Antioxidant Activity of Elytraria acaulis Aerial Extracts

Suresh Babu Bastipati. Int. Journal of Engineering Research and Application www.ijera.com
ISSN : 2248-9622, Vol. 7, Issue 3, ( Part -3) March 2017, pp.55-59
www.ijera.com DOI: 10.9790/9622- 0703035559 55 | P a g e
Evaluation of Anti-oxidant Activity of Elytraria acaulis Aerial
Extracts
Suresh Babu Bastipati1*
and M. Lakshmi Narasu1
1
Centre for Biotechnology, IST, JNTUH, Kukatpally, Hyderabad – 500 085, T.S, India
ABSTRACT
Elytraria acaulis, a stem less perennial herb of Acantheceae family has many medicinal and therapeutic
properties. Anti oxidative activity of the aerial parts of this Elytraria acaulis were assessed in the present study.
The aerial parts of the plant (Stem & Leaves) were extracted in different organic solvents such as n-Hexane,
Ethanol, Methanol, Ethyl Acetate and Chloroform. Initially, Total Phenolic & Total Flavonoids content in
different solvent plant extracts were estimated. The free radical scavenging and antioxidant activity of the
Elytraria acaulis aerial extracts in different organic solvents were also assayed by DPPH assay, FRAP assay.
The aerial extracts of Elytraria acaulis have shown significant anti oxidant activity. Hence, further studies on
this plant will enable elucidation of its therapeutic properties and medicinal applications.
Keywords: Aerial parts, phytochemical analysis, Antioxidant assays, Elytraria acaulis.
I. INTRODUCTION
Antioxidants are substances that protect
cells from the damage caused by free radicals.
Antioxidants interact with and stabilize free
radicals and prevent the damage, which may lead to
cancer. Examples of antioxidants include beta-
carotene, glutathione, lycopene, vitamins C, E, A
well as enzymes such as catalase, superoxide
dismutase and various peroxidases and other
substances (Blot et al., 1993). Low levels of
antioxidants, or inhibition of the antioxidant
enzymes, causes oxidative stress and may damage
or kill cells. As oxidative stress might be an
important part of many human diseases, the use of
antioxidants in pharmacology is intensively
studied, particularly for treatments of stroke and
neurodegenerative diseases (Xianquanet al., 2005).
Antioxidants are also widely present as ingredients
in dietary supplements maintaining health and
preventing diseases such as cancer and coronary
heart disease. Although, initial studies suggested
that antioxidant supplements might promote health,
later, large clinical trials did not detect any benefit
and suggested instead that excess supplementation
may be harmful (Steinmetz et al., 1996). Free
radicals are very unstable and react quickly with
other compound by abstracting its electron to attain
stability. When the attacked molecule loses its
electron, it becomes a free radical itself, resulting in
the disruption of the substance especially in fatty
foods. Environmental factors such as pollution,
radiation, cigarette smoking and herbicides can also
spawn free radicals in the body (Borek, 1991).
Elytraria acaulis is a stem less perennial
herb belonging to acantheceae family of plant
kingdom, which is a small shrub that grows in
shady dry places. This plant is often found on often
on rocky or sandy soils. It is a traditional herb, is
the whole plant is used for medicinal purposes.
Practitioners of traditional medicine have described
therapeutic efficacy of many indigenous plants for
several disorders. The World Health Organization
estimates that plant extracts or their active
constituents are used as folk medicine in traditional
therapies of 80% of the world's population. The
pharmacological industries have produced a
number of new antibiotics; however, resistance to
these drugs by microorganisms has increased due
to their genetic ability to transmit and acquire
resistance to synthetic drugs. The present study
was designed to analyze the phytochemicals
present in the plant extract as well its free radical
scavenging property (invitro anti-oxidant assay).
II. MATERIALS & METHODS
Collection and preparation of Plant Material:
Fresh leaves and stem of Elytraria acaulis were
procured from Seshachalam forest, Tirupathi, India.
The dried samples were cut into small pieces and
ground into fine powder using a dry grinder. The
ground samples were sieved to get uniform particle
size, then kept in an airtight container and stored in
a freezer (-20°C)until further analysis.
