TWI747121B - Use of fermented product of camellia sinensis leaves for regulating expressions of cct gene, pink1 gene, parp2 gene, mitf gene, ung gene, ercc6 gene, terc gene, cd40 gene, dynll2 gene, grk5 gene, relb gene, tnfsf14 gene, il4r gene, and rela gene, and anti-aging, whitening, enhancing immunity and reducing fat accumulation - Google Patents

Abstract

The present disclosure provides a use of a fermented product of Camellia sinensis leaves for regulating expressions of CCT gene, Pink1 gene, PARP2 gene, MITF gene, UNG gene, ERCC6 gene, TERC gene, CD40 gene, DYNLL2 gene, GRK5 gene, RELB gene, TNFSF14 gene, IL4R gene, and RELA gene, and anti-aging, whitening, enhancing immunity and reducing fat accumulation.

Description

The fermented product of tea leaves is used to regulate CCT gene, Pink1 gene, PARP2 Gene, MITF gene, UNG gene, ERCC6 gene, TERC gene, CD40 gene, DYNLL2 gene, GRK5 gene, RELB gene, TNFSF14 gene, IL4R gene and RELA gene expression level and anti-aging, whitening, improving immunity and reducing fat accumulation Purpose

The present invention relates to a fermented product of tea leaves for regulating CCT gene, Pink1 gene, PARP2 gene, MITF gene, UNG gene, ERCC6 gene, TERC gene, CD40 gene, DYNLL2 gene, GRK5 gene, RELB gene, TNFSF14 gene, The expression level of IL4R gene and RELA gene and the use of anti-aging, whitening, improving immunity and reducing fat accumulation.

Since ancient times, human beings have always dreamed of pursuing the way of eternal youth and immortality. In recent years, with the development and progress of medicine and biotechnology, not only can the front-end medical technology be used to combat diseases, but many products demanding anti-aging have also been gradually developed. With the advent of research and development, the anti-aging trend has slowly spread to the world in recent years. Take Japan as an example. More than 70% of the people have anti-aging awareness, and Taiwanese people's concern about anti-aging continues to increase. This anti-aging trend The trend will drive the sales of anti-aging related products, and it is expected that the global market will continue to expand.

Telomerase is a ribonucleoprotein complex composed of RNA and protein, which is closely related to the regulation mechanism of telomeres. Telomerase can extend the repair capacity of telomeres, reduce the loss of telomeres due to cell division, and increase the number of cell divisions and replications. Telomerase activity is also related to cell proliferation and survival. Cells will continue to lose telomerase activity, and telomerase DNA will also be lost during the DNA repair process. Therefore, telomerase activity is related to cell aging.

Skin tissue is composed of epidermis, dermis and subcutaneous tissue. The dermis contains a lot of collagen and hyaluronic acid, which are closely related to the water retention and elasticity of the skin. Human skin will experience aging, rough skin or wrinkles with age, physiological factors or environmental factors. For example, the skin of normal young people has a certain degree of elasticity and tension. When the expression muscles relax, the skin will quickly become Recover and make wrinkles disappear; but after entering middle age, the skin begins to age significantly, and the skin becomes thinner, harder, dry, and reduced in tension; dermal collagen decreases, elastic fibers are denatured, and broken, which reduces the tension and elasticity of the skin. Therefore, When the expression muscles relax, the skin cannot recover quickly, and wrinkles will form over time. With the increase of age, the skin and subcutaneous tissues become more relaxed, coupled with the atrophy or loss of facial support tissues, and the softness of muscles, the skin will become weaker. Slippage occurs under the action of gravity, forming deeper wrinkles. Rough skin is caused by externally important factors such as dryness, ultraviolet rays, cleansers, chemical substances and other irritating substances, or internally important factors such as disturbance of hormone balance, and is accompanied by a decline in the barrier function of the stratum corneum. Phenomenon such as the decrease of water content, the increase of epidermal metabolism, the production of scales and the roughening of keratin. Therefore, if the cells on the skin lose their elasticity and moisturizing function, it will cause skin wrinkles, dryness, loss of luster and aging.

Melanogenesis (that is, melanin synthesis) refers to when the skin melanocytes (dermal melanocytes) are exposed to environmental factors (such as ultraviolet (UV)) or physiological factors (such as fatigue, stress ( After induction of stress, chronic inflammation and abnormal release of α-melanocyte stimulating hormone (α-MSH) in the body, tyrosine in melanocytes The process of being converted into melanin by catalysis by tyrosinase (which is the rate-limiting step of melanin production) and a series of redox reactions. Melanin can protect the hypodermis of the skin from photodamage caused by ultraviolet rays. However, when melanin is accumulated on the skin in a large amount or is abnormally distributed, it may cause skin diseases. disorders, such as lentigines, freckle, melasma, age spots, and hyperpigmentation.

