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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.
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RESEARCH ARTICLE
Table 1: Percent Inhibition of Injected Hind Paws Edema by AESE in Freund’s Adjuvant Arthritic Model in Rats
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
study. Animals were procured from the animal house of water ad libitum. The experiments were designed and
AISSMS College of Pharmacy, Pune and kept under conducted in accordance with ethical norms approved by
standard laboratory condition of temperature (25 ± 2°C) Committee for the Purpose of Control and Supervision on
and humidity (55 ± 5 %) under 12:12 light dark cycle. The Experiments on Animals (CPSCEA) and Institutional Animal
animals were fed with standard pellet diet and drinking Ethical Committee (IAEC).
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RESEARCH ARTICLE
Table 6: Graded Scores of the Joints of Hind Limb of Various Treatment Groups
Table 7: Graded Scores of the Hind Paw Histopathology of Various Treatment Groups
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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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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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Figure 6: Radiographical analysis of the joints of hind limb of various treatment groups
In the preliminary phytochemical screening, the aqueous be safe at 2000 mg/kg, after 24 h and 14 days observation
stem bark extract of Ailanthus excelsa showed positive period in mice.
results for steroids, triterpenoids, saponins, tannins, x Effect on Change in Paw Volume of Injected and
glycosides and flavonoids. Non-injected Paw: Normal control rats showed little
paw volume change over the course of the study
Acute Toxicity Study (Figure 1). CFA injection resulted in progressive
The single oral dose of AESE did not produce any signs of swelling of the injected (primary) hind paw upto 28 th
toxicity during the 4 hour continuous observation period in day. AESE (at all dose levels) and methotrexate
all animals. No mortality was seen and AESE was found to demonstrated a significant decrease (P<0.01) in paw
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RESEARCH ARTICLE
CI
LCI
LCI
Methotrexate /k AESE
100 mg/kg
LCI LCI
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. while methotrexate was effective on 14th as well as 28th day
Treatment was continued up to the 28 th day and all (Figure 2).
the drugs showed significant inhibition of swelling on x Effect on Body Weight: Decrease in body weight was
21st and 28th day (P < 0.01) compared to arthritic observed during the arthritic condition. The AESE 200
control. AESE 100, 200 and 400 mg/kg produced mg/kg and standard drug methotrexate significantly
33.54 %, 27.09 % and 29.70 % of inhibition (P<0.01) increased the body weight as compared to the arthritic
respectively, on 28 th day as compared with the control (Figure 3).
arthritic control. Methotrexate produced a significant x Effect on Joint Diameter: There was a significant
inhibition of the rat paw edema by about 36.08 % increase in the ankle joint diameter in all the CFA
(P<0.01) (Table 1). induced groups when compared to the normal control
AESE 400 mg/kg counteracted secondary inflammation (Figure 4). AESE 100 mg/kg and methotrexate showed
significantly on 21st & 28th day as compared to arthritic significantly lesser joint diameter (P<0.05) as compared
control. AESE 200 mg/kg showed significant effect on 28th to the arthritic control group from 14th day while AESE
200 and 400 mg/kg were effective from 21st day. On cartilage and bone (Figure 7). The group receiving AESE
28th day all the groups with treatment showed at 100 mg/kg dose demonstrated a significant effect on
significant results. cellular infiltration. However, at the dose levels of 200
x Effect on Mechanical Hyperalgesia: AESE at all dose and 400 mg/kg, AESE demonstrated a significant and
levels and methotrexate produced a significant increase dose dependent improvement in all of the above
(P<0.01) in pain threshold from 21st day onwards mentioned parameters of joint histology. Cellular
(Figure 5). infiltration was significantly reduced and there was
x Effect on Haematological Parameters: AESE (100 attenuation of periosteal thickening and cartilage and
mg/kg, 200 mg/kg and 400 mg/kg) and methotrexate bone damage compared to arthritic control. The
treated group significantly increased (P<0.01) the RBC various parameters were graded and are represented
count which was diminished in arthritic group. Also, in Table 7.
