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Antioxidant activity of selected India

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Article in Prostaglandins Leukotrienes and Essential Fatty Acids March 2000

DOI: 10.1054/plef.1999.0128 Source: PubMed

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 Prostaglandins, Leukotrienes and Essential Fatty Acids (2000) 62(2), 107110
2000 Harcourt Publishers Ltd
doi:10.1054/plef.1999.0128, available online at http://www.idealibrary.com on

Antioxidant activity of selected

Indian spices
S. Shobana, K. Akhilender Naidu
Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570 013, India

Summary Spices and vegetables posses antioxidant activity that can be applied for preservation of lipids and
reduce lipid peroxidation in biological systems. The potential antioxidant activities of selected spices extracts (water
and alcohol 1:1) were investigated on enzymatic lipid peroxidation. Water and alcoholic extract (1:1) of commonly
used spices (garlic, ginger, onion, mint, cloves, cinnamon and pepper) dose-dependently inhibited oxidation of fatty
acid, linoleic acid in presence of soybean lipoxygenase. Among the spices tested, cloves exhibited highest while onion
showed least antioxidant activity. The relative antioxidant activities decreased in the order of cloves, cinnamon,
pepper, ginger, garlic, mint and onion. Spice mix namely ginger, onion and garlic; onion and ginger; ginger and garlic
showed cumulative inhibition of lipid peroxidation thus exhibiting their synergistic antioxidant activity. The antioxidant
activity of spice extracts were retained even after bioling for 30 min at 100C, indicating that the spice constituents
were resistant to thermal denaturation. The antioxidant activity of these dietary spices suggest that in addition to
imparting flavor to the food, they possess potential health benefits by inhibiting the lipid peroxidation. 2000 Harcourt
Publishers Ltd

INTRODUCTION tion and thermo-stability of antioxidant potential of spice

Spices are common food adjuncts that impart flavor,
aroma or piquancy to foods. Spices are consumed in a
variety of combinations depending on taste preferences.
Spices or aromatic vegetable products are used in cooked
or semi-cooked foods, sauces, dressings and soups, and
some of the vegetable spices like onion/capsicum are con-
sumed raw. Spice principles like curcumin and capsicin
prevent oxidation of oils and fats.1,2 Active principles of
spices such as curcumin (turmeric), capsaicin (red chill-
ies), eugenol (cloves), linalool (coriander), piperine (black
pepper), zingerone (zinger) and cuminaldehyde (cumin)
are reported to inhibit lipd peroxidation.3,4,5 As spices are
usually added as flavoring agents to food preparations as
either raw or crushed paste and cooked at high tempera-
tures, the present study was undertaken to evaluate the
crude aqueous plus alcoholic extracts (1:1) of selected
individual spices/spice mix on enzymatic lipid peroxida-
Received 3 November 1999
Accepted 16 November 1999
Correspondence to: K. Akhilender Naidu, Department of Biochemistry and
Nutrition, Central Food Technological Research Institute, Mysore 570 013,
India. Fax: +91 821 517233; E-mail:kanaidu@mailcity.com
extracts.
MATERIAL AND METHODS
Chemicals
Linoleic acid, Soybean Type IV lipoxygenase, Tween 80,
Tris were purchased from Sigma chemical and co. USA.
Other chemicals used were of analar grade. Ginger
(Zingiber officinale), garlic (Allium sativum), onion
(Allium cepa), mint (Mentha spicata), cloves (Syzigium
aromaticum), cinnamon (Cinnamommum zeylanicum)
and pepper (Piper nigrum) were purchased from the local
market.
Spice extracts
One gram each of ginger, garlic, onion, mint leaves, cin-
namon, cloves and pepper were weighed separately and
each one of them were crushed finely and ground to
paste in mortar and pestle and extracted in 5 ml of dis-
tilled water and ethanol mixture (1:1). This extract was
centrifuged at 5000 rpm and the supernatant was used as
source of spice extract. The spice extract was used direct-

