Plant Foods for Human Nutrition 62: 13–17, 2007. c 2007 Springer Science+Business Media, Inc. DOI: 10.1007/s11130-006-0035-3 13 Antioxidant in Mango (Mangifera indica L.) Pulp ˆ ´ HUMBERTO de QUEIROZ,2 MARIA ELIANA LOPES SONIA MACHADO ROCHA RIBEIRO,1, ∗ JOSE 3 ´ RIBEIRO de QUEIROZ, FLAVIA MILAGRES CAMPOS1 & HELENA MARIA PINHEIRO SANT’ANA1 1 Departamento de Nutric ˜ o e Sa´ ¸a ude, Universidade Federal de Vic ¸ osa, Vic ¸ osa, MG, Brazil; 2 Departamento de Bioqu´ ımica e Biologia Molecular, Universidade Federal de Vic ¸ osa, Vic ¸ osa, MG, Brazil; 3 Departamento de Qu´ ımica, Universidade Federal de Vic ¸ osa, Vic ¸ osa, MG, Brazil (∗ author for correspondence; e-mail:
[email protected]) Published online: 23 January 2007 Abstract. This work was carried out to investigate the pulp composition of four mango cultivars (Haden, Tommy Atkins and Ub´ a) at the ripening stage in relation to three components with antioxidant potential (total phenolics, carotenoids and ascorbic acid). Total phenolic compound content was estimated by the Folin-Ciocalteu reagent and total carotenoid content by spectrophotometry at 450 nm. The contents of β -carotene and total vitamin C (ascorbic acid and dehydroascorbic acid) were quantified by high performance liquid chromatography. Differences were found among the four mango cultivars in all the components analyzed. The content of phenolic compounds ranged from 48.40 (Haden) to 208.70 mg/100 g (Ub´ a); total carotenoid from 1.91 (Haden) to 2.63 mg/100 g (Palmer); β -carotene from 661.27 (Palmer) to 2,220 µg/100 g (Ub´ a) and total ascorbic acid ranged from 9.79 (Tommy Atkins) to 77.71 mg/100 g (Ub´ a). These results corroborated previous information that mangoes are a good source of antioxidants in human diet. Key words: Ascorbic acid, β -carotene, Dehydroascorbic acid, Mango cultivars, Phenolic compounds. Abbreviations AA DHAA GAE HPLC TSS = = = = = Ascorbic acid Dehydroascorbic acid Gallic acid equivalents High performance liquid chromatography Total soluble solid. Introduction High consumption of fruits and vegetables has been associated with a lower incidence of degenerative diseases. Such protective effects are thought to be partially associated with the various antioxidant compounds contained in these foods [1]. Fruits from tropical and subtropical climates contain different antioxidant components. Mango (Mangifera indica L.) is one of the most important commercial crops worldwide in terms of production, marketing and consumption. Brazil ranks among the major mango producing countries [2]. Mangoes can be considered a good source of dietary antioxidants, such as ascorbic acid, carotenoids and phenolic compounds [3]. β -carotene is the most abundant carotenoid in several cultivars. The nutritional value of mango as a source of vitamin C and provitamin A should also be emphasized. Vitamin C is an essential human diet component, required for scurvy prevention, presents biological functions in collagen formation, inorganic iron absorption, inhibition of nitrosamine formation, and immune system enhancement. Ascorbic acid acts as an antioxidant and therefore offers some protection against oxidative stress-related diseases [4]. Among the carotenoid pigments widely distributed in plant tissues, β -carotene provides the highest vitamin A activity. Vitamin A and its metabolites are essential for vision, reproduction, and immune function, besides performing other important physiological functions, including the deactivation of reactive oxygen species [5]. The action of phenolic compounds in foods has been drawn a lot of attention because of their biological activity in cancer and heart diseases prevention [6]. These compounds are preferably oxidized in biological medium and function as antioxidant nutrient economizer, protecting organisms against the oxidative stress [7]. Studies carried out with rats fed diet supplemented with 10% mango confirmed reduction in cytotoxic effects induced by dimethyldrazine through the optimization of enzymatic oxidative mechanisms [8]. Despite mango’s importance and wide acceptance by the consumer, little has been studied on the content of antioxidant components in mango varieties. The studies have been focused the nutritional value as source of vitamin C and