RESEARCH ARTICLE

Experimental Investigation of Anti-rheumatoid Activity of

Ailanthus excelsa in Adjuvant-induced Arthritic Rats
Smita Pillewan1*, Neeraj Vyawahare1, Tina Saldanha1, Sujata Taware1, Neha Piarchand1

Abstracts: The present work was performed to evaluate the effects of aqueous stem bark extract of Ailanthus excelsa (AESE) for
anti-rheumatoid activity. Three doses of AESE (100, 200 & 400 mg/kg orally) were selected. Arthritis was induced by injecting
0.1 ml (0.1 % w/v) of complete Freund’s adjuvant in subplanter region of left hind paw of Wistar albino rats. Different
paramaters like paw edema (inflammation), body weight, ankle joint diameter, mechanical hyperalgesia, hematological studies,
biochemical studies, spleen weight, radiological and histological analysis of bone damage were assessed in Freund’s adjuvant
induced paw inflammatory model. Methotrexate (0.75 mg/kg, p.o) was the standard reference drug. Paw volume changes were
estimated using plethysmometer and Randall-selieto apparatus used to determine the mechanical hyperalgesia. All dose levels
of Ailanthus excelsa aqueous stem bark extract (AESE) showed significant and dose-dependent anti-rheumaroid effects
compared to arthritic control group. Altogether these results suggest that the aqueous stem bark extract of Ailanthus excelsa
could be considered as a potent anti-rheumatoid agent.

INTRODUCTION investigate the rationality of their use in modern scientific

Rheumatoid arthritis is a type of chronic, progressive,
systemic, inflammatory disorder of unknown etiology
causing persistent synovitis, pain, joint destruction, and
functional disability. Hence it leads to significant morbidity
and increased mortality. Rheumatoid arthritis is the 31st
leading cause of YLD (Years lived with disability) at global
level, accounting for 0.8 % of total global YLD’s as per
World Health Report, 2002. [1] The life expectancy
decreases by about 3-10 years. The morbidity and
mortality caused by Rheumatoid Arthritis (R.A) has a
substantial socioeconomic impact. [2] Approximately 1% of
world population is suffering from RA and it is 2.5 times
more common in women. [3] R. A therapy involves use of
non-steroidal anti-inflammatory drugs (NSAIDS),
Immunomodulatory agents, disease modifying anti-
rheumatic drugs (DMARDS) and Biological therapies. These
well established treatments have certain serious
drawbacks like lack of efficacy in all cases, excessive side
effects and high cost. Treatment for Arthritis is mostly a
lifetime process and hence above mentioned drawbacks
need to be addressed. Some side effects are bone marrow
suppression, cardiovascular complications, hepatotoxicity,
renal impairment, etc.
Though a large number of new drugs and therapies
have been developed over the past few decades, even
today, no ideal drug treatment is available to completely
cure or check the progress of this disease. Hence, many of
arthritic patients commonly prefer complementary and
alternative medicines [4] which emphasize the need of a
cost effective drug with minimal side effects.
Herbals are used in the art of healing since the time
immemorial. According to an estimate of WHO nearly 80
% of the population of developing countries rely on
traditional medicine, mostly plant drugs for their primary
health care needs. [5] The belief that herbal medicines are
more easily available, affordable and have less side
effects, promote its use. Hence, it is essential to
1AISSMS College of Pharmacy, Near RTO, Kennedy Road, Pune-01,
Maharastra, India.
E-mail: smita_25us@yahoo.com
*Corresponding author
terms.
Ailanthus excelsa Roxb. a plant used in the Indian
system of medicine have great importance for treating
different ailments. In the indigenous system of medicine
the bark of A. excelsa is used as febrifuge, anthelmintic,
appetizer, in conditions of diarrhoea and dysentery. [6]
Infusion of the bark is used as bitter tonic, carminative,
expectorant, antispasmodic, especially useful in bronchitis,
dyspepsia and asthma. Also infusion of bark and leaves are
reputed as tonic in debility after child birth. [7]
Ailanthus excelsa is a large deciduous fast growing
tree, 18-25 m tall, belonging to Simourbeaceae family. It is
also called as tree of heaven. It is indigenous to central
and southern India and Srilanka. The presences of
quassinoids, alkaloids, terpenoids, glycosides and
caumarin have been reported [8, 9, 10] in the stem bark of
tree. Many of the medicinal properties are demonstrated
for extracts of Ailanthus excelsa bark like antifertility, [11]
Antifungal, [12] Bronchodilatory activity, [13]
Antiplasmodial, [14] Gastro protective and anti secretary,
[15] methanolic extract for analgesic activity, [16] petroleum
ether, ethyl acetate and methanolic extract for antifungal,
anti-inflammatory and antinociceptive activity. [17] The
decoction of stem bark of A. excelsa is used in folklore
medicinal system for anti- arthritic activity by tribals. [18]
Hence, it has been decided to investigate the anti-
rheumatoid activity of the Ailanthus excelsa. Traditionally,
infusion of bark is used widely for the treatment of
different ailments. Infusions are mostly the aqueous
extract of the plant. Hence we have selected the aqueous
extract of Ailanthus excelsa stem bark to evaluate its anti-
rheumatoid potency.
MATERIALS AND METHODS
Plant Extract
Ailanthus excelsa aqueous extract was obtained as a gift
sample from Garlico Herbal, Indore, India.
Animals
Swiss albino mice weighing 25-30 gm and Wistar rats
weighing 250–300 gm of either sex were used for the

