World Journal of Pharmaceutical Research

Rucha et al. World Journal of Pharmaceutical Research

SJIF Impact Factor 5.990

Volume 4, Issue 7, 1301-1309. Research Article ISSN 2277– 7105

IN VITRO EVALUATION OF FREE RADICAL SCAVENGING

ACTIVITIES OF EPIPHYLLUM OXYPETALUM

Rucha Dandekar*, Bharti Fegade and V H Bhaskar

Gahlot Institute of Pharmacy, Plot no-59, Sector 14, Koparkhairane, Navi Mumbai- 400709.

ABSTRACT
Article Received on
27 April 2015, Objective: The main aim of the study is to determine the free radical

Revised on 22 May 2015,

Accepted on 14 June 2015
*Correspondence for
Author
Rucha Dandekar
Gahlot Institute of
Pharmacy, Plot no-59,
Sector 14, Koparkhairane,
Navi Mumbai- 400709.
scavenging activity of Epiphyllum oxypetalum by in vitro method.
Method: DPPH radical scavenging assay and Hydrogen peroxide
scavenging assay were the methods used to determine the antioxidant
activity Result: The maximum percent inhibition by DPPH radical
and Hydrogen peroxide scavenging method was seen at 2000 µg/ml
and 500 µg/ml concentration in both alcohol and aqueous extract
respectively. Conclusion: Epiphyllum oxypetalum exhibits antioxidant
activity
KEYWORDS: Epiphyllum oxypetalum, DPPH radical scavenging
assay, Hydrogen peroxide scavenging assay, Antioxidant activity.

INTRODUCTION
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) includes free radicals
and other non radical reactive derivatives. Reactivity of radicals is generally stronger than
non-radical species though radicals are less stable. ROS and RNS includes radicals such as
hydroxyl (OH•), superoxide (O2•−), peroxyl (RO2•), alkoxyl (RO•), hydroperoxyl (HO2•),
nitrogen dioxide (NO2•) nitric oxide (NO•) and lipid peroxyl (LOO•); and non radicals like
hydrogen peroxide (H2O2), nitrogen dioxide (NO2•), hypochlorous acid (HOCl), ozone
(O3), singlet oxygen , peroxynitrate (ONOO−), nitrous acid (HNO2), lipid peroxide (LOOH),
dinitrogen trioxide (N2O3).[1]

Free radicals are the species capable of independent existent that contains one or more
unpaired electrons in its outer shell. Free radicals having single electron in their outer shell
and becomes more reactive. They are unstable and try to become stable, either by accepting
or donating an electron.[2] The free radicals are generated in living systems as a part of the

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normal physiological process.[3] Generation of free radicals or reactive oxygen species (ROS)
during metabolism and other activities beyond the antioxidant capacity of a biological system
gives rise to oxidative stress.[4,5] The ROS and free radicals mediated reactions are involved
in various pathological conditions such as anaemia, asthma, arthritis, inflammation, neuro
degeneration, cancer, mutagenesis, Alzheimer, AIDS, ageing process and perhaps dementia,
malaria.[6-8]

Antioxidant may be defined as radical scavengers which protect the human body against free
radical that cause pathological condition. Higher concentrations of oxygen supply is very
toxic and it can damage tissues and antioxidant system of body thus resulting in the
production of free radicals. An imbalance between the production of free radicals and natural
antioxidants could cause damage to the proteins, DNA and genetic material within the cells.
The present day concepts of toxicity are due to the involvement of oxygen free radicals or
reactive oxygen species. Therefore, it becomes essential to test the antioxidant potential of
the plant material.[9]

Antioxidants are the substance that when present in low concentrations compared to those of
an oxidisable substrate significantly delays or prevents oxidation of that substance.[10] Plants
are the potential source of natural antioxidants. It has been reported in the scientific reports
and experimental that plants contain a large variety of phytochemicals having antioxidant
property.[11] Natural antioxidants or phytochemical antioxidants are the secondary metabolites
of plants.[12] Examples include carotenoids, flavonoids, tocopherols (delta> gamma>
beta>alpha), Beta carotene, Lycopene, Sesamol, Anthocyanins, Catechins, Ellagic acid,
Lutein, Resveratrol, cinnamic acids, benzoic acids, folic acid, ascorbic acid, tocotrienols etc.,
are some of the antioxidants produced by the plants.[13]

