Source: https://russianpatents.com/patent/255/2557885.html
Timestamp: 2019-04-19 01:26:46+00:00

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The invention relates to medicine, specifically to pharmacology, and relates to funds that can be used in the treatment of inflammatory diseases of different etiology.
Today the range of official anti-inflammatory fitosredstv insufficient to address the problem of therapy of inflammatory diseases. The difficulty is that, first, inflammation is a highly dynamic process with many alternative and overlapping pathways , which impact systemically rather difficult. Secondly, long-term administration of synthetic anti-inflammatory drugs contributes to the occurrence of side effects . The decisive criterion in the choice of anti-inflammatory drugs is not only their effectiveness, but also safety. Therefore, alternative synthetic drugs with anti-inflammatory activity can serve as drugs of plant origin, which have a broad spectrum of biological activity, a mild effect on the body, well tolerated, with no toxic effects with prolonged use and a limited range of contraindications . Tincture of Echinacea - most widely currently used herbal preparation possessing immunostimulating and anti-inflammatory properties�I. It is known that biologically active substances (BAS), part of Echinacea can reduce the synthesis of proinflammatory cytokines, to inhibit lipoxygenase and ziklooksigenazny metabolism of arachidonic acid, and can also act as antioxidants and thus reduce inflammatory processes in tissues [3, 4, 5]. As the comparison drug, we used a Tincture of Echinacea purpurea ("Galenopharm", St. Petersburg, permitted for use on the territory of Russia, reg. No. 000167.01-2000). However, its disadvantage is that it is a concentrated extract of medicinal plants, a complex of biologically active components, to obtain the necessary therapeutic effect which requires careful selection and adjustment of the dose and duration of use, possible allergic reactions. Besides alcohol tinctures may be contraindicated for persons driving the vehicles, persons with alcohol dependence, children, adolescents.
Promising biologically active compounds possessing anti-inflammatory properties may be the polyphenolic compounds (PFCs) isolated from fruits of mountain ash. Thanks to a rich complex of biologically active substances the fruits of mountain ash are widely used in folk medicine for inflammatory diseases of the liver, gallbladder,�EC connective tissue [2, 6]. Recent studies of the mechanisms of action of natural polyphenols showed that the pharmacological activity is due to the ability to influence the activity of many regulatory proteins, thereby reducing the severity of inflammatory reactions .
The object of the invention is to expand the Arsenal fitosredstv anti-inflammatory effect.
This object is achieved in vivo by intragastric application of the 5-day course at a dose of 50 mg/kg polyphenol complex (PFC), isolated from the hydroalcoholic extract of the fruits of mountain ash by cleaning the water-alcohol extract of mountain ash from the protein-polysaccharide complex 95% ethyl alcohol. In vitro by the addition of PFC Rowan at doses 50 and 500 µg/ml in culture of mononuclear peripheral blood of volunteer donors with further yield assessment of anti-inflammatory cytokines.
According to the literature Rowan berries contain the following groups of biologically active compounds: polyphenol complex - flavonoids, phenolic acids, anthocyanins, catechins, tannins; carotenoids, polysaccharides (pectin) and sugar free, vitamin C, organic acids, etc. [8, 9]. The main phenolic compounds of fruits of mountain ash are rutin, cyanidin-3-o-glucoside and chlorogenic �Isleta. These substances have pronounced anti-inflammatory and antioxidant activity .
SFC have a broad spectrum of pharmacological activity - antioxidant, antihypoxic, anti-viral, antitumor activity [10, 11]. They are not addictive, the effect of "drug" and can be used for a long time. The biological activity of PFS allows to use them as medicaments.
The main mechanism of anti-inflammatory non-steroidal anti-inflammatory drugs (NSAIDs) is the inhibition of cyclooxygenase (COX) is a key enzyme involved in the conversion of arachidonic acid to endoperoxide, used for synthesis of prostaglandins and thromboxane. Prostaglandins are mediators and modulators of inflammation, pain and fever. Inhibition of COX reduces the level of prostaglandins that are responsible for the main symptoms of inflammation: swelling, vasodilation, fever, pain .