In order to achieve the purpose of skin whitening, many melanogenesis inhibitors have been used to lighten or remove the accumulated melanin or dark spots on the skin. Most of these melanogenesis inhibitors are through the following The mechanism of action to regulate the production of melanin: (1) Before melanin production: for example, inhibition of tyrosinase mRNA transcription (mRNA transcription) (such as C2-ceramide, tretinoin and tretinoin) Vanillic acid (vanillic acid) and glycosylation (such as calcium D-pantetheine-S-sulfonate (PaSSO3Ca)); (2) During the period of melanin production: such as inhibition of tyrosinase Activity (such as hydroquinone, arbutin, and kojic acid), accelerate the degradation of tyrosinase (such as linoleic acid) and phenylthiourea ( phenylthiourea)), and promote the reduction of dopaquinone (such as ascorbic acid); and (3) after melanin production: for example, promote the decomposition of melanin (such as linoleic acid), Inhibit melanosome transfer (such as Niacinamide and serine protease inhibitor), and accelerate skin turnover (such as liquiritin and glycolic acid) acid)).

Fat is an essential component in the human body, but excessive fat content can cause damage to the human body. In countries where the national economy is taking off, the problem of obesity (also known as metabolic syndrome) is only increasing. Therefore, from the perspective of Asia, China will be the next wave of countries where the increase in obesity problems will increase sharply.

In addition, Taiwan has now become the "fattest country in Asia." Among citizens over 18 years old, the proportion of men overweight is 51.1%, and the proportion of women is 35.8%, and the proportion is still rising, even overweight. The age group is also declining year by year, and the proportion of overweight students in elementary and middle schools has risen to more than 25%, which is the eighth-highest region in the world. Therefore, obesity is a problem that needs to be solved urgently.

The immune system of the organism is extremely important for the maintenance of the health of the organism. The immune system contains a large number of immune cells. Under normal circumstances, immune cells can produce an appropriate immune response. Externally, they can be invaded by foreign pathogens (such as bacteria, molds, viruses, microorganisms, and various antigens). Help the body to resist pathogens; internally, it can break down the aging cells in the body that cannot function normally, or abnormal cancer cells produced by genetic mutations. Therefore, the improvement of immunity helps organisms The body is maintained in a healthy state. Among them, the function of immune cells will gradually decrease as the cells age, which is one of the main reasons for the decline in immunity. Therefore, if the aging of immune cells can be delayed, immunity can be effectively enhanced.

Abnormal immune cell function, also known as immune dysfunction, includes conditions such as low immune function, allergic reactions, and autoimmune reactions, which can cause harm to the health of the organism. Among them, individuals with low immune function are more susceptible to infection by foreign pathogens, and cancer may occur because the cancer cells in the body cannot be cleared in time. Therefore, if the immunity can be effectively improved, the function of the immune system can be improved to maintain the health of the individual.

However, most of the medicines, food products, or skin care products currently used to solve the above problems are made of chemical ingredients. Long-term use is not only harmful to human health, but also these products are often expensive and not affordable for ordinary users. In order to solve the above-mentioned problems, those skilled in the art urgently need to develop novel medicines, food products or skin care products with anti-aging, reducing fat accumulation, whitening, and improving immunity to benefit the majority of people in need.

In view of this, the object of the present invention is to provide a fermented product of tea tree (Camellia sinensis ) leaves for preparing a chaperonin containing T-complex protein complex containing T-complex protein primary unit α (TCP1) complex 1 subunit alpha (TCP1) complex, CCT ) gene, type 1 PTEN-induced kinase 1, Pink1 gene, poly [ADP-ribose] polymerase 2 (Poly [ADP-ribose] polymerase 2, PARP2 ) Genes, Microphthalmia-associated transcription factor ( MITF ) gene, uracil DNA glycosylase ( UNG ) gene, ERCC cut-type repair 6, endonuclease non-catalytic subunit (ERCC excision) repair 6,endonuclease non-catalytic subunit, ERCC6 ) gene, telomerase RNA component ( TERC ) gene, CD40 gene, tubulin, light chain, type 2 LC8 (Dynein, Light Chain, LC8- Type 2, DYNLL2 ) gene, G Protein-Coupled Receptor Kinase 5 ( GRK5 ) gene, transcription factor RelB ( RELB ) gene, tumor necrosis factor superfamily member 14 , TNFSF14 ) gene, interleukin-4 receptor (Interleukin-4 receptor, IL4R ) gene, and transcription factor P65 ( RELA ) gene expression composition, wherein the fermented product of the tea leaves is obtained by a It is prepared by a method including the following steps: extracting tea leaves with a solvent to obtain a tea leaf extract, and then using a Saccharomyces cerevisiae , a Bifidobacterium lactis , and a Lactobacillus gasseri ( Lactobacillus gasseri ) and a lignoacetobacter xylinus (Gluconacetobacter xylinus) simultaneously ferment the tea leaf extract to obtain the fermented product of the tea leaf, wherein the The solvent is water, alcohol, or a mixture of alcohol and water.

