KR101980624B1 - Composition for preventing, ameliorating or treating prostate cancer comprising fermented silkworm extract as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating prostate cancer, which contains a fermented silkworm extract as an active component. A silkworm extract fermented by using Bacillus subtilis enhances an antioxidant effect compared to before fermentation and since the fermented silkworm extract, especially the fermented silkworm extract of a Daeseongjam variety has an excellent effect of inhibiting proliferation of prostate cancer cells, the composition of the present invention can be effectively used as a therapeutic agent for prostate cancer or a health functional food for alleviating prostate cancer.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, ameliorating or treating prostate cancer comprising fermented silkworm extract as an active ingredient.

The present invention relates to a composition for preventing, improving or treating prostate cancer, which comprises, as an active ingredient, a silkworm extract fermented with Bacillus subtilis.

Prostate cancer is one of the most common malignant diseases in men, with 346,000 patients in Europe reporting in 2006 and 87,000 deaths. This is the second most common cancer-diagnosed cancer in the United States and the second leading cause of cancer-related mortality in men. In addition, the incidence of prostate cancer is increasing in Korea, and it has been reported that the incidence rate is 5th among all kinds of cancer, and the rate of increase is 1.

Prostate cancer can be detected early due to the increased sensitivity of prostate specific antigen (PSA) monitoring assay, but PSA is not produced only in prostate cancer cells but is also produced in normal prostate cells. Therefore, prostate cancer, acute urinary retention, The numerical value can also be increased by massage or the like.

In addition to surgical therapy, external radiation therapy and androgen-insemination (also called hormone ablation) are the treatment of prostate cancer. However, both external radiotherapy and androgen-insemination have been associated with a number of adverse effects (increased risk of cardiovascular disease, increased risk of osteoporosis, weight gain, decreased brain cognition, reduced incidence of urethral stricture, loss of sexual desire and infertility, reduced risk of skeletal calcium salt associated with osteoporosis, ). ≪ / RTI > In addition, the androgen block may cause early and early development of androgen-independent neoplastic clones, which may further accelerate the progression of long-term tumors. Therefore, there is a growing need for the development of a new prostate cancer treatment using natural products, which are known to have few side effects and are safe.

On the other hand, the importance of insects as new biological resources has recently been used in various fields. The domestic insect industry started inspecting insect resources and finding insect insects through the establishment of 'the first insect industry five-year plan (2011-2015)' from the announcement of 'Insect Industry Industry Promotion and Support Act' in 2010 , Insecticide, functional biomaterials, industrial new materials, biomimetic engineering, etc., by strengthening the R & D of insect resources, supporting the cultivation of insect farmers, It is expected that the use of insect resources will be greatly expanded into the fusion and complex area of

Among them, silkworm ( Bombyx mori L.) is a larval larva of Bombycidae. It is also recorded in the records of Dongbokgwon and Bonchogangmok. It has been used as a folk remedy for malnutrition, pulmonary tuberculosis, encephalopathy, rhinorrhea, and stroke. In the past, it has mainly been used only in the sleeping industry, but recently it has been treated as a cosmetic, food, and medical material due to the development of functional food market and biotechnology. In addition, silkworms have high nutritional value due to abundance of proteins, free amino acids, monounsaturated fatty acids or polyunsaturated fatty acids, minerals, fibers and deoxynojirimycin, and they are effective in inhibiting active oxygen, improving hyperlipidemia, Prevention, and hypoglycemia have been reported.

On the other hand, fermentation is a process in which microorganisms, especially useful microorganisms such as probiotics, produce alcohols, organic acids, and CO ₂ as products using saccharides. The fermentation of foods leads to destruction of toxic substances, enhancement of digestibility, And other various physiologically active substances, as well as hydrolytic enzymes and other enzymes that decompose carbohydrates, proteins, lipids, etc., thereby producing nutritional value and enhancing health functionalities. Popularity is getting higher and higher. Therefore, we intend to further improve the functionality by fermenting various silkworm silkworms.

