KR102304419B1 - Multifunctional cosmetic composition that contains amino acids and peptides derived from Moringa seeds, effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles - Google Patents

Abstract

The present invention provides a multifunctional cosmetic composition which is effective in skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles. More specifically, the multifunctional cosmetic composition according to the present invention is a hydrolysate obtained by hydrolyzing a protein extracted from moringa seeds with a proteolytic enzyme, the multifunctional cosmetic composition comprising 5-15 wt% of a free amino acid, 70-90 wt% of an oligopeptide, and 5-15 wt% of a polypeptide.

Description

Multifunctional cosmetic composition that contains amino acids and peptides derived from Moringa seeds, effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles by containing amino acids and peptides derived from Moringa seeds , skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles}

The present invention relates to a multifunctional cosmetic composition containing amino acids and peptides derived from moringa seeds, which is effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles.

The genus Moringa has several 14 plant species (Moringa peregrina, M. aptera, M. concanensis, M. drouhardii). , including M. hildebrandtii, M. Longituba), among which Moringa pterygosperma (Moringa oleifera) ) are best known, these are fast-growing trees that are very well adapted to a variety of conditions, growing throughout the tropics of Asia, Africa and South America. The fruit, 30-50 cm long, hangs like a drumstick, hence its English name "drumstick tree", and its green pods are recognized as a vegetable. As a result, seeds are rarely left ripe for oil production.

Other parts of this tree (leaf, root, root bark, flower and seed) are used in traditional medicine in the country where it is grown.

Moringa seeds are characterized by the presence of oils whose content varies between 21 and 53% depending on the species and maturity of the seed. The content of Moringa oleifera species mentioned in the literature ranges from 21 to 34%.

Due to its excellent oxidative stability and excellent flavor fixing properties, moringa oil, also called Bhen or Ben oil, was the most used oil in ancient civilizations for cosmetic and religious uses. This oil was used by cosmetic manufacturers until the last century, and its use has recently been "rediscovered".

More recently, besides oil, moringa seeds have attracted researchers' attention for protein extracts. Accordingly, European Patent Application No. 1064008 describes the use of Moringa seed protein extract on the skin and mucous membranes for its softening, physiological conditioning, moisturizing, restructuring, restorative effects, and as an anti-wrinkle and anti-fouling agent. have.

In addition, Republic of Korea Patent No. 10-191566 discloses a composition for external application for skin whitening containing an extract of germinated Moringa seeds as an active ingredient and a composition for external application for skin whitening containing a subcritical extract of Moringa seeds as an active ingredient This is disclosed.

However, there is no mention of improving the skin cosmetic effect through appropriate adjustment of the content ratio of amino acids, polypeptides and oligopeptides derived from Moringa seeds.

1. Republic of Korea Patent No. 10--191566

Therefore, the problem to be solved by the present invention is a multifunctional cosmetic that contains moringa seed-derived amino acids, oligopeptides, and polypeptides in appropriate proportions to moisturize the skin, whiten the skin, maintain and improve skin elasticity, and prevent skin wrinkles to provide a composition.

In order to achieve the above technical problem, the present invention

As a hydrolyzate obtained by hydrolyzing proteins extracted from Moringa seeds with proteolytic enzymes,

5 to 15% by weight of free amino acids;

oligopeptide 70 to 90% by weight; and

Polypeptide 5 to 15% by weight

It provides a multifunctional cosmetic composition effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles by containing it.

In the composition according to the present invention as described above, the Moringa seeds are Moringa seeds germinated after immersion in 1 wt% fulvic acid aqueous solution for 24 hours.

In the composition according to the present invention as described above, the germinated Moringa seeds are mixed with tourmaline powder in a volume ratio of 1:1 before hydrolysis, and then maintained for 24 hours.

In the composition according to the present invention as described above, the oligopeptide has a molecular weight of 300 to 1,500 Daltons (Da), and the polypeptide has a molecular weight of 3,000 to 15,000 Daltons (Da).

In addition, the present invention, after immersion in 1 wt% fulvic acid aqueous solution for 24 hours, the germinated Moringa seeds are mixed with tourmaline powder in a volume ratio of 1:1 and maintained for 24 hours, followed by hydrolysis with proteolytic enzyme Provided is a multifunctional cosmetic composition effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles by containing decomposition products.

The composition of the present invention contains amino acids, oligopeptides, and polypeptides derived from moringa seeds in an appropriate ratio to show multifunctionality effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles, so its value as a cosmetic raw material is very high. high.

