RU2755312C1 - Biologically active dietary supplement from holothuria and method for its preparation - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: inventions relate to the food industry, namely, to a method for obtaining a biologically active dietary supplement (dietary supplement) from Far Eastern commercial holothurias, which can be used to activate the immune system and mental and physical performance. The method for obtaining dietary supplements from holothuria includes grinding of raw materials and their double extraction with ethyl alcohol, separation of the alcohol extract. At the same time, holothurians are used completely, the extraction of crushed raw materials with ethyl alcohol is carried out at a ratio of 1:1-1:1.3 kg/l, at a temperature of 30°-45°C for 5-6 hours, the residue after extraction with ethyl alcohol is squeezed out and re-extracted with ethyl alcohol under the same conditions. The resulting water-alcohol filtrates are combined and filtered. Ethyl alcohol is distilled from the combined filtrate under vacuum to its concentration of no more than 10%. Next, the resulting water-alcohol extract containing triterpene glycosides is used to prepare a gelatin mass, from which the capsule shell is formed, and fish oil is injected into the capsules. Dietary supplements from holothuria, which has the form of gelatin capsules, contains 0.015-0.028 mg of triterpene glycosides in the gelatin shell of the capsule and 0.3 g of fish oil inside the capsule.

EFFECT: invention makes it possible to expand the range of dietary supplements obtained by combining triterpene glycosides from holothuria and omega-3 polyunsaturated fatty acids from fish oil.

4 cl, 4 dwg, 2 tbl

Description

The inventions relate to the food industry, namely to methods of obtaining biologically active products from the Far Eastern commercial sea cucumbers, which can be used as a fortifying, biologically active additive (BAA), designed to activate the immune system and mental and physical performance.

The resulting preparation based on omega-3 polyunsaturated fatty acids and triterpene marine glycosides represents a new important step in the development of biologically active natural complexes with enhanced pharmacological action.

The main active biologically active substances of sea cucumbers are triterpene glycosides, which have prophylactic and therapeutic properties on the human body, help restore the functioning of vital organs, including the functions of the endocrine glands, activate the immune system, creating the prerequisites for inhibiting the growth of malignant neoplasms, and contribute to the restoration of the reproductive system of the body ( Bordbar S., Anwar F., Saari N. High-Value Components and Bioactives from Sea Cucumbers for Functional Foods - A Review // Mar. Drugs. 2011. Vol. 9, P. 1761-1805; doi: 10.3390 / md9101761) ...

Polyunsaturated fatty acids (PUFAs) of the omega-3 family have long been known as a means of preventing and reducing the risk of cardiovascular diseases and as a means of affecting the overall reduction of inflammatory processes in the body. And with the latest views on omega-3 acids, they are classified as drugs that significantly increase the body's resistance to Covid-19 (Hathaway D. "Omega 3 Fatty Acids and COVID-19: A Comprehensive Review" // Infect Chemother. 2020. V. 52 (4): 478-495, (https://doi.org/10.3947/ic.2020.52.4.478).

A known method of obtaining a product [RF Patent No. 2562595, IPC A61K 35/56, A61P 37/02, publ. 09/10/2015], which has biologically active properties from sea cucumbers, which consists in freeing sea cucumbers from internal organs, washing the skin-muscular sac, grinding it to a homogeneous mass, isolating biologically active components from it, which is carried out in two stages, at the first stage crushed The musculoskeletal mass is mixed with water, then sonicated, then alkaline hydrolysis is carried out, then neutralized to pH = 7.0, the resulting hydrolyzate is dried, the dry product is ground to a powdery state, and at the second stage, the powder is mixed with sunflower or rapeseed oil and kept under constant stirring, the resulting fat suspension is stabilized with a dispersant. The above method allows you to obtain a biologically active drug with chondroprotective and immunomodulatory properties.

