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1、Contents lists available at ScienceDirectLWT-Food Science and Technologyjournal homepage: of ultrasound-assisted extraction of natural antioxidants fromPiper betle using response surface methodologyAmeena Alia,Xiao Yien Limc,Chien Hwa Chongd,Siau Hui Mahb,Bee Lin ChuaaaSchool of Engineering,Taylors

2、University,Lakeside Campus,No 1,Jalan Taylors,47500 Subang Jaya,Selangor,MalaysiabSchool of Biosciences,Taylors University,Lakeside Campus,No 1,Jalan Taylors,47500 Subang Jaya,Selangor,MalaysiacSchool of Engineering and Physical Sciences,Heriot-Watt University Dubai Campus,Dubai International Academ

3、ic City,P.O.Box 294345,Dubai,United Arab EmiratesdSchool of Engineering and Physical Sciences,Heriot-Watt University Malaysia Campus,No 1 Jalan Venna P5/2,Precinct 5,62200 Putrajaya,MalaysiaA R T I C L E I N F OKeywords:Piper betleUltrasound extractionOptimizationTotal phenolic contentAntioxidant ac

4、tivityChemical compounds studiedin this article:HydroxyChavicol(PubChem CID:70775)Eugenol(PubChem CID:3314)Isoeugenol(PubChem CID:853433)4-Allyl-1,2-Diacetoxybenzene(PubChem CID:166872)A B S T R A C TNatural antioxidants are excellent substitute for their synthetic counterparts in dietary supplement

5、s.Presentstudy employed three-level Box-Behnken design through RSM to optimize the recovery of natural antioxidantsfrom Piper Betle via ultrasound-assisted extraction(UAE).The influence of three extraction parameters,tem-perature(5070 C),ethanol concentration(7090%)and solute to solvent ratio(1:101:

6、30 g/mL)on the ex-traction yield(EY),total phenolic content(TPC)and antioxidant capacity was investigated.The optimumconditions were determined to be 51.60 C with 78.74%ethanol and ratio of 1:21.85 g/mL.Experimental va-lidation showed a maximum EY of 13.88%with TPC of 311.21 mgGAE/gDW and 97.57%anti

7、oxidant capacitythat were all within 95%confidence level of predicted values.Additionally,UAE gave significantly better yield(13.71%),TPC(289.05 mgGAE/gDW),total flavonoid content(21.50 mgRE/gDW)and antioxidant activity(94.99%)than maceration which gave lower yield(10.96%),TPC(246.98 mgGAE/gDW),tota

8、l flavonoid con-tent(13.48 mgRE/gDW)and antioxidant activity(78.12%)respectively.General phytochemical screening ex-posed the presence of additional saponins and tannins in the UAE extracts.Chemical composition of the opti-mized extract via GC/MS indicated the presence of four major phenolic compoun

9、ds,hydroxychavicol,eugenol,isoeugenol and 4-allyl-1,2-diacetoxybenzene with peak areas of 66.55,11.92,2.90 and 3.21%respectively.1.IntroductionPiper betle,belonging to the Piperaceae family,are the leaves of awoody plant that is widely distributed mainly across Asian regions.Intraditional Asian medi

10、cine,Piper betle is known as one of the mostcommon medicinal plants utilized as contemporary and alternativemedicine among cancer patients(Farooqui et al.,2016).The herbseffective antioxidant potential has been demonstrated via multiple ra-dical scavenging activities(Sazwi,Nalina,&Rahim,2013).Furthe

11、r-more,the extract of Piper betle has been proven to reduce and inhibitlipid peroxidation together with enhancing the levels of natural anti-oxidants such as Vitamin C and E(Saravanan,Prakasam,Ramesh,&Pugalendi,2004).The reason behind the antioxidative nature of Piperbetles extract is due to the exi

