Free Radical Scavenging Activity of
Leaves of Memecylon edule Roxb
S Sheela*, N
Dora Babu and K Ilango
SRM College of Pharmacy, SRM University, Kattankulathur-603203, Tamil
Nadu.
Free
radical scavenging potential of leaves of Memecylon edule. Roxb
was studied using superoxide radical scavenging effect, hydroxyl radical scavenging effect, lipid peroxide assay, nitric oxide scavenging
effect. The content of superoxide
radicals was studied by NBT reduction method, hydroxyl radical by Deoxy-ribose degradation method, lipid peroxide level by
estimating the thiobarbituric acid releasing
substances (TBARS) and the nitric oxide radical by Griess
method. The ethanolic and aqueous leaf extracts were
used for the study with reference to the ascorbic acid as standard drug. It was
found that the aqueous extract showed significant free radical scavenging
potential compared to the ethanolic extract. The
IC50 for aqueous extract was found to be 502.9µg/ml, 493.78µg/ml,
469.6µg/ml and 227.8 µg/ml for superoxide
radical scavenging effect, hydroxyl
radical scavenging effect, lipid
peroxide assay and nitric oxide scavenging effect respectively. The extracts studied can
provide efficient antioxidant protection by complementary mechanisms, such as
free radical scavenging and metal ions reduction with
the non polar extracts.
KEYWORDS: Memecylon edule, superoxide radical scavenging effect, hydroxyl radical scavenging effect, lipid peroxide assay, nitric oxide scavenging effect.
INTRODUCTION:
Free
radicals having broad ranges of effects in biological systems have drawn on the
attention of many experimental works. Highly reactive free radicals, especially
oxygen-derived radicals, which are formed by exogenous chemicals or endogenous
metabolic processes in the human body, are capable of oxidizing biomolecules, resulting in cell death and tissue damage.
Oxidative damage plays a significantly pathological role in human diseases.
Cancer, emphysema, cirrhosis, atherosclerosis and arthritis have all been
correlated with oxidative damage 1.Almost all organisms are well
protected against free radical damage by enzymes such as superoxide dismutase
and catalase or compounds such as ascorbic acid, tocopherol and glutathione 2. When the mechanism
of antioxidant protection becomes unbalanced by factors such as ageing, deterioration
of physiological functions may occur resulting in diseases and accelerating
ageing. However, antioxidant supplements may be used to help the human body to
reduce oxidative damage4. The safety of natural antioxidants have
been much focused for its use to inhibit
lipid peroxidation or to protect the human body from
the oxidative damage by free radicals.Therefore our
aim in this study is to evaluate the scavenging potential of extracts of Memecylon edule Roxb leaf.
Memecylon edule Roxb (Melastomataceae) is a small evergreen tree
native to India, especially the Deccan
Plateau, including most of Karnataka,
Andhra
Pradesh, and parts of Tamil Nadu.
The common names include kaayam, delek bangas, delek air, miat, and nemaaru.
Leaves and roots are used as medicine for dysentery and as astringent. The
decoction of the wood is a powerful
astringent. It is stated that the leaves of the plant Memecylon edule Roxb is used as powerful astringent., emmengogue. It
contains tartaric and malic acids. Tonic
for diarrhea4, 5.
The anti-inflammatory, analgesic and antioxidant activities
of the leaves of Memecylon edule Roxb. was determined using in
vitro for their interleukin-10 production; the most active fraction was
further studied in vivo for its anti-inflammatory and analgesic
activities using the ethylphenylpropiolate
(EPP)-induced mouse ear edema and the writhing test with mice. All fractions
except Hex were tested for their radical scavenging activity towards
1′-diphenyl-2-picrylhydrazyl radical (DPPH). The results provided support
for the traditional claim of Memecylon edule Roxb leaves in
relieving inflammation and pain6.
MATERIALS AND METHODS:
Plant material:
The
leaves of the plant Memecylon edule Roxb were collected from Chittoor
district of Andhra Pradesh, India. The plant material was taxonomically
identified by Prof. P.Jayaraman, Director, Plant
Anatomy Research Center, Chennai, Tamil Nadu, India. A
voucher specimen has been preserved in our laboratory for future reference. The
collected plant material was dried under shade and then powdered with a
mechanical grinder and stored in an air-tight container. The dried powder
material of the leaves was defatted with petroleum ether and the marc thus
obtained was then extracted separately with distilled water (by cold
maceration) and 75% ethanol (using Soxhlet
apparatus). The solvent was completely removed under reduced pressure and a
semisolid mass was obtained. The dried MWE and MAE (water and ethanolic extracts of Memecylon
leaves respectively) were dissolved in distilled water and used for the present
study.
