KR101754218B1 - Composition for improving skin conditions and preventing or treating proliferative skin diseases containing fermented extracts of lactic acid bacteria complex and portulaca oleracea as active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin, a pharmaceutical composition for preventing or treating proliferative skin diseases, a prophylactic skin disease prevention and skin-improving food additive or a health food .

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for improving skin and a composition for preventing or treating a proliferative skin disease, the composition comprising a complex lactic acid bacterium disruption solution and a fermented extract of marigold as an active ingredient, and a composition for preventing or treating a proliferative skin disease. PORTULA OLERACEA AS ACTIVE INGREDIENT}

The present invention relates to a cosmetic composition for skin improvement and a pharmaceutical composition for preventing or treating a proliferative skin disease, comprising a complex lactic acid bacterium disruption solution and a fermented extract of marigold as an active ingredient.

In addition to rapid industrialization, the generation of strong oxidizing substances such as active oxygen species due to air pollution and the increase of UV amount due to ozone layer destruction cause skin aging and pigmentation in humans. Various antioxidants such as vitamin C, tocopherol and coenzyme Q10 have been used for the production of cosmetics for the purpose of protecting the skin from these various oxidizing substances. Recently, it has been found that various physiological active substances such as whitening, anti-aging, anti-wrinkle and antioxidant exist in various plants including herbal medicines, and thus their extracts are widely used in the production of natural cosmetics or herbal cosmetics.

An abnormality in the rate of cell proliferation within keratinocytes can sometimes lead to hyperproliferation, manifesting in many proliferative skin diseases, including various forms of psoriasis, coupled with an abnormal rate of inflammation and / or apoptosis. For psoriasis, which is a typical proliferative skin disease, there is currently no superior therapy, and only inhibition therapy is present.

Portulaca oleracea L. is a yellowish brown or yellowish white plant belonging to the genus Porphyra, and has a mild, unusual odor, mucinous and salty taste. It has been known to be used in the treatment of bacterial dysentery, boils, hemorrhoids, cervical lymphadenitis, eczema, adenomyosis, and uterine bleeding in folk remedies because it has fever, detoxification and hemostasis.

Fermentation refers to the process by which microorganisms degrade organic matter using their own enzymes. It is known that raw materials after fermentation through microorganisms are at least two times to several tens of times more effective than before fermentation. Fermented metabolites contain various amino acids, organic acids and antioxidants that are good for the skin, promoting skin metabolism and making skin texture resilient and smooth. In addition, it has been reported that the fermentation process reduces the particle size, decomposes toxic substances such as heavy metals, so that it has good absorption rate and alleviates skin troubles and allergy side effects.

 There have been active researches on skin condition improvement and proliferative skin disease treatment, but there is still a need for a new method for these. Accordingly, the present invention is intended to apply a new natural fermented extract to the above-mentioned necessity.

Korean Patent Publication No. 10-2014-0045893 Korean Patent Publication No. 10-2011-0040174

The present invention relates to a cosmetic composition for improving skin, a pharmaceutical composition for preventing or treating proliferative skin diseases, a prophylactic skin disease prevention and skin-improving food additive or a health food .

An embodiment of the present invention provides a cosmetic composition for skin improvement comprising a complex lactic acid bacterium disruption solution and fermented extract of marigold as an active ingredient.

As used herein, the " marjoram extract " may be prepared by mixing marjoram, which has been crushed by an extraction method known in the art, in an extraction solvent and then extracting it. Wherein the extraction solvent is at least one aqueous solution selected from the group consisting of water, an anhydrous or a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane and 1,3- Preferably, it may be sterilized water or 60% ethanol, and two or more different solvents may be mixed or used sequentially, but the present invention is not limited thereto.

The term " complex lactic acid bacteria " used in the present invention may be one or more kinds of mixed bacteria selected from the group consisting of lactic acid bacteria generally present in fermented foods such as cheese, kimchi, yogurt, etc. which can be used for food. According to one embodiment of the present invention, the complex lactic acid bacteria are selected from the group consisting of Lactobacillus, Leuconostoc, Lactococcus, Pediococcus, bifidobacterium, , Streptococcus sp., And the like.

The "multiple lactic acid bacterial disruption solution and fermentation extract of marigold" used in the present invention can be prepared by drying the extract of marigolds naturally or by using an apparatus, specifically a rotary vacuum concentrator and a freeze drier, , May be a fermentation extract prepared by diluting the purified water with a certain concentration, preferably 1 to 20% by weight, inoculating the lactic acid bacteria for fermentation, fermenting the lactic acid bacteria for fermentation, and pulverizing the lactic acid bacteria through physical and chemical methods. Specifically, the "crushing" is performed by treating the fermentation broth of the complex lactic acid bacterium with lysozyme, reacting the mixture at 37 ° C. for 3 to 9 hours, preferably 6 hours, using an ultrasonic mill or a homogenizer .

According to one embodiment of the invention, the compound lactic acid bacteria lysate and the combined lactic acid bacterium cells in a fermentation extract content of Portulaca Oleracea may be 10 ⁸ to ¹⁰ 10 CFU / ml, preferably ¹⁰ 10 CFU / ml.

The fermentation microorganism refers to fermentation microorganisms commonly used in the fermentation related industry, and specifically includes, but is not limited to, lactic acid bacteria, enzymes, yeast, fungi, actinomycetes and the like.

The lactic acid bacteria for fermentation refers to lactic acid bacteria for fermentation which are generally known in the fermentation related industry. Specifically, lactic acid bacteria belonging to the genus Lactobacillus, Leuconostoc, Lactococcus, Pediococcus, Leucine, streptococcus, and the like.

According to one embodiment of the present invention, the fermentation extract may be contained in an amount of 2 to 10% by weight, preferably 5% by weight based on the total weight of the composition.

