ES2235655B1 - ISOLATED EXTRACT FROM A CYCLAMEN EUROPAEUM L. PLANT AS A THERAPEUTIC AGENT. - Google Patents

Abstract

Extract isolated from a Cyclamen europaem L. plant as a therapeutic agent. Said extract isolated from the wild plant Cyclamen europaeum L. is used for the manufacture of a medicament for the treatment of a disease associated with an insufficiency of secretion in the interferon system in a human and / or animal. Said extract isolated from the wild plant Cyclamen europaeum L. can be administered repeatedly and for prolonged periods of time, maintaining appropriate levels of interferon.

Description

Isolated extract of a Cyclamen europaeum L plant . as a therapeutic agent

Field of the Invention

The present invention is related to diseases associated with deficiencies in the immune system.

In particular, the present invention relates to an isolated extract of Cyclamen europaeum L. for the manufacture of a medicament for the treatment of a disease associated with an insufficiency in the secretion of the interferon system.

Background of the invention

It is complex to assess the role of the system immune and your immunocompetence level to maintain the homeostasis of the organism in a normal state. The slightest Immune surveillance disorder is capable of carrying serious consequences, including cancer diseases, autoimmune processes or cytogenic alterations of greater incidence. The continuous environmental deterioration at scale global (the so-called ecological stress), as well as the conditions of social and economic life, cause a serious immune deficit of well-known consequences. Therefore, one of the objectives of modern medicine and pharmacology lies in the search and incorporation into practice of new medications that Maintain the optimal functioning of the immune system.

Currently, many products with the indicated properties, both natural and synthetic, are already used in the clinic: immunoglobulins, interferons, interleukins, cytokines, hormone analogs, etc. Due to their immunotropic capacity they can be general or specific, and vary in their effectiveness in preventing and treating the pathologies of the immune system. Among them, immunomodulators of natural origin stand out: the eleutherococcus, the Chinese magnolia ( Schisandra chinensis ), the echinacea, the pelargonium ( Pelargonium reniforme / sidoides),
etc.

On the other hand, the wild Cyclamen europaeum L. Plant, referred to in the present invention as a cyclamino plant, belongs to the Primulaceae family, and although it is a very popular plant in many countries of the world, its use is strictly limited to decorative purposes. . The current information on its medicinal properties is scarce and refers to the use of the juice or powder obtained from the plant to treat the pain of
head.

In this sense, the Spanish patent ES2170726, describes the use of an isolated extract of Cyclamen europaeum L. for the manufacture of a medicine for the treatment of sinusitis. This application describes the fact that the therapeutic action of a preparation comprising the isolated extract is based on the stimulation of the secretory activity of the integumentary mucosa epithelium in general, as well as the sero- and submucosal glands, which facilitates intense drainage of the paranasal sinuses (mechanical action).

Summary Description of the Invention

The present invention is directed to the use of an isolated extract of the wild plant Cyclamen europaeum L. for the manufacture of a medicament for the treatment of a disease associated with an insufficiency in the secretion of the interferon system.

Next, the object of the present invention

Description of the invention

The present invention relates to the use of an isolated extract of the wild plant Cyclamen europaeum L. for the manufacture of a medicament for the treatment of a disease associated with an insufficiency of the secretion of the interferon system.

In the present invention, by "disease associated with an insufficiency in the secretion of the system interferon "means a pathological state of the organism due to to a lack of interferon formation, being responsible of said lack of secretion, factors such as environmental (for example stress) or the presence of a foreign agent for the host organism (for example a bacteria, viruses, etc.)

In the present invention by "system interferon "means the family of proteins found involved in the inflammatory process and in the production of reactive oxygen species. Once activated, they induce generation of mediating molecules; and these in turn, activate a set of cells that are recruited at the process site inflammatory.

The isolated extracts of the wild plant Cyclamen europaeum L. They can be obtained by conventional procedures. An example is the Spanish patent ES2170726, in particular in column 5, line 21-column 6, line 54, where a method of obtaining an isolated extract of Cyclamen europaeum L is described .

Surprisingly, it has been discovered that isolated extracts of the wild Cyclamen europaeum L. plant stimulate the secretion of interferons, which allows, in this way, the treatment of diseases caused by an insufficiency in the secretion of the interferon system.

