RU2324491C1 - Antiallergic agent - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: antiallergic agent is produced by toluene extraction of the birch bark and contains the active components such as betulin, lupeol and O-caffeate taken in particular quantities.

EFFECT: agent is efficient against allergy and doesn't reveal side effects.

11 tbl, 9 ex

Description

Technical field

The invention relates to medicine, and more specifically to the search for new anti-allergenic drugs.

State of the art

According to socio-economic damage, the impact on the level of health and quality of life, allergic diseases occupy one of the leading places in the structure of human pathology and represent a global medical and social problem. According to the literature, at present the allergy problem is a national security problem, 25-40% of the population of Russia have various allergic diseases, over the past 30 years the number of allergic complications in economically developed countries doubles every ten years. The socio-economic damage from allergic diseases in Russia is $ 2.6 billion per year.

There are a fairly large number of well-known antihistamines used in modern medicine, but the problem of finding new effective drugs against various diseases of allergic origin continues to be relevant. Especially important is the task of determining new antiallergenic agents of natural, in particular plant origin, with a minimum of side effects when applied.

In folk medicine, a number of plants have anti-inflammatory and anti-allergenic properties [http://eastbook.by.ru/minaeva/g14/d24.htm], including warty birch, which indicates the possibility of using anti-allergens from birch leaves for preparation and birch sap.

The birch bark has anti-inflammatory properties due to the pentacyclic triterpenoids included in its composition: betulin, as well as lupeol, uveol, betulinic and oleanolic acids and other minor components. The anti-allergenic activity of the products obtained from this plant is associated with the content of betulin in them (see patent RU 2254032), as well as another triterpenoid - oleanolic acid.

The antiallergenic activity of triterpenoids (as histamine H ₁ receptor blockers) is disclosed by Japanese authors (JP 10-287582), who investigated birch bark extract, consisting of triterpenoids, with a predominance of betulin among them. However, the composition of the studied extracts is not disclosed in this work, and another mechanism of action is indicated.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an antiallergenic agent based on a triterpenoid composition having a different mechanism of action than the known antiallergenic antihistamines and the compositions studied in the above patents.

Another objective of the present invention is to obtain such an anti-allergenic agent, which, not inferior in certain respects to the most effective drugs such as suprastin and clarithin (loratadine), does not give their side effects and is more active than betulin.

These problems are solved by the present invention by obtaining a composition of triterpenes of a certain composition, namely (wt.%):

betulin 65-71 lupeol 12-16 3-O- betulin coffee 5-15 related substances to one hundred%

The composition is obtained by extraction of birch bark - the upper part of the birch bark by the method according to patent RU No. 2192879, owned by the applicant of this application.

Example 1

The extraction of crushed birch bark was carried out with toluene for about 1.5 hours with constant stirring and a temperature of 90-110 ° C. After completion of the extraction process, the mass was filtered through tissue at a temperature of 40-50 ° C.

Toluene was partially distilled from the meal, and the remaining mass was cooled for 6-10 hours to a temperature of 15-5 ° C, after which repeated tissue filtration and washing of the mass on the filter with toluene were carried out. Then, vacuum distillation of toluene was carried out and the final product was obtained in the form of yellowish crystals. The obtained extract was subjected to high performance liquid chromatography (HPLC) under the following conditions: Separon S 6 × C ₁₈ column (3.3 × 150 mm), eluent: acetonitrile / methanol / water + phosphoric acid 40/40/20 + 0.6 ml per 100 ml of the mixture, flow rate of eluent 0.5 ml / min.

Detection was carried out using a UV detector at 210 nm. The composition of the extract according to the results of HPLC (%):

betulin - 65

Lupeol - 12

3-O-coffee betulin - 15

related substances - the rest is up to 100%

Example 2

The extract was obtained according to example 1 and subjected to additional purification with ethyl alcohol, then washing with water and drying. In this case, crystals were obtained from cream to white. (With additional purification, the content of betulin and lupeol increased and the content of betulin caffeate, which gave the crystals a yellow color, decreased). According to the results of HPLC, the extract had the following composition (%):

betulin - 71

Lupeol - 16

3-O-betulin coffee - 5

related substances - the rest is up to 100%

Examples of studies of antiallergenic activity

Evaluation of the specific antiallergenic effect of the composition according to the invention was carried out according to the guidelines approved by the Russian Pharmacological Committee “Guidelines for assessing the allergenic properties of pharmacological substances” and “Guidelines for the study of immunotropic activity of pharmacological substances” (2000). Albino male guinea pigs were used in the work. from the nursery of the Russian Academy of Medical Sciences (RAMS) "Central" weighing 250-300 g and mice of mice CBA line weighing 18-20 g from after a two-week quarantine in the vivarium of the Research Institute of Pharmacology of the Russian Academy of Medical Sciences, animals were kept under standard conditions with free access to food and water. Briquetted granular feeds were used as the main feed. Two effective preparations with different mechanisms of antiallergenic pharmacological action: clarithin (loratadine) and suprastin (chloropyramine).

Research methods

General anaphylaxis in albino guinea pigs

Taking into account modern ideas that allergic sensitization, stimulation of IgE antibody production is caused by the selective development of Th2 helpers, as well as experimental evidence of the allergenic properties of ovalbumin (13), a sensitization model of intact guinea pigs with a 0.6% solution of chicken egg protein (CJ) was chosen , the main allergenic component of which is ovalbumin.

To obtain an anaphylaxis reaction, animals of the control and experimental groups were orally immunized with a 0.6% solution of chicken egg protein (CJA) at a dose of 1 ml per 250 g of body weight for three days (dissolved in physiological solution of CJA to guinea pigs was administered orally through a soft plastic probe) (6).

In the first series of experiments, 12 days after the start of the immunization with CJD, the experimental composition was administered iv for the first three experimental groups of guinea pigs at a dose of 50 mg / kg for three days in animals of the experimental groups; at a dose of 50 mg / kg, 1% starch gel was administered to control animals. On the next day, the 1st experimental group of guinea pigs was injected with the test composition at a dose of 50 mg / kg, the 2nd experimental group with suprastin, at a dose of 50 mg / kg, a similar volume of solvent was administered to control animals. After 2 hours, animals of all groups were intracardially injected with CJD at a dose of 1 mg per 300 g of body weight, after which the development of an anaphylactic reaction was recorded.