In an embodiment of the present invention, the CCT gene is a chaperonin containing TCP1 subunit 2 ( CCT2 ) gene and a chaperonin containing TCP1 subunit 5 ( CCT5 ) gene. Or a chaperonin containing TCP1 subunit 8, CCT8 gene.

In an embodiment of the present invention, the expression levels of the CCT gene, the Pink1 gene, the PARP2 gene, the UNG gene, the ERCC6 gene, and the TERC gene are up-regulated, and the MITF gene, the CD40 gene, and the DYNLL2 The expression levels of the genes, the GRK5 gene, the RELB gene, the TNFSF14 gene, the IL4R gene, and the RELA gene are down-regulated.

Another object of the present invention is to provide a fermented product of Camellia sinensis leaves for preparing a composition that reduces fat accumulation, wherein the fermented product of tea leaves is prepared by a method including the following steps : Extract tea leaves with a solvent to obtain a tea leaf extract, and then with a brewer's yeast ( Saccharomyces cerevisiae ), a Leiter B bacteria ( Bifidobacterium lactis ), a Lactobacillus gasseri (Lactobacillus gasseri) and a wood acetic acid The bacterium ( Gluconacetobacter xylinus ) simultaneously ferments the tea leaf extract to obtain the fermented product of the tea leaf, wherein the solvent is water, alcohol, or a mixture of alcohol and water.

In an embodiment of the present invention, the fermented product of the tea leaves is further used to prepare an anti-aging, whitening and immunity-boosting composition.

In an embodiment of the present invention, the whitening is to inhibit melanin production.

In an embodiment of the present invention, the anti-aging is to increase telomerase activity, increase DNA repair ability, increase skin moisture content, increase skin elasticity, and reduce skin wrinkles.

In an embodiment of the present invention, the ratio of the solvent to the tea leaves is 50~125:1 (w/w), and the extraction step is performed at 50~100°C.

In an embodiment of the present invention, the concentration of the saccharomyces cerevisiae is between 0.01 and 0.5% (v/v), and the concentration of the Rettler B bacterium is between 0.01 and 0.25% (v/v). The concentration of Lactobacillus is between 0.01 and 0.25% (v/v), and the concentration of the lignoacetic acid is between 10 and 15% (v/v).

In an embodiment of the present invention, the fermentation time of the Saccharomyces cerevisiae, the Leiter B bacterium, the Lactobacillus gasseri, and the lignoacetic acid bacterium is 12-25 days.

In an embodiment of the present invention, the tea leaf is a red jade black tea leaf.

In an embodiment of the present invention, the effective concentration of the fermented product of the tea leaves is at least 0.5 μg/mL.

In an embodiment of the present invention, the composition is a medicine, a food product, or a skin care product.

To sum up, the effect of the fermented product of tea leaves of the present invention is that it can regulate CCT gene, Pink1 gene, PARP2 gene, MITF gene, UNG gene, ERCC6 gene, TERC gene, CD40 gene, DYNLL2 gene, GRK5 gene, The expression levels of RELB gene, TNFSF14 gene, IL4R gene and RELA gene can achieve the effects of anti-aging, whitening, improving immunity and reducing fat accumulation. In addition, the fermented product of the tea leaves of the present invention also has the effects of increasing the moisture content of the skin, elasticity, and anti-wrinkle smoothing texture. Experiments have also confirmed that the fermented product of the tea leaves has the effects of slimming and beautifying skin, while effectively reducing body fat and weight, reducing waist circumference, and It can moisturize and anti-wrinkle, achieve slim and beautiful effect.

The following will further explain the embodiments of the present invention. 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 scope of the present invention. Some changes and modifications can be made, so the scope of protection of the present invention shall be subject to the scope of the attached patent application.

Figure 1 is a data diagram of the efficacy of the fermented product of the tea leaves of the present invention in reducing fat accumulation, where "**" indicates that compared with the control group, p <0.01.

Figure 2 is a staining diagram of the effect of fermented tea leaves of the present invention on reducing fat accumulation.

Figure 3 is a data diagram of the utility of the fermented product of the tea leaves of the present invention in enhancing the expression of anti-aging-related genes, where "*" means comparison with the control group, p <0.05;"**" means comparison with the control group, p <0.01.

Fig. 4 is a data diagram of the utility of the fermented product of the tea leaves of the present invention in reducing the expression of genes related to melanin production, where "***" means compared with the control group, p <0.001.

Figure 5 is a data diagram of the utility of the fermented product of the tea leaves of the present invention in improving the expression of DNA repair-related genes, where "*" means comparison with the control group, p <0.05;"**" means comparison with the control group , P <0.01.

Fig. 6 is a data diagram of the utility of the fermented product of the tea leaves of the present invention in enhancing the expression of the TERC gene, wherein "*" represents the comparison with the control group, p <0.05.

