Use of imperata cylindrica ferment for promoting collagen proliferation and method for manufacturing imperata cylindrica ferment

Abstract

The present application relates to the use of a plant fermentation product, in particular the use of a Imperata cylindra fermentation product for increasing the expression of genes, promoting the proliferation of collagen and elastin, and increasing the antioxidant capacity. The present application provides an Imperata cylindra fermentation product prepared by two-stage fermentation of an Imperata cylindra water extract with yeast and lactobacillus. The present application also provides the use of an Imperata cylindra fermentation product for preparing a composition for increasing the expression of keratin gene, polykeratin microfilament gene and hyaluronic acid synthase gene, promoting the proliferation of collagen and elastin, and increasing the antioxidant capacity. The Imperata cylindra fermentation product can effectively increase the skin moisturizing capacity, maintain the arrangement of keratinocytes, maintain the integrity of the stratum corneum structure, improve the skin barrier function, make the skin firm and elastic, and prevent skin aging.

Description

Technical Field

[0001] This invention relates to the use of Imperata cylindrica root ferment, and more particularly to the use of Imperata cylindrica root ferment for enhancing gene expression, promoting collagen and elastin production, and improving antioxidant capacity. Background Technology

[0002] The epidermis is the outermost layer of the skin, consisting of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale from the outside in. The epidermis is mainly formed by the continuous upward differentiation of undifferentiated cylindrical keratinocytes from the stratum basale, a process called keratinization. Keratinocytes have a high water content. As cells differentiate upwards, their shape gradually becomes flattened, and the nucleus and organelles begin to degenerate and atrophy, forming dead cells without nuclei and organelles in the stratum corneum. The main function of the epidermis is to retain moisture and form a skin barrier to resist various external damages. The outermost layer of the epidermis consists of a weakly acidic sebum film and a brick-wall-like stratum corneum. This barrier locks in the skin's moisture and oils, resists the invasion of pathogens on the skin surface, and protects against external foreign substances and ultraviolet light, playing a vital protective role for the human body.

[0003] The stratum corneum, the outermost layer of the epidermis, contains dead cells, but its main component is keratin. Keratin absorbs moisture to keep the skin hydrated. Keratinocytes also secrete substances like hyaluronic acid as intercellular matrix to maintain the integrity of the epidermal barrier, preventing moisture loss and forming a protective barrier. When skin is exposed to excessively cold or hot environments, or irritated by ultraviolet light, keratinocytes cannot maintain normal metabolic cycles, and the skin's water-retention capacity decreases. This damages the epidermal barrier, making the skin rough, dry, flaky, fragile, easily irritated, sensitive, and prone to redness. Therefore, the health and water-retention capacity of the stratum corneum are crucial for resisting external damage.

[0004] In summary, in response to the modern trend of emphasizing natural and healthy products, and to improve the problem of skin becoming fragile and sensitive due to damage to keratinocytes and decreased water retention capacity, it is indeed necessary to develop a comprehensive skin care composition that can effectively enable keratinocytes to secrete more moisturizing factors, maintain the arrangement of keratinocytes, maintain the integrity of the stratum corneum structure, and thus enhance the skin barrier function. Summary of the Invention

[0005] Therefore, one object of the present invention is to provide the use of Imperata cylindrica root fermentation product for preparing a composition that enhances the expression levels of keratin (KRT) gene, filaggrin (FLG) gene, and / or hyaluronan synthase (HAS) gene.

[0006] Another object of the present invention is to provide the use of Imperata cylindrica root ferment for preparing compositions that promote the proliferation of collagen and / or elastin in skin cells.

[0007] Another object of the present invention is to provide the use of Imperata cylindrica root ferment for preparing compositions that enhance the antioxidant activity of skin cells.

[0008] Another object of the present invention is to provide a method for manufacturing Imperata cylindrica root ferment, comprising: fermenting an Imperata cylindrica root aqueous extract sequentially with a yeast and a lactobacillus; wherein the Imperata cylindrica root ferment is obtained by a two-stage fermentation of an Imperata cylindrica root aqueous extract and a microbial community, wherein the microbial community is composed of a yeast (Saccharomyces cerevisiae) and a lactobacillus (Lactobacillus plantarum).

[0009] In one embodiment of the present invention, the Imperata cylindrica root water extract is prepared by mixing Imperata cylindrica root with water at a solid-liquid ratio of 1:18-30, and then... o The fermentation process involves sterilization and extraction at C for 0.5-3 hours; the yeast strain is BCRC20271 and the lactobacillus strain is BCRC910805; the yeast addition amount is 0.01-0.5% (v / v) and the lactobacillus addition amount is 0.01-0.25% (v / v); the culture time for both the yeast and the lactobacillus is 1-3 days; and the fermentation process increases the total polysaccharide content of Imperata cylindrica root, wherein the total polysaccharide content of the fermented Imperata cylindrica root is at least 20%.

[0010] In another embodiment of the present invention, the KRT gene is the keratin14 (KRT14) gene; and the HAS gene includes the hyaluronan synthase 2 (HAS2) gene and the hyaluronan synthase 3 (HAS3) gene.

