This document summarizes a study that analyzed the phenolic profile and antioxidant activity of extracts from different parts (roots, leaves, fruits) of the Citrullus colocynthis plant. Reverse phase high performance liquid chromatography was used to simultaneously quantify phenolic acids and flavonoids in ethanol and hexane extracts. The major phenolic compounds identified were ferulic acid, vanillic acid, p-coumeric acid, gallic acid, p-hydroxy benzoic acid, chlorogenic acid, quercetin, myricetin and catechin. Total phenolic and flavonoid contents were highest in ethanol extracts of the leaves. Ethanol extracts of the leaves also exhibited the highest antioxidant and DPPH radical sc

1. Industrial Crops and Products 45 (2013) 416–422
Contents lists available at SciVerse ScienceDirect
Industrial Crops and Products
journal homepage: www.elsevier.com/locate/indcrop
Phenolic profile and antioxidant activity of various extracts from Citrullus
colocynthis (L.) from the Pakistani flora
Abdullah I. Hussaina,b,∗
, Hassaan A. Rathorea,∗∗
, Munavvar Z.A. Sattara
, Shahzad A.S. Chathab
,
Fiaz ud din Ahmada
, Ashfaq Ahmada
, Edward J. Johnsc
a
School of Pharmaceutical Sciences, University Sains Malaysia, Penang, Malaysia
b
Institute of Chemistry, Government College University, Faisalabad, Pakistan
c
Department of Physiology, University College Cork, Cork, Ireland
a r t i c l e i n f o
Article history:
Received 30 June 2012
Received in revised form
21 December 2012
Accepted 1 January 2013
Keywords:
Quercetin
Ferulic acid
Gradient elusion
RP-HPLC
DPPH radical scavenging
a b s t r a c t
The aim of the present study was to quantify phenolic acids and flavonoids from Citrullus colocynthis
roots (CCR), leaves (CCL) fruits (CCF) and compare the antioxidant and free radical scavenging activities
of their extracts and major compounds. Reverse phase high performance liquid chromatography method
was developed and validated for the simultaneous quantification of phenolic acids and flavonoids from
ethanol and hexane extracts of CCR, CCL and CCF. The antioxidant activity of CCR, CCL and CCF extracts
was investigated by measuring total phenolics, total flavonoids contents, DPPH free radical scavenging
activity, inhibition of linoleic acid peroxidation and reducing power. The RP-HPLC analysis of C. colocynthis
extracts revealed the presence of ferulic acid, vanillic acid, p-coumeric acid, gallic acid, p-hydroxy benzoic
acid and chlorogenic acid being the major phenolic acids and quercetin, myricetin and catechin being
the most prominent flavonoids compounds. The amounts of TP (3.07–18.6 mg/g of dry plant material,
measured as gallic acid equivalent) and TF (0.51–13.9 mg/g of dry plant material, measured as catechin
equivalent) were higher in ethanol extracts of CCL, followed by CCR and CCF. Ethanol extract of CCL
exhibited the highest antioxidant and DPPH radical scavenging activities followed by ethanol extracts of
CCR and CCF. However, only hexane extract of CCF showed considerable DPPH radical scavenging activity.
The results suggested that CCR, CCL and CCF extracts showed a wide variability of their phenolic acids
and flavonoids composition and antioxidant activity.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Flavonoids and other phenolic compounds have been suggested
to play preventive role against the incidence of some common
diseases like cancer, cardiovascular and neurodegenerative disor-
ders (Hussain et al., 2008). Some phenolic compounds including
flavonols and hydroxycinnamic acids have been widely distributed
in plants and among them flavonols are of particular impor-
tance in the human diet as antioxidants. Due to large number
of phenolic compounds in plant materials, quantitative determi-
nation of individual flavonoids and phenolic acids is a tedious
job. Therefore, these compounds are normally hydrolyzed and the
∗ Corresponding author at: Institute of Chemistry, Government College University
Faisalabad, Pakistan. Tel.: +92 41 9200037; mobile: +92 300 7631058.
∗∗ Corresponding author at: School of Pharmaceutical Sciences, University Sains
Malaysia, Pulau Penang, Malaysia. Tel.: +66 4 6533188.
