Journal of the Science of Food and Agriculture J Sci Food Agric 80:522±526 (2000)

Effect of antioxidant principles isolated frommango ( Mangifera indica L) seed kernels onoxidative stability of buffalo ghee (butter-fat)D Puravankara,* V Boghra and RS SharmaDairy Chemistry Department, SMC College of Dairy Science, Gujarat Agricultural University, Anand Agricultural Campus, Anand 388110,India

(Rec

* CoV3LCont

# 2

Abstract: A method was developed to extract and isolate the antioxidant principles, ie mainly phenolic

and phospholipid classes, from mango (Mangifera indica L) seed kernels using organic solvents. The

presence of at least six phenolic compounds and eight phospholipids in the isolates was con®rmed by

chromatographic techniques. A phenolic preparation and a phospholipid preparation were prepared

separately by dissolving the isolated compounds from mango seed kernels in buffalo ghee. The phenolic

preparation contained 9.6mg% water-extractable phenolics, 69.5mg% total phenolics and 6.39mg%

phospholipids. The phospholipid preparation contained 155.8mg% phospholipids, 0.11mg% water-

extractable phenolics and 0.19mg% total phenolics. The addition of these preparations to buffalo ghee

at 5, 10 and 20% levels individually and in combination signi®cantly increased the levels of phenolics

and phospholipids respectively. Samples of buffalo ghee with added BHA contained levels of these

compounds similar to that of a control sample without any other additives. The antioxidant indices

calculated from the induction period of ghee samples stored at 80�2°C. in comparison with the control

were, in order, 10.11 (20% phospholipid and phenolic preparation) > 8.88 (10% phospholipid and

phenolic preparation) > 8.66 (20% phenolic preparation) > 6.44 (5% phospholipid and phenolic

preparation) > 5.44 (10% phenolic preparation) > 4.88 (20% phospholipid preparation) > 3.00 (5%

phenolic preparation) > 2.77 (10% phospholipid preparation) > 2.22 (5% phospholipid preparation) >1.44 (0.02% BHA). This demonstrated that the phenolics and phospholipids isolated from mango seed

kernel, when added jointly to buffalo ghee, helped in extending the shelf-life of ghee.

# 2000 Society of Chemical Industry

Keywords: antioxygenic indices; butylated hydroxy anisole (BHA); chromatography; extraction; buffalo ghee(butter-fat); isolation; mango seed kernel; natural antioxidant; peroxide; phenolic; phospholipid; syntheticantioxidant

INTRODUCTIONThe mango (Mangifera indica L), commonly referred

to as the king of fruits,1 is an important fruit crop

cultivated in tropical regions. After consumption or

industrial processing of the fruits, considerable

amounts of mango seeds are discarded as waste.2

About 3�105tons of dry kernels from these mango

seeds are available annually in India.3 These kernels,

depending on the variety, contain on average 5.7%

protein, 9.3% fat, 79.9% carbohydrate, 2.0% crude

®bre and 3.11% ash.4

In times of scarcity and famine, mango seed kernels

are consumed, after boiling, by poor people. The fat

from mango seed kernel is also a promising source of

edible oil.5 If properly collected and used in India the

total estimated production of mango seed kernel lipids

is about 3�104tons. In addition, this fat from mango

seeds has attracted the attention of scientists in recent

years as a cocoa butter substitute, because the former

has a fatty acid and triglyceride pro®le similar to that of

eived 22 January 1998; revised version received 4 November 1999; a

rrespondence to: D Puravankara, Lucerne Foods (A Division of Can4Y6, Canadaract/grant sponsor: Indian Council of Agricultural Research, New Delh

000 Society of Chemical Industry. J Sci Food Agric 0022±5142/2

cocoa butter.6,7 Parmar8 initiated work on the addition

of mango seed kernel to buffalo ghee. It was later

inferred that there was a signi®cant increase in the

shelf-life of ghee and that mango seed kernel can be a

good source of natural antioxidants.

Continuous use of synthetic antioxidants may cause

health hazards such as teratogenic and carcinogenic

effects in experimental animals and primates.9 Other

studies have suggested the involvement of high doses

of synthetic antioxidants in cancer tissue initiation and

propagation, chromosomal aberrations and tissue

damage. Food legislators in many countries are

hesitant to allow the addition of synthetic antioxidants

to foods and wish to protect consumers from the risk of

such chemicals.10 This scienti®c evidence and public

mistrust of synthetic food additives are suf®cient

reason to search for alternative natural antioxidants

for food systems.