Chemicals:
Deoxy ribose, nitro blue tetrazolium,
thiobarbituric acid, trichloro
acetic acid, EDTA, was purchased from Sigma Chemical Co.
Ltd, USA. All other unlabeled chemicals and reagents were of analytical grade
and were used without further purification.
Superoxide
radical scavenging effect:
Superoxide radical scavenging activity was studied
by. Alkaline DMSO method7. The
alkaline DMSO (1% in 5mM NaOH) was added to the
reaction mixture containing nitro-blue tetrazolium
(NBT 0.1mg) and MWE and MAE at various concentrations (100,
250, 500, 750, 1000μg). The absorption was determined at 560nm. The reduction of NBT by the
superoxide radical generated was calculated in presence and absence of the test
drugs. In this study, thiourea (20mM) was used as the
reference standard.
Hydroxyl
radical scavenging effect:
This study was conducted by measuring the inhibition
of deoxyribose degradation in presence of the leaf
extracts. Hydroxyl radical was generated by Fe-EDTA and H2O2
in presence of ascorbic acid. MWE and MAE were added in various concentrations (100,
250, 500, 750, 1000μg) to a reaction mixture containing deoxyribose
(3mM); FeCl3 (0.1 mM), EDTA (0.1mM),
ascorbic acid (0.1mM) H2O2 (2mM) in phosphate buffer(20 mM/pH- 7.4) to make a final volume of 3ml. To this mixture trichloroacetic acid and thiobarbituric
acid (0.5ml each) were added and measured the absorbance at 532nm. The
percentage of hydroxyl radical inhibition and IC50 were determined
by the method of Halliwell et.al8. Catechol solution was used as a reference standard.
Lipid peroxide
scavenging effect:
In this study the liver tissue homogenate was
adjusted to 10mg/ml. The effect of leaf extracts on lipid peroxides was
estimated as malondialdehyde by thiobarbituric
acid (TBA) method9, 10. To the reaction mixture
containing MWE and MAE at various concentrations (100, 250, 500, 750, 1000μg). 1ml of liver
tissue homogenate and 1 ml of “HCl- trichloroacetic acid – thiobarbituric
reagent” was added. The mixture was heated for 5 minutes in a water bath at 370C,
and after cooling, the flocculent precipitate was removed by centrifugation at
1000rpm for 10 min. The absorbance of the supernatant liquid was measured at
532nm against blank and the lipid peroxide content was determined using the
extinction coefficient 1.5l 10-6m-1
cm-1. The final result was expressed as nanomoles
of malondialehyde per mg of protein. Vitamin E at a
concentration of 50mcg/ml was used for comparing the results.
Nitric oxide
radical scavenging effect:
Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH
interacts with oxygen to produce nitrite ions which were measured by the Griess reaction (11, 12). The reaction mixture
(3mL) containing sodium nitroprusside (10 mM) in phosphate buffered saline (PBS) and the MWE and MAE
in different concentrations (10, 25, 50, 75, 100 and 125μg) were incubated
at 25oC for 150 minutes. Every 30 minutes, 0.5 ml of the incubated
sample was removed and 0.5 ml of Griess reagent (1% sulphanilamide, 0.1% naphthylethylene
diamine dihydrochloride in
2% H3PO4) was added. The absorbance of the chromophore formed was measured at 546 nm. All the analyses
were performed in triplicate and the results were averaged. The percentage
inhibition of nitric oxide generated was measured by comparing the absorbance
values of control and test. Curcumin was used as a
reference compound.
RESULTS:
The Table: 01
shows the superoxide scavenging effect of MWE and MAE and Thiourea
on the alkaline DMSO-NBT system. The decrease of absorbance at 560nm with
antioxidants thus indicates the consumption of superoxide anion in the reaction
mixture. MWE at concentration from 100-1000 μg/ml
inhibited the production of superoxide anion radicals by 19.45 -63.75 %.. The IC50 value of MWE on superoxide radical
scavenging activity was found to be 502.9μg/ml and for MAE was 910.5μg/ml.