Hereinafter, specific contents for easily carrying out the present invention will be described in detail through examples and the like.

The composition of the present invention optimizes the skin cosmetic effect by appropriately adjusting the content ratio of amino acids, oligopeptides, and polypeptides derived from Moringa seeds. It is characterized by using linga seeds, and it is characterized in that the hydrolyzate using moringa seeds that have undergone pre-treatment, germination, and post-treatment is used as an active ingredient.

Specifically, the present invention

the present invention

As a hydrolyzate obtained by hydrolyzing proteins extracted from Moringa seeds with proteolytic enzymes,

5 to 15% by weight of free amino acids;

oligopeptide 70 to 90% by weight; and

Polypeptide 5 to 15% by weight

It provides a multifunctional cosmetic composition effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles by containing it.

In the composition according to the present invention as described above, the Moringa seeds are Moringa seeds germinated after immersion in 1 wt% fulvic acid aqueous solution for 24 hours.

In the composition according to the present invention as described above, the germinated Moringa seeds are mixed with tourmaline powder in a volume ratio of 1:1 before hydrolysis, and then maintained for 24 hours.

In the composition according to the present invention as described above, the oligopeptide has a molecular weight of 300 to 1,500 Daltons (Da), and the polypeptide has a molecular weight of 3,000 to 15,000 Daltons (Da).

In addition, the present invention is a hydrolyzate hydrolyzed with a proteolytic enzyme after mixing the germinated Moringa seeds with tourmaline powder in a volume ratio of 1:1 after immersion in 1 wt% fulvic acid aqueous solution for 24 hours and maintaining for 24 hours. It is characterized in that it uses as an active ingredient.

The Moringa seed used in the present invention is a seed of the Moringa tree, and the Moringa tree is a plant of the Moringa family (Moringaceae Family) and the genus Moringa (Moringa Genus), and the scientific name is Moringa Oleifera (synonym Moringa Pterygosperma), and the scientific name is derived. Also known as Murunggai in one Tamil language. Moringa is a legume plant and is a tropical tree that eats the whole tree as well as the leaves and fruits.

In particular, the water-soluble amino acids contained in Moringa seeds play a role in purifying contaminants in water. When these amino acids are applied to the skin, they show a detoxifying effect that purifies the skin of contaminants and makes the skin clean.

In addition, since it contains a large amount of carotenoids such as beta-carotene and rutin, as well as vitamins and minerals, it can brighten dull skin and realize a whitening effect.

In the present invention, germinated Moringa seeds are used, and the germinated Moringa seeds contain more effective ingredients necessary for life phenomena compared to the pre-germination state, so that stronger efficacy can be provided.

The germination conditions of Moringa seeds are not particularly limited, but examples that can be used by germinating in the following way include soaking in room temperature water for about 24 hours and then transferring them to an incubator and incubating them for 48 to 72 hours, in which case the room temperature is 25℃ It should be kept warmer than that.

However, the present invention provides a novel and advanced composition with improved effect, characterized in that it is carried out after soaking for about 24 hours in an aqueous solution of fulvic acid containing 1.0 wt% of fulvic acid before germination, unlike in the conventional germinating stage of Moringa seeds can do.

In addition, the degree of germination is not particularly limited, but it is most preferable to germinate up to 0.5 to 2 cm in length of shoots according to various tests of the present inventors.

The fulvic acid is extracted by a purified water extraction method of extracting using purified water from a natural humic substance. To be more specific, purified water is added so that the concentration of the natural humic substance is 5-10%, and then in a hot water of 60-70 ℃. The supernatant obtained by stirring is filtered/concentrated and extracted.

In addition, fulvic acid may be extracted by an extraction method by strong acid/strong base treatment, which is a general extraction method set by the International Humic Substances Society, an international society. In more detail, natural humic material is extracted by treatment with strong acid (0.1M HCl), or dissolved fulvic acid by precipitation of humic acid in strong acid (6M HCl, pH 1) after treatment with strong base (0.1M NaOH, pH 10) is separated and extracted.

In the present invention, the fulvic acid may be extracted by various methods other than the above methods, and the separated fulvic acid is added with filtration, adsorption chromatography, cation exchange resin chromatography and dialysis to remove foreign substances and increase the purity. It can also be carried out, and freeze-drying can be carried out to further increase the concentration of fulvic acid.

In addition, the fulvic acid of the present invention may be used by purchasing a commercially available known material, and if the known material has low purity, additional purification methods such as the above may be added.