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Table 1: Percent Inhibition of Injected Hind Paws Edema by AESE in Freund’s Adjuvant Arthritic Model in Rats

Percent Inhibition of Hind Paw Edema by AESE

Duration Control AESE 100 AESE 200 AESE 400 Methotrexate
0th Day 0.00 0.59 0.89 0.74 1.19
4th Day 0.00 0.91 0.55 -1.37 -1.28
7th Day 0.00 0.82 -0.64 -0.36 -1.00
14th Day 0.00 18.14 11.52 15.08 19.42
21st Day 0.00 37.36 34.47 35.09 42.12
28th Day 0.00 33.54 27.09 29.70 36.08
Values are expressed as mean; n=6. AESE – Ailanthus excelsa stem bark extract

Table 2: Effect of AESE on Haematological Parameters

Haematological Parameters
Treatments (mg/kg)
WBC Count (1000/cu. mm) RBC Count (million/cu.mm) Hb (gm%) ESR (mm/hr)
Normal control 7.64 ± 0.44 8.45 ± 0.45 15.13 ± 0.48 2.73 ± 0.35
Arthritic control 14.67 ± 0.66 6.07 ± 0.43 10.55 ± 0.68 5.98 ± 0.62
Methotrexate 7.76 ± 0.72** 8.39 ± 0.39** 15.24 ±0.54** 3.15 ± 0.52**
AESE 100 12.55 ± 0.93 7.62 ± 0.19* 14.45 ± 0.53** 3.88 ± 0.53*
AESE 200 12.11 ± 0.79 7.57 ± 0.23* 13.32 ± 0.76* 4.40 ± 0.42
AESE 400 8.66 ± 0.79** 8.34 ± 0.39** 14.07 ± 0.77** 2.92 ± 0.44**
Values are expressed as mean ± SEM, n=6 in each group; *P<0.05 and **P<0.01 (ANOVA followed by dunnett’s ‘t’ test). All treatment groups compared to
arthritic control. AESE – Ailanthus excelsa stem bark extract

Table 3: Effect of AESE on Biochemical Parameters

Biochemical parameters (Mean)

Treatments (mg/kg)
Total Protein (IU/L) Total Albumin (IU/L) Total Globulin (IU/L)
Normal Control 7.22 ± 0.28 4.22 ± 0.31 2.99 ± 0.46
Vehicle Control 5.46 ± 0.50 2.52 ± 0.27 6.75 ± 0.42
Methotrexate 8.17 ± 0.45** 4.10 ± 0.20** 4.06 ± 0.56**
AESE 100 6.87 ± 0.51 3.28 ± 0.23 3.69 ± 0.60**
AESE 200 8.23 ± 0.56** 3.66 ± 0.19** 4.57 ± 0.56*
AESE 400 6.63 ± 0.41 3.37 ± 0.20 3.15 ± 0.45**
Values are expressed as Mean ± SEM, n=6 in each group; * P<0.05 and **P<0.01 (ANOVA followed by dunnett’s ‘t’ test). All treatment groups compared
to arthritic control. AESE – Ailanthus excelsa stem bark extract

Table 4: Effect of AESE on SGOT, SGPT and ALP levels

Drug Treatment (mg/kg) SGOT (IU/L) SGPT (IU/L) ALP (IU/L)

Normal Control 134.00 ± 2.44 38.67 ± 2.12 280.43 ± 36.84
Arthritic Control 291.67 ± 3.10 79.33 ± 3.93 686.38 ± 0.71
Methotrexate 231.67 ± 6.76** 49.5 ± 3.34** 359.13± 0.28**
AESE 100 274.00 ± 6.34 70.00 ± 4.82 529.0 ± 0.99
AESE 200 234.50 ± 5.20** 48.0 ± 2.48** 487.15±0.57*
AESE 400 274.33 ± 5.57 62.67 ± 4.92* 485.0 ± 0.63*
Values are expressed as Mean ± SEM, n=6 in each group; * P<0.05 and **P<0.01 (ANOVA followed by dunnett’s ‘t’ test). All treatment groups compared
to arthritic control. AESE – Ailanthus excelsa stem bark extract

Table 5: Effect of AESE on Tissues Weight Change

Drug Treatment (mg/kg) Spleen Weight (g)

Normal Control 0.63 ± 0.02
Arthritic Control 1.02 ± 0.05
Methotrexate 0.64 ± 0.03**
AESE 100 0.75 ± 0.03**
AESE 200 0.84 ± 0.05*
AESE 400 0.75 ± 0.03**

study. Animals were procured from the animal house of
AISSMS College of Pharmacy, Pune and kept under
standard laboratory condition of temperature (25 ± 2°C)
and humidity (55 ± 5 %) under 12:12 light dark cycle. The
animals were fed with standard pellet diet and drinking
water ad libitum. The experiments were designed and
conducted in accordance with ethical norms approved by
Committee for the Purpose of Control and Supervision on
Experiments on Animals (CPSCEA) and Institutional Animal
Ethical Committee (IAEC).

