TWI702055B - Pachyrhizus erosus fermented extracts and the use thereof for enhancing the gene expression of col, timp, lox, eln, has, sod, tcp1 and ung, and for reducing the skin melanin content - Google Patents

Abstract

The invention provides a Pachyrhizus erosus fermented extracts, and the use thereof for enhancing the gene expression of COL, TIMP, LOX, ELN, HAS, SOD, TCP1 and UNG, and for reducing the skin melanin content. The Pachyrhizus erosus fermented extracts can effectively increase skin anti-aging activity, increase skin firmness, and increase skin whitening ability, and achieve comprehensive skin care effect. The Pachyrhizus erosus fermented extracts is prepared by fermenting the Pachyrhizus erosus extracts to yeast, lactic acid bacteria and acetic acid bacteria for three-stage fermentation.

Description

Cooled potato fermented product and its use for preparing a composition for enhancing COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and reducing skin melanin content

The present invention relates to a cold potato fermented product and its use, in particular to a cold potato fermented product used to prepare and increase COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and reduce Use of the composition for skin melanin content.

The epidermal layer is the outermost layer of the skin. From the outside to the inside, it is the stratum corneum, the granular layer, the spinous layer and the basal layer. The epidermal layer is mainly formed by undifferentiated cylindrical keratinocytes in the basal layer. It is called keratinization. The water content in keratinocytes is high. As the cells metabolize and differentiate, the shape of keratinocytes will gradually become flat, and the nucleus and organelles will begin to degenerate and shrink, and dead cells without nuclei and organelles will be formed in the stratum corneum. The main function of the epidermis is to keep the skin water and form a skin barrier to resist various external damages. The outermost layer of the epidermis is composed of a weakly acidic sebum membrane and a stratum corneum like a brick wall. This barrier can lock the skin. Moisture and grease, resistance to the invasion of bacteria on the skin surface, and resistance to damage from foreign bodies and ultraviolet light, have a very important protective effect on the human body.

However, as the age gets older, the skin will gradually age (Ageing). The causes and processes of skin aging are very complicated and involve countless physiological phenomena, including ultraviolet rays, free radicals, Reduced collagen, slowed cell renewal, appearance of abnormal cells, reduced skin fat, lack of intercellular substance, cessation of cell growth, decreased hormones, etc. are more common factors. But so far, most of the skin anti-aging products on the market can only increase the antioxidant activity, and cannot directly and effectively improve or delay the occurrence of skin aging.

In addition, in recent years, in order to meet people's demand for whitening skin and fewer spots, various whitening products that inhibit the formation of melanin or lighten spots have been continuously developed on the market. However, these products have added too many carcinogenic products to achieve special effects. Chemical substances or environmental hormones cause consumers to use ingredients that can harm the skin and affect their health without their knowledge.

Therefore, based on the above, in response to the modern age that emphasizes natural health, it is really necessary to develop a comprehensive skin health care composition that can directly and effectively improve or delay the occurrence of skin aging, while also taking into account the requirements of skin whitening.

For this reason, one object of the present invention is to provide a cold potato fermented product, in which the water extract of cold potato is fermented sequentially by yeast ( Saccharomyces cerevisiae ), Lactobacillus plantarum , and Acetobacter aceti And get.

Another object of the present invention is to provide a cold potato fermented product as described above for preparing a collagen (Collagens, COL) gene, a matrix metallopeptidases (Tissue inhibitor of metallopeptidases, TIMP) gene, and lysine oxidase Use of a combination of expression levels of (Lysyl oxidase, LOX) gene, elastin (ELN) gene, and Hyaluronan synthase (HAS) gene.

Another object of the present invention is to provide a cold potato fermented product as described above for the preparation of a superoxide dismutase (SOD) gene, a TCP1 folding protein (Chaperonin containing TCP1, CCT) gene, and Uracil DNA glycosylase (UNG) gene expression level composition use.

Another object of the present invention is to provide a use of the fermented cold potato as described in item 1 of the patent application for preparing a composition for reducing the melanin content of the skin.

