Accepted Manuscript

Optimization of ultrasound-assisted extraction of antioxidant compounds from

Tunisian Zizyphus lotus fruits using response surface methodology
Khaoula Mkadmini hammi, Ahmed Jdey, Chedly Abdelly, Hatem Majdoub,
Riadh Ksouri
PII:
DOI:
Reference:

S0308-8146(15)00410-0
http://dx.doi.org/10.1016/j.foodchem.2015.03.047
FOCH 17294

To appear in:

Food Chemistry

Received Date:
Revised Date:
Accepted Date:

5 December 2014
11 March 2015
14 March 2015

Please cite this article as: Mkadmini hammi, K., Jdey, A., Abdelly, C., Majdoub, H., Ksouri, R., Optimization of
ultrasound-assisted extraction of antioxidant compounds from Tunisian Zizyphus lotus fruits using response surface
methodology, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.03.047

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Optimization of ultrasound-assisted extraction of

antioxidant compounds from Tunisian Zizyphus lotus

fruits using response surface methodology

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Optimization of ultrasound-assisted extraction of antioxidant compounds from Tunisian

Zizyphus lotus fruits using response surface methodology

Khaoula MKADMINI HAMMIa*, Ahmed JDEYa, Chedly ABDELLYa,

Hatem MAJDOUBb, Riadh KSOURIa

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2050 Hammam-lif, Tunisia.

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Monastir, Universit de Monastir, Bd. de lenvironnement, 5019 Monastir, Tunisia.

Laboratoire des Plantes Extrmophiles, Centre de Biotechnologie de Borj- Cdria, BP 901,

Laboratoire des Interfaces et des Matriaux Avancs (LIMA), Facult des Sciences de

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*Corresponding author: Khaoulafayrouzahammi@gmail.com

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Abstract

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The optimization of antioxidant extraction conditions from a ripe edible fruits of Zizyphus

31

lotus (L.) with an ultrasound-assisted system was achieved by response surface methodology.

32

The central composite rotatable design was employed for optimization of extraction

33

parameters in terms of total phenolic content and antioxidant activities using 2,2-diphenyl-1-

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picrylhydrazyl radical scavenging activity and phosphomolybdenum assay. The optimum

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operating conditions for extraction were as follows: ethanol concentration, 50%; extraction

36

time, 25 min; extraction temperature, 63C and ratio of solvent to solid, 67 mL/g. Under these

37

conditions, the obtained extract exhibited a high content of phenolic compounds (40.782 mg

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gallic acid equivalents/g dry matter) with significant antioxidant properties (the total

39

antioxidant activity was 75.981 mg gallic acid equivalents /g dry matter and the 2,2-diphenyl-

40

1-picrylhydrazyl radical scavenging activity was 0.289 mg/mL).

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Keywords: Optimization, response surface methodology, central composite rotatable design,

45

ultrasound-assisted system, total phenolic content, phosphomolybdenum assay, 2,2-diphenyl-

46

1-picrylhydrazyl radical scavenging activity.

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1. Introduction

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The genus Zizyphus belonging to the family Rhamnaceae is widespread in tropical and sub-

58

tropical regions: Asia, Africa, North America, South America, Oceania and Europe with the

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center of diversity in Asia (Richardson et al., 2004). There are 135-170 species of Zizyphus

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(Maraghni et al., 2010). Only Z. spina-christi (L.) Willd, Z. vulgaris Lam. and Z. lotus (L.)

61

Lam are found in Tunisia. Recently, several pharmacological researches (Borgi & Chouchane,

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2009; Borgi et al., 2007) have been reported on the various species of Zizyphus (Rhamnaceae)

63

particularly Zizyphus lotus which is commonly known as sedra and the edible fruit called

64

nbeg. In Tunisia, this species grows under a variety of environmental conditions. So, it is

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localized in different regions mainly in arid zone such as Tozeur. Zizyphus lotus is dormant

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from October through March and mature plant flowers in May and June and produces fruits in

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August (Maraghni et al., 2010). This plant is used in folklore medicine for the treatment of

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various diseases such as diabetes, bronchitis, diarrhea and abscess (Le-Floch, 1983).

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Previous studies have been reported on Zizyphus lotus from the locality of Cherahil (Monastir,

70

Tunisia). So, anti-spasmodic activities of leaves and root barks of this plant were

71

demonstrated in rodents (Borgi & Chouchane, 2009). Besides, Borgi et al. (2007)

72

demonstrated that root barks of Z. lotus, showed a significant and dose-dependent anti-

73

inflammatory and analgesic activity in carrageenan-induced paw oedema in rats. In the same

74

paper, it was mentioned that the presence of flavonoids in the Zizyphus lotus extracts was

75

responsible for these beneficial effects. Also, chemical analysis of these species has led to the

76

isolation of four dammarane type saponins from root bark of Z. lotus collected in March 1994

77

at the locality of Cherahil (Renault et al., 1997). Fruit of Zizyphus lotus of North Africa is

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delicious and consumed directly due to its high nutritional value. In fact, Abdeddaim et al.

