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Proceedings S.Z.P.G.M.I. Vol: 32(1): pp. 35-39, 2018. PSZMC-667-32-1-2018
Prophylactic Anti-Arthritic Effect of Cassia fistula
in Murine Rheumatoid Arthritis Model
Hassan Farooq1, Mariyam Iftikhar Piracha2 and Saadia Shahzad Alam2
1Department of Pharmacology, Khawaja Muhammad Safdar Medical College, Sialkot,
2Department of Pharmacology, Shaikh Zayed Postgraduate Medical Institute, Lahore
ABSTRACT
Introduction: Rheumatoid arthritis (RA) is a major autoimmune disease and an important cause of
potentially preventable disability. Many drugs are available for its treatment, however, we need an
improved remedy to prevent its onset and worsening in patients afflicted by this disease. Natural substances
including plants have been researched for their anti-arthritic potential. Cassia fistula could have similar
ability. Aims and Objective: Evaluation of prophylactic anti-arthritic effect of Cassia fistula in Complete
Freund’s Adjuvant (CFA) induced murine model of RA. Material and Methods: The study was carried out
for 15 days on 48 male rats divided into 6 groups of 8 rats each. Group 1 (negative control), group 2
(positive control), group 3-6 were given a anthraquinone and methanolic extracts of Cassia fistula orally
BD on days 1,2 &3, the first dose being given 30min prior to CFA injection (0.2ml). Caliper measurement
of right ankle joint and RA factor was used to evaluate the anti-arthritic effect of Cassia fistula on days 1, 9
and 15. Results: Prophylactic groups showed 36-50% lesser ankle swelling and reduction in rheumatoid
factor by 62.5% and 87.5% as compared to that of the diseased control (group 2) in a dose dependent
manner (500mg>250mg/kg) for both methanolic and anthraquinone extracts of Cassia fistula.
Conclusion: Overall difference among groups was significant with p-value < 0.05. Cassia fistula showed a
prophylactic potential in RA treatment due to its anti-inflammatory and anti-oxidant properties.
Keywords: Cassia fistula, anthraquinone, CFA, RA factor.
INTRODUCTION
Rheumatoid Arthritis (RA) is a chronic, crippling,
debilitating, painful autoimmune disorder which
leads to the deformity and immobility of particularly
small joints of hands, fingers, knees and feet1(Fig-
1). Its prevalence varies between 0.3% and 1%
globally between the ages of 20 and 40 2.
Cassia fistula commonly known as
Amaltas, popularly called “Indian Laburnum” in
English has been extensively used in Ayurvedic
medicine for various ailments3 (Fig-2). Every part of
this plant is recognized for its medicinal properties
but most importantly the fruit pulp has shown useful
application in gout and rheumatism and possesses
anti-inflammatory activity4.
The plant is rich in phenolic antioxidants
such as anthraquinones, flavonoids and flavan-3-ol
derivatives, of which anthraquinones and their
variants appear to be the common active principle in
all parts of the Cassia fistula plant5. Bark showed
the highest antioxidant potential6. Cassia fistula
fruit pulp active principle, has anti-inflammatory
ability by inhibiting super oxide anion production
from human neutrophils contributing to the overall
therapeutic activity of the anthraquinone7,8.
To investigate these claims further, the
current research was conducted, which differed
from older researches as it employed the Complete
Freund’s Adjuvant (CFA) murine model of
rheumatoid arthritis to determine prophylactic anti-
arthritic effect of anthraquinone derived from
Cassia fistula bark and fruit pulp. Whereas
previously the carrageenan model had been
employed.
36
Prophylactic Anti-arthritic effect of Cassia fistula in murine Rheumatoid Arthritis model
Fig-1: Rheumatoid Arthritis Fig-2: Cassia fistula (Amaltas)
effecting metacarpophalangeal
and interphalangeal joint
MATERIAL AND METHODS
This experimental study was conducted in
University of Veterinary and Animal Sciences
(UVAS) Lahore, after approval from Institutional
Review Board (IRB) and was completed in batches
of 15 days for a period of 3 months.
Fruit pulp and bark of Cassia fistula was collected
in the month of April from University of the Punjab,
Botany Department, Lahore.
Preparation of Cassia fistula extracts:
Anthraquinone and methanolic extracts of Cassia
fistula were made in Applied Chemistry Research
Centre, PCSIR Laboratories, Lahore. Fruit pulp and
bark of Cassia fistula was dried and finely
powdered. Extraction was done by the method
described below9. The extract was used after the
confirmatory anthraquinone test.
