Simultaneous Optimization of Microwave-Assisted Extraction of...

Research ArticleSimultaneous Optimization of Microwave-Assisted Extraction ofPhenolic Compounds and Antioxidant Activity of Avocado(Persea americana Mill) Seeds Using ResponseSurface Methodology

Alexander Weremfo 1 Felix Adulley1 and Martin Adarkwah-Yiadom2

1Department of Biochemistry School of Biological Sciences University of Cape Coast Cape Coast Ghana2Drugs Cosmetics and Forensic Department Ghana Standards Board Accra Ghana

Correspondence should be addressed to Alexander Weremfo aweremfouccedugh

Received 12 February 2020 Revised 16 June 2020 Accepted 30 July 2020 Published 17 August 2020

Academic Editor Rongda Xu

Copyright copy 2020 Alexander Weremfo et al )is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

)is study was designed to optimize three microwave-assisted extraction (MAE) parameters (ethanol concentration microwavepower and extraction time) of total phenolics total flavonoids and antioxidant activity of avocado seeds using response surfacemethodology (RSM) )e predicted quadratic models were highly significant (plt 0001) for the responses studied )e extractionof total phenolic content (TPC) total flavonoid content (TFC) and antioxidant activity was significantly (plt 005) influenced byboth microwave power and extraction time )e optimal conditions for simultaneous extraction of phenolic compounds andantioxidant activity were ethanol concentration of 583 (vv) microwave power of 400W and extraction time of 48min Underthese conditions the experimental results agreed with the predicted values MAE revealed clear advantages over the conventionalsolvent extraction (CSE) in terms of high extraction efficiency and antioxidant activity within the shortest extraction timeFurthermore high-performance liquid chromatography (HPLC) analysis of optimized extract revealed the presence of 10phenolic compounds with rutin catechin and syringic acid being the dominant compounds Consequently this optimized MAEmethod has demonstrated a potential application for efficient extraction of polyphenolic antioxidants from avocado seeds in thenutraceutical industries

1 Introduction

Avocado (Persea americanaMill) belongs to the family ofLauraceae and is an important fruit crop endemic to thetropical and subtropical regions but presently cultivatedworldwide )e food industry has shown remarkable in-terest in processing and enhancing the value of this cropdue to its high economic importance In addition to itspleasant sensory properties there has been growing in-terest in the consumption of avocado-derived productsowing to its high nutritional value and reported health-promoting andor disease-preventing properties [1 2])e seed is a major by-product of avocado industry and isusually discarded with no further application [3] In

addition this important by-product represents an envi-ronmental and waste management problem )e avocadoseed constitutes up to 16 of the weight of the fruit [4] andis a rich source of polyphenols with antioxidant andantimicrobial properties [4ndash7] Recent studies havedemonstrated the antioxidant anticancer antidiabeticanti-inflammatory blood pressure reducing antimicro-bial insecticidal and dermatological activities of seedpreparations [4 8] Due to their beneficial effects avocadoseeds can be an alternative inexpensive source of bioactivecompounds and an efficient extraction of importantphenolics from the avocado waste could improve theeconomics of the avocado industry and minimize envi-ronmental impact

HindawiJournal of Analytical Methods in ChemistryVolume 2020 Article ID 7541927 11 pageshttpsdoiorg10115520207541927

)e extraction of phenolic compounds from avocadoseeds has been investigated in the last decades focusingmainly on conventional extraction methods such as mac-eration Soxhlet and heat reflux extraction methodsHowever these methods are very time-consuming and re-quire large quantities of solvents [9 10] Recently severalefficient and advanced extraction techniques includingaccelerated solvent extraction [6] ultrasound-assisted ex-traction [11] and supercritical fluid extraction [12] havebeen developed for the extraction of phenolic compoundsfrom avocado seeds Microwave-assisted extraction (MAE)is a green and effective extraction technique that uses mi-crowave energy to heat polar solvent in contact with sam-ples by ionic conduction and dipole rotation whichimproves cell wall destruction and increases solubility ofcompounds such as flavonoids [13ndash15] MAE has gainpopularity in recent times due to it benefits of improvedefficiency reduced extraction time low solvent consump-tion higher extraction rate and high potential for auto-mation [16 17] MAE technique has been used for theextraction of bioactive compounds from a wide variety ofmatrices such as grapes [18] tomatoes [19] apple [20] andcoffee [21] However the extraction of phenolic bioactivecompounds from avocado seeds has not been evaluatedusing MAE

)e efficiency of the MAE process is usually affected byseveral variables such as extraction power time solventcomposition and solvent-to-sample ratio [18 22ndash24] It istherefore important to optimize these process variables toachieve maximum yield of bioactive compounds from theraw materials In this study a response surface method-ology (RSM) was used to determine the effect of MAEprocess variables and their interactions to ensure maximalextraction efficiency )is method allows the optimizationof all variables simultaneously and predicts the most effi-cient conditions with the use of a minimal number ofexperiments [25] RSM has recently been used to optimizethe extraction conditions of phenolics from various plants[26ndash28] )us the objective of this study was to optimizeMAE conditions to obtain maximum yield of phenolicantioxidants from avocado seeds RSM was used to predictthe effects of microwave power extraction time and eth-anol concentration on total phenolic content (TPC) totalflavonoid content (TFC) and antioxidant activity of avo-cado seed extract

2 Materials and Methods

21 Chemicals FolinndashCiocalteu phenol reagent alumin-ium chloride (AlCl3middot6H2O) sodium carbonate (Na2CO3)22-diphenyl-1-picrylhydrazyl radical (DPPH) 22prime-azi-nobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)sodium nitrite (NaNO2) sodium hydroxide (NaOH)ethanol and phenolic compounds (gallic acid catechinrutin quercetin 4-hydroxybenzoic acid syringic acidferulic acid vanillic acid caffeic acid and p-coumaricacid) were purchased from Sigma-Aldrich Corp (StLouis MO USA) All chemicals and solvents were ofanalytical grade

22 Sample Preparation Avocado fruits (Persea americanaMill var Hass) with adequate ripeness for consumptionwere obtained from a local market at Bonyere (Ghana) inFebruary 2019 )e seeds were manually removed from thefruits cleansed sliced into small and thin size and sun-driedfor 12 days until no more weight loss was observed )edried seeds were milled into fine powder using a blenderand the particle size was standardized using a 250 μm sieve)e moisture content of the dried avocado seeds was 89)e powdered sample was stored at minus20degC in airtight bagsuntil being used

23 Experimental Design A face-centred central compositedesign was used to optimize three independent microwaveparameters ethanol concentration ( X1) microwavepower (W X2) and extraction time (min X3) of four de-pendent variables total phenolic content (YTPC) total fla-vonoid content (YTFC) DPPH scavenging activity (YDPPH)and ABTS scavenging activity (YABTS) )ese independentmicrowave parameters were selected due to their significantinfluence on the efficiency of MAE [18 22ndash24] Generallyethanol and methanol are better solvents for extraction ofphenolic compounds Considering the potential use of thisproduct in food industry ethanol was selected as the solventin this study )e independent variables were coded at threelevels and their actual values selected based on literaturedata and preliminary experimental results )e independentvariables and their related codes and levels are displayed inTable 1 A total of 17 experimental runs were performedrandomly which included three replicates at the centre point(Table 2) and all the experiments were replicated thrice toimprove the analysis Regression analysis for the experimentdata was performed and was fitted into a second-orderpolynomial model

Y βo + 1113944k

i1βixi + 1113944

k1

i1iltj1113944

k

j2βijxj + 1113944

k

i2βiix

2ii (1)

where β0 βi βii and βij are the regression coefficients xi andxj are the coded levels of independent variables affecting thedependent response Y and k is the number of parameters

