RU2587755C1 - Method of producing active substance from maral antlers - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to a method of producing active substance of maral antlers. Method of producing active substance of maral antlers involves extraction of stem cells and their cultivation, as a source of stem cells is maral antlers, which are finely dispersed and treated in collagenase II type, cells are cultured in working medium DMEM, FBS, L-glutamine, penicillin and streptomycin, when culture of 75-80 % of monolayer is changed and cultivated repeatedly, obtained conditioned medium is cleaned and filtered under certain conditions.

EFFECT: method described above enables to efficiently select stem cells of maral antlers, to produce conditioned medium with high concentration of active biomolecules, cleaning and concentrating fraction of active substance.

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Description

The present invention relates to medicine and is a pharmacological composition containing a purified and concentrated fraction of active substances from an air-conditioned environment obtained by cultivation of maral antlers stem cells.

Antlers of the maral are characterized by rapid growth, up to 2 mm per day, have a tubular non-keratinized structure with a network of blood vessels. Red deer antlers grow, getting the maximum amount of nutrients and macro / microelements. From 22 amino acids in antlers contains from 20 to 16, depending on the species. In addition, antlers contain phospholipids, esters of unsaturated fatty acids, macro and micro elements (Fe, Mn, Mg, Co, Zn. Cu, Ca, F, I), a balanced set of fat and water soluble vitamins, regulators of hormones and peptides, normalizing the reactivity of the immune system [Sheina N.E. Renewable bio-raw materials of Yakutia: composition, properties, biotechnological aspects of processing (review). Part 1. Development based on animal raw materials, 2011].

The use of antlers in scientific medical practice in Russia dates back to the beginning of the 18th century. The list of forms of drugs in which antlers are used is quite wide [I.V. Chernova. Antlers and blood in folk medicine of the population of the Sayan-Altai region at the end of the XIX-XX centuries, 2009]. In Russia, 4 drugs are officially approved for therapeutic and prophylactic use: antler of maral alcohol extract “pantocrine”, tablets and aqueous extract in ampoules, “pantohematogen” (based on the blood of maral); from antlers of a reindeer - "Rantarin" in tablets and alcohol extract "Velkornin" [I.N. Vinokurov. Marketing activities promoting the antler products of reindeer husbandry in Russia and PC (Y), 2014]. In addition, preparations from deer antlers are also used as an external agent, in particular antler baths.

In recent years, cellular technologies have been actively developing, some methods have been successfully introduced into clinical practice. The use of antler stem cells in their pure form as a cell product is limited by ethical considerations and legislative prohibitions on the use of xenogenic cells in humans. However, it is possible to use individual growth factors and peptides produced by these cells. The specified pharmacological composition can be used to stimulate and regulate the regeneration of organs and tissues. The composition of the nutrient medium used for stem cell cultivation includes most of the essential amino acids, trace elements, nucleic acids, vitamins and minerals [R.Ya. Freshness. Culture of animal cells. Practical Guide, 2011]. During the cultivation process, the cell culture produces and secretes into the nutrient medium growth factors that function as signaling molecules, providing interactions between cells, due to which cell growth and proliferation are stimulated.

A known method of preserving antlers of maral [Lunitsyn V.G. A method of preserving antlers of maral. Patent No. 2314688, September 14, 2006]. The preservation method includes group cooking of antlers mounted on hangers in water with a temperature of 96-98 ° C at 9-fold immersion, wind drying after each cooking and heat drying at a temperature of 70-80 ° C, while the antlers are wiped after cutting dry clay powder and without sorting them by weight categories make cooking for two days by immersing antlers, mounted on hangers in the position of the butt down, in water with a duration of immersion of 1.5 minutes and cooling between immersions of 3 minutes, while wind and heat drying are carried out the position of the initial consolidation of antlers on veshalah. The disadvantage of this method is the heat treatment of antlers, which leads to the destruction of thermolabile substances, in connection with which of the active substances in the final product there are only macro- and microelements.

Closest to the technical nature of the proposed method is a method of obtaining a composition for the regeneration of skin cells and mucous membranes [Kolesnikova A.I. Composition for the regeneration of skin cells and mucous membranes. Patent No. 2455354, July 10, 2012]. A method of obtaining a composition for the regeneration of skin cells and mucous membranes includes the cultivation of human bone marrow mesenchymal stem cells (KMSC) in a nutrient medium. Cultivation is carried out in an atmosphere of a gas mixture containing 5% carbon dioxide and 95% air, at 37 ° C.

The disadvantage of this method is the low proliferative potential of human KMSC compared with the stem cells of deer antlers, and, as a result, obtaining a conditioned medium takes longer, and the concentration of active substances in it is much lower compared to any antler products. In addition, in addition to the growth factors produced by the cells, the composition contains additives (antibiotic / antimycotic, fetal bovine serum), which can affect the development of adverse events during treatment.

