| In the present paper,a high efficient,fast,easy and accurate method was developed for the analysis the phenolic compounds from Geranium sibiricum Linne.Seasonal variation of corilagin,geraniin as well as total phenolic contents during the growth period,where were from the whole grass,roots,stems and leaves,were investigated and then,the antioxidant capacity were evaluated to confirm the optimum picking part and stage.The feasibility of MAEE(microwave-assisted enzymatic extraction)were evaluated and the important parameters involved in the process were optimized,whilst,the antioxidant capacity of the crude extracts of MAEE under optimal conditions were evaluated though FRAP and DPPH assays.The feasibility and the effect of acid or alkali hydrolysis in microwave-assisted extraction as well as antibacterial activity of the extracts were evaluated to selected appropriate phenols extraction method,after the MAE acidic hydrolysis method was selection,single factor,CCD(central composition design)and first order equation experiment were employed to optimize the conditions and then,the SEM(scanning electron microscope)experiment was conducted to verify the destructive effects of the method.Antioxidant capacity of the different fraction of the crude extracts were evaluated to select the best fraction as well as the antioxidant property of corilagin and geraniin,the main phenolic compounds from G.sibiricum Linne,were investigated by reducing antioxidant power(FRAP),1,1-diphenyl-2-picrylhydrazyl(DPPH),NBT(Superoxide Radical Scavenging Assay[Nitroblue Tetrazolium Chloride(NBT)Assay]),?-carotene-linoleic acid bleaching and reduce power assays.1.The HPLC-UV method was developed to determinate the phenolic compounds from G.sibiricum Linne,The HPLC-UV method was developed for simultaneous determination corilagin and geraniin contents:Chromatographic conditions:HiQ Sil C18V reversed-phase column(250 mm × 4.6 mm I.D.),mobile phase consisted of acetonitrile and 0.1%formic acid aqueous solution,flow rate 1.0 mL/min,injection volume 10 ?L and column temperature 30?,the UV detector was set at the wavelength of 220 nm.2.Seasonal variation in antioxidant compositions of Geranium sibiricum Linne.The results were as follows:(1)The seasonal variation of weights of the whole grass,leaves and stems of G.sibiricum Linne during the growth period were similar,which existed the fast growth(from May to June),slow growth(from June to July)and withering period(from August to September)against the weight of the root with fast growth period(from May to July)and slow growth period(from July to September).(2)Corilagin(CG),geraniin(GE)and total phenolic contents increased dramatically in the vegetative growth period and decreased gradually during the reproductive growth and withering period,while the total flavonoid contents presented the reverse phenomenon.(3)The optimum picking part and time were the whole grass and July,respectively,in terms of the evaluation of total antioxidant capacity assay,which was 11.24±0.28 mmol Fe(?)/g extracts.(4)High relationship between corilagin,geraniin and total phenolic contents from G.sibiricum Linne as well as good relationship between the antioxidant capacity evaluated by DPPH,FRAP and ABTS assay were obtained.(5)Correlation of the data obtained from PCA(principal composition analysis)showed that corilagin,geraniin and total phenolic contents were major contributors to the antioxidant capacity and they could be responsible for the property.3.Optimization of microwave-assisted enzymatic extraction of corilagin and geraniin from G.sibiricum Linne and evaluation of antioxidant activity.The results were as follows:(1)MAE and enzyme can be combined to enhance the extraction yields of corilagin and geraniin.In addition,any organic solvent was not used in the method.(2)The optimal extraction conditions were as follows:irradiation power 500 W,ratio of solvent to material 40 mL/g,irradiation temperature 33?,pH 5.2,amount of cellulase 3600 U/g and irradiation time 9 min.Under these conditions,the extraction yields of CG and GE achieved 6.79 and 19.82 mg/g,which increased 64.01%and 72.95%,respectively,as compared with the control ones.(3)Through the verification of the model,the extraction conditions obtained by response surface methodology were not only accurate and reliable,but also with practical value reflecting the expected optimization.(4)Applying the MAEE method,the antioxidant activities of crude extracts were 2.61 mmol FeS04/g DW and 0.118 mg/mL(IC50),according to the FRAP and DPPH assays,respectively.(5)Our results show that MAEE is an alternative method for the natural products extraction from plants because of its higher efficiency and environmental friendship.4.Effect of acid hydrolysis in the microwave-assisted extraction of phenolic compounds from G.sibiricum Linne with the guidance of antibacterial activity.The results were as follows:(1)MAE with HCl hydrolysis method can not only enhance the extraction yields of corilagin,geraniin as well as total phenolic content from G.sibiricum Linne,but also improve its antoxidant activity.(2)The optimal conditions were as follows:irradiation power 500 W,ratio of solvent to material 25 mL/g,irradiation temperature 33?,ethanol concentration 22%,concentration of HCl 0.03 mol/L and irradiation time 8 min during the extraction process,the extraction yields of TPC,CG and GE were 201.51 mg GAE/g,14.75 and 45.17 mg/g,which represented an increase of 28.60%,53.80%and 35.65%,respectively,as compared with only 20%in MAE experiments.(3)Through the verification of the model,the extraction conditions obtained by response surface methodology were not only accurate and reliable,but also with practical value reflecting the expected optimization.(4)From SEM micrograph observation,HCl hydrolysis had more destructiveness effect on plant cells than other extraction methods,which can be a confirmation for its strong extraction efficiency.(5)Our results showed that HCl hydrolysis in MAE was an alternative method for the natural products extraction from plants because of its higher efficiency and shorter extraction time.The research also provides a basis for further exploitation and use of the nature resources on human health.5.Antioxidant capacity of different fraction and main phenolic compounds from G.sibiricum Linne.The results were as follows:(1)The total antioxidant capacity experiments demonstrated that the ethyl acetate and n-butyl fractions from the ethanol extraction of G.sibiricum Linne had better antioxidant capacity,which were 17.32 and 16.71 mmol Fe(?)/g extracts,respectively.(2)After partition the crude extraction with petroleum ether,ethyl acetate and n-butanol,successively,the contents of corilagin,geraniin and total phenolic of each fraction were detected,the result indicated that the ethyl acetate and n-butanol fraction had more contents,which were 67.71±0.72 mg/g?211.91 ±3.42 mg/g?450.21 mg GAE/g extracts and 50.32 mg/g?137.36 mg/g,372.15mg GAE/g extracts,respectively.(3)The antioxidant capacity of different fractions were detected in terms of FRAP,DPPH,NBT,nitric oxide(NO)scavenging,?-carotene-linoleic acid bleaching,and reducing power assays,the results showed that the ethyl acetate fraction had the best antioxidant capacity,the corresponding IC50 value were 0.03822?0.063?0.0061?0.047 and 0.00606 mg/mL,respectively.(4)The correlation between corilagin,geraniin and total phenolic contents and antioxidant capacity,obtained from different antioxidant assays,indicated that the major contributors of the property were corilagin and geraniin.(5)The antioxidant property assays demonstrated that the antioxidant capacity of geraniin was better than that of corilagin at all respect of superoxide anion radicals,hydroxyl radicals and peroxyl radicals scavenging,on the base of FRAP,DPPH,NBT,NO,?-carotene-linoleic acid bleaching assays,and the corresponding IC50 value were 0.032?0.0337?0.0023?0.0387 and 0.0034 mg/mL. |
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