RU2257909C1 - Bilberry sprout concentrate and compositions based on thereof eliciting geriatric-protecting activity - Google Patents

Abstract

FIELD: medicine, chemical-pharmaceutical industry.

SUBSTANCE: invention relates to manufacturing compositions eliciting complex geriatric-protecting effect. Concentrate of bilberry sprouts (Vaccinium myrtillus) comprises flavonoids in the amount 5 weight %, not less, that elicits geriatric-protecting activity. The composition eliciting geriatric-protecting activity comprises bilberry sprouts concentrate in the amount from 10 to 90 weight % and pharmaceutically acceptable carrier. The composition comprises additionally at least one concentrate prepared from plants taken among the following: water parsnip (Sium latifolium), sorrel (Rumex acetosa), burnet (Sanguisorba officinalis), agrimony (Agrimonia asiatica), dropwort (Filipendula ulmaria), fallopia (Fallopia convulvus) and/or their mixtures wherein the content of these components is from 10 to 90 weight %. The composition can be made in liquid formulation, pills, tablets, capsules and granules. Compositions act on processes of free-radical oxidation, stabilize calcium homeostasis in cells and normalize function of cellular protective systems with effect on estrogen receptors and protect actively brain neurons against degenerative processes.

EFFECT: valuable medicinal properties of composition.

4 cl, 10 tbl, 13 ex

Description

The invention relates to medicine and can be used in food or pharmaceutical compositions with a complex geroprotective effect, namely with antioxidant, neuroprotective activities and the ability to regulate calcium homeostasis in the cell.

According to modern concepts, human aging is the result of a whole range of changes in the biochemical status of the human body. Particular importance is attached to the processes of free-radical oxidation and dysregulation of calcium homeostasis in cells, initiating the development of degenerative processes that occur in an aging organism against the background of a sharp weakening of natural defense systems, a decrease in the level of growth factors and hormones [Increasing healthy life span: convential measures and slowing the innate aging. Ann. N Y Acad. Sci. 2002, v. 959]. Currently, active work is underway in the world to search for and introduce into practice new pharmacological preparations, biologically active substances and simply nutritional supplements, the action of which is aimed at compensating for the deficiency of endogenous protective factors that occurs with age. In the world market there is a huge demand for such drugs. For example, the market for drugs for the treatment and prevention of senile dementia (including Alzheimer's disease) is estimated at 10 mln. dollars [National Institute on Aging, National Institutes Of Health. Progress Report on Alzheimer's Disease: Taking the Next Steps, Alzheimer's Disease. Education and Referral Center, Silver Spring, Maryland, 2002]. In recent years, geroprotective drugs based on herbal components that have a milder effect on the body compared with synthetic drugs have gained particular importance and demand. In particular, extracts from the Ginkgo biloba plant are actively used as an active component of medicines and numerous food additives recommended for improving cognitive functions and body resistance in normal aging [McKenna DJ, Jones K, Hughes K. Efficacy, safety, and use of ginkgo biloba in clinical and preclinical applications. Altern Ther Health Med. 2001; 7: 70-86]. According to the total sales of herbal preparations in the USA in 1998, Ginkgo preparations took the first place [Agriculture Business Profile, Jun. 2001 (web.site:http//www.agric.gov.ab.ca/agdex/200/263_830-2.html)].

However, the rather high cost of such imported drugs sharply limits the possibilities of their wide use in domestic preventive and therapeutic practice.

It is known that in plants there are biologically active substances, the so-called phytoestrogens that interact with human estrogen receptors. These include various flavonoids, such as quercetin, apigenin, and kempferol. As recently established, it is these components that determine the spectrum of positive effects of extracts from Ginkgo biloba [Karyn M. Maclennan, Cynthia L. Darlington, Paul F. Smith. The CNS effects of Ginkgo biloba extracts and ginkgolide B. Progress in Neurobiology 67 (2002) 235-257; Krish Chandrasekaran, Zara Mehrabian, Brigitte Spinnewyn, Katy Drieu, Gary Fiskum. Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia. Brain Research 922 (2001) 282-292].

However, this plant does not grow in Russia and is a product of import, and obtaining an effective extract from the leaves of Ginkgo biloba is a complex, high-tech and expensive process.

It is known to use the biologically active food supplement “Glutapiron” based on disodium salt of 2- (2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine-4-carboxamido) glutaric acid, which has anticarcinogenic and geroprotective effects (RU, No. 2112512, C1, class A 61 K 31/44, 06/10/98, bull. No. 16). However, it is most pronounced when the radiation exposure to the body is eliminated.

The task to which the invention is directed is to expand the arsenal of agents that can be used as biologically active additives in food and pharmaceutical compositions with a wide biological effect, in particular geroprotective.

The technical result that can be obtained by carrying out the invention is an expanded spectrum of biological effects on the body of the claimed substances, namely the prevention and protection of the human body from premature aging and increasing its resistance to the development of neurodegenerative diseases of old age, which are based on a complex neuroprotective effect as well as antioxidant activity and the ability to regulate cell calcium homeostasis.

