JOURNAL OF FOOD COMPOSITION AND ANALYSIS (2000) 13, 149d156 doi:10.1006/jfca.1999.

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ORIGINAL ARTICLE Mango (Mangifera indica) Kernel: Chromatographic Analysis of the Tannin, and Stability Study of the Associated Polyphenol Oxidase Activity
Sunday S. Arogba
Department of Food Science & Technology, The Federal Polytechnic, P.M.B. 1037, Idah, Kogi State, Nigeria Received May 4, 1998, and in revised form July 24, 1999

The composition of polyphenols (tannins) and the properties of an associated enzyme, polyphenol oxidase (PPO), in the Nigerian mango (M. indica) kernel were investigated. Tannins were qualitatively and quantitatively analysed. PPO activity of "ltered extract of the fresh mango kernel suspension (40% w/v) was studied spectrophotometrically at 420 nm using catechol as substrate. Results showed that a 6.4% (w/v) gravimetric yield of tannin from dry mango kernel meal contained tannic acid, gallic acid, and epicatechin in the ratio 17 : 10 : 1, respectively. Acid hydrolysis of the condensed tannins indicated the presence of ellagic acid, gallocatechin, and an acylated cyanidin in the ratio 11 : 7 : 5. Moist heat at 903C inactivated PPO by 50% of its maximum activity within 3 min. Furthermore, up to 90% inactivation could be achieved after 5 min of water blanching, thereby reducing signi"cantly the rate of undesired browning leading to tannin formation. Water-blanching would advantageously leach soluble tannic substances into the soak-water. 2000 Academic Press Key =ords: tannin, characterization, heat, PPO, mango kernel, Mangifera indica.

INTRODUCTION Mango (Mangifera indica) trees are tropical fruit-bearing plants of Asia and Africa. However, the fruits are popular world-wide and are relished for the tasty #eshy mesoscarp (Vandrendriessche, 1976). For several decades, the seeds (shell plus kernel) have been discarded as waste in Africa whereas in Asia, interest has mainly been on the lipid component of the kernel because of its potential application in the confectionery industry as a source of cocoa-butter substitute (Lakshminarayana et al., 1983; Rukmini and Vijayaraghavan, 1984; Gaydou and Bouchet, 1984; Ali et al., 1985; Hemavathy et al., 1987). The composition of several varieties of mango kernel has been reported (Lakshminarayana et al., 1983; Hemavathy et al., 1987; Dhingra and Kapoor, 1985; Augustin and Ling, 1987; Arogba, 1997). However, under-utilization of the whole mango kernel could be partly due to the limited knowledge of its toxicological properties (Arogba, 1997). Tannin was implicated as the bitter principle present in the kernel. Tannins are known to form a complex with protein and minerals, thereby reducing the biological value of protein-rich foods signi"cantly (Narasinga Rao and Prabhavathi, 1982; Adewusi and Osuntogun, 1991; Millic, 1972). The extent of complex formation depends on the relative concentration of the reactants (Millic, 1972). Furthermore, the toxicological e!ect of tannin depends on its composition (Millic and Stojanovic, 1972).
0889}1575/00/020149#08 $35.00/0 2000 Academic Press

