Experimental work was carried out departmental laboratories of Technology of food and processing industries, biotechnology and standardization, the scientific center of radioecological research" of the Shakarim State University of Semey, Kazakhstan and Research Institute of Biotechnology" of Kemerovo State University Russia. The selected plants were collected in the East Kazakhstan region, Katon-Karagai district - levsey, and Shemonaikha district- echinacea. Echinacea purpurea (L.) was sampled from the Shemonaikha area of the East Kazakhstan region and Rhaponticum carthamoides root from the Katon- Karagay area of East Kazakhstan region. The authenticity of medicinal plant raw materials was carried out by a specialist biologist on macroscopic (external) and microscopic (anatomical) features under the requirements of General Pharmacopoeia Article 1.5.3.0003.15 "Technique of microscopic and microchemical research of medicinal plant raw materials and medicinal plant preparations".

For the preparation of a tincture, an aqueous solution of ethyl alcohol of different concentrations was used. It is recommended to use a 35% aqueous solution of ethyl alcohol to make a medicinal plant tincture. To prepare the tincture used pharmacopeia medicinal plants that are not poisonous: for one part of the plant mass used 5 parts of the extractant. The infusion temperature of the infusion was within 20 – 25 °C (Kowalczyk et al., 2012).

Echinacea purpurea (L.) and Rhapónticum carthamoídes root were used as plant materials. To prepare the tincture, selected plant materials were shade dried and grinded to small particles of 3 – 5 mm size.

Powdered material of both plants (100 g each) was added to an aqueous solution of 35% ethyl alcohol at herb: extractant 1:5 ratios. The resultant mixture was loaded into a mesh basket, which was placed in a percolator. The percolator was equipped with a jacket, for the possibility of maintaining the temperature of maceration in the range from 20 to 30 °C for 24 hrs. Subsequently, the temperature of the percolator was increased up to 40 – 50 °C with continuous stirring for at least one hr. The solvent was removed and a fresh extraction solvent was added to the treated herbs in the ratio of the herb: extractant 1:1. Infusion with periodic mixing was carried out for 12 hrs and drained all the extract into an intermediate container. The solvent extracts were combined in the tank. To purify the resulting mixture of ballast substances was carried out via the process of sedimentation at a temperature of 5 – 8 °C for 5 days, then carry out the process of filtration of alcoholic infusion through a cotton-grass filter. The resulting tincture was stored in a dark glass jar (Figure 2).

Heavy metal contents in the tincture were quantitatively analyzed using atomic absorption spectrophotometer following the previously reported standard procedure (Ayaz et al., 2014; Khan et al., 2016). All chemicals and reagents used were of high-quality analytical grade. Plants powdered material 0.2 g was added to a crucible and ignited at 55 °C for six hrs using a muffle furnace. The resultant ash was digested via concentrated HCL 0.5 mL and evaporated using hot plate apparatus. A small quantity of double distilled water was added to the mixture followed by filtration and the final volume was increase to 30 mL using double distilled water. The solution formed was analyzed using Shimadzu single quadrupole GCMS-QP2010 SE gas chromatograph-mass spectrometer (Shimadzu Corporation, Japan) for the number of heavy metals according to the standards available in our laboratory including lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) following standard operating procedure and were compared with Permissible levels of Technical Regulations of the Customs Union in herbal preparation for human use.

Flavonoid quantification was determined by previously reported standard protocol (Ayaz et al., 2017b; Pandey and Tripathi, 2014; Rajani Kanaki, 2008; Ab Halim et al., 2012).

Briefly, 1 g of powdered (1 mm) sample was added to 100 mL grinding flask and 30 mL of 70% alcohol was added to it. Flask was heated in a boiling water bath for 30 minutes. Subsequently, the flask was cooled to room temperature under a shower of cold water and filtered through a paper filter into a measuring flask with a capacity of 100 mL. The filtrate was flushed with 70% alcohol and added the alcohol to increase volume up to the mark of the flask. The obtained solution was named "solution A". Thereafter, 4 mL of "solution A" was transferred to a 25 mL flask and 2 mL of 2% solution of aluminum chloride in 95% alcohol was added to it, followed by the addition of 95% alcohol into the flask until the mark. The resultant solution was kept for 20 min and optical density was measured using a spectrophotometer at a wavelength of 410 nm in a 10 mm thick cuvette. For comparison solution, 4 mL of "solution A" was added to a flask, volume was increased to 25 mL and 1 drop of diluted hydrochloric acid was added to it. Finally, volume increased using 95% alcohol. Flavonoids (X) was quantified using the formula (1);

(1) X = D × 100 × 100 × 25 330 × m × ( 100 − W )

Where:

D – optical density of the tested solution; 330 – Specific absorption index of the complex with aluminum chloride at 410 nm; m – a mass of the sample (g); W – mass loss on drying (%).

