Quantitative determination of the total content of flavonoids in Flavozid and Immunoflazid syrups

Abstract

New syrup compositions Flavozid and Immunoflazid that contain 2% Proteflazid (ethanol extract of wild Deschampsia caespitosa L. and Calamagrostis epigeios L.) have been developed. The total content of flavonoids in both syrups is of the level of 4 – 6 gmL, which complicates the development of an analytical method. A new method for quantitative determination of flavonoids in the aforementioned syrups has been developed on the basis of differential UV spectrophotometry of their complexes with aluminum chloride. The proposed method is introduced into the Manufacturer’s Pharmacopoeic Article for the syrups. The existing formulation of Flavozid and Immunoflazid syrups is based on the active pharmaceutical ingredient Proteflazid, which was first developed and registered in Ukraine in 2001 and is the alcohol extract of wild Deschampsia caespitosa L. and Calamagrostis epigeios L. Proteflazid prepared using the original technology was recommended for use in medical practice as a composition with direct antiviral and immunomodulating action with properties of an interferon activator and DNA protease inhibitor in virus-infected cells [1 – 3]. The principal biologically active compounds of Proteflazid are flavonoids similar to quercetin (rutin), the molecules of which are based on a flavone oxygen-containing heterocycle. However, the mixture of Proteflazid flavonoids did not contain those that are frequently used as standards. Therefore, the development of a quantitative determination method for flavonoids in the syrup, where the content of Proteflazid and the corresponding flavonoids is 2% (or 4 – 6 gmL) of the starting extract, is of definite scientific interest. The goal of our work was to develop a method that would enable quantitative determination of total flavonoids, which absorb in the UV and visible spectral regions, using aluminum chloride as a complex-forming reagent. The glycoside species were converted to the aglycons by preliminary boiling of the syrup in the presence of HCl in order to increase the total amount of flavonoids in the analytes. Acid hydrolysis of the glucosides in the syrup was carried out in an excess of conc. HCl taken at 1% of the volume of the investigated syrup. The optimum conditions for acid hydrolysis with time were determined from the degree of conversion of the glycoside component into aglycons with TLC monitoring and UV spectrophotometric data. Aglycons from the syrup were extracted by ethylacetate. Solvent was vacuum distilled until production of a pasty residue that was dissolved in ethanol (96%). Studies of the optimum conditions for complexation using freshly prepared aluminum chloride (5%) in ethanol (96%) showed that increasing the volume of the solution to greater than 1 mL in the analyte did not improve the optical density parameters of the aluminum-chloride complex of the flavonoids. The maximum optical density was retained over 25 – 40 min at wavelength 422 nm. A solution with all components of the test solution with the exception of aluminum chloride was used as a reference to compensate for absorption at 420 – 424 nm of the starting extract before complex formation with aluminum chloride. Considering the complex flavonoid composition of the starting material, known standards from various flavonoid groups were used for the quantitative determination. However, the absorption maximum of the complex with aluminum chloride of none of them agreed with that for the com