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 RESEARCH ARTICLE

Table 6: Graded Scores of the Joints of Hind Limb of Various Treatment Groups

Necrosis of Bones – Periosteum Adjacent Tissue Reaction

Group Joint Space Narrowing
erosions Inflammation Edema and Inflammation
Normal 00 00 00 00
Arthritic +++ +++ ++++ ++++
AESE100 ++++ +++ +++ +++
AESE200 +++ +++ ++ +++
AESE400 ++ ++ ++ ++
Methotrexate ++ ++ + ++
0: no abnormality detected, +: damage/ active changes up to less than 25 %, ++: damage/ active changes up to less than 50 %, +++: damage/ active
changes up to less 75 %, ++++: damage/ active changes up to more than 75 %

Table 7: Graded Scores of the Hind Paw Histopathology of Various Treatment Groups

Joint Space Infiltration of Connective Tissue Periosteal

Group Degree of Erosion
Narrowing Cells Proliferation Thickening
Normal 00 00 00 00 00
Arthritic +++ ++++ ++++ +++ +++
AESE100 +++ +++ ++ +++ ++
AESE200 ++ ++ ++ ++ ++
AESE400 + ++ + ++ +
Methotrexate ++ ++ + ++ +++
0: no abnormality detected , +: damage changes up to more than 25 %, ++: damage changes up to more than 50 %, +++: damage changes up to more than
75 %, ++++: damage changes up to more than 75 %

Drugs and Chemicals Group VI served as normal control 1 % (w/v) of CMC in

x Ailanthus excelsa aqueous extract was obtained as a gift
sample from Garlico Herbal, Indore, India.
x Complete Freunds adjuvant: F5881 (10ml) lot Sigma
Aldrich, USA.
x Methotrexate Tabs: Mext 7.5, Wallace Pharmaceutical
Ltd, Mumbai. Batch No. MHG1101 was purchased from
local market.
double distilled water p.o] with no arthritic induction.
Arthritis was induced in all groups except normal control
by injecting 0.1 ml (0.1 % w/v) Complete Freund’s adjuvant
(which is a suspension of killed Mycobacterium
tuberculosis bacteria homogenized in liquid paraffin) into
the left hind paw. Treatment was started from 14th day of
induction of arthritis up to 28th day.

Phytochemical Screening Assessment of Arthritis

Phytochemical screening of the methanolic root extract of
Ailanthus excelsa was carried out using standard
procedures.
Acute Toxicity Study
Acute oral toxicity assay was performed in healthy
nulliparous and non-pregnant adult female albino Swiss
mice (25–30 gm) as per the OECD guidelines-423 (OECD,
2001). 2 groups of three Swiss albino mice were treated
with Ailanthus excelsa aqueous stem bark extract 2000
mg/kg, orally. The mice were continuously observed for
first 5 hours for any gross behavioral, neurological or
autonomic toxic effect and then periodically up to 24 h for
any toxic symptoms and mortality. Further, for next 14
days animals were kept under supervision for any signs of
delayed toxicity.
Complete Freund’s Adjuvant Induced Arthritis in Rats
[19, 20]
Animals were randomly divided into 6 groups of 6 animals
each (n=6). Group II served as arthritic control [1% (w/v)
of CMC in double distilled water p.o] with induced arthritis.
Group II-IV served as test groups of AESE [100, 200 and
400 mg/kg p.o in double distilled water containing carboxy
methyl cellulose (1 %, w/v) respectively]. Group V was
given reference standard (Methotrexate 0.75 mg/kg p.o.)
x Paw Volume: [21] The paw volumes of injected and non-
injected paws, were measured using a digital
plethysmometer (Panlab, Italy), after which adjuvant
was administered. Thereafter, paw volumes were
measured again on the 0th, 4th, 7th, 14th, 21st and 28th
days. The hind paws of rats were dipped upto ankle
joint. The mean changes in injected paw edema with
respect to initial paw volume, were calculated on
respective days and % inhibition of paw edema with
respect to arthritic control group was calculated using
following formula
Percent Inhibition = [(C– T) / C] ൈ 100
Where, ‘T’ represents increase in edema volume of drug
treated animal’s paw and ‘C’ represents increase in edema
volume of arthritic control animals.
x Determination of Body Weight (in gm) & Ankle Joint
Diameter (in inches): [21] Rats were placed
individually in the 0.1 g precision balance (Gill real
weight, India) on 0th, 4th, 7th, 14th, 21st and 28th day
respectively and body weights were noted down. The
ankle joint diameters of left hind paw were measured
using a digital vernier caliper on the above-mentioned
testing days after induction of arthritis.
x Mechanical Hyperalgesia: [22] Mechanical hyperalgesia
of left hind paw was evaluated by Randall-Selitto paw

