A randomized, double-blind placebo-controlled study on acceptability, safety and efficacy of oral administration of sacha inchi oil (Plukenetia volubilis L.) in adult human subjects

Food and Chemical Toxicology 65 (2014) 168–176

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A randomized, double-blind placebo-controlled study on acceptability, safety and efficacy of oral administration of sacha inchi oil (Plukenetia volubilis L.) in adult human subjects Gustavo F. Gonzales ⇑, Carla Gonzales Department of Biological and Physiological Sciences, Faculty of Sciences and Philosophy Alberto Cazorla Tálleri, Universidad Peruana Cayetano Heredia, Lima, Peru Insituto de Investigaciones de la Altura, Universidad Peruana Cayetano Heredia, Lima, Peru

a r t i c l e

i n f o

Article history: Received 24 October 2013 Accepted 22 December 2013 Available online 2 January 2014 Keywords: Lipid profile Toxicity Adverse effects Clinical trial Linolenic acid Linoleic acid

a b s t r a c t The study was designed to assess acceptability and side-effects of consumption of sacha inchi oil, rich in a-linolenic acid and sunflower oil, rich in linoleic acid, in adult human subjects. Thirty subjects received 10 or 15 ml daily of sacha inchi or sunflower oil for 4 months. Acceptability was assessed with daily selfreport and with a Likert test at the end of the study. Safety was assessed with self- recording of sideeffects and with hepatic and renal markers. Primary efficacy variables were the change in lipid profile. Subjects reported low acceptability of sacha inchi oil at week-1 (37.5%). However, since week-6, acceptability was significantly increased to 81.25–93.75%. No differences were observed in acceptability with respect to sex or oil volume (P > 0.05). Most frequent adverse effects during first weeks of consuming sacha inchi oil or sunflower oil were nauseas. The side-effects were reduced with time. Biochemical markers of hepatic and kidney function were maintained unchanged. Serum total cholesterol and LDL cholesterol levels and arterial blood pressure were lowered with both oils (P < 0.05). Higher HDL-cholesterol was observed with sacha inchi oil at month-4. In conclusion, sacha inchi oil consumed has good acceptability after week-1 of consumption and it is safety. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The genus Plukenetia belongs to the Euphorbiaceae family and is composed of nineteen species. It is a climbing, monoecious, deciduous plant (Gillespie,1993). In Peru, sacha inchi (Plukenetia volubilis L.) is a wild and cultivated plant, which grows between 100 m above sea level in the low jungle and 2000 m above sea level in the high jungle of the Amazon, in the departments of Amazonas, Cusco, Junín, Pasco, San Martin, Loreto, and Madre de Dios (Dostert et al., 2009). Abbreviations: ALA, alpha linolenic acid; ALP, alkaline phosphatase; ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; BMI, body mass index; CRP, C reactive protein; CV, coefficient of variation; DBP, diastolic blood pressure; DHA, docosahexaenoic acid; EDTA, ethylenediaminetetraacetic acid; EPA, eicosapentaenoic acid; GGT, gamma glutamyl transferase; 5-HEPE, 5-hydroxy-eicosapentaenoic acid; IRB, Institutional Review Board; LA, linoleic acid; LC, long chain; LDL cholesterol, low density lipoprotein cholesterol; FAs, fatty acids; HDL-cholesterol, high density lipoprotein cholesterol; HOMA, homeostasis model assessment; HRQL, health-related quality of life; IR, insulin resistance; x-3 FAs, omega-3 fatty acids; x-6 FAS, omega-6 fatty acids; PUFA, polyunsaturated fatty acids; SBP, systolic blood pressure; SREBPs, sterol regulatory element-binding proteins; VLDL, very low density lipoprotein. ⇑ Corresponding author. Address: Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430 Lima 31, Peru. Tel.: +51 1 3190000x2535. E-mail addresses: [email protected] (G.F. Gonzales), carla.gonzales@ upch.pe (C. Gonzales). 0278-6915/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.12.039

Sacha inchi is a potential oilseed crop because the seeds of this plant are rich in unsaturated fatty acids (FAs), particularly a-linolenic acid (ALA) (18:3n-3), a kind of omega-3 fatty acids (x-3 FAs) and in less proportion linoleic acid (LA), a kind of omega-6 fatty acids (x-6 FAS) (18:2n-6)(Fanali et al., 2011; Bondioli et al., 2006). Sacha inchi also contains proteins and sterols (Guillen et al., 2003; Bondioli et al., 2006). Sacha inchi seed oil has approximately 7:10 ratio of (omega-6) x-6: (omega 3) x-3 FAs (Wang et al., 2012). It is reported that optimal ratio between linoleic acid (LA) and ALA in the diet ranges between 4:12 and 5:1 without exceeding 10:1 (Russo, 2009). ALA through elongation and desaturation processes is converted to long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) as eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids (Brenna et al., 2009). LA competing with the same enzymes used by ALA converts LA to arachidonic acid (eicosatrienoic acid), a fatty acid of the omega-6 class (Monteiro et al., 2013). Flaxseed/linseed oil is characterized by high content in ALA but not in LA. This oil inhibited arachidonic acid (AA) metabolism (Kaithwas et al., 2011) due to the fact that ALA is preferentially producing EPA and DHA (Monteiro et al., 2013) by competition with the same enzymes that produce AA from LA. Although is suggested that conversion of ALA to DHA is limited in humans, a recent study have demonstrated that oral

