Effect of extraction conditions on the antioxidant activity of Vernonia amygdalina Del. (Asteraceae)

TẠP CHÍ PHÁT TRIỂN KHOA HỌC VÀ CÔNG NGHỆ - 37<br /> KỸ THUẬT & CÔNG NGHỆ, TẬP 1, SỐ 3, 2018<br /> <br /> <br /> <br /> <br /> Effect of extraction conditions on the<br /> antioxidant activity of Vernonia amygdalina<br /> Del. (Asteraceae)<br /> Dinh Chung Duong*, Ngoc Yen Nguyen Thi, Hung Lam Hoa<br /> <br />  are evidences that the regression model can<br /> Abstract—In this study, the effect of represent the experimental data well. HPLC<br /> extraction conditions on the antioxidant activity showed that leaves contain at least six<br /> of Vernonia amygdalina Del. (Asteraceae) was flavonoids, two of which are apigenin and<br /> evaluated by Response surface methodology luteolin. The flavonoids apigenin and luteolin<br /> and central composite design (RSM-CCD) to were identified in the extract from Vernonia<br /> predict the content of phenolic compounds with amygdalina with high levels of apigenin (2.72<br /> maximum antioxidant activity. Total phenol mg/g dw), luteolin (3.76 mg/g dw).<br /> and flavonoid contents were determined by Keywords—Vernonia amygdalina Del.,<br /> spectrophotometry method, especially the extraction conditions, polyphenol, antioxidant<br /> flavonoid content was identified by HPLC-DAD activity, oxidative stress.<br /> system. The antioxidant activity was estimated<br /> by the DPPH and the FRAP method. Results 1 INTRODUCTION<br /> showed that extracting time, extracting<br /> temperature and solvent-to-material ratio had a<br /> significant effect on phenolic content (p <<br /> F ree radicals play important roles and necessary<br /> for life. It was produced continuously in all<br /> cells as part of a normal cellular function. Free<br /> 0.001). The interactions between the three radicals and oxidants contain both toxic and<br /> factors were also found to be significant at 0.05 beneficial compounds. Oxidative stress, arising as<br /> level of probability. After re-estimating a result of an imbalance between free radical<br /> predicted variables on the experiment, we production and antioxidant defenses [1] but cannot<br /> found that the polyphenol content was 137.15 ± gradually be destroyed, following their<br /> 1.36 mg gallic acid /g dry weight (dw), the accumulation in the body. This process is partly<br /> flavonoid content was 96.78 ± 1.39 mg reposible for the development of diseases such as<br /> quercetin/g dw, the total antioxidant activity arthritis, vasculitis, lupus erythematous, adult<br /> was 1.95 ± 0.09 mg ascorbic acid/g dw and iron respiratory diseases syndrome, hypertension, heart<br /> reduction activity was 5.90 ± 0.12 mg FeSO 4/g diseases, stroke, intestinal is chemianeurological<br /> dw at optimum conditions of 34.82 hours at disorder (Alzheimer's disease, Parkinson's disease,<br /> 53.09 °C with solvent to material ratio is 43.64 muscular dystrophy) [2, 3].<br /> (ml/g). The correlation coefficients were greater Antioxidants act as a radical scavenger, a<br /> than 0.995 observed between the predicted and hydrogen donor, electron donor, peroxide<br /> actual values for the response variables, which decomposer, singlet oxygen quencher, a enzyme<br /> inhibitor, synergist, and metal chelating agents.