EVALUATION OF ANTIINFLAMMATORY EFFECT OF Capparis Aphylla Roth. USING EXPERIMENTAL ANIMAL MODELS

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7.      Serum A/G ratio [Mythilypriyaa Rajendran,1997,61, 1861 – 1878.]

Estimation of Protein Contents:

Principle:
Proteins, in an alkaline medium, bind with the cupric ions present in the biuret reagent to form a blue-violet coloured complex; the intensity of the color formed is directly proportional to the amount of protein present in the sample.

Reagents:
Reagent A: Biuret reagent.
Reagent B: Protein standard.
Pipette into clean dry test tubes labeled as blank, standard and test.

Addition Sequence

Blank

Standard

Test

Biuret Reagent

1.0

1.0

1.0

Distilled water

0.02

-

-

Protein standard

---

0.02

-

Sample

---

---

0.02

Mix well and incubate at 37oC for 10 min. or at room temperature for 30 min. Measure the absorbance of the standard, test sample against the blank within 60 min at 550nm.

Calculation:
Total proteins in gm/ dl = (Abs T/ Abs S) X 8
Normal Range: 6-8gm/dl

Determination of Serum Albumin: (Godkar. 2005)

Name of the method: Bromocresol green method.

Principle: Albumin present in serum binds specifically with bromocresol green at pH 4.1 to form green colored complex, intensity of which can be measured colorimetrically by using 640 nm.

Normal Range: 3.3-4.8 g/dL.

Preparation of reagents:

1) Albumin reagent (ready to use): It is prepared by mixing following chemicals in 900    ml distilled water.
a) Succinic acid: 8.85 gm
b) Bromocresol green: 108 mg
c) Sodium azide: 100 mg
d) Brij-35: 4.0 ml

pH of this solution is adjusted by using 1 N sodium hydroxide to 4.1. Final volume is    made to one litre by using distilled water. It is stable at room temperature for one year.

2) Albumin standard (4gm/dl): Bovine albumin 4.0 gm in 100 ml of normal saline    containing 0.1 gm/dl sodium azide. It is stable at 2-8°C for one year.

Procedure:

Mono step method

  • Take three test tubes and labeled them as test, standard and blank.
  • Add 5 ml of albumin reagent in each of the test tube.
  • Then add 0.05 ml of serum, albumin standard and distilled water respectively.
  • Mix thoroughly and keep at room temperature (25°C+ 5° C) for exactly 10 minutes.
  • Measure the intensities of the test and standard by setting at 100% T, by using 640 nm (red filter).

Calculation:
Serum albumin g/dl = O.D. test/ O.D. Std. x 4

Determination of Globulins:
Normal range = 1.8-3.6 g/dl

A/G ratio = serum albumin (g/dl) / serum globulins (g/dl)
Normal range = 1.2:1 to 2:1

8.       Histopathological analysis [chemico biologicalinteraction, [ijpd, curative effect on rat]
The proximal interphalangeal joint of the hind paw of the rats were removed and separated from the surrounding tissues and weighed. The joints fixed in 10% formalin were decalcified, sectioned and finally stained with haematoxylin and eosin to examine the histopathological changes during the experimental period in all the groups under light microscope.

Cartilage and bone destruction by pannus formation was scored ranging from 0, no change; 1, mild change (pannus invasion within cartilage);  2, moderate change (pannus invasion into cartilage/subchondral bone); 3, severe change (pannus invasion into the subchondral bone); and vascularity (0, almost no blood vessels; 1, a few blood vessels; 2, some blood vessels; 3, many blood vessels). Histopathological  changes in the knee joints were scored in the femur region on 5 semiserial sections of the joint, spaced 70  μm apart. Scoring was performed on decoded slides by two observers, as described earlie

9.       Radiological analysis[chemico biologicalinteraction, [ijpd, curative effect on ra]
Before sacrificing the animals, X-rays were taken at the joints of the hind paw of the animals for evaluating the bone damage. Radiographs were taken using X-ray apparatus and industrial X-ray ?lm. The X-ray apparatuswas operated at 220Vwith a 40V peak, 0.2 s exposure time, and a 60 cm tube-to-?lmdistance for anterior–posterior projection.

The following radiograph criteria were considered: These scores (destroyed or intact joint) were used as a quantal test for bone necrosis. Radiographs were carefully examined and abnormalities were graded as follows:
(i)  Periosteaic reaction, 0 - 3 (none, slight, moderate, marked);
(ii)  Erosions, 0 - 3 (none, few, many small, many large);
(iii) Joint space narrowing, 0  - 3 (none, minimal, moderate, marked);
(iv) Joint space destruction, 0 - 3 (none, minimal, extensive, ankylosis).
Bone destruction was scored on the patella as described previously.

RESULTS

A.    Body Weight Change
The body weight of the control rats progressively increased while the gain in body weight of arthritic rat was retarded significantly. Drug treated group showed progressive increase in body weight.


Figure: Effect of methanolic extract of Capparis aphylla Roth. on % body   weight change.

Values are expressed as mean ± SEM. N=6.Values are statistically evaluated using ANOVA analysis followed by Dunnett’s test. Significant values were compared with #P<0.05 normal control Vs model control & *P<0.05 model control Vs all MECA groups

B.     Arthritic Index
In this study, the incidence of inflammation was 100% on administration of freund’s adjuvant (i.e all the rats responded with an arthritic scored >1), if the rats were not treated with anti inflammatory agent. There was a difference in the severity of  inflammation being less in treated group and this could be attributed to the action of standard or MECA.


Figure :Effect of methanolic extract of Capparis aphylla Roth. on arthritic index.

Values are expressed as mean ± SEM. N=6.Values are statistically evaluated using ANOVA analysis followed by Dunnett’s test. Significant values were compared with #P<0.05 normal control Vs model control & *P<0.05 model control Vs all MECA groups 

C.    Paw Volume
An intraplantar  injection of  CFA into the left hind paw produced a more marked inflammatory response in the model control animals than the normal control(Fig 4.4.3).Seven days after CFA injection, the edema volume were significantly higher in model control animal than normal control animals


Figure : Effect of methanolic extract of Capparis aphylla Roth. on paw edema.

Values are expressed as mean ± SEM. N=6.Values are statistically evaluated using ANOVA analysis followed by Dunnett’s test. Significant values were compared with #P<0.05 normal control Vs model control & *P<0.05 model control Vs all MECA groups

D.    Erytrocyte Sedimentation Rate (ESR)
The indomethacin, MECA 190 mg/kg, MECA 240 mg/kg and MECA 300 mg/kg  treated groups ESR was significantly lower as compared to model control  group (Fig4.4.4).


Figure :Effect of methanolic extract of Capparis aphylla Roth. on ESR.

Values are expressed as mean ± SEM. N=6.Values are statistically evaluated using ANOVA analysis followed by Dunnett’s test. Significant values were compared with #P<0.05 normal control Vs model control & *P<0.05 model control Vs all MECA groups

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