KR102579105B1 - Compositions for skin whitening, UV protection, and anti skin cancer through enzymatic conversion of Luffa aegyptiaca extracts using probioticst - Google Patents

Abstract

The present invention relates to a composition containing a loofah cucumber fermented extract as an active ingredient. By including the loofah cucumber fermented extract as an active ingredient, it has excellent antioxidant effect, is effective in improving skin whitening and blocking ultraviolet rays, and is also effective in preventing or preventing skin cancer. It can be treated.

Description

Compositions for skin whitening, UV protection, and anti skin cancer through enzymatic conversion of Luffa aegyptiaca extracts using probioticst }

The present invention relates to a cosmetic composition containing fermented loofah cucumber extract fermented with probiotics as an active ingredient and a composition for preventing, improving, or treating skin cancer.

Recently, in the case of cosmetics, the use of chemical compounds has become more and more repulsive to consumers due to the side effects of skin irritation, so the use of chemical compounds is being reduced and natural ingredients are being explored.

Among various natural materials, animal-derived materials have been actively developing functional cosmetics using plant-based ingredients due to increasing consumer resistance to animal-derived materials due to animal welfare, mad cow disease, swine cholera, and avian influenza, and anti-oxidation, whitening, and Consumer demand for complex functional cosmetics such as wrinkle improvement and UV protection is increasing.

Functional cosmetics refer to cosmetics in various stages of use, such as essence, serum, cream, powder, and base, with specific functions such as whitening, wrinkle improvement, and UV protection. Multifunctional cosmetics that perform only one function have been developed to increase ease of use, and specialized products for various purposes, such as pore care, nutrition supply, strengthening elasticity, acne treatment, whitening, and UV protection, are also being released.

Whitening materials are rapidly emerging as it is difficult for new single substances to enter the country and large cosmetics companies are focusing on developing multi-functional products for whitening and UV protection.

To date, the development and commercialization of cosmetic materials with skin whitening effects have been actively progressing, and the demand for and commercialization of sunscreens is increasing due to increased exposure to ultraviolet rays due to ozone layer destruction.

Meanwhile, skin cancer is a general term for all malignant tumors formed in skin tissue, and is mainly caused by environmental external stimuli such as ultraviolet rays or genetic factors. It is known that a family history of several genes, including the KIT gene, has an effect on skin cancer. However, the factors known to have the greatest influence are external stimuli such as ultraviolet rays. Outdoor activities have become more frequent due to the introduction of Western lifestyles, the use of suntans and tanning equipment has increased, and the amount of transmitted ultraviolet rays has decreased due to the weakening of the ozone layer. The incidence of skin cancer is gradually increasing due to the increase in skin cancer.

Exposure to large amounts of ultraviolet rays can cause thymidine dimers to form in the DNA inside cells or double-strand breaks. If damaged DNA is not properly repaired, the primary oncogene may change into an oncogene, and the tumor suppressor gene may not function properly. In this case, the division control mechanism inside the skin cells does not work normally, so proliferation is not restricted and normal cells eventually mutate into tumor cells. Cancerized tissue attracts blood vessels in the surrounding area through hormone secretion and receives nutrients, while the cancerous cells themselves also metastasize to other parts of the body through lymph nodes and blood vessels.

Cases where a tumor begins to develop in the skin are classified as 'primary' skin cancer, and cases where the tumor begins to develop in another organ and then metastasizes and settles in the skin are classified as 'metastatic' skin cancer. The commonly used definition of skin cancer is primary. It means skin cancer. Although it is a fairly common cancer in people of Caucasian descent, it is not yet actively studied because the mortality rate is generally lower than that of cancer of internal organs.

Based on 2012 statistics, skin cancer accounts for 2.0% of all cancers in Korea. Diagnosis can be made not only through examination devices such as CT and ultrasound, but also through the naked eye or biopsy, but the symptoms are often overlooked and early detection is delayed, so the cure and survival rates are not high. Skin cancer is divided into squamous cell carcinoma, basal cell carcinoma, and melanoma, depending on which cells it originates from among the cells that make up skin tissue. Squamous cell carcinoma, a malignant tumor derived from keratinocytes of the epidermis, and basal cell carcinoma, which is a malignant tumor of cells in the lowest layer of the epidermis or hair follicles, are quite common among skin cancers prevalent in Korea, but have a relatively high mortality rate. low. On the other hand, malignant melanoma is a cancer derived from melanocytes that determine the color of the skin, and is a dangerous cancer with the highest degree of malignancy and a low survival rate due to easy invasion and metastasis.