(1967) and Leung et al. describes for the "rst time the characteristics of tannin and the e!ect of moist heat on the activity of an associated catalyst of tannin-formation (that is PPO) endogenous to mango (M. indica) kernel. Acid Hydrolysis of Condensed ¹annins Before the acid treatment was given. The powdered sample (10. and then dried in the conventional air-oven at 1053C until a constant weight was attained.. 1993. indica) which ripens from late March until late May was obtained from about 290$10 km south-east of the centrally located Federal Capital Territory. Singh and Ravindranath. and rarely does a single dose cause lethal e!ect (Singleton and Kratzer. instead. Seymour et al.. PPO activity varies with oxygen concentration. "ltered through Whatman no. The ethanol extract was treated with anhydrous sodium sulphate.150 AROGBA Tannins as natural phenolic substances di!er remarkably in chemical structure depending on the plant source. This paper. The moist heat conditions necessary to arrest enzymic browning in mango kernel during handling were examined in this experiment. 1996).. 1971).3. like peroxidase and catalase.5% w/v) in 96% ethanol was soaked for 16 h. plant source. 1998).10. 1973). Nigeria. Abuja. 1994) but PPO (EC 1. A brown solid obtained as the total tannin was weighed and expressed as percentage (w/w) of the kernel sample. 1973).. This information could be helpful to potential processors in order to improve the utilization of mango kernel. and the treatment given. Some properties of PPO in mango kernel have been reported (Arogba et al. employed the leucoanthocyanin reagent (5% v/v of concentrated HCl in n-butanol) for 30 min in similar tannin studies. 1972) was observed and separated after several recrystallization in ethyl acetate..1) participates in redox reactions involving phenolic or #avonoid compounds resulting in enzymic browning (Seymour et al. and concentrated to dryness using a rotary evaporator. 1975. The weight was equivalent to a dry matter content of 91% (w/v). is widely distributed in plants (Reeds. A white amorphous precipitate of gallotannins (Millic. Extraction of ¹otal ¹annin The pulp of ripe mango fruit was peeled o! manually using a stainless-steel knife. We . MATERIALS AND METHODS Sample Collection The Ikanekpo variety of mango (M. So far the composition of mango kernel tannin has not been critically analysed except for the measurement of the melting point of gallotannin extracted from Asian mango kernel (El Ansari et al. The supernatants were combined and evaporated to dryness using a rotary evaporator en vacuo. 4 "lter paper. A suitable amount of the brown residue was re#uxed with 10% (v/v) concentrated HCl in n-butanol for 10 min in this study. therefore. the brown solid obtained earlier was redissolved in ethyl acetate. (1979) had. The methods of Luh et al. Aurand and Woods. Tannins in wine have potent antioxidancy against low-density lipoprotein (LDL) of which the oxidized form is a precursor of coronary heart disease (Teissedre et al. and the shell cracked open to obtain the whitish semi-dry kernel. Polyphenol oxidase (PPO). 1993. The latter was pulverized using a pestle and mortar.

0 74. The constituents of the total tannin. colour reactions (Table 4) before and after exposure to UV light (360 and 254 nm). 6% HOAc. secBuOH}HOAc}H O (BAW 4 : 1 : 5). Characterization of the Kernel ¹annins Empirical qualitative tests (Table 1) were conducted.75 2. Adewoye and Ajayi.5 2.   1972.17 1. Zaria.9 25. before and after acid hydrolysis. ether.7 22. The melting point of the white amorphous precipitate was recorded.9 4. 1988a. 1979. and comparisons of results with relevant literature data (Millic. were separated on 18 cm. HOAc}HCl}H O (Fore  stal 30 : 3 : 10). b. Luh et al.45 0. Nigeria).TANNIN AND EFFECT OF HEAT ON PPO OF MANGO KERNEL TABLE 1 Properties of tannic substances freshly extracted from dry mango kernel meal Solubility Colour of aqueous solution UV (254 nm) Forestal formulation#UV (254 nm) 1% gelatin Neutral lead acetate FeCl }K Fe(CN) (1%)    151 Water.70 0.8 11. treatments with ammonia fumes.62 0.31 observed a resulting dark-green solution which was concentrated on a rotary evaporator for characterization. ethyl acetate (in decreasing order) Pink at neutral pH Non#uorescent Fluorescent upper band White precipitate White precipitate Blue TABLE 2 Content of individual polyphenols in dry mango kernel meal Mango kernel meal Constituents Before hydrolysis Total tannin-related substances Gallotannin Tannic acid Gallic acid Condensed tannin-related polyphenols Epicatechin After acid hydrolysis Condensed tannin-related polyphenols Ellagic acid Gallocatechin n-butyl-cyanidin % of total tannins 100. neutral lead acetate spray. 2% HOAc.97 1. 1989).44 0.6 27. 1967.  Indices used for the identi"cation of the constituents include reference phenolic compounds (courtesy of NARICT.5 46. namely. and 1% FeCl }  K Fe(CN) spray. Adewoye and Ajayi. The polyphenolic constituents were "rst estimated as percentage ratios of the total tannins using the mean values of the relative lengths of the chromatographic bands . acetone.. and 88% HCOOH}HCl}H O (FHW 5 : 2 : 3).9 % (w/w) of dry matter 6.78 1.37 4. absolute alcohol. Leung et al.18 cm Whatman 3MM chromatographic paper essentially by a two-way technique using combinations of the following solvent systems..0 6. calculated R values (Table 3).

