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International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 98
Phytochemical analysis, antimicrobial and
antioxidant activities of different parts
of Pleocaulus sessilis (Nees) Bremek
(Acanthaceae)
H. L. Raghavendra1, T. R. Prashith Kekuda2, S. Akarsh2, M. C. Ranjitha2
H. S. Ashwini3
1Department of Biochemistry, School of Medicine, Wollega University, Nekemte, Ethiopia, 2Department
of Microbiology, S.R.N.M.N College of Applied Sciences, N.E.S Campus, Shivamogha, Karnataka, India,
3Department of PG Studies and Research in Applied Botany, Jnana Sahyadri, Kuvempu University,
Shankaraghatta, Karnataka, India
Abstract
Aim: This study aims to investigate antimicrobial and antioxidant activity of leaf, stem, and inflorescence of
Pleocaulus sessilis (Nees) Bremek belonging to Acanthaceae. Materials and Methods: The leaves, inflorescences,
and stems were separated, dried under shade, powdered, and extracted using methanol by maceration process.
Preliminary phytochemical analysis was carried out by standard phytochemical tests. Antibacterial and antifungal
activity was carried out by agar well diffusion and poisoned food technique, respectively. Antioxidant activity was
evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, 2,2-azinobis 3-ethylbenzothiazoline
6-sulfonate (ABTS) radical scavenging and ferric reducing assays. Folin-Ciocalteu reagent method was employed
to estimate the total phenolic content of extracts. Results and Discussion: Flavonoids, saponins, steroids, and
phenols were present in all three extracts. Extracts were inhibitory to all test bacteria with maximum activity against
Klebsiella pneumoniae. Overall, inflorescence extract exhibited high inhibition of test bacteria when compared to
other extracts. Extracts were effective in reducing mycelial growth of test fungi. Leaf extract was more effective
against test fungi followed by inflorescence and stem extracts. All extracts exhibited dose-dependent radical
scavenging and ferric reducing activity. Leaf extract exhibited marked antioxidant activity when compared to
other two extracts. The leaf extract scavenged DPPH and ABTS radicals with an inhibitory concentration value
of 27.16 µg/ml and 9.16 µg/ml, respectively. Total phenolic content was high in leaf extract (112.13 mg gallic
acid equivalents [GAE]/g) followed by inflorescence (85.65 mg GAE/g) and stem (42.42 mg GAE/g) extracts.
Conclusion: The plant can be used to treat diseases caused by pathogenic bacteria, prevention, and control of
phytopathogens and oxidative damage caused by free radicals. Further studies are to be carried out to isolate and
characterize active principles from the plant and to determine their biological activities.
Key words: Antimicrobial, antioxidant, phytochemical, Pleocaulus sessilis, Strobilanthes sessilis
Address for correspondence:
T. R. Prashith Kekuda, Department of Microbiology,
S.R.N.M.N College of Applied Sciences, N.E.S Campus,
Balraj Urs Road, Shivamogha - 577 201, Karnataka,
India. Phone: +91-9739864365.
E-mail: p.kekuda@gmail.com
Received: 27-02-2017
Revised: 23-03-2017
Accepted: 05-04-2017
INTRODUCTION
Plants are an integral part of daily life as
humans depend on many plants for food,
shelter, cloth, timber, dyes, and medicine.
The use of herbal medicine for therapy is as
old as humanity itself, and it is estimated that
80% of world’s population depend on plant
based formulations for healthcare needs. The
traditional medicine that involves the utilization
of plants plays a significant protective role in
humans and animals particularly in developing
and under-developing countries. Worldwide, the
traditional medicinal practitioners use the plants
for treatment of several diseases and disorders. Traditional
medicine is commonly practiced in various countries such
ORIGINAL ARTICLE
Raghavendra, et al.: Antioxidant activities of different parts of Pleocaulus sessilis (Nees) Bremek
International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 99
as China, India, Japan, Pakistan, Sri Lanka, and Thailand.
Knowledge of medicinal plants and their use by indigenous
culture are useful for conservation of cultural traditions and
biodiversity and healthcare as well as drug development.
Plants represent an integral part of several systems of
medicine such as Ayurveda, Sidda, and Unani. Plants are
known to be the sources lead compounds for the development
of modern drugs. Several drugs such as aspirin, digoxin,
quinine, vincristine, vinblastine, reserpine, and morphine
have been derived from plants. Nowadays, immense interest
on medicinal plants and exploration of medicinal values of
plants is increased because of several complications such as
the development of resistance and side effects associated with
the use of modern drugs such as antibiotics and anticancer
agents.[1-9]
The genus Strobilanthes belongs to the family Acanthaceae.
The genus includes perennial flowering herbs and shrubs
with about 350 species out of which at least 46 are native
to India. Strobilanthes is the second largest genus in the
family Acanthaceae. The name Strobilanthes is derived
from the Latin words “strobilus” meaning cone and
“anthos” meaning flower or shoot.[10,11] Pleocaulus sessilis
(Nees) Bremek (synonym Strobilanthes sessilis Nees
var. sessilioides) is a small perennial shrub with hardy
tetragonous stem (30-45 cm) covered with brownish hairs.
Leaves are up to 4-5 cm long, as broad as long, ovate,
acute, coriaceous and bullate-hairy on both surfaces;
crenate-serrate, rounded, or narrowed at base. Flowers
blue, in terminal pedunculate, densely hairy bracteates
spikes 4-6 cm long. It is distributed in peninsular India and
is commonly found on rocky slopes among grasses in Baba
Budangiri.[12] In the previous study, Patil et al.[13] screened
solvent extracts of P. sessilis leaves for phytoconstituents,
antioxidant and antimicrobial activities. Phytochemical
analysis revealed constituents such as phenols, flavonoids,
saponins, and tannins. Antioxidant activities and
antimicrobial activities of extracts were found to be good.
