Orange beverage ameliorates high-fat-diet-induced metabolic disorder in mice

Journal of Functional Foods 24 (2016) 254–263

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Orange beverage ameliorates high-fat-diet-induced metabolic disorder in mice B. Escudero-López a, M.S. Fernández-Pachón a,b, G. Herrero-Martín a, Á. Ortega a,c, I. Cerrillo a,b, F. Martín a,c, G. Berná a,c,* a

Area of Nutrition and Food Sciences, Department of Molecular Biology and Biochemical Engineering, Universidad Pablo de Olavide, ES-41013 Sevilla, Spain b Investigador Asociado, Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Av. Pedro de Valdivia 641, Santiago de Chile, Chile c CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM-Instituto de Salud Carlos III, 28029 Madrid, Spain

A R T I C L E

I N F O

A B S T R A C T

Article history:

Metabolic syndrome (MetS) refers to a group of disorders that includes insulin resistance,

Received 26 December 2015

central obesity, arterial hypertension and hyperlipidaemia. Regular consumption of bioactive

Received in revised form 13 April

compounds has consistently been associated with a reduced risk of these disorders. The

2016

aim of this study was to determine if an orange beverage with high concentrations of bioactive

Accepted 18 April 2016

compounds (flavanones, carotenoids, melatonin, and ascorbic acid) and low alcohol content

Available online

(<1%, v/v) improves metabolic parameters through modulation of oxidative stress, lipid profile and inflammatory response in a rodent model of high fat diet (HFD)-induced obesity. Mice

Keywords:

with HFD-induced MetS were fed the orange beverage for 12 weeks (volume equivalent to

Fermented orange juice

250 mL/day in human). Long-term intake of the orange beverage decreased plasma TAG, oxi-

Bioactive compounds

dized LDL and C-reactive protein levels. The present data provide evidence of a beneficial

HFD

effect of orange beverage intake on some outcome parameters related to HFD-induced MetS.

Metabolic syndrome

© 2016 Elsevier Ltd. All rights reserved.

Antioxidant status Lipid profile

1.

Introduction

Metabolic syndrome (MetS) is characterized by a group of metabolic abnormalities, including obesity, dyslipidaemia, hyperglycaemia and hypertension (Huang, 2009). These con-

ditions refer to the clusters of risk factors that increase the prevalence of type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) (Shin et al., 2013). Chronic inflammation and oxidative stress are recognized as major factors involved in the pathogenesis of MetS (Hotamisligil, 2006; Hutcheson & Rocic, 2012). Lifestyle also plays a pivotal role in the development

Chemical compounds: Melatonin (PubChem CID: 896); Ascorbic acid (PubChem CID: 54670067); Ethanol (PubChem CID: 702); Hesperidin (PubChem CID: 3594); Naringenin-7-O-rutinoside (PubChem CID: 25244529); Beta-cryptoxanthin (PubChem CID: 182237). * Corresponding author. Genoveva Berná Amorós, Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo Olavide, Carretera de Utrera Km 1, 41013 Sevilla, Spain. Tel.: +34 954977943; fax: +34 954349813. E-mail address: [email protected] (G. Berná). Abbreviations: AI, atherogenic index; AS, aqueous alcohol solution; AUC, area under the curve; CAT, catalase; CTL, control; CRP, C-reactive protein; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; HFD, high fat diet; IPGTT, intraperitoneal glucose tolerance test; MDA, malondialdehyde; OB, orange beverage; OJ, orange juice; oxLDL, oxidized lowdensity lipoprotein; PAC, plasma antioxidant capacity; SOD, superoxide dismutase; TC, total cholesterol http://dx.doi.org/10.1016/j.jff.2016.04.013 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

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Journal of Functional Foods 24 (2016) 254–263