42}0. . Assay for PPO Activity PPO activity was determined using a colorimetric method (Lee and Smith. The spots were detected using the ferrocyanide spray reagent described earlier. 4) paper into a Erlenmeyer #ask (250 ml) placed on ice.44 Solvent system a"BAW 415.30 c d 0.58 0. d"&&Forestal'' formulation. m.p.78 0. The procedure was observed to be reproducible and reliable with 3% standard deviations.p (Gallic acid!the observed) m.152 AROGBA TABLE 3 R values of individual polyphenols in dry mango kernel meal R values a b 0.78 0.85 0.74 0.58}0. developed. obtained after cracking the shell manually.01 M phosphate bu!er (pH 6.0 0.17 0. by following the steps described thus: fractions which were resolved in the two-way chromatography were cut from the papers.83 0. correction factor was further applied to the gallic acid ratio due to some overlap and melting point attenuation by tannic acid. b"2% acetic acid.42}0. the relative concentrations (% w/w) of the constituents in the dry matter of the mango kernel were computed using the above percentage ratios and the gravimetric yield of the total tannin obtained earlier.0. No brown colour was observed in the crude extract before use. Five hundred microliters each of the ethyl acetate extracts of all the constituents were spotted on the same Whatman 3MM paper and developed using the &&Forestal formulation'' solvent system. and made up to 100 ml with bu!er in a measuring cylinder. were crushed with pre-chilled (03C) 0.40 0.85 0. each eluate was concentrated to dryness on a rotary evaporator en vacuo and rediluted with 5 ml ethyl acetate. On "ltering. 1979) based on the initial rate of increase in absorbance at 420 nm.58}0.42}0. e"FHW 523.0}0.84 0.0 0. thus: Gallic acid"%+Gallic acid#Tannic acid.0}0. and separately redissolved in methanol.17 0.46 e Before hydrolysis Tannic acid Gallic acid Epicatechin After acid hydrolysis Ellagic acid Gallocatechin Uncon"rmed compound n-butyl-cyanidin 0. (Gallic acid!Tannic acid as impurity) Secondly. c"6% acetic. Triplicate determination was carried out. 10 ml of .80 0.50}0. and measurements of band lengths for each constituent were averaged and expressed as percentage ratio of total constituents. In this study.74 0.58}0. The macerate was immediately "ltered using suction through coarse "lter (Whatman No. Preparation of Crude Mango Kernel PPO Extract Forty grams of white kernel. In this study.44 0. 60 ml) using a pestle and mortar.84 0.21 0.

