HK1033097B - Fermented vegetal material, process for producing it, the use thereof, the pharmaceutical composition and dietary supplement containing the same - Google Patents

Description

Plant fermented substance, its preparation method and application, and medicinal composition and dietary additive containing the same

The present invention relates to fermented and dried vegetal material having immunostimulating and metastasis inhibiting properties, to pharmaceutical compositions containing it, to a process for preparing it and to the use of said dried vegetal material as dietary supplement in the production of immunostimulating and, in particular, metastasis inhibiting pharmaceutical compositions.

One of the main goals of treating tumors is to inhibit metastasis, since a primary tumor due to malignant growth of cells may spread to neighboring cells and organs by metastasis and lead to subsequent secondary tumors, which cannot be removed by surgical means and may be resistant to chemotherapy.

Researchers have increasingly focused on developing immunomodulatory substances and have conducted intensive research on substances of natural origin-mainly of plant origin. It is well known that compounds having a quinone structure play an important biological role. Several quinone derivatives are found in plants, such as ubiquinone, plastoquinone, menaquinone, which play a role in photosynthesis, but also in the cellular respiratory system and in blood coagulation in vertebrates, and thus in humans. Several benzoquinone derivatives are also used for pharmaceutical use. Doxorubicin, daunomycin and mitomycin c. are quinone derivatives with a cell growth inhibiting function, while other benzoquinones and hydroquinones have an antimicrobial effect and are active ingredients of antibiotics suitable for treating bacterial infections, such as Tetran-B, Metacyclin and doxycycline.

The literature reports that 2, 6-dimethoxy-p-benzoquinone (2, 6-DMBQ) and 2-methoxy-p-benzoquinone (2-MBQ) have fungicidal and bacteriostatic effects, and they are also cytotoxic to malignant 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.agric.food chem.39,. 266 (1991). It has been shown in the literature that a mixture of 2, 6-dimethoxy-p-benzoquinone and ascorbic acid inhibits the growth of Ehrlich ascites tumor cells in mice. [ Proc.Natl.Acad.Sci.USA 81, 2088-2091(1984) and 82. 1439-. 2, 6-dimethoxy-p-benzoquinone and 2-methoxy-p-benzoquinone were found and isolated in several plants [ Magy. Kem. Folyorat 102/7.,. 320-325(1996) ]. In wheat (Tricitumvulgaris), more precisely in malt, these two compounds are found in large amounts in the form of glycosides. These compounds were isolated from malt fermented with yeast and identified to determine the extent of spoilage of bread quality upon addition to the malt. [ Helv. HimActa 33., 433 (1950); j.chem Soc.London 1952, 4821-.

The amount of 2-MBQ in malt is approximately 0.05% by weight and 2, 6-DMBQ is 0.01% by weight (as glycoside) according to the literature. Quinones, especially methoxy-substituted-p-benzoquinones, are reactive and their hydroquinone-glycosides are more stable and inert, which explains the presence of quinones as glycosides.

In our studies on malt fermentation we concluded that: the dried extract obtained from the fermentation liquor produced when malt is fermented with Saccharomyces cerevisiae (Saccharomyces cerevisiae) has surprising immunostimulating and metastasis inhibiting effects, and it can be successfully used as an active substance in immunostimulating and metastasis inhibiting pharmaceutical compositions.

This finding is surprising, since although there are reports in the literature that mixtures of 2, 6-DMBQ and ascorbic acid inhibit ehrlichescopes tumor cells-which are well known for their suitability for quantitative analysis of tumor growth and for studying their biochemical function-the above-mentioned extracts described in the present invention, which contain 2, 6-DMBQ and 2-MBQ in addition to unidentified components, proved to have no effect on these primary tumor cells, even when used in combination with ascorbic acid. This extract acts on all primary tumour cells and has been shown to be very effective only in treating metastases. The treatment of primary tumors and metastases developing from primary tumors varies in nature, and it is not self-evident to researchers that plant extracts containing components known to be effective in primary tumor treatment, but whose chemical composition is not yet fully understood, have immunostimulatory and metastasis-inhibiting effects [ Cancer Principles and Practice of Oncology, Vol 1.4, part.j.b.lippicott company.

The invention therefore relates to a fermented and dried plant material having immunostimulating and metastasis-inhibiting properties, which is obtainable by fermenting malt with Saccharomyces cerevisiae in an aqueous medium, filtering the fermentation broth to remove cells and drying the liquid.

