Method for producing pectin hydrolysis products

Process for the manufacture of pectin hydrolysis products

Description

The invention relates to a method for producing pectin hydrolyzates, in particular a pharmaceutical or dietetic preparation for reducing and / or preventing the adhesion of pathogenic substances and organisms to eukaryotic cells, in particular mammalian cells, or for inhibiting galectin 3-mediated cell-cell and / or cell-matrix interactions that lead to the development of tumor diseases, Process for blocking the accumulation of pathogenic substances or organisms on eukaryotic cells, process for the inhibition of galectin-3 mediated cell-cell and / or cell-matrix interactions, as well as the pectin hydrolysates and preparations produced by these processes.

Pathogenic organisms but also cell-damaging substances first have to attach themselves to the surface of the target cell in order to be able to cause infection or damage to the attacked cell. This attachment or adhesion^' is mediated, for example, by a liganol-receptor relationship, in which glycostructures play an important role. Infection can be prevented by blocking these glycostructures on the target cell surface or on the ligand. Glycostructures also play an important role in tumor formation and metastasis formation (Liotta et al., Annu. Rev. Cell Biol., 55 (1986), 1037-1057). The formation of tumors includes cellular interactions that are mediated by cell surface components, in particular carbohydrate-binding proteins. The adhesion of tumor cells is mediated by cellular adhesion molecules. Many stages of metastasis formation include cell-cell interactions or interactions between cells and the extracellular matrix (ECM), which are mediated by cell surface components. The extracellular matrix (ECM) consists mainly of laminin, fibronectin and proteoglycans, many of which are glycosylated and whose oligosaccharide side chains offer recognition determinants for cellular adhesion molecules. Laminin is an N-linked glycoprotein that has poly-N-acetyllactosamine sequences. Metastases colonize when circulating agglomerates of tumor cells, platelets and lymphocytes in capillaries make contact with the endothelium via adhesive molecules. This contact provides the signal for opening the endothelial cell functions. This enables the tumor cells to bind to receptors on the basal membrane via additional adhesion molecules. After the basement membrane has been destroyed, the tumor cells are given direct access to the stoma, with interactions between laminin and fibronectin and the corresponding receptors taking place, as in the primary tumor invasion. Important representatives of the carbohydrate-binding proteins are the galactoside-binding lectins galectin-1 and galectin-3 (Raz and Lotan, Cancer Metastasis Rev. 6 (1987), 433; Gabius, Biochim. Biophys. Acta, 1071 (1991), 1). Galectin 3 is known to promote embolic tumor disintegration in the bloodstream and increase metastasis. Galectin-3 is expressed on the cell surface of many tumor cells, with galectin-3 expression increasing as the tumor develops. Galectin-3 is also expressed by activated macrophages and oncogenically transformed cells or metastatic cells. Galectin-3 has a high affinity for oligosaccharides which comprise polylactosamines, whereby galectin-3 binds in particular to two glycoproteins which occur in several cell types, for example human colon carcinoma cells and human breast carcinoma cells. Another ligand for Galectin 3 is, for example, laminin. Galectin 3, which is also expressed on the surface of endothelial cells, is also involved in the adhesion of tumor cells to endothelial cells.

No. 5,834,442 describes methods for the treatment of cancer in mammals, in particular for the treatment of prostate cancer, the treatment of cancer, including the inhibition of metastasis, being carried out by oral administration of modified pectin, preferably water-soluble pH-modified citrus pectin. To produce pH-modified pectin, a pectin solution is depolymerized by increasing the pH to 10.0 and then lowering the pH to 3.0. The modified pectin has a molecular weight of about 1 to 15 kd. Rats given modified citrus pectin in drinking water showed a significantly reduced formation of lung metastases compared to untreated control groups. In vitro experiments showed that the adhesion of galectin-3 expressing MLL endothelial cells to rat aortic endothelial cells (RAEC) was almost completely inhibited in the presence of modified citrus pectin. The effect of pH-modified citrus pectin on the colony formation of MLL endothelial cells was investigated in further experiments. The ability of cells to grow in semi-solid medium (anchorage independence) can be used as a criterion for cell transformation and cell invasion potential, since cell growth in a semi-solid medium requires cell migration and colonization. It was found that modified citrus pectin could significantly reduce both the number of MLL colonies formed and their size. Modified citrus pectin appears to have a cytostatic effect rather than a cytotoxic effect. The effect of modified citrus pectin on cell-cell interactions and cell-matrix interactions that are based on carbohydrate-mediated mechanisms, in particular galectin-3-mediated interactions, was also examined. It shows that modified citrus pectin in the

^"Contrary to unmodified citrus pectin

Adhesion of B16-Fl melanoma cells to laminin inhibited. Laminin is known to serve as a ligand for soluble galectin-3.

