Accepted Manuscript

Impact of extraction techniques on antioxidant capacities and phytochemical

composition of polyphenol-rich extracts

Cecilia Castro-López, Janeth M. Ventura-Sobrevilla, María D. González-

Hernández, Romeo Rojas, Juan A. Ascacio-Valdés, Cristóbal N. Aguilar,
Guillermo C.G. Martínez-Ávila

PII: S0308-8146(17)31014-2
DOI: http://dx.doi.org/10.1016/j.foodchem.2017.06.032
Reference: FOCH 21254

To appear in: Food Chemistry

Received Date: 14 February 2017

Revised Date: 1 June 2017
Accepted Date: 5 June 2017

Please cite this article as: Castro-López, C., Ventura-Sobrevilla, J.M., González-Hernández, M.D., Rojas, R.,
Ascacio-Valdés, J.A., Aguilar, C.N., Martínez-Ávila, G.C.G., Impact of extraction techniques on antioxidant
capacities and phytochemical composition of polyphenol-rich extracts, Food Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.foodchem.2017.06.032

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1 Title and Authorship Information

2 Paper title

4 IMPACT OF EXTRACTION TECHNIQUES ON ANTIOXIDANT CAPACITIES

5 AND PHYTOCHEMICAL COMPOSITION OF POLYPHENOL-RICH EXTRACTS

7 Full author name

8 Cecilia Castro-Lópeza,b, Janeth M. Ventura-Sobrevillaa, María D. González-Hernándezb,

9 Romeo Rojasb, Juan A. Ascacio-Valdésa, Cristóbal N. Aguilara, and Guillermo C. G.

10 Martínez-Ávilab*

11

12 Full institutional mailing addresses

a
13 Autonomous University of Coahuila, Department of Food Science and Technology.

14 School of Chemistry. 25280, Saltillo, Coahuila, Mexico.

b
15 Autonomous University of Nuevo Leon, Laboratory of Chemistry and Biochemistry

16 School of Agronomy. 66050, General Escobedo, Nuevo León, Mexico.

17

18 Email addresses

19 c.castro@uadec.edu.mx; janethventura@uadec.edu.mx; lolis.90.6@gmail.com;

20 romeo.rojasmln@uanl.edu.mx; alberto_ascaciovaldes@uadec.edu.mx;

21 cristobal.aguilar@uadec.edu.mx and guillermo.martinezavl@uanl.edu.mx*

22

23 Dra. Ventura-Sobrevilla and Dr. Martínez-Ávila had the same responsibility in this manuscript.

24
25 Abstract

26 In this work, impact of extraction methods (maceration, decoction, MAE, and UAE) on

27 TPC, antioxidant activity, and the mass fraction of phenolics in several plant extracts

28 (Punica granatum, Juglans regia, Moringa oleifera, and Cassia fistula) was investigated.

29 The results showed that, despite the nature of matrix, the highest values of TPC in all

30 samples were obtained by MAE as follows: PP (18.92 ± 0.11), ML (15.19 ± 0.11), HL

31 (12.69 ± 0.16), and WS (12.80 ± 0.11) mg GAE g-1 respectively, and exhibited potent

32 antioxidant activity (from 0.28 ± 0.01 to 5.34 ± 0.02 mg GAE g-1), representing sources of

33 powerful antioxidants. The LC-MS2 analysis revealed a wide range of phenolics,

34 highlighting their content in phenolic acids, flavonoids and lignans. The presence of

35 different phenol molecules demonstrated that the extraction method had influence on

36 phytochemical profile. Finally, due to its high extraction efficiency, MAE was the more

37 effective extraction technique.

38

39 Keywords: polyphenols; extraction technique; antioxidant; LC-MS2

40

41 Chemical compounds studied in this article

42 Punicalin (PubChem CID: 5464368); Pedunculagin (PubChem CID: 442688); Casuarinin

43 (PubChem CID: 101601178); Cinnamic acid (PubChem CID: 444539); Ellagic acid

44 (PubChem CID: 5281855); Medioresinol (PubChem CID: 181681); Caffeoyl tartaric acid

45 (PubChem CID: 6440397); Diosmetin 7-O-rutinoside (PubChem CID: 5281613); 3-

46 Caffeoylquinic acid (PubChem CID: 1794427)

47

48
49

50 1. Introduction

51 In food industry, synthetic antioxidants are mainly used to prevent oxidative changes of

52 food constituents that can cause repugnant flavors, and destruction of valuable nutrients.

53 However, these synthetic antioxidants have the disadvantage of being very volatile and

54 according to some authors are suspected to be detrimental to health because such

55 compounds may cause liver swelling, influence liver enzyme activities, and provoke

56 carcinogenicity (Zhang, Yang, Zu, Chen, Wang, & Liu, 2010). In this sense, the interest in

57 the investigation of plant-derived compounds or antioxidants from natural origin, especially

58 polyphenols, has evoked considerable interest among nutritionists, food manufacturers, and

59 consumers because of their safety and potential therapeutic value (Calabrese & Maines,

60 2006). Recently, medicinal plants (such as Moringa oleifera, Cassia fistula, Centella

61 asiatica, Amaranthus viridis, among others), as well as agricultural and industrial residues

62 (i.e. pomegranate peel, walnut shell, apple and grape pomace, banana peel, watermelon

63 seeds, strawberry waste) have escalated much interest in recovery of phytochemicals which

64 could be used mainly in food industry, but also with pharmaceutical and cosmetic

65 applications (Lizcano et al., 2012; Na, Thuong, & Bae, 2011). In this context, recovery of

66 antioxidant compounds from these materials is typically accomplished through different

67 extraction techniques considering their chemistry and uneven distribution in the plant

68 matrix.

69 Previous studies revealed that the yield and bioactivity of extract were correlated with the

70 applied extraction methods. So, the used method in this procedure becomes essential for the

71 accurate quantification and determination of antioxidant capacity (Zhang et al., 2015).

72 Several extraction conditions are reported in the literature and solid-liquid extraction is
73 most frequently used technique for isolation of plant antioxidant compounds. However,

74 typical methods of solid-liquid extraction such as hot water bath, maceration, soxhlet

75 extraction, and percolation, which have been used for many decades, are very time-

76 consuming, and require relatively large quantities of solvents besides the extract yields, and

77 resulting antioxidant activities of the plant materials are low and poor (Rodríguez-Rojo,

78 Visentin, Maestri, & Cocero, 2012). Because of this, there is an increasing demand for new

79 green non-conventional methods to shorten the extraction time, reduce organic solvent

80 consumption, and create a selective recovery. Novel extraction methods including

81 ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), supercritical

82 fluid extraction (SFE), accelerated solvent extraction (ASE), and enzyme-assisted

83 extraction are fast and efficient for extracting chemicals from solid plant matrixes (Biesaga,

84 2011; Martínez-Correa, Magalhaes, Queiroga, Peixoto, Oliveira, & Cabral, 2011).

85 Each vegetable material has its own unique properties in terms of phenolic components.

86 Thus, it is important to study an optimal extraction method and provide a better

87 understanding for the potential application of different technologies in the processing, and

88 effective utilization of natural products (Zhang et al., 2015). Based on the above rationale,

89 the study was carried out to evaluate the influence of extraction method and solid-liquid

90 ratio on total phenolic content, as well as the antioxidant abilities of four plant materials

91 (Punica granatum peels, Juglans regia shells, Moringa oleifera and Cassia fistula leaves).

