Preparation of plant extracts
Fresh-ripended fruits of Jujube and petals of Saffron were collected from Birjand, South Khorassan, Iran from September to October, 2014. These were dried in shadow then ground with a grinder (Hamilton Beach Brand, Canada). Aqueous extracts were prepared using 5 g of dried powder in 100 ml of boiling water for 2 h. The mixtures were filtered through a No. 1 Whatman filter paper and thus oven-dried at 40 °C for 24 h. Eventually, the samples lyophilized by freezing at − 80 °C for 24 h [13]. The crude extracts were dissolved in distilled water just prior to oral administration. Each extract was administered in five doses equal to 150, 500, 1000, 2000 and 5000 mg/kg body weight. The voucher number of specimen for Saffron (H. No. 2669) and for Jujube (H. No. 2470) was deposited in the Herbarium of Birjand University, Iran.
Ferric Reducing Antioxidant Power (FRAP) assay
The total antioxidant capacity of herbal extracts was determined by FRAP assay [14]. The results were expressed in MFe (II)/g dry weight of plant extracts (DW).
Folin–Ciocalteu assay
The total phenolic contents of herbal extracts were measured using the Folin Ciocalteu method [15]. The data were expressed as milligram Gallic acid equivalents (GAE)/g dried weight of plant extracts (DW).
Determination of Total monomeric anthocyanins (TMA)
The TMA have been estimated by a pH differential method [16] using UV–Vis spectrophotometer. Results were expressed as mg of cyanidin-3-glucoside equivalents per liter of herbal extracts.
Animal experiments
Male Wistar rats (6–8-old-week; 200 ± 20 g) were purchased from animal house of Birjand University of Medical Sciences, Iran. All animal procedures were approved by the Animal Ethical Committee in accordance with the Guidelines for the Care and Use of Laboratory Animals prepared by this university. Rats were individually housed in a thermally controlled (25 ± 2 °C) room free from any sources of chemical contamination. Light was maintained on a 12 h dark–light cycle, and rats were fed a standard laboratory diet of rat chow (Javaneh Khorasan Co, Mashhad, Iran) with free access to tap water.
Study design
Animals were acclimatized to the laboratory conditions for the duration of ten days before the commencement of the experiment. 77 Rats were randomly assigned to 11 groups (n = 7/cage) and were weighed weekly. Experimental groups J1 and S1, J2 and S2, J3 and S3, J4 and S4 and J5 and S5 were received extracts at 150, 500, 1000, 2000 and 5000 mg/kg by gavage, respectively, for 14 days. Healthy control group (C) was gavaged with normal saline during this period. The used doses of herbs were based on Ahmad study [17].
Sample preparation
At the end of study blood was collected in sterile vial with and without anticoagulant for whole blood and serum separation respectively.
Hematology analysis
Red Blood Cells (RBC), Hemoglobin (Hb), Packed Cell Volume (PCV), Total White Blood Cells (WBC), Leukocyte Count (DLC) and thrombocyte count was estimated using an automated blood analyzer (Cell Dyn®3700, Abbott Dignostic, USA).
Biochemical analysis
Sera samples were analyzed for biochemical parameters such as Fasting Blood Suger (FBS), lipid profile (cholesterol, triglyceride, LDL, HDL), total protein, albumin, total bilirubin, using standard commercial kits (Pars Azmoon, Tehran, Iran).
Assessment of liver function
Serum Alanine Amino Teransferases (ALT/SGPT), Aspartate Amino Teransferases (AST/SGOT), Lactate Dehydrogenase (LDH), Alkaline Phosphatase (ALP), were determined to check the function of liver.
Assessment of kidney function
Blood Urea Nitrogen (BUN), cratinine and uric Acid using standard commercial kits (Pars Azmoon, Tehran, Iran) were evaluated to control the function of kidneys.
Statistical analysis
Results were expressed as the mean ± SD of the indicated number of independent experiments. The Student-T test was used to compare the mean values of different parameters obtained in various groups by employing SPSS (version 18) software and P < 0.05 were considered as significant.