Comparative study on effects of Xiaoyao Powder and its modified prescription on plasma metabolomics of rats with chronic immobilization stress.

OBJECTIVE: To determine the changes in the levels of endogenous metabolites in rats with chronic immobilization stress (CIS) taking Xiaoyao Powder (XYP) and its modified prescription version, which lacks the volatile oils extracted from Herba Menthae. METHODS: Twenty-four experimental male SD rats were randomly divided into 4 groups of 6 rats each: control, model, XYP-1 (containing volatile oils from Herba Menthae), and XYP-2 (lacking volatile oils). All rats except control group rats were subjected to CIS 3 h per day for 21 consecutive days. Groups XYP-1 and XYP-2 were given the extracted XYS with or without volatile oils (3.854 g/kg; suspended in distilled water) via gavage 1 h before CIS each day for 21 days. Rats were anesthetized using intraperitoneal injection of pentobarbital sodium (40 mg/kg) on the 22nd day. Observations were made using a Varian INOVA 600 MHz NMR spectrometer at 27 °C. Carr-Purcell-Meiboom-Gill (CPMG) and longitudinal eddy-delay (LED) were applied, resulting in spectra showing only the signals from micro- and macro-metabolites. RESULTS: Compared to controls, rats subjected to CIS showed increased levels of plasma metabolites, such as acetic acid, choline, N-glycoprotein (NAC), saturated fatty acid, and blood sugars. Levels of low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and unsaturated fatty acids were decreased. The biochemical effects of XYS were characterized by elevated levels of VLDL, LDL, threonine, methionine, and glutamic acid in plasma. CONCLUSION: Some common and characteristic metabolites on the anti-CIS of XYP and its modified prescription were obtained. The metabolomics technology is a valuable tool and may be used to identify the specific metabolites and potential biomarkers of therapeutic effect of Chinese medicinal prescriptions.

[Metabonomic study on the regulatory effect of xiaoyao powder on chronic immobilization stressed rats].

OBJECTIVE: To provide a scientific basis for systematic research on the mechanism of chronic immobilization stress (CIS) induced metabolic network change in rats, through detecting the changes of endogenous metabolites in rats with CIS, treated or un-treated with Xiaoyao Powder (XYP), for determining the small molecule marker compound that closely associated with the metabonomical specificity of CIS and acting mechanism of XYP. METHODS: Thirty-six experimental male SD rats were divided into 3 groups, the normal control group, the model group and the XYP group. And all the three groups were subdivided into two subgroups respectively on day 7 and day 21 of the experiment. The stress rat model of CIS was made by chronic restraining method for 3 h every day. Starting from the first day of modeling, XYP 3.854 g/kg in volume of 1 mL/100 g body weight was administered 1 h before restraining via gastrogavage to rats in the XYP group, while equal volume of distilled water was given to rats in the other two groups instead. Blood samples were collected on the 8 th day and 22 th day for detection in the following procedure: at 27 degrees C, 300 microL supernate of blood plasma was taken, calling the Carr-Purcell-Meiboom-Gill (CPMG) and longitudinal eddy-delay (LED) sequence respectively on a Fourier variable nuclear magnetic resonance (NMR) spectrometer, pre-saturated inhibition of the water peak was performed; free induction decay (FID) signals were transferred via 32 k Fourier transformation to gain one-dimensional NMR spectrogram; by taking TSP as the chemical migration reference peak, the segmental integral calculus (0.04 ppm per segment) was performed from 4.5 - 0.5 ppm (CPMG) and 6.0 - 0 ppm (LED) within the peak ranges in 1H spectra using the VNMR software; after normalization, centering and scaling were conducted on data, then used for pattern recognition of principal component analysis (PCA) using the SIMCA-P 10.0 software, or if necessary, the partial least squares discriminate analysis (PLS-DA) was performed. RESULTS: (1) The metabolites in the model group were significantly different from those in the control group, suggesting that the animal model was successfully established with the metabolic network different to that of control. The model group and the XYP group could be differentiated from the control group by the differences of metabolites and metabolic networks between groups; XYP could intervene the metabolites or the metabolic path to cause changes in final metabolites. (2) The serum contents of lactic acid (1.4, 4.16), choline (3.24), N-acetylgalactosamine (NAC) and saturated fatty acids (1-3) increased, but unsaturated fatty acids (1.99,4-5), blood sugar (34), HDL (0.83), etc. reduced in the CIS rats. XYP showed obvious regulatory effects on final metabolites, causing decrease of lactic acid, choline, NAC, saturated fatty acids and blood sugar, and increase of unsaturated fatty acids, blood sugar, HDL, 3.44 ppm compound, etc. CONCLUSIONS: The metabolic phenotype in CIS rats includes the increase of lactic acid, choline, NAC, saturated fatty acid, and the decrease of blood sugar contents, unsaturated fatty acid, HDL, 3.44 ppm compound, etc., these may be the markers of the metabolites. The final metabolites changes induced by CIS are primarily the lipid substances. XYP markedly regulates the contents of final metabolites, showing the regulatory effects on final metabolites, but what is the metabolite or metabolic pathways it interferes to alter the final metabolites should be confirmed by further studies.

[Metabonomic study of syndrome of liver qi stagnation and spleen deficiency in rats].

OBJECTIVE: To determine the changes of the plasma metabolic phenotype in rats with chronic restraint stress (rats with syndrome of liver qi stagnation and spleen deficiency), so as to reveal the biological features of the syndrome of liver qi stagnation and spleen deficiency, and to examine potential application of metabonomic analysis in studies of syndromes in traditional Chinese medicine. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into four groups: group A, 7 d normal control group; group B, 21 d normal control group; group C, 7 d stress group; and group D, 21 d stress group, with 6 rats in each group. Chronic restraint was used to induce stress in rats. Blood was collected from the cardio-ventricle under anesthesia on the 8th (groups A and C) or 22nd day (groups B and D) and detected by using the Fourier variable superconducting nuclear magnetic resonance (NMR) spectrometer (Varian UnityInova 600 M). Free induction decay signals were transferred into one-dimensional NMR spectrogram via 32 k Fourier transformation. Segmental integral calculus (0.04 ppm per segment) was performed from 4.5-0.5 ppm (Carr-Purcell-Meiboom-Gill, CPMG) or 6.0-0 ppm (longitudinal eddy-delay, LED) as defaulted 1H spectra values by using the VNMR software. Data were saved as text or excel files after normalization and then used for pattern recognition analyses. All the data were analyzed by principal component analysis (PCA) using the SIMCA-P 10.0 software (Umetrics AB, Umea, Sweden). RESULTS: The PCA analysis of rat plasma (1)H NMR spectra revealed different metabolic spectra between stress and control groups, which were consistent with alterations of in vivo metabolisms in rats under stress stimuli. Compared with the normal control group, rats with repeated stress displayed significant changes in spectral peak shapes of acetate, lactate, tyrosine, low-density lipoprotein, and unknown compounds (3.44 ppm). These altered metabolites can be used as biomarkers of syndrome of liver qi stagnation and spleen deficiency for further studies. CONCLUSION: The (1)H NMR spectra of metabolites in the rat blood are differentially changed after chronic stress. Specific, characteristic metabolic products can be identified by analyses of metabonomics, which lead to interpretation of biological feature of Chinese medicine syndromes. Therefore, metabonomic analysis is an approach with good development prospects to studies of TCM syndromes.