Agent for treatment and prevention of parkinson's disease

FIELD: medicine; pharmacology.

SUBSTANCE: extract of the birch bark containing the specific amount of betulin is used in prevention and treatment of Parkinson's disease.

EFFECT: extract of birch bark is efficient for prevention and treatment of Parkinson's disease.

19 dwg, 7 tbl

 

The technical field

The invention relates to the field of medicine. More specifically it relates to research new tools for the prevention and treatment of Parkinson's disease.

The level of technology

Parkinson's disease is a chronic progressive degenerative disease of the Central nervous system, clinically manifested violation of voluntary movements.

Parkinson's disease belongs to the primary, or idiopathic, parkinsonism. There are also secondary parkinsonism, which may be due to various reasons, such as encephalitis, excessive medication, atherosclerosis of cerebral vessels. The term "parkinsonism" refers to any syndrome in which there are characteristic for Parkinson's disease neurological disorders.

In Russia, according to various estimates, there are between 117000 to 338000 patients with Parkinson's disease. After dementia, epilepsy and cerebrosides disease Parkinson's disease is the most common problem of older people, as evidenced by its prevalence in Russia (1996):

- 1.8:1000 in the General population

- 1.0:100 in the population of those over 70

- 1.0:50 in the population of those over 80

The origin of Parkinson's disease remains to be elucidated, however, as the cause of the disease is considered the combination of the W several factors:

- aging

- heredity

some toxins and substances.

The average age of onset of Parkinson's disease - 55 years. At the same time in 10% of patients the disease occurs at a young age, 40 years. The incidence of Parkinson's disease does not depend on gender, race, social status and place of residence. Assuming that the increase in the average age of the population in the coming years, the prevalence of Parkinson's disease in the population will increase.

Drug therapy of Parkinson's disease is aimed at making up for the deficiency of dopamine, developing Parkinson's disease result from the death of brain cells, and the slow down disease progression, or death of brain neurons is neuroprotective therapy.

For pharmacotherapy of parkinsonism used cholinolytic drugs derived aminoadamantana, DOPA-containing means, inhibitors of monoamine oxidase (MAO) type b inhibitors, catechol-O-methyltransferase (COMT) and agonists of dopamine receptors.

Anticholinergic

This group of drugs began to be used for the treatment of Parkinson's disease for more than 100 years ago, but now is not completely lost its importance. In clinical practice anticholinergic drugs are used both in isolation and in combination with other antiparkinsonian and means however, in recent years there is quite restrained in their use due to the relatively low efficiency and a wide range of side effects. Anticholinergic drugs do not compensate for deficiency of dopamine and do not stop the progression of the disease, but only inhibit the increased activity of cholinergic systems. The use of these drugs is shown in the initial stages of the disease and individual clinically diagnosed positive dynamics. This group of drugs contraindicated with glaucoma, prostatic hypertrophy, intellectual-mnestic disorders.

Anticholinergic drugs have the greatest influence on the rigidity and autonomic manifestations of parkinsonism. Only some of them are effective against tremor.

Among the commonly used anticholinergic drugs include trihexyphenidyl, biperiden.

Derivatives amantadine

There are several assumptions about the mechanism of action of amantadine and its analogues. Drugs in this group have mild anticholinergic action and inhibit the reuptake of dopamine, and also give amphetamine-like effect, promoting the release of dopamine from the presynaptic terminala. In recent years it was found that amantadine acts as an antagonist of the NMDA receptor, h is about given the proven violations of glutamatergic neurotransmission in Parkinson's disease and its significance in the change corticosterone, thalamocortical and other relationships is also one of the possible mechanisms of action of drugs in this group.

Amantadina quite effective in rigid and akineticheskih forms a smaller impact on tremor. Their distinctive feature is the low toxicity, rare side effects, no contraindications, however, in General the efficacy of these drugs is low, and the improvement is not more than half of the patients (Walker et al., 1972). Among the side effects usually include dizziness, dry mouth, anxiety, visual hallucinations. Drugs in this group should not be used in elderly patients with intellectual disability disorders.

Among the drugs in this group include amantadine hydrochloride and sulfate.

DOPA - containing drugs

The role of dopamine as a possible basic therapeutic tool in the treatment of Parkinson's disease, studied with the 50-ies, when Carlsson noted that the symptoms of parkinsonism are caused by reserpine. In subsequent years, it was found reduced levels of dopamine in the striatum in Parkinson's disease (Behringer, Hornykiewicz, 1960), which has led to the emergence of a significant number diaminobenzoic drugs, components, and now the "gold standard" therapy of Parkinson's disease.

Dopamine does not pass through the blood-brain barrier. Therefore, replacement therapy is used metabolic precursor of dopamine, l-DOPA (L-dioxyphenilalanin), which passes through the blood-brain barrier and in dopaminergic neurons under the influence of cerebral DOPA-decarboxylase (MCM) is converted into dopamine (N.Quin, 1987).

One tablet clean levodopa contains 250 or 500 mg of active substance; the maximum daily dose of 3 g But due to the peculiarities of the metabolism of L-DOPA, which is "premature" biochemical changes outside of the brain by the decarboxylation, transamination and methylation, clean levodopa currently little used.

Pathogenetically justified and therefore widespread in the world is a combination of levodopa with a peripheral inhibitors L-MCM-carbidopa or benserazide. This allows you to reduce the dose and increase the duration of action of the drug (Vinstar, 1995; Wengyuan et al., 1999, N.Quinn, C.Olanow, 1996). The most frequently used drugs in this group - madopar (levodopa / benserazide in the ratio 4:1), Naka (levodopa / carbidopa - 10:1), sinemet (levodopa / carbidopa - 10:1).

Side effects of levodopa are manifested in three areas: in the autonomic nervous system (nausea, vomiting, orthostatic hypotension, the Arde), the motor system (psoriasis, clinical fluctuations), mental health problems (anxiety, agitation, hallucinations, depression with risk of suicide), in addition, marked teratogenic effects of the drug. Be careful while taking other medicines that may increase therapeutic and adverse effects of levodopa (adrenoreceptor agonists, tricyclic antidepressants) or, on the contrary, mitigate their (vitamin B6, neuroleptics, reserpine, methyldopa).

MAO inhibitors (IN)

MAO inhibitors (IN) according to their pharmacological action inhibit the metabolism of newly formed dopamine towards oxidative deamination by turning it end up in HVA acid. It is shown that selegiline is also inhibits the reuptake of dopamine. MAO inhibitors increase, thus, the content of dopamine in the brain tissue, preventing its collapse. In addition, there is evidence of the antioxidant effects of MAO inhibitors (IN), their neuroprotective capacity, which justifies the use of selegilina in the early stages of Parkinson's disease (J.Knoll, 1996; Myllyla et al., 1992).

