Impact of Colombian yellow fruits and tropical fruits drinks consumption on the antioxidant status of healthy women

Reactive oxygen species are responsible for causing different types of damage in the body which are associated with the onset of chronic non-communicable diseases. A strategy to counteract these effects is by a diet rich in antioxidants compounds found in fruits. The aim of this study was to determine the antioxidant capacity in plasma and the content of total phenolic compounds in healthy women who consumed a drink prepared with Nativanox® Colombian tropical fruits or a drink prepared with Nativanox® Colombian yellow fruits during a short period of time. Nineteen healthy women received a daily 200 mL drink with Nativanox® Colombian tropical fruits or a drink with Nativanox® Colombian yellow fruits for 14 days. Before and after the intervention period blood and plasma were obtained to analyze C-reactive protein levels, lipid profile, total phenolic content and antioxidant status through FRAP, ABTS and EROS methods. Regular consumption of a drink with Nativanox® Colombian tropical fruits had a positive impact on the lipid profile and the antioxidant capacity on plasma of healthy women. The consumption of a drink with Nativanox® Colombian yellow fruits diminished diastolic blood pressure. These results showed that a Nativanox® Colombian yellow and tropical fruit contains bioactive compounds that can improve oxidative status in plasma and contribute to reduce the risk of cardiovascular disease.

that mitochondrion produce reactive oxygen species (ROS) or free radicals from electron transporter chain (Harman, 1956).Free radicals are also produced by enzymatic reactions such as NADPH oxidase reactions of phagocytes to destroy invading microbes or xanthine oxidase.In addition, external sources such as pollution, cigarette smoke and sunlight produce ROS which are incorporated into organisms and cells.Excessive production of ROS leads to damage of lipids, proteins, carbohydrates and DNA (Dean et al., 1997).The membranes exposed to free radicals lose their ability to transport nutrients properly; lipoproteins become oxidized forms; and DNA damage has the potential to accumulate consecutive mutations, which can lead to carcinogenesis (Ames et al., 1993).Therefore, the oxidative damage theory strictly recalls the concept that antioxidant molecules are capable to slow oxidative process and very important for homeostasis normal body metabolism.
An antioxidant with biological function is defined like a substance present at low concentrations able to decreases or prevents oxidation of a substrate.Such substances may have direct action by the neutralization of free radicals and non-radical reactive species or indirect, through induction of enzyme systems such as glutathione reductase, catalase and superoxide dismutase (Halliwell and Whiteman, 2004;Hicks et al., 2006).Among the antioxidants of dietary origin, that is, present in fruits and vegetables are carotenoids and polyphenols, the phenolic acids and flavonoids comprise 60% of the phenolic compounds obtained from diet (Ramos, 2007).Polyphenols are secondary plan metabolites that contribute to the organoleptic qualities, color and defense against pathogens attacks.The chemical structure of phenols having one or more aromatic rings with one or more hydroxyl groups which confer activity radical scavenging inactivating directly ROS or by binding to pro-oxidant metal ions through their groups OH (Rice-Evans et al., 1997).In recent years, it has been shown that a diet rich in polyphenols can improve health and reduce the incidence of cardiovascular disease (Shroeter et al., 2006).These effects are primarily due to its antioxidant properties that can usually justify their vasodilatory and vasoprotective actions and their antithrombotic, antilipemics, antiarterosclerotics, anti-inflammatory and anti-apoptotic (Potenza et al., 2007) shares.In addition, some studies have shown that these compounds can also inhibit angiotensin converting enzyme (ACE) and inhibition of this enzyme would justify its vasodilating and cardioprotective effects (Ojeda et al., 2010;Andriambeloson et al., 1997).One of the most studied properties of polyphenols is the ability to improve the lipid profile (Aviram and Rosenblat,1994), thus, can prevent the development and occurrence of atherosclerosis, a disease characterized by progressive clogging of the arteries as a result of lipid accumulation in the arterial wall.These compounds are capable of attenuating the onset and progression of this disease due to their ability to mitigate LDL oxidation by increasing the concentration of HDL-cholesterol and inhibit the proliferation of vascular smooth muscle (Osakabe et al., 2001).
Phenolic compounds are the largest non-energy plant substances group, so that products derived from fruit can be consumed to improve health.Furthermore, the combined food can have more benefits to consume the active components alone, because the set of nutrients can have a synergistic effect (Betoret et al., 2009).Andean berries and yellow Colombian native fruits with trade name Nativanox® prepared by the company Tecnas SA, rich in flavonoids, anthocyanins, tannins, polyphenols and carotenoids were determined ORAC value (Oxygen Radical Absorbance capacity) whose values were 55.000 μmol Trolox/100 g for Nativanox® tropical fruits, 40,000 μmol Trolox/100 g for Nativanox® Andean berries and 40,000 μmol Trolox/100 g for fruits Nativanox® yellow (Peñaloza and Rojano, 2014).These results suggested that the consumption of a drink rich in bioactive compounds such as polyphenols, with high antioxidant capacity determined by ORAC can improve antioxidant status after consumption regularly for a shorttime in healthy women.
According to data reported by the National Survey of Nutritional Status of Colombia (ENSIN) in 2010, 33.2 and 71.9%, a low percentage of Colombians consume daily fruits and vegetables, respectively.Only 1 in 5 Colombians consume whole foods, and just 25% of the population eats fruits and vegetables daily.Therefore, as a strategy to encourage the consumption of fruits and vegetables, has been proposed based on drinks fruit as an option accepted by the population (Wootton-Beard and Ryan, 2011).Fruits or fruit juices are good sources of antioxidants such as polyphenols.These could fulfill the role of mediating biological processes, resulting in the prevention of non-communicable chronic diseases such as dyslipidemia, hypertension, atherosclerosis and cancer (Morton et al., 2000).The aim of this study was to determine the impact on the antioxidant status of healthy women who consumed a drink prepared with Nativanox® colombian tropical fruits or consumed a drink prepared with Nativanox® colombian yellow fruits, obtaining a significant antioxidant activity for 2 weeks.

