Food

Chemistry
Food Chemistry 100 (2007) 1100–1105
[Link]/locate/foodchem

Evaluation of antioxidant activity of some plant extracts

and their heat, pH and storage stability
Saeedeh Arabshahi-D, D. Vishalakshi Devi, Asna Urooj *

Department of Food Science and Nutrition, University of Mysore, Manasagangotri, Mysore 570006, India

Received 24 January 2005; received in revised form 1 November 2005; accepted 15 November 2005

Abstract

In the present study, three plant foods, namely, drumstick leaves (Moringa oleifera), mint leaves (Mentha spicata) and carrot tuber
(Daucus carota) were extracted with ethanol and analyzed for their antioxidant activity. The antioxidant activity of extracts was evalu-
ated according to the amount of malonaldehyde (MDA) formed by the FeSO4-induced oxidation of linoleic acid and a high PUFA oil
(sunflower oil) at 37 C in Trizma-buffer (pH 7.4). At a concentration of 1.5 mg/ml of linoleic acid ,the extracts from drumstick and car-
rot had a higher antioxidant activity (83% and 80%) than a-tocopherol (72%). In sunflower oil, the extracts from drumstick leaves and
mint leaves were found to exhibit a similar activity( 46% and 44%). The extract from drumstick exhibited the highest activity in both lipid
systems. In addition, the stability of extracts to pH (4 and 9) and temperature (100 C, 15 min) was investigated. The antioxidant activity
of the extracts from mint leaves and carrot was higher at pH 9 than pH 4, while that of drumstick extract remained the same under both
pH conditions. The extract from carrot was more heat-stable than other extracts. The three extracts stored in the dark at 5 and 25 C
after a 15 day period did not show any significant change (p 6 0.05) in their antioxidant activity. These data indicate that selected plant
extracts are potential sources of dietary antioxidants.
 2005 Elsevier Ltd. All rights reserved.

Keywords: Antioxidant activity; Plant extract; Processing; Stability

1. Introduction 2002). The search for safe and effective naturally occurring
antioxidants is now focused on edible plants, especially
Lipid oxidation is a highly deteriorative process in spices and herbs (Nakatani, 1997). A large number of
foods, as it leads to unacceptable properties for the cus- plants have been screened as a viable source of natural
tomer and a loss in nutritional value. In addition, oxidation antioxidants including tocopherols, vitamin C, carotenoids
leads to health disorders such as atherosclerosis and can- and phenolic compounds which are responsible for mainte-
cerogenesis among others. Hence the presence of antioxi- nance of health and protection from coronary heart dis-
dants in foods is essential for their quality, retention and eases and cancer (Castenmiller et al., 2002; Javanmardi,
safety (Koleva et al., 2003; Pizzale, Bortolomeazzi, Vichi, Stushnoff, Locke, & Vivanco, 2003; Kaur & Kapoor,
Uberegger, & Conte, 2002). Toxicological effects of syn- 2001). However, food composition tables, which are neces-
thetic antioxidants and consumer preference for natural sary tools for epidemiological and nutritional studies, are
products have resulted in increased interest in the applica- really only representative of food stuffs consumed in their
tion of natural antioxidants (Castenmiller et al., 2002; raw state, without considering the fact that concentration
Kaur & Kapoor, 2001; Koleva et al., 2003; Pizzale et al., of nutrients and their biological activity may be changed
by environmental variables as well as processing (Nicoli,
*
Corresponding author. Tel.: +91 821 249 5789; fax: +91 821 2419301.
Anese, & Parpinel, 1999).
E-mail addresses: azarabshahi2003@[Link] (S. Arabshahi-D), The consequence of food processing and preservation
asnaurooj@[Link] (A. Urooj). procedure on the overall antioxidant activity of foods are