The present study was carried to evaluate antimicrobial
and antioxidant activity of leaf, stem, and inflorescence of
P. sessilis [Figure 1].
MATERIALS AND METHODS
Collection and Identification of Plant
The plant materials were collected at Baba Budangiri,
Chikkamagalure district, Karnataka during February 2016.
The plant was authenticated by referring standard flora.[12]
Extraction
The plants were washed to remove dirt and other extraneous
matter. Different parts, namely, leaves, stem, and inflorescence
were separated, dried under shade and were powdered
separately. For extraction, we employed maceration process
in which 20 g of each powder was transferred into separate
conical flasks containing 100 ml of methanol. The flasks
were left for 48 h (during which the flasks were stirred
occasionally) followed by filtering the contents of flasks
through 4-fold muslin cloth followed by Whatman No. 1
filter paper. The filtrates were evaporated to dryness at 40°C
and the extracts obtained were stored in refrigerator until
use.[8,14]
Phytochemical Screening of Extracts
The leaf, stem, and inflorescence extracts were subjected to
preliminary phytochemical analysis. The presence of various
phytoconstituents namely alkaloids, flavonoids, tannins,
steroids, saponins, glycosides, terpenoids, and phenols were
detected by standard phytochemical tests.[5,6,15]
Antibacterial Activity of Extracts
The potential of different extracts of P. sessilis to inhibit
Gram-positive bacteria (Bacillus subtilis and Staphylococcus
aureus) and Gram-negative bacteria (Pseudomonas
aeruginosa and Klebsiella pneumoniae) was determined by
Agar well diffusion method as described in our previous
study.[8] In this method, the test bacteria were aseptically
inoculated into sterile nutrient broth tubes and incubated at
37°C for 24 h. The broth cultures were swab inoculated on
sterile nutrient agar plates. With the help of a sterile gel borer,
wells of 6 mm diameter were punched in the inoculated
plates. Extracts (20 mg/ml of dimethyl sulfoxide [DMSO]),
reference antibiotic (chloramphenicol, 1 mg/ml of sterile
distilled water), and DMSO were transferred aseptically in
labeled wells. The plates were left undisturbed for 30 min and
then incubated in upright position for 24 h at 37°C. Using a
ruler, zones of inhibition formed around wells was measured.
The presence of zone of inhibition around the wells is the
indication of antibacterial activity of extracts.
Figure 1: Pleocaulus sessilis
Raghavendra, et al.: Antioxidant activities of different parts of Pleocaulus sessilis (Nees) Bremek
International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 100
Antifungal Activity of Extracts
Poisoned food technique employed in our previous
studies[8,16] was used to assess the antifungal effect of extracts
of P. sessilis against test fungi, namely, Colletotrichum
capsici, Fusarium oxysporum f.sp. Zingiberi, and Alternaria
alternata. In brief, control (without extract) and poisoned
(0.5 mg extract/ml of medium) potato dextrose agar plates
were aseptically inoculated with the test fungi followed by
incubating the plates at 28°C for 96 h in upright position.
Later, the colony diameter of test fungi was measured using
a ruler in mutual perpendicular directions. Antifungal effect
(in terms of reduction in mycelial growth of test fungi) of
extracts was determined using the formula:
Inhibition of mycelial growth (%) = (A-B/A) ×100, where
“A” and “B” refers to colony diameter of test fungi in control
and poisoned plates, respectively. Reduction in mycelial
growth of test fungi in poisoned plates is indication of
antifungal potential of extracts.
1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Radical
Scavenging Activity of Extracts
In this assay, 1ml of different concentrations (6.25-200 µg/ml
of methanol) of extracts and ascorbic acid (reference standard)
was mixed with 3 ml of DPPH radical solution (0.004% in
methanol) in labeled test tubes. The tubes were then incubated
in dark for 30 min at room temperature followed by measuring
the absorbance of reaction mixture in spectrophotometer at
517 nm. Methanol replacing the extract/ascorbic acid served
as control (i.e., 1 ml methanol + 3 ml DPPH radical solution).
Inhibition of DPPH radicals (%) was calculated using the
formula:
Inhibition of DPPH radicals (%) = (A-B/A) ×100, where
“A” and “B” refers to the absorbance of DPPH control and
absorbance of DPPH in the presence of extract/ascorbic acid.
The inhibitory concentration (IC50) value was calculated. IC50
value denotes the concentration of extract/standard required
to scavenge 50% of free radicals.[16,17]
2,2-Azinobis 3-Ethylbenzothiazoline 6-Sulfonate
(ABTS) Scavenging Activity of Extracts
Unlike DPPH assay, the assay that involves scavenging
of ABTS radicals requires generation of the radicals. The
ABTS radical was generated by mixing ABTS stock solution
(7 mM) with potassium persulfate (2.45 mM). The reaction
mixture was left in the dark for 16 h at room temperature and
the resulting dark colored solution was diluted using distilled
water to an absorbance of 0.7 at 730 nm. 1 ml of different
concentrations (6.25-200 µg/ml of methanol) of extracts and
ascorbic acid (reference standard) was mixed with 3 ml of
ABTS radical solution in clean and labeled test tubes. The
tubes were incubated in dark for 30 min at room temperature
followed by measuring the absorbance of reaction mixture
in spectrophotometer at 730 nm. Methanol replacing the
extract/ascorbic acid served as control (i.e., 1 ml methanol +
3 ml ABTS radical solution). The ABTS radical scavenging
activity of extracts was calculated using the formula:
Scavenging activity (%) = (A-B/A) ×100, where “A” is
the absorbance of the ABTS solution without extract/
ascorbic acid and “B” is the absorbance of ABTS solution
in the presence of extract/ascorbic acid. The IC50 value was
calculated. IC50 denotes the concentration of extract required
to scavenge 50% of the radicals.[16]
Ferric Reducing Activity of Extracts
The reducing potential of extracts from different parts of
P. sessilis was determined by ferric reducing assay.[17,18] The
initial reaction mixture consisted of different concentrations
(6.25-200 µg/ml) of extracts and ascorbic acid (reference
standard) in 1 ml of methanol, 2.5 ml of phosphate buffer
(pH 6.6), and 2.5 ml of potassium ferricyanide (1%). The
tubes were incubated at 50°C for 20 min in a water bath.
