KR101177915B1 - Anti-aging composition for skin external application containing yeast fermented solution of spinach extracts - Google Patents

Abstract

The present invention relates to an anti-wrinkle and anti-aging skin composition containing the yeast fermentation solution of the spinach extract obtained by inoculating and incubating the yeast in the spinach extract as an active ingredient. The yeast fermentation solution of the spinach extract has the effect of promoting the synthesis of procollagen (procollagen) and inhibit the production of collagenase (collagenase).

Wrinkle improvement, anti-aging, antioxidant, spinach extract, yeast fermentation

Description

Anti-wrinkle and anti-aging skin external composition comprising yeast fermentation solution of spinach extract {ANTI-AGING COMPOSITION FOR SKIN EXTERNAL APPLICATION CONTAINING YEAST FERMENTED SOLUTION OF SPINACH EXTRACTS}

The present invention relates to an external skin composition for preventing wrinkles and preventing aging using spinach extract.

As human skin ages, the secretion of various hormones that regulate metabolism internally decreases, and the function and activity of immune cells and skin cells decreases, resulting in reduced biosynthesis of immune proteins and bioconstituent proteins necessary for living organisms. Intrinsic aging occurs.

In addition, aging occurs due to the generation of blemishes, fine gold and dry skin due to the increase in wrinkles, decrease in elasticity, and dry skin due to photoaging by various pollutants and ultraviolet rays externally.

The skin is composed of epidermis, dermis, and subcutaneous. Among them, the dermis is composed of fibrous and matrix components, and collagen, which occupies most of the dermis, gives strength and tension to the skin and protects the skin. .

With age, the synthesis of fibroblasts (fibroblast) and the decrease of cell number, which decreases the number of collagen, the loss of moisture in the skin cells and the structure of the horny layer Will change.

In addition, the action of collagenase (collagenase) that breaks down the collagen, a major structural component of the skin is increased to increase the cross-linked form of collagen to reduce the smoothness, moisturizing, swelling.

Therefore, it inhibits the biosynthesis of matrix metalloproteinase (MMPs), which breaks down collagen, which is a major structural component of skin, and promotes skin aging by using a substance that can promote the production of procollagen, a precursor of collagen. There is a growing interest in mitigating natural products.

For example, various natural products such as Cordyceps sinensis, spiny ginseng, ganoderma lucidum, deer antler, ginseng, hyssop, Angelica, wolfberry, earthenware, tea grass and green-yellow vegetables are used.

Spinach extract of greenish yellow vegetables is used as a raw material for functional cosmetics because it functions to inhibit oxidative stress of cells, prevent and reduce DNA damage, repair and prevent damage to mitochondria and loss of membrane fluidity.

Recently, as consumers' aesthetic desire for anti-aging rises, the consumer's desire for purchasing a product containing an anti-aging component is increasing. In addition, the demand for these functional products is increasing rapidly.

Therefore, companies producing functional products are investing a lot of interest and investment in the development of natural materials that can alleviate skin aging by using compositions having improved efficacy and anti-aging effects for wrinkles.

Therefore, the present inventors, while studying the skin external composition for improved wrinkle and anti-aging effect using natural products, it was found that the yeast fermentation solution obtained by cultivating spinach extract with yeast has excellent wrinkle improvement and anti-aging effect. The invention has been completed.

An object of the present invention is to provide an external skin composition having a wrinkle prevention and anti-aging effect using a spinach extract.

Another object of the present invention is to provide a skin external preparation composition having an anti-aging effect and anti-wrinkle comprising a yeast fermentation solution of spinach extract.

Still another object of the present invention is to provide a method for efficiently extracting spinach extract.

In order to achieve the above object, the present invention provides a skin external composition for anti-wrinkle and anti-aging containing the yeast fermentation solution of the spinach extract obtained by inoculating and incubating the yeast in the spinach extract as an active ingredient.

The spinach extract may be extracted using a extraction method or a supercritical fluid extraction method using a specific solvent, such as water, ethanol, glycol, the yeast It may be selected from the group consisting of Hansenula polymorpha or Pichia pastoris .

The spinach extract can be obtained using spinach young leaves.

The yeast is GRAS (Generally Recognized As Safe) grade in the safety evaluation, spinach extract and yeast fermentation solution is a natural substance with less side effects and high stability.

The spinach extract is added in an amount of 0.1 to 10% by volume based on the total amount of the culture solution including the spinach extract and yeast, and may be incubated at 30 ° C. to 37 ° C. for 48 hours. When the spinach extract is added in less than 0.1% by volume it is difficult to make a yeast fermentation solution of the spinach extract, if it exceeds 10% by volume it is preferable to incubate within the above range because it affects the growth of yeast.

In addition, the temperature is a good temperature for yeast growth Pichia pastoris (30 ° C), Hansenula polymopa ( Hansenula polymorpha ) is the optimal growth condition at 37 ℃. In addition, when the incubation time exceeds 48 hours, it may not be desirable to incubate beyond this time since there is no change in the fermentation product.

