| 1. Objectives and significanceTraditional Chinese medicine is treasure of the Chinese nation, it has made an indelible contribution for the health and breeding of the Chinese nation. The composition of traditional Chinese medicine is complicated, not only effective components to diseases but also adjuvant therapy or inactive ingredients were included, It is different from the general chemical medicine, traditional Chinese medicine rely on a variety of ingredients work together. So its quality control mode is essential different from single component chemical medicine. It is based on a series of quality standards, to monitor, evaluate and control each process in the production of traditional Chinese medicine. How to better control the quality of traditional Chinese medicine is one of the biggest challenge we are facing now. Along with the advance of modern chemical analysis technology, a variety of analysis methods are used to the quality control of the Chineseherbal pieces and its compound.Lianggesan (contained Song "He Ji Ju Fang") has the effect of clearing away heat and toxic material, for the treatment of pneumonia, bronchitis, sinusitis, headaches and other diseases such as rubella, stroke. Single herbs with the Chinese medicine formulae have cathartic, diuretic, choleretic, anti-inflammatory and antibacterial effects respectively. Acute lung injury is one of the clinical common respiratory critical diseases with alveolar epithelial cells and capillary endothelial cell damage which caused by various directly or indirectly factors, causing diffuse pulmonary interstitial and alveolar edema, at the same time the release of a variety of inflammatory mediators, causing pulmonary inflammatory reaction too much out of control, then lead to acute hypoxic respiratory insufficiency. The clinical treatment of acute lung injury with cooperation of Chinese and western medicine has certain advantages and characteristics. In previous study, we found Lianggesan can reduce rat lung tissue damage induced by LPS. To study the fingerprint of Lianggesan Decoction, assign the chemical composition represented by fingerprint peaks, establish the content determination method of Forsythoside A, Baicalin and Rhein in Lianggesan Decoction, provide a scientific evidence for material basis research and quality control of Lianggesan Decoction, and to establish a quality evaluation method for the overall features of the decoction. At the same time, do the preliminary anti-inflammatory experiments on a cellular level using the concentrated dry powder of Lianggesan water extraction liquid and its main ingredient Forsythoside A and Baicalin.2. Methods2.1To research the fingerprint of Lianggesan Decoction and the content determination of its main ingredients Forsythoside A, Baicalin and Rhein.The fingerprint was established by HPLC with Diode Array Detector (DAD), Zorbax Eclipse XDB-C18column (250mm×4.6mm,5μm), gradient eluted with methanol-0.1%phosphoric acid at flow rate of lmL/min. The detection wavelength was at235nm, and column temperature was at38℃. The injection volume was10μL, detection time was85min. Using baicalin peak as a reference, the chromatograms of10batches of Lianggesan and negative control decoctions were established to analysis the overall quality of Lianggesan Decoction. Using the fingerprint similarity evaluation software to evaluate the similarity of10batches of samples. According to the characteristic peaks to analysis the herb attribution, to a certain extent indicate the material base of Lianggesan Decoction. Determination the content of Forsythoside A, Baicalin and Rhein in5batches of herbs at the same time under this chromatographic conditions, establishing its content determination method to provide a reference for the formation of quality standardof Lianggesan. 2.2The cell toxicity studies of Lianggesan, Forsythoside A and Baicalin on RAW264.7macrophagesRAW264.7macrophages incubated with low glucose DMEM in37℃and5%CO2in the environment until cell growth to90%, passage as1:3. Take the cell in logarithmic growth phase, adjust the cell concentration of1×104per well in96well plates, divided into normal control group and treatment groups. Treatment groups were given by Baicalin, Forsythoside A and Lianggesan with different concentrations. Detecting the cell toxicity of Forsythoside A, Baicalin and Lianggesan on RAW264.7macrophages by MTT assays. To determine the appropriate drug concentration by calculate cell survival rate, Experiment was divided into normal control group, model control group and LPS+drug groups, the OD value determined by MTT assays, calculate cell survival rate to test the effects of drugs on cell proliferation.2.3The effect of Lianggesan, ForsythosideA and Baicalinin LPS-stimulated RAW264.7macrophages.RAW264.7macrophages was treated by LPS to establish inflammatory model, Experiment is divided into normal control group, model control group and LPS+drug group.24h after drug treatment, collect the cell culture supernatant, The content of NO in supernatant was measured in NO assay kit. The content of IL-6and TNF-a in supernatant were measured in Enzyme-linked immunosorbent assay (ELISA) kits.2.4Statistical MethodsStatistical software SPSS13.0was used to analyze the experimenial data. The experimental data was indicated by mean ((?))