This activity is due to its pharmacodynamics as well as to the fact that Cyclamen extracts are rich in saponins, for example Cyclamine and Escina (active substances that are glycosides, mainly triterpenoids or flavonoids as aglycone), although, as is known to experts in the matter, the amount of saponins and the maintenance of its pharmacodynamic activity in the pharmaceutical preparation depend, to a large extent, on the procedure followed to obtain it. Furthermore, it has been observed that said activity is a secretory modulating activity in the interferon system; when interferon levels are very low, the administration of said extract stimulates the secretion of interferons to a level that is considered by the expert as normal and vice versa, when interferon levels are high, the administration of said extract makes the level of interferons return to a level that is considered by the expert as normal.

Advantageously, and due to this modulating activity, said Cyclamen extract can be administered repeatedly and for prolonged periods of time, maintaining appropriate levels of interferon (unlike the immunostimulators known in the state of the art which, having no such modulating activity, are administered for short periods of time, to prevent interferon levels from being excessively
elevated).

The inventors of the present invention believe that other extracts from other plants belonging to the Primulaceae family could be found which, because they contain saponins structurally similar or equal to cyclamine, can give rise to effects similar or equal to those described in the present invention.

In an embodiment of the present invention, said extract isolated from Cyclamen europaeum is lyophilized.

Surprisingly, the inventors of the present invention have discovered that isolated extracts from the wild plant of Cyclamen europaeum L. they act in all the above and below mentioned indications, especially if these extracts are lyophilized immediately after the preparation of the extract, since in this way the efficiency and stability in the galenic formulations is improved.

An embodiment of the present invention relates to the use of an isolated extract of Cyclamen europaeum L. for the manufacture of a medicament for the treatment of a disease associated with an insufficiency of secretion of the interferon system, said medication being administered by orally.

For oral administration, said medicament can be prepared in the form of normal, sucking or coated tablets, dragees or other suitable formulations (among others, sublingual or buccal), with a release of the active substance (i.e., of Cyclamen extract ) normal, sustained or with controlled diffusion. Formulations with sustained release or with controlled diffusion of the active substance in appropriate dosages are advisable to avoid possible disorders and irritations of the gastrointestinal tract that could arise from saponins after oral ingestion of normal tablets, drops or syrups.

Optionally, for oral administration, said medicament can be prepared in the form of a mouthwash. The inventors of the present invention have proven that gargling with a mouthwash comprising an isolated extract of Cyclamen manages to treat the infection in angina.

Another embodiment of the present invention relates to the use of an isolated extract of Cyclamen europaeum L. for the manufacture of a medicament for the treatment of a disease associated with an insufficiency of secretion of the interferon system, said medication being administered topically.

For topical administration, especially intranasally, this medication can be prepared in the form of ointments, jellies or drops that are applied to each nasal meatus. All these presentations can be muco-
Adhesive

Preferably, said medicament is administered in the form of a nasal spray. These formulations can be administered, for example, by dissolving them in sterile water ( water for injections ) and dispersing them with a spray-doser suitable for each nasal meatus.

The authors of the present invention have proven, for the first time, that intranasal administration of a medicament according to the present invention (extract isolated from Cyclamen ), induces a stimulation in the secretion of the interferon system, in particular interferon \ alpha and γ (IFN-? and IFN-?). This new possibility is very important and surprising because the intranasal administration of chemically or biologically defined immunostimulants or immunomodulators, for example interferons of various types, such as IFN-? Or IFN-?, Was unknown until
now.

In an embodiment of the present invention, said extract is used for the manufacture of a medicine intended for the treatment of a disease associated with a insufficiency of interferon? secretion.

In another embodiment of the present invention, said extract is used for the manufacture of a medicine intended for the treatment of a disease associated with a insufficiency of interferon? secretion.

The inventors of the present invention have found that the administration of an isolated extract of Cyclamen europaeum L. causes a significant increase in interferon levels (α and γ), of the active fraction of T lymphocytes and a decrease in IgG, among others. The fact that there is an increase in the secretion of interferons, leads to an activation of the immune system, taking place the immune response of the organism to the appearance of the foreign agent.

In an embodiment of the present invention, the isolated extract is used for the manufacture of a medicine intended for the treatment of a viral disease selected from group comprising: colds; breast swelling recurrent acute or chronic paranasal such as catarrhal highmoritis or purulent, frontitis, ethmoiditis, sphenoiditis or sinusitis combined; purulent sinusitis or orbital complications associated to these; otitis; pharyngitis; laryngitis; flu.

