TW202017585A - Use of imperata cylindrica fermented extract for enhancing the gene expression of keratin, filaggrin and hyaluronan synthase, promoting the proliferation of collagen and elastin, and enhancing antioxidant capacity of skin cells - Google Patents

Abstract

The present invention provides a use of Imperata cylindrica fermented extract for enhancing the gene expression of Keratin, Filaggrin and Hyaluronan synthase, promoting the proliferation of collagen and elastin, and enhancing antioxidant capacity of skin cells. The Imperata cylindrica fermented extract can effectively increase the moisturizing ability of the skin, maintain the keratinocyte arrangement and the integrity of the stratum corneum structure to enhance the skin barrier function, and make the skin firm and elastic, and prevent skin aging. The Imperata cylindrica fermented extracts is prepared by fermenting the Imperata cylindrica extracts to yeast, and lactic acid bacteria two-staged fermentation.

Description

The ferment of Pakchoi root is used to enhance the expression of skin cell keratin gene, polykeratin microfilament gene and hyaluronic acid synthase gene, promote the proliferation of collagen and elastin, and enhance the antioxidant capacity

The present invention relates to the use of a fermented pakchoi root, especially a fermented pakchoi root for enhancing the expression of skin cell keratin gene, polykeratin filament gene and hyaluronic acid synthase gene, promoting the proliferation of collagen and elastin, And the use of enhancing antioxidant capacity.

The epidermal layer is the outermost layer of the skin, which is the stratum corneum, the granular layer, the spinous layer and the basal layer in sequence from the outer to the inner. The epidermal layer is mainly formed by the continuous differentiation of the undifferentiated cylindrical keratinocytes in the basal layer. This process Called keratinization. Keratinocytes have a high water content. As the cells metabolize and differentiate, the shape of the keratinocytes will gradually become flat, and the nuclei and organelles will begin to degenerate and shrink, and dead cells without nucleus and organelles will form in the stratum corneum. The main function of the epidermal layer is to keep the skin water-retaining and form a skin barrier to resist various external damages. The outermost layer of the epidermal layer is composed of a weakly acidic sebum film and the stratum corneum like a brick wall structure. This barrier can lock the skin. Moisture and oil, resist the invasion of bacteria on the skin surface, and resist the damage of foreign bodies and ultraviolet light, etc., have a very important protective effect on the human body.

In the stratum corneum of the epidermal layer, although the keratinocytes are dead cells, the main component is keratin. Keratin can absorb water to keep the skin moist. Keratinocytes also secrete substances such as hyaluronic acid as the intercellular substance. To maintain the structural integrity of the epidermal skin barrier to prevent skin water Disperse and form complete protection. When the skin is exposed to excessively cold or overheated environments and irradiated with ultraviolet light and other stimuli, the keratinocytes cannot maintain the normal metabolic cycle, and the skin's ability to retain water is also reduced, resulting in damage to the skin's epidermal barrier, making the skin rough, Dry and desquamated, fragile and susceptible to irritation, sensitive redness, so the health of the stratum corneum and the ability to retain water are very important to resist external damage.

Based on the above, in response to the modern trend of natural health, in order to improve the problem that the skin becomes fragile and sensitive due to the damage of keratinocytes and the decline in water retention capacity, develop a kind of effective keratinocytes to secrete more moisturizing factors, and A comprehensive skin health care composition that maintains the arrangement of keratinocytes and maintains the structural integrity of the stratum corneum to enhance the skin barrier function is indeed necessary.

Therefore, one object of the present invention is to provide a fermented Pakchoi root for preparing keratin (Keratin, KRT ) gene, polykeratin microfilament (Filaggrin, FLG ) gene, and/or hyaluronan synthase (Hyaluronan synthase, HAS ) Use of a gene expression amount composition.

Another object of the present invention is to provide a use of fermented Pakchoi root to prepare a composition for promoting collagen and/or elastin in skin cell proliferation.

Another object of the present invention is to provide a use of the fermented Pakchoi root for preparing a composition for enhancing the antioxidant activity of skin cells.

Another object of the present invention is to provide a method for producing fermented pakchoi root, which comprises: obtaining a water extract of pakchoi root through a yeast and a lactobacillus in order to ferment it in sequence; It is obtained by two-stage fermentation of a water extract of Rhizoma Imperatae and a microbial group, wherein the microbial group is composed of a yeast ( Saccharomyces cerevisiae ) and a Lactobacillus plantarum .

In an embodiment of the present invention, wherein the water extract of Phellodendron arundinacea root is obtained by mixing Phellodendron chinense with water at a solid-liquid ratio of 1:18-30 and sterilized extraction at 50-100°C for 0.5-3 hours; and The yeast strain is a strain of BCRC20271, and the lactobacillus strain is a strain of BCRC910805; the yeast The added amount is 0.01-0.5% (v/v), the added amount of the Lactobacillus is 0.01-0.25% (v/v); and the cultivation time of the yeast and the Lactobacillus are respectively 1-3 days; Moreover, the fermentation system increases the total polysaccharide content of Pakchoi root, wherein the total polysaccharide of Pakchoi root ferment is at least 20%.