92 Finally, extracts with the highest antioxidant activity were sequentially fractionated using

93 UPLC-ESI-Q/TOF-MS2 to identify and characterize their principal chemical constituents

94 (phenolic compounds).

95

96 2. Materials and methods

97 2.1 Chemicals and reagents

98 Gallic acid, linoleic acid, ethanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2′-azino-bis

99 (3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS •+), (±)-6-Hydroxy-

100 2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), Folin-Ciocalteu’s phenol reagent,

101 trichloroacetic acid, and other reagents were purchased from Sigma-Aldrich (Toluca,

102 Mexico). While acetonitrile, methanol, water, and formic acid were all LC-MS grade and

103 purchased from Fisher Scientific Chemicals (Fair Lawn, NJ, USA).

104

105 2.2 Plant materials

106 Four plant materials were used to obtain polyphenolic compounds. As a by-product of the

107 fruit juice industry and regional trade, pomegranate peels (Punica granatum), and walnut

108 shells (Juglans regia) were provided by a local producer (Saltillo, Mexico). While two

109 medicinal plants of popular use in the region: moringa (Moringa oleifera), and hojasen

110 (Cassia fistula) leaves were obtained in February 2016. Immediately after the materials

111 were recollected, they were washed with distilled water and peeled or leafed off manually

112 (as applicable). Peels and leaves were dried in a forced air oven at 60 °C for 24 h or to

113 constant weight and then grounded using an electrical grinder. The ground powder was

114 collected and stored in airtight bags in darkness in a dry place at room temperature until

115 their treatment. The following coding was used: PP, pomegranate peel; WS, walnut shell;

116 ML, moringa leaves; and HL, hojasen leaves.

117

118 2.3 Methods for extracting phenolic compounds

119 Several extraction methods were performed using two different solid: liquid ratios (1:25

120 and 1:50, w/v) with water as solvent. Conventional solid-liquid extraction (maceration and
121 decoction), and the use of emerging technologies (microwave-assisted extraction and

122 ultrasound-assisted extraction) were tested as extraction methods and are described below.

123

124 2.3.1 Conventional solid-liquid extractions

125 Maceration procedure was employed for the extraction of polyphenols following the

126 procedures recommended by Rodríguez-Pérez, Quirantes-Piné, Fernández-Gutiérrez, &

127 Segura-Carretero (2015). Briefly, 0.2 g of plant material was extracted with 5 mL or 10 mL

128 of deionized water (as appropriate to maintain the solid: liquid ratio) at room temperature

129 (30 °C) for 2 h with magnetic stirring at 180 rpm away from light. The extracts were

130 centrifuged at 10, 000 rpm for 10 min at 4 °C and stored at 4 °C until testing.

131 For the decoction method, as described by Kaneria, Kanani, & Chanda (2012), 0.2 g of

132 dried powder of the samples were mixed with 5 mL or 10 mL of deionized water (as

133 appropriate to maintain the solid: liquid ratio). For propitiate the extraction this mixture was

134 allowed to stand in an oven at 60 °C for 2 h with magnetic stirring at 180 rpm. After this

135 period, all samples were centrifuged at 10, 000 rpm for 10 min at 4 °C and the supernatant

136 was recovered and stored at -20 °C until further use.

137

138 2.3.2 Ultrasound-assisted extraction (UAE)

139 Subsequently, ultrasound-assisted extraction was carried out as described by Wong-Paz,

140 Muñiz-Márquez, Martínez-Ávila, Belmares-Cerda, & Aguilar (2015). First, 0.2 g of each

141 material were mixed with 5 mL or 10 mL of deionized water (as appropriate to maintain the

142 solid: liquid ratio). All the samples were placed in dark brown-colored reagent bottles with

143 narrow necks and immersed for 60 min at room temperature (25 °C) in an ultrasonic water

144 bath (2510, Branson, USA) at 40 KHz (100% power). Then, the obtained extracts were
145 centrifuged at 10, 000 rpm for 10 min at 4 °C to remove solids and stored under

146 congelation (-20 °C) prior to the analysis.

147

148 2.3.3 Microwave-assisted extraction (MAE)

149 A MARS 6-Microwave Digestion System (CEM Corporation, UK) was used for extraction

150 of phenolic compounds. The apparatus was equipped with a 40-Place starter set (Teflon

151 digestion vessels with a volume of 75 mL) and every vessel in the batch was controlled via

152 CEM’s floor mounted IR sensor technology. In this method, extractions of 1 g of each

153 material were accomplished with 25 mL or 50 mL of deionized water (as appropriate to

154 maintain the solid: liquid ratio). The MAE extraction parameters were microwave power:

155 550 W, extraction time: 90 s, and controlled temperature: 70 °C. After the treatment, the

156 plant extracts were centrifuged at 10, 000 rpm for 10 min at 4 °C and the supernatant was

157 collected and stored at -20 °C until further use.

158

159 2.4 Determination of total phenolic content (TPC)

160 The total phenolic content of vegetal extracts was investigated by Folin-Ciocalteu’s method

161 following the procedures of Georgé, Brat, Alter, & Amiot (2005). Exactly, 25 μL of

162 properly diluted supernatant was mixed with 25 μL of Folin-Ciocalteu’s reagent and after 1

163 min, 25 μL of sodium carbonate (75 g L-1) was added. The obtained solution was mixed

164 thoroughly and incubated at 40 °C for 30 minutes in a water bath. Subsequently the mixture

165 was adjusted with 200 µL of distilled water and the absorbance was recorded at 750 nm

166 with a microplate reader (Synergy HT Multi-Detection Microplate Reader, BioTek

167 Instruments, USA). The absorbance of the extract was compared with a gallic acid standard

168 curve for estimating concentration of TPC in the sample. The phenol content was calculated
169 as mean ± SD and expressed as milligrams of gallic acid per gram (mg GAE g-1) of raw

170 material.

171

172

173 2.5 Antioxidant activities

174 2.5.1 DPPH• radical scavenging activity

175 The antioxidant activity in the extracts was evaluated as the DPPH • free radical-scavenging

176 activity. The activities were determined by the methodology proposed by Brand-Williams,

177 Cuvelier, & Berset (1995). The hydrogen atom or electron donation abilities of the samples

178 and some pure compounds were measured from a light-purple colored DPPH• methanol

179 solution (60 mM). 5 µl of each extract was added to a 295 µL DPPH• radical solution. After

180 a period of incubation in the dark for 30 minutes, the absorbance of the samples was

181 recorded at a wavelength of 517 nm with the above-mentioned microplate reader. The

182 ability to inhibit was calculated by the following equation (1) and expressed as percent

183 inhibition of DPPH• radical:

184 Inhibition (%) = [(A control – A sample) / A control] *100

185 Eq. (1)

186 where A control is the absorbance of the control reaction (containing all reagents except the

187 test compound) and A sample is the absorbance with the test compound. Gallic acid was used

188 as reference. The DPPH• inhibition was expressed as the average of three replications in

189 gallic acid equivalent expressed in milligrams per gram (mg GAE g -1) of raw material.