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 RESEARCH ARTICLE

3.50

3.00
Paw edema volume (ml)
2.50 0th
*
** ** ** ** ** 4th
2.00 **
** ** **
** ** 7th
1.50
14th
1.00 21st

0.50 28th

0.00
Arth Ctr AESE100 AESE200 AESE400 MTX0.75 Norm Ctr
Treatment (mg/kg)
Figure1: Effect of AESE on volume of injected paw. The mean paw edema counts were expressed as Mean ± SEM, n=6 in each
group; *P<0.05 and **P<0.01 (ANOVA followed by Dunnett’s ‘t’ test). All treatment groups compared to arthritic control. Arth Ctr –
Arthritic control, AESE – Ailanthus excelsa Stem Bark Extract, Norm Ctr – Normal control

1.6

1.4
* *
Paw edema volume(ml)

1.2 *
** **
* **
1 * 4th
**
0.8 7th

0.6 14th
21st
0.4
28th
0.2

0
Arth Ctr AESE100 AESE200 AESE400 MTX0.75 Norm Ctr

Treatment (mg/kg)
Figure 2: Effect of AESE on volume of Non–injected paw. Values were expressed as Mean ± SEM, n=6 in each group; *P<0.05 and
**P<0.01 (ANOVA followed by Dunnett’s ‘t’ test). All treatment groups compared to arthritic control. Arth Ctr – Arthritic control,
AESE – Ailanthus excelsa Stem Bark Extract, Norm Ctr – Normal control

15.0 13.1

10.0 7.8
Change in Body weight

5.2
5.0 3.2
1.5
0.0
ArthCtr AESE100 AESE200 AESE400 MTX0.75 NormCtr
-5.0

-10.0

-15.0

-20.0 -18.2
Treatment mg/kg
Figure 3: Effect of AESE on change in body weight. The mean paw volume (ml) were expressed as Mean ± SEM, n=6 in each group;
*P<0.05 and **P<0.01 (ANOVA followed by Dunnett’s ‘t’ test). All treatment groups compared to arthritic control. Arth Ctr –
Arthritic control, AESE – Ailanthus excelsa Stem Bark Extract, Norm Ctr – Normal control

withdrawal test using analgesiometer (UGO Basile, x Biochemical Analysis: [23] On 28th day, blood was
Italy) on 0th, 4th, 7th, 14th, 21st and 28th. The cut-off withdrawn by retro-orbital puncture and serum was
pressure was 450 gm to avoid injury. separated which was used for estimation of total

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 RESEARCH ARTICLE

0.500
0.450
Ankle joint Diameter (inches)

0.400
*
0.350 ** ** ** 0th
0.300 ** **
** ** ** 4th
0.250
** 7th
0.200
14th
0.150
0.100 21th

0.050 28th
0.000
Arth Ctr AESE 100 AESE 200 AESE 400 MTX 0.75 Norm Ctr

Treatment (mg/kg)
Figure 4: Effect of AESE on ankle joint diameter. The mean of joint diameter were expressed as Mean ± SEM, n=6 in each group;
*P<0.05 and **P<0.01 (ANOVA followed by Dunnett’s ‘t’ test). All treatment groups compared to arthritic control. Arth Ctr –
Arthritic control, AESE – Ailanthus excelsa Stem Bark Extract, Norm Ctr – Normal control

140.0
0th
Mechanical Withdrawal Pressure

120.0 **
** 4th
** ** **
100.0 ** ** **
7th
80.0 *
60.0 14th

40.0 21th

20.0 28th
0.0
Arth Ctr AESE 100 AESE 200 AESE 400 MTX 0.75 Norm Ctr

Treatment mg/kg

Figure 5: Effect of AESE on mechanical withdrawal threshold. Values are expressed as Mean ± SEM, n=6 in each group; *P<0.05
and **P<0.01 (ANOVA followed by Dunnett’s ‘t’ test). All treatment groups compared to arthritic control. Arth Ctr – Arthritic
control, AESE – Ailanthus excelsa Stem Bark Extract, Norm Ctr – Normal control

protein, albumin, globulin, SGOT, SGPT and alkaline paraffin embedding by making sections of 5 μ thickness.
phosphatase (ALP). The sections were stained with haematoxylin and eosin
x Radiological Analysis of Arthritic Rats: [24] On 28th and then evaluated under light microscope for the
day X-rays were taken at the joints of the hind paw of presence of periosteal thickening, joint space
the animals for evaluating the bone damage. narrowing, cellular infiltration and destruction of joint
Radiographs were taken using X-ray apparatus space.
(Siemens- 60 MA, Germany) and industrial X-ray film At the end of the study, spleen of all the animals were
(Fuji photo film, Japan). The X-ray apparatus was removed and weighed to evaluate the change in organ.
operated at 220 V with a 40 V peak, 0.2 second exposure
time, and a 60 cm tube-to film distance for anterior- Data Analysis
posterior projection. (Omega Laboratories, Pune.) The observations are represented as Mean ± SEM. The data
x Haematological Determinations in Arthritic Rats: [25] were processed by one-way analysis of variance (ANOVA)
On the 29th day of the study, blood was withdrawn followed by Dunnett’s‘t’ test. P<0.05 was considered
through retro orbital vein puncture of all groups and the significant.
biochemical parameters like haemoglobin content, WBC
count, erythrocyte sedimentation rate (ESR) and RBC Statistical Analysis
count were noted. The results are expressed as mean + SEM. Comparison
x Histopathological Analysis of Arthritic Rats: [26] The between the groups were made by one way analysis of
rats were sacrificed on day 29 by cervical dislocation. variance (ANOVA) followed by Dunnett’s‘t’ test.
Hind limbs were removed and immersed in 10%
buffered formalin for 24 h. Further limbs were RESULTS
decalcificed in 15 % of formic acid and processed for Phytochemical Screening

