KR20110122389A - Fermented plant germ extract which inhibits the growth of cancer cells and method for preparing the same - Google Patents

Abstract

PURPOSE: A plant embryo fermentation extract and a food containing the same for health is provided to suppress the growth of cancer cells and to prevent and treat cancer. CONSTITUTION: A method for preparing plant embryo fermentation extract for suppressing cancer cells comprises: a step of inoculating bacteria or fungi into a plant embryo medium and fermenting; a step of extracting fermentation, filtering, and concentrating. The plant embryo medium is wheat, soybean, barley, brown rice, or mixture thereof. A strain for fermentation is Aspergillus oryzae, Aspergillus awamori, Aspergillus niger, Aspergillus kawachii, Rhizopus delemar, Bacillus subtilis, Bacillus subtilis natto, Bacillus licheniformis, or mixture thereof. A health food for suppressing cancer cell growth contains the plant embryo fermentation extract as an active ingredient.

Description

Plant embryo fermentation extract that inhibits the growth of cancer cells and a method of manufacturing the same {FERMENTED PLANT GERM EXTRACT WHICH INHIBITS THE GROWTH OF CANCER CELLS AND METHOD FOR PREPARING THE SAME}

The present invention relates to a plant embryo fermentation extract having cancer cell growth inhibitory activity and a method for producing the same.

Despite the development of modern medicine, cancer is still one of the most difficult diseases to treat, and cancer in Korea is increasing every year. Cancer forms abnormal tissues in organs, skeletal and skin tissues due to excessive proliferation of cytologically abnormal cells, and is characterized by infiltration and metastasis of surrounding tissues. Cancer causes 80-90% of environmental factors such as smoking, diet, air pollution, ultraviolet rays, radiation, and viruses (Lim, Hyo-Kwon et al . , 38 (2), 262 (2006)). . Current methods used to treat cancer include chemotherapy, radiation therapy, surgical operations, and the like. However, these treatment methods have a lot of problems due to side effects, so researches on chemical cancer preventive agents having few side effects are being actively conducted. Chemical cancer preventive agents are used to prevent and treat cancer using natural or synthetic compounds. However, the synthesized compounds have many problems than the natural product, it is carried out a lot of foods and prevention of cancer research component which inhibit or delay the occurrence of cancer in the natural products are for the current are natural products according to that known contain multiple [Park Geun-hyung et al., Korea Biotechnology and Bioengineering paper, 22 (3), 139 (2007), bangmyeonghui et al., Journal of Korea and nutrition, 39 (8), 756 (2006)].

 Prevention and treatment of cancer using natural products is possible because the active ingredient contained inhibits tumor growth and metastasis by activating the immune function. Immunotherapy for cancer includes non-specific methods using natural killer cells, lymphokine active killer cells, or macropharge, and specific methods using CTL cells (cytokine T lymphocytes). Nonspecific immune enhancers include mushroom polysaccharide lentinan, thymus thymosin, rice bran-soluble fiber arabinoxylane, and AHCC (active hexose correlated) isolated from cell wall fibers in mushroom mycelium. compound), flavonoids of plant components, and Avemar (trade name: Avemar) using fermented wheat germ extract.

Abe Mar (Avemar) is the supernatant was filtered, and then by mixing and fermenting yeast and wheat germ in an appropriate ratio to the dry product, U.S. Patent No. 6,355,474 No. and Articles [Mate Hidvegi et al., Cancer Biotherapy & Radiopharmaceuticals, 14 (4 , 278 (1999). Wheat germ contains 2-methoxy-ρ-benzoquinone (2-MBQ) and 2,6-dimethoxy-ρ-benzoquinone (2,6-DMBQ) in glycoside form, which are anticancer and antibacterial It is known to be active (Pething R. et al., Proc Natl Acad Sci USA , 82, 1439 (1985)).

