Chemoprotective and antiobesity effects of tocols from seed oil of Maqui-berry: Their antioxidative and digestive enzyme inhibition potential

Journal Pre-proof Chemoprotective and antiobesity effects of tocols from seed oil of Maqui-berry: Their antioxidative and digestive enzyme inhibition potential José Miguel Bastías-Montes, Karen Monterrosa, Ociel Muñoz-Fariña, Olga García, Sergio M. Acuña-Nelson, Carla Vidal-San Martín, Roberto Quevedo-Leon, Isao Kubo, Jose G. Avila-Acevedo, Mariana Domiguez-Lopez, Zhao-Jun Wei, Kiran Thakur, Carlos L. Cespedes-Acuña PII:

S0278-6915(19)30826-9

DOI:

https://doi.org/10.1016/j.fct.2019.111036

Reference:

FCT 111036

To appear in:

Food and Chemical Toxicology

Received Date: 14 November 2019 Revised Date:

19 November 2019

Accepted Date: 3 December 2019

Please cite this article as: Bastías-Montes, José.Miguel., Monterrosa, K., Muñoz-Fariña, O., García, O., Acuña-Nelson, S.M., Vidal-San Martín, C., Quevedo-Leon, R., Kubo, I., Avila-Acevedo, J.G., DomiguezLopez, M., Wei, Z.-J., Thakur, K., Cespedes-Acuña, C.L., Chemoprotective and antiobesity effects of tocols from seed oil of Maqui-berry: Their antioxidative and digestive enzyme inhibition potential, Food and Chemical Toxicology (2020), doi: https://doi.org/10.1016/j.fct.2019.111036. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Chemoprotective and antiobesity effects of tocols from seed oil of Maqui-

2

berry: their antioxidative and digestive enzyme inhibition potential.

3

José Miguel Bastías-Montes1*, Karen Monterrosa1, Ociel Muñoz-Fariña2, Olga García2, Sergio

4

M. Acuña-Nelson1, Carla Vidal-San Martín1, Roberto Quevedo-Leon3, Isao Kubo4, Jose G.

5

Avila-Acevedo5a, Mariana Domiguez-Lopez5b, Zhao-jun Wei6, Kiran Thakur6, Carlos L.

6

Cespedes-Acuña7*.

7

1

8

Chile.

9

2

Instituto de Ciencia y Tecnología en Alimentos, Universidad Austral de Chile, Valdivia. Chile.

10

3

Departamento de Acuicultura y Recursos Agroalimentarios, Universidad de Los Lagos, Osorno,

11

Chile. 4ESPM Department, UC-Berkeley, CA 94720-3112, USA.

12

5a

13

5b

14

Celular. Universidad Nacional Autónoma de Mexico, Mexico D.F., Mexico.

15

6

16

People's Republic of China

17

7

18

Natural Products, Faculty of Sciences, University of Bio-Bío, Andrés Bello Avenue, Chillan,

19

Chile.´

20

*Authors for correspondence and reprints requests: 1J.M.Bastías-Montes, Departamento de

21

Ingeniería en Alimentos, Phone +56-42-2463042, E-mail:[email protected] / 3C.L.Cespedes-

22

Acuña, [email protected], Department of Basic Sciences, Research Group in Chemistry and

Departamento de Ingeniería en Alimentos, Universidad del Bío-Bío, P.O. Box 447, Chillán,

Unidad UBIPRO, Laboratorio de Fitoquimica, FES-Iztacala, Tlalnepantla, Estado de Mexico. Departamento de Biología Celular y Desarrollo, Laboratorio 305-Sur, Instituto de Fisiología

School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009,

Department of Basic Sciences, Research Group in Chemistry and Biotechnology of Bioactive

1

23

Biotechnology of Bioactive Natural Products, Universidad del Bío- Bío, Andrés Bello Av. #720,

24

Chillan 3780000, Chile.

25

Abstract

26

Maqui-berry (Aristotelia chilensis) is the emerging Chilean superfruit with high nutraceutical

27

value. Until now, the research on this commodity was focused on the formulations enriched with

28

polyphenols from the pulp. Herein, contents of tocols were compared in the seed oil of Maqui-

29

berry obtained through three different extraction methods followed by determining their

30

antioxidative and enzyme inhibitions in-vitro. Firstly, oilseed was extracted with n-hexane

31

(Soxhlet method), chloroform/methanol/water (Bligh and Dyer method) and pressing (industrial).

32

These samples were used to access their effects against DPPH, HORAC, ORAC, FRAP, Lipid-

33

peroxidation (TBARS), α-amylase, α-glucosidase, and pancreatic lipase. All the isomers of

34

tocopherol and tocotrienol were identified, and β-sitosterol was the only sterol found in higher

35

amounts than other vegetable oils. The Bligh and Dyer method could lead to the highest

36

antioxidative capacity compared to Soxhlet and press methods likely because the latter have a

37

higher amount of tocopherols. Further, seed oil from Maqui berry and their tocols (α, β, γ, δ-

38

tocopherols, tocotrienols, and β-sitosterol) warrant clinical investigation for their antioxidative

39

and antiobesity potential. Taken together, these findings provide relevant and suitable conditions

40

for the industrial processing of Maqui-berry.

41

Keywords: Tocols, sterols, antioxidant, fatty acids, maqui seeds oil, Aristotelia chilensis.

42 43

1. Introduction

44

Antioxidants compounds delay the oxidation process by interrupting the development of

45

free radicals, inhibiting their polymerization chain reactions, and other subsequent oxidizing

2

46

reactions (Halliwell and Aruoma, 1991). Since long, synthetic and natural antioxidants like α-

47

tocopherol have long been used for food processing which protect against damage caused by the

48

oxidation process. Natural antioxidants have been known to be effective against many

49

communicable and non-communicable diseases. Several environmental factors (smog including

50

volatile chemicals and UV-radiation, as well as diets rich in saturated fatty acids) are responsible

51

for the enhanced oxidative damage in the body. The antioxidants can protect the body from

52

serious oxidative damage and can lead to a better life (Roberts et al., 2003).

53

The vast research on various free radicals in aging and chronic non-communicable diseases

54

has directed many researchers to evaluate the antioxidant potential of different plant samples

55

(Fernandez et al., 2004).

56

In addition to enzyme inhibitions (α-amylase, α-glucosidase, pancreatic lipase, and others),

57

the antioxidants are also recognized as nutraceuticals (Schinella et al., 2002). In the past, our

58

research group has evaluated Chilean flora and their biological activities (Cespedes et al., 2006a;

59

2006b) and especially with nutraceuticals activities of Maqui Berry (Cespedes et al., 2017a;

60

2017b; Cespedes et al., 2018). Also, an assessment of the extracts and compounds from seeds of

61

Maqui-berry Aristotelia chilensis (Mol.) Stuntz (Elaeocarpaceae) has been initiated.

62 63

The search for novel plant based nutraceuticals and agrochemicals culminated into various research projects focusing on natural compounds (Cespedes et al., 2006a).

64

The increasing global demand for berries makes it possible to commercialize a diverse

65

array of native Chilean fruits (maqui, murtilla, and white strawberry) given the added value of

66

their high antioxidant and dietary fiber levels (ODEPA, 2013; Anderson et al., 2009). A.

67

chilensis, commonly called maqui-berry, is a native tree in Chile and its fruits are excellent

68

source for polyphenols and anthocyanins with beneficial and health-promoting properties

3

69

(Fuentealba et al., 2012; Cespedes et al., 2008; Cespedes-Acuña et al., 2018; Cespedes et al.,

70

2009; Cespedes et al., 2017a; 2017b; Fuentes et al., 2015; Vasconcelos et al., 2013). At present,

71

the industrialization of maqui berry mainly involves obtaining juice and extracts from the pulp,

72

and these processes generate a by-product that contains a big proportion of Maqui seed

73

(approximately 50%) (Brauch et al., 2016), which could be a good source of nutrients. The

74

utilization of waste products is the current focus of agricultural research (Prado et al., 2012). The

75

previous studies showed that the seed oils from blackberry, red raspberry, and blueberry contain

76

high levels of linolenic acid, tocopherols, polyphenols, and carotenoids (Parry and Yu, 2004;

77

Parry et al., 2005). The best-known case is grape seeds, from which oils and pomace contain high

78

levels of essential fatty acids, vitamin E, and polyphenols (Baydar et al., 2007; Wettasinghe and

79

Shahidi, 1999). Most of the berry seed oils that have been studied to date have the higher content

80

of polyunsaturated fatty acids that can provide essential fatty acids (Van Hoed et al., 2009). It has

81

been reported that the lipid content of the dry Maqui berries is 8.13%, which is due to its low

82

content of fruit pulp concerning the seed content with the seed to pulp ratio of 1: 1 (w/w) (Brauch

83

et al., 2016). Also, the sterol content in Maqui leaves is around 10-15% (Cespedes, 1996; Muñoz

84

and Ramos, 2016). Nevertheless, there are still other concerns related to nutraceuticals obtaining

85

methods and that need to be solved and tangible advantages to be demonstrated before the

86

industrial implementation of these novel technologies. Therefore, selecting a suitable extracting

87

technology is crucial in the food industry.