190

191 2.5.2 ABTS•+ radical scavenging activity

192 The inhibition assay of the ABTS•+ radical was conducted according to the methodology

193 proposed by Van den Berg, Haenen, Van den Berg, & Bast (1999). The ABTS•+ radical

194 cation was generated by mixing an aqueous solution of potassium persulfate (2.45 mM) and

195 ABTS•+ (7 mM); these reagents react stoichiometrically in a ratio of 1:2 respectively and

196 must be kept in the dark at room temperature for 12 h before use. Diluted solutions of

197 ABTS•+ were prepared in ethanol until a value of 0.700 ± 0.002 nm absorbance was

198 obtained. Later, the samples were treated as follows: were mixed 95 μL of the dilute

199 solution of ABTS•+ with 5 μL of sample and the absorbance was measured at a wavelength

200 of 734 nm with the above-mentioned microplate reader. The capacity to inhibit the radical

201 was calculated according to the following equation (2) and results were expressed as

202 percent inhibition of the ABTS•+ radical:

203 Inhibition (%) = [(A control – A sample) / A control] *100

204 Eq. (2)

205 where A control is the absorbance of the control reaction (containing all reagents except the

206 test compound) and A sample is the absorbance with the test compound. The ABTS•+

207 scavenging ability of extract was calculated according to the standard curve plotted with

208 trolox and expressed as Trolox equivalent in milligrams per gram (mg TE g -1) of raw

209 material.

210

211 2.5.3 Ferric reducing power assay (FRAP)

3+
212 The ferric ion (Fe ) reducing power was determined as described by Benzie & Strain

213 (1996) with a slight modification. Exactly, to a 5 μL of each sample were added 12 μL of

214 phosphate buffer (pH 7) prepared by the premixing of 6.15 mL of potassium phosphate di-

215 basic (1M) plus 3.85 mL of potassium phosphate mono-basic (1 M) and graduated to 100
216 mL with distilled water. Subsequently, it was added to the mixture 22 μL of potassium

217 ferrocyanide 1%, homogenized and incubated in a boiling water bath at 50 °C for 20

218 minutes. After cooling, 12 μL of trichloroacetic acid 10% were added. Following, 45 μL of

219 distilled water and 10 μL of ferric chloride 0.1% were added and shaken thoroughly. The

220 absorbance was recorded at a wavelength of 700 nm with the above-mentioned microplate

221 reader. Finally, the results were reported as gallic acid equivalents in milligrams per gram

222 (mg GAE g-1) of raw material.

223

224 2.5.4 Lipid peroxidation inhibition assay

225 This test system was developed to determine the ability of substances to inhibit the

226 generation of hydroxy peroxides at the early stages of the oxidation of linoleic acid, and

227 this oxidation is monitored by measuring the values of conjugated dienes

228 spectrophotometrically. The assay was determined as described by Zou, Lu, & Wei (2004)

229 with a slight modification. First, the linoleic acid solution was prepared by mixing 0.6 g of

230 linoleic acid and 1.5 g of Tween 20 in 8 mL of ethanol. Then, the plant extract (50 µL) was

231 mixed with linoleic acid solution (100 µL) and acetate buffer (1500 µL, 0.02 M, pH 4).

232 Controls contained 50 µL of distilled water. The samples were homogenized in vortex and

233 incubated at 37 °C for 1 min. Once achieved 1 min, 750 µL of 50 M FeCl2 solution (0.01 g

234 FeCl2 and 0.017 g EDTA diluted to 100 mL with distilled water) were added to induce the

235 lipid oxidation and incubated for 24 h at 37 °C. Two aliquots (250 µL) were withdrawn

236 during this period, at 0 and 24 h. Each aliquot obtained was handled as follows: the aliquot

237 was added to NaOH solution (1 mL, 0.1 M, in ethanol at 10%, v/v) to stop the oxidation

238 process; after ethanol (2.5 mL, 10%, v/v) was placed to dilute the sample. Then, the

239 absorbance of the samples was measured at 232 nm (SmartSpec Plus Spectrophotometer,
240 Bio-Rad Laboratories, USA). Ethanol (10%, v/v) was used as blank. Percent inhibition of

241 linoleic acid oxidation was calculated with the following equation (3):

242 Lipid oxidation inhibition (%) = [(A-B) / A] *100

243 Eq. (3)

244 where A is the difference between the absorbance of distilled water (as control) after 24 h

245 and 0 h of incubation, and B is the difference between the absorbance of each extract

246 sample after 24 h and 0 h of incubation.

247

248 2.6 UPLC-ESI-Q/TOF-MS2 analysis

249 The system used was an Acquity ultra-performance liquid chromatography (UPLC)

250 consisting of an auto-sampler and a binary pump equipped with a 10 µL loop (partial loop

251 injection mode). Qualitative identification of polyphenols was carried out using a BEH

252 PHENYL (2.1 mm x 100 mm, 1.7 µm; WATERS, UK) analytical column operated at 40 °C

253 and the chromatographic separation was conducted according to the methodology proposed

254 by Kumari, Elancheran, Kotoky, & Devi (2016) with minor modifications. Gradient

255 separation was performed for each sample using a mobile phase of solvent A: 0.1% (v/v)

256 formic acid water and solvent B: 100% acetonitrile, with a constant flow rate of 0.3 mL

257 min-1. Samples (3 µL) were injected by an auto sampler with a rapid screening runtime of

258 10 min, started with the gradient program, 97% A for 1.10 min, followed by multiple

259 gradients from 5% B to 15% B from 1.10 to 4.40 min, holding 15% B for 4.60 min, getting

260 back to the initial conditions (3% B) in 1 min and re-equilibration of the column. The

261 UPLC system was coupled to a quadrupole-time-of-flight (Q-TOF™, WATERS, UK)

262 orthogonal accelerated Q-TOF mass spectrometer, equipped with an electrospray ionization

263 source (ESI). Mass spectra were recorded within 10 min. The full screen mass spectra
264 detection was carried out in the negative ion mode in a mass range m/z of 50-1200 Da and

265 using a capillary voltage of -3.5 and +4.0 kV, a dry gas temperature of 210 °C, a dry gas

266 flow of 8.0 L min-1, a nebulizer pressure of 2.0 bar, and spectra rate of 1 Hz. Moreover,

267 automatic MS/ MS experiments were performed using a ramp collision energy of 15-35 V

268 with argon as collision gas and adjusting the scan time every 1 second. The identification of

269 the phenolic compounds in the various extracts was obtained by using the full mass

270 spectrum and its unique mass fragmentation spectrum. Comparison of the observed MS 2

271 spectra with those found in the literature and Databases, such as SciFinder-Scholar

272 (https://scifinder.cas.org), Phenol-Explorer (www.phenol-explorer.eu), MassBank

273 (http://www.massbank.jp/), ChemSpider (http://www.chemspider.com), and PubChem

274 (https://pubchem.ncbi.nlm.nih.gov), were the main tool for identification of the compounds.

