MXPA00001557A - Immunostimulatory and metastasis inhibiting fermented vegetal material - Google Patents

Abstract

The invention concerns an immunostimulatory and metastasis inhibiting fermented, dried vegetal material, pharmaceutical compositions containing the latter, the production process and the use of the dried material in the production dietary supplement and of immunostimulatory and metastasis inhibiting pharmaceutical compositions. The material according to the invention can be obtained by fermentation of wheat germ in an aqueous medium inthe presence of Saccharomyces cerevisiae and by drying the fermented liquid.

Description

FERMENTED VEGETABLE MATERIAL IMMUNOSTIMULATOR AND METASTASIS INHIBITOR FIELD OF THE INVENTION The present invention relates to a dry and fermented plant material immunostimulatory and metastasis inhibitor, pharmaceutical compositions containing the same, to the process for the preparation of the material and the use of the dry plant material as a dietary supplement and in the production of pharmaceutical compositions in a stimulators and especially inhibitors of metastasis. BACKGROUND OF THE INVENTION One of the main objectives of the treatment of tumors is the inhibition of metastasis, since the primary tumor caused by the malignant growth of the cells is dispersed via metastasis to the surrounding cells and organs and causes in these secondary tumors, which they can not be removed surgically and can become resistant to chemotherapy. Researchers are concentrating more and more on the development of materials in unomodulators and materials of origin -mainly plant-natural- have been intensively studied.

It is a well-known fact that compositions with a quinone structure play an important biological role. Various quinone derivatives are found in plants, such as ubiquinones, plastoquinones, menaquinones, which play a role in photosynthesis, but also in the cellular respiratory system of vertebrates, and therefore also of humans and in the coagulation of the blood, etc. Various derivatives of benzoquinone are also used for medicinal purposes. Adria icine, daunurobicin and mitomycin C are derivatives of quinone with a cytostatic effect, while other benzo- and hydroquinones have an antimicrobial effect and are the active components of antibiotics suitable for treatment of bacterial infections such as Tetran-B, Metacycline. and Doxycycline. The literature reports that 2,6-dimethoxy-p-benzoquinone (2,6-DMBQ) and 2-methoxy-p-benzoquinone (2-MBQ) have a fungicidal and bacteriostatic effect, and are also cytotoxic for malignant tumor cells . [Int. J. Quant. Chem .: Quant. Biol. Symp. 7., 217-222 (1980), 9., 27-30 (1982) and 12., 257-261 (1985); Phytochemistry 27., 225 (1971) and J. Agrie.