24 Extraction Process

241 Microwave-Assisted Extraction (MAE) MAE wasperformed using a domestic microwave oven system(Kenwood K30CSS14 Microwave China) operating at800W maximum power and a frequency of 2450MHz )eapparatus was equipped with a digital control system forirradiation time and microwave power )e oven wasmodified in order to condense the vapor generated duringextraction into the sample 1 g of avocado seeds powder wasstirred in 20mL aqueous ethanol and the mixture was ir-radiated using the microwave system )e MAE extractionparameters were microwave power (80ndash400W) extractiontime (1ndash5min) and ethanol concentration (40ndash80))ereafter the sample was filtered using a vacuum pump

2 Journal of Analytical Methods in Chemistry

and the liquid extract was collected and stored at 4degC untilfurther use

242 Conventional Solvent Extraction (CSE) Phenoliccompounds in avocado seeds were extracted using a CSEmethod optimized by Gomez et al [29] Briefly 1 g of av-ocado seeds powder was mixed with 60mL of 56 ethanol(vv) and the mixture was kept in a thermostatic water bath(Grant W14 Cambridge England) at 63degC with shaking for23min After cooling the mixture was centrifuged at2500 rpm for 10min and the supernatant was recoveredthrough filtration and stored at 4degC until further use

25 Phytochemical Analysis

251 Determination of Total Phenolic Content (TPC)TPC of the avocado seed extract was determined using theFolinndashCiocalteu method [16] )e extract (100 μL) wasmixed with 750 μL of a 10-fold diluted FolinndashCiocalteureagent followed by 750 μL of sodium carbonate (75 wv))e mixture was incubated in dark at room temperature(27degC) for 90min and its absorbance measured at 725 nmusing an UV-Vis spectrophotometer (Labomed SpectroUVD 3200 USA) against the blank Gallic acid was used forthe calibration curve (Figure S1) )e results were expressedas mg of gallic acid equivalent (GAE) per gram of dry weight(dw) of avocado seeds

252 Total Flavonoid Content (TFC) )e flavonoid contentin the extract was determined by the aluminium chloridemethod [30] Briefly 05mL of extract was diluted with15mL of distilled water 05mL of 10 (wv) aluminiumchloride and 01mL of potassium acetate (1M) )e finalvolume was made up to 5mL with distilled water and themixture kept at room temperature for 30min )e absor-bance was measured at a wavelength of 415 nm against blank(AlCl3 solution) after 30min of equilibrium )e TFC wasquantified using quercetin standard curve (Figure S2) andestimated as mg of quercetin equivalent (QE) per gram ofdry weight (dw) of avocado seeds

26 Antioxidant Activity

261 DPPH Radical Scavenging Activity )e DPPH assaywas performed as described by Pandey et al [30] )eextract (1 mL) was mixed with 3mL of DPPH solution(4 mL of stock DPPH solution in 96mL of 80methanol)and the mixture was kept in dark for 30min at roomtemperature )e absorbance of the mixture was mea-sured at 520 nm using UV-Vis spectrophotometer(Labomed Spectro UVD 3200 USA) A mixed solution of1 mL ethanol and 3mL DPPH solution was used as theblank Antioxidant activity of the extract was expressed asthe percent inhibition according to the followingequation

Table 2 Central composite design (CCD) with observed response of the dependent variables from MAE of avocado seeds

Independent variables Phenolic compounds Antioxidant activityRun order X1 () X2 (W) X3 (min) TPC (mgmiddotGAEg) TFC (mgmiddotQEg) DPPH (inhibition) ABTS (inhibition)1 40 80 1 5299 098 2293 17592 80 80 1 4725 066 2486 11213 40 400 1 7464 889 4431 3964 80 400 1 6476 590 3988 35635 40 80 5 6594 559 3823 37626 80 80 5 6634 914 3252 35477 40 400 5 7629 1970 7976 60668 80 400 5 7783 1665 7395 56189 40 240 3 7706 1092 4964 464010 80 240 3 7871 970 4756 406611 60 80 3 6873 643 325 507712 60 400 3 8939 1570 6211 803213 60 240 1 7279 1014 398 448114 60 240 5 7916 2145 6866 731815 60 240 3 8352 1548 5435 689216 60 240 3 8045 1523 5259 674417 60 240 3 8466 1610 5768 7033X1 ethanol concentration X2 microwave power X3 extraction time TPC total phenolic content TFC total flavonoid content DPPH 22-diphenyl-1-picrylhydrazyl radical scavenging ability ABTS 22prime-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) scavenging ability

Table 1 )ree levels of the three variables of the extraction process

Independent variables SymbolsCoded levels

minus1 0 1Ethanol concentration () X1 40 60 80Microwave power (W) X2 80 240 400Extraction time (min) X3 1 3 5

Journal of Analytical Methods in Chemistry 3

inhibition Acontrol minus Asample

Acontroltimes 100 (2)

where Acontrol is the absorbance value of the blank andAsample is the absorbance of extract and DPPH solution

262 ABTS Radical Scavenging Activity ABTS radicalscavenging ability of the avocado seed extract was evaluatedusing a spectrophotometric method as described by Dah-moune et al [16] A radical solution (7mM ABTS and245mM potassium persulfate in equal proportions) wasprepared and left to stand in the dark at room temperature(27degC) for 16 h until the reaction was completed and theabsorbance was stable at 734 nm )is solution was dilutedwith ethanol (80) till an absorbance value of 070plusmn 002 at734 nm was obtained )e extract (01mL) was mixed with39mL of diluted ABTS solution and kept in dark for 15minat room temperature )e absorbance was measured at734 nm against blank (diluted ABTS solution) using UV-Visspectrophotometer (Labomed Spectro UVD 3200 USA))e antioxidant activity of the extract was expressed aspercent inhibition according to



where Acontrol is the absorbance value of the blank andAsample is the absorbance of extract and ABTS solution

27 HPLC Analysis Phenolic compounds present in theoptimized extract were analyzed using Shimadzu UFLCchromatographic system (Shimadzu Corporation KyotoJapan) equipped with two LC-20AD pumps and SPD-20AVultraviolet-visible detector )e separation of the com-pounds was performed using Luna C18 column(150mmtimes 46mm 3 μm) at a column temperature of 30degC)e mobile phase consisted of A (1 acetic acid in aceto-nitrile) and B (1 acetic acid in water) with gradient elution0ndash3min (9 A) 3ndash37min (9ndash68 A) 37ndash39min (68 A)and 39ndash40min (69ndash9 A) )e flow rate was 08mLminand the injection volume was 5 μL Each standard solutionand sample was analyzed in triplicate )e peaks were de-tected by UV at wavelength of 280 nm according to thescanning mode of the UV detector )e phenolic com-pounds were identified by comparing their retention timeswith corresponding standards All the identified compoundswere quantified by external standard method using cali-bration curves and their concentrations were expressed asmg100 gmiddotdw

28 Statistical Analysis Statistical analysis and responsesurface plots were performed using the Design-Expertsoftware (version 110 Stat-Ease Inc MN USA) Data wereanalyzed using analysis of variance (ANOVA) at 95confidence level

