Simultaneous Optimization of Microwave-Assisted Extraction of Phenolic Compounds and Antioxidant Activity of Avocado (Persea americana Mill.) Seeds Using Response Surface Methodology

Alexander Weremfo; Felix Adulley; Martin Adarkwah-Yiadom

doi:10.1155/2020/7541927

Simultaneous Optimization of Microwave-Assisted Extraction of Phenolic Compounds and Antioxidant Activity of Avocado (Persea americana Mill.) Seeds Using Response Surface Methodology

Weremfo, Alexander;Adulley, Felix;Adarkwah-Yiadom, Martin 2020-08-17 00:00:00 Hindawi Journal of Analytical Methods in Chemistry Volume 2020, Article ID 7541927, 11 pages https://doi.org/10.1155/2020/7541927 Research Article Simultaneous Optimization of Microwave-Assisted Extraction of Phenolic Compounds and Antioxidant Activity of Avocado (Persea americana Mill.) Seeds Using Response Surface Methodology 1 1 2 Alexander Weremfo , Felix Adulley, and Martin Adarkwah-Yiadom Department of Biochemistry, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana Drugs, Cosmetics and Forensic Department, Ghana Standards Board, Accra, Ghana Correspondence should be addressed to Alexander Weremfo; aweremfo@ucc.edu.gh Received 12 February 2020; Revised 16 June 2020; Accepted 30 July 2020; Published 17 August 2020 Academic Editor: Rongda Xu Copyright © 2020 Alexander Weremfo et al. )is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. )is study was designed to optimize three microwave-assisted extraction (MAE) parameters (ethanol concentration, microwave power, and extraction time) of total phenolics, total ﬂavonoids, and antioxidant activity of avocado seeds using response surface methodology (RSM). )e predicted quadratic models were highly signiﬁcant (p < 0.001) for the responses studied. )e extraction of total phenolic content (TPC), total ﬂavonoid content (TFC), and antioxidant activity was signiﬁcantly (p< 0.05) inﬂuenced by both microwave power and extraction time. )e optimal conditions for simultaneous extraction of phenolic compounds and antioxidant activity were ethanol concentration of 58.3% (v/v), microwave power of 400 W, and extraction time of 4.8 min. Under these conditions, the experimental results agreed with the predicted values. MAE revealed clear advantages over the conventional solvent extraction (CSE) in terms of high extraction eﬃciency and antioxidant activity within the shortest extraction time. Furthermore, high-performance liquid chromatography (HPLC) analysis of optimized extract revealed the presence of 10 phenolic compounds, with rutin, catechin, and syringic acid being the dominant compounds. Consequently, this optimized MAE method has demonstrated a potential application for eﬃcient extraction of polyphenolic antioxidants from avocado seeds in the nutraceutical industries. addition, this important by-product represents an envi- 1. Introduction ronmental and waste management problem. )e avocado Avocado (Persea americana Mill.) belongs to the family of seed constitutes up to 16% of the weight of the fruit [4] and Lauraceae and is an important fruit crop endemic to the is a rich source of polyphenols with antioxidant and antimicrobial properties [4–7]. Recent studies have tropical and subtropical regions but presently cultivated worldwide. )e food industry has shown remarkable in- demonstrated the antioxidant, anticancer, antidiabetic, terest in processing and enhancing the value of this crop anti-inﬂammatory, blood pressure reducing, antimicro- due to its high economic importance. In addition to its bial, insecticidal, and dermatological activities of seed pleasant sensory properties, there has been growing in- preparations [4, 8]. Due to their beneﬁcial eﬀects, avocado terest in the consumption of avocado-derived products seeds can be an alternative inexpensive source of bioactive owing to its high nutritional value and reported health- compounds, and an eﬃcient extraction of important promoting and/or disease-preventing properties [1, 2]. phenolics from the avocado waste could improve the )e seed is a major by-product of avocado industry and is economics of the avocado industry and minimize envi- usually discarded with no further application [3]. In ronmental impact. 2 Journal of Analytical Methods in Chemistry )e extraction of phenolic compounds from avocado 2.2. Sample Preparation. Avocado fruits (Persea americana seeds has been investigated in the last decades focusing Mill. var. Hass), with adequate ripeness for consumption, mainly on conventional extraction methods such as mac- were obtained from a local market at Bonyere (Ghana) in eration, Soxhlet, and heat reﬂux extraction methods. February 2019. )e seeds were manually removed from the However, these methods are very time-consuming and re- fruits, cleansed, sliced into small and thin size, and sun-dried quire large quantities of solvents [9, 10]. Recently, several for 12 days until no more weight loss was observed. )e eﬃcient and advanced extraction techniques including dried seeds were milled into ﬁne powder using a blender, accelerated solvent extraction [6], ultrasound-assisted ex- and the particle size was standardized using a 250 μm sieve. traction [11], and supercritical ﬂuid extraction [12] have )e moisture content of the dried avocado seeds was 8.9%. been developed for the extraction of phenolic compounds )e powdered sample was stored at −20 C in airtight bags from avocado seeds. Microwave-assisted extraction (MAE) until being used. is a green and eﬀective extraction technique that uses mi- crowave energy to heat polar solvent in contact with sam- 2.3. Experimental Design. A face-centred central composite ples, by ionic conduction and dipole rotation, which improves cell wall destruction and increases solubility of design was used to optimize three independent microwave compounds such as ﬂavonoids [13–15]. MAE has gain parameters: ethanol concentration (%, X ), microwave power (W, X popularity in recent times due to it beneﬁts of improved ), and extraction time (min, X ) of four de- 2 3 eﬃciency, reduced extraction time, low solvent consump- pendent variables: total phenolic content (Y ), total ﬂa- TPC vonoid content (Y ), DPPH scavenging activity (Y ), tion, higher extraction rate, and high potential for auto- TFC DPPH mation [16, 17]. MAE technique has been used for the and ABTS scavenging activity (Y ). )ese independent ABTS microwave parameters were selected due to their signiﬁcant extraction of bioactive compounds from a wide variety of matrices, such as grapes [18], tomatoes [19], apple [20], and inﬂuence on the eﬃciency of MAE [18, 22–24]. Generally, ethanol and methanol are better solvents for extraction of coﬀee [21]. However, the extraction of phenolic bioactive compounds from avocado seeds has not been evaluated phenolic compounds. Considering the potential use of this product in food industry, ethanol was selected as the solvent using MAE. )e eﬃciency of the MAE process is usually aﬀected by in this study. )e independent variables were coded at three several variables such as extraction power, time, solvent levels, and their actual values selected based on literature data and preliminary experimental results. )e independent composition, and solvent-to-sample ratio [18, 22–24]. It is therefore important to optimize these process variables to variables and their related codes and levels are displayed in Table 1. A total of 17 experimental runs were performed achieve maximum yield of bioactive compounds from the raw materials. In this study, a response surface method- randomly, which included three replicates at the centre point (Table 2), and all the experiments were replicated thrice to ology (RSM) was used to determine the eﬀect of MAE process variables and their interactions to ensure maximal improve the analysis. Regression analysis for the experiment data was performed and was ﬁtted into a second-order extraction eﬃciency. )is method allows the optimization of all variables simultaneously and predicts the most eﬃ- polynomial model: cient conditions with the use of a minimal number of k k�1 k k experiments [25]. RSM has recently been used to optimize Y � β + 􏽘 β x + 􏽘 􏽘 β x + 􏽘 β x , (1) o i i ij j ii ii the extraction conditions of phenolics from various plants i�1 i�1,i<j j�2 i�2 [26–28]. )us, the objective of this study was to optimize MAE conditions to obtain maximum yield of phenolic where β , β , β , and β are the regression coeﬃcients; x and 0 i ii ij i antioxidants from avocado seeds. RSM was used to predict x are the coded levels of independent variables aﬀecting the the eﬀects of microwave power, extraction time, and eth- dependent response Y; and k is the number of parameters. anol concentration on total phenolic content (TPC), total ﬂavonoid content (TFC), and antioxidant activity of avo- cado seed extract. 