E-mail addresses: abdullahijaz@gcuf.edu.pk, ai.hussain@yahoo.com
(A.I. Hussain), hassaan@usm.my (H.A. Rathore).
resulting aglycones are identified and quantified. Different meth-
ods for extraction, hydrolysis and analysis of phenolics by high
performance liquid chromatography (HPLC) have been published
(Obmann et al., 2012; Wei et al., 2012). However, few HPLC meth-
ods are available for the simultaneous analysis of various classes
of phenolics which do not contain adequate separation (Lin et al.,
2010). Present study was carried out to investigate phenolics in the
different extracts of Citrullus colocynthis.
C. colocynthis L. Schrad. (C. colocynthis), an underutilized cucurbit
plant, is wildly distributed in hot arid areas of the world, including
Pakistan, India and Saudi Arabia (Asyaz et al., 2010; Sawaya et al.,
1983). The dried pulp of unripe fruit and leaves are used medici-
nally for its drastic purgative and hydragogue cathartic action on
the intestinal tract and a folk remedy for cancerous tumors as well
(Kumar et al., 2009). Roots may also be used as purgative against
ascites, for jaundice, urinary diseases, rheumatism, and for snake-
poison (Kumar et al., 2009). Few reports are available in literature
on phytochemistry and traditional used of C. colocynthis to treat
diabetes, cancer and arterial hypertension (Aly and Naddaf, 2006;
Delazar et al., 2006; Eddouks et al., 2002; Huseini et al., 2009;
0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.01.002

2. A.I. Hussain et al. / Industrial Crops and Products 45 (2013) 416–422 417
Tahraoui et al., 2007) but no any report available on the pheno-
lic profile and antioxidant activity of extracts from different parts
of C. colocynthis from Pakistan and elsewhere.
Therefore, the potential of C. colocynthis plant as an industrial
crop and its folk uses in curing different diseases from different
part of the world encouraged us to explore bioactive constituents
from different parts of the plant responsible for antioxidant activity.
In the present study extraction of phenolic compounds from roots,
leaves and fruits of C. colocynthis has been done using two differ-
ent solvents (n-hexane and ethanol). Total phenolic contents, total
flavonoid contents, antioxidant activity and free radical scavenging
capacity of different extracts were evaluated. Moreover, identifica-
tion and quantification of phenolic acids and flavonoids were also
carried out simultaneously in single run using reverse phase high
performance liquid chromatography (RP-HPLC).
2. Materials and methods
2.1. Collection, identification and pre-treatment of plant
materials
Whole plants of C. colocynthis were collected from the desert
area (Hasalpur) of South Punjab, Pakistan in May–June 2011. The
plant specimens were identified and authenticated by Dr. Muham-
mad Naeem (Taxonomist), Department of Botany, Government
College University, Faisalabad, Pakistan [voucher specimen code, C.
colocynthis (2734), University of Agriculture, Faisalabad, Pakistan].
The plant materials were segregated into roots, leaves and fruits
and dried at 35 ◦C and grinded to fine powder using grinder
(TSK-949, Westpoint, France). The materials that passed through
80-mesh sieve were used for extraction purposes.
2.2. Reference compounds, reagents and chemicals
Standards and reference chemicals used in this study including
gallic acid, chlorogenic acid, ferulic acid, vanillic acid, p-coumeric
acid, sinapic acid, p-hydroxy benzoic acid, caffeic acid, quercetin,
myricetin, kaempferol, catechin, ascorbic acid, linoleic acid
(60–74%), Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH•), Tween 80, butylated hydroxytoluene (BHT), buty-
lated hydroxylanisole (BHA) were obtained from Sigma Chemical
Co. (St Louis, MO, USA). All other chemicals (analytical grade) i.e.
ferrous chloride, ammonium thiocyanate, hydrochloric acid, chlo-
roform, hexane, ethanol, and methanol used in this study were
purchased from Merck (Darmstadt, Germany), unless stated oth-
erwise.
2.3. Sample preparation
Two different solvents systems i.e. n-hexane and ethanol were
selected for the preparation of non-polar and polar C. colocynthis
roots (CCR), C. colocynthis leaves (CCL) and C. colocynthis fruits
(CCF) extracts. Briefly, the ground plant materials (600 g for each
sample) were extracted with 3000 mL of each of the solvent in a
Soxhlet unit (5000 mL capacity) for 18 h. The extracts were then
filtered through Whatman filter paper (No. 1). The solvents were
removed under reduced pressure, using a rotary evaporator (EYELA,
SB-651, Rikakikai Co. Ltd., Tokyo, Japan). The dried, crude concen-
trated extracts were weighed to calculate the yield and stored in a
refrigerator (−4 ◦C), until used for analyses.