On the other hand, the germ of the plant contains not only high-quality nutrients but also various bioactive substances, but is discarded as a by-product during milling or processing, or sold as a feed material rather than a food material. In recent years, many researchers have been interested in the bioactive substances contained in these plant embryos, which is expected to greatly recycle resources and use them as high value-added food materials.

Accordingly, the present inventors, while searching for a new functional material using the excellent physiological activity of the plant embryo, by confirming that the plant embryo fermentation extract extracted by inoculating and fermenting the fermentation strain in the plant embryo medium effectively inhibit the growth of various cancer cell lines The present invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing plant embryo fermentation extract for inhibiting cancer cell growth.

Another object of the present invention is to provide a plant embryo fermentation extract for cancer cell growth prepared by the above method.

Still another object of the present invention is to provide a health promoting food for inhibiting cancer cell growth, containing the plant embryo fermentation extract as an active ingredient.

In order to achieve the above object, the present invention comprises the steps of inoculating and fermenting strains for fermentation selected from the group consisting of bacteria and fungi in the plant embryo medium; And it provides a method for producing a plant embryo fermentation extract for inhibiting cancer cell growth, comprising the step of extracting and filtering the fermentation obtained in the above.

In order to achieve the above another object, the present invention provides a plant embryo fermentation extract for inhibiting cancer cell growth prepared by the above method.

In order to achieve the above another object, the present invention provides a plant embryo fermentation extract as an active ingredient, providing a health promoting food for inhibiting cancer cell growth.

Plant embryo fermented extract prepared in the present invention and health-promoting food containing the same can be usefully used for the prevention and treatment of cancer.

Figure 1 shows the manufacturing process of the plant embryo fermentation extract of the present invention,
2 to 6 are Abemar and cervical cancer cell line (HeLa) in the treatment of hepatic cancer cell line (HepG2), breast cancer cell line (MCF7), colon cancer cell line (HCT116), skin cancer cell line (B16) and cervical cancer cell line (HeLa), respectively. It shows the survival rate of the cancer cell line according to,
FIG. 7 is an HPLC chromatogram showing the result of 2,6-DMBQ analysis of the plant embryo fermentation prepared in Example 2. FIG.

Hereinafter, the present invention will be described in more detail.

The present invention comprises inoculating and fermenting a fermentation strain selected from the group consisting of bacteria and fungi in the plant embryo medium; And it provides a method for producing a plant embryo fermentation extract for inhibiting cancer cell growth, comprising the step of extracting and filtering the fermentation obtained in the above.

The production method of the present invention is characterized in that the fermented product obtained by inoculating a fermentation strain selected from the group consisting of bacteria and fungi in a plant embryo medium containing plant embryos and then fermenting is extracted and concentrated by filtration. The plant embryo fermentation extract thus prepared may be used as a raw material for health promoting foods to inhibit cancer cell growth by processing into plant embryo fermentation extract powder by drying and powdering in a conventional manner. The manufacturing and processing step can be preferably carried out according to the method shown in FIG.

Specifically, the production method of the present invention is mixed with 1 to 300% by weight of purified water in a plant embryo, and then sterilized at 60 to 130 ℃, preferably 121 ℃ 1 to 60 minutes, preferably 30 minutes Prepare fermented plant embryo medium. In this case, the plant embryo may be used alone or in a suitable ratio of the embryos of plant seeds such as wheat (wheat) embryo, soybean embryo, brown rice embryo, barley (macro) embryo, wheat embryo, or wheat embryo and soybean Mixtures of embryos are preferred.

Subsequently, the medium is inoculated with a strain for fermentation to 0.1 to 30% by weight, preferably 1 to 5% by weight, and it is 15 to 40 ° C, preferably 24 to 168 hours, preferably 48 to 48 ° C. Plant embryo fermentation is made by incubation and fermentation for 72 hours.