Korean Patent No. 1246266 discloses a method for producing fermented silkworm powder and a composition for prevention or treatment of hyperlipemia and fatty liver including fermented silkworm powder. Korean Patent Publication No. 2015-0083604 discloses a composition for preventing and treating prostate cancer A composition for preventing, improving or treating prostate cancer containing the fermented silkworm extract of the present invention as an active ingredient has not been disclosed.

The present invention provides a composition for preventing, improving or treating prostate cancer, which comprises an extract of fermented silkworm cocoa fermented by using Bacillus subtilis as an active ingredient, Promoted and inhibited proliferation of prostate cancer cells, thereby completing the present invention.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating prostate cancer, which comprises a fermented silkworm extract as an active ingredient.

The present invention also provides a health functional food composition for preventing or ameliorating prostate cancer, which comprises a fermented silkworm extract as an active ingredient.

The present invention relates to a composition for preventing, ameliorating or treating prostate cancer, which contains fermented silkworm extract as an active ingredient. The extract of Bacillus subtilis fermented silkworm silkworm, especially the fermented extract of Daesung junhoe, does not affect the survival of skin cancer and colon cancer cells , And the effect of inhibiting the proliferation of prostate cancer cells was excellent. Therefore, the therapeutic agent for prostate cancer; Or as a material for a health functional food for preventing or ameliorating prostate cancer.

Fig. 1 shows the results of confirming the antioxidative effect of the silkworm extract and the fermented silkworm extract of the present invention. BW (Butylated hydroxytoluene) is a positive control, BU is an extract of Bauanthus quinquinone, Quercus mongolica extract of Namwon, Bacillus subtilis silkworm extract, BBO is an extract of silkworm mung bean fermented silkworm. The letters a, b, c, d and e in the figure indicate that there is a significant difference between them, and p < 0.05.
2 is a graph showing the effect of the silkworm extract and fermented silkworm extract of the present invention on B16-F10 cells (skin cancer cell line), HT-29 cells (colorectal cancer cell line), LNcaP cells (prostate cancer cell line) and MCF- Which is the result of confirming the effect of inhibiting proliferation. Con is a non-treated control group, BU is an extract of Bauanthus quinquinones, NW is Namwonan mugwort extract, Bok is mulberry extract, BBu is bauanic acid mugwort fermented silkworm extract, The silkworm extract of silkworm, BBO, is an extract of silkworm mungbean fermented silkworm.

The present invention relates to a pharmaceutical composition for preventing or treating prostate cancer containing an extract of fermented silkworm as an active ingredient.

In one embodiment, the fermentation silkworms silkworm powder Bacillus subtilis (Bacillus subtilis), and the mixture to be preferred, and even more preferably silkworm powder was fermented at 35 ~ 39 ℃ for 24-72 hours Bacillus subtilis (Bacillus subtilis ) and fermented at 37 DEG C for 48 hours, but the present invention is not limited thereto.

The silkworm varieties are preferably mature, but are not limited thereto.

Daesung junnu is produced in Boeun, Chungcheongbuk-do. It consumes mulberry leaves produced in Boeun area in Chungbuk, but it is not limited to this.

The fermented silkworm extract of the present invention is preferably extracted using water, a C ₁ to C ₄ lower alcohol or a mixture thereof, more preferably water, but not limited thereto .

In one embodiment of the present invention, the composition inhibits the proliferation of prostate cancer cells and has an effect of enhancing antioxidant activity.

The pharmaceutical composition according to the present invention may be formulated into oral formulations such as capsules, powders, granules, tablets, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method .

Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Various compounds or mixtures including cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include various excipients such as a wetting agent, a sweetening agent, a fragrance, a preservative, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, solution, emulsion and syrup . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

A suitable dose of the pharmaceutical composition of the present invention may be variously prescribed by factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient .

The pharmaceutical composition of the present invention may be administered orally or parenterally. In the case of parenteral administration, the composition may be administered topically to the skin, intravenously, subcutaneously, intramuscularly, intraperitoneally, or transdermally.

The present invention also provides a health functional food composition for preventing or ameliorating prostate cancer, which comprises a fermented silkworm extract as an active ingredient.