Therefore, there is a need for the development of natural substances that can improve antioxidant properties, skin whitening, block ultraviolet rays, and further prevent, improve, or treat skin cancer.

Republic of Korea Patent No. 2141575 Republic of Korea Patent Publication No. 2020-0089040

The purpose of the present invention is to provide a cosmetic composition containing fermented loofah cucumber extract fermented with probiotics as an active ingredient.

In addition, another object of the present invention is to provide a health functional food containing fermented loofah cucumber extract fermented with probiotics as an active ingredient.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating skin cancer containing fermented loofah cucumber extract fermented with probiotics as an active ingredient.

The cosmetic composition for improving skin whitening and blocking ultraviolet rays of the present invention to achieve the above-described other purposes may contain fermented loofah cucumber extract as an active ingredient.

The fermented luffa cucumber extract may be an extract obtained by fermenting the luffa cucumber solvent extract with a strain.

The strains include Bacillus tequilensis , Lactobacillus plantarum , Lactobacillus sakei , Leuconostoc mesenteroides , and Lactobacillus buchneri . It may be one or more types selected from the group consisting of.

The scrub cucumber solvent extract may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The loofah cucumber solvent extract may be an extract extracted with water.

The loofah cucumber solvent extract may be treated with an ultrasonicator with a frequency of 20 to 50 kHz and a power of 50 to 700 W for 5 to 60 minutes.

The extraction temperature during the ultrasonic treatment may be 50 to 90 °C.

In addition, the health functional food for improving skin whitening and improving or preventing skin cancer of the present invention to achieve the other purposes described above may contain fermented loofah cucumber extract as an active ingredient.

In addition, the pharmaceutical composition capable of preventing or treating skin cancer of the present invention to achieve the above-described other purpose may include fermented loofah cucumber extract as an active ingredient.

The skin cancer may be melanoma.

The composition containing the fermented loofah cucumber extract of the present invention as an active ingredient has excellent antioxidant effect through fermentation using probiotic strains and is effective in improving skin whitening and blocking ultraviolet rays.

In addition, the composition containing the fermented loofah cucumber extract of the present invention as an active ingredient can improve, prevent, or treat skin cancer and can be used as a health functional food or pharmaceutical composition.

Figure 1 is a graph measuring the viability of cells when using the loofah cucumber fermented extract prepared according to Example 1 of the present invention at different concentrations.
Figure 2 is a graph measuring cell viability when using the loofah cucumber fermented extract prepared according to Example 2 of the present invention at different concentrations.

The present invention relates to a cosmetic composition containing fermented loofah cucumber extract fermented with probiotics as an active ingredient and a composition for preventing, improving, or treating skin cancer.

Hereinafter, the present invention will be described in detail.

The composition of the present invention contains fermented loofah cucumber extract as an active ingredient.

The loofah cucumber is a vine plant of the cucurbit family and is an herbaceous plant that grows in tropical and subtropical climates. When the fruit of the loofah cucumber is boiled and the skin is removed, a soft substance like a sponge is released, which is why it is called a loofah. The natural sap of loofah cucumber is rich in vitamins, calcium, and minerals, and is known to have a high content of flavonoids, which have antioxidant properties, and play a role in removing oxygen radicals from the human body. According to Donguibogam, it is mentioned that loofah cucumber and bellflower root contain triterpenoid saponin, which is effective in strengthening bronchial and lung function and thus is effective in relieving hypersensitivity respiratory disease caused by asthma. In addition, in oriental medicine, it is used to treat fever, galactorrhea, enteritis, and intestinal inflammation, and the sap extracted from living vines is used for cosmetics and medicinal purposes. Seeds contain about 40% oil, so the oil is squeezed out and the seed cake is used as fertilizer or feed.

The fermented loofah cucumber extract of the present invention is obtained by fermenting a solvent extract with a strain.

