Antioxidant and Free Radical Scavenging Activities of Ervatamia coronaria
Stapf. leaves
Iranian Journal of
Pharmaceutical Research (2004) 2: 119-126
Received: March 2004
Accepted: April 2004 |
Copyright ? 2004 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services |
Original Article
Antioxidant and Free Radical Scavenging
Activities of Ervatamia coronaria Stapf. leaves
Malaya Gupta*, Upal Kanti Mazumdar,
Periyasamy Gomathi, Ramanathan Sambath Kumar.
Division of Pharmacology &
Pharmaceutical Chemistry,Department of Pharmaceutical
Technology,Jadavpur University,Kolkata. India.
Abstract
The present study was carried
out to evaluate the antioxidant and free radical scavenging activity of
methanolic extract of Ervatamia
coronaria leaves (Apocynaceae) in various
systems. DPPH radical, superoxide anion radical, nitric oxide radical
and hydroxyl radical scavenging assays were carried out to evaluate the
antioxidant potential of the extract. The antioxidant activity of
methanolic extract increased in a dose dependent manner. About 50, 100,
250 and 500 ?g of methanol extract of
Ervatamia coronaria (MEEC)
showed 61.33, 66.21, 72.04 and 76.83% inhibition respectively on
peroxidation of linoleic acid emulsion. Like antioxidant activity, the
effect of MEEC on reducing power increases in a dose dependent manner.
In DPPH radical scavenging assay the IC50 value of the extract was found to be 167.09
?g/ml. MEEC was found to inhibit the nitric oxide radicals generated
from sodium nitroprusside. The IC50 value
was found to be 83.375 ?g/ml, whereas the IC50 value
of curcumin was 20.4 ?g/ml. Moreover, the MEEC was found to scavenge the
superoxide generated by PMS/NADH-NBT system. MEEC was also found to
inhibit the hydroxyl radical generated by Fenton's reaction, where the
IC50 value of MEEC was found to be more than 1000
?g/ml and for catechin the IC50
value was found to be 5 ?g/ml, which
indicates the prooxidant activity of MEEC. The amounts of total phenolic
compounds were also determined in this study. The results obtained in
the present study indicate that the MEEC can be a potential source of
natural antioxidant.
Keywords: Ervatamia
coronary; Antioxidant activity; Lipid peroxidation; Free radical
scavenging; DPPH assay.
Introduction
The effects of free radicals on human
beings are closely related to toxicity, disease and aging (1).
Most living species have an efficient defense system to protect
themselves against the oxidative stress induced by Reactive
Oxygen Species (ROS) (2). Recent investigations have shown that
the antioxidant properties of plants could be correlated with
oxidative stress defense and different human diseases including
cancer, atherosclerosis and the aging process (3-5). The
antioxidants can interfere with the oxidation process by
reacting with free radicals, chelating free catalytic metals
and also by acting as oxygen scavengers.
Ervatamia coronaria Stapf (Synonym : Tabernaemontana
divaricata) (6) belongs to the
family Apocynaceae, is a glabrous, evergreen tree indigenous to
India and is cultivated in gardens for its ornamental and
fragrant flowers. This species has been extensively
investigated and a number of chemical constituents such as
alkaloids (7-10), triterpenoids (11-13), steroids (12, 14),
flavonoids (15), phenyl propanoids (14, 15) and phenolic acids
(8) were isolated from leaves, roots and stems of the plant. In
Indian traditional system of medicine the plant material is
widely used as a purgative, tonic to the brain, the spleen and
the liver; in the treatment of cancer, wounds and inflammations
(16, 17). The plant extract was also found to possess
analgesic, antipyretic, vasodilator and CNS depressant effects
(18), antispasmodic, hypotensive activity (19),
antiinflammatory (8), uterine stimulant effect (20) and
cytotoxic activity (21).
Furthermore, literature survey of E. coronaria
revealed that no researcher has yet reported antioxidant
activities of this plant. Therefore, it is worth conducting an
investigation on the in vitro antioxidant activities of methanolic extract
of E. coronaria leaves (MEEC).
