RU2233868C2 - Method of production of biologically active agents from fir-tree green - Google Patents

Abstract

FIELD: production of biologically active agents from fir-tree green.

SUBSTANCE: proposed method includes grinding starting material, extraction by organic solvent and subsequent distillation. Fir-tree green dried in air at moisture content not exceeding 10% is used as starting material. Raw material is extracted by azeotropic mixture of isopropyl alcohol: water of 88:12. Then, extractant is distilled and total extractant is settled for 48-72 h at temperature of 60-70 C, after which lower water organic layer is drained, cooled, washed with non-polar solvent which is removed and fraction of polar agents is obtained. Sum of triterpen acids os separated from upper organic layer of total extractant. Proposed method makes it possible to separate two agents of different chemical composition and biological activity in one cycle.

EFFECT: extended functional capabilities; enhanced efficiency.

5 cl, 1 dwg, 2 tbl, 3 ex

Description

The invention relates to agriculture and biotechnology, in particular to methods for producing biologically active extractive substances from woody fir green.

Of the variety of extractives extracted from coniferous wood, the most widely known and used in agriculture are triterpene acids with growth-promoting and fungicidal activity.

There are various methods for producing a biologically active amount of triterpenic acids from woody fir green, which are widely used in agriculture to increase plant productivity. Known, for example, is a method for producing a biologically active amount of triterpenic acids from dried woody green Siberian fir (RF Patent No. 2108803, class A 61 K 35/78, publ. 04/20/98), according to which the chopped woody greenery is extracted with tert-butyl methyl ether, the extract is treated with an aqueous solution of an alkaline solution, acidified to pH 2 and the desired product is isolated from this solution by extraction with tert-butyl methyl ether.

The disadvantages of this method are its multi-stage and functional limitations associated with obtaining only one biologically active drug, since tert-butyl methyl ether does not extract a group of polar substances that also have high biological activity. For their extraction, subsequent extraction with a more polar extractant is necessary. The sequential extraction of green wood with various extractants creates significant difficulties in the industrial use of this method due to their constant mixing, the formation of azeotropic mixtures and the large volume of distilled flammable liquids.

Closest to the claimed method, the prototype is a method for producing a biologically active amount of triterpenic acids from woody fir fir by extraction with aqueous alcohols or acetone (RF Patent No. 2179393, class A 01 N 65/00, publ. 02.20.2000). The method consists in the extraction of chopped freshly picked fir needles of Siberian aliphatic alcohol with the number of carbon atoms 1, 2, 3 or acetone with a water content of not more than 50%. The solvent is distilled off from the obtained extract, then water is obtained under reduced pressure, and a viscous dark green mass is obtained. The distilled solvent-water mixture is then again used as an extractant of a new portion of fir green wood.

The yield of the target product is 6.3-6.5%.

The known method has several disadvantages. Firstly, the quantitative determination of the amount of triterpenic acids in this way, without a standard, is practically impossible, and using the standard, the accuracy of determining the yield of the target product does not exceed 30% (TU 2449-003-03533895-01). Thus, in this method, the output is not the sum of triterpene acids, but the total yield of extractive substances (the actual yield of the target product is not more than 4%). Secondly, when extracting freshly harvested raw materials or when using a heavily watered solvent, the yield of the group of neutral substances, which also have high biological activity, is significantly reduced. Thirdly, a homogeneous extract is obtained only when using woody greens with a moisture content of not more than 10% and a single use of solvents with a water content of not more than 10%. Fourthly, with a constant return of the distilled solvent for subsequent extraction, an increase in water content occurs due to the extraction of residual moisture from the feedstock. This leads to a decrease in the total yield of extractable substances. When the solvent is distilled off from such an extract, it is stratified into two layers, and depending on the amount of water in the still residue, the aqueous layer containing polar substances can be both the upper and lower layers. With a high water content, the upper layer is aqueous, and the lower one contains neutral substances and triterpene acids, as described in example 2 of the prototype. With a small amount of residual moisture (10-20% of the weight of the bottom residue), the group of polar compounds with residual solvent and excess moisture contains the lower layer.

The aim of the invention is to increase the functionality of the method by isolating from woody green fir in one technological process two preparations, different in chemical composition and biological activity.

