KR102250928B1 - Cosmetic composition containing Trapa japonica Flerov callus extracts - Google Patents

Abstract

The present invention relates to a cosmetic composition, food composition, and pharmaceutical composition containing the extract of Neungsil callus as an active ingredient, and has excellent antioxidant, anti-inflammatory, and stretch marks relief effects without cytotoxicity. It can be usefully used as an appropriate composition.

Description

Cosmetic composition containing Trapa japonica Flerov callus extracts {Cosmetic composition containing Trapa japonica Flerov callus extracts}

The present invention relates to a cosmetic composition, a food composition, and a pharmaceutical composition containing a neungsil callus culture extract, and more particularly, a cosmetic composition for pregnant women containing a neungsil callus culture extract having excellent antioxidant effect, whitening effect and stretch marks removal effect, It relates to a food composition, a pharmaceutical composition.

Due to pregnancy, women change their skin and body rapidly due to hormonal effects and metabolic changes. During pregnancy, the skin becomes dry and rough, and the body undergoes various changes depending on the area. Among the various changes that appear during pregnancy, especially those on the skin, changes in skin appendages, including pigmentation, dilation and increase of blood vessels, changes in the skin structure including the occurrence of striae glands, and the occurrence or worsening of acne due to the development of sebaceous glands. Etc.

Pigmentation such as spots, freckles, and blemishes is experienced by over 90% of women during pregnancy, and these symptoms are caused by increased secretion of estrogen and progesterone hormones. Among the causes of pigmentation, hormones or genetic factors cannot be removed artificially, but they can be prevented by blocking UV rays or eating enough fruits and vegetables rich in vitamins.

Stretch marks began to be known by Troisier in 1889 and occurred mainly in the abdomen of pregnant women and were called stretch marks. Also, depending on the cause and color, it is classified into striae, white, and purple striae. Stretch marks have been reported to appear in a variety of environments, including rapid weight gain due to pregnancy and obesity, and in general, it is most common to accompany pregnancy. Although the exact cause of changes in collagen tissue such as stretch marks has not been identified, it is known to be due to an increase in the activity of adrenal cortical hormones, and the morphology and quantitative changes of elastic fibers or collagen fibers. In the case of applying drugs for the treatment of stretch marks, drugs that can mainly help collagen fiber proliferation are used. Mainly, artificial synthesis of vitamin A is dominated, and fruit acids, vitamin C, and vitamin K are used as auxiliary. However, although these drugs are somewhat different depending on the individual, most of them have side effects because they irritate the epidermis. In the case of stretch marks cosmetics, it is possible to condition the skin by using it in combination with other main treatments to help suppress the severe progression of early stretch marks, and by using it in advance on areas where stretch marks can occur. The use of cosmetics is done as a preventive measure for stretch marks, but it is best to start treatment at least as early as possible, especially when it turns red. In parallel with the use of stretch marks, do moderate abs and chest exercises to double the stretch marks relief effect, take nutritional supplements containing vitamins that can regenerate the skin, refrain from exposure to UV rays or alcohol and smoke, and lose weight. It is suggested to try to control it and give it a massage that helps blood circulation (Cosmetic Dermatology, 2001).

Cosmetics for pregnant women are desirable to alleviate pigmentation and stretch marks, which are skin problems of pregnant women, while excluding ingredients that are harmful to the human body such as formaldehyde and isoflavone. In particular, as problems due to genetic modification of cosmetic ingredients or environmental hormones have emerged in recent years, expectations for functional cosmetics containing extracts of natural substances that are not harmful to the human body and can be safely used in the long term are increasing.

Patent Document: Domestic Patent No. 10-0927970

Therefore, the present inventors were screening for a cosmetic composition material for pregnant women from materials derived from natural products that were proven to be safe, and there was no research on the use of Neungsil extract, known for its efficacy, such as Geonwi (健胃) and Geonbi (健脾) as a cosmetic. With attention, the antioxidant, anti-inflammatory, pigmentation relief, and stretch marks improvement effects of Neungsil extract were confirmed. As a result, compared to the fruit kernels, peels, kernels, and peel extracts of Neungsil fruit, the cultured extract of Neungsil callus exhibited excellent antioxidant and anti-inflammatory effects at various concentrations.As a result, it was confirmed that it is safe for pregnant women and has the effect of improving skin problems of pregnant women. , Has completed the present invention.

Therefore, the problem to be solved by the present invention is to provide a cosmetic composition containing a culture extract of neungsil callus as an active ingredient.

Another problem to be solved by the present invention is to provide a food composition containing a culture extract of neungsil callus as an active ingredient.

Another problem to be solved by the present invention is to provide a pharmaceutical composition having an antioxidant effect and inhibiting melanin synthesis, containing a culture extract of Neungsil callus as an active ingredient.

