Food Chemistry 122 (2010) 1205–1211

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Food Chemistry
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Evaluation of antioxidant activities of various solvent extracts of Carissa opaca fruits

Sumaira Sahreen, Muhammad Rashid Khan *, Rahmat Ali Khan
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 4400, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: The chloroform and aqueous fractions of Carissa opaca fruit, a traditional medicinal fruit in Pakistan pos-
Received 3 December 2009 sessed a high amount of total phenolic and flavonoid contents as compare to other solvent fractions with
Received in revised form 1 February 2010 potent antioxidant activities in scavenging DPPH, superoxides, hydroxyl, hydrogen peroxide, ABTS radi-
Accepted 30 March 2010
cals, and had strong iron chelating activity. On the other hand, the ethyl acetate fraction showed the high-
est inhibition of b-carotene linoleic acid peroxidation and phosphomolybdate assay. A high correlation
coefficient existed between EC50 values of DPPH, superoxides, hydroxyl, hydrogen peroxide, ABTS radi-
Keywords:
cals, total phenolics and flavonoids, but a non significant correlation was found in the case of iron chelat-
Carissa opaca
Antioxidant activity
ers, b-carotene and phosphomolybdate assay. This study verified that the chloroform and aqueous
Solvent extraction fractions have strong antioxidant activities which were correlated with its high level of phenolics and
Phenolics flavonoids. These fractions can be used as a source of potential antioxidant or functional food material.
DPPH Ó 2010 Elsevier Ltd. All rights reserved.
Fruit

1. Introduction and degenerative diseases is continuously advancing. In fact, the

Free radicals, chemical reactions, and several redox reactions of
various compounds may cause protein oxidation, DNA damage,
and lipid peroxidation in living cells (Morissey & O’Brien, 1998). In-
creased consumption of whole grains, fruits, and vegetables is re-
lated to a reduced risk of chronic diseases, diabetes, cancer,
cardiovascular and neurodegenerative diseases such as Alzhei-
mer’s disease (Kaur & Kapoor, 2001). Different fruits exhibit differ-
ent antioxidant capacities according to their polyphenol content,
vitamin C, E, carotenoids and flavonoids (Saura-Calixto & Goni,
2006).
Polyphenols are common constituents of the human diet, with
fruits and vegetables being the major dietary source of these bioac-
tive compounds. The possible health benefits of polyphenol con-
sumption have been suggested to derive from their antioxidant
properties Evidence for their role in the prevention of degenerative
diseases is emerging. There are huge varieties of antioxidants con-
tained in fruits. Several methods have been developed to estimate
the antioxidant capacity of different plant materials (Guo et al.,
2003). Phenolic compounds are ubiquitous in plants, and when
plant foods are consumed, these phytochemicals contribute to
the intake of natural antioxidants in the human diets and has to
be resolved to obtain the optimum antioxidant efficiency.
The scientific basis for the statement that plants and their active
constituents play an important role in the prevention of chronic
* Corresponding author. Tel.: +92 51 90643086; fax: +92 51 9205753.
E-mail address: mrkhanqau@yahoo.com (M.R. Khan).
origin of many therapeutic substances is due to secondary metab-
olism in the plant (Maganha et al., 2010).
Carissa opaca is an evergreen shrub which can grow up to 3.5 m
tall, branches glabrous or puberulous, leaves glabrous, opposite
and ovate, spines arising between the petiole, hard and sharp,
2.5–3.5 cm long. Flowers white, corolla tube slender 8–12 mm
long. Fruit berry some what ellipsoid, dark purple when ripe, with
milky juice, edible. The plant is distributed in drier parts of India
and Pakistan (from Punjab-Himalayas up to 6000 ft, in Murree)
Burma and Sri Lanka (Nazimuddin & Qaiser, 1983). The whole
plant is commonly used as a treatment in worm infested sores of
animals, fly repellent, stimulants and in asthma (Jabeen, Khan,
Ahmad, Zafar, & Ahmad, 2009). Researchers have reported that
fruits and leaves are used for the treatment of jaundice and hepa-
titis (Abbasi et al., 2009). Adhikari, Babu, Saklani, and Rawat (2007)
reported that the fruit is a purgative, used in fever cough and
diarrhea.
Recently, researchers have sought to isolate powerful and non-
toxic natural antioxidants from edible plants not only to prevent
autoxidation and lipid peroxidation, but also to replace synthetic
antioxidants. The fruit of C. opaca has been more often used as a
food than for medicinal purpose, which attracted our curiosity to
investigate the antioxidant activity of the fruit. To our knowledge,
there is no report in the literature on the antioxidant potential of C.
opaca fruit. The main objectives of this study were (i) to determine
the phenolic content and antioxidant capacity of fruit and (ii) to
examine the efficiency of different solvents for the extraction of
phenolics.

