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Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 6 pp. 1601ñ1608, 2016 ISSN 0001-6837
Polish Pharmaceutical Society
Traditional system of medicines or herbal care
is the original source for most of the medicines and
is used worldwide. Medicinal plants possess a wide
variety of chemical constituents which are helpful
for curing different diseases. Recent discovery of
medicinal plants have led to improvement of human
health care systems. Plant based drugs are used as
medicines either as a whole or their principle con-
stituents are separated by different chemical tech-
niques. This practice continues because of different
biological advantages, pharmacological activities
and fewer side effects associated with medicinal
plants (1).
Inflammation is a primary defense mechanism
that helps the body to protect itself against various
types of diseases, allergens, chemicals and other
toxic reactions. Inflammation is a process which
involves a large variety of inflammatory mediators
and prominent increase in localized leucocytes.
Prostaglandins are the substances that modulate the
cell and tissue response during inflammation. Their
biosynthesis also continues during the cardiovascu-
lar disease, cancers and colonic adenomas. Although
it is a mechanism of protection for the body, an
uncontrolled inflammation and various mediators of
inflammation can induce or maintain a disease and
can even aggravate it (2). Plants are widely used for
treating inflammation because the adverse effects
produced by these plant based drugs are very few. In
addition, they are easily affordable and lower in cost
than the synthetic drugs (3).
Pyrexia or fever occurs as bodyís natural
defense mechanism in response to any infection or
disease. In pyrexia, an environment is created inside
the body in which the infectious pathogens and dam-
aged tissues are unable to survive (4). Normal body
temperature is maintained and regulated by hypo-
thalamus that maintains a balance between heat loss
and production. Fever occurs due to some distur-
bances in this thermostat of body and as a result
bodyís temperature rises. The temperature regulat-
ing mechanisms then works to normalize the body
EVALUATION OF ANTI-INFLAMMATORY, ANALGESIC AND
ANTIPYRETIC ACTIVITIES OF AQUEOUS AND ETHANOLIC EXTRACTS OF
SEEDS OF BUCHANANIA LANZAN SPRENG. IN ANIMAL MODELS
AMIR MEHMOOD1, IRFAN HAMID2, ALI SHARIF1, MUHAMMAD FURQAN AKHTAR1,
BUSHRA AKHTAR3, AMMARA SALEEM4, JAVED IQBAL1, MARYAM SHABBIR1
and SAJID ALI1*
1Faculty of Pharmacy, The University of Lahore, Lahore, Pakistan
2Akson College of Health Sciences, Mirpur University of Science and Technology, AJK, Pakistan
3Institute of Pharmacy, Physiology and Pharmacology, University of Agriculture, Faisalabad, Pakistan
4Department of Pharmacy, Government College University, Faisalabad, Pakistan
Abstract: The present study was designed to evaluate the anti-inflammatory, analgesic and antipyretic activi-
ties of the aqueous and ethanolic extracts of seeds of Buchanania lanzan Spreng. Albino mice were used as
experimental animals to evaluate these activities. The study was performed in three phases; Phase-I for evalu-
ation of anti-inflammatory activity, Phase-II for antipyretic and Phase-III for analgesic activities were evaluat-
ed. Carrageenan induced paw edema, brewer yeast induced pyrexia and acetic acid induced writhing methods
were used to evaluate anti-inflammatory, antipyretic and analgesic activities, respectively. Tests were per-
formed by dividing the animals in five groups. First group was negative control, second group was positive con-
trol, third, fourth and fifth groups were treated with 125, 250 and 500 mg/kg of extracts. respectively. The data
were statistically analyzed using ANOVA where p < 0.05 were considered significant. The results suggested
that seeds of Buchanania lanzan Spreng. possess anti-inflammatory, analgesic and antipyretic activity.
Keywords: Buchanania lanzan Spreng., analgesic, anti-inflammatory, antipyretic
1601
* Corresponding author: e-mail: sajidalichishti@gmail.com; phone: 0092-333-4404880
1602 AMIR MEHMOOD et al.
temperature. These mechanisms include dilation of
superficial blood vessels and sweating etc. When
any infectious agent enters into the body through a
break in its barriers, it will interact with immune
cells and will promote the release of endogenous
mediators like cytokines, prostaglandins and
endothelins. In pre-optic area of anterior hypothala-
mus, PGE2 play a vital role in fever induction (5).
Pain is a sensorial modality and sometimes it is
the only feature for diagnosis of various diseases.
