Mangiferin content, carotenoids, tannins and oxygen radical absorbance capacity (ORAC) values of six mango (Mangifera indica) cultivars from the Colombian Caribbean

Mango is one of the tropical fruits of greater production and consumption in the world, and a rich source of bioactive compounds, with various functional properties such as antioxidant activity. In Colombia, mango’s market is very broad and diverse. However, there are very few studies that determined the content of bioactive secondary metabolites. The objective of this study was to evaluated the content of different metabolites like Mangiferin, carotenoids, tannins, and the antioxidant capacity by oxygen radical absorbance capacity (ORAC) methodology of six cultivars from the Colombian Caribbean region, with total carotenoid values ranging from 24.67 to 196.15 mg of β-carotene/100 g dry pulp; 84.30 to 161.49 mg Catequine eq./100 g dry pulp for the content of condensed tannins, and 91.80 to 259.23 mg/100 g dry pulp for mangiferin content. The ORAC methodology showed important antioxidant activity results, such as the Chancleta variety with the highest value (2163.78 μmol Trolox/100 g dry pulp). In conclusion, the evaluated mango varieties had promising results as functional food of high nutraceutical value, being Chancleta, Criollo and Jobo varieties, the fruits with highest content in bioactive compounds that expressed the best antioxidant activity.


INTRODUCTION
Mango (Mangifera indica L.) from Anacardiaceae Family, is a tree 15 to 30 m high, with quick growth in shallow and well drained soils, and pH oscillating from 5.5 to 7.5 (Shah et al., 2010).In tropical zones of Colombia, it grows up to 1200 m high, with a high fruit production within 3 months of the year in low rain seasons (Bally, 2006).In Colombia, mango has more than 200 ecotypes or genetically differentiated cultivars.However, there is *Corresponding author.E-mail: brojano@unal.edu.co.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License no much information on its properties as functional food.
In other hand, mango in currently known as one of the most important tropical fruit.It has been cultivated since prehistoric times and its tree has been object of high veneration in India.Different countries cultivate this fruit, among them are, Indonesia, Florida, Hawaii, Mexico, South Africa, Egypt, Israel, Brazil, Cuba and Philippines.Probably, India has more commercial plantations than the rest of the world.However, mango's economic importance is due to the great local consumption in Caribbean countries, especially in Colombia (Michel et al., 2000).
The consumption of fruits rich in biologically active compounds brings important benefits for human health; mango is considered a fruit with nutraceutical properties due to presence of carotenoids, ascorbic acid, fiber, polyphenols, among others; which are associated to antioxidant and anti-inflammatory properties, important in treatments of metabolic disorders such as obesity and diabetes (Ibarra- Garza et al., 2015;Septembre-Malaterre et al., 2016).Also, knowledge of concentration of bioactive structures in fruits, allows obtaining information to implement extraction and drying techniques to give it greater added value as a functional food and to compete in the international market with high quality products.
One of the bioactive compounds of greatest interest in the species M. indica is Mangiferin, a polyphenol type glycosyl xanthone that presents pharmacological actions with antioxidant, anti-inflammatory, and neuroprotective properties (Takeda et al., 2007).Studies have suggested that Mangiferin also has an in vitro anticancer effect in cellular lines of acute myeloid leukemia (AML) (Shoji et al., 2011).Matkowski et al. (2013) reported Mangiferin as a promising natural product with benefits as analgesic, antidiabetic, anti-sclerotic, antimicrobial, antiviral, cardio and hepatoprotective, anti-allergic, monoamine oxygenase (MAO) inhibitor, and protector against UV radiation.
Carotenoids are pigments found in fruits and vegetables such as mango.Chemically, carotenoids are classified as tetraterpenes, of which more than 600 natural structures of different size, shape and polarity have been identified.Carotenoids are associated with various biological processes like antioxidant defense, photosynthesis and, some of them, as precursors of vitamin A. Also, they determine the orange and yellow colors of mango fruits.Many studies reported that an adequate intake of carotenoids is related to the decrease of various types of cancer (Melendez-Martínez et al., 2007).
In other hand, condensed tannins are polymers formed by flavan-3-ol units.Also, they are one of the principal antioxidant metabolites in products like tee and cocoa which content can be up to 35% of total polyphenols.Tannins are associated to the astringent flavor of fruits and enzymatic browning reactions in mango bark (Pierson et al., 2014).
In Colombia, mango is consumed mostly fresh and it is used in pulps, nectar, juices, jellies and jams elaboration (Mora et al., 2002).Due to the low agro-industrial development, sale in fresh is privileged.However, postharvest losses reach approximately 40% of annual production (Sumaya-Martinez et al., 2012).Mango is one of the most consumed tropical fruit in the world, especially for its flavor, texture and versatility (Oliveira et al., 2016).However, the studies that have characterized its nutraceutical power in Caribbean Colombian zone cultivars are few.That is why the present work wants to know the metabolites content, specifically Mangiferin, carotenoids and total polyphenols, associated with antioxidant activity in six mango cultivars from Córdoba, Colombia.