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.03.120
1206 S. Sahreen et al. / Food Chemistry 122 (2010) 1205–1211
2. Materials and methods
2.1. Fruit collection
The fruit was collected in May 2009 from Islamabad, Pakistan
and the plant was identified by its vernacular name and later val-
idated by Dr. Mir Ajab Khan, Department of Botany, Quaid-i-Azam
University, Islamabad. A voucher specimen was deposited at the
Herbarium of Pakistan Museum of Natural History, Islamabad.
2.2. Extract preparation
The fruits (1.0 kg fresh) of uniform size (7–8 mm long) at matu-
rity were collected and dried under shade to obtain 530 g dry sam-
ple. The dried samples were powdered in a Willy Mill to 60-mesh
size and used for solvent extraction. For extract preparation, 500 g
of dried sample was extracted twice with 2 l of 95% methanol at
25 °C for 48 h. The extracts were filtrated through Whatman No.
1 and combined followed by concentration using a rotary evapora-
tor (Panchun Scientific Co., Kaohsiung, Taiwan) under reduced
pressure at 40 °C. The filtrate obtained was suspended in water
(50 ml) and n-hexane (100 ml) was added, shaked well and the lay-
ers were allowed to separate for 6 h in a separating funnel. N-hex-
ane layer was separated and again n-hexane (100 ml) was added to
obtain the n-hexane fraction. Similarly, the protocol was repeated
with ethyl acetate and the remaining solvents i.e., chloroform,
butanol and water. Each of the fractions obtained were dried using
a rotary evaporator (Kil et al., 2009). The dry extract obtained with
each solvent was weighed. The percentage yield was expressed in
terms of air dried weight of plant material.
2.3. Chemicals
Ascorbic acid, aluminium chloride, 2,2-azino-bis-(3-ethylbenzo-
thiazoline-6-sulphonic acid) (ABTS), Ferric chloride (FeCl3), Tween
80, b-carotene, (+)-catechin, Potassium persulphate, Folin–Ciocal-
teu’s phenol reagent, ferrozine, gallic acid, rutin, linoleic acid, 2,2-
diphenyl-1-picrylhydrazyl (DPPH), nitro blue tetrazolium (NBT),
phenazine methosulphate (PMS), thiobarbituric acid (TBA) and tri-
chloroacetic acid (TCA) were purchased from Sigma Co. (St. Louis,
MO, USA). Sulphuric acid, deoxyribose, riboflavin, sodium carbon-
ate (Na2CO3), sodium hydroxide (NaOH), sodium nitrite (NaNO2),
disodium hydrogen phosphate (Na2HPO4) and hydrogen peroxide
(H2O2) were obtained from Wako Co. (Osaka, Japan). Ferrous chlo-
ride (FeCl2), potassium ferricyanide (K3Fe(CN)6), sodium dihydro-
gen phosphate (NaH2PO4) and all solvents used were of analytical
grade were purchased from Merck Co. (Darmstadt, Germany). Dis-
tilled deionized water (dd. H2O) was prepared by Ultrapure TM
water purification system (Lotun Co., Ltd., Taipei, Taiwan).
2.4. Determination of total phenolics
The total phenolics were determined by the spectrophotometric
method (Kim, Jeong, & Lee, 2003). In brief, a 1 ml portion (dissolved
in methanol) of appropriately diluted extracts, was added to 9 ml
of deionised distilled water. Deionised distilled water was used
as blank. 1 ml of Folin–Ciocalteu’s phenol reagent was added to
the mixture and then shaken. After 5 min, 10 ml of a 7% Na2CO3
solution was added with mixing. The mixed solution was then
immediately diluted to volume (25 ml) with deionised distilled
water and mixed thoroughly. After 90 min at 23 °C, the absorbance
was read at 750 nm. The standard curve for total phenolics was
made using gallic acid standard solution (0–100 mg/l) under the
same procedure as above. The total phenolics were expressed as
milligrams of gallic acid equivalents (GAE) per g of dried sample.
2.5. Determination of total flavonoids
Total flavonoid content was determined following Park et al.
(2008). Fifty milligram of each fraction was dissolved in 10 ml of
80% aqueous methanol and filtered through Whatman filter paper
No. 42 (125 mm). In a 10 ml test tube, 0.3 ml of extracts, 3.4 ml of
30% methanol, 0.15 ml of 0.5 M NaNO2 and 0.15 ml of 0.3 M
AlCl36H2O were added and mixed. After 5 min, 1 ml of 1 M NaOH
was added. The mixture was measured at 506 nm. The standard
curve for total flavonoids was made using rutin standard solution
(0–100 mg/l) under the same procedure as above. The total flavo-
noids were expressed as milligrams of rutin equivalents per gram
of dried sample.
2.6. Antioxidant assays
Each sample was dissolved in 95% methanol at a concentra-
