RU2540487C1 - Product based on alga laminaria angustata for improvement of regeneration and proliferation of cells, method of its obtaining and application - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method of obtaining product for improvement of regeneration and/or proliferation of cells by processing alga Laminaria Angustata, water-isobutanol extract of alga Laminaria Angustata is obtained, supernatant is taken, centrifuged, supernatant is re-taken and subjected to vacuum drying, after which obtained powder is diluted with water and obtained solution is re-centrifuged with further re-taking of supernatant, which is subjected to ultrafiltration, with separation of fraction with molecular weight 5-10 kDa from ultrafiltrate. Product for improvement of regeneration and/or proliferation of cells. Medication for improvement of regeneration and/or proliferation of cells.

EFFECT: improvement of regeneration and proliferation of cells.

4 cl, 4 tbl

Description

The invention relates to the field of pharmaceuticals and medicine and relates to a product based on Laminaria Angustata algae for improving cell regeneration and proliferation, a method for its preparation and use as a part of a medicinal product for restoring and improving the state of an organism in complex therapy of immunodeficiency states of various origins, including the state of an organism after chemotherapy with oncological diseases.

State of the art

Algae preparations are widely used for clinical nutrition as a source of trace elements, iodine and biologically active substances, which have a general strengthening effect, increase working capacity, regulate immunity and have other beneficial effects. It is also noted that in areas where the diet includes a lot of seafood, including algae, higher life expectancy.

The positive effect of the use of algae and preparations based on them on the human body is observed in immunodeficiency states, oncological diseases, and aging. Numerous studies have been aimed at obtaining biologically active preparations from algae (Bespalov V.G., 2005, Bespalov V.G., Nekrasova V.B., 2000).

It is known that obtaining a number of preparations from algae is associated with their heat treatment in the presence of aggressive acids (patent RU No. 2030885 C1, 03.20.1995). Such treatment destroys many biologically active compounds and leads to the accumulation of relatively inert polysaccharides and agarose-related compounds.

A known method of processing algae Laminaria Angustata to obtain the food product "Lamifaren" with a maximum content of free alginate in the form of calcium and sodium salts (36%) and macro- and microelements in the most bioavailable form for the body (Patent RU No. 2230464, 20.06. 2004). Lamifaren food product is used as a source of macro- and microelements and as a sorbent. The method of obtaining "Lamifarene" includes the preparation of algae, processing them in an acidic environment at pH 6 with a ratio of the mass of algae and a solution of 1: 1.5 and keeping for 5-6 hours, then the mass of algae is washed 4 times with fresh water in temperature 20 ° C for 10-30 minutes with a weight ratio of algae and water of 1: 3. Then water is drained, treated with dehydrated water vapor at a water temperature of 40-60 ° C at a weight ratio of algae to water of 1: 1 for 16-24 hours with continuous stirring, after which the resulting algae mass is homogenized to obtain a finely dispersed gel mass. The use of an acidic environment in the preparation of the food product "Lamifaren" also leads to the destruction of biologically active compounds and the multicomponent composition.

There is evidence that many proteins in algae are firmly bound to the polysaccharides of the cell walls and they do not pass into solution during aqueous extraction, but remain in the cell pellets.

It is known that the effectiveness of the treatment of various immunodeficiency conditions, antitumor chemotherapy and the prognosis of the disease largely depends on the state of the body and the ongoing maintenance therapy. Recently, the practice of the use of biologically active additives (BAA) and preparations based on natural biologically active compounds has become widespread as an accompanying therapy in the prevention and treatment of immunodeficiency states and oncological diseases. However, the lack of scientifically based criteria for the use of such drugs, the inability to clearly determine the necessary doses of the drugs used due to their multicomponent nature, leads to the empirical prescription of such drugs, sometimes with the opposite result of use, which is associated with the occurrence of side effects due to their multicomponent nature.

The use of biologically active drugs with a narrow component, established by biochemical and / or chemical methods, would increase the effectiveness of the treatment of a wide range of diseases, including accompanying therapy for cancer.

Thus, the objective of the present invention is to obtain a biologically active product from the alga Laminaria Angustata, which improves cell regeneration and proliferation and does not cause side effects due to the multicomponent composition.