79

(2014) demonstrated that this organ was rich in minerals (calcium, magnesium, sodium,

80

potassium and phosphorus), carbohydrates, fatty acids and proteins that are essential for some

81

physicochemical processes necessary for a good health. Besides, Benammar et al. (2010)

82

showed that the fruit pulp of this plant contained a higher vitamin A and C compared to the

83

other parts. These vitamins are responsible for human cell T-prolifiration. In the same paper,

84

it was revealed that the fruit pulp was the richest source of linoleic acid (18:2n6), a precursor

85

of n6 fatty acids. In addition, it was mentioned that the fruit pulp exhibited a higher

86

antioxidant capacity than other parts of Z. lotus. Moreover, Alhakmani et al. (2014)

87

demonstrated that fruits of Zizyphus spina-christi are rich in phenolic compounds responsible
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for the antioxidant activity. Also, it was mentioned that this organ could be used as a natural

89

source of antioxidants to prevent the progression of some diseases. In this context, secondary

90

metabolites like flavonoids, phenolic acids, saponins and alkaloids are a specific antioxidants

91

compounds that protect plant, animals, food and human from oxidative stress which is a result

92

of the production of reactive oxygen species (ROS), including superoxide anion radical,

93

hydrogen peroxide, hydroxyl radical as well as reactive nitrogen species (RNS) which can

94

cause inflammation and contribute to tissue damage (Mothana, 2011). Besides, in food

95

products, free radicals react with lipids and cause lipid peroxidation which affects the quality

96

of product (taste, color and flavor) (Biglari et al., 2008). In humans, oxidative stress causes

97

many current diseases including inflammation, cataract, cancer, arteriosclerosis, autoimmune,

98

Parkinson and neurodegenerative syndromes (Ksouri et al., 2011). As demonstrated by

99

Lacopini et al. (2008), antioxidants, such as phenolic acids and flavonoids, have a great

100

capacity for scavenging free radicals like peroxide, hydroperoxide of lipid hydroxyl, and thus

101

inhibit the oxidative mechanisms that lead to cardiovascular diseases and cancer. Besides, it is

102

well known that flavonoids and phenolic acids could preserve the quality of food products

103

from chemical oxidation due to exposure to air (oxygen) or to the effects of light

104

(Maisuthisakul et al., 2007).

105

In most food industries, synthetic antioxidants for example, butylated hydroxyanisole (BHA),

106

butylated hydroxytoluene (BHT), tertiary butyl. hydroquinone (TBHQ) and propyl gallate

107

(PG) are used in order to prevent the rancidity of processed food. Because of their potential

108

carcinogenicity and other dangerous effects, they have been replaced by natural antioxidants

109

extracted from various plants (Suhaj, 2006).

110

The present study investigates the antioxidant effect of the ethanolic extract of the edible fruit

111

part of Z. lotus collected from an arid zone (Tozeur, Tunisia).

112

Various novel extraction techniques have been developed for the extraction of antioxidant

113

secondary metabolites including ultrasound-assisted extraction, supercritical fluid extraction,

114

microwave-assisted extraction, and accelerated solvent extraction. Among these techniques,

115

ultrasound-assisted extraction is a simple, efficient and inexpensive alternative. It is more

116

effective at extracting secondary metabolites due to the acoustic cavitation effect produced in

117

the solvent by the passage of ultrasonic waves which can lead to the destruction of cells and

118

enhance the contact surface area between solid and liquid phases. These effects permit better

119

penetration of the solvent into the sample increasing the extraction yield of secondary

120

metabolites (Wang et al., 2008).

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The efficiency of an extraction process is influenced by many factors such as solvent

122

composition, extraction temperature, extraction time, and solvent to solid ratio (Cacace &

123

Mazza, 2002). Response Surface Methodology (RSM) is a useful tool for optimizing the

124

chemical process; it is an efficacy mathematical and statistical technique for analysis of

125

empirical models that describes the effect of independent variables and their interactions on

126

response variables (Myers & Montgomery, 2002).

127

In the present study, optimization of extraction temperature, extraction time, ethanol

128

concentration, ratio of liquid to dry matter through the ultrasound bath equipment to

129

determine the best extraction conditions of antioxidants from Z. lotus fruits using RSM and

130

employing central composite rotatable design (CCDR) was investigated.

131

2. Materials and methods

132

2.1. Plant material

133

Zizyphus lotus (L.) fruits were collected from Tozeur, South of Tunisia, in August 2013. In

134

order to minimize the degradation of phenolic compounds, fruits were dried in lyophilizer and

135

stored at 4C until use.

136

2.2. Chemicals and reagents

137

Gallic acid was purchased from Fluka (Buchs, Switzerland). Sulfuric acid, ammonium

138

molybdate and ethanol (HPLC grade) were purchased from Merck (Darmstadt, Germany).

139

DPPH (2,2-diphenyl-1-picrylhydrazyl), FolinCiocalteu reagent, sodium phosphate and

140

sodium carbonate were purchased from Sigma-Aldrich (Brazil). Ultrapure water was obtained

141

from the Millipore system (Billerica, USA).

142
143

2.3. Ultrasound-assisted extraction of antioxidant compounds

144

Extractions were carried out in an ultrasonic bath (Sonorex Digital 10 P, Bandelin) at 35 KHz.

