Screening Antioxidant Composition From Ziziphora Clinopodioides Lam. By On-line Hplc Method And Study On The Separation Of Chemical Constituents And Their Antioxidant Activity

The formation of chronic disease is closely related to free radicals in the body, includingdiabetes, AIDS, cardiovascular disease, aging and so on. Due to the side effects of syntheticantioxidants, more and more people have a tendency to choose efficient non-toxic naturalantioxidant. At present there are many methods used to measure plant antioxidants, but the rapiddetection methods is not enough. This article takes the Ziziphora clinopodioides Lam. as theresearch material, set up a new online screening methods to rapidly detect antioxidant activitycompounds in plant extracts and definitely choose the compounds after screening, thus provided anew way to search and find new drugs from natural products. Because the research of Ziziphoraaromatic is not thorough, therefore we build up a systematic approach to separate its chemicalcomposition and studied the active ingredients of the separated compounds. The main researchcontents and results are as follows:1. It has optimized the ceric sulfate method to test the antioxidant activity ingredient indifferent acidic media、temperature、reaction time、pH、methanol and acetonitrile volume fractionwhich may have diverse influences on test results. The optimal conditions were: the acidicmedium is sulfuric acid, the pH value is1, the temperature is room temperature, the reaction istwenty seconds, in fact there are almost no differences on the system between methanol andacetonitrile. Based on the preliminary study we have determined the antioxidant activity ofdifferent antioxidant activity material, which can provide reference data for the simulation of cericsulfate to study on the antioxidant activity of ingredients in natural products.2. It has established a rapid detection online system of antioxidant active ingredients incomplex mixtures using HPLC-Ce(SO4)2, the detection part optimization settings are as follows:the flow rate of mobile phase is1mL/min, the flow rate of Ce(SO4)2is0.2mL/min, the length ofreaction coil is3meter. Applying this system to analyze the antioxidant components of Ziziphoroaromatic, and managed separating the compounds which obtained after online screening. It hassucceeded in getting nine antioxidant activity substances through using a variety of means andafter using the NMR technique to analyze it can be confirmed these compounds are:protocatechuic acid(FT-1), rosmarinic acid(FT-2), caffeic acid(FT-3), luteolin(FT-4), pinocembrin-7-O-rutinoside(FT-5), baicalein(FT-6), kaempferide(FT-7), quercetin(FT-8), apigenin(FT-9).Among these FT-4, FT-5, FT-6and FT-9were first time isolated from this plant. It has a broaderapplicability compared with the existing online screening method.3. It has set up the method by using recycling preparative HPLC to segregate the chemicalcomposition in Ziziphora aromatic, and14compounds were isolated from Ziziphora aromatic bythe joint utilization of various chromatography techniques with silicagel medium voltagepreparation of liquid chromatography preparation of high pressure liquid chromatography andpope appraisal methods with1H NMR、13C NMR、HSQC、HMBC、ROESY、UV. Thesecompounds were respectively: picein(FT-10),4’,5,7-trihydroxyisoflavone(FT-11), irisdichototinD(FT-12), acacetin-7-O-rutinoside(13), chrysin-7-O-rutinoside(FT-14),4-hydroxyacetophenone-4-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside(FT-15),4-hydroxy-3-methoxy-acetophenone-4-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside(FT-16), Polybotrin(FT-17),1-O-p-coumaroyl glycerol(FT-18),5-(hydroxymethyl)-2-furancarboxaldehyde(FT-19), acacetin(FT-20), chrysin(FT-21),6,8-di-C-β-D-glucosylchrysin(FT-22), dibutyl phthalate(FT-23) and the FT-10~12, FT-15~19,FT-22~23were for the first time isolated from these compounds.4. It has adopted the96-well plate method then used the isolated monomeric compounds tomeasure the minimum inhibition concentration against the Staphylococcus aureus and theEscherichia coli. And then used the isolated target substance which with antioxidant activity toconduct in vitro antioxidant activity experiment. The results show that: quercetin,4’,5,7-trihydroxyisoflavone, kaempferide, rosmarinic acid, baicalein, acacetin-7-O-rutinoside,luteolin, protocatechuic acid,4-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside they can inhibitthe growth of Staphylococcus aureus and Escherichia coli. And the scutellarein has the strongestantibacterial activity, it can separately inhibit the growth of Staphylococcus aureus andEscherichia coli with the MIC of12.5μg/mL and25μg/mL；all of the online screened compoundshave some antioxidant activity, the difference between these compounds was just the strength ofthe activity and the quercetin shows the strongest antibacterial activity, the IC50values of DPPHand ABTS were17.25μg/mL and5.64μg/mL.