DE19732855C2 - Special soy extract, its preparation, formulations containing it and their use for the prevention or treatment of cancer - Google Patents

Description

The invention relates to new extracts obtained by extraction of ripe, whole soybeans or oil-free soybean meal (Glycine max. (L.) MERRIL, legume family), her position and formulations containing these extracts. The new extracts are due to their content of isoflavones and sapo ninen marked in defined proportions.

It is known that soybean saponin and isoflavone components to in addition to saccharide and amino acid components as well as proteins and mineral salts in quantities different from their geographical Origin and conditions under which the plant is grown and was harvested, depend.

The Saponingehalte were divided into three classes depending on divided into the chemical structure of their triterpene components: Soybean saponins of Groups A, B and E (Okubo K. et al., ACS Symp., Ser. 546, 330, 1994).  

Isoflavone components consist of glucosidosoflavones (daidzin, Genistin and glycitin) linked to the saccharine chain Acyl radicals - z. B. Malonsäurereste - may contain.

Corresponding to biomedical literature and epidemiological information published in recent years, mainly on the population in the East, which feeds on a large scale with soy-based foods, the use of these foods greatly reduces breast cancer in women and men Male Prostate Cancer (A. Nomura, BE, Henderson J. Lee, American Journal of Clinical Nutrition, 1978, 31 , 2020-2025, T. Hirayama in Diet, Nutrition and Cancer, 1986 pp. 41-53, Y. Hayashi M. Nagao, T. Sugimura, S. Takayama, L. Tomatis, LW Wattenberg and GN Wogan eds. Tokyo: Japanese Scientific Society Press; RK Severson, AMY Nomura, JS Grove, GN Stemmerman, Cancer Research, 1989, 49 , 1857-1860). These data, which lack a clear scientific basis, are usually attributed to the isoflavone agonists genistein, daidzein and glycitein found in various soy-based foods.

These isoflavones were used in in vitro models in terms of their Capability with protein kinases, in particular with tyrosine kinase - Enzymes that play a role in tumor cell proliferation play - interact, studied.

There have been a number of recent attempts to obtain pharma Zeutische preparations based on soy extracts for the Präven tive treatment of cancer.

Some patents report preparations of new soy extrak  obtained by chemical or enzymatic hydrolysis of in Soybeans or germs contain isoflavone glycosides (Kikkoman Corp. J-08291191; Kikkoman Corp. J-07173148; Kelly GE WHERE 9323069; Kikkoman Corp. J-05170756). All these patents are correct focusing exclusively on the production of isoflavones higher Concentration and these substances become antitumor activities attributed.

It has now been found that in contrast to the previously described extracts containing glucosidisoflavones and soybean saponins Group B in defined proportions, significantly effective samer are as isoflavones alone or as extracts with other Weight ratio of glucosidosoflavones to soybean saponins Group B. This also applies in particular to that from the US A-4,428,876 known product obtained by isolation of saponi and flavonoids from legumes. This one for applied procedures differs from the inventions to the invention, inter alia, by an extraction of the vegetable material with aqueous alkali solution and by the absence of step c of the method according to the invention.

WO 96/10341 A1 relates to food and medicinal products, the we include substantial hypocotyls of soybean seeds. A Hin note the present extraction method and the hereafter The extract available can not be found in this document men.

JP-59088064-A relates to the use and isolation of soya Saponins, among others, an adsorption of saponins on a polystyrene-type ion exchange resin for use comes. The extract according to the invention, let alone its The properties shown below are also in this document not remove.

Accordingly, the invention relates to an extract which is 0.6 to 1.5 Parts by weight, preferably 1 part by weight, of soybean saponins Group B contains 1 part by weight of glucosidosoflavones and is obtainable by the method described below.

As shown by HPLC-MS analyzes, Group B soybean saponins, which have a positive influence on the activity of the isoflavone components, have the following structures:

The invention further relates to a process for the preparation of the extract as defined above which is characterized in that it comprises the following steps:

• (a) the extraction of mature, whole soya beans or of oil-free soybean meal containing Group B soybean saponins and glucoside Isoflavones in the reciprocal ratio of 3: 2 to 2: 3, with aliphatic Alcohols or a mixture of these alcohols with water,
• b) concentrating the extract of step a)
• c) purification of the concentrated extract from step b) of oily and lipophilic substances by treatment with aliphatic hydrocarbons;
• d) the extraction of the active components with water immiscible aliphatic alcohols;
• e) concentrating the extract of step d) and its Drying.