The problems to be solved by the present invention are not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a cosmetic composition containing a culture extract of neungsil callus as an active ingredient.

Trapa japonica Flerov is a fruit of dryweed, which is a side leaf plant among aquatic plants growing in cold and warm temperate zones, and is distributed in Japan, Taiwan, Manchuria, China, Ussuri, and all over the northern hemisphere. Rhizoma is a year-old that grows by floating on the surface of the water. The leaves are triangular rhombus, there are many serrations on the edge, and the tips of the leaves are sharp. Neungsil ripens black in early autumn, and both ends become sharp and become sharp thorns. It is written in <Medicinal Plant Dictionary> that eating dried dried fruit not only heals the pock, relieves alcohol poisoning, brightens eyes, but also heals stomach cancer and uterine cancer. It is written that if you drink it three times a day, it is effective against various types of cancer, eliminates alcohol poisoning and tare poison, and facilitates digestion. Neungsil is composed of about 15% starch. In addition, it contains ingredients such as glucose, protein, vitamin C, and vitamin B. The energy is flat and has a slightly sweet taste. In oriental medicine, it is recommended as a nutritional diet for the elderly or poorly malnourished due to weak constitution and weak gastrointestinal function because it has mainly geonwi (健胃) and geonbi (健脾) functions. Neungsil can be eaten as porridge or just boiled. In Lee Sijin's <Bonchogangmok>, dry porridge improves the stomach, removes heat from the inside, and if adults or the elderly use dry porridge, it makes the stomach and spleen healthy. It is mentioned that it strengthens and gives energy. In addition, it is known from previous studies that it has an effect of inhibiting photoaging.

Callus refers to a special tissue or cell mass that occurs when a tissue cut from a plant is cultured in a medium containing auxin, a kind of plant is wounded, or a wound site is treated with auxin. Usually callus is an amorphous tissue or cell mass that has lost its ability to cause normal organ formation or tissue differentiation, and is mostly made up of flow cells. In a broad sense, it also includes plant tumor tissues caused by infections such as agrobacterium. Callus can be derived from any tissue, such as the root, stem, or leaf of a plant. In the present invention, a callus derived from a neungsil leaf was used, but the present invention is not limited thereto.

In the present invention, the "extraction" may be obtained by extraction, separation and fractionation from nature using a method for extraction, separation and fractionation known in the art. "Extract" as defined in the present invention is obtained by extraction treatment from the fruit using a suitable solvent, for example, fruit kernels, fruit peel, fruit kernels and peels, crude extract of callus, polar solvent soluble extract Or non-polar solvent-soluble extracts.

In the present invention, the neungsil extract may be extracted according to various extraction solvents and extraction methods, and suitable solvents for extracting the extract from neungsil callus include, for example, water, methanol, ethanol, propanol, C1-C4 alcohols such as isopropanol and butanol, or a mixed solvent thereof may be used. Ethanol, 50 to 90% (v/v) ethanol, or 60% to 80% (v/v) ethanol may be used as the extraction solvent of the extract of the present invention, but is not limited thereto. In the case of preparing a culture extract of neungsil callus using ethanol, both the fat-soluble and water-soluble active ingredients in the culture extract of neungsil callus can be sufficiently dissolved, and an extract exhibiting the optimum effect can be prepared. According to one embodiment of the present invention, the culture extract of neungsil callus is extracted with an alcohol having 1 to 4 carbon atoms. According to an embodiment of the present invention, the culture extract of Neungsil callus is characterized in that it is extracted using 60 to 80% (v/v) ethanol as an extraction solvent.

The extraction temperature is preferably room temperature, but is not limited thereto. In addition, as the extraction method, methods such as hot water extraction, cold precipitation extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, compression, and the like may be used.

After obtaining the extract using water or an organic solvent as described above, a liquid product may be obtained by cooling, heating, and filtration at room temperature by a conventional method known in the art, or further evaporating the solvent, spray drying, or freeze drying. You may.

In the present invention, the prepared neungsil callus culture extract can be obtained by fractionating after additionally distilling, specifically silica gel column chromatography, thin layer chromatography, high performance liquid chromatography (High performance liquid chromatography) can be obtained as an additionally purified fraction by using various chromatography, preferably thin layer chromatography, but the present invention is not limited thereto, and an appropriate solvent is used. Any method that can be used to obtain additional fractions may be used.

In the present invention, the solvent used to obtain the fraction may be any one or more selected from the group consisting of chloroform, ethyl acetic acid, butanol and water, and chloroform, ethyl acetic acid, butanol may be used, and ethyl acetic acid may be used. It is not limited.

According to an embodiment of the present invention, the culture extract of neungsil callus is treated with starch degrading enzyme on neungsil callus, extracted with ethanol, and then heated and concentrated two to three times by heating and pressing at 70-100° C. and 400-850 mbar, and It is characterized in that produced by a method comprising heating and concentrating once to twice by decompressing temperature at 40 to 80° C. and 100 to 350 mbar.