2.4. Extraction Process 2.4.1. Microwave-Assisted Extraction (MAE). MAE was 2. Materials and Methods performed using a domestic microwave oven system (Kenwood K30CSS14 Microwave, China) operating at 2.1. Chemicals. Folin–Ciocalteu phenol reagent; alumin- 800 W maximum power and a frequency of 2450 MHz. )e ium chloride (AlCl ·6H O); sodium carbonate (Na CO ); 3 2 2 3 apparatus was equipped with a digital control system for 2,2-diphenyl-1-picrylhydrazyl radical (DPPH); 2,2′-azi- irradiation time and microwave power. )e oven was nobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); modiﬁed in order to condense the vapor generated during sodium nitrite (NaNO ); sodium hydroxide (NaOH); extraction into the sample. 1 g of avocado seeds powder was ethanol; and phenolic compounds (gallic acid, catechin, stirred in 20 mL aqueous ethanol, and the mixture was ir- rutin, quercetin, 4-hydroxybenzoic acid, syringic acid, radiated using the microwave system. )e MAE extraction ferulic acid, vanillic acid, caﬀeic acid, and p-coumaric parameters were microwave power (80–400 W), extraction acid) were purchased from Sigma-Aldrich Corp. (St. time (1–5 min), and ethanol concentration (40–80%). Louis, MO, USA). All chemicals and solvents were of )ereafter, the sample was ﬁltered using a vacuum pump, analytical grade. Journal of Analytical Methods in Chemistry 3 Table 1: )ree levels of the three variables of the extraction process. Coded levels Independent variables Symbols −1 0 1 Ethanol concentration (%) X 40 60 80 Microwave power (W) X 80 240 400 Extraction time (min) X 1 3 5 Table 2: Central composite design (CCD) with observed response of the dependent variables from MAE of avocado seeds. Independent variables Phenolic compounds Antioxidant activity Run order X (%) X (W) X (min) TPC (mg·GAE/g) TFC (mg·QE/g) DPPH (%inhibition) ABTS (%inhibition) 1 2 3 1 40 80 1 52.99 0.98 22.93 17.59 2 80 80 1 47.25 0.66 24.86 11.21 3 40 400 1 74.64 8.89 44.31 39.6 4 80 400 1 64.76 5.90 39.88 35.63 5 40 80 5 65.94 5.59 38.23 37.62 6 80 80 5 66.34 9.14 32.52 35.47 7 40 400 5 76.29 19.70 79.76 60.66 8 80 400 5 77.83 16.65 73.95 56.18 9 40 240 3 77.06 10.92 49.64 46.40 10 80 240 3 78.71 9.70 47.56 40.66 11 60 80 3 68.73 6.43 32.5 50.77 12 60 400 3 89.39 15.70 62.11 80.32 13 60 240 1 72.79 10.14 39.8 44.81 14 60 240 5 79.16 21.45 68.66 73.18 15 60 240 3 83.52 15.48 54.35 68.92 16 60 240 3 80.45 15.23 52.59 67.44 17 60 240 3 84.66 16.10 57.68 70.33 X � ethanol concentration; X � microwave power; X � extraction time; TPC � total phenolic content; TFC � total ﬂavonoid content; DPPH � 2,2-diphenyl- 1 2 3 1-picrylhydrazyl radical scavenging ability; ABTS � 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) scavenging ability. and the liquid extract was collected and stored at 4 C until 2.5.2. Total Flavonoid Content (TFC). )e ﬂavonoid content further use. in the extract was determined by the aluminium chloride method [30]. Brieﬂy, 0.5 mL of extract was diluted with 1.5 mL of distilled water, 0.5 mL of 10% (w/v) aluminium 2.4.2. Conventional Solvent Extraction (CSE). Phenolic chloride, and 0.1 mL of potassium acetate (1 M). )e ﬁnal compounds in avocado seeds were extracted using a CSE volume was made up to 5 mL with distilled water, and the method optimized by Gomez ´ et al. [29]. Brieﬂy, 1 g of av- mixture kept at room temperature for 30 min. )e absor- ocado seeds powder was mixed with 60 mL of 56% ethanol bance was measured at a wavelength of 415 nm against blank (v/v), and the mixture was kept in a thermostatic water bath (AlCl solution) after 30 min of equilibrium. )e TFC was (Grant W14, Cambridge, England) at 63 C, with shaking for quantiﬁed using quercetin standard curve (Figure S2) and 23 min. After cooling, the mixture was centrifuged at estimated as mg of quercetin equivalent (QE) per gram of 2500 rpm for 10 min, and the supernatant was recovered dry weight (dw) of avocado seeds. through ﬁltration and stored at 4 C until further use. 2.6. Antioxidant Activity 2.5. Phytochemical Analysis 2.6.1. DPPH Radical Scavenging Activity. )e DPPH assay 2.5.1. Determination of Total Phenolic Content (TPC). was performed as described by Pandey et al. [30]. )e TPC of the avocado seed extract was determined using the extract (1 mL) was mixed with 3 mL of DPPH solution Folin–Ciocalteu method [16]. )e extract (100 μL) was (4 mL of stock DPPH solution in 96 mL of 80% methanol), mixed with 750 μL of a 10-fold diluted Folin–Ciocalteu and the mixture was kept in dark for 30 min at room reagent followed by 750 μL of sodium carbonate (7.5%, w/v). temperature. )e absorbance of the mixture was mea- )e mixture was incubated in dark at room temperature sured at 520 nm using UV-Vis spectrophotometer (27 C) for 90 min, and its absorbance measured at 725 nm (Labomed Spectro UVD 3200, USA). A mixed solution of using an UV-Vis spectrophotometer (Labomed Spectro 1 mL ethanol and 3 mL DPPH solution was used as the UVD 3200, USA) against the blank. Gallic acid was used for blank. Antioxidant activity of the extract was expressed as the calibration curve (Figure S1). )e results were expressed the percent inhibition, according to the following as mg of gallic acid equivalent (GAE) per gram of dry weight equation: (dw) of avocado seeds. 4 Journal of Analytical Methods in Chemistry A − A 3. Results and Discussion control sample % inhibition � × 100, (2) control 3.1. Model Fitting. A central composite design (CCD) was used to study the eﬀects and interactions of MAE parameters where A is the absorbance value of the blank and control (ethanol concentration, microwave power, and extraction A is the absorbance of extract and DPPH solution. sample time) on TPC, TFC, DPPH, and ABTS. )e experimental design matrix with corresponding responses is presented in Table 1. )e experimental values obtained ranged from 47.25 2.6.2. ABTS Radical Scavenging Activity. ABTS radical to 89.39 mg·GAE/g for TPC, 0.66 to 21.45 mg·QE/g for TFC, scavenging ability of the avocado seed extract was evaluated 22.93 to 79.76% DPPH inhibition, and 11.21 to 80.32% ABTS using a spectrophotometric method as described by Dah- inhibition (Table 2). )e values showed considerable de- moune et al. [16]. A radical solution (7 mM ABTS and pendence on the extraction conditions, which suggests the 2.45 mM potassium persulfate in equal proportions) was need to optimize the extraction process. Quadratic poly- prepared and left to stand in the dark at room temperature nomial models were developed, and the adequacy and ﬁtness (27 C) for 16 h until the reaction was completed, and the of the models were evaluated by ANOVA. )e ANOVA absorbance was stable at 734 nm. )is solution was diluted results revealed that the four models were highly signiﬁcant with ethanol (80%) till an absorbance value of 0.70± 0.02 at (P< 0.0001) for TPC, TFC, DPPH, and ABTS (Table 3). )e 2 2 2 734 nm was obtained. )e extract (0.1 mL) was mixed with respective values of R , Adj-R and Pred-R for TPC (0.9758, 3.9 mL of diluted ABTS solution and kept in dark for 15 min 0.9446, and 0.8086, respectively), TFC (0.9875, 0.9715, and at room temperature. )e absorbance was measured at 0.8679, respectively), DPPH (0.9912, 0.9800, and 0.9372, 734 nm against blank (diluted ABTS solution) using UV-Vis respectively), and ABTS (0.9899, 0.9770, and 0.9255, re- spectrophotometer (Labomed Spectro UVD 3200, USA). spectively) were all close to 1, indicating good correlation )e antioxidant activity of the extract was expressed as between the predicted and the actual results [31]. Moreover, percent inhibition, according to the low values of coeﬃcient of variation (CV, %: 3.55, 9.28, 4.83, and 5.98) suggested that the experimental values were A − A control sample reliable and reproducible [32, 33]. Furthermore, the lack of % inhibition � × 100, (3) control ﬁt values were not signiﬁcant (P> 0.05), indicating the adequacy of the model in predicting MAE of phenolic where A is the absorbance value of the blank and compounds and antioxidant activity of avocado seeds. control A is the absorbance of extract and ABTS solution. sample 3.2. Inﬂuence of the Extraction Parameters