88

To date, there is very little information on the chemical composition of the Maqui berry

89

seed or any product derived from the seed, such as oil and their enzyme inhibition and

90

antioxidant activities. After the thorough review, we present the first ever report to evaluate the

91

enzymatic inhibition and antioxidant activities of Maqui berry seed oil extracted using three

4

92

extraction methods. This information could be of interest for the future exploitation of this

93

Chilean natural resource.

94

2. Materials and methods

95

2.1. Materials and reagents

96

The Maqui fruit samples were obtained from a cultivar in “Coihueco town” into the Region

97

of Ñuble, Chile, the year 2015. HPLC grade reagents, such as methanol, n-hexane, glacial acetic

98

acid, chloroform, sulfuric acid, and ethanol, were purchased from Merck (Darmstadt, Germany).

99

Sodium hydroxide, ammonium chloride, sodium sulfate, and potassium hydroxide were procured

100

from Sigma-Aldrich Inc. (St. Louis, MO, USA). A certified fatty acid methyl ester (FAME)

101

reference standard mixture (37 fatty acids from C4 to C24) and Sylon BTZ were obatined from

102

Supelco (Bellefonte, PA, USA). Reagents also included 2, 2′-Azobis (2-amidinopropane)

103

dihydrochloride 97% (AAPH), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic

104

acid), and DPPH (2,2-diphenyl-1-picrylhydrazyl). Standard α-tocopherol was purchased from

105

Sigma (St. Louis, MO); β, γ, and δ-tocopherols from Supelco (Bellefonte, PA), and tocotrienols

106

from Davos Life Science (Singapore). Sterol standards, such as campesterol (65%), stigmasterol

107

(95%), β-sitosterol (≥ 97%) and sitostanol (≥ 95%), as well as the internal standard 5a-cholestane

108

(≥ 95.0%), were purchased from Sigma–Aldrich. Quercetin, rutin and ursolic acid were available

109

from our previous work (Kubo et al., 1994). Rosmarinic acid was purchased from Cayman

110

Chemical Co. (Ann Arbor, MI). Nordihydroguaiaretic acid (NDGA) and trifluoroacetic acid

111

(TFA) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Dimethyl sulfoxide

112

(DMSO), soybean lipoxygenase-1 (EC 1.13.11.12, Type I) and linoleic acid were purchased from

113

Sigma Chemical Co. (St. Louis, MO). Tris was obtained from Fisher Scientific Co. (Fair Lawn,

114

NJ).

115

Hydroperoxyoctadecadienoic acid (13-HPOD: λmax = 234 nm, ε = 25 mM-1cm-1) was prepared

Ethanol

was

purchased

from

Quantum

5

Chemical

Co.

(Tuscola,

IL).

13-

116

enzymatically by the previously described procedure (Gibian & Galaway, 1976) and stored in

117

ethanol at -18 ºC.

118

2.2. Recovery of Maqui berry seed

119

The pomace residue containing seeds from Maqui berry was subjected to chemical washing

120

using a sodium hydroxide 3% solution at 90 ° C for 6 minutes, and then after, neutralization was

121

performed by adding 5% citric acid.

122

2.3. Seed oil extraction

123

2.3.1. Soxhlet extraction

124

For extraction by the Soxhlet method, Maqui seeds were dried until 8% water in a forced-

125

air oven (Memert, UF 750) at 70 °C for 5 hours. Then, the seeds were crushed in a screw mill

126

(Thomas Wiley L20) to obtain homogeneous flour. Fifteen grams of milled seeds were soaked

127

with solvent for 1 hour at room temperature without exposure to light. Then, the seeds were

128

extracted with n-hexane (300 mL) using a Soxhlet extractor for 3 hours at 70 °C. After extraction,

129

the hexane-oil mixture was passed through a layer of anhydrous magnesium sulfate placed over a

130

filter paper in a funnel. Solvent was removed using a rotary evaporator (Rotavapor R-210, Büchi

131

Labortechnik, Switzerland) at 40 °C (Fiori et al., 2014).

132

2.3.2. Obtaining oils by Bligh & Dyer method and pressing

133

Seventy grams of fresh Maqui berry seeds were ground in a blender and then placed in

134

centrifuge tubes (7 g ground seed in each tube). After wards, chloroform/methanol/water (2:1:0.8

135

v/v) solution was added at room temperature with shaking for 1 hour. Then, the ratio was

136

changed (2:2:1.8 v/v) and 7 mL anhydrous sodium sulfate 3% was added to each tube. After

137

gentle shaking, the mixture was centrifuged (Supra 25K; Hanil Science Industrial Co., Incheon,

138

Korea) at 4000 rpm for 10 min to obtain two separate phases. The lower phase was collected and

139

filtered through Whatman N° 1 with a small number of anhydrous Na2SO4 (1.5–2.5 g) at the 6

140

bottom. Solvents were evaporated under reduced pressure at 40 °C (Rotavapor R-210, Büchi

141

Labortechnik, Switzerland). The extracted oil was weighed and stored at -20 °C until further

142

analysis (Bligh and Dyer, 1959).

143

Finally, the pressed oil from this sample was provided by Granasur oil Company,

144

Santiago de Chile.

145

2.4. Physicochemical analysis

146

The refractive index (RI) was determined according to the norm AOCS 28 Cc 7-25

147

(AOCS28, 2002). Color characteristics of oil were determined using the method described by

148

Shao et al., 2015. The iodine value was performed according to the ISO 3961 (ISO3961, 1996)

149

procedure. The density was determined following the methodology proposed by AOCS Cc 10c-

150

95 (AOCS, 2000). To determine the free fatty acids, the methodology described by ISO 660

151

(ISO660, 1996) was used, and the peroxide index was determined following the procedure

152

described by the AOCS Cd 8-53 (AOCS, 1980).

153

2.5.Fatty acid analysis

154

The fatty acids methyl esters (FAME) were analyzed according to AOAC 996.06 (AOAC,

155

2006). Samples were converted to methyl esters by esterification with NH4Cl–H2SO4–methanol

156

solution for GC–MS analysis (Hartman and Lago, 1973). The free fatty acids were extracted with

157

0.5 N NaOH–methanol solution, which was followed by the addition of hexane to perform the

158

fatty acid separation according to AOAC 996.06 (AOAC 2006) after some modifications, with a

159

gas chromatograph (Shidmadzu, GC-2010) coupled with an FID detector (Agilent, MSD 5975C).

160

The operation conditions of the chromatographic analysis were the following: initial temperature

161

of 60 °C (2 min) up to 100 °C (5 min), heating at 8 °C/min up to 200 °C (2 min), heating at 4

162

°C/min up to 240 °C (5 min), and heating at 3 °C/min and 240 °C up to the final time. The

7

163

compounds were separated in a capillary column (Rt-2560, with 100 m x 0.25 mm x 0.20 μmol)

164

consisting of 5%-phenyl-methylpolysiloxane, using helium as carrier gas with a flow of 1.2

165

mL/min. The injected samples were in a Split mode (ratio 1:100). FAME was identified through

166

a comparison between retention times from the standard mix of fatty acids and quantified by the

167

area normalization using ChemStation Software Agilent Technologies, Inc. 07/09, Germany.