tion1 mg/ml and then diluted to prepare the series concentrations
for antioxidant assays. Reference chemicals were used for compar-
ison in all assays.
2.6.1. DPPH radical scavenging activity assay
The DPPH assay was done according to the method of Brand-
Williams, Cuvelier, and Berset (1995) with some modifications.
The stock solution was prepared by dissolving 24 mg DPPH with
100 ml methanol and then stored at 20 °C until needed. The work-
ing solution was obtained by diluting DPPH solution with metha-
nol to obtain an absorbance of about 0.980 (±0.02) at 517 nm
using the spectrophotometer. A 3 ml aliquot of this solution was
mixed with 100 ll of the fractions at varying concentrations (25–
250 lg/ml). The solution in the test tubes were shaken well and
incubated in the dark for 15 min at room temperature. Then the
absorbance was taken at 517 nm. The scavenging activity was esti-
mated based on the percentage of DPPH radical scavenged using
the following equation:
Scavenging effect ð%Þ
¼ ½ðcontrol absorbance  sample absorbanceÞ
=ðcontrol absorbanceÞ  100:
EC50 value is the effective concentration that could scavenge 50% of
the DPPH radicals. Ascorbic acid and rutin standard were used as
positive controls.
2.6.2. Superoxide anion radical scavenging assay
The assay for superoxide anion radical scavenging activity was
based on a riboflavin-light-NBT system (Beauchamp & Fridovich,
1971). The reaction mixture contained 0.5 ml of phosphate buffer
(50 mM, pH 7.6), 0.3 ml riboflavin (50 mM), 0.25 ml PMS (20 mM),
and 0.1 ml NBT (0.5 mM), prior to the addition of 1 ml sample solu-
tion at varying concentrations (25–250 lg/ml). Reaction was
started by illuminating the reaction mixture with different concen-
trations of the methanolic extracts using a fluorescent lamp. After
20 min of incubation, the absorbance was measured at 560 nm.
Ascorbic acid was used as standard. The percent inhibition of super-
oxide anion generation was calculated using the following formula:
Scavenging activity ð%Þ
¼ ð1  absorbance of sample=absorbance of controlÞ  100:
2.6.3. Phosphomolybdate assay
The antioxidant activity of samples was evaluated by the
phosphomolybdenum method according to the procedure of
Umamaheswari and Chatterjee (2008). An aliquot of 0.1 ml of sam-
 S. Sahreen et al. / Food Chemistry 122 (2010) 1205–1211
ple solution was mixed with 1 ml of reagent solution (0.6 M sul-
phuric acid, 28 mM sodium phosphate and 4 mM ammonium
molybdate). The test tubes were capped with silver foil and incu-
bated in a water bath at 95 °C for 90 min. After the samples had
cooled to room temperature, the absorbance of the mixture was
measured at 765 nm against a blank. Ascorbic acid was used as
standard. The antioxidant capacity was estimated using the follow-
ing formula:
Antioxidant effect ð%Þ
¼ ½ðcontrol absorbance  sample absorbanceÞ
=ðcontrol absorbanceÞ  100:
2.6.4. Hydroxyl radical scavenging assay
Hydroxyl radical scavenging activity of extracts was assayed by
the method of Halliwell and Gutteridge (1981). The reaction mix-
ture contained 500 ml of 2-deoxyribose (2.8 mM) in phosphate buf-
fer (50 mM, pH 7.4), 200 ml of premixed ferric chloride (100 mM)
and EDTA (100 mM) solution (1:1; v/v), 100 ml of H2O2 (200 mM)
without or with the extract solution (100 ml). The reaction was trig-
gered by adding 100 ml of 300 mM ascorbate and incubated for 1 h
at 37 °C. A solution of TBA in 1 ml (1%; w/v) of 50 mM NaOH and
1 ml of 2.8% (w/v; aqueous solution) TCA was added. The mixture
was heated for 15 min on a boiling water bath and then cooled.
The absorbance was measured at 532 nm. The scavenging activity
of the hydroxyl radical was calculated as follows:
Scavenging activity ð%Þ
¼ ð1  absorbance of sample=absorbance of controlÞ  100:
2.6.5. Hydrogen peroxide-scavenging activity
The ability of the extracts to scavenge hydrogen peroxide was
determined according to the method of Ruch, Cheng, and Klaunig
(1989). A solution of hydrogen peroxide (2 mM) was prepared in
50 mM phosphate buffer (pH 7.4). Aliquots (0.1 ml) of the ex-
tracted sample were transferred into the test tubes and their vol-
umes were made up to 0.4 ml with 50 mM phosphate buffer (pH
7.4). After addition of 0.6 ml hydrogen peroxide solution, tubes
were vortexed and absorbance of the hydrogen peroxide at
230 nm was determined after 10 min, against a blank. The abilities
