KR102405511B1 - Composition for skin regeneration, improvement or treatment damaged by ultraviolet light containing blackberry fermented product as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for skin regeneration and improvement or treatment damaged by ultraviolet rays containing blackberry fermented product as an active ingredient. Since the effect is remarkably superior to the previously known blackberry extract, it can be used more usefully in the development of skin wrinkles or skin inflammation-related inner beauty food, pharmaceuticals, etc.

Description

Composition for skin regeneration, improvement or treatment damaged by ultraviolet light containing blackberry fermented product as an active ingredient}

The present invention relates to a composition for skin regeneration and improvement or treatment damaged by ultraviolet rays containing blackberry fermented product as an active ingredient.

The skin is the most external organ of the body, and at the same time has various functions and is an important organ that has a great influence on appearance and image. As it covers the outer surface of the body, it can be said to be an organ that plays the role of the body's primary defense barrier, such as preventing the evaporation of moisture, controlling body temperature, protecting the human body from external stimuli such as ultraviolet rays, and preventing the intrusion of harmful microorganisms.

In addition, the skin protects the human body from physical and chemical injury, infection of microorganisms and parasites such as bacteria or fungi, injury by ultraviolet rays, drying, etc., while also acting as a nerve receptor for various external stimuli, and recognizes external antigens. It serves to provide immune cells. This skin can be damaged by various external factors including sunlight. Sunlight is composed of visible light, ultraviolet light, and infrared light. Among them, ultraviolet light plays a beneficial role such as synthesizing vitamin D in the body and sterilizing it, and at the same time, it prevents skin aging, skin cancer, dryness, dermatitis, fine lines, blemishes, and freckles. also make it happen UV rays are divided into three types, A, B, and C, depending on the wavelength. Among them, UVC is blocked by the ozone layer, and UVA and UVB affect the skin. In particular, UVB (290-320 nm), a medium-wavelength ultraviolet ray, causes many side effects such as skin cancer, suppression of the immune system, and photoaging.

On the other hand, black berry is a plant group belonging to the dicotyledonous plant Rosaceae Rosaceae and strawberries. It is rich in various vitamins and can enhance immunity. It is good for eyestrain with high vitamin A content, is rich in dietary fiber, and contains anthocyanins. It is widely known for its abundance. In particular, among the anthocyanins contained in blackberries, C3G (Cyanidin-3-O-β-d-glucopyranoside) is known to have the best antioxidant effect. It is strong, and the highest C3G content of blackberries is 3 times that of mulberry, 69 times that of grapes, and 6.9 times that of colored rice. C3G prevents brain aging and physical aging by removing free radicals, and is also effective in treating retinal disorders and improving eyesight. There is also a report that delays the growth of tumor cells in mouse skin and reduces the growth and migration of human lung cancer cells.

On the other hand, Korean Patent Publication No. 2019-0058091 discloses 'Anti-inflammatory composition containing blueberry extract', and Korean Patent Publication No. 2009-0105492 discloses 'Whitening cosmetic composition containing acai berry extract as an active ingredient. ' is initiated. However, a composition for skin regeneration and improvement or treatment damaged by ultraviolet rays containing the blackberry fermented product of the present invention as an active ingredient has not yet been disclosed.

The present invention was derived by the above requirements, and in the present invention, when a fermented product was prepared using a Lactobacillus plantarum JBMI F5 (KACC91638P) strain in a blackberry extract, blackberry extract before fermentation The present invention was completed by confirming that the content of total polyphenols and total flavonoids was increased and the antioxidant activity was excellent, and the improvement effect on skin wrinkles and skin inflammation induced by UV rays was significantly increased.

In order to solve the above problems, the present invention also provides a health functional food composition for preventing or improving skin damage caused by ultraviolet rays containing blackberry fermented product as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammation caused by ultraviolet rays containing blackberry fermented product as an active ingredient.

Since the blackberry fermented product of the present invention is significantly superior to the previously known blackberry extract in inhibiting skin wrinkles and inflammation induced by ultraviolet irradiation, inner beauty foods, pharmaceuticals, etc. It can be used more usefully for development.