More effective action, especially in the later stages, is the combination of MAO inhibitors (IN) with DOPA-containing drugs, which can significantly reduce the dose of L-DOPA, and therefore the severity of side effects.

Agonists of dopamine receptors

An important and promising direction was the search for medicines, capable to act in the "bypass" degenerating neurons, bypassing the presynaptic part of it, directly on dopamine receptors. So a new class of anti-Parkinsonian drugs are agonists of dopamine receptors.

Agonists of dopamine receptors is shown as monotherapy in the early stages of the disease and later in combination with levodopa preparations. In the early stages of the disease the effectiveness of dopamine agonists compared with that of levodopa preparations. However, after a few years the effectiveness of treatment with dopamine agonists is reduced and you will need to attach the drug levodopa. In the literature widely discusses how the use of agonists of dopamine receptors allows you to delay the development of complications when using drugs L-DOPA.

In patients with moderately or significantly pronounced symptoms joining a dopamine agonist drugs levodopa allows in many cases to increase the effectiveness of treatment and reduce the severity of motor fluctuations. (A.Lieberman et al., 1983; C.Goetz et al., 1985; R.McDonald, R.Horowski, 1983; Y.Mizuno et al., 1995).

Side effects of dopamine agonists are nausea, vomiting, orthostat the ical hypotension, which is caused by stimulation of peripheral dopaminergic receptors. Among the side effects due to Central dopaminergic receptors, include hallucinations, nightmares, changes in higher mental functions. Some patients experience constipation.

Currently, the company has accumulated considerable experience in the application ergalieva agonists of dopamine receptors (bromocriptine, pergolid, e.g.).

Inhibitors of COMT

One of the most promising and significant achievements in recent years was the opening of the brand new, highly efficient class antiparkinsonian drugs - inhibitors COMT. COMT is one of the main enzymes for metabolism of levodopa, dopamine and other catecholamines and their metabolites. COMTE is a common enzyme found in different parts of the body, including neurons and stroma, except nigrostriatum dopaminergic neurons (A.Kastner et al., 1994). Peripheral COMT inhibitors reduce the metabolism of levodopa and thereby increase its content in the striatum.

It should be noted that the drugs, which both Central and peripheral COMT inhibitors (tolkapon), according to most authors, give significantly more pronounced side effects (K.Holm, .Spencer, 1999). Currently, C is nesobrannosti application tolcapone discussed.

The invention

Despite a fairly broad spectrum actually used and under research drugs for treatment of Parkinson's disease, continues to be an urgent task of finding new means of this assignment that is due not only to lack of efficacy known means, but their side effects. It is to this task and the present invention is directed.

The essence of it is unexpectedly discovered by the inventors possible use for the prevention and treatment of Parkinson's disease and, respectively, for the manufacture of medicines for this purpose extract of the bark of the upper white part of birch bark. The extract is a set of triterpene compounds of the series, with a predominance (70%) of betulin (Betulinol).

Disclosure of inventions

In previously conducted by the authors of the present invention studies of birch bark extract (application for invention of the Russian Federation No. 2005138574) found that behavioral tests of General and targeted screening in mice, the drug demonstrates a psychotropic effect, the profile of which can be characterized as a manifestation of dopamine-positive activity, in particular, due to the fact that:

1. the extract at a dose of 50 mg/kg increases the horizontal locomotor activity is in hectometre Ugo Basile;

2. extract enhances timolepticeski effect of antidepressant Pirazidola combined with the introduction testing "despair";

3. the extract causes a neuroprotective effect in tests "maze" and "conditioned reflex of passive avoidance".

In addition, other researchers have obtained experimental evidence in favor of the antioxidant properties of the extract of birch bark.

The combination of the above experimental data allowed us to propose the hypothesis that the combination of the described properties meets the requirements that can be presented to the antiparkinsonian drug with potential (Tillerson J.L, Caudle W.M., Reveron M.E., Miller G.W. Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Exp. Neurol., 2002, 178:80-90).

Further research, a set of experiments using standard behavioral and pharmacological tests recommended for detection of anti-Parkinsonian activity (Voronina T.A., Waldman EA, Neronova LN. Guidelines for the study of the antiparkinsonian activity of pharmacological substances. In the book: "Manual on experimental (preclinical) study of new pharmacological substances", Moscow, 2000, 147-152), the nature and the results of which are outlined below, support this assumption.

Research the research was conducted using "birch Bark extract dry (BAS), produced by LLC "Birch world", Russia, Moscow and used for the manufacture of dietary supplements. The experiments were conducted on 196 mice-males line C57/BI weighing 20-25 g contained in laboratory vivarium on a standard diet.

BAS was injected into mice for 1.5 hours before testing using intragastric probe in the form of a suspension in 1%solution of potato starch in a dose of 50 mg/kg of animal weight. Mice of the control groups were treated with equivalent volumes of 1%starch.

The research results are reflected in the drawings, which depict:

figure 1 - the Effect of BAS on the total time catalepsy caused by haloperidol.

figure 2 - the Effect of BAS on the latent period of tremor caused by Arecoline, at 90 min after ingestion.

figure 3 - Dynamics of the effect of BAS on the latent period of the beginning of tremor in mice.

figure 4 - the Effect of BAS on the duration arecoline tremor in mice.

figure 5 - the Effect of BAS on duration in mice caused by Arecoline, at 90 min after ingestion.

6 - Effect of 5-fold prior to the introduction of BAS (50 mg/kg/day) on oligosynthesis effect of MPTP (24 mg/kg) in hectometre (**/* - significant difference from gr).

7 - the Effect of a single injection BAS (50 mg/kg, orally), Midantana (20 mg/kg, in, b) and their combinations on horizontal locomotor activity we who she C57BI/6 in the open-field test (arithmetic mean ± error standard deviation).

Fig. 8 - the Effect of a single injection BAS (50 mg/kg, orally), Midantana (20 mg/kg, in, b) and their combination on locomotor activity of mice C57BI/6 hectometre Ugo Basile (arithmetic mean ± error is standard deviation).

Determining the ability of the birch bark extract (BAS) to change the catalepsy induced in mice by haloperidol

Haloperidol is a typical neuroleptic - derived butyrophenones. It comes quickly and lasts a long time. The mechanism of action of haloperidol is associated with the blockade of dopamine receptors, Central alpha-adrenoceptor blocking action and disruption of neuronal reuptake and storage of catecholamines. The most frequent side effect of haloperidol are extrapyramidal disorders.