145

Samples (2g) were powdered and placed into Erlenmeyer flasks (250 mL). Samples were

146

exhaustively extracted with different proportions of ethanol-water (from 0 to 100%) at

147

different extraction time (from 5 to 45 min), in different ratios of aqueous ethanol to raw

148

material (from 10 to 70 mL/g) and at extraction temperature varying from ambient

149

temperature to 65C. In this study, ethanol was selected as extraction solvent due to its low

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toxicity. The mixtures were centrifuged at 2000 x g for 20 min at 4C and the supernatants

151

were collected for the antioxidant activity determination.

152

2.4. Analytical methodology

153

2.4.1. Determination of total antioxidant activity

154

The total antioxidant activity (TAA) of the extract was evaluated by the phosphomolybdenum

155

method according to the procedure described previously (Prieto et al., 1999). The assay is

156

based on the reduction of Mo (VI)Mo (V) by the extract and subsequent formation of a green

157

phosphate/Mo (V) complex at acidic pH. A 0.2 mL extract was combined with 2 mL of

158

reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium

159

molybdate).

160

The tubes containing the reaction solution were incubated at 95C for 90 min. Then the

161

absorbance of the solution was measured using a UVvis spectrophotometer (Perkin Elmer

162

Lambda 40 UV/Vis Spectrophotometer) at a wavelength of 695 nm. The antioxidant activity

163

was expressed as milligram gallic acid equivalents (GAE) per gram of dry weight material

164

(mg GAE/g Z.lotus). Measurements were performed in triplicate.

165

2.4.2. DPPH radical scavenging activity

166

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activities were performed

167

using a method described elsewhere (Hanato et al., 1988); 1mL of extract with different

168

concentrations were mixed with 250 L of freshly prepared DPPH solution (0.2 mM in

169

methanol). The mixtures were shaken vigorously and stand for 30 min at room temperature in

170

the dark and then the absorbance values were measured at 517 nm against the blank reagent.

171

The radical scavenging activity (percent inhibition) was expressed as percentage of DPPH

172

radical elimination calculated according to the following equation:

173

sample
Percent Inhibition (%) = 1
100
A

control

(1)

174

where Acontrol is the absorbance of the control (DPPH solution with no sample), and Asample is

175

the absorbance of the samples (DPPH solution with sample). All tests were run in triplicate

176

and the average value was calculated. Antiradical DPPH bleaching activity is expressed as

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IC50 in (mg/mL) which denoted the concentration of sample required to scavenge 50% of

178

DPPH free radicals. The lower value of IC5O corresponded to the highest antiradical activity.

179
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2.4.3. Determination of the total phenolic compounds

181

Total phenolic content (TPC) of different extracts were determined using FolinCiocalteu

182

reagent slightly modified by Dewanto et al. (2002) using gallic acid as a standard. Briefly,

183

125 L of each suitable diluted extract was added to 500 l of distilled water and 125 l of

184

the FolinCiocalteu reagent. The mixture was shaken and allowed to stand for 6 min, before

185

addition of 1250 l of Na2CO3 (70 g. L-1). The solution was then adjusted with distilled water

186

to a final volume of 3 mL and mixed thoroughly. After 90 min of incubation at room

187

temperature (20C), the absorbance was measured at 760 nm using a UVvis

188

spectrophotometer (Perkin Elmer Lambda 40 UV/Vis Spectrophotometer). The calibration

189

curve was performed with gallic acid (concentrations ranging from 50 to 200 g/mL) and

190

total phenolic content was expressed as milligram of gallic acid equivalents (GAE) per gram

191

of dry weight material (mg GAE/g Z.lotus). Measurements were performed in triplicate.

192
193

2.5. Statistical analyses

194

2.5.1. Experimental Design

195

Responses surface methodology (RSM) was employed to determine the optimum levels of the

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extraction time (min) (X1), the ethanol concentration (v/v, %) (X2), the extraction temperature

197

(C) (X3) and the solvent to solid ratio (mL/g) (X4) related to three responses yields of total

198

antioxidant activity (YTAA) , DPPH free radical scavenging activity (YIC50) and total phenolic

199

content (YTPC). The operating conditions were given in Table 1. We evaluated the effects of

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extraction time that varied from 5 to 45 min, ethanol concentration was ranged from 0 to

201

100%, the range of temperature extraction was selected from ambient temperature to 65C

202

avoiding the boiling temperature of ethanol at 78C and the ratio of solvent to solid was

203

evaluated from 10 to 70 mL/g. All these conditions were selected based on preliminary

204

experimental results.

205

The variation of total antioxidant activity (YTAA) , DPPH free radical scavenging activity

206

(YIC50) and total phenolic content (YTPC) versus the four retained variables X1, X2, X3, and X4,

207

were studied using a polynomial second degree model given by the following equation:

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4
Y = + X + X 2 + X X (2)
k
0
i i
ii i
ij i j
i =1
i =1
i j =1

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Where Yk represents the measured response variables, 0 is a constant, i, ii and ij are the

210

linear, quadratic and interactive coefficients of the model, respectively. Xi and Xj are the

211

levels of the independent variables.