The health functional food composition of the present invention may be a single formulation selected from powders, granules, pills, tablets, capsules, candies, syrups and beverages, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The active ingredient may be suitably used depending on its intended use (prevention or improvement). Generally, the health functional food composition of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight based on the raw material, when the food or beverage is produced. However, in the case of long-term consumption intended for health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount of more than the above range.

There is no particular limitation on the kind of the health functional food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

In addition, the health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention may contain ingredients that are conventionally added. For example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of a drink, a natural carbohydrate or a flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, , Xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above-mentioned health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like. Although the ratio of the above-mentioned ingredients is not critical, it is generally selected in the range of 0.01 to 0.1 part by weight based on 100 parts by weight of the health functional food composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples. It is to be understood by those skilled in the art that these preparations and examples are merely intended to explain the present invention more specifically and that the scope of the present invention is not limited thereto.

Manufacturing example  1. Preparation of fermented silkworm extract

The present invention was carried out in March 2017 by the Rural Development Administration (Jeonju, Jeonju, Jeonju, Korea), and the silkworm powder (Buan Jeonnam, Namwon, Chungbuk, Korea) was distributed. The silkworm varieties of Buan in Jeonnam and Namwon in Jeonbuk are sleeping in Baekmok.

As the fermentation strains, Bacillus ( Bacillus sp. Subtilis KACC 91157 strain) was used, and 5% (v / w) of the silkworm powder was inoculated to the hot-air dried silkworm powder at each locality where the pre-cultured Bacillus was sterilized and fermented at 37 ° C for 48 hours, Silkworm powder was obtained.

10 parts by weight of purified water per 1 part by weight of silkworm powder was added and the mixture was stirred for 3 hours in a constant temperature water bath at 37 ° C. to obtain a silkworm powder extract and a powdery extract of fermented silkworm by the mountain area. The extract was collected and filtered through a filter paper (Whatman NO. 2, Toyo 2A; Toyo Roshi, Tokyo, Japan) to obtain a filtrate.

Example 1. Antioxidant  Active measurement

The antioxidant activity of the silkworm extract and fermented silkworm extract of Preparation Example 1 was measured according to the Blois method (1958). That is, DPPH (α, α'-diphenyl-β-picrylhydrazyl) solution was prepared by dissolving 16 mg of DPPH in 100 ml of ethanol, mixing 100 ml of distilled water, and filtering with a filter paper (Whatman NO.2). 5 ml of the DPPH solution was mixed with 1 ml of a 0.5% (w / v) sample solution, reacted at room temperature for 30 minutes, and the absorbance was measured at 528 nm. (BHT) was added at a concentration of 0.05% (v / v) to measure the absorbance reduction in the same manner as described above. The DPPH free radical scavenging activity was expressed as the percentage difference (%) between the absorbance of the sample and the sample without addition.

As a result, as shown in FIG. 1, the extract of silkworm silage showed an increase in antioxidative effect by fermentation. In particular, in the BO group, the activity was 29.83%, while the BBO group had an activity of 49.03% It was the biggest.

It was confirmed that silkworms had antioxidative activity on their own and fermented silkworms had the highest activity in fermented silkworms, especially BBO group. Through the antioxidant effect, silkworm silkworms, especially mudwax fermented silkworms, It is confirmed that it can be usefully used.

Example 2. Cancer cells  multiplication Inhibition  Measure

The B16-F10 cells (skin cancer cell line), HT-29 cells (colorectal cancer cell line) and LNcaP cells (prostate cancer cell line) used in the present invention were purchased from ATCC. The cells were cultured in DMEM supplemented with 100 units / ml of Antibiotic Antimycotic (GIBCO® / Invitrogen ™, Gran Island, NY, USA) and 10% (v / v) FBS (Fetal Bovine Serum) And subcultured in a 5% CO ₂ incubator.

On the other hand, MCF-7 cells (breast cancer cell lines) were purchased from ATCC and cultured in the same conditions as above using RPMI 1640 medium supplemented with 100 units / ml of Antibiotic Antimycotic and 10% (v / v) FBS .

