Pharmacokinetic and metabolism study of ginsenoside Rb2 in rat by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

In this study, a simple and rapid ultra-fast liquid chromatography tandem mass spectrometry method was established and validated to determine ginsenosides Rb2 in rat plasma. Acetonitrile-mediated protein precipitant was applied to the sample preparation. Chromatographic separation was carried out on an Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.8 µm). The analytes were monitored using multiple reactions monitoring mode with precursor-to-product ion transitions at m/z 1077.4-945.3 and m/z 799.8 → 637.8 for ginsenoside Rb2 and internal standard, respectively. The mobile phase was composed of 0.1% formic acid aqueous solution and acetonitrile. The assay showed excellent linearity over the concentration range of 2-1,000 ng/ml, with correlation coefficient >0.995. The method was further validated for selectivity, precision, accuracy, recovery, and stability according to the US Food and Drug Administration guidelines. The validated method was successfully applied to pharmacokinetic and bioavailability studies of ginsenoside Rb2 in rat plasma. Based on the pharmacokinetic results, ginsenoside Rb2 showed slow clearance and low oral bioavailability (0.15%). In addition, the metabolites of ginsenoside Rb2 in rat urine and feces were characterized according to their accurate masses and fragment ions. The proposed metabolic pathway was deglycosylation.

Stimulation of Epithelial Sodium Channels in Endothelial Cells by Bone Morphogenetic Protein-4 Contributes to Salt-Sensitive Hypertension in Rats.

Previous studies have shown that high salt induces artery stiffness by causing endothelial dysfunction via increased sodium influx. We used our unique split-open artery technique combined with protein biochemistry and in vitro measurement of vascular tone to test a hypothesis that bone morphogenetic protein 4 (BMP4) mediates high salt-induced loss of vascular relaxation by stimulating the epithelial sodium channel (ENaC) in endothelial cells. The data show that high salt intake increased BMP4 both in endothelial cells and in the serum and that exogenous BMP4 stimulated ENaC in endothelial cells. The data also show that the stimulation is mediated by p38 mitogen-activated protein kinases (p38 MAPK) and serum and glucocorticoid-regulated kinase 1 (Sgk1)/neural precursor cell expressed developmentally downregulated gene 4-2 (Nedd4-2) (Sgk1/Nedd4-2). Furthermore, BMP4 decreased mesenteric artery relaxation in a benzamil-sensitive manner. These results suggest that high salt intake stimulates endothelial cells to express and release BMP4 and that the released BMP4 reduces artery relaxation by stimulating ENaC in endothelial cells. Therefore, stimulation of ENaC in endothelial cells by BMP4 may serve as another pathway to participate in the complex mechanism of salt-sensitive (SS) hypertension.

Dietary salt blunts vasodilation by stimulating epithelial sodium channels in endothelial cells from salt-sensitive Dahl rats.

BACKGROUND AND PURPOSE: Our recent studies show that the reduced activity of epithelial sodium channels (ENaC) in endothelial cells accounts for the adaptation of vasculature to salt in Sprague-Dawley rats. The present study examines a hypothesis that enhanced ENaC activity mediates the loss of vasorelaxation in Dahl salt-sensitive (SS) rats. EXPERIMENTAL APPROACH: We used the cell-attached patch-clamp technique to record ENaC activity in split-open mesenteric arteries. Western blot and immunofluorescence staining were used to evaluate the levels of aldosterone, ENaC, eNOS and NO. Blood pressure was measured with the tail-cuff method and the artery relaxation was measured with the wire myograph assay. KEY RESULTS: High-salt (HS) diet significantly increased plasma aldosterone and ENaC activity in the endothelial cells of Dahl SS rats. The endothelium-dependent artery relaxation was blunted by HS challenge in these rats. Amiloride, a potent blocker of ENaC, increased both phosphorylated eNOS and NO and therefore prevented the HS-induced loss of vasorelaxation. As, in SS rats, endogenous aldosterone was already elevated by HS challenge, exogenous aldosterone did not further elevate ENaC activity in the rats fed with HS. Eplerenone, a mineralocorticoid receptor antagonist, attenuated the effects of HS on both ENaC activity and artery relaxation. CONCLUSIONS AND IMPLICATIONS: These data suggest that HS diet blunts artery relaxation and causes hypertension via a pathway associated with aldosterone-dependent activation of ENaC in endothelial cells. This pathway provides one of the mechanisms by which HS causes hypertension in Dahl SS rats. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

ZNF307 (Zinc Finger Protein 307) Acts as a Negative Regulator of Pressure Overload-Induced Cardiac Hypertrophy.

