KR100554346B1 - A new anti-cancer polysaccharide from the genus bifidobacterium - Google Patents

Abstract

The present invention is an anticancer active polysaccharide having a molecular weight of 1.4 million to 1.5 million Daltons isolated from Bifidobacteria known as beneficial bacteria among human and animal intestinal bacteria, a method for separating and purifying the same, and a functional food and pharmaceutical composition comprising the polysaccharide. It is about. Specifically, the method for separating and purifying anticancer active polysaccharides including homogenization heat treatment, phenol extraction, and precipitation using ethanol for polysaccharides showing anticancer activity derived from Bifidobacterium bifiderm BGN4, and various polysaccharides as an active ingredient. It relates to food ingredients or pharmaceutical preparations.

Anticancer activity, Bifidobacterium, polysaccharide

Description

A novel anti-cancer polysaccharide derived from Bifidobacterium {A NEW ANTI-CANCER POLYSACCHARIDE FROM THE GENUS BIFIDOBACTERIUM}

Figure 1 is a graph comparing the anticancer activity between the bifidus bacteria derived from the human body.

2 is a graph comparing the titers of anticancer active polysaccharides derived from BGN4 against various cancer cell lines.

Figure 3 shows the results of identification of anticancer activity bifidus BGN4 by 16S rRNA sequencing.

Figure 4 shows the process of separating and purifying the anticancer active polysaccharides in Bifidobacterium.

Figure 5 shows the GPC results for determining the molecular weight of the anti-cancer active polysaccharide derived from Bifidobacterium.

Figure 6 shows the results of BIO-LC analysis (using PA1 column) to analyze the components of the anti-cancer active polysaccharide derived from Bifidobacterium.

FIG. 7 shows another analysis result of BIO-LC (using MA1 column) for analyzing the components of the anti-cancer active polysaccharide derived from Bifidobacterium.

FIG. 8 shows a method for determining binding between constituent sugars of an anti-cancer active polysaccharide derived from Bifidobacterium.

9 is a TCL result analyzing the binding between the constituent sugar of the anticancer active polysaccharide.

10 shows anticancer activity against colorectal cancer cell lines by anticancer active polysaccharides.

The present invention relates to anticancer active polysaccharides isolated from Bifidobacteria known as beneficial bacteria among human and animal intestinal bacteria, separation methods thereof, and food and pharmaceutical compositions comprising the same. More specifically, in the Bifidobacterium bifiderm BGN4 strain, a method for preparing the polysaccharide and separating the polysaccharide by homogenization heat treatment, phenol extraction and precipitation using ethanol, and the like, including the polysaccharide as an active ingredient It relates to food and pharmaceutical compositions.

Bifidobacterium was first discovered by Tissier as a microorganism that lives in the intestines of humans, animals and insects. Bifidobacterium has the characteristics of gram positive, absolute anaerobic, GC-rich, etc. and is found in the intestinal dominant flora of the mother's infant. Bifidobacteria is a family of Actinomycetes different from the well-known lactobacillus lactobacilli , lactococci , etc., and Corynebacteria , Streptomyces , and Mycobacteria. ( Mycobacteria ) is located close to the taxonomy. It also produces lactic acid: acetic acid in a 3: 2 ratio as a metabolite as a lactic acid-producing strain, and plays a role in preventing foreign invading pathogens by adjusting pH. These properties are known to play a key role in maintaining the balance of normal intestinal flora. Ingesting milk fermented with Bifidobacterium has the effect of alleviating lactose intolerance. Recent studies have revealed useful functions such as lowering cholesterol, enhancing immune function, and anticancer activity. In addition, the adsorption power to the intestinal mucosa is excellent, and there is increasing interest in the world, and the heat of development for dairy products using Bifidobacterium is increasing.

Until now, the research direction of Bifidobacterium has been to identify and count strains, develop screening methods, screen for strains that secrete acid, bile, and bacteriocins, analyze their own biochemical properties, and plasmids derived from Bifidobacterium. Analysis, cloning and biochemical analysis of characteristic enzymes are the mainstream.

