RU2182174C2 - Method of isolation of avermectin complexes - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: invention relates to the microbiological synthesis and isolation of avermectins from biomass of actinomycete Streptomyces avermitilis used for antiparasiting preparations preparing. Method involves extraction of avermectins from biomass of S. avermitilis, purification of extract, separation of lipid fraction and isolation of the end product. Extraction of avermectins from biomass is carried out with a mixture consisting of water and low- and high-boiling solvents mixing with water in unlimited ratio. The proposed method ensures to isolate avermectin complexes with different content of individual avermectins showing the biological activity. EFFECT: improved method of isolation. 5 cl, 5 tbl, 5 ex

Description

 The invention relates to the field of protection of animals and plants, and more particularly to microbiological synthesis and isolation from biomass of avermectins used for the production of antiparasitic drugs.

State of the art
It is known that Streptomyces avermitilis actinomycete, when cultured in the presence of carbon, nitrogen, and phosphorus sources under constant aeration, produces eight structurally similar substances called avermectins, which have a pronounced antiparasitic effect (GB 1573955, 1980). All avermectins in chemical structure are 16-membered macrolides and differ among themselves with substituents at 5 and 26 carbon ring atoms. Individual avermectins, designated A _1a , A _1b , A _2a , A _2b , B _1a , B _1b , B _2a and B _2b , have different biological activity. The most active are avermectins B ₁ , especially B _1a .

A known strain of Streptomyces avermitilis ATCC 31272, producing an average of 500 μg / ml avermectins (US patent 4310519, 1982). The productivity of the strain Streptomyces avermitilis VNIISKHM-51 (patent RU 2048250, 1995) is 700 μg / ml. The strain Streptomyces avermitilis BHIISXM-54 already produces 1500 μg / ml avermectins (patent RU 2054483, 1996), and the content of avermectin B _1a reaches 35% or more. The strain Streptomyces avermitilis CCM 4697, deposited in the Czech collection of microorganisms in Brno, produces an average of 2300 μg / ml avermectins with a variability of 2000-3000 μg / ml.

 In all the above cases, avermectins are isolated from the mycelium by extraction with an individual organic solvent miscible with water, followed by separation, for example, chromatographic, and obtaining individual avermectins.

 Patent RU 2054483 (publ. 1996) describes a method for producing an avermectin complex by extraction from a dried mycelial mass using an aqueous ethanol solution, followed by freezing, treatment with charcoal and ion-exchange resins. From the obtained alcohol solution containing 8 individual avermectins, various dosage forms of veterinary use are prepared.

SUMMARY OF THE INVENTION
The present invention allows to isolate complexes of avermectins with different contents of individual avermectins, differing in biological activity.

 To isolate avermectin complexes of various component composition, dry biomass obtained by growing the producer in deep conditions on known nutrient media is used.

Extraction of avermectins from biomass is carried out with a mixture consisting of water and low and high boiling solvents, miscible with water in an unlimited ratio. Low boilers mean solvents with a boiling point not exceeding the boiling point of water, for example ethyl, methyl and isopropyl alcohols or acetone, as high boiling solvents with a boiling point above 150 ^° C are used: polyethylene glycol with a molecular weight of 200 (PEG-200), polyethylene glycol with molecular weight 400 (PEG-400) or dimethyl sulfoxide (DMSO) and others. The extracts are purified from pigments and inorganic impurities using cation exchange and anion exchange resins. Water and a low boiling solvent are then distilled off. By settling the remainder of the extract in a high-boiling solvent, lipid impurities are separated.

Further, in one case, all the avermectins present immediately can be precipitated from the solution using water and a total avermectin complex can be obtained. In another case, in the presence of water and ethyl alcohol, only a part of avermectins with a predominant content of avermectins B _1a and B _{1b is} precipitated. The mother liquor after separation of part of the avermectins is mixed with water, precipitating the remaining avermectins.

At the same time receive:
1) the total avermectin complex, called aversectin C, containing 8 avermectin components in the ratio,%:
Avermectin A _1a - 6-12
Avermectin A _1b - Traces
Avermectin A _2a - 15-25
Avermectin A _2b - 2-4
Avermectin B _1a - 30-40
Avermectin B _1b - 2-12
Avermectin B _2a - 14-24
Avermectin B _2b - 1-2
or
2) a complex called aversectin B, containing mainly avermectins B _1a and B _1b ,%:
Avermectin B _1a - 76-83
Avermectin B _1b - 12-16
Other Avermectins - Traces
Avermectin B _2a - 0.8-6
Avermectin A _1a - 1-6
3) the complex, called aversectin AB, of the following composition,%:
Avermectin A _1a - 4-10
Avermectin A _1b - Traces
Avermectin A _2a - 25-45
Avermectin A _2b - 2.1-4.5
Avermectin B _1a - 12-18
Avermectin B _1b - 3-9
Avermectin B _2a - 25-45
Avermectin B _2b - 2.5-5.5
The biological properties of the resulting preparations are presented in tables 1 and 2.