And the yeast fermentation solution of the spinach extract is an external skin composition for preventing wrinkles and aging, characterized in that it comprises 0.001 to 30% by weight based on the total weight of the composition.

If the yeast fermentation solution of the spinach extract is less than 0.001% by weight may cause a problem that can not achieve the object of the present invention, if it exceeds 30% by weight is limited to the above range because it affects the growth of yeast .

In addition, the culture may use a batch culture, a continuous culture, a fed-batch culture.

Batch culture is a method in which a cell is initially fermented once and then fermented without supplying any more nutrients or removing products. Continuous culture is a way in which new nutrient media are continuously fed into the reactor, allowing cells to flow out of the fermenter and sustaining growth and material production for a long time.

In the fed-batch culture, the initial sugar concentration is lowered so that the growth of the cells is not inhibited. After the start of the cultivation, the fermentation proceeds to add the required amount of sugar to keep the sugar concentration low during the entire fermentation process. It is a method of culturing microorganisms.

It is widely used in fermentation industry (bakery yeast, amino acid antibiotics) due to less substrate inhibition. In the present invention, it is preferable to produce a yeast fermentation solution using oil culture, but is not necessarily limited thereto.

The yeast fermentation solution of the spinach extract has the effect of promoting the synthesis of procollagen (procollagen), a precursor of collagen (collagen), and inhibits the production of collagenase (collgenase), a degradation enzyme of collagen. This can be confirmed through an embodiment to be described later.

In addition, the yeast fermentation solution of the spinach extract is added to the active substance having a known skin aging inhibitory effect and skin wrinkle suppression effect, as long as it does not deleterious effect on the skin aging inhibitory effect and skin wrinkle suppression effect other than the yeast fermentation solution of spinach extract It may be included. In addition, the use of yeast has the advantage of being more environmentally friendly and mass production of active ingredients.

On the other hand, the spinach extract is a step of obtaining the extract through an extraction solvent or supercritical extraction method; And passing the obtained extract through a reverse osmosis membrane to decolorize.

Extraction by the extraction solvent may use at least one solvent selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, trimethylene glycol, dichloromethane and hexane.

In particular, when the trimethylene glycol (1,3-Propanediol) is used as a solvent, even if water is added thereto, the active ingredient does not elute or precipitate, resulting in higher extraction efficiency than ethanol. Thus, the present invention used trimethylene glycol (1,3-Propanediol), but is not necessarily limited thereto.

Another extraction method is supercritical fluid extraction. Here, "supercritical fluid" refers to a gaseous state at room temperature but a fluid above the critical temperature and the critical pressure.

More specifically, carbon dioxide, nitrogen, methane, ethylene, propane or propylene may be used as the supercritical fluid during supercritical fluid extraction. In particular, carbon dioxide can be purchased in large quantities, relatively inexpensive, non-explosive, and has the advantage of being sufficiently safe for food processing.

However, carbon dioxide having nonpolar properties has a limit in extracting polar materials. On the other hand, glycol has a characteristic of the polarity is characterized by more easily extract the polar material in the spinach.

Therefore, in the present invention, supercritical extraction was performed using glycol as a co-solvent in carbon dioxide to increase the extraction efficiency of the polar and nonpolar substances in spinach.

The glycol used as the co-solvent may be selected from the group consisting of butylene glycol, propylene glycol and trimethylene glycol. In the present invention, trimethylene glycol (1,3-Propanediol) is particularly preferred, but is not necessarily limited thereto.

The critical extraction temperature is 40 ℃ to 80 ℃, the extraction pressure is 200 to 400 bar, the concentration of glycol is characterized in that 0.1 to 5% by volume.

More specifically, when the temperature is less than 40 ° C, supercritical carbon dioxide may not be formed well, and when it exceeds 80 ° C, the heat-sensitive active ingredient may be destroyed and thus extracted within the above range. When the extraction pressure is less than 200 bar, there is a concern that the supercritical carbon dioxide may not be formed well, and when the extraction pressure exceeds 400 bar, there is a problem that the process cost increases as a result of unnecessarily increasing the pressure regardless of the increase in extraction yield. Therefore, it is preferable to extract in the said range.

In addition, when the glycol concentration is less than 0.1% by volume, it is difficult to extract the polar substance in the spinach, and when it exceeds 5% by volume, it is preferable to extract within the above range because there is no change in the maximum active ingredient extraction amount. That is, the range is a range in which the amount of the co-solvent is minimized and the active ingredient can be extracted to the maximum.

In other words, the spinach extract may use a specific extraction solvent or supercritical fluid extraction. Next, the spinach extract according to the present invention should be decolorized in order to produce a product of the natural material, through the reverse osmosis-membrane separation process, the spinach extract can be discolored.