[0011] The Imperata cylindrica root ferment of this invention, after undergoing the microbial fermentation step of this invention, can effectively increase the content of its active ingredient, total polysaccharides, thereby enhancing skin moisturizing effects and improving the integrity of the stratum corneum structure. It can also effectively increase the expression levels of the KRT14, FLG, HAS2, and HAS3 genes, thereby effectively promoting the secretion of more moisturizing factors by the stratum corneum, increasing skin moisturizing capacity, maintaining keratinocyte arrangement, and maintaining the integrity of the stratum corneum structure, thus enhancing skin barrier function. It can also effectively promote collagen proliferation in skin cells, restoring smoothness and firmness to improve skin hydration and make the skin plump and smooth. Furthermore, it can effectively promote elastin proliferation in skin cells to maintain skin firmness and elasticity, reduce wrinkles, and make the skin plump and smooth. Finally, it can effectively scavenge free radicals in skin cells, enhancing the antioxidant capacity of skin cells and preventing skin aging. Therefore, the Imperata cylindrica root ferment of the present invention can be used to prepare a composition that enhances the expression of skin cell keratin genes, polykeratin microfilament genes and hyaluronic acid synthase genes, promotes collagen and elastin proliferation, and enhances antioxidant capacity. This composition is a pharmaceutical product, a food product or a health care product, which can be administered to an individual by oral administration, skin application or other means.

[0012] The following will further illustrate the embodiments of the present invention. The examples listed below are for illustrative purposes only and are not intended to limit the scope of the present invention. Anyone skilled in the art can make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims. Attached Figure Description

[0013] Figure 1 This is a bar chart comparing the total polysaccharide content in Imperata cylindrica root ferment and Imperata cylindrica root water extract according to an embodiment of the present invention.

[0014] Figure 2 This is a bar graph illustrating the effect of Imperata cylindrica fermentation on enhancing the expression levels of the KRT14, FLG, HAS2, and HAS3 genes according to an embodiment of the present invention; ** p < 0.01;

[0015] Figure 3 This is a bar graph illustrating the effect of Imperata cylindrica root ferment on promoting collagen proliferation according to an embodiment of the present invention; ** p < 0.01; ### p < 0.001;

[0016] Figure 4 This is a bar graph showing the effect of Imperata cylindrica root ferment on promoting elastin proliferation according to an embodiment of the present invention; ** P < 0.01; ## p < 0.01;

[0017] Figure 5This is a bar graph showing the effect of Imperata cylindrica root ferment on enhancing the antioxidant capacity of cells according to an embodiment of the present invention; ***p<0.001; ### p<0.001. Detailed Implementation

[0018] The values ​​used in this article are approximate. All experimental data are expressed within 20%, the better range is within 10%, and the optimal range is within 5%.

[0019] Statistical analysis was performed using Excel software. Data are expressed as mean ± standard deviation (SD), and differences between individuals were analyzed using Student's t-test.

[0020] Imperata cylindrica, a perennial herb belonging to the genus Imperata in the family Gramineae, is also known as thatch grass, white thatch, or thatch root. The plant grows to 20-80 cm tall, with white, creeping rhizomes that develop scales at the nodes and have a sweet taste at the tips. The leaves are simple, alternate, and clustered at the base; older plants often have broken, fibrous leaf sheaths at the base. The leaves are flat, linear or linear-lanceolate. It flowers in summer with silvery-white flowers and dense branching. Currently, Imperata cylindrica is known to have effects such as clearing heat and moistening the lungs, promoting body fluid production and detoxification, strengthening the liver and spleen, reducing internal heat and promoting body fluid production, cooling the blood, and promoting urination.

[0021] As used in this article, the term "Imperata cylindrica fermentation product" means that the water extract of Imperata cylindrica is obtained by fermentation of yeast and lactobacillus in a one- or two-stage process. The water extract of Imperata cylindrica is obtained by extracting Imperata cylindrica and solvent at a ratio of 1:18-30 (w / w) for a period of time and temperature.

[0022] According to the present invention, pharmaceutical products can be manufactured using techniques known to those skilled in the art into dosage forms suitable for parenterally or topically administration, including, but not limited to: injections [e.g., sterile aqueous solutions or dispersions], sterile powders, external preparations, and the like.

[0023] According to the present invention, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may comprise one or more reagents selected from the following: solvent, buffer, emulsifier, suspending agent, decomposer, 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 scope of professional competence and routine practice of those skilled in the art.

[0024] According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of: water, normal saline, phosphate buffered saline (PBS), an aqueous solution containing alcohol, and combinations thereof.

[0025] According to the present invention, the pharmaceutical product can be administered via a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, intradermal injection, and intralesional injection.

[0026] According to the present invention, pharmaceutical products can be manufactured using techniques known to those skilled in the art as external preparations suitable for topical application to the skin, including, but not limited to: emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions, salves, and bandages.

[0027] According to the present invention, the external formulation is prepared by mixing the pharmaceutical product of the present invention with a base known to those skilled in the art.