Figure 7 is a data diagram of the utility of the fermented product of tea leaves of the present invention in reducing the expression of genes related to inducing immune response, where "*" means comparison with the control group, p <0.05;"**" means comparison with the control group , P <0.01;"***" means that compared with the control group, p <0.001.

Fig. 8 is a data chart of the human body efficacy of the fermented product of tea leaves of the present invention.

Fig. 9 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention.

Fig. 10 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention.

Fig. 11 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention.

Figure 12 is another data diagram of the human body efficacy of the fermented tea leaves of the present invention, where "**" indicates that compared with before drinking (week 0), p <0.01.

Figure 13 is another data diagram of the human body efficacy of the fermented tea leaves of the present invention, where "**" indicates that compared with before drinking (week 0), p <0.01.

Figure 14 is another data diagram of the human body efficacy of the fermented tea leaves of the present invention, where "**" indicates that compared with before drinking (week 0), p <0.01.

Figure 15 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention, where "*" indicates that compared with before drinking (week 0), p <0.05.

Figure 16 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention, where "**" indicates that compared with before drinking (week 0), p <0.01.

Fig. 17 is another data graph of the human body efficacy of the fermented product of tea leaves of the present invention.

Fig. 18 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention.

definition

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%.

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.

According to the invention, tea (Camellia sinensis) is a Theaceae (Theaceae) Camellia (Camellia) and is a perennial evergreen woody plants, deciduous shrub or tree. In tropical regions, there are also tree-type tea trees as high as 15-30 meters, and the base tree circumference is more than 1.5 meters. Cultivated tea trees are often pruned to inhibit vertical growth, so the tree height is usually between 0.8 and 1.2 meters.

As used herein, the term "anti-aging" means to prevent and slow down the appearance of aging of human skin, such as the generation of wrinkles and loss of elasticity. The evaluation of the extent to which this goal is achieved will be determined based on many factors known to those who are familiar with the art, such as the general state of the consumer, age, gender, etc.

According to the present invention, the operating procedures and parameter conditions related to fermentation culture fall within the scope of professionalism and routine technology of those who are familiar with this technology.

As used herein, the terms " Saccharomyces cerevisiae ", " Bifidobacterium lactis ", " Lactobacillus gasseri " and " Gluconacetobacter xylinus " It is intended to cover those saccharomyces cerevisiae, Leiter B bacteria, Lactobacillus gasseri, and lignoacetic acid bacteria that can be easily obtained by those skilled in the art (for example, those that can be purchased from domestic or foreign depository institutions), or use Strains of Saccharomyces cerevisiae, Rettler B, Lactobacillus gasseri, and Lignoacetic acid bacteria isolated and purified from natural sources by the usual microbial isolation method used in this art.

As used in this article, the terms "inhibition of melanogenesis" and "inhibition of melanin synthesis", "depigmenting", "lightening the melanin", " "Whitening", "skin color lightening", "bleaching", "whitening", "brightening", "blackening" and "blackening" can be used interchangeably .

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenterally or orally administration by using techniques well known to those skilled in the art. This includes, but is not limited to, injections. (injection) [for example, sterile aqueous solution (sterile aqueous solution) or dispersion (dispersion)], sterile powder (sterile powder), lozenge (tablet), tablet (troche), pill (pill), capsule ( capsule) and the like.

According to the present invention, the medicine can be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection, Intravenous injection, sublingual administration, and transdermal administration.

According to the present invention, the medicine may further include a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of 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 professionalism and routine techniques of those who are familiar with this technique.

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

According to the present invention, the composition can be used as a food additive, which is added during the preparation of raw materials by a conventional method, or added during the preparation of food, and is formulated with any edible material for supply 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.

Example 1. Preparation of fermented product of tea tree ( Camellia sinensis) leaves

In a preferred embodiment of the present invention, the tea used in the present invention is Hongyu black tea (purchased from Taiwan Agriculture and Forestry Co., Ltd.). First, mix the tea leaves of Hongyu black tea with water in a ratio of 1:50~125, sterilize and extract them at 50~100℃ for 0.5~3 hours. Then, it is cooled to room temperature for subsequent fermentation. After that, 0.01~0.5% (v/v) Saccharomyces cerevisiae (Saccharomyces cerevisiae) BCRC20271, 0.01~0.25% (v/v) Bifidobacterium lactis (Bifidobacterium lactis) BCRC910887, 0.01~0.25% (v/v) v) Lactobacillus gasseri BCRC910886, and 10-15% (v/v) Gluconacetobacter xylinus BCRC12335 fermentation for 12-25 days (the above strains are all purchased from the Food Industry Development Institute in Taiwan (Food Industry Research and Development Institute, FIRDI) Biosource Collection and Research Center (BCRC)). After that, it was concentrated under reduced pressure at 45°C to 70°C, and filtered with a mesh of 200 to 400 mesh to obtain a fermented product of tea leaves.