In the second series of experiments, 12 days after the start of immunization with CJD, the experimental composition was administered intravenously to the experimental group of guinea pigs at a dose of 50 mg / kg for three days; the experimental group of animals was injected at a dose of 50 mg / kg, 1% starch gel was administered to control animals. On the next day, the first experimental group of guinea pigs was injected with the test composition at a dose of 50 mg / kg, the second experimental group was injected with betulin at a dose of 50 mg / kg, the control animals were given the same volume of solvent. After 2 hours, animals of all groups were intracardially injected with CJD at a dose of 1 mg per 300 g of body weight, after which the development of an anaphylactic reaction was recorded.

Additionally, the effect of the intravenous administration of the reference drug, clarithin, at a dose of 30 mg / kg, on the anaphylactic reaction of guinea pigs immunized with a 6% solution of CJD was studied.

The Weigle Anaphylactic Index was calculated using the following formula:

,

,

where N is the number of guinea pigs that have died;

N ₁ - the number of guinea pigs that developed severe shock;

N ₂ - the number of guinea pigs that developed moderate shock;

N ₃ - the number of guinea pigs that developed a mild shock;

N ₄ - Guinea pigs in which there was no shock.

With the death of all animals in the group, the Weigle index is 4 (++++). In severe shock - 3 (+++), in moderate shock - 2 (++), in mild shock - 1 (+), in the absence of anaphylactoid reactions in guinea pigs - the index is 0 (3).

Inflammation reaction to concanavalin A

The inflammatory response to Con A (a pseudo-allergic reaction) is based on Con A's ability non-specifically, i.e. without the participation of reagins with allergens on the membranes of target cells, as a result of a direct action on mast cell membranes and basophilic leukocytes, they release inflammatory mediators (histamine, serotonin, leukotrienes, etc.).

The experiments were carried out on mice of male CBA line weighing 18-20 g. Two routes of administration of the test substances were used - intraperitoneal and oral, the introduction was carried out in 1% starch gel. With intraperitoneal administration, the test composition was administered at doses of 5 mg / kg, 50 mg / kg and 100 mg / kg, betulin was administered at doses of 10 mg / kg and 100 mg / kg, reference preparations, suprastin and clarithin, were administered at doses of 5 mg / kg and 50 mg / kg. With the oral route of administration, the test composition was administered at doses of 5 mg / kg, 50 mg / kg and 100 mg / kg, betulin was administered at doses of 50 mg / kg and 100 mg / kg, suprastin and clarithin at doses of 5 mg / kg and 50 mg / kg The animals in the control group were similarly injected with an appropriate volume of starch gel. Taking into account the pharmacokinetics of the studied pharmacologically effective compounds, Con A (20 μl of the solution at a concentration of 5 mg / ml) was administered to mice of the experimental and control groups after 2 hours subplanarly (into the pads of the back foot), and the same volume of physiological saline was applied to the contralateral limb. After an hour, the mice were sacrificed, the mass of paws was determined, and the inflammation reaction index (I _p ) was calculated by the formula:

,

,

where P _op is the mass of the foot, into the pad of which Con A was injected;

P _to - the mass of the foot, in the pillow which was injected with saline.

The results of studies of the antiallergenic effect of the composition according to the invention showed the presence of antiallergenic biological activity, which is confirmed by the following examples.

The effect of the composition of the invention and betulin on exudative swelling of the foot of rats on carrageenan in comparison with dexamethasone

Outbred white rats of males weighing 170-200 were used in the experiments. Animals of the three experimental groups 60 minutes before the administration of carrageenan were injected with the composition at a dose of 50 mg / kg, betulin at a dose of 50 mg / kg or dexamethasone at a dose of 1.4 mg / kg, which is calculated for rats according to EJFreirech et al. (5) corresponds to a daily dose for a person of 15 mg. Control animals were similarly injected with 1% starch solution. Then, animals of the experimental and control groups were injected with 0.1 ml of a 1% solution of carrageenan (Sigma) in the right cushion of the hind foot, and 0.1 ml of solvent (physiological solution) in the left control paw. Then, for 4 hours, the animals of the control and experimental groups every hour measured the edema of the feet of the hind limbs with a micrometer. In addition, after 4 hours of experiment, immediately after the slaughter of the animals, both hind legs were cut off along the protrusion of the bones below the junction of the tibia and tibia and above the heel joint. The severity of acute exudative inflammation was assessed at the end of the experiment by the difference in the mass of the experimental (P _op ) and control (P _k ) paws according to the generally accepted method.

Study of the effect of the composition of the invention on anaphylaxis of isolated organs on a model of anaphylactic contracture of the smooth muscles of an isolated ileum Guinea pig

Albino guinea pigs weighing 250-300 g were sensitized with triply recrystallized ovalbumin (Sigma). Ovalbumin was administered three times a day in the form of a mixture consisting of 0.25 ml of physiological saline containing 5 mg of ovalbumin and 0.25 ml of complete Freund's adjuvant (Difco). The first injection was made subcutaneously in the hind limb; two subsequent intramuscularly into the thigh. The experiments were performed 4 weeks after the last sensitizing injection.

To prepare segments of the intestines, the animals were killed with a cervical dislocation, the abdominal cavity was opened and the ileum was isolated, which was placed in Krebs solution. Then sections of the ileum were cut out about 2 cm long, the intestinal contents were washed with Krebs solution and the upper layer of the longitudinal muscles was isolated. Thus obtained sample of the longitudinal ileum muscles was fixed at one end on a holder with platinum electrodes, transferred to a working chamber with Krebs solution, with a temperature of 37 ° C and constant transmission through a solution of carbogen (95-96% O ₂ , 5-4% CO ₂ ). The second end of the sample was fixed on the sensitive element of the mechanotron. After a 20-minute stabilization period, with periodic renewal of the Krebs solution in the working chamber, rectangular pulses with a frequency of 0.1 Hz, a duration of 1 ms and a voltage of 60 V. mumps caused by histamine solution (10 ^-7 M) (7) in the absence or presence of test compounds. Drug contractions were recorded using a mechanotron, converted electrical signals were recorded on a tape recorder. After registration of the effect, the incubation solution in the cuvette was replaced using a flow system. Measurements were taken on at least three segments of the ileum of each animal.

