International Journal of Advanced Research and Publications

ISSN: 2456-9992

Comparative Study Of Free Radical Scavenging

Potential Of Selected Myanmar Medicinal Plants
Mya Thida, Khin Mar Mya, Chan Myae Nyein, Khin Nyein Chan, War War Lwin
Cell Culture Lab, Pharmaceutical Research Department, Biotechnology Research Department, Ministry of Education,
Kyaukse, Mandalay Division, Myanmar, Phone-+959 - 5189081
myathida09@gmail.com

Cell Culture Lab, Pharmaceutical Research Department, Biotechnology Research Department, Ministry of Education,
Kyaukse, Mandalay Division, Myanmar, Phone-+959 - 797325273
Khin.821@gmail.com

Cell Culture Lab,Pharmaceutical Research Department, Biotechnology Research Department, Ministry of Education,
Kyaukse, Mandalay Division, Myanmar, Phone-+959 -797607043
chanmyaenyein@gmail.com

Cell Culture Lab,Pharmaceutical Research Department, Biotechnology Research Department, Ministry of Education,
Kyaukse, Mandalay Division, Myanmar, Phone-+959 -2174049
mijoester@gmail.com

Pharmaceutical Research Department, Biotechnology Research Department, Ministry of Education,

Kyaukse, Mandalay Division, Myanmar, Phone-+959 -402511601
warwarlwin@gmail.com

Abstract: The present investigation was to compare the effect of radical scavenging activity between the selected ethanol extracts of
Phyllanthus maderaspatensis L., Croton oblongifolius Roxb and Cynodon dactylon Pers. The effective antioxidant activity of selected
extracts on DPPH radical was showed in following orders: Phyllanthus maderaspatensis L.> Croton oblongifolius Roxb > Cynodon
dactylon Pers with their IC50 values of 48µg/ml, 64µg/ml and116µg/ml respectively. Texted extracts were compared with the standard
ascorbic acid and its IC50 value was 5.6µg/ml. For the scavenging effect of nitric oxide radical, Cynodon dactylon Pers showed the most
effective with IC50 value of 5.2µg/ml than Croton oblongifolius Roxb and Phyllanthus maderaspatensis L. with their IC50 value were
44.1µg/ml and 61.9µg/ml respectively. Three tested extracts were less scavenging activity than the standard Gallic acid IC 50 value of
3.58µg/ml. In the super oxide radical scavenging assay, Croton oblongifolius Roxb possessed the strongest scavenging activity with IC 50
value of 15.5µg/ml comparing the standard Gallic acid IC 50 of 1.79µg/ml. The IC50 value of Phyllanthus maderaspatensis L. and Cynodon
dactylon Pers were 22.3µg/ml and 32.9µg/ml respectively. The radical inhibition (%) activity of tested extracted showed the concentration-
dependent elevation with the smallest IC50 value. In this observation, each texted extracts possessed the antioxidant activity and each have
the different radical scavenging effects.