Sulforhodamine B (SRB) (Sigma Aldrich Co., St. Louis, Mo., USA), a method of confirming the degree of apoptosis by measuring the total amount of proteins in living cells by absorbance, The surviving cells were attached to a well plate by trichloroacetic acid (TCA) (Sigma Aldrich Co.), SRB was bound to the basic amino acid residues in the protein of the cells, and the final treated Tris solution And the absorbance of SRB dissolved by Tris buffer was measured. More specifically, the cells were diluted so as to be inoculated at ² × 10 ⁴ per well, and the cells were divided into 48 well plates and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. 500 and 1000 占 퐂 / ml) and reacted for 24 hours. After completion of the reaction, 12% (v / v) TCA was added to fix cells at 4 ° C, and the wells were washed and then stained with 0.4% (w / v) SRB solution. After completion of the staining, the cells were washed with 1% (v / v) acetic acid, and a 10 mM Tris solution was added to dissolve the SRB. The dissolved SRB solution was transferred to a 96-well plate and absorbance was measured at 540 nm using a microplate reader (SpectraMax Plus 384, Molecular Devices Corp., Sunnyvale, Calif.).

As a result, when B16-F10 cells were diluted with the sample of Preparation Example 1 as shown in Fig. 2, there was no significant difference in cell survival rate regardless of the concentration as compared with the control (Con). In addition, when HT-29 cells were diluted and treated with the samples of Preparation Example 1, the BB, BB, and BNW groups had no significant effect on cell viability, But there was no significant difference in the survival rate between the two groups. In the LNcaP cells, the BU and NW groups showed no cell growth inhibitory effect regardless of the fermentation, whereas the BO and BBO groups significantly decreased the cell survival rate compared to the control group and the effect was enhanced by fermentation . In particular, it was confirmed that the cell viability was reduced to 68.27% in the BBO group at a concentration of 1000 μg / ml, and the cell growth of LNcaP cells was most effectively inhibited. When MCF-7 cells were treated with BBO at a concentration of 1000 μg / ml, the cell viability was reduced to 81.67% and the cell growth was significantly inhibited. The results showed that BU and NW group showed no growth inhibitory effect on B16-F10, HT-29, LNcaP and MCF-7 cells regardless of fermentation. F10 and HT-29 cells was more effective in inhibiting the growth of LNcaP and MCF-7 cells, and it was confirmed that LNcaP cells were superior to LNcaP cells in growth inhibition.

Example 3: , pH  And total acidity change

The sugar content of the silkworm extract and the fermented silkworm extract according to Production Example 1 was determined by taking 300 쨉 l of a water-soluble extract containing 1% (w / v) of the sample of Preparation Example 1 using a sugar refractometer (Kruss, Germany) ° Brix). The pH was measured using a pH meter (SevenCompact ™ pH / Ion S220, Mettler-Toledo AG, Switzerland) taking 10 ml of the sample and titrating 5 ml of the sample with 0.1 N NaOH solution until pH 8.3 Was calculated by the following equation (1).

[Formula 1]

Total acidity (w / v,%) = (0.006 x V x F) / S x 100

V is consumption of 0.1N NaOH (ml), F is a factor of 0.1N NaOH, S is the amount of sample (ml)

As a result, it was confirmed that the sugar content of Buan silkworm (BU), Namwon silkworm (NW) and Boun silkworm (BO) group increased as compared with before fermentation through fermentation as shown in Table 1.

On the other hand, the pH of the BU group decreased to 6.72, while the pH of the BU group decreased to 6.72. The pH of the NW and BO groups were 6.60 and 6.69, respectively, and increased to 6.79 and 6.95, respectively, by fermentation.

On the other hand, acidity of BU group and BBU group decreased by fermentation at 0.76 and 0.60%, respectively. Acidity of NW group and BNW group increased by fermentation to 0.76 and 0.80%, respectively, Both BO and BBO groups showed 0.85% acidity and showed no difference due to fermentation.

The results showed that the fermentation of silkworm increased the sugar content in all treatments, but the pH and total acidity of the silkworm were different depending on the silkworm production and the variety of silkworm.

A, b, c, d and e in the column indicate that there is a significant difference between them, and p < 0.05.