to scavenge the hydrogen peroxide were calculated using the fol-
lowing equation:
Hydrogen peroxide scavenging activity
¼ ð1  absorbance of sample=absorbance of controlÞ  100:
2.6.6. ABTS radical cation scavenging activity
The ABTS radical cation scavenging activity was performed with
slight modifications (Re et al., 1999). Briefly, ABTS solution (7 mM)
was reacted with potassium persulfate (2.45 mM) solution and
kept overnight in the dark to yield a dark coloured solution con-
taining ABTS radical cation. Prior to use in the assay, the ABTS rad-
ical cation was diluted with 50% methanol for an initial absorbance
of about 0.700 (±0.02) at 745 nm, with the temperature control set
at 30 °C. Free radical scavenging activity was assessed by mixing
300 ll of test sample with 3.0 ml of ABTS working standard in a
microcuvette. The decrease in absorbance was measured exactly
1 min after mixing the solution, then up to 6 min. The final absor-
bance was noted. The percentage inhibition was calculated accord-
ing to the formula:
Scavenging effect ð%Þ
¼ ½ðcontrol absorbance  sample absorbanceÞ
=ðcontrol absorbanceÞ  100:
The antioxidant capacity of test samples was expressed as EC50, the
concentration necessary for a 50% reduction of ABTS.
1207
2.6.7. b-Carotene bleaching assay
The assay was performed as given by Elzaawely, Xuan, Koyama,
and Tawata (2007) and modified slightly. First, 2 mg of b-carotene
dissolved in 10 ml of chloroform was mixed with 20 mg of linoleic
acid and 200 mg of Tween80 followed by removal of chloroform un-
der nitrogen and the addition of 50 ml of distilled water with vigor-
ous shacking to prepare the b-carotene linoleate emulsion. An
aliquot of each sample (30 ll) was mixed with 250 ll of the emul-
sion, and then the absorbance was determined at 470 nm at 45 °C
for 2 h. b-Carotene bleaching inhibition was estimated using the fol-
lowing equation:
Bleaching inhibition ð%Þ
¼ ðb-carotene content after2 h of assay
=initial b-carotene contentÞ  100:
The EC50 value is the sample concentration that gives 50% anti-
oxidant ability. Catechin standard was used as the positive control.
2.6.8. Chelating power
The ability of the extract to chelate iron (II) was estimated
according to the method of Dastmalchi et al. (2008). The extracts
were dissolved with methanol to prepare various sample solutions
at 8, 6, 4, 2, 1 mg/ml and 500, 250 lg/ml. An aliquot of each sample
(200 ll) was mixed with 100 ll of FeCl22H2O (2.0 mM/l) and
900 ll of MeOH. After 5 min incubation, the reaction was initiated
by the addition of 400 ll of ferrozine (5.0 mM/l). After 10 min incu-
bation, the absorbance at 562 nm was recorded. The chelating
activity (%) was calculated using the following equation:
Chelating activity ð%Þ
¼ ½ðcontrol absorbance  sample absorbanceÞ
=ðcontrol absorbanceÞ  100:
EC50 value is the effective concentration that chelates 50% of iron
(II). Catechin was used as the positive control.
2.6.9. Reducing power assay
The reducing power was determined according to the method of
Oyaizu (1986). Extract solution (2 ml), phosphate buffer (2 ml,
0.2 M, pH 6.6) and potassium ferricyanide (2 ml, 10 mg/ml) were
mixed, and then incubated at 50 °C for 20 min. Trichloroacetic acid
(2 ml, 100 mg/l) was added to the mixture. A volume of 2 ml from
each of the above mixtures was mixed with 2 ml of distilled water
and 0.4 ml of 0.1% (w/v) ferric chloride in a test tube. After 10-min
reaction, the absorbance was measured at 700 nm. Increased absor-
bance of the reaction mixture indicated a high reducing power.
2.7. Statistical analysis
All assays were carried out in triplicates and results are ex-
pressed as mean ± SD. ANOVA test was used to analyze the differ-
ences among EC50 of various fractions for different antioxidant
assays with least significance difference (LSD) P < 0.01 as a level
of significance. Experimental results were further analyzed for
Pearson correlation coefficient of phenolic, flavonoids with differ-
ent antioxidant assays and tested for significance by Student’s t-
test (P < 0.05; P < 0.01). The EC50 values were calculated using the
Graph Pad Prism 5 software.
3. Results and discussion
3.1. Extraction yield, total phenolics and flavonoid contents
The percentage yields of the methanol extract of fruit of C. opaca
and its different fractions are shown in Table 1. The extraction
1208 S. Sahreen et al. / Food Chemistry 122 (2010) 1205–1211

