Effect of the emulsion of Sacha Inchi (Plukenetia huayabambana) oil on oxidative stress and inflammation in rats induced to obesity

Journal of Functional Foods 64 (2020) 103631

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Effect of the emulsion of Sacha Inchi (Plukenetia huayabambana) oil on oxidative stress and inflammation in rats induced to obesity

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Johnny P. Ambulaya, , Percy A. Rojasc, Olga S. Timoteob, Teresa V. Barretoc, Ana Colarossia a Micronutrients Laboratory, Laboratories for Research and Development, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, San Martin de Porres, Lima 31, Peru b Bioincuba, Calle José Antonio N°310 Urb. Camacho, La Molina, Lima 12, Peru c Unit of Molecular Biotechnology, Laboratories for Research and Development, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, San Martin de Porres, Lima 31, Peru

A R T I C LE I N FO

A B S T R A C T

Keywords: Obesity Lipid profile Oxidative stress Inflammation Sacha Inchi

Sacha Inchi (Plukenetia huayllabambana), an oleoleguminosae with a high content ω-3, could help normalize alterations of obesity. We evaluated the effect of emulsion of Sacha Inchi (SI) oil on the lipid profile, oxidative stress and inflammation in serum and liver tissue of obesity-induced rats. Five groups induced to obesity by highfat diet were analyzed. One group without treatment, two groups with the emulsion of SI oil (OSI1:0.25 ω-3/day, OSI2:0.5 ω-3/day), one group with atorvastatin and one group with atorvastatin plus the emulsion of SI oil. The treated groups lowered the values of total cholesterol, triglycerides, and LDLc, and increased HDLc. The lipid and protein oxidation marker, IL-6 and leptin decreased in serum, as well as TNF-α hepatic. In contrast, serum adiponectin, antioxidant capacity, and liver catalase activity increased in the OSI2 group. Therefore, the emulsion of SI oil normalizes the lipid profile and decreases oxidative stress and inflammation in an obesity model.

1. Introduction Obesity caused by the excessive accumulation of body fat is considered a global epidemic in both developed and developing countries, with figures bordering 13% of the world population (Hill, Melanson, & Wyatt, 2000; Organización Mundial de la Salud, 2018; Phillips et al., 2012). Obesity is a high-risk factor for the development of several metabolic disorders such as hyperlipidemia, nonalcoholic fatty liver disease, hypertension, insulin resistance, and cardiovascular diseases, the latter being the first cause of death worldwide (Kopelman, 2000; Mann, 2002; National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation & III), 2002; Organizacion Mundial de la Salud, 2017). The chronic consumption of a diet rich in fats and cholesterol can induce hyperlipidemia, accumulation of lipids in the liver, lipid peroxidation, inflammation and hepatotoxicity (Phillips et al., 2012; van der Heijden et al., 2015; Zou et al., 2006). In individuals with a diet rich in saturated fats, alterations in the lipid profile have been observed, such as an increase in triglycerides (TG), total cholesterol (TC), LDL cholesterol (LDLc) and a decrease in HDL cholesterol (HDLc) (Quehenberge & Dennis, 2011; Zou et al., 2006). In the same way, the

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alteration of the levels of these lipoproteins in serum could cause an accumulation of lipids and change of cellular metabolism, which is expressed in inflammation, oxidative stress (EO) and cellular atrophy (De Ferranti & Mozaffarian, 2008; Savini, Catani, Evangelista, Gasperi, & Avigliano, 2013). Oxidative stress in obesity may be due to the excess production of reactive oxygen species (ROS) or a lower antioxidant capacity, due to the decrease of antioxidant molecules or activity of antioxidant enzymes such as superoxide dismutase and catalase (Furukawa et al., 2004; Savini et al., 2013; Zhu, Zhang, Wang, Xiao, & Zhou, 2006). Oxidative stress due to fat accumulation in tissues (De Ferranti & Mozaffarian, 2008; Fan, Zirpoli, & Qi, 2013; Vincent & Taylor, 2006) can produce malonaldehyde (MDA), a marker of lipid oxidation and advanced oxidation products (AOPP), a marker of protein oxidation (Codoñer-Franch, Valls-Bellés, Arilla-Codoñer, & Alonso-Iglesias, 2011; Dalle-Donne, Rossi, Colombo, Giustarini, & Milzani, 2006; Furukawa et al., 2004; Krzystek-Korpacka et al., 2008; Savini et al., 2013; Yagi, 1987). These metabolites can react with proteins and cause an inflammatory response by the activation of monocytes and/or macrophages (Basu, 2004; Dalle-Donne et al., 2006; Liu et al., 2006). Obesity is considered as a pro-inflammatory state since an increase

Correspondence author. E-mail address: [email protected] (J.P. Ambulay).

https://doi.org/10.1016/j.jff.2019.103631 Received 20 February 2019; Received in revised form 11 October 2019; Accepted 12 October 2019 Available online 01 November 2019 1756-4646/ © 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Journal of Functional Foods 64 (2020) 103631