Generally, the dissolved material can be separated from the solvent by using various kinds of selective membranes. Examples of such membranes include a microfiltration membrane, an ultrafiltration membrane, a nano separation membrane, and a reverse osmosis membrane. Among them, the separation using the reverse osmosis membrane has the advantage of low energy consumption, easy operation, and easy automation.

Thus, the present invention uses a reverse osmosis-membrane separation process. As a result, the active ingredient having a small molecular weight in the spinach extract may be separated and concentrated, and decolorization and desalination may also be performed to more efficiently manufacture the target substance.

As a result, the yeast fermentation solution obtained by inoculating yeast in the spinach extract extracted by the extraction method can provide an external skin composition for improving wrinkles and preventing aging.

In more detail, the composition is a flexible cosmetics, astringent cosmetics, nourishing cosmetics, nutrition cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body It can be used as a cosmetic composition having a formulation selected from the group consisting of oils, body essences, makeup bases, foundations, hair dyes, shampoos, rinses and body cleansers.

In addition, the composition may be used as a pharmaceutical composition having a formulation selected from the group consisting of ointment gel, cream, patch and spray.

When used as a composition in the cosmetic composition further to match the formulation of the cosmetic Can be added. For example, purified water, moisturizers, oils, surfactants, higher alcohols, low and low viscosity agents, chelating agents, pigments, fatty acids, antioxidants, preservatives, waxes, pH adjusters, fragrances and the like can be added.

As the moisturizing agent, glycerin, propylene glycol, butylene glycol, hexylene glycol, diglycerin, sorbitol, betaine, glycerin-26 and the like may be used.

The oils include mineral oil, cyclomethicone, squalane, octyldodecyl myristate, olive oil, macadamia nut oil, glyceryl octanoate, castor oil, ethylhexyl isononanoate, dimethicone, cyclopentasiloxane, Sunflower seed oil and the like can be used.

The surfactant includes sorbitan sesquinolate, polysorbate 60, glyceryl stearate, lipophilic glyceryl stearate, sorbitan oleate, sorbitan stearate, die-cetyl phosphate, sorbitan stearate / shoe Cross-Cocoate, Glyceryl Stearate / Polyethylene Glycol-100 Stearate, Ceteareth-6 Oliveate, Lakydyl Alcohol / Behenyl Alcohol / Arachidyl Glucoside, Polypropylene Glycol-26-butes-26 / Polyethylene glycol-40 hydrogenated castor oil and the like can be used.

Cetyl alcohol, stearyl alcohol, octyldodecanol, isostearyl alcohol and the like may be used as the higher alcohol.

Xanthan gum, xanthan gum, carbomer, magnesium aluminum silicate, cellulose gum, dextrin palmitate may be used.

As the chelating agent, disodium idieti, tetrasodium idieti and the like may be used. As the dye, Blue No. 1, Red No. 40, Yellow No. 4, Yellow No. 5, etc. may be used.

As the fatty acid, stearyl acid, mystic acid, palmitic acid, isostearic acid, lauric acid, or the like may be used.

Tocopheryl acetate, butylated hydroxytoluene, etc. may be used as the antioxidant.

As the preservative, methyl paraben, butyl paraben, propyl paraben, phenoxyethanol, imidazolidinyl urea, chlorphenesin and the like may be used.

Carnauba wax, candenilla wax, beeswax, braze and the like may be used as the wax.

As the pH adjusting agent, triethanolamine, citric acid, or the like may be used.

As the fragrance, natural fragrance, combination fragrance, or the like may be used.

The formulation of the cosmetic prepared with the composition of the present invention is not particularly limited, such as lotion, essence, lotion, gel, cream.

The present invention has the effect of providing an external composition for skin having anti-wrinkle and anti-aging effects using spinach extract and yeast fermentation thereof.

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example  1: Preparation of Spinach Extract

1-1: Preparation of Spinach Extract Using Trimethylene Glycol (1,3-Propanediol)

To 1 g of commercially available spinach powder, 100 ml of trimethylene glycol (1,3-Propanediol, PD, Dupont, USA) was added and stirred at room temperature for 20 hours. The stirred material was filtered using filter paper, and the pattern of the extract was compared using high performance liquid chromatography (HPLC) (C-18 / 258 nm / Acetonitrile, Agilent).

1-2: Hot water export method  Preparation of Spinach Extract Using

A spinach extract was obtained in the same manner as in Example 1-1, except that 100 ml of hot water (40 ° C.) was added instead of trimethylene glycol (1,3-Propanediol), and the same method as in Example 1-1. By using high-performance liquid chromatography, the pattern of the extract was compared.

As shown in Figure 1, it can be seen that the characteristic components appeared in the glycol (1,3-Propanediol) extract of Example 1-1 and the hot water extraction of Example 1-2, respectively. That is, 32-1 (Spinch1 + DWext) and 33-1 (Spinch 2 + DWext) analyzed by extracting hot water; And the peak patterns of 30-1 (Spinch2 + PDext) and 31-1 (Spinch2 + PDext) extracted by glycol (1,3-Propanediol) component analysis can be confirmed. In the present invention, all of these extracts are used as raw materials for the yeast fermentation reaction. And all yeast fermentation extract of spinach extract is effective in preventing wrinkles and aging.