168

2.6. Tocols and Sterols analysis

169

Tocopherols were separated and quantified by HPLC according to the AOCS Ce 8-89

170

(AOCS, 1990) method with a WatersTM 717 plus Merck Hitachi L-6200A HPLC, Hibar 150-4, 6

171

Puropher Star RP-18e column (250 mm x 4.6 mm x 5 µm) and fluorescence detector

172

(Spectrophotometer F-1050, software Clarity) at an excitation wavelength set at 290 nm and

173

emission wavelength set at 330 nm. The mobile phase used was methanol: water (99.5:0.5 v/v) at

174

a flow rate of 1.0 mL/min with an injection of 20 µL. Tocol content was determined by the

175

external standard method. Areas were converted to concentrations using the standard curve of α-

176

tocopherol in n-hexane. Determinations were performed in triplicates. The content and

177

composition of the sterols were determined by GC following the procedure described by AOCS

178

Ce 2-66 (AOCS, 1997). Each seed oil (50 mg) was saponified with 1 M KOH in methanol for 18

179

hours at refrigeration temperature. Then, water was added and unsaponifiable materials were

180

extracted three times with n-hexane (1:1, v/v). The solvent was evaporated under a stream of

181

nitrogen. Dry residues were dissolved in 0.2 mL of ethanol/hexane. Sterol derivatives were

182

separated on a gas chromatograph (Shimadzu GC-2010, Japan) equipped with a fused silica Rtx-

183

5 Sil-MS capillary column (30 m x 0.25 mm x 1 µm; Restek Corporation, U.S). A sample of 1.0

184

μL was injected in a split mode (ratio 1:100). The column temperature was held at 100 °C for 5

185

min and then increased to 250 °C at a rate of 25° C/min, which was held for 1 min and then

8

186

further increased to 290 °C at a rate of 3 °C/min for 20 min. The FID detector (Agilent, MSD

187

5975C) with a temperature set at 300 °C was used. Helium was used as a carrier gas at a flow rate

188

of 1.5 mL/min. An internal standard, 5α-cholestane was used for sterol quantification. Sterols

189

were identified by comparing their retention times (relative to 5α-cholestane) with those of

190

commercially available standards and the results were expressed as mg/kg of oil.

191

2.7. Antioxidant capacity

192

These procedures were carried out with mixtures as follow: M1 (Tocols + tocotrienols); M2

193

(only tocols); M3 (only tocotrienols); M4 (α + γ tocols); M5 (tocols + tocotrienols + β-

194

sitosterol); S6 (γ-tocopherol); S7 (δ-tocopherol); S8 (α-tocopherol); S9 (β-tocopherol); S10

195

(tocotrienols + fatty acids).

196

2.7.1. ORAC assay

197

The ORAC assay was based on the previous procedure described by Davalos et al., 2004

198

with some modifications from Ou et al., 2002. The reaction was performed in 75 mM phosphate

199

buffer (pH 7.4), and the final volume of the reaction mixture was 270 μL. Antioxidants (45 μL of

200

oil sample with a concentration of 10.0 ppm; for pure chemicals, 0.43 mg of each tocopherol was

201

dissolved in 1 mL and 10 µL to adjust a concentration of 10 µM) and fluorescein (175 μL; 70

202

nM, final concentration) solutions were placed in the micro plate wells. The mixture was

203

preincubated for 15 min at 37 °C. AAPH solution (50 μL; 20 mM, final concentration) was

204

rapidly added using a multichannel pipet. The microtiter plate was immediately placed in a

205

Biotek™ Model FLx800 (Biotek Instruments, Inc., Winooski, VT) fluorescence plate reader set,

206

and the fluorescence was recorded every minute for 120 min, using an excitation λ = 485/20 nm

207

and emission λ = 582/20 nm, to reach a 95% loss of fluorescence. The microplate was

208

automatically shaken before each reading. A blank (FL + AAPH) using phosphate buffer instead

9

209

of the antioxidant solution and eight calibration solutions using Trolox and α-tocopherol as

210

antioxidants were performed in each assay. All reaction mixtures were prepared in triplicates, and

211

at least three independent assays were performed for each sample. ORAC values were expressed

212

as Trolox equivalents using the standard curve calculated for each assay. Values were expressed

213

in μmol of Trolox equivalent/g of sample.

214

2.7.2. Reduction of the 2,2-Diphenyl-1-Picrylhydrazyl Radical (DPPH).

215

Extracts were chromatographed on TLC and examined for antioxidant effects by spraying

216

the TLC plates with DPPH reagent. Specifically, the plates were sprayed with 0.2% DPPH in

217

methanol (Torres et al., 2003; Cespedes et al., 2001). Further, each sample was analyzed with

218

DPPH in microplates of 96 wells as follows: extracts (50 µL) were added to 150 µL DPPH (100

219

µM, final concentration) in methanol (The microtiter plate was immediately placed in a Biotek™

220

Model ELx808, Biotek Instruments, Inc., Winooski, VT) and the absorbance was read at 515 nm

221

after 30 min (Dominguez et al., 2005; Cespedes et al., 2010). Quercetin and α-tocopherol were

222

used as standards.

223

2.7.3. Ferric reducing antioxidant power estimation (FRAP).

224

The FRAP assay was performed as previously described by Benzie and Strain (Benzie and

225

Strain, 1999). Reagents were freshly prepared and mixed in the proportion 10:1:1, for A:B:C,

226

where A ¼ 300 mM sodium acetate trihydrate/glacial acetic acid buffer pH 3.6; B ¼ 10 mM

227

TPTZ in 40mMHCl and C ¼ 20mMFeCl3. Catechin was used to make a standard curve (5e40

228

mM final concentration) with all solutions including samples dissolved in sodium acetate

229

trihydrate/glacial acetic acid buffer. The assay was carried out in 96-well plates, at 37 °C at pH

230

3.6, using a 10 mL sample or standard plus 95 mL of the mixture of reagents shown above. After

10

231

10 min incubation at RT, absorbance was read at 593 nm. Results are expressed as mmol catechin

232

equivalents (CatE) per gram of sample. All tests were conducted in triplicates.

233

2.7.4. Estimation of lipid peroxidation through rat brain

234

As an index of lipid peroxidation, TBARS levels were measured using rat brain

235

homogenates according to the method described by Ng et al., 2000 with some modifications.

236

Adult male Wistar rats (200 - 250 g) were provided by the Instituto de Fisiología Celular,

237

UNAM, and the study was approved by the Animal Care and Use Committee (PROJ.-NOM 087-

238

ECOL-SSA 1-2000). Rats were maintained at 25 °C on a 12/12 hours light-dark cycle with free

239

access to food and water and sacrificed under mild ether anesthesia. Cerebral tissue was rapidly

240

dissected from the whole brain and homogenized in phosphate-buffered saline (0.2 g KCl, 0.2 g

241

KH2PO4, 8 g NaCl and 2.16 g NaHPO4 x 7 H2O/L, pH 7.4) to use one in 10 homogenates, w/v

242

(Rossato et al., 2002). The homogenate was centrifuged for 10 min at 3400 rpm, and the resulting

243

pellet was discarded. The protein content of the supernatant was measured by the method of

244

Lowry et al. (Lowry et al., 1951), and samples were adjusted to 2.5 mg protein/mL with PBS.

245

The supernatant (400 mL, 1 mg protein) was pre-incubated with Maqui berry seed oil compounds

246

(50 mL) at 37 °C for 30 min, then peroxidation was initiated by the addition of 50 mL freshly

247

prepared FeSO4 solution (final concentration 10 mM), and incubated at 37 °C for an additional 1

248

hour (Ng et al., 2000).

249

The TBARS assay was performed as described by Ohkawa et al., 1979 with few

250

modifications. 0.5 mL TBA reagent (1% thiobarbituric acid in 0.05 N NaOH and 30%

251

trichloroacetic acid, 1:1) was used, and the final solution was cooled on ice for 10 min,

252

centrifuged at 10, 000 rpm for 5 min, and then heated at 95 °C in a boiling water bath for 30 min.

253

After cooling on ice, the absorbance was read at 532 nm in a Spectronic Genesys 5

11

254

spectrophotometer. Quercetin and BHT were used as positive controls. Concentrations of

255

TBARS were calculated using a TMP standard curve (Esterbauer and Cheeseman, 1990). Results

256

are presented as nmoles TBARS per mg of protein, with percent inhibition after 30 min

257

calculated as the inhibition ratio (IR), where

258

IR(%) = (C – E)/C x 100%

259

Where C is the absorbance of the control and E is the absorbance of the test sample. These

260

values were plotted against the log10 of the concentrations of individual extracts, and a decrease

261

of 50% in peroxidation was defined as the EC50.

