International Research Journal of Biotechnology (ISSN: 2141-5153) Vol. 2(5) pp. 119-127, May, 2011
Available online http://www.interesjournals.org/IRJOB
Copyright © 2011 International Research Journals
Full Length Research Paper
Antioxidative and free radical scavenging activities of
terminalia species
Sundar S. Mety and Pratima Mathad*
Plant Biotechnology Laboratory, Department of Post-Graduate Studies and Research in Botany
Gulbarga University, Gulbarga-585 106, Karnataka, India
Accepted 10 May, 2011
The present investigation was undertaken to analyze the antioxidative and free radical scavenging
activities of crude methanolic and ethanolic extracts in three different parts such as leaves, stem bark
and fruit pulp of four species of Terminalia viz., T. alata, T. arjuna, T. bellarica and T. catappa belonging
to Combretaceae family. The phytochemical screening examination indicated the presence of alkaloids,
phenols, flavonoids, terpenoids, saponins, steroids, tannins, quinines and glycosides. In which phenols
and flavonoids were recorded as highly reactive and further, it was confirmed by quantitative estimation
and their content ranged from 0.28 ± 0.01 - 1.95 ±0.01 and 0.12 ± 0.01 - 0.90 ± 0.03 mg/100 gm
respectively. It was observed from results that the DPPH free radical assay, Ascorbic acid and Ferric
reducing power were highly reactive for antioxidative and free radical scavenging activities. However, it
was interesting to record that the maximum activities were recorded in methanolic extract of T. arujan
and T. alata stem bark and the minimum was recorded in stem bark of T. catappa, when compared to
ethanolic extracts of all the plant parts. It is evident from the experimental results that all the plant
extracts have promising antioxidative and free radical scavenging activities, thus justifying their
traditional use.
Key words: Antioxidants, Free radicals, Terminalia and DPPH scavenging
INTRODUCTION
In oxidation process highly reactive and harmful chain
reactions of oxygen species are generated which cause
damage to living organism. The oxygen centered free
radicals and other reactive oxygen species, which are
continuously produced has resulted in cell death or tissue
damage. This oxidative damage caused by free radical is
related to pathogenesis of many chronic degenerative
diseases like cancer, diabetes, neurodegenerative
disease, antherosclerosis, cirrhosis, malaria and AIDS
(Azizova et al., 2002; Sian et al., 2003; Quinteo et al.,
2006, Nagler et al., 2006). Reactive oxygen species
(ROS), including superoxide free radical, hydrogen
peroxide, hydroxyl free radical and singlet oxygen play a
key role in the oxidative damage of these diseases.
Which in turn resulted in DNA mutation, protein
*Corresponding author Email: unipm@rediffmail.com
inactivation, rapid peroxidation and cell death (Vertunani
et al., 2004) cell apoptosis or abnormal proliferation,
eliciting the occurrence of diseases from cellular and
molecular level (Nardberg, 2001).
Antioxidant is a molecule, which terminate the chain
reaction by removing free radical intermediates. Plants
and animals maintain complex system of multiple type of
antioxidant, the natural plant based antioxidants are
playing an important role in the maintenance of human
health for the past three decades (Devasagayam et al.,
2004). This has attracted a great deal of research
interest in natural antioxidants.
A worldwide trend
towards the use of natural phytochemicals present in
various plant foods, berry crops, tea, herbs, oilseeds,
beans, fruits and vegetables are used for the production
of
raw
materials
or
preparations
containing
phytochemicals with significant antioxidant capacities and
health benefits (Exarchou et al., 2002). The plant fruits,
vegetables, medicinal herbs etc., may contain a wide
120 Int. Res. J. Biotechnol.
variety of free radical scavenging molecules including
rosemary, sage, thyme, nutmeg, turmeric, white pepper,
chili pepper, ginger and several Chinese medicinal plants
extract (Lee et al., 2003). The majority of the active
compounds such as phenolic compounds (phenolic acid,
flavonoids, quinines, cumarines, lignans, stilbenes,
tannins, etc.,), nitrogen compounds (alkaloids, amines,
betalains, vitamins, terpenoids and carotenoids) are rich
in antioxidants (Prior, 2003; Cai et al., 2004). A direct
relationship between antioxidant activity and phenolic
content of plant extracts (Kaur and Kapoor, 2002;
Ivanova et al., 2005).