In yet another embodiment of the present invention, the KRT gene is a keratin 14 (Keratin14, KRT14 ) gene; and the HAS gene includes a Hyaluronan synthase 2 (Hyaluronan synthase 2, HAS2 ) gene and a Hyaluronan synthase 3 (Hyaluronan synthase 3 , HAS3 ) gene.

After passing through the microbial fermentation step of the present invention, the fermented chrysanthemum root of the present invention can effectively increase the content of the total polysaccharide in the effective component to improve the skin moisturizing effect and enhance the integrity of the skin cuticle structure; it can also be effectively improved The expression levels of KRT14 gene, FLG gene, HAS2 gene and HAS3 gene can effectively make the skin keratin secret more moisturizing factors, and increase the skin's moisturizing ability, maintain the arrangement of keratinocytes and maintain the integrity of the stratum corneum structure to enhance the skin barrier function; It can also effectively promote the proliferation of collagen in skin cells, and can restore skin smoothness and firmness, so as to increase skin saturation and make the skin plump and smooth; it can also effectively promote the proliferation of elastin in skin cells to maintain skin firmness and elasticity And reduce the generation of wrinkles on the skin to make the skin plump and smooth; and can effectively remove free radicals in skin cells, can enhance the antioxidant capacity of skin cells to prevent the occurrence of skin aging. Therefore, the fermented roots of the present invention can be used to prepare a composition for enhancing the expression of skin keratin gene, polykeratin filament gene and hyaluronic acid synthase gene, promoting the proliferation of collagen and elastin, and enhancing the antioxidant capacity Use, the composition The composition is a medicinal product, a food or a care product, and can be administered to a body by oral administration, skin application, or the like.

The embodiments of the present invention will be further described below. The examples listed below are used to clarify the present invention, and are not intended to limit the scope of the present invention. Anyone who is familiar with this art, without departing from the spirit and scope of the present invention, Some changes and retouching can be done, so the scope of protection of the present invention shall be deemed as defined by the scope of the attached patent application.

FIG. 1 is a bar graph showing the comparison of the total polysaccharide content in the water extract of Phellodendron chinense L. fermented and P. chinensis root according to an embodiment of the present invention.

FIG. 2 is a bar graph of the effect of the fermented Phellodendron amurense L. on raising the expression level of the KRT14 gene, FLG gene, HAS2 gene and HAS3 gene; ** p<0.01.

FIG. 3 is a bar graph of the effect of fermented Phellodendron amurense promoting collagen proliferation according to an embodiment of the present invention; ** p<0.01; ### p<0.001.

FIG. 4 is a bar graph of the effect of fermented Phellodendron amurense promoting elastin proliferation according to an embodiment of the present invention; ** P<0.01; ## p<0.01.

FIG. 5 is a bar graph of the effect of the fermented Phellodendron amurense L. cellulite to enhance the antioxidant capacity of cells according to an embodiment of the invention; *** p<0.001; ### p<0.001.

The numerical values used in this article are approximate values, and all experimental data are expressed within a range of 20%, preferably within a range of 10%, and most preferably within a range of 5%.

Use Excel software for statistical analysis. Data mean ± standard deviation (SD) represented by the difference between the two herein by Student t test (student's t -test) analysis.

Imperata cylindrica is a perennial herbaceous plant of the Gramineae genus Imperata . It is also known as thatch, chrysanthemum, and chrysanthemum. The plant is 20-80 cm tall. The rhizome is white and runs horizontally under the ground. Scales, sweet tip, alternate leaves, concentrated on the base, when old, the base often has broken and fibrous leaf sheaths, flat leaves, strips or strips lanceolate, bloom in summer, flowers are silvery white, densely branched . At present, it is known that Baimaogen has the effects of clearing away heat and nourishing the lung, regenerating the body and detoxifying, strengthening the liver and nourishing the spleen, lowering the fire and regenerating the body, cooling blood and diuresis.

As used in this article, the term "fermenta root ferment" means that the water extract of Pakchoi root is fermented by yeast and Lactobacillus in one or two stages, wherein the water extract of Pakchoi root is Pakchoi root and the solvent is 1: The ratio of 18-30 (w/w) is obtained by extraction with time and temperature.

According to the present invention, pharmaceutical products can be manufactured into a dosage form suitable for parenterally or topically administration using techniques well known to those skilled in the art, including, but not limited to: injections (injection) [for example, sterile aqueous solution or dispersion], sterile powder, external preparation, and the like.

According to the present invention, the pharmaceutical product may further include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, and decomposers ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The selection and quantity of these reagents fall within the professional qualities and routine techniques of those who are familiar with this technology.

According to the present invention, the pharmaceutically acceptable carrier contains a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), Aqueous solution containing alcohol and their combination.

According to the present invention, the medicinal product can be administered by parenteral routes selected from the group consisting of subcutaneous injection, intradermal Injection (intraepidermal injection), intradermal injection (intradermal injection) and intralesional injection (intralesional injection).