After cooling, 2.5 ml of trichloroacetic acid (10%) followed
by 0.5 ml of ferric chloride (0.1%) was added to each of the
tubes and the tubes were left for 10 min at room temperature.
The absorbance of reaction mixture of each tube was
measured at 700 nm spectrophotometrically. An increase in
the absorbance with increase in concentration of extracts/
standard indicated increasing reducing power.
Total Phenolic Content of Extracts
Folin-Ciocalteu reagent (FCR) method is employed to
determine the content of phenolics in extracts and this method
is widely used method for estimating the content of total
phenolics in various samples including plant extracts. In this
method, a dilute concentration of each extract (0.5 ml) was
mixed with 0.5 ml of FC reagent (1:10) and 2 ml of sodium
carbonate (2%) in separate tubes. The tubes were incubated
at room temperature for 30 min. The absorbance of reaction
mixtures of each tube was determined spectrophotometrically
at 765 nm. Gallic acid was used as standard and a standard
curve was plotted using different concentrations of gallic
acid (0-1000 µg/ml). The total phenolic content in different
extracts was estimated as mg GAE from the graph.[8,19]
RESULTS AND DISCUSSION
Phytoconstituents Detected in Extracts of P. sessilis
The medicinal and pharmacological properties exhibited
by plants are due to the presence of secondary metabolites
such as alkaloids, flavonoids, tannins, saponins, and
terpenoids that are distributed in the various parts of the
plants. These chemicals are studied under the concept called
Raghavendra, et al.: Antioxidant activities of different parts of Pleocaulus sessilis (Nees) Bremek
International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 101
phytochemistry. Most of these phytochemicals have profound
physiological effects on the health. Hence, it is important to
detect these phytoconstituents in medicinal plants so as to
correlate the possible therapeutic role played by them.[5,6,15,19-22]
There are several protocols to extract phytochemicals from
plants, such as maceration, Soxhlet extraction, supercritical
fluid extraction, and microwave-assisted extraction.[17] In
the present study, we followed maceration process to get
an extract from various parts of P. sessilis using methanol
as the extraction solvent. It has been shown that methanol
can dissolve many phytochemicals including polyphenolic
compounds present in the plants.[17,20,23-25]
The yield and color of extracts of P. sessilis is shown in
Table 1. The yield was high in case of stem followed by
leaf and inflorescence. The color of inflorescence and stem
extracts was light green whereas leaf extract was dark
green. In the present study, we screened the presence of
various phytochemicals in leaf, stem, and inflorescence
extract of P. sessilis by standard tests. Table 2 shows the
phytochemicals which were detected in extracts of P. sessilis.
Flavonoids, saponins, steroids, and phenols were present in
all three extracts. Phytoconstituents, namely, alkaloids and
glycosides were not detected in all three extracts. Terpenoids
and tannins were detected only in inflorescence and leaf
extract, respectively. The study of Shende et al.[11] revealed
the presence of glycosides, steroids, and flavonoids in the
leaf of S. sessilis. The study of Patil et al.[13] showed the
presence of phenols, flavonoids, saponins, and tannins in leaf
extract of P. sessilis.
Antibacterial Activity of Extracts of P. sessilis
One of the major milestones in the field of medicine is the
discovery of antibiotics. The use of these wonder drugs
resulted in prevention and control of huge number of deaths.
However, indiscriminate use of these antibiotics resulted
in the emergence of resistant pathogens. Antibiotic therapy
not only affects the target pathogen but also commensal
inhabitants of the human host. Moreover, the ability of
these pathogens to transmit the resistance to susceptible
ones created major problem in the treatment of diseases.
These antibiotic-resistant pathogens are of serious concern
in the community as well as hospital settings. High cost,
side effects, and the resistance problems associated with
these antibiotics triggered immense interest in scientific
community to search alternatives for disease control. Plants,
their extracts, and the purified compounds from them are
shown to be effective in inhibiting pathogenic bacteria
including resistant strains.[20,22,26-30]
In this study, we screened the efficacy of extracts of P. sessilis
by agar well diffusion assay. The result of the inhibitory
activity of extracts against Gram positive and Gram negative
bacteria is shown in Table 3. The presence of inhibition zone
around the wells was considered positive for antibacterial
activity. Extracts were shown to inhibit all test bacteria.