According to one embodiment of the present invention, the skin improvement is characterized in that the skin is selected from the group consisting of antioxidant, dermal cell growth promotion, collagen increase, collagenase reduction, immune cell activity inhibition, inflammation inhibition, skin hydration control, Improvement of skin barrier, improvement of skin texture, reduction of skin erythema and reduction of pigmentation.

Another embodiment of the present invention provides a cosmetic comprising the cosmetic composition for skin improvement.

The cosmetic may have formulations of ointments, solutions, suspensions, emulsions, pastes, gels, creams, lotions, serums, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays , Preferably a formulation of a skin toner, a water cream or a cream, but is not limited thereto.

One embodiment of the present invention provides a pharmaceutical composition for preventing or treating a proliferative skin disease comprising a complex lactic acid bacterium disrupted solution and a fermented extract of marigold as an active ingredient.

According to one embodiment of the present invention, the complex lactic acid bacterium lysate and the fermentation extract of marigolds are prepared by diluting the dried product of the extract obtained from the marigold extract to a certain concentration, preferably 1 to 10% by weight, in a solvent, The fermentation broth prepared by inoculating the complex lactic acid bacterium and preferably fermenting to 10 ⁹ to 10 ¹⁰ CFU / ml or higher is subjected to a disruption method known in the art, specifically, enzyme treatment and sonication to remove lactic acid bacteria And may be a fermentation extract prepared by shredding.

According to one embodiment of the invention, the fermentation extract of the lactic acid compound lysate and Portulaca Oleracea content of the composite may be a lactic acid bacteria lysate is 10 ⁸ to ¹⁰ 10 CFU / ml, preferably ¹⁰ 10 CFU / ml.

According to one embodiment of the present invention, the fermentation extract may be contained in an amount of 2 to 10% by weight, preferably 5% by weight, based on the total weight of the composition.

According to one embodiment of the present invention, the proliferative dermatosis is selected from the group consisting of actinic keratosis, basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma, elevated dermatofibrosis, angioma, flammei, A malignant melanoma, metastatic carcinoma, and psoriasis, in a mammal, including a human, a human,

The pharmaceutical composition of the present invention may be formulated into various oral or parenteral dosage forms, preferably parenteral dosage forms, more preferably ointments.

Examples of formulations for oral administration include, but are not limited to, tablets, pills, hard, soft capsules, liquids, suspensions, emulsions, syrups, granules and the like. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, a lubricant such as silica, talc, stearic acid and its magnesium or calcium salt and / Or polyethylene glycol). The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid Or a disintegrating or boiling mixture such as its sodium salt and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The formulations may be prepared by conventional mixing, granulating or coating methods.

Examples of the formulations for parenteral administration include injections, ointments, suppositories, and the like, but are not limited thereto. Depending on the characteristics of each formulation, appropriate additives such as solvents can be selected according to common knowledge of those skilled in the art.

Another embodiment of the present invention provides a pharmaceutical preparation containing the pharmaceutical composition for preventing or treating the proliferative skin disease.

Another embodiment of the present invention provides a food additive for preventing and treating a proliferative skin disease and containing a fermented extract of a complex lactic acid bacterium as an active ingredient.

Another embodiment of the present invention provides a health functional food for preventing proliferative skin diseases and improving skin comprising an extract of a complex lactic acid bacterium and a fermented extract of marigold as an active ingredient.

According to an embodiment of the present invention, the health functional food may be, for example, but not limited to beverage, gum, tea, vitamin complex, and other health supplement foods. The health functional food may be in the form of a capsule, tablet, powder, granule, liquid, ring, flake, paste, syrup, gel, beverage, jelly or varnish.

According to an embodiment of the present invention, the complex lactic acid bacterium lysate and the fermented extract of marigold may contain 0.05 to 3% by weight based on the total weight of the health functional food.

According to an embodiment of the present invention, the health functional food may include a food supplementary additive suitable in view of common knowledge of those skilled in the art, for example, various nutrients, vitamins, electrolytes, flavors, colorants, , Alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like.

The present invention provides new uses of compositions containing the complex lactic acid bacterium lysate and fermented extract of marigold as an active ingredient for skin improvement or proliferative skin disease prevention and treatment. The fermented extract of the present invention is useful as an antioxidant, dermal cell growth promoting, collagen increasing, collagenase reduction, immune cell activity inhibition, inflammation inhibition, skin hydration control, skin wrinkle improvement, dermis intimacy increase, skin elasticity improvement, Improvement of skin texture, reduction of skin erythema and reduction of pigment deposition. In addition, the fermented extract of the present invention can be used for the treatment and / or prophylaxis of diseases such as actinic keratosis, basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma, hypertrophic dermatofibroma, hemangioma, flamma, hematoma, Kaposi sarcoma, mastocytosis, And proliferative skin diseases such as dysplasia, nevus cellulata, malignant melanoma, malignant melanoma, metastatic carcinoma and psoriasis.

Fig. 1 shows the effect of decomposition of active oxygen of the complex-type lactic acid bacteria disruptant of the present invention, fermented extract of mashin, alpha -tocopherol and BHT.
FIG. 2 shows the cell activity of human dermal fibroblasts (nHDF) according to the concentration of the fermented extract of the complex lactic acid bacterium of the present invention and marigold.
FIGS. 3 and 4 show changes in expression of COL1A1 and collagenase MMP1 in human dermal fibroblasts, respectively.
FIG. 5 shows the cell activity of mouse macrophages (RAW 264.7) according to the concentration of the complex lactic acid bacteria disruptant of the present invention and the fermentation extract of marigold.
Figure 6 shows the activity of luciferase in immune cells according to the concentration of the complex lactic acid bacterial disruptant of the present invention and fermented extract of marigold.
FIG. 7 and FIG. 8 show changes in IL-1β and TNF-α gene expression according to the concentrations of the fermented extract of the complex lactic acid bacteria and the marine algae of the present invention, respectively.
9 to 17 are graphs showing skin moisture content, skin oil content, degree of skin wrinkles, dermal density, skin elasticity, and skin elasticity of a subject to be tested according to the duration of use of the cream and toner containing the complex lactic acid bacterium disruptant of the present invention and the fermented extract of marigold, , Transdermal water loss, skin texture improvement, skin erythema degree, and pigmentation area.