Many forms of therapy are available for curing this
condition, among them medicinal herbs are widely
used (6). Pain is a sensation which is unpleasant and
is localized to a part of body. In response to tissue
injury visceral distensions, peripheral nociceptors
and some other factors are stimulated, which devel-
op pain. Perception of pain is a normal physiologi-
cal response and it is mediated by the healthy nerv-
ous system (7).
Different experimental models are used for eval-
uating anti-inflammatory, analgesic and antipyretic
activities of herbal plants. For studying anti-inflam-
matory activity, a large number of experimental mod-
els have been developed. These models are basically
of two types; acute inflammatory model and chronic
inflammatory model. Acute models are developed to
study and evaluate the drugs which are involved in
erythema, leucocyte migration and chemotaxis,
changes in vascular permeability, phagocytosis-poly-
morphonuclear leucocytes, measurement of local
pain and rat paw edema (8). Acute-inflammatory
models are carrageenan induced paw edema, xylene
Table 1. Experimental design.
Groups Drug Dose Route No. of animals
Group 1 (negative control) Normal saline 1 mL/kg Oral 5
Group 2 (positive control) Indomethacin 10 mg/kg Oral 5
Group 3 Plant extract 125 mg/kg Oral 5
Group 4 Plant extract 250 mg/kg Oral 5
Group 5 Plant extract 500 mg/kg Oral 5
Table 2. Anti-inflammatory activity of Buchanania lanzan Spreng. (aqueous extract).
Average paw volume (mm)
Groups
1 h 2 h 3 h
Normal saline 3.70 ± 0.01 3.76 ± 0.01 3.97 ± 0.01
Indomethacin 2.93 ± 0.03 2.13 ± 0.01 1.91 ± 0.01
125 mg/kg 3.41 ± 0.02 3.16 ± 0.05 2.86 ± 0.05
250 mg/kg 3.37 ± 0.02 2.94 ± 0.01 2.48 ± 0.04
500 mg/kg 3.25 ± 0.41 2.71 ± 0.01 2.39 ± 0.08
Note: All specified doses showed a statistically significant (p < 0.05) effect.
Table 3. Percentage inhibition of carrageenan induced paw edema (aqueous extract).
Inhibition of carrageenan Average
Groups induced paw edema (%) inhibition
1 h 2 h 3 h (%)
Indomethacin 20.8 43 51.8 38.2
125 mg/kg 7.8 15.9 27.9 17.2
250 mg/kg 8.9 21.8 37.5 22.7
500 mg/kg 12.1 27.9 39.7 26.5
Evaluation of anti-inflammatory, analgesic and antipyretic activities of... 1603
induced ear edema, egg albumin induced edema,
arachidonic acid induced ear edema and croton oil
induced edema in mice/rats. Chronic models are cot-
ton pellet induced granuloma and adjuvant arthritis in
mice/rats (9). Analgesic activity is determined in two
ways; central analgesic activity and peripheral anal-
gesic activity. Peripheral analgesic activity is evaluat-
ed by acetic acid induced writhing and hot plate
method (10). Antipyretic activity is studied by brew-
erís yeast induced pyrexia, vaccines induced pyrexia
and D-amphetamine induced pyrexia (11).
Buchnania lanzan Spreng. (family Anacardia-
ceae) is commonly known as Char, chironji,
Almondette Tree, Murkali, Nurkale. It is an indige-
nous Hill Plant, Plain Land commonly found
throughout the greater part of India in dry deciduous
forests up to an altitude of 1200 m. The decoction of
its bark is useful in abdomen disease, cough and
bronchitis. Fruit is used to treat nervous debility,
cardiac debility, constipation, fever, ulcer,
oligospermia and general debility (12). The parts of
plant like roots, leaves, fruits, seeds and gum are
used for various medicinal applications like cure for
blood disorder, fever, ulcer, burning sensation of
body, diarrhea, dysentery, asthma and snakebite
(13). Aqueous and ethanolic extracts of seeds of
Char were used to evaluate anti-inflammatory, anal-
gesic and anti-pyretic activity.
The present study was undertaken to assess the
anti-inflammatory, antipyretic and analgesic activity
of aqueous and ethanolic extracts of seeds of
Buchnania lanzan Spreng.
MATERIALAND METHOD
Collection of plant material
Seeds of Buchnania lanzan Spreng. were pur-
chased from local market of Lahore, Pakistan. It was
identified and authenticated by Prof. Dr.
Zaheerudin, Botany Department of Government
College University, Lahore, Pakistan. The seeds
were preserved in herbarium of Government
College University, Lahore, Pakistan.