The invention also relates to immunostimulatory and metastasis inhibiting pharmaceutical compositions of active substances including the fermented dried botanical described in the invention.

The invention also relates to the use of the fermented dried plant material in the production of a pharmaceutical composition for immunostimulation and metastasis inhibition.

The invention also relates to methods of treating mammals by stimulating the immune system and inhibiting metastasis using the pharmaceutical compositions described herein.

The invention further relates to a food additive comprising the fermented dried plant matter maltodextrin described in the present invention.

The fermented dried plant matter according to the invention has been identified and tested on the basis of its 2, 6-DMBQ content. We would like to emphasize here that it is not possible to identify the chemical composition of the extract completely by the available methods, so all data are based on 2, 6-DMBQ.

Malt is a by-product of flour processing and is available in large quantities, and milled defatted or virgin malt has proven useful in the production of the fermented dry plant matter of the present invention. The use of defatted malt is not particularly advantageous. Fermentation was carried out with Saccharomyces cerevisiae. Such yeasts are commercially available. The fermentation period is about 10 to 24 hours, preferably 15 to 20 hours or about 18 hours. The fermentation temperature is about 25-35 deg.C, preferably about 30 deg.C. The weight ratio of malt and yeast is 4: 1 to 2: 1, preferably about 3: 1, and the ratio of dry matter to water is 1: 6 to 1: 12, preferably about 1: 9.

Fermentation-laboratory scale-can be achieved by adding an aqueous suspension of freshly ground malt and yeast to a glass fermenter, then stirring and shaking the mixture. The fermented liquid is easily foamed.

The mixture is centrifuged at 2000-4500 rpm, preferably about 3000 rpm, for 5-15 minutes after fermentation. The supernatant is boiled and cooled and then dried in a suitable manner, for example by lyophilization or spray drying.

This product, a reddish-brown powder, is a material according to the invention. Due to its hygroscopic nature, it is necessary to keep it in a closed container at low temperatures until further use. The resulting dried material had a2, 6-DMBQ content of 0.4 mg/g. Continuous aeration, e.g., 0.5L of air/L of broth/min, and slow agitation should be used for large scale fermentation (e.g., 4 cubic meters). The fermentation time was about 18 hours. Usual additives can be used in order to prevent foam formation-the use of sunflower oil is preferred. At the end of the fermentation process aeration and agitation are interrupted and the fermentation broth is separated from the malt-yeast suspension by conventional means, for example first by means of a screw decanter and then by means of a separator and pressure filter (filter press). If necessary, a filter aid can be added. It is suitable to use 5-10kg of filtered perlite per cubic meter of fermentation broth. The fermentation broth was filtered clear and the effect of the filtration was examined microscopically. The filtered fermentation broth is essentially free of cells, meaning less than 1 yeast cell per 10 fields of view. The fermentation broth, which gives about 1.5% by weight of dry matter, is evaporated in a vacuum concentrator, preferably at 40-50 ℃, and boiled at atmospheric pressure for about 15 minutes after the vacuum concentration is finished. This reduces the activity of harmful enzymes. The mixture is then dried by spray drying, for example in a rotary spray apparatus. If the fermentation process described above is used, the resulting fermented dry plant matter has a2, 6-DMBQ content of 0.12-0.52mg/g dry matter and a 2-MBQ content of 0.05-0.28mg/g dry matter, depending on the benzoquinone content of the malt used.

Since the end product is hygroscopic, a commonly used additive such as maltodextrin, gum arabic, guar gum, xanthan gum, locust bean flour (locustbean flours), and the like may be added at the time of spraying in order to enhance the efficacy of spray drying and utilization of the end product. Preferably maltodextrin is used. A suitable method is to measure the dry matter content of the mixture evaporated in the vacuum concentrator, boil it and add a large amount of maltodextrin to bring the dry matter content of the mixture to approximately 30% by weight. A suitable method is to dissolve maltodextrin in hot water and add the concentrated mixture after cooling. The dried end product-a powder-contains 60% by weight of fermented dried plant matter and 40% by weight of maltodextrin.

The stability of the resulting material was verified by detecting changes in the concentration of 2, 6-DMBQ. Quantitative analysis can be performed by HPLC. The powder obtained by the above described process is stable at room temperature for 3 years. The fermented dried vegetable matter according to the invention can be used as active substance in pharmaceutical compositions for immunostimulation and metastasis inhibition. The pharmaceutical compositions may contain ascorbic acid or other cytostatic substances in addition to the active substances mentioned above.

