1

An integrated approach for the valorization of mango seed kernel: efficient extraction 1

solvent selection, phytochemical profiling and antiproliferative activity assessment. 2

3

Diego Ballesteros-Vivas1,2a, Gerardo Alvarez-Rivera2a, Sandra Johanna Morantes Medina3 4

Andrea del Pilar Sánchez Camargo1, Elena Ibánez2, Fabián Parada-Alfonso1, Alejandro 5

Cifuentes2* 6

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1 High Pressure Laboratory, Department of Chemistry, Faculty of Science, Universidad 8

Nacional de Colombia, Carrera 30 #45-03, Bogotá D.C., 111321, Colombia. 9

2 Laboratory of Foodomics, Institute of Food Science Research, CIAL, CSIC, Nicolás Cabrera 10

9, 28049 Madrid, Spain. 11

3 Unit of Basic Oral Investigation (UIBO), School of Dentistry, Universidad El Bosque, Av. 12

Carrera 9 #131 A-02, Bogotá D.C., 110121, Colombia. 13

14 a These two authors contributed equally to this work. 15

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*Corresponding author: 17

Prof. Dr. Alejandro Cifuentes, Laboratory of Foodomics, Institute of Food Science Research, 18

CIAL (CSIC), Nicolás Cabrera 9, 28049 Madrid, Spain, e-mail: a.cifuentes@csic.es, Tel.: +34 19

910017955; fax: +34 910017905. 20

21

Keywords: 22

Mangifera indica L.; fruit by-products; Hansen solubility parameters; Pressurized-liquid 23

extraction; LC-Q-TOF; GC-Q-TOF; High-resolution mass spectrometry; antiproliferative 24

activity; HT-29 cell line; CCD-18Co cell line. 25

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2

ABSTRACT 27

A novel valorization strategy is proposed in this work for the sustainable utilization of a major 28

mango processing waste (i.e. mango seed kernel, MSK), integrating green pressurized-liquid 29

extraction (PLE), bioactive assays and comprehensive HRMS-based phytochemical 30

characterization to obtain bioactive-rich fractions with high antioxidant capacity and 31

antiproliferative activity against human colon cancer cells. Thus, a two steps PLE procedure 32

was proposed to recover first the non-polar fraction (fatty acids and lipids) and second the polar 33

fraction (polyphenols). Efficient selection of the most suitable solvent for the second PLE step 34

(ethanol/ethyl acetate mixture) was based on the Hansen solubility parameters (HSP) approach. 35

A comprehensive GC- and LC-Q-TOF-MS/MS profiling analysis allowed the complete 36

characterization of the lipidic and phenolic fractions obtained under optimal condition (100% 37

EtOH at 150°C), demonstrating the abundance of oleic and stearic acids, as well as bioactive 38

xanthones, phenolic acids, flavonoids, gallate derivatives and gallotannins. The obtained MSK-39

extract exhibited higher antiproliferative activity against human colon adenocarcinoma cell line 40

HT-29 compared to traditional extraction procedures described in literature for MSK utilization 41

(e.g. Soxhlet), demonstrating the great potential of the proposed valorization strategy as a 42

valuable opportunity for mango processing industry to deliver a value-added product to the 43

market with health promoting properties. 44

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1. INTRODUCTION 52

Mango (Mangifera indica L.) is one of the most important tropical fruit crops, with an annual 53

production of more than 38 million tonnes (Mitra, 2016). The commercial importance of mango 54

fruit is due, among other reasons, to its sensorial quality attributes, high nutritional value and 55

functional compounds content (Ediriweera, Tennekoon, & Samarakoon, 2017; Gentile et al., 56

2019; Ribeiro & Schieber, 2010). Colombia plays an increasing role in world mango 57

production with cultivars such as ‘Sugar mango’, recognized by its sensorial qualities 58

(Corrales-Bernal, Maldonado, Urango, Franco, & Rojano, 2014). The industrial mango 59

processing generates about 40–60% of fruit wastes (12–15% of peels and 15–20% of kernels 60

seeds); none of them currently used for commercial purposes (Nawab, Alam, Haq, & Hasnain, 61

2016). Recently, several researches about the chemical composition and bioactive potential of 62

mango seed kernel (MSK) have been reviewed (Jahurul et al., 2015; Torres-León et al., 2016). 63

MSK contains important families of health-promoting compounds including fatty acids and 64

triacylglycerols (Lieb et al., 2018), gallotanins (Luo et al., 2014), xanthones (e.g. mangiferin) 65

(Barreto et al., 2008), flavonoids and phenolic acids, among others (Dorta, González, Lobo, 66

Sánchez-Moreno, & de Ancos, 2014; Lopez-Cobo et al., 2017). Polyphenolic compounds from 67

mango have been reported to have a strong antioxidant activity (Barreto et al., 2008; Soong & 68

Barlow, 2006; Sultana, Hussain, Asif, & Munir, 2012), and exhibit bioactivity in cancer cell 69

line models, including breast, liver, leukemia, cervix, prostate, lung and colon (Abdullah, 70

Mohammed, Rasedee, & Mirghani, 2015; Abdullah, Mohammed, Rasedee, Mirghani, & Al-71

Qubaisi, 2015; Luo et al., 2014; Timsina & Nadumane, 2015). In particular, mangiferin (2-β-72

D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one) has been reported as one of the 73

most bioactive phytochemicals in mango; in both in vitro and in vivo models (Imran et al., 74

2017). 75

4

Considering the bioactive potential of MSK, the development of green valorization strategies 76

to obtain polyphenolic-rich extracts from this valuable biowaste, pose a great challenge and a 77

unique opportunity for mango processing industry to deliver a value-added product to the 78

market with health promoting properties. Thus, strategies based on efficient extraction solvent 79

selection and use of new green extraction processes can help fulfilling the goals of the green 80

extraction of natural products (Chemat, Vian, & Cravotto, 2012). Hansen solubility parameters 81

(HSP) was shown to be a useful predictive model to ascertain the solubility of solutes, such as 82

secondary metabolites, in different solvents through their affinity and miscibility estimation. 83

In terms of new green extraction processes, Pressurized Liquid Extraction (PLE) is a 84

recognized environmentally friendly technique due to its higher extraction efficiency, lower 85

solvent consumption, short extraction time and the possibility of using green solvents (Ameer, 86

Shahbaz, & Kwon, 2017; Herrero, Castro-Puyana, Mendiola, & Ibañez, 2013). Several 87

research works have been conducted employing the joint strategy involving HSP+PLE to 88

target bioactive compounds recovery from natural sources (Ballesteros-Vivas et al., 2019; 89

Damergi et al., 2017; Sánchez-Camargo et al., 2017; Srinivas, King, Monrad, Howard, & 90

Hansen, 2009). 91

In this context, the present research aimed to develop an integrated valorization strategy, 92

involving HSP approach and sequential PLE procedure, in vitro antioxidant assays and 93

comprehensive characterization with advanced analytical techniques (liquid chromatography 94

and gas chromatography coupled to high resolution mass spectrometry) to obtain mangiferin 95

and other phenolic compounds from ‘sugar MSK’ with selective antiproliferative activity 96

against human colon adenocarcinoma cell line HT-29. An integrated process scheme of the 97

proposed MSK valorization strategy is shown in Figure 1. 98

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2. MATERIAL AND METHODS 101

2.1 Samples and reagents 102

Sugar mango fruits were purchased from a local market in Bogotá D.C., Colombia in February 103

2018. Mango fruit by-products were obtained after mechanical pulping process. Seeds were 104

split into coat and kernel (endosperm). ‘Sugar MSK’ (5.3% moisture content) was dried at 105

room temperature in the darkness during 48 h, subsequently ground to fine powder and stored 106

at -20 °C until its use. 107

Gallic acid, quercetin, trolox, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 108

2,2-diphenyl-1-picrylhydrazyl (DPPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 109

bromide (MTT), RPMI-1640 cell culture medium, streptomycin (0.1 mg/mL), penicillin (100 110

U/mL) potassium acetate, ammonium acetate, sodium carbonate, formic acid, potassium 111

persulfate, aluminum chloride, were purchased from Sigma-Aldrich (Madrid, Spain). Fetal 112

bovine serum (Gibco) and 0.05% trypsin-EDTA (Gibco) were purchased from Thermo Fisher 113

Scientific (Rockford, IL). Merck (Darmstadt, Germany) provided the Folin-Ciocalteu phenol 114

reagent. Solvents employed were HPLC-grade. Acetonitrile, chloroform, ethanol and methanol 115

were acquired from VWR Chemicals (Barcelona, Spain), whereas ethyl acetate by Scharlau 116

(Barcelona, Spain). Ultrapure water was obtained from a Millipore system (Billerica, MA, 117

USA). For the UPLC-q-TOF-MS analyses, MS grade ACN and water from LabScan (Dublin, 118