According to an embodiment of the present invention, the culture extract of neungsil callus is based on the total weight of the composition. It is characterized in that it is contained in a concentration of 0.03 to 10% by weight. This is because the effect cannot be expected if it is less than 0.03%, and if it exceeds 10%, it may affect skin stability and formulation stability in cosmetic formulations, and economic feasibility is deteriorated.

According to one embodiment of the present invention, the cosmetic composition containing the culture extract of neungsil callus of the present invention as an active ingredient has an antioxidant effect on the skin. In addition, it is known that scavenging active oxygen is effective in inhibiting the formation of melanin pigments. Compared to fruit kernels, peels, kernels, and peel extracts, cultured callus extracts showed superior antioxidant effects without cytotoxicity at various concentrations.

According to one embodiment of the present invention, the cosmetic composition containing the culture extract of neungsil callus of the present invention as an active ingredient has a skin whitening effect. The term "whitening" refers to whitening the skin. Melanin is produced from tyrosine through a complex process by the action of tyrosinase (Ty) present in pigment cells. The produced melanin is delivered to the skin cells, and it exhibits a circulating action in which melanin is lost and disappeared along with epidermal detachment. That is, when the skin is exposed to ultraviolet rays, tyrosinase is activated, and the tyrosinase acts on tyrosine present in the skin tissue to induce an oxidation process that generates dopa and dopaquinone, and thereby, in melanocytes, which are skin pigment cells. Melanin is synthesized in melanosomes, and the synthesized melanin is delivered to keratinocytes, which are keratinocytes of the skin, and reaches the skin surface by the keratinization process to protect the skin from ultraviolet rays. However, when melanin is excessively synthesized locally, or when the physiological function of the skin decreases due to skin lesions and aging, melanin is deposited on the skin surface to cause various pigmentation such as spots and freckles. Is characterized by having a whitening effect by inhibiting the mechanism by which melanin synthesis is inhibited from melanosomes, and the present invention is not limited thereto.

According to one embodiment of the present invention, the cosmetic composition containing the culture extract of Neungsil callus of the present invention as an active ingredient has an anti-aging effect. Body absorption of antioxidants that remove free radicals, which are considered important causes of skin aging, are important in preventing and delaying skin aging.

According to one embodiment of the present invention, the cosmetic composition containing the extract of the neungsil callus culture of the present invention as an active ingredient has a stretch marks relief effect.

According to one embodiment of the present invention, the cosmetic composition containing the culture extract of neungsil callus of the present invention as an active ingredient has an anti-inflammatory effect on the skin. Compared to fruit kernels, peels, kernels, and peel extracts, the cultured callus extracts showed superior anti-inflammatory effects without cytotoxicity at various concentrations.

According to one embodiment of the present invention, the cosmetic composition is a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray. It has a formulation selected from the group consisting of.

The health functional food according to the present invention is to provide a health functional food for skin whitening, comprising a culture extract of neungsil callus as an active ingredient.

According to one embodiment of the present invention, the neungsil callus culture extract is a health functional food for skin whitening that is extracted with C1-C4 alcohol.

According to one embodiment of the present invention, it is to provide a health functional food for antioxidants, comprising a culture extract of neungsil callus as an active ingredient.

According to one embodiment of the present invention, the culture extract of neungsil callus is a health functional food for antioxidants extracted with C1-C4 alcohol.

The health functional food according to the present invention may be provided in the form of powder, granule, tablet, capsule, syrup or beverage, and the health functional food is an active ingredient, other foods or food additives other than the neungsil callus culture extract according to the present invention. They are used together, and can be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health or therapeutic treatment.

There are no specific restrictions on the types of health functional foods, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, and drinks. , Alcoholic beverages and vitamin complexes.

The therapeutic pharmaceutical composition according to the present invention is a pharmaceutical composition for preventing or treating pigmentation disorders, having an inhibitory effect on melanin synthesis and an antioxidant effect, comprising a culture extract of Neungsil callus as an active ingredient.

In the present invention, the skin pigmentation disease may include various diseases caused by skin pigmentation, but preferably may be a disease that occurs locally on the skin due to increased synthesis of melanin pigment, and more preferably May be characterized as one or more diseases selected from the group consisting of spots, freckles, black spots, birthmarks, pigmentation by drugs, pigmentation after inflammation, and hyperpigmentation occurring in dermatitis.

As mentioned above, when the present invention is used as a medicine, the composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, Lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, syrup, arabic rubber, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnaubanap, synthetic Aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, and the like can be used.