In the first series of experiments, the studied drugs, composition, suprastin and loratadine were introduced into the solution after receiving stable reactions to histamine 5-15 minutes before the next administration of the agonist. The composition was dissolved in 96.6% ethanol and added so that the final concentration of the drug was 2 × 10 ⁻³ mg / ml. In the interval between the addition of the test substance, the Krebs solution in the chamber was changed every 5 min.

In the second series of experiments, the composition was incubated with the ileum for an hour and for a day (in Krebs solution, the concentration of the studied drug was 4 × 10 ^-2 mg / ml) at room temperature, an appropriate volume of solvent was added to the control ileum samples. Next, we determined the contractile activity of the longitudinal ileum muscles caused by histamine.

Determination of NF-kB activity

U937 cells were cultured in DMEM cell medium with a bovine serum content of 10% and, prior to the experiment, were plated in 24-well plates at 200 cells / well. Using the TransFast transfection reagent (Promega, USA) according to the protocol of the manufacturer, the cells were transfected with the luciferase reporter gene under the promoter containing NF-kB binding elements (pNF-KB-LUC; Stratagene, USA) and the vector containing the gene β-galactosidase (pSV-β-galactosidase control vector; Promega, USA; to assess the effectiveness of transfection).

48 hours after transfection with the pSV-β-galactosidase vector, the cells were washed with PBS and fixed in a 2% formaldehyde / 0.2% glutardehyde solution prepared in phosphate-buffered saline (pH 7.4) for 5 min at 4 ° C. To analyze β-galactosidase activity, the cells were washed with PBS and incubated with o-nitrophenyl-β-D-galactopyranoside β-galactosidase substrate at 37 ° C until a yellow color developed. The degree of staining, which determines the transfection efficiency, was calculated on a microplate reader (PerkinElmer Wallac 1420 VICTOR2 Multilabel Counter) at a wavelength of 420 nm. After transfection of the plasmid pNF-kB-LUC, the claimed composition was added to the cells at a final concentration of 50 μg / ml, 10 μg / ml, 5 μg / ml and 1 μg / ml and incubated with it at 37 ° C and 5% CO ₂ . After 1 hour, TNFα (100 ng / ml) was added to the cells and incubated with it for another 3 hours under the same conditions. At the end of this period, the cells were washed with PBS, destroyed with lysis buffer (Promega, USA) and centrifuged at 12000g (1 min, 4 ° C). The supernatant was collected in sterile 0.5 ml microtubes and frozen to -70 ° C. To evaluate luciferase activity, the supernatant (5 μg of dissolved protein) was added to a 96-well polysterine plate (PerkinElmer Wallac) containing 10 μl / well of reporter lysis buffer. A lumen meter (PerkinElmer Wallac 1420 VICTOR2 Multilabel Counter) was programmed to add 50 μl Luciferase Assay Reagent (Promega) to each well and measure the luminous intensity 2 seconds after application. All samples were placed in three parallels. The activity of the nuclear factor kV was expressed in relation to the activity of β-galactosidase (which determines the efficiency of transfection) to the number of conventional light units obtained from the evaluation of luciferase activity. In order to correctly evaluate the experimental results, two controls were set: cells transfected with pFC-MEKK plasmid with a constitutively active luciferase gene (positive control), and cells transfected with pCIS-CK plasmid (Staratagene, USA) with no NF-kB binding elements in luciferase gene (negative control).

Statistical data processing

Initial data preparation and calculations were carried out in the statistical software package (PSP) STATISTICA (version 6.0) for WINDOWS. The results are presented as arithmetic mean and its average error. Significance of differences - by t-student test.

Example 3

The data presented in table 1 show that the intravenous administration of the composition at a dose of 50 mg / kg in 2 times inhibited the intensity of the systemic anaphylaxis reaction in animals of the experimental groups compared to the control. The reaction index in the control group of animals was 2.1 (in 4 guinea pigs severe anaphylactic shock was determined, in 3 - moderate shock, in 3 - mild shock). When a dose of 50 mg / kg was administered, 3 guinea pigs developed moderate shock, 4 weak mild shock, 3 anaphylactic reaction was not detected (Weigle reaction index was 1.0). With the intravenous administration of a comparison drug, in this case suprastin (the most effective drug in the treatment of severe forms of anaphylaxis), at a dose of 50 mg / kg, moderate anaphylactic shock was determined in 2 animals, weak shock developed in 2 pigs, and in 6 no anaphylactic reaction was detected (Weigle reaction index was 0.6).

Example 4

In a repeated experiment, it was also found that the intravenous administration of the composition according to the invention at a dose of 50 mg / kg (Table 2) halved the intensity of the anaphylaxis systemic reaction in guinea pigs. The reaction index at a dose of 50 mg / kg was 0.8 (in 1 guinea pigs severe anaphylactic shock was determined, in 2 - moderate shock, in 1 - mild shock, in 6 anaphylactic reaction was absent). With the intravenous administration of betulin at a dose of 50 mg / kg, 2 pigs developed severe shock, 2 - mild shock, 5 - no anaphylactic reaction (Weigle reaction index was 0.9). In the control group of animals, 1 guinea pig died, in 2 - a severe shock, in 2 animals a moderate anaphylactic shock was determined, in 5 pigs a weak shock developed (Weigle reaction index was 1.9).

When studying the effect of intravenous administration of clarithin at a dose of 30 mg / kg on the intensity of anaphylactic shock in guinea pigs immunized with 6% CJD, it was found that in the experimental group of 7 animals, 2 guinea pigs died and 2 developed severe shock , moderate shock was determined in 3 pigs, the reaction index was 2.9. In the control group, 7 out of 11 animals died, 2 animals developed severe shock, 2 animals showed moderate shock, and the reaction index was 3.5 (table 3).