[0028] According to the present invention, the substrate may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons [such as petroleum jelly and white petrolatum], waxes [such as paraffin and yellow wax], preserving agents, antioxidants, surfactants, absorption enhancers, stabilizing agents, and gelling agents [such as carbomer]. ® 974P (carbopol ®The additives include 974P, microcrystalline cellulose, and carboxymethyl cellulose; active agents; humectants; odor absorbers; fragrances; pH adjusting agents; chelating agents; emulsifiers; occlusive agents; emollients; thickeners; solubilizing agents; penetration enhancers; anti-irritants; colorants; and propellants. The selection and quantity of these additives fall within the scope of professional expertise and routine techniques of those familiar with this technology.

[0029] According to the present invention, the skincare product may further comprise an acceptable adjuvant that is widely used in skincare product manufacturing techniques. For example, the acceptable adjuvant may comprise one or more reagents selected from the following: solvents, gelling agents, surfactants, preservatives, antioxidants, screening agents, chelating agents, surfactants, coloring agents, thickening agents, fillers, fragrances, and odor absorbers. The selection and quantity of these reagents fall within the scope of professional competence and routine practice for those skilled in the art.

[0030] According to the present invention, skincare products can be manufactured in a form suitable for skincare or makeup using techniques known to those skilled in the art. This includes, but is not limited to: aqueous solutions, aqueous-alcohol solutions, or oily solutions; oil-in-water type, water-in-oil type, or complex emulsions, gels, ointments, creams, masks, patches, packs, liniments, powders, aerosols, sprays, lotions, emulsions, pastes, foams, dispersions, drops, mousses, sunblocks, tonic water, foundations, makeup remover products, soaps, and other body cleansing products.

[0031] According to the present invention, the skincare product may also be used in combination with one or more external use agents selected from the following known active ingredients: whitening agents [such as tretinoin, catechin, kojic acid, arbutin, and vitamin C], moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts [such as aloe extract], skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, and antiwrinkle agents. Topical agents include antiseptic agents, wound-healing agents, corticosteroids, and hormones. The selection and dosage of these topical agents fall within the scope of the professional competence and routine techniques of those skilled in this field.

[0032] According to the present invention, food products can be used as food additives, added during the preparation of raw materials or during the production of food by conventional methods, and formulated with any edible material to form food products for human and non-human animal consumption.

[0033] According to this invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

[0034] This invention provides the use of Imperata cylindrica root fermented product for preparing compositions that enhance the expression levels of keratin genes, polykeratin microfilament genes, and hyaluronic acid synthase genes in skin cells, promote collagen and elastin proliferation, and improve antioxidant capacity. The Imperata cylindrica root fermented product of this invention is obtained by two-stage fermentation of Imperata cylindrica root water extract with yeast and lactobacillus. The Imperata cylindrica root water extract is obtained by extracting one Imperata cylindrica root with a solvent, which is water, alcohol, or a mixture of alcohol and water. The Imperata cylindrica root fermented product of this invention can be used to enhance the expression levels of KRT14, FLG, HAS2, and HAS3 genes, promote the proliferation of collagen and elastin in skin cells, increase skin moisturizing ability, maintain keratinocyte arrangement, maintain the integrity of the stratum corneum structure, and improve the antioxidant capacity of skin cells.

[0035] Meanwhile, the present invention is used to prepare a composition that enhances the expression levels of skin cell keratin genes, polykeratin microfilament genes and hyaluronic acid synthase genes, promotes collagen and elastin proliferation, and enhances antioxidant capacity. It may also contain an effective amount of Imperata cylindrica root ferment and a pharmaceutically acceptable carrier. The composition is a pharmaceutical product, a food product or a health product.

[0036] The following details the preparation method of the Imperata cylindrica root ferment, and tests on the ferment's effects on total polysaccharide content, KRT, FLG, and HAS gene expression, collagen and elastin production in skin cells, and cellular antioxidant capacity. These tests demonstrate the efficacy of the Imperata cylindrica root ferment in increasing the expression of KRT14, FLG, HAS2, and HAS3 genes, promoting collagen and elastin production in skin cells, enhancing skin hydration, maintaining keratinocyte arrangement, maintaining stratum corneum structure integrity, and improving skin cell antioxidant capacity.

[0037] Example 1: Preparation method of Imperata cylindrica root fermentation product of the present invention