of several features commonly recognized as MetS (Brown et al., 2009). In fact, chronic consumption of a HFD is strongly associated with the development of obesity in humans and rodents (Buettner, Scholmerich, & Bollheimer, 2007), promotes hepatic oxidative stress (Milagro, Campion, & Martinez, 2006) and triggers some inflammatory processes (Innis & Jacobson, 2007). Increases in MetS incidence has resulted in an increased need for therapeutic and preventative strategies. Experimental and epidemiological data indicate that consumption of energy diluted foods with naturally occurring phytochemicals, in particular fruits and vegetables, prevents obesity and is associated with a lower risk of coronary artery disease (Liu, 2013). Orange juice is known to be a rich source of bioactive compounds such as flavonoids, carotenoids and melatonin (Fernández-Pachón et al., 2014; Stinco et al., 2012; Tounsi et al., 2011). Accumulating evidence suggests that orange juice consumption could beneficially modulate some of the pathophysiological features associated with MetS, such as obesity (Titta et al., 2010), dyslipidaemia (Aptekmann & Cesar, 2013), hyperglycaemia (Ghanim et al., 2007) and oxidative and inflammatory stress (Coelho, Hermsdorff, & Bressan, 2013; Sánchez-Moreno et al., 2003). Recently, alcoholic fermentation processes have been carried out in fruit juices (Mena et al., 2014; Pérez-Gregorio, Regueiro, Alonso-González, Pastrana-Castro, & Simal-Gándara, 2011), resulting in products with higher concentrations of bioactive compounds than the respective substrates. Moreover, the fermentation process involves a final product with alcohol. Our group has previously described the profile of bioactive compounds in alcoholic fermented orange juice (orange beverage), showing increases in flavanone, carotenoid and melatonin contents in relation to the original juice (Cerrillo, Escudero-López, Hornero-Méndez, Martín, & Fernández-Pachón, 2014; Escudero-López et al., 2013; Fernández-Pachón et al., 2014). To assess the potential functional benefits of the orange beverage, a 12-week intervention study was carried out in healthy mice. The results showed that orange beverage intake could exert greater protection against cardiovascular risk factors than the original orange juice (Escudero-López et al., 2015). Based on these results and the evidence from the literature, the aim of the present study was to investigate whether regular consumption of orange beverage reverses the metabolic parameters by modulating the inflammatory response, lipid profile and oxidative stress in a rodent model of HFD-induced obesity (a physiological model of MetS).

2.

Materials and methods

2.1.

Chemicals and reagents

Chemicals were purchased from Sigma-Aldrich Quimica (Alcobendas, Spain).

2.2.

Characteristics of the orange beverage

The company Grupo Hespérides Biotech S.L. carried out the controlled alcoholic fermentation and subsequent pasteurization of commercial orange juice made from Citrus sinensis L. var. Navel late (Huelva, Spain). The beverage obtained was rich in bioactive

Table 1 – Quality parameters, bioactive compounds content and antioxidant activity of orange juice (OJ) and orange beverage (OB). Composition

OJ

OB

pH Total carbohydrates (g/L) Alcohol (% v/v) Total flavanones (mg/L) Naringenin-7-O-glucoside (mg/L) Naringenin-7-O-rutinoside (mg/L) Hesperetin-7-O-rutinoside (mg/L) Hesperetin-7-O-glucoside (mg/L) Isosakuranetin-7-O-rutinoside (mg/L) Total carotenoids Neochrome (mg/L) Auroxanthin (mg/L) Mutatoxanthin (mg/L) All-trans-zeaxanthin (mg/L) All-trans-lutein (mg/L) β-Crytoxanthin (mg/L) Carotene (mg/L) Provitamin A (RAEs/L) Ascorbic acid (mg/L) Melatonin (ng/mL) ORAC (µmol/L) FRAP (µmol/L) TEAC (µmol/L) DPPH (% inhibition)

3.48 ± 0.2 78.2 ± 5.64 0 698.9 ± 20.5 0.6 ± 0.0 363.7 ± 8.4 274.9 ± 10.2 11.5 ± 0.7 47.9 ± 1.3 5.37 ± 0.21 0.37 ± 0.01 0.65 ± 0.03 0.32 ± 0.01 0.40 ± 0.02 0.30 ± 0.01 0.71 ± 0.03 0.37 ± 0.01 75.3 ± 3.58 409 ± 1.8 3.15 ± 0.03 6044 ± 247 10.3 ± 0.4 5.4 ± 0.1 58.1 ± 26

3.43 ± 0.2 53.7 ± 4.65 0.85 ± 0.01 806.2 ± 5.1 0.7 ± 0.0 412 ± 0.8 310.5 ± 1.7 22.0 ± 3.0 60.7 ± 3.1 6.41 ± 0.23 0.44 ± 0.01 0.78 ± 0.04 0.38 ± 0.02 0.47 ± 0.02 0.35 ± 0.02 0.85 ± 0.04 0.46 ± 0.02 90.7 ± 3.97 394 ± 5.4 16.88 ± 1.42 8169 ± 652 9.9 ± 0.1 5.4 ± 0.0 77.4 ± 1

Values are expressed as the mean ± SD. RAEs, retinol activity equivalents; ORAC, oxygen radical absorbance capacity; FRAP, ferric reducing antioxidant power assay; TEAC, Trolox equivalent antioxidant capacity; DPPH radical scavenging assay. Modified from Escudero-López et al. (2013) and Fernández-Pachón et al. (2014) with permission from the authors.

compounds with high antioxidant activity. The quality parameters, bioactive compound contents and antioxidant activities of the commercial orange juice and the final orange beverage were previously evaluated in Escudero-López et al. (2013) and Fernández-Pachón et al. (2014) (Table 1). Briefly, the orange beverage has: (i) 806 ± 5 mg/L flavanones, among which the most abundant are naringenin-7-O-rutinoside (412 ± 0. 8 mg/L) and hesperidin (hesperetin 7-O-rutinoside) (310 ± 1.7 mg/L); (ii) 6.4 ± 0.26 mg/L carotenoids, among which the most abundant is cryptoxanthin (0.85 ± 0.04 mg/L); (iii) 394 ± 5.4 mg/L ascorbic acid; and (iv) 16.8 ± 1.5 ng/mL melatonin (Table 1). In addition, the orange beverage has moderate alcohol content (0.85 ± 0.01%, v/v).