Disadvantages:

1. The method provides for obtaining from the hydrolyzate of sea cucumbers a complex preparation containing many biological components - polyunsaturated fatty acids, proteins, hexosamines, triterpene glycosides. Moreover, triterpene glycosides, in this case, are not the most important component in the opinion of the authors.

2. The implementation of the method is multistage, includes the use of solutions of alkali and acid, which is undesirable for food production.

3. The authors in the manufacture of the drug use the skin-muscular sac, neglecting the viscera.

4. The best way to quantitatively recover triterpene glycosides from the cellular matrix is to treat the tissue with an organic solvent such as ethanol. Other methods of extracting these substances are not as effective.

5. The authors offer an opaque emulsion with color characteristics. The use of this preparation is impossible to obtain high-quality soft gelatin capsules, since the impurities present will not ensure the transparency of the gelatin capsule and make it unattractive to the consumer.

6. The final form of dietary supplements is presented in the form of an emulsion in vegetable oil (sunflower). The argument that the PUFAs of sunflower oil are very important are not justified.

7. The inventors do not even consider the possibility of a synergistic effect of the possibility of using fish oil as a source of omega-3 polyunsaturated fatty acids and marine triterpene glycosides.

A known method of complex processing of sea cucumbers [RF Patent No. 2236155, IPC A23L 1/30, A23L 1/305, A23L 1/333, Publ. 20.09.2004], in which the raw material (trepang or cucumaria) is cut, the entrails, corollas and intra-abdominal film are removed and boiled in sea, fresh or salted water. Drying of thermally processed cucumaria is carried out at a temperature of 10 to 65 ° C to a water content in the product equal to 5-12%, minerals 4-8% to obtain a semi-finished product for the production of a food product. The insides of the sea cucumbers are drained, lightly pressed, frozen, chopped and dried. The resulting feed biologically active additive is used as a means of increasing the resistance of animals to infectious diseases, in the form of an additive to feed. The cooking media are collected and dried to a water content of 5-12% to obtain a biologically active food supplement "Akmar". The resulting product is used as an adaptogenic agent in the form of powder, tablets, and also in encapsulated form. The biologically active additive "Akmar" is a gray powder with various shades of brown, obtained from dried waters during the complex processing of holothurians, and consists of protein - 32.0-42.0%, water - 5.0-12.0%, mineral substances - 50.0-55.0% (the predominant elements are calcium, potassium, sodium, magnesium), lipids - 0.05-0.2%, the amount of triterpene glycosides is in the range of 5000-7000 μg / g, amino sugars - 0.5-2.0%.

Disadvantages:

1. The implementation of the method does not provide a complete extraction of triterpene glycosides from tissues.

2. The use of residues (concentrates) from the processing of sea cucumbers for the benefit of animal nutrition, of course, is acceptable, but it is just an attempt to hide the impossibility of obtaining real concentrates of triterpene glycosides for human consumption.

3. The processing technology is not deep and only involves the use of holothurians in various directions - both for humans (preferably) and for animals.

4. The content of lipids in the preparation (BAA) - 0.1% - cannot be the basis for recommending the preparation as a source of omega-3 polyunsaturated fatty acids.

A known method of obtaining dry hydrolyzate from sea cucumbers [RF Patent No. 2538393, MCP A23L 1/30, A23L 1/305, A23L 1/33, A23J 1/04, publ. 10.01.2015, bul. No. 1], including the stages of hydrolysis of the prepared raw material with an enzyme preparation for at least 2 hours, drying the hydrolyzate and grinding the dry hydrolyzate into powder. What is new is that holothurian carcasses, peeled from entrails and crushed, are used as prepared raw materials; at the stage of hydrolysis, a proteolytic enzyme preparation isolated from marine aquatic organisms and having a proteolytic activity of 3.5-15 PE / g is used as an enzyme preparation, with a ratio of the enzyme preparation to the prepared raw material by weight of 1: 50-1: 200, while hydrolysis is carried out within 2-8 hours at a temperature of 18-35 ° C; and at the stage of drying, freeze drying is used until the moisture content of the dry hydrolyzate is more than 12% wt. Dry hydrolyzate in the amount of 0.14-0.50 g is packed in gelatin capsules.

