Evaluation of the hypoglycemic, hypolipidemic and anti alpha amylase effects of extracts of the twigs and fruits of ficus ovata vahl (moraceae)

II.5. Biochemical experimentation

II.5.1. Determination of plasmatic lipid profile

II.5.1.1. Quantitative determination of cholesterol (chronolab kits).

Fatty acid

HO

Cholesterol

+

Cholesterol ester

R - C - O

= O

Cholesterol esterase

Principle The cholesterol present in the sample generates a coloured complex as shown.

Cholesterol + O₂ ^{cholesterol oxidase} 4-cholestenona + H₂O₂

2H₂O₂ + Phenol + 4-Aminophenazone ^peroxidase Quinoneimine + 4H₂O

The intensity of the colour formed is proportional to the cholesterol level (Naito, 1984).

Reagents (see annex 2)

Procedure: Total cholesterol was determined by first preparing the working reagent (mixture of 2 reagents). The blank (1mL WR), standard (1 mL WR and 10 uL standard) and the samples (1 mL WR and 10 uL sample) were prepared. The solution was mixed and incubated for 10 min at room temperature. The spectrometer was adjusted to zero with distilled water. The absorbance (A) of samples and standard were read against the blank at 500nm. The colour was stable for at least 60 minutes.

Calculation: cholesterol in the sample mg/dL =

II.5.1.2- Quantitative determination of Triglycerides (chronolab kits).

Principle Sample triglycerides incubated with lipoprotein lipase (LPL) liberate glycerol and free fatty acids. Glycerol is converted to glycerol- 3-phosphate (G3P) and adenosine -5-diphosphate (ADP) by glycerol kinase and ATP. Glycerol-3-phosphate (G3P) is then converted by glycerol phosphate dehydrogenase (GPO) to dehydroxyacetone phosphate (DAP) and hydrogen peroxide (H₂O₂). In the last reaction, hydrogen peroxide (H₂O₂) reacts with 4-aminophenazone (4-AP) and p-chlorophenol in the presence of peroxidise (POD) to give a red colour dye: The intensity of the colour formed is proportional to the Triglycerides concentration in the sample (Fossati et al., 1982).

Glycerol + ATP Mg2+ glycerol-3-phosphate + ADP

GK

Glycerol-3-phosphate + O₂ dihydroxyacetone phosphate + H₂O₂

GPO

2H₂O₂ + 4- aminoantipyrine + 4- chlorophenol quinoneimine + HCl + 4H₂O

POD

R, R₁, R₂ et R₃ = variable radicals

+

CH₂ - OH

CH₂ - OH

CH - OH

Glycérol

Lipoprotein lipase

CH₂ - O - C - R₁

CH₂ - O - C - R₃

R₂ - C - O - CH

= O

= O

= O

Triglycerides

R - C - OH

= O

Fatty acids

.

Reagents (see annex 3)

Procedure: Triglycerides were determined by first preparing the working reagent (mixture of 2 reagents). The blank (1mL WR), standard (1 mL WR and 10 uL standard) and the samples (1mL WR and 10 uL sample) were prepared. The solutions were mixed and incubated for 10 min at room temperature. The spectrometer was adjusted to zero with distilled water. The absorbance (A) of the samples and standard were read against the blank at 500nm. The colour was stable for at least 60 minutes.

Calculation: Triglycerides in the sample mg/dL =

II.5.1.3- Quantitative determination of HDL Cholesterol (chronolab kits)

Principle: The very low density (VLDL) and low density (LDL) lipoproteins from serum or plasma are precipitated by phosphotungstate in the presence of magnesium ions. After removed by centrifugation the clear supernatant containing high density lipoproteins (HDL) is used for the determination of HDL cholesterol (Young, 2001).

Reagents (see annex 4)

Procedure: Precipitation was done by pipeting 100 uL of the reagent and 1mL of sample into the centrifuge tube. The solution was well mixed and allowed to stand for 10 minutes at room temperature followed by centrifuge at 4000 rpm for 10 minutes. The supernatant was collected and tested for HDL cholesterol (HDLc).

Calculations: HDLc in the sample mg/dL =