Wednesday, 5 September 2012

Antiarthritis Activity of Aristolochia Bracteata Extract in Experimental Animals

6 The Open Natural Products Journal, 2009, 2, 6-15
1874-8481/09 2009 Bentham Open
Open Access
Antiarthritis Activity of Aristolochia Bracteata Extract in Experimental
Havagiray R. Chitme* and Nitin P. Patel
Department of Pharmacology, H.S.K. College of Pharmacy, Bagalkot-587101, Karnataka, India
Abstract: The Aristolochia bracteata is well known for its antiarthritis properties in Indian system of medicine and folk
medicine. The objective of the present study was to evaluate its folk claim in rheumatoid arthritis (RA) and propose a
probable mechanism of action. Anti arthritic activity was evaluated using Freund’s complete adjuvant in rats, the course
of treatment was followed for over and 4 weeks post inoculation period using health parameters, clinical and behavioral
methods of study. Estimation of blood Hb, ESR and change in body weight were considered as health parameters and
clinical observations included paw edema volume, thermal hyperalgesia, radiological and histomarphological analysis and
exploratory behavior was studied in behavioral observations. The results indicates that, regular treatment of adjuvant induced
arthritic rats with A. bracteata extracts improves ESR, Hb value and also restores body weight. Significant (P<0.01)
inhibitory effect was observed with A. bracteata extract on Freund’s complete adjuvant induced paw edema throughout
the study (P<0.001). The latency to thermal stimuli and inhibitory effect on xylene induced ear edema was significantly
(P<0.05) affected by oral treatment of A. bracteata, irrespective of solvent used for extraction. Treatment of FCA induced
rats with A. bracteata extracts shown (P<0.05) increase in pain threshold, weight bearing ability, ambulation and also decline
in scratching, defecation and urination, were observed as a sign of improvement in behavioral condition. The results
obtained in this study showed promising effect on FCA modulated health status, clinical observations and behavioral
Keywords: Anti-arthritis, arthritis, Aristolochia bracteata, Freund’s complete adjuvant.
Immune system is vital to survive, because a hyperactive
immune system may cause fatal disease due to over whelming
allergic reaction leading to series of derangements, loss
of normal capacity to distinguish self from non-self resulting
in immune reactions against one’s own tissues and cells
called autoimmune diseases. These autoimmune changes are
receiving increased attention in drug discovery and development
as the progress has been made in understanding immune
and inflammatory processes. Several autoimmune diseases
including myasthenia gravis, serum sickness, pernicious
anaemia, pemphigus vulgaris, SLE, reactive arthritis,
etc. are the severe concern of medical and pharmaceutical
community because of unknown etiology. Rheumatoid Arthritis
(RA) is one of the most common autoimmune inflammatory
conditions of unknown etiology characterized by
symmetric, erosive synovitis and in same cases extraarticular
involvement [1].
The presently available pharmacological treatments in
the market not only causing economical exploitation, but
also associated with severe adverse effects [2-3]. Despite
extensive use of currently available therapy, most RA patients
are suffering from declined functional ability because
of inability in preventing cartilage breakdown and joint destruction
[4]. Recently, efforts have been focused on using
the class of drugs called biologics (antibodies or soluble re-
*Address correspondence to this author at the Oman Medical College, P. O.
Box 620 Postal Code 130 Azaiba, Muscat-Sultanate of Oman, Oman; Tel:
968-24504608-194; Fax: 968-24504820; E-mail:
ceptors for IL-1, IL-6 and TNF-􀀁) for the treatment of RA.
Although these agents reduce the inflammation and joint
destruction, their long-term risks and benefits are not yet
clear. Additionally, higher costs and the findings that they
are not effective universally and severe side effects such as
life threatening infections, increased risk of malignancies
and require continuous and careful monitoring [5-10].
However, to develop a proper medication which will be
ecofriendly and having very less side effects that can be used
for prophylactic and therapeutic purpose to control this dangerous
disease is still a big challenge to a scientific community
working in this area. The use of complementary and
alternative medicine (CAM) therapies, such as acupuncture,
physiotherapy, yoga and extract of medicinal herbs, and is
on rise. According to reports 60-90% of dissatisfied arthritis
patients are likely to seek the option of CAM therapy [11,
Herbal medicine is the root of various traditional medicine
systems around the world. Ayurvedic medicine in India
has proven track record of 5000 years and forms part of the
National Health Service, offered along side conventional
medicine. The ayurvedic, National Formulary lists some
8000 well proven ayurvedic formulations described in
Dravyaguna (Ayurvedic Pharmacology). Remedies are made
from single or multiple herbs and minerals for various medical
conditions like asthma, flu, diabetes, arthritis, heart disease,
digestive problems, mental health and skin problems.
Herbal medicines yielding about 25% of currently used
crude drugs with another 25% derived from chemically altered
natural products [13].
Antiarthritis Activity of Aristolochia Bracteata Extract The Open Natural Products Journal, 2009, Volume 2 7
In ancient texts about 500 plants have been indicated in
the treatment of arthritis, however only few number of plants
have been evaluated scientifically (<50). Aristolochia bracteata
is most commonly known as kidamari, wildly distributed
in Deccan Gujarat, western and southern India, Bihar,
Sindh, Bundelkhand and Bengal. It has been found most
commonly in ancient texts for important medicinal properties
including anthelmentic, fever, purgative and painful
joints. Recently, it has been reported for hypotensive, hypothermia,
antioxidant and anti-inflammatory properties. However,
no study has been carried out to evaluate its antiarthritis
property. Therefore, the present study was carried
out with an objective to evaluate anti-arthritis activity of
Aristolochia bracteata whole plant extract prepared by successive
solvent extract. This study will evaluate its folk
claims and also propose its probable mechanism of action.