It is known from EP 0 716 605 Bl that the adherence of pathogenic germs, such as E. coli, to cells, in particular to epithelial cells of the gastrointestinal and genitourinary tract, is essential (that is to say through a specially prepared carrot soup, bladder tea, coconut milk etc.) can be reduced to 90%). According to this publication, this effect can be attributed to the pectins present in the plant products, which are essentially chains of 1,4-α-glycosidically linked galacturonides, the acid groups of which are 20 to 80% esterified with methanol, and besides Galacturonic acid can optionally contain other sugar components, for example glucose, galactose, xylose and arabinose. From the publication it can further be seen that monogalacturonic acid shows no blocking of the adhesion, whereas with digalacturonide and trigalacturonide there is a blocking of 91.7% and 84.6% respectively. This publication clearly states that the monomeric galacturonic acid has no blocking of the adhesion and that the desired blocking effect decreases with increasing molecular weight of the galacturonides. It follows that the degree of polymerization of the desired galacturonides is DP 2 or 3. It is also required that the degree of esterification is <2%. However, the pectin hydrolysis products produced by the method described therein contain only very low proportions of the active^' designated di- and trigalacturonides (approx. 12% based on the raw material). This production method wastes resources and leads to environmental problems, since the unusable by-products that are present in large proportions have to be disposed of.

The technical problem underlying the present invention consists in further methods and means for combating infections and for reducing and / or preventing the adhesion of harmful, in particular pathogenic, substances and organisms to eukaryotic cells, in particular mammalian cells, and for blocking interactions between Mammalian cells, especially tumor cells, which are mediated by carbohydrate-binding galectin 3 molecules on the cell surface and are responsible for the development of, in particular, tumor diseases, and for the treatment of tumor diseases, in particular for preventing metastasis, in mammals.

This technical problem is solved by the processes according to the invention for the production of pectin hydrolysis products which lead to the production of oligogalacturonides with the lowest possible proportion of monomers, a high proportion of molecules with at least one double bond each and with a degree of esterification of> 20% and which can be carried out with a substantially higher yield than described in the prior art. The problem is solved in particular in that an aqueous solution or suspension of a pectin or pectin-containing, in particular vegetable, material, preferably a pectin with a high degree of esterification, in a first process step with a first pectin-hydrolyzing enzyme A and then in a second process step treated with a second pectin hydrolyzing enzyme B. A pectin hydrolysis product as defined above is obtained, which has excellent properties as an agent for reducing or preventing the adhesion to the vital and / or reproductive functions of cells which are more harmful, for example pathogenic, allergenic, infectious or toxic. Has substances or organisms, for example microorganisms such as yeasts, fungi, germs, bacteria, viruses, spores, viroids, prions.

The enzyme A used can be, for example, a pectin lyase (EC 4.2.2.10) or an endopolygalacturonase (EC 3.2.1.15), but preferably a pectin lyase. The enzyme B used can be an endopolygalacturonase or a pectin lyase, but preferably an endopolygalacturonase.

According to the invention, it was surprisingly found that galacturonides which carry double bonds in the molecule are particularly effective in blocking the adhesion of, for example, pathogenic germs and cell-damaging substances to epithelial cells of the gastrointestinal and urogenital tract. In addition, for a particularly efficient prevention and / or reduction, for example blocking, a higher degree of esterification, in particular> 20, preferably> 30,> 40,> 50, particularly preferably> 60,> 65,> 70 or> 71% required. However, the procedure described in the prior art only leads to galacturonides which have no double bonds and are virtually completely desesterified.

In connection with the present invention, the term degree of esterification is understood to mean the proportion of the acid groups of a galacturonide which are basically available for an esterification and which is esterified with an alcohol, in particular methanol.