Mango seed kernel can be a potential source of

natural antioxidants. It was earlier inferred that the

ccepted 17 November 1999)

ada Safeway Limited), Milk Plant, 7650 18th Street, Burnaby, BC

i

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Antioxidant principles from mango seed kernel

enhancement of the shelf-life of ghee may be due to

various types of phospholipid and phenolic com-

pounds present in mango seed kernels. However, no

efforts have been made to isolate the naturally

available antioxidant principles from the abundantly

available waste material, mango seed kernel. More-

over, it remained ambiguous whether the increase in

stability of buffalo ghee against autoxidation was

exclusively due to phospholipid or phenolic com-

pounds or to a combined synergistic action of these

compounds.

MATERIALS AND METHODSButter samples from fresh raw cream obtained from

fresh raw buffalo milk were heat clari®ed to ghee until

the temperature reached 100°C. Mango seed kernel

powder (MSKP) passing through a 100-mesh sieve

was obtained from sun-dried kernels collected from

ripened mangoes of varieties such as Rajpuri, Langra,

Kaiser, Alphonso and Desi. The MSKP was analysed

for moisture, fat, protein and ash following the

methods speci®ed by the Indian Standards Institu-

tion.11 The carbohydrate content was calculated by

difference. For the phospholipids of MSKP, appro-

priate methods were followed for extraction,12 estima-

tion,13,14 resolution15 and detection on thin layer

chromatograms. The total phenolic content was also

determined using the method of Swain and Hills.16

For extraction and isolation of phenolics and phos-

pholipids, 50g of MSKP was mixed with 500ml of

chloroform/methanol mixture (2:1 v/v), shaken for 1h

and stored overnight. It was then ®ltered through

Whatman ®lter paper No 41. To this ®ltered chloro-

form/methanol mixture, ethyl acetate and water were

added (5:1:0.5 v/v/v) in a separating funnel and mixed

well. Two layers were allowed to separate and were

collected separately. The chloroform/methanol mix-

ture which remained in the bottom layer was again

mixed with ethyl acetate and water in the same

proportion as before, and this procedure was repeated

three times. The top layers were pooled together and

vacuum evaporated in a rotary ¯ask evaporator to

about 20ml. The two concentrated liquids obtained

from vacuum evaporation of the pooled top and

bottom layers were subsequently passed through

anhydrous sodium sulphate columns (2.8cm�30cm)

separately. Both columns were washed with ethyl

acetate to elute the phenolic compounds, until the

ethyl acetate washings were free from phenolics as

indicated by the absence of blue colour development

according to the method of Swain and Hills.16 The

ethyl acetate washings were combined and concen-

trated in vacuum to about 20ml in a rotary ¯ask

evaporator, and a suitable aliquot of this extract was

used for two-dimensional paper chromatographic

resolution of phenolic compounds by the method

described by Roberts and Woods,17 and visualized

using appropriate dip reagents by method of Barton etal. 18 Subsequently, both sodium sulphate columns

J Sci Food Agric 80:522±526 (2000)

were washed with methanol to elute the phospho-

lipids, and the methanol washings were combined and

concentrated in a rotary ¯ask evaporator to about

20ml. A suitable aliquot of this extract was used for

thin layer chromatographic separation of phospho-

lipids by the methods described for resolution and

detection by Morrison et al15 and Stahl19 respectively.

The combined concentrated ethyl acetate washings

were tested for the presence of phospholipids by thin

layer chromatography, and the concentrated methanol

washings were tested for the presence of phenolic

compounds by the method described earlier. Finally,

the ethyl acetate and methanol washings were com-

pletely evaporated in a vacuum oven maintained at a

temperature of not more than 60°C and 600mmHg

vacuum to obtain crude phenolics and phospholipids.

These crude antioxidant concentrates were mixed with

100g buffalo ghee and ®ltered through a four-layer

muslin cloth to obtain phenolic and phospholipid

preparations respectively. These preparations were

added at levels of 5, 10 and 20% (v/v) individually and

in combination to freshly prepared buffalo ghee

samples. For comparison, the permitted synthetic

antioxidant butylated hydroxyanisole (BHA) was

added at the 0.02% (w/v) level.