<br /> Both enzymatic and nonenzymatic antioxidants<br /> Received: Sep 19th, 2018; Accepted: Dec 17th, 2018; exist in the intracellular and extracellular<br /> Published: Dec 30th, 2018 environment to detoxify ROS (reactive oxygen<br /> species) [4]. The human body has several<br /> “This study was sponsored by The Science and Research<br /> Development Fund of Nguyen Tat Thanh University.” mechanisms to counteract oxidative stress by<br /> Dinh Chung Duong, Ngoc Yen Nguyen Thi is with Falculty producing antioxidants, such as the superoxide<br /> of Pharmacy in Nguyen Tat Thanh Univeristy, 298-300A dismutase, catalase, glutathione peroxidase and<br /> Nguyen Tat Thanh Street, Ward 13, District 4, Ho Chi Minh glutathione reductase which are either naturally<br /> City (e-mail: ddchung@ntt.edu.vn).<br /> Hung Lam Hoa is with Department of Physiochemical, produced or externally supplied through foods<br /> Faculty of Chemical Engineering, Ho Chi Minh City University and/or supplements such as vitamin A, C , E [5, 6],<br /> of Technology, VNU-HCM.<br /> 38 SCIENCE & TECHNOLOGY DEVELOPMENT JOURNAL -<br /> ENGINEERING & TECHNOLOGY, VOL 1, ISSUE 3, 2018<br /> <br /> glutathione [7] and polyphenol antioxidants Nguyen Tat Thanh University. The leaves of the<br /> originated from plants [8-11]. plant were air-dried in shade and finely powdered.<br /> Vernonia amygdalina is a shrub that grows<br /> 2.2 Experimental design<br /> predominantly in Africa and Asia. That is a plant<br /> widely used for application in natural medicine. It Experimental variables of extraction process<br /> is commonly known as “bitter leaf” which is due were performed based on RSM combined with<br /> to its bitter taste [12]. It is characterized by a soft- Box-Behnken design for extraction of polyphenols<br /> wooded tree of 2 to 5 m with an elliptical leaf from and antioxidant activity from V. amygdalina<br /> the genus Vernonia [2]. The phytochemical leaves. The variables were designed of three levels<br /> screening of the plants studied showed that the (lower, middle and higher value, being coded as<br /> presence of flavonoids, saponins, alkaloids, −1, 0 and +1) (Table 1) and a total of 15 runs<br /> tannins, phenolics, terpenes, steroidal glycosides, including 3 at central experiments were carried out<br /> sesquiterpene lactones, triterpenoids [13, 14] was to optimize the level of chosen variables, such as<br /> represented by polysaccharides[15], luteolin, extraction temperature (X1, oC), extraction time<br /> luteolin 7-O-β-glucoside luteolin 7-O-glucuronide (X2, hour) and solvent to sample ratio (X3, g/ml)<br /> [12], vernolide, vernolepin, vernodalin, (Table 2). The total polyphenol content (Y1), total<br /> hydroxyvernolide, vernodalol, vernomygdin, flavonoid content (Y2), radical scavenging activity<br /> vernomenin, 4,15-dihydrovernodalin, 1,2,11,12ʹ,3ʹ (DPPH) (Y3) and ferric ion reducing antioxidant<br /> hexahydrovernodalin, 1,2,4,15,11,13,2ʹ,3ʹ power (Y4) were expressed individually as a<br /> octahydrover nodalin, epivernodalol, and function of the independent variables. The<br /> vernonioside [16-19]. The pharmacological generalized second-order polynomial model used<br /> properties of V. amygdalina have been reported to in the response surface analysis as follows:<br /> 3 3 2 3<br /> Y   0    i X i    ii X i2    X X<br /> following antidiabetic [20], antioxidant [12, 21],<br /> antimicrobial[22], antifungal[23], antiplasmodial ij i j <br /> i 1 i 1 i 1 j i 1<br /> [24], cathartic [25], hepatoprotective [26], and<br /> (1)<br /> antitumor activity [27, 28].