The fermented dried plant matter can be processed into common solid or liquid pharmaceutical compositions for oral or parenteral administration. The hygroscopic properties of the fermented dried vegetable matter should be taken into account when producing the pharmaceutical composition. A suitable form is a capsule that isolates the active substance from moisture in the air.

Auxiliary substances commonly used in pharmaceutical practice can be used in the manufacture of pharmaceutical compositions. Since these substances and their possible uses are well described in the pharmaceutical literature, the selection and preparation of suitable forms is a matter of routine practice. A single dose of the active substance can be varied over a wide range, depending on the condition of the patient. The selection of the appropriate dosage is always the responsibility of the physician. In general, suitable effects are achieved if the dose is in the range from 0.001 to 100g, preferably from 0.01 to 50g, more preferably from 0.1 to 40g, per kg of body weight, for example from 0.1 to 10g, from 1 to 25 or from 10 to 30 g.

The fermented dried vegetable matter according to the invention may also be mixed with ascorbic acid (vitamin C). According to our experiments ascorbic acid enhanced the metastasis inhibiting effect of the substances of the invention. The weight ratio of fermented dry plant matter to ascorbic acid may be, for example, from 10: 1 to 1: 1, preferably from 6: 1 to 2: 1, more preferably 3: 1, 4: 1 or 5: 1.

The invention also relates to immunostimulatory and/or metastasis-inhibiting treatments with fermented dried vegetal material according to the invention. The key point of treatment is to administer an effective amount of fermented dry plant matter to a patient.

The fermented dried plant matter can also be used as a dietary supplement for mammals. In this case, it is preferred to use a mixture containing maltodextrin together with auxiliary substances commonly used in the food industry, such as aromas, sweeteners, pigments and the like, and the dietary supplement can be produced, for example, by granulating a mixture containing 60% by weight of dry matter and 40% by weight of maltodextrin plus aroma substances and sweeteners on a hydraulic bed (fluid bed) and then filling the granules, for example, into bags.

The drawings are described below:

FIG. 1 Standard chart for HPLC measurement of 2, 6DMBQ

FIG. 2 HPLC chromatogram of chloroform extract of the above-mentioned substances

FIG. 3 HPLC chromatogram of ethanol extract of the above substance

FIG. 4 binding of B16 tumor cells in the presence of different concentrations of lyophilisate at 60 th and 90 th minute after implantation (each value represents the mean. + -. standard deviation of 8 replicates measured by spectrophotometry)

Figure 5 effect of different doses of lyophilisate on B16 cell proliferation after 24 and 48 hours of initial treatment (each value represents mean ± standard deviation of 8 replicates measured by spectrophotometry).

Figure 6 progression of human a2058 melanoma after 24 hours of initial treatment in the presence of different doses of lyophilizate. The evaluation of the cultures was carried out spectrophotometrically in parallel on the basis of the protein (SRB) and the dehydrogenase activity (MTT) (each value represents the mean. + -. standard deviation of 8 parallel experiments).

Figure 7 quantification of apoptotic cells following treatment of a2058 human melanoma in vitro cultures with different doses of lyophilisate.

The following examples are provided to illustrate the production and medicinal efficacy of the fermented dried plant matter according to the present invention.

Example 1.

Laboratory-scale malt fermentation

To 100g of ground fresh malt (according to Hungarian standard MSZ-081361-80) was added 1000ml of drinking water and a suspension of 33.3g of yeast (Saccharomyces cerevisiae). The mixture was shaken on a shaker at 30 ℃ for 18 hours. During this time the broth produced a lot of foam and reached three times its initial volume. After fermentation the mixture was centrifuged at 3000/min for 15 min. After boiling and cooling the supernatant was dried by lyophilization. The resulting lyophilized material was stored in a freezer (-10 ℃) until further use. The lyophilizate obtained had a2, 6DMBQ content of 0.4mg/g dry matter (0.04% by weight).

