The following chemicals were used: Diphenyl-1-picrylhydrazyl (DPPH), Potassium phosphate, Catechin hydrate, Butylated hydroxytoluene (BHT), Sodium phosphate dibasic, Sodium phosphate monobasic, Potassium ferricyanide(III), Trichloroacetic acid (TCA), Iron(III) chloride, L-Ascorbic Acid, L-Glutathione reduced (GSH), Sodium tetraborate decahydrate, Boric Acid, 5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB), Phosphate buffered saline (PBS), Methanol, Sodium carbonate, meta-Phosphoric acid, Ethylenediaminetetraacetic acid (EDTA), Phenylmethanesulfonyl fluoride (PMSF), Acrylamide, N,N’-Methylenebis (acrylamide), (hydroxymethyl) aminomethane (Tris), Sodium dodecyl sulfate (SDS), Ammonium persulfate (APS), N,N,N^’,N^’-Tetramethylethylenediamine (TEMED), Glycine, Red Ponceau, Bovin Serum Albumins (BSA), Tween 20, Whatman^® 3 MM paper, Protran^® nitrocellulose membranes 0,45 µm, were purchased from Sigma Chemical Co. (St.Lous, MO). Potassium ferricyanide, Ferric chloride, Sodium Chloride, and Folin-Ciocalteu phenol reagent were acquired from Merck (Milan, Italy). Mouse monoclonal anti-Band 3 and anti- β-actin antibodies were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Biotinylated antihorse IgG and Avidin-biotin HRP visualization systems (Vecstain^® ABC kit PK-6100) were procured Vector Laboratories, Inc (California, USA). 7315 UV/V spectrophotometer (Jenway, Staffordshire, United Kingdom) was used for spectrophotometric assay. Centrifuge centric 200/R (Techtnica, Železniki, Slovenia) was used for centrifugations.

Phenolic substances, known to be responsible for the antioxidant activity of plant extracts, are mostly extracted by organic solvents but as plants are commonly consumed as water extracts by people, the aqueous extract of Aloe vera leaves were preferably used in our investigation. Mature Aloe vera leaves was collected from Drogo farm, Rocca Imperiale, Cosenza (Italy).

The gel (100 g = 0.75 g dry matter) was homogenized in a electric blender, then diluted with an equal volume of PBS and homogenized for a second time. The extract was kept at 4˚C overnight, then filtered through cloth. The clear filtrate was kept at –20˚C in aliquots.

Aliquot of 0.1 ml of Aloe vera gel extract was made up to 4.6 ml with distilled water in a tube. After addition of 0.1 ml Folin-Ciocalteu reagent (previously diluted 3-fold with distilled water) and 0.3 ml 2% aqueous sodium carbonate solution, tubes were vortexed and the absorbance of the blue color that developed in each assay mixture was recorded after 2 h at 760 nm, against a blank containing 0.1 ml of the extraction solvent [14]. Catechin hydrate (0.1 - 0.8 mg/g) was used for calibration of a standard curve. The results were expressed as grams Catechin Equivalents (CEs)/L of gel extract. The data were presented as the average of triplicate analyses.

Free radical scavenging activity was determined using DPPH assay [15]. When DPPH reacts with an antioxidant compound which can donate hydrogen, it is reduced. The change in color from deep violet to golden/light yellow can be measured at 517 nm. Briefly, 1 mL of 0.3 mM of DPPH solution was added to 1 mL of Aloe vera gel extract (5 - 30 µM CEs), and incubated in the dark at room temperature for 30 min. Inhibition of free radical DPPH in percent (I%) was calculated from the absorption according to the following equation:

Ten independent experiments were carried out and results were expressed as mean values ± standard error of the mean (S.E.M.). The extract concentration providing 50% inhibition (EC50%) was calculated from the graph of scavenging effect percentage against the extract concentration. Ascorbic acid and BHT were used as standards.

Aloe vera gel extract (5 - 30 µM CEs) were mixed with 200 mM PBS (pH 6.6) and 1% K₄[Fe(CN)₆]. The mixture was incubated at 50˚C for 20 min and 10% TCA was then added to the mixture and centrifuged 650 g for 10 min. The upper layer of solution was mixed with distilled water and 0.1% FeCl₃. The increase in absorbance at 700 nm of the reaction mixture indicated reducing power [16]. BHT and Ascorbic Acid were used as standards.

