| Aim:Abnormal Savda Munziq is commonly used to clinical preparation of Uygur medicine, is a combination of extensive clinical practice developed with Uighur medicine featuring classic formulation. The formulation composed of ten medicinal herbs, namely Alhagi pseudoalhagi Desv., Cordia dichotoma Forst.f, Anchusa italica Retz., Ziziphus jujuba Mill., Glycyrrhiza uralensis Fisch., Lavandula angustifolia Mill, et al. The preparation has proven simple savda abnormal body fluids to the body, diuretic, anti-inflammatory and detoxifying functions, primarily for the treatment of abnormal savda caused by cancer, diabetes and cardiovascular disease adjuvant therapy.The purpose of this paper is to establish Abnormal Savda Munziq and quality control methods, and improve the prescription herbs Cordia dichotoma fruits substances basic research; establishing Abnormal Savda Munziq and the prescription herbs Cordia dichotoma fruits qualitative and quantitative analysis; to determine Abnormal Savda Munziq and the prescription herbs Cordia dichotoma fruits in vitro antioxidant activity; Evaluation of herbs compound and the prescription herbs Cordia dichotoma fruits different extracts of mouse spleen lymphocyte proliferation and human colon cancer HCT116cells; providing the basis research data for the clinical application of Abnormal Savda Munziq and its prescription herbs Cordia dichotoma fruits.Methods:1. Qualitative identification method using thin-layer chromatography, the Abnormal Savda Munziq and its single herbs of the prescriptions qualitative analysis by TLC, summed Abnormal Savda Munziq and its single herbs in the thin-layer chromatographic behavior.Carried out of the herbs Cordia dichotoma fruits on the Medicine fruit morphology, traits, microscopic, TLC study. Phytochemical system using pre-test method Cordia dichotoma fruits in the chemical composition of the pre-experimental study, initially identified Cordia dichotoma fruits in chemical composition type. Using the Folin-Ciocalteu colorimetric determination of total phenolic content; With5%vanillin-acetic acid and perchloric acid colorimetry triterpene acid content; The use of2%ninhydrin and phosphate buffer (pH=6.80) colorimetric determination of amino acids.2. Application TLCS, HPLC-DAD, GC-MS method for Abnormal Savda Munziq (decoction) and the prescription herbs Cordia dichotoma fruits qualitative and quantitative analysis. Determined by TLCS Abnormal Savda Munziq (decoction) of rutin, quercetin, rosmarinic acid, glycyrrhizic acid content; HPLC-DAD determination six phenolic compounds including gallic acid, protocatechuic acid, caffeic acid, rutin, rosmarinic acid, luteolin and glycyrrhizic acid in the Abnormal Savda Munziq (decoction). According to the corresponding peaks in the chromatogram retention time and UV spectral data of the standard products test sample and spiked samples, the abnormal black Munziq (decoction) contains six kinds of phenolic compounds and glycyrrhizin qualitative identification and quantitative analysis. Qualitative and quantitative analysis six phenolic compounds and glycyrrhizin in Abnormal Savda Munziq (decoction). By a thin layer of qualitative analysis shows Abnormal Savda Munziq in Cordia dichotoma Forst.f, Melissa officinalis L., Foeniculum vulgare Mill., Anchusa italica Retz., Lavandula angustifolia Mill, contain rosmarinic acid, therefore, HPLC-MS" qualitative, HPLC quantitative determination rosmarinic acid of Cordia dichotoma fruits. Using C6-C20n-alkanes series of standard samples mixed with the Abnormal Savda Munziq (decoction) volatile oil, measured by GC-MS, get Abnormal Savda Munziq (decoction) volatile oil total ion current through U.S. NIST07and NIST07s mass spectral library and retention index retrieval, analysis to determine the chemical composition, the use of peak area normalization method to calculate the content of each component.3. Establish four kinds of anti-oxidation model including scavenging ability, DPPH· radical scavenging capacity、superoxide anion radical scavenging effect、the SOD activity rate、the hydroxyl radical scavenging for Abnormal Savda Munziq (decoction) and Cordia dichotoma fruits extracts, compared the differences between the different extracts and different antioxidant models models.Using concanavalin ConA (stimulating T lymphocytes specific reagents) and LPS (stimulated B lymphocytes proprietary reagent) reagent model to study Abnormal Savda Munziq (decoction), Cordia