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Vol 2|Issue 2| 2012 | 59-61.
59 | P a g e
e-ISSN 2248 - 9169
Print ISSN 2248 - 9150
International Journal of
Experimental Pharmacology
www.ijepjournal.com
ASSESSMENT OF THE ANTI-ARTHRITIC EFFECTS OF BRASSICA
NIGRA SEED EXTRACTS IN EXPERIMENTAL MODELS IN ALBINO
RATS
M. Vinyas1*, Shiv Kumar1, Bheemachari1, K. Sivaiah1, G. Avinash Kumar Reddy2
1NET Pharmacy College, Department of Pharmacology, Raichur, India.
2Department of Pharmacognosy, Sree Vidyaikethan College of Pharmacy, Tirupathi, India.
INTRODUCTION
Brassica nigra Linn. (Cruciferae) commonly
called as black mustard has traditionally used as simple
rubefacient, diuretic, emetic, pneumonia, bronchitis, nerve
stimulant and vesicant [1]. All the three extracts contain
alkaloids, saponins and flavonoids etc. In our laboratory it
was observed that crude extract of BN exert anti-arthritic
activity in rats. Pharmacological studies reported on the BN
are anti-microbial [2], insulinotropic and anti-
hyperglycemic [3]. Brassica nigra has been traditionally
used in India, Sri Lanka, Bangladesh and Pakistan for the
treatment of inflammation and rheumatism. However,
systematic study of this plant has not been carried out for the
anti-arthritic activity.
MATERIALS AND METHODS
The seeds of fresh unadulterated B.nigra were
collected from the fields of Vikarabad and authenticated by
renowed botanist Professor, Vedavyas, L.V.D College,
Corresponding Author
M. Vinyas
E.mail:- vinyasamayasa7@gmailcom
Raichur, Karnataka. The air dried and powdered seeds of
B.nigra were successively extracted with alcohol in a
soxhlet apparatus and macerated with water. The extract
obtained was made free of any solvent by rotary evaporator
at 400 C under reduced pressure, and dried in a vacuum
oven.
Test animals
Albino rats of either sex weighing between (150-
200 g) were procured from central animal house of N.E.T.
Pharmacy College, Raichur for experimental purpose. The
animals were acclimatized to laboratory conditions for 7
days. The animals were supplied with commercially
available standard diet. Water was allowed ad libitum under
hygienic conditions. The animals were grouped in cages in
an air conditioned room at the temperature of 22 + 1°C
with12 h light and dark cycle. The ethical guidelines for the
investigation of the animals used in experiment were
followed in all the tests.
Acute oral toxicity studies
The acute toxicity of seeds extracts of Brassica
nigra were determined by using female albino rats of
weight between (180-200) g, maintained under standard
conditions. The animals were fasted for 12 hr prior to the
experiments. Animals were administered with single dose
ABSTRACT
The aim of this study was to assess the anti-arthritic activity of Brassica Nigra. Arthritis is induced in the albino rats
by inducing the freunds complete adjuvant. The seeds of freunds adjuvant were coarsely powdered and extracted with ethanol
(95%) and water using soxhlet. The effect of these plant extracts on arthritic rats were assessed by the various blood
parameters and also taking the changes in paw volume. The BN suppressed the anti –arthritic changes induced in rats and
results were statically significant.
Keywords: Brassica Nigra, Anti-arthritic activity, Seed extract.
Vol 2|Issue 2| 2012 | 59-61.
60 | P a g e
of seeds extracts of Brassica nigra (Linn.) and observed for
its mortality up to 48 hr study period (short term toxicity).
Based on the short-term toxicity profile, the next dose was
decided as per OECD guidelines No 425. From the LD50
dose 1/5th dose was selected and considered as high dose.
Induction of arthritis
Rats were injected with Freunds complete
adjuvant, containing 10 mg of heat killed Mycobacterium
tuberculosis in 1ml paraffin oil (0.1ml) into the left paw
intra dermally. The paw volume was measured using
plethysmograph apparatus [4].