The loofah cucumber solvent extract is extracted through ultrasonic treatment under an extraction solvent. Specifically, the loofah cucumber and the extraction solvent are mixed at a weight ratio of 1:5 to 25, preferably 1:10 to 20, and extracted at 20 to 50 kHz, preferably. Preferably, an ultrasonicator with a frequency of 30 to 40 kHz and a power of 50 to 700 W, preferably 150 to 400 W, is used at 50 to 90° C., preferably 70 to 80° C., for 5 to 60 minutes, preferably 15 to 30 minutes. It has been processed for some time.

If loofah cucumbers are extracted with a solvent or with ultra-high pressure rather than with ultrasound, not only a small amount of active ingredients are extracted, but the antioxidant, skin whitening, UV protection, and skin cancer prevention or treatment effects may be low.

If the weight ratio of the loofah cucumber and the extraction solvent is outside the above range, the active ingredient of the loofah cucumber may be extracted in a small amount in the extract.

In addition, if the frequency and power of the ultrasonicator are less than the lower limit, the active ingredients of the loofah cucumber may be extracted in a small amount, and if it exceeds the upper limit, a large amount of substances other than the active ingredients may be extracted, reducing the effectiveness.

In addition, if the extraction temperature and extraction time are below the above lower limit, the active ingredients of the loofah cucumber may be extracted in a small amount, and if the extraction temperature and extraction time are above the above upper limit, the functionality may be reduced due to thermal transformation of polyphenols and tyrosinase. .

The extraction solvent for extracting the extract is water, lower alcohol having 1 to 4 carbon atoms, ethylene glycol, ethyl ether, or a mixed solvent thereof. The lower alcohol may include an aqueous solution of 20 to 99% by volume of methanol, ethanol, butanol, or propanol, and preferably water may be used for excellent antioxidant effect, improved skin whitening, UV protection, and skin cancer prevention or treatment effect. there is.

The fermented loofah cucumber extract of the present invention is obtained by inoculating the strain into the loofah cucumber ultrasonic solvent extract, preferably the loofah cucumber ultrasonic hot water extract, and then fermenting at 25 to 50°C at 100 to 300 rpm for 1 to 10 days.

The strains include Bacillus tequilensis , Lactobacillus plantarum , Lactobacillus sakei , Leuconostoc mesenteroides , and Lactobacillus buchneri . ), and when strains other than the above strains known to produce beta-glucosidease are used, the antioxidant effect, skin whitening improvement, UV protection, and skin cancer prevention or treatment effect are reduced. It may be absent or low.

If the temperature, mixing speed, and time during fermentation are less than the lower limit, the active ingredients of the loofah cucumber may be extracted in a small amount, and if it exceeds the upper limit, the desired effect may not be achieved at all due to decomposition of the bioactive substances. .

The fermented loofah cucumber extract of the present invention can be used in health functional foods and pharmaceutical compositions in addition to cosmetic compositions.

The term 'extract' used in this specification while referring to loofah cucumbers includes not only crude extracts obtained by treatment with an extraction solvent, but also processed products of loofah cucumber fermented extracts. For example, loofah cucumber fermented extract can be prepared in powder form by additional processes such as reduced pressure distillation and freeze-drying or spray-drying.

Meanwhile, in this specification, the term 'containing as an active ingredient' means containing a sufficient amount to achieve the efficacy or activity of the loofah cucumber fermented extract. As an example, the fermented luffa cucumber extract is used at a concentration of 10 to 1500 μg/ml, preferably 100 to 1000 μg/ml. Since the loofah cucumber fermented extract is a natural product and has no side effects on the human body even when used in excessive amounts, the upper quantitative limit of the loofah cucumber fermented extract contained in the composition of the present invention can be selected and implemented within an appropriate range by a person skilled in the art.

In addition to the above cosmetic composition, the cosmetic composition of the present invention can be blended with other ingredients that are usually blended in cosmetics as needed. These ingredients include fat ingredients, moisturizers, emollients, surfactants, organic and inorganic pigments, Organic powder, ultraviolet absorber, preservative, disinfectant, antioxidant, pH adjuster, alcohol, colorant, fragrance, blood circulation promoter, coolant, antiperspirant, purified water, water-soluble vitamin, fat-soluble vitamin, polymer peptide, polymer polysaccharide, sphingolipid and seaweed. Extracts, etc. can be mentioned.