Table 1 cfc
Total phenolics, flavonoid and extraction yield of methanol extract and soluble
hfc efc bfc
fractions of Carissa opaca fruit.
mfc afc asa rt
Plant Total phenolics Total flavonoids Extraction
extracts (mg gallic acid (mg rutin yield (%) 100
equivalent/g) equivalent/g)

n-Hexane 25.8 ± 2.8a 6.4 ± 0.43a 4.5 ± 0.5a
fraction
Ethyl 32.3 ± 5.6b 14.2 ± 1.2b 5.7 ± 0.43a
acetate
fraction
Chloroform 62.7 ± 2.1d 38.3 ± 2.8e 9.2 ± 0.28b
fraction
Butanol 46.2 ± 2.2c 21.6 ± 2.9c 4.9 ± 1.11a
fraction
Methanol 78.9 ± 1.7e 32.5 ± 1.2d 12.5 ± 1.45c
extract
Aqueous 58.4 ± 3.4d 33.4 ± 2.8d 10.7 ± 0.99b
fraction
Each value in the table is represented as mean ± SD (n = 3).
Means not sharing the same letter are significantly different (LSD) at P < 0.01
probability level in each column.
yield of these samples varied from 4.5 ± 0.5% to 12.5 ± 1.45% with a
descending order of methanol > aqueous > chloroform > ethyl ace-
tate > butanol > n-hexane fraction. So the extractions with metha-
nol, water and chloroform resulted in the highest amount of total
extractable compounds, whereas the extraction yield with ethyl
acetate, butanol and n-hexane was significantly less (P < 0.01) in
comparison with that of the other solvents. Phenolics or polyphe-
nols are plant secondary metabolites and are very important by
virtue of their antioxidant activity by chelating redox-active metal
ions, inactivating lipid free radical chains and preventing hydro-
peroxide conversions into reactive oxyradicals. Table 1 summa-
rizes the total phenolic compounds in fractions expressed as
gallic acid equivalents (GAE), varied between 25.8 ± 2.8 mg and
78.9 ± 1.7 mg/g dry weight of fraction. The methanolic extract
exhibited the highest total phenolics content (78.9 ± 1.7 mg gallic
acid equivalent/g fraction), whereas the contents obtained with
n-hexane were much smaller (P < 0.01) (25.8 ± 2.8 mg gallic acid
equivalent/g), which is in agreement with the report of Ao, Li,
Elzaawely, Xuan, and Twata (2008). The content of total flavonoids
expressed as rutin equivalents, varied from 6.4 ± 0.43 to 38.3 ± 2.8
mg as rutin equivalent/g fraction. These amounts were comparable
with results described in the literature for other extracts of plant
products (Ao et al. 2008; Manian, Anusuya, Siddhuraju, & Manian,
2008). The rich-flavonoid plants could be a good source of antiox-
idants that would help to increase the overall antioxidant capacity
of an organism and protect it against lipid peroxidation (Sharififar,
Dehghn-Nudeh, & Mirtajaldini, 2009). Phenolic acids and flavo-
noids have been reported to be the main phytochemicals responsi-
ble for the antioxidant capacity of fruits and vegetables. Plant
derived polyphenols display characteristic inhibitory patterns to-
ward the oxidative reaction in vitro and in vivo (Bahramikia, Arde-
stani, & Yazdanparast, 2009).
3.2. DPPH radical scavenging activity
Fig. 1 shows that the DPPH radical scavenging ability of samples
can be ranked as chloroform > aqueous > methanol > buta-
nol > ethyl acetate > n-hexane fractions. The observed differential
scavenging activities of the extracts against the DPPH system could
be due to the presence of different compounds in the fractions. The
EC50 values of scavenging DPPH radicals for the chloroform and
aqueous fractions were 54.13 ± 2.3 and 59.56 ± 0.9 lg/ml, respec-
tively, while the n-hexane fraction had an EC50 of >>250 lg/ml
(Table 2). Though the DPPH radical scavenging activities of the
DPPH % scavanging
80
60
40
20
0
0 50 100 150 200 250 300
Concentration (µg/ml)
Fig. 1. DPPH radical scavenging activity of different extracts from the methanol
extract of Carissa opaca fruit by different solvents at different concentrations. Each
value represents a mean ± SD (n = 3). –d–, n-hexane fraction; –j–, ethyl acetate
fraction; –N–, chloroform fraction; –.–, butanol fraction, ––, methanol fraction;
, aqueous fraction, , ascorbic acid; , rutin.
fractions were less (P < 0.01) than those of ascorbic acid and rutin,
the study revealed that chloroform and aqueous fractions has free
radical scavengers or inhibitors, acting possibly as primary
antioxidants.
3.3. Superoxide radical scavenging activity
Superoxide is a reactive oxygen species, which can cause dam-
age to the cells and DNA leading to various diseases. It was, there-
fore, proposed to measure the comparative interceptive ability of
the antioxidant extracts to scavenge the superoxide radical (Vani,
Rajani, Sarkar, & Shishoo, 1997). The superoxide radical scavenging
effect of different fractions of methanol extract of fruit of C. opaca
was compared with the same doses of ascorbic acid ranging from
25 to 250 lg/ml as shown in Fig. 2. EC50 values in superoxide scav-
enging activities were in the order of aqueous > chloro-
form > methanol > butanol > ethyl acetate > n-hexane fraction
(Table 2). All of the fractions had a scavenging activity on the
superoxide radicals in a dose dependent manner. Nonetheless,
when compared to ascorbic acid, the superoxide scavenging activ-
ity of the extract was found to be low (P < 0.01). This could be due
to the presence of flavonoids and other antioxidants in the extract.
3.4. Phosphomolybdate assay
The phosphomolybdate method has been routinely used to
evaluate the antioxidant capacity of extracts (Prieto, Pineda, &
Aguliar, 1999). In the presence of extracts, Mo (VI) is reduced to
Mo (V) and forms a green coloured phosphomolybdenum V com-
plex, which shows a maximum absorbance at 700 nm. The anti-
oxidant capacity of different fractions of fruits of C. opaca can
be ranked in the order of ethyl acetate > chloroform > aque-
ous > methanol > butanol > n-hexane fraction. The EC50 values of
the antioxidant capacity for the ethyl acetate and chloroform
fractions were 86.34 ± 3.4 and 168.05 ± 3.2 lg/ml, respectively,
while for the n-hexane fraction it was > 500 lg/ml (Table 2).
However, the scavenging activity of all the extracts were found
to be much low (P < 0.01) when compared to ascorbic acid. The
results obtained imply that the ethyl acetate fraction has a strong
ability (P < 0.01) to act as antioxidant as compared to other
fractions.
 S. Sahreen et al. / Food Chemistry 122 (2010) 1205–1211 1209