Example  2: Supercritical  Preparation of Spinach Extract Using Fluid Extraction Method

Supercritical fluid extraction conditions were progressed to 8 types as shown in Table 1 below, and extracted with carbon dioxide and glycol (1,3-Propanediol) according to temperature and pressure changes as supercritical fluids. Under the conditions of # 1 to # 8, 2.5 g of a sample (spinach powder) was extracted and the extract was dissolved using ethanol and MC (methylene chloride) and dried.

[Table 1] Primary Supercritical Fluid Extraction Conditions

sample T (℃) P ( bar ) Modifier
( PD ,% v / v) Extraction  Time ( min ) Sample
weight #One
40 250 - 180 2.5 #2 400 - 180 2.5 # 3 400 - 180 2.5 #4 400 5 180 2.5 # 5
80 250 - 180 2.5 # 6 400 - 180 2.5 # 7 400 5 180 2.5 #8 60 400 - 180 2.5

Through the results in Table 1, it can be seen that the extraction efficiency is high at the extraction temperature of 40 ℃ to 80 ℃, extraction pressure 200 to 400 bar. In addition, it can be seen that the extraction efficiency is relatively higher when using glycol as an auxiliary solvent.

In order to confirm the reproducibility of the above primary extraction conditions and to secure more specific supercritical extraction conditions, the secondary extraction was performed under the conditions shown in Table 2 below using the sample (spinach powder) used during the primary extraction.

In Table 2 below, “1-1, 1-2” means that the sample number is extracted twice in the same condition under the same condition because a large amount of sample (spinach powder) is used. . The meaning of the sample number of "2-1, 2-1, 3-1, and 3-2" is also the same. As a result of the second extraction, the condition of obtaining an extract of about 2% level compared to the raw material used for the supercritical extract was obtained. Figure 2 is a supercritical fluid extract primary (a), secondary (b) sample photograph.

[Table 2] Second Supercritical Fluid Extraction Conditions

# Samples T (℃) P (bar) CO ₂
flowrate
(ml / min) Modifier
(PD,% v / v) CO ₂
Used (g) Extract
Weight (g) Extraction sample
Weight (g) Extract (g) / Sample (g) 1-1 40 400 2.85 0.15 564 0.1697 9 0.0298 1-2 40 400 2.85 0.15 564 0.0985 2-1 60 400 3 - 593.7 0.1252 12 0.0210 2-2 60 400 3 - 593.7 0.1265 3-1 80 250 3 - 572 0.1337 12 0.0213 3-2 80 250 3 - 572 0.1222 4 60 250 3 - 572 0.0627 3 0.0209 5 60 250 0.5 - 95.4 0.0514 3 0.0171

Example  3: Efficacy Test of Spinach Extract

3-1: Procollagen Synthesis Promoting Activity Test of Supercritical Fluid Extracts

Human dermal fibroblast (HDF) cells (Modern Cell & Tissue Technologies) were prepared using Dulbecco's Modified Eagle Medium (Delbecco's Modified Eagle Medium) containing penicillin (100 units / ml), streptomycin (100 μg / ml), 10% FBS. , Inc. Korea) was added and cultured in a 37 ℃ incubator containing 5% carbon dioxide. The cultured fibroblasts were inoculated at 200 μl at a concentration of 5 × 10 ⁴ cells / ml in a 48 well plate and cultured for 24 hours.

Fibroblasts cultured for 24 hours were washed once with Dulbecco's phosphate buffered saline (PBS) and exchanged with serum-free DMEM medium (serum free-Dulbecco's Modified Eagle Medium) (WelGENE, Inc. Korea) for 6 hours. The fibroblast medium was removed and washed once with PBS, and then diluted in serum-free DMEM medium (WelGENE, Inc. Korea), supercritical fluid extract dissolved in DMSO to a concentration of 10 μg / ml and 100 μg / ml and 200 Treated by μl and incubated for 72 hours. Cultures were harvested and procollagen was measured.

Procollagen (procollagen) is measured according to instructions using a procollagen type I-peptide EIA kit (procollagen Type I C-Peptide EIA, Takara Biomedical Co.), which can measure procollagen biosynthesis.

As a control, fibroblasts were cultured in a medium containing fibroblast culture and a growth promoter (TGF-beta) without adding a supercritical fluid extract.

3, it can be seen that the # 1 to # 8 supercritical fluid extract of the primary extract according to the present invention has the effect of promoting procollagen synthesis.

Specifically, # 4, # 7 and # 8 It can be seen that the fibroblasts of the sample synthesized more procollagen.