High anti-inflammatory effect of polyphenols is due to the many "points of application" of their actions to the evolving process of inflammation; it is believed that one of the main mechanisms are the inhibition of Pro-inflammatory enzymes (COX, lipoxygenase, NADP-H-oxidase and phospholipase A2)  and in�the impact on intracellular signaling molecule, leading to decreased production of proinflammatory cytokines IL-1, IL-6, IFNγ and TNF-α . The closest analogue of the proposed tools are agents of plant origin "Kamada", "kolhuri", "Kalenjin". However, the known tools are basically the total of medicines, to obtain the necessary therapeutic effect which requires careful selection and adjustment of the dose, the duration of application of the drug. On these drugs (or their components) possible allergic reactions. In the case of aqueous-alcoholic tinctures and extracts there are contraindications for persons with intolerance to alcohol, persons requiring attention in the workplace.
To increase the effectiveness of treatment and reduce the risk of side effects is the remedy in which the impact is amplified through the use of PFC official medicinal raw material, selected from water-alcohol extract of the fruits of mountain ash by purification from protein-polysaccharide complex 95% ethyl alcohol.
The technical result consists in the creation of new bioactive agents that have anti-inflammatory effect on the basis of pharmacologically active substances from the fruit of the Rowan tree.
New in the present invention to obtain�asanoha result is used as anti-inflammatories PFC, isolated from water-alcohol extract of the fruits of mountain ash by purification from protein-polysaccharide complex 95% ethyl alcohol, at a dose of 50 mg/kg a course of 5 days in vivo and in vitro at doses 50 and 500 µg/ml.
The invention will be clear from the following description and the attached drawing. In Fig.1 shows a flowchart of receiving 40% of the liquid extract of the fruit of the Rowan tree.
To establish the anti-inflammatory properties of PFC Rowan had studied their market impact (for 5 days) on cell response mice of the CBA/CaLac. And were tested in vitro to study the effect of PFC Rowan, on synthesis of cytokines by mononuclear cells of peripheral blood of healthy volunteer donors.
PFC ashberries isolated from the hydroalcoholic extract of the fruits of mountain ash by purification from protein-polysaccharide complex 95% ethyl alcohol.
To receive 40% of the extract used official medicinal plant material (mprm), collected in August 2012 near the city of Tomsk.
40% liquid extract of fruits of mountain ash received in the laboratory of pharmaceutical technology REC "pharmacy" method of fractional Remus�erachi a dynamic process . To obtain the extract used a laboratory reactor with a steam jacket ("the Radleys", Germany) connected to a submerged circulation unit temperature control "IO 1" ("Thermax", Russia), overhead stirrer RZR 2020 ("Heidolph, Germany) and ball the refrigerator.
The extract was prepared in a ratio of 1:5 in 40% ethanol and the extract received 500 ml of liquid extract. Process flow diagram is presented in figure 1. The dry residue liquid extract were determined using the gravimetric moisture meter MS-70 ("AND", Japan).
Rowan berries were crushed with a grinder and separated from dust by using a sieve with d=0.25 mm. the Crushed raw material is sieved through a sieve with d=5 mm. From the fraction of raw materials with a particle size of 5 mm with an electronic scale HL-200 ("AND", Japan) were weighed three sample raw weight by 100.0.
Counting the required number of source of water and ethanol. Mixed alcohol and water. Alcohol 96,5% - 440,2 ml of purified water - 653,5 ml.
The extract was obtained in the laboratory of pharmaceutical technology REC "pharmacy". To obtain the extract used a laboratory reactor with a steam jacket ("the Radleys", G�Romania), connected to submersible circulation unit temperature control "IO 1" ("Thermax", Russia), overhead stirrer RZR 2020 ("Heidolph, Germany) and ball the refrigerator.
In a laboratory reactor was loaded 100,0 g mprm and 640,5 ml of 40% ethanol. The unit controlling the temperature of the exposed temperature for extraction 80°C. the Overhead stirrer was connected to the reactor.
A laboratory reactor was left at 80°C and vigorous stirring for 1 hour.
After 1 hour of extraction of the stirrer and the temperature control was disconnected from the network. Opening the valve of the reactor was lowered working volume of the reactor in a beaker. At the same time from above into the reactor filed 140,5 ml of fresh extractant. Emaciated raw wrung out and then not used.
Stage download - maceration was repeated 2 more times. To do this each time in the reactor was uploaded and 100.0 g of fresh raw materials. The extractant used the previously obtained extract. During the extraction received 500 ml of the extract.
For treatment of related substances was performed upholding the extract for 3 days at a temperature of 8°C.
Supernatant extract was filtered through several layers of gauze.
The solids content in the liquid extract of the fruits of mountain ash amounted 18,25%.
