The method of obtaining biologically active peptides caudal neurosecretory system (cnss) vertebrate

 

The invention relates to medicine and relates to a method of production of the active substances, namely peptides CNSS from raw materials of animal origin with antiandrogenna and antiestrogenic activity. The lumbosacral spinal cord of animals homogenized in a solution of sodium chloride at a ratio of 1:10, 1:15, extracted with the extract, double-saturated ammonium sulfate and gradually centrifuged for 25-30 min at 6000-9000 rpm, when the first precipitate is discarded, and the second is when it is dissolved in 0.8 to 1% solution of sodium chloride, absoluut, chromatographic in the UV spectrum in the range 220-280 nm and target product lyophilizer. Technical result: method is environmentally friendly, easy, economical, reduces the time of extraction and receive sediment and allows you to get active peptides of high purity. table 2.

The invention relates to medicine and relates to a method of obtaining biologically active substances, namely peptides CNS [1] from raw materials of animal origin.

A method of obtaining biologically active substances from raw materials of animal origin with antiandrogenna and antiestrogenic activity [2]. It includes homogeniser aqueous suspensions of the brain, deposition of substances ammonium sulfate, dissolve the precipitate in a solution of sodium chloride, desalination, chromatography was carried out and freeze drying of a substance.

The disadvantage of this method is the mixture of other substances with biologically active peptides CNSS that in their application in medical practice can cause unwanted side effects. Additionally, getting active substances from the precipitate was obtained by filtration, significantly lengthening the time allocation of the final product.

The objective of the invention: production of biologically active peptides CNS vertebrates, purified from other substances.

This is achieved by homogenization of the lumbosacral spinal cord of vertebrates, extraction of biologically active peptides by solution of sodium chloride, precipitation of peptides ammonium sulfate, dissolving the precipitate in a solution of sodium chloride, desalination, chromatography was carried out in the UV spectrum and lyophilized peptides.

The method is as follows: homogenize the lumbosacral spinal cord of vertebrates and the extracted peptides for 10-15oFrom within, for example, 25-30 minutes in the 0.8-1% solution of sodium chloride in combination 1:10-1:15 to sagaut ammonium sulfate at 5-10oC.

During the first 9% of the saturation precipitated high molecular weight proteins, throw them away and re-saturation of the extract with ammonium sulfate up to 39-40% precipitated biologically active peptides, dissolve the precipitate in a 0.85-1% solution of sodium chloride, absoluut, chromatographic in the UV spectrum with obtaining purified peptides, lyophilizer them. The output of the active peptides is 250 μg per 1 kg of raw material. Obtained in this way, the peptides have a pronounced antiandrogenna (PL. 1) and antiestrogenic (PL.2) activity. They have a molecular weight of 1300, which is confirmed by thin layer chromatography. Peptides are broken down by proteases are inactivated by boiling or keeping them in a thermostat at 37oC for 1 hour. They are deposited deproteinization means: sodium chloride, ammonium sulfate, tetraoxane acid.

When using as a raw material of the lumbosacral spinal cord offer in the way the parameters of temperature, environment, concentrations of estrogen it is thought that, its correlation with brain homogenate, speed centrifugation are optimally valid. So using brain throughout increases the proportion of roughage, energy, volume shodaime extraction of peptides over 1.5 hours, and above the 15oWith is partial inactivation.

The concentration of the solution of sodium chloride is less than 0.8% reduces the degree of extraction of the peptides, their output and increase this process, but above 1% contributes to the deposition of part of the peptides, reducing their output.

The ratio of estrogen it is thought that with brain homogenate less than 1:10 reduces the degree of extraction of the peptides and their output, and an increase of more than 1:15 does not give a positive effect.

Centrifugation of the suspension of the brain at speeds below 6000 rpm affects the deposition of suspended particles of the brain and increase the time to 1 hour, and the speed of 9000 rpm does not increase the efficiency of separation of suspended particles of the brain.

Centrifugation at 5-10oWith the need to preserve the activity of thermolabile peptides. The temperature decrease of less than 5oWith lengthens the time of deposition of suspended particles up to 1 hour, more than 10oWith - leads to decreased activity of peptides.

