KR102448457B1 - Method for producing Saccharomyces cerevisiae extract using ultrasonication having potent inhibiting activity for skin cancer and composition for inhibiting skin cancer comprising the same - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating skin cancer comprising a yeast ultrasonic extract, and a method for preparing the same, wherein the ultrasonic yeast extract has remarkable activity of inhibiting the proliferation of skin cancer cells, and is used in cosmetics, functional foods and pharmaceuticals. It can be useful.

Description

A method for producing an ultrasonic yeast extract having excellent skin cancer inhibitory activity, and a composition for inhibiting skin cancer containing an ultrasonic yeast extract prepared by the method described above the same}

The present invention relates to a composition for improving, preventing, or treating skin cancer comprising an ultrasonic yeast extract.

Skin cancer is caused by frequent exposure to various harmful substances and UV rays due to the increase in the amount of UV radiation due to the destruction of the ozone layer due to changes in life patterns such as increased outdoor activities and environmental pollution. As the number of elderly people increases, the number of patients continues to increase worldwide.

On the other hand, many skin cancer patients simply overlook dermatitis or moles or boils when they first develop skin cancer. Among skin cancers, skin cancer that accounts for a large proportion is basal cell carcinoma (BCC), which accounts for about 80%, and squamous cell carcinoma, which accounts for about 16% of other skin cancers, SCC), and about 4% of melanomas. Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are collectively referred to as nonmelanoma skin cancer (NMSC). Melanoma arises from melanocytes in the epidermis, and most of them are metastatic cancer or cancer causing death. In 2000, 47,000 new melanomas were identified, of which 7,700 died (R. T. Greenlee et al. al. Cancer J. Clin., 50: 7-33, 2000).

In particular, melanoma is also induced by chronic exposure to ultraviolet rays, but is expected to be caused by intermittent exposure such as extreme tanning. Because melanoma has a poorer prognosis than other skin cancers, recurrence is common even if detected early, and life can be shortened. Skin lesions appear as yellowish-brown or black spots or nodules, and are usually caused by sun-exposed areas after middle age. Sometimes it occurs on the hands and toes, especially the tips of the thumbs and toes, and black vertical lines can be seen on the toenails.

However, until now, the treatment drugs for skin cancer only relieve the symptoms of skin cancer, and the development of a fundamental therapeutic drug has not been made, and many side effects are known. .

Therefore, there is a need for the development of a new skin cancer therapeutic agent that is more fundamental and has excellent therapeutic or improvement effects, as well as fewer side effects.

Republic of Korea Patent 10-1365735 Republic of Korea Patent 10-1069686

It is an object of the present invention to provide a cosmetic composition for preventing or improving skin cancer comprising an ultrasonic yeast extract as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving skin cancer comprising an ultrasonic yeast extract as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating skin cancer comprising an ultrasonic yeast extract as an active ingredient.

Another object of the present invention is to provide a method for producing an ultrasonic yeast extract comprising adding an aqueous solution of sodium hydroxide (NaOH) to yeast, and treating the ultrasonic wave.

In order to achieve the above object, the present invention provides a cosmetic composition for preventing or improving skin cancer comprising a yeast ultrasonic extract as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving skin cancer comprising an ultrasonic yeast extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating skin cancer comprising an ultrasonic yeast extract as an active ingredient.

In addition, the present invention provides a method for producing an ultrasonic yeast extract comprising the step of adding an aqueous solution of sodium hydroxide (NaOH) to yeast, and treating the ultrasonic wave.

The yeast ultrasonic extract according to the present invention has excellent activity of inhibiting the proliferation of skin cancer cells, and thus can be usefully used in the manufacture of cosmetics, functional foods, and pharmaceuticals for the prevention, improvement or treatment of skin cancer.

1 is a result of analyzing the cell proliferation inhibitory ability of the yeast ultrasonic extract prepared according to each extraction condition against skin cancer cell lines.
2 is a result of Western blot analysis of the protein expression levels of c-myc and cyclin D1 in yeast ultrasonic extract prepared according to each extraction condition.
3 is a result showing tumors formed in mice after 0, 2, 4 and 6 days after intraperitoneal administration of yeast ultrasound extract after tumor formation by injecting B16F10 melanoma cell line into mice (n=2).
Figure 4 is the result of measuring the size of the tumor formed in the mouse after 0, 2, 4 and 6 days after intraperitoneal administration of yeast ultrasound extract after tumor formation by injecting B16F10 melanoma cell line into the mouse (n=2) .
5 is a result of western blot measurement of the expression change of Cyclin D1 protein in the tumor tissue of the mouse after 6 days after intraperitoneal administration of the yeast ultrasound extract after forming a tumor by injecting the B16F10 melanoma cell line into the mouse (n) =3).