Many Indian medicinal plants are considered as
potential
sources
of
antioxidant
compounds
(Surveswaran et al., 2007). Terminalia is one of the
important genera, all the members of this genera are
used to treat a number of ailments. The bark of
Terminalia alata is considered to be bitter, styptic, diuretic
and cardiotonic and in Ayurveda it is used for the
treatment of bone fractures hemorrhages and bronchitis
(Kirtikar et al., 1935). The stem of Terminalia arjuna is
used as cardio-protective agent in hypertension, heart
diseases, hypocholesterolaemic and antioxidants effect in
humans (Gupta et al., 2001). Terminalia bellarica is used
in the treatment of fever, cough, asthma, urinary
diseases, piles, chronic diarrhea, dysentery, flatulence,
vomiting, colic and enlarged spleen and liver (Cheng et
al., 2003). Similarly, the leaf juice of Terminalia catappa
is used to relieve rheumatic joint pain, leprosy and skin
diseases and bark is used as astringent, diuretic and
cardiotonic (Parrota 2000).
Infact, there is an increased quest for natural
antioxidants with action of broad-spectrum. The majority
of the rich diversity of Indian medicinal plants are yet to
be scientifically evaluated for such properties. Also the
phenols, flavonoids and tannins relationship to
antioxidants activity are yet to be completely examined in
Indian medicinal plants.
There are no reports on
Terminalia alata especially in antioxidant activities.
Therefore, the present study was undertaken to evaluate
and compare the antioxidative and free radical
scavenging activities in different plant parts and in
different solvent extracts in four species of Terminalia,
which are extensively used in Indian traditional system of
medicine.
MATERIALS AND METHODS
Collection of plant materials
The leaves, stem bark and fruit pulp of Terminalia alata,
T. arjuna, T. bellarica and
T. catappa were
collected from Khanapur forest of Bidar district,
Konchavaram forest of Gulbarga district in Karnataka,
India. They are authentically identified with the help of
Flora of Gulbarga District (Seetharam et al., 2000) and
Flora of Karnataka. The voucher specimens were
deposited in the Departmental Herbarium, Gulbarga
University, Gulbarga (HGUG).
Preparation of crude extract
The collected materials were washed with alcohol (70%
v/v) to remove the dust particles and microbes. Then
materials were shade dried in a tray dried at 400C. The
dried plant materials were pulverized to a coarse powder
using a mechanical pulverizer. The obtained powder was
stored in an air tight container for further use. The powder
materials were separately extracted using Soxhlet
extractor in absolute ethanol and methanol for 24 h
(Wang and Curtis, 2006). The extracts thus obtained
were dried and stored at 300 C for further use.
Phytochemical Screening
The phytochemical screening of the Terminalia species
was conducted according to Materia Medika Indonesia
procedures (Ditjen and Depkes, 1989).
Antioxidant Activities
Analysis of Total Phenols
The amount of total phenolic content in the selected
Terminalia species was determined by Folin-Ciocalteu’s
method as described by Lister and Wilson (2001). 50µg
of each plants extracts, 2.5 mL (1/10 dilution) of FolinCiocalteu’s reagent and after 3 min. 2 ml. of 7.5 %
sodium carbonate solution (w/v) were added and mixed
0
thoroughly and incubated at 45 C for 15 min. The
absorbance of all samples was measured at 765 nm
using UV-Vis spectrophotometer against blank. The
results were expressed as milligrams of gallic acid
equivalent per gram of dry weight (GAE g/dw).
Chemical reagents
All the required chemical and solvents used were from
SD-Fine and Hi-Media Mumbai and 2,2-diphenyl-1pricrylhydrazyl (DPPH) was obtained from Sigma Aldrich
Bangalore.