2.3.

Experimental design and HFD protocol

Forty-five male OF1 mice (eight weeks old at the beginning of the experiment, Charles River Laboratories, Barcelona, Spain) were housed individually in cages in a controlled environment (12 h daylight cycle, 22 °C) with free access to food and water. After one week of acclimatization, the mice were randomly divided into two groups: control (CTL) group (n = 10), fed with a control diet (standard diet; 4% of total calorie intake from fat; Scientific Animal Food and Engineering, Spain), and HFD group (n = 35), fed with a HFD (45% of total calorie intake from fat), for 12 weeks, respectively (Fig. 1).

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Journal of Functional Foods 24 (2016) 254–263

Fig. 1 – Schematic diagram of the in vivo experiment. All mice were fed a high fat diet based on lard for 12 weeks and were then divided into 4 groups for the following 12 weeks. First group (n = 7) continued with the same high-fat diet (HFD-CTL), the second group (n = 7) was fed with the same HFD plus orange juice (HFD-OJ), the third group (n = 7) was fed with the same HFD plus orange beverage (HFD-OB), and the fourth group (n = 7) was fed with the same HFD plus alcohol solution (HFD-AS).

After MetS was induced in the HFD over 12 weeks, the control group (CTL) and seven mice of the HFD group were used to determine the biochemical parameters related to MetS. The rest of the mice in the HFD group (n = 28) were divided into four groups (n = 7 per group) as follows: (1) control group (HFDCTL) mice fed with HFD and water; (2) HFD-OJ group, mice fed with HFD and drank commercial orange juice diluted 1:10 in tap water (this dilution factor was calculated based on a proportional intake of 250 mL/day of orange juice – one serving – in human and assuming that mice drink 5 mL/day water; (3) HFD-OB group, mice fed with HFD and drank the orange beverage diluted 1:10 in tap water; (4) HFD-AS group, mice fed with HFD and drank aqueous alcohol solution diluted 1:1000 from a 96% ethanol stock (this dilution factor provides the equivalent amount of alcohol of a proportional intake of 250 mL of orange beverage/day in human). Treatments continued for 12 more weeks (see Fig. 1). At the end of the experimental period, the animals were sacrificed by cervical dislocation. The HFD and experimental solutions, as well as tap water, were available ad libitum to the mice, and bottles were replaced every 2 days to avoid oxidation and precipitate formation. Body weights were recorded once/week throughout the study period and their liquid volume consumption was controlled every 2 days. For glucose determination, blood was collected from the tail vein of conscious animals. Fasting blood glucose was measured using a glucometer (Accu-Chek Aviva, Roche, Indianapolis, IN, USA). Animals were maintained according to a protocol approved by the Pablo de Olavide University Ethical Committee and following the international rules for animal research.

2.4.

Samples collection and preparation

Mice were fasted overnight prior to sacrifice by cervical dislocation. Blood samples were collected by intracardiac puncture, and plasma was separated by centrifugation at 3000 g (10 min, 4 °C). Plasma samples were stored at −80 °C for subsequent analysis. Livers and visceral adipose tissue were carefully dissected, weighed using an electronic balance and immersed in liquid N2 before storage at −80 °C for subsequent analysis.

2.5.

Intraperitoneal glucose tolerance test (IPGTT)

IPGTT was performed after the 12-week HFD period. After overnight fasting, mice were injected intraperitoneally with glucose (2 g/kg body weight), and blood samples (20 µl) were obtained by tail snipping at 0, 15, 30, 60 and 120 min. Glucose was measured with a portable glucometer (AccuCheck II; Roche, Castle Hill, New South Wales, Australia). The glucose responses during the IPGTT were calculated via estimation of the total area under the glucose curves (AUC) using the trapezoidal method (Turner et al., 1990).

2.6.

Plasma antioxidant capacity (PAC)

2.6.1.

Oxygen radical absorbance capacity (ORAC) assay

Plasma samples were diluted (1:2000) in phosphate buffer (75 mM, pH 7.4). ORAC assay was performed according to Ou, Hampsch-Woodill, and Prior (2001).

Journal of Functional Foods 24 (2016) 254–263

2.6.2.

Ferric reducing antioxidant power (FRAP) assay

Plasma samples were diluted (1:10) in distilled water. The ferric reducing ability was estimated according to Delgado-Andrade, Rufián-Henares, and Morales (2005).

2.6.3.

Trolox equivalent antioxidant capacity (TEAC) assay

Plasma samples were diluted (1:10) in water/methanol (1:1). TEAC assay was performed following the procedure described by Delgado-Andrade et al. (2005). All plasma antioxidant capacity measurements were performed after 6 months of nutritional intervention.