The buffalo ghee samples with and without addi-

tives were analysed for moisture, free fatty acids, total

phenolics, peroxide value20 and water-extractable

phenolic compounds.16 The samples were stored in

an oven maintained at 80�2°C and their peroxide

development was monitored at intervals of 48h to

record the time taken in hours to reach a peroxide

value of 5. The peroxide value of 5 has been used by

Pruthi et al21 and Parmar and Sharma22 in their

storage studies of ghee at 80�2°C. In this accelerated

storage study at 80�2°C the ghee was presumed to be

deteriorated after a peroxide value of 5. To test the

effectiveness of the additives, antioxygenic indices or

protection factors were calculated according to Pruthi

et al. 21 Three replications were carried out in a

completely randomized design, with comparison of

treatments by Duncan's multiple-range test and

statistical analysis of data according to Snedcor and

Cochran.23

RESULTS AND DISCUSSIONIt is reported that ghee made from buffalo milk is more

prone to oxidative deterioration than that made from

cow's milk;24 and irrespective of the source of the raw

material, it is accepted that ghee made by the creamery

butter method is more susceptible to oxidative

deterioration than that made by other methods.25

Hence, in this experiment, ghee was made from

buffalo milk using the creamery butter method. The

use of MSKP from ripened mangoes of mixed varieties

was preferred for the isolation of antioxidative prin-

ciples because of its ease of availability and because it is

a potential source of waste.

The MSKP contained on average 8.6% moisture,

523

Figure 1. Two-dimensional paper chromatograms of phenolic compoundsisolated from mango seed kernel powder. The chromatogram wereobtained with the following solvent systems: (a) n-butanol/acetic acid/water(4:1:5 v/v/v); (b) 2% acetic acid (in water). .

Figure 2. Thin layer chromatograms of (A) chloroform/methanol extract ofmango seed kernel powder and (B) phospholipids isolated from mangoseed kernel powder representing total number of phospholipid fractions.The chromatograms were obtained with the following solvent system:chloroform/methanol/ammonia/water (65:35:5:2.5 v/v/v/v).

D Puravankara, V Boghra, RS Sharma

11.64% fat, 5.38% protein, 1.37% ash and, by

difference, an assumed 73.01% carbohydrates. These

results are in general agreement with those reported by

Bhatnagar and Subramaniam.4 Besides these con-

stituents, the MSKP also contained 5.68% total

phenolics and 0.29% phospholipids. The phospho-

lipids on the basis of fat of MSKP were found to be

2.4%. These values are in close conformity to those

reported by Vanpee et al26 for Gholek variety. The

value of total phenolics is considerably lower than that

reported by Das and Banerjee.27 This difference could

be due to variations in the nature of the raw material

and the methods of extraction and estimation.

The method used to extract and isolate the phenolic

compounds from MSKP separates six major phenolic

compounds, as revealed by the two-dimensional paper

chromatographic pattern (Fig 1). The presence of

phenolic compounds in MSKP of the same pattern has

previously been reported by Sharma28 and Parmar and

Sharma.22 These were assumed to be mainly gallic

acid and ellagic acid and gallates. Similarly, the

method described for extraction and isolation of

phospholipids from MSKP yields eight different

fractions, as revealed by the thin layer chromato-

graphic pattern (Fig 2B). The appearance of all the

fractions of phospholipids in the chloroform/methanol

extract of MSKP (Fig 2A) and the phospholipids

isolated by the present method reveals the presence of

all phospholipid classes naturally occurring in MSKP.

This pattern of phospholipids was in close resem-

blance to that identi®ed earlier by Moharram and

Moustafa.29 Tentative identi®cation of the phos-

pholipid fractions showed phosphatidyl serine, lyso-

phosphatidyl choline, phosphatidyl inositol,

sphingomyelin, phosphatidyl choline, phosphatidyl

ethanolamine, phosphatidic acid and glycerophos-

phatidyl compounds.

Vacuum drying of ethyl acetate washings gave on

average 1.165g of a dark brown material with a strong

524

phenolic smell at 2.33% yield and about 41.02%

recovery, if it is considered to be recovered from 50g of

MSKP. The low recovery may be due to incomplete

extraction of phenolic compounds from MSKP by

chloroform/methanol mixture. Similarly, vacuum dry-

ing of methanol washings gave on average 12.8g of a

light brown viscous material similar to that of mango

seed kernel fat at 6.4% yield, if it is considered to be

recovered from 50g of MSKP. The higher yield may

be due to the presence of other lipid constituents, eg

neutral lipids, glycolipids, etc, which may be simulta-

neously extracted by the chloroform/methanol mix-

ture. These vacuum-dried materials were mixed with

buffalo ghee and ®ltered to obtain preparations rich in

phenolic and phospholipid compounds respectively.

The buffalo ghee used for the preparation of a

mixture rich in antioxidative principles contained on

average 6.9mg% phospholipids, 0.11mg% total

phenolics and 0.187mg% water-extractable phenolics.

The phenolic preparation contained 6.39mg% phos-

pholipids, 9.6mg% water-extractable phenolics and

69.5mg% total phenolics. Similarly, the phospholipid

preparation contained 155.8mg% phospholipids,

J Sci Food Agric 80:522±526 (2000)

Figure 3. Peroxide value of buffalo ghee as affected by addition of various preparations and BHA.