275

276 2.7 Experimental design and statistical analysis

277 The influence of the extraction method and solvent-to-solid ratio were investigated using a

278 full factorial design for each material (4 extraction process x 2 solid: liquid ratios). All

279 experiments were conducted at three levels of measurements and results reported as

280 mean ± standard deviation (SD). The data were analyzed by analysis of variance

281 (ANOVA), followed by Tukey test to detect significant differences among means of each

282 factor and level at p < 0.05. Statistical analysis was performed using Minitab 17 Statistical

283 Software (State College, PA: Minitab, Inc.).

284

285 3. Results and discussion

286 3.1. Influence of solid: liquid ratios

287 It is well known from literature that extraction conditions and characteristics of the sample

288 can affect the efficiency of the extraction, independently or interactively (Liyana-Pathirana

289 & Shahidi, 2005). Effects of solid: liquid ratio on antioxidant compounds (Figure 1,

290 phenolics), and antioxidant activity (Table 1) of plant extracts were investigated. It can be

291 seen that regardless of the sample studied, total phenolic content, and antioxidant activities,

292 respectively, increased generally when solid: liquid ratio increased.

293 We noted in the first part (ratio 1:50), when the amount of all plants powder increases, the

294 chance of bioactive components coming into contact with the solvent goes up, which leads

295 to higher leaching-out rates. In the other hand, for solid: liquid ratio 1:25, content

296 decreasing may be basically due to the saturation phenomenon. In fact, when the solvent is

297 saturated on bioactive components, the cellular phenomenon of diffusion stops and there

298 has stabilization rate of extracted compounds or decreased. This is consistent with mass

299 transfer principles; the driving force during mass transfer is the concentration gradient

300 between the solid and the bulk of the liquid, which is greater when a higher solvent-to-solid

301 ratio is used (Xi, 2009).

302 The solid: liquid ratio has a positive effect; in fact, the higher the solid: liquid ratio, the

303 higher the total amount of solids obtained. Also, interactions of the extracted compounds

304 with the solvent could have modified the activity coefficients and thus the solubility of the

305 compounds. Similar results about the effect of solid: liquid ratio on the extraction of

306 phenolic compounds were also reported for grape pomace by Pinelo, Rubilar, Jerez,

307 Sinerio, & Nuñez (2005), who also found similar relationship of solid: liquid ratio with

308 extraction yields.

309

310 3.2 Comparison of extraction methods on recovery of TPC

311 Effect of maceration, decoction, microwave-assisted extraction, and ultrasound-assisted

312 extraction on the content of total phenolics of pomegranate peel, walnut shell, moringa and

313 hojasen leaf extracts is shown in Figure 1. Significant differences (p <0.05) in TPC were

314 found among differently extraction methods. As shown in Figure 1, the levels of phenolics

315 varied widely, ranging from 6.40 to 18.92 mg GAE g-1 for PP extracts (Figure 1a), from

316 1.17 to 12.80 mg GAE g-1 for WS extracts, from 2.73 to 15.19 mg GAE g -1 for ML extracts,

317 and from 1.68 to 12.69 mg GAE g-1 for HL extracts (Figure 1b, 2c, 2d, respectively) and

318 were depending on the solid: liquid ratio and extraction technique. Importantly, these levels

319 are almost similar or higher than those previously reported for MAE extracts of

320 pomegranate peel (Sood & Gupta, 2015), for UAE and maceration extracts of Moringa

321 oleifera lam leaves (Rodríguez-Pérez et al., 2015).

322 The highest amount of polyphenols was obtained by using microwave-assisted extraction

323 technology. However, the recovery of polyphenols were also satisfactory in samples

324 obtained by decoction, followed by UAE and maceration. This highest extraction efficiency

325 of MAE could be attribute to the powerful shear, high-frequency vibration, high-velocity

326 impaction and cavitation involved in the processing (Pak-Dek et al., 2011). Bioactive

327 compounds are accumulated, in most cases, in vacuoles located inside the plant cell,

328 surrounded by rigid cell wall. During MAE, the high temperature and microwave energy

329 may burst the cell wall and release the bioactive compounds into the water. The energy

330 from the microwave dissipates into polar molecule such as phenolic compounds generating

331 heat thus leading to enhancing their release from cell walls. As a consequence of heating

332 effect, the components in the cell dissolve into the solvent more effectively, in accordance

333 to the disruption theory (Kaufmann, Christen, & Veuthey, 2001).

334
335 3.3 Effect of extraction methods on the antioxidant activity

336 For evaluating the effectiveness of antioxidants, different methods specific to their

337 chemical properties have been used. In this study four complementary methods were

338 followed to evaluate the antioxidant activity due to their simplicity, stability and accuracy.

339 DPPH• scavenging activity of different plant materials as affected by extracting methods is

340 shown in Table 1. As can been see the extracts with the highest DPPH• radical-scavenging

341 capacity were in this order: PP (decoction = 5.34 mg GAE g-1), WS (MAE = 5.35 mg GAE

342 g-1), ML (MAE = 5.24 mg GAE g -1), and HL (decoction = 5.14 mg GAE g -1). These results

343 are in good agreement with the previous findings of Hemat, Elsheshetawy, & Mahdy

344 (2016) for walnut extracts obtained by decoction, while extracts of moringa leaf were

345 higher than those of earlier findings of Nouman, Anwar, Gull, Newton, Rosa, &

346 Domínguez-Perles (2016) using decoction as extraction method. It has well established that

347 free radical scavenging activity of plant extracts is mainly due to phenolic compounds. By

348 increasing the concentrations of phenolic compounds, the number of hydroxyl groups

349 available in the reaction medium increased. So, the possibility of hydrogen donation to free

350 radicals will increase.

351 Another evaluation of the antioxidant activity of the samples studied is given by their

352 ability to scavenge the cation radical ABTS •+. Results of this assay are summarized on

353 Table 1 for all extractions methods and plant materials. The sequence of ABTS •+

354 scavenging ability was decoction > MAE > maceration > UAE for pomegranate peel

355 extracts, decoction > MAE > UAE > maceration for walnut shell extracts, decoction >

356 MAE > UAE > maceration for moringa leaf extracts, and decoction > MAE > UAE >

357 maceration for hojasen leaf extracts. As shown in Table 1, pomegranate peel and walnut

358 shell extracts obtained by decoction and MAE had the best antioxidant capacity. While,
359 UAE and maceration extracts had the lower antiradical capacity (mg TE g -1 per sample).

360 The superiority of the two first extraction methods over UAE, are agree with those reported

361 by Abdelfadel, Khalaf, Sharoba, & Assous (2015) who suggested that, thermal treatment

362 might destroy the cell wall and the subcellular compartments of vegetables to liberate

363 greater amounts of components that can influence antioxidant activity.

364 FRAP assay provides a simple and effective method for measuring the ability of

365 antioxidants in plant samples to act as reducing agents. The reducing potential of the tested

366 extracts was recorded over a concentration range from 0.28 (solid: liquid ratio 1:25) to 3.74

367 (solid: liquid ratio 1:50) mg GAE g -1 and followed the order of effectiveness as:

368 Pomegranate MAE extracts (3.74) > moringa MAE extracts (3.10) > walnut MAE extracts

369 (3.09) > hojasen MAE extracts (2.81) mg GAE g -1, respectively. From the results, it was

370 evident that there were large variations in ferric reducing antioxidant power among the

371 extraction methods influenced significantly (p < 0.05). Compared with decoction, MAE,

372 and UAE extracts, maceration extracts exhibited again the lowest antioxidant capacity in

373 reducing power assay. The highest reducing power activity of MAE extracts, followed by

374 decoction, may be attributed to their higher contents of total phenolic due to the action of

375 their hydroxyl group which might act as electron donors (Sood & Gupta, 2015). It has also

376 been reported that several compounds with reducing activity can be generated during MAE,

377 including thermolysis, heterocyclization and caramelization of sugars which contribute to

378 the significantly increased reducing power (Charurin, Ames, & Del Castillo, 2002).