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consumption of a single dose of 10 or 15 ml sacha inchi oil (Plukenetia volubilis L.) was able to increase plasma DHA levels within 24 h (Gonzales et al., 2014). The existing literature supports the protective effects of LC n-3 PUFA in maternal and offspring health, cardiovascular health, insulin sensitivity, the metabolic syndrome, cancer, critically ill patients, immune system disorders (Kremmyda et al., 2011) and obesity (Buckley and Howe, 2010). Consumption of LA results in a lower plasma total and LDL cholesterol (Sacks and Katan, 2002). Similarly, consumption of oil (5 or 10 ml) from the species Plukenetia huallabamba, which is rich in ALA (4 g/10 ml) resulted in reduction in total and LDL cholesterol after 4 months of treatment (Garmendia et al., 2011). Plukenetia huallabamba is a different species than sacha inchi (P. volubilis L.). The interest in sacha inchi has increased during the last years and this is appreciated in the evolution of exportations from Peru. During 2005 was exported USD 28,811.90 and in 2012 USD 3’168,285.43 (Promperu, 2013). Actually, sacha inchi oil is an alternative for fish oil which also is rich in omega-3. Traditionally the taste of fish oil has been perceived as unpleasant (Yaxley et al., 2011) and this is enough to subject choose to refuse the use of fish oil. Palatability and enjoyment of foods are often tied to their energy density and therefore fat content. Energy-dense foods that are rich in fat are more palatable than are many low-energy–density vegetables and fruit (Drewnowski, 1998). It is not known how pleasant the perception is after sacha inchi oil consumption. Since sacha inchi oil has increased as alternative of food in different parts of the world, it is necessary to know about acceptability, and safety of its consumption. The present study has been designed to assess the acceptability of two dose (10 and 15 ml) of sacha inchi oil and compared with sunflower oil; describe the frequency and extent of self-reported side effects of sacha inchi or sunflower oils. The study also describes efficacy of sacha inchi oil orally consumed during 4 months in a randomized, doubleblind, placebo (sunflower oil)-controlled study. 2. Materials and methods 2.1. Subjects Eligible subjects were no vegetarian, men and women, 25–55 years of age, BMI (>20-<35 kg/m2), without history of hyperlipidemia or any other disease likely to affect lipid metabolism, or to consume fatty acid, lipid or antioxidant supplements, or any medication likely to interfere with the objectives of this study. Subjects were recruited through local advertisement. None of them was referred directly by a physician. The sample size has been calculated with a 20% of error, confidence level at 95% and 50% heterogeneity. According to Food and Drug Administration (FDA), the total number of subjects and patients included in Phase 1 studies varies with the drug, but is generally in the range of 20–80. FDA (2012). Thirty-four subjects were enrolled, 4 of whom withdrew the study within the first month. The study was approved by the Institutional Review Board (IRB) of the Universidad Peruana Cayetano Heredia and from the National Institute of Health from the government of Peru. All subjects were informed on the purpose and procedures of the study and signed a consent form. The participation of the volunteers included, among other things the filling out of a questionnaire and allowing sampling of venous blood. The questionnaire explored sociodemographic characteristics, health status, personal and family medical history and lifestyle information, including dietary and smoking habits. Six subjects declare to smoke but less than one pack a year. All participants also underwent anthropometric and body composition measurements.

2.2. Design This was a 4-months randomized, double-blind, placebo-controlled study comparing acceptability, safety and efficacy of oral administration of sacha inchi (Plukenetia volubilis L.) oil compared with sunflower oil in adult human subjects (Phase I clinical trial). A group consumed sacha inchi oil and the other group sunflower oil. Men and women were randomly assigned to each group.

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The oils were consumed each day in the morning in dose of 10 or 15 ml. Subjects were randomly assigned according dose of oil to be consumed. For this purpose, the oil contained in a dark bottle of 250 ml was placed in a volumetric cup to obtain the required volume. The sacha inchi oil contains 47.7% ALA and 10 ml sacha inchi oil contains 4.4 g ALA whereas 15 ml contains 6.6 g ALA. The linoleic acid content was 34.9% compared with 57.2% from sunflower oil (Gonzales et al., 2014). Meat, poultry, eggs, dairy products, fruits and vegetables, cereals and grains and all food containing no fat were unrestricted throughout the study. The subjects were asked to consume no fish, salad or cooking oils, mayonnaise, sauces, salad dressings, desert, bakery foods throughout the period of the study. 2.3. Oil products Sacha inchi (Plukenetia volubilis L.) seed was identified as San Martin ecotype (P vol-00115052007), and the voucher of the specimen was deposited at Herbarium Amazonense of the Universidad Nacional de la Amazonia Peruana UNAP)-Iquitos, Peru. Sacha inchi oil (Plukenetia volubilis L.) was obtained from San Martin-Peru. Oils were produced by cold pressed method. For this research, sacha inchi oil (EcolineÒ) (Sanitary Register is C1300710N/UESASA) was used. Premium PrimorÒ is an edible refined oil obtained from sunflower seed. According the protocol, the oil in dark bottles (250 ml) with labels 1 or 2 had been kept under appropriate storage conditions at Research and Development Laboratory at the Universidad Peruana Cayetano Heredia (LID, UPCH). 2.4. Blood samples and analyses Blood was drawn from forearm vein and collected in two tubes, one containing EDTA as the anticoagulant to obtain plasma, and the second without anticoagulant to obtain serum. Blood was obtained after an overnight fast between 08:30 and 10:30 h at the beginning of the study, and again every 4 weeks throughout the study. Plasma and serum were separated by centrifugation at 2000g for 10 min at 4 °C. Then, samples were split into aliquots and frozen at 70 °C before assays. 2.5. Anthropometric measurements Anthropometric measurements were also performed, namely body weight, and height. Body weight was measured while the participants were wearing light clothing and no shoes. Height was determined with a stadiometer to the nearest of 0.1 cm. Body mass index (BMI) was calculated as kg/m2. These data were obtained every month throughout the study. Subjects were weighed at the same time of day at the start the study and after 1, 2, 3 and 4 months of interventions. 2.6. Acceptability Subjects daily fill a notebook indicating about acceptability of the oil. In addition, a monitor called daily to each participant asking for taste of the oils. Participants were also asked at the end of the study to score taste on a 5-point Likert scale ranging from ‘‘extremely unpleasant’’ (score 1) to ‘‘extremely pleasant’’ (score 5), as this scale has been reported to be reliable for assessment of taste preference (McGough et al., 2006). 2.7. Safety 2.7.1. Self-recording This part of the study was assessed through a daily survey by phone call. Subjects were asked about experience of adverse side-effects. For safety assessment, adverse events were documented at all visits, after general and specific patient questioning. 2.7.2. Health status score A generic health-related quality of life (HRQL) questionnaire was used for assessment of health status. The health questionnaire based on 20 questions or items about health included 7 dimensions or domains: Physical functioning scale, Role Physical scale, Bodily pain scale, general health, Vitality scale, Role emotional scale and mental health scale. All items are scored on a scale of 0–100, with higher scores indicating better health-related quality of life. This questionnaire has been previously validated in our population (Gonzales et al., 2013). 2.7.3. Biochemical markers Blood samples were taken for routine hematology and biochemistry screens: hemoglobin, hematocrit, serum C reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, bilirubin, gamma glutamyl transferase, serum albumin, serum total proteins, serum creatinine, and serum uric acid levels. These markers were assessed before and at months 1, 2, 3 and 4 of sacha inchi or sunflower oil consumption.