Analysis of Total Flavonoids
The total flavonoids were quantitatively determined by
Swain and Hills method (1959). Each of the sample (50
µg) of both methanol and ethanol extracts of leaves, stem
Mety and Mathad 121
bark and fruit pulp were homogenate with 10 ml of
methanol using pestle and mortar and centrifuged at
5000 rpm for 10 min. The obtained supernatant was
collected and evaporated to dryness keeping in a hot
water bath thus the residue obtained was re-dissolved in
5 ml of distilled water. 0.1 and 0.2 ml are diluted to 2ml
with distilled water and 4 ml of vanillin reagent was added
rapidly after 15 min. The absorbance of all samples was
measured at 599 nm using UV-vis spectrophotometer
against blank. The results were expressed as milligrams
of phloroglucinol equivalent per gram of dry weight.
Total Ascorbic Acid Content
Ascorbic acid content in plant material was estimated as
per the method described by Sadashivam and Manikam
(1992). 50 µg all plant material was homogenized in 10
ml 4% oxalic acid and centrifuged at 5000 rpm for 15 min.
The supernatant were collected and bromine water was
added drop wise with constant stirring to give a yellow
color. The excess bromine was expelled by blowing in air
with a pipette. Final volume 25 ml with 4% oxalic acid, 2
ml of brominated extract was adjusted to 3 ml with
distilled water. This was allowed to react with 1 ml of 2%
DNPH filtered and used, followed by 1-2 drops of
Thiourea (10%). Blank was prepared as above with
distilled water in the place of Ascorbic acid or extract and
0
incubated at 37 C for three hours. The orange red
Osazone crystals were dissolved by adding 7 ml of 80%
H2SO4. The absorbances were measured at 540 nm
using UV-vis spectrophotometer.
The results were
expressed as milligrams of ascorbic acid equivalent per
gram of dry weight.
Determination of Reducing Property
Reducing properties of each sample were determined by
Potassium Ferricyanide method (Pulido et al., 2000).
50µg of each extract made up to 2.5 ml of 200 mM of
sodium phosphate buffer (pH 6.6), 1% (w/v) potassium
ferricyanide and 2.5ml of Trichloroacetic acid (TCA)
added, then heated at 50°C for 20min. Further, it was
centrifuged at 5000 rpm for 10min. The supernatant (5
mL) was dissolved in 10 ml of distilled water. Finally
0.1% (w/v) of FeCl3 (Freshly prepared) was added. The
absorbances of all samples were measured at 700 nm
using UV-vis spectrophotometer. Reducing property was
expressed in terms of OD700 and higher OD is indicative
of high reducing power.
Free radical Scavenging Assay
DPPH radical scavenging spectrophotometric assay was
used in order to determine the inhibition concentration
(IC50) which is described as the amount of antioxidant
necessary to decrease the initial concentration of the
DPPH radical by 50%. Inhibition percentage (IP)
descripts as the percentage of total DPPH radical which
reacts with the antioxidant at the steady state of all the
extract. The experiments were performed using UV-Vis
spectrophotometer (Farrukh et al., 2006). An aliquots of
50µg of each samples extract (1 mg/ml) was mixed
separately with 100 mM ethanolic solution of DPPH
radical and the final volume was of 3 ml, ethanol served
as a control. The decrease in the colour of the solution
which is indicative of decrease in the concentration of
DPPH radical was monitored by the decrease in the
absorbance at 516nm for a period of 30min. during which
time the radical stabilizes (Urishi et al., 1994). The
percentage of remaining DPPH (% DPPHRem) were
calculated by formula
DPPHREM% = (DPPH)t / (DPPH)t0 X 100
Where (DPPH)t is the absorbance measured at any time t
and (DPPH)to is the absorbance measured at time zero.
IC50 was calculated by plotting % DPPHREM at the steady
state against various concentrations of each extract. The
Result was expressed as mg antioxidant g/DPPH (Argolo
et al., 2003).
Statistical analysis
All the experiments were performed in triplicate and the
results were expressed as mean±SD (standard
deviations). Statistical analysis was performed using
SPSS 12.0 and MS-Excel 2007. The significant
difference was considered at p values ≤ 0.05.