Extraction of anthraquinone
30gm powdered cassia fruit pulp + 150ml ethanol
(1:5) in Soxhlet apparatus
Heated for 24hrs at solvent’s boiling point
Anthraquinone (rhein) extract
Concentrated, dried and stored with desiccator
Preparation of methanolic extract
Powdered cassia bark + petroleum ether
Extraction with methanol and double distilled water
in soxhlet extractor
Extract concentrated under reduced pressure in
rotary vacuum evaporator
Refrigerated for further use
Test for Anthraquinones
fruit pulp extract
10ml of 1% HCl +
bark extract
Boil for 5 minutes
Filter and cool the sample
Partition of the cool filtrate was done twice
using equal volumes of chloroform and 10%
ammonia and then the layer was allowed to
separate. Rose pink color indicated the presence of
combined anthraquinones9.
Preparation and assessment of rat model of
rheumatoid arthritis:
Arthritis was induced in right hind paw foot pad of
each rat using a single dose (0.2 ml) of CFA which
contains killed Mycobacterium tuberculosis and
non-metabolizable oils, provides continuous release
of antigens and thus stimulates a strong persistent
immune response10. Within a few hours a swelling
was noticed around the injection site whereas
clinical evidence of the arthritis was noted on the 9th
post CFA injection day. The swelling grew
gradually and was associated with declining rat
mobility over a 15day period as the arthritis
progressed. Treatment was initiated on day 1 11.
Assessment of disease progression was made by
caliper measurements of ankle joint and RA factor
on day 1, 9 and 15.
A total of 48 adult male wistar albino rats
weighing 170–200 gm was placed in animal house
of UVAS, Lahore. They were acclimatized for a
week and maintained in polypropylene cages at 25 ±
2° C, with relative humidity 45-55% under 12h light
and dark cycles and fed with standard laboratory
diet with water ad libitum.
They were divided into six groups having
eight rats each. Every rat was clearly numbered.
The test extracts were administered in suspension
form in water using 1% carboxymethyl cellulose as
suspending agent as per experimental requirement:
Group 1: Healthy control group; it was not treated
and was given equal quantity of normal saline.
Group 2: Experimental control rats were given a
single 0.2ml dose of CFA injection in right hind paw
foot pad sub-cutaneously and left for self-recovery.
37
Prophylactic Anti-arthritic effect of Cassia fistula in murine Rheumatoid Arthritis model
Group 3: Anthraquinone extract 250mg/kg orally BD
on day 1, 2 and 3.
Group 4: Anthraquinone extract 500mg/kg orally BD
on day 1, 2 and 3.
Group 5: Methanolic extract 250mg/kg orally BD
on day 1, 2 and 3.
Group 6: Methanolic extract 500mg/kg orally BD
on day 1, 2 and 3.
First dose of extracts was given 30min before
CFA injection to each group.
On day 1, 9 and 15 caliper measurement of
right ankle joint was done and blood samples were
collected by cardiac puncture for RA factor (Figs-
3,4).
Statistical Analysis:
Data was analyzed with SPSS version 20.0 and was
described by using Mean + SD for each group.
Comparison between groups was done by using
one-way ANOVA test and p-value < 0.05 was taken
as statistically significant.
RESULTS
Mean+ SD
Day 1
Day 9
Day 15
Group 1
0.24 ±0.05
0.24 ± 0.05
0.24 ± 0.05
Group 2
0.58 ± 0.05
0.54 ± 0.07
0.53 ± 0.07
Group 3
0.50 ± 0.00
0.46 ± 0.05
0.45 ±0.05
Group 4
0.29 ± 0.04
0.26 ± 0.05
0.25 ± 0.05
Group 5
0.55 ± 0.05
0.54 ± 0.05
0.50 ± 0.08
Group 6
0.40 ± 0.00
0.35 ± 0.05
0.34 ± 0.05
Table-1: Caliper measurement of right ankle joint (cm)
The difference among groups was significant with
p-value 0.000.
Rheumatoid factor
Day 1
Day 9
Day 15
A
P
A
P
A
P
Group 1
8
(100%)
-
8
(100%)
-
8
(100%)
-
Group 2
8
(100%)
-
1
(12.5%)
7
(87.5%)
1
(12.5%)
7
(87.5%)
Group 3
8
(100%)
-
4
(50%)
4
(50%)
5
(62.5%)
3
(37.5%)
Group 4
8
(100%)
-
6
(75%)
2
(25%)
7
(87.5%)
1
(12.5%)
Group 5
8
(100%)
-
4
(50%)
4
(50%)
5
(62.5%)
3
(37.5%)
Group 6
8
(100%)
-
6
(75%)
2
(25%)
7
(87.5%)
1
(12.5%)
p-value
0.011
0.005
Table-2: Comparison of Rheumatoid factor
A: Absent P: Present
Fisher’s exact test revealed that there was
significant association between rheumatoid factor
and study groups (p-value = 0.011). The proportion
of animal with rheumatoid factor was higher in
groups 2, 3 and 5.