Example 1
3.5 kg of S. avermitilis SSM 4697 mycelium, dried on a spray dryer, was extracted for 1 hour with stirring and at room temperature with a mixture of 0.5 L of PEG-400 and 6.5 L of 75% ethanol. The precipitate was separated by suction filtering under vacuum and washed with 0.8 L of 96% ethanol. The filtrate and washings were combined to obtain 7.0 L of alcohol extract containing 500 ml of PEG-400 with a concentration of 7.7 mg / ml avermectins, which was then passed sequentially through columns with 400 g of KU-2x8 cation exchanger in H ⁺ form and anion exchanger EDE-10P in OH ^- form. The columns were washed with 500 ml of 96% ethanol. Received 7.15 l of purified alcoholic extract (avermectins 7.5 mg / ml, yield 99.4%), distilled off the alcohol and water on a rotary evaporator at 55 ^o C in vacuum. The residue in the flask, heated to 65 ^{° C.} , was placed in a separatory funnel to separate the lipid fraction.

The lower layer with a volume of 580 ml (avermectins 76 mg / ml) was separated after 3.2 hours and poured into 1750 ml of distilled water, heated to 40 ^{° C.} with vigorous stirring. The mixture was stirred for 3-5 hours to form a precipitate, the main part of the supernatant was separated by decantation, and the rest by filtration through a glass filter in vacuo. The filter cake was washed with 500 ml of cold distilled water and dried in a convection stream of warm air.

 The dry sediment contained 41 g of aversectin C.

 To assess the biological activity of aversectin C for warm-blooded animals, acute toxicity was studied in outbred white rats and male mice with a single intragastric (oral) administration in the form of an aqueous suspension on Tween 21 using a gastric tube. For rats weighing 150-200 g, the drug was administered in the following doses of 50, 70, 84, 90, 110 and 150 mg / kg body weight. Outbred white mice weighing 25-30 g aversectin C was administered in doses of 7, 10, 25, 40, 50 and 60 mg / kg. Each dose in rats and mice was tested on 6 individuals. To calculate the average lethal dose, the Litchfield and Wilcoxon probit analysis method was used (see, for example, M. Belenky. Elements of a quantitative assessment of the pharmacological effect. L.: Gosmedizdat, 1963, p. 152). The results of the determination of acute toxicity of aversectin C for rats and mice are shown in table 3.

To assess the biological activity of aversectin C for invertebrates, SC ₅₀ for Aphelenchoides bessei (rice nematode) was studied according to the method described in patent RU 2013053 on "Method for determining the nematicidal activity of avermectins", 1994. It was found that SC ₅₀ for Aphelenchoides bessei is 0.7 μg / ml

Example 2
500 ml of the extract (extractant is a mixture of 75% alcohol and DMSO in a ratio of 3: 1) with an aversectin concentration of 20 mg / ml was purified as described in example 1, the alcohol and water were distilled off, as in example 1. The lipid fraction on a separatory funnel separated after 3 hours. Received 120 ml of the lower layer containing DMSO and aversectin C - 56 mg / ml (a total of 6.72 g of aversectin C - 67.2%).

 A solution of aversectin C in DMSO was poured into 360 ml of distilled water, stirred for 4 hours and aversectin C was isolated as described in Example 1. 8.2 g of a dry preparation were obtained containing 79.0% (6.48 g) of aversectin C - yield 64.8%.

 Next, we evaluated the biological activity of aversectin C for warm-blooded and invertebrates in the same way as in example 1. The same results were obtained.

Example 3
3.4 l of the extract containing 3 l of 75% alcohol and 400 ml of PEG-200 were purified from pigments and inorganic impurities, passing sequentially through columns with KU-2x8 and EDE-10P ion-exchange resins. Received 3.2 l of purified extract.

 The extract was concentrated as described in Example 1. After distilling off the alcohol and water, the residue was transferred to a separatory funnel. After settling, two layers were obtained: the lower layer — PEG-200, containing 40 g of aversectin C, and the upper — lipid fraction.

The bottom layer was slowly poured into 1.37 L of water with stirring. The precipitate was separated on a filter, washed with cold distilled water and dried in a vacuum oven. Received 38.4 g of aversectin C of the following composition,%:
Avermectin
A _1a - 11
A _1b - Traces
A _2a - 23
A _2b - 2.1
In _1a - 38
B _1b - 3.1
B _2a - 21
B _2b - 1.8
Verification of the biological activity of aversectin C for warm-blooded and invertebrates is the same as in example 1, showed the same results.

 Obtaining a complex with a predominance of group B avermectins (aversectin B) and assessing its biological activity.