The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

In the present invention, the composition may be administered in various oral or parenteral dosage forms at the time of actual clinical administration.When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. It is preferable to use those disclosed in Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA, as a suitable formulation known in the art. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, various excipients in addition to water and liquid paraffin, which are commonly used simple diluents, include suspensions, liquid solutions, emulsions, syrups, etc. as the liquid preparations for oral administration, such as wetting agents, sweeteners, fragrances, preservatives, etc. May be included.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The parenteral administration may be performed using external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

The composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, age, sex, type of infected virus, and Sensitivity to the active drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and can be easily determined by a person skilled in the art.

The term "administering" as used herein means providing a given composition of the present invention to a subject in any suitable manner.

In the present invention, the pharmaceutical composition may be administered to an individual by various routes. All modes of administration can be expected and may be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection.

The pharmaceutical composition of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods of using biological response modifiers for the treatment of skin pigmentation disorders.

The description of the composition of the present invention described above is omitted in order to avoid excessive complexity due to repeated description of the contents, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention pertains. To have.

Cosmetic compositions, food compositions, and pharmaceutical compositions containing the extract from the culture of Neungsil callus according to the present invention have excellent antioxidant effects, whitening effects, anti-inflammatory effects, and stretch marks alleviating effects without cytotoxicity. It can be usefully used as a composition.

1 is (a) fruit kernel extract, (b) fruit fruit peel extract, (c) fruit kernel and peel extract, (d) confirming the cytotoxicity at a concentration of 0,015 to 50 ㎍ / mL of the fruit callus culture extract It is a diagram showing the results of CCK-8 analysis of RAW 264.7 cells.
Figure 2 is (a) fruit kernel extract, (b) fruit fruit peel extract, (c) fruit kernel and peel extract, (d) confirming the cytotoxicity at 0.015 to 50 ㎍ / mL concentration of the callus culture extract It is a diagram showing the results of CCK-8 analysis of HaCaT cells.
Figure 3 is a diagram showing the results of MTT analysis to confirm the cytotoxicity at a concentration of 12.5 to 100 ㎍ / mL of Neungsil callus culture extract.
Figure 4 shows the results of DPPH removal at a concentration of 0.005 to 1,000 μg/mL of (a) fruit kernel extract, (b) fruit peel extract, (c) fruit kernel and peel extract, (d) callus culture extract It is a degree.
Figure 5 is a diagram showing the DPPH removal ability results at a concentration of 12.5 to 100 ㎍ / mL of Neungsil callus culture extract.
Figure 6 shows the results of NO scavenging activity at a concentration of 5 to 50 μg/mL of (a) fruit kernel extract, (b) fruit fruit peel extract, (c) fruit kernel and peel extract, (d) callus culture extract It is a degree.
7 is a diagram showing the results of NO scavenging ability at a concentration of 12.5 to 100 μg/mL of Neungsil callus culture extract

Hereinafter, the present invention will be described in detail by way of Preparation Examples, Examples and Experimental Examples.

However, the following Preparation Examples, Examples, and Experimental Examples are only to specifically illustrate the present invention, and the content of the present invention is not limited by the Preparation Examples, Examples, and Experimental Examples.

Example 1: Preparation of Neungsil extract

1-1) Preparation of Neungsil fruit extract

Neungsil fruit was purchased from Hongik Jewon (Cheonan, KOREA). _{500 mL of 70% ethanol (C 2} H ₅ OH) was mixed with each of 50 g of husks, kernels, husks and grains of fruit fruit and extracted under reflux for 3 hours to obtain a filtrate, and the obtained filtrate was used in a rotary vacuum concentrator (Buchi B- 480 Co., Flawil, Switzerland) under reduced pressure. The concentrated solution was freeze-dried in a freeze dryer (EYELA FDU-540 Co., Tokyo, Japan) to make a powder, and the obtained powder was stored in a cryogenic freezer (-80℃) and diluted in distilled water to the required concentration according to the experiment. Was used.

1-2) Preparation of Neungsil callus culture extract

Induction of good callus

The leaf tissue of the sterilized fruit was 2 to 3% sucrose, 0.4% gelrite, kinetin, a-naphthaleneacetic acid (NAA), benzyl adenine (BA), and indole as plant growth regulators. It was placed in MS medium containing acetic acid (IAA) at 5.5 to 5.9 pH, and treated with cancer in a 25° C. plant incubator (JSCC-250 cp, JSResearch, Korea) to induce callus. The induced callus was subcultured at least 4 times at intervals of 2 weeks to proliferate the cells, and after 8 weeks, the cells were subcultured at intervals of 4 weeks to induce somatic cells. For suspension culture, 1 g of callus was inoculated into a 500 mL flask containing 5.5 pH, 250 mL of MS base culture medium containing 2 mg/L of NAA and 1 mg/L of BA selected as the optimal medium, sealed with sterilized aluminum foil, and then at 120 rpm in a stirrer. Incubated at a rate. The temperature of the culture chamber was adjusted to 19 to 21° C., and the photoperiod was controlled by light treatment for 14 hours and dark treatment for 10 hours, followed by primary culture for 1 to 2 weeks. Thereafter, the suspension medium was first increased to 250 mL under the same culture conditions, and 250 mL of the suspension medium was secondarily increased in accordance with the callus growth rate, followed by a total of 4 to 6 weeks to induce neungsil callus.