Foof Chem.39., 266 (1991)]. It has been shown that a mixture of 2,6-dimethoxy-p-benzoquinone and ascorbic acid inhibits the growth of Ehrlich ascites tumor cells in mice. [Proc. Nati Acad. Sci. USA 81., 2088-2091 (1984) and 82, 1439-1442 (1985)]. It has been found and isolated 2, 6-dimethoxy-p-benzoquinone and 2-methoxy-p-benzoquinone in various plants [Magy. Ké. Folyóirat 102/7., 320-325 (1996)]. These two compositions can be found in the largest amounts in the form of glycosides in wheat (Tricitum vulgaris), to be more exact in wheat germ. The compositions were isolated in the 1950s from wheat germs fermented with yeast and were identified for the purpose of examining the quality deterioration of the bread to which the wheat germ has been added. [Helv. Him. Acta 33., 433 (1950); J. Chem. Soc. London 1952, 4821-4823]. According to the wheat germ literature, the amount of 2-MBQ is approximately 0.05% by weight, that of 2,6-DMBC of approximately 0.01% by weight (as glycosides). The presence of quinones as glycosides is explained by the fact that quinones, especially p-benzoquinones substituted by methoxy are reactive while their hydroquinone glycosides are more stable and inert. During our experiments concerning the study of wheat germ fermentation, we concluded that the dry extract derived from the fermented liquid produced while fermenting wheat germ with flour yeast (Saccharomyces cerevisiae) has a surprising immunostasising and metastasis inhibiting effect, and can successfully applied as the active agent of the immunosorbent and metastasis inhibiting pharmaceutical compositions. This finding is surprising, because even though literature reports [Proc. Nati .Acad. Sci. USA 80., 129 (1983)] that a mixture of 2,6-DMBQ and ascorbic acid inhibits the growth of Ehrlich's ascites tumor cells - these are eminently suitable for quantitative analyzes of tumor growth and for the study of its biochemical functions - the extract described in the invention, which also contains unidentifiable components 2,6-DMBQ and 2-MBQ - proved to be practically ineffective in these primary tumor cells, even combined with ascorbic acid - generally in all tumors primary, and proved to be only very effective in the treatment of metastases. The aspects of the treatment of primary tumors and the developed metastases of those are basically different, so it is not obvious to the researcher that a plant extract containing components known to be effective in the treatment of primary tumors and not yet fully identifiable as its chemical composition has an immunostimulatory and metastasis inhibiting effect. [Cancer, Principles and Practice of Oncology, Vol 1, 4th edition, J.B. Lippincott Company, Philadelphia, 1993.]. SUMMARY OF THE INVENTION Accordingly, the present invention relates to dry and fermented plant material immunostimulatory and metastasis inhibitor, which is obtainable by fermenting wheat germ with yeast flour in an aqueous medium, filtering the cell-free fermented liquid and dry it The present invention also relates to an immunostimulatory and metastasis inhibiting pharmaceutical composition, the active agent of which contains the dry, fermented plant material described in the invention. The present invention also relates to the use of the dry and fermented plant material mentioned above in the production of pharmaceutical compositions in a stimulatory and metastasis inhibiting. The present invention also relates to processes for treating mammals for the stimulation of the immune system and inhibition of metastasis with the pharmaceutical composition described in the invention. The invention further relates to a dietary supplement comprising the dry, fermented plant material described in the invention and maltodextrin. According to the invention, the dried, fermented plant material has been identified and examined based on its 2,6-DMBQ content. We would like to note here that it was not possible to fully identify the chemical composition of the extract with the available methods, so that all the data refer to the contents of 2,6-DMBQ as a base. The wheat germ proved suitable for use in the production of the dry, fermented plant material described in the present invention, which is a by-product of flour milling and available in large quantities - ground as flour, either in its state original or defatted. The use of defatted wheat germ has no specific advantages. The fermentation was done with flour yeast (Saccharomyces cerevisiae). This type of yeast is commercially available. The fermentation period was about 25-35 ° C, preferably about 30 ° C. The proportion by weight of the wheat germ and the yeast is 4: 1 - 2: 1, preferably of about 3: 1, the ratio by weight of the dry matter and the water is 1: 6 - 1:12, preferably of approximately 1: 9. The fermentation - on a laboratory scale - can be carried out, for example, by adding the yeast suspension in water to a freshly ground wheat germ in a glass fermenter and stirring or shaking the mixture. The fermented liquid becomes foamy. After fermentation, the mixture is centrifuged for 5-15 minutes at 2000-4500 / min, preferably at approximately 3000 / min. The supernatant is boiled, cooled and dried appropriately, for example, by lyophilization or spray drying.

