WORKING OUT AND VALIDATION OF THE METHOD OF QUANTITATIVE DETERMINATION OF FLAVONOIDS IN THE HERB OF LEONTODON AUTUMNALIS L

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Leontodon autumnalis L. from Asteraceae family is broadly used in folk medicine as antitussive agent in the Caucasus and Karelia, for treatment of digestive organs diseases in Ukrainian and Belarus Polesye. There are also recommendations in folk medicine for newborn children bathe in decoction of Leontodon autumnalis L. when they cry or do not sleep. Previously we have established that the herb of Leontodon autumnalis L. contains flavonoids, which are represented by aglycones, luteolin, and apigenin and their glycosides. Flavonoids exhibit a wide spectrum of phar- macological activity. Flavonoids and drugs on their basis have anti-infl, antioxidant, diuretic, anti-diabetic, and other types of action [3, 5]. Wide application of flavonoids in medicinal practice creates the necessity of working out and improvement of their analysis methods. The purpose of the works is to work out a methodology of a quantitative determination of flavonoids in the herb of Leontodon autumnalis L., and its validation. The herb of Leontodon autumnalis L. stocked up in Kursk, Belgorod, and Voronezh Oblasts in 2013-2014 in a mass bloom was the object of the study. The most widespread method of quantitative determination of flavonoids is differential spectrophotometry of determination in a visible spectrum [1, 4]. We have suggested a method of spectrophotometry determination for the quantitative determination of flavonoids sum in raw materials of Leontodon autumnalis L. after preliminary conduction of reaction of complex- ation with solution of aluminum chloride in SF- 2000 spectrophotometer. While choosing for a standard for the calculation of flavonoids sum we have measured spectrums of adsorption of an alcohol extract from the raw materials of Leontodon autumnalis L. with aluminum chloride and cynaroside with aluminum chloride. The obtained spectrums coincided, and maximum adsorption was at the wavelength 395 nm. With that we have studied the stages like extraction of flavonoids and complexation conditions with aluminum chloride solution. The working out of quantitative determination provides the validation. Thus we have offered a cynaroside (standard sample of Fitopanacea CAS no. 5373-11-5) - 7-glycoside of luteolin for a standard substance for the calculation of flavonoids sum. While establishing the quantitative content of flavonoids in the grass of Leontodon autumnalis L. we have studied and revealed optimal conditions for the analysis: raw materials fineness factor, extraction time, extracting agent type, volume, and concentration of aluminum chloride solution (table 1) [2]. We have established that the most complete extract of flavonoids from the grass of Leontodon autumnalis L. is achieved with the 75% ethanol extraction within 45 minutes with raw materials fineness factor at 1.0 mm, extraction before the balance. Optimal condition for the complexation is reaction with 2 ml of 2% aluminum chloride within 30 minutes. Based on the studies conducted we have worked out a method of quantitative determination of flavonoids in the herb of Leontodon autumnalis L. About 1.0 g (precisely weighed quantity) of raw materials which pass through the sieve with holes diameter 1 mm, are placed into a flavonoids with friction lid, with the volume of 250 ml. The raw materials are flavonoids with 100 ml of 70% ethanol and weighed with ±0.01 error. The flavonoid is attached to the return condenser and placed on a boiling water bath for 45 minutes. The flavonoids was periodically shaken to wash away the particles of raw materials from the walls. After that, the fl with extract is cooled under the jet of flavonoids water and is weighed. If necessary the weight is brought to the initial with 70% ethanol. Cooled and brought to the initial weight ex- tract is filtered through the paper filter rejecting the first 10 ml of filtrate necessary for drench- ing the filter. 10.0 ml of filtrate are taken from the extract obtained which are placed into a measuring flask with a capacity of 25 ml, add- ed with 2 ml of 2% aluminum chloride solution dissolved in 70% ethanol. And after that in 10 minutes it is added with two drops of dissolved acetic acid. Solution volume is brought to the mark with 70% ethanol. In the next 30 minutes the solution’s optical density is measured in a spectrophotometer at the wavelength 395 nm in a ditch with working layer thickness 10 mm. Solution with the same components as the analyzed solution but without aluminum chlo- ride was used as a comparing solution. The content of flavonoid sum in percent concentration (X) in terms of cynaroside was calculated following the formula: where D - optic density of the solution under analysis at the wavelength 395 nm; V - the extract volume, ml; m - raw materials weighing mass, g; W - wetness of the raw materials, %. Validation of the worked out method of quantitative determination of flavonoids was conducted following the indices of linearity, range of use, repetition, reproducibility, correctness in accordance with the ICH recommendations on “Validation of analytic methods. Content and methodology” [5]. When checking the linearity of the method we have prepared 6 colored solutions of luteolin-7-glycoside complex with aluminum glycoside of different concentration, measured their optical densities at the wavelength 395 nm and build a graph of dependence of optical density on the flavonoid concentration (figure 1). Correlation coefficient amounted to 0.999614, therefore this method can be used for the analysis of flavonoids of the herb of Leontodon autumnalis L. in terms of cynaroside in a stipulated range of concentrations (table 2). To check the range of the method application we have obtained extracts from the herb of Leontodon autumnalis L. of different concentration. For this purpose extracts were obtained from the weighings 0.4, 0.6, 0.8, 1.0, 1.2 (precise weighing) and the flavonoids sum determination in these extracts was obtained with the worked out method (figure 2). According to the obtained experimental data we have calculated the correlation coefficient, angle coefficient of linear regression, free term of a graph (table 3). Correlation coefficient amounted to 0.999954, thus, the worked out method can be used for the quantitative determination of flavonoid sum in terms of cynaroside within a given range of concentrations. To prove the repetitiveness of the method we analyzed one sample of raw materials of Leontodon autumnalis L. in six replications. Acceptance criterion was expressed by the value of relative standard deviation, which should not exceed 10% (table 4). The reproducibility of the method was determined using one sample of raw materials in 6 replications. The acceptance criterion was expressed by the value of a relative standard deviation, which should not exceed 15%. It amounted to 6.98 (table 5). The correctness of the method was established by means of measuring of quantitative content of flavonoid in terms of cynaroside in extracts, obtained by the addition of a standard solution of cynaroside 0.05% - 0.25 ml, 0.5 ml, 0.75 ml to the extract under study. Acceptance criterion is an average percent of recovery with the use of solutions of the targeted concentrations; its average value should be limited by 100±5%. Recovery percent in the worked out method fluctuates between 97.12% and 102.69%, its average value amounts to 100.17% with a percentage error 4.37% (table 6). Following the worked out method we have established a content of flavonoids in the herb of Leontodon autumnalis L., stored up in different habitats (table 7). Analyzing the data from the table, we should note that the quantity of flavonoids in the herb of Leontodon autumnalis L. fluctuates between 0.55% and 0.71%. Conclusions 1. For the first time we have worked out the method of spectrophotometric determination of the flavonoid sum in the herb of Leontodon autumnalis L. Content of flavonoids in the raw materials of Leontodon autumnalis L. fluctuates between 0.55% and 0.71%. 2. We have conducted the validation of the worked out method by the indices of linearity, range of application, repetition, reproducibility, and correctness.

About the authors

R. A Bubenchikov

Kursk State Medical University

Email: bubenchikova.ksmu@yandex.ru
Kursk

N. N Goncharov

Kursk State Medical University

Kursk

References

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