<br /> where Y is the predicted response, β0, βi, βii, and<br /> Vernonia amygdalina Del. is a plant widely<br /> βij are the regression coefficients for the intercept,<br /> used for application in natural medicine. The study<br /> linearity, square, and interaction, respectively, Xi<br /> of medicinal plants starts with the pre-extraction<br /> and Xj (i=1–3, j=1–3 and i≠j) are the independent<br /> and the extraction procedures, which is an<br /> variables.<br /> important step in the processing of the bioactive<br /> The analysis of variance (ANOVA) using<br /> constituents from plant materials. Hence, selection<br /> Design Expert trial version 7.0.0 (State Ease, Inc.)<br /> of proper extraction method needs meticulous<br /> was carried out to determine maximal values of<br /> evaluation. Traditional methods such as<br /> reponses. The significance of all the terms of<br /> maceration and soxhlet extraction are commonly<br /> polynomial equation was analyzed statistically by<br /> used in the laboratory research. However,<br /> computing the P-value < 0.05.<br /> extensive extraction time, experimental numbers<br /> with low extraction productivity and unstable Table 1. Independence factors and corresponding levels<br /> results [29]<br /> Response surface methodology is commonly Values of coded<br /> used to reduce experimental numbers and evaluate Independent variables Unit levels<br /> the interaction between the design factors for -1 0 +1<br /> improving materials and methods for further Extraction temperature (X1) o<br /> C 45.0 52.5 60.0<br /> application in many industries. In this study, Extraction time (X2) hour 16.0 32.0 48.0<br /> optimal conditions for extraction were determined Solvent-to-material ratio (X3) ml/g 20.0 40.0 60.0<br /> by RSM to predict the content of phenolic<br /> 2.3 Chemicals and Reagents<br /> compounds with maximum antioxidant activity<br /> from V. amydalina Del. leaves. Folin-ciocalteu, gallic acid, quercetin, 2,2-<br /> diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-<br /> 2 MATERIALS AND METHODS tripyridyl triazine (TPTZ), luteolin, apigenin,<br /> aluminium chloride (AlCl3), and sodium carbonate<br /> 2.1 Plant Material (Na2CO3) were purchased from Sigma Aldrich<br /> Leaves of V. amygdalina were collected at Cu (Singapore). All the chemicals were analytical<br /> Chi ward, Ho Chi Minh city in November 2017 grades.<br /> and were identified by Botanical department of<br /> TẠP CHÍ PHÁT TRIỂN KHOA HỌC VÀ CÔNG NGHỆ - 39<br /> KỸ THUẬT & CÔNG NGHỆ, TẬP 1, SỐ 3, 2018<br /> <br /> Table 2. Box–Behnken design matrix and experimental responses<br /> <br /> Variables Polyphenol Flavonoid Antioxidant Ferous reducing<br /> Content content activity activity<br /> Runs (Y1) (Y2) (Y3) (Y4)<br /> X1 X2 X3<br /> (oC) (hour) (ml/g)<br /> 1 52.5 32.0 40.0 135.18 97.85 1.91 5.87<br /> 2 45.0 16.0 40.0 84.59 69.70 1.20 2.05<br /> 3 52.5 32.0 40.0 139.07 98.93 1.97 5.96<br /> 4 60.0 32.0 60.0 106.96 67.57 1.51 3.52<br /> 5 45.0 32.0 20.0 81.55 68.47 1.15 2.08<br /> 6 52.5 16.0 20.0 88.21 67.39 1.25 3.41<br /> 7 52.5 32.0 40.0 137.14 98.68 1.94 5.94<br /> 8 45.0 32.0 60.0 89.30 75.13 1.26 2.67<br /> 9 60.0 32.0 20.0 88.77 59.47 1.25 2.65<br /> 10 60.0 16.0 40.0 87.22 55.66 1.23 2.55<br /> 11 60.0 48.0 40.0 114.75 66.49 1.62 2.96<br /> 12 52.5 48.0 20.0 94.83 77.15 1.34 3.19<br /> 13 52.5 16.0 60.0 88.21 80.21 1.25 3.66<br /> 14 45.0 48.0 40.0 86.37 71.06 1.22 2.24<br /> 15 52.5 48.0 60.0 116.08 83.70 1.64 4.46<br /> Y1 = mg gallic acid/g dw; Y2 = mg quercetin/g dw; Y3 = mg