379 Finally, the antioxidant principles of plant extracts can also be explained as their ability to

380 inhibit lipid peroxidation. Therefore, inhibition of linoleic acid oxidation determined for

381 extracts of different plant materials as affected by extracting techniques are shown in Table

382 1. The level of inhibition of linoleic acid peroxidation of the extracts was between 37.56
383 and 92.02 % indicating significant differences (p <0.05). Among plant materials, maximum

384 inhibition was noted by MAE pomegranate peel extract (92.02 %), followed by decoction

385 walnut shell extract (81.84 %), MAE hojasen leaf extract (80.21 %), and decoction moringa

386 leaf extract (61.09 %). As expected, the present data revealed that, regardless of the solid:

387 liquid ratio, the extracts of all plant materials, prepared using the decoction extracting

388 technique and microwave-assisted extraction, exhibited higher levels of inhibition of

389 linoleic acid oxidation than those obtained by ultrasound-assisted extraction. UAE is a

390 process that uses high intensity, high frequency sound waves and solvents to extract

391 targeted compounds from various matrices. Physical and chemical properties of materials

392 under ultrasound extraction conditions are altered due to the propagation and interaction of

393 sound waves as they disrupt the plant cell walls. This can explain antioxidant tendency on

394 UAE extracts (Dhanani, Shah, Gajbhiye, & Kumar, 2013).

395 Overall, the antioxidant activities of samples were related to the variety of materials and

396 extraction methods employed. Pomegranate peel extracts had much better antioxidant

397 activities than the others plant extracts, this could be attributed to their related higher total

398 phenolic content (Figure 1). Among the applied extraction methods, MAE extracts showed

399 the highest antioxidant activities, followed by decoction and UAE. The lower bioactivity of

400 extracts prepared by UAE and maceration techniques could be due to their relatively low

401 amount of phenolics (Kosanić, Ranković, & Vukojević, 2011). The above results indicated

402 that extraction technologies such as MAE could be a promising method for extracting

403 antioxidants from natural sources.

404

405 3.4 Compound identification by UPLC-Q/TOF-MS2

406 UPLC fingerprinting provides the chemical characterization of the extracts. The phenolic

407 compounds detected in this work were tentatively characterized by means of MS data,

408 together with the interpretation of the observed MS 2 spectra in comparison with those

409 found in the literature. To determine which compounds could be responsible for the

410 observed antioxidant activity, the polyphenol composition of the different extractions was

411 identified by UPLC-Q/TOF-MS2. For the purposes of this study, the characterization of two

412 of the four plant samples selected based on their behavior of antioxidant activity in general

413 and total polyphenol content will be presented below.

414 Figure 2 showed the chromatogram of pomegranate peel extracts obtained by two different

415 extraction methods. The corresponding compounds were identified by the interpretation of

416 their fragmentation patterns obtained from mass spectra (MS 2 experiment). The retention

417 times and mass spectrum data along with peak assignments for compounds identified using

418 negative ionization are described in Table 2. The pomegranate peel extracts obtained by

419 decoction (Figure 2a) and MAE (Figure 2b) showed important differences among

420 themselves. A total of 19 phenolic compounds were identified in the both extracts using

421 UPLC-Q/TOF-MS2 (Table 2). Cinnamic acid (m/z 146.9279, peak 7), and granatin B

422 (Galloyl-HHDP-DHHDP-hexoside) (m/z 950.7491, peak 13), which forms part of type III-

423 tannins (dehydroellagitannins) previously described (Fischer, Carle, & Kammerer, 2011),

424 were identified only in decoction extract. While secoisolariciresinol di-O-glucoside (m/z

425 540.8878, peak 7), and pedunculagin I (m/z 782.8182, peak 12) were detected in MAE

426 extracts. Furthermore, peak 7 have not generally been described in the pomegranate

427 literature, thus demonstrating that the phenolic profile of pomegranates may be much more

428 complex. The presence of specific phenolics may be influenced by different cultivars and

429 factors such as soil quality, climate, and stress conditions where plant foods are grown
430 (Alshikh, de Camargo, & Shahidi, 2015); therefore, some differences with the literature are

431 expected. In general, 14 compounds, such as ellagitannins and some flavonols, were found

432 in both pomegranate peel extracts (decoction and MAE) as previously reported (Li, He, Li,

433 Zhao, Liu, & Kong, 2015). Peak 2 and 3 were identified as isomers of

434 hexahydroxydiphenoyl (HHDP) hexoside. These compounds have a molecular ion [M-H]-

435 at m/z 480.9497, and eluted at 1.16 and 1.34 min, respectively, with their MS 2 fragment ion

436 at m/z 300.9503, characteristic deprotonated ellagic acid, by losing one hexose moiety [M-

437 H-162]-. Peak 9 exhibited an [M-H]- ion at m/z 782.8182. The loss of water and ellagic acid

438 in the MS2 experiment produced fragments at m/z 765 and m/z 480.8762, respectively.

439 Based on this fragmentation pathway and the occurrence of further typical fragments as

440 described above, peak 9 was identified as pedunculagin I (Figure 4) (Seeram, Lee, Hardy,

441 & Heber, 2005). Furthermore, peak 16 showed an [M-H]- ion at m/z 934.7589 and typical

442 fragment ion at m/z 632.7493 was identified as casuaricitin (galloyl-bis-HHDP hexoside).

443 Peak 10 and 14 were identified as punicalagin and were detected as doubly charged ion

444 species displaying an [M-2H]2- ion at m/z 540.9286, which is equivalent to a molecular

445 weight of 1084 Da. The fragment at m/z 780.9607 in the MS2 experiment indicated the loss

446 of a gallagic acid moiety. Punicalagin occurs in two isomeric forms, the α and β anomers

447 (Lu, Ding, & Yuan, 2008), which were confirmed in the present study as illustrated by

448 different retention times of this compound. These compounds are representative in P.

449 granatum L. and have been detected previously during a large-scale purification of

450 pomegranate husk polyphenols (Seeram et al., 2005).

451 On the other hand, Figure 3 showed the chromatogram of moringa leaf extracts obtained by

452 two different extraction methods. The corresponding compounds were identified by the

453 interpretation of their fragmentation patterns obtained from mass spectra (MS2 experiment).
454 The retention times and mass spectrum data along with peak assignments for compounds

455 identified using negative ionization are described in Table 3. It shows the list of 23 phenolic

456 compounds identified in M. oleifera extracts, which includes lignans, anthocyanins,

457 flavones, hydroxycinnamic acids, and chalcones (Figure 3; Table 3). It is clear from UPLC

458 chromatogram (Figure 3) that for each extraction method, there are nine peaks (with the

459 molecular mass and fragmentation patterns) suggesting that differences are present in each

460 case. For the characterization of decoction extract, 18 molecules were tentatively identified.