Ireland) were employed. 119

120

2.2 Hansen Solubility Parameters estimation 121

HSP for mangiferin and green solvents, including ethanol, ethyl acetate, ethyl lactate and (+)-122

limonene, were estimated using HSPiP® software v 5.0 at normal conditions, following the 123

methodology previously reported by Sánchez-Camargo et al (Sánchez-Camargo et al., 2017). 124

Briefly, the SMILES (Simplified molecular input line syntax) of mangiferin 125

6

[C1=C2C(=CC(=C1O)O)OC3=CC(=C(C(=C3C2=O)O)C4C(C(C(C(O4)CO)O)O)O)O] was 126

break into corresponding functional groups using Yamamoto-molecular break (Y-MB) method 127

and then HSP parameters were estimated by “Do It Yourself” tool. Subsequently, the affinity 128

between the mangiferin and the green solvents was measured by Ra or “distance” term using 129

their HSPs values through “Solvent optimizer” tool (the smaller Ra corresponding to the greater 130

affinity between solvent and solute). The variation of Ra at different temperatures (25-150 °C) 131

was also studied. For this purpose, the temperature dependence of mangiferin solubility 132

parameters was estimated by Jayasri and Yaseen method (Jayasri & Yaseen, 1980), employing 133

the critical data obtained by Marrero & Gani group contribution method (Marrero & Gani, 134

2001). The temperature effect on HSPs of green solvents was evaluated by the Gunn-Yamada 135

(Pereira, Silva, & Rodrigues, 2011) and Williams et al. (Williams, Rubin, & Edwars, 2004) 136

methods. Finally, Ra between mangiferin and green solvents were estimated at different 137

temperatures. 138

139

2.3 Pressurized liquid extraction (PLE) 140

A commercial ASE 200 device (11 mL stainless steel cells) was used for PLE process in two 141

steps. For each extraction, ‘sugar MSK’ samples and sea sand were mixed in a 1:2 w/w 142

proportion. The mixture was extracted on static mode at 100 bar. After the extraction, the 143

solvent was removed by evaporation with continuous stream of gaseous nitrogen. Extraction 144

yield was expressed as g of extract/100 g dry weight basis of sample (mean of duplicate). 145

146

2.3.1. PLE- first step evaluation 147

Due to ‘sugar MSK’ fat content, a first defatting step was required in order to recover the fat 148

while cleaning the sample for polyphenolics’ extraction. Three “alternative and usable” 149

solvents were tested to avoid the use of n-hexane: n-heptane, cyclohexane and (+)-limonene. 150

7

n-Hexane was used as reference nonpolar solvent. In order to achieve the maximum defatting 151

of ‘sugar MSK’, kinetics extraction curves for each nonpolar solvent were studied at 100 °C 152

and 100 bar for 90 min. 153

154

2.3.2. PLE-second step optimization 155

The polyphenolic compounds recovery, including mangiferin, from ‘sugar MSK’ after the 156

defatting process was optimized using a three-level face-centered central composite design 157

(CCD). The effect of temperature (50-150 °C) and green solvent composition (according to 158

HSP results) were investigated on mangiferin content, extraction yield, total phenolic content, 159

total flavonoid content and antioxidant activity. Experimental data was fitted with the following 160

second order polynomial equation: 161

, , , Eq. (1) 162

where is the response variable, and , , , , , , and , are regression 163

coefficients of variables for intercept, linear, quadratic, and interaction terms, respectively, and 164

and are the independent variables, representing solvent composition and temperature, 165

respectively. The adequacy of the model was determined by coefficient of regression (R2) and 166

the F-test value obtained from the analysis of variance (ANOVA) by the statistical software 167

STATISTICA 12 (Stat Soft, Inc., Tulsa, OK 74104, USA). Pareto charts for the standardized 168

effects of independent variables on response factors were also generated. A multiple response 169

optimization was carried out by combining the experimental factors, looking for maximizing 170

the desirability function (Ballesteros-Vivas et al., 2019). 171

172

2.4 Conventional extractions 173

Conventional solvent extractions Bligh & Dyer (Bligh & Dyer, 1959; Breil, Abert Vian, Zemb, 174

Kunz, & Chemat, 2017) (B&D) and dynamic maceration (DM) were used for comparison as 175

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standards to determine the efficiency of the PLE-first and -second steps, respectively. For the 176

B&D method, a ‘sugar MSK’ sample and 2:1 chloroform:methanol (v/v) mixture were 177

homogenized for 2 min. Then, chloroform and water were added to get a final ratio of 2:2:1.8 178

chloroform:methanol:water (v/v/v). The mixture was shaken vigorously for 2 min and the final 179

biphasic system was then separated by centrifugation (10 min at 2000 rpm). The lower 180

chloroform phase layer was collected and the solvent was evaporated under a stream of 181

nitrogen, in order to recover the lipid content. Moreover, DM was performed using green 182

solvents according to HSPs results. Samples of ‘sugar MSK’ and solvent were mixed in a 183

proportion of 1:5 (w/v) and kept in agitation at 750 rpm, 25 ºC for 24 h. Subsequently, the 184

extract was separated from the sample by centrifugation (20 min at 5000 rpm) and the solvent 185

was evaporated under a stream of nitrogen. 186

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2.5 Determination of total phenolic content (TPC) 188

TPC was determined by the Folin–Ciocalteu (Hosu, Cristea, & Cimpoiu, 2014) method with 189

slight modifications. Gallic acid (0–100 µg/mL) was used for calibration of a standard curve. 190

10 µL of extract solution, 600 µL of water and 50 µL of Folin-Ciocalteu reagent (0.2 M), were 191

mixed. After 5 min, 150 μL of Na2CO3 (20% w/v) and 190 µL of water were added. After 120 192

min for allowing the reaction to take place, the absorbance was measured at 760 nm using a 193

microplate spectrophotometer reader (Synergy HT, BioTek Instruments, Winooski, VT, USA). 194

The results were expressed as milligrams of gallic acid equivalents per gram of dry weight 195

basis (mg GAE/g Db) as mean of three replicates. 196

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2.6 Determination of total flavonoid content (TFC) 198

TFC was estimated by Aluminium chloride colorimetric method (Hosu et al., 2014) with slight 199

modifications. Quercetin (0–100 µg/mL) was used for calibration of a standard curve. 100 µL 200

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of extract solution, 30 µL of AlCl3 (10% w/v), 30 µL of potassium acetate (1 M), 300 µL of 201

EtOH and 540 µL of water were mixed and incubated for 30 min. Then, the absorbance was 202

measured at 415 nm using a microplate spectrophotometer reader. The results were expressed 203

as milligrams of quercetin equivalents per gram of dry weight basis (mg QE/g Db) as mean of 204

three replicates. 205

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2.7 Antioxidant capacity assays 207

2.7.1 DPPH assay 208

DPPH scavenging activity was performed according to procedure previously described by 209

(Brand-Williams, Cuvelier, & Berset, 1995) with some modifications. The EC50 value was 210

defined as the concentration of the extract sufficient to reduce to 50% the maximum absorption 211

value estimated in the blank DPPH. To this end, 25 µL of different methanolic solutions of 212

extracts and 975 µL DPPH solution (60 µM) were mixed and incubated for 4 h. After, the 213

absorbance was measured at 516 nm in a microplate spectrophotometer reader. EC50 was 214

expressed as µg/mL of extract solution (mean of three replicates). 215

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2.7.2 TEAC assay 217

The TEAC assay was performed following Re et al. procedure (Re et al., 1999). Trolox (0.25-218

2.0 mM) was used for calibration of a standard curve. The ABTS•+ radical cation was produced 219

by reacting ABTS solution (7.00 mM) with K2S2O8 solution (2.45 mM) in dark for 16 h. The 220

ABTS•+ radical was diluted to an absorbance of 0.7 at 734 nm. Next, 10 µL of different 221

solutions of extracts were added to 990 µL of ABTS•+ solution. Absorbance of mixture was 222

recorded at 734 nm every 5 min for 45 min in a microplate spectrophotometer reader. The 223

extracts were analysed in triplicate and results expressed as TEAC values (mM trolox/g 224

extract). 225

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2.8 Cell lines and cell culture 226

HT-29 (human colon adenocarcinoma) and CCD-18Co (normal human colon fibroblast) cells 227

were purchased from the American Type Culture Collection. Cell lines were cultured in RPMI 228

1640 medium, supplemented with Hepes 25 mM, L-glutamine 2.05 mM, 10% fetal bovine 229

serum and 25 μg/mL gentamicin, and incubated at 37 °C under 5% CO2 in a humidified 230

atmosphere. When the cell achieved 80%–90% confluent, it was detached by trypsin-EDTA 231

and sub-cultured into new sterile culture flasks for further propagation. 232

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2.9 Antiproliferative activity assay 234