RESULTS AND DISCUSSION
Evaluation of Antioxidant properties
The antioxidant activities (Tables 2 and 3; Figure 1-6)
were determined for the methanol and ethanol extracts of
all the plant parts investigated. The results of the total
phenolics determination (Table 1) for all the extracts
varied from 0.28 ± 0.01 to 1.95 ± 0.01 mg/100 gm of
extracts. The highest amount was found in the
methanolic extracts of T. arjuna stem bark (1.95 ± 0.01
mg/100 gm) and the least values in ethanolic extracts of
T. catappa fruit pulp (0.28 ± 0.01 mg/100 gm). From the
results, it is clear that the methanolic extracts have higher
phenolics content when compared to the ethanolic
extracts (Table 2). Similar, studies have been carried out
on the phenolic content in the different parts like leaf,
bark and fruits of a few members of Terminalia spp and
high content of phenols were observed in the stem parts
than the other parts (Bajpai et al., 2005).
122 Int. Res. J. Biotechnol.
Table 1. Preliminary screening of secondary metabolites in methanolic extracts of Terminalia species
Phytochemical
content
Alkaloids
Phenol
Flavonoids
Triterpenoids
Saponins
Steroids
Tannins
Quinines
Glycosides
T. alata
T. arjuna
T. bellarica
T. catappa
+
+++
+++
+
+
+
++
+
+
+
+++
+++
+
+
+
++
+
+
+
+++
++
+
+
+++
+
+
++
++
+
+
++
+
+ weak, ++ moderate, +++ strong and –no activity
Table 2. Estimation of phenolic content in different species of Terminalia (mg/100 g)
Species/
parts
T. alata
T. arjuna
T. bellarica
T. catappa
Methanol
Leaves
1.318±0.003
1.395±0.007
1.394±0.047
1.116±0.010
Stem bark
1.874±0.011
1.9543±0.013
0.759±0.008
1.242±0.005
Fruit pulp
1.843±0.008
0.355±0.004
Ethanol
Leaves
0.705±0.006
0.565±0.029
0.833±0.013
0.586±0.010
Stem bark
0.595±0.002
1.037±0.030
0.505±0.000
0.394±0.008
Fruit pulp
0.469±0.007
0.289±0.010
The significant difference was considered at p value ≤ 0.05
Table 3. Estimation of flavonoids content in different species of Terminalia (mg/100gm)
Species/
parts
T. alata
T. arjuna
T. bellarica
T. catappa
Methanol
Leaves
0.350±0.004
0.304±0.002
0.331±0.004
0.219±0.012
Stem bark
0.517±0.007
0.901±0.033
0.484±0.006
0.393±0.004
Fruit pulp
0.236±0.006
0.123±0.005
Ethanol
Leaves
0.321±0.007
0.414±0.006
0.850±0.015
0.150±0.004
Stem bark
0.509±0.012
0.822±0.005
0.440±0.004
0.256±0.007
Fruit pulp
0.182±0.004
0.173±0.010
The significant difference was considered at p value ≤ 0.05
Bushra et al., (2009) observed a high phenolic content in
methanolic extracts, hence this is clearly indicating the
fact that phenolics are often extracted in higher amounts
in more polar solvents such as methanol and ethanol.
The flavonoids content were determined in all the plant
parts in both the methanolic and ethanolic extracts. High
flavonoids content were observed in methanolic extracts
compared to ethanolic extracts (Table-3). It was observed
from the results that the methanolic extract had higher
content of flavonoids in stem bark of T. arjuna (0.90 ±
0.33 mg/100 gm), least value was recorded in the
ethanolic extracts of T. catappa Leaves (0.15 ± 0.00) and
T. alata recorded as the second highest content. Similar
studies were carried out by Bushra sultana
et al.(
2009) on Terminalia arjuna and in other plants such as
Moringa oleifera, Eugenia jambolana, Acacia nilotica,
Azadirachta indica, , Ficus religiosa and Aloe
barbadensis and recorded the high content of flavonoids
in methanolic extracts of leaves followed by stem. The
concentration of the flavonoids in plant extracts depends
on the polarity of solvents used for the extracts (Milan et
al., 2010). Flavonoids play an important role in curing
many chronic diseases as antioxidants.