the detection of free gallic acid and gallocatechin strongly suggests that gallic acid could have existed in other forms in the mango kernel.p. The samples contained in test tubes were then immediately placed on ice to cool before conducting the assay as described above. 1 ml of 0. epicatechin mentioned above. Adewoye and Ajayi. 8. 18 and 20 min. partly or wholly could have arisen at the extraction stage of the experiment (Millic. 15. respectively. 1981). which on acid hydrolysis would justify the ellagic acid detected. 16. epicatechin and gallocatechin were characteristically resolved in aqueous acetic acid (2 or 6%) . The gravimetric yield of 6. 1972) or much earlier during the dehydration of the fresh kernel. 1988a. E+ect of Blanching ¹ime The crude enzyme extract (3 ml) was heated at 903C in a controlled temperature circulating water bath for 0.p. Chromatographic separation using particularly the Forestal (AHW 30 : 3 : 10) solvent formulation followed by irradiation with UV light at 254 nm appeared to identify the tannin fraction of mango kernel. 1981: gallic acid m. 1997) of 4. Each assay mixture was replicated twice. suggested that gallic acid (cf.5 ml of the crude enzyme extract were pipetted in this sequence into a test tube. therefore. Ellagitannin is one such example. was not observed in this study. The e!ect of solvent system composition could.5% w/w) and m. The R values and colour characteristics which aided the identi"cation of the tannin constituents are presented in Tables 3 and 4. catechin. 12. However. 10.p. One unit of PPO activity caused a change in absorbance of 0. Ellagic acid and gallocatechin were the main constituents of the condensed tannin-related polyphenols in the mango kernel. RESULTS AND DISCUSSION Mango Kernel ¹annin Composition Table 1 shows some general properties of the isolated tannin of mango kernel. 4. 6.5% (w/w) expressed as catechin equivalent for the same variety of mango kernel. 1971). The possible presence of the epimer. Ellagic acid is a dilactone comprising two molecules of gallic acid (Sharp. 1981) (cf. epicatechin which constituted about 3% of the total tannins.5 M catechol.TANNIN AND EFFECT OF HEAT ON PPO OF MANGO KERNEL 153 0. 19xx: 2353C) attenuated by tannic acid m. mixed. 2503C) were the main constituents. explain why gallic acid. (2383C) of gallotannin of African mango kernel were compared with those of Asian origin (El Ansari et al. Di!erence in varietal composition became apparent when the yield (4. In this study. Table 2 indicates the composition of mango kernel tannin.001/min at 420 nm. Gallocatechin was identi"ed after acid hydrolysis and could justify the earlier detection of the monomeric aglycone.0).. It was implied that stronger protonation of the hydroxyl groups of tannin constituents by HCl of a solvent system appears to weaken the resolution of spots. of 2183C (Sharp. Sharp. The observed m. sec-BAM 415 gave better resolution of spots than the Forestal (AHW 30 : 3 : 10) formulation when employed in the "rst run of a two-way chromatography. and absorbance recorded at 30 s intervals. and 0. Dean. Prior to acid treatment of the dry kernel meal.p. Gallic acid constituted one-third of the gallotannin fraction (Table 2). Furthermore.01 M phosphate bu!er (pH 6. respectively.4% (w/w) compares favourably with a titrimetric yield (Arogba. 2. the choice of solvent system for the chromatographic separation was of paramount importance. 5. The &&Forestal'' solvent system and the colour reactions observed in this study sharply distinguished gallic acid from ellagic acid.

Furthermore. The role of pH in the #uorescence of some tannin constituents of mango kernel needs further investigation.. ¹hermal Inactivation of a Browning Enzyme (PPO) The e!ect of moist heat on PPO activity is shown in Table 5. 1967)..154 AROGBA TABLE 4 Colour characteristics of individual polyphenols in dry mango kernel meal Colour FeCl #  K Fe(CN)   Neutral lead acetate White (#) # # # # # # Visible Brown Brown Grey Faint yellow Brown Brown Absorption UV Pink Fluorescent purple Faint pink Fluorescent bluish green Green Absorption Greyish UV#NH  Pink Fluorescent purple Pink Fluorescent blue Pink Pink Grey Before hydrolysis Tannic acid Gallic acid Epicatechin After acid hydrolysis Ellagic acid Gallocatechin Uncon"rmed compound n-butyl-cyanidin Blue (#) # # # # # # TABLE 5 Variation of residual PPO activity of mango kernel extract with blanching times at 903C Blanching time (min) % Residual PPO activity 0 100 3 48 (5}20) 10 PPO activity was determined at 420 nm using catechol as substrate. and values shown were extrapolated from the graph of blanching time versus PPO activity. n-butyl-cyanidin was characterized (Tables 3 and 4) using the techniques highlighted in the methodology section. Similar chromatographic behaviour and colour changes of the acylated cyanidin were observed during the analysis of the condensed tannins of rapeseed hulls. 1979) using the same leucoanthocyanin reagent (n-butanol}concentrated HCl. The Forestal formulation showed that gallic acid had purple #uorescence before treatment. the choice of 903C was the maximum attainable temperature of the enzyme extract at atmospheric pressure which potential processors of mango kernel at cottage level could readily employ during blanching without undue monitoring. and bluish #uorescence for ellagic acid after acid hydrolysis and illumination with UV light at 254 nm (Table 4).. 95 : 5 v/v) (Luh et al. 1998). 1978). Fifty per cent of the maximum enzyme activity is a measurement criterion commonly used in enzyme studies (White et al. faba bean hulls. It is independent of the absolute maximum value in di!erent sources and treatment conditions. (Table 3).. the optimal pH of 6.0 was employed in the assay procedure given in the Experimental section. and sorghum grains (Leung et al. Based on our previous work (Arogba et al. .