For the qualitative assessment of tannins content, powder material (300 mg) of the plants was used following the previously reported procedure (Ejikeme et al., 2014). The required powder material was transferred to a test tube and boiled in a water bath for 30 minutes using a water bath. After boiling, the mixture was filtered through Whatman filter paper 42 and a few drops of ferric chloride (0.1%) were added to the filtrate. A brownish-green color was resulted indicating the presence of tannins. Subsequently, quantitative analysis was done (Geetha and Geetha, 2014). In 500 mL of conical flask put 2 g of shredded and sieved through a sieve with a hole diameter of 3 mm sample and added 250 mL of boiling water to it. Then the flask was weighted using an electronic scale and boiled the contents with periodic stirring. Thereafter, the flask was placed at room temperature to get the contents cool and weighted again, freshly boiled water was added to fill the missing weight. About 100 mL of the resulting extraction was filtered into a conical flask of 200 – 250 mL through absorbent cotton so that particles of samples do not get into the flask. Subsequently, 25 mL of filtered solution was poured into another conical flask with a volume of 750 – 1000 mL, then 500 mL of water was added, 25 mL of indigo-sulphonic acid and titrated against potassium permanganate solution (0.02 mol.L^-1; reagent grade 99.5%, LLP “Nikitin”, Semey, Kazakhstan ) until golden yellow color appeared. At the same time, conducted a control experiment. The content of tannins (X) in percent converted to absolute dry raw materials, was calculated by the formula (2);

(2) X   =   ( V-V 1 ) ×0.004157×250×100×100 M×25× ( 100-W )

Where:

V – the amount of potassium permanganate solution (0.02 mol.L^-1) used for titration of extraction (mL); V1 – the amount of potassium permanganate solution (0.02 mol.L^-1), used for titration in the control experiment (mL); 0.004157 – amount of tannins equivalent to 1mL of potassium permanganate solution (0.02 mol.L^-1) per tannin (g); M – sample weight (g); W – mass loss on drying (%); 250 – total extraction volume (mL); 25 – extraction volume took for titration (mL).

The amino acid composition was determined using GCMS-QP 2010 Ultra chromatomass spectrometer (Shimadzu Corporation, Japan), M-04-38-2009 test method (FR.1.31.2010.07015) (Bashari et al., 2019). Plants sample weighing 1 g was transferred to a conical flask and a 1000 cm³ extraction solution was added. The mixture was shaken for 60 min using a mechanical shaker and 100 cm³ of supernatant liquid was transferred from it into a beaker. About 5.0 cm³ of sulfosalicylic acid (reagent grade 99%, LLP “Kazbiohim”, Almaty, Kazakhstan) was added to it and continued mixing with a magnetic mixer for 5 min. The mortar was filtered to remove the sludge. The pH of the obtained solution was adjusted to 2.20 using sodium hydroxide solution (assay percentage ≥98%, LLP “Kazbiohim”, Almaty, Kazakhstan). The solution was transferred into a measuring flask and increased volume with a citrate buffer. The chromatography was performed following SOPs available for the equipment. It is important that the same amount (±0.5%) of standard solution and sample is entered into the column unless an internal standard is used (for chromatographic systems requiring a low sodium concentration) and unless the amino acid ratio in standard solutions and the sample should be as close as possible.

The amino acid content of the test sample, g.kg^-1, is calculated using the formula (3);

(3) w   =   A b ×c×M×V b A c ×m×1000

Where;

A_b – amino acid peak area in hydrolyzate and extract; c – molar concentration of amino acid in standard solution, mol.dm^-1; M – amino acid molecular weight; V_b – total hydrolyzate volume or calculated total dilution volume of the extract cm; A_c – amino acid peak area in a standard solution.

Previously reported standard protocol was used to quantitatively analyze saponins content (Ayaz et al., 2016; Zeb et al., 2014). The sample was milled to the size of the particles passing through a sieve with 1 mm diameter holes. About 0.5 g of the sample was packed in a chuck from filter paper and was placed in the extractor of the Soxhlet device. Chloroform extraction (reagent grade 99%, LLP LenReaktiv, Moscow, Russia) was discarded and the holder with the plant sample was dried. The sample was placed on the cartridge in a round bottom flask with a capacity of 100 mL. Subsequently, 50 mL of 90% alcohol (LLP “Nikitin”, Semey, Kazakhstan) was poured in and heated with the back cooler in a boiling water bath for 1 hour. The extraction was filtered through a dry paper filter into a distillation flask with a capacity of 200 mL, the rest on the filter is twice washed with 90% alcohol in portions of 5 mL. Extractions were combined and the solvent was distilled under vacuum drying.

The residue was dissolved in 20 mL of 96% alcohol (LLP “Nikitin”, Semey, Kazakhstan) during heating, cooled, quantitatively transferred with 25 mL of 96% alcohol into a flask with 50 mL capacity, filled up with 96% alcohol till the mark. Two mL of the solution obtained was placed into a 50 mL conical flask, 8 mL of concentrated sulfuric acid (mass fraction of H₂SO₄, 93.6 – 95.6%, LLP “Nikitin”, Semey, Kazakhstan) was added to it and stirred. After 30 min the optical density of the obtained solution was measured on the spectrophotometer SF-46 (Lomo Company, Russia) at a wavelength of 405 nm in a cuvette with a layer thickness of 10 mm. The mixture consisting of 2 mL of 96% alcohol and 8 mL of concentrated sulfuric acid was used for comparison. Content of saponins (X) in 1 g of raw material was calculated by the formula (4);

(4) x =  D   ×   m 0   ×   50   ×   10   ×   6   ×   2   ×   100   ×   100 D 0   ×   m   ×   1   ×   2   ×   100   ×   25   ×   6   ×   100   ×   ( 100   -   W )               = D   ×   m 0   ×   20 D 0   ×   m   ×   ( 100-W )

Where:

D – optical density of the test solution; D₀ – optical density of Pharmacopoeia standard sample of Escin; m – sample weight, g; m₀ – the mass of Pharmacopoeia Standard Sample Escin (g); W – mass loss on drying (%).

Organoleptic and physicochemical indicators including consistency, appearance, taste, color, taste, and odor were analyzed in the obtained tincture following American Herbal Products Association (AHPA) guidelines for organoleptic analysis of herbal ingredients (Upton et al., 2016; Dentali, 2013).