Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2013, 3 (3):32-40
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ISSN : 2231 3168

CODEN (USA) : JMBRB4

Evaluation of the antioxidant activity of Cucumis dipsaceus

S. K. Ramya Urs, H. N. Krishna Kumar*, E. Chandana and Jyoti Bala Chauhan

Department of Studies in Biotechnology, Microbiology & Biochemistry, Pooja Bhagavat Memorial Mahajana Post
Graduate Centre, Affiliated to University of Mysore, Metagalli, Mysore, Karnataka, India
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ABSTRACT

The present work on the antioxidant activity of aqueous and methanolic extracts of Cucumis dipsaceous fruit is the
first investigation. The results obtained in the in vitro models such as Reducing power assay, DPPH radical
scavenging activity, Superoxide anion radical scavenging activity, Nitric oxide scavenging assay, and Metal ion
chelating activity clearly suggest that, both the aqueous and methanolic extracts showed strong antioxidant activity
when compared with different standards such as BHA, EDTA and Curcumin. In the present investigation, the
sample and BHA showed significant effects on the DPPH radical scavenging at all amounts. The IC50 values of
methanolic, aqueous extract and BHA were 66, 77.5 and 7g/ml respectively. Inhibition of superoxide scavenging
by aqueous and methanolic extracts showed concentrations dependent manner with an IC50 values of 137 and
287g/ml respectively. methanolic extract was a potent superoxide anion scavenger than the aqueous extract. The
reducing capacity was exhibited by both aqueous and methanolic extracts at all amounts. The reducing power of
aqueous extract was higher than the methanolic extract. The ferrous ion chelating effect was shown by both of
aqueous and methanolic extracts with IC50 values of 251 and 282g/ml respectively. The positive control EDTA
showed the IC50 value of 178g/ml. The test samples were investigated for their inhibitory effects on nitric oxide
production. The metahnolic extract showed significant free radical scavenging action against nitric oxide (NO)
induced release of free radicals (IC50 = 32 g/ml) than aqueous extract (IC50 = 44 g/ml). In aqueous and
methanolic extracts of Cucumis dipsaceous, 31.5 2 and 43.1 3.1 mg (gallic acid equivalent per gram) of phenols
were detected respectively. There is little correlation between antioxidant activity and phenol content.

Key words: Cucumis dipsaceous, antioxidant activity, reducing power, metal ion chelation, free radical scavenging
activity.
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INTRODUCTION

Many plants contain antioxidant compounds and these compounds protect cells against the damaging effects of
reactive oxygen species (ROS), such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and
peroxynitrite [1, 2]. ROS are produced from endogenous sources within the living organisms via various
mechanisms i.e. normal aerobic respiration, stimulated poly-morpho-nuclear leukocytes and macrophages and
peroxisomes or from exogenous sources which includes tobacco smoke, ionizing radiation, certain pollutants,
organic solvents, and pesticides [3,4,5]. When the balance between ROS production and antioxidant defenses is lost
oxidative stress results which induce some oxidative damage to biomolecules like lipids, nucleic acids, proteins
and carbohydrates. Their damage causes various pathological conditions including ageing, arthritis, asthama, cancer,
diabetes, rheumatism, heart disease and artherosclerosis [6, 7]. Many antioxidant compounds, naturally occurring in
plant sources have been identified as free radical or active oxygen scavengers [8, 9]. Synthetic antioxidants such as
butylated hydroxy toluene (BHT), butylated hydroxy anisole (BHA) etc., have restricted use in food industry as they

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are suspected to be carcinogenic [10]. Hence, the studies on natural antioxidant have gained increasingly greater
importance.

The genus Cucumis L. is belonging to the family cucurbitaceae with ca. 35 species is distributed in the world [11].
There are 6 species in India [12]. Medicinally the genus is very important. Traditionally, Cucumis is used for
headaches, dysentery etc. The seeds are cooling and diuretic, the fruit juice is used as a nutritive and as a demulcent
in anti-acne lotions. The fruits contain Vitamin B1, ascorbic acid, rutin, an enzyme erepsin, oxidase, succinic and
maleic dehydrogenases and so on. The seeds contain - and -amyrin, sitosterols and cucurbitasides, whereas, the
leaves contain free cucurbitasides B and C and ferredoxin [13]. Cucumis dipsaceus is a annual climber a native of
Sudan and Southern Egypt Africa and widely spread in Ethiopia, Kenya, Somali, Tanzania, Uganda [11]. There is
no information about antioxidant activities of the fruits of this plant. The objective of this study was to evaluate the
antioxidant properties of aqueous and methanolic extracts of the fruits of Cucumis dipsaceous with the
measurements including the reducing capacity, metal ion chelating activity and the scavenging activity of the free
radicals.