12、stence of natural antioxidants like hy-droxychavicol and eugenol(Chakraborty&Shah,2011;Pin et al.,2010).Due to its efficacy,researchers have proposed the possible uti-lization of Piper betle as a source of natural antioxidants in food andpharmaceutical products(Dwivedi&Tripathi,2014;Venkadeswaraet a

13、l.,2014).Conventional extraction methods such as distillation and solventextraction(maceration,soxhlet,percolation,infusion extraction)andnon-conventional ones including supercritical fluid extraction,ac-celerated solvent are typically implemented in the recovery of naturalantioxidants(Azwanida,2015

14、).As effective as they may be,high sol-vent and energy consumption and prolonged extraction period makesthem undesirable from the economics perspective(Gonzlez-Centeno,Comas-Serra,Femenia,Rossell,&Simal,2015).The use of ultrasoundin the recovery of desired compounds has been proven to be an effectiv

15、eand efficient extraction technique in terms of garnering more yield withreduced solvent usage and extraction time(Vilkhu,Mawson,Simons,&Bates,2008).Ultrasound-assisted extraction(UAE)relies on the phe-nomenon of acoustic cavitation and mechanical effects for the extrac-tion of compounds from plants

16、 sources.Collapse of the cavitationalbubbles on the plant matrixs surface causes the cell walls to rupture,resulting in higher and faster penetration of the solvent into the plantmaterial.Thus,due to enhanced overall mass transfer,the extraction ofhttps:/doi.org/10.1016/j.lwt.2017.11.033Received 27

17、September 2017;Received in revised form 14 November 2017;Accepted 15 November 2017Corresponding author.E-mail addresses:(A.Ali),l.xiao_yienhw.ac.uk(X.Y.Lim),chien_hwa.chonghw.ac.uk(C.H.Chong),SiauHui.Mahtaylors.edu.my(S.H.Mah),beelin.chuataylors.edu.my(B.L.Chua).LWT-Food Science and Technology 89(20

18、18)681688Available online 17 November 20170023-6438/2017 Elsevier Ltd.All rights reserved.Tthe desired compounds are accelerated(Tomik et al.,2016;Vilkhuet al.,2008).The use of ultrasound for extraction applications in food andpharmaceutical industries is promising.However,the utilization of ul-tras

19、ound for the recovery of natural antioxidants from the medicinalherb,Piper betle,is yet to be fully explored.Thus,the primary aim ofthis paper is to optimize the ultrasound-assisted extraction of naturalantioxidants from Piper betle.This was achieved by investigating theimpact of three extraction pa

20、rameters(temperature,solute to solventratio and solvent concentration)for optimum extraction yield,TPC and2,2-diphenyl-1-picrylhydrazy(DPPH)antioxidant capacity.The statis-tical approach of response surface methodology(RSM)was employedfor the optimization of the extraction parameters.This paper also

21、 aimsto draw comparison between the phenolic content and antioxidantsactivities of Piper betle using UAE and conventional maceration method.Lastly,this paper aims to identify and quantify the predominant phe-nolic compounds present in the optimized Piper betles extract thatcontributes to the high an

22、tioxidant activity of Piper betle via Gas chro-matographymass spectrometry(GC/MS).2.Materials and methods2.1.Plant materialsA total of 10 kg of fresh leaves of Piper betle were purchased in asingle batch from a local shop in Chow Kit market,Kuala Lumpur,Malaysia.The washed and cleaned leaves were pr

23、e-treated(dried)in anair forced convection oven(FAC-350,Protech,USA)at 50 C for a day.The dried leaf samples were then crushed into powdered form andconceded through 800 m-mesh sieve before being used for actual ex-traction.2.2.Chemicals and reagentsThe two reagents Fast blue BB(FBBB)and DPPH(1,1-di

24、phenyl-2-picrylhydrazyl)of analytical grade were purchased from Sigma-aldrich(Sigma-aldrich GmbH,Steinheim,Germany).The solvents used in thisresearch include 95%ethanol,99.9%methanol and chloroform(HPLCgrade).The remaining chemicals used include Gallic acid standard andsodium hydroxide pellets.All o