1 is a result of confirming the cell viability after pre-treating various samples to Hs68 cells and irradiating UVB. Blank is the group in which nothing was treated; Con is the UVB-only treatment group; NAC is a group treated with N-acetylcysteine; LP1 and LP2 are groups treated with Lactobacillus plantarum JBMI F5 strain culture medium (LP1: 1×10 ⁷ CFU/ml and LP2: 5×10 ⁷ CFU/ml); BB1 and BB2 are groups treated with blackberry extract at 0.008 and 0.016 mg/ml, respectively; BBL1 and BBL2 are a group treated with a mixture of unfermented blackberry extract and Lactobacillus plantarum JBMI F5 strain culture (BBL1: 1×10 ⁷ CFU/ml and BBL2: 5×10 ⁷ CFU/ml); And FBB1 and FBB2 are produced by inoculating the Lactobacillus plantarum JBMI F5 strain in the blackberry extract, each of the blackberry fermented products containing 1×10 ⁷ CFU/ml and 5×10 ⁷ CFU/ml of the JBMI F5 strain. The treated group is shown.
Figure 2 is a result of analyzing the production amount of procollagen type I and MMP-1 by ELISA when various samples were pretreated to Hs68 cells and irradiated with UVB. Blank is a group treated with distilled water and not irradiated with UVB, Con is a group treated with distilled water and irradiated with UVB, NAC is a group treated with N-acetylcysteine and irradiated with UVB, LP is Lactobacillus Plantarum JBMI F5 strain culture is treated and UBV is irradiated, BB is blackberry extract treated and UVB is irradiated, BBL is blackberry extract and Lactobacillus plantarum JBMI F5 strain culture mixture is treated and UBV is treated. , and FBB refers to a group in which blackberry extracts were inoculated with Lactobacillus plantarum JBMI F5 strain, processed blackberry fermented products, and UBV was irradiated.
3 is a Western blot result (A) confirming the expression levels of procollagen and MMPs when Hs68 cells were pretreated with blackberry ferment (FBB) and irradiated with UVB, and a graph (B) quantified and shown.
Figure 4 shows that when Hs68 cells were pretreated with fermented blackberries (FBB) and irradiated with UVB, phosphorylation levels of mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cell (NFκB) were reduced. Western blot results (A) and graphs (B) showing quantification thereof.
5 is a photograph (A) and H&E (hematoxylin- eosin) is a photograph showing histological changes through staining (B) and a graph showing the thickness of the epidermis measured by (C). CON is a group fed distilled water and not irradiated with UVB, UVB is a group fed distilled water and irradiated with UVB, RA is a group fed with retinoic acid and irradiated with UVB, LP is Lactobacillus plantarum A group fed JBMI F5 strain culture and irradiated with UBV, BB was a group fed blackberry extract and irradiated with UVB, and BBL was a group fed blackberry extract and Lactobacillus plantarum JBMI F5 strain culture mixture and irradiated with UBV. , and FBB refers to the group that ate blackberry fermented product and irradiated with UBV.
6 shows the amount of MMP-1 (A) and procollagen (B) produced in the mouse serum and tissue when the blackberry fermented product (FBB) of the present invention was fed to an animal model mouse and irradiated with UVB for 4 weeks. is the result
7 is a photograph showing the immunohistochemical changes of MMP-1 (A) and quantification of procollagen and MMPs proteins when an animal model mouse was fed with the blackberry fermented product (FBB) of the present invention and irradiated with UVB for 4 weeks. This is the graph (B) shown.
8 is an animal model mouse fed with the blackberry fermented product (FBB) of the present invention and irradiated with UVB for 4 weeks, ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinases) and p38 protein Western blot results confirming the decrease in phosphorylation level of
9 is an animal model mouse fed with the blackberry fermented product (FBB) of the present invention and irradiated with UVB for 4 weeks, iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) expression decrease and These are Western blot results confirming the decrease in phosphorylation level of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha).