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Compliance was monitored by empty bottle counting and by questioning the subjects at the treatment end. C-reactive protein (CRP), an inflammatory marker, was assessed using the NycocardR CRP single test system using NycoCard READER II (Axis-Shield PoC AS, Oslo, Norway). The test is a solid phase immunometric assay. Intra-assay coefficient of variation (CV) was <7%. The reference range was 65 mg/L. Hemoglobin (Hb) concentration was measured in situ using the HemoCue system (Anglholm, Sweden). Hematocrit measurements were done using the microhematocrit method. Hb values were expressed as g/dL and hematocrit in percentage. AST and ALT were measured by kinetic tests (Cypres diagnostics, Langdorp, Belgium) using a spectrophotometer at 340 nm. Normal values are up to 26 U/L and 29 U/L, respectively in men and up to 22 U/L in women. Intra-assay CV was 3.91% for AST and 1.84% for ALT. Inter-assay CV was 4.20% for AST and 3.04% for ALT. Sensitivity was 1.20 U/L and 5.44 U/L for ALT and AST, respectively. Gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) were measured by kinetic tests (Cypres diagnostics, Langdorp, Belgium) using a spectrophotometer at 405 nm. Normal values for GGT were <50 U/L (Oh et al., 2011) and for alkaline phosphatase were 73–203 U/L. Intra-assay CV was 1.44% for GGT and 1.30% for ALP. Inter-assay CV was 2.28% for GGT and 2.61% for ALP. Sensitivity was 2.56 U/L and 4.26 U/L for GGT and ALP, respectively. Serum bilirubin was measured by a commercial kit (Cypres diagnostics, Langdorp, Belgium) using a spectrophotometer at 555 nm. Normal values are up to 1.10 mg/dL. Detection limit was 0.04 mg/dL. Intra-assay CV was 3.55% and inter-assay CV was 4.12%. Serum creatinine and uric acid levels were determined using commercial kits (Cypres diagnostics, Langdorp, Belgium). Measurements were done using spectrophotometer at 505 nm and 520 nm, respectively. References values for serum creatinine are 0.7–1.4 mg/dL for males and 0.6–1.1 mg/dL in females, whereas for uric acid were 3.6–7.7 mg/dL in males and 2.5–6.8 mg/dL in females. Limit of detection was 0.11 mg/dL for creatinine and 0.03 mg/dL for uric acid. Intra-assay CV was 1.99% and 0.63% for creatinine and uric acid, respectively. Between-assay CV was 1.68% and 1.58% for creatinine and uric acid, respectively.

2.8. Efficacy Blood glucose, serum lipid profile and levels of serum insulin, testosterone and estradiol were measured to assess efficacy. The concentrations of these compounds were assessed before and at months 1, 2, 3, and 4 of the oils consumption. Serum triacylglycerol, total cholesterol, and HDL-cholesterol levels were determined using commercially available enzymatic colorimetric assays (Cypres diagnostics, Langdorp, Belgium) on spectrophotometer at 505 nm. Serum triacylglycerol levels and serum total cholesterol levels were measured by an enzymatic-colorimetric test. HDL-cholesterol was measured by phosphotungstic precipitation method. Normal range for triacylglycerol was 40–160 mg/dL in men and 35–135 mg/dL in females; for serum total cholesterol was less than 200 mg/ dL and for HDL-cholesterol were 45–65 mg/dL in women and 35–55 mg/dL in men. Intra-assay CV was 0.60%, 0.71% and 1.18% for triacylglycerol, total cholesterol and HDL-cholesterol, respectively. Between-assay CV was 1.83%, 1.24% and 2.72% for triacylglycerol, total cholesterol and HDL-cholesterol, respectively. LDL-cholesterol was calculated according to the Friedewald formula: LDL-cholesterol = [Total cholesterol – VLDL cholesterol-HDL cholesterol] (Gazi and Elisaf, 2007). VLDL cholesterol is calculated as triacylglycerol/5. For glucose measurement a portable glucometer Accu-Chek (ROCHE, USA) was used. This method is based in the activity of the glucose oxidase enzyme. Serum testosterone (Cat.TKTT1), serum estradiol (Cat. TKE21) and serum insulin (Cat. TKIN2) levels were measured by a solid-phase 125I radioimmunoassay (RIA), (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Normal values for testosterone in men were 245–1836 ng/dL and for females were not detectable (ND) to 81 ng/dL. Sensitivity of the assay was 4 ng/dL. Within assay CV ranges from 5% to 10% and Inter-assay precision ranges from 5.9% to 12%. Normal values for estradiol were ND-44 pg/mL in normal adult men and from <20 to 375 pg/mL in women depending of the cycle phase. Sensitivity was 8 pg/mL, within-assay coefficient of variation was 4–7% and between-assay coefficient of variation was 4.2–8.1%. Normal values for insulin were <29.4 mIU/L; sensitivity was 1.2 mIU/L, intra-assay coefficient of variation was 3.3–12.7% and inter-assay coefficient of variation was 6.6–12.4%. Insulin resistance (IR) was estimated using the homeostasis model assessment (HOMA-IR) with the following formula: HOMA-IR = fasting insulin (mIU/L)  fasting glucose (mg/dL)/405 (Matthews et al., 1985). Serum albumin and total proteins were measured by colorimetric test. The test for albumin uses bromocresol reagent whereas for total protein was measured using the Biuret reagent (Cypres diagnostics, Langdorp, Belgium). Albumin was measured at 630 nm and normal values ranges from 3.5 to 5.0 g/dL. Serum total protein was measured at 546 nm and normal range varies from 6.6 to 8.3 g/dL. Limit of detection was 0.04 g/dL and 0.008 g/dL for albumin and total protein, respectively. Intra-assay CV was 2.78% for albumin and 1.92% for total protein. Inter-assay CV was 2.77% for albumin and 2.39% for total protein.

Sitting BP was measured in the left arm using an aneroid sphygmomanometer. Systolic (SBP) and diastolic (DBP) blood pressure were obtained from each subject and results were expressed in mm Hg. 2.9. Statistical analysis Data were analyzed by using the Stata 10 version software (Stata Corp LP, College Station, TX, USA). Continuous variables are presented as means ± standard error of the mean and categorical variables as absolute frequencies. The chi-square test evaluated associations between the categorical variables and the Student’s t test and the ANOVA were applied to evaluate differences in mean values of normally distributed data between two means or more than two means, respectively. Furthermore, to compare differences between two means after ANOVA, post hoc analyses were performed. In cases of non homogeneous variables, the Kruskal–Wallis test and Mann–Whitney U test were used. Multiple regression analysis were also performed for biochemical markers after controlling for age, sex, type of oil consumed, volume of oil consumed and time of treatment. The level of significance was defined at P < 0.05.