Washing, drying, grinding and storage of plant
The seeds were washed with distilled water to
remove dirt particles and contamination. Washed
seeds were dried at room temperature under shade
for appropriate time period. Fully dried seeds were
then grinded with mortar and pestle until a coarse
powder was obtained. The powder was passed
through sieve #20 and stored in air tight, labeled
containers till further processing.
Extract preparation
Aqueous and ethanolic extracts of Buchnania
lanzan Spreng. seedís powder were prepared by
triple maceration process. For the preparation of
aqueous extract, 500 g of powdered material was
soaked in 1000 mL of distilled water in an amber
Table 5. Percentage inhibition of carrageenan induced paw edema (ethanolic extract).
Inhibition of carrageenan Average
Groups induced paw edema (%) inhibition
1 h 2 h 3 h (%)
Indomethacin 7.4 47.0 51.2 41.8
125 mg/kg 13.0 26.0 30.0 23.0
250 mg/kg 16.0 35.0 40.0 30.3
500 mg/kg 21.0 36.0 46.0 34.3
Table 4. Anti-inflammatory activity of Buchanania lanzan Spreng. (ethanolic extract).
Groups
Mean values of paw edema (mm) ± SEM
1 h 2 h 3 h
Normal saline 3.72 ± 0.01 3.88 ± 0.01 3.98 ± 0.01
Indomethacin 2.70 ± 0.07 2.10 ± 0.01 1.94 ± 0.01
125 mg/kg 3.21± 0.01 2.84 ± 0.01 2.78 ± 0.01
250 mg/kg 3.11 ± 0.01 2.52 ± 0.01 2.38 ± 0.01
500 mg/kg 2.93 ± 0.01 2.48 ± 0.01 2.14 ± 0.04
Note: All specified doses showed a statistically significant (p < 0.05) effect.
1604 AMIR MEHMOOD et al.
colored glass container for 3 days. After stated time,
the solvent was removed using muslin cloth and the
remaining plant material was again soaked in 500
mL of distilled water and whole process was repeat-
ed thrice. Ethanolic extract was prepared by the
same method using ethanol as solvent.
Drying of extracts
The extracts were filtered through Whatman
filter paper and then dried in rotary vacuum evapo-
rator until a semi-solid mass was obtained. This
semi-solid mass was spread in Perti dishes to com-
pletely dry the extract in oven. The dried extracts
were stored in refrigerator at 4OC until further pro-
cessing.
Grouping of animals
Albino male mice were obtained from
Department of Theriogenology, University of
Veterinary and Animal Sciences, Lahore, Pakistan.
The weight of each mouse ranged in 20-25 g. The
animals were of the same breed and batch. The mice
were divided into 5 groups with each group having
five mice. They were kept in polycarbonate cages
under controlled temperature (22-25OC) and had free
access to feed and water.
Pharmacological evaluation
Pharmacological evaluation of the seed
extracts of Buchanania lanzan Spreng. was done in
three phases. In phase-I anti-inflammatory activity
was evaluated. In phase-II analgesic activity and in
phase-III anti-pyretic activity were evaluated. The
treatment strategy for animal groups is given in
Table 1.
Phase-I - Evaluation of anti-inflammatory activity
Inflammation was induced by injecting 1% car-
rageenan solution (dose of 0.05 mL) to the right hind
paw of mice. Test drug and standard drug were given
1 h before the administration of carrageenan in
respective groups. In negative control group, the
mice were treated with normal saline and in positive
control, indomethacin was given. A mark was made
at the paw of each mouse up to the ankle joint. Paw
volume was measured up to ankle joint in drug treat-
ed and drug untreated groups before and after admin-
istration at a time interval of 0, 1, 2 and 3 h (14).
Table 6. Brewer's yeast induced antipyretic activity (aqueous extract).
Mean values of temperature (∞F) ± SEM
Groups Before drug administration After drug administration
Normal temp. 18 h 1 h 2 h 3 h
Normal saline 97.51 ± 0.01 102.95 ± 0.03 102.14 ± 0.02 102.78 ± 0.04 102.98 ± 0.01
Indomethacin 97.58 ± 0.09 102.71 ± 0.03 101.34 ± 0.04 100.31 ± 0.09 97.02 ± 0.01
125 mg/kg 97.96 ± 0.02 102.38 ± 0.08 101.86 ± 0.01 99.73 ± 0.01 99.04 ± 0.01
250 mg/kg 96.85 ± 0.03 102.74 ± 0.02 101.43 ± 0.01 99.34 ± 0.01 98.55 ± 0.02
500 mg/kg 97.20 ± 0.10 102.12 ± 0.02 101.14 ± 0.03 99.03 ± 0.03 98.19 ± 0.19
Note: All specified doses showed a statistically significant (p < 0.05) effect.