262

2.7.5. Determination of malondialdehyde (MDA) an index of lipid peroxidation of liposomes

263

Liposomes were prepared as described by El-Hafidi and Baños, 1997. Two hundred

264

miligrams of phosphatidylcholine from soybean (Sigma-Aldrich-Mexico) was sonicated in 2mL

265

phosphate buffer for 30 min. The clear final solution was centrifuged at 12,000 rpm filtered

266

through a column of Sephadex G50 to eliminate all the traces of metal resulted from the tip

267

during the sonication. Lipid peroxidation of liposomes was induced by 5 mM of CuSO4 and 1.0

268

mM of ascorbic acid to generate hydroxyl radicals by Fenton reaction in the presence and

269

absence of different oil extracts (oil samples and tocopherols).

270

The determination of the MDA-equivalent by the formation of thiobarbituric acid reactive

271

substances (TBARS) was used to evaluate the lipid peroxidation in a liposome. The fluorescence

272

method was used for TBARS quantification according to the method described by El-Hafidi and

273

Baños, 1997; and the procedure was carried out with 10 mg of phosphatidylcholine liposome.

274

The samples were treated with 0.05 mL of ethanol containing 4% of BHT in 1mL KH2PO4 (0.15

275

M, pH 7.4), the mixture was agitated in vortex for 5 s, and incubated for 30 min at 37 °C with

276

constant agitation. At the end of incubation, 1.5mL of 0.8% thiobarbituric acid and 1mL of 20%

12

277

acetic acid at pH 3.5 were added. The mixture was heated with boiling water for 1 hour, and

278

immediately the samples were placed in ice.

279

The TBARS was extracted by adding 1 mL of KCl (2%) and 5 mL of n-butanol. The n-

280

butanol phase was separated after centrifugation for 2 min at 755 x g and it was carried out to

281

measure the fluorescence in Fluorimeter (Perkin Elmer Luminescence LS-50B) at the excitation

282

wavelength of 515 nm and the emission wavelength of 553 nm. The concentration of the MDA

283

equivalent (TBARS) was determined using a calibration curve obtained from standard 1,1,3,3-

284

tetraethoxypropane (Sigma-Aldrich).

285

2.7.5. HORAC (Hydroxyl Radical Averting Capacity).

286

The Hydroxyl Radical Averting Capacity (HORAC) is used to measure the capability of a

287

substance to neutralize the hydroxyl radical (HO˙) generated by Fenton-like reactions employing

288

a Co(II) complex using fluorescein FS as a probe. HORAC assays were performed by the method

289

developed by Ou et al., 2002, modified for the FL-800 microplate (BioTek) reader and tested

290

successfully in several reports. HORAC values were calculated using a regression equation

291

between the caffeic acid concentration and the area under the decay of the FS curve (AUC)

292

according to the calibration curve for caffeic acid (between the range of 0 - 250µmol/Lcaffeic acid):

293

y = 0.0695x - 2.9111; R2 = 0.9856

294

where y is the net AUC and x is the concentration of caffeic acid in µmol/L. The results are

295

presented in µmol of equivalents of caffeic acid (CAE) per g of dry sample (µmolCAE/gDE) ± SE.

296

2.8 α-Amylase, α-Glucosidase and Pancreatic Lipase Inhibition Assays of tocols mixtures

297

The porcine pancreatic lipase inhibitory assay was adapted from Jaradat et al., 2017. 1

298

mg/mL (1000 µg/mL) of each tocols mixture stock solution was used in 10% DMSO; from which

299

five different solutions were prepared with the following concentrations: 50, 100, 200, 300, and

13

300

400 µg/mL. 1 mg/mL stock solution of pancreatic lipase enzyme was prepared immediately

301

before being used.

302

A stock solution of PNPB (p-nitrophenyl butyrate) was prepared by dissolving 20.9 mg of

303

PNPB in 2 mL of acetonitrile. 0.1 mL of porcine pancreatic lipase (1 mg/mL) was added to test

304

tubes containing 0.2 mL of the various concentrations (50, 100, 200, 300, and 400 µg/mL) of

305

mixture sample. The resulting solutions were then made up to 1 mL by adding Tri-HCl solution

306

(pH 7.4) and incubated at 25 °C for 15 min. After the incubation period, 0.1 mL of PNPB

307

solution was then added to each test tube. Each solution was again incubated for 30 min at 37 °C.

308

Pancreatic lipase activity was determined by measuring the hydrolysis of p-nitrophenyl butyrate

309

to p-nitrophenol at 405 nm using a UV-visible spectrophotometer. The same procedure was

310

repeated with Orlistat (a positive control) using the same concentrations as mentioned above. The

311

established tests were performed in triplicates.

312

Additionally, the α-amylase and α-glucosidase inhibitory effect of each tocols sample were

313

assayed according to the procedure described previously by Nowicka et al., 2018. Acarbose was

314

included in the case of α-amylase and α-glucosidase as a positive control, while the Orlistat was

315

used as a positive control for pancreatic lipase. The results were expressed as IC50 values.

316

These procedures were carried out with mixtures as follow: M1 (Tocols + tocotrienols); M2

317

(only tocols); M3 (only tocotrienols); M4 (α + γ tocols); M5 (tocols + tocotrienols + β-

318

sitosterol); S6 (γ-tocopherol); S7 (δ-tocopherol); S8 (α-tocopherol); S9 (β-tocopherol); S10

319

(tocotrienols + fatty acids).

320

2.9 Statistical analysis

321

The analysis of the studied samples was performed in three independent experiments. The

322

results were presented as the mean ± standard deviation (SD). The statistical analysis was

14

323

performed in STATGRAPHICS Centurion XVI (Statgraphics, 2009) and Origin 8.0. One-way

324

analysis of variance (ANOVA) was used to compare the composition between the extractive

325

methods of seed oils. Differences between samples were examined using a Tukey’s test and were

326

considered significant at p < 0.05. Pearson’s test was used to find the correlation between total

327

tocols content and the antioxidant capacity of the studied oils. All the data were analyzed by one-

328

way ANOVA followed by Dunnett’s test for comparisons against control. Values of p < 0.05 (*)

329

and p < 0.01 (**) were considered statistically significant and the significant differences between

330

means were identified by GLM Procedures. Also, differences between treatment means were

331

established with a Student–Newman-Keuls (SNK) test. The IC50 values for DPPH and TBARS

332

were calculated by the Probit analysis. Complete statistical analyses were performed using the

333

MicroCal Origin 8.0 statistical and graphs PC program. Data are presented as a means ± SEM of

334

five different experiments. At P < 0.05, the difference was statistically significant. Multiple

335

comparisons between the experimental groups were performed by one-way ANOVA with a

336

Tukey post hoc test.

337

3. Results and discussion

338

3.1. Physicochemical characterization

339

The iodine value or iodine index is the mass of iodine in grams that is consumed by 100 grams of

340

a chemical substance. Iodine numbers are often used to determine the amount of unsaturation in

341

fatty acids. Table 1 shows the iodine index in the Maqui berry oils obtained by the Soxhlet and

342

Bligh & Dyer methods, and there was no significant difference between these two methods.

343

According to Chile’s Food Sanitary Regulation (Sanitary Regulations of the Food, Chile., 2015),

344

the iodine index in sunflower oil must oscillate between 120 and 140; iodine index for olive oil is

345

80-90 and that for oil of the grapes seed is 115-140. Therefore, the results for Maqui oil are close

15

346

to the established ranges for the other vegetable oils. The value of the refractive index obtained in

347

the press was significantly higher than that for the oils from the Soxhlet and Bligh and Dyer

348

methods, very close to those reported by previous study (Baydar et al., 2007) in grape seed oils.

