Research On The Study Of The Flavonoid Composition And End-To-End Standardization Of The Raw Material And Dosage Form Of Calendula Officinalis L. Growing In Uzbekistan

Abstract

Herbal preparations have been used since ancient times as medicines to treat a range of ailments. Each plant synthesizes an unlimited number of very complex and unusual chemicals, the structure of which is different. There are over 120 different plant-derived chemicals currently considered important medicines.

The World Health Organization (WHO) emphasizes the importance of qualitative and quantitative methods for the characterization of herbal preparations, the quantification of biomarkers and/or chemical markers.

If the main active ingredient is known, it is most logical to quantify that compound. Where active ingredients are known to contribute to therapeutic efficacy, herbal preparations should be standardized for these compounds. If the active ingredients are not yet known, a marker substance can be selected for analytical purposes, which should be specific to the herbal preparation.

Phytochemical standardization covers all possible information obtained with respect to the chemical constituents present in a herbal medicinal product. Therefore, phytochemical evaluation for standardization purposes includes the following:

• Preliminary testing for the presence of various chemical groups.
• Quantification of chemical groups of interest (e.g. total alkaloids, total flavonoids or phenols, total triterpene acids, total tannins, etc.).
• Creation of fingerprint profiles.
• Quantification of important chemical components.

The objective of the study: to study the chemical composition and standardization of raw materials and dosage forms of Calendula officinalis L., growing in Uzbekistan.

To achieve this goal, the following tasks were defined:

- to study the content of the main group of biologically active compounds in the raw materials of Calendula officinalis L.;

-to determine the main component for standardization;

- to develop a method for qualitative and quantitative analysis of the content of the main substance using chromatography methods.

To study the main group of biologically active substances, dried and crushed raw materials of Calendula officinalis L. were extracted with 70% alcohol. The alcoholic extracts were thickened and the extract was successively treated with various solvents, in particular chloroform, ethyl acetate and n-butanol. In order to optimally separate flavonoids in the obtained ethyl acetate fractions of calendula, studies were carried out on the choice of a chromatographic system for TLC, the results of which showed that the solvent system consisting of ethyl acetate, formic acid and chloroform in a ratio of 2:1:2 has the most optimal separating ability. The data obtained by TLC showed that the largest number of flavonoid compounds pass into the ethyl acetate fraction of calendula - 8 adsorption zones of yellow color, flavonoid character. Also, for the separation and identification of calendula flavonoids, the HPLC method was used on HPLC by Agilent Technologies (USA) "1100 series", with the “ChemStation 3D” software, equipped with a four-gradient pump with a vacuum degasser and an autosampler, a column thermostat and a UV/VIS diode array spectrophotometric detector with a wavelength from 190 to 800 nm. We used a chromatographic column 150x3.0 mm in size filled with Zorbax Eclipse XDB C-18 sorbent with a particle size of 3.5 µm from Agilent Technologies. The analyzes were carried out in isocratic mode with a mobile phase flow rate of 0.5 ml/min, detection was performed at 250-400 nm, while simultaneously recording the spectra of each peak. Mobile phase: 50 mM phosphate buffer (pH=3) - methanol (80:20), the volume of the sample injected into the chromatograph injector was 10 μL, the duration of the analysis was 40 min. Flavonoids were identified by the retention time and the UV-correspondence factor of the spectra of standard samples solutions of the studied substances (SSS). The results of the identification of flavonoids of Calendula officinalis L. raw materials showed that it contains: hyperoside, luteolin, luteolin-7-glycoside, luteolin-3'-O-β-D-glucopyranoside, kaempferol, quercetin, isoquercetin, isorhamnetin-3-O-β-D -glycoside. With the use of this method, a method of end-to-end standardization of raw materials was developed - flowers and tincture of Calendula officinalis L., growing in Uzbekistan. Quercetin was used as a standard substance. The results of the studies show that the content of quercetin in flowers and tincture of calendula is on average 0.0146 and 0.0028%, respectively, and the relative error of the average result of the proposed method reaches up to ±4.99%.