dichotoma fruits extracts, and rosmarinic acid standard on mouse spleen lymphocyte proliferation. Using human colon cancer cell line (HCT116) as an object of study, MTT assay detect different concentrations of Abnormal Savda Munziq (decoction), Cordia dichotoma fruits extracts, and rosmarinic acid standard on cell proliferation.Results:1. After repeated TLC experimental results show Abnormal Savda Munziq (decoction) with various herbs in the developing solvent ethyl acetate-formic acid-acetic acid-water (15:1:1:1.5) expand, expand more fully, and bands were clear obviously, where Abnormal Savda Munziq (decoction) bands with relative good agreement herbs. Glycyrrhizin (Rf=0.11) and liquiritin (Rf=0.54) is Abnormal Savda Munziq (decoction) prescription herbs Glycyrrhiza uralensis Fisch. provided features ingredients, and its chromatographic bands were yellow-brown and yellow; rutin (Rf=0.17) by the prescription herbs Adiantum capillus-veneris L., Lavandula angustifolia Mill., Foeniculum vulgare Mill., Euphorbia humifusa Willd., Alhagi pseudoalhagi Desv. provided; chlorogenic acid (Rf=0.18) by the prescription herbs Adiantum capillus-veneris L. provided,; rosmarinic acid (Rf=0.81) by the prescription herbs Cordia dichotoma Forst.f, Anchusa italica Retz., Melissa officinalis L., Lavandula angustifolia Mill., Foeniculum vulgare Mill, provided.Cordia dichotoma fruits conical ovate, often significant longitudinal shrinkage or mesh-like irregular edges, peel long oval cross-section, tangent extended outer cuticle; mesocarp of the parenchyma, scattered with stone cells, and scattered small vascular bundles. Stone cells were polygonal, round or oblong, endosperm cells polygonal, and a lot of fluids containing intends crystal; catheter pitted more. The variation coefficient of the fruit length, fruit diameter, fruit shape index were0.0720%,0.1298%,13.3800%, respectively. Carried out on Cordia dichotoma fruits TLC, the results show that:Anhui Huoshan G plate production with toluene:ethyl acetate:formic acid (3:7:1) as developing solvent separation is better. Cordia dichotoma fruits contain carbohydrates, amino acids and proteins, alkaloids, tannins, organic acids, volatile oil, polyphenols, myxoid stroma. There were undetected coumarin, steroids and terpenes, lactones, flavonoids. Measured by colorimetry Cordia dichotoma fruits of total polyphenols, total triterpene acids, total amino acids, the results showed that:80%ethanol extract total polyphenol contents and total triterpene acid contents was1.00%,0.075%, respectively. Aqueous extract of total amino acids was1.39%.2. TLC scanning method from Abnormal Savda Munziq identified four kinds of chemical components, namely rutin (Rf=0.16), rosmarinic acid (Rf=0.79), glycyrrhizin (Rf=0.09), licorice glycosides (Rf=0.54), the results showed that:GF254plate in accordance with developing ethyl acetate-formic acid-acetic acid-water (15:1:1:1.5) solvent expand and the separation effect is good, the separation is high, compared to silica gel G plate and H plate, no color, and the spots were clear focus.In the methodological study, a standard curve was linear relationship, and the regression coefficient R were greater than0.999; precision, stability and reproducibility, and the recovery well, limit of determination (LOD) in the27.5-38.17ng between the limit of quantitation (LOQ) in between91.80-127.26ng. In the determination of the three batches abnormal black Munziq in rosmarinic acid (1.8983±0.097mg/g) as the most common ingredients in herbs highest, followed by glycyrrhizin (1.3847±0.047mg/g), rutin content of0.3887±0.014mg/g, liquiritin content of0.3253±0.007mg/g.RP-HPLC-DAD gradient elution techniques from Abnormal Savda Munziq were identified six kinds of phenolic compounds, namely gallic acid, protocatechuic acid, caffeic acid, rutin, rosmarinic acid, luteolin. In order to obtain a better resolution and shorter analysis time, the chromatographic conditions were optimized, including the column, the type of reverse-phase column, the extraction solvent, the detection wavelength and the mobile phase.The results showed that the optimal separation conditions were:Column Agilent TC-C18column (4.6mm×250mm,5μm); mobile phase A phase of0.3%formic acid in methanol, B phase containing0.3%formic acid solution, a linear gradient program starts100%B;0-15min,100%～92%B;30min～50min,83%～75%B;50min～80min,75%～55%B;80min～100min,55%～40%B; flow1mL/min; column