Example 2

Large-scale malt fermentation

300kg of ground malt (according to Hungarian standard) and 100kg of yeast were placed in a 5 cubic meter fermenter and drinking water was added until the volume reached 40001. The fermentation time was 18 hours, during which continuous aeration (0.51 air/1 broth/min) and slow stirring (30 rpm) were carried out. To suppress foam formation 11 sunflower oil/cubic meter was added to the mixture. After termination of the fermentation aeration and agitation, the fermentation broth was first separated in a screw decanter, then in a separator and finally in a pressure filter with woven filter membranes. 10kg of auxiliary substance perlite for filtration per cubic meter were added. The fermentation broth was filtered to clear and the clarity examined microscopically. The filtered fermentation broth is essentially free of cells, meaning less than 1 yeast cell per 10 fields of view. The resulting fermentation broth, which is about 1.5% by weight of the dry matter, is evaporated in a vacuum concentration unit at 40-50 ℃. After completion of the vacuum concentration boiling is carried out for about 15 minutes at atmospheric pressure. The dry matter content of the solution was then determined and maltodextrin was added-dissolved first with hot water and then cooled-to a dry matter content of 30% by weight of the solution. The solution was then spray dried in a shear nozzle rotary spray dryer (shear nozzle rotary spray dryer) with an outflow air temperature of 90 ℃. The end product obtained, a powder, contains 60% by weight of fermented vegetable matter 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 examples below-was 0.4mg/g dry matter.

Example 3

Characterization of materials according to the invention

The substances according to the invention can be characterized in two ways, namely by determining the 2, 6DMBQ content or by so-called fingerprint chromatography (fingerprint chromatography). In both cases HPLC was used.

The analysis was carried out on the substances obtained by the method described in example 1 and the method described in example 2, the results being identical in both cases.

A. Quantitative and qualitative analysis of benzoquinone derivatives

Sample preparation

It was necessary to increase the benzoquinone concentration in the frozen material prior to analysis. For this purpose the lyophilisate is diluted with distilled water to the initial concentration (dry matter content 1% by weight) - (0.5g lyophilisate. 50ml distilled water). The solution was extracted 3 times with 3X 25ml of chloroform. The residual water in the chloroform phase was removed with anhydrous sodium sulfate. After filtration the chloroform phase was evaporated and chloroform was added to the residue to make a total volume of 5 ml. The sample was injected for HPLC analysis.

Qualitative and quantitative analysis of the benzoquinone derivatives was performed by HPLC method.

Description of the HPLC method

The HPLC equipment used consisted of a Beckman model 114M pump, a LaborMim UV lamp along with a Merk-Hitachi-DAD model.4500 diode array detector and a Water 740 type integrator unit. A10 μ column chromatography of Chromsil C18 (250X 4mm) was used for the measurement. UV detection at290nmThe flow rate was 2 ml/min. The eluent used had the following composition: na (Na)₂HPO₄25mmol NaH₂PO₄ 25mmol，Na₂EDTA 25mmol，NH₂OH HCl 20mmol, 10 vol% methanol, pH 6.05. Three different benzoquinones and hydroquinones were analyzed using the methods described above. There is only a small difference between the residence times of these compounds. The selectivity was greatly enhanced by reducing the intensity of the elution wash (organic phase down to 10%). Analysis of the retention data showed that p-benzoquinone showed a longer retention than the corresponding hydroquinone and that the retention time increased with increasing methoxy. All three standards were at a concentration of 0.1mg/ml table 2 shows the retention time (tR) and capacity factor (K') data for the standards. The purpose of the measurement is to detect and quantify 2, 6-dimethoxy-p-benzoquinone, so this section will be discussed in detail below. It was examined whether the method described in detail above is suitable for the quantitative analysis of 2, 6-dimethoxy-p-benzoquinone. The standard diagram used is shown in fig. 1. The result is a straight line with a correlation constant of 0.99. It was also tested whether the method could be repeated, each sample being runThree replicates were measured and the dispersion of the results calculated. Table 1 shows the parallels measured on the samples over six months and the dispersion between them. Our results show that the measurement method is suitable for the accurate and reproducible measurement of 2, 6-dimethoxy-p-benzoquinone.