Antioxidant principles from mango seed kernel

0.113mg% water-extractable phenolics and

0.187mg% total phenolics. This comparison shows

that the addition of vacuum-dried phenolic com-

pounds increased the level of phenolic constituents

without affecting the phospholipid content, while the

addition of vacuum-dried phospholipid compounds

increased the level of phospholipids without affecting

the phenolic content. By the addition of phenolic

preparation at 5, 10 and 20% (v/v) to the buffalo ghee

samples, there were gradual increases in water-

extractable phenolic content and total phenolic con-

Table 1. Changes in water-extractable phenolics (mg%), total phenolics (mg%), phaddition of various preparations at different levels (v/v) and BHA at 0.02% (w/v)

Treatment Water-extractable phenolics Total phenoli

Control 0.113 0.187

Phenolic preparation

5% v/v (PH5) 0.355 5.07

10% v/v (PH10) 0.652 9.30

20% v/v (PH20) 1.000 11.95

Phospholipid preparation

5% v/v (PL5) 0.122 0.187

10% v/v (PL10) 0.155 0.187

20% v/v (PL20) 0.122 0.187

Blend (50:50) of phenolic and phospholipid preparations

5% v/v (PLPH5) 0.373 5.140

10% v/v (PLPH10) 0.622 9.430

20% v/v (PLPH20) 1.217 11.46

0.02% w/v BHA 0.108 4.28

Average of three replications.a Hours to reach a peroxide value of 5m eq of peroxide oxygen per kg of ghee.b Ratio of induction period of treated sample to induction period of control sample

J Sci Food Agric 80:522±526 (2000)

tent over the control and BHA-treated samples.

Similarly, the addition of phospholipid preparation at

5, 10 and 20% (v/v) elevated the level of phospholipids

in buffalo ghee. However, when both preparations

were added together, there were signi®cant increases

in both phenolic and phospholipid contents of buffalo

ghee samples.

The peroxide values (Fig 3) of buffalo ghee samples

with and without additives indicated that the devel-

opment of peroxide occurred at a faster rate in control

samples than in those with additives. The addition of

ospholipids (mg%), induction period and antioxidant index of buffalo ghee on

cs Phospholipids Induction period a Antioxidant index b

6.9 144

5.93 432 3.00

7.01 784 5.44

6.58 1248 8.66

12.73 320 2.22

16.83 400 2.77

22.77 704 4.88

12.21 928 6.44

17.69 1280 8.88

22.98 1456 10.11

6.58 210.6 1.44

.

525

D Puravankara, V Boghra, RS Sharma

preparations offered resistance against autoxidation of

buffalo ghee, as evident from the increases in induc-

tion period and antioxygenic index (Table 1).

The induction periods time taken in hours to reach a

peroxide value of 5meq of peroxide oxygen per kg of

buffalo ghee) were, in order, 144h (control)<210.6h

(BHA)<320h (5% phospholipid preparation)<400h

(10% phospholipid preparation)<432h (5% phenolic

preparation)<704h (20% phospholipid prepara-

tion)<784h (10% phenolic preparation)<928h (5%

phenolic and phospholipid preparation)<1248h

(20% phenolic preparation)<1280h (10% phenolic

and phospholipid preparation)<1456h (20% pheno-

lic and phospholipid preparation). The addition of

preparations offered resistance against autoxidation of

buffalo ghee, as evident from the increases in induc-

tion period. Further, the effectiveness is clearly seen

when the antioxygenic indices (ratio of induction

period of treated sample to induction period of control

sample) are calculated (Table 1).

It is revealed from the data (peroxide values,

induction periods and antioxygenic indices) that the

addition of preparations helps to extend the stability of

buffalo ghee against autoxidation. The results also

show that the phenolics are more effective than the

phospholipids in increasing the induction period of

buffalo ghee.

However, the combination of both phenolic and

phospholipid compounds, when added to buffalo

ghee, increased the induction period to its maximum

value, suggesting a synergistic action of the two types

of compounds. Addition of preparations at a level of

5% or above is more effective in prolonging the

stability of buffalo ghee than addition of BHA at the

permitted level in ghee. Besides these two major

classes of compounds, other factors such as toco-

pherols, carotenoids4 and sugar/amino acid browning

reaction products30 may also be involved in the

effectiveness of MSKP in extending the shelf-life of

buffalo ghee.

ACKNOWLEDGEMENTSD Puravankara is grateful to the Indian Council of

Agricultural Research, New Delhi for providing

®nancial assistance through a junior fellowship.

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J Sci Food Agric 80:522±526 (2000)