Phytochemical profile, anti-inflammatory, antimutagenic and antioxidant properties of Acrocomia aculeata (Jacq.) Lodd. pulp oil

as laxative, whereas its oil has analgesic and healing properties. However, the beneficial pharmacological effects of A . aculeata oil to human health are still scarce. Nunes that the A. aculeata kernel oil presents hypoglycemic effects, decrease in weight gain and deposition of medium chain fatty acids in the epididymal adipose tissue in diabetic rats, suggesting that A. aculeata oil is a promising source of energy in diets to type 2 diabetes mellitus patients On addition, Silva also recently demonstrated that A. aculeata pulp oil decreased glucose levels induced by a rich fructose-diet or by streptozotocin administration in rats Supporting evidence to the beneficial effects of MPD shows that it can reduce total cholesterol levels and exhibits antioxidant activities These observations suggest that pharmacological properties of MPD should be further explored to provide new basis for its nutritional and medical use. On this study, we showed the physicochemical profile of MPD and determined its anti-inflammatory, antimutagenic and antioxidant properties in a mice model. smoreno@ucdb.br Abstract Acrocomia aculeata , known as macauba, is extensively distributed in the Southeast and Midwest of Brazil. Macauba fruit is used as food; however, its therapeutic properties are still poorly understood. The goal of the study was to investigate the chemical composition of macauba pulp oil (MPD) and anti-inflammatory, antimutagenic and antioxidant properties of MPD. The anti-inflammatory and antimutagenic properties were evaluated in Swiss mice treated with MPD per gavage or incorporated in the diet for 10 days. The serum of mice was subjected to lipid peroxidation evaluation and the in vitro MPD antioxidant activity was assessed. The organoleptic profile of MPD showed it was suitable for consumption and this composition showed unsaturated fatty acids with oleic acid the most abundant. The MPD presented anti-inflammatory properties, inducing a decrease in the migration of neutrophil and mononucleate cells to the peritoneal cavity of mice; it also inhibited the formation of micronuclei in mice. Furthermore, MPD presented antioxidant activity and high carotenoid content. Dur data support the hypothesis that MPD has anti-inflammatory and antimutagenic properties, and these effects can correlate with fatty acid profile and antioxidant compounds. There is great potential for the application of MPD for nutritional


Introduction
The benefits of plant oils for human health have long been the focus of investigations, especially in recent decades when they replaced some animal oils in food. Vegetable oils such as olive, palm, soy, corn, sunflower and canola are a common dietary constituent and consumed in high quantities worldwide (Esfahani et al., 2011). Some of these oils, such as olive oil, have presented beneficial properties for human health, due to the presence of antioxidant compounds, such as polyphenols, tocopherols and fatty acids Poveda et al., 2005;Ju et al., 2010), such as oleic and linoleic acids (Estruch et al., 2013).
On Brazil, there are 119 species of palms, distributed in 39 genera (Scariot et al., 1995). The genus Acrocomia comprises many species, such as Acrocomia aculeata (Jacq.) Lodd., which grows in the Southeast and Midwest of Brazil (Henderson et al., 1995). The fruit of A. aculeata, known as macauba, is widely consumed; however, its therapeutic properties are poorly understood. A. aculeata fruits are rich in flavonoid antioxidants, β-carotene and tocopherols, and the fatty acid profile in macauba pulp oil (MPD) is composed mostly of monounsaturated, such as oleic acid (Coimbra & Jorge, 2011;Lescano et al., 2015). This profile suggests that the consumption of the oil obtained from the macauba can be healthy. However, oil composition may be different according to genetic factors and abiotic factors such as light, temperature, precipitation, nutrition, season and time of collection and harvesting techniques (Singer et al., 2016).
A. aculeata pulp fruit has long been used in folk medicine as laxative, whereas its oil has analgesic and healing properties. However, the beneficial pharmacological effects of A. aculeata oil to human health are still scarce. Nunes et al. (2018) demonstrated that the A. aculeata kernel oil presents hypoglycemic effects, decrease in weight gain and deposition of medium chain fatty acids in the epididymal adipose tissue in diabetic rats, suggesting that A. aculeata oil is a promising source of energy in diets to type 2 diabetes mellitus patients (Nunes et al., 2018). On addition, Silva et al. (2019) also have recently demonstrated that A. aculeata pulp oil decreased glucose levels induced by a rich fructose-diet or by streptozotocin administration in rats (Silva et al., 2019). Supporting evidence to the beneficial effects of MPD shows that it can reduce total cholesterol levels and exhibits antioxidant activities (Nunes et al., 2018;Arena et al., 2018). These observations suggest that pharmacological properties of MPD should be further explored to provide new basis for its nutritional and medical use. On this study, we showed the physicochemical profile of MPD and determined its anti-inflammatory, antimutagenic and antioxidant properties in a mice model.