According to the present invention, pharmaceutical products can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, which include, but are not limited to: emulsion, gel Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, milk Serums, pastes, foams, drops, suspensions, salves and bandages.

According to the present invention, the external preparation is prepared by mixing the pharmaceutical product of the present invention with a base well known to those skilled in the art.

According to the present invention, the substrate may include one or more additives selected from the group consisting of water, alcohols, glycols, hydrocarbons [such as petroleum, jelly, and White petrolatum (white petrolatum,)], wax [such as paraffin and yellow wax], preserving agents (antiserving agents), antioxidants (antioxidants), surfactants (surfactants), absorption enhancers (absorption enhancers), stabilizers, gelling agents [such as carbopol ^® 974P (carbopol ^® 974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethylcellulose (carboxymethylcellulose)] , Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusions Occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants and propellants Agent (propellants) and so on. The selection and quantity of these additives fall within the scope of professional qualities and routine skills of those who are familiar with this technology.

According to the present invention, the maintenance product may further include an acceptable adjuvant widely used in the maintenance product manufacturing technology. For example, the acceptable adjuvant may contain one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, dyes Coloring agent, thickening agent, filler, fragrance and odor absorber. The selection and quantity of these reagents fall within the professional qualities and routine techniques of those who are familiar with this technology.

According to the present invention, skin care products can be manufactured into a form suitable for skincare or makeup using techniques well known to those skilled in the art. This includes, but is not limited to: aqueous solution (aqueous solution), water -Aqueous-alcohol solution or oily solution, oil-in-water type, water-in-oil type or compound emulsion, gel Glue, ointment, cream, mask, patch, pack, wipe, powder, aerosol, spray, emulsion, emulsion, paste, foam, dispersion, drops, mousse ( mousse, sunblock, tonic water, foundation, foundation remover products, makeup remover products, soap, and other body cleansing products.

According to the present invention, the skin care product can also be used in combination with one or more external use agents of known activity selected from the following: whitening agents [such as tretinoin, catechins (catechin), kojic acid, arbutin and vitamin C], humectants, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts[plant extracts][ Such as aloe extract], skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, anti-dermatitis agents (antidermatitis agents), antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, Antiseborrheic agents, wound-healing agents, cortex Corticosteroids and hormones. The selection and quantity of these external agents fall within the professional qualities and routine technologies of those who are familiar with this technology.

According to the present invention, a food product can be used as a food additive, which is added during the preparation of raw materials by conventional methods, or added during the production of food, and formulated with any edible material for Food products consumed by humans and non-human animals.

According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

The invention provides a use of a fern of Phellodendron chinense to prepare a composition for enhancing the expression of skin keratin gene, polykeratin microfilament gene and hyaluronic acid synthase gene, promoting the proliferation of collagen and elastin, and enhancing the antioxidant capacity The fermented radix chrysanthemum of the present invention is a fermented radix chrysanthemum fermented by two-stage fermentation of the water extract of chrysanthemum root with yeast and Lactobacillus. The solvent is water, alcohol, or a mixture of alcohol and water. The fermented Pakchoi root of the present invention can be used to enhance the expression of KRT14 gene, FLG gene, HAS2 gene and HAS3 gene, promote the proliferation of collagen and elastin of skin cells, and make Increased skin moisturizing ability, maintain the arrangement of keratinocytes, maintain the integrity of the stratum corneum structure, and enhance the antioxidant capacity of skin cells.

At the same time, the present invention is used to prepare a composition for enhancing the expression of skin cell keratin gene, polykeratin microfilament gene and hyaluronic acid synthase gene, promoting the proliferation of collagen and elastin, and enhancing the antioxidant capacity, and may also contain an effective The amount of fermented Pakchoi root and a pharmaceutically acceptable carrier, the composition is a medicinal product, a food or a maintenance product.

The detailed preparation method of the fermented Pakchoi root of the present invention will be described in detail below, together with the test of the fermented Pakchoi root fermented substance in the increase of total polysaccharide content, the test of the elevated KRT gene, FLG gene, HAS gene, and the promotion of skin cell hyperplasia collagen Tests, tests to promote the proliferation of elastin in skin cells, and tests to enhance the antioxidant capacity of cells to confirm that the fermented Pakchoi root promotes the expression of KRT14 gene, FLG gene, HAS2 gene and HAS3 gene, and promotes the collagen of skin cells Proliferation of protein and elastin, increase the skin's moisturizing ability, maintain the arrangement of keratinocytes, maintain the integrity of the stratum corneum structure, and enhance the antioxidant capacity of skin cells.