Thus, in all of the above experiments, the composition according to the invention halves the severity of the systemic reaction of general anaphylaxis compared with the data of the control groups. The suppression of anaphylactic shock with the introduction of the composition is more pronounced than with the introduction of betulin and the comparison drug clarithin, which is widely used in various allergic diseases.

Example 5

From the data presented in table 4, it follows that a single intraperitoneal administration of the composition according to the invention in doses of 5 mg / kg, 50 mg / kg and 100 mg / kg to CBA mice caused a significant suppression of the pseudo-allergy reaction to Con A by 45.3%, 56.0% and 71.7%, respectively. With a single intravenous injection of betulin at doses of 5 mg / kg and 50 mg / kg, a pseudo-allergy reaction to Con A was suppressed by 50.3% (p <0.01) and 39.0% (p <0.01), respectively. The introduction of similar drugs of comparison, clarithin and suprastin, statistically significantly suppressed the reaction of inflammation to Con A. With the introduction of clarithin at doses of 5 mg / kg and 50 mg / kg, the difference with the control values reached 79.9% and 90.6%, with the introduction of suprastin in the same doses - 78.6% and 66.0%.

Example 6

A single oral administration of the composition of the invention at a dose of 50 mg / kg to CBA mice resulted in a pronounced suppression of the inflammatory response to Con A by 72.7% (Table 5). A single administration of per os betulin at doses of 50 mg / kg and 100 mg / kg and suprastin at doses of 5 mg / kg and 50 mg / kg did not suppress the inflammation response to Con A. Comparison drug, clarithin, administered orally at doses of 5 mg / kg and 50 mg / kg, also caused a dose-dependent suppression of the inflammatory response to Con A by 45.5% and 83.2%, respectively.

Thus, the established data make it possible to indicate the presence of an anti-allergenic anti-inflammatory effect in the composition according to the invention upon intraperitoneal and oral administration, betulin and suprastin did not have anti-inflammatory effect upon oral administration.

Example 7

A single intraperitoneal administration of the composition of the invention at a dose of 50 mg / kg to white outbred male rats led to a reliable suppression of exudative edema caused by carrageenan throughout the experiment (table 6, see drawing). In rats of the control group, the local inflammatory response maximized 3 hours after administration of carrageenan, by this time in animals that were administered the composition according to the invention, there was a significant suppression of exudative edema by 25.9%. The intraperitoneal administration of a dexamethasone comparison drug resulted in a 31.5% reduction in edema compared with the control group 3 hours after the administration of carrageenan. With the introduction of betulin, a significant decrease in the severity of exudative edema by 12.0% was observed only 2 hours after the administration of carrageenan. The data obtained indicate the presence in the composition of a more pronounced anti-inflammatory effect compared with betulin, comparable to the action of one of the most effective anti-inflammatory drugs - dexamethasone.

Example 8

There was no significant change in contractile activity of the ileum of guinea pigs when the composition of the invention was exposed to various concentrations with ileum longitudinal muscle samples for 5 minutes, 1 hour and 1 day, which indicates the absence of a direct effect of the composition on H1 receptors (table 8). H1 receptor blockers, suprastin and loratadine, used in various concentrations, caused a pronounced suppression of the spasmogenic effect of histamine on the ileum of guinea pigs.

Example 9

Table 9 shows the initial data obtained during the formulation of the experiment.

It can be seen from the table that the composition of the invention suppresses both spontaneous and induced activity of nuclear factor kB. A significant decrease in the activity of NF-kB is observed when 5 μg / ml or more is added to the cells, while doses of 5 and 10 μg / ml have similar biological activity and reduce the activity of NF-kB by 2-2.5 times, while 50 μg / ml bring it to almost zero. When recalculating the tested doses of the claimed composition to betulin, it is seen that 50 μg / ml of the composition is 80 μM betulin, that is, the dose is not reachable in vivo. A more acceptable dose is 5 μg / ml, i.e. 8 μM betulin. It 2 times suppresses the basal activity of the nuclear factor kB and 2.5 times TNFa-induced. The results obtained indicate a high activity of the claimed composition against nuclear factor kB.

The data presented allow us to conclude that the composition of the present invention is effective as an antiallergenic agent, comparable with the action of drugs such as suprastin, claritin and dexamethasone, and thus on the possibility of using it as an antiallergenic agent and for the manufacture of antiallergenic agents. It is important to note that the antiallergenic activity of the composition in the experiments was higher compared to that of its main ingredient - betulin, which indicates the joint action of the ingredients of the composition.

The mechanism of action of the claimed antiallergenic drugs

The pathogenesis of allergic diseases developing according to type 1 hypersensitivity is based on the activation of Th2 helper cells and the production of IL-4, IL-5 and IL-13 cytokines, followed by the synthesis of IgE antibodies with high affinity for mast cells and basophils. The antigen interacts with IgE antibodies fixed on mast cells, which leads to cell activation and the secretion of allergy mediators (histamine, serotonin, etc.). However, according to the classification of PGHGell and PRACoombs, there are 4 more types of hypersensitivity, which are based on other mechanisms. The mechanism of action of classical antihistamines anti-allergenic drugs is based primarily on the blockade of the binding of histamine to the receptor H ₁ expressed on the cell surface. Antihistamines have a higher affinity for the H ₁ receptor, which leads to preferential binding of the receptor to the drug rather than to histamine (1,4).

It has been experimentally established that the mechanism of action of the antiallergenic composition according to the invention is different from that of classical antihistamines and indicated in previously published patents, and the authors make certain assumptions about the actual mechanism of action.

When studying the effect of the composition of the invention on the anaphylaxis of isolated organs on the model of anaphylactic contracture of the smooth muscle of the ileum (ileum) of guinea pigs, no significant changes in the contractile activity caused by histamine, ileum of the guinea pigs were detected at various concentrations of the temporal exposure of the composition with ileum longitudinal muscular samples , which indicates the absence of direct action of the claimed composition on H ₁ receptors (15).

In accordance with various mechanisms of the pathogenesis of allergic diseases, corticosteroids are one of the main classes in the treatment of allergies in addition to antihistamines. Steroids have proven highly effective against allergic and other types of inflammation, inhibiting both its early and late manifestations. For the anti-inflammatory effects of corticosteroids, their ability to inhibit the synthesis of pro-inflammatory cytokines, prostaglandins, cellular phospholipases, and adhesion molecules is most important (1,4). In particular, upon activation of phospholipase A _{2, the} cleavage of arachidonic acid from phospholipids of cell membranes by cyclooxygenase leads to the synthesis of prostaglandins, lipoxygenases to the synthesis of the leukotriene family, some of which were previously called the slow-acting substance of anaphylaxis (1). A search is currently underway for steroid-like compounds with the ability to suppress the activity of phospholipase A ₂ , thereby reducing the presence of a substrate for the development of various forms of inflammation and having less toxicity compared to classical corticosteroids (4, 7, 9, 14). A number of authors attribute lupane triterpenoids to the so-called phytoecdysteroids (phytoecdysteroids) due to the fact that the mechanism of action and chemical structure of triterpenoids and steroids are quite close (15, 16). In chronic use, corticosteroid antiallergenic drugs reduce resistance to infections, disrupt metabolism, in particular the metabolism of carbohydrates, fats and proteins, inhibit the activity of the hypothalamus-pituitary-adrenal system, cause liver damage, neurological disorders and have a number of other side effects. Steroid-like compounds of plant origin do not have such pronounced side effects (16).

The anti-inflammatory activity of the composition according to the invention is primarily due to the fact that, according to the literature, betulin, like corticosteroids, is an inhibitor of phospholipase A ₂ (7). In addition, according to the literature, betulin and betulinic acid have affinity for glucorticosteroid receptors, have anti-inflammatory activity comparable to dexamethasone, and the anti-inflammatory activity of betulinic acid on the carrageenan edema model is more pronounced than that of betulin (17). In the experiments presented, the composition according to the invention had a more pronounced anti-inflammatory effect compared to betulin, comparable to the hydrocortisone homolog dexamethasone.

The composition of the present composition according to the invention contains a large number of triterpenoids of caffeates, which have antioxidant, anti-tumor, antimalarial and other properties (8, 10, 16, 19). In particular, the antioxidant properties of birch bark extracts are explained by the presence of betulin caffeates, oleanic and betulinic acids in their composition (5). The caffeoyl group has been proven to enhance biological activity (18), in this case anti-allergenic.

According to modern concepts, the selective stimulation of Th ₁ helpers producing interferon γ (IFN-γ) delays the production of Th ₂ cells secreting interleukin IL 4, which stimulates the synthesis of IgE antibodies (13). It is possible that the suppression of the systemic anaphylaxis reaction is also associated with the ability to selectively induce γ-interferon and thus inhibit the production of Th ₂ helper cells and the synthesis of IgE antibodies (18). Currently, this is one of the promising areas of immunotherapy for allergic diseases.

It is possible to suggest another mechanism of action based on the results of a study to identify the composition according to the present invention the ability to suppress both spontaneous and induced activity of nuclear factor kB. Adding to the cells of the U937 line a composition in an amount of 5.10 μg / ml reduced the activity of NF-kB by 2-2.5 times. High activity against nuclear factor kB may explain the pronounced anti-inflammatory and some forms of anti-allergenic effect.

At the next stage of the study, the clinical efficacy and safety of the use of the composition according to the invention was studied. For this purpose, the funds “Superantitox 50” and “Superantitox 25” were used for bronchial asthma (BA). Both dietary supplements basically contain a composition according to the present invention. Supplement "Superantitox 50" contains 50 mg of the composition and fructose, "Superantitox 25" - 25 mg of the composition and fructose.

Research Methods and Scope

Clinical testing of dietary supplements "Superantitox 25" and "Superantitox 50" was carried out on the basis of the 5th therapeutic (allergological) department of the Municipal Clinical Hospital No. 57 of Moscow.

The criteria for inclusion of patients in the study were:

age from 18 to 60 years old,

confirmation of the diagnosis of "Bronchial asthma" by clinical and instrumental methods of examination, consent to treatment and examination,

the absence of concomitant diseases that have a significant impact on the course of the underlying pathology and require additional prescriptions (diabetes, tuberculosis, malignant tumors).

39 patients with bronchial asthma were under observation.

In order to assess the clinical effectiveness of the use of drugs in the complex treatment of patients, original schemes of medical observation diaries and questionnaires for patients were developed in which the clinical manifestations of the disease were scored.

The use of drugs was agreed with all patients; informed consent was obtained from each patient.

In accordance with the Protocol, patients were examined:

Laboratory:

1. Standard clinical blood test (general).

2. Determination of class E immunoglobulins.

3. Cytological examination of induced sputum and nasal secretion (with bronchial asthma and allergic rhinitis).

Instrumental:

- Spirography, incl. test using bronchodilators.

- Peakfluometry.

- Determination of nitric oxide in exhaled air.

Studies were conducted before and after treatment.

Brief description of patients with asthma

Under observation were 39 patients with bronchial asthma of varying severity in the acute stage.

Upon admission to the hospital, a typical clinic of bronchial asthma (BA) was observed in all patients with impaired general condition (to a state of moderate severity), an increase in respiratory rate (from 21 to 30 per min), a change in percussion sound (boxed, boxed) and auscultatory changes (weak, hard breathing; wheezing with various quantitative characteristics).

Laboratory examination of patients was carried out in accordance with general standards and the research protocol. A general blood test in 10 cases (5 in each group) revealed an increase in the level of leukocytes to 12.9-13.4 · 10 ⁶ , mainly in those receiving glucocorticosteroids, less often (4 cases) - moderate eosinophilia - up to 8%.

The number of immunoglobulins E at admission was increased in 25 of 39 patients; the level of increase was different - from 187 units to exceeding 1000 units (method threshold).

Patients are divided into two subgroups depending on the planned treatment with the studied dietary supplements: “Superantitox 50” (21 patients) or “Superantitox 25” (18 patients). Then each subgroup is divided taking into account the clinical indications of systemic glucocorticosteroids (cGCS).

