RU2257907C2 - Method for obtaining biologically active substance to regulate energy balance - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: the present innovation deals with obtaining biologically active substances of protein nature out of cereals, moreover it is necessary to isolate a stress separating protein 310 and/or protein being immunochemically similar to that as coarse and/or finely purified extract of the target product. For this purpose one should destruct cellular walls of 3-7-d-aged sprouts due to homogenization to isolate cell-free extract due to centrifuging which should be supplemented with a reagent-precipitator at the first stage under certain conditions to obtain residue followed by isolating a liquid phase which should be subjected to the second of impact with reagent-precipitator under certain conditions, then residue should be separated against a liquid phase due to centrifuging, and an obtained coarsely purified extract of target product should be separated against reagent-precipitator due to dialysis due to the impact of fine purification by gel filtration, if necessary. Moreover, one should conduct this procedure at certain temperature mode, and , if necessary, the extract of target product should be freeze dried. The suggested biologically active substance provides efficient regulation of body energy balance.

EFFECT: higher efficiency.

8 cl, 3 ex

Description

The invention relates to the production of biologically active substances of protein nature from plants and their use for the regulation of energy metabolism of living organisms.

Known methods for extracting active substances from plants with antihypoxic and antioxidant activity, by preparing from them preparations in the form of infusions, decoctions, juices and tinctures. A known method of strengthening the immune system and regulating metabolism by taking these drugs [Pastushenkov L.V., Lesiovskaya E.E. Plants - antihypoxants (herbal medicine) / St. Petersburg Chemical-Pharmaceutical Institute. - S.Pb., 1991. - 96 p.] [1].

The disadvantage of this method is the presence in the resulting preparation of simultaneously recoverable substances that can impede the necessary active effect of the desired component.

A known method of isolating an extract from a rosemary leaf having the properties of a natural antioxidant for its use in cosmetic dermatology [Biochemical studies of natural antioxidant isolated from rosemary and its application in cosmetic dermatology / Calabrese V. et.al.// Int. J. Tissue React. - 2000. - V. 22. - N 1. - 5-15.] [2].

A known method of obtaining the active substance in the form of an extract of wheat germ and other plants and the possibility of using it as an ingredient in cosmetic preparations [Effect of germinated seeds extract on the respiratory activity of human skin fibroblasts and sheep liver mitochondria. Influence on cell viability and proliferation and their usefulness as active cosmetic ingredient / Benaiges A., Armengol R. et.al. // International Journal of Cosmetic Science. - 2001. - V.23. - P.243-255.] [3].

Known methods of using extractive antioxidant substances are limited in one direction and solve a highly specific problem in the field of cosmetic dermatology.

Adverse environmental and climatic conditions, insufficiently high-quality and unbalanced nutrition cause weakening of the body's defenses, the rapid development of various pathological changes in the body, and the disruption of normal physiological processes. To achieve an increase in the body's tolerance of adverse factors, to eliminate the decrease in the functional activity of the body in existing conditions is a difficult task. Its solution is directed to the creation of biologically active substances containing, as a rule, vitamins, microelements, amino acid complexes, flavonoids and other substances of a low molecular nature. Known biologically active substances allow to varying degrees physiological processes, increase the body's defenses to the action of adverse factors, but they require prolonged use and in many cases are ineffective.

Currently, there are practically no preparations for removing plants from the oppressed state that have been exposed to unfavorable factors, such as a short-term temperature drop during the growing season, absence or insufficient supply of moisture for plant nutrition, etc.

The objective of the invention is to obtain the active substance of a qualitatively new level, effectively affecting the restoration of the functioning of compensatory mechanisms in order to ensure the normal flow of energy processes in the body in extreme conditions.

The technical result of the invention is the allocation of a substance in quantities sufficient to be used as an active substance, affecting the dynamics of energy processes, reducing damage caused by oxidative stress in the body, and for restorative regulation of the vital processes of the body.

The technical result ensures the isolation from plants of the stress uncoupling protein BCS 310 and / or an immunochemically related protein and its use.