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A central composite rotatable design was composed of 29 experiments: 16 of which

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corresponding to a complete factorial design 24, eight experiments as star points (2) and

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five in the middle factors fields. Statistical analysis was performed using the software

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STATISTICA (version 7.0) for the experimental design and regression analysis of the

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experimental data. Students t-test permitted the checking of the statistical significance of the

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regression coefficient and Fishers F-test determined the second-order model equation at a

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probability (p) of 0.001, 0.01 or 0.05. Model adequacy was evaluated using the lack of fit, the

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coefficient of determination (R2) and the F-test value obtained from the analysis of variance

220

(ANOVA). Regression analysis and three and two dimensional response surface plots were

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plotted to determine optimum conditions for total antioxidant capacity (YTAA) , DPPH free

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radical scavenging activity (YIC50) and total phenolic content (YTPC).

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The test of statistical significance was based on the total error criteria with a confidence level

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of 95.0%

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3. Results and discussion

227

3.1. Fitting the models:

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Total antioxidant activity (YTAA) , DPPH scavenging activity (YIC50) and total phenolic

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content (YTPC) in Zizyphus lotus (L.) extracts obtained from 29 experiments were listed in

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Table 1. The data obtained from the central composite design (24) were fitted to second order

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polynomial equations. The significance of the coefficients of the models was determined by

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analysis of variance (ANOVA). Table 2 illustrates only significant coefficients and

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corresponding P values which are inferior to 0.05, indicating the considerable effect of these

234

coefficients on respective response variables. In fact, the results showed that for three

235

responses, extraction time and ethanol concentration have a significant quadratic effects

236

(p<0.05) (negative effect on TAA and TPC and positive effect on DPPH scavenging ability).

237

All the linear coefficients were significant (p<0.05) on DPPH scavenging ability and TPC
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238

determination, whereas, no significant effect of ethanol concentration was found on TAA

239

response.

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For three responses, a considerable interaction effect was established between variables as

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follows; extraction time-ethanol concentration, ratio of solvent to dry matter-ethanol

242

concentration and extraction temperature - ratio of solvent to dry matter. Only for the

243

response of DPPH scavenging activity, the interaction between extraction time and extraction

244

temperature had a significant effect. Also, interaction between ethanol concentration and

245

extraction temperature showed an important effect on TAA and TPC responses.

246

The validity of models are confirmed using lack of fit testing as summarized in Table 3;

247

ANOVA for the lack of fit test for three responses were insignificant (p>0.05) indicating that

248

the model adequately fitted the experimental data.

249

determination (R2) of 0.8255, 0.8466 and 0.8303 were obtained for the response of total

250

antioxidant activity, DPPH scavenging ability and total phenolic content, respectively and

251

revealed that there are a good correlations between responses and independent variables.

Also, the coefficients of multiple

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253

3.2. Response surface analysis of total antioxidant activity

254

Taking into account only the significant factors (Table 2), the obtained model that shows the

255

relationship between the TAA and the extraction parameters with a satisfactory correlation

256

coefficient (R2=0.8255) is given below:

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258

YTAA = 34.23498 + 5.75767X 1 + 6.68286X

+ 7.52313X

- 3.91092X 12 - 4.53330 X 22

(3)

+ 7.00243X 1 X 2 + 4.57351X 2 X 3 - 5.54372X 2 X 4 + 5.34818X 3 X 4

259
260

The statistical significance of regression equation was checked by Fishers F-test. As shown

261

in Table 3, the F Value of regression coefficients is superior to the tabulated value (Fregression =

262

9.99052 > Ftabulated(9,19,0.05) = 2.42) and the P Value was smaller than 0.0001 which indicated

263

that the variables of the model have a significant effect on the total antioxidant activity

264

response at 95% confidence level. Also, the ratio of the mean square of lack-of-fit and pure

265

error is inferior to the tabulated value (F

266

means that the lack of fit statistic was not significant (p > 0.05) hence the model is valid.

267

Two and three dimensional response surfaces were plotted for the results of total antioxidant

268

activity in Zizyphus lotus (L.) extracts as presented in Fig.1 which shows the interaction

269

between all the significant combined factors.

lack -of- fit

=4.78466 < Ftabulated(15,4,0.05) = 5.86) which

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According to Fig.1.A, the increase of both ethanol concentration and extraction time increases

271

the response of TAA (extraction temperature and ratio of liquid to solid were fixed at the

272

center). Fig.1.B demonstrated that the TAA increased significantly and can surpass the value

273

of 50 with the increase of both temperature and ethanol concentration at a fixed extraction

274

time and ratio (liquid to dry matter) of 25 min and 40 mL/g, respectively. Fig1.C showed that

275

the TAA increased considerably with the increase of ratio and the decrease of ethanol

276

concentration at a fixed extraction time and extraction temperature.

277

It could be seen from Fig.1.D that there is an area in which the value of TAA can be superior

278

than 80 at a high ratio and extraction temperature when ethanol concentration and extraction

279

time were kept at 50% and 25 min, respectively.