The antiproliferative activity of ‘sugar MSK’ extracts were evaluated by MTT assay. Cells in 235

exponential growth phase (70-80% confluence) were trypsinized, counted and seeded in 96-236

well plates at a density of 1.0 ×104 (HT-29) and 4.5 ×103 (CCD-18Co) cells/well. The plates 237

were incubated for 24 hours at 37°C to allow the cell adhesion. Cells were treated with the 238

vehicle (DMSO 0.1% v/v) regarded as untreated controls or with different concentrations of 239

extracts (6.25 – 100 μg/mL) and incubated at three different time points 24, 48, and 72 h. After 240

the incubation, the medium was removed and 100 μL of MTT solution (0.25 mg/mL RPMI 241

1640 medium) was added to each well and the plate was incubated for 4 h. After, medium was 242

discarded and cells were washed with 200 μL of phosphate buffer saline (PBS). 100 μL of 243

DMSO were added to each well to dissolve the formazan crystals. The absorbance was 244

measured at 570 nm using a microplate reader (Tecan, Infinite® 200 PRO). Triton X-100 245

(1.0%) was used as a positive control. The cell viability was expressed as percentage of live 246

cells relative to controls. The IC50 values (concentration of extract that causes 50% inhibition 247

or cell death) were determined based on the dose-dependent response curves of extract using 248

GraphPad Prism 7.0 software (GraphPAD Corp., San Diego, CA, USA). Each experiment was 249

performed as three independent test with minimum three replicated. 250

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2.10 Phytochemical profiling of ‘sugar MSK’ extracts and mangiferin quantification 252

2.10.1 Gas chromatography-mass spectrometry (GC-q-TOF-MS) 253

Fat composition of ‘sugar MSK’ extract obtained in the PLE- first step was studied using GC-254

q-TOF-MS after derivatization according to Fiehn method, with some variations (Ibáñez, Simó, 255

Palazoglu, & Cifuentes, 2017). For this purpose, fat samples were subjected a two-step 256

derivatization process by methoxyamination and silylation reactions. 5 µL of fat solution (20 257

mg/mL n-heptane) were dried in SpeedVac Concentrator (SC200, Savant Instrument, Inc., 258

Farmingdale, NY, USA). Subsequently, 10 µL of methoxyamine (CH3ONH2•HCl) solution (40 259

mg/mL pyridine) were added to dried sample and the mixture was shaken at 750 rpm for 60 260

min and 30 °C. Then 90 µL of MSTFA (N-methyl-N-(trimethylsilyl) trifluoroacetamide) with 261

1% TMCS (trimethylchlorosilane) and 2 µL of d27-myristic acid were added to mixture and 262

shaken again (750 rpm for 30 min and 37 °C). Derivatized samples were analysed employing 263

a 7890B Agilent system (Agilent Technologies, Santa Clara, CA, USA) coupled to a 264

quadrupole time-of-fight (q-TOF) 7200 (Agilent Technologies, Santa Clara, CA, USA) 265

equipped with an electronic ionization (EI) interface. An Agilent Zorbax DB5- MS + 10 m 266

Duragard Capillary Column (30 m × 250 μm x 0.25 μm) was used for chromatographic 267

separation. Sample injection volume was 1 µL. The injector operated in split mode (ratio of 268

10:1 and a split flow of 8.4 mL/min) at 250 °C. Helium was used as carrier gas at a constant 269

flow (0.8 mL/min). The oven temperature was programmed to start at 60 °C, heated to 325 °C 270

at 10 °C/min and held at this temperature for 10 min. MS parameters were the following: 271

electron impact ionization at 70 eV, filament source temperature of 250 °C, quadrupole 272

temperature of 150 °C, m/z scan range 50–600 amu at a rate of 5 spectra per second. Systematic 273

mass spectra deconvolution of chromatographic signals and tentative identification of 274

12

unknowns was performed using the Agilent Mass Hunter Unknown Analysis tool and mass 275

spectral databases (i.e. NIST MS Search v.2.0 and Fiehn Lib). 276

277

2.10.2 Liquid chromatography-tandem mass spectrometry (UHPLC-q-TOF-MS/MS) 278

The mangiferin content determination and phytochemical profiling of ‘sugar MSK’ extracts 279

obtained during the PLE- second step were studied using an Agilent 1290 UHPLC system 280

(Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 6540 quadrupole-time-281

of-flight mass spectrometer (q-TOF MS). A Zorbax Eclipse Plus C18 column (2.1 × 100 mm, 282

1.8 µm particle diameter, Agilent Technologies, Santa Clara, CA) was used for 283

chromatographic separation at 30 °C. The mobile phases were as follows: eluent A, H2O 284

(0.01% v/v formic acid), and eluent B, acetonitrile (0.01% v/v formic acid). The linear gradient 285

program was 0-30% B in 0-7 min, 30-80% B in 7-9 min, 80-100% B in 9-11 min, 100% B in 286

11-13 min and 0% B in 13-14 min at a flow rate of 0.5 ml/min and the sample injection volume 287

was 5 µL. The MS and MS/MS analyses were obtained in the negative ion mode using an 288

orthogonal ESI source (Agilent Jet Stream, AJS, Santa Clara, CA, USA). MS parameters were 289

the following: capillary voltage, 4000 V; nebulizer pressure, 40 psi; drying gas flow rate, 10 290

L/min; gas temperature, 350 ºC; skimmer voltage, 45 V; fragmentor voltage, 110 V. The MS 291

and Auto MS/MS modes were set to acquire m/z values ranging between 50-1100 and 50-800, 292

respectively, at a scan rate of 5 spectra per second. Agilent Mass Hunter Qualitative analysis 293

software (B.07.00) was used for post-acquisition data processing. The accurate mass data, 294

isotopic patterns, ion source fragmentation, MS/MS fragmentation patterns, MS databases (i.e., 295

HMDB, Metlin, MassBank) and bibliographic search were employed for tentative 296

identification of ‘sugar MSK’ phytochemicals. Quantitative data for mangiferin were obtained 297

by calibration curve constructed with the standard compound in the range of 0.1-100 µg/mL. 298

299

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3. RESULTS AND DISCUSSION 300

3.1 Theoretical selection of green solvents for phenolics recovery by HSP approach: 301

mangiferin as target compound 302

Using Y-MB method (HSPiP®), HPSs of mangiferin were obtained at room conditions (25 °C-303

1.01 bar) as can be seen in Table 1. The dispersive interaction parameter, D, for mangiferin 304

(20.3 MPa1/2) showed a higher influence on solubility parameters due to geometry (very 305

flattened boat conformation) and aromatic character of three-ring system (benzenoid-pyraoind-306

benzenoid) (Gales & Damas, 2005). On the other hand, polar (P), and hydrogen-bonding (H) 307

parameters were affected by high hydroxylation degree of the xanthone and the -D-308

glucopyranosyl moiety. These HSP data were used to predict the mangiferin solubility in green 309

solvents calculating the “distance” or Ra value (Table 1). Ra scores showed greater miscibility 310

of mangiferin with ethyl lactate (9.1 MPa1/2) in comparison with ethanol (11.5 MPa1/2), ethyl 311

acetate (11.8 MPa1/2) and (+)-limonene (13.6 MPa1/2). However, the physicochemical 312

properties of ethyl lactate (boiling point 154 °C at 1.01 bar) make difficult its evaporation to 313

obtaining dry extracts, thus limiting its application. For this reason, the HSPs of ethanol-ethyl 314

acetate mixtures were calculated in order to obtain a similar ethyl lactate affinity (Table 1). The 315

ethanol:ethyl acetate 50:50 v/v mixture showed a close distance to ethyl lactate respect to 316

mangiferin (Ra = 9.7 MPa1/2). This can be explained by the decrease of polar and hydrogen-317

bonding parameters of ethanol due to ethyl acetate addition. Therefore, ethanol, ethyl acetate 318

and their mixture (50:50 v/v) were preferred as extraction solvents for the PLE-second step 319

optimization process. The temperature effect on Ra is also presented in Table 1. As can be seen, 320

Ra value increases with the temperature showing a lower affinity for mangiferin, however this 321

can be different in practice, because HSP approach is based on thermodynamic data and the 322

kinetic phenomena, such as mass transfer and solubility increase due to temperature, are not 323

14

considered. These effects have been previously demonstrated for the extraction of natural 324

compounds from algae (Sánchez-Camargo, Montero, Cifuentes, Herrero, & Ibáñez, 2016). 325

326

3.2 Selection of solvent for the first step of the PLE procedure: defatting step 327

Kinetic experiments were performed to select the most suitable non-polar solvent for fat 328

extraction from ‘sugar MSK’. Four kinetic curves using n-heptane, cyclohexane, (+)-limonene 329

and n-hexane (reference solvent in PLE process) were obtained considering times from 10 to 330

