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B.R. Srilatha et al.

/ Journal of Pharmacy Research 2012,5(6),3296-3303

Research Article
ISSN: 0974-6943

Available online through


www.jpronline.info

In Vitro Antioxidant and Free Radical Scavenging Activities


of Mukia Maderaspatana (Linn.) M.Roem
B.R. Srilatha* and S. Ananda
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore-570006, Karnataka, India

Received on:14-02-2012; Revised on: 22-03-2012; Accepted on:08-04-2012


ABSTRACT
Mukia maderaspatana (Linn.) M. Roem, is an annual monoecious, climbing vine or prostrate herb, and is an edible plant. It is also extensively used in
Folklore medicine. In this study the antioxidant and free radical scavenging activities of the plant extract are characterized. Methanol extract of the edible part
of the whole plant was prepared and studied for its total phenolics and flavonoid content, invitro antioxidant activities lipoxygenase and cyclooxygense
inhibiting activities and free radical scavenging activities. Hydroxyl radical, nitric oxide, superoxide and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging activities were determined by standard methods and compared with appropriate reference compounds like ascorbic acid, catechin or butylated
hydroxyl anisole (BHA).The antioxidant activities of M. maderaspatana were observed in a dose-dependent manner. The total antioxidant and reducing
power activities were comparable to those of ascorbic acid and BHA. Cyclooxygenase (COX), lipoxygenase (LOX) inhibiting activity and the hydroxyl
radical, nitric oxide, superoxide and DPPH radical scavenging activities were also comparable to the reference compounds like ascorbic acid, catechin,
quercetin and BHA. The cyclooxygenase activity and lipoxygenase activity of macrophages and the lipoxygenase activity of soybean extract were also
inhibited. The plant extract had phenolics and flavonoids.M. maderaspatana has a powerful antioxidant activity against various in vitro oxidative systems
and would probably be equally effective against in vivo radicals and oxidants. Since M. maderaspatana is an edible plant, it can be a source of natural
antioxidants and be useful as potential food supplement.
Keywords: Mukia maderaspatana, antioxidant activity, LOX, COX, free radical scavenging.
INTRODUCTION
In healthy individuals, the production of free radicals is balanced by the
antioxidative defence system. Free radical generation is one of the antibacterial defences and hence a natural process of the host cells. When the equilibrium favours the generation of free radicals, it results in depletion of antioxidants and results in oxidative stress. Oxidative stress contributes to diverse
disorders including diabetes, cardiovascular diseases, cancer and injury to the
central nervous system.[1] Free radicals especially oxygen derived radicals are
formed by one electron reduction of molecular oxygen and are collectively
called as Reactive Oxygen Species (ROS). ROS cause membrane lipid
peroxidation.[2] In addition they can cause the oxidation of protein, DNA and
other biological molecules.[3]

hydroperoxy endoperoxide (PGG2 ), and the peroxidase component reduces


the endoperoxide to the corresponding alcohol (PGH2 ), the precursor of
Prostaglandins, thromboxanes, and prostacyclins.[5]

In an attempt to overcome ROS induced cellular damage antioxidant supplements and food rich in antioxidants are included in the diet. Synthetic antioxidants are suspected to have side effects like liver damage and carcinogenesis
in laboratory animals.[6] Hence, natural antioxidants from plant extracts like
green tea, spice extracts and herbal extracts are studied extensively for non
toxic molecules. Plants and plant extracts have been extensively used because
of their medicinal properties to prevent and cure diseases. 80 % of the worlds
population still depends on natural products for treatment. Indian medicinal
The lipoxygenase (LOX) enzymes catalyze the oxidation of polyunsatu- system also depends on the use of plants. Phytochemicals present in plants
rated fatty-acids such as linoleic acid or arachidonic acid (AA) containing the have been shown to have diverse biological activities like cardioprotective,[7]
cis-methylene interrupted diene structure yielding conjugated hydroperox- cancer prevention[8] and inhibiton of bone resorption.[9] One of the most
ides are non-heme, non-sulfur iron dioxygenases.[4] They are widely distrib- common activities of the phytochemicals is the antioxidant and free radical
uted throughout plants, animals, fungi and some bacteria. This family of scavenging activity.
enzymes plays a major role in polyunsaturated fatty acid metabolism by
catalyzing the incorporation of molecular oxygen into certain polyunsatu- Mukia maaderaspatana (L.) M. Roem (Cucurbitaceae) is an annual
rated fatty acids, producing hydroperoxide products. These lipoxygenases monoecious, climbing vine or prostrate herb, densely covered with white
are mediators of inflammation along with another oxygenase the hairs. M. maderaspatana is traditionally used as a leafy vegetable and it is
Cyclooxygenase (COX). Cyclooxygenase (also called Prostaglandin H Syn- extensively used in Folklore medicine as good diuretic, stomachic (a digestive
thase or PGHS) is a bifunctional enzyme exhibiting both COX and peroxi- tonic), gentle aperient, antipyretic, antiflatulent, antiasthmatic and
dase activities. The COX component converts arachidonic acid to a antibronchitis. In scientific literature M. maderaspatana has been shown to
be hepatoprotective, [10] anti-inflammatory, [11] antiarthiritic, [ 1 2 ]
Immunomodulatory,[13] anti platelet [14] and antimicrobial.[15]The consumption
*Corresponding author.
of M. maderaspatana leaf tea decreased the blood pressure and showed
B.R.Srilatha
beneficial effects on lipid profile, fibrinogen, bilirubin and body mass index in
Department of Studies in Chemistry,
human volunteers. [16] This plant is edible, and is grown in the wild as well as
University of Mysore,
in the kitchen garden, and since it has many health promoting activities, the
Manasagangotri,
present study was carried out to evaluate the antioxidant and radical scavenging
Mysore-570006,
activities of methanolic extract of the edible part of the plant.
Karnataka, India

Journal of Pharmacy Research Vol.5 Issue 6.June 2012

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B.R. Srilatha et al. / Journal of Pharmacy Research 2012,5(6),3296-3303