[0038] In one embodiment of the present invention, the Imperata cylindrica root is thoroughly washed, and the washed Imperata cylindrica root is mixed with water at a solid-liquid weight ratio of 1:18-30, and then stirred at 50-100 ml. o Sterilize and extract at C for 0.5-3 hours to obtain Imperata cylindrica root aqueous extract. This extract is then cooled to room temperature for subsequent two-stage fermentation. First, 0.01-0.5% (v / v) of yeast (Saccharomyces cerevisiae, purchased from the Center for Biological Resources Conservation and Research, Taiwan, patent number BCRC20271) is introduced into the Imperata cylindrica root aqueous extract and fermented at 20-37°C. oAfter fermentation at C for 1-3 days, 0.01-0.25% (v / v) of Lactobacillus plantarum TCI028 (patent registered with the Center for Biological Resources Conservation and Research, Taiwan, patent number BCRC910805) is added directly at 20-37°C. o Fermentation takes 1-3 days. Lactobacillus TCI028 is registered in Taiwan Patent Application No. 106145146. The fermentation sequence of these two bacteria is: yeast, then lactobacillus, and the order cannot be reversed. Finally, without removing these two bacteria, the fermentation is completed using a set sugar content range of 35-45°, pH 2-4, and alcohol <3%. If the test results meet these specifications, the fermentation is considered complete, and a fermentation broth is obtained, with a total polysaccharide content of at least 20%. The fermentation broth is then sterilized at 45-70°C. o C is concentrated under reduced pressure, filtered through a 200-400 mesh sieve, and then sterilized after adjusting the specifications with 0.5-1.5% preservative (Phenoxyethanol) to obtain the Imperata cylindrica root ferment of the present invention. It is produced by microbial fermentation, which releases a large amount of effective substances from Imperata cylindrica root.

[0039] Example 2: The effect of the fermentation step of the present invention on increasing the total polysaccharide content in Imperata cylindrica root water extract.

[0040] In this embodiment, to compare whether the total polysaccharide content in the fermented Imperata cylindrica root of the present invention is higher than that in the water extract of Imperata cylindrica root, the phenol-sulfuric acid assay is used to quantify the concentration of total polysaccharides in the sample. When sugars encounter strong acids, the hydroxyl groups on the structural formula combine with phenols to produce an orange-yellow liquid. Therefore, the concentration of total polysaccharides can be detected by colorimetry (especially the absorbance value of the sample at 490 nm). First, accurately weigh 10 mg of D-glucose (purchased from JT Baker, USA, catalog number 1916-01) and place it in a 10 mL volumetric flask. Add ddH2O to a total volume of 10 mL to prepare the D-glucose standard (D-Glucose stock, 1 mg / mL). Then, serially dilute the standard with ddH2O according to the formulations in Table 1 to obtain D-Glucose concentrations of 0 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL. Separately, take 1.25 g of Phenol (purchased from Merck, Germany, catalog number 1.00206.0250) and place it in a volumetric flask. Add ddH2O to a total volume of 25 mL to prepare the 5% phenol reaction solution.

[0041] Table 1. Formulation of serially diluted d-glucose standards

[0042]

[0043] Next, regression curves for the standard solutions were plotted. 100 μL of each concentration of standard solution was placed in a glass test tube, and 500 μL of 5% phenol solution was added to each tube. Then, 2.5 mL of sulfuric acid solution (95.5% H2SO4, purchased from Showa, Japan, catalog number 1970-5250) was slowly added to each test tube. The mixture was vortexed and allowed to stand for 20 minutes. Then, 200 μL of each mixture was placed in a 96-well culture dish, and the absorbance was measured at 490 nm to plot the regression curve formula for the standard solutions. Next, 100 μL of Imperata cylindrica root water extract and Imperata cylindrica root ferment of the present invention were placed in glass test tubes, and 500 μL of 5% phenol solution was added to each test tube. Then, 2.5 mL of sulfuric acid solution was slowly added to each test tube. After mixing with Vortex, the mixture was allowed to stand for 20 minutes. Then, 200 μL of each mixture was placed in a 96-well culture dish, and the absorbance was measured at 490 nm. The concentration was calculated by the regression curve formula of the standard solution and the concentration was then multiplied back by the dilution factor to obtain the concentration of total polysaccharides in the original Imperata cylindrica root water extract and the Imperata cylindrica root ferment of the present invention.

[0044] The results of the increase in total polysaccharide content in the fermented Imperata cylindrica root of the present invention are as follows: Figure 2 As shown, after undergoing the two-stage fermentation process of this invention, the total polysaccharide content of Imperata cylindrica root is 21% higher than that of the pure Imperata cylindrica root water extract. This result shows that the fermented Imperata cylindrica root product of this invention can effectively increase the total polysaccharide content after undergoing the microbial fermentation step of this invention. This fermentation step can enhance the skin moisturizing effect and improve the integrity of the skin's stratum corneum structure.

[0045] Example 3: The efficacy of the Imperata cylindrica root ferment of the present invention in enhancing the expression levels of the KRT14 gene, FLG gene, HAS2 gene, and HAS3 gene.

[0046] This invention analyzes the expression levels of the TGM1, KRT1, KRT10, KRT14, AQP3, FLG, HAS2, and HAS3 genes regulated by the Imperata cylindrica root fermentation product. The human primary epidermal keratinocytes (HPEK) were purchased from CELLnTEC (Switzerland), catalog number HPEK-50. These cells were cultured in serum-free keratinocyte-SFM medium (Gibco, catalog number #17005042, USA) at 37°C in a cell culture incubator containing 5% CO2.