90 min with sample collection every 10 min. In addition, B & D was employed for total lipid 331

recovery as standard method. Figure 2 shows the comparison of the B & D results and the 332

kinetic curves. As can been seen, B & D method and (+)-limonene presented close extraction 333

efficiency (16.01% and 15.32%, respectively). However, (+)-limonene exhibited a slow 334

extraction rate with a maximum accumulated recovery at 90 min, requiring large solvent- and 335

time-consuming, consequently (+)-limonene was discarded as solvent for this PLE-step. The 336

profiles of the n-heptane, n-hexane and cyclohexane kinetic curves showed that the extraction 337

yields increased rapidly in the first 10 min, reaching the equilibrium after 20 min of extraction. 338

The rapid increase of extraction yields in the initial stage is usually attributed to washing of 339

components located on the external surface of the matrix particles (Okiyama et al., 2018), in 340

this case the cellular lipid matrix from MSK. After this stage, the extraction rate decreases and 341

the lipophilic compounds are principally recovery from plastids in broken cells by a diffusion 342

process (Xi, Yan, & He, 2014). The n-heptane provided the extraction yield (13.82%) nearest 343

to B & D method followed by n-hexane (13.19%) and cyclohexane (8.83%). The performance 344

differences among n-heptane and B & D method can be explained by the high selectivity of 345

chloroform/methanol/water system for lipids recovery due to the partial miscibility of the 346

chloroform in the aqueous and organic phases, which ensures that neutral and polar lipids 347

independent of their molecular volume, are solubilized in the coexisting phases (Breil et al., 348

15

2017). In this sense, n-heptane can be limited to medium polar and non-polar lipids recovery. 349

In addition, n-heptane is considered as usable and alternative to more toxic non-polar solvents 350

such as n-hexane and petroleum ether (Calvo-Flores, Monteagudo-Arrebola, Dobado, & Isac-351

Garcia, 2018). For these reasons, n-heptane was selected as solvent for the PLE-first step and 352

20 min were considered as appropriated extraction time. 353

354

3.3 Optimization of the second step of the PLE procedure 355

The effects of solvent composition (percentage of EtOH in the mixture EtOH/EtOAc: 0, 50 and 356

100 % v/v) and temperature (50, 100 and 150 ºC) were investigated on mangiferin content, 357

extraction yield, TPC, TFC and antioxidant activity (EC50 and TEAC) to optimize the PLE-358

second step. CCD was used to study the best possible combination of independent extraction 359

parameters for each response. Table 2 shows the codified and real levels of independent 360

variables and the resulting responses. The experimental data were fitted to linear, interaction 361

and quadratic regression models. Polynomial equation coefficients were calculated through 362

response surface methodology (RSM) and are provided in Table S1 (Supplementary material). 363

The analysis of variance (ANOVA) was performed to confirm the adequacy, significance level 364

and predictive value of regression models (Table S1). The values from the lack-of-fit test were 365

not significant (p > 0.05), consequently the models fit to experimental data. Most of terms 366

showed high F-values and low p-values (<0.0001) indicating that the regression models were 367

significant. The coefficients of determination (R2) were close to 1 (0.756-0.933), indicating a 368

high degree of correlation between the experimental and predicted values of the models 369

(Briones-Labarca, Giovagnoli-Vicuna, & Canas-Sarazua, 2019). Pareto charts and surface 370

responses were plotted to display the influence of the extraction parameters on response 371

variables (See Figure 3). Mangiferin content was principally influenced by negative effect of 372

solvent composition followed by positive effect of temperature (Figure 3A). In this sense, the 373

16

highest mangiferin content (13.27 ± 1.67 mg mangiferin/g Db) was observed using 100% EtOH 374

at 150 °C, however this amount is equivalent to that obtained by DM using 50% EtOH (13.60 375

± 2.22 mg mangiferin/g Db) and 100% EtOH (12.34 ± 1.67 mg mangiferin/g Db) at 25 °C, 376

since no statistical significant differences between these values were observed (p > 0.05). These 377

results are consistent with HSP data prediction, given that mangiferin showed affinity by 378

EtOH:EtOAc mixture (50:50 v/v) and EtOH solvents. The mangiferin content from M. indica 379

organs, including seed kernel, has been an object of study in several investigations, due to the 380

high bioactivity of this compound. Mangiferin content in MSK varies depending on the 381

cultivars’ nature and its geographical origin, with values ranging between 0.22 and 8.98 mg 382

mangiferin/g Db (Barreto et al., 2008; Lopez-Cobo et al., 2017; Luo et al., 2012; Ruales et al., 383

2018). Solid-liquid extraction at room conditions or ultrasound-assisted extraction using 384

methanol and methanol-water mixtures have been the most employed extraction techniques for 385

mangiferin recovery from MSK. 386

On the other hand, extraction yield was mainly affected by both solvent composition and 387

temperature, with a minor contribution of the interaction and the quadratic effects (Figure 3B). 388

Thus, the highest extraction yield (12.15 ± 0.90%) was obtained employing 150 ºC and 100 % 389

EtOH. The TPC and TFC responses showed a very similar behaviour according to Pareto charts 390

(Figures 3C and 3D, respectively). In both cases, TPC and TFC were influenced by linear effect 391

of temperature and by negative and quadratic effect of solvent composition. As for TPC and 392

TFC, the best results (143.79 ± 2.09 mg GAE/g Db and 1.21 ± 0.16 mg QE/g Db and, 393

respectively) were obtained using 100% EtOH and 150 °C. Comparatively, TPC values were 394

within the ranges reported in previous studies (2.19-740 mg GAE/g Db), in contrast TFC results 395

were higher than those previously reported (0.72-1.31 g (+)-catechin 100/g Db) for MSK 396

(Torres-León et al., 2016). TFC obtained by DM at 25 °C and 50 % EtOH was higher (3.72 ± 397

0.08 mg QE/g Db) than the obtained by PLE extractions. This difference can be explained by 398

17

the low temperatures employed in DM that ensure the thermolabile flavonoids recovery, as 399

well as by the longer extraction time (24 h), which allows higher contact between the sample 400

and the solvent. 401

EC50 response was influenced mainly by negative and quadratic effect of solvent composition 402

and by the negative and linear effect of temperature (Figure 3E), obtaining the highest free 403

radical scavenging capacity (14.34 ± 0.19 µg/mL) employing 100% EtOH and 100 °C. As for 404

TEAC the principal effect was the temperature followed by solvent composition (Figure 3F) 405

and the best value (2.31 mM trolox/g) was observed using 100% EtOH at 150 ºC. 406

Comparatively, the ‘sugar MSK’ extracts had a moderate antioxidant capacity respect to the 407

one reported previously for other MSK cultivars: 0.56-14.00 µg/mL and 0.44-1.03 mmol 408

trolox/g, for EC50 and TEAC, respectively (Luo et al., 2014; Maisuthisakul & Gordon, 2009). 409

Due to the notably high mangiferin and phenolic contents as well as the moderate antioxidant 410

power of ‘sugar MSK’, PLE extraction was optimized for all responses studied. To attain this, 411

desirability function combining mangiferin content, extraction yield, TPC, TFC, EC50 and 412

TEAC responses was calculated. Profiles for predicted values estimated by desirability 413

function are shown in Figure 4. Optimal conditions were 100% EtOH v/v and 150 °C at 0.92 414

desirability value, corresponding to experimental run number 10 of the CCD. The desirability 415

value was very close to 1, indicating a high maximisation degree for multi-response 416

optimization. Predicted response values obtained by global desirability function under 417

optimum conditions were checked with those of experiment 10. The observed and predicted 418

data were within the confidence intervals (Figure 4). Despite the optimum conditions were at 419

the experimental region limit, the proximity between predictive and experimental data 420

confirmed that selected RSM model was successfully applied for PLE of ‘sugar MSK’ to obtain 421

extracts with maximum mangiferin content, phenolic content and antioxidant activity. 422

423

18

3.4 UHPLC-q-TOF-MS/MS profiling analysis of ‘sugar MSK’ extracts 424

As a result of the phytochemical profiling of MSK polar fractions obtained by PLE, a total of 425

71 compounds were tentatively identified on the basis of their accurate mass, MS/MS 426

fragmentation patterns, MS databases (i.e., HMDB, Metlin, MassBank) and previously 427

reported data in literature. Table 3 summarizes the phytoconstituents identified by ESI-q-TOF-428

MS/MS analysis in negative ionization mode, including the retention time (min), molecular 429

formula, experimental deprotonated molecular ions ([M-H]-), calculated mass error (ppm), and 430

MS/MS product ions. 431

Although the composition of the edible part of mango and some by-products such as peel, seed 432

husk and seed kernel has been described in literature for several cultivars, information about 433

the composition of mango seed kernel for ‘sugar mango’ cultivar is limited. In this regard, 434

typical phenolic acid and flavonoids, along with characteristic xanthones and benzophenone 435

derivatives were identified in sugar MSK, as reported in literature for other mango cultivars. 436

