Сhemical analysis of dense-gas extracts from lime flowers

Abstract

The purpose of this work was to make qualitative and quantitative analysis of phenolic biologically active substances (BAS) in the extracts produced from lime flowers with condensed gases, using method of high-performance liquid chromatography (HPLC).

Materials and methods: materials for this study were the extracts obtained by consequent processing of the herbal drug and marcs thereof with various condensed gases: difluorochloromethane (Freon R22), difluoromethane (Freon R32), azeotropic mixture of difluoromethane with pentafluoroethane (Freon 410A) and freon-ammonium mixture. Extracts obtained with the latter were subjected to further fractionation by liquid-liquid separation into hexane, chloroform, ethyl acetate and aqueous-alcohol phases. Besides, the supercritical СО₂ extract, obtained from the herbal drug under rather strong conditions (at temperature 60°С and pressure 400 bar), was studied in our previous research. Presence of phenolic BAS and their quantity in the researched samples were determined by method of HPLC with UV-spectrometric detection.

Results and discussion: It has been found that Freon R22 extracted trace amounts of rutin from lime flowers – its content was only 0.08% of the total extract weight. On the other hand, Freons R32 and R410А showed good selectivity to moderately polar BAS of lime flowers (derivatives of flavonoids and hydroxycinnamic acids): in particular, the extract obtained with freon R32 contained about 1.3% of the total phenolic substances, and it was the only one of the investigated condensed gases used by us which took the basic flavonoid of lime flowers tiliroside – its content was 0.42% of extract weight. Also Freons R32 and R410А were able to withdraw another compound dominating among phenolic substances in the yielded extracts. Its quantity was rather noticeable – up to 0.87% of extract weight. This substance was not identified by existing database, but its UV-spectrum was similar to those of apigenin glucoside. Total quantities of phenolic compounds in extracts obtained with Freons R32 and R410А were 1.29% and 0.90% respectively. Freon-ammoniac extracts (hexane, chloroform, ethylacetate and aqueous-alcoholic phases in total) contained appreciable quantity of total phenolic BAS – 4.48%, among them 1.31 % was occupied by methoxycoumarins, which came into the hexane fraction. The Freon R410А modified by ammonia successfully extracted apigenin glucoside which was found in ethylacetate fraction in quantity of 0.78% that approximately 15 times exceeded content of this BAS in the extract obtained with non-modified Freon R32. Caffeic and p-cumaric acids with the total content of 0.50% (0.30+0.20% respectively) were revealed only in ethylacetate phase of freon-ammoniac extract. Besides, in chloroform fraction of this extract it was found unidentified substance in quantity of 1.02%. In total after consequent processing of lime flowers with studied condensed gases and their mixtures combined extracts contained 6.75% of phenolic compounds. Supercritical СО₂ was unable to take phenolic substances (this extract was previously analyzed by thin-layer chromatography and therefore was excluded from this work).

Conclusions: Thus, some kinds of freons and also their mixtures modified with ammonia are promising extractive solvents for rather polar BAS of lime flowers, and consecutive infusion with different condensed gases allows to provide full processing of the specified herbal drug and to fractionate the substances different in their chemical composition and polarity.