RU2764439C1 - Method for obtaining an extract from quinoa grains enriched with phytoecdysteroids - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to a method for obtaining an extract from quinoa grains. The method for obtaining an extract from quinoa grains includes grinding and sieving the grains through a sieve with a pore size of 0.35 mm, then 2-stage extraction: at the first stage, the crushed grains are extracted with 500 ml of 20-60% ethyl alcohol solution and at a ratio of 1:20 g/ml, respectively, at room temperature for 1 hour, clarified by centrifugation for 30 minutes on a BEKMAN J-6B centrifuge at 4000 rpm and the supernatant is selected, at the second stage the resulting precipitate is extracted with 250 ml of 20-60% solution of ethyl alcohol under similar conditions, at a ratio of 1:10 g/ml, respectively, at room temperature for 45-60 minutes, followed by centrifugation, the supernatant is selected and combined with the one obtained at the first stage, ultrafiltration is carried out through a membrane with a pore size of 10 kD, with the collection of a low-molecular fraction, which is then subjected to reverse osmotic concentration, followed by removal of ethanol under vacuum on a rotary evaporator, passing this concentrate through a column with a hydrophobic sorbent, washing the sorbent with water and subsequent desorption of phytoecdysteroids, flavonoids and polyphenols with 70-75% ethyl alcohol, removal of ethanol from the alcohol flush under vacuum on a rotary evaporator and lyophilic drying.

EFFECT: method makes it possible to obtain an extract (concentrate) from quinoa grains enriched with phytoecdysteroids (mostly 20-hydroxyecdysone) and flavonoids.

1 cl, 3 ex

Description

The invention relates to food biotechnology, namely to obtaining enriched food products to increase the adaptive capacity of the body under stress, increase mental and physical performance, improve short-term and long-term memory, improve carbohydrate and lipid metabolism, prevent and compensate for abdominal obesity. The invention finds application in functional nutrition, restorative medicine, geriatrics and sports.

It is known that a modern person is exposed to stress factors of various nature, strength and duration of action. The stress hormone cortisol takes part in the regulation of stress, which, in response to fatigue, physical exertion, starvation, fear and other emergency stressful situations, mobilizes metabolic processes in the body: the breakdown of proteins (including muscle proteins) into amino acids, the launch of gluconeogenesis processes in the liver, an increase in blood glucose levels, and fat accumulation. Stress of low intensity and duration has a protective effect and helps the human body overcome the challenges of stress. At the same time, after the removal of the stress load, the level of cortisol returns to the base level. Under conditions of chronic stress, when cortisol levels are constantly elevated, under its influence, thyroid function is suppressed, blood pressure rises, hyperglycemia occurs, immunity decreases, the amount of abdominal fat increases with normal body weight, and bone density decreases. In turn, this can lead to various diseases, such as diabetes, myocardial infarction, stroke, atherosclerosis, and others. Many well-known drugs aimed at the treatment of certain cardiovascular diseases or metabolic disorders do not eliminate their main cause due to chronic stress [Dilman V.M. Large biological clock (Introduction to integral medicine). M: Knowledge, 1981. 208 p.].

Currently, much attention is paid to anti-stress phytoregulation using medicinal plants, which are effective not only for the treatment of various diseases, but also as a method of normalizing the physiological functions and psychophysiological state of healthy people. Their effectiveness has been shown to adapt the body in situations of both acute stress and prolonged exposure to stressors, to increase efficiency and accelerate recovery after stress. For the prevention and correction of stressful conditions, which include fatigue, asthenia, decreased performance, adaptogens are used - substances of predominantly plant origin that increase the body's nonspecific resistance to adverse stress effects. Adaptogens act on the centers of hormonal regulation, optimizing the secretion of cortisol and providing a tonic and stimulating effect on the functions of the nervous system and the body as a whole. They are effective both at the early stage of stress and at the stage of stress exhaustion, activating metabolic processes. Adaptogens are also characterized by antioxidant action and provide protection against damage to cellular structures by free radicals. They have an immunostimulating effect, increase physical and mental performance, resistance to various physiological and psychological stress factors, and reduce the time for adaptation to them.