In addition, it should be noted the relative cheapness of the preparations obtained due to the wide distribution in our climatic zone of the plants that make up their basis.

According to the invention, a blueberry shoot concentrate and compositions based on it are disclosed containing 10 to 90% by weight of blueberry shoot concentrates with a flavonoid content of at least 5%.

Bilberry has long been used in folk medicine as an astringent, gastrointestinal agent in the form of an infusion or a decoction of its fruits (Mashkovsky MD, M .: Medicine, 1977, vol. 1, p. 385. Big Encyclopedia of Traditional Medicine, M .: "ANS Publishing House", 2003, p. 1075, Medicines used in medical practice. Edited by MA Klyuyev, M .: Medicine, 1989, p. 460). Widely used preparations based on blueberries in ophthalmology. These are such drugs as Mirtilene forte, Striks, Mirtikam. In particular, blueberry anthocyanosides contribute to the regeneration of rhodopsin, the photosensitive retinal pigment, thereby increasing the sensitivity of the retina to changes in light intensity (Mironova E.M., Stavitskaya T.V., Shormaz I.N. KOF, Volume 4, No. 2, 2003 )

The use of blueberry shoot concentrates was not found by us.

When creating the present invention, it was found that blueberry shoot concentrates and compositions based on it with a flavonoid component content of at least 5% have a wide range of active biological effects on the body and can be used as agents with geroprotective and neuroprotective activity in the form of biologically active additives in food and pharmaceutical compositions, due to the effects we discovered on free radical oxidation processes, the ability to stabilize calcium homeostasis in cells and normalize the functioning of cellular protective systems with action on estrogen receptors, the functional activity of which decreases with age.

The term "concentrate" used in this application means (from Latin con ... (cum) - together and centrum - focus, center) a product with a high content of necessary (depending on its purpose) substances (Ukrainian Soviet Encyclopedic Dictionary of 3 T. / Editorial: A.V. Kudrinsky et al., K., Editor-in-Chief of the SSE, 1988, vol. 2, p. 127). The term "extract" used in this application means (from Latin extractum - extraction) condensed extract from plant material (Ukrainian Soviet Encyclopedic Dictionary in 3 volumes / Editorial: A.V. Kudrinsky et al., K., Chap. ed. USE, 1988, v. 3, p. 703). Receive with the help of solvents (extractants) - water, alcohol, etc. There are dry extracts (residual humidity not higher than 5%, condensed extracts - residual humidity 25% (Big Medical Encyclopedia, Ch. Ed. A.N. Bakulev, Moscow: “Soviet Encyclopedia”, 1964, Volume 35, p. 60) .

The concentrates of the plants indicated in the application may be in liquid form or in solid form, for example, granular or powder.

Suitable plant concentrates are obtained, for example, by drying or freezing by freezing freshly cut plants, followed by grinding and / or granulating the dried material, or by squeezing the freshly cut plants and collecting the liquid fraction.

A suitable method for producing concentrates of the claimed plants are various extraction methods known in the art, for example, by extracting freshly cut or dried plants with water or one or more solvents with suitable purity for food, or a mixture of water with one or more solvents suitable for inclusion in food cleanliness. Suitable solvents with suitable edible purity include propane, butane, butyl acetate, ethyl acetate, ethanol, carbon dioxide, acetone, nitrous oxide and propan-2-ol, or mixtures thereof. Preferred ones are water, ethanol, carbon dioxide, or mixtures thereof. After the extraction step, the liquid phase is optionally concentrated or dried by evaporation, or dried by freezing. In addition, concentrates can be obtained by introducing freshly cut or dried material of the listed plants into water and distilling the mixture with water vapor. The distillate is collected and preferably concentrated by evaporation to dryness. To obtain a concentrate with the claimed characteristics — a content of flavonoid components of at least 5 wt.% — Two or more concentration stages can be combined.

The content of flavonoid components is controlled using the method of quantitative determination of the amount of flavonoids used in the pharmaceutical industry (Chemical analysis of medicinal plants, under the editorship of N.I. Grinkevich, L.N. Safronich. M .: Higher school, 1983, pp. 91-92 ) The amount of the concentrate used according to the invention and the compositions containing it can vary widely depending on the patient to be treated and his needs.

The term "geroprotective activity" used in this application means a biologically active effect that slows down aging and prolongs life by preventing degenerative processes that accompany premature aging, and improving the quality of life by reducing the number and degree of intensity of life-compatible pathologies characteristic of the elderly and senile age.

The term “composition” means the use of any dosage or food form containing a concentrate of blueberry shoots or compositions based on it, which could find prophylactic or therapeutic use in medicine as a means with geroprotective activity for the prevention and prevention of degenerative processes that accompany premature aging.