Extraction of sample: Each ground sample was
weighed and transferred into a beaker. Solvents like
n-Hexane, Ethyl acetate, Ethanol, Methanol and
Chloroform in the increasing order of polarity were
added in the ratio of 1:10 and stirred with the aid of
a magnetic stirrer. The extraction mixture was left
to sediment for at least 72 h before the extract was
separated from the residue by filtration through
Whatman No. 1 filter paper. The solvent plant
RESEARCH ARTICLE OPEN ACCESS

extracts were lyophilized using a freeze dryer and
the residual solvent of each extract was removed
under reduced pressure using a rotary evaporator.
Extracts were produced in duplicates and used to
assay the antioxidant activity.
2.1 Total Phenolic content of Elytraria acaulis
extracts
The total phenolic contents of Elytraria
acaulis leaves & stem extracts were determined
according to the method described by Malik and
Singh. Aliquots of the extracts were taken in a 10
ml glass tube and made up to a volume of 3 ml with
distilled water. Then 0.5 ml folinciocalteau reagent
(1:1 with water) and 2 ml Na2CO3 (20%) were
added sequentially in each tube. A blue color was
developed in each tube because the phenols
undergo a complex redox reaction with
phosphomolibdic acid in FolinCiocalteau reagent in
alkaline medium which resulted in a blue colored
complex, molybdenum blue. The test solutions
were warmed for 1 minute, cooled and absorbance
was measured at 650 nm against the reagent used as
a blank. A standard calibration plot was generated
at 650 nm using known concentrations of catechol.
The concentrations of phenols in the test samples
were calculated from the calibration plot and
expressed as mg catechol equivalent of phenol/g of
sample (Malik et.al. 1980).
2.2 Total Flavonoid content of Elytraria acaulis
extracts
The aluminum chloride method was used
for the determination of the total flavonoid content
of the sample extracts
5
. Aliquots of extract
solutions were taken and made up the volume 3ml
with methanol. Then 0.1ml AlCl3 (10%), 0.1ml
Na-K tartarate and 2.8 ml distilled water were
added sequentially. The test solution was
vigorously shaken. Absorbance at 415 nm was
recorded after 30 minutes of incubation. A standard
calibration plot was generated at 415 nm using
known concentrations of quercetin. The
concentrations of flavonoid in the test samples were
calculated from the calibration plot and expressed
as mg quercetin equivalent /g of sample (Mervat et.
Al., 2009).
2.3. DPPH Assay of Elytraria acaulis extracts
Effect of Elytraria acaulis solvent extracts
on DPPH radical was measured based on the
method modified by (Patel Rajesh et al., 2011;
ArunaPrakash, 2000). An aliquot of 200 μl of
extract of various concentrations andascorbic acid
as standard were mixed with 800 μl of 100 mMTris
- HCl buffer (pH 7.4). The mixture was then added
to 1 ml of 500 μM DPPH. This was made up to a
DPPH final concentration of 250 μM. The mixture
was shaken vigorously and left to stand at room
temperature for 20 min in a dark room. Absorbance
at 517 nm was measured using a UV-Vis
spectrophotometer until the reading reached a
plateau. The capability of seaweeds extracts to
scavenge the DPPH radical was calculated by using
the following equation:
% scavenging = [Absorbance of control-
Absorbance of test sample/Absorbance of control]
X 100.
IC50 value was determined from the plotted graph
of scavenging activity versus the concentration of
seaweed extracts, which is defined as the amount of
antioxidant necessary to decrease the initial DPPH
radical concentration by 50%. Triplicate
measurements were carried out and their activity
was calculated by the percentage of DPPH
scavenged.
2.4. FRAP Assay of Elytraria acaulis extracts
Ferric ions reducing power was measured
according to the method of Oyaizu with a slightest
modification (Oyaizu 1986). Hydroalcoholic
extract of Kalanchoepinnatain different
concentrations ranging from 100 μl to 500 μl were
mixed with 2.5 ml of 20 mM phosphate buffer and
2.5 ml 1%, w/v potassium ferricyanide, and then
the mixture was incubated at 50 °C for 30 min.
Afterwards, 2.5 ml of 10%, w/v trichloroacetic acid
and 0.5 ml 0.1%, w/v ferric chloride were added to
the mixture, which was kept aside for 10 min.