The stress uncoupling protein BCS 310 or immunochemically related proteins are isolated from seedlings of cereal crops in the form of a coarse and / or highly purified extract of the target product.

The target product in the form of the BCS 310 protein and / or an immunochemically related protein is obtained by destroying the cell walls of 3-7 daily seedlings, followed by separation of media and isolation of cell-free extract. The selection of the target product is carried out using a precipitating reagent in two stages. A precipitating reagent is added to the acellular extract in the first stage of exposure to form a precipitate, followed by isolation of the liquid phase. The isolated liquid phase is subjected to the second stage of exposure to the reagent precipitator. The precipitate formed is separated from the liquid phase and then from the precipitating reagent, obtaining a crude extract of the target product.

The selection of the extract of the target product is carried out at a temperature of from 0 to + 4 ° C.

A highly purified stress protein extract is obtained by fine-cleaning the crudely purified extract of the target product.

The resulting target product is dried if necessary.

The destruction of the cell walls of seedlings is carried out, in particular, by homogenization.

The precipitating reagent in the first stage of exposure is added to the acellular extract to a concentration of 30%, with holding for 6-18 hours.

The precipitating reagent in the second stage of exposure is added to the isolated liquid phase to a concentration of it (in solution) equal to 50%, with holding for 4-24 hours.

Separation of media and isolation of cell-free extract after destruction of the cell walls of seedlings and further separation of the liquid phase from the sediment is carried out by centrifugation at 10000 g and above.

The crude extract of the target product is separated from the precipitating reagent. The separation of the target product from the precipitating reagent is carried out by dialysis.

Fine purification of the extract of the target product to obtain a highly purified extract is carried out using gel filtration on a column with Sephadex grades G-100 - G-200.

Ammonium sulfate is used as the precipitating reagent.

Drying of the target product is carried out lyophilically.

The resulting crudely and / or highly purified extract of the stress protein BHSh 310 and / or an immunochemically related protein is used to regulate the energy balance of the body.

For the first time, the stress protein BCS 310 was isolated from seedlings of winter rye.

The isolated protein has a molecular weight of about 310 kD and consists of two types of subunits with a molecular weight of 66 and 56 kD. [Kolesnichenko A.V. et al. Characterization of winter rye protein accumulating during hypothermia // Plant Physiology. - 1996.- T.43. - No. 6 - S.894-899] [4].

Highly purified stress protein extract has a higher degree of activity. Infiltration of body tissue with a form based on a highly purified stress protein causes a significant increase in the rate of oxygen uptake by the tissue, an increase in the rate of substrate oxidation and thermogenesis, a significant decrease in the level of peroxidation of biological macromolecules.

The target product obtained as a result of carrying out the invention has a strongly pronounced antioxidant activity and can be used to reduce damage caused by oxidative stress by actively regulating the degree of conjugation of oxidation and phosphorylation in mitochondria upon penetration into body tissue.

The target product in the form of a crude or highly purified protein extract is used in the form of a powder, various types of solutions, emulsions, granules, suspensions, suspensions, ointments, with food and / or pharmacologically based excipient, as well as an integral part of a complex preparation.

A complex preparation may contain the target product in various percentages, depending on the type of drug, food, cosmetology, agrochemical and other forms of use of the drug.

The drug based on the stress-releasing vegetable protein BCS 310 or on the basis of immunochemically related proteins causes regulated dissociation of oxidation and phosphorylation in mitochondria in the tissue. As a result, the energy balance of the cell is restored and improved, the body acquires resistance to the effects of adverse factors.

Under the influence of the drug, tissue cell respiration is increased, not associated with phosphorylation, and as a result, a decrease in the formation of reactive oxygen species in mitochondria, a decrease in the level of peroxidation of biological macromolecules.

The drugs penetrate well into the tissue through the cell walls and have no toxic effect even at a concentration of 10 mg / ml.

Based on a highly purified and / or crudely purified stress protein extract, it is possible to create a qualitatively new line of active forms, including food, medicine, cosmetic, agrochemical, etc. Their action is due to the selective effect of the biologically active substance obtained on the energy processes in the cell. They have a positive effect under stressful conditions of a human, animal, plant organism and do not cause unwanted side effects.

