Screening of active ingredients for estrogenic effect of total flavonoids from Cuscutae Semen / 中国药房

OBJECTIVE To screen the active ingredient with estrogenic effect from total flavonoids of Cuscutae Semen. METHODS The estrogenic effect of total flavonoids from 10 batches of Cuscutae Semen was evaluated with mouse uterus coefficient and endometrial thickness as evaluation indexes， establish its fingerprint and calibrate the common peak. Common peak and spectrum-effect relationship of the above two indicators were analyzed by bivariate relationship analysis and grey correlation analysis to screen active components with estrogenic effect. UPLC-Q-TOF-MS technology was used to characterize the active components. RESULTS The estrogenic effect of total flavonoids from 10 batches of Cuscutae Semen was good. Twenty-eight and thirty-three common peaks of total flavonoids in Cuscutae Semen were obtained in the positive and negative ion modes respectively. The constituents represented by peaks 7，10，12-16，26 in positive ion mode and peaks 2，5，8，9，12，16，19，22-26 in negative ion mode were highly correlated with the estrogenic effect of total flavonoids from Cuscutae Semen. Further identification showed that the active substances with estrogenic effect from the total flavonoids of Cuscutae Semen were 5，7，3′， 4′-tetramethoxyflavone， 6- O-（trans） p-coumarin-furanfructose-（2→1）-glucopyranoside， rutin， kaempferol-3，7-diglucoside， apigenin-7-O-glucoside， hyperoside， baicalin， quercitin， quercetin， apigenin， kaempferol， isorhamnetin， rhododendron， isoquercetin， kaempferol-3-furan arabinoside， 2，6-octadecanediacetylic acid. CONCLUSIONS A total of 16 chemical components with estrogenic effect are screened from total flavonoids of Cuscutae Semen in the study， which can provide reference for the development of phytoestrogens.

Study on Metabolic Transformation of Total Glycosides of Cistanche deserticola in Artificial Gastric and Intestinal Juice / 中国药房

ABSTRACT OBJECTIVE：To study the metabolic transformation of total glycosides of Cistanche deserticola in artificial gastric and intestinal juice，and to speculate its metabolic transformation pathway in vivo. METHODS：UPLC/Q-TOF-MS was adopted. The determination was performed on ACQUITY UPLC BEH column with mobile phase consisted of 0.2％ formic acid water-acetonitrile（gradient elution）at the flow rate of 0.2 mL/min. The detection wavelength was set at 330 nm，and column temperature was 25 ℃. The ion source was electrospray ion source，and mass to charge ratio（m/z）was 50→1 000. In the positive and negative ion mode，the metabolic components of the total glycosides of C. deserticola in artificial gastric and intestinal juice were identified analysis，and combined with the literature，the metabolic pathway of total glycosides of C. deserticola in artificial gastric and intestinal juice was speculated. RESULTS：After the total glycosides of C. deserticola were metabolized by artificial gastric juice，and a total of 69 components were estimated，including 14 prototype components （such as Mustard aldehyde glucoside，daucosstorol） and 55 metabolic components （such as Methyl-O-Kankanoside J，Methyl-O-Kankanoside E），it is speculated that its metabolic pathways were methylation，demethylation，hydroxylation，methoxylation，acetylation，sulfation，and glucuronidation. After the total glycosides of C. deserticola were metabolized by artificial intestinal juice，a total of 90 components were estimated，including 4 prototype components（such as Kankanoside M，Kankanoside L）and 86 metabolic components（such as Methyl-O-Kankanoside， Methyl-O-Kankanoside E）. It was speculated that its metabolic pathways were methylation， demethylation，hydroxylation，dehydroxylation，methoxylation，acetylation，sulfation and glucuronidation. CONCLUSIONS：This study preliminarily speculates that the total glycosides of C. deserticola may be metabolized by methylation，demethylation， hydroxylation and other metabolic pathway in artificial gastrointestinal juice，which may provide reference for the in vivo metabolic transformation of total glycosides of C. deserticola.

Studies on influence factors of gnsenoside Rg1 and Rb1 absorption in intestines of rats / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the absorption of gnsenoside Rg1 and Rb1 in Radix Gngseng at different intestine segments of rats and the influence of the drug solution concentration, pH, P-gp inhibitor.</p><p><b>METHOD</b>The intestine cannulation was performed for in situ recirculation. Gnsenoside Rg1, Rb1 and phenol red concentration in the flux were separately measured by the reversed phase HPLC and UV.</p><p><b>RESULTS</b>When the concentration was raised from 0.075-0.75 g L(-1) and 0.03-0.3 g L(-1), the uptake of ginsenoside Rg1 and Rb1 was separately linearly increased (r >0.999), and no changes of K(a) absorption fraction and t(1/2) are found. The pH of flux has no effect on drug absorption. Ginsenoside Rg1 can be absorbed in the whole intestine and no changes of K(a), absorption fraction and t(1/2) refound and all the parameters of ginsenoside Rb1 at jejunum are higher than that at ileum and duodenum (P <0. 5). Further more, P-gp inhibitor verapamil has obvious effect on the intestinal absorption of ginsenoside Rb1 (P <0.5) while it has no effect on ginsenoside Rg1.</p><p><b>CONCLUSION</b>The absorption of ginsenoside Rg1 and Rb1 in intestine of rat are first-order kinetics, the absorption mechanism is infered the passive diffusion. Ginsenoside Rg1 has no specific absorption locus in intestine of rat and ginsenoside Rb1 has specific absorption locus of jejunum. Meanwhile, ginsenoside Rb1 is the P-gp substrate, and could increase its fraction of bioavailability by corporation with P-gp inhibitor.</p>

Transient gene expression of soluble VEGFR2: I-IV / 生物工程学报

The extracelluar domain I-IV of target gene VEGFR2 (Vascular endothelial growth factor receptor 2) was cloned from villus of trimester abortion by RT-PCR, and linked to the expression vectors. Then, the transfection conditions were optimized in serum-free suspension culture HEK293 using GFP (Green fluorescence protein) as the report gene. The results showed that the optimal transfection efficiency and cell number were obtained when the ratio of foreign DNA: PEI = 1:2 (W/W), DNA = 1.5 g /10(6) cells and shaking speed (120 r/min) in serum free medium in the beginning 4 hours of transfection. After optimizing the transfection conditions, the expression vector was successfully constructed for transient gene expression in HEK293, COS-7, and CHO-K1. The result shows that the target protein was only detected in CHO-K1 supernatant. Because of the C-terminal 8-His tag of target protein, target protein was subsequently purified using Ni2+-IDA and 5 mg purified protein was obtained in 1.5 L supernatant of CHOK1.