At this time, the strain for fermentation may be selected from the group consisting of bacteria and fungi, and specific examples of the fungus , Aspergillus orisae , Aspergillus awamori , Aspergillus niger ( Aspergillus niger) ), Aspergillus kawachii and Rhizopus delemar , Bacillus subtilis as bacteria, Bacillus subtilis natto and Bacillus licheniformis licheniformis ), of which Aspergillus oryzae is preferred.

Thereafter, the plant embryo fermented product of the present invention may be prepared by extracting the plant embryo fermented product prepared above using a conventional extraction method, for example, hot water extraction or an organic solvent and filtering. Specifically, the plant embryo fermentation prepared above is about 1 to 20 times the weight of water, an organic solvent (example C _1-4 alcohol) or a mixed solvent thereof, preferably 10 to 100% (v / v) Extracted with ethanol one to five times by extraction methods such as hot water extraction, cold sediment extraction, ultrasonic extraction or reflux cooling extraction for 10 hours to 100 ℃ extraction temperature, preferably at room temperature for about 1 hour to 10 days The extract is then filtered and concentrated under reduced pressure.

The plant embryo fermentation extract prepared above may be dried and powdered by conventional methods such as freeze drying or vacuum drying. At this time, the fermentation extract may be spray dried for more effective powdering.

The plant embryo fermentation extract prepared according to the present invention can ferment plant embryos with useful microorganisms to release physiologically active ingredients, and further increase the bioavailability by converting the components of the glycoside structure into nonglycoside forms. In addition, when fermented using the yeast fungus Aspergillus oryzae , the active ingredient is freed and enhanced as much as possible compared to the conventional Avemar fermented using yeast, liver cancer, breast cancer and colon cancer. It has been confirmed that the survival rate of skin cancer and cervical cancer cell lines can be effectively suppressed.

Therefore, the plant embryo fermentation extract prepared according to the method of the present invention and health-enhancing foods comprising the same as an active ingredient can be usefully used as food for preventing and treating various cancers.

The present invention also provides a health promoting food for inhibiting cancer cell growth, containing the plant embryo fermentation extract as an active ingredient.

The health promoting food of the present invention comprises the plant embryo fermentation extract in an amount of 0.1 to 100% by weight, preferably 20 to 80% by weight of the total composition.

The health promoting food of the present invention may be in the form of powder, granules, tablets, capsules or beverages, and may further include other natural or synthetic substances for health function, additives for commercialization, and the like.

Health drink of the present invention is the plant embryo fermentation extract powder 0.1 based on 100ml To 50 g, preferably 1 to 10 g. In addition, there is no particular limitation on the liquid component except for containing the extract as an essential ingredient in the ratio indicated, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like, and conventional sugars such as polysaccharides such as dextrin, cyclodextrin, And sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the health-promoting food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. Others may contain pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination.

Health-promoting foods to which the fermented extract of the present invention may be added include, for example, various foods, beverages, gums, vitamin complexes, health supplements, and the like.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Reference Example: Culture of Fermented Strains

Aspergillus oryzae (KCTC 6377, Gene Bank of Korea Biotechnology Center, Korea Research Institute of Bioscience and Biotechnology) was used as a strain for fermenting plant embryos, and MEB (malt extract broth) medium was used as a culture medium for strain culture. The medium was added to purified water, mixed, and sterilized at 121 ° C. for 15 minutes.

One colony was taken from the plate medium in which the fermented strains were cultured, inoculated into the sterilized MEB medium and shaken at 30 ° C. for 24 hours to obtain a seed culture, and then the culture solution was inoculated into the MEB medium to 3% by volume. Strain culture was obtained by shaking culture for 24 hours.

Example 1 Preparation of Wheat Germ Fermentation

20 wt% of purified water was added to the wheat germ, mixed and left to stand at room temperature for 4 hours, and then, sterilized in a sterilized container for 30 minutes at 121 ℃ to prepare a medium. After inoculating the culture medium prepared in the reference example in the amount of 5% by weight, and cultured at 24 ℃ for 72 hours to prepare a wheat germ fermentation.