Date	2, 6-DMBQ content (mg/50g sample)			(σ％)
					04.27	3.45	3.54	3.66	0.08-2
04.28	3.27	3.68	3.31	0.18-5
					05.10	1.56	1.49	1.48	0.03-1.9
05.10	3.65	3.42		0.12-3
					06.08	3.00	2.98	2.86	0.06-2
06.09	2.92	3.06	2.87	0.08-2.3
					06.10	3.34	3.23	3.36	0.05-1.5

TABLE 1

Parallel numerical value and dispersion result of 2, 6-DMBQ qualitative analysis

Standard of merit	tR	k’
			2，6-DMBQ	13.4	5.0
2，6-DMBQ	5.1	1.2
			2，-MBQ	8.5	2.7

TABLE 2

Retention time (t) to receive test substance_R) And capacity factor (k')

Figure 2 shows an HPLC chromatogram of a chloroform sample prepared as described above. The chromatogram showed a single peak with the 2, 6-DMBQ characteristic. The determination of the 2, 6-DMBQ content is based on the latter.

B. Fingerprint chromatography

Sample preparation

50ml of 96% by volume ethanol were added to 5g of the spray-dried material (prepared as described in example 2). The mixture was shaken for 30 minutes at 50 ℃ 200/min, after which the mixture was filtered, evaporated to dryness and the remaining material was dissolved in 10ml of methanol. The filtered solution was injected to the column.

HPLC method

The HPLC equipment, chromatography column, and conditions used were as described above. The eluent, however, had the following composition: na (Na)₂HPO₄ 1.25mmol.NaH₂PO₄ 1.25mmol，Na₂EDTA 1.25mmol，NH₂OHHCl 2.50mmol, 5 vol% methanol.

FIG. 3 shows the results of HPLC chromatogram. The results show that under such conditions two characteristic peaks are separated, one at 4.7 minutes (5) and the other at 5.8 minutes (6). At retention times 7.3 and 7.7 minutes two peaks (7, 8) appeared next to each other, which could not be completely separated. A less intense peak appears at 9.8 minutes (9), followed by a characteristic intense peak (11) at 13.1 minutes. One smaller peak (12) appeared at 15 minutes and another strong peak (14) appeared at 18 minutes. The spectrum showed a smaller peak (15) at 21.8 minutes and a flatter peak at 31 minutes, containing several substances.

C. Stability test

The decomposition of the substance according to the invention was carried out by detecting the change in the concentration of 2, 6-dimethoxy-p-benzoquinone we carried out storage experiments at 3 different temperatures (room temperature 20 ℃, 40 ℃ and 60 ℃). Lyophilizate-about 1 g-was stored in airtight tubes. The duration of the experiment was 8 weeks. Three replicates of three different storage temperature series of samples were run each week. The quantitative analysis of the benzoquinone derivatives was performed by HPLC.

Experiments have shown that the dried material according to the invention remains stable, i.e. 2, 6DMBQ remains unchanged, even after 3 years of storage at room temperature. At the same time, the substance is unstable at 40 ℃ and 2, 6DMBQ decomposes within a few weeks, and the 2, 6DMBQ content rapidly decreases within a few days at 60 ℃.

The decomposition of 2-methoxy-p-benzoquinone was also investigated in two series of experiments. This compound is less stable than 2, 6-dimethoxy-p-benzoquinone and its presence cannot be detected after one week of storage of the sample at 40 ℃ or 60 ℃. While the concentration of this compound remains unchanged at room temperature.

Example 4

Efficacy test

The lyophilisate prepared as described in example 1 and the spray-dried material prepared as described in example 2 were tested for efficacy. As a standard, a dried substance having a2, 6DMBQ content of 0.4mg/g and an HPLC curve as shown in FIG. 2 was used. Both substances give the same result-so the substance according to the invention will in turn be referred to simply as a lyophilisate. The results of biological and toxicological experiments on this lyophilisate will be discussed below, with emphasis on immune reconstitution and tumor growth and metastasis suppression effects.

I. Inhibitory Effect on tumor growth and metastasis (in vivo experiment)

The following injectable tumor types grown in mice or rats were used for testing: high metastasis of Lewis lung tumor (mouse lung cancer) resulted in a variant (3LL-HH), B16 mouse melanoma, and HCR-25 human colon tumor xenografts.

3LL-HH (LLT-HH) and B16 tumors were maintained in C57B1/6 inbred mice. HCR-25 xenografts were injected into CBA/CA mice previously immunosuppressed by thymectomy and systemic irradiation. Tumor cells of 3LL-HH and HCR-25 tumors were transplanted into the spleen and tumor cells of B16 melanoma were transplanted into the muscle of the left lower limb. The treatment group injected with HCR-25 tumor and the control group were subjected to splenectomy 21 days after tumor transplantation.

