2HOD, Department of Anatomy and Physiology, Gokaraju Rangaraju College of Pharmacy, Osmania University, Hyderabad, India
3Professor, Department of Pharmacology, Gokaraju Rangaraju College of Pharmacy, Osmania University, Hyderabad, India
Key words: Tecoma stans; Antiarthritic activity; Complete freund’s adjuvant
If inflammation goes unchecked, it can damage cartilage, the elastic tissue that covers the ends of bones in a joint, as well as the bones themselves. Over time, there is loss of cartilage, and the joint spacing between bones can become smaller. Joints can become loose, unstable, painful and lose their mobility. Joint deformity also can occur. Joint damage cannot be reversed, and because it can occur early, doctors recommend early diagnosis and aggressive treatment to control RA.
Rheumatoid arthritis most commonly affects the joints of the hands, feet, wrists, elbows, knees and ankles. The joint effect is usually symmetrical. That means if one knee or hand if affected, usually the other one is, too. Because RA also can affect body systems, such as the cardiovascular or respiratory systems, it is called a systemic disease. [1]
It affects about 1% of the population of world in a female and male ratio of 2.5:1. (R1) It caused by number of pro inflammatory molecules released by macrophages including reactive oxygen species and Eicosanoids such as prostaglandins, leukotrienes and cytokines. The regulation of these mediators secreted by macrophages and other immune cells and modulation of Arachidonic acid metabolism by inhibiting enzymes like COX and LOX are the potential target for chronic inflammatory conditions. [2]
Even though various categories like immunosuppressants, NSAIDs, steroidal anti-inflammatory drugs are being used till now, the potential side effects give a limitation for their use. Now it is a growing concern allover for the development of new safe, potent, less toxic Antiarthritic drug. Hence, there is a need to explore for more naturally available alternatives, so that their therapeutic values can be assessed and expanded. [3]
Plants are one of the most important sources of medicines. The use of natural remedies for the treatment of inflammatory and painful conditions has a long history, starting with Ayurvedic treatment, and extending to the European and other systems of traditional medicines. Plant drugs are known to play a vital role in management of inflammatory diseases. [4]
Tecoma stans Linn belonging to family Bignoniaceae commonly known as Yellow bells in India. The plant has been reported to have anti-inflammatory, antibacterial, antidiabetic, antispasmodic, anti-nociceptive, nephroprotective and anticancer properties. Tecoma stans plant have traditional claim for use in arthritic disorder. But no pharmacological work has been done on evaluation of its Antiarthritic activity. So the present study was carried out to evaluate Antiarthritic effect of methanolic stem extract of Tecoma stans in wistar albino rats. [5]
Healthy adult Swiss mice (20-30 gm) were subjected to acute oral toxicity studies as per Organization for Economic Cooperation and Development (OECD) guidelines. Animals were observed individually after dosing at least once during the first 30 min, periodically during the first 24 h, with special attention given during the first 4 h, and daily thereafter, for a total of 14 days. The changes in skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic, central nervous system, somatomotor activity and behaviour pattern were noted.
The percentage inhibition of protein denaturation was calculated by the following formula:
Groups |
Treatment |
I |
Rats received normal saline (s.c) |
II |
Rats received 0.1 mL of egg albumin (s.c) |
III |
Rats received METS (200 mg/kg bd. wt, p.o) + 0.1 mL of Egg albumin (s.c) |
IV |
Rats received METS (400 mg/kg bd. wt, p.o) + 0.1 mL of Egg albumin (s.c) |
V |
Rats received diclofenac sodium (15 mg/kg bd. wt, p.o) + 0.1 mL of Egg albumin (s.c) |
The in vivo anti-arthritic study was evaluated using egg albumin induced edema (Phlogistic agent) model. 0.1 mL of egg albumin from fresh egg was subcutaneously injected into the right hind paw of the rats. The rats were administered the test sample and reference drug 30 minutes before inducing inflammation.