Haemoglobin (gm %), which was lowered in arthritic
control group, was also increased by the AESE and DISCUSSION
standard, restoring it back to normal. WBC count and Rat complete Freund’s adjuvant arthritis is an experimental
Erythrocyte sedimentation rate (ESR) raised during model of polyarthritis which has been widely used for
arthritic condition were effectively restored by AESE preclinical testing of numerous anti-arthritic agents. [28]
400 mg/kg and methotrexate (Table 2). The mycobacteria (bacterial peptidoglycan and muramyl
x Effect on Biochemical Parameters: Biochemical dipeptide) in Complete Freund’s Adjuvant (CFA) attract
parameters like serum Total protein, albumin and macrophages and other cells to the injection site which
globulin showed significant changes in the arthritic enhances the immune response. [29, 30]
control group compared to normal control. The changes In the present work, we have studied the effect of AESE
in total and individual protein levels are presented in on complete Freund’s adjuvant induced arthritis. The
Table 3. In CFA induced arthritic rats, there was treatment was started after development of arthritis i.e
significant decrease (P<0.01) in total protein and from the 14th day of induction of arthritis which indicates
albumin levels while significant (P<0.01) increase in whether AESE has the ability to cure the disease.
globulin level as compared with the normal control Paw edema is a major factor in assessing the degree of
group. On treatment with AESE and methotrexate all inflammation and therapeutic efficacy of the drugs. Paw
these changes were brought back to normal. All the edema in adjuvant induced arthritis in rats is known due to
treatment groups significantly counteracted the involvement of inflammation. After adjuvant injection in
increase in serum globulin levels. AESE 200 mg/kg the rat hind paw, a pronounced swelling and hyperalgesia
increased the total protein and albumin level (P < 0.01) appeared with no involvement of contralateral paw and is
which was altered in arthritic control group. The rise in caused by the irritant effect of the adjuvant. This response
serum alkaline phosphatase was significantly inhibited is considered as primary response. The appearance of
(P < 0.01) in the groups treated with AESE 200 mg/kg, secondary lesions that is, swelling of the non-injected paw
AESE 400 mg/kg and Methotrexate (P < 0.05). SGOT and is a manifestation of cell-mediated immunity. The
SGPT levels which were elevated in arthritic condition suppression of such paw edema by a drug shows its
were determined to check the effect on liver functions. immunosuppressive activity. [31, 32] AESE at all dose levels
AESE 200 and 400 mg/kg showed significant decrease and reference standard methotrexate treated rats showed
as compared to arthritic control as shown in Table 4. significantly decreased paw volume from 7 th day after
x Effect on Tissue Weight Changes: In arthritic control inoculation of CFA in preventive protocol. Secondary
group the increase in spleen weight was observed. inflammation was significantly inhibited by 400 mg/kg of
AESE at the dose of 100, 200 and 400 mg/kg AESE. This reveals potent suppression by AESE on cell-
significantly decreased the weight of the spleen as mediated immunity in arthritic rats. The increase in joint
shown in Table 5. diameter is due to inflammation at the ankle joint.
x Radiographic Analysis: There was no evidence of joint Increased joint diameter is significantly reduced by AESE.
pathology in normal control rats. Radiography of the Many kinds of triterpenoids are known to impair
hind paw of rats in which arthritis was induced by CFA histamine release from mast cells and exert anti-
revealed intense periarticular inflammation, soft tissue inflammatory activity. [33] This might be involved here
swelling, bone resorption and joint erosion (Figure 6). also.
In groups treated with the AESE, a dose dependant The changes in the body weight were monitored as
reduction in joint damage was observed. Similarly, indicator of arthritic symptoms and the loss of body weight
methotrexate also demonstrated a significant reduction usually begins to appear at the onset stage of arthritis. The
in joint damage. The various parameters were graded decreased body weight of arthritis induced animals might
and are represented in Table 6. also be linked to the systemic or local action of cytokines
x Effect on Histology of Inflamed Joint: The such as TNF-α, since TNF-α has been closely related to the
histopathological observation of hind paws of rats loss in body weight occurring in animals suffering from
exposed to CFA revealed intense periosteal thickening, chronic inflammation. [34] The weight loss in arthritic rats
joint space narrowing, connective tissue proliferation was significantly recovered by AESE and methotrexate
with a significant cellular infiltration, erosion of treatment.
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RESEARCH ARTICLE
In adjuvant treated rats, the affected articulations are plant as an anti-arthritic agent. Also it paves way for
infiltrated by blood-derived cells, mainly neutrophils, further investigation of the chemical constituents
macrophages and dendritic cells. [34] Besides producing responsible for the activity.
inflammation, it is associated with pain and motor
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