Epiphyllum oxypetalum is plant belonging to cactaceae family. It has many traditional uses
since people have been using this plant for the treatment of various ailments. The
phyllocaldes contain some active ingredients and show antibacterial activity.[14] The stem is
also used medicinally to cure dropsy and cardiac affections. Vietnamese people use petals of
the faded blooms to make soups which are supposed to have tonic and aphrodisiac medicinal
properties.[15]

The uses of medicinal plants as traditional medicine is wide spread and represent a large
source of natural anti-oxidants that might serve as leads for the development of the novel

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drugs.[16] Recently, much attention has been directed towards the development of ethno-
medicines with strong antioxidant properties.[17]

MATERIAL AND METHODS

Chemicals
1, 1-diphenyl-2-picrylhydrazyl DPPH was obtained from Sigma Aldrich. Hydrogen peroxide
and ascorbic acid were obtained from Loba Chemie. All other chemicals and reagents used
during this test were of analytical grade.

Plant material
Collection of plant material
The fresh leaves of Epiphyllum oxypetalum were collected from the nusery and local garden
in Ratnagiri, Maharashtra. The collected leaves were identified and authenticated by Dr.
Harshad Pandit from Guru Nanak Khalsa College, Matunga.

Preparation of plant extract

The fresh leaves were washed under running tap water, shed dried and coarsely powdered in
a mechanical grinder.

Preparation of alcohol extract - The powder was extracted with absolute ethanol in soxhlet
extractor at temperature 40-50°C. The extract was dried on water bath at 60°C.

Preparation of aqueous extract - The powder was weighed approximately and was added in
distilled water. The extraction process was carried out at 80°C for 4-5 hrs. Then the extract
was filtered hot using a muslin cloth and dried on water bath at 60°C.

METHODS
1) DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging assay method[18]
The free radical scavenging activities of alcohol and aqueous extracts and the standard L-
ascorbic acid (vitamin C) were measured in terms of hydrogen donating or radical scavenging
ability, using the stable radical DPPH. 0.1mM solution of DPPH in alcohol was prepared and
it was protected from light influence by maintaining the dark condition and was folded with a
aluminum foil and 3 ml of this solution was added to 1 ml various concentrations of (100–
2000μg/ml) extracts or standard solution of 10–100μg/ml. Absorbance was taken after 30
minutes at 517 nm. The percentage inhibition activity was calculated using the following
formula - % Inhibition = [(Acontrol – Atest)/Acontrol] ×100

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Where, Acontrol- Absorbance of the control

Atest - Absorbance of extract/standard taken as ascorbic acid.

2) Hydrogen peroxide scavenging assay method[19]

The ability of the extract to scavenge hydrogen peroxide was determined in this assay
method. The extracts (4ml) were prepared using distilled water at various concentrations
(100μg - 500 μg). Then extracts were mixed with 0.6 ml of 4mM H2O2 solution prepared in
phosphate buffer (0.1 M with pH 7.4) and incubated for 10 min. The absorbance was
measured at 230 nm against blank solution containing the plant extract without H2O2.
Ascorbic acid was used as standard reference. The percentage inhibition activity was
calculated using the following formula -
% Inhibition = [(Acontrol – Atest)/Acontrol] ×100
Where, Acontrol- Absorbance of the control
Atest - Absorbance of extract/standard taken as ascorbic acid.