To obtain compositions corresponding to the invention, the proposed blueberry shoot concentrate is mixed as an active ingredient with a pharmaceutical carrier according to the pharmaceutical compounding methods.

The invention also claims compositions additionally containing a concentrate of at least one of a number of plants: a cadastre - Sium latifolium, a sorrel - Rumex acetosa, a blood soup - Sanguisorba officinalis, a repetition - Agrimonia asiatica, meadowsweet - Filipendula ulmaria, fallopia - Fallopia convolvulus and mixtures, and the content of these components is from 10 to 90 wt.%, and the total content of flavonoid components is at least 5%.

The composition may further include edible components selected from the group consisting of vitamins, minerals, trace elements, fiber (including microcrystalline cellulose), flavoring agents, preservatives, colorants, emulsifiers, starch, calcium carbonate (chalk), sweeteners substances (including lactose, sucrose, fructose) while maintaining an effective dose of the composition.

The term "effective dose of a composition" used in this application means the use of that amount of blueberry shoot concentrate or compositions based on it, which, in combination with its activity and toxicity indicators, as well as on the basis of specialist knowledge should be effective in this dosage form.

Any composition of the foregoing may be in liquid or solid form, in particular in the form of pills, tablets, capsules and granules.

Figures 1 and 2 show data on the effect of a blueberry shoot concentrate on neurons, where Fig. 1 shows the effect of preliminary and subsequent incubation with substances on glutamate toxicity (100 μm) for cerebellar neurons; figure 2 - the effect of preliminary and subsequent incubation with substances on the toxicity of glutamate (100 μm) in a culture of neurons of the cerebral cortex.

The possibility of carrying out the invention with the implementation of the claimed purpose and obtaining a technical result is confirmed, but not limited to the following examples.

Example 1. Obtaining a concentrate of blueberry shoots.

The extracts of blueberry shoots were carried out on a capacitive apparatus (5 l) with a paddle mixer, a heat-exchange jacket and an integrated filter. In capacitive apparatuses, the extraction of plant materials was carried out according to the method of maceration. The ratio of solid and liquid phases at all stages of extraction was 1:10, the amount of extractant used was 3 l for each stage of extraction. As extractant, water or a 40% solution of ethyl alcohol in an amount of 3 L was taken, which was poured into the container of the extractor. In the apparatus, 300 g of pre-crushed vegetable raw materials of 0.63-3 mm fractions, bulk density of 316 kg / m ³ were poured into the apparatus, the bulk density was 316 kg / m ³ , the lid was turned on and the flow of coolant circulating through the jacket of the apparatus, which was supplied from preheated to 75 °, was turned on FROM. The extraction process was carried out at a temperature of 50 ± 5 ° C. Next, the extraction mass can withstand 0.5 hours. A pressure of up to 0.05 MPa is applied to the apparatus and the filtering process is underway, while the extract enters the receiving tank. The solid phase (raw material) remains on the filter screen of the apparatus. The described process is repeated three times. During the second and third extraction, 3 l of distilled water is loaded into the apparatus. After the third extraction, meal (waste raw materials) is discharged from the apparatus.

The resulting extract was combined, filtered and then concentrated in vacuo. The extract from the tank is sequentially fed in portions to the evaporator of the vacuum evaporator. Concentrated extracts were carried out under vacuum at a temperature of 45 ± 2 ° C and at a residual pressure of 76-38 mm RT. Art. up to about 1/13 of the original volume. At the end of the evaporation, an aqueous concentrated extract with a density of 1.106 g / cm ³ and distillation of water are obtained. Water distillation is used later on in the extraction stage, it is transported from the condensate collector to the extraction stage by a pump. The concentrated extract is sent to a measuring device and then to separation and drying.

The yield of extractive substances is A) aqueous extraction of 18%, B) extraction of 40% aqueous-alcoholic solution of 22.2%.

The determination of the amount of flavonoids was carried out according to the methodology used in the pharmaceutical industry (Chemical analysis of medicinal plants, under the editorship of N.I. Grinkevich, L.N. Safronich. M .: Higher school, 1983, pp. 91-92).

1 g of the test extract is placed in a bag of filter paper and extracted in a Soxhlet apparatus (with a capacity of 100 ml) with chloroform for 3 hours. The chloroform extract is discarded, and the bag with a hinge is dried and extracted in a round bottom flask with a reflux condenser in a water bath of 30 ml of methyl alcohol for 3 hours, stirring occasionally. Take 20 ml of extraction and methyl alcohol is distilled off to dryness. The dry residue is dissolved in 20 ml of distilled water, quantitatively transferred to a separatory funnel with a capacity of 50 ml and purified twice with 15 ml of carbon tetrachloride. The purified aqueous extract is treated five times with 20 ml of ethyl acetate, waiting each time for complete phase separation. The combined ethyl acetate extract was evaporated to dryness in vacuo and the residue was dried for 1 h in an oven at t = 65 ° C. The dried residue is dissolved in a small volume of ethyl alcohol and transferred quantitatively to a volumetric flask with a capacity of 25 ml, the volume is brought to the mark with ethyl alcohol (solution A), 0.5 ml of solution A is transferred to a volumetric flask with a capacity of 50 ml and the volume is adjusted to the mark with ethyl alcohol ( solution B).

