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Therapeutic potential of Polyalthia cerasoides

stem bark extracts against oxidative stress and
nociception

Article October 2015

DOI: 10.4103/0257-7941.171667

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Original Article

Therapeutic potential of Polyalthia cerasoides stem

bark extracts against oxidative stress and nociception
B. C. Goudarshivananavar, V. Vigneshwaran1, Madhusudana Somegowda1, Kattepura K. Dharmappa2,
Siddanakoppalu N. Pramod1
Department of Studies in Chemistry, Sahyadri Science College (Autonomous), Kuvempu University, 1Department of Studies and
Research in Biochemistry, Laboratory of Immunomodulation and Inflammation Biology, Sahyadri Science College (Autonomous),
Kuvempu University, Shimoga, 2Department of Studies in Biochemistry, PG Centre of Mangalore University, Madikeri, Karnataka, India

ABSTRACT Conclusion: The ethyl acetate fraction enriched with

Background: Polyalthia cerasoides is a medicinal plant flavinoids and steroids from Polyalthia cerasoides stem
known for its ethnopharmacological importance. Despite bark has potent bioactivity to combat inflammation, ROS
this, investigation related to its therapeutic benefit is still and pain. This needs further characterization for potential
unexplored. therapeutic applications.
Aim: To evaluate the stem bark extracts of Polyalthia
KEYWORDS: Analgesia, antiinflamatory, carageenan,
cerasoides for pharmacological activities relating to
catalase, lipid peroxidation, nociception, reactive oxygen
inflammation, nociception and oxidative stress using invivo
species
and invitro models.
Materials and Methods: Pet ether, ethyl acetate and
chloroform fractions of the stem bark were evaluated for
antiinflammatory activity by carrageenaninduced hind paw
edema in rats. Antinociceptive activity in mice was assessed
using thermally and chemically induced analgesic models.
The free radical quenching potential of the extracts was INTRODUCTION
initially analyzed using the invitro DPPH photometric assay,
Hydroxyl radical scavenging and Lipid Peroxidation assays.
Then modulatory effect of the extracts on invivo antioxidant
I nflammation is a tissue response to stimuli such
as: Pathogens, damaged cell and irritants. It is the
protective attempt by the organism to remove the
system was evaluated by carbon tetrachloride induced
hepatotoxicity and subsequent measurements of antioxidant injurious stimuli as well to initiate healing process of the
enzymes such as Superoxide dismutase, Catalase and tissue.[1] Acute inflammation is characterized by edema,
Peroxidase from the liver homogenate. erythema, pain, heat and above all, loss of function.
Results: Among the tested fractions, ethyl acetate extract had These important signs of inflammation are triggered
substantially inhibited the inflammation by 68.5% that was by the infiltration mediators which include histamine,
induced by subcutaneous carrageenan injection whereas pet tryptase, prostaglandins, leukotrienes and white blood
ether and chloroform extract showed only minimal inhibitory corpuscles(leucocytes).[2,3]
effect. Investigation of the antinociceptive activity revealed
that the ethyl acetate fractions had significantly repressed the
Reactive oxygen species(ROS) are natural by products
algesia in both the analgesic experimental models. In vitro
of body metabolism and are charged molecules that
and invivo individual antioxidant assays demonstrated that
the ethyl acetate fraction has strong free radical quenching attack cells, tear through cellular membranes, react and
potential which also restores the endogenous hepatic
enzymes. This is an open access article distributed under the terms of the Creative Commons
AttributionNonCommercialShareAlike 3.0 License, which allows others to remix,
tweak, and build upon the work noncommercially, as long as the author is credited
and the new creations are licensed under the identical terms.
Access this article online
Quick Response Code:
Website: For reprints contact: reprints@medknow.com

www.ancientscienceoflife.org
DOI:
10.4103/0257-7941.171667
How to cite this article: Goudarshivananavar BC, Vigneshwaran V,
Somegowda M, Dharmappa KK, Pramod SN. Therapeutic potential
of Polyalthia cerasoides stem bark extracts against oxidative stress
and nociception. Ancient Sci Life 2015;35:70-8.

