Plant Foods for Human Nutrition 62: 1317, 2007. c 2007 Springer Science+Business Media, Inc. DOI: 10.

1007/s11130-006-0035-3

13

Antioxidant in Mango (Mangifera indica L.) Pulp

HUMBERTO de QUEIROZ,2 MARIA ELIANA LOPES SONIA MACHADO ROCHA RIBEIRO,1, JOSE 3 RIBEIRO de QUEIROZ, FLAVIA MILAGRES CAMPOS1 & HELENA MARIA PINHEIRO SANTANA1
1 Departamento de Nutric o e Sa a ude, Universidade Federal de Vic osa, Vic osa, MG, Brazil; 2 Departamento de Bioqu mica e Biologia Molecular, Universidade Federal de Vic osa, Vic osa, MG, Brazil; 3 Departamento de Qu mica, Universidade Federal de Vic osa, Vic osa, MG, Brazil ( author for correspondence; e-mail: sribeiro@ufv.br)

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 -carotene and total vitamin C (ascorbic acid and dehydroascorbic acid) were quantied 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); -carotene from 661.27 (Palmer) to 2,220 g/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, -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]. -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, -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 conrmed reduction in cytotoxic effects induced by dimethyldrazine through the optimization of enzymatic oxidative mechanisms [8]. Despite mangos 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 -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, -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 rst

14 three varieties were randomly collected in commercial plantations in northern and northwestern Minas Gerais state, at the intermediary ripening stage. The mangoes were stored at room temperature until complete ripening, by subjective observation considering softness and peel color [10]. Pulp from 20 completely ripe mangoes of each variety was homogenized without peel and pureed. The pulp from variety Ub a was acquired directly from the regional farm industry (Zona da Mata Mineira). The mango puree was lyophilized, stored at 20 C and protected from light until analysis. Pulp moisture content was determined by drying at 105 C, and the results expressed in 100 grams of fresh weight (edible portion). Total Carotenoids and -Carotene Carotenoid extraction followed the procedure described in Rodriguez et al. [13] with minor modications. Before the extraction, the lyophilized samples were reconstituted in distilled water and all the procedures performed in dim light. Samples (5.0 g) were ground with cold acetone in a Marconi microgrinder MA 102, and vacuum-ltered using a B uchner funnel until the residue became colorless. The extract was partitioned with petroleum ether, each fraction was washed with distilled water for complete acetone removal. The extract was concentrated in a rotary evaporator (<35 C) to a nal volume of 10 ml. The volume was then completed to 25.0 ml with petroleum ether to obtain the concentrated extract. Total carotenoid content was estimated in triplicates according to methodology described by Higby [14]. For -carotene quantication, aliquots (2.0 ml) of concentrated extracts were evaporated under nitrogen, redissolved in acetone (2.0 ml), ltered through a 0.45 m Millipore membrane, and 30 l aliquots were injected into the liquid chromatographic system. The HPLC analysis system consisted of high-pressure pump (Shimadzu LC10 ATVP, Tokyo, Japan), an auto-sampler injector, and a UV-visible photodiode array detector (Shimadzu SPD-10 AVP), controlled by Multi System, Class VP 6.12. Separation was performed in a RP18 columm (Microsorb, 5 m, 4 nm 250 nm). Wavelength detection was 450 nm and mobile phase consisted of methanol, ethyl acetate and acetonitrile (70:20:10) at a ow rate of 2.0 ml min1 . Quantication was performed by external standardization, using -carotene to prepare the standard curve and the peak area for the calculation. Ascorbic Acid (AA) and Dehydroascorbic Acid (DHAA) Lyophilized pulp of each mango variety was reconstituted with distilled water, on the basis of their moisture content. The extraction was performed according to Vinci et al. [15], with minor modications. Five grams of reconstituted pulp were stirred in ultra pure water (15.0 ml) for 5 min with a micro grinder. The mixture was ltered and the total nal volume was adjusted to 25.0 ml with ultra pure water and centrifuged at 1000 g for 15 min. Two milliliters of the supernatant were ltered in a 0.45 m Millipore membrane and a 30 l aliquot was immediately used for HPLC analysis. HPLC analysis used the same equipment of -carotene quantication. Other chromatographic conditions were: ultra pure water with metaphosphoric acid at pH 2.2 as mobile phase; detection at 238 nm and ow rate of 1.0 ml min1 . Reduction of dehydroascorbic acid (DHAA) was performed with dithiothreitol (DTT). An aliquot (1.0 ml) of the sample supernatant was mixed with 0.1 ml phosphate