[0047] 1.5×10 5 HPEK-50 cells were cultured in six-well culture dishes containing 2 mL of the above-mentioned cell culture medium and cultured at 37°C for 24 hours. After the culture medium was removed without disturbing the attached cells, the cells were divided into three groups: (1) a control group containing only cell culture medium, (2) a comparison group containing 0.125% Imperata cylindrica root water extract, and (3) an experimental group containing 0.125% Imperata cylindrica root ferment of the present invention. The cells were cultured for 6 hours. Then, the keratinocytes were recovered with cell lysis buffer (RB buffer, purchased from Geneaid, Taiwan, Cat No. RBD300). RNA was collected from the cells of the two groups using an RNA extraction reagent kit (purchased from Geneaid, Taiwan, Cat No. RBD300). Then, the RNA was extracted using SuperScript. ® III reverse transcriptase (purchased from Invitrogene, USA, catalog number 18080-051) was used with 2000 ng of extracted RNA as a template and a primer to generate the corresponding cDNA product from the reverse transcription of mRNA. Then, ABI StepOnePlus was used... TMThe Real-Time PCR system (Thermo Fisher Scientific, USA) and KAPA SYBR FAST (Sigma, USA, catalog number 38220000000) were used to perform quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) experiments on the two sets of reverse transcription products using the primer combinations listed in Table 2. The conditions were 95°C for 1 second, 60°C for 20 seconds, for a total of 40 cycles. This was used to quantify the mRNA expression levels of the TGM1, KRT1, KRT10, KRT14, AQP3, FLG, HAS2, and HAS3 genes. The quantitative values ​​were derived from the threshold cycle number (Ct), and the relative mRNA levels of the target genes were derived from Equation 2. -△Ct Where △Ct=Ct 目标基因 -Ct ACTB (β-actin) was then used to perform an unpaired one-tailed student t-test using Excel software to determine the coefficient of variation and whether there was a statistically significant difference (*p value < 0.05; **p value < 0.01; ***p value < 0.001).

[0048] Table 2. Combinatorial primers for quantitative real-time reverse transcription polymerase chaining reactions

[0049]

[0050] The results of the Imperata cylindrica root fermentation product of the present invention in increasing the expression levels of the KRT14 gene, FLG gene, HAS2 gene, and HAS3 gene are as follows: Figure 2 As shown. Previous studies have indicated that TGM enables strong bonds to form between the cell membrane and structural proteins of keratinocytes, increasing the strength and stability of the epidermis; KRT forms keratin microfilaments, while FLG helps these microfilaments assemble into a robust network, providing strength and elasticity to the skin; AQP increases the permeability of water within keratinocytes, thereby increasing their water content; HAS promotes the ability of keratinocytes to secrete hyaluronic acid, maintains the integrity of the stratum corneum structure, and enhances the skin barrier function, thus improving the skin's water retention capacity. Specifically, increasing the expression levels of the AQP and HAS genes, especially the HAS gene, can effectively enable keratinocytes to secrete more moisturizing factors.

[0051] After treatment with the Imperata cylindrica ferment of this invention, the expression levels of the KRT14 gene, FLG gene, HAS2 gene, and HAS3 gene in human primary keratinocytes were approximately 1.2 times higher than those in the control group, approximately 1.4 times higher, approximately 1.5 times higher, and approximately 1.2 times higher. Furthermore, the expression levels of the KRT14, FLG, HAS2, and HAS3 genes were all higher than in the control group. However, the expression levels of the TGM1, KRT1, KRT10, and AQP3 genes were not significantly different from those in the control group. These results indicate that the Imperata cylindrica ferment of this invention, after undergoing microbial fermentation, can effectively increase the expression levels of the KRT14, FLG, HAS2, and HAS3 genes, thereby effectively promoting the secretion of more moisturizing factors by the keratinocytes, enhancing skin hydration, maintaining keratinocyte arrangement, and preserving the integrity of the stratum corneum structure, thus improving the skin barrier function.

[0052] Example 4: The efficacy of the fermented Imperata cylindrica root extract of the present invention in promoting collagen production in dermal fibroblasts.

[0053] In this embodiment of the invention, human skin fibroblasts (CCD-966sk) were used to analyze the effect of the Imperata cylindrica root ferment on promoting collagen proliferation. These human skin fibroblasts were purchased from the American Type Culture Collection (ATCC®), catalog number CRL-1881, and cultured in a medium containing 10% fetal bovine serum and 90% Minimum Essential Medium (MEM) (purchased from Gibco, USA), which contained 1 mM sodium pyruvate and 1% penicillins / streptomycin.

