Studies on antioxidant activities of grape pomace using in vitro, ex vivo, and in vivo models

Grape pomace (GP) is a by-product resulting from the wine industry and can be considered raw material for animal nutrition, mainly due to its richness in polyphenolic substances. The present study, determined the antioxidant activity of GP by different in vitro assays including 1,1-Diphenyl-2-picrylhydrazyl (DPPH), superoxide anion, and hydroxyl radical and hydrogen peroxide scavenging activity and the inhibitory effect on iron-induced lipid peroxidation system. The estimated IC50 value (the concentration required to scavenge 50% of the radicals) of GP methanolic extract was 53.49 mg/L for DPPH; 57.37 mg/L for hydroxyl radical; 29.06 mg/L for superoxide radical and 102.15 mg/L for hydrogen peroxide. The effect of grape pomace supplements in broiler diets on oxidative stability of meat was tested in an experiment on 80 broiler chicks, 1-day-old Cobb 500, divided into 2 groups (C and E) reared on permanent wood shaves litter (10–12 cm thick). Compared to the control diet C, during the grower (14–28 days) and finisher (29–42 days) stages, the experimental diet (E) was supplemented with 6% GP. At the end of the experiment, 6 chicks aged 42 days from each group were slaughtered and samples of thigh meat were collected for further analysis. When the iron-induced lipid peroxidation system was applied, no significant differences were noticed between ex vivo groups’ lipid peroxidation inhibition percentage (24.71% inhibition when GP was added to meat samples and 24.10% inhibition when GP was ingested by animals) and in vivo data (26.92% inhibition) obtained.


Introduction
Grape is a well-known phenol-rich fruit, which mainly includes anthocyanins, flavanols, stilbenes, and phenolic acids [1]. Due to their important bioactivity, the antioxidant properties have been intensively studied. Several methods were used to evaluate the antioxidant activity of different parts of grapes (table grapes, grape seeds or skin, grape pomace, etc.): DPPH method [2]; oxygen radical absorbance capacity assay (ORAC); ferric reducing antioxidant power (FRAP); 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay [3]. In the wine industry, large quantities of residues are generated, by these by-products causing studies, grape by-products were used for their properties in improving the redox status of meat [8,9]. Other authors considered that using such natural antioxidants in animal nutrition could be limited due to the low bio-availability of polyphenols and also the fact that many types of polyphenols can lose part of their antioxidant capacity in vivo [10].
The antioxidant activity of bioactive compounds by in vitro, ex vivo, or in vivo models' studies, depending on the biomarker used, is considered a useful tool to assess oxidative stress, being necessary to demonstrate the mechanisms of action [11,12]. Although in vitro methods are inexpensive and simple to apply, to achieve the final objectives, such as the enrichment of animal products in certain compounds or their shelf-life extension, animal trials are required [13]. Ex vivo experiments are the middle variants between in vitro and in vivo ones, in the sense that they offer the possibility of a fairly accurate simulation of in vivo conditions, but unlike the latter, there is a certain flexibility, although everything is much better controlled [14].
The objective of this study was to evaluate and compare, through in vitro, ex vivo, and in vivo experiments, the antioxidant activity and inhibitory effect of grape pomace extract on lipid peroxidation of broilers meat.

Plant material and extraction procedure of polyphenols
Grape pomace was obtained from the Research and Development Station for Viticulture and Vinification in Pietroasa, Buzau County, Romania, and derived from red grapes (Merlot variety). Grape pomace raw material was dried in stove BMT model Ecocell Blueline Comfort (Nuremberg, Germany) according to Commission Regulation (EC) No 152/2009 and ground with a Grindomix GM 200 mill (Retsch, Germany). For analytical determinations, 1 g of dried GP was mixed with 10 mL methanol 80% and incubated for 24 h, in the dark. The extracted sample was centrifuged at 1500 g for 10 min. The supernatant was considered for analysis.

