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Research Article
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Evaluation of Ethanolic Seed Extract of Lagenaria siceraria for Their Therapeutic Potential |
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N.S. Gill,
S. Singh,
R. Arora
and
M. Bali
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ABSTRACT
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During the past few decades, the drugs from natural products have gained importance
in the field of medicine. Many plants and their products exhibit marked pharmacological
activities. Lagenaria siceraria fruit is one of the important natural
plant which is used in the treatment of various disorders. The present study
was designed to investigate the antioxidant, anti-inflammatory and analgesic
potential of ethanolic extract of Lagenaria siceraria seeds. The Lagenaria
siceraria seeds were evaluated for their antioxidant potential by 1,1-diphenyl-2-picrylhydrazyl
method. The extract showed significant antioxidant potential in a dose dependent
manner as compared to ascorbic acid. The extract showed maximum scavenging activity
i.e., 75.19% at 200 μg by 1,1-diphenyl-2-picrylhydrazyl free radical scavenging
method. Thus, it was further studied for anti-inflammatory activity by Carrageenan
induced rat paw edema and analgesic activity by Tail immersion method and Hot
plate method at different concentrations i.e., 100, 200 and 300 mg kg-1.
The results showed that ethanolic extract of Lagenaria siceraria seeds
possess good therapeutic potential and may be useful as a natural antioxidant
and it can be used in the treatment of inflammation and pain.
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Received: July 16, 2012;
Accepted: September 19, 2012;
Published: October 04, 2012
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INTRODUCTION
Plants have been important source of medicine for thousands of years. Natural
plants are rich source of potent medicinal compounds like morphine, cocaine,
silymarin and digitalis. Many pharmacologically active compounds which showed
antioxidant activity have been obtained from various plant parts and it is not
possible to synthesize these compounds in laboratories. There is a large amount
of evidence that phenylpropanoids and their glycosylated forms are powerful
anti oxidants. They act either by direct scavenging of reactive oxygen and nitrogen
species or by acting as chain-breaking peroxyl radical scavengers (Korkina,
2007). Among the natural compounds, phenolic substances such as phenolic
acids are known for their high anti-oxidative and anti-bacterial activity. Phenolic
compounds possess marked pharmacological activities like anti-oxidant, free-radical
scavenging, analgesic, anti-inflammatory and anti-ulcer activity (Kaur
and Arora, 2009).
Oxidation reactions inside the biological systems can produce free radicals.
Free radicals are the group of atoms having one unpaired electron and include
hydroxyl, super oxide, nitric oxide and peroxide radicals. In our body many
diseases are produced due to over production of free radicals (Sharma,
1995). These can cause damage to the cell or even cell death and can cause
the diseases such as arthritis, cancer and AIDS (Dutra et
al., 2008). To protect body cells from harmful effects of free radicals
certain defensive substances are required which can neutralize the free radicals
(Jacob, 1995). Antioxidants are agents which scavenge
these free radicals and prevent the damage caused by them (Lobo
et al., 2008). These are used in the treatment of various diseases
like cancer, congestive heart failure, Parkinsons disease. These, themselves
get oxidized and neutralize free radicals and they inhibits the oxidation of
the substrate (Valko et al., 2007). Antioxidants
are of great importance these days and several plants of Cucurbitaceae family
are known for their therapeutic potential.
Cucurbitaceae family is commonly known as family of gourds, melons or pumpkins
composed of more than 110 genera and 650-850 species. Cucurbits are a major
source of secondary metabolites. Therefore they are the largest and the most
diverse plant families (Gill and Bali, 2011). In Cucurbitaceae
family, several plants such as Cucumis anguria, Momordica dioica
and Zehneria scabra are known for their medicinal benefits because they
show antimicrobial activities (Kumar and Kamarj, 2010).
The leaves of plant Coccinia grandis are used to control hyperglycemia
and to treat various diseases such as biliary disorders, anorexia, cough, diabetic
wounds and hepatic disorders (Bhattacharya and Samanta,
2010; Yadav et al., 2010). An earlier study
showed that fruits of Momordica charantia show larvicidal activity against
three mosquito species Anopheles stephensi, Culex quinquefasciatus
and Aedes aegypti (Singh et al., 2006).
Lagenaria siceraria is another medicinally important plant of Cucurbitaceae
family which is found in India, Moluccas and Ethiopia. The cultivated form of
Lagenaria siceraria is considered to be of African and Asian origin.