Preparation of Neungsil Callus Culture Extract

The Neungsil callus culture extract was treated with starch degrading enzyme on Neungsil callus, extracted with ethanol, and then heated and pressurized at 70 to 100°C and 400 to 850 mbar to heat and concentrate 2 to 3 times, 40 to 80°C and 100 to 350 It was prepared by a method comprising heating and concentrating once to twice by reducing temperature at mbar.

Example 2: Confirmation of Cytotoxicity of Neungsil Callus Culture Extract

1-1) CCK-8 analysis

To confirm cytotoxic and anti-inflammatory effects, RAW264.7 macrophages and HaCaT cells were cultured and used. RAW264.7 macrophages and HaCaT cells were purchased from Korean Cell Line Bank (Korea), and DMEM medium supplemented with fetal bovine serum (FBS; Welgene, Korea) and penicillin/streptomycin (Invitrogen, USA) ( Welgene) was used for the culture of RAW264.7 macrophages and HaCaT cells. The cells were cultured in an incubator under conditions of 5% CO _{2, 37° C. and 100% humidity.}

To determine the maximum treatment concentration of HaCaT cells and RAW264.7 cells without cytotoxicity, a CCK-8 assay (cell counting kit-8) was performed, and the cell viability was measured by treating at various concentrations for 24 hours. After dispensing the cells into a 24 well-plate at 5.0×10 ⁴ cells/well, the cells were stabilized for one day. After removing the existing culture medium, ficin was treated on the cells by concentration and reacted at 37°C for 24 hours. After removing the culture medium, a solution of DMEM:EZ-Cytox diluted 10:1 was treated with 100 µl for each well and reacted for about 30 minutes, and then the absorbance was measured at 450 nm using an ELISA reader (BioTek Instruments Inc., USA). And, the cell viability was calculated based on the absorbance of the control group to which no cells were added. Cell viability was expressed as a percentage of the control.

Cell viability (%) = (Abssample / Abscontrol) × 100

Abscontrol: absorbance of the reaction solution without adding a sample

Abssample: absorbance of the reaction solution to which the sample was added

The control group was not treated with a sample, and each extract was treated at a concentration of 0.015 to 250 μg/mL to measure cell viability. As a result, in RAW 264.7 macrophages, cytotoxicity was observed at a concentration of 15 μg/mL or higher in the case of the extract obtained by extracting the shell and kernel at the same time, and cytotoxicity was observed at a concentration of 0.15 μg/mL or higher, which is a lower concentration in the case of the peel extract. On the other hand, it was observed that the extract from which the skin was removed and only the kernel was extracted had no cytotoxicity even at a high concentration of 150 μg/mL. Neungsil callus culture extract was confirmed that the cytotoxicity does not appear even at a concentration of 250 μg / mL (Fig. 1).

In HaCaT cells, cytotoxicity was observed at a concentration of 150 μg/mL or higher in the case of the extract obtained by extracting the shell and kernel at the same time, and cytotoxicity was observed at a concentration of 50 μg/mL or higher, which is a lower concentration in the case of the peel extract. On the other hand, it was observed that the extract from which only the shell was removed was not cytotoxic even at a high concentration of 150 μg/mL. In addition, it was confirmed that no cytotoxicity appeared even at a concentration of 250 μg/ml in the culture extract of Neungsil callus (FIG. 2).

As a result, it was confirmed that the cellulite fruit peel extract exhibited cytotoxicity at the lowest concentration among the extracts, and the neungsil callus cultured extract had no cytotoxicity even at a high concentration.

1-2) MTT analysis

RAW264.7 cells purchased from Korea Cell Line Bank were inoculated with 2 × 10 ^{6 cells/dish in a 100 mm 2} culture dish, and then DMEM containing 100 IU/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal calf serum (FBS). The medium was added and cultured in an incubator (Sanyo, Japan) containing 5% carbon dioxide at 37°C. RAW264.7 cells were subcultured at intervals of 2 days.

The MTT assay measures the ability of mitochondria to reduce MTT tetrazolium, a yellow water-soluble substrate, to red-purple formazan by the action of cell dehydrogenase. Reduction of MTT occurs only in cells with active metabolism. Therefore, it is widely used to evaluate the cytotoxicity of samples.