3-2: Supercritical  Cytotoxicity Test of Fluid Extracts ( Cytotoxicity Test )

Human dermal fibroblast (HDF) cells (Modern Cell & Tissue Technologies) were prepared using DMEM medium containing penicillin (100 units / ml), streptomycin (100 μg / ml), and 10% fetal bo-vine serum (FBS). Dulbecco's Modified Eagle Medium) was added and incubated in a 37 ° C. incubator containing 5% carbon dioxide. The cultured fibroblasts were inoculated at 200 μl at a concentration of 1 × 10 ⁴ cells / ml in a 96 well plate and incubated for 24 hours.

Supercritical fluid diluted to 10 μg / ml and 100 μg / ml in serum-free DMEM® medium (serum free-Dulbecco's Modified Eagle Medium, WelGENE, Inc. Korea) on fibroblasts washed once with PBS after removing the medium. The extracts were treated with 200 μl and incubated for 20 hours. The cell culture medium of each treatment group was removed, washed once with PBS, and then diluted 1/10 of the WST-1 solution used in the Western blot analysis to serum-free DMEM medium. Add 200 μl and incubate for 1-6 hours.

Then, using a microplate-reader (TECAN Group Ltd, Switzerland), which is a device that can analyze protein quantification and the method of confirming antigen-antibody reactions by binding enzymes to antibodies (ELISA) at once. The absorbance at 450-590 nm was measured and the results are shown in FIG. 4.

Referring to Figure 4, the cytotoxicity level of the culture medium produced by fibroblasts grown in the presence of the control medium (medium) and TGF-beta (growth promoter) that plays an important role in determining the differentiation of cells When comparing the cytotoxicity level of the samples with # 1 to # 8, it can be seen that the similarity of the control and # 1 to # 8 samples. That is, the extract according to the present invention can be seen that there is little cytotoxicity.

Example  4: reverse osmosis Membrane separation  Test the efficacy of extracts obtained through the process

4-1: Reverse Osmosis Membrane separation  Extract Preparation Using Process

The extract obtained in Example 1-1 was passed through a reverse osmosis-membrane (PHILOSEP Membrane System, PHILOS, Korea) to obtain a discolored extract (see the right fraction of Figure 5). like this The collected extracts were subjected to distillation under reduced pressure, a method well known in the art. Concentrated.

4-2: Procollagen Synthesis Promoting Activity Test

In order to determine the efficacy of the extract obtained in 4-1, the following experiment was performed. Human dermal fibroblast, Modern Cell & Tissue Technologies (HDF) cells were penicillin (100 units / ml), streptomycin (100 μg / ml). DMEM medium containing 10% FBS was added and cultured in a 37 ° C. incubator containing 5% carbon dioxide. The cultured fibroblasts were inoculated at 200 μl at a concentration of 5 × 10 ⁴ cells / ml in a 48 well plate and cultured for 24 hours.

Fibroblasts cultured for 24 hours were washed once with PBS and exchanged with serum free DMEM medium (WelGENE, Inc. Korea) for 6 hours. After removing the fibroblast medium and washing once with PBS, the membrane-separated fraction dissolved in DMSO was diluted to 10 μg / ml and 100 μg / ml in serum-free DMEM® medium (WelGENE, Inc. Korea) and 200 μl. Treat and incubate for 72 hours.

Cultures are harvested to measure procollagen. Procollagen was measured by procollagen type I-peptide EIA kit (Takara Biomedical Co., Japan), which can measure the degree of procollagen biosynthesis, according to the method of use of the machine.

In addition, the degree of procollagen biosynthesis was measured after culturing fibroblasts as described above using the spinach extract obtained in Example 1-1.

As a control, fibroblasts were added to media containing only fibroblasts without the addition of spinach extract, media containing DMSO 0.1% media, pyridine 0.1% media and growth promoter (TGF-beta). Cultured ones were used.

When the fibroblasts were cultured through the spinach extract or the reverse osmosis-membrane separation process in FIG. 6, the synthesis of procollagen was high. Thus, it can be seen that the extract obtained through the reverse osmosis-membrane separation process contains relatively more substances capable of promoting procollagen synthesis.

4-3: Cytotoxicity Test

Human dermal fibroblast, Modern Cell & Tissue Technologies (HDF) cells were added with DMEM medium containing penicillin (100 units / ml), streptomycin (100 μg / ml), 10% FBS to contain 5% carbon dioxide. Cultured in a 37 ℃ incubator. The cultured fibroblasts were inoculated in a 200 well of 96 X well plate (well plate) at a concentration of 1 X 10 ⁴ cells / ml for 24 hours. After the medium was removed, washed once with PBS, and then diluted in serum-free DMEM medium (WelGENE, Inc. Korea), supercritical fluid extract dissolved in DMSO to a concentration of 10 μg / ml and 100 μg / ml and treated with 200 μl. Incubated for 20 hours.

Cell cultures of each treatment group were removed, washed once with PBS, and then diluted WST-1 solution to 1/10 of serum-free DMEM medium and added 200 μl and incubated for 1 to 6 hours. Thereafter, the absorbance at 450-590 nm was measured using a microplate reader, and the results are shown in FIG. 7.