MWE had strong superoxide radical scavenging activity compared to that of MAE.
Table: 02 shows the hydroxyl radical scavenging
activity of MWE and MAE along with positive control Catechin.
The concentration of MWE and MAE needed for 50% inhibition was found to be 493.78 and 963.2
μg/ml respectively. Table: 03 exhibits
the reduction of lipid peroxide level in terms of thiobarbituric
acid releasing substances by MWE and MAE with reference to ascorbic acid. It
was found that the concentration required for reducing the lipid peroxide level
for MWE and MAE are 469.6 and 982.7μg/ml respectively. Table:
04 represents the percentage inhibition of nitric
oxide generation by MWE and MAE. Curcumin was used as
a reference compound. The IC50 value of MWE was found to be 227.8 and 940.9
μg/ml respectively. The results reveal that MWE
shows significant free radical scavenging effect and comparable with that of
standard drugs, whereas the MAE does not have potential effect in scavenging
the free radicals.
TABLE:
01 Superoxide radical scavenging effect
of leaves of Memecylon edule Roxb.
|
SL.NO. |
CONCENTRATION
OF DRUG µg/ml |
PECENTAGE
SCAVENGING EFFECT |
IC50µg/ml |
||
|
MWE |
MAE |
MWE |
MAE |
||
|
1.
|
1000 |
63.75± 0.31 |
54.91± 0.33 |
502.9 |
910.5 |
|
2.
|
750 |
59.74± 0.29 |
40.47± 0.27 |
||
|
3.
|
500 |
49.71± 0.22 |
27.90± 0.26 |
||
|
4.
|
250 |
39.82± 0.19 |
23.80± 0.28 |
||
|
5.
|
100 |
19.45± 0.09 |
17.76± 0.15 |
||
|
6.
|
Thio urea
(20 mM) |
55.06
± 0.306 |
- |
||
n=
5. MWE = Memecylon edule water
extract. MAE=
Memecylon edule ethanolic extract.
TABLE:
02 Hydroxyl radical scavenging effect
of leaves of Memecylon edule Roxb.
|
SL.NO. |
CONCENTRATION
OF DRUG µg/ml |
PERCENTAGE
SCAVENGING EFFECT |
IC50µg/ml |
||
|
MWE |
MAE |
MWE |
MAE |
||
|
1.
|
1000 |
77.75± 1.73 |
51.91± 1.34 |
493.78 |
963.2 |
|
2.
|
750 |
56.74± 2.12 |
36.47± 0.98 |
||
|
3.
|
500 |
50.71± 0.56 |
27.90± 0.45 |
||
|
4.
|
250 |
39.82± 0.12 |
23.80± 0.36 |
||
|
5.
|
100 |
23.76± 0.34 |
19.45± 0.21 |
||
|
6.
|
Catechin
(0.01mM) |
54.46
± 1.63 |
- |
||
n=
5. MWE = Memecylon edule water
extract. MAE= Memecylon edule ethanolic extract.
TABLE: 03 Lipid peroxide scavenging effect of leaves of Memecylon edule Roxb.
|
SL.NO. |
CONCENTRATION OF DRUG µg/ml |
TBARS(105 nano moles) |
PERCENTAGE SCAVENGING EFFECT |
IC50µg/ml |
|||
|
|
|
|
|||||
|
MWE |
MAE |
MWE |
MAE |
MWE |
MAE |
||
|
1.
|
1000 |
1.680± 0.21 |
1.208± 0.49 |
57.62 |
50.88 |
469.6 |
982.7 |
|
2.
|
750 |
1.745± 0.19 |
1.107± 0.54 |
55.98 |
38.57 |
||
|
3.
|
500 |
1.854± 0.14 |
1.098± 0.43 |
53.23 |
21.27 |
||
|
4.
|
250 |
2.287± 0.22 |
1.987± 0.26 |
42.32 |
16.58 |
||
|
5.
|
100 |
3.008± 0.15 |
2.487± 0.21 |
24.13 |
14.96 |
||
|
6.
|
Control |
3.956 |
- |
- |
|||
|
7.
|
Ascorbic acid (50 µg/ml) |
1.91 ± 0.27 |
51.82 ± 0.92 |
- |
|||
n=
5. MWE = Memecylon edule water
extract. MAE=
Memecylon edule ethanolic extract.