2.7.

Antioxidant enzyme activities

Activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were evaluated in liver samples according to Carmagnol, Sinet, and Jerome (1983), Cohen and Somerson (1969), Cribb, Leeder, and Spielberg (1989), and McCord and Fridovich (1969)), respectively. Samples were homogenized in a 1:4 (w:v) buffered solution (100 mM Tris-HCl with 0.1 mM EDTA, 0.1% triton X-100) using a Miccra D-1 homogenizer (Miccra, Germany). The homogenates were centrifuged at 20,800 g (30 min, 4 °C). The resulting supernatants were collected and stored at −80 °C until analysis. Total protein content was assessed by standard Bradford’s procedure (Bradford, 1976). Determination of antioxidant enzyme activities was performed after 6 months of nutritional intervention.

2.8.

Endogenous antioxidant compounds

Albumin, bilirubin and uric acid contents were measured in plasma samples using the reagents and manufacturer’s protocols established by Roche Diagnostics. Measurements were recorded in a COBAS Integra 400 Plus biochemistry analyser (Roche Diagnostics, España). The total glutathione, oxidized (GSSG) and reduced (GSH) contents were determined in liver samples using a commercial kit (EnzoLife Sciences, Plymouth Meeting, PA, USA). Measurements were recorded on a Synergy HT-multimode microplate reader (Biotek Instruments, Winooski, VT, USA). Liver samples were homogenized using a Thomas-Teflon homogenizer in a solution of 5% metaphosphoric acid 1:20 (w/v) and kept on ice. Homogenates were centrifuged at 15,700 g (10 min, 4 °C) and the supernatants were used for the analysis. Measurements of endogenous antioxidant compounds were performed after 6 months of nutritional intervention.

2.9.

Lipid profile analysis

Total cholesterol (TC), high-density lipoprotein cholesterol (HDLc), and triglycerides (TAG) were measured in plasma samples using commercial kits (Invitrogen Life Technologies, Waltham, MA, USA; Roche Diagnostics Systems Inc., Branchburg, NJ, USA and Thermo Scientific, Middletown, WY, USA, respectively). Measurements were recorded on a Synergy HT-multimode microplate reader (Biotek Instruments). Low-density lipoproteins cholesterol (LDL-c) was obtained using the Friedewald’s formula (Friedewald, Levy, & Fredrickson, 1972). Atherogenic

257

index (AI) was obtained using the formula AI = (total cholesterol − HDL cholesterol)/HDL cholesterol.

2.10.

Lipid peroxidation and serum oxLDL measurements

The lipid peroxidation was evaluated by the thiobarbituric acid reactive substance (TBARS) level. TBARS was measured in the liver homogenates according to Buege and Aust (1978), and the results were expressed as malondialdehyde (MDA) equivalents concentration. The oxidized low-density lipoprotein (oxLDL) content was measured in plasma samples using a commercial kit (Cusabio Biotech, Wuhan, China). Measurements were recorded on a Synergy HT-multimode microplate reader (Biotek Instruments). These measurements were performed after 6 months of nutritional intervention.

2.11.

Inflammation status

The plasma levels of C-reactive protein (CRP) were measured by ELISA kits purchased from USCN Life Science Inc. (Wuhan, China). Measurements were recorded on a Synergy HTmultimode microplate reader (Biotek Instruments). These measurements were determined after 6 months of nutritional intervention.

2.12.

Statistical analyses

Data are expressed as the mean ± standard error of the mean (SEM). Student’s t test was applied to establish differences between CTL and HFD groups. One-way analysis of variance (ANOVA) followed by Tukey’s test were used to establish differences between HFD-CTL, HFD-OJ, HFD-OB and HFD-AS groups. A probability value of p < 0.05 was adopted as the criteria for significant differences. These analyses were carried out using GraphPad Instat 3 Software.

3.

Results and discussion

3.1. Effect of the HFD on the body weight and metabolic parameters of adult male mice The HFD group displayed a significant increase in body weight compared with the CTL group (p < 0.05). The HFD induced a weight gain of 11.5% (p < 0.05) after 12 weeks compared with the CTL group (Table 2). A significant increase (p < 0.001) in visceral adipose tissue was observed in the HFD group compared with that at the start of the intervention (Table 2). On the other hand, a decrease (p < 0.01) was observed in the liver weight of the HFD mice group compared with the CTL group (Table 2). After 3 months, the plasma concentrations of TC, LDL-c, TAG, atherogenic index and fasting blood glucose levels were significantly increased (p < 0.001) in the HFD group compared with the CTL group (Table 2). A significant decrease (p < 0.05) in HDL-c levels was observed in the HFD group compared with the CTL group (Table 2). To address the response to glucose administration, we performed an IPGTT. The HFD group presented significantly higher levels of blood glucose than the CTL group at 60 (p < 0.05) and 120 (p < 0.001) min after i.p. glucose injection