The study has been designed according to the guidelines
published as guidance for industry by U.S. Food and Drug
Administration on ethical standards for investigations of
experimental arthritis in animals using in vivo methods [14].
All the procedures were carried out on Wistar albino rats
of either sex weighing 200-250gm for anti-arthritic study and
Swiss albino mice of body weight15-30 gm for acute toxicity
study and xylene-induced ear edema were bred and raised
under the animal facility of H.S.K. College of Pharmacy,
Bagalkot, Karnataka. Food and water were supplied ad libitum
and the animals were kept in a 12 h light: 12h dark cycle
and environmental temperature (23 ± 1 °C) in standard propylene
cages. Cage cleaning consisted of daily change of
rinse husk bedding. All the animals were monitored over 28
days and sacrificed for the histological assessment of inflammation.
All the experiments were conducted in accordance
with the Institutional Animal Ethics Committee
(821/01/a/CPCSEA/HSKCP/IAEC/2004-2005). Due to the
painful condition imposed on the animals, the number of
subjects used was restricted to the minimum that allowed
reliable statistical analysis of the results. Each group was
composed of 6 animals.
Aristolochia bracteata was collected during November
2005 from village Devla, Amreli District, Gujarat, and was
authentified by Taxonomist Prof. V.V. Siddhulingappanavar,
HOD, Dept. of Botany, Basaveshwar Science College,
Bagalkot, Karnataka, India.
The whole plant Aristolochia bracteata was collected
after authentification and dried under shade and powdered.
To get uniform size passed it through sieve no. 44# and was
subjected to extraction with petroleum ether, chloroform and
methanol in a soxhlet extractor successively with 12 hrs cycle
[15]. The extract was concentrated by distillation and by
using flash evaporator to yield a semisolid residue. The percentage
yields of petroleum ether extract, chloroform extract
and methanolic extract was calculated and found 4.73%,
3.693%, and 3.66% respectively. All the extracts were preserved
in a refrigerator till further use.
Preparation of Test Solutions
The test solution of methanolic extract was prepared by
dissolving it in water. The suspension of petroleum ether and
chloroform extract was prepared by suspending it in a 5%
Span80 using mechanical shaker. Above prepared test solution
and suspensions of Petroleum ether, Chloroform, and
Metnanolic extract were tested in doses of 100, 200, and 400
mg/kg p.o. for its anti- arthritic, leukotriene infiltration inhibitory
and anti-nociceptive properties.
Acute Oral Toxicity Study
Healthy Swiss albino mice of either sex weighing 15-30
gm, starved overnight were divided into 3 groups (n=9) and
were fed with increasing doses (1, 2, and 4 gm/kg) of each
extract and the toxicity was evaluated as per the Guidelines
for non-clinical toxicity Investigation of Herbal Medicine
(Annexure-I) given by the Ministry of Health and Family
Welfare, Govt. of India [16]. The total drug extracts administered
orally in doses of up to 4 gm/kg, did not produce any
evident sign of toxicity and mortality in rats, and were observed
upto 14 days after administration.
Freund’s complete adjuvant (FCA) [17] composed of 1
mg/ml heat killed Mycobacterium tuberculosis, mineral oil
and mannide monooleate, an inducing agent for arthritis was
purchased from Sigma Aldrich Co, St Louis, USA. Indomethacin
was obtained from the U-Medico Laboratories Pvt.
Ltd., G.I.D.C., Vapi, Gujarat, India as a complementary
sample and was used as a standard drug. All the other
chemicals and solvents used were of AR grade. Instruments
that were used in the present study are, ESR Stands and Top
pipettes, Sahli’s Haemometer, UGO BASILE Digital
Plethysmometer (Italy), Dental X-ray machine (Siemens
Multiphos 10), Spencer type Wes wax Microtome, Metzer
Biomedical Research Microscope, Eddy’s Hot Plate and
Cork borer of 8 mm diameter.
Induction of Anesthesia
For the induction of arthritis, rats were anaesthetized with
40-mg/kg thiopentane injected intraperitoneally, for acute
terminal experiments. Once anaesthetized, the animals were
constantly kept under observation to ensure that breathing is
slow and regular. Sign of deep anesthesia was indicated by
the abolition of withdrawal reflex when the hind paw of the
rat was squeezed [18].
Induction of Monoarthritis
For the induction of arthritis injection of the left ankle
joint was performed under anesthesia: the tarsial area of the
hind paw was grasped and the fossa distal and medial to the
‘lateral malleolus’ of the fibula was palpated. A 26 gauge
needle was introduced into the capsule of the tibiotarsal joint
percutaneously by directing it cephalad, mesiad and superiorly
from the midpoint of the ‘inframalleolar fossa,’ until a
distinct loss of resistance was felt approximately 4 mm and
complete adjuvant or vehicle injected. With a true intracapsular
injection, a firm resistance to injection was characteristically
felt after the injection of 0.05 ml of fluid [18, 19].
Baseline (pre-induction) behavioral and clinical observations
were made prior to injection of vehicle or complete adjuvant,
and then at each week up to 28 days (4 weeks).