Keywords: antioxidant, radical scavenging activity, nitric oxide, super oxide

1. Introduction researches informed that different kinds of antioxidant

Endogenous and exogenous sources of biological processes
in the body for energy- generation also produced reactive
oxygen species (ROS) and reactive nitrogen species (RNS)
as byproducts. [1] Super oxide, hydrogen peroxide, alkoxyl,
peroxyl, hydroxyl radical and hypochlorous acid are named
ROS as well as nitric oxide and peroxynitrite named RNS are
at low or moderate levels are beneficial for human health.
Redox imbalance between the production of ROS and the
antioxidant defense systems leading to a phenomenon called
oxidative stress. [2] Oxidative stress to tissues and other
biomolecules such as lipids, proteins and DNA eventually
associated with chronic diseases consisting cancer, diabetes,
aging, Alzheimer’s diseases, Parkinson’s diseases,
artherosclerosis, kidney disorders, liver and pancreatic
diseases.[3] Natural antioxidant substances from diet and
different plant extracts are presumed to counteract these
oxidative stress and prevention to diseases. [4] The major
enzymatic antioxidants such as: super oxide dismutase
(SOD), catalase(CAT), glutathione peroxidase (GPx), etc.,
and other enzymatic antioxidants including vitamin E,
vitamin C, carotenoids, flavonoids, omega-3 fatty acid, etc.
from nutrient are effective against free radicals and
associated with disease prevention.[5],[6] Many scientific
compounds possessed anti-inflammatory, antiatherosclerotic,
antitumor, antimutagenic, anticarcinogenic, antibacterial, and
antiviral activities [7],[8]. Therefore, investigation of the
natural antioxidants from plant materials may be major
importance to replace the synthetic one. Various methods are
used to evaluate the antioxidant property for tested samples
and radical scavenging assays are rapid, reliable and useful.
In this research work, 1, 1-diphenyl-2-picrylhydrazyl
(DPPH)-Assay, nitric oxide (NO) radical scavenging assay
and super oxide (SO) radical scavenging assay were used for
investigation of the total antioxidant activity of selected
extracts and evaluation of their free radical scavenging
effects.
2. MATERIALS AND METHODS
2.1 Plant materials
Phyllanthus maderaspatensis L.(P.maderaspatensis), Croton
oblongifolius Roxb(C.oblongifolius) and Cynodon dactylon
Pers(C.dactylon) were selected as tested plants depending on
their traditional medicinal usage and also based on the
literature [9 ] (The Wealth of India,1951). Collection was
made during April & May, 2017 in Ta-soe & Katae village,