There are many reports on the anticancer effects of lactic acid bacteria and yogurt, but few reports on the anticancer effects of polysaccharides. Oda et al. (1983) found that Lactobacillus helvetticus polysaccharides were composed of glucose and galactose (2: 1), and 20 mg / kg and 80 mg / kg polysaccharides were intraperitoneally administered to mice that developed S-180 ascites cancer. 144% and 233% lifespan prolonged, respectively, and the polysaccharide reported no cytotoxicity in vitro. Kitazawa et al. (1990) conducted anticancer experiments using viscous fermented milks consumed in Finland and Iceland, and reported that the polysaccharides consisted of glucose, galactose, and rhamnose. The mechanism of the anticancer effect by such lactic acid bacteria is not clear, but it is reported that it is host mediated and due to the activity of the cellular immune system. As described above, some anti-cancer effects by lactic acid bacteria and bifidus cells have been reported, but specific anticancer activity by polysaccharides of bifidus and its specific substance have not been reported.

The present invention is to provide an anticancer active polysaccharide derived from Bifido bacterium Bifidum BGN4.

Another object of the present invention is to provide a method for separating and purifying the anticancer active polysaccharides from bifidus.

Still another object of the present invention is to provide a functional food and pharmaceutical composition comprising the anticancer active polysaccharide.

In order to achieve the above object, the present invention measured anticancer activity against colon cancer cells in order to isolate the bifidus strain from the stool and to isolate a strain of high anticancer activity. As a result, Bifido bacterium Bifidum BGN4, a strain having high anticancer activity, was isolated.

The bacteriological characteristics of Bifidobacterium bifiderm BGN4 isolated from the present invention are shown in Table 1 below.

Characteristics of Bifidobacterium Bifidum BGN4 shape Club or Y Gram dyeing + F6PPK +

To isolate and purify anti-cancer polysaccharides from this strain, the optimal medium was 50 g / L lactose, 10 g / L bee extract, 10 g / L peptone, 5 g / L yeast extract, 7 g / L sodium acetate, 2 g / L ammonium citrate, 2 g / L disodium phosphate, 1 g / L tween 80, 0.2 g / L magnesium sulfate, 0.5 g / L L-cysteine It is preferable to include. More preferably, the medium is maintained at pH 5.0 at 37 ° C. and immobilized with calcium carbonate and 2 g / L of phytic acid and 1 g / L of vitamin C.

Polysaccharide separation was obtained by cell culture at 35-40 ° C., centrifuged, homogenized after cell crushing, cell extracts were obtained once again, centrifuged, heat treated at about 80 ° C., and then precipitated through another centrifugation. The supernatant is removed to obtain a supernatant, and the supernatant is phenol extracted to remove the protein remaining in the supernatant. Finally, the anticancer polysaccharide is obtained through ethanol precipitation.

The molecular weight of the anticancer active polysaccharide was in the range of 1.4-1.6 million daltons, and each component was analyzed by hydrolysis.

These polysaccharides were confirmed to have anti-cancer activity as a result of the experiment, the pharmaceutical composition comprising them may include pharmaceutically acceptable excipients, carriers, diluents, and the like, tablets, pills, dispersants, granules, elixirs, suspensions , Emulsifiers, solutions, syrups and the like. It can also be administered orally or parenterally, and the daily dosage ranges from 1 to 20 mg / kg body weight.

Hereinafter, the invention will be described in more detail with reference to Examples.

Example 1

Isolation of Bifidus from Stool

After collecting the stool of the volunteer, the stool was diluted 10 times with sterile physiological saline, and then, as described in Korean Patent No. 135780, TP medium (trypticase), which is a selective medium of pH 7.0. ) 10 g, proteose peptone No. 3 (3.5 g), ammonium sulfate 3 g, KH ₂ PO ₄ 2 g, K ₂ HPO ₄ 1 g, cysteine-HCl (L-cystein-HCl) 0.5 g of H ₂ O, 0.2 g of MgSO ₄ , 15 g of agar, 50 ml of TOS solution (transgalactoologosaccharide) and 50 ml of 30% sodium propionate solution (prepared with distilled water to make 1 L total) And incubated. Subsequently, single colonies generated in TP medium were taken to investigate morphological characteristics under a microscope, and gram staining and F6PPK (fructose 6 phosphate phosphokeolase) tests were performed to confirm that they were bifidus. BHI medium or MRS medium was used for culturing and storing the isolated bifidus strains.

Example 2

Isolation and Identification of Bifidus Bacteria with High Anticancer Activity

In order to search for high anticancer activity among the bifidus strains isolated in Example 1, anticancer activity against the colorectal cancer cell line was measured. That is, cancer cell lines HT-29, HCT116, K-ras-transformed NIH3T3, Chang, NIH3T3, HeLa, etc. were inoculated in DMEM or RPMI1640 medium with 10% FBS and incubated at 37 ° C in an incubator filled with 5% CO ₂ . And the anticancer activity was measured by the MTT method which is generally performed in a laboratory by adding the cell lysate.