2.4. HPLC analysis of phenolic acids and flavonoids
2.4.1. Hydrolysis of sample
The hydrolysis of C. colocynthis extracts was done as reported
previously (Nuutila et al., 2002) with slight modification. Briefly,
10 mL of 50% aqueous methanol solution containing 1.2 M HCl and
0.04% (w/v) ascorbic acid as antioxidant was added to 1000 mg
of crude extract. The hydrolysis was performed at 80 ◦C under
reflux for 2 h. After refluxing, the extracts were allowed to cool and
were made up to 10 mL with methanol. The extracts were filtered
through 0.45 ␮m non-pyrogenic filter (Minisart, Satorius Stedim
Biotech GmbH, Goettingen, Germany) prior to injection.
2.4.2. Preparation of calibration curves
Stock solutions of the standards (gallic acid, p-hydroxy ben-
zoic acid, chlorogenic acid, caffeic acid, vanillic acid, p-coumeric
acid, sinapic acid, ferulic acid, catechin, myricetin, quercetin,
kaempferol) were freshly prepared by dissolving authentic com-
pounds in methanol (100 ␮g/mL). Working standards solutions
were made by gradual dilution with methanol to the required con-
centration 0.4–100 ␮g/mL. The calibration curve was constructed
for each standard by plotting the concentration of standard against
peak area.
2.4.3. Chromatographic conditions
The HPLC analysis was performed with Shimadzu CBM-20A sys-
tem (Shimadzu Corporation, Kyoto, Japan) equipped with gradient
model LC-20AD pumps system, a SPD 20A UV/Visible detector, CTO-
10AS VP column oven, an auto injection (SIL-20AHT) and degasser
(DGU-20A5) systems. A hypersil GOLD C18 column (250 × 4.6 mm
internal diameter, 5 ␮m particle size) (Thermo Fisher Scientific
inc) and a non-linear gradient consisting of solvent A (acetoni-
trile:methanol, 70:30) and solvent B (water with 0.5% glacial acetic
acid). Following gradient program was used for the separation of
phenolic acids and flavonoids; 10–15% A from 0 to 5 min; 15–20%
A from 5 to 18 min; 20–40% A from 18 to 40 min and kept at 40% A
from 40 to 45 min; 40–10% A from 45 to 50 min and kept at 10% A
from 50 to 55 min). UV spectra were recorded at 275 nm. The ana-
lytes were identified by matching the retention times and spiking
samples with standards and quantification was based on an exter-
nal standard method. HPLC separation efficiency was assessed by
the separation factor (˛) and resolution (Rs). The reproducibility of
each compound was measured and the standard deviation was cal-
culated from six measurements through run-to-run and day-to-day
basis.
2.5. Evaluation of in vitro antioxidant activity
Following antioxidant assays were employed for the determi-
nation of antioxidant activity and free radical scavenging capacity
of CCR, CCL and CCF extracts.
2.5.1. Determination of total phenolics (TP) contents
Amounts of TP from CCR, CCL and CCF extracts were assessed
using Folin-Ciocalteu reagent, reported earlier (Hussain et al.,
2012). Briefly, 50 mg of crude extract was mixed with 0.5 mL of
Folin–Ciocalteu reagent and 7.5 mL deionized water. The mixture
was kept at room temperature for 10 min, and then 1.5 mL of 20%
sodium carbonate (w/v) was added. The mixture was heated in
a water bath at 40 ◦C for 20 min and then cooled in an ice bath.
Absorbance was measured at 755 nm using a spectrophotometer
(Bio Tek Instrument, Inc., VT, USA). Amounts of TP were calculated
using gallic acid calibration curve (0.195–3.125 mg/mL) (Fig. 1) and
reported in mg/g of dry plant material, measures as gallic acid
equivalent (GAE).
2.5.2. Determination of total flavonoids (TF) contents
Total flavonoid contents of CCR, CCL and CCF were determined
following the procedure reported previously (Hussain et al., 2012).
Briefly, extract solution (1 mL) containing 10 mg extract was placed
in a 10 mL volumetric flask and then 5 mL of distilled water was

3. 418 A.I. Hussain et al. / Industrial Crops and Products 45 (2013) 416–422
Fig. 1. Calibration curves of gallic acid (a) and catechin (b).
added followed by 0.3 mL of 5% NaNO2. After 5 min, 600 ␮L of 10%
A1C13 was added. After another 5 min 2 mL of 1 M NaOH was added
and volume was made up to 10 mL with distilled water. Absorbance
was measured at 510 nm using spectrophotometer (Bio Tek Instru-
ment, Inc., VT, USA). Total flavonoid contents were calculated using
a calibration curve for catechin (0.195–3.125 mg/mL) (Fig. 2). The
amounts of TF were calculated and reported in mg/g of dry plant
material, measured as catechin equivalent (CE).
