Effects of Grape Extracts on the In Vitro Activity of Enzymes Involved in Oxidative Stress Regulation

Materials and Methods

Chemicals and reagents. XO from bovine milk and pyrogallol were purchased from Sigma-Aldrich (St Louis MO, USA). H₂O₂ was purchased from Merck (Darmstadt, Germany). All the other chemicals and solvents were of the highest quality commercially available.

Plant material. The two Greek Vitis vinifera varieties Mandilaria (red grapes) and Asyrtiko (white grapes) are cultivated in the Greek island of Santorini. The methanolic and aqueous extracts were obtained as follows: the juice was removed from the grapes and the solid residue was air-dried. Part of the residue was then extracted three times with methanol for 48 h and another part was extracted with water at 45°C for 48 h. The solvents were evaporated under reduced pressure and the residues were diluted in water.

The other extracts derived from grape (berry), stem (fruits talk) and skin (pomace) were obtained as vinification by-products from common red (Mandilaria, Mavrotragano, Voidomato, and Batiki) and white (Vilana, Asyrtiko and Athiri) varieties cultivated in Greek regions. The date accompanying some extracts refers to the year of vinification. The respective samples were air-dried, mill-powdered and stored at room temperature. Part of the dried samples was mixed with MeOH/H₂O/HCl 1.0 N (90:9.5:0.5 v/v) and sonicated in an ultrasonic bath for 10 min. The solvent was filtered and the remaining solid was re-extracted for an additional three times, using the same solvent system and procedure. The combined extracts were evaporated under vacuum and the resulting slurry was dissolved in MeOH/H₂O (1:1) and centrifuged for 10 min (3,000×g). The supernatant was extracted with petroleum ether (3×30 ml) to remove the lipids and concentrated under vacuum. The remaining residue was poured into brine and extracted repetitively with ethyl acetate (EtOAc, 4×30 ml). Thus, all sugars present remained in the aqueous layer. The combined organic layers were dried over anhydrous MgSO₄ and evaporated under vacuum. The resulting dried residues were diluted in water. To avoid polyphenol degradation, all the aforementioned processes were performed in the absence of direct sunlight and at temperatures below 35°C.

The polyphenolic composition of the tested grape extracts is presented in Tables I and II. The identification and quantification of the polyphenolic compounds has been previously described using HPLC analysis (20).

Assessment of total polyphenol content of the extracts. The phenolic content of the different extracts was determined using the Folin-Ciocalteu reagent (21). Each sample (0.1 ml at an appropriate dilution) was added to 5 ml of deionized water and 0.5 ml of Folin-Ciocalteu reagent. After mixing, the samples were left for 3 min at room temperature and 1.4 ml of a 25% w/v solution of sodium carbonate (Na₂CO₃) and 3 ml of deionized water were added. The mixture was left for 1 h at room temperature in the dark and the absorbance at 765 nm was measured. Each sample was tested in triplicate. Blank samples contained only 5.1 ml of deionized water and 0.5 ml of Folin-Ciocalteu reagent. The optical density of the samples (0.1 ml) alone in 1.4 ml of a 25% w/v solution of sodium carbonate (Na₂CO₃) and 8 ml of deionized water at 765 nm was also measured. The total phenolic content was determined by a standard curve of absorbance values using standard concentration solutions of 0, 50, 150, 250 and 500 μg/ml of gallic acid (GA). The total polyphenol content (TPC) is presented as mg of GA per g of extract (mg GA/g extract).

Red blood cell lysate preparation. Human blood was drawn from a forearm vein of healthy, resting, male volunteer of 28 years of age. Blood was collected in EDTA tubes, centrifuged immediately at 1,370×g for 10 min at 4°C and the plasma was discarded. The packed erythrocytes were lysed with 1:1 (v/v) distilled water, inverted vigorously and centrifuged at 4,020×g for 15 min at 4°C. Red blood cell lysate (RBCL) was collected as supernatant and was used as the source of CAT and total SOD. Typically, 4-5 ml of RBCL were obtained from 10 ml of whole blood. The lysate samples were freshly used.