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150 and 200 g obtained from the bioterium of Peruvian University Cayetano Heredia were randomized. The emulsion oil made of the Sacha Inchi species called Plukenetia huayllabambana, (LMH-E1410) was developed by HERSIL S.A. (Lima, Perú) from the lipids part of the seed. The emulsion was obtained from the mixture of Sacha Inchi oil and excipients; for each 1 unit of SI oil, 1.74 units of excipients were added. The excipient was used to stabilize the oil (avoid oxidation), but it did not affect the study treatments because the control groups (NOC, OC) received the excipient and it does not contain lipids. Two presentations of SI emulsion oil with different content of ω-3 (0.5 g/day and 0.25 g/day) were obtained for subsequent treatments. The composition of the Sacha Inchi oil is mainly composed of 54.79% linolenic acid (C18: 3), 26.69% linoleic acid (C18: 2), 9.71% oleic acid (C18: 1), 5.23% palmitic acid (C16: 0) and 1.91% stearic acid (C18:0). The animals were fed a normal diet (LFD, D12450B, 10 kcal% lipids, Research Diets, Inc USA) and a high-fat diet (HFD; D12451, 45 kcal% lipids, Research Inc, USA) for 16 weeks for induction to obesity. The experimental groups formed by the obese rats had high levels of total cholesterol, triglycerides, increase in body weight and Lee and Roher index to be defined as obese rats (Carroll, Zenebe, & Strange, 2006; Ricci & Levin, 2015; Simson & Gold, 1982). These animals that continued with the diet of 45% Kcal of lipids were randomized for the different treatments. The group that received the normal diet was assigned non-obese control (NOC, n = 6) and the obese rats were grouped into 5 groups: obese control group (OC, n = 6), 2 obese groups of 7 rats with doses 2.5 ml of emulsion SI oil (OSI1 (0.25 g ω-3/day) and OSI2 (0.5 g ω-3/ day)), obese group treated with atorvastatin 10 mg/Kg (OAT, n = 6) and obese group atorvastatin plus 2.5 ml of SI emulsion oil (SIO2). All groups were treated by orogastric route for a period of 11 weeks. Once the various experimental treatments were finished, the fasting rats were sacrificed. The blood samples were obtained by cardiac puncture and the serum was prepared by centrifugation to be frozen and stored at −20 °C until further analysis (Donovan & Brown, 2006b, 2006a). The livers were collected from each rat then frozen in liquid nitrogen and stored at −80 °C for further analysis.

of various markers of inflammation has been observed (Sánchez-Lozada et al., 2010). For example TNF-α, a pro-inflammatory cytokine produced mainly by macrophages, it has been positively correlated with adiposity, triglyceridemia and insulin resistance, and negatively with HDLc levels (Bastard et al., 2006; Svegliati-Baroni et al., 2006). IL-6, another pro-inflammatory cytokine that can be produced by adipose tissue and contribute up to 30% of the amount present in serum (Bastard et al., 2006; Zulet, Puchau, Navarro, Martí, & Martínez Hernández, 2007). IL-6 is capable of affecting the metabolism of the liver, increasing VLDL and hypertriglyceridemia (Nonogaki et al., 1995). Leptin increases as the lipid content increases in the adipose tissue and has the ability to inhibit the appetite by inhibiting the production of neuropeptide Y (Lago, Dieguez, Gómez-Reino, & Gualillo, 2007; Zulet et al., 2007). However, it also has other functions such as stimulating the maturation of monocytes, activating lymphocytes, inducing the expression of cytokines IL-6 and TNF-α and stimulating the oxidation of lipids (Curat et al., 2004; Fan et al., 2013; Lago et al., 2007). In obesity models, the increase in leptin has been associated with inflammation as mentioned above (Bastard et al., 2006; Dourmashkin et al., 2006; Ghibaudi et al., 2015). Adiponectin has a different function to leptin, as this improves insulin sensitivity and has an anti-inflammatory effect by the increase of IL-10 and IL-1, and decreased IL-6, TNF-α, and NF-kB (Bastard et al., 2006; Lago et al., 2007). However, it is decreased in obesity, diabetes mellitus type 2 and nonalcoholic fatty liver (Bastard et al., 2006; Fan et al., 2013; SvegliatiBaroni et al., 2006; Xu, Wang, & Lam, 2007). Some food components with the suppressive capacity of adipogenesis can be useful in the prevention of problems associated with obesity (Hwang et al., 2005; H. Kim, Della-fera, Lin, & Baile, 2006). One of these components is the long chain omega 3 polyunsaturated fatty acids (ω-3 LCPUFA). Several studies with ω-3 LCPUFA in people with problems associated with obesity, as well as obesity models in experimental animals show a beneficial effect on the lipid profile (Devarshi, Jangale, Ghule, Bodhankar, & Harsulkar, 2013; Harris, 1997; Schmidt, Kristensen, De Caterina, & Illingworth, 1993; Valenzuela, Tapia, Gonzalez, & Valenzuela, 2011), inflammation (Al-Azzawi et al., 2011; Calder, 2003; De Lorgeril & Salen, 2004; García-Ríos, Meneses María, Pérez-Martínez, & Pérez-Jiménez, 2009; Zamaria, 2004), oxidative stress (Fan et al., 2013; Fernández-Sánchez et al., 2011; Vincent & Taylor, 2006) and gene expression (Devarshi et al., 2013; Ide, 2000; Svegliati-Baroni et al., 2006). α-linolenic acid, known as ω-3, is an essential long-chain fatty acid found in high amounts in many nuts. Some Peruvian species that contain this compound in high percentage are, the Plukenetia volubilis and Plukenetia huayllabambana (Muñoz Jáuregui, Ortiz Ureta, Castañeda Castañeda, Lizaraso Caparó, Barnett Mendoza, Cárdenas Lucero, & Emma, 2013; Ruiz, Díaz, Anaya, & Rojas, 2013). Both species in the form of oil as nuts to have a positive effect on the lipid profile, the first one being the most studied (Garmendia, Pando, & Ronceros, 2013; Gorriti et al., 2010; Huamán et al., 2008), however, Plukenetia huayllabambana has a higher content of ω-3 (53.24% vs 44.7%) (Muñoz Jáuregui et al., 2013; Ruiz et al., 2013) and it has not been shown if it has an effect on oxidative stress and inflammation in the obesity model. Therefore, the objective of the study was to evaluate the effect of Plukenetia huayllabambana emulsion oil on the lipid profile, oxidative stress and inflammation in serum samples and liver tissue of rats induced to obesity.