The product, a reddish-brown powder is the material according to the invention. It is convenient to keep the material cooled until later use, and due to its hygroscopic nature in sealed containers. The 2,6-DMBQ content of the resulting dry material is about 0.4 mg / g. In case of large scale fermentation (for example 4 cubic meters) it is suitable to apply continuous aeration, for example 0.5 1 air / 1 fermented liquid / minute and slow stirring. The fermentation period is approximately 18 hours. In order to inhibit foaming, the usual additives can be applied - it is preferable to use sunflower oil. At the end of the fermentation process the aeration and stirring are interrupted and the fermented liquid is separated from the wheat germ-yeast suspension in the usual manner, for example in a tapped decanter, subsequently in a separator and a filter press. If necessary, filter aid material can be added. It is appropriate to use 5-10 kg of perlite filtration per cubic meter of fermented liquid. The fermented liquid is severely filtered and the quality of the filtration is verified by a microscope. The filtered fermented liquid contains virtually no cells, which means that no more than 1 yeast cell is found per 10 views. The resulting fermented liquid, which contains about 1.5% by weight of dry material is evaporated in a vacuum condenser, preferably at a temperature of about 40-50 ° C and after completion of the vacuum it is boiled at atmospheric pressure for about 15 minutes. This results in a decrease in harmful enzymatic activities. Subsequently the mixture is dried by spray drying, for example in a rotary spray apparatus. If the aforementioned fermentation process is used, the 2.6 DMBQ content of the resulting dry, fermented plant material is 0.12-0.52 mg / g dry material and the content of 2 MBQ is 0.05-0.28 mg / g of dry material - depending on the benzoquinone content of the wheat germ used. As the final product is hygroscopic in order to improve the efficiency of spray drying and the use of the final product, one of the usual additives can be used, for example, maltodextrin, acacia gum, guar gum, xanthene, grain flour of false acacia, etc. during the spray drying. It is preferable to use maltodextrin. The proper process is to determine the dry material content of the mixture, evaporated in the vacuum condenser and boiled and adding so much maltodextrin that the dry material content of the mixture to be dried is about 30% by weight. It is appropriate to dissolve the maltodextrin in hot water and add it cooled to the condensed mixture. After drying the final product - a powder contains approximately 60% by weight of dry, fermented plant material and approximately 40% by weight of maltodextrin. The stability of the material obtained can be verified by monitoring the changes in the concentration of 2.6 DMBQ. The quantitative analysis can be performed by HPLC. The powder produced according to the process described above remains stable for about 3 years at room temperature. The dry, fermented plant material according to the invention can be used as the active agent of immunostimulatory and metastasis inhibiting pharmaceutical compositions. The pharmaceutical composition may also contain ascorbic acid or other cytostatic materials in addition to said active agent. The dried, fermented plant material can be processed for the usual solid or liquid pharmaceutical compositions for peroral or parenteral administration. During the production of the pharmaceutical compositions the hygroscopic character of the dry, fermented plant material must be taken into account. A suitable form is a capsule that protects the active agent from moisture in the air. In the production of the pharmaceutical compositions, auxiliary materials normally applied in pharmaceutical practice can be used. Because these materials and their possible uses are described in detail in the pharmaceutical literature, the selection and preparation of the proper form is a routine task. The single dose of the effective agent can vary between wide limits depending on the condition of the patient, and the selection of the appropriate effective dose should always be the responsibility of the doctor. Generally, adequate results can be obtained if the dose is 0.001-100 g, preferably 0.01-50 g, even more preferably 0.1-40 g per kg body weight, for example in doses of 0.1-10, 1-25 or 10-30. g. The dry, fermented plant material according to the invention can also be mixed with ascorbic acid (Vitamin C). According to our experiments, ascorbic acid can improve the metastasis inhibiting effect of the material according to the invention. The weight ratio of the dry, fermented plant material and the ascorbic acid can be for example 10: 1-1: 1, preferably 6: 1, 2: 1, more preferably 3: 1, 4: 1 or 5: 1. The invention also relates to the immunostimulatory and / or metastasis inhibiting treatment with the dry, fermented plant material according to the invention. The point of treatment is to give the patient an effective dose of dry, fermented plant material. The dry, fermented plant material can also be used as a dietary supplement for mammals. In this case it is preferable to apply the mixture containing maltodextrin and the usual auxiliary materials applied in the food industry, for example aromatic materials, sweeteners, coloring agents, etc. The dietary supplement can be produced for example by granulating the mixture containing 60% by weight of dry matter and 40% by weight of maltodextrin plus aromatic materials and sweeteners in a fluid bed, and packaging the instant granulate in, for example, bags. BRIEF DESCRIPTION OF THE DRAWINGS The adjacent figures are described below: Figure 1. Calibration diagram of HPLC measurement of 2.6 DMBQ. Figure 2. HPLC chromatogram of the chloroform extract of the material. Figure 3. HPLC chromatogram of the ethanol extract of the material. Figure 4. B16 tumor cell binding in the presence of lyophilized at different concentrations in the 60th and 90th minute after placement. (Each value represents the average of 8 parallel measurements ± SD, evaluation by means of econometric spectrophotometric measurement). Figure 5. The effect of different doses of lyophilisate on the proliferation of B16 cells after 24 and 48 hours after the start of treatment. (Each value represents the average of 8 parallel measurements ± SD, evaluation by means of spectrophotometric measurement). Figure 6. The development of human melanoma A2058 in the presence of different doses of lyophilisate 24 hours after the start of treatment. The evaluation of the cultures was carried out based on the protein (SRB) and dehydrogenase activity (MTT) by means of parallel spectrophotometric measurements. (Each value represents the average of 8 parallel measurements ± SD). Figure 7. Proportion of apoptotic cells (FACS analysis) in human A2058 melanoma in in vitro cultures after treatment with different doses of lyophilisate. The following examples serve to illustrate the dried plant material, fermented according to the invention, its production and pharmacological effects. DETAILED DESCRIPTION OF THE PREFERRED MODALITY Example 1 Laboratory-scale fermentation of the wheat germ. A suspension of 33.3 g of yeast (Saccharomyces cerevisiae) and 1000 ml of drinking water has been added to lOOg. of fresh wheat germ (according to the Hungarian standard MSZ-081361-80) ground to flour quality. The mixture was stirred on a stirrer for 18 hours at 30 ° C. During this period, the fermented liquid became foamy and reached approximately three times its original volume.

