TWI734058B - Use of chimonanthus salicifolius extract for delaying skin cell aging - Google Patents

Abstract

The present invention provides a use of Chimonanthus salicifolius extract for enhancing the gene expression of Chaperonin containing TCP1, PTEN-induced putative kinase 1, Parkinson juvenile disease protein, Autophagy-related gene, Sirtuin 1, Glutamine-dependent NAD⁺ synthetase, Mitochondrial ribosomal protein S5, and/or Ubiquitin-like protein. The Chimonanthus salicifolius extract can effectively delay skin and systemic aging, maintain mitochondrial activity and normal metabolism, and increase the concentration of NAD+ in cells, and has strong anti-glycation activity. The Chimonanthus salicifolius extract is prepared by extracting Chimonanthus salicifolius using water, alcohols, or mixtures of water and alcohols as solvents.

Description

Use of Chimonanthus praecox extract for delaying skin cell aging

The present invention relates to the use of Chimonanthus chinensis extract, in particular to the use of Chimonanthus chinensis extract for preparing and enhancing the expression of CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, NADSYN gene, MRPS5 gene, and Ubl gene. The amount of use of the composition.

In biology and medicine, aging refers to the phenomenon that the physiological state of an organism deteriorates over time. Aging will cause the decline or weakening of the functions of various parts of the body’s tissues or organs, and make the health of the organism worse. Eventually lead to the death of organisms, modern people's lives are under great pressure, and the accumulation of long-term stress will accelerate the aging of body functions, and many chronic diseases will also be derived.

In addition, with the rising awareness of health care and the advancement of medical technology, the average life expectancy has increased, but the functional degradation caused by aging is still aggravated with age. Therefore, in addition to the treatment and prevention of diseases, the prevention of aging is also an important aspect of the elderly society. One of the important topics.

However, although many genes related to aging and related mechanisms have been studied, such as the SIRT1 gene called longevity gene, the protein compiled by it is an important NAD ⁺ -dependent deacetylation in mammals. Enzymes are involved in many important physiological and pathological processes in the human body; in addition, the length of telomeres has also been confirmed to be related to the lifespan of an individual. However, there are still no drugs or methods that can be effectively used to delay aging. Most of them can only recommend intake of foods with antioxidant active substances from the diet, and maintain proper exercise and maintain a normal routine and mood.

Based on the above, in order to achieve the effect of delaying aging conveniently and effectively, we have developed a method that can effectively enhance the performance of anti-aging genes, regulate the performance of NAD ⁺ related genes, regulate the performance of genes related to mitochondrial activity, and improve the perception of cognitive function. The combination with executive ability is really necessary.

For this reason, one of the objectives of the present invention is to provide a Chimonanthus praecox extract for preparing Chaperonin containing TCP1 ( CCT ) genes, PTEN-induced putative kinase 1, Pink1 genes, Parkinson's disease juvenile protein (Parkinson juvenile disease protein, Parkin ) gene, autophagy-related gene ( Atg ) gene, anti-aging enzyme 1 (Sirtuin 1, SIRT1 ) gene, glutamine-dependent tobacco Glutamine-dependent NAD ⁺ synthetase ( NADSYN ) gene, mitochondrial ribosomal protein S5 ( MRPS5 ) gene, and/or Ubiquitin-like protein protein, Ubl ) gene expression composition, wherein the Chimonanthus praecox extract is obtained by extracting a Chimonanthus praecox with a solvent, and the solvent is water, alcohol, or a mixture of alcohol and water. .

Another object of the present invention is to provide a use of Chimonanthus chinensis extract for preparing a composition for delaying aging, wherein the Chimonanthus chinensis extract is obtained by extracting a Chimonanthus chinensis with a solvent, and the solvent is water and alcohol. , Or a mixture of alcohol and water.

In an embodiment of the present invention, the weight ratio of the solvent to the Chimonanthus praecox is 10-20:1-5, and the extraction temperature is 50-100°C.

In another embodiment of the present invention, the Chimonanthus praecox extract enhances the expression level of CCT gene and/or Atg gene in a monocyte.

In another embodiment of the present invention, the Chimonanthus chinensis extract enhances the CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, NADSYN gene, MRPS5 gene, and/or Ubl gene in a skin cell. Expressiveness.