2.2. Analysis of serum lipids The serum concentration of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDLc) and high-density lipoprotein cholesterol (HDLc) was measured using the commercial enzyme kits (Wiener Lab) in the VALTEK-50 spectrophotometer. 2.3. Malonaldehyde (MDA) Malonaldehyde is a product of lipoperoxidation that was determined in serum by the thiobarbituric acid method (Estepa, Ródenas, & Martín, 2001). The samples were mixed with 0.1 ml of BHT (butylated hydroxytoluene, Sigma), 0.1 ml of FeCl3 6H2O (Merk), 1.5 ml of glycine buffer and 1.5 ml of thiobarbituric acid (Sigma), then cooled and heated in boiled water for 60 min. The levels of the thiobarbituric acid reactive species (TBARS) were determined at an absorbance of 532 nm. For the determination of TBARS in the liver tissue, slight modifications were made to the described; for the test, acetate buffer was used and it was not refrigerated (Ohkawa, Ohishi, & Yagi, 1979). The concentration in samples of serum (μmol/L) and liver (nmol/g protein) was evaluated using a calibration curve with 1,3,3-tetraethoxypropane (Sigma).

2. Materials and methods 2.4. Advanced protein oxidation products (AOPP) 2.1. Animals and diet To determine the AOPP, a marker of oxidative stress, the serum samples were previously centrifuged on a filter plate at 10,000g at 4 °C for 60 min. 200 μl of the supernatant was taken and mixed with 10 μl of potassium iodate (Sigma) and 20 μl of acetic acid (Merk). The concentrations of AOPP were determined using a calibration curve with

All procedures were performed according to the recommendations of the ethics committee of Peruvian University Cayetano Heredia and to the National Institutes of Health guide for the care and use of Laboratory animals. One hundred male Sprague Dawley rats between 2

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Table 1 Effect of the emulsion of Sacha Inchi (Plukenetia huayllabambana) oil on the serum lipids of rats induced to obesity with a diet high in lipids. NOC −1

Cholesterol (mg dl ) Triglycerides (mg dl−1) LDL-C (mg dl−1) HDL-C (mg dl−1)

82.9 52.3 39.6 33.1

± ± ± ±

11** 6.3 7.3* 4.9*

OC

OSI1

102.5 ± 3 62.4 ± 3 56.7 ± 8.3 21.8 ± 6.4

80.9 48.5 30.9 26.3

OSI2 ± ± ± ±

13.7* 10* 8.8** 5.8

88.5 41.7 36.5 37.6

OAT ± ± ± ±

7** 7.6** 9.7** 3.6**

76.7 43.4 23.6 37.3

OATSI2 ± ± ± ±

5.5** 8.6** 7** 3.3**

85.8 ± 4.6** 42.3 ± 7** 33 ± 5** 36.1 ± 7.2**

NOC: non-obese control, OC: obese control group, OSI1: Obese group with the emulsion of Sacha Inchi oil 1, OSI2: Obese group with the emulsion of Sacha Inchi oil 2, OAT: obese group with atorvastatin, OATSI2: obese group with the emulsion of Sacha Inchi oil plus atorvastatin. *P < 0.05 compared with OC. **P < 0.01 compared with OC.

chloramine-T (0–100 μmol/L, Sigma) (Liu et al., 2006; Witko-Sarsat et al., 1996).

2.9. Statistical analysis The statistical analyses were performed with the STATA 12 program. The results were expressed as mean ± standard deviation (SD). The data were analyzed with the one-way ANOVA test. When the pvalue in the ANOVA was < 0.05, then the Bonferroni multiple comparison tests were performed. The Kruskal-Wallis test was performed when the variances were not equal.

2.5. Total antioxidant capacity The antioxidant power reducing ferric (FRAP) and ABTS+ tests were used to quantify the antioxidant capacity in serum and liver tissue (Benzie & Strain, 1996). The TPTZ-Fe+3 complex or the ABTS+ radical can be reduced by the antioxidant of the biological samples by measuring the absorbance changes at 593 nm and 660 nm respectively. For the antioxidant measurement of the liver tissues, they were previously homogenized (10% w/v) at 4 °C and centrifuged at 1500 rpm for 10 min (Katalinic, Modun, Music, & Boban, 2005). For the standard curves, ferrous sulphate at concentrations of 62.5–2000 μmol/L and trolox (Calbiochem) at 1–5 mM were used for the FRAP and ABTS+ assays (Benzie & Strain, 1996; Erel, 2004).

3. Results 3.1. Effect of emulsion of Sacha Inchi (Plukenetia huayllabambana) oil on serum lipids At the end of the 16 weeks of feeding with the HFD diet, lipid profile values were significantly changed in comparison to the LFD control diet (NOC vs OC), with the exception of triglycerides. However, the treatments with the emulsion of Sacha inchi oil (OSI1 and OSI2) significantly decreased the levels of LDLc in 45% and 36% respectively in comparison to the OC group. In the same way, this can be observed for total cholesterol levels. Triglyceride levels also decreased significantly by 22% and 33% for the OSI1 and OSI2 groups respectively compared to the OC group. On the other hand, the level of HDLc increased in the OSI2 group by 72% with respect to the OC group. The group OAT (used as an internal control) and OATSI2 had a similar degree of significance to OSI2 with respect to the OC group (Table 1).