Mango Physicochemical Characteristics Twenty units of each variety were analyzed for weight and rmness. Pulp quality of each variety (20 units pooled and homogenized) was evaluated for the parameters color and total soluble solids (TSS). Firmness was determined using a Fruit Pressure Tester (FT 327, Italy) equipped with an 11 mm-diameter plunger tip. Two measurements were taken from the opposite sides of each fruit after removing the peel. Pulp color was evaluated in a ColorQuest II Sphere colorimeter (Hunter Lab Reston, VA), using the L , a , b scale. Total soluble solids (TSS) was determined using a hand refractometer TECNAL, model AR200.

Total Phenolic Content Pulp extracts containing phenolic compounds were obtained as previously reported by Bloor [11]. Lyophilized material (1.0 g) was extracted using a 10.0 mL mixture of methanol: water (60:40; v/v), shaken at 180 rpm at room temperature for 30 min. Only one extraction was performed since the 1:10 (sample: solvent) ratio had extraction efciency close to 100%. The mixture was centrifuged at 1000 g for 10 min. The supernatant fractions were used immediately for analysis. Total phenolic contents in the extracts were estimated using the Folin-Ciocalteu reagent, according to Singleton [12]. The ascorbic acid reaction prior to alkali addition was monitored and the total phenol values were corrected. Absorbance (max = 765 nm) was measured using a UV/VIS spectrophotometer SHIMADZU UV-VIS 1601 (Kyoto, Japan). 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. All the measurements were evaluated in triplicate.

15 buffer pH 7.4 to obtain a nal pH of 6.0, and 100 mM DTT was added to obtain a nal concentration of 8.0 mM. The mixture was kept in the dark for 10 min to convert DHAA into AA, followed by sample analysis for total vitamin C content under the same chromatographic conditions described for the ascorbic acid analysis. DHAA was calculated from the difference in contents between total vitamin C and AA before conversion, using a L-ascorbic acid standard curve. The peaks of interest were identied by comparing the retention time of -carotene and ascorbic acid standards and samples; and mainly through the absorption spectrum, since a diode array detector was used. All determinations were performed in triplicates. + a (indicating red), -a (indicating green), + b (indicating yellow) and -b (indicating blue) [14]. Positive values of a and b , as observed in this work (Table 1), are attributed to the carotenoids present in the pulp. The pooled pulp sample of the evaluated varieties presented total soluble solids (TSS) indices above the minimum acceptable for consumption (12.0 o Brix). Overall, the physicochemical characteristics of the studied mango varieties were similar. However, if it is considered that the mango ripening curve can differ among varieties, the use of these values is not suitable for comparing ripe stages. The main use of such indices is for sample characterization.

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). The mean weight of the varieties Haden, Tommy Atkins and Palmer ranged from approximately 450 to 500 grams, being the fruits classied from medium to large. These sizes meet the market requirements for fresh mango consumption [16]. Small fruits of variable weight characterize the variety Ub a. Such characteristic does not interfere with consumer acceptance, since this variety is mainly used in agro industry for juice and pulp production. Firmness is widely used as a ripeness test for many fruits. Pulp rmness decreases with increasing ripening as the pectin content decreases and the soluble solids content increases [17]. It was veried that ripening, based on rmness, was more uniform for varieties Haden and Tommy Atkins, and more variable among Palmer fruits, resulting in a higher standard deviation for the latter. The determination of the coordinates L , a , b characterizes pulp color. At this scale, L measures luminosity that varies from zero (black) to 100 (pure white); a e b values represent the levels of tonality and saturation, with In this study, pulp composition of four mango commercial varieties grown in Brazil was evaluated for the contents of total phenolics, total carotenoids, -carotene and Vitamin C (ascorbic acid and dehydroascorbic acid), considering that all the components present bioactivity related with antioxidant mechanism. The two forms of vitamin C (AA and DHAA) were quantied, since little attention has been given to determining the dehydroascorbic acid content in foods. It is known that the oxidized form (DHAA) is converted into ascorbic acid in the human body, and thus it has vitamin C value [18].

Total Phenolics Total phenolic content was different among the four varieties, being higher in Ub a mango pulp (Fig. 1). The Palmer cultivar showed an intermediary value and Haden and Tommy Atkins presented lower values. The analyzed mango cultivars contain expressive total phenolic concentrations that may contribute to increase antioxidant intake in human diet, since the intake of polyphenolic compounds in the diet was estimated to range between 0.15 and 1.0 g/day [19, 20].