The intense peak observed in the TIC at 2.0 min (see Figure 5A), confidently identified as 437

gallic acid (m/z 169.0142 [M−H]−), was one of the main compounds in the phytochemical 438

profile of the polar PLE extracts. The widespread presence of this phenolic acid in the seed 439

kernel of sugar mango is evidenced by the broad variety of gallic acid derivatives identified in 440

sugar MSK extracts (see Table 3). The presence of other relevant phenolic acids such as quinic 441

acid (compound 1, m/z 191.0561), protocatechuic acid (compound 11, m/z 153.0193), p-442

hydroxybenzoic acid (compound 19, m/z 137.0244), ferulic acid (compound 47, m/z 193.0506) 443

and ellagic acid (compound 52, m/z 300.9990) could also be confirmed by commercial 444

standards. 445

Gallates and gallotannins were the main family of compounds identified in the polar MSK 446

extracts (see Table 3 and Figure 5E). The presence of gallic acid derivatives can be identified 447

by MS/MS product ions at m/z 169.0142 and m/z 125.0239 or 124.0160. According to this 448

19

fragmentation pattern, the methyl and ethyl esters of gallic acid were identified at m/z 183.0299 449

(compound 24) and m/z 197.0455 (compound 43), respectively, being ethylgallate the most 450

abundant compound in the analysed extract. Digallic acid (compound 29), galloyl 451

methylgallate (compound 59), galloyl ethylgallate (compound 67), and ethyl trigallate 452

(compound 70) could be identify due to the loss of the galloyl moiety (C7H4O4, 152.0110 Da). 453

Two minor galloyl derivatives (compound 7 and 9, m/z 343.0671) were assigned as 454

galloylquinic acid isomers, showing fragment ions at m/z 169 and 127, as representative 455

diagnostic ions of both galloyl and quinic acids, respectively. 456

Gallotannins represent the main family of compounds identified in polar MSK extracts. Since 457

the composition of these hydrolysable tannins is based on a core structure of glucose esterified 458

with gallic acid residues, the MS/MS fragmentation pattern of these polyphenolic biopolimers 459

is mainly characterized by the successive loss of galloyl (Gall, 152.0110 Da) and glucose (Glu, 460

162.0529 Da) subunits. Thus, a set of gallotannin isomers containing up to 6 galloyl subunits 461

were identified. Galloyl glucose (compound 3), the simplest isomer, shows [M-H]- ion at m/z 462

331.0671, exhibiting m/z 169.0142 [M−H−Glu]− as the major product ion. The identity of 463

deprotonated molecular ions m/z 483.0780 (compounds 13, 17, 26, 27), m/z 635.0890 464

(compounds 23, 34, 35, 38, 39, 42, 44), m/z 787.1000 (compounds 45, 51, 56, 60, 63), m/z 465

939.1109 (compound 64) and m/z 1091.1219 (compound 71) could be confidently assigned to 466

digalloyl-, trigalloyl-, tetragalloy-, pentagalloyl- and hexagalloyl glucose isomers, 467

respectively, with Δm/z below 5 ppm, as determined by HRMS. 468

Gallotannins identified at m/z 493.1199 (Compounds 2, 4, 6 and 10) and m/z 645.1309 469

(Compounds 8, 14, 15, 18, 20, 22, 28, 32) share product ions at m/z 331 [493−Glu]− and m/z 470

169 [331−Glu]−, suggesting the presence of two glucose subunits. These compounds were 471

tentatively assigned as galloyl diglucoside and digalloyl diglucose isomers, respectively. Other 472

group of gallotanins with [M-H] − ion at m/z 493.0991 (compounds 49, 62, 55, 65, 66, 68) were 473

20

tentatively identified as feruloyl galloyl glucose isomers. This assignation is supported by 474

characteristic product ions at m/z 295.0450 [M–H–Gall–CH2O2]− and m/z 169.0142 [M–H–475

Glu–Feruloyl]−. 476

Unlike in mango peel, where a wide variety of quercetin and rhamnetin derivatives have been 477

reported (Gomez-Caravaca, Lopez-Cobo, Verardo, Segura-Carretero, & Fernandez-Gutierrez, 478

2016), the content of flavonoids in sugar MSK is mainly based on catechin and epicatechin 479

(compounds 31 and 37, m/z 289.0718 [M−H]−), as well as on (epi)catechin gallate (compound 480

57, m/z 289.0718). In addition, quercetin and quercetin glucoside (compounds 69, m/z 481

289.0718) and 54, m/z 289.0718) were also present, although in minor extent. The identity of 482

common flavonoids such as (epi)catechin and quercetin was confirmed by commercial 483

standard. The glycosylated and galloylated derivatives were identified based on the 162.0528 484

(C6H10O5) and 152.0110 (C7H4O4) Da neutral loss in the MS/MS spectrum, respectively. 485

Seven xanthone-like structures were detected in MSK extracts. Mangiferin (m/z 421.0776, 486

compound 36) was the most abundant xanthone, being one of the most relevant phytochemicals 487

reported in mango. The presence of this xanthone C-glycoside in MSK was confirmed by 488

commercial standards and by typical MS/MS fragment ions at m/z 301.0361 and m/z 331.0468. 489

The same diagnostic ions were observed for compounds 50 and 61 (m/z 421.0776 [M−H]−), 490

tentatively assigned as mangiferin isomers. Similar fragmentation pattern was shown for 491

compounds 46 (m/z 573.0886 [M−H]−), readily identified as mangiferin gallate with a major 492

product ion at m/z 403 [M−H−170]−, indicating the neutral loss of gallic acid. An extra methyl 493

group was observed in MS/MS spectra of compound 41, tentatively identified as O-methyl 494

mangiferin, also called as homomangiferin. 495

The presence of benzophenones, major intermediates in the biosynthetic pathway of xanthones, 496

could also be confirmed in MSK polar extracts. Compounds 16, 30 and 40, were tentatively 497

identified as maclurin-C-glucoside derivatives. These phytochemicals contain a 2,3',4,4',6-498

21

pentahydroxybenzophenone as core structure, sharing the typical fragmentation pattern 499

characterized by the loss of 120 and 90 Da neutral fragments corresponding to C-glycosilated 500

derivatives. Similar neutral loses were observed for compound 24, tentatively identified as 501

iriflophenone glucoside, a C-glycosylated tetrahydroxybenzophenone. Typical fragment ions 502

at m/z 333, 303, and 193 were consistent with data reported in literature (Dorta et al., 2014). 503

Hexahydroxybenzophenone isomers (peaks 21, 48 and 53) were also detected at m/z 421.0776 504

[M−H]−, exhibiting m/z 125.0239 [C6H5O3]− as the main product ion after the loss of a the 505

galloyl moiety (152 Da). 506

507

3.5 GC-q-TOF-MS profiling analysis of ‘sugar MSK’ extracts 508

The non-polar PLE extracts obtained from MSK were analysed by GC-q-TOF-MS to 509

characterize the main lipidic components. A derivatization procedure described section 2.10.1 510

was applied in order to improve detectability of fatty acids (FAs) and other lipids (e.g. 511

phytosterols), leading to the identification of the corresponding trimethylsilyl derivatives. 512

Table 4 summarizes the tentatively identified metabolites, including their corresponding 513

characteristic GC-HRMS parameters (e.g. retention time, match factor values given by NIST 514

database, monoisotopic mass, calculated mass error (∆m/z) and main HR-MS/MS fragments), 515

that confirm their unambiguous identification. As shown in Table 4, five major fatty acids 516

including palmitic acid, linoleic acid, oleic acid, stearic acid, and eicosapentaenoic acid were 517

positively identified as trimethylsilyl derivatives, showing the characteristic [M-H+TMS-518

CH3]+ ions GC-(EI+)-MS spectra. Unlike fatty acids, the terpenoid β-sitosterol could be 519

clearly detected by the [M-H+TMS]+ ion of the terpenoid-TMS derivatives. 520

Figure 6 illustrates the lipidic profile of MSK extracts, showing palmitic acid, oleic acid and 521

stearic as the most abundant compounds. The presence of stearic acid and oleic acid as major 522

fatty acids in non-polar MSK extracts is consistent with data reported in literature (Lieb et al., 523

22

2018). The predominance of these fatty acids provides more stability compare to other oils rich 524

in polyunsaturated fatty acids. Several studies report mango seed kernel fat with typical 525

characteristics of a vegetable butter, and these oils are suitable for mixing together with 526

vegetable oils for use in the confectionery industry. The absence of “trans” fatty acids is another 527

advantage of the lipids from mango seed, as they are responsible for the development of various 528

diseases and adverse effects on human health (Torres-León et al., 2016). 529

530

3.6 Antiproliferative activity 531

HT-29 cell line is considered as paradigm of the colon carcinogenesis and is one the most 532

refractory colon cancer line against the antiproliferative activity of natural compounds (Castro-533