Extraction procedure for analysis of liposoluble compounds
For extraction of liposoluble compounds, the sample considered (2 g) was mixed with 130 mL ethanol, 100 mg BHT, 2 mL sodium ascorbate solution, 50 mg EDTA, and 25 mL potassium hydroxide solution 50%. The sample solution obtained was boiled for 30 min at 80 °C [15] and quantitatively transferred by rinsing with water into a separating funnel. The extraction of liposoluble compounds was performed with petroleum ether and the extract was evaporated under vacuum until dry. The residue was dissolved in 10 mL ethanol.
Method parameters for vitamin E were: isocratic conditions, 1.5 mL/min flow rate, and 4% water and 96% methanol mobile phase.

Content of total polyphenols
For total polyphenols content, the sample solution was formed by mixing the extracted sample (0.5 mL) with Folin Ciocalteu reagent (0.5 ml) and distilled water (7 ml). Sodium carbonate 20% (2 mL) was added and the sample solution was incubated at room temperature, for an hour in the dark. The absorbance was measured at 732 nm and the results are expressed as mg Gallic acid equivalents per gram sample [16].

DPPH free radical scavenging assay
A mixture of 0.4 mL methanolic extract of GP, 2 mL of 0.2 mM DPPH solution, and 1.6 mL distilled water was vortexed and left to stand at room temperature for 30 min in the dark and the absorbance was recorded at 517 nm (spectrophotometer Jasco V-530, Japan Servo Co.Ltd., Japan) [12]. The scavenging activity of the sample against DPPH radical was expressed as a percentage and was calculated using the following formula: Where AS is the absorbance of the sample and AC is the absorbance of the control.

Superoxide radical scavenging assay
The superoxide radicals were generated in 3 ml of Tris-HCl buffer (16 mM, pH 8.0) containing 1 ml of NBT (50 mM) solution, 1 ml NADH (78 mM) solution, and 1 mL sample solution of GP extract with various concentrations were mixed [12]. One ml of PMS solution (10 mM) was added for starting the reaction and after 5 min the absorbance was measured at 560 nm (spectrophotometer Jasco V-530, Japan Servo Co. Ltd., Japan). Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. The superoxide anion generation inhibition percentage was calculated using the following formula: Where AS is the absorbance of the sample and AC is the absorbance of the control (ascorbic acid). All determinations were done in triplicate.

Hydroxyl radical scavenging assay
For hydroxyl radical scavenging assay of GP, the reacting mixture used contained: 200 µL KH2PO4-KOH (100 mM), 200µL deoxyribose (15 mM), 200µL FeCl3 (500µM), 100 µL EDTA (1 mM), 100 µL ascorbic acid (1 mM), 100 µL H2O2 (10 mM) and 100 µL sample solution (11). The sample solutions were incubated at 37 °C for 1 h. At the end of the incubation period, 1mL TBA (1% w/v) was added to each mixture followed by the addition of 1mL TCA (2.8% w/v). After incubation at 80 °C for 20 min, the absorbance was recorded at 532 nm. The rate constants of reaction were calculated from competition plots obtained with varying concentrations of the antioxidant in the assay mixtures.
The results were expressed as the inhibition of deoxyribose attack percentage using the following formula: Where AS is the absorbance of the sample and AC is the absorbance of the control (ascorbic acid). All determinations were done in triplicate.

Determination of hydrogen peroxide scavenging activity
For determination of hydrogen peroxide scavenging activity, 3.4 ml of extract sample prepared in phosphate buffer (0.1 mM, pH 7.4) with various concentrations was mixed with 600 µl of a hydrogen peroxide solution and the absorbance was recorded at 230 nm after 10 min incubation at room temperature, against a blank solution containing phosphate buffer without hydrogen peroxide [12]. The percent of scavenging of hydrogen peroxide of extracts and standard was calculated according to the formula: Where AS is the absorbance of the sample and AC is the absorbance of the control (ascorbic acid). All determinations were done in triplicate.

In vivotest (experimental design).
The experiment complied with Directive 2010/63/EU on the protection of animals used for scientific purposes and the experimental procedures were approved by the Ethical

Statistical analysis
All the data are given as the mean ± standard deviation of three individual measurements. 50% inhibitory concentrations (IC50) were calculated by plotting the data in the graph as concentration versus percentage inhibition of free radical formation using the XLSTAT software (Addinsoft, Paris, France).
For iron-induced lipid oxidation results, the data obtained were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey comparison procedure to calculate the interrelation between the groups, using the XLSTAT software (Addinsoft, Paris, France). A probability level of < 5% was considered significant.