280
281

3.3. Response surface analysis of DPPH radical scavenging activity

282

Analysis of variance (Table 3) demonstrates the relationship between DPPH scavenging

283

ability and significant extraction parameters with a satisfactory regression coefficient

284

(R2=0.8466). Equation (4) shows the mathematical model that describes the relationship

285

between the significant independent variables and response of DPPH scavenging activity.

286
287

Y IC 50 = 1.21817 - 0.11592 X 1 + 0.07822 X 2 - 0.16075 X 3 - 0.16945 X 4 + 0.08642 X 12

+ 0.10804 X 22 - 0.09425 X 1 X 2 - 0.08443 X 1 X 3 + 0.18906 X 2 X 4 - 0.09635 X 3 X 4

(4)

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289

According to Fishers F-test, the F value of regression coefficients is superior to the tabulated

290

value (F

291

smaller than 0.0001. This indicated that the independent variables listed in Table 2 have a

292

significant effect on the response of DPPH scavenging ability. Also, the ratio of the mean

293

square of lack-of-fit and pure error is inferior to the tabulated value (F

294

fit=4.22161<Ftabulated (14, 4, 0.05) =

295

fit indicated that this one was insignificant and the model is valid.

296

Fig.2 presents the two and three dimensional responses surface of all significant interaction

297

effects of factors mentioned in Table 2.

298

The effect of ethanol concentration, extraction time and their mutual interaction on the DPPH

299

scavenging ability (IC50) is illustrated in Fig.2.A. At 50% of ethanol concentration, the

300

increase in the antiradicalair activity was observed with the increase in extraction time until

301

35 min, and then a decrease in this activity was observed with the increase of extraction time.

regeression

= 9.93938 > Ftabulated(10, 18, 0.05) = 2.41) and the P value corresponding was

lack

of-

5.88) and the P value of 0.08715 corresponding to the lack- of-

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However, in Fig.2.B similar linear increase in the DPPH scavenging capacity was observed

303

with the increase in extraction temperature and extraction time at a fixed ratio and ethanol

304

concentration. The effect of ratio and ethanol concentration shown in Fig.2.C demonstrated

305

that the inhibition concentration at 50% (IC50) could be inferior to the value of 0.4 at a high

306

level of ratio and a low level of ethanol concentration variable.

307

As can be seen in Fig.2.D, a good inhibition of DPPH free radical was observed with an

308

increase in both ratio and extraction temperature.

309
310

3.4. Response surface analysis of total phenolic content

311

The mathematical model correlating the content of total polyphenols (TPC) in term of

312

significant independent variables is given below:

313
314

TPC

= 20.39293 + 2.96212 X 1 - 1.94096 X 12 - 1.95963 X 2 - 2.66846 X 22

(5)

+ 3.44121 X 3 + 3.51871 X 4 + 3 .18569 X 1 X 2 + 1.84431 X 2 X 3 - 4.60069 X 2 X 4 + 2.65806 X 3 X 4

315
316

As given in Table 3, the analysis of variance (ANOVA) of total phenolic content pointed out a

317

strong positive correlation (R2=0.8303). In addition, according to Fishers F-test, it can be

318

observed that the F-value model is superior to the tabulated one (Fregression=8.07704 >

319

Ftabulated(10,18,0.05) = 2.41). This result indicates that the model have a significant effect on the

320

total phenolic content response (P Value < 0.0001).

321

The lack of fit test determines the adequacy of selected model describing the effect of

322

extraction parameters on TPC response. In fact, results showed that the ratio of the mean

323

square of lack-of-fit and pure error is inferior to the tabulated value (Flack

324

=4.64275<Ftabulated

325

significant (p > 0.05), and hence, the model is valid.

326

As clearly shown from Fig.3.A, at 50% of ethanol concentration, the extraction of TPC

327

increased with extraction time until 41min, while. Above this extraction time (> 41min), the

328

TPC response started to decrease.

329

Nevertheless, as shown in Fig.3.B, an increase in extraction temperature at 50% of ethanol

330

concentration enhanced significantly the TPC extraction when the extraction time and the

331

ratio (liquid to dry matter) were fixed at 25 min and 40 mL/g, respectively.

(14,4,0.05)

-of- fit

= 5.88), which means that the lack of fit statistic was not

12

332

The effect of ratio (liquid to dry matter) and ethanol concentration shown in Fig.3.C

333

demonstrated that the TPC value could be superior to 30 mg GAE/g dry matter for a high ratio

334

(liquid to dry matter) and a low level of ethanol concentration variable when the extraction

335

temperature and the extraction time were fixed at 45C and 25 min, respectively.

336

As it can be seen from Fig.3.D, total phenolic content increase dramatically and could reach

337

more than 40 mg GAE/g dry matter with the increase of both ratio (liquid to dry matter) and

338

extraction temperature at 50% of ethanol concentration and an extraction time of 25 min.