In order to achieve the object of the present invention, the present invention provides a health functional food composition for preventing or improving skin damage caused by ultraviolet rays containing blackberry fermented product as an active ingredient.

In the health functional food composition of the present invention, the skin damage caused by the ultraviolet rays may be skin wrinkles or skin inflammation, but is not limited thereto.

In addition, the UV light may be UV-B, but is not limited thereto.

In addition, the composition is preferably an inner beauty food, but is not limited thereto.

Since the health functional food composition according to the present invention inhibits apoptosis that may be caused by ultraviolet rays, increases procollagen production, and inhibits matrix metalloproteinase (MMP) activity, it is possible to protect skin damage caused by ultraviolet rays.

In addition, the blackberry fermented product may be prepared by inoculating a blackberry extract with a Lactobacillus plantarum JBMI F5 (KACC91638P) strain, but is not limited thereto. The Lactobacillus plantarum JBMI F5 ( Lactobacillus plantarum JBMI F5) (KACC91638P) strain is a strain deposited with the Institute of Agricultural Biotechnology on March 10, 2011.

In addition, the fermentation conditions of the blackberry fermented product may be performed at 32 to 42° C. for 4 to 15 hours, and preferably, may be performed at 37° C. for 7 hours, but is not limited thereto.

In addition, the blackberry extract used to prepare the blackberry fermented product may be extracted using various extraction methods such as solvent extraction, physical extraction, and enzymatic extraction, but may preferably be extracted using a physical extraction method. not limited The extraction method does not destroy the active ingredient, and it is possible to obtain a blackberry fermented product with high activity from the blackberry.

In addition, the blackberry extract shall include any one of an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude product or a purified product.

In the health functional food composition for preventing or improving skin damage caused by ultraviolet rays of the present invention, the type of the food is not particularly limited. Examples of foods to which the above substances can be added include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, There are beverages, alcoholic beverages, vitamin complexes, dairy products, and dairy products, and includes all health functional foods in the ordinary sense. The health functional food may be manufactured or processed in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto, and may be manufactured or processed in any form according to the law. .

The blackberry fermented product of the present invention may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the intended use thereof. In general, the amount of the blackberry fermented product in the health food may be added in an amount of 0.1 to 90 parts by weight based on the total weight of the food. However, in the case of long-term intake for health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The health functional beverage composition of the present invention is an essential ingredient in the indicated ratio, and there is no particular limitation on other ingredients contained in addition to the blackberry fermented product. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above, the blackberry fermented product of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like may be contained. In addition, the blackberry fermented product of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammation caused by ultraviolet rays containing blackberry fermented product as an active ingredient.

In the pharmaceutical composition of the present invention, the inflammation may be skin inflammation, but is not limited thereto.

In addition, the fermented blackberry may increase procollagen production and inhibit the activity of matrix metalloproteinase (MMP), but is not limited thereto.

The pharmaceutical composition of the present invention preferably further comprises a pharmaceutically acceptable carrier, excipient and diluent in addition to the blackberry ferment, but is not limited thereto.

The pharmaceutical composition comprising the fermented blackberry of the present invention as an active ingredient may be in various oral or parenteral formulations.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycero gelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and it is preferable to select a skin external application method for parenteral administration, but is not limited thereto.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art.

The dosage of the pharmaceutical composition of the present invention can be used in various ranges depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Materials and Methods

Blackberry Fermentation Manufacturing

After the blackberries were weighed, water was added to prepare the blackberry content to be 66% (w/v), and the blackberry extract was obtained by grinding with a grinder, and then filtered using a cotton cloth. The filtered blackberry extract was sterilized at 121° C. for 15 minutes, adjusted to pH 6, and then cultured for one day in Lactobacillus plantarum JBMI F5 (KACC91638P) strain culture solution 5% (6×10 ⁹ CFU) /ml) was inoculated and fermented at 37°C at 230 rpm for 7 hours to obtain a blackberry fermented product (hereinafter referred to as 'sample'). Dextrin was added to the obtained fermented blackberry and then freeze-dried.