In connection with the present invention, unsaturated galacturonic acid molecules are understood to mean, in particular, 4,5-unsaturated galacturonic acid molecules.

In one embodiment of the invention, treatment with another, third enzyme C is provided after treatment with enzyme B. The enzyme C used here can be a pectin esterase (EC 3.1.1.11). This allows the degree of esterification of the products to be set in a particularly targeted manner.

In a further embodiment of the invention, it is provided that after the enzymatic treatment according to the invention has taken place, the remaining undissolved constituents are separated from the solution by centrifugation and / or ultrafiltration. In a further embodiment it is provided that the solution obtained after the enzyme treatment according to the invention and clarification by centrifugation or ultrafiltration is converted into dry form, for example into ground, granular, granulated or powder form, by one of the methods known per se.

The solution obtained after the enzymatic treatment according to the invention or the dry product obtained therefrom have very good effects with regard to blocking the adhesion of, for example, pathogenic germs and cell-damaging substances to, for example, epithelial cells of the gastrointestinal ^* and urogenital tract in humans and animals. They can therefore be used in animal feed, for example, to prevent diarrhea during piglet rearing.

The solution or suspension of the pectin or of the pectin-containing, preferably vegetable, material used according to the invention has a pH in a range from 3.5 to 5.5 or / and in a further preferred embodiment a concentration of the pectin from 3% to 25% on.

Treatments with the enzymes A, B and optionally C take place at a pH value of 3.5 to 5.5 over a period of 2 hours to 24 hours at a temperature of 25 ° C to 60 ° C and a concentration of Enzyme A, B optionally C from 10 to 30 ml / kg pectin instead. In a further preferred embodiment it is provided that the proportion of the galacturonides in the pectin hydrolyzate (% by weight in relation to dry substance) is at least 60,> 70, 75,> 80 or particularly preferably at least 85% by weight.

In a further preferred embodiment it is provided that the proportion of carbohydrates with a DP-1 (monomers) in the total carbohydrates of the pectin hydrolyzate in the pectin hydrolyzate is <25, <20, <10, <8, <5, particularly preferably <4% by weight. -% (based on dry matter).

In a further preferred embodiment it is provided that the proportion of carbohydrates, in particular galacturonides with a degree of polymerization DP> 10, based on the total carbohydrate content of the pectin hydrolyzate, is less than 10, <8, particularly preferably <5% by weight (based on Dry matter).

In a further preferred embodiment it is provided that the proportion of the unsaturated galacturonides in the total content of the galacturonides in the pectin hydrolyzate is at least 10, preferably> 15,> 20,> 25,> 30, in particular 36.5% by weight to 46% by weight (based on dry matter).

In a preferred embodiment, the pectin hydrolyzates produced according to the invention have a proportion of at least 60,> 70,> 75,> 80 or particularly preferably at least 85% (on dry substance) of carbohydrates, in particular galacturonides, with a degree of polymerization of 2 to 10. preferably from 3 to 8, particularly preferably 4.5 to (% by weight of dry substance, based on the total carbohydrate content of the pectin hydrolyzate).

In a particularly preferred embodiment, the pectin used is citrus pectin, apple pectin or sugar beet pectin.

In a preferred embodiment, the pectin-containing material used, in particular vegetable pectin-containing material, is apple pomace, sugar beet pulp or citrus pellets, that is to say dried residues, for example from the production of orange juice, lemon juice and / or lemon juice.

The pectin hydrolysates produced according to the invention, that is to say pectin hydrolysis products, are outstandingly suitable as pharmaceutical or dietetic preparations for combating infectious diseases and / or for combating the accumulation of harmful substances and / or organisms on mammalian cells, in particular human cells.

The pectin hydrolyzates produced according to the invention are also outstandingly suitable as a pharmaceutical preparation for inhibiting cell-cell interactions and / or interactions between cells and the extracellular matrix in a human or a mammal, in particular those interactions caused by galectin 3 molecules on the cell surface mediated. The pectin hydrolysis products according to the invention are therefore particularly suitable for inhibiting cell-cell and / or cell-matrix interactions in which tumor cells are involved and which are therefore responsible for the development of diseases, in particular tumor diseases in a human or a mammal. The pectin hydrolyzates according to the invention are therefore also suitable as a pharmaceutical preparation for the treatment of tumor diseases, in particular for reducing tumor growth and / or for reducing the formation of metastases in cancer diseases, since they prevent galectin-3-mediated tumor cell adhesion and / or the invasion potential of the tumor cells .