on Total Phenolic Content. )e TPC in avocado seed extract varied from 47.25 2.7. HPLC Analysis. Phenolic compounds present in the to 89.39 mg·GAE/g (Table 2). )e lowest yield was achieved optimized extract were analyzed using Shimadzu UFLC at ethanol concentration of 80% and microwave power of chromatographic system (Shimadzu Corporation, Kyoto, 80 W after 1 min of extraction, while the highest yield was Japan), equipped with two LC-20AD pumps and SPD-20AV obtained at ethanol concentration of 60% and microwave ultraviolet-visible detector. )e separation of the com- power of 400 W after 3 min of extraction time. Table 3 shows pounds was performed using Luna C18 column that microwave power (X ) and extraction time (X ) had (150 mm × 4.6 mm, 3 μm) at a column temperature of 30 C. 2 3 signiﬁcant (P< 0.05) positive eﬀect on TPC and the most )e mobile phase consisted of A (1% acetic acid in aceto- nitrile) and B (1% acetic acid in water) with gradient elution signiﬁcant factor is microwave power. )e quadratic eﬀect 2 2 2 (X , X and X ) also had signiﬁcant (P< 0.05) inﬂuence on 0–3 min (9% A), 3–37 min (9–68% A), 37–39 min (68% A), 1 2 3 and 39–40 min (69–9% A). )e ﬂow rate was 0.8 mL/min, TPC under MAE. )ere was a signiﬁcant (P < 0.05) inter- action between ethanol concentration and extraction time and the injection volume was 5 μL. Each standard solution and sample was analyzed in triplicate. )e peaks were de- (X X ), as well as microwave power and extraction time 1 3 (X X ). )e second-order polynomial equation for TPC was tected by UV at wavelength of 280 nm according to the 2 3 scanning mode of the UV detector. )e phenolic com- expressed as pounds were identiﬁed by comparing their retention times Y � 83.19 − 1.20X + 8.17X + 5.31X − 0.38X X with corresponding standards. All the identiﬁed compounds TPC 1 2 3 1 2 2 2 were quantiﬁed by external standard method using cali- + 2.19X X − 2.17X X − 5.55X − 4.37X 1 3 2 3 1 2 bration curves, and their concentrations were expressed as mg/100 g·dw. − 7.46X . (4) 2.8. Statistical Analysis. Statistical analysis and response )e eﬀects of the independent variables and their mutual surface plots were performed using the Design-Expert interactions on TPC can be seen on the three-dimensional software (version 11.0, Stat-Ease, Inc., MN, USA). Data were response surface curves shown in Figures 1(a)–1(c). MAE of analyzed using analysis of variance (ANOVA) at 95% TPC from avocado seeds increased initially and decreased as conﬁdence level. the ethanol concentration increased (Figures 1(a) and 1(b)). X2: power (W) X2: power (W) X3: time (min) X3: time (min) X3: time (min) X3: time (min) Journal of Analytical Methods in Chemistry 5 Table 3: Regression coeﬃcient (β) and analysis of variance (ANOVA) of the predicted second-order polynomial models for phenolic compounds and antioxidant activity. Coeﬃcient (β) Factor TPC TFC DPPH ABTS Intercept 83.19 15.04 53.93 67.44 Linear X -conc −1.20 −0.40 −1.61 −2.27 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ X -power 8.17 4.40 14.90 11.97 ∗∗ ∗∗∗ ∗∗∗ ∗∗∗ X -time 5.31 4.60 12.13 11.43 Interaction X X −0.3750 −1.16 −0.8075 0.01 1 2 X X 2.19 0.48 −1.13 0.47 1 3 ∗ ∗ ∗∗ X X −2.17 1.06 5.82 −0.34 2 3 Quadratic 2 ∗∗ ∗∗ ∗ ∗∗∗ X −5.55 −4.32 −4.63 −22.82 2 ∗ ∗∗ ∗∗ X −4.37 −3.56 −5.93 −0.80 2 ∗∗ ∗∗ X −7.46 1.17 0.9999 −7.35 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ F-value (model) 31.33 61.66 87.93 76.60 F-value (lack of ﬁt) 1.58 7.04 0.74 5.41 R 0.9758 0.9875 0.9912 0.9899 Adj-R 0.9446 0.9715 0.9800 0.9770 Pred-R 0.8086 0.8679 0.9372 0.9255 CV (%) 3.55 9.28 4.83 5.98 ∗ ∗∗ ∗∗∗ p< 0.05, p< 0.01, p< 0.001. 90 90 80 80 70 70 60 60 50 50 40 40 5 400 400 80 5 80 4 320 320 70 4 70 3 240 240 60 3 60 2 160 160 50 2 50 1 80 80 40 1 40 (a) (b) (c) 25 25 20 20 15 15 5 5 0 0 5 80 400 80 5 400 4 70 320 70 4 320 3 60 240 60 3 240 2 50 160 50 2 160 1 40 80 40 1 80 (d) (e) (f) Figure 1: Response surface plot showing the interactive eﬀect of MAE variables on TPC ((a)–(c)) and on TFC ((d)–(f)). Similar observation was reported for MAE of polyphenols explained by the heightened degree of sample cell membrane from Coriolus versicolor mushroom [22], from chokeberries breakage and improved phenolic compounds solubility by [23], from Myrtus communis L. leaves [16] and from the initial increase in ethanol concentrations [35, 36]. blueberry leaves [34]. )is signiﬁcant (p < 0.01) quadratic However, as ethanol concentration continues to increase, the eﬀect of ethanol concentration on TPC (Table 3) could be polarity of the solvent changes, which may lead to increased X1: conc (%) X1: conc (%) X1: conc (%) X2: power (W) X1: conc (%) X2: power (W) TFC (mg·QE/g) TPC (mg·GAE/g) TPC (mg·GAE/g) TFC (mg·QE/g) TFC (mg·QE/g) TPC (mg·GAE/g) 6 Journal of Analytical Methods in Chemistry impurities being extracted [35], therefore reducing the As shown in Table 3, microwave power and extraction time amount of total phenolic compounds extracted. Also in- exhibited a highly signiﬁcant (p< 0.001) positive linear eﬀect, creased diﬀusion resistance due to coagulation of proteins at while the quadratic terms of ethanol concentration and mi- high ethanol concentrations may prevent dissolution of crowave power showed signiﬁcant (p< 0.01) negative eﬀect on polyphenols and inﬂuences the extraction rate [36]. the extraction of TFC from avocado seeds. )e same linear and As shown in Figure 1(a), microwave power had sig- quadratic eﬀects were observed for TPC extraction, which niﬁcant inﬂuence on TPC than ethanol concentration and suggests that similar factors aﬀected the extraction of TFC from this may be attributed to the increased solubility of phenolic avocado seeds. )is is expected as ﬂavonoids represent a compounds as a result of increasing power which promotes subgroup of polyphenols. )e interaction of microwave power cell rupture and enhances exudation of phenolic compounds and extraction time (X X ) had a signiﬁcant (p< 0.05) positive 2 3 into the extracting solvent [37]. Ozbek et al. [38] reported eﬀect on TFC. At lower microwave powers, increasing ex- similar behaviour for MAE of TPC from pistachio hull. )e traction powers gradually increased TFC value over time extraction time was an important parameter that inﬂuenced (Figure 1(f)). )is signiﬁcant (p < 0.05) interaction of mi- the extraction of TPC. As shown in Figures 1(a) and 1(b)), crowave power and extraction time (X X ) is tentatively 2 3 the extraction of TPC increased with increasing extraction explained by the low rate of mass transfer at low microwave time to about 4 min, beyond which a decrease in TPC was powers, which would require more time for the phenolic observed. )is result is in agreement with that reported from compounds to dissolve from the avocado seeds into the so- Calop pulp [39]. Extended extraction time was expected to lution. At higher microwave powers, the dissolution of phe- favour the extraction of phenolic compounds, since enough nolic compounds can reach equilibrium in a relatively short time is required for solvent penetration into the plant tissue, time, hence the extraction of TFC are not readily aﬀected by dissolving the compounds and subsequently diﬀusing out to changes in the extraction time. Ethanol concentration was the the extraction medium [40]. However, at longer extraction least important factor as it did not show a signiﬁcant eﬀect on time, the extracted yields decreased due to increased dissolu- TFC (Table 3). However, the signiﬁcant (p< 0.05) negative tion of polymer matrix, which causes an increase in viscosity interaction of ethanol concentration and microwave power and thereby encapsulating the extracted compounds [41]. In (X X ) on the extraction of TFC suggested that optimal mi- 1 2 addition, long extraction time may increase exposure to light crowave power values increase as ethanol concentration de- and oxygen which will eventually result in the oxidation of creases (Figure 1(d)). phenolic compounds [42]. According to ANOVA analysis (Table 3), the interactive eﬀect of ethanol concentration and 3.4. Inﬂuence of the Extraction Parameters on Antioxidant extraction time (X X ) had signiﬁcant positive inﬂuence 1 3 (p< 0.05) on TPC. As shown in Figure 1(b), the extraction of Activity. )e antioxidant activity of the avocado seed extract was determined using ABTS and DPPH assays. )e results in TPC increased with increasing ethanol concentration and extraction time to about 60% and 4 min, respectively, after Table 3 show that the ABTS scavenging activity was inﬂu- enced by ethanol concentration, microwave power and which increasing ethanol concentration and extraction time caused a decrease in the recovery of TPC. Figure 1(c) illustrates extraction time, while DPPH activity depended on micro- wave power and extraction time. )e model equation for the eﬀect of microwave power and extraction time on TPC. )is signiﬁcant (p < 0.05) positive interaction (Table 2) is in antioxidant activity can be represented as follows: agreement with earlier reports [43, 44]. Increasing microwave Y � 67.44 − 2.27X + 11.97X + 11.43X + 0.01X X ABTS 1 2 3 1 2 power increased TPC as extraction time increases (1–3 min). 