Known adaptogens are tinctures and preparations based on ginseng, eleutherococcus, aralia, magnolia vine, rhodiola and some other plants, however, they have contraindications for insomnia, increased nervous excitability and hypertension [Dyrdymov I.V. Ginseng, Eleutherococcus (to the mechanism of biological action). M., 1976. 184 p.; Yaremenko K.V. Optimal body condition and adaptogens. SPb., 2007. 129 p.]. These shortcomings are deprived of drugs and dietary supplements based on plants containing phytoecdysteroids [Volodin V.V., Mataev S.I. Ecdysteroid-containing plants - sources of new adaptogens. Bulletin of Biotechnology and Physico-Chemical Biology. Yu.A. Ovchinnikova, 2011. V. 7. No. 2, S. 52-59.].

Phytoecdysteroids - polyhydroxylated sterols, which are structural analogs of insect molting hormones, are found in many species of wild plants. Phytoecdysteroids are not toxic to mammals. A pronounced actoprotective effect was revealed (patent RU No. 2276991, 2005 A61K 36/21).

A hypolipidemic and anti-ischemic agent is known, which is an ecdysteroid-containing substance of a mixture of 20-hydroxyecdysone in an amount of at least 75% and 258-inocosterone in an amount of at least 10%, isolated from the aerial part of the plant Serratula coronata L. (Asteraceae). (RU, patent No. 2337701, 2007, A61K 36/28).

Ecdysteroids are introduced into the body in pure form or in the form of plant extracts. For this, medicinal plants with a relatively high content of ecdysteroids are used, for example, safflower-like leuzea, from the rhizomes of which liquid and dry extracts are obtained and purified 20-hydroxyecdysone is isolated as the substance of the tonic drug ecdysten [Kurakina I.O., Bulaev V.M. Ekdisten-tonic agent in tablets of 0.005 g // New drugs. M., 1990. Issue. 6. S. 16-18.].

To obtain biologically active food supplements, the use of plants belonging to the list of medicinal plants has not been recommended recently. Food plants can be used for this purpose, however, examples of known food plant species containing phytoecdysteroids are few. These include, for example, spinach and quinoa (Chenopodium quinoa Willd.). (Bakrim A., Maria A., Sayah F. et al. Ecdysteroids in spinach (Spinacia oleracea L.): biosynthesis, transport and regulation of levels // Plant Physiology and Biochemistry, 2008. V. 46. P. 844-854 .; Foucault et al A.-S., Even P., Lafont R. et al. Quinoa extract enriched in 20-hydroxyecdysone affects energy homeostasis and intestinal fat absorption in mice fed a high-fat diet // Physiology and Behavior, 2014 V. 128. P. 226-231.).

Quinoa or rice quinoa (Chenopodium quinoa Willd.), is a dicotyledonous plant (a class of angiosperms in which the seed embryo has two lateral opposite cotyledons), belonging to the Chenopodiaceae family, is one of the oldest crops in the Andean region of South America, where about 7000 years. Quinoa contains in its composition a large number of secondary metabolites (fatty acids, flavonoids, terpenoids, steroid and nitrogen-containing compounds). The high content of these compounds in quinoa determines its antidiabetic, antitumor, antimicrobial, anti-inflammatory, and immunomodulatory activity.

The quinoa plant has a high nutritional value and medicinal properties. Quinoa grains contain many useful substances for the human body. For example, vitamins D, C, A, K, E and all B vitamins, magnesium, manganese, iron, copper, zinc and phosphorus. The diet of a modern person should contain not only a balanced amount of known macronutrients (proteins, fats and carbohydrates), but also the so-called micronutrients or minor components that come with plant foods.