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Journal of Functional Foods 24 (2016) 254–263

Table 2 – Effects of 12 weeks HFD on body and visceral weight, fasting blood glucose, lipid profile and atherogenic index of mice. 41.6 ± 0.39

91.8 ± 5.4

54.4 ± 1.1* 40.9 ± 3.2***

50.7 ± 2.4

42.0 ± 1.3**

103.8 ± 6.2

145.7 ± 3.3***

61.5 ± 4.6 88.4 ± 2.8 39.9 ± 0.6 36.3 ± 3.2 1.2 ± 0.1

199.8 ± 8.3*** 205.7 ± 4.4*** 36.7 ± 0.6* 129.2 ± 3.1*** 4.6 ± 0.2***

Results are expressed as the mean ± S.E.M. (CTL n = 10; HFD = 7). * p < 0.05, **p < 0.01, ***p < 0.001 HFD group vs CTL group T0: baseline body weight and fasting blood glucose at the beginning of the nutritional intervention.

(Fig. 2A). Fig. 2B shows a significant increase (p < 0.01) in the AUC of HFD animals. This suggests that HFD animals had impaired glucose management. As expected, these mice presented changes in multiple clinical chemistry parameters. They developed obesity, dyslipidaemia and glucose intolerance; therefore, they can be considered a convenient rodent model for MetS. These results were consistent with those previously reported by Fraulop, Ogg-Diamantino, Fernandes-Santos, Barbosa, and Mandarim-de-Lacerda (2010) and Podrini et al. (2013) who used mouse models after consumption of a HFD. All subsequent analyses presented in this paper will now compare HFD mice given water (HFD-CTL Group) with the HFD fed mice supplemented with orange juice, orange beverage or aqueous alcoholic solution (HFD-OJ, HFD-OB and HFD-AS groups, respectively).

3.2. Effects of OJ, OB and AS intake on body and visceral weights in mice with HFD-induced MetS Mice appear to perfectly tolerate the replacement of drinking water for diluted orange juice (1:10, v/v), orange beverage (1:10, v/v) or alcohol solution (1:100, v/v). At the end of the nutritional intervention, final body weight, liver weight and visceral fat were similar in the four groups of mice, with differences not statistically significant (Table 3). Thus, the new orange beverage was not able to significantly modify body and visceral fat weight. Other studies, such as Titta et al. (2010) in which mice are subjected to HFD with ad libitum intake of blond orange juice observed similar effects. In addition, a review of scientific evidence on the association between alcohol and obesity showed that light-to-moderate alcohol intake does not lead to weight gain during short periods of time (Traversy & Chaput, 2015). According to Reagan-Shaw, Nihal, and Ahmal (2008) the alcohol human equivalent dose consumed by our mice, after the orange beverage intake, was 8.4 mg/kg body weight, which is lower than the moderate alcohol ingestion (0.3–0.6 g ethanol per kg body weight). However, although not significant, in all groups between a 13%

CTL group

350

HFD group

48.8 ± 1.1 16.9 ± 2.4

400 *

300

HFD group

250

**

200 150 ***

100 50 0 0

15

30

45

60

75

90

105

120

Time (min)

B 35000 AUC (mg/dLx120 min)

Body weight (g) Visceral fat (mg/g body weight) Liver weight (mg/g body weight) Fasting blood glucose (mg/dL) TAG (mg/dL) TC (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) IA

CTL group

Blood glucose (mg/dL)

T0

A

**

30000 25000 20000

CTL group

15000

HFD group

10000 5000 0

Fig. 2 – Effects of 12 weeks of the HFD on (A) glucose tolerance and (B) the area under the curve of IPGTT. The data are expressed as the mean ± SEM (n = 7). Symbols represent mean values, and the error bars represent the S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001.

and 28% decrease in visceral fat was observed when compared with control group. In HFD-OJ and HFD-OB groups this reduction could be due to flavonones. In this regard, some authors have evaluated the effects of flavanones in reducing adipose tissue, detailing the possible mechanisms involved. A study of mice fed HFD has shown that intake of naringin, a phenolic compound isolated from the Citrus sinensis, for ten weeks significantly decreased visceral adiposity (19.5%) most likely by activation of AMP-activated protein kinase (AMPK), which increased the expression of peroxisome proliferatoractivated receptor-α (PPAR-α) and inhibited PPAR-γ, decreasing lipid synthesis and promoting oxidation, respectively (Pu et al., 2012). In HFD-AS group, the visceral fat decrease could be due to moderate alcohol intake. Robinson, Prins, and Venkatesh (2011) observed that moderate alcohol intake significantly increase hepatic and skeletal muscle fatty acid oxidation, thereby decreasing visceral fat.