After fermentation, the mixture was centrifuged for 15 minutes at 3000 / min. After boiling and cooling, the supernatant was dried by lyophilization and the resulting lyophilized material was kept in the freezer (-10 ° C) until further use. The 2.6 DMBQ content of the resulting lyophilisate was 0.4 mg / g dry material (0.04% by weight). Example 2. Fermentation of wheat germ on a large scale 300 kg of wheat germ milled to flour quality (according to the Hungarian standard) and 100 kg of yeast were placed in a fermenter of 5 cubic meters and drinking water was added until the volume became 4000 1. The fermentation period was 18 hours, during which continuous aeration was used (0.5 1 of air / 1 of fermented liquid / minute) and slow agitation (30 rev / min). In order to inhibit foaming, 1 1 μm cubic of sunflower oil was added to the mixture. After fermentation, the aeration and stirring were interrupted and the fermented liquid was separated, first in a threaded decanter, then in a separator and finally in a filter press with textile filter. As auxiliary material, 10 kg of filtration perlite / cubic m was added. The fermented liquid was severely filtered and the acuity was verified by microscope. The filtered fermented liquid contained virtually no cells, which means that for every 10 views a maximum of 1 yeast cell was found. The resulting fermented liquid, containing about 1.5% by weight of dry material, was evaporated in a vacuum condenser at a temperature of 40-50 ° C and, after interrupting the vacuum, boiled at atmospheric pressure for about 15 minutes. After determining this dry matter content of the solution and adding as much maltodextrin dissolved first in hot water and then cooled - the dry matter content of the solution became approximately 30 mass%. After this, the solution was spray-dried in a rotating nozzle spray dryer in which the exit air temperature was 90 ° C. The resulting final product - a powder - contained 60% by weight of the fermented plant material according to the invention and 40% by weight of maltodextrin. The content of 2.6 DMBQ - determined by HPLC according to the method described in the following example - was 0.4 mg / g dry material.

EXAMPLE 3. CHARACTERISTICS OF THE MATERIAL ACCORDING TO THE INVENTION The material according to the invention can be characterized in two ways, either by determining its content of 2, 6 DMBQ or by the so-called fingerprint chromatogram. In both cases, HPLC chromatography is used. The analysis was carried out both on the material produced as described in Example 1 and on that obtained as described in Example 2 and the results were identical in both cases. A. Quantitative and qualitative analysis of benzoquinone derivatives Preparation of the sample Before analysis, it is necessary to increase the concentration of benzoquinones in the lyophilisate. In order to achieve this, the lyophilisate was diluted to the original concentration with distilled water (1% by weight of the dry matter content) (0.5 g of lyophilisate, 50 ml of distilled water).

The solution was extracted with 3 x 25 ml of chloroform in three stages. The eventual moisture remaining in the chloroform phase was removed with water-free Na sulfate. After filtration, the chloroform phase was evaporated, to the remainder chloroform was added to a total volume of 5 ml. This sample was injected during the HPLC analysis. The qualitative and quantitative analysis of the benzoquinone derivatives was carried out by the HPLC method. Description of the HPLC method The applied HPLC equipment consists of a Beckman 114 M pump, a LaborMim UV, respectively a Merck-Hitachi-DAD mod. 4500 and an integrating unit type Waters 740. A column of 10 μ Chromsil C18 (250 x 4 mm) was used for the measurements. UV detection was carried out at a frequency of 290 mm, the flow rate was 2 ml / min. The composition of the applied eluent was as follows: 25 mmol of Na2HP0, 25 mmol of NaH2P04, 25 mmol of Na2EDTA, 20 mmol of NH20H.HC1, methanol to 10% of the volume, pH = 6.05. Three different benzo- and hydroquinones were analyzed by the method described above. There is, however, a small difference between the retention time of the compounds. By decreasing the resistance of the eluent - the organic phase to 10% - the selectivity was improved to a good degree. The analysis of the retention data has shown that the p-benzoquinones show a higher retention than the corresponding hydroquinones and that retention increases with the number of methoxy groups. The concentration of the three standards was 0.1 mg / ml. Table 2 shows the retention times (tR) of the standards and the capacity factor data (k '). The purpose of the measurements was the detection and quantitative definition of 2,6-dimethoxy-p-benzoquinone, so this part of the measurements will be discussed in detail below. It was examined whether the method developed is suitable for the quantitative analysis of 2,6-dimethoxy-p-benzoquinone. The calibration diagram used is shown in Figure 1. With a correlation coefficient of 0.99, the result is a straight line. It was also verified if the measurements can be reproduced, in each sample three parallel measurements were conducted and the dispersion of the results was calculated. Table 1 shows the parallel results of the measurements conducted in the samples during a period of six months and their dispersions. Our results show that the measurement method is suitable for the accurate and reproducible measurement of 2,6-dimethoxy-p-benzoquinone.

