Male CF1 mice weighing 25 g were purchased from the Institute of Public Health of Chile. Animals were quarantined for environmental adjustment, at standard conditions (22˚C ± 2˚C) and at a regular light-dark cycle (12 h - 12 h), with free access to food and water before testing. Thereafter they were divided into groups of 8 mice according to the experimental groups. Animals were kept in group during quarantine and experimental period (40 days) at the Biotery of Universidad de Talca and supervised daily by trained staff.

6 apple extract were obtained (organic Fuji (OF) and traditional Fuji (TF); and organic Granny Smith (OGS) and traditional Granny Smith (TGS), as well as apple skin at harvest time (Fuji (FS) and Granny Smith (GSS)) (detail of the extracts found in Table 2) using the following procedure: cutting the waste/peel into small pieces and homogenized with 80% aqueous EtOH for 6 hours.

In an early stage of patent procedure. The six extracts obtained were administered in the drinking water of the animals as performed by He [17] .

Total phenolics were determined by the Folin-Ciocalteu method. Briefly, 0.1 ml of extract was mixed with 0.5 ml of the Folin-Ciocalteu phenol reagent (Merck, Darmstadt, Germany). The mixture was incubated for 5 min and then 0.5 ml of sodium carbonate (Na₂CO₃; 10%, w/v) was added and incubated for 15 min at room temperature. Absorbance was measured at 640 nm with the spectrophotometer. Total phenolic concentrations in the peel and diets were expressed as mg of chlorogenic acid equivalents (CAE) g⁻¹ FW.

Two types of diet were used, normal (ND) and fat diet (FD). The increased calories of FD were supplemented

with vegetable oil and animal fat. Vitamins and proteins (as peptone) were added to balance the amount of vita- mins, minerals and proteins. FD was prepared once a week and kept frozen until use following Moore-Carrasco et al. [18] .

Random samples of the diet were taken to the Instituto de Química de los Recursos Naturales de la Universidad de Talca for proximal chemical analysis (percentages of humidity, ashes, lipids or fats, proteins, fibre and elements free of nitrogen) using standardized methods for AOAC [19] .

Animals were randomized and divided into groups of 8 animals per experimental group (FO, FP, FT, GSO, GSP, GST), normal diet (ND) control and fat diet (FD) control. Each experimental group received FD plus water with the corresponding extract in drinking water as explained in Table 2. The extract was standardized 400 mg phenols per kilogram of animal weight (400 mg/kg), according to Hogan [20] and toxicology recommendation from Shoji [21] (see antioxidant consumption).

Water and food intake was quantified daily and the mice weight was controlled weekly. The study length was 40 days; at the end of this period animals were anesthetized in fasting condition with an intraperitoneal injection containing a mixture of Ketamine (Richmond, Argentina), Acetopromacine and Xylazine (Centrovet, Chile). Then, blood obtained with 1% heparin from aorta was centrifuged, separated and frozen at −70˚C until analysis to determine plasma: glycemia (GLI), total cholesterol (T-CHOL), cholesterol low density lipoprotein (CHOL LDL) and cholesterol high density lipoprotein (CHOL HDL) by commercial kit ROCHE following manufacturer’s instruction in autoanalyzer ROCHE, Elecsys 1010. Organs were also obtained: heart, liver, white adipose tissue (WAT), kidney and gastrocnemius, which were weighed on a precision scale (Shimadzu UX-UW) and frozen at −70˚C.

Animal handling met all the rules established by the National Commission for Scientific and Technological Research of Chile (CONICYT). All protocols carried out in this work have the approval of the bioethics and bio security committee of Universidad de Talca.

Statgraphics centurion was used for the analysis of the results with an ANOVA statistical test. p < 0.05 was considered statistically meaningful.

We characterized extract and diet. Table 3 shows the results for each extract obtained, as well as its characterization according to phenol content in each of them. Additionally, characterization of diets to which animals were subjected is shown in Table 3. Difference between both diets was based on lipid contents in ND was 3% and in FD was 28%. The lipid content of the diets are within the values obtained in accordance previously reported by Moore [18] .

Evolution of the weight was expressed in mean % of weight gain in relation to mean % initial weight. There is a significant difference between mean % of weight gained in the FD group (32.8%) and the rest of the study groups and including ND (Figure 1).

In heart, liver, kidney and gastrocnemius there are no significant difference (Table 4); only visceral white adipose tissue (WAT) showed a significant difference of weight (Figure 2) expressed in mean % of weight gain of WAT in relation to mean % initial weight of WAT. Mean weight of WAT in relation to the mean of initial weight shows the difference between the group with FD and ND. Statistical analysis shows that WAT weight decreased considerably in all study groups reaching similar levels to ND group (Figure 2) it means that all groups are statistically different compared to ND group.

There have been studies showing that apple extracts are one of the few fruits that have demonstrated anti- obesity effect. Boque et al. conducted a screening of different plants (almond, apple, cinnamon, orange, witch hazel, lime blossom, grape, birch) activities anti-obesity seeking in Wistar rats [22] , finding that only extracts of apple and cinnamon were considered anti-obesity because of their effects in reducing body fat (body fat-lower- ing) in the animals.