The optical density of solution B was determined on a spectrophotometer in a cuvette with a layer thickness of 1 cm at λ = 330 nm.

The percentage of the sum of flavonoids X is calculated by the formula

- удельный показатель поглощения, равный 560,8; m - навеска сырья в расчете на взятый аликвот, г; w - потеря в массе сырья при высушивании (в %); L - толщина слоя, см.where D is the optical density of the test solution; 25 - the volume of solution A, ml; K is the dilution coefficient of solution A, equal to 100;

- specific absorption rate equal to 560.8; m is a sample of raw materials per aliquot taken, g; w is the loss in mass of raw materials upon drying (in%); L is the thickness of the layer, see

The content of flavonoid components was 23% and 21%, respectively.

Example 2. Obtaining a concentrate of blueberry shoots.

Obtaining a concentrate is analogous to example 1, but a hydro-pulse apparatus for extraction is used. The yield of extractive substances: A) water extraction - 19%; B) extraction with a 40% aqueous-alcoholic solution — 23.3%. The extraction time is 1 hour. Hydro module 1: 7. The amplitude of the ripple is 10 mm. The ripple frequency is 0.4 Hz. The extraction process was carried out at a temperature of 50 ± 5 ° C.

The content of flavonoid components was 25% and 23%, respectively.

Example 3. Obtaining a concentrate of blueberry shoots.

Obtaining a concentrate is analogous to example 1, but the concentration is carried out by lyophilization in freeze drying.

The content of flavonoid components was 23% and 21%, respectively.

Example 4. Obtaining a concentrate of shoots of blueberries.

Obtaining a concentrate is analogous to example 2, but the concentration is carried out by lyophilization in freeze drying.

The content of flavonoid components was 25% and 23%, respectively.

Example 5. Obtaining a concentrate of blueberry shoots.

Obtaining a concentrate is analogous to example 1, but acetone, ethyl acetate or propan-2-ol are used as extractant. The yield of extractive substances: A) extraction with acetone - 17.4%; B) extraction with ethyl acetate - 19.3%; C) extraction with propanol - 16.8%.

Example 6. Obtaining a concentrate of blueberry shoots.

Obtaining a concentrate is analogous to example 1, but acetone, ethyl acetate or propan-2-ol are used as extractant and a hydro-pulse apparatus for extraction is used. The yield of extractive substances: A) extraction with acetone - 19.4%; B) extraction with ethyl acetate - 20.3%; C) extraction with propanol - 18.6%. The extraction time is 1 hour. Hydro module 1: 7. The ripple amplitude is 10 mm. The ripple frequency is 0.4 Hz. The extraction process was carried out at a temperature of 50 ± 5 ° C.

Example 7. Antioxidant activity of a blueberry shoot concentrate.

The study of primary antioxidant activity was carried out using the so-called FRAP assay, based on the reduction of iron ions Fe ³⁺ in Fe ²⁺ . Thymol was used as a standard. The measurements were carried out at 505 nm. For calibration, an aqueous solution of 1000 μm / L Fe ₂ SO ₄ × 7H ₂ O was used.

Table 1.
Antioxidant activity of blueberry shoot concentrate. No. Name of concentrate % color development 1 Blueberry water concentrate - Vaccinium myrtillus, flavonoid content - 25 wt.% 32.1 2 Blueberry water concentrate - Vaccinium myrtillus, flavonoid content - 5 vol.% 50.1 3 Blueberry water concentrate - Vaccinium myrtillus, flavonoid content - 4 vol.% 59.3 4 Aqueous-alcoholic concentrate of blueberries Vaccinium myrtillus, the content of flavonoids - 23 wt.% 42,4 5 Water-alcoholic concentrate of blueberries Vaccinium myrtillus, flavonoid content - 5 wt.% 52.3 6 Aqueous-alcoholic concentrate of blueberries Vaccinium myrtillus, the content of flavonoids - 4 wt.% 62.1 7 Blueberry Acetone Concentrate - Vaccinium myrtillus, flavonoid content - 19 wt.% 43,4 8 Vaccinium myrtillus blueberry ethyl acetate concentrate, flavonoid content 20 wt.% 41.8 nine Vaccinium myrtillus blueberry propanol concentrate, flavonoid content -18 wt.% 48.8 10 Thymol 56.8

As can be seen from the table, the studied concentrates have a pronounced antioxidant activity that exceeds the activity of the standard - thymol. Moreover, with a total content of flavonoid components of less than 5%, the antioxidant activity is lower than the activity of the standard.

The effect of bilberry shoot concentrate on lipid peroxidation in mitochondria.