The present invention provides a composition for preventing, improving or treating skin cancer comprising an ultrasonic yeast extract as an active ingredient.

In the above, the yeast may be Saccharomyces cerevisiae .

In the above, skin cancer consists of malignant melanoma, papilloma, carcinoma, squamous cell carcinoma, basal cell carcinoma and spindle cell carcinoma. It may be selected from the group, but is not limited thereto.

In addition, the present invention provides a method for producing an ultrasonic yeast extract having an activity capable of effectively inhibiting the proliferation of skin cancer cells.

The yeast ultrasonic extract is prepared by treating yeast with ultrasonic waves.

In the present invention, it was confirmed that the ultrasonic extract of yeast obtained by performing the sodium hydroxide (NaOH) aqueous solution treatment and the ultrasonic treatment together has excellent skin cancer inhibitory activity. It was confirmed that the extract obtained by ultrasonic treatment at 95° C. for 1.5 hours to 2.5 hours had the best skin cancer inhibitory activity.

Therefore, the present invention can provide a method for producing an ultrasonic yeast extract having skin cancer inhibitory activity, preferably, the method includes adding 0.1M to 0.2M sodium hydroxide (NaOH) aqueous solution to yeast, and 85°C to 95°C It may include the step of treating ultrasonic waves for 1.5 hours to 2.5 hours.

More preferably, 0.2M sodium hydroxide (NaOH) aqueous solution is added to yeast and ultrasonicated at 90° C. for 2 hours to obtain an ultrasonic yeast extract having excellent skin cancer suppression activity.

In the method of the present invention, if the concentration of sodium hydroxide (NaOH) is less than 0.1M, there is a slight problem in the extraction of the extract containing the physiologically active substance having skin cancer inhibiting activity from yeast, whereas it exceeds 0.2M There is a problem that can cause the transformation of the physiologically active substances contained in the extract due to the nature of the highly basic solution and loss of activity. Therefore, the concentration of sodium hydroxide (NaOH) should be used in the range of 0.1M to 0.2M, and most preferably, it can be used in a concentration of 0.2M.

In addition, when the extraction temperature during ultrasonic extraction of the present invention is carried out at less than 85° C., as confirmed in one embodiment of the present invention, there is a problem that the skin cancer inhibitory activity of the yeast ultrasonic extract is insufficient, and at a temperature exceeding 95° C. When carried out, the destruction or denaturation of the active ingredient may be induced, which may also lead to a decrease in the activity of inhibiting skin cancer.

In addition, in the ultrasonic extraction of the present invention, the extraction time may be performed in 1.5 hours to 2.5 hours.

If the ultrasonic treatment is performed for less than 1.5 hours, the extraction of useful extracts having skin cancer inhibitory activity from yeast may be insufficient, and even when treated for more than 2.5 hours, there is a problem in that the skin cancer inhibitory activity of the yeast ultrasonic extract is reduced. .

Therefore, in order to obtain an extract with the best inhibitory activity of skin cancer from yeast, it is necessary to use 0.1M to 0.2M sodium hydroxide (NaOH) aqueous solution, a temperature of 85°C to 95°C, and ultrasonication for 1.5 hours to 2.5 hours. must be carried out under the conditions.

In addition, the yeast ultrasonic extract obtained by the method of the present invention has excellent anticancer activity against skin cancer.

In particular, the yeast ultrasonic extract obtained under the conditions according to the present invention has significantly superior anticancer activity against skin cancer compared to other types of cancer.

In one embodiment of the present invention, after treating the yeast ultrasonic extract obtained under different conditions to a melanoma cell line, a type of skin cancer, the cell proliferation inhibitory ability of the melanoma cell line was analyzed. The extract obtained by ultrasonic treatment at 90 ° C.

This effect was confirmed to be superior to the extract obtained by treating the ultrasonic wave for 1 hour or 3 hours under the same conditions.

Furthermore, the present inventors analyzed changes in intracellular protein expression of c-myc and cyclin D1 according to the treatment of the yeast ultrasonic extract of the present invention in order to confirm by what mechanism the skin cancer inhibitory activity of the yeast ultrasonic extract of the present invention occurs. did.

As a result, the expression level of c-myc in the group treated with the yeast ultrasonic extract of the present invention did not show a change in expression level compared to the group that was not treated with the extract, but the expression level of the cyclin D1 protein was significantly reduced.