2.5.3. DPPH radical scavenging assay
2,2-Diphenyl-1-picrylhydrazyl radical (DPPH•) assay was car-
ried out to measure the free radical scavenging activity as
described previously (Hussain et al., 2008). C. colocynthis extract
and pure phenolic acid and flavonoids compounds concentra-
tions in methanol (1–100 ␮g/mL) were mixed with 2 mL of 90 ␮M
methanol solution of DPPH. After 30 min incubation period at
Fig. 2. Extracts yields (g/100 g) of hexane and ethanol extracts of C. colocynthis roots,
leaves and fruits.
room temperature, the absorbance was read at 517 nm. Butylated
hydroxytoluene (BHT) and butylated hydroxyl anisol (BHA) were
used as positive control for comparison and 90 ␮M DPPH solu-
tion was taken as blank. The percent scavenging was calculated
by following formula:
Scavenging (%) = 100 ×
Ablank − Asample
Ablank
where Ablank is the absorbance of the DPPH solution and Asample is
the absorbance of the extract solution. Extract concentration pro-
viding 50% scavenging (IC50) was calculated from the graph-plotted
inhibition percentage against extract concentration.
2.5.4. Inhibition of linoleic acid peroxidation
The antioxidant activity of CCR, CCL, and CCF extracts and pure
compounds were also determined in terms of measurement of per-
cent inhibition of linoleic acid peroxidation following a method
reported before (Hussain et al., 2011). Briefly, 5 mg of each extract
and pure compounds was added to a solution mixture of linoleic
acid (130 ␮L), 99.8% ethanol (10 mL) and 10 mL of 0.2 M sodium
phosphate buffer (pH 7). Total mixture was diluted up to 25 mL
with distilled water. The solutions were incubated at 40 ◦C for 175 h
and the degree of oxidation was measured before and after incuba-
tion, following thiocyanate method. Briefly, 10 mL of ethanol (75%),
200 ␮L of an aqueous solution of ammonium thiocyanate (30%),
200 ␮L of sample solution and 200 ␮L of ferrous chloride (FeCl2)
solution (20 mM in 3.5% HCl) were mixed sequentially. After 3 min
of stirring, the absorption values of the mixtures were determined
at 500 nm. A negative control was performed with linoleic acid
but without extracts. Synthetic antioxidants; BHT and BHA were
used as positive control. Increases in absorbance values of samples,
negative and positive controls were calculated by subtracting first
value (0 h) from second value (175 h) and inhibition of linoleic acid
peroxidation was calculated using following formula:
Percent inhibition
= 100 −
Abs. increase of sample
Abs. increase of negative control
× 100
2.5.5. Determination of reducing power
The reducing power of CCR, CCL, and CCF extracts was deter-
mined according to the procedure reported earlier, with little
modification (Anwar et al., 2009). Briefly, concentrated extract
(0.625–10.0 mg) was mixed with sodium phosphate buffer (5.0 mL,
0.2 M, pH 6.6) and potassium ferricyanide (5.0 mL, 1.0%); the
mixture was incubated at 50 ◦C for 20 min. Then 5 mL of 10%
trichloroacetic acid was added and the mixture centrifuged at
980 × g for 10 min at 5 ◦C in a refrigerated centrifuge (CHM-17;
Kokusan Denki, Tokyo, Japan). The upper layer of the solution
(5.0 mL) was decanted and diluted with 5.0 mL of distilled water
and ferric chloride (1.0 mL, 0.1%), and absorbance read at 700 nm
using spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo,
Japan). BHT and BHA were used as positive control.
2.6. Statistical analysis
All the experiments were carried out in triplicate and the data
are presented as mean values ± standard deviation (SD). Statistical
analysis of the data was performed by Analysis of Variance (ANOVA)
and Duncan’s multiple range tests using STATISTICA 5.5 (Stat Soft
Inc., Tulsa, Ok, and USA) software and a probability value of P ≤ 0.05
was considered to represent a statistical significance difference
among mean values.

4. A.I. Hussain et al. / Industrial Crops and Products 45 (2013) 416–422 419
Fig. 3. Typical HPLC chromatogram showing the separation of phenolic acids and flavonoids in a single run.