339
340

3.5. Optimization of extraction conditions

341

The objective of this procedure is to determine the levels of experimental factors which would

342

allow obtaining an extract with a high antioxidant activity. Response surface methodology

343

was a good tool for optimization. In fact, a compromise was found in choosing the optimal

344

area according to the following criteria: IC50 < 0.4 mg/mL and TAA > 70 mg (GAE)/g dry

345

matter and TPC > 40 mg (GAE)/g dry matter. These criteria are satisfied if one can explore

346

the results of Fig.1.D of total antioxidant activity, Fig.2.D of IC50 and Fig.3.D of TPC. For the

347

three responses, respectively, the optimum point was marked in the area which corresponds to

348

the following conditions: ethanol concentration, 50%; extraction time, 25 min; extraction

349

temperature, 63C; and ratio of liquid to dry matter, 67 (mL/g). Under these optimal

350

conditions of extraction, the experimental values of total antioxidant activity, DPPH free

351

radical scavenging activity (IC50) and total phenolic content were, 0.2895 mg/mL, 75.9815

352

mg (GAE)/g dry matter and 40.782 mg (GAE)/g dry matter, respectively. Compared with

353

Zizyphus lotus from other region of Tunisia and other countries, the DPPH scavenging ability

354

of southern Tunisian Z.lotus fruits (IC50=0.289 mg/mL) evaluated in this study was similar to

355

Z. lotus fruits localized in North-Western region of Tunisia (Jendouba) (IC50=0.310.005

356

mg/mL) (Ghazghazi et al.,2014). Also, our results were approximately 1.65 times higher than

357

those obtained in Moroccan Zizyphus lotus fruits (IC50= 477.6 47.6 g/mL) using 70%

358

aqueous methanol (Bakhtaoui et al.,2014). In addition to that, the content of phenolic

359

compounds in this specie was 1200 g p-Coumaric acid equivalents per gram of dry matter.

360

Furthermore, previous studies have been reported on other species of Zizyphus. In fact, Okala

361

et al. (2014) investigated the antioxidant properties of Zizyphus mauritiana fruits from Nigeria

362

and reported that the IC50 value of DPPH was found to be 338.45g/mL, which was

363

approximately 1.17 times lower than the value obtained in the present work. Concerning the
13

364

measurement of total phenolic contents, the value was about 4.02353.6 mg gallic acid

365

equivalents/g dry matter. This result was lower than the one reported for southern Tunisian

366

Z.lotus fruits. In fact, these variability of antioxidant activities and total phenolic contents

367

values of Zizyphus genus could be explained by various factors such as, the geographical

368

provenance, the type of species and the nature of solvent extraction as demonstrated by

369

Ksouri et al. (2008).

370

In comparison with other exotic fruits, antioxidant compound content in southern Tunisian

371

Z.lotus fruits were higher than Mangosteen (TPC= 54 7 mg/100g fresh fruit,

372

IC50=11.5 3.6 mg/mL), and Dragon fruit (TPC= 21 6 mg/100g fresh fruit, IC50=27.5 3.9

373

mg/mL) (Lim et al., 2007).

374
375

4. Conclusion

376

In this work, response surface methodology (RSM) employing central composite rotatable

377

design (CCDR) was successfully used to determine the optimal conditions for the extraction

378

of antioxidant compounds from Zizyphus lotus (L.) fruits under ultrasonication. In this

379

context, the same statistical methodology was successfully used by Zhao et al. (2014) to

380

determine the optimal extraction conditions of antioxidant compounds from Epimedium

381

brevicornum. In fact, only three extraction variables were optimized including ethanol

382

concentration, extraction time and ratio of aqueous ethanol to raw material for the

383

achievement of high extraction yield of the phenolic compounds with high antioxidant

384

activity. The selected range of each independent variables and obtained results of optimum

385

conditions depending on plant material.

386

The experimental results adequately fitted with second-order polynomial models and showed

387

significant linear, quadratic and interaction effects of the independent variables. The

388

regression coefficients (R2) of 0.8255, 0.8466 and 0.8303 were obtained for the response of

389

total antioxidant activity, DPPH scavenging ability and total phenolic content, respectively.

390

These values revealed good correlations between responses and independent variables. The

391

same optimal conditions could be used to get an extract from Zizyphus lotus (L.) fruits with a

392

high phenolic content, a high total antioxidant activity and a lower IC50 of scavenging activity

393

on DPPH free radical. In fact, a compromise between these three responses (YIC50, YTAA and

394

YTPC), according to the response surface analysis was determined. The optimum operating

395

conditions for extraction were as follows: ethanol concentration, 50%; extraction time, 25
14

396

min; extraction temperature, 63C and solvent to solid ratio, 67 mL/g. Under these conditions,

397

the obtained extract exhibited a high content of phenolic compounds (40.782 mg gallic acid

398

equivalents/g dry matter) with significant antioxidant properties (the total antioxidant activity

399

was 75.981 mg gallic acid equivalents /g dry matter and the 2,2-diphenyl-1-picrylhydrazyl

400

radical scavenging activity was

401

responses exhibited that experimental values are in agreement with the predicted ones (TPC

402

of 42.081 mg (GAE)/g dry matter, TAA of 77.134 mg (GAE)/g dry matter and IC50 value of

403

0.311 mg/mL). The results also showed that Zizyphus lotus (L.) fruits present potential

404

antioxidant activities that make it beneficial for human health by preventing or reducing

405

oxidative damage.