The subcultured RAW264.7 cells were dispensed into a 96-well plate at 1×10 ⁵ cells/well and cultured in an incubator containing 5% carbon dioxide at 37° C. for 24 hours, and then the culture medium was replaced with a serum-free medium. Subsequently, the cells were treated with Neungsil callus culture extract diluted so that the final treatment concentrations were 12.5, 25, 50, and 100 μg/mL, and the positive control was SDS diluted so that the final treatment concentrations were 1, 10, 100, and 1000 μg/mL. Each of the cells was treated and cultured in cell culture conditions for 24 hours. After incubation, MTT solution was added to each well and reacted for 2 hours in a light-shielding state, and then the supernatant was removed, and the resulting formazan was completely dissolved in DMSO, and the absorbance was measured at 540 nm with a microplate reader (Biotek Synergy-HT, USA). I did.

As a result, cytotoxicity of -3.00% at 12.5 μg/mL, -2.64% at 25 μg/mL, -1.02% at 50 μg/mL, -0.50% at 100 μg/mL in the treated group of Neungsil callus culture extract compared to the untreated group As shown, it was confirmed that the culture extract of Neungsil callus did not affect cytotoxicity at all treatment concentrations (FIG. 3).

Example 3: Measurement of polyphenol content

The polyphenol content of each of the fruit husks, kernels, husks + grains extracts, and cultured extracts of fruit callus were measured by applying the Gutfinger method (Gutfinger T., Journal of the American Oil Chemists Society, Vol. 58, No. 11, 1981). I did. 0.5 ml of 50% Foiln-Ciocalteu's phenol reagent was added to 1 ml of the extracted sample solution, and reacted at room temperature for 3 minutes. The reaction solution was _{sequentially mixed with 1 ml of saturated Na 2} CO ₃ solution and 7.5 ml of distilled water, allowed to stand for 30 minutes, centrifuged at 12,000 rpm for 10 minutes, and then the supernatant was taken and the absorbance was measured at 760 nm. The total polyphenol content was calculated according to the calibration curve prepared by using gallic acid as a standard material, and GAE (Gallic acid equivalent)/g was used as the unit of measurement.

The total polyphenol content in each extract was tested three times, and the average value and standard deviation of the values were calculated, and the standard calibration curve of the gallic acid standard solution contained per 1 g of the weight of the extract and solvent fraction. It was measured in terms of the amount of gallic acid (GAE; gallic acid equivalent). As a result of measurement using gallic acid as a standard material, it is shown in Table 1 below.

In the fruit of the fruit, a lot of polyphenol, 213.20±15.78mg/g GAE, was contained in the skin, and the polyphenol content of the culture extract of the fruit callus was also very high. Polyphenol is a cosmetic ingredient related to antioxidant and whitening effects, and the results related to polyphenol content are related to the antioxidant and whitening effects of Neungsil extract.

sample Total polyphenol content (mg/g ext.) Neptune fruit kernel extract 19.04±0.58 Fruit Fruit Peel Extract 213.20±15.78 Fruit kernel + peel extract 174.33±10.69 Neungsil callus culture extract 205.2±18.97

Example 4: Measurement of flavonoid content

The flavonoid content of each of the fruit peel, kernel, peel + kernel extract, and the culture extract of Neungsil callus were measured by applying the Nieva Moreno method (Mihye Kim, Ph.D. Thesis, Nambu University, 2012). To 0.5 ml of a mixture of 0.1 ml of each sample and 0.9 ml of 80% ethanol, 0.1 ml of 10% aluminum niatate, 0.1 ml of 1M potassium acetate, and 4.3 ml of 80% ethanol were added and allowed to stand at room temperature for 40 minutes, and the absorbance was measured at 415 nm. The content was calculated from the standard curve prepared using (quercetin).

The total flavonoid content in each extract was tested three times, and the average value and standard deviation of the values were calculated, and the quercetin contained per 1 g of the weight of the extract and solvent fraction was calculated through the standard calibration curve of the quercetin standard solution. It was measured in terms of quantity (QE; quercetin equivalent). Table 2 shows the results of measurement using quercetin as a standard material. Like the total polyphenol content, the total flavonoid content was higher in the skin of the fruit extract. Flavonoids are cosmetic ingredients related to antioxidant and anti-aging effects, and the results related to flavonoid content are related to the antioxidant and anti-aging effects of Neungsil extract.

sample Total flavonoid content (mg/g ext.) Neptune fruit kernel extract 8.24±0 Fruit Fruit Peel Extract 29.30±3.24 Fruit kernel + peel extract 27.24±1.98 Neungsil callus culture extract 37.4±7.43

Example 5: Measurement of antioxidant activity by DPPH assay

Since the whitening activity is related to free radicals acting directly on melanocytes, it is known that the antioxidant activity is closely related to the whitening effect. Therefore, it was confirmed through DPPH analysis whether or not the antioxidant activity of the extract of neungsil fruit (bark, kernel, husk + kernel, respectively) and the culture of neungsil callus.