3. Results and discussion
3.1. Extracts yields
The yields of ethanol and hexane extracts of CCR, CCL, and CCF
are given in Fig. 1. The amount of components extracted from
different parts of C. colocynthis plant using different solvents var-
ied widely. Generally, the highest extracts yields were obtained
with ethanol except in case of fruit extract, which give maximum
yield with hexane (10.8 g/100 g). Because CCR contains 200–300
seeds/fruit (75% of the weight of fruit) and C. colocynthis seeds
have high amount of oil (hexane extract). Maximum extract yield
with ethanol was obtained from CCL (17.4 g/100 g) followed by CCR
(14.9 g/100 g) and CCF (7.74 g/100 g). The significant (P < 0.05) dif-
ferences in the yield of extracts from different plant parts might be
attributed to the availability of different extractable components,
define by the chemical composition of plant materials. Ethanol has
been proven as effective solvent to extract phenolic compounds
(Siddhuraju and Becker, 2003). Ethanol is mostly preferred for the
extraction of antioxidant compounds mainly because of its less
toxicity (Sultana et al., 2007).
3.2. HPLC separation of phenolic acids and flavonoids
The developed HPLC method using binary gradient solvent sys-
tems (acetonotrile:methanol, 70:30 and glacial acetic acid:water,
0.5:99.5) and C18 column (250 × 4.6 mm internal diameter, 5 ␮m
particle size) could simultaneously separate eight phenolic acids
and four flavonoids within 50 minutes at flow rate of 0.8 mL/min
(Fig. 3). The separation factors (˛) of all the separated compounds
were >1.0 and the resolutions (Rs) were higher than 1.5 (data not
shown). The reproducibility for separation of the phenolic acids and
flavonoids was also good with RSD < 2.00% (run-to-run) and 2.70%
(day-to-day) for integrated areas basis. The developed method
could be used to separate phenolic acids and flavonoids in one run
from samples with varied matrixes. It was used to determine the
phenolic acids and flavonoids in roots, fruits and leaves extracts of
C. colocynthis.
Table 1 shows the amount (mg/100 g of dry plant material)
of eight phenolic acids including gallic acid, chlorogenic acid, p-
hydroxy benzoic acid, caffeic acid, vanillic acid, p-coumeric acid,
sinapic acid and ferulic acid, and four flavonoids including cate-
chin, myricetin, quercetin and kaempfrol in different extracts of
CCR, CCL and CCF. The RP-HPLC analysis of C. colocynthis extracts
revealed the presence of ferulic acid, vanillic acid, sinapic acid, p-
coumeric acid, gallic acid, p-hydroxy benzoic acid and chlorogenic
acid, quercetin and myricetin being the most prominent polyphe-
nolic components. Ferulic acid was found to be major phenolic acid
in ethanol extract of CCL (193.8 mg/100 g of dry plant material) fol-
lowed by valillic acid (13.78 mg/100 g of dry plant material), sinapic
acid (12.98 mg/100 g of dry plant material), p-coumaric acid (12.91
193.8 mg/100 g of dry plant material), gallic acid (11.05 mg/100 g
of dry plant material), p-hydroxy benzoic acid (10.57 mg/100 g of
dry plant material), caffeic acid (4.92 mg/100 g of dry plant mate-
rial) and chlorogenic acid (4.42 mg/100 g of dry plant material). The
main phenolic acid identified from the ethanol extract of CCR was
gallic acid (3.71 mg/100 g of dry plant material) and the major phe-
nolic acid found in ethanol extract of CCF was chlorogenic acid
(9.93 mg/100 g of dry plant material).
Flavonoids were identified at higher level than phenolic acids
in the C. colocynthis leaves and roots. Quercetin was the major
flavonoid (579.9 mg/100 g of dry plant material) in the leaves
followed by myricetin (381.7 mg/100 g of dry plant material),
catechin (95.45 mg/100 g of dry plant material) and kaempferol
(5.95 mg/100 g of dry plant material). From the ethanol extracts
of CCR, catechin was found to be major flavonoid (65.31 mg/100 g
of dry plant material) followed by myricetin (25.81 mg/100 g of
dry plant material), quercetin (7.05 mg/100 g of dry plant mate-
rial) and kaempfrol (6.79 mg/100 g of dry plant material). Ethanol
extract of CCF contained 16.45, 2.97, 1.33 and 2.37 (mg/100 g of
dry plant material) catechin, myricetin, quercetin and kaempfrol,
respectively. Significant (P < 0.05) variations were observed in the
contents of phenolic acids and flavonoids with respect to different
C. colocynthis plant part.
Leaves due to accumulation of phenolic compounds with the
maturity of the plant possess relatively higher amounts of TF

5. 420 A.I. Hussain et al. / Industrial Crops and Products 45 (2013) 416–422
Table 1
Contents of phenolic acids and flavonoids identified from ethanol and hexane extracts of C. colocynthis roots, leaves and fruits.