Leaf and inflorescence extracts inhibited K. pneumoniae
to high extent while stem extract caused high inhibition
of B. subtilis and S. aureus. B. subtilis and K. pneumoniae
were inhibited to high extent among Gram-positive and
Gram-negative bacteria, respectively. Overall, stem extract
displayed least inhibitory activity. Inhibitory activity of
reference antibiotic was higher than that of extracts. No
Table 1: Yield and color of extracts of P. sessilis
Extract Yield (%) Color
Leaf 4.33 Dark green
Inflorescence 4.10 Light green
Stem 6.41 Light green
P. sessilis: Pleocaulus sessilis
Table 2: Phytoconstituents detected in extracts of
P. sessilis
Constituents Leaf Inflorescence Stem
Alkaloids ‑ ‑ ‑
Flavonoids + + +
Saponins + + +
Terpesnoids ‑ + ‑
Glycosides ‑ ‑ ‑
Tannins + ‑ ‑
Steroids + + +
Phenols + + +
P. sessilis: Pleocaulus sessilis
Table 3: Antibacterial activity of extracts of P. sessilis
Treatment Zone of inhibition in cm
B. subtilis S. aureus P. aeruginosa K. pneumoniae
Leaf extract 1.6 1.5 1.5 1.8
Inflorescence extract 1.7 1.5 1.6 1.9
Stem extract 1.4 1.4 1.2 1.4
Antibiotic 2.8 2.9 2.6 2.4
DMSO 0.0 0.0 0.0 0.0
B. subtilis: Bacillus subtilis, S. aureus: Staphylococcus aureus, P. aeruginosa: Pseudomonas aeruginosa, K. pneumonia: Klebsiella
pneumonia, P. sessilis: Pleocaulus sessilis
Raghavendra, et al.: Antioxidant activities of different parts of Pleocaulus sessilis (Nees) Bremek
International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 102
inhibition of test bacteria was observed in case of DMSO.
Extracts and purified compounds from Strobilanthes species
have shown to possess antibacterial activity. Taraxerol,
isolated from Strobilanthes callosus exhibited high reduction
of edema but the lower antimicrobial effect at doses
employed.[31] A compound 4-acetyl-2,7-dihydroxy-1,4,8-
triphenyloctane-3,5-dione isolated from dichloromethane
extract of Strobilanthes crispus was shown to possess
marked inhibitory activity against Gram-positive and Gram-
negative bacteria.[32] Methanol extract of leaf of Strobilanthes
cusia exhibited marked activity against S. aureus followed
by B. subtilis, Enterobacter aerogenes, Escherichia coli,
and K. pneumoniae.[33] Venkatachalapathi and Ravi[34]
evaluated the antibacterial activity of the petroleum ether
and methanolic extracts from the Strobilanthes ciliatus. Both
extracts exhibited inhibitory activity against Gram-positive
and Gram-negative bacteria. The leaf extract of S. crispus
was shown to exhibit inhibitory activity against S. aureus and
Streptococcus pneumoniae whereas no visible inhibition was
observed against K. pneumoniae and P. aeruginosa.[35]
Antifungal Activity of Extracts of P. sessilis
Several biological agents such as insects, bacteria, viruses,
and fungi attack plants at various stages of growth and
development resulting in a reduction of productivity and
economic loss to farmers. As compared to other agents,
the impact of fungi on crop production losses is highest.
Agrochemicals such as pesticides and fungicides have
been routinely used to prevent and control plant diseases
and crop loss. However, many fungicides are toxic and
have undesirable effects on non-target organisms present
in the environment. Some synthetic fungicides are non-
biodegradable and accumulate in the soil, plants, and water
and consequently affect humans through the food chain.
Besides, the development of resistance that has been noticed
in phytopathogenic fungi toward the synthetic fungicides
is another great challenge. Hence, it is desirable to use
alternative approaches that are eco-friendly for controlling of
plant diseases. Plants appear to be promising alternatives for
plant disease management. It is known that the use of natural
products can reduce the population of pathogens and control
the development of diseases. Plants have been considered as
potential agents in integrated pest management programs.
The use of plant-based formulations is cheap, eco-friendly,
and free of toxic effect on humans. A number of plants have
been reported to cause inhibition of several phytopathogenic
fungi.[23,36-41]
Table 4 and Figure 2 depict the antifungal effect of extracts
against test fungi. Poisoning of medium with the leaf,
inflorescence, and stem extracts revealed a considerable
reduction in the size of colonies of test fungi. Extracts
exhibited varied inhibitory activity against test fungi
(inhibitory activity ranged between 20% and 60%). Leaf
extract inhibited C. capsici (60.52%) to higher extent
followed by A. alternata (45.45%) and F. oxysporum (40%).
Both inflorescence and stem extracts inhibited A. alternata to
a maximum extent followed by C. capsici and F. oxysporum.
Earlier studies have shown the efficacy of extracts of
Strobilanthes species against fungi. Inhibitory effect of
petroleum ether and methanol extract of S. ciliatus was tested
against fungi, namely, Trichophyton rubrum, Microsporum
gypseum, Monascus purpureus by Venkatachalapathi and
Ravi.[34] The petroleum ether extract exhibited significant
antifungal activity against the test fungi. The leaf extract
of S. crispus did not show any visible inhibition against
Aspergillus brasiliensis and Candida albicans.[35]
DPPH Radical Scavenging Activity of Extracts
of P. sessilis
The method of DPPH radical scavenging was first developed
by Blois[42] to determine the antioxidant activity using a stable
DPPH-free radical. The assay is based on the measurement of
the scavenging capacity of substances termed as antioxidants.
In DPPH, the odd electron of nitrogen atom is reduced when
it receives a hydrogen atom from antioxidants resulting in
the formation of corresponding hydrazine (DPPH). DPPH
radical is a stable, organic and nitrogen-centered free radical
having a strong absorption at 517 nm (in alcoholic solution).