3. Results 3.1. General characteristics of the population Four subjects, all females, left the study, one subject from the sunflower oil group due to a surgical intervention no associated with the study or the ingestion of the oil. A second female, from the sacha inchi oil group, was retired after 5 days of the beginning the study due to no tolerance of the oil taste, and headache. The third subject (sacha inchi oil) was retired from the study after 17 days for personnel reasons, and the fourth female (sacha inchi oil) abandoned after 18 days of beginning the study due to personal reasons. The final sample size included 30 subjects (13 men and 17 women). The groups of study were comparable, as sex proportion (male/ female), age, weight, height, BMI, SBP, DBP, hemoglobin/hematocrit, and blood glucose were similar between intervention groups (P > 0.05). 3.2. Acceptability Subjects report low acceptability of sacha inchi oil at first week of consumption (37.5%). However, since week-6, the acceptability was significantly increased such as 81.25–93.75% of subjects accepted the consumption of the sacha inchi oil. Sunflower oil had higher acceptability at week-1 (71.43%) and this value was maintained throughout the duration of the study (P > 0.05) (Table 1).

Table 1 Acceptability of the oral ingestion sacha inchi oil or sunflower oil.

a

Weeks

Sacha inchi oil⁄

Sunflower oil⁄⁄

P

1 2 3 4 5 6 7 8 9 10 11 12 13–17

6/16 (37.50) 11/16 (68.75) 11/16 (68.75) 11/16 (68.75) 11/16 (68.75) 15/16 (93.75)a 13/16 (81.25)b 14/16 (85.50)c 14/16 (85.50)c 14/16 (85.50)c 14/16 (85.50)c 14/16 (85.50)c 13/16 (81.25)b

10/14 (71.43) 10/14 (71.43) 10/14 (71.43) 10/14 (71.43) 9/14 (64.29) 8/14 (57.14) 8/14 (57.14) 10/14 (71.43) 10/14 (71.43) 10/14 (71.43) 10/14 (71.43) 10/14 (71.43) 10/14 (71.43)

0.06 >0.1 >0.1 >0.1 >0.1 0.03 >0.1 >0.1 >0.1 >0.1 >0.1 >0.1 >0.1

P = 0.002; bP = 0.03; cP = 0.009 with respect to values at week 1. P = Significance between interventions. ⁄ Chi square test = 37.34; P < 0.002 between weeks. ⁄⁄ Chi square test = 2.48; P = 1.00 between weeks.

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At week-1, there was a trend for higher acceptability to sunflower oil than to sacha inchi oil (P = 0.06). From weeks 2 to 5, no differences were observed between subjects consuming sacha inchi or sunflower oil (P > 0.05). At week-6, a higher acceptability was observed with sacha inchi oil than with sunflower oil (P = 0.03). From weeks 7 to 17 no differences in acceptability were observed between interventions (Table 1). No differences were observed in acceptability of men and women (P > 0.05). No differences were also observed when compared consumption of 10 ml with 15 ml of sacha inchi oil (P > 0.05) (Data do not shown). No differences were observed between sacha inchi oil and sunflower oil for different taste rating in the Likert test (P > 0.05). At all, 62.5% of subjects consuming sacha inchi oil and 85.7% of

Table 2 Self-report of side-effects during the consumption of sacha inchi oil and sunflower oil. Weeks

Sacha inchi oil

Sunflower oil

P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

11/16 (68.75) 9/16 (56.25) 10/16 (62.50) 7/16 (43.75) 5/16 (31.25)a 4/16 (25.00)a 4/16 (25.00)a 2/16 (12.50)b 2/16 (12.50)b 1/16 (6.25)c 1/16 (6.25)c 1/16 (6.25)c 1/16 (6.25)c 2/16 (12.50)b 1/16 (6.25)c 2/16 (12.50)b 2/16 (12.50)b

8/14 6/14 4/14 1/14 2/14 1/14 0/14 1/14 0/14 0/14 1/14 1/14 0/14 0/14 0/14 0/14 1/14

>0.1 >0.1 0.08 0.04 >0.1 >0.1 0.1 >0.1 >0.1 >0.1 >0.1 >0.1 >0.1 >0.1 >0.1 >0.1 >0.1

(57.14) (42.85) (28.57) (7.14)e (14.28)d (7.14)e (0.00)f (7.14)e (0.00)f (0.00)f (7.14)e (7.14)e (0.00)f (0.00)f (0.00)f (0.00)f (7.14)e

Sacha inchi oil; chi square: 61.90; P < 0.0001. Sunflower oil; chi square: 63.26; P < 0.0001. P = 0.03; bP = 0.0032; cP = 0.0006; dP = 0.046; eP = 0.01; fP = 0.0019 with respect to values at week 1.

a

subjects consuming sunflower oil reported that taste was pleasant (P > 0.05 between intervention groups) (data do not shown). No differences in acceptability were observed with different volume of oils (10 or 15 ml) (data do not shown).

3.3. Side effects Sacha inchi oil produced more side-effects within first 4 weeks of consumption. Thereafter, the reports of side-effects with sacha inchi oil were significantly reduced. Sunflower oil produced more side-effects within the first three-weeks of consumption and thereafter a significantly reduction in side-effects were observed (Table 2). Women reported more side-effects than men (P < 0.05). The side-effects described by subjects were nausea, headache, tiredness, sleep, vomiting, flatulence, belching, heartburn, higher frequency of depositions and constipation. The most frequent adverse effects described during first weeks of consuming sacha inchi oil were nausea (14 subjects) followed by eructation (5 subjects). Other minor adverse effects were hot flash, headache, cramping, and constipation, whereas in those consuming sunflower oil were nauseas (3 subjects), and flatulence (2 subjects). Minor adverse effects were cramping, flatulence, and high frequency of depositions. Hot flashes observed in one woman disappeared completely at week-9 of treatment with sacha inchi. The side-effects were reduced with time such as at week-17 after sacha inchi oil, only one subject with headache + nausea and one with nausea were observed. With sunflower oil one subject reported adverse effect at week-17 of consumption. The assessment of health status score showed similar basal values, in the two groups of intervention (P > 0.05). Throughout the period of interventions, health status scores based in the generic HRQL questionnaire remained unchanged overtime in the groups consuming sacha inchi oil (P > 0.05) or sunflower oil (P > 0.05) and no differences were observed between interventions (P > 0.05) (Data do not shown). Data on biochemical markers of hepatic and kidney function are shown in Table 3. Serum ALT and AST levels, markers of the

