WO2005065699A1

WO2005065699A1 - Isolated extract comprising saponins of a plant as therapeutic agent

Info

Publication number: WO2005065699A1
Application number: PCT/IB2004/003948
Authority: WO
Inventors: Vakhtang Mtchedlidze
Original assignee: Hartington Business SL
Current assignee: Hartington Business SL
Priority date: 2003-12-29
Filing date: 2004-12-02
Publication date: 2005-07-21
Anticipated expiration: 2006-06-29
Also published as: ES2235655B1; ES2235655A1

Abstract

Said isolated extract which comprises saponins as active substance is used for manufacturing a medicament for the treatment of an illness associated with an insufficiency of interferon system secretion in a human and/or animal. Said isolated extract can be administered repeatedly and over lengthy periods of time, maintaining appropriate interferon levels.

Description

ISOLATED EXTRACT COMPRISING SAPONINS OF A PLANT AS THERAPEUTIC AGENT

FIELD OF THE INVENTION

The present invention is related with the illnesses associated with immune-system deficiencies. In particular, this invention relates to an isolated extract of a plant which comprises saponins as active substance for manufacturing a medicament for treating an illness associated with an insufficiency of interferon system secretion.

BACKGROUND OF THE INVENTION

Evaluating the role played by the immune system and its immunoco petence level in maintaining the ho eostasis of the organism in a state of normality is a complex task. The least disturbance of immunological vigilance can lead to serious consequences, including the most widespread oncological diseases, autoimmune processes and cytogenic alterations. Ongoing environmental deterioration on a global scale (so-called ecological stress) , together with social and economic living conditions, give rise to a serious immunological deficit whose consequences are all too well known. One of the aims of modern medicine and pharmacology therefore lies in seeking and incorporating into practice new medicaments that maintain optimal functioning of the immune system. Many products having such properties, both natural and synthetic, are already used in clinical practice: immunoglobulins, interferons, interleukins, cytokines, hormone analogues, etc. Their immunotropic capacity makes them general or specific, and they vary in their effectiveness in preventing and treating immune-system pathologies. Outstanding among them are the immuno odulators of natural origin: eleuterococo, Schizandra or Wu-wei-zi (Schisandra chinensis) , Equinacea, Palargonium (Pelargonium reni forme/ ^'sidoldes) , etc. Moreover, the wild plant Cyclamen europaeum L., called in the present invention cyclamen plant, belongs to the Pri ulaceae family and, while it is a very popular plant in many countries of the world, its use is limited to decorative purposes. Little information is currently known about its medicinal properties, and that relates to use of the juice or powder obtained from the plant to treat headaches. In this sense, Spanish patent ES2170726 describes the use of an isolated extract of Cyclamen europaeum L. for manufacturing a medicament for the treatment of sinusitis. Said application discloses the fact that the therapeutic action of a preparation which includes the isolated extract is based on stimulating the secretory activity of the integumentary eptithelium of the mucous membrane in general, together with the serum and submucous glands, thereby facilitating intense drainage of the paranasal sinuses (mechanical action) .

SUMMARISED DESCRIPTION OF THE INVENTION

This invention is directed at the use of an isolated extract of a plant which comprises saponins as active substance for manufacturing a medicament for treating an illness associated with an insufficiency of interferon system secretion.