2.6. Enzymatic activity of superoxide dismutase (SOD) and catalase (CT) in liver tissue The activity of superoxide dismutase in the liver was determined by the ability to inhibit autoxidation of pyrogallol (Marklund & Marklund, 1974). The liver tissue was homogenized (10% w/v) at 4 °C with triscacodylic acid buffer containing 1 mM EDTA, pH 8.2. It was immediately centrifuged at 6000g at 4 °C for 10 min and the supernatant was used to evaluate the activity of SOD at 420 nm (Luostarinen, Laasonen, & Calder, 2001). The activity of catalase was determined using the solution K2Cr2O7/acetic acid and H2O2 to form chromic acetate, a colorimetric component that can be read at 570 nm (Sinha, 1972). For this CT test, the liver tissue was previously homogenized (10% w/v) in phosphate buffered saline at 4 °C and centrifuged at 10000 rpm at 4 °C for 15 min. The obtained supernatant was used to evaluate said activity (Noeman, Hamooda, & Baalash, 2011).

3.2. Effect of the emulsion of Sacha Inchi (Plukenetia huayllabambana) oil on markers of oxidative stress

The liver tissues were homogenized (10% w/v) with RIPA buffer (Sigma-Aldrich) and protease inhibitor at 4 °C. Homogenized crude tissues were incubated at 4 °C for 1 h and then centrifuged at 16000g at 4 °C for 60 min (Sánchez-Lozada et al., 2010). The supernatants and sera were used for the immunoassay tests of TNF-α, IL-6, IL-10, and IL-4 according to the procedures of the DB OptEIA commercial kit (BD Biosciences). Millipore ELISA kit was used for serum leptin and adiponectin measurements (catalog number: EZRADP-62K, EZRL-83K respectively). All samples were read with the microplate reader VERSAmax, CA 94089, Molecular Devices.

The lipid oxidation marker (MDA) decreased significantly by 27% in the OSI2 group with respect to the OC group. Similarly, the protein oxidation marker (AOPP) also decreased significantly in 41% and 33% for the OSI1 and OS2 groups, respectively, compared to the OC group. With respect to the total antioxidant capacity in serum, there were no changes for any of the groups that were treated with the emulsion of Sacha Inchi oil (Table 2). The hepatic MDA that was significantly increased in the OC group compared to the NOC group was decreased by 35% and 22% (OSI1 and OSI2, respectively), although it was not significant. On the other hand, the liver antioxidant capacity that was decreased in the OC with respect to the NOC, was significantly increased in the OSI2 group in 63% and 65% for the FRAP and ABTS+ respectively. Similarly, the enzymatic activity of catalase increased by 75% in the OSI2 group when compared with the OC group, approaching the values of the NOC group. Unlike the groups treated with the emulsion of Sacha Inchi oil, the OAT group increased the enzymatic activity of superoxide dismutase by more than 100% (Table 2).

2.8. Determination of protein levels

3.3. Effect of the emulsion of Sacha Inchi (Plukenetia huayllabambana) oil on markers of inflammation

Protein levels were determined using the Pierce® BCA commercial kit (Thermo Scientific).

The serum IL-6 level was not significantly increased by 20% in the OC group compared to the NOC, however, it was significantly

2.7. Immunoassay of cytokines

3

Journal of Functional Foods 64 (2020) 103631

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Table 2 Effect of the emulsion of Sacha Inchi (Plukenetia huayllabambana) oil on MDA, AOPP, antioxidant capacity (FRAP y ABTS+), the activity of SOD y CT on serum and hepatic tissue of rats induced to obesity with a diet high in lipids. NOC

OC

OSI1

OSI2

OAT

OATSI2

Serum MDA (μmol L−1) AOPP (μmol L−1) FRAP (μmol L−1) ABTS (mmol L−1)

11.8 ± 1.8 65.4 ± 11.7** 2117 ± 550 1.8 ± 0.3

12.8 ± 1.7 96.3 ± 20 2323.8 ± 535.6 1.97 ± 0.4

14.8 ± 5.9 56.4 ± 16** 2087 ± 232 2.1 ± 0.4

9.3 ± 2.4* 64.8 ± 16* 2393 ± 501.6 2 ± 0.7

11 ± 3.1 90.5 ± 42 2610 ± 508.4 2.13 ± 0.3

9.9 ± 3.8 61.7 ± 34.2 2260.2 ± 602.6 2.1 ± 0.5

Liver MDA (nmol per mg of protein) FRAP (μmol/mg of protein) ABTS (mmol per mg of protein) SOD (U per mg of protein) CAT (μmol H2O2 per mg of protein)

3.4 ± 1* 205.4 ± 23** 0.64 ± 0.09** 9.8 ± 7.7 2832 ± 718**

5.7 ± 1.5 99.4 ± 23 0.4 ± 0.02 9.4 ± 7.3 1427 ± 428

3.7 ± 1.8 127 ± 46 0.44 ± 0.07 7.9 ± 5.4 1717 ± 308

4.4 ± 1.4 162 ± 18* 0.63 ± 0.06** 13.5 ± 8.5 2481 ± 491**

6.5 ± 2.2 130 ± 38 0.56 ± 0.07** 22.9 ± 11.1* 1721 ± 432

5.8 ± 2.4 140.5 ± 26 0.66 ± 0.01** 11.3 ± 8.1 1589 ± 537

NOC: non-obese control group, OC: obese control group, OSI1: obese group with emulsion of Sacha Inchi oil 1, OSI2: obese group with the emulsion of Sacha Inchi oil 2, OAT: obese group with atorvastatin, OATSI2: obese group with the emulsion of Sacha Inchi oil 2 plus atorvastatin. *P < 0.05 compared with OC. **P < 0.01 compared with OC. Table 3 Effect of the emulsion of Sacha Inchi (Plukenetia huayllabambana) oil on adipokines (leptin and adiponectin) and cytoquines (TNF-α, IL-6, IL-4, and IL-10) on serum and hepatic tissue of rats induced to obesity with a diet high in lipids. NOC

