Synergism and rules of the new combination drug Yiqijiedu formulae (YQJD) on ischemic stroke based on amino acids (AAs) metabolism.

The use of combination drugs is considered to be a promising strategy to control complex diseases such as ischemic stroke. The detection of metabolites has been used as a versatile tool to reveal the potential mechanism of diverse diseases. In this study, the levels of 12 endogenous AAs were simultaneously determined quantitatively in the MCAO rat brain using RRLC-QQQ method. Seven AAs were chosen as the potential biomarkers, and using PLS-DA analysis, the effects of the new combination drug YQJD, which is composed of ginsenosides, berberine, and jasminoidin, on those 7 AAs were evaluated. Four AAs, glutamic acid, homocysteine, methionine, and tryptophan, which changed significantly in the YQJD-treated groups compared to the vehicle groups (P < 0.05), were identified and designated as the AAs to use to further explore the synergism of YQJD. The result of a PCA showed that the combination of these three drugs exhibits the strongest synergistic effect compared to other combination groups and that ginsenosides might play a pivotal role, especially when combined with jasminoidin. We successfully explored the synergetic mechanism of multi-component and provided a new method for evaluating the integrated effects of combination drugs in the treatment of complex diseases.

[Determination of endogenous amino acids in brain tissues after cerebral ischemia by RRLC-QQQ].

OBJECTIVE: To establish a method to determine underivatized endogenous amino acids in brain tissues after cerebral ischemia based on RRLC-QQQ. METHOD: Diamonsil chromatographic column C18 (4.6 mm x 250 mm, 5 microm) was adopted to determine 12 amino acids in 15 min, with acetonitrile-0.1% formic acid for gradient elution. The flow rate was set at 0.5 mL x min(-1). With ESI as the ion source, positive ion scanning mode was adopted for multi-reaction monitoring. RESULT: Each amino acid standard curve (AAs) showed good linear relationship within the detection range (r > 0.996), with the limit of detection of less than 11%, the limit of quantitation of less than 3.09 microg x L(-1). The RSD of intra- and inter-day precisions at high, middle and low concentrations were less than 11%. CONCLUSION: The determination results of actual samples showed that compared with the levels of AAs of the sham operation group, all of the remaining amino acids apart from N-acetyl-aspartate increased in brain tissues. Some amino acids showed significant changes in a time-dependent manner after the operation. The method is so simple, rapid and sensitive that it can be used for finding biological metabolite markers of cerebral ischemia, and exploring cerebral ischemia molecular mechanisms and synergistic mechanism of combined administration of multi-component traditional Chinese medicines.

[Advance of studies on metabolic fingerprinting analytical techniques and data processing methods].

Metabolomics is an emerging discipline subsequent to genomics, transcriptomics and proteomics, aiming for systematically studying the regularity of changes in metabolite to revealing organism's nature of movement and metabolism. It is especially important in modern pharmacological studies. Metabolic fingerprinting analysis is a method for metabolic analysis on high throughput of all metabolites, studying changes in drugs, organisms and endogenic metabolites caused by drugs and finding out related biomarkers to reflect dynamic changes inside organisms more directly and explain the mechanism of drugs and their effects on diseases. This essay summarizes some new metabolic fingerprint analytical methods and data processing methods used for metabolic fingerprint, elaborates their advantages and disadvantages and looks ahead to their combination with studies on traditional Chinese medicines, providing room for the development of new methods and new approaches for studies on complexity theory system of traditional Chinese medicines.

Response surface modeling and optimization of accelerated solvent extraction of four lignans from fructus schisandrae.

A new method based on accelerated solvent extraction (ASE) combined with response surface methodology (RSM) modeling and optimization has been developed for the extraction of four lignans in Fructus Schisandrae (the fruits of Schisandra chinensis Baill). The RSM method, based on a three level and three variable Box-Behnken design (BBD), was employed to obtain the optimal combination of extraction condition. In brief, the lignans schizandrin, schisandrol B, deoxyschizandrin and schisandrin B were optimally extracted with 87% ethanol as extraction solvent, extraction temperature of 160 ° C, static extraction time of 10 min, extraction pressure of 1,500 psi, flush volume of 60% and one extraction cycle. The 3D response surface plot and the contour plot derived from the mathematical models were applied to determine the optimal conditions. Under the above conditions, the experimental value of four lignans was 14.72 mg/g, which is in close agreement with the value predicted by the model.