The object of this invention is detailed below. DESCRIPTION OF THE INVENTION The present invention relates to the utilization of an isolated extract of a plant which comprises saponins as active substance for manufacturing a medicament for treating an illness associated with an insufficiency of interferon system secretion. In the present invention, ^'"illness associated with an insufficiency of interferon system secretion" means a pathological condition of the organism due to a deficiency in the formation of interferons, with factors such as environmental factors (e.g., environmental stress) or the presence of an agent foreign to the host organism (such as a bacterium, virus, etc.) being responsible for said secretion deficiency. In this invention, "interferon system" means the family of proteins involved in the inflammatory process and in the production of reactive species of oxygen. Once activated, they induce the generation of mediator molecules, which in turn activate a set of cells that are recruited at the site of the inflammatory process. In a preferred embodiment of the present invention, said saponins are triterpenoid saponins. En another preferred embodiment of the present invention, said isolated extract is from Cyclamen europaeum L. The isolated extracts of the wild plant Cyclamen europaeum L. can be obtained by means of conventional procedures. One example is disclosed in Spanish patent ES2170726, and particularly in column 5, line 21-column 6, line 54, where there is a description of a procedure for obtaining an isolated extract of Cyclamen europaeum L. Surprisingly, it has been discovered that isolated extracts of plants which comprise saponins as active substance, such as those obtained from The wild plant of Cyclamen europaeum L., stimulate the secretion of interferons, thereby permitting the treatment of illnesses caused by an insufficiency in the interferon system secretion. This activity is due to their pharmacodynamics as well as to said extracts (such as those of Cyclamen) which are rich in saponins, such as Cyclamine and Escina (active substances which are glycosides, mainly triterpenoids or flavonoids and aglycones) , although, as it is known to those skilled in the art, the amount of saponins and maintenance of their pharmacodynamic activity largely depends on the process followed to obtain them. It has further been observed that said activity is a modulating activity of interferon system secretion; when interferon levels are very low, administration of said extract stimulates the secretion of interferons up to a level considered by experts to be normal, while on the other hand, when interferon levels are high, administration of said extract achieves a return of interferon levels to the levels considered normal by experts. Advantageously, due to this modulating activity, said extract can be administered repeatedly and over lengthy periods of time, maintaining appropriate levels of interferon (unlike the immunostimulators known in the state of the art which, as they do not have said modulating activity, are administered over short periods of time in order to prevent interferon levels becoming excessively high) . The inventors of the present invention believe that other extracts from different plants belonging to the Primulaceae family could be found which, due to their containing saponins structurally identical or similar to cyclamine, might give rise to effects similar or identical to those described in this invention. In one embodiment of this invention, said isolated extract is freeze-dried. Surprisingly, the inventors of this invention have discovered that the isolated extracts such as those obtained from the wild plant of Cyclamen europaeum L. act on all the indications mentioned above and below, especially if these extracts are freeze-dried immediately following preparation of the extract, as the efficacy and stability of the galenical formulations are improved thereby. One embodiment of this invention relates to the use of an isolated extract which comprises saponins as active substance for manufacturing a medicament for treating an illness associated with an insufficiency of interferon system secretion, the medicament being administered orally. For oral administration said medicament can be prepared in the form of normal suckable or coated tablets, pills or other suitable formulations (including sublingual or buccal) , with normal, sustained or controlled-diffusion release of the active substance (i.e. saponins) . The sustained-release or controlled-diffusion formulations for the active substance in suitable doses are advisable for preventing potential gastrointestinal tract upsets and irritations that could arise due to the saponins following the 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 this invention have found that the treatment of infection in anginas can be achieved by carrying out gargles with a mouthwash including an isolated extract from a plant according to the first aspect of the present invention, such as Cyclamen. Another embodiment of this invention relates to the utilization of an isolated extract of which comprises saponins as active substance for manufacturing a medicament for treating an illness associated with an insufficiency of interferon system secretion, by administering the medicament topically. For topical administration, especially intranasally, said medicament can be prepared in the form of ointments, jellies or drops which are applied into each nasal meatus. All these preparations can be muco-adhesive. Preferably, said medicament is administered in the form of a nasal spray. Such formulations can be administered, for example, by dissolving them in sterile water ( water for injections) and dispersing them with a suitable dispensing spray into each nasal meatus. The authors of the present invention have found, for the first time, that intranasal administration of a medicament in accordance with this invention induces stimulated secretion of the interferon system, and particularly of interferon α and γ (IFN-α and IFN-γ) . This new possibility is very important and surprising because intranasal administration of chemically or biologically defined immunostimulants or immunomodulators, such as interferons of various types, like IFN-α or IFN-γ, was unknown until now. In one embodiment of this invention, said extract is used for manufacturing a medicament for treating an illness associated with an insufficiency of interferon α secretion. In another embodiment of this invention, said extract is used for manufacturing a medicament for treating an illness associated with an insufficiency of interferon γ secretion. The inventors of this invention have found that the administration of an isolated extract according to the first aspect of the invention and as it is exemplified below using Cyclamen europaeum L., leads to a significant increase in the levels of interferon (α and γ) , in the active fraction of T lymphocytes, and a reduction of IgG, among other effects. The fact that there is increased secretion of interferons gives rise to an activation of the immune system, with the immune response of the organism activating due to the appearance of a foreign agent. This activity is due to their pharmacodynamics as well as to said extracts (such as those from Cyclamen) which are rich in saponins, such as Cyclamine and Escina (active substances which are glycosides, mainly triterpenoids or flavonoids and aglycones) , although, as it is known to those skilled in the art, the amount of saponins and maintenance of their pharmacodynamic activity largely depends on the process followed to obtain them. It has further been observed that said activity is a modulating activity of interferon system secretion; when interferon levels are very low, administration of said extract stimulates the secretion of interferons up to a level considered by experts to be normal, while on the other hand, when interferon levels are high, administration of said extract achieves a return of interferon levels to the levels considered normal by experts . In one embodiment of this invention, the isolated extract is used for manufacturing a medicament for treating a viral illness selected from the group that includes: colds; relapsing acute or chronic inflammation of the paranasal sinuses such as catarrhal or purulent high oritis, frontitis, ethmoiditis, sphenoiditis or combined sinusitis; purulent sinusitis or the orbital complications associated therewith; otitis; pharyngitis; laryngitis; influenza. In another embodiment, said viral illness caused by the influenza virus is flu. The antiviral activity of an isolated extract of Cyclamen europaeum L. is described below by following as a model an influenza infection in white mice (see Example 3) . The influenza infection was developed within a context of a serious depression of immunological homeostasis indicators such as the interferon system or phagocytosis. The administration of said isolated extract rendered possible an improvement of parameters such as the interferon system or phagocytosis, reaching statistical significance in the late phases of the infection. The "protective" effect of the isolated extract was fully confirmed by means of a generalised infection (influenza infection parameter) . The mortality of the mice from generalised infection was reduced from 90% to 75%.