ascorbic acid/g dw; Y4 = mg FeSO4/g dw<br /> <br /> double-distilled water. The mixture was allowed to<br /> 2.4 Determination of total phenolic content<br /> stand for 15 min, and absorbance was measured at<br /> The total phenolic content of the extract was 415 nm. The total flavonoid content was calculated<br /> determined by the Folin–Ciocalteu method [30]. from a calibration curve established by quercetine<br /> Samples (0.5 ml) were introduced into test tubes, solution 20 – 200 µg/ml, and the result was<br /> mixed thoroughly with 2.5 ml of Folin–Ciocalteu expressed as mg rutin equivalent per g dry weight.<br /> reagent for 5 min, followed by the addition of 2 ml CxFxV<br /> of 20% (w/v) sodium carbonate. The mixture was TFC <br /> Wx(1 - h)<br /> allowed to stand for a further 90 min in the dark at<br /> room temperature, and absorbance was measured Where C: sample concentration calculated from<br /> at 760 nm. The total phenolic content was calibration curve (mg/ml), F: dilution factor; V:<br /> calculated from the calibration curve, and the total volumn of ethanol extract (ml), W: sample<br /> results were expressed as mg of gallic acid weight (g), h: sample moiture content.<br /> equivalent per g dry weight.<br /> CxFxV 2.6 DPPH method of antioxidant assay<br /> TFC <br /> Wx(1 - h) The antioxidant activity of the extract was<br /> Where C: sample concentration calculated from determined by the 1,1-diphenyl-2-picryl-hydrazyl<br /> calibration curve (mg/ml), F: dilution factor; V: (DPPH) assay of Yuvaraj (2013) [32] with some<br /> total volumn of ethanol extract (ml), W: sample modifications. Briefly, 0.5 ml of each extract (was<br /> weight (g), h: sample moiture content. diluted with ethanol to suitable concentration)<br /> were mixed with 2,5 ml DPPH solution (0.25 µM)<br /> 2.5 Determination of total flavonoid content and incubated in the dark at room temperature for<br /> The total flavonoid content of crude extract was 30 min. A blank containing 2.5 ml of DPPH and<br /> determined by the aluminium chloride colorimetric 0.5 ml methanol was prepared and treated as the<br /> method of Thaipong (2006) [31]. In brief, 1 ml of test samples. The absorbance of the mixture was<br /> crude extract (1 mg/ml ethanol) were mixed with then measured at 517 nm. The ability of the<br /> 4 ml of distilled water and then 0.3 ml of 5% sample to scavenge DPPH radical was determined<br /> NaNO2 solution; 0.3 ml of 10% AlCl3 solution was from:<br /> added after 5 min of incubation, and the mixture DPPH radical scavenging activity (%) = [(Abscontrol<br /> was allowed to stand for 2 min. Then, 2 ml of – Abssample)/ Abscontrol]x100<br /> 1 mol/L NaOH solution were added, and the final Ascorbic acid with concentrations of 3 – 15<br /> volume of the mixture was brought to 10 ml with µg/ml was used as a positive control to set up<br /> 40 SCIENCE & TECHNOLOGY DEVELOPMENT JOURNAL -<br /> ENGINEERING & TECHNOLOGY, VOL 1, ISSUE 3, 2018<br /> <br /> calibration curve and the result was expressed as 2.9 Statistical Analysis<br /> mg ascorbic acid equivalent per g dry weight. Data were expressed as mean ± SD. Statistical<br /> 2.7 Ferric ion Reducing Antioxidant Power significance was determined by one-way analysis<br /> (FRAP) Assay of variance followed by the Tukey test. was<br /> considered significant.