Known functional food product for reducing excess body weight, containing phytoecdysteroids from the food plant quinoa, and a method for its production. The content of phytoecdysteroids in quinoa grains is not higher (0.01-0.02)%. It is necessary to consume at least 100 g of unprocessed dry grains in order for the human body to receive an effective concentration of phytoecdysteroids. The authors of this invention propose to use an enriched product containing 50 times more phytoecdysteroids than the original grains of quinoa. According to the claimed method, to obtain an enriched product, quinoa grains are first extracted with boiling water in a ratio of 1:4 to remove saponins, which give the extract a bitter taste. Ecdysteroids are extracted from quinoa grains by secondary extraction with a mixture of ethanol-water (1:1) at a temperature of 80°C. The dry extract is treated with absolute ethanol. The ethanol fraction is filtered and the final product is dried. The method also involves the use of butanol instead of ethanol. The content of phytoecdysteroids in the enriched product is from 2% to 7%, which is two orders of magnitude higher than in the feedstock. This enriched extract is added to food products such as yogurt, other dairy products and drinks, or formulated into gelatin capsules to form dietary supplements. According to the patent, one capsule contains 7.5 mg of phytoecdysteroids. The recommended dose for the prevention or compensation of abdominal obesity is 4 capsules or 30 mg of phytoecdysteroids per day. This method has a number of disadvantages, such as the use of imported plant materials (quinoa grains), the inevitable loss of phytoecdysteroids at the stage of primary water extraction when removing saponins (the solubility of phytoecdysteroids in water is about 2 g/l), the use of large volumes of ethanol increases the cost of production, and the use butanol is undesirable when receiving food additives (see patent No. 2491083, 2008, A61K 36/185).

The claimed method, thanks to the selected methods and modes of conduct, makes it possible to obtain an extract from quinoa grains enriched with phytoecdysteroids, with a content of 20-hydroxyecdysone up to 50 mg/g of dry extract, flavonoids up to 240 mg/g in dry extract.

The technical objective of the claimed method is to develop an effective method for obtaining an extract from quinoa grains, which allows increasing the content of 20-hydroxyecdysone to 50 mg/g of dry extract, flavonoids to 240 mg/g of dry extract. The present invention makes it possible to expand the arsenal of means for increasing the adaptive capacity of the body under stress, increasing mental and physical performance, improving short-term and long-term memory, improving carbohydrate and lipid metabolism, preventing and compensating for abdominal obesity.

In addition, the method for obtaining an extract from quinoa seeds is less laborious and cost-effective while maintaining the qualitative and quantitative characteristics of the final product.

The technical result of the claimed method is to obtain an extract (concentrate) from quinoa grains enriched with phytoecdysteroids (mostly 20-hydroxyecdysone) and flavonoids.

The technical result is achieved by obtaining an extract from quinoa grains by a method including grinding and sieving the grains through a sieve with a pore size of 0.35 mm, then 2-stage extraction at the first stage, the crushed grains are extracted with 500 ml of a 20-60% ethanol solution and in a ratio of 1:20 (g/ml), respectively, at room temperature for 1 hour, clarified by centrifugation for 30 minutes (centrifuge BEKMAN J-6B) at a speed of 4000 rpm and the supernatant is taken. in the second stage, the precipitate obtained is extracted with 250 ml of a 20-60% solution of ethyl alcohol in a ratio of 1:10 (g/ml), respectively, at room temperature for 45-60 minutes, followed by centrifugation, the supernatant is taken and combined with that obtained at the first stage, ultrafiltration through a membrane with with a pore size of 10 kD, with the collection of a low molecular weight fraction, which is then subjected to reverse osmosis concentration, followed by removal of ethanol under vacuum on a rotary evaporator , passing this concentrate through a column with a hydrophobic sorbent "Diasorb-100 C16" with an elution rate that ensures the passage of the front of the applied sample through the sorbent for 50-60 minutes, washing the sorbent with water and subsequent desorption of phytoecdysteroids, flavonoids and polyphenols with 70-75% ethyl alcohol , removal of ethanol from the alcohol wash under vacuum on a rotary evaporator and freeze drying.

The resulting extract (concentrate) can be used as a prophylactic agent to overcome the negative effects of stress, accelerate recovery after strong physical and mental stress, in particular, for people of extreme professions, athletes and for people engaged in heavy physical labor, exposed to harmful natural and industrial factors, older people experiencing a state of chronic stress.