Table 1. Parallel values of the quantitative analysis of 2,6-DMBQ and the dispersion of the results Table 2. Values of retention time (tR) and capacity factor (k ') of the materials examined Figure 2 shows the HPLC chromatogram of the chloroform sample prepared as described above. The chromatogram shows only a peak characteristic of 2.6 DMBQ. The DMBQ 2.6 contents of the sample were determined on the basis of the latter. B. Fingerprinting chromatogram Sample preparation 50 ml 96% volume of ethanol was added to 5 g of spray dried material (prepared as described in example 2). The mixture was stirred for 30 minutes at a temperature of 50 ° C and 200 / rain. After this, the mixture was filtered, dried by evaporation and the remaining material was dissolved in 10 ml of methanol. The filtered solution was injected into the column. HPLC Method The HPLC instruments, the column and the conditions described above were also used here, but the composition of the eluent was as follows: 1.25 mmol of Na2HP0, 1.25 mmol of NaH2P04, 1.25 mmol of Na2EDTA, 2.50 mmol of NH2OH.HCl , methanol at 5% volume. Figure 3 shows the resulting HPLC chromatogram. It is shown that under these conditions two peak characteristics appeared, one at approximately 4.7 minutes (5) and one at 5.8 minutes (6). At a retention time of 7.3 minutes resp. 7.7 minutes, two peaks quickly followed each other (7.8), which could not be separated completely. A peak of lower intensity appeared at 9.8 minutes (9), followed by a characteristic intensive peak (11) at approximately 13.1 minutes. At about 15 minutes a smaller peak (12) and another intensive peak (14) at 18 minutes. At 21.8 minutes, the chromatogram showed a lower peak (15) and at about 31 minutes a more uniform peak containing several materials. C. Stability Tests According to the invention, the decomposition of the material was verified through changes in the concentration of 2,6-diraethoxy-p-benzoquinone. Storage experiments were conducted at three different temperatures (ambient temperature of 20 ° C, 40 ° C and 60 ° C). The lyophilisate - approximately 1 g - was stored in test tubes that were sealed in airtight manner. The duration of the experiments was 8 weeks, each week three parallel measurements were conducted on samples taken from the three series maintained at different temperatures. Quantitative analysis of the benzoquinone derivatives was conducted by HPLC * According to the invention, tests have shown that the dry material remained stable even after three years at room temperature, ie the content of 2.6 DMBQ remained practically without changes. At the same time, the material is not stable at 40 ° C - 2.6 DMBQ decomposes in a few weeks, and at 60 ° C the contents of 2,6 DMBQ decrease rapidly within a few days. The decomposition of 2-methoxy-p-benzoquinone was also studied in both series of measurements. This compound is more unstable than 2, 6-dimethoxy-p-benzoquinone and, as a consequence, its presence could not be shown after one week of storage in the samples kept at 40 or 60 ° C. At room temperature, the concentration of this compound also remained unchanged. Example 4. Efficacy tests The efficacy of both lyophilizate prepared as described in the example has been examined. 1 as the spray dried material, prepared as described in example 2. A standardized dry material was used with a DMBQ content of 2.6, 0.4 rag / g dry material and an HPLC curve as shown in the figure 2. Both materials gave the same results - so that the material according to the invention will be called in the subsequent simply lyophilized. Below we will discuss the results of the biological and toxicological tests concerning this lyophilized, with special emphasis on the development of tumors and immune reconstitution and the inhibitory effects of metastasis. I. Tumor growth and metastasis inhibitory effect (in vivo tests) For the tests, the following types of injectable growth tumors were used in mice or rats: a highly metastatic forming variant (3LL-HH) of Lewis lung carcinoma (lung cancer in mice), B16 mouse melanoma and human colon carcinoma xenograft HCR-25. The tumors 3LL-HH (LLT-HH) and B16 were maintained in inbred C57B1 / 6 mice. The HCR-25 xenograft was injected into CBA / CA mice, previously immunosuppressed by ti ectomy and with complete body irradiation. In the case of tumor 3LL-HH and HCR-25, the tumor cells were transplanted into the spleen, in the case of B16 melanoma in the muscles of the left lower extremity. A control group and a treated group of animals injected with HCR-25 tumors were also splenectomized 21 days after tumor transplantation. The treatment with the lyophilisate described in the invention was started 24 hours after the injection of the tumor. Daily doses of 3g / kg of body weight per os were administered. with a stomach probe, in the form of 0.6g / ml aqueous suspension. The tests were completed 14 days after the transplant in the case of the 3LL-HH tumor, 21 days after the transplant in the case of the B16 tumor and 51 days after the transplantation in the case of the HCR-25 tumor 5 when bleeding the animals under narcosis. Tables 3, 4 and 5 summarize the results of the tests. p < 0.01 each test group contained 10 animals Table 3. Effect of the treatment with the lyophilisate according to the invention on the number of liver metastases in the case of mouse lung cancer 3LL-HH injected into the spleen * Splenectoria was carried out 21 days after the tumor had been injected into the spleen. Each test group contained 12 animals.