Antioxidant composition of grape pomace
The bioactive compounds with antioxidant properties from GP are presented in Table 1. Total polyphenol content is the major contributor to the antioxidant activity of GP, but other nutrients with antioxidant properties were determined and their activity must be considered.
The plant extracts are complex mixtures, containing compounds with antioxidant and prooxidant properties, sometimes with synergic actions by comparison with individual compounds [20]. Polyphenols are the major antioxidant component of GP, but their composition varies depending on the variety, with some authors estimating that only 2% of total polyphenols are extractable [21].
Exogenic antioxidants, supplied by foods and feeds are essential for counteracting oxidative stress. They act by different pathways, like inactivating or scavenging free radicals in the initiation and propagation phase of lipid peroxidation [22]. For example, tocopherols are free radical scavengers and prevent the formation of lipid hydroperoxides by breaking the chain of lipid peroxidation [23]. Polyphenols are also "chain-breaking" antioxidants, and carotenoids are the most efficient molecules for singlet oxygen quenching [24]. Zinc does not directly attack free radicals but is important in the prevention of their formation and can protect membranes from iron-initiated lipid oxidation [25].
Commission of the National Research and Development Institute for Biology and Animal Nutrition.
A total of 80, one-day-old Cobb 500 broiler chicks were purchased from a commercial hatchery and housed in an environment-controlled experimental hall (16 broilers/m 2 capacity). The broilers were reared on permanent wood shave litter (10-12 cm thick), in 3 m 2 boxes (each group was housed in a single box). During the first 14 days, all broilers received a control diet (C) with corn, soybean meal, gluten, and sunflower oil, as the main ingredients. After 14 days, the chicks were weighed individually and were assigned randomly to 2 groups (C and E) homogenous in terms of body weight. The broilers had free access to the feed and water. Compared to the control diet (C), during the grower (14-28 days) and finisher (29-42 days) feeding stages, the experimental diet (E) was supplemented with 6% GP [9]. At the end of the experiment, 6 chicks aged 42 days from each group were slaughtered, and samples of thigh meat were collected for further analysis. The tissue sampled was cryogenic with liquid nitrogen, ground (IKA A11 Basic Analytical Mill, Werke, Germany), and stored in plastic tubes at -80 °C.