SUMMARY OF THE INVENTION

The present invention relates to a biologically active product for improving cell regeneration and proliferation, which is a fraction with a molecular weight of 5-10 kDa, subjected to ultrafiltration of an aqueous-isobutanol extract of algae Laminaria Angustata.

The present invention also relates to a method for producing said product by preparing a water-isobutanol extract of Laminaria Angustata algae, which is subjected to ultrafiltration, followed by selection of a fraction with a molecular weight of 5-10 kDa.

In addition, the present invention relates to a medicament for improving cell regeneration and / or proliferation, comprising said product and pharmaceutically acceptable additives.

The specified drug can be used to restore and improve the condition of the body in the complex therapy of immunodeficiency states of various origins, including the condition of the body after chemotherapy for cancer.

DETAILED DESCRIPTION OF THE INVENTION

The biologically active product according to the present invention is obtained under conditions ensuring the conservation of the biological activity of the extracted substances in the following way. The dried and granular preparation of algae Laminaria Angustata is mixed with isobutanol, incubated, then water is added, the resulting mixture is heated, preferably to 55-65 ° C, and incubated for one day. Then the supernatant is taken, centrifuged, the supernatant is taken again and subjected to vacuum drying, after which the obtained powder is diluted with water and the resulting solution is again centrifuged, followed by ultrafiltration, taking a fraction with a molecular weight of 5-10 kDa from the ultrafiltrate.

A medicament for improving cell regeneration and / or proliferation is obtained by combining the product of the present invention in an amount and pharmaceutically acceptable additives effective for cell regeneration and / or proliferation. Depending on the choice of pharmaceutically acceptable additives, the drug can be in the form of suppositories for rectal administration, tablets or capsules for oral administration, solutions for intravenous and intramuscular injections. Preferably, the content of the product of the present invention in a unit dosage form is 0.1-10 mg. As acceptable additives for the preparation of suppositories, solid fat is usually used; to obtain tablets, lactose monohydrate, potato starch, calcium stearate are usually used as auxiliary substances. When using the product in capsules, adjuvants are usually added to it, such as talc, magnesium stearate, silicon oxide, capsules usually include gelatin, titanium dioxide, preservatives such as sodium lauryl sulfate, methyl parahydroxybenzoate, and various food colors can also be used. For intravenous and intramuscular injections, the product is diluted in saline. Treatment regimens (regimen and dose of drug administration) are chosen by specialists based on the severity of the disease, the condition and age of the patient and are specified during use.

The obtained drug can be used to improve the regeneration and / or proliferation of cells, in particular, to restore and improve the state of the body in the complex therapy of immunodeficiency states of various origins, including the state of the body after chemotherapy for cancer.

Production Example

Laminaria Angustata kelp powder (1 part, 100 g) is added to isobutanol (2.5 parts, 250 ml), mixed until homogeneous (a homogeneous dark green mixture is obtained), the mixture is incubated for 10 minutes at a temperature of 18-25 ° C, after which 250 ml of distilled water are added to the mixture, and all contents are thoroughly mixed until a homogeneous solution is obtained. The resulting mixture is heated in a water bath or in a thermostat for 1 hour at a temperature of 55-65 ° C, after which it is incubated for 22-28 hours at a temperature of 18-25 ° C, then the supernatant is collected and centrifuged for 30 minutes at 3000 rpm at a temperature of 2-8 ° C, then again select the supernatant, which is placed in a rotary evaporator and purged with argon. Vacuum drying in a rotary evaporator is carried out for 1 hour at a temperature of 18-25 ° C, gradually lowering the pressure, after which the temperature in the rotary evaporator is increased to 55-65 ° C and the product is left at this temperature until it is completely dry. The resulting powder is diluted in distilled water to obtain a 3% solution, followed by centrifugation at 5000 rpm, and then at 3000 rpm at 4-8 ° C and selection of the supernatant. The resulting solution was subjected to ultrafiltration and a fraction with a molecular weight of 5-10 KDa was selected.

The resulting product is a solution having a faint light yellow hue. Its composition, established using chromatography and filtration methods, includes low molecular weight proteins, polypeptides and individual peptide fragments.