Finally, the absorbance was measured at 700 nm.
Ascorbic acid was used as positive reference
standard. All assays were run in triplicate way and
averaged.
Statistical analysis
Experimental results are expressed as
means ± standard deviation (SD). All
measurements were replicated three times. IC50
values were also calculated by linear regression
analysis. Experiments results were further analyzed
for Pearson correlation coefficient(r) between total
phenolic, flavonoid and DPPH radical scavenging
assay using the Microsoft Excel 2007 software and
two-way analysis of variance (ANOVA) was
applied to investigate the differences among means
by using the Microsoft Excel 2007. The values
were considered to be significantly different at P <
0.05.
III. RESULTS AND DISCUSSION
Natural antioxidants that are present in
herbs and spices are responsible for inhibiting or
preventing the deleterious consequences of
oxidative stress. Spices and herbs containfree
radical scavengers like polyphenols, flavonoids and

phenolic compounds. Several reports have
conclusively shown close relationship between
total phenolic contents and antioxidative activity of
the different plants. Since the chemical composition
and structures of active extract components are
important factors governing the efficacy of natural
antioxidants, the antioxidant activity of an extract
could not be explained on the basis of their
phenolic content, which also needs their
characterization. These results from various free
radical-scavenging systems revealed that the
Elytraria acualis had significant antioxidant
activity and free radical-scavenging activity.
3.1 Total Phenolic content of Elytraria acaulis
Phenolic compounds are a class of
antioxidant agents, which act as free radical
terminators. Total phenols were measured by
FolinCiocalteu reagent in terms of Gallic acid
equivalent. According to our study, the high
contents of this Phytochemical in aqueous extract
of Elytraria acaulis can explain its high radical
scavenging activity (Table 1). Figure 1 shows the
activity for Elytraria acaulis plant extract in all
solvents was found to be lower than the standard
(0.078) at 200 µg/ml concentration. It was found
that the activity of Elytraria acaulis plant extract in
ethanol was found to be higher (0.075) followed by
methanol, ethyl acetate, chloroform and hexane
solvents respectively at 1000 µg/ml concentration.
3.2 Total Flavonoid content of Elytraria acaulis
According to our study, the high contents
of this Phytochemical in aqueous extract of
Elytraria acaulis can explain its high radical
scavenging activity (Table 2). Figure 2 shows the
activity for Elytraria acaulis plant extract in all
solvents was found to be lower than the standard
(0.866) at 200 µg/ml concentration. It was found
that the TFC of Elytraria acaulis plant extract in
methanol was found to be higher (0.506) followed
by ethanol, ethyl acetate, hexane and chloroform
solvents respectively at 1000 µg/ml concentration
but less when compares to that of standard
Quercetin.
3.3 DPPH Assay of Elytraria acaulis extracts
DPPH is a stable free radical at normal
temperature. It shows specific absorbance at 517nm
due to color of methanolic solution of DPPH. Body
also contains man free radicals, which assumed
same as DPPH. Decrease in absorbance of mixture
indicates the radical scavenging activity (Table no:
3). Inhibition activity for Elytraria acaulis plant
extract in Methanol solvent was found to be higher
(82.0) when compared with the other solvents at
1000 µg/ml concentration and it is lower than
standard Ascorbic acid (86.0). Inhibitory activity
was found to be higher for Methanol followed by
Hexane, Ethanol and Chloroform extracts (Fig. 3).
3.4 FRAP Assay of Elytraria acaulis extracts
FRAP assay is based on a redox-linked
reaction, whereby antioxidants present in plant
extracts act as reductants while ferric ions in
reagents serve as oxidants. Reduction of ferric-
tripyridyltriazine to ferrous complex forms an
intense blue color with maximum absorption at 593
nm. The ferric reducing ability of extracts of
Elytraria acaulisis shown in Table 6. Figure 6
showed that Methanol, Ethanol, Ethyl acetate,
Chloroform and Hexane extract reduced ferric ions
efficiently and had reducing activity in the range of
0.127+0.02– 0.153+0.01. Hexane and Chloroform
extracts showed least reducing activity with
0.119+0.01 and 0.127+0.02 respectively. Methanol
extract of Elytraria acaulis displayed significant
reducing ability (0.153+0.01). Reducing activity of
all the extracts was lesser than the standard
quercetin (0.236+0.02).