Example 1. Take 100 grams of fresh or frozen spinal cord of the animal during lumbosacral, homogenize on the top. The homogenate was poured in 10 parts of 0.8% sodium chloride solution with the addition of 0.05% EDTA. The extraction is carried out at 10oC for 30 min at Kee is about the speed of 9000 rpm, the precipitate discarded, and the supernatant double-saturated ammonium sulfate, 15oC. the First 9% saturation of ammonium sulfate precipitated high molecular weight proteins, which are separated by centrifugation at 6000 rpm for refrigerated centrifuge at 10oC for 20 min and discard. The supernatant again saturate to 39% ammonium sulfate, precipitating biologically active peptides CNS. The precipitate obtained by centrifugation at 10oWith refrigerated centrifuge for 30 min with a speed of 9000 Rev/min the Supernatant discarded. The precipitate is dissolved in 0.85% sodium chloride solution, absoluut, chromatographic in the UV spectrum in the range from 220 to 280 nm and lyophilized pure peptides. The output of the active substance is 25 μg, i.e., 250 μg per 1 kg of raw material.

Example 2. Take 100 grams of fresh or frozen spinal cord of the animal during lumbosacral, homogenize on the top. The homogenate was poured 15 parts of 0.9% sodium chloride solution with the addition of 0.05% EDTA. The extraction is carried out at the 12oC for 30 min with constant shaking. Centrifuged the suspension cord for 30 min in refrigerated centrifuge at 5oWith speeds of 800 10% saturation of ammonium sulfate precipitated high molecular weight proteins, which is separated by centrifugation at 6000 rpm for refrigerated centrifuge for 25 min and discard. Second - 40% saturation ammonium sulfate precipitated bioactive peptides CNS. The precipitate obtained by centrifugation at 8000 rpm in 7oWith refrigerated centrifuge for 30 minutes the Supernatant discarded. The precipitate was dissolved in 0.9% sodium chloride solution, absoluut, chromatographic in the UV spectrum in the range from 230 to 280 nm and lyophilized pure peptides. The output of the active substance is 25 μg, i.e., 250 μg per 1 kg of raw material.

Example 3. Take 100 grams of fresh or frozen spinal cord of the animal during lumbosacral, homogenize on the top. The homogenate was poured 15 parts of 1% aqueous solution of sodium chloride with the addition of 0.05% EDTA. The extraction is carried out at 15oWith over 25 min with constant shaking. Centrifuged the suspension of brain tissue within 28 min in refrigerated centrifuge at 8oWith a speed of 7000 rpm and the precipitate discarded, and the supernatant double-saturated ammonium sulfate at the 14oC. the First 10% saturation of ammonium sulfate precipitated high molecular weight proteins that separate centrifugual the offered liquid ammonium sulfate precipitated bioactive peptides CNS. The precipitate obtained by centrifugation at 9000 rpm at 10oC for 25 min, the Supernatant discarded. The precipitate is dissolved in a 0.85% solution of sodium chloride, absoluut, chromatographic in the UV spectrum in the range from 250 to 280 nm and lyophilized pure peptides. The output of the active peptides is 25 μg, i.e., 250 μg per 1 kg of raw material.

Thus, the proposed method allows to reduce the time selection, can save energy and to obtain biologically active peptides CNS vertebrates in high purity.

Sources of information 1. Bahtinov A. Endocrine function of the caudal spinal cord and its physiological significance // Honey. Newspaper - 1997. - 22 - C. 7 2. Bahtinov A. P. RF Patent 2074726 for the invention "Method of obtaining biologically active substances with antiandrogenna and antiestrogenic activity.

Claims

The method of obtaining biologically active peptides caudal neurosecretory system of vertebrates, with antiandrogenna and antiestrogenic activity, namely, that the lumbosacral spinal cord of animals homogenized with 0.8-1% solution of sodium chloride at a ratio of 1:10, 1:15, e and gradually centrifuged 25-30 min at 6000-9000 rpm, when the first precipitate is discarded, and the second is when it is dissolved in 0.8 to 1% solution of sodium chloride, absoluut, chromatographic in the UV spectrum in the range 220-280 nm and target product lyophilizer.

 

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