3 Results and Discussion

31 Model Fitting A central composite design (CCD) wasused to study the effects and interactions ofMAE parameters(ethanol concentration microwave power and extractiontime) on TPC TFC DPPH and ABTS )e experimentaldesign matrix with corresponding responses is presented inTable 1)e experimental values obtained ranged from 4725to 8939mgmiddotGAEg for TPC 066 to 2145mgmiddotQEg for TFC2293 to 7976DPPH inhibition and 1121 to 8032ABTSinhibition (Table 2) )e values showed considerable de-pendence on the extraction conditions which suggests theneed to optimize the extraction process Quadratic poly-nomial models were developed and the adequacy and fitnessof the models were evaluated by ANOVA )e ANOVAresults revealed that the four models were highly significant(Plt 00001) for TPC TFC DPPH and ABTS (Table 3) )erespective values of R2 Adj-R2 and Pred-R2 for TPC (0975809446 and 08086 respectively) TFC (09875 09715 and08679 respectively) DPPH (09912 09800 and 09372respectively) and ABTS (09899 09770 and 09255 re-spectively) were all close to 1 indicating good correlationbetween the predicted and the actual results [31] Moreoverthe low values of coefficient of variation (CV 355 928483 and 598) suggested that the experimental values werereliable and reproducible [32 33] Furthermore the lack offit values were not significant (Pgt 005) indicating theadequacy of the model in predicting MAE of phenoliccompounds and antioxidant activity of avocado seeds

32 Influence of the Extraction Parameters on Total PhenolicContent )eTPC in avocado seed extract varied from 4725to 8939mgmiddotGAEg (Table 2) )e lowest yield was achievedat ethanol concentration of 80 and microwave power of80W after 1min of extraction while the highest yield wasobtained at ethanol concentration of 60 and microwavepower of 400W after 3min of extraction time Table 3 showsthat microwave power (X2) and extraction time (X3) hadsignificant (Plt 005) positive effect on TPC and the mostsignificant factor is microwave power )e quadratic effect(X2

1 X22 and X2

3) also had significant (Plt 005) influence onTPC under MAE )ere was a significant (Plt 005) inter-action between ethanol concentration and extraction time(X1X3) as well as microwave power and extraction time(X2X3) )e second-order polynomial equation for TPC wasexpressed as

YTPC 8319 minus 120X1 + 817X2 + 531X3 minus 038X1X2

+ 219X1X3 minus 217X2X3 minus 555X21 minus 437X

22

minus 746X23

(4)

)e effects of the independent variables and their mutualinteractions on TPC can be seen on the three-dimensionalresponse surface curves shown in Figures 1(a)ndash1(c) MAE ofTPC from avocado seeds increased initially and decreased asthe ethanol concentration increased (Figures 1(a) and 1(b))


Similar observation was reported for MAE of polyphenolsfrom Coriolus versicolor mushroom [22] from chokeberries[23] from Myrtus communis L leaves [16] and fromblueberry leaves [34] )is significant (plt 001) quadraticeffect of ethanol concentration on TPC (Table 3) could be

explained by the heightened degree of sample cell membranebreakage and improved phenolic compounds solubility bythe initial increase in ethanol concentrations [35 36]However as ethanol concentration continues to increase thepolarity of the solvent changes which may lead to increased

Table 3 Regression coefficient (β) and analysis of variance (ANOVA) of the predicted second-order polynomial models for phenoliccompounds and antioxidant activity

FactorCoefficient (β)

TPC TFC DPPH ABTSIntercept 8319 1504 5393 6744LinearX1-conc minus120 minus040 minus161 minus227lowastX2-power 817lowastlowastlowast 440lowastlowastlowast 1490lowastlowastlowast 1197lowastlowastlowastX3-time 531lowastlowast 460lowastlowastlowast 1213lowastlowastlowast 1143lowastlowastlowastInteractionX1 X2 minus03750 minus116lowast minus08075 001X1 X3 219lowast 048 minus113 047X2 X3 minus217lowast 106lowast 582lowastlowast minus034QuadraticX2

1 minus555lowastlowast minus432lowastlowast minus463lowast minus2282lowastlowastlowastX2

2 minus437lowast minus356lowastlowast minus593lowastlowast minus080X2

3 minus746lowastlowast 117 09999 minus735lowastlowast

F-value (model) 3133lowastlowastlowast 6166lowastlowastlowast 8793lowastlowastlowast 7660lowastlowastlowastF-value (lack of fit) 158 704 074 541R2 09758 09875 09912 09899Adj-R2 09446 09715 09800 09770Pred-R2 08086 08679 09372 09255CV () 355 928 483 598lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001

90

80

70

60

TPC

(mgmiddot

GA

Eg)

X2 power (W) X1 conc ()

50

40

400320

240160

80

8070

6050

40

(a)

90

80

70

60

TPC

(mgmiddot

GA

Eg)

X3 time (min) X1 conc ()

50

40

54

32

1

8070

6050

40

(b)

90

80

70

60

TPC

(mgmiddot

GA

Eg)

X3 time (min) X2 power (W)

50

40

54

32

1

400320

240160

80

(c)

25

20

15

10

TFC

(mgmiddot

QE

g)


5

0

400320

240160

80

8070

6050

40

(d)

25

20

15

10

TFC

(mgmiddot

QE

g)

5

0


54

32

1

8070

6050

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(e)

25

20

15

10

TFC

(mgmiddot

QE

g)

5

0


54

32

1

400320

240160

80

(f )

Figure 1 Response surface plot showing the interactive effect of MAE variables on TPC ((a)ndash(c)) and on TFC ((d)ndash(f ))


impurities being extracted [35] therefore reducing theamount of total phenolic compounds extracted Also in-creased diffusion resistance due to coagulation of proteins athigh ethanol concentrations may prevent dissolution ofpolyphenols and influences the extraction rate [36]

As shown in Figure 1(a) microwave power had sig-nificant influence on TPC than ethanol concentration andthis may be attributed to the increased solubility of phenoliccompounds as a result of increasing power which promotescell rupture and enhances exudation of phenolic compoundsinto the extracting solvent [37] Ozbek et al [38] reportedsimilar behaviour for MAE of TPC from pistachio hull )eextraction time was an important parameter that influencedthe extraction of TPC As shown in Figures 1(a) and 1(b))the extraction of TPC increased with increasing extractiontime to about 4min beyond which a decrease in TPC wasobserved )is result is in agreement with that reported fromCalop pulp [39] Extended extraction time was expected tofavour the extraction of phenolic compounds since enoughtime is required for solvent penetration into the plant tissuedissolving the compounds and subsequently diffusing out tothe extraction medium [40] However at longer extractiontime the extracted yields decreased due to increased dissolu-tion of polymer matrix which causes an increase in viscosityand thereby encapsulating the extracted compounds [41] Inaddition long extraction time may increase exposure to lightand oxygen which will eventually result in the oxidation ofphenolic compounds [42] According to ANOVA analysis(Table 3) the interactive effect of ethanol concentration andextraction time (X1X3) had significant positive influence(plt 005) on TPC As shown in Figure 1(b) the extraction ofTPC increased with increasing ethanol concentration andextraction time to about 60 and 4min respectively afterwhich increasing ethanol concentration and extraction timecaused a decrease in the recovery of TPC Figure 1(c) illustratesthe effect of microwave power and extraction time on TPC)is significant (plt 005) positive interaction (Table 2) is inagreement with earlier reports [43 44] Increasing microwavepower increased TPC as extraction time increases (1ndash3min))is phenomenon could be explained by the enhanced masstransfer rate and solubility of phenolic compounds due todecreasing surface tension and solvent viscosity with increasingmicrowave power which improve sample wetting and matrixpenetration respectively thereby enhancing extraction effi-ciency [16 45 46] However at high levels of microwave power(320ndash400W) increasing the extraction time after 4min de-creased TPC which may be due to degradation of certainphenolic compounds [16]

33 Influence of the Extraction Parameters on Total FlavonoidContent )e predictive equation for the relationship be-tween TFC and the extraction parameter was expressed asfollows