Hereinafter, the present invention will be described in more detail with reference to one or more embodiments. However, these embodiments are illustrative of one or more embodiments, and the scope of the present invention is not limited to these embodiments.

1. Preparation of sample

1.1. Preparation of Fermented Extract of Lactic Acid Bacteria and Lactic Acid Bacteria

The crude lactic acid bacteria disruption solution and the fermentation extract of marigold were obtained using an ethanol extraction method commonly used in the art. Specifically, the crushed marjoram was mixed with 60% ethanol so as to have a concentration of 10% by weight. The mixed raw materials were extracted by stirring at room temperature for 48 hours. The crude extract was filtered through 250 mesh and 0.5 mu m filter paper to obtain a liquid extract. Extraction dried material was obtained from the filtered liquid extract using a rotary vacuum concentrator and a freeze dryer. The extract was diluted to 5% by weight with purified water, and then fermented by inoculating Lactobacillus plantarum, Lactobacillus acidophilus, Leuconostoc mesenteroides, Bifidobacterium longum and Streptococcus thermophilus, which are known lactic acid bacteria, at the same ratio. The fermented extract was mixed with 0.001% by weight of lysozyme and reacted with the compound lactic acid bacteria at 37 ° C for 3 to 9 hours, and the lactic acid bacteria were disrupted using an ultrasonic mill. The pulverized liquid was filtered through 500 mesh and 0.2 mu m filter paper to obtain a liquid fermentation extract. As a result of carrying out a related test example of the composition according to the present invention thus formed, it was confirmed that there is no abnormality in the skin stability.

1.2. Manufacture of skin toner

The composition of the ingredients for making a skin toner containing the composition of the present invention is shown in the following Table 1 and is prepared by the following procedure. The A phase (water phase) was completely dissolved at room temperature and the B phase (alcohol phase) was heated to 50 DEG C using a heater. The phase B was mixed with the phase A and solubilized at 1000 rpm for 10 minutes using a disperser. The mixture was cooled, filtered, and maintained at a pH of 5.2 to complete a skin toner. The crude lactic acid bacterium lysate and the fermented extract of mariginase were prepared by adding 5 wt% of the extract.

Prize Raw material Content (% by weight) Control group Positive control group Experimental group A phase Purified water 5 - - Aloe - 5- - Eternal life and sea bird
Complex fermentation extract - - 5 Disodium EDTA 0.01 0.01 0.01 1,3-butylene glycol 4 4 4 Citric acid 0.04 0.04 0.04 Sodium citrate 0.1 0.1 0.1 B phase Hyaluronic acid 2 2 2 Hydrogenated 0.4 0.4 0.4 Castor oil Suitable amount Suitable amount Suitable amount C phase Purified water Up to 100 Up to 100 Up to 100

1.3. Manufacture of moisture cream

The composition of the composition for making the water cream containing the composition of the present invention is shown in Table 2 below. Concretely, the A phase was heated to 80 DEG C and stirred, and the B phase was heated to 80 DEG C to dissolve, and then the mixture was put into the phase A and emulsified at 2000 rpm for 10 minutes. The C phase was evenly mixed, and the mixture was put on the A + B phase at 80 ° C and neutralized at 2000 rpm for 5 minutes. The solution was cooled to 30 DEG C while stirring slowly, and adjusted to pH 6.15. The crude lactic acid bacterium lysate and the fermented extract of mariginase were prepared by adding 5 wt% of the extract.

Phase Raw material Content (% by weight) Control group Positive control group Experimental group A phase Purified water 5 - - Aloe - 5 - Extracts of fermented Marchia-prey containing complex lactic acid bacteria - - 5 Disodium EDTA 0.01 0.01 0.01 Paraoxybenzoic acid ester 0.2 0.2 0.2 Clofenine 0.2 0.2 0.2 Hyaluronic acid 5 5 5 Aloe gel 5 5 5 1% aqueous solution of xanthan gum 5 5 5 Carboxyvinyl polymer 15 15 15 B phase Squall 7 7 7 Dimethicone 3 3 3 Cyclomethicone 3 3 3 Polysorbate 60 2 2 2 Sorbitolate 0.5 0.5 0.5 Glyceryl stearate One One One Stearic acid One One One C phase Triethanolamine One One One Purified water Up to 100 Up to 100 Up to 100

1.4. Manufacture of cream

The constituents for preparing the cream containing the composition of the present invention are shown in Table 3 below. Concretely, the A phase was heated to 80 DEG C and stirred, and the B phase was heated to 80 DEG C to dissolve, and then the mixture was put into the phase A and emulsified at 2000 rpm for 10 minutes. The C phase was evenly mixed, and the mixture was put on the A + B phase at 80 ° C and neutralized at 2000 rpm for 5 minutes. Subsequently, the mixture was gradually cooled to 30 DEG C while being mixed and stirred, and adjusted to pH 6.23. The crude lactic acid bacterium lysate and the fermented extract of mariginase were prepared by adding 5 wt% of the extract.

