Method to produce ecdysteroids

 

The invention relates to the field of biologically active substances from plant material, in particular to a method of simultaneously receiving 20-hydroxyecdysone but also of inokosterone, ecdysone, magisteria And having the following structural formula:

20-hydroxyecdysone - R1=OH, R2=H, R3=OH, R4=H. Inokosterone - R1=OH, R2=H, R3=H, R4=OH. Addison - R1=H, R2=H, R3=OH, R4=H. Magistero A - R1=OH, R2=CH3, R3=OH, R4=H. The method allows to increase the yield of target products and completeness of the allocation of ecdysteroids from vegetable raw materials due to the fact that in the process comprising grinding plant and water extraction of the target products, plant material is subjected to milling and mechanochemical treatment in a ball mill in the presence of abrasive material and water-soluble adsorbent in the following ratio, wt.%: water-soluble adsorbent 5-30; abrasive material 5-25; vegetable raw materials - the rest. As ball mills use a planetary mill, a vibration and vibratory centrifugal types, enabling acceleration acting balls from 60 to 400 m/s2. Preferably, the abrasive material used quartz sand, and in sobienie relates to the field of biologically active substances from plant material, namely 20-hydroxyecdysone (ecdysterone,-Edison, 2, 3, 14, 20, 22, 25 hexahydroxy-(20R, 22R)-5-cholesterol-7-EN-6-one), ecdysone (-Edison, 2, 3, 14, 22, 25 pentahydroxy-(20S, 22R)-5-cholesterol-7-EN-6-one), inokosterone (2, 3, 14, 20, 22, 26 hexahydroxy-(20R, 22R, 25S)-5-cholesterol-7-EN-6-one) and magisteria And (2, 3, 14, 20, 22, 25 hexahydroxy-24-methylene-(20R, 22R)-5-cholesterol-7-EN-6-one).

A method of obtaining of ecdysone, according to which the air-dried leaves of plants of the genus Serratula crushed and extracted with water at a temperature not exceeding 100C for 1 hour. The extract obtained is passed through a layer of solid hydrophobic sorbent, for example, activated carbon, diasorb, Polisorb, while concurrently separation of related substances and purification of the target product. Then the product exgo of this method is the low yield of the target product, 0.49% of air-dry raw material, the use of large amounts of distilled water and organic solvents.

A method of obtaining the ecdysone from vegetable raw materials by crushing the air-dried leaves of plants of the genus Serratula, extraction with water at a temperature not exceeding 100And filtering, the filtrate concentrated, and the concentrate is extracted with ethyl acetate, the extract was partially evaporated the solvent, the concentrated extract is cooled and filtered off the precipitated crystals of ecdysone. According to this method, the yield of the target product is 0.65 to 0.7% by weight of dry plant materials (2. Pat. Of the Russian Federation No. 2151598, class a 61 K 31/575, 01 J 75/00, 2000).

The disadvantage of this method is the large amount of ethyl acetate, the low yield of the target product. When using this method selects only-Edison, other ecdysteroids are not allocated and not used.

The closest technical solution chosen for the prototype is a way of simultaneously receiving 20-hydroxyecdysone and inokosterone from the aerial parts of the plant Serratula coronata, including the grinding of raw materials, extraction of the target product water, Reis water extract zinc sulfate, solid-phase extraction of ecdysteroids by filtering the clarified aqueous extract through the sorbent “diasorb With 16 Tons”, selective elution of ecdysteroids with sorbent solvent, concentrated fractions obtained with ecdysteroids, the recrystallization of the target substances and vacuum drying of ecdysteroids (3. Punegov Century, Mishurov B. N., Nikitin, E. N., Konareva Century. And. features of the technology of production of ecdysteroids plants Serratula coronata L by the method of solid-phase extraction. Proc. Dokl. The second international. proc. New and nontraditional plants and prospects of their practical use”. - Pushchino, 1997, so 2, S. 79-81).

The disadvantage of this method is the low yield of the target components in the calculation of the air-dry raw material: 1,1% 20-hydroxyecdysone and 0.2% inokosterone.

The problem solved by the claimed technical solution is to increase the yield of target products at the expense of a more complete selection of ecdysteroids from vegetable raw materials.