In addition, in order to further improve the above-described effect, the present invention may further include the step of mixing the germinated Moringa seeds with tourmaline powder in a 1:1 volume ratio before hydrolysis and then aging for 24 hours.

The tromaline is also called tourmaline, and the powder used as a complex borosilicate mineral containing iron, magnesium, alkali metals, and aluminum as a chemical component is more effective as it is finer because it is good if there is a lot of contact with Moringa seeds.

In addition, hydrolysis using a protease in the present invention can be used without any particular limitation as long as it is widely known in the art to which the present invention pertains. Examples thereof include hydrolysis using papain, peptidase R, Flavozyme, ProteAX, and Alcalase.

In addition, in the hydrolyzate obtained through hydrolysis, undigested proteins, polypeptides, oligopeptides, and free amino acids are mixed in a certain ratio. 5 to 15% by weight of amino acids; oligopeptide 70 to 90% by weight; And by adjusting to contain 5 to 15% by weight of the polypeptide, it provides a multifunctional cosmetic composition in which the effect of skin moisturizing, skin whitening, skin elasticity maintenance and improvement, and skin wrinkle prevention is significantly enhanced. In addition, in order to enhance the effect in the detailed test of the present invention, the oligopeptide preferably has a molecular weight of 300 to 1,500 Daltons (Da), and the polypeptide has a molecular weight of 3,000 to 15,000 Daltons (Da).

The composition of the present invention may be contained in an amount of 0.01 to 95.0% by weight, preferably 5.0 to 95.0% by weight, more preferably 10.0 to 90.0% by weight based on the total weight of the cosmetic. If the content of the peptide is less than 0.01% by weight, it is difficult to expect the skin efficacy of the component, and if it exceeds 95.0% by weight, the efficacy is not increased, and the efficiency is lowered.

In addition, the composition of the present invention may further contain a known substance exhibiting a cosmetic effect. Specifically, it may further contain one or more selected from the group consisting of ascorbyl glucoside, licorice extract, arbutin, ascorbic acid, kojic acid and niacinamide, which is contained in an amount of 0.5 to 5.0% by weight based on the total weight of the composition. . This is because if it is less than 0.5% by weight, it is difficult to show efficacy by being absorbed into the skin, and if it is more than 5.0% by weight, it is difficult to show the value as a cosmetic because it is toxic to the skin or the stickiness of the skin is greatly increased.

The composition of the present invention provides excellent antioxidant power, skin whitening, skin elasticity enhancement or skin wrinkle improvement, etc. skin aging prevention effect, skin moisturizing power improvement effect, anti-inflammatory effect, skin trouble improvement effect such as acne and atopy, sebum control It is possible to provide an overall improvement effect of the overall skin condition, such as effect, pore contraction effect, keratin improvement effect, skin convergence or preparation effect, complexion improvement by improving blood circulation, skin irritation alleviation effect, homeostasis improvement effect, and the like.

The composition according to the present invention may be formulated containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example, solutions, gels, solids, kneaded dry products, emulsions obtained by dispersing an oily phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) and non It may be provided in the form of an ionic vesicle dispersant, or in the form of a cream, skin, lotion, powder, ointment, spray or concealer stick. In addition, it may be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions can be prepared according to conventional methods in the art.

In addition, the composition according to the present invention is a fatty substance, an organic solvent, a solubilizer, a thickening agent, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic or non Ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredients commonly used in cosmetics; It may contain adjuvants commonly used in the field of cosmetology or dermatology. The adjuvant is introduced in an amount generally used in the field of cosmetology or dermatology.

In addition, the composition of the present invention may contain a skin absorption promoting material in order to increase the skin cosmetic effect.

Hereinafter, the configuration and effect of the present invention will be described in more detail through Examples and Test Examples. However, these Examples and Test Examples are provided only for the purpose of illustration to help the understanding of the present invention, and the scope and scope of the present invention are not limited by the following examples.

< Example : Composition Preparation>

One. Example One

Moringa oleifera seeds were powdered and then fat was removed to about 0.5 wt% using hexane, and the fat removed powder was dried at room temperature for 24 hours.

0.5 g of the powder was mixed in a 0.4M NaCl solution for extracting protein from the powder at a 20:1 ratio (v/w) for 1 hour and then centrifuged (6000g, 15 minutes, room temperature) to dissolve the suspension and insoluble The material was isolated.