In an embodiment of the present invention, the addition amount of the yeast is 0.01-0.5% (v/v), the addition amount of the lactobacillus is 0.01-0.25% (v/v), and the addition amount of the acetobacter is It is 3-10% (v/v); and the fermentation time of the yeast is 1 to 2.5 days, the fermentation time of the lactobacillus is 1 to 3 days, and the fermentation time of the acetobacter is 3 to 10 days.

In another embodiment of the present invention, the collagen gene is collagen type 23 A1 (Collagen, type XXIII, alpha 1; COL23A1) gene, collagen type 25 A1 (Collagen, type XXV, alpha 1; COL25A1) ) Gene, collagen type 6 A2 (Collagen, type VI, alpha 2; COL6A2) gene, and collagen type 4 A4 (Collagen, type IV, alpha 4; COL4A4) gene; the matrix metalloproteinase gene is matrix metalloproteinase 1 (Tissue inhibitor of metallopeptidases 1, TIMP1) gene, and matrix metalloproteinase 3 (Tissue inhibitor of metallopeptidases 3, TIMP3) gene; the hyaluronan synthase gene is Hyaluronan synthase 2, HAS2 gene and hyaluronic acid synthesis Enzyme 3 (Hyaluronan synthase 3, HAS3) gene.

In another embodiment of the present invention, the cold potato fermentation product enhances the production of elastin.

In another embodiment of the present invention, the cold potato fermented product improves skin firmness and hyaluronic acid production.

In another embodiment of the present invention, the superoxide dismutase gene is a superoxide dismutase 2 (SOD2) gene and a superoxide dismutase 3 (SOD3) gene ; The folding protein gene of TCP1 is the Chaperonin containing TCP1 2 (CCT2) gene of TCP1, the Chaperonin containing TCP1 4 (CCT4) gene of TCP1, and the Chaperonin containing TCP1 4 (CCT4) gene of TCP1. Containing TCP1 5, CCT5) gene, and TCP1's Chaperonin containing TCP1 7, CCT 7 gene.

In another embodiment of the present invention, the cold potato fermentation system promotes DNA repair ability.

In the present invention, the cold potato fermented product obtained by three-stage fermentation of the cold potato with yeast, lactobacillus and acetobacter can effectively increase the total polyphenol content in the cold potato, enhance the ability of skin cells to resist ultraviolet light, and can also effectively enhance the skin The expression level of COL4A4 gene, TIMP1 gene, LOX gene, HAS2 gene, HAS3 gene, CCT2 gene, CCT5 gene, CCT7 gene, and SOD3 gene in the cell, and has better effect than cold potato water extract, so it can be more effective It is used to prepare pharmaceutical compositions that increase skin antioxidant activity, skin anti-aging activity, skin firmness, and skin whitening ability, and achieve comprehensive skin health effects.

The following will further illustrate the embodiments of the present invention in conjunction with the drawings. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone familiar with the art will not depart from the spirit and spirit of the present invention. Within the scope, some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

Figure 1 is a bar graph showing the increased total polyphenol content of the fermented cold potato of the present invention.

Figure 2 is a bar graph showing the reduction of melanin production in the fermented cold potato of the present invention.

Fig. 3 is a bar graph showing the increased expression of COL gene, TIMP gene, ELN gene, and HAS gene in the DNA microarray analysis of the fermented cold potato of the present invention.

Fig. 4 is a bar graph showing the expression of SOD gene, CCT gene, and UNG gene in the DNA microarray analysis of the cold potato fermentation product of the present invention.

Figure 5 is a bar graph showing the enhancement of COL4A4 gene expression in the fermented cold potato of the present invention.

Fig. 6 is a bar graph showing the improvement of TIMP1 gene expression in the fermented cold potato of the present invention.

Fig. 7 is a bar graph showing the enhancement of LOX gene expression in the fermented cold potato of the present invention.

Fig. 8 is a bar graph showing the enhancement of HASs gene expression in the fermented cold potato of the present invention.

Fig. 9 is a bar graph showing the enhancement of CCTs gene expression in the fermented cold potato of the present invention.

Fig. 10 is a bar graph showing the enhancement of SOD3 gene expression by the fermented cold potato of the present invention.