Example 1 The preparation method of the fermented Pakchoi root of the present invention

In an embodiment of the present invention, the roots of Rhizoma Imperatae are thoroughly washed, and the washed Rhizoma Imperatae is mixed with water at a solid-liquid weight ratio of 1:18-30 and sterilized and extracted at 50-100°C for 0.5-3 hours to Obtained the water extract of Phellodendron chinense, which was cooled to room temperature for subsequent two-stage fermentation. First, 0.01-0.5% (v/v) yeast ( Saccharomyces cerevisiae ) was planted and purchased for biological resource preservation and research Center, Taiwan, No. BCRC20271) After fermenting in the water extract of Phellodendron chinense at 20-37°C for 1-3 days, then directly add 0.01-0.25% (v/v) Lactobacillus plantarum TCI028, patent custody At the Center for Biological Resources Conservation and Research, Taiwan, numbered BCRC910805), fermented at 20-37°C for 1-3 days; of which, the Lactobacillus TCI028 series has been patented at Republic of China Patent Application No. 106145146, and the fermentation of these two strains is in order It is: yeast and lactobacilli, which cannot be adjusted back and forth. Finally, without removing these two bacteria, use the set sugar content range of 35~45°, pH2~4, alcohol <3% and other specifications, if the inspection meets With this specification, it is judged that the fermentation is completed and the fermentation broth is obtained, and its total polysaccharide content accounts for at least 20%. Next, the fermentation broth is sterilized at high temperature, the fermentation broth is concentrated under reduced pressure at 45-70°C, and filtered through a 200-400mesh mesh screen, and then adjusted by 0.5-1.5% preservative (Phenoxyethanol) and then sterilized to obtain this product. The invented fermented pakchoi root is made by microbial fermentation, so that the effective substance of pakchoi root is released in large quantities.

Example 2 The effect of the fermentation step of the present invention to increase the total polysaccharide content in the water extract of Phellodendron chinense

In this embodiment, in order to compare whether the content of total polysaccharide in the fermented Pakchoi root of the present invention is higher than that of the water extract of Pakchoi root, the Phenol-sulfuric acid assay is used to quantify the concentration of total polysaccharide in the sample. When encountering a strong acid, the hydroxyl group in the structural formula will combine with phenol and produce an orange-yellow liquid, so the colorimetric method (especially the absorbance of the sample at 490nm) can be used to detect the concentration of the total polysaccharide. First, accurately weigh 10 mg of D-glucose (D-Glucose, purchased from JTBaker, USA, number 1916-01) into a 10 mL volumetric flask, then add ddH ₂ O to a total volume of 10 mL, and complete D- Glucose standard product (D-Glucose stock, 1 mg/mL), then serially dilute the above standard product according to the formula in Table 1, and dilute with dd H ₂ O to 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/ mL, 150μg/mL, and 200μg/mL of D-Glucose. In addition, 1.25g of Phenol (purchased from Merck, Germany, No. 1.0206.0250) was taken into a volumetric flask, and then ddH ₂ O was added to a total volume of 25mL to complete a 5% Phenol solution.

Then draw the regression curve of the standard solution, take 100 μL of each standard solution in a glass test tube, add 500 μL of 5% Phenol solution, and slowly add 2.5 mL of sulfuric acid solution (95.5% H ₂ SO4, purchased from Showa, Japan , No. 1970-5250) In each test tube, mix with Vortex and let stand for 20 minutes, then take 200μL of each group of mixed solution in a 96-well culture dish and measure the absorbance at 490nm to draw the regression curve formula of the standard solution . Next, take 100 μL of Pakchoi root water extract and the Pakchoi root fermentation product of the present invention in glass test tubes, respectively add 500 μL of 5% Phenol solution, and slowly add 2.5 mL of sulfuric acid solution to each test tube, mix with Vortex After homogenization, let stand for 20 minutes, then take 200μL of each group of mixed solution in a 96-well culture dish, measure the absorbance at 490nm, and use the regression curve formula of the above standard solution to calculate the concentration by the internal difference method and then multiply the dilution factor In order to obtain the concentration of total polysaccharides in the raw water extract of Pakchoi root and the fermented Pakchoi root of the present invention.

The result of the increase in the total polysaccharide content in the fermented Pakchoi root of the present invention is shown in FIG. 2. After the two-stage fermentation of the present invention, the total polysaccharide content of Pakchoi root is 21% higher than that of the pure Pakchoi root water extract. This result shows that the pakchoi root fermented product of the present invention can be effective after the microbial fermentation step of the present invention. To increase the total polysaccharide content, this fermentation step can enhance the skin's moisturizing effect and enhance the integrity of the skin's stratum corneum structure.

Example 3 The efficacy of the fermented Pakchoi root of the present invention in enhancing the expression level of KRT14 gene, FLG gene, HAS2 gene and HAS3 gene

The present invention uses human primary epidermal keratinocytes (HPEK) to perform expression of TGM1 gene, KRT1 gene, KRT10 gene, KRT14 gene, AQP3 gene, FLG gene, HAS2 gene, and HAS3 gene expression of Pakchoi root fermented material of the present invention. Quantitative analysis. The human primary skin keratinocytes were purchased from CELLnTEC (Switzerland) No. HPEK-50, and the cells were cultured in serum-free keratinocyte-SFM (Gibco, No. #17005042, United States), and contained Incubate in a 37°C cell incubator with 5% CO ₂ .