The cosmetic composition of the present invention can be prepared in the form of emulsified formulations and solubilized formulations commonly used in the art.

In addition, the ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the ingredients as active ingredients, for example, conventional auxiliaries such as stabilizers, pigments, and natural fragrances, and It may further contain a carrier.

Products to which the composition of the present invention can be added include, for example, mist, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, and UV protection cream. , cosmetics such as moisture cream, hand cream, foundation, essence, nutritional essence, mask pack, press powder, loose powder, eye shadow, lip protector, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, etc. There is.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. , butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

Additionally, the present invention provides a health functional food composition containing fermented loofah cucumber extract as an active ingredient.

Health functional foods are foods made by adding fermented loofah cucumber extract to food ingredients such as beverages, teas, spices, gums, and confectionery, or by encapsulating, powdering, or suspending them, and have specific health effects when consumed. However, unlike regular drugs, it has the advantage of not having any side effects that may occur when taking the drug for a long time since it is made from food. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of loofah cucumber fermented extract added in such health functional foods cannot be uniformly specified as it depends on the type of health functional food being targeted, but it can be added within the range that does not damage the original taste of the food. It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets, or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule, or beverage.

In addition, the present invention provides a new use of the fermented loofah cucumber extract for the manufacture of medicine for the prevention or treatment of skin cancer or medicine for animals.

The 'pharmaceutical composition' or 'medicine' may further include appropriate carriers, excipients, and diluents commonly used in the production of pharmaceutical compositions, etc., in addition to the fermented loofah cucumber extract as the active ingredient.

The 'carrier' is a compound that facilitates the addition of the compound into cells or tissues. The 'diluent' is a compound diluted in water that not only stabilizes the biologically active form of the target compound, but also dissolves the compound.

There is no need to specifically limit the carrier, excipient, and diluent, but for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The amount of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may vary depending on the age, gender, and weight of the patient or animal to be treated, and, above all, the condition of the subject to be treated, the specific category of the disease to be treated, or It will depend on the type, route of administration, and properties of the therapeutic agent used.

The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals is appropriately selected depending on the absorption rate of the active ingredient in the body, the excretion rate, the age, weight, sex, and condition of the patient or animal to be treated, and the severity of the disease to be treated. , it is generally preferred to administer 0.1 to 1,000 mg/kg per day, preferably 1 to 500 mg/kg, more preferably 5 to 250 mg/kg, and most preferably 10 to 100 mg/kg. The unit dosage form formulated in this way can be administered several times at regular time intervals as needed.

The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may be administered individually as a preventive or therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The pharmaceutical compositions, medicines, pharmaceutical compositions for animals, or medicines for animals can be used by formulating them into oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods. there is. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations include the compound with at least one excipient, such as starch, calcium carbonate, sugar or lactose, and gelatin. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .

The method of treating skin cancer involves administering the composition to humans or non-human animals, especially mammals, for example, administering the composition to an individual to be treated with skin cancer.

The dosage, administration method, and frequency of administration for the treatment may refer to the dosage, administration method, and frequency of administration of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is clear to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the attached patent claims.

Example 1. Ultrasonic hot water extract fermentation

culture medium

Bacillus tequilensis was inoculated into MRS broth at 1% and cultured in a shaking incubator at 35°C at 150 rpm for 24 hours, then crushed with a homogenizer to obtain a crude enzyme solution, and then incubated with 0.1 M HCl and 0.1 M NaOH. The pH was adjusted to 7.

Fermentation of ultrasonic hydrothermal extract

Loofah cucumber and water were mixed at a weight ratio of 1:20, placed in an ultrasonicator (JAC Ultrasinic, Hwaseng, Korea), and extracted for 30 minutes using ultrasound (40 kHz, 200 W) at 80°C. Then, the extract was washed at 1000 rpm for 5 minutes. An ultrasonic hot water extract of loofah cucumber was obtained by recovering the supernatant by centrifuging for 1 minute. The obtained loofah cucumber ultrasonic hot water extract was cooled at room temperature, then mixed with the Bacillus tequilensis crude enzyme solution at a solid-liquid ratio (w/v) of 1:10, the pH was adjusted to 6.95-7.05, and the enzyme was incubated at 50°C for 48 hours. After conversion, the supernatant was recovered by centrifugation to obtain a loofah cucumber fermented extract.