In another embodiment of the present invention, the CCT gene comprises a Chaperonin containing TCP1 subunit 2, CCT2 gene, and a Chaperonin containing TCP1 subunit 2 (Chaperonin containing TCP1 subunit 2, CCT2) gene. subunit 5, CCT5 ) gene, Chaperonin containing TCP1 subunit 6A ( CCT6A ) gene, Chaperonin containing TCP1 subunit 7, CCT7 gene, TCP1 subunit 7 A group consisting of Chaperonin containing TCP1 subunit 8, CCT8 genes and any combination thereof; the Parkin gene is the Parkinson juvenile disease protein 1 (Parkinson juvenile disease protein 1, Parkin1 ) gene; and Ubl Gene is Ubiquitin-like protein 5 (Ubiquitin-like protein 5, Ubl-5 ) gene; and Atg gene includes autophagy-related gene 1 (Autophagy-related gene 1, Atg1 ) gene, autophagy-related gene 8 (Autophagy-related gene 8, Atg8 ) gene and any combination thereof.

In another embodiment of the present invention, the Chimonanthus chinensis extract has anti-glycation activity, can increase a mitochondrial activity, and/or regulate an NAD ⁺ activity; and the concentration of the Chimonanthus chinensis extract is At least 0.125mg/mL.

The Chimonanthus praecox extract of the present invention can effectively increase the expression levels of CCT2 gene, CCT6A gene, CCT7 gene, and Atg8 gene related to anti-aging and rejuvenation in peripheral blood mononuclear cells, and can effectively delay the aging of whole body cells and reach the whole body. Anti-aging effect, and enhance the mitochondrial activity of whole body cells, so that whole body cells maintain normal operation and prevent cell senescence. At the same time, the Chimonanthus praecox extract of the present invention can also effectively increase the CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg8 gene, SIRT1 gene, related to anti-aging and rejuvenation in skin cells. The expression levels of NADSYN gene, MRPS5 gene, and Ubl-5 gene can effectively delay the aging of skin cells, enhance the activity of mitochondria in skin cells, and maintain the normal metabolic function of mitochondria to promote skin repair, Prevent the death of skin cells and extend the life of skin cells. In addition, the Chimonanthus chinensis extract of the present invention also has potent activity in inhibiting glycation reaction, which helps delay the progression of chronic diseases and aging. Therefore, the Chimonanthus chinensis extract of the present invention can be used to prepare a composition for enhancing the expression of anti-aging-related genes and/or delaying aging, wherein the composition is a medicine, a food or a skin care product. , Can be administered to a body by oral administration, skin application, etc.

The following will further illustrate the implementation 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 who is 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 protection scope of the present invention shall be subject to those defined by the attached patent scope.

Fig. 1 is a bar graph showing the increase of CCT gene expression in monocytes by the extract of Chimonanthus praecox according to an embodiment of the present invention. **p value<0.01; ***p value<0.001.

Fig. 2 is a bar graph showing the increase of Atg8 gene expression in monocytes by the extract of Chimonanthus praecox according to an embodiment of the present invention. ***p<0.001.

Fig. 3 is a bar graph showing the enhancement of CCT gene expression in skin cells by the Chimonanthus chinensis extract according to an embodiment of the present invention. *p<0.05, ***p<0.001.

Fig. 4 is a bar graph showing the expression of Pink1 gene, Parkin gene, and Atg8 gene in skin cells enhanced by Chimonanthus chinensis extract according to an embodiment of the present invention. *p<0.05, **p<0.01.

Fig. 5 is a bar graph showing the improvement of the expression of SIRT1 gene in skin cells by the Chimonanthus praecox extract according to an embodiment of the present invention. *p<0.05.

Fig. 6 is a bar graph showing the increase of NADSYN gene, MRPS5 gene, and Ubl-5 gene expression in skin cells by the extract of Chimonanthus praecox according to an embodiment of the present invention. **p<0.01, ***p<0.001.

Fig. 7 is a bar graph showing the anti-glycation activity of the Chimonanthus chinensis extract according to an embodiment of the present invention.

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.

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%.