1.5×10 ⁵ HPEK-50 cells were cultured in a six-well culture dish containing 2 mL of the above cell culture solution. After culturing at 37° C. for 24 hours, the culture solution was removed without disturbing the attached cells, and then the The cells are divided into the following three groups: (1) a control group containing only cell culture fluid, (2) a comparative group with 0.125% of Pakchoi root water extract, and (3) an experimental group with 0.125% of Pakchoi root fermented material of the present invention After 6 hours of operation, the keratinocytes were recovered in cell lysate (RB buffer, purchased from Geanaid, Taiwan, Cat No. RBD300), and then used RNA extraction reagent kit (purchased from Geneaid, Taiwan, Cat No. (RBD300) Collect RNA in two groups of cells separately, and then use SuperScript ^® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) to extract RNA with 2000 ng as a template and use primers to generate corresponding cDNA products for reverse transcription of mRNA Then, use ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific, USA), and KAPA SYBR FAST (purchased from Sigma, USA, No. 38220000000) to carry out the two sets of reverse transcription products using the combination primers in Table 2 Quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time reverse transcription polymerase chain reaction) test, the conditions are 95 ℃ for 1 second, 60 ℃ for 20 seconds, a total of 40 cycles. Used to quantify the mRNA expression of TGM1 gene, KRT1 gene, KRT10 gene, KRT14 gene, AQP3 gene, FLG gene, HAS2 gene, and HAS3 gene, where the quantitative value is taken from the threshold cycle number (Ct), and the target gene mRNA The relative quantity system is derived from Equation 2 ^-△Ct , where △Ct=Ct _{target gene-} Ct _ACTB (β-actin, beta-actin), and then use Excel software to perform unpaired single-tailed student t-test to determine the mutation The coefficient is statistically significantly different (*p value <0.05; **p value <0.01; ***p value <0.001).

The results of enhancing the expression level of KRT14 gene, FLG gene, HAS2 gene, and HAS3 gene by the fermented material of Phellodendron chinense in the present invention are shown in FIG. 2. Previous studies have pointed out that TGM will form strong bonds between the cell membrane of keratinocytes and structural proteins, and can increase the strength and stability of the epidermal layer; KRT will form keratin egg filaments, and FLG will help keratin filaments assemble into a strong The network provides strength and elasticity to the skin; AQP will increase the permeability of water in the keratinocytes to increase the water content of the keratinocytes; HAS will promote the ability of the keratinocytes to secrete hyaluronic acid, and complete and enhance the structure of the skin horny layer The skin barrier function can improve the skin's water retention capacity. Among them, the expression level of AQP gene and HAS gene, especially HAS gene, can effectively make keratinocytes secrete more moisturizing factors.

After the primary human keratinocytes were treated with the fermented Pakchoi root of the present invention, the KRT14 gene expression was about 1.2 times higher than the control group, the FLG gene expression was about 1.4 times higher than the control group, and the HAS2 gene expression was about 1.5 times higher than the control group. times, and HAS3 expression levels of genes than the control group a height of about 1.2 times, and KRT14 gene, the FLG gene, the HAS2 gene, and expression levels of HAS3 genes of both than comparative group is high; Further, TGM1 gene, KRT1 gene, KRT10 gene, The AQP3 gene is not much different from the control group. This result shows that the fermented Pakchoi root of the present invention can effectively increase the expression of the KRT14 gene, FLG gene, HAS2 gene and HAS3 gene after the microbial fermentation step. It can make the skin keratin secret more moisturizing factors, and increase the skin's moisturizing ability, maintain the arrangement of keratinocytes, maintain the integrity of the stratum corneum structure, and enhance the skin barrier function.

Example 4 The effect of the fermented chrysanthemum root of the present invention on promoting the proliferation of collagen in skin fibroblasts

In the embodiment of the present invention, human skin fibroblast (CCD-966sk) is used to analyze the fermented matter of Phellodendron chinense in promoting collagen hyperplasia. The human skin fibroblasts were purchased from the American Type Culture Collection (ATCC®), number CRL-1881, and the cells were cultured in 10% fetal bovine serum (Fetal Bovine Serum) and 90% Minimum Essential Medium (MEM ) Culture medium (purchased from Gibco, USA), which contains 1 mM sodium pyruvate and 1% penicillins/streptomycin.

