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 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, poly-
phenol 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 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 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

150 AROGBA

Tannins as natural phenolic substances di!er remarkably in chemical structure

depending on the plant source, and rarely does a single dose cause lethal e!ect
(Singleton and Kratzer, 1973). 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., 1996). 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., 1971).
Polyphenol oxidase (PPO), like peroxidase and catalase, is widely distributed in
plants (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 treatment
given. Some properties of PPO in mango kernel have been reported (Arogba et al.,
1998). The moist heat conditions necessary to arrest enzymic browning in mango
kernel during handling were examined in this experiment. This information could be
helpful to potential processors in order to improve the utilization of mango kernel.
This paper, therefore, 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.

MATERIALS AND METHODS

Sample Collection

The Ikanekpo variety of mango (M. 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, Abuja, Nigeria.

Extraction of ¹otal ¹annin

The pulp of ripe mango fruit was peeled o! manually using a stainless-steel knife, and
the shell cracked open to obtain the whitish semi-dry kernel. The latter was pulverized
using a pestle and mortar, and then dried in the conventional air-oven at 1053C until
a 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 Whatman
no. 4 "lter paper, and concentrated to dryness using a rotary evaporator. A brown
solid obtained as the total tannin was weighed and expressed as percentage (w/w) of
the kernel sample.

Acid Hydrolysis of Condensed ¹annins

Before the acid treatment was given, the brown solid obtained earlier was redissolved
in 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 of Luh 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
 TANNIN AND EFFECT OF HEAT ON PPO OF MANGO KERNEL 151

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 pH
UV (254 nm) Non#uorescent
Forestal formulation#UV (254 nm) Fluorescent upper band
1% gelatin White precipitate
Neutral lead acetate White precipitate
FeCl }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 hydrolysis
Total tannin-related substances 100.0 6.37
Gallotannin 74.5 4.75
Tannic acid 46.6 2.97
Gallic acid 27.9 1.78
Condensed tannin-related polyphenols 25.5 1.62
Epicatechin 2.7 0.17
After acid hydrolysis
Condensed tannin-related polyphenols 22.8 1.45
Ellagic acid 11.0 0.70
Gallocatechin 6.9 0.44
n-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 ¹annins

Empirical qualitative tests (Table 1) were conducted. The melting point of the white
amorphous precipitate was recorded.
The constituents of the total tannin, before and after acid hydrolysis, were separated
on 18 cm;18 cm Whatman 3MM chromatographic paper essentially by a two-way
technique 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 and
Ajayi, 1989).
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
152 AROGBA

TABLE 3
R values of individual polyphenols in dry mango kernel meal

R values

a b c d e

Before hydrolysis
Tannic acid 0.58}0.78 0.17 0.58}0.74
Gallic acid 0.50}0.78 0.17 0.44 0.58}0.74
Epicatechin 0.44 0.21 0.42}0.58
After acid hydrolysis
Ellagic acid 0.0}0.80 0.0 0.0}0.40 0.0
Gallocatechin 0.84 0.30
Uncon"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"FHW
523.

developed, by following the steps described thus: fractions which were resolved in the
two-way chromatography were cut from the papers, and separately redissolved in
methanol. 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 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. The
spots were detected using the ferrocyanide spray reagent described earlier. Triplicate
determination was carried out, and measurements of band lengths for each constitu-
ent were averaged and expressed as percentage ratio of total constituents. The
procedure 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 some
overlap 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 matter
of the mango kernel were computed using the above percentage ratios and the
gravimetric 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 crushed
with pre-chilled (03C) 0.01 M phosphate bu!er (pH 6.0, 60 ml) using a pestle and
mortar, and made up to 100 ml with bu!er in a measuring cylinder. The macerate was
immediately "ltered using suction through coarse "lter (Whatman No. 4) paper into
a 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
 TANNIN AND EFFECT OF HEAT ON PPO OF MANGO KERNEL 153

0.01 M phosphate bu!er (pH 6.0), 1 ml of 0.5 M catechol, and 0.5 ml of the crude
enzyme 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 of
0.001/min at 420 nm.

E+ect of Blanching ¹ime

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

RESULTS AND DISCUSSION

Mango Kernel ¹annin Composition

Table 1 shows some general properties of the isolated tannin of mango kernel.
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.
Table 2 indicates the composition of mango kernel tannin. The gravimetric yield of
6.4% (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 (El
Ansari 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 acid
constituted one-third of the gallotannin fraction (Table 2).
Prior to acid treatment of the dry kernel meal, epicatechin which constituted about
3% of the total tannins, partly or wholly could have arisen at the extraction stage of
the experiment (Millic, 1972) or much earlier during the dehydration of the fresh
kernel. 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 is
one 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 sharply
distinguished gallic acid from ellagic acid. Ellagic acid and gallocatechin were the
main constituents of the condensed tannin-related polyphenols in the mango kernel.
The R values and colour characteristics which aided the identi"cation of the tannin

constituents 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 (AHW
30 : 3 : 10) formulation when employed in the "rst run of a two-way chromatography. 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. The e!ect of
solvent system composition could, therefore, explain why gallic acid, epicatechin
and gallocatechin were characteristically resolved in aqueous acetic acid (2 or 6%)
154 AROGBA

TABLE 4
Colour characteristics of individual polyphenols in dry mango kernel meal

Colour

FeCl # Neutral

K Fe(CN) lead acetate Visible UV UV#NH
  
Before hydrolysis
Tannic acid Blue (#) White (#) Brown Pink Pink
Gallic acid # # Brown Fluorescent Fluorescent
purple purple
Epicatechin # # Grey Faint pink Pink
After acid hydrolysis
Ellagic acid # # Faint yellow Fluorescent Fluorescent
bluish green blue
Gallocatechin # # Brown Green Pink
Uncon"rmed
compound # # Brown Absorption Pink
n-butyl-cyanidin # # Absorption Greyish Grey

TABLE 5
Variation of residual PPO activity of mango kernel extract with blanching times at 903C

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 extrapolated
from the graph of blanching time versus PPO activity.

(Table 3). The Forestal formulation showed that gallic acid had purple #uorescence
before treatment, and bluish #uorescence for ellagic acid after acid hydrolysis and
illumination with UV light at 254 nm (Table 4). The role of pH in the #uorescence of
some 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 colour
changes of the acylated cyanidin were observed during the analysis of the condensed
tannins 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).

¹hermal Inactivation of a Browning Enzyme (PPO)

The e!ect of moist heat on PPO activity is shown in Table 5. Fifty per cent of the
maximum enzyme activity is a measurement criterion commonly used in enzyme
studies (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 et
al., 1998), the optimal pH of 6.0 was employed in the assay procedure given in the
Experimental section. Furthermore, 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.
 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, 1960). In this study, 50% of PPO activity was
observed at about 3 min at 903C and 5 min for 90% inactivation (Table 5). Further
inactivation of PPO, however, was di$cult beyond 5 min and was attributed to the
matrix e!ects of other major sample components in the extract, that is, the in#uence of
the degree of purity of the enzyme extract on PPO activity.

CONCLUSION

The study has shown that mango kernel contained hydrolysable tannin which was
about 75% of the total tannin content. These tannins would require treatment during
processing 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-blanching
of mango kernel at 903C for 5 min. This is a simple and e!ective process that can be
carried out at a cottage industry level. The additional advantage of water-blanching is
the 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 authentic
samples of tannic substances at the Laboratory of the National Research Institute for Chemical Technology
(NARICT), Zaria.

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