DEVELOPMENT OF STANDARDIZATION METHODOLOGY OF ELECAMPANE RHIZOMES AND ROOTS (INULA HELENIUM L.) FOR THE HYDROXYCINNAMIC ACIDS CONTENT

Abstract

Introduction. The elecampane rhizomes and roots are an officinal medicinal plant raw material (MP), which belongs to a group of medicines that stimulate expectoration with indication in the instruction for use - for upper respiratory tract and lungs diseases (bronchitis, tracheitis, catarrh of the upper respiratory tract); diseases of the gastrointestinal tract (gastritis, enterocolitis, lack of appetite, poor digestion). According to the literature about the analysis and standardization of elecampane rhizomes and roots, this usage is associated with high content of inulin and other fruitosans [1-7]. Numerous studies indicate other kinds of activities of MP and its various extracts - antidiabetic, antioxidant, bacteriostatic, antifungal [8-11], which is associated with the content of polyphenolic compounds, flavonoids, hydroxycinnamic acids. Considering insufficient level of elecampane rhizomes and roots standardization - absence of a monograph in State Pharmacopoeia of Ukraine and leading world pharmacopoeias, it is time to revise quality criteria for this raw material. Therefore, the object of this study was to develop a methodology of elecampane rhizomes and roots standardization for the hydroxycinnamic acids content.

Materials and methods. Research of the quality and quantity content of hydroxycinnamic acids was carried out on three industrial series of MP (JSC "Liktravy" Zhytomyr, Ukraine, samples 1-3 correspond to the series 20315, 30915, 10116) and three growing wild samples of MP (samples 4-6 , collected in Ternopil region). The study of the quality content of hydroxycinnamic acids was carried out in methanol extracts of raw materials. The methods of thin layer chromatography (TLC) and absorption spectrophotometry in the UV and visible region of the spectrum were used in this work.

Results and discussion. On the TLC-chromatogram of methanol extract of the elecampane roots four intensive zones appear: very intense zone with blue fluorescence and intense with greenish-blue fluorescence appear that are at chlorogenic and chicoric acids levels respectively on the chromatogram of reference solution. The two other zones with intense greenish-blue fluorescence are detected in the chromatogram of test solutions between chlorogenic and chicoric acids zones. Weak zones of blue fluorescence in the bottom of the chromatogram, located below the zone of chlorogenic acid and near the starting line were observed additionally besides the already described. The samples of raw materials have been generally similar in the composition of hydroxycinnamic acids - the number, position, colour and relative intensity of fluorescence (within one sample) of hydroxycinnamic acids zones are similar for all samples. Thus, based on the results of chromatographic researches, taking into account the literature data on the hydroxycinnamic acids composition [1, 5, 10, 11] and biological activity of aqueous-alcoholic extracts of this MP [8] and hydroxycinnamic acids, such as [12-15] it was proposed to identify these substances during the standardization of rhizomes with roots elecampane raw materials. In the electronic absorption spectra of water-alcohol extracts of different series elecampane rhizomes and roots the characteristic for hydroxycinnamic acids absorbance curve with maximum at a wavelength of 325 ± 2 nm was observed. This fact may be another additional identification marker when establishing identity of the studied raw materials. The nature of the spectrum, in particular the presence of clear absorption maximum at 325 nm wavelength, gives for hydroxycinnamic acid determination by direct measurement of absorption in the maximum followed by calculation of content by using specific absorption index. Chlorogenic acid was chosen as the standard for calculating the amount of hydroxycinnamic acids content, despite the fact that it is present in elecampane rhizomes with roots both according to the literature data and to the results of the chromatographic research of industrial and growing wild raw samples.

In developing the methodology of quantitative determination of the hydroxycinnamic acids amount it was found that in order to ensure the fullness of their extraction ethanol should be used as extractant (50% V / V). The investigated samples contain the hydroxycinnamic acid in an amount of slightly more than one percent. These data are generally lower than reported by the authors [6] – 2%. The difference in the content can be explained from different perspectives - the results of hydroxycinnamic acids determination of wild raw growing in central regions of Ukraine are shown in the work [6], but the extractant used, the conditions of sample preparation and method of final determination are not listed. The total content of hydroxycinnamic acids defined in local raw material is comparable to the content of hydroxycinnamic acids and their derivatives, obtained in elecampane callus culture [11].

Conclusions. As a result of the development of standardisation methodology of elecampane rhizomes and roots additional indicators of their quality - the identification and quantification of hydroxycinnamic acids are proposed.

1. It was proposed to identify the investigated raw material comparing TLC profiles of elecampane methanol extract and standard substances solution. Chlorogenic and chicoric acids were selected as identification markers. In the test solution chromatogram two intense zones of greenish-blue fluorescence should be detected between chlorogenic and chicoric acids.

2. The spectrophotometric methodology of hydroxycinnamic acids quantification, calculated into a chlorogenic acid, was developed. The content of hydroxycinnamic acids for industrial raw materials and wild samples is in the range of 1,10-1,35%. To establish the quality criterion of the hydroxycinnamic acids content results of more samples research for elecampane samples from different areas and different year of growth for taking into account possible effects of environmental factors on their content are needed.

3. The application of the proposed methods of identification and quantification of hydroxycinnamic acids allow to distinguish, by comparison of TLC profiles and electronic absorption spectra, two raw materials that are rich in inulin, identified by its presence, and in the crushed state are similar - rhizomes with roots elecampane and roots of chicory.