The study of the organic acids in dry extract of ledum palustre shoots

Labrador tea shoots has long been used in folk medicine as an antispasmodic, diuretic, diaphoretic, disinfectant, anti-inflammatory, soothing and antitussive remedy. In general it is due to the presence of terpene compounds, phenolic compounds, tannins and other substances in the plant material. “Ledin” was the only one medicine that was produced from this raw material. It based on the essential oil and used as antitussive remedy. But for now, just only packed raw materials remain on drugstores shelves. That is why it is necessary to obtain standardized, dry extract from this raw material. We have already studied phenolic compounds essential oil, amino acids and polysaccharides in this plant. So the next step of our research was to investigate the composition of organic acids in dry extract of labrador tea shoots. By PC method the malonic, malic and citric acids were identified in dry extract. By chromatographymass spectrometry method the 26 organic acids were identified in dry extract. Total content of them was 296.16 g/kg. The dominant compounds were levulinic acid (9.62 %), malic acid (4.48 %), citric acid (8.46 %) and 4 oksy benzoic acid (2.03 %). Obtained results of the investigations of the qualitative composition and quantitative content of organic acids in dry extract from Ledum palustre shoots show prospects for further research and the possibility of developing new drugs and will be used for future standardization of the extract.


INTRODUCTION
Labrador tea (Ledum palustre, Erisaseae family) -evergreen, squat shrub that has a significant area in northern Ukraine. The infusion and decoction of the young shoots of the plant has long been used in folk medicine as an antitussive, bactericidal and diaphoretic remedy in treatment of acute and chronic bronchitis, pneumonia, tuberculosis, whooping cough and other diseases that accompanied by cough. And also in treatment of spastic enterocolitis, endometritis and liver disease. Antiinflammatory properties of labrador tea used in arthritis treatment. The wide spectrum of pharmacological activities is due to the presence of terpenoids 1.6-4.6 %, phenolic compounds -4.9 %, tannins -1.6-4.6 % and other biologically active compounds [1]. "Ledin" was the only one medicine that was produced from this raw material. It based on the essential oil and used as antitussive remedy in tablet form in dose for 0.5 g. But for now, just only packed raw materials remain on drugstores shelves. We have already studied phenolic compounds [4] essential oil [3], amino acids [2] and polysaccharides [9] in this plant. So the next step of our research was to investigate the composition of organic acids in dry extract of labrador tea shoots.
So the aim of our research was to investigate the composition of organic acids of dry extract of Labrador tea shoots.

MATERIALS AND METHODS
The object of our research was a dry extract from labrador tea shoots. 500 g of chopped by way of rolling raw material was placed in 5000 ml flask and 1000 ml of 50 % ethyl alcohol was added. Extraction was carried out at room temperature for one day. Extraction was performed four times with a new portion of extractant. Then extracts were combined, filtered and evaporated in the vacuum-circulation apparatus at 100 °C and 340 mmHg till the dry extract was obtained.
Primary research of organic acids was carried out by paper chromatography (PC) method with use as the solvent system ethylacetate -formic acid -water (3 : 1 : 1) and presence of the reference solutions of organic acids. The dried chromatograms was processed with bromphenol blue and methyl red reagents (0.3 g of bromphenol blue and 0.1 g of methyl red dissolved in 100 ml of methanol) [5,6].
Determination of qualitative and quantitative composition of organic acids in dry extract was carried out by chromatography-mass spectrometry method on Agilent Technologies 6890 chromatograph with mass spectrometric detector 5973 and chromatography column -DB-5 with diameter 0.25 mm and a length of 30 m. As methylating agent 14 % BCl3 in methanol was used. Methylation carried out during 8 hours at 65 °C [7]. The analysis was carried out at the following conditions: thermostat temperature was programmed from 50 to 250 °C with © Upyr T. V., 2016 [72] Фармацевтична хімія та фармакогнозія УКРАЇНСЬКИЙ БІОФАРМАЦЕВТИЧНИЙ ЖУРНАЛ, № 6 (47) 2016 ISSN 2311-715X a speed of 4 °C/min; the rate of carrier gas (helium): 1 ml/min; transfer from GC to MS carried out till 230 °C; source temperature maintained at 200 °C; electron ionization conducted at 70 eV. For components identification, library of mass spectra NIST05 and WILEY 2007 with a total number of spectra more than 470,000 in conjunction with AMDIS and NIST programs were used [7,8].

RESULTS AND DISCUSSION
The yield of extractable substances from labrador tea shoots in calculation on dry raw material was 13.7 %. By PC method the malonic, malic and citric acids were identified in dry extract.
The results of the chromatography-mass spectrometry analysis of dry extract are presented in the Table and Figure. Total content of organic acids in dry extract of labrador tea shoots was 296.16 g/kg. 26 organic acids were identified: 2 monocarboxylic acids, 7 dicarboxylic acids, 1 tricarboxylic acids, 9 aromatic acids and 7 fatty acids. The dominant compounds were levulinic acid (9.62 %), malic acid (4.48 %), citric acid (8.46 %) and 4-oksy benzoic acid (2.03 %).

CONCLUSIONS
Obtained results of the investigations of the qualitative composition and quantitative content of organic acids in dry extract from Ledum palustre shoots show prospects for further research and the possibility of developing new drugs and will be used for future standardization of the extract.