2 2 )is phenomenon could be explained by the enhanced mass + 0.47X X − 0.34X X − 22.82X − 0.80X 1 3 2 3 1 2 transfer rate and solubility of phenolic compounds due to − 7.35X , decreasing surface tension and solvent viscosity with increasing microwave power, which improve sample wetting and matrix Y � 53.93 − 1.61X + 14.90X + 12.13X − 0.81X X DPPH 1 2 3 1 2 penetration, respectively, thereby enhancing extraction eﬃ- 2 2 − 1.13X X + 5.82X X − 4.63X − 5.93X 1 3 2 3 1 2 ciency [16, 45, 46]. However, at high levels of microwave power (320–400 W), increasing the extraction time after 4 min de- + 1.00X . creased TPC which may be due to degradation of certain (6) phenolic compounds [16]. )e linear eﬀects of microwave power and extraction time showed a highly signiﬁcant (p< 0.001) positive eﬀect on ABTS scavenging activity, while ethanol concentration 3.3. Inﬂuence of the Extraction Parameters on Total Flavonoid exhibited signiﬁcant (p< 0.05) negative eﬀect on ABTS. Content. )e predictive equation for the relationship be- Moreover, the quadratic eﬀects of ethanol concentration tween TFC and the extraction parameter was expressed as 2 2 (X ) and extraction time (X ) showed highly signiﬁcant 1 3 follows: (p< 0.001) and moderately signiﬁcant (p< 0.01) negative Y � 15.04 − 0.40X + 4.40X + 4.60X − 1.16X X eﬀects on ABTS activity, respectively (Table 3). As shown in TFC 1 2 3 1 2 2 2 2 Figure 2(e), increasing ethanol concentration above 60% + 0.48X X + 1.06X X − 4.32X − 3.56X + 1.17X . 1 3 2 3 1 2 3 resulted in a quadratic decrease in ABTS activity. Inter- (5) estingly, there was no signiﬁcant interactive impact X2: power (W) X2: power (W) X3: time (min) X3: time (min) X3: time (min) X3: time (min) Journal of Analytical Methods in Chemistry 7 90 90 90 80 80 70 70 60 60 60 50 50 40 40 40 30 30 30 20 20 5 80 5 400 400 80 4 320 4 70 320 70 60 3 3 240 240 60 2 50 2 160 160 50 40 1 80 (a) (b) (c) 100 100 100 80 80 60 60 40 40 40 20 20 0 0 5 80 400 400 80 5 70 4 4 320 3 60 240 60 3 240 2 50 160 50 160 80 40 1 80 (d) (e) (f) Figure 2: Response surface plot showing the interactive eﬀect of MAE variables on DPPH and ABTS activity. (p> 0.05) of X X , X X , or X X on ABTS scavenging 11.0 (Stat-Ease, Inc.). )e optimal microwave extraction 1 2 1 3 2 3 activity (Table 3). )is indicates that the ABTS scavenging conditions for optimum TPC, TFC, DPPH, and ABTS in a activity of the extract was individually aﬀected by ethanol single experiment were determined to be as ethanol con- concentration, microwave power, and extraction time and centration of 58.3%, microwave power of 400 W, and ex- not by their interaction. traction time of 4.8 min with desirability of 0.955. )e In case of DPPH antioxidant activity, both microwave numerical optimization provided the maximum predicted power and extraction time showed highly signiﬁcant values of 83.90 mg·GAE/g for TPC, 21.84 mg·QE/g for TFC, (p< 0.001) positive linear eﬀect. )e quadratic eﬀects of the 75.67% DPPH inhibition, and 82.66% ABTS inhibition. ethanol concentration (X ) (p< 0.05) and microwave power Experiments were performed under the optimized condi- tions, and the results are presented in Table 4. )e exper- (X ) signiﬁcantly (p< 0.01) inﬂuenced DPPH scavenging activity (Table 3). Moreover, increasing both microwave imental values agreed with the predicted values, conﬁrming power and extraction time resulted in signiﬁcant positive the reliability of the model obtained by CCD in predicting interactive eﬀect on DPPH activity (Figure 2(c)). )us, the the contents of phenolic compounds and antioxidant activity longer the extraction time, the better the DPPH scavenging using MAE. activity of the extract. Similar observation was reported by Garrido et al. [47] from chardonnay grape marc. 3.6.ComparisonofMAEwithCSE. )e results of TPC, TFC, Although both ABTS and DPPH scavenging activities and antioxidant activity from avocado seeds by MAE and exhibited relatively similar patterns, the minor diﬀerences CSE are shown in Table 5. )e MAE method signiﬁcantly could be due to the present of various phenolic compounds (p< 0.05) enhanced the extraction of phenolic compounds in the extract, which exert diﬀerent kinetics and reaction and antioxidant activity as compared to CSE. In addition mechanisms to diﬀerent antioxidant activity [30]. Similar to improved extraction eﬃciency, solvent consumption ﬁndings have been reported from vine pruning residues [48] and extraction time were signiﬁcantly reduced by MAE in and from rhizomes of Rheum moorcroftianum [30]. comparison with CSE. Using ultrasound-assisted ex- traction, a TPC value of 57.3 mg·GAE/g was obtained 3.5. Optimization of Extraction Conditions and Veriﬁcation of from avocado seeds [11]. )e fast and eﬃcient extraction Predictive Model. )e optimal conditions for simultaneous of phenolic compounds from avocado seeds by MAE extraction of maximum phenolic compounds (TPC and could be explained by the rapid heat generation by mi- TFC) and antioxidant activity (DPPH and ABTS) from dry crowave energy which causes destruction of the cellular avocado seeds were predicted by maximizing the desirability matrix and enhances the release of phenolic compounds of the responses using Design-Expert software trial version [13, 14] and hence antioxidant activity. X1: conc (%) X1: conc (%) X1: conc (%) X2: power (W) X1: conc (%) X2: power (W) ABTS (% inhibition) DPPH (% inhibition) ABTS (% inhibition) DPPH (% inhibition) ABTS (% inhibition) DPPH (% inhibition) 8 Journal of Analytical Methods in Chemistry Table 4: Experimental and predicted values of response variables at optimum extraction conditions. Optimum extraction conditions Maximum value Response variables X (%) X (W) X (min) Experimental value Predicted value 1 2 3 TPC (mg·GAE/g) 82.36± 1.05 83.90 TFC (mg·QE/g) 19.93± 2.50 21.84 58 400 5 DPPH (%) 73.61± 0.57 75.67 ABTS (%) 80.20± 3.23 82.66 X : ethanol concentration (%); X : microwave power (W); X : extraction time (min). Experimental results were expressed as average values± standard 1 2 3 deviation (n � 3). Table 5: Comparison of MAE with CSE. Extraction method Ethanol (%) Time (min) Power (W) Temp ( C) TPC (mg·GAE/g) TFC (mg·QE/g) DPPH (%) ABTS (%) a a a a MAE 58 5 400 – 82.36± 1.05 19.93± 2.50 73.61± 0.57 80.20± 3.23 b b b b CSE 56 23 – 63 51.86± 2.40 11.14± 1.90 60.56± 2.85 63.82± 3.45 )e results are expressed as mean ± SD (n � 3). Values within the same column with diﬀerent letters are signiﬁcantly diﬀerent at p< 0.05. UV Channel 1 280 nm 0 5 10 15 20 25 30 35 40 Min (a) 0 5 10 15 20 25 30 35 40 Min (b) Figure 3: HPLC chromatograms of (a) mixed phenolic standards and (b) avocado seed extract recorded at 280 nm. UV Journal of Analytical Methods in Chemistry 9 Table 6: HPLC quantiﬁcation of phenolic compounds in avocado extract include rutin, catechin, and syringic acid. )us, seed extract under optimal MAE conditions. this optimized MAE method could be beneﬁcial for the extraction and analysis of polyphenolic antioxidants Content Compounds Retention time (min) from avocado seeds for industrial purposes. (mg/100g·dw) Gallic acid 3.32 6.89± 0.04 Catechin 9.65 52.46± 0.15 Data Availability 4-Hydroxybenzoic acid 10.45 12.47± 0.05 Vanillic acid 12.61 6.71± 0.01 )e data used to support the ﬁndings of this study are Caﬀeic acid 12.90 4.18± 0.51 available from the corresponding author upon request. 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Publisher: Hindawi Publishing Corporation
Copyright: Copyright © 2020 Alexander Weremfo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN: 2090-8865
eISSN: 2090-8873
DOI: 10.1155/2020/7541927
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Abstract