Table 7. Brewer's yeast induced antipyretic activity (ethanolic extract).
Mean values of temperature (∞F) ± SEM
Groups Before drug administration After drug administration
Normal temp. 18 h 1 h 2 h 3 h
Normal saline 97.52 ± 0.07 102.83 ± 0.27 102.04 ± 0.01 102.84 ± 0.01 102.96 ± 0.01
Indomethacin 97.72 ± 0.04 102.73 ± 0.03 101.50 ± 0.05 100.37 ± 0.03 97.23 ± 0.04
125 mg/kg 97.26 ± 0.02 102.54 ± 0.01 101.96 ± 0.01 100.73 ± 0.01 99.74 ± 0.01
250 mg/kg 97.15 ± 0.03 102.32 ± 0.02 101.44 ± 0.01 100.34 ± 0.01 99.56 ± 0.02
500 mg/kg 96.94 ± 0.03 102.34 ± 0.02 101.64 ± 0.03 100.03 ± 0.03 99.12 ± 0.12
Note: All specified doses showed a statistically significant (p < 0.05) effect.
Evaluation of anti-inflammatory, analgesic and antipyretic activities of... 1605
Quantification of anti-inflammatory activity
The anti-inflammatory activity of the seed
extract of Buchanania lanzan Spreng. was quantified
by measuring the paw edema (in mm) using digital
Vernier caliper. The results were expressed in terms
of mean values of paw edema of each group ± SEM.
Reduction in paw edema was measured by the
percentage inhibition of the positive control and
extract treated groups as compared to the negative
control. The percentage inhibition was calculated
using the following formula:
Percentage inhibition = (Control ñ Treated) /
(Control) ◊ 100
Phase-II - Evaluation of antipyretic activity
Brewerís yeast solution was used to induce
fever in mice by subcutaneous administration of
solution below the nape of the neck. Initial tempera-
ture was recorded by using digital clinical ther-
mometer. After 18 h of administration, animal which
showed a mean rise of 0.3-0.5OC body temperature
were selected. Animals were treated with normal
saline, standard drug and drug extracts as per exper-
iment design. Temperature of mice was recorded
after an interval of 1, 2 and 3 h post dosing (15).
Quantification of antipyretic activity
Antipyretic activity was quantified by check-
ing the decrease in body temperature at different
time intervals. The results were calculated in F ±
SEM for all groups.
Phase-III - Evaluation of analgesic activity
Acetic acid induced writhing method was used
to evaluate analgesic activity of Buchanania lanzan
Spreng. seed extract. The mice were treated with
intraperitoneal injection of 1% acetic acid solution
(0.1 mL) and number of writhing movements was
counted for 20 min (16).
Quantification of analgesic activity
The reduction in number of writhing after
administering positive control and drug extracts
with respect to negative control group was quanti-
fied by calculating percentage inhibition using fol-
lowing formula:
Percentage inhibition = (Ncñ Nt) / (Nc) ◊ 100
where, Ncis mean number of writhing in control
group, Ntis mean number of writhing in treated
group
Statistical analysis
For statistical analysis, the data were evaluated
using Statistical Package of Social Sciences (SPSS).
The results were expressed as the mean ± SEM and
analysis of variance (ANOVA) was applied to the
data.
Table 8. Acetic acid induced analgesic activity of Buchanania lanzan Spreng. (aqueous extract).
Groups Drug Dose Number of writhing Inhibition of
(mean) ± SEM writhing (%)
Group 1 Normal saline 1 mL/kg 66.8 ± 0.44 N/A
Group 2 Indomethacin 10 mg/kg 17.6 ± 0.54 73.65
Group 3 Test extract 125 mg/kg 44.4 ± 0.54 33.53
Group 4 Test extract 250 mg/kg 36.0 ± 0.04 46.12
Group 5 Test extract 500 mg/kg 29.4 ± 0.54 55.99
Note: All specified doses showed a statistically significant (p < 0.05) effect.
Table 9. Acetic acid induced analgesic activity of Buchanania lanzan Spreng. (ethanolic extract).
Groups Drug Dose Number of writhing Inhibition of
(mean) ± SEM writhing (%)
Group 1 Normal saline 1 mL/kg 65.0 ± 0.70 N/A
Group 2 Indomethacin 10 mg/kg 16.8 ± 0.83 74.15
Group 3 Test extract 125 mg/kg 40.0 ± 0.70 38.46
Group 4 Test extract 250 mg/kg 32.4 ± 0.54 49.37
Group 5 Test extract 500 mg/kg 25.6 ± 0.54 60.00
Note: All specified doses showed a statistically significant (p < 0.05) effect.