Chimonanthus salicifolius (Chimonanthus salicifolius) is a deciduous or evergreen shrub of the Chimonanthus family (Calycanthaceae) Chimonanthus. It is produced in Xiushui and Guangfeng, Jiangxi, China. The branchlets are square to nearly cylindrical; the leaves are paired It is raw, papery or nearly leathery, and the leaf surface is rough with pinnate veins; the flowers are yellow or yellow-white with purple-red stripes, and the flowering period is from August to October each year.

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenterally or topically by using techniques well known to those skilled in the art. This includes, but is not limited to: injections (injection) [e.g., sterile aqueous solution or dispersion], sterile powder, external preparation, and the like.

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 includes 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 drug can be administered by a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, intradermal injection Injection (intradermal injection) and intralesional injection (intralesional injection).

According to the present invention, the medicine can be manufactured into an external preparation suitable for topical application to the skin using techniques well-known to those skilled in the art. This includes, but is not limited to: emulsion, coagulation Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, milk Serum, paste, foam, drop, suspension, salve and bandage.

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

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 (white petrolatum,)], wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers (absorption enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ^® 974P (carbopol ^® 974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)] , Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusion Occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants (Propellants) and so on. The selection and quantity of these additives fall within the scope of professionalism and routine technology of those who are familiar with this technology.

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

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

According to the present invention, skin care products can also be used in combination with one or more external use agents with known activities selected from the following: whitening agents (such as tretinoin, catechins) (catechin), kojic acid, arbutin and vitamin C), moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers 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 ( Antiaging agents, antiwrinkle agents, antiseborrheic agents, wound-healing agents, corticosteroids and hormones. The selection and quantity of these topical agents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, a food product can be used as a food additive, which can be added during the preparation of raw materials by a conventional method, or added during the production process of the food, and 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.

The present invention provides a Chimonanthus Willow extract for preparing and enhancing CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, NADSYN gene, MRPS5 gene, and Ubl gene and delaying aging use. The Chimonanthus Willow extract of the present invention The substance is obtained by extracting a waxy chinensis with a solvent, the solvent is water, alcohol, or a mixture of alcohol and water, which can be used to enhance the CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 in monocytes or skin cells Gene, Pink1 gene, Parkin gene, Atg8 gene, SIRT1 gene, NADSYN gene, MRPS5 gene, and/or Ubl-5 gene expression, and reduce the formation of glycation end products, so as to achieve the body's systemic anti-aging effect.

At the same time, the present invention is used to prepare a composition for enhancing the expression levels of CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, NADSYN gene, MRPS5 gene, and Ubl gene, and may also include an effective amount of Chimonanthus praecox extract And a pharmaceutically acceptable carrier, the composition is a medicine, a food or a skin care product.

The following will describe in detail the extraction method of the Chimonanthus praecox extract of the present invention, and the test of the Chimonanthus praecox extract in enhancing the expression of CCT2 gene, CCT6A gene, CCT7 gene, and Atg8 gene in peripheral blood mononuclear cells; and The Willow Leaf Chimonanthus extract is used to enhance the expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg8 gene, SIRT1 gene, NADSYN gene, MRPS5 gene, and Ubl-5 gene in skin cells And the use of in vitro anti-glycation test tube experiments to test the anti-glycation activity of the Chimonanthus praecox extract of the present invention to confirm that the Chimonanthus praecox extract is effective in enhancing CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, NADSYN gene, MRPS5 gene, and/or Ubl gene expression level, and the effect of reducing the formation of glycation end products.

Example 1 Preparation method of Chimonanthus chinensis extract of the present invention

In an embodiment of the present invention, the leaves of Chimonanthus praecox and the extraction solvent of water, alcohol, or alcohol-water mixture are mixed at a solid-liquid ratio of 1-5:10-20. The extraction solvent is preferably water. After homogenization After extraction in a solvent at 50-100°C for 0.5-3 hours, a crude extract is obtained. After the crude extract is cooled to room temperature, it is filtered with a 400 mesh filter to obtain a filtrate. Finally, the filtrate is concentrated under reduced pressure at 45-70°C to obtain the Chimonanthus chinensis extract of the present invention. In another embodiment of the present invention, the extraction solvent of the Chimonanthus chinensis leaves and water mixture is mixed at a solid-to-liquid ratio of 1:20, and after homogenization, the extraction is performed in the solvent at 100°C for 0.5 hours to obtain a crude extraction. After cooling the crude extract to room temperature, filter with a 400 mesh filter to obtain a filtrate. Finally, the filtrate is concentrated under reduced pressure at 45-70°C to obtain the Chimonanthus chinensis extract of the present invention.