349

As for the density, which is one of the most important physical properties indicating the purity of

350

a substance as oil (Knothe and Steidley, 2014), the results in Maqui seed oil had a range similar

351

to those for the sunflower oil at a density of 0.913-0.923; olive oil at 0.909-0.160, and grape seed

352

oil at 0.923-0.926 as indicated (Sanitary Regulations of the Food, Chile., 2015). This value is for

353

the oils obtained from blackberry and raspberry seeds with reported density values of 0.997-

354

0.999 and 0.968-0.967, respectively (Dimić et al., 2012). The peroxide values of seed oil from

355

Maqui berry were in the international range and showed good performance when compared with

356

those of olive oil (Sanitary Regulations of the Food, Chile., 2015; Knothe and Steidley, 2014;

357

Dimić et al., 2012).

358

Regarding the color analysis for the L* parameter (Table 2), the oil extracted with the Bligh

359

and Dyer method was the clearest, presenting significant difference (p < 0.05) to those extracted

360

by Soxhlet and pressing (Table 2); with the latter obtained lower values, indicating greater

361

opacity. When comparing the a* values, pressed and Soxhlet seed oils were greener, while the oil

362

obtained by the Bligh & Dyer method was more orange. The b* values showed that all oils had

363

some degrees of yellowish color; however, the oil obtained by the Bligh and Dyer method

364

resulted in a higher b* value (p < 0.05). The Chroma parameter (C*ab) indicated saturation, and

365

the Bligh and Dyer method had the highest value (p < 0.05) compared to the other two methods,

366

which coincided with the visual observation. Regarding h*ab, all the values are close to 90°,

367

indicating a tendency toward yellow and brown. It has been observed that there are some

368

differences in the presence of subtle red-orange color in oils extracted using methanol and the

369

presence of tocopherols and the yellow color when using chloroform (Payal et al., 2016). In a 16

370

study of eight cultivars of conventional Spanish olive oils, these values were observed, ranging

371

from 61.94 to 99.28 for L*, from −14.96 to 9.96 for a*, from 11.98 to 128.68 for b*, from 12.20

372

to 128.96 for C*ab and from 85.03 to 100.84 for h*ab (Moyano et al., 2008), similar to those

373

explored in this study.

374

3.2. Fatty acids profile

375

The fatty acid profile of the Maqui seed oils was presented in Table 3. There was no

376

significant difference between the Soxhlet and Bligh and Dyer methods; however, when

377

compared with the press, there was a difference (p > 0.05), which could be due to the different

378

origin of the seeds used. The polyunsaturated fatty acids were found to be the most abundant

379

fatty acids in Maqui berry seed oils obtained by all three extractive methods, and the largest one

380

was linoleic acid, which accounted for 45%, followed by monounsaturated fatty acids, in which

381

oleic (39-40%) and saturated (8%) palmitic acid were found; and there was no significant

382

difference between the two. The values were in a similar trend to those found previously (Brauch

383

et al., 2016) in dried Maqui berries with 8.7, 33.28, 46.31, and 2.09% palmitic, oleic, linoleic, and

384

linolenic acid, respectively. The content of oleic acid in Maqui berry seed oils is comparable to

385

other oils, such as corn oil (25-50%), sunflower oil (14-34%), and sesame oil (35-50%) as

386

described (Sanitary Regulations of the Food, Chile., 2015) in Chile’s Food Sanitary Regulation.

387

Additionally, in terms of the presence of linoleic acid, it was similar to those in corn oil (40-

388

60%), soybean oil (45-60%), sesame oil (40-50%), and rosehip oil (40-46%). As well as, the

389

content of linolenic acid was similar to those in oil of grape seed < 2%, olive oil < 1%, and

390

sesame oil < 1% (Sanitary Regulations of the Food, Chile., 2015).

391

3.3 Tocols and sterol composition

17

392

The composition of tocols in the Maqui seed oils (Table 4) from the press oil, blight & dyer

393

and Soxhlet methods were different (p < 0.05); the α-γ-tocopherol content was higher for the

394

Bligh and Dyer extraction method compared with pressing (169.33 and 56.76 mg/kg).

395

The levels of α- and γ-tocopherols in oils extracted by the Soxhlet method were significantly

396

lower (p < 0.05) than those extracted with the Bligh and Dyer method, which contrasts with that

397

reported previously (Adhikari et al., 2008). The differences in the results obtained in this study

398

could be associated with several factors such as the extraction process, a solvent used, quality and

399

the origin of the raw material, storage conditions, and pretreatment (Louli et al., 2004). As for the

400

tocols content in other oils, canola oil contains 184 mg/kg, as indicated by Guinazi, 2004.

401

According to Baydar et al. 2007, the values of α-tocopherol in grape seed oil varied depending on

402

the variety and crop areas that can vary from 128.14 to 325.39 mg/kg. Regarding raspberry seed

403

oil, Xu et al. (2006) reported a value of 330.8 mg/kg with a value of 117 mg/kg for blackberry

404

seed oil. Oomah et al. (2000) reported similar results to those obtained in this study. Regarding γ-

405

tocopherol content in oil, results like those found in this study were reported by Baydar et al.

406

(2007) in grape seed oil extracts, ranging from 14.37 to 31.73 mg/kg oil. In the crude oil of two

407

varieties of blueberries (Vaccinium macrocarpon and Vaccinium corymbosum), a study by Van

408

Hoed et al. (2011) obtained 62.8 and 59.3 mg/kg of oil, respectively. In sunflower oil, 92.3 mg/kg

409

γ-tocopherol has been reported and at a higher amount in raspberry seed (794 mg/kg and 704

410

mg/kg) (Grilo et al., 2014). Oomah et al. (2000) reported raspberry seed oil extracted with hexane

411

in the Soxhlet system contained 2720 mg/kg γ-tocopherol. Determination of the minor

412

compounds in vegetable oils, including the tocopherol group, is essential for the analytical

413

evaluation of the quality, origin, method of extraction, refining, and possible adulteration which

414

could be possible in oil-seed products (Cert et al., 2000). α-Tocopherol exhibits anti-

415

inflammatory activity and modulates the expression of proteins involved in cholesterol 18

416

metabolism (Wallert et al., 2014). γ-Tocopherol is considered as the most potent free radical

417

scavenger among vitamin E isomers; besides, it has a strong anti-inflammatory activity and is

418

related to the inhibition of carcinogenesis (Ju et al., 2010), given that γ-tocopherol has greater

419

antioxidant capacity than other tocopherols (Hwang and Winkler-Moser, 2017). Tocopherols and

420

tocotrienols in this study were found at higher concentrations (Table 4) than in a study by Casal

421

et al. (2010) who reported that olive oil had 1-2 mg/kg β-tocopherol and 5.7 mg/kg β-tocotrienol

422

and sunflower oil had 21 mg/kg β-tocopherol, 5.6 mg/kg β-tocotrienol, 3.2 mg/kg γ-tocotrienol,

423

and 1.2 mg/kg δ-tocotrienol. On the other hand, Oomah et al. (2000) reported values higher than

424

those found in this study of which raspberry seed oil contained 174 mg/kg δ-tocopherol extracted

425

with hexane and 71 mg/kg δ-tocopherol in raspberry cold-pressed oil, and tocotrienols were not

426

detected. Rather, high levels of tocopherols in berry seed oils could explain their oxidation

427

capacity despite an unsaturation level of fatty acids of more than 90% (Oomah et al., 2000)

428

(Table 4).

429

The only phytosterol found in the studied Maqui berry seed oils was β-sitosterol (Table 4).

430

According to CODEX Alimentarius (1999), the levels were 64-70 mg/kg for grape seed oil, 42-

431

70 mg/kg for sunflower oil, 45-57 mg/kg for canola oil, and 47-60 mg/kg for soybean oil. Olive

432

oil had 279.14 to 846.25 mg/kg β-sitosterol (Xiang et al., 2016). Others have reported 666.8

433

mg/kg and 2266.2 mg/kg β-sitosterol in olive pomace oil (Cañabate-Diaz et al., 2007). Therefore,

434

compared to the vegetable oils mentioned above, Maqui berry seed oil could be considered a

435

good source of β-sitosterol.

436

3.4 Antioxidant capacity

437

With respect to the antioxidant activity found in oils from seeds of Maqui Berry, with the

438

ORAC test (Table 5), the oil obtained by the Bligh and Dyer method showed higher values for

439

ORAC 9724.1 µmol TE/100 g and 21289.58 µmol α-tocopherol/100 g oil (data not show) 19

440

compared to Soxhlet and pressed oil methods. The antioxidant activity depends not only on the

441

extraction method but also on the solvent used for extraction because various antioxidant

442

compounds with different chemical characteristics and polarities may or may not be soluble in a

443

solvent (Turkmen et al., 2066). It has been found that methanol is more efficient for extracting

444

lower molecular weight polyphenols (Dai and Mumper, 2010); this could explain the differences

445

in the values obtained in this study that were mainly in the oil obtained by the Bligh & Dyer

446

method (an extraction method that used methanol).