After adding 100 μl of ficin solution to 200 μl of 100 μM DPPH solution, it is reacted at 37° C. for about 30 minutes. Thereafter, absorbance was measured at 517 nm to observe the degree of decrease in absorbance. The DPPH radical scavenging ability was expressed as a percentage of the absorbance difference between the sample itself and the sample-free group (control) and the sample-added group (sample).

DPPH inhibition rate (%) =

As a result of measuring the DPPH scavenging rate of each extract, when compared to the control vitamin C, the fruit kernel extract showed better scavenging ability at a concentration of 500 μg/ml. In the case of the fruit peel extract, it was confirmed that it exhibited more than 75% of radical scavenging activity even at a low concentration of 0.005㎍/㎖, indicating excellent antioxidant activity. This is believed to be related to the content of antioxidants such as polyphenols and flavonoids. In the case of the fruit peel extract, it shows cytotoxicity at a concentration of 0.15µg/ml or more, so it is required to be used at a low concentration, but exhibits excellent antioxidant activity even at a low concentration (Fig. 4).

In the case of the simultaneous extract of fruit kernel + peel, as shown in the cytotoxicity test results, the median value between the kernel fruit kernel extract and the peel extract of the fruit fruit was shown. At a concentration of 50µg/ml or more, it was confirmed that the radical scavenging activity was similar to that of vitamin C, and there was no radical scavenging activity at a concentration below that.

In the case of the culture extract of Neungsil callus, the radical scavenging ability is improved in a concentration-dependent manner, and even at a concentration of 0.05 μg/ml or more, the radical scavenging ability is much better than that of the control vitamin C, and thus the antioxidant effect is evaluated to be very excellent. In the cytotoxicity test, it was confirmed that the extract of Neungsil callus culture had no cytotoxicity even at a high concentration of 250㎍/㎖ or more, and had excellent radical scavenging ability even at a low concentration.

In addition, an experiment was performed to confirm the DPPH radical scavenging ability of the culture extract of Neungsil callus using ascorbic acid, which is known to have an antioxidant effect. To each well of a 96-well plate, 100 μL of 0.15 mM DPPH solution prepared using methanol and 100 μL of cultured callus extract diluted with ultrapure water to a final treatment concentration of 12.5, 25, 50, and 100 μg/ml were added, respectively, In the positive control group (ascorbic acid), 100 μL of diluted ascorbic acid was added to the final treatment concentrations of 0.1, 1, 10, and 100 μg/ml. After the light-shielding reaction at room temperature for 30 minutes, the absorbance of the reaction solution was measured at 540 nm using a microplate reader and shown in the figure. Neungsil callus culture extract showed excellent DPPH radical scavenging ability compared to the control (FIG. 5).

Example 6: Measurement of whitening effect

In melanoma cultured cells, the effect of inhibiting melanin synthesis was confirmed by the extract of cultured callus of Neungsil. In order to induce melanin synthesis, 50nM a-MSH (melanin stimulating hormone) was used, and as a positive control, arbutin used as a whitening agent was treated. B16-F10 melanoma cells were dispensed into 6 well plates at 2×10 ⁵ cells per well and then stabilized for 24 hours. Cells were treated with 50nM a-MSH, diluted with 0.1% callus culture extract or 0.1% arbutin, and cultured for 5 days in DMEM medium containing 1% FBS and 1% PS. After cultivation, the supernatant was taken and the absorbance was measured at 405 nm to measure the amount of melanin synthesis, and the inhibitory effect of melanin synthesis by the culture extract of Neungsil callus was measured.

In melanoma cells, in the experimental group treated with a-MSH alone, melanin synthesis increased significantly, but in the experimental group treated with Neungsil callus culture extract, the melanin content decreased to almost the same level as the positive control arbutin. Through these results, it was confirmed that the neungsil callus culture extract has an excellent whitening effect that effectively inhibits melanin.

Example 7: Measurement of anti-inflammatory effect by Nitric Oxide (NO) analysis

In order to investigate the anti-inflammatory activity of the extracts of Neungsil fruit (shell, kernel, husk + kernel, respectively) and Neungsil callus culture extract, the inhibitory ability of NO production by LPS stimulation was investigated in RAW 264.7 cells. Neungsil fruit kernel extract showed a significant decrease at the concentration of 25㎍ / ㎖ or more. In addition, Neungsil fruit peel extract showed a significant decrease at a concentration of 5㎍/㎖ or more. Neungsil fruit kernel + bark extract showed a significant decrease at concentrations of 5㎍/㎖ or more.

Neungsil callus culture extract showed a significant decrease at a concentration of 5㎍ / ㎖ or more. Both the extract for each site of neungsil and the callus culture extract had the effect of inhibiting NO production, and in particular, the neungsil bark extract and the neungsil callus culture extract had excellent NO inhibitory effects even at low concentrations (Table 3, FIG. 6).

