mango_polyphenol

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JOURNAL OF FOOD COMPOSITION AND ANALYSIS (2000) 13, 149d156

doi:10.1006/jfca.1999.0838

Available online at http://www.idealibrary.com on

ORIGINAL ARTICLE

Mango (Mangifera indica) Kernel: Chromatographic Analysis ofthe 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, poly-phenol oxidase (PPO), in the Nigerian mango (M. indica) kernel were investigated. Tannins werequalitatively and quantitatively analysed. PPO activity of"ltered extract of the fresh mangokernel suspension (40% w/v) was studied spectrophotometrically at 420 nm using catechol assubstrate. Results showed that a 6.4% (w/v) gravimetric yield of tannin from dry mango kernelmeal 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 anacylated cyanidin in the ratio 11:7:5. Moist heat at 903C inactivated PPO by 50% of itsmaximum activity within 3 min. Furthermore, up to 90% inactivation could be achieved after5 min of water blanching, thereby reducing signi"cantly the rate of undesired browning leadingto tannin formation. Water-blanching would advantageously leach soluble tannic substancesinto 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 #eshymesoscarp (Vandrendriessche, 1976). For several decades, the seeds (shell plus kernel)have been discarded as waste in Africa whereas in Asia, interest has mainly been onthe lipid component of the kernel because of its potential application in the confec-tionery 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 (Lak-shminarayana et al., 1983; Hemavathy et al., 1987; Dhingra and Kapoor, 1985;Augustin and Ling, 1987; Arogba, 1997). However, under-utilization of the whole mangokernel 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 formationdepends on the relative concentration of the reactants (Millic, 1972). Furthermore, thetoxicological e!ect of tannin depends on its composition (Millic and Stojanovic, 1972).

0889}1575/00/020149#08 $35.00/0 2000 Academic Press


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Tannins as natural phenolic substances di!er remarkably in chemical structuredepending on the plant source, and rarely does a single dose cause lethal e!ect

(Singleton and Kratzer, 1973). Tannins in wine have potent antioxidancy againstlow-density lipoprotein (LDL) of which the oxidized form is a precursor of coronaryheart disease (Teissedre et al., 1996). So far the composition of mango kernel tanninhas not been critically analysed except for the measurement of the melting point ofgallotannin extracted from Asian mango kernel (El Ansari et al., 1971).

Polyphenol oxidase (PPO), like peroxidase and catalase, is widely distributed inplants (Reeds, 1975; Seymour et al., 1993; Singh and Ravindranath, 1994) but PPO(EC 1.10.3.1) participates in redox reactions involving phenolic or #avonoid com-pounds resulting in enzymic browning (Seymour et al., 1993; Aurand and Woods,1973). PPO activity varies with oxygen concentration, plant source, and the treatmentgiven. Some properties of PPO in mango kernel have been reported (Arogba et al.,

1998). The moist heat conditions necessary to arrest enzymic browning in mangokernel during handling were examined in this experiment. This information could behelpful to potential processors in order to improve the utilization of mango kernel.

This paper, therefore, describes for the "rst time the characteristics of tannin andthe e!ect of moist heat on the activity of an associated catalyst of tannin-formation(that is PPO) endogenous to mango (M. indica) kernel.

MATERIALS AND METHODS

Sample Collection

The Ikanekpo variety of mango (M. indica) which ripens from late March until lateMay was obtained from about 290$10 km south-east of the centrally located FederalCapital Territory, Abuja, Nigeria.

Extraction ofotal annin

The pulp of ripe mango fruit was peeled o!manually using a stainless-steel knife, andthe shell cracked open to obtain the whitish semi-dry kernel. The latter was pulverizedusing a pestle and mortar, and then dried in the conventional air-oven at 1053C untila constant weight was attained. The weight was equivalent to a dry matter content of

91% (w/v).The powdered sample (10.5% w/v) in 96% ethanol was soaked for 16 h. The etha-

nol extract was treated with anhydrous sodium sulphate, "ltered through Whatmanno. 4 "lter paper, and concentrated to dryness using a rotary evaporator. A brownsolid obtained as the total tannin was weighed and expressed as percentage (w/w) ofthe kernel sample.