Table 3 Biochemical markers in adult human subjects during sacha inchi oil or sunflower oil consumption.

a

OIL

ALT (U/L)

Sacha inchi Sunflower

30.99 ± 8.05 29.62 ± 4.51

30.35 ± 3.46 28.40 ± 1.79

28.33 ± 4.22 27.81 ± 2.51

25.89 ± 2.02 28.98 ± 2.62

24.14 ± 2.26 28.39 ± 2.18

>0.05 >0.05

AST (U/L)

Sacha inchi Sunflower

26.27 ± 5.86 23.32 ± 4.80

32.03 ± 6.02 32.13 ± 3.53

28.45 ± 6.80 28.38 ± 5.26

25.58 ± 2.80 27.29 ± 3.21

26.59 ± 2.92 30.20 ± 5.10

>0.05 >0.05

GGT (U/L)

Sacha inchi Sunflower

26.07 ± 6.38 44.19 ± 10.18

29.19 ± 4.41 44.72 ± 9.33

31.28 ± 4.22 49.84 ± 13.43

27.04 ± 2.66 44.38 ± 10.01

30.59 ± 3.31 4.09 ± 12.25

>0.05 >0.05

Alk. phosphatase (U/L)

Sacha inchi Sunflower

152.17 ± 16.71 197.83 ± 18.44

194.49 ± 21.33 215.72 ± 27.45

192.54 ± 16.09 217.14 ± 19.39

197.60 ± 16.61 187.21 ± 12.25

179.32 ± 11.92 176.68 ± 9.72

>0.05 >0.05

Total Bilirubin (mg/dL)

Sacha inchi Sunflower

1.05 ± 0.13 0.76 ± 0.15

0.98 ± 0.08 1.39 ± 0.25

1.03 ± 0.12 1.02 ± 0.11

1.08 ± 0.08 1.12 ± 0.07

1.02 ± 0.11 0.92 ± 0.12

>0.05 >0.05

CRP (mg/L)

Sacha inchi Sunflower

5.00 ± 0.00 10.14 ± 4.91

5.00 ± 0.00 5.14 ± 0.10

6.88 ± 1.88 6.64 ± 1.64

5.13 ± 0.12 5.00 ± 0.00

5.00 ± 0.00 5.00 ± 0.00

>0.05 >0.05

Creatinine (mg/dL)

Sacha inchi Sunflower

0.93 ± 0.05 1.05 ± 0.06

1.02 ± 0.03 1.04 ± 0.04

1.05 ± 0.04 1.17 ± 0.07

1.02 ± 0.05 1.11 ± 0.04

1.06 ± 0.05 1.16 ± 0.05

>0.05 >0.05

Uric acid (mg/dL)

Sacha inchi Sunflower

4.26 ± 0.33 5.15 ± 0.47

4.61 ± 0.32 4.88 ± 0.48

4.94 ± 0.27 5.41 ± 0.50

4.98 ± 0.35 5.44 ± 0.37

4.63 ± 0.39 5.30 ± 0.43

>0.05 >0.05

Albumin (g/dL)

Sacha inchi Sunflower

4.59 ± 0.18 4.66 ± 0.18

4.11 ± 0.08b 4.14 ± 0.10a

4.22 ± 0.18 4.08 ± 0.10a

5.00 ± 0.15b 4.88 ± 0.15

5.15 ± 0.15b 4.78 ± 0.14

<0.05 <0.05

Total protein (g/dL)

Sacha inchi Sunflower

6.65 ± 0.27 7.83 ± 0.33*

7.39 ± 0.46 8.01 ± 0.62

7.80 ± 0.21a 7.82 ± 0.11

8.60 ± 0.22a 8.35 ± 0.27

8.44 ± 0.18a 8.67 ± 0.22

<0.01 >0.05

P < 0.01 with respect to data at week 0. P < 0.05 with respect to data at week 0. P < 0.05 with respect to sacha inchi oil.

b *

Biochemical marker

0-Month

1-Month

2-Month

3-Months

4-Months

P

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integrity of the hepatocytes, were not affected during 4 months of treatment with sacha inchi oil or sunflower oil (P > 0.05). Serum GGT and alkaline phosphatase levels, markers of biliary systems, were not modified during 4 months of intervention with sacha inchi or sunflower oils. In addition, serum total bilirubin levels were similar after 4 months of consumption of sacha inchi oil or sunflower oil compared with values before consumption. Levels of serum creatinine and uric acid, markers of kidney function, were maintained unchanged during 4 months of intervention with sacha inchi oil or sunflower oil (P > 0.05). In addition, no differences were observed between both interventions at any time (P > 0.05). Serum CRP, an inflammatory marker, was also not modified by interventions (P > 0.05) (Table 3).

Fig. 1. Levels of serum (a) cholesterol, (b) LDL-cholesterol and (c) HDL-cholesterol in adult subjects receiving sacha inchi (}) or sunflower oil (h) for 4 months. Data are mean ± SEM. (a) and (b) Sacha inchi: P < 0.05, basal vs months 3 and 4. Sunflower: P < 0.05, basal vs months 3 and 4. (c) P < 0.05 between group with sacha inchi and sunflower oil at month 4.