Disadvantages:

1. Raw materials of sea cucumbers are used without further fractionation according to the classes of biological substances, thereby definitely narrowing the field of application.

2. Use of holothurian carcasses, without viscera, when receiving the preparation.

3. The proteolytic enzyme "Krusenzyme" is known to be obtained from the hepatopancreas of the king crab. This enzyme is very resistant to temperature effects - up to + 70 ° C and above. Enzyme inactivation carried out according to this method - 60-65 ° C - does not guarantee the end of the hydrolysis process in the final product.

4. The use in the freeze drying process unambiguously increases the cost of products, because it is a costly energy and time process.

5. The final product obviously contains a small amount of triterpene glycosides, and also cannot be a full-fledged source of omega-3 polyunsaturated fatty acids.

A known method of obtaining a biologically active product from the commercial sea cucumber Cucumaria japonica [RF Patent No. 2426453, IPC A23L 1/30, A23L 1/325, A61K 35/56, A61P 1/16, publ. 08/20/2011], which consists in freeing the sea cucumber from internal organs, crushing the musculocutaneous sac and extracting the homogenate with 96% ethyl alcohol at a ratio of 1: 2 and heating for 5-6 hours. The extract is separated by decantation, the residue is re-extracted with the same portion of alcohol under the same conditions. The combined aqueous-alcoholic extract is used to obtain the Kumazid product, as described in RF patent No. 2271820. The biological material remaining after extraction is dried in air at room temperature until the smell of alcohol is absent, and then evacuated to the maximum alcohol concentration in food products, but not more than 0.5% of the weight of the product obtained (according to GLC data). A gray solid material is obtained, which is subjected to mechanical grinding on a ball-type crusher for 3-5 hours and sieved through a sieve to a finished product particle size of no more than 0.08 mm. The resulting product has the name "Kukumariovit".

Disadvantages of the resulting product:

1. The drug is a protein concentrate (70-75%), carbohydrates (4.5-4.9%), minerals, etc.

2. Despite the fact that the preparation is prepared from sea cucumbers, the authors do not even indicate the residual amounts of triterpene glycosides, although their presence, even residual, should be indicated.

3. According to the technochemical composition, there are no lipids in the preparation. Therefore, this product is not a source of marine omega-3 fatty acids.

The closest technical solution [RF Patent No. 2271820, IPC A61K 35/56, A61P 37/02, publ. 20.03.2006, bul. No. 8] to the claimed technical solution in terms of the number of essential features is a method of obtaining a biologically active additive, in which the raw material - gutted cucumaria Cucumaria japonica is crushed (example 1) and extracted with ethyl alcohol (ratio 1: 2, kg / l) when heated for 4 hours, the extract is separated, the residue is re-extracted with alcohol under the same conditions, the combined extract is centrifuged, the supernatant is separated, extracted with butanol, a solution of cholesterol in water-saturated butanol is added to the upper phase, the solution is concentrated, centrifuged, the precipitate is washed three times with organic solvents (alcohol , ether, etc.), centrifuged, washed with diethyl ether, dried.

Disadvantages:

1. The method makes it possible to effectively concentrate triterpene glycosides from sea cucumbers, however, the technology of obtaining is multistage, laborious and cannot be the basis for widespread production of the drug.

2. Unfortunately, the authors cannot refuse to use diethyl ether in the process. This sharply limits the possibilities of placing technology in pharmaceutical production, not to mention food production.

3. The approach to the concentration of triterpene glycosides in the form of their complex with cholesterol (molar ratio 1: 2) is not a good formula for food use, since cholesterol is still considered as a risk factor for the cardiovascular system

4. The product according to the method is practically an individual substance that does not contain lipids, including omega-3 polyunsaturated fatty acids.