0308-8146/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/[Link].2005.11.014
 S. Arabshahi-D et al. / Food Chemistry 100 (2007) 1100–1105 1101

generally the result of different events, hence the evaluation & Zubic, 1998) and analyzed by Folin-Ciocalteu micro
of processing factors influencing the antioxidant activity is method (Slinkard & Singleton, 1977). Results were
imperative to increase or preserve their efficacy and bio- expressed as grams of gallic acid per 100 g of dried plant
availability (Kaur & Kapoor, 2001; Nicoli et al., 1999). material.
In addition, from a nutritional point of view, the under-
standing of the consequence of food processing is one of 2.2. Preparation of antioxidant extracts
the most important steps in correct interpretation and eval-
uation of study results regarding dietary habits and human A weighed portion (15 g) of each dried sample was
health (Kaur & Kapoor, 2001). extracted with 50 ml of 95% (v/v) ethanol for 6 h in a
The present study addresses the utilization of some plant mechanical shaker. The extracts were filtered and filtrates
materials such as drumstick leaves (Moringa oleifera), mint were evaporated at 40 C to dryness in a rotary evaporator
leaves (Mentha spicata) and carrot tuber (Daucus carota) as (Buchi Laboratoriums-Technik, Flawil/ Schweiz, Switzer-
sources of natural antioxidants. Our objectives were to land). The dried extract obtained from each plant
evaluate the antioxidant activity and stability (pH, heat material was stored in an airtight container at 4 C until
and storage) of these extracts. further use.

2. Materials and methods 2.3. Determination of antioxidant activity

Selected plant materials (drumstick leaves, mint leaves (a) Oxidation system of lipid by ferrous sulphate: The oxi-
and carrot tuber) were bought in bulk, cleaned, grated (car- dation of linoleic acid was conducted according to the
rot) and dried in a hot air oven at 50 C. The dried plant method of Tamura and Yamagami (1994). An aque-
materials were ground separately and passed through a ous solution containing linoleic acid (1.5 mg/ml) was
60 mesh sieve and kept in air tight containers at 4 C until poured into a 30 ml test tube and diluted with 4.85 ml
further use. of Trizma-buffer solution (0.25 mM, pH 7.4) contain-
5,5 0 -Dithio(bis)nitrobenzoic acid (DTNB) was obtained ing 0.2% sodium dodecyl sulphate (w/v) and 0.75 mM
from Sigma Staishaim, Germany, b-carotene from Sigma potassium chloride. Trizma buffer was prepared by
chemicals Co. (St. Louis, MO, USA) and a,a 0 -Bipyridyl diluting 6.075 g of Tris(hydroxymethyl)aminometh-
from E-Merck Ltd. (Bombay, India). Tocopherol and ane and 11.184 g of potassium chloride with distilled
ascorbic acid were obtained from SD fine chemicals (Bom- water to 1 L after adjusting the pH of the solution to
bay, India). Glutathione and linoleic acid were obtained 7.4. Lipid peroxidation was initiated by adding
from Sisco Research Lab. Pvt. Ltd. (Bombay, India). 0.05 ml ferrous sulphate (20 mM). Incubation was
continued for 16 h at 37 C in the dark. The reaction
2.1. Determination of antioxidant components was stopped by adding 50 ll of 1% butylated
hydroxytoluene (BHT) in ethanol. The solution
Ascorbic acid was determined according to reduction in obtained (9.9 ml) was used for antioxidant activity
the absorbance of 2,6-dichlorophenol indophenol dye on assay.
reaction with ascorbic acid (AOAC, 1970). (b) Antioxidant activity assay: The dried plant extract
a-Tocopherol was extracted by direct saponification of (100 lg) in 100 ll of 0.5% ethanol/water was mixed
dried sample and estimated based on the formation of a with the solution (9.9 ml) mentioned above when nec-
red complex from the reaction of a ,a 0 -bipyridyl with fer- essary. a-Tocopherol was used as a standard to eval-
rous ion due to reduction of ferric ion by tocopherol uate the antioxidative activity of samples. The reacted
(Freed, 1996). solution obtained (1 ml) was used for TBA assay.
b-Carotene was separated by liquid column chromatog- (c) TBA assay: The degree of oxidation of oil was mea-
raphy, followed by measuring the absorbance of the eluate sured by thiobarbituric acid (TBA) assay described
at 450 nm against standard b-carotene (Ranganna, 1999). by Ohkawa, Ohishi, and Yagi (1979).The reacted solu-
A column of 30 · 1 cm was packed with 6 g of alumina; tion (1 ml) mentioned above was incubated with 0.2%
about 1–2 g of sodium sulphate (anhydrous) was placed (w/v) thiobarbituric acid (3 ml) and 0.05 M sulfuric
on top of the packed column. After loading the sample acid (2.5 ml) for 30 min in 95 C water bath. The solu-
on top of the column, a mixture of acetone–hexane (3:7, tion was then cooled in ice for 5 min. The coloured
v/v) was used as the developing solvent. substances were extracted with 4.0 ml of 1-butanol.
Reduced glutathione was determined based on the The absorbance of 1-butanol layer was measured at
development of a yellow compound due to reaction of 532 nm. A calibration curve was constructed by using
5,5 0 -dithio(bis)nitrobenzoic acid with compounds contain- malonaldehyde-bis-diethyl-acetal and results were
ing sulphydryl groups (Beutler & Kelly, 1963). expressed as malonaldehayde equivalents. Antioxi-
Total phenols were extracted by heating a weighed por- dant activity (AOA) was expressed as percentage inhi-
tion (50–500 mg) of dried sample with 5 ml of 1.2 M HCl in bition of lipid peroxidation relative to the control
50% aqueous methanol for 2 h at 90 C (Vinson, Hao, Su, using the following equation:
1102 S. Arabshahi-D et al. / Food Chemistry 100 (2007) 1100–1105