7 shows that the cytotoxicity of the fractions subjected to the reverse osmosis-membrane separation process is similar to the degree of cytotoxicity shown in the other controls, and the spinach extract fraction obtained through the reverse osmosis-membrane separation process has a cytotoxicity. It can be seen that the stability is recognized.

Example  5: Efficacy Test of Yeast Fermentation Solution of Spinach Extract

5-1: Yeast Fermentation Solution of Spinach Extract

Pichia, a GRAS-grade industrial yeast sold by Invitrogen (USA) pastoris ) (Invitrogen, Cat. No. K1740-01) was purchased and cultured in medium (glycerol 10 g / l, yeast nitrogen base without amino acid 13.4 g / l, biotin 4 g / l). Next, the spinach hot water extract obtained in Example 1-2 was added to the complex medium containing the culture yeast (containing 2% by weight of yeast extract and 1% by weight of glycerol) so as to contain 10% (v / v) of the total culture solution. Incubated for 48 hours at 30 ℃ using a fed-batch system.

5-2: Yeast Fermentation Solution of Spinach Extract Collagenase ( Collagenase ) Expression inhibition effect measurement

Human-derived keratinocyte cell lines HaCaT cells (Amorepacific, Inc., Korea) were placed in DMEM medium (Dulbecco's Modified Eagle Medium) containing penicillin (100 IU / ml), streptomycin (100 g / ml), 10% FBS. The cells were cultured in a 37 ° C. incubator containing% carbon dioxide. The cultured keratinocytes were inoculated at 1 × 10 ⁴ cells / ml in 96 well plates and cultured for 1 day.

After incubation, the medium was removed, and the yeast fermentation solution and the lactic acid bacteria lysate (1 to 3 of FIG. 8) of the spinach extract produced through the process of Example 5-1 were added to the DMEM medium at concentrations of 10 / ml and 100 / ml, respectively. Incubated for 20 hours.

Then, in order to induce the expression of collagenase (MMP-1), after removing the medium was added DPBS (Dulbecco's Phosphate Buffered Saline) used for cell culture, UV-B was irradiated with 15 mJ.

After incubation for 24 hours, the medium was harvested. Protein Extraction Experiment After quantifying the total protein amount through Bradford Assay, the total protein amount of each treatment group was corrected to prepare a sample for Western blotting. The prepared sample was separated according to the size of the protein in 10% protein electrophoresis (SDS-PAGE), which was transferred to the membrane to obtain an antibody (MMP-1 antibody). Human MMP-1 Affinity Purified Polyclonal Ab, Goat Ig, R & D systems and donkey anti-goat IgG-HRP, Santa cruz biotechnology) as secondary antibodies were visualized on X-ray film.

Referring to FIG. 8, it can be seen that the generation of collagenase was suppressed in all samples # 1 to # 3 when comparing the thickness of the bands of the control samples with the samples # 1 to # 3.

Through the above embodiment, the yeast fermentation solution of the spinach extract promotes the synthesis of procollagen (procollagen) and inhibits the production of collagenase (Collagenase) to implement a skin external composition for preventing wrinkles and preventing aging, which has further improved efficacy. Able to know.

Manufacturing example  1: containing yeast fermentation solution Cosmetics

A. Lotion with Yeast Fermentation Solution

a) flexible lotion

Examples of the preparation of the flexible lotion containing the yeast fermentation solution are shown in Table 1 below. Purified water is added to the aqueous component and dissolved at room temperature to form an aqueous phase. The yeast fermentation solution, oil phase component, emulsifier, preservative, and fragrance are dissolved at room temperature, then added to the aqueous phase, mixed and filtered.

[Table 3]

ingredient Content (% by weight) Yeast Fermentation Solution 0.10 EDTA 0.05 PIZZY 1500 4.00 Carbomer 0.16 1,3 Butylene Glycol 3.00 Polyoxyethylene Hydrogenated Castor Oil 0.45 Triethanolamine 0.12 Propylparabens and Artificial Flavors and Yellow No.4 0.10 Purified water 92.02 Sum 100

b) converging lotion

A preparation example of the flexible lotion containing the yeast fermentation solution is shown in Table 2 below. Purified water is added to the aqueous component and dissolved at room temperature to form an aqueous phase. Ethanol is added to the yeast fermentation solution, oil phase component, emulsifier, preservative, and fragrance to dissolve at room temperature to prepare an alcohol phase, which is then added to the aqueous phase, mixed and filtered.