In another embodiment, said viral disease, caused by the influenza virus, is the flu.

The antiviral activity of an isolated extract of Cyclamen europaeum L. described below, as a model of a flu infection in white mice (See Example 3). Influenza infection developed in a context of serious depression of indicators of immunological homeostasis as important as the interferon system or phagocytosis. The administration of an isolated extract of Cyclamen europaeum L. made possible the improvement of parameters such as the interferon system or phagocytosis, reaching statistical significance in the late stages of infection.

The "protective" effect of the isolated extract of Cyclamen europaeum L. It was fully confirmed by a generalized infection (parameter of influenza infection). The mortality of mice due to generalized infection was reduced from 90% to 75%. Histological analysis of the liver showed an improvement of the pathomorphological picture in liver tissue. The immunofluorescence analysis showed an intensification of the specific illumination of Kupffer liver cells, which demonstrates their activation, with the consequent protective action of parenchymal cells (hepatocytes).

In this way, based on the model of influenza infection in white mice, we have verified the complex protective capacity of the isolated extract of Cyclamen europaeum L. , both in general terms (in relation to the immune system), and in its quality as antiviral substance (specifically: anti-flu). This ability includes gene protection that, in turn, influences the repair and functioning of immune cells. One of the mechanisms of the protective action of the isolated extract of Cyclamen europaeum L. derives from its antioxidant properties, which result in the recovery of breathing at the intracellular level (including cells
immune).

Optionally, said disease, associated with a insufficiency of the secretion of the interferon system, is caused by a pathogenic bacteria.

In the present invention, by "bacteria pathogenic "means a bacterium that causes a lack of secretion of the interferon system in the host organism, giving place to a disease or pathology.

Nitrogen oxide (NO) is a universal biological messenger, with a broad spectrum of action, which is synthesized when many of the non-adrenergic and non-cholinergic branches of the autonomic nervous system are put into operation. Endogenous nitrogen oxide is an essential part of calcium homeostasis in cells and, therefore, of the activity of Ca2 +-dependent protein kinases. Nitrogen oxide also plays a specific role in the formation of the secretions of the mucous membranes of the oral cavity and upper respiratory tract. The presence in the salivary glands of nitrogen oxide synthetase, both neuronal type (nNOS) and inducible type (iNOS) has been demonstrated. The activation of nNOS in cells with the help of methacholine and substance P enables the opening of ion channels and the start of secretions (Gillespic J, Liu X, Martin W, "The effect of L-arginine on the response of the rat muscle to nerve stimulation ", Br. J. Pharmacol., 1989, 88 , 1080-1082; Lomniczi A. et al ," Role of nitric oxide in salivary secretion ", Neuroimmunomodulation, 1998, 5 , 226-233. Nitrogen oxide is considered a regulator of lymphocyte proliferation; the synthesis of NO by phagocytic cells forms the basis of its bactericidal action (Springall D. et al ., "Immunological detection of nitric oxide synthetase in human tissue", Histo-chemistry, 1992, 98 , 259-266).

As shown below, the Cyclamen europaeum L. extract, in accordance with the present invention, has the ability to inhibit active forms of oxygen such as nitrogen oxide generators, which enables a decrease in the intensity of the oxidative stress, which indicates that it can be used for the treatment of a bacterial type disease.

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In another embodiment, an isolated extract of Cyclamen europaeum is used for the manufacture of a medicament for the treatment of a disease associated with an insufficiency of secretion of the interferon system, which is caused by a fungus or yeast, preferably by a fungus of the genus Candida

In the present invention, by "protective effect" or "protective" it is understood that the administration of the isolated extract of Cyclamen results in the secretion of interferons which trigger a response against a particular agent (eg, virus or bacteria) foreign to the organism.

A possible mechanism of action that would explain the immunotropic action of an extract of Cyclamen europaeum L. , would be the ability to amplify the activity of the immune cells of the mucous membranes of the nasal and oral sinuses with the consequent chain activation of the systemic factors of the immune system, improved free radical elimination defenses and antitoxic effect.

The isolated extract of Cyclamen europaeum L. It allowed the decrease of hypoxia as described in Example 3. It also did not cause harmful effects on the organism of pregnant mice or fetuses.

The following is included, by way of illustration and Non-limiting, the following examples.

Examples Example 1 Obtaining an isolated extract of Cyclamen europaeum L.