70 2015 Ancient Science of Life | Published by Wolters Kluwer - Medknow

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Goudarshivananavar, et al.: Therapeutic potential of stem bark of Polyalthia cerasoides
create havoc with nucleic acids, proteins and enzymes
present in the body. Various reports have demonstrated
the role of ROS in inflammation and it is also evident
from the antiinflammatory effects of the antioxidants. As
inflammatory conditions induce pain and oxidative stress,
drug products with combinational treatment plans are most
preferred for inflammation therapy.[3,4] Drugs currently
used for management of pain and inflammatory conditions
are either narcotics such as opioids or nonnarcotics. Both
of them have toxic side effects on chronic administration.[5]
On the other hand many medicines of plant origin have
been used since a long time without any adverse effects.
Plants represent a large untapped source of structurally
novel compounds that may serve as leads for the
development of novel drugs.[6]
Plants belonging to the family Annonaceae have
long been used as a major source of medicines
for the prevention and treatment of a variety of
diseases in India and many Asian countries. To date,
ethnopharmacological claims for Annonaceae include
the use of its bark to control blood pressure, diabetes and
its use as a febrifuge.[79] Polyalthia cerasoides(Roxb.)
Bedd.(Annonaceae) is a medium sized tree distributed
in almost all forests of Deccan India up to 3000 ft. In
southern India, the plant is almost exclusively used
for its edible fruits and seeds.[10,11] The stem bark of
this plant is used as tonic to combat stress and pain by
local medicinal practitioners and experimental studies
have demonstrated its normalizing activity on brain
neurotransmitters, moderate cytotoxicity and antioxidant
activity invitro. [11,12] Various studies have reported
the antiproliferative, apoptotic and antimutagenic
activity of the seed extract.[13] Pharmacognostic reports
relating to the extracts of stem bark of P.cerasoides
is limited. The present study has been attempted to
understand the pharmacological activities, particularly
antiinflammatory, analgesic and invitro and invivo
antioxidant potential of the stem bark extracts of
P.cerasoides to identify new potential phytoconstituents
which may further provide information to develop novel
drugs to manage pain, stress and inflammations.
MATERIALS AND METHODS
Chemicals and reagents
Carrageenan suspension, formalin, acetic acid, ethyl
acetate, chloroform, petroleum ether, ascorbic acid were
purchased from Himedia Laboratories Pvt. Ltd., Mumbai,
India. The reagent 1,1diphenyl1picrylhydrazyl(DPPH)
was procured from SigmaAldrich(St. Louis, MO, USA).
Ancient Science of Life / Oct-Dec 2015 / Vol 35 / Issue 2
Pentazocine and Diclofenac sodium injections were
purchased from Novartis and Ranbaxy, India, respectively.
All other solvents and chemicals used in this experiment
were of analytical grade.
Animals
Swiss albino mice, 68weeks old (2025g) and Wistar
rats (150200 g) of either sex were used in the study.
The animals had free access to food and water, and
were housed at room temperature, 242C in a natural
(12h each) lightdark cycle. The animals were acclimatized
for at least 5days to the laboratory conditions before
conducting experiments. The experimental protocol was
approved by the Institutional Animal Ethics Committee
(IAEC) and the care of the laboratory animals was taken
as per the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA)
regulations.
Plant collection and solvent extraction
The bark of P.cerasoides was collected from the forests
of Kuvempu University campus area. The plant was
authenticated with the taxonomist from Department of
Botany, Kuvempu University, India. Fresh plant material
was collected and sprayed with alcohol in order to arrest
any enzymatic degradation. The collected materials were
washed with running Water and subsequently tapped dry
and chopped into pieces. The material was then shade dried
and coarsely powdered.
The weighed amount of material was successively extracted
using soxhlet apparatus with solvents of varying polarity
starting from petether, chloroform and ethyl acetate.
Each extraction was carried out for 18h(approximately
45cycles) at room temperature. The extracts were
concentrated after evaporating the solvent using flash
evaporator, under reduced pressure and controlled
temperature. The extracts so obtained were air dried,
weighed, packed and stored at 4 deg. C.[14]
Phytochemical analysis of the extracts and acute toxicity
studies
Qualitative analysis for the phytochemical constituents in
the extracts of stem bark of P.cerasoides was performed
based on the specific qualitative tests as reported
previously.[15] All the three extracts, i.e.pet ether(PEPCF),
chloroform(CLPCF) and ethyl acetate(EAPCF) fractions
were evaluated for the presence of medicinally active
phytochemicals. The tests include detection of alkaloids,
saponins, tannins, glycosides, steroids or terpinoids,