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

The present invention provides a cold potato fermented product and its use for preparing a composition for enhancing COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and reducing skin melanin content. The cold potato fermentation product of the invention is obtained by three-stage fermentation of cold potato with yeast, lactobacillus and acetobacter, which can be used to increase the total polyphenol content, enhance the anti-ultraviolet ability of skin cells, and also effectively improve The expression level of COL4A4 gene, TIMP1 gene, LOX gene, HAS2 gene, HAS3 gene, CCT2 gene, CCT5 gene, CCT7 gene, and SOD3 gene in skin cells.

At the same time, the present invention is used to prepare a composition for enhancing COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and reducing skin melanin content, and can also contain an effective amount of cold potato The fermented product and a pharmaceutically acceptable carrier, the composition is in the form of powder, granular, liquid, gel or paste, and the dosage form is food, drink, medicine, reagent or nutritional supplement Provided in the form.

The detailed preparation method of the cold potato fermentation product of the present invention will be described in detail below, and the total polyphenol content test of the cold potato fermentation product, UVA resistance test, preliminary test of regulatory genes, test of the ability to regulate elastin genes, and regulatory resistance Test of aging gene ability to confirm that the cold potato ferment Enhance the expression of COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and enhance the effect of skin anti-aging ability.

Cell culture

The present invention uses human skin fibroblasts (CCD-966sk) to carry out the cell experiment of the cold potato fermentation product of the present invention. This human skin fibroblast cell line was purchased from the American Type Culture Collection (United States) under the number CRL-1881. The cell line was cultured in MEM (Minimum essential medium, purchased from Gibco, USA, 12100-046) containing 10% Fetal Bovine Serum, and 1 mM sodium pyruvate and 1 % Of penicillin/streptomycin.

Example 1 Preparation method of cold potato fermented product of the present invention

Pachyrhizus erosus ( Pachyrhizus erosus ) is a perennial vine-like herb of the leguminous ( Legumen ) genus Pachyrhizus , also known as jicama, pueraria lobata, and pueraria lobata . Pachyrhizus erosus is a legume named after its roots are like potato pieces. The root tuber can be eaten raw or cooked. The diameter is about 20-30 cm. It is rich in sugar and protein. It can be processed into sago powder, which has the effect of cooling and removing heat.

In an embodiment of the present invention, the cold potato is thoroughly washed, the washed cold potato is mixed with water in a weight ratio of 1:5-10, and sterilized and extracted at 50-100°C for 0.5-1.5 hours to obtain the cold Potato water extract. The cold potato water extract is cooled to room temperature for subsequent three-stage fermentation. It is first colonized with 0.01-0.5% yeast ( Saccharomyces cerevisiae , purchased from the Biological Resources Conservation and Research Center, Taiwan, numbered BCRC20271 ) Fermented in the cold potato water extract for 1-2.5 days, then directly add 0.01-0.25% Lactobacillus ( Lactobacillus plantarum TCI028, patent deposited at the Center for Biological Resources Conservation and Research, Taiwan, numbered BCRC910805) for fermentation 1- For 3 days, add 3-10% Acetobacter aceti (purchased from Biological Resources Conservation and Research Center, Taiwan, numbered as BCRC11688) and ferment for 3-10 days. Among them, the Lactobacillus TCI028 system has been registered in the Republic of China Patent Application No. 106145146 Completed the patent deposit, the fermentation sequence of these three bacteria is: yeast, lactobacillus, acetobacter, and cannot be reversed. Finally, without removing these three bacteria, use the set sugar content range 2-10°, pH2 -4. No alcohol, polyphenols and other specifications. If the inspection meets the specifications, it is determined that the fermentation is completed and the fermentation broth is obtained, and the total polyphenol content is at least 20%. Next, the fermentation broth was concentrated under reduced pressure at 45-70°C, filtered through a 200-400 mesh screen, and then added 1-3% citric acid and 40-70% isomalt-oligosaccharides to adjust the specifications and then sterilized. The cold potato fermented product of the present invention is produced by microbial fermentation to release a large amount of effective substances of the cold potato.

