## 2 Materials and Methods
### Methods
#### 2.2.1 Qualitative Phytochemical Screening  
The dried raw plant materials were subjected to repeated hot ethanol extraction. The obtained alcoholic extracts were then concentrated to a certain volume. The active principles of the extracts were identified by means of specific reactions. Qualitative chemical analysis was performed on the initial alcoholic extractive solutions [47; 48], with particular emphasis on such methods as reduction with [PERSON]'s and [PERSON] reagents and the use of the Molisch reaction and Styassny's reagent. _Identification of Reducing Compounds_[47]:  
1. The reaction with [PERSON]'s reagent was based on the ability of these compounds to reduce a solution of [PERSON]'s reagent to copper oxide in a strongly alkaline medium. A solution of 1 mL of alcoholic extract diluted with 2 mL of distilled water was treated with 1 mL of a mixture of [PERSON]'s solutions and heated for 30 min. The appearance of a red-brick precipitate of copper oxide indicates the presence of reducing compounds. 2. Reaction with [PERSON] reagent: 3-4 drops of [PERSON] reagent were added to 1 mL of alcoholic extract solution and heated. We then observed the deposition of the silver mirror on the test tube. _Identification of Carbohydrates_  
The carbohydrates were identified using the Molisch reaction. A total of 2 mL of alcoholic extract solution was concentrated to dryness. Then, 1-2 drops of concentrated sulfuric acid and 3-4 drops of 1% thymol were added to the residue. The presence of a red-orange color indicated the presence of such compounds. _Identification of Tannins_[49]  
According to the chemical nature of tannins and their classification into gallic tannins and catechin tannins [49], the following methods of identification were used:  
1. Identification with 1% ferric chloride: 1 mL of alcoholic extract solution was diluted with 2 mL of distilled water and 2-3 drops of ferric chloride were added until a green (for the catechin tannin) or blue (for the gallic tannin) color was observed. 2. Identification with [PERSON]'s reagent (a hydrochloric formaldehyde solution formed by formaldehyde and concentrated hydrochloric acid in a molar ratio of 2:1): 5 mL of alcoholic extract solution was treated with 4-5 mL of [PERSON]'s reagent; then, the solution was heated for at least 30 min. The appearance of a red-brown precipitate indicates the presence of the catechin tannin. 3. The final step was the separation of the catechin tannin from the gallic tannin. 4. For the blue-black color that indicates the presence of the catechin tannin, 10 mL of extract solution was treated with 10 mL of [PERSON]'s reagent then heated for at least 30 min. The condensed catechin tannin was separated by filtration. The solution was neutralized with Na2 CO3. When the blue color appeared, 1-2 drops of [PERSON]'s reagent were added. The blue color was a signal of the presence of gallic tannins. _Identification of Flavonosides_  
Flavorosides were identified directly in the alcoholic solution if they were not mixed with anthocyanins or were identified after hydrolysis [49]. 1. The Shibata reaction (cyanidol reaction): under the reducing action of nascent hydrogen, the pyranic nucleus undergoes a reduction of the ketone group with the redistribution of electrons inside the nucleus and the formation of a pyrrole salt. A 3-5 mL solution was evaporated to a residue. The residue was dissolved in 1-2 mL of hot methanol. The solution was brought to a test tube, and fragments of metallic magnesium and 0.5 mL of concentrated hydrochloric acid were added. Within 5 min, an orange (flavones), a red (flavonols), and a red-purple (flavanones) color appeared. 2. Chelation reaction with salts of bi- and trivalent metals: 3-5 mL of solution was evaporated to dryness. The residue was dissolved in 1-3 mL of alcohol and then 1-2 mL of aluminum chloride was added. The intensification of the yellow coloration was observed. 3. Formation of phenoxides: 3-5 mL of solution was evaporated.