In one case, the patient on the 6th day of taking "Superantitox 50" appeared urticaria rash, which died away after discontinuation of the drug. It is difficult to establish the exact relationship with taking the drug, because on the same days the patient consumed various foods, including and allergenic in nature. This patient was not included in the observation group.

Thus, the following observed groups were identified:

I gr (10 patients) received "Superantitox 50" on the background of cGCS,

II gr. (10 patients) received "Superantitox 50" without cGCS,

III gr. (9 patients) received "Superantitox 25" on the background of cGCS,

IV gr. (9 patients) received "Superantitox 25" without cGCS.

Patients of groups I and II from the first day of their stay in the hospital were prescribed “Superantitox 50”: 1 capsule 2 times a day, 15-20 minutes before meals; daily dose - 100 mg. The course of treatment ranged from 15 to 20 days, depending on the length of stay in the hospital.

From the first day of their stay in the hospital, “Superantitox 25” was prescribed to patients of group III and IV: 1 capsule 2 times a day, 15-20 minutes before meals; daily dose - 50 mg. The course of treatment ranged from 15 to 20 days.

Depending on the severity of the course of the disease, patients received bronchodilators and inhaled glucocorticosteroids (IGCs) (beclamethasone dipropionate, fluticasone propionate at the doses recommended by GINA 2002) as the basic therapy. Against the background of exacerbation and hospitalization in a hospital, 19 patients received cGCS.

In group II, 8 out of 10 patients received only "Superantitox 50", two patients also inhaled bronchodilators; IGKS were not appointed. Thus, this group can be considered a monotherapy group.

In group IV only “Superantitox 25” received 1 patient, a combination of it with bronchodilators - three, a combination with ICS and bronchodilators - five patients.

The control group (16 patients, 8 with and without cGCS) received only basic therapy.

To assess the clinical effect, medical observation diaries and an original questionnaire for patients were used.

In the observation diary, the doctor daily evaluated the available leading symptoms of bronchial asthma in points:

I General condition:

- Satisfactory - 3 points

- Moderate - 2 points

- Severe - 1 point

II Respiratory rate:

- Up to 20 - 0 points

- 21-25 - 1 point

- 26-30 - 2 points

- 31 and more - 3 points

III Percussion sound:

- pulmonary - 1 point

- with boxed shade - 2 points

- boxed - 3 points

IV Breath:

- vesicular - 1 point

- weakened - 2 points

- hard - 3 points

V wheezing:

- no - 0 points

- single whistling with forced expiration - 1 point

- a few whistling on the exhale - 2 points

- multiple whistling, humming - 3 points

Patients also assessed their well-being, marking it in questionnaires for 1, 5, 10 days of treatment and at discharge (15-20 days) (in points):

I. General condition

1 point: very poor

2 points: poor

3 points: satisfactory

4 points: good

5 points: excellent

II. Frequency of asthma attacks (... once a day, week, month)

III. Cough

0 points: no

1 point: occasional cough

2 points: daily moderate

3 points: paroxysmal rare

4 points: paroxysmal daily

5 points: persistent, round-the-clock cough that interferes with sleep

IV. Labored breathing

0 points: no

1 point: rarely on exertion

2 points: rarely alone

3 points: constantly

V. Wheezing wheezing

0 points: no

1 point: only a limited period of time (morning, evening), with physical activity

2 points: often, several episodes during the day

3 points: constantly

VI. Feeling of stuffiness in the chest

0 points: no

1 point: only a limited period of time (morning, evening), with physical activity

2 points: often, several episodes during the day

3 points: constantly

In assessing the clinical effectiveness of the studied dietary supplements, objective manifestations of asthma, the results of questioning patients, reflecting the well-being and functionality of patients while taking drugs, and laboratory and instrumental data were taken into account.

RESULTS OF THE STUDY OF THE EFFICIENCY OF BES DRUGS IN COMPLEX THERAPY OF BRONCHIAL ASTHMA

"Superantitox 50"

"Superantitox 50" was prescribed in combination therapy for 20 patients with bronchial asthma. The monitoring group included 16 women and 4 men aged 18 to 60 years; the average age was 45.3 ± 2, 87 years.

The forms of bronchial asthma were diagnosed: atopic - in 8, endogenous - in 6, mixed - in 6 patients. Current: persistent mild - in 5, moderate - in 12, severe - in 3 patients (Table 10).

When contacting patients, a typical clinical picture of AD of the corresponding form and severity was noted.

In group I — in patients with initially expressed abnormalities requiring the appointment of cGCS — the positive dynamics of the clinical manifestations of asthma, due mainly to hormone therapy, came quickly. The average period of normalization of the general condition (from moderate to satisfactory) was 3.0 ± 0.31 days, the disappearance of shortness of breath was 3.1 ± 0.90 days. Percussion sound became pulmonary, on average, after 6.3 ± 1.47 days, the auscultatory picture returned to normal a little later: vesicular breathing was heard after 3-6-10 days (average: 7.6 ± 1.69 days), dry rales disappeared after 5-17 days (on average - after 9.7 ± 1.76 days).

In the control group of patients receiving cGCS (8 people), these indicators were: normalization of the general condition - 3.6 ± 0.45 days, the disappearance of shortness of breath - 4.0 ± 0.97 days, normalization of percussion sound - 7.4 ± 1 , 51 days, auscultatory picture 8.2 ± 1.51 days for breathing, 10.2 ± 1.84 days for the disappearance of wheezing. Thus, indicators in the main group returned to normal somewhat faster, which can be noted as a trend (without statistical significance).

In group II, the initial state of patients was slightly better: a moderate severity was found in 3 out of 10 patients, its normalization occurred in 1.4 ± 0.28 days (in the control - in 2.4 ± 0.30 days; significance of differences with p <0.05). The respiration rate in 6 cases was initially normal, in the rest it was normalized, on average, in 6 days (in the control - in 6, 2 days). Percussion sound with a boxed tinge was noted in 8 patients, normalization occurred within 6-11 days, and in 4 patients the boxed tint was preserved until discharge from the hospital (in the control - similarly). Vesicular respiration was initially noted in 3 patients; in the rest, it became vesicular during the treatment - on average, by 13.4 ± 0.18 days (in the control - by 14.1 ± 0.31 days). Dry rales were auscultated in all patients upon admission; terms of their disappearance ranged from 7-8 to 15 days, on average - 10.3 ± 0.81 days; in the control - 11.6 ± 0.92 days.