6.3. Qualitative detection and quantitative determination of major BAS liquid extract of fruits of mountain ash.
6.3.1. Qualitative detection of biologically active compounds.
To determine the qualitative composition of biologically active substances in aqueous-alcoholic extract under vacuum distilled alcohol. The condensed extract was diluted with hot purified water, stirred and the precipitate was filtered. For the separation of complex mixtures of substances of the filtrate was applied the methods of selective extraction, using as extractants hexane, chloroform, ethyl acetate and n-butanol. Thus we received 4 fractions: hexane (GF), chloroform (HF), ethylacetate (EAF) and buchanania (BB).
From the obtained fractions under vacuum on a rotary installation was distilled organic solvents. The extractives residue was dissolved in 95% ethanol and analyzed using the methods of one-dimensional chromatography on paper (BH) and in a thin layer of sorbent (TLC "Sorbfil") in parallel with the known substances witnessed in different solvents (n-butanol - acetic acid - water 4:1:2, 4:1:5; 15-30% acetic acid). Detection of the substances was carried out in filtered UV-light at wavelengths� 254 nm before and after processing chromatograms of 2% ethanol solution of aluminum chloride [15, 16, 17]. The results of the determination are presented in table 2.
Thus, the qualitative composition of the extract of the fruit of the Rowan tree are represented by flavonoids (quercetin and rutin), phenolic acids (FC) (coffee, p-coumaric, chlorogenic, ferulic and vanillic) and anthocyanins (cyanide-3-O-glucoside).
We determined the quantitative content of flavonoids, FC, anthocyanins and sugars (table.3).
The amount of flavonoids in terms of rutin was determined spectrophotometrically with the use of complexing reaction with aluminum chloride [9, 18].
The quantitative content of the sum of FC acids in terms of chlorogenic acid was determined by direct spectrophotometric analysis of aqueous-alcoholic extract .
The content of total anthocyanins in terms of cyanidin-3-O-glucoside was determined spectrophotometrically according to your own color in the visible region .
The quantitative content of total free sugars in conversion to glucose was determined spectrophotometrically, based on the color reaction of monosaccharides with picric acid to form aminopyrine acid in sugar recovery group NO2in N 2.
The resulting aqueous-alcoholic extract pressed to ½ volume (250 ml) on a rotary evaporator Hei - VAP Value, Hei - VAP Advantage ("Heidolph, Germany). The condensed extract was placed in a conical flask of 2000 ml and slowly using a magnetic stirrer were added into the flask with 95% ethanol in the ratio 1:3 (750 ml) and left for 12 hours in a cool dark place. After 12 hours the contents of the flask were centrifuged with a speed of 3000 rpm for 30 minutes. The obtained supernatant was filtered and pressed on a rotary evaporator - PFC.
Received PFC was a thick viscous liquid of brown color, sour-bitter taste and odor. The solids content in the PFC amounted 53,71%.
To study the qualitative composition of biologically active substances PFC under vacuum distilled alcohol. Condensed PFC diluted with hot purified water, stirred and filtered. For the separation of complex mixtures of substances obtained aqueous solution was used the methods of selective extraction, using as extractants g�Xan, chloroform, ethyl acetate and n-butanol. Thus we received 4 fractions: hexane (GF), chloroform (HF), ethylacetate (EAF) and buchanania (BB).
From the obtained fractions under vacuum on a rotary installation was distilled organic solvents. The extractives residue was dissolved in 95% ethanol and analyzed using the methods of one-dimensional chromatography on paper (BH) and in a thin layer of sorbent (TLC "Sorbfil") in parallel with the known substances witnessed in different solvents (n-butanol - acetic acid - water 4:1:2, 4:1:5; 15-30% acetic acid). Detection of the substances was carried out in filtered UV-light at a wavelength of 254 nm before and after processing chromatograms of 2% ethanol solution of aluminum chloride [15, 16, 17]. The results of the determination are presented in table 4.
Thus, the qualitative composition of the PFC Rowan, represented by flavonoids (quercetin and rutin), FC (coffee, p-coumaric, chlorogenic, ferulic and vanillic) and anthocyanins (cyanide-3-O-glucoside).
We have the quantitative determination of free sugars, flavonoids, FC and anthocyanins (table.5).
The quantitative content of total free sugars in terms of glitch�zu was determined spectrophotometrically .
The quantitative content of the sum of FC acids in terms of chlorogenic acid was determined by direct spectrophotometric analysis .