3.4. Efficacy A significant reduction of serum total cholesterol levels (Fig. 1a) and C-LDL levels (Fig. 1b) were observed since month-2 of sacha inchi or sunflower oil consumption (P < 0.01). Serum C-HDL levels were significantly higher at month-4 after sacha inchi oil consumption than with sunflower oil consumption (Fig. 1c). Serum triacylglycerol levels were maintained unchanged throughout the period of study with sacha inchi and sunflower oils. Levels of triacylglycerol were 94.94 ± 11.67 (mean ± sem) mg/dL at time-0 and 98.34 ± 10.44 mg/dL at month-4 with sacha inchi oil (P > 0.05) and 101.46 ± 8.52 mg/dL at time-0 and 121.71 ± 13.18 mg/dL at month-4 with sunflower oil (P > 0.05). Serum albumin and total protein levels were increased after 4 months of treatment with sacha inchi oil or sunflower oil (P < 0.05). However, the increase in total protein after sacha inchi oil consumption was higher than with sunflower oil at months 2, 3 and 4 (P < 0.05) (Fig. 2). Blood glucose levels through the period of intervention with sacha inchi oil or sunflower oil remained without changes. In addition, no differences in fasting glucose levels were observed between interventions (sacha inchi vs sunflower oil) at the different times of the study (Fig. 3). Serum Insulin levels were increased during treatment with sacha inchi or sunflower oil, without differences between interventions (Fig. 3). Similarly, HOMA index increased after sacha inchi and sunflower oil without differences between interventions (Fig. 3). According to the multiple regression analysis, values of HOMA increased with time of oil consumption (0.22 ± 0.06; Beta coefficient ± SE, P = 0.01) and it was higher with sacha inchi oil consumption (0.38 ± 0.18; P = 0.039) and lower in women (0.42 ± 0.18; P = 0.023). Systolic blood pressure was reduced significantly at month-2 of sacha inchi oil or sunflower oil consumption. No differences in SBP were observed between groups consuming sacha inchi or sunflower oils (P > 0.05) (Table 4). Diastolic blood pressure was reduced significantly at month-2 of consumption of sacha inchi oil or sunflower oil and it was maintained lower up to the end of the study. No differences were observed between groups consuming sacha inchi or sunflower oils (P > 0.05) (Table 4). Multiple regression analysis showed that SBP was associated with age (0.21 ± 0.08; Beta coefficient ± SE; P = 0.018) and it was lower in women (4.44 ± 1.75; P = 0.012).

Fig. 2. Percent changes of serum Total protein (mg/dL) levels in adult subjects receiving sacha inchi (}) or sunflower oil (h) for 4 months. Data are mean ± SEM. P < 0.05 at months 2, 3 and 4 between sunflower and sacha inchi groups.

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Fig. 3. Levels of (upper) blood glucose, (middle) serum insulin and (bottom) HOMA index in adult subjects receiving sacha inchi (}) or sunflower oil (h) for 4 months. Data are mean ± SEM. Glucose: P > 0.05 between times and between interventions. Insulin: P < 0.05 between basal and month 3 and for sacha inchi and for sunflower oil. P > 0.05 between interventions. HOMA: P < 0.05 between basal and months 2, 3 and 4 of sacha inchi oil consumption and between basal and months 3 and 4 of sunflower oil consumption. P > 0.05 between interventions.

Table 4 Systolic blood pressure (SBP) in mm Hg and diastolic blood pressure (DBP) in mm Hg in adult human subjects who consumed sacha inchi oil or sunflower oil during 16 weeks. Time of oil consumption (months)

SBP (mm Hg) after sacha inchi oil#

SBP (mm Hg) after sunflower oil#

DBP (mm Hg) after sacha inchi oil#

DBP (mm Hg) after sunflower oil#

0 1 2 3 4

124.4 ± 2.62 123.1 ± 2.30 113.7 ± 2.29⁄ 104.1 ± 2.85⁄ 112.8 ± 1.82⁄

129.23 ± 2.65 128.54 ± 3.79 117.2 ± 1.72⁄ 109.2 ± 3.24⁄ 114.8 ± 1.74⁄

79.73 ± 2.04 82.26 ± 2.61 72.33 ± 2.14⁄⁄ 69.26 ± 2.81⁄ 70.40 ± 1.82⁄⁄

80.23 ± 1.39 83.77 ± 3.36 73.54 ± 1.87⁄⁄ 71.92 ± 2.25⁄ 73.00 ± 1.48⁄

Data are mean ± standard error of the mean. #P < 0.05 with respect to the time of intervention. ⁄P < 0.01; inchi oil group and sunflower oil group.

⁄⁄

P < 0.05 with respect to values at time 0. P > 0.05 between sacha

Fig. 4. Levels of serum (upper) testosterone, (middle) estradiol and (bottom) the ratio T/E2 levels in adult subjects receiving sacha inchi (}) or sunflower oil (h) for four months. Data are mean ± SEM. P > 0.05 between time and between interventions.

Similarly, DBP was associated with age (0.19 ± 0.07; P = 0.018) and it was lower in women (6.44 ± 1.37; P = 0.000). Serum testosterone, estradiol or T/E2 levels did not change with time after consumption of sacha inchi oil or sunflower oil (P > 0.05) (Fig. 4).

after sunflower oil consumption in adult men and women in a double-blind placebo-controlled clinical assay (Phase-I). For this study, it was used liquid sacha inchi or sunflower oil at dose of 10 or 15 ml ingested in a single daily dose during 4 months. These volumes are equivalent in sacha inchi oil to 4.4 and 6.6 g ALA/day.

4. Discussion

4.1. Acceptability

This study was designed to determine acceptability and tolerance of sacha inchi oil and compared results with those obtained

The present study showed a good acceptability of sacha inchi oil at the end of 4 months of consumption. This was observed despite

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an initial low acceptability rate at week-1 (37.50% of subjects accepted the taste of sacha inchi oil). The taste rating assessed by a Likert test (McGough et al., 2006) at the end of the study showed that most of subjects after 4 months of the liquid oils consumption reported they were mainly pleasant with taste of the oils. No differences were observed between taste of those subjects consuming sacha inchi oil or sunflower oil. A female subject decided to stop the study within the first week of the study due to the taste of sacha inchi oil. Since 85% of subjects accepted sacha inchi oil after week-8 of the study, then, data suggest that different volume of liquid oil (10 or 15 m)(4.4 or 6.6 g ALA/day) could be adequate for subjects who want to increase intake of omega 3. After a period of habituation no longer than a week the product is easily accepted. It is important to mention that consumption was directly in the liquid form as a drink. However, most of the time people consume oils accompanying the salads or foods. Then, the acceptability will increase if sacha inchi oil is mixed with vegetable or other foods as normally used. Generally, fish oil containing omega-3 fatty acids are tolerated, and their adverse effects are limited to gastrointestinal complaints (discomfort, upset stomach) and a fishy odor (Chan and Cho, 2009). Sacha inchi oil containing omega-3 fatty acids could be an alternative if an omega 3 product from a plant source is required.

score was similar to that observed in the group receiving sunflower oil. In addition, specific adverse effects were analyzed assessing hepatic and kidney function through biochemical markers. Results demonstrated that consumption of sacha inchi or sunflower oil for 4 months did not affect different variables assessed as hepatic or kidney markers. In addition, C reactive protein, an inflammatory marker, was unchanged throughout the period of oil consumption suggesting that consumption of sacha inchi oil or sunflower oil were unable to induce inflammation during the period of study. Moreover hemoglobin levels were maintained within the normal range throughout the study. Study in rats comparing linseed and sacha inchi oils (0.5 ml/kg) showed that no toxicity was observed and Letal Dose 50 (LD50) for sacha inchi oil and linseed oil were above 37 g/kg body weight (Gorriti et al., 2010). Taking together all of these data, it is possible to suggest that adverse events reported by subjects are of minor importance and they do not affect general health status. These data also suggest that consuming 10 or 15 ml of sacha inchi oil during 4 months is safety. There is a report of a case of occupational allergic rhinoconjunctivitis and bronchial asthma related to exposure to seeds of P. volubilis in a cosmetic company (Bueso et al., 2010). However, there is not description of allergies in case of consumption of the oil of P. volubilis.