MATERIALS AND METHODS

Plant material and Extraction

The fruits of Cucumis dipsaceous Ehrenb. ex Spach. were collected between November and January-2009 from
Mysore, Karnataka, India. The plant materials were identified and authenticated from the Department of Botany,
University of Mysore. The collected materials were washed thoroughly in water, shade dried for a week and ground
to coarse powder in electric grinder. The powder thus obtained was extracted in water and methanol. For aqueous
extraction, 20 g of sample was homogenized with 60 ml of distilled water and the homogenate was kept in a shaker
at 40C for 24 hrs. Then, the extract was filtered over Whatman No. 1 paper. The extract was dried under reduced
pressure at a yield of 12 % (w/w). For methanol extraction, 20 g of sample was mixed with 40 ml of methanol and
the obtained extracts were filtered over Whatman No. 1 paper. The filtrate was collected and the solvent was
evaporated to obtain dry extract with yield of 10.5% (w/w). Both the extracts were stored at 20C until use.

Chemicals
Nitroblue tetrazolium (NBT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4-triazine (ferrozine), butylated
hydroxyanisole (BHA), xanthine, xanthine oxidase and dimethyl sulfoxide (DMSO) were purchased from Sigma
(St. Louis, USA). Ethylene diamine tetra acetic acid (EDTA), potassium ferricyanide, trichloro acetic acid, gallic
acid, curcumin, Greiss reagent, Folin-Ciocalteu reagent etc. were purchased from M/S Sisco Research laboratories,
Mumbai, India. All chemicals and solvents used were of analytical grade.

DPPH radical scavenging activity

The free radical scavenging activity was measured by 1, 1-diphenyl-2-picrylhydrazil (DPPH) using the method of
Shimada et al., [14] with slight modifications. Briefly, 2ml of DPPH solution (0.1mM in methanol) and 2ml of both
aqueous and methanolic extracts of different concentrations (50-200g) was incubated at room temperature for 30
minutes. After 30 minutes absorbance was measured at 517 nm in a spectrophotometer against a blank. Lower
absorbance of the reaction mixture indicated higher free radical scavenging activity. The scavenging of DPPH
radical in percentage was calculated by the following equation:

Scavenging activity (%) = (1- A1/A0) X 100

Where A0 was the absorbance of the control and A1 was the absorbance in the presence of sample. BHA was used as
positive control.

Superoxide anion radical scavenging activity

The method described by Okamura et al., [15] was used to investigate the superoxide anion radical scavenging
activity with some modification. This assay is based on the removal rate of xanthine/xanthine oxidase generated
superoxide by measuring the reduction of nitro blue tetrazolium (NBT). The reaction mixture contained different
concentrations of both aqueous and methanolic extracts in 5% DMSO (100-500 g/ml), 1ml of a mixture of
xanthine(0.1mM) and NBT(0.2mM) in potassium phosphate buffer (50mM, pH 7.5) containing EDTA(0.05mM),
0.1ml xanthine oxidase (0.8 unit/ml diluted in 50mM phosphate buffer, pH 7.5) was incubated at 37C for 20 min.
Addition of 2 mL of 2.5 N HCl to the mixtures terminated the reaction, followed by increase of coloration of NBT,

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which was measured at 560nm against a blank. The scavenging of superoxide anion in percentage was calculated
using the equation described as in the case of DPPH. BHA was taken as the positive control.

Reducing power assay

The reducing power was determined according to the method of Oyaizu [16]. Different concentrations of test
samples (200-1000 g for both aqueous and methanolic extract) in 1 ml of distilled water were mixed with 2.5 ml of
phosphate buffer (0.2 M, pH 6.6) and 2.5ml of 1% potassium ferricyanide. The mixture was incubated at 50 C for
20 minutes. Following incubation, 2.5 ml of 10% trichloroacetic acid was added to the mixture, which was then
centrifuged at 3000 rpm for 10 minutes and the absorbance was measured at 700 nm in a spectrophotometer.
Increased absorbance of the reaction mixture indicates increased reducing power. BHA was used as a positive
control.

Metal ion chelating activity

The chelating of ferrous ions by the aqueous and methanolic extracts was estimated by the method of Dinis et al.,
[17]. Briefly, extracts (100 500 g) were added to a solution of 0.05 ml FeCl2 (2 mM). The reaction was initiated
by the addition of 0.2ml of ferrozine (5 mM). The mixture was shaken vigorously and left standing at room
temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562 nm against the
blank. The percentage of inhibition of ferrozineFe2+ complex formation was calculated using the formula described
for DPPH. EDTA was used as a positive control.