Example 2: Preparation of Plant Embryonic Fermentation

A plant embryo fermented product was prepared in the same manner as in Example 1, except that wheat embryos and wheat soybean embryos were mixed and used instead of wheat embryos.

Example 3: Wheat Germ Fermentation Extract Preparation

1 kg of wheat germ fermented product prepared in Example 1 and 9 kg of 70% (v / v) ethanol were added to a stainless steel extraction container, and then extracted at room temperature for 10 hours using a stirrer. When the extraction process was completed, filtered, concentrated using a vacuum rotary concentrator to prepare an extract concentrate, and freeze-dried to prepare a wheat germ fermentation extract.

Example 4: Preparation of plant embryo fermentation extract

A plant embryo fermentation extract was prepared in the same manner as in Example 3, except that the plant embryo fermentation product prepared in Example 2 was used instead of the wheat germ fermentation product.

Example 5: Plant Embryonic Fermentation Extract Powder Preparation

Before freeze-drying the extract concentrate prepared in Example 4, 15% by weight of dextrin (product name: Max 1000, obtained by Matsutani Co., Ltd.) was mixed based on the solid content contained in the extract concentrate. It was then spray-dried to prepare plant embryo fermented extract powder.

Preparation Example: Preparation of health promoting food containing plant embryo fermentation extract

The plant embryo fermentation extract powder prepared in Example 5 was used as a main raw material to prepare health promoting foods in the form of tablets, capsules, concentrates, and health functional drinks.

1) Preparation of Tablets

60 wt% of the plant embryo fermentation extract powder prepared in Example 5 based on the total weight of the tablet, an excipient such as lactose, crystalline cellulose, white sugar, magnesium stearate, and the like, according to a conventional tablet preparation method Tablets were prepared after mixing, tableting and coating.

2) Preparation of Capsule

Using the appropriate amount of excipients, such as 60% by weight of the plant embryo fermentation extract powder prepared in Example 5, lactose, crystalline cellulose, white sugar, magnesium stearate and the like, according to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules. To prepare a capsule.

3) Preparation of concentrate

1000 mg of plant embryo fermented extract powder prepared in Example 5, 600 mg of oligosaccharide, 50 mg of mixed vitamins and 30 ml of strawberry flavor were added thereto, followed by mixing the above components according to a conventional method for preparing a liquid solution. Filled with polyethylene foam and sterilized to prepare a concentrate.

4) Manufacture of health functional drinks

600 mg of plant embryo fermented extract powder prepared in Example 5, 100 g of oligosaccharide, 1000 mg of citric acid, 1 g of taurine, 50 mg of mixed vitamin, and 2 g of plum concentrate were added and mixed, and mixed for about 1 hour. After stirring and heating at ℃, the resulting solution was collected by filtration in a sterilized 2ml container, sealed sterilization and refrigerated.

Test Example 1: Measurement of survival rate of cancer cell line using MTT assay

The growth inhibitory effect (cancer cell line survival rate) on the cancer cell lines of the wheat germ fermentation extract and the plant embryo fermentation extract prepared in Examples 3 and 4 were confirmed by MTT analysis.

The MTT assay was performed by MTT ((3- (4,5-dimethylthiazol-2-yl) -2, a yellow tetrazolium) by mitochondrial dehydorgenase enzyme, which occurs during metabolism of living cells. By the method of converting 5-ditenyltetrazolium bromide)) into insoluble purple formazan, the viability of living cells can be measured by the concentration of purple formazan. Specifically, the cancer cell line of the appropriate concentration was pre-cultured for 24 hours, and after adding a proper amount of Avemar as a sample and a comparative group of the present invention and incubating for 24 hours, 5 mg / mL MTT reagent was added to each well 1 Time incubated further. After removing the medium, each well was washed twice with 1 mL of phosphate saline (PBS, pH 7.4). Subsequently, 400 μL of DMSO was added to each well to dissolve the insoluble formazan produced above, and then 200 μL was transferred to a 96-well clear plate, and the absorbance was measured at 570 nm using an ELISA reader. In the case of the control group, the same experiment was performed without adding a sample. Cell viability of cancer cell lines was calculated using the following equation, and specific experimental methods for each cancer cell line are described in Test Examples 1-1 to 1-5 below.