Source of chemicals: plant materials and extraction
Ripe Acrocomia aculeata fruits were collected in a cultivation area of this species in Mato Grosso do Sul State, Central-West region of Brazil (coordinates 22° 24' 59" N, 54° 14' 16" W). The formal identification of the plant material used in your study was confirmed by Dr. Simone Palma Favaro (no voucher specimen was deposited in herbarium). The access to botanical material is registered in the Management System of Genetic Patrimony and Associated Traditional Knowledge -SOSGEN, Brazil (nº. A623CA8). Acrocomia aculeata pulp oil was provided by the Biotechnology Laboratory of the UCDB. Macauba fruits were harvested in December in the mid-western region of Brazil (coordinates 22° 24' 59" N, 54° 14' 16" W). Fruits were gathered from five plants, around 100 kg, and the bulk was used to the pulp oil extraction process. Rotten fruits were discarded. A sample of 20 kg of the selected fruits were washed with distilled water and immediately dried in a forced air circulation oven at 40 °C for 3 h. The fruits were depulped using a stainless-steel knife. The pulp was dried in a forced air circulation oven at 60 °C until moisture around 10% was attained. The yield of the oil was 32% in dry basis. Macauba Pulp Dil (MPD) was obtained using Soxhlet apparatus with hexane as organic solvent, which was removed in a rotary evaporator at 40 °C.

Physicochemical properties of the MPO
The pulp oil was characterized by determining the acidity (oleic acid %), iodine and peroxide values, according to the ADCS method (American Dil Chemists' Society, 2004). As an indication of the formation of primary compounds of oxidation, we determined the molar absorptivity at 232 and 270 nm in an AquaMate spectrophotometer, model v.4.55, according to the OUPAC method (Paquot, 1979) using isooctane as solvent (UV-OR-HPLC). The fatty acid profile was determined by gas chromatography with a flame ionization detector (Agilent Technologies 6890N). The fatty acids methyl esters produced with the boron trifluoride catalyst (BF3) were evaluated under the following conditionsI: injector temperature of 225 °C, detector temperature of 285 °C, column temperature (HP-88 100 mm × 0.250 mm) initial 170 °C for 2 min, ramp 7 °C/min to a final temperature of 210 °C/29 min, split ratio 200I:1, hydrogen flow 40 mL/min, air flow 450 mL/min, helium flow 40 mL/min, injection volume 2 μL.
Quantification of total carotenoids was performed by diluted oil in petroleum ether, and absorbance was measured in 450 nm, using the absorptivity of β-carotene in petroleum ether (2592) to determine concentration. The analyses were performed in triplicate (Rodriguez-Amaya & Kimura, 2004).

Animals and animal care
For the execution of the experimental procedures, male and female Swiss mice weighing 18-22 g (6 weeks) were obtained at the Animal Facility of Universidade Católica Dom Bosco (UCDB). The animals were kept in ventilated rack at constant temperature (23 °C) in a light/dark cycle, with free access to feed and water and were acclimated for a week before experiments (Ethics Committee authorizationI: 005/2011.

MPO incorporation into feed
The MPD was incorporated into conventional food (Labina, Purina, Brazil) in three concentrations (5, 10 and 20% w/w). Briefly, the food was macerated and the oil (5, 10 and 20%) was incorporated. After, the moisture was dry (40°) and pelletizing. The modified feed was offered to the mice over 10 days, ad libitum.

Experimental design
Animals (n = 5) were treated with MPD (50 μL, gavage) or saline, for 10 days and fed with the conventional diet. Aiming to evaluate the effect of MPD on diet, mice were fed with conventional or modified feed (5, 10 and 20% MPD) for 10 days. Dn the 11th day, the animals underwent antimutagenic and anti-inflammatory assays. For the antimutagenic evaluation, mice were pre-treated with colchicine (0.5 mg/kg, intraperitoneally), a mutagenic drug (Paquot, 1979) or vehicle (saline). After 24 hours, the micronucleus number was evaluated in erythrocytes (caudal vein). For the investigation of the anti-inflammatory effect of MPD, mice were injected (i.p.) with thioglycolate 4% (500 µL; Merck, USA) or vehicle (saline). After 6 hours, the leukocyte migration evaluation was performed as described below. The euthanasia of the animals in the procedures was done by increasingly graded anesthesia (Ketamine 150 mg. kg -1 (Dopalen, CEVA/Brazil) and Xilazine 15 mg. kg -1 (Anasedan, CEVA/Brazil) and subsequently animals were submitted to cervical dislocation. The death was confirmed by cessation of respiration and heartbeat, and absence of reflexes.

