(2.1)--药为什么这样用药为什么这样用.pdf

《(2.1)--药为什么这样用药为什么这样用.pdf》由会员分享，可在线阅读，更多相关《(2.1)--药为什么这样用药为什么这样用.pdf（8页珍藏版）》请在得力文库 - 分享文档赚钱的网站上搜索。

1、Analytical MethodsOptimisation of the extraction of phenolic compounds from apples usingresponse surface methodologyAline Albertia,Accio Antonio Ferreira Zielinskia,Danianni Marinho Zardoc,Ivo Mottin Demiateb,Alessandro Nogueirab,Luciana Igarashi MafraaaFood Engineering Graduate Programme,Federal Un

2、iversity of Paran,Francisco H.dos Santos Street,CEP 81.531-990 Curitiba,Paran,BrazilbFood Science and Technology Graduate Programme,State University of Ponta Grossa,4748 Carlos Cavalcanti Av.,Uvaranas Campus,CEP 84.030-900 Ponta Grossa,Paran,BrazilcPharmaceutical Sciences Department,State University

3、 of Ponta Grossa,4748 Carlos Cavalcanti Av.,Uvaranas Campus,84030-900 Ponta Grossa,Paran,Brazila r t i c l ei n f oArticle history:Received 24 July 2013Received in revised form 30 September 2013Accepted 18 October 2013Available online 31 October 2013Keywords:Antioxidant capacityPhenolic profileHPLCS

4、olvent extractionBoxBehnken experimental designa b s t r a c tThe extraction of phenolic compounds from apples was optimised using response surface methodology(RSM).A BoxBehnken design was conducted to analyse the effects of solvent concentration(methanolor acetone),temperature and time on the extra

5、ction of total phenolic content,total flavonoids and anti-oxidant capacity(FRAP and DPPH).Analysis of the individual phenolics was performed by HPLC in opti-mal extraction conditions.The optimisation suggested that extraction with 84.5%methanol for 15 min,at28?C and extraction with 65%acetone for 20

6、 min,at 10?Cwere the best solutions for this combination ofvariables.RSM was shown to be an adequate approach for modelling the extraction of phenolic com-pounds from apples.Most of the experiments with acetone solutions extracted more bioactive com-pounds,and hence they had more antioxidant capacit

7、y,however,chlorogenic acid and phloridzin hadhigher yields(32.4%and 48.4%,respectively)in extraction with methanol.?2013 Elsevier Ltd.All rights reserved.1.IntroductionApples are the second most important fruit in the world(70 million tons)and are produced in temperate climate countries(Tropics of C

8、ancer and Capricorn).They are consumed throughoutthe year in most countries of the world,not only for their organo-leptic qualities,but also due to technological advancements in areaof conservation(Braga et al.,2013).Apples and their products contain significant amounts of phe-nolic compounds(Khaniz

9、adeh et al.,2008),which play an impor-tant role in maintaining human health,since they have apreventive effect against various types of diseases such as cancer,cardiovasculardiseases,neuropathiesanddiabetes(Shahidi,2012).Chlorogenic acid and p-coumaroylquinic acid are the mainphenolic acids found in

10、 apples;epicatechin,catechin,procyanidins(B1 and B2),quercetins glycosides,anthocyanins and phloridzinare the major flavonoids(Khanizadeh et al.,2008;Tsao,Yang,Xie,Sockovie,&Khanizadeh,2005).Tsao et al.(2005)reported thatamong the main phenols found in apples,cyanidin-3-galactosideand procyanidins h

11、ave antioxidant activity three times higherand twice as high,respectively,than epicatechin and glycosidesof quercitins.There is growing interest in the study of these bioactive com-pounds(Kchaou,Abbs,Blecker,Attia,&Besbes,2013;Spigno,Tramelli,&De Faveri,2007;Wijekoon,Bhat,&Karim,2011),andfor this pu

12、rpose,the first step is extracting them from the vacuolarstructures and other tissues where they are found(Wink,1997).The extraction conditions may not be the same for different plantmaterials since they are influenced by several parameters,suchas the chemical nature of the sample,the solvent used,a