Experimental
Plant material
The plant Ervatamia
coronaria was collected in
March 2003 from the Kolli Hills, Tamil Nadu, India. The plant
material was taxonomically identified by the Botanical Survey
of India, Shibpur, Kolkata, India, and the Voucher specimen
(No. GMG1) was retained in our laboratory for future reference.
The leaves were dried under shade with occasional shifting and
then powdered with a mechanical grinder and stored in an
airtight container. The dried and powdered leaves (500 g) were
defatted with petroleum ether (60-80°C) in a Soxhlet
apparatus. The defatted powder material thus obtained was
further extracted with methanol in the Soxhlet for 72 h. The
solvent was removed by distillation under suction and the
resulting semisolid mass was dried using the rotary flash
evaporator to yield (14.51%) a solid residue (methanolic
extract). The dried MEEC was suspended in distilled water and
used for further studies.
Materials
Ammonium thiocyanate was purchased from
E. Merck, Germany. Ferrous chloride, ferric chloride, 1,
1-diphenyl-2-picryl-hydrazyl (DPPH), nicotinamide adenine
dinucleotide (NADH), EDTA, butylated hydroxy toluene (BHT),
butylated hydroxy anisole (BHA), a-tocopherol, ascorbic acid, quercetin,
catechin, pyrocatechol, curcumin, nitroblue tetrazolium,
thiobarbituric acid, 2-deoxy-2-ribose, trichloroacetic acid,
phenazine methosulphate and potassium ferricyanide were all
purchased from Sigma Chemical Co. Ltd, USA. All the other
unlabeled chemicals and reagents were of analytical grade.
Antioxidant activity
The antioxidant activity of MEEC was
determined according to the thiocyanate method (22). About 10
mg of MEEC was suspended in 10ml water. Various concentrations
(50, 100, 250 and 500 µg/ml) of MEEC were added to
linoleic acid emulsion (2.5 ml, 0.04 M, pH 7.0) and phosphate
buffer (2 ml, 0.04 M, pH 7.0). The linoleic acid emulsion was
prepared by mixing 0.2804 g of linoleic acid, 0.2804 g of Tween
20 as emulsifier and 50 ml phosphate buffer and then the
mixture was homogenized. The final volume was adjusted to 5 ml
with potassium phosphate buffer (0.04 M, pH 7.0). Then the
mixed samples were incubated at 37 °C in a glass flask for
60 h to accelerate the oxidation process (23, 24). Each 12 h, 1
ml of the incubated sample was removed and 0.1 ml of FeCl2
(0.02 M) and 0.1ml of ammonium thiocyanate (30%) were added.
The amount of peroxide was determined by measuring the
absorbance at 500 nm. Alpha tocopherol was used as the
reference compound. In order to eliminate the solvent effect,
the control sample, which contains the same amount of solvent
added into the linoleic acid emulsion in the test sample and
reference compound was used. All the data are the average of
triplicate analysis. The percentage inhibition of lipid
peroxide generation was measured by comparing the absorbance
values of control and those of test samples.
Reducing power
The reducing power of MEEC was determined
according to the method of Oyaizu (25). 10 mg of MEEC
extract in 1ml of distilled water was mixed with phosphate
buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide
[K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated
at 500C for 20 min. A portion (2.5 ml) of trichloroacetic acid
(10%) was added to the mixture, which was then centrifuged at
3000 g for 10 min. The upper layer of the solution (2.5 ml) was
mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%)
and the absorbance was measured at 700 nm. BHT was used as the
reference material. All the tests were performed in triplicate
and the graph was plotted with the average of three
observations.
Inhibition of DPPH radical
The free radical scavenging activity of
MEEC was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH)
using the method of Blois (26). 0.1 mM solution of DPPH in
methanol was prepared and 1ml of this solution was added to 3
ml of various concentration of MEEC and the reference compound
(50, 100, 150, 200 and 250 µg). After 30 min, absorbance
was measured at 517 nm. BHA was used as the reference material.