Total polyphenol content

2L of methanol was added to a 100 mg sample, homogenized at 30° C. for 60 minutes, centrifuged for 10 minutes, filtered, and the supernatant was used. In 100 μl of the supernatant, 500 μl of tertiary distilled water and 50 μl of Paulin-Siocaltu reagent were mixed, reacted at room temperature for 5 minutes, and then 600 μl of 2% (w/v) sodium carbonate was added and mixed. After the mixture was reacted in a dark place at 37°C, it was measured at 765 nm using a spectrophotometer, and a calibration curve was prepared using gallic acid (Sigma Chemical Co., St.Louis, MO, USA) as a standard material, and the total The polyphenol content was expressed as mg GAE (qgarllic acid equivalent) for 1 g of a dry sample.

Total flavonoid content

2L of methanol was added to a 100 mg sample, homogenized at 30° C. for 60 minutes, centrifuged for 10 minutes, filtered, and the supernatant was used. 1ml of the supernatant and 10% (w/v) aluminum chloride (AlCl ₃ ) were mixed in the same amount and reacted at room temperature for 5 minutes, measured at 415 nm using a spectrophotometer, and quercetin (qercetin, A calibration curve was prepared using Sigma Chemical Co.). Flavonoid content was expressed as mg QE (quercetin equivalent) for 1 g of dry sample.

L-Ascorbic Acid Content

L-ascorbic acid (L-ascorbic acid) content was measured using a Megazyme kit. Specifically, 3% (w/v) metaphosphoric acid and 10 mM EDTA (ethylenediaminetetraacetic acid) were added to a 100 mg sample, homogenized at room temperature for 60 minutes, centrifuged for 10 minutes, and then filtered supernatant was used. Mix 152 μl of tertiary distilled water and buffer 1 (please indicate the amount to be added) to 10 μl of sample, and after 3 minutes, MTT[3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] buffer 2 was added and reacted for 3 minutes, the primary absorbance (A1) was measured at 578 nm using a spectrophotometer, and a PMS (5-methylphenazinium methyl sulfate) buffer was added and reacted for 8 minutes. Then, the secondary absorbance (A2) at 578 nm was measured.

antioxidant activity

DPPH (2,2-Diphenyl-1-picrylhydrazyl) analysis is 20 μl of sample and 80 μl of DPPH (2,2-Diphenyl-1-picrylhydrazyl, Sigma-Aldrich, St Louis, MO, USA) solution were mixed in a 96-well plate and reacted in the dark for 30 minutes at room temperature, It was measured at 517 nm using a spectrophotometer (Multiskan ELISA, Thermo scientific Co. Ltd). Ascorbic acid (Sigma-Aldrich) was used as a positive control.

ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) analysis was 80mM ABTS solution and 30mM potassium persulfate (Potassium persulfate, K ₂ S ₂ O ₈ ) solution were mixed and reacted in a dark place at 30 ° C for about 1 hour, and then the diluted ABTS solution was used so that the absorbance at 734 nm was 1 ± 0.1. . 90 μl of the reacted diluted ABTS solution and 10 μl of each concentration sample were mixed and reacted for 30 minutes in a dark place at 37°C to measure absorbance at 734 nm. As a positive control, Trolox ^® (Sigma-Aldirch), a water-soluble vitamin E derivative, was used. did

Skin fibroblast culture

Human skin fibroblast line Hs68 was added to DMEM (Dulbecco's modified Eagle medium, Gibco, Grand Isaland, NY) with 10% (v/v) fetal bovine serum, Gibco, Grand Isaland, NY) and 1% penicillin. , 37° C. and 5% CO ₂ , and incubated in an incubator. Subcultures were maintained by culturing at intervals of 4 to 5 days.