Table 2
Antioxidant effect (EC50) on DPPH radicals, superoxide radicals, total antioxidant capacity and hydroxyl radicals of methanol extract and soluble fractions of Carissa opaca fruit.

Plant EC50 lg/ml

extracts
Scavenging Scavenging Phospho Scavenging ability Scavenging ability Scavenging b-carotene Chelating
ability on DPPH ability on super molybdate on hydroxyl on hydrogen ability on ABTS bleaching power
radicals oxide assay radicals peroxide radicals inhibition
n-Hexane >>250f >250e >500f >>250f >>250h >>500f >8000f 42.35 ± 3.1d
fraction
Ethyle >250e >250e 86.34 ± 3.4b >250e >250g >500e 233.89 ± 4.3b 46.39 ± 1.9d
acetate
fraction
Chloroform 54.13 ± 2.3b 48.23 ± 5.6b 168.05 ± 3.2c 36.24 ± 1.1b 47.28 ± 0.7c 80.49 ± 1.1b 1007.41 ± 8.6 27.48 ± 0.5b,c
fraction
Butanol 90.23 ± 0.7d 84.78 ± 4.8d 174.28 ± 4.8c 82.34 ± 0.6d 133.22 ± 2.0f 140.26 ± 2.2d 867.71 ± 5.8e 30.42 ± 0.3b,c
fraction
Methanol 77.76 ± 1.2c 53.27 ± 2.6c 228.46 ± 3.5e 60.17 ± 1.4c 86.31 ± 1.9e 122.31 ± 2.8c 433.45 ± 4.9c 34.21 ± 0.9c
fraction
Aqueous 59.56 ± 0.9b 33.43 ± 2.1b 206.18 ± 2.4d 59.72 ± 0.9c 61.21 ± 1.3d 74.14 ± 0.8b 733.90 ± 7.6d 25.14 ± 0.2a,b
fraction
Ascorbic 19.59 ± 0.8a 21.86 ± 0.6a 24.09 ± 2.4a 29.78 ± 1.1a 23.04 ± 0.7a 64.63 ± 1.5a – –
acid
a
Catechin – – – – – – 133.81 ± 2.8 21.74 ± 0.5a
Rutin 19.31 ± 0.7a – – – 29.04 ± 1.5b – – –

Each value in the table is represented as mean ± SD (n = 3).

Means not sharing the same letter are significantly different (LSD) at P < 0.01 probability level in each column.