Iron-induced lipid oxidation
The method used for inducing lipid oxidation was described by Yadav and Bhatnagar [18]. The frozen meat samples were homogenized for 15s in nine volumes of 0.15 M, pH 7.4, and ice-cold Tris HCl buffer. The solution obtained was centrifuged at 14,000 g for 20 min and the supernatant was considered for analysis. Two mL of supernatant were incubated with or without the GP methanolic extract (1000 mg/L) in a total volume of 4 ml. Peroxidation was initiated by adding 0.2 mL FeCl2 (100 µM) and 0.2 mL Ascorbic acid (500 µM). The mixture was incubated at 37 °C for 0, 60, 120, and 180 min.
Third derivative spectrophotometry described by Botsoglou et al. [19], was used to determine the TBARS values in thigh meat samples. A mixture containing 2 mL of sample solution, 5 mL TCA (7.5%), and 2.5 mL BHT in ethanol (0.8%) were centrifuged at 3000 g for 3 min., and an aliquot part reacted with TBA 0.8% solution for 50 min at 80 °C. Following incubation, the sample was cooled under running water and the absorbance was read at 540 nm (third derivative spectra) using a spectrophotometer (Jasco V-530, Japan Servo Co. Ltd., Japan). TBARS values were calculated against a standard curve obtained with 1,1,3,3-tetra methoxy propane (TMP). at 517 nm in the presence of different concentrations of GP extract. The hydroxyl radical scavenging activity of GP methanolic extract at different concentrations (7.5-45 mg/L) compared with AA as a standard is presented in Fig. 2. The superoxide radical scavenging activity of GP is presented in Fig. 3 and the scavenging activity of GP on hydrogen peroxide is presented in Fig. 4. In different analytical assays, the antioxidant activity of vegetal material can vary depending on phenolic or nonphenolic antioxidant constituents [26]. Some researchers consider that a single method for determining an antioxidant profile is not enough for providing a comprehensive picture and that a multimethod approach is more appropriate [27]. Superoxide anion, hydroxyl radical, and hydrogen peroxide are oxygen free radicals, being part of a larger group of molecules called reactive oxygen species (ROS) [28].
The DPPH assay is used for screening the proton-donating ability of plant extracts [29]. In our study, a dose-dependent increasing inhibition percentage was observed (Fig. 1). Other authors [30], reported that 50 mg/L methanolic extracts of GP exhibit 67.3% free radical scavenging activities. The inhibition activity of GP extract can be attributed to its ability to hydrogen donate [2]. In our study, the estimated IC50 value (the concentration required to scavenge 50% of the radicals) of GP methanolic extract was 53.49 mg/L and the calculated result was 10 times higher than that of the synthetic antioxidant, AA (5.58 mg/L). In another study, Zhu et al. [31] found a direct relationship between total polyphenols content and the DPPH scavenging ability of GP and this could explain the strong antioxidant activity of GP (scavenging rate of DPPH radical, higher than 70%).
The hydroxyl radical has a short lifetime and is the most reactive free radical [28]. It is considered to be capable of damaging almost every molecule found in living cells [30]. Also, this radical is considered to be one of the initiators of lipid peroxidation by abstracting hydrogen atoms from unsaturated fatty acids. In the presented results, a dosedependent radical scavenging activity was noticed and

Free radical scavenging activity of grape pomace methanolic extract
The methanolic extract of GP was screened for its free radical scavenging activities by using DPPH, hydroxyl radical, superoxide anion, and hydrogen peroxide assays. Figure 1 shows the radical scavenging activity of different concentrations of GP extract. The radical scavenging activity was evaluated by the decrease in absorbance of DPPH radical    as an antioxidant and iron-induced lipid oxidation was carried out using the Fe 2+ /AA oxidation system (E1). The third group of samples was collected from the experimental group of animals (dietary supplemented with 6% GP) and iron-induced lipid oxidation was carried out using the Fe 2+ /AA oxidation system (E2). The sample solutions were incubated at different times. The extent of lipid peroxidation was measured by the TBARS assay, using derivative spectral analysis for interference elimination from other reactive compounds.
The effect of treatments on lipid peroxidation of thigh muscle tissues is presented in Fig. 5. The E1 group recorded MDA values below the control group, with significant differences (P < 0.05) being recorded only after 2 and 3 h of incubation but at the same incubation times no significant differences were registered between experimental groups. Further, the lipid peroxidation preventive properties of GP can also be observed from Fig. 6. The inhibition percentage of experimental groups was measured at every incubation periods and compared to the control group. All ex vivo obtained results (0 to 180 min incubation time) were compared to inhibition percentage calculated based on MDA concentrations observed at the end of the experiment for in vivo collected samples. The results showed no significant differences between the in vivo group and ex vivo data obtained after 2 h of incubation.
Lipid peroxidation is a complex process, involving the formation and propagation of lipid radicals, the uptake of oxygen, and a rearrangement of double bonds in unsaturated lipids, producing the breakdown products. In the ex vivo model, the Fe 2+ /AA system induced the production of peroxyl radicals, and the GP extracts proved their ability to scavenge peroxyl radicals and delay lipid peroxidation.
In our study, the effect of GP extracts on the lipid peroxidation process was proved from the first step of measurements 45 mg/kg grape pomace extract exhibited 41.88% inhibition while AA exhibited 52.87% scavenging activity on hydroxyl radicals. The IC50 value of the extract and standard were found to be 57.37 mg/L and 39.64 mg/L, respectively (Fig. 2). The scavenging effect on hydroxyl radical is usually investigated using the 2-deoxyribose oxidation method. Some authors [32] considered that molecules that inhibit deoxyribose degradation are those that can chelate the iron ions becoming inactive in a Fenton reaction.
Superoxide radical is considered a precursor of more reactive species, such as single oxygen and hydroxyl radicals, and is an initiator of lipid peroxidation [33,34]. Superoxide anion can be generated in enzymatic systems by autoxidation reactions or by nonenzymatic electron transfer which reduces molecular oxygen [32]. The current results presented an inhibition effect that increased with increasing concentrations. GP at the final concentration of 150 mg/L exhibited an 83.98% scavenging effect (Fig. 3). The IC50 value of GP (29.06 mg/L) was almost 2 times lower than that of the synthetic antioxidant AA (50.47 mg/L). GP scavenged hydrogen peroxide in a concentration-dependent manner. The IC50 value of the extract was 102.15 mg/L and for AA it was found to be 20.02 mg/L. Similar results were reported regarding the scavenging effect of grape seeds compared with synthetic antioxidants like BHA or quercetin [27]. A possible explanation might be that flavonoids can scavenge the superoxide anion [35]. GP is recognized as an economical source for the recovery of a large number of biologically active compounds such as flavonoids, phenolic acids, phenolic alcohols, and stilbenes [36].
The production of hydrogen peroxide is a result of the dismutation of superoxide radicals [37]. Hydrogen peroxide is a reactive nonradical, which can penetrate biological membranes. Hydrogen peroxide itself is not very reactive, but in the presence of Fe 2+ , it can generate hydroxyl radicals. Hydrogen peroxide can be converted into water by phenolic compounds, which are good electron and protons donors [38]. GP scavenges hydrogen peroxide in a concentrationdependent manner, with a 45 mg/L GP inhibiting it at a rate of 26.25%. The IC50 value of the extract was 102.15 mg/L and for AA it was found to be 20.02 mg/L (Fig. 4).