β -carotene but information on the total phenolic content are scarce. Mango cultivars differ in their antioxidant component content due to genotypic variation, and preharvest factors, including climatic conditions, agricultural practices, and ripening stage [9]. This article describes the results of the study on the contents of total phenolic compounds, total carotenoids, β -carotene and vitamin C (ascorbic acid and dehydroascorbic acid) in the pulp of four mango varieties, cultivated in Brazil. Materials and Methods Sample Collection and Preparation Four mango cultivars grown in Brazil were used in this study: Haden, Tommy Atkins, Palmer and Ub´ a. The first with minor modifications.0 ml min−1 .0 ml) for 5 min with a micro grinder. and the results expressed in 100 grams of fresh weight (edible portion). and 30 µl aliquots were injected into the liquid chromatographic system. 5 µm. the lyophilized samples were reconstituted in distilled water and all the procedures performed in dim light.0 g) was extracted using a 10. The extract was partitioned with petroleum ether. b∗ scale. Pulp quality of each variety (20 units pooled and homogenized) was evaluated for the parameters color and total soluble solids (TSS). Pulp from 20 completely ripe mangoes of each variety was homogenized without peel and pureed.45 µm Millipore membrane and a 30 µl aliquot was immediately used for HPLC analysis. Lyophilized material (1. at the intermediary ripening stage.0 ml with ultra pure water and centrifuged at 1000 × g for 15 min. v/v). The mixture was filtered and the total final volume was adjusted to 25. aliquots (2.0 ml). An aliquot (1. All the measurements were evaluated in triplicate. Tokyo. Japan). Other chromatographic conditions were: ultra pure water with metaphosphoric acid at pH 2.45 µm Millipore membrane.0 ml min−1 . Wavelength detection was 450 nm and mobile phase consisted of methanol. Pulp color was evaluated in a ColorQuest II – Sphere colorimeter (“Hunter Lab Reston”. VA). a∗ . The mango puree was lyophilized.0 ml) of the sample supernatant was mixed with 0. Total Carotenoids and β -Carotene Carotenoid extraction followed the procedure described in Rodriguez et al. Total carotenoid content was estimated in triplicates according to methodology described by Higby [14]. Total soluble solids (TSS) was determined using a hand refractometer TECNAL.0 ml with petroleum ether to obtain the concentrated extract.0 mL mixture of methanol: water (60:40. Two milliliters of the supernatant were filtered in a 0. an auto-sampler injector. Firmness was determined using a Fruit Pressure Tester (FT 327. Separation was performed in a RP18 columm (Microsorb. on the basis of their moisture content. using β -carotene to prepare the standard curve and the peak area for the calculation. Five grams of reconstituted pulp were stirred in ultra pure water (15. Pulp moisture content was determined by drying at 105 ◦ C. 4 nm × 250 nm). Gallic acid was used as a spectrophotometer standard and total phenolic content was determined on the basis of calibration equations with values expressed as Gallic Acid Equivalents (GAE) in milligrams per 100 grams of pulp. The supernatant fractions were used immediately for analysis. model AR200. [15]. by subjective observation considering softness and peel color [10]. Total Phenolic Content Pulp extracts containing phenolic compounds were obtained as previously reported by Bloor [11].12. Quantification was performed by external standardization. each fraction was washed with distilled water for complete acetone removal. controlled by Multi System. stored at –20 ◦ C and protected from light until analysis. ethyl acetate and acetonitrile (70:20:10) at a flow rate of 2. The extract was concentrated in a rotary evaporator (<35◦ C) to a final volume of 10 ml.0 ml) of concentrated extracts were evaporated under nitrogen. . and a UV-visible photodiode array detector (Shimadzu SPD-10 AVP). Class VP 6. The pulp from variety Ub´ a was acquired directly from the regional farm industry (‘Zona da Mata Mineira’). shaken at 180 rpm at room temperature for 30 min. [13] with minor modifications. For β -carotene quantification. using the L∗ . The mangoes were stored at room temperature until complete ripening. The ascorbic acid