In particular, the extracts of the invention may be prepared by extracting mature, whole soybeans or oil-free soybean meal containing Group B soybean saponins and glucosidisoflavones in the reciprocal ratio of 3: 2 to 2: 3 with aliphatic alcohols alone or in admixture with water. preferably with a mixture of ethanol / water, such as 95% ethanol. After concentrating the extract and purifying oily and lipophilic substances by treatment with aliphatic hydrocarbons (eg n-hexane or n-heptane), the active components become water-immiscible aliphatic alcohols such as n-butanol, isobutanol and isoamyl alcohol , extracted. After concentration to a reduced volume, the organic phase is dried under vacuum. The invention further relates to a modification of the above process, in which, after stage b) or c), the concentrated alcohol extract is subjected to the following stage d '), followed by stage e):

• 1. the adsorption of the active components on an adsorbent polystyrene-based resin; rinsing the resin with Water; and elution of the active ingredients with etha nol.

According to this amendment, a typical extract of Invention be prepared by adsorption of the active Kom components (isoflavones and saponins) concentrated in the Alcohol extract of the plant material are included a polystyrene-based adsorption resin such as Duolite or any  Lich XAD, especially XAD1180 due to its easy clean rem ph; and elution of the mixture of isoflavones and soya Group B saponins again with ethanol after careful Rinse the resin column with water to remove salts and other non-active components.

The extract obtained under these conditions contains 13 to 17 % By weight of isoflavones and 0.6 to 1.5 parts by weight of soybean saponins Group B, according to the quality of the plant used 1 part by weight of glucoside isoflavones. This Extract also contains a large amount of polyphenolic sub punching, which are essential for the extract inherent de activity.

One embodiment of the above method and modification thereof comprises the following steps:

• a) suspending the extract from step e) in a Mi of a water-miscible alcohol and water and diluting with a water immiscible aprotic solvents;
• b) heating the mixture from step f) to complete the resolution and leaving at room temperature;
• c) collecting the failed soy saponins of the group B by filtration;  
• d) the separation of the organic phase from the water phase se; and concentrating the organic phase and drying for obtaining the isoflavone components; and
• e) mixing the saponins of step h) and the isoflavones from step i) to form the extract.

Accordingly, the extracts of the invention may be preferred also those caused by a very high content of glucosidiso flavones and soybean saponins of group B in the above be characterized ratio are also manufactured are from the according to the Ver described above or its modification obtained extract. For this purpose can proceed as follows: The said extract is in a water miscible alcohol, such as ethanol or methanol, with a water content of 10 to 50% by volume and suspended with a water-immiscible aprotic solvent, such as Methylene chloride or ethyl acetate. The obtained hete rogene mixture is used to complete the dissolution of the Extract heated and left at room temperature, so that the Soy saponins of group B can fail. The one purity over 90% saponins are obtained by filtration collects. The isoflavone components that have a purity of more than 80% are from the aqueous mother liquor through Separating the organic phase and evaporating and drying the the latter received. The isoflavones and saponins can then be mixed to an extract with the highest possible Content of glucosidosoflavones and containing soya sapo  Group B is from 0.6 to 1.5 parts by weight per 1 weight part to obtain glucosidisoflavones.

Preferred conditions for carrying out the individual Ver driving steps of the method according to the invention are the consequences the. Here are the units of measure for parts by volume l (liters) and those for parts by weight kg (kilograms).

Stage a: The extraction of the plant material is approaching added with 12 to 17 volumes of solvent per 1 part (weight) Biomass performed. The extraction temperature is appropriate higher than 55 ° C. Each extraction is conveniently within performed less than 4 hours. Suitable solvents Apart from ethanol, methanol, propanol and isopro are among others propanol. These solvents can contain up to 10% water.

Stage b: Concentration of the extract is expedient in case of a temperature below 50 ° C under vacuum. The extract will appropriately concentrated to an alcohol content of 65 to 75%.

Step c: Purification is convenient with 0.3 to 0.6 volumes to 1 part (by weight) of aliphatic hydrocarbons Lich material performed. A suitable procedure is that of extraction of oily and lipophilic substances.

Stage d: The extraction of the active ingredients is expediently carried out with Do not mix 0.2 to 0.4 volumes of alcoholic with water solvent per extraction, calculated to 1 part (Ge  weight) of vegetable matter; preferably three ex carried out tractions.

Stage e: The concentration of the extract of stage d takes place expedient at a temperature below 50 ° C under vacuum.

Step f: The extract of step e) is useful in 5 to 10 Volumes (1 part extract) of water-soluble alcohol with an alcohol to water ratio in the range of 2: 8 to 3: 7 vol / - Vol extract suspended. The aprotic, not mixed with water bare solvent is useful in an amount of 2 to 5 Volumes, based on 1 part (weight) of the extract from step e used.

Level g: The mixture is allowed to complete dissolution reach, appropriately heated and refluxed. to closing the mixture is preferably 15 to 24 h at room temperature.

Stage i: Evaporation of the organic phase is expedient at a temperature of less than 30 ° C under vacuum.