Undamaged tubers are chosen (no clues of rot). Then they are cleaned in running water with a brush, removing the remains of dirt and sand. Following, the tubers are cleaned with desalinated water and allowed to air dry free.

The tubers, once dried, get into a bath and cover with 95% ethyl alcohol, keeping them that way for 1 hour.

Then the tubers are cut obtained previously, they are placed in a steel press stainless, and the juice is extracted.

The crushing obtained is placed in a reactor, cover with desalinated water, heat until the temperature is reached from 40-70 ° C, and it stays that way for 1 hour.

After it cools, it is squeezed and the extract obtained in the press is mixed with the juice. Then for extract ballast substances, it is added to the extraction obtained (juice + extract) 95% ethyl alcohol in an amount of 15% of the total volume.

Then, the extraction obtained is subjected to a normal filtration and then sterile filtration. Be bottle this sterile solution in 0.5 ml vials.

Finally lyophilization takes place.

Example 2 Study of the immunostimulatory capacity of an isolated extract of Cyclamen europaeum L.

The study of the immunostimulatory capacity of the extract of Cyclamen europaeum L. It was carried out based on non-specific resistance and humoral immunity values: the percentage of B lymphocytes and T lymphocytes and their subpopulations (active T, helper T, suppressor T, self-regulation index), the number of immunoglobulins of the three main classes G (IgG), M (IgM) and A (IgA), the phagocytic activity of neutrophils in the blood (TF = phagocyte number, IF = phagocytic index, FF = final phase of phagocytosis), and leukocyte reaction to interferons in vitro (IFN-? and IFN-?). (Kipiani Va et al . "Oxidative processes in peridontitis", Int. J. Immunorehabilitation, 2000, 2 , 291). The immunological analysis of the blood was carried out by micromethods (a maximum of 0.2 ml of blood extracted from the fingertip).

First, the immunostimulatory capacity of the medication was checked in healthy volunteers who had not suffered from influenza, respiratory diseases or other diseases Infectious or inflammatory processes in the last six months.

Said extract was administered by applying 0.13 ml of a solution containing 50 mg of a lyophilized extract (obtained according to Example 1) in 5 ml of water for injections (corresponding to 1.3 mg of lyophilized extract), with a nebulizer-doser in each nasal meatus. This Dosage corresponds to twice 1.3 mg (total 2.6 mg) of lyophilized extract per day. The application was made for 10 days, which corresponds to a total dose of 26 mg of the extract lyophilized

The blood test was performed twice: before of the study (1st sample) and between days 10 and 12 (2nd sample). Be Five volunteers between 25 and 44 years of age were studied age.

As can be seen in Table 1, all background parameters studied (mean values) were found within the numerical limits registered in the volunteers healthy, coinciding with the data of other authors. The results obtained show that the administration of said extract improved and activated some immunological parameters, being especially relevant a significant increase (p <0.05) of the IgA of secretion - 0.51 g / l. (initially 0.38 g / l.) in the wash nasal.

After this study it was verified how the administration of said extract in homeostasis indices of patients with various forms of breast inflammation paranasal First, it should be noted that all these patients (in total, 24 people) showed a severe depression of the immunocompetence of the organism and, above all, of the system interferon (IFN-? - 19.3 units / ml vs 43.4 of control; IFN-? - 8.2 pcs / ml (31.8 pcs), as well as the active fraction of the T-22.0% lymphocytes (32.6% of control), of the immunoregulation index - 1.71 (2.5), and of the phagocytic values: phagocytic number (TF) - 52.9% (75.5%), phagocytic index (IF) - 2.53 (6.3), final phase of phagocytosis (FF) - 52.4% (72.6%)). As for other parameters, your changes do not were statistically significant (you can only highlight the increase in blood of the IgG value, with an average of 14.1 g / l. versus  12.5 g / l. of control, which points to the allergic background characteristic for this pathology.