In the experiment we used 48 male mice C57BL/6 at the age of about 2 months (weighing 18-23 g), distributed randomly into 4 experimental groups:

1. Oral - 1% starch;/b - saline (FR);

2. Oral - BAS;/b - FR;

3. Oral - 1% starch;/b 1 mg/kg haloperidol;

4. Oral - BAS;/b 1 mg/kg haloperidol.

The duration of catalepsy each mouse were fixed for 2 min sessions after 30, 60 and 90 min after injection haloperi the La. Suspension BAS was introduced using intragastric probe inside for 1.5 hours before injection neuroleptic. The results of statistical processing of data groups is presented in figure 1.

A statistically significant effect catalepsy evolved to 90 minutes after administration of neuroleptic (right chart), occupying 112 seconds of the 120-second session (p<0.02). The action of the BAS after oral administration at a dose of 50 mg/kg did not differ from the placebo effect, however, is much higher (more than twice, p<0.05) reduced the duration of catalepsy induced by haloperidol.

Consequently, BAS in these experimental conditions, shows the dopamine-positive activity, counteracting the effect of the blocker DA receptors haloperidol.

Determining the ability of the birch bark extract (BAS) to influence the tremor caused by arecoline in mice.

The acetylcholine (ach) is a neurotransmitter numerous interneurons caudate nucleus, and that in this structure there is the greatest concentration of AX (Guttman M., S.J. Kish, Furukawa Y. Current concepts in the diagnosis and management of PA's disease. CMAJ, 2003, 168 (3), 293-301). Cholinergic model parkinsonism syndrome plays a primary activation of cholinergic neurons in the caudate nucleus of the system by the introduction of arecoline or oxotremorine. This experience is used agonist m-cholinergic receptors, arecoline in standard tremo agendas dose of 25 mg/kg, subcutaneously.

In the experiment we used 24 male C57BL/6 at the age of about 2 months (weighing 18-23 g), distributed randomly into 2 experimental groups:

1. Oral - 1% starch; p/K - 25 mg/kg arecoline;

2. Oral - BAS; p/K - 25 mg/kg arcolin.

Suspension BAS (50 mg/kg) was administered in accordance with the Protocol presented above. After 30, 60, 90, 120 and 150 minutes after administration of arecoline mice were recorded: a)latent period beginning tremor (s); b)the duration of the tremor (s).

The results of the statistical data presented in figure 2, 3, 4 and 5. Found that the introduction of arecoline on the background of the placebo causes of tremor at 33 seconds, while the same on the background of 50 mg/kg BAS sets the beginning of the tremor to 73 sec (figure 2; p<0.00000).

Moreover, the magnitude of the latent period under the action of BAS increases within 2 hours after administration of arecoline, and then begins to decline (figure 3).

The dynamics of the duration arecoline tremor after the introduction of BAS are presented in figure 4, from which it follows that the action of the extract leads to the reduction in the duration of up to one and a half hours after the introduction of arecoline. In later periods (2 and 2.5 h) the effectiveness of BAS disappears.

Thus, the maximum efficiency and effect on the duration of the tremor caused by Arek is Lin, accounts for 90 min (F(1, 22)=4.9959 when p=0.03588) (figure 5).

Thus, BAS demonstrates the presence of anticholinergic properties, manifested in the reduction of latent period and duration arecoline tremor.

Determining the ability of the birch bark extract (BAS) to influence the reduction of motor activity in mice hectometre Ugo Basils and "open field", on the model parkinsonism syndrome, caused by a systemic injection of neurotoxin MPTP mode prophylactic use BAS

Model parkinsonism syndrome, caused by injection of the neurotoxin MPTP (4-phenyl-1,2,3,6-tetrahydropyridine), is the most adequate model of parkinsonism, as MPTP selectively damages dopaminergic neurons of the black substance. The toxin under the action of MAO-b in astrocytes turns into a 1-methyl-4-phenylpyridine (MFP+), using dopamine Transporter is captured YES-ergicheskie neurons. Effects of MPTP caused toxic effect MFP+causing power failure through inhibition of complex 1 of the mitochondrial respiratory chain. This effect is most pronounced in primates and mice is in line C57BI/6 (Feldman RS, Meyer J.S., Quenzer, L.F. PA's disease and Alzheimer's disease. In: Principles of neuropsychopharmacology., Sinauer Associates, Inc., Publishers, Massachusetts, 1997, 861-909).

The present experiment was aimed at detecting presumably, the anti-toxin activity BAS, when MPTP was administered once on the background of preliminary 5-fold application of the suspension of the extract. In this variant of the experiment was to evaluate the possible "preventive" the importance of BAS.

In the experiment we used 36 male mice C57BI/6. Suspension BAS was administered orally daily dose of 50 mg/kg /day for 5 days. The toxin at a dose of 25 mg/kg or placebo was administered 30 min before a 10-min observation of physical activity in hectometre Ugo Basile.

Animals were randomly divided into 3 experimental groups:

1. Inside the 1% starch 5-fold; in/b - saline (FR) 1 times;

2. Inside the 1% starch 5-fold; in/b - MPTP to 1-fold;

3. Inside - BAS 5-fold; in/b - MPTP to 1-fold.

The General behavior of the animals after administration of BAS or placebo did not show significant differences from the usual. However, after the introduction of the toxin in mice was observed deviations from normal autonomic and behavioral signs: piloerection, salivation, metropulse, breathless.

Aggregated indicators of horizontal locomotor activity, automatically measured in hectometre Ugo Basile 10-min session presented on Fig.6.

The findings suggest that the introduction of BAS mice C57BI/6 for 5 days at 50 mg/kg / day leads to the restoration of the total number of horizontal lane is the sites in hectometre Ugo Basile 10-min session by 50% (p< 0.01), reduced with a single injection of MPTP on the 5th day of the experiment.

The result indicates that the extract has protective properties against Oligocene caused by the toxic effect of MPTP on DA ergicheskie neurons.

Determining the ability of the birch bark extract (BAS) to influence the reduction of motor activity in mice hectometre and "open field", on the model parkinsonism syndrome, caused by a systemic injection of neurotoxin MPTP in the mode of therapeutic action BAS

The present experiment was aimed at the discovery of the alleged anticarcinoma activity BAS on the background of two preliminary application of MPTP. In this variant of the experiment was supposed to: a) assess their own anticarcinoma activity BAS; b) to assess the ability of the extract to modulate the effect known antiparkinsonian drug Midantana (Morozov I.S., Petrov V.I., Sergeev S.A. Pharmacology of adamantanes, Volgograd, 2001).

