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

Pharma courses

Pharma Admission

pharma courses

pharma admission



An arthritic index for each animal was be calculated as the sum of these scores. The average scores for each group of drug treated animals was be compared with that of disease control animals

3.       Paw edema[Magari Katsue, 2003 139, 927–934.]
Paw inflammation was be quantified based on paw swelling . The volume of the left hind  paw was be measured before and after arthritis induction by a mercury displacement method using a plethysmometer for rats. Paw swelling was be expressed as % inhibition of using following formula:


Evaluation:
a).For primary lesions:The percent inhibition of paw volume of the injected left paw over vehical control was be measured at day 7.
b).For secondary lesions:The percent inhibition of paw volume of the non-injected right paw over  control was be measured at day 21.

4.      Erythrocyte Sedimentation Rate ( ESR) [Mythilypriyaa Rajendran,1997,61, 1861 – 1878]
Higher erythrocyte sedimentation rate is indicator of inflammatory disorder.

Method:  WESTERGREN’S METHOD

Requirements:
1)Westergren’s pipette and Westergren’s stand (2) Oxalate bulb, (3) Sodium Citrate  solution

Westergren’s pipette: It has a bore of 3mm and a length of 300 mm. It is marked with graduations from 0 to 200. It is open from both the ends.

Westergren’s Stand: It can accommodate six pipettes. There is base containing six   grooves each containing a rubber cushion to prevent leakage of blood from Westergren’s pipette. The stand allows the pipettes to remain in exactly vertical position.

Procedure:
1)A sample of blood (about 3 ml) was be obtained by puncturing retro-orbital plexus and  was be mixed with 3.8% sodium citrate solution in proportion of four parts of blood to one  part of citrate soon. The mixing of blood was be done by rotating the sample gently between the palms of hands.
2) The blood was be sucked slowly up to the mark zero in the Westergren’s tube.
3) The tube was be set upright in the Westergren’s stand, taking care that no blood escapes.  The tube was be fixed with the help of screw cap.
4)At the end of one hour and two hours, the upper level of red blood cell column was be     read. It was be indicator of mm of clear plasma or ESR.

5.      Serum Rheumatoid Factor (RF) [Jung  Hyun-Ju,2005, 359–367]

Principle Of The Method:
The RF-Turbilatex is a quantitative turbidimetric test for the measurement of RF in human serum or plasma. Latex particles coated with human gammaglobulin are agglutinated when mixed with samples containing RF. The agglutination causes an absorbance change, dependent upon the RF contents of sample that can be quantified by comparison from a calibrator of known RF concentration.

Preparations:

Diluent (R1)

Tris buffer 20 mmol/L, pH 8.2

Sodium azide 0.95g/L

Latex (R2)

Latex particles coated with human gamma globulin, pH 7.4

Sodium azide 0.95g/L

RF-CAL

Calibrator. Human serum. The RF concentration is stated on the vial   label.

Optional

Ref.:  1102114 Control serum ASO/CRP/RF level L

Ref.: 1102115

Control serum ASO/CRP/RF Level H.

Reagents for RF test

For RF Calibrator:
RF Calibrator was be reconstituted with 2.0 ml of distilled water. It was be mixed gently and brought to room temperature for 10 minutes before use.

For Calibration curve (range from 20 to 160 IU/ml):
The following RF calibrator dilutions was be prepared in the normal saline. The concentration of the RF calibrator was be multiplied by the corresponding factor stated in table below to obtain the RF concentration of each dilution.

Calibrator dilution

1

2

3

4

5

6

Calibrator RF (ml)

  ---

100

10

90

25

75

50

50

75

25

100

---

Factor

0

0.1

0.25

0.5

0.75

1.0

For one point calibration (linear range up to 100 IU/ml):
Preparation for RF calibrator dilution: 30ml RF calibrator + 70 ml normal saline The RF calibrator concentration was be multiplied by 0.33 to obtain the RF concentration of the diluted calibrator.

Procedure:
1. The reagents and the photometer (cuvette holder) was be brought to 37°C.