The invention therefore also relates to the pectin hydrolysates obtained according to the invention, that is to say pharmaceutical preparations and dietary preparations containing pectin hydrolysis products, which can be designed, for example, as foods or luxury foods, for example milk products, yoghurt, cereals, baked goods, etc.

The invention also relates to the use of the abovementioned pectin hydrolysis products for the production of medicaments for preventing the accumulation or adhesion of harmful substances and / or organisms on mammalian cells, in particular itial cells, in particular for combating, ie prophylaxis and therapy, of infectious diseases. Poisoning, allergies etc.

The invention also relates to the use of the aforementioned pectin hydrolysis products to prevent the accumulation or adhesion of harmful substances and / or organisms on mammalian cells, in particular human cells, in particular for combating infectious diseases, poisoning, allergies etc.

The infections combated according to the invention can in particular be infections of the blood system, the respiratory tract, the urogenital tract, the nasopharynx or the gastrointestinal tract. .

Another area of ​​application is in human nutrition, where they help, for example, to prevent diarrhea in infants as well as adults.

The invention also relates to the use of the aforementioned pectin hydrolysis products for inhibiting cell-cell interactions and / or interactions between cells and the extracellular matrix, in particular those interactions mediated by carbohydrate-binding galectin 3 molecules located on the cell surface and which are responsible for the development of human or mammalian diseases, in particular tumor diseases. These diseases are, in particular, prostate carcinomas, kidney carcinomas, Kaposi's sarcoma, forms of chronic leukemia, breast carcinomas, breast adenocarcinomas, sarcomas, ovarian carcinomas, rectal carcinomas, pharyngeal carcinomas, melanomas, small intestine tumors, colon tumors , Bladder tumors, mastocytomas, lung carcinomas, bronchial carcinomas, pharyngeal squamous cell carcinomas, gastrointestinal carcinomas and gastric carcinomas. The pectin hydrolysis products according to the invention can be used in particular to reduce the invasion potential of metastatic tumor cells and / or to inhibit the adhesion of tumor cells. The pectin hydrolysis products produced according to the invention are preferably administered orally.

A further preferred embodiment of the invention relates to the use of the pectin hydrolysis products according to the invention for the treatment of tumor diseases of a human or a mammal. The pectin hydrolyzates according to the invention can preferably be used for the treatment of such tumors which are based on cell-cell interactions and / or interactions between cells and the extracellular matrix, in particular those interactions which are caused by carbohydrate-binding galectin-3 located on the cell surface. Molecules are conveyed. Using the pectin hydrolyzates according to the invention, therefore, prostate carcinomas, kidney carcinomas, Kaposi's sarcoma, forms of chronic leukemia, breast carcinomas, breast adenocarcinomas, sarcomas, ovarian carcinomas, rectal carcinomas, pharyngeal carcinomas, small arms cancer, melanoma cancer, thin carcinomas , Bladder tumors, mastocytomas, lung carcinomas, bronchial carcinomas, pharyngeal squamous cell carcinomas, gastrointestinal carcinomas and gastric carcinomas are treated. The use of the pectin hydrolyzates according to the invention for tumor treatment aims in particular at inhibiting the adhesion of tumor cells and / or reducing the invasion potential of metastatic tumor cells. The invention also relates to the use of the aforementioned pectin hydrolysis products for the production of a pharmaceutical preparation for inhibiting cell-cell interactions and / or interactions between cells and the extracellular matrix, in particular those interactions caused by carbohydrate-binding agents located on the cell surface Galectin 3 molecules are mediated and used for the development of human or mammalian diseases, in particular the tumor diseases described above are responsible. The pharmaceutical preparation for inhibiting cell-cell interactions and / or interactions between cells and the extracellular matrix is ​​preferably administered orally.