Traditionally, it is used as medicine in India, China, Brazil, Hawaiian island
etc. It is commonly known as lauki and bottle gourd. Fruits of this plant are
traditionally used for their cardioprotective, cardiotonic, diuretic and nutritive
properties. These are also used in treatment of pain, ulcer, fever, pectoral
cough, asthma and jaundice (Shah and Seth, 2010a). A
decoction of Lagenaria siceraria is employed in the treatment of anasarca,
ascites and beriberi (Deshpande et al., 2008).
The extract of flowering plant of Lagenaria siceraria shows the presence
of flavones-C glycosides. It contains more proportions of soluble dietary fibres
which helps in lowering serum cholesterol levels (Milind
and Kaur, 2011). The seed oils are good source of lipids and proteins and
are used as protein supplement in human nutrition (Chinyere
et al., 2009). The seeds can be used for their therapeutic potential.
The present study was carried out to evaluate antioxidant, anti inflammatory
and analgesic potential of Lagenaria siceraria seeds.
MATERIALS AND METHODS
Plant material: Lagenaria siceraria seeds were purchased from
Local Grain Market Kharar, Mohali in August 2011. The seeds were authenticated
under the voucher specimen No: 1213 which 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 two days
and coarsely powdered. The sample was kept in light-protected conditions.
Drugs and chemicals: Ascorbic acid and carrageenan were obtained from
Central Drug House Pvt. Ltd., Mumbai, India. Hydrogen peroxide was obtained
from E-Merck Ltd., Mumbai. 1,1-diphenyl-2-picrylhydrazyl (DPPH) was obtained
from Sigma Chemical Co., USA. All other chemical reagents used were of analytical
grade which were procured from different companies (Loba Chem, Mumbai and Merck
Limited, Mumbai).
Animals: The Wistar albino rats (200-250 g) and Swiss albino mice of
either sex were obtained from Punjab Agricultural University (PAU) Ludhiana.
They were acclimatised in standard laboratory conditions like diet, environmental
temperature and humidity. The experimental protocol was 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).
Extraction: The powdered seeds were extracted for 72 h with ethanol
at room temperature and stirred occasionally. Initially, the solvent was filtered
off using muslin cloth and then with Whatman filter paper and the filtrate obtained
was concentrated under reduced pressure on a rotary evaporator at 40°C.
The concentrated filtrate was defatted successively with hexane. The aqueous
layer was separated and concentrated on the water bath. The extract was stored
at 4°C for further use for various investigations.
Phytochemical screening: The crude extract of Lagenaria siceraria
seeds was analyzed for the presence of various phytochemical constituents like
alkaloids, carbohydrates, proteins, tannins, saponins, flavonoids, steroids,
terpenoids, coumarin glycosides and phenolic acids using standard procedures
of analysis (Harborne, 1973).
Free radical scavenging activity
Quantitative scavenging activity on, 1,1-diphenyl-2 picrylhydrazyl radical:
The quantitative free radical scavenging activity of ethanolic extract of Lagenaria
siceraria was determined by using 1,1-diphenyl-2 picrylhydrazyl method according
to Gill et al. (2011).
Anti-inflammatory activity
Carrageenan-induced rat paw edema: The carrageenan-induced rat paw edema
experiment was carried out according to Jain et al.
(2011). Total five groups of rats were used and different doses were given
to them as follows:
• |
Group I: Control: Carboxy methyl cellulose (1% CMC,
p.o.)+carrageenan |
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Group II: Standard: Diclofenac sodium (12.5 mg kg-1,
p.o.)+carrageenan |
• |
Group III: EELS 100: Ethanolic extract (100 mg kg-1,
p.o.)+carrageenan |
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Group IV: EELS 200: Ethanolic extract (200 mg kg-1,
p.o.)+carrageenan |
• |
Group V: EELS 300: Ethanolic extract (300 mg kg-1, p.o.)+carrageenan |
where, EELS is ethanolic extract of Lagenaria siceraria.
Analgesic activity: In both models, groups of six mice in each group
of either sex were used with different doses as follows:
• |
Group I: Control group: Carboxy methyl cellulose suspension
(1% CMC, p.o.) |
• |
Group II: Standard group: Diclofenac sodium at a dose of 10 mg
kg-1, p.o. |
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Group III: EELS 100: Ethanolic extract at a dose of 100 mg kg-1,
p.o. |
• |
Group IV: EELS 200: Ethanolic extract at a dose of 200 mg kg-1,
p.o. |
• |
Group V: EELS 300: Ethanolic extract at a dose of 300 mg kg-1,
p.o. |
Where, EELS is Ethanolic extract of Lagenaria siceraria.
Tail immersion method: The analgesic activity was determined by tail
immersion method according to Ahmad et al. (1992).