Example 2. Evaluation of the effectiveness of fermented tea leaves in reducing fat accumulation

In this example, mouse bone marrow stromal cell OP9 was used to test the effect of the fermented product of tea leaves of the present invention in reducing fat accumulation. The mouse bone marrow stromal cell OP9 line was purchased from the American Type Culture Collection (ATCC ^® ), numbered CRL-2749 ^TM . The cells are cultured in pre-adipocyte expansion medium (Pre-adipocyte Expansion Medium) before differentiation, which contains 90% of the minimum essential medium Alpha medium (minimum essential medium alpha medium, purchased from Gibco, USA, 12100-046) , 20% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, USA), and 1% penicillin/streptomycin (Penicillin-streptomycin, purchased from Gibco, USA); and use differentiation medium (Differentiation Medium) Differentiate mouse bone marrow stromal cells, which contains 90% minimum essential medium Alpha medium, 20% fetal bovine serum, and 1% penicillin/streptomycin. The lipids in the cells are stained with Oil Red O reagent (purchased from Sigma, USA), in which a 3 mg/mL Oil Red O stock solution is prepared with 100% isopropanol, and the stock solution is prepared with ddH ₂ O to 60% The role of the solution.

In order to verify that the fermented product of tea leaves of the present invention has the effect of reducing fat accumulation, firstly, mouse bone marrow stromal cells were differentiated into adipocytes. First, 8×10 ⁴ mouse bone marrow stromal cells OP9 and 500 μL of pre-adipocytes were expanded The culture solution was inoculated in a 24-well culture dish and cultured at 37°C for 7 days, with fresh differentiation medium replaced every 3 days. Next, use a microscope (ZEISS) to observe the formation of lipid droplets to ensure that the cells have fully differentiated.

After that, OP9 cells were divided into 2 groups, including a control group and an experimental group. The fermented product of 0.0625% tea leaves was added to the cells of the experimental group, while the cells of the control group were left untreated. Then, the cells of each group were cultured for 7-10 days, and the medium was changed every 3 days.

Next, oil red O was used to stain the lipids in the cells to evaluate whether the fermented product of the tea leaves of the present invention can indeed reduce fat accumulation. PBS) wash the cells twice, then add 1mL of 10% formaldehyde (purchased from Echo chemical, Taiwan, Cat.TG1794-4-0000-72NI) and react at room temperature for 30 minutes to fix the cells, then remove the formaldehyde and use Wash the cells gently with 1mL of PBS twice, then add 1mL of 60% isopropanol (purchased from Echo chemical, Taiwan, PH-3101) to each well of the cells. After reacting for 1 minute, remove the isopropanol and add 1mL The oil red O action solution was reacted at room temperature for 1 hour, then the oil red O action solution was removed and quickly decolorized with 1 mL of 60% isopropanol for 5 seconds, and then photographed and quantified with a microscope. Next, add 100% isopropanol to the stained cells, and place on a shaker to react for 10 minutes to dissolve the oil droplets, and then take 100 μL into a 96-well culture plate, and read each group with a measuring ELISA reader (BioTek) The OD _{510nm is} read to quantify the oil red O.

The results of this example are shown in Figs. 1 and 2. Figure 1 is a data graph showing the effect of fermented tea leaves of the present invention on reducing fat accumulation. Figure 2 is a staining diagram of the effect of fermented tea leaves of the present invention on reducing fat accumulation. It can be seen from Figure 1 and Figure 2 that compared with the control group, the relative fatty oil droplet volume of the experimental group has decreased. Among them, compared with the control group, the relative fatty oil droplet volume of the experimental group was reduced by about 33.8%. The results of this example show that the fermented product of the tea leaves of the present invention has the effect of reducing fat accumulation.

Example 3. Evaluation of the effectiveness of fermented tea leaves in enhancing the expression of anti-aging-related genes

First, the human peripheral blood white blood cell (PBMC) (purchased from ATCC ^® PCS-800-011 ^TM ) was used to test the efficacy of the fermented product of tea leaves of the present invention in enhancing the expression of anti-aging-related genes.

1.5×10 ⁵ human peripheral blood white blood cells were cultured in a six-well culture dish containing 2 mL of X-VIVOTM 10 cell culture. Next, the cultured cells were divided into a control group and an experimental group. The fermented product of 0.5μg/mL tea leaves was added to the cells of the experimental group, and the culture solution was added to the cells of the control group. After 6 hours of treatment of the cells of each group, the cells of each group were recovered with cell lysate (purchased from Geanaid Company, Taiwan) and used for gene expression analysis.