A further preferred embodiment of the invention therefore also relates to the use of the pectin hydrolysis products according to the invention for the manufacture ^■ Development of a pharmaceutical preparation which can be used for the treatment of the tumor diseases described above, that is to say for the treatment of tumors which are based on cell-cell interactions and / or interactions between cells and the extracellular matrix, in particular those interactions which are mediated by carbohydrate-binding galectin 3 molecules on the surface of the cell. It is provided according to the invention that the pharmaceutical preparation can be used to reduce tumor growth and / or to reduce the formation of metastases, the pharmaceutical preparation according to the invention in particular preventing the adhesion of tumor cells and / or reducing the invasion potential of tumor cells. The pharmaceutical preparation according to the invention is preferably administered orally.

The present invention also relates to a method for blocking the accumulation of harmful, in particular pathogenic, substances or organisms on cells of a human or mammalian body, comprising the administration of the pectin hydrolysis products produced according to the invention to a human or a mammal in an amount which is sufficient block the accumulation of harmful substances or organisms on mammalian cells and prevent the development of an infection. The pectin hydrolysis products produced according to the invention are preferably administered orally.

The invention also relates to a method for inhibiting cell-cell interactions and / or interactions between cells and the extracellular matrix which are mediated by carbohydrate-binding galectin-3 molecules on the cell surface and for the development of human or mammal Diseases, in particular the tumor diseases mentioned above, are responsible, comprising the administration of the pectin hydrolysis products produced according to the invention to a human or a mammal with a tumor disease in an amount sufficient to reduce and / or inhibit galectin-3-mediated cell-cell and / or cell-matrix interactions. The pectin hydrolysis products produced according to the invention are preferably administered orally. The present invention also relates to pharmaceutical preparations which contain the pectin hydrolysis products according to the invention in pharmaceutically or therapeutically effective amounts. In connection with the present invention, a “pharmaceutical preparation” is understood to mean a mixture used for diagnostic, therapeutic and / or prophylactic purposes, which contains the pectin hydrolysis products according to the invention as active substances in a form which can be administered well to a patient or a mammal. The pharmaceutical preparation can be both a solid and a liquid mixture. "In pharmaceutically or therapeutically effective amounts" means that the active ingredients contained in the pharmaceutical preparation are contained in a dose sufficient to prevent the onset of a disease, for example an infectious disease or a tumor, to cure the condition of such a disease Stop progression of such a disease and / or relieve the symptoms of such a disease. In a preferred embodiment, the pharmaceutical preparations according to the invention, in addition to the pectin hydrolysis products according to the invention, have pharmaceutically compatible carriers and, if appropriate, diluents, mold release agents, lubricants, auxiliaries, fillers, sweeteners, flavorings, colorants, flavorings or other pharmaceutically active substances.

The pectin hydrolysis products are also used in a pharmaceutically effective amount in the dietary preparations provided according to the invention.

Further preferred embodiments are listed in the subclaims.

The invention is explained in more detail with the aid of the following examples.

Example 1 :

To 1 1 of a citrus pectin solution (30 g highly esterified pectin in 1 1 water) was added 0.3 ml of a pectin lyase (for example Rohapect PTE from Röhm) and the solution with stirring at .pH 5.0 and 45 ° C incubated for 2 hours. Then 0.75 ml of an endopolygalacturonase (for example Pectinase PL from Amano) was added and incubated for a further 3 hours under unchanged reaction conditions. Then the enzymes were inactivated by heating to 95 ° C. The insoluble residue was removed by centrifugation, the clear solution was evaporated to dryness and the solid obtained was weighed. The weight was 25.8 g (corresponding to a yield of 75.6% based on the raw material used).

The product obtained was examined according to generally known analytical methods and the following composition was found: carbohydrates DP 1 3.6

Galacturonides 83.9 of which unsaturated 46.0 o

(assumed mean DP = 4.5)

DP 2 - 10 80.4 Q. O

DP> 10 16.0 o

Degree of esterification 72.0 Q. o

Salinity 3.0

Crude protein 1.7

Water content 4.6

Example 2:

0.3 ml of a pectin lyase (for example Rohapect PTE from Röh) was added to 1 l of a citrus pectin solution (30 g highly esterified pectin in 1 l water) and the solution was stirred at pH 5.0 and 45 ° C for Incubated for 2 hours. Then 0.75 ml of an endopolygalacturonase (for example Pectinase PL from Amano) was added and incubated with the reaction conditions unchanged for a further 3 hours. Then the enzymes were inactivated by heating to 95 ° C.