Then, the suspension is diluted 10-fold with cold water at 8 to 10 ° C., and the solution is prepared by mixing the protein precipitate obtained by centrifugation (12,000 g, 10 minutes, 10 ° C) with purified water to a concentration of 25 wt %, papain (papain), peptidase R (peptidase R), flavozyme (Flavourzyme), protease (ProteAX), and Alcalase (Alcalase) was treated by mixing 1% by weight of each enzyme.

The treated solution was adjusted to a final pH of 6.0 using 1M NaOH, and the reaction was carried out at 50 ° C. under 40 rpm conditions. After 24 hours, the solution was boiled for 10 minutes to stop the enzymatic reaction, suspended, and dried after centrifugation. A powdered moringa seed hydrolyzate was obtained.

The powder was dissolved at a concentration of 20 mg/mL using distilled water, and then separated into free amino acids, oligopeptides, and polypeptides through membrane filtration, and dried to powder, respectively, wherein the oligopeptide had a molecular weight of 300 to 1,500 Daltons (Da). and the polypeptide has a molecular weight of 3,000 to 15,000 Daltons (Da).

Then, 10% by weight of the free amino acid, 85% by weight of the oligopeptide; and 5% by weight of the polypeptide to complete the composition according to the present invention.

2. Example 2

After immersing Moringa oleifera seeds in a 1% by weight aqueous solution of fulvic acid for 24 hours, transfer them to an incubator and incubate while keeping them warm at 25°C or higher. It is different from Example 1 in that linga oleifera seeds were mixed with the same volume of tourmaline powder and used after aging for 24 hours.

3. Example 3

After immersing Moringa oleifera seeds in a 1% by weight aqueous solution of fulvic acid for 24 hours, transfer them to an incubator and incubate while keeping them warm at 25°C or higher. Ringa oleifera seeds were mixed with the same volume of tourmaline powder, aged for 24 hours, and then powdered. Then, about 0.5% by weight of fat was removed using hexane, and the fat removed powder was dried at room temperature for 24 hours.

0.5 g of the powder was mixed in a 0.4M NaCl solution for extracting protein from the powder at a 20:1 ratio (v/w) for 1 hour and then centrifuged (6000g, 15 minutes, room temperature) to dissolve the suspension and insoluble The material was isolated.

Then, the suspension is diluted 10-fold with cold water at 8 to 10 ° C., and the solution is prepared by mixing the protein precipitate obtained by centrifugation (12,000 g, 10 minutes, 10 ° C) with purified water to a concentration of 25 wt %, papain (papain), peptidase R (peptidase R), flavozyme (Flavourzyme), protease (ProteAX), and Alcalase (Alcalase) was treated by mixing 1% by weight of each enzyme.

The treated solution was adjusted to a final pH of 6.0 using 1M NaOH, and the reaction was carried out at 50 ° C. under 40 rpm conditions. After 24 hours, the solution was boiled for 10 minutes to stop the enzymatic reaction, suspended, and dried after centrifugation. The composition of Example 3 was completed by obtaining a hydrolyzate of Moringa seeds in a powder state.

4. comparative example

Moringa oleifera seeds were powdered and then fat was removed to about 0.5 wt% using hexane, and the fat removed powder was dried at room temperature for 24 hours.

0.5 g of the powder was mixed in a 0.4M NaCl solution for extracting protein from the powder at a 20:1 ratio (v/w) for 1 hour and then centrifuged (6000g, 15 minutes, room temperature) to dissolve the suspension and insoluble The material was isolated.

Then, the suspension is diluted 10-fold with cold water at 8 to 10 ° C., and the solution is prepared by mixing the protein precipitate obtained by centrifugation (12,000 g, 10 minutes, 10 ° C) with purified water to a concentration of 25 wt %, papain (papain), peptidase R (peptidase R), flavozyme (Flavourzyme), protease (ProteAX), and Alcalase (Alcalase) was treated by mixing 1% by weight of each enzyme.

The treated solution was adjusted to a final pH of 6.0 using 1M NaOH, and the reaction was carried out at 50 ° C. under 40 rpm conditions. After 24 hours, the solution was boiled for 10 minutes to stop the enzymatic reaction, suspended, and dried after centrifugation. A composition of Comparative Example was completed by obtaining a hydrolyzate of Moringa seeds in a powder state.

< test example 1: Evaluation of in-vitro whitening efficacy>

1) Measurement of tyrosinase inhibitory activity

● Experimental method

In 0.5 ml of 0.175M sodium phosphate buffer (pH 6.8), 0.2 ml of 10 mM L-DOPA, a substrate solution, and 0.1 ml of a sample solution are added to a 96 well plate and mixed. Add 0.2 ml of mushroom tyrosinase (110 U/ml) to the mixture, react at 25°C for 2 minutes, and measure the DOPA chrome generated in the reaction solution at 475 nm. Tyrosinase inhibitory activity is indicated by the decrease in absorbance of the group with and without the sample solution.