Prize Raw material Content (% by weight) Control group Positive control group Experimental group A phase Purified water 5 - - Aloe - 5 - Extracts of fermented Marchia-prey containing complex lactic acid bacteria - - 5 Disodium EDTA 0.01 0.01 0.01 Paraoxybenzoic acid ester 0.2 0.2 0.2 Clofenine 0.2 0.2 0.2 Hyaluronic acid 5 5 5 1% aqueous solution of xanthan gum 5 5 5 Carboxyvinyl polymer 15 15 15 B phase Squall 7 7 7 Dimethicone 3 3 3 Cyclomethicone 3 3 3 Polysorbate 60 2 2 2 Sorbitolate 0.5 0.5 0.5 Sheer butter 5 5 5 Glyceryl stearate One One One Cetostearyl alcohol One One One Stearic acid One One One C phase Purified water Up to 100 Up to 100 Up to 100

2. Experimental methods and results

Example 1. Confirmation of the effect of decomposition of active oxygen by the fermented extract of the complex lactic acid bacterium disruptant and marigold

In order to confirm the effect of improving the beauty of skin by the combined lactic acid bacteria disintegration liquid and fermented extract of marigold, it was investigated whether active oxygen species, which plays a major role in skin aging, were removed by the mixed lactic acid bacteria disintegration liquid and fermentation extract of marigold.

(1, 1-diphenyl-2-picryl hydrazyl, DPPH) was used in order to confirm the effect of decomposition of active oxygen in fermented extracts of Lactobacillus sp. Free radical scavenging activity. 4 mL of methanol was added to the test tube, and the crude lactic acid bacteria disruption solution and the fermented extract of mashin were added by concentration. Then, 1 mL of 0.15 mM DPPH solution was added and reacted at room temperature for 30 minutes and absorbance was measured at 520 nm. Initial (Ai) and blank (Ab) without substrate and DPPH were measured, and positive control group compared α-tocopherol and BHT.

As a result of the analysis, the complex lactic acid bacterium disruption solution and the fermented extract of marigold according to the present invention showed a higher active oxygen decomposition ability at a lower concentration than the positive control groups, -tocopherol and BHT (see Table 4 and FIG. 1). Therefore, it was confirmed that the decomposition of active oxygen was promoted by the complex lactic acid bacterial disruption solution and the fermentation extract of marigold, thereby being effective in improving skin beauty.

Concentration (mg / L) 0 One 10 100 extract 0 32.5 41.2 76.5 alpha -tocopherol 0 25.3 37.9 68.5 BHT 0 39.4 53.1 82.2

Example  2. Complex lactic acid bacteria  The crushing liquid and Machi  By fermentation extract Of human dermal fibroblast  Confirmation of growth promoting effect

In order to confirm the skin cosmetic improvement effect of the mixed lactic acid bacteria disruption liquid and the fermented extract of marigold, the fibroblast growth to synthesize collagen, which is a constituent of the dermis, was promoted by the fermentation extract of the complex lactic acid bacteria and the marigold.

Human dermal fibroblast (nHDF) was purchased from Lonza. Cell culture medium was supplemented with 10% fetal bovine serum (FBS; Gibco), 10% penicillin (100 units / ml) and streptomycin (100 g / ml) in Dulbecco's modified Eagle's sediment (DMEM; Gibco) mL) was added. The cultivation was carried out under the conditions of 37 ° C, 95% humidity, 5% CO ₂ incubator, and cells between 3 and 10 generations were used for the experiment. In order to investigate the degree of cytotoxicity of human lactic acid bacteria bacterium nHDF cells treated with the mixed lactic acid bacterium and fermented extract of marigold, Cells were cultured for a period of time, and viability was then measured using MTT of the cells.

As a result, the cell growth was increased in a concentration-dependent manner after treatment of the mixed lactic acid bacterium disruptant and the fermented extract of marigold, compared to the control (no treatment) group (see Table 5 and Fig. 2 Reference).

Time (hr) Concentration (mg / L) 0 One 10 100 0 100 100 100 100 24 99.48 104.11 107.65 111.15 48 100.41 105.14 110.61 113.61 72 103.16 109.61 113.15 123.15

Example  3. Complex lactic acid bacteria  The crushing liquid and Machi  By fermentation extract Col1A1  Increased expression and Of MMP1  Confirm expression reduction

In order to confirm the skin cosmetic improvement effect of the mixed lactic acid bacteria disruption liquid and the fermentation extract of marigold, the expression of COL1A1, one of typical collagen expressed by human dermal fibroblast, and MMP1, a collagenase, was measured. In order to confirm the degree of collagen expression change by the combined lactic acid bacteria disruption liquid and the fermentation extract of marigold, the human lipid fibroblast was treated with each of the complex lactic acid bacterium disruption liquid and the fermentation extract of marigolds, and then cultured for 24 hours.

Herein, quantitative real-time PCR (qRT-PCR), which is a method of continuously detecting a fluorescent substance by attaching a fluorescent substance to a DNA product amplified during a polymerase chain reaction (PCR) PCR). To quantitatively determine the expression of COL1A1 and MMP1 gene expression by the combined lactic acid disruption and fermentation extracts of marigolds, SYBR Green I (Invitrogen) was used to confirm the results of the fluorescence values emitted by the PCR products Respectively. The reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ and 1 × green. The denaturation, annealing and polymerization were carried out at 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds, respectively, after primary denaturation at 94 ° C for 3 minutes using Linegene K (BioER) 40 cycles and fluorescence intensity was measured after each cycle. PCR results were verified by melting curve for each result. The threshold cycle (Ct) value of each gene was normalized to the Ct value of β actin, and then the change amount of the Ct value was compared and analyzed. The Ct value was the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reached a constant reference value above the basal value, and the amount of gene expression was confirmed through the Ct value. The respective genes used in the experiment are shown in Table 6 below.

gene Forward primer Reverse primer beta -actin 5-GGATTCCTATGTGGGCGACGA-3 5-CGCTCGGTGAGGATCTTCATG-3 COL1A1 5-AGGGCCAAGACGAAGACATC-3 5-AGATCACGTCATCGCACAACA-3 MMP1 5-TCTGACGTTGATCCCAGAGAGCAG-3 5-CAGGGTGACACCAGTGACTGCAC-3

As a result of analysis, the expression of Col1A1 mRNA was increased in a concentration-dependent manner after treatment of the mixed lactic acid bacterial disruptant and the fermented extract of marigold compared to the control (see Table 7 and FIG. 3). In addition, the expression of MMP1 mRNA was decreased in a concentration-dependent manner after the treatment of the complex lactic acid bacterial disruption solution and the fermentation extract of marigold compared to the control (see Table 8 and FIG. 4).