Histological analysis of the liver showed improved pathomorphological symptoms in the hepatic tissue. Immunofluorescence analysis revealed an intensification of the specific illumination of Kupffer's liver cells, thereby demonstrating their activation, with the resultant protective action of parenchyma cells (hepatocytes) . Thus, on the basis of the model of influenza infection in white mice, we found a complex protection capacity of the isolated extract de Cyclamen europaeum L., both on a general level (in relation to the immune system) and in its capacity as an antiviral substance (and specifically, anti-influenzal) . This capacity includes genie protection, which in turn has an influence on the repair and functioning of the immune cells. One of the protective-action mechanisms of the isolated extract of Cyclamen europaeum L. derives from its antioxidant properties, which give rise to a recovery of respiration at intracellular level (including the immune cells) . Optionally, said illness, associated with an insufficiency of interferon system secretion, is caused by a pathogenic bacterium. In this invention, "pathogenic bacterium" is taken to mean a bacterium which causes a deficiency in the secretion of the interferon system in the host organism, giving rise to a disease or pathology. Nitric oxide (NO) is a universal biological messenger, with a broad spectrum of action, which is synthesised when many organs of the non-adrenergic and non-cholinergic branches of the autonomous nervous system come into operation. Endogenous nitric oxide is a fundamental part of calcium homeostasis in the cells and, therefore, of the activity of the dependent protein- kinases of Ca²⁺. Nitric oxide also plays a specific role in the formation of secretions from the mucous membranes of the mouth cavity and of the upper airways. The presence of nitric oxide synthetase, both of neuronal (nNOS) and inducible (iNOS) types in the salivary glands has been demonstrated. The activation of nNOS in the cells with the help of metacolin and substance P enables the opening of ionic 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. Nitric oxide is considered to be a regulator of lymphocyte proliferation; NO synthesis by the phagocyte cells constitutes 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 this invention has the capacity to inhibit the active forms of oxygen as nitric oxide generators, thus enabling a reduction in the intensity of oxidation stress, thereby indicating that it can be used for the treatment of a bacterial-type disease. In another embodiment, an isolated extract of a plant comprising saponins as active substance is used for manufacturing a medicament for treating an illness associated with an insufficiency of interferon system secretion, which is caused by a fungus or yeast, preferably by a fungus of the Candida genus. In this invention, "protective effect" or "protective" means that administration of the isolated extract gives rise to the secretion of interferons which trigger a response against a particular agent (for example, a virus or bacterium) foreign to the organism. One possible action mechanism that would explain the immunotropic action of an extract according to the present invention and as it is exemplified below, would be the ability to increase the activity of the immune cells of the nasal and buccal sinuses with the resulting chain- activation of the immune system systemic factors, improved defences for the elimination of free radicals and an antitoxic effect. The isolated extract of Cyclamen europaeum L. enabled a reduction of hypoxia as described in Example 3. Moreover, it did not produce harmful effects on the organism of gestating mice or on the foetuses. Included below by way of non-restrictive illustration are the following examples.

EXAMPLES

Example 1 : Preparing an isolated extract of Cyclamen europaeum L. Undamaged tubercles (without signs of rotting) are selected. They are then cleaned with a brush under running water, removing remains of earth and sand. The tubercles are then washed with desalinated water and left to dry in the open air. 2005/065699

11 Once dry, the tubercles are placed in a bath and covered with 95% ethyl alcohol, remaining thus for 1 hour. The tubercles so obtained are then placed in a stainless steel press and the juice is extracted. The triturate so obtained is placed in a reactor, covered with desalinated water, heated until a temperature of 40-70^°C is attained, and kept thus for 1 hour. It is then cooled and squeezed, and the extract obtained in the press is mixed with the juice. Then, in order to remove residual substances, 95% ethyl alcohol is added to the extraction obtain (juice+extract) to an amount of 15% of the total volume. The extraction obtained is then submitted to normal filtering, followed by sterile filtering. This sterile solution is then bottled in 0.5 ml vials. Finally, freeze-drying is carried out.