Based on the invention, it is possible to obtain preparations with varying degrees of exposure to living organisms to remove them from a stressful state.

The invention makes it possible to obtain biologically active substances of various degrees of exposure to regulate the energy metabolism of the body in metabolic disorders.

A method of obtaining a biologically active substance is as follows.

Example 1

To obtain the extract of the stress protein BCS 310, three-day-old wheat seedlings (Triticum aestivum L.) are homogenized to break down the cell walls and then centrifuged at a speed of 10,000 g to obtain a cell-free seedling extract. First, the first stage of exposure to the reagent precipitator is carried out in order to fractionate the seedling extract. For this, a precipitating reagent is added to the obtained extract in the form, for example, of ammonium sulfate, bringing the solution concentration to 30% saturation. After 6 hours, the precipitate formed is separated in a centrifuge at 10,000 g to isolate the supernatant liquid phase. In the second stage of exposure, ammonium sulfate is added to the supernatant until it reaches a concentration in solution of 50%. After standing for 12 hours, the resulting substance is centrifuged to obtain a coarse extract of the stress protein BCS 310, precipitated. The resulting extract is separated from the precipitating reagent by dialysis. All technological operations from the destruction of the cell walls of seedlings to the selection of the target product in the form of a crudely purified extract of stress protein is carried out at a temperature of 0 to + 4 ° C.

Example 2

The selection of the target product in the form of a crude extract of the stress uncoupling protein BCS 310 is carried out as follows. Five-day-old rye seedlings are ground to a homogeneous mass for the destruction of cell walls. The liquid extract is separated from the cell membranes using a centrifuge.

First, a precipitating agent in the form of ammonium sulfate is added to the obtained seedling extract, while providing a concentration of the precipitating agent in the seedling extract equal to 30%. The precipitation reaction in the first stage of exposure is 18 hours. The precipitate was separated in a centrifuge in the manner described in Example 1. The precipitating reagent was again added to the separated liquid phase, bringing its concentration in the solution to 50%. Then produce aging for 24 hours.

Increasing the aging time of more than 24 hours is not rational, as it does not provide greater precipitation of the target product.

Next, a crude extract of the expected product is obtained as in Example 1. The resulting extract is freeze-dried.

Example 3

The target product is obtained by the method described in example 1, using three-day-old seedlings of winter rye (Secale cereale L.) for this. During the first stage of precipitation, when the precipitating reagent in the form of ammonium sulfate is added to the acellular extract of the seedlings, the precipitation reaction is carried out for 12 hours at a concentration of the precipitating reagent equal to 30%. In the second stage of introducing the precipitating reagent to the obtained supernatant at a concentration of 50%, the precipitation reaction is carried out for 15 hours.

The obtained crudely purified extract is subjected to fine purification by gel filtration on a G-200 Sephadex column, isolating the highly purified extract of the target product in the form of immunochemically pure stress-releasing protein BCS 310 or immunochemically related protein.

The authors conducted a series of studies to study the effect of the stress uncoupling protein BCS 310 on lipid peroxidation and respiratory activity of plant mitochondria.

Studies have noted that the use of the selected target product in the form of an aqueous solution for processing cereal seedlings increases the cold resistance of plants.

For example, keeping the protein of cereal seedlings in an aqueous solution increases the amount of stress protein BCS 310 in the tissues of the seedlings. The rate of oxygen consumption increases to 533.39 nmol O ₂ / (min g of tissue), which is 40% higher than infiltration with water (391.61 nmol O ₂ / (min g of raw tissue). The temperature difference between living and dead seedlings when cooled, it was 2 ° C. After the infiltration of these seedlings with the obtained active substance, this value increases and amounts to 2.5 ° C.

During low-temperature stress, tissue infiltration with the drug leads to a decrease in the level of lipid peroxidation, i.e. pronounced antioxidant activity of the protein preparation is manifested [J. Immunoassay Immunochem. - 2003. - V.24. - N 1. - P.41-55.] [5].