Asorbate is a powerful reducing agent capable of
rapidly scavenging a number of reactive oxygen species.
L-Ascorbate (Vitamin C) is considered as the strong
Mety and Mathad 123
T.alata.leaf
Fig-1: Total ascorbic acid content in different parts of Terminalia species
T.alata.stem
Figure 1. Total ascorbic acid content in different parts of Terminalia species
T.arjun.leaf
0.14
T.arjun.stem
T.bel.leaf
T.bel.stem
0.12
T.bel.f ruit
T.cat.leaf
T.cat.stem
Ab sorp tion at 540nm
0.1
T.cat.fruit
0.08
0.06
0.04
0.02
0
Methanol
Ethanol
Figure 1. Total ascorbic acid content in different parts of Terminalia species
Fig-2: Total reducing power in different species of Terminalia
Fig-2: Total reducing power in different parts of Terminalia species
T.alata.leaf
T.alata.stem
T.arjun.leaf
1.2
T.arjun.stem
T.bel.leaf
T.bel.stem
1
T.bel.fruit
Absorption at 700nm
T.cat.leaf
0.8
T.cat.stem
T.cat.fruit
0.6
0.4
0.2
0
Methanol
Ethanol
Figure 2. Total reducing power in different parts of Terminalia species
reducing agent carrying out the reducing function which
may reduce the risk of chronic diseases such as cancer,
cardiovascular and helps make collagen, a tissue needed
for gums and blood vessels (Singh, 2010). High amount
of total ascorbic acid content was observed in methanolic
extract of T. arjuna stem bark and lower values were
observed in T. catappa stem bark when compared to the
ethanolic extracts of all the plant parts (Figure 1). T. alata
showed high ascorbic acid content next to T. arjuna stem.
Similarly, Sunday and Gboyega (2010) have compared
the antioxidant potential of different parts of Ipomea
asarifotia, Guiera senegalensis, Ansiopus mannii and
124 Int. Res. J. Biotechnol.
Fig-3: Free radical scavenging properties of Terminalia species in methanolic extracts
100
T.alata.leaf
Fig-3: Free radical scavenging properties of Terminalia
species in methanolic extracts
90
T.alata.stem
T.arjun.leaf
T.arjun.stem
T.bel.leaf
80
T.bel.stem
T.bel.f ruit
Remaining DPPH %
70
T.cat.leaf
T.cat.stem
T.cat.f ruit
60
DPPH
50
40
30
20
10
0
0
5
10
15
20
25
30
Time in minute s
Figure 3. Free radical scavenging properties of Terminalia species in methanolic extracts
Fig-4: Free radical scavenging properties of Terminalia species in ethanolic extracts
Fig-4: Free radical scavenging properties of Terminalia
species in ethanolic extracts
120
T.alata.leaf
T.alata.stem
T.arjun.leaf
T.arjun.stem
T.bel.leaf
100
T.bel.stem
T.bel.f ruit
Remaining DPPH %
T.cat.leaf
T.cat.stem
80
T.cat.f ruit
DPPH
60
40
20
0
0
5
10
15
Time in minutes
20
25
30
Figure 4. Free radical scavenging properties of Terminalia species in ethanolic extracts
observed the higher content of ascorbic acid in stem
bark, leaves and root barks of G. senegalensis than any
parts of Ipomea asarifotia and Ansiopus mannii.
The total reducing power was determined in all the
plant parts in both the methanolic and ethanolic extracts.
The methanolic extracts had given maximum amount of
reducing power when compared ethanolic extract. The
methanolic extract of T. arjuna stem bark had more
reducing power and least reducing power was recorded
in T. catappa stem bark of ethanolic extracts (Figure 2).
