Study on Cucumis melo var. utilissimus Seeds for the Therapeutic Potential
The present study was designed to investigate Cucumis melo var. utilissimus seeds for their antioxidant and therapeutic potential. The antioxidant activity of methanolic extract was measured by DPPH method and hydrogen peroxide free radical scavenging activity. The methanolic extract of Cucumis melo var. utilissimus seeds (MECU) showed maximum antioxidant potential. Hence, it was further evaluated for anti-inflammatory activity by carrageenan induced rat paw edema and analgesic activity by tail immersion method and tail flick method at different concentrations, i.e., 100, 200 and 300 mg kg-1. The significant results was found at maximum dose. The results suggested that methanolic seed extract possess great therapeutic potential.
Received: February 01, 2010;
Accepted: April 12, 2010;
Published: July 27, 2010
Natural products, including plants, animals and minerals have been the basis
to treat human diseases. The future of natural products drug discovery will
be more holistic, personalized and involve use of ancient and modern therapeutic
skills in a complementary manner so that maximum benefits can be accrued to
the patients (Patwardhan and Hooper, 1992). There is
emerging interest in the use of naturally occurring antioxidants for the management
of a number of pathophysiological conditions, most of which involve free radical
damage. The implication of oxidative stress in the etiology and progression
of several acute and chronic clinical disorders has led to the suggestion that
antioxidants can have health benefits as prophylactic agents (Soobrattee
et al., 2005).
The Cucurbitaceae family includes several species of cultivated plants that
have great economical importance like Watermelon (Citrullus lanatus L.),
Squash (Cucurbita maxima L.), Cucumber (Cucumis sativus L.) and
Cantaloupe (Cucumis melo L.) (Ritschel et al.,
2004). Earlier studies on the cucurbitaceae family showed that cantaloupe
pulp extract possesses high antioxidant and anti-inflammatory properties (Vouldoukis
et al., 2004). Antioxidant properties of seeds extract on streptozotocin
induced diabetic rats has been reported by Sathishsekar
and Subramanian (2005). The active principles in these vegetal extracts
are principally water soluble or lipophilic antioxidant molecules. Most of these
plant extracts contain various amounts of vitamin E, C, carotenes, triterpenoids
and other flavonoids (Aruoma, 2003) and were used as
potential antioxidant prophylactic agents for both health and disease management
(Peng et al., 2000; Clarkson
and Thompson, 2000). Cucumis melo var. utilissimus is an annual
creeping herb, cultivated in many parts of the country, especially in upper
India and particularly in Uttar Pradesh and Punjab. Fruit has been traditionally
used for the diuretic, antihelmentic and cooling effect (Nadkarni,
2002). But the seeds have not been explored yet. Increased attention has
been paid in recent years on utilization of waste and by-products to regain
their therapeutic potential. Thus components and properties of seeds are valuable
and need to be examined (Brouns, 2002). The present study
was undertaken to investigate the antioxidant and therapeutic potential of Cucumis
melo var. utilissimus seeds.
MATERIALS AND METHODS
Cucumis melo var. utilissimus seeds were purchased from Local
Grain Market Sector 26, Chandigarh (UT) in 2009. The seeds were authenticated
and the voucher specimen No. 0387 has been deposited in the Botanical and Environmental
Science Department, Guru Nanak Dev University, Amritsar, Punjab. The seeds were
cleaned, washed, dried at room temperature for 2 days and coarsely powdered.
The sample was kept in light-protected air tightened containers.
Chemicals and Drugs
Diclofenac sodium obtained from Jackson Laboratories Pvt. Ltd. Amritsar,
Morphine obtained from Government Medical College, Patiala, Carrageenan, ascorbic
acid, methanol and ethyl acetate obtained from Central Drug House Pvt. Ltd.
Mumbai, Silica Gel 60-120, potassium dihydrogen phosphate, sodium hydroxide,
silica gel G and acetone obtained from E-Merk Pvt. Ltd. Mumbai, Hexane and chloroform
obtained from Loba Chemie Pvt. Ltd. Mumbai.
The wistar albino rats of either sex were obtained from NIPER, Mohali. They
were kept at standard laboratory diet, environmental temperature and humidity.
The experimental protocol was duly approved by Institutional Animal Ethics Committee
(IAEC) and care of the animals was carried out as per the guidelines of Committee
for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA),
Ministry of Environment and Forest, Government of India (Reg. No. 874/ac/05/CPCSEA).