TABLE: 04 Nitric oxide radical
scavenging effect of leaves of Memecylon edule Roxb.
|
SL.NO. |
CONCENTRATION
OF DRUG µg/ml |
PERCENTAGE
SCAVENGING EFFECT |
IC50
µg/ml |
||
|
MWE |
MAE |
MWE |
MAE |
||
|
1.
|
1000 |
68.95± 0.39 |
53.14± 1.96 |
227.8 |
940.9 |
|
2.
|
750 |
62.83± 0.33 |
47.91± 1.65 |
||
|
3.
|
500 |
61.29± 0.25 |
45.91± 0.87 |
||
|
4.
|
250 |
54.85± 0.17 |
39.54± 0.59 |
||
|
5.
|
100 |
51.82± 0.013 |
37.85± 0.34 |
||
|
6.
|
Curcumin
(50µg/ml) |
78.5
± 0.036 |
- |
||
n=
5. MWE = Memecylon edule water
extract. MAE=
Memecylon edule ethanolic extract.
DISCUSSION:
The
present study has shown the confirmation of considerable free radical scavenging
potential of the leaf extracts of Memecylon edule Roxb especially
water extract. Superoxides
are produced from molecular oxygen due to oxidative enzymes 13 of
body as well as via non enzymatic reaction such as autoxidation
by catecholamines 14. In the present
study, superoxide radical reduces NBT to a blue colored formazan
that is measured at 560 nm 15. Table 01 shows the superoxide
scavenging effect of MWE and MAE and curcumin on the
alkaline DMSO-NBT system. The decrease of absorbance at 560nm with antioxidants
thus indicates the consumption of superoxide anion in the reaction mixture. MWE
had strong superoxide radical scavenging activity compared to MAE.
Hydroxyl radicals are the major active
species causing lipid oxidation and enormous biological damage 16, 17.
The deoxyribose method is a simple assay to determine
the rate constants for reactions of hydroxyl radicals 18.
Ferric-EDTA was incubated with H2O2 and ascorbic acid at
pH 7.4. Hydroxyl radicals were formed in free solution and were detected by
their ability to degrade 2-deoxy-2-ribose into fragments that on heating with
TBA at low pH form a pink chromogen 19, 20.
Any hydroxyl radical scavenger added to the reaction would compete with Deoxy ribose for the availability of hydroxyl radicals, thus
reducing the amount of MDA formation. In this study it was found that MWE had
good scavenging effect compared to that of MAE and the results were comparable
with the standard anti-oxidant.
Lipid peroxidation (LPO)
is a free radical-related process that in biologic systems may occur under
enzymatic control, e.g., for the generation of lipid-derived inflammatory
mediators, or non enzymatically.
This latter form is associated mostly with cellular damage as a result of
oxidative stress, which also involves cellular antioxidants in this process. In this study, measurement of malondialdehyde (MDA) is a marker of free radical induced lipid
peroxidation which may provide possible information
on radicals accentuated structural membrane injury. The detection of MDA was
measured as MDA-thiobarbituric acid adduct from the
tissue homogenate used. This study shows that MWE had good effect on reducing
lipid peroxidation on the tissue homogenate used. The
results were comparable with the standard ascorbic acid.
Nitric oxide (NO) is an important
chemical mediator generated by endothelial cells, macrophages, neurons, etc.
and is involved in the regulation of various physiological processes21.
Excess concentration of NO is associated with several diseases 22, 23.
Oxygen reacts with the excess nitric oxide to generate nitrite and peroxynitrite anions, which act as free radicals24, 25.
In the present study, the extract competes with oxygen to react with
nitric oxide and thus inhibits generation of the anions. Table 04 represents
the percentage inhibition of nitric oxide generation by MWE and MAE. Curcumin was used as a reference compound. MWE showed
significant scavenging effect on nitric oxide radicals than the MAE.
Our study reveals that the water extract
of the leaves of Memecylon edule Roxb
has potential capacity in eradicating the free radicals than the ethanolic extract. Further studies are needed to better
characterize the active constituents of the plant responsible for eradicating
free radical scavenging activity.
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Received on 12.07.2009
Accepted on 14.08.2009
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Research Journal of Pharmacognosy and Phytochemistry.
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