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Goudarshivananavar, et al.: Therapeutic potential of stem bark of Polyalthia cerasoides
carbohydrates and flavonoids in the fractions and are
carried out following the standard protocols as described.
The presence of the phytochemicals were assessed and
graded as, +, ++ based on the absence and abundance
in the fractions.
Acute toxicity study was carried out using Swiss albino
mice(2530g) by up and down staircase method as
per CPCSEA guidelines as described previously. [3]
Suspension of pet ether, chloroform and ethyl acetate
fractions of P.cerasoides was orally administered to
different groups of mice at doses of 50, 300, 1000 and
2000mg/kg body weight following standard intraoral
protocol. Animals were observed for 48h to study their
general behavior, signs of discomfort and nervous
manifestations. The death and behaviour of the animals
in each group were recorded and were used for the
assessment of approximate Lethal Dose(LD50) and acute
toxicity level and also to fix the dose for the further
pharmacological studies.
Analgesic activity
Analgesic activity for the fractions was assayed in mice by
inducing pain chemically and thermally using acetic acid
induced writhing model and hot plate assay respectively.
Acetic acid induced writhing assay
Acetic acid writhing assay was performed in accordance
with the methods described earlier. [3] Briefly, five
groups of mice(n=6) were randomly formed. The
groups were treated as(i) control(distilled water, p.o.)
(ii) standard(Diclofenac, 5mg/kg i.p.) while the three test
groups received suspensions of pet ether, chloroform and
ethyl acetate extract fractions of stem bark of P.cerasoides
(100 and 200mg/kg p.o.) respectively. Acetic acid solution
0.6%v/v(10ml/kg) was injected by intraperitoneal route
one hour after treatment and number of writhes(i.e.,index
of pain reaction against chemical stimuli characterized
by abdominal muscle contraction together with turning
of trunk and extension of hind limbs) was counted over a
period of 20min. Analgesic activity was also expressed as
a percentage of inhibition of writhes with respect to the
control group.
Eddys hot plate assay
Eddys Hot plate assay was performed by the method as
described previously.[16] Briefly, a hot plate was maintained
at 551C. Albino mice were divided in six groups(n=6).
The animals were placed on the hot plate and the basal
reaction time taken to cause a discomfort(licking of paw or
jumping response whichever appeared first) was recorded
72 Ancient Science of Life / Oct-Dec 2015 / Vol 35 / Issue 2
at 0min. Acutoff period 15sec. was established to prevent
damage to the paws. The treatment and groupings of mice
was done in the same manner as mentioned in the acetic
acid induced writhing model except that in this case, the
standard group received pentazocine (5 mg/kg i.p.). The
reaction time in seconds was reinvestigated at 30, 60, and
120min after the treatment. Changes in mean reaction
time were noted.
Antiinflammatory activity
Acute antiinflammatory activity of suspensions of pet
ether, chloroform and ethyl acetate extracts of stem bark
of P.cerasoides(100 and 200mg/kg p.o.) respectively,
was evaluated using carragenan induced paw edema in
rats as described previously with slight modifications.[17]
Briefly, six groups of albino rats(n=4) were randomly
distributed as control, standard and test groups. The initial
paw volumes of each animal were measured by means of a
mercury plethysmometer. The standard group was treated
with Diclofenac injection(5mg/kg, i.p.) while suspensions of
pet ether, chloroform and ethyl acetate extracts of stem bark
of P.cerasoides(100 and 200mg/kg p.o.) was administered
to test groups and distilled water(10ml/kg, i.p.) was given
to the control group, 0.1ml of 1% carrageenan solution
was injected in the plantar region of the left hind paw of
rats thirty minutes after treatment. Paw volumes were again
measured 3h after carrageenan injection. The difference
in edema volume was calculated in each control, test and
standard group and compared with the control group for
determination of the percentage of inhibition of the paw
edema.
Invitro antioxidant activity of EAPCF from stem bark of
P. cerasoides
Reaction with DPPH radical
The DPPH radical scavenging potential of P.cerasoides
extracts was evaluated by previously described
methods with slight modifications.[18] Briefly, 2.5 ml of
200 mM DPPH in methanol was mixed with 0.5ml of
different concentrations of ethyl acetate P.cerasoides
fraction(1100mg/ml) in methanol and kept in dark for
30min. The absorbance at 517nm was measured. Ascorbic
acid(ASC) was used as a standard for comparison. Plotting
the percentage DPPH scavenging against ASC and EAPCF
concentration gave the standard cuve from which the IC50
value is determined.
Reaction with hydroxyl radical
Hydroxyl radical scavenging activity was measured
by the ability of the ethyl acetate extract of
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Goudarshivananavar, et al.: Therapeutic potential of stem bark of Polyalthia cerasoides