Therefore, it was found that the yeast ultrasound extract of the present invention exhibits anticancer activity against skin cancer by inhibiting the proliferation of cancer cells by inhibiting the expression and activity of cyclin D1 involved in the cell cycle of skin cancer cells.

Therefore, the yeast ultrasonic extract prepared by the method of the present invention can be usefully used in the manufacture of cosmetics, health functional foods and pharmaceuticals for the prevention, improvement or treatment of skin cancer.

Accordingly, the present invention provides a cosmetic composition for preventing or improving skin cancer comprising the yeast ultrasonic extract as an active ingredient.

In the cosmetic composition of the present invention, the composition may be prepared in liquid or solid form using bases, adjuvants and additives commonly used in the cosmetic field. Cosmetics in liquid or solid form, for example, but not limited thereto, may include, but are not limited to, the form of lotion, cream, lotion, bathing agent, and the like.

Bases, adjuvants and additives commonly used in the cosmetic field are not particularly limited, and examples thereof include water, alcohol, propylene glycol, stearic acid, glycerol, cetyl alcohol, and liquid paraffin.

When the composition of the present invention is prepared as a cosmetic composition, the composition of the present invention may include not only the yeast ultrasonic extract, but also ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, and pigments. and conventional adjuvants such as perfumes, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, It may be formulated as oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

In addition, in the cosmetic composition for preventing or improving skin cancer of the present invention, the content of the active ingredient of the present invention is preferably 0.0001 to 20% by weight relative to the total weight of the composition. If the content of the active ingredient is less than 0.0001% by weight, it is not preferable because the original desired skin cancer improvement or prevention effect cannot be sufficiently achieved. There is a risk of causing a problem in the appearance of the composition, thereby.

Furthermore, the present invention provides a food composition for preventing or improving skin cancer comprising an ultrasonic yeast extract as an active ingredient.

The food composition of the present invention includes all types of functional foods, nutritional supplements, health foods, and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the yeast ultrasonic extract of the present invention can be prepared and consumed in the form of tea, juice and drink, or it can be consumed by granulation, encapsulation and powdering. In addition, it may be prepared in the form of a composition by mixing with a known substance or active ingredient known to have an effect of preventing or improving skin cancer.

In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (eg, canned fruit, bottled, jam, marmalade, etc.), fish, meat and their processed foods (eg, ham, corned sausage, etc.) ), breads and noodles (eg udon noodles, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable protein, It can be prepared by adding the active ingredient of the present invention to retort food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.).

The preferred content of the active ingredient of the present invention in the food composition of the present invention is not limited thereto, but may preferably be included in an amount of 0.01 to 50 wt % of the solid content in the final prepared food. In addition, in order to be used in the form of a food additive, the composition of the present invention may be prepared and used in the form of a powder or a concentrate.

In addition, the present invention provides a pharmaceutical composition for preventing or treating skin cancer comprising the yeast ultrasound extract as an active ingredient.

The pharmaceutical composition may be in the form of tablets, capsules, pills, granules, powders, extracts, suspensions, emulsions, syrups, oral forms of aerosols, formulations, ointments, liniments, infusions, or external forms of intravenous, intramuscular, subcutaneous, intracerebrovascular It may be prepared in the form of a sterile injectable solution or suppository for administration.

The pharmaceutical dosage form of the pharmaceutical composition of the present invention may contain, alone or other therapeutically useful pharmaceutically active substances, and may be combined with an appropriate carrier conventionally acceptable for the manufacture of the pharmaceutical composition in a conventional manner. It can be formulated in various forms by

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include dextrose, lactose, sucrose, starch, sorbitol, mannitol, xylitol, gum acacia, calcium silicate, calcium phosphate, alginate, gelatin, cellulose , methyl cellulose, microcrystalline cellulose, water, propylhydroxybenzoate, mineral oil, methylhydroxybenzoate, talc, magnesium stearate, and the like. In the case of formulation, it can be prepared using a diluent or excipient such as a binder, disintegrant, filler, extender, surfactant, wetting agent, etc. usually used.

Solid preparations for oral administration include tablets, capsules, pills, granules, powders, etc., and such solid preparations include at least one excipient, for example, starch, sucrose or lactose, gelatin, etc., to the active ingredient of the present invention. It is prepared by mixing.

In addition to simple excipients, lubricants such as talc and magnesium stearate may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are frequently used simple diluents, various excipients such as fragrances, preservatives, wetting agents, sweeteners, etc. may be included. .

Injections in the form of solutions or suspensions for parenteral administration may be administered subcutaneously, intravenously, intramuscularly, or intraperitoneally.