The method is carried out as follows

Quinoa grains are crushed and sifted through a sieve with a pore size of 0.35 mm. Then a 2-stage extraction is carried out with stirring with a 20-60% solution of ethanol at room temperature for 1 hour. At the first stage, the crushed grains are extracted with 500 ml of a 20-60% ethanol solution in a ratio of 1:20 (g / ml), respectively, at room temperature for 1 hour. Clarified by centrifugation for 30 minutes (BEKMAN J-6B centrifuge ) at a speed of 4000 rpm and the supernatant was taken. At the second stage, the resulting precipitate is extracted with 250 ml of a 20-60% ethanol solution in a ratio of 1:10 (g/ml), respectively, at room temperature for 45-60 minutes, followed by centrifugation. The supernatant is taken and combined with that obtained in the first step. Next, ultrafiltration of the combined supernatant is carried out in a tangential flow on a membrane filtration unit based on an ASF-018 filter holder (VLADISRT, RF) through a membrane with a pore diameter of 10 kD with the collection of a low molecular weight fraction, which is then subjected to reverse osmosis concentration followed by removal of ethanol under vacuum on a rotary evaporator , passing this concentrate through a column with a hydrophobic sorbent "Diasorb-100 C16" with an elution rate that ensures the passage of the front of the applied sample through the sorbent for 50-60 minutes, washing the sorbent with water and subsequent desorption of phytoecdysteroids, flavonoids and polyphenols with 70-75% ethyl alcohol , removal of ethanol from the alcohol flush under vacuum on a rotary evaporator and an aqueous solution of phytoecdysteroids, flavonoids are lyophilized (freeze-drying LS-500, produced by PROINTECH, RF).

The content of 20-hydroxyecdysone in the final product is up to 50 mg/g of dry preparation, flavonoids up to 240 mg/g of dry preparation.

EXAMPLES OF OBTAINING EXTRACTS

EXAMPLE 1.

Quinoa grains (25 g) after grinding and sifting through a sieve with a pore size of 0.35 mm in the first stage, the grains were extracted with 500 ml of 20% ethyl alcohol in a ratio of 1:20 (g/ml), respectively, at room temperature for 1 hour, clarified by centrifugation for 30 minutes (BEKMAN J-6B centrifuge) at 4000 rpm, and the supernatant was collected. At the second stage, the precipitate obtained at the first stage was re-extracted with 250 ml of 20% ethyl alcohol in a ratio of 1:20 (g/ml), respectively, under similar conditions, centrifuged, the supernatant was taken and combined with that obtained at the first stage.

The combined supernatant was subjected to ultrafiltration in a tangential flow on a membrane filtration unit based on an ASF-018 filter holder (VLADISRT, RF) through a membrane with a pore diameter of 10 kD with the collection of a low molecular weight fraction. The resulting filtrate was concentrated by reverse osmosis on a unit with a URF-1812 roll membrane filter, and ethanol was removed under vacuum on a rotary evaporator.

After that, the resulting concentrate was passed through a column with a hydrophobic sorbent "Diasorb-100 C 16" (CJSC "BioKhimMak", volume 100 ml), with an elution rate that ensured the passage of the front of the applied sample through the sorbent for 60 minutes, further washing the column with water and washing product with 75% ethyl alcohol. After collecting the product, the alcohol was removed on a rotary evaporator and an aqueous solution of phytoecdysteroids, polyphenols, and flavonoids was lyophilized (freeze drying LS-500, PROINTECH, RF).

The content of 20-hydroxyecdysone in the dry extract was 36.9±1.8 mg/g, flavonoids 204.1±10.2 mg/g. The concentration of 20-hydroxyecdysone relative to the feedstock in the final dry product was 238 times.

EXAMPLE 2.

Quinoa grains (25 g) after grinding and sieving through a sieve with a pore size of 0.35 mm in the first stage, the grains were extracted with 500 ml of 60% ethyl alcohol in a ratio of 1:20 (g/ml), respectively, at room temperature for 1 hour, then clarified by centrifugation for 30 minutes (BEKMAN J-6B centrifuge) at 4000 rpm, and the supernatant was collected. At the second stage, the precipitate obtained at the first stage was re-extracted with 250 ml of 60% ethanol under similar conditions, centrifuged, the supernatant was taken and combined with that obtained at the first stage.