5. The use of this product for the production of dietary supplements with omega-3 fatty acids is impossible, since this complex is completely insoluble either in fat or in an aqueous medium and will only cause a negative attitude of the consumer towards its external, unattractive data.

The technical problem solved by the invention is the expansion of the range of dietary supplements (biologically active additives) with high consumer characteristics, due to the production of a drug that combines the presence of marine triterpene glycosides in the form of a shell of soft gelatin capsules containing fish oil inside it, as a source of polyunsaturated fatty acids. omega-3 acids (PUFA).

The technical problem posed is solved by the fact that in the known method of obtaining a biologically active supplement from sea cucumbers, including grinding raw materials, its double extraction with ethyl alcohol, separation of the alcohol extract, according to the invention, both the muscular sac and the insides of the sea cucumbers are used as raw materials, extraction with ethyl alcohol crushed raw materials are carried out at a ratio of 1: 1-1: 1.3 kg / l, at a temperature of 30 ° -45 ° C for 5-6 hours. The residue after extraction with ethyl alcohol is squeezed out and re-extracted with ethyl alcohol under the same conditions, the resulting aqueous-alcoholic extracts are combined and filtered, ethyl alcohol is distilled off from the resulting combined filtrate under vacuum until its residual concentration in the product is not more than 10%; then the obtained aqueous-alcoholic extract containing triterpene glycosides is used to prepare a gelatinous mass with further encapsulation of fish oil into it.

Grinding the sea cucumber is imperative as it facilitates the glycoside extraction process, mainly saving time. Grinding can be carried out both on an industrial grinder and on a meat grinder used in everyday life. This is enough to keep the process going intensively.

The ratio of components in the extraction of holothurian-alcohol is in the range of 1/1 –1 / 1.3 kg / l. This proportion allows you to extract the maximum amount of glycosides without consuming unnecessary amounts of alcohol. A decrease in the volume of alcohol less than the declared norm does not provide the required extraction efficiency, and its increase is slightly reflected in the extraction of glycosides.

Carrying out repeated extraction of the squeezed sediment with alcohol (after the first extraction) makes it possible to extract residual amounts of triterpene glycosides from the tissues of sea cucumbers, which, in the end result, makes it possible to obtain a dietary supplement with high consumer properties.

The process of extraction of crushed sea cucumbers with alcohol must be carried out at a temperature of 30 ° C to 45 ° C. The increased (above room) temperature of the extraction accelerates the extraction of triterpene glycosides from the cellular matrix and thereby narrows the duration of the extraction process (up to 5-6 hours).

If the temperature is reduced to less than 30 ° C, then the extraction process may increase not even by hours, but by a day, and, conversely, an increase in temperature above 45 ° C does not give advantages for the implementation of the process - the temperature causes negative chemical reactions in the biological environment, and most importantly, does not increase the accumulation of glycosides in the extract and reduce the holding time of the mixture.

In combination with the extraction temperature, the optimal process time was experimentally found - 5-6 hours, which ensures the production of dietary supplements with good consumer properties. Deviation from the obtained parameters did not ensure the efficiency of the process. Carrying out the extraction process for less than 5 hours led to a decrease in the extraction of glycosides from the raw material, and carrying out the process for more than 6 hours did not lead to an increase in the saturation of the extract with glycosides, but only to unnecessarily spent time.

The distillation of alcohol to a residual concentration of no more than 10% ensures its complete removal in the future and is not contained in the final product, which has a positive effect on its consumer characteristics.

The use of the obtained aqueous-alcoholic extract of sea cucumbers containing triterpene glycosides for the preparation of a gelatinous mass ensures the inclusion of triterpene glycosides in the capsule shell, which not only increases the nutritional value of the target product, but also provides it with good organoleptic qualities, allowing to obtain the final product of an extremely transparent consistency and containing two different types of biologically active substances - triterpene glycosides and omega-3-series polyunsaturated fatty acids.