1606 AMIR MEHMOOD et al.
RESULTS AND DISCUSSION
In previous literature, phytochemical investi-
gation revealed the presence of new glycoside,
myricetin-3-rhamnoside-3-galactoside (17). The
component fatty acids of the seed fat were found to
be myristic, palmitic, stearic, oleic and linoleic
acids. The moisture, crude protein, pentosan and
water soluble mucilage contents of seeds were
reported as 5.2, 6.95, 3.8 and 2.8 percent, respec-
tively. The mucilage showed absence of protein and
pentosan (18).
The results of anti-inflammatory effect of
aqueous and ethanolic extract of Buchanania lanzan
Spreng. on carrageenan-induced edema in paw of
mice are presented in Tables 2 and 4, respectively,
whereas percentage inhibition by aqueous and
ethanolic extract is depicted in Tables 3 and 5,
respectively. Edema development due to inflamma-
tion is a biphasic event in which the initial phase is
attributed to the release of histamine and serotonin.
The second phase of edema is due to the release of
protease, prostaglandins and lysosomes. Most of the
anti-inflammatory drugs are clinically effective in
second phase of inflammation (19). The data repre-
sented that the ethanolic extract at dose of 500
mg/kg showed significant reduction (p < 0.01) in
edema and faster rate of inhibition as compared to
other doses. However, the aqueous and ethanolic
extracts showed moderate reduction in edema when
compared with the extract of standard drug (Fig. 1).
The carrageenan induced paw edema model is
known to be sensitive to the effect of NSAIDs which
primarily inhibits cyclooxygenase involved in syn-
Figure 1. Percentage inhibition of carrageenan induced paw edema by standard drug, aqueous and ethanolic extract of Buchanania lanzan
Spreng.
Figure 2. Percentage inhibition of Brewerís yeast induced pyrexia by standard drug, aqueous and ethanolic extract of Buchanania lanzan
Spreng.
Evaluation of anti-inflammatory, analgesic and antipyretic activities of... 1607
thesis of prostaglandins (20). Therefore, it can be
reasonably concluded that the inhibitory effect of
aqueous and ethanolic extract of Buchanania lanzan
Spreng. on inflammation in mice may be due to the
inhibition of cyclooxygenase enzyme.
The antipyretic activity of aqueous and ethano-
lic extract is given in Tables 6 and 7, respectively. An
increase in temperature was evident after 18 h of
Brewerís yeast administration. After the administra-
tion of indomethacin and drug extracts, a significant
decrease in temperature was noted. The extracts
showed dose dependant decrease in temperature with
increasing potency from 125 to 500 mg/kg. The aque-
ous extract of seed at dose of 500 mg/kg had evident
antipyretic activity but it was moderate as compared
to the positive control after 3 h of drug administration.
Therefore, aqueous extract proved to contain more
antipyretic activity as compared to ethanolic extract
but lesser than standard antipyretic drug (Fig. 2).
The peripheral analgesic activity of drug
extract was tested by acetic acid induced writhing
test. Acetic acid induced writhing test is a standard
test to check the pain sensitivity of opiates and non-
opiates analgesics. The pain sensation is due to
abdominal constriction due to irritation of peritoneal
cavity caused by acetic acid. Prolonged irritation
leads to an increase in level of prostaglandins (PGE2
and PGF2α) biosynthesis and lipoxygenase products
in peritoneal fluids. The increase in these products
enhances the level of free arachidonic acid which
subsequently develops inflammatory pain by
increasing capillary permeability in peritoneal cavi-
ty of abdomen (21). The reference drug used was
indomethacin which is non-opiate analgesic. This
NSAID involves blockade of production of
prostaglandins by inhibition of both COX-1 and
COX-2. The aqueous and ethanolic seed extract
produced a significant decrease in the writhing
counts (Tables 8 and 9). Minimum number of
writhing was observed with 500 mg/kg ethanolic
extract. The percentage inhibition of pain induced
by acetic acid was dose dependent i.e., with an
increase in dose of aqueous and ethanolic extract the
number of writhing in groups decreased in contrast
to negative control group (Fig. 3). Analgesic activi-
ty of aqueous and ethanolic seed extract indicates
that peripherally active analgesic property might be
present (p < 0.01).
CONCLUSION
The result of biological screening on the frac-
tions from aqueous and ethanolic extract of
Buchanania lanzan Spreng. confirms consistent
activities in anti-inflammatory, antipyretic and anal-
gesic tests.
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Received: 5. 12. 2015