Anti-inflammatory assay: leukocyte migration into mice peritoneal cavity
The mice were euthanized by deepening anesthesia; the peritoneal cavity was washed with 3 mL of PBS plus EDTA (5%w/v) and the exudate collected. The total cell count took place in a Neubauer chamber. The differential leukocyte counts were performed in panotic stained slides (Laborclin, Brazil), and were examined in optical microscope (Nikon  Eclipse 80i, Dil immersion lens, 100/1.25). The results were expressed as number of mononucleate cells and neutrophils x 10 6 /mL (Moreno et al., 2006).

Evaluation of antimutagenic activity of MPO: micronucleus test
The micronuclei were analyzed in erythrocytes in peripheral blood. Two slides were prepared by drawing a drop of mouse blood over the slide to form a thin smear which was air-dried for 24 h, fixed in methanol for 10 min and stained for 40 min with 5% (w/v) Giemsa stain. For each slide, 2000 erythrocytes were examined at 1000 X magnification and scored under light microscope (Nikon  Eclipse 80i, Dil immersion lens, 100/1.25). For the scoring of micronuclei, the criteria from Al-Sabti & Metcalfe (1995) were adopted.

Lipid peroxidation by TBARS assay
Lipid peroxidation was studied in incubations (2 mL final volume in PBS, pH 7.4, 37 °C), containing plasma (200 μL) obtained from blood collected through the caudal vein of treated and non-treated mouse groups. Control samples were treated with the same volume of DMSD used to dilute antioxidants (the final dilution of DMSD was 1/1000). After 30 minutes' incubation at 100 °C, lipid peroxidation was measured as the formation of TBARS, as previously described (Atmaca, 2004). Results were expressed in μmoles of MDA/mg serum.

Hydroxyl radical scavenging activity assay
On order to evaluate the hydroxyl radical scavenging activity of MPD, oil and standard ascorbic acid solutions were diluted in DMSD and methanol, respectively, at a concentration of 50 μg/mL, diluted to 5, 10 and 25 μg/mL. The assay was carried out as described by Smirnoff & Cumbes (1989). The absorbance of hydroxylated salicylate complex was measured at 562 nm. Analyses were performed in triplicate and the results were expressed as percentage of antioxidant activity (AA%).

Antioxidant activity by β-carotene/linoleic acid system
Onitially, the MPD and BHT standard solutions were prepared in DMSD and ethanol at a concentration of 50 μg/mL as per the methodology described by Marco (1968) modified by Hammerschmidt & Pratt (1978). BHT was used as a positive control at the same concentration, and ethanol was used as a blank. Analyses were performed in triplicate and the absorbance recorded at intervals of 20 min (20, 40, 60 min).

Statistical analysis
Data analysis and graphic representation was performed by GraphPad Prism 6.0 program. The data are reported as the means ± standard errors of the means (SEM) of values obtained from two different experiments. The means of different treatments were compared by analysis of variance (ANDVA). Of significance was determined, individual comparisons were subsequently tested with Bonferroni's t test for unpaired values. A p value of 0.05 or less was considered significant.

Physicochemical properties, stability, quality and fatty acid profile of MPO
Physical-chemical properties of a given vegetable oil can vary according to the region of cultivation, maturation stadium, storage length and conditions, genetic variability, processing method, among other factors. The MPD assessed in this study showed acidity and peroxide indexes of 2.47% and 9.28 mEq/kg, respectively (Table 1). These values indicate that MPD is within the range established to other similar oils, though the high peroxide value. Also, the MPD iodine value found in this work was 72.01 ± 2.22, as demonstrated in Table 1. On order to provide information about their identity, conservation status and changes caused by processing, spectrophotometric analysis of MPD using ultraviolet wavelength was performed. The MPD showed absorptivity at 232 and 270 0of the MPD, and the values were 2.49 ± 0.04 and 0.64 ± 0.01 respectively (Table 1) Fatty acid profile of MPD is shown in Table 2. Twelve fatty acids were identified, the majority of them unsaturated fatty acids with oleic acid the most abundant (72%). There was also demonstrated that MPD contains high concentrations of total carotenoids expressed as β-carotene (694 ± 8.31 μg/g) ( Table 2).