Determination of carotenoids
A representative portion of the sample was taken in a test tube.4 ml of acetone was added and stirred in a vortexer for 2 min.The mixture was centrifuged at 4000 rpm/10 min and the supernatant was collected in another test tube.The absorbance of the solution was determined at 449 nm.

Determination of tannins
The analysis was performed according to the methods of the pharmacopoeia and the AOAC method after some modifications.25 ml of the Nativanox extract were measured in a 1 L conical flask, and 25 ml of indigo solution and 750 ml of deionized water were added.Titration was performed with an aqueous solution of potassium permanganate (KMnO4) 0.1 N until the blue solution turned green.The target of the test was made with 25 ml of indigo carmine solution in 750 ml of deionized water.The samples were analyzed in triplicate.

Determination of flavonoids
Determining flavonoids was performed following the method described by Debnath et al. (2011).A standard curve using catechin was constructed.Results were expressed as mg catechin/100 g Nativanox®.Readings were taken at 510 nm.
The mixture was allowed to stand for 6 min with constant aeration and at room temperature (26°C), and then the fluorescence intensity was measured.Results are expressed as mg of DMSO/100 g of Nativanox®, by constructing a standard curve using different concentrations of DMSO.

Evaluation of the total capacity to trap reactive oxygen species (ROS) of Nativanox®
Capacity of Nativanox® to trap reactive oxygen species (ROS) was evaluated.ROS are generated by the azo compound, 2,2'-diazobis (2-amidinopropane) dihydrochloride) (AAPH), which in aqueous medium free radicals produced at a constant speed (Martín- Romero, 2008).The reaction was carried out in phosphate buffer (pH 7.4).50 ml of a solution of AAPH, 50 ml of an ethanolic solution of dichlorofluorescein diacetate, 2850 ml of buffer and 50 µl of Nativanox® were mixed.Immediately, fluorescence intensity was emitted during the first 10 min and compared to the intensity emitted in the absence of the sample which was read.Results are expressed as values TEAC (µmol Trolox/100 g Nativanox®), by constructing a standard curve using different concentrations of Trolox.

Preparation of drinks
A powder enriched in polyphenolic compounds was obtained, packaged in foil pouch protected from light, with trade name Nativanox® produced by the company TECNAS S.A. Passion fruit (Passiflora edulis), Anana (Ananas comosus) smooth cayenne variety, Granadilla (Passiflora ligularis), Common Guava (Psidium guajava L.) and Feijoa (Acca sellowiana) were used.The edible portion of each fruit was obtained and homogenized in a vegetable processor Black & Decker model FP1550S and homogenized in Ultraturrax T-50 Basic IKA-WERK containing the bioactive compounds in Table 1.The drink was prepared from the powdered extract.Following the recommendations of USDA and FDA for ORAC units/d indicated for preventing degenerative diseases, 5 g of Nativanox® colombian tropical fruits or 8.2 g of Nativanox® colombian yellow fruits representing 5,000 ORAC which were diluted in 200 mL of a Colombian blend mineral water and stored at 4°C until consumption were used.These preparations were made daily and consummed 1 h after dilution.