13、gitation,extraction time,solute/solvent ratio and temperature(Haminiuk,Maciel,Plata-Oviedo,&Peralta,2012;Luthria,2008).In addition,the oxidation of phenolic compounds should be avoided,since theyare involved in the enzymatic browning reaction and consequentlylose their phenol function and antioxidan

14、t capacity(Nicolas,Richard-Forget,Goupy,Amiot,&Aubert,1994).It is advisable touse dry,frozen or lyophilised samples to avoid enzyme action(Escribano-Bailn&Santos-Buelga,2004).The optimisation of the extraction of phenolic compounds isessential to reach an accurate analysis.Response surface method-ol

15、ogy(RSM)is an effective tool for optimising this process.More-over,it is a method for developing,improving and optimisingprocesses,and it can evaluate the effect of the variables and theirinteractions(Farris&Piergiovanni,2009;Wettasinghe&Shahidi,1999).Thus,this study aimed to evaluate the effect of

16、concentrationsof the solvents,methanol and acetone,time and temperature on0308-8146/$-see front matter?2013 Elsevier Ltd.All rights reserved.http:/dx.doi.org/10.1016/j.foodchem.2013.10.086Corresponding author.Tel.:+55 42 32203775.E-mail address:.br(A.Nogueira).Food Chemistry 149(2014)151158Contents

17、lists available at ScienceDirectFood Chemistryjournal homepage: extraction of apple phenolic compounds and their antioxidantcapacity using RSM as the optimisation technique.2.Materials and methods2.1.MaterialsGala apples(10 kg)used in the experiments were obtained inthe city of Ponta Grossa(25?05042

18、00S 50?0904300O),Paran,Brazil.The reagents FolinCiocalteau,Trolox(6-hydroxy-2,5,7,8-tet-remethychroman-2-carboxylic acid),TPTZ(2,4,6-Tri(2-pyridyl)-s-triazine),DPPH(2,2-diphenyl-2-picrylhydrazyl),chlorogenicacid,p-coumaric acid,phloridzin,phloretin,(+)-catechin,(-)-epi-catechin,procyanidin B1,procya

19、nidin B2,quercetin,quercetin-3-D-galactoside,quercetin-3-b-D-glucoside,quercetin-3-O-rhamno-side,quercetin-3-rutinoside,caffeic acid and gallic acid werepurchased from SigmaAldrich(St.Louis,MO,USA).Methanol,ace-tone,acetic acid and acetonitrile were purchased from J.T.Baker(Phillipsburg,NJ,USA)and s

20、odium nitrite and aluminium chloridefrom Vetec(Rio de Janeiro,RJ,Brazil)and Fluka(St.Louis,MO,USA),respectively.The liquid nitrogen(99%)used was producedwith StirLIN-1(Stirling Cryogenics,Dwarka,New Delhi,India).The aqueous solutions were prepared using ultra-pure water(Milli-Q,Millipore,So Paulo,SP

21、,Brazil).2.2.Methods2.2.1.Extraction of phenolic compoundsThe apples were fragmented in a microprocessor(Metvisa,Brusque,SC,Brazil),immediately frozen with liquid nitrogen(1:2,w/v)to avoid the oxidation of the phenolic compounds(Guyot,Marnet,Sanoner,&Drilleau,2001),and lyophilised(LD1500,Terroni,So

22、Paulo,SP,Brazil).The freeze-dried material(without seeds)was homogenised by crushing in a mortar.1 g ofthe crushed apple was extracted with 60 mL of methanol or ace-tone in different concentrations,followed by incubation at differ-ent temperatures and times(Table 1).Then,the mixture wascentrifuged(8

23、160g,20 min at 4?C)(HIMAC CR-GII,Hitachi,Ibaraki,Japan),concentrated by evaporation under vacuum(40?C)in a ro-tary evaporator(Tecnal TE-211,Piracicaba,SP,Brazil),and freeze-dried.The samples were reconstituted with 2 mL of 2.5%aceticacid and methanol(3:1,v/v)and filtered through a 0.22lm(Nylon)syrin

24、ge filter(Waters,Milford,MA,USA)prior to analysis.2.2.2.Total phenolic content(TPC)The total phenolic content(TPC)was determined by colorimet-ric analysis using FolinCiocalteau reagent,as described bySingleton and Rossi(1965).In a test tube,8.4 mL of distilled water,100lL of sample,and 500lL of Foli