8 The Open Natural Products Journal, 2009, Volume 2 Chitme and Patel
Measurement of Health Status
The health status parameters included (i) Body Weight,
(ii) Hemoglobin (Hb) level, and (iii) Erythrocyte Sedimentation
Rate (ESR) measurements. The body weights of all the
animals were recorded in grams on weekly basis by using
single pan weighing balance. Haemoglobin levels of all the
animals were evaluated on 29th day of study using Sahli Hellige
Haemometer and the results are expressed in gm % unit.
Erythrocyte sedimentation rate were estimated by Westergren
pipettes having 2.5 mm internal diameter, 300 mm
length, and 1 ml capacity and ESR stands. Blood was collected
from all the arthritic and non-arthritic animals used in
the study by retro orbital [20].
Behavioral Observations- Open Field Test
For behavioral observations all the animals were subjected
to open field test before the induction of arthritis and
there after every week up to 4 weeks (28-days). Briefly rat
was placed in an open field in the sound-attenuated room.
The floor was white polyvinyl with a black grid dividing
open field into 84 squares (10 x 10 cm). Illumination was
provided by a bulb (60 W) placed above the center of the
field, while the rest of the room was in darkness. The rat was
initially placed in the corner or in the center of the field and
observed for 5 min. in all tests latency time to start explore
the open field (seconds), horizontal locomotor activity (grid
lines crossed), vertical locomotor activity (rearing), grooming
(rubbing the nose with its porepaws and preening), instance
of defecation (number of boluses), and number of
urinations were recorded. Between the trials the box was
cleaned with wet sponge and paper tissue [21, 22]. All the
observations took between 8.00 and 12.00 h.
Assessment of Arthritis
Assessing joint swelling may not merely reflect disease
activity but may indicate a chronic phenomenon reflecting
joint damage. Clinical severity of arthritic inflammation was
measured by the quantification of the paw volume changes;
measurement was carried out by using UGO BASILE Digital
Plethysmometer (Italy) [23]. The paw volumes were recorded
on 0 day, 7th day, 14th day, 21st day and, 28th day
(each week up to 4 weeks).
Anti-Nociceptive Activity
The apparatus consists of a hot plate on which the rats
were placed for testing (Eddy’s Hot Plate Method). The apparatus
consists of a 20 cm diameter metal-hot plate surface
set at 50 °C, a plexiglass cage that fits the hot metal surface,
and a timer operated by stop watch. Pain threshold was determined
by the latency for nociceptive response (withdrawal
of any paw) with a maximum cut-off time 15 sec for all
groups on the last day of experiment [24]. All the extracts of
plant Aristolochia bracteata and standard drug (Indomethacin)
were administered prior to 1 hr of the test.
Xylene-induced Ear Edema in Mice
Overnight-starved Swiss albino mice were divided into
11 different groups of 6 each. The extracts under study were
administered orally 30 min prior to the application of xylene
(0.03 ml) to the anterior and posterior surfaces of the left ear
of the mice. The right ears of all the mices were remained
untreated, and control group was received only normal saline.
All the animals were sacrificed after the induction of
inflammation (after 2 hours) i.e. after xylene application,
both ears were removed. The circular sections of ears of the
treated and untreated animals were taken using 8 mm diameter
cork borer and weighed. The edematous response was
measured as weight difference between the two plugs and
the anti-inflammatory activity expressed as percentage of
edema reduction in treated mice with regard to the control
mice. All experiments were uniformly started between 11:00
and 14:00 h in order to avoid variations in the inflammatory
response due to circadian fluctuations in the levels of corticosteroids
[25, 26].
Histological Assessment
All the animals were sacrificed at the end of the experiments.
Left hind paws were removed of all the animals and
post fixed in formal saline (7 days) and then decalcified in
5% formic acid. Joints were then trimmed, embedded and
sectioned at 6 ìm. Sections were then stained with haematoxyline
and eosin. Histological study was carried out by 0,
infiltrate in skin and overlying tissues; 2, dense inflammatory
infiltrate or arthritis; 3, synovitis; 4, hyperplastic synovium,
inflammatory infiltrate in the joint; 5, arthritis with
destruction of catilage, pannus formation, using research
microscope [27, 28].
Radiographic evaluation was performed on the basis of
radiographs and coned down views of lower limbs. Radiographs
were taken with Siemens, Multiphos 10 (version 1.0)
dental X-ray machine [29].
Statistical Evaluation
A one-way analysis of variance with Dunnett’s comparisons
to control and unpaired Student‘t’ test were used to determine
statistical significance of the preclinical data collected
from behavioral observations. The data collected from
the clinical observations, hot plate study, and ear edema
study (mean ± SEM) were analysed statistically for differences
using the student t-test.
Health Status
The body weight in normal group animal rats remains
same during 4 weeks study. In, FCA injected group of animals,
body weight of animals was declining after 1st week of
study and significant loss (P<0.001) in weight was in 3rd and
4th week. Blood haemoglobin content was significantly
(P<0.001) declined to 8.667± 0.8 from 17.33± 0.76 and ESR
was significantly (P<0.01) increased to 3.15±4.113 from
13.17±1.35 in FCA injected group when compared to normal
on 29th day of study. Indomethacin treatment significantly
(P<0.05) restored loss in body weight on 3rd and 4th week
and decreased level of blood haemoglobin due to FCA injection.