Hindawi Journal of Analytical Methods in Chemistry Volume 2020, Article ID 7541927, 11 pages https://doi.org/10.1155/2020/7541927 Research Article Simultaneous Optimization of Microwave-Assisted Extraction of Phenolic Compounds and Antioxidant Activity of Avocado (Persea americana Mill.) Seeds Using Response Surface Methodology 1 1 2 Alexander Weremfo , Felix Adulley, and Martin Adarkwah-Yiadom Department of Biochemistry, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana Drugs, Cosmetics and Forensic Department, Ghana Standards Board, Accra, Ghana Correspondence should be addressed to Alexander Weremfo; aweremfo@ucc.edu.gh Received 12 February 2020; Revised 16 June 2020; Accepted 30 July 2020; Published 17 August 2020 Academic Editor: Rongda Xu Copyright © 2020 Alexander Weremfo et al. )is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. )is study was designed to optimize three microwave-assisted extraction (MAE) parameters (ethanol concentration, microwave power, and extraction time) of total phenolics, total ﬂavonoids, and antioxidant activity of avocado seeds using response surface methodology (RSM). )e predicted quadratic models were highly signiﬁcant (p < 0.001) for the responses studied. )e extraction of total phenolic content (TPC), total ﬂavonoid content (TFC), and antioxidant activity was signiﬁcantly (p< 0.05) inﬂuenced by both microwave power and extraction time. )e optimal conditions for simultaneous extraction of phenolic compounds and antioxidant activity were ethanol concentration of 58.3% (v/v), microwave power of 400 W, and extraction time of 4.8 min. Under these conditions, the experimental results agreed with the predicted values. MAE revealed clear advantages over the conventional solvent extraction (CSE) in terms of high extraction eﬃciency and antioxidant activity within the shortest extraction time. Furthermore, high-performance liquid chromatography (HPLC) analysis of optimized extract revealed the presence of 10 phenolic compounds, with rutin, catechin, and syringic acid being the dominant compounds. Consequently, this optimized MAE method has demonstrated a potential application for eﬃcient extraction of polyphenolic antioxidants from avocado seeds in the nutraceutical industries. addition, this important by-product represents an envi- 1. Introduction ronmental and waste management problem. )e avocado Avocado (Persea americana Mill.) belongs to the family of seed constitutes up to 16% of the weight of the fruit [4] and Lauraceae and is an important fruit crop endemic to the is a rich source of polyphenols with antioxidant and antimicrobial properties [4–7]. Recent studies have tropical and subtropical regions but presently cultivated worldwide. )e food industry has shown remarkable in- demonstrated the antioxidant, anticancer, antidiabetic, terest in processing and enhancing the value of this crop anti-inﬂammatory, blood pressure reducing, antimicro- due to its high economic importance. In addition to its bial, insecticidal, and dermatological activities of seed pleasant sensory properties, there has been growing in- preparations [4, 8]. Due to their beneﬁcial eﬀects, avocado terest in the consumption of avocado-derived products seeds can be an alternative inexpensive source of bioactive owing to its high nutritional value and reported health- compounds, and an eﬃcient extraction of important promoting and/or disease-preventing properties [1, 2]. phenolics from the avocado waste could improve the )e seed is a major by-product of avocado industry and is economics of the avocado industry and minimize envi- usually discarded with no further application [3]. In ronmental impact. 2 Journal of Analytical Methods in Chemistry )e extraction of phenolic compounds from avocado 2.2. Sample Preparation. Avocado fruits (Persea americana seeds has been investigated in the last decades focusing Mill. var. Hass), with adequate ripeness for consumption, mainly on conventional extraction methods such as mac- were obtained from a local market at Bonyere (Ghana) in eration, Soxhlet, and heat reﬂux extraction methods. February 2019. )e seeds were manually removed from the However, these methods are very time-consuming and re- fruits, cleansed, sliced into small and thin size, and sun-dried quire large quantities of solvents [9, 10]. Recently, several for 12 days until no more weight loss was observed. )e eﬃcient and advanced extraction techniques including dried seeds were milled into ﬁne powder using a blender, accelerated solvent extraction [6], ultrasound-assisted ex- and the particle size was standardized using a 250 μm sieve. traction [11], and supercritical ﬂuid extraction [12] have )e moisture content of the dried avocado seeds was 8.9%. been developed for the extraction of phenolic compounds )e powdered sample was stored at −20 C in airtight bags from avocado seeds. Microwave-assisted extraction (MAE) until being used. is a green and eﬀective extraction technique that uses mi- crowave energy to heat polar solvent in contact with sam- 2.3. Experimental Design. A face-centred central composite ples, by ionic conduction and dipole rotation, which improves cell wall destruction and increases solubility of design was used to optimize three independent microwave compounds such as ﬂavonoids [13–15]. MAE has gain parameters: ethanol concentration (%, X ), microwave power (W, X popularity in recent times due to it beneﬁts of improved ), and extraction time (min, X ) of four de- 2 3 eﬃciency, reduced extraction time, low solvent consump- pendent variables: total phenolic content (Y ), total ﬂa- TPC vonoid content (Y ), DPPH scavenging activity (Y ), tion, higher extraction rate, and high potential for auto- TFC DPPH mation [16, 17]. MAE technique has been used for the and ABTS scavenging activity (Y ). )ese independent ABTS microwave parameters were selected due to their signiﬁcant extraction of bioactive compounds from a wide variety of matrices, such as grapes [18], tomatoes [19], apple [20], and inﬂuence on the eﬃciency of MAE [18, 22–24]. Generally, ethanol and methanol are better solvents for extraction of coﬀee [21]. However, the extraction of phenolic bioactive compounds from avocado seeds has not been evaluated phenolic compounds. Considering the potential use of this product in food industry, ethanol was selected as the solvent using MAE. )e eﬃciency of the MAE process is usually aﬀected by in this study. )e independent variables were coded at three several variables such as extraction power, time, solvent levels, and their actual values selected based on literature data and preliminary experimental results. )e independent composition, and solvent-to-sample ratio [18, 22–24]. It is therefore important to optimize these process variables to variables and their related codes and levels are displayed in Table 1. A total of 17 experimental runs were performed achieve maximum yield of bioactive compounds from the raw materials. In this study, a response surface method- randomly, which included three replicates at the centre point (Table 2), and all the experiments were replicated thrice to ology (RSM) was used to determine the eﬀect of MAE process variables and their interactions to ensure maximal improve the analysis. Regression analysis for the experiment data was performed and was ﬁtted into a second-order extraction eﬃciency. )is method allows the optimization of all variables simultaneously and predicts the most eﬃ- polynomial model: cient conditions with the use of a minimal number of k k�1 k k experiments [25]. RSM has recently been used to optimize Y � β + 􏽘 β x + 􏽘 􏽘 β x + 􏽘 β x , (1) o i i ij j ii ii the extraction conditions of phenolics from various plants i�1 i�1,i<j j�2 i�2 [26–28]. )us, the objective of this study was to optimize MAE conditions to obtain maximum yield of phenolic where β , β , β , and β are the regression coeﬃcients; x and 0 i ii ij i antioxidants from avocado seeds. RSM was used to predict x are the coded levels of independent variables aﬀecting the the eﬀects of microwave power, extraction time, and eth- dependent response Y; and k is the number of parameters. anol concentration on total phenolic content (TPC), total ﬂavonoid content (TFC), and antioxidant activity of avo- cado seed extract. 