447

Plant oils contain several antioxidants that can inhibit oxygen radicals in biological systems

448

and maintain oil quality and stability during prolonged storage (Papadopoulos and Boskou, 1991;

449

Montedoro et al., 1992). One Study on olive oils showed that the concentrations of these

450

substances vary greatly depending on the area of production, crops, climate, harvest time, and

451

storage (Montedoro et al., 1992). According to Szydłowska-Czerniak et al. (2008), the ORAC

452

values in extra virgin olive oil were in the range of 433 to 902 µmol TE/100 g. Haytowitz and

453

Bhagwat, (2010) reported the ORAC value of 372 µmol TE/100 g for extra virgin olive oil. These

454

values were lower than those found in the present study. In the case of canola oil obtained by

455

pressing, ORAC values from 640 to 682 µmol TE/100 g and 994 to 1106 µmol TE/100 g in

456

canola oil extracted with solvent have been reported (Yang et al., 2011). Studies using blueberry,

457

strawberry, and raspberry seed oils (Buschman et al., 2004), 975.29 and 2315 µmol α-

458

tocopherol/100 g oil were reported; these values were similar with those from the Soxhlet

459

extraction, minor to Bligh and Dyer extraction and pressing methods. Tocols content and

460

antioxidant capacity were correlated using Pearson's positive correlations and they showed P =

461

0.7430 and P = 0.6710 for the Bligh and Dyer method (Figure 1-C) and pressing method (Figure

462

1-A) respectively, indicating positive correlations between the total tocols content and

463

antioxidant capacity. For the oil extracted by Soxhlet, the correlation was negative (P = -0.3070). 20

464

Results with the same trend have been found by Szydłowska-Czerniak et al. (2008) who reported

465

a linear but not significant correlation in the antioxidant capacity and total content of tocopherols

466

in canola and olive oils extracted by pressing and solvent (R = 0.9053, P = 0.09470). In

467

blackberry seed oil, the ORAC values indicate the presence of antioxidants in oils, but there was

468

no correlation between the tocopherol levels and ORAC values (Buschman et al., 2004).

469

The anti-oxidative activity could be due to the presence of other lipophilic antioxidants, such

470

as the fatty acids, carotenoids, and the phenolic compounds because of a relationship between

471

total phenols and antioxidant capacity has been reported in olive oils (Ninfali et al., 2001).

472

Antioxidant potential radical scavenging activity assay can be determined by measuring the

473

free-radical inhibitory ability of antioxidants by using stable free radicals such as DPPH. For this,

474

assay mixtures of isolated tocols and tocols with tocotrienols were tested between 1.0 to 25.0

475

ppm (Table 6).Three samples had a high inhibitory activity (more than 85%) against DPPH

476

radical formation. The highest DPPH scavenging activity was M1 (a mix with tocols +

477

tocotrienols). The IC50 values were obtained for mix and compounds that showed strong

478

inhibition (more than 80%, Table 6). The highest activity was shown by M1 (1.9 ppm), M2 (7.1

479

ppm), and M3 (4.6 ppm) mixes, although the other mixtures also had a similar IC50 values (M4:

480

11.3; M5: 15.2; S6: 6.1; S7: 19.0; S8: 19.9; S9: 10.6; S10: >25.0 ppm) (Table 6). Several samples

481

assayed using DPPH radical scavenging test showed IC50 values similar or higher than the

482

standards

483

tetramethylchromane-2-carboxylic acid (Trolox), quercetin and α-tocopherol).

compounds

(such

as

β-carotene,

lycopene,

(±)-6-hydroxy-2,5,7,8-

484

Table 6 depicted the inhibition of the formation of TBARS by measuring lipid peroxidation.

485

The thiobarbituric acid (TBA) assay measures the total peroxide content at the later stage of lipid

486

oxidation, involving the quantification of the secondary products formed. The results of the

21

487

TBARS assay showed that lipid peroxidation inhibition values in all the mixed compounds,

488

tested between 1.0 to 25.0 ppm, were higher than 80%. TBARS IC50 values were established for

489

every active sample. The highest activity was shown by M1 (2.8 ppm), M2 (12.9 ppm), and M3

490

(6.9 ppm) mixtures, although the other mixtures also had a similar IC50 values (M4: 9.6; M5:

491

35.2; S6: 6.6; S7: 15.9; S8: 25.4; S9: 19.7; S10: >25.0 ppm). The IC50 values for all samples were

492

shown in Table 6. The highest percentage of inhibition was shown by M1 (IC50: 2.8 ppm).

493

Concerning the antioxidant evaluation by DPPH and TBARS tests, the most active sample was

494

M1. All samples assayed (mixtures and single compounds) showed antioxidant activity. Based on

495

our testing, we cannot establish which are the substances responsible for this bioactivity.

496

However, the presence of fatty acids, tocopherols, and tocotrienols is the key active component in

497

Maqui berry seeds oils. The antioxidant activity of tocols has been widely reported in the

498

literature, and they are also recognized as powerful antioxidants (Lu et al., 2018).

499

Hydroxyl HO are highly reactive free radicals generated through Fenton reaction. Since HO

500

radicals have a short life and a high constant rate, it is unlikely that antioxidants present at

501

biological concentrations will be able to scavenge this kind of radicals. However, antioxidants

502

can act as metal chelators and prevent the formation of hydroxyl radicals, therefore acting as

503

preventative antioxidants (Ou et al., 2002). The hydroxyl radical averting capacity (HORAC)

504

assay measures the ability of the antioxidant to chelate Co(II) before the occurring of Fenton

505

reaction (Ou et al., 2002). HORAC values of tocols were shown in Table 5. HORAC values were

506

ranged between 766.9 and 3280.1 µmol CAE/g in our samples. There was no significant

507

difference observed among the different samples assayed. M1 had the highest and S10 had the

508

lowest value (Table 5) (Prior et al., 2016).

509

The ORAC evaluation was performed for extracts and isolated compounds and ferric

510

reducing antioxidant power (FRAP) was performed only for the isolated compounds. The 22

511

capacity for a compound to scavenge peroxyl radicals generated by spontaneous decomposition

512

of AAPH was estimated by Trolox Equivalents, using the ORAC assay [11, 88?]. The samples

513

assayed in our study showed values in the range of 844.9–19101.8 µmol TE/g sample for ORAC

514

and from 719.9 to 15937.2 µmol Cat E/g sample for the FRAP assay, respectively. The ORAC

515

and FRAP values for compounds presenting in Maqui berry seed oils were given in Table 5. Like

516

our earlier measurements with extracts, M1 had the highest activity in both assays. Similarly, M2

517

showed a very good potency with values of 18295.3 µmol TE/g and 14877.7 µmol Cat E/g for

518

ORAC and FRAP assays, respectively. Other mixtures (M3, M4, and M5) showed 10894.5,

519

9641.1 and 9669.4 µmol TE/g sample for ORAC assay, and 13888.9, 15541.9, and 9910.8 µmol

520

Cat E/g for FRAP assay, respectively (Table 5). There was greater variability observed in FRAP

521

assay which might be due to the fact that reaction of the ferric-TPTZ complex was only partially

522

completed within the 10 min reaction period. In agreement with the ORAC assay, mixtures M1,

523

M2, M3, and M4 showed the greatest values of FRAP. Those data correlated well with the

524

ORAC values for these mixtures.

525

Antioxidant activities show a direct relationship with the content of phenolic moieties

526

present in the mixtures and extracts. Same as DPPH and TBARS activities, M1 was the most

527

active in both the ORAC and FRAP assays (Table 5). These values correlated very well between

528

ORAC and total polyphenolic composition of all extracts and mixtures as well as between FRAP

529

and total phenolic composition of mixtures.