Compounds Concentration of compounds (mg/100 g of dry plant material)
Ethanol extracts Hexane extracts
Roots Leaves Fruits Roots Leaves Fruits
Gallic acid 3.71 ± 0.20d
11.05 ± 0.56e
2.71 ± 0.15c
– 0.16 ± 0.01a
0.37 ± 0.02b
p-Hydroxy-benzoic acid 1.85 ± 0.09c
10.57 ± 0.49e
5.80 ± 0.12d
0.02 ± 0.01a
0.04 ± 0.01a
0.30 ± 0.03b
Chlorogenic acid 2.06 ± 0.08d
4.42 ± 0.18e
9.93 ± 0.42f
0.08 ± 0.01a
0.20 ± 0.01b
0.84 ± 0.04c
Caffeic acid 2.79 ± 0.15c
4.92 ± 0.19d
2.95 ± 0.11c
0.05 ± 0.01a
0.04 ± 0.01a
0.15 ± 0.01b
Vanillic acid 1.53 ± 0.09d
13.78 ± 0.93f
2.79 ± 0.22e
0.02 ± 0.00a
0.66 ± 0.04c
0.19 ± 0.01b
p-Coumaric acid 0.96 ± 0.08a
12.91 ± 0.05c
1.26 ± 0.07b
– – –
Sinapic acid 0.20 ± 0.02a
12.98 ± 0.65c
3.38 ± 0.29b
– – –
Ferulic acid 1.55 ± 0.08a
193.8 ± 5.70c
2.10 ± 0.10b
– – –
Catechin 65.31 ± 2.70d
95.45 ± 3.36e
16.45 ± 1.01c
0.11 ± 0.01a
0.59 ± 0.04b
–
Myricetin 25.81 ± 1.7e
381.7 ± 7.33f
2.97 ± 0.18d
0.05 ± 0.01a
0.16 ± 0.01b
0.75 ± 0.04c
Quercetin 7.05 ± 1.3d
579.9 ± 16.3e
1.33 ± 0.06c
0.10 ± 0.08a
0.62 ± 0.03b
0.69 ± 0.0.4b
Kaempferol 6.79 ± 0.44f
5.95 ± 0.25e
2.37 ± 0.09d
0.07 ± 0.01a
0.20 ± 0.01b
1.82 ± 0.11c
Values are mean ± SD of triplicate determinations. Different letters in superscript represent significant difference (P < 0.05) between different extracts.
contents than other plant organs (Siddhuraju et al., 2002). Our
results are in agreement of literature report on gas chromatogra-
phy mass spectrometry (GC–MS) analyses of ethyl acetate extract
of C. colocynthis roots, which showed the presence of caffeic acid
and ferulic acid as major phenolic acid followed by vanillic acid and
p-coumaric acid (Hsouna and Alayed, 2012).
3.3. Total phenolic, total flavonoids contents and antioxidant
activity
The amount of TP and TF contents extracted from CCR, CCL and
CCF extracts in hexane and ethanol solvents ranged from 0.17 to
18.6 GAE, mg/g of dry plant material and from 0.12 to 13.9 CE, mg/g
of dry plant material, respectively (Table 2). Ethanol extract of all
the plant materials showed significantly (P < 0.05) higher TP and TF
contents than hexane extracts. TP contents from ethanol extracts
of CCL, CCR and CCF were found to be 18.6, 6.35 and 3.07 GAE, mg/g
of dry plant materials, while, TF contents were found to be 13.9,
2.52 and 0.51 CE, mg/g of dry plant material, respectively. Hexane
extracts of CCR, CCL and CCF contained very less amount of TP and
TF contents. Significant variation (P < 0.05) in TP and TF contents
of CCR, CCL and CCF were recorded. TP and TF contents from
extracts of varied plants were reported in literature as a marker for
antioxidant potential (Hussain et al., 2012; Sultana et al., 2007).