First, 2×10 ⁴ human dermal fibroblasts were planted in a 24-well culture dish containing 500 μL of the above-mentioned culture solution, and after incubation at 37° C. for 24 hours, with 1x Phosphate buffered saline, 1xPBS ) Rinse the cells without disturbing the cells, and then divide the cells into the following two groups: (1) a comparison group added with 0.125% Pakchoi root water extract, and (2) an experiment with 0.125% Pakchoi root fermented substance of the present invention Groups were dissolved in 500 μL of MEM without fetal bovine serum, and the group without any extract added was used as a blank control group, and the group containing only culture medium without cells was used as the background value for subsequent reading of absorbance. After incubating for 48 hours at 37°C, without disturbing the cells, collect 1 mL of culture solution in each group of cells and place it in a 1.5 mL centrifuge tube. Use the soluble collagen analysis kit (Sircol ^TM Soluble Collagen Assay kit, Bicolor Life Science Assays, Notherm Ireland, United Kingdom), to detect the amount of collagen secreted by skin fibroblasts. First add 200μL of collagen isolation & concentration reagent (collagen isolation & concentration reagent) and invert upside down 6-8 times to mix evenly, then place the centrifuge tube at 4℃ for 16-18 hours, take out after the time is up, avoid shaking to the tube Centrifuge at 12000rpm for 10 minutes at 4°C. After centrifugation, carefully remove 1mL of supernatant, add 1mL of Sircol dye reagent (Sircol dye reagent) to stain collagen, and gently shake for 30 minutes to mix Homogenize, then centrifuge at 12000 rpm for 10 minutes at 4°C, remove the supernatant, and then add 750 μL of acid-salt washing reagent to wash away excess dye, and at 12,000 rpm at 4°C After centrifuging for 10 minutes, remove the supernatant, and carefully remove the liquid remaining on the tube wall and lid with a cotton ball. Then add 250 μL of strong alkali reagent (alkali reagent) and dissolve the collagen evenly with a test tube shaker. Finally, the absorbance value of the products of each group and the blank control group at 555 nm is measured with an enzyme immunoassay analyzer (ELISA reader, BioTek), and after deducting the background value, the percentage of the difference between the reading value of each group and the blank control group is calculated Then, student t-test is performed in Excel software to determine whether the coefficient of variation is statistically significant (compared with the blank control group: *p value <0.05; **p value <0.01; ***p value <0.001. Compared with the water extract of Phellodendron amurense: #p value <0.05;##p value <0.01;###p value <0.001).

The experimental results of the fermented chrysanthemum root of the present invention in promoting the proliferation of collagen in skin cells are shown in FIG. 3. The experimental group of human primary keratinocytes treated with the fermented Pakchoi root of the present invention had a collagen content significantly higher than that of the blank control group by 23%, and the comparison group treated with Pakchoi root water extract was only 2% higher than the blank control group Among them, the experimental group and the comparative group also have significant differences. This result shows that the fermented chrysanthemum root fermented product of the present invention can effectively promote the collagen proliferation of skin cells after the microbial fermentation step, and can restore the skin to be smooth and firm, so as to increase the saturated amount of skin to make the skin plump and smooth.

Example 5 The effect of the fermented Pakchoi root of the present invention on promoting the proliferation of elastin in skin fibroblasts

In the embodiment of the present invention, human skin fibroblast (CCD-966sk) is used to analyze the fermented matter of Pakchoi root of the present invention to promote elastin proliferation. The human skin fibroblasts were purchased from the American Type Culture Collection (ATCC®), number CRL-1881, and the cells were cultured in 10% fetal bovine serum (Fetal Bovine Serum) and 90% Minimum Essential Medium (MEM ) Culture medium (purchased from Gibco, USA), which contains 1 mM sodium pyruvate and 1% penicillins/streptomycin.

First, plant 1×10 ⁵ human dermal fibroblasts in a 6-well culture dish containing 2 mL of the above culture medium, and incubate at 37°C for 24 hours, using phosphate buffered saline (Phosphate buffered saline, PBS) in Rinse the cells without disturbing the cells, and then divide the cells into the following two groups: (1) a comparison group with 0.125% Pakchoi root water extract, and (2) an experimental group with 0.125% Pakchoi root fermentate of the present invention, Dissolve in 2mL of MEM without fetal bovine serum, and use the group without any extract as the blank control group, and the group containing only the culture medium without cells as the background value of the subsequent absorbance reading. After culturing the cells in the above groups for 48 hours at 37°C, remove the culture solution without disturbing the cells and wash the cells once with 1xPBS, then add 200 μL of trypsin (Trypsin) for 3 minutes to make the cells detach from the culture plate. Add 1mL of cell culture solution to stop the reaction, and collect the cells together with the culture solution into a 1.5mL test tube, and centrifuge at 300g for 5 minutes to remove the supernatant, wash the cells once with 1xPBS, then centrifuge at 300g for 5 minutes and then move The supernatant was removed, and about 300 μL of 1xPBS was added to resuspend the cells, followed by an elastin assay kit (Fastin ^™ Elastin Assay kit, purchased from Biocolor, UK) to detect the amount of elastin secreted by skin fibroblasts. First add 100 μL of 1.0M oxalic acid solution and place on a hot plate at 100°C for 1 hour, then add the same volume of Elastin Precipitating Reagent to each tube, close the lid tightly and fully use vortex Mix well and let stand for 15 minutes to completely precipitate the elastin. After centrifuging at 10,000g for 10 minutes, drain the liquid in the centrifuge tube and invert the centrifuge tube and tap on the paper towel to remove most of the tube. The remaining liquid, then add 1mL of TPPS (5,10,15,20-tetraphenyl-21H,23H-porphine tetra-sulfonate) dye reagent, and invert the centrifuge tube several times to mix evenly and place it on the mechanical shaker Set the reaction at 6 rpm for 90 minutes to remove the unbound dye. The bottom of the centrifuge tube and the reddish brown precipitate of the elastin-dye complex can be observed, and then add 250 μL to each tube Dye Dissociation Reagent, close the lid tightly and mix thoroughly with vortex to release the dye into the solution to ensure that all the bound dye has entered the solution, and then transfer the contents of each tube to the 96-well culture plate Measure the absorbance of the products of each group and the blank control group at 513nm with an enzyme immunoassay analyzer (ELISA reader, BioTek), and after subtracting the background value, calculate the difference between the reading value of each group and the blank control group %, and then conduct student t-test with Excel software to determine whether the coefficient of variation is statistically significant (compared with the blank control group: *p value<0.05; **p value<0.01; ***p value< 0.001. Compared with the water extract of Phellodendron chinense: #p value<0.05;##pvalue<0.01;###pvalue<0.001).