The rat paw volume was measured at intervals of 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min and 24 hrs. The % inhibition of the edema was determined by the following equation;
% Inhibition = Io –Ii/ Io X 100
Io = change in paw volume in control group;
Ii = change in paw volume in treated group
Groups |
Treatment |
I |
Rats received normal saline (p.o) |
II |
Rats received 0.1 mL of Complete Freund's adjuvant (s.p) |
III |
Rats received 0.1 mL of Complete Freund's adjuvant (s.p) + METS (200 mg/kg bd. wt (p.o), 8-21 days) |
IV |
Rats received 0.1 mL of Complete Freund's adjuvant (s.p) + METS (400 mg/kg bd. wt (p.o), 8-21 days) |
V |
Rats received 0.1 mL of Complete Freund's adjuvant (s.p) + diclofenac sodium (15 mg/kg bd. wt (p.o), 8-21 days) |
Rat adjuvant arthritis is widely used for evaluation of various anti-arthritic agents. The hallmarks of this model are reliable onset and progression of robust easily measurable, periarticular inflammation, marked bone resorption and periosteal bone proliferation. In this model wistar albino rats (150-200, grams 6/group) are generally used in studies of adjuvant arthritis. Induction of adjuvant arthritis is done with Complete Freund’s Adjuvant (CFA).
Rats were injected, subcutaneously, 0.1 mL of complete Freund’s adjuvant into the plantar region of the left hind paw. The changes in the paw volume (left and right) were measured on various days up to 21 days. The herbal product at (200, 400 mg/kg/day) and diclofenac sodium at (15 mg/kg) doses were administered orally for 14 days after 7 days of Freund’s adjuvant administration.
On the 21st day rats were sacrificed by cervical dislocation and ankle joints were isolated from the injected hind paw. [8]
The percentage yield of the extract was found to be.
% yield of extract = Amount of extract obtained/ Amount of powder used ×100
= 15g / 250g × 100
= 6 % w/w
Phytochemical constituents |
Inference |
Alkaloids |
++ |
Glycosides |
++ |
Saponins |
++ |
Steroids |
++ |
Flavonoids |
++ |
Phenols |
+ |
Tannins |
+ |
Fats |
_ |
Proteins and amino acids |
+ |
The Antiarthritic activity of the extract was further confirmed by using albumin denaturation method. Standard diclofenac sodium at a dose of 400 mg/mL produced prominent % stabilization (95.20%). The methanolic extract of the plant showed marked increase in the % stabilization. The activity of 400 mg/mL of extract (85.60%) was found to be better than that of 100 mg/kg (56.10%). The activity of the 400 mg/mL of the extract was comparable to that of standard.
S. No |
Treatment |
Concentration(µg/mL) |
Absorbance (660 nm) |
% Inhibition |
1 |
Saline |
_ |
4.1 |
_ |
2 |
METS |
100 |
1.8 |
56.1 |
3 |
METS |
200 |
1.2 |
70.2 |
4 |
METS |
300 |
0.9 |
78.1 |
5 |
METS |
400 |
0.6 |
85.6 |
6 |
Diclofenac sodium |
400 |
0.2 |
95.2 |
Groups |
Treatment |
Mean paw volume (mL) |
||||||
Time in minutes |
||||||||
0 |
30 |
60 |
90 |
120 |
180 |
1440 |
||
I |
Saline |
0.22±0.04 |
0.22±0.03 |
0.23±0.04 |
0.24±0.02 |
0.24±0.05 |
0.24±0.08 |
0.24±0.03 |
II |
0.1 mL egg albumin |
0.23±0.03 |
0.66±0.05a |
0.74±0.02a |
0.85±0.04a |
0.89±0.04a |
0.92±0.06a |
0.93±0.04a |
III |
METS 200 mg/kg + 0.1 mL egg albumin |
0.22±0.02 |
0.54±0.03*,a |
0.52±0.03**,B |
0.50±0.09**,A |
0.50±0.02**,A |
0.48±0.02**,A |
0.48±0.04*,A |
IV |
METS 400 mg/kg + 0.1 mL egg albumin |
0.24±0.04 |
0.50±0.02*,B |
0.46±0.03**,B |
0.42±0.02**,B |
0.39±0.03**,a |
0.38±0.06**,a |
0.36±0.09**,B |
V |
Diclofenac sodium (15 mg/kg) + 0.1 mL egg albumin |
0.21±0.03 |
0.46±0.03**,b |
0.43±0.02**,b |
0.40±0.04**,b |
0.36±0.05**,b |
0.34±0.03**,b |
0.31±0.05**,b |
Groups |
Treatment |
Dose (mg/kg) |
Mean paw volume (mL) |
|||
0th Day |
7th Day |
14th Day |
21st Day |
|||
I |