Studies have confirmed this trend in animal models, Cho [12] testing pome apple (PA) and apple juice concentrate (AJC) on different groups with a high fat diet. They found that the groups which were supplied with PA and AJC in the presence of high-fat diet had a lower weight, lower total CHOL, lower CHOL LDL, while CHOL HDL was significantly higher in the PA group compared with AJC. The results suggest that products supplemented with PA and AJC can help suppress the increase in WAT and improve the lipid profile in obese rats, supporting our results.

NFE: nitrogen free elements. FO: organic Fuji. FP: Fuji skin. FT: traditional Fuji. GSO: organic Granny Smith. GSS: Granny Smith skin. GST: traditional Granny Smith. ND: normal diet. FD: fat diet.

NFE: nitrogen free elements. FO: organic Fuji. FP: Fuji skin. FT: traditional Fuji. GSO: organic Granny Smith. GSS: Granny Smith skin. GST: traditional Granny Smith. ND: normal diet. FD: fat diet.

The administration of phenols of apple peel/discard to animals was 400 mg/kg mouse weight according to their initial weight. The amount of food that was consumed during the assay per group was between 1.311 gr (FP and GSP) and 1.451 gr (FO).

Table 5 shows biochemical results obtained in plasma of test mice dosed 400 mg of phenol/kg. In mice sub- jected to FD and the contribution of different apple extracts, a decrease of CHOL TOT, CHOL LDL and an increase of CHOL HDL was observed with all extracts studied, at similar level to the ND group. Levels of CHOL LDL and CHOL HDL are in Table 5. No difference was found between the extract from discarded fruit or apple skin. Our obtained results allow inferring that the extract consumed by animals in the study (400 mg phenols/kg) decreases the content of total cholesterol and LDL cholesterol, and increases HDL cholesterol, with WAT decrease playing a key role. Results confirm the findings of other researchers, in which cholesterol accumulation in rabbits in the ascending aorta decreased with the administration of quercetin glucoside [23] . Also, in obese Zucker rats that consumed apples, cholesterol level decreased [24] . Another study which confirms apple use for plasma cholesterol decrease in model animals was carried out by Leontowicz et al. [13] in which skin and pulp

CHOL TOT: total cholesterol. CHOL HDL: HDL cholesterol. CHOL LDL: LDL cholesterol. NFE: nitrogen free elements. FO: organic Fuji. FP: Fuji skin. FT: traditional Fuji. GSO: organic Granny Smith. GSS: Granny Smith skin. GST: traditional Granny Smith. ND: normal diet. FD: fat diet. ^*Different letters indicate higher significant difference with p > 0.05. Results are found with the mean error.

was used at the ripeness peak. In this report we used fruits discarded in orchard and skin waste from fruit processing. Components that are present in apple presenting the beneficial effect in reducing CHOL are complex polysaccharides such as pectin and different polyphenol. Aprikian [24] separated these compounds and demonstrated that they produce a greater decrease in the lipid profile in Wistar rats acting together than separately. A high correlation has been found between anti-hypercholesterolemic effects and certain compounds present in apple like catechin, epicatechin, procyanidin B1 and β-carotene which would be primarily responsible phytocompounds by lowering cholesterol by this fruit [25] . It is necessary to explain the mechanisms by which metabolites present in apples as well as in its extracts produce a decrease in plasmatic cholesterol levels in mammals. In this respect, two hypotheses have been proposed: 1) The component or components present in apples as well as in its extracts can inhibit cholesterol absorption, as pointed out by Koo et al. [26] , who details decrease in plasma cholesterol for the effect of catechin present in green tea. This effect would be due to the inhibition of lipid absorption in the intestine and the inhibition of lipid hydrolysis, mostly pancreatic lipase and pancreatic phospholipase A2. These findings have been reaffirmed by meta-analysis reviews [27] . In future trials, lipid concentrations in the faeces of animals during the experiment might be quantified, and studies might focus on transporters and enzymes in the intestines, either in animal models or cellular cultures that are related to cholesterol absorption.

2) According to another hypothesis, there is evidence that compounds present in apples and their extracts interfere in cholesterol metabolism; this was demonstrated by Osada et al. [28] . It has been proposed that Ursolic acid, present in fruit, is capable of modulating cholesterol catabolism, producing increase of muscle mass and brown adipose tissue (BAT), decreasing obesity, glucose intolerance and fatty liver [29] . This work would explain, in turn, WAT decrease in studied mice [30] . This effect has also been confirmed in another animal model such as the Wistar rats, showing improved glucose tolerance to a diet high in fructose and improved lipolytic response of adipocytes, proposing that the anti-obesity effects are achieved through the regulation of genes included in adipogenesis, lipolysis and oxidation of fatty acids, reducing the expression of mRNA of SREBF1 and increasing aquaporin 7 (AQP7), adipocyte binding enhancer protein 1 (Aebp1) and peroxisome proliferator activator receptor gamma co-1 alpha (PPARGC1A) [31] .

Important efforts have been made in the search of functional ingredients from apple pulp and skin, in order to incorporate them in healthier food for the population [32] . In this study, supplies to prepare the fruit extracts obtained from fruit discarded in orchard and the apple skin waste from fruit processing, both considered discards of low commercial value.

The contribution of mediterranean diet to the health of people consuming it has been widely studied and associated to a decrease of cardiovascular diseases [33] -[35] . Of foods that are consumed, one of the main candidate compounds are the phenols which have high antioxidant capacity [36] .