The leading role in the process of triggering cell death belongs to the mitochondria and the specific process of changing the permeability of the inner membrane of the mitochondria as a result of the opening of special mitochondrial pores (OMP). Aging of the body is accompanied by numerous violations in the functioning of mitochondria. In particular, with age, the sensitivity of mitochondria increases both to factors causing lipid peroxidation in mitochondria and to inducers of the discovery of WMD.

The experiments were conducted on preparations of isolated rat liver mitochondria in accordance with the methods described in the article: “Shevtsova EF, Kireeva EG, Bachurin S.O. The effect of a fragment of β-amyloid peptide 25-35 on the non-selective permeability of mitochondria. Bulletin of Experimental Biology and Medicine. 2001, v. 132 (12), pp. 652-656. "

Table 2.
The effect of blueberry concentrate on lipid peroxidation in mitochondria. No. Plant name IC50, mcg / ml 1 Blueberries Vaccinium myrtillus, contents 12 flavonoid components - 25 wt.% 2 Blueberries Vaccinium myrtillus, flavonoid content 5.7% by weight 16 3 Blueberries Vaccinium myrtillus, flavonoid content 4.1% 22 4 Quercetin 17 5 Ginkgo biloba fifteen

It has been reliably shown that the blueberry shoot concentrate reduces the formation of substances interacting with thiobarbituric acid (which is an indicator of lipid peroxidation) in rat liver mitochondria when they are incubated under standard energy conditions regardless of the respiration substrates used (succinate / rotenone or glutumate / malate) as without inducers of the discovery of WMD and lipid peroxidation (spontaneous activity or "mitochondrial aging in vitro"), and in the presence of tert-butyl hydroxyperoxide, calcium and inorganic th phosphate. Moreover, the action of the blueberry shoot concentrate is more pronounced and manifests itself at a lower effective concentration than the standard Ginkgo biloba extract.

Example 8. The effect of blueberry shoot concentrate on calcium cell homeostasis.

We studied the effect of blueberry shoot concentrate and Ginkgo biloba Egb 761 as a standard for capture of ⁴⁵ Ca ⁺² in synaptosomes of the cerebral cortex of newborn rat pups (9-10 days) upon stimulation of capture with 200 mM glutamic acid. Synaptosomes were isolated according to the standard Hayos method (Hajos F., Brain Res., 1975, 93, No. 3, 485-489). To accumulate the radioactive label, the P ₂ fraction of synaptosomes was suspended in incubation buffer A (mM): NaCl 132, KCl 5, HEPES 5, pH 7.4. Synaptosomes were incubated with the analyte for 5 minutes (37 ° C), then the capture of ⁴⁵ Ca ^{+2 was} stopped by filtration through GF / B filters (Whatman, England), washing three times with cold buffer B (145 mM KCl, 10 mM Tris, 54 mM Trilon B), and the amount of radioactive label on the scintillation counter was counted. The amount of captured ⁴⁵ Ca ⁺² in synaptosomes was determined as the difference between the label content in synaptosomes with and without glutamate stimulation and expressed as a percentage of control (control was taken as 100%).

Table 3.
The effect of blueberry concentrate (flavonoid content 25%) on the uptake of ⁴⁵ Ca ⁺² in rat cortex synaptosomes upon stimulation with glutamate (in% Ca from the control). Concentration, mcg / ml 0.5 1 2,5 5 10 25 fifty 100 200 Blueberry Concentrate - 100,2 76.7 64.5 44.7 65.9 75.8 84.9 87.5 Ginkgo biloba Egb 761 Extract 89.3 79.3 - 65.7 60.0 52,4 74.8 138.7 215.0

It was shown that the bilberry shoot concentrate at concentrations of 1-200 μg / ml inhibits the uptake of ⁴⁵ Ca ⁺² into rat synaposomes of the cerebral cortex (maximum inhibition of uptake is 55.3 ± 7.1% at a concentration of 10 μg / ml). IC ₅₀ = 9.1 μg / ml. Indicative is the effect of the blueberry shoot concentrate on calcium homeostasis of the cell at lower concentrations than Ginkgo biloba extract, and with greater efficiency.

Example 9. The neuroprotective effect of a concentrate of blueberry shoots on neurons under conditions of increased concentration of glutamate in the medium.

Glutamate is an exciting neurotransmitter in the brain that plays an important role in learning and memory. However, with an excess of glutamate, it can exhibit neurotoxic properties and cause neuronal degeneration. It is assumed that the neurotoxic effect of glutamate may play a role in age-related changes in the brain and in the pathogenesis of neurodegenerative diseases. A number of studies have shown the protective effect of plant extracts, especially Ginkgo biloba, as well as some of its individual components (quercetin) against glutamate toxicity in model experiments on cell culture, including in single studies on cultured neurons.

Cerebral grain-cell neurons (CZM) were obtained from the brain of rats of 7-8 days of age by trypsinization followed by pipetting [Schousboe A. et al., 1989; Lermontova et al., 2001].