In the experiment we used 88 male mice C57BI/6. The toxin at a dose of 30 mg/kg or placebo was administered intraperitoneally once a day for 2 days in a row. BAS was introduced on the second day a single dose of 50 mg/kg 1.5 hours before injection of MPTP. Midantan were administered a single dose of 20 mg/kg/W for 30 min prior to injection of the toxin. Preliminary animal was randomly divided into 5 exp the pilot groups:

Table 1
The Protocol of the experiment
no group1 dayDay 2
1 (n=12)Saline,/bPlacebo inside

Saline,/b
2 (n=19)MPTP, a/bPlacebo inside

MPTP, a/b
3 (n=19)MPTP, a/bMidantan,/b

MPTP, a/b
4 (n=19)MPTP, a/bBAS, inside

MPTP, a/b
5 (n=19)MPTP, a/bBAS, inside

Midantan,/b

MPTP, a/b

After 30 min after the second injection of MPTP in mice was consistently rated behavior in hectometre (5 min) and in the "open field" (3 min.)

After 30-40 min after MPTP injections on the second day of the horizontal locomotor activity (number of crossed squares) significantly decreased with 101,17±6,74 in group 1 to 4.58±1,21 in group 2 (p=0,000000, t-student test).

The results of the introduction of BAS, midantana and their combination on vertical activity presented on Fig.7. It is seen that the acute effect of 50 mg/kg BAS almost equipotential action of the drug comparison Midantana (20 mg/kg): the first increased the number of movements up to 331% (p=0.21, t-student test), and the second up to 327% (p=0.008, t-student test) relative to that in the group with MPTP.

The efficiency of their combination also differed from the effect of the toxin - 413% (p=0.007, t-student test), but statistical significance from the effects of the drugs individually marked.

Similar results were obtained when measuring the motor activity of the same groups of mice using actometry (Fig):

Double introduction of the toxin led to a fall in the number of movements with 234,8±32,2 in group 1 to 11.2±3,2 in group 2 (p=0,000; t-student test). In turn, acute application of BAS, Midantana and combinations thereof partially recovered induced locomotor deficit to 265% (p=0.013; t-student test), 275% (p=0.033; t-student test) and 311% (p=0,040; t-student test) respectively, relative to the values of group 2 taken 100%.

It should be noted that under the influence of MPTP and drugs were changed not only the amount of movement, but also the qualitative composition of motor activity: the introduction of the toxin has also led to a decrease in vertical activity (hours) and search activity (looking at holes in the open-field test with 31:1:1.7 in group 1 to 0.11±0,11 in group 2 (p=0,000; t-student test) and 5.3±0.1 to 0.6±0,2 (p=0,000; t-test Student the and), respectively.

The introduction of BAS and its combination with Midantana partially, but not statistically significantly restored these parameters locomotor activity selectively. Thus, the extract and the combination of increased reduced rate "horizontal activity" with a statistical accuracy of 0.04 and 0.03 on the criterion of Mann-Whitney, respectively.

Thus, experimentally selected conditions allowed us to discover two versions of the test (open field and automatic octomer Ugo Basile)that the bark extract at the dose of 50 mg/kg in a single oral administration has the ability to partially, but not statistically significantly to compensate for locomotor deficits in male mice C57BI/6 caused a twofold introduction of the toxin MPTP.

This effect was quantitatively comparable to the acute action of clinically common antiparkinsonian drug Midantana (Amantadine)is used in intraperitoneal dose of 20 mg/kg

The combination of the above-mentioned substances also increase the number and improve patterns of motor activity in mice with MPTP-induced parkinsonism syndrome, multiple best in comparison with the effects of both drugs separately.

In order to further research was further set of experiments in vitro and ex vivo aimed at from the value of the alleged dopamine-positive properties of BAS using the common neurochemical approaches, characterize the pharmacodynamics specific effects BAS.

To achieve the goal the following tasks were defined:

1. To evaluate the possibility of interaction with BAS receptors of dopamine, serotonin, glutamate and acetylcholine using membrane preparations and labelled by tritium ligands of these receptors.

2. To study the effect of BAS on functional indices of presynaptic activity: synaptosomal capture, biosynthesis and secretion of dopamine, as well as the metabolism of dopamine and serotonin in the homogenates of the competent structures of the brain of mice and rats.

Materials and animals

The experiments were conducted on mice-males line C57/BI weighing 20-25 g and rats male Wistar with weighing 180-200 g contained in laboratory vivarium on a standard diet.

In experiments ex vivo BAS was administered to animals for 1.5-2 hours before decapitate using probe inside in the form of a suspension in 1%solution of potato starch in a dose of 50 mg/kg the Animals of the control groups were treated with equivalent volumes of 1%starch.

Results studies

Experiment 1. Study of the effect of BAS on specific binding to receptors of neurotransmitters in the brain structures of rats

The statement of this series of experiments pursued 2 goals: (1) to examine whether the receptors are the main neurotransmitters of the brain is possible molecular the mi targets for primary (direct) pharmacological actions of BAS - in vitro; and (2) to study, not does the system introduction BAS significant indirect effects on receptor characteristics after 2 and 24 hours under the conditions ex vivo.

The extent of the affinity of the extract to receptors in vitro experiments assessed the value of IC50 concentrations of BAS, inhibiting the binding of radioligand 50%.

Possible dynamic changes of receptors under the influence of BAS in experiments ex vivo expressed in terms of Utah and Kd, reflecting the number of binding sites and the degree of affinity for the receptor, respectively.

In the study of receptor binding used the following ligands: for dopamine D1-, D2 - and D3-subtypes in the striatum - [G-3H]-SCH-23390, [G3H]-spiperone, 7-OH-[G-3H]-DPAT, respectively; for serotonin 5-HT2-subtype in the frontal cortex - [G-3H]-ketanserin; for glutamate NMDA subtype in the hippocampus - [3H-G](+)MK-801; for nicotinic acetylcholine receptors subtypes in brain [3H-G]-(±)Nicotine. All labeled ligands were obtained by the method of solid-phase catalysis in OHVV IMG RAS (head of. - academician of RAS Nfeed). Selection of specific ligands was carried out in accordance with the recommendations of IUPHAR ("Guide to Receptors and Channels", S.P.H.AIexander, A.Mathie and J.A.Peters, BJP, v.147, Suppl.3, 2006).