2.4. Extraction Process 2.4.1. Microwave-Assisted Extraction (MAE). MAE was 2. Materials and Methods performed using a domestic microwave oven system (Kenwood K30CSS14 Microwave, China) operating at 2.1. Chemicals. Folin–Ciocalteu phenol reagent; alumin- 800 W maximum power and a frequency of 2450 MHz. )e ium chloride (AlCl ·6H O); sodium carbonate (Na CO ); 3 2 2 3 apparatus was equipped with a digital control system for 2,2-diphenyl-1-picrylhydrazyl radical (DPPH); 2,2′-azi- irradiation time and microwave power. )e oven was nobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); modiﬁed in order to condense the vapor generated during sodium nitrite (NaNO ); sodium hydroxide (NaOH); extraction into the sample. 1 g of avocado seeds powder was ethanol; and phenolic compounds (gallic acid, catechin, stirred in 20 mL aqueous ethanol, and the mixture was ir- rutin, quercetin, 4-hydroxybenzoic acid, syringic acid, radiated using the microwave system. )e MAE extraction ferulic acid, vanillic acid, caﬀeic acid, and p-coumaric parameters were microwave power (80–400 W), extraction acid) were purchased from Sigma-Aldrich Corp. (St. time (1–5 min), and ethanol concentration (40–80%). Louis, MO, USA). All chemicals and solvents were of )ereafter, the sample was ﬁltered using a vacuum pump, analytical grade. Journal of Analytical Methods in Chemistry 3 Table 1: )ree levels of the three variables of the extraction process. Coded levels Independent variables Symbols −1 0 1 Ethanol concentration (%) X 40 60 80 Microwave power (W) X 80 240 400 Extraction time (min) X 1 3 5 Table 2: Central composite design (CCD) with observed response of the dependent variables from MAE of avocado seeds. Independent variables Phenolic compounds Antioxidant activity Run order X (%) X (W) X (min) TPC (mg·GAE/g) TFC (mg·QE/g) DPPH (%inhibition) ABTS (%inhibition) 1 2 3 1 40 80 1 52.99 0.98 22.93 17.59 2 80 80 1 47.25 0.66 24.86 11.21 3 40 400 1 74.64 8.89 44.31 39.6 4 80 400 1 64.76 5.90 39.88 35.63 5 40 80 5 65.94 5.59 38.23 37.62 6 80 80 5 66.34 9.14 32.52 35.47 7 40 400 5 76.29 19.70 79.76 60.66 8 80 400 5 77.83 16.65 73.95 56.18 9 40 240 3 77.06 10.92 49.64 46.40 10 80 240 3 78.71 9.70 47.56 40.66 11 60 80 3 68.73 6.43 32.5 50.77 12 60 400 3 89.39 15.70 62.11 80.32 13 60 240 1 72.79 10.14 39.8 44.81 14 60 240 5 79.16 21.45 68.66 73.18 15 60 240 3 83.52 15.48 54.35 68.92 16 60 240 3 80.45 15.23 52.59 67.44 17 60 240 3 84.66 16.10 57.68 70.33 X � ethanol concentration; X � microwave power; X � extraction time; TPC � total phenolic content; TFC � total ﬂavonoid content; DPPH � 2,2-diphenyl- 1 2 3 1-picrylhydrazyl radical scavenging ability; ABTS � 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) scavenging ability. and the liquid extract was collected and stored at 4 C until 2.5.2. Total Flavonoid Content (TFC). )e ﬂavonoid content further use. in the extract was determined by the aluminium chloride method [30]. Brieﬂy, 0.5 mL of extract was diluted with 1.5 mL of distilled water, 0.5 mL of 10% (w/v) aluminium 2.4.2. Conventional Solvent Extraction (CSE). Phenolic chloride, and 0.1 mL of potassium acetate (1 M). )e ﬁnal compounds in avocado seeds were extracted using a CSE volume was made up to 5 mL with distilled water, and the method optimized by Gomez ´ et al. [29]. Brieﬂy, 1 g of av- mixture kept at room temperature for 30 min. )e absor- ocado seeds powder was mixed with 60 mL of 56% ethanol bance was measured at a wavelength of 415 nm against blank (v/v), and the mixture was kept in a thermostatic water bath (AlCl solution) after 30 min of equilibrium. )e TFC was (Grant W14, Cambridge, England) at 63 C, with shaking for quantiﬁed using quercetin standard curve (Figure S2) and 23 min. After cooling, the mixture was centrifuged at estimated as mg of quercetin equivalent (QE) per gram of 2500 rpm for 10 min, and the supernatant was recovered dry weight (dw) of avocado seeds. through ﬁltration and stored at 4 C until further use. 2.6. Antioxidant Activity 2.5. Phytochemical Analysis 2.6.1. DPPH Radical Scavenging Activity. )e DPPH assay 2.5.1. Determination of Total Phenolic Content (TPC). was performed as described by Pandey et al. [30]. )e TPC of the avocado seed extract was determined using the extract (1 mL) was mixed with 3 mL of DPPH solution Folin–Ciocalteu method [16]. )e extract (100 μL) was (4 mL of stock DPPH solution in 96 mL of 80% methanol), mixed with 750 μL of a 10-fold diluted Folin–Ciocalteu and the mixture was kept in dark for 30 min at room reagent followed by 750 μL of sodium carbonate (7.5%, w/v). temperature. )e absorbance of the mixture was mea- )e mixture was incubated in dark at room temperature sured at 520 nm using UV-Vis spectrophotometer (27 C) for 90 min, and its absorbance measured at 725 nm (Labomed Spectro UVD 3200, USA). A mixed solution of using an UV-Vis spectrophotometer (Labomed Spectro 1 mL ethanol and 3 mL DPPH solution was used as the UVD 3200, USA) against the blank. Gallic acid was used for blank. Antioxidant activity of the extract was expressed as the calibration curve (Figure S1). )e results were expressed the percent inhibition, according to the following as mg of gallic acid equivalent (GAE) per gram of dry weight equation: (dw) of avocado seeds. 4 Journal of Analytical Methods in Chemistry A − A 3. Results and Discussion control sample % inhibition � × 100, (2) control 3.1. Model Fitting. A central composite design (CCD) was used to study the eﬀects and interactions of MAE parameters where A is the absorbance value of the blank and control (ethanol concentration, microwave power, and extraction A is the absorbance of extract and DPPH solution. sample time) on TPC, TFC, DPPH, and ABTS. )e experimental design matrix with corresponding responses is presented in Table 1. )e experimental values obtained ranged from 47.25 2.6.2. ABTS Radical Scavenging Activity. ABTS radical to 89.39 mg·GAE/g for TPC, 0.66 to 21.45 mg·QE/g for TFC, scavenging ability of the avocado seed extract was evaluated 22.93 to 79.76% DPPH inhibition, and 11.21 to 80.32% ABTS using a spectrophotometric method as described by Dah- inhibition (Table 2). )e values showed considerable de- moune et al. [16]. A radical solution (7 mM ABTS and pendence on the extraction conditions, which suggests the 2.45 mM potassium persulfate in equal proportions) was need to optimize the extraction process. Quadratic poly- prepared and left to stand in the dark at room temperature nomial models were developed, and the adequacy and ﬁtness (27 C) for 16 h until the reaction was completed, and the of the models were evaluated by ANOVA. )e ANOVA absorbance was stable at 734 nm. )is solution was diluted results revealed that the four models were highly signiﬁcant with ethanol (80%) till an absorbance value of 0.70± 0.02 at (P< 0.0001) for TPC, TFC, DPPH, and ABTS (Table 3). )e 2 2 2 734 nm was obtained. )e extract (0.1 mL) was mixed with respective values of R , Adj-R and Pred-R for TPC (0.9758, 3.9 mL of diluted ABTS solution and kept in dark for 15 min 0.9446, and 0.8086, respectively), TFC (0.9875, 0.9715, and at room temperature. )e absorbance was measured at 0.8679, respectively), DPPH (0.9912, 0.9800, and 0.9372, 734 nm against blank (diluted ABTS solution) using UV-Vis respectively), and ABTS (0.9899, 0.9770, and 0.9255, re- spectrophotometer (Labomed Spectro UVD 3200, USA). spectively) were all close to 1, indicating good correlation )e antioxidant activity of the extract was expressed as between the predicted and the actual results [31]. Moreover, percent inhibition, according to the low values of coeﬃcient of variation (CV, %: 3.55, 9.28, 4.83, and 5.98) suggested that the experimental values were A − A control sample reliable and reproducible [32, 33]. Furthermore, the lack of % inhibition � × 100, (3) control ﬁt values were not signiﬁcant (P> 0.05), indicating the adequacy of the model in predicting MAE of phenolic where A is the absorbance value of the blank and compounds and antioxidant activity of avocado seeds. control A is the absorbance of extract and ABTS solution. sample 3.2. Inﬂuence of the Extraction Parameters on Total Phenolic Content. )e TPC in avocado seed extract varied from 47.25 2.7. HPLC Analysis. Phenolic compounds present in the to 89.39 mg·GAE/g (Table 2). )e lowest yield was achieved optimized extract were analyzed using Shimadzu UFLC at ethanol concentration of 80% and microwave power of chromatographic system (Shimadzu Corporation, Kyoto, 80 W after 1 min of extraction, while the highest yield was Japan), equipped with two LC-20AD pumps and SPD-20AV obtained at ethanol concentration of 60% and microwave ultraviolet-visible detector. )e separation of the com- power of 400 W after 3 min of extraction time. Table 3 shows