Vegetal material
Vegetal material was collected in the Department of Córdoba, Colombia, especially in Monteria's local market (18 M. ASL and a mean temperature of 28°C in June 2016).Specimens in optimum conditions of each variety were collected randomly in mature state.After being stored in perforated polypropylene bags, they were taken to the Laboratory Food Science, Universidad Nacional de Colombia, Medellin, for analysis.

Sample preparation
3.0000 g of pulp were homogenized with 20 ml of deionized water in Ultra-Turrax (IKA-WERK © ) homogenizer.The extract obtained from that was centrifuged at 5000 rpm for 10 min at room temperature.The supernatant recovered was stored at 4°C until the analysis.

Color determination
The color was measured with a colorimeter Minolta (Minolta Co. Ltd., Osaka, Japan) on the basis of the color system CIELAB (L*, a*, b*).In this system L*, a* and b* describe a tridimensional space, where L* is the vertical axis and its value ranges from 100 for perfect white to zero for black.Values a* and b* specify the axis green-red and yellow-blue, respectively.That values range from -60 to +60 or from -a (green) to +a (red) and from -b (blue) to +b (yellow).To determinate the color, a portion of one side of each fruit was obtained and measured longitudinally in three equidistant points (Garza et al., 1996).

Assessment of reducing power by FRAP method
This method assesses the reducing power of a sample according to its capacity to reduce ferric iron (Fe +3 ) with TPTZ to its ferrous form (Fe +2 ), which has its maximum absorbance at 593 nm, according to the design done by Benzie and Strain (1996).50 µL of sample were added to 900 µL of a FRAP solution, acetate buffer pH 3.4, TPTZ and FeCl3 in relation 10:1:1.The results were expressed as ascorbic acid equivalent antioxidant capacity (AEAC: mg of ascorbic acid/100 g dry weight).

Oxygen radical absorbance capacity (ORAC) assay
The method described by Prior et al. (2005) and Romero et al. (2010) was used.30 µL of sample were added to 21 µL of fluorescein 1x10 -2 M in PBS (75 mM), 2.899 µL of PBS (75 mM), and 50 µL of AAPH 0.6 M in PBS (75 mM), temperature was controlled to 37°C and pH was kept at 7.4.The readings were done at an excitation ʎ 493 nm and excitation slit 10 nm.It was compared to the primary pattern Trolox® curve.The results were expressed as TEAC, µmol trolox equivalent /100 g of dry weight according to Equation 1. (1) Where AUC is the area under the curve of the sample, AUC° is the area under the curve for control, AUCTrolox is the area under the curve for Trolox, and f is the dilution factor for the extracts.

Total phenols
Determination of phenols was done by the colorimetric method Folin-Ciocalteu designed by Singleton and Rossi (1965).50 µL of sample were added to 125 µL of Folin reagent and 400 µL of sodium carbonate 7.1% (w/v), adjusting with distilled water until 1000 µL.The spectrophotometric reading was done at 760 nm and it was compared to the pattern curve using gallic acid as standard.The results were expressed as mg of gallic acid equivalent: GAE/100 g dry weight.