461 Four of them were different polyphenols in comparison of the sample extracted by MAE

462 and yielded the following fragmentation patterns: peak 2 exhibited a precursor ion at m/z

463 312.0504 [M-H]- and fragment ion at m/z 133.0135 and is suggested as caffeoyl tartaric

464 acid. Peak 7 was identificate as a glucuronated form of quercetin (quercetin 3-O-

465 glucuronide) and had a precursor ion at m/z 477.0809 [M-H]- and fragment ion at m/z

466 301.0509, which is due to the loss of glucoronic acid [M-H-176]- and the presence of

467 quercetin (Abu-Reidah, Arráez-Román, Segura-Carretero, & Fernández-Gutiérrez, 2013).

468 Peak 9, assigned as 3-p-coumaroylquinic acid, presented a precursor ion at m/z 337.0423

469 [M-H]- and fragment ion at m/z 163.0420 (coumaric acid). Peak 11 was determined as

470 kaempferol hexose and has a precursor ion at m/z 447.0736 [M-H]- and fragment ion at m/z

471 285.0560, product due to a loss of a hexosyl moiety (Justesen, 2000) (Figure 5; Table 3).

472 Some of these phenols have been previously identified as one of the major compounds in

473 M. oleifera leaves (Coppin et al., 2013). While, interestingly, five peaks were detected only

474 in MAE extracts and presented the following fragmentation patterns: peak 3 had a

475 precursor ion at m/z 569.9882 [M-H]- and fragment ion at m/z 273.0764 (phloretin 2-O-

476 xylosyl-glucoside). Peak 6 had a precursor ion at m/z 611.9875 [M-H]- and fragment ion at

477 m/z 449.7196 (cyanidin 3,5-O-diglucoside) which corresponds to the loss of one glucose
478 (162 Da) (Abu-Reidah, Ali-Shtayeh, Jamous, Arráez-Román, & Segura-Carretero, 2015).

479 Peak 9 (myricetin-3-O-glucoside) had a precursor ion at m/z 479.0826 [M-H]- and fragment

480 ion at m/z 316.0243 which corresponded to myricetin in structure after the neutral loss of

481 287 Da (hexose-malic acid moiety loss). Peak 12 was identificate as delphinidin 3-O-

482 rutinoside and had a precursor ion at m/z 611.9875 [M-H]- and fragment ion at m/z

483 303.0196. Peak 21 with a precursor ion at m/z 505.0067 [M-H]- and fragment ion at m/z

484 303.0912 (delphinidin 3-O-galactoside) indicates methyl-delphinidin aglycone yielded after

485 the neutral loss of galloyl-galactoside moiety (Figure 5; Table 3). These compounds are

486 part of the M. oleifera composition and were tentatively identified in agreement with

487 Jaiswal et al. (2013). Finally, as expected, the predominant group of phenolic compounds

488 in M. oleifera leaf extracts was the flavonoid group, with 3-Caffeoylquinic acid, peonidin

489 3-O-glucoside, luteolin 7-O-rutinoside, delphinidin 3-O-galactoside, methyl-

490 dihydroquercetin hexoside, apigenin-7-O-(6”-O-galloyl)-β-D-glucopyranoside, and

491 diosmetin 7-O-rutinoside being the most predominant flavonoids in both extracts

492 (decoction and MAE extracts). To the best of our knowledge the only lignan found in this

493 study (medioresinol, m/z 387.0521) has not yet been reported in moringa leaf extracts.

494

495 3.4.1 Possible causes of the differences in the polyphenolic content

496 Although environmental and genetic factors have been attributed to be some of the causes

497 of plant metabolome differences, in this study where the extraction method was the main

498 variant to compare the type of extracted compounds among plant materials, it can be

499 established that the extraction technique could influence the phenolic profile. For MAE

500 technology, it has been reported that microwave radiation has the property of transferring

501 energy directly to the reactants, causing the instantaneous superheating that promotes
502 chemical transformations and reactions which result in the organic synthesis of new

503 compounds (Hernandez & Leyva, 2011). Also, the observed difference in phenolic

504 fingerprint as a result of microwave treatment compared to decoction method could be

505 related to break of covalence bonds between phenolic components and an increase in free

506 phenolic components with low molecular weight, which could be associated to heat

507 generated by absorption of electromagnetic rays during microwave treatment (Jamshidi,

508 Barzegar, & Sahari, 2014).

509 While, in conventional extraction (decoction), heat is transferred through convection and

510 conduction from the surface, here, the extractability of solvents depends mainly on the

511 solubility of the compound in the solvent, the mass transfer kinetics of the product and the

512 strength of solute/matrix interaction with corresponding limitations on heat and mass

513 diffusion rate impacting on the distribution and presence of different compounds (Dhanani

514 et al., 2013).

515

516 4. Conclusion

517 The total phenolic content, and antioxidant activity were affected by the solid: liquid ratio,

518 and the best results were obtained with 1: 50 w/v. Also, the results presented in this study

519 show that the microwave-assisted extraction is a simple alternative for extraction of several

520 phenolic and flavonoid compounds from different plant sources, and more efficient than

521 maceration and decoction extraction method and ultrasound, and thus demonstrating that

522 the extraction method has major influences on type of compounds recovered. MAE,

523 provides extracts with a largest amount of phenolic compounds with several potential

524 properties for food and pharmaceutical industries. However, it is necessary, in-depth
525 optimization studies for cost reduction, process time, energy, raw materials and therefore

526 environmental impacts.

527

528 Acknowledgments

529 Cecilia Castro-López thanks to Mexican Council for Science and Technology (CONACYT)

530 for the postgraduate scholarship. Authors thank to PAICYT-UANL (CT254-15) for the

531 financial support given. Finally, authors sincerely thank M. Sc. Edgar T. Vazquez Ramos

532 from Waters Corporation for his technical support.

533

534 Competing interest statement

535 The authors declare no conflict of interest.

536

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 657 Table 1. Comparison among antioxidant activity assays and different extraction methods

Extraction DPPH• (mg GAE g-1) ABTS•+ (mg ET g-1) FRAP (mg GAE g-1) LPO (%)
al method 1:25* 1:50* 1:25* 1:50* 1:25* 1:50* 1:25* 1:5
Maceration 1.85 ± 0.01 b, C 4.61 ± 0.03 a, C 2.13 ± 0.01 b, D 4.51 ± 0.04 a, B 1.13 ± 0.01 b, C 3.54 ± 0.01 a, C 80.40 ± 0.5 b, B 82.51 ±
Decoction 2.64 ± 0.03 b, A 5.34 ± 0.02 a, A 2.49 ± 0.01 b, A 4.75 ± 0.01 a, A 1.39 ± 0.02 b, A 3.58 ± 0.01 a, B 70.79 ± 0.3 b, D 81.36 ±
MAE 2.23 ± 0.01 b, B 4.84 ± 0.03 a, B 2.38 ± 0.02 b, B 4.66 ± 0.01 a, B 1.21 ± 0.04 b, B 3.74 ± 0.05 a, A 82.13 ± 0.2 b, A 92.02 ±
UAE 1.77 ± 0.03 b, C 4.20 ± 0.04 a, D 2.26 ± 0.01 b, C 4.36 ± 0.02 a, C 1.16 ± 0.01 b, C 3.54 ± 0.01 a, C 73.67 ± 0.6 b, C 81.55 ±