The insoluble residue was removed by centrifugation and the clear solution was ultrafiltered (cut-off 10,000). The permeate was dried and gave 22.8 g of solid (yield 66.8% based on the raw material used). Carbohydrates DP 1 3.0%

Galacturonides 84.1% of which unsaturated 36.5%

(assumed mean DP = 4.5) DP 2 - 10 93.0%

DP> 10 4.0%

Degree of esterification 72.0%

Salinity 6.7%

Crude protein 1.3% water content 4.4%

Example 3

0.3 ml of a pectin lyase (for example Rohapect PTE from Röhm) was added to 1 1 of a citrus pectin solution (30 g of highly esterified pectin in 1 1 of water) and the solution was stirred at pH 5.0 and 45 ° C. for Incubated for 2 hours. Then 0.75 ml of an endopolygalacturonase (for example Pectinase PL from Amano) was added and incubated with the reaction conditions unchanged for a further 3 hours. Then 0.5 ml of a pectin esterase (for example Rheozyme from Novo Nordisk) was added and incubated for a further 45 minutes. Then the enzymes were inactivated by heating to 95 ° C. The insoluble residue was removed by centrifugation, the clear solution evaporated to dryness.

The product obtained was examined by generally known analytical methods. In contrast to Example 1, a degree of esterification of 35% was found. Example 4:

Dried orange peel or citrus pellets were crushed to a particle size of approx. 1-5 mm and 100 g of this was stirred into 400 ml of water and allowed to swell. Then concentrated nitric acid (10 g) was added and the suspension was heated to 85 ° C. and stirred at this temperature for 1.5 hours. The mixture was then cooled to 45 ° C., the pH was raised to 4.5 with NaOH and, after addition of 0.3 ml of a pectin lyase (for example Rohapect PTE from Röhm), ​​incubated for 2 hours. Then 0.75 ml of an endopolygalacturonase (for example Pectinase PL from Amano) was added and incubated with the reaction conditions unchanged for a further 3 hours. Then the enzymes were inactivated by heating to 95 ° C., concentrated and the suspension was dried on a drum dryer.

Example 5:

Dried orange peel or citrus pellets were crushed to a particle size of approx. 1-5 mm and 100 g of this was stirred into 400 ml of water and allowed to swell. Then concentrated HC1 (8 g) was added and the suspension was heated to 85 ° C. and stirred at this temperature for 1.5 hours. The mixture was then cooled to 45 ° C., the pH was raised to 4.5 with NaOH and, after addition of 0.3 ml of a pectin lyase (for example Rohapect PTE from Röhm), ​​incubated for 2 hours. Then 0.75 ml of an endopolygalacturonase (for example Pectinase PL from Amano) was added and incubated with the reaction conditions unchanged for a further 3 hours. Then the enzymes were inactivated by heating to 95 ° C., concentrated and the suspension was dried on a drum dryer.

Example 6:

Prevention of the adhesion of pathogenic germs in human epithelial cells

For this test, human uroepithelial cells obtained by centrifugation from the morning urine and two strains of Staphylococcus aureus and E. coli, each as a suspension with 10⁹ Keimen / mL inserted.

Test execution

Epithelial cells and germ suspension were incubated together at 37 ° C for 30 minutes. Then the epithelial cells were separated from the non-adherent germs by membrane filtration (8μ). The filters were washed several times, placed in physiological saline and the epithelial cells suspended in them. After centrifuging the suspension in saline, the pellet was applied to slides and stained according to May-Grünwald and Giemsa. The number of germs adhering to 50 epithelial cells was counted. The number gave the blank. Epithelial cells without the addition of a germ suspension served as a control.

By doing. In the main experiment, epithelial cells were first incubated with aqueous solutions of different concentrations from pectin hydrolysis products prepared according to the invention (corresponding to Example 1) for 1, 2 or 3 hours. Then they were brought together with the germ suspension and further treated as described above. The count of the adhering germs on 50 epithelial cells gave the measured value.

Result

With the "neutral" carbohydrates used as a comparison, such as raffinose, nystose and isomelecitis, no reduction in the germ attachment to the epithelial cells was found. With the pectin hydrolysis products according to the invention, the adhesion of all tested microorganisms was almost completely prevented (blocking:> 95%).