The powdered seeds were extracted for 72 h with methanol and stirred every
18 h using a sterile glass rod. The filtrate obtained was concentrated on a
rotary evaporator at 40°C. The concentrated filtrate was suspended in distilled
water and partitioned successively with hexane. The aqueous layer was separated
and concentrated on the water bath. The extract was stored at 4°C for further
used for various investigations.
The crude extracts were analyzed for alkaloids, tannins, saponins, flavonoids,
steroids, terpenoids and phenolic acids using standard procedures of analysis
Quantitative Scavenging Activity on DPPH Radical
The antioxidant activity was measured by DPPH method (Gill
et al., 2009). About 1 mL (500-700 μg mL-1) solution
of methanolic seed extract was added to 1.5 mL of freshly prepared methanolic
solution of DPPH (0.05 mM). The change in absorbance at 517 nm was measured
after 30 min by a spectrophotometer against a blank solution. A methanolic solution
of DPPH was used as negative control. Ascorbic acid was used as reference drugs.
Each reading was performed in triplicate. Percentage inhibition was calculated
by using the following equation:
||Absorbance of the negative control
||Absorbance of the sample
Hydrogen Peroxide Radical Scavenging Activity
The methanolic seed extract 1 mL (50-250 μg mL-1) solution
was mixed with 2.4 mL, phosphate buffer (0.1 M, pH 7.4) and 0.6 mL of H2O2
solution (43 mM). After 10 min the absorbance was measured at 230 nm using spectrophotometer
against a blank solution. The percentage inhibition was calculated. Each reading
was performed in triplicate (Buyukbalci and Sedef Nehir,
The carrageenin-induced rat paw edema assay was carried out according to
Winter et al. (1962). Extract as well as standard
drug were prepared with 1% Carboxy Methyl Cellulose (CMC) to administration
in experimental animals. The animals were divided into 5 groups of 6 animals
Experimental Design for Anti-Inflammatory Action:
Group I (Disease Control): Carrageenan (1%) was administered in the
plantar surface of rat
Group II (Standard): Diclofenac sodium (12.5 mg kg-1, p.o.)
Group III (MECU 100): Methanolic extract (100 mg kg-1, p.o.)
Group IV (MECU 200): Methanolic extract (200 mg kg-1, p.o.)
Group V (MECU 300): Methanolic extract (300 mg kg-1, p.o.)
Paw edema was induced by injecting 0.1 mL carrageenan (1%) prepared in physiological saline into the sub plantar tissues of the left hind paw of each rat. The methanolic seed extract was administered orally 30 min before carrageenan administration. The paw volume was measured at intervals of 1, 2 and 3 h by the mercury displacement method using a plethysmometer. The percentage inhibition of paw volume in drug treated group was compared with the carrageenan control group. Diclofenac sodium was used as reference drug.
||The average increase in paw volume of control
||The average increase in paw volume after the administration of test and
Extract as well as standard drug were prepared with 1% Carboxy Methyl Cellulose
(CMC). The animals were divided into 5 groups of 6 animals each.
Experimental Design for Analgesic Action:
Group I (Control Group): Vehicle (1% CMC, p.o.)
Group II (Standard Group): Morphine (10 mg kg-1 p.o.)
Group III (MECU 100 Group): Methanolic extract (100 mg kg-1
Group IV (MECU 200 Group): Methanolic extract (200 mg kg-1
Group V (MECU 300 Group): Methanolic extract (300 mg kg-1
Tail Flick Test
The tail of mice was placed on the radiant heat source (1 cm distance from
the nichrome wire) of an analgesiometer and time taken by the animals to withdraw
its tail from the radiant heat source was recorded using tail flick analgesiometer
at 0, 30, 60, 90 and 120 min time interval after the drug administration. A
cut off time was maintained 15 sec to avoid injury of tail in mice. The temperature
was maintained at 52±0.5°C.
Tail Immersion Test
The tail of mice (1 cm from the terminal part of tail) was immersed in warm
water kept constant at 52.5±0.5°C. The reaction time of the tail-flick
response was determined at 0, 30, 60, 90 and 120 min after the administration
of drugs. A cut off time was maintained 15 sec to avoid injury of tail in mice
(Palanichamy and Nagarajan, 1999).
All the results were expressed as Mean±Standard Error of Means (SEM).
The data was statistically analyzed by one way Analysis of Variance (ANOVA)
followed by Tukeys multiple range tests by using Sigmastat Version-2.0
Software. The p<0.05 was considered to be statistically significant.