Example 2 The total polyphenol content of the fermented cold potato of the present invention is increased

In this example, in order to compare whether the total polyphenol content in the cold potato fermentation of the present invention is higher than that in the cold potato water extract, the Folin-Ciocalteu colorimetric method is used to determine the total polyphenol content in the cold potato fermentation. The determination method uses tungsten in the reagent Molybdic acid quantifies the total polyphenol content. After it is reduced (from Mo ⁶⁺ to Mo ⁵⁺ ), a blue compound is generated, and the intensity of the blue compound is positively correlated with the total polyphenol content. First, dilute the cold potato fermentation product and cold potato water extract of the present invention by 1 time with water and take 100 mL into a centrifuge tube, then add 500 μL of Folin-Ciocalteu phenol reagent to mix and let stand for 3 minutes, then add 400 μL of 7. After mixing with 5% sodium carbonate and letting it stand for 30 minutes, take 200μL of the reaction solution in a 96-well plate and measure the absorbance at 750nm. Among them, gallic acid is used as the standard substance to make the standard curve. 10g of gallic acid is dissolved in water, and standard 0, 20, 40, 60, 80, and 100μL/mL gallic are prepared. Add 100μL of the standard solution of each concentration to a 10mL centrifuge tube, and add 500μL of Folin-Ciocalteu phenol reagent, mix and let stand for 3 minutes, add 400μL of 7.5% sodium carbonate, mix well and let stand for 30 minutes, take 200 μL of the reaction solution in a 96-well plate, and measure the absorbance at 750 nm to obtain a standard curve, and then convert the total polyphenol content in the cold potato fermentation product and cold potato water extract of the present invention through the standard curve.

The result of the cold potato fermented product of the present invention increasing the total polyphenol content is shown in FIG. 1. Polyphenols are an antioxidant. Previous studies have pointed out that polyphenols can prevent oxidative damage caused by ultraviolet light, increase the antioxidant stress of cells, help remove free radicals, and prevent DNA damage. Cool potato water extract After the three-stage fermentation of the Ming Dynasty, the total polyphenols were 13 times as high as the pure cold potato water extract. This result shows that the cold potato fermentation product of the present invention can effectively increase the total content of the cold potato fermented product after specific microbial fermentation steps. The content of polyphenols, this specific fermentation step can improve the antioxidant capacity of cold potatoes, and can be used to prepare a composition for improving the antioxidant capacity of the skin, so as to enhance the skin's anti-aging and anti-ultraviolet damage ability.

Example 3 The ability of the cold potato fermented product of the present invention to reduce the melanin content of human skin fibroblasts

This example is to compare whether the cold potato fermented product of the present invention has a higher ability to reduce the melanin content than the cold potato water extract. Therefore, melanoma cells (B16F10, number ATCC ^® CRL-6475 ^TM ) are used to test, which contains 10% Fetal Bovine Serum, 1% penicillin-streptomycin (purchased from Gibco, USA), and 90% DMEM (Dulbecco's Modified Eagle Medium, purchased from Gibco, USA, 12100-046) medium for cell to cultivate. First, add 3 mL of culture medium to each well of the 6-well culture plate and implant 1.5×10 ⁵ cells. After culturing the cells at 37°C for 24 hours, remove the culture medium without disturbing the attached cells. The cells were divided into the following four groups (n=4 in each group): (1) blank control group, (2) 0.25% koji acid as positive control group, (3) 0.25% cold potato water extract, and (4) 0.25% The cold potato fermented product of the present invention is treated in 3 mL of fresh culture medium at 37°C for 48 hours, the culture medium is removed and the cells are washed twice with 1x Phosphate buffered saline (1x PBS), and then added 200μL Trypsin was reacted for 3 minutes to make the cells fall off the culture plate, 200μL of culture medium was added to stop the reaction, and the cells and culture medium were collected into a 15mL centrifuge tube, centrifuged at 400g for 5 minutes, and the supernatant was removed. After washing the cells twice with 1mL of 1xPBS, resuspend the cells with 200μL of 1xPBS, then place the cell solution in liquid nitrogen and quickly freeze for 10 minutes, and then let the cells stand at room temperature for 30 minutes to thaw. Then, centrifuge at 12,000g for 30 minutes and remove the supernatant, then add and mix 120μL of 1N NaOH (configured with ddH ₂ O) in a dry bath at 60°C for 1 hour, then transfer 100μL of the test solution to a 96-well culture plate Measure the OD _450nm in the medium, and the melanin content is determined by the following formula: Melanin content (%)=(%)=(experimental group OD value/control group OD value)×100% and then use Excel software to perform student t-test to determine the coefficient of variation And whether there is a statistically significant difference (*p value<0.05; **p value<0.01; ***p value<0.001).