Concentration (mg / L) 0 One 10 100 Expression of Col1A1 (%) 100 107.45 149.56 212.56

Concentration (mg / L) 0 One 10 100 MMP1
Expression (%) 100 99.56 85.61 60.16

Example 4. Confirmation of growth inhibition effect of immune cells by the fermented extracts of the complex lactic acid bacteria disrupted liquid and marigold

In order to confirm the effect of improving the skin cosmetic effect of the combined lactic acid bacteria disintegrating liquid and the fermentation extract of marigold, mouse macrophage (RAW 264.7) growth affecting the inflammation and erythema of the skin was observed in the fermented extract of the complex lactic acid bacteria and mashin Respectively. Mouse immune cells were purchased from Korean Cell Line Bank (KCTC). Cell culture medium was prepared by adding 10% FBS (Gibco), 10% penicillin (100 units / mL) and streptomycin (100 g / mL) to DMEM (Gibco). The cultivation was carried out at 37 ° C, 95% humidity, 5% CO ₂ incubator conditions, and cells between 3 and 10 generations were used for the experiment. To investigate the degree of cytotoxicity of RAW 264.7 cells, which were mouse immune cells treated with the Lactobacillus spp. And the fermentation extracts of the mashin, the mixed lactic acid bacteria lysate and the fermented extract of the mashin were treated at respective concentrations Cells were cultured for 24 hours, and then cell activity was measured.

As a result of the analysis, the cell growth was decreased in a concentration-dependent manner after treatment of the mixed lactic acid bacterium and the fermented extract of marigold compared to the control (untreated group) (see Table 9 and FIG.

Time (hr) Concentration (mg / L) 0 One 10 100 0 100 100 100 100 24 100.12 98.64 94.88 91.44 48 99.46 94.64 93.64 88.84 72 98.64 92.96 89.96 82.48

Example 5 Confirmation of Inhibitory Effect of NF-κB Transcription by Mixed Lactobacillus Lysate and Fermented Extracts of Marchia

In order to investigate the effect of NF-κB transcription factor, which plays an important role in the immune response, on NF-κB transcription ability of the complex lactic acid bacterial disruption solution and fermented extract of mashin Respectively.

NF-κB reporter analysis was performed to determine the degree of inhibition of NF-κB transcription by the Lactobacillus spp. The promoter region of the gene known to be transcribed by NF-κB was cloned into a vector containing the luciferase gene and inserted into the immune cells. NF-κB transcription-inducing LPS was treated to activate the NF-κB transcriptional activity, followed by lysate fermentation of the complex lactic acid bacterium and the fermentation extract of the mash. The luciferase activity is determined by the presence of the IgG kappa NF- kappa B region (SEQ ID NO: 5-GGGGACTTTCC-3) oligonucleotides upstream of the minimal IL-8 promoter (positions 67 to +44), the NF- (Gibco-BRL), and the gene was inserted into the immune cells according to the reagent manual. The lipofectamine plus (Gibco-BRL) The transfection ability of the NF-κB was activated by treating LPS with the gene-inserted immune cells, and then the complex lactic acid bacterium disruption solution and the fermented extract of the mashing were treated at the concentrations of 0, 1, 10, and 100 mg / Lt; / RTI > The cultured immune cells were directly added with 0.1 mL dissolution buffer (0.1 M HEPES, pH 7.6, 1% Triton-X, 1 mM DTT, and 2 mM EDTA) at the time of collection to collect cell lysates, The cells were centrifuged at rpm to obtain cell proteins. Protein concentration was measured using a Bradford assay (Bio-Rad Laboratories), and then 20 μg of protein was added to a luciferase assay mixture (25 mM glycylglycine, 15 mM MgSO ₄ , 1 mg / mL BSA, ATP and 1 mM D-luciferin (Analytical Luminescence Laboratory), and the luminescence was measured three times repeatedly for 20 seconds using Monolight 2010 (Analytical Luminescence Laboratory) to obtain an average value.

As a result of the analysis, the transactivity of NF-κB increased by LPS after the treatment of the complex lactic acid bacterial disruption solution and the fermentation extract of marigold decreased in a concentration-dependent manner as compared with the LPS-treated group (see Table 10 and FIG.

Extract concentration (mg / L) 0 0 One 10 100 LPS - + + + + Luciferase activity
(Drainage) One 4.56 4.21 3.41 1.48

Example  6. Complex lactic acid bacteria  The crushing liquid and Machi  Identification of inhibitory effect of fermented extract on inflammatory cytokine synthesis

In order to confirm the skin cosmetic improvement effect of the combined lactic acid bacteria disruption liquid and the fermentation extract of marigold, it was confirmed that the synthesis of the inflammatory cytokine inducing the immune response was inhibited by the fermentation extract of the complex lactic acid bacteria and the marigold.