Inhibition rate (%) = 1-(absorbance in the sample added group / absorbance in the non-sample group) x 100

● Experiment result

Tyrosinase plays a very important role in the production of melanin in the skin. It is a tyrosine hydroxylase that oxidizes tyrosine in melanosome to make DOPA. It oxidizes DOPA to make DOPA quinone and acts as an important enzyme for synthesizing melanin polymer. The whitening effect can be seen through the tyrosinase inhibitory activity, and the tyrosinase inhibitory activity was measured and the results are shown in Table 1. Arbutin was used as a positive control.

Sample Concentration (μg/ml) 125 250 500 1000 2000 comparative example 29.1±2.2 37.7±1.2 40.4±1.5 47.5±0.6 55.5±2.5 Example 1 55.2±0.8 63.9±0.9 81.1±0.7 87.6±0.8 90.2±2.1 Example 2 60.2±2.5 72.3±0.8 87.1±1.2 92.5±1.7 95.4±2.3 Example 3 69.4±2.2 70.2±0.4 84.3±0.8 90.2±0.9 92.1±2.1 Arbutin 53.2±1.0 60.3±1.5 78.7±0.6 84.6±1.2 88.7±2.5

Looking at the results in Table 1, as a result of measuring the tyrosinase inhibitory activity of the comparative example, it was found that the tyrosinase inhibitory activity according to the concentration of 125 µg/ml to 2000 µg/ml was concentration-dependent, but lower than that of Arbutin, a positive control. It was confirmed to have a level of tyrosinase inhibitory activity. However, Examples 1 and 2 exhibited higher tyrosinase inhibitory activity than Arbutin.

And it was able to confirm the more excellent effect in Example 2 and Example 3 rather than Example 1.

2) Measurement of melanin biosynthesis inhibitory activity

● Experimental method

The melanoma cell line, B16F10 cell, was purchased from the Korean Cell Line Bank, and Dulbecco's modified Eagle's containing 1% antibiotic (Gibco, USA) and 10% fetal bovine serum (FBS; Gibco, Grand Island, USA) medium (DMEM) medium was used at 37° C., 5% CO ₂ Incubated in an incubator, and subculture was performed once every 2 days.

B16F10 cells cultured in DMEM medium are inoculated in a 24-well plate at 2.0 X 10 ⁴ cells/well. After incubation for 24 hours at 37°C, 5% CO _{2 in an} incubator, samples are prepared by concentration. In order to stimulate the samples and cells for each concentration, α-MSH, a stimulant, is mixed and added at 200 nM. After culturing for 48 hours at 37°C, 5% CO _{2 in an} incubator, remove the culture medium and wash each well with phosphate buffer (pH 7.4). Add 100 μl of 1N NaOH at a time to remove the cells and transfer them to a 1.5 ml tube. After reacting at 80° C. for 1 hour, absorbance is measured at 405 nm in a spectrophotometer. Inhibition of melanin biosynthesis is indicated by the decrease in absorbance in the group with and without the sample solution.

● Experiment result

Melanin biosynthesis inhibitory activity measurement results are shown in Table 2 below.

sample Melanin contents (%) α-MSH(-) - α-MSH (+, 200 nm) 100 α-MSH (+, 200 nM) + Comparative Example 50 μg/ml 44.2 ± 3.4 α-MSH (+, 200 nM) + Example 1 50 μg/ml 21.7 ± 1.8 α-MSH (+, 200 nM) + Example 2 50 μg/ml 14.1 ± 2.5 α-MSH (+, 200 nM) + Example 3 50 μg/ml 15.9 ± 1.2

Looking at the results of Table 2, when compared to the α-MSH alone treatment group, it was possible to confirm the effect of more than twice the effect of the peptide of Example than the peptide of Comparative Example, and in particular, more excellent effects were confirmed in Examples 2 and 3 than in Example 1. could

< test example 2: in- in vivo Whitening Effect>

● Experimental method

An in vivo experiment was performed on the pigmentation inhibitory effect for the Examples and Comparative Examples.

After receiving a 6-week-old HRM2 hairless mouse from Central Laboratory Animals Co., Ltd. and acclimatizing it in the SPF animal breeding room for 1 week, the experiment was conducted, and water and feed were freely supplied.