It is advisable to prepare a gelatinous mass for the manufacture of capsules by mixing the obtained extract containing triterpene glycosides with gelatin, glycerol, sorbic acid and water at a temperature of 60 ° C and stirring for 4 hours, with the following ratio of components:

hydroalcoholic extract of sea cucumbers 300 - 800 g gelatin 850 - 1650 g glycerol 300 - 550 g sorbic acid 5.0 g water up to 5000 l

The introduction of sorbic acid into the gelatinous mass allows not only to lengthen the shelf life of the obtained dietary supplement, but also prevents the growth and activity of fungi and yeast. The use of the components in the stated ranges ensures further obtaining of gelatin capsules of a marketable form, transparent and durable.

The raw material can be various species of sea cucumber, in particular, Apostichopus japonicus, Apostichopus parvimensis, Apostichopus californicus, as well as cucumaria, in particular Cucumaria japonica, Cucumaria glacialis, Cucumaria fraudatrix, Cucumaria calcigera.

It is advisable to use the most widespread commercial sea cucumbers living in the Far Eastern seas as a raw material - sea cucumber Apostichopus japonicus or cucumaria Cucumaria japonica.

The claimed technical result is also achieved by obtaining a biologically active supplement from sea cucumbers, characterized in that it is made in the form of gelatin capsules obtained by the method according to any one of claims 1 to 3, while inside the gelatin shell of the capsule containing 0.015 - 0.028 mg of triterpene glycosides is introduced 0.3 g of fish oil, which is a source of polyunsaturated fatty acids.

Biomedical experiments have shown that the use as a dietary supplement containing both PUFA and triterpene glycosides not only has a positive effect on the mental and physical performance of a person, but also improves alertness, memory, psychomotor coordination; vascular tone of capillaries is regulated, normalizing blood pressure (hypotensive effect); the functional state of nails, skin, hair improves. Moreover, it is known that marine triterpene glycosides are very powerful natural potency stimulants that rejuvenate and restore the male body by increasing testosterone levels, improving blood supply to the pelvic organs, reducing inflammation in the prostate, increasing the number and mobility of sperm.

It has been experimentally established that the introduction of 0.015 - 0.028 mg of triterpene glycosides into the shell of one capsule, and the placement of 0.3 g of fish oil inside the capsule, when taking 6 capsules per day, provides the daily intake of these biologically active substances. This is the level that will help maintain the health of the body.

The invention is carried out as follows.

Freshly caught or frozen sea cucumbers (trepang or cucumaria) 1.0 kg are crushed on a top with entrails and poured with 96% ethyl alcohol 1.0-1.3 liters (ratio 1 / 1-1 / 1.3, kg / l ).

The extraction process with ethyl alcohol is provided by heating the crushed mass from 30 ° C to 45 ° C for 5-6 hours in a sealed container.

The resulting alcoholic extract is filtered, the precipitate is poured with the same amount of alcohol and the procedure is repeated again.

Alcohol is removed from the combined extract containing triterpene glycosides by distillation in vacuum (100 mm Hg) to an alcohol concentration of 10% (according to GLC data).

The solution obtained after evacuation is subjected to volume decantation, followed by filtration.

After filtration, an aqueous-alcoholic solution containing triterpene glycosides is used instead of distilled water when preparing a gelatin solution (for encapsulating fish oil). at a temperature of 60 ^about C, stirring for 4 hours, while the alcohol residue completely evaporates.

The production of soft gelatin capsules with the introduction of fish oil into them is carried out on the Capsulator - B3 (Belarus). The resulting product is a dietary supplement (a source of marine triterpene glycosides and omega-3 PUFAs).

