Research On Antioxidant Activity And Antioxidant Functional Beverage Of Masson Pine Needles

Background and purposePine needles (Pinus massoniana Lamb.) are needle-shaped leaves of pinaceae plant.In recent years, it is found that pine needle extracts contain a variety of natural antioxidantactive substances, such as flavonoids, wood, vitamin, polysaccharides and trace elements.Besides, it has non-toxic side effects. Therefore, masson pine needles are the goodmaterials of antioxidant and functional food ingredients.At present, the study of masson pine needle antioxidant activity mainly concentratedon some components may have antioxidant activity. Research on using antioxidantactivity as a quality monitoring parameter and obtaining the main antioxidant componentsfrom complex material system has not been reported. So our team used antioxidantactivity as a quality monitoring parameter to study the antioxidant activity and antioxidantfunctional beverage of masson pine needles.Before this research, we have studied the method of extraction, separation,purification and composition analysis of antioxidant system from masson pine needles,and the antioxidant extract function evaluation of anti-fatigue activity. On this basis, thisstudy improves the extraction and separation methods of antioxidant activity andevaluates the antioxidant function. At the meanwhile, expandly research series of masson pine needles functional beverage.Methods1The extraction processAntioxidant active ingredient of pine needles was extracted by solvent extractionmethod. Reasonable ranges of parameters were achieved through single factorexperiments, in which ratio of material to solvent, extracting solvent, extracting time andextracting temperature were investigated. Optimal conditions of extraction weredetermined by orthogonal tests.2Isolation and purificationAntioxidant active ingredient of pine needles was isolated and purified bymacroporous adsorption resin column chromatography method. Through static adsorptionand desorption experiment, screening suitable macroporous adsorption resin. After agraded elution, evaluate the total antioxidant capacity of fraction, choosing the highestone to the next research.3Antioxidant function evaluationAntioxidant function of masson pine needles was evaluated in vitro and in vivotests.The indexes in vitro test including total antioxidant capacity, hydroxyl radicalscavenging ability and super oxygen anion removal ability while In vivo tests are lipidperoxide (MDA) content, various antioxidant enzyme activity such as SOD, GSH-Px andCAT.4Composition analysesUsing ultraviolet-visible spectrum, Chemical chromogenic reaction and highperformance liquid chromatography-mass spectrometry (LC-MS) to get compoundretention time, molecular weight and other information, then combining database andliterature to analysis of pine needles antioxidant active ingredients.5Antioxidant function beverage developmentThe optimum technology parameters for producing the pine needles and green teacompound beverage that with higher antioxidant activity were investigated by orthogonaltest and sensory evaluation. Results1The extraction processBy single factor analysis and orthogonal experiment, the optimum extraction processof pine needles antioxidant active ingredients were as follows:0%ethanol extractionsolvent (ie, ultra-pure water), solid to liquid ratio was1:40,extraction temperature was80℃and extraction time was90min. Validation tests showed that total antioxidant capacityof the extract under optimal conditions of183.293U/ml. In this part, we also verified ourgroup in the study process that the water is the best solvent extraction.2Isolation and purificationCompared with AB-8, D101macroporous resin, NKA-9macroporous resin was bestsuited for separation and purification of pine needles antioxidant active ingredients. Basedon the study of sample volume, eluent concentration, eluent flow rate and eluentconsumption, we identified the NKA-9macroporous resin column chromatographyseparation and purification of pine needles antioxidant active ingredients conditions: thesample amount was9BV, eluent was40%ethanol elution rate3BV/h and the elutionvolume was3BV.By means of high performance liquid chromatography analysis, the purification ofpresent purified extract was better than the previous purified extract and the presentextract before purified.3Antioxidant function evaluationAt the same concentration, the total antioxidant capacity, the ability of removinghydroxyl radical and superoxide anion of PNPM-2were better than those of PNPM-1.In addition, the total antioxidant capacity of PNPM-2was close to Vc. Though thehydroxyl radical scavenging ability of PNPM-2is slightly lower than that of Vc, theability to remove superoxide anion free radicals at low concentration significantly higherthan that of Vc.In vivo experiment, compared with model group, masson pine needles antioxidantsand Vc can reduce serum MDA content, increase the mice serum SOD, GSH-Px and CATactivity level. The difference was statistically significant. The increase of mass concentration of PNPM-2, the more significant of their antioxidant power.4Composition analysesThough LC-MS/MS analysis, the chemical constituents of the main antioxidantcomponents from the strongest antioxidant fractions was certificated, which mainlycontain Catechin. It was consistent with previous results, that belong to flavonoidglycosides.5Antioxidant function beverage developmentThe optimum extraction conditions of compound beverage were at90℃for30minute，1:80(g: ml) mass ratio (pine to green tea was1:1(g: g)/water). The optimumrecipe of compound beverage was as follows:3%sugar,0.01%citric acid,0.1%honey.Finally, we can get the pine needles and green tea compound beverage that not only withspecial fragrance flavor but also with higher antioxidant activity. The total antioxidantcapacity of compound beverage was149.492U/ml, the hydroxyl radical scavengingcapacity was27.860U/ml and the tea polyphenols content was1941.183mg/kg.ConclusionUnder optimum conditions，we get the highest total antioxidant capacity extract frompine needles. Then by NKA-9macroporous adsorption resin column chromatography,and evaluate the antioxidant activity of purified liquid. Though LC-MS/MS analysis, thechemical constituents of the main antioxidant components from the strongest antioxidantfractions was certificated, which mainly catechins.Though orthogonal test and sensory evaluation, we can get the pine needles andgreen tea compound beverage that not only with special fragrance flavor but also withhigher antioxidant activity.