4.2. Adverse effects 4.3. Efficacy The second objective of the study was to determine the adverse effects after use of sacha inchi oil and compare data with those obtained after sunflower oil consumption during 4 months. Consumption of sacha inchi oil was associated in some cases with nausea but this side effect was reduced such at the end of the study only two subjects reported nauseas as side effect. This side-effect seems to be associated more to the taste of sacha inchi oil rather than an adverse effect directly associated with the product. In fact, at week-one when acceptability of sacha inchi oil was lower, 10 out of 16 subjects consuming sacha inchi oil presented nauseas as adverse symptoms and this rate was reduced to 2 of 16 subjects at week 17 of oil consumption. Previous reports showed that use of liquid fish oil was also associated to gastrointestinal discomfort (Villani et al., 2013), or that, consumption of capsules of fish oil was associated with almost 54% of presence of eructation and nauseas as side effects (Holguin et al., 2005). In these studies, it was suggested that side effects were of minor relevance and probably more associated to the taste rather than an adverse effect of the oils. This suggestion is based to the fact that side adverse reports disappeared with time of consumption. A woman aged 47 years old consuming sacha inchi oil reported hot flashes as symptom. However, this could not be associated with sacha inchi oil ingestion and probably is a symptom associated with perimenopausal status. However, symptoms disappeared completely at week 9. We cannot assure that elimination of hot flushes was due to consumption of sacha inchi oil; however, data in the literature using omega-3 support this statement (Pruthi et al., 2007; Bourre, 2007; Lucas et al., 2009). The generic health-related quality of life (HRQL) questionnaire used for assessment health status showed that score was maintained unchanged throughout 4 months of consumption of sacha inchi oil or sunflower oil. This test is useful to assessment of health status from a general perspective (Gonzales et al., 2010, 2013) and it correlates with tests measuring specific diseases (Alonso et al., 1998). In such sense, if sacha inchi oil consumption produced adverse effects, this could be detected using this test. Results demonstrate that consumption of sacha inchi oil during 4 months did not change the health status score observed before the study and this

During the period of the study, body weight was maintained unchanged after use of sacha inchi oil or sunflower oil. This is in accordance with results using long chain n-3 PUFA (LCn-3PUFA). In fact, Dietary LCn-3PUFA supplementation during a weight loss program does not appear to assist weight loss (Munro and Garg, 2013). Serum total cholesterol and serum low density lipoprotein cholesterol levels were reduced with both sacha inchi oil and sunflower oil. However, no differences were observed between interventions. A similar pattern has been observed when compared consumption of 15 ml flaxseed oil rich in ALA with 15 ml olive oil rich in monounsaturated fat (Kontogianni et al., 2013). ALA is likely to inhibit cholesterol and fatty acid biosynthesis pathway by suppressing the expression of transcriptional factor SREBPs (Fukumitsu et al., 2012). The fact that content of ALA is lower in sunflower oil suggest that the effect of this oil on serum total cholesterol and LDL-cholesterol levels is using another pathway. Other studies show contrasting results. In one study, the ALA (4.4 g/day), EPA, or DHA intake did not affect fasting serum concentrations of total and LDL cholesterol, but fasting serum triacylglycerol concentrations significantly decreased in the EPA (0.14 mmol/L) and DHA (0.30 mmol/L) interventions and also in the ALA intervention (0.17 mmol/L) (Egert et al., 2009). In the present study, serum HDL cholesterol concentration was improved with sacha inchi oil consumption at month-4 of consumption. Previous study showed that DHA intake significantly increased serum HDL cholesterol, whereas no changes were found with ALA or EPA intake (Egert et al., 2009). Moreover, the n-3 polyunsaturated fats in fish oils suppress cardiac arrhythmias and reduce triacylglycerols, but they have little effect on LDL or HDL cholesterol levels (Sacks and Katan, 2002). It is still unknown the reasons why in some studies oil rich in omega 3 was associated with different responses in serum triacylglycerol and HDL-C levels. It is necessary studies with high number of participants. Fasting glucose levels were maintained unchanged during 4 months of period of study with sacha inchi or sunflower oil. Other study showed that higher intake of polyunsaturated fatty acid, n-6 fatty acid, linoleic acid, and oleic acid were significantly associated