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Next, the 24 patients were divided into two groups: 12 patients treated with Cyclamen extract (called "Pac. Cycl." In table 1) and 12 patients treated with traditional treatment (called "Pac. Trad." In table one). The traditional treatment included the administration of antibiotics "amoxiclav, cyprinol, doxycycline" for 5 days together with antihistamine antibiotics "suprastine, zodak, claritin, semperx", nasal drops "naftisin, flasolin, nazivine", mucolytics "sinupret, bromhexine", sulfanilamides , decongestants, punctures, physiotherapy, and about 7 or 10 sinus washes. Between days 13 and 16 from the start of the treatment, the patients were taken the 2nd blood sample for immunological analysis and analyzed according to standard procedures (Jondal et al ., Surface markers human T and B lymphocytes. A large population of lymphocytes forming nonimmune rosstette with sheep red blood cells, J. Exp. Med, 1972, 136 (3), 207-290; Novikov DK, Clinical Immunology and Allerology Guide, Minsk "Bielorus", 1987, p. 223; Manzini G et al ., Immunochemical quantification of antigens by single radical immunodiffusion, Immunochemistry, 1965, 2, p. 235-254; Kost EA, Stenko MI, Methodology guide for clinical studies, Moscow, "Medicine", 1975, 185-187 ; Motavkina HC, and others, Micromethods in immunology, Vladivistok, 1987, p. 181; Soloviev VD, Bektimirov TA Interferons in medical theory and practice, Moscow, "Medicine", 1981, 315.). The data obtained from the analysis showed good immunocorrective properties of said extract in these patients (see table 1 above).

First, a statistically significant increase in the α and γ interferons (p <0.01), of the active fraction of the T-lymphocytes (p <0.05), of the immunoregulation index (p < 0.05), the phagocytic index (p <0.02) and the final phase of phagocytosis (p <0.05). It also highlights the decrease in IgG value (p <0.05) related to the neutralization of allergic factors in the body. The clinical observation data show an improvement in the objective and subjective symptoms of these patients, which confirms the clinical and immunostimulatory effect of the Cyclamen extract.

As for group A (that is, the treatment group) traditional), it was also possible to verify the improvement of these parameters Here the significant change only experienced it interferon-? activity (p <0.05) and the immunoregulation index (p <0.05).

As conclusions of the present example, the isolated extract of Cyclamen europaeum L .:

1. Did not produce allergenic, pyrogenic or local irritations to healthy volunteers or patients. No temperature changes were observed in patients (measurements taken daily at night), or manifestations of local edema, pruritus, hyperemia, spasms or cough.

2. It did not produce a significant activation of the Immune homeostasis rates in healthy volunteers, such as It is typical of "authentic" medications immunomodulators

3. Correct and activate some parameters immunological (especially interferon systems and the phagocytosis). The administration favored the decline of IgG levels in the blood indicating the decrease in load antigenic and smoothing the allergic background of patients

Example 3 Study of the efficacy of an extract of Cyclamen europaeum L. in the treatment of a viral disease

Starting from a model of influenza infection in white mice, viral reproduction in the lungs was studied of animals and the accumulation of the virus in the torrent blood based on antibody production, phagocytosis and the interferon was assessed for homeostasis immunological

The influence of influenza infection on the phagocytic activity of liver Kupffer cells, on the synthesis of nitrogen oxide (NO) and on the state of the chromosomal apparatus of bone marrow cells was also verified. All the parameters referred to were studied in mice infected with the influenza virus, treated with the lyophilized extract isolated from Cyclamen europaeum L. (obtained according to Example 1).

In this way, under the aforementioned conditions experimental the following parameters were studied Related to influenza infection:

A) animal mortality;

B) the reproduction dynamics of the virus flu in the lungs of mice and their accumulation in the bloodstream; Y

C) the immune status of mice.

Influenza virus type A0 was inoculated (obtained from the museum of viral preparations of the Institute of Medical Biotechnology of the Georgia Academy of Sciences) (seal PR8) intranasally, under mild ether anesthesia with a dose of 10 4 EID 50 / 0.1 ml. The content of the virus in a suspension of the lungs and blood serum in different moments after infection, through analysis volumetric samples in ten-day chicken embryos, of as described in ZDRODOVSKI PF, SOKOLOV MI, "Guide for the laboratory diagnosis of viral diseases and rickettiosis ", chapter 96, Medicine, Moscow, 1965. All samples for virological, immunological and genetic analyzes were taken from three mice, sacrificed at the same time after a specific period (indicated in Table 2), obtaining the average values.

The animals were distributed in three groups Main (40 mice in each group):

1. Control of the influenza virus (spread of untreated animals)

2. Effect of the isolated extract of Cyclamen europaeum L. To this end, the preparation was administered to the animals by means of an oral probe, at a rate of 0.5 ml of solution, as defined above, daily for 5 days; being the first administration:

a) 48 hours before virus inoculation; or

b) 24 hours after inoculation of the virus.

