THE STUDY OF FATTY AND ORGANIC ACIDS OF VACCINIUM ULIGINOSUM LEAVES

Using chromato-mass-spectrometry the analysis of the qualitative composition and the quantitative content of organic and fatty acids in Vaccinium uliginosum leaves has been conducted. The analysis of methyl esters of fatty and organic acids has been performed using a gas chromatograph/mass spectrometer (5973N/6890N MSD/DS Agilent Technologies, USA). Identification of methyl esters of acids has been performed on the basis of the calculation of the equivalent length of the aliphatic chain using the data from the NIST 05 and Willey 2007 mass spectra library with the total number of spectra more than 470000 in combination with AMDIS and NIST programmes for identification; the retention time has been also compared with the retention time of standard compounds (Sigma). In Vaccinium uliginosum leaves 36 organic and fatty acids have been identified, their total content is 5867.84 mg/kg. In leaves 18 fatty acids and 18 organic acids have been found. Dominant components of Vaccinium uliginosum leaves among fatty acids are palmitic acid (1566.74 mg/kg), myristic acid (273.46 mg/kg), tetracosanoic acid (219.73 mg/kg) and arachic acid (214.82 mg/kg), and among organic acids – levulinic acid (600.32 mg/kg) and malonic acid (493.17 mg/kg). Based on the research conducted it has been proven that Vaccinium uliginosum leaves are the promising raw material for further pharmacognostic study.

The family of Ericaceae has a special place among small fruits, one of its main representative is Vaccinium uliginosum (bog blueberry) [3], which is interesting not only for its biology, ecology, geography and history, but also for its practical value in pharmacy.
The leaves of Vaccinium uliginosum increase the activity of the stomach, intestines and heart [1].In folk and scientific medicine bog blueberry is popular as an antiscorbutic and antidysentric agent [1,2].A decoction of branches with leaves in the ratio of 1:10 is used in folk medicine in heart diseases and colitis.Infusion and decoction of leaves are recommended in diabetes, anemia, and to improve metabolism [1].Moreover, Vaccinium uliginosum leaves are also used in various herbal teas and decoctions for the treatment of heart diseases, hypertension, diabetes mellitus, constipations [1,2].
Earlier the qualitative composition and the quantitative content of some classes of BAS were studied in Vaccinium uliginosum leaves, namely simple phenols, derivatives of hydroxycinnamic acids, flavonoids and tannins [4].Continuing the study of BAS from Vaccinium uliginosum leaves and products of their processing our attention was drawn to the fact that the composition of organic acids and fatty acids has been almost unstudied.
Therefore, the aim of our work was to study the qualitative composition and the quantitative content of organic and fatty acids of Vaccinium uliginosum.

Materials and Methods
The study of Vaccinium uliginosum leaves was conducted in the following way: to 0.50 mg of the dried powdered raw material in a 2 ml vial the internal standard (50 µg of tridecane in hexane) and 1.0 ml of methylating agent -14% methylene chloride in methanol, Supelco No.3-3033 were added.The mixture was kept in a sealed vial for 8 hours at 65°С.During this time fatty oil was completely extracted from Vaccinium uliginosum leaves, and there was re-esterification of fatty and organic acids.The reaction mixture was decanted from the precipitate and diluted with 1 ml of distilled water.To obtain methyl esters of fatty and organic acids 0.2 ml of methylene chloride was added, shaken for 1 hour and subjected to chromatography.The sample injection of 2 µl was carried out to the chromatographic column in a splitless mode, it allowed to introduce the sample without loss in splitting, as well as to increase the sensitivity of the chromatographic method significantly up to 20 times.The sample injection rate was 1 ml/min, the time -0.2 min.The analysis of methyl esters of fatty and organic acids was performed using a gas chromatograph/mass spectrometer (5973N/6890N MSD/DS Agilent Technologies, USA).
The detector of the mass spectrometer was quadrupole, the ionization method was electron impact (EI), and the ionization energy was 70 eV.The mode of recording the full ion current was used for analysis.For splitting a HP-INNOWAX capillary column (30 m×250 μm) was used.The stationary phase was INNOWAX.The mobile phase was helium, the flow rate -1 ml/min.The temperature of the heater of the sample injection was 250°С.The thermostat temperature was programmed from 50 to 250°С.Identification of methyl esters of acids was performed on the basis of the calculation of the equivalent length of the aliphatic chain using the data from the NIST 05 and Willey 2007 mass spectra library with the total number of spectra more than 470000 in combina- tion with AMDIS and NIST programmes for identification; the retention time was also compared with the retention time of standard compounds (Sigma).

Results and Discussion
The content of organic acids in Vaccinium uliginosum leaves was 5867.84 mg/kg.The relative content of acids was calculated as a percentage of their total content.The retention time of acids is given in Table .Using chromato-mass-spectrometry the analysis of the qualitative composition and the quantitative content of organic and fatty acids in Vaccinium uliginosum leaves was conducted.In Vaccinium uliginosum leaves 36 organic and fatty acids were identified.Palmitic acid dominated among fatty and organic acids; its content was 26.7% of the total amount of acids, and the relative content of fatty acids was 56.76%.

CONCLUSIONS
The qualitative composition and the quantitative content of organic and fatty acids in Vaccinium uliginosum leaves have been studied.In all, 36 substances have been identified.Dominant components of Vaccinium uliginosum leaves were palmitic acid, levulinic acid and malonic acid.Further pharmacognostic study of Vaccinium uliginosum leaves is promising for creation of new medicines.