To study the biological activity of the product, sodium chloride is added to the resulting solution to a final concentration of 0.09% or it is mixed with saline in a ratio of 1: 1.

Examples of the study of the biological activity of the obtained product

Study of the effect of the obtained product on cell proliferation

We used H-9 cells, which were placed in a 24-well culture plate at a concentration of 250 × 103 / ml at 1.0 ml per well. The resulting solution of the test product was added to the wells at a concentration of 10 μl / ml. An appropriate amount of culture medium was added to the control wells, which included RPMI-1640 containing 10% fetal calf serum, glutamine (146 mg per 500 ml) and gentamicin (20 mg per 500 ml).

Cells with or without added product were incubated for 24 hours in a CO ₂ incubator at 37 ° C. At the end of the incubation, the number and percentage of dead cells were counted. The results are presented in table 1.

Table 1
Study of the effect of the obtained product on cell proliferation A drug Results after 24 hr incubation Number of cells per well, mln / ml % dead cells The control 0.25 4.3 Product received 0.30 3.0

As can be seen from table 1, the resulting product at a concentration of 10 μl / ml causes cell proliferation.

It was found that when the resulting product is stored at -80 ° C for 1 year, its ability to cause cell proliferation is fully preserved.

In contrast to the product according to the present invention, similar studies conducted with the well-known drug "Lamifaren" showed that this drug does not have the ability to cause cell proliferation.

Study of the effect of the obtained product on bone marrow mesenchymal stem cells

The effect of the obtained product on donor bone marrow mesenchymal stem cells (MSCs) was studied. Material was collected from donors in sterile bags with an anticoagulant. Isolation of the mononuclear fraction was carried out on ficoll. The cells obtained after isolation were divided into 3 parts: 1. control (cultivation of bone marrow cells without the studied product, 2. cultivation of bone marrow cells together with the studied product used at a dose of 10 μl / ml, 3. cultivation of bone marrow cells together with the studied substance (preparation) used at a dose of 50 μl / ml.

The cultivation of bone marrow cells was carried out in sterile vials at a temperature of 37 ° C under conditions of absolute humidity and 5% CO ₂ in air. To do this, 80-100 million bone marrow mononuclear cells in 30 ml of complete culture medium were placed in a culture bottle with a bottom area of 175 mm. After a certain time, the vial was washed from non-adherent cells. Passaging of stromal precursors was performed 1 time per week.

During the study, the resulting product was used at a concentration of 10 and 50 μl / ml. The product was introduced into the culture at the initiation, as well as at each passage during the whole time of cell cultivation. The culture was passaged on day 14 from the start of cultivation. Cultivation results were evaluated by counting the number of MSCs collected from passage.

EC (cloning efficiency) of the primary culture was determined on day 14 by the number of stromal precursor colonies per 10 ⁵ explanted cells. The growth dynamics of the cell population was determined by the rate of cell growth as the ratio of the number of cells obtained from this passage to the number of cells planted in the previous passage.

The results of studies evaluating the effect of the obtained product on proliferation and the percentage of early umbilical cord blood precursors during prolonged cultivation are presented in table 2.

table 2
The effect of the obtained product on proliferation and the percentage of early umbilical cord blood precursors during prolonged cultivation EC Ok at the end of the cultivation phase 14 day The control 15.6 2.1 × 106 Substance, 10 μl 21,4 9.6 × 106 Substance, 50 μl 25.6 13.2 × 106 EC - cloning efficiency
OK - the total number of cells in the group

The results presented in the table show that the introduction of the obtained product into the cell culture has a stimulating effect on the proliferation of stromal precursors. The studied product increases the number of MSC precursors in primary culture.

The study of colony-stimulating activity of the obtained product

When studying the effect of the obtained product on the proliferation and percentage of early umbilical cord blood precursors, the following cellular elements were used as target cells: stem cells of cord blood of a full-term newborn with a well-known basic colony-forming activity isolated on ficoll.

In an in vitro study, the resulting product was used at an optimal concentration of 10 μl / ml.

The study of the effect of the obtained product on long-term cultures of hematopoietic precursors was performed using hematopoietic stem cell expansion (HSC) kits from CellGenix. Cultivation in this system is always carried out with a standard set of colony-stimulating factors, the action of the studied funds is implemented against their background.

