RU2215532C2 - Method for complex processing sea cucumber viscera and preparing biologically active food supplements and biologically active food supplements "tingol-2" and "erogol" - Google Patents

Abstract

FIELD: medicine, food industry. SUBSTANCE: sea cucumber viscera are milled to particle size 0.5 mm, not above, extracted with ethyl alcohol in the ratio from 1:1 to 1:3 at temperature from 5 C to +60 C for from 5 h to 10 days and liquid part is separated to obtain an aqueous-alcoholic extract. Then residue of sea cucumber viscera with water content from 55 to 75% is dried at temperature 60-75 C for 2.5-4 h, milled to obtain powdered state, tableted or capsulated. Biologically active supplement "TINGOl-2" represents an aqueous-alcoholic extract from sea cucumber viscera containing triterpene glycosides in the amount 700-3 500 mcg/ml in alcohol content 10-50% and lipids content 0.5-4%. Biologically active supplement "Erogol" represents dried powder containing 3-12% of water, triterpene glycosides in the amount 800-4 500 mcg/ml; 35-45% of protein; 13.5-24.5 mg/kg of manganese; 44.0-82.0 mg/kg of zinc; 481.2-893.5 mg/kg of iron; 5.6-10.4 mg/kg of copper; 2.4-4.2 mg/kg of nickel, and also sum of elements of calcium, magnesium and potassium in the amount 6 500-12 000 mg/kg. Invention provides the simple and effective method for preparing two kinds of biologically active supplements enhancing concentration and motility of human and animal spermatozoids and their physical working ability. Invention can be used for preparing biologically active supplements from sea cucumbers. EFFECT: improved and enhanced processing method. 4 cl, 7 ex

Description

 The invention relates to the food industry and can be used to process the entrails of holothurians with the production of biologically active food additives “TINGOL-2” and “Erogol” as general strengthening agents at high physical loads and increased sexual activity (potency) and the function of reproduction of humans and animals.

A known method of obtaining funds for stimulating the body's defenses from marine aquatic organisms (application 97106885/13 from 04/22/97), including the primary extraction of marine aquatic organisms. Whole freshly caught specimens of Japanese trepangs are taken as a source, the funds are extracted with diluted ethanol solutions at a temperature of 35 ^° C, the obtained one is evaporated and purified by step hydrophobic chromatography using water and an ethanol concentration gradient of 10, 20, 40, 50, 96%.

 A known method of preparing a food treatment and prophylactic product from sea urchin caviar (US Pat. RF 2116039 from 07/08/97). The method includes extracting caviar, washing, drying, salting, grinding, mixing with the components. The therapeutic and prophylactic product includes sea urchin caviar in an amount of 15-25% and a mixture of vegetable oils.

 A known method of obtaining a lipid and carotenoid fraction from holothurians, mainly Cucumaria frondosa (US Pat. US 6055936 from 05/02/2000), including extracting the insides of the "sea cucumber", acidification to pH between 4 and 5.5 of the insides of the "sea cucumber", drying the acidified insides extraction of the total lipid fraction with either hexane, or liquefied propane, or supercritical carbon dioxide, or a combination of the above substances.

Received several compositions of substances:
- a composition of substances comprising lipid fractions of a sea cucumber without carotenoid material obtained by the following methods: a) contacting the red carotenoid-containing lipid fractions of a sea cucumber with activated bleaching clay, or a similar suitable material, or material that sorb pigments; b) separating this bleaching clay from the remaining lipid fraction, which is essentially freed from carotenoids.

 - a composition of substances comprising a carotenoid lipid fraction obtained by acetone extraction of fresh acidified viscera of a "sea cucumber".