pounds was performed using Luna C18 column that microwave power (X ) and extraction time (X ) had (150 mm × 4.6 mm, 3 μm) at a column temperature of 30 C. 2 3 signiﬁcant (P< 0.05) positive eﬀect on TPC and the most )e mobile phase consisted of A (1% acetic acid in aceto- nitrile) and B (1% acetic acid in water) with gradient elution signiﬁcant factor is microwave power. )e quadratic eﬀect 2 2 2 (X , X and X ) also had signiﬁcant (P< 0.05) inﬂuence on 0–3 min (9% A), 3–37 min (9–68% A), 37–39 min (68% A), 1 2 3 and 39–40 min (69–9% A). )e ﬂow rate was 0.8 mL/min, TPC under MAE. )ere was a signiﬁcant (P < 0.05) inter- action between ethanol concentration and extraction time and the injection volume was 5 μL. Each standard solution and sample was analyzed in triplicate. )e peaks were de- (X X ), as well as microwave power and extraction time 1 3 (X X ). )e second-order polynomial equation for TPC was tected by UV at wavelength of 280 nm according to the 2 3 scanning mode of the UV detector. )e phenolic com- expressed as pounds were identiﬁed by comparing their retention times Y � 83.19 − 1.20X + 8.17X + 5.31X − 0.38X X with corresponding standards. All the identiﬁed compounds TPC 1 2 3 1 2 2 2 were quantiﬁed by external standard method using cali- + 2.19X X − 2.17X X − 5.55X − 4.37X 1 3 2 3 1 2 bration curves, and their concentrations were expressed as mg/100 g·dw. − 7.46X . (4) 2.8. Statistical Analysis. Statistical analysis and response )e eﬀects of the independent variables and their mutual surface plots were performed using the Design-Expert interactions on TPC can be seen on the three-dimensional software (version 11.0, Stat-Ease, Inc., MN, USA). Data were response surface curves shown in Figures 1(a)–1(c). MAE of analyzed using analysis of variance (ANOVA) at 95% TPC from avocado seeds increased initially and decreased as conﬁdence level. the ethanol concentration increased (Figures 1(a) and 1(b)). X2: power (W) X2: power (W) X3: time (min) X3: time (min) X3: time (min) X3: time (min) Journal of Analytical Methods in Chemistry 5 Table 3: Regression coeﬃcient (β) and analysis of variance (ANOVA) of the predicted second-order polynomial models for phenolic compounds and antioxidant activity. Coeﬃcient (β) Factor TPC TFC DPPH ABTS Intercept 83.19 15.04 53.93 67.44 Linear X -conc −1.20 −0.40 −1.61 −2.27 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ X -power 8.17 4.40 14.90 11.97 ∗∗ ∗∗∗ ∗∗∗ ∗∗∗ X -time 5.31 4.60 12.13 11.43 Interaction X X −0.3750 −1.16 −0.8075 0.01 1 2 X X 2.19 0.48 −1.13 0.47 1 3 ∗ ∗ ∗∗ X X −2.17 1.06 5.82 −0.34 2 3 Quadratic 2 ∗∗ ∗∗ ∗ ∗∗∗ X −5.55 −4.32 −4.63 −22.82 2 ∗ ∗∗ ∗∗ X −4.37 −3.56 −5.93 −0.80 2 ∗∗ ∗∗ X −7.46 1.17 0.9999 −7.35 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ F-value (model) 31.33 61.66 87.93 76.60 F-value (lack of ﬁt) 1.58 7.04 0.74 5.41 R 0.9758 0.9875 0.9912 0.9899 Adj-R 0.9446 0.9715 0.9800 0.9770 Pred-R 0.8086 0.8679 0.9372 0.9255 CV (%) 3.55 9.28 4.83 5.98 ∗ ∗∗ ∗∗∗ p< 0.05, p< 0.01, p< 0.001. 90 90 80 80 70 70 60 60 50 50 40 40 5 400 400 80 5 80 4 320 320 70 4 70 3 240 240 60 3 60 2 160 160 50 2 50 1 80 80 40 1 40 (a) (b) (c) 25 25 20 20 15 15 5 5 0 0 5 80 400 80 5 400 4 70 320 70 4 320 3 60 240 60 3 240 2 50 160 50 2 160 1 40 80 40 1 80 (d) (e) (f) Figure 1: Response surface plot showing the interactive eﬀect of MAE variables on TPC ((a)–(c)) and on TFC ((d)–(f)). Similar observation was reported for MAE of polyphenols explained by the heightened degree of sample cell membrane from Coriolus versicolor mushroom [22], from chokeberries breakage and improved phenolic compounds solubility by [23], from Myrtus communis L. leaves [16] and from the initial increase in ethanol concentrations [35, 36]. blueberry leaves [34]. )is signiﬁcant (p < 0.01) quadratic However, as ethanol concentration continues to increase, the eﬀect of ethanol concentration on TPC (Table 3) could be polarity of the solvent changes, which may lead to increased X1: conc (%) X1: conc (%) X1: conc (%) X2: power (W) X1: conc (%) X2: power (W) TFC (mg·QE/g) TPC (mg·GAE/g) TPC (mg·GAE/g) TFC (mg·QE/g) TFC (mg·QE/g) TPC (mg·GAE/g) 6 Journal of Analytical Methods in Chemistry impurities being extracted [35], therefore reducing the As shown in Table 3, microwave power and extraction time amount of total phenolic compounds extracted. Also in- exhibited a highly signiﬁcant (p< 0.001) positive linear eﬀect, creased diﬀusion resistance due to coagulation of proteins at while the quadratic terms of ethanol concentration and mi- high ethanol concentrations may prevent dissolution of crowave power showed signiﬁcant (p< 0.01) negative eﬀect on polyphenols and inﬂuences the extraction rate [36]. the extraction of TFC from avocado seeds. )e same linear and As shown in Figure 1(a), microwave power had sig- quadratic eﬀects were observed for TPC extraction, which niﬁcant inﬂuence on TPC than ethanol concentration and suggests that similar factors aﬀected the extraction of TFC from this may be attributed to the increased solubility of phenolic avocado seeds. )is is expected as ﬂavonoids represent a compounds as a result of increasing power which promotes subgroup of polyphenols. )e interaction of microwave power cell rupture and enhances exudation of phenolic compounds and extraction time (X X ) had a signiﬁcant (p< 0.05) positive 2 3 into the extracting solvent [37]. Ozbek et al. [38] reported eﬀect on TFC. At lower microwave powers, increasing ex- similar behaviour for MAE of TPC from pistachio hull. )e traction powers gradually increased TFC value over time extraction time was an important parameter that inﬂuenced (Figure 1(f)). )is signiﬁcant (p < 0.05) interaction of mi- the extraction of TPC. As shown in Figures 1(a) and 1(b)), crowave power and extraction time (X X ) is tentatively 2 3 the extraction of TPC increased with increasing extraction explained by the low rate of mass transfer at low microwave time to about 4 min, beyond which a decrease in TPC was powers, which would require more time for the phenolic observed. )is result is in agreement with that reported from compounds to dissolve from the avocado seeds into the so- Calop pulp [39]. Extended extraction time was expected to lution. At higher microwave powers, the dissolution of phe- favour the extraction of phenolic compounds, since enough nolic compounds can reach equilibrium in a relatively short time is required for solvent penetration into the plant tissue, time, hence the extraction of TFC are not readily aﬀected by dissolving the compounds and subsequently diﬀusing out to changes in the extraction time. Ethanol concentration was the the extraction medium [40]. However, at longer extraction least important factor as it did not show a signiﬁcant eﬀect on time, the extracted yields decreased due to increased dissolu- TFC (Table 3). However, the signiﬁcant (p< 0.05) negative tion of polymer matrix, which causes an increase in viscosity interaction of ethanol concentration and microwave power and thereby encapsulating the extracted compounds [41]. In (X X ) on the extraction of TFC suggested that optimal mi- 1 2 addition, long extraction time may increase exposure to light crowave power values increase as ethanol concentration de- and oxygen which will eventually result in the oxidation of creases (Figure 1(d)). phenolic compounds [42]. According to ANOVA analysis (Table 3), the interactive eﬀect of ethanol concentration and 3.4. Inﬂuence of the Extraction Parameters on Antioxidant extraction time (X X ) had signiﬁcant positive inﬂuence 1 3 (p< 0.05) on TPC. As shown in Figure 1(b), the extraction of Activity. )e antioxidant activity of the avocado seed extract was determined using ABTS and DPPH assays. )e results in TPC increased with increasing ethanol concentration and extraction time to about 60% and 4 min, respectively, after Table 3 show that the ABTS scavenging activity was inﬂu- enced by ethanol concentration, microwave power and which increasing ethanol concentration and extraction time caused a decrease in the recovery of TPC. Figure 1(c) illustrates extraction time, while DPPH activity depended on micro- wave power and extraction time. )e model equation for the eﬀect of microwave power and extraction time on TPC. )is signiﬁcant (p < 0.05) positive interaction (Table 2) is in antioxidant activity can be represented as follows: agreement with earlier reports [43, 44]. Increasing microwave Y � 67.44 − 2.27X + 11.97X + 11.43X + 0.01X X ABTS 1 2 3 1 2 power increased TPC as extraction time increases (1–3 min). 