25、nCiocalteau reagent wereadded.After 3 min,1.0 mL of 20%sodium carbonate was addedinto each tube,which was agitated in a vortex(Vision ScientificCO.LTD.,Korea).After 1 h,the absorbance(720 nm)was measuredby spectrophotometer(model Mini UV 1240,Shimadzu,Kyoto,Ja-pan).The measurement was compared to a

26、calibration curve ofchlorogenic acid total phenolic concentration=1473.3?absor-bance;R2=0.998;p 0.001 and the results were expressed as mil-ligrams of chlorogenic acid equivalents(CAE)per kilogram of applemg CAE/100 g.2.2.3.Total flavonoid content(TFC)The total flavonoid content(TFC)of the phenolic

27、extracts wasdetermined using a method described by Zhishen,Mengcheng,and Jianming(1999)with modifications.250lL of the sampleswas mixed with 2.72 mL of ethanol(30%,v/v)and 120lL of sodiumnitrite solution(0.5 mol/L).After 5 min,120lL of aluminum chlo-ride(0.3 mol/L)was added.The mixture was stirred a

28、nd was al-lowed to react for 5 min.Then,800lL of sodium hydroxide(1 mol/L)was added and the absorbance was measured at 510 nmusing a spectrophotometer(model Mini UV 1240,Shimadzu,Kyoto,Japan).The measurement was compared to a calibration curve ofcatechin(CT)flavonoidconcentration=755.37?absorbance;R

29、2=0.996;p 0.001 and the results were expressed as milligramsof catechin equivalents(CTE)per kilogram of apple mg CTE/100 g.2.2.4.Measurement of in vitro antioxidant capacityFree-radical scavenging activity of the extracts was determinedin triplicate by the DPPH assay according to the Brand-Williamsm

30、ethod,Brand-Williams,Cuvelier,and Berset(1995)with minoradaptations.This method determines the hydrogen donatingcapacity of molecules and does not produce oxidative chain reac-tions or react with free radical intermediates.Diluted samples(100lL)were mixed with 3.9 mL of 60lmol/L methanolic DPPH.Tabl

31、e 1BoxBehnken design applied for apple phenolic compounds extraction.RunFactorsTime(min)Temperature(?C)Solvent concentration(%)1?1?102+1?103?1+104+1+105?10?16+10?17?10+18+10190?1?1100+1?1110?1+1120+1+1130001400015000True valuesaMethanolAcetone?110107050015258565+1204099.980aValues adopted for each f

32、actor in the phenolic extraction experiment.152A.Alberti et al./Food Chemistry 149(2014)151158The absorbance was measured at 515 nm using a spectrophotom-eter(model Mini UV 1240,Shimadzu,Kyoto,Japan)after the solu-tion had been allowed to stand in the dark until stabilisation(timepreviously determin

33、ated).Antiradical capacity was defined as theamount of apple necessary to decrease the DPPH concentrationby 50%,EC50.The lower the EC50,the higher the antioxidant power.The total antioxidant potential of the extracts was determinedin triplicate using the ferric reducing antioxidant power(FRAP)as-say

34、 as described by Benzie and Strain(1996)with minor modifica-tions.The assay is based on the reducing power of antioxidantspresent in extracts,in which a potential antioxidant reduces theferric ion(Fe3+)to ferrous ion(Fe2+);the latter forms a blue com-plex(Fe2+/TPTZ).Absorbance of the FRAP reagent(3

35、mL)was takenat 593 nm and after sample addition(100lL);it was monitored forup to 6 min.To calculate the antioxidant capacity,the change inabsorbance between the FRAP reagent and the mixture after6 minofreaction,wascorrelatedwithacalibrationcurve(FRAP=805.81?absorbance;R2=0.999;p 0.001)ofTrolox(0.11.

