The study of fatty acids in Anchusa officinalis L. herb of the Ukrainian flora

The presence of different groups of biologically active substances in the medicinal plant raw material determines its wide use in medical practice. The study of the chemical composition and possible pharmacological effects of medicinal plants are priorities in the process of their research. Aim. To study the content and the composition of fatty acids in Anchusa officinalis L. herb of the Ukrainian flora using the gas chromatography/mass spectrometry method (GC/MS). Materials and methods. The samples of Anchusa officinalis L. herb were harvested in June 2017 in the IvanoFrankivsk region. The study was performed using the GC/MS method. To identify the components, the spectra obtained were considered on the basis of general laws of fragmentation for molecules of organic compounds under the action of the electron impact, as well as by comparing the results with data from NIST05 and WILEY 2007 mass spectrum libraries. The data analysis was performed using Statistic 7.0, Excel 7.0 statistical program package for Microsoft Office for Windows. Results and discussion. This study presents the results the fatty acids investigation in Anchusa officinalis L. herb of the Ukrainian flora. For the first time, 8 fatty acids were identified. They were myristic acid (163.36 mg/kg), palmitic acid (2324.39 mg/kg), linoleic acid (1498.33 mg/kg), linolenic acid (2696.36 mg/kg), stearic acid (410.16 mg/kg), arachidic acid (240.43 mg/kg), behenic acid (409.23 mg/kg), lignoceric acid (498,38 mg/kg). The total mass of unsaturated fatty acids (linoleic and linolenic acid) was 4045.95 mg/kg, while the total mass of saturated fatty acids (myristic, palmitic, stearic, arachidic, behenic and lignoceric acid) was 4194.69 mg/kg. It indicates an insignificant predominance of unsaturated acids in the plant raw material. The total content of fatty acids in Anchusa officinalis L. herb was 8240.64 mg/kg (0.8 % by weight of the plant raw material). Conclusions. The study of fatty acids in Anchusa officinalis L. herb of the Ukrainian flora has been conducted for the first time. In the raw material, the following 8 fatty acids have been identified: unsaturated fatty acids (linoleic and linolenic acid) and saturated fatty acids (myristic, palmitic, stearic, arachidic, behenic and lignoceric acid). The total content of fatty acids in Anchusa officinalis L. herb is 0.8 % by weight of a dry raw material. The dominant compound among fatty acids is linolenic acid (2696.36 mg/kg of a dry raw material). Taking into account the above the further search for the method of obtaining the fatty acid complex from Anchusa officinalis L. herb is promising in order to develop the effective and safe herbal medicines.

Ключевые слова: Anchusa officinalis L.; трава; ГХ/МС; биологически активные вещества; жирные кислоты Introduction. Despite significant advances in the study of medicinal plants, all the possibilities associated with their use in medicine have not yet been exhausted. Currently, the selection and study of individual biologically active substances from plants are the main areas of research when developing new drugs. Often, when using medicinal plants, not just any substance, but a complex of substances is active. At the same time, substances that do not have a self-expressed pharmacological effect can contribute to the therapeutic effect. The presence of different groups of biologically active substances in the medicinal plant raw material determines its wide use in medical practice. Biologically active substances of plants are inorganic and organic compounds; their common characteristic is high activity in relatively small quantities. The study of the chemical composition and possible pharmacological effects of medicinal plants are priorities in the process of their research.
Fatty acids are not synthesized by the human body. Different types and concentrations of fatty acids are presented in many plant and animal sources. Unsaturated fatty acids, in particular linoleic and linolenic, regulate the lipid metabolism, provide the growth and development of organisms and increase their resistance to harmful environmental factors [1,2].
Anchusa L. species are a valuable source of natural compounds with potential therapeutic effects. Due to the variety of biologically active substances, Anchusa genus species show diverse pharmacological actions [9].
Anchusa officinalis L. is the most common type and it is widespread in muddy and sandy places and in fields almost all over Ukraine [9].
There is limited number of reports in the world on studying fatty acids in Anchusa genus species. Al-Salihi F.  dry flowers. The highest content was recorded for n-hexadecanoic and octadecanoic acids -3.6404 µgm and 4.6040 µgm /100 g of a dry raw material, respectively [2]. Kukukboyaci N. et al. (2013) determined the content of fatty acids in the seed oils of Anchusa species of the Turkish flora -Anchusa azurea Miller var. azurea, Anchusa leptophylla Roemer & Schultes subsp. leptophylla, Anchusa arvensis (L.) Bieb. subsp. orientalis (L.) Nordh. and Anchusa undulata L. subsp. hybrida (Ten.) Coutinho. The composition of fatty acids for all species was the same, and it was represented by oleic, palmitic, linolenic, ɣ-linolenic and α-linolenic acids [1]. Ozcan T. (2008) studied the fatty acid composition in seeds of two informal groups of Anchusa officinalis L. of the Turkish flora. It was found that predominant acids were linoleic, oleic, and α-and ɣ-linolenic [10]. Thus, the study of fatty acids of Anchusa officinalis L. herb of the Ukrainian flora is relevant and is conducted for the first time.
The aim of this work was to study the content and the composition of fatty acids in Anchusa officinalis L. herb of the Ukrainian flora using the gas chromatography/mass spectrometry method (GC/MS).

