Chemical Composition of Essential Oil from Seven Ocimum basilicum L. Accessions, Brine Shrimp Lethality Bioassay and Inhibitory Activities Against GAPDH and APRT

Journal of Essential Oil Research: JEOR, Jan/Feb 2007 by Alves, Péricles B, Filho, Pedro S Freire, Moraes, Valéria R S, Blank, Arie F, Et al

Inhibitory activity against glycossomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH)from Trypanosoma cruzi: TcGAPDH activity was determined according to a modification of a previously reported procedure (18). Reduced NADH was measured spectrophotometrically at 340 nm at 30-s interval. The reaction medium was 50 mM Tris-HCl pH 8.6 buffer, 1 mM EDTA, 1 mM β-mercapto-ethanol, 30 mM Na^sub 2^H sO^sub 4^,2.5 m M NAD ,0.3mMglyceraldehyde-3-phosphate ' and 4-9 μg protein, in a total volume of 1000 μL. The reaction was initiated by the addition of enzyme.

The specific activity (unit = U) of the enzyme was calculated as below:

(U/mg) = {(Δ absorbance/Δ t) × volume of cell}/6.22 × volume of enzyme × [enzyme]

Where Δt = 0.5 min; volume of cell = 1.00 mL; ξNADH = 6.22 (μMol/cm3)-1/cm; volume of enzyme = 0.005 mL; [enzyme] concentration of enzyme in mg/mL.

Trypanosoma cruzi GAPDH-inhibitory activity: The inhibitory activity was recorded using the reaction medium as above, in a total volume of 1000 μL. Absorbance was read at 340 nm at 30s intervals. The oils were tested at 100 μg/mL in 10% DMSO using 5 μL of GAPDH at 0.90 mg/mL. In each case, a blank experiment was performed with 10% DMSO alone in the reaction medium and was used as the positive control. The specific activity of TcGAPDH was not significantly affected by 10% DMSO alone.

Data were means of 3 repetitions and values as percent of control were used as follows:

% inhibitory activity = {(U/mg control - U/mg compound)/U/mg control} × 100

Inhibitory activity against adenine phosphoribosyltransferase (APRT) from Leishmania tarentolae: The L. tarentolae APRT enzyme was purified from Escherichia coli in a recombinant system, as described by Silva et al. (19). The purified protein was stocked at -80°C in 100 mM Tris-HCl pH 7.4, 5 mM MgCl2, glycerin 10 % (v/v) at 1 μg/mL. The enzymatic assay was developed by modification from spectrophotometric protocol previously reported by Tutle and Krenitsky (20). Adenosine monophosphate (AMP) was measured spectrophotometrically at 259 nm. The reaction medium was 100 mM Tris-HCl, pH 7.4, 200 μM 5'-phospho-α-D-ribosyl1'-pyrophosphate (PRPP), 100 μM Adenine, 5 mM MgCl^sub 2^, in a total volume of 500 μL. The reaction was initiated by the addition of enzyme.

The specific activity (unit = U) of the enzyme was calculated as below:

(U/mg) = {(Δ absorbance/Δ t) × volume of cell}/1.24 × volume of enzyme × [enzyme]

Where Δt = 1 min; volume of cell = 1.00 mL; ξAMP = 1.24 (ΔMol/cm^sup 3^)-1/cm; volume of enzyme = 0.005 mL; [enzyme] = concentration of enzyme in mg/mL.

L. tarentolae APRT- inhibitory activity: The inhibitory activity was recorded using the reaction medium as above, in a total volume of 1000 μL. Absorbance was read at 259 nm at 60 s interval. The oils were tested at 50 μg/mL in 10% DMSO using 5 μL of APRT at 0.90 mg/mL. In each case, a blank experiment was performed with 10% DMSO alone in the reaction medium and was used as the positive control. The specific activity of APRT was not significantly affected by 10% DMSO alone.