The composition of dietary supplements (final product):

- the content of triterpene glycosides (1 capsule, 0.3 g fat, net) 0.015-0.028 mg; - content of omega-3 polyunsaturated fatty acids (1 capsule, 0.3 g fish oil) 80-85 mg

The resulting capsules are a soft transparent gelatinous shell containing marine triterpene glycosides and an internal content of sea fat in the capsule. Shelf life of capsules (according to the recommendation of Rospotrebnadzor - 2 years).

The invention is illustrated by the following figures and tables.

Table 1 shows the quantitative yield of glycosides from the sea cucumber Apostichopus japonicus.

As follows from the presented results, the use of the viscera content for the extraction of glycosides significantly increases the yield of triterpene glycosides (according to the claimed method) from the mass sea cucumbers Apostichopus japonicus.

The amount of triterpene glycosides was calculated per holotoxin A1.

Analyzes of the obtained extracts for the content of holotoxin A1 were carried out at the National Research Center for Marine Biology named after V.I. A.V. Zhirmunsky "of the Far Eastern Branch of the Russian Academy of Sciences (Vladivostok).

Holotoxin A1 standard provided by the Pacific Institute of Bioorganic Chemistry. G.B. Elyakov FEB RAS (Vladivostok).

The device used is a Shimadzu LC-30Nexera X2 liquid chromatograph (Japan).

Analysis conditions: column "Shodex C18P" (2 mm diameter, 150 mm length, 5 µm granulation), temperature 40C; eluents: A-methanol, B-water (both with the addition of +25 mM formic acid and +22 mM ammonia), gradient: 0 min- 50% A + 50% B, linear rise to 100% A by 15 minutes, detector - low-temperature light-scattering detector ELSD-LT II (Shimadzu, Japan), temperature 35 ° С, nitrogen pressure 350 kPa.

Calculation of holotoxin A1 in the sample was carried out using the calibration curves of standard solutions.

Table 2 shows the hematological parameters of patients.

FIG. 1 shows the formula for holotoxin A1.

FIG. 2 shows the chromatogram of the analysis of the standard holotoxin A1, 100 μg / ml, the volume of the stab is 5 μl.

Figure 3 is a chromatogram of the analysis of an extract sample. Sample, the volume of the speck is 10 μL.

FIG. 4 shows their combination on the same scale for further calculation of the amount of triterpene glycosides (Example 1).

The invention is illustrated by the following examples.

Example 1.

Freshly caught sea cucumber, Apostichopus japonicus, 1.0 kg, grind on a top entirely with entrails and fill with 96% ethyl alcohol 1.0 l (ratio 1/1, kg / l).

The extraction process with ethyl alcohol is carried out by heating the crushed mass at 30 ° C for 6 h in a sealed container.

The resulting alcoholic extract is filtered, and the precipitate is poured with the same amount of alcohol (1.0 l) and the procedure is repeated again.

Alcohol is removed from the combined extract by distillation in vacuum (100 mm Hg) to an alcohol concentration of 10% (according to GLC data).

The solution obtained after evacuation, containing triterpene glycosides, with a volume of 1.1 l, is subjected to decantation followed by filtration.

After filtration, an aqueous-alcoholic solution containing triterpene glycosides is used in the preparation of a gelatinous mass (for encapsulating fish oil). 800.0 g of the obtained aqueous-alcoholic extract of sea cucumber L is poured into a measuring container, 850.0 g of gelatin, 300.0 g of glycerin and 5.0 g of sorbic acid are added, and the volume is brought to a total volume of 5.0 L with water, the solution is thoroughly mixed within 4 hours, at a temperature of 60 ° C, while the alcohol residue completely evaporates. Manufacturing of soft gelatin capsules with fish oil content is carried out on Capsulator-B3 (Belarus). The resulting product is a dietary supplement (a source of marine triterpene glycosides and omega-3 PUFAs).

The composition of dietary supplements (final product):

- the content of triterpene glycosides (holotoxin A1) -

0.015 mg / 1 capsule;

- content in 1 capsule of 0.3 g of fish oil, net, which includes in terms of omega-3 polyunsaturated fatty acids - 0.82 mg.