Cortical neurons were obtained from the brain of 1-2-day-old rats by trypsinization followed by pipetting and centrifugation based on the previously described methods.

Cells suspended in the culture medium were added 1 ml to the wells of a 24-well plate (Corning) pre-coated with polylysine. The cell concentration was 2.5 × 10 ⁶ -5 × 10 ⁶ cells / ml. The culture medium consisted of a mixture of minimal Eagle medium and Ham modified Dulbecco's medium F12 (1: 1), supplemented with 10% calf serum, glutamine (2 mM), antibiotics, glucose (15 mM) and KCl to 25 mM, the pH of the medium was adjusted to 7-7.2 NaHCO ₃ . All media components from Sigma. Tablets with cultures were placed in a CO ₂ incubator at 37 ° C and 100% humidity. After a day, 80% of the culture medium was replaced with a similar fresh one with Supplement N1, but without serum and with the addition of cytosine arabinoside to suppress division of non-neuronal cells. 8-11 days after the maturation of neurons and their respective receptors, the cultures were exposed to 100 μM glutamate and test substances in conjunction or with preliminary incubation with the substances. Before incubation with glutamate, the culture medium was replaced with a minimal Eagle medium (without phenol red) supplemented with 10 mM HEPES, glucose, antibiotics, pH 7.4-7.5, and cultures were incubated for 30-40 min in a CO ₂ incubator before dropping the test substances or in conjunction with them. Then glutamate and test substances were added, if they were not already. Glutamate incubation was carried out for 30 min at room temperature. Then the medium was replaced with a standard culture medium with the corresponding test substances and the cultures were placed in an incubator for 18-20 hours. The state of the cells in the culture was assessed daily using an Aksiovert phase contrast inverted microscope with a video image source on a computer monitor with software that allows you to compare photos of the fields of cultured neurons obtained at different times of the experiment. Cultures were photographed before the experiment and compared with photographs of the corresponding visual fields after the experiment by the number of surviving neurons. For each concentration of the substance, cells were counted in 3 fields of view in 2-4 cultures before and 18-20 hours after exposure.

Incubation with bilberry shoot concentrate (flavonoid component content - 25%) before glutamate instillation, during and after incubation with glutamate revealed more reliable protective properties of bilberry shoot concentrate. The protective effect of the flavonoid component of the bilberry shoot concentrate quercetin is also shown (Fig. 1). In the culture of neurons of the cerebral cortex, which have more glutamate receptors compared with cerebellar neurons, the effect of glutamate was more pronounced and the protective properties of the bilberry shoot concentrate and quercetin manifested themselves more clearly (Fig. 2). A specific inhibitor of glutamate receptors NMDA-type MK-801 in the conditions of our experiment protected neurons by almost 100%, the protective properties of blueberry shoot concentrate are also well defined (Fig. 2). A well-known mechanism of action of plant extracts is their antioxidant properties. The need for preliminary incubation for the manifestation of a neuroprotective effect, along with antioxidant properties, may indicate a more specific effect of quercetin and components of the blueberry shoot concentrate on glutamate neurotoxicity, for example, through action on glutamate receptors. From an analysis of the results obtained, it follows that the blueberry shoot concentrate and the flavonoid component of the blueberry shoot concentrate quercetin exhibit pronounced neuroprotective properties and prevent neuronal degeneration.

Example 10. Obtaining plant extracts.

Obtaining plant extracts: cadastre, sorrel, blood pot, burr, meadowsweet, fallopia, was carried out as described in example 1.

The yield of extracts is presented in table 4.

Table 4.
The output of extractive substances. Plant name Extractive yield (%) water extraction extraction with a 40% aqueous-alcoholic solution Sium latifolium 12.1 8.8 Agrimonia eupatoria 20,2 26.8 Filipendula ulmaria 16,4 22.4 Fallopia convolvulus 15,2 20.1 Rumex acetosa 16,2 16.6 Sanguisorba officinalis 21.1 24.2

The determination of the amount of flavonoids was carried out as described in example 1a), according to the methodology used in the pharmaceutical industry (Chemical analysis of medicinal plants, under the editorship of N.I. Grinkevich, L.N. Safronich. M .: Higher school, 1983, p. 91-92).

The content of flavonoid components is presented in table 5.

Table 5.
The content of flavonoid components in plant extracts. Plant name The content of flavonoid components (%) water extraction extraction with a 40% aqueous-alcoholic solution Sium latifolium 15.4 12.5 Agrimonia eupatoria 24.4 23.6 Filipendula ulmaria 19.8 21,4 Fallopia convolvulus 23.1 21,2 Rumex acetosa 28,2 27.9 Sanguisorba officinalis 22.1 23.6

Example 11. Obtaining plant extracts.

a) 300 g of pre-crushed vegetable raw materials of 0.63-3 mm fractions in a rotary knife mill, bulk density of 316 kg / m ³ , was poured into an oven with forced ventilation. Drying and concentration were carried out at 45 ° С with constant air blowing at the same temperature.

b) 300 g of pre-crushed vegetable raw materials of 0.63-3 mm fractions in a rotary knife mill, bulk density of 316 kg / m ³ , was poured into freeze drying. The raw materials were frozen to a temperature of -25 ° C, then the foreline pump was turned on. Upon reaching a vacuum of 10 ^-5 MPa, heating of the freeze-drying plates was included, a temperature of 30 ° C. Drying and concentration were carried out until the sample temperature reached 30 ° C.