The absorption decreases as the electron pairs off. The
Table 4: Colony diameter of test fungi on control and
poisoned plates
Treatment Colony diameter in cm
C. capsici F. oxysporum A. alternata
Control 3.8 4.0 3.3
Leaf extract 1.5 2.4 1.8
Inflorescence
extract
2.4 3.2 1.8
Stem extract 3.0 3.2 2.1
C. capsici: Colletotrichum capsici, F. oxysporum: Fusarium
oxysporum, A. alternate: Alternaria alternata
Figure 2: Inhibition of test fungi (%) by extracts of Pleocaulus
sessilis
Raghavendra, et al.: Antioxidant activities of different parts of Pleocaulus sessilis (Nees) Bremek
International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 103
decolorization is stoichiometric with respect to the number
of electrons taken up. The method involving scavenging of
DPPH radicals is simple, rapid, inexpensive, and widely used
method to measure the capacity of compounds to behave
as free radical scavengers or hydrogen donors. This assay
has been widely employed for investigating antioxidant
properties of various kinds of samples including plant
extracts. One more advantage is that the radical is stable and
need not be generated as in case of ABTS radicals.[42-47]
In the present study, we evaluated the effect of extracts of
P. sessilis to scavenge-free radicals by DPPH assay. Bleaching
of color of DPPH radical solution in the presence of varying
concentrations of extracts and standard was monitored at
517 nm. Extracts and ascorbic acid scavenged DPPH radicals
in a dose-dependent manner [Figure 3]. Among extracts,
leaf extract exhibited stronger scavenging potential with
IC50 value 27.16 µg/ml followed by inflorescence extract
(IC50 value of 37.15 µg/ml) and stem extract (IC50 value of
58.56 µg/ml). Ascorbic acid exhibited stronger scavenging
effect (IC50 value of 6.17 µg/ml) when compared to extracts.
Although scavenging effect caused by extracts was low when
compared to ascorbic acid, it is evident from the study that
the extracts possess hydrogen donating efficacy; and hence,
the extracts can act as potent-free radical scavengers. It has
been shown that Strobilanthes species exhibit antioxidant
activity. The water soluble vitamins and catechins of
S. crispus contributed to high antioxidant activity of leaves
of S. crispus.[48] Solvent extracts of Strobilanthes kunthiana
have been shown to possess scavenging effect against DPPH
radicals.[49] In another study, Ghasemzadeh et al.[9] observed
DPPH radical scavenging potential of aqueous and ethanol
extract of S. crispus.
ABTS Radical Scavenging Activity of Extracts
of P. sessilis
Similar to DPPH assay, the ABTS assay is another widely
used in vitro radical scavenging assay. However, this method
needs the generation of ABTS radicals which can be easily
done by reacting ABTS salt with potassium persulfate. The
ABTS radical cation is reactive towards most antioxidant
compounds. ABTS radical is soluble in aqueous as well
as organic solvents. The method is a useful in determining
the antioxidant potential of both lipophilic and hydrophilic
antioxidants in a variety of samples including plant extracts.
A compound having electron donating property will reduce the
blue-green ABTS radical solution to colorless neutral form.
This reduction is indicated by suppression of its characteristic
long wavelength absorption spectrum.[14,43,45,46,50-53]
In the present study, we evaluated the effect of extracts of
P. sessilis to scavenge ABTS radicals and the result is shown
in Figure 4. The extracts scavenged ABTS radicals in a dose-
dependent manner. Among extracts, leaf extract displayed
marked scavenging activity (IC50 value 9.16 µg/ml) followed
by inflorescence extract (IC50 value 15.11 µg/ml) and stem
extract (IC50 value 39.30 µg/ml). At extract concentration
100 µg/ml and higher, a scavenging activity of >90% was
observed in case of all extracts. Reference compound ascorbic
acid scavenged ABTS radicals more efficiently (IC50 value of
5.69 µg/ml) than extracts of P. sessilis. Extracts scavenged
ABTS radicals more effectively than DPPH radicals.
Although the extracts have displayed low radical scavenging
effect when compared to ascorbic acid, it is evident that the
extracts possess the electron donating property due to which
the extracts could serve as potent scavengers of free radicals.
Ferric Reducing Activity of Extracts of P. sessilis
Several assays are designed and used to determine the overall
antioxidant activity as an indication of total capacity to
withstand adverse effect of stress induced by the formation
of free radicals. The reducing potential reflects the electron
donating capacity which is associated with antioxidant
activity. The presence of reductants (antioxidants) in samples
results in the reduction of ferric complex to ferrous form and
this reducing potential of sample can be determined by the
direct reduction of Fe[(CN)6]3 to Fe[(CN)6]2. The addition
of free Fe3+ to the reduced product results in the formation
of the intense Perl’s Prussian blue complex, Fe4[Fe(CN)6]3,
which possess a strong absorbance at 700 nm. An increase in
absorbance of the reaction mixture indicates an increase in
Figure 3: 1,1‑diphenyl‑2‑picrylhydrazyl radical scavenging
activity of extracts and reference standard
Figure 4: 2,2‑azinobis 3‑ethylbenzothiazoline 6‑sulfonate
radical scavenging activity of extracts and reference standard
Raghavendra, et al.: Antioxidant activities of different parts of Pleocaulus sessilis (Nees) Bremek
International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 104
the reducing capacity of extract.[14,17,54-58] In the present study,
we screened the extracts of P. sessilis for ferric reducing
activity. An increase in the absorbance of reaction mixtures
was observed on increasing the concentration [Figure 5].
This indicated reducing power of the extracts. Among
extracts, marked potential was observed in case of leaf
extract followed by inflorescence and stem extracts. Ascorbic
acid displayed higher reducing potential than that of extracts.
Although the reducing potential of extracts observed was
low when compared to reference standard, it is evident that
the extracts possess reductive ability which could serve as
electron donors, terminating the radical chain reactions.