P.cerasoides (6500mg/ml) to scavenge the hydroxyl
radicals generated by the Fe3+ascorbateEDTAH2O2
system(Fenton reaction). [19] The reaction mixture
containing deoxyDribose(3 mM), ferric chloride(0.1 mM),
EDTA(0.1 mM), hydrogen peroxide(2 mM) in phosphate
buffer(20 mM, pH=7.4), with different concentrations
of the EAPCF in a volume of 0.3ml were added, to give
a final volume of 3.0ml. After incubation for 30min at
ambient temperature, 1.0ml of TCATBA reagent(equal
volumes of TCA2.8% and TBA0.5% in 4mM NaOH) was
added, followed by heating the tubes in a water bath for
30min. The tubes were then cooled and the absorbance
was measured at 532nm. Mannitol was used as standard
for comparison. Different concentrations(0.54.5mg/ml)
of mannitol were mixed as explained above. Plotting the
percentage inhibition of hydroxyl radical scavenging
against that of mannitol and EAPCF concentration gave
the standard curve from which IC50 value was calculated.[19]
Lipid peroxidation(LPO) assay
Egg phospatidylcholine(20mg) in chloroform(2ml) was
dried and further dispersed in normal saline(5ml). The
mixture was sonicated to get a homogeneous suspension
of liposomes. Lipid peroxidation was initiated by adding
0.05 mM trolox to a mixture containing liposome(0.1ml),
150 mM potassium chloride, 0.2 mM ferric chloride,
EAPCF(0.10300mg/ml) in a total volume of 0.4ml. The
reaction mixture was incubated for 40min at 37C. After
incubation, the reaction was terminated by adding 1ml
of ice cold 0.25 M hydrochloric acid containing 20%w/v
of trichloroacetic acid, 0.4%w/v of thiobarbituric acid
and 0.05%w/v of butylated hydroxytoluene. After heating
at 80C for 20min, the samples were cooled. The pink
chromogen was extracted with a constant amount of
nbutanol, and the absorbance of the upper organic layer
was measured at 532nm. Trolox was used as standard
for comparison. Plotting the percentage inhibition of LPO
scavenging against trolox concentration gave the standard
curve and IC50 value is caluculated for the samples.[20]
Invivo antioxidant activity for EAPCF in mice
The invivo antioxidant activity of ethyl acetate fraction
of P.cerasoides was carried out using Swiss albino
mice(68weeks old) as described previously.[3] Briefly,
animals were divided into groups(n=6). GroupI: Served as
control(administered PBS, 5ml/kg, p.o.). GroupII: Served as
negative control(CCl4/olive oil(1:1), 1ml/kg, i.p on 3rdand
4thday). GroupIII: Treated with Silymarin 100mg/kg, p.o
for successive five days. Test groupsIV and V: Suspensions
of ethyl acetate fraction at the dose of 250 and 500mg/kg,
p.o. respectively for five days. All the animals except
Ancient Science of Life / Oct-Dec 2015 / Vol 35 / Issue 2
control group received CCl4 (1ml/kg, i.p.) on 3rdand
4thday. On the fifth day 2hr after the administration of
the last dose, livers were isolated to measure the levels of
antioxidant enzymes.
Five percent liver homogenate was prepared with 0.15 M
KCl and centrifuged at 1000rpm for 10min. The cell free
supernatant was used for the estimation of Super oxide
dismutase(SOD), Catalase and Peroxidase by the methods
described previously.[2123]
SOD assay
0.5ml of liver homogenate was taken, and 1ml of 50 mM
sodium carbonate, 0.4ml of 24 M NBT, and 0.2ml of
0.1 mM EDTA were added. The reaction was initiated by
adding 0.4ml of 1 mM hydroxylamine hydrochloride.
Zero time absorbance was taken at 560nm followed by
recording the absorbance after 5min at 25 C. The control
was simultaneously run without liver homogenate. Units
of SOD activity were expressed as the amount of enzyme
required to inhibit the reduction of NBT by 50%. The
specific activity was expressed in terms of units per mg
of proteins.
Catalase assay
1ml of liver homogenate was taken with 1.9ml of phosphate
buffer in test tubes (50 mM, pH 7.4). The reaction was
initiated by the addition of 1ml of H2O2(30 mM). Control
without liver homogenate was prepared with 2.9ml of
phosphate buffer and 1ml of H2O2. The decrease in optical
density due to decomposition of H2O2 was measured at the
end of 1min against the blank at 240nm. Units of catalase
were expressed as the amount of enzyme that decomposes
1 M H2O2 per min at 25 C. The specific activity was
expressed in terms of units per mg of proteins.
Peroxidase assay
0.5ml of liver homogenate was taken, and to this were
added 1ml of 10 mM KI solution and 1ml of 40 mM
sodium acetate. The absorbance of potassium iodide
was read at 353nm, which indicates the amount of
peroxidase. Then 20 l of H2O2(15 mM) was added, and
the change in the absorbance in 5min was recorded. Units
of peroxidase activity were expressed as the amount of
enzyme required to change the optical density by 1 unit
per min. The specific activity expressed in terms of units
per mg of proteins.
Statistical analysis
Data were expressed as MeanStandard Deviation(SD).
The values were then subjected to oneway ANOVA
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Goudarshivananavar, et al.: Therapeutic potential of stem bark of Polyalthia cerasoides