Example 2 The effect of the Chimonanthus praecox of the present invention in enhancing the expression of systemic anti-aging genes

In the embodiment of the present invention, human peripheral blood mononuclear cell (PBMC) is used to test the ability of the Chimonanthus praecox extract of the present invention to regulate the expression of aging-related genes in cells, wherein the cell line is derived from fresh blood. It was isolated from, and cultured in X-VIVO ^TM 10 Serum-free hematopoietic cell medium (purchased from Lonza, Switzerland, number BE02-055Q). First, the above-mentioned human peripheral blood mononuclear cells were cultured in a six-well culture dish containing 2 mL of the above-mentioned culture medium at an amount of ^{1×10 5 cells per well, and cultured at 37°C for 16 hours, and then the cells were divided into the following two} Group: (1) Control group containing only the above cell culture solution, (2) Experimental group added with 0.25mg/mL Chimonanthus praecox extract of the present invention, and after treating the cells for 6 hours, the cells were recovered with cell lysate After the cells, use the RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No.FC24015-G) to collect the RNA in the two groups of human peripheral blood mononuclear cells, and then use SuperScript ^® III reverse transcriptase (purchased from Invitrogene Corporation, United States, No. 18080-051) Use 2000ng of extracted RNA as a template to perform reverse transcription with the primers in Table 1 to generate the corresponding cDNA, and then use the ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific, United States ), and KAPA SYBR FAST (purchased from Sigma Company, U.S., No. 38220000000). The two sets of reverse transcription products were respectively subjected to quantitative real-time reverse transcription polymerase chain reaction with the combination primers in Table 1. ) Test, the conditions are 95°C for 1 second, 60°C for 20 seconds, a total of 40 cycles. It is used to quantify the mRNA expression level of CCT2 gene, CCT6A gene, CCT7 gene, and Atg8 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), in which, since blood circulates throughout the body, human peripheral blood mononuclear spheres are used as the experimental cell line Anti-aging gene testing is designed to extend to the systemic effect. 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).

CCT gene is a kind of Chaperonin. Its main function is to correct misfolded proteins and send the proteins that cannot be repaired successfully into the proteasome for hydrolysis. Previous studies have pointed out that if this protein regulates Failure of the repeated use system will cause misfolded proteins to accumulate in the cells, causing cell function decline and accelerating the aging and death of cells, so it is considered to be related to the aging regulation of the individual.

The Atg gene is an autophagy-related gene, which participates in the degradation of waste products in the cell and the autophagy of circulation, especially to remove the mutated DNA in the mitochondria and restore the normal metabolic function of the mitochondria. And previous studies have pointed out that the overexpression of this gene can prolong the lifespan of mice, so it is considered to be related to the aging effect of cells.

The results of the experiment that the Chimonanthus chinensis extract of the present invention enhances the expression of CCT2 gene, CCT6A gene, and CCT7 gene in human peripheral blood mononuclear cells are shown in FIG. 1. After human peripheral blood mononuclear cells were treated with the Chimonanthus praecox extract of the present invention, the expression levels of CCT2 gene, CCT6A gene, and CCT7 gene were about 1.09 times, 1.25 times, 1.8 times, and 1.28 times that of the control group, respectively, and The expression level of CCT6A gene and CCT7 gene was significantly higher than that of the control group. This result shows that the Chimonanthus praecox extract of the present invention can effectively increase the expression of CCT2 , CCT6A , and CCT7 genes related to anti-aging and rejuvenation in peripheral blood mononuclear cells, and can effectively delay cell aging and achieve systemic effects. It has anti-aging effects and has the potential to restore mature cells to the state of stem cells.