Maceration 2.60 ± 0.01 b, A 5.26 ± 0.04 a, A 2.12 ± 0.01 b, B 4.55 ± 0.03 a, B 0.82 ± 0.01 b, D 2.62 ± 0.01 a, C 75.79 ± 0.5 b, B 78.00 ±
Decoction 2.67 ± 0.01 b, A 5.34 ± 0.01 a, A 2.31 ± 0.01 b, AB 4.77 ± 0.01 a, A 1.08 ± 0.01 b, B 2.85 ± 0.01 a, B 77.23 ± 0.4 b, A 81.84 ±
MAE 2.59 ± 0.01 b, B 5.35 ± 0.04 a, A 2.65 ± 0.01 b, A 4.71 ± 0.03 a, A 1.28 ± 0.01 b, A 3.09 ± 0.05 a, A 62.63 ± 0.4 b, D 79.05 ±
UAE 2.34 ± 0.02 b, C 5.13 ± 0.03 a, B 2.19 ± 0.01 b, C 4.71 ± 0.03 a, A 0.87 ± 0.02 b, C 2.46 ± 0.01 a, D 70.12 ± 0.4 b, C 71.89 ±

Maceration 2.53 ± 0.05 b, A 5.17 ± 0.03 a, A 1.72 ± 0.02 b, C 3.19 ± 0.02 a, D 1.19 ± 0.01 b, B 2.84 ± 0.01 a, C 40.73 ± 0.3 b, B 49.56 ±
Decoction 2.60 ± 0.02 b, A 5.23 ± 0.01 a, A 1.93 ± 0.02 b, A 4.24 ± 0.03 a, A 1.04 ± 0.01 b, C 2.91 ± 0.01 a, B 45.43 ± 0.3 b, A 61.09 ±
MAE 2.49 ± 0.02 b, A 5.24 ± 0.03 a, A 1.84 ± 0.02 b, B 4.14 ± 0.02 a, B 1.55 ± 0.05 b, A 3.10 ± 0.05 a, A 46.10 ± 0.5 b, A 54.56 ±
UAE 1.87 ± 0.01 b, B 5.03 ± 0.03 a, B 1.55 ± 0.06 b, D 3.85 ± 0.02 a, C 0.70 ± 0.01 b, D 2.43 ± 0.03 a, D 37.56 ± 0.6 b, C 42.36 ±

Maceration 1.10 ± 0.01 b, D 3.19 ± 0.03 a, B 1.31 ± 0.02 b, C 3.42 ± 0.01 a, C 0.36 ± 0.01 b, C 1.63 ± 0.01 a, C 66.28 ± 0.2 b, C 67.62 ±
Decoction 2.50 ± 0.01 b, A 5.14 ± 0.01 a, A 2.03 ± 0.01 b, A 4.65 ± 0.03 a, A 0.68 ± 0.01 b, B 2.62 ± 0.01 a, B 68.01 ± 0.2 b, B 70.12 ±
MAE 2.39 ± 0.02 b, B 5.13 ± 0.04 a, A 1.76 ± 0.01 b, B 4.32 ± 0.03 a, B 0.87 ± 0.06 b, A 2.81 ± 0.01 a, A 72.62 ± 0.4 b, A 80.21 ±
UAE 1.25 ± 0.03 b, C 2.78 ± 0.04 a, C 1.13 ± 0.04 b, D 3.52 ± 0.02 a, C 0.28 ± 0.01 b, D 1.54 ± 0.01 a, D 62.63 ± 0.5 b, D 64.45 ±
658

659 *Solid: liquid ratio. MAE, microwave-assisted extraction and UAE, ultrasonic-assisted extraction. Data

660 presented as mean ± standard deviation (n= 3, each plant). Mean values followed by different superscript

661 letters within the same row indicate significant (p <0.05) differences of means within the solid: liquid ratio;

662 and values followed by different superscript capital letters within the same column indicate significant (p

663 <0.05) differences of means within the extraction method according to Tukey’s multiple range test

664
665 Table 2. Phytochemical compounds detected and characterized in pomegranate peel extracts obtained by decoction and
666 microwave-assisted extraction (MAE) with water (1:50) by using UPLC-Q/TOF-MS2 in negative ionization mode
MS2 Occurrence
Pea Rt [M-H]- Tentative Polyphen Polyphenol Molecul Domina
k (min (m/z) assignment ol class family ar Decocti MA
nt
N° ) formula on E
fragmen
t ion
1 0.82 181.064 Dihydrocaffeic acid Phenolic Hydroxyphenyl C9H10O4 136.9508 √ √
6 acid propanoic acids
2 1.16 480.949 Hexahydroxydiphe Phenolic Ellagitannins - 300.9557 √ √
7 noyl (HHDP)- acid
Hexoside
3 1.34 480.949 Hexahydroxydiphe Phenolic Ellagitannins - 300.9503 √ √
9 noyl (HHDP)- acid
Hexoside
4 1.89 780.812 Punicalin α Phenolic Ellagitannins C34H22O2 600.7992 √ √
 3 acid 2
5 1.96 780.813 Punicalin β Phenolic Ellagitannins C34H22O2 600.8007 √ √
5 acid 2
6 2.47 305.019 (+)-Gallocatechin Flavonoid Flavonols C15H14O7 179.0057 √ √
9
2.88 146.927 Cinnamic acid Phenolic Hydroxycinna C9H8O2 144.9542 √
7 9 acid mic acids
2.88 540.887 Secoisolariciresinol Lignan Lignans C26H38O1 360.9482 √
8 di-O-glucoside 2

8 3.06 706.852 Epicatechin 3-O- Flavonoid Proanthocyanid C36H34O1 393.9573 √ √

4 galactoside ins dimers 5

9 3.25 782.818 Pedunculagin I Phenolic Ellagitannins C34H24O2 480.8762 √ √

2 acid 2
10 3.64 1083.88 Punicalagin α Phenolic Ellagitannins C48H28O3 780.9008 √ √
72 acid 0
11 3.95 146.927 Cinnamic acid Phenolic Hydroxycinna C9H8O2 144.9542 √
9 acid mic acids
12 4.17 782.818 Pedunculagin I Phenolic Ellagitannins C34H24O2 480.8762 √
2 acid 2
13 4.29 950.749 Granatin B Phenolic Ellagitannins C41H28O2 932.8765 √
1 (Galloyl-HHDP- acid 7
DHHDP-hexoside)
14 4.71 1083.88 Punicalagin β Phenolic Ellagitannins C48H28O3 780.9607 √ √
45 acid 0
15 5.0 798.803 Ellagic acid Phenolic Ellagitannins 478.9257 √ √
4 derivative acid
16 5.14 934.758 Casuarinin Phenolic Ellagitannins C41H28O2 632.7493 √ √
9 (Galloyl-bis- acid 6
HHDP-hexoside)
17 5.43 462.939 Ellagic acid Phenolic Ellagitannins - 300.9497 √ √
4 hexoside acid
18 5.51 146.927 Cinnamic acid Phenolic Hydroxycinna C9H8O2 144.9542 √
9 acid mic acids
19 7.11 300.957 Ellagic acid Phenolic Hydroxybenzoi C14H6O8 300.5834 √ √
2 acid c acid dimers
667 Table 3. Phytochemical compounds detected and characterized in M. oleifera leaf extracts obtained by decoction and microwave-assisted
668 extraction (MAE) with water (1:50) by using UPLC-Q/TOF-MS2 in negative ionization mode