Pathological cardiac hypertrophy is a key risk factor for heart failure. We found that the protein expression levels of the ZNF307 (zinc finger protein 307) were significantly increased in heart samples from both human patients with dilated cardiomyopathy and mice subjected to aortic banding. Therefore, we aimed to elucidate the role of ZNF307 in the development of cardiac hypertrophy and to explore the signal transduction events that mediate the effect of ZNF307 on cardiac hypertrophy, using cardiac-specific ZNF307 transgenic (ZNF307-TG) mice and ZNF307 global knockout (ZNF307-KO) mice. The results showed that the deletion of ZNF307 potentiated aortic banding-induced pathological cardiac hypertrophy, fibrosis, and cardiac dysfunction; however, the aortic banding-induced cardiac hypertrophic phenotype was dramatically diminished by ZNF307 overexpression in mouse heart. Mechanistically, the antihypertrophic effects mediated by ZNF307 in response to pathological stimuli were associated with the direct inactivation of NF-κB (nuclear factor-κB) signaling and blockade of the nuclear translocation of NF-κB subunit p65. Furthermore, the overexpression of a degradation-resistant mutant of IκBα (IκBαS32A/S36A) reversed the exacerbation of cardiac hypertrophy, fibrosis, and dysfunction shown in aortic banding-treated ZNF307-KO mice. In conclusion, our findings demonstrate that ZNF307 ameliorates pressure overload-induced cardiac hypertrophy by inhibiting the activity of NF-κB-signaling pathway.

AMP-Activated Protein Kinase Attenuates High Salt-Induced Activation of Epithelial Sodium Channels (ENaC) in Human Umbilical Vein Endothelial Cells.

Recent studies suggest that the epithelial sodium channel (ENaC) is expressed in the endothelial cells. To test whether high salt affects the NO production via regulation of endothelial ENaC, human umbilical vein endothelial cells (HUVECs) were incubated in solutions containing either normal or high sodium (additional 20 mM NaCl). Our data showed that high sodium treatment significantly increased α-, ß-, and γ-ENaC expression levels in HUVECs. Using the cell-attached patch-clamp technique, we demonstrated that high sodium treatment significantly increased ENaC open probability (P O ). Moreover, nitric oxide synthase (eNOS) phosphorylation (Ser 1177) levels and NO production were significantly decreased by high sodium in HUVECs; the effects of high sodium on eNOS phosphorylation and NO production were inhibited by a specific ENaC blocker, amiloride. Our results showed that high sodium decreased AMP-activated kinase (AMPK) phosphorylation in endothelial cells. On the other hand, metformin, an AMPK activator, prevented high sodium-induced upregulation of ENaC expression and P O . Moreover, metformin prevented high salt-induced decrease in NO production and eNOS phosphorylation. These results suggest that high sodium stimulates ENaC activation by negatively modulating AMPK activity, thereby leading to reduction in eNOS activity and NO production in endothelial cells.

[Study on secondary structural changes of protein in the formation of microcapsule by FTIR spectroscopy].

The mixture of whey protein and isolated soy protein with maltodextrin as microcapsules wall material was adopted. The secondary structure constitution of these two kinds of proteins in the process of heating and spray drying together with maltodextrin were studied by Fourier transform infrared spectra. The result showed that both whey protein and isolated soy protein exhibited differences in the secondary structures. The rate of alpha-helix in whey protein decreased 1.9% and beta-turn content of the protein remains virtually unchanged, beta-sheet increased 8.19%, and random coil decreased 7.18%. At the same time the content of alpha-helix in isolated soy protein decreased 1.64%, the change of beta-sheet is not obvious while beta-turn increased 10.20% and random coil decreased 9.03%. Meanwhile, the amide I bands of these two proteins both shifted to the lower wave number direction, indicating that in the formation process of the microcapsule wall structure there are complex interactions between maltodextrin and proteins, and the hydrogen bonds which comes into existence are comparatively strong. With the help of scanning electron microscope, it was discovered that the surface is more smooth and integrated when the microcapsules wall material contains whey protein, which is high in alpha helix.

[Study on the microstructure of fluorescent labelling ghee microcapsules by Tomoscan imaging].