The technical result of the proposed method for the preparation of the active substance from deer antlers is the isolation of stem cells from deer antlers, the production of a conditioned medium with a high concentration of active biomolecules in a short time, the purification and concentration of the active substance fraction.

The technical result is achieved in that for the isolation of stem cells, finely chopped maral antlers are treated in 0.15% type II collagenase for 2 hours at 37 ° C, the cells are cultured in DMEM with 10% FBS, 2 mM L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin. When the culture reaches 75-80% of the monolayer, the medium is changed and cultured for 4 days, the resulting conditioned medium is purified and filtered using the Lab Scale filtration system through 10 and 50 kDa membranes.

Description of the method.

The biomaterial (maral antlers) is taken in a nursery that mimics the natural habitat of animals. Using a disposable scalpel, the biomaterial is divided into equal parts, 2 × 2 cm in size, and placed in sterile tubes with medium with a high content of antibiotics (DMEM with the addition of 2% FBS, 2 mm L-glutamine, 200 units / ml penicillin and 200 μg / ml streptomycin, 200 units / ml amphotericin, 100 units / ml gentamicin). Material is delivered to the laboratory within 6 hours after collection.

Using disposable scalpels, antlers are finely dispersed. The resulting homogenizate is incubated in 0.15% type II collagenase at 37 ° C for 2 hours. The enzyme is inactivated with 10 ml of DMEM medium with the addition of 2% FBS, the homogenizate is precipitated at 300 g for 7 minutes, and the supernatant is removed. Counting and assessment of cell viability is carried out on an automatic cell counter Countess (Invitrogen, USA). After counting, the cells are transferred to a culture vial, at a rate of 5 × 10 ⁵ cells / cm ² , and cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin. The expansion of cells is carried out according to the standard method of culturing KMSC. Cells are cultured up to 2 passages with a change of medium once every three days. Cryopreservation of cell culture is carried out according to standard methods.

To obtain a conditioned medium, the required number of cells is thawed according to a standard procedure, and then passaged onto culture plastic at a rate of 10 × 10 ³ cells / cm ² . The change of environment is carried out when the culture reaches 75-80% of the monolayer. Cells are cultured for 4 days. The conditioned medium is collected in a sterile bottle.

To obtain the active substance, the Lab Scale filtration system (Millipore, USA), Pellicon XL membranes (Millipore, USA) of 10 and 50 kDa are used. Filtration is carried out in accordance with the manufacturer's instructions. After filtration, a composition of the active substance with a size of 10-50 kDa and amino acids, nucleic acids, trace elements, vitamins contained in the growth medium is obtained. The resulting composition is concentrated 2 times.

The application of the proposed method allows to obtain in a short time a stem cell culture of maral antlers using a relatively small amount of biomaterial, and to isolate a pharmacological substance from an air-conditioned medium containing pro-angiogenic, anti-inflammatory, immunomodulating and stimulating cytokine regeneration.

The developed method allows you to create a composition of the active substance for use in order to restore and improve the working capacity of people working or living in ecologically polluted territories, exposed to excessive emotional and mental stress while reducing physical activity, working in conditions of great physical overload, patients with chronic inflammatory diseases ( including with a hidden course), postoperative patients.

Information sources

1. Vinokurov I.N., Alekseev E.D., Mandarov A.E. Marketing activities for the promotion of antler products of reindeer husbandry in Russia and PC (Y). Modern problems of science and education. 2014. No1. S. 382.

2. Chernova I.V. Antlers and blood in folk medicine of the population of the Sayan-Altai region at the end of the XIX-XX centuries. News of Altai State University. 2009. No. 4-3. S. 250-252.

3. Sheina N.E., Shein A.A., Kuzmina V.F., Vedekind V.Α., Kershengolts B.M. Renewable bio-raw materials of Yakutia: composition, properties, biotechnological aspects of processing (review). Part 1. Development based on animal raw materials. 2011. No4. S. 59-64.

4. Freshni R.Ya. Culture of animal cells. Practical Guide, 2011.

5. Lunitsyn V.G. A method of preserving antlers of maral. Patent number 2314688, September 14, 2006.

6. Kolesnikova A.I. Composition for the regeneration of skin cells and mucous membranes. Patent number 2455354, July 10, 2012.

Claims (1)

A method of obtaining the active substance from maral antlers, including the isolation of stem cells and their cultivation, characterized in that maral antlers are used as a source of stem cells, which are finely dispersed and treated in 0.15% type II collagenase for 2 hours at 37 ° C , the cells are cultured in DMEM working medium, 10% FBS, 2 mM L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin, when the culture reaches 75-80% monolayer, the medium is changed and cultured for 4 days, obtained conditioned medium clean and fi truyut using Lab Scale filtration system through membranes 10 and 50 kDa.