Many studies confirmed that amounts and composition of phenolic
compounds is diversified at sub-cellular level and within the tissues
(Shi et al., 2005; Sultana et al., 2007). Polyphenols are a class of nat-
ural compounds that exhibited antioxidants activity and act as free
radical terminators (Huang et al., 2005). C. colocynthis extracts are
rich source of phenolic antioxidants and exhibited good antioxi-
dant activity (Dallak, 2011; Gill et al., 2011; Kumar et al., 2008;
Sebbagh et al., 2009). Our results are in accordance of Hsouna and
Alayed (2012) in a sense that methanol extracts of C. colocynthis
roots contained higher TP and TF contents than hexane extracts,
however, at the same time contrary with the amount of TP and TF
contents. TP and TF contents in our study were lower than reported
(Hsouna and Alayed, 2012). These variations might be attributed to
the varied phenological status and agroclimatic conditions of the
regions.
In the DPPH assay, the radical scavenging capacity of CCR, CCL
and CCF increased in a concentration dependent manner. The per-
centage scavenging provided by extracts concentration 10 ␮g/mL
and extract concentration provided 50% scavenging (IC50) are given
in Table 2. All the ethanol extracts of C. colocynthis exhibited appre-
ciable radical scavenging activity ranging from 56.8% to 67.2% (IC50
5.97–6.42 ␮g/mL). Among them, ethanol extract of CCL exhibited
Table 2
Total phenolic contents, total flavonoid contents, DPPH free radical scavenging capacity and antioxidant activity of hexane and ethanol extracts of C. colocynthis roots, leaves
and fruits and major compounds.
Extracts and major compoundsa
Antioxidant assays
Total phenolic
contents (mg/g)b
Total flavonoid
contents (mg/g)c
DPPH radical
scavenging (%)d
DPPH, IC50
(␮g/mL)
Inhibition of
linoleic acid
peroxidation (%)e
CCR (hexane) 0.17 ± 0.01a 0.12 ± 0.01a 39.8 ± 2.0b 15.9 ± 0.77f 46.3 ± 3.9a
CCL (hexane) 0.82 ± 0.33b 0.51 ± 0.03b 35.7 ± 1.4a 16.7 ± 0.98f 46.9 ± 2.9a
CCF (hexane) 0.84 ± 0.06b 0.46 ± 0.05b 60.2 ± 2.9d 8.15 ± 0.39e 43.8 ± 4.0a
CCR (ethanol) 6.35 ± 0.45d 2.52 ± 0.03c 56.8 ± 2.2c 6.42 ± 0.43c 79.2 ± 4.3b
CCL (ethanol) 18.6 ± 0.55e 13.9 ± 0.69d 67.2 ± 3.5e 5.97 ± 0.49bc 80.9 ± 2.9b
CCF (ethanol) 3.07 ± 0.11c 0.51 ± 0.04b 58.1 ± 2.3c 7.14 ± 0.25d 76.5 ± 3.3b
Quercetin – – 83.7 ± 4.5f 2.95 ± 0.13a 85.9 ± 3.3b
Myricetin – – 78.4 ± 3.0f 3.27 ± 0.19a 85.7 ± 3.7b
Catechin – – 77.5 ± 2.3f 3.25 ± 0.19a 87.6 ± 3.0b
Ferulic acid – – 69.7 ± 1.4e 6.12 ± 0.23c 80.9 ± 2.8b
BHT – – 70.9 ± 1.2e 5.39 ± 0.26b 81.3 ± 3.0b
BHA – – 72.8 ± 2.2e 5.35 ± 0.24b 83.6 ± 4.2b
Values are mean ± standard deviation of three independent experiments. Different letters in the same column represent significant difference (P < 0.05).
a
Citrullus colocynthis roots (CCR), Citrullus colocynthis leaves (CCL), Citrullus colocynthis fruits (CCF).
b
Total phenolic contents, mg/g of dry plant material, measured as gallic acid equivalent.
c
Total flavonoid contents, mg/g of dry plant material, measured as catechin equivalent.
d
DPPH scavenging (%) provided by extract concentration, 10 ␮g/mL.
e
Inhibition of linoleic acid peroxidation (%) provided by extract concentration, 200 ␮g/mL.