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 RESEARCH ARTICLE
Normal control
Methotrexate
AESE 200mg/kg
Figure 6: Radiographical analysis of the joints of hind limb of various treatment groups
In the preliminary phytochemical screening, the aqueous
stem bark extract of Ailanthus excelsa showed positive
results for steroids, triterpenoids, saponins, tannins,
glycosides and flavonoids.
Acute Toxicity Study
The single oral dose of AESE did not produce any signs of
toxicity during the 4 hour continuous observation period in
all animals. No mortality was seen and AESE was found to
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Arthritic Control
AESE 100mg/kg
AESE 400mg/kg
be safe at 2000 mg/kg, after 24 h and 14 days observation
period in mice.
x Effect on Change in Paw Volume of Injected and
Non-injected Paw: Normal control rats showed little
paw volume change over the course of the study
(Figure 1). CFA injection resulted in progressive
swelling of the injected (primary) hind paw upto 28 th
day. AESE (at all dose levels) and methotrexate
demonstrated a significant decrease (P<0.01) in paw
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CI

Normal Control Arthritic Control

LCI
LCI

Methotrexate /k AESE
100 mg/kg

LCI LCI

AESE 200 mg/kg /k

AESE 400 mg/kg

Figure 7: Effects of AESE, on Histology joints of control and experimental animals. Photograph showing necrosis of bone (black
arrow), periosteal thickness (yellow arrow), CI- cellular infiltration, LCI – low cellular infiltration H&E stain 100X

edema after single dose on the 14 th day itself.
Treatment was continued up to the 28 th day and all
the drugs showed significant inhibition of swelling on
21st and 28th day (P < 0.01) compared to arthritic
control. AESE 100, 200 and 400 mg/kg produced
33.54 %, 27.09 % and 29.70 % of inhibition (P<0.01)
respectively, on 28 th day as compared with the
arthritic control. Methotrexate produced a significant
inhibition of the rat paw edema by about 36.08 %
(P<0.01) (Table 1).
AESE 400 mg/kg counteracted secondary inflammation
significantly on 21st & 28th day as compared to arthritic
control. AESE 200 mg/kg showed significant effect on 28th
while methotrexate was effective on 14th as well as 28th day
(Figure 2).
x Effect on Body Weight: Decrease in body weight was
observed during the arthritic condition. The AESE 200
mg/kg and standard drug methotrexate significantly
increased the body weight as compared to the arthritic
control (Figure 3).
x Effect on Joint Diameter: There was a significant
increase in the ankle joint diameter in all the CFA
induced groups when compared to the normal control
(Figure 4). AESE 100 mg/kg and methotrexate showed
significantly lesser joint diameter (P<0.05) as compared
to the arthritic control group from 14th day while AESE