Example 2. Fermentation of hot water extract_ultrasonic omitted

culture medium

Bacillus tequilensis was inoculated into MRS broth at 1% and cultured in a shaking incubator at 35°C at 150 rpm for 24 hours, then crushed with a homogenizer to obtain a crude enzyme solution, and then incubated with 0.1 M HCl and 0.1 M NaOH. The pH was adjusted to 7.

Fermentation of thermal water extract

Luffa cucumber and water were mixed at a weight ratio of 1:20, extracted for 50 minutes at 80°C, and the extract was centrifuged at 1000 rpm for 5 minutes to recover the supernatant, thereby obtaining luffa cucumber and hot water extract. After cooling the loofah cucumber and hot water extract obtained above at room temperature, the loofah cucumber hot water extract and Bacillus tequilensis crude enzyme solution were mixed at a solid-liquid ratio (w/v) of 1:10 to adjust the pH to 6.95-7.05 and then cooled at 50°C. After enzymatic conversion for 48 hours, centrifugation was performed to recover the supernatant to obtain a loofah cucumber fermented extract.

Example 3. Direct fermentation of loofah cucumbers

culture medium

Bacillus tequilensis was inoculated into MRS broth at 1% and cultured in a shaking incubator at 35°C at 150 rpm for 8 hours. The pH of the culture medium was adjusted to 6.95-7.05 using 0.1 M HCl and 0.1 M NaOH under sterile conditions.

Direct fermentation of loofah cucumbers

The above culture medium and the sterilized (121°C, 15 minutes) loofah cucumber powder were mixed at a weight ratio of 20:1 and cultured at 35°C for 48 hours. Cell growth and enzymatic decomposition were performed in parallel, followed by centrifugation. By recovering the supernatant, fermented loofah cucumber was obtained.

Comparative Example 1. Ultrasonic hot water extract_fermentation omitted

The same procedure as in Example 1 was carried out, except that loofah cucumbers and water were mixed at a weight ratio of 1:20, placed in an ultrasonicator (JAC Ultrasinic, Hwaseng, Korea), and then subjected to 30 cycles using ultrasonic waves (40 kHz, 200 W) at 80°C. After extraction for 5 minutes, the extract was centrifuged at 1000 rpm for 5 minutes to recover the supernatant, thereby obtaining an ultrasonic hot water extract of loofah cucumber without fermentation.

Comparative Example 2. Hot water extract_fermentation omitted

The same procedure as in Example 2 was carried out, except that loofah cucumber and water were mixed at a weight ratio of 1:20, extracted at 80° C. for 50 minutes, and then the extract was centrifuged at 1000 rpm for 5 minutes to recover the supernatant, thereby omitting fermentation. A hot water extract of loofah cucumber was obtained.

<Test example>

Test Example 1. Measurement of total polyphenol content

Total polyphenol content (mg GAE/g DW) was measured by modifying the Folin-Denis method, and folin-ciocalteu's phenol solution (Folin &Ciocalteu'sphenol; Sigma-Aldrich, USA) was added to the sample to reduce polyphenol compounds. It was based on the molybdenum blue color reaction that occurs. A 0.2 N FC solution was added to 0.14 ml of the sample and left for 10 minutes. Then, 0.56 ml of 7.5% Na ₂ CO ₃ was added and reacted for 1 hour, and the absorbance value was measured at 756 nm. Gallic acid (Sigma-Aldrich, USA) was used as a standard material, and the unit was expressed as mg gallic acid equivalent (GAE)/g dry weight (DW) compared to the prepared gallic acid calibration curve.

division Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Total polyphenols
(mg GAE/g DW) 30.2 22.4 13.3 10.10 8.42

As shown in Table 1 above, the loofah cucumber fermentation extract prepared according to Example 1 of the present invention was confirmed to have a higher total polyphenol content compared to Example 2, Example 3, Comparative Example 1, and Comparative Example 2.

In addition, it was confirmed that the total polyphenol content of the directly fermented loofah cucumber product of Example 3 was low as it was fermented directly without extracting the loofah cucumber.