The experimental results of the fermented chrysanthemum root of the present invention in promoting elastin proliferation in skin cells are shown in FIG. 4. The experimental group of human primary keratinocytes treated with Pakchoi root fermented material of the present invention had significantly higher elastin content of 34% than the blank control group. The comparison group treated with Pakchoi root water extract was only about 2 higher than the blank control group %, of which the experimental group is also significantly different from the comparison group. This result shows that the fermented chrysanthemum root fermented product of the present invention can effectively promote the proliferation of elastin of skin cells after the microbial fermentation step, and can maintain the skin firm and elastic, so as to reduce the generation of wrinkles and make the skin plump and smooth.

Example 6 The effect of the fermented Pakchoi root of the present invention on enhancing the antioxidant capacity of cells

In the embodiment of the present invention, human skin fibroblast (CCD-966sk) is used to analyze the fermented matter of Phellodendron chinense in the invention to enhance the antioxidant capacity of the cell. The human skin fibroblasts were purchased from the American Type Culture Collection (ATCC®), number CRL-1881. The cell line was cultured in 90% MEM (Minimum essential medium, purchased from Eagle, USA, No. 6100-061) cell culture medium containing 0.1 mM of non-essential amino acids, 1.5 g/L of sodium bicarbonate and 1 mM sodium pyruvate , Which contains 10% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, USA).

2×10 ⁵ CCD-966sk cells were cultured in a 6-well culture dish containing 2 mL of the above-mentioned cell culture fluid. After culturing at 37° C. for 24 hours, the culture fluid was removed without disturbing the attached cells, and then the The cells were divided into the following four groups (n=2 for each group): (1) blank control group with cell culture medium added for 2 hours, (2) 1 mM H ₂ O ₂ (purchased from Sigma, USA, stock solution concentration 35) %) positive control group, (3) a comparative group with 1 mM H ₂ O ₂ and water extract of Pakchoi root simultaneously, and (4) an experiment with 1 mM H ₂ O ₂ and fermented Pakchoi root of the present invention Group, of which group (2), group (3), and group (4) were all reacted at 37°C for 1 hour. Then each group was added with 5 μg/mL of DCFH-DA at 37°C for 15 minutes, and then treated with H ₂ O ₂ at 37°C for 1 hour, then washed the cells twice with 1 mL of 1xPBS, and added 200 μL of trypsin in the dark After 5 minutes of reaction, the cells are detached from the culture plate. Add appropriate culture medium to stop the reaction. Collect the cells and culture medium into a 1.5 mL centrifuge tube. Centrifuge at 400 g for 10 minutes to remove the supernatant. After washing the cells once with 1xPBS, centrifuging at 400g for 10 minutes, removing the supernatant, then resuspending the cells with 1mL of 1xPBS and adding DCFH-DA (purchased from Sigma, USA, number SI-D6883-50MG, 5mg/mL) In DMSO) label the cells, and use flow cytometry to detect the fluorescence values of the cells at the excitation wavelength of 450-490nm and the emission wavelength of 510-550nm, and calculate the number of cells containing the fluorescent signal. Then use Excel software to conduct student t-test to determine whether there are statistically significant differences between the two sample groups (compared with the blank control group: *p value <0.05; **p value <0.01; ***p value< 0.001. Compared with the water extract of Phellodendron chinense: #p value<0.05;##pvalue<0.01;###pvalue<0.001).

Dichloro-dihydro-fluorescin diacetate (DCFH-DA) is a stable non-polar compound that can freely penetrate the cell membrane. When DCFH-DA enters the cell, it will be hydrolyzed by the lipase in the cell to form polar DCFH and stay in Reactive Oxygen Species (ROS) and DCFH produce redox reaction to form Dichloro-fluorescin (DCF), which will produce green fluorescence after excitation at 450-490nm wavelength It can be detected at a wavelength of 510-550nm, so detecting the fluorescence intensity of cells treated with DCFH-DA can reflect the content of reactive oxygen species in the cells.