MATERIALS AND METHODS
Chemicals
Quercetin, catechin, Linoleic acid, Adenosine-5-triphosphate (ATP),
Dithiothreitol (DTT), Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine),
1, 1-Diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Chemical Co. (Bangalore, India). Butylated hydroxyl anisole (BHA), thiobarbituiric
acid (TBA), 4- dimethylaminocinnamaldehyde were purchased from MERCK
(Mumbai, India). Folin-Ciocalteuss phenol reagent, 2-deoxy-2-ribose, phloroglucinol, ascorbic acid, gallic acid (GA),vanillin, ammonium molybdate
,Sodium nitroprusside , -napthyl-ethylenediamine dihydrochloride, ethyl
ene diamine tetra acetic acid(EDTA), nicotinamide adenine dinucleotide
(NADH), nitroblue tetrazolium (NBT), phenazonium methosulphate (PMS)
and trichloroacetic acid(TCA) were purchased from SRL(Mumbai, India).
All other chemicals and solvents used are of analytical grade.
Plant material
M. maderaspatana were collected from Hassan district, Karnataka state,
India during the month of June, authenticated by botanist Dr. M.S. Sudarshana,
University of Mysore. A voucher specimen (BOT-003-2010) deposited in
the Botany Herbarium, University of Mysore. Mysore.
Preparation of Methanolic Extract
The edible part of the whole plant was shade dried and pulverized using a
mechanical grinder. The powdered plant material (50g) was extracted with
methanol (200ml) by soxhlet apparatus for 24 hours. The extract was evaporated using a rotary vaccum-evaporator at 40o C to provide dry extract, with
a yield of 10% w/w. The extract was kept at -200 C until use.
Preliminary Phytochemical Analysis
The extracted plant material was subjected to qualitative phytochemical
screening. The presence or absence of the phytochemical constituents of
extracted plant material was analyzed by using the standard methods.[17]
Estimation of total phenolic content
The total phenolic content was estimated by using Folin-Ciocalteu reagent
according to the method of Singleton and Rossi with the slight
modification.[18]Briefly,0.1ml of the plant extract was mixed with 1ml of FC
reagent(1:1 with water) and incubated at RT for 5 min.1ml of 20%
Na2 CO3 solution was added to the reaction mixture which was incubated at
RT for 30 min, and the absorbance was read at 750nm.Gallic acid was used as
standard and the total phenolic content was expressed as gallic acid equivalents in milligrams per gram of extract.
Estimation of total flavonoids content
The total flavonoids were estimated using quercetin, phloroglucinol or catechin as standards by three different methods as follows:
Aluminium chloride (ALCL3 ) reagent method of Chang et al [19]: The plant
extract (0.1ml) was mixed with 1.5ml of methanol, 0.1ml of 10% aluminium
chloride, 0.1 ml of 1M potassium acetate and 2.8ml of distilled water. After
30 min incubation at RT, the absorbance of the reaction mixture was measured at 415nm. Quercetin was used as standard. The flavonoid content was
expressed as mg quercetin equivalent per gm of extract.
Vanillin reagent method of Swan-Hillis [20]. The plant extract (0.1ml) was
added to 1 ml of 50% methanol followed by 1.5 ml of vanillin reagent .After
incubation for 15min at RT, the absorbance of the reaction mixture was
measured at 500nm.Phloroglucinol was used as standard. The flavonoid content was expressed as mg phloroglucinol equivalent per gm of extract.
Chromogen reagent method of Delcour and de Varebeke [21]. The plant extract

(0.1ml) was added to 1ml of methanol followed by 5 ml of chromogen reagent


(1g 4 dimethylaminocinnamaldehyde dissolved in a cooled mixture of 250ml
of concentrated HCL and 750 ml of methanol, completed to 1L with methanol). After 10 min incubation at RT, the absorbance of the reaction mixture
was measured at 640nm. Catechin was used as a standard. Total flavonoid
content was expressed as mg catechin equivalent per gm of extract.
IN VITRO ANTIOXIDANT ASSAY
Total antioxidant activity
The total antioxidant activity of sample was evaluated by the method of
Prieto et al.,[22] based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds and formation of green phosphomolybdenum complex at
acidic pH.Various concentrations (20-100g/ml) of plant extract were mixed
with 1ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammoniummolybdate). The reaction mixture was incubated
in a water bath at 95C for 90 min. After cooling to room temperature, the
absorbance of the mixture was measured at 695 nm against blank. The relative activity of the sample was compared with that of standard ascorbic acid.
Reducing power assay
The reductive potential of the extract was determined according to the method
of Oyaizu.[23] Different concentrations of plant extract in 0.5 ml of MeOH
were mixed with 2.5 ml of Phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of
1% potassium ferricyanide. The mixture was incubated for 20 minutes at 50
C. At the end of the incubation, 2.5 ml of 10% trichloroacetic acid was added
to the mixture and centrifuged at 3000 rpm for 10 minutes. The upper 2.5 ml
layer was mixed with 2.5ml of distilled water and 0.5 ml of 0.1% ferric
chloride, and the absorbance was measured at 700 nm. A higher absorbance of
the reaction mixture indicated greater reducing power. BHA was used as a
positive control.
Cyclooxygenase inhibiting activity
Cycloxygenase enzyme activity was assayed using rat peritoneal macrophages. The rat peritoneal macrophages were isolated essentially by the method
described by Suzuki and Murachi.[24] The macrophages were lysed and the
lysate was used as an enzyme source. Cycloxygenase was assayed using
Amplex Red reagent by the method of Zhou et al.[25] In the presence of
cycloxygenase the non fluorescent reagent is converted to resorufin, a fluorescent molecule. The assay reaction was carried out in final volume of 1ml
containing 100 M linoleic acid and 10 M Amplex red reagent(prepared in
pure DMSO and stored at -20o C) in 50mM Tris HCl buffer pH 9.0. The
reaction mixture was incubated for 5 min at 37o C and the relative fluorescence intensity was measured in a fluorimeter using appropriate blanks. The
excitation was at 563nm and emission was at 587nm. The cycloxygenase
inhibitor assay was carried out by pre incubating the enzyme with the plant
extract for 15 min prior to determining its COX activity. The results are
expressed as percent inhibition of the COX activity. The results were compared with standard phytochemicals.
Lipoxygenase inhibiting activity
Lipoxygenase enzyme activities were assayed using rat peritoneal macrophages as source of enzyme for 5- Lipoxygenase and soybean extract as a
source for 12- Lipoxygenase.
5-Lipoxygenase inhibition:
Rat peritoneal macrophages were isolated as described above. Macrophage
lysate was used as a source of the 5- Lipoxygenase. The enzyme assay was
carried out essentially by the method of Aharony and Stein.[26] Briefly, the
reaction was carried out in a final volume of 2ml containing 50 M DTT, 200
M ATP, 300 M CaCl2, and 150 M Linoleic acid. The reaction was started