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200 and 400 mg/kg were effective from 21st day. On
28th day all the groups with treatment showed
significant results.
x Effect on Mechanical Hyperalgesia: AESE at all dose
levels and methotrexate produced a significant increase
(P<0.01) in pain threshold from 21st day onwards
(Figure 5).
x Effect on Haematological Parameters: AESE (100
mg/kg, 200 mg/kg and 400 mg/kg) and methotrexate
treated group significantly increased (P<0.01) the RBC
count which was diminished in arthritic group. Also,
Haemoglobin (gm %), which was lowered in arthritic
control group, was also increased by the AESE and
standard, restoring it back to normal. WBC count and
Erythrocyte sedimentation rate (ESR) raised during
arthritic condition were effectively restored by AESE
400 mg/kg and methotrexate (Table 2).
x Effect on Biochemical Parameters: Biochemical
parameters like serum Total protein, albumin and
globulin showed significant changes in the arthritic
control group compared to normal control. The changes
in total and individual protein levels are presented in
Table 3. In CFA induced arthritic rats, there was
significant decrease (P<0.01) in total protein and
albumin levels while significant (P<0.01) increase in
globulin level as compared with the normal control
group. On treatment with AESE and methotrexate all
these changes were brought back to normal. All the
treatment groups significantly counteracted the
increase in serum globulin levels. AESE 200 mg/kg
increased the total protein and albumin level (P < 0.01)
which was altered in arthritic control group. The rise in
serum alkaline phosphatase was significantly inhibited
(P < 0.01) in the groups treated with AESE 200 mg/kg,
AESE 400 mg/kg and Methotrexate (P < 0.05). SGOT and
SGPT levels which were elevated in arthritic condition
were determined to check the effect on liver functions.
AESE 200 and 400 mg/kg showed significant decrease
as compared to arthritic control as shown in Table 4.
x Effect on Tissue Weight Changes: In arthritic control
group the increase in spleen weight was observed.
AESE at the dose of 100, 200 and 400 mg/kg
significantly decreased the weight of the spleen as
shown in Table 5.
x Radiographic Analysis: There was no evidence of joint
pathology in normal control rats. Radiography of the
hind paw of rats in which arthritis was induced by CFA
revealed intense periarticular inflammation, soft tissue
swelling, bone resorption and joint erosion (Figure 6).
In groups treated with the AESE, a dose dependant
reduction in joint damage was observed. Similarly,
methotrexate also demonstrated a significant reduction
in joint damage. The various parameters were graded
and are represented in Table 6.
x Effect on Histology of Inflamed Joint: The
histopathological observation of hind paws of rats
exposed to CFA revealed intense periosteal thickening,
joint space narrowing, connective tissue proliferation
with a significant cellular infiltration, erosion of
cartilage and bone (Figure 7). The group receiving AESE
at 100 mg/kg dose demonstrated a significant effect on
cellular infiltration. However, at the dose levels of 200
and 400 mg/kg, AESE demonstrated a significant and
dose dependent improvement in all of the above
mentioned parameters of joint histology. Cellular
infiltration was significantly reduced and there was
attenuation of periosteal thickening and cartilage and
bone damage compared to arthritic control. The
various parameters were graded and are represented
in Table 7.
DISCUSSION
Rat complete Freund’s adjuvant arthritis is an experimental
model of polyarthritis which has been widely used for
preclinical testing of numerous anti-arthritic agents. [28]
The mycobacteria (bacterial peptidoglycan and muramyl
dipeptide) in Complete Freund’s Adjuvant (CFA) attract
macrophages and other cells to the injection site which
enhances the immune response. [29, 30]
In the present work, we have studied the effect of AESE
on complete Freund’s adjuvant induced arthritis. The
treatment was started after development of arthritis i.e
from the 14th day of induction of arthritis which indicates
whether AESE has the ability to cure the disease.
Paw edema is a major factor in assessing the degree of
inflammation and therapeutic efficacy of the drugs. Paw
edema in adjuvant induced arthritis in rats is known due to
involvement of inflammation. After adjuvant injection in
the rat hind paw, a pronounced swelling and hyperalgesia
appeared with no involvement of contralateral paw and is
caused by the irritant effect of the adjuvant. This response
is considered as primary response. The appearance of
secondary lesions that is, swelling of the non-injected paw
is a manifestation of cell-mediated immunity. The
suppression of such paw edema by a drug shows its
immunosuppressive activity. [31, 32] AESE at all dose levels
and reference standard methotrexate treated rats showed
significantly decreased paw volume from 7 th day after
inoculation of CFA in preventive protocol. Secondary
inflammation was significantly inhibited by 400 mg/kg of
AESE. This reveals potent suppression by AESE on cell-
mediated immunity in arthritic rats. The increase in joint
diameter is due to inflammation at the ankle joint.
Increased joint diameter is significantly reduced by AESE.
Many kinds of triterpenoids are known to impair
histamine release from mast cells and exert anti-
inflammatory activity. [33] This might be involved here
also.
The changes in the body weight were monitored as
indicator of arthritic symptoms and the loss of body weight
usually begins to appear at the onset stage of arthritis. The
decreased body weight of arthritis induced animals might
also be linked to the systemic or local action of cytokines
such as TNF-α, since TNF-α has been closely related to the
loss in body weight occurring in animals suffering from
chronic inflammation. [34] The weight loss in arthritic rats
was significantly recovered by AESE and methotrexate
treatment.