1.1. Binding to dopamine receptors in vitro.

a) Method

The study radioreceptor binding YES-R is the receptors was carried out according to the method of Sun et al. (2003) with modifications. Striatum rats was homogenized in 10 ml ice-cold (0-4° (C) 50 mm Tris-HCl buffer (pH 7.4 at 4° (C)using a homogenizer (Teflon-glass". The resulting suspension was centrifuged at 40000 g for 20 min in an ultracentrifuge Optima L-70K" (Beckman Coulter). After centrifugation the supernatant was decanted, the precipitate resuspendable re-homogenization in the same volume of buffer, then re-centrifuged. The washing procedure was performed three times, the precipitate resuspendable in 10 ml of 50 mm Tris-HCl incubation buffer (pH 7.4 at 37° (C) with the addition of salts (120 mm NaCl, 5 mm KCl, 2 mm CaCl2-2H2O, 1 mm MgCl2-6N2On) [11] and used in 250 μl in procedure binding [G3H]-ligands of dopamine receptors. The obtained membrane fraction was incubated with [G-3H]-SCH23390 (specific activity 60 CI/mmol), [G-3H]-spiperone (95 CI/mmol), 7-OH-[G-3H]-DPAT (120 CI/mmol). When the binding of 7-OH-[G-3H]-DPAT, to increase the selectivity for D3 receptors, the reaction mixture was excluded divalent cations and added 1 mm Na4EDTA.

[G-3H]-ligand was added to the incubation mixture in a volume of 50 μl at a final concentration of 0.1 nm, and incubated for 20 min at 37 ° °using solid state thermostat "Termite" (NGO "DNA-technology"). Nonspecific binding was defined by the presence of 50 μl of unlabeled ligand (20 μm), which was 12-14% of the total. Specific binding was calculated as the difference between total and nonspecific binding.

IC50in relation to the binding of [G-3H]-ligands was determined when added to the incubation medium 50 µl sample compounds in the final concentrations in the range 10-3-10-8M. the Volume of the incubation mixture was 500 µl. The binding process was stopped by adding 2 ml of ice-cold 50 mm Tris-HCl buffer and rapid filtration through glass fiber filters type a GF/B (Whatman), followed by washing with ice buffer total volume of 14 ml Filters pre-moistened before the experiment in ice-cold 50 mm Tris-HCl buffer for 3 hours.

The filters were dried for 12 hours at room temperature, then placed in scintillation fluid (reagent Shaving) with a volume of 4 ml and used for scintillation counting. The radioactivity of each sample was measured for 2 min in a scintillation counter Wallac 1411. The effectiveness of the account amounted to 40-45%.

b) Results

D1-dopamine receptors. The concentration dependence of the effects of BAS on dopamine receptors D1-type presented in Fig.9.

Conclusion: BAS has no affinity for dopamine receptors D1-type.

D2-dopamine receptors. The concentration dependence of the effects of BAS on dopamine receptors D2-type who redstavlena figure 10.

It is shown that the influence of common antagonist of this type of receptor Haloperidol describes the classical curve (dotted line) and is characterized by the value of IC50=0,57 µmol/L. Making BAS does not detect the effect of competition for receptor binding sites up to the maximum used a concentration of 1 mm (solid curve).

Conclusion: BAS has no affinity for dopamine receptors D2-type.

D3-dopamine receptors. The concentration dependence of the effects of BAS (Betulin) on dopamine receptors DS-type represented by figure 11.

The impact of the standard antagonist of this type of receptor (+)-7-OH-DPAT describes the classical curve (dotted line) and is characterized by the value of IC50=14.6 nmol/L. Making BAS found no effect of competition for receptor binding sites up to the maximum used a concentration of 1 mm (solid curve).

Conclusion: BAS has no affinity for the dopamine D3 receptor-type.

1.2. Binding to serotonin receptors in vitro

a) Method

The study radioreceptor binding to CT-receptors was carried out according to the method of Sun et al. (2003)described for the study of D2-dopamine receptors (see 1.1.) with the exception that the study took membrane preparations frontal cortex of the brain, and as a specific ligand used [G-3H]-ketanserin with the specific radioactivity of the 60 curies/mmol.

b) Results

Serotonin 5-HT2 receptors

The concentration dependence of the effects of BAS on serotonin receptors 5-HT2-type presented on Fig.

It is seen that the effect of common antagonist of this type of receptor ketanserina describes the classical curve (dotted line) and is characterized by the value of IC50=0.70 µmol/L. Making BAS found no effect of competition for receptor binding sites up to the maximum used a concentration of 1 mm (solid curve).

Conclusion: BAS has no affinity for serotonin receptors 5-HT2type.

1.3. Binding to NMDA receptors in vitro.

a) Method

The study of binding to the NMDA-receptor subtypes were produced according to method L.-M.Zhou et al. (1996) with modifications. Used tritium-labeled MK-801 (dizocilpine) with specific activity of 210 Curie/mol. The fabric of the hippocampus or cerebellum was homogenized in a Potter homogenizer (Teflon-glass") in 10 volumes of 5 mm HEPES/4.5 mm Tris buffer (pH 7.6)containing 0.32 M sucrose (buffer No. 1). The homogenate was diluted to 50 volumes of buffer to survey No. 2 (5 mm HEPES/4.5 mm Tris buffer (pH 7.6) and centrifuged 10 min at 1000 g. The supernatant was collected and again centrifuged 20 min at 25000 g. Sediment homogenized in 50 volumes of buffer # 2 and centrifuged 20 min at 8000 g. Supernatant and his soft, shaky ndcleu selected and the center of what was fugereville 20 min at 25000 g. The precipitate suspended in buffer No. 3 (5 mm HEPES/4.5 mm Tris buffer containing 1 mm Na4EDTA (pH 7.6), and the suspension is again centrifuged. This washing procedure was performed four times, with the last wash EDTA was removed from the composition. The final precipitate resuspendable in 5 volumes of buffer # 2 and kept in liquid nitrogen. The reaction mixture (final volume 0.5 ml) contained 200 ál of buffer No. 2, 50 µl of labeled ligand (50 nm p.p.) and 250 µl of the protein suspension. Nonspecific binding was determined in the presence of 50 μl of unlabeled ligand.

