Plant material

The subject of the study was fresh samples of the aboveground part of Salvia deserta Schangin collected in the foothills of Kaskasu in southern Kazakhstan. The produced herbarium of the harvested plant raw material was transferred to the ethnobotanical collection of the botanical garden of the Institute of Botany and Phytointroduction of the Forestry and Wildlife Committee of the Ministry of Ecology and Natural Resources of the Republic of Kazakhstan, where the species was confirmed (No. 01-05/280, August 2, 2023). Plant collection was carried out from late June to mid-July, during the peak of the plant’s flowering period, in accordance with the requirements of Good Agricultural and Collection Practices (GACP). The raw material was cleaned of foreign objects and dried in shade at room temperature (25°C±5°C) with ventilation. After drying, the raw material was ground to a size of 1–3 mm using an IKA M20 laboratory mill [13].

Obtaining the lyophilized extract

The pharmacological properties of the Tartarian sage were studied using ultrasonic extraction. Ground raw material in an amount of 100 g was subjected to extraction with a 40% water-alcohol solution in a 1:5 raw material to extractant ratio. Extraction was performed 3 times for 30 minutes in an ultrasonic bath (Elmasonic S100H, Germany) with a reflux refrigerator. The extracts were then filtered and refrigerated at +2–8°C for 2 days to precipitate ballast. The resulting alcohol extract was evaporated in a rotary vacuum evaporator (IKA RV-10, Germany) to a thick extract and then lyophilized in a freeze dryer (Harvest Right, USA).

The obtained lyophilized extract of the Tartarian sage is a lyophilized dry powder, greenish-brown in color, with a slightly sweet, astringent taste and a specific aroma. The particles of the extract have an anisodiametric, plate-like shape, with an inhomogeneous and complex surface.

To study the pharmacological properties of the obtained lyophilized extract of Tartarian sage, non-clinical tests were conducted on laboratory animals.

Non-clinical studies of the pharmacological properties of lyophilized extract of Tartarian sage were carried out in the laboratory of the Centre for Experimental Pharmacology and Toxicology (CEPT) of the JSS Academy of Higher Education and Research (JSS AHER, JSS College of Pharmacy, Mysore), which was GLP certified (CPCSEA Central Animal Facility Registration Number 261/PO/ReBi/S/2000/CPCSEA).

Acute oral toxicity

Acute oral toxicity of the aqueous solution of the lyophilized extract was determined and analyzed according to OECD guidelines, test No. 423:2001, IDT, scheme 1 (GOST 32644-2014). Per the procedure, two groups of animals were used, and each group was divided into subgroups of 3 mice each. Since there was no prior information on the toxicity of the test substance, a dose of 500 mg/kg was used as the starting dose, and the maximum dose was 2,000 mg/kg of body mass. The lyophilic extract was dissolved in purified water and administered to animals at a dose of 0.2 ml orally. The animals were clinically monitored prior to administering the substance, then 30 minutes, 1, 2, 3, and 6 hours after dosing, and at least once a day until the end of the study to assess toxicity and the general condition of the animals. Body weight was recorded before dosing, on day 7, and on day 14. The animals were observed for 14 days. After the studies were completed, the animals were euthanized to test for macroscopic anomalies of internal organs [14].

Antinociceptive properties

Test animals were divided into five groups of 6 mice. The control group was administered saline solution (10 ml/kg). An hour before the administration of acetic acid, a 100 mg/kg solution of diclofenac sodium was administered to the standard group. The experimental groups of animals received an aqueous solution of the lyophilized extract of Tartarian sage at a minimum dose of 100 mg/kg and a maximum dose of 400 mg/kg. All doses were calculated taking into account the weight of the mice and the volume of the injected solution. The preparations were administered by oral gavage. The total amount of writhing movements was calculated after intraperitoneal administration of 0.7% acetic acid solution in a volume of 0.2 ml. Twists and stretches were observed and counted for 15 minutes [15].

Anti-inflammatory activity

Anti-inflammatory activity was assessed by the method of carrageenan-induced paw edema. The tested animals were weighed and divided into five groups of 6 individuals each. The lyophilic extracts and the reference preparation, diclofenac sodium, were dissolved in purified water and administered per os one hour before the injections. All doses were calculated based on the weight and volume of the therapeutic solution administered to rats. The preparations were administered by oral gavage. The carrageenan solution was administered through a subplantar injection (0.1 ml) to the inside of the right hind paw. Rats in group 1 received saline and served as negative controls. In group 2, the rats were injected with a 1% suspension of carrageenan in saline in the subplantar region of the right hind leg. Group 3 rats were administered diclofenac sodium (50 mg/kg of body mass), which was considered the standard. Increasing doses of the lyophilic extract of Salvia deserta Schangin (100 mg/kg, 400 mg/kg of body mass) were administered to rats in groups 4 and 5 [16].

Antioxidant activity by the DPPH (2,2-diphenyl-1-picrylhydrazyl) method

The DPPH test is based on the interaction of a stable 1,1-diphenyl-2-picrylhydrazyl free radical with a hydrogen donor. This test relies on the reduction of a radical solution with an antioxidant (AH) or a radical (R •), and the reaction proceeds as follows: DPPH• + AH → DPPH• -H + A• ; DPPH• + R• → DPPH• -R [17]. During the reaction, the DPPH solution is discolored from violet, and the color change serves as an indicator of antioxidant activity. The absorbance of the test solutions is measured with an ultraviolet spectrophotometer (UV-1800 Shimadzu, Japan) at a wavelength of 517 nm. The antioxidant property determined by the DPPH reduction method (Sigma Aldrich, USA) is denoted as EC50, which is the concentration of extract consumed to absorb 50% of DPPH free radicals [18]. Methanol of 99.8% purity (Sisco Research Laboratories, India) was used as the solvent, and ascorbic acid solution (10 mg/ml) was used as the reference standard. Antioxidant activity (AOA) was calculated [19]. Next, a diagram is plotted, and the EC50 is determined from the data of this figure (2) [20].

Determination of antioxidant activity by the Ferric Reducing Antioxidant Power (FRAP) method

The FRAP test is based on the reduction of the 2,4,6-tripyridyl-s-triazine complex (TPTZ) with iron chloride hexahydrate (FeCl₃·6H₂O). Over the course of the reaction, they become colorless and change color to brown over time [17,18].

To conduct the study, a 10 mg/ml aqueous-alcoholic solution of lyophilized extract was prepared. Aliquots of the extract solution (5, 10, 20, 40, 60, 80, and 100 μl) were transferred into test tubes and diluted to 1 ml with a 40% aqueous alcohol solution. Next, a phosphate buffer solution (0.2 M pH 6.6) and a 1% potassium ferricyanide solution were added and incubated in a water bath at 50°C for 30 minutes. A 10% trichloroacetic acid solution was added to the resulting solution and stirred in a centrifuge (6,500 rpm) for 5 minutes. To 2.5 ml of the resulting solution, 1 ml of purified water and 0.2 ml of 0.1% FeCl₃ solution were added, and the absorbance of the solution was measured with a spectrophotometer (UV-1800 Shimadzu, Japan) at λ = 700 nm. For comparison with the test solution, only the solvent was used in the control group. As a comparative reference group, an ascorbic acid solution (10 mg/ml) prepared in dimethyl sulfoxide (DMSO) was used. The reference standard was prepared in the same way, only using an ascorbic acid solution instead of the extracts [21]. Antioxidant activity and EC50 were determined in the same manner as described above [20].