3.3. Effects of OJ, OB and AS intake on antioxidant status in mice with HFD-induced MetS Table 4 shows PAC values (ORAC, FRAP and TEAC), endogenous antioxidant contents [albumin, bilirubin, uric acid and

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Table 3 – Effects of orange juice, orange beverage and aqueous alcoholic solution consumption in body and visceral weights in mice with HFD-induced MetS.

Body weight (g) Visceral fat (mg/g body weight) Liver weight (mg/g body weight)

HFD-CTL

HFD-OJ

HFD-OB

HFD-AS

64.8 ± 3.4a 59.6 ± 5.7a 30.5 ± 1.5a

57.6 ± 2.1a 46.3 ± 5.6a 30.9 ± 1.6a

65.7 ± 2.5a 51.1 ± 5a 32.0 ± 2a

59.4 ± 3.1a 42.8 ± 3.3a 31.8 ± 0.8a

Results are expressed as the mean ± S.E.M. (n = 7). Values with different roman letters in the same line indicate means that are significantly different by one-way analysis of variance followed by Tukey’s test (p < 0.05).

glutathione (total, GSSG and GSH)] and antioxidant enzyme activities (CAT, SOD, GPx and GR) in the four mice groups (HFDCTL, HFD-OJ, HFD-OB, and HFD-AS). Regarding ORAC assay, an increase in values was observed when orange juice (104%) and orange beverage (53.9%) were administered; this change was only significant in the first case (p < 0.05) compared to the HFDCTL group. In contrast, ORAC was not affected in the HFD-AS group after the intervention period, showing similar levels to the HFD-CTL group. These results suggest that the positive effect observed is mainly due to the bioactive compounds. In this way, intervention studies showed that intake of orange juice has a beneficial effect on plasma antioxidant capacity (PAC). Thus, the consumption of 200 mL of orange juice for 26 days increased by 500% the PAC measured by the ORAC assay (Álvarez-Parrilla et al., 2010). The intake of 600 mL for three months also significantly increased CAP subjects with hypercholesterolaemia and hypertriacylglycerolaemia (Foroudi, Potter, Stamatikos, Patil, & Deyhim, 2014). On the other hand, moderate alcohol intake does not exert any effect on PAC, as was also observed by Torres et al. (2015). They observed that PAC modification was due to phenolic compounds contained in wine and not by alcohol. In relation to the endogenous antioxidants, albumin, bilirubin and glutathione concentrations were not affected in any group after the intervention period. Only the uric acid concentration was significantly decreased in the HFD-OJ group (p < 0.01) and the HFD-OB group (p < 0.05) with respect to the HFD-CTL group. It has been observed that at physiological concentrations, uric acid has antioxidant effects, but high plasmatic levels of uric acid is considered as a risk

factor for metabolic diseases (Kanbay et al., 2016; Yu et al., 2016). We observed that mice with HFD-induced MetS had high plasmatic levels of uric acid that significantly decreased with orange juice and orange beverage, but not with alcohol intake. Thus, we propose that the uric acid decrease is due to bioactive compounds found in both orange juice and orange beverage, particularly naringenin (Wang et al., 2012) and melatonin (Cano Barquilla et al., 2014). Regarding the antioxidant enzyme activities, CAT, SOD, GPx and GR levels did not present significant changes after orange juice, orange beverage or aqueous alcoholic solution intake (Table 4).

3.4. Effects of OJ, OB and AS intake on lipid profile and AI in mice with HFD-induced MetS Fig. 3A shows the TAG, TC, HDL-c and LDL-c concentrations in the four mice groups with induced MetS. A significant decrease in TAG levels were observed in HFD-OJ (34.2%; p < 0.01), HFD-OB (36.3%; p < 0.001) and HFD-AS (31.5%; p < 0.01) groups with respect to control group. However, although not significant, we observe an increase in HDL-c levels in the three interventional groups. This increase was higher in HFD-OB group. A not significant, but considerable decrease (44%) was observed in HFD-OB atherogenic index when compared with control group (Fig. 3B). Thus, the three interventional groups improved their lipid profile; however, in the HFD-OB the improvement is higher. This would suggest a synergy effect of bioactive components of orange beverage and its moderate alcohol levels.

Table 4 – Effects of orange juice, orange beverage and aqueous alcoholic solution consumption on antioxidant markers in mice with high fat diet-induced metabolic syndrome.