Total Phenolic Content of Extracts of P. sessilis
Plants are known to produce a wide array of secondary
metabolites. Out of many phytochemicals, the phenolic
compounds are considered to be the most important plant
secondary metabolites as they are beneficial to the plants
and exhibit a range of bioactivities including antioxidant
activity. These compounds are shown to exhibit strong
antioxidant activity due to their ability to scavenge-free
radicals, break radical chain reactions, and to chelate metal
ions. Consumption of foods containing phenolic compounds
is proven to be associated with reduced risk of cardiovascular
diseases and certain types of cancer.[56,59-63] FCR method is
widely used to estimate the content of total phenolics in
plants. The method is simple, oldest, and the results are
reproducible. The phenolic compounds react with FCR under
basic conditions and results in the formation of a blue colored
complex which exhibit absorption maxima near 750 nm.[14,64]
In this study, we estimated the content of total phenolics in
extracts of P. sessilis by FCR method. The phenolic content
was found to be high in leaf extract followed by inflorescence
extract and stem extract [Table 5]. A positive correlation
was observed between the phenolic content of extracts and
antioxidant activity observed, i.e., extracts containing high
phenolic content exhibited stronger antioxidant activity. Such
observations were made in earlier studies of Tilak et al.,[59]
Vivek et al.,[64] Coruh et al.[65] and Poornima et al.[66] where
extracts containing high phenolic content exhibited marked
antioxidant activity.
CONCLUSIONS
Plants have been used traditionally for the treatment of
various ailments throughout the world. Extracts and purified
metabolites from plants exhibit a range of bioactivities which
can be exploited for drug development. In the present study,
we observed antimicrobial and antioxidant activity in leaf,
inflorescence, and stem extracts of P. sessilis. Overall, leaf
extract exhibited stronger bioactivities when compared
to other two extracts. The observed bioactivities could be
ascribed to the presence of secondary metabolites detected in
the extracts. The plant can be used against microbial infections
and oxidative damage. Further studies on isolation of active
principles from extracts and their bioactivity determinations
are to be carried out.
ACKNOWLEDGMENTS
Authors would like to thank Principal, S.R.N.M.N College
of Applied Sciences and N.E.S, Shivamogga for providing
facilities and moral support to conduct research.
REFERENCES
1. Vedavathy S. Scope and importance of traditional
medicine. Indian J Tradit Knowl 2003;2:236-9.
2. Williams LA. Ethnomedicine. West Indian Med J
2006;55:215-6.
3. Idu M, Erhabor JO, Efijuemue HM. Documentation on
medicinal plants sold in markets in Abeokuta, Nigeria.
Trop J Pharm Res 2010;9:110-8.
4. Rath S, Dubey D, Sahu MC, Debata NK, Padhy RN.
Surveillance of multidrug resistance of 6 uropathogens
in a teaching hospital and in vitro control by 25
ethnomedicinal plants used by an aborigine of India.
Asian Pac J Trop Biomed 2012;2:S818-29.
5. Bhandary SK, Kumari S, Bhat VS, Sharmila KP,
Bekal MP. Preliminary phytochemical screening of
various extracts of Punica granatum peel, whole fruit
and seeds. Nitte Univ J Health Sci 2012;2:34-8.
Figure 5: Ferric reducing activity of extracts and reference
standard
Table 5: Content of total phenolics (mg GAE/g) in
extracts
Extract Total phenolic content
Leaf 112.13
Inflorescence 85.65
Stem 42.42
GAE: Gallic acid equivalents
Raghavendra, et al.: Antioxidant activities of different parts of Pleocaulus sessilis (Nees) Bremek
International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 105
6. Mir AM, Sawhney SS, Jassal MM. Qualitative and
quantitative analysis of phytochemicals of Taraxacum
officinale. Wudpecker J Pharm Pharmocol 2013;2:15.
7. Swamy SH, Asha MM, Chaithra M, Vivek MN,
Kambar Y, Kekuda PT. Antibacterial activity of flower
extract of Caesalpinia pulcherrima, Delonix regia
and Peltaphorum ferrugineum against urinary tract
pathogens. Int Res J Biol Sci 2014;3:80-3.
8. Kekuda PT, Akarsh S, Darshini SM, Prafulla D,
Raghavendra HL. Antiradical and antimicrobial activity
of Atylosia lineata Wt. And Arn. Sci Technol Arts Res J
2015;4:180-3.
9. Ghasemzadeh A, Jaafar HZ, Rahmat A. Phytochemical
constituents and biological activities of different extracts
of Strobilanthes crispus (L.) Bremek leaves grown in
different locations of Malaysia. BMC Complement
Altern Med 2015;15:422.
10. Preethi F, Suseem SR. A comprehensive study on an
endemic Indian genus - Strobilanthes. Int J Pharmacogn
Phytochem Res 2014;6:459-66.
11. Shende VS, Jadhav SD, Aloorkar NH, Kulkarni AS,
Suryavanshi SV. Pharmacognostic and phytochemical
evaluation of Strobilanthes sessilis nees. Leaves. Int J
Pharmacogn 2015;2:310-4.
12. Yoganarasimhan SN, Subramanyam K, Razi BA. Flora
of Chikmagalur District. Dehra Dun: International Book
Distributors; 1981. p. 250.
13. Patil SV, Mane RP, Anbhule PV, Shimpale VB,
Deshmukh MB. Phytochemical estimation, antioxidant
and antimicrobial properties of Pleocaulus sessilis: An
endemic plant from Western Ghats of India. J Appl
Chem 2015;3:1-10.
14. Hazra B, Biswas S, Mandal N. Antioxidant and free
radical scavenging activity of Spondias pinnata. BMC
Complement Altern Med 2008;8:63.
15. Yusuf AZ, Zakir A, Shemau Z, Abdullahi M, Halima SA.
Phytochemical analysis of the methanol leaves extract
of Paullinia pinnata Linn. J Pharmacogn Phytother
2014;6:10-6.
16. Kekuda PT, Raghavendra HL, Solomon T, Duressa D.
Antifungal and antiradical potential of Moringa
stenopetala (Baker f.) Cufod (Moringaceae). J Biosci
Agric Res 2016;11:923-9.
17. Do QD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH,
Soetaredjo FE, Ju Y. Effect of extraction solvent on total
phenol content, total flavonoid content, and antioxidant
activity of Limnophila aromatica. J Food Drug Anal
2014;22:296-302.