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 International Journal of Advanced Research and Publications
ISSN: 2456-9992
Kyaukse Township, Mandalay Division, Myanmar.
Botanical identification was made by an authorized botanist
of Mandalay University, Myanmar. (Table 1)
Table 1: Selected Medicinal Plants
The
No Botanical Name Family Name Parts
Used
The
1
Phyllanthus
Phyllanthaceae whole
maderaspatensis L.
part
Croton oblongifolius
2 Roxb. Uphorbiaceae leaves
The
3 Cynodon dactylon Pers. Gramineae whole
part
2.2 Preparation of plant extracts
The selected plant samples were collected, cleaned, air-dried,
powdered and stored in air-tight containers for further use.
Each of air dried powdered samples was percolated with
95% ethanol for one month. The solvent of each extract
material were filtered and filtrates were concentrated by
using rotary evaporator. The concentrated plant extracts were
stored in refrigerator for further experiments. All
experiments were done at Pharmaceutical Research
Department, Biotechnology Research Department (BRD),
Kyaukse, Ministry of Education.
2.3 Phytochemical Examination of Plant Samples
Phytochemical examination of plant samples was carried out
to screen the presence of 11 types of compounds such as
alkaloids, glycosides, reducing sugar, phenolic compound,
carbohydrates, amino acids, flavonoids, tannin, saponin
glycosides and cyanogenic glycosides and the acidity
whether acidic or basic or neutral. [10]. (Trease and Evans,
1980).
2.4 DPPH Scavenging Activity Assay
The ability of tested plant extracts to inhibit the DPPH
radical was evaluated in a rapid and quantified using 96-well
micro titer plate and measured at a spectrophotometer. [11]
The different concentration volumes of extracts (100μl) were
added to 100µl of methanolic solution of DPPH (0.2mM).
The reaction mixture was allowed to react in dark at room
temperature for 30 minutes. Methanol served as the blank
and DPPH in methanol, without the extracts, served as the
control. After 30 minutes of incubation, the reaction mixture
was measured at 517nm in a spectrophotometer (Eliza micro
plate reader). The radical scavenging activity was calculated
as follows:
% scavenging = [Absorbance of Control- Absorbance of
Sample/ Absorbance of Control] ×100
Graphical representation of radical scavenging activity (%)
was plotted against concentrations of tested samples. IC50
value was calculated the inhibitory concentration at which
radicals were scavenged by 50%. Ascorbic acid was used
for positive control. Tests were carried out in triplicate
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2.5 Nitric Oxide Radical Scavenging Assay
The quantities of nitrite can be determined using Griess
reagent. Scavenger of nitric oxide competes with oxygen to
reduce the production of nitrite oxide. [12] The assay was
performed in a standard flat-bottomed 96-well micro titer
plate. The desired concentrations of extracts were diluted
with methanol from the stock 10mg/ml (previous dilute with
DMSO). Gallic acid was served as standard and it was
diluted with distilled water in same concentration. The
reaction mixture is consisting of 10µl different
concentrations of each extract, 20µl of potassium phosphate
buffer (0.1M) and 70µl of sodium nitroprusside (10mM)
were placed in each well and incubated at 25°C for (90-100)
min. The same amount of reaction mixture without extract
with DMSO served as blank as control. After incubation,
plate was pre-read at 540nm at spectrophotometer. After that
50µl of [sulphanilic acid (0.33%) dissolved in 20% glacial
acetic acid] was added. And then plate was stranded for 5min
to complete of diazotization. After 5min, 50ul of naphthalene
diamine dihydrochloride (0.1%) was added and shake. After
that plate was incubated again at 25°C for 30min. After
complete incubation, plate was shaken again and final-
measured at 540nm by spectrophotometer. Same procedure
was done with Gallic acid which was standard compare with
tested extracts. Percent inhibition was calculated by the
following equation:
% scavenging = [Absorbance of Control- Absorbance of
Sample/ Absorbance of Control] ×100
IC50 which is an inhibitory concentration of each extract that
reduce 50% of nitric oxide formation was determined. IC50
was calculated from the plot of inhibition percentage against
extract concentration. [13], [14] Tests were carried out in
triplicate.
2.6 Superoxide Radical Scavenging Assay
Super oxide radical, major biological source of ROS
contribute oxidative stress to biomolecules leading to chronic
diseases. [15] Radical scavenging assay is effective way for
the antioxidant screening of plant extracts as a rapid and
sensitive. [16] Assay was assessed by Winter Bourn et al.,
(1975). [17] The assay was based on the ability of extracts to
inhibit formazan formation by scavenging the super oxide
radicals generated in riboflavin- light- nitro blue tetrazolium
(NBT) system. For this research work, it was a little
modified in a reagents concentration and tested volume to
adjust for 96-microtitter plate. 10µl of different
concentration of tested extracts, 15µl of EDTA (12mM),
10ul of NBT (1.2mM), and 5ul of riboflavin (0.5mM) were
added in each well and reaction mixture was diluted up to
200 µl with phosphate buffer (50mM). Then DMSO was
added instead of extracts for control. Plate was shaked and
pre-measured at 560nm in spectrophotometer. After that the
plate was illuminated for 30min and measured again for final
read at 560nm. [17], [18] Super oxide scavenging activity
was indicated that the difference in absorbance before and
after illumination value. Gallic acid was used as standard
control. IC50 values for each extracts as well as Gallic acid
standard were calculated.
% scavenging = [Absorbance of Control- Absorbance of
Sample/ Absorbance of Control] ×100
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 International Journal of Advanced Research and Publications
ISSN: 2456-9992

3. Results and Discussion

3.1 Phytochemical Screening
Three selected plants were qualitatively tested for the
presence of chemical constituents. According to the results,
cyanogenic glycoside of tested extracts was not detected.
Thus theses extracts could be safe for further experiments. In
these samples, alkaloid, phenolic compound, tannin and
flavonoid were detected in large amount. Therefore, these
extracts should have antioxidant activity and these could be
inhibiting the free radical generation.