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B.R. Srilatha et al. / Journal of Pharmacy Research 2012,5(6),3296-3303


by adding 50l of the enzyme preparation. The formation of 5-HETE was
followed for 3min at 234nm at room temperature. The 5-LOX inhibiting
activity of the plant extract was determined by pre incubating the enzyme
with the plant extract prior to determining its 5-LOX activity. The results are
expressed as percent inhibition of the 5-LOX activity. The reference
phytochemicals were also used for their inhibitory activity on 5-LOX.
Preparation of Soybean Lipoxygenase:
The soybean lipoxygenase was partially purified by the method of Axelrod
et al.[27] Briefly soybean seeds were obtained from National Seed Collection
Centre and powdered with a pestle and mortar. The powder was suspended
in petroleum ether (60-80) in cold and extracted three times. The defatted
powder was dried in air and suspended in 0.2M sodium acetate buffer pH 4.5
at 10% concentration (w/v) and stirred at 4o C for 1 hr. The suspension was
allowed to settle and then decanted. It was then centrifuged in a refrigerated
centrifuge at 4o C for 10 min at 5000rpm. The clear supernatant was used as
the source of enzyme.
12- Lipoxygenase inhibition:
Soybean lipoxygenase activity was assayed by the procedure of Axelrod et
al,.[27].Briefly the reaction was carried out in a final volume of 3 ml containing
2.9 ml of 0.1M borate buffer pH 9.0 and 50 l of 10mM Linoleic acid. The
reaction was started by the addition of 50 l of the soybean enzyme extract.
The enzyme activity was measured by following the formation of the product, 12-HETE at 234nm for up to 1min. The enzyme inhibition was determined by pre incubating the enzyme with the plant extract or standard
phytochemicals prior to determining its 12-LOX activity. The results are
expressed as percent inhibition of the 12-LOX activity.
Hydroxyl radical scavenging assay
The hydroxyl radical (OH) scavenging activity was determined by the method
of Halliwell et al,.[28] In this assay, OH is produced by reduction of H2 O2 by
the transition metal (iron) in the presence of ascorbic acid. The generation of
OH is detected by its ability to degrade deoxyribose to form products, which
on heating with thiobarbituric acid (TBA) forms a pink coloured chromogen.
Addition of different concentrations of plant extract competes with deoxyribose for OH and diminishes the colour formation. The reaction mixture in
a final volume of 2 ml, contained 0.1 ml of EDTA (1mM),0.01 ml of
FeCl3 (10mM), 0,1 ml of H 2 O2 (10mM),0.36 ml of deoxyribose (10mM),1 ml
of plant extract (concentrations from 20-200g/ml), 0.33 ml of phosphate
buffer(50 mM, pH 7.4) and 0.1 ml of ascorbic acid(1mM) added in sequence.
The mixture was incubated at 37o C for 1 hour. 1 ml of the incubated mixture
was mixed with 1 ml of 10% trichloroacetic acid and 1 ml of TBA (1% in
0.025 M NaOH) and heated for one hour on water bath at 80o C when a pink
chromogen developed, which was measured at 532 nm. The scavenging activity was compared using BHA as a standard. Decreased absorbance of the
reaction mixture indicated increased hydroxyl radical scavenging activity.
The percentage radical scavenging activity was calculated by using the formula:
% inhibition [OH] =

A0 A 1
A
x 100

(Equation 1)

Where A 0 was the absorbance of the control and A 1 was the absorbance in the
presence of the samples or standard.
DPPH radical scavenging assay
The DPPHradical scavenging activity of the plant extract was determined
spectrophotometrically by the method of Williams et al.[29] DPPH reacts
with an antioxidant compound that can donate hydrogen radical and thereby