Absorbance of Control  Absorbance of sample the MDA equivalent value was highest for carrot extract,
AOAð%Þ ¼
Absorbance of control indicating the inability of this extract to inhibit lipid
 100. oxidation.
(d) Heat, pH and storage stability: The extracts were Antioxidant activities (AOA) of the extracts from
heated in a boiling water bath for 15 min and the selected plant materials and a-tocopherol are presented in
residual antioxidant activity was determined as previ- Fig. 1. The extracts from DL and CT exhibited good
ously described. For pH stability, antioxidant AOA in the linoleic acid peroxidation system. At a concen-
extracts were pre-incubated at pH 4 and 9 and the tration of 1.5 mg/ml, the two extracts inhibited 80–83%
residual antioxidant activity was determined. The peroxidation of linoleic acid after 16 h incubation, which
antioxidant extract from each material was divided was significantly (p 6 0.05) higher than the value obtained
into two aliquots. The first aliquot was stored in the for a-tocopherol (72%). In another system, using sunflower
dark under refrigeration (5 C) and the second one oil as a rich source of PUFA, both DL and ML extracts
was stored in the dark at room temperature (25 C). exhibited similar activities, 46% and 44%, respectively.
Antioxidant activity was determined after 15 days These results indicated the relative efficiency of extracts
for each aliquot. in inhibiting lipid oxidation.
The AOA of the three extracts was found to vary
with temperature (Fig. 2). Heating at 100 C for 15 min
3. Statistical analysis increased (p 6 0.05) the antioxidant potency of ML extract
by 15%. Perhaps heat processing improved the bioavailabil-
Data were recorded as means ± standard deviation of ity of the antioxidants present in ML extract. Incubating
duplicate measurements. Analyses of variance were per- DL extract at 100C for 15 min resulted in a significant
formed by ANOVA test and significance differences decrease (p 6 0.05) in AOA by 17%. Heat processing may
between the means were determined by Duncan’s New have resulted in degradation of antioxidants present in
Multiple Range Test (Steele & Torrie, 1980). DL extract, thereby decreasing the activity. However, heat