However, in the case of Neungsil fruit peel extract, since cytotoxicity is a concern, it is preferable to use Neungsil kernel extract and Neungsil callus culture extract showing excellent results in terms of cytotoxicity as a cosmetic material requiring safety.

sample Extract content (㎍ / ㎖) NO generation amount
(%, 100% control) NO generation amount by LPS
(%, control 100%) Neptune fruit kernel extract 6.25 179.2±4.93, 189.0±8.29 12.5 172.5±4.36 25 162.6±4.58 50 154.1±7.14 Fruit Fruit Peel Extract 5 158.1±4.25 205.5±9.43 10 151.5±4.04 25 100.0±3.51 50 92.9±1.96 Fruit kernel + peel extract 5 183.3±5.69 202.4±0.95 10 167.6±7.79 25 110.7±4.87 50 95.3±1.02 Neungsil callus culture extract 5 160.1±2.89 203.8±5.61 10 146.8±2.56 25 110.8±4.98 50 101.5±3.45

In addition, an experiment was performed to confirm the inhibitory ability of NO production of the conventionally known anti-inflammatory substance (NG-Methyl-L-arginine acetate salt, L-NMMA) and Neungsil callus culture extract as a positive control. RAW264.7 cells were cultured under the same conditions as in Example 2. Cells were treated with Neungsil callus culture extract diluted so that the final treatment concentrations were 12.5, 25, 50, and 100µg/ml and 1ppm LPS, and then diluted so that the final treatment concentrations were 5, 10, 20, and 40µg/ml. The cells were treated with a positive control (L-NMMA) and 1 ppm of LPS, and cultured for 24 hours under cell culture conditions. After cultivation, 50 μL of griess reagent was added to 50 μL of the cell culture supernatant, followed by a light-shielding reaction for 10 minutes, and absorbance was measured at 540 nm. Nitric oxide production inhibitory ability test results As a result of measuring the NO production inhibitory ability induced by LPS of the cultured callus extract of Neungsil callus, it was confirmed that the inhibitory ability was improved in a concentration-dependent manner (FIG. 7 ).

Example 8: Measurement of the effect of removing stretch marks of a cosmetic composition

Measurement of the effect of improving stretch marks

In order to measure the stretch marks improvement effect of the cream prepared according to Preparation Example 1, 40 experimenters (20 to 35 years old women) were divided into two groups of 20 people, and for each group, Formulation Example 1 ( The cream of Table 4) was applied twice a day for 3 consecutive months.

ingredient density(%) Neungsil callus culture extract 1.00 Cabbage extract 10.0 Spirulina Maxima Extract 0.10 Butylene Glycol 30.0 1,2-hexanediol 2.00 Purified water 56.9

The effect of improving stretch marks was evaluated by measuring changes in the length (mm), width (mm), and chromaticity of the stretch marks over time. Before applying the cream to the skin, the area and length of the stretch marks were measured and measured at 0, 4, 8, and 12 weeks, respectively, to observe the change pattern. The change in the color of the stretch marks was measured using a video microscope equipped with image anasoftware.

division 0 weeks 4 weeks 8 weeks 12 weeks Manufacturing Example 1 Length(mm) 40.5 38.9 37.1 34 Width(mm) 4.2 4.1 3.9 3.5 Chromaticity 4.8 4.5 3.6 3.0

Referring to Table 5, the skin stretch marks of the test subject to which the cream of Formulation Example 1 containing the culture extract of Neungsil callus was applied was alleviated.

Claims (11)

A cosmetic composition comprising a culture extract of neungsil callus as an active ingredient. The cosmetic composition of claim 1, wherein the composition is for skin antioxidant. The cosmetic composition of claim 1, wherein the composition is for skin whitening. The cosmetic composition according to claim 1, wherein the neungsil callus culture extract is extracted with C1-C4 alcohol. The cosmetic composition according to claim 1, wherein the neungsil callus culture extract is extracted using 50 to 90% (v/v) ethanol as an extraction solvent. The method of any one of claims 1 to 5, wherein the composition is a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing containing surfactant, oil, powder foundation, emulsion foundation, A cosmetic composition having a formulation selected from the group consisting of wax foundation and spray. A health functional food for skin whitening, comprising the extract of neungsil callus culture as an active ingredient. The health functional food for skin whitening according to claim 7, wherein the neungsil callus culture extract is extracted with C1-C4 alcohol. A health functional food for antioxidants that contains a cultured extract of neungsil callus as an active ingredient. The antioxidant health functional food of claim 9, wherein the neungsil callus culture extract is extracted with C1-C4 alcohol. A pharmaceutical composition for the prevention or treatment of pigmented diseases, having an inhibitory effect on melanin synthesis and an antioxidant effect, comprising the extract of neungsil callus as an active ingredient.

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