Step j: Preferably, one prepares from the saponins of step h and the glucosidisoflavones of stage i an alcoholic Lö which relates the saponins and glucosidisoflavones contains 1: 1, which then expediently at a temperature is concentrated to dryness below 50 ° C under vacuum.  

The amounts of isoflavones and soybean saponins of group B are determined by HPLC analysis using a Supelco-Sil LC-ABZ column (250 mm x 4.6 mm), 5 μm, and a ternary gradient eluent from A) H ₂ O (CF ₃ COOH 0.01%), B) acetonitrile (CF ₃ COOH 0.01%) and C) methyl alcohol (CF ₃ COOH 0.01%). The individual components can be identified and characterized by mass spectrometry combined with HPLC over a thermal spray interface.

The extracts of the invention have a pronounced anti proliferative activity. Table 1 gives the antiproliferative Activity against an ovarian tumor cell line (OVCA 433) such as the.

connection IC ₅₀ μM Soy saponins of group B 6.2 Soy Isoflavones 4.5 Soya extract prepared according to Example 1 1.6 Soya extract prepared according to Example 3 1.1

Cells were grown in a monolayer culture to a minimum of essential medium supplemented with calf serum and 200 units / ml of penicillin to keep the medium sterile. For reproducibility of the assay, the cells were trypsinized each week and placed on plates at a density of 8x10 ⁴ cells / ml and incubated at 37 ° C under an air atmosphere containing 5% CO ₂ and moisture. To evaluate the activity of the compounds, the cells were placed in wells (Falcon 3046, Becton Dickinson NY) at a concentration of 1 x 10 ⁵ / ml in the minimum amount of substrate. After 24 hours, the substrate was replaced with fresh substrate and the compounds dissolved in absolute ethanol were added. Controls were treated analogously with the vehicle in the absence of the drug to be tested. The above-described treatment was repeated at intervals of 24 hours during a test period of 72 hours. The inhibition of cell proliferation was evaluated by directly counting the lines, comparing the growth of the controls to that of the "treated" tests. As shown, the extracts according to the invention have an antiproliferative activity which is stronger than the sum of the antiproliferative activity of their components (synergistic action). The compounds of the present invention inhibited cell proliferation in vivo, as measured by measuring the size of tumors transplanted into naked athymic mice, according to the usual literature conditions. The treatment of the animals with doses in the range of 10 to 500 mg / kg led to a pronounced regression of the examined tumors until their disappearance in a high percentage of the individuals.

Table 2 below shows analogously obtained subsequent results for further extracts A to D having the following composition:

Extract A: weight ratio of isoflavones to saponins of 1: 0.5;
Extract B: weight ratio of isoflavones to saponins of 1: 2;
Extract C: weight ratio of isoflavones to saponins of 1: 1;
Extract D: weight ratio of isoflavones to saponins of 1: 1.5.

The extracts A to C were in this case by the method of Bei obtained game 3; Extract D corresponds to the extract of Bei game 1.

connection IC ₅₀ μM Extract A (comparison) 4.2 Extract B (comparison) 5.3 Extract C 1.4 Extract D 1.2

The invention therefore also relates to a combination  tion, the active component as defined above Contains extract. In particular, the invention relates to this Extract containing pharmaceutical composition for the Prevention or treatment of cancer. Above all, the concerns Invention to a pharmaceutical extract containing this extract composition for the prevention and treatment of breast cancer in the female and prostate cancer in the male.

The products or extracts according to the invention may be suitable ter way in tablets, soft or hard gelatin capsules, granu laren powders for the preparation of ready-to-use solutions or fluids or liquids, which kom kom with their solubility patibel are formulated. The doses of the invention Extracts range from 30 mg to 500 mg with or without repeated administration per day, preferably at 200 mg at twice daily. The appropriate administration The oral form is the oral form.

The following examples illustrate the invention.

Example 1 - Preparation of soybean extract with an isoflavone content of 15% by weight and a ratio of glucosidisoflavones / Group 1 soybean saponins of 1: 1.5 by purification with Lö sungsmitteln

10 kg of oil-free soybean meal containing 0.2% glucosidiso Flavone and 0.3% Group B soybean saponins are co-administered five times 30 l of 95% ethyl alcohol refluxed. The alcoholsx  are mixed and concentrated in vacuo to 5 l. you diluted with 1.5 l of water and extracted four times with 5 l of n-He xan. The hexane phase is discarded and the concentrated alkoxide extracted four times with 2.5 l of n-butanol. The organic Phase is concentrated and dried under vacuum. You get 133 g extract with an isoflavone content of 15 wt .-% and a Content of Group B soybean saponins of 22.5% by weight.

An HPLC chart of an extract obtained by the method of this example is shown in FIG .