[ Equation ]

Test Example  1-1: liver cancer cell line ( HepG2 Growth inhibitory effect on

Liver cancer cell line (HepG2) was preincubated for 24 hours to be 5 × 10 ⁵ cells / well in 12-well plates. 2 mg / mL of Avemar (Biromedicina, Hungary; comparative) and 1 mg / mL of the fermented extracts of Examples 3 and 4 were treated with 10 μL of each well and incubated for 24 hours. It was. At this time, since Avemar contained 50% of the excipient contained in itself, 2 mg / ml was used to compare the sample of the present invention under the same concentration. Then, 5 mg / mL of MTT reagent (Sigma, USA) was added to each well 12 μL and incubated for 1 hour. After removing the medium, each well was washed twice with 1 mL of phosphate saline (PBS, pH 7.4). 400 μL of DMSO was added to each well to dissolve insoluble formazan, and then 200 μL was transferred to a 96-well clear plate and the absorbance was measured at 570 nm with an ELISA reader.

Survival rate of cancer cell lines showing growth inhibitory effect on hepatic cancer cell line (HepG2) was calculated using the above equation, and the results of the comparative group and Examples 3 and 4 are shown graphically in FIG. 2, the comparative group and Example 3 and The results of 4 are shown in Table 1.

Test Example 1-2: Growth Inhibitory Effect on Colorectal Cancer Cell Line (HCT116)

Colon cancer cell line (HCT116) was pre-incubated for 24 hours to be 5 × 10 ⁵ cells / well in a 12-well plate and subjected to MTT analysis by treating the sample under the same conditions as in Test Example 1-1.

Survival rate of cancer cell lines showing growth inhibitory effect on colorectal cancer cell line (HCT116) was calculated using the above equation and the results of Comparative Group and Examples 3 and 4 are shown graphically in Figure 3, Comparative Group and Example 3 and The results of 4 are shown in Table 1.

Test Example 1-3: Growth Inhibitory Effect on Breast Cancer Cell Line (MCF-7)

The breast cancer cell line (MCF-7) was pre-incubated for 24 hours to be 5 × 10 ⁵ cells / well in a 12-well plate and subjected to MTT analysis by treating the sample under the same conditions as in Test Example 1-1.

Survival rate of cancer cell line showing growth inhibitory effect on breast cancer cell line (MCF-7) was calculated using the above equation and the results of the comparative group and Examples 3 and 4 are shown in the graph in Figure 4, Comparative group and Example 3 And the results of Table 4 are shown in Table 1.

Test Example 1-4: Growth Inhibitory Effect on Skin Cancer Cell Line (B16)

The skin cancer cell line (B16) was pre-incubated for 24 hours to be 5 × 10 ⁴ cells / well in a 12-well plate, and the samples were treated under the same conditions as those of Test Example 1-1 to perform MTT analysis.

Survival rate of the cancer cell line showing the growth inhibitory effect on skin cancer cell line (B16) was calculated using the above equation and the results of the comparative group and Examples 3 and 4 are shown graphically in Figure 5, the comparative group and Examples 3 and 4 The results are shown in Table 1.

Test Example 1-5: Growth Inhibitory Effect on Cervical Cancer Cell Line (HeLa)

Cervical cancer cell line (HeLa) was pre-incubated for 24 hours in a 6-well plate at 1.5 × 10 ⁵ cells / well, and the samples were treated under the same conditions as in Test Example 1-1 to perform MTT analysis.