followed by Turkeys multiple comparison tests for Toxicity and antinociceptive activity of the P. cerasoids
significant difference. The level of significance was stem bark
considered at P0.05 and P0.01.
Fractions were checked for toxicity in mice using the
staircase method after administration at different doses
RESULTS ranging from 502000mg/kg(p.o.). No toxicity was
observed for all the fractions at 50, 100, 200, 500 and
Stem bark fractions and phytochemical constituents of 1000mg/kg with no change in behavior or movements
P. cerasoides among the mice. However, at 2000mg/kg dose five mice
Three different fractions were obtained after solvent based showed movement reduction and suffered shock initially
sequential extraction. The yields are shown in Table1. for 56h. and recovered completely to normalcy after 24h.
The pet ether fraction(PEPCF) was semisolid(3.5%) It appears that the fractions obtained from P.cerasoides
with light brown color and was moderately positive for were not toxic to mice even at 2000mg/kg.
the content of saponins, terpenoids and sterols, the
chloroform fraction(CFPCF) was light green, solid(2%) In the acetic acid induced writhing method, Pet ether, and
with a moderate content of terpenoids, sterols and ethyl acetate fractions showed a significant analgesic
alkaloids. The ethyl acetate(EAPCF) fraction was black activity against chemically induced pain. Both the fractions
semisolid with a maximum yield(5.5%) and was positive have shown a reduction in the number of writhes as
moderately for contents, tannins or phenols, saponins, compared to standard[Figure1a]. PEPCF at 100 and
quinones and it showed abundance for sterols and 200mg/kg doses are effective in reducing pain by 28.82%,
flavonoids. The results of phytochemical constituents 48.48% respectively and EAPCF at the same dose levels
are shown in Table2. shows reduction in pain by 61.73%, 63.68%, whereas
CFPCF fraction showed less than 10%[Figure1b]. The
Table1: Percentage yield of crude fractions from the EAPCF fraction is more significant(P<0.001) compared
stem bark of P. cerasoides with the solvent extraction of to standard drug diclofenac(63.7%). On hotplate test,
petroleum ether, chloroform and ethyl acetate PEPCF(6.6sec) and EAPCF(4.5sec) showed significant
Name of the Color Consistency Percentage elevation in pain threshold when compared to standard
fraction yield(w/w)
drug pentozocin (7.6 sec) and control (3.4 sec) as
Petroleum ether Light brown Semi solid 3.5
fraction(PEPCF) represented in Figure2. The thermal sensitivity which was
Chloroform Light green Solid 2.0 reduced by EAPCF fraction at 200mg/kg dose was very
fraction(CFPCF) significant(P<0.01) and comparable with the standard
Ethyl acetate Black Semi solid 5.5 drug pentazocine.
fraction(EAPCF)