reaction prior to alkali addition was monitored and the total phenol values were corrected. The mixture was centrifuged at 1000 × g for 10 min. Only one extraction was performed since the 1:10 (sample: solvent) ratio had extraction efficiency close to 100%. The extraction was performed according to Vinci et al.0 g) were ground with cold acetone in a Marconi microgrinder MA 102. Two measurements were taken from the opposite sides of each fruit after removing the peel. Total phenolic contents in the extracts were estimated using the Folin-Ciocalteu reagent. according to Singleton [12]. HPLC analysis used the same equipment of β -carotene quantification.14 three varieties were randomly collected in commercial plantations in northern and northwestern Minas Gerais state. The HPLC analysis system consisted of high-pressure pump (Shimadzu LC10 ATVP. and vacuum-filtered using a B¨ uchner funnel until the residue became colorless. Ascorbic Acid (AA) and Dehydroascorbic Acid (DHAA) Lyophilized pulp of each mango variety was reconstituted with distilled water.2 as mobile phase. Japan).1 ml phosphate Mango Physicochemical Characteristics Twenty units of each variety were analyzed for weight and firmness. detection at 238 nm and flow rate of 1. Samples (5. redissolved in acetone (2. Italy) equipped with an 11 mm-diameter plunger tip. Before the extraction. Absorbance (λmax = 765 nm) was measured using a UV/VIS spectrophotometer SHIMADZU UV-VIS 1601 (Kyoto. The volume was then completed to 25. Reduction of dehydroascorbic acid (DHAA) was performed with dithiothreitol (DTT). filtered through a 0. using a L-ascorbic acid standard curve. are attributed to the carotenoids present in the pulp.3 54. resulting in a higher standard deviation for the latter. the physicochemical characteristics of the studied mango varieties were similar. β -carotene and Vitamin C (ascorbic acid and dehydroascorbic acid). The two forms of vitamin C (AA and DHAA) were quantified. pulp composition of four mango commercial varieties grown in Brazil was evaluated for the contents of total phenolics.7 Note.51 (Kg/cm2 ) 4. since this variety is mainly used in agro industry for juice and pulp production.0. Physicochemical characteristics of fruit and pulp Firmnessb Cultivar Haden Tommy Atkins Palmer Ub´ a Weighta (g) 486. a∗ .9 39. The pooled pulp sample of the evaluated varieties presented total soluble solids (TSS) indices above the minimum acceptable for consumption (12. Firmness is widely used as a ripeness test for many fruits.15 buffer pH 7.3 45. b∗ characterizes pulp color. a. The main use of such indices is for sample characterization.1 14. TSSc ◦ Brix 14.22 11.9 59. evaluated in the 20 pooled fruits.80 22.7 14. and more variable among Palmer fruits. The mixture was kept in the dark for 10 min to convert DHAA into AA. since the intake of polyphenolic compounds in the diet was estimated to range between 0. Positive values of a∗ and b∗ . The Palmer cultivar showed an intermediary value and Haden and Tommy Atkins presented lower values. 20].4 b∗ 48.64 477. However.3 58. .0 o Brix). since a diode array detector was used. L∗ measures luminosity that varies from zero (black) to 100 (pure white). the use of these values is not suitable for comparing ripe stages. n. Pulp firmness decreases with increasing ripening as the pectin content decreases and the soluble solids content increases [17].4 to obtain a final pH of 6. All determinations were performed in triplicates. d Evaluated in the 20 pooled fruits. being the fruits classified from medium to large. At this scale.d.5 11.7 14. It was verified that ripening.20 493. and 100 mM DTT was added to obtain a final concentration of 8.89 2. These sizes meet the market requirements for fresh mango consumption [16]. Overall. Small fruits of variable weight characterize the variety Ub´ a.1 a∗ 13.0 mM.68 20. if it is considered that the mango ripening curve can differ among varieties. The determination of the coordinates L∗ .: not determined. Total Phenolics Total phenolic content was different among the four varieties.4 15.6 ± 2. Tommy Atkins and Palmer ranged from approximately 450 to 500 grams. since little attention has been given to determining the dehydroascorbic acid content in foods.9 ± 1. and thus it has