As the comparison drug used Tincture of Echinacea purpurea ("Galenopharm", St. Petersburg, permitted for use on the territory of Russia, reg. No. 000167.01-2000), which before use put dealcoholization (diluted with the necessary amount of tincture 2 times in purified water, was evaporated in a thermostat at 37°C to reduce the amount of 4 times).
The influence of PFC Rowan, on the functional activity of immunocompetent cells was evaluated in experiments in vivo and in vitro. In vivo studies were conducted on 60 mice-males line CBA/CaLac weighing 18-20 g, obtained from the laboratory EBM, research Institute of pharmacology named after E. D. Goldberg" SB RAMS. Animals corresponded to 1 category (certified), contained in an incomplete barrier system, had constant access to water and food. Feeding the mouse�th was made complete granulated feed PC 121-10 (GOST-R 50258-92). Watering animals was carried out with distilled water from a sterilized plastic bottles with metal spouts-drinkers. The feed and water samples were periodically analyzed for microbiological contamination. The influence of PFC investigated plants on the functional activity of immunocompetent cells in vitro was determined by the ability of mononuclear cells in spontaneous and mitogen-stimulated test to synthesize proinflammatory (IL-1, IL-2, IFNγ) and anti-inflammatory (IL-4) cytokines in the presence of PFC Rowan. The obtained data were processed statistically using the software Statistica 6.0. For all samples tested the hypothesis of normality of the distribution in magnitude of the coefficient of skewness and coefficient of kurtosis. For samples obeying normal distribution, we calculated the mean value of the trait X and the error average value m. For samples that are not subject to normal distribution, we calculated the median Me and quartiles Q1-Q3. The hypothesis test of equality of averages was performed using t-student criterion, when the mismatch distribution to the normal law used the nonparametric Mann-Whitney test. The significance level of tests was set equal to 0.1%, 1% or 5%.
The animals were divided into 3 groups: 10 mice control 1, received within 5 days in 0.2 ml of distilled water intragastrically; 10 - experiment 1 received dealcoholizing tincture of Echinacea purpurea (ne), which was administered to the animals for 5 days at a dose of 50 mg/kg in a volume of 0.2 ml of distilled water intragastrically prototype; 10 - experience 2, received PFC Rowan, which was administered to the animals for 5 days at a dose of 50 mg/ml. the Effect of PFC Rowan on cellular immunity in animal line CBA/CaLac was evaluated in the reaction of delayed-type hypersensitivity (DTH). For this, mice were subjected to sensitization by subcutaneous injection of 1×107sheep red blood cells in a volume of 100 µl. On day 5 after sensitization under aponeurotic plate of one hind limb was injected with a resolving dose of antigen (1×108in a volume of 20 µl). In the contralateral paw as a control received saline in the same volume. Studied the effect of PFC Rowan on the process of formation of a clone of antigen-specific T lymphocytes, and they introduced a 5-day course to mice before sensitization (scheme I DTH), as well as the effect of plant compounds on the production of proinflammatory cytokines antigen specific T-lymphocytes: PFC Rowan introduced a 5-day course from the day of sensitization before the introduction of the resolving dose (II CX�mA DTH) .
As a result of experiments, it was found that course the introduction of PFC obtained from the fruit of mountain ash, contributed to the decline in the production by lymphocytes of proinflammatory cytokines when meeting with the antigen (table.6).
The influence of PFC Rowan on the functional activity of immunocompetent cells in vitro was determined by the ability of mononuclear cells in spontaneous and mitogen-stimulated test to synthesize proinflammatory (IL-1, IL-2, IFNγ) and anti-inflammatory (IL-4) cytokines.
Mononuclear cells were isolated by centrifugation of stabilized heparin (25-30 U/ml) peripheral blood of 18 healthy volunteer donors on a density gradient ficoll-Pak (ρ=1,077, "Amersham Biosciences AB, Sweden). Cell viability was counted in the camera Goryaeva with trypan blue and then volume was adjusted to the desired concentration of 2 million/ml using complete culture medium (PRS), consisting of medium RPMI-1640, 10% FBS, 2 mm L-glutamine and 40 mg/ml gentamicin.
