Comparison of Medicinal Part Quality of Typha angustifolia / 中国药房

OBJECTIVE：To compare the quality between Stamen typhae and pollen of T. angustifolia ，and provide scientific evidence for the improvement of quality standard of T. angustifolia . METHODS ：Fifteen batches of S. typhae were collected. Pollen minus sieve ，impurity plus sieve （filament and anther ）were sift out from S. typhae according to the identification method of T. angustifolia in Chinese Pharmacopoeia （2015 edition）. The characteristics and components of S. typhae and pollen ，filament and anther of T. angustifolia were comfirmed by impurity ， character examination and microscope ， TLC. The contents of isorhamnetin-3-O-neohesperidoside and typhaneoside in S. typhae and pollen ，impurities plus sieve （filament and anther ）of T. angustifolia were determined by HPLC. RESULTS ：S. typhae was a mixture of pollen ，anther and filament of T. angustifolia ，in the form of brownish yellow flocculent. The pollen of S. typhae was yellow powder with delicate hand feel ，slight smell and light taste；the surface of cells was slightly striped. The filaments and anthers were filiform and short-term ，rough and astringent ，and the cell surface were long strip. TLC chromatogram of S. typhae ，pollen and impurity of T. angustifolia had the same color spots at the same location. The contents of isorhamnetin- 3-O-neohesperidoside，typhaneoside and their aggregate were the highest in pollen （0.42％，0.24％，0.64％）；the second in S. typhae （0.22％，0.17％，0.39％）；the lowest in the impurities plus sieve （0.19％， 0.14％，0.33％）. The total contents of isorhamnetin- 3-O-neohesperidoside and typhaneoside in S. typhae and in impurities plus sieve did not reach the content limit stipulated in Chinese Pharmacopoeia （not less than 0.50％）. CONCLUSIONS：The medicinal components of T. angustifolia mainly exist in pollen. It is suggested that S. typhae should be used as the raw material to obtain pollen，and should not be used directly.

Simultaneous determination of eight flavonoids in plasma using LC-MS/MS and application to a pharmacokinetic study after oral administration of Pollen Typhae extract to rats.

A sensitive, reliable and validated LC-MS/MS method was developed to determine the presence of eight flavonoids (catechin, typhaneoside, isorhamnetin-3-O-neohesperidoside, astragalin, isorhamnetin-3-O-ß-d-glucoside, naringenin, kaempferol and isorhamnetin) in rat plasma. Puerarin was selected as the internal standard. Precipitation of the protein method with acetonitrile was used to extract these flavonoids from the rat plasma samples. The analysis was carried out on an Eclipse plus C18 column (4.6 mm×100mm, 1.8µm) when acetonitrile and formic acid aqueous solution (0.1%) was used as the mobile phase at a flow rate of 0.3mLmin-1. A tandem mass spectrometer having an electrospray ionization (ESI) source was used to detect eight flavonoids using multiple reaction monitoring (MRM) in the negative ionization mode. The LLOQs for catechin, typhaneoside, isorhamnetin-3-O-neohesperidoside, astragalin, isorhamnetin-3-O-ß-d-glucoside, naringenin, kaempferol and isorhamnetin are 4, 4, 4, 0.8, 1, 0.4, 2 and 0.2ngmL-1, respectively. The precision, accuracy and recovery were all within acceptable limits and the analytes were stable in plasma for all conditions tested. The method was successfully applied to pharmacokinetic study of four flavonoids in rat plasma after administering Pollen Typhae extract orally to rats.

Wenshen Xiaozheng Tang induces apoptosis and inhibits migration of ectopic endometriotic stromal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Wenshen Xiaozheng Tang (WXT), a traditional Chinese medicine prescription, exerted a good therapeutic effect on endometriosis. However, the underlying mechanism is unclear. In the present study, we sought to evaluate the effect of WXT on the proliferation and migration of ectopic endometriotic stromal cells and explore the potential molecular mechanism. MATERIALS AND METHODS: Primary stromal cells derived from ectopic endometriotic lesions of patients with endometriosis were isolated and cultured. The inhibition effect of WXT on cell proliferation was determined by MTT. Apoptosis of ectopic endometriotic cells treated with WXT was analyzed with Annexin V-FITC/7-AAD staining. The activation of caspases was detected by western blot analysis. The influence of WXT on migration of ectopic endometriotic cells was measured by scratch wound healing assay and Transwell assay. The DNA binding activity of NF-κB and the expression of nuclear p65 protein were determined by electrophoretic mobility shift assay and western blot analysis, respectively. The impact of WXT on the expression of NF-κB regulated gene products involved in apoptosis and migration was determined by western blot analysis. RESULTS: WXT inhibited the proliferation of ectopic endometriotic cells in a time- and dose-dependent manner. In addition, WXT treatment resulted in significant induction of apoptosis through the activation of caspases and inhibition of migration in ectopic endometriotic cells. WXT notably suppressed constitutive NF-κB-DNA-binding activity as well as TNF-α induced nuclear translocation of NF-κB p65 subunit in ectopic endometriotic cells. Moreover, WXT diminished the expression of NF-κB regulated gene products involved in apoptosis and migration, including c-IAP1, c-IAP2, XIAP, survivin, Mcl-1, COX-2 and MMP-9. CONCLUSIONS: Our results indicate that WXT induces apoptosis and inhibits migration of ectopic endometriotic stromal cells.