temperature was30℃; detection wavelength290nm; injection volume was10μL. The content of gallic acid, protocatechuic, caffeic acid, rutin, rosmarinic acid, luteolin in Abnormal Savda Munziq was:0.481,0.0675,0.119,0.437,0.836,0.198mg/g. In methodological study, the standard curve was linear relationship, and the regression coefficient R were higher than0.999; precision, stability, repeatability, and the recovery is good.Experiment standard "Chinese Pharmacopoeia"(2010edition) reproduces method, during the experimental conditions explored, the use of RP-HPLC-DAD gradient elution method was modified gradient experiments to measure glycyrrhizic acid. Experimental results show that glycyrrhizin in Abnormal Savda Munziq in an amount of2.71mg/g, this method is rapid, simple, accurate, reproducible, and can be used to Abnormal Savda Munziq glycyrrhizic acid determination.The method of reversed-phase high-performance liquid chromatography-tandem mass spectrometry used to qualitatively and quantitatively measured rosmarinic acid in Cordia dichotoma fruits by confirmed relative retention with the standard value and the two fragments on the spectrum. By orthogonal design optimize the extraction of rosmarinic acid in Cordia dichotoma fruits to obtain the optimal extraction conditions: ethanol concentration of70%, solid-liquid ratio of1:35g/mL, ultrasonic time was30min, ultrasonic power of300W. Based on the obtained optimal extraction rate0.1071%. Iquid chromatography-tandem mass spectrometry qualitative usage sensitive, accurate and reliable, liquid chromatography assay method is simple and repeatable.The Abnormal Savda Munziq volatile oil chemical components analyzed by capillary chromatography, by gas chromatography data processing area normalization method measured the relative percentage of each component, and the method of gas chromatography-mass spectrometry techniques conjunction used in volatile oil analyzed by gas chromatography tandem mass spectrometry total ion chromatography. The computer database searches with the standard mass spectrum control, analytical and standards were identified18major compounds, wherein the trans-anethole (83.97%) or more, which determine the Abnormal Savda Munziq is the main component of volatile oil trans-anethole. According to the literature, anethole is Abnormal Savda Munziq prescription medicines Foeniculum vulgare Mill.’s main ingredient, known the main volatile oil component of Abnormal Savda Munziq from prescription medicines Foeniculum vulgare Mill.3. Experiments established Abnormal Savda Munziq and the prescription herbs Cordia dichotoma fruits scavenging ability, DPPH· radical scavenging capacity, superoxide anion radical scavenging effect, t the SOD activity rate、the hydroxyl radical scavenging effect four kinds of anti-oxidation model. Abnormal Savda Munziq antioxidant capacity compared with the positive control Vc weak.The different of Cordia dichotoma fruits extracts antioxidant capacity is as follows:Hydroxyl radical scavenging general trend is:80%ethanol extract>Vc>purified80%ethanol extract. On DPPH· radical scavenging general trend is:When the solution concentration<40μg/mL time, Vc> purified80%ethanol extract>80%ethanol extract. When the solution concentration>80μg/mL, the80%ethanol extract>Vc> purified80%ethanol extract. On superoxide anion radical scavenging overall trend:Vc>80%ethanol extract>purified80%ethanol extract. SOD activity rate on the overall trend:Vc>80%ethanol extract> purified80%ethanol extract. Cordia dichotoma fruits80%ethanol extracted antioxidant capacity of the overall trend:The DPPH· radical scavenging effect> The hydroxyl radical scavenging effect> The superoxide anion radical scavenging>The SOD activity rate. Cordia dichotoma fruits purified80%ethanol extract antioxidant capacity of the overall trend is:The DPPH· radical scavenging effect>The hydroxyl radical scavenging effect> The SOD activity rate> The superoxide anion radical scavenging. Vc antioxidant capacity of the overall trend is:The superoxide anion radical scavenging> The SOD activity rate> The DPPH· radical scavenging effect> The hydroxyl radical scavenging effect.Experiments established concanavalin ConA (stimulated T lymphocytes specific reagents) and LPS (stimulated B lymphocytes proprietary reagent) reagent model to investigate Abnormal Savda Munziq, Cordia dichotoma fruits extract and rosmarinic acid standard on splenic lymphocyte proliferation. The