3.3.4 Determination of the effects of Xancus pyrum on alpha esterase activity in cisplatin treated animals (azodye coupling method, Bancroft and Cook, 1984)

## 3.3.4.1 Principle

Esterase enzyme present or absent in monocytes hydrolyses the substrate alphanaphthyl acetate to form an invisible primary reaction product (PRP). The complex is coupled with the diazonium salt to produce coloured final reaction product under the microscope [12].

#### 3.3.4.2 Reagents

1.Fixative solution. Formaldehyde (HCHO 37%, 25 ml) acetone (45 ml), double distilled water (30 ml), disodium hydrogen phosphate (Na2HPO4-20 mg) and potassium dihydrogen phosphate (KH2PO4-100 mg).

Solution A. Pararosaline was prepared by dissolving 1 g powder in 20 ml double distilled water and 5 ml HCl. Gently warmed solution is filtered and stored in dark at 4°C.

Reitman and Frankel method is an end-point colorimetric method for the esti-

2. 2,4-DNPH Colour Reagent 2,4-Dinitrophenyl hydrazine

3. Sodium hydroxide, 4 N Sodium hydroxide 4. Working pyruvate standard, 8 mM (150 IU/L) Sodium pyruvate

• Solution I: Dilute 1 mL of Reagent 3 to 10 mL with purified water.

Pipette into tube marked Blank Standard Test Control

Reagent 1 0.25 0.25 0.25 0.25 Serum — — 0.05 — Standard — 0.05 — —

Reagent 2 0.25 0.25 0.25 0.25 Deionized water 0.05 — —— Serum — —— 0.05

Solution I 2.5 2.5 2.5 2.5

Mix well and read the optical density against purified water in a photometer at

<sup>¼</sup> absorbance of test � absorbance of control � concentration of standard absorbance of standard � absorbance of blank

Mix well and allow to stand at room temperature (15–30°C) for 20 min

where concentration of standard = 150 IU/L.

Phosphate buffer Preservative Stabilizer

Preservative Stabilizer

Preservative Stabilizer

(3)

Reagent no. Reagents Composition 1. Buffered alanine-α-KG substrate, pH 7.4 L-alanine α-KG

Chemoprotective Effect of Edible Gastropod, Xancus pyrum and Its Usefulness…

mation of enzyme activity.

DOI: http://dx.doi.org/10.5772/intechopen.88655

Working reagent preparation:

Mix well and incubate at 37°C for 30 min

• Reagent 1, 2 and 4 are ready to use.

3.4.1.2 Reagents

3.4.1.3 Procedure

Volume in mL

505 nm, within 15 min. Calculation:

57

AST GPT ð Þ activity IUð Þ =L


#### 3.3.4.3 Procedure

Bone marrow from both femurs of mice was collected in PBS, washed thrice and smeared over the slides. Air dried slides were fixed in freshly prepared fixative 30 s at 4°C and dipped in double distilled water thrice. Air dried slides were incubated at room temperature in the following freshly prepared filtered solution. 1.2 ml solution A and 1.2 ml solution B was mixed well and allowed to react for 1 min after which solution C was added and was made up to 50 ml solution by phosphate buffer (pH 7.4).

Slides were incubated in above solution for 45 min at 37°C. After incubation slides were washed in double distilled water for 10 min and counter stained with haematoxylin for 1 min. After staining slides were washed in water for long time and observed under microscope (100�, oil immersion) for scoring positive and negative alpha esterase cells out of 4000 cells.

#### 3.4 Determination of the effect of Xancus pyrum on enzyme levels in cisplatin treated animals

Liver homogenates were made in ice cold Tris buffer (0.1 M pH 7.4) and was used for the estimation of SGOT, SGPT, urea and creatinine. Serum was also used to estimate all the above parameters.

#### 3.4.1 Estimation of SGPT (Span Diagnostics Ltd., Surat, India)

#### 3.4.1.1 Principle

Alanine aminotransferase (ALT) catalyses the transamination of L-alanine and α-ketoglutarate to form pyruvate and L-glutamate. Pyruvate so formed is coupled with 2,4-dinitrophenyl hydrazine (2,4-DNPH) to form a corresponding hydrazone, a brown coloured complex in alkaline medium and this can be measured colorimetrically [13].

L � alanine þ α � ketoglutarate ⇄ pyruvate þ L � glutamate (1)

Pyruvate þ 2, 4 � DNPH ⇄ corresponding hydrazone brown colour ð Þ (2)

Chemoprotective Effect of Edible Gastropod, Xancus pyrum and Its Usefulness… DOI: http://dx.doi.org/10.5772/intechopen.88655

Reitman and Frankel method is an end-point colorimetric method for the estimation of enzyme activity.
