Method for obtaining biologically active substance to regulate energy balance

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

 

The invention relates to the production of biologically active proteins from plants and use their tlapehuala energy metabolism of living organisms.

Known methods of extraction of active substances from plants having antihypoxic and antioxidant activity by preparation of these drugs in the form of infusions, decoctions, juices and liqueurs. There is a method of strengthening the immune system and regulation of metabolism by receiving these drugs [Pastushenkov " L.V., Lesiovskaja E.E. Plants - antihypoxants (phytotherapy) / S.-Petersburg chemical-pharmaceutical Institute. - St. Petersburg., 1991. - 96 S.] [1].

The disadvantage of this method is the presence in the resulting drug simultaneously extractable substances that can interfere with the necessary activity of the target component.

A known method of separation of the extract from leaf of rosemary with an antioxidant, for its application 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 method of obtaining the active substance in the form of extract, wheat germ and other plants and the possibility of its use as an ingredient in cosmetic preparations [Effect of germinated seeds extract n 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 supported. - P.243-255.] [3].

Known methods of applying extractives of antioxidant action is limited to one direction and decide Skopelitis task in the field of cosmetic dermatology.

Adverse environmental and climatic conditions, poor and unbalanced diet causes weakening the body's defenses, the rapid development of various pathological changes in the body, disruption of normal physiological processes. To increase the tolerability of the body of unfavorable factors, to eliminate the reduction in the functional activity of the organism under existing conditions is a difficult task. Her decision directed the creation of biologically active substances, containing, as a rule, vitamins, minerals, amino acids, flavonoids and other substances of low molecular weight nature. Known biologically active substances help in varying degrees to adjust physiological processes, increase protective forces of an organism to the action of unfavorable factors, but they require long-term use and in many cases are ineffective.

Currently, there are almost no drugs to derive the C oppressed condition of the plants, affected by adverse factors, such as short-term reduction in the temperature during the vegetation period, the absence or insufficient moisture supply to power plants and other

The task of the invention to provide an active substance of a qualitatively new level, effectively influencing the recovery of compensatory mechanisms to ensure the normal flow of energy processes in the body in extreme conditions.

The technical result of the invention is the separation of substances in quantities sufficient for use as the active substance, affecting the dynamics of the energy processes that reduce damage caused by oxidative stress in the body, and rehabilitation regulation necessary life processes of the organism.

The technical result provides a selection of plant stress uncoupling protein BHS 310 and/or immunochemically related protein and its application.

Stress uncoupling protein BHS 310 or immunochemically related proteins extracted from seedlings of cereals in the form of crude and/or purified extract of the target product.

The target product in the form of protein BHS 310 and/or immunochemically related protein get, ruin the cell wall 3-7 daily seedlings, with the subsequent separation of the media and the release of cell-free extract. The selection of the target product are using reagent-precipitator in two stages. To the cell-free extract in the first phase impact type reagent precipitator for sediment, followed by separation of the liquid phase. The selected liquid phase is subjected to the second stage of exposure to the reagent-precipitator. The formed precipitate was separated from the liquid phase and then from reagent-precipitator, getting robocity extract the target product.

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

Highly purified extract of stress protein get, exposing fine cleaning robocity extract the target product.

The obtained target product, if necessary, dried.

The destruction of cell walls of seedlings is carried out, in particular, gomogenizirovannom.

Reagent precipitator at the first stage of impact is added to the cell-free extract to a concentration of 30%, maturing within 6-18 hours.

Reagent precipitator in the second stage effects add to the selected liquid phase to its concentration (in solution)equal to 50%, keeping within 4-24 hours.

The division of media and isolation of cell-free extract after the destruction of the cell walls prorostka and further separation of the liquid phase from the precipitate carried out by centrifugation at 10000 g and above.

Robocity extract the target product is separated from the reagent-precipitator. The separation of the target product from reagent-precipitator perform dialysis.

Fine purification of the extract of the target product to obtain a purified extract is administered by gel filtration on a column of Sephadex G brands-100 - G-200.

As reagent-precipitator apply ammonium sulfate.

Drying of the target product carry out a freeze.

The resulting robocity and/or highly purified extract of stress protein BHS 310 and/or immunochemical related protein used for the regulation of the energy balance of the body.

First of stress protein BHS 310 isolated from winter rye seedlings.