[Table 4]

ingredient Content (% by weight) Yeast Fermentation Solution 0.10 EDTA 0.05 glycerin 2.00 ethanol 7.00 1,3 Butylene Glycol 3.00 Polyoxyethylene Hydrogenated Castor Oil 0.40 Propylparabens and Artificial Flavors and Yellow No.4 0.10 Purified water 86.35 Sum 100

B. Lotion

A preparation example of a lotion containing a yeast fermentation solution is shown in Table 3 below. The aqueous phase purified water, triethanol amine and butylene glycol are dissolved by heating to 70 占 폚, and the oily fatty acid, oily component, emulsifier and preservative are heated to 70 占 폚 to dissolve the solution. After the emulsification is completed, a 2% solution of xanthan gum, a hydrophilic thickener, is added to adjust the amount to 0.05% by weight. After cooling the solution to 45 ° C., yeast fermentation solution, flavor and pigment are added and mixed and then cooled to 30 ° C.

[Table 5]

ingredient Content (% by weight) Yeast Fermentation Solution 0.10 glycerin 3.00 Carbomer 0.10 Xanthan gum 0.05 1,3 Butylene Glycol 3.00 Polyglycerin-3 Methylglucose Distearateyl 1.50 Glycerin Stearate 0.50 Cetearyl Alcohol 0.30 Jojoba oil 3.00 Liquid paraffin 2.00 Squalane 3.00 Dimethicone 0.50 Tocopheryl acetate 0.20 Triethanolamine 0.10 Propylparabens and Artificial Flavors and Yellow No.4 0.10 Purified water 82.55 Sum 100

C. gel

Preparation of the gel containing the yeast fermentation solution is shown in Table 4 below.

TABLE 6

ingredient Content (% by weight) Yeast Fermentation Solution 0.10 glycerin 5.00 Carbomer 0.60 EDTA 0.02 1,3 Butylene Glycol 4.00 Hydroxyethylcellulose 0.15 Triethanolamine 0.50 Triclosan 0.20 ethanol 5.00 Polyoxy Ethylene Hydrogenated Castor Oil 4.00 Propylparabens and Artificial Flavors and Yellow No.4 0.10 Purified water 80.33 Sum 100

D. Essence

The preparation example of the essence containing the yeast fermentation solution is shown in Table 5 below. The thickener is added to purified water with slow stirring to disperse uniformly. The aqueous phase is mixed to prepare the aqueous phase. Ethanol is added to the emulsifier, skin softener, fragrance and preservative to dissolve. The alcohol phase is added to the aqueous phase with stirring and an alkali agent is added. Finally, yeast fermentation solution and pigment are added.

[Table 7]

ingredient Content (% by weight) Yeast Fermentation Solution 0.10 EDTA 0.05 Potassium-cetylphosphate 0.30 glycerin 5.00 1,3 Butylene Glycol 4.00 Glyceryl Stearate / Pig-100 Stearate 0.50 Sorbitan stearate 0.30 Macadamia Nut Oil 0.50 Jojoba oil 0.50 Tocopheryl Esate 0.50 Butylated hydroxy toluene 0.50 Dimethicone 3.00 Polyacrylamide / C13-14 Isoparaffin / Laureth-7 1.50 Glyceryl Polymethacrylate / Propylene Glycol 5.00 ethanol 3.00 Propylparabens and Artificial Flavors and Yellow No.4 0.10 Purified water 75.15 Sum 100

E. Cream containing yeast fermentation solution

A preparation example of the nourishing cream containing the yeast fermentation solution is shown in Table 6 below. Water-soluble purified water, triethanolamine and glycol (1,3-Propanediol) were heated and dissolved at 70 ° C, and oily fatty acids, oily ingredients, emulsifiers and preservatives were heated to 70 ° C to add a dissolved solution to emulsify them. Let's do it. After the emulsification is completed, the solution is cooled to 45 ° C., the yeast fermentation solution and the flavor are added and dispersed, and then cooled to 30 ° C.

[Table 8]

ingredient Content (% by weight) Yeast Fermentation Solution 0.10 Jojoba oil 5.00 Liquid paraffin 7.00 Cetylaryl alcohol 2.00 Polyglyceryl-3 Methyl Glucose Disteare 2.00 Glyceryl stearate 0.50 Squalane 3.00 Propylene glycol 4.00 glycerin 5.00 Triethanolamine 0.30 Carboxy Vinyl Polymer 0.30 Tocopheryl acetate 0.20 Propylparabens and Artificial Flavors and Yellow No.4 0.10 Purified water 70.5 Sum 100

Simple modifications and variations of the present invention can be easily made by those skilled in the art, and all such modifications or changes can be seen to be included in the scope of the present invention.

Figure 1 compares the high-speed liquid chromatography (HPLC) pattern of spinach hot water extract and glycol extract.

Figure 2 is a photograph (a) of the samples # 1 to # 8 under the first supercritical fluid extraction conditions and (b) of a sample # 1-1 to # 5 under the second supercritical fluid extraction conditions.

3 shows the results of experiments promoting procollagen synthesis of supercritical fluid extracts.

Figure 4 shows the cytotoxicity test results of the supercritical fluid extract.

5 is a comparison of fractions before (left) and after (right) membrane separation processes.

6 is a result of procollagen synthesis promoting activity test of the reverse osmosis membrane transmembrane fraction.