Enzyme activity experiments. The effect of the extracts on the inhibition or induction of enzymatic activity was assessed at the substrate concentration where maximum velocity was obtained. The Michaelis-Menten model was applied to all enzymatic experiments. Many polyphenols exhibit maximal absorption at the tested wavelengths, therefore it is possible that extracts increase the optical density of the samples. However, the majority of the tested extracts did not affect the optical density. Control samples were prepared identically to the test samples without the extract. It should be noted that the addition of the extracts to the reaction mixture did not affect the pH of the reaction. All initial reaction rates were in the linear range and were measured over the first 2 to 4 min of the reaction, depending on the enzyme. Each assay was performed in triplicate in three independent experiments and the optical density was measured using a Hitachi U-1500 Spectrophotometer (San Jose, USA).

Assessment of XO activity. XO activity was determined by measuring the formation of uric acid from xanthine within a set time. Changes in the levels of uric acid produced through the presence of the test samples allow the potency of inhibitors/activators to be assessed. The reaction mixture (500 μl) contained sodium phosphate buffer (33 mM, pH 7.5 with EDTA 0.1 mM), xanthine (4.8 μM) and the extract at different concentrations (1-300 μg/ml). The reaction was initiated through the addition of 43 mU XO diluted in water. The production of uric acid was determined at room temperature at 295 nm for 4 min. IC₅₀ values were determined as the concentration of the extract required to inhibit XO activity by 50% as monitored through the decrease in uric acid production. Allopurinol was used as positive control since it is a known inhibitor of XO (15).

Assessment of CAT activity. CAT activity was determined using the method of Aebi (22). Changes in the levels of H₂O₂ decomposed through the presence of CAT in the test samples allow the potency of inhibitors/activators to be assessed. Briefly, 4 μl RBCL (diluted 1:40) in 67 mM sodium potassium phosphate (pH 7.4) and the extract at different concentrations (1-300 μg/ml) were incubated at 37°C for 10 min. Afterwards, H₂O₂ at a final concentration of 30 mM was added to each reaction mixture and the change in absorbance was immediately measured at 240 nm for 2 min. IC₅₀ values were determined as the concentration of sample required to inhibit CAT activity by 50% as monitored through H₂O₂ decomposition. Sodium azide was used as positive control since it is a known inhibitor of CAT (23).

Assessment of total SOD activity. SOD activity was determined using the method of Dieterich et al. (24). Pyrogallol autoxidation by O₂^•− present in the air can be inhibited by the SOD scavenging of O₂^•−. Therefore, changes in pyrogallol autoxidation rate through the presence of SOD in the test samples allow the potency of inhibitors/activators to be assessed. Briefly, 1 ml of reaction contained 770 μl Tris-HCl diethylenetriaminepenta-acetic acid (DTPA) buffer pH 8.2 (0.05 mM Tris-HCl, 1 mM DTPA), 100 μl test extract at different concentrations (50-800 μg/ml) and 30 μl RBCL diluted 1:10. The samples were mixed and incubated for 5 min at room temperature. The reaction was initiated by the addition of pyrogallol (final concentration 80 μM) and the absorbance was immediately measured at 420 nm for 3 min. Control samples were prepared identically to the test samples without the extracts.

Many polyphenols are potential scavengers of O₂^•− (25). Therefore, we tested the possible inhibitory effects of extracts on pyrogallol autoxidation without the presence of SOD. The reaction mixture (1 ml) contained 800 μl Tris-HCl DTPA buffer pH 8.2 (0.05 mM Tris-HCl, 1 mM DTPA) and 100 μl test extract at the IC₅₀ concentration. The reaction was initiated by the addition of pyrogallol (final concentration 80 μM) and the absorbance was immediately read at 420 nm for 3 min. Control samples were prepared identically to the test samples without the extracts.

The concentration of extract increasing SOD activity by 50% (PC₅₀) was determined through decrease in pyrogallol autoxidation rate. The percentage of possible inhibition of pyrogallol autoxidation by the extracts at concentrations equal to PC₅₀ values was also determined.

Statistical analysis. Statistical computations were carried out using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). For statistical analysis of the results, one-way ANOVA was applied followed by Dunnett's test for multiple pair-wise comparisons. Differences were considered significant at p<0.05.