it is reduced. A change in colour, from deep violet to light yellow, was


measured. DPPH is a stable free radical and accepts an electron or hydrogen
radical to become a stable diamagnetic molecule. The intensity of the yellow
colour depends on the amount and nature of radical scavenger present in the
sample and standard compounds. The reaction mixture containing 0.5 ml of
0.5 mM DPPH, and various concentrations of plant extract (10- 80g/ml),
were made up to 2 ml with methanol. Then the tubes were incubated at 37o C
for 30 minutes and the absorbance of this solution was measured at 517 nm.
BHA was used as the standard for comparison. The percentage radical scavenging activity was calculated by using equation 1 as described above.
Nitric oxide radical scavenging assay
Nitric oxide radical (NO) generated from sodium nitroprusside in aqueous
solution at physiological pH reacts with oxygen to produce nitrite ion, which
is measured by the Griess- Illosvoy reaction.[30] Briefly, 3ml of the mixture
containing 10mM sodium nitroprusside and different concentrations of plant
extract (100-600g/ml) in phosphate buffer (pH 7.4) were incubated at 25oC
for 60 min. After incubation, 0.5 ml of the reaction mixture was mixed with
0.5 ml of sulfanilic acid reagent (0.33% in 20 % glacial acetic acid) and
allowed to stand for 5 min for complete diazotization. Then 1ml of naphthyl
ethylene diamine dihydrochloride (0.1% in water) was added and the solution
mixed and allowed to stand for 30 min at 25o C. A pink coloured chromophore
was formed in diffused light. The absorbance was measured at 540nm.Ascorbic
acid was used as a standard. The percentage radical scavenging activity was
calculated by using equation 1 as described above.
Superoxide anion radical scavenging assay
Superoxide anion radical (O2 - ) scavenging activity was determined by
Nishimikis method[31] with slight modifications. The assay was based on the
oxidation of NADH by phenazine methosulphate (PMS) in order to liberate
PMSred . PMSred converts oxidized nitroblue tetrazolium (NBT oxi) to the reduced form NBT red , which forms a violet coloured complex. The formation of
violet colour indicated the generation of superoxide anions. A decrease in the
formation of colour in the presence of the antioxidant was a measure of its
superoxide scavenging activity. 1 ml of NBT (156 M NBT in 100 mM
phosphate buffer, pH 7.4), 1 ml of NADH (468 M in 100 mM phosphate
buffer, pH 7.4), and various concentrations of plant extract (240-800g/ml),
were mixed well in a final volume of 3 ml. The reaction was started by the
addition of 100 l of PMS (60 M PMS in 100 mM phosphate buffer, pH
7.4). The reaction mixture was incubated at 25C for 5 minutes, and the
absorbance was measured at 560 nm. Catechin was used as the standard.
Decreased absorbance of the reaction mixture indicated increased superoxide
anion scavenging activity. The percentage radical scavenging activity was
calculated by using equation 1 as described above.
RESULTS
Preliminary Phytochemical Analysis
The phytochemical analysis of the total methanolic extract revealed the
presence of various bioactive components, of which flavonoids and phenolics were most prominent, while it did not contain any saponins.The result of
the phytochemical test has been summarized in Table 1.
Table 1: Qualitative phytochemical screening of Mukia maderaspatanaextract
Plant Constituents Tested

Result

Alkaloids
Flavonoids
Tannins
Saponins
Glycosides
Steroids
Phenolics
Aminoacids
Quinones

+
+
+
+
+
+
+
+

Phytochemicals were qualitatively detected by the standard


methods.+ Present; - Absent

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B.R. Srilatha et al. / Journal of Pharmacy Research 2012,5(6),3296-3303

Table 2: Total phenolic and flavonoid contents of Mukia maderaspatanaextract

0.8
0.6
0.5

Ph
lor
C
og
luc ont
ro
ino
l
l5
0u
g

0.4
0.3
Ascorbic acid
Mukia

0.1
0

Figure 1: Total antioxidant activity of methanolic extract of M. maderaspatana and ascorbic acid.
The total antioxidant capacity was assayed based on the reduction of Mo (VI) to Mo (V) by the
antioxidant compounds and formation of green phosphomolybdenum as described in the methods.

Reducing power assay


The total reducing power of M.maderaspatana methanolic extract compared
with BHA is shown in Figure 2.Both the extract and BHA showed a linear
increase in the reducing activity in a dose dependent manner. The plant
extract could reduce the Fe3+ ions, but had a lesser reductive activity than
corresponding concentration of BHA.
0.9
0.8
OD at 700 nm

0.7
0.6
0.5
0.4
0.3
BHA
Mukia

0.2
0.1
0
-0.1 0

20

40
60
Concentration g/ml

Qu
erc
eti
n5
0u
g

50
ug
Ph
lor
og
luc
ino
l50
ug

Ca
tec
hin

120

80

Figure 2: Reducing power activity of methanolic extract of M. maderaspatana and BHA.


The total reducing power was assayed based on the ability of the antioxidant compounds to
reduce Fe+++ to Fe++ as described in methods.

Figure 4: 5-Lipoxygenase inhibiting activity of methanolic extract of M. Maderaspatana and


Catechin, Phloroglucinol and Quercetin.
5-LOX activity was assayed using linoleic acid as substrate in rat peritoneal macrophages. Plant extract
and phytochemicals were tested for their ability to inhibit formation of 5-HETE as described in methods.

The M .maderaspatana extract inhibited 12-lipoxygenase and is shown in


Figure 5. When 50 g each of M.maderaspatana extract and standard
phytochemicals were assayed for their enzyme inhibition activity , the extract gave the highest inhibition of 35.5% compared with quercetin which
shows only 21.7% inhibition.
100
90
80
70
60
M
uk
ia
50
ug

100

Qu
erc
eti
n5
0u
g

40
60
80
Concentration g/ml

50
ug

20

Percent
Percent
Activity
Activity

Ca
tec
hin

OD at 695 nm

0.7

120
100
80
60
40
20
0
M
uk
ia 5
ug
M
uk
ia
15
ug

0.9

-0.1

El
lag
ic
ac
id
50
ug

Lipoxygenase inhibiting activity


The M. maderaspatana extract inhibited 5-lipoxygenase in a dose dependent
manner (Figure 4). The amount of extract causing 50% inhibition of activity
was 4.63g. Under identical conditions 5g quercetin showed 58.9% inhibition and 10 g of catechin showed 50.1% inhibition.

5u
g

Total antioxidant capacity


The total antioxidant activity of the M.maderaspatana methanolic extract is
shown in Figure 1 and compared with ascorbic acid. Both the extract and
ascorbic acid showed a linear increase in absorbance with increase in concentration. However, M.maderaspatana extract showed a significant change at
20 to 80g/ml concentration and more powerful antioxidant activity than
ascorbic acid by showing a higher absorbance than the corresponding ascorbic acid concentration.

Figure 3: Cyclooxygenase inhibiting activity of methanolic extract of M. Maderaspatana and


Catechin, Phloroglucinol, Quercetin and Ellagic acid.
Cyclooxygenase activity was assayed by a fluorimetric method using Amplex Red reagent. Rat peritoneal
macrophages were the source of enzyme and linoleic acid was used as the substrate. Plant extract and
phytochemicals were tested for their ability to inhibit the formation of fluorescent resorufin from non
fluorescent Amplex red reagent as described in methods.

