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
Animals
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
changes.
Keywords:
Anti-arthritis, arthritis, Aristolochia bracteata, Freund’s
complete adjuvant.
INTRODUCTION
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: hrchitme@rediffmail.com
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,
12].
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].
MATERIALS
AND METHODS
Animals
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.
PLANT
EXTRACT PREPARATION
Collection
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.
Extraction
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.
Materials
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].
Radiography
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.
RESULTS
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 **
Indomethacin
(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***
Indomethacin
(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
Animal
No.
Normal
(mineral
oil
+ 5%
span)
Control
(FCA
+
5%
span)
Standard
(Indomethacin,
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).
Histomorphology
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)
Control
(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
infiltration.
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
infiltration.
Radiography
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
joints.
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
infiltration.
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.
DISCUSSION
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)
[22,32].
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
studies.
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
infiltration.
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
infiltration.
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.
CONCLUSION
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
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