2000 Harcourt Publishers Ltd Prostaglandins, Leukotrienes and Essential Fatty Acids (2000) 62(2), 107110
108 Shobana and Akhilender Naidu

ly or diluted with distilled water depending on the antiox-
idant potency of spice. The spice extracts were mixed at
1:1 ratio and the spice mix was checked for antioxidant
activity.
Boiled spice extracts
The spice extract was boiled in a water bath at 100C for
30 min and then allowed to cool at room temperature to
check the antioxidant activity.
ly insoluble in water. The main objective of this study is
to evaluated antioxidative potential of crude spice extract
(water plus alcohol 1:1) and thermal stability during the
processing of cooking.
Aqueous plus ethanolic extracts of all the spices tested
showed significant inhibition of lipoxygenase-dependent
enzymatic lipid peroxidation and the inhibitory effect
of spices were found to be concentration dependent
(Figs 14). Among the spices tested, cloves showed the
highest antioxidant activity while onion showed the least
antioxidant activity. The degree of antioxisant effect of

Lipid peroxidation assay

Enzymatic lipid peroxidation was measured spectropho-

tometrically following an increase in absorbance of lipid
hydroperoxides formation at 234 nm as described else-
where.5 The reaction medium (3.0 ml final volume) in the
sample cuvette contained 50.0 M linoleic acid, 5.0 nM
soybean lipoxygenase enzyme in 50 mM Tris buffer
pH 7.4. Increase in absorbance at 234 nm was monitored
in Shimadzu UV 160A Spectrophotometer. Extinction
coefficient of 25 mM1 cm1 was used for quantification of
lipid hydro-peroxides. The enzyme activity was expressed
as moles or hydroperoxide formed/min/ nmole of
enzyme.

Inhibitory studies

Soybean lipoxygenase enzyme was pre-incubated for

2 min with different concentrations of spice extracts
(unboiled and boiled) prior to initiation of the reaction
with substrate, linoleic acid. Positive controls were main-
Fig. 1 Effect of cloves on lipid peroxidation.
tained with spice extracts and substrate to check the
effect of spices on non-enzymatic lipid peroxidation. The
IC50 value (the concentration which causes 50.0% inhibi-
tion of enzyme activity) was determined by plotting a
graph with spice concentration versus percent inhibition
of lipid peroxidation. The spice mixes, namely ginger and
garlic; onion and ginger; ginger, garlic and onion were
evaluated at different concentrations to find out the syn-
ergistic effect of spices on lipid peroxidation.

RESULTS AND DISCUSSION

Ginger, garlic, onion, pepper, mint leaves, cloves and cin-

namon are some of the commonly used dietary spices in
vegetarian and non-vegetarian preparations. Spices are
added to food in raw chopped form or ground paste either
individually or as a mix of spices in various Indian food
preparations. In some food preparations they are con-
sumed raw, without any cooking, and in most dishes they
are cooked at temperatures of 100C. Most of the studies
on antioxidant properties of spices were concentrated on
spice active principles which are fat soluble and practical- Fig. 2 Effect of cinnamon on lipid peroxidation.

Prostaglandins, Leukotrienes and Essential Fatty Acids (2000) 62(2), 107110 2000 Harcourt Publishers Ltd
 Antioxidant activity of selected Indian spices 109

Table 1 The inhibitory effect of spices on lipid peroxidation.

Spices/Synthetic IC50 Value (mg)

antioxidants

Cloves 0.280.005
Cinnamon 1.000.02
Pepper 5.500.03
Ginger 7.500.05
Mint 11.600.04
Onion 18.000.02
BHA 0.0150.001
BHT 0.0280.002

Antioxidant activity related to flavonoids quercetin,

luteolin, capsaicin and ascorbic acid were reported in
fresh pepper (Capsicum annuum) extract.7 Similarly, gin-
ger is reported to contain antioxidantsgingerol and
hexahydrocurcumin.8 In this study the crude extracts
(50% alcohol extract) of fresh spices might contain more
than one antioxidant and could be responsible for
significant inhibition of enzymatic lipid peroxidation.
Fig. 3 Effect of garlic, ginger and pepper on lipid peroxidation.
These results are in agreement with the reported inhibi-
tion of lipid peroxidation by spice principles like eugenol,
zingerone and piperine.36 The spice mixes (garlic, ginger
and onion) showed cumulative effect indicating synergis-
tic antioxidative activity of spices (Fig. 5). The interesting
observation in this study was that boiling garlic, ginger
cloves, cinnamon and pepper extracts at 100C for 30 min
did not only retain the antioxidant activity but also
showed significantly higher antioxidant activity, indicat-
ing that the spice constituents were resistant to thermal
denaturation. The possible release of bound antioxidant