Hot plate method: Analgesic activity of the extract was determined by
using hot plate method according to the method of Shukla
et al. (2010).
Statistical analysis: 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.
RESULTS
Preliminary phytochemical screening for ethanolic seed extract of Lagenaria
siceraria showed the maximum presence of chemical constituents flavonoids,
amino acids, triterpenoids, carbohydrates, sterols and alkaloids (Table
1). Qualitatively, discoloration of DPPH indicates scavenging potential
of the compound tested i.e., yellow color over purple. Quantitatively, antioxidant
potential of EELS was evaluated by DPPH and H2O2 radical
scavenging activity.
Table 1: |
Phytochemical screening |
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+: Present, -: Absence, ++: Maximum presence of chemical constituents |
Table 2: |
Percentage scavenging by DPPH method |
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Values are Mean±SE of triplicate experiments, DPPH:
1,1-diphenyl-2-picrylhydrazyl |
Table 3: |
Effect of EELS on carrageenan induced paw edema in rats |
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CMC: Carboxy methyl cellulose, EELS: Ethanolic extract of
Lagenaria siceraria, Values are Mean±SEM of 6 animals in each
group, ap<0.05 compared with disease control group, bp<0.05
compared with diclofenac sodium treated group |
The maximum scavenging effect of ethanolic extract of Lagenaria siceraria
extract on the DPPH radical was 75.19% at a concentration of 200 μg mL-1
and was comparable to the scavenging effects of ascorbic acid (Table
2). Carrageenan administration was shown to significantly rise in the paw
volume as compared to normal control group. The ethanolic 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 control group. At a maximum dose of 300 mg kg-1 ethanolic extract
of Lagenaria siceraria showed maximum percentage inhibition of about
56.52% after 3 h. The other doses of 100 and 200 mg kg-1 of extract
showed 14.49 and 43.46% inhibition of edema (Table 3). All
the values were significant (p<0.05) in comparison with control and standard.
The ethanolic seed extract were exhibited marked central analgesic effect as
evidenced by significant increase in reaction time when compared to the normal
control group. The ethanolic extract of Lagenaria siceraria extract (300
mg kg-1) showed significant central analgesic activity by tail immersion
method in which the reaction time of 14.43±0.08 sec was noticed after
3 h (Table 4). At dose of 100 mg kg-1 and 200 mg
kg-1 the extract showed the reaction time of 7.13±0.01 and
7.13±0.01 sec, respectively after 3 h. Using hot plate method the extract
showed better analgesic activity at a dose of 300 mg kg-1 with the
reaction time of 12.20±0.01 sec after 3 h (Table 5).
Table 4: |
Analgesic effect of EELS by tail immersion test |
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CMC: Carboxy methyl cellulose, EELS: Ethanolic extract of
Lagenaria siceraria, Values are Mean±SEM of 6 animals in each
group, ap<0.05 compared with disease control group, bp<0.05
compared with diclofenac sodium treated group |
Table 5: |
Analgesic effect of EELS by hot plate method |
 |
CMC; Carboxy methyl cellulose, EELS: Ethanolic extract of
Lagenaria siceraria, Values are Mean±SEM of 6 animals in each
group, ap<0.05 compared with disease control group, bp<0.05
compared with diclofenac treated group |
At dose of 100 and 200 mg kg-1 the reaction time of 5.14±0.09
and 11.30±0.15 sec, respectively was observed after 3 h. All the values
were significant (p<0.05) in comparison with control and standard.
DISCUSSION
The fruit of Lagenaria siceraria plant has been already described as
a prophylactic agent for the treatment of various cardiac disorders and used
as antioxidant, antihyperlipidemic, antihyperglycemic, cardiotonic and as hepatoprotective
(Deshpande et al., 2008). The leaves of Lagenaria
siceraria are also evaluated as emetic, anthelmintic and as antimicrobial
agent (Badmanaban and Patel, 2010). The pulp of the
fruit is considered as cooling agent, diuretic and antibilious (Shah
and Seth, 2010b). The fruits, leaves, oil and seeds are edible and are used
as folk medicines in the treatment of jaundice, diabetes, ulcer, piles, colitis,
insanity, hypertension, congestive cardiac failure and skin diseases. The flowers
are an antidote to poison. The stem bark and rind of the fruit are diuretic.
The seed is vermifuge. Extracts of the plant have shown antibiotic activity.
Leaf juice is widely used for baldness (Rahman, 2003).