The resulting capsules are a soft gelatinous shell containing sea triterpene glycosides, and inside the capsules there is sea fat, which is a source of PUFA.

Appearance: soft gelatin capsule with a transparent shell and light yellow fish oil.

Shelf life of capsules (according to the recommendation of Rospotrebnadzor - 2 years).

Example 2.

Freshly caught sea cucumber, Apostichopus japonicus 1.0 kg, is grinded entirely with entrails and poured with 96% ethyl alcohol 1.3 liters (ratio 1 / 1.3 kg / l).

The extraction process with ethyl alcohol is carried out by heating the crushed mass at 45 ° C for 5 h in a sealed container.

The resulting aqueous-alcoholic extract is filtered, and the precipitate is poured with the same amount of alcohol (1.3 l) and the procedure is repeated again.

Further, the process continues as in example 1. However, to prepare the gelatinous mass, 300 g of the obtained aqueous-alcoholic extract are placed in a measuring container, 1650 g of gelatin, 550 g of glycerin, 5.0 g of sorbic acid are added and water is added to a total volume of 5 liters. The solution is thoroughly mixed for 4 hours at a temperature of 60 ⁰ C. The encapsulation is carried out as described in example 1.

The composition of dietary supplements (final product):

- the content of triterpene glycosides (holotoxin A1) 0.022 mg / 1 capsule; - content of omega-3 polyunsaturated fatty acids (1 capsule of 0.3 g fish oil, net) 0.82 mg

The resulting capsules are soft gelatinous shells containing marine triterpene glycosides and with an internal content of sea fat in the capsule.

Appearance: soft gelatin capsule with a transparent shell and light yellow fish oil.

Example 3.

Freshly harvested cucumaria, Cucumaria japonica, 1.0 kg, is grinded entirely with entrails and poured with 96% ethyl alcohol 1.0 liters (ratio 1/1, kg / l).

All other manipulations are done as in example 1.

In the composition of cucumaria triterpene glycosides, glycosides other than holotoxin A1 are the main ones, so they were measured in total by a different method (Aminin D.L. et al. “Spectrophotometric determination of lymphoposide A from sea cucumber Stichopus japonicus S. // Antibiotics. 1981. T 26, No. 8, pp. 585-588).

The composition of dietary supplements (final product):

- the content of total triterpene glycosides 0.028 mg / 1 capsule; - content of omega-3 polyunsaturated fatty acids (1 capsule of 0.3 g fish oil, net) 0.84 mg.

The resulting capsules are a soft gelatinous shell containing marine triterpene glycosides, inside of which sea fat is placed.

Appearance: soft gelatin capsule with a transparent shell and light yellow fish oil.

Example 4.

Gelatin capsules with fish oil obtained as in example 1 were prescribed to 11 patients without chronic diseases and acute pathologies. The group consisted of male volunteers from 18 to 57 years old. The daily intake consisted of six gelatin capsules of 0.3 g of fish oil and 0.15 mg of triterpene glycosides. Duration of the course is 21 days.

The program of the experiment was agreed with the Ethics Committee of the National Scientific Center for Marine Biology. A.V. Zhirmunsky "of the Far Eastern Branch of the Russian Academy of Sciences (Vladivostok).

Table 2 shows the results of the effectiveness of taking a biologically active food supplement to correct lipid metabolism and increase the immune status. Before the beginning and after the end of the experiment, a study of the lipid spectrum of blood was carried out, namely, the content in the blood plasma of the level of total cholesterol (TC), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), very lipoprotein cholesterol was assessed. low density (VLDL), triglycerides (TG), and also studied the level of testosterone in the blood and determined the spermogram. The general condition of the subjects (mood, sleep, performance, libido, etc.) was monitored daily.

Volunteers, after receiving a daily intake of dietary supplements as part of a diet that satisfies 25% of the human body's daily need for PUFAs of the omega-3 family and with a content of at least 0.15 mg of triterpene glycosides, showed a tendency towards correction of lipid metabolism and an increase in sexual activity, physical and mental performance. It was found that against the background of a decrease in the level of total cholesterol, there is a significant decrease in LDL cholesterol, as well as an increase in HDL cholesterol, the significance of differences in these values was 95% (t> 2). This indicates a reduced risk of developing atherosclerosis.

The subjects had an increase in the number of sperm in 1 ml of sperm by 20.1%, sperm motility increased by 19.3%, the level of testosterone in the blood increased by 11.6%. Thus, the developed dietary supplement stimulates spermatogenesis and increases sexual activity. All subjects noted cheerfulness during the day against the background of an increase in their general mental and physical performance and sexual desire.

The results obtained revealed an obvious beneficial effect of the inclusion in the diet of patients with impaired lipid metabolism of dietary supplements with an increased content of polyunsaturated fatty acids of the omega-3 family and triterpene glycosides.

Thus, a technology has been developed for producing a two-component dietary supplement that combines a high presence of triterpene glycosides from Far Eastern sea cucumbers, valuable polyunsaturated acids of the omega-3 series and an attractive delivery of the final product for the consumer - extremely transparent soft gelatin capsules with a distribution of glycosides in gelatin, and omega -3 acids in the capsule itself, which is impossible with other approaches. A dietary supplement containing several biologically active substances at the same time can contribute to the significant promotion of such drugs on the market. The method also makes it possible to expand the range of obtained dietary supplements by introducing capsules of various types of sea fat or valuable vegetable oil, or a herbal extract of a medicinal plant.

On the basis of this invention, the technology of obtaining a biologically active food supplement (Omega-3 with Trepang extract) was finally improved. The dietary supplement was approved by the Federal Food and Drug Administration and Rospotrebnadzor and is registered in Russia as an additional source of triterpene glycosides and omega-3 polyunsaturated fatty acids (Certificate of state registration SGR No. AM.01.07.01.003.R.000154.07.20 dated 14.07.2020).

Claims (5)

1. A method of obtaining a biologically active supplement from sea cucumbers, including grinding raw materials and its double extraction with ethyl alcohol, separation of alcohol extract, characterized in that holothurians are used as raw materials completely, extraction of crushed raw materials with ethyl alcohol is carried out at a ratio of 1: 1-1: 1 , 3 kg / l, at a temperature of 30 ° -45 ° C for 5-6 hours, the residue after extraction with ethyl alcohol is squeezed out and re-extracted with ethyl alcohol under the same conditions, the resulting aqueous-alcoholic filtrates are combined and filtered from the resulting combined filtrate ethyl alcohol is distilled off under vacuum until its concentration is not more than 10%; then the obtained aqueous-alcoholic extract containing triterpene glycosides is used to prepare a gelatinous mass, from which the capsule shell is formed, and fish oil is introduced into the capsules. 2. The method according to claim 1, characterized in that the preparation of the gelatinous mass is carried out by mixing a water-alcohol extract containing triterpene glycosides with gelatin, glycerin, sodium benzoate, sorbic acid and water, at a temperature of 60 ° C and stirring for 4- x hours, with the following ratio of components: hydroalcoholic extract of sea cucumber 300-800 g gelatin 850-1650 g glycerol 300-550 g sorbic acid 5.0 g water up to 5.0 l 3. The method according to claim 1, characterized in that sea cucumbers are used as raw materials - trepang Apostichopus japonicus or cucumaria Cucumaria japonica. 4. Dietary supplement from sea cucumbers, characterized in that it has the form of gelatin capsules obtained by the method according to any one of claims 1-3, while inside the gelatin shell of the capsule containing 0.015-0.028 mg of triterpene glycosides, 0.3 g of fish fat.

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