25、f the chemicals mentioned with theexception of the reagents were purchased from Sigma-aldrich(Sigma,St.Louis,MO,USA).2.3.Extraction procedures2.3.1.Ultrasound-assisted extraction(UAE)Ultrasound-assisted extraction of phenolic compounds from Piperbetle was performed using an ultrasonic bath system(P1

26、20 H,Elmasonic,Germany).1 g of powdered leaf with the designed volume ofethanol concentration were placed in an ultrasonic bath that isequipped with digital control system for sonication time,temperatureand frequency.Based on the experimental design,UAE was performedat a frequency of 37 kHz with a c

27、onstant power of 400 W.Extractiontemperature and time were continuously monitored from the controlpanel of the equipment.Distilled water was added to maintain a con-stant desired temperature with 2 C in the ultrasonic bath.Extractionperiod of 30 min was applied based on preliminary trial studies asp

28、rolong extraction time can lead to structural alteration and disin-tegration of the bioactive compounds(Moorthy et al.,2017).The im-pact of extraction temperature(50,60 and 70 C),solvent concentration(70%,80%and 90%v/v)and solute to solvent ratio(1:10,1:20 and1:30 g/mL)were investigated.Following th

29、e extraction,samples werefiltered and dried at 50 C using a vacuum rotary evaporator(Hei-VAPPlatinum 3,Heidolph,Germany)to obtain the crude extract.The crudeextracts were stored at 4 C prior to consequent analysis.2.3.2.Maceration extractionMaceration extraction of the phenolic antioxidants from Pip

30、er betlewas performed in a water bath system(Copens Scientific Sdn Bhd,Malaysia).1 g of powdered and sieved leaf samples were extracted with80%ethanol at 50 C for 30 min.The extracts obtained were dried inthe same manner as above and stored at 4 C before further analysis.2.4.Total phenolic content(T

31、PC)and extraction yieldExtraction yield(EY)of the crude extract was obtained using Eq.(1).TPC was quantified as described by Medina(2011)with slightmodifications.1:20 mg/mL of crude extract in deionized water wasadded to 0.1 mL of 0.1%FBBB reagent which was kept aside for a min.This was followed by

32、the addition of 0.1 mL of 5%sodium hydroxidesolution.The mixtures were kept at room temperature for 90 min be-fore transferring 200 L of the sample mixtures to a 96-well plate.Theabsorbance of the samples were read at 420 nm by means of a micro-plate spectrophotometer(Epoch 2,BioTek,USA)(Medina,2011

33、).TPCis expressed in terms of mg gallic acid equivalent/g of dried extractaccording to the regression equation of gallic acid calibration curve(r2=0.9899)that was procured in the same manner as above.=EYWW100%ds(1)Where Wdand Wsare the weight of the crude extract and Piper betlepowder sample in gram

34、s respectively.2.5.Total flavonoid content(TFC)and phytochemical screeningTotal flavonoid content(TFC)assay was conducted according toAyoola et al.(2008)with minor modifications.2 mL of extract sampleswith concentration of 1 mg/mL was added to 2 mL of 2%aluminiumtrichloride ethanolic solution.The sa

35、mple mixtures were kept at roomtemperature for an hour before measuring their absorbance at 420 nmvia a microplate spectrophotometer(Ayoola et al.,2008).TFC is ex-pressed in terms of mg rutin equivalent/g of dried extract according tothe regression equation of rutin calibration curve(r2=0.9839).Theg

36、eneral phytochemical screening of alkaloids,steroids,polysaccharide,condensed tannins and saponins were performed as elaborated byAdline and Devi(2014)and Evans(2009).2.6.DPPH antioxidant assayA modified version of DPPH radical scavenging assay was followedas described by Pin et al.(2010).Samples mi