OC

Serum IL-6 (pg ml−1) TNF-α (pg ml−1) IL-10 (pg ml−1) Leptin (ng ml−1) Adiponectin (ng ml−1)

18.1 ± 4.6 27.5 ± 1.4 26.9 ± 20 8.3 ± 2.9** 85.5 ± 13.2

21.8 25.5 20.5 28.9 80.3

Liver TNF-α (pg per mg of protein) IL-4 (pg per mg of protein) IL-10 (pg per mg of protein)

584 ± 61 * 131 ± 20 269 ± 27*

974 ± 158 86.3 ± 9.6 195 ± 33

± ± ± ± ±

6.1 1.6 14 3.7 5.6

OSI1

OSI2

OAT

OATSI2

15.43 ± 8.8 24.56 ± 2.4 25.1 ± 8.4 8.5 ± 3.9** 90.9 ± 6.5

12.45 ± 4.32* 24.23 ± 2.4 28.5 ± 8.24 9.2 ± 5.6** 99.9 ± 6.7**

19.34 ± 13.8 25.6 ± 3.1 20.75 ± 3.1 16.6 ± 8.7** 99.2 ± 5.7**

18.2 ± 5.6 25.3 ± 1.3 22.74 ± 14 18.2 ± 3.6* 98.3 ± 6.3**

727 ± 161 110 ± 35 211 ± 42

638 ± 72** 115.3 ± 36 251.8 ± 51.3

867 ± 147 133.9 ± 36 201.6 ± 18

923 ± 225 189 ± 50** 204.5 ± 27

NOC: non-obese control group, OC: obese control group, OSI1: obese group with emulsion of Sacha Inchi oil 1, OSI2: obese group with the emulsion of Sacha Inchi oil 2, OAT: obese group with atorvastatin, OATSI2: obese group with the emulsion of Sacha Inchi oil 2 plus atorvastatin. *P < 0.05 compared with OC. **P < 0.01 compared with OC.

treatments of the emulsion of Sacha Inchi (Plukenetia huayllabambana) oil they came to normalize (Table 1), therefore, they would be decreasing the cardiovascular risk, which is the leading cause of death worldwide (Mann, 2002; Organizacion Mundial de la Salud, 2017). In a similar study, a group of rats were treatment with Sacha Inchi oil (Plukenetia volubilis) for a period of 8 weeks, and decrease in TC, TG levels and increased the HDLc level. Nevertheless, there were no statistically significant differences, probably because animals were not induced to obesity (Gorriti et al., 2010). In other experiment using chia oil (Salvia hispanica L.), which is high in ω-3 (Bushway, Wilson, Houston, & Bushway, 1984), result in decrease level of TG and increase in HDLc in the serum of not obese rats compared with the control group, but in this case, significant differences were found (Ayerza & Coates, 2005). In another study, in obese rats, linseed oil (Linum usitatissimum) rich in ω-3, decreased levels of CT, TG and LDLc and increased the level of HDLc compared to the obese group without treatment, although the increase in HDLc was not significant (Vijaimohan et al., 2006). A similarity between the 2 mentioned studies and the present study was the high content of ω-3 (chia 57%, flax ≈51-55% and the emulsion of Sacha Inchi oil 54.79%) and the difference could be found in the different proportions between ω -6 and ω-3 (Chirinos et al., 2013), which could have a different impact on the lipid profile (Rodriguez-Leyva, Bassett, McCullough, & Pierce, 2010; Simopoulos, 2008; Simopoulos, 2011; Valenzuela et al., 2014). It has been reported that excessive amounts of ω-6 or a high ω-6/ω-3 ratio, as found in Western diets, promotes cardiovascular diseases and inflammatory processes while increasing levels of ω-3 (a low ratio of ω-6/ω-3) suppresses these effects (Simopoulos, 2008). Studies conducted in humans

decreased by 43% in the SIO2 group. Serum IL-10 levels were also increased by 40% in the OSI2 group with respect to the OC group, although it was not statistically significant. Serum leptin levels increased dramatically by more than 200% in the OC group with respect to NOC, but were decreased in the groups treated with the emulsion of Sacha Inchi oil in 71% and 68% (OSI1 and OSI2, respectively) in comparison to OC (p < 0.01). Likewise, they were also significantly reduced in the OAT and OATSI2 groups with respect to the OC. The opposite happened with the serum adiponectin level, significantly increased by 24% in the OSI2 group with respect to the OC. It also happened with the OAT and OATSI2 groups (Table 3). The TNF-α level of the liver tissue increased significantly in 67% in the OC group with respect to the NOC, which was reduced by 34% in the OSI2 group with respect to the OC (p < 0.01). With regard to IL-4, it increased by more than 100% in the group treated with atorvastatin plus the emulsion of Sacha Inchi oil (OATSI2) compared to the OC group (p < 0.01). The IL-10 levels of the liver tissue were also increased by 8% and 29% for the OSI1 and OSI2 groups respectively with respect to the OC, although they were not statistically significant (Table 3).