3.6. Hydrogen peroxide radical scavenging activity

hfc efc cfc bfc
Hydrogen peroxide itself is not very reactive, but it can some-
mfc afc asa times be toxic to cells, since it may give rise to hydroxyl radicals
100 inside the cell (Halliwell, 1991). Extracts from C. opaca fruits were
capable of scavenging hydrogen peroxide in a concentration-
Superoxide % scavanging

dependent manner (25–250 lg/ml). As compared with the EC50

80
values, the hydrogen peroxide- scavenging activities of chloroform
(47.28 ± 0.7 lg/ml) and aqueous (61.21 ± 1.3 lg/ml) fractions were
60 more (P < 0.01) effective (P < 0.01) than that of ethyl acetate
(>250 lg/ml) and n-hexane(>>250 lg/ml) fractions. EC50 values
40 of all the extracts, in scavenging abilities on hydrogen peroxide,
were significantly different (P < 0.05) from the EC50 values ob-
20 tained for ascorbic acid (Table 2). The scavenging abilities on
hydrogen peroxide were in descending order of chloroform > aque-
0 ous > methanol > butanol > ethyl acetate > n-hexane fraction.
0 50 100 150 200 250 300
Concentration (µg/ml) 3.7. ABTS radical scavenging activity

Fig. 2. Super oxide radical scavenging activity of different extracts from the
methanol extract of Carissa opaca fruit by different solvents at different concen-
trations. Each value represents a mean ± SD (n = 3). –d–, n-hexane fraction; –j–,
ethyl acetate frcation; –N–, chloroform fraction; –.–, butanol fraction; ––,
methanol fraction; , aqueous fraction; , ascorbic acid.
3.5. Hydroxyl radical scavenging activity
Hydroxyl radical scavenging capacity of an extract is directly re-
lated to its antioxidant activity (Babu, Shylesh, & Padikkala, 2001).
The hydroxyl radical scavenging activity can be ranked as chloro-
form > aqueous > methanol > butanol > ethyl acetate > n-hexane
fraction. All results showed antioxidant activity in a dose depen-
dent manner at concentrations of 25–250 lg/ml. The EC50 values
of scavenging hydroxyl radicals for the chloroform fraction was
36.24 ± 1.1 lg/ml, while for the n-hexane fraction it was >250 lg/
ml (Table 2). The ability of extracts/fractions to quench hydroxyl
radicals seems to be directly related to the prevention of propaga-
tion of the process of lipid peroxidation and they seem to be good
scavengers of active oxygen species, thus reducing the rate of
reaction.
ABTS radical scavenging ability of samples can be ranked as
aqueous > chloroform > methanol > butanol > ethyl acetate > n-
hexane fraction. The results obtained clearly imply that all the
tested samples inhibit or scavenge the radical in a concentration-
dependent manner. The aqueous and chloroform fractions from
the fruit of C. opaca exhibited the highest radical scavenging activ-
ities when reacted with the ABTS radicals. In contrast, the n-hexane
and ethyl acetate fractions did not show a leveling effect at the
highest concentration, however their radical scavenging effects
were much less (P < 0.01) than that of the aqueous and chloroform
fractions. The EC50 values obtained for the aqueous fraction
(74.14 ± 0.8 lg/ml) was significantly different (P < 0.05) from the
EC50 values obtained for the ethyl acetate (>500 lg/ml) and n-hex-
ane (>>500 lg/ml) fractions. The ABTS radical scavenging activity
of ascorbic acid was followed by aqueous and chloroform fractions
(Table 2), however, a significance difference (P < 0.01) in EC50 was
present between fractions and ascorbic acid.
3.8. b-Carotene bleaching assay
With regard to the b-carotene bleaching assay, the antioxidant
activity of fractions can be ranked as ethyl acetate > metha-
1210 S. Sahreen et al. / Food Chemistry 122 (2010) 1205–1211
nol > aqueous > butanol > chloroform > n-hexane. b-carotene
bleaching assay showed the dose dependent response curve for
all the fractions at concentrations ranging from 50 to 8000 lg/ml.
The EC50 values of for catechin was 133.81 ± 2.8 lg/ml and was fol-
lowed by ethyl acetate 233.89 ± 4.3 lg/ml and methanol fraction
433.45 ± 4.9 lg/ml, respectively (Table 2). This data suggested that
ethyl acetate and methanol fractions have a considerable ability to
react with free radicals to convert them into more stable non-reac-
tive species and to terminate radical chain reaction.
3.8. Chelating activity
An important mechanism of antioxidant activity is the ability to
chelate/deactivate transition metals, which possess the ability to
catalyze hydro peroxide decomposition and Fenton-type reactions.
Therefore, it was considered of importance to screen the iron (II)
chelating ability of extracts (Manian et al., 2008). All the fractions