results showed Abnormal Savda Munziq, Cordia dichotoma fruits crude extract, Cordia dichotoma fruits polysaccharides can significantly improve the in vitro lymphocyte proliferation. On splenic lymphocyte proliferation overall trend:Cordia dichotoma fruits crude extract> rosmarinic acid standard> Cordia dichotoma fruits polysaccharides> Abnormal Savda Munziq (decoction).In this study, human colon cancer cell line (HCT116) as an object of study, MTT assay at different concentrations Abnormal Savda Munziq, Cordia dichotoma fruits water extract and rosmarinic acid standard on cell proliferation. The results showed that: Abnormal Savda Munziq, Cordia dichotoma fruits aqueous extract and rosmarinic acid standard on human colon cancer cell line HCT116inhibition of proliferation has a certain role. The general trend of the inhibition of rosmarinic acid standard> Abnormal Savda Munziq> Cordia dichotoma fruits aqueous extract, and Cordia dichotoma fruits polysaccharide HCT116human colon cancer cell lines in vitro proliferation without inhibition.Conclusion:1. TLC carried out on the Abnormal Savda Munziq and its various medicinal prescriptions studies, established Abnormal Savda Munziq and its various medicinal prescriptions TLC material composition assessment base. Research methods and results of Abnormal Savda Munziq of component separation and quality control methods provide a foundation. In this study, thin-layer chromatography Abnormal Savda Munziq chemical substances based on a preliminary identification of the ingredients by Abnormal Savda Munziq with the single taste herbs, medicinal herbs negative control and standard Rf value and color contrast, good separation can be identified, higher levels of chemical composition. By thin layer chromatography can be directly identified Abnormal Savda Munziq contains rutin, rosmarinic acid, glycyrrhizin, glycyrrhizin and chlorogenic acid; preparations quercetin, gallic acid, luteolin, ferulic acid, caffeic acid temporarily can not be identified, need to HPLC-UV, HPLC-MS, GC-MS and other more sophisticated equipment identification.Cordia dichotoma fruits after pre-test chemical composition, can be initially judged contains carbohydrates, amino acids and proteins, alkaloids, tannins, organic acids, volatile oil, polyphenols, myxoid stroma. Undetected coumarin, steroids and terpenes, lactones, flavonoids. Cordia dichotoma fruits carried on TLC research shows that its chemical composition at a wavelength of365run UV lamp is mostly blue-purple. Measured by colorimetry Cordia dichotoma fruits of total polyphenols, total triterpene acids, total amino acids.2. Experiments using TLCS qualitative and quantitative analysis of the Abnormal Savda Munziq of glycyrrhizin, glycyrrhizic acid, rutin and rosmarinic acid content, RP-HPLC-DAD gradient elution techniques qualitative and quantitative analysis the of the six kinds of phenolic compounds and glycyrrhizin in Abnormal Savda Munziq. By gas chromatography tandem mass spectrometry capillary chromatography method analyzed essential oils in Abnormal Savda Munziq, identified a total of18kinds of major compounds, trans-anethole (83.97%) or more.3. Experimental studied the Abnormal Savda Munziq and the prescription herbs Cordia dichotoma fruits antioxidant capacity and found that, Abnormal Savda Munziq water extract, Cordia dichotoma fruits80%ethanol extract and purified80%ethanol extract all have antioxidant capacity.Experiment closely integrated Chinese chemical and immunology, to study Cordia dichotoma fruits on splenic lymphocyte proliferation. The results showed Abnormal Savda Munziq, Cordia dichotoma fruits crude extract, Cordia dichotoma fruits polysaccharides can significantly improve the in vitro lymphocyte proliferation.Inhibiting the proliferation of human colon cancer cells was found Abnormal Savda Munziq extract, Cordia dichotoma fruits aqueous extract and rosmarinic acid standard showed some inhibition,Based on the Abnormal Savda Munziq and the prescription herbs Cordia dichotoma fruits quality control studies and in vitro activity tests, the establishment of Abnormal Savda Munziq qualitative and quantitative analysis methods, perfect set of prescription herbs Cordia dichotoma fruits quality standards, research component in vitro activity for Abnormal Savda Munziq and group parties herbs Cordia dichotoma fruits provide the basis for the clinical application of research data. |
PDF Full Text Request