4. Discussion It has been reported that increased levels of CT, TG, LDLc and decreased serum HDLc increase cardiovascular risk (Lozano et al., 2008; National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation & III), 2002). In our study in an obesity model with high lipid diet (HFD) these alterations were found and with the 4

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have also shown that the consumption of oils with high ω-3 content has a protective effect on the lipid profile. The consumption of 5 or 10 ml (2 and 4 g ω-3/day) of oil of the species Plukenetia huayllabambana in patients with hypercholesterolemia significantly decreased the levels of CT, LDLc and increased the HDLc after a treatment of 16 weeks (Garmendia et al., 2013). Similar results were observed with oil of the specie Plukenetia volubilis, which also produced a significant decrease in the values of CT, LDLc and an increase in HDLc in healthy people compared with their baseline values (Gonzales & Gonzales, 2014). The suppression of VLDL secretion has been reported in cultures of hepatocytes treated with remnant chylomicrons that were enriched (QRE) with ω-3 LCPUFA (QRE ω-3 LCPUFA), but not with saturated fatty acids. A decrease in the expression of the hepatic 4α nuclear factor (HNF-4α) was also observed; HNF-4α is responsible for the activation of the TG microsomal transfer protein (MTP) and MTP participates by transferring TG and cholesterol to the endoplasmic reticulum for the assembly of VLDL (López-Soldado, Avella, & Botham, 2009). Therefore, it is possible that the decrease of TG in the serum of the groups treated with the emulsion of Sacha Inchi oil is due to a decrease in the secretion and formation of VLDL and LDLc respectively, due to the lower expression of HNF-4α and MTP. This hypothesis should be confirmed in future studies since ω-3 of emulsion of SI oil could be metabolized in ω3 LCPUFA as EPA and DHA. In another study, mice supplemented with ω-3 LCPUFA increased Apo AI (apoprotein AI) (Ahmed, Balogun, Bykova, & Cheema, 2014). In the present study, HDLc increase, possibly associated with an increase in Apo AI, which favors the reverse transport of cholesterol from the extrahepatic tissues by the activation of lecithin cholesterol acyl transferase (LCAT) (Kapur, Ashen, & Blumenthal, 2008). In addition, it has been reported that the concentration of HDLc and LCAT can be modulated by fatty acids in the diet; the consumption of ω-3 can increase up to 83% the activity of the LCAT (Vaysse-Boué et al., 2007). In the present study, LCAT activity was not measured, so it would be important to measure this activity for future studies. MDA is a marker of lipid peroxidation that can cause an immunogenic response, as well as the inactivation of cellular constituents such as enzymes, membranes, proteins, among others (Ayala, Muñoz, & Argüelles, 2014; Dalle-Donne et al., 2006). The levels of serum MDA that was increased in the OC group were decreased by 27% in the OSI2 group, this decrease is possibly due not only to the high ω-3 content but also to tocopherols and other metabolites of the emulsion of SI oil that has been reported in the Plukenetia species (Chirinos et al., 2013; Chirinos, Pedreschi, Domínguez, & Campos, 2015), since it was demonstrated that alpha-tocopherol decreases MDA in vitro and in vivo in microsomes and erythrocytes respectively (Gonzalez Flecha, Repetto, Evelson, & Boveris, 1991; Jain, McVie, & Smith, 2000). A higher content of ω-3 in the form of eicosapentaenoic acid (EPA) in the tissues and serum by the replacement with arachidonic acid (ARA) could have contributed to the decrease of MDA in serum and liver tissue, because the ω-3 and the EPA has a greater capacity to eliminate the superoxide radical and a lower susceptibility to oxidation, unlike other fatty acids (Ander et al., 2010; Lluís et al., 2013; Nassar, Marot, Ovidio, Castro, & Jordao, 2014; Richard, Kefi, Barbe, Bausero, & Visioli, 2008; Valenzuela et al., 2014; Vial et al., 2011). AOPP, a marker of protein oxidation, rises when there are obesity and diabetes and correlates positively with TG and negatively with HDLc in serum; also its increase is associated with inflammatory processes and deterioration of carbohydrate metabolism (Atabek et al., 2006; Codoñer-Franch et al., 2011; Korkmaz et al., 2013; KrzystekKorpacka et al., 2008; Rajendran et al., 2014). Serum AOPP levels were significantly reduced by any of the doses of the emulsion Sacha Inchi oil, similar results were also found with soybean and flaxseed oils in experimental animals with respect to the control. Therefore, the decrease in MDA along with AOPP would favor the improvement of carbohydrate metabolism, the reverse transport of total cholesterol by HDL, decreased cardiovascular risk and oxidative stress (Atabek et al.,