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 RESEARCH ARTICLE
In adjuvant treated rats, the affected articulations are
infiltrated by blood-derived cells, mainly neutrophils,
macrophages and dendritic cells. [34] Besides producing
inflammation, it is associated with pain and motor
dysfunction [35] as a result of which hyperalgesia is induced.
The dose dependant analgesic effect of AESE in animals
with adjuvant arthritis is marked as evident by the increase
in the threshold for mechanical hyperalgesia i.e anti-
nociceptive effect. ESR is elevated during the inflammation,
stress and cell necrosis. [36] The reduction in the ESR and
increase in the Hb count which was significantly altered in
arthritic control group was remarkably counteracted by
AESE and reference standard methotrexate, restoring back
to near normal in both protocols. Thus justifying its
significant role in arthritic conditions.
In the present study there was a significant decrease in
albumin level and increase in globulin level in arthritic rats.
It was also postulated that during inflammation, the
mediators released, histamine, bradykinin and
prostaglandins increase the permeability of vascular
tissues to albumin leading to reduction in its serum levels.
Treatment with AESE increased the albumin and decreased
the globulin level in arthritic rats at 400 mg/kg of dose
which indicates that AESE might have a suppressive action
on the mediators of inflammation.
To evaluate liver function, SGOT and SGPT levels were
estimated. [36] Alkaline phosphtase, SGOT and SGPT levels
were significantly reduced in arthritic animals after the
administration of AESE and methotrexate in dose
dependent manner. The decreased enzyme levels by AESE
treatment emphasized a decreased bone loss and organ
protective role of AESE in adjuvant induced rats.
Spleen is another vital organ involved in immune
responses. Subplantar administration of CFA significantly
increased weight of spleen which is in accordance with
previous studies. [36] AESE effectively reduced the Weight
in both the protocols.
The anti-arthritic effect of AESE was confirmed by
radiological and histological evaluations. In adjuvant
induced arthritic group, soft tissue swelling along with
narrowing of the joint spaces were observed which implies
the bony destruction in arthritic condition. In both the
protocols, AESE showed significant anti-arthritic effect in a
dose dependant manner. It was found that 400 mg/kg dose
of AESE was more effective.
AESE normalized the various biochemical and
haematological abnormalities in CFA induced arthritis in
rats. AESE also significantly attenuated CFA induced bone
deformity and cartilage destruction. The effect might be
due to potential phytochemicals found to be present in
preliminary phytochemical analysis such as phytosterols,
triterpenoids, saponins, tannins and flavonoids.
CONCLUSION
The present study has revealed the significant anti-
rheumatoid effects of aqueous AESE. It suggests that
Ailanthus excelsa aqueous extract could be a valuable
addition to the current R. A therapies. Moreover, these
studies strongly validate the claims of the tribal use of this
Inventi Impact: Ethnopharmacology Vol. 2013, Issue 1
[E-ISSN 0976-7568, P-ISSN 2229-4155]
plant as an anti-arthritic agent. Also it paves way for
further investigation of the chemical constituents
responsible for the activity.
REFERENCES AND NOTES
1. Deborah Symmons, Colin Mathers, Bruce Pfleger. The global
burden of rheumatoid arthritis, 2000.
2. Buch M, Emery P. The etiology and pathogenesis of
Rheumatoid Arthritis. Hospital Pharmacy 9, 5-10.
Cardiovascular mortality is increased in seropositive patients,
Arthritis Rheum., 46 (8) 2002, 2010–2019.
3. David M Lee, Michael E Weinblatt .Rheumatoid arthritis-
Review. The Lancet, Vol 358, 2001.
4. Shankaranarayanan J, Christina A J M, Kalyan S Betanabhatla
Jagan Athimoolam, Sundara Saravanan K,
Balakumar Mahalingam. Evaluation of Glycine max Merill.
Seeds for Antiarthritic Activity in Male Wistar Rats,
International Journal of Pharmaceutical Sciences and
Nanotechnology, 1(4):363-366, 2009.
5. Inamdar Nazma, Shima Edalat, Vikram B Kotwal, Sunita
Pawar. Herbal drugs in milieu of modern drugs. International
Journal of Green Pharmacy, 2(1):2-8, 2008.
6. Kirtikar K R and Basu B D. Indian Medicinal Plants”, Reprinted
Edn., Lalit Mohan Basu Allahabad. Vol IV, 1664, 1996.
7. Nadkarni A K. Indian Materia Medica. 1st Edn, Popular
Prakashan, Mumbai, India, 1976.
8. Bayçu Gülriz, Ataa Said, Ahmed Farag, Khaled Rashed. Protein
patterns and chemical constituents of Ailanthus altissima
(Miller) Swingle and Ailanthus excelsa Roxb. IUFS Journal of
Biology, 67(1):81-88, 2008.
9. Yoganandam G. Prakash, k.periyanayagam and K.ilango.
Pharmacognostical and phytochemical studies on stem bark of
ailanthus excelsa roxb. (Simaroubaceae). International journal
of pharma and bio sciences.1 (2), 2010.
10. V Ravichandran, B Suresh, M N Sathishkumar, K Elango, R
Srinivasan. Antifertility activity of hydroalcoholic extract of
Ailanthus excelsa (Roxb): An ethnomedicines used by tribals
of Nilgiris region in Tamilnadu. Journal of