The studied product was introduced into the culture upon initiation (day 0).

The culture was removed on day 0, then from bag 1 on day 14, 28, from bag 2 on day 42 and 56 and from bag 3 on day 70 and 84 from the start of cultivation. Colonies were counted under an inverted microscope (40x) and CD34 + cell counts were evaluated on a FACSCalibur ™ flow cytometer, manufactured by Becton Dickinson (USA).

The study of the effect of the obtained product on hematopoietic stem cells was carried out in a culture system - methyl cellulose. Cultivation in methyl cellulose makes it possible to study the nature of the effect of the obtained product on the proliferation of the entire spectrum of hematopoietic precursors and the direction of differentiation: early, giving mixed colonies (CFU mix), intermediate - granulocyte / macrophage colonies (CFU g / m) and late - granulocyte, macrophage and erythroid colonies (CFU g, CFU m and CFU er). Cultivation in this system is always carried out with a standard set of colony-stimulating factors, the action of the studied substances is implemented against their background.

The culture was removed on day 14. Colony counting was performed under an invert microscope (40x).

The evaluation of the cultivation results was carried out with the calculation of the number of colony forming precursors of each species per 10 ⁵ explanted cells. The following indicators were analyzed:

1. CFU mix - mixed colonies (early predecessors);

2. CFU er - erythroid precursors;

3. CFU um - granulocyte-macrophage precursors;

4. CFU g - granulocytic precursors;

5. CFU m - macrophage precursors.

EC (cloning efficiency) was determined by the sum of colony forming precursors of all types per 10 ⁵ explanted cells.

The results of evaluating the effect of the obtained product on proliferation and the percentage of early umbilical cord blood precursors during prolonged cultivation are presented in table 3.

OK - total cellularity (number of nuclear cells);

C - mixed colonies;

G - granulocytic colonies;

M - macrophage colonies;

G / M - granulocyte-macrophage colonies;

E - erythroid colonies;

EC - cloning efficiency.

Reliability of differences from control (by student criterion): * p <0.05

As can be seen from the data presented in table 3, with prolonged contact (for 14, 28, 42 and 56 days of constant exposure), the resulting product at a concentration of 10 μl / ml has a statistically significant significant stimulating effect on the proliferation of early hematopoiesis precursors (to the greatest extent CD34 + and CD133 +), CFU mix. and CFU g / m. The degree of expansion of hematopoietic cells (total cellularity of the culture) is significantly higher when the product obtained is added to the culture medium than in the control.

The resulting product provides a delayed natural loss of surviving cells compared with the control (56 days). With prolonged cultivation, the content of stem cells and their proliferative potential decrease, however, under the action of the obtained product, delayed degradation of the culture is observed (70 and 84 days), while in the control this effect is observed much earlier (after 56 days).

The study of hemostatic activity of the obtained product

To study the haemostimulating activity of the obtained product, the umbilical cord blood stem cells of a full-term newborn with well-known basic colony-forming activity isolated on ficoll were used as target cells. In the study, the resulting product was used at a concentration of 10 μl / ml. The study of the effect of the obtained product on long-term cultures of hematopoietic progenitor cells was performed using hematopoietic stem cell expansion (HSC) kits from CellGenix. Cultivation in this system is always carried out with a standard set of colony-stimulating factors, the action of the studied substances is implemented against their background.

The resulting product was introduced into the culture at initiation (day 0) and again on 84 day. The culture was removed on day 14 from the start of cultivation. Colonies were counted under an inverted microscope (40x), and the content of CD34 + cells was estimated on a FACSCaliburTM flow cytometer, manufactured by Becton Dickinson (USA).

The effect of the obtained product on hematopoietic stem cells was studied in the culture system: methyl cellulose, which makes it possible to study the nature of the effect of the obtained product on the proliferation of the entire spectrum of hematopoietic precursors and to determine the differentiation directions: early, giving mixed colonies (CFU mix), intermediate - granulocyte / macrophage colonies (CFU g / m) and later - granulocytic, macrophage and erythroid colonies (CFU g, CFU m and CFU er). Cultivation in this system is always carried out with a standard set of colony-stimulating factors, the action of the studied substances is implemented against their background.

