The purpose of this study was to evaluate the ability of aqueous extract of Aloe barbadensis Miller (Aloe vera) on oxidative damage and Anion Exchanger 1 (AE1, also known as Band 3) expression in human erythrocytes exposed to the water soluble free radical initiator 2.2’-azobis-2-amidinopropano dihydrochloride (AAPH). In addition, total phenolic compounds in the extracts were determined as catechin equivalent and the various antioxidant activities were compared to natural and synthetic standard antioxidants such as BHA and ascorbic acid. Since Aloe vera extract did not cause a consumption of the cytosolic antioxidant, glutathione (GSH) when it was direct incubated with GSH in basic aerated aqueous solution, this indicates that Aloe vera extract does not proceed auto oxidation at this experimental condition. Furthermore, Aloe vera extract prevent the consumption of GSH, in radical treated RBCs. It also inhibit consumption of GSH when it was direct incubated with AAPH. Aloe vera gel extract inhibits the generation of diphenyl-2-picrylhy-drazyl (DPPH) and the scavenging activity was increased in a dose dependent manner. Aloe vera extract was shown the similar reducing power than standards BHT and ascorbic acid. Biochemical analysis by SDS-PAGE and western blotting showed that AAPH-induced oxidative stress increased the susceptibility of AE1 to proteolytic degradation. Of note, our data evidenced that Aloe vera treatment was able to partially restore the normal RBC membrane protein profiles in a dose-dependent manner. These results clearly demonstrate the antioxidative activity of Aloe vera gel extract that might be ascribed to a synergistic action of the bioactive compounds contained therein.
Aloe vera (L.) BURM. fil. (synonym A. barbadensis MILLER) (Liliaceae), is a perennial succulent plant belonging to the Aloeaceae family, a sub-family of the Asphodelaceae [
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 [
Free radical scavenging activity was determined using DPPH assay [
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 [
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 [
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. [
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 [
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 [
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.
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.
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 (
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, (
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 (
DPPH scavenging activity showed the effectiveness of the plant extract in donating hydrogen proton to the lone pair electron of the radical. Since Aloe vera gel extract inhibits the generation of DPPH radical in a dose dependent manner, it could be assumed that gel extract contains compounds capable of donating protons to the free radicals thus demonstrating its reducing power. This property is generally associated with the presence of reductones which have been reported react with certain precursors of peroxide, thus preventing their formation [
GSH content in RBCs. It is well known that AAPH can exert formation of High Molecular Weight (HMW) proteins with a concomitant decrease of Low Molecular Weight (LMW) proteins [
The present study demonstrates that Aloe Vera gel extract induces beneficial effects in terms of protection against reduction of GSH content, RBCM protein breakdown and band-3 degradation caused by AAPH. Our results also underline the important role of Aloe vera gel extract in the maintenance of the antioxidant status and antioxidant defense in RBCs. This scavenging activity might be due to the synergistic actions of bioactive compounds present in the plant extracts. We suggest that dietary Aloe vera supplementation can help to prevent oxidative stress and might be also useful for the treatment of oxidative stress-related human disorders by virtue of its antioxidant activity.