530

The chemical characterization suggests that the different phytochemicals can be antioxidant

531

components (tocopherols and tocotrienols, mainly) in the active extracts and mixtures,

532

determined using ORAC, HORAC, and FRAP methods which give us a direct measure of

533

hydrophilic chain-breaking antioxidant capacity against peroxyl radicals. Thus, the highest

534

ORAC values of our extracts and mixtures showed a strong antioxidant potential (Table 5). 23

535

Besides, the ORAC values of mixtures showed a strong positive correlation with polyphenols

536

content (R > 0.95) (data not shown). The same level of correlation was observed between the

537

FRAP values and phenolic composition of the mixtures.

538

3.5 Digestive Enzyme inhibition

539

Pancreatic lipase (PL) plays a transcendental role in the fat absorption during the digestive

540

biochemical processes, so the inhibition of this enzyme is key for obesity control. The samples

541

used in this study showed an important inhibition level; almost all mixtures used show an

542

inhibitory effect with IC50 values between 71.33 µg/mL and 255.98 µg/mL (Table 6), being the

543

more active M2 and M4 with 71.3 and 90.12 µg/mL, respectively. The obesity is associated with

544

the incidence of type 2 diabetes, so the inhibition of α-amylase and α-glucosidase was assessed,

545

showing a good activity, which could serve as a complement to the use of fruits of Maqui berry

546

(Table 6).

547 548

4. Conclusions

549

To summarize, values found in the determination of the refractive index, iodine index, and

550

density in the seed oils were similar to other commonly consumed vegetable oils. Quality

551

parameters, such as the free acidity and peroxide index were significantly different (p < 0.05) in

552

oils from different extraction methods. However, they were within the parameters established

553

according to the current regulations for other vegetable oils. Concerning tocols, the presence of

554

tocopherols and tocotrienols was identified and they contain more α and γ-tocopherol. β-sitosterol

555

was the only phytosterol present in all the three extracted Maqui berry seed oils higher than other

556

vegetable oils. The oils showed higher antioxidant capacity compared to the reported values for

557

other common vegetable oils (canola and olive). The correlation analysis revealed a positive

24

558

correlation between the total tocols, and antioxidant activity measured with the ORAC test when

559

the oils were extracted using pressing and Bligh & Dyer extraction method; however, there was a

560

negative correlation with the Soxhlet extraction method. Because the oils studied may be

561

considered important sources of beneficial components, it requires additional investigation on

562

Maqui berry seeds. The presence of these compounds could certainly explain their antioxidative

563

activities, but further detailed analysis of the bioactive constituents is warranted.

564 565 566

Acknowledgements

567

The authors gratefully acknowledge support from University of Bío-Bío grant GI

568

152322/EF and Foundation for Agricultural Innovation (Fundación para la Innovación Agraria,

569

FIA) for providing partial support grant FIA PYT-2015-0219. SMAN acknowledgment to Center

570

CRHIAM-FONDAP-CONICYT grant # 15130015 and Research Group CATOXINAL GI

571

172122 / VC Universidad del Bío-Bío. JMBM acknowledge to Fondecyt Program Conicyt grant

572

# 1191127, for partial support. Besides, the authors thanks to Prof David S. Seigler, curator

573

Herbarium of University of Illinois at Urbana-Champaign, USA, for botanical identification of

574

the plant and the English grammar correction of the text. Also, we thank Anne Murray (ESPM,

575

UC, Berkeley, CA, USA); Ana Ma. Garcia-Bores and Antonio Nieto (UBIPRO FES-Iztacala, and

576

Instituto de Quimica, respectively, UNAM, Mexico D.F., Mexico); Kiran Thakur (School of

577

Food and Biological Engineering, Hefei University of Technology, Hefei, P.R. China), for

578

technical assistance. CLCA and IK acknowledge to Seed Funds Program of Conicyt-Chile and

579

UC-Berkeley “2013 UC Berkeley-Chile Seed Grants”, grant (# 2013-02): A New Connection:

580

Potential Cancer Treatment Agents.

581 25

582 583

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804 805 806 807 808

35

809

Tables and Figures

810

Table 1. Physicochemical and quality parameters in Maqui berry seed oils Extraction

Iodine-index

Refractive

Density

Free Fatty acids Peroxide-index

method

(%)

index (nD 40°C) (g/cm3)

(% oleic acid)

(meq de O2 /kg)

Soxhlet

88.10 ± 1.90a

1.469 ± 0.01a

0.92 ± 0.00a

2.20 ± 0.20a

9.94 ± 0.94c

Bligh & Dyer

85.88 ± 1.16a

1.469 ± 0.00a

0.899 ± 0.01a

2.07 ± 0.06a

6.65 ± 0.40b

Press oil

109.53 ± 2.46b

1.473 ± 0.02b

0.921 ± 0.00a

2.27 ± 0.06a

3.4 7 ± 0.31a

(a-c)

811

Values are means values ± SD (standard deviation) (n = 3). Values with different superscript letters

812

indicate significant differences with P < 0.05 according to Tukey. Iodine index result expressed as per cent of iodine absorbed per

813

100 g of Oil. Relative density (20/20°C expressed in g / cm3)

in the same column they

814 815 816

Table 2. Color parameters in Maqui berry seed oils Extraction

L*

Soxhlet

39.61 ± 0.55a .-0.58 ± 0.04b 11.48 ± 0.57a 11.49 ± 0.57a 93.0 ± 0.33b

a*

b*

C*ab

h*ab

Bligh & Dyer 51.88 ± 0.71b 1.16 ± 0.13c

14.15 ± 0.41b 14.20 ± 0.41b 85.2 ± 0.13a

39.39 ± 0.78a -0.9 ± 0.03a

10.16 ± 0.62a 10.20 ± 0.62a 95.2 ± 0.42c

Prensado 817 818

Mean values ± SD (standard deviation) (n = 3). Different superscript letters in the same column indicate

819

significant differences with P < 0.05 according to the Tukey Test. L*: Measures brightness and varies

820

from 100 for the perfect white to zero for black. a*: when positive it is close to red, gray when it is zero

821

and green when negative. b *:measures yellow when positive, gray when zero and blue when negative

822 823

36

824

Table 3. Fatty acid composition (%) of the studied maqui seeds oils Compound

Press Oil

Soxhlet

Bligh & Dyer

C12:0

0.23 ± 0.03a

0.49 ± 0.04b

0.49 ± 0.02b

C14:0

0.65 ± 0.02a

0.96 ± 0.02b

0.96 ± 0.02b

C16:0

8.55 ± 0.01a

8.64 ± 0.05a

8.61 ± 0.08a

C16:1

0.26 ± 0.01b

0.18 ± 0.02a

0.18 ± 0.01a

C18:0

2.63 ± 0.05a

2.78 ± 0.06a

2.78 ± 0.03a

40.6 ± 0.07b

40.63 ± 0.11b

C18:1n9

39.71 ± 0.04a

C18:2n6

45.81 ± 0.05b

45.38 ± 0.19a

45.42 ± 0.03a

C18:3n3

2.35 ± 0.02b

0.98 ± 0.05a

0.98 ± 0.03a

12.87 ± 0.17b

12.82 ± 0.14b

SFA

12.06 ± 0.13a

MUFA

39.97 ± 0.08a

40.77 ± 0.06b

40.81 ± 0.12b

PUFA

48.16 ± 0.08b

46.36 ± 0.17a

46.38 ± 0.03a

3.99 ± 0.14a

3.60 ± 2.31b

PUFA/SFA ratio

3.61 ± 1.40b

825 826

Data represents means ± SD (standard deviation) (n = 3); n.d: not detected. SFA: sum of saturated fatty

827

acids; MUFA: sum of monounsaturated fatty acids; PUFA: sum of polyunsaturated fatty acids. Values

828

with different superscript letters (a-c) within each row are significantly different at P < 0.05.