 The closest method of processing holothuria is the method of obtaining a complex of biologically active products from holothuria Cucumaria frondosa (US Pat. RF 2112527 dated 07/06/95), including homogenization of raw materials, multiple extraction, combining extracts, purification and sequential isolation of target products. The extraction of biomass is carried out with a mixture of ethanol-chloroform at a ratio of respectively (1: 2: 2) - (1: 1: 2), repeated extraction with ethanol at room temperature and a ratio of raw materials: ethanol (1: 1) - (1: 3) and the following with ethanol at a boiling point and the ratio of raw materials: ethanol (1: 3) to (1: 5), the solid residue obtained after repeated extraction is dried, crushed and a protein feed additive is obtained, and the extracts are combined, filtered, evaporated, the residue thus obtained is extracted repeatedly with acetone at a ratio of the residue K: acetone (1: 5) - (1:10) and a precipitate is obtained containing a complex of saponins and an acetone extract containing carotenoids, then the acetone extract is filtered and evaporated to a dry residue, the residue is dissolved in ethanol at a ratio of residue: ethanol 1: 3 , filtered and evaporated to obtain a carotenoid concentrate, and a dense precipitate containing a saponin complex was dissolved at a precipitate: ethanol ratio of 1: 3, evaporated to one third of the initial volume, filtered hot, the filtrate was dissolved in butanol at a filtrate: butanol ratio (1 : 3) - (1: 5 ), filtered while hot and the filtrate is evaporated to a dry residue, then the residue is dissolved in ethanol with a ratio of residue: ethanol (1: 8) - (1:10), the solution is filtered while hot and the filtrate is evaporated to a dry residue - a concentrate of a complex of saponins while the resulting product additionally contains carotenoids.

 This method of obtaining a complex of biologically active products from holoturia Cucumaria frondosa involves the use of flammable, environmentally harmful substances (chloroform, acetone, butanol), a long, laborious, energy-intensive, multiple, multi-stage extraction, involves the use of fresh raw materials. In addition, this method involves the preparation of chemically extracted, purified carotenoids and saponins and therefore lacks the synergistic action of micro and macro elements, glycosides, vitamins, amino sugars and other natural composition present in holothuria.

 Known agent for the treatment of radiation sickness (US Pat. RF 2141833 from 07.16.97), where it is proposed to use triterpene glycosides (cucumariosides) from the Far Eastern holothuria Cucumaria japonica for the treatment of radiation sickness. Substances increase the survival of irradiated animals, increase the body's resistance to the effects of exposure to ionizing radiation.

 Pat. US 5993797 from 11/30/99 receiving sulfated polysaccharide from the Far Eastern trepang and the use of this substance for the treatment of vascular hyperplasia.

 Pat. USA 5985330 dated 11.16.99, 5989572 dated 11.23.99, 5770205 dated 23.06.98, 5876762 dated 02.03.99, including the preparation of fractions B1000 and T2000 from the epithelial layer and the “corolla” of the “sea cucumber” Cucumaria frondosa and the use of these fractions in various concentrations and variations for the treatment of various arthritic inflammatory rheumatoid processes.

 Known fortifying agents obtained from raw materials of animal origin. Pantocrine and Rantarin - extracts from antler sika deer, red deer, deer or extract from antler deer (Mashkovsky M.D. Medicines, vol. 1., M .: Novaya Volna LLC: Publisher S.B.Divov, 2001 p. 136). Pantogem (TU 429-021-001-92), the active principle of which is the extraction of sika deer from whole blood.

 Known treatment and prophylactic balm Vitagen (TU 9169-010-20785204-94), containing 37 components of plant and animal origin, including ginseng, eleutherococcus, magnolia vine, preparations from kelp, salmon milk, mummy.

 These funds are used in the rehabilitation of various past diseases, a decrease in the functions of the central nervous system, and a decrease in muscle and mental performance.