Aloe vera gel (50 - 500 µM CEs) was incubated with GSH (100 µM) in a borate buffer (50 mM, pH 9.25). An appropriate volume of the reaction mixture was removed at intervals and added to an aqueous solution of DTNB (0.2 mM, pH 9.0). After 3 min incubation, absorbance at 412 nm was measured and thio concentrations calculated by using [17]:

Blood was obtained from donor volunteers (10 men aged 47 ± 11 years) by venipuncture, and collected into tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. The samples were centrifuged at 1100 g/ min for 10 minutes; plasma and buffy coat were carefully removed and discarded. RBCs were washed three times with PBS pH 7.4. RBCs used on the day were preincubated with AAPH at final concentration of 50 mM. In all sets of experiments incubations of RBCs were carried out at 37˚C for 180 minutes in the presence of AAPH with and without Aloe vera gel under gentle shaking. Untreated RBCs has been used as control.

Blood GSH level was measured spectrophotometrically using Ellmans reagent (DTNB) as a coloring reagent as the method described by Beutler et al. [18]. Briefly, 0.2 ml blood was mixed with 1.8 ml double distilled water and glacial meta-phosphoric acid (a precipitating solution) was added, centrifuged and supernatant was collected. Lastly, the supernatant was mixed with 300 mM disodium hydrogen sulphate and DTNB reagent and was allowed to stand for 2 min at room temperature. The absorbance was read at 412 nm. GSH values were determined using the molar extinction coefficient of 13,600 cm^–1·M^–1.

Performed RBCs were lysed in 20 volumes of lysis buffer (5 mM sodium phosphate, 1 mM EDTA, pH 7.9), in presence of the protease inhibitor phenylmethylsulfonyl fluoride (final concentration of 0.1 mM) were centrifuged (20 min at 4˚C, 13500 g). To removal from the button granulocyte debris, the membrane pellet was re-suspended in the above buffer and centrifuged once more. The ghosts were washed 5 - 7 times in a similar manner until white [19]. The protein concentration of the RBC membrane suspensions was determined by Bradford method [20].

Lysates (25 µg) were equal loaded for 5 min with β-actin and then subjected to 10% SDS-PAGE and transferred to nitrocellulose. The membrane was blocked in low-fat (1%) milk diluted 1:20 into TBST (10 mM Tris HCl [pH 7.5], 50 mM NaCl, 0.1% Triton X-100) for 30 min, followed by incubation for 1.5 h with the anti-Band 3 antibody. Membranes then were washed extensively with TBST and immuneoreactive protein was detected by incubation with biotinylated anti-horse IgG. Protein was visualized using Vecstain^® ABC kit.

Statistical analyses of the results were performed using one and two-way ANOVA followed by Bonferroni’s test. P value < 0.05 was considered statistically significant.

The antioxidant activity of plant materials closely correlated with the content of their phenolic compounds [21]. The Aloe vera gel extract was shown to contain 1.34 ± 0.087 grams of CEs/L.

The presence of reductants in the samples tested would result in the reducing of Fe³⁺ to Fe²⁺ by donating an electron. Amount of Fe²⁺ complex can then be monitored by measuring the formation of Perl’s blue at 700 nm. Figure 1 shows the reducing power of Aloe vera gel and the reference compounds, BHT and Ascorbic Acid. Here, Aloe Vera gel (10 µM CEs) was shown the same reducing power than standards.

Aloe vera gel extract inhibits the generation of DPPH radical in a dose dependent manner and its IC₅₀ value was found to be 2.9 ± 0.1 µg/ml (10 µM CEs), which is defined as the concentration of substrate that causes 50% loss of the DPPH activity (color). A lower value of IC₅₀ indicates the greater antioxidant activity of a test substance. Figure 2 shows the radical scavenging ability of Aloe vera gel and the reference compounds.

AAPH induce a decrease in sulfhydryl groups after incubation with glutathione in basic aerated aqueous solution (borate buffer, pH 9.25). Aloe vera gel extract did not cause a consumption of GSH when it was direct incubation with AAPH (Figure 3).

The GSH content of untreated RBCs at 37˚C after 180 min was 0.37 ± 0.029 µM. AAPH (50 mM) induced a rapid consumption of cytosolic GSH (0.25 ± 0.015 µM). Addition of different concentrations Aloe vera gel extract (5, 10, 20 and 30 µM CEs), prevented the GSH consumption induced by AAPH, (Figure 4).

Western Blot analysis of RBCM isolated after AAPH in-

cubation with and without Aloe vera gel extract (5 - 30 µM CEs) were performed with mouse monoclonal antibody anti-Band 3 (Figure 5). Our dates showed: (a) an increased susceptibility to proteolysis breakdown of AE1 transporter during oxidative damage (lane 2) compared with the intact Band 3 in untreated, control sample (lane 1); (b) a reduction of Band-3 degradation after incubation with Aloe vera gel extract in a dose dependent manner (lanes 3-6); β-actin was used as loading control.