[0054] First, 2×10 4 Human skin fibroblasts were seeded in 24-well culture dishes containing 500 μL of the above-mentioned culture medium and incubated at 37°C. o After culturing at 37°C for 24 hours, the cells were washed with 1x phosphate-buffered saline (PBS) without disturbing the cells. The cells were then divided into two groups: (1) a control group containing 0.125% Imperata cylindrica root water extract, and (2) an experimental group containing 0.125% Imperata cylindrica root ferment of this invention. Each group was dissolved in 500 μL of MEM without fetal bovine serum. The group without any extract was used as a blank control, and the group containing only culture medium without cells was used as the background value for subsequent absorbance readings. oAfter culturing at 48°C, without disturbing the cells, 1 mL of culture medium was collected from each group and placed in a 1.5 mL centrifuge tube. The amount of collagen secreted by dermal fibroblasts was detected using a soluble collagen assay kit (Sircol™ Soluble Collagen Assay kit, Bicolor Life Science Assays, Northern Ireland, UK). First, 200 μL of collagen isolation and concentration reagent was added and the tubes were inverted 6-8 times to mix thoroughly. Then, the centrifuge tubes were incubated at 4°C. o Place in a container at room temperature for 16-18 hours. After this time, remove the container, avoiding shaking it, and then place it in a container at room temperature. o Centrifuge at 12000 rpm for 10 minutes at C. After centrifugation, carefully remove 1 mL of supernatant, then add 1 mL of Sircol dye reagent to stain the collagen, and gently shake for 30 minutes to mix thoroughly. Then, at 4°C... o After centrifuging at 12000 rpm for 10 minutes at C, the supernatant was removed, and then 750 μL of acid-salt washing reagent was added to wash away excess dye. o After centrifugation at 12000 rpm for 10 minutes at C, the supernatant was removed, and the liquid remaining on the tube wall and cap was carefully removed with a cotton ball. Then, 250 μL of strong alkali reagent was added, and the collagen was dissolved evenly using a shaker. Finally, the absorbance of the products of each group and the blank control group at 555 nm was measured using an ELISA reader (BioTek). After subtracting the background value, the percentage difference between the readings of each group and the blank control group was calculated. A student t-test was then performed using Excel software to determine the coefficient of variation and whether the difference was statistically significant (compared to the blank control group: *p < 0.05; **p < 0.01; ***p < 0.001. Compared with Imperata cylindrica root water extract: #p < 0.05; ##p < 0.01; ###p < 0.001).

[0055] The experimental results of the Imperata cylindrica root ferment of the present invention in promoting collagen proliferation in skin cells are as follows: Figure 3As shown, the experimental group of human primary keratinocytes treated with the Imperata cylindrica root ferment of this invention had a significantly higher collagen content than the control group (23%), while the control group treated with Imperata cylindrica root water extract had only a 2% higher collagen content than the control group. The experimental group also showed a significant difference from the control group. These results demonstrate that the Imperata cylindrica root ferment of this invention, after undergoing microbial fermentation, can effectively promote collagen proliferation in skin cells, restore smoothness and firmness to the skin, and increase skin hydration to make the skin plump and smooth.

[0056] Example 5: The efficacy of the fermented Imperata cylindrica root extract of the present invention in promoting the proliferation of elastin in dermal fibroblasts.

[0057] In this embodiment of the invention, human skin fibroblasts (CCD-966sk) were used to analyze the effect of the Imperata cylindrica root fermentation product of the present invention on promoting elastin proliferation. The human skin fibroblasts were purchased from the American Type Culture Collection (ATCC®), catalog number CRL-1881, and cultured in a medium containing 10% fetal bovine serum and 90% Minimum Essential Medium (MEM) (purchased from Gibco, USA), which contained 1 mM sodium pyruvate and 1% penicillins / streptomycin.

[0058] First, 1×10 5 Human skin fibroblasts were seeded in 6-well culture dishes containing 2 mL of the above-mentioned culture medium and incubated at 37°C. o After culturing at 37°C for 24 hours, the cells were washed with phosphate-buffered saline (PBS) without disturbing them. The cells were then divided into two groups: (1) a control group containing 0.125% Imperata cylindrica root water extract, and (2) an experimental group containing 0.125% Imperata cylindrica root ferment of this invention. Each group was dissolved in 2 mL of MEM without fetal bovine serum. A control group without any extract was used, and a group containing only culture medium without cells was used as the background value for subsequent absorbance readings. The cells in each group were cultured at 37°C. oAfter culturing at C for 48 hours, the culture medium was removed without disturbing the cells, and the cells were washed once with 1xPBS. Then, 200 μL of trypsin was added and reacted for 3 minutes to detach the cells from the culture dish. 1 mL of cell culture medium was added to stop the reaction, and the cells and culture medium were collected into a 1.5 mL test tube. After centrifugation at 300 g for 5 minutes, the supernatant was removed, and the cells were washed once with 1xPBS. After centrifugation at 300 g for 5 minutes, the supernatant was removed, and about 300 μL of 1xPBS was added to resuspend the cells. Then, the amount of elastin secreted by skin fibroblasts was detected using the Fastin™ Elastin Assay kit (purchased from Biocolor, UK). First, add 100 μL of 1.0 M oxalic acid solution and incubate on a 100°C hot plate for 1 hour. Then, add the same volume of elastin precipitation reagent to each tube, tighten the cap, and vortex thoroughly. Let stand for 15 minutes to allow complete elastin precipitation. Centrifuge at 10,000 g for 10 minutes, drain the liquid from the centrifuge tubes, invert the tubes, and tap them gently on a paper towel to remove most of the remaining liquid. Next, add 1 mL of TPPS (5,10,15,20-tetraphenyl-21H,23H-porphine tetrasulfonate) dye reagent, invert the centrifuge tubes several times to mix thoroughly, and then incubate on a mechanical shaker at 6 rpm for 90 minutes. Remove unbound dye; a reddish-brown precipitate of the elastin-dye complex can be observed at the bottom of the centrifuge tubes. Finally, add 250 mL of TPPS solution to each tube. μL of dye dissociation reagent was added, and after tightening the cap, the mixture was thoroughly vortexed to release the dye into the solution, ensuring that all bound dye had entered the solution. The contents of each tube were then transferred to a 96-well culture dish, and the absorbance of the products of each group and the blank control group at 513 nm was measured using an ELISA reader (BioTek). After subtracting the background value, the percentage difference between the readings of each group and the blank control group was calculated. A student t-test was then performed using Excel software to determine the coefficient of variation and whether the difference was statistically significant (compared to the blank control group: *p value < 0.05; **p value < 0.01; ***p value < 0.001. Compared with Imperata cylindrica root water extract: #p value < 0.05; ##p value < 0.01; ###p value < 0.001).