Carotenoids determination
It was done by Ultraviolet-Visible (UV-Vis) spectrophotometry.In a test tube, 1.0000 g of sample was added to 5.0 mL of cold acetone and it was left to stand for nearly 15 min in refrigeration (4°C).The mixture was centrifuged at 1370 gravities for 10 min, the supernatant was collected in another test tube.The pellet was reextracted with 5.0 mL of cold acetone.Both acetone extracts were mixed, and then, were filtered in Whatman paper No. 42 and its absorbance was determined at 449 nm.Carotenoid concentration was obtained by the respective calibration curve, with β-carotene as pattern substance.Data was obtained by the Software SkanIt 2.4.2RE for Multiskan Spectrum (Biswas et al., 2011).The results were expressed as mg of β-carotene equivalent/100 g dry weight.

Ascorbic acid determination
It was established by HPLC.The aqueous supernatant was filtered (pore size 0.45 µm) and dilutions in super pure water, before injection to chromatographer.A liquid chromatographer Shimadzu® model LC-20AD was used, equipped with an auto-injector SIL-20A/HT, a communication module CBM-20A and a (PDA) SPD-M20A, calibrated to 245 nm.Ascorbic acid quantification was done with a C-8 (5 µm, 250 mm x 4.6 mm) column.Formic acid 0.1% was used as mobile phase, at a flow rate of 0.8 mL min -1 , at 35°C in isocratic conditions (Kelebek et al., 2009).The results were expressed as mg of ascorbic acid/100 g dry weight.

Condensed tannin determination
This method is based on reaction of condensed tannins with vanillin under acid conditions, using catechin as standard.A 2 ml aliquot of freshly prepared vanillin solution (1 g/100 mL) in sulfuric acid 70% was added to 500 µL of mango extract.The mixture was incubated at 20°C for 15 min and its absorbance was read at 500 nm.(+) Catechin was used to build the reference curve, and the results were expressed as mg catechin/100 g dry weight (Hagerman and Butler, 1989).

Statistical analysis
All the experiments were performed in triplicate.The regressions were calculated with a 95% significance level (p<0.05),using the Statgraphics Plus program version 5.0 (Statistical Graphics Corp., Rockvilee, MD).

Color determination
Color measure is a fundamental parameter in climacteric fruit analysis, given that the changes observed by the naked eye are associated with variations in the fruit's chemical composition.According to Nambi et al. (2015) in the mango's mature state, the coordinates of CIELAB system are characterized by presenting values between 51 to 60, 16 to 29, and 48 to 62 for L*, a* and b* respectively (Table 1).
Mango's pulp composition varies depending on many factors such as variety, location, weather, and maturity stage.However, previous studies have shown that the stage of maximum expression of bioactive compounds is presented in mature mango.Polyphenols, reducing agents like ascorbic acid, and carotenoids found in fruits and vegetables are the most studied metabolites due to its antioxidant potential, which are associated with health benefits such as protection against cardiovascular diseases and cancer (Masibo and He, 2009).Total phenols and Mangiferin contents were superior in the Criollo variety.Tannins were higher in Criollo and Corazón varieties.While the ascorbic acid content was expressed better in Chancleta variety (99.6 mg ascorbic acid/100 g dry base).The variety Jobo reported the highest carotenoid content (196.15mg β-carotene/100 g dry base) (Table 2).
Polyphenolic compounds present in fruits, have antioxidant activity because of their property to interact with different oxygen radical species by a reducing action.The mechanisms to capture free radicals are classified into two types.In both cases, they are blockers of the initial stage in the oxidative process of lipid or protein.The first mechanism is the transferability of a hydrogen atom, called HAT, which is measured by ORAC methodology; and in the second case, it is the transfer of a polyphenol electron, SET mechanism, which is measured by FRAP technique.In Table 3, it is observed that Criollo mango has high values for ORAC and FRAP, in such way that these antioxidant properties by both mechanisms are due to its composition of phenols and condensed tannins fundamentally.Chancleta mango has the highest values for ORAC in accordance with its total phenol content.

DISCUSSION
Various studies report similar values of color measurement to those found in this research, concluding that the carotenoid content like β-carotene, 9-cisviolaxanthin and lutein, is correlated positively (r 2 > 0.9) to the measurements of these parameters (Ayour et al., 2016).Thus, the studied mango varieties could have important bioactive pigment content.During maturation, climacteric fruits suffer different physiological changes at physical and biochemical level.Among the phenomena associated with fruit maturation, there is color change from green to yellow, orange, or even red.This change is caused by carotenoids' synthesis and chlorophyll's degradation as the first observable sign of maturation (Merzlyak et al., 1999).