YTFC 1504 minus 040X1 + 440X2 + 460X3 minus 116X1X2

+ 048X1X3 + 106X2X3 minus 432X21 minus 356X

22 + 117X

23

(5)

As shown in Table 3 microwave power and extraction timeexhibited a highly significant (plt 0001) positive linear effectwhile the quadratic terms of ethanol concentration and mi-crowave power showed significant (plt 001) negative effect onthe extraction of TFC from avocado seeds)e same linear andquadratic effects were observed for TPC extraction whichsuggests that similar factors affected the extraction of TFC fromavocado seeds )is is expected as flavonoids represent asubgroup of polyphenols )e interaction of microwave powerand extraction time (X2X3) had a significant (plt 005) positiveeffect on TFC At lower microwave powers increasing ex-traction powers gradually increased TFC value over time(Figure 1(f)) )is significant (plt 005) interaction of mi-crowave power and extraction time (X2X3) is tentativelyexplained by the low rate of mass transfer at low microwavepowers which would require more time for the phenoliccompounds to dissolve from the avocado seeds into the so-lution At higher microwave powers the dissolution of phe-nolic compounds can reach equilibrium in a relatively shorttime hence the extraction of TFC are not readily affected bychanges in the extraction time Ethanol concentration was theleast important factor as it did not show a significant effect onTFC (Table 3) However the significant (plt 005) negativeinteraction of ethanol concentration and microwave power(X1X2) on the extraction of TFC suggested that optimal mi-crowave power values increase as ethanol concentration de-creases (Figure 1(d))

34 Influence of the Extraction Parameters on AntioxidantActivity )e antioxidant activity of the avocado seed extractwas determined using ABTS andDPPH assays)e results inTable 3 show that the ABTS scavenging activity was influ-enced by ethanol concentration microwave power andextraction time while DPPH activity depended on micro-wave power and extraction time )e model equation forantioxidant activity can be represented as follows

YABTS 6744 minus 227X1 + 1197X2 + 1143X3 + 001X1X2


22

minus 735X23

YDPPH 5393 minus 161X1 + 1490X2 + 1213X3 minus 081X1X2

minus 113X1X3 + 582X2X3 minus 463X21 minus 593X

22

+ 100X23

(6))e linear effects of microwave power and extraction

time showed a highly significant (plt 0001) positive effecton ABTS scavenging activity while ethanol concentrationexhibited significant (plt 005) negative effect on ABTSMoreover the quadratic effects of ethanol concentration(X2

1) and extraction time (X23) showed highly significant

(plt 0001) and moderately significant (plt 001) negativeeffects on ABTS activity respectively (Table 3) As shown inFigure 2(e) increasing ethanol concentration above 60resulted in a quadratic decrease in ABTS activity Inter-estingly there was no significant interactive impact


(pgt 005) of X1X2 X1X3 or X2X3 on ABTS scavengingactivity (Table 3) )is indicates that the ABTS scavengingactivity of the extract was individually affected by ethanolconcentration microwave power and extraction time andnot by their interaction

In case of DPPH antioxidant activity both microwavepower and extraction time showed highly significant(plt 0001) positive linear effect )e quadratic effects of theethanol concentration (X2

1) (plt 005) and microwave power(X2

2) significantly (plt 001) influenced DPPH scavengingactivity (Table 3) Moreover increasing both microwavepower and extraction time resulted in significant positiveinteractive effect on DPPH activity (Figure 2(c)) )us thelonger the extraction time the better the DPPH scavengingactivity of the extract Similar observation was reported byGarrido et al [47] from chardonnay grape marc

Although both ABTS and DPPH scavenging activitiesexhibited relatively similar patterns the minor differencescould be due to the present of various phenolic compoundsin the extract which exert different kinetics and reactionmechanisms to different antioxidant activity [30] Similarfindings have been reported from vine pruning residues [48]and from rhizomes of Rheum moorcroftianum [30]

35 Optimization of Extraction Conditions andVerification ofPredictive Model )e optimal conditions for simultaneousextraction of maximum phenolic compounds (TPC andTFC) and antioxidant activity (DPPH and ABTS) from dryavocado seeds were predicted by maximizing the desirabilityof the responses using Design-Expert software trial version

110 (Stat-Ease Inc) )e optimal microwave extractionconditions for optimum TPC TFC DPPH and ABTS in asingle experiment were determined to be as ethanol con-centration of 583 microwave power of 400W and ex-traction time of 48min with desirability of 0955 )enumerical optimization provided the maximum predictedvalues of 8390mgmiddotGAEg for TPC 2184mgmiddotQEg for TFC7567 DPPH inhibition and 8266 ABTS inhibitionExperiments were performed under the optimized condi-tions and the results are presented in Table 4 )e exper-imental values agreed with the predicted values confirmingthe reliability of the model obtained by CCD in predictingthe contents of phenolic compounds and antioxidant activityusing MAE

36ComparisonofMAEwithCSE )e results of TPC TFCand antioxidant activity from avocado seeds by MAE andCSE are shown in Table 5 )e MAE method significantly(plt 005) enhanced the extraction of phenolic compoundsand antioxidant activity as compared to CSE In additionto improved extraction efficiency solvent consumptionand extraction time were significantly reduced by MAE incomparison with CSE Using ultrasound-assisted ex-traction a TPC value of 573 mgmiddotGAEg was obtainedfrom avocado seeds [11] )e fast and efficient extractionof phenolic compounds from avocado seeds by MAEcould be explained by the rapid heat generation by mi-crowave energy which causes destruction of the cellularmatrix and enhances the release of phenolic compounds[13 14] and hence antioxidant activity

90D

PPH

( in

hibi

tion)


203040506070

400320

240160

80

8070

6050

40

80

(a)


54

32

1

8070

6050

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90

DPP

H (

inhi

bitio

n)

20304050607080

(b)


54

32

1

400320

240160

80

90

DPP

H (

inhi

bitio

n)

20304050607080

(c)

100

ABT

S (

inhi

bitio

n)


0

20

40

60

400320

240160

80

8070

6050

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80

(d)


54

32

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8070

6050

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100A

BTS

( in

hibi

tion)

0

20

40

60

80

(e)


54

32

1

400320

240160

80

100

ABT

S (

inhi

bitio

n)

0

20

40

60

80

(f )

Figure 2 Response surface plot showing the interactive effect of MAE variables on DPPH and ABTS activity


Table 5 Comparison of MAE with CSE

Extraction method Ethanol () Time (min) Power (W) Temp (degC) TPC (mgmiddotGAEg) TFC (mgmiddotQEg) DPPH () ABTS ()MAE 58 5 400 ndash 8236plusmn 105a 1993plusmn 250a 7361plusmn 057a 8020plusmn 323a

CSE 56 23 ndash 63 5186plusmn 240b 1114plusmn 190b 6056plusmn 285b 6382plusmn 345b

)e results are expressed as meanplusmn SD (n 3) Values within the same column with different letters are significantly different at plt 005

500000

400000

300000

200000

100000

00 5 10 15 20

Min

UV

25 30 35

UV Channel 1 280nm

40

(a)

50000

60000

70000

40000

30000

20000

10000

00 5 10 15 20

Min25 30 35 40

(b)

Figure 3 HPLC chromatograms of (a) mixed phenolic standards and (b) avocado seed extract recorded at 280 nm

Table 4 Experimental and predicted values of response variables at optimum extraction conditions

Response variablesOptimum extraction conditions Maximum value

X1 () X2 (W) X3 (min) Experimental value Predicted valueTPC (mgmiddotGAEg)