Table 4. Effect of the treatment with the lyophilisate according to the invention on the number of liver metastases 51 days after injected HCR-25 human colon carcinoma in the spleen of immunosuppressed CBA / CA mice * p < 0.01 Each test group contained 10 animals Table 5. Effect of the treatment with the lyophilisate according to the invention on the weight of the extremity injected with the tumor and on the number of lung metastases in the case of B16 melanoma injected into the muscles of the limb 21 days after having Injected the tumor The above results demonstrated that the lyophilizate described in the invention significantly decreased - by 71% - the number of lung metastases when the 3LL-HH tumor had been injected into the spleen. (Table 3). In the case of HCR-25 human colon carcinoma, the 50-day treatment decreased both the spleen weight with the tumor and the number of liver metastases. The number of metastases compared to the control group was approximately 50% in both the splenectomised and non-splenectised groups. (Table 4). In the case of B16 melanoma injected into the muscle, the weight of the growing tumor in the muscle did not change to treatment effect, but the number of lung metastases decreased significantly, by 85% compared to the control group (Table 5 ). II. In vitro tests As the previous tests have not shown ambiguously that the lyophilisate described in the invention can significantly decrease the metastasis of malignant tumors, the effects of the products in the different stages of metastasis formation were also examined. The formation of metastasis consists of several phases in which, beyond the proliferation and apoptotic activity of the primary tumor cells, the potential adhesion of the tumor cells and the protective mechanisms of the organism against the tumor cells play a role. In the in vi tro tests, the effect of lyophilisate on cell proliferation and apoptosis and adhesion was studied. As a large part of the in vi ve tests was carried out on B16 mouse melanoma cells, in the first stage this tumor was used to examine the lyophilizate. 1. Adhesion test The adhesion of the tumor cells was examined on a 96-well microplate. The lyophilized doses were 300, 3000 and 30000 μg / ml. An RPMI culture medium was used, both in the absence of serum and in the presence of 10% FCS. Adhesion was examined 10, 30, 60, 90 and 120 minutes after incubation under normal circumstances. The evaluation was carried out by means of a colorimetric process based on the SRB test. (Mossmann, T .: J. Immunol. Neth. < 6¿, 55-63 / 1983 /). The test is based on the staining of sulforhodamine of the total protein content of the culture, the absorbance was read at 570 nm with a spectrophotometer. Figure 4 only shows two moments in time, which, however, adequately represent the effect of the lyophilisate. It was shown that if the dose of the lyophilizate was 3000 μg / ml or ten times greater, the adhesion of the tumor cells dramatically decreased, both in the presence and in the absence of serum. If the dose is 300 μg / ml, no such effect can be observed. 2. Proliferation test In this test, the tumor cells were placed for 24 hours before treatment in the 96 perforation raicroplaca. After treatment with appropriate doses of lyophilisate, the proliferation activity of the cells was also tested by SRB analysis, 24, 48 and 72 hours after treatment. The results of the repeated tests have shown that as a treatment effect in the range 900-15,000 μg / ml, the tumor cells reach the surface of the monolayer and, as shown by the trypan-blue ink exclusion method. , they die. (Kaltenbach, J. P. et al.: Exp. Cells Res. 15., 112-117 / 1985 /). (Figure 5). Our tests in human amelanotic melanoma (A2058 tumor cells) - Todaro, G. J. et al. : Proc. Nati Acad. Sci. USA 7_7. 5258-5262 / 1980 /), have shown similar results to those in mouse melanotic melanoma (Figure 6). In the case of this tumor parallel to the SRB analysis, the MTT analysis representing the metabolic activity of the cell was also performed (Colé, S. P. C: Cancer Chemother, Pharmacol, 1_7_, 259-263 / 1986 /). The test is based on the phenomenon in which a metabolically active cell transforms tetrasolium salt into a colored glaze product, mainly through its dehydrogenase activity. The color reaction, which is proportional to the activity, can be read by a spectrometer at 570 nm, the MTT analysis clearly showed that functional activity of the reduced tumor cells, even if the dose was 300 μg / ml (Figure 4) . As in the case of the cells that reach the surface the cause of apoptosis is unknown, the apoptotic activity of the entire cell population was tested by flow cytometric analysis (FACS) (Figure 7). As shown in Figure 7 the tumor cells were apoptotic, depending on the dose, to an unusually higher degree. III. Test of effects on immune reaction The effect of the lyophilisate described in the invention was examined in two different models. In a series of tests, the possibility of transforming in a burst of mononuclear cells obtained from the spleen of animals treated with lyophilisate was studied, while in the other, in the allograft skin transplant model, the total union of the transplanted skin was studied. in the region of the back of mice. 1. T-lymphocyte burst transformation test The treatment with the lyophilisate described in the invention, significantly increases the burst transplantation of the T-lymphocytes that play an important role in the immune reaction.