Experimental design:
Albino rats of either sex weighing between (150-
200g) were divided into following 5 groups of 6 animals
each. Group I: Negative control (receives vehicle).Group II:
Positive control Group III: Standard Indomethacin (10
mg/kg). Group IV: High dose of alcoholic extract of
Brassica nigra seeds. Group V: High dose of aqueous
extract of Brassica nigra seeds. Groups were treated orally
with the extracts of Brassica nigra, distilled water and
standard respectively until 14 days from the day of
injection of Freunds Complete Adjuvant. During the 14
days of the treatment, the paw volume, body weight, ankle
diameter, rectal temperatures were measured on alternative
days. At the end of the 14th day animals were sacrificed by
cervical dislocation. The serum was separated and for the
further biochemical studies.
Preliminary phytochemical screening:
The extracts were subjected to chemical tests to
detect the chemical constituents like saponins, alkaloids,
glycosides, proteins flavonoids etc., using standard
procedures.
Biochemical assays
Hemoglobin is estimated by using Sahil`s method.
Red blood cells and white blood cells were estimated by the
according to the method of chesbrough and Mc Arthur in
an improved Naeubauer chamber. For biochemical
determination, blood was collected from the retro-orbital
route without any anti-coagulant. After one hour serum was
separated by centrifugation and maintained at -4oc until
further use. The serum was subjected for determination of
Blood Urea Nitrogen (BUN), Total protein, Albumin, and
Calcium by using Semi auto-analyzer.
Statistical analysis
It was performed using Student's unpaired ' t ' test
and P values less than 0.05were considered significant.
Data are represented as mean+SEM
RESULTS
Paw volume:
In arthritic rats, inflammation reached maximum
on day 3 and maintained till day 9. Paw maintained its
inflammation till day 14. A significant reduction was
observed by the standard and extracts treated groups as
compared to group II.
Body weight
The changes in the body weights were compared
on day1 and the day 14. The mean percentage increase in
different groups were calculated and compared with group
2. The changes in body weights for group 1 (14.88%),
group 2 (6.91%), group 3 (12.57%), group 4 (16.91%) and
group 5 (17.39%).
Rectal temperature
There was no significant variation of rectal
temperature from day 1 to day 14 in all the groups they
maintained the normal rectal temperature.
Vernier scale
In arthritic rats, ankle diameter reached maximum
on day 7. Ankle maintained its inflammation till day 14. A
non-significant reduction was observed by the standard and
extracts treated groups as compared to group II.
Table 1.
Parameter
Group I
Group II
Group III
Group IV
Group V
RBCx106/ mm3
5.56±0.20
4.30±0.27
5.80±0.28**
5.79±0.29**
6.217±0.26**
WBC x103/mm3
7679±362
12980±616
7726±296***
8431±519***
7516±476***
Hb (cc/100ml)
13.62±0.41
10.87±0.41
13.54±0.48*
11.63±0.88
11.68±0.79
BUN (mg/dl)
23.22±0.10
29.69±0.99
24.03±0.58***
26.85±0.75*
25.18±1.11**
Total protein (g/dl)
5.26±0.41
4.05±0.37
6.69±0.41***
6.42±0.31***
5.88±0.43**
Albumin (g/dl)
3.48±0.40
3.79±0.39
3.15±0.16*
3.31±0.31
3.54±0.15
calcium (mg/dl)
81.03±2.23
145.19±3.19
84.73±1.14***
101.92±2.80***
93.96±0.53***
DISCUSSION
Adjuvant arthritis in rats is considered to be an
immunologically mediated and most frequently
investigated model of chronic inflammation. This model
depicts the very close similarity with the clinical RA. Due
to inoculation of CFA, there was an increase in the ankle
diameter which signs as an inflammation for the ankle joint.
On the 13th day, there was decrease in the ankle joint in
both the extracts as well as the standard. Among them,
alcoholic extract has given better result than that of the
Vol 2|Issue 2| 2012 | 59-61.