The treatment with the lyophilisate according to the invention starts 24 hours after the injection of the tumour. The daily dose was 3g/kg body weight and was administered orally in the form of a 0.6g/ml aqueous suspension by means of a gastric feeding tube (stomachprobe).

Testing in experiments with 3LL-HH tumors ended 14 days after transplantation. The B16 tumor was 21 days post-transplantation and the HCR-25 tumor was 51 days post-transplantation, and was terminated by bleeding the anesthetized animals to death.

Tables 3, 4 and 5 summarize the results of the tests.

Treatment of	Number of cells injected	Number of liver metastases
			Control	3×104	104.0±28.2
Freeze-dried substance	3×104	19.8±16.4*

P＜0.01

Each group contained 10 animals

Table 3.

Spleen injection of 3LL-HH mice Lung cancer

Therapeutic Effect of lyophilisates according to the invention on liver metastasis count

Treatment of	Spleen weight (g) of growing tumor	Number of liver metastases
			Control, no splenectomy	1.02±0.59	42.0±25.8
Lyophilized product, without splenectomy	0.62±0.47	19.5±19.0
			Control, splenectomy	0.10±0.02	19.1±13.5
Lyophilisate, splenectomy	0.08±0.02	10.6±11.6

Splenectomy was performed 21 days after injection of tumors into the spleen

Each group contained 12 animals

TABLE 4

Therapeutic Effect of lyophilisate according to the invention on liver turnover number 51 days after HCR-25 human Colon cancer injection into spleen of immunosuppressive CBA/CA mice

Treatment of	Weight of limbs from which tumors grow (average)	Number of lung metastases (average)
			Control	7.6±0.43	42.4±10.2
Freeze-dried substance	7.2±0.38	6.2±3.7*

*P＜0.01

Each group contained 10 animals

Table 5.

B16 therapeutic Effect of lyophilisate according to the invention on Limb weight and Lung metastasis number of injected tumors 21 days after Limb muscle injection of melanoma

The above results show that treatment with the lyophilisate according to the invention significantly-71% reduced the number of lung metastases when 3LL-HH tumors were injected into the spleen. (Table 3)

50 days of treatment in HCR-25 human colon cancer experiments reduced both the weight of the spleen growing the tumor and the number of liver metastases. The number of metastases was reduced by 50% in both the splenectomized and non-splenectomized groups compared to the control. (Table 4)

In the intramuscular melanoma experiment, the weight of the tumor growing in muscle was not changed by the treatment, but the reduction in the number of lung metastases was very significant, and was reduced by 85% compared to the control group (Table 5)

II in vitro testing

The above tests clearly show that the lyophilisate according to the invention significantly reduces the metastasis of malignant tumours and that the efficacy of the product in developing different stages of metastasis is also examined. The formation of metastases consists of several phases in which, in addition to the proliferative and apoptotic activity of the primary tumor cells, the adhesion capacity of the tumor cells and the protective mechanisms of the tissues against the tumor cells play a role. The effect of lyophilisate on cell proliferation, apoptosis and adhesion was studied in vitro tests.

Since a significant portion of in vivo testing was performed on B16 mouse melanoma cells, lyophilisates were first tested with such cells.

1. Adhesion test

Tumor cell adhesion was performed in 96-well plates. The dose of lyophilizate was 300, 3000, and 30000. mu.g/ml. RPMI medium without serum and with 10% FCS was used. Adhesion was tested after 10, 30, 60, 90 and 120 minutes incubation in the conventional environment. The test was carried out colorimetrically on the basis of the detection of SRB (Mossmann. T.; J.Immunol. Neth.65, 55-63/1983). The assay was based on the visualization of total protein in culture by thiocyanate B, with absorbance read at 570nm on a spectrophotometer. Figure 4 shows only two time points, but is sufficient to fully represent the potency of the lyophilizate. If the lyophilisate is dosed at 3000. mu.g/ml or 10 times higher, the adhesion of the tumour cells is significantly reduced, both in the presence and in the absence of serum. Such an effect is not observed if the dose is 300. mu.g/ml.

2. Proliferation assay

In this assay tumor cells were placed in 96-well plates 24 hours prior to treatment. The proliferative activity of the cells was tested by SRB assay 24, 48 and 72 hours after treatment with the appropriate dose of lyophilisate. The results of the repeated tests showed that treatment in the range of 900-15000. mu.g/ml resulted in tumor cells reaching the surface of the monolayer and that-Trypan blue exclusion experiments showed-death. (Kaltenbach, J.P.et al: Exp Cell Res.15, 112-117/1985/) (FIG. 5).