This is shown by the following experiment. C57B116 mice were treated for six weeks, five times a week. with a stomach probe with the lyophilisate described in the invention in a dose of 3 g / kg (0.6 g / ml aqueous suspension). After completing the treatment, the lymphocytes obtained by perfusion of the spleen of the animals, were transferred to a cell culture, treated with 1 μg / ml of Con A. After 48 hours, the cells carrying out DNS synthesis were marked by 0.4 μCi 3H of thymidine. The degree of labeling was defined with a liquid scintillation counter (Beckman). In Table 6, the treatment with Con A significantly increased, compared to the control, the incorporation of 3H-TdR, ie burst transformation.

Table 6 Incorporation of 3H-thymidine in the spleen lymphocytes of the mice treated with lyophilisate and control 2. Test of the immunostimulatory effect in the allograft skin transplant model The best model for the illustration of the immunodeficient reaction restitution is the test of transplantation of allograft skin in mice, partially immunodeficient by thymectomy (operational removal of the thymus). The mouse breeds C57B110 and B10LP differ only in the H-3 site, so skin transplanted from members of one race in the other is not rejected within 7 days, but only after approximately 3 weeks. If the recipient has been thymectomized, the rejection occurs after an average of 50 days. All materials that accelerate maturity and differentiate from marrow lymphocytes, such as thymus hormones, reduce the time necessary for rejection of transplanted skin. (J. I munpharmacology, 1_, 67-78 / 1985 /). The same effect could be observed in our tests when the treatment is applied with the lyophilisate described in the invention. C57B110 mice were used as a receptor, and B10LP mice as a donor. The recipient mice were thymectomized and skin transplantation continued after 7 weeks. For os - 5 times a week, 30 mg / kg - treatments with lyophilisate were started one week after thymectomy. The treatment was concluded 70 days after the skin transplant. Eventual rejection of the skin was observed daily. Table 7 shows that the rejection time in mice without thymectomizing was 21 days (males) resp. 28.7 days (females). In erectomized mice the rejection time was prolonged to 52.4 days resp. 41.6 days The treatment with the lyophilisate according to the invention significantly decreased, in thymectomized and treated mice, the survival of the skin transplants (grafts). This shows that the immunodeficiency caused by thymectomy was significantly reduced as a result of the treatment, which means that the lyophilisate has an immunostimulating effect. * 0.001 < p < 0.01 vs control ectomized ** 0.01 < p < 0.05 vs thymectomized control Table 7 The effect of the treatment with the lyophilisate according to the invention in the rejection of skin grafts (recipient, mice C57B11Ó, donor, B10LP mice) The tests in vi v and in vi tro with lyophilized according to the invention show that this product has a significant anti-metastatic effect in several animal test models. This effect is probably related to the immunostimulatory effect observed in both the in vi ve and the in vi tro tests, but it is possible that the reduction in the number of metastases is also influenced by the antiproliferative effects that cause apoptosis, the effect of the material in adhesion and the effect that causes the creation of free radicals. IV. Test of radical binding activity How benzoquinones have obtained a well-known effect on the formation of free radicals, the binding activity of the lyophilized radical was also tested. Both the superoxide radical (SSA) and hydroxyl (OH-SA) junction were measured with the electron rotation resonance method. The lyophilisate has a significant SSA, the binding activity of the free radical of 1 mg corresponds to the activity of 5.64 μg of superoxide dismutase (SOD). The lyophilisate has no OH-SA activity, but interrupts the hydrogen-peroxide / Fe hydroxyl radical formation system, so it can be assumed that it has the so-called non-chelating activity. V. Toxicological tests (sub-acute) On day 77 toxicological tests were carried out according to the recommendations of the Registry of Industrial Toxicology Animal-data (RITA).