Embodiment 4. Configuration  Amino acid content change

For the analysis of the constituent amino acids, 15 ml of performic acid and 15 ml of 6N HCl were added to 0.2 g of the sample of Preparation Example 1 and acid hydrolyzed in a dryer at a temperature of 110 ° C for more than 24 hours. The digested samples were concentrated under reduced pressure in a 55 ° C water bath and then diluted to 25 ml in a volumetric flask with a pH 2.2 diluted citric acid dilution buffer and a fixed amount was analyzed using an amino acid automatic analyzer Biochrom 30 (Biochrom, UK) Respectively.

As a result, as shown in Table 2, the total content of constituent amino acids in the BU group was 305,826 ppm, and glutamic acid, glycine, alanine and aspartic acid were contained in the order of BBU group Total content of amino acids was 213,553ppm and most amino acids including glutamic acid and glycine were all decreased. The total amino acid content of NW group was detected as 252,164ppm, but the total amino acid content including alanine, glycine and glutamic acid was decreased and total amino acid content was decreased to 75,119ppm by fermentation.

On the other hand, the total amino acid content of BO group was found to be 214,160 ppm. The amino acid concentration was in the order of alanine, glycine, valine, leucine and lysine, Was 271,900 ppm, and the content of most amino acids including aspartic acid, glutamic acid and glycine was increased. In particular, it was confirmed that serine (4,164 ppm), which was not detected in the BO group, was generated.

Example 5. Minerals  Content analysis

Mineral contents were measured according to the AOAC analysis method (1975). That is, 1 g of each dry powder of Preparation Example 1 was precisely taken and stirred at 550 ° C for 3 hours, completely dissolved in 6N HCl to completely dissociate the acid, and then the acid was completely removed in a water bath. To the dried product, 3N HCl , And the contents were diluted to a certain ratio according to the kind of element, and the mineral content was measured using an atomic absorption spectrophotometer (Analyst 300, Perkin Elmer, Norwalk CT, USA).

The results of measurement of the mineral content of the sample prepared in Preparation Example 1 are shown in Table 3. The composition of minerals is represented by Ca, Cu, Fe, K, Mg, Mn, Na, Zn, Calcium, and magnesium, respectively. Potassium and calcium were the most abundant in the BBU group at 379.27 and 71.43 ppm, respectively, and magnesium was the highest in the BBO group at 40.67ppm. In particular, sodium content was low at 4.47, 1.40, and 1.32 ppm in BU, NW, and BO groups, respectively, but increased significantly at 22.40, 20.47, and 18.10 ppm after fermentation, respectively. Zinc, iron, manganese and copper were contained in small amounts. The fermented silkworm increased the content of fermented silkworm larvae after fermentation compared with that before fermentation. The fermented silkworms showed higher mineral content by fermenting silkworms. Respectively.

A, b, c, d and e in the column indicate that there is a significant difference between them, and p < 0.05.

Claims (8)

A pharmaceutical composition for the prophylaxis or treatment of prostate cancer, which contains an extract of fermented silkworm as an active ingredient of a varietal of Daesung. [Claim 7] The method according to claim 1, wherein the fermented silkworm is fermented at 35 to 39 DEG C for 24 to 72 hours by adding Bacillus subtilis to silkworm powder of a mosquito's sleeping variety. Composition. delete The pharmaceutical composition for preventing or treating prostate cancer according to claim 1, wherein the silkworm of the marshmallow variety is produced in Boeun, Chungbuk. The pharmaceutical composition for preventing or treating prostate cancer according to claim 1, wherein the solvent of the fermented silkworm extract of the marshmallow variety is water, a C ₁ -C ₄ lower alcohol or a mixture thereof. The pharmaceutical composition for preventing or treating prostate cancer according to claim 1, wherein the composition inhibits the proliferation of prostate cancer cells. A health functional food composition for preventing or ameliorating prostate cancer, which contains fermented silkworm extract of Daesung sleeping varieties as an effective ingredient. [Claim 7] The composition according to claim 7, wherein the fermented silkworm extract of the marshmallow varieties has an antioxidative effect enhanced by fermentation.

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