Example 2 : study of the immunostimulant capacity of an isolated extract of Cyclamen europaeum L.

The study of the immunostimulant capacity of the extract of Cyclamen europaeum L. was carried out on the basis of the non-specific resistance and humoral immunity values: the percentages of B lymphocytes and T lymphocytes and their subpopulations (active Ts, adjuvant Ts, suppressor Ts, autorregulation index) , the number of immunoglobulins of the three main classes, G (IgG) , M (IgM) and A (IgA) , the phagocytary activity of neutrophils in the blood (TF = phagocyte number, IF = phagocyte index, FF = final phase of phagocytosis) , and reaction of the leukocytes to interferons in vitro (IF -α and IFN-γ) . (Kipiani Va et al. "Oxidative processes in peridontitis", Int. J. Immunorehabilitation, 2000, 2, 291) . The immunological analysis of the blood was carried out by means of micromethods (a maximum of 0.2 ml of blood extracted from the fingertip) . Firstly, the immunostimulant capacity of the medicament was checked in healthy volunteers who had not suffered from influenza, respiratory illnesses or other infectious diseases 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 freeze-dried extract (prepared as in Example 1) in 5 ml of water for injections (corresponding to 1.3 mg of freeze-dried extract), with a dispensing spray device into each nasal meatus. This dosage corresponded to twice 1.3 mg (a total of 2.6 mg) of the freeze-dried extract a day. Application was continued for 10 days, corresponding to a total dosage of 26 mg of the freeze-dried extract. The blood analysis was carried out twice: before the study (1st sample) and between days 10 and 12 (2nd sample) . Five volunteers aged between 25 and 44 years were submitted to study. As can be observed in Table 1, all the background parameters studied (mean values) lay within the numerical values recorded in healthy volunteers, coinciding with the data from other authors. The results obtained show that administration of said extract improved and activated some immunological parameters, the most notable effect being a significant increase (p<0.05) of secreted IgA - 0.51 g/1 (initially 0.38 g/1) in the nasal lavage. Following this study, an examination was undertaken of how administration of said extract affected homeostasis indexes in patients with various forms of inflammation of the paranasal sinuses. It should be noted, firstly, that all these patients (24 persons in total) showed a severe depression of the organism' s immunocompetence and, particularly, of the interferon system (IFN-α - 19.3 uds/ml as compared with 43.4 for control; IFN-γ - 8.2 uds/ml (31.8 uds) , and of the active fraction of the T lymphocytes- 22.0% (32.6% for control), of the immunorregulation index - 1.71 (2.5), and of the phagocyte values: phagocyte number (TF) - 52.9% (75.5%), phagocyte index (IF) - 2.53 (6.3), final phase of phagocytosis (FF) - 52.4% (72.6%)). In relation to other parameters, the changes therein were not statistically significant (the only one of note being an increase of the IgG value in the blood, with a mean of 14.1 g/1 compared to 12,5 g/1 for control), which points to the allergic background characteristic of this pathology.

Table 1 Influence of the isolated extract of Cyclamen on the immunological parameters in healthy volunteers (n = 5) and patients (n = 24) with diseases of the paranasal sinuses, treated with extract of Cyclamen: n = 12, and traditionally treated: n = 12.

Note: All the values are averages of all the volunteers or patients respectively.

1 = initial sample blood

2 = following the treatment

Cycl. Pat. 1 = blood sample from the 12 patients before the treatment with Cyclamen extract

Cycl. Pat. 2 = blood sample from the 12 patients after the treatment with Cyclamen extract Trad. Pat. 2 = blood sample from 12 patients after the traditional treatment with ?? (The initial blood of the extract group is analogous to the blood of the traditional treatment group) .

Ta = active T lymphocytes. Th = adjuvant T lymphocytes. Ts - suppressor T lymphocytes. Ii = immunorregulation index. B = No. of immunoglobulinas of the 3 main classes.