As shown in Table 1, Treatment of FCA injected group
of animals with petroleum ether extract of A. bracteata
shown dose dependent effect in improvement of haemoglobin
level 11.83±0.79, 12.5±0.56 and 12.17±0.6, and maintain
ESR at 4.5±0.56, 3.8±0.36, and 4.8±0.48, it also significantly
restores loss in body weight on 4th week of study. MethanoAntiarthritis
Activity of Aristolochia Bracteata Extract The Open Natural Products Journal, 2009, Volume 2 9
lic extract treatment of arthritic rats significantly decreased
(P<0.01) ESR level 5.83±0.75, 6±1.13 and 4.5±0.56, also
improved body weight in 4th week study. Lower dose of
methanolic extract 100 mg/kg, significantly (P<0.01) increased
blood haemoglobin content to 12.33 ± 0.72, as compared
to control group. In this study more significant
(P<0.01) effect on Hb and ESR was shown by chloroform
extract treatment without significantly altering body weight
of animals throughout the study (Table 1).
FCA Induced Paw Edema
In mineral oil injected normal group rats, increase in paw
volume was observed but not significant. FCA injection in
tibiotarsal joint significantly (P<0.001) increases paw volume
from first week and almost same volume was maintained
throughout study. Significant (P<0.05, P<0.01, and
P<0.001) inhibition of FCA induced increased paw volume
was noted in indomethacin treated group of animals from 2nd
week to 4th week of study (1.408± 0.35, 1.04±0.29 and
0.8±0.25 respectively). Petroleum ether extract of A. bracteata
shown significant (P<0.05, P<0.01) inhibition on FCA
induced increase paw edema on 1st week of study and maintained
the effect significantly till the completion of study.
Chloroform extract shown more promising results in inhibiting
paw edema volume from 1st week (P<0.01), and maintained
significantly inhibitory effect (P<0.001) from 2nd
week of study. Similarly, methanolic extract if A. bracteata
at low dose (100 mg/kg) and moderate dose (200 mg/kg), but
results were varying at higher (400 mg/kg) dose of extract
(Table 2).
FCA Induced Thermal Hyperalgesia
No significant change in hot plate reaction time was
noted in FCA injected group of animals on 28th day of study
when compared to control group. Intra peritoneal indomethacin
treatment significantly (P<0.001) increases basal
reaction time (7.35±0.34 from 3.38±0.05) as compared to
control group. Petroleum ether extract treatment group has
no significant change in reaction to thermal stimuli, except
moderate dose (200 mg/kg), (P<0.05). More surprising results
were obtained when animals were treated with all doses
of chloroform extract (100, 200 and 400 mg/kg), it significantly
(P<0.01, P< 0.001) increases time for basal reaction
when compared to control group. Methanolic extract shown
more significant (P<0.01) results in lower doses than higher
dose (P<0.05) when compared to control group (Table 3).
Open Field Test
In non-treated group of rats, no significant change in behavior
was observed on day FCA injection. In 1st week significant
decrease in rearing and defection, 2nd week significant
decrease in rearing, grooming, defecation and ambulation,
in 4th week of study significant decrease in latency to
explore and decrease in rearing, grooming and ambulatory
behavior were noted. Indomethacin treatment shown its effect
in 2nd week of study by decreasing grooming in 3rd week
significantly increases number of defecation, and decrease
latency. However, in 4th week there was no change in behavior
except increased ambulation. The significant change in
exploratory behavior of animals treated with A. bracteata
extract was observed on 1st day of dose administration may
indicate effect of handling in behavior. No significant effect
on behavior was seen up to 7th day of observation. However,
in 2nd week significant effects were seen on latency to explore
and ambulation. Methanolic extract at 400 mg/kg,
chloroform extract at100 and 200 mg/kg significantly decreased
grooming behavior. In 3rd week of the study, petroleum
ether and methanolic extracts shown significant reduction
in latency time, increase in rearing, defecation and
Table 1. Effect of Aristolochia bracteata Extract on FCA Induced Change in the Haemoglobin (Hb) Level, Erythrocyte Sedimentation
Rate (ESR) and, Body Weight
Treatment and Dose ESR (mm/hr) Hb (gm %) Body Weight
0 week 1st week 2nd week 3rd week 4th week
Normal 13.17 ± 1.352 17.33 ± 0.76 250 ± 18.26 250 ± 18.26 250 ± 18.26 250 ± 18.26 250 ± 18.26
FCA+Saline 31.5 ± 4.1 ** 8.7 ± 0.8 *** 233.3 ± 21.08 233.3 ± 21.08 208.3 ± 15.37 191.7 ± 15.37* 158.3 ± 8.3 **
(10 mg/kg i.p.)