Table 6.
Flavonoid content (%) in dried and frozen plant concentrate. Plant name Dried concentrate Frozen Concentrate warrant officer 7.8 8.8 repeshka 6.4 7.2 meadowsweet 7.4 8.3 fallopia 7.7 8.1 sorrel 8.1 9.2 burnet 6.2 6.8

Example 12. Obtaining plant extracts.

Obtaining plant extracts: cadastre, sorrel, blood pot, burr, meadowsweet, fallopia, was carried out as described in example 1, but acetone, ethyl acetate or propan-2-ol were used as extractant. The yield of extracts is presented in table 7.

Table 7.
The output of extractive substances. Plant name Extractive yield (%) acetone extraction ethyl acetate extraction propanol extraction Sium latifolium 11.2 7.9 9.9 Agrimonia eupatoria 18,4 25.1 24.7 Filipendula ulmaria 17.3 19.7 23,4 Fallopia convolvulus 16.1 21,2 19.1 Rumex acetosa 18,4 15.9 17.8 Sanguisorba officinalis 22.2 23.1 25.6

The determination of the amount of flavonoids was carried out as described in example 1a), according to the methodology used in the pharmaceutical industry (Chemical analysis of medicinal plants, under the editorship of N.I. Grinkevich, L.N. Safronich. M., Higher School, 1983, p. 91-92).

The content of flavonoid components is presented in table 8.

Table 8.
The content of flavonoid components in plant extracts. Plant name The content of flavonoid components (%) acetone extraction ethyl acetate extraction propanol extraction Sium latifolium 13.5 13,4 13.6 Agrimonia eupatoria 22.1 21.9 22.9 Filipendula ulmaria 17.9 20.1 21.1 Fallopia convolvulus 22.5 22.2 20,2 Rumex acetosa 27.4 29.1 28.8 Sanguisorba officinalis 20.7 20.1 22.8

Example 13. The antioxidant activity of the compositions based on a blueberry concentrate and plant extracts: cadastre, sorrel, blood pot, burr, meadowsweet, fallopia.

The study of primary antioxidant activity was carried out using the so-called FRAP assay, based on the reduction of iron ions Fe ³⁺ in Fe ²⁺ . Thymol was used as a standard. The measurements were carried out at 505 nm. For calibration, an aqueous solution of 1000 μm / L Fe ₂ SO ₄ × 7H ₂ O was used.

Table 9.
Antioxidant activity of compositions based on blueberry concentrate. No. Composition % color development 1 blueberry concentrate - 90 wt.%
extract of the huntsman - 10 wt.% 42.3 2 blueberry concentrate - 50 wt.%
extract of the huntsman - 50 wt.% 44.7 3 blueberry concentrate - 90 wt.%
Repeshka extract - 10 wt.% 36,2 4 blueberry concentrate - 50 wt.%
repeshka extract - 50 wt.% 31.1 5 blueberry concentrate - 90 wt.%
meadowsweet extract - 10 wt.% 41,4 6 blueberry concentrate - 50 wt.%
meadowsweet extract - 50 wt.% 41.1 7 blueberry concentrate - 90 wt.%
fallopia extract - 10 wt.% 40.1 8 blueberry concentrate - 50 wt.%
fallopia extract - 50 wt.% 47.3 nine blueberry concentrate - 90 wt.%
sorrel extract - 10 wt.% 39.5 10 blueberry concentrate - 50 wt.%
sorrel extract - 50 wt.% 41.2 eleven blueberry concentrate - 90 wt.%
Hemophilus extract - 10 wt.% 44.6 12 blueberry concentrate - 50% by weight 41.2 thirteen blueberry concentrate - 60 wt.%
extract of the huntsman - 10 wt.%
microcrystalline cellulose - 30 wt.% 47.4 14 blueberry concentrate - 60 wt.%
Repeshka extract - 10 wt.%
microcrystalline cellulose - 30 wt.% 39.8 fifteen blueberry concentrate - 60 wt.% meadowsweet extract - 10 wt.% microcrystalline cellulose - 30 wt.% 48.3 16 blueberry concentrate - 60 wt.%
fallopia extract - 10 wt.%
microcrystalline cellulose - 30 wt.% 45.6 17 blueberry concentrate - 60 wt.%
sorrel extract - 10 wt.%
microcrystalline cellulose - 30 wt.% 42,4 eighteen blueberry concentrate - 60 wt.%
Hemophilus extract - 10 wt.%
microcrystalline cellulose - 30 wt.% 47.8 19 blueberry concentrate - 60 wt.%
extract of the huntsman - 10 wt.%
calcium carbonate (chalk) - 30 wt.% 49.3 twenty blueberry concentrate - 60 wt.%
Repeshka extract - 10 wt.%
calcium carbonate (chalk) - 30 wt.% 41.8 21 blueberry concentrate - 60 wt.%
meadowsweet extract - 10 wt.%
calcium carbonate (chalk) - 30 wt.% 47.6 22 blueberry concentrate - 60 wt.%
fallopia extract - 10 wt.%
calcium carbonate (chalk) - 30 wt.% 44.8 23 blueberry concentrate - 60 wt.%
sorrel extract - 10 wt.%
calcium carbonate (chalk) - 30 wt.% 41.7 24 blueberry concentrate - 60 wt.%
Hemophilus extract - 10 wt.%
calcium carbonate (chalk) - 30 wt.% 48.7 25 thymol 56.8