0.289 mg/mL). The modeling adequacy of these three

406
407

Acknowledgements

408

This work was supported by a grant from Ministry of Higher Scientific Research and

409

Information and Communication Technologies of Tunisia. The authors would like to thank

410

the technical team of the National Research Centre of Materials Sciences in Borj Cedria

411

Technological Park.

412
413
414

415

416

417

418

419

420

421
15

422

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537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
18

557

Table captions

558

Table 1. Coded levels, Conditions runs and measured responses used in experimental design

559

for response surface methodology.

560

Table 2. Regression coefficients of the predicted second-order polynomial models for the total

561

antioxidant activity, the DPPH scavenging activity and the total phenolic content.

562

Table 3. Analysis of variance (ANOVA) of the second order polynomial models for the total

563

antioxidant activity (TAA), the DPPH scavenging activity (IC50 ) and the total phenolic

564

content (TPC).

565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
19

582

Figure captions

583

Fig.1.Response surface plots (3D,right) and contour plots (2D,left) of total antioxidant activity

584

as a function of significant interactions between factors : (A) ethanol concentration and

585

extraction time; (B) extraction temperature and ethanol concentration; (C) solvent to solid

586

ratio and ethanol concentration; (D) solvent to solid ratio and extraction temperature.

587

Fig.2. Response surface plots (3D,right) and contour plots (2D,left) of DPPH free radical

588

scavenging activity as a function of significant interactions between factors : (A) ethanol

589

concentration and extraction time ; (B) extraction temperature and extraction time; (C)

590

Solvent to solid ratio and ethanol concentration; (D) solvent to solid ratio and extraction

591

temperature.

592

Fig.3. Response surface plots (3D,right) and contour plots (2D,left) of total phenolic content

593

as a function of significant interactions between factors : (A) ethanol concentration and

594

extraction time ; (B) extraction temperature and ethanol concentration; (C) Solvent to solid

595

ratio and ethanol concentration; (D) solvent to solid ratio and extraction temperature.

596

20

597
598

21

599
600

22

601
602

23

603

Table 1.
Experimentsa
Time
(min), X1

Independent variables

Responses

(C), X3

Solvent/solid
Ratio
(mL/g), X4

YTAAb
(mg
GAE/g
dry
matter)

Ethanol
Concentration
(%), X2

Temperature

YIC50c
(mg/mL)

YTPCd
(mg
GAE/g
dry
matter)

15(-1)

25(-1)

35(-1)

25(-1)

17.840

1.587 12.590

35(+1)

25(-1)

35(-1)

25(-1)

21.650

1.575 14.870

15(-1)

75(+1)

35(-1)

25(-1)

14.562

1.621 10.700

35(+1)

75(+1)

35(-1)

25(-1)

23.007

1.517 15.340

15(-1)

25(-1)

55(+1)

25(-1)

9.507

1.758

6.980

35(+1)

25(-1)

55(+1)

25(-1)

4.507

1.840

2.450

15(-1)

75(+1)

55(+1)

25(-1)

18.793

1.583 13.100

35(+1)

75(+1)

55(+1)

25(-1)

41.859

0.891 28.230

15(-1)

25(-1)

35(-1)

55(+1)

33.505

1.269 19.200

10

35(+1)

25(-1)

35(-1)

55(+1)

26.434

1.455 16.600

11

15(-1)

75(+1)

35(-1)

55(+1)

0.253

12

35(+1)

75(+1)

35(-1)

55(+1)

25.386

1.508 15.980

13

15(-1)

25(-1)

55(+1)

55(+1)

42.513

1.057 25.900

14

35(+1)

25(-1)

55(+1)

55(+1)

46.705

0.543 31.700

15

15(-1)

75(+1)

55(+1)

55(+1)

14.106

1.670

16

35(+1)

75(+1)

55(+1)

55(+1)

65.430

1.107 25.230

17

5(-;-2)

50(0)

45(0)

40(0)

10.843

1.716

18

45(+;+2)

50(0)

45(0)

40(0)

27.986

1.337 17.760

19

25(0)

0(-;-2)

45(0)

40(0)

30.068

1.325 18.890

20

25(0)

100(+;+2)

45(0)

40(0)

3.782

1.900

1.700

21

25(0)

50(0)

25(-;-2)

40(0)

7.782

1.780

6.430

22

25(0)

50(0)

65(+;+2)

40(0)

47.586

1.915

0.160

8.899

8.650

0.850 29.200

24

23

25(0)

50(0)

45(0)

10(-;-2)

10.646

1.750

7.980

24

25(0)

50(0)

45(0)

70(+;+2)

49.620

0.640 30.500

25

25(0)

50(0)

45(0)

40(0)

41.395

1.157 21.530

26

25(0)

50(0)

45(0)

40(0)

44.728

0.985 26.700

27

25(0)

50(0)

45(0)

40(0)

38.986

1.211 21.230

28

25(0)

50(0)

45(0)

40(0)

37.677

1.193 21.400

29

25(0)

50(0)

45(0)

40(0)

32.988

1.245 20.870

604
605

a: Standard order

606

b: Total antioxidant activity

607
608

c: Concentration of sample required to scavenge 50% of 2,2-diphenyl-1-picrylhydrazyl free

radical

609

d: Total Phenolic content

610
611

25

612

Table 2.