Total GSH Liver (mmol/g) GSSG GSH Albumin (g/dL) Bilirubin (mMol/L) Uric acid (mg/dL) CAT Liver (U/mg prot) SOD Liver (U/mg prot) GPX Liver (mU/mg prot) GR Liver (U/mg prot) CAP:ORAC (µmol/L) CAP:TEAC (mmol/L) CAP: FRAP (mmol/L)

HFD-CTL

HFD-OJ

HFD-OB

HFD-AS

7.2 ± 0.9a 0.52 ± 0.03a 6.6 ± 1.0a 3.47 ± 0.13a 1.32 ± 0.31a 1.51 ± 0.14a 18.68 ± 1.4a 34.7 ± 3.2a 117.18 ± 5.4a 2.63 ± 0.15a 4484 ± 849a 2.21 ± 0.10a 1.31 ± 0.06a

6.8 ± 0.7a 0.54 ± 0.03a 6.3 ± 0.7a 3.25 ± 0.11a 1.57 ± 0.35a 0.89 ± 0.1b 22.46 ± 2.6a 35 ± 2.8a 127.5 ± 17.2a 2.87 ± 0.17a 9149 ± 1115b 2.22 ± 0.10a 1.20 ± 0.11a

8.6 ± 0.8a 0.53 ± 0.03a 8.0 ± 0.8a 3.39 ± 0.11a 1.3 ± 0.32a 1.07 ± 0.08b 19.7 ± 1.2a 36.3 ± 3.6a 129.6 ± 18.2a 2.72 ± 0.19a 6902 ± 1493ab 2.07 ± 0.12a 1.13 ± 0.07a

6.2 ± 0.8a 0.52 ± 0.03a 5.6 ± 0.8a 3.33 ± 0.1a 1.69 ± 0.34a 1.17 ± 0.12ab 22.59 ± 4.7a 34.6 ± 3.6a 133.1 ± 21.4a 3.09 ± 0.33a 4516 ± 698a 2.24 ± 0.07a 1.23 ± 0.09a

Results are expressed as the mean ± S.E.M. (n = 7). Values with different roman letters in the same line indicate means significantly different by one-way analysis of variance followed by Tukey’s test (p < 0.05).

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A

A 250

a

a

a

150

b b

25

HFD- CTL

a

a

HFD-OJ

a

HFD-OB

b a

a

a

100 a a a a

50

a

a

a

HFD-CTL

HFD-OJ

20

HFD-AS

nmol /mL

mg/dL

200

HFD-OB HFD-AS

a

15 10

0 TAG

TC

HDL-c

LDL-c

5

HFD- CTL

0

B 7 6

a

HFD-OJ a

HFD-OB

5

a

B

HFD-AS

3,5

4

3

AI

a 3

2,5

1

2

0

Fig. 3 – (A) Lipid profiles of the HFD-CTL (control: water), HFD-OJ (orange juice 1:10), HFD-OB (orange beverage 1:10) and HFD-AS (aqueous alcoholic solution 1:1000) mice groups with HFD-induced MetS. (B) Atherogenic index in mice with HFD-induced MetS. The data are expressed as the mean ± S.E.M. (n = 7). Values with different letters indicate significant difference by one-way analysis of variance followed by Tukey’s test (p < 0.05).

Experimental studies have shown that citrus the flavonoids naringenin and hesperidin have hypolipidaemic properties and can reduce TAG (Choi, Yokozawa, & Oura, 1991a, 1991b). Their primary mechanism of action is the inhibition of both acyl CoA:cholesterol acyltransferase and microsomal triglyceride transfer protein, thereby reducing the availability and transfer of lipids to apoB (Wilcox, Borradaile, de Dreu, & Huff, 2001). Moreover, mutant Wrn (Deltahel/Deltahel) mice that show high fasting blood triglyceride levels reversed the hypertriacylglycerolaemia when treated with ascorbic acid (Lebel, Massip, Garand, & Thorin, 2010). On the other hand, in a cross-sectional survey and a study over time, the authors observed that moderate alcohol drinkers with a history of CVD had lower TAG levels than nondrinkers. The relationship between lower TAG levels and increased alcohol intake has been previously well described (Albert, Glynn, & Ridker, 2003). However, no significant differences were observed in TC, LDL-c and HDL-c levels among groups. The current data support findings from Daher, Abou-Khalil, and Baroody (2005) that assessed the effects of a six-month period of chronic orange juice intake in a normolipidaemic rat model, showing that orange juice use did not have a significant effect on plasma TC, LDL-c and HDL-c concentrations compared with the control group. In other studies with humans (Morand et al., 2011; Rangel-Huerta et al., 2015) receiving 500 mL/day or 250 mL/ day of orange juice for 4 and 12 weeks, respectively, no significant differences in TC, LDL-c, HDL-c were found. In an

uMol/L

2

HFD-CTL a

HFD-OJ ab ab

HFD-OB HFD-AS

b

1,5 1

0,5 0

Fig. 4 – (A) Lipid peroxidation as MDA equivalents in the HFD-CTL (control: water), HFD-OJ (orange juice 1:10), HFDOB (orange beverage 1:10) and HFD-AS (aqueous alcoholic solution 1:1000) mice groups with HFD-induced MetS. (B) Oxidized low-density lipoproteins cholesterol (oxLDL) values (µmol/L) in HFD-CTL (control: water), HFD-OJ (orange juice 1:10), HFD-OB (orange beverage 1:10) and HFD-AS (aqueous alcoholic solution 1:1000) groups of mice with HFD-induced MetS. The data are expressed as the mean ± S.E.M. (n = 7). Values with different letters indicate significant difference by one-way analysis of variance followed by Tukey’s test (p < 0.05).

uncontrolled, step-wise increased-dose study in hypercholesterolaemic patients, Kurowska et al. (2000) reported a 21% significant increase in HDL-c but only after the third and highest dose of 750 mL/day. Overall, the evidence of a beneficial effect of citrus juice on cardiovascular health is rather inconsistent. Improvements in blood lipids in some studies are not confirmed in others.