Stimulated cytokine production IL-1, IL-2, IL-4, IFNγ was examined in a 24-hour cell cultures after the addition of lipopolysaccharide (LPS "Sigma", USA) for production of IL-1 or concanavalin A (Con A, Sigma, USA), IL-2, IL-4 and IFNγ. Spontaneous production of cytokines was determined by cultivating mononuclear cells in DRC without stimulants. For OTS�nki synthesis of the major immunoglobulins cell culture, activated the mitogen lacunosa (ML, "Sigma", USA), were incubated for 10 days. The cultivation was carried out in CO2incubator at 37°C in an atmosphere with 5% CO2. In the experimental samples were studied added PFCR in 2 concentrations of 50 and 500 μg/ml immediately prior to cultivation of mononuclear cells. The reference drug (Echinacea tincture) put dealcoholization and put in nutrient medium at a dose of 50 μg/ml. the Control samples were considered cells incubated without PFC Rowan.
The concentration of the studied cytokines in the supernatants of mononuclear cells was assessed by ELISA, using the appropriate kits (Vector-best, Novosibirsk).
In experiments in vitro PFC Rowan, at selected concentrations (50 and 500 μg/ml) without mitogenic stimulation had no effect on the synthesis of IL-1.
Incubation mitogenstimulated cells with PFC Rowan, at a dose of 50 μg/ml resulted in a decrease in the synthesis of the cytokine (374,73 (371,18-376,57) PG/ml) compared with control (393,15 (392,30-394,43) PG/ml; p<0.05) and tincture of Echinacea (381,24 (372,59-384,93) PG/ml; p<0,05).
In spontaneous test PFC Rowan had no effect on the synthesis of IL-2. Adding to mitogenstimulated cultures of cells of PFC in Rowan d�ze 50 and 500 μg/ml resulted in decreased production of the studied cytokine in comparison with control and drug comparisons (p< 0.05) (table.7).
In experiments in vitro PFC Rowan, contributed to the decrease in the concentration of IFNγ in cultures of mononuclear cells stimulated in the test. Level mitogenstimulated synthesis of IFNγ after making PFC Rowan in a dose of 500 µg/ml was decreased relative to the control and comparison drug and did not differ from performance in the spontaneous test. PFC Rowan dose of 50 μg/ml had no effect on stimulated synthesis of this cytokine (table.8).
PFC Rowan had no effect on spontaneous and stimulated production of anti - inflammatory cytokine IL-4 in vitro. Echinacea tincture contributed to a decrease in the synthesis of IL-4 in stimulated test (tab.9).
Thus it was experimentally established that the PFC, isolated from the hydroalcoholic extract of the fruits of mountain ash by purification from protein-polysaccharide complex 95% ethyl alcohol, applied 5-day course at a dose of 50 mg/kg intraperitoneally, decreases the production of proinflammatory cytokines antigen-activated T-lymphocytes unlike the prototype - tincture of Echinacea. In experiments in vitro PFC Rowan did not affect the production of anti-inflammatory t�of tokine IL-4 mitogen-activated cells, and to a greater extent than the reference drug (Echinacea tincture), contributed to a decrease in mitogen-induced synthesis of proinflammatory cytokines IL-1, IL-2 at a concentration of 50 μg/ml and IL-2, IFNγ in a dose of 500 μg/ml.
PFC, isolated from the hydroalcoholic extract of the fruits of mountain ash by purification from protein-polysaccharide complex 95% ethyl alcohol, has anti-inflammatory activity and may expand the Arsenal of drugs used in inflammatory diseases.
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The use of polyphenolic complex, obtained by extraction of the crushed fruits of mountain ash 40% ethyl alcohol at a ratio of 1:5 with subsequent condensation of the resulting aqueous-alcoholic extract to ½ of the volume of added�eat 95% of ethyl alcohol in the ratio 1:3, by centrifugation of the precipitate after 12 hours, filtration and concentration of the supernatant with obtaining polyphenolic complex, as a means of anti-inflammatory effect.
SUBSTANCE: invention represents a mixture of two structural isomers: 2,6-di(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)-4-methylphenol and its diastereomers, and 2-(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)-6-(2,2,1-trimethylbicyclo[2.2.1]hept-5-yl)-4-methylphenol, and their diastereomers with the ratio of the first and second structural isomer isomers from 60:40 wt % to 95:5 wt %.
EFFECT: extension of the arsenal of means, possessing simultaneously haemorheological, anti-aggregate, anti-thrombogenic, retinoprotecting, endothelium-protecting, neuroprotecting, anti-arrhythmia and anti-ischemic activity, enhancing the cerebral blood flow.

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