Experimental groups were normal control group (purified water application), UV irradiation control group (UV + purified water application), positive control group (UV + Arbutin application), Example 1 application group (UV + Example 1 application), Example 2 application group (UV + Example 2 application), Example 3 application group (UV + Example 3 application) and Comparative Example application group (UV + Comparative example application), divided into 6 groups of 3 animals each, from 3 days before UV irradiation until the end of the experiment The drug was applied.

After anesthesia with ketamine (ip 2 ml/kg) for animal control, 100 mg of cream was applied to the entire back and absorbed for 30 minutes. It was carried out using a lamp emitting light, and the amount of ultraviolet light was measured with a UV-meter, and the irradiation amount was gradually increased to 100 mJ/cm, 150 mJ/cm, and 200 mJ/cm, and irradiated once a day for 5 days each.

The degree of pigmentation according to the coating material was measured using a colorimeter as the center of the L value in the same area, and it was performed once a day starting from 1 day before the sample application and until the end of the experiment. Measurements were made after anesthesia before sample application.

● Experiment result

The L* value (lightness value) measured with a colorimeter was calculated according to the following equation by setting the control group to which purified water was applied and UVB was not irradiated and the UVB group to which purified water was applied and irradiated with UVB was set as a control group, and the results are shown in Table 3 .

<Equation>

sample L* value Normal control group (purified water application) 1.2 ± 0.3 UV irradiation control group (UV + purified water applied) -3.5 ± 0.5 Positive control group (UV + Arbutin application) 0.3 ± 0.2 Comparative example application group (UV + comparative example application) -2.9 ± 0.3 Example 1 application group (UV + Example 1 application) 0.7 ± 0.2 Example 2 application group (UV + Example 2 application) 0.9 ± 0.4 Example 2 application group (UV + Example 2 application) 0.8 ± 0.2

As a result of the experiment in Table 3, when looking at the UV irradiation control group, it was found that the L* value was lowered due to pigmentation during UVB irradiation. It could be confirmed that the anti-pigmentation was prevented, but there was a slight difference from the normal control group, indicating that the pigmentation could not be completely suppressed.

However, when the Example according to the present invention was applied, it showed a difference compared with the UV irradiation control group, and a significant difference could be confirmed, and even more, it showed a better effect than the Arbutin application used as a positive control group, confirming the possibility of commercialization.

In addition, in Examples 2 and 3, it was more excellent than Example 1, and the in-vivo effect was hardly confirmed in the comparative example.

< test example 3: Skin Moisturizing>

1. Cytotoxicity experiments

The cytotoxicity test was performed by Moseman's method (Moseman TJ, Immuno Methods, 65, 55 (1983)) and Skaper et al. (Skaper SD, et al, Cell Culture, 2, 17 (1990)) using MTT (3-4). ,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide) was measured.

Human keratinocytes HaCaT were purchased from the Korean Cell Line Bank (Seoul, Korea) and used. HaCaT was cultured in an animal cell incubator at 37°C and 5% CO2 supply condition in a laboratory using DMEM medium containing antibiotics of 10% (V/V) FBS, penicillin 100U/mL, and streptomycin 100g/mL.

The compositions of Examples and Comparative Examples were dissolved in DMSO and added to the culture medium at 100 g/mL, and the cells were cultured in the culture medium for 24 hours. After treatment with MTT solution (5 mg/mL) and further incubated for 4 hours, the amount of the produced blue crystal formazan was measured for absorbance at 595 nm using an ELISA reader. Toxicity to cells was expressed as a percentage of the average absorbance value of each control group.

To investigate cytotoxicity in HaCaT keratinocytes, when treated at a concentration of 100 g/mL, cytotoxicity was investigated by calculating the degree of change in cell proliferation compared to that treated only with DMSO, a control.

In all the results, cell viability showed a result of 95 to 98%, and it was found that both the compositions of Examples and Comparative Examples had no cytotoxicity.

2. Evaluation of water retention capacity when applied to the human body

In order to evaluate the skin moisturizing effect of the compositions of Examples and Comparative Examples, the moisture retention ability was measured as follows for skin coated with each test substance of Examples and Comparative Examples. The test substance dissolved in purified water at 15% by weight was used.