O. Ali. ACKNOWLEDGEMENT The author thanks the Polytechnic Research Committee of the National Board for Technical Education (NBTE). These tannins would require treatment during processing in order to reduce their in vivo toxic e!ect. Amer. 50% of PPO activity was observed at about 3 min at 903C and 5 min for 90% inactivation (Table 5). ¸eather Chem. Oil Chem. 1589}1593. 83. Properties of polyphenol oxidase in mango (Mangifera indica) kernel. Ugboko. Nutritive value of mango seed kernel. M. R. Soc. Assoc. Sci. Sterols. Adewoye. the rate of tannin formation through enzyme activity could be signi"cantly reduced by water-blanching of mango kernel at 903C for 5 min. trypsin inhibitor activity and in-vitro digestibility of some legume seeds in Nigeria. 139}145. 459}462. M. ¸eather ¹ech. Food J.. and Bouchet. O. S. and Woods. S. K. 1960). L. T. L. Physical.. 36. however. #avones and tannins of Parkia clappertoniana. W. O. S. J. N. In this study. 153}156. J. Food Agric.. Phytochemistry 10.TANNIN AND EFFECT OF HEAT ON PPO OF MANGO KERNEL 155 It has been shown that a very sharp change occurs in PPO activity when a critical temperature of 823C is reached (Schultz. 72. Oil Chem. Nigeria for the "nancial grant. A. K. Ajiboye. Agbaji and Dr O. Arogba. A.. A. and Ajayi. 110}121. Sci. O. 2239}2241. Sci. O. L. REFERENCES Adewoye. 53}59. 520}523. A.. A. was di$cult beyond 5 min and was attributed to the matrix e!ects of other major sample components in the extract. Nig. Rahman. 321}328. Ajayi for assisting with authentic samples of tannic substances at the Laboratory of the National Research Institute for Chemical Technology (NARICT). that is.. Vol. Soc. C. Soc. 752}756. Chem. New York. Sastry. Y. P. M. 62(3). R.. 11th edn. P. Food Chemistry. E. Variations in fat content and lipid class composition in ten di!erent varieties. Soc. M. M.... Chemical and Functional Properties of Nigerian Mango (Mangifera indica) Kernel and its Processed Flour. O. 27}29. A. E!ects of cooking on tannin content. A. (1973). (1991). and Ahmed. J. J. O.. 61(10). (1987). Amer. and Afolabi. the in#uence of the degree of purity of the enzyme extract on PPO activity. and Ajayi. The additional advantage of water-blanching is the possible leaching of soluble tannic substances into the soak-water. J. S. J. S. (1984). and Osuntogun.) (19xx) ¸ange1s Handbook of Chemistry. R. and Ling. . (1971).. Aurand. and Kapoor. Am. G. Essienette. J. Polyphenols of Mangifera indica. M. 77. Dean. E. E. CONCLUSION The study has shown that mango kernel contained hydrolysable tannin which was about 75% of the total tannin content. Y. J. Phenolic constituents of Parkia clappertoniana fruit husk. AVI. Westport. Composition of mango seed kernel.. pp. El Ansari. Gaydou. Pertanika 10(1). (1989). S. Dhingra. J. S. (1997). O. A. B. (1985). and Ajayi. Gafur. K. A. Chem. O. 73. and Nayudamma. Reddy.. S. triterpene alcohols and fatty acids of the kernel fat of di!erent Malagasy mango (Mangifera indica) varieties. Adewoye.. 73. (1988b). R. McGraw-Hill. Connecticut. O. Augustin.. Adewusi. (1985). O. (1998). Furthermore. S. ¸eather ¹ech. and Dr A.. 6223. Zaria. Further inactivation of PPO. This is a simple and e!ective process that can be carried out at a cottage industry level. Anthocyanidins of Parkia clappertoniana. S. 9. 7}378. A. Flavonols. Food Agric. (Ed.. Arogba.. methyl sterols. O. M. Food Agric. (1988a).

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