 Known drug MIGI-K-LP, with non-specific immunomodulating properties (TU 15-16-28-94), which is a hydrolyzate of mussel meat containing melanoidins with a molecular weight of up to 5000 and more than 5000 at a quantitative ratio (mg / ml solution) equal to 28 - 50: 2-8, having a pH of 5.0 - 5.8, containing 28-33 wt.% solids and containing amine nitrogen of 0.85-1.2% of the total weight of the product. The hydrolyzate from mussels and the method for its preparation are protected by RF patent 2017439, cl. A 23 L 1/333 A 23 G 1/04. The mussel hydrolyzate contains a complete set of essential amino acids, low molecular weight nitrogen compounds, trace elements (Co, Cu, Zn, Mn, Fe, Mo, etc.), free fatty acids, as well as a number of substances of a melanoid nature.

 MIGI-K-LP is recommended for people in contact with ionizing radiation, receiving chemotherapy, patients with chronic inflammatory processes, immunodeficiency, metabolic disorders, burns, for wound healing in the postoperative period.

 MIGI-K-LP take 15-45 ml (1-3 tablespoons) a day before, after or during meals.

 Known means for the prevention and treatment of Aleutian mink disease (application RU 4891144/15 from 12/13/1990). Cucumarioside is used - the total fraction of triterpene glycosides of the Far Eastern holothuria - Japanese cucumaria. The drug is administered subcutaneously in 1 ml of physical. solution in a dose of 1 μg / kg of animal mass once during treatment 2 weeks before the start of the rut and again in the 1st half of pregnancy, with prophylaxis - a single subcutaneous injection in the same dose.

The closest is a food fortifying treatment and prophylactic additive (US Pat. RF 2117435 from 08.20.98), obtained from the muscle tissue of cucumaria, containing the amount of triterpene glycosides in an amount of at least 100 μg / cm ³ , with a pH of 6.3-7.0, mass fraction of solids not less than 20%, mass fraction of amine nitrogen not less than 0.7%, mass fraction of salt not more than 5%. This nutritional supplement is derived from cucumari muscle tissue.

 However, when cutting holothurians, losses reach 65-70%, with the main ones falling to the share of viscera (35-40%). In the industrial processing of animals, the entrails are considered waste and their use is considered irrational. Meanwhile, as shown by numerous literature data and studies conducted by us, the holothurium interiors are material whose biological activity is rather high (Kalinin V.I. et al., 1994; Zaitsev et al., 1980, Anisimov M.M. 1987; Savvateeva, Maslova, Volodarsky, 1983).

 TINGOL-2 and Erogol are new products. The technology for their production was developed at the TINRO Center, their application is unknown.

 The essence of the method of complex processing of the internals of holothuria to obtain biologically active food additives "TINGOL-2" and "Erogol" is as follows.

The crushed (to a particle size of no more than 0.5 mm) holothuria interiors (cucumaria, trepanga) are extracted with 95-96% ethanol in the ratio ethanol: interiors from 1: 1 to 1: 3 at a temperature of minus 5 to plus 60 ^o С within 5 hours to 10 days, the liquid part is separated by centrifugation (rotor speed of 3000-5000 rpm for 10-25 minutes) or by filtration to obtain a water-alcohol extract - biologically active food supplement "TINGOL-2", then the remainder of the internals of holothuria with a water content of 55 to 75% is dried at a temperature of 6 0-75 ^o C for 2.5 to 4 hours, crushed to a powder state (tabletted or encapsulated) to obtain a biologically active food supplement "Erogol".

 Biologically active food supplement "TINGOL-2", which is an extract from the entrails of holothuria, containing triterpene glycosides in an amount of 700-3500 μg / ml, with an alcohol content of 10-50% and lipids of 0.5-4%, with biological activity, consisting in increasing the physical performance of the body and sexual activity (potency) of humans and animals.

 Biologically active food supplement "Erogol", which is a dry powder obtained by drying the material after alcohol extraction of the internals of holothurium to a water content of 3-12% and containing triterpene glycosides in the amount of 800-4500 μg / g, protein 35-45%, manganese 13.5 - 24.5 mg / kg, zinc 44.0 - 82.0 mg / kg, iron 481.2 - 893.5 mg / kg, copper 5.6 - 10.4 mg / kg, nickel 2, 4 - 4.2 mg / kg, as well as the sum of the elements (calcium, magnesium, potassium) that regulate the water-salt balance of the body 6500 - 12000 mg / kg, which has biological activity, which consists in increasing physical the health of the body and the sexual activity (potency) of humans and animals.