Qu
erc
etin

Phenolics were assayed by the method of Singleton and Rossi using Gallic acid
as the standard. Flavonoids were assayed using three methods and three
different standards as described in methods. b Results are expressed as milligram
equivalent per gram of extract.

M
uk
ia
10
ug

0.2

20

10
ug

41
8.8
0.1
3

40

Ca
tec
hin

Gallic acid
Quercetin
Catechin
Phloroglucinol

60

M
uk
ia5
ug

Phenolics
Flavonoids

80

con
tro
l

Total content
(mg equivalent/gm)b

100

Co
ntr
Ph
lor
ol
og
luc
ino
l5
0u
g

Standard

120

Percent Activity

Phytochemicala

Cyclooxygenase inhibiting activity


M. maderaspatana extract showed Cyclooxygenase inhibiting activity in a
dose dependent manner. The amount of extract which caused 50% inhibition
was 8.42g. Under identical conditions 50 g of the standard phytochemicals
inhibited COX activity from 82% to 97% which is shown in Figure 3.
Percent Activity

Total phenolic and flavanoid content


Total phenolics and flavanoids content in the methanolic extract are shown in
Table 2.The phenolic content was very high compared with flavanoid content. Among the flavanoids, the three different assay methods with three
different standards, namely Quercetin, Phloroglucinol and Catechin yielded
different quantities of the flavanoids. The highest amount was detected by
the Aluminium chloride reagent using quercetin as standard and the least
amount was detected by the dimethyl amino cinnamaldehyde reagent using
catechin as standard.

Figure 5:12-Lipoxygenase inhibiting Activity of methanolic extract of M. Maderaspatana and


Catechin, Phloroglucinol and Quercetin.
12-LOX activity was assayed using linoleic acid as substrate. Soybean extract was used as a source of the
enzyme. Plant extract and phytochemicals were tested for their ability to inhibit formation of 12-HETE as
described in methods.

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120
Percent Inhibition

nitrite release may be attributed to a direct NO scavenging effect and is


shown in Figure 8 compared with Ascorbic acid. The results show that
extract and reference compound, ascorbic acid had scavenging activity with
IC50 values of 269.0g/ml and 155.7g/ml respectively (Table 3). The Ascorbic acid showed higher NO scavenging activity than the extract.
100
Percent Inhibition

Hydroxyl radical scavenging assay


The scavenging activity of M.maderaspatana extract on OH is shown in
Figure 6 and compared with BHA. The IC50 value (Table 3) of scavenging
effect was 22.9g/ml and 37.6g/ml for the extract and standard BHA respectively. M.maderaspatana extract showed inhibition of OH formation
during incubation period and the percentage of inhibition was higher than
that of BHA at all concentrations.

100
80
60
40

80
60
Ascorbic Acid

40

Mukia

20
0

BHA
Mukia

200

20

50

100
150
Concentration g/ml

200

250

600

800

Figure 6: Hydroxyl radical scavenging activity of methanolic extract of M. maderaspatana and


BHA.
The Hydroxyl radical was generated from H2O2 and reacted with deoxydibose. The degradation products
of deoxyribose were reacted thiobarbituric acid. The ability of the antioxidant compounds was tested by
their ability of scavenge OH radicals as described in the methods.

Table 3: Radical scavenging activities of Mukia maderaspatana extract


Radical scavenging assay Reference
Standards

IC 50 values(g/ml) of
Standard Mukia extract

Hydroxyl radical
DPPH radical
Nitric oxide
Superoxide

37.6
8.1
155.7
400

BHA
BHA
Ascorbic acid
Catechin

22.9
15.9
269
300

The radical scavenging assays were carried out using the methanolic extract of M.
maderaspatana and compared with reference standards. The IC50 values were calculated from
linearized plots of concentration versus activity.

DPPH radical scavenging assay


The DPPH assay is based on the ability of DPPH, a stable free radical, to
decolourize in the presence of antioxidants. The scavenging activity of
M.maderaspatana extract on DPPH is shown in Figure 7 and compared with
BHA. The IC50 values (Table 3) of the standard BHA and extract was found
to be 8.1g/ml and 15.9g/ml respectively. The results show that BHA had
the higher DPPH scavenging activity than the extract. The scavenging activity was found to be concentration dependent.

Figure 8: Nitric oxide radical scavenging activity of methanolic extract of M. maderaspatana and
Ascorbic acid.

NO generated from sodium nitroprusside reacts with oxygen to produce nitrite ion, which is measured by
the Griess reaction. The ability of the antioxidant molecule to prevent the formation of nitrite was used as
a measure of nitric oxide scavenging activity as described in the methods.

Superoxide radical scavenging assay


The superoxide anion derived from dissolved oxygen by Phenazine
methosulphate/NADH coupling reaction reduces nitro blue tetrazolium. Figure 9 shows the ability of M.maderaspatana and the standard catechin to
quench superoxide radicals in this scavenging activity assay. As mentioned in
Table 3, the extract as well as standard catechin showed the scavenging
activity with IC50 values of 300g/ml and 400g/ml respectively. The percentage of inhibition by the extract was greater than that of catechin.

Percent Inhibition

90
80
70
60
50
40
30
20
10
0

Catechin
Mukia

200

400

600

800

1000

1200

Concentration g/ml
Figure 9: Superoxide radical scavenging activity of methanolic extract of M. maderaspatana and
Catechin.
The superoxide anion scavenging activity was determined by the ability of the antioxidant molecules to
decrease the formation of reduced NBT as described in the methods.

120
100

Percent Inhibition

400

Concentration g/ml

80
60
BHA
Mukia

40
20
0
0

20

40
60
Concentration g/ml

80

100

Figure 7: DPPH radical scavenging activity of methanolic extract of M. maderaspatana and BHA.
The DPPH radical scavenging activity was determined by the ability of the antioxidant molecules to
donate an electron or hydrogen radical to DPPH to make it a stable diamagnetic molecule as described in
the methods.