Antimutagenic effects
On order to evaluate the antimutagenic effect of MPD, mice were pre-treated with 50 µL of oil or saline by gavage for 10 days. The results showed that MPD was able to protect the animals against DNA damage induced by colchicine, since the micronucleus number in erythrocytes from mice treated with MPD was 44% lower when compared with control group (p < 0.05) (Figure 2A). On order to confirm the protective effect of MPD, the oil was incorporated in the food and mice were fed for 10 days with a diet containing MPD at different rates of supplementation (5, 10 and 20%) and then challenged with colchicine ( Figure 2B). Results showed that micronucleus frequency was lower (45%) in all MPD concentrations compared to the control (p < 0.05), corroborating the chemoprotective effect of oil consumption.

Antioxidant activity and decrease of lipid peroxidation induced by MPO
On order to test the antioxidant effect of MPD, a combination of in vitro assays was performedI: firstly, we used the method of sequestration of hydroxyl radicals, followed by β-carotene/linoleic acid measurements. Both assays demonstrated remarkable antioxidant properties of MPD when compared to the ascorbic acid and BHT of the control group (Figure 3). The antioxidant activities (% AA) of the MPD and ascorbic acid, used as control, are described in Table 3. The results show that MPD presented Figure 1. Anti-inflammatory effect of MPD. The anti-inflammatory effect of macauba pulp oil (MPD) was assessed by leukocyte migration into the mice peritoneal cavity. Mice (n = 5) were pre-treated with MPD (25 and 50 µL, gavage) for 10 days. Dn the 11 th day, the inflammatory stimulus (Thioglycolate 4%, i.p.) was injected. Control groups received saline 0.9% (200 μL i.p). After 6 h, total and differential cell counts were performed. The results are expressed as number of neutrophils and mononuclear cells x 10 6 (mean ± SEM) *p < 0.05 when compared to the group treated with Thioglycolate. ANDVA followed by Bonferroni post-test. (Panel B) Mice received diet incorporated with MPD (5, 10 and 20%, ad libitum) for 10 days. The control group was fed with standard diet. Dn the 11 th day, the mice were treated with colchicine (0.5 mg/kg, i.p.). After 24 hours the micronucleus frequency in erythrocytes was evaluated. 2000 cells/mouse (n = 5) were counted. Results are expressed as mean ± SEM (standard error of the mean) of the number of micronucleus. *p < 0.05 when compared to the control group. ANDVA followed by Bonferroni post-test.
antioxidant activity similar to ascorbic acid in the range of 5 to 50 μg/mL. On addition, an evaluation of the antioxidant properties of MPD as well as oxidation-associated β-carotene and linoleic acid were also conducted ( Table 4). All doses of MPD exhibited antioxidant activity similar to that observed in the control group (BHT).
We also performed lipid peroxidation in the plasma from mice subjected to diet with MPD or MPD by gavage. Mice treated with MPD per gavage presented a decrease in lipid peroxidation (25%) when compared with control group ( Figure 3A). Dn the other hand, only the animals that received a diet supplemented with MPD 20% showed reduced (67%) lipid peroxidation ( Figure 3B).