Our testing of human melanoma (A2058 tumor cells-Todaro, G.J.et al: Proc.Natl.Acad.Sci USA 775258-5262/1980 /) showed similar results to mouse melanoma (FIG. 6). MTT assays of the melanin activity of the responder cells were also performed in parallel with the SRB assays in this tumor assay. (Cole, S.P.C.: Cancer Chemother. Pharmacol.17, 259-263/1986 /). The test is based on the following phenomena: the metabolically active cells convert the tetrasolium salt into a colored formazan (formazan) product primarily by their dehydrogenase activity. The color response corresponding to the above activity was read by a spectrophotometer at 570nm and the MTT assay clearly showed a decrease in the functional activity of the tumor cells even at doses of 300. mu.g/ml (FIG. 4). Since the cause of apoptosis was unclear when the cells reached the surface, apoptotic activity of the entire cell population was tested by flow cytometry (FACS) analysis (fig. 7). Figure 7 shows that apoptosis of tumor cells-dependent on dose-reached an unusual degree.

Testing the Effect on immune response

The effect of the lyophilisate according to the invention was examined in two different models. The possibility of a mononuclear cell transformation burst (blast transformation) from the spleen of animals treated with lyophilisate was investigated in a series of tests; the total binding of transplanted skin in the dorsal region of mice in the allogeneic skin graft model was studied in another series of tests.

1. Testing for outbreaks of T-lymphocyte transformation

Treatment with the lyophilisate according to the invention significantly increases the transformation burst of T-lymphocytes which play an important role in the immune response. This is shown by the following experiment.

C₅₇Bl₁₆Mice were treated with the lyophilisate described in the present invention for six weeks, 5 times a week, orally administered by a gastric feeding tube, at a dose of 3g/kg (0.6g/ml aqueous suspension). After completion of the treatment, lymphocytes obtained from the spleen by perfusion were subjected to cell culture and treated with 1. mu.g/ml ConA. After 48 hours, the cells undergoing DNS synthesis were labeled with 0.4. mu. Ci of 3H timidin. The extent of marking was measured with a liquid scintillation counter (Beckman). As shown in Table 6, treatment with ConA was significantly improved-compared to the control-³Incorporation of H-TdR, i.e., conversion burst.

Treatment of	1μg/ml Con A
			Average (cpm)	SEM
	Control	3760.6			583.3
Freeze-dried substance			8041.8	957.1

Table 6.

Freeze-dried treatment and control mouse spleen lymphocyte pairs³Incorporation of H-timidins

2. Detection of immunostimulatory effects in allogeneic skin graft models

The best model to demonstrate the recovery of a defective immune response is an allogeneic skin transplant experiment in mice that are partially immunodeficient by thymectomy (surgical removal of the thymus). C₅₇Bl₁₀And B₁₀LP blood systemThe mice of (2) differ only in the H-3 locus, so that the skin graft from one mouse of blood origin to another does not cause rejection within 7 days, but only after 3 weeks. If the recipient has undergone a thymectomy, rejection occurs on average after 50 days. Any substance that promotes the maturation and differentiation of lymphocytes and bone marrow, such as thymic hormone, reduces the time required to reject transplanted skin (j7,67-78/1985 /). The same effect was observed in our experiments when treated with lyophilisate according to the invention.

C₅₇Bl₁₀Mouse as receptor B₁₀LP mice served as donors. Recipient mice were subjected to a thymectomy and skin was transplanted 7 weeks later. The thymus was treated with a 30mg/kg body weight dose of the lyophilisate 5 times per week after 1 week of thymectomy and administered orally. Treatment was terminated 70 days after skin transplantation. Possible rejections were observed daily. Table 7 shows that the rejection time was 21 days (male) and 28.7 days (female) in mice without thymectomy. The rejection time was extended to 52.4 days and 41.6 days in thymectomized mice, respectively. Treatment with the lyophilisate according to the invention greatly shortens the survival time of the skin grafts in thymectomized and treated mice. This indicates that the result of the treatment is a significant reduction in the immunodeficiency due to thymus excision, which means that the lyophilisate has an immunostimulating effect.