 Examples illustrating the invention.

 Example 1

To 1 kg of crushed internals of holothuria (Cucumaria japonica) add 1 liter of 95-96% ethanol, extracted at a temperature of minus 5 ^o С for 10 days, centrifuged or filtered to obtain a biologically active food supplement "TINGOL-2".

 "TINGOL-2", containing 50% alcohol, 0.5% lipids, 700 mcg / ml triterpene glycosides, has biological activity, which consists in increasing the physical performance of the body and increasing the sexual activity (potency) of humans and animals.

 "TINGOL-2" is a clear liquid of light straw color with the smell and taste of seafood, is stable in storage for 12 months.

The rest of the entrails of holothuria with a water content of 55% is dried at a temperature of 60 ^o C for 2.5 hours, crushed (tabletted or encapsulated), 190 g of the therapeutic product “Erogol” are obtained. "Erogol" is a loose brown powder with a water content of 3%, triterpene glycosides 800 μg / g, protein 45%, manganese 13.5 mg / kg, zinc 44.0 mg / kg, iron 481.2 mg / kg, copper 5.6 mg / kg, nickel 2.4 mg / kg and the amount of elements that regulate the water-salt balance of 6500 mg / kg. It has biological activity, which consists in increasing the physical performance of the body and increasing the sexual activity (potency) of humans and animals.

 Example 2

1 liter of 95-96% ethanol is added to 2 kg of crushed holothuria internals (Cucumaria frondosa), extracted at a temperature of 10 ^° C for 3 days, centrifuged or filtered to obtain a biologically active food supplement “TINGOL-2”.

 "TINGOL-2", containing 30% alcohol, 2.5% lipids, triterpene glycosides 2100 μg / ml, has biological activity, which consists in increasing the physical performance of the body and increasing sexual activity (potency) of humans and animals.

 "TINGOL-2" is a clear straw-colored liquid with the smell and taste of seafood, is stable in storage for 12 months.

The rest of the entrails of holothuria with a water content of 65% is dried at a temperature of 65 ^o C for 3 hours, crushed (tabletted or encapsulated), 190 g of the therapeutic product “Erogol” are obtained. "Erogol" is a loose brown powder with a water content of 7.5%, triterpene glycosides 2700 μg / g, protein 40%, manganese 18.8 mg / kg, zinc 63.0 mg / kg, iron 690.0 mg / kg, copper 8.0 mg / kg, nickel 3.3 mg / kg and the amount of elements that regulate the water-salt balance of 9300 mg / kg. It has biological activity, which consists in increasing the physical performance of the body and increasing the sexual activity (potency) of humans and animals.

 Example 3

To 3 kg of crushed internals of holothuria (Stichopus japonicus) add 1 liter of 95-96% ethanol, extracted at a temperature of 60 ^o C for 5 hours, centrifuged or filtered to obtain a biologically active food supplement "TINGOL-2".

 "TINGOL-2", containing alcohol 10%, lipids 4.0%, triterpene glycosides 3500 μg / ml, which has biological activity, which consists in increasing the physical performance of the body and increasing sexual activity (potency) of humans and animals.

 "TINGOL-2" is a transparent yellow liquid with the smell and taste of seafood, is stable in storage for 12 months.

The rest of the entrails of holothuria with a water content of 75% is dried at a temperature of 75 ^o C for 4 hours, crushed (tabletted or encapsulated), 380 g of the therapeutic product “Erogol” are obtained. "Erogol" is a loose brown powder with a water content of 12%, triterpene glycosides 4500 μg / g, protein 35%, manganese 24.5 mg / kg, zinc 82.0 mg / kg, iron 893.5 mg / kg, copper 10.4 mg / kg, nickel 4.2 mg / kg and the amount of elements that regulate the water-salt balance of 12000 mg / kg. It has biological activity, which consists in increasing the physical performance of the body and increasing the sexual activity (potency) of humans and animals.

 Example 4

 The influence of TINGOL-2 on the reproductive system of mammals was studied at the Department of Biology, Voronezh State Medical University.

All animals from birth were kept under standard vivarium conditions. Studies were conducted on four groups of non-linear male mice:
Group 1 (control, adults) received a normative food mixture;
2nd group - adult males received "TINGOL-2" in the form of one daily supplement to the main feed in the amount of 0.03 ml per 1 kg of body weight;
3rd group (control, young) received a normative food mixture;
4th group - young males received "TINGOL-2" in the form of a daily individual supplement to the main feed of 0.03 ml per 1 kg of body weight;
During the entire period of the experiment, the animals were kept under normal light and temperature conditions, dynamic monitoring of the condition and behavior of animals was carried out. After 2 months, all animals were killed by decapitation and carried out morphometric, morphological and biochemical tests. A study of the testes of experimental animals, both adults and young, treated with TINGOL-2, revealed an increase in mitotically dividing spermatogonia in comparison with the control by 15% (in young animals) and by 26% (in adult animals). In males, the number of seminiferous tubules with mature spermatids (stage 12 of spermatogenesis) as a result of taking TINGOL-2 significantly increases compared with the control.

 There were no deviations in the behavior of adult and young animals treated with TINGOL-2 compared with the control.

 Morphometric parameters of adult and young mice (body weight, liver, spleen, testes) after taking "TINGOL-2" did not differ from those in the control groups.

 Example 5

 Studies on the use of "TINGOL-2" in the treatment of patients suffering from impaired sexual function were carried out on the basis of the clinic of the Internal Affairs Directorate of Primorsky Krai and the Center for Family Planning and Reproduction of Vladivostok.

 The selection criterion was men (10 people) with functional disturbances in the erection component and a decrease in libido. The age of patients ranged from 24 to 55 years. All subjects filled out anonymous questionnaires about the development of the disease, were tested.

 The following preliminary laboratory tests were carried out: clinical blood test, general urine test, blood protein test, cholesterol, spermogram study, testosterone level determination.

 After a preliminary laboratory study, the subjects used TINGOL-2 in a dose of 5 ml 3 times a day for 21 days. The specified dose is 0.15 ml per 1 kg of human weight daily. The general condition of the subjects was monitored daily (mood, sleep, performance, libido, etc.). After using TINGOL-2, repeated laboratory tests were carried out: a general analysis of blood, cholesterol, blood proteins, spermogram, and the level of testosterone in the blood was determined.

 After treatment, it was found that “TINGOL-2” has a pronounced biological activity, which consists in improving the physical activity of the subjects, working capacity, mood, increasing libido, spontaneous and adequate erection, increasing the number of sexual intercourse, and improving sleep. The subjects increased the number of sperm in 1 ml of sperm by 80.3%, sperm motility increased by 61.0%, the level of motionless sperm decreased by 2 times. Blood testosterone levels increased by 18.3%.

 Example 6

 Studies on the use of "TINGOL-2" in the treatment of patients suffering from impaired sexual function were carried out on the basis of the clinic of the Internal Affairs Directorate of Primorsky Krai and the Center for Family Planning and Reproduction of Vladivostok.

 The selection criterion was men (15 people) with functional disturbances in the erection component and a decrease in libido. The age of patients ranged from 23 to 61 years. All subjects filled out anonymous questionnaires about the development of the disease, were tested.

 A blood test, a general urine test, a study of blood proteins, the amount of cholesterol, testosterone, and a spermogram were performed.

 After a preliminary laboratory study, patients (5 people) used TINGOL-2, 0.05 ml per 1 kg of body weight daily for 42 days (1 teaspoon of the drug 1 time per day. The general condition of the subjects was monitored daily (mood, physical and mental performance, libido, etc.).

 It was established that "TINGOL-2" has activity, which consists in increasing physical and mental performance, increasing libido, spontaneous and adequate erection, increasing the number of sexual intercourse. The level of testosterone in the blood increased by 7.7%, the number of spermatozoa with rapid translational movement increased by 22.3%.

 Patients (10 people) receiving “TINGOL-2” 5 teaspoons daily (0.25 ml per 1 kg of a person’s weight) noted in some cases (3 people) a slight overexcitation in the evening, which was sometimes expressed in difficulty falling asleep . After 20 days of treatment, the testosterone level in these patients increased by 20.1% compared with the control indicators, the number of sperm with the absence of translational motion decreased by 40%, the number of sperm with a slow translational motion decreased by 49%, the number of sperm with a rapid translational motion increased 1.5 times, the total amount of ejaculate increased by 63%.

 Example 7

 The use of Erogol as a therapeutic and prophylactic supplement in the treatment of patients suffering from impaired sexual function.

 A group of patients (15 people) was formed on the basis of the clinic of the Internal Affairs Directorate of Primorsky Krai and the Center for Family Planning and Reproduction of Vladivostok. All subjects filled out anonymous questionnaires about the development of the disease, were tested. The following preliminary laboratory studies were carried out: a clinical blood test, a general urinalysis, a study of blood proteins, cholesterol, a spermogram study, determination of the level of testosterone, prolactin, FSH, hypertension. After a preliminary laboratory study, the subjects used Erogol at a dose of 2 capsules 2 times a day to 3 capsules 3 times a day, i.e. from 0.03 to 0.06 g per 1 kg of body weight for 21 days. The general condition of the subjects was monitored daily (mood, sleep, performance, libido, etc.). After the use of Erogol, repeated laboratory tests were carried out: a general analysis of blood, cholesterol, blood proteins, urine, the level of testosterone, prolactin, FSH, hypertension in serum, and spermogram were determined.

 There was an improvement in physical activity, performance, mood, spontaneous and adequate erection, increased libido, increased number of sexual intercourse, improved sleep. Sperm motility increased by 78%, the number of motionless sperm decreased by 2.3 times; the total sperm count in 1 ml of sperm increased from 146.4 million to 164.7 million. The level of testosterone in the blood increased by an average of 38.9%.

Claims (3)

1. The method of complex processing of the internals of holothuria to obtain a biologically active food supplement that increases the concentration and mobility of sperm in humans and animals and their physical performance, characterized in that the internals of holothuria are crushed to particles no larger than 0.5 mm, extracted 95-96 % ethanol in the ratio of ethyl alcohol: holothuria internals from 1: 1 to 1: 3 at a temperature of minus 5 to plus 60 ^o C for 5 hours to 10 days, the liquid part is separated by centrifugation or filtration with by treating with an aqueous-alcoholic extract, then the remainder of the internals of holothuria with a water content of 55 to 75% is dried at 60-75 ^° C for 2.5-4 hours, ground to a powder state, tabletted or encapsulated.  2. A biologically active food supplement that increases the concentration and motility of spermatozoa in humans and animals and their physical performance, characterized in that it is a water-alcohol extract from the entrails of holothuria, obtained according to claim 1, containing triterpene glycosides in the amount of 700- 3500 μg / ml with an alcohol content of 10-50% and lipids of 0.5-4%.  3. A biologically active food supplement that increases the concentration and motility of sperm in humans and animals and their physical performance, characterized in that it is a dry powder obtained according to claim 1, containing 3-12% water, triterpene glycosides in an amount of 800 -4500 μg / ml, protein 35-45%, manganese 13.5-24.5 mg / kg, zinc 44.0-82.0 mg / kg, iron 481.2-893.5 mg / kg, copper 5 , 6-10.4 mg / kg, nickel 2.4-4.2 mg / kg, as well as the sum of the elements of calcium, magnesium, potassium 6500-12000 mg / kg.