The 24 patients were then divided into two groups: 12 patients treated with the extract of Cyclamen (termed "Cycl. Pat." in Table 1) and 12 patients treated with the traditional treatment (termed "Trad. Pat." in Table 1) . 5 The traditional treatment included the administration of antibiotics (amoxiclav, ciprinol, doxicillin) for 5 days together with antihistamine antibiotics (suprastina, zodak, claritin, semprex) , nosedrops (naftisina, flasolina, nazivina) , mucolytics (sinupret, bromhexina) ,

10 sulphanylamides, decongestants, punction treatments, physiotherapy treatments, and some 7 to 10 nasal sinus lavages. Between days 13 and 16 from the start of the treatment, the 2nd sample of blood was taken from the patients for immunological analysis and was analysed using

15 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 D. K., Guia de immunologia and alerologia clinica, Minsk "Bielorus",

201987, p. 223; Manzini G et al., Immunochemical quantification of antigens by single radical immunodiffusion, Immunochemistry, 1965, 2, pp. 235-254; Kost E. A., Stenko M. I., Methodology Guide for Clinical Studies, Moscow, "Medicina", 1975, 185-187; Motavkina H.

25 C, and others, Micromethods in Immunology, Vladivistok, 1987, p. 181; Soloviev V. D., Bektimirov T. A. Interferons in medical theory and practice, Moscow, "Medicina", 1981, 315.). The data obtained from the analysis show good immunocorrective properties for said extract in these

30 patients (see Table 1 above) . Firstly, a statistically significant increase of interferons α and γ (p<0.01), of the active fraction of the T-lymphocytes (p<0.05), of the immunorregulation index (p<0.05), of the phagocyte index (p<0.02) and of the final

35 phase of phagocytosis (p<0.05) was observed. Likewise notable was a reduction of the IgG value (p<0.05) related with the neutralisation of the allergising factors of the organism. The clinical observation data show an improvement in these patients' subjective and objective symptoms, thus confirming the clinical and immunostimulant effect of the extract of Cyclamen. Group A (i.e. the traditional treatment group) also showed an improvement of said parameters. Here, significant change was undergone only in interferon-α activity (p<0.05) and in the immunorregulation index (p<0.05) . As conclusions to this example, the isolated extract of Cyclamen europaeum L. : 1. Did not produce allergenic or pyrogenic effects nor local irritation in either healthy volunteers or patients. No temperature alterations were observed in the patients (measurements taken daily at night) , nor manifestations of the local oedema, pruritis, hyperaemia, spasm or cough types. 2. Did not produce significant activation of immunological homeostasis indices in the healthy volunteers, as inherent to "true" immunomodulator medicaments . 3. Corrects and activates some immunological parameters (the interferon and phagocytosis systems, particularly) . Administration boosted a fall in levels of blood IgG, showing reduction of antigen load and an alleviation in the allergic background of the patients. Example 3 : Study of the efficacy of Cyclamen europaeum L. extract on the treatment of viral disease.

Taking as starting point a model of influenza infection in white mice, a study was made of viral reproduction in the animals' lungs and accumulation of the virus in the bloodstream; an immunological evaluation of homeostasis was carried out on the basis of antibody production, phagocytosis and interferon. The influence of the influenza infection was likewise found in the phagocytary activity of the Kupffer' s liver cells, in nitric oxide (NO) synthesis and in the state of the bone marrow cells' chromosome apparatus. All these parameters were studied in mice infected with the influenza virus, treated with the isolated freeze-dried extract of Cyclamen europaeum L. (prepared as in Example 1) . Accordingly, and under the aforesaid experimental conditions, the following parameters related with the influenza infection were studied: A) mortality of the animals; B) the influenza virus' reproduction dynamics in the mice's lungs and its accumulation in the bloodstream; and C) the immunological status of the mice.

Type A0 influenza virus (obtained from the viral preparations museum of the Medical Biology Institute of the Georgia Academy of Sciences) (seal PR8) was inoculated intranasally under a mild anaesthetic of ether at a dose of 10⁴ EID₅₀/0.1 ml. A check was made for virus content in a suspension from the lungs and in blood serum at different times following the infection, using volumetric analysis of the samples in ten-day-old chicken embryos, as described in ZDRODOVSKI PF, SOKOLOV MI, "Guide to Laboratory Diagnosis of Viral Diseases and Rickettsiosis", chapter 96, Medicine, Moscow, 1965. All the samples for the virological, immunological and genetic analyses were taken from three mice, killed at the same time after a specific period of time (stated in Table 2) , to obtain the mean values. The animals were distributed into three main groups (40 mice in each group) : 1. Control of the influenza virus (infection of untreated animals) 2. Effect of the isolated extract of Cyclamen europaeum L. For this purpose the preparation was administered to the animals using an oral tube, at the rate of 0.5 ml of solution daily, as set out above, for 5 days; the first administration taking place: a) 48 hours before inoculation of the virus; or b) 24 hours after inoculation of the virus. The dosage of the isolated extract of Cyclamen europaeum L. was determined on the basis of the following calculations: For an adult man (mean body weight of 60 kg) there corresponds a monodose of 0.25 ml of the preparation solution, containing 25 mg of the freeze-dried extract (prepared as in Example 1) . In proportion to the mean body weight of a mouse (20 grams) , the corresponding monodose of isolated freeze-dried extract of Cyclamen is 0.8 μg per mouse. The preparation of the freeze-dried extract of Cyclamen (50 mg of freeze-dried substance in 5.0 ml of water for injections) was used to prepare a solution 100 ml, which contains 0.8 μg of active substance (i.e. freeze-dried extract) per 0.5 ml.