Example 2 - Preparation of a soy extract with an Isofla content of 15% by weight and a ratio of glucosidisoflavo ne / S soybean saponins of 1: 1.5 by purification on poly styrene resin

The aqueous concentrate prepared in Example 1 does not extracted with n-butanol, but with polyethoxylated Rhizi Nut oil (Cremophor®) is treated to remove the resinous residues the concentration of alcohol phase dissolve. You then bring it in one column on XAD1180 resin suspended in purified water (5 l). The column is allowed to completely remove Salts, sugars and surface-active agents with water rinsed and then eluted with about 10 l of 95% ethyl alcohol. To Concentrating and drying the ethanol eluate gives 130 g Extract of the same composition as that of the Example 1 obtained extract.  

Example 3 - Preparation of a Soya Extract with an Isofla content of 43% by weight and a ratio of glucosidisofla vone / soybean saponins of group B of 1: 1

200 g of the ex obtained in Example 1 or 2 are suspended in 1 l of aqueous 20% ethyl alcohol and diluted with 0.5 l of ethyl acetate. The suspension is subjected to countercurrent heating with vigorous stirring until complete dissolution and then left during a night. The failed Saponi ne (38 g, purity 93%) are recovered by filtration and the Ethyl acetate and water-containing aqueous mother liquor separated. The organic phase is concentrated under vacuum and dried. The isoflavone residue - 37 g with one purity of 81% - is dissolved in 1 l of ethyl alcohol and crystallized with 32 g lysed saponins to arrive at a product, the isoflavones and saponins in a weight ratio of 1: 1 contains. The alcohol solution is turned to dryness under vacuum concentrated and yields 69 g of extract containing 43 wt .-% gluco sidisoflavones and 43% by weight soybean saponins of group B.

Example 4 - Production of hard gela containing soybean extract gelatin capsules Soya extract prepared according to Example 1 200.0 mg lactose 67.5 mg Microcrystalline cellulose 22.5 mg Colloidal silica 3.0 mg Croscarmellose sodium (crosslinked polymer of carboxymethyl cellulose sodium) 21.0 mg talc 8.0 mg magnesium stearate 3.0 mg

Example 5 - Preparation of tablets containing soybean extract Soya extract prepared according to Example 3 400.0 mg Soy polysaccharides 155.5 mg Microcrystalline cellulose 57.0 mg hydroxypropyl methylcellulose 12.0 mg Hydrogenated vegetable oil 19.5 mg Colloidal silica 3.0 mg magnesium stearate 3.0 mg

Claims (9)

A process for producing a soy extract having a content of 0.6 to 1.5 parts by weight of soybean saponins of group B per 1 part by weight of glucosidisoflavones, characterized in that it comprises the following steps:

• a) the extraction of mature, whole soybeans or oil-free soybean meal containing Group B soybean saponins and glucosidisoflavones in the reciprocal ratio of 3: 2 to 2: 3, with aliphatic alcohols or a mixture of these alcohols with water;
• b) concentrating the extract of step a);
• c) purifying the concentrated extract of step b) of oily and lipophilic substances by treatment with aliphatic hydrocarbons;
• d) extracting the active components with water immiscible aliphatic alcohols;
• e) concentrating the extract of step d) and drying it.

2. Modification of the process according to claim 1, wherein after stage b) or stage c) the concentrated alcohol extract is subjected to the following stage d '), followed by stage e)

• 1. adsorption of the active components on a polystyrene-based adsorption resin; rinsing the resin with water; and elution of the active ingredients with ethanol.

3. The method according to claim 1 or 2, comprising the following additional steps:

• a) suspending the extract from step e) in a mixture of a water-miscible alcohol and water and diluting with a water-immiscible aprotic solvent;
• b) heating the mixture of step f) for complete dissolution and leaving at room temperature;
• c) collecting the failed group B soybean saponins by filtration;
• d) separating the organic phase from the water phase; and concentrating the organic phase and drying to obtain the isoflavone components; and
• e) mixing the saponins of step h) and the isoflavones of step i) to form the extract.

4. Soya extract containing 0.6 to 1.5 parts by weight Group B soybean saponins per 1 part by weight of glucosidisofla vone, obtainable by a process according to one of the claims che 1 to 3. 5. Soya extract according to claim 4, characterized by a Content of 1 part by weight of group B soybean saponins, 1 Ge each Weight fraction glucosidisoflavones. 6. Pharmaceutical composition containing as effective Kom Component an extract according to claim 4 or 5. 7. Use of the pharmaceutical composition according to An claim 6 for the prevention or treatment of cancer. 8. Use of the pharmaceutical composition according to An claim 6 or 7 for the prevention or treatment of breast cancer in the woman. 9. Use of the pharmaceutical composition according to An claim 6 or 7 for the prevention or treatment of Prosta takrebs by the man.