Study design
Double-blind trial with an intervention period of 14 days (2 weeks) was used.Twenty healthy women were recruited and given 200 ml/day a drink with tropical fruits (n = 9) or a drink with yellow fruits (n =11).During the study, the diet or lifestyle of the participants did not change though they were instructed to avoid the consumption of fruits or similar elements contained in the juices.Each subject's weight and height were measured for calculating the body mass index (BMI).The subjects' diets were assessed before and at the end of the intervention using 72 h recall.Women received the drink packed in plastic bottles sterilized for consumption weekend, they were asked refrigerated, protected from direct light and heat.Fasting venous blood samples were collected before and after 2 weeks of intervention from each subject.Blood was collected in heparinized tubes and centrifuged to obtain plasma for antioxidants analysis and non-heparinized tubes to obtain serum for lipid profile, glucose and C reactive protein analysis.

Subjects
Twenty healthy women (aged 18 to 60 years) from the Universiy of Antioquia at Eastern Region were recruited in the study.The study protocol was approved by the Human Ethics Committee of Denstistry Faculty from the University of Antioquia University, and informed consent was obtained from each subject.All the subjects were in good health, with a regular medical history, and none of the subjects were on any medication, smoking, heavy physical exercise or took vitamin/mineral, antioxidant or herbal supplements.

Antropometrics and blood pressure
Before (initial time, day 1) and after the intervention (final time, day 14), weight, size, body mass index (BMI), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using Tanita BC-1500 Ironman Radio Wireless, measuring tape Seca 201 and digital sphygmomanometer Welch Allyn, respectively.

Total phenols in blood plasma
The total phenolic content was determined according to the adapted Folin-Ciocalteu method (Singleton, 1999).Plasma deproteinized by perchloric acid (50 µl) were mixed with 125 µl of Folin-Ciocalteu reagent and 400 µl of sodium carbonate solution (7.1% p/v), and the resulting solution was brought to a final volume of 1000 µl.The mixture was stirred and stored at room temperature for 30 min in the dark.The absorbance was measured at 760 nm against a control sample.Aqueous solutions of gallic acid were used to build a calibration curve.The results were expressed as gallic acid equivalents (GAE)/ml.

FRAP assay (Ferric Reducing/Antioxidant Power) in blood plasma
The antioxidant capacity of wine was estimated according to the procedure described by Benzie and Strain (1996), with some modifications.This method is based on the increase of absorbance due to the formation of 2, 4, 6-tripyridil-s-triazine (TPTZ)-Fe (II) in the presence of reducing agents.A volume of 50 μl of deproteinized plasma was mixed with 950 μl FRAP reagent previously dissolved in acetate buffer (pH 3.6).The absorbance increase was measured at 590 nm.The FRAP values were expressed as ascorbic acid equivalent antioxidant capacity (AEAC: mg ascorbic acid per ml) using an ascorbic acid standard curve.

ABTS assay in blood plasma
ABTS radical was produced through an oxidative reaction of ABTS using potassium persulphate.The capacity of plasma samples to scavenger ABTS radical was evaluated by reducing the absorbance after 30 min at 732 nm.Results were presented as TEAC values/L solution using a Trolox standard curve (Van der Berg et al., 1999).

Evaluation of ROS scavenging capacity in blood plasma
The method described by Rojano (2008) was used.2.7 dichlorodihydrofluorescein (DCFH) reacts with ROS produced by 2,2'-diazobis (2-amidinopropane dihydrocloruro) (AAPH) in an aqueous medium and forms the compound 2,7 dichlorofluorescein (DCF) fluorescence.50 µl of 0.3 M AAPH solution were mixed with 50 µl of 2,7-dichlorofluorescein diacetate ethanolic solution 2.4 mM, 2850 µl of 75 mM phosphate buffer, pH 7.4 and 50 µl of the sample were be evaluated.The intensity of fluorescence emitted during the first 10 min was analyzed and compared to the intensity emitted in the absence of the sample (λ excitation: 326 nm, one λ emission: 432 nm and 10 nm slit).Results were expressed as % relative value.

Statistical analysis
Data are presented as mean ± standard deviation (SD) for normally distributed data or median and p25-p75 for data with non-normal distribution.For statistical differences, Student's t-test for repeated samples and the Wilcoxon test were used.To determine the correlation between antioxidant activity values, blood biomarkers and the total content of polyphenolic compounds, the Pearson correlation coefficient was calculated.ANOVA with repeated measures was used to evaluate interaction between time and type of drink, this type of ANOVA was choosen beacause participants of the study were measured at two times to see the changes to the intervention with Nativanox® Colombian yellow fruits or Nativanox® Colombian tropical fruits.A post-test analysis was performed for significant differences.The level of significance was p<0.05.All analyses were performed using SPSS version 22.