A) Mortality of the mice The A.0/PR8 influenza virus is highly pathogenic for white mice, proving fatal in 100% of cases. In our study, mortality was observed to start on the fourth day from the infection, with all the infected animals having died by the eighth day of the experiment (group 1) . In the second group the mortality of the animals was : -92% (23 of the 25 mice) and took place between 4 and 10 days, when the extract was administered 24 h after inoculation of virus; and -76% (that is, 19 of the 25 mice died) and took place between 6 and 11 days, when the extract was administered 48 h before inoculation of virus. Analysis of the mortality of the white mice by influenza infection shows that the freeze-dried extract of

Cyclamen permits treatment of the influenza infection.

B) Reproduction of the virus in the lungs and viremia. Active reproduction of the virus in the lungs began in untreated animals right from the first day of the infection, reaching a maximum on the third day (6.3 lg EID) (lg EID means the decimal logarithm of the Embryonary Infection Dose (in hens) ) . The reproduction intensity then diminished progressively down to this parameter, at the moment prior to the death of the animal (7th day), to 3.0 lg EID. In parallel to reproduction of the virus in the lungs, viremia was observed (with a maximum of 2.3 lg EID) . (S. Zdrodovski "Manual of Diagnosis of Viral Infections in the Laboratory" Moscow, Ed. «Medicina», 1967.) In the second group of mice, with the same dynamics, the maximum on the third day attained 5.3 lg EID, while by the 10th day the amount had reduced to 1.6 lg EID. It is important to point out that the viremia (1.3 - 0.5 lg EID) was recorded only on days 3 and 5 of the experiment . In the third group, all the indices of reproduction of the virus in the lungs and of viremia were lower than the figures for the control group. The analysis of the results of this series of investigations shows the effect of the isolated extract of Cyclamen europaeum in treatment of the influenza virus . The effect showed itself in a reduction of the principal indicator of influenza infection (reproduction of the virus in the lungs) . No less important was the reduction in viremia indices.

C. Immunological status of the mice

The comparative immunological analysis showed that the model of influenza infection in white mice is accompanied by a depression of factors as important as interferon and phagocytosis. In the second animal group, i.e. with administration of the isolated freeze-dried extract of Cyclamen, a tendency towards improvement in all immunological indicators was observed in the late phase of the infection, with some of these indicators attaining statistical significance (Table 2) .

Table 2. Influence of the influenza infection and of the freeze-dried extract of Cyclamen on the immunological indicators in white mice

A: influenza infection; B: influenza infection + treeze- dried extract of Cyclamen; Control: untreated animals; TF: phagocyte number; IF: phagocyte index; FF: final phase of phagocytosis .

Convincing data exist (A.G. Bukrinskaia, V.M Zhdanov «Virology» Moscow, 1992; A. . Smorodintsev - «Influenza», Leningrad, «Medicina» 1975) to suggest that influenza infection is not limited to reproduction of the virus in the upper respiratory airways and in the lungs, but that it also provokes serious infection throughout the entire organism. Experimental virology has a convenient experimental model of generalised influenza infection, by which the virus is inoculated intravenously in large dosages, instead of via the natural intranasal route (R. R. Jachapuridze «Study of the immune system of guinea pigs under influenza infection conditions for experimental purposes», «Expertnaia klinicheskaia medicina» journal 2002, Wl-2, pp. 38-40) . Under these conditions, the virus accumulates in the liver, leading to mass death of the animals 2 or 3 days after the infection, i.e. prior to intensive reproduction of the virus in the lungs. This methodology is simple and notable for its high level of reproducibility. On the basis of these data, we found mortality of the animals, the presence of fragments of the virus in the liver of the white mice and functional activity of the