Isolated protein has a molecular weight of about 310 KD and consists of two types of subunits with a molecular mass of 66 and 56 KD. [A.V. Kolesnichenko and other Characteristics of the protein from winter rye, accumulate during hypothermia // plant Physiology. - 1996. - T. No. 6 - S-899] [4].

Highly purified extract of stress protein has a higher degree of activity. Infiltration of the tissues of the body shape based on highly purified stress protein causes a significant increase in the rate of oxygen consumption of tissue, the increase in the rate of consumption of substrate oxidation and thermogenesis, significant with irenie level of peroxidation of biological macromolecules.

The resulting implementation of the invention, the target product has strong antioxidant activity and can be used to reduce damage caused by oxidative stress due to active regulation of the degree of coupling of oxidation and phosphorylation in the mitochondria when the penetration into the tissue of the body.

The target product in the form of groupbooking or purified protein extract used in the form of powder, various types of solutions, emulsions, granules, suspensions, suspensions, ointments, food and/or pharmacologically informed filler, but also as an integral part of the integrated product.

The integrated product may contain the target product in different percentages depending on the type of drug, food, cosmetic, agrochemical and other forms of the drug.

Preparation on the basis of stressful divisive vegetable protein BHS 310 or on the basis of immunochemically related proteins causes fabric adjustable separation of oxidation and phosphorylation in mitochondria. In the restored and improved energy balance of the cell, the organism acquires resistance to adverse factors.

When the drug effect is the amplification of cellular respiration fabric, non-phosphorylation, and as a consequence reduce the formation of reactive oxygen species in mitochondria, reducing lipid peroxidation of biological macromolecules.

Preparations are well penetrate into the tissue through the cell wall and do not have a toxic effect even at a concentration of 10 mg/ml

On the basis of high-purity and/or groupbooking extract stress protein possible the creation of a new line of active forms, including food, pharmaceutical, cosmetic, agrochemical and other Their action is due to a selective effect derived biologically active substances on the energy processes in the cell. They have a positive effect in stress conditions of the human body, animals, plants and do not cause unwanted side effects.

On the basis of the invention can be obtained drugs with varying degrees of impact on living organisms to derive them from stress.

The invention enables to obtain biologically active substances of different degrees of exposure to the regulation of energy metabolism in the body during metabolism.

The method of obtaining biologically active substances is as follows.

Example 1

To extract stress protein BHS 310 three-day seedlings of wheat (Triticum aestivm L.) homogenize for the destruction of cell walls and then centrifuged at a speed of 10000 g, receiving a cell-free extract of seedlings. First carry out the first stage of exposure to the reagent-precipitator order fractionation of the extract of seedlings. To do this, to the obtained extract add reagent precipitator in the form of, for example, ammonium sulfate, bringing the concentration of the solution up to 30% saturation. After 6-hour incubation, the precipitate separated in a centrifuge at 10,000 g with the separation of liquid phase (supernatant. In the second stage of exposure to the supernatant add ammonium sulfate to achieve its concentration in the solution is equal to 50%. After standing for 12 hours the formed substance centrifuged, getting in the sediment robocity extract stress protein BHS 310. The extract obtained is separated from the reagent-precipitator dialysis. All process operations from the destruction of the cell walls of seedlings to the selection of the target product in the form of groupbooking extract stress protein is carried out at temperatures from 0 to +4°C.

Example 2

The selection of the target product in the form of groupbooking extract stress uncoupling protein BHS 310 carried out as follows. Pachystachya seedlings of rye grind until a homogeneous mass for the destruction of the cell walls. The liquid extract is separated from cellular membranes by centrifugation.

First to the floor the Chennai extract sprouts add reagent precipitator in the form of ammonium sulfate, while the concentration of the reagent-precipitator extract seedlings equal to 30%. The deposition reaction in the first stage of exposure to lead 18 hours. The precipitate was separated in a centrifuge so as described in example 1. To separate the liquid phase again add reagent precipitator, bringing the concentration in the solution up to 50%. Then make keeping within 24 hours.

The increase in time-keeping for more than 24 hours is not rational because it does not provide greater deposition of the target product.

Next get robocity extract the target product as in example 1. The obtained extract is freeze-dried.

Example 3

The target product is obtained in the manner described in example 1, using a three-day winter rye seedlings (Secale cereale L.). When carrying out the first stage of deposition, adding reagent-precipitator in the form of ammonium sulfate to the cell-free extract seedling reaction deposition are 12 hours at a concentration of reagent-precipitator equal to 30%. In the second stage of the introduction of the reagent-precipitator to get to the supernatant at a concentration equal to 50%, the deposition reaction are within 15 hours.