had high levels of ferrous ion chelating activity but were signifi-
cantly low (P < 0.01) as compared to catechin. The chelating activ-
ity increased with the concentration of each fraction. The sequence
for chelating power was aqueous > chloroform > butanol > metha-
nol > n-hexane > ethyl acetate fraction. The iron chelating data
measured at different concentrations (25–250 lg/ml) suggested
that ferrous ion chelating effects of all the fractions of fruit of C.
opaca would be somewhat beneficial to protect against oxidative
damage. The EC50 value of iron chelating activity for the aqueous
fraction was 25.14 ± 0.2 lg/ml, ethyl acetate fraction was
46.39 ± 1.9 lg/ml, while for catechin it was 21.74 ± 0.5 lg/ml (Ta-
ble 2).
3.9. Reducing power activity
In the reducing power assay, the presence of reductants (antiox-
idants) in the fractions would result in the reduction of Fe+3/ferric
cyanide complex to the ferrous form by donating an electron.
Increasing absorbance at 700 nm indicates an increase in reducing
ability. It was found that the reducing power increased with the
concentration of each sample. The sequence for reducing power
was aqueous > methanol > ethyl acetate > butanol > n-hex-
ane > chloroform fraction. The various solvent fractions from fruit
of C. opaca exhibited a good reducing power of 1.3 ± 0.15 and
1.244 ± 0.17 at 0.25 mg/ml for aqueous and methanol fractions,
respectively (Table 3).
3.10. Correlation with EC50 values of antioxidant activities and
phytochemical contents
Through correlation analysis for phytochemical contents with
EC50 values of radical scavenging activity and/or antioxidant ability
of extract of C. opaca fruit and its various soluble fractions, the con-
tents of phenolics and flavonoids exhibited good correlation
Table 3
Reducing power of methanol extract and soluble fractions of Carissa opaca fruit.
Plant extracts Reducing power absorbance at 700 nm (250 lg/ml)
n-Hexane fraction 1.125 ± 0.25b
Ethyl acetate fraction 1.161 ± 0.04d
Chloroform fraction 0.982 ± 0.10a
Butanol fraction 1.129 ± 0.54bc
Methanol extract 1.244 ± 0.17b
Aqueous fraction 1.3 ± 0.15c
Ascorbic acid 2.6 ± 0.23e
Each value in the table is represented as mean ± SD (n = 3).
Means not sharing the same letter are significantly different (LSD) at P < 0.01
probability level in each column.
Table 4
CorrelationsA between the EC50 values of antioxidant activities and phenolic and
flavonoids content of Carissa opaca fruit.
Assays Correlation R2
Phenolics Flavonoids
EC50 of DPPH radical scavenging ability 0.7280a 0.8527b
EC50 of superoxide radical scavenging ability 0.7919a 0.8853b
EC50 of antioxidant capacity 0.1446 0.2694
EC50 of Hydroxyl radical scavenging ability 0.7598a 0.8639b
EC50 of hydrogenperoxide radical scavenging ability 0.7656a 0.9359b
EC50 of ABTS radical scavenging ability 0.7160a 0.8352b
EC50 of b-carotene bleaching inhibition 0.3518 0.4420
EC50 of chelating power 0.4455 0.6854b
A
Carissa opaca fruit methanolic extract and its soluble fractions were used in the
correlation.
a
Indicate significance at P < 0.05.
b
Indicate significance at P < 0.01.
(R2 > 0.7159) with DPPH, superoxide, hydroxyl, hydrogen peroxide
and ABTS radical scavenging activities (Table 4). However, a non
significant correlation was found between antioxidant capacity
and b-carotene bleaching inhibition. In addition, EC50 of chelating
power presented a significant correlation with flavonoids while
non significant with phenolics. Our results are in agreement with
Ao et al. (2008) who reported a strong relationship with DPPH
and ABTS as compare to b-carotene.
4. Conclusion
The results obtained in this work have considerable value with
respect to the antioxidant activities of chloroform and aqueous
fractions of C. opaca fruits. The activity of these fractions is attrib-
uted to the phenolic and flavonoid contents. Consequently, our re-
sults suggested that the extract can be utilized as an effective and
safe antioxidant source, although the antioxidant activities of chlo-
roform and aqueous fractions were lower than that of ascorbic acid
and rutin. It can be concluded that, fruit of C. opaca consumed as a
foodstuff in different areas of Pakistan, can be used as an accessible
source of natural antioxidants with consequent health benefits. In-
deed, there is a current need for availability of new plant derived
bioactive molecules; thus C. opaca fruits may be a great natural
source for the development of new drugs.
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