Kupffer' s cells when the isolated extract of Cyclamen was administered. The model of generalised influenza infection in white mice was carried out by intravenous inoculation of 1.0 ml of the influenza virus of reference A0/PR8, from the Viral Preparations Museum of the Medical Biology Institute of the Georgia Academy of Sciences, with a content of 10⁷ EID. Under these conditions, high hepatropy of the virus was observed, as denoted by the indicators of mortality, viral hemagglutinin in the liver, histological analysis, the immunofluorescence test and evaluation of phagocyte activity in the Kupffer's cells of the liver (AVTSIN AP, SHAECHLAMOV VA - Ultrastructural bases of cell pathology// Moscow, "Medicina", 1979, cl98; GVAMICHAVA TA - Parenchymo-stromatic interrelation in benign and malignant tumours of the large intestine//Doctoral thesis abstract in medical sciences, Tiflis.1993, c44; LUPPA J. Histoche ical Principles// Moscow, "Mir", 1980, cl80) . The analyses demonstrated that over the course of the first three days mass mortality occurred in 90% of the animals. It should be noted that the mice's death occurred in the absence of virus in the bloodstream, and that it was unrelated with reproduction of the virus in the lungs . The isolated freeze-dried extract of Cyclamen europaeum L. was administered to the mice (following the dosage described in Example 2) on the two days preceding inoculation of the virus and for three days thereafter. The mortality of the animals in this group was 75%, that is, 15% lower than that of the control group (p<0.1) . In comparison with the control group, it is interesting to note that in these mice a reduction of the amount of hematogglutins (viral antigen) was recorded in the liver, although it did not attain statistical significance. Immunofluorescence analysis by means of the indirect Kuns method (A.S. Zdrodovski «Manual of Diagnosis of Viral Infections in the Laboratory » Moscow, Ed. «Medicina», 1967) revealed viral antigen localisation in the hepatocytes and in the Kupffer' s cells . Strong illumination of the Kupffer's cells and a specific fluorescence in the cytoplasm of some hepatic A cells occurred twelve hours from infection. By 24 hours some 70% of the hepatocytes (in a cut of 35-40 cells) had illuminated, indicating an accumulation of the influenza antigen in the hepatic parenchyma, that is, propagation of the virus throughout the entire liver. In the mice which had been administered the freeze-dried extract of Cyclamen prior to inoculation of virus the luminosity intensity of the Kupffer' s cells did not alter over this period, while at 24 hours slightly fluorescent hepatocytes (in a cut of 21-29 cells) were observed in the parenchyma. The isolated freeze-dried extract of Cyclamen europaeum L. thus prevented the hepatic cells from being damaged by a massive viral infection, thanks to stimulation of the phagocytosis processes of the Kupffer' s cells and subsequent elimination. In order to obtain supplementary confirmation of these findings, a study was undertaken of the acid phosphatase (indicator enzyme) that shows cellular digestion processes (AVTSIN AP, SHAKHLAMOV VA, "Ultrastructural bases of cell pathology", Medicina, chapter 198, Moscow, 1979; LUPPA J. "Histochemical Principles", Mir, chapter 180, 1980) . The amount of this enzyme clearly indicates the intensity of phagocytosis, i.e. of digestion of the phagocyted viral particles in the Kupffer' s cells . An "Opton" cytophotometer was used to obtain a histogram print-out of the hepatic cuts, counting the number of points (due to the acid phosphatase) distributed into classes. In the untreated white mice the variation curves were distributed in the limits of 4-10 classes. Administration of the isolated freeze-dried extract of Cyclamen gave rise to a statistically insignificant increase in the activity of the acid phosphatase (3-11 classes) in the untreated mice. Generalised influenza infection was characterised by a strong depression of fermentation activity, that is, of the function of the cellular lisosomes (variation curves) within the limits of 2-7 classes (monitoring using an Opton spectrophotometer) . The important aspect to note is that the isolated freeze-dried extract of Cyclamen europaeum L. enabled an activation of this enzyme in the infected mice (2-9 classes) . Moreover, it is well-known that influenza infection and generalised infection give rise to serious cell and tissue hypoxy. Bearing in mind the special "involvement" of the lungs in this action of the viruses, we studied the intensive synthesis of nitric oxide (NO) in the lungs of the white mice during the experimental influenza infection. In order to determine the amount of free nitric oxide (NO) in the pulmonary tissues, once, on the third day from the infection (at the time of maximum reproduction of the virus in the mice) , sodium dimethyldithiocarbamate was used as marker. Firstly, in brief outline, lung samples were taken from mice killed on the third day from the influenza infection (from a total of 5 mice, at the time of maximum reproduction of the virus in the lungs of the mice) . 0.2 ml of dimethyldithiocarbamate was then added to 1.0 ml of lung suspension, following which the samples were placed into polyethylene tubes (2 cm x 5 cm) and frozen in liquid hydrogen (-196°) . The electroparamagnetic resonance spectrum was taken with a 33 1317 radiospectrometer (Russia) with the aid of a quartz Dewar flask. At the same time the nitric oxide was studied in intact mice (group 1) , infected mice (2) , infected mice treated with an isolated freeze-dried extract of Cyclamen europaeum L. (3), and also in non-infected mice which had been administered the freeze-dried extract of Cyclamen (4) . For the intact animals (group 1) a non-significant signal of nitric oxide was marked in the electroparamagnetic resonance (12.6 mmol/mg). On the third day of the influenza infection this parameter rose to 24.6 mol/ml, p<0.01 (group 2). Under the effects of administration of the isolated freeze-dried extract of Cyclamen europaeum L. (group 3) the intensity of the nitric oxide (NO) signal in the electroparamagnetic resonance was 16.9 mmol/mg (p<0.05), that is, an improvement was shown in the antioxidant reparative mechanisms of the lung cells. It is interesting to note that in the intact mice treated with said isolated freeze- dried extract of Cyclamen (group 4) there was a certain reduction of the amount of NO (10.8 mmol/ml) . As in other pathologies, during (generalised) influenza infection serious hypoxia occurs in certain tissues. In our trial, the lungs of the infected white mice showed an alteration of electronic transportation in the mitochondria, a reduction of transferrin activity in the blood plasma, alterations of the integrity of the membrane structures and an activation of nitric oxide synthesis . As evidenced by the foregoing, the isolated freeze-dried extract of Cyclamen europaeum L. showed a capacity to inhibit the active forms of oxygen as nitric oxide generators, thus enabling a reduction of the oxidative stress intensity. Analysis of the data relating 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, Neuroimmuno odulation, 1998, 5, 226-233; SPRINGALL D. et al . - Immunological detection of nitric oxide synthase in human tissue, Histo-che istry, 1992, 98, 259-266) . While in the healthy volunteers this indicator did not exceed 10 mmol/mg, in the patients with sinusitis a concentration of NO several times higher (between 3 and 5 times) was recorded, with an intensification observed in the electroparamagnetic resonance signal proportional to the seriousness of the pathological process (see Table 4) . With the improvement of the clinical symptoms a reduction of the NO indicators was observed, which in the case of the group to which the freeze-dried extract of Cyclamen had previously been administered almost reached the control levels (-16.5 mm/mg) .