[0059] The experimental results of the Imperata cylindrica root ferment of the present invention in promoting the proliferation of elastin in skin cells are as follows: Figure 4 As shown, the experimental group of human primary keratinocytes treated with the Imperata cylindrica root ferment of this invention had a significantly higher elastin content than the control group (34%), while the control group treated with Imperata cylindrica root water extract only had about a 2% higher content than the control group. The experimental group also showed a significant difference from the control group. These results demonstrate that the Imperata cylindrica root ferment of this invention, after undergoing microbial fermentation, can effectively promote elastin proliferation in skin cells and maintain skin firmness and elasticity, thereby reducing wrinkles and making the skin plump and smooth.

[0060] Example 6: The efficacy of the fermented Imperata cylindrica root of the present invention in enhancing cellular antioxidant capacity.

[0061] This invention utilizes human skin fibroblasts (CCD-966sk) to analyze the effect of the Imperata cylindrica root ferment on enhancing the antioxidant capacity of cells. The human skin fibroblasts were purchased from the American Type Culture Collection (ATCC®), catalog number CRL-1881. The cells were cultured in 90% MEM (Minimum essential medium, Eagle, USA, catalog number 61100-061) containing 0.1 mM non-essential amino acids, 1.5 g / L sodium bicarbonate, and 1 mM sodium pyruvate, with 10% fetal bovine serum (Gibco, USA).

[0062] 2×10 5 One CCD-966sk cell was cultured in a 6-well culture dish containing 2 mL of the above cell culture medium and cultured at 37°C for 24 hours. After the culture medium was removed without disturbing the attached cells, the cells were divided into the following four groups (n=2 per group): (1) a blank control group with only cell culture medium added for 2 hours, (2) a positive control group with 1 mM H2O2 (purchased from Sigma, USA, stock solution concentration of 35%) added, (3) a comparison group with 1 mM H2O2 and Imperata cylindrica root water extract added simultaneously, and (4) an experimental group with 1 mM H2O2 and Imperata cylindrica root fermentation product of the present invention added simultaneously. Among them, groups (2), (3), and (4) were all cultured at 37°C for 24 hours. oThe reaction was carried out at C for 1 hour. Then, each group was treated with 5 μg / mL DCFH-DA at 37°C for 15 minutes, followed by treatment with H2O2 at 37°C for 1 hour. Cells were then washed twice with 1 mL of 1xPBS, and 200 μL of trypsin was added. The cells were reacted in the dark for 5 minutes to allow them to detach from the culture dish. Appropriate culture medium was added to terminate the reaction, and the cells, along with the culture medium, were collected into 1.5 mL centrifuge tubes. After centrifugation at 400 g for 10 minutes, the supernatant was removed. Cells were then washed once with 1 mL of 1xPBS, centrifuged at 400 g for 10 minutes, and the supernatant was removed. Cells were then resuspended in 1 mL of 1xPBS, and DCFH-DA (purchased from Sigma, USA, catalog number SI-D6883-50MG, 5 mg / mL stored in DMSO) was added to label the cells. Flow cytometry was used to detect the fluorescence values ​​of the cells at excitation wavelengths of 450-490 nm and emission wavelengths of 510-550 nm, and the number of cells containing fluorescent signals was calculated. Then, a student t-test was performed using Excel software to determine whether there was a statistically significant difference between the two sample groups (compared to the blank control group: *p value < 0.05; **p value < 0.01; ***p value < 0.001. Compared with the water extract of Imperata cylindrica root: #p value < 0.05; ##p value < 0.01; ###p value < 0.001).

[0063] Dichloro-dihydro-fluorescin diacetate (DCFH-DA) is a stable, nonpolar compound that can freely permeate cell membranes. When DCFH-DA enters a cell, it is hydrolyzed by intracellular lipases to form polar DCFH, which remains inside the cell and cannot leave. It then undergoes a redox reaction with reactive oxygen species (ROS) to form dichloro-fluorescin (DCF). When excited at a wavelength of 450-490 nm, the resulting green fluorescence can be detected at a wavelength of 510-550 nm. Therefore, detecting the fluorescence intensity of cells treated with DCFH-DA can reflect the content of reactive oxygen species in the cell.