58 400 5

8236plusmn 105 8390TFC (mgmiddotQEg) 1993plusmn 250 2184DPPH () 7361plusmn 057 7567ABTS () 8020plusmn 323 8266X1 ethanol concentration () X2 microwave power (W) X3 extraction time (min) Experimental results were expressed as average valuesplusmn standarddeviation (n 3)


37 HPLC Analysis of Phenolic Compounds in Avocado SeedExtract Ten phenolic compounds contained in the opti-mized extract of avocado seeds were identified by HPLC atwavelength of 280 nm (Figure 3) )e identified compoundswere gallic acid catechin 4-hydroxybenzoic acid vanillicacid caffeic acid syringic acid p-coumaric acid rutinferulic acid and quercetin Most of these compounds havepreviously been identified in avocado seeds [3 49 50] )econtent of each phenolic compound in this extract wasquantified (Table 6) )e most abundant compounds in thisextract were rutin (7167mg100 g) catechin (5246mg100 g) and syringic acid (4587mg100 g) )e concentra-tion of catechin which is one of the most abundant phenoliccompounds in avocado seeds was higher than the previouslyreported value of 2584mg100 g [50] )is may be due toamong other things the extraction technique employedMost of the identified phenolic compounds have shownsignificant free radical scavenging activity [30 51] hence thecombined effects of these phenolic compounds may bepartly responsible for the antioxidant activity observed in theextract obtained by MAE

4 Conclusion

In this study three parameters of MAE were successfullyoptimized for the maximum extraction of polyphenoliccompounds and antioxidant activity of avocado seedsusing RSM )e results indicate that both microwavepower and extraction time significantly influenced theextraction of phenolic compounds (TPC and TFC) andantioxidant activity (DPPH and ABTS) )e optimalconditions for simultaneous extraction of maximumphenolic compounds (TPC and TFC) and antioxidantactivity (DPPH and ABTS) from avocado seeds wereethanol concentration of 583 microwave power of400W and extraction time of 48 min Under theseconditions the experimental results agreed with thepredicted values MAE revealed clear advantages overCSE in terms of high extraction efficiency and antioxi-dant activity of extract within the shortest extractiontime Furthermore ten phenolic compounds have beenidentified and quantified from this extract )e pre-dominant phenolic compounds in the avocado seed

extract include rutin catechin and syringic acid )usthis optimized MAE method could be beneficial for theextraction and analysis of polyphenolic antioxidantsfrom avocado seeds for industrial purposes

Data Availability

)e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

)e authors declare no conflicts of interest regarding thepublication of this paper

Supplementary Materials

Figure S1 gallic acid calibration curve Figure S2 quercetincalibration curve (Supplementary Materials)

References

[1] M L Dreher and A J Davenport ldquoHass avocado compositionand potential health effectsrdquo Critical Reviews in Food Scienceand Nutrition vol 53 no 7 pp 738ndash750 2013

[2] V L Fulgoni M Dreher and A J Davenport ldquoAvocadoconsumption is associated with better diet quality and nu-trient intake and lower metabolic syndrome risk in US adultsresults from the national health and nutrition examinationsurvey (NHANES) 2001ndash2008rdquo Nutrition Journal vol 12no 1 p 1 2013

[3] B Melgar M I Dias A Ciric et al ldquoBioactive character-ization of Persea americanaMill by-products a rich source ofinherent antioxidantsrdquo Industrial Crops and Productsvol 111 pp 212ndash218 2018

[4] D Dabas R Shegog G Ziegler and J Lambert ldquoAvocado(Persea americana) seed as a source of bioactive phyto-chemicalsrdquo Current Pharmaceutical Design vol 19 no 34pp 6133ndash6140 2013

[5] M Calderon-Oliver H B Escalona-Buendıa O N Medina-Campos J Pedraza-Chaverri R Pedroza-Islas and E Ponce-Alquicira ldquoOptimization of the antioxidant and antimicrobialresponse of the combined effect of nisin and avocadobyproductsrdquo LWTmdashFood Science and Technology vol 65pp 46ndash52 2016

[6] J G Figueroa I Borras-Linares J Lozano-Sanchez andA Segura-Carretero ldquoComprehensive characterization ofphenolic and other polar compounds in the seed and seed coatof avocado by HPLC-DAD-ESI-QTOF-MSrdquo Food ResearchInternational vol 105 pp 752ndash763 2018

[7] J G Rodrıguez-Carpena D Morcuende and M EstevezldquoAvocado by-products as inhibitors of color deterioration andlipid and protein oxidation in raw porcine patties subjected tochilled storagerdquo Meat Science vol 89 no 2 pp 166ndash1732011

[8] R G Araujo R M Rodriguez-Jasso H A RuizM M E Pintado and C N Aguilar ldquoAvocado by-productsnutritional and functional propertiesrdquo Trends in Food Scienceamp Technology vol 80 pp 51ndash60 2018

[9] S A Heleno P Diz M A Prieto et al ldquoOptimization ofultrasound-assisted extraction to obtain mycosterols fromAgaricus bisporus L by response surface methodology and

Table 6 HPLC quantification of phenolic compounds in avocadoseed extract under optimal MAE conditions

Compounds Retention time (min) Content(mg100gmiddotdw)

Gallic acid 332 689plusmn 004Catechin 965 5246plusmn 0154-Hydroxybenzoic acid 1045 1247plusmn 005Vanillic acid 1261 671plusmn 001Caffeic acid 1290 418plusmn 051Syringic acid 1336 4587plusmn 005p-Coumaric acid 1672 713plusmn 022Rutin 1706 7167plusmn 204Ferulic acid 1792 476plusmn 045Quercetin 2428 672plusmn 002


comparison with conventional Soxhlet extractionrdquo FoodChemistry vol 197 no Part B pp 1054ndash1063 2016

[10] C-N Zhao J-J Zhang Y Li X Meng and H-B Li ldquoMi-crowave-assisted extraction of phenolic compounds fromMelastoma sanguineum fruit optimization and identifica-tionrdquo Molecules vol 23 no 10 p 2498 2018

[11] M A Tremocoldi P L Rosalen M Franchin et al ldquoEx-ploration of avocado by-products as natural sources of bio-active compoundsrdquo PLoS One vol 13 no 2 Article IDe0192577 2018

[12] P Paramos M Corazza and H Matos ldquoStudies on extractionfrom avocadorsquos waste biomass to generate process designalternatives of valuable productsrdquo Belgarian Chemical Com-munications vol 51 no Special Issue B pp 049ndash051 2019

[13] C-H Chan H K Yeoh R Yusoff and G C Ngoh ldquoA first-principles model for plant cell rupture in microwave-assistedextraction of bioactive compoundsrdquo Journal of Food Engi-neering vol 188 pp 98ndash107 2016

[14] H K Kala R Mehta K K Sen R Tandey and V MandalldquoCritical analysis of research trends and issues in microwaveassisted extraction of phenolics have we really done enoughrdquoTrAC Trends in Analytical Chemistry vol 85 pp 140ndash1522016

[15] Y Sun X Liao Z Wang X Hu and F Chen ldquoOptimizationof microwave-assisted extraction of anthocyanins in redraspberries and identification of anthocyanin of extracts usinghigh-performance liquid chromatographyndashmass spectrome-tryrdquo European Food Research and Technology vol 225 no 3-4 pp 511ndash523 2007

[16] F Dahmoune B Nayak K Moussi H Remini andKMadani ldquoOptimization ofmicrowave-assisted extraction ofpolyphenols from Myrtus communis L leavesrdquo Food Chem-istry vol 166 pp 585ndash595 2015