The reaction mixture was incubated at room temperature for 2 hours. After incubation the samples were filtered through glass fiber filters GF/B (Whatman)pre-soaked in 0.3% polyethylenimine for 2 hours at 4°C. Each tube was washed once with cold buffer No. 2, then the filters were washed three times with the same volume of buffer. The filters were dried in air until dry, and transferred to scintillation vials. The filters were filled with 5 ml of scintillation mixture containing 4 g PPO, 0.2 g ERROR on 1 l of toluene. Radioactivity was determined by scintillation counter Wallac 1411, with efficiency accounts for about 45%.

b) Results

Glutamate NMDA receptors

The concentration dependence of the effects of BAS (Betulin) on glutamate receptors of the NMDA type of submissions is and Fig.

It is seen that the effect of common antagonist of this type of receptor MK-801 (dizocilpine) describes the classical curve (solid) and is characterized by the value of IC50 of 7.3 nmol/L. Making BAS (Betulin) finds the effect of competition for receptor binding sites with IC50=8.5 µmol/L.

Conclusion: BAS has micromolar affinity for the NMDA glutamate receptors.

1.4. The binding of n-acetylcholine receptors in vitro

a) Method

The study radioreceptor binding to nicotinic receptors was carried out according to the method (Michael et al., 1986) with modifications. The final volume of the incubation mixture contained 50 μl of [3H-G](±)Nicotine, 250 ál of buffer and 200 ál of protein a suspension of membranes. To determine nonspecific binding was added unlabeled nicotine in the concentration range from 10-10up to 10-4M Tubes thermostatically in a shaker-incubator at 37°C for 40 minutes (Charles et al., 1997). Then the tubes were cooled in ice for 20 minutes (Regina et al., 2002). Filtration was performed through the filters GF/C, pre-soaked for days in a 0.3% solution of polyethylenimine at 4°With subsequent washing with cold buffer. The filters are transferred to scintillation vials and added to 5 ml scintillation fluid on the basis of dioxane. Radioactivity was determined by scintillation counter Wallac 1411" efficiency accounts for about 42%.

(b)

Acetylcholine receptors nicotinic type

The concentration dependence of the effects of BAS on n-holinoretseptory presented on Fig.

It is seen that the effect of common antagonist of this type of receptor - Nicotine describes the classical curve (solid) and is characterized by the value of IC50=13,4 nmol/L. Making BAS (Betulin) finds no effect of competition for receptor binding sites up to 10-4mol/L.

Conclusion: BAS shows no affinity for nicotinic holino-receptors.

2.1. Binding to dopamine receptors ex vivo

The results of studying the effect of a single injection of a suspension BAS (50 mg/kg, orally) for constants of receptor binding of labeled spiroperidol after 2 and 24 hours compared with placebo effect presented on Fig.

From the data in the drawing, it follows that the introduction of BAS reduces the binding of labeled ligand in the striatum as 2 hours and one day after injection. Moreover, these changes do not occur by reducing the affinity to dopamine receptors (KD), a thanks induced BAS decrease in the number of binding sites (Bmax): two hours, their density decreases with 904 fmol/mg protein in the control of up to 670 fmol/mg protein, while continuing to decrease after 24 hours up to 625 fmol/mg protein.

Conclusion: acute introduction BAS dose of 50 mg/kg induces a gradual decrease tightly the ti D2-dopamine receptors in the striatum of rats.

2.2 Binding to serotonin receptors ex vivo

The results of studying the effect of a single injection of a suspension BAS (50 mg/kg, orally) for constants of receptor binding labeled ketanserina after 2 and 24 hours compared with placebo effect presented on Fig.

From the data in the drawing, it follows that the introduction of BAS leads to an increase in the density of serotonin receptors in the frontal cortex after 2 hours with 106 fmol/mg protein to 164 fmol/mg protein, while the next day there is some reduction of this indicator (130 fmol/mg protein), not reaching, however, values with placebo. Draws attention to itself and the observed 2 hours after the introduction of BAS reduced affinity of the ligand to the receptor from 5 to 8 nm, disappearing through the day (5,9 nm).

Conclusion: acute introduction BAS dose of 50 mg/kg induces a transient increase in binding to serotonin receptors in the cortex of the brain.

2.3 Linking NMDA glutamate receptors ex vivo

The results of studying the effect of a single injection of a suspension BAS (50 mg/kg, orally) for constants of receptor binding labeled MK-801 after 2 and 24 hours compared with placebo effect presented on Fig.

From the presented data it follows that the introduction of 50 mg/kg BAS leads to a significant increase in the density of NMDA receptors in the hippocampus of rats after 24 hours, 3154 fmol/mg protein to 8276 fmol/mg protein b is C change affinity. However, 2 hours after the introduction of BAS notes and transient worsening of affinity to receptors on 22%.

Conclusion: acute introduction BAS dose of 50 mg/kg induces an increase in the binding to NMDA receptors in the hippocampus of the rat brain after 24 hours.

2.4 Linking with n-acetylcholine receptors ex vivo

The results of studying the effect of a single injection of a suspension BAS (50 mg/kg, orally) for constants of receptor binding of labeled nicotine after 2 and 24 hours compared with placebo effect presented on Fig.

From the presented data it follows that the introduction of 50 mg/kg BAS leads to a significant increase in the density of nicotinic receptors in the frontal cortex of rats after 24 hours, 232 protein to 334 fmol/mg protein without changing the affinity. 2 hours after the introduction of BAS significant changes in receptor characteristics not observed.

Conclusion: acute introduction BAS dose of 50 mg/kg induces a significant increase in the binding of n-acetylcholine receptors in the cerebral cortex of rat brain.

Experiment 2. Study of the effect of BAS on the functional system of the reuptake of dopamine uptake in rat brain

System reverse takeover (presynaptic transport) neurotransmitters of the Central nervous system is an effective way of regulating synaptic neuropterida, therefore, is the target of many psychotropic the drugs: psychoactive, ANTIPARKINSONISM, antidepressant.

With regard to the pharmacological mode of action BAS, including these activities in this series of experiments investigated the effects of BAS on the system reuptake of dopamine in isolated nerve endings of the striatum of rats.

a) Method

Synaptosomal capture [3N]-YES

Obtaining coarse fraction synaptosomes structures: then from the brain of rats striatum homogenized using a homogenizer glass-Teflon" in 10 volumes of chilled solution of 0.32 M sucrose, equilibrated with 10 mm Tris-HCl at 0-4°C. the Resulting homogenate was centrifuged in a centrifuge at 1000 g for 10 minutes Then the supernatant (S1) decantation and again centrifuged at 12000 g for 25 minutes the precipitate P2 ("coarse fraction by synaptosomes structures") resuspendable in a modified Krebs buffer (125 mm NaCl, 5 mm KCl, 1.2 mm MgSO4, 1.2 mm CaCl2, 10 mm HEPES, 10 mm D-glucose; pH 7.4), at a rate of 5 ml buffer per 1 g of original tissue.