7 shows the cytotoxicity test results of the reverse osmosis membrane transmembrane fraction.

8 is an X-ray film of visualizing MMP-1 expression according to the strain in the yeast fermentation broth added: 1 is L. bulcaricus (KCTC 3188) , 2 is L.casei (KCTC3109), and 3 is P. pastoris (Invitrogen, Cat. No. K1740-01).

Claims (20)

Wrinkle prevention and anti-aging cosmetic composition containing the yeast fermentation solution of the spinach extract obtained by inoculating and incubating the yeast in the spinach extract. The method of claim 1, wherein the yeast fermentation solution of the spinach extract is to prevent wrinkles and anti-aging cosmetic composition, characterized in that to promote the synthesis of collagen and inhibit the production of collagenase. The method of claim 1, wherein the spinach extract comprises: obtaining the extract through an extraction solvent or a supercritical fluid extraction method; And decolorizing the obtained extract by passing it through a reverse osmosis membrane. A cosmetic composition for preventing and aging of wrinkles, characterized in that obtained. The method of claim 3, wherein the spinach extract is extracted by using one or more solvents selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, trimethylene glycol, dichloromethane and hexane as an extraction solvent A cosmetic composition for preventing wrinkles and aging, which is obtained. According to claim 3, The spinach extract is characterized in that obtained through the supercritical fluid extraction method using glycol as supercritical carbon dioxide and co-solvent, anti-wrinkle and anti-aging cosmetic composition. According to claim 5, In the supercritical fluid extraction method extraction temperature is 40 ℃ to 80 ℃, the extraction pressure is 200 to 400 bar and the concentration of glycol is characterized in that 0.1 to 5% by volume, anti-wrinkle and anti-aging cosmetics Composition. According to claim 1, wherein the yeast Hansenula polymorpha ( Hansenula polymorpha ) or Pichia pastoris (pichia pastoris ) at least one member selected from the group consisting of anti-wrinkle and anti-aging cosmetic composition. According to claim 1, wherein the spinach extract is added to 0.1 to 10% by volume based on the total amount of the culture solution containing the spinach extract and yeast, wrinkles, characterized in that cultured for 6 to 48 hours at a temperature of 30 ℃ to 37 ℃ Preventive and anti-aging cosmetic composition. According to claim 1, wherein the yeast fermentation solution of the spinach extract is characterized in that the cosmetic composition for preventing and aging wrinkles, characterized in that it comprises 0.001 to 30% by weight based on the total weight of the composition. According to claim 1, wherein the spinach extract is wrinkles prevention and anti-aging cosmetic composition, characterized in that using the spinach young leaf extract. Wrinkle prevention and anti-aging pharmaceutical composition containing the yeast fermentation solution of the spinach extract obtained by inoculating and incubating the yeast in the spinach extract. The method of claim 11, wherein the yeast fermentation solution of the spinach extract is anti-wrinkle and anti-aging pharmaceutical composition, characterized in that to promote the synthesis of collagen and inhibit the production of collagenase. The method of claim 11, wherein the spinach extract comprises: obtaining the extract through an extraction solvent or a supercritical fluid extraction method; And decolorizing the obtained extract by passing it through a reverse osmosis membrane. A pharmaceutical composition for preventing wrinkles and preventing aging, characterized in that obtained. The method of claim 13, wherein the spinach extract is extracted by using at least one solvent selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, trimethylene glycol, dichloromethane and hexane as an extraction solvent Wrinkle prevention and anti-aging pharmaceutical composition, characterized in that obtained. The method of claim 13, wherein the spinach extract is characterized in that obtained through the supercritical fluid extraction method using a supercritical carbon dioxide and glycol as a co-solvent, anti-wrinkle and anti-aging pharmaceutical composition. The method of claim 15, wherein the extraction temperature in the supercritical fluid extraction method is 40 ℃ to 80 ℃, the extraction pressure is 200 to 400 bar and the concentration of glycol is 0.1 to 5% by volume, anti-wrinkle and anti-aging agent Pharmaceutical composition. 12. The method of claim 11, wherein the yeast is Hansenula polymorpha ( Hansenula polymorpha ) or Pichia pastoris (pichia pastoris ) at least one selected from the group consisting of anti-wrinkle and anti-aging pharmaceutical composition. The method according to claim 11, wherein the spinach extract is added to 0.1 to 10% by volume based on the total amount of the culture solution containing the spinach extract and yeast, wrinkles, characterized in that incubated for 6 to 48 hours at a temperature of 30 ℃ to 37 ℃ Prophylactic and anti-aging pharmaceutical compositions. The method of claim 11, wherein the yeast fermentation solution of the spinach extract is anti-wrinkle and anti-aging pharmaceutical composition, characterized in that it comprises 0.001 to 30% by weight based on the total weight of the composition. The method of claim 11, wherein the spinach extract is anti-wrinkle and anti-aging pharmaceutical composition characterized in that using the spinach young leaf extract.

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