Accordingly, it was confirmed that the fermentation result of the loofah cucumber ultrasonic hot water extract of Example 1 had superior antioxidant effect compared to other groups.

Test Example 2. Measurement of Tyrosinase Activity Inhibition Effect

The ability to inhibit tyrosinase activity was tested by modifying Flurkey's method. A buffer was prepared by dissolving 0.8039 g of sodium phosphate monobasic anhydrous and 0.9511 g of sodium phosphate dibasic anhydrous, respectively, in 100 mL of distilled water and adjusting the pH to 6.8. The prepared buffer was used as a solvent to prepare the substrate 10 mM 3,4-dihydroxy phenylanin (I-dopa) (20 mg/mL) and the enzyme 1250 unit tyrosinase. Distilled water was mixed and 1250 unit tyrosinase was diluted 10 times. 0.2 g of substrate, 0.2 g of sample, and 0.2 g of enzyme were added in that order, stirred, and reacted at 25°C for 30 minutes. 0.2 g of substrate, 0.2 g of sample, and 0.2 g of enzyme were sequentially added to the microtube, stirred, and reacted at 25°C for 30 minutes. After the reaction, the absorbance was measured at a wavelength of 475 nm. At this time, kojic acid was prepared at a concentration of 2 mg/mL as a positive control.

[Equation 1]

Tyrosinase inhibition rate (Inhibitory activity, %) = [1-sample OD/control group OD]*100

division Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Tyrosinase inhibition rate (%) 52.2 31.3 22.0 7.3 5.2

As shown in Table 2 above, the loofah cucumber fermented extract prepared according to Example 1 of the present invention has a higher tyrosinase inhibition rate than Example 2, Example 3, Comparative Example 1, and Comparative Example 2, and is therefore effective in skin whitening. It was confirmed that it was effective.

Test Example 3. Measurement of UV blocking rate

The UV blocking rate was measured in the ultraviolet ray B (280-320 nm) wavelength range using a UV/Vis spectrophotometer to measure the UV blocking rate of the loofah cucumber extract prepared according to Examples and Comparative Examples, and this was measured as a representative polyphenol. The UV blocking rate compared to tannin was measured by comparing the absorption of tannin with 1 mg/mL.

division Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 UV-B blocking rate (%)
Tannin contrast 53.9 23.3 20.6 11.1 2.4

As shown in Table 3 above, the loofah cucumber fermented extract prepared according to Example 1 of the present invention has a higher ultraviolet ray blocking rate than Example 2, Example 3, Comparative Example 1, and Comparative Example 2, so it blocks ultraviolet rays that reach the skin. It is effective.

In other words, it was found that the cosmetic composition applied to the skin blocks ultraviolet rays at a high level, thereby reducing the ultraviolet rays that directly reach the skin by more than 50%.

Test Example 4. Measurement of cell viability by MTT assay

Human-derived HEK-293 cells and B16F0 cancer cell line, a melanoma cell, were purchased from the Biological Resource Center (KCTC, Korea) and cultured in DMEM medium containing 10% Fetal Bovine Serum and 1% Penicillin, respectively, at 37°C and 5% CO. It was cultured under ₂ conditions and subcultured every 3 to 4 days at a culture density of 80 to 85% and used in experiments. For cell culture, a 37°C, 5% CO ₂ incubator was used. The HEK-293 is obtained by harvesting and transforming aborted fetal kidney cells, and unlike normal cells that die after a certain period of time, it can continue to proliferate rapidly like cancer cells and can cause chromosomal instability.

To measure cell viability, MTT assay, a cell viability test, was performed to evaluate the effect of extracts and fermented products on the survival of normal cells and cancer cells. The cell line was dispensed at 1X10 ⁴ cells/well (180 uL each) into a 96 well plate and cultured for 24 hours. Afterwards, the medium was removed, the extract was diluted by concentration, 20 uL was dispensed onto a plate, and the extract was cultured in a 5% CO ₂ incubator at 37°C for 72 hours. Remove the medium, dissolve the MTT reagent at 0.25 mg/mL in PBS, mix with the medium, dispense onto the plate, and incubate for 4 hours. Then, remove all reagents to dissolve the formazon crystals, dispense 100 uL of DMSO, and use a microplate reader. Absorbance was measured at 540 nm.