In general, injectable solutions or suspensions are prepared in an effective amount in a pharmaceutically acceptable liquid carrier such as water, saline, aqueous dextrose and related sugar solutions, nonvolatile oils, ethanol, glycerin, polyethylene glycol, glycols such as propylene glycol, and the like. It can be prepared by homogeneously mixing the active ingredient of the present invention. In addition, if necessary, adjuvants such as solubilizers, antibacterial agents, chelating agents, buffers, and preservatives may be included.

The coating agent can be easily prepared in various forms such as creams, ointments, suspensions, and emulsions according to conventional manufacturing methods. When prepared in the form of an ointment or cream, the active ingredient of the present invention is contained in a general oil-in-water or water-in-oil type base, and fragrances, chelating agents, pigments, antioxidants, preservatives, etc. can be used as needed, and for the purpose of improving physical properties It can be combined with vitamins, proteins, and minerals.

In the present invention, the dosage of the active ingredient may be changed according to the weight of the patient, the severity of the disease, the drug formulation, the health status, the route of administration and the administration period. For a desirable effect, it is good to administer 0.001 to 10 g/kg per day, preferably 0.01 to 0.1 g/kg, but is not limited thereto. Administration can be administered once or divided into several times a day.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of yeast ultrasonic extract

The present inventors prepared a yeast ultrasonic extract by varying the extraction conditions as follows in order to prepare a yeast extract having skin cancer inhibitory activity.

First, an aqueous solution in which NaOH is dissolved at each concentration (0.15 and 0.2M) is added to yeast in powder form (Saccharomyces cerevisiae; purchased from Celltech Co., Ltd.) addition), by time (1 hr, 3 hr, 5 hr), ultrasonication was performed to prepare an ultrasonic extract, wherein the sonication conditions were 20 kHz, 80% amplitude, 750 W, pulse 20 s on/10 s off. were extracted at different temperatures of 90°C, 70°C and 50°C, respectively.

Thereafter, each obtained extract was neutralized with HCl, desalted and freeze-dried, and then the following experiment was performed. As a control group, a yeast extract obtained using only an alkaline solution without sonication was used.

Example 2. Analysis of antiproliferative ability of yeast ultrasound extract against skin cancer cell lines

In order to determine whether each of the yeast ultrasonic extracts obtained in Example 1 has skin cancer inhibitory activity, the B16F10 melanoma cell line was treated with each yeast ultrasonic extract, and then the degree of cell proliferation was analyzed. Each sample used for cell proliferation analysis is shown in Table 1 below.

Cell proliferation analysis of the B16F10 melanoma cell line was performed as follows. Briefly, the B16F10 melanoma cell line was seeded at 4 x 10 ³ cells per well in a 96-well plate, and each ultrasonic extract was treated at various concentrations 3 hours after cell dispensing, followed by 3 days at 37°C under 5% CO ₂ conditions. incubated during After culturing for 3 days, living cells were developed using Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) reagent to measure the degree of cell proliferation.

As a result, it was confirmed that the ultrasonic yeast extracts (#2 ~ #13) obtained by ultrasonication had a superior effect on inhibiting the proliferation of melanoma cells compared to the control group (control, #1) obtained by the alkali extraction method without sonication. (Fig. 1). In particular, the yeast ultrasonic extracts (#10, #11) obtained by ultrasonication at 90° C. for 2 hours in an aqueous solution of NaOH at a concentration of 0.2M showed a remarkable cell proliferation inhibitory effect of melanoma cell lines compared to extracts obtained under other conditions. was confirmed (Fig. 1).

On the other hand, the yeast ultrasonic extract of the present invention obtained under the above conditions is compared to the extract obtained using the 0.15M NaOH aqueous solution having a concentration lower than 0.2M and the extract obtained using the 0.5M aqueous NaOH solution having a concentration higher than 0.2M. It was confirmed that the cell proliferation inhibitory activity of the skin cancer cell line was remarkable.

From the above results, the present inventors found that the extract obtained by ultrasonication at a concentration of 0.2M NaOH aqueous solution, 20 kHz, 80% amplitude, 750 W, pulse 20 s on/10 s off conditions at a temperature of 90° C. for 2 hours, is skin cancer. It was confirmed that there is a significant effect on the anticancer activity.

Example 3. Cyclin D1 expression level analysis for yeast ultrasonic extract

From the results of Example 2, it was confirmed that the yeast ultrasonic extract had a significant effect on anticancer activity against skin cancer. Therefore, the present inventors performed an experiment to confirm the effect of the yeast ultrasound extract on the intracellular mechanism for skin cancer inhibition. c-myc is known to be an oncogene, and Cyclin D1 is known to be overexpressed in cancer cells. The present inventors confirmed the change in the expression level of c-myc and Cyclin D1 protein in melanoma cell lines when the yeast ultrasonic extract was treated through western blot.