14.0 ± 2.696 11.67 ± 0.92 * 275 ± 33.54 233.3 ± 38.01 258.3 ± 27.13 291.7 ± 27.1 * 291.7 ± 27.1 **
CE, 100 mg/kg, p.o. 3.67 ± 0.49 ** 14.5 ± 1.5 * 200 ± 18.26 200 ± 18.26 200 ± 18.26 200 ± 18.26 200 ± 18.26
CE, 200 mg/kg, p.o. 5 ± 0.9661 ** 13.17 ± 0.65 ** 200 ± 18.26 200 ± 18.26 200 ± 18.26 191.7 ± 15.37 191.7 ± 15.37
CE, 400 mg/kg, p.o. 4.3 ± 0.49 ** 13.67 ± 0.9 ** 166.7 ± 10.5 * 166.7 ± 10.5 * 166.7 ± 10.54 166.7 ± 10.54 166.7 ± 10.54
PEE, 100 mg/kg, p.o. 4 ± 0.86 ** 11.83 ± 0.79 * 258.3 ± 27.13 258.3 ± 27.13 225 ± 17.08 225 ± 17.08 225 ± 17 **
PEE, 200 mg/kg, p.o. 3.8 ± 0.31 ** 12.5 ± 0.56 ** 258.3 ± 24 258.3 ± 23.86 225 ± 21.41 225 ± 21.41 225 ± 21.4 *
PEE, 400 mg/kg, p.o. 4.8 ± 0.48 ** 12.17 ± 0.6 ** 216.7 ± 21.08 216.7 ± 21.08 216.7 ± 21.08 225 ± 25.00 225 ± 25 *
ME, 100 mg/kg, p.o. 5.83 ± 0.75 ** 12.33 ± 0.72 ** 233.3 ± 38 233.3 ± 38.01 208.3 ± 27.13 208.3 ± 27.13 225 ± 21.41 *
ME, 200 mg/kg, p.o. 6 ± 1.13 ** 11.17 ± 0.87 283.3 ± 25 283.3 ± 24.72 283.3 ± 24.7 * 275 ± 21.41 * 258.3 ± 15 ***
ME, 400 mg/kg, p.o. 4.5 ± 0.56 ** 10.17 ± 0.65 225 ± 33.4 225 ± 33.54 208.3 ± 27.13 208.3 ± 27.13 225 ± 21.41*
Effect of Aristolochia bracteata extract on FCA induced change in ESR and Hb was studied on 29th day of study. Change in body weight of rats was recorded as weekly basis. Blood
was collected form rat’s retro orbital on 29th day, ESR and Hb were estimated by Westergern pipette and Sahli’s Haemometer. Data collected was analysed by student‘t’ test expressed
as mean ± SE. P value less than 0.05 was considered as significant. *P<0.05, **P<0.01, ***P<0.001.
10 The Open Natural Products Journal, 2009, Volume 2 Chitme and Patel
Table 2. Effect of Aristolochia bracteata Extract on FCA Induced Rat Paw Edema
Treatment and Dose Paw Edema (ml)
0 week 1st week 2nd week 3rd week 4th week
Normal 0.02 ± 0.005 0.037 ± 0.005 0.037 ± 0.005 0.038± 0.004 0.04 ± 0.006
FCA+Saline 0.01 ± 0.02 2.375 ± 0.164*** 2.355 ± 0.173*** 2.67 ± 0.145*** 2.597 ± 0.168***
(10 g/kg i.p.)
0.01 ± 0.02 2.715 ± 0.076 1.408 ± 0.348* 1.04 ± 0.29** 0.86 ± 0.249***
ME, 100 mg/kg, p. o. 0.02 ± 0.02 1.875 ± 0.099* 1.063 ± 0.15*** 1.14 ± 0.18*** 0.968 ± 0.18***
ME, 200 mg/kg, p.o. 0.01 ± 0.01 1.868 ± 0.286 0.575 ± 0.154*** 0.09 ± 0.077*** 0.025 ± 0.03***
ME, 400 mg/kg, p.o. 0.005 ± 0.006 2.278 ± 0.244 2.123 ± 0.235 1.457 ± 0.328* 1.45 ± 0.326*
EE, 100 mg/kg, p.o. 0.125 ± 0.053 1.447 ± 0.296 * 1.172 ± 0.261** 0.936 ± 0.15*** 0.746 ± 0.1***
EE, 200 mg/kg, p.o. 0.045 ± 0.055 1.205 ± 0.24** 0.9 ± 0.293** 0.7 ± 0.258*** 0.57 ± 0.28***
EE, 400 mg/kg, p.o. -0.003 ± 0.006 1.965 ± 0.78 0.88 ± 0.258** 0.59 ± 0.229*** 0.3 ± 0.11***
CE, 100 mg/kg, p.o. 0.006 ± 0.011 0.93 ± 0.25** 0.596 ± 0.19*** 0.635 ± 0.214*** 0.621 ± 0.177***
CE, 200 mg/kg, p.o. 0.02 ± 0.01 1.167 ± 0.267** 0.967 ± 0.307** 0.7 ± 0.2*** 0.558 ± 0.23***
CE, 400 mg/kg, p.o. 0.01 ± 0.007 1.533 ± 0.1** 0.858 ± 0.17*** 0.7 ± 0.145*** 0.596 ± 0.1***
Effect of Aristolochia bracteata in FCA induced increase in rat paw edema was studied every week by using UGO-BASILE Plethysmometer. Change in paw volume was calculated
from difference in FCA injected and saline injected paw volume. Data collected from the present study, are expressed as mean ± SE and analysed by student‘t’ test. FCA injected
group was compared with mineral oil injected group and extract and indomethacin treated group was compared with FCA injected group for coming to conclusion. P value less than
0.05 was considered as significant. *P<0.05, **P<0.01, ***P<0.001.
Table 3. Effect of Aristolochia bracteata Extract on FCA Induced Thermal Hyperalgesia (Sec) Using Eddy’s Hot Plate
Extract and Dose of Extract (mg/kg, p.o.)
Ether Extract Chloroform Extract Methanolic Extract
oil + 5%
(FCA +
5% span)
10mg/kg i.p.)