2. The following assay conditions was be described:
Wavelength: 650 nm (600-650 nm)
Temperature: 37°C
Cuvette light path: 1 cm

3. The instrument was be adjusted to zero with distilled water.

4. The following reagents was be pipette into a cuvette and mixed.

5. Absorbance was be read after 2 minutes (A2) of the sample addition.


Ablank

A2 Calibrator/ Sample

NaCl 9.0 g/l (ml)

7.0

-

Calibrator or sample (ml)

-

7.0

R1: Diluent (ml)

0.9

0.9

R2: Latex (ml)

0.1

0.1

Calculations:

For calibration curve:
Calculated the absorbance difference (A2- Ablank reagent) of each point of the calibration curve and plotted the values obtained against the RF concentration of each calibrator dilution. Rheumatoid factor concentration in the sample was be calculated by interpolation of its (A2-Ablank reagent) in the calibration curve.

For one point calibration:
[(A2-Ablank reagent) sample] / [(A2-Ablank reagent) calibrator] X Diluted calibrator concentration = IU/mL RF

The reading of RF factor of all the groups obtained was be compared with the control animals and was be expressed as IU/mL RF.

Reference Values :
Normal values up to 20 IU/mL. Each laboratory should establish its own reference range.

6.      Serum C - reactive protein (CRP) [Mythilypriyaa Rajendran,1997,61, 1861 – 1878.]

Principle:
C-reactive protein is measured by turbidimetry method of analysis. Turbidimetry is the measurement of the reduction in light transmission caused by particle formation. Light transmitted in forward direction is detected. The amount of light absorbed by suspension of particles depends on specimen concentration and on the particle size. Solution requiring quantitation by turbidimetry is measured using visible spectrophotometers.

Reagents:

Reagent 1: Diluent
Tris Buffer 20 mmol/L
Sodium Azide 0.95g/L pH 8.2

Reagent 2: Latex reagent
Suspension of Latex particles coated with anti human CRP
Sodium Azide 0.95g/L

Reagent 3: CRP Calibrator
Lyophillized Human Serum
CRP concentration on label

Calibration:
The assay was be calibrated to the reference material CRM 470/RPPHS.

Reagent Preparation & Stability:

Working Reagent:  Contents of the Latex Reagent vial was be gently mixed and working reagent was be prepared as,
1 ml Latex Reagent + 9 ml Diluent
This working reagent was be stable for 30 days at 2-8° C.

CRP Calibrator: Contents of vial was be reconstituted with 1.0 ml of distilled water and kept for 10 minutes before use.
This CRP calibrator was be stable for 30 days at 2-8° C or 3 months at -30° C.

Samples:
Fresh serum is preferred, though samples stored for 8 days at 2-8° C or 3 months at -20° C may also be used.

Equipments required:
Photometer,
Liquid dispensing system
General laboratory equipments.

Procedure:

Assay protocol

Wavelength : 540 nm (530-550nm)
Blank : Distilled water
Temperature : 37° C
Cuvette Path Length : 1 cm

Dispense

Calibrator

Samples

Working Reagent

500 µL

500 µL

Calibrator

Sample

3 µL

3 µL

Reagents for CRP test
Blank reading was be adjusted with distilled water. Working reagent and Calibrator/Sample was be pipetted out as per the pipetting system. Absorbance was be measured immediately (A1) and exactly after 2 minutes (A2) of sample addition.

Auto analyzer was be suitably programmed in fixed time mode with a nominal delay of 10 sec and a read interval of 120 sec.

Calculation:
(A2-A1) sample       X Calibrator Concentration
(A2-A1) Calibrator

Reference Values
Normal values up to 6 mg/L.
Each laboratory should establish its own reference range.

NOW YOU CAN ALSO PUBLISH YOUR ARTICLE ONLINE.

SUBMIT YOUR ARTICLE/PROJECT AT articles@pharmatutor.org

Subscribe to Pharmatutor Alerts by Email

FIND OUT MORE ARTICLES AT OUR DATABASE


 

Pages

FIND MORE ARTICLES