Nitric oxide radical scavenging assay


M. Maderaspatana extract decreased the amount of nitrite generated from
the decomposition of sodium nitroprusside. In this assay, the suppression of

DISCUSSION
Reactive oxygen species( ROS), such as superoxide anions, hydrogen peroxide, and hydroxyl, nitric oxide and peroxynitrite radicals, play an important
role in oxidative stress related to the pathogenesis of various disease.[32]
Most of the natural products contain abundant and potent antioxidants.
They exert their beneficial effects through the additive action of several
chemical compounds acting at single or multiple target sites associated with
a physiological process in contrast to synthetic antioxidants based upon a
single chemical. The antioxidant activity of the 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. [33]
Our phytochemical analysis (Table 1) is consistent with literature, although

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we have not identified the individual components, the class of molecules
reported in literature are shown to be present in the methanolic extract. In the
total phenolics and flavonoids estimation our result show M. maderaspatana
contained significant amount of flavonoids and phenolics (Table 2). Phytochemical studies of the entire leaf extract of M. maderaspatana report the
presence of coumarins, phenolic compounds, flavonoids, steroid, triterpene,
multiple glycosides (22,23-dihydrospinasterol3-O--D-glucoside),
decosanoic acid and mixture of fatty acids.[34] The polyphenolic compounds
are major plant constituents having potential antioxidant activity mainly due
to their redox properties. The mechanism of action of flavonoids is through
scavenging or chelating process and phenolic contents are also very important plant constituents because of their scavenging ability due to their hydroxyl groups.[1] Both of these compounds have good antioxidant potential
and their effects on human nutrition and health are considerable.
The total antioxidant activity of M.maderaspatana increased with increasing
concentration of the extract indicating the reduction of Mo (VI) to Mo (V)
and subsequent formation of phosphomolybdate complex by donating an
electron. The higher reducing power shown by the methanolic extract in
comparison with ascorbic acid(Figure 1) suggests that the methanolic extract
is able to reduce water soluble oxidants and that it is a better anti oxidant than
ascorbic acid. Antioxidant activity has been reported to go together with the
development of reducing power and is a significant indicator of its potential
antioxidant activity. [35] For measurements of the reductive ability, we also
studied the Fe3+ to Fe2+ transformation in the presence of a methanolic extract
using the method of Oyaizu. [23] The reducing capacity of M. maderaspatana
might be due to its hydrogen donating ability. The reducing power of M.
maderaspatana increased in a concentration dependent manner, indicating
that some compounds are both electron donors and could react with free
radicals to convert them into more stable products. They can also terminate
radical chain reactions and this activity was comparable with that of BHA
(Figure 2).
There are three main LOX enzymes that are known to be involved in the
leukotriene pathway, 5-LOX, 12-LOX, and 15-LOX. Most attention has
been focused on 5-LOX, which appears to be the key enzyme when it comes
to blocking leukotriene synthesis.[36] The direct 5-LO inhibitors can be subdivided into three groups: redox-active compounds, iron-ligand inhibitors
with weak redox-active properties, and (non-redox) competitive 5- LO inhibitors. Natural products which are 5-LO inhibitors, reduce the active site
iron thereby uncoupling the catalytic cycle of the enzyme. Thus, phenols,
flavonoids, and coumarins, are efficient 5-LO inhibitors in vitro and in vivo.[37]
The key regulatory enzyme of the pathway of conversion of arachidonic acid
to prostaglandin (PG) G2 and PGH2.is COX (PGH synthase). PGH2 is
subsequently converted to a variety of eicosanoids that include PGE2, PGD2,
PGF2a, PGI2, and thromboxane A2. Prostaglandins are formed by the oxidative cyclization of the central 5 carbons within 20 carbon polyunsaturated
fatty acids It is now well established that there are two distinct isoforms of
COX, namely COX-1 and COX-2. Using the Fluorescent Activity Assay
provides a convenient fluorescence-based method for detecting COX-1 or
COX-2 activity in both crude cell lysates and purified enzyme. preparation
Ideally, one would like to be able to inhibit both COX-2 and 5-LOX simultaneously, to maximize anti-inflammatory effects.[38] Interestingly, the
M.maderaspatana extract had both COX inhibitory activity and 5-LOX
inhibitory activity higher than the standard phytochemicals(Figure 3and 4).
The inhibition was dose dependent. Current understanding of the biological
significance of these enzymes and products in humans health and disease is
still limited.
The hydroxyl radical has a short half-life and is the most reactive and damaging of ROS. It causes oxidative damage to DNA, lipids and proteins.[39] The