Example 7:

1.5 g of the dried permeate obtained in Example 2 were dissolved in 100 ml of 50 mM Na acetate solution, pH 5.0, and then on a column (2.6 × 30 cm), which was equipped with the anion exchanger AG 1 X2 (from BioRad) and filled with 50 mM Na acetate solution, pH 5.0, had been added. The flow from the column was analyzed using HPAEC (high performance anion exchange chromatography) and hydrolyzed using 1N HCl at 95 ° C. for one hour.

Result:

In comparison with Raftiline (from Orafti) as standard, the oligosaccharides eluted from the column had a DP distribution of '2-12. The analysis of the hydrolyzates using a sugar analyzer (from Biotronik) showed mainly galactose (70%) on monosaccharides, in addition arabinose (23%) and traces of glucose and mannose. Overall, 8.3% of the products containing galacturonides were obtained as neutral sugar-containing oligosaccharides in the preliminary run .

Example 8:

Growth of colon carcinoma cell lines on extracellular matrix (ECM) in the presence of pectin hydrolyzate

The human colon carcinoma cell lines HT-29 and Caco-2 were in a cell density of 1 x 10⁴ Cells / ml sown on 15 mm petri dishes and in the medium RPMI 1640 + 10% fetal calf serum (FCS) (HT-29) or MEM + 10% FCS (Caco-2) at 37 ° C under a 5% CO₂ containing cultivated atmosphere. The cells were grown to confluence for 1 to 2 days. The dishes were then washed once with PBS, then incubated with PBS and 0.5% Triton X-100 for 30 min at room temperature on a shaker and then washed 3 times with PBS. The cell lines described above were then sown again on the dishes with the ECM layers prepared in this way. The influence of pectin hydrolyzate on cell growth was determined by measuring the cell number. For this purpose, the cells were detached again after 48 hours using trypsin / EDTA solution in HBSS (10 min) and washed in PBS solution. The number of living cells was then determined by staining with tryphan blue solution (650 mg tryphan blue in 400 ml 0.9% NaCl, 1: 1 (v / v)) in a Neubauer counting chamber. As a control, experiments with glucose were carried out. The results are shown in Table 1.

Table 1 shows that glucose had no influence on the growth of the HT 29 and Coco-2 cell lines, while pectin hydrolyzate reduced the growth of the cells depending on the concentration used by up to 75%.

Table 1

<img file="WO0242484A2_D0001.tif" />Example 9:

Decrease in the invasion capacity of Caco-2 cells by pectin hydrolyzate

The effect of pectin hydrolyzate on the invasion capacity of Caco-2 cells was examined using the invasion test described by Erkell and Schirrmacher (Cancer Research, 48 (1988), 6933-6937). The test is based on the migration of cells through the pores of a Nucleopore polycarbonate filter into a protein gel that contains various ECM proteins such as collagen types I and III, fibronectin and laminin, on a nitrocellulose filter. The cells were added to the test system together with the pectin hydrolyzate and then the cells that had migrated through the protein layer in the lower nitrocellulose layer were evaluated quantitatively. As a control, the effect of glucose on the invasion capacity of Caco-2 cells was examined.

The results of these tests are shown in Table 2. The results show that the pectin hydrolyzate according to the invention was able in some cases to significantly reduce the invasion capacity of Caco-2 cells depending on the concentration used, while glucose only caused a slight reduction in the invasion capacity of Caco-2 cells in higher concentrations. Table 2

<img file="WO0242484A2_D0002.tif" />

Example 10:

Anti-galectin-3 antibody binding by pectin hydrolyzate

Expression of galectin-3 on colon carcinoma cells was determined using an anti-galectin-3-specific monoclonal antibody (mouse Ig) and a corresponding anti-mouse FITC-coupled secondary antibody by means of immunofluorescence / flow cytometry methods ^' certainly. Increasing concentrations of the pectin hydrolyzate and of glucose as a control were incubated together with the primary antibody on the target cells and then the inhibitory influence of the soluble sugar substance on the anti-galectin-3 binding was measured. The influence of pectin hydrolyzate on the anti-galectin 3 binding is shown in Table 3. Table 3 shows that glucose does not, or only slightly, reduces the binding reaction of the monoclonal anti-galectin-3 antibody, while pectin hydrolyzate sometimes significantly reduces the binding of the antibody, depending on the concentration used.

Table 3

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