Received robocity extract is subjected to fine purification using gel filtration on a column of Sephadex brand G-200, highlighting the highly purified extract of the desired product in the form of immunochemically net stress uncoupling protein BHS 310 or immunochemical related protein.

The authors conducted a series of studies of the impact of stress uncoupling protein BHS 310 on lipid peroxidation and respiratory activity of mitochondria of plants.

The study noted that the use of the selected target product in the form of an aqueous solution for treatment of seedlings of cereals increases cold tolerance of plants.

For example, keeping in aqueous solution of the protein of cereal seedlings increases the amount of stress protein BHS 310 in the tissues of the seedlings. The rate of oxygen consumption increases to 533,39 nmol O2/(min g tissue), which is 40% more compared to the infiltration of water (391,61 nmol O2/(min g wet tissue). The difference of the temperature of the living and the dead seedlings when cooling was 2°C. After infiltration of these seedlings received the active ingredient in this value increases and is 2.5°C.

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

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

The feedback obtained of the target product segregation increases the function of the cells, i.e. its ability to protect against the damaging effects of hostile factors.

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

The present invention opens the possibility of creating a new class of selectively acting substances to another level. The resulting implementation of the invention, the target product can be widely used in various fields related to ensuring optimal performance of the vital functions of living organisms in adverse conditions.

The method provides a selection of substances in quantities sufficient for use as the basis for creating a new line of substances that affect the dynamics of the energy processes for the recovery of the regulation of essential life processes of the organism.

Sources of information

1. The Pastushenkov " L.V., Lesiovskaja E.E. Plants - antihypoxants (phytotherapy) / S.-Petersburg chemical-pharmaceutical Institute. - SPb., 1991. - 96 S.

2. Biochemical studies of natural antioxidant isolated from rosemary and its application in cosmetic dermatology / V. Calabrese 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 cosmetic active igredient / Benaiges A., Armengol R. etc. // International Journal of Cosmetic Science. - 2001. - V.23 supported. - P.243-255.

4. A.V. Kolesnichenko and other Characteristics of the protein from winter rye, accumulate during hypothermia // plant Physiology. - 1996. - T. No. 6. - S-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/ A.V. Kolesnichenko, Grabelnych O.I., Tourchaninova V.V., Zykova V.V., Koroleva N.A., Pobezhimova T.P., Voinikov V.K. //J. Immunoassay Immunochem. - 2003. - V.24. - N 1. - P.41-55.

1. The method of obtaining biologically active substances for the regulation of the energy balance of the body, including the use of cereal seedlings and obtaining the extract, characterized in that the seedlings produce stress uncoupling protein BHS 310 and/or protein, immunochemically related to him in the form of crude and/or purified extract of the target product, for this destroys the cell wall of 3-7 per diem seedlings and produce cell-free extract, which in the first phase impact type reagent precipitator for sediment, followed by separation of the liquid phase, which is subjected to the second stage of exposure to the reagent-precipitator, and then the precipitate was separated from the liquid phase, and the obtained robocity extract the target product is separated from the reagent-precipitator, exposing it for fine cleaning, however, the selection of the extract of the target product is carried out at temperature R is the bench from 0 to +4° With, if necessary, extract the target product is dried.

2. The method according to claim 1, characterized in that the cell wall destroy gomogenizirovannom.

3. The method according to claim 1, characterized in that the reagent-precipitator at the first stage of impact is added to the cell-free extract to a concentration of 30%, maturing within 6-18 hours

4. The method according to claim 1, characterized in that the reagent-precipitator in the second stage effects add to the selected liquid phase to its concentration in the solution is equal to 50%, keeping within 4-24 hours

5. The method according to claim 1, characterized in that the reagent-precipitator using ammonium sulfate.

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 seedlings and further separation of the liquid phase from the precipitate carried out by centrifugation at 10000g and above.

7. The method according to claim 1, characterized in that robocity extract the target product is separated from the reagent-precipitator dialysis.

8. The method according to claim 1, characterized in that the fine purification of the extract of the target product is administered by gel filtration on a column of Sephadex G brands-100 - G-200.

9. The method according to claim 1, characterized in that the extract of the target product freeze-dried.



 

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