Total phenols
Phenolic compounds constitute a group of secondary metabolites that are considered natural antioxidants with multiple biological benefits for human health.According to the results from Table 3, the varieties with greater polyphenol content are Chancleta, Criollo and Jobo,with 238.14,196.58 and 160.44 mg of gallic acid/100 g dry matter, respectively.Different authors say that the main phenols found in mango are chlorogenic, gallic, vanillin and protocatechuic acid, in order of abundance (Wall-Medrano et al., 2014).On the other hand, in the mango's peel and seed, values between 3.8 and 13 GAE/100 g dry matter have been found (Dorta et al., 2012).Chong et al. (2013) reported values between 120.70 and 210.24 mg gallic acid/100 g for mango cultivated in Malaysia.Also, Lobo et al. (2017) showed that there is a great oscillation in total phenol content in the Tommy Atkins mango pulp cultivated in Brazil (46.18 -116.93 mg gallic acid/100 g).Thanaraj et al. (2009) reported 1055 and 1691 mg gallic acid/100 g dry weight as the interval for five mango varieties cultivated in Sri Lanka.A high variability in total phenol content was reported for mango, due to the different weather and growing conditions of each region.However, the evaluated Colombian pulps presented a significant content of phenolic compound as compared to what was reported by most authors.

Total carotenoids
Carotenoids are the compounds responsible for coloration in most food, some of them are provitamin A, like α and β-carotene, and β-cryptoxanthin.Recent studies have made these pigments' antioxidant properties manifest, as well as their effectiveness in the prevention of certain human diseases, such as atherosclerosis or even cancer (Müller et al., 2011).Criollo and Jobo varieties presented the higher total carotenoid content with 105.88 and 196.15 mg βcarotene equivalent/100 g dry matter, respectively.Silva et al. (2014), who quantified bioactive compounds in different fruit from Brazil, found 0.954 mg β-carotene equivalent/100 g dry matter in mango; Seok et al. (2010) reported that the carotenoid content in Malaysia's mango pulp was 0.650 equivalent β-carotene/100 g dry matter, which are much lower values than the ones found in this study.It is worth noting that there are 17 important carotenoids found in mango, among them βcryptoxanthin, zeaxanthin, isomers of luteoxanthin, violaxanthin, neoxanthin and β-carotene, which is the last one, the one with the highest prevalence (Rungpichayapichet et al., 2015).The above, suggests Colombian mango as an important source of these pigments, not only for fresh consumption, but for its use as a colorant additive in food matrices.

Vitamin C
Mango is recognized for its important contribution in vitamins like ascorbic acid, thiamin, riboflavin, and niacin.In this study, the varieties with the most vitamin C content were Chancleta and Jobo.However, the reported content for the six cultivars was lower than the one for other fruits studied in countries like Ecuador, Brazil and Colombia (Contreras-Calderón et al., 2011;Vasco et al., 2008).Seok et al. (2010) found that ascorbic acid content in fresh mango from Malaysia was 136.8 mg/100 g dry matter, a value close to the one found for Chancleta.The variations in vitamin C content are due to the fact that this compound is cataloged as the most unstable nutrient and prone to immediate loss after harvest.Therefore, its content depends on the changes during the post-harvest handling, storage conditions, transformation and elaboration (Spínola et al., 2013).

Condensed tannins
Polyphenols present in fruits and vegetables can include simple structured compounds (phenolic acids), oligomers (flavonoids, xanthones, stilbenes), or polymers (condensed tannins) (Pierson et al., 2014).Condensed tannins are oligomeric and polymeric proanthocyanidins that are widely distributed in the vegetable kingdom.It has been reported that they are used to stop small local bleeding, decrease buccal cavity inflammations, colds, bronchitis, burns, hemorrhoids, etc. (Piovesan et al., 2017;Kasay et al., 2013).Also, the condensed tannins' chemical nature makes them a natural source of organic compounds, with an application potentially wide for medicinal and industrial uses (Aguilar- López et al., 2012).According to the results in Table 2, all mango fruits presented a high condensed tannin content, highlighting the varieties Corazón and Criollo.Gorinstein et al. (2011) evaluated the bioactive compound content in different exotic fruits, reporting 27.0 mg of catechin equivalent/100 g dry weight as the condensed tannin content in mango.Arogba (2014) reported a content of 135.0 mg of catechin equivalent/100 g dry weight in mango seed.The above shows that the evaluated mango varieties presented an important condensed tannin content, compounds whose antioxidant activity has been widely reported (Dobrecky et al., 2014;Ocampo et al., 2014).