Hypoglycemic effect of protopanaxadiol-type ginsenosides and compound K on Type 2 diabetes mice induced by high-fat diet combining with streptozotocin via suppression of hepatic gluconeogenesis.

Compound K (CK) is a final intestinal metabolite of protopanaxadiol-type ginsenosides (PDG) from Panax ginseng. Although anti-diabetic activity of CK have been reported with genetic mouse models (db/db mice) in recent years, the therapeutic usefulness of CK and PDG in type 2 diabetes, a more prevalent form of diabetes, remains unclear. In the present investigation, we developed a mouse of non-insulin-dependent diabetes mellitus that closely simulated the metabolic abnormalities of the human disease. For this purpose, type 2 diabetes was induced in male ICR mice by combining of streptozotocin. The male ICR mice fed with HFD for 4 weeks received 100mg/kg of STZ injected intraperitoneally. After 4 weeks, mice with fasting (12h) blood glucose levels (FBG) above 7.8 mmol/L were divided into 3 groups (n=12) and treated with vehicle (diabetes model, DM), 300 mg/kg/day of PDG and 30 mg/kg/day of CK for 4 weeks while continuing on the high-fat diet. Hypoglycemic effects of CK and PDG were consistently demonstrated by FBG levels, and insulin-sensitizing effects were seen during oral glucose tolerance testing (OGTT). Moreover, the mechanism of hypoglycemic effect in type 2 diabetic mice was examined. Gluconeogenic genes, Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase), were decreased in two treatment groups with CK showing greater effects. These findings demonstrated the hypoglycemic and insulin-sensitizing capabilities of CK on type 2 diabetes induced by HFD/STZ via down-regulation of PEPCK and G6Pase expression in liver.

[Fluorescence resonance energy transfer quenching method for determination of arsenic with acridine orange-rhodamine B].

A new method for the determination of trace arsenic was developed by using fluorescence resonance energy transfer from acridine orange (AO) to rhodamine B (RB). It was found that under the condition of lambda(ex)/lambda(em) = 470/580 nm, effective energy transfer could occur between AO and RB in the dodecyl benzene sodium sulfonate solution. The fluorescence intensity of RB was diminished by molybdoarsenide which was formed by the reaction of arsenic (V) with molybdate in sulfuric acid medium. The detection limit of this method was 2. 56 microg x L(-1). This method was used for the determination of trace arsenic in tea. The range of determination for arsenic was 0.01-0.25 mg x L(-1). The relative standard deviation for the determination of arsenic was 0.48%-0.64%. The recoveries for the addition of 0.01-0.03 mg x L(-1) arsenic were 98%-103%. The method has been applied to the determination of arsenic with satisfactory results.

[Determination of eliminating ratio of Chinese traditional medicine for hydroxyl radical by fluorescent spectrophotometry].

Benzoic acid with weak fluorescence may react on .OH, and products with intense fluorescence are made. Extractives of Chinese traditional medicine may eliminate .OH in solution, and make amounts of the products to reduce. Then, increase level of fluorescence of products in solution will be lowered. Based on this principle, a new method is developed to determine eliminating ratio of Chinese traditional medicine for .OH. It is shown that productivity of .OH tends to saturation when H2O2 is shown more than 20 min by 280 nm UV light; .OH may react on benzoic acid completely when molar ratio of H2O2 and benzoic acid is 30:1; linear response range of products fluorescence is 2.2-80 mmol.L-1 with concentration of H2O2. IC50 of elimination .OH with magnoliae and eucommia are 1.025 and 515.3 mg.L-1 respectively. There are no remarkable difference between these results and that of spectrophotometry.