Discussion
Physical-chemical properties of a virgin plant oil can vary according to the region of cultivation, maturation stadium, storage length and conditions, genetic variability and processing method, among other factors, and may lead to the deterioration of the oil, affecting properties such as sensory quality, nutritional function and toxicity (Ballus et al., 2015;Beltrán et al., 2005). Therefore, MPD was characterized with regard to parameters of quality and identity. The MPD assessed in this study showed acidity and peroxide indexes within the standards for crude palm oil, taken as a reference for MPD, since there is no legal compliance for this oil. Thus, the reference was 5% oleic acid and 10 mEq/kg peroxide value (Beltrán et al., 2005). Another reference could be olive oil, whose acidity is established according to the obtaining process (mechanical extraction and / or solvent extraction), whether refined or blended. There are two levels for maximum acidity of olive oilI: 3.3% for virgin olive oil and 0.3% for refined olive oil (Beltrán et al., 2005;Brasil, 2005). The iodine value is an identity standard of vegetable oils because it accounts for the degree of oil unsaturation. On addition, the iodine value can be used to track oxidation since this reaction changes the number of double bonds of the fatty acids chains. The MPD iodine value found in this work (72.01 ± 2.22) was very close to the one reported by Prates-Valério et al. (2019). This value is within the recommended standards for olive oil. According to Resolution 22/77/MS, the iodine value for olive oil is supposed to be between 75 and 90 (Brasil, 2005). The similarity of the iodine value of MPD and olive oil is due to the fatty acid profile, which is rich in oleic acid, as discussed below.
Spectrophotometric analysis of oils using ultraviolet wavelength can provide information about their identity, conservation status and changes caused by processing (Tilahun et al., 2019). Molar absorptivity usually correlates positively to peroxide value and is a measurement of conjugated dienes (at 232 nm) and trienes (at 270 nm) that arose due to the primary oil oxidation process. The MPD showed absorptivity in accordance with the high peroxide value reported in stored macauba fruits (Nunes et al., 2015) and with the trend observed during the refining of MPD (Kiritsakis & Markakis, 1987). Dur results indicated that the MPD had absorptivity (2.49 at 232 nm and 0.64 at 270 nm) similar to that recommended for extra virgin olive oil (2.5 at 232 nm and 0.22 at 270 nm) (Gallardo-Guerrero et al., 2005). The data show that the MPD utilized in this study had high quality and stability, despite its extraction with hexane. We suggest that innovative extraction techniques can produce MPD with improved quality and edible properties.
The fatty acid profile of MPD showed twelve fatty acids, the majority of them unsaturated fatty acids with oleic acid the most abundant (72%). These results are in agreement with previous reports which describes the presence of oleic and palmitic acid at around 73% and 16%, respectively, in macauba oil obtained from the fruit of other Brazilian regions (Coimbra & Jorge, 2011;Lescano et al., 2015;Tilahun et al., 2019). Also, it has Determinations were performed after 60 min. The results represent the mean ± EPM of the analysis performed in triplicate. Significantly different when compared with the respective Ascorbic acid control (p ≤ 0.05). ANDVA followed by Bonferroni post-test. been reported that several plant oils, such as olive oil, present high levels of monounsaturated acids, similarly observed in MPD (Gallardo-Guerrero et al., 2005;Kiritsakis & Markakis, 1987;Nissensohn et al., 2016). The consumption of this oil source may be associated with the reduction of risk factors for coronary heart disease, in particular, amelioration of the lipid profile, inhibition of lipid and DNA oxidation, decrease of insulin resistance and inflammation (Estruch et al., 2006;Gillingham et al., 2011;Calder, 2017).
The results observed in this study support that MPD efficiently affects acute inflammation by inhibiting leukocyte migration, which is possibly mediated by one of its main phytochemicals, phenolic compounds and fatty acids, which are able to reduce inflammatory mediators. On this context, several studies demonstrated that polyunsaturated fatty acids exhibit a protective role in health by decreasing pro-inflammatory mediators (Wiktorowska-Dwczarek et al., 2015;Ventro et al., 2017). Murumalla et al. (2012) showed that polyunsaturated fatty acids inhibit LPS-induced cytokine secretion, such as OL-6, TNF-α, and MCP-1 in both adipose tissue and mature adipocyte models [36]. Similarly, it has also been observed that vegetable oil blend, rich in monounsaturated fatty acids with oleic acid as the major lipid, can exert analgesic and anti-inflammatory activities by down-regulation of the expression of pro-inflammatory cytokines such as TNF-α, OL-6 and ND, as well as the production of free radicals in lipopolysaccharide-stimulated macrophage cells (Guidoni et al., 2019;Zadeh-Ardabili & Rad, 2019). We showed that 100 µL was able to inhibit neutrophil migration to the peritoneal cavity, however, less efficiently than either 25 or 50 µL of MPD (Data not shown). The observation that 25 µL of MPD is more effective as an anti-inflammatory treatment than higher volumes (50 or 100 µL) might be explained by the fact that high doses of lipid, such as palm oil, administered per gavage in mice could damage intestinal barrier integrity, improved intestinal permeability, altered the intestinal microbiota and increased inflammatory cytokine expression (Araújo et al., 2017;Gulhane et al. 2016;Lam et al., 2015;Cani et al., 2008). On addition, increasing levels of saturated palmitic acid, but not unsaturated oleic (Ghezzal et al., 2020), promotes a dual effect of palm oil consumption in metabolic and inflammatory diseases, suggesting that the benefit of palm oil is associated with their moderate dietary intake.