2006; Codoñer-Franch et al., 2011; Liu et al., 2006; Marsche et al., 2009). Antioxidants are important for protection against cellular oxidative damage since it is present under different conditions, such as Alzheimer's disease, cancer and chronic inflammation (Rajendran et al., 2014). An indirect way to measure antioxidants is through antioxidant capacity, which decreases in obese people (Amirkhizi, Siassi, Djalali, & Foroushani, 2004; Karaouzene et al., 2011). The OSI2 group that was treated with the emulsion of SI oil increased the hepatic antioxidant capacity by ABTS+ and FRAP with respect to the OC group, this has probably been contributed by the increase in the activity of catalase, an enzyme that neutralizes hydrogen peroxide and antioxidant capacity of the emulsion of Sacha Inchi oil (Chirinos et al., 2013, 2015). Other studies in experimental animals treated with ω-3 or ω-3 LCPUFA have observed an increase in antioxidant capacity, enzymes antioxidants and decrease in MDA and oxidative stress (de Assis et al., 2012; Marsman et al., 2011). The results of the emulsion of Sacha Inchi oil and the reported results demonstrate the protective effect against oxidative stress by indirectly measuring the antioxidant capacity, which would be protecting against inflammation and EO in the liver. The activity of catalase decreases in animals induced to obesity (Furukawa et al., 2004) and in our study decreased in the obese group induced by a diet high in lipids. The increase in the activity of hepatic CAT by the emulsion of Sacha Inchi oil in OSI2 is probably due to the decrease in nitric oxide (NO) in the liver (data not shown) since NO has the ability to bind to the heme group of catalase to inhibit it (Brown, 1995). The increase in activity could also be attributed to the higher peroxisomal oxidation of the fatty acids, as was demonstrated with perilla oil (61.49% ω-3, 13.92% ω-6), Sacha Inchi (Plukenetia volubilis) or ω-3 LCPUFA (Castillo, Arias, & Farías, 2014; Kim & Choi, 2005; Rincón-Cervera et al., 2016). Inflammation, obesity, and alterations in the metabolism of fatty acids are a triad in the origin of the metabolic syndrome (Robinson, Buchholz, & Mazurak, 2007) and this was found in the obese group when the lipid profile was altered, when IL-6 and leptin in serum were increased, and TNF-α in the liver; also by the decrease of hepatic IL-10. IL-6, a pro-inflammatory cytokine response in the acute phase (Castell, Andus, Kunz, & Heinrich, 1989; Koj, 1989), increases as the number of adipose tissue increases, suggesting that adipose tissue is an important source of increased IL-6 (Fried, Bunkin, & Greenberg, 1998). The level of IL-6 in serum was decreased by the emulsion of Sacha Inchi oil in the OSI2 group, which suggests that the effect could be by the dependence of the content of ω-3 (0.5 g/day) against the OSI1 group (0.25 g ω-3/ day). The decrease of IL-6 in serum could also have favored the decrease of hepatic TNF-α since IL-6 is capable of stimulating its production and targeting the liver by 80% (Castell et al., 1989; Heinrich, Behrmann, Müller-Newen, Schaper, & Graeve, 1998). On the other hand, the ability of IL-6 to inhibit lipoprotein lipase increases serum triglycerides (Nonogaki et al., 1995), therefore, the decrease in IL-6 could have favored the decrease in TG. In a study in diabetic rats, a decrease in the level of IL-6 was found after they were treated with ω-3 intraperitoneally (75 mg/kg/day) for a period of 5 days (Shen, Ma, Shen, Xu, & Das, 2013), which supports the mentioned thing. It has been reported that low levels of EPA were associated with high levels of IL-6 (Ferrucci et al., 2006), and supplementation with flaxseed oil (15 ml /day/3 months) is able to decrease the levels of IL-6 in the serum of dyslipidemic patients (Rallidis et al., 2003), with linseed being a source of high ω-3 content. . In addition, the consumption of oil of the species Plukenetia volubilis (10–15 ml) increases the content of αlinolenic acid and EPA in plasma and tissues of humans and rodents (Gonzales, Gonzales, & Villegas, 2014; Rincón-Cervera et al., 2016; Valenzuela et al., 2014). Therefore, sources with a high content of ω-3 as the emulsion of Sacha Inchi oil decreases IL-6 and reduces inflammation. TNF-α, another pro-inflammatory cytokine produced mainly by macrophages, plays an important role in the pathogenesis of insulin resistance and overexpression has been found in adipose tissue 5

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2012). All the above mentioned would be evidencing a protective effect to the inflammation and in the development of diseases associated with obesity such as cardiovascular, atherosclerosis and insulin resistance (Ohashi, Shibata, Murohara, & Ouchi, 2014; Ouchi & Walsh, 2007).

of obesity models (Bastard et al., 2006). In our obesity model, a decrease in the hepatic TNF-α cytokine was observed in the OSI2 group, probably due to the increase in EPA, since the increase in this was related to its lower production, which favored the anti-inflammatory effect (González-Mañán et al., 2012; James, Gibson, & Cleland, 2000; Mayer et al., 2002; Tappia, Shabraili, Clark, & Grimble, 1997; Valenzuela et al., 2014). Circulating leptin correlates strongly with adiposity (Yan et al., 2012) and in the study, the OC group that did not receive treatment had a high-fat diet (45 Kcal%), showing hyperlipidemia that is associated with macrophage activation and pro-cytokine expression inflammatory (Bastard et al., 2006; Curat et al., 2004; Fan et al., 2013). The increase of TG in adipose and hepatic tissues can lead to loss of sensitivity or resistance to leptin in obesity models or nonalcoholic fatty liver (Ding, Wang, & Fan, 2014; Guzmán-Ruiz et al., 2012; Jung & Kim, 2013; Pan, Guo, & Su, 2014). The emulsion of Sacha Inchi oil treatments decreased significantly the leptin, probably by regulating PPARγ in adipose tissue, as reported with oil perilla or flaxseed supplement, which also have a high ω-3 content (Cammisotto, Gélidas, Deshaies, & Bukowiecki, 2003; Ide, 2000; McCullough et al., 2011). These results suggest that the emulsion o SI oil would be decreasing leptin resistance and inflammation. A different behavior to leptin was found with adiponectin when the obese group received the emulsion of SI oil (OSI2), significantly increased with respect to the OC group. Reduced levels of adiponectin have been reported in obesity models (Younan, Rashed, & Abd El Aziz, 2013). Studies similar to the present, in Sprague Dawley rats treated with linseed oils or ω-3 LCPUFA, also showed an increase in the concentrations of adiponectin in plasma and adipose tissue (Sekine, Sasanuki, Murano, Aoyama, & Takeuchi, 2008; Younan et al., 2013). As mentioned above, the high content of ω-3 in the emulsion of SI oil or oils of the mentioned studies would have changed the proportion of ω6/ω-3 that could have similar physiological effects with different intensities. Rats supplemented with Plukenetia volubilis oil, reported a decrease in the ratio of ω-6/ω-3 in erythrocytes and tissues (RincónCervera et al., 2016; Valenzuela et al., 2014). Likewise, in a study of the nutritional intervention in healthy individuals, they reported a reduction in the ω-6/ω-3 ratio and an increase in serum adiponectin (GuebreEgziabher et al., 2008). Adiponectin can activate lipoprotein lipase and, in our study, its activation would have contributed to the decrease of TG in serum. Additionally, adiponectin is able to favor the oxidation of fat through the activation of Acyl-CoA oxidase and in the present study the probable increase in lipid oxidation would have also contributed to the decrease of TG in serum and increased activity of catalase (De Oliveira, De Mattos, Silva, Mota, & Zemdegs, 2012; Ide, 2000; Kim & Choi, 2005; Rincón-Cervera et al., 2016; Xu et al., 2007). This statement would be attributed to the high ω-3 content of the emulsion of SI oil, as reported in a group of rats that were fed a high-fat diet (45% fat Kcal) for 10 weeks and then ω-3 LCPUFA. Their serum adiponectin levels were increased by 66% compared to their control, as well as mRNA synthesis and adiponectin protein content in the adipose tissue (Younan et al., 2013). The increase of adiponectin by the emulsion of SI oil would also have shown anti-inflammatory effects, which together with the increased ω-3 LCPUFA decreased the production of IL-6 in serum and hepatic TNF-α (Svegliati-Baroni et al., 2006; Valenzuela et al., 2014). Likewise, adiponectin and EPA would have favored the increase, although not significant, of IL-10 in serum and liver tissue. These actions were reported in different studies (Fasshauer et al., 2003; Matsuzawa, 2007; Satoh-Asahara et al., 2012), suggesting that the expression of the adiponectin gene is regulated by IL-6 or by the adiponectin 1 receptor in macrophages (Luo et al., 2013; Wanders, Plaisance, & Judd, 2012). IL-4, an anti-inflammatory cytokine, which increased but not significantly, could have contributed to the increase in adiponectin and decrease in leptin, as reported in mice fed a diet high in lipids and treated intraperitoneally with IL-4 (Chang, Ho, Lu, Huang, & Shiau,