The survival rate of pre-infiltrated cereal seedlings after exposure to cold increases by 20%.

As a result of exposure to the resulting target product, the segregation function of the cell increases, i.e. its ability to defend itself against the damaging effects of hostile factors.

The target product resulting from the implementation of the method can be widely used in medical practice for the prevention and treatment of metabolic disorders.

The present invention opens up the possibility of creating a new class of selectively active active substances of a qualitatively different level. The resulting target product obtained as a result of the invention can be widely used in various fields related to the optimal functioning of the vital functions of living organisms in adverse conditions.

The method provides the allocation of a substance in sufficient quantities to use it as a basis for creating a new line of substances that affect the dynamics of energy processes, for restorative regulation of the necessary vital processes of the body.

Sources of information

1. Pastushenkov L.V., Lesiovskaya E.E. Plants - antihypoxants (herbal medicine) / St. Petersburg Chemical-Pharmaceutical Institute. - SPb., 1991. - 96 p.

2. Biochemical studies of natural antioxidant isolated from rosemary and its application in cosmetic dermatology / Calabrese V. et. al.// Int. J. Tissue React. - 2000. - V.22. - N 1. - 5-15.

3. Effect of germinated seeds extract on the respiratory activity of human skin fibroblasts and sheep liver mitochondria. Influence on cell viability and proliferation and their usefulness as active cosmetic ingredient / Benaiges A., Armengol R. etc. // International Journal of Cosmetic Science. - 2001. - V.23. - P.243-255.

4. Kolesnichenko A.V. et al. Characterization of winter rye protein accumulating during hypothermia // Plant Physiology. - 1996.- T.43. - No. 6. - S. 894-899.

5. Influence of stress protein CSP 310 and antiserum against this protein on oxygen uptake, lipid peroxidation and temperature of winter wheat seedling shoots during cold stress / Kolesnichenko A.V., Grabelnych O.I., Tourchaninova V.V., Zykova V.V., Koroleva N.A., Pobezhimova T.P., Voinik V. // J. Immunoassay Immunochem. - 2003. - V.24. - N 1. - P.41-55.

Claims (9)

1. A method of obtaining a biologically active substance for regulating the energy balance of the body, including the use of cereal seedlings and extract, characterized in that the stress isolating protein BCS 310 and / or a protein immunochemically related to it in the form of a crude and / or highly purified target extract are isolated from the seedlings product, for this, the cell walls of 3-7 daily seedlings are destroyed and cell-free extract is isolated, to which, at the first stage of exposure, a precipitating reagent is added to form adka with subsequent isolation of the liquid phase, which is subjected to the second stage of exposure to the precipitating reagent, then the precipitate is separated from the liquid phase, and the obtained crude extract of the target product is separated from the precipitating reagent, subjecting it to fine purification if necessary, while the extraction of the target product extract is carried out at temperature conditions from 0 to + 4 ° C, if necessary, the extract of the target product is dried. 2. The method according to claim 1, characterized in that the cell walls are destroyed by homogenization. 3. The method according to claim 1, characterized in that the precipitating reagent in the first stage of exposure is added to the cell-free extract to a concentration of 30%, with holding for 6-18 hours 4. The method according to claim 1, characterized in that the precipitating reagent in the second stage of exposure is added to the selected liquid phase to a concentration of 50% in the solution, with holding for 4-24 hours 5. The method according to claim 1, characterized in that as the precipitating reagent, ammonium sulfate is used. 6. The method according to claim 1, characterized in that the separation of the cell-free extract after the destruction of the cell walls of the seedlings and the further separation of the liquid phase from the precipitate is carried out by centrifugation at 10000 g and above. 7. The method according to claim 1, characterized in that the crude extract of the target product is separated from the precipitating agent by dialysis. 8. The method according to claim 1, characterized in that the fine purification of the extract of the target product is carried out using gel filtration on a column with Sephadex grades G-100 - G-200. 9. The method according to claim 1, characterized in that the extract of the target product is freeze-dried.