IV. CONCLUSION
In conclusion, the present study was
undertaken to analyze Antioxidant activity of the
aerial extracts of Elytraria acaulis and the findings
clearly indicated that Elytraria acaulis has
profound antioxidant activity. These results are
supportive to the traditional use of Elytraria
acaulis as antioxidative agent. The selected plant
has varieties of phytochemicals such as phenolics,
flavonoids and tannins and possesses various
activities such as radical scavenging, antioxidant
and reducing activities. Therefore, this plant
species may be attempted to derive the drugs of
antioxidant properties proving the efficacy of this
research work whichis highly valuable for
identification of newer antioxidant principles. The
scientific community has begun to interest in the
identification, recovery and enhanced performance
of natural antioxidant principles from plant sources.
The purpose of this present study is also guide to
the invention of new antioxidant sources
from Elytraria acaulis.
Conflict of Interest
We declare that we have no conflict of interest.
ACKNOWLEDGEMENTS
The authors would like to acknowledge CBT,
JNTUH for providing me the laboratory facilities.
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S.No. Concentration
(μg/ml)
Methanol Ethanol Ethyl
Acetate
Hexane Chloroform
1. 200 271+0.35 227+0.32 258+0.48 174.0+0.24 194.0+0.24
2. 400 421+0.35 376+0.43 351+0.25 242+0.35 242+0.35
3. 600 532+0.24 489+0.35 482+0.56 324+0.25 314+0.25
4. 800 642+0.45 557+0.21 576+0.48 452+0.57 478+0.57
5. 1000 752+0.63 632+1.32 672+0.56 526+0.26 575+0.26
*Each Value represents mean value+ standard deviation of three replicates
Table 1: Total Phenolic Content (Expressed as mg Gallic Acid/g dry weight) of Elytraria acaulis
Fig 1: Total Phenolic Content of Elytraria acaulis extracts
Concentration
(μg/ml)
Methanol Ethanol Ethyl
Acetate
Hexane Chloroform
200 32.02+1.25 54.5+0.32 83.17+1.35 74.0+0.24 94.0+0.24
400 74.45+1.35 108.3+1.23 173.4+1.56 142.2+0.35 162.3+0.35
600 103.67+1.3 284.4+1.35 216.82+1.3 224.3+0.25 214.4+0.25
800 142.34+0.45 357.1+1.21 376.32+1.48 352.2+0.57 378.5+0.57
1000 178.34+0.63 432.2+1.32 421.23+1.56 426.4+0.26 475.6+0.26
Table 2: Total Flavanoid content (Expressed as mg quercetine/g dry weight) of Elytraria acaulis

Fig 2: Total Flavonoid Content of Elytraria acaulis extracts
S.No. Concentration Ascorbic Acid Methanol Ethanol Chloroform Hexane
1. 50 50.42+0.50 34+0.60 32+0.59 33+0.58 30+0.62
2. 100 56.64+0.52 46+0.58 45+0.50 43+0.53 42+0.54
3. 150 63.52+0.43 62+0.40 59+0.43 60+0.48 58+0.42
4. 200 70.42+0.35 68+0.48 65+0.39 66+0.42 67+0.39
5. 250 78.62+0.28 73+0.34 70+0.30 72+0.35 69+0.30
6. 300 86.0+0.22 82.0+0.20 79+0.24 76+0.23 81+0.22
IC50 52.3 118.58 129.27 127.67 135.38
*All data were average (+SD) of three replicates
Table 3: DPPH Assay of Elytraria acaulis extracts
Fig 3: DPPH Assay of Elytraria acaulis extracts
S.No. Elytraria Acaulis
1. Quercetin 0.236+0.02
2. Methanol 0.153+0.01
3. Ethanol 0.142+0.02
4. Ethyl Acetate 0.139+ 0.01
5. Hexane 0.119+0.01
6. Chloroform 0.127+0.02
*Each value represents mean values+standard deviation of three replicates.
Table 6: Ferric Reducing Ability – FRAP (Expressed as mM FeSO4/g dry weight) of Elytraria Acaulis

Antioxidant Activity of Elytraria acaulis Aerial Extracts

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