Cell protective effect evaluation

To analyze the cytoprotective effect of fermented blackberries on Hs68 cells, the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide) assay was used. Cells at 1×10 ⁴ cells/ml were dispensed into a 96-well cell culture vessel and stabilized for 24 hours, and then cultured in serum-deficient DMEM medium for 24 hours to equalize the cell cycle. After 24 hours of treatment with each concentration of blackberry fermented product (Lactobacillus plantarum JBMI F5 strain contains 1×10 ⁷ CFU/ml or 5×10 ⁷ CFU/ml), washed twice with PBS, and then a small amount of PBS and UVB 100 mJ/cm 2 was irradiated using a UV crosslinker (Analytik Jena AG, Jena, Germany). After UV irradiation, serum-deficient DMEM medium was added to the cells and incubated for 24 hours. Control (Blank) cells were exposed to the same medium conditions without UVB irradiation only. 5 mg/ml MTT reagent was put into each well, incubated for 4 hours, the supernatant was removed, 100 ml of DMSO (dimethyl sulfoxide) was added to dissolve the formazan crystal, and then absorbance was measured at 590 nm using a microplate reader. did

laboratory animal

6-week-old male SKH-1 hairless mice (20-24 g) were purchased from Samtaco (Osan, Korea). Mice were randomly divided into 3 mice per cage and bred at a temperature of 22±2° C. and a humidity of 55±5% with a 12-hour day/night cycle. Mice were acclimatized to the surrounding environment for a week before the start of the experiment, and allowed to freely consume food and water. All procedures for animal experiments were reviewed and approved by the Experimental Animal Management Committee of the Jeonju Agricultural and Biomaterials Research Institute, and the guidelines of the Animal Experimental Ethics Committee (IACUC) were strictly followed. After one week of adaptation period, mice were divided into 7 groups, 6 mice in each group. Blank is a group fed with distilled water and not irradiated with UVB, Con is a group fed with distilled water and irradiated with UVB, NAC is a group fed with retinoic acid and irradiated with UVB, LP is Lactobacillus plantarum A group fed JBMI F5 strain culture and irradiated with UBV, BB was a group fed blackberry extract and irradiated with UVB, and BBL was a group fed blackberry extract and Lactobacillus plantarum JBMI F5 strain culture mixture and irradiated with UBV. , and FBB refers to the group that ate blackberry fermented product and investigated UBV. Each group was orally administered with the samples 30 minutes before UVB irradiation as shown in Table 1 below.

group classification group sample Treatment concentration (/mouse) UVB irradiation CON Vehicle (Distilled tap water) 200 μl X UVB Vehicle 200 μl O RA retinoic acid 0.01% (w/v) O LP Lactobacillus plantarum JBMI F5 strain culture medium 1.0E+10 CFU O bb blackberry extract 3.16 mg O BBL Blackberry extract and Lactobacillus plantarum JBMI F5 strain culture mixture 3.16 mg + 1.0E+10 CFU O FBB Blackberry Fermentation 1.0E+10 CFU O

UVB irradiation

For the production of an animal model of skin damage by UV irradiation, a UV lamp device (Waldmann Medizintechnik, Schwenningen, Germany) was used. Mice were exposed to UVB once daily at 30 mJ/cm ² for the first week, 60 mJ/cm ² for the second week, 90 mJ/cm ² for the third week, and 120 mJ/cm ² for the last week. The control group was not irradiated with ultraviolet rays, and 24 hours after the last irradiation, using SILFLO (Silflo, J&S Davis Ltd, UK), a duplicate image of the dorsal skin folds of the mouse was taken, and the skin tissue was collected from the central dorsal side of the mouse. .

Analysis of procollagen type I and MMP-1 production amount

Procollagen is a component of collagen, synthesized in fibroblasts, and is related to skin adhesion and elasticity. Matrix metalloproteinases-1 (MMP-1) is associated with skin aging and is known to be increased by UV rays. In the present invention, in order to investigate the efficacy of blackberry fermented product for reduced procollagen type I and increased MMP-1 by ultraviolet light, human MMP-1 and human MMP-1 and Changes in the production amount of procollagen type I and MMP-1 were confirmed using the Human Pro-Collagen Type I ELISA kit (Abcam, Cambridge, UK), and MMP-1 (CUSABIO, Wuhan, China) and procollagen type I (Abcam, Cambridge, UK) ELISA kits were used to confirm the change in production amount of procollagen type I and MMP-1. The blood was centrifuged at 3,000 rpm for 15 minutes and then centrifuged at 12,000 rpm for 10 minutes to separate the serum, and the skin tissue was homogenized using a homogenizer, and then centrifuged at 4°C at 12,000 rpm for 20 minutes to separate the supernatant. The experiment was performed according to the manufacturer's instructions.