Figure 1 is a graph measuring the viability of cells when using the loofah cucumber fermented extract prepared according to Example 1 of the present invention at different concentrations, and Figure 2 is a graph measuring the concentration of the loofah cucumber fermented extract prepared according to Example 2 of the present invention. This is a graph measuring the viability of cells when used separately.

As shown in Figure 1, the fermented loofah cucumber extract of Example 1 inhibited the growth of B16F0 cancer cells at all concentrations, and it was confirmed that growth inhibition of HEK-293 cells also occurred from 4 mg/ml. In particular, it was confirmed that the cell survival rate of B16F0 cancer cells was significantly lower than that of HEK-293 cells.

In addition, as shown in Figure 2, the loofah cucumber fermented extract of Example 2 slightly inhibited the growth of HEK-293 cells and cancer cells at all concentrations, and in particular, the cell viability of B16F0 cancer cells was lower than that of the loofah cucumber fermented extract of Example 1. It was confirmed that it was 2 to 3 times higher than that.

That is, in terms of cell viability of HEK-293 cells, the loofah cucumber fermented extract of Example 1 and the loofah cucumber fermented extract of Example 2 are similar at all concentrations, but in terms of cell viability of B16F0 cancer cells, the loofah cucumber fermented extract of Example 1 It was confirmed that as the concentration increased, the cell viability of B16F0 cancer cells was significantly reduced compared to the fermented loofah cucumber extract of Example 2.

Accordingly, the fermented loofah cucumber extract of Example 1 can be effectively used in the treatment, improvement, or prevention of skin cancer.

Below, a formulation example of a composition containing the extract of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.

Formulation Example 1: Preparation of powder

20 mg of extract powder of Example 1

100 mg lactose

Talc 10 mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

Formulation Example 2: Preparation of tablets

10 mg of extract powder of Example 1

Corn starch 100 mg

100 mg lactose

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

Formulation Example 3: Preparation of capsules

10 mg of extract powder of Example 1

3 mg crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a typical capsule manufacturing method.

Formulation Example 4: Preparation of granules

1,000 mg of extract powder of Example 1

Vitamin mixture dosage

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Monobasic Potassium Phosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a mixture of ingredients suitable for health functional foods in a preferred embodiment, but the mixing ratio may be modified arbitrarily, and the above ingredients are mixed according to a normal health functional food manufacturing method. Next, granules can be prepared and used to manufacture a health functional food composition according to a conventional method.

Formulation Example 5: Preparation of beverage formulation

1,000 mg of extract powder of Example 1

1,000 mg citric acid

100 g oligosaccharides

2 g plum concentrate

1 g taurine

Add purified water to make a total of 900 mL.

After mixing the above ingredients according to a typical beverage production method, stirring and heating at 85° C. for about 1 hour, the resulting solution was filtered, placed in a sterilized 2 L container, sealed, sterilized, and stored in the refrigerator. Then, the solution according to the present invention was mixed. It is used in the production of functional beverage compositions.

An example of manufacturing a cosmetic composition suitable for applying the present invention will be presented.

Preparation Example 6: Toner

The preparation example of lotion among cosmetics containing the ultrasonic extract of Example 1 is shown in Table 4 below.

ingredient Content (% by weight) Extract of Example 1 5.0 glycerin 6.0 1,3-butylene glycol 3.0 PEG 1500 1.0 allantoin 0.1 DL-panthenol 0.3 EDTA-2Na 0.02 Benzophenone-9 0.04 Sodium Hyaluronate 5.0 ethanol 10.0 Polysorbate 20 0.2 Preservatives, fragrance, coloring a very small amount Distilled water remaining amount Sum 100

Preparation Example 7: Lotion

The preparation example of lotion among cosmetics containing the ultrasonic extract of Example 1 is shown in Table 5 below.

ingredient Content (% by weight) Extract of Example 1 3.0 propylene glycol 6.0 glycerin 4.0 Triethanolamine 1.2 Tocopheryl Acetate 3.0 liquid paraffin 5.0 squalane 3.0 macadami nut oil 2.0 Polysorbate 60 1.5 Sorbitan sesquioleate 1.0 Carboxy vinyl polymer 1.0 Preservatives, fragrance, coloring a very small amount Distilled water remaining amount Sum 100