Survival rate of cancer cell lines showing growth inhibitory effect on cervical cancer cell line (HeLa) was calculated using the above equation and the results of the comparative group and Examples 3 and 4 are shown graphically in Figure 6, the comparative group and Example 3 and The results of 4 are shown in Table 1.

As shown in Table 1, when the plant embryo fermentation extract of the present invention is treated to cancer cell lines, it showed up to 10 times better cancer cell growth inhibitory effect than Avemar. In particular, the plant embryo fermentation extract prepared by mixing wheat embryos and soybean embryos of Example 4 inhibited the growth of female cancer cell lines breast cancer (MCF7), cervical cancer (HeLa) and skin cancer (B16).

Test Example 2: Analysis of 2,6-DMBQ content of plant embryo fermentation

The content of 2,6-dimethyl-p-benzoquinone (2,6-DMBQ) of the plant embryo fermentation product prepared in Example 2 was analyzed under the following conditions using HPLC. Indicated in:

<Analysis condition>

Detector-UV detector (275 nm),

Column-Spelco alpi-amide (SUPELCO, RP-AMIDE) C16 (4.0 × 250 mm),

Flow rate-0.70 ml / min,

Temperature-70 ℃,

Injection volume-20 μl,

Travel time- 20 minutes;

Mobile phase-0.025 M potassium dihydrogen phosphate solution: acetonitrile = 90: 10 (V / V).

As shown in FIG. 7, 2,6-DMBQ was not detected in the plant embryo itself, whereas 0.033 mg per gram (g) of the sample was detected in the plant embryo fermentation product of the present invention. It can be confirmed that it is advantageous.

Test Example 3: Analysis of isoflavone content of plant embryo fermentation

The isoflavone content of the plant embryo fermentation prepared in Example 2 was analyzed using HPLC under the following conditions:

Detector-UV detector (254 nm),

Column- ODS 80-TM, TOSOH (5.5 × 150 mm),

Flow rate-1.0 ml / min,

Temperature-225 ℃,

Injection volume-20 μl,

Elution time-45 minutes,

Eluent-A solution acetonitrile, B solution 0.02% phosphoric acid (V / V)

Concentration gradient

0-15 minutes: A solution 10-19%

15-28 minutes: A solution 19-32%

28-38 minutes: A solution 32-55%

As a result of HPLC analysis, some of the isoflavones were detected due to the soybean embryos contained in the fermented plant embryos. It was confirmed that the conversion.

Claims (8)

Inoculating and fermenting a fermentation strain selected from the group consisting of bacteria and fungi on the plant embryo medium; And extracting and filtering and concentrating the fermented product obtained as described above. The method of claim 1,
The plant embryo medium is selected from the group consisting of wheat (wheat) embryos, soybean embryos, brown rice embryos, barley (macro) embryos and mixtures thereof. The method of claim 1,
The plant embryo medium is prepared by sterilizing after mixing 1 to 300% by weight of purified water into the plant embryo. The method of claim 1,
The strains for fermentation are Aspergillus oryzae , Aspergillus awamori , Aspergillus niger , Aspergillus kawachii , Aspergillus kawachii , Rhizopus delemar ), Bacillus subtilis , Bacillus subtilis natto , Bacillus licheniformis , and a mixed strain thereof. The method of claim 4, wherein
The fermentation strain is a production method, characterized in that the Aspergillus oliase ( Aspergillus oryzae ). The method of claim 1,
The fermentation is obtained by adding a fermentation strain of 0.1 to 30% by weight to the medium and fermentation at 15 to 40 ℃ for 24 to 168 hours. Plant embryo fermentation extract for inhibiting cancer cell growth prepared by the method of any one of claims 1 to 6. A health promoting food for inhibiting cancer cell growth comprising the plant embryo fermentation extract of claim 7 as an active ingredient.

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