vitamin C value [18]. a∗ e b∗ values represent the levels of tonality and saturation.0 11. as observed in this work (Table 1). -a (indicating green). total carotenoids. based on firmness. The analyzed mango cultivars contain expressive total phenolic concentrations that may contribute to increase antioxidant intake in human diet. was more uniform for varieties Haden and Tommy Atkins. Such characteristic does not interfere with consumer acceptance. It is known that the oxidized form (DHAA) is converted into ascorbic acid in the human body. Antioxidant Constituents Results and Discussion Mango Physicochemical Characteristics A number of parameters related to fruit quality were evaluated in order to characterize the samples (Table 1).27 118. Table 1.15 and 1.3 Colorimetryd L∗ 61.5 ± 1. and mainly through the absorption spectrum.58 n.33 ± ± ± ± 26.0 g/day [19. considering that all the components present bioactivity related with antioxidant mechanism. 1). + a (indicating red).6 49. followed by sample analysis for total vitamin C content under the same chromatographic conditions described for the ascorbic acid analysis.56 7. DHAA was calculated from the difference in contents between total vitamin C and AA before conversion. The mean weight of the varieties Haden. being higher in Ub´ a mango pulp (Fig. + b (indicating yellow) and -b (indicating blue) [14].d.b Values expressed as means ± standard deviation of determinations in twenty mangoes. with In this study. c Total Soluble Solids at 25 ◦ C. The peaks of interest were identified by comparing the retention time of β -carotene and ascorbic acid standards and samples. The aqueous extraction was a convenient procedure.7 and 56. Different letters are significantly different (p < 0. Comparison of total phenolic content in mango pulp. Comparison of total vitamin C in mango pulp. Such differences can be attributed C Total Carotenoids and β -Carotene The variety Haden showed total carotenoids significantly lower than the other three varieties. mg GAE/ 100 g fresh weight carotenoids ß-carotene mg/ 100 g fresh weight 3500 80 AA Total vit. Varieties Tommy Atkins and Palmer showed no difference in β -carotene contents.05.8 µg/100 g). although the bioavailability is unknown. Tukey’s test). 51. The latter showed the highest content of β -carotene. ranging from 10. The RAE values expressed as Retinol Activity Equivalents (RAE) in micrograms/100 grams were 74.32 to 17. respectively. agree with reports on its low content in fruits [9].71%. Dehydroascorbic acid values. Atkins Palmer Ubá Figure 2. but were significantly lower than varieties Haden and Ub´ a. Tommy Atkins and Palmer mangoes was lower than those described for the same varieties (36.16 250 200 150 100 50 0 b Figure 1.Atkins Palmer Ubá Haden T.6. originated from another country [22]. since the mango pulp pH is acid and therefore preserves the ascorbic acid because it has pH below the optimum for the oxidase ascorbate activity [24]. which did not differ among themselves for this constituent (Fig. The ascorbic acid content and total vitamin C in the pulp of the four mango varieties was variable among the cultivars. Figure 3. the four mango cultivars are regarded as a very good source of provitamin A. Values obtained in the present study for total carotenoids for varieties Tommy Atkins and Palmer were lower than the content reported previously with the same mango cultivars obtained under experimental field conditions in Brazil [16]. However.209–1. The authors also reported a much higher β -carotene content than that quantified in the present study for the Tommy Atkins mango (1. .05. Palmer and Ub´ a cultivars. Considering the vitamin A value expressed in Retinol Activity Equivalents -RAE [23]. Tommy Atkins. 55 and 185 to Haden. 3).405 µg/100 g). 2). β -carotene content of Haden mango was similar to the one described in studies with the same variety cultivated in another region of Brazil (494– 82 µg/100 g) [21]. GAE: Gallic Acid Equivalents.05. Comparison of carotenoid and β -carotene contents in mango pulp. Different letters are significantly different (p < 0. Ascorbic Acid. 31. C c 60 µg/ 100g fresh weight 3000 2500 2000 1500 1000 500 0 A b a b b C 40 A B B 20 a b B b B 0 Haden T .7 mg/100 g. β carotene content of the evaluated mango pulps was higher than the one described for a non-reported mango variety (49. Different letters are significantly different (p < 0. Tukey’s test). Tukey’s test). which provides only an estimate rather than an accurate determination. being higher in the variety Ub´ a (Fig. The total ascorbic acid content obtained for Haden. 