The results are as shown in Figure 1 BGN4 strain (Professor Ji Geun Eok, Department of Food and Nutrition, Seoul National University; Reference Journal of Food Protection, Vol. 63, (2000) 1133-1136 / International Journal of Food Microbiology Vol. 46 (1999) 231-241.) Was found to have the best anticancer activity. For culturing BGN4 strain, BHI medium or MRS medium was used. As shown in FIG. 2, the anticancer activity of BGN4 against various cell lines showed strong proliferation inhibitory effect in HT-29 (colon cancer cell line) and oncogenic ras-transformed cells. The BGN4 strain was identified as Bifidobacterium bifidum BGN4 through 16S rRNA sequencing (see FIG. 3).

Example 3

Isolation and Purification of Polysaccharides with Anticancer Activity of Bifidobacterium Bifidum BGN4

In this strain, the composition of the optimum medium for producing anticancer active polysaccharides was completed. The optimum composition of the medium was 50 g / L lactose, 10 g / L bee extract, 10 g / L peptone, 5 g / L yeast extract, 7 g / L sodium acetate, 2 g / L Ammonium citrate, 2 g / L disodium phosphate, 1 g / L tween 80, 0.2 g / L magnesium sulfate, 0.5 g / L L-cysteine, maintained at pH 5.0 at 37 ° C. and immobilized Calcium carbonate was added and 2 g / L phytic acid and 1 g / L of vitamin C were added. At this time, the anticancer active polysaccharide of 60 mg / L could be produced. The method for separating and purifying the anticancer active polysaccharide is as shown in FIG. 4.

Example 4

Molecular weight confirmation of anticancer active polysaccharides

The molecular weight of the polysaccharide obtained by cell wall extraction of Bifidobacterium Bifidum BGN4 was confirmed using GPC (Agilent, USA). Dextran (molecular weight (dalton); 5,200, 48,500, 273,000, 830,000, Fluka) was used as a standard sample, and the molecular weight size was compared by adding dextran (molecular weight 830,000 daltons) having the largest molecular weight in the sample. As a result, as shown in FIG. 5, the molecular weight of the anticancer polysaccharide was estimated to about 1.5 million.

At this time, the analysis conditions are as follows.

Column: PL aquagel-OH Mixed, 7.5 ㅧ 300mm, 8㎛

Column Temperature: 25 ℃

Flow rate: ml / min

Mobile phase: water

Detector: Refractive Index Detector

Correction: dextran standard (purchased from Fluka)

Example 5

Identification of Components of Anticancer Active Polysaccharides

In order to identify the constituent sugars of the anticancer active polysaccharides extracted by the above-described method from the cell wall of Bifidobacterium Bifidum BGN4, hydrolysis was performed using TFA and analyzed by Bio-LC. That is, 1 mg of 500 mM TFA (Trifluroacetic acid) was added to 10 mg of anticancer active polysaccharide, and then reacted for 30 minutes at room temperature, and then decomposed for 150 minutes at 100 ° C. and analyzed by Bio-LC.

As standards, myo-inositol, rhamnose, glucose, galactose, ribose, fructose, adonitol (ribitol), glycerol, mannose, arabinose, fucose, xylose, sorbose, tagatose, inositol, mannitol, sorbitol, dulcitol, xylitol, allose, (others Disaccharides: sucrose, lactose, melibiose, trisaccharides and raffinose) were used, and the analysis conditions of each PA1 column and MA1 column are shown in Table 2.

column PA1 column MA1 column Condition Separation liquid-200 mM NaOH Flow rate-1 ml / min Pressure-1550-1580 bar Separation liquid-600 mM NaOH, flow rate-0.4 ml / min, pressure-1440 ~ 1460 Standard material Myo-inositol, ribose, rhamnose, glucose, galactose Myo-inositol, ribitol, rhamnose, glucose, galactose

Table 3 shows Bio-LC results of polysaccharides derived from BGN4 using a PA1 column, and Table 4 shows Bio-LC results of polysaccharides derived from BGN4 using a MAI column. Corresponding graphs are FIGS. 6 and 7.