Pea Rt [M-H]- MS2 Occurrence

Tentative Polypheno Polyphenol Molecula Dominan
k N° (min (m/z) l class family r formula Decoctio MA
assignment t
) n E
fragment
ion
1 0.78 387.052 Medioresinol Lignan Lignans C21H24O7 180.8399 √ √
1
2 1.00 312.050 Caffeoyl tartaric Phenolic Hydroxycinnami C13H12O9 133.0135 √
4 acid acid c acids
3 1.05 569.988 Phloretin 2-O- Flavonoid Dihydrochalcon C26H32O14 273.0764 √
2 xylosyl- es
glucoside
4 1.37 Peonidin 3-O- Flavonoid Anthocyanins C22H23O11 301.0493 √ √
462.711 glucoside
4
5 1.53 353.034 3- Phenolic Hydroxycinnami C16H18O12 191.0006 √ √
2 Caffeoylquinic acid c acids
acid
6 1.56 611.987 Cyanidin 3,5-O- Flavonoid Anthocyanins C27H31O16 449.7196 √
5 diglucoside
7 1.61 477.080 Quercetin 3-O- Flavonoid Flavonols C21H18O13 301.0509 √
9 glucuronide
8 1.75 611.987 Cyanidin 3-O- Flavonoid Anthocyanins C27H31O16 287.0396 √
 5 sophoroside
9 1.98 337.042 3-p- Phenolic Hydroxycinnami C16H18O8 163.0420 √
3 Coumaroylquini acid c acids
c acid
1.98 479.082 Myricetin-3-O- Flavonoid Flavonols C20H18O12 316.0243 √
6 glucoside
10 2.07 353.034 3- Phenolic Hydroxycinnami C16H18O12 191.0006 √ √
2 Caffeoylquinic acid c acids
acid
11 2.20 447.073 kaempferol Flavonoid Flavones C21H20O11 285.0560 √
6 hexose
12 2.34 611.987 Delphinidin 3- Flavonoid Anthocyanins C27H31O16 303.0196 √
5 O-rutinoside
13 2.56 447.073 kaempferol Flavonoid Flavones C21H20O11 285.0560 √
6 hexose
14 2.63 593.037 Luteolin 7-O- Flavonoid Flavones C27H30O15 284.9632 √ √
1 rutinoside
15 3.95 439.111 (+)-Catechin 3- Flavonoid Catechins C22H18O10 289.0983 √ √
2 O-gallate
16 4.14 463.005 Quercetin 3-O- Flavonoid Flavonols C21H20O12 303.0520 √ √
8 galactoside
17 4.75 505.006 Delphinidin 3- Flavonoid Anthocyanins C21H21ClO1 303.0912 √ √
7 O-galactoside 2

18 5.05 449.185 Dihydroquerceti Flavonoid Dihydroflavonol C21H22O11 305.1634 √ √

3 n 3-O- s
rhamnoside
19 5.16 505.006 Delphinidin 3- Flavonoid Anthocyanins C21H21ClO1 303.0912 √ √
7 O-galactoside 2

20 5.36 479.119 Methyl- Flavonoid Flavonols C22H24O12 299.0574 √ √

0 dihydroquerceti
n
hexoside
21 5.56 505.006 Delphinidin 3- Flavonoid Anthocyanins C21H21ClO1 303.0912 √
7 O-galactoside 2

22 5.91 438.975 Apigenin-7-O- Flavonoid Flavones C28H24O14 271.0618 √ √

3 (6”-O-galloyl)-
β-D-
glucopyranoside
23 6.34 607.012 Diosmetin 7-O- Flavonoid Methoxyflavone C28H32O15 563.0753 √ √
3 rutinoside s
669 Figure captions

670 Figure 1. Total phenolic content (TPC) of (a) pomegranate, (b) walnut, (c) moringa and (d)

671 hojasen extracts obtained from various extraction techniques (MC, maceration; DC,

672 decoction; MAE, microwave-assisted extraction; and UAE, ultrasonic-assisted extraction).

673 Data represents mean ± standard deviation. Different lowercase letter indicates significant

674 differences (p <0.05) between different solid: liquid ratio and different capital letter

675 indicates significant differences (p <0.05) between extraction method according to Tukey’s

676 multiple range test

677 Figure 2. Base-peak chromatogram of phenolic compounds in pomegranate peel extracts

678 obtained by (a) decoction and (b) microwave-assisted extraction by UPLC-Q/TOF-MS2 in

679 negative ionization mode. For peak assignment see Table 2

680 Figure 3. Base-peak chromatogram of phenolic compounds in M. oleifera leaf extracts

681 obtained by (a) decoction and (b) microwave-assisted extraction by UPLC-Q/TOF-MS2 in

682 negative ionization mode. For peak assignment see Table 3

683

684

685

686

687

688

689

690

691
692 Figure 1.

(b)

TPC (mg GAE g-1)

20
15 aA
aB aC
10 aD
5 bA bC bB
bD
0
MC DC MAE UAE
Extraction method

Solid: liquid ratio (1:25) Solid: liquid ratio (1:50)

aA (d)
TPC (mg GAE g-1)

20
aC
15 aD TPC (mg GAE g-1) 20
15 aA aB
10 bB bA
bD
5 10 aC
aC bA bB
0 5 bD bC
MC DC MAE 0
Extraction method MC DC MAE UAE
Extraction method
693 Solid: liquid ratio (1:25) Solid: liquid ratio (1:50)
Solid: liquid ratio (1:25) Solid: liquid ratio (1:50)
TPC (mg GAE g-1)

20
aA
15 aB

10 aD
bA
bC bB
5
0
MC DC MAE
Extraction method

Solid: liquid ratio (1:25) Solid: liquid ratio (1:50)

694

695

696 Figure 2.
 100
5 14
10
4
Absolute Intensity (%)

2 8 15

1 3 6
7
9 11 13 16 17
19
0

100
14
10

5 15
4 89 17
2 12 16
1 3 6 7 18 19
0
0 1 2 3 4 5 6 7 8 9 10

Retention time (min)

697
698

699

700 Figure 3.

34
 100
5 17
16

14

22
Absolute Intensity (%)

10
7 9 18
2 4 13 19 20
1 23
11 15
0
100
12

16 17

14
3
1 5
10 18 19 22
6
8 20 21
4 9 15 23
0
0 1 2 3 4 5 6 7 8 9 10

Retention time (min)

701
702

703

35
704 Highlights

705

706 ● Several extraction methods are applied for the obtention of polyphenol extracts.

707 ● Significant influence on TPC, and antioxidant activity is established.

708 ● Performance of the antioxidant activities is affected by the solid: liquid ratio.

709 ● LC-MS2 is applied for identification of phenolics in plant extracts.

710 ● MAE is the more effective extraction technique for polyphenols recovery.

711

712

36