Table 4. Influence of the isolated freeze-dried extract of Cyclamen on the nitric oxide (mmol/mg) content in saliva and in nasal secretions Before Cyclamen extract Traditional treatment treatment After treatment After treatment 43.5 - (4.1)* 16.5 - (1.7)* 31.8 - (2.9)* (*) shows how many times greater the amount of nitric oxide (NO) was in comparison with controls Example 4 : Assay of the inmunomodulatory ability with extracts obtained from tea seeds .

Said extracts, obtained following procedures well- known for those skilled in the art, were tested following the same protocols than those described above. The results were similar to those obtained using a Cyclamen europaeum extract: reduced response time (latency) , character of secretions, absence of, or negligible pain and salivatory response, amon others

Claims

C A I MS 1. Use of an isolated extract of a plant which comprises saponins as active substance for manufacturing a medicament for treating an illness associated with an insufficiency of secretion in the interferon system in a human and/or animal. 2. Use of an isolated extract according to claim 1, wherein said saponins are triterpenoid saponins. 3. Use of an isolated extract according to claim

1, wherein said isolated extract is obtained from Cyclamen europaeum L. 4. Use of an extract according to Claim 1, in which said extract is freeze-dried. 5. Use of an extract according to either of the preceding claims, in which said medicament is administered orally. 6. Use of an extract according to either of the preceding claims, in which said medicament is administered topically. 7. Use of an extract according to either of claims 1-5, in which said medicament is administered intranasally. 8. Use according to Claims 1 to 4 for manufacturing a mouthwash for the treatment of an infection in the anginas . 9. Use according to Claim 1, in which said illness is associated with an insufficiency of interferon α secretion. 10. Use according to Claim 1, in which said illness is associated with an insufficiency of interferon γ secretion. 11. Use according to Claim 1, in which said illness is a viral disease selected from the group which includes: colds; relapsing acute or chronic inflammation of the paranasal sinuses such as catarrhal or purulent highmoritis, frontitis, ethmoiditis, sphenoiditis or combined sinusitis; purulent sinusitis or the orbital complications associated therewith; otitis; pharyngitis; laryngitis; influenza. 12. Use according to Claim 11, in which said illness consists in a supurant inflammatory infection. 13. Use according to Claim 11, in which said viral disease, caused by the influenza virus, is flu. 14. Use according to any of Claims 1 to 10, in which said illness, associated with an insufficiency of interferon system secretion, is caused by a pathogenic bacterium. 15. Use according to any of Claims 1 to 10, in which said illness, associated with an insufficiency of interferon system secretion, is caused by a fungus or yeast. 16. Use according to Claim 15, in which said fungus is of the Candida genus.