Peak No. ComponentName RetentionTime AreanC * min HeightnC Amount% RelativeAmount% One myo-inositol 1.37 68.156 1062.328 0.0041 26.32 2 rhamnose 2.47 12.498 137.168 0.0014 4.83 3 glucose, galactose 3.63 115.807 739.041 0.0031 44.72 4 Ribose 4.30 62.499 445.558 0.0036 24.13

Peak No. ComponentName RetentionTime AreanC * min HeightnC Amount% RelativeAmount% One N.A 7.83 144.53 597.54 N.A 26.32 2 rhamnose 12.30 14.12 35.76 0.0010 3.87 3 glucose 20.67 114.76 175.21 0.0043 31.52 4 galactose 22.97 39.94 45.79 0.0018 10.97 5 ribose 26.73 86.47 103.84 0.0721 23.76

Example 6

Analysis of binding between constituent sugars of anticancer active polysaccharide

Methylation was performed to confirm the binding between the constituent sugars of the anticancer polysaccharide. The method is shown in FIG.

As a result, GC / MS did not match the database due to the diversity of components and the variety of coupling structures. In addition, TLC results (conditions were developed solvent: Hexane: Ethyl Acetate = 5: 2), as shown in Figure 9, it was detected in a number of locations was estimated to be a novel material formed of various components and structures.

Example 7

Antitumor Activity Animal Experiment Using BGN4

The anticancer activity confirmed in vitro was confirmed in vivo using experimental animals. In other words, soymilk (soybean soymilk) was used as a medium, and BGN4 was incubated therein, and lyophilized powder was administered to cancer-induced mice to compare the number and size of cancer cells. As a result, it was confirmed that the number and size of cancer cells is significantly reduced compared to the control group, which is shown in Table 5.

Example 8

Preparation of Health Functional Food Using Bifidobacterium Bpi4

It was recovered by culturing in the BGN4 production medium and lyophilized BGN4 powder was prepared. The BGN4 powder was mixed with functional food materials to prepare a formal product with the composition of Table 6.

ingredient content Bifidus Raw (BGN4) Lactic Acid Bacteria Raw Starch Fructooligosaccharide Yogurt Powder Total 4% 1% 56% 30% 9% 100%

Since the bifidus bacteria according to the present invention are present in the large intestine, a small amount of Lactobacillus acidophilus powder, a lactobacillus known to exist in the small intestine, is added to balance the small intestine and increase the intestinal effect. Manufactured suit product and can contain Bifidobacterium bipyridinium bonus BGN4 about 2 × 10 ⁹ / g according to the present invention, a filler is also seuneun contained 2 × 10 ⁸ / g Lactobacillus know.

Example 9

Anticancer Activity of Bifidobacterium Bifi4 BGN4 Activity of Polysaccharides against Colon Cancer Cell Line

Separation and purification of polysaccharides having anticancer activity of Bifidobacterium Bifidum BGN4 was carried out by the method shown in Example 3. Colorectal cancer cell lines for analyzing anticancer activity were used HT29 and HCT116 and the results are shown in FIG.

As described above, it was confirmed that the novel polysaccharides produced by the known Bifidobacterium bifidum BGN4 exhibited excellent anticancer activity. Food and functional fermented milk can be manufactured.

In addition, if only the anticancer active polysaccharides produced in this strain is purified and developed as a raw material of a new anticancer agent may be used as a medicine and pharmaceutical raw materials.

Claims (8)

delete delete Bifidobacterium bifiderum BGN4 was cultured and centrifuged, followed by cell crushing and homogenization. Cell extracts were obtained and centrifuged. And a step of removing the protein remaining in the supernatant by phenol extraction of the supernatant, followed by ethanol precipitation reaction. The method of claim 3, wherein the growth medium of Bifidobacterium Bifi4 BGN4 is 50 g / L lactose, 10 g / L bee extract, 10 g / L peptone, 5 g / L yeast extract, 7 g / L sodium acetate, 2 g / L ammonium citrate, 2 g / L disodium phosphate, 1 g / L tween 80, 0.2 g / L magnesium sulfate, 0.5 g / L L-cysteine Method for producing an anticancer active polysaccharide, characterized in that it comprises a. 5. The anticancer active polysaccharide according to claim 4, wherein the medium has a temperature of 37 DEG C, a pH of 5.0, and further comprises immobilized calcium carbonate, 2 g / L phytic acid and 1 g / L of vitamin C. Manufacturing method. delete delete delete

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