**2.6. Anti-inflammatory activity by antidenaturation activity**

The anti-inflammatory activity of *Opuntia elatior* was studied by using inhibition of albumin denaturation technique [15].

#### *2.6.1. Preparation of test sample and standard solution*

Stock solution is prepared by dissolving 5 mg of methanolic, hexanoic and distilled water extract in to 5 ml of methanol, hexane and distilled water respectively to give the concentration of 1 mg/ 1 ml. From this stock solution 100, 200, 300, 400, 500 μl of each solution was taken and added in to 900, 800, 700, 600, 500 μl of their respective solvents, to make 1 ml of working standard solution. 5 mg of Diclofen tablet was dissolved in 5 ml of methanol water to make 1 mg/ 1 ml of concentration. From this stock solution 100, 200, 300, 400, 500 μl of solution was taken and added in to 900, 800, 700, 600, 500 μl of methanol respectively to make 1 ml of working standard. **Procedure:** 0.05 μl of test samples were taken from working stand and 0.45 ml of 5% BSA solution was added to it. The tubes were incubated for 30 min at 37°C in an incubator. A 2.5 ml of PBS buffer was then added and the O.D was taken at 660 nm. Distilled water was served as a blank. Distilled water and BSA solution was taken as a control.

Calculation

**2.5. Antioxidant activity by DPPH method**

128 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

ger added to DPPH reagent solution [14].

Calculation

denaturation technique [15].

*2.5.1. Preparation of standard solution, test sample and DPPH solution*

antiradical activity is calculated by using the following equation.

**2.6. Anti-inflammatory activity by antidenaturation activity**

*2.6.1. Preparation of test sample and standard solution*

% *of antiradical activity* <sup>=</sup> *Control absorbance* <sup>−</sup> *Sample absorbancex* \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *Control absorbance*! <sup>×</sup> <sup>100</sup>

served as a blank. Distilled water and BSA solution was taken as a control.

This method is simple and sensitive. The assay is based on the theory of hydrogen donor is an antioxidant. It measures compounds that are total radicle scavengers. DPPH accept hydrogen from an antioxidant. The antioxidant effect is proportional to disappearance of DPPH in the test sample. These methods involve measurement of decrease in absorbance of DPPH at its absorption maxima of 516 nm, which is proportional to concentration of free radicle scaven-

Standard solution is prepared by dissolving 10 mg of ascorbic acid in 10 ml of methanol to give the concentration of 1 mg/1 ml. Stock solution is prepared by dissolving 5 mg of methanolic, hexanoic and distilled water extract in to 5 ml of methanol, hexane and distilled water respectively to give the concentration of 1 mg/1 ml. 0.1 mM DPPH is prepared by dissolving 11 mg of DPPH in 8.46 ml of distilled water. It is protected from light by covering the tubes by aluminum foil. **Procedure:** (1) In 3 ml of methanol, 150 μl DPPH is added and reading is taken at 516 nm as control. (2) 0.2, 0.4, 0.6, 0.8, and 1.0 ml aliquots are taken from the test sample. (3) The test sample is diluted by adding methanol up to 3 ml. (4) 150 μl DPPH is added to each of the tubes. (5) Absorbance is taken by UV–visible spectrophotometer at 516 nm. (6) The % of

The anti-inflammatory activity of *Opuntia elatior* was studied by using inhibition of albumin

Stock solution is prepared by dissolving 5 mg of methanolic, hexanoic and distilled water extract in to 5 ml of methanol, hexane and distilled water respectively to give the concentration of 1 mg/ 1 ml. From this stock solution 100, 200, 300, 400, 500 μl of each solution was taken and added in to 900, 800, 700, 600, 500 μl of their respective solvents, to make 1 ml of working standard solution. 5 mg of Diclofen tablet was dissolved in 5 ml of methanol water to make 1 mg/ 1 ml of concentration. From this stock solution 100, 200, 300, 400, 500 μl of solution was taken and added in to 900, 800, 700, 600, 500 μl of methanol respectively to make 1 ml of working standard. **Procedure:** 0.05 μl of test samples were taken from working stand and 0.45 ml of 5% BSA solution was added to it. The tubes were incubated for 30 min at 37°C in an incubator. A 2.5 ml of PBS buffer was then added and the O.D was taken at 660 nm. Distilled water was % *of anti* <sup>−</sup> *denaruration activity* <sup>=</sup> *Control absorbance* <sup>−</sup> *Sample absorbancex* \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *Control absorbance*! <sup>×</sup> <sup>100</sup>

#### **2.7. Qualitative phytochemical analysis**

The qualitative phytochemical analysis of test for alkaloids, test for flavonoids, test for phytosterols, test for saponin, test for phenol and test for tannin perform by standard method reported by Parekh and Chanda (2008) [16].

#### **2.8. Thin Layer chromatography**

The separate chemical mixtures using the thin layer chromatography (TLC). TLC is performed on a sheet of aluminum foil, thin layer coated with of adsorbent material use the silica gel. This thin layer of adsorbent is known as the stationary phase. The solvent mixture (mobile phase) is drawn up the plate via capillary action and after the sample has been applied on the plate. Separation is achieved based on the different ascends the TLC plate at different rates [17]. **Procedure:** 10 ml of Methanol: chloroform (6: 4) was taken in glass beaker as a stationary phase. Test sample was applied on thin layer of absorbent material using capillary tube. This absorbent sheet was placed in beaker containing methanol: chloroform solvents and allowing it to run. After running of sample with solvent mixture absorbent sheet was removed from the beaker and was allowed it to dry. After drying of sheet, it was observed in UV light at high and short wavelength. Spray of concentrated sulfuric acid was applied to it to detect presence of alkaloids in the sample.

Calculation

Convexation 
$$Rf \text{ value} = \frac{\text{Distance travelled by solute}}{\text{Distance travelled by solvent}}$$

#### **2.9. HPTLC analysis**

HPTLC analysis based on the principles of adsorption the separation of compound. The mobile phase solvent flows based upon the principle of capillary action. The chemical components separation according to their affinities toward the absorbent. The stationary phase travels slower because component with more affinity. Travels faster the chemical molecule component with lesser chemical charge toward the stationary phase. Thus, the component separated on a chromatographic plate. A 10 mg of methanolic extract was dissolved in 1 ml of methanol. This test solution was used for HPTC analysis. HPTLC aluminum sheet precoated with silica gel was used as the adsorbent. The chromatographic development chamber was saturated with mobile phase to place the plates. The plates were run and derivatized. The derivatized plates were heated, bands were observed and scanned at 254 nm and photographs were taken for record.