Protein Expression Analysis

Cold RIPA buffer (10mM Tris-HCl, pH 7.5, 0.1%(v/v) NP-40, 0.5% sodium deoxycholate, 0.1%(w/v) SDS, 1mM sodium orthovanadate, 120mM sodium in Hs68 cell line and mouse skin tissue chloride, 1 mM phenylmethylsulfonyl fluoride, 10 g/mL leupeptin, and 1 g/mL aprotinin) were added and dissolved, and the supernatant was separated by centrifugation at 10,000 x g for 10 minutes. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Lab., Hercules, CA, USA) after electrophoresis using SDS-polyacrylamide gel. After blocking non-specific binding sites in 5% skim milk (0.1% Tween 20 containing PBS, PBST) solution for 1 hour, primary antibodies [anti-iNOS, COX-2, MAPK, NFκB, IκBα, MMPs, COLs (Cell Signaling) , Beverly, MA)] and incubated for 2 hours at room temperature with a secondary antibody [anti-rabbit IgG or anti-mouse IgG linked with horseradish peroxidase, (SantaCruz Biotechnology, Inc. USA)], followed by ECL solution. Thus, the protein expression level was measured with amersham imager 600 (GE Healthcare, Buckinghamshire, UK).

Immunohistochemical test

The dorsal skin tissues of UV-irradiated mice were fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin. After slicing the paraffin block using a cryotome, the tissue section was placed on the slide and stained with H&E (Hematoxylin and eosin), MT (Masson's trichrome) and mouse MMP-1 antibody (Abcam, Cambridge, UK). H&E staining was used to evaluate general histopathological changes and epidermal thickness, and MT staining was used to measure the degree of change in collagen fibers. The stained tissue slides were observed and photographed using an optical microscope (Olympus, Tokyo, Japan). In order to evaluate the epidermal hyperplasia in UV-irradiated mouse skin tissue, the vertical thickness of the epidermis was measured at 200× magnification (H&E staining) at 5 randomly selected positions per slide. Data were evaluated through Image J 1.52 (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), an image analysis program.

Example 1. Increased content of physiologically active substances and antioxidant activity of blackberry fermented product

The content of physiologically active substances and antioxidant activity were measured for the blackberry extract and the blackberry fermented product prepared using the Lactobacillus plantarum JBMI F5 strain. As a result, as shown in Table 2 below, when the blackberry extract was inoculated with the Lactobacillus plantarum JBMI F5 strain to prepare a fermented product, the culture of the blackberry extract and the blackberry extract and the Lactobacillus plantarum JBMI F5 strain was The total polyphenol content (TPC, total polyphenol content), total flavonoid content (TFC, total flavonoid content) and vitamin content (VC, Vitamin C) were significantly increased compared to the mixture (unfermented), and DPPH (2,2 -Diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) radical scavenging activity were also significantly increased. Through the above results, it was found that the content of bioactive substances and antioxidant activity were remarkably increased as the blackberry extract was inoculated with Lactobacillus plantarum JBMI F5 strain and fermented.