All the tests were performed in triplicate and the graph was
plotted with the mean values. The percentage of inhibition was
calculated by comparing the absorbance values of the control
and test samples.
Inhibition of Nitric oxide radical
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 (27, 28). The reaction mixture (3 ml)
containing sodium nitroprusside (10 mM) in phosphate buffered
saline (PBS) and MEEC and the reference compound in different
concentrations (10, 25, 50, 75 and 100 µg) were incubated
at 25°C for 150 min. Each 30 min, 0.5 ml of the incubated
sample was removed and 0.5 ml of the 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 tests were performed in
triplicate and the results averaged. The percentage inhibition
of nitric oxide generated was measured by comparing the
absorbance values of control and test sampels Curcumin was used
as a positive control compound.
Inhibition of Superoxide anion radical
Measurement of superoxide anion
scavenging activity of MEEC was performed based on the method
described by Nishimiki (29) and slightly modified. About 1 ml
of nitroblue tetrazolium (NBT) solution containing 156 µM
NBT which is dissolved in 1.0 ml of phosphate buffer (100 mM,
pH 7.4), 1 ml of NADH solution containing 468 µM of NADH
which is dissolved in 1 ml of phosphate buffer (100 mM, pH 7.4)
and 0.1 ml of various concentration of MEEC and the reference
compounds (10, 25, 50, 75 and 100 µg) were mixed and the
reaction started by adding 100 µl of phenazine
methosulphate (PMS) solution containing 60 µM of PMS 100
µl of phosphate buffer (100 mM, pH 7.4). The reaction
mixture was incubated at 250C for 5 min and the absorbance at
560 nm was measured against the control samples. BHT and
quercetin were used as the reference compounds. All the tests
were performed in triplicate and the results averaged. The
percentage inhibition was calculated by comparing the results
of control and test samples.
Inhibition of Hydroxyl radical
Hydroxyl radical scavenging activity was
measured by studying the competition between deoxyribose and
the test compounds (MEEC) for hydroxyl radical generated by
Fe3+-Ascorbate-EDTA-H2O2 system (Fenton reaction) according to
the method of Kunchandy and Rao (30). The reaction mixture
contained in a final volume of 1.0 ml, 100 µl of
2-deoxy-2-ribose (28 mM in KH2PO4-KOH buffer, 20 mM, pH 7.4),
500mcl of the various concentrations of MEEC and the reference
compound (1, 100 and 1000 µg) in KH2PO4-KOH buffer (20
mM, pH 7.4), 200 µl of 1.04 mM EDTA and 200 µM
FeCl3 (1:1 v/v), 100 µl of 1.0 mM H2O2 and 100 µl of
1.0 mM ascorbic acid was incubated at 370C for 1h. 1.0 ml of
thiobarbituric acid (1%) and 1.0 ml of trichloroacetic acid
(2.8%) were added to the test tubes and incubated at 100°C
for 20 min. After cooling, absorbance was measured at 532 nm
against a control sample containing deoxyribose and buffer.
Catechin was used as a positive control. Reactions were carried
out in triplicate. The percentage inhibition was determined by
comparing the results of the test and control compounds.
Amount of total phenolic compounds
Total soluble phenolics present within in
the MEEC were determined using the Folin-Ciocalteu reagent,
according to the method of a Slinkard and Singleton (31). 0.1
ml of suspension of 1mg of MEEC in water was totally
transferred into 100 ml Erlenmeyer flask. Then the final volume
was adjusted to 46 ml by the addition of distilled water.
Afterwards, 1 ml of Folin - Ciocalteu reagent (FCR) was added
to this mixture and after 3 min, 3 ml of Na2CO3 (2%) was added.
Subsequently, the mixture was shaken on a shaker for 2 h at
room temperature and then its absorbance measured at 760 nm.