DISCUSSION
Rheumatoid arthritis is a progressive
inflammatory autoimmune disease with articular
and systemic effects. T cells, B cells and pro-
inflammatory cytokines play key roles in the
pathophysiology of rheumatoid arthritis12.
Phytotherapy has been recognized as
valuable and readily resource for primary health
care and WHO has endorsed its safe and effective
use. It is estimated that 80% of the population living
in the developing countries rely exclusively on
traditional medicine for their primary health care
needs13. Plants have a great importance in treating
arthritis. Cassia fistula, Amaltas an indigenous plant
was previously researched and showed beneficial
effects in Carrageenan induced in-vitro murine
model of arthritis14. Since prevention is better than
cure, patients need a drug that can either prevent the
disease or reduce its morbidity.
Bearing this in mind the present research
work was conducted on Cassia fistula bark
(methanolic extract) and fruit pulp (anthraquinone)
for preventing rheumatoid arthritis which was
generated in-vitro using the CFA model in rats.
Fig-3: Caliper measurement
of ankle thickness
Fig-4: 3ml blood was drawn by
cardiac puncture &
serum was separated for
assessment of RA factor
38
Prophylactic Anti-arthritic effect of Cassia fistula in murine Rheumatoid Arthritis model
The following parameters were analyzed in detail.
Rat Ankle Thickness (Caliper measurement):
In the present investigation the arthritic rats showed
a soft tissue redness, warmth and swelling 8 days
after CFA induction around the right ankle joint and
immobility on day 9. This reflected the acute phase
of arthritis and was due to edema of tissues such as
ligaments and joint capsules which was measured
through vernier calipers.
On day 1 pre-treatment with anthraquinone
and methanolic extract in doses of 500mg/kg BD
(groups 4 &6) before induction of rheumatoid
arthritis and subsequently for the next two days as
the disease was developing resulted in a 36-50%
lesser ankle swelling in the affected ankle as
compared to the that of the diseased control (group
2). Later on, caliper measurements of ankle
thickness showed similar changes. Our results
revealed a significant reduction on day 9 and 15 in a
dose dependent manner (500mg>250mg/kg) for
both methanolic and anthraquinone extracts of
Cassia fistula as shown in Table-1.
These results concurred with a previous
study in which carrageenan induced arthritis model
was used to evaluate the effect of orally
administered extracts of Cassia fistula and
significant ameliorative activity of methanolic
extract was noted15. Similarly, here too the reduction
in inflammation seen could be due to inhibition of
mediators of inflammation such as histamine,
serotonin and prostaglandin due to inhibitory
hydroxyl scavenging activity16 and an antioxidant
effect by inhibiting lipid peroxidation17.
Rheumatoid factor:
Rheumatoid factor is intimately linked with the
pathology of rheumatoid arthritis. Initially all the
rats were normal and rheumatoid factor was absent
while it remained absent throughout the experiment
in the healthy control group.
At the end of the study on day 15, 87.5% of
group 2 rats had rheumatoid factor, while in group 3
and 5 62.5% of the rats showed a reduction in
rheumatoid factor after administration of 250mg/kg
BD of anthraquinone and methanolic extracts of
Cassia fistula respectively.
However, dose dependent effect was seen in groups
4 and 6 which were administered 500mg/kg BD
doses of anthraquinone and methanolic extracts,
which gave much better results and presence of
rheumatoid factor was 87.5% lower than that of
group 2. This action could be due to inhibition of
inflammatory mediators and lipid peroxidation by
the Cassia fistula extracts16,17. Overall difference
among groups was significant with p-value < 0.05.
Our findings on the lowering of rheumatoid
factor using methanolic extract and rhein could not
be substantiated as no similar study existed to
support or refute them.
CONCLUSION
This was a novel research which showed
prophylactic anti-arthritic effect of Cassia fistula
(Amaltas) in a CFA induced murine model of
rheumatoid arthritis. The CFA model used was
unique as well, as it allowed to develop a form of
rheumatoid arthritis which greatly mimicked clinical
rheumatoid arthritis.
In conclusion, an extremely interesting,
dose dependent prophylactic anti-arthritic potential
of anthraquinone and methanolic extracts of Cassia
fistula 250mg/kg BD and 500mg/kg BD emerged.