The experimental results of the fermented material of Phellodendron arundinaceum in the present invention to improve the antioxidant capacity of cells are shown in FIG. 5, wherein the fluorescence value measured by the blank control group is taken as 0%, and the positive control group treated with H ₂ O ₂ is used The measured fluorescence value is 100%. The experimental group of human primary keratinocytes treated with the fermented Pakchoi root of the present invention can significantly reduce the ROS content by 15%. The comparative group treated with the water extract of Pakchoi root can only reduce about 3.5%, among which the experimental group is also compared with the comparative group There are significant differences. This result shows that the fermented chrysanthemum root fermentation product of the present invention can effectively remove free radicals in skin cells after the microorganism fermentation step, and can enhance the antioxidant capacity of skin cells to prevent the occurrence of skin aging.

In summary, after fermented by microbial fermentation, the fermented chrysanthemum root of the present invention can effectively increase the content of the total polysaccharide in the active ingredient, so as to enhance the moisturizing effect of the skin, and enhance the integrity of the skin stratum corneum structure; Effectively improve the expression of KRT14 gene, FLG gene, HAS2 gene and HAS3 gene, and effectively make the skin keratin secret more moisturizing factors, and increase the skin's moisturizing ability, maintain the arrangement of keratinocytes, and maintain the integrity of the stratum corneum to enhance the skin Barrier function; It can also effectively promote the collagen proliferation of skin cells, and can restore skin smoothness and firmness, so as to increase skin saturation and make the skin plump and smooth; it can also effectively promote the proliferation of elastin of skin cells to maintain skin tightness It is elastic and reduces the generation of wrinkles on the skin to make the skin plump and smooth; and it can effectively remove free radicals in skin cells and enhance the antioxidant capacity of skin cells to prevent skin aging. Therefore, the fermented roots of the present invention can be used to prepare a composition for enhancing the expression of skin keratin gene, polykeratin filament gene and hyaluronic acid synthase gene, promoting the proliferation of collagen and elastin, and enhancing the antioxidant capacity Use, the composition The composition is a medicinal product, a food or a care product, and can be administered to a body by oral administration, skin application, or the like.

<110> Dajiang Biomedical Co., Ltd.

<120> The ferment of Pakchoi root is used to enhance the expression of skin keratin gene, polykeratin microfilament gene and hyaluronic acid synthase gene, promote the proliferation of collagen and elastin, and enhance the antioxidant capacity

<130> 107B0475-I1

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Claims (15)

Use of fermented Pakchoi root to prepare a composition for enhancing the gene expression level of keratin ( KRT ) gene, polykeratin filament (Filaggrin, FLG ) gene, and/or hyaluronan synthase ( HAS ) gene . The use as described in item 1 of the patent application scope, wherein the KRT gene is a keratin 14 (Keratin14, KRT14 ) gene. The use as described in item 1 of the patent application scope, wherein the HAS gene comprises a Hyaluronan synthase 2 (Hyaluronan synthase 2, HAS2 ) gene and a Hyaluronan synthase 3 (Hyaluronan synthase 3, HAS3 ) gene. A use of the fermented Pakchoi root to prepare a composition for promoting collagen and/or elastin in skin cell proliferation. The use of fermented Pakchoi root to prepare a composition for enhancing the antioxidant activity of skin cells. The use as described in the first, fourth, or fifth item of the patent application scope, wherein the fermented Pakchoi root is obtained by two-stage fermentation of a water extract of Pakchoi root and a microbial group, wherein the microbial group The system consists of a yeast ( Saccharomyces cerevisiae ) and a lactobacillus ( Lactobacillus plantarum ). The use as described in Item 6 of the patent application range, wherein the two-stage fermentation is obtained by sequentially fermenting the yeast and the Lactobacillus. The use as described in item 1, item 4, or item 5 of the patent application scope, wherein the total polysaccharide of the fermented Pakchoi root is at least 20%. The use as described in item 1, item 4, or item 5 of the patent application scope, wherein the composition is a medicine, a food or a maintenance product. A method for manufacturing fermented radix chrysanthemum roots comprises: fermenting a water extract of radix chrysanthemum roots through a yeast and a lactobacillus in sequence. The manufacturing method as described in item 10 of the patent application scope, wherein the yeast strain is a strain of BCRC20271; the lactobacillus strain is a strain of BCRC910805. The manufacturing method as described in item 10 of the patent application scope, wherein the addition amount of the yeast is 0.01-0.5% (v/v); and the addition amount of the Lactobacillus is 0.01-0.25% (v/v). The manufacturing method as described in item 10 of the patent application scope, wherein the cultivation time of the yeast and the lactobacilli are respectively 1-3 days. The manufacturing method as described in item 10 of the patent application scope, wherein the water extract of Pakchoi root is a Pakchoi root mixed with water at a solid-liquid ratio of 1:18-30 and sterilized and extracted at 50-100°C for 0.5-3 Hourly income. The manufacturing method as described in item 10 of the patent application scope, wherein the fermentation system increases the total polysaccharide content of Pakchoi root by at least 20%.

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