The removal of cultures was carried out on day 14. Colony counting was performed under an invert microscope (40x).

The evaluation of the cultivation results was carried out with the calculation of the number of colony forming precursors of each species per 10 ⁵ explanted cells. The following indicators were analyzed:

CFU mix - mixed colonies (early predecessors);

CFU er - erythroid precursors;

CFU um - granulocyte-macrophage precursors;

CFU g - granulocytic precursors;

CFU m - macrophage precursors.

EC (cloning efficiency) was determined by the sum of colony forming precursors of all types per 10 ⁵ explanted cells.

The effect of the obtained product on the viability of hematopoietic precursors during thawing of the culture after prolonged cultivation in bags was also studied.

The results of a study of the effect of the obtained product on proliferation and the percentage of early umbilical cord blood precursors during prolonged cultivation are presented in table 4.

Table 4
The effect of the obtained product on proliferation and the percentage of early umbilical cord blood precursors during prolonged cultivation

OK - total cellularity (number of nuclear cells) of the colony;

C - mixed colonies;

G - granulocytic colonies;

M - macrophage colonies;

G / M - granulocyte-macrophage colonies;

E - erythroid colonies;

EC - cloning efficiency.

As can be seen from the data presented in table 4, the repeated introduction of the obtained product into the culture does not have an additional stimulating effect on the proliferation of hematopoiesis precursors.

Adding the resulting product to thawed culture leads to better preservation of cells in the culture compared to the control.

Thus, as a result of the studies, data were obtained on the expressed ability of the obtained product to improve the regeneration and proliferation of cells, especially the early precursors of hematopoiesis.

It was established that the resulting product at a concentration of 10 μl / ml causes cell proliferation in culture.

When storing the obtained product at a temperature of -80 ° C for 1 year, its ability to cause cell proliferation in the culture is fully preserved.

The introduction of the resulting product into cell culture has a stimulating effect on the proliferation of stromal precursors. Under its influence, the number of precursors of bone marrow mesenchymal stem cells in primary culture increases.

With prolonged contact with the cell culture (for 14, 28, 42 and 56 days of constant exposure), the resulting product even at a concentration of 10 μl / ml has a pronounced stimulating effect on the proliferation of early hematopoiesis precursors. Moreover, under the action of the obtained product, the overall cellularity of the cultures increases.

The resulting product provides delayed natural loss of surviving cells. Under the action of the obtained product, a significant slowdown in the degradation of cell culture is observed. The data obtained are of fundamental importance for the preparation of cell preparations for their further use for such purposes as slowing down aging and, possibly, rejuvenation; they indicate the fundamental possibility of using this product to restore and improve the state of the body, which is extremely important in the treatment of immunodeficiency states of various origins, including the state of the body of cancer patients after chemotherapy.

Repeated introduction of the obtained product into the culture does not have an additional stimulating effect on the proliferation of hematopoietic precursors, however, the addition of the obtained product to a thawed culture leads to an improvement in the preservation of cells in this culture. This effect seems very important when using the obtained product for the preparation of cell preparations for the purposes of further therapeutic use.

Claims (4)

1. A method of obtaining a product to improve cell regeneration and / or proliferation by processing the Laminaria Angustata algae, characterized in that under conditions ensuring the biological activity of the extracted substances is preserved, an isobutanol extract of the Laminaria Angustata algae is obtained, then the supernatant is selected, centrifuged, the supernatant is re-selected and subjected to vacuum drying, after which the resulting powder is diluted with water and the resulting solution is again centrifuged, followed by selection with upernatant, which is subjected to ultrafiltration, selecting from the ultrafiltrate fraction with a molecular weight of 5-10 kDa. 2. Product for improving the regeneration and / or proliferation of cells obtained by the method according to claim 1. 3. A medicament for improving cell regeneration and / or proliferation, comprising the product of claim 2 and pharmaceutically acceptable additives. 4. The drug according to claim 3, designed to restore and improve the condition of the body in the complex therapy of immunodeficiency states of various origins, including the condition of the body after chemotherapy of cancer.