4. Results Table 2
Antioxidant activity of plant extracts in different lipid systems
The analyzed antioxidant compounds in dried powder Lipid system Antioxidant activity (mmol MDA equivalents/kg oil)
of drumstick leaves (DL), mint leaves (ML) and carrot DLa MLa CTa
tuber (CT) are shown in Table 1. Among the samples b a
Linoleic acid 0.12 ± 0.02 0.23 ± 0.03 0.14 ± 0.03b
examined, DL was a rich source of ascorbic acid. All the Sunflower oil 7.63 ± 0.53b 7.86 ± 0.65b 12.02 ± 0.86a
three samples were found to have appreciable amount of
Values bearing different superscripts a, b in rows (comparison between
a-tocopherol, b-carotene and polyphenols. The total samples) differ significantly (p 6 0.05).
amount of phenolics (gallic acid equivalents) ranged from a
DL, drumstick leaves; ML, mint leaves; CT, carrot tuber.
1.68 g% in dehydrated CT-powder to 4.5 g% in dehydrated
DL-powder. The estimated value for drumstick is in good
agreement with reported data by Siddhuraju and Becker linoleic acid sunflower oil
100
(2003). a a
A comparison of secondary products of lipid peroxida-
tion measured as malonaldehayde (MDA) equivalents is 80
Antioxidant activity (%)*

b c
shown in Table 2. The MDA equivalent values were
found to be lower in linoleic acid compared to sunflower a'
oil. In a system comprising of sunflower oil as substrate, 60
b' b'

Table 1 40
Contents of ascorbic acid, tocopherol, b-carotene, glutathione and total
phenols in selected plant materials
Compound DLa MLa CTa 20 c'
b a b b
Ascorbic acid (mg) 431.30 ± 15.34 40.25 ± 1.54 20.0 ± 0.81
a-Tocopherol (mg)b 24.65 ± 0.21a 18.70 ± 0.56b 3.98 ± 0.21c
0
b-Carotene (lg)b 14,412 ± 898a 12,180 ± 668a 9667 ± 627b
Glutathione (mmol)b 129.53 ± 1.91a 70.6 ± 0.82b 129.53 ± 1.35a Std DL ML CT
Total phenols (g)b 4.50 ± 0.25a 4.30 ± 0.29a 1.68 ± 0.12b Fig. 1. Antioxidant activity of extracts in different lipid substrates: Std, a-
Values bearing different superscripts a, b in rows (comparison between Tocopherol; DL, drumstick leaves; ML, mint leaves; CT, carrot tuber. a,
samples) differ significantly (p 6 0.05). b, c: Values with the same letter in the same lipid system are not
a
DL, drumstick leaves; ML, mint leaves; CT, carrot tuber. significantly different (p 6 0.05). * Percentage inhibition of lipid peroxida-
b
Per 100 g dry basis. tion relative to control.
 S. Arabshahi-D et al. / Food Chemistry 100 (2007) 1100–1105 1103

No treatment Heat treatment Day 0 Day 15, ~5˚C Day 15, ~25˚C
100 1.8

a a' a'' a''

90 a" 1.6
a''
a a'

mmol MDA equivalents/ kg oil

a" a'
1.4
80 a
b'
a a'
Antioxidant activity (%)*

b 1.2
70
1
60
0.8
50
0.6
40
0.4
30
0.2
20
0
10 DL ML CT

0 Fig. 4. Effect of storage on antioxidant activity of extracts in sunflower

DL ML CT oil: DL, drumstick leaves; ML, mint leaves; CT, carrot tuber. Values with
the same letter are not significantly different (p 6 0.05).
Fig. 2. Effect of heat treatment (100 C, 15 min) on antioxidant activity
of extracts in linoleic acid: DL, drumstick leaves; ML, mint leaves; CT,
carrot tuber. a, b: Values with the same letter are not significantly
different (p 6 0.05). * Percentage inhibition of lipid peroxidation relative 5. Discussion
to control.