Various extracts of fruit of Lagenaria siceraria were found to have anti-inflammatory,
analgesic, hepatoprotective, antihyperlipidemic, diuretic and antibacterial
activities (Shah et al., 2010a). In foot and
mouth diseases, green fruit slices are rubbed inside mouth (Mohale
et al., 2008). The fruits of this plant species are used for medicinal
and culinary purposes. Medicinally, fruits are also used in the treatment of
diabetes. Other uses include the treatment of cough, asthma, jaundice, kidney
stone, colds and measles. Furthermore, the fruits have laxative and diuretic
properties (Shah et al., 2010b). Leaves of Lagenaria
siceraria have been evaluated for analgesic and Central Nervous System (CNS)
depressant activity (Pawar et al., 2010). Aerial
parts of this plant is quite safe and can be used in the treatment of the chronic
diseases like diabetes without any toxicity (Saha et
al., 2011). Extracts of the plant Lagenaria siceraria were also
found to possess significant hepatoprotective activity (Lakshmi
et al., 2011). Since fruit, stem bark, rind, aerial parts and leaves
of the plant Lagenaria siceraria showed various pharmacological properties,
so in the present study, ethanolic seed extract of Lagenaria siceraria was
evaluated for its antioxidant activity followed by in vivo analgesic
and anti-inflammatory activity. The phytochemical screening indicates the presence
of flavonoids and phenolic compounds in ethanolic seed extract of Lagenaria
siceraria. The presence of flavonoids is the basis for the confirmation
of Lagenaria siceraria seeds as antioxidant agents. Antioxidant properties
are revealed by in-vitro DPPH and H2O2 method. When a
solution of DPPH is mixed with a substance that can donate a hydrogen atom,
then this gives rise to the reduced form with the loss of violet colour. The
decolourisation is due to the acceptance of electrons from antioxidant compound
(Kalpna et al., 2011). Hydrogen Peroxide is an
oxidizer and under certain catalytic conditions it can degrade into water and
oxygen by accepting electrons (Koop, 2006). Triterpenoids
isolated from the various species of Cucurbitaceae family are responsible for
anti-inflammatory activity (Saleem, 2009). Carrageenan
induced rat paw edema method was performed to determine the anti-inflammatory
activity of the extract. Carrageenan is a strong chemical use for the release
of inflammatory and proinflammatory mediators like prostaglandins, leukotrienes,
histamine, bradykinin, TNF-α, etc. (Amdekar et al.,
2012). In this model of inflammation ethanolic extract of Lagenaria siceraria
had very consistent anti-inflammatory activity and thus showed significant decrease
in the paw thickness of rat. Lagenaria siceraria inhibited the leukocyte
influx and raised LTB4 levels and decreases edema. It also cause
significant free radical scavenging activity. Tail immersion and Hot plate methods
were carried out to evaluate the analgesic potential of seed extract which show
dose dependent results. Free radicals are involved during pain stimulation and
antioxidants show reduction in such pain. Thus ethanolic extract of seeds possess
analgesic property by inhibiting the free radical formation.
There are many limitations associated with the above mentioned methods with
which various pharmacological activities have been estimated. Owing to the complexity
of the antioxidants and their mechanism of actions, no single testing method
is capable of providing a antioxidant profile of a studied sample and a combination
of different methods is necessary. Another limitation of DPPH method was the
false negative reaction with the SH-group compounds, such as Glutathione (GSH).
Despite such limitations, DPPH free radical scavenging assay can be helpful
for primary screening and finding of novel antioxidants. In the methods of estimation
of analgesic and anti-inflammatory activity, various Non-steroidal anti-inflammatory
drugs are used. Though these drugs are effective in controlling pain, their
wide range of adverse effects are the biggest limitations in their use. About
34-46% of the users of NSAIDs usually sustain some gastrointestinal damage due
to the inhibition of the protective cylco-oxygenase enzyme in gastric mucosa.
The triterpenoids isolated from various species of cucurbita family have been
reported to possess anti-inflammatory activities. The cucurbitacins from Lagenaria
siceraria were evaluated for their anti inflammatory and inhibitory effects
on the growth of cancer cell lines. Cucurbitacin glucosides are reported to
possess antioxidant and free-radical scavenging activities. Thus the triterpenoids
might be responsible for the free radical scavenging, analgesic and anti-inflammatory
activity. Thus, the extract of Lagenaria siceraria seeds can be employed
as antioxidant, analgesic, anti-inflammatory agent for human body.
CONCLUSION
The present study on extract of Lagenaria siceraria has demonstrated
that this plant has significant analgesic and anti-inflammatory properties and
it justifies the traditional use of this plant in the treatment of various types
of pains and inflammations.
ACKNOWLEDGMENTS
The authors are highly thankful 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
help to carry out this project.
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