The dose of the isolated extract of Cyclamen europaeum L. It was determined from the following calculations:

an adult man (average body weight of 60 kg) corresponds to a single dose of 0.25 ml of the solution of the preparation, which contains 25 mg of the lyophilized extract (obtained according to Example 1). In proportion to the average body weight of a mouse (20 grams), the single dose of lyophilized extract isolated from corresponding Cyclamen is 0.8 µg per mouse. From the preparation of the lyophilized extract of Cyclamen (50 mg of lyophilized substance dissolved in 5.0 ml of water for injections) a solution was prepared in 100 ml, containing 0.8 µg of active ingredient (ie lyophilized extract ) per 0.5 ml.

A) Mortality of the mice

The A0 / PR8 flu virus is highly pathogenic for white mice, resulting in 100% fatal cases. In our study it was observed that mortality begins at 4th day of infection, all animals dying infected by the eighth day of the experiment (group 1).

In the second group the mortality of the animals It was from:

-92% (23 of the 25 mice) and took place between 4 and 10 days, when the extract was administered 24 h after virus inoculation; Y

-76% (that is, 19 of the 25 mice died) and it took place between 6 and 11 days, when the extract 48 h before virus inoculation.

The analysis of the mortality of white mice due to influenza infection shows that lyophilized extract isolated from Cyclamen allows the treatment of influenza infection.

B) Reproduction of the virus in the lungs and viremia

In untreated animals, from day one of the infection, active reproduction began in the lungs of the virus, reaching its maximum on the third day (6.3 lg EID) (lg EID means the decimal logarithm of the Embryonic Infection Dose (in chickens)). Subsequently the intensity of reproduction was decreasing progressively reaching this parameter, at the moment before the death of the animal (7th day), at 3.0 lg EID. Parallel to the reproduction of the virus in the lungs observed viremia (with a maximum of 2.3 lg EID). (S. Zdrodovski "Manual of diagnosis of viral infections in the laboratory "Moscow, Ed." Medicine ", 1967.)

In the 2nd group of mice, with the same dynamics, the maximum on the third day reached 5.3 lg EID and on the 10th day the amount It was reduced to 1.6 lg EID. It is important to note that viremia (1,3 - 0.5 lg EID) was registered only on days 3 and 5 of experiment.

In the third group all the indices of virus reproduction in the lungs and viremia were lower than the control group figures.

The analysis of the results of this series of investigations show the effect of the isolated extract of Cyclamen europaeum in the treatment of influenza virus. The effect was manifested in the reduction of the main indicator of influenza infection (the reproduction of the virus in the lungs). No less important is the decrease in viremia rates

C. Immune status of mice

The comparative immunological analysis showed that the model of influenza infection in white mice is accompanied by the depression of factors as important as interferon or phagocytosis

In the second group of animals, that is, with the administration of the lyophilized extract isolated from Cyclamen , a tendency was observed to improve all the immunological indicators in the late phase of the infection, some of these indicators reaching statistical significance (Table 2).

TABLE 2 Influence of influenza infection and lyophilized Cyclamen extract on immunological indicators in white mice

2

There are convincing data (A.G. Bukrinskaia, V.M Zhdanov "Virology" Moscow, 1992; A.A. Smorodintsev - "The Influenza ", Leningrad," Medicine "1975) suggesting that the Influenza infection is not limited to virus reproduction in the upper airways and in the lungs, but causes a serious infection of the whole organism. In experimental virology there is a comfortable model of generalized influenza infection, according to the which virus is inoculated in large doses intravenously, in place of the intranasal natural pathway (R. R. Jachapuridze "Study of the immune system of guinea pigs in conditions of infection flu for experimental purposes ", magazine" Expertnaia klinicheskaia medicine "2002, No. 1-2, p. 38-40). Under these conditions, the virus accumulates in the liver, causing the mass death of animals 2 or 3 days after infection, that is, before reproduction Intensive virus in the lungs. This methodology is simple and It stands out for its high level of reproducibility.

Based on these data, we have verified the mortality of the animals, the presence of fragments of the virus in the liver of white mice and the functional activity of Kupffer cells when the isolated extract of Cyclamen is administered.