2). it should be taken into consideration that the values in the present study were obtained by the spectrophotometric method. which was the prevailing type of carotene in the pulp (Fig. respectively) cultivated in experimental field in Brazil [16]. Dehydroascorbic Acid and Total Vitamin C Ascorbic acid (AA) and dehydroascorbic acid (DHAA) were quantified in this study using dithiothreitol as reducing agent. However. National Academy Press. but the variety Ub´ a showed a better performance. Moacir Brito Oliveira (Technical Assistant of ABANORTE. v. Rosseto CJ. Bravo L (1998) Polyphenols: chemistry.66. 2005.org. 5. Kader A (2000) A Preharvest and postharvest factors influencing vitamin C content of horticultural crops. 6. Crozier-Willi G. Free Radic Biol Med 17: 333–349. 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Acknowledgments The authors thank the “Tropical Ind´ ustria de Alimentos” (Visconde do Rio Branco . et al (2002) Bioavailability and metabolism. Krishna KRS. Bonamone A. Winkler BS.1.).71. and 86. Burns J. Nagy S (1980) Vitamin C contents of citrus fruit and their products: a review. Raymundo LC. thus various ripening stages can be assumed. and 20. http://apps. Lee T. Chichester CO (1976) Carotenoids pigment changes in ripening Mamordica charantia fruits. Santhanam K (2003) Reduction of dimethylhydrazine-induced cytotoxicity by mango fruit bar: changes in antioxidant enzymes in rats. Ascorbic acid content varies according to cultivars and tissues. 8. Charleux Jl. Stahl W. climatic conditions. Diplock AT.87. 10. Ruggieri G (1995) Ascorbic acid in exotic fruits: a liquid chromatographic investigation. 4. 6. Methods Enzymol 299: 152–177. Dainly J. Phytochemistry 62: 939–947. Rev Bras Frutic 26: 264–271. C (1994) Stone Fruit maturity indices: a descriptive review. Tommy Atkins. Chytil F (1999) Vitamin A: Not for vision only. Shieber A. 506p. Fraser PD. Rex TS (1994) The redox couple between glutathione and ascorbic acid: A chemical and physiological perspective. 19. Bramley PM (2003) Identification and quantification of carotenoids. 2000. n. Lamuela-Ravent´ os RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Higby WKA (1962) A simplified method for determination of some aspects of the carotenoid distribution in natural and carotene fortified orange juice. In RH. Hecker KD.17 to several factors. Brazil) for supplying the mango fruits. and its several antioxidant compounds give its products (juice and pulp) characteristics that favor preservation without the need for synthetic antioxidants. Anilakumar KR.55% of the Recommended Dietary Allowance of Vitamin A [23].. Postharvest News and Information. Rodriguez DB. 23. 11. Botr´ e F. Conclusions This study shows that although all the analyzed fruits contained phenolic compounds. tocopherols and chlorophylls in commonly consumed fruits and vegetables. van den Berg H. Williamson G (2000) Dietary intake and bioavailability of polyphenols. 6. Washington.33. cultural practices. 3. Simpson KL. Mol Aspects Med 23: 39–100.55 mg of ascorbic acid per 100 grams of pulp. 16.fao. 2. Mantovani DMB. 14. Acessed: February. 15. 11. Jana´ uba . Bloor SJ (2001) Overview of methods for analysis and identification of flavonoids. Mele G. IOM . Singleton VL. Van Buren JP (1984) Function of pectin in plant tissue structure and firmness. Vitamin E. Bast A. Dietary Reference Intakes for Vitamin C. Variety Ub´ a can be considered a rich source of dietary antioxidants. Nutritionally. using different quantification methods [15]. References 1. Carle R (2000) Characterization of polyphenols in mango puree concentrate by HPLC with diode array and mass spectrometric detection. 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Orthofer R. Arthur J. Carvalho CRL. Lee SK. . Kapoor HC (2001) Antioxidants in fruits and vegetables– the millenium’s health. 9. Palmer and Ub´ a supplies to a reference adult man respectively 17. Kok Fj. metabolism.