Preparation Example 8: Nutritional Cream

The manufacturing example of the nutritional cream among cosmetics containing the ultrasonic extract of Example 1 is shown in Table 6 below.

ingredient Content (% by weight) Extract of Example 1 1.0 Lipophilic glycerin monostearate 1.5 cetearyl alcohol 1.5 stearic acid 1.0 Polysorbate 60 1.5 Sorbitan Stearate 0.6 Isostearyl isostearate 5.0 squalane 5.0 mineral oil 35.0 Dimethicone 0.5 Hydroxyethylcellulose 0.12 glycerin 6.0 Triethanolamine 0.7 Preservatives, fragrance, coloring a very small amount Distilled water remaining amount Sum 100

Preparation Example 9: Essence

Preparation examples of essences in cosmetics containing the ultrasonic extract of Example 1 are shown in Table 7 below.

ingredient Content (% by weight) Extract of Example 1 1.5 glycerin 10.0 Betaine 5.0 PEG 1500 2.0 allantoin 0.1 DL-panthenol 0.3 EDTA-2Na 0.02 Benzophenone-9 0.04 Hydroxyethylcellulose 0.1 Sodium Hyaluronate 8.0 Carboxy vinyl polymer 0.2 Triethanolamine 0.18 octyldodecanol 0.3 Octyldodeceth-16 0.4 ethanol 6.0 Preservatives, fragrance, coloring a very small amount Distilled water remaining amount Sum 100

Preparation Example 10: Emulsion for mask pack

The preparation example of the emulsion for a mask pack among the cosmetics containing the ultrasonic extract of Example 1 is shown in Table 8 below.

ingredient Content (% by weight) Extract of Example 1 1.0 polyvinyl alcohol 15.0 cellulose gum 0.15 glycerin 3.0 PEG 1500 2.0 Cyclodextrin 0.15 DL-panthenol 0.4 allantoin 0.1 Monoammonium glycyrrhizinate 0.3 nicotinamide 0.5 ethanol 6.0 PEG 40 Hydrogenated Castor Oil 0.3 Preservatives, fragrance, coloring a very small amount Distilled water remaining amount Sum 100

Claims (10)

Containing loofah cucumber fermented extract as an active ingredient,
The loofah cucumber fermented extract is a fermented extract obtained by fermenting the loofah cucumber solvent extract with a strain,
The strains include Bacillus tequilensis , Lactobacillus plantarum , Lactobacillus sakei , Leuconostoc mesenteroides , and Lactobacillus buchneri . A cosmetic composition, characterized in that it is one or more selected from the group consisting of. delete delete The cosmetic composition according to claim 1, wherein the solvent extract of loofah cucumber is extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The cosmetic composition according to claim 1, wherein the loofah cucumber solvent extract is an extract extracted with water. The cosmetic composition according to claim 1, wherein the solvent extract of loofah cucumber is treated with an ultrasonicator with a frequency of 20 to 50 kHz and a power of 50 to 700 W for 5 to 60 minutes. The cosmetic composition according to claim 6, wherein the extraction temperature during ultrasonic treatment is 50 to 90°C. Containing loofah cucumber fermented extract as an active ingredient,
The loofah cucumber fermented extract is a fermented extract obtained by fermenting the loofah cucumber solvent extract with a strain,
The strains include Bacillus tequilensis , Lactobacillus plantarum , Lactobacillus sakei , Leuconostoc mesenteroides , and Lactobacillus buchneri . A health functional food for improving skin whitening and improving or preventing skin cancer, characterized in that it contains one or more types selected from the group consisting of. Containing loofah cucumber fermented extract as an active ingredient,
The loofah cucumber fermented extract is a fermented extract obtained by fermenting the loofah cucumber solvent extract with a strain,
The strains include Bacillus tequilensis , Lactobacillus plantarum , Lactobacillus sakei , Leuconostoc mesenteroides , and Lactobacillus buchneri . A pharmaceutical composition for preventing or treating skin cancer, characterized in that it contains at least one selected from the group consisting of. The pharmaceutical composition according to claim 9, wherein the skin cancer is melanoma.