The generalized influenza infection model in white mice was carried out by inoculation via 1.0 ml intravenous influenza virus with reference A0 / PR8, from the Museum of viral preparations of the Institute of Medical Biotechnology of the Georgia Academy of Sciences, with a content of 10 7 EID. Under these conditions a high hepatotropy of the virus, as indicated by the indicators of mortality, viral hemagglutinin in the liver, analysis histological, immunofluorescence test and assessment of phagocytic activity of liver Kupffer cells (AVTSIN AP, SHAKHLAMOV VA - Ultrastructural bases of the pathology of cell // Moscow., "Medicine", 1979, c198; GVAMICHAVA TA - Parenchymal-stromatic interrelation in tumors benign and malignant of the large intestine // Thesis summary PhD in medical sciences, Tbilisi, 1993, c44; LUPPA J. Principles of histochemistry // Moscow, "Mir", 1980, c180).

The analyzes showed that in the course of first three days there was a massive mortality that reached 90% of the animals. It should be noted that the death of the mice took place in the absence of virus in the torrent blood, and that was not related to virus reproduction In the lungs.

Lyophilized extract isolated from Cyclamen europaeum L. It was administered to the mice (following the dosage described in Example 2), the two days prior to the inoculation of the virus and for three days after it. The mortality of the animals in this group was 75%, that is, 15% lower than that of the control group (p <0.1). It is interesting to note that in these mice a reduction in the amount of hemagglutinins (viral antigen) in the liver was recorded in comparison to the control group, without reaching statistical significance.

The immunofluorescence analysis by Kuns indirect method (A.S. Zdrodovski "Diagnostic Manual of viral infections in the laboratory "Moscow, Ed. "Medicine", 1967) revealed the location of viral antigen in hepatocytes and in Kupffer cells. TO the twelve hours of the infection there was a strong illumination of Kupffer cells and a specific fluorescence in the cytoplasm of some liver cells. At 24 hours he had illuminated about 70% of hepatocytes (in a cut of 35-40 cells), which indicates an accumulation of influenza antigen in the liver parenchyma, that is, a spread of the virus throughout the liver.

In mice to which the lyophilized Cyclamen extract had been administered prior to virus inoculation, the intensity of the luminosity of the Kupffer cells was not modified during this period and, at 24 hours in the parenchyma, hepatocytes were observed slightly fluorescent (in a 21-29 cell cut). In this way, the lyophilized extract isolated from Cyclamen europaeum L. it prevented liver cells from being damaged by a massive viral infection, thanks to the stimulation of phagocytosis processes of Kupffer cells and subsequent elimination.

To obtain a complementary confirmation of these facts, acid phosphatase (indicator enzyme) that reflects the processes of cellular digestion (AVTSIN AP, SHAKHLAMOV VA, "Ultrastructural bases of cell pathology", Medicine , chapter 198, Moscow, was studied. 1979; LUPPA J. "Principles of histochemistry", Mir , chapter 180, 1980). Obviously, the amount of this enzyme indicates the intensity of phagocytosis, that is, of the digestion of the phagocytic viral particles in Kupffer cells.

With the help of an "Opton" cytophotometer, a printed histogram of the liver sections was obtained, in which the number of points (due to acid phosphatase) distributed in classes is counted. In untreated white mice the variation curves were distributed in the limits of 4-10 classes. Administration of the lyophilized extract isolated from Cyclamen resulted in a statistically insignificant increase in acid phosphatase activity (3-11 classes) in untreated mice. Generalized influenza infection was characterized by a strong depression of fermentation activity, that is, of the function of cell lysosomes (variation curves) within the limits 2-7 classes (monitoring by an Opton spectrophotometer).

The important aspect is that the lyophilized extract isolated from Cyclamen europaeum L. made an activation of this enzyme possible in infected mice (2-9 classes).

On the other hand, it is well known that the infection Influenza and generalized infection cause serious hypoxia cellular and tissue. Given the special "interest" of the lungs for this action of viruses, we have studied the intensive synthesis of nitrogen oxide (NO) in the lungs of White mice during experimental influenza infection.

In order to determine the amount of free nitrogen oxide (NO) in lung tissues, in a only, on the third day of infection (at the time of maximum virus reproduction in mice) was used sodium dimethyldithiocarbamate as a marker. Briefly, first instead, lung samples were obtained from sacrificed mice on the third day of the flu infection (of 5 mice in total, in the moment of maximum reproduction of the virus in the lungs of the mice). Then 0.2 ml of dimethyldithiocarbamate was added to 1.0 ml of lung suspension, after which they introduced the samples in a polyethylene tube (2 cm x 5 cm) and they were frozen in liquid hydrogen (-196 °). The spectrum of the Electroparamagnetic resonance was obtained with a radio spectrometer \ exists \ ni 1317 (Russia) with the help of a quartz Dewar.