On addition to its anti-inflammatory effects, several in vivo and in vitro reports have shown that A. aculeata oil also exhibited absence of acute and sub-acute toxicity in terms of the cytotoxicity, genotoxicity, and mutagenicity (Silva et al., 2019;Traesel et al., 2014Traesel et al., , 2015. These observations were also observed in the present study, which showed that MPD prevents antimutagenic effects induced by colchicine. Dxidative stress induced by free radicals and reactive oxygen/nitrogen species play an important role in several disturbances of health and in pathogenesis (Kehrer & Klotz, 2015), such as DNA damage and inflammatory disease (Sanjinez-Argandoñ & Chuba, 2011; Rocha et al., 2013). Since a diet including antioxidants is important for the inhibition of oxidative stress (Sanjinez-Argandoñ & Chuba, 2011;Rocha et al., 2013), the antioxidant properties of MPD were investigated. We verified that MPD contains high concentrations of total carotenoids expressed as β-carotene (694 ± 8.31 μg/g) than observed in previous reports (Coimbra & Jorge, 2011;Nunes et al., 2015), which is a well-recognized natural antioxidant with potential health benefits (Kehrer & Klotz, 2015). Dur data are supported by previous observation that show that A. aculeata pulp is rich in antioxidants such as β-carotene (Sanjinez-Argandoñ & Chuba, 2011;Rocha et al., 2013), α-tocopherol (Coimbra & Jorge, 2011;Schex et al., 2018), and monounsaturated fatty acids such as oleic acid (Ciconini et al., 2013;Mariano et al., 2011). Another line of evidence of the anti-inflammatory properties of A. aculeate oil is based on lipid peroxidation in the plasma from mice subjected to diet with MPD or MPD by gavage. Mice treated with MPD per gavage presented a decrease in lipid peroxidation (25%) when compared with control group ( Figure 3A). On addition, only the animals that received a diet supplemented with 20% MPD showed reduced (67%) lipid peroxidation ( Figure 3B). These effects can be due to carotenoids and other antioxidant contents (Rodriguez-Concepcion et al., 2018;Russell, 2004;Duthie et al., 1996) and high concentration of oleic acid .
Onflammatory disease and DNA damage have been widely associated with oxidative stress (Czarny et al., 2018;Zhang et al., 2019). Therefore, the anti-inflammatory and antimutagenic effects of MPD may be associated with the high quantity of antioxidant compounds, such as carotenoids. On fact, several studies have shown that β-carotene suppressed LPS-induced release of OL-1β, OL-6, and TNF-α by inhibition of the NF-κB, JAK2/STAT3, and JNK/p38 MAPK signaling pathways in macrophages (Li et al., 2019). Regarding mutagenesis, it was demonstrated that supplementation with α-tocopherol and β-carotene may protect DNA against oxidative damage, supporting the hypothesis that plant oils, rich in these antioxidants, may have antimutagenic properties (Pool-Zobel et al., 1997;Anter et al., 2010). Additionally, it has been shown that A. aculeata oil has chemopreventive effects against damage caused by cyclophosphamide in the male reproductive system of rats, possibly by acting as an antioxidant (Arena et al., 2018). Ot is important to note that the major dietary sources of tocopherols are vegetable oils, from corn, soybean, sesame, cottonseed, and nut . Previous reports with other vegetable oils support our results and suggest that the antimutagenic effect of MPD might be correlated with the presence of carotenoids. For example, Umegaki et al. (1994) demonstrated that β-carotene was able to reduce the formation of micronuclei in human lymphocytes irradiated with low doses of X-rays. Besides, Evangelista et al. (2004) verified the anticlastogenic effect of antioxidant compounds in olive and canola oils, which can act by inhibiting oxidative damage induced by RDS (Evangelista et al., 2004). The antioxidant protection of β-carotene is mediated by scavenging oxidants such as superoxide and this may result in the decrease in RDS. Thus, we suggest that the antioxidant effects of MPD, which is rich in β-carotene, may be partly due to its role in the prevention of the free radicals in the testis.

Conclusions
MPD presents anti-inflammatory and antimutagenic properties and these effects might be associated with its phytonutrient profile, such as its fatty acids, which are rich in oleic acid and antioxidant compounds, such as carotenoids. MPD shows a promising potential as an innovative plant oil with improved pharmacological properties and versatile nutritional purposes.