Corroborating this aspect was a demonstrable
improvement in the right ankle joint caliper
measurement and serum rheumatoid factor levels.
Based upon our research it can be deduced
that prophylactic administration of 500mg/kg BD
doses of anthraquinone and methanolic extracts
revealed greater efficacy in preventing rheumatoid
arthritis in a CFA model. We therefore suggest that
Cassia fistula has the potential to prevent
rheumatoid arthritis and prophylaxis reduces the
morbidity of the disease to a great extent.
The CFA Model used simulated the clinical
picture of RA to a large extent but did not replicate
it exactly. Further research can be done regarding
the use of this herb for treating the complications of
arthritis for example twisted joints, and deformity.
REFERENCES
1. M. Shipley, A. Rehman. Rheumatology and
Bone disease. Kumar and Clark Clinical
Medicine. 2005; 6: 523-532.
2. Chronic Diseases and Health Promotion by
WHO for Rheumatic diseases; CHP
Department 2016.
3. Kala CP. Ethnobotany and ethno conservation
of Aegle marmelos (L.) Correa. Indian J
Traditional Knowledge. 2006; 5(4): 537-540
39
Prophylactic Anti-arthritic effect of Cassia fistula in murine Rheumatoid Arthritis model
4. Indian Herbal Pharmacopoeia revised new
edition, 2002, Indian Drug Manufacturers
Association Mumbai, page no.106-113.
5. Nadkarni KM. Indian Materia Medica-2, 3rd
ed. Popular Prakashan, Bombay, Gupta.
Pharm Biol. 2009; 47(3): 195-202.
6. Luximon-Ramma, Bahorun-T and Aruoma
OI. Antioxidant activities of phenolic,
proanthocyanidin, and flavonoid components
in extracts of Cassia fistula. J Agric Food
Chem. 2002 Aug 28: 50(18):5042-7.
7. Kannampalli P, Chandrasekaran VRM,
Kuppannan GA, Sivanesan K. Effect of
pretreatment of Cassia fistula Linn leaf
extract against subacute CCl4 induced
hepatotoxicity in rats, Indian J. Exp. Biol. 43:
526–30.
8. Kannampalli P, Chandrasekaran, VRM,
Kuppannan GA, Sivanesan K (2007). Effect
of Cassia fistula Linn leaf extract on
diethylnitrosamine induced hepatic injury in
rats. Chemico-Biological Interaction. 167:12-
8.
9. J.Anitha and S.Miruthula. Anti inflammatory
and phytochemical analysis of cassia fistula
fruit pulp extracts. IJP (210-212)
10. L.M. Lichtenberger, J.J.Romoero, E.J.Dial.
Naproxen-PC: A GI safe and highly effective
anti-inflammatory. (2-4).
11. A.M Bendele. Animal models of rheumatoid
arthritis. J. musculoskeletal Neuron Interact
2001:1(4);377-385).
12. Koenders MI. Marijnissen RJ, Abdollahi-
Roodsaz S, et al. Interleukin-1 drives
pathogenic Th17 cells during spontaneous
arthritis in interleukin-1 receptor antagonist–
deficient mice. Arthritis Rheum. 2008; 58:
3461–70.
13. Gupta RK. Medicinal & Aromatic plants. 1st
ed. Bombay: CBS Publishers & Distributors,
2010, 116-7.
14. Gupta AK, Tondon N, Sharma M. Quality
Standards of Indian Medicinal Plants,
Medicinal Plants Unit. Indian Council Med
Res 2008; 2: 47-53.
15. Conner, E. M., Grisham, M. B. Inflammation,
free radicals and antioxidants, Nutrition.
1996: 12: 274-277.
16. Comporti, M. Three models of free radical
induced cell injury, Chem Biol Interact. 1989;
72(1-2): 1-56.
17. Raju I, Moni M, Subramanian V. Anti-
Inflammatory and Antioxidant activities of
Cassia fistula Linn Bark Extracts. Afr. J. Trad.
CAM; 2005: 2 (1): 70–85.
The Authors:
Dr. Hassan Farooq
Assistant Professor
Department of Pharmacology
Khawaja Muhammad Safdar Medical College
Sialkot
Dr. Mariyam Iftikhar Piracha
M. Phil trainee
Department of Pharmacology
Shaikh Zayed Postgraduate Medical Institute Lahore
Dr. Saadia Shahzad Alam
Professor of Department of Pharmacology
PGMI Federal Shaikh Zayed Hospital Lahore
Corresponding author:
Dr. Hassan Farooq
Assistant Professor
Department of Pharmacology
Khawaja Muhammad Safdar Medical College
Sialkot
hassan_oldravian@yahoo.com