Acid Hydrolysis of Condensed annins

Before the acid treatment was given, the brown solid obtained earlier was redissolvedin ethyl acetate. A white amorphous precipitate of gallotannins (Millic, 1972) was

observed and separated after several recrystallization in ethyl acetate.The supernatants were combined and evaporated to dryness using a rotary evapor-

ator en vacuo. 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. The methods ofLuh et al.(1967) and Leung et al. (1979) had, instead, employed the leucoanthocyanin reagent(5% v/v of concentrated HCl in n-butanol) for 30 min in similar tannin studies. We

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TABLE 1

Properties of tannic substances freshly extracted from dry mango kernel meal

Solubility Water, acetone, ether, absolute alcohol, ethyl acetate(in decreasing order)

Colour of aqueous solution Pink at neutral pHUV (254 nm) Non#uorescentForestal formulation#UV (254 nm) Fluorescent upper band1% gelatin White precipitateNeutral lead acetate White precipitateFeCl

}K

Fe(CN)

(1%) Blue

TABLE 2

Content of individual polyphenols in dry mango kernel meal

Mango kernel meal

Constituents % of total tannins % (w/w) of dry matter

Before hydrolysisTotal tannin-related substances 100.0 6.37Gallotannin 74.5 4.75

Tannic acid 46.6 2.97Gallic acid 27.9 1.78

Condensed tannin-related polyphenols 25.5 1.62Epicatechin 2.7 0.17

After acid hydrolysisCondensed tannin-related polyphenols 22.8 1.45

Ellagic acid 11.0 0.70Gallocatechin 6.9 0.44n-butyl-cyanidin 4.9 0.31

observed a resulting dark-green solution which was concentrated on a rotary evapor-ator for characterization.

Characterization of the Kernel annins

Empirical qualitative tests (Table 1) were conducted. The melting point of the whiteamorphous precipitate was recorded.

The constituents of the total tannin, before and after acid hydrolysis, were separatedon 18 cm;18 cm Whatman 3MM chromatographic paper essentially by a two-waytechnique using combinations of the following solvent systems, namely, sec-BuOH}HOAc}H

O (BAW 4:1:5), 2% HOAc, 6% HOAc, HOAc}HCl}H

O (Fore-

stal 30:3:10), and 88% HCOOH}HCl}H

O (FHW 5:2:3).Indices used for the identi"cation of the constituents include reference phenolic

compounds (courtesy of NARICT, Zaria, Nigeria), calculated R

values (Table 3),colour reactions (Table 4) before and after exposure to UV light (360 and 254 nm),

treatments with ammonia fumes, neutral lead acetate spray, and 1% FeCl}

K

Fe(CN)

spray, and comparisons of results with relevant literature data (Millic,1972; Luh et al., 1967; Leung et al., 1979; Adewoye and Ajayi, 1988a, b; Adewoye andAjayi, 1989).

The polyphenolic constituents were "rst estimated as percentage ratios of the totaltannins using the mean values of the relative lengths of the chromatographic bands

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TABLE 3

R

values of individual polyphenols in dry mango kernel meal

R

values

a b c d e

Before hydrolysisTannic acid 0.58}0.78 0.17 0.58}0.74Gallic acid 0.50}0.78 0.17 0.44 0.58}0.74Epicatechin 0.44 0.21 0.42}0.58

After acid hydrolysisEllagic acid 0.0}0.80 0.0 0.0}0.40 0.0Gallocatechin 0.84 0.30Uncon"rmed compound 0.84 0.42}0.85 0.83

n-butyl-cyanidin 0.42}0.85 0.46

Solvent system a"BAW 415, b"2% acetic acid, c"6% acetic, d"&&Forestal'' formulation, e"FHW523.

developed, by following the steps described thus: fractions which were resolved in thetwo-way chromatography were cut from the papers, and separately redissolved inmethanol. On "ltering, each eluate was concentrated to dryness on a rotary evapor-ator en vacuo and rediluted with 5 ml ethyl acetate. Five hundred microliters each ofthe ethyl acetate extracts of all the constituents were spotted on the same Whatman3MM paper and developed using the &&Forestal formulation'' solvent system. The

spots were detected using the ferrocyanide spray reagent described earlier. Triplicatedetermination was carried out, and measurements of band lengths for each constitu-ent were averaged and expressed as percentage ratio of total constituents. Theprocedure was observed to be reproducible and reliable with 3% standard deviations.In this study, correction factor was further applied to the gallic acid ratio due to someoverlap and melting point attenuation by tannic acid, thus:

Gallic acid"%+Gallic acid#Tannic acid,

;

m.p (Gallic acid!the observed)

m.p. (Gallic acid!Tannic acid as impurity)

Secondly, the relative concentrations (% w/w) of the constituents in the dry matterof the mango kernel were computed using the above percentage ratios and thegravimetric yield of the total tannin obtained earlier.