Moisture retention capacity was measured by applying a certain amount (0.1g/4cm2) to the inside of the forearms of 20 subjects in a constant temperature and humidity room with a room temperature of 22°C and a relative humidity of 45%, and the skin before, 1 hour after, and 2 hours after application. of the water content was measured. As the measuring device, a moisture content meter (corneometer CM820, Courage + Khazaka, Cologne, Germany) that measures the moisturizing power by measuring the electric capacity of the skin according to the moisture content of the skin was used, and the results are shown in Table 4 below.

electrical conductivity before application 1 hour after application 2 hours after application Example 1 50 83 77 Example 2 50 90 85 Example 3 50 88 81 comparative example 50 56 51

From Table 4, it was confirmed that the composition according to the present invention has a remarkably superior water retention ability compared to the composition of Comparative Example, which proves that it is a material having a very excellent moisturizing effect. In particular, looking at the results of Examples 2 and 3, the synergistic effect of fulvic acid and tromarin can be confirmed.

3. Aquaporin-3 and involucrin synthesis promoting effect experiment

In order to confirm the effect of increasing the synthesis of involucrin, which plays an important role in the barrier and moisturizing functions of skin cells, and aquaporin-3, which plays an important role in water movement, the compositions of Examples and Comparative Examples were applied to the cells as follows. After each treatment, it was confirmed by western blotting.

First, human keratinocytes were inoculated with HaCaT, and after 24 hours, the compositions of Examples and Comparative Examples were added, respectively, and cultured in DMEM medium, 37° C., 5% CO 2 . After culturing for 24 hours, the cells were recovered, disrupted, and only proteins were extracted and stored at -40°C. The extracted protein was quantified by BCA saasy. After dilution with 30 g of protein amount of each sample, electrophoresis on 12% SDS-gel was carried out, and then it was transferred to PVDF membrane. After reacting with the primary antibody (rabbit anti-aquaporins-3 antibody), the remaining antibody was washed and removed and reacted with the secondary antibody (rabbit HRP-coupled anti-IgG antibody).

After removing the remaining antibody, a reagent (peroxidase substrate and chromogen solution) that reacts with the antibody to cause a color reaction was added. The colored band was measured and quantified through image analysis (BioRad, Universal Hood, USA), and the results are shown in Tables 5 and 6 below.

division Aquaporin-3 expression level (%) control 100 Example 1 148 Example 2 163 Example 3 162 comparative example 103

As shown in Table 5, the comparative example showed little effect compared to the control, and the example according to the present invention showed an effect of 50% or more, and in particular, a significant synergistic effect could be confirmed in Examples 2 and 3 there was.

division Expression level of involucrin (%) control 100 Example 1 149 Example 2 164 Example 3 158 comparative example 102

The expression of involucrin, which plays an important role in skin barrier function and moisturizing, when treated with HaCaT keratinocytes, as shown in Table 6, the comparative example showed little effect compared to the control, and the examples according to the present invention showed little effect. It showed an effect of 50% or more, and in particular, a significant synergistic effect was confirmed in Examples 2 and 3.

Through the above experimental results, the composition of the present invention was able to confirm the excellent effect on the skin barrier function and moisturizing.

< test example 4: Prevents skin wrinkles and maintains elasticity>

1. Measurement of Elastase Inhibition Rate

The inhibitory effect on the activity of Elastase, an enzyme decomposing elastin, was confirmed as follows.

Elastase used elastase derived from human leukocyte cells, and a synthetic substrate MeOSuc-Ala-Ala-Pro-Val-pNA was used as a substrate for elastase. A buffer solution of 100 mM Tris (pH 7.5) was used. Elastase was finally used at 0.2 mU using a buffer solution. In addition, the synthetic substrate of elastase was diluted with a buffer solution to a final concentration of 05 mM after making a 100 mM solution using DMSO.

At this time, the positive control was set to put quercetin (Quercetin) known as an elastase inhibitor at a concentration of 100ppm. Samples of Examples and Comparative Examples were also added so as to be 100 ppm. The reaction was carried out in a 96-well plate, and the reaction was carried out at room temperature for 20 minutes. The absorbance was measured at 405 nm at intervals of 1 minute using a spectrophotometer, and the slope of the absorbance versus time was obtained and determined as the enzyme activity. The elastase inhibition rate was calculated as follows and the results are shown in Table 7.

sample Elastase inhibition rate (%) positive control 43.12% Example 1 77.29% Example 2 88.21% Example 3 84.25$ comparative example 42.18%

As can be seen from the results of Table 7, it was found that the composition of the present invention had a very good elastase inhibitory ability, and although it showed some effect in the comparative example, it was inferior to the composition of the present invention, especially in Example 2 and A significant synergistic effect was confirmed in the composition of Example 3.