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with a decreased prevalence of impaired glucose metabolism (P for trend = 0.03, 0.01, 0.02, and 0.04, respectively). Alpha-linolenic acid was marginally significantly associated with a decreased prevalence of impaired glucose metabolism (P for trend = 0.12) (Kurotani et al., 2013). Moreover, we have not observed differences in glucose levels when compared the group using sunflower or sacha inchi oil. More recent study showed that insulin, glucose, and the homeostatic model assessment (HOMA) of insulin resistance were not altered by the interventions during eight weeks with low-fat diet with omega-3 fatty acids (LFn3) (Young et al., 2013). However, 5-hydroxy-eicosapentanoic acid (5-HEPE), an omega-3 unsaturated fatty acid metabolite, enhanced glucose-dependent insulin secretion (Kogure et al., 2011). In our study, we have demonstrated that subjects consuming sacha inchi oil increased serum levels of insulin and HOMA. A similar finding was reported in a previous study using 5 and 10 ml oil obtained from another species of Plukenetia (Plukenetia huallabamba) (Garmendia et al., 2011). It is important to note that mean values of HOMA index were not in the range of insulin resistance (HOMA > 2.6) (Ascaso et al., 2003). In healthy elderly subjects, levels of glycemia, insulin or HOMA index were maintained unchanged when ratios n-6/n-3 PUFAs were 11.4–2.4 (Griffin et al., 2006). However, this is not the case of the present study, in which sacha inchi oil contains a ratio omega-6/ omega-3 of 0.73 whereas sunflower oil a ratio omega-6/omega-3 of 95.3. These results indicate that changes in insulin levels and HOMA index observed after sacha inchi oil or sunflower oil are due to other factors different to ALA or LA. It is interesting that a study in patients with metabolic syndrome, treatment with n-3 fatty acids from fish oil resulted in significant decrease in levels of triacylglycerol, but a significant increase in LDL, glucose and insulin resistance (Simão et al., 2010). A meta-analysis of 11 randomized controlled trials showed that omega-3 polyunsaturated fatty acid intervention has a lack of effect on insulin sensitivity (Akinkuolie et al., 2011). Arterial blood pressures were also reduced with both interventions (sacha inchi and sunflower oil). The flaxseed-derived polyunsaturated fatty acids including the omega-3 and omega-6 essential fatty acids have been shown to blunt the effects of hypertension (Al-Bishri, 2013; Lorente-Cebrián et al., 2013). Omega3 polyunsaturated fatty acids (n3-PUFAs) are thought to have multiple cardiovascular benefits, including prevention of arterial stiffness (Losurdo et al., 2013). The effect on blood pressure could be a response to effect on LDL levels. In fact, when LDL cholesterol levels were not modified after long chain omega-3 fatty acids from fish oils, arterial blood pressure was not modified too (Root et al., 2013). N-6 fatty acid intake also reduced arterial blood pressure (Czernichow et al., 2011). As, sacha inchi and sunflower oil reduced LDL cholesterol, it is suggested that this reduction may be in part responsible of the reduction of arterial blood pressure. Serum albumin levels increase with time after consumption of sacha inchi or sunflower oil and serum total protein increased with time after sacha inchi oil consumption more than with sunflower. This has been observed assessing the impact of fish oil-based lipid emulsion in children (Lee et al., 2009). Increased levels of albumin generally indicate adequate nutrition via protein turnover shifted towards an anabolic state. In addition, it can also reflect the synthetic function of the liver. Albumin has antioxidant property and binds fatty acids. Previously, it has been demonstrated that albumin modulates DHA protecting against human hepatocellular carcinoma cell lines (Kanno et al., 2011). These are further evidences to the overall beneficial effects that sacha inchi oil supplementation has on liver processes and nutrition. Since use of medicinal plants has increased world-wide is important to know adverse effects of herbal medicines. One study on 50 systematic reviews of 50 different herbal medicines showed severe adverse effects in liver and kidney, colon perforation, carci-

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noma, coma and death observed in 4 plants. Moderately severe effects were noted for 15 herbal medicines and minor adverse effects were noted for 31 herbal medicines (Posadzki et al., 2013). We have not observed severe effects with sacha inchi oil. Most of the adverse effects are of minor importance, and they disappeared despite the consumption of sacha inchi oil was maintained. As the study, a clinical trial of phase I was mainly oriented to acceptability and safety, one limitation of the study is that efficacy could better be observed in higher sample size as that in clinical trials of phase II. Despite of this, there is an indication that sacha inchi oils may reduce serum total cholesterol and LDL cholesterol, increase HDL cholesterol, and increase total protein. In conclusion the present study has demonstrated that sacha inchi oil administered as liquid in volume of 10 (4.4 mg ALA/day) or 15 ml (6.6 mg ALA/day) for 16 weeks has good acceptability after first week of consumption and it is safety after 16 weeks of consumption. 5. Conflict of Interest The authors declare that there are no conflicts of interests. Transparency document associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.fct.2013.12.039. Acknowledgments The present study was granted by Perubiodiverso project, an initiative supported by Swiss Cooperation SECO and the German Cooperation (implemented by GIZ) through the grant N° 398. GF participated in the design of the study, statistical analysis and writing the paper. CG participated in the development of the study. All the authors revised the manuscript. CP Diana Flores participated as monitor of the research. The authors acknowledge the support from Katherine de Yanac, Gissell Alessandra Manrique, Julio Rubio and Manuel Gasco during the work of field and analysis of data. Shanantina provided the sacha inchi (P. volubilis L.) oil from productive region of San Martin – Peru and ALICORP provided an edible refined oil of sunflower. References Akinkuolie, A.O., Ngwam, J.S., Meigs, J.B., Djoussé, L., 2011. Omega3 polyunsaturated fatty acid and insulin sensitivity: a meta-analysis of randomized controlled trials. Clin. Nutr. 30 (6), 702–707. Al-Bishri, W.M., 2013. Favorable effects of flaxseed supplemented diet on liver and kidney functions in hypertensive wistar rats. J. Oleo Sci. 62 (9), 709–715. Alonso, J., Prieto, L., Ferrer, M., Vilagut, G., Broquetas, J.M., Roca, J., Batlle, J.S., Antó, J.M., 1998. Testing the measurement properties of the Spanish version of the SF36 health survey among male patients with chronic obstructive pulmonary disease. Quality of life in COPD study group. J. Clin. Epidemiol. 51 (11), 1087– 1094. Ascaso, J.F., Pardo, S., Real, J.T., Lorente, R.I., Priego, A., Carmena, R., 2003. Diagnosing insulin resistance by simple quantitative methods in subjects with normal glucose metabolism. Diab. Care 26, 3320–3325. Bondioli, P., Della Bella, L., Rettke, P., 2006. Alpha linolenic acid rich oils. Composition of Plukenetia volubilis (sacha Inchi) oil from Peru. Riv. It. Sostan. Grasse 83, 120–123. Bourre, J.M., 2007. Dietary omega-3 fatty acids for women. Biomed. Pharmacother. 61 (2–3), 105–112. Brenna, J.T., Salem Jr., N., Sinclair, A.J., Cunnane, S.C., 2009. Alpha-linolenic acid supplementation and conversion to n-3 long-chain polyunsaturated fatty acids in humans. Prostaglandins Leukot. Essent. Fatty Acids 80, 85–91. Buckley, J.D., Howe, P.R.C., 2010. Long-chain omega 3 polynsaturated fatty acids may be beneficial for reducing obesity - a review. Nutrients 2, 1212–1230. Bueso, A., Rodríguez-Perez, R., Rodríguez, M., Dionicio, J., Pérez-Pimiento, A., Caballero, M.L., 2010. Occupational allergic rhinoconjunctivitis and bronchial asthma induced by Plukenetia volubilis seeds. Occup. Environ. Med. 67 (11), 797–798. Chan, E.J., Cho, L., 2009. What can we expect from omega-3 fatty acids? Cleve. Clin. J. Med. 76 (4), 245–251.

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