Fig. 4 Effect of onion and mint on lipid peroxidation.

spices decreased in the order of cloves > cinnamon > pep-

per > ginger > garlic > mint > onion (Table 1). However,
spice crude extracts were less potent antioxidants com-
pared to synthetic antioxidants BHT and BHA. The spice
extracts showed much higher antioxidant activity com-
pared to fresh extract even after boiling them for 30 min
Fig. 5 Effect of spice mix on lipid peroxidation.
(except in onion) at 100C (Table 2). Spice extract mix, A: Onion (4 mg) plus ginger (2 mg);
namely onion (412 mg) and ginger (28 mg); ginger B: Onion (8 mg) plus ginger (4 mg);
(24 mg) and garlic (24 mg) showed cumulative inhibi- C: Onion (12 mg) plus ginger (6 mg);
D: ginger (2 mg) plus garlic (2 mg);
tion of lipid peroxidation and the synergistic per cent E: ginger (4 mg) plus garlic (4 mg);
inhibition was around the expected value (Fig. 5). F: ginger (2 mg) plus garlic (2 mg) and onion (4 mg).

2000 Harcourt Publishers Ltd Prostaglandins, Leukotrienes and Essential Fatty Acids (2000) 62(2), 107110
110 Shobana and Akhilender Naidu

Table 2 Effect of boiling of spice extracts on lipid peroxidation. titate different phenolic antioxidants in crude water
extracts of above spices.
Spice Spice Fresh Boiled inhibition
Concentration (mg) %

Cloves 0.33 61
Cinnamon 0.67 21
0.99 48
Ginger 6.00 41
Garlic 6.00 26
8.00 46
10.00 65
Pepper 4.00 24
6.00 64
Onion 16.00 47
principles during heat treatment could be responsible for
the higher antioxidant activity compared to fresh spice
extract (Table 2). Thermal stability studies on spices,
namely garlic and crude gingerol, in oils (soybean and
ground nut) heated at 105165C showed significant
antioxidant activity.9,10 However, boiling of spice mixes in
presence of souring agent tamarind was reported to pro-
duce significant quantitative loss in the active principles
of turmeric, capsicum and pepper.11 Similarly, oleoresin
from red pepper (capsaicin) showed moderate antiodiant
activity at 100C and no antioxidant effect at 150C due
to degradation of capsaicin.12 These observations imply
that thermal stability of spices varies with the individual
spice and the cooking conditions.
Spices are known to exert their effect on lipid peroxida-
tion by scavenging reactive oxygen species1315 or chelate
metal ions needed for initiation of lipid peroxidation.7
Similarly, spice extracts in this study might contain more
than one antioxidant and thus scavenge the hydroxy rad-
ical or quench the fatty acid radicals and thus inhibit the
propagation of lipid peroxidation.4,5
In conclusion, this study clearly demonstrated that
crude (water and alcoholic) extracts of spices exhibit
(individually and as a mix) significant antioxidant activity
similar to that of spice active principles and some of these
spice extracts are resistant to thermal denaturation.
Although the spice crude extracts are less potent antioxi-
dants compared to BHA and BHT, these dietary spices are
much safer than the synthetic antioxidants. Thus the
spices, which are widely used in Indian food preparations
in addition to imparting flavor and taste to the food,
might act as good antioxidants and thus prevent oxida-
tion of lipids and adverse effects of lipid peroxidation.
Further studies are in progress to characterize and quan-
76
ACKNOWLEDGMENTS
35 The authors gratefully acknowledge Dr V. Prakash, Director,
65 CFTRI, and Dr S. G. Bhat, Head Department of Biochemistry and
85
Nutrition, for their encouragement and support.
36
79
100
70
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100
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