5. Conclusions The main objective of the study was to evaluate the effect of emulsion (Plukenetia huayllabambana) SI oil on the lipid profile, oxidative stress, and inflammation in the serum and liver tissue samples of rats induced to obesity. Therefore, the emulsion of SI oil showed an anti-lipemic effect due to the decrease in TC, TG, LDLc, and HDLc. Also in the contribution to the reduction of oxidative stress and inflammation; the first due to the decrease in MDA and AOPP in serum, and the increase of capacity antioxidant (FRAP, ABTS), and CAT of liver tissue. And in the second, by the decrease in IL-6, TNF-α and leptin, and an increase in adiponectin. The results show that the emulsion of Plukenetia huayllabambana oil would help to normalize the metabolic alterations of the lipids by the lipid profile, oxidative stress, and inflammation that manifest itself in obesity. Ethics statements All procedures were performed according to the recommendations of the ethics committee of Peruvian University Cayetano Heredia and to the National Institutes of Health guide for the care and use of Laboratory animals. Disclosure statement The authors declare that they do not have conflict of interest. Funding source This work was supported by the FINCyT (National Innovation Science and Technology funds) [grant number 046-FINCyT-PITEI]. References Ahmed, A. A., Balogun, K. A., Bykova, N. V., & Cheema, S. K. (2014). Novel regulatory roles of omega-3 fatty acids in metabolic pathways: A proteomics approach. Nutrition & Metabolism, 11(1), 6. https://doi.org/10.1186/1743-7075-11-6. Al-Azzawi, H. H., Wade, T. E., Swartz-Basile, D. A., Wang, S., Pitt, H. A., & Zyromski, N. J. (2011). Acute pancreatitis in obesity: Adipokines and dietary fish oil. Digestive Diseases and Sciences, 56(8), 2318–2325. https://doi.org/10.1007/s10620-0111626-x. Amirkhizi, F., Siassi, F., Djalali, M., & Foroushani, A. R. (2004). Evaluation of oxidative stress and total antioxidant capacity in women with general and abdominal adiposity. Obesity Research & Clinical Practice, 4(3), e163–e246. https://doi.org/10.1016/j.orcp. 2010.02.003. Ander, B. P., Edel, A. L., McCullough, R., Rodriguez-Leyva, D., Rampersad, P., Gilchrist, J. S. C., ... Pierce, G. N. (2010). Distribution of omega-3 fatty acids in tissues of rabbits fed a flaxseed-supplemented diet. Metabolism: Clinical and Experimental, 59(5), 620–627. https://doi.org/10.1016/j.metabol.2009.09.005. Atabek, M. E., Keskin, M., Yazici, C., Kendirci, M., Hatipoglu, N., Koklu, E., & Kurtoglu, S. (2006). Protein oxidation in obesity and insulin resistance. European Journal of Pediatrics, 165(11), 753–756. https://doi.org/10.1007/s00431-006-0165-5. Ayala, A., Muñoz, M. F., & Argüelles, S. (2014). Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity, 2014. https://doi.org/10.1155/2014/ 360438. Ayerza, R., & Coates, W. (2005). Ground chia seed and chia oil effects on plasma lipids and fatty acids in the rat. Nutrition Research, 25(11), 995–1003. https://doi.org/10. 1016/j.nutres.2005.09.013. Bastard, P. J., Maachi, M., Lagathu, C., Kim, J. M., Caron, M., Vidal, H., ... Feve, B. (2006). Recent advances in the relationship between obesity, inflammation, and insulin resistance. European Cytokine Network, 17(1), 4–12. Basu, S. (2004). Isoprostanes: Novel bioactive products of lipid peroxidation. Free Radical Research, 38(2), 105–122. https://doi.org/10.1080/10715760310001646895. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of ‘“antioxidant power”’: The FRAP assay. Analytical Biochemistry, 239(1), 70–76. Brown, G. (1995). Reversible binding and inhibition of catalase by nitric oxide. European Journal of Biochemistry, 232(1), 188–191. https://doi.org/10.1111/j.1432-1033.

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