In this example, the genes used to analyze anti-aging related genes include the chaperonin containing TCP1 subunit 2 ( CCT2 ) gene, and the chaperonin containing TCP1 subunit 5 ( CCT5 ) gene. Gene, chaperonin containing TCP1 subunit 8, CCT8 gene, PTEN-induced kinase 1, Pink1 gene, and poly[ADP-ribose] polymerase 2 (Poly [ADP-ribose] polymerase 2, PARP2 ) gene.

Use RNA extraction reagent kit (purchased from Geneaid Company, Taiwan) to collect RNA in each group of cells, and then use NanoString nCounter (purchased from NanoString Company, USA) to use 75ng of extracted RNA as a template, with customized barcode (barcode) ) (157 genes in total) for direct digital detection (direct digital detection) direct quantification, expression of each gene, quality control (Quality Control, QC), background value correction, data normalization (data normalization), ratio (ratios) ), fold change (fold-changes), differences in expression levels (differential expression) is automatically calculated by nSolver ^TM analysis software (nSolver ^TM analysis Software) (Version 4.0), to change the performance of multiple standard deviations were calculated using STDEV formula Excel software of, and A one-tailed Student’s t test was used in Excel to analyze whether there are statistically significant differences. The results of this example are shown in FIG. 3.

Fig. 3 is a data diagram showing the utility of the fermented product of the tea leaves of the present invention in enhancing the expression of anti-aging-related genes. It can be seen from Figure 3 that whether it is CCT2 gene, CCT5 gene, CCT8 gene, Pink1 gene or PARP2 gene, compared with the control group, the relative expression of genes measured in the experimental group has been significantly improved. The results of this example show that the fermented product of tea leaves of the present invention can achieve anti-aging effects by enhancing the expression of anti-aging-related genes.

Example 4. Evaluation of the effectiveness of fermented tea leaves in reducing the expression of genes related to melanin production

In this example, human peripheral blood leukocyte cells (PBMC) were used to test the efficacy of the fermented product of tea leaves of the present invention in reducing the expression of melanin production-related genes. The experimental procedure is roughly the same as that described in Example 3, except that it is used to analyze melanin production-related genes as microphthalmia-associated transcription factor ( MITF ) genes. The results of this example are shown in FIG. 4.

Figure 4 is a data diagram showing the effectiveness of the fermented product of tea leaves of the present invention in reducing the expression of genes related to melanin production. It can be seen from Figure 4 that compared with the control group, the relative expression of MITF gene measured in the experimental group has a significant decrease. The results of this example show that the fermented product of the tea leaves of the present invention can achieve the whitening effect by reducing the expression of genes related to melanin production.

Example 5. Evaluation of the utility of fermented tea leaves in enhancing the expression of DNA repair-related genes

In this example, human peripheral blood leukocyte cells (PBMC) were used to test the efficacy of the fermented product of the tea leaves of the present invention in enhancing the expression of genes related to DNA repair. The experimental procedure is roughly the same as that described in Example 3, except that it is used to analyze DNA repair related genes including uracil DNA glycosylase ( UNG ) gene and ERCC cut-off repair 6, internal nucleic acid Enzyme non-catalytic subunit (ERCC excision repair 6, endonuclease non-catalytic subunit, ERCC6 ) gene. The results of this example are shown in Figure 5.

Fig. 5 is a data diagram showing the utility of the fermented product of tea leaves of the present invention in enhancing the expression of genes related to DNA repair. It can be seen from Figure 5 that whether it is the UNG gene or the ERCC6 gene, compared with the control group, the relative expression of the gene measured in the experimental group has been significantly improved. The results of this example show that the fermented product of the tea leaves of the present invention can achieve anti-aging effects by enhancing the expression of genes related to DNA repair.

Example 6. Evaluation of the utility of the fermented product of tea leaves in enhancing the expression of telomerase RNA component (TERC) gene

In this example, human peripheral blood leukocyte cells (PBMC) were used to test the efficacy of the fermented product of tea leaves of the present invention in enhancing the expression of TERC gene. The experimental procedure is roughly the same as that described in Example 3, except that the gene used for analysis is the TERC gene. The results of this example are shown in FIG. 6.

Fig. 6 is a data diagram showing the utility of the fermented product of the tea leaves of the present invention in enhancing the expression level of the TERC gene. It can be seen from Figure 6 that compared with the control group, the relative expression of the TERC gene measured in the experimental group has been significantly improved. The results of this example show that the fermented product of the tea leaves of the present invention can achieve the anti-aging effect by enhancing the expression of the TERC gene.

Example 7. Evaluation of the effectiveness of fermented tea leaves in reducing the expression of genes related to inducing immune response

In this example, human peripheral blood leukocyte cells (PBMC) were used to test the efficacy of the fermented product of tea leaves of the present invention in reducing the expression of genes related to inducing immune response. The experimental procedure is roughly the same as that described in Example 3, but the difference is that it is used to analyze the genes involved in inducing immune response, including CD40 gene, tubulin, light chain, and type 2 LC8 (Dynein, Light Chain, LC8-Type). 2, DYNLL2 ) gene, G Protein-Coupled Receptor Kinase 5 ( GRK5 ) gene, RELB gene, tumor necrosis factor superfamily member 14 (tumor necrosis factor superfamily member 14, TNFSF14 ) gene, mediator Interleukin-4 receptor ( IL4R ) gene, and RELA gene. The results of this example are shown in FIG. 7.