Treatment of	Time of repulsion
						Male sex		Female
	Average (time)	SEM	Average (time)	SEM
					Control (without thymus excision)	21.0	3.1	28.7	4.5
Control (thymus excision)	52.4	5.0	41.6	5.5
					Freeze-dried substance (30mg/kg)	28.8*	8.6	32.6**	4.5

0.001 < p < 0.01 vs. thymectomized mice

0.01 < p < 0.05 relative thymectomized mice

TABLE 7

Effect of treatment of lyophilisates according to the invention on rejection of skin grafts (recipient mice)

C₅₇Bl₁₀Donor mouse B₁₀LP)

In vivo and in vitro tests carried out in several animal experimental models using lyophilisates according to the invention show that this product has a significant anti-metastatic effect. This effect may be related to the immunostimulatory effect observed in vivo and in vitro experiments, but the reduction in the number of metastases may also be influenced by antiproliferative effects, apoptosis-inducing effects, the effect of the substance on adhesion and the effect leading to the generation of free radicals.

Radical binding Activity

Since benzoquinone is well known to have an effect on the formation of free radicals, we also tested the binding activity of the lyophilisate described in the present invention on free radicals. The binding of peroxide (SSA) and hydroxyl radical (OH-SA) was measured simultaneously by electron spin resonance. The above lyophilisate has significant SSA, 1mg net free radical binding activity equivalent to that of 5.64 μ g superoxide dismutase (SOD). The lyophilisate has no OH-SA activity, but it is capable of destroying the hydrogen peroxide/Fe hydroxyl radical formation system, so it can be suspected of having so-called non-chelator activity.

Toxicology testing (subacute)

According to the Registry of Industrial diagnostics Animal-data (RITA) (exp. Toxic. Pathol).47247-266/1995/) was tested for 77 days toxicology in F344 rats and C57Bl10 mice. The animals were treated daily with a dose of 3g/kg (0.6mg/ml suspension). Changes in animal body weight, eventual pathological physical changes and spontaneous death were observed during the treatment. At the end of the experiment, the weights of heart, lung, thymus, spleen, liver, kidney, and testis were measured and pathological examination was performed on 34 organs listed by RITA. No spontaneous death was observed. The change in body weight of the animals was similar to that of the control group. The weight of the different organs at the end of the experiment was unchanged compared to the control group. No changes that could be caused by lyophilisate were observed during the pathological treatment of the treated animals.

The above results indicate that the fermented vegetable matter according to the invention is not toxic and can be treated with it in case of damage to all the immune system due to its immunostimulating effect. Due to its biological properties described above, it can be used as a complementary means in the medical treatment of malignant tumors, mainly for inhibiting metastasis.

Claims (12)

1. A method for producing a fermentative dried substance with immunostimulating and metastasis-inhibiting properties, characterized in that ground malt is fermented in an aqueous medium at 25-35 ℃ for 10-24 hours in the presence of Saccharomyces cerevisiae, and the fermentation broth is separated, filtered to be clear, evaporated, boiled and dried as such or in the presence of an auxiliary drying substance.

2. A process according to claim 1, characterized in that the fermentation is carried out for 15 to 20 hours.

3. A process according to claim 2, characterized in that the fermentation is carried out at 30 ℃ under continuous aeration and stirring for 18 hours.

4. A process according to claim 3, characterized in that the drying is carried out in the presence of maltodextrin.

5. A fermented dried material having immunostimulating and metastasis inhibiting effects, which is obtained by the method of any one of claims 1 to 4.

6. The substance according to claim 5, having a2, 6-dimethoxy-p-benzoquinone content of 0.12 to 0.52mg/g dry matter and a 2-methoxy-p-benzoquinone content of 0.05 to 0.28mg/g dry matter, measured by HPLC.

7. A material according to claim 6, which has a2, 6-dimethoxy-p-benzoquinone content of 0.4mg/g dry matter.

8. An immunostimulating and metastasis suppressing pharmaceutical product, the active ingredient of which is the fermented dried substance according to claim 1.

9. A mammalian dietary supplement comprising the fermented dry matter of claim 1.

10. A dietary supplement according to claim 9, comprising a dried substance obtained by the method of claim 4, said dried substance comprising 60% by weight of fermented substance and 40% by weight of maltodextrin.

11. Use of the fermented dry matter of claim 5 for the production of a pharmaceutical composition for immunostimulation and metastasis inhibition.

12. Use of the fermented dry matter of claim 5 in the manufacture of a dietary supplement for mammals.