18. Kekuda PT, Manasa M, Poornima G, Abhipsa V,
Rekha C, Upashe SP, et al. Antibacterial, cytotoxic and
antioxidant potential of Vitex negundo var. negundo and
Vitex negundo var. purpurascens- A comparative study.
Sci Technol Arts Res J 2013;2:59-68.
19. Zlotek U, Mikulska S, Nagajek M, Swieca M. The effect
of different solvents and number of extraction steps on
the polyphenol content and antioxidant capacity of basil
leaves (Ocimum basilicum L.) extracts. Saudi J Biol Sci
2016;23:628-33.
20. Cowan MM. Plant products as antimicrobial agents. Clin
Microbiol Rev 1999;12:564-82.
21. Omar S, Lemonnier B, Jones N, Ficker C, Smith ML,
Neema C, et al. Antimicrobial activity of extracts of
eastern North American hardwood trees and relation to
traditional medicine. J Ethnopharmacol 2000;73:161-70.
22. Gobalakrishnan R, Kulandaivelu M, Bhuvaneswari R,
Kandavelb D, Kannan L. Screening of wild plant species
for antibacterial activity and phytochemical analysis of
Tragia involucrata L. J Pharm Anal 2013;3:460-5.
23. Gurjar MS, Ali S, Akhtar M, Singh KS. Efficacy of
plant extracts in plant disease management. Agric Sci
2012;3:425-33.
24. Naz F, Qamarunnisa S, Shinwari ZK, Azhar A, Ali SI.
Phytochemical investigations of Tamarix indica Willd.
and Tamarix passernioides Del. ex Desv. leaves from
Pakistan. Pak J Bot 2013;45:1503-7.
25. Iloki-Assanga SB, Lewis-Luján LM, Lara-Espinoza CL,
Gil-Salido AA, Fernandez-Angulo D, Rubio-Pino JL,
et al. Solvent effects on phytochemical constituent
profiles and antioxidant activities, using four different
extraction formulations for analysis of Bucida buceras L.
and Phoradendron californicum. BMC Res Notes
2015;8:396.
26. Lenski RE. Bacterial evolution and the cost of antibiotic
resistance. Int Microbiol 1998;1:265-70.
27. Jernberg C, Löfmark S, Edlund C, Jansson JK. Long-
term impacts of antibiotic exposure on the human
intestinal microbiota. Microbiology 2010;156:3216-23.
28. Shakeri A, Hazeri N, Vlizadeh J, Ghasemi A, Tavallaei FZ.
Phytochemical screening, antimicrobial and antioxidant
activities of Anabasis aphylla L. Extracts. Kragujevac J
Sci 2012;34:71-8.
29. Dahiya P, Purkayastha S. Phytochemical screening and
antimicrobial activity of some medicinal plants against
multi-drug resistant bacteria from clinical isolates.
Indian J Pharm Sci 2012;74:443-50.
30. Naz R, Bano A. Phytochemical screening, antioxidants
and antimicrobial potential of Lantana camara in
different solvents. Asian Pac J Trop Dis 2013;3:480-6.
31. Singh B, Sahu PM, Sharma MK. Anti-inflammatory and
antimicrobial activities of triterpenoids from Strobilanthes
callosus nees. Phytomedicine 2002;9:355-9.
32. Koay YC, Wong KC, Osman H, Eldeen IM, Asmawi MZ.
Chemical constituents and biological activities of
Strobilanthes crispus L. Rec Nat Prod 2013;7:59-64.
33. Shahni R, Handique PJ. Antibacterial properties of leaf
extracts of Strobilanthes cusia (Nees) Kuntze, a rare
ethno-medicinal plant of Manipur, India. Int J PharmTech
Res 2013;5:1281-5.
34. Venkatachalapathi S, Ravi S. Antimicrobial activity
of Strobilanthes ciliatus nees. Indo Am J Pharm Res
2013;3:3124-8.
35. Lim V, Yap CS, Chong HW, Shukkoor MS, Priya M.
Antimicrobial evaluation and GC-MS analysis of
Strobilanthes crispus ethanolic leaf extract. Eur J Med
Raghavendra, et al.: Antioxidant activities of different parts of Pleocaulus sessilis (Nees) Bremek
International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 106
Plants 2015;10:1-8.
36. Tapwal A, Nisha, Garg S, Gautam N,
Kumar R. In vitro antifungal potency of plant extracts
against five phytopathogens. Braz Arch Biol Technol
2011;54:1093-8.
37. Nashwa SM, Abo-Elyousr KA. Evaluation of various
plant extracts against the early blight disease of tomato
plants under greenhouse and field conditions. Plant Prot
Sci 2012;48:74-9.
38. Al-Aksar AA. In vitro antifungal activity of three Saudi
plant extracts against some phytopathogenic fungi.
J Plant Prot Res 2012;52:458-62.
39. Kambar Y, Manasa M, Vivek MN, Kekuda PT. Inhibitory
effect of some plants of Western Ghats of Karnataka
against Colletotrichum capsici. Sci Technol Arts Res J
2014;3:76-82.
40. Neela FA, Sonia IA, Shamsi S. Antifungal activity of
selected medicinal plant extract on Fusarium oxysporum
Schlechtthe causal agent of fusarium wilt disease in
tomato. Am J Plant Sci 2014;5:2665-71.
41. Sales MD, Costa HB, Fernandes PM, Ventura JA,
Meira DD. Antifungal activity of plant extracts with
potential to control plant pathogens in pineapple. Asian
Pac J Trop Biomed 2016;6:26-31.
42. Blois MS. Antioxidant determinations by the use of a
stable free radical. Nature 1958;181:1199-200.
43. Antolovich M, Prenzler PD, Patsalides E, McDonald S,
Robards K. Methods for testing antioxidant activity.
Analyst 2002;127:183-98.