Iron-induced lipid peroxidation
The GP potential in counteracting the oxidation process was studied by inducing lipid peroxidation on thigh muscle samples collected from an in vivo experiment. Meat samples (n = 6) collected from the control group (without GP supplements) were considered and iron-induced lipid oxidation was carried out using the Fe 2+/ AA oxidation system (C). Other meat samples (n = 6) were collected from the same group and GP methanolic extract was added to each sample tissues. In a study on broiler chicks, the results showed a significantly lower lipid peroxidation, measured by MDA formation in refrigerated breast samples after 1 and 7 days of storage, under 6% dietary GP influence [41]. In another study [42], it was proved that GP supplementation at 1.5 and 3% levels in chicken diets can delay the lipid peroxidation in breast and thigh meat.

Conclusion
GP is an important source of antioxidant compounds, including phenolics, vitamins, and minerals and a potential synergism may provide the inhibition effect on lipid peroxidation. GP can be considered a free radical inhibitor in different in vitro assays, including DPPH, superoxide anion, hydroxyl radical, and hydrogen peroxide scavenging activity. It can be used for minimizing or preventing lipid oxidation in the meat tissues in an iron-induced lipid peroxidation system. The ex vivo prooxidant/antioxidant model system proved to be a predictable tool for estimating the antioxidant behavior of GP in, in vivo conditions.
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In a review study [39] it was reported that GP presents strong inhibition against the secondary lipid peroxidation phase. Other authors cited in the same article, considered that GP had higher activity than isolated compounds and suggested a synergistic effect of phenolic compounds. In a study on piglets [40] fed a 9% GP dietary supplement, there was an increase in hydrogen peroxide decomposition and a significant decrease in TBARS values in seven of nine tested Fig. 6 In vivo and ex vivo data comparison of inhibition percentage of lipid peroxidation under GP influence. All values were statistically different (P < 0.05) except those marked with the * In vivo experiment: meat samples collected from animals fed with GP supplements related to meat samples collected from animals fed without GP supplements Ex vivo experiments: meat samples collected from animals fed without GP supplements, ex vivo oxidized and treated with GP methanolic extract (black columns) and meat samples collected from animals fed with GP supplements, and ex vivo oxidized (grey columns) related to meat samples collected from animals fed without GP supplements and ex vivo oxidized