[0064] The experimental results of the Imperata cylindrica root ferment of the present invention in enhancing the antioxidant capacity of cells are as follows: Figure 5As shown, the fluorescence value measured in the blank control group was taken as 0%, and the fluorescence value measured in the positive control group treated with H2O2 was taken as 100%. In the experimental group of human primary keratinocytes treated with the Imperata cylindrica root ferment of this invention, the ROS content was significantly reduced by 15%, while the control group treated with Imperata cylindrica root water extract only reduced it by about 3.5%. The experimental group also showed a significant difference from the control group. This result shows that the Imperata cylindrica root ferment of this invention, after undergoing a microbial fermentation step, can effectively scavenge free radicals in skin cells, enhance the antioxidant capacity of skin cells, and prevent skin aging.

[0065] In summary, the Imperata cylindrica root ferment of the present invention, after undergoing microbial fermentation, can effectively increase the content of its active ingredient, total polysaccharides, thereby enhancing skin moisturizing effects and improving the integrity of the stratum corneum structure. It can also effectively increase the expression levels of the KRT14, FLG, HAS2, and HAS3 genes, thereby promoting the secretion of more moisturizing factors by the stratum corneum, increasing skin moisturizing capacity, maintaining keratinocyte arrangement, and maintaining the integrity of the stratum corneum structure, thus enhancing skin barrier function. Furthermore, it can effectively promote collagen proliferation in skin cells, restoring smoothness and firmness to improve skin hydration and make the skin plump and smooth. It can also effectively promote elastin proliferation in skin cells to maintain skin firmness and elasticity, reduce wrinkles, and make the skin plump and smooth. Finally, it can effectively scavenge free radicals in skin cells, enhancing the antioxidant capacity of skin cells and preventing skin aging. Therefore, the Imperata cylindrica root ferment of the present invention can be used to prepare a composition that enhances the expression of skin cell keratin genes, polykeratin microfilament genes and hyaluronic acid synthase genes, promotes collagen and elastin proliferation, and enhances antioxidant capacity. This composition is a pharmaceutical product, a food product or a health care product, which can be administered to an individual by oral administration, skin application or other means. [Sequence List] <110> TCI Biotech Co., Ltd. <120> Uses of Imperata cylindrica root ferment for promoting elastin production and methods for manufacturing Imperata cylindrica root ferment. <130> 107B0475-I1 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 1 gatcgcatca cccttgagtt ac 22 <210> 2 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 2 gcaggttcag attctgccc 19 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 3 agagtggacc aactgaagag t 21 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 4 attctctgca tttgtccgct t 21 <210> 5 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 5 tcctacttgg acaaagttcg gg 22 <210> 6 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 6 cccctgatgt gagttgcca 19 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 7 ttctgaacga gatgcgtgac 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 8 gcagctcaat ctccaggttc 20 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 9 ggggagatgc tccacatcc 19 <210> 10 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 10 aaaggccagg ttgatggtga g 21 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 11 ggcaaatcct gaagaatcca 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 12 tgctttctgt gcttgtgtcc 20 <210> 13 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 13 aagaacaact tccacgaaaa ggg 23 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 14 ggctgggtca agcatagtgt 20 <210> 15 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 15 cgcagcaact tccatgagg 19 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 16 agtcgcacac ctggatgtag t 21 <210> 17 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 17 catgtacgtt gctatccagg c 21 <210> 18 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 18 ctccttaatg tcacgcacga t 21

Claims

1. Use of a fermented Imperata cylindrica root extract for preparing a composition that promotes collagen proliferation in skin cells, wherein the fermented Imperata cylindrica root extract is obtained by sequentially fermenting an aqueous extract of Imperata cylindrica root with a yeast strain and a lactobacillus strain, wherein the yeast strain is BCRC20271 and the lactobacillus strain is BCRC910805; the fermented Imperata cylindrica root extract increases the expression levels of the keratin 14 gene, the polykeratin microfilament gene, and / or the hyaluronic acid synthase gene, and the total polysaccharide content of the fermented Imperata cylindrica root extract is at least 20%.

2. Use according to claim 1, characterized in that, The composition is a pharmaceutical product, a food product, or a health product.

3. A method for producing a fermented product of Imperata cylindrica root, comprising: A water extract of Imperata cylindrica root is fermented sequentially with a yeast and a lactobacillus to obtain an Imperata cylindrica root ferment that promotes collagen production in skin cells. The yeast is strain BCRC20271 and the lactobacillus is strain BCRC910805. The Imperata cylindrica root ferment increases the expression levels of the keratin 14 gene, the polykeratin microfilament gene, and / or the hyaluronic acid synthase gene. The total polysaccharide content of the Imperata cylindrica root ferment is at least 20%.

4. The manufacturing method according to claim 3, characterized in that, The amount of yeast added is 0.01-0.5% v / v; and the amount of lactobacillus added is 0.01-0.25% v / v.

5. The production method according to claim 3, wherein The culture time for the yeast and lactobacillus is 1-3 days, respectively.

6. The production method according to claim 3, wherein The Imperata cylindrica root water extract is obtained by mixing Imperata cylindrica root with water at a solid-liquid ratio of 1:18-30, and then... o The product is obtained by sterilization and extraction at C for 0.5-3 hours.

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