44. Molyneux P. The use of the stable free radical
diphenylpicrylhydrazyl (DPPH) for estimating
antioxidant activity. Songklanakarin J Sci Technol
2004;26:211-9.
45. Prior RL, Wu X, Schaich K. Standardized methods for
the determination of antioxidant capacity and phenolics
in foods and dietary supplements. J Agric Food Chem
2005;53:4290-302.
46. Thaipong K, Boonprakob U, Crosby K,
Cisneros-Zevallos L, Byrne DH. Comparison of
ABTS, DPPH, FRAP, and ORAC assays for estimating
antioxidant activity from guava fruit extracts. J Food
Comp Anal 2006;19:669-75.
47. Kedare SB, Singh RP. Genesis and development of
DPPH method of antioxidant assay. J Food Sci Technol
2011;48:412-22.
48. Ismail M, Manickam E, Danial AM, Rahmat A,
Yahaya A. Chemical composition and antioxidant
activity of Strobilanthes crispus leaf extract. J Nutr
Biochem 2000;11:536-42.
49. Brijyog, Singh B, Das S, Maithi A. Antioxidant property
for lipophilic extract of Strobilanthes kunthiana flowers.
Indian J Res Pharm Biotechnol 2014;2:1005-9.
50. Wan C, Yu Y, Zhou S, Liu W, Tian S, Cao S. Antioxidant
activity and free radical-scavenging capacity of Gynura
divaricata leaf extracts at different temperatures.
Pharmacogn Mag 2011;7:40-5.
51. Martysiak-Zurowska D, Wenta W. A comparison of
ABTS and DPPH methods for assessing the total
antioxidant capacity of human milk. Acta Sci Pol
Technol Aliment 2012;11:83-9.
52. Rakesh KN, Junaid S, Dileep N, Vinayaka KS,
Kekuda PT, Raghavendra HL. Antibacterial and
antioxidant activity of Fahrenheitia zeylanica (Thw.)
Airy. Sci Technol Arts Res J 2013;2:27-33.
53. Shalaby EA, Shanab SM. Comparison of DPPH and
ABTS assays for determining antioxidant potential
of water and methanol extracts of Spirulina platensis.
Indian J Geo Mar Sci 2013;42:556-64.
54. Gulcin I, Topal F, Sarikaya SB, Bursal E, Bilsel G,
Goren AC. Polyphenol contents and antioxidant
properties of Medlar (Mespilus germanica L.). Rec Nat
Prod 2011;5:158-75.
55. Meir S, Kanner J, Akiri B, Hadas SP. Determination
and involvement of aqueous reducing compounds in
oxidative defense systems of various senescing leaves.
J Agric Food Chem 1995;43:1813-7.
56. Chung Y, Chien C, Teng K, Chou S. Antioxidative and
mutagenic properties of Zanthoxylum ailanthoides Sieb
and Zucc. Food Chem 2006;97:418-25.
57. Kekuda PT, Vinayaka KS, Swathi D, Suchitha Y,
Venugopal TM, Mallikarjun N. Mineral composition,
total phenol content and antioxidant activity of a
macrolichen Everniastrum cirrhatum (Fr.) Hale
(Parmeliaceae). E J Chem 2011;8:1886-94.
58. Kiran R, Kekuda PT, Kumar PH, Hosetti BB,
Krishnaswamy K. Biological activities of Sarcanthus
pauciflorus. J Appl Pharm Sci 2013;3:105-10.
59. Tilak JC, Adhikari S, Devasagayam TP. Antioxidant
properties of Plumbago zeylanica, an Indian medicinal
plant and its active ingredient, plumbagin. Redox Rep
2004;9:219-27.
60. Kusirisin W, Jaikang C, Chaiyasut C, Narongchai P.
Effect of polyphenolic compounds from Solanum
torvum on plasma lipid peroxidation, superoxide anion
and cytochrome P450 2E1 in human liver microsomes.
Med Chem 2009;5:583-8.
61. Sudharshan SJ, Valleesha NC, Chinmaya A, Kekuda PT,
Murthuza S, Rajeshwara AN. Radical scavenging
activity, phenol and flavonoid content of selected
traditionally used Indian medicinal plants. Asian J Exp
Sci 2010;24:11-5.
62. Pavithra GM, Siddiqua S, Naik AS, Kekuda PT,
Vinayaka KS. Antioxidant and antimicrobial activity
of flowers of Wendlandia thyrsoidea, Olea dioica,
Lagerstroemia speciosa and Bombax malabaricum.
J Appl Pharm Sci 2013;3:114-20.
63. Sayari N, Saidi MN, Sila A, Ellouz-Chaabouni S,
Bougatef A. Chemical composition, angiotensin
I-converting enzyme (ACE) inhibitory, antioxydant and
antimicrobial activities of Ononis natrix leaves extracts.
Free Radic Antioxid 2016;6:23-33.
64. Vivek MN, Kambar Y, Manasa M, Kekuda PT,
Vinayaka KS. Radical scavenging and antibacterial
activity of three Parmotrema species from Western Ghats
Raghavendra, et al.: Antioxidant activities of different parts of Pleocaulus sessilis (Nees) Bremek
International Journal of Green Pharmacy • Apr-Jun 2017 • 11 (2) | 107
of Karnataka, India. J Appl Pharm Sci 2014;4:86-91.
65. Coruh N, Celep AG, Ozgokce F, Iscan M. Antioxidant
capacities of Gundelia tournefortii L. Extracts and
inhibition on glutathione-S-transferase activity. Food
Chem 2007;100:1249-53.
66. Poornima G, Kekuda PT, Vinayaka KS. Antioxidant
efficacy of Olea dioica Roxb (Oleaceae) leaves.
Biomedicine 2012;32:506-10.
Source of Support: Nil. Conflict of Interest: None declared.