Antiinflammatory potential of P. cerasoides stem bark

Table2: Qualitative phytochemical analysis of crude fractions
obtained from the extraction of stem bark of P.cerasoides Acute antiinflammatory potential of P. cerasoides
Name of the phytoconstituent PEPCF CFPCF EAPCF fractions were investigated at its minimal dose producing
Carbohydrates analgesia against carrageenan induced rat paw edema. The
Amino acids and proteins Figure3a shows the edema volume in control and treated
Tannins and phenols + groups. The standard drug Diclofenac(5mg/kg i.p.) showed
Saponins + + 75.4% inhibition of edema whereas the corresponding
Triterpenoids + + values for PEPCF fraction at 100 and 200mg/kg were
Quinones + 26.22% and 36.06% respectively and for EAPCF, they
Sterols + + ++ were 57.38% and 68.85%. EAPCF extract exhibits
Glycosides significant antiinflammatory activity with respect
Flavonoids ++ to control(P<0.001) and is comparable to the drug.
Alkaloids +
However, the inhibitory effect of CFPCF fraction
The presence of the phytochemicals were assessed and graded as, +,
++ based on the absence and abundance in the fractions. PEPCF:Pet was<15% and is not significant. The results were shown
ether fraction, CFPCF: Chloroform fraction, EAPCF: Ethyl acetate fraction in Figure3b.

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Goudarshivananavar, et al.: Therapeutic potential of stem bark of Polyalthia cerasoides

a b
Figure1: Antinociceptive activity of P.cerasoides stem bark extracts in acetic acid induced writhing in Swiss albino mice.(a) P.cerasoides stem
bark fractions(100 and 200mg/kg, p.o.). Control distilled water(10ml/kg, p.o.), standard drug diclofenac sodium(5mg/kg. i.p.).(b) Percentage
reduction of writhing in groups treated with the extracts and standard drug. One way ANOVA followed by multiple Tukeys comparison test. Values
are presented as the meanSEM(standard error); n=6 for all groups(Statistically significant values are *P<0.05; **P<0.01)

radicals(4200g/mL) with IC50 value of 89.88g/ml and

standard Mannitol(15g/mL) was found to be 4.89mg/ml.
The inhibition of Lipid peroxidation(LPO; 1200g/mL)
by EAFPC was found to be IC50=75.64g/ml as compared
to that of standard trolox(IC50=8.75g/ml). The EAFPC
exhibited significant antioxidant potential with IC50 value
much greater compared to their respective pure standard
components. The purified active component from the
EAFPC fraction may be potential with lower IC50 value.

In vivo antioxidant potential of EAPCF

Figure2: Antinociceptive activity of P.cerasoides stem bark
determined on Eddys hot plate test in Swiss albino mice. P. cerasoides
stem bark fractions(PEPCF, CFPCF, EAPCF, 100 and 200mg/kg,
p.o.). Control distilled water (10ml/kg, p.o.), standard drug pentazocine
(5 mg/kg, i.p .). One way ANOVA followed by multiple Tukeys
comparison test. Values are the meanSEM, n=6 in each group,
(statistically significant values are *P<0.05; **P<0.01)
Antioxidant property of EAFPC
The antioxidant potential of ethyl acetate fraction of
P. cerasoides stem bark(EAFPC) was evaluated and
was found to be effective in reducing nociception and
inflammation. The invitro and invivo antioxidant potential
of EAFPC is shown in Figure4 and Table3 respectively.
In vitro antioxidant potential of EAPCF
The invitro antioxidant activity is measured as the ability
to scavenge free radicals as compared to the standard
antioxidant molecule. The method is represented in
Figure4. EAFPC demonstrated DPPH radical(1100g/mL)
scavenging activity with IC50 levels of 42.43g/ml, whereas
the IC50 value of the standard ascorbic acid(15g/mL)
was 3.19g/ml. The ability to scavenge hydroxyl
Pretreatment of rats with suspensions of ethyl
acetate extract of P.cerasoides at a dose of 250 and
500mg/kg was compared with control group administered
with PBS(Negative control) and Silymarin(positive
control) for the activity of liver enzymes, Catalase, SOD,
and Peroxidase. Results showed a significant improvement
in the activity of enzymes in EPC treated group compared
to the hepatotoxic group(Group injected with CCl4 alone)
which showed decreased levels of hepatic enzymes induced
by CCl4[Table3]. These findings indicate that P.cerasoides
stem bark extracts could significantly protect against
hepatic injury by normalizing the endogenous antioxidant
enzymes that are involved in combating ROS.
DISCUSSION
Reactive oxygen species(ROS) and their intermediates have
been found to be the mediators of inflammation and also
responsible for the pathogenesis of various inflammatory
diseases and tissue damage, which is associated with the
stimulation of pain.[4,6] Pain is a complex process mediated
by many physiological mediators e.g.,prostaglandins,
bradykinins, substancep etc., The petroleum ether,
chloroform and ethyl acetate extracts of P.cerasoides