[17] J A Perez-Serradilla and M D Luque de Castro ldquoMicro-wave-assisted extraction of phenolic compounds from winelees and spray-drying of the extractrdquo Food Chemistry vol 124no 4 pp 1652ndash1659 2011

[18] K Krishnaswamy V Orsat Y Gariepy and K )angavelldquoOptimization of microwave-assisted extraction of phenolicantioxidants from grape seeds (Vitis vinifera)rdquo Food andBioprocess Technology vol 6 no 2 pp 441ndash455 2013

[19] J Pinela M A Prieto A M Carvalho et al ldquoMicrowave-assisted extraction of phenolic acids and flavonoids andproduction of antioxidant ingredients from tomato anutraceutical-oriented optimization studyrdquo Separation andPurification Technology vol 164 pp 114ndash124 2016

[20] B Pavlic A Naffati T Hojan J Vladic Z Zekovic andS Vidovic ldquoMicrowave-assisted extraction of wild apple fruitdustmdashproduction of polyphenol-rich extracts from filter teafactory by-productsrdquo Journal of Food Process Engineeringvol 40 no 4 p e12508 2017

[21] M Pettinato A A Casazza P F Ferrari D Palombo andP Perego ldquoEco-sustainable recovery of antioxidants fromspent coffee grounds by microwave-assisted extractionprocess optimization kinetic modeling and biological vali-dationrdquo Food and Bioproducts Processing vol 114 pp 31ndash422019

[22] J H Maeng H Muhammad Shahbaz K Ameer Y Jo andJ H Kwon ldquoOptimization of microwave-assisted extractionof bioactive compounds from Coriolus versicolor mushroomusing response surface methodologyrdquo Journal of Food ProcessEngineering vol 40 no 2 p e12421 2017

[23] V M Simic K M Rajkovic S S Stojicevic et al ldquoOpti-mization of microwave-assisted extraction of total

polyphenolic compounds from chokeberries by responsesurface methodology and artificial neural networkrdquo Separa-tion and Purification Technology vol 160 pp 89ndash97 2016

[24] Z Zekovic J Vladic S Vidovic D Adamovic and B PavlicldquoOptimization of microwave-assisted extraction (MAE) ofcoriander phenolic antioxidantsmdashresponse surface method-ology approachrdquo Journal of the Science of Food and Agri-culture vol 96 no 13 pp 4613ndash4622 2016

[25] M A Bezerra R E Santelli E P Oliveira L S Villar andL A Escaleira ldquoResponse surface methodology (RSM) as atool for optimization in analytical chemistryrdquo Talanta vol 76no 5 pp 965ndash977 2008

[26] T Belwal P Dhyani I D Bhatt R S Rawal and V PandeldquoOptimization extraction conditions for improving phenoliccontent and antioxidant activity in Berberis asiatica fruitsusing response surface methodology (RSM)rdquo Food Chemistryvol 207 pp 115ndash124 2016

[27] W Setyaningsih I E Saputro C A Carrera and M PalmaldquoOptimisation of an ultrasound-assisted extraction methodfor the simultaneous determination of phenolics in ricegrainsrdquo Food Chemistry vol 288 pp 221ndash227 2019

[28] U J Vajic J Grujic-Milanovic J Zivkovic et al ldquoOptimi-zation of extraction of stinging nettle leaf phenolic com-pounds using response surface methodologyrdquo IndustrialCrops and Products vol 74 pp 912ndash917 2015

[29] F Gomez S Sanchez M Iradi N Azman and M AlmajanoldquoAvocado seeds extraction optimization and possible use asantioxidant in foodrdquo Antioxidants vol 3 no 2 pp 439ndash4542014

[30] A Pandey T Belwal K C Sekar I D Bhatt and R S RawalldquoOptimization of ultrasonic-assisted extraction (UAE) ofphenolics and antioxidant compounds from rhizomes ofRheum moorcroftianum using response surface methodology(RSM)rdquo Industrial Crops and Products vol 119 pp 218ndash2252018

[31] O R Alara N H Abdurahman and O A Olalere ldquoOpti-mization of microwave-assisted extraction of flavonoids andantioxidants from Vernonia amygdalina leaf using responsesurface methodologyrdquo Food and Bioproducts Processingvol 107 pp 36ndash48 2018

[32] L S Badwaik P K Borah and S C Deka ldquoOptimization ofmicrowave assisted extraction of antioxidant extract fromGarcinia pedunculata Robxrdquo Separation Science and Tech-nology vol 50 no 12 pp 1814ndash1822 2015

[33] F Dahmoune G Spigno K Moussi H Remini A Cherbaland K Madani ldquoPistacia lentiscus leaves as a source ofphenolic compounds microwave-assisted extraction opti-mized and compared with ultrasound-assisted and conven-tional solvent extractionrdquo Industrial Crops and Productsvol 61 pp 31ndash40 2014

[34] W Routray and V Orsat ldquoMAE of phenolic compounds fromblueberry leaves and comparison with other extractionmethodsrdquo Industrial Crops and Products vol 58 pp 36ndash452014

[35] H Li Z Deng T Wu R Liu S Loewen and R TsaoldquoMicrowave-assisted extraction of phenolics with maximalantioxidant activities in tomatoesrdquo Food Chemistry vol 130no 4 pp 928ndash936 2012

[36] B Yang X Liu and Y Gao ldquoExtraction optimization ofbioactive compounds (crocin geniposide and total phenoliccompounds) from Gardenia (Gardenia jasminoides Ellis)fruits with response surface methodologyrdquo Innovative FoodScience amp Emerging Technologies vol 10 no 4 pp 610ndash6152009


[37] M Mendes A P Carvalho J M C S Magalhatildees et alldquoResponse surface evaluation of microwave-assisted extrac-tion conditions for Lycium barbarum bioactive compoundsrdquoInnovative Food Science amp Emerging Technologies vol 33pp 319ndash326 2016

[38] H N Ozbek D K Yanık S Fadıloglu and F GogusldquoOptimization of microwave-assisted extraction of bioactivecompounds from pistachio (Pistacia vera L) hullrdquo SeparationScience and Technology vol 55 no 2 pp 289ndash299 2020

[39] F Saci H Louaileche M B Bey and L Meziant ldquoOptimi-zation of phenolic compound recovery and antioxidant ac-tivity from carob pulp using response surface methodologyrdquoInternational Food Research Journal vol 24 no 3 p 10942017

[40] G E Viacava S I Roura andM V Aguero ldquoOptimization ofcritical parameters during antioxidants extraction frombutterhead lettuce to simultaneously enhance polyphenolsand antioxidant activityrdquo Chemometrics and IntelligentLaboratory Systems vol 146 pp 47ndash54 2015

[41] C S Eskilsson E Bjorklund L Mathiasson L Karlsson andA Torstensson ldquoMicrowave-assisted extraction of felodipinetabletsrdquo Journal of Chromatography A vol 840 no 1pp 59ndash70 1999

[42] P Tay C Tan F Abas H Yim and C Ho ldquoAssessment ofextraction parameters on antioxidant capacity polyphenolcontent epigallocatechin gallate (EGCG) epicatechin gallate(ECG) and iriflophenone 3-C-β-glucoside of agarwood(Aquilaria crassna) young leavesrdquo Molecules vol 19 no 8pp 12304ndash12319 2014

[43] I T Karabegovic S S Stojicevic D T VelickovicN C Nikolic and M L Lazic ldquoOptimization of microwave-assisted extraction and characterization of phenolic com-pounds in cherry laurel (Prunus laurocerasus) leavesrdquo Sep-aration and Purification Technology vol 120 pp 429ndash4362013

