to alleviate neuropathic pain. The potential analgesic effects of catalpol were evaluated by chronic constriction injury (CCI) and lumbar 5 spinal nerve ligation (L5 SNL) model. In addition, we explored whether catalpol altered the degree of microglia activation and neuroinflammation in rat spinal cord after CCI induction. Repeated administration of catalpol (1, 5, 25, and 125 mg/kg) reversed mechanical allodynia induced by CCI and L5 SNL in a dose-dependent manner in rats. Levels of activated microglia, activated NF-κB, and proinflammatory cytokines (IL-1ß, IL-6, TNF-α) in lumber spinal cord were elevated in rats following CCI induction, and catalpol significantly inhibited these effects. Our results demonstrated that catalpol produces significant antinociceptive action in rodent behavioral models of neuropathic pain and that this effect is associated with modulation of neuroinflammation in spinal cord.

Effects of catalpol on ATPase and amino acids in gerbils with cerebral ischemia/reperfusion injury.

This study aimed to investigate the effects of catalpol on ATPase and amino acids in gerbils following cerebral ischemia/reperfusion (CI/R) injury. Gerbil model of CI/R was prepared by bilateral common carotid occlusion for 10 min followed by 6 h of reperfusion. Catalpol (5, 10 or 20 mg/kg per day) was injected intraperitoneally for 3 days before the carotid occlusion. Stroke index was measured during the reperfusion. ATPase activity, glutamate (Glu) and aspartate contents in brain tissue homogenate were examined. The results showed that catalpol significantly improved the stroke index compared with sham group (P < 0.05 or P < 0.01). Catalpol markedly increased the activities of Na(+)-K(+)-ATPase and Ca(2+)-ATPase (P < 0.05 or P < 0.01), and significantly decreased the content of Glu in brain tissue (P < 0.05 or P < 0.01). These data suggest that the efficacy of catalpol pretreatment on CI/R injury is associated with the enhancement of ATPase activity and the inhibition of excitatory amino acid toxicity.

Iridoid glycosides extracted from zhizi (fructus gardeniae) decrease collagen-induced platelet aggregation and reduce carotid artery thrombosis in an in vivo rat model.

OBJECTIVE: To investigate the anti-platelet aggregation and antithrombotic effects in rats of iridoid glycosides extracted from Zhizi (Fructus Gardeniae). METHODS: The present study evaluated the antithrombotic activity of iridoid glycosides (IGs) in a rat model of carotid artery thrombosis. The effects on coagulation, such as thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT), and the effect on collagen-induced platelet aggregation in vivo were investigated. Rats were intragastrically administered IGs (50, 100 or 200 mg/ kg) twice daily for 3 days. RESULTS: IGs were shown for the first time to have an antithrombotic action through the inhibition of platelet aggregation, with little effect on the coagulation time of peripheral blood. Our results also showed that IGs may significantly and dose-dependently reduce arterial thrombus load in a model of carotid artery thrombosis and inhibit collagen-induced platelet aggregation in rats. IGs (100 or 200 mg/kg) had no significant effect on APTT and PT, but did lengthen TT at a higher dose. CONCLUSION: These data, together with the previously reported neuroprotective effects of IGs in rats with cerebral ischemia, suggest that the antithrombotic action of IGs may potentially contribute to the treatment of cerebral ischemic diseases, including cerebral apoplexy.

Validated LC-MS method for simultaneous quantitation of catalpol and harpagide in rat plasma: application to a comparative pharmacokinetic study in normal and diabetic rats after oral administration of Zeng-Ye-Decoction.

A simple and efficient liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for simultaneous quantitation of catalpol and harpagide in normal and diabetic rat plasma. Protein precipitation extraction with acetonitrile was carried out using salidroside as the internal standard (IS). The LC separation was performed on an Elite C18 column (150 × 4.6 mm, 5 µm) with the mobile phase consisting of acetonitrile and water within a runtime of 12.0 min. The analytes were detected without endogenous interference in the selected ion monitoring mode with positive electrospray ionization. Calibration curves offered satisfactory linearity (r > 0.99) at linear range of 0.05-50.0 µg/mL for catalpol and 0.025-5.0 µg/mL for harpagide with the lower limits of quantitation of 0.05 and 0.025 µg/mL, respectively. Intra- and inter-day precisions (RSD) were <9.4%, and accuracy (RE) was in the -6.6 to 4.9% range. The extraction efficiencies of catalpol, harpagide and IS were all >76.5% and the matrix effects of the analytes ranged from 86.5 to 106.0%. The method was successfully applied to the pharmacokinetic study of catalpol and harpagide after oral administration of Zeng-Ye-Decoction to normal and diabetic rats, respectively.