<br /> The FRAP assay was conducted according to<br /> the method reported by Benzie and Strain (1999)<br /> 3 RESULTS AND DISCUSSION<br /> [33]. FRAP reagent was prepared freshly by<br /> mixing three solutions, sodium acetate buffer at 3.1 Effect of extraction variables on total<br /> pH = 3, 6, 10 mM TPTZ solution in 40 mM polyphenol content (TPC)<br /> HCl solution and 20 mM ferric chloride (FeCl3) The experimental data showing the total<br /> solution in proportions of 10:1:1 (v/v/v). For the phenolic content was 81.55 – 139.07 mg gallic<br /> assay, 0.5 ml of plant extracts was mixed with acid equivalents/g dry weight. The ANOVA<br /> 2.5 ml of FRAP reagent. The samples were showed the model F value of 182.21 with<br /> vortexed for 1 min and incubated in dark for probability (p < 0.0001) which implied that the<br /> 30 min at 40°C. The absorbance of reaction model was significant and there was only 0.01%<br /> mixture was measured at 593 nm. The standard chances that this large F value could occur due to<br /> ferrous sulfate solution (FeSO4) of 10 – 100 µg/ml noise. The coefficient of determination R2 was<br /> was used for calibration curve. The results of 0.9970 expressing the strong correlation between<br /> FRAP activity expressed as ferrous equivalent per input variables and TPC. Indeed, phenolic content<br /> g dry weight (mg FeSO4/g dw) were then of extracts was significantly influenced (p <<br /> extrapolated from the standard curve. 0.05) by linear (X1, X2, X3), interaction parameters<br /> 2.8 High pressure liquid chromatography test (X1X2, X1X3, X2X3) and quadratic parameters (X12,<br /> condition X22, X32) (Table 3). The curved surface plot<br /> (Figure 1a-c) demonstrated the role of three<br /> The sample (10 mg crude extract) was added<br /> extraction variables effect positively on TPC at<br /> 100 ml of methanol: water (1: 1) solution,<br /> medium levels of these factors. The final empirical<br /> ultrasonic extraction in 15 minutes (no heat) and<br /> regression model of their relationship between<br /> after that centrifuge 6000 rpm for 10 minutes, take<br /> responses and the three tested variables could be<br /> solution, add 100 ml of 20% acid HCl hydrolyzed<br /> expressed by the following quadratic polynomial<br /> in 3 hours at 85C. Then, the aglycon flavonoids<br /> equation:<br /> were extracted by 20 ml of ethyl acetate (x3), Y1= 137.13 + 6.99X1 + 7.98X2 + 5.90X3 + 6.44X1X2 + 2.61X1X3<br /> combine the extract, and rotate the solvent. The + 5.31X2X3 - 24.54X12 - 19.35X22 - 20.94X32 (2)<br /> residue is dissolved in 3 ml mobile phase. The<br /> sample washed with column Bond Elut C18 SPE 3.2 Effect of extraction variables on total<br /> (Agilent - USA) activated by 3ml water. Wash flavonoid content (TFC)<br /> diluted solution of 5 ml with mobile phase, filter The experimental data showing the total<br /> through PTFE membrane 0.45 µm for flavonoid content was 55.66 – 98.93 mg rutin<br /> chromatography injection. Condition equivalents/g dry weight. The ANOVA showed<br /> chromatography analysis was performed using an the model F value of 369.62 with probability (p <<br /> Agilent Technologies 1260 infinity I, with a 0.0001) which implied that the model was<br /> photodiode array detector (PDA - G1315D) and an significant and there was only 0.01% chances that<br /> automatic injector. Stationary phase was used a this large F value could occur due to noise. The<br /> Zorbax XDB reversed phase (SB-C18 150 x 4.6 coefficient of determination R2 was 0.9985<br /> mm), 5 μm particle size. The mobile phase expressing the strong correlation between input<br /> composed of acetonitrile and 1 % phosphoric acid variables and TPC. Indeed, phenolic content of<br /> aqueous solution (68:32, v/v) at a flow rate of 0.7 extracts was significantly influenced (p < 0.05) by<br /> ml/min. The injection volume was 50 μL and the linear (X1, X2, X3), interaction parameters (X1X2,<br /> temperature was maintained at 40°C during the X1X3, X2X3) and quadratic parameters (X12, X22,<br /> analysis. Detection was realized at wavelength X32) (Table 3). The curved surface plot (Figure 1a-<br /> 384 nm. Two reference standards, luteolin and c) demonstrated the role of three extraction<br /> apigenin [12, 34], were simultaneously used in this variables effect positively on TPC at medium<br /> experiment as markers. levels of these factors. The final empirical<br /> regression model of their relationship between<br /> responses and the three tested variables could be<br /> TẠP CHÍ PHÁT TRIỂN KHOA HỌC VÀ CÔNG NGHỆ - 41<br /> KỸ THUẬT & CÔNG NGHỆ, TẬP 1, SỐ 3, 2018<br /> <br /> expressed by the following quadratic polynomial (X1, X2, X3), three quadratic effects (X12, X22, X32),<br /> equation: and three interactive effects (X1X2, X1X3, X2X3).<br /> Y2= 98.49 - 4.40X1 + 3.18X2 + 4.27X3 + 2.27X1X2 The model P value of 0.0001 obtained for the<br /> + 0.36X1X3 - 1.571X2X3 - 21.11X12 - 11.65X22 - antioxidant capacity implied that the model is<br /> 9.72X32 (3) hingly significant (Table 3). The regression<br /> equation predicted by mathematical models for Y3,<br /> 3.3 Effect of extraction variables on antioxidant<br /> Y4 were given below:<br /> capacity<br /> Y3 =1.94 + 0.097X1 + 0.11X2 + 0.084X3 +<br /> The antioxidant capacity of the extract was 0.093X1X2 + 0.037X1X3 + 0.075X2X3 – 0.35X12 –<br /> determined by two methods: DPPH and FRAP 0.27X22 – 0.30X32 (4)<br /> assay. The results of ANOVA analysis showed Y4 = 5.92 + 0.33X1 + 0.15X2 + 0.37X3 +<br /> that the antioxidant activity significantly affected 0.055X1X2 – 0.007X1X3 + 0.26X2X3 – 2.21X12 –<br /> by the extraction temperature, extraction time, and 1.26X22 – 0.98X32 (5)<br /> solvent-to-material ratio with three linear effects<br /> Table 3. ANOVA analysis for model<br /> <br /> Source TPC content TFC content Antioxidant Ferrous reducing power<br /> (Y1) (Y2) activity (FRAP) (Y4)<br /> (DPPH) (Y3)<br /> <br /> F-Value P-Value F-Value P-Value F-Value P-Value F-Value P-Value<br /> <br /> Model 182.21 < 0.0001 369.62 < 0.0001 175.58 < 0.0001 1501.80 < 0.0001<br /> X1 106.29 0.0001 195.15 < 0.0001 99.41 0.0002 437.06 < 0.0001<br /> X2 138.51 < 0.0001 102.11 0.0002 129.43 < 0.0001 87.32 0.0002<br /> X3 75.78 0.0003 183.78 < 0.0001 73.35 0.0004 556.88 < 0.0001<br /> X1 X2 45.13 0.0011 28.29 0.0031 44.74 0.0011 6.07 0.0470<br /> X1 X3 7.42 0.0416 0.65 0.4553 7.35 0.0422 9.83 0.0258<br /> X2 X3 30.73 0.0026 12.41 0.0169 29.41 0.0029 130.48 < 0.0001<br /> X12 605.44 < 0.0001 2075.91 < 0.0001 591.25 < 0.0001 9060.61 < 0.0001<br /> X22 376.55 < 0.0001 632.85 < 0.0001 358.40 < 0.0001 2948.54 < 0.0001<br /> X32 440.85 < 0.0001 440.36 < 0.0001 427.18 < 0.0001 1785.03 < 0.0001<br /> Lack of Fit 0.950 0.549 3.465 0.2320 0.750 0.6148 0.820 0.5901<br /> R2 0.9970 0.9985 0.9968 0.9990<br /> Adj R2 0.9915 0.9958 0.9912 0.9960<br /> Pre R2 0.9686 0.9793 0.9699 0.9964<br /> <br /> The effect of the variables and their interaction The three-dimensional surface response in<br /> on the antioxidant capacity of the V. amygdalina Figure 1 evaluated the relationship between three<br /> leaf extracts is shown in three-demensional input variables and the contribution of each<br /> response surface in Figure 1. A higher antioxidant parameter on the values of responses.