±standard deviation (±s). The single-factor analysis of variance(ANOVA) was used to analyze the data. LSD method was used in multiple comparisons between groups when variance is homogeneous. Dunnett’sT3method was used when variance is not homogeneous. Chi-square test was used to deal with count data. P<0.05indicates that the data have statistical significance.3. Results 3.1The HPLC fingerprint of Lianggesan Decoction was established, the similarity of the10batches of samples were above0.9,22common peaks were marked and attributed to the herbs.8chromatographic peaks were identified. While established the method that simultaneous determination the content of main components Forsythoside A, Baicalin and Rhein in Lianggesan Decoction. Under this method, the linear relationship of Forsythoside A, Baicalin and Rhein is good, were in the range of10-160μg/mL,20.5-328μg/mL,3.125-50μg/mL respectively, the average recoveries (n=6) were98.8%,107.1%and102.5%respectively.3.2Detect the cell toxicity of different concentrations of Lianggesan, Forsythoside A and Baicalin on RAW264.7macrophages by MTT assays. According to the survival rate, the treatment group vs normal control group, P<0.05, the statistically difference was significant. The cell survival rate assumes the dose-response relationship. Forsythiaside A(100,10,5μg/mL), Baicalin(40,20,1μg/mL) and Lianggesan(40,10,1μg/mL) were the ultimately determined drug concentration. Give LPS(1μg/mL) stimulation induced RAW264.7cell inflammatory model, after treat with the concentration of drugs, the OD value determined by MTT assays. The model control group vs normal control group, P<0.05, the statistically difference was significant, the cell survival rate assumesproliferation effects of LPS on RAW264.7cell. Baicalin and Lianggesantreatment group vs model control group, P<0.05, the statistically difference was significant, the cell survival rate shows that the drugs could dose dependent inhibit the proliferation effects of LPS on RAW264.7cell. Forsythiaside A treatment group vs model control group, P<0.05, the statistically difference was significant, according to the cell survival rate, within a certain range of concentrations of Forsythiaside A appear proliferation effects to RAW264.7cells.3.3Give LPS stimulation induced RAW264.7cell inflammatory model, the NO content in supernatant of model control group was significantly higher than that of normal control group.The high, medium and low dose of three treatment groups could significantly reduce the level of NO in the cell culture supernatant. The IL-6and TNF-a level of model control group in cell culture supernatant significantly higher than that of normal control group, The high, medium and low dose of three treatment groups could significantly reduced the level of IL-6in the cell culture supernatant. The TNF-a level compared with model control group significantly reduced in high dose group of Forsythiaside A, high and low dose group of Baicalin, high and medium dose group of Lianggesan.4. Conclusions4.1The established HPLC-DAD fingerprint is accurate, stable, and reproducible, which could reflect the overall characteristics of Lianggesan Decoction and improve the specificity to quality control of Chinese herbal formula.4.2Established the method that simultaneous determination the content of main components Forsythoside A, Baicalin and Rhein in Lianggesan Decoction. The method is stable, have good linearity and repeatability, could be used as one of the quality control index of Lianggesan Decoction, lay a foundation for further study of its material basis and mechanism.4.3The MTT assays show that Forsythoside A, Baicalin and Lianggesan could inhibit the proliferation effects of LPS on RAW264.7cell.4.4After the success of LPS stimulated inflammatory model, the levels of inflammatory cytokines IL-6, TNF-a and NO increased significantly. It can significantly reduce these levels by dealing with Forsythoside A, Baicalin and Lianggesan. This may be preliminary indication of anti-inflammatory mechanism of Forsythoside A, Baicalin and Lianggesan. |
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