2. Measurement of Collagenase Inhibition Rate

The inhibitory effect on the activity of collagenase, an enzyme that decomposes collagen, was confirmed as follows.

Collagenase derived from mouse pancreas was used, and a synthetic substrate, N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala, was used as the collagenase substrate. A buffer solution in which 50 mM Tricine, 10 mM CaCl ₂ ·H ₂ O, and 400 mM NaCl (pH7.5) were dissolved was used. The synthetic substrate was dissolved at a concentration of 10 mM using a buffer and finally diluted to 75 μM. Collagenase was dissolved in 4 degree cold tertiary distilled water to a concentration of 1 unit/ml and finally diluted with a buffer solution to 0.05 unit/ml.

The samples of Examples and Comparative Examples were tested at a final concentration of 100 ppm. At this time, the positive control group was set to have a final concentration of 100 ppm of epigallocatechin gallate (EGCG), which is known to have a collagenase inhibitory effect. The reaction was carried out in a 96-well plate, and reacted at room temperature for 10 minutes. The absorbance was measured at 345 nm at intervals of 1 minute using a spectrophotometer, and the maximum slope of the absorbance versus time was obtained and determined as the activity of the enzyme. The collagenase inhibition rate was calculated as follows and the results are shown in Table 8.

sample Collagenase inhibition rate (%) positive control 42.12% Example 1 68.18% Example 2 80.12% run 3 78.42% comparative example 39.15%

As can be seen from the results of Table 8, the composition of the present invention was found to have a very good collagenase inhibitory ability, and although it showed some effect in the comparative example, it was inferior to the composition of the present invention, especially in Example 2 and A significant synergistic effect was confirmed in the composition of Example 3.

3. Measurement of cell proliferation effect

To confirm the activation of skin cell proliferation, cell proliferation activity was measured using human fibroblasts.

After adding the compositions of Examples and Comparative Examples at 10 μg/ml to the culture medium, the MTT reduction method was performed by culturing human fibroblasts, and the formazan product was extracted with isopropyl ethanol, and absorbance was measured at 570 nm. By doing so, the cell proliferation activity was compared. At this time, as a negative control, a culture medium without the sample was used. The measured results were numerically calculated using the following formula, and the results are shown in Table 9 below.

Cell proliferative capacity (%) = absorbance in each sample group / absorbance in control group

sample Cell proliferative activity (%) Example 1 137.28% Example 2 144.14% Example 3 142.28% comparative example 112.15%

As can be seen from the results of Table 9, the composition of the present invention showed very excellent cell proliferation activity compared to the cell proliferation activity (100%) of the untreated control group, and showed some effect in the comparative example. It was less than that of Example 1, and in particular, in Examples 2 and 3, a significant synergistic effect was shown.

As a result of the above examination, it was found that the cosmetic composition according to the present invention is excellent in maintaining and improving skin elasticity and preventing skin wrinkles, and is ultimately the best cosmetic composition capable of preventing skin aging.

In summary of the above-described test examples, the composition according to the present invention has remarkably excellent advantages in skin moisturizing, skin whitening, maintaining or improving skin elasticity, skin wrinkle prevention effect, and ultimately skin anti-aging effect.

Claims (5)

As a hydrolyzate obtained by hydrolyzing proteins extracted from Moringa seeds with proteolytic enzymes,
5 to 15% by weight of free amino acids;
oligopeptide 70 to 90% by weight; and
Polypeptide 5 to 15% by weight
contains,
The moringa seed is a multifunctional cosmetic effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles, characterized in that the moringa seeds are germinated after being immersed in 1 wt% fulvic acid aqueous solution for 24 hours. composition.
According to claim 1, wherein the germinated Moringa seeds are mixed with tourmaline powder in a volume ratio of 1:1 before hydrolysis and maintained for 24 hours. Skin moisturizing, skin whitening, skin elasticity maintenance and improvement, skin A multifunctional cosmetic composition effective for anti-wrinkle.
The method of claim 1, wherein the oligopeptide has a molecular weight of 300 to 1,500 Daltons (Da), and the polypeptide has a molecular weight of 3,000 to 15,000 Daltons (Da). A multifunctional cosmetic composition effective in preventing skin wrinkles.
Moringa seeds germinated after being immersed in 1 wt% fulvic acid aqueous solution for 24 hours are mixed with tourmaline powder in a volume ratio of 1:1 and maintained for 24 hours and then hydrolyzed with a proteolytic enzyme. Multifunctional cosmetic composition effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles.


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