In order to confirm the extent of expression of inflammatory cytokines by the combined lactic acid bacteria-containing lysate and the fermented extract of marigolds, the LPS-treated immune cells were treated with the disrupted lactic acid bacteria lysate and the fermented extract of marigolds at respective concentrations, Lt; / RTI > The gene expression changes were then measured by qRT-PCR. In order to quantitatively determine the IL-1β and TNF-α gene expression changes by the combined lactic acid bacterial disruption solution and the fermentation extract of the mashin, SYBR Green I (Invitrogen) was used to confirm the results by the fluorescence values emitted by the PCR products. Were used. The reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ and 1 × green. The denaturation, annealing and polymerization were carried out at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds, respectively, after primary denaturation at 94 ° C for 3 minutes using Linegene K (BioER) 40 cycles and fluorescence intensity was measured after each cycle. PCR results were verified by melting curve for each result. The Ct value of each gene was normalized to the Ct value of β - actin, and the change of the Ct value was compared and analyzed. The Ct value was the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reached a constant reference value above the basal value, and the amount of gene expression was confirmed through the Ct value. Primers for each gene used in the experiment are shown in Table 11 below. L-NG-monomethyl arginine and acetate salt (L-NG-Monomethylarginine, Acetate Salt; L-NMMA) were used as the positive control.

gene Sequential primer Reverse primer beta -actin 5-CCAAGGCCAACCGCCGC-3 5-AGGGTACATGGTGCCGCC-3 IL-1? 5-CTCAATGGACAGAATATCAACCAACA-3 5-ACAGGACAGGTATAGATTCTTTCCTTTG-3 TNF-a 5-TGCCTATGTCTCAGCCTCTTC-3 5-GAGGCCATTTGGGAACTTCT-3

As a result of the analysis, the expression of IL-1β mRNA increased by LPS in a concentration-dependent manner after the treatment of the complex lactic acid bacterium disruption solution and the fermentation extract of marigold was decreased as compared with the LPS treatment group (see Table 12 and FIG. In addition, the expression of TNF-α mRNA increased by LPS in a concentration-dependent manner after treatment of the complex lactic acid bacterium disruption solution and the fermentation extract of marigold was decreased as compared with the LPS treatment group (see Tables 13 and 8 below).

Extract concentration (mg / L) 0 0 One 10 100 LPS - + + + + IL-1? MRNA expression
(Drainage) One 3.74 3.15 2.45 1.36

Extract concentration (mg / L) 0 0 One 10 100 LPS - + + + + Expression of TNF-α mRNA
(Drainage) One 3.85 3.25 2.45 1.65

Example  7. Complex lactic acid bacteria  The crushing liquid and Machi  Skin by fermentation extract Water content  Confirmation of index change

In order to confirm the improvement effect of the skin cosmetic effect of the mixed lactic acid bacteria disintegrating liquid and the fermented extract of marigold, the skin water content was analyzed using a corneometer and a sebumeter.

First, in order to examine the effect of the mixed lactic acid bacterium disruption liquid and the fermented extract of marigold on the improvement of skin oiliness, 30 adult women over 30 years old were selected to use the cream and toner prepared above. Cream and toner were used for the control, the positive control and the experimental group for 2 weeks and 4 weeks, respectively, and skin moisture index was measured according to the instrument manual using a moisture analyzer (MPA5, Courage-Khazaka Electronic GmbH). Using a moisture analyzer, the same test person measured the right ball portion of the subject to be measured three times in succession. Skin oil index was measured according to the instrument manual using a sebum analyzer (MPA5, Courage-Khazaka Electronic GmbH). Using the sebum analyzer, the same person in charge of the test measured the area of the forehead which is the measurement site of the subject.

As a result of analysis, the skin moisture was increased in a time-dependent manner after treatment of the complex lactic acid bacterium and the fermented extract of marigold compared to the control (no treatment group) (see Table 14 and FIG. 9). In addition, in the case of oil, the skin moisture was increased in a concentration-dependent manner after treatment of the complex lactic acid bacterium disruption solution and the fermented extract of marigolds, as compared with the control (no treatment group) (see Tables 15 and 10 below).

Before experiment after 2 weeks After 4 weeks Control group 265 271 272 Positive control group 259 276 290 Experimental group 266 315 330

Before experiment after 2 weeks After 4 weeks Control group 315 320 323 Positive control group 324 335 345 Experimental group 322 341 355

Example 8 Confirmation of Effect of Combined Lactic Acid Bacterial Lactate and Fermented Extract of Mashishi on Improving Skin Wrinkles

Skin wrinkles were analyzed by using a skin analyzer in order to confirm the effect of improving the wrinkles of the complex lactic acid bacterium and the fermented extract of marigold.

First, in order to confirm the effect of the combined lactic acid bacterium disruption liquid and the fermentation extract of marigolds on the improvement of skin wrinkles, 30 adult women over 30 years old were selected and the cream and toner were used. After using the cream and toner for 2 weeks and 4 weeks for the control, the positive control and the experimental group respectively, the degree of facial wrinkles was measured according to the machine manual using PRIMOS Lite (field of view 18 flexible 3D measuring, GFMesstechnik GmbH) Respectively. Using the PRIMOS light, the same test person fixes the face of the subject on a specially prepared PRIMOS facial fixation kit and adjusts the tip of the eye, the measurement site, to match the focus pattern of the PRIMOS light Respectively.

As a result of analysis, the wrinkles of the eye tail were decreased in a time-dependent manner after the treatment of the complex lactic acid bacterium disruption solution and the fermentation extract of marigold in a time-dependent manner (see Table 16 and Fig.

Before experiment after 2 weeks After 4 weeks Control group 32.15 31.56 31.01 Positive control group 33.48 31.41 28.27 Experimental group 31.24 29.27 27.01

Example 9. Confirmation of Skin Thickening Effect by Fermented Extract of Lactic Acid Bacterium and Lactic Acid Bacteria

Increased proliferation of dermal fibroblasts and increased expression of collagen by fermented extracts of multiple lactic acid bacteria and mashishi were confirmed. Thus, it was analyzed whether the thickness of the dermal layer was increased by the collagen produced, and the effect of improving the skin thickness by the fermented extract of the complex lactic acid bacteria disruption solution and the marigold was confirmed.

First, in order to examine the effects of the mixed lactic acid bacteria disruption liquid and the fermented extract of marigolds on skin thickness, 30 adult women over 30 years old were selected and the cream and toner were used. The control, positive control and experimental groups were allowed to use cream and toner for 2 and 4 weeks, respectively, and then measured according to the instrument manual using a DUB-skin scanner (tpm taberna pro medicum). A DUB-skin scanner was applied with a gel for ultrasound examination and the probe was placed at right angles to the skin, and then the same test person was measured with the same pressure on the right side of the subject's right eye.