[44] G Spigno and D M De Faveri ldquoMicrowave-assisted ex-traction of tea phenols a phenomenological studyrdquo Journal ofFood Engineering vol 93 no 2 pp 210ndash217 2009

[45] M A Al-Farsi and C Y Lee ldquoOptimization of phenolics anddietary fibre extraction from date seedsrdquo Food Chemistryvol 108 no 3 pp 977ndash985 2008

[46] C S Eskilsson and E Bjorklund ldquoAnalytical-scale micro-wave-assisted extractionrdquo Journal of Chromatography Avol 902 no 1 pp 227ndash250 2000

[47] T Garrido M Gizdavic-Nikolaidis I Leceta et al ldquoOpti-mizing the extraction process of natural antioxidants fromchardonnay grape marc using microwave-assisted extrac-tionrdquo Waste Management vol 88 pp 110ndash117 2019

[48] M S Jesus Z Genisheva A Romanı R N PereiraJ A Teixeira and L Domingues ldquoBioactive compoundsrecovery optimization from vine pruning residues usingconventional heating and microwave-assisted extractionmethodsrdquo Industrial Crops and Products vol 132 pp 99ndash1102019

[49] J C Rosero S Cruz C Osorio and N Hurtado ldquoAnalysis ofphenolic composition of byproducts (seeds and peels) ofavocado (Persea americana Mill) cultivated in ColombiardquoMolecules vol 24 no 17 p 3209 2019

[50] A E-H Z Shafika A E-A I Ferial H A SomiaR A E-M Nehal and A H Shahinaz ldquoBioactive compoundsand antioxidant activities of avocado peels and seedsrdquoPakistan Journal of Biological Sciences vol 23 no 3pp 345ndash350 2020

[51] K Dhalwal V M Shinde Y S Biradar and K R MahadikldquoSimultaneous quantification of bergenin catechin and gallicacid from Bergenia ciliata and Bergenia ligulata by using thin-layer chromatographyrdquo Journal of Food Composition andAnalysis vol 21 no 6 pp 496ndash500 2008


)e extraction of phenolic compounds from avocadoseeds has been investigated in the last decades focusingmainly on conventional extraction methods such as mac-eration Soxhlet and heat reflux extraction methodsHowever these methods are very time-consuming and re-quire large quantities of solvents [9 10] Recently severalefficient and advanced extraction techniques includingaccelerated solvent extraction [6] ultrasound-assisted ex-traction [11] and supercritical fluid extraction [12] havebeen developed for the extraction of phenolic compoundsfrom avocado seeds Microwave-assisted extraction (MAE)is a green and effective extraction technique that uses mi-crowave energy to heat polar solvent in contact with sam-ples by ionic conduction and dipole rotation whichimproves cell wall destruction and increases solubility ofcompounds such as flavonoids [13ndash15] MAE has gainpopularity in recent times due to it benefits of improvedefficiency reduced extraction time low solvent consump-tion higher extraction rate and high potential for auto-mation [16 17] MAE technique has been used for theextraction of bioactive compounds from a wide variety ofmatrices such as grapes [18] tomatoes [19] apple [20] andcoffee [21] However the extraction of phenolic bioactivecompounds from avocado seeds has not been evaluatedusing MAE

)e efficiency of the MAE process is usually affected byseveral variables such as extraction power time solventcomposition and solvent-to-sample ratio [18 22ndash24] It istherefore important to optimize these process variables toachieve maximum yield of bioactive compounds from theraw materials In this study a response surface method-ology (RSM) was used to determine the effect of MAEprocess variables and their interactions to ensure maximalextraction efficiency )is method allows the optimizationof all variables simultaneously and predicts the most effi-cient conditions with the use of a minimal number ofexperiments [25] RSM has recently been used to optimizethe extraction conditions of phenolics from various plants[26ndash28] )us the objective of this study was to optimizeMAE conditions to obtain maximum yield of phenolicantioxidants from avocado seeds RSM was used to predictthe effects of microwave power extraction time and eth-anol concentration on total phenolic content (TPC) totalflavonoid content (TFC) and antioxidant activity of avo-cado seed extract

2 Materials and Methods

21 Chemicals FolinndashCiocalteu phenol reagent alumin-ium chloride (AlCl3middot6H2O) sodium carbonate (Na2CO3)22-diphenyl-1-picrylhydrazyl radical (DPPH) 22prime-azi-nobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)sodium nitrite (NaNO2) sodium hydroxide (NaOH)ethanol and phenolic compounds (gallic acid catechinrutin quercetin 4-hydroxybenzoic acid syringic acidferulic acid vanillic acid caffeic acid and p-coumaricacid) were purchased from Sigma-Aldrich Corp (StLouis MO USA) All chemicals and solvents were ofanalytical grade

22 Sample Preparation Avocado fruits (Persea americanaMill var Hass) with adequate ripeness for consumptionwere obtained from a local market at Bonyere (Ghana) inFebruary 2019 )e seeds were manually removed from thefruits cleansed sliced into small and thin size and sun-driedfor 12 days until no more weight loss was observed )edried seeds were milled into fine powder using a blenderand the particle size was standardized using a 250 μm sieve)e moisture content of the dried avocado seeds was 89)e powdered sample was stored at minus20degC in airtight bagsuntil being used

23 Experimental Design A face-centred central compositedesign was used to optimize three independent microwaveparameters ethanol concentration ( X1) microwavepower (W X2) and extraction time (min X3) of four de-pendent variables total phenolic content (YTPC) total fla-vonoid content (YTFC) DPPH scavenging activity (YDPPH)and ABTS scavenging activity (YABTS) )ese independentmicrowave parameters were selected due to their significantinfluence on the efficiency of MAE [18 22ndash24] Generallyethanol and methanol are better solvents for extraction ofphenolic compounds Considering the potential use of thisproduct in food industry ethanol was selected as the solventin this study )e independent variables were coded at threelevels and their actual values selected based on literaturedata and preliminary experimental results )e independentvariables and their related codes and levels are displayed inTable 1 A total of 17 experimental runs were performedrandomly which included three replicates at the centre point(Table 2) and all the experiments were replicated thrice toimprove the analysis Regression analysis for the experimentdata was performed and was fitted into a second-orderpolynomial model

Y βo + 1113944k

i1βixi + 1113944

k1

i1iltj1113944

k

j2βijxj + 1113944

k

i2βiix

2ii (1)

where β0 βi βii and βij are the regression coefficients xi andxj are the coded levels of independent variables affecting thedependent response Y and k is the number of parameters

24 Extraction Process

241 Microwave-Assisted Extraction (MAE) MAE wasperformed using a domestic microwave oven system(Kenwood K30CSS14 Microwave China) operating at800W maximum power and a frequency of 2450MHz )eapparatus was equipped with a digital control system forirradiation time and microwave power )e oven wasmodified in order to condense the vapor generated duringextraction into the sample 1 g of avocado seeds powder wasstirred in 20mL aqueous ethanol and the mixture was ir-radiated using the microwave system )e MAE extractionparameters were microwave power (80ndash400W) extractiontime (1ndash5min) and ethanol concentration (40ndash80))ereafter the sample was filtered using a vacuum pump



























1 X22 and X2




22

minus 746X23

(4)












90

80

70

60

TPC

(mgmiddot

GA

Eg)


50

40

400320

240160

80

8070

6050

40

(a)

90

80

70

60

TPC

(mgmiddot

GA

Eg)


50

40

54

32

1

8070

6050

40

(b)

90

80

70

60

TPC

(mgmiddot

GA

Eg)


50

40

54

32

1

400320

240160

80

(c)