3.2 DPPH Scavenging Activity

The assay was based to show free radical scavenging activity
of tested extracts on DPPH radical. The scavenging reaction
between (DPPH) and an antioxidant potential was measured
at absorbent 517nm. The degree of discoloration indicates
the scavenging potential of the selected extracts as their
hydrogen donating ability. Table (2) showed the antioxidant
activity of selected extracts in percent inhibition (%) of
different concentration with their IC50 values compared with
standard ascorbic acid at same concentration. According to
this investigation, Phyllanthus maderaspatensis L. showed
the highest scavenging activity of 78.8% at its maximum
concentration 200µg/ml than Croton oblongifolius Roxb and
Cynodon dactylon Pers for their scavenging activity of
63.2% and 61% respectively at same concentration. IC50
values of Phyllanthus maderaspatensis L., Croton
oblongifolius Roxb and Cynodon dactylon Pers were
48µg/ml, 64µg/ml and 116µg/ml respectively. The
scavenging effect of Ascorbic acid standard was 86.5% at the
highest concentration of 200µg/ml with IC50 of 5.6µg/ml.
Radical inhibition activity of standard and tested extracted
showed concentration-dependent for highest inhibition at
maximum dosage (Fig-1) and lowest IC50 value. The DPPH
radical scavenging activities of all tested samples were less
effective than standard Ascorbic acid.
Figure1: Extracts concentration (µg/ml) Vs. Percent
inhibition of Selected Extracts by DPPH Scavenging Assay
Method
3.4 Super Oxide Scavenging Activity
Among the free radicals, super oxide is one of the powerful
reactive oxygen species and harmful to the human health.
[22] In this observation, all tested extracts possessed the
higher scavenging effect on super oxide radical. (Table 4)
clearly presented that percent inhibition of super oxide
radical at different extracts concentration with each IC50
value. The highest scavenging activity was found in Croton
oblongifolius Roxb extract and lowest in Cynodon dactylon
Pers (91.6% with IC50 value 15.5µg/ml and 77.3% with IC50
value of 32.9µg/ml) respectively. For Phyllanthus
maderaspatensis L., scavenging percent was 84% with IC50
value of 22.3µg/ml. (Table 4) Therefore, Croton
oblongifolius Roxb presented the significant scavenging
effect compared with the capacity of standard Gallic acid
complete inhibition with IC50 value of 1.79µg/ml. All tested
extracts and Gallic acid standard exhibited concentration-
dependent radical scavenging activity, which is the higher
the concentration, the greater the scavenging effects. (Fig- 3)

3.3 Nitric Oxide Scavenging Activity Table2: DPPH Scavenging Effects of Selected Extracts
Despite the nitric oxide involve in normal physiological Compared with Ascorbic Acid
processes, excessive generation of nitric oxide is harmful to
human health. [19] In addition, nitric oxide is implicated in Inhibition Activity (%)
inflammation, cancer and other pathological conditions. Concentra Phyllanthus Croton .Cynodon
Ascorbic
-tion maderaspatenis oblongifolls dactylon
[20],[ 21] Thus it is an essential to screen the natural (µg/ml) L. Roxb. Pers.
Acid
antioxidant that could be inhibit nitric oxide generation. The 200 78.88 63.23 61 86.46
assay was performed with three tested extracts were 100 74.26 55.37 47.64 82.1
compared with Gallic acid as standard to find out the 50 51.51 47.21 35.25 76.99
25 26.78 40.35 30.40 72.26
scavenging potential. Among the tested extracts, Cynodon 12.5 14.37 31.03 27.09 71.2
dactylon Pers showed the highest activity for 66.3% 6.25 8.58 18.08 26.46 53.76
scavenging at the highest concentration (200µg/ml) with IC50 3.125 4.83 6.67 23.96 24.25
value of 5.19µg/ml comparing to the standard Gallic acid IC50 (µg/ml) 48.2±0.45 64.2±3.4 116±5.6 5.6±0.07
was 89.8% scavenging effect at the same concentration with
IC50 value of 3.58µg/ml. The scavenging percent of Croton
oblongifolius Roxb and Phyllanthus maderaspatensis L. were
63.2% and 60.6% respectively with IC50 value of 44.1µg/ml
and 61.9µg/ml respectively. Table (3) The degree of
inhibition for tested extracts and Gallic acid exhibited the
higher the concentration, the greater the scavenging
potential. (Fig 2) This observation indicated that the tested
extracts may contain compound that capable to inhibit and
reduce the nitric oxide generation.