Nitric oxide scavenging assay

The method of Sreejayan & Rao [18] was followed for this assay. This assay is based on the principle that, Sodium
nitroprusside solution spontaneously generates nitric oxides, which reacts with oxygen to produce nitrite ions that
can be estimated using Greiss reagent. Scavengers of nitric oxides compete with oxygen leading to reduce
production of nitrite ions. For the experiment, sodium nitroprusside (10mM) in phosphate buffered saline was mixed
with different concentrations of both aqueous and methanolic extracts (25-100g). The resulting solutions were
then incubated at room temperature for 2 hrs. After incubation, 0.5 ml of Griess reagent was added. The
absorbance of the chromophore formed was measured at 546 nm. Curcumin was used as positive control.

Estimation of total phenolic compounds

Total soluble phenolic compounds were determined with the Folin-Ciocalteu reagent, according to the method
suggested by Slinkard & Singleton [19]. To 0.1 ml of extract solution (1 mg/ml in distilled water), one ml of Folin-
Ciocalteu reagent was added. After three minutes, 3 ml of Na2 CO3 (2%) was added. The mixture was allowed to
stand for 2 hrs at room temperature with intermittent shaking. The absorbance was measured using a
spectrophotometer at 760 nm. The concentration of total phenolic compounds in samples was determined as mg of
gallic acid equivalents per gram dry weight.

Statistical analysis
Results were expressed as mean standard deviation of the three measurements. The IC50 (amount of extract needed
to inhibit 50% of free radicals) values were graphically determined by a linear regression analysis.

RESULTS AND DISCUSSION

DPPH radical scavenging activity

DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become a stable
diamagnetic molecule [20]. One of the antioxidant mechanisms is to provide hydrogen atoms to free radicals and to
stop the chain reaction [21]. The reduction capability of DPPH radicals was determined by the decrease in its
absorbance at 517 nm, which is induced by antioxidants. Hence, DPPH is often used as a substrate to evaluate
antioxidative activity of antioxidants [22]. The free radical scavenging by the antioxidant samples are credited to
their hydrogen donating ability [23]. In the present investigation, the sample and BHA showed significant effects on
the DPPH radical scavenging at all amounts. The IC50 values of methanolic, aqueous extract and BHA were 66, 77.5
and 7g/ml respectively (Fig. 1). The scavenging effect of water and methanol extracts of Cucumis dipsaceous and
standard on the DPPH radical decreased in that order: BHA>methanol extract>aqueous extract. The result indicates
that both the extracts have a noticeable effect on scavenging free radical.

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Super oxide anion radical scavenging activity
Superoxide anion plays an important role in the formation of reactive oxygen species such as hydrogen peroxide,
hydroxyl radical and singlet oxygen, which induces oxidative damage in lipids, protein, and DNA [3, 24]. The study
showed, both methanolic and aqueous extracts were found to possess scavenging effects on superoxide anions at
concentrations dependent manner with an IC 50 values of 137 and 287 g/ml respectively (Fig. 2). As shown in
Fig.2, methanolic extract was a potent superoxide anion scavenger than the aqueous extract. The decrease of
absorbance at 560 nm with antioxidants indicates the consumption of superoxide anion in the reaction mixture.
Superoxide radical scavenging activity of those samples showed the following order: BHA> mehanolic extract >
water extract.

Reducing Power assay

The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity [25].
The antioxidant activity of putative antioxidants have been attributed to various mechanisms, among which are
prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of
continued hydrogen abstraction, reductive capacity and radical scavenging [21, 4]. In this study, both aqueous and
methanolic extracts of Cucumis dipsaceous exhibited effective reducing capacity at all amounts (Fig. 3). The
reducing power of aqueous extract was higher than the methanolic extract. The reducing capacity of sample
increased with increasing amount, indicating some compounds present in extracts were electron donors and could
react with free radicals to convert them into more stable products and terminate the radical chain reactions.
Reducing power of water and methanol extracts and standard compound exhibited the following order: BHA> water
extract >methanol extract.