Fig. 7 is a data diagram showing the utility of the fermented product of tea leaves of the present invention in reducing the expression level of genes related to inducing immune response. It can be seen from Figure 7 that whether it is CD40 gene, DYNLL2 gene, GRK5 gene, RELB gene, TNFSF14 gene, IL4R gene or RELA gene, compared with the control group, the relative expression of the gene measured in the experimental group has a significant decrease. The results of this example show that the fermented product of the tea leaves of the present invention can achieve the effect of enhancing immunity by reducing the expression of genes related to inducing immune response.

Example 8. Human body efficacy test of fermented tea leaves

First, recruit 8 subjects to drink a bottle of fermented tea leaves of the present invention every day at a dose of 10 mL/day for 4 weeks. Weight, waist circumference, and body fat were measured before and on the 4th week after drinking. And skin phase measurement. The subjects did not make dietary adjustments. The conditions of the subjects were 24≦BMI<27; body fat: male>25%, female>30%, female and male between 20 and 55 years old. Testing items include: 1. Body weight, waist circumference and body fat percentage; 2. Skin (wrinkles, texture, transepidermal water loss (TEWL), water content and elasticity); 3. Questionnaire. The results of this example are shown in Figures 8 to 18 and Table 1.

Fig. 8 is a data chart of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Fig. 8 that compared with before drinking (week 0), the weight of the subject decreased by 0.9 kg in the 4th week after drinking.

Fig. 9 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Fig. 9 that compared with before drinking (week 0), the body mass index (BMI) of the subjects at the 4th week after drinking decreased by 0.3.

Fig. 10 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Figure 10 that compared with before drinking (week 0), the body fat percentage of the subjects decreased by 0.5% in the 4th week after drinking.

Fig. 11 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Figure 11 that compared with before drinking (week 0), the body fat rate of the subjects' trunk decreased by 0.6% in the fourth week after drinking.

Fig. 12 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Fig. 12 that compared with before drinking (week 0), the waist circumference of the subject decreased by 3.8 cm in the 4th week after drinking.

Fig. 13 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Figure 13 that compared with before drinking (week 0), the subject's transdermal water loss (TEWL) decreased by 16.1% in the 4th week after drinking.

Fig. 14 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Fig. 14 that compared with before drinking (week 0), the moisture content of the skin of the subjects increased by 12.7% in the 4th week after drinking.

Fig. 15 is another data chart of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Fig. 15 that compared with before drinking (week 0), the skin elasticity of the subjects increased by 19.5% in the 4th week after drinking.

Fig. 16 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Fig. 16 that compared with before drinking (week 0), the subjects’ skin wrinkles were reduced by 23.9% in the 4th week after drinking.

Fig. 17 is another data graph of the human body efficacy of the fermented product of tea leaves of the present invention. It can be seen from Fig. 17 that compared with before drinking (week 0), the skin texture of the test subjects improved by 10.9% in the 4th week after drinking.

Fig. 18 is another data diagram of the human body efficacy of the fermented product of tea leaves of the present invention. Table 1 shows the improvement of the body weight, body fat, waist circumference, lower abdomen or hip circumference of the fermented product of the tea leaves of the present invention Effect. It can be seen from Figure 18 and Table 1 that compared with before drinking (week 0), the improvement effect of subjects on body weight, body fat, waist circumference, lower abdomen or hip circumference reached 50~87.5% in the 4th week after drinking.

The results of this example show that the fermented product of the tea leaves of the present invention can reduce weight, lower BMI, lower body fat percentage, lower trunk body fat percentage, reduce waist circumference, reduce TEWL, increase skin moisture content, increase skin elasticity, and reduce skin The effect of wrinkles and skin texture improvement, thereby achieving the effect of anti-aging and reducing fat accumulation.

In summary, the fermented product of tea leaves of the present invention can be regulated by CCT gene, Pink1 gene, PARP2 gene, MITF gene, UNG gene, ERCC6 gene, TERC gene, CD40 gene, DYNLL2 gene, GRK5 gene, RELB gene, TNFSF14 Gene, IL4R gene and RELA gene expression levels to achieve anti-aging, whitening, improve immunity and reduce fat accumulation. In addition, the fermented product of the tea leaves of the present invention also has the effects of increasing the moisture content of the skin, elasticity, and anti-wrinkle smoothing texture. Experiments have also confirmed that the fermented product of the tea leaves has the effects of slimming and beautifying skin, while effectively reducing body fat and weight, reducing waist circumference, and It can moisturize and anti-wrinkle, achieve slim and beautiful effect.