As a result of analysis, the skin thickness was increased in a time-dependent manner after treatment of the complex lactic acid bacterium disruption solution and the fermentation extract of marigold (see Table 17 and Fig. 12 below), as compared with the control (no treatment group).

Before experiment after 2 weeks After 4 weeks Control group 54.65 54.89 55.41 Positive control group 54.56 55.15 55.65 Experimental group 53.51 54.41 56.65

Example 10 Confirmation of Skin Elasticity Improvement Effect by Combined Lactic Acid Bacterial Lysate and Fermented Extract of Mashin

The effect of improving the skin elasticity was confirmed in order to confirm the skin cosmetic improvement effect of the mixed lactic acid bacteria disintegrating liquid and the fermented extract of marigold. To investigate the effect of multiple lactobacillus disruption and fermented extracts of marigolds on skin thickness, 30 adult women over 30 years old were selected and cream and toner were used. After using cream and toner for 2 weeks and 4 weeks for control, positive control and experimental group respectively, the degree of change of skin elasticity was measured with DermaLab USB elasticity probe. The DermaLab USB elastic probe shows the skin change and resilience with time of inhalation and inhalation time. The same test person measured the probe three times after fixation to the left ball with tape.

As a result of analysis, skin elasticity was increased in a time-dependent manner after treatment of the mixed lactic acid disruptant and the marine fermented extract, as compared to the control (no treatment group) (see Tables 18 and 13 below).

Before experiment after 2 weeks After 4 weeks Control group 11.56 11.61 11.41 Positive control group 12.13 12.65 12.98 Experimental group 12.10 12.89 13.51

Example 11 Confirmation of Improvement of Skin Barrier Effect by Combined Lactic Acid Bacterial Lysate and Fermented Extract of Mashishi

The skin barrier improvement effect was confirmed in order to confirm the skin cosmetic improvement effect of the mixed lactic acid bacteria disintegrating liquid and the fermented extract of marigolds.

First, in order to confirm the effect of the complex lactic acid bacterium disruption liquid and the fermentation extract of marigold on the skin barrier improvement, 30 adult women over 30 years old were selected and the cream and toner were used. Cream and toner were used for 2 weeks and 4 weeks for the control, the positive control and the experimental group, respectively. Then, the TEWL (TransEpidermal Water Loss) was measured using a DermaLab USB TEWL probe (Cortex Technology, Inc.) It was measured according to the manual of the apparatus.

 As a result of analysis, the amount of transdermal water loss was reduced in a time-dependent manner after treatment of the complex lactic acid bacterium disruption solution and the fermentation extract of marigold in a time-dependent manner (see Table 19 and FIG.

Before experiment after 2 weeks After 4 weeks Control group 32.89 32.56 32.01 Positive control group 33.51 32.51 31.84 Experimental group 33.15 32.12 31.02

Example 12. Confirmation of improvement of skin texture by fermented extract of multiple lactic acid bacterium disruption liquid and marigold

In order to confirm the skin cosmetic improvement effect of the mixed lactic acid bacteria disintegrating liquid and the fermented extract of marigold, the skin texture improving effect was confirmed.

First, in order to examine the effect of the combined lactic acid bacterium disruption liquid and the fermented extract of marigolds on the improvement of skin texture, 30 adult women over 30 years old were selected to use the cream and toner prepared above. After 2 and 4 weeks of cream and toner were applied to the control, positive control, and experimental groups respectively, skin texture was measured according to the instrument manual using PRIMOS light (field of view 45, flexible 3D measuring, GFMesstechnik GmbH). Using the PRIMOS light, the same test person measured the area of the forehead of the subject three times in succession and measured the same area.

As a result of analysis, the depth of the wrinkles generated on the skin to determine the texture of the skin was decreased in a time-dependent manner after the treatment of the complex lactic acid bacterium disruption solution and the fermentation extract of the marigold (in the following Tables 20 and 15 Reference).

Before experiment after 2 weeks After 4 weeks Control group 16.16 15.98 15.76 Positive control group 15.85 15.43 14.85 Experimental group 16.15 15.36 14.78

Example  13. Complex lactic acid bacteria  The crushing liquid and Machi  Identification of erythema improvement effect by fermentation extract

The inhibition of the proliferation of the immune cells and the decrease of the expression of the inflammatory cytokines by the fermented extracts of the complex lactic acid bacteria and the marigold were confirmed through Examples 5, 6 and 7 above. Therefore, it was analyzed whether the erythema was reduced by the reduced cytokine, and the erythema improvement effect of the fermented extract of the mixed lactic acid bacteria and the marigold was confirmed.

First, in order to examine the effect of the mixed lactic acid bacteria disintegrating liquid and the fermented extract of marigold on the erythema improvement, 30 adult women over 30 years old were selected and the cream and toner were used. The control, positive control, and experimental groups were allowed to use cream and toner for two and four weeks, respectively, followed by spectrophotometer (Spectrophotometer CR-2600D, Konica Minolta) according to the instrument manual. The same test person measured the right side of the subject's right side of the ball three times in succession, and the mean value was calculated and used for the analysis. The values measured in the spectrophotometer are L *, a *, and b *, and the red value indicates a lot of red as the value of a * increases to a value indicating erythema.

As a result of analysis, erythema was reduced in a time-dependent manner after the treatment of the complex lactic acid bacterium disruption solution and the fermentation extract of marigold in a time-dependent manner (see Table 21 and FIG. 16).

Before experiment after 2 weeks After 4 weeks Control group 12.52 12.41 12.12 Positive control group 12.79 12.31 11.89 Experimental group 13.16 12.15 11.61

Example 14 Confirmation of Improvement of Pigmentation Deposition by Combined Lactic Acid Bacterial Disruption Solution and Fermented Extract of Marigold

In order to confirm the skin cosmetic improvement effect of the mixed lactic acid bacteria disintegrating liquid and the fermented extract of marigold, the effect of improving pigment deposition was confirmed.