Materials and methods.
The samples of Anchusa officinalis L. herb were harvested in June 2017 in the Ivano-Frankivsk region. The plant was identified at the Department of Pharmacy, Ivano-Frankivsk National Medical University, Ivano-Frankivsk, Ukraine, and the authenticated voucher specimens were deposited there. All reagents used were of the highest purity (Sigma-Aldrich, St. Louise, USA). The study was performed by the GC/MS method using an Agilent Technologies 6890 chromatograph with a 5973 mass spectrometric detector and a HP-5ms capillary column (diameter -0.25 mm, length -30 m). The speed of the carrier gas (helium) was 1.0 ml/min. The temperature of the sample heater was 250 °C. The temperature of the thermostat was programmed from 60 °C to 320 °C with a speed of 7 °/min.
To obtain fatty acid methyl esters, 0.05 g of the crushed herb was placed in 2 ml vials, adding n-tridecane as an internal standard and 2 ml of 2 % solution of acetyl chloride in methanol. The vials were incubated for 2 h at 37 °C. Fatty acid methyl esters were extracted with 500 μl of hexane.
To identify the components, the spectra obtained were considered on the basis of general laws of fragmentation for molecules of organic compounds under the action of the electron impact, as well as by comparing the results with data from NIST05 and WILEY 2007 mass spectrum libraries with a total of more than 470000. The quantitative content of components (X, mg/kg) was determined relative to the internal standard by the formula: X = (P 1 × 50) / (P 2 × m), where: P 1 -is the peak area of the substance studied; 50 -is the mass of the internal standard introduced into the sample, μg; P 2 -is the peak area of the standard; m -is the weight of the raw material, g.
The data analysis was performed using Statistic 7.0, Excel 7.0 statistical program package for Microsoft Office for Windows.
Results and discussion. This study presents the results the fatty acids investigation in Anchusa officinalis L. herb of the Ukrainian flora. For the first time, 8 fatty acids were identified: 2 polyunsaturated -linoleic, ɣ-linolenic and 6 saturated -palmitic, stearic, myristic, arachidonic, behenic, lignocerinic acids. However, 6 compounds were not identified, but it was determined that 4 of them belonged to alkanes. The data obtained are presented in Fig. The results of the analysis of fatty acids in Anchusa officinalis L. herb are given in Table. According to data of Tab. 1, 15 compounds were determined in Anchusa officinalis L. herb; 8 of them were identified as fatty acids. They were myristic acid (163.36 mg/kg), palmitic acid (2324.39 mg/kg), linoleic acid (1498.33 mg/kg), linolenic acid (2696,36 mg/kg), stearic acid (410.16 mg/kg), arachidic acid (240.43 mg/kg), behenic acid (409,23 mg/kg), lignoceric acid (498.38 mg/kg). The total mass of unsaturated fatty acids (linoleic and linolenic acid) was 4045.95 mg/kg, while the total mass of saturated fatty acids (myristic, palmitic, stearic, arachidic, behenic and lignoceric acid) was 4194.69 mg/kg. It indicates an insignificant predominance of unsaturated acids in the plant raw material. The total content of fatty acids in Anchusa officinalis L. herb was 8240.64 mg/kg (0.8 % by weight of the plant raw material).

Conclusions and prospects of further research
The study of fatty acids in Anchusa officinalis L. herb of the Ukrainian flora has been conducted for the first time.
In the raw material, the following 8 fatty acids have been identified: unsaturated fatty acids (linoleic and linolenic acid) and saturated fatty acids (myristic, palmitic, stearic, arachidic, behenic and lignoceric acid).
It has been determined that unsaturated acids are predominant in the plant raw material. The dominant compound among fatty acids is linolenic acid (2696.36 mg/kg of a dry raw material). The total content of fatty acids in Anchusa officinalis L. herb is 0.8 % by weight of a dry raw material.
Taking into account the above the further search for the method of obtaining the fatty acid complex from Anchusa officinalis L. herb is promising in order to develop the effective and safe herbal medicines.
Conflict of interest: the author has no conflict of interests to declare.