RU2161149C1 - Method of isolation of biologically active sum of acids from fir wood greens - Google Patents

Abstract

FIELD: biotechnology, agriculture. SUBSTANCE: invention relates to preparing biologically active substances from fir (Abies) wood greens used for protection and increased productivity of plants. Method ensures to obtain concentrate of triterpene acids from fir wood greens with yield 3.0-6.8% of absolutely dry raw. Method involves milling fir wood greens, preparing acid salts by direct treatment of milled raw with humidity 45% and less with 0.5-50% alkali aqueous solution or 1.0-50% alkaline metal carbonate aqueous solution followed by separation of wood green solid part from obtained solution of salts, acidification of alkali aqueous solution or alkaline metal carbonate aqueous solution to pH 2-3, extraction of triterpene acids with organic solvent and the following removal of solvent and preparing the end product. EFFECT: improved method of isolation, increased yield of acids.

Description

 The invention relates to the production of biologically active substances with fungicidal and growth-promoting activity, which can be used in agriculture to protect and increase plant productivity.

 Known [RF Patent N 2108803, publ. 08.04.97] a method for producing a biologically active amount of triterpene acids with a yield of 6.2% of absolutely dry raw materials from dried and ground woody fir fir by extraction with tert-butyl methyl ether, treating the extract with an aqueous solution of an alkaline agent with further processing of the aqueous-alkaline solution of the target product.

 Known [RF Patent N 2108107, publ. 03/06/97] a method of obtaining a biologically active salt of triterpenic acids from dried and ground wood green fir by extracting it with an organic solvent - a mixture of petroleum ether (nefras) and ethyl acetate, treating the extract with aqueous alkali, separating the aqueous solution from the organic phase after spontaneous neutralization to pH 7-8 and drying it to a powder.

 However, this method involves pre-drying the woody greens, which entails additional labor and energy costs or dependence on natural conditions, if the drying is carried out without heating. In addition, the evaporation of an aqueous-alkaline solution of the target product in vacuum requires significant energy and does not always guarantee the preservation of the composition of the target product.

 The objective of the invention is to develop a new effective method for the isolation of biologically active amounts of triterpenic acids from woody fir green.

 The technical result consists in the development of a fundamentally new method for the isolation of triterpenic acids with a yield of the target product of 4.0-6.8% of absolutely dry raw materials (a.s.). The method allows to simplify the process and to maintain the content of triterpenic acids in the product with optimal parameters of the isolation process.

 The result is achieved in that wood greens chopped up to 0.5-1 mm with a moisture content of up to 45% or less are subjected to treatment with an aqueous alkali solution or an aqueous solution of alkali metal carbonates, the amount of acids is extracted by extraction with an organic solvent after the destruction of their salts. To destroy salts, an aqueous solution of a mineral or organic acid is added to the obtained aqueous-alkaline solution of the target product until a pH of 2-3 is reached.

 To increase the content of triterpenic acids in the target product from the resulting emulsion in an aqueous alkaline solution, neutral substances can be extracted with petroleum ether.

 The essential features of the invention are: grinding wood green fir with a moisture content of 45% or less to a size of 0.5-1.0 mm, obtaining salts of acids directly by processing the crushed raw materials with 0.5-50.0% aqueous alkali solution or 1.0 -50.0% aqueous solution of alkali metal carbonate, separation of the solid part of green wood from the resulting salt solution, acidification of an aqueous alkali solution or an aqueous solution of alkali metal carbonate to pH 2-3, extraction of triterpene acids with an organic solvent, subsequent removal solvent and obtaining the target product.

 The method is as follows.

Example 1. Wood green fir fir with a moisture content of 45% is crushed on a screw shredder or mixer. 63 g of chopped greens are mixed with 780 g of a 2% aqueous sodium hydroxide solution (needles: alkaline solution ratio 0.5: 1) at room temperature (t = 20-25 ^° C). The mixture is stirred with a mechanical stirrer (speed 150-300 rpm.) For 30 minutes. The resulting heterogeneous mixture is separated: the solid phase is separated by filtration, the liquid phase is transferred to an aqueous-alkaline solution in a separatory funnel, and a 12% aqueous solution of sulfuric acid is added until a pH of 2-3 is reached. After that, acids are extracted three times with diethyl ether (diethyl ether: 1: 1 solution). The extracts are washed with water, dried, the diethyl ether is distilled off and an acid concentrate is obtained with a yield of 6.0-6.8% of a.s.

 The isolated acids according to TLC and HPLC coincide with the labels of triterpenic acid in fir. The maximum of the selected acids in the UV spectrum corresponds to absorption in the region of 236–238 nm. The acid chromatogram obtained by HPLC on a Milichrome liquid chromatograph with a Lichrosorb column and an eluent consisting of a mixture of 0.05 M orthophosphoric acid and methanol (15:85) with UV detection of 200 and 240 nm corresponds to a chromatogram of the sum of triterpene acids, isolated from fir by a known method.

 Example 2. The selection of acids is carried out as in example 1. The treatment of needles is carried out with a 10% aqueous solution of sodium carbonate. The ratio of needles: an alkaline solution of 0.5: 1. The yield of acid concentrate 3.5 - 4.0% of a.s. The isolated acids according to TLC and HPLC coincide with the labels of triterpenic acid in fir. The maximum of the selected acids in the UV spectrum corresponds to absorption in the region of 236–238 nm. The acid chromatogram obtained by HPLC on a Milichrome liquid chromatograph with a Lichrosorb column and an eluent consisting of a mixture of 0.05 M orthophosphoric acid and methanol (15:85) with UV detection of 200 and 240 nm corresponds to a chromatogram of the sum of triterpene acids, isolated from fir by a known method.

 Thus, the new proposed method allows to obtain a concentrate of triterpenic acids from woody fir green with a yield of 4.0-6.8% of absolutely dry raw materials (acid yield of 6.2% of a.s. by a known method).

Claims (1)

 A method of isolating a biologically active amount of acids from chopped wood fir green, including the selection of the target product by extraction with an organic solvent, characterized in that chopped up to 0.5-1 mm wood green fir with a moisture content of up to 45% or less is pre-treated with an aqueous alkali solution with a concentration of 0 , 5 - 50% or 1.0 - 50% aqueous solution of alkali metal carbonate, the aqueous-alkaline solution of salts is separated and the salts are destroyed by acidification of the aqueous-alkaline solution or an aqueous solution of alkali metal carbonate alla to pH 2 - 3.