Table 1. Physicochemical characteristics of fruit and pulp Firmnessb Cultivar Haden Tommy Atkins Palmer Ub a Weighta (g) 486.64 477.20 493.27 118.33 26.80 22.68 20.22 11.51 (Kg/cm2 ) 4.9 1.56 7.6 2.89 2.5 1.58 n.d. TSSc Brix 14.1 14.4 15.7 14.3 Colorimetryd L 61.3 58.3 54.9 59.1 a 13.0 11.5 11.7 14.4 b 48.3 45.9 39.6 49.7

Note. a,b Values expressed as means standard deviation of determinations in twenty mangoes; c Total Soluble Solids at 25 C, evaluated in the 20 pooled fruits; d Evaluated in the 20 pooled fruits; n.d.: not determined.

16

250 200 150 100 50 0

b

Figure 1. Comparison of total phenolic content in mango pulp. GAE: Gallic Acid Equivalents. Different letters are signicantly different (p < 0.05; Tukeys test).

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 eld conditions in Brazil [16]. However, it should be taken into consideration that the values in the present study were obtained by the spectrophotometric method, which provides only an estimate rather than an accurate determination. Considering the vitamin A value expressed in Retinol Activity Equivalents -RAE [23], the four mango cultivars are regarded as a very good source of provitamin A, although the bioavailability is unknown. The RAE values expressed as Retinol Activity Equivalents (RAE) in micrograms/100 grams were 74; 51; 55 and 185 to Haden, Tommy Atkins, Palmer and Ub a cultivars, respectively. Ascorbic Acid, Dehydroascorbic Acid and Total Vitamin C Ascorbic acid (AA) and dehydroascorbic acid (DHAA) were quantied in this study using dithiothreitol as reducing agent. The aqueous extraction was a convenient procedure, 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]. The ascorbic acid content and total vitamin C in the pulp of the four mango varieties was variable among the cultivars, being higher in the variety Ub a (Fig. 3). Dehydroascorbic acid values, ranging from 10.32 to 17.71%, agree with reports on its low content in fruits [9]. The total ascorbic acid content obtained for Haden, Tommy Atkins and Palmer mangoes was lower than those described for the same varieties (36.6; 31.7 and 56.7 mg/100 g, respectively) cultivated in experimental eld in Brazil [16]. Such differences can be attributed
C

Total Carotenoids and -Carotene The variety Haden showed total carotenoids signicantly lower than the other three varieties, which did not differ among themselves for this constituent (Fig. 2). Varieties Tommy Atkins and Palmer showed no difference in -carotene contents, but were signicantly lower than varieties Haden and Ub a. The latter showed the highest content of -carotene, which was the prevailing type of carotene in the pulp (Fig. 2). -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 g/100 g) [21]. The authors also reported a much higher -carotene content than that quantied in the present study for the Tommy Atkins mango (1,2091,405 g/100 g). carotene content of the evaluated mango pulps was higher than the one described for a non-reported mango variety (49.8 g/100 g), originated from another country [22].

mg GAE/ 100 g fresh weight

carotenoids -carotene
mg/ 100 g fresh weight
3500

80

AA Total vit. C
c

60

g/ 100g fresh weight

3000 2500 2000 1500 1000 500 0

A

b a

b b
C

40
A

20

Haden

T .Atkins

Palmer

Ub

Haden

T. Atkins

Palmer

Ub

Figure 2. Comparison of carotenoid and -carotene contents in mango pulp. Different letters are signicantly different (p < 0.05; Tukeys test).

Figure 3. Comparison of total vitamin C in mango pulp. Different letters are signicantly different (p < 0.05; Tukeys test).

