Ascorbic acid, Deoxyribose, Tiobarbitoric acid (TBA), ferric chloride, methanol, butylated hydroxytoluene (BHT) and trichloroacetic acid (TCA) were obtained from Merck (Germany).
Plant material and Preparation of extracts
P. vera L. was collected from (Khorasan- Gonabad region) I.R Iran. It was identified by Mr. Ahi in the Herbarium of School of Pharmacy, Mashhad University of Medical Sciences (MUMS), IR. Iran. For the decoction extract, 1 L water was added to 100 g plant material and boiled for 15 min and percolation was performed till the solvent become colorless. Afterwards, the solution was filtrated and evaporated in a water bath (maintained at 30-40°C). The extract was stored in a refrigerator at 4°C.
To obtain the ethanolic extract, the leaves and fruits of plant were prepared by defatted powder via Soxhlet with petroleum ether giving as dry residue and was macerated in ethanol 80° (v/v) for 72 h. Then, the macerated mixture was filtered and evaporated as mentioned previously.
The gum was extracted from the resin by cold maceration by hydrodistillation with ethanol. The combined hydroalcoholic extract was filtered through filter paper and evaporated to dryness on water bath.
Characterization of extract by HPLC
The separation was carried out on a millipore column (5 μm, 1.5 X 3.9 mm) using gradient elution. Gradient was performed using water-phosphoric acid (0.1 N, 99:1) and acetonitrile-phosphoric acid (0.1 N, 99:1) at a total flow rate of 1 mL/min; gradient composition (min, % acetonitrile-phosphoric acid): 0.0, 5; 5.0, 7.5; 10, 10; 15, 12.5; 20, 15; 25, 17.5; 30, 20; 35, 22.5; 40, 25). The extracts were dissolved in methanol and filtered through a membrane filter (0.45 μm). 1.0 μL sample of 10 g/L of extract was injected in to a reversed-phase column (RPC-18). The peaks were momtored at 236 nm.
Animals
Male Wistar rats weighing 200-250 g were used for the study. The animals were bred and housed in the Animal House of the Faculty of Pharmacy Mashhad University of Medical Sciences accordance with ethical committee Acts.
Deoxyribose assay
In this method, after mixing the materials, hydroxyl radicals were produced by ascorbic acid, H
2O
2 and Fe
3+-EDTA thus deoxyribose degradation take placed and produced malondialdhyde. The reaction mixture contained 100 μL of 28 mM 2-deoxy- 2-ribose 500 μL solution of various concentration of the material test (aqueous and ethanolic extracts of pistachio fruits and leaves and hydroalchoholic extracts of gum in buffer), 200 μL of 200 μM FeCl
3 and 1.04 mM EDTA (1:1 v/v), 100 μL H
2O
2 (1 mM) and 100 μL ascorbic acid (1 mM). All solutions were prepared freshly. After an incubation period of 1 h at 37°C, 1 mL of TBA (1%in 50 mM NaOH) and 1 mL of TCA were added to the reaction Mixture and the tubes were heated at 100°C for 20 min. The degree of deoxyribose degradation was measured by the TBA reaction. Absorbance was read at 532 nm (
18,
19). The percentage Absorbance was read at 532 nm. The percentage of inhibition of deoxyribose degradation was calculated using the following equation:
%Inhibition = (A0 - A1) / A0 × 100
Here, A
0 is the absorbance of the control in the absence of samples; A
1 is the absorbance in the presence of samples. DMSO was used as a positive control (
20) and the negative control was all of the reaction mixture without extracts.
Site-specific reactions assay
This assay was prepared in the following three ways:
This test was done as mentioned previous to except that 100 mL of FeCl3 instead of 200 mL of Fe3+-EDTA Solution was extra, for evaluated potency of sample radical scavenging and Fe chelating.
The evaluation was performed without ascorbic acid as a starter for oxidation.
Deoxyribose itself was omitted from the test (
18).
Erythrocyte membrane peroxidation
According to the method that was described before (
19,
21), the evaluation of protective effects of pistachio extracts, were done with modification. After anesthizing the Wistar rats (200-250 g) with chloroform, whole blood were collected via a cardiac puncture to heparinized tubes. The RBC was separated from plasma by centrifugation at 1500 g for 15 min. Packed RBC was washed twice with NaCl 0.15 M, and preincubated with phosphate buffer (pH = 7.4) containing sodium azide (1 mM) to inhibit catalase. Then H
2O
2 (10 mM) was added, peroxidation was initiated H
2O
2 (10 mM). 100 μL solutions of various concentrations of the material test were added and the mixture was incubated at 37°C for 60 min. The addition of 28% (w/v) trichloroacetic acid terminated reaction. Lipid peroxidation was measured using thiobarbitoric method. The quantity of MDA was determined by measuring the absorbance at 532 nm BHT was used as a positive control (
21,
22).
Liver microsomal preparation and lipid peroxidation induced by Fe2+/ascorbat
This test was carried out according to the method that was described previously (
23). Wistar rats were anesthetized and liver was perfused with ice-cold saline through the portal vein until getting uniformly pale and were immediately removed. Then, pieces of liver were homogenized with 4 volume of ice-cold 0.1 M potassium phosphate buffer (pH = 7.4) containing 1.15% (w/v) KCl. The homogenate was centrifuged at 10000 g for 60 min. The supernatant was used for the study. According to the mentioned previous method, (
23) for measuring antioxidant activity, rat liver microsome (2 mg/ mL) was mixed with 0.1 mL of FeSO4 (26% mM), 0.1 mL of ascorbate (0.13 mM), 0.1 mL of the sample in 150 mM KCl/Tris-HCl buffer solution (pH = 7.4). The mixture was incubated at 37°C for 60 min in a water bath; 0.75 mL of 2 M trichloroacetic acid/1.7 M HCl was added to stop trhe reaction, then tubes were centrifuged (4000 rpm, 10 min) and 0.5 mL of the supernatant was mixed with 0.15 mL TBA and was heated at 95°C for 10 min. The level of malondialdehyde was determined by measuring the absorbance at 532 nm. The percent of lipid peroxidation inhibition was calculated by following Equation:
%I = (A0 - A1 / A0) × 100
Here, A
0 is the absorbance of the control reaction; A
1 is the absorbance in the presence of the agents. BHT was used as a positive control (
20).
Phytochemical test
Phytochemical screening of the extract was performed using the following reagents and chemicals (
24): Alkaloids with Dragendorff’s reagent, flavonoids by the use of Mg and HCl; tannins with 1% gelatin and 10% NaCl solutions and saponins with ability to produce hemolysis of RBC.
Statistical analysis
Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. The p-value less than p < 0.05 were considered to be statistically significant. PCS software was used to calculate IC50 value.