37、xtures were prepared inconcentrations of 0.5 mg/mL in 80%ethanol.Aliquots of 160 L ofPiper betle samples mixture were transferred to 96-well plate which wasfollowed by the addition of 40 L of working 1 mM DPPH methanolicsolution.The plates were kept in the dark for 3 min in ambient tem-perature.The

38、absorbance of the sample solutions were read at 520 nmwith a microplate spectrophotometer.The radical scavenging activity,is expressed as%inhibition activity with the following Eq.(2)(Pinet al.,2010):=AAADPPH%Inhibitionactivity100cSc(2)Where Acis the absorbance of blank solution containing DPPH only

39、 andAsis the absorbance of the solution containing DPPH with Piper betleextract.2.7.Gas chromatography/mass spectroscopy(GC/MS)assayChemical composition of the optimized extract samples were per-formed as elaborated by Foo,Salleh,and Mamat(2015)using GC/MS(7890A,Agilent Technologies,Malaysia)with sl

40、ight modifications.In-itial temperature of the oven was programmed at 70 C that was raisedA.Ali et al.LWT-Food Science and Technology 89(2018)681688682to 305 C at a rate of 20 C/min.Helium(carrier gas)was injected at arate of 1.2 mL/min 1 mL of 0.1 mg/mL samples were injected into thecapillary colum

41、n in split mode for run time of approximately 17 min(Foo et al.,2015).Identification of the individual compounds was doneby library match with NIST Mass Spectral library(version 2).2.8.Response surface methodology(RSM)In present study,a three-factor,three-level Box-Behnken design(BBD)was employed to

42、 obtain the optimum UAE conditions for theextraction of antioxidants from Piper betle.BBD was selected for currentresearch as it is particularly effective when three variables are con-cerned in the experimental domain with reduced number of experi-ments allowing for a more efficient and economic app

43、roach(Granato&Ares,2013).The extraction variables with their respective levels andcoded factors are displayed in Table 1.The complete design matrix ofBBD with a total of 17 experiments is presented in Table 2.Experi-mental data of predicted and actual responses were collected in theform of extractio

44、n yield,TPC and DPPH antioxidant activity(Table 2).The experimental data for the three responses were fitted into second-order polynomial model as in the following equation:=+=Y x x x xoikiiikiiijikijij1122(3)Where Y is the response,xiandxjare the independent variables(iand j range from 1 to k),ois

45、a constant,i,ii,andijare the regressioncoefficients of linear,quadratic and interactive terms respectively,k isthe number of number of parameters(3 for current study)(Moorthyet al.,2017).2.9.Statistical analysisAll of the analysis above were carried out in triplicates and valuesexpressed as mean.Reg

46、ression analysis of the experimental data wasperformed using Design expert software v.10(Stat-Ease,Minneapolis,Minnesota,USA).Analysis of variance(ANOVA),different statisticalparameters including coefficient of determination(R2),adjusted coef-ficient of determination(R2a)and predicted coefficient of

47、 determination(R2p)were all employed to check the adequacy of the developed models.Coefficient of variation(CV)and adequate precision were also ex-amined to further evaluate the precision of the developed models.Significance of each term was considered when 0.05.In additionto the quadratic models,re

48、sponse surface plots were generated to es-tablish the relationship between the independent variables and theresponses.2.10.Optimization and validation of RSM extraction modelsNumerical optimization technique was performed to determine theoptimum conditions for maximum EY,TPC and DPPH antioxidant ac-

49、tivity.The prime conditions were identified with the desirability valueof 1 for each respective response.Validation of the developed modelswere done by performing triplicate experiments under the optimizedparameters.Finally,the average experimental results and 95%predic-tion interval range of predic

50、ted values were compared.This is essentialto evaluate the accuracy and precision of the optimized conditions.3.Results and discussion3.1.Determination of extraction parameters for RSM optimizationThe selection of appropriate solvent is as pivotal as any other ex-traction parameter when solvent extra