"Superantitox 25"

"Superantitox 25" was prescribed in complex therapy for 18 patients with bronchial asthma. The observation group included 11 women and 7 men aged 18 to 60 years; the average age was 44.3 ± 3.22 years.

The forms of bronchial asthma were diagnosed: atopic - in 5, endogenous - in 4, mixed - in 9 patients. The course: persistent mild - in 6, moderate - in 8, severe - in 4 patients (Table 11).

When contacting patients, a typical clinical picture of AD of the corresponding form and severity was noted.

In the III group of patients receiving cGCS and Superantitox 25 against moderate to severe AD, the general condition was initially assessed as moderate, normalization occurred after 2-8 days, on average, after 3.7 ± 0.31 days. At the same time, the respiratory rate returned to normal. The normalization of the percussion picture took 8.6 ± 2.02 days, the normalization of respiration - 10.7 ± 1.84 days, the disappearance of wheezing - 10.7 ± 1.62 days. In the control group, similar phenomena were observed (see above).

In patients of group IV (similar to group II), the general condition, respiratory rate, and percussion picture were initially little changed, their normalization took 5-6 days. However, in 2 patients, the box-like shade during percussion, as in group II, was preserved until discharge - as well as dry rales.

The average duration of clinical manifestations was: general condition disorders - 1.9 ± 0.29 days, changes in respiration rate - 5.0 ± 1.91 days, changes in percussion sound - 6.5 ± 1.67 days, respiratory pattern - 11, 2 ± 2.14 days, dry wheezing in the lungs - 8.9 ± 1.21 days.

The clinical results of monotherapy with drugs were analyzed. In group II, patients actually received monotherapy "Superantitox 50" (only in 2 cases out of 10 supplemented with bronchodilator foradil). An individual assessment of the well-being and objective state of patients before and after treatment made it possible to state positive dynamics in half of the patients (5 cases), moderate positive dynamics in 3, and the absence of objective dynamics in 2 patients. In group IV (“Superantitox 25”), true monotherapy can be discussed only in one case; positive dynamics of clinical manifestations of AD was noted. In other patients, bronchodilators were used (3; all with a positive dynamics of symptoms) or a combination of bronchodilators with ICS (5, 2 of them with positive shifts of all parameters, 3 with positive dynamics of subjective parameters).

Changes in the well-being and condition of patients during treatment were monitored daily by both the attending physician and the patients themselves.

The analysis of objective and subjective parameters in AD patients receiving “Superantitox 50” and “Superantitox 25” on the background of the basis treatment with the inclusion of cGCS (groups I and III) showed that the main therapeutic effect, of course, was provided by cGCS. Some differences in the rate of normalization of general well-being were noted with prolonged use of dietary supplements: for 10-20 days of treatment, patients' self-esteem of their condition was slightly higher than in the control group (p <0.05).

In groups of patients treated without the use of cGCS, certain differences were observed in the onset of positive changes in objective and subjective parameters. With superantitox 50 monotherapy (group II, 10 patients), on the 20th day of treatment (before discharge from the hospital), the best auscultatory picture (normalization of breathing, the disappearance of wheezing (p <0.05) was noted. Patients on the 20th day of treatment noted the best Compared to the control, on the 10th and 20th days of treatment — the disappearance of breathing difficulties (p <0.05).

In group IV, it is not possible to consider the use of “Superantitox 25” as monotherapy, since most patients received bronchodilators, and half of the patients also received beclomethasone. Nevertheless, it should be noted that certain significant differences between the clinical parameters and the control in these patients concerned the same parameters: in a larger number of patients, vesicular breathing was already auscultated by day 5, health was normal before (judging by the scores of the 5th, 10th and 20th day of treatment). Similar to the data of group II, shortness of breath, wheezing and chest congestion disappeared at an earlier date (p <0.05; p <0.01).

Thus, the clinical analysis showed that the use of BES "Superantitox 50" and "Superantitox 25" in the treatment of AD patients contributes to a somewhat earlier, compared with the control, normalization of well-being, and when used for 10-20 days - and some indicators characterizing breathing (auscultatory and subjective data).

Tolerance "Superantitox 50" and "Superantitox 25". The preparations "Superantitox 50" and "Superantitox 25", in general, were well tolerated by patients. The aforementioned case of an allergic reaction (urticaria), not significantly associated with the use of "Superantitox 50", was noted.

The results of the clinical and laboratory-functional observation allow us to recommend the dietary supplements "Superantitox 50" and "Superantitox 25" for use in the treatment of patients with bronchial asthma. According to clinical observation, both agents contributed to an earlier normalization of well-being, as well as some indicators that characterize breathing (auscultatory and subjective data). Analysis of laboratory data did not reveal a negative effect of taking BAS drugs on controlled parameters; in some cases, positive dynamics of indicators such as eosinophilia, the level of total immunoglobulin E, the level of nitric oxide in exhaled air, and respiratory function indicators (these laboratory parameters tended to normalize) were observed. not statistically confirmed).