The above description is only illustrative, and not restrictive. Any equivalent modifications or alterations that do not depart from the spirit and scope of the present invention shall be included in the scope of the appended patent application.

Claims (12)

A fermented product of tea tree ( Camellia sinensis ) leaves is used to prepare a chaperonin containing T-complex protein 1 subunit alpha (TCP1) complex, CCT ) gene that regulates the T-complex protein 1 subunit alpha (TCP1) complex , The first type PTEN-induced kinase (PTEN-induced kinase 1, Pink1 ) gene, poly[ADP-ribose] polymerase 2 (Poly[ADP-ribose] polymerase 2, PARP2) gene, microphthalmia- related transcription factor (Microphthalmia- associated transcription factor ( MITF ) gene, uracil DNA glycosylase ( UNG ) gene, ERCC excision repair 6, endonuclease non-catalytic subunit ( ERCC6 ) Gene, telomerase RNA component ( TERC ) gene, CD40 gene, tubulin, light chain, type 2 LC8 (Dynein, Light Chain, LC8-Type 2, DYNLL2 ) gene, G protein coupling accepts G Protein-Coupled Receptor Kinase 5 ( GRK5 ) gene, transcription factor RelB ( RELB ) gene, tumor necrosis factor superfamily member 14 (tumor necrosis factor superfamily member 14, TNFSF14 ) gene, interleukin-4 receptor The use of the composition of the expression level of the body (Interleukin-4 receptor, IL4R ) gene and the transcription factor P65 ( RELA ) gene, wherein the fermented product of the tea leaves is prepared by a method including the following steps: A tea leaf extract is obtained by solvent extraction of the tea leaves, and then a Saccharomyces cerevisiae , a Bifidobacterium lactis , a Lactobacillus gasseri and a Gluconacetobacter xylinus ) simultaneously fermenting the tea leaf extract to obtain the fermented product of the tea leaf, wherein the solvent is water, alcohol, or a mixture of alcohol and water. The use described in item 1 of the scope of patent application, wherein the CCT gene is a chaperonin containing TCP1 subunit 2, CCT2 gene, and a chaperonin containing TCP1 subunit 5 (chaperonin containing TCP1 subunit 5). , CCT5 ) gene or chaperonin containing TCP1 subunit 8, CCT8 gene. The use as described in item 1 of the scope of patent application, wherein the expression levels of the CCT gene, the Pink1 gene, the PARP2 gene, the UNG gene, the ERCC6 gene, and the TERC gene are up-regulated, the MITF gene, the CD40 The expression levels of the gene, the DYNLL2 gene, the GRK5 gene, the RELB gene, the TNFSF14 gene, the IL4R gene, and the RELA gene are down-regulated. A fermented product of tea tree ( Camellia sinensis ) leaves is used to prepare a composition for reducing fat accumulation, anti-aging, whitening and improving immunity, wherein the fermented product of tea leaves is prepared by a method including the following steps Obtained: A tea leaf extract is obtained by extracting tea leaves with a solvent, and then a Saccharomyces cerevisiae , a Bifidobacterium lactis , a Lactobacillus gasseri and a wood Acetic acid bacteria ( Gluconacetobacter xylinus ) simultaneously ferment the tea leaf extract to obtain the fermented product of the tea leaf, wherein the solvent is water, alcohol, or a mixture of alcohol and water. The use described in item 4 of the scope of patent application, wherein the whitening is to inhibit the production of melanin. The use described in item 4 of the scope of patent application, wherein the anti-aging is to enhance telomerase activity, enhance DNA repair ability, enhance skin moisture content, enhance skin elasticity, and reduce skin wrinkles. For the use described in item 1 or item 4 of the scope of patent application, the ratio of the solvent to the tea leaves is 50~125:1 (w/w), and the extraction step is performed at 50~100°C. For the use described in item 1 or item 4 of the scope of the patent application, the concentration of the beer yeast is between 0.01 and 0.5% (v/v), and the concentration of the Rettler B bacteria is between 0.01 and 0.25% ( v/v), the concentration of Lactobacillus gasseri is between 0.01 and 0.25% (v/v), and the concentration of the lignoacetic acid bacteria is between 10 and 15% (v/v). The use according to item 1 or item 4 of the scope of patent application, wherein the fermentation time of the saccharomyces cerevisiae, the Rettler B bacterium, the Lactobacillus gasseri, and the lignoacetic acid bacterium is 12 to 25 days. Such as the use described in item 1 or item 4 of the scope of patent application, wherein the tea leaf is a red jade black tea leaf. For the use described in item 1 or item 4 of the scope of the patent application, the effective concentration of the fermented product of the tea leaves is at least 0.5 μg/mL. Such as the use described in item 1 or item 4 of the scope of patent application, wherein the composition is a medicine, a food product or a skin care product.

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