6. A.I. Hussain et al. / Industrial Crops and Products 45 (2013) 416–422 421
Fig. 4. Reducing potential of hexane and ethanol extracts of C. colocynthis roots, leaves and fruits.
the highest radical scavenging capacity (67.2%, IC50 5.97 ␮g/mL)
which is comparable to activity of pure compounds phenolic acid
and flavonoids compounds and synthetic antioxidants like BHT
and BHA. Hexane extracts of CCR and CCF showed 39.8 and 35.7%
(IC50 15.9 and 16.7 ␮g/mL) radical scavenging activity, respectively,
which is comparatively poor than respective ethanol extracts. How-
ever, hexane extract of CCF showed good radical scavenging activity
(60.2%, IC50 7.25 ␮g/mL), which is comparable to ethanol extracts
of CCF. Major flavonoid (Quercetin, myriscetin and catechin) and
ferulic acid showed excellent radical scavenging activity and high
radical scavenging activity of ethanol extract might be attributed to
the presence of these components in CCR and CCL extracts. Excel-
lent DPPH radical scavenging activity of methanol extract of C.
colocynthis seeds and fruits was reported earlier in literature (Gill
et al., 2011; Hsouna and Alayed, 2012; Kumar et al., 2008). DPPH
radical scavenging capacity of plant extracts could be explained by
the presence of phenolic acids and flavonoids (Siddhuraju et al.,
2002).
The antioxidants activity of CCR, CCL and CCF extracts was also
assessed by ability to prevent from oxidation. Inhibition of linoleic
acid peroxidation was used to assess the antioxidant activity of
different C. colocynthis extracts. All the ethanol extracts exhibited
appreciable inhibition of peroxidation ranging from 76.5% to 81.3%
and were comparable with activity of pure compounds and syn-
thetic antioxidants (Table 2). The best activity was observed with
ethanol extract of CCL (80.9%) followed by ethanol extracts of CCR
(79.2%) and CCF (76.5%). All the hexane extracts of C. colocynthis
showed poor antioxidant activity in this assay. High percentage
inhibition of linoleic acid peroxidation by methanol extract of C.
colocynthis roots is also reported in literature (Hsouna and Alayed,
2012). Considerable inhibition of peroxidation of C. colocynthis
ethanol extracts could be attributed to presence of high flavonoids
contents, such as quercetin potentially responsible for the consid-
erable activity of the plant extracts (Boots et al., 2008).
Measurement of reducing potential also reflects some aspects
of antioxidant activity of plant extracts (Oueslati et al., 2012). In
this method a ferric ion are reduced to ferrous ions with change
in color and the intensity of color depends on the reducing poten-
tial of the compounds/extracts. Greater the intensity of the color,
greater should be the absorption; consequently, greater should be
the reducing power. The data for the reducing potential of different
C. colocynthis extracts is presented in Fig. 4. The reducing potential
of the CCR, CCL and CCF extracts measured for the concentration up
to 10.0 mg/mL, showed general increase in activity when concen-
tration increased. Reducing potential of CCR, CCL and CCF hexane
and ethanol extracts at 10 mg/mL ranged from 0.13 to 3.71. As in
above results, again the ethanol extracts showed better reducing
potential than hexane extracts. Among ethanol extract, CCR extract
provided the highest reducing power, comparable with BHT and
BHA, followed by CCL and CCF extracts. No earlier reports are avail-
able regarding the reducing potential of C. colocynthis extracts with
which to compare the results of our present analysis. However,
Anwar et al. (2009) reported the good correlation index (>0.94)
between concentration of fennel extract and absorbance in the
reducing potential assay.
4. Conclusion
In conclusion, this study first time reports the composition of
phenolic acids and flavonoids in different parts of C. colocynthis
along with total phenolic, total flavonoid contents and in vitro
antioxidant activity. The results of the present study would cer-
tainly help to ascertain the potency of the crude CCR, CCL and
CCF extracts as potential source of natural antioxidants. Among all
extracts, ethanol extracts of CCR and CCL contained the high TP,
TF contents and showed the excellent antioxidant and free radi-
cal scavenging activities. However, further research is needed to
investigate these extracts in vivo using different diseased models
and develop their application for pharmaceutical and nutraceuti-
cals industries.

7. 422 A.I. Hussain et al. / Industrial Crops and Products 45 (2013) 416–422
Acknowledgements
We would like to extend our special gratitude to Professor Dr.
Zahri Ismail, School of Pharmaceutical Sciences, Universiti Sains
Malaysia, for allowing us to work in Pharmaceutical Chemistry Lab.
This work is a part of Postdocoral project funded by the Universiti
Sains Malaysia and TWAS under the scheme of USM-TWAS Post-
doctoral Fellowship Award.
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