Example 4
To a 200 ml solution of aversectin C in PEG-400, prepared as in Example 2 (76 mg / ml aversectin C), 400 ml of 40% ethanol were added in portions with constant stirring. The mixture was stirred for another 1 hour and left to crystallize at room temperature for 10 hours. The precipitated crystals were filtered through a glass filter under vacuum, the crystals on the filter were washed with 50 ml of a 70% alcohol solution in water cooled to 10 ^° C and dried at 60 ^° C to constant weight in an oven.

Received 4.1 g of aversectin B with the content of the main components,%:
In _1a - 82.2
In _1b - 15.2
B _2a - 0.8
A _1a - 1.3
Next, we evaluated the biological activity, as described in example 1. The following data were obtained with a single intragastric (oral) administration to rats and male mice (Table 4).

When assessing the biological activity of aversectin B for invertebrates using the example of Aphelenchoides bessei according to the method specified in Example 2, SK ₅₀ was 0.4 μg / ml.

Obtaining a complex with a predominance of avermectins of group A ₂ and B ₂ (aversectin AB) and assessment of its biological activity
Example 5
The mother liquor from crystallization of aversectin B and washing (example 4) were evaporated in vacuo. The remainder was transferred to a separatory funnel. After settling, two layers formed. The lower layer - a solution of aversectin A in PEG-400 was poured into 600 ml of water. The precipitate was separated on a filter and dried. Received 12.6 g of the drug - Aversectin AB.

The composition of the drug,%:
A _1a - 7.1
A _1b - Traces
A _2a - 27.1
A _2b - 2.1
In _1a - 12.1
B _1b - 3.7
In _2a - 44.4
In _2b - 3.5
Assessment of biological activity was carried out as described in example 1. When studying acute toxicity in rats and male mice, the following results were obtained (Table 5).

The CK ₅₀ for aversectin AB when using the test with Aphelenchoides bessei was 1.0 μg / ml.

Industrial applicability
The resulting aversectins can be used to prepare drugs for the prevention and treatment of various parasitic diseases of animals and humans.

 Aversectin C and aversectin B have a longer protective effect. Aversectin B is more slowly excreted from the body, and its duration of action against ectoparasites will be approximately 7-10 days longer than that of aversectin AB.

 Aversectin C provides the same period of protective action against ectoparasites as aversectin B, but its waiting time (use of dairy and meat products after using the drug) is at the level of 14-21 days, and at aversectin B - at the level of 21-28 days .

 Aversectin AB has a high activity against larvae of the subcutaneous gadfly of cattle. It is faster than aversectin B and aversectin C are excreted from animals and cannot be found in milk 3 days after administration, which allows it to be used for the prevention and treatment of hypodermatosis in the dairy herd.

 Based on aversectins, various dosage forms (injection, ointment, oral, powder, tablet, irrigation, paste) intended for the treatment of animals, as well as liquid, paste and powder for controlling pests of crops, can be made. These aversectin-based formulations may include various excipients and excipients.

Claims (3)

 1. The method of separation of avermectin complexes, including the extraction of avermectins from the biomass of S. avermitilis, purification of the extract, separation of the lipid fraction and the isolation of the target product, characterized in that the extraction is carried out with a mixture of high and low boiling organic solvents with added water, the extract is purified on ion exchange resin, the lipid fraction is separated by sedimentation of the extract after distillation of the low boiling solvent and water, and the selection of the target product from a solution in a high boiling solvent is carried out by m precipitation with water or an aqueous solution of a low boiling solvent.  2. The method according to p. 1, characterized in that methanol, ethanol, isopropanol, acetone or acetonitrile are used as low boiling solvents, polyethylene glycol, dimethyl sulfoxide or polypropylene glycol are used as high boiling solvents, and the amount of water added during extraction is 20-25 % of the volume of the extracting mixture. 3. The method according to p. 1 or 2, characterized in that the total avermectin complex with the following composition of the components is precipitated from a solution in a high boiling solvent,%:
Avermectin A _1a - 6-12
Avermectin A _1b - Traces
Avermectin A _2a - 15-25
Avermectin A _2b - 2-4
Avermectin B _1a - 30-40
Avermectin B _1b - 2-12
Avermectin B _2a - 4-24
Avermectin B _2b - 1-2
4. The method according to p. 1 or 2, characterized in that from the solution in a high boiling solvent, after adding an aqueous solution of a low boiling solvent, an avermectin complex is isolated with the following composition of components,%:
Avermectin B _1a - 76-83
Avermectin B _1b - 12-16
Avermectin B _2a - 0.8-6
Avermectin A _1a - 1-6
Other Avermectins - Traces
5. The method according to p. 4, characterized in that from the solution in a high boiling solvent remaining after separation of avermectin B, an avermectin complex is isolated by water precipitation with the following composition,%:
Avermectin A _1a - 4-10
Avermectin A _1b - Traces
Avermectin A _2a - 25-45
Avermectin A _2b - 2.1-4.5
Avermectin B _1a - 12-18
Avermectin B _1b - 3-9
Avermectin B _2a - 25-45
Avermectin B _2b - 2.5-5.5

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