As a result, it was confirmed that the yeast ultrasonic extract had the effect of significantly inhibiting the expression of Cyclin D1 protein compared to the control ( FIG. 2 ). In particular, the extract obtained by sonication at 90° C. for 2 hours in an aqueous NaOH solution of 0.15 M and 0.2 M concentration is an extract obtained under different conditions (0.2 M NaOH aqueous solution, 3 hours, ultrasonic treatment at 70° C.; or 0.2 M NaOH It was confirmed that there is an effect of remarkably inhibiting the expression of Cyclin D1 protein compared to aqueous solution, 3 hours, ultrasonic treatment at 50° C. (FIG. 2). However, there was no effect on the expression of c-myc (Fig. 2).

From the above results, the present inventors confirmed that the yeast ultrasound extract suppressed the expression of Cyclin D1, thereby inhibiting cell proliferation of skin cancer cell lines, and having a remarkable effect on anticancer activity against skin cancer.

Example 4. in vivo Analysis of anticancer activity of yeast ultrasound extract against skin cancer in

In order to confirm the anticancer activity of the yeast ultrasound extract against skin cancer, the present inventors performed an experiment to analyze the size of the tumor and the Cyclin D1 expression level in the tumor tissue by treating the yeast ultrasound extract in an animal model. Briefly, 6-week-old BALB/c-nu mice were used for the experiment, and the mice were acclimatized for 1 week after purchase. Then, the B16F10 melanoma cell line (number of cells: 5x10 ⁵ ) was injected into the dorsal skin tissue of each mouse. After confirming that a tumor was formed in the dorsal skin tissue of the mouse, the yeast ultrasound extract (500 mg/kg·body weight) was intraperitoneally administered once every 2 days. The tumor size was analyzed 0, 2, 4, and 6 days after the administration of the yeast ultrasound extract, and the change in the expression of Cyclin D1 protein in the tumor tissue 6 days after the administration of the yeast ultrasound extract was measured by Western blot.

As a result, it was confirmed that in mice treated with the yeast ultrasound extract, the tumor size was significantly reduced compared to the control group not treated with the extract (FIG. 3). In particular, in mice treated with yeast ultrasound extract for 6 days, the tumor size was confirmed to be about 593 mm ³ , which confirmed that the tumor size was reduced by about 63% compared to the control group that was not treated with the extract (about 945 mm ³ ). (Fig. 4).

In addition, it was confirmed that the expression of Cyclin D1 protein was significantly reduced in the tumor tissue of the mouse treated with the yeast ultrasound extract ( FIG. 5 ).

From the above results, the present inventors confirmed that the yeast ultrasound extract had a remarkable effect on anticancer activity even in an animal model of skin cancer.

Claims (10)

As a cosmetic composition for preventing or improving skin cancer comprising an ultrasonic yeast extract as an active ingredient,
The yeast ultrasonic extract is prepared by a manufacturing method comprising adding 0.1M to 0.2M sodium hydroxide (NaOH) aqueous solution to yeast, and treating the ultrasonic waves at 85°C to 95°C for 1.5 hours to 2.5 hours. , a cosmetic composition for preventing or improving skin cancer. The method of claim 1,
The yeast is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) The composition. delete delete The method of claim 1,
The skin cancer is from the group consisting of malignant melanoma, papilloma, carcinoma, squamous cell carcinoma, basal cell carcinoma and spindle cell carcinoma. composition of choice. As a food composition for preventing or improving skin cancer comprising an ultrasonic yeast extract as an active ingredient,
The yeast ultrasonic extract is prepared by a manufacturing method comprising adding 0.1M to 0.2M sodium hydroxide (NaOH) aqueous solution to yeast, and treating the ultrasonic waves at 85°C to 95°C for 1.5 hours to 2.5 hours. , a food composition for preventing or improving skin cancer. As a pharmaceutical composition for the prevention or treatment of skin cancer comprising a yeast ultrasonic extract as an active ingredient,
The yeast ultrasonic extract is prepared by a manufacturing method comprising adding 0.1M to 0.2M sodium hydroxide (NaOH) aqueous solution to yeast, and treating the ultrasonic waves at 85°C to 95°C for 1.5 hours to 2.5 hours. , a pharmaceutical composition for preventing or treating skin cancer. A method for producing an ultrasonic yeast extract comprising adding 0.1M to 0.2M sodium hydroxide (NaOH) aqueous solution to yeast, and ultrasonicating at 85°C to 95°C for 1.5 hours to 2.5 hours.
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