Comparative pharmacokinetics of swertiamarin in rats after oral administration of swertiamarin alone, Qing Ye Dan tablets and co-administration of swertiamarin and oleanolic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Qing Ye Dan is a well-known herbal drug that is widely used to treat viral hepatitis in the Yi and Hani minority regions in the Yunnan province of China. MATERIALS AND METHODS: An LC-MS/MS method was developed to determine the levels of swertiamarin in rat plasma. Swertiamarin and naringin (internal standard, IS) were extracted from rat plasma using solid-phase extraction (SPE) to purify the samples. The pharmacokinetics of the following different administration methods of swertiamarin in rats were studied: oral administration of swertiamarin alone, a Qing Ye Dan tablet (QYDT) and co-administration of swertiamarin and oleanolic acid, with each method delivering approximately 20mg/kg of swertiamarin. Non-compartmental pharmacokinetic profiles were constructed by using the software DAS (version 2.1.1), and the pharmacokinetic parameters were compared using an unpaired Student's t-test. RESULTS: The results showed that the pharmacokinetic parameters Cmax, AUC0-∞, Vz/F and CLz/F were significantly different (P<0.05) among the three types of swertiamarin administration. CONCLUSIONS: The data indicate that oleanolic acid and the other ingredients present in QYDT could affect the pharmacokinetic behaviour of swertiamarin in rats.

Comparative pharmacokinetic profiles of picrosides I and II from kutkin, Picrorhiza kurroa extract and its formulation in rats.

Picrosides I and II are the active chemical constituents, present in the roots and rhizomes of Picrorhiza kurroa Royle (family: Scrophulariaceae). The plant is ethnomedically claimed for the treatment of liver and upper respiratory tract infection, fever, dyspepsia and scorpion sting. This study attempts to determine the in vivo pharmacokinetic profile of picrosides I and II in rats after oral administration of three different preparations namely, kutkin (a mixture of picrosides I and II), P. kurroa extract and Picrolax® capsule (marketed formulation). A simple, precise, specific and sensitive method was developed for simultaneous quantification of picrosides I and II in rat plasma and was applied for the pharmacokinetic study. Pharmacokinetic parameters were calculated from the observed plasma concentration of picrosides I and II. The results showed a significant difference (p≤0.05) in oral bioavailability of picrosides I and II from different preparations. Both the compounds were found to be more bioavailable from P. kurroa extract followed by Picrolax® capsule and kutkin. Moreover, we also developed a novel method for isolation of kutkin from roots of P. kurroa with a high yield of 2.4% w/w. The information gained from this study provides a meaningful basis for clinical application and mechanistic study of such phytochemicals.

Characterization of multiple absorbed constituents in rats after oral administration of Paederia scandens decoction.

Paederia scandens (Lour.) Merri. (Jishiteng in Chinese) is a Chinese traditional medicine widely used in treating various diseases. However, its active components have remained unknown. In the present study, a rapid and sensitive method by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-MSn) techniques was employed to investigate the absorbed constituents in rats after oral administration of Paederia scandens decoction. By comparing their MS data with those of authentic compounds and published data, a total of six compounds (paederosid, 1; paederosidic acid, 2; paederosidic acid methyl ester, 3; 6-hydroxy geniposide, 4; asperuloside, 5; and deacetyl asperuloside, 6) were identified in the P. scandens decoction samples. In addition, a total of seven compounds, including three iridoid glucosides and four of their metabolites, were identified in rat urine samples after administration. In addition, six compounds, including four iridoid glucosides and two of their metabolites, were identified in rat serum samples after administration. Our results significantly narrow the range of potentially active compounds in P. scandens decoction, and build a solid foundation for future research on its mechanism.