The result of the cold potato fermented product of the present invention in reducing the amount of melanin production is shown in FIG. 2. After human skin fibroblasts are treated with the cold potato fermented product of the present invention, the melanin content of the cells is reduced by 14%, which is similar to the positive control group value of kojic acid, while the unfermented cold potato water extract only reduces about 8% Cell melanin content. This result shows that the cold potato fermented product of the present invention can effectively reduce the melanin content of cells after a specific microbial fermentation step. This specific fermentation step can enhance the effect of cold potato to reduce the cell melanin content and can be used for preparation The composition for improving skin whitening makes the skin clear and bright.

Example 4 Analysis of genes regulated by the fermented cold potato of the present invention

In order to preliminarily detect which genes of the cold potato fermentation product of the present invention will affect the expression of which genes in the cells, DNA microarray is used to analyze the genes whose expression levels increase in the cells after the cold potato fermentation product of the present invention is processed. First, after collecting human skin fibroblasts stimulated by 2% cold potato fermentation for 24 hours, the cells were recovered with cell lysate (RB buffer, purchased from Geanaid, Taiwan, Cat.No.RBD300), and RNA was used The extraction reagent kit (purchased from Geneaid, Taiwan, Cat.No.RBD300) is used to extract RNA from human skin fibroblasts, and then SuperScript ^® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-044) 1μg of extracted RNA was used as a template and reverse transcription reaction (42°C, 2 hours) with T7 Oligo (dT) primers to produce the corresponding cDNA product with T7 promoter (promoter), then 1μL of DNA synthetase (Polymerase) and 0.5μL of RNA degrading enzyme (RNase H) decomposes the original RNA template of the cDNA product, and uses the single-stranded cDNA as a template to perform a DNA synthesis reaction (16°C, 2 hours) to form a double-stranded cDNA product. The cDNA product is purified to remove RNA, primers, enzymes, and salts that may inhibit in vitro transcription. In the purification system, add 50μL of cDNA product into 50μL of Nuclease-free water and mix well, then add 250μL of cDNA binding buffer to mix well, put it in the cDNA spin column, centrifuge at 10,000g for 1 minute, and then transfer the liquid under the centrifuge tube. Then put the cDNA spin column in a centrifuge, centrifuge at 10,000g for 3 minutes to completely remove the residual alcohol in the spin column, and then move the spin column to a new 1.5mL centrifuge tube, and add it to a centrifuge tube that has been preheated at 55℃ 12μL of Nuclease-free water, stand for 5 minutes at room temperature, and centrifuge at 10,000g for 2 minutes to obtain purified double-stranded cDNA. Then use Amino Allyl MessageAmp ^TM II aRNA Amplification Kit (purchased from Ambion, USA, number AM1753), and Amino Allyl UTP (aaUTP) with the double-stranded cDNA as a template for in vitro transcription reaction to synthesize amino-allyl modified aRNA (amino allyl-modified antisensesRNA) to amplify the amount of RNA to be tested, and then use the Amino Allyl MessageAmp ^TM II aRNA Amplification Kit to purify the aRNA product (purchased from the United States, number AM1753) to remove NTP that is not embedded in the product , Salts, enzymes and inorganic phosphates to improve the stability of aRNA, and then use the Labeling Reagent and Hybridization Kit (purchased from Phalanx Biotech Group, USA, number HOA v7) to add Cy5 NHS ester dye (purchased from AAT Bioquest, USA, Cat.151) Dye coupling reaction with UTP residues modified by amino allyl groups on aRNA. After removing the remaining dye, replace the buffer solution with water without nuclease (Nuclease-free Water, purchased from Ambion, USA, number AM1753), and established Whole Genome Experssion microarray hybridization system (purchased from Phalanx Biotech Group, USA, number HOA v7), and used Agilent Microarray Scanner for scanning and signal segmentation. After standardizing the original data, the relative expression level of different genes was determined by using the log2 ratio method. Then use Excel software to perform unpaired one-tailed student t-test to determine whether the coefficient of variation is statistically significant (*p value<0.05; **p value<0.01; ***p value<0.001).