At the same time, nitrogen oxide was studied in intact mice (group 1), infected (2), in infected mice treated with a lyophilized extract isolated from Cyclamen europaeum L. (3), and also in non-infected mice to which the lyophilized Cyclamen extract was administered (4).

In intact animals (group 1) a non-significant signal of labeled nitrogen oxide was recorded on electroparamagnetic resonance (12.6 mmol / mg). On the third day of influenza infection this parameter was increased to 24.6 mmol / ml, p <0.01 (group 2). Under the effects of the administration of the lyophilized extract isolated from Cyclamen europaeum L. (group 3) the intensity of the signal of the nitrogen oxide (NO) in the electroparamagnetic resonance was 16.9 mmol / mg (p <0.05), that is, an improvement of the antioxidant repair mechanisms was revealed in the cells of the lungs. It is interesting that in intact mice treated with said lyophilized extract isolated from Cyclamen (group 4) there was a certain reduction in the amount of NO (10.8 mmol / ml).

As in other pathologies, serious hypoxia occurs in certain tissues during influenza (generalized) infection. In our test, in the lungs of infected white mice an alteration of the electronic transport in the mitochondria, a decrease in the activity of transferrin in the blood plasma, alterations of the integrity of the membrane structures and an activation of the nitroxide synthesis
Geno

As evidenced by the above, the lyophilized extract isolated from Cyclamen europaeum L. It has shown the ability to inhibit active forms of oxygen such as nitrogen oxide generators, which makes it possible to reduce the intensity of oxidative stress.

Analysis of the data related to the concentration of NO showed a direct correlation between the severity of the process and the amount of NO in the samples (LOMNICZI A. et al . - Role of nitric oxide in salivary secretion, Neuroimmunomodulation, 1998, 5, 226-233; SPRINGALL D. et al . - Immunological detection of nitric oxide synthase in human tissue, Histo-chemistry, 1992, 98, 259-266). If this indicator did not exceed 10 mmol / mg in healthy volunteers, a concentration of NO was recorded several times higher in patients with sinusitis (between 3 and 5 times), with an intensification of the signal in electroparamagnetic resonance proportional to the severity of the pathological process (See Table 4).

With the improvement of the clinical picture, a decrease in the NO indicators was observed, and in the case of the group in which the lyophilized extract of Cyclamen was previously administered, it reached almost the control levels (-16.5 mm / mg) .

TABLE 4 Influence of lyophilized extract isolated from Cyclamen on the content of nitrogen oxide (mmol / mg) in saliva and nasal secretions

3

Claims (14)

1. Use of an isolated extract of the wild plant Cyclamen europaeum L. for the manufacture of a medicament for the treatment of a disease associated with an insufficiency of secretion in the interferon system in a human and / or animal. 2. Use of an extract according to claim 1, wherein said extract is lyophilized. 3. Use of an extract according to any of the claims, wherein said medicament is administered by orally. 4. Use of an extract according to any of claims 1 or 2 wherein said medicament is administered topically 5. Use of an extract according to any of claims 1 or 2, wherein said medicament is administered intranasally. 6. Use according to claim 1 or 2 for the manufacture of a mouthwash for the treatment of a angina infection 7. Use according to claim 1 wherein said disease is associated with an insufficiency of the interferon? secretion. 8. Use according to claim 1, in where said disease is associated with an insufficiency of the γ interferon secretion. 9. Use according to claim 1, in where said disease is a viral disease selected from among the group that includes: cold; breast swelling recurrent acute or chronic paranasal such as catarrhal highmoritis or purulent, frontitis, ethmoiditis, sphenoiditis or sinusitis combined; purulent sinusitis or orbital complications associated to these; otitis; pharyngitis; laryngitis; flu. 10. Use according to claim 9 in where said disease consists of an inflammatory infection suppurative 11. Use according to claim 9 in where said viral disease, caused by the influenza virus, It's the flu 12. Use according to any of the claims 1 to 8 wherein said disease, associated with a insufficiency of the secretion of the interferon system, is caused by a pathogenic bacteria. 13. Use according to any of the claims 1 to 8, wherein said disease, associated with a insufficiency in the secretion of the interferon system, is caused by a fungus or yeast. 14. Use according to claim 13, wherein said fungus is of the genus Candida.