In the present study, three plant materials, namely

drumstick leaves, mint leaves and carrot tuber, were used
treatment did not make a significant (p 6 0.05) change in as sources of natural antioxidants. Samples were subjected
AOA of CT extract indicating stability of this extract. to oven drying (50 C) before solvent extraction as it was
The AOA of extracts varied with pH (Fig. 3). Results observed that controlled oven drying of the selected sam-
indicate that the activity of ML and CT extracts was higher ples resulted in maximum retention of antioxidant nutri-
at pH 9 than at pH 4, while that of extract from DL was ents such as ascorbic acid, a-tocopherol, b-carotene and
unchanged in both alkaline and acid media. glutathione (Reddy, Urooj, & Kumar, 2005; Vishalakshi,
The three extracts stored in the dark at 5 and 25 C after 2003).
15 days did not show any significant change (p 6 0.05) in Under the experimental conditions described, the three
their AOA (Fig. 4). Results indicate that the extracts stored selected samples exhibited antioxidant activity. The ability
at 25 C, were quite stable compared to those stored at to inhibit lipid oxidation differed according to the type of
5 C, even after 15 days. vegetable extract, oxidizable substrate used, thermal and
pH treatment to which the extracts were subjected. In an
earlier study, the AOA of both DL leaves extract and pow-
pH 4.0 pH 9.0 der was evaluated using a linoleic acid–b-carotene system
0.9
(Reddy et al., 2005). It was interesting to note that the
0.8 a"
AOA of extract was 98% and that of dehydrated powder
mmol MDA equivalents/ kg oil

a a' was 87% compared to the AOA of synthetic antioxidants

0.7
a b" butylated hydroxyanisole (85%) and butylated hydroxy tol-
0.6 uene (92%). Various solvent extracts of DL from different
0.5 b'
agroclimatic regions are reported to exhibit marked antiox-
idant activity (Siddhuraju & Becker, 2003). Also, few
0.4 reports are available on AOA of different extracts from
0.3
M. spicata (Dorman, Kosar, Kahlos, Holm, & Hiltunen,
2003; Marinova & Yanishlieva, 1997; Tarwadi & Agte,
0.2 2003; Triantaphyllou, Blekas, & Boskou, 2001), carrot
0.1
puree (Talcott, Howard, & Brenes, 2000) and carrot juice
(Gazzani, Papetti, Massolini, & Daglia, 1998). However,
0 the effect of processing such as heat, pH and storage on
DL ML CT the AOA of plant extracts studied has not been reported
Fig. 3. Effect of pH on antioxidant activity of extracts in linoleic acid: DL, so far.
drumstick leaves; ML, mint leaves; CT, carrot tuber. a, b: Values with the It is well known that many factors such as antioxi-
same letter are not significantly different (p 6 0.05). dant concentration, temperature and pH of the media,
1104 S. Arabshahi-D et al. / Food Chemistry 100 (2007) 1100–1105

processing treatment and storage strongly influence the 6. Conclusions

antioxidant activity (Gazzani et al., 1998). In the present
study, the three extracts were subjected to heat treatment In the present study it was found that drumstick, mint
at 100 C (15 min), which resulted in a significant and carrot are potential sources of antioxidant compo-
decrease in AOA in DL extract while no difference was nents. They exhibit potent antioxidant activity in different
observed in AOA of CT extract. The decrease in AOA lipid systems. The antioxidant activity of extracts varied
of DL extract might be due to the loss of naturally with pH, heat treatment and storage. These findings con-
occurring antioxidants present in the extract or forma- firm that anti- and prooxidant properties of vegetables are
tion of novel compounds having prooxidant activity strongly influenced by a number of processing factors and
upon heat processing. However, a significant increase in by the reaction conditions. Therefore, it is important to
AOA due to thermal processing occurred in ML extract. consider the optimum technological condition and pro-
Thermal processing can induce the formation of com- cessing factors influencing activity and bioavailability of
pounds with antioxidant properties or improve the plant antioxidants for utilization in food and biological
AOA of naturally occurring antioxidants (Nicoli et al., systems. In addition to being consumed as healthy antiox-
1999). The AOA of a number of vegetable juices is idants, the compounds present in these plants that are
reported to be stabilized by boiling, suggesting that the responsible for antioxidant activity could be isolated
initial prooxidant activity is due to prooxidases which and then used as food additives to delay the oxidative
are inactivated at high temperatures (Gazzani et al., deterioration of foods. Further studies in this area are
1998). It is reported that crucifer extracts exhibit either in progress.
a prooxidant or an antioxidant activity depending on
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