Mangiferin
Mangiferin is the main polyphenol that constitutes mango's leaves, fruit and bark.The literature has reported its wide pharmacological, antidiabetic, antitumor, immunomodulatory and antioxidant activity (López et al., 2015).According to the results presented in Table 2, mangos cultivated in Córdoba present similar values and even higher for Mangiferin content reported in different research.For example, Luo et al. (2012) reported that the Mangiferin content in 11 varieties of mango pulp from China, was between 0.2 and 20 mg/100 g dry matter.
Other authors suggested that Mangiferin is mainly found in mango's peel, leaves and bark, and in smaller measure, in pulp, reporting values from 169 mg/100 g dry matter in peel, and 4.2 mg/100 g dry matter in seeds (Rymbai et al., 2015).This shows that the evaluated Colombian mangos have a promising Mangiferin content, highlighting their nutraceutical quality and industrialization of this fruit in functional products.The high Mangiferin content in the studied varieties can be explained since this xanthone is synthesized by the phenylpropanoid pathway in presence of high solar radiation, which is a representative condition of the Caribbean Colombian region (Ruiz and Romero, 2001).

FRAP and ORAC-Total
Different methodologies have been proposed to evaluate fruit's antioxidant capacity, of which ferric ion reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) methods are widely used.These methods measure different mechanisms of antioxidant activity; while ORAC is related to capacity for neutralizing a free radical using HAT mechanism, FRAP assay measures sample's capacity for reducing the ion Fe 3+ to Fe 2+ (SET mechanism) (Rodríguez et al., 2010;Botero et al., 2007).In this research, the variety with the best reducing capacity was Chancleta with 296.06 mg of ascorbic acid/100 g dry sample, followed by Criollo and Azúcar.In general, low values of reducing capacity have been reported for mango (Paz et al., 2015).

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FRAP values found in the evaluated mango pulps, match with low quantity of vitamin C analyzed by HPLC.However, ORAC unities from Chancleta, Criollo and Jobo varieties are higher than the ones reported by Singh et al. (2015) in dried Tommy mango chunks (408 to 651 µmol TE/100 g dry sample).In other hand, ORAC values between 0.46 and 1.33 mmol Trolox/100 g dry simple were found in a study that was done on 10 cultivars of Mediterranean diet fruits (Wojdylo et al., 2016).This shows that the analyzed varieties in this research exhibit a considerable antioxidant capacity measured with ORAC methodology.It is worth to mention that this fluorometric technique is the one endorsed by the United States Department of Agriculture (USDA) for measuring antioxidant capacity in food and nutritional supplements (Rojano et al., 2012).

Correlations between polyphenols and antioxidant activity
As shown in Figures 1 and 2, a positive correlation was found between total phenol content and antioxidant capacity evaluated with ORAC and FRAP (r 2 = 0.87 and r 2 = 0.84), which shows that polyphenols are the main contributors to mango's antioxidant activity.Also, the synergism between antioxidants could explain why the fruit's antioxidant capacity is higher than the individual content in each antioxidant, like vitamin C. Similar results have been found in other researches (Thaipong et al., 2006;Silva and Sirasa, 2016).

Conclusion
The present study showed the antioxidant potential of six mango varieties cultivated in the Department of Córdoba, being Chancleta, Criollo and Jobo, the varieties that showed the highest antioxidant activity in vitro.As regards mangiferin content, all varieties stood out above fruits from other countries.These results prove that Colombian mangoes have a high nutraceutical potential due to its Mangiferin content and to the antioxidant expression of the other bioactive compounds like carotenoids and tannins.It is necessary to conduct studies in vivo to determine bio-accessibility and/or bioavailability of mango's extracts with higher antioxidant potential and Mangiferin as the main bioactive.

Table 2 .
Secondary metabolite results in mango samples.

Table 3 .
Antioxidant activity results in mango samples.