This goal is achieved in that air-dried and shredded woody greens (needles and shoots) of fir with a moisture content of not more than 10% are extracted with an azeotropic mixture: isopropyl alcohol: water (88:12), the extractant is distilled off and returned to production, the total extract is left to stand for 48-72 hours at a temperature of 60-70 ° C to separate it into two layers, after which the lower aqueous-organic layer is drained, cooled, washed with a non-polar solvent (tert-butyl methyl ether, white spirit, gasoline) and removed in vacuo from him leftover solution of Tell. The result is a fraction of polar substances in the form of a viscous red-brown mass with a solids content of 69-77%, soluble in organic polar solvents (acetone, alcohol). From the upper organic layer containing the non-polar part of the total extract, the sum of triterpenic acids is isolated in a known manner.

The output of the amount of triterpenic acids is 5-6% of the weight of the feedstock.

The yield of the fraction of polar substances (the conditional name of the preparation is Terpenol) depends on the moisture content of the raw materials and solvent and is 4.7-6% by weight of dry woody greens.

Thus, in one technological process two biologically active preparations are isolated at once: the sum of triterpenic acids and Terpenol.

The drug is a natural terpenes complex phenolic substances and carbohydrate, which gives a positive color reaction on the phenols with ferric chloride and showing in the NMR spectrum ^{of 13} C nuclei in signals carbohydrate carbon atoms. The biological activity of the preparation Terpenol, according to field trials, is different from the properties of the sum of triterpenic acids obtained in a known manner (RF Patent No. 2108803, 1998).

Distinctive features of the proposed method in comparison with the prototype are:

- as the feedstock, air-dried wood green fir with a moisture content of not more than 10% is used, which prevents the extraction of residual moisture from the feedstock and dilution with water of the extractant;

- an azeotropic mixture is used as extractant: isopropyl alcohol: water in a ratio of 88:12, which allows extraction of not only ether-soluble substances (triterpene acids and related neutral substances), but also qualitatively different, more polar components;

- after distillation of the extractant, the total extract is subjected to sedimentation for 48-72 hours at a temperature of 60-70 ° C, which allows you to divide it into two layers: the upper organic layer containing the sum of triterpene acids, and the lower aqueous-organic layer containing the fraction of polar components ;

- the lower layer of the extract is drained, cooled, washed with a solvent, mainly tert-butyl methyl ether, the solvent is removed and the polar fraction of the components is obtained (Terpenol preparation), which allows to increase the functionality of the method by obtaining a new valuable biologically active product.

The invention is illustrated by the following examples of specific performance.

Example 1

Dried in air and chopped wood green fir with a moisture content of 5% (12.2 kg) is poured with 50 l of aqueous 88% isopropyl alcohol. From the obtained extract at a temperature of 65 ° C and a residual pressure of 300 mm Hg, the extractant is distilled off in an amount of 50 l, which is constantly returned to the extraction. The bottom residue in the amount of 2.3 kg is left at a temperature of 60 ° C for three days (72 hours) to separate the mixture into two layers, then 1 liter of the lower aqueous-organic layer is poured through the lower pipe into a flask, cooled to 45 ° C and add 0.5 l of tert-butyl methyl ether. After stirring with a mechanical stirrer at 60 rpm for 30 minutes, the mixture was allowed to stand (5 minutes) and the upper (ether) layer was separated by decantation. Get 0.74 kg fractions of the polar components of the extract in the form of a red-brown resinous mass with a solids content of 77%. In terms of dry matter, its weight is 0.57 kg, which is 4.7% of the weight of the original woody green.

The decanted ether layer is mixed with the upper layer of the original stratified bottoms (1.3 kg), 10 l of tert-butyl methyl ether and 10 l of a 2% aqueous sodium hydroxide solution are added to it. The resulting solution was vigorously stirred and allowed to separate for 0.5 hours, after which the lower (water-alkaline) layer was drained and the upper (ether) was re-treated with a new portion (5 L) of a 2% aqueous sodium hydroxide solution, which this aqueous-alkaline layer is combined with that obtained during the first aqueous-alkaline treatment, acidified with hydrochloric acid to pH 2, vigorously stirred with 12 l of tert-butyl methyl ether and left for 15 minutes to delaminate. The ether layer was separated, washed with a saturated aqueous solution of sodium chloride and the solvent was distilled from it. 0.698 kg (5.7%) of the amount of triterpenic acids is obtained.

Thus, the total yield of extractive substances upon extraction with isopropyl alcohol by an azeotrope of water is 1.27 kg or 10.4% of the weight of dry raw materials.

Example 2

Dried in air and chopped wood green fir (3 kg) with a moisture content of 10% is poured with 20 l of aqueous 88% isopropyl alcohol. From the resulting extract at a temperature of 70 ° C and a residual pressure of 400 mm Hg the extractant is distilled off in an amount of 20 l, which is constantly returned to the extraction. VAT residue in the amount of 0.65 kg is defended at a temperature of 70 ° C for 48 hours. The upper layer of the stratified bottoms (0.42 kg) is decanted, and the lower layer is drained, cooled to 35 ° C and washed with 0.2 liters of white spirit. Obtain 0.22 kg of viscous brown mass (Terpenol preparation) with a solids content of 68% (yield 0.15 kg or 5% of the feedstock.

The white spirit wash solution is mixed with the top layer of the bottom residue (0.42 kg) and the amount of triterpene acids is isolated from it, as in Example 1. The yield of the amount of triterpene acids is 0.18 kg (6% of the feedstock).

The drawing shows a chromatogram obtained by HPLC for samples of the fraction of polar components obtained under the conditions of example 1. Analysis conditions: microcolumn liquid chromatograph "Millichrom", column "Lichrosorb RP-18", grain - 5 μm, eluent - a mixture of 0.05 M aqueous phosphoric acid and 20% methanol in a ratio of 85:15 by volume, respectively, UV detection at 200 and 240 nm.

Thus, the use of an aqueous azeotrope of isopropyl alcohol as an extractant of woody greens, followed by settling of the bottom residue, allows one to isolate a fraction of polar substances along with a preparation containing the amount of triterpene acids simply and practically without additional costs.

Example 3

Field tests of the biological activity of the Terpenol preparation were carried out in the Pervomaisky district of the Altai Territory on spring wheat “Altayskaya-92”. Pre-sowing seed treatment was carried out per day of sowing with a preparation containing the amount of triterpenic acids (CTTK) - a consumption rate of 50 g / t, and Terpenol - a consumption rate of 5 g / t, at a flow rate of 10 l / t. The effectiveness of the preparations for pre-sowing seed treatment of spring wheat “Altayskaya-92” is presented in table 1. The table shows that the greatest increase was obtained using the Terpenol preparation - 5 c / ha.

Vegetative plants of spring wheat were treated in the tillering phase with preparations (CTTK) (consumption rate of 50 g / ha) and Terpenol (consumption rate of 5 g / ha) at a flow rate of 300 l / ha. Control plants were sprayed with water.

The processing results are presented in table 2. It can be seen from the table that the largest yield increase was obtained using the Terpenol preparation - 7.3%. Thus, Terpenol has a high biological activity on the growth and development of plants (spring wheat), not inferior, and sometimes even superior, to the activity of a well-known preparation containing the sum of triterpenic acids.

Claims (5)

1. A method of producing biologically active substances from woody fir green, including grinding the feedstock, extraction with an organic solvent and subsequent distillation of the latter, characterized in that the wood-dried woody greens with a moisture content of not more than 10% are used as raw materials, the raw material is extracted with an isopropyl azeotropic mixture alcohol: water (88:12), the extractant is distilled off, the total extract is defended for 48-72 hours at a temperature of 60-70 ° C, the lower aqueous-organic layer is drained, cooled, washed with a non-polar solution With the help of a solvent, the latter is removed and a fraction of polar substances is obtained, and the sum of triterpenic acids is isolated from the upper organic layer of the total extract. 2. The method according to claim 1, characterized in that the extraction is carried out at a ratio of raw materials: extractant equal to 1: (4-7). 3. The method according to claim 1, characterized in that the extractant is distilled off at a temperature of 65-70 ° C and a residual pressure of 300-400 mm Hg 4. The method according to claim 1, characterized in that the lower aqueous-organic layer after draining is cooled to 35-45 ° C. 5. The method according to claim 1, characterized in that tert-butyl methyl ether or white spirit or gasoline is used as a non-polar solvent.