Preparation of Crude Mango Kernel PPO Extract

Forty grams of white kernel, obtained after cracking the shell manually, were crushedwith pre-chilled (03C) 0.01 M phosphate bu!er (pH 6.0, 60 ml) using a pestle andmortar, and made up to 100 ml with bu!er in a measuring cylinder. The macerate wasimmediately "ltered using suction through coarse "lter (Whatman No. 4) paper intoa Erlenmeyer #ask (250 ml) placed on ice. No brown colour was observed in the crude

extract before use.

Assay for PPO Activity

PPO activity was determined using a colorimetric method (Lee and Smith, 1979)based on the initial rate of increase in absorbance at 420 nm. In this study, 10 ml of

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0.01 M phosphate bu!er (pH 6.0), 1 ml of 0.5 M catechol, and 0.5 ml of the crudeenzyme extract were pipetted in this sequence into a test tube, mixed, and absorbance

recorded at 30 s intervals. One unit of PPO activity caused a change in absorbance of0.001/min at 420 nm.

E+ect of Blanching ime

The crude enzyme extract (3 ml) was heated at 903C in a controlled temperaturecirculating water bath for 0, 2, 4, 5, 6, 8, 10, 12, 15, 16, 18 and 20 min, respectively. Thesamples contained in test tubes were then immediately placed on ice to cool beforeconducting the assay as described above. Each assay mixture was replicated twice.

RESULTS AND DISCUSSION

Mango Kernel annin Composition

Table 1 shows some general properties of the isolated tannin of mango kernel.Chromatographic separation using particularly the Forestal (AHW 30:3:10) solventformulation followed by irradiation with UV light at 254 nm appeared to identify thetannin fraction of mango kernel.

Table 2 indicates the composition of mango kernel tannin. The gravimetric yield of6.4% (w/w) compares favourably with a titrimetric yield (Arogba, 1997) of 4.5% (w/w)expressed as catechin equivalent for the same variety of mango kernel. Di!erence in

varietal composition became apparent when the yield (4.5% w/w) and m.p. (2383C)of gallotannin of African mango kernel were compared with those of Asian origin (ElAnsari et al., 1971). The observed m.p. suggested that gallic acid (cf. Dean, 19xx:2353C) attenuated by tannic acid m.p. of 2183C (Sharp, 1981) (cf. Adewoye and Ajayi,1988a; Sharp, 1981: gallic acid m.p. 2503C) were the main constituents. Gallic acidconstituted one-third of the gallotannin fraction (Table 2).

Prior to acid treatment of the dry kernel meal, epicatechin which constituted about3% of the total tannins, partly or wholly could have arisen at the extraction stage ofthe experiment (Millic, 1972) or much earlier during the dehydration of the freshkernel. The possible presence of the epimer, catechin, was not observed in this study.Gallocatechin was identi"ed after acid hydrolysis and could justify the earlier detec-

tion of the monomeric aglycone, epicatechin mentioned above.Furthermore, the detection of free gallic acid and gallocatechin strongly suggests

that gallic acid could have existed in other forms in the mango kernel. Ellagitannin isone such example, which on acid hydrolysis would justify the ellagic acid detected.Ellagic acid is a dilactone comprising two molecules of gallic acid (Sharp, 1981). The&&Forestal'' solvent system and the colour reactions observed in this study sharplydistinguished gallic acid from ellagic acid. Ellagic acid and gallocatechin were themain constituents of the condensed tannin-related polyphenols in the mango kernel.