Normal control |
Saline |
0.20±0.04 |
0.20±0.06 |
0.20±0.01 |
0.22±0.08 |
II |
CFA |
0.1mL |
0.25±0.02 |
0.75±0.02a |
0.94±0.01a |
0.92±0.09a |
III |
CFA +METS |
0.1mL+200 mg/kg |
0.30±0.04 |
0.68±0.02,*,a,A |
0.63±0.02**,a |
0.60±0.10*,a |
IV |
CFA +METS |
0.1mL+400 mg/kg |
0.30±0.02 |
0.60±0.04*, a |
0.50±0.03**, B |
0.46±0.02**, B |
V |
CFA +Diclofenac sodium |
0.1mL+15 mg/kg |
0.25±0.05 |
0.55±0.02** |
0.46±0.01**, b |
0.40±0.02**,b |
Rheumatoid arthritis, an autoimmune disease, involves denaturation of proteins and hence production of auto-antigens. Invitro Antiarthritic activity of the methanolic extract was carried out by the protein denaturation method.
Denaturation of protein is one of the causes of rheumatoid arthritis. Production of autoantigens in certain rheumatic diseases may be due to in vivo denaturation probably involves alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding. Several anti-inflammatory drugs have shown dose dependent ability to inhibit internally induced protein denaturation. In our present study, METS (Methanolic stem extract of Tecomastans) inhibited heat induced protein denaturation and may be one of the reason of possessing anti-arthritic activity. Inflammation is the tissue response to injury and involves a complex process of enzymatic reactions. [10]
In the present study, the Antiarthritic activity of METS was evaluated by using two in vivo experimental models of arthritis, viz. Egg albumin and Complete Freund’s Adjuvant induced arthritis. Egg albumin induced model is an acute method of screening of anti-arthritic potential. It involves tissue mediated response and localized inflammation is produced by Egg albumin injection into the rat paw. In the present study the methanolic extract of Tecoma stans at doses 200 and 400 mg/kg bd.wt significantly inhibited the paw edema induced by Egg albumin. The effect may be due to certain changes in inflammatory response which are comparable with Diclofenac sodium. Thus, to further confirm the activity of the extract, its efficacy in reducing joint inflammation in CFA induced arthritis in rats was evaluated.
CFA – induced arthritis is a chronic model used to study the pathogenesis of rheumatoid arthritis for testing therapeutics. One of the reasons for the wide utilization of this model is a strong correlation between the efficacy of therapeutic agents in this model and in rheumatoid arthritis in humans, and this model is characterized by swelling in joints with the influence of inflammatory cells, erosion of joint cartilage and bone destruction and remodeling.
Also, soft tissue swelling around the ankle joints was appeared during the progress of arthritis in CFA injected rats, which was considered as oedema of the exacting tissues. Bacterial peptidoglycan and muramyl dipeptide are responsible for the induction of adjuvant arthritis. Determination of paw oedema is according to the grapevine simple, susceptible and rapid procedure to evaluate the degree of inflammation and assess the therapeutic effects of drugs.
CFA induced significant inflammation of the ankle joint of the rats. METS at doses 200 and 400 mg/kg bd.wt significantly inhibited paw swelling in arthritic rats. The inhibition of inflammation by the extract might be due to inhibition of bacterial peptidoglycan and muramyl dipeptide which are responsible for the swelling, bone and cartilage erosion. There was significant inhibition of weight loss in the extract treated group and it was comparable to standard diclofenac. This inhibition might be due to prevention of production of cytokine such as TNF-α and interleukin-1B by the phytoconstituents present in the extract. [11-15]
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