Comparative analysis of freon extracts from lime flowers cropped in 2016 and 2017 years

Abstract

Introduction. The article is devoted to gas-liquid-chromatography analysis of the freon extracts taken from lime flowers croppedin 2016 and 2017 years. It’s well-known that quantitative content of a sum of extractives or active substances (and even their structure) can varyconsiderably in plants depending onharvesting place (and, hence, soils), harvest time, and, especially, weatherconditions. This is the main reasons for difficulties in standardization of herbal drugs. Often they are standardizedby total extractives, for some plant species there are no techniques of quantitative analysis at all in pharmacopoeias, such an example is lime flowers. Recently, in 2007, 2008, 2009 we studied lipophilic fractions from lime flowers and found slight differences in their quantitative and qualitative compositions. The main aim was to compare chemical composition of volatile compounds in lipophilic freon extracts from lime flowers considering that springs and beginning of summers in 2016 and 2017 was characterized by abnormal weather conditions. Materials and methods. The herbal raw materials were lime flowers cropped from linden Tilia cordata within Kharkivregion in 2016 and 2017, comminuted to particle size 0,5-2,0 mm. They were extracted with condensed difluorochloromethane (freon R22) in two parallel connected percolators of the experimental equipment. The extraction was two-stage at raw material : extracting solvent ratio 1:8 on each stage under constant circulation of a liquid phase. The minimum pressure and temperaturewere 12 atm and +30°С accordingly, maximum ones – 17 atm and +45°С. Totalexposure of the process took about 2 hours on each stage. The extracts were dissolved in methylene chloride to make 1,00 % solution which was entered into the front injector of gas-chromato-mass-spectrograph «Agilent Technology 6890» (USA) including two consequently connected columns: Agilent 19091S-433HP-5MS, filled with adsorbed5 % Phenyl-Methyl-Silox, and Agilent 165-6626 USB369911A, and also mass selective detector 5973N. The oven was programmed as follows: equilibration time – 0.5 min, temperature – from 50°C to 250 °C with rate 4°C/min, then 30°C/min to 300 °C, maximum temperature was 325°C, runtime – 51,667 min. Carrier gas was helium: in the first column the flow rate - 1 ml/min under 14,313 psi, and in the second one – 3,8 ml/min at initial pressure 3,8 psi, and in the outlet vacuum was provided. Transferring from gas-liquid chromatograph (GLC)to mass spectrometer occurredat 230°С. The detector and evaporator temperature was supported at 150°С. Electron ionization was spent at 70 eV in range of masses from m/z 29 to 450. Identification of compounds was carried out by comparisonof the acquired mass spectra with the data of existing libraries (about 500000 substances). Pentadecane was usedas the internalstandard. Quantitative content of each substance expressed in percentage of total assay weight of the samplewas determined by areas of corresponding peaks considering that 10⁹standard unitsof an area corresponded to 0,002 mg of a substance in the assay. Besides, relative percentage content of each substance in the assays was calculated. All solvents and chemicals were of analytical grade. Chromatographic experiments were reproduced in triplicate. Results and discussion. On the chromatograms of the difluorochloromethane extracts from lime flowers cropped in 2016 and 2017 on the territory of Kharkiv region 80 and 68 volatile substances were found accordingly. Most of them have retention times more than 35 minutes. Samples of 2017 contained more volatile substances with shorter retention times if compare with extracts obtained from 2016 crop. Total yield of the freon extract in 2016 was 2,75 %, and in 2017 – 1,42 % recalculated on dryweight of raw materials studied. Prevailing chemical groups of substances were aliphatic hydrocarbons and fatty acids, also there werealcohols and terpenoids in appreciable quantities. In the sample of 2016 total quantitative contents of aliphatichydrocarbons, aromaticalcohols and terpenoids were approximately 3 times more in comparison with 2017. At the same time, fatty acids, especially unsaturated, and also aldehydes and ketones were found to be much less, and esters were absent at all in 2016 samples. The freon extract of lime flowers cropped in 2016 consisted of 14 substances which relative content exceeded 1 % with total contain 78,7 % of the whole assay, where the dominating ones were: hexatriacontane - 18,73 %, n-hexadecanoic acid - 12,75 %, linoleic alcohol - 10,68 %, 1-pentadecene - 10,57 %. The freon extract of the raw material of 2017 crop included 13 substances with total contain 84,14 % of the whole assay weight from which the prevailing ones are: oleic, octadecanoic, hexadecanoic acids with concentrations 27,68 %, 15,02 % and 12,91 % respectively. Conclusions. The comparative analysis of the freon extracts from lime flowers cropped in 2016 and 2017 years using method of gas-liquid chromatographyhas been carried out. Basing on results of gas-liquid chromatographyit has been established that the samplesobtained from raw materials of 2016 and 2017 years considerably differ in their quantitative and qualitativecharacteristics of essential oil fractions: the studied samples contained 80 and 68 compoundsaccordingly, total percentage of dominating substances was78,66% and 84,14% of the assays weight respectively. Thus, weather conditions which can change dramatically in spring from year to year in Ukraine, especially in its Northeast part, strongly influence accumulation of volatile compounds in lime flower.