DEVELOPMENT OF THE METHOD OF QUANTITATIVE ANALYSIS FOR POT MARIGOLD FLOWERS

Abstract

Extract from pot marigold flowers (Calendula officinalis L.) has been used for a long time in both folk and traditional medicine as antiinflammatory, wound-healing, and carcinostatic preparations administered in various cases. The list of pot marigold preparations currently in use includes carophyllenic ointment (containing carotenoids extracted from pot marigold flowers), caleflon (containing the sum of flavonoids extracted from this plant), and pot marigold tincture [1, 2]. At present, the quality of pot marigold flowers used as raw material for tincture preparations is evaluated only with respect to the extracted substances. The purpose of this study was to develop a method for the quantitative analysis of biologically active substances in pot marigold flowers and related preparations. We suggest that the raw plant material should be quantitatively characterized with respect to isorhamnetin diglycoside – a flavonoid which is one of the main active components responsible for the pharmacological activity [1]. The proposed procedure includes isolation of the sum of flavonoids from the raw plant material, separation of isorhamnetin diglycoside from other components by TLC, elution of the target compound from a sorbent by 40% ethanol solution, and spectrophotometric analysis. EXPERIMENTAL PART Previously we have determined the optimum conditions for isolating biologically active substances from pot marigold flowers [3], which are also employed for the quantitative analysis of isorhamnetin diglycoside in the raw material. For the TLC separation of isorhamnetin diglycoside, we have performed experiments with various solvent systems, including chloroform – methanol – water, butanol – acetic acid – water, and hydrochloric acid – butanol taken in various proportions. The best separation was achieved using a mobile phase representing the 4 :1:5 n-butanol – acetic acid – water mixture (upper phase). The results of the TLC experiments are summarized in Table 1. The flavonoid band in the TLC pattern was detected by characteristic fluorescence under UV irradiation and by the R f value. Then we have studied the dependence of the amount of isorhamnetin diglycoside extracted from the sorbent on the duration of elution with aqueous ethanol solutions. Experiments with the solutions of various concentrations showed that the optimum eluent is a 40% ethanol solution. The results of these tests are presented in Fig. 1 by a plot of the