Metal ion chelating activity

Ferrozine can quantitatively form complexes with Fe2+. In the presence of chelating agents, the complex formation
is disrupted resulting in a decrease in the red colour of the complex. Measurement of colour reduction therefore
allows estimating the metal chelating activity of the coexisting chelator [26]. In this assay both extracts of Cucumis
dipsaceous and standard compound are interfered with the formation of ferrous and ferrozine complex, suggesting
that they have chelating activity and are able to capture ferrous ion before ferrozine. The ferrous ion-chelating effect
was shown by both of aqueous and methanolic extracts of Cucumis dipsaceous with IC50 values of 251 and
282g/ml respectively (Fig. 4). The positive control EDTA showed the IC50 value of 178g/ml. The data obtained
from Fig. 4 reveal that both the extracts demonstrate a marked capacity for iron binding, suggesting that their action
as peroxidation protector may be related to its iron binding capacity. Metal chelating capacity was significant, since
it reduced the concentration of the catalysing transition metal in lipid peroxidation [22]. It was reported that
chelating agents, which form s-bonds with a metal are effective as secondary antioxidants because they reduce the
redox potential thereby stabilizing the oxidized form of the metal ion [27].

Nitric oxide scavenging assay

It was observed that the nitric oxide scavenging activity of extracts was a dose dependent fashion, the concentration
of nitrite after spontaneous decomposition of sodium nitroprusside indicating that both the extracts may contain
compounds that are able to scavenge nitric oxide. In the present study, the aqueous and methanolic extracts were
investigated for their inhibitory effects on nitric oxide production. The metahnolic extract showed significant free
radical scavenging action against nitric oxide (NO) induced release of free radicals (IC50 = 32 g/ml) than aqueous
extract (IC50 = 44 g/ml) as shown in Fig.5. Nitric oxide (NO) exhibits numerous physiological properties and it is
also implicated in several pathological states [28]. However, the specificity of this assay has been questioned since
nitrite is one final product of the reaction of nitric oxide with oxygen, through intermediates such as NO3, N2O4 and
N2O3 [29]. Therefore the decrease in the nitrite production could also be due to interaction of the extract with other
nitrogen oxides [30].

Estimation of total phenolic compounds

Phenols are very important plant constituents because of their radical scavenging ability due to their hydroxyl
groups [31]. In aqueous and methanolic extracts of Cucumis dipsaceous, 31.5 2 and 43.1 3.1 mg (gallic acid
equivalent per gram) of phenols were detected respectively. It is suggested that polyphenolic compounds have
inhibitory effects on mutagenesis and carcinogenesis in humans, when up to 1.0 g daily ingested from a diet rich in
fruits and vegetables [32]. In addition, it has been reported that phenolic compounds are associated with antioxidant
activity and play a crucial role in stabilizing lipid peroxidation [33]. In our study, there is little correlation between
antioxidant activity and phenol content. Aqueous extract with less phenol content showed higher activity in DPPH

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radical, superoxide anion scavenging and nitric oxide scavenging compared to methanolic extract, which may have
been due to nonphenolic compounds present in the extract. There are reports that antioxidant activity could also be
from nonphenolic compounds [34].

Fig.1 Shows DPPH radical scavenging activity. a. BHA b. Aqueous and methanolic extracts of Cucumis dipsaceus
Each value represents mean SD of three replicates.

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IC 50= 26g/ml

b
Fig.2 Shows Super oxide anion radical scavenging activity. a. BHA b. Aqueous and methanolic extracts of Cucumis dipsaceus
Each value represents mean SD of three replicates.

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Fig.3 Shows reducing power assay. a. BHA b. Aqueous and methanolic extracts of Cucumis dipsaceus
Each value represents mean SD of three replicates.

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Fig.4 To show metal ion chelating activity of aqueous and methanolic extracts of Cucumis dipsaceus and positive control EDTA
Each value represents mean SD of three replicates.

IC50= 16g/ml (curcumin)

IC50= 32g/ml (methanolic extract)
IC50= 44g/ml (aqueous extract)

Fig.5 To show nitric oxide scavenging assay of aqueous and methanolic extracts of Cucumis dipsaceus and positive control curcumin.
Each value represents mean SD of three replicates.

CONCLUSION

The results obtained in the in vitro models such as reducing power, DPPH radical, superoxide anion scavenging,
nitric oxide scavenging, and metal ion chelating activities clearly suggest that, both the aqueous and methanolic
extracts of Cucumis dipsaceous showed strong antioxidant activity when compared with different standards such as
BHA,EDTA and Curcumin. The results of this study showed that the extracts can be used as easily accessible source
of natural antioxidants and as a possible food supplement or in pharmaceutical industry. The fruits of Cucumis
dipsaceous could serve as a new source of natural antioxidants or nutraceuticals with potential applications to
reducing the level of oxidative stress and related health benefits.

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Acknowledgements
The authors are thankful to Prof. C.K. Renukarya, Director, Pooja Bhagavat Memorial Mahajana Post Graduate
Centre, Mysore for providing necessary facilities to carry out the research work.

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