RESULTS
After two weeks (14 days) of consumption of a drink prepared with Nativanox® Colombian tropical fruits and other prepared with Nativanox® Colombian yellow fruits, no significant changes were observed in the anthropometric variables weight and BMI, but significant decrease in diastolic blood pressure in the group of yellow fruits was observed (Table 2).
An improvement in lipid profile parameters was evidenced in both groups, but only a statistical significance was reached (p <0.05) in the group of Nativanox® colombian tropical fruits for total cholesterol and LDL-cholesterol (Table 3).
The antioxidant capacity measured in plasma using ABTS and ROS methods, and the total concentration of phenols in both groups showed no significant difference between day 0 and day 14 of intervention.However, in the group of participants who received the drink prepared with Nativanox ® Colombian tropical fruits, a significant difference in reducing activity of plasma determined by FRAP assay was observed (Table 4).There was an inverse Pearson correlation between the total phenolic compounds content and triglyceride, VLDLcholesterol, total cholesterol, FRAP and ROS values in the group Nativanox® Colombian tropical fruits.On the contrary, the group that received drink prepared with Nativanox® Colombian yellow fruits did not show significant Pearson correlation (Table 5).
A significant interaction time vs. intervention was found for cholesterol-LDL and FRAP values (p <0.05) (Table 6), indicating a different in time variation in every group, being significant for the group that received the drink prepared with Nativanox® Colombian tropical fruits.
Additionally, the values of DBP, total cholesterol, HDLcholesterol and LDL-cholesterol were significantly different between the two groups, likewise significant changes of FRAP values after the intervention period were evidenced (Table 6).