Puyana et al., 2017; Fearon & Vogelstein, 1990; Valdes et al., 2013). For these reasons and 534

considering the mangiferin content, phenolic content and antioxidant capacity of the PLE-535

extract obtained from ‘sugar MSK’ under optimum conditions (100% EtOH-150 °C), its 536

antiproliferative activity was also tested on HT-29. Thus, HT-29 cells were incubated with 537

different concentrations of the optimum-PLE extract (from 6.25 to 100 μg/mL) during 24, 48 538

and 72 h, and cell proliferation was measured by the MTT assay. Comparatively, 539

antiproliferative activity of the DM extract (100% EtOH at 25 °C) was studied under the same 540

conditions. In addition, the PLE-extract was also tested on CCD-18Co cell line to determine 541

its potential toxicity in non-cancer colon cells. Antiproliferative activity was expressed as IC50 542

value and the results are shown in Figure 7. As can be seen, the viability of HT-29 cells was 543

reduced in response to treatment with PLE-extract after 48 and 72 h of exposition (IC50 = 56.37 544

± 3.45 and 28.67 ± 5.35 μg/mL, respectively). PLE-extract was most active at 72 h of treatment, 545

decreasing the cell viability in a dose-dependent manner. In contrast, DM extract did not induce 546

any cytotoxic effect at the concentrations and times tested. On the other hand, PLE-extract did 547

not exert inhibition on CCD-18Co cell proliferation at 48 h, but it showed an antiproliferative 548

23

effect at 72 h (IC50 = 85.19 ± 5.26 μg/mL). According to these results, the selectivity index (SI) 549

of PLE-extract on HT-29 respect to CCD-18Co cells was calculated, revealing a value of 2.97 550

(Figure 7). According to (Badisa et al., 2009) a compound with SI value > 2 exhibits selective 551

toxicity toward cancer cells but gives minimal toxicity or no harm to normal cells, while a 552

compound with SI value < 2 is considered toxic even to normal cells. This approach has also 553

been applied to establish the selectivity degree of extracts from vegetable sources towards 554

cancer cells (Asif et al., 2017; Asif et al., 2016); following this criteria, it is possible to state 555

that PLE-extract has selectivity toward HT-29 cells. 556

In a recent study, the antiproliferative potential of a methanolic extract from ‘sugar MSK’ 557

obtained using Soxhlet was evaluated against a panel of human cancer cell lines that included 558

MDA-MB-231 (breast adenocarcinoma), PC-3 (prostate adenocarcinoma), A-549 (lung 559

adenocarcinoma) and HT-29 (Castro-Vargas et al., 2019). Results showed a decrease of HT-560

29 cells viability (~75%) at 125 µg/mL of methanolic extract and the authors related the 561

phenolic composition with its antiproliferative activity. This antiproliferative potential was 562

comparatively lower than the present study and this difference can be explained by the 563

extraction technique employed in each case, since unlike the Soxhlet method, the PLE can 564

more efficiently concentrate the polyphenolic compounds responsible for the antiproliferative 565

properties of the extract. 566

The antioxidant, antiproliferative and chemopreventive properties of polyphenolics and other 567

compounds identified in ‘sugar MSK’ PLE-extract have been described previously in the 568

scientific literature. Mangiferin is one of the most important compounds from Anacardeciae 569

and its bioactivity has been reported in several review works (Adam, Piotr, Edyta, & Dorota, 570

2013; Imran et al., 2017; Khurana, Kaur, Lohan, Singh, & Singh, 2016; Rajendran, Rengarajan, 571

Nandakumar, Divya, & Nishigaki, 2015). According to (Gold-Smith, Fernandez, & Bishop, 572

2016) mangiferin is involved in different molecular mechanisms, including cell protection 573

24

against oxidative stress and DNA damage, as well as down-regulation of inflammation, cell 574

cycle arrest, apoptosis promotion and proliferation reduction of malignant cells. Overall, 575

previous studies showed that the anticancer effect of mangiferin is more pronounced when used 576

as a chemopreventive agent against induced colon carcinogenesis (Khurana et al., 2016). 577

However, the well-known antioxidant power of mangiferin and its capacity to induce apoptosis 578

through inhibition of NF-κB activation in different cancer cell lines (Gold-Smith et al., 2016), 579

allows thinking that this compound contributes to the antiproliferative activity of PLE-extract 580

observed on HT-29 cells. 581

Gallic acid was also identified in the PLE-extract and its antiproliferative activity has been 582

previously reported both in vitro and in vivo models (Verma, Singh, & Mishra, 2013). The 583

antiproliferative effects of gallic acid are mediated via gene modulation of cell cycle, 584

metastasis, angiogenesis and apoptosis, as well as by the inhibition of NF-κB and Akt 585

activation. In the cancer colon cell lines HT-29 (Verma et al., 2013), LS180 (Velderrain-586

Rodriguez et al., 2018) and Caco-2 (Salucci, Stivala, Bugianesi, & Vannini, 2002) the 587

antiproliferative activity of gallic acid has been related with apoptosis and antioxidant 588

mechanisms. 589

Gallic acid structurally related compounds such as gallates and galloyl glycosides are also 590

present in the PLE-extract. Ethyl gallate is the most abundant compound in the extract and its 591

anticancer activity against different cancer cell lines through induction of apoptosis has been 592

established (Kim et al., 2012; Mohan, Thiagarajan, & Chandrasekaran, 2014). Likewise, 593

galloyl glycosides may regulate the production of reactive oxygen species by altering the redox 594

balance in the cell, which activates the intrinsic mitochondrial apoptosis pathway (Banerjee, 595

Kim, Krenek, Talcott, & Mertens-Talcott, 2015). Ellagic acid was another bioactive compound 596

found in the optimum PLE-extract. The antiproliferative activity of ellagic acid has been 597

studied in the cancer colon cell lines HTC116 and Caco-2, whose cell viability was altered by 598

25

cell cycle modulation, Bax translocation, caspase-8 activation and PCNA expression reduction 599

(Yousef, El-Masry, & Yassin, 2016). 600

The antiproliferative activity observed for PLE-extract could not be attributed to a single 601

component but to the possible synergistic effect of some of the compounds present in the 602

extract. In this respect, previous studies have shown the synergic behavior of various 603

polyphenolics for antioxidant and antiproliferative activities. García-Rivera et al. (García-604

Rivera, Delgado, Bougarne, Haegeman, & Berghe, 2011) observed significant antiproliferative 605

effects with the Vimang® (a standardized extract derived from mango bark) compounds 606

mangiferin and gallic acid, against the MDA-MB-231, HT-1080 and Caco-2 cancer cells and 607

upon measuring cytotoxic activities, the authors found that Vimang® and gallic acid, but not 608

mangiferin, were able to kill MDA-MB-231 cells, suggesting that minor amounts of gallic acid 609

in Vimang® are sufficient to trigger significant antiproliferative effects. Likewise, 610

hydroxybenzoic acids and hydroxycinnamic acids have a potential inhibitory effect on cancer 611

cells proliferation by synergistic interactions arresting cell cycle and inducing apoptosis 612

(Rocha, Monteiro, & Teodoro, 2012). In this way, the potentiated effects by polyphenolic 613

compounds’ combination in the reduction of cancer cell viability and apoptosis induction has 614

been confirmed by isobolographic analysis of cell proliferation data for ellagic acid and 615

quercetin (Mertens-Talcott, Talcott, & Percival, 2003). 616

617

4. CONCLUSIONS 618

In this work, an integrated valorization strategy was proposed through an optimized PLE 619

procedure in two sequential steps to obtain bioactive-rich fractions with high antioxidant 620

capacity and demonstrated antiproliferative activity. The lipidic fraction was firstly recovered 621

with n-heptane, whereas a mixture of ethanol/ethyl acetate was selected as a suitable green 622

extraction solvent for the subsequent recovery of mangiferin and other phenolic compounds, 623

26

on the basis of preliminary studies applying the HSP approach. Phenolic extracts obtained 624

under optimal PLE conditions after RSM optimization showed satisfactory extraction yield and 625

good antioxidant activity with notably high mangiferin and phenolics concentration levels. The 626

profiling analysis of the lipidic and phenolic MSK fractions by GC and UHPLC coupled to q-627

TOF-MS/MS revealed the presence of abundant oleic and stearic acids, as well as typical 628

phenolic acids, flavonoids, characteristic xanthones, as well as a broad family of gallate 629

derivatives and gallotannins with demonstrated in vitro bioactivity, as evidenced by the 630

selective antiproliferative activity exhibited against human colon adenocarcinoma cell line HT-631

29. The proposed valorization strategy represents a powerful multiplatform of integrated 632

analytical technologies to improve the sustainability of mango processing industry. 633