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Goudarshivananavar, et al.: Therapeutic potential of stem bark of Polyalthia cerasoides

a b
Figure3: Inhibitory effect of P.cerasoides stem bark on carageenan induced paw edema model in rats.(a) P.cerasoides stem bark fractions(PEPCF,
CFPCF, EAPCF, 100 and 200mg/kg p.o.). Control distilled water(10ml/kg, p.o.), standard drug diclofenac sodium(5mg/kg. i.p.).(b) Percentage
reduction of paw volume(ml) in treated groups. PEPCF and EAPCF at 200mg/kg had exhibited 36.06% and 68.85% inhibition respectively.
One way ANOVA followed by multiple Tukeys comparison test. Values are presented as the meanSEM(standard error); n=6 for all groups,
(statistically significant values are *P<0.05; **P<0.01)

that pet ether, and ethyl acetate extracts also significantly

elevate the response latency period suggesting centrally
mediated analgesic effect.

P.cerasoides stem bark extracts have also exhibited a

Figure4: Free radical scavenging ability of P.cerasoides stem
bark(EAPCF) by DPPH radical, Hydroxyl radical and Lipid Peroxidation
by invitro assays. IC50 value of EAPCF on ROS scavenging potential
is compared to the respective standards. Values are meanSEM,
n=6, one way ANOVA followed by Tukeys multiple comparison test.
Significant values are *P<0.05; **P<0.01)
stem bark was evaluated for its analgesic potential in
both peripheral(nonnarcotic) and central(narcotic)
type pain models. Pretreatment with pet ether, and ethyl
acetate extracts(100 and 200mg/kg) markedly reduced
the pain response produced by acetic acid, manifested as
writhing at the employed doses. In the acetic acid induced
writhing model the contractions induced by acetic acid
in the mice results from an acute inflammatory reaction
with production of PGE2 and PGF2 in the peritoneal
fluid.[24] Therefore, it is likely that pet ether, and ethyl
acetate extracts at both the tested doses might suppress
the formation of these substances or antagonize their
action, thus exerting analgesic activity. The hotplate test
is commonly used to assess narcotic analgesics or other
centrally acting drugs[25,26] and the present results showed
76 Ancient Science of Life / Oct-Dec 2015 / Vol 35 / Issue 2
moderate antiinflammatory potential at the employed
doses. It has been proposed that inflammatory reaction
occurs in two phases: Via release of histamine, serotonin
and bradykinin in the early or first phase, followed by the
release of prostaglandin in the late or second phase.[3,27]
A significant antiinflammatory activity against carrageenan
induced inflammation was also observed in pet ether, and
ethyl acetate extracts, suggesting influence of the fraction on
release, synthesis or action of the inflammatory mediators.
We conclude that the pet ether, and ethyl acetate extracts
of the plant contains active herbal principles which are
moderately polar in nature and possess potential analgesic
and antiinflammatory activities.
Free radical induced lipid peroxidation is believed to be
one of the major causes of cell membrane damage leading
to a number of pathological situations. The hepatic damage
induced by CCl4 is well known to be mediated by its free
radical metabolites such as CCl3 and CCl3COO, which
interact with unsaturated lipid membrane to produce lipid
peroxidation and other cellular macromolecules leading to
cell damage and decreased activity of hepatic antioxidant
enzymes such as catalase, Super oxide dismutase(SOD) and
peroxidase.[28,29] The results of the present work indicate
that ethyl acetate extract of P.cerasoids(250mg/kg,
500mg/kg, p.o) increased the depleted antioxidant enzyme
levels induced by CCl4, thus protecting the structural
integrity of hepatocyte cell membrane or by causing
regeneration of damaged liver cells. The extract was found
to be capable of enhancing or maintaining the activity
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Goudarshivananavar, et al.: Therapeutic potential of stem bark of Polyalthia cerasoides