Hugan Qingzhi medication ameliorates hepatic steatosis by activating AMPK and PPARα pathways in L02 cells and HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hugan Qingzhi tablet (HQT), a lipid- lowering traditional Chinese medicine formula, has been used for the prevention and treatment of nonalcoholic fatty liver (NAFLD). AIM OF THE STUDY: This study was realized to evaluate the effects of HQT-medicated serum on hepatic steatosis using in vitro experiments with cells and explore the relevant mechanisms with method of serum pharmacology. MATERIALS AND METHODS: A model of hepatic steatosis in the L02 and HepG2 cells was induced by free fatty acid (FFA). The components in the HQT-medicated serum were assayed by high-performance liquid chromatography. Intracellular lipid droplets were detected by Oil Red O staining, and their ultrastructure was examined by transmission electron microscope. The biochemical parameters, including triglyceride (TG), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), total antioxidant capacity (T-AOC), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH), were measured with commercial kits. Furthermore, the expression of adiponectin, AMP-activated protein kinase (AMPK) phosphorylation, sterol regulatory element-binding protein 1 (SREBP-1), peroxisome proliferator activated receptor-α (PPARα), carnitine palmitoyltransferase 1 (CPT-1), and acetyl-CoA oxidase 1 (ACOX1) was analyzed by Western blot and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Moderate- and high-dose HQT-medicated serum reduced (P<0.05 or P<0.01) the accumulation of lipid droplets and the cellular TG content in L02 and HepG2 cells. They caused significant reductions (P<0.01) in LDH, AST, ALT and MDA and significant increase (P<0.05 or P<0.01) in T-AOC in the culture medium. They also caused increase (P<0.05 or P<0.01) in GSH level and SOD activity in FFA-induced steatotic L02 and HepG2 cells. Furthermore, moderate- and high-dose HQT-medicated serum enhanced (P<0.01) adiponectin expression in a concentration-dependent manner and increased (P<0.05 or P<0.01) the phosphorylation of AMPK and the expression of PPARα, CPT-1, and ACOX1, and reduced (P<0.05 or P<0.01) the expression of SREBP-1. CONCLUSION: The results suggested that HQT-medicated serum exerts a preventive effect against hepatic steatosis, and the potential mechanism might be activation of AMPK and PPARα pathways.

Analysis of isorhamnetin-3-O-neohesperidoside in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry and its application to pharmacokinetic studies.

AIM: To establish an LC-MS/MS method for determination of isorhamnetin-3-O-neohesperidoside and investigate its application on pharmacokinetic study in rats. METHODS: Eight rats were given 5 mg·kg(-1) isorhamnetin-3-O-neohesperidoside after intravenous administration. A highly sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of isorhamnetin-3-O-neohesperidosidein rat plasma using rutin as internal standard. The analytes and rutin (internal standard) were extracted with methanol followed by a rapid isocratic elution with 10 mmol·L(-1) ammonium acetate buffer/methanol (20 : 80, V/V) on a C18 column (150 mm × 2.1 mm, I.D., 5 µm) and subsequent analysis by mass spectrometry in the multi-eaction-monitoring mode. RESULTS: The assays were linear over the concentration range of 0.01-10 µg·mL(-1) for isorhamnetin-3-O-neohesperidosidein rat plasma. The lower limit of quantifications for isorhamnetin-3-O-neohesperidoside was 0.01 µg·mL(-1). CONCLUSION: The validated method is successfully applied to determine the plasma concentrations of isorhamnetin-3-O-neohesperidosidein in rats.

Analysis of isorhamnetin-3-O-neohesperidoside in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry and its application to pharmacokinetic studies / 中国天然药物

AIM@#To establish an LC-MS/MS method for determination of isorhamnetin-3-O-neohesperidoside and investigate its application on pharmacokinetic study in rats.@*METHODS@#Eight rats were given 5 mg·kg(-1) isorhamnetin-3-O-neohesperidoside after intravenous administration. A highly sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of isorhamnetin-3-O-neohesperidosidein rat plasma using rutin as internal standard. The analytes and rutin (internal standard) were extracted with methanol followed by a rapid isocratic elution with 10 mmol·L(-1) ammonium acetate buffer/methanol (20 : 80, V/V) on a C18 column (150 mm × 2.1 mm, I.D., 5 μm) and subsequent analysis by mass spectrometry in the multi-eaction-monitoring mode.@*RESULTS@#The assays were linear over the concentration range of 0.01-10 μg·mL(-1) for isorhamnetin-3-O-neohesperidosidein rat plasma. The lower limit of quantifications for isorhamnetin-3-O-neohesperidoside was 0.01 μg·mL(-1).@*CONCLUSION@#The validated method is successfully applied to determine the plasma concentrations of isorhamnetin-3-O-neohesperidosidein in rats.

Study on quality standard for Puhuang Dispensing Granule / 中成药

Objective: To study the quality standard for Puhuang Dispensing Granule (Pollen Typhae). Methods : Puhuang Dispensing Granule was identified by TLC and isorhamnetin-3-O-neohesperidoside and typhaneoside in Puhuang Dispensing Granule were determined by HPLC. Results : The linear relationship was at the range of 0.4～1.21?g for isorhamnetin-3-O-neohesperidoside and 0.42～1.25?g for typhaneoside, respectively. The average recovery was 98.88% for isorhamnetin-3-O-neohesperidoside and 99.68% for typhaneoside, respectively. Conclusion : The method is available with a good reproducibility and can control the quailty of Puhuang Dispensing Granule.