The study of the capture of [3N]-YES. Procedure capture was carried out according to Protocol [13]. For the reaction of capture used the incubation medium containing 125 mm NaCl, 5 mm KCl, 1.2 mm MgSO4, 1.2 mm CaCl2, 10 mm HEPES, 10 mm D-glucose, 200 μm solution of pargyline (inhibitor of MAO-b) (pH of incubation medium = 7.4). To determine the percentage of nonspecific processes instead of the environment Inc is Bali used a 0.32 M sucrose solution, containing 10 mm Tris-HCl (pH 7.4), in which synaptosome incubated at 0-4°C.

The capture reaction was started with the introduction to the incubation medium containing the desired concentration of [3H]-YES, pargyline and analyte, 50 μl of the suspension by synaptosomes structures. The tubes were incubated in solid-state thermostat "Termite" (NGO "DNA-technology") 3 min at 37°C. the Reaction was stopped by rapid ultrafiltration through glass-fiber filters Whatman GF/C Filters were washed twice with 5 ml ice-cold saline solution, dried at room temperature, transferred to scintillation vials and added to 7 ml of scintillation fluid Shaving, and then measured their levels of radioactivity using a scintillation counter Wallac 1411 efficiency account tritium 40-45%.

b) Results

The study of drug action BAS on synaptosomal reuptake of dopamine in vitro was launched with the introduction to the incubation medium containing the desired concentration of [3H]-YES, pargyline (MAO inhibitor) and analyte, 50 μl of the suspension by synaptosomes structures. Previously it was shown that PPP Midantan able to inhibit the reuptake of dopamine with a concentration of proinvestirovany with IC50 ranging concentrations ˜200-250 microns. In this regard, we measured the reuptake of dopamine at a concentration of Midantana (250 μm) in incubations mixture.

Determination of the influence of BAS on the reuptake of dopamine in the brain tissue of rats was performed in 4 groups: group 1 - physical R-R. (0.9% NaCl); group 2 - BAS (Betulin) 500 µm; group 3 - Midantan (250 μm); group 4 - Midantan (250 μm) + BAS (Betulin) 500 ám.

It was found a statistically significant inhibition of reuptake of dopamine (40%). When the joint making Midantana (250 μm) + BAS(Betulin 500 μm) was found more pronounced in comparison with the introduction of Midantana (250 μm), reuptake inhibition is about 60%, which proves the ability of BAS to potentiate 30% inhibitory effect Midantana on the reuptake of dopamine (Fig).

Conclusion: BAS has no direct effect on the dopamine Transporter, however, can potentiate the inhibitory effect Midantana additional 30%.

Experiment 3. Study of the effect of BAS on the process of regulation of the biosynthesis of dopamine and serotonin in the brain structures in mice ex vivo

The biosynthesis of neurotransmitters into the presynaptic nerve endings is the second of the functional links of neuropterida under the regulatory influence of membrane receptors and intracellular factors. It was therefore explored the effects of BAS on the activity of the biosynthesis of dopamine and serotonin in the homogenates of cerebral cortex, striatum and hippocampus of mice.

and IU is the odik

The content of monoamines was determined by high performance liquid chromatography with electrochemical detection (HPLC-ED).

Deciding on the content of monoamines and their metabolites in brain tissue of mice was performed in 2 groups. The analyte was injected intraperitoneally in a volume of 2 ml/kg NSD1015 for 30 minutes, BAS 2 hours before decapitate animals.

The first group of animals was injected physiologic solution oral and NSD 1015 (50 mg/kg) intraperitoneally. The second group of animals was injected BAS (50 mg/kg) orally and NSD 1015 (50 mg/kg) intraperitoneally.

The need to use NSD1015 to assess biosynthesis due to the high intensity of the processes of biosynthesis of monoamines, resulting in a relatively low content of their predecessors in the brain. NSD1015 blocks the synthesis of dopamine from l-DOPA due to inhibition of the enzyme L-iminodicarboxylate aromatic acids (DAAC). When this occurs, the accumulation of DOPA and accordingly a decrease in the concentration of dopamine and its metabolites, also there is an accumulation of tyrosine, leading to increased concentration of 5-GTP (precursor to serotonin).

b) Results

Presented in Tables 2-4 data indicate that systemic injection of BAS in the dose of 50 mg/kg every two hours leads to a decrease in the activity of the biosynthesis of neurotransmitters in the striatum of mouse brain: synthesis of l-DOPA is in (the concentration of the precursor of dopamine DOPA) is suppressed by 22%, and the synthesis of serotonin (the concentration of the precursor of serotonin 5-STES) - 32% (Table 2).

Table 2
The effect of BAS on the content of biogenic amines in the striatum of mice Was/6 background NSD 1015 (m±S.E.M)
DovONDOFUS5-STESYES5-GIUCGVKARTICLE
Phys. p-p + NSD10152,87±0,311,95±0,181,43±0,181,06±0,0645,37±2,752,30±0,274,30±0,623,23±0,20
BAS + NSD10152,24±0,08*1,82±0,141,26±0,180,72±0,07*52,98±2,602,43±0,323,85±0,753,78±0,19
*- statistically significant difference, U-test Mann-Whitney, p<0,05.

In the cortex BAS had no effect neither on the synthesis of YES or synthesis ARTICLE (Table 3)and in the hippocampus was suppressed only the synthesis of serotonin (Table 4):

Table 3
The effect of BAS on the content of biogenic amines in to the re of mice 57l/6 background NSD 1015 (m± S.E.M)
DovONDOFUS5-STESYES5-GIUCGVKARTICLE
Phys. p-p + NSD10150,98±0,093,45±0,360,10±0,041,00±0,150,37±0,091,86±0,200,68 0,065,61±0,24
BAS + NSD10150,86±0,114,21±0,370,10±0,030,82±0,050,36±0,071,79±0,190,41±0,07*7,98±0,90*
*- statistically significant difference, U-test Mann-Whitney, p<0,05

Table 4
The effect of BAS on the content of biogenic amines in the hippocampus of mice C57Black/6 background NSD 1015 (m±S.E.M)
DovONDOFUS5-STESYES5-GIUCGVKARTICLE
Phys. p-p + NSD10150,51±0,122,39±0,360,12±0,041,39±0,271,58±0,713,05±0,460,48±0,203,42 0,58
B3C + NSD10150,33±0,101,89±0,370,06±0,020,78±0,14*1,28±0,612,72±0,600,28±0,132,96±0,59
* - statistically significant difference, U-test Mann-Whitney, p<0,05

Conclusion: a single injection of 50 mg/kg BAS leads to the suppression of the biosynthesis of DA in the striatum tissue, as well as to inhibition of the biosynthesis of the ARTICLE in the tissue of the striatum and hippocampus.