36、0 mmol/L).The results were expressed inlmoL Troloxequivalents per kilogram of apple(lmoL TE/100 g).2.2.5.Experimental designIn order to evaluate the extraction parameters and optimise theconditions of apple phenolic extraction,a Box and Behnken(1960)design was used.The effect of the independent vari

37、ables extractiontime(min),X1,extraction temperature,X2,and the concentration ofthe solvent,X3,at three variation levels were evaluated in theextraction process(Table 1).The fifteen experiments were con-ducted to analyse the response pattern and to establish modelsfor phenolic extraction,with methano

38、l and acetone solutions sep-arately.All experiments were carried out randomly.A second-order polynomial equation was used to fit the experi-mental data of the studied variables.The generalised second-orderpolynomial model used in the response surface analysis is shownin Eq.(1):Y b0X3i1biXiX3i1biiX2i

39、X2i1X3ji1bijXiXj1where Y is the predicted response,b0,bi,biiand bijare the regres-sion coefficients for intercept,linear,quadratic and interactionterms,respectively,and Xi,and Xjare the independent variables(Bruns,Scarmino,&Barros Neto,2006).The statistical significanceof the terms in the regression

40、 equations was examined by ANOVAfor each response.The terms statistically found as non-significantwere excluded from the initial model and the experimental datawere re-fitted only to the significant(p 6 0.05)parameters.Thesimultaneous optimisation was obtained by the desirability func-tion proposed

41、by Derringer and Suich(1980).The optimised condi-tions of the independent variables were further applied to validatethe model,using the same experimental procedure as made previ-ously,in order to verify the prediction power of the models bycomparing theoretical predicted data to the experimental dat

42、a.Triplicate samples of the optimised proportion were prepared andanalysed.2.2.6.HPLC analysis of phenolic compounds in optimum conditionsThe HPLC apparatus was a 2695 Alliance(Waters,Milford,MA,USA),with photodiode array detector PDA 2998(Waters,Milford,MA,USA),quaternary pump and auto sampler.Sepa

43、ration was per-formed on a Symmetry C18(4.6?150 mm,3.5lm)column(Waters,Milford,MA,USA)at 20?C.The mobile phase was composed of solvent A(2.5%acetic acid,v/v)and solvent B(acetonitrile).The following gradient was ap-plied:39%B(05 min),916%B(515 min),1636.4%B(1533 min),followed by an isocratic run at

44、100%of B(5 min)and reconditioning of the column(3%of B,10 min).The flow ratewas 1.0 mL/min.Identification of phenolic compounds was per-formed by comparing their retention time and spectra with thoseof standards.The runs were monitored at 280 nm(flavan-3-olsand dihydrochalcones),320 nm(hydroxycinnam

45、ic acids)and350 nm(flavonols).Quantification was performed using calibra-tion curves of standards(at least seven concentrations were usedto build the curves)(Table 2).2.2.7.Statistical analysisData were presented as mean and standard deviation(SD)orpooled standard deviation(PSD).All variables had th

46、eir varianceanalysed using the F test(two groups)orby Hartleys test(p P 0.05).Differences among groups were assessed by means ofStudent-t test for independent samples(two groups)or one-wayANOVA followed by Fisher LSD test.Pearson products(r)were usedto evaluate the strength of correlation among the

47、parameters eval-uated.A p-value below 0.05 was considered significant.All statis-tical analyses were performed using Statistica 7.0(StatSoft Inc.,USA).3.Results and discussion3.1.Optimisation of extraction using methanol as solventThe mean values of the total phenols,flavonoids,DPPH andFRAP of the e

48、xtraction performed on apples with methanol areshown in Table 3.The total phenols of the methanol extraction ran-ged statistically(p 0.001)from 457.93(assay number 8)to599.09 mg/100 g(central point).The highest values for total phe-nols were observed at the central point of the experimental designwi

49、th 85.0%methanol for 15 min at 25?C(central point).The multiple regression analysis of total phenol values showedthat the model was significant(p 0.001),did not present lack offit(p=0.16)and it could explain 80.91%of all variance in data(R2adj=0.80).The quadratic regression coefficient of concentrat

50、ion(X3)was negative and significant.The predicted model can be de-scribed by the(Eq.2)in terms of coded values.Y 578:93?80:83X232The results suggested that time and temperature had negligibleeffects on the yield of total phenols.The extraction of flavonoids ranged significantly(p 0.001)from 106.81(a