First, in order to confirm the effect of the combined lactic acid bacterium disruption solution and fermented extract of marigolds on improvement of pigment deposition, 30 adult women over 30 years old were selected and cream and toner were used. The control, positive control and experimental groups were allowed to use cream and toner for 2 and 4 weeks, respectively, and then measured according to the instrument manual using ANTERA 3D (Miravex). The same examiner used the left ball area three times in a row for analysis.

As a result of analysis, the area of pigmentation decreased in time-dependent manner after treatment of the complex lactic acid bacterium disruption solution and the fermentation extract of marigold compared to the control (no treatment) (see Table 22 and Fig. 17 below).

Before experiment after 2 weeks After 4 weeks Control group 222.16 218.64 214.13 Positive control group 220.16 201.61 196.61 Experimental group 218.61 184.65 165.64

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and equivalents thereof are included in the scope of the present invention. .

Claims (16)

Claims 1. A cosmetic composition for improving skin wrinkles, comprising a complex lactic acid bacterium disruption solution and fermented extract of marigold as an active ingredient,
The fermented extract is prepared by diluting the dried product of the extract of the complex lactic acid bacteria and the fermented extract of the marigold with the solvent, inoculating the complex lactic acid bacteria,
Wherein the complex lactic acid bacteria comprises Lactobacillus plantarum , Lactobacillus acidophilus , Leuconostoc mesenteroides , Bifidobacterium longum and Streptococcus thermophilus .
delete delete The method according to claim 1,
Wherein the complex lactic acid bacterium cells have a content of 10 ⁸ to 10 ¹⁰ CFU / ml or more in the crude lactobacillus lysate and fermentation extract of marigold.
The method according to claim 1,
Wherein the fermentation extract is contained in an amount of 2 to 10% by weight based on the total weight of the composition.
The method according to claim 1,
Wherein the improvement in skin wrinkles is caused by an increase of collagen due to promotion of dermal cell growth and reduction of collagenase.
7. A cosmetic comprising the cosmetic composition for improving skin wrinkles according to any one of claims 1 to 6.
A pharmaceutical composition for preventing or treating a proliferative skin disease comprising a complex lactic acid bacterium disrupted solution and a fermented extract of marigold as an active ingredient,
The fermented extract is prepared by diluting the above-mentioned complex lactic acid bacteria lysate and the fermented extract of the marigold with the extract of the extract obtained from the marinade in a solvent, inoculating the complex lactic acid bacteria,
Wherein said complex lactic acid bacteria include Lactobacillus plantarum , Lactobacillus acidophilus , Leuconostoc mesenteroides , Bifidobacterium longum and Streptococcus thermophilus ,
Wherein the proliferative skin disease is selected from the group consisting of actinic keratosis, basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma, hypertrophic dermatosarcoma, hemangioma, flamma, hematoma, Kaposi sarcoma, mastocytosis, A malignant melanoma, a metastatic carcinoma, and psoriasis, wherein the composition is at least one selected from the group consisting of benign cell nevus, malignant melanoma, malignant melanoma, metastatic carcinoma and psoriasis.
delete delete 9. The method of claim 8,
A pharmaceutical composition for preventing or treating a proliferative skin disease, wherein the complex lactic acid bacterium cells have a content of 10 ⁸ to 10 ¹⁰ CFU / ml or more in the lactic acid bacteria-containing lysate and fermented extract of marigold.
9. The method of claim 8,
Wherein the fermentation extract is contained in an amount of 2 to 10% by weight based on the total weight of the composition.
delete A pharmaceutical preparation comprising the pharmaceutical composition for the prophylactic or therapeutic treatment of proliferative skin diseases according to any one of claims 8, 11 and 12.
The present invention relates to a food additive for preventing proliferative skin diseases and improving wrinkles of skin, which contains a complex lactic acid bacterium disruption solution and fermented extract of marigold as an active ingredient,
The fermented extract is prepared by diluting the above-mentioned complex lactic acid bacteria lysate and the fermented extract of the marigold with the extract of the extract obtained from the marinade in a solvent, inoculating the complex lactic acid bacteria,
Wherein said complex lactic acid bacteria include Lactobacillus plantarum , Lactobacillus acidophilus , Leuconostoc mesenteroides , Bifidobacterium longum and Streptococcus thermophilus ,
Wherein the proliferative skin disease is selected from the group consisting of actinic keratosis, basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma, hypertrophic dermatosarcoma, hemangioma, flamma, hematoma, Kaposi sarcoma, mastocytosis, Wherein the food additive is at least one selected from the group consisting of nevus cell carcinoma, malignant melanoma, malignant melanoma, metastatic carcinoma and psoriasis.
A health functional food for preventing proliferative skin diseases and improving skin wrinkles containing a complex lactic acid bacterium lysate and a fermented extract of marigold as an active ingredient,
The fermented extract is prepared by diluting the above-mentioned complex lactic acid bacteria lysate and the fermented extract of the marigold with the extract of the extract obtained from the marinade in a solvent, inoculating the complex lactic acid bacteria,
Wherein said complex lactic acid bacteria include Lactobacillus plantarum , Lactobacillus acidophilus , Leuconostoc mesenteroides , Bifidobacterium longum and Streptococcus thermophilus ,
Wherein the proliferative skin disease is selected from the group consisting of actinic keratosis, basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma, hypertrophic dermatosarcoma, hemangioma, flamma, hematoma, Kaposi sarcoma, mastocytosis, A malignant melanoma, a metastatic carcinoma, and psoriasis, which is characterized in that it is at least one selected from the group consisting of benign cell nevi, malignant melanoma, malignant melanoma, metastatic carcinoma and psoriasis.

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