Measurement results of TPC, TFC, VC and antioxidant activity of blackberry fermented product TPC (mg/g) TFC (mg/g) VC (mg/100g) DPPH radical
Scavenging activity (%) ABTS radical
Scavenging activity (%) bb 18.96±0.25 0.24±0.09 51.05±3.36 18.82±0.60 18.94±0.19 BBL 16.55±0.09 0.32±0.04 254.72±18.19 ^*** 20.57±1.20 39.31±0.174 ^*** FBB 28.86±0.25 ^*** 0.38±0.02 335.56±36.63 ^*** 33.55±2.50 ^*** 64.20±2.53 ^*** AA - - - 91.46±0.18 - Trolox - - - - 94.15±0.25

BB is blackberry extract; BBL is a mixture of blackberry extract and Lactobacillus plantarum JBMI F5 strain culture (not fermented); FBB is a blackberry fermented product prepared by inoculating a Lactobacillus plantarum JBMI F5 strain into a blackberry extract; AA is ascorbic acid, ***; Compared to blackberry extract, it means that the effect of increasing TPC, TFC, VC, DPPH radical scavenging activity or ABTS radical scavenging activity is statistically significant, meaning that the p value is less than 0.001.

Example 2. Cell protective effect of fermented blackberry

In order to confirm the protective effect of the blackberry fermented product prepared by inoculating the blackberry extract with the Lactobacillus plantarum JBMI F5 strain, the blackberry fermented product was pre-treated to Hs68 cells, irradiated with UVB, and then MTT analysis was performed. As a result, as shown in Figure 1, the cell viability decreased in the UVB-only treatment group (Con), while the cell viability was maintained in all groups treated with the blackberry fermented product, confirming that there was a cytoprotective effect from UVB rays. Therefore, in the present invention, the following examples were performed on blackberry fermented product (JBMI F5 strain included 1×10 ⁷ CFU/ml).

Example 3. Analysis of changes in production amount of procollagen and MMPs according to blackberry fermentation

Inhibition of procollagen production and increased expression of matrix metallopeptidases (MMPs) in skin damage caused by UV irradiation are known as target markers. Therefore, the efficacy of the blackberry fermented product for suppressing the production of procollagen induced by UVB irradiation and increasing the expression of MMPs in Hs68 cells was confirmed. As a result, as shown in FIG. 2, the production of procollagen type I was inhibited by UVB irradiation and the production of MMP-1 was increased, whereas when the blackberry fermented product was pre-treated and irradiated with UVB, the production of procollagen type I was It was found that the production was increased and the production of MMP-1 was decreased, and there was a significant difference in the production of procollagen type I and the degree of reduction of MMP-1 when compared with the blackberry extract. In addition, in the protein expression analysis results, it was found that the expression of collagen I and III, which was decreased due to UVB irradiation, was increased by pre-treatment of the blackberry fermented product, and the expression of MMP-1 and MMP-2, which was increased due to UVB irradiation, was decreased. was found (FIG. 3).

Example 4. Analysis of MAPK and NFκB Activity by Blackberry Fermentation Treatment

Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in regulating immune response, matrix metalloproteinases (MMPs) expression, and inflammation. In this example, it was observed whether the MAPK and NFκB activities of blackberry fermentation (FBB) were inhibited using Western blotting. As a result, phosphorylation activity of NFκB, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK), and p38 proteins was increased when UVB was alone irradiated as shown in FIG. Phosphorylation of ERK, JNK, p38 and NFκB was effectively inhibited when the blackberry ferment was pretreated. On the other hand, there was no change in the phosphorylation level of MAPKs and NFκB when the blackberry extract was pretreated. According to these results, it is judged that the improvement effect of the blackberry fermented product on skin damage caused by ultraviolet rays is achieved through inhibition of the signaling pathways of MAPKs and NFκB.

Example 5. Efficacy of skin wrinkle improvement by ultraviolet light according to blackberry fermented product treatment in model mice

The effect of fermented blackberry on the formation of wrinkles by UVB irradiation in the dorsal skin of mice was confirmed. As a result, as shown in FIG. 5A , it was confirmed that the wrinkle formation of the mouse skin was significantly increased by UVB exposure for 4 weeks, and even if the blackberry extract was pre-treated, there was no effect on the wrinkle formation change. On the other hand, when the blackberry fermented product of the present invention was pre-treated and irradiated with UVB, it was confirmed that the wrinkle formation was significantly reduced. UVB irradiation is known to induce skin roughness by excessive epidermal proliferation and increase in skin thickness. Therefore, in order to measure the thickness of the skin after UVB irradiation, a histological examination was performed through hematoxylin-eosin (H&E) staining. As a result, compared with the control group (CON), the thickness of the epidermis was significantly increased due to proliferation and hypertrophy of keratinocytes in the UVB-only treatment group, but the thickness of the epidermis was significantly increased when the blackberry fermented product of the present invention was pretreated. decreased and it was confirmed that it was significantly reduced compared to the blackberry extract ( FIGS. 5B and 5C ).