All the tests were performed in triplicate and the results
averaged. The concentration of total phenolic compounds in MEEC
was determined as microgram of pyrocatechol equivalent by using
an equation that was obtained from the standard pyrocatechol
graph. The equation is given below;
Absorbance = 0.001 x Pyrocatechol (
µg) + 0.0033
Statistical analysis
Experimental results were mean±SD
of three parallel measurements. Statistical analysis was
performed according to the student t-test. and ANOVA procedure.
The values for P < 0.05 were regarded as significant and the
values for P < 0.01 as highly significant.
Results And Discussion
Lipid peroxidation has been defined as
the biological damage caused by free radicals, wich are formed
under oxidative stress (32). The antioxidative activity of
natural sources is due to the active compounds present in the
plants. Most natural antioxidants are found in wood, bark,
stem, leaf, fruit, root, flower and seed (33). Most of these
compounds are normally phenolic or polyphenolic in nature eg,
tocopherols, flavonoids and derivatives of cinnamic acid,
phosphatidic and other organic acids.
Antioxidant activity
In this study the antioxidative activity
of the leaf extract of Ervatamia
coronaria was measured using the
ammonium thiocyanate method. This method was used to measure
the peroxide level during the initial stages of lipid
oxidation. The antioxidant activity of MEEC might be due to the
reduction of hydroperoxide, inactivation of free radicals or
complexation with metal ions, or combinations thereof. This
good antioxidant activity of MEEC might be attributed to the
presence of phytochemicals, such as flavonoids and biflavones
(34). Figure 1 illustrates the antioxidative activities of
various concentrations of MEEC (50, 100, 250 and 500
µg/ml). The different concentrations of MEEC (50, 100,
250 and 500 µg/ml) showed antioxidant activities in a
dose dependent manner and had 61.33, 66.21, 72.04 and 76.83%
inhibition respectively on the lipid peroxidation of linoleic
acid system. MEEC, at a concentration of 500 µg/ml showed
76.83% inhibition, which is more or less equal to the
antioxidant activity of 500 µg/ml of a-tocopherol (77.13%).
The IC50 value of MEEC on lipid peroxidation was found
to be 40.76 µg/ml. The results indicate that methanolic
extract of Ervatamia coronaria significantly (P<0.05) inhibits the
linoleic acid peroxidation. The antioxidative activity of the
leaves of Ervatamia coronaria may be due to the reduction of
hydroperoxides, inactivation of free radicals, chelation of
metal ions or combinations thereof.
Reductive ability
The antioxidant activity has been
reported to be concomitant with the development of reducing
power (35). However this pattern was not observed in this
research. Okuda et al. reported that the reducing power of
tannins prevents liver injury by inhibiting the formation of
lipid peroxides. The reducing capacity of a compound may serve
as a significant indicator of its potential antioxidant
activity (36). Figure 2 shows the reductive capabilities of
MEEC compared with BHT. The reducing capacity of a compound may
serve as a significant indicator of its potential antioxidant
activity (27). Like antioxidant activity, the reducing power of
MEEC increased with increasing amount of sample. All MEEC
showed concentrations higher activities than the control and
these differences were statistically highly significant (P <
0.01).
Inhibition of DPPH radical
The DPPH radical is considered to be a
model for a lipophilic radical. A chain reaction in lipophilic
radicals was initiated by the lipid autoxidation. The radical
scavenging activity of the crude plant extract was determined
from the reduction in the optical absorbance at 517 nm due to
scavenging of stable DPPH free radical. Positive DPPH test
suggests that the samples are free radicals scavengers. The
scavenging effects of MEEC and BHA on DPPH radical are compared
and shown in Figure 3. MEEC had significant scavenging effects
on the DPPH radical and the effects increasing with increasing
concentration in the range of 50-250 µg/ml. Compared with
that of BHA, the scavenging effect of MEEC was lower. The IC50 value of MEEC on DPPH radical scavenging
assay was found to be 167.09 µg/ml. The results were
found to be statistically significant (P<0.05).