634

Acknowledgements 635

This research was supported by COOPA20145, project from CSIC (Programa de Cooperación 636

Científica para el Desarrollo “i-COOP+”). G.A.-R. would like to acknowledge Ministerio de 637

Ciencia Investigacion y Universidades (MICINN) for a “Juan de la Cierva” postdoctoral grant. 638

The authors also thank the support from the AGL2017-89417-R project (MICINN). 639

27

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860

861

862

863

864

865

866

867

868

869

870

871

872

873

874

875

876

877

878

879

880

881

37

Figure captions 882

Figure 1. Workflow of the proposed mango seed kernel (MSK) valorization strategy. 883

Figure 2. Kinetic behaviour of the extraction yield employing different solvents during the 884

defatting step. PLE extractions performed at 100 °C and 100 bar. 885

Figure 3. Standardized Pareto charts for the response variables studied and their 886

corresponding response surfaces. 887

Figure 4. Desirability value and predicted response variables in multi-response optimization. 888

Figure 5. TIC (A) and HREICs (B-E), corresponding to the phenolic fraction of MSK extracts 889

analysed by UHPLC-ESI(-)-q-TOF-MS/MS. (B) Mangiferin isomers; (C-D) Other phenolic 890

compounds; (E) Gallates and gallotannins. 891

Figure 6. GC-q-TOF(MS) profile of the non-polar fraction of MSK extracts obtained by 892

developed PLE procedure. 893

Figure 7. IC50 values and percentage of growth of HT-29 and CCD-18Co cells incubated for 894

(A) 48 and (B) 72 h, with different concentrations of ‘sugar MSK’ DM- and PLE-extracts. SI 895

of PLE-extract was expressed as

ratio. (*) indicates significant differences 896

between the treated and control samples, p < 0.05. Error bars represent standard error of the 897

mean. 898

899

900

901

902

903

904

905

906

38

Table 1. Hansen Solubility Parameters and distance for mangiferin and green solvents at different temperatures

Compound/Solvent T (°C) Ra*

(MPa1/2) (MPa1/2) (MPa1/2) (MPa1/2)

Mangiferin

25 20.3 10.7 12.5 0 50 20.2 10.6 12.4 0 100 19.9 10.5 12.2 0 150 19.6 10.3 12 0

Ethyl lactate

25 16 7.6 12.5 9.1 50 15 7.4 11.8 10.9 100 13 7 10.4 14.9 150 11.2 6.6 9.2 18.6

Ethanol

25 15.8 8.8 19.4 11.5 50 14.7 8.5 18.2 12.6 100 12.6 8 16 15.2 150 10.5 7.5 14 18.4

Ethyl acetate

25 15.8 5.3 7.2 11.8 50 14.9 5.2 6.8 13.1 100 13.1 4.9 6.1 15.9 150 11.3 4.6 5.3 18.8

(+)-Limonene

25 17.2 1.8 4.3 13.6 50 16.4 1.8 4.1 14.3 100 14.9 1.7 3.7 15.7 150 13.5 1.6 3.3 17.2

Ethanol:ethyl acetate 50:50 v/v

25 15.8 7.1 13.3 9.7

50 14.8 6.9 12.5 11.4

100 12.8 6.5 11 14.6

150 10.9 6.1 9.7 18 *Distance respect to mangiferin

39

Table 2. Experimental design conditions (experiments 1 to 10) and results for each response variable studied for the optimization of the PLE-second step and macerations (experiments I, II and III) of ‘sugar MSK’.

ExperimentCodified variables Real variables

Mangiferin content

Extraction yield

TPC TFC EC50 TEAC

%EtOHTemperature

(°C) %EtOH

Temperature(°C)

(mg/g) (%) (mg GAE/g) (mg QE/g) (g/mL) (mM trolox/g)

1 -1 -1 0 50 0.48 ± 0.05 0.33 ± 0.01 5.05 ± 0.75 0.08 ± 0.01 42.51 ± 1.90 0.03 ± 0.01

2 -1 0 0 100 1.41 ± 0.33 0.89 ± 0.11 11.17 ± 0.72 0.14 ± 0.01 29.10 ± 0.16 0.10 ± 0.02

3 -1 +1 0 150 0.79 ± 0.16 2.87 ± 0.36 23.98 ± 1.78 0.60 ± 0.06 31.61 ± 1.55 1.25 ± 0.10

4 0 -1 50 50 3.44 ± 0.43 2.81 ± 0.23 39.40 ± 0.58 0.35 ± 0.04 48.56 ± 1.51 0.06 ± 0.02

5, 6 (CPa) 0 0 50 100 10.51 ± 0.55 8.19 ± 0.22 94.86 ± 3.38 1.10 ± 0.11 40.53 ± 2.59 1.74 ± 0.21

7 0 +1 50 150 10.60 ± 0.40 9.54 ± 0.50 93.42 ± 0.04 1.40 ± 0.13 30.01 ± 0.30 1.23 ± 0.31

8 +1 -1 100 50 3.00 ± 0.26 2.31 ± 0.01 31.12 ± 0.98 0.20 ± 0.01 15.89 ± 1.07 0.75 ± 0.15

9 +1 0 100 100 10.06 ± 0.63 8.93 ± 0.10 88.64 ± 1.70 0.74 ± 0.03 14.34 ± 0.19 1.41 ± 0.37

10 +1 +1 100 150 13.27 ± 1.67 12.15 ± 0.90 143.79 ± 2.09 1.21 ± 0.16 17.59 ± 1.19 2.31 ± 0.26

I 0 25 0.13 ± 0.06 9.24 ± 1.06 3.69 ± 0.30 2.75 ± 0.22 78.06 ± 2.65 0.21 ± 0.24

II 50 25 13.60 ± 2.22 11.96 ± 0.27 14.06 ± 0.32 3.72 ± 0.08 61.07 ± 0.72 0.05 ± 0.01

III 100 25 12.34 ± 0.37 6.82 ± 0.12 26.00 ± 1.75 1.61 ± 0.08 66.99 ± 1.02 0.08 ± 0.02 a Central point of the experimental design

Values presented are mean ± sd

40

Table 3. Tentatively identified compounds from ‘sugar MSK’ extract by LC-q-TOF-MS/MS analysis.

Peak No

Ret. Time (min)

Family Tentative identif. Formula [M-H]- (m/z)

(measured)

[M-H]- (m/z)

(theoretical)

Error (ppm)

MS2 product ions (-) (m/z)

1 0.652 Phenolic acid Quinic acid C7H12O6 191.0570 191.0561 -4.6 127, 93, 85 2 0.825 Gallotannin Galloyl diglucoside isomer 1 C19H26O15 493.1200 493.1199 -0.2 313, 169, 125 3 1.737 Gallotannin Galloyl glucose isomer C13H16O10 331.0680 331.0671 -2.8 271, 241, 169, 125 4 1.997 Gallotannin Galloyl diglucoside isomer 2 C19H26O15 493.1200 493.1199 -0.2 313, 169, 125 5 2.090 Phenolic acid Gallic acid * C7H6O5 169.0139 169.0142 2.1 125, 79 6 2.214 Gallotannin Galloyl diglucoside isomer 3 C19H26O15 493.1200 493.1199 -0.2 313, 169, 125 7 2.301 Gallate Galloylquinic acid isomer 1 C14H16O10 343.0680 343.0671 -2.7 127, 169 8 2.691 Gallotannin Digalloyl diglucoside isomer 1 C26H30O19 645.1315 645.1309 -1.0 465, 313, 169 9 2.765 Gallate Galloylquinic acid isomer 2 C14H16O10 343.0680 343.0671 -2.7 127, 169

10 2.865 Gallotannin Galloyl diglucoside isomer 4 C19H26O15 493.1200 493.1199 -0.2 313, 169, 125 11 3.219 Phenolic acid Protocatechuic acid * C7H6O4 153.0197 153.0193 -2.4 109, 91 13 3.299 Gallotannin Digalloyl glucose isomer 1 C20H20O14 483.0790 483.0780 -2.0 331, 313, 169, 125 14 3.299 Gallotannin Digalloyl diglucoside isomer 2 C26H30O19 645.1315 645.1309 -1.0 465, 313, 169 15 3.559 Gallotannin Digalloyl diglucoside isomer 3 C26H30O19 645.1315 645.1309 -1.0 465, 313, 169 16 3.776 Benzophenone Maclurin-C-glucoside C19H20O11 423.0937 423.0933 -1.0 193, 303 17 3.907 Gallotannin Digalloyl glucose isomer 2 C20H20O14 483.0790 483.0780 -2.0 331, 313, 169, 125 18 3.907 Gallotannin Digalloyl diglucoside isomer 4 C26H30O19 645.1315 645.1309 -1.0 465, 313, 169 19 4.173 Phenolic acid p-Hydroxybenzoic acid * C7H6O3 137.0246 137.0244 -1.3 93 20 4.254 Gallotannin Digalloyl diglucoside isomer 5 C26H30O19 645.1315 645.1309 -1.0 465, 313, 169