Example 6. Changes in procollagen and MMPs synthesis ability according to blackberry fermentation in model mice

Matrix metalloproteinases-1 (MMP-1) is known to decrease collagen synthesis by inducing the breakdown of collagen fibers. Procollagen is produced by fibroblasts of the skin and its expression is reduced due to UVB irradiation, which causes photoaging. In order to confirm the synthesis ability of MMP-1 and procollagen after UVB exposure to the serum and tissue homogenate of a model mouse, it was measured using an ELISA kit. As a result, UVB irradiation in serum and tissue homogenates significantly increased MMP-1 production, and when the blackberry fermented product (FBB) of the present invention was pre-treated and irradiated with UVB, MMP-1 production was significantly reduced. In addition, there was a significant difference between the blackberry extract (BB) treatment group and the blackberry fermented product (FBB) treatment group. Similarly, in the measurement result of procollagen production, it was found that the procollagem content, which was significantly reduced due to UVB irradiation, increased when the blackberry fermented product (FBB) was pretreated, and the blackberry extract (BB) treated group and the blackberry fermented product ( There was a significant difference between the FBB) treatment groups (FIG. 6).

In addition, as a result of observing immunohistochemical changes after UVB irradiation in the dorsal skin tissue of hairless mice, MMP-1 expression was increased by UVB irradiation as shown in FIG. 7 , whereas blackberry fermented (FBB) pretreatment In the results of quantification of procollagen and MMPs proteins, the contents of MMP-1 and MMP-2 increased and the contents of procollagen 1 and 3 decreased due to UVB irradiation, but blackberry fermented ( FBB), it was confirmed that the contents of MMP-1 and MMP-2 were decreased and the contents of procollagen 1 and 3 increased. It was confirmed that there was a significant difference in the change.

Example 7. Analysis of MAPK and NFκB pathway protein expression following blackberry fermentation in model mice

The pathway of mitogen-activated protein kinase (MAPK) signaling plays an important role in regulating immune responses, matrix metalloproteinases (MMPs) expression, and inflammation. In this example, it was observed whether fermented blackberry (FBB) inhibited MAPK and NFκB activity in mouse skin tissues using Western blotting. As a result, phosphorylation levels of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinases), and p38 proteins were increased after UVB irradiation as shown in FIG. 8, but blackberry fermented (FBB) When pre-treatment and UVB irradiation were performed, the phosphorylation levels of the increased ERK, JNK and p38 proteins were significantly reduced, and it was confirmed that the efficacy was superior to that of the blackberry extract (BB). In addition, the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was significantly increased due to UVB irradiation, but the expression of iNOS and COX-2 increased by pretreatment of blackberry fermented product (FBB) The amount was reduced and it was confirmed that there was a significant difference between the blackberry extract (BB) and blackberry fermented product (FBB) groups (FIG. 9A).

On the other hand, activation of MAPKs is known to cause activation of several transcription factors, such as NFκB (nuclear factor kappa light chain enhancer of activated B cell). Fermented blackberry (FBB) effectively reduced phosphorylation levels of NFκB and IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) increased by UVB irradiation (FIG. 9B).

Agricultural Biotechnology Research Institute KACC91638P 20110310

Claims (7)

Health function for preventing or improving skin wrinkles and skin inflammation caused by UV rays containing blackberry fermented product prepared by inoculating blackberry extract with Lactobacillus plantarum JBMI F5 (KACC91638P) strain as an active ingredient food composition. delete delete The health functional food composition for preventing or improving skin wrinkles and skin inflammation caused by ultraviolet rays according to claim 1, wherein the composition is an inner beauty food. delete delete delete

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