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

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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