The results of the DNA microarray analysis of the cold potato fermentation product of the present invention are shown in Figs. 3 and 4. It can be seen in Figure 3 that the expression levels of COL23A1, COL25A1, COL6A2, TIMP3, ELN, and HAS2 genes in skin fibroblasts increased significantly by 2 ^0.56 -2 ^1.37 times after being treated with the cold potato fermented product of the present invention. Among them, COL23A1, COL25A1, and COL6A2 are the structural proteins of collagen; TIMP3 is known to inhibit the activity of MMP (Matrix metalloproteinases) enzymes related to cancer metastasis; ELN is Elastin; HAS can express hyaluronic acid synthase, Can promote the ability of keratinocytes to secrete hyaluronic acid.

It can be seen in Fig. 4 that the expression levels of SOD2, CCT2, CCT4, and UNG genes in skin fibroblasts also increased significantly by 2 ^0.37 -2 ^1.46 times after the cold potato fermented product of the present invention is processed. Among them, SOD It is an enzyme that can catalyze the disproportionation reaction of superoxide and convert it into oxygen and hydrogen peroxide. It is an important antioxidant in the organism and protects cells exposed to oxygen; the CCT gene is a chaperonin One type of protein whose main function is to correct misfolding is considered to be related to the regulation of aging of the individual; UNG is an important protein in the DNA repair mechanism and is also considered to be related to the regulation of aging.

These results can preliminarily observe that the cold potato fermented product of the present invention may be related to the production of collagen, elastin and hyaluronic acid, and may also be related to the effects of anti-oxidation, anti-aging and DNA repair. In order to understand the cooling of the present invention in more detail The effect of fermented potato on cells will be further discussed in the following examples for the effect of the fermented cold potato of the present invention in regulating the expression of various elastin-related genes and various anti-aging-related genes.

Example 5 The effect of the cold potato fermented product of the present invention to enhance the expression of elastin gene

Human skin fibroblasts were used to test the expression of elastin gene in the cold potato fermented product of the present invention. Culture 1.5x10 ⁵ human skin fibroblasts in a six-well culture dish containing 2 mL of the above culture medium, and divide them into the following three groups: (1) Control with 2% (v/v) of the aforementioned cold potato water extract Group, (2) the experimental group added 2% (v/v) of the cold potato fermented product of the present invention, and (3) the blank control group containing only the culture solution, cultured at 37°C for 6 hours, and then fibroblasts were Cell lysate (RB buffer, purchased from Geanaid, Taiwan, No. RBD300) was used to recover the cells, and the RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No.FC24015-G) was used to collect the three groups of fibroblasts. Then use SuperScript ^® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) with 2000ng of extracted RNA as a template and primers to generate the corresponding cDNA product of mRNA reverse transcription, and then use ABI StepOnePlus ^TM Real- Time PCR system (Thermo Fisher Scientific Company, USA), and KAPA SYBR FAST (purchased from Sigma Company, USA, No. 38220000000) to perform quantitative real-time reverse transcription polymerase chain reaction of the three sets of reverse transcription products with the combination primers in Table 1. (Quantitative real-time reverse transcription polymerase chain reaction) test, the conditions are 95°C for 1 second, 60°C for 20 seconds, a total of 40 cycles. To quantify COL1A1 gene, COL1A2 gene, COL4A1 gene, COL4A3 gene, COL4A4 gene, COL4A5 gene, MMP1 gene, MMP9 gene, MMP2 gene, TGM1 gene, TIMP1 gene, ELN gene, FBN1 gene, LOX gene, HAS2 gene, and HAS3 The mRNA expression level of the gene, where the quantitative value is taken from the threshold cycle number (Ct), and the relative amount of mRNA of the target gene is derived from the equation 2 ^-△Ct , where △Ct=Ct _{target gene-} Ct _GAPDH (glyceraldehyde-3 -Phosphate dehydrogenase, Glyceraldehyde-3-phosphate dehydrogenase), and then use Excel software to perform unpaired one-tailed student t-test to determine whether the coefficient of variation is statistically significant (*p value<0.05;** p value<0.01; ***p value<0.001).