The combined supernatant was subjected to ultrafiltration in a tangential flow on a membrane filtration unit based on an ASF-018 filter holder (VLADISRT, RF) through a membrane with a pore diameter of 10 kD with the collection of a low molecular weight fraction. The resulting filtrate was concentrated by reverse osmosis on a unit with a URF-1812 roll membrane filter, and ethanol was removed under vacuum on a rotary evaporator.

After that, the resulting concentrate was passed through a column with a hydrophobic sorbent "Diasorb-100 C 16" (CJSC "BioKhimMak", volume 100 ml), with an elution rate that ensured the passage of the front of the applied sample through the sorbent for 60 minutes, further washing the column with water and washing product with 75% ethyl alcohol. After collecting the product, the alcohol was removed on a rotary evaporator and an aqueous solution of phytoecdysteroids, polyphenols, and flavonoids was lyophilized (freeze drying LS-500, PROINTECH, RF).

The content of 20-hydroxyecdysone in the dry extract was 30.8±1.5 mg/g, the amount of flavonoids was 170.1±8.2 mg/g. The concentration of 20-hydroxyecdysone relative to the feedstock in the final dry product was 200 times.

EXAMPLE 3.

Quinoa grains (200 g) were crushed and sifted through a sieve with a pore size of 0.35 mm, after which extraction was carried out in two stages. At the first stage, the grains were extracted with 500 ml of 40% ethyl alcohol in a ratio of 1:20 (g/ml) respectively at room temperature for 1 hour, Then they were clarified by centrifugation for 30 minutes (centrifuge BEKMAN J-6B) at a speed of 4000 rpm and the supernatant was collected. At the second stage, the precipitate obtained in the first stage was re-extracted with 250 ml of 40% ethanol under similar conditions, centrifuged, the supernatant was taken and combined with that obtained in the first stage, as described in examples 1 and 2. The combined supernatant was subjected to ultrafiltration through a membrane with dimensions pores of 10 kDa with the collection of the low molecular weight fraction, which was concentrated by reverse osmosis on an installation with a roll membrane filter "URF-1812" and ethanol was removed under vacuum on a rotary evaporator.

After that, the resulting concentrate was passed through a column with a hydrophobic sorbent "Diasorb-100 C 16" (CJSC "BioKhimMak", volume 100 ml), with an elution rate that ensured the passage of the front of the applied sample through the sorbent for 60 minutes, and after washing the column with water, it was washed off polyphenols, phytoecdysteroids and flavonoids with 75% ethyl alcohol. After collecting the product, the alcohol was removed on a rotary evaporator, and the aqueous solution was lyophilized (freeze drying LS-500, PROINTECH, Russia).

The content of 20-hydroxyecdysone in the resulting final product is 42.9 mg/g of dry matter, flavonoids 212.3±7.1 mg/g of dry matter, the concentration of 20-hydroxyecdysone relative to the feedstock is 168 times.

Claims (1)

A method for obtaining an extract from quinoa grains, including grinding and sifting the grains through a sieve with a pore size of 0.35 mm, then 2-stage extraction: at the first stage, the crushed grains are extracted with 500 ml of a 20-60% ethanol solution and in a ratio of 1: 20 g / ml, respectively, at room temperature for 1 h, clarified by centrifugation for 30 min on a BEKMAN J-6B centrifuge at a speed of 4000 rpm and the supernatant is taken, in the second stage, the resulting precipitate is extracted with 250 ml of 20-60% ethyl alcohol solution under similar conditions, in a ratio of 1:10 g/ml, respectively, at room temperature for 45-60 minutes, followed by centrifugation, the supernatant is taken and combined with that obtained at the first stage, ultrafiltration is carried out through a membrane with a pore size of 10 kD , with the collection of a low molecular weight fraction, which is then subjected to reverse osmosis concentration, followed by removal of ethanol under vacuum on a rotary evaporator, passing this th concentrate through a column with a hydrophobic sorbent, washing the sorbent with water and subsequent desorption of phytoecdysteroids, flavonoids and polyphenols with 70-75% ethyl alcohol, removing ethanol from the alcohol wash under vacuum on a rotary evaporator and freeze drying.