2 2 )is phenomenon could be explained by the enhanced mass + 0.47X X − 0.34X X − 22.82X − 0.80X 1 3 2 3 1 2 transfer rate and solubility of phenolic compounds due to − 7.35X , decreasing surface tension and solvent viscosity with increasing microwave power, which improve sample wetting and matrix Y � 53.93 − 1.61X + 14.90X + 12.13X − 0.81X X DPPH 1 2 3 1 2 penetration, respectively, thereby enhancing extraction eﬃ- 2 2 − 1.13X X + 5.82X X − 4.63X − 5.93X 1 3 2 3 1 2 ciency [16, 45, 46]. However, at high levels of microwave power (320–400 W), increasing the extraction time after 4 min de- + 1.00X . creased TPC which may be due to degradation of certain (6) phenolic compounds [16]. )e linear eﬀects of microwave power and extraction time showed a highly signiﬁcant (p< 0.001) positive eﬀect on ABTS scavenging activity, while ethanol concentration 3.3. Inﬂuence of the Extraction Parameters on Total Flavonoid exhibited signiﬁcant (p< 0.05) negative eﬀect on ABTS. Content. )e predictive equation for the relationship be- Moreover, the quadratic eﬀects of ethanol concentration tween TFC and the extraction parameter was expressed as 2 2 (X ) and extraction time (X ) showed highly signiﬁcant 1 3 follows: (p< 0.001) and moderately signiﬁcant (p< 0.01) negative Y � 15.04 − 0.40X + 4.40X + 4.60X − 1.16X X eﬀects on ABTS activity, respectively (Table 3). As shown in TFC 1 2 3 1 2 2 2 2 Figure 2(e), increasing ethanol concentration above 60% + 0.48X X + 1.06X X − 4.32X − 3.56X + 1.17X . 1 3 2 3 1 2 3 resulted in a quadratic decrease in ABTS activity. Inter- (5) estingly, there was no signiﬁcant interactive impact X2: power (W) X2: power (W) X3: time (min) X3: time (min) X3: time (min) X3: time (min) Journal of Analytical Methods in Chemistry 7 90 90 90 80 80 70 70 60 60 60 50 50 40 40 40 30 30 30 20 20 5 80 5 400 400 80 4 320 4 70 320 70 60 3 3 240 240 60 2 50 2 160 160 50 40 1 80 (a) (b) (c) 100 100 100 80 80 60 60 40 40 40 20 20 0 0 5 80 400 400 80 5 70 4 4 320 3 60 240 60 3 240 2 50 160 50 160 80 40 1 80 (d) (e) (f) Figure 2: Response surface plot showing the interactive eﬀect of MAE variables on DPPH and ABTS activity. (p> 0.05) of X X , X X , or X X on ABTS scavenging 11.0 (Stat-Ease, Inc.). )e optimal microwave extraction 1 2 1 3 2 3 activity (Table 3). )is indicates that the ABTS scavenging conditions for optimum TPC, TFC, DPPH, and ABTS in a activity of the extract was individually aﬀected by ethanol single experiment were determined to be as ethanol con- concentration, microwave power, and extraction time and centration of 58.3%, microwave power of 400 W, and ex- not by their interaction. traction time of 4.8 min with desirability of 0.955. )e In case of DPPH antioxidant activity, both microwave numerical optimization provided the maximum predicted power and extraction time showed highly signiﬁcant values of 83.90 mg·GAE/g for TPC, 21.84 mg·QE/g for TFC, (p< 0.001) positive linear eﬀect. )e quadratic eﬀects of the 75.67% DPPH inhibition, and 82.66% ABTS inhibition. ethanol concentration (X ) (p< 0.05) and microwave power Experiments were performed under the optimized condi- tions, and the results are presented in Table 4. )e exper- (X ) signiﬁcantly (p< 0.01) inﬂuenced DPPH scavenging activity (Table 3). Moreover, increasing both microwave imental values agreed with the predicted values, conﬁrming power and extraction time resulted in signiﬁcant positive the reliability of the model obtained by CCD in predicting interactive eﬀect on DPPH activity (Figure 2(c)). )us, the the contents of phenolic compounds and antioxidant activity longer the extraction time, the better the DPPH scavenging using MAE. activity of the extract. Similar observation was reported by Garrido et al. [47] from chardonnay grape marc. 3.6.ComparisonofMAEwithCSE. )e results of TPC, TFC, Although both ABTS and DPPH scavenging activities and antioxidant activity from avocado seeds by MAE and exhibited relatively similar patterns, the minor diﬀerences CSE are shown in Table 5. )e MAE method signiﬁcantly could be due to the present of various phenolic compounds (p< 0.05) enhanced the extraction of phenolic compounds in the extract, which exert diﬀerent kinetics and reaction and antioxidant activity as compared to CSE. In addition mechanisms to diﬀerent antioxidant activity [30]. Similar to improved extraction eﬃciency, solvent consumption ﬁndings have been reported from vine pruning residues [48] and extraction time were signiﬁcantly reduced by MAE in and from rhizomes of Rheum moorcroftianum [30]. comparison with CSE. Using ultrasound-assisted ex- traction, a TPC value of 57.3 mg·GAE/g was obtained 3.5. Optimization of Extraction Conditions and Veriﬁcation of from avocado seeds [11]. )e fast and eﬃcient extraction Predictive Model. )e optimal conditions for simultaneous of phenolic compounds from avocado seeds by MAE extraction of maximum phenolic compounds (TPC and could be explained by the rapid heat generation by mi- TFC) and antioxidant activity (DPPH and ABTS) from dry crowave energy which causes destruction of the cellular avocado seeds were predicted by maximizing the desirability matrix and enhances the release of phenolic compounds of the responses using Design-Expert software trial version [13, 14] and hence antioxidant activity. X1: conc (%) X1: conc (%) X1: conc (%) X2: power (W) X1: conc (%) X2: power (W) ABTS (% inhibition) DPPH (% inhibition) ABTS (% inhibition) DPPH (% inhibition) ABTS (% inhibition) DPPH (% inhibition) 8 Journal of Analytical Methods in Chemistry Table 4: Experimental and predicted values of response variables at optimum extraction conditions. Optimum extraction conditions Maximum value Response variables X (%) X (W) X (min) Experimental value Predicted value 1 2 3 TPC (mg·GAE/g) 82.36± 1.05 83.90 TFC (mg·QE/g) 19.93± 2.50 21.84 58 400 5 DPPH (%) 73.61± 0.57 75.67 ABTS (%) 80.20± 3.23 82.66 X : ethanol concentration (%); X : microwave power (W); X : extraction time (min). Experimental results were expressed as average values± standard 1 2 3 deviation (n � 3). Table 5: Comparison of MAE with CSE. Extraction method Ethanol (%) Time (min) Power (W) Temp ( C) TPC (mg·GAE/g) TFC (mg·QE/g) DPPH (%) ABTS (%) a a a a MAE 58 5 400 – 82.36± 1.05 19.93± 2.50 73.61± 0.57 80.20± 3.23 b b b b CSE 56 23 – 63 51.86± 2.40 11.14± 1.90 60.56± 2.85 63.82± 3.45 )e results are expressed as mean ± SD (n � 3). Values within the same column with diﬀerent letters are signiﬁcantly diﬀerent at p< 0.05. UV Channel 1 280 nm 0 5 10 15 20 25 30 35 40 Min (a) 0 5 10 15 20 25 30 35 40 Min (b) Figure 3: HPLC chromatograms of (a) mixed phenolic standards and (b) avocado seed extract recorded at 280 nm. UV Journal of Analytical Methods in Chemistry 9 Table 6: HPLC quantiﬁcation of phenolic compounds in avocado extract include rutin, catechin, and syringic acid. )us, seed extract under optimal MAE conditions. this optimized MAE method could be beneﬁcial for the extraction and analysis of polyphenolic antioxidants Content Compounds Retention time (min) from avocado seeds for industrial purposes. (mg/100g·dw) Gallic acid 3.32 6.89± 0.04 Catechin 9.65 52.46± 0.15 Data Availability 4-Hydroxybenzoic acid 10.45 12.47± 0.05 Vanillic acid 12.61 6.71± 0.01 )e data used to support the ﬁndings of this study are Caﬀeic acid 12.90 4.18± 0.51 available from the corresponding author upon request. 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Journal of Analytical Methods in Chemistry – Hindawi Publishing Corporation

Published: Aug 17, 2020

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Simultaneous Optimization of Microwave-Assisted Extraction of Phenolic Compounds and Antioxidant Activity of Avocado (Persea americana Mill.) Seeds Using Response Surface Methodology

Simultaneous Optimization of Microwave-Assisted Extraction of Phenolic Compounds and Antioxidant Activity of Avocado (Persea americana Mill.) Seeds Using Response Surface Methodology

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Simultaneous Optimization of Microwave-Assisted Extraction of Phenolic Compounds and Antioxidant Activity of Avocado (Persea americana Mill.) Seeds Using Response Surface Methodology

Simultaneous Optimization of Microwave-Assisted Extraction of Phenolic Compounds and Antioxidant Activity of Avocado (Persea americana Mill.) Seeds Using Response Surface Methodology

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