(Exp. Toxic, Pathol, 1, 247-266 / 1995 /) in F344 rats and C57B110 mice. The animals were treated daily with a dose of 3 g / kg (0.6 g / ml aqueous suspension). During the treatment, weight changes of the animals were observed, eventual physical changes, spontaneous death of the animals. At the conclusion of the test the weight of the heart, lungs, thymus, spleen, liver, kidneys and testes were measured and the 34 organs prescribed by the RITA were examined pathologically. No spontaneous death was observed, the weight of the animals changed as that of the control group. At the conclusion of the test the weight of the different organs showed no changes compared to the control group. During the pathological processing of the treated animals, no changes were observed, which could have been caused by the lyophilisate. The above results show that the fermented plant material according to the invention is non-toxic, and due to its immunostimulatory effect, it is indicated in all the states in which the immune system is damaged. Due to its biological characteristics described above it can have its complementary uses in the medical treatment of malignant tumors, mainly in the inhibition of metastasis.

Claims (12)

1. CLAIMS 1. A dry, fermented, immunostimulatory and metastasis inhibiting material derived from the fermented liquid obtained by fermenting wheat germ with Saccharomyces cerevisiae in an aqueous medium. The material according to claim 1, characterized in that it has an HPLC chromatogram corresponding substantially to that shown in Figure 3. 3. The material according to claim 1, characterized by the content of 2,6-dimethoxy-p-benzoquinone. , of which 0.12-0.52 mg / g is dry material and the content of 2-methoxy-p-benzoquinone, of which 0.05-0.28 mg / g is dry material, determined by HPLC. 4. The material according to claim 3, characterized by the content of 2,6-dimethoxy-p-benzoquinone, of which 0.4 mg / g is dry material. 5. A process for producing a dry, fermented, immunostimulatory and metastasis inhibiting material, characterized in that the ground wheat germ is fermented in an aqueous medium in the presence of Saccharomyces cerevisiae and the fermented liquid is separated, severely filtered, evaporated, boiled and dry as such or in the presence of auxiliary drying materials. 6. The process according to claim 5, characterized in that the fermentation is carried out at a temperature of about 30 ° C, for about 18 hours under agitation and continuous aeration. The process according to claim 6, characterized in that the drying is carried out in the presence of maltodextrin. 8. An immunostimulatory and metastasis inhibiting pharmaceutical product containing as active agent a dry material derived from the fermented liquid obtained by the fermentation of wheat germ in an aqueous medium in the presence of Saccharomyces cervisiae. 9. A dietary supplement for mammals, characterized in that it contains a dry material derived from the fermented liquid obtained by the fermentation of wheat germ in an aqueous medium in the presence of Saccharomyces cervisiae. The dietary supplement according to claim 9, characterized in that it contains the dry material obtained by the process of claim 5 which consists of 60% by weight of fermented material and 40% by weight of maltodextrin. 11. The use of a dry material derived from the fermented liquid obtained by the fermentation of wheat germ in an aqueous medium in the presence of Saccharomyces cervisiae in the production of an immunostimulatory and metastasis inhibiting pharmaceutical composition. 12. The use of a dry material derived from the fermented liquid obtained by the fermentation of wheat germ in an aqueous medium in the presence of Saccharomyces cervisiae in the production of a dietary supplement for mammals.