100 200 400 100 200 400 100 200 400
1 3.4 3.4 8.4 2.6 3.3 7.5 5 6 5 4.0 5 7.3
2 3.4 3.2 8.7 3.9 2.6 9.5 6 6.5 5.6 6.5 5.6 4
3 3.5 3.6 8 3.8 3.5 3.5 3.8 6.2 5.9 6 4.3 5
4 3.3 3.4 7.5 4.1 2.9 2.7 5 5.7 5.5 4.4 7.2 5.5
5 2.9 3.4 7.6 5.1 2.6 5.5 5.6 5.4 4.2 4.9 5.8 5.9
6 2.6 3.3 6.3 5.2 2.9 3.6 5.3 5.5 5.6 7 4 4
Mean 3.18 3.38 7.75 *** 4.12 2.97 * 5.38 5.12 ** 5.88 *** 5.3 *** 5.47 ** 5.32 ** 5.28 *
S.D. 0.35 0.13 0.84 0.96 0.37 2.66 0.75 0.43 0.61 1.21 1.16 1.25
SEM 0.14 0.05 0.34 0.39 0.15 1.09 0.31 0.17 0.25 0.49 0.47 0.51
t-value -------- 1.29 12.5 1.86 2.61 1.84 5.58 13.72 7.48 4.19 4.0 1 3.69
Effect of Aristolochia bracteata extract on FCA induced thermal hyperalgesia was studied by using Eddy’s Hot Plate. Basal reaction time (sec) was recorded after placing the rat in
plexiglass covered hot plate at 50 C, cut off time was 15 seconds. Results obtained are expressed as mean ± SE and analysed by student‘t’ test. FCA injected group was compared
with mineral oil injected group and extract and indomethacin treated group was compared with FCA injected group for coming to conclusion. P value less than 0.05 was considered as
significant. *P<0.05, **P<0.01, ***P<0.001.
ambulation. In 4th week, significant increase in ambulation
behavior was observed with all doses of methanolic extract,
petroleum ether (400 mg/kg), and chloroform extract (400
mg/kg). Methanolic extract (200 and 400 mg/kg) and petroleum
ether (100 and 400 mg/kg), have shown significant
effect on rearing behavior.
Xylene-Induced Ear Edema
Application of xylene to anterior and posterior surface
significantly (P<0.05) increased ear edema weight up to
5.18±1.14 from 1± 0.1. xylene-induced ear edema was significantly
(P<0.05) inhibited by indomethacin by intraperitoAntiarthritis
Activity of Aristolochia Bracteata Extract The Open Natural Products Journal, 2009, Volume 2 11
neal treatment to 1.57 ± 0.33. All doses of petroleum ether
and methanolic extract significantly (P<0.05) inhibited xylene-
induced ear edema. However the inhibitory effect of
chloroform extract was only observed at high dose (400
mg/kg) but not at low doses 100 and 200 mg/kg (Table 4).
In FCA treated group of rats eosinophill infiltration,
synovitis and damage of cartilage was evident. Treatment
with indomethacin significantly inhibited infiltration of inflammatory
cells and synovitis and also maintains integrity
of cartilage tissues. And treatment with petroleum ether and
chloroform extract of A. bracteata successfully inhibited all
these inflammatory processes. However, methanolic extract
treatment at lower dose and moderate dose inhibited synovitis
and pannus formation but not inflammatory cells infiltration.
High dose (400 mg/kg) of methanolic extract has no
effect on any aspects of FCA induced arthritic reactions
(Figs. 1-12).
Fig. (1). Effect of Span 80 (5%) on mineral oil induced rats knee
joint histopathology (H & E, X 100). Normal joint arachitecure.
Fig. (2). Effect of Span 80 (5%) on FCA induced monoarthritic rats
knee joint histopathology (H & E, X 100). Joint cartilage destruction
and high inflammatory cellular infiltration.
Fig. (3). Effect of Indomethacin (10 mg/kg i.p.) on FCA induced
monoarthritic rats knee joint histopathology (H & E, X 100). Joint
cartilage protection and low inflammatory cellular infiltration.
Table 4. Effect of Aristolochia bracteata Extract on Xylene-Induced Ear Edema (mg) in Mice
Extract and Dose of Extract (mg/kg, p.o.)
Ether Extract Chloroform Extract Methanolic Extract
Animal No. Normal
(mineral oil +
5% span)
(FCA +
5% span)
Standard (Indomethacin,
10mg/kg i.p.)
100 200 400 100 200 400 100 200 400
1 0.8 4.4 0.7 0.6 0.7 0.6 1.9 2.3 1.9 0.7 1.7 1.9
2 0.7 6.6 1 0.4 0.9 0.9 1.7 2.2 1.2 4.1 1.3 0.4
3 1.2 9.8 1.8 3 3 0.7 3.3 2.7 1.2 2 3 1.6
4 1.3 4.3 1.3 1.3 1.2 1.3 4.2 2.9 0.9 1.8 0.1 0.6
5 1.2 1.5 1.6 1.6 0.9 0.9 2.9 2.4 1 1.9 0.4 1.4
6 0.9 4.5 3 2.9 0.8 0.6 2.3 3.1 1 0.7 2.1 3
Mean 1 5.18 * 1.57 * 1.63 * 1.25 * 0.83 2.72 2.6 1.2 * 1.87 * 1.43 * 1.48 *
S.D. 0.25 2.78 0.8 1.11 0.87 0.27 0.94 0.36 0.36 1.24 1.08 0.94
SEM 0.1 1.14 0.33 0.45 0.36 0.11 0.38 0.15 0.15 0.51 0.44 0.38
t-value ------- 3.65 3.06 2.9 3.3 3.8 2.06 2.25 3.47 2.66 3.08 3.08
Effect of Aristolochia bracteata extract on xylene-induced ear edema was studied as a part of illustration of mechanism of action. Extract and drug were administered 1 hr prior to
the topical application of xylene, both ear were separated and bored with 8 mm diameter borer. The difference in weight of ear are represented as mean ± SE and analysed by student‘
t’ test for coming to conclusion. P value less than 0.05 was considered as significant. *P<0.05, **P<0.01, ***P<0.001.