Mety and Mathad 125
Fig-5: Inhibition Percentage and conce ntration in methonolic extra cts of Terminalia species
100
Figure 5. Inhibition Percentage and Concentration in methanolic extracts of Terminalia
90
80
70
60
50
40
30
20
10
0
IP
T.alata.leaf
T.alata.stem
T.arjun.leaf
IC50
T.arjun.stem
T.bel.leaf
T.bel.stem
T.bel.f ruit
T.cat.leaf
T.cat.stem
T.cat.f ruit
Figure 5. Inhibition Percentage and Concentration in methanolic extracts of Terminalia
Fig-6: Inhibition Percentage and concentration in ethonolic extracts of Terminalia species
100
Figure 6. Inhibition Percentage and Concentration in ethanolic extracts of Terminalia
90
80
70
60
50
40
30
20
10
0
IP
T.alata.leaf
T.alata.stem
T.arjun.leaf
IC50
T.arjun.stem
T.bel.leaf
T.bel.stem
T.bel.f ruit
T.cat.leaf
T.cat.stem
T.cat.fruit
Figure 6. Inhibition Percentage and Concentration in ethanolic extracts of Terminalia
These results were also supported by earlier studies in
reducing power is expected to be high in polar fractions
of extracts of medicinal plants as they principally contain
effective H+ donors (Repetto Llesuy, 2001). Similar
studies were carried out by Bushra sultana et al.( 2009)
on Terminalia arjuna and in other plants such as Moringa
oleifera, Eugenia jambolana, Acacia nilotica, Azadirachta
indica, , Ficus religiosa and Aloe barbadensis, and
recorded the maximum activity of reducing power in
methanolic extracts of leaves followed by stem. The
reducing power plays an important role in assessing the
antioxidant activity.
The Inhibition percentage (IP) and Inhibition
concentration (IC50) values are considered to be a good
measure of the antioxidant efficiency of pure compounds
and extracts (Argolo et al., 2003). The results of free
radical scavenging capacity in all the extracts of
Terminalia species were evaluated. The DPPH free
radical scavenging activity had more in methanolic
extracts of T. arjuna in stem bark and least activity were
recorded in ethanolic extract of T. catappa stem bark
(Figure 3-6). The antioxidant property increases with the
126 Int. Res. J. Biotechnol.
increase in polarity of the solvent used for extraction. The
DPPH free radical scavenging activity was assessed by
calculating Inhibition Percentage (IP) of methanolic and
ethanolic extracts in Terminalia species. From the data, it
was recorded that the maximum IP value was found in
methanolic extract of T. arjuna stem bark and least in
ethanolic extract of T. catappa stem bark (Figure 3 and
4). Further, it was confirmed by IC50 values, the
concentration of antioxidant needed by 50% the initial
concentration of DPPH radicals (Figure 5 and 6). The
time taken for reaching a steady state for free radical
scavenging was faster in methanolic extracts (Padma
Sree et al., 2007) which shows that polar fractions like
methanol and ethanol gave high free radical scavenging
activity (93.95 - 95.42 IP). Similarly, highest (IP and IC50
mg/DPPH) have been already reported in leaf extracts of
Bauhinia monandra (Argolo et al., 2003).
Based on the IP and IC50 values, it was inferred that
antioxidant properties increased with increase in polarity
of the solvent used for the extraction. High IP values
were obtained as expected for methanolic and ethanolic
plant extracts as observed in previous studies (Badami et
al., 2005). The IC50 and IP values obtained from the
crude extracts allowed us to categorize the antioxidant
sources and the present results showed that methanolic
extracts of all the Terminalia species bark and fruit pulp is
preferable sources for extracting pure antioxidant
compounds as they showed high IP values.
CONCLUSION
The present investigation on the antioxidative and free
radical scavenging activities of crude methanolic and
ethanolic extracts in three different parts of four
Terminalia species, the phenols and flavonoids were
found to be potent antioxidants. The investigation
indicates that the T. alata, a less studied plant is
considered as one of the best source for natural
antioxidant. The DPPH free radical assay, Ascorbic acid
and Ferric reducing power of T. alata was highly reactive
for both antioxidative and free radical scavenging
activities. Further, there is a need to isolate and identify
these natural antioxidant compounds present in T. alata
an important medicinal plant, which is extensively used in
an Indian system of medicine.
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