In the present paper, fluorescein isothiocyanate was chosen as a fluorescence probe to mark casein protein in alkaline conditions. The interaction of the casein protein marked or not marked and fluorescein isothiocyanate was preliminarily discussed by the spectrum changes of UV-absorption and fluorescence spectrometry. Fluorescent marker was separated from SephadexG-50 chromatography column. With it as an emulsifier, the fluorescently-labeled ghee microcapsules were prepared by spray drying. And using laser scanning confocal microscope by tomoscan imaging to detect the microstructure of ghee microcapsules with the excitation of 488 nm argon-ion laser, the results showed that the casein protein assembled in the membrane surface of oil-water interface and microcapsules. The ghee microcapsules had two forms, namely mononuclear and multinuclear. The microcapsule was spherical. Its surface was smooth with no crack and no hollow. Its wall surface was intact and wall structure was relatively dense. The particle size showed obvious difference. Small particles attached to large particles, forming partial agglomerating powders to contribute to enhancing the solubility of microcapsules. These prove that the ghee microcapsule is an ideal microcapsule product.

Near-infrared spectroscopy and partial least-squares regression for determination of arachidonic acid in powdered oil.

Near-infrared (NIR) spectroscopy was evaluated as a rapid method of predicting arachidonic acid content in powdered oil without the need for oil extraction. NIR spectra of powdered oil samples were obtained with an NIR spectrometer and correlated with arachidonic acid content determined by a modification of the AOCS Method. Partial Least-Squares regression was applied to calculate models for the prediction of arachidonic acid. The model developed with the raw spectra had the best performance in cross-validation (n = 72) and validation (n = 21) with a correlation coefficient of 0.965, and the root mean square error of cross-validation and prediction were both 0.50. The results show that NIR, a well-established and widely applied technique, can be applied to determine the arachidonic acid content in powdered oil.

[Studies on the characters and microstructure of enzyme and octenyl succinic anhydride modified starch].

Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), UV-Vis spectra, laser nano size detector (LNSD) and scanning electron microscope (SEM) were employed to analyze the characters and structure of enzyme and octenyl succinic anhydride modified starch. The results indicated that the enzymatic starch reacted with octenyl succinic anhydride, bringing only octenyl succinic anhydride groups but not any other groups. The esterification of enzymatic starch only took place in amorphous region, but had no effect on the crystal form of starch granule. The clarity of EOSS increased with the increase in substitution degree. The particle size of oil emulsion made by EOSS was fine and well-distributed, meaning that the emulsion has excellent emulsibility and emulsifying stability. The embedding of oil encapsulated with EOSS is fine. It can be concluded that the properties of EOSS is excellent, and can be used as emulsifier and wall material of microcapsule.

Rapid determination of docosahexaenoic acid in powdered oil by near-infrared spectroscopy.

Near-infrared spectroscopy (NIRS) was used as a rapid and nondestructive method to determine the content of docosahexaenoic acid (DHA) in powdered oil samples. A total of 82 samples were scanned in the diffuse reflectance mode by Nicolet 5700 FTIR spectrometer and the reference values for DHA was measured by gas chromatography. Calibration equations were developed using partial least-squares regression (PLS) with internal cross-validation. Samples were split in two sets, one set used as calibration (n = 66) whereas the remaining samples (n=16) were used as validation set. Two mathematical treatments (first and second derivative), none (log(1/R)) and standard normal variate as scatter corrections and Savitzky-Golay smoothing were explored. To decide upon the number of PLS factors included in the PLS model, the model with the lowest root mean square error of cross-validation (RMSECV=0.44) for the validation set is chosen. The correlation coefficient (r) between the predicted and the reference results which used as an evaluation parameter for the models is 0.968. The root mean square error of prediction of the final model is 0.59. The results reported in this article demonstrate that FT-NIR measurements can serve as a rapid method to determine DHA in powdered oil.

[Analysis of primary elemental speciation distribution in mungbean during enzymatic hydrolization].

In the present paper, trace elements contents of cuprum, zincum, manganese and ferrum in mungbean and their primary speciation distribution during enzymatic hydrolization were investigated with ICP-AES OPTIMA 5300DV plasma emission spectroscopy. The trace elements were separated into two forms, i.e. dissolvable form and particulate form, by cellulose membrane with 0.45 microm of pore diameter. All the samples were digested by strong acid (perchloric acid and nitric acid with 1 : 4 ratio ). The parameters of primary speciations of the four elements were calculated and discussed. The results showed: (1) Contents of cuprum, zincum, manganese and ferrum in mungbean were 12.77, 31.26, 18.14 and 69.38 microg x g(-1) (of dry matter), respectively. Different treatment resulted in different elemental formulation in product, indicating that more attention should be paid to the trace elements pattern when producing mungbean beverage with different processes. (2) Extraction rates of cuprum, zincum, manganese and ferrum in extract were 68.84%, 51.84%, 63.97% and 30.40% with enzymatic treatments and 36.22%, 17.58%, 7.85% and 22.99% with boil treatment, respectively. Both boil and enzymatic treatments led to poor elemental extraction rates, which proved that it was necessary to take deep enzymatic hydrolysis treatment in mungbean beverage process as the trace element utilization rate was concerned. (3) Amylase, protease and cellulose showed different extraction effectiveness of the four trace elements. Generally, protease exhibited highest efficiency for the four elements extraction. All of the four trace elements were mostly in dissolvable form in all hydrolysates and soup. (4) Relative standard deviations and recovery yields are within 0.12%-0.90% (n = 11) and 98.6%-101.4%, respectively. The analysis method in this paper proved to be accurate.

