Chapter 11: anti-arthritic activity

Chapter 11: anti-arthritic activity

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 Chapter 11: anti-arthritic activity

11. Evaluation of in-vitro anti-arthritic activity of Grewia nervosa by

protein denaturation method

11.1 Introduction

Inflammation is a complex biological response of vascular tissue to harmful stimuli,

pathogens, irritants characterized by redness, warmth, swelling & pain.Prolonged
inflammation leads to rheumatoid arthritis ,atherosclerosis, hey fever, ischemic heart diseases
etc& inflammation is a common manifestation of infectious disease like leprosy, tuberculosis,
syphilis, asthma inflammatory bowel syndrome, nephritis,vascularitis,celiac diseases,
autoimmune diseases etc.

Anti-inflammatory drugs like NSAIDs used to reduce the swelling and pain of inflammation.
But these agents carry the risk of gastro-intestinal toxicity, cardiovascular and other toxicity
for prolonged use . For these reason, there is a need for ant-inflammatory drugs havingless
severe side effects to use for chronic inflammatory disease as well. Therefore, in recent time,
more interest isshown in alternative and natural drugs for treatment ofvarious diseases, but
there is a lack of proper scientific evidences. ( HabiburRahman, 1,2 1M. ChinnaEswaraiah and 2A.M.
Dutta)

The in-vitro anti-arthritic activity was studied using bovine serum albumin(BSA) protein
denaturation method. When BSA is heated it undergoes denaturation & expressed antigens
associated with type-III hypersensitivity reaction & that is related to diseases such as serum
sickness, glomeralonephritis, rheumatoid arthritis & system lupus eruthematosus.
(PHYSALIS ANGULATA)

The methanolic leaves extract of G.nervosa was investigated here very first time for
evaluation of anti-arthritic activity and different concentrations of it were so prepared for this
experiment

11.2 Materials & method

11.2.1 Chemicals &Instruments:

 Diclofenac Sodium (Square Pharmaceuticals,Bangladesh),

 Bovine serum albumine,
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 Chapter 11: anti-arthritic activity

 Phosphate buffer analytical grade.

 Instruments UV spectroscopy, centrifuge.

11.2.2 Preparation test reagents:

0.5% Bovine serum albumin (BSA):

250mg of BSA was dissolved in 50 ml of water.

Preparation of sample:

At first 150 mg of Methanolic leaves extract of G.nervosa in 50ml of distilled water to

prepare 3000µg/ml concentration. This solution is serial diluted to prepare 1500µg/ml,750
µg/ml, 375µg/ml, 187.5µg/ml concentration.

Preparation of test solution (0.5ml): 0.5% w/v aquous solution of BSA (0.45ml) and test
solution(0.05ml) of different concentration were used.

Preparation of test control solution (0.5ml): 0.5% w/v aquous solution of BSA(0.45ml) and
distilled water(0.05ml) were used.

Preparation of product control (0.5ml): 0.45 ml distilled water and test solution (0.05ml) of
different concentrations were used.

Preparation of standard solution (0.5ml): 0.5% w/v aquous solution of BSA(0.45 ml) and
Diclofenac Sodium (0.05 ml) of different concentrations were used.

11.2.3 Procedure:

Test solution (0.05ml) of different concentrations (3000, 1500.750,375 &187.5µg/ml) and

standard drug Diclofenac sodium (0.05ml) of different concentrations (3000, 1500.750,375
&187.5µg/ml) were mixed with 0.5% of w/v aqueous solution of BSA( 0.45ml). Then the
samples were incubated at 370C for 20 min followed by incubation at 57 0C for 3 min. 2.5 ml
of Phosphate buffer (PH 6.3) was added to all the above sample after cooling. UV visible
spectrophotometer was used to measure the absorbance at 255 nm. The control represents
100% protein denaturation. The percentage inhibition of protein denaturation was calculated
by following formula:

Percentage of inhibition = 100-[{(optical density of test solution- optical density of product

control)/ optical density of test control} × 100].

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 Chapter 11: anti-arthritic activity

11.3 Result

Table 8.1: Effect of methanolic leaves extract of G.nervosa on Bovine serum albumin ptotein
denaturation

Treatment Dose (µg/ml) % Inhibition

MLF 3000 56.98
MLF 1500 54.40
MLF 750 47.95
MLF 375 33.11
MLF 187.5 27.74
Diclofenac 3000 80.43
Diclofenac 1500 78.27
Diclofenac 750 65.3
Diclofenac 375 62.7
Diclofenac 187.5 54.83
MLF= Methanolic leaves extract

90
80
70
60
% Inhibition

50
40 Standard
30 G.N
20
10
0
3000 1500 750 375 187.5
Dose (µg/ml)

Fig (8.1): Effect of methanolic leaves extract of G.nervosa on Bovine serum albumin.

Methanolic leaves extract of G.nervosa at a concentrations of 3000, 1500.750,375

&187.5µg/ml showed inhibition of BSA denaturation by 56.98%, 54.4%,47.95%,33.11% &
27.74 % respectively whereas standard drug, Diclofenac sodium at a concentrations of 3000,
1500.750,375 &187.5µg/ml showed inhibition of protein denaturation by 80.43%,
78.27%,65.37%,62.79%, & 54.83% respectively (Table 8.1)

11.4 Discussion

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 Chapter 11: anti-arthritic activity

Rheumatoid arthritis, an autoimmune disease, involves denaturation of protein & hence

production of auto-antigens. Heat induced denaturation of BSA involves presentations of
antigens that are related to diseases such as rheumatoid arthritis .Denaturation of BSA was
inhibited by methanolic leaves extract of G.nervosa which suggested that G.nervosa might
prevent denaturation of protein in rheumatoid arthritis & hence a potential anti-arthritic agent.

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