21 4.297 Benzophenone Hexahydroxylated benzophenone isomer 1

C13H10O7 277.0356 277.0354 -0.8 125

22 4.514 Gallotannin Digalloyl diglucoside isomer 6 C26H30O19 645.1315 645.1309 -1.0 465, 313, 169

23 4.557 Gallotannin Trigalloyl glucose isomer 1 C27H24O18 635.0907 635.0890 -2.7 483, 465, 423, 313, 295, 169, 125

24 4.644 Benzophenone Iriflophenone glucoside C19H20O10 407.0991 407.0984 -1.8 317, 287 25 4.688 Gallate Methylgallate C8H8O5 183.0300 183.0299 -0.6 168, 124 26 4.688 Gallotannin Digalloyl glucose isomer 3 C20H20O14 483.0790 483.0780 -2.0 331, 313, 169, 125 27 4.861 Gallotannin Digalloyl glucose isomer 4 C20H20O14 483.0790 483.0780 -2.0 331, 313, 169, 125

41

28 4.861 Gallotannin Digalloyl diglucoside isomer 7 C26H30O19 645.1315 645.1309 -1.0 465, 313, 169 29 4.948 Gallate Digallic acid C14H10O9 321.0259 321.0252 -2.2 169, 125 30 4.948 Benzophenone Maclurin-C-(O-galloyl)-glucoside C26H24O15 575.1047 575.1042 -0.8 193, 303, 333 31 4.999 Flavonoid Catechin * C15H14O6 289.0720 289.0718 -0.8 245, 203, 109 32 5.035 Gallotannin Digalloyl diglucoside isomer 8 C26H30O19 645.1315 645.1309 -1.0 465, 313, 169

34 5.686 Gallotannin Trigalloyl glucose isomer 2 C27H24O18 635.0907 635.0890 -2.7 483, 465, 423, 313, 295, 169, 125

35 5.859 Gallotannin Trigalloyl glucose isomer 3 C27H24O18 635.0907 635.0890 -2.7 483, 465, 423, 313, 295, 169, 125

36 3.000 Xanthone Mangiferin* C19H18O11 421.0787 421.0776 -2.5 403, 331, 301, 259 37 6.033 Flavonoid Epicatechin C15H14O6 289.0720 289.0718 -0.8 245, 203, 109

38 6.120 Gallotannin Trigalloyl glucose isomer 4 C27H24O18 635.0907 635.0890 -2.7

483, 465, 423, 313, 295, 169, 125

39 6.337 Gallotannin Trigalloyl glucose isomer 5 C27H24O18 635.0907 635.0890 -2.7 483, 465, 423, 313, 295, 169, 125

40 6.337 Benzophenone Maclurin-C-(O-digalloyl)-glucoside C33H28O19 727.1157 727.1152 -0.7 169, 303, 575 41 6.423 Xanthone Homomangiferin C20H20O11 435.0940 435.0933 -1.6 345, 315, 285, 272

42 6.467 Gallotannin Trigalloyl glucose isomer 6 C27H24O18 635.0907 635.0890 -2.7 483, 465, 423, 313, 295, 169, 125

43 6.684 Gallate Ethyl gallate C9H10O5 197.0471 197.0455 -7.9 169, 124

44 6.771 Gallotannin Trigalloyl glucose isomer 7 C27H24O18 635.0907 635.0890 -2.7 483, 465, 423, 313, 295, 169, 125

45 6.901 Gallotannin Tetragalloyl glucose isomer 1 C34H28O22 787.1019 787.1000 -2.5 617, 465, 295, 169, 125 46 6.944 Xanthone Mangiferin gallate C26H22O15 573.0888 573.0886 -0.4 403, 331, 301 47 7.344 Phenolic acid Ferulic acid * C10H10O4 193.0506 193.0506 0.2 178, 134

48 7.378 Benzophenone Hexahydroxylated benzophenone isomer 2

C13H10O7 277.0356 277.0354 -0.8 125

49 7.465 Gallotannin Feruloyl galloyl glucose isomer 1 C22H22O13 493.0991 493.0988 -0.7 323, 295, 169, 125 50 7.508 Xanthone Mangiferin isomer 1 C19H18O11 421.0787 421.0776 -2.5 403, 331, 301, 259 51 7.638 Gallotannin Tetragalloyl glucose isomer 2 C34H28O22 787.1019 787.1000 -2.5 617, 465, 295, 169, 125 52 7.725 Phenolic acid Ellagic acid C14H6O8 300.9990 300.9990 0.0 229, 145

53 7.812 Benzophenone Hexahydroxylated benzophenone isomer 3

C13H10O7 277.0356 277.0354 -0.8 168, 124

54 7.812 Flavonoid Quercetin glucoside C21H20O12 463.0884 463.0882 -0.4 301

42

55 7.899 Gallotannin Feruloyl galloyl glucose isomer 3 C22H22O13 493.0991 493.0988 -0.7 323, 295, 169, 125 56 7.942 Gallotannin Tetragalloyl glucose isomer 3 C34H28O22 787.1019 787.1000 -2.5 617, 465, 295, 169, 125 57 8.029 Flavonoid (Epi)catechin gallate C22H18O10 441.0830 441.0827 -0.6 289, 169 59 8.159 Gallate Galloyl methylgallate C15H12O9 335.0410 335.0409 -0.4 183, 124 60 8.246 Gallotannin Tetragalloyl glucose isomer 4 C34H28O22 787.1019 787.1000 -2.5 617, 465, 295, 169, 125 61 8.333 Xanthone Mangiferin isomer 2 C19H18O11 421.0787 421.0776 -2.5 403, 331, 301, 259 62 8.376 Gallotannin Feruloyl galloyl glucose isomer 2 C22H22O13 493.0991 493.0988 -0.7 323, 295, 169, 125 63 8.463 Gallotannin Tetragalloyl glucose isomer 5 C34H28O22 787.1019 787.1000 -2.5 617, 465, 295, 169, 125 64 8.506 Gallotannin Pentagalloyl glucose C41H32O26 939.1120 939.1109 -1.2 787, 768, 617, 465, 169 65 8.767 Gallotannin Feruloyl galloyl glucose isomer 4 C22H22O13 493.0991 493.0988 -0.7 323, 295, 169, 125 66 8.940 Gallotannin Feruloyl galloyl glucose isomer 5 C22H22O13 493.0991 493.0988 -0.7 323, 295, 169, 125 67 9.070 Gallate Galloyl ethylgallate C16H14O9 349.0573 349.0565 -2.3 197, 169, 124 68 10.290 Gallotannin Feruloyl galloyl glucose isomer 6 C22H22O13 493.0991 493.0988 -0.7 323, 295, 169, 125 69 11.126 Flavonoid Quercetin * C15H10O7 301.0353 301.0354 0.3 178, 151 70 11.500 Gallate Ethyl trigallate C23H18O13 501.0681 501.0675 -1.3 359, 197 71 12.010 Gallotannin Hexagalloyl glucose C48H36O30 1091.1230 1091.1219 -1.0 787, 768, 617, 465, 169

* Identification confirmed by commercial standard

43

Table 4. Tentatively identified compounds from ‘sugar MSK’ extract by GC-TOF-MS analysis.

Peak No

Ret. Time (min)

Family Tentative identif.

Match Factor

Formula Monoisotopic

mass

m/z [M+R]+ (measured)

*

m/z [M+R]+ (calculated)

*

Error (ppm)

Main fragments (m/z)

1 15.978 Tricarboxylic acid Citric acid* 79 C6H8O7 192.0270 - - - 396, 357, 145, 105, 74

2 18.286 FA (16:0) Palmitic acid* 86 C16H32O2 256.2402 313.2567a 313.2557a -3.1 129, 117, 75 3 19.803 FA (C18:2Δ9,12) ω-6 Linoleic acid * 79 C18H32O2 280.2402 337.2574a 337.2557a -4.9 262, 149, 117, 95 4 19.858 FA (C18:1Δ9) ω-9 Oleic acid* 89 C18H34O2 282.2559 339.2720a 339.2714a -1.8 145, 129, 117, 75

5 20.090 FA (C18:0) Stearic acid * 82 C18H36O2 284.2715 341.2877a 341.2870a -1.9 145, 129, 117, 75 6 23.234 FA (C20:5Δ5,8,11,14,17) ω-3 Eicosapentaenoi

c acid* 69 C20H30O2 302.2246 - - - 361, 217, 169,

147, 73 7 28.133 Terpenoid β-Sitosterol * 77 C29H50O 414.3862 486.4264b 486.4251b -2.6 396, 357, 145,

105, 73 * Trimethylsilyl (TMS) derivative: a R = (-H+TMS-CH3); b R = (-H+TMS)