<br /> capacity was obtained in the extraction by The RSM model and ANOVA analysis showed<br /> increasing extraction temperature, time and that the values of TPC and TFC content and<br /> solvents. However, the yield of antioxidant antioxidant activity were affected proportionally<br /> compounds tended to reduce at elevated by three variables: extraction temperature,<br /> temperature and elongated time because of the rate extraction time, and solvent-to-material ratio. By<br /> of decomposition of these compounds. The increasing these parameters, the results of<br /> temperature utilized during extraction influenced responses tended to decrease due to the<br /> the stability of antioxidant compounds due to decomposition of phenolic compounds. The<br /> chemical and enzymatic degradation; these factors maximum level was determined under the<br /> have been suggested to be the main mechanisms following experimental conditions: a temperature<br /> underlying reduction of the polyphenol content in of 53.09°C, extraction time of 34.82 hours, and a<br /> the extraction. Besides, further increase of the solvent-to-material ratio of 43.64 (ml/g). In order<br /> solvent to material ratio may dilute the extraction to validate the suitability of the mathematical<br /> solution thereby lowering the antioxidant activity. model for predicting the optimal response value,<br /> 42 SCIENCE & TECHNOLOGY DEVELOPMENT JOURNAL -<br /> ENGINEERING & TECHNOLOGY, VOL 1, ISSUE 3, 2018<br /> <br /> verification experiments were carried out under the dw. 1.95 ± 0.09 mg ascorbic/g dw và 5.90 ± 0.12<br /> optimal conditions. The values of TPC, TFC mg FeSO4/g dw, respectively. Based on the results,<br /> content, antioxidant power (DPPH and FRAP the experimental values of responses were found to<br /> assay) obtained from experiment were 137.15 ± be quite comparable with predicted values at 95%<br /> 1.36 mg gallic/g dw, 96.78 ± 1.39 mg quercetin/g confidence level.<br /> <br /> (a) (b) (c)<br /> Design-Expert® Software Design-Expert® Software Design-Expert® Software<br /> <br /> Total Polyphenol Total Polyphenol Total Polyphenol<br /> 139.07 139.07 139.07<br /> <br /> 81.55 81.55 81.55<br /> 140 140 140<br /> Total Polyphenol = 139.07 Total Polyphenol = 139.07 Total Polyphenol = 139.07<br /> Std # 14 Run # 3 Std # 14 Run # 3 Std # 14 Run # 3<br /> X1 = A: Nhiet do = 52.50 126 X1 = B: Thoi gian = 32.00 127 X1 = A: Nhiet do = 52.50 125.25<br /> Total Polyphenol<br /> <br /> <br /> <br /> <br /> Total Polyphenol<br /> <br /> <br /> <br /> <br /> Total Polyphenol<br /> X2 = B: Thoi gian = 32.00 X2 = C: Ty le DM/Dl = 40.00 X2 = C: Ty le DM/Dl = 40.00<br /> 112 114 110.5<br /> Actual Factor Actual Factor Actual Factor<br /> C: Ty le DM/Dl = 40.00 A: Nhiet do = 52.50 B: Thoi gian = 32.00<br /> 98 101 95.75<br /> <br /> <br /> <br /> 84 88 81<br /> <br /> <br /> <br /> <br /> 48.00 60.00 60.00 48.00 60.00 60.00<br /> 40.00 56.25 50.00 40.00 50.00 56.25<br /> 32.00 52.50 40.00 32.00 40.00 52.50<br /> 24.00 48.75 30.00 24.00 30.00 48.75<br /> B: Thoi gian A: Nhiet do C: Ty le DM/Dl B: Thoi gian C: Ty le DM/Dl A: Nhiet do<br /> 16.00 45.00 20.00 16.00 20.00 45.00<br /> <br /> <br /> <br /> <br /> (d) (e) (f)<br /> Design-Expert® Software Design-Expert® Software Design-Expert® Software<br /> <br /> Total Flavonoid Total Flavonoid Total Flavonoid<br /> 98.93 98.93 98.93<br /> <br /> 55.66 55.66 55.66<br /> 99 100 100<br /> Total Flavonoid = 98.68 Total Flavonoid = 98.68 Total Flavonoid = 98.93<br /> Std # 13 Run # 7 Std # 13 Run # 7 Std # 14 Run # 3<br /> X1 = A: Nhiet do = 52.50 88 91.75 X1 = A: Nhiet do = 52.50 89.5<br /> X1 = B: Thoi gian = 32.00<br /> Total Flavonoid<br /> <br /> <br /> <br /> <br /> Total Flavonoid<br /> Total Flavonoid<br /> <br /> <br /> <br /> <br /> X2 = B: Thoi gian = 32.00 X2 = C: Ty le DM/Dl = 40.00 X2 = C: Ty le DM/Dl = 40.00<br /> 77 83.5 79<br /> Actual Factor Actual Factor Actual Factor<br /> C: Ty le DM/Dl = 40.00 A: Nhiet do = 52.50 B: Thoi gian = 32.00<br /> 66 68.5<br /> 75.25<br /> <br /> <br /> 55 58<br /> 67<br /> <br /> <br /> <br /> 48.00 60.00 60.00 60.00<br /> 60.00 48.00<br /> 40.00 56.25 50.00 56.25<br /> 50.00 40.00<br /> 32.00 52.50 40.00 52.50<br /> 40.00 32.00<br /> 24.00 48.75 30.00 48.75<br /> B: Thoi gian A: Nhiet do 30.00 24.00 C: Ty le DM/Dl A: Nhiet do<br /> 16.00 45.00 C: Ty le DM/Dl B: Thoi gian 20.00 45.00<br /> 20.00 16.00<br /> <br /> <br /> <br /> <br /> (g) (h) (i)<br /> Design-Expert® Software Design-Expert® Software Design-Expert® Software<br /> <br /> Total antioxydase Total antioxydase Total antioxydase<br /> 1.97 1.97 1.97<br /> <br /> 1.15 1.15 1.15<br /> 1.97 1.97 1.97<br /> Total antioxydase = 1.97 X1 = B: Thoi gian Total antioxydase = 1.97<br /> Std # 14 Run # 3 X2 = C: Ty le DM/Dl Std # 14 Run # 3<br /> X1 = A: Nhiet do = 52.50 1.76<br /> 1.78 1.79<br /> Total antioxydase<br /> <br /> <br /> <br /> <br /> X1 = A: Nhiet do = 52.50<br /> Total antioxydase<br /> <br /> <br /> <br /> <br /> Total antioxydase<br /> <br /> <br /> <br /> <br /> X2 = B: Thoi gian = 32.00 Actual Factor X2 = C: Ty le DM/Dl = 40.00<br /> A: Nhiet do = 52.50 1.56<br /> 1.59 1.61 Actual Factor<br /> Actual Factor<br /> B: Thoi gian = 32.00<br /> C: Ty le DM/Dl = 40.00<br /> 1.35<br /> 1.39 1.43<br /> <br /> <br /> 1.14<br /> 1.20 1.25<br /> <br /> <br /> <br /> 60.00 60.00<br /> 48.00 60.00 60.00 48.00<br /> 50.00 56.25<br /> 40.00 56.25 50.00 40.00<br /> 40.00 52.50<br /> 32.00 52.50 40.00 32.00<br /> 30.00 48.75<br /> 24.00 48.75 30.00 24.00 C: Ty le DM/Dl A: Nhiet do<br /> B: Thoi gian A: Nhiet do C: Ty le DM/Dl B: Thoi gian 20.00 45.00<br /> 16.00 45.00 20.00 16.00<br /> <br /> <br /> <br /> <br /> (k) (m) (l)<br /> Design-Expert® Software Design-Expert® Software Design-Expert® Software<br /> <br /> Frap value Frap value Frap value<br /> 5.96 5.96 5.96<br /> <br /> 2.05 2.05 2.05<br /> 6.00 6.00 6.00<br /> Frap value = 5.96 Frap value = 5.96 Frap value = 5.94<br /> Std # 14 Run # 3 Std # 14 Run # 3 Std # 13 Run # 7<br /> 5.00 X1 = B: Thoi gian = 32.00 5.28 X1 = A: Nhiet do = 52.50 5.00<br /> X1 = A: Nhiet do = 52.50<br /> X2 = B: Thoi gian = 32.00 X2 = C: Ty le DM/Dl = 40.00 X2 = C: Ty le DM/Dl = 40.00<br /> Frap value<br /> <br /> <br /> <br /> <br /> Frap value<br /> Frap value<br /> <br /> <br /> <br /> <br /> 4.55 4.00<br /> 4.00 Actual Factor Actual Factor<br /> Actual Factor<br /> A: Nhiet do = 52.50 B: Thoi gian = 32.00<br /> C: Ty le DM/Dl = 40.00<br /> 3.83 3.00<br /> 3.00<br /> <br /> <br /> 3.10 2.00<br /> 2.00<br /> <br /> <br /> <br /> 60.00 48.00 60.00 60.00<br /> 48.00 60.00<br /> 50.00 40.00 50.00 56.25<br /> 40.00 56.25<br /> 40.00 32.00 40.00 52.50<br /> 32.00 52.50<br /> 30.00 24.00 30.00 48.75<br /> 24.00 48.75 C: Ty le DM/Dl B: Thoi gian C: Ty le DM/Dl