Ethnopharmacology, 112; 189–191, 2007.
11. Joshi B C (a), A Pandey, R P Sharma and A Khare. Quassinoids
from Ailanthus excelsa, Phytochem., 62 (4); 579-584, 2003.
12. Kumar D, S S Bhujbal, R S Deoda and S C Mudgade.
Bronchodilator activity of aqueous extract of stem bark of
Ailanthus excelsa Roxb. Phcog Res., 2; 102-106, 2010.
13. Dell'Agli Mario, Germana V Galli, Silvia Parapini, Nicoletta
Basilico, Donatella Taramelli, Ataa Said, Khaled Rashed, Enrica
Bosisio. Anti-plasmodial activity of Ailanthus excelsa.
Fitoterapia, 79; 112–116, 2008.
14. Melanchauski L S, A P G S Broto, T M Moraes, A L M Nasser
and A Said. Gastroprotective and antisecretory effects of
Ailanthus excelsa (Roxb). J. Nat. Med., 64:109-113, 2010.
15. Chauhan Khushbu, Parmar Lalkrushn, Solanki Roshni,
Sundariya Dhruvil1, Dr. Adeshara SP. Evaluation of analgesic
activity of Alianthus excelsa Roxb. Using rat as an
experimental animal. Journal of Pharmacy Research, 4(5);
1336-1337, 2011.
16. Patil Sharanabasappa B, Upender Reddy C H and N M
Goudgaon. Antifungal, Anti-Inflammatory and Analgesic
Activity of Ailanthus Excelsa Bark, Ijpsr, 1(12):119-122, 2010.
17. Sandeep Biradar, V.A.Kangralkar, Yuvaraj Mandavkar, Megha
Thakur, Nilesh Chougule.Anti-Inflammatory, Anti-Arthritic, An
algesic and Anticonvulsant Activity of Cyperus Essential Oils.
Int J Pharm Sci, Vol 2, Issue 4, 112-115
18. Sharad M Malia, Arulmozhi Sinnathambia, Chinmay
U Kapasea, Subhash L Bodhankara, Kakasaheb R Mahadik.
 2013 pep 723, CCC: $10 © Inventi Journals (P) Ltd
Published on Web 15/01/2013, www.inventi.in
 RESEARCH ARTICLE
Anti-arthritic activity of standardised Extract
of Phyllanthus amarus in Freund's complete adjuvant induced
arthritisb Biomedicine & Aging Pathology, 2011.
19. Kalpesh ramdas patil; anti-arthritic activity of bartogenic acid;
evidence-based complementary and alternative medicine,
2011.
20. L O Randall and J J Selitto. A method for measurement of
analgesic activity on inflamed tissue. Arch Int
Pharmacodyn, 61, 409–419, 1957.
21. A Gomes, S Bhattacharya, M Chakraborty, P Bhattacharjee, R
Mishra and A Gomes. Anti-arthritic activity of Indian
monocellate cobra (Naja kaouthia) venom on adjuvant
induced arthritis. Toxicon, 55:670–673, 2010.
22. Trivedi H R. Aegle Marmelos suppressess Inflamation
&Cartilage destruction in Collagen induced Arthritic rat..
IJPRD, 2011.
23. Patil K S, Jayaprakash S. Effect of Celastrus paniculatus Willd.
Seed on adjuvant induced arthritis in Rats. Pharmacognosy
Magzine, 3(11):177–183, 2007.
24. Mythilypriya R, Shanthi P, Sachdanandam P. Therapeutic
effect of Kalpaamruthaa, an herbal preparation on adjuvant
induced arthritis in wistar rats. Inflammopharmacology 16:
21–35, 2008.
25. Carlson R P, Datko L J, Blazek E M, Delustro F, Lewis A J.
Comparison of inflammatory changes in established type II
collagen and adjuvant induced arthritis using out bred Wistar
rats. International Journal of Immunopharmacology, 7:811-
826, 1985.
26. Bennett B, Irene J Check, Margaret, R Olsen and Robert L
Hunter. A comparison of commercially available adjuvants for
use in research. Journal of Immunopharmacological Methods,
153:31-40, 1992.
27. Deep B J. Comparison of Freund’s and Ribi adjuvants for
inducing to the synthetic antigen (TG)-AL in rabbits. Journal of
Immunological Methods, 152:105-113, 1992.
Inventi Impact: Ethnopharmacology Vol. 2013, Issue 1
[E-ISSN 0976-7568, P-ISSN 2229-4155]
28. Singh P, Yadav R J & Pandey A. Utilization of indigenous
systems of medicine & homeopathy in India. Indian Journal of
Medical Research, 122: 137–142, 2005.
29. Singh B, Bani S, Gupta D K, Chandan B K, Kaul A. Anti-
inflammatory activity of ‘TAF’: an active fraction from the
plant Barleria prionitis Linn. Journal of Ethnopharmacology,
85:187–193, 2003.
30. Mehta, Neeraj K Sethiya, Chetan Mehta, G B Shah. Anti-arthritic
activity of roots of Hemidesmus indicus R.Br. (Anantmul) in
rats. Asian Pacific Journal of Tropical Medicine, 130-135, 2012.
31. A VanderBorght, P Geusens, J Raus and P Stinissen. The
autoimmune pathogenesis of rheumatoid arthritis: role of
autoreactive T cells and new immunotherapies. Semin
Arthritis Rheum, 31:160–175, 2001.
32. M De Castro Costa, P De Sutter, J Gybels and J Van Hees,
Adjuvant induced arthritis in rat: a possible animal model of
chronic pain. Pain, 10:173–186, 1981.
33. William J K, Arthritis, allied condition. A textbook of
rheumatology. Baltimore, Tokyo: Waverlay Company; 1207,
1996.
34. A C A Moura, E L F. Silva, A G Fraga & M C A Wanderley,
Phytomedicine, 12:138, 2005.
35. Pedernera A M, Guardia T, Calderon C G, Rotelli A E, Rocha N E,
Genaro S D. Antiulcerogenic and anti-inflammatory activity of
the methanolic extract of Larrea divaricata Cav. in rat. J
Ethnopharmacol, 105(3):415–20, 2006.
Cite this article as: Smita Pillewan, Neeraj
Vyawahare, Tina Saldanha, Sujata Taware, Neha
Piarchand. Experimental Investigation of Anti-
rheumatoid Activity of Ailanthus excelsa in Adjuvant-
induced Arthritic Rats. Inventi Impact:
Ethnopharmacology, 2013(1): 35-44, 2013.
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Published on Web 15/01/2013, www.inventi.in