Preliminary phytochemical screening of methanolic seed extract showed the maximum
presence of chemical constituents such as protein and amino acid, triterpenes,
carbohydrates, sterols and tannins (Table 1). The maximum
scavenging effect of MECU extract on the DPPH radical was 73.7% at a concentration
of 300 μg mL-1 was comparable to the scavenging effects of ascorbic
acid (Table 2). The scavenging effect of MECU extract on the
H2O2 radical was 57.52% at a concentration of 200 μg
mL-1 was comparable to the scavenging effects of ascorbic acid (Table
3). Carrageenan administration was shown to significantly rise in the paw
volume as compared to normal control group. The methanolic seed extract (100,
200 and 300 mg kg-1) exhibited statistically significant reduction
of percentage inhibition of paw volume at dose dependent manner as compared
to carrageenan control group (Table 4).
|| Phytochemical screening of Cucumis melo var. utilissimmus
|+: The presence of compounds, -: The absence of compounds
|| H2O2 radical scavenging activity of
the Cucumis melo var. utilissimus seeds extract
|| Effect of methanolic extract of Cucumis melo var.
utilissimus seeds on carrageenan induced rat paw edema
|Values are Mean±SEM of 6 animals in each group. ap<0.05
vs. Control, bp<0.05 vs. Diclofenac sodium
|| Analgesic effect of the methanolic extract of Cucumis
melo var. utilissimus seeds by tail flick
|Values are Mean±SEM of 6 animals in each group. ap<0.05
vs. Control, bp<0.05 vs. Morphine
The methanolic seed extract were exhibited marked central analgesic effect
as evidenced by significant increase in reaction time when compared to the normal
control group. The MECU extract (300 mg kg-1) were shown significant
central analgesic activity by tail flick method (Table 5).
The MECU extract (200 and 300 mg kg-1) were shown significant results
as compared to the 100 mg kg-1 pre-treated group by tail immersion
method (Table 6).
|| Analgesic effect of methanolic extract of Cucumis melo
var. utilissimus seeds by tail immersion test
|Values are Mean±SEM of 6 animals in each group. ap<0.05
vs. Control, bp<0.05 vs. Morphine
Intensive research was conducted over the last 10 years to promote the antioxidant
nutritional medicine in the nutraceutical field (Lee et
al., 2003; Dugas et al., 1999). The antioxidant
and anti-inflammatory effects of melon extracts have been already described
(Murcia et al., 2001; Campanella
et al., 2003) but little was known on the potent pharmacological
effect of the MECU extract. In the present work we demonstrated that the MECU
extract possesses significant antioxidant properties as revealed by DPPH and
H2O2 method. The DPPH radical contains an odd electron,
which is responsible for the absorbance at 515-517 nm and also for a visible
deep purple color. When DPPH accepts an electron donated by an antioxidant compound,
the DPPH is decolorized, which can be quantitatively measured from the changes
in absorbance (Lee et al., 2003). Scavenging
of H2O2 by antioxidants may be due to donation of electrons
to H2O2, thus neutralizing it to water (Bhalodi
et al., 2008). Carageenan induced inflammation is a useful model
to detect oral action of anti-inflammatory agents (Di Rosa
et al., 1971). The development of oedema in the paw of the rat after
the injection of carageenan is due to release of histamine, serotonin and prostaglandin
like substances (Vinegar et al., 1969). The MECU
extract showed significant anti-inflammatory effect. Triterpenoids isolated
from the various species of Cucurbitaceae family has been reported to have anti-inflammatory
activity (Mohammad, 2009). Tail immersion and tail methods
were carried out to evaluate the analgesic potential of seed extract. The duration
as well as the intensity of analgesia of MECU extract was dose dependent. The
analgesic effect was clearly greater at concentration of 300 mg kg-1
than the lower concentration of 100 mg kg-1. Thermal stimuli induced
fall in tail withdrawal latency is most sensitive to central acting analgesics
like narcotic drugs (Furst et al., 1988). In
the present study, we concluded that the significant antioxidant activity in
the MECU extract is essential for its therapeutic potential.
Thus from the above study it may be concluded that the methanolic extract of Cucumis melo var. utilissimus seeds has significant antioxidant activity which may be responsible for its great therapeutic potential.
Thanks to Professor A. C. Rana and all faculty members of Rayat Institute of Pharmacy for their encouragement and support. We are also grateful to Rayat and Bahra Educational and Research Trust for their unconditional helps to carry out this project.
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