M.maderaspatana extract was investigated for its ability to scavenge OH


radicals generated by Fenton reaction. As the methanolic extract or standard
BHA is added to the Fenton reaction mixture, the hydroxyl radicals are
scavenged and prevent the degradation of 2-deoxyribose. Like many other
free radicals, OH can be neutralized if it is provided with hydrogen atoms.Our
results shown in Figure 6 and Table 3, indicate that the scavenging activities
of the methanolic extract was higher than that of BHA. This may be because
of the presence of phenolic substances which can have active hydrogen
donating ability of hydroxyl substitutions. DPPH is stable nitrogen centered
free radical which can be effectively scavenged by antioxidants and can accept an electron or hydrogen radical to become a stable diamagnetic molecule.[40] It has been widely used for rapid evaluation of the antioxidant
activity of plant extracts relative to other methods. In its radical form,
DPPHdisappears on reduction by methanolic extract resulting in a colour
change from purple to yellow. Figure 7 and Table 3 illustrate the effect of M.
maderaspatana on DPPHscavenging, is thought to be due to their hydrogen
donating ability which was comparable to that of BHA. In addition, the
ability to scavenge the DPPH radicals is related to the inhibition of lipid
peroxidation since the first attack of the free radicals is on the membrane
lipids. Nitric oxide radicals play important roles in various types of inflammatory conditions including juvenile diabetes, multiple sclerosis, arthritis and ulcerative colitis. [41]
Nitric oxide radical scavenging study proved that M. maderaspatana extract
is a potent scavenger of nitric oxide. The extract inhibits nitrite formation by
competing with oxygen to react with nitric oxide directly and also inhibit its
synthesis. The scavenging activities of the extracts and ascorbic acid showed
that the nitric oxide scavenging activity of the former is comparable to that of
the latter (Figure 8 and Table 3). Further, the lethal consequence of NO
increases significantly upon reaction with superoxide radical resulting in the
formation of highly reactive peroxynitrite anion (ONOO-), especially its
protonated form, peroxynitrous acid (ONOOH). ONOO- has added to the
pathogenesis of diseases such as heart disease, Alzheimers disease, and
atherosclerosis.Superoxides are produced from molecular oxygen due to oxidative enzymes [42] of body as well as via non enzymatic reaction such as auto
oxidation by catecholamines. In the present study, superoxide anion derived
from dissolved oxygen by PMS/NADH coupling reaction reduces NBT. The
figure 9 and Table 3 shows the scavenging effect of M. maderaspatana. The
decrease in absorbance at 560 nm with various concentration of extract indicates the consumption of superoxide anion in the reaction mixture. Superoxide anions indirectly initiate the lipid oxidation as a result of superoxide and
hydrogen peroxide, serving as precursors of singlet oxygen and OH. Inhibition of superoxide generation by M. maderaspatana may be due to the
presence of flavonoids. Robak [43] reported that the antioxidant properties of
flavonoids are effective primarily by the scavenging of superoxide anion. In
our study the superoxide radical scavenging activity of the extract was better
than that of Catechin (Figure 9).

CONCLUSION
The antioxidative data presented in this study demonstrates that M.
maderaspatana exhibits free radical scavenging activity as well as a primary
antioxidant activity that reacts with free radicals, which may limit free radical
damage occurring in the human body. The antioxidative effects of M.
maderaspatana were accomplished in a dose-dependent manner. The various antioxidant properties of the extract may be attributed to strong hydrogen donating ability and their effectiveness as scavengers of OH, superoxide,
Nitric oxide, DPPH and other free radicals. Simultaneous inhibition of COX
and LOX by extract suggests that it is potentially anti inflammatory. Thus,
M. maderaspatana has a powerful antioxidant activity against various in
vitro oxidative systems and would probably be equally effective against in
vivo radicals and oxidants. Since M. maderaspatana is an edible plant it can

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be a source of natural antioxidants and anti inflammatory molecules and
useful as potential food supplement.
ACKNOWLEDGEMENTS
The financial assistance from the Institution of Excellence (IOE) project of
the University of Mysore is gratefully acknowledged. B.R.Srilatha is thankful to the IOE for the award of Fellowship.
REFERENCES
1. Cook NC, Samman S, Flavonoids-chemistry, metabolism,
cardioprotective effects and dietary sources, Nutr Biochem, 7,
1996, 66-76.
2. Pryor WA, Free radical reactions and their importance in
biochemical systems, Fed Proc, 32, 1973, 1862- 1869.
3. Adelman R, Saul RL, Ames B, Oxidative damage to DNA: relation
to species metabolic rate and life-span, Proc Nal Acad Sci, 85,
1998, 2706-2708.
4.
Bergstrom S, Holman RT, Total conjugation of linoleic acid in
oxidation with lipoxidase, Nature, 161(4080), 1948, 55. [Pub
Med: 18899663]
5. Mead, J. F., Alfin-Slater, R. B., Howton, D. R., and Popjak, G,
Prostaglandins, thromboxanes, andprostacyclin, In
Lipids:Chemistry, Biochemistry, and Nutrition (Mead, J. F., ed),
Plenum Press, New York. 1986, pp, 149-216.
6. Branen A, Toxicology and biochemistry of butylated hydroxyanisole and butylated hydroxytoluene, J Am Oil Chem Soc, 52, 1975,
59-63.
7. Sadzuka Y, Sugiyama T, Shimoi K, Kinae N, Hirota S, Protective
effect of flavonoids on doxorubicin induced cardiotoxicity, Toxicology Letters, 92,1997, 1-7.
8. Marchand LL, Cancer preventive effects of flavonoids: a review,
Biomedicine and Pharmacotherapy
56, 2002, 296-301.
9.
Yamaguchi M, Gao YH, Inhibitory effect of genistein on bone
resorption in tissue culture,
Biochemical Pharmacology, 55,
1998, 71-76.
10. Thabrew MIRA, Jayatilaka PAPW, Perera DJB, Evaluation of
efficacy of Melothira madaraspatana in the alleviation of carbon
tetrachloride induced liver dysfunction, J Ethnopharmacol, 23,
1988, 305-312.
11. Sinha BN, Sasnal D, Basu SP, Pharmacological studies on Melothria
maderaspatana, Fitoterapia, 68, 1997, 75-78.
12. Ramakrishnamacharya CH, Krishnaswamy MR, Rao RB,
Vishwanathan S, Anti- inflammatory efficacy of Melothria
maderaspatana in active rheumatoid arthritis, Clin Rheumatol,
15(2), 1996, 214-215.
13. Thabrew MI, De Silva KTD, Labadio RP, De Bio PAF, Vander
Berg B,Immunomodulatory activity of three SriLankan medicinal
plants used in hepatic disorders, J Ethanopharmacol, 33(12),1991,63-66.
14. Iman RA, Lakshmi Priya B, Chithra R, Shalini K, Sharon V,
Chamundeeswari D, Vasantha J,et al, In vitro antiplatelet activity
guided fractionation of aerial parts of Melothria maderaspatana,
Indian J Pharm Sci, 68(5),2006, 668-670.
15. Hemamalini K, Varma VK, Antimicrobial activity of methanolic
leaves extract of Melothria maderaspatana Linn,
Pharmacologyonline, 3, 2007, 323-326.
16. Raja B, Kaviarasan K, Arjunan MM, Pugalendi KV,Effect of
Melothria Maderaspatana leaf-tea consumption on blood pressure, lipid profile, anthropometry, fibrinogen, bilirubin and albumin levels in patients with hypertension, J Altern Complement
Med, 13(3),2007,349-354.