The problem is solved due to the fact that in the inventive production method of ecdysteroids from plant material, comprising a grinding plant and water extraction of the target products, plant material previously exposed rogo adsorbent in the following ratio of components, wt.%:

Water-soluble adsorbent 5-30

Abrasive material 5-25

Plant material the Rest

Preferably, as ball mills use a planetary mill, a vibration and vibratory centrifugal types, enabling acceleration acting balls from 60 to 400 m/s2.

Preferably, the abrasive material used quartz sand.

Preferably, as water-soluble adsorbent used sucrose.

Salient features of the claimed invention that are in causal relationship with achievable technical result are:

- vegetable raw material is subjected to mechanochemical treatment by grinding it in ball mills;

- milling and mechanochemical processing is carried out in the presence of abrasive material and water-absorbent;

- mechano-chemical activation is carried out at the following ratio, wt.%:

Plant material 50-70

Water-soluble adsorbent 5-30

Abrasive Else

The proposed method is as follows.

The air-dried plant material containing phytoecdysteroids, mixed with water-soluble adscam the use of any other solid water-soluble adsorbent, consisting of polyfunctional compounds containing 4 or more hydroxyl groups, and an abrasive material, for example quartz sand, which does not exclude the use of other abrasives that do not change the chemical nature of the target product, is subjected to mechanochemical activation by grinding in a planetary ball mills, vibration and vibratory centrifugal types, enabling acceleration of the acting bodies (balls) from 60 to 400 m/s2. In the mechanochemical treatment is the fine crushing of raw material to particle sizes 4-3,5 μm. Introduction abrasive material dramatically increases the efficiency grinding plant raw material and leads to the destruction of cell walls. The cellular content of processed so plants in the process of mechanochemical activation in contact with a water-soluble adsorbent. This ecdysteroids plants are concentrated on the surface of the water-soluble adsorbent due to the formation of molecular complexes.

The resulting mixture is subjected to a twofold extraction with water at a temperature of 50-70C. the duration of the extraction is 1.0-1.5 hours.

The secretion of ecdysteroids from an aqueous extract of the MOU.

Mechanochemical treatment of the mixture of the above composition provides the following:

the destruction of cell walls in the particles of plant materials allows better allocate ecdysteroids from plants allocated to the mixture contains 20-hydroxyecdysone, Edison, inokosterone and magistero And structural formula of which is shown in the drawing;

- formation of composite particles consisting of plant material, in which are embedded particles of abrasive material, such as quartz sand, leads to an increase in the weight, which facilitates subsequent sedimentation, filtration or centrifugation of aqueous extracts;

- due to the formation of soluble molecular complexes is the binding of biologically active substances, ecdysteroids with a water-soluble adsorbent that allows you to easily and adequately perform water extraction of ecdysteroids.

The inventive method is illustrated by the following examples.

Example 1.

Dry aerial part of plants Serratula coronata mixed with quartz sand and sucrose in the following ratio, wt.%: 60:20:20 respectively, is treated in a flow mechanical activator type VCM when accelerating balls 120 m/s2and uragiru twice with hot water at a temperature of 50-70C for 1.0 to 1.5 hours, the water ratio 6,0-7,0. After settling sludge press through filtrating or centrifuged. United water portion is cooled to a temperature of 3-6With after which gradually crystallizes from solution 20-hydroxyecdysone, which is collected by centrifugation or filtration.

The filtrate is extracted three times with ethyl acetate at a ratio of water - organic phase 3:1. After removal from the extract 3/5 solvent receive the concentrate, which is cooled again to 3-6With and sucked the remnants of the drop-down 20-hydroxyecdysone. From the obtained extract secrete ecdysteroids by sorption filter by a known method. The fractions containing according to HPLC (high efficient liquid chromatography) target components, collect. Analysis by HPLC on ecdysteroids carried out according to [Century. Century. Volodin and other Inokosterone and magistero And Serratula coronata // plant Physiology, Volume 45, №3 (1998), 378-381.]. From the fractions containing the target components, the solvent is removed under vacuum. If necessary, cleaning of ecdysteroids carried out by recrystallization by a known method.

The output of products in terms of the

Magistero And 0,018-0,02

Example 2.