61 | P a g e
standard. Adjuvant rats treated with BN and Indomethacin
either in prophylactic or therapeutic protocol showed
decrease in ankle diameter as compared with that of the
toxicant group. Rectal temperature is an index of
inflammation which is measured by rectal thermometer on
alternate days till 14th day. All the groups excluding
Indomethacin group have shown no significant variation in
rectal temperature on all the alternate days. Whereas in
Indomethacin group there was a little decrease in the rectal
temp throughout the experiment because of its cox
inhibitory action [5]. Paw volume is an index of paw
inflammation which is measured by plethysmograph on
alternate days till 14th day. Both the aqueous and the
standard decreased the paw volume on day 13. Alcoholic
extract produced little effect on paw volume. The ability of
BN to reduce a response to AA is consistence with the
findings of present study on this plant, as traditionally it
was used as an immunosuppressant for arthritis at least in
the early phase of disease. The extract treated group and the
standard increases their body weight on day 13 as that of
the normal animals. Where as in the toxicant group, there is
slight increase in body weight. Both the extracts have
shown an increase in body weight more than that of the
standard and the normal animals signifies its protection
from the body weight loss in arthritic condition.
In the present study, the arthritic rats (group II)
showed a reduced RBC count and Hb level. All these
conditions indicating the anaemic condition and is
commonly noted in patients with chronic arthritis. The two
most common reasons for anaemia in arthritic patients are
gastrointestinal blood loss from arthritic medication and
bone-marrow changes in patients with inflammatory
arthritis which prevents the release of iron for incorporation
into RBCs [6]. Both the extract treated groups and the
standard showed a significant recovery from anaemia. The
increase in WBC count in arthritic rats is the indication for
inflammation which was significantly suppressed in BN
extracts treated rats and Indomethacin treated rats. BUN
parameter is selected to know about the kidney function.
There was increase in the BUN levels in the toxicant group.
There was significant decrease in BUN levels in all the
extracts and Indomethacin treated group of rats. Among
both the extract, the aqueous gave better results than
alcohol extract and comparable with the standard
Indomethacin. The absence of liver damage induced by AA
treated rats was indicated by significant difference in serum
total protein and no increase in serum albumin. But there is
an increased level of total protein and there is no significant
variation in albumin levels indicating no liver damage has
occurred in extracts treated groups and Indomethacin.
Calcium reabsorbed from the bone, enters the blood stream
is enormously excreted through the urine resulting in
hypercalemia and hypercalcuria conditions which was in
line with previous reports. There was significant decrease
in Ca levels in all the extracts and Indomethacin treated
group of rats
The phytochemical analysis of the extracts
revealed that it contains flavonoids, carbohydrates,
glycosides, proteins and alkaloids. Of these flavonoids and
alkaloids are well known for there ability to inhibit pain and
inflammation. Flavonoids also have anti-inflammatory
properties due to their inhibitory effects on enzymes
involved in the production of chemical mediators of
inflammation. Flavonoids are widely utilized in medicine as
anti-osteoporotic agents are shown to increase the density
of the diaphysis in rats induced with osteoporosis.
Experimental studies showed that flavonoids act by
inhibiting osteoclastic bone reabsorption with in vitro and
in vivo. Flavonoids have been known to be responsible
osteoporosis where they increase the bone mineral density
CONCLUSION
This study clearly demonstrates that the anti-
arthritic activity of the B.nigra seed extracts and the results
were comparable to the indomethacin. Further studies are
needed for the isolation for the other compounds
responsible for the isolation for the anti-arthritic potential.
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pepo and Brassica nigra. J Med Plants Res, 3(5), 2009, 429-432.
3. Anand P, Murali KY, Tandon V, Chandra R, Murthy PS. Preliminary studies on anti-hyperglycemic effect of aqueous
extract of Brassica nigra (Linn) Koch. Indian J. Exp.Biol, 45(8), 2007, 696-701.
4. Shivanad Pandey. Various techniques for the evaluation of anti-arthritic activity in animal models. J Adv Pharm Tech Res,
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