12 The Open Natural Products Journal, 2009, Volume 2 Chitme and Patel
Fig. (4). Effect of Petroleum ether extract (100 mg/kg p.o.) on FCA
induced monoarthritic rats knee joint histopathology (H & E, X
100). Low cartilage destruction and high inflammatory cellular
Fig. (5). Effect of Petroleum ether extract (200 mg/kg p.o.) on FCA
induced monoarthritic rats knee joint histopathology (H & E, X
100). Low cartilage destruction and low inflammatory cellular infiltration.
Fig. (6). Effect of Petroleum ether extract (400 mg/kg p.o.) on FCA
induced monoarthritic rats knee joint histopathology (H & E, X
100). Very low cartilage destruction and low inflammatory cellular
FCA injected group of animals shown deformation and
abnormality in toes. Treatment with petroleum ether and
chloroform extract of A. bracteata restored normal architecture,
whereas methanolic extract fails to maintain integrity of
Fig. (7). Effect of Chloroform extract (100 mg/kg p.o.) on FCA
induced monoarthritic rats knee joint histopathology (H & E, X
100). Low cartilage destruction and high inflammatory cellular
Fig. (8). Effect of Chloroform extract (200 mg/kg p.o.) on FCA
induced monoarthritic rats knee joint histopathology (H & E, X
100). No cartilage destruction and low inflammatory cellular infiltration.
Fig. (9). Effect of Chloroform extract (400 mg/kg p.o.) on FCA
induced monoarthritic rats knee joint histopathology (H & E, X
100). No cartilage destruction and no inflammatory cellular infiltration.
The Freund’s complete adjuvant (FCA) induced arthritis
model in rats is the most common model used by several
Antiarthritis Activity of Aristolochia Bracteata Extract The Open Natural Products Journal, 2009, Volume 2 13
scientists to evaluate potential anti-arthritic agents. This preclinical
model predicted the activities of a number of compounds
that are currently used in the treatment of rheumatoid
arthritis are being tested in clinical trials. Recently, Baumgartner
et al. distinguished 4 phases of arthritis on the basis
of biochemical markers of arthritis [30] and different from
clinical phases described by Houssay et al. (i) Day 1-4, ,
with acute local inflammation and systemic effects (liver);
(2) Days 7-12, with remission of acute inflammation and
periarthritis ; (3) Days 12-28, with chronic inflammation,
periarthritis and osteogenic activity; (4) Day 35 onwards
(indefinitely), with permanent articular deformity and minimal
(burn-out) inflammation [31]. A general increase in 5-
HT synthesis within the whole central nervous system during
the acute phase of the disease (2-3 weeks postinoculation)
with a specific, further enhancement restricted to the spinal
cord during the post acute phase (4-6 weeks postinoculation)
The results obtained in our study and indomethacin is
similar to the results as predicted by earlier studies [33-35].
In various animal models to evaluate the effect of compounds
upon the development of adjuvant induced arthritis.
Body weight was considered as an indirect index of health
status and recovery from disease [36-38]. In our study we
followed US FDA guidelines on preclinical evaluation and
considered ESR, Hb and body weight as an indirect index in
restoration of health [14]. A dramatic cessation of growth
and decline in body weight was indicated in control group of
animals from first week of study similar to earlier study.
Significant restoration and gain in body weight was evident,
when treated with indomethacin as shown by previous study.
It also improved blood Hb level without of significantly affecting
ESR. All doses of petroleum ether, chloroform and
methanolic extracts significantly restored ESR, blood Hb
content and body weight change in 4th week of study. However,
more promising results were obtained with chloroform
extract indicating more efficacies in recovering from FCA
induced arthritis. The results support the involvement of antioxidant
and anti-anaemic properties in maintenance [39].
FCA induced arthritis have been used as a model of subchronic
or chronic inflammation in rats and of considerable
relevance for the study of pathophysiology and pharmacological
control of inflammatory processes. In our study, the
monoarthritis was very stable in inflammatory signs. The
initial inflammatory response was developed within few
hours, but more critical clinical signs were seen on 1st week
of postinoculation and there after for several week. Previous
studies demonstrate that A. bracteata appears to be efficient
in acute and sub-acute inflammatory processes [22, 40, 41].
Our study on mice ear edema supports the above study as all
doses and extract of A. bracteata inhibited xylene-induced
ear edema, which has been used as an inflammation model
with leukotriene inhibition. The tibiotarsal joint and paw
volume was significantly increased in 1st week, and maintained
throughout 4th week of study, but shown some decline
in paw volume on last day. Effect of indomethacin was seen
from 2nd week of treatment, whereas A. bracteata extracts
shown its onset from 1st week of study and more significant
effects were seen in petroleum ether and chloroform extracts
than indomethacin, probably through the same mechanism as
indomethacin by inhibition of phospholipase A2 and increased
vascular permeability followed by excess infiltration
of cytokines and leukotriene at the inflamed sites possibly by
its active constituent aristolochic acid present in the extracts
[39]. These results are also supported by our histomarphology
Fig. (10). Effect of Methanolic extract (100 mg/kg p.o.) on FCA
induced monoarthritic rats knee joint histopathology (H & E, X
100). High cartilage destruction and absence of inflammatory cellular
Fig. (11). Effect of Methanolic extract (200 mg/kg p.o.) on FCA
induced monoarthritic rats knee joint histopathology (H & E, X
100). Low cartilage destruction and low of inflammatory cellular
Fig. (12). Effect of Methanolic extract (400 mg/kg p.o.) on FCA
induced monoarthritic rats knee joint histopathology (H & E, X
100). No change in cartilage architecture and low of inflammatory
cellular infiltration.
