Collecting and Identifying the Plant
Leaves and twigs of the wild thyme plant were collected from the areas near Shiraz, Iran, in March-April 2019, dried at 25˚C in the shade, and then powdered by a mechanical mill. The dried powder was kept in plastic bags in a freezer until testing.
Twenty grams of the obtained powder was poured into an Erlenmeyer flask and 200 ml of ethyl alcohol at 70˚C was added. Next, the flask was closed by its cap and the solution was held for 48 hours, while the content of the flask was shaken once every 12 hours. Following 48 hours, the content of the flask was filtered into a beaker using a filter paper and a funnel glass. Then, the filtered solution was poured into a flask and placed in a rotary device at 75˚C with average rotation speed. After solvent evaporation, the resultant concentrated liquid was spread on the glass surface and left to dry. The obtained powder containing approximately 2.59 percent of the concentrated extract was collected after drying. Finally, the yielded powder was used to prepare doses of 100, 200, and 400 mg/kg. All the solutions were prepared by distilled water.
First, thymus vulgaris essential oil was prepared and the compounds were then isolated by GC/MS device (HP-6840/5973) in the central laboratory of Ferdowsi University of Mashhad. The constituting elements were identified by comparing their mass spectra with the existing standard spectra.
Small rats, each with an approximate weight of 200±5 g, were kept in clean cages at 25±2˚C and in a diurnal cycle of 12 hours of light and 12 hours of darkness, with relative humidity of 40-60 percent. The animals had access to water and food.
Preparation of Diabetic Animals
Streptozotocin (Pharmacia & Upjophn, USA) (60 mg/kg) solved in sterile saline just shortly prior to the test was intraperitoneally injected into rats. Animals with up to 180 milligrams/deciliter of glucose level of serum were used in the test five days after the injection.
The wild thyme hydroalcoholic extract was inraperitoneally used as treatment in different doses for 18 days. The number of animals in each group was 10. The first control group (Group 1) received merely regular food and water. The second control group (Group 2) received saline daily and the three experimental groups (Groups 3-5) daily and intraperitoneally received a low dose (100 mg), a medium dose (200 mg), and a high dose (400 mg) of wild thyme hydroalcoholic extract in two groups of healthy and diabetic for 18 days.
Assay of Hepatic Marker Enzymes
The hepatic marker enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in serum were measured using diagnostic kits (Parsazmon, Iran). As such, glucose, cholesterol, HDL cholesterol in serum, as well as LDL and VLDL cholesterol were measured .
The liver tissue was fixed in 10% formalin for 48 h. It was then followed by dehydration by passing through a series of graded alcohol, beginning with 50% alcohol and progressing in graded steps to 100% (absolute) alcohol, and was finally embedded in paraffin. Liver slices (5–6 μm thick) were prepared using a semi-automated rotator microtome, stained with Hematoxylin and Eosin dyes, and observed microscopically.
Total RNA from liver tissues was extracted using the Column RNA Isolation Kit (DENAzist Asia Co., Iran) and reverse-transcribed with cDNA synthesis kit (Thermo Fisher Scientific., USA).
The designed primers (Beacon Designer v8) were as follows: for BCL2, F: 5′- GAGCGTCAACAGGGAGA-3′ and R: 5′- GCCAGGAGAAATCAAACA-3′; for BAX, F: 5′- ACTAAAGTGCCCGAGCTGA-3′ and R: 5′- ACTCCAGCCACAAAGATGGT-3′; for C3, F: 5′- GGAGCTTGGAACGGTACGCT-3′ and R: 5′- AGTCCACTGACTTGCTCCCA-3′; for C9, F: 5′- AGCCAGATGCTGTCCCATAC-3′ and R: 5′- CAGGAGACAAAACCTGGGAA-3′; for β-actin: F: 5′-ATCAGCAAGCAGGAGTACGAT-3′ and R: 5′- AAAGGGTGTAAAACGCAGCTC-3′.
The normalization and analyses of the qPCR data were performed using Genex Version 6 software (MultiD, Göteborg, Sweden) and Relative Expression Software Tool (REST; QIAGEN, Hilden, Germany).
Statistical Data Analysis
The collected data were analyzed using SPSS statistical software, and one-way analysis of variance (one-way ANOVA) and Tukey test at the significant level of p<0.05 were run to investigate between-group differences. All results were presented as Standard Error of Mean (SEM).