DISCUSSION
The results of several studies show the beneficial effect of polyphenols on the cardiovascular system, through mechanisms that improve endothelial function, platelet aggregation and anti-inflammatory function (Vita, 2005).
In this study, a positive effect of consumption drink prepared with Nativanox® Colombian yellow fruits on the diastolic blood pressure after ingesting daily for 14 days was observed, suggesting a health benefit of this product on the vascular homeostasis.Similar findings after consumption of a diet rich in berries showed a high antioxidant content (Erlund et al., 2008) or after consumption during 4 weeks of rich tomato lycopene (Engelhard et al., 2006).Taking account of the bioactive compounds present in Nativanox® Colombian yellow fruits (Table 1), this preparation has an important content of tannins and carotenoids.Tannins are present in pommegranate juice (Ignarro et al., 2006;Stowe, 2011) and Sumac berries (Rhus coriaria) which are able to reduce diastolic pressure and the vascular smooth muscle cell (VSMC) migration by 62% (Zargham and Zargham, 2008).It has been shown that hydrolisable tannins are able to increase nitric oxide (NO) a vasoactive molecule endothelialderived whose alteration is involved in the ocurrence of atheroesclerosis (Moncada and Higgs, 2006).In relation to the carotenoids present, only in Nativanox® Colombian yellow fruits, β-carotene is widely distributed in fruits in vegetables and is considered beneficial to endothelial functions and vascular health because of the ability to increase NO that leads to a downregulation of the expression of NF-kB-dependent adhesion molecules in endothelial cells involved in the proinflammatory response, by which (Aizawa et al., 2003;Gammone et al., 2015).Thus, it will be interesting to investigate the type of tannins (hydrolysable or condensed) and carotenoids present in Nativanox® Colombian yellow fruits and to determine its effects on nitric oxide metabolites after consumption.
In other hand, the findings in this study regarding lipid profile show that consumption of Nativanox® Colombian tropical fruits rich in polyphenols such as flavonoids and tannins, decreased the concentration of total cholesterol   and LDL-cholesterol.A study shows similar findings of the relationship between cocoa catechins and epicatechins with the LDLcholesterol concentration (Baba et al., 2007).Beneficial effects have been observed in humans after intake of chocolate (Baba et al., 2007;Mursu et al., 2004), grape juice (Freedman et al., 2001) and black tea (Duffy et al., 2001) with high content of phenolic compounds.Epidemiological studies suggest that the rate of cardiovascular events is reduced by about 1% for every 1% decrease in LDL (Brown et al., 2006), which is important in the prevention and management of these diseases.A possible mechanism involved in the results observed here on LDL-cholesterol levels is the evidenced obtained with phenolics of cocoa that decrease levels of LDL-cholesterol by reducing protein and mRNA expression of ApoB protein and mRNA which is contained in LDL-cholesterol lipoprotein, whereas cocoa polyphenols increase apolipoprotein (Apo) A1 and mRNA expression present in HDL-cholesterol lipoprotein (Andújar et al., 2012).With regard to total phenol content in the drink prepared with Nativanox® Colombian tropical fruits and triglycerides and VLDL-cholesterol, a negative correlation was found, indicating that the higher concentration of total phenols lower levels of these parameters.These effects on triglycerides and VLDL has been observed in people following diets naturally rich in polyphenols (2776 to 2903 mg) for 8 weeks (Annuzzi et al., 2014) that significantly reduced fasting triglyceride concentrations (2-factor ANOVA) in plasma (P = 0.023) and large very-low-density lipoproteins (VLDLs) (P = 0.016) and postprandial triglyceride total area under the curve in plasma (P = 0.041) and large VLDLs (P = 0.004) (Anuzzi et al., 2014).
In the case of hydroxyl radicals, they are generated by the Fe 2+ -EDTA/H 2 O 2 system.The mechanism is carried out in three stages: first, the oxidation of the pair Fe 2+ EDTA occurs with molecular oxygen to form Fe 3+ -EDTA and superoxide radical.In the second, the superoxide radical in the presence of hydrogen is dismutated to H 2 O 2 ; and in the last stage, the Fe (II)-EDTA catalyzes the decomposition of H 2 O 2 to •OH (Yang and Guo, 2001).After being generated, hydroxyl radicals react with terephthalic acid to form a highly fluorescent product monohydric, 2-hidroxitereftalato acid.The capacity of Nativanox® to catch hydroxyl radicals decreases the amount of product 2-hidroxitereftalato, which can be evidenced on the decreased fluorescence intensity.Values for Nativanox® of 36147 mg DMSO/100 g dry weight have a considerable value as a bioactive expression.
Similarly, some bioactive of Nativanox have a similar behavior like anthocyanins.This actives compounds might provide a lot of effects such as the reactive oxygen species scavenger capacity, chelate metals, stimulating the expression of enzymes, reducing the formation of oxidative DNA adduct, reducing lipid peroxidation inhibiting toxins and environmental mutagenesis carcinogens, and reducing cell proliferation by modulating the signal transduction pathways (Wang and Jiao, 2000;Wang and Stoner, 2008).Nativanox contains bioactive with a high capacity to trap peroxyl free radicals (ROO•), hydroxyl (OH •), and generally reactive oxygen and nitrogen species (ROS and RNS).An intake of 200 ml Nativanox drinks provides about 4,000 IU ORAC, enough to maintain a good oxidative balance.
The study of antioxidant status in plasma of participants after 14 days of intake 200 ml of a drink prepared with Nativanox® colombian tropical fruits led to a significant increase in the reducing capacity of plasma determined by FRAP method, a trend also reported by Pedersen et al. (2000) in a group of 9 healthy women who consumed a cranberry juice for 1 week.This effect was attributed to the phenolic content and vitamin C of juice.
The correlation between the total phenol content in the beverage prepared with Nativanox® Colombian tropical fruits and FRAP value was inversely significant, suggesting that this antioxidant activity in plasma could be attributed to other polyphenolic compounds presents in drink such as tannins, carotenoids and vitamin C.

CONCLUSION
Results of this study show favorable changes in diastolic blood pressure, total cholesterol and LDL-cholesterol as well as improvement in antioxidant status in plasma measured by the FRAP method, and an inverse relationship between total phenolic content and triglycerides and VLDL-cholesterol levels after consumption of a drink containing Nativanox® Colombian tropical fruits and other drink with Nativanox® Colombian yellow fruits for a short period of time in healthy women.These findings are important because they can explain the protective role against cardiovascular risk of a diet that includes fruits, and propose the development of food matrices containing active ingredients mixtures of yellow and tropical fruits for the prevention of non-communicable chronic diseases associated with oxidative stress.It is also demonstrated that the combination of fruits has a synergistic effect on the antioxidant capacity, which opens the door to the development of nutraceuticals or functional foods from the Nativanox® Colombian yellow or tropical fruits.Additional studies are needed to identify the bioactive compounds and the mechanisms responsible for these observed effects and their effect on cardiovascular risk population.

Table 2 .
Effect of treatment on antropometrics and blood pressure values.

Table 3 .
Lipid profile, reactive C protein and blood glucose.

Table 6 .
ANOVA repeated measures anthropometric, blood and plasma markers.