14 The Open Natural Products Journal, 2009, Volume 2 Chitme and Patel
Using arthritic rats as a model of chronic pain, several
studies evaluated hyperalgesia in different ways. It has been
stated that the method of nociceptive thermal stimulus, such
as the hot plate provides a quantitative measurement of hyperalgesia
related to behaviors [22, 24, 36]. The alteration in
response to an acute pain stimulus in the non- affected limb
probably reflects involvement of inhibitory controls caused
by obvious long-standing nociceptive input from the contra
lateral arthritic limb. An earlier study shows that reduction of
the latency for the animal’s reaction correspondingly augmented
sensitivity to pain [24]. In our study thermal hyperalgesia
was tested on the last day to avoid tissue lesion.
Similar to earlier reports indomethacin significantly increased
latency time to thermal stimulus. A. bracteata also
shown significant increase in basal reaction time, and chloroform
extract produced more prominent effects (P<0.001) on
thermal hyperalgesia similar to indomethacin, indicating its
similar analgesic property. Hence, arthritic pain is associated
with increased level of 5-HT in central nervous system and
subsequently in spinal cord [42]. The A. bracteata may be
acting on these sites and possibly abolishing spinal reflex
and producing analgesic effects. However, more studies are
required in this line to establish.
With respect to earlier studies on arthritic rat’s exploratory
behavior and stress, we observed reduced ambulatory
movement, rearing, grooming, and increased itching,
scratching, defecation and urination supporting the previous
studies on behavior of arthritic rats [19, 22, 36, 42]. In this
group of rats we observe an attempt of protection of the affected
paw, as evidenced by causing an elevation, as well as
avoidance to support its own weight. Indomethacin decreased
latency to explore and rearing increased ambulatory
movements behaviors supporting its analgesic property.
However it fails in overcoming from aggression, stress and
irritability as evidenced in defecation, urination, scratching,
and grooming behaviors.
The change in behavior of rats in 1st day of treatment by
the extracts may be due to mishandling of animals while
administration. Treatments of animals with A. bracteata
have shown no significant effect on exploratory behavior of
arthritic rats indicating need of long-term treatment in overcoming
from clinical signs. In 2nd week, all type of extracts
significantly decrease grooming behavior indicating the efficiency
of these extracts in overcoming from arthritic discomfort
including itching, irritation, and scratching, also showing
their onset of action.
The methanolic extract of A. bracteata shown significant
increase in latency to explore, rearing, and ambulation, with
significantly increase in defecation. Possibly this action may
be mediated by flavanoids present in this extract improving
weight bearing capacity and feeling of wellness. Petroleum
ether extract significantly decrease total latency time to explore
and increase number of defecations indicating failure
of this extract in overcoming from stress and intension
movement associated with healing of arthritis as evidence in
other parameters of this study. In 4th week methanolic extract
has shown significant increase weight bearing threshold and
ambulation may be associated with improvement in health
status supported by our study of Hb, ESR, and body weight.
In order to characterize the severity of disease more accurately,
a quantification of lower body radiograph was taken
of two animals from each group. In control group abnormality
and deformation were observed but have no clinical significance.
Treatment with indomethacin, petroleum ether and
chloroform extract of A. bracteata restored the abnormalities
seen in control group. But, methanolic extract failed to produce
inhibitory effect, supporting efficacy and potency of
chloroform extract use in the treatment of arthritis.
On the basis of the results obtained in this study we conclude,
and propose that possibly, the potent anti-arthritic
effect of Aristolochia btracteata chloroform extract may be
through maintenance of synovial membrane and vascular
permeability, thereby inhibiting cytokines and leukotriene
infiltration inhibition as evidenced in paw edema volume and
xylene-induced ear edema. In turn, protecting synovial
membrane and destruction of cartilage and improving health
status through antioxidant and haematonic properties. The
similarity in the extract and indomethacin may propose the
inhibitory effect on phospholipase A2 and prostaglandin.
Eddy’s hot plate test indicates its possible analgesic effect
may be mediated through central and spinal serotonergic
neurons inhibitory effect. The results obtained in the present
study indicate that Aristolochia bracteata is having a potent
anti-arthritic property. This study also demonstrates not only
its ability in overcoming arthritis and its complications but
also clinical signs as evidenced in paw edema, thermal hyperalgesia
and histomarphological examinations. Improvement
in health parameters consider in this study including
HB, ESR, and body weight indicating its beneficial effects
while recovery from arthritis. The open field test considered
for the present study to evaluate effect of A. bracteata on
behavior of animals and clinical symptoms while long term
treatment for arthritic condition have shown in appreciable
results by impairing intension for movement, weight bearing
capacity, lack of irritation and stress and increase in threshold
of pain in overcoming from the disease and its symptoms.
However, further fractionation and isolation of chloroform
extract is required to observe safety, efficacy and potency
of Aristolochia btracteata against arthritis.
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Received: October 10, 2008 Revised: January 02, 2009 Accepted: January 17, 2009
© Chitme and Patel; Licensee Bentham Open.
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