829

37

830 831

832 833 834

Table 4. Tocols obtained from the studied Maqui berry seeds oils extracted using different methods. Compound α-tocopherol (mg/kg) β-tocopherol (mg/kg) γ- tocopherol (mg/kg) δ- tocopherol (mg/kg) α-tocotrienol (mg/kg) β- tocotrienol (mg/kg)

Press Oil 169.33 ± 11.39c 7.78 ± 2.34a 56.76 ± 2.98c 13.58 ± 3.5a 323.80 ± 20.3 20.20 ± 5.99a

Soxhlet 60.88 ± 22.66a 13.21 ± 6.04a 16.91 ± 1.13a 34.46 ± 1.89b n.d 10.62 ± 2.72a

Bligh & Dyer 123.94 ± 9.60b 14.49 ± 4.52a 29.99 ± 6.81b 27.11 ± 2.44b n.d 17.25 ± 0.57a

γ-tocotrienol (mg/kg)

5.74 ± 1.05a

9.84 ± 2.84a

5.92 ± 0.62a

δ- tocotrienol (mg/kg)

53.9 ± 7.42ab

69.68 ± 6.65b

38.07 ± 9.83a

Total tocols (mg/kg)

327.3 ± 19.32b

215.59 ± 13.68a

256.79 ± 16.87a

β-sitosterol (mg/kg)

3584.60 ± 17.12a

4520.94 ± 51.18b

4180.10 ± 6.38ab

Data represents means ± SD (standard deviation) (n = 3); n.d.: not detected. Values with different superscript letters (a-c) within each row are significantly different at P < 0.05.

835 836

38

Table 5. Antioxidant Capacity of A. chilensis fruit extracts, measured with the ORAC, HORAC and FRAP assays*.

837 838 839 840

Sample

a

HORACc

ORACb µmol TE/g sample

µmol Cat E/g extract

2537.19 ± 152.6a

n.d

A

1116.17 ± 110.3b

n.d.

B

9724.21 ± 552.7c

n.d.

C

M1

19101.8 ± 127.9d

3280.4 ± 96.6a

15937.2 ± 44.1a

M2

18295.3 ± 135.9d

2101.2 ± 55.6b

14877.7 ± 23.3b

M3

10894.9 ± 122.8c

1091.9 ± 23.4c

13888.9 ± 11.5c

948.2 ± 15.6a 1240.7 ± 15.1d 895.5 ± 6.6a 959.9 ± 9.3a 2459.9 ± 87.2b 950.2 ± 7.1a 766.9 ± 5.4e

15541.9 ± 36.4a 9910.8 ± 19.3d 6939.9 ± 12.7e 6878.1 ± 10.9e 10719.9 ± 12.9f 8899.5 ± 19.3d 7425.1± 96.6g

M4 M5 S6 S7 S8 S9 S10 841 842 843 844 845 846 847 848 849 850 851

µmol CAE/g sample

FRAPd

c

9641.8 ± 99.7 9699.9 ± 97.1c 10536.8 ± 190.1c 9800.8 ± 79.7c 11555.9 ± 113.6e 15889.5 ± 111.2f 12654.9 ± 119.6e

a

n.d. n.d. n.d.

Extracts A (Press oil); B (Soxhlet); C (Bligh&Dyer); from seeds of A. chilensis. M1 (Tocols + tocotrienols); M2 (only tocols); M3 (only tocotrienols); M4 (α + γ tocols); M5 (tocols + tocotrienols + β-sitosterol); S6 (γ-tocopherol); S7 (δ-tocopherol); S8 (α-tocopherol); S9 (βtocopherol); S10 (tocotrienols + fatty acids), for detail see Material and Methods. b Expressed as µmol TE/g extract, (µmol of Trolox Equivalents / gram sample). Mean ± SD, n=3. Different letters show significant differences at (P < 0.05), using Duncan’s multiple-range test. c Expressed as µmol Caffeic acid Equivalents (CAE/g sample / gram extract). d Expressed as µmol CatE/g extract, (µmol of Catequin Equivalents / gram sample). * Mean ± SD, n = 3. Values with the same letter are not significantly different (P < 0.05).

39

852

Table 6. IC50 values [µg/mL (ppm)] of mixtures and compounds from seeds oils of A. chilensis

853

needed to inhibit oxidative damage and digestive enzymes*.

3

TBARS4 5.1 ± 0.5b

DPPH

TBARS

M1

1.9 ± 1.3b

2.8 ± 0.9b

c

M2

7.1 ± 1.9c

12.9 ± 1.8c

20.1 ± 2.8

M3

4.6 ± 1.5b

6.9 ± 1.2b

10.9 ± 1.9d

M4 M5 S6 S7 S8 S9 S10 β-carotene lycopene quercetin orlistat acarbose 854 855 856 857 858 859 860 861 862 863 864 865 866

2

Sample

1

11.3 ± 2.2a 15.2 ± 2.8c 26.1 ± 0.9d 21.0 ± 2.1e 11.9 ± 2.8f 20.6 ± 2.5e 25.0 ± 3.8g 10.1 ± 1.1a 12.1 ± 1.9a 7.2 ± 1.7c -

9.6 ± 1.5a 35.3 ± 3.9d 6.6 ± 1.2e 15.9 ± 1.8c 10.4 ± 2.2f 19.7 ± 6.1g 38.9 ± 6.9h 13.1± 2.2c 10.8± 1.9f 1.9 ± 0.9b -

a

5.4 ± 0.6 9.7 ± 1.9d 4.1 ± 0.8a 30.2 ± 2.4e 5.8 ± 1.2f 31.8 ± 2.8g 39.8 ±3.3g 9.9 ± 2.3d 8.9 ± 1.8d 1.2 ± 0.8h -

1

Pancreatic lipase 121.99

α-amylase α-glucosidase >400.0

>400.0

71.33

212.3

343.2

255.98

>400.0

>400.0

90.12 253.66 138.98 142.89 138.01 152.12 989.99 101.88 139.57 119.98 17.0 -

216.9 >400.0 322.9 299.2 198.5 288.6 n.d. 67.8 144.1 35.8 50.5 0.8

346.2 >400.0 388.2 301.1 265.9 377.3 n.d. 165.7 188.9 89.3 36.2 1.5

See Material and Methods explanation of extracts. M1 (Tocols + tocotrienols); M2 (only tocols); M3 (only tocotrienols); M4 (α + γ tocols); M5 (tocols + tocotrienol + β-sitosterol); S6 (γtocopherol); S7 (δ-tocopherol); S8 (α-tocopherol); S9 (β-tocopherol); S10 (tocotrienols + fatty acids), for detail see Material and Methods. 2 IC50 for inhibition of diphenyl picryl hydrazyl radical formation. 3 IC50 for inhibition of peroxidation of lipids, estimated as thiobarbituric acid reactive substances for rat brain procedures. 4 IC50 for inhibition of peroxidation of lipids, estimated as thiobarbituric acid reactive substances for liposomes procedures. * Values are expressed as µg/mL (ppm), See Methods for details. Mean ± SD, n = 3. Different letters show significant differences at (P < 0.05), using Duncan’s multiple-range test.

867 868 869

40

870 871

Figure 1. Pearson's correlations between the total tocol content and antioxidant capacity, (A:

872

Press Oil; B: Soxhlet; C: Bligh and Dyer).

873 874 875

Figure 1A, 1B and 1C in attached files

41

► Phytochemicals from seed oils of A. chilensis showed antioxidant activities ► The IC50 concentration was between 1.8 – 39.8   ml of samles ► These samles showed very ood activities in ORAC, HORAC and FRAP measurements, resectively ► Tocoherols, tocotrienols and -sitosterol were the most active metabolites ► At low concentrations these comounds showed a ood effect on diestive enzymes inhibitory activity: aainst Pancreatic liase, -amylase, and -lucosidase.

Chemoprotective and antiobesity effects of tocols from seed oil of Maquiberry: their antioxidative and digestive enzyme inhibition potential. José Miguel Bastías-Montes1*, Karen Monterrosa1, Ociel Muñoz-Fariña2, Olga García2, Sergio M. Acuña-Nelson1, Carla Vidal-San Martín1, Roberto Quevedo-Leon3, Isao Kubo4, Jose G. Avila-Acevedo5a, Mariana Domiguez-Lopez5b, Kiran Thakur6, Carlos L. Cespedes-Acuña7*.

ALL AUTHORS CONTRIBUTED TO THE ARTICLE IN EQUALITY

*Authors for correspondence and reprints requests: 1J.M.Bastías-Montes, Departamento de Ingeniería en Alimentos, Phone +56-42-2463042, E-mail:[email protected] / 3

C.L.Cespedes-Acuña, [email protected], Department of Basic Sciences, Research Group

in Chemistry and Biotechnology of Bioactive Natural Products, Universidad del Bío- Bío, Andrés Bello Av. #720, Chillan 3780000, Chile.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: