RU2336889C2 - Method of obtaining of biologically active lipid fraction of siberian fir (abies sibirica) wood green extract - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention is referred to pharmaceutical industry, in particular, to a method of obtaining of biologically active lipid fraction of a fir Siberian (Abies Sibirica) wood green extract. The method of obtaining of biologically active lipid fraction of a fir Siberian (Abies Sibirica) wood green extract, including extraction of dry crushed raw materials under certain conditions, washing by a solution of the alkaline agent, separation of layers into organic and water-alkaline phases. Extract an extracting agent from an organic phase under certain conditions with obtaining of lipid fraction; treat the water-alkaline layer with acid, separate the creating organic phase and obtain the amount of triterpene acids. Further accumulate lipid fraction of an extract, hydrolyse it with water, remove volatiles and obtain partially hydrolysed lipid fraction; further process partially hydrolysed lipid fraction with the aqueous-alcoholic alkali while boiling, concentrate using stripping, extract two times, dry up the extract, filter the dehydrating agent, concentrate the solution and obtain perhydrolysed lipid fraction.

EFFECT: obtaining of effective biologically active lipid fraction using simplified technology.

2 tbl, 4 ex

Description

The invention relates to methods for producing biologically active substances (BAS) from plant materials, specifically to extracting, fractionating and modifying extractive substances of green wood fir (DZ) of fir to obtain a biologically active lipid fraction of the extract (partially or fully hydrolyzed), the sum of triterpene acids and fir oil .

Inventions related to methods for producing extractive biologically active substances from plant materials have, as a rule, two objectives. Firstly, economic feasibility achieved by using waste from the forest and crop industries as raw materials. Secondly, the separation of the sum of extractive substances into target groups of chemically similar bioactive compounds by simple technological methods.

So, for example, the known method [A.T. Diordiychuk, V.G. Kharchenko. A method of obtaining feed from wood coniferous species. RU 2032359, C1, 04/10/1995] extraction of biologically active substances from coniferous DZ, consisting in processing crushed fresh DZ with a water-gasoline steam mixture, multi-stage processing of condensate with the release of “provitamin concentrate” and “vitamin-containing concentrate” and subsequent combination of the above products when applied to the mixture fresh and steamed DZ. The use of a mixture of saturated hydrocarbons (gasoline) in combination with water vapor lowers the temperature of treatment of DZ and, according to the authors, preserves the complex of valuable substances of DZ. The disadvantage of this method is the incompleteness of the extraction of biologically active substances from DZ during extraction with a low-polar hydrocarbon extractant, as well as the complexity and multi-stage technology.

The known method [S. M. Repyakh, N. A. Chuprova, N. D. Barabash, G. V. Tikhomirova, V. G. Novik, O.S. Apartsev. A method of obtaining products from green wood fir. SU 1375226, A1, 12/28/1984] extraction of biologically active substances from fir DZ, which consists in treating crushed fresh DZ with water vapor (to remove fir oil), followed by infusion in aqueous isopropanol at a temperature of 30-60 ° C, cooling the filtered infusion with the formation of three immiscible between the phases - solid wax and two liquid layers, from which by separation and evaporation of isopropanol get "chlorophyll-carotene paste" (fat-soluble substances) and "natural fir extract" (concentrate of water-soluble substances), respectively. The disadvantage of this method is the use of 55% aqueous isopropanol as an extractant to obtain a feed additive, since this mixed extractant, due to its high polarity, extracts a mixture of strong resin and higher acids from the DZ fir, which reduce the consumer properties of the product.

Closest to the claimed method — the prototype — is the method [V.M. Chekurov, O.V. Mishukova, A.G. Druganov, O.V. Mikhaylapova. A method of obtaining a biologically active amount of neutral isoprenoids. RU 2199861, C1, 10.26.2001] obtaining a "biologically active amount of neutral isoprenoids" (the active substance of the drug "RastStim"). The method involves grinding air-dry DZ fir, extracting it with a water-immiscible organic solvent (tert-butyl methyl ether or a mixture of ethyl acetate and benzene in a ratio of 1: 1), separating the extract from the needles, treating it with an aqueous solution of an alkaline agent, separating the aqueous-alkaline and organic layers, separation from the organic layer of the sum of neutral isoprenoids by successively distilling off the extractant and the volatile components of fir oil in vacuum at a residual pressure of 0.1 mm Hg and a temperature of 70-80 ° C, and from the water-alkaline layer - the sum of triterpenic acids by acidification and extraction with an organic solvent. The amount of neutral isoprenoids is active as a biostimulator of growth processes and tomato yields. The technological disadvantage of the prototype method is the use of high vacuum (0.1 mm Hg) to separate volatile components, which requires the use of expensive special devices, oil vacuum pumps, special refrigerants to trap the extractant and volatile components of the extract.

The tasks to which the present invention is directed are:

~ increase the functionality, complexity and rationality of the processing of extractive substances DZ fir;

~ diversification of fractions of extractive substances by types of biological activity for the purpose of efficient use in the production of goods for agriculture and medicine;

~ improvement of technology using standard and affordable technological equipment, ensuring industrial scale production.

Problem solving is achieved by the fact that:

~ target products are obtained by hydrolysis of the lipid fraction of the extract of DZ fir with treatment with hot water (partial hydrolysis) or with water-alcohol alkali (exhaustive hydrolysis);

~ they use hydrodistillation at a residual pressure of 0.1-0.3 atm and a temperature of 50-90 ° C to remove volatile components from the lipid fraction of the extract using standard and affordable processing equipment that provides an industrial scale of production.

The main difference of the present invention from the prototype method is the hydrolysis (partial or complete) of the lipid fraction of the extract DZ fir. The target product obtained in example 1 is a partially hydrolyzed lipid fraction of the extract of DZ fir, exhibits previously unknown high biological activity (growth-promoting and immunomodulating effects) when used in poultry farming.

Partial hydrolysis of the components of the lipid fraction - acetates and other esters, including glycerides - is carried out by treating the lipid fraction with hot water in the presence of minimal amounts (residues) of an alkaline agent used to wash the amount of triterpenic acids from the total extract of DZ fir. Partial hydrolysis is combined with hydrodistillation, which, in addition to removing the volatile components of fir oil, ensures the removal of acetic acid formed during the hydrolysis of acetates. The presence of the mentioned substances in the target product is undesirable - they adversely affect the animal organism and, thereby, reduce the quality of the feed.

Exhaustive hydrolysis of the ether components of the lipid fraction is carried out by heating a mixture of an alcohol solution of a partially hydrolyzed lipid fraction and aqueous alkali. Hydrolysis provides the complete removal of free and bound carboxylic acids, glycerol, as well as chlorophylls and free phenolic compounds. The target product obtained in example 2 is an exhaustively hydrolyzed lipid fraction of the extract from DZ fir.

The target product obtained in example 1 is a non-volatile green-brown waxy mass containing a mixture of polyprenols and their acetates, phytosterols and their esters, triterpene alcohols, partially hydrolyzed fatty acid glycerides, chlorophyll and carotenoids, phospholipids, higher fatty acids and other native substances. The target product obtained in example 2 is a non-volatile mass of yellow-brown or dark-orange colored liquid honey consistency containing a mixture of polyprenols, phytosterols, carotenoids, alkanols, neutral sesqui-, di- and triterpenoids and a number of other substances.

The target products obtained in examples 1 and 2, have high bioactivity when used as feed additives for poultry. Target products easily form emulsions of various concentrations in water with emulsifiers. Emulsions are easy to dose and convenient when applied to food. Production tests of feed additives obtained in this way revealed a significant positive effect of the products obtained according to examples 1 and 2 on the development and zootechnical indices of poultry and their advantages in biological activity in comparison with the "sum of neutral isoprenoids" obtained by the prototype method (see examples 3, 4).

The technological advantage of the proposed method is the removal of volatile components by hydrodistillation in a moderate technological vacuum of 0.1-0.3 ata, which allows the use of standard large-scale technological equipment (typical industrially manufactured equipment and liquid ring pumps for chemical plants), without the need for special traps and refrigerants . The distillation of volatile components by hydrodistillation is carried out with high productivity and in mild conditions - in the cube does not occur polymerization and / or thermal decomposition and resinification of the native components of the lipid fraction of the extract. The products obtained in examples 1 and 2 are readily soluble in most organic solvents - hexane, tert-butyl methyl ether, alcohol, isopropanol, acetone, ethyl acetate.

An advantage of the proposed method is that the method allows to obtain valuable products (partially or fully hydrolyzed lipid fraction) that have high biological activity from waste products of the active ingredient of the drug "Novosil" (amount of triterpene acids).

The method is illustrated by the following examples.

Example 1

Air-dried chopped fir greens (80 kg) in bags of coarse calico are placed in an extractor, tert-butyl methyl ether (350 L) is poured from a measuring device and infused at room temperature for 8 hours. The extract was drained (220 L), fresh tert-butyl methyl ether (260 L) was added and insisted again for 8 hours. A second portion of the extract is drained (260 L). The extracts are combined and treated with a 2% aqueous solution of sodium hydroxide (150 l) with stirring in an apparatus with a stirrer. The mixture is transferred to a separatory separator funnel, wait for complete separation and separate the layers.

The organic extract is portionwise loaded into the distillation apparatus and tert-butyl methyl ether is distilled off at a dilution of 0.1-0.3 ata (vacuum ring pump) and the temperature in the cube is not higher than 50 ° C. Get the lipid fraction of the extract (4.8 kg), freed from the extractant and containing volatile components (fir oil).

The aqueous-alkaline layer in the separatory funnel is acidified with 5% hydrochloric acid to pH 2, the formed organic phase is separated, by concentration (drying) of which in a technical vacuum the amount of triterpene acids (4.9 kg) is obtained in the form of a gray-green glassy mass after cooling .

The lipid fraction of the extract is accumulated (48 kg), placed in a hydrodistillation vessel, water (48 L) is added, it is heated to 50-80 ° С with stirring, and the volatile components are distilled off with steam for 1-3 hours at a pressure of 0.1 -0.3 ata (vacuum ring pump). The hydrodistillate is separated into aqueous and organic (fir oil, 12 kg) layers, and 31 kg of the target product (partially hydrolyzed lipid fraction) green-brown are drained from the cube hot. The product, when cooled to room temperature, acquires a wax consistency.

Example 2

The partially hydrolyzed lipid fraction (20 kg) obtained under the conditions of Example 1 was dissolved in ethanol (25 L), an aqueous KOH solution (6.5 kg in 7 L of water) was added, heated under stirring in a reflux apparatus and refluxed for 2 hours, the mixture was concentrated by distillation of the solvents on a rotary evaporator in a vacuum ring pump at a temperature not exceeding 80 ° C, the residue was extracted twice with tert-butyl methyl ether (100 l each), the extract was dried with anhydrous sodium sulfate, the desiccant was filtered off, and the solution was concentrated by distillation of the ether in a vacuum of a water-jet pump, 11.8 kg of the expected product (an exhaustively hydrolyzed lipid fraction) of a thick yellow-brown or dark orange honey consistency are obtained.

The study of the effectiveness of the application of the target products according to examples 1 and 2, as well as a comparative study of the effects of the target products and the "sum of neutral isoprenoids" [V.M. Chekurov, O. V. Mishukova, A. G. Druganov, O. V. Mikhailailapova. A method of obtaining a biologically active amount of neutral isoprenoids. RU 2199861, C1, 10.26.2001] on the development and zootechnical indicators of poultry - laying hens (example 3) and broiler chickens (example 4) - was carried out jointly with SibAgroPharm LLC (Novosibirsk Region, Naukograd Koltsove).

Example 3

By the principle of analogues, four groups of laying hens were formed with 10,000-20000 heads in each group for repetition (4-fold repetition of the experiment). From 1 to 20 days of life, the chickens were asked in the feed: experimental group No. 1 - the target product according to example 1; experimental group No. 2 - the target product according to example 2; experimental group No. 3 - "the amount of neutral isoprenoids"; the control group was given food without additives. Consumption rates of the active substance were 50, 100 and 250 g / t of feed. The results are presented in table 1.

Example 4

By the principle of analogues, four groups of broiler chickens were formed with 20,000-30000 heads in each group for repetition (4-fold repetition of the experiment).

From 1 to 20 days of life, the chickens were asked in the feed: experimental group No. 1 - the target product according to example 1; experimental group No. 2 - the target product according to example 2; experimental group No. 3 - "the amount of neutral isoprenoids"; the control group was given food without additives. The consumption rate of the active substance is 250 g / t of feed. The results are presented in table 2.

An analysis of the results shown in examples 3 and 4 indicates that the feeding of lipid fractions of Siberian fir extract obtained by various technologies positively affects the zootechnical performance of broiler chickens and laying hens. An increase in the safety and average daily gain of young animals, an increase in the productivity of laying hens, and an increase in feed conversion were noted. However, the best results were recorded when using the claimed feed additives — partially and completely hydrolyzed lipid extract fractions (experimental groups 1 and 2), while the use of “sum of neutral isoprenoids” shows a relatively low effect.

Given the differences in the yields of the claimed products and the technologies for their production, it is economically preferable to use a partially hydrolyzed lipid fraction of the extract DZ fir - the product of example 1. The emulsion of this product in the emulsifier "Twin" is registered with the trademark "Vitaprinol". The effectiveness of the use of the feed additive "Vitaprinol" (the consumption rate of the preparation is 0.7 kg / t of feed) was demonstrated on broiler chickens and quails. Thus, the safety of broiler chickens on the 20th day of life was 99.1% versus 98.5% in the control group with an increase in average individual weight by 9.1% (data of Angarsk Poultry Farm CJSC, Irkutsk Region). The increase in the safety of young quail was 14%, the average daily gain in live weight was 10.96–13.81%, and the reduction in feed costs per 1 kg of gain was 6.0–12.0%. During the reproductive period, an increase in the egg production of quail amounted to 7.90-19.94%, a decrease in feed consumption - 12.28-24.56% (data from the State Scientific Institution of Siberian Scientific Research Institute of Siberian Agricultural Sciences).

Thus, the inventive method allows waste-free processing of organic extract of DZ fir to produce three products for agriculture (feed production and crop production) and medicine, respectively: the lipid fraction, hydrolyzed completely or partially (the active substance of the drug Vitaprinol), the amount of triterpene acids (the active substance of the drug Novosil ) and fir oil.

Table 1 Indicators
Experience Options Preservation,% Feed conversion Productivity in 140 days,% Productivity in 170 days,% Notch in 140 days,% Notch in 170 days,% control group 92 ± 2.2 1.91 ± 0.05 52.4 ± 4.3 78.1 ± 4.3 0.3 ± 0.02 1.7 ± 0.5 Experimental group No. 1 fifty 95 ± 1,5 1.89 ± 0.03 59.9 ± 2.4 87.3 ± 3.2 0.1 ± 0.01 0.9 ± 0.25 one hundred 97 ± 1.3 1.84 ± 0.07 61.7 ± 2.1 89.5 ± 2.1 0.05 ± 0.005 0.5 ± 0.05 250 99.5 ± 0.7 1.81 ± 0.06 62.3 ± 1.4 90.0 ± 1.4 - 0.1 ± 0.03 Experimental group No. 2 fifty 96 ± 1.7 1.88 ± 0.04 61.9 ± 1.7 89.2 ± 1.5 0.1 ± 0.01 0.5 ± 0.1 one hundred 98 ± 1.5 1.81 ± 0.08 63.4 ± 1.0 90.0 ± 1.7 0.06 ± 0.02 0.2 ± 0.03 250 99.9 ± 0.9 1.79 ± 0.04 64.1 ± 0.8 91.2 ± 0.9 - - Experimental group No. 3 fifty 91.1 ± 3.4 1.90 ± 0.04 55.1 ± 3.1 81.5 ± 3.4 0.4 ± 0.19 1.9 ± 0.23 one hundred 90.4 ± 2.7 1.89 ± 0.06 58.9 ± 2.7 83.6 ± 1.3 0.35 ± 0.01 1.1 ± 0.14 250 95.1 ± 1.6 1.87 ± 0.04 61.5 ± 1.7 85.1 ± 1.9 0.25 ± 0.03 0.5 ± 0.14

table 2 Indicators
Experience Options Preservation,% The average weight of 1 head, g The average daily gain, g / day The output of carcasses of the 1st category,% Feed conversion Productivity index Control group 83.4 ± 0.1 1791.0 ± 164.0 48.0 ± 3.7 79.4 ± 4.4 1.64 ± 0.6 263.0 ± 14.0 Experimental group No. 1 95.3 ± 0.4 2032.0 ± 88.0 52.9 ± 2.8 94.1 ± 2.4 1.56 ± 0.2 293.6 ± 2.2 Experimental group No. 2 94.7 ± 0.2 2114.7 ± 92.3 55.7 ± 3.6 94.0 ± 3.0 1.55 ± 0.5 321.0 ± 1.6 Experimental group No. 3 91.1 ± 0.3 1898.1 ± 102.4 50.1 ± 2.1 90.2 ± 3.5 1,60 ± 0,4 269.3 ± 2.8

Claims (1)

A method of obtaining a biologically active lipid fraction from an extract of woody green Siberian fir (Abies Sibirica), including double extraction of air-dried chopped wood green fir with tert-butyl methyl ether, combining the extracts, washing the extract with 2% aqueous sodium hydroxide solution, separation of aqueous alkaline and the organic phase, acidification of the aqueous-alkaline phase to pH 2 with the allocation of the sum of triterpene acids, distillation of the extractant from the organic phase, characterized in that as a biologically active lipid of the second fraction, two fractions are obtained: a partially hydrolyzed lipid fraction and an exhaustively hydrolyzed lipid fraction, while tert-butyl methyl ether is distilled off from the organic extract at a dilution of 0.1-0.3 at and at a temperature of no higher than 50 ° C to obtain a lipid fraction, then obtained the lipid fraction is hydrolyzed with water at a temperature of 50-80 ° C and freed from volatile components by hydrodistillation at a residual pressure of 0.1-0.3 ata, a partially hydrolyzed lipid fraction is obtained, then to obtain an exhaustively hydrolyzed of the lipid fraction, part of the partially hydrolyzed fraction is treated with water-alcohol alkali during boiling, concentrated by distillation of the solvents under reduced pressure and a temperature of no higher than 80 ° C, extracted twice with tert-butyl methyl ether, dried, the drying agent is filtered off, the solution is concentrated and an exhaustively hydrolyzed lipid fraction is obtained .