As can be seen from the table, the studied compositions based on blueberry concentrate have a pronounced antioxidant activity that exceeds the activity of the standard - thymol.

The effect of blueberry concentrate compositions on lipid peroxidation in mitochondria.

The experiments were conducted on preparations of isolated rat liver mitochondria in accordance with the methods described in the article: “Shevtsova EF, Kireeva EG, Bachurin S.O. The effect of a fragment of β-amyloid peptide 25-35 on the non-selective permeability of mitochondria. Bulletin of Experimental Biology and Medicine. 2001, v. 132 (12), pp. 652-656. ”

Table 10.
The effect of blueberry concentrate compositions on lipid peroxidation in mitochondria. No. Composition IC50, mcg / ml 1 blueberry concentrate - 90 wt.%
extract of the huntsman - 10 wt.% 28 2 blueberry concentrate - 90 wt.%
Repeshka extract - 10 wt.% 25.8 3 blueberry concentrate - 90 wt.%
meadowsweet extract - 10 wt.% 38 4 blueberry concentrate - 90 wt.%
fallopia extract - 10 wt.% 44.7 5 blueberry concentrate - 90 wt.%
sorrel extract - 10 wt.% 70 6 blueberry concentrate - 90 wt.%
Hemophilus extract - 10 wt.% > 50 7 blueberry concentrate - 60 wt.%
extract of the guardian - 10 wt.%
microcrystalline cellulose - 30 wt.% 32 8 blueberry concentrate - 60 wt.%
Repeshka extract - 10 wt.%
microcrystalline cellulose - 30 wt.% 27 nine blueberry concentrate - 60 wt.%
meadowsweet extract - 10 wt.%
microcrystalline cellulose - 30 wt.% 40 10 blueberry concentrate - 60 wt.%
fallopia extract - 10 wt.%
microcrystalline cellulose - 30 wt.% 45 eleven blueberry concentrate - 60 wt.%
sorrel extract - 10 wt.% 65 microcrystalline cellulose - 30 wt.% 12 blueberry concentrate - 60 wt.%
Hemophilus extract - 10 wt.%
microcrystalline cellulose - 30 wt.% 51 thirteen Quercetin 17 14 Ginkgo biloba fifteen

As can be seen from the table, the studied compositions based on blueberry concentrate have a pronounced antioxidant activity. It has been reliably shown that extracts of the studied plants reduce the formation of substances interacting with thiobarbituric acid (which is an indicator of lipid peroxidation) in rat liver mitochondria when incubated under standard energy conditions and can be considered as active antioxidants of lipid peroxidation in mitochondria.

Thus, the studies showed that blueberry shoot concentrates and compositions based on it with a flavonoid content of at least 5% and the above composition have a wide range of active biological effects on the body and can be used as agents with geroprotective and neuroprotective activity in the form of biologically active additives in food and pharmaceutical compositions, due to the effects we discovered on free radical oxidation processes, the ability to stabilize to normalize calcium homeostasis in cells and normalize the functioning of cellular protective systems with action on estrogen receptors, the functional activity of which decreases with age, actively protecting brain neurons from degenerative processes.

Claims (4)

1. Blueberry shoot concentrate (Vaccinium myrtillus) containing flavonoids in an amount of at least 5 wt.% And having geroprotective activity. 2. A composition having geroprotective activity, characterized in that it contains the concentrate according to claim 1 in an amount of 10-90 wt.% And a pharmaceutically acceptable carrier. 3. The composition according to claim 2, additionally contains a concentrate of at least one of the plants selected from the series - a cadastre (Sium latifolium), sorrel (Rumex acetosa), a blood pot (Sanguisorba officinalis), a burr (Agrimonia asiatica), meadowsweet (Filipendula) ulmaria), fallopia (Fallopia convolvulus) and / or mixtures thereof, the content of these components being 10-90 wt.%. 4. The composition according to PP.2 and 3, made in liquid form, in the form of pills, tablets, capsules, granules.

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