The R

values and colour characteristics which aided the identi"cation of the tanninconstituents are presented in Tables 3 and 4, respectively. However, the choice of sol-vent system for the chromatographic separation was of paramount importance. In

this study, sec-BAM 415 gave better resolution of spots than the Forestal (AHW30: 3 : 10) formulation when employed in the"rst run of a two-way chromatography. Itwas implied that stronger protonation of the hydroxyl groups of tannin constituentsby HCl of a solvent system appears to weaken the resolution of spots. The e!ect ofsolvent system composition could, therefore, explain why gallic acid, epicatechinand gallocatechin were characteristically resolved in aqueous acetic acid (2 or 6%)



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TABLE 4

Colour characteristics of individual polyphenols in dry mango kernel meal

Colour

FeCl

# NeutralK

Fe(CN)

lead acetate Visible UV UV#NH

Before hydrolysisTannic acid Blue (#) White (#) Brown Pink PinkGallic acid # # Brown Fluorescent

purpleFluorescentpurple

Epicatechin # # Grey Faint pink Pink

After acid hydrolysisEllagic acid # # Faint yellow Fluorescent

bluish green

Fluorescent

blueGallocatechin # # Brown Green PinkUncon"rmedcompound # # Brown Absorption Pinkn-butyl-cyanidin # # Absorption Greyish Grey

TABLE 5

Variation of residual PPO activity of mango kernel extract with blanching times at 90 3C

Blanching time (min) 0 3 (5}20)% Residual PPO activity 100 48 10

PPO activity was determined at 420 nm using catechol as substrate, and values shown were extrapolatedfrom the graph of blanching time versus PPO activity.

(Table 3). The Forestal formulation showed that gallic acid had purple #uorescencebefore treatment, and bluish #uorescence for ellagic acid after acid hydrolysis andillumination with UV light at 254 nm (Table 4). The role of pH in the #uorescence ofsome tannin constituents of mango kernel needs further investigation.

n-butyl-cyanidin was characterized (Tables 3 and 4) using the techniques high-

lighted in the methodology section. Similar chromatographic behaviour and colourchanges of the acylated cyanidin were observed during the analysis of the condensedtannins of rapeseed hulls, faba bean hulls, and sorghum grains (Leung et al., 1979)using the same leucoanthocyanin reagent (n-butanol}concentrated HCl, 95:5 v/v)(Luh et al., 1967).

hermal Inactivation of a Browning Enzyme (PPO)

The e!ect of moist heat on PPO activity is shown in Table 5. Fifty per cent of themaximum enzyme activity is a measurement criterion commonly used in enzymestudies (White et al., 1978). It is independent of the absolute maximum value in

di!erent sources and treatment conditions. Based on our previous work (Arogba etal., 1998), the optimal pH of 6.0 was employed in the assay procedure given in theExperimental section. Furthermore, the choice of 903C was the maximum attainabletemperature of the enzyme extract at atmospheric pressure which potential processorsof mango kernel at cottage level could readily employ during blanching withoutundue monitoring.

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It has been shown that a very sharp change occurs in PPO activity when a criticaltemperature of 823C is reached (Schultz, 1960). In this study, 50% of PPO activity was

observed at about 3 min at 903C and 5 min for 90% inactivation (Table 5). Furtherinactivation of PPO, however, was di$cult beyond 5 min and was attributed to thematrix e!ects of other major sample components in the extract, that is, the in#uence ofthe degree of purity of the enzyme extract on PPO activity.

CONCLUSION

The study has shown that mango kernel contained hydrolysable tannin which wasabout 75% of the total tannin content. These tannins would require treatment duringprocessing in order to reduce their in vivo toxic e!ect. Furthermore, the rate of tannin

formation through enzyme activity could be signi"cantly reduced by water-blanchingof mango kernel at 903C for 5 min. This is a simple and e!ective process that can becarried out at a cottage industry level. The additional advantage of water-blanching isthe possible leaching of soluble tannic substances into the soak-water.

ACKNOWLEDGEMENT

The author thanks the Polytechnic Research Committee of the National Board for Technical Education(NBTE), Nigeria for the "nancial grant, and Dr A. S. Agbaji and Dr O. O. Ajayi for assisting with authenticsamples of tannic substances at the Laboratory of the National Research Institute for Chemical Technology(NARICT), Zaria.

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