17. Kokate CK, Textbook of pharmacognosy, Nirali Publ, Pune, 1985,


112.
18. Singleton VL, Rossi JA, Colorimetry of total phenolics with phosphomolybdic- phosphotungstic acid reagents, Am J Eno Vitic,16,
1965,144-158.
19. Chang C, Yang M, Wen H, Chem J, Estimation of total flavonoid
content in propolis by two complementary colorimetric methods,
J Food Drug Analysis, 10, 2002, 178-182.
20. Swain T, Hillis WE, The phenolic constituents of Prunus
domestica.I. The quantitative analysis of phenolic constituents, J Sci Fd Agri, 10, 1995, 63-68.
21. Delcour JA, DeVarebeke DJ, A new colorimetric assay for flavonoids in Pilsner Beers, J Inst Brew, 91, 1985, 37-40.
22. Prieto P, Pineda M, Aguilar MM, Spectrophotometric quantitation
of antioxidant capacity through the formation of a
phosphomolybdenum complex: specific application to the determination of vitamin E, Anal Biochem, 269,1999,337-341.
23. Oyaizu M, Studies on product of browning reaction prepared
from glucose amine, Japan J Nutr, 44, 1986, 307-315.
24. Suzuki Y and Murachi T, The Occurrence of a Neutral Protease
and Its Inhibitor in Rat Peritoneal Macrophages,J.Biochem,82,
1977, 215-220.
25. Zhou, M., Diwu, Z., Panchuk-Voloshina, N., and Haugland, R, A
stable nonfluorescent derivative of resorufin for the fluorometric
determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases,
Anal. Biochem, 253, 1997, 162-168.
26. D. Aharony, R.L. Stein, Kinetic mechanism of guinea pig neutrophil 5-lipoxygenase, J. Biol. Chem, 261, 1986, 1151211519.
27. Axelrod, B., Cheesebrough, T.M., & Laasko, S, Lipoxygenase from
soybeans, Methods in Enzymology, 71, 1981, 441451.
28. Halliwell B, Gutteridge JMC, Arnoma OL, The deoxyribose
method: A simple test tube assay for the determination of rate
constant for reaction of hydroxyl radical, Anal
Biochem,165,1987,215-219
29. Brand Williams W, Cuvelier ME, Berset C, Use of free radical
method to evaluate antioxidant activity, Lebensmittel-Wissenschaft
and Technologi, 1995, 28:25-30.
30. Green LC, Wanger DA, Glogowski J, Analysis of nitrate, nitrite
and (15 N) Nitrate in biological fluids, Anal Biochem, 126, 1982,
131-138.
31. Nishimiki M, Appaji N, Yagi K, The occurrence of superoxide
anion in the reaction of reduced phenazine methosulphate and
molecular oxygen, Biochem Biophys Res Commun,46, 1972,849854.
32. Finkel, T and Holbrook, N.-J, Oxidants, oxidative stress and the
biology of aging, Nature, 408, 2000, 239-247.
33. Oktay M,Gulcin I, Kufrevioiglu OI, Determination of in vitro
antioxidant activity of fennel (Foeniculum vulgare) seed extracts,
Lebensm Wiss Technol, Food Sci Technol, 36, 2003, 263-271.
34. Sinha BN, Sasmal D, Basu SP, Pharmacological studies on Melothria
maderaspatana,Fitoterapia,1, 1997,75-78.
35. Yen GC, Duh PD, Tsai CL, Relationship between antioxidant activity and maturity of peanut hulls, J Agric Food Chem, 41, 1993,
67-70.
36. Smith W. L. and Murphy R. C, The eicosanoids: cyclooxygenase,
lipoxygenase and epoxygenase pathways D.E. Vzmcc and J.E.
Vance (Eds.), Biochemistry of Lipids. Lipoproteiils apul Membranes (4th Edn), Elsevier Science B.V.2002, CHAPTER 13, pp341371.
37. Yoshimoto T, Furukawa M, Yamamoto S, Horie T and Watanabe-

Journal of Pharmacy Research Vol.5 Issue 6.June 2012

3296-3303

B.R. Srilatha et al. / Journal of Pharmacy Research 2012,5(6),3296-3303


Kohno S, Flavonoids: potent inhibitors of arachidonate 5lipoxygenase, Biochem Biophys Res Commun, 116,1983, 612-8.
38. Altavilla D, Squadrito F, Bitto A, Polito F, Burnett BP, Di Stefano
V. and Minutoli L, Flavocoxid, a dual inhibitor of cyclooxygenase
and 5-lipoxygenase, blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages, Br J Pharmacol, 157,
2009, 1410-8.
39. Spencer JPE, Jenner A, Aruoma OI, Intensive oxidative DNA damage promoted by L-DOPA and its metabolites implications for
neurodegenerative disease, FEBS Lett, 353,1994,246-250.

40. Villano D, Fernandez-Pachona MS, Moyab ML, Troncosoa AM,


Garcia- Parrilla MC, Radical scavenging ability of polyphenolic
compounds towards DPPH free radical,Talanta,71(Suppl
1),2007,230-235.
41. Miller MJ, Sadowska-Krowicka H, Chotinaruemol S, Kakkis JL,
Clark DA, Amelioration of chronic ileitis by nitric oxide synthase
inhibition, The JPharmac and Experi Thera, 264,1993,11-16.
42. Hemmani T, Parihar MS, Reactive oxygen species and oxidative
DNA damage, Ind J Physiol Pharmacol, 42, 1998, 440-444.
43. Catherine A,Rice E, Nicholas J, George P, Structureantioxidant
activity relationships of flavonoids and phenolic acids, Free Radic
Biol Med, 20, 1996,933-956.

Source of support: Nil, Conflict of interest: None Declared

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