Table3: The invivo effect of P. cerasoides stem bark extracts(EAPCF) on liver antioxidant enzymes in CCl4 induced
hepatotoxicity in rats
Treatment group Dose(mg/kg) Super oxide dismutase(units/mg) Catalase(units/mg) Peroxidase(units/mg)
Control(negative controls) + PBS 5 ml/kg 24.00.54 436.433.07 145.23.21
CCl4 alone(hepatotoxic) 1.0 8.480.12c 121.541.53c 48.432.70c
Silymarin(positive control) + CCl4 100+1.0 19.71.33c 407.472.43c 138.21.40b
Ethyl acetate extract(EAPCF) + CCl4 250+1.0 15.111.58 b
368.21.54 b
118.785.12b
Ethyl acetate extract(EAPCF) + CCl4 500+1.0 19.543.22c 398.45.23c 131.324.30c
Values are meanSEM, n=6, oneway ANOVA followed by Tukeys multiple comparison test. bP<0.01 when compared with control, cP<0.001 when
compared with control and CCl4 group. All the animals except control group received CCl4/olive(1:1) 1 ml/kg, i.p. on 3rd and 4th day. Positive control
and test samples were administered for 5 days

Figure5: Schematic representation of the pharmacological activities of P.cerasoides stem bark extracts

of hepatic enzymes which are involved in combating
ROS.[30] The P.cerasoides extracts effect on liver antioxidant
enzymes can also be confirmed by correlating it with its
inhibitory effect on the lipid peroxidation invitro. Further
preliminary phytochemical investigation of ethyl acetate
extract revealed the presence of polyphenols, flavonoids
and coumarins. The mode of action of ethyl acetate extract
in affording the potential antioxidant activity against CCl4
may be due to cell membrane stabilization, hepatic cell
regeneration and activation of antioxidant enzymes such
as SOD, catalase and peroxidase by these active principles.
Taken together, our findings indicate that P.cerasoides
stem bark extracts apart from alleviating pain may also
significantly protect against hepatic injury by normalizing
the endogenous antioxidant enzymes that are involved in
combating ROS[Figure5].
CONCLUSION
Ethyl acetate extract of P.cerasoides stem bark demonstrated
a significant analgesic and antiinflammatory activity with
ROS scavenging potential. This study indicates a positive
correlation between the ROS scavenging potential and
antiinflammatory activity. Considering the results of
experimental parameters, our study provides strong scientific
evidence for considering the P.cerasoides stem bark extracts
Ancient Science of Life / Oct-Dec 2015 / Vol 35 / Issue 2
as natural antioxidants targeting algesia and inflammation.
Hence we conclude that P.cerasoides stem bark could
be used as a therapeutic drug in oxidative stress induced
pathological conditions. But an in depth investigation with the
pure compounds is necessary to understand the mechanism
behind its significant medicinal importance.
Acknowledgements
The authors gratefully acknowledge the Sahyadri
Science College, Shimoga(A constituent College of
Kuvempu University) for the supporting this study.
Dr.Siddanakoppalu N. Pramod and V. Vigneshwaran
acknowledges the University Grants Commission,
Government of India.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Address for correspondence:
Dr. Siddanakoppalu N. Pramod,
Laboratory of Immunomodulation and Inflammation Biology,
Department of Studies and Research in Biochemistry,
Sahyadri Science College (Autonomous), Kuvempu University,
Shimoga - 577 203, Karnataka, India.
E-mail: snpramod2029@gmail.com