25

20

15

10

TFC

(mgmiddot

QE

g)


5

0

400320

240160

80

8070

6050

40

(d)

25

20

15

10

TFC

(mgmiddot

QE

g)

5

0


54

32

1

8070

6050

40

(e)

25

20

15

10

TFC

(mgmiddot

QE

g)

5

0


54

32

1

400320

240160

80

(f )








22 + 117X

23

(5)





22

minus 735X23



22

+ 100X23














90D

PPH

( in

hibi

tion)


203040506070

400320

240160

80

8070

6050

40

80

(a)


54

32

1

8070

6050

40

90

DPP

H (

inhi

bitio

n)

20304050607080

(b)


54

32

1

400320

240160

80

90

DPP

H (

inhi

bitio

n)

20304050607080

(c)

100

ABT

S (

inhi

bitio

n)


0

20

40

60

400320

240160

80

8070

6050

40

80

(d)


54

32

1

8070

6050

40

100A

BTS

( in

hibi

tion)

0

20

40

60

80

(e)


54

32

1

400320

240160

80

100

ABT

S (

inhi

bitio

n)

0

20

40

60

80

(f )







500000

400000

300000

200000

100000

00 5 10 15 20

Min

UV

25 30 35

UV Channel 1 280nm

40

(a)

50000

60000

70000

40000

30000

20000

10000

00 5 10 15 20

Min25 30 35 40

(b)





58 400 5




4 Conclusion



Data Availability






References





















































































1 X22 and X2




22

minus 746X23

(4)












90

80

70

60

TPC

(mgmiddot

GA

Eg)


50

40

400320

240160

80

8070

6050

40

(a)

90

80

70

60

TPC

(mgmiddot

GA

Eg)


50

40

54

32

1

8070

6050

40

(b)

90

80

70

60

TPC

(mgmiddot

GA

Eg)


50

40

54

32

1

400320

240160

80

(c)

25

20

15

10

TFC

(mgmiddot

QE

g)


5

0

400320

240160

80

8070

6050

40

(d)

25

20

15

10

TFC

(mgmiddot

QE

g)

5

0


54

32

1

8070

6050

40

(e)

25

20

15

10

TFC

(mgmiddot

QE

g)

5

0


54

32

1

400320

240160

80

(f )








22 + 117X

23

(5)





22

minus 735X23



22

+ 100X23














90D

PPH

( in

hibi

tion)


203040506070

400320

240160

80

8070

6050

40

80

(a)


54

32

1

8070

6050

40

90

DPP

H (

inhi

bitio

n)

20304050607080

(b)


54

32

1

400320

240160

80

90

DPP

H (

inhi

bitio

n)

20304050607080

(c)

100

ABT

S (

inhi

bitio

n)


0

20

40

60

400320

240160

80

8070

6050

40

80

(d)


54

32

1

8070

6050

40

100A

BTS

( in

hibi

tion)

0

20

40

60

80

(e)


54

32

1

400320

240160

80

100

ABT

S (

inhi

bitio

n)

0

20

40

60

80

(f )







500000

400000

300000

200000

100000

00 5 10 15 20

Min

UV

25 30 35

UV Channel 1 280nm

40

(a)

50000

60000

70000

40000

30000

20000

10000

00 5 10 15 20

Min25 30 35 40

(b)





58 400 5




4 Conclusion



Data Availability






References





































































90

80

70

60

TPC

(mgmiddot

GA

Eg)


50

40

400320

240160

80

8070

6050

40

(a)

90

80

70

60

TPC

(mgmiddot

GA

Eg)


50

40

54

32

1

8070

6050

40

(b)

90

80

70

60

TPC

(mgmiddot

GA

Eg)


50

40

54

32

1

400320

240160

80

(c)

25

20

15

10

TFC

(mgmiddot

QE

g)


5

0

400320

240160

80

8070

6050

40

(d)

25

20

15

10

TFC

(mgmiddot

QE

g)

5

0


54

32

1

8070

6050

40

(e)

25

20

15

10

TFC

(mgmiddot

QE

g)

5

0


54

32

1

400320

240160

80

(f )








22 + 117X

23

(5)





22

minus 735X23



22

+ 100X23














90D

PPH

( in

hibi

tion)


203040506070

400320

240160

80

8070

6050

40

80

(a)


54

32

1

8070

6050

40

90

DPP

H (

inhi

bitio

n)

20304050607080

(b)


54

32

1

400320

240160

80

90

DPP

H (

inhi

bitio

n)

20304050607080

(c)

100

ABT

S (

inhi

bitio

n)


0

20

40

60

400320

240160

80

8070

6050

40

80

(d)


54

32

1

8070

6050

40

100A

BTS

( in

hibi

tion)

0

20

40

60

80

(e)


54

32

1

400320

240160

80

100

ABT

S (

inhi

bitio

n)

0

20

40

60

80

(f )







500000

400000

300000

200000

100000

00 5 10 15 20

Min

UV

25 30 35

UV Channel 1 280nm

40

(a)

50000

60000

70000

40000

30000

20000

10000

00 5 10 15 20

Min25 30 35 40

(b)





58 400 5




4 Conclusion



Data Availability






References

































































22 + 117X

23

(5)





22

minus 735X23



22

+ 100X23














90D

PPH

( in

hibi

tion)


203040506070

400320

240160

80

8070

6050

40

80

(a)


54

32

1

8070

6050

40

90

DPP

H (

inhi

bitio

n)

20304050607080

(b)


54

32

1

400320

240160

80

90

DPP

H (

inhi

bitio

n)

20304050607080

(c)

100

ABT

S (

inhi

bitio

n)


0

20

40

60

400320

240160

80

8070

6050

40

80

(d)


54

32

1

8070

6050

40

100A

BTS

( in

hibi

tion)

0

20

40

60

80

(e)


54

32

1

400320

240160

80

100

ABT

S (

inhi

bitio

n)

0

20

40

60

80

(f )







500000

400000

300000

200000

100000

00 5 10 15 20

Min

UV

25 30 35

UV Channel 1 280nm

40

(a)

50000

60000

70000

40000

30000

20000

10000

00 5 10 15 20

Min25 30 35 40

(b)





58 400 5




4 Conclusion



Data Availability






References




































































90D

PPH

( in

hibi

tion)


203040506070

400320

240160

80

8070

6050

40

80

(a)


54

32

1

8070

6050

40

90

DPP

H (

inhi

bitio

n)

20304050607080

(b)


54

32

1

400320

240160

80

90

DPP

H (

inhi

bitio

n)

20304050607080

(c)

100

ABT

S (

inhi

bitio

n)


0

20

40

60

400320

240160

80

8070

6050

40

80

(d)


54

32

1

8070

6050

40

100A

BTS

( in

hibi

tion)

0

20

40

60

80

(e)


54

32

1

400320

240160

80

100

ABT

S (

inhi

bitio

n)

0

20

40

60

80

(f )







500000

400000

300000

200000

100000

00 5 10 15 20

Min

UV

25 30 35

UV Channel 1 280nm

40

(a)

50000

60000

70000

40000

30000

20000

10000

00 5 10 15 20

Min25 30 35 40

(b)





58 400 5




4 Conclusion



Data Availability






References
































































500000

400000

300000

200000

100000

00 5 10 15 20

Min

UV

25 30 35

UV Channel 1 280nm

40

(a)

50000

60000

70000

40000

30000

20000

10000

00 5 10 15 20

Min25 30 35 40

(b)





58 400 5




4 Conclusion



Data Availability






References





























































4 Conclusion



Data Availability






References




























































Simultaneous Optimization of Microwave-Assisted Extraction of...

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