RESULT AND DISCUSSION

1) DPPH radical scavenging assay
Absorbance of control = 0.59±0.007

Table 1: DPPH scavenging activity of alcohol extract and aqueous extract of leaves
Alcohol extract Aqueous extract
Sr. Concentration
Absorbance % Inhibition Absorbance % Inhibition
no. in μg/ml
at 517 nm ± SD at 517 nm ± SD
1 100 0.481±0.008 18.47±0.76 0.531±0.01 9.77±1.67
2 200 0.421±0.004 28.74±0.89 0.524±0.004 11.51±1.19
3 300 0.362±0.007 38.73±1.73 0.492±0.005 16.65±1.23
4 400 0.315±0.005 46.58±0.88 0.476±0.004 19.41±0.75
5 2000 0.234±0.007 60.37±1.67 0.388±0.002 34.23±0.88

Table 2: DPPH scavenging activity of standard drug ascorbic acid

Sr. no. Concentration in μg/ml Absorbance at 517 nm % Inhibition± SD
1 10 0.055±0.002 90.63±0.51
2 20 0.054±0.0008 90.91±0.1
3 30 0.050±0.001 91.48±0.19
4 50 0.045±0.002 92.33±0.33
5 80 0.028±0.002 95.26±0.43

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Figure 1: Graphical representation of DPPH radical scavenging assay of alcohol and

aqueous extract

Figure 2: Graphical representation of DPPH radical scavenging assay of standard drug

ascorbic acid

The model of scavenging the stable DPPH radical is a widely used method to evaluate the
free radical scavenging ability of various samples. DPPH is a stable nitrogen-centered free
radical the colour of which changes from violet to yellow upon reduction by either the
process of hydrogen- or electron- donation. Substances which are able to perform this
reaction can be considered as antioxidants and therefore radical scavengers. It was found that
the radical-scavenging activities of all the extracts increased with increasing concentration.
Maximum inhibition by alcohol extract (60.37±1.67) and aqueous extract (34.23±0.88) was

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shown at 2000μg/ml concentration whereas ascorbic acid showed maximum inhibition

(95.26±0.43) at 80μg/ml concentration.

2) Hydrogen peroxide scavenging assay

Absorbance of control= 0.734±0.02

Table 3: Hydrogen peroxide scavenging assay of alcohol extract and aqueous extract of
leaves
Alcohol extract Aqueous extract
Sr. Concentrati
Absorbance % Inhibition± Absorbance % Inhibition±
no. on in μg/ml
at 230 nm SD at 230 nm SD
1 100 0.617±0.02 15.92±0.72 0.653±0.02 11.03±0.25
2 200 0.534±0.02 27.25±1.04 0.622±0.02 15.24±0.65
3 300 0.500±0.01 31.8±1.15 0.594±0.01 18.93±1.43
4 400 0.442±0.02 39.78±1.83 0.567±0.01 22.72±1.16
5 500 0.412±0.009 43.76±0.97 0.521±0.01 27.07±0.16

Table 4: Hydrogen peroxide scavenging assay of standard drug ascorbic acid

Concentration in Absorbance % Inhibition±
Sr. no.
μg/ml at 230 nm SD
1 10 0.131±0.003 82.03±1.83
2 20 0.122±0.005 83.34±2.04
3 40 0.107±0.007 85.37±2.33
4 60 0.094±0.006 87.11±2.09
5 100 0.068±0.003 90.64±1.15

Figure 3: Graphical representation of hydrogen peroxide scavenging assay of alcohol

and aqueous extract of Epiphyllum oxypetalum leaves

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Figure 4: Graphical representation of hydrogen peroxide scavenging assay of standard

drug ascorbic acid

Typical phenolics that possess antioxidant activity have been characterized as phenolic acids
and flavonoids. Phenolic acids have repeatedly been implicated as natural antioxidants in
fruits, vegetables, and other plants. Maximum inhibition by alcohol extract (43.76±0.97) and
aqueous extract (27.07±0.16) was shown at 500μg/ml concentration whereas ascorbic acid
showed maximum inhibition (90.64±1.15) at 100μg/ml concentration. In the present study the
antioxidant activity test by DPPH radical scavenging assay and Hydrogen peroxide
scavenging assay was found to be positive for both the extracts which can be attributed to the
presence of phenols and flavonoids. The higher antioxidant activity was shown by alcohol
extract.

CONCLUSION
The alcohol extract and aqueous extract exhibited antioxidant activity. The maximum
antioxidant activity was shown by the alcohol extract than the aqueous extract. Thus it can be
concluded that the Epiphyllum oxypetalum exhibits antioxidant activity.

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