[Analysis of speciation distribution of se in mungbean during enzymatic hydrolization].

In the present paper, selenium content in mungbean and selenium speciation distribution in mungbean during enzymatic hydrolization was investigated with AFS-230E atomic fluorescence photometer. Selenium in the decoction and enzymatic hydrolysates samples were separated into two species including primary speciation and secondary speciation. The primary speciation included the soluble and the suspended forms and was divided by 0.45 microm filter membrane. The secondary speciation included the inorganic and the organic forms and was divided by D101 macroreticular resin. The speciation parameters of selenium such as extractive rate, remain rate, residue rate, immerse-residue ratio and speciation distribution coefficients, etc in different samples were calculated. The results showed: (1) Selenium content in mungbean was 54.79 microg x g(-1) (of dry matter). (2) Over 90% selenium in mungbean was extracted by enzymatic treatment, but only 19.26% selenium came out in water when only treated by hot water. The extraction rates of Se in the process of amylase, protease and cellulase were 33. 64%, 55.96% and 5.189%, respectively. It was inferred that most selenium was in conjugate or complexation form in mungbean protein. (3) The distribution coefficient of selenium in organic form was 59.87% in the total enzymatic product and 3.64% in the mungbean soup. Organic form distribution coefficients of selenium in amylase and protease hydrolysates were 15.51% and 44.36%, respectively. No organic selenium was detected in cellulase hydrolysate. It was inferred that selenium was in complexation form in mungbean cellulose. All the results showed that enzymatic hydrolization treatment did not only improve the total content of selenium greatly, but also materially improved the organic form content of selenium in water. The recovery for the method was 97.8% and RSD was 1.1% (n=11).

[Determination of nutrition elements in Yak milk powder by atomic absorption spectrometry].

In the present paper, orthogonal design test was planed by changing cinefaction temperature, cinefaction time, and the concentration of hydrochloric acid. Yak milk powder and milk powder were digested by dry method. The contents of elements Ca, Mg, Fe, Cu, Zn, Mn, K and Na were determined by flame atomic absorption spectrometry. The best conditions for the digestion were obtained as follows: the temperature was 510 degrees C, cinefaction time 4 hours, and hydrochloric acid concentration 1 : 5. Meanwhile, under the best experimental conditions, the recovery ratio of the method in the range of 95.2%-107.3%, and the relative standard deviations were 0.38%-3.86% (n=6). The experimental results proved that Yak milk powder is valuable and nutritious food, and has rich nutrition elements.

[The interaction of acridine orange with anionic surfactant and its application to the determination of protein].

The change in the UV-absorption spectrum and fluorescence spectrum of acridine orange(AO) due to the addition of surfactant dodecyi benzene sulfonic acid sodium sait (SDBS) and bovine serum albumin(BSA) was studied. Meanwhile, the effects of the in situ formed AOAO dimer in SDBS as a fluorescence probe and BSA were discussed. A new method for the determination of BSA using fluorescence is presented. The results indicate that the method is sensitive and rapid. The linear range of determination is 0-4. 17 x 10(-7) mol x L(-1). The relative standard deviation is 1.9%, and the detection limit is 8.73 x 10(-10) mol x L(-1).

[Spectrometry study on the interaction of Magdala red and bovine serum albumin].

In the present paper, the interaction of Magdala red and bovine serum albumin(BSA) was studied by UV-absorption and fluorescence spectrometry. The results of UV-absorption spectrometry show that the way of the interaction of Magdala red and BSA should obey the Scatchard Model. In other words, Magdala should be combined to some certain regions of BSA. Meanwhile, taking advantage of fluorescence quenching, the reaction mechanism of Magdala with BSA was explored. Protein molecule conformation change is discussed by synchronous fluorescence spectrometry.