Inhibition of Nitric oxide radical
It is well known that nitric oxide has an
important role in various types of inflammatory processes in
the animal body. In the present study, the crude extract of the
leaves was checked for its inhibitory effect on nitric oxide
production. Figure 4 illustrates the percentage inhibition of
nitric oxide generation by MEEC. Curcumin was used as a
reference compound. The concentration of MEEC needed for 50%
inhibition was found to be 83.38 µg/ml, whereas 20.4
µg/ml was needed for curcumin. The results were found to
be statistically significant (P<0.05).
Inhibition of Superoxide anion radical
Superoxide anions indirectly initiated
lipid oxidation as a result of superoxide and hydrogen peroxide
serving as precursors of singlet oxygen and hydroxyl radicals
(37). Robak and Glyglewski reported that the antioxidant
properties of flavonoids are effective mainly via the
scavenging of superoxide anion. MEEC was found to possess good
scavenging activity on superoxide anions at all the tested
concentrations (38). MEEC at concentrations from 10-100
µg/ml, inhibited the production of superoxide anion
radicals by 11.09-42.67%. MEEC also showed a strong superoxide
radical scavenging activity. The results are presented in
Figure 5. The IC50 value of
MEEC on superoxide radical scavenging activity was found to be
118.77 µg/ml, whereas the IC50 value
of BHT and quercetin was found to be 22.77 and 31.58
µg/ml respectively. The results were found to be
statistically significant (P<0.05).
Inhibition of hydroxyl radical
Hydroxyl radicals are the major active
oxygen species causing lipid oxidation and enormous biological
damage (39). Ferric-EDTA was incubated with H2O2 and ascorbic
acid at pH 7.4. Hydroxyl radicals were formed in free solution
and detected by their ability to degrade 2-deoxy-2-ribose into
fragments that on heating with TBA at low pH form a pink
chromogen (40, 41). When MEEC and the reference compound,
catechin, added to the reaction mixture they removed hydroxyl
radicals from the sugar and prevented degradation. The results
are shown in Figure 6. MEEC was also capable of reducing DNA
damage at all concentrations used. Catechin used as a standard
was highly effective in inhibiting the oxidative DNA damage.
The IC50 value of MEEC on hydroxyl radical
scavenging assay was found to be 1833.73 µg/ml. The
results were found to be statistically significant (P<0.05).
Amount of total phenolic compounds
Phenols are very important plant
constituents because of their scavenging ability, which is due
to their hydroxyl groups (42). In MEEC (1 mg) 64.7 µg
pyrocatechol equivalent of phenols was detected. The phenolic
compounds may contribute directly to the antioxidative action
(43). The result indicates strong association between
antioxidative activities and phenolic compounds (r2 = 0.9983),
suggesting that phenolic compounds are probably responsible for
the antioxidative activities of Ervatamia
coronaria. Phenolic compounds are
effective hydrogen donors, making them good antioxidants (44).
Similarly Shahidi and Naczk reported that naturally occurring
phenolics exhibit antioxidative activity (45). Thus,
therapeutic properties of E. Coronaria may be possibly
attributed to the phenolic compounds present.
Conclusion
It is well known that free radicals are
one of the causes of several diseases, such as Parkinson
disease (46), Alzheimer type dementia (47) etc. The production
of free radicals and the activity of the scavenger enzymes
against those radicals, such as superoxide dismutase (SOD) is
correlated with the life expectancies (48). Polyphenols,
tannins and flavonoids are very valuable plant constituents in
the scavenging action due to their several phenolic hydroxyl
groups (49). The exact constituents of MEEC, which shows free
radical scavenging action, are unclear. However, the
phytoconstituents like polyphenol and flavonoids present in the
plant extract may be responsible for antioxidant and free
radical scavenging activities.
Thus, the radical scavenging activity,
reductive capability and anti-lipoperoxidant activity strongly
suggests that MEEC has antioxidant and anti-lipoperoxidant
activities. Further studies are needed to evaluate the in vivo antioxidant
potential of this extract in various animal models and to
isolate the active component.
Acknowledgement
One of the authors, P. Gomathi, is
grateful to AICTE, New Delhi, India, for providing the
financial support to this work.
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