Experiment 4. Study of the effect of BAS on the intracellular metabolism and the secretion of dopamine in the striatum tissues. frontal cortex and hippocampus of mice in ex vivo

Study of influence of BAS (50 mg/kg, orally, 2 hours) on the metabolism of monoaminergic neurotransmitters and secretion of dopamine were carried out in the brain of mice C57BI/6.

a) Method

The content of monoamines was determined by high performance liquid chromatography with electrochemical detection (HPLC-ED).

Deciding on the content of monoamines and their metabolites in brain tissue of mice was performed in 2 groups. Injections of substances was performed for 2 hours before decapitate animals.

The first group of animals was given a placebo, while the second group was injected BAS (50 mg/kg) orally.

b) Results

The metabolism of catecholamines (dopamine and noradrenaline) and Indo the amines in the presynaptic endings is carried out by the enzymes monoamine oxidase type A (serotonin) and (catecholamines), in the result of which is formed from the first 5-hydroxyindoleacetic acid (GIUK), and second - deoxypyridoxine acid (DOHUK)detectable intracellular. Extracellular synaptic active dopamine is exposed to the enzyme COMT (catecholamine-o-methyltransferase) and is metabolized in HVA acid (GVK), extracellular metabolite of dopamine.

Therefore, the level of GVK possible to judge the intensity of secretory processes in the brain ex vivo. So, two hours after the introduction of BAS in the studied brain structures is not observed changes in the intensity of the secretion of dopamine (Table 5, 6 and 7).

Table 5
The effect of BAS on the content of biogenic amines in the striatum of mice C57Black/6 (m±S.E.M)
DovONDOFUS5-STESYES5-GIUCGVKARTICLE5-GIUC/ST
Control0,74±0,153,01±0,103,54±0,330

0
64,57±7,372,08±0,206,20±0,655,33±0,230,39±0,05
BAS0,74±0,081,88 0,43*4,78±0,43*0

0
63,55±2,84of 3.77±0,22*of 5.82±0,404,59±0,180,82±0,04*
* - statistically significant difference, U-test Mann-Whitney, p<0,05

However, in the striatum decreases the level of norepinephrine (-37%), accompanied by a corresponding increase in the concentration of intracellular metabolite DOFUS (+35%), which indicates the activation of the metabolism of norepinephrine (Table 4). The effect of the acceleration of intracellular metabolism of catecholamines is also observed in the cortex, where DAFUK increases by 52% (Table 6).

Table 6
The effect of BAS on the content of biogenic amines in the cortex of mice C57Black/6. (m±S.E.M)
ONDOFUS5-STESYES5-GIUCGVKARTICLE5-GIUC/ST
Control3,53±0,320,29±0,050,07±0,010,74±0,171,56±0,180,75±0,115,90±0,090,27±0,03
BAS4,15±0,340,44±0,07*/td> 0,07±0,010,73±0,083,21±0,36*0,90±0,166,46±0,640,50±0,02*
* - statistically significant difference, U-test Mann-Whitney, p<0,05

Draws attention to the effects of BAS on the metabolism of serotonin - in all three brain structures is logged significant activation of the metabolism of the neurotransmitter, as measured by the increase in concentration of the main metabolite of serotonin, 5-treated acid.

td align="center"> 0,44±0,15
Table 7
The effect of BAS on the content of biogenic amines in the hippocampus of mice C57Black/6. (m±S.E.M)
ONDOFUS5-STESYES5-GIUCGVKARTICLEDOHUK/YESGIUK/ST
Control4,27±0,250,35±0,040,05±0,011,20±0,553,05±0,28*0,47±0,06of 4.44±0,180,37±0,06*0,68±0,05*
BAS2,09±0,470,51±0,190,05±0,011,05±0,284,20±0,73*3,62±0,541,18±0,25*1,32±0,10*

Conclusion: the Introduction of BAS does not lead to significant changes in the secretion of dopamine, but activates the metabolism of DA. However, little has been studied earlier ARTICLE-ergicheskie component plays a significant role as a functional antagonist of dopamine and independent. Range of pharmacological activity BAS confirms the importance of the last statement.

The use of birch bark extract with prevailing up to 70%content of betulin for the prevention and treatment of Parkinson's disease.



 

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1 tbl, 3 ex

FIELD: medicine; cosmetology.

SUBSTANCE: invention relates to the agents for elimination of grey hair. The agent contains the chromogenic agent, which contains the beer yeast, hop and water in the following correlation, in mass%: 5-10 of the beer yeast, 2.5-5 of hop, water - the rest.

EFFECT: these components are harmless and provide efficient restoration of dark color in grey hair.

1 tbl, 3 ex

FIELD: medicine; cosmetology.

SUBSTANCE: invention relates to the cosmetic agents, namely cosmetic agent for care and maintenance of the natural hair function; contains the routine cosmetic raw materials, differs in containing the 2-furanon derivative , at least one polymeric compound and at least one compound of protein hydrolysates and their derivatives.

EFFECT: invention efficiently maintains natural hair function.

6 cl, 32 ex, 1 dwg

FIELD: medicine; cosmetology.

SUBSTANCE: invention relates to the cosmetic agents, namely cosmetic agent for care and maintenance of the natural hair function; contains the routine cosmetic raw materials, differs in containing the 2-furanon derivative , at least one polymeric compound and at least one compound of protein hydrolysates and their derivatives.

EFFECT: invention efficiently maintains natural hair function.

6 cl, 32 ex, 1 dwg

FIELD: medicine, oncology, amino acids.

SUBSTANCE: invention relates, in particular, to the development of an antitumor preparation based on natural substances. Invention relates to an amino acid preparation comprising at least one modified essential amino acid obtained by treatment of amino acid by ultraviolet radiation (UV) at wavelength 250-350 nm for 12-80 h at temperature 15-30oC or with ozone at temperature 15-25oC. The modified amino acid has no toxicity for health cells. Also, invention relates to a method for preparing such preparation. Invention provides the development of an antitumor preparation based on modified amino acids and expanded assortment of antitumor preparations being without cytotoxicity for normal cells.

EFFECT: valuable medicinal antitumor properties of preparation.

8 cl, 4 tbl, 2 dwg, 4 ex

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