17 to several factors. Ascorbic acid content varies according to cultivars and tissues, climatic conditions, cultural practices, maturity stage and postharvest factors [9]. Studies with several mango cultivars have reported a range from 25.3 to 182.55 mg of ascorbic acid per 100 grams of pulp, using different quantication methods [15]. However, it should be considered that authors usually fail to present the physicochemical characteristics of the fruits investigated in their studies, thus various ripening stages can be assumed. Nutritionally, the daily intake of one serving of pulp equivalent to 150 grams of varieties Haden, Tommy Atkins, Palmer and Ub a supplies to a reference adult man respectively 17.43; 10.87; 11.71, and 86.34% of the Recommended Dietary Allowance of Vitamin C and 12.33; 5.66; 6.1, and 20.55% of the Recommended Dietary Allowance of Vitamin A [23].
4. Diplock AT, Charleux Jl, Crozier-Willi G, Kok Fj, Rice-Evans C, Roberfroid M et al. (1998) Functional food science and defense against reactive oxidative species. Br J Nutr 80: s77s112. 5. Chytil F (1999) Vitamin A: Not for vision only. Br J Nutr 82: 161 162. 6. Kris-Etherton PM, Hecker KD, Bonamone A, Coval SM, Binkoski AE, Hilpert KF, Griel AE, Etherton TD (2002) Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. Am J Med 113: 71S81S. 7. Bravo L (1998) Polyphenols: chemistry, dietary sources, metabolism, and nutritional signicance. Nutr Rev 56: 317333. 8. Anilakumar KR, Khanum f, Krishna KRS, Santhanam K (2003) Reduction of dimethylhydrazine-induced cytotoxicity by mango fruit bar: changes in antioxidant enzymes in rats. Plant Foods Hum Nutr 58: 111. 9. Lee SK, Kader A (2000) A Preharvest and postharvest factors inuencing vitamin C content of horticultural crops. Postharvest Biol Technol 20: 207220. 10. Crisoto, C (1994) Stone Fruit maturity indices: a descriptive review. Postharvest News and Information. v. 5, n. 6, 65N68N. 11. Bloor SJ (2001) Overview of methods for analysis and identication of avonoids. Methods Enzymol 335: 314. 12. Singleton VL, Orthofer R, Lamuela-Ravent os RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol 299: 152177. 13. Rodriguez DB, Raymundo LC, Lee T, Simpson KL, Chichester CO (1976) Carotenoids pigment changes in ripening Mamordica charantia fruits. Annals of Botany 40: 615624. 14. Higby WKA (1962) A simplied method for determination of some aspects of the carotenoid distribution in natural and carotene fortied orange juice. J Food Sci 27: 4249. 15. Vinci G, Botr e F, Mele G, Ruggieri G (1995) Ascorbic acid in exotic fruits: a liquid chromatographic investigation. Food Chem 53: 211 214. 16. Carvalho CRL, Rosseto CJ, Mantovani DMB, Morgano MA, Castro JV, Botoletto N (2004) Evaluation of mango cultivars selected by Instituto Agron omico de Campinas compaired to others of commercial importance. Rev Bras Frutic 26: 264271. 17. Van Buren JP (1984) Function of pectin in plant tissue structure and rmness. In RH. Walter (Ed.), The chemistry and technology of pectin (pp. 122). New York: Academic Press. 18. Winkler BS, Orselli SM, Rex TS (1994) The redox couple between glutathione and ascorbic acid: A chemical and physiological perspective. Free Radic Biol Med 17: 333349. 19. Stahl W, van den Berg H, Arthur J, Bast A, Dainly J, et al (2002) Bioavailability and metabolism. Mol Aspects Med 23: 39100. 20. Scalbert A, Williamson G (2000) Dietary intake and bioavailability of polyphenols. J Nutr 130: 2073S2085S. 21. Godoy TH, Rodriguez-Amaya DB (1989) Carotenoid composition of commercial mangoes from Brazil. Libensm-Wissu-Techonol 22: 100103. 22. Burns J, Fraser PD, Bramley PM (2003) Identication and quantication of carotenoids, tocopherols and chlorophylls in commonly consumed fruits and vegetables. Phytochemistry 62: 939947. 23. IOM - INSTITUTE OF MEDICINE. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. National Academy Press, Washington, DC., 2000, 506p. 24. Nagy S (1980) Vitamin C contents of citrus fruit and their products: a review. J Agric Food Chem 28: 818.

Conclusions This study shows that although all the analyzed fruits contained phenolic compounds, ascorbate and -carotene, their contents are markedly different among cultivars. All mango varieties represent a potential source of natural antioxidants, but the variety Ub a showed a better performance. Variety Ub a can be considered a rich source of dietary antioxidants, and its several antioxidant compounds give its products (juice and pulp) characteristics that favor preservation without the need for synthetic antioxidants. Acknowledgments The authors thank the Tropical Ind ustria de Alimentos (Visconde do Rio Branco - Minas Gerais, Brazil) and Mr. Moacir Brito Oliveira (Technical Assistant of ABANORTE, Jana uba - Minas Gerais, Brazil) for supplying the mango fruits. References
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