Carried out under the conditions of example 1. As extractant aqueous filtrate obtained after deposition of 20-hydroxyecdysone but also use a mixture of n-butanol-ethanol in a ratio of 3:1. The product yield, wt.%:

20-Hydroxyecdysone 1,7-1,85

Inokosterone 0,24-0,29

Edison of 0.28-0.31 in

Magistero And 0,017-0,019

Example 3.

Is carried out under the conditions of example 1. The resulting aqueous extract previously degreased with petroleum ether or gasoline (B-1 or BD-2). Then conduct a continuous countercurrent extraction with ethyl acetate mixture of ecdysteroids from the aqueous extract. Then remove 4/5 organic solvent, precipitated upon cooling of the concentrate crystals 20-hydroxyecdysone collected by centrifugation or filtration.

From the obtained organic extract allocate the amount of ecdysteroids in the conditions of example 1. The product yield in terms of dry plant material is, wt.%:

20-Hydroxyecdysone 2,0-2,1

Inokosterone 0,29-0,32

Edison 0,31-0,34

Magistero And 0,02-0,022

The products obtained are: 20-hydroxyecdysone - colorless or pink crystals with so pl. 241,5-242,0With magistero And - colorless amorphous rc="https://img.russianpatents.com/chr/176.gif">-+60,5(methanol), Addison - bright amorphous powder, 235-240With inokosterone - colorless powder decomposes at a temperature of more than 255C []37D=+59,0-+60,1(methanol).

The stated ratio of the concentrations of the components of mechanochemical treated mixture is determined with the following effects:

The introduction of quartz sand at a concentration of less than 5% leads to a sharp increase in the time of mechanochemical processing to technologically unacceptable values. The use of quartz sand at a concentration of more than 25 wt.% impractical due to the fact that large concentrations do not affect the efficiency of grinding and mechanical activation and unproductive cost of grinding sand grow.

Introduction sucrose in amounts less than 5 wt.% not ensures complete removal of product from plant material. The application for processing mixtures containing more than 30 wt.% sucrose becomes inefficient from an economic point of view - the cost of increasing the yield of the product does not pay off due to the cost, additionally consumed sucrose.

ohimicheskie processing the mixture of air-dried plant materials with water-soluble adsorbent, and abrasive material in the above-mentioned ratio of components allows the destruction of cell walls of plants, and the contents of the cell comes in contact with a water-soluble adsorbent.

An additional advantage of the presence in the processed mixture of abrasive material is the inclusion of small particles it into particles of plant materials. This facilitates subsequent sedimentation, filtration or centrifugation of aqueous extracts. The interaction of ecdysteroids with a water-soluble adsorbent, for example, sucrose, leads to the formation of soluble molecular complexes.

Thus, through the application of mechanochemical activation is achieved a more complete extraction of the raw materials of the target components at lower cost solvent.

Compared with the prototype of the proposed technical solution allows to increase the output of ecdysteroids: 20-hydroxyecdysone with 1.08 wt.% the prototype up to 2.1 wt.%. In addition, the claimed solution allows you to find not only these ecdysteroids as Edison, inokosterone, but also magistero A.

The use of the claimed technical solution allows to significantly increase the concentration of 20-hydroxyecdysone in water extracts, resulting manages to hold its crystallization directly from the aqueous phase. Consumption of ethyl acetate at the stage extracts the>/p>1. Method to produce ecdysteroids from vegetable raw materials, including grinding plant, water extraction of target components at a temperature not exceeding 100°C and reextraction related hydrophobic substances hydrocarbon solvent, wherein the plant material previously subjected to milling and mechanochemical treatment in high-stressed mill in the presence of abrasive material and water-soluble adsorbent in the following ratio, wt.%:

Water-soluble adsorbent 5-30

Abrasive material 5-25

Plant material the Rest

2. The method according to p. 1, characterized in that as a high-stressed mill used ball mills planetary, vibration and vibratory centrifugal type acceleration affecting tel 60 - 400 m/s2.

3. The method according to p. 1, characterized in that the abrasive material used quartz sand.

4. The method according to p. 1, characterized in that as the water-soluble adsorbent used sucrose.

5. The method according to p. 1, characterized in that the aqueous extraction of target components is carried out at a temperature of 50-70°C.

 

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