3.5. Effects of OJ, OB and AS intake on lipid peroxidation and inflammatory markers in mice with HFD-induced MetS Fig. 4 shows the lipid peroxidation marker (TBARS) and plasma oxLDL in the four mice groups (HFD-CTL, HFD-OJ, HFD-OB and HFD-AS). The TBARS concentrations were not significantly different among the four mice groups (Fig. 4A). However, a decreasing tendency (31.1%) was observed in the HFD-OB group with respect to the HFD-CTL group. Concerning oxLDL levels,

Journal of Functional Foods 24 (2016) 254–263

900

HFD-CTL

a

800

HFD-OJ

700

ng/mL

(2008) suggested a U-shaped relationship between alcohol consumption and CRP in healthy humans.

HFD-OB b

600 500

261

b

HFD-AS

4.

Conclusion

b

400 300

200 100

0

Fig. 5 – Inflammation status in the HFD-CTL (control: water), HFD-OJ (orange juice 1:10), HFD-OB (orange beverage 1:10) and HFD-AS (aqueous alcoholic solution 1:1000) mice groups with HFD-induced MetS. The data are expressed as the mean ± S.E.M. (n = 7). Values with different letters indicate significant difference by one-way analysis of variance followed by Tukey’s test (p < 0.05).

We developed a mouse model to study MetS by feeding male OF1 mice a saturated fat-HFD (45% of caloric intake coming from lard saturated fats) for 3 months. We used a non-inbred mouse line with high genetic diversity to induce the HFDMetS to be as close as possible to human MetS, because MetS has several causes that act together. In this mouse model, we observed that consumption of our new orange beverage, with high concentrations of bioactive compounds and low alcohol content is able to reduce plasmatic TAG, oxLDL and CRP plasmatic levels. These outcomes are beneficial for the metabolic consequences of obesity.

Acknowledgements a significant decrease (38.5%; p < 0.01) in the HFD-OB group was found compared with the HFD-CTL group (Fig. 4B). Only HFD-OB group had a significant decrease on oxLDL levels. This could be due to the low alcohol content and higher bioactive compounds present in the orange beverage (Table 1). In the orange beverage, a 36% increase of melatonin levels with respect to orange juice was observed (Fernández-Pachón et al., 2014). It has been demonstrated that melatonin has antioxidant effects throughout the increase in glutathione peroxidase (GSH-PX) and paraoxonase (PON 1) activity. The last one is an antiatherogenic enzyme able to inhibit LDL oxidation as well as to convert oxLDL in inactive compounds (Demirtas, Pasaoglu, Bircan, Kantar, & Turkozkan, 2015). In addition, Rao et al. (2003) showed that light to moderate ethanol consumption increases PON1 activity and the level of liver PON1 mRNA in rats and humans. Thus, the decrease observed in oxLDL, in HFDOB group, could be due to a higher activity and expression of PON1 originated by the synergic action of melatonin and moderate alcohol intake. Fig. 5 shows CRP levels in the four mice groups. The CRP levels in the HFD-OJ (49.1%; p < 0.001), HFD-OB (32.4%; p < 0.01) and HFD-AS (34.5%; p < 0.001) groups were significantly lower than those in the HFD-CTL group. Several studies have shown that hesperidin (Rizza et al., 2011), cryptoxanthin (Gammone, Riccioni, & D’Orazio, 2015) and melatonin (Agil et al., 2013) reduced CRP levels in humans and rats. This implies that both bioactive compounds and moderate alcohol consumption could be responsible for the observed beneficial effect. Intervention studies have noted the beneficial effects of regular consumption of orange juice on various biomarkers that indicate the inflammatory state (Buscemi et al., 2012; Dalgard et al., 2009). On the other hand, alcohol could also exert an antiinflammatory effect (immunomodulation) reducing the prevalence of cardiovascular disease (Athyros et al., 2008). During the past 3 decades, numerous epidemiological studies have indicated that moderate alcohol consumption was associated with lower markers of systemic inflammation, including CRP. In this way, Imhof, Blagieva, Marx, and Koenig

We are grateful to Grupo Hesperides Biotech S.L. for providing samples of fermented orange juice. Authors are grateful for the support of the “Junta de Andalucía” and “Ministerio de Ciencia e Innovación” through the Projects P09-AGR4814M and IPT-20111008 respectively. The Research Project grant of BEL is supported by the Junta de Andalucía. Authors belong to PAIDI Research Group BIO311 from Junta de Andalucia.

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