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 International Journal of Advanced Research and Publications
ISSN: 2456-9992

Values are expressed as mean ± standard deviation (n=3) Table 3: Nitric Oxide Scavenging Activity of Selected Plant
Extracts Compared with Gallic Acid

Inhibition Activity (%)

Concentra Phyllanthus Croton .Cynodon Gallic
-tion maderaspa oblongifolls dactyln Acid
(µg/ml) Tenis L. Roxb. Pers.
200 60.66 63.26 66.33 89.85
100 52.87 56.08 62.02 87.86
50 48.9 50.62 61.22 86.26
25 45.6 46.68 58.19 83.56
12.5 39.67 46.35 58.14 81.03
6.25 38.03 39.9 54.25 69.93
3.125 31.86 37.68 39.99 44.93
IC50 (µg/ml) 61.9±2.9 44.1±3.6 5.2±0.7 3.58±0.1

Values are expressed as mean ± standard deviation (n=3)

Figure 2: Concentration (µg/ml) Vs % Inhibition of Tested
Extracts by Nitric Oxide Scavenging Assay Method Table 4: Super Oxide Scavenging Activity of Tested Extracts
Compared with Gallic Acid
Inhibition Activity (%)
Concenta Phyllanthus Croton .Cynodon Gallic Acid
-tion maderaspa oblongifol dactylon
(µg/ml) tensis. ius Roxb pers
200 84 91.65 77.3 100
100 71.23 72.27 73.62 100
50 60.23 66.68 56.14 91.79
25 53.49 56.67 46.67 91.62
12.5 35.46 47.53 35.17 87.78
6.25 27.25 32.72 28.86 80.03
3.125 20.24 28.08 25 69.24
1.562 - - - 45.55
IC50 22.3±0.5 15.5±1.2 32.9±3.2 1.79±0.01
(µg/ml)

Figure: 3 Concentration (µg/ml) Vs. Percent Inhibition of
Tested Extracts by Super Oxide Scavenging Assay Method
4. Conclusion
All texted extracts contained the large amount of flavonoid,
tannin and phenolic compound by the phytochemical test.
These phenolic compounds are a major group of antioxidant
or act as free radical scavengers. [23] And then all tested
extracts inhibited free radicals such as DPPH, NO and SO,
indicating their antioxidant activity with concentration-
dependent manner. In addition, the capacity of each tested
extract indicated the different types of radical scavenging
activity from the results of radical scavenging assays.
Based on these finding, these texted extracts would be a
potential source of natural antioxidants as a therapeutic
agent.
Values are expressed as mean ± standard deviation (n=3)
Acknowledgement
The authors would like grateful to Dr Aye Aye Khai,
Professor and Head of Biotechnology Research Department
for kindly supported us the study, Dr Thaesu Moe and Dr
Htet Htet Win for their constructive helpful and Dr Win Win
Khai for botanical identification.
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Author Profile
Mya Thida received Ph.D
(Biotechnology) in 2009 from Yangon
Technological University. From 2007 to
2013, she worked at Pharmaceutical
Research Department, Kyaukse
Technological University. During 2013
to 2015, she studied at Natural Research
Center, Korea Institute of Sciences and
Technology, Gangneung, South Korea,
as a visiting scientist. From 2015 to now, she has been
working at Biotechnology Research Department, Kyaukse,
Ministry of Education, Myanmar.
109