Table 1 Dose The number of animals in the group WEIGLE Reaction Index The control 10 2.0 Composition:
100 mg / kg
1000 mg / kg 10
10 1,1
1.3 table 2 Dose The number of animals in the group WEIGLE Reaction Index The control 10 2.1 Composition 50 mg / kg 10 1,0 Suprastin: 50 mg / kg 10 0.6 Table 3 Dose The number of animals in the group WEIGLE Reaction Index The control 10 1.9 Composition:
50 mg / kg 10 0.8 Betulin: 50 mg / kg 9 0.9 Table 4 Dose The number of animals in the group WEIGLE Reaction Index The control eleven 3,5 Claritin 30 mg / kg 7 2.9

Table 5 Dose of a pharmacologically effective compound The number of animals in the group Reaction index % of control The control 10 15.9 ± 1.7 one hundred Composition: 5 mg / kg 10 8.7 ± 1.9 (p <0.05) 54.7 50 mg / kg 10 7.0 ± 1.1 (p <0.01) 44.0 100 mg / kg 10 4.5 ± 1.4 (p <0.01) 28.3 Betulin: 5 mg / kg 10 7.9 ± 1.2 (p <0.01) 49.7 50 mg / kg 10 9.7 ± 1.3 (p <0.01) 61.0 Claritin: 5 mg / kg
50 mg / kg 10
10 3.2 ± 1.1 (p <0.01)
1.5 ± 0.5 (p <0.01) 20.1
9,4 Suprastin: 5 mg / kg 10 3.4 ± 1.1 (p <0.01) 21,4 50 mg / kg 10 5.4 ± 1.7 (p <0.01) 34.0

Table 6 Dose The number of animals in the group Reaction index The control 10 13.8 ± 1.6 Composition: 5 mg / kg 10 12.4 ± 2.2 The control 10 14.3 ± 2.0 Composition: 50 mg / kg
100 mg / kg 10
10 3.9 ± 1.3 (p <0.01)
10.6 ± 2.1 Betulin: 50 mg / kg 10 14.6 ± 2.5 100 mg / kg 10 14.0 ± 2.5 Claritin: 5 mg / kg
50 mg / kg 10
10 7.8 ± 2.2 (p <0.05)
2.4 ± 0.8 (p <0.01) Suprastin: 5 mg / kg
50 mg / kg 10
10 16.4 ± 1.9
13.7 ± 3.3

Table 7 Edema measurement time (in hours) Swelling of the foot × 10 ^-1 mm Control (n = 8) Composition 50 mg / kg w / b (n = 8) Betulin 50 mg / kg, w / b (n = 8) Dexamethasone 1.4 mg / kg, ip (n = 8) 0 33.3 ± 1.4 34.8 ± 1.6 32.0 ± 0.9 29.8 ± 0.7 one 43.5 ± 1.9 37.3 ± 1.6 * 38.3 ± 0.8 * 32.0 ± 0.9 ** 2 49.0 ± 4.0 36.8 ± 1.6 * 41.8 ± 2.8 36.0 ± 0.8 ** 3 53.7 ± 5.9 39.8 ± 1.4 * 44.8 ± 2.3 36.8 ± 1.1 * four 52.3 ± 5.7 37.0 ± 1.6 * 41.5 ± 2.0 30.7 ± 1.1 ** Note: significance of differences with control: * - p <0.05; ** - p <0.01; n is the number of animals in the group

Table 8 Group Compound Exposure Time Contractile activity of the longitudinal muscles of the ileum (%) The control one hundred Composition 5 minutes 89.1 ± 3.8 Suprastin 1 × 10 ^-10 mg / ml 5 minutes 46.5 ± 4.1 Suprastin 2 × 10 ^-3 mg / ml 5 minutes 26.5 ± 1.5 Suprastin 0.2 mg / ml 5 minutes Complete suppression Loratadine 0.01 mg / ml 15 minutes 28.4 ± 3.2 Control (alcohol) 1 hour one hundred BES 1 hour 90.1 ± 17.1 Control (alcohol) 24 hours one hundred BES 24 hours 108.3 ± 11.2

Table 9

Samples Luciferase (luminosity) β-galactosidase (OD 420 nm) Luciferase / β-galactosidase Luciferase / β-gal. Hole numbers one 2 3 one 2 3 one 2 3 (M ± m; n = 3) Cells without stimulation 470 560 507 0,07 0,062 0.058 6714 9037 8747 8166 ± 1201 Composition (1 μg / ml) 222 356 615 0.036 0.039 0.064 6175 9123 9614 8304 ± 1766 Composition (5 μg / ml) 153 108 183 0.035 0.033 0.05 4360 3272 3660 3764 ± 523 Composition (10 μg / ml) 153 122 157 0.05 0.037 0.04 3060 3305 3935 3433 ± 428 Composition (50 μg / ml) 3.7 3.4 1.7 0.002 0.002 0.001 1843 1722 1652 1739 ± 92 Control (TNFα) 731.5 909 1254 0.059 0.069 0.058 12398 13174 21621 15731 ± 4858 BAS (1 μg / ml) + TNFα 758 600 390 0.057 0.05 0.05 13300 12002 7949 11084 ± 2650 Composition (5 μg / ml) + TNFα 187 606 413 0.048 0.073 0.064 3890 8296 6458 6214 ± 2102 Composition (10 μg / ml) + TNFα 1107 733 149 0.139 0.101 0.041 7964 7256 3644 6288 ± 2200 Composition (50 μg / ml) + TNFα 3 5 3 0.002 0.004 0.003 1327 1155 1154 1212 ± 94 Positive control 1824 1368 776 0.075 0.065 0.035 24320 21046 22163 22510 ± 1580 Negative control 0.9 1.1 1.4 0.049 0.048 0.044 19 23 31 24 ± 6

Table 10 Group I (n = 10); Superantitox 50 + basis treatment with the inclusion of cGCS Group II (n = 10) Superantitox 50 + basis treatment without the use of cGCS Forms of BA: Atopic four four Endogenous 2 four Mixed four 2 The severity of asthma: Light persistent flow one four Moderate 6 6 Heavy 3 -

Table 11 Group III (n = 9); Superantitox 25 + basic treatment with the inclusion of cGCS Group IV (n = 9) Superantitox 25 + basis treatment without the use of cGCS Forms of BA: Atopic 2 3 Endogenous 3 one Mixed four 5 The severity of asthma: Light persistent flow - 6 Moderate 5 3 Heavy four -

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Claims (1)

An allergy product obtained by toluene extraction of birch bark, containing betulin, lupeol, 3-O-betulin caffeate as active components, as well as related substances in the following amount, wt.%: betulin 65-71 lupeol 12-16 3-O-betulin coffee 5-15 related substances the rest is up to 100%