The result of the cold potato fermented product of the present invention in increasing the expression of COL4A4 gene is shown in Fig. 5; the result of increasing the expression of TIMP1 gene is shown in Fig. 6; the result of increasing the expression of LOX gene is shown in Fig. 7, where LOX The main function is to cross-link collagen and elastin to stabilize the glue The integrity and elasticity between fibrils and mature elastin; the results of improving the expression of HAS gene are shown in Figure 8. Among them, the group treated with cold potato water extracts are similar to the data of the blank control group; however, after the cold potato fermentation product of the present invention is treated, the expression level of the COL4A4 gene is 1.78 times that of the blank control group, and the TIMP1 gene The expression level is 1.37 times that of the blank control group, the expression level of LOX gene is 1.24 times that of the blank control group, the expression level of HAS2 gene is 2.24 times that of the blank control group, and the expression level of HAS3 gene is 2.74 times that of the blank control group . This result shows that the cold potato fermented product of the present invention can effectively increase the expression level of COL4A4 gene, TIMP1 gene, LOX gene, HAS2 gene, and HAS3 gene after specific microbial fermentation steps, and make collagen, elastin, and hyaluronic acid Its production is improved, and it can be used to prepare a composition for improving skin firmness to make the skin smooth and elastic.

Example 6 The effect of the cold potato fermented product of the present invention to enhance the performance of anti-aging genes

The human skin fibroblasts were used to test the anti-aging gene expression of the cold potato fermented product of the present invention. Culture 1.5x10 ⁵ human skin fibroblasts on a six-well culture plate containing 2 mL of the above culture medium, and divide them into the following three groups: (1) control group with 2% of the aforementioned cold potato water extract, (2) add The experimental group of 2% cold potato fermented product of the present invention, and (3) the blank control group containing only the culture solution, were cultured at 37°C for 24 hours, and the method of Example 5 was used to analyze the cold potato fermented product of the present invention. CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO gene, PARP1 gene, PARP2 gene, NADSYN gene, MRPS5 gene in skin fibroblasts , Ubl-5 gene, and SOD3 gene performance.

The results of the cold potato fermented product of the present invention in increasing the expression of CCT2 gene, CCT5 gene, and CCT7 gene are shown in FIG. 9; the result of increasing the expression of SOD3 gene is shown in FIG. Among them, the data of the group treated with cold potato water extract is similar to or lower than that of the blank control group; however, after the cold potato fermentation product of the present invention is treated, the expression level of CCT2 gene is 1.1 times that of the blank control group , The expression level of CCT5 gene was 1.3 times that of the blank control group, the expression level of CCT7 gene was 2.5 times that of the blank control group, and the expression level of SOD3 gene was 1.5 times that of the blank control group. This result shows that the cold potato fermented product of the present invention can effectively increase the expression level of CCT2, CCT5, CCT7, and SOD3 genes after specific microbial fermentation steps, and can effectively enhance the ability of cell metabolism and effectively improve It has the efficiency of scavenging free radicals in cells, and can be used to prepare a composition that improves the skin's anti-aging and anti-oxidation ability, so that the skin can restore its youthful state.