613
614

TAA
model

Term

Regression
coefficientsa

Standard
error

t Value

p Value

34.23498

1.31708

25.99299

< 0.00001

5.75767

0.89167

6.45717

0.00296

6.68286

0.89167

7.49476

0.00166

7.52313

0.89167

8.43712

0.00108

11

-3.91092

0.83137

-4.70419

0.00928

22

-4.53330

0.83137

-5.45281

0.00549

12

7.00243

1.09207

6.41207

0.00304

23

4.57351

1.09207

4.18793

0.01383

24

-5.54372

1.09207

-5.07634

0.00710

34

5.34818

1.09207

4.89729

0.00806

1.21817

0.03075

39.60603

< 0.0001

-0.11592

0.02082

-5.56692

0.005100

0.07822

0.02082

3.75648

0.01983

-0.16075

0.02082

-7.72010

0.00151

-0.16945

0.02082

-8.13771

0.00124

11

0.08642

0.01941

4.45124

0.01123

22

0.10804

0.01941

5.56509

0.00510

12

-0.09425

0.02550

-3.69586

0.02091

13

-0.08443

0.02550

-3.31066

0.02963

24

0.18906

0.02550

7.41334

0.00176

IC50 model

26

34

-0.09635

0.02550

-3.77806

0.01947

20.39293

0.737658

27.64552

< 0.0001

2.96212

0.499397

5.93141

0.004049

-1.95963

0.499397

-3.92398

0.017190

3.44121

0.499397

6.89073

0.002325

3.51871

0.499397

7.04592

0.002139

11

-1.94096

0.465624

-4.16851

0.014048

22

-2.66846

0.465624

-5.73093

0.004591

12

3.18569

0.611634

5.20849

0.006478

23

1.84431

0.611634

3.01539

0.039341

24

-4.60069

0.611634

-7.52197

0.001672

34

2.65806

0.611634

4.34584

0.012197

TPC
model

615
616

a: only terms with p < 0.05 were included

617

TAA: Total antioxidant activity

618
619

IC50: Concentration of sample required to scavenge 50% of 2,2-diphenyl-1-picrylhydrazyl

free radical.

620

TPC: Total phenolic content

621
622

27

623

TAA (R2=0.8255)
Source of

SS

DF

MS

F Value

P Value

Regression

6284.03800

698.22646

9.17556

<0.0001

Residuals

1445.83063

19

76.09634

Lack of fit

1369.50325

15

91.30021

4.78466

0.07059

Pure error

76.32738

19.08184

Total

7729.86863

28

9.00137

<0.0001

4.22161

0.08715

8.07704

<0.0001

4.64275

0.07451

variation

624

Table 3.
IC50 (R2=0.8466)
Regression

3.28372

10

0.32837

Residuals

0.65664

18

0.03648

Lack of fit

0.61502

14

0.04393

Pure error

0.04162

0.0104

Total

3.94036

28

Regression

1853,20231

10

185.32023

Residuals

412,99337

18

22.94407

Lack of fit

389,05125

14

27.78937

Pure error

23,94212

5.98553

Total

2266,19568

28

TPC(R =0.8303)

625
626

DF: Degree of freedom; SS: sum of squares; MS: mean square

627

TAA: Total antioxidant activity

628
629

IC50: Concentration of sample required to scavenge 50% of 2,2-diphenyl-1-picrylhydrazyl

free radical.

630

TPC: Total phenolic content

631
632
633
28

List of abbreviations

634
635

DPPH: 2,2-diphenyl-1-picrylhydrazyl

636

TAA: Total antioxidant activity

637

TPC: Total phenolic content

638

RSM: Responses surface methodology

639
640

IC50: Concentration of sample required to scavenge 50% of 2,2-diphenyl-1-picrylhydrazyl

free radical.

641

X1: Extraction time (min)

642

X2 : Ethanol concentration (v/v, %)

643

X3 : Extraction temperature (C)

644

X4 : Solvent to solid ratio (mL/g)

645

YTAA: Total antioxidant activity response

646

Y IC50: 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity response

647

YTPC: Total phenolic content response

648

ANOVA: Analysis of variance

649

Fregression : Fishers F-test Value of regression

650

F lack -of- fit : Fishers F-test Value of lack -of- fit

651

Ftabulated: Tabulated value of Fishers F-test

652

mg GAE/g dry matter: milligram of gallic acid equivalents per gram of dry matter

653

Yk : The measured response variables

654

0: constant of the model

655

i: Linear coefficients of the model

656

ii: Quadratic coefficients of the model

657

ij: Interactive coefficients of the model

658

Xi and Xj : The levels of the independent variables

659

R2: Coefficient of determination

660
29

661

-Ultrasound-assisted extraction of antioxidants compounds

662

-Optimization of antioxidant extraction conditions from fruits of Zizyphus lotus

663

-Response surface methodology (RSM) using a central composite rotatable design

664

- Determination of total antioxidant activity and DPPH radical scavenging capacity

665

- Use of ethanol as solvent extraction (low toxicity)

666

30