In summary, the cold potato fermented product obtained by three-stage fermentation of cold potato with yeast, lactobacillus and acetobacter in the present invention can effectively increase the total polyphenol content therein, enhance the ability of skin cells to resist ultraviolet light, and It can also effectively increase the expression of COL4A4 gene, TIMP1 gene, LOX gene, HAS2 gene, HAS3 gene, CCT2 gene, CCT5 gene, CCT7 gene, and SOD3 gene in skin cells, and has a better effect than cold potato water extract. Therefore, it can be more effectively used to prepare pharmaceutical compositions that increase skin antioxidant activity, skin anti-aging activity, skin firmness, and skin whitening ability, and achieve comprehensive skin health effects.

<110> Dajiang Biomedical Co., Ltd.

<120> Cold potato fermented product and its use in preparing a composition for enhancing COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and enhancing skin anti-aging ability

<130> 107B0099-I1

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Claims (9)

A cold potato fermented product, which is a cold potato water extract obtained by sequentially fermenting yeast ( Saccharomyces cerevisiae ), Lactobacillus plantarum , and Acetobacter aceti ; wherein, the addition of the yeast The amount is 0.01-0.5% (v/v); the added amount of Lactobacillus is 0.01-0.25% (v/v); and the amount of Acetobacter is 3-10% (v/v); and the yeast The fermentation time is 1 to 2.5 days, the fermentation time of the lactobacillus is 1 to 3 days, and the fermentation time of the acetic acid bacteria is 3 to 10 days. A use of the cold potato fermented product as described in item 1 of the scope of patent application for the preparation of a pharmaceutical composition that increases skin firmness, wherein the cold potato fermented product promotes collagen (Collagens, COL) gene, lysine Lysyl oxidase (LOX) gene, elastin (ELN) gene, matrix metallopeptidases (TIMP) gene expression level, in order to achieve the effect of increasing skin firmness. A use of the cold potato fermented product described in item 1 of the scope of patent application for preparing a pharmaceutical composition that increases skin firmness, wherein the cold potato fermented product improves the performance of the hyaluronan synthase (HAS) gene To achieve the effect of increasing skin firmness. A use of the fermented cold potato as described in item 1 of the scope of patent application for preparing a medicinal composition for reducing the production of melanin. A use of the cold potato fermented product described in item 1 of the scope of patent application for the preparation of a pharmaceutical composition for delaying aging, wherein the cold potato fermented product promotes superoxide dismutase (SOD) gene and TCP1 Folding protein (Chaperonin containing TCP1, CCT) gene, and/or Uracil DNA glycosylase (UNG) gene expression level, in order to achieve the effect of delaying aging. The use described in item 2 of the scope of patent application, wherein the collagen gene is collagen type 23 A1 (Collagen, type XXIII, alpha 1; COL23A1) gene, collagen 25 type A1 (Collagen, type XXV, alpha 1) COL25A1) gene, collagen type 6 A2 (Collagen, type VI, alpha 2; COL6A2) gene, collagen type 4 A4 (Collagen, type IV, alpha 4; COL4A4) gene, or any combination thereof. The use described in item 2 of the scope of patent application, wherein the matrix metalloproteinase gene is matrix metallopeptidases 1 (Tissue inhibitor of metallopeptidases 1, TIMP1) gene, matrix metalloproteinase 3 (Tissue inhibitor of metallopeptidases 3, TIMP3) gene, Or any combination thereof. The use described in item 3 of the scope of patent application, wherein the hyaluronan synthase gene is Hyaluronan synthase 2 (Hyaluronan synthase 2, HAS2) gene, Hyaluronan synthase 3 (Hyaluronan synthase 3, HAS3) gene, or any combination thereof . The use described in item 5 of the scope of patent application, wherein the superoxide dismutase gene is superoxide dismutase 2 (SOD2) gene, superoxide dismutase 3 (SOD3) Gene, or any combination thereof; the folding protein gene of TCP1 is the folding protein 2 (Chaperonin containing TCP1 2, CCT2) gene of TCP1, the folding protein 4 (Chaperonin containing TCP1 4, CCT4) gene of TCP1, the folding protein 5 of TCP1 (Chaperonin containing TCP1 5, CCT5) gene, TCP1 folding protein 7 (Chaperonin containing TCP1 7, CCT 7) gene, or any combination thereof.

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