The experimental results of the Chimonanthus praecox extract of the present invention in enhancing the expression of Atg8 gene in human peripheral blood mononuclear cells are shown in FIG. 2. After the human peripheral blood mononuclear cells were treated with the Chimonanthus praecox extract of the present invention, the expression level of Atg8 gene was about 1.57 times that of the control group, and was significantly higher than that of the control group. This result shows that the Chimonanthus chinensis extract of the present invention can also effectively increase the expression of Atg8 gene in peripheral blood mononuclear cells, promote the elimination of mutant mitochondrial DNA, and maintain the normal metabolic function of mitochondria. Make cells maintain normal operation and prevent cell senescence.

Example 3 The effect of the Chimonanthus praecox of the present invention in enhancing the expression of anti-aging genes in skin cells

In the embodiment of the present invention, human skin fibroblast (CCD-966sk) was used to test the anti-aging effect of the Chimonanthus chinensis extract of the present invention on the skin, wherein the cell line was purchased from the Food Industry Development Institute (Taiwan) and Numbered as BCRC 60153, this cell line was cultured in Minimum Essential Medium (MEM, purchased from Thermo Fisher Scientific, USA) containing Earle's Balanced Salt Solution (EBSS). First, the above-mentioned human skin fibroblasts were cultured in a six-well culture dish containing 2 mL of the above-mentioned culture medium at a cell amount of ^{1×10 5 cells per well, and cultured at 37°C for 16 hours, and then the cells were divided into the following two groups:} (1) The control group containing only the above-mentioned cell culture solution, (2) The experimental group adding 0.5 mg/mL Chimonanthus praecox extract of the present invention, and after treating the cells separately for 48 hours, the cells were recovered with cell lysate , Use the RNA extraction reagent kit to collect the RNA in the two groups of human skin fibroblasts, and then use the SuperScript ^® III reverse transcriptase to use 2000ng of the extracted RNA as the template to perform reverse transcription with the primers in Table 1 and Table 2 The corresponding cDNA was then used ABI StepOnePlus ^TM Real-Time PCR system and KAPA SYBR FAST to perform quantitative real-time reverse transcription polymerase chain reaction tests with the combination primers in Table 1 and Table 2 for the two sets of reverse transcription products, respectively, under the condition of 95 React at °C for 1 second, and at 60 °C for 20 seconds, a total of 40 cycles. It is used to quantify the mRNA expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg8 gene, SIRT1 gene, NADSYN gene, MRPS5 gene, and Ubl-5 gene. The value is taken from the threshold cycle number (Ct), and the relative amount of target gene mRNA is derived from the equation 2 ^-△Ct , where △Ct=Ct _{target gene-} Ct _GAPDH , and then use Excel software to perform unpaired single-tail 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 SIRT1 gene is an important NAD ⁺ -dependent deacetylase in mammals. Previous studies have pointed out that the decreased expression of the SIRT1 gene will lead to a decrease in the biosynthesis of mitochondria and lead to aging-related diseases. Therefore, this gene It has been recognized as a longevity gene and is inseparable from the effect of aging.

The Pink1 gene is a serine/threonine protein kinase located in the mitochondria. Its function is to protect the mitochondria from malfunctioning during cellular stress. It is known that the mutation of this gene is related to Parkinson's Previous studies have pointed out that the overexpression of this gene can increase the lifespan of mice, so it is also considered to be related to the regulation of individual aging.

Parkin gene is an enzyme that exists in the Ubiquitin-proteasome system, and acts as a regulator of protein breakdown, especially to help break down abnormal mitochondria in cells. It is known that the mutation of this gene is associated with Parkinson's It is related to the disease, and previous studies have also pointed out that increasing the expression of this gene can delay the aging effect of cells, so it is considered to be related to the aging effect of cells.

Previous studies have pointed out that compared with old mice, young mice contain more NAD ⁺ ; and increasing the NAD ⁺ concentration in old mice can make their physiological conditions younger, so NAD ⁺ is It is believed to be related to delaying individual aging, and it is known that SIRT1 gene, NADSYN gene, MRPS5 gene, and UBL-5 gene are related to regulating NAD ⁺ .

The experimental results of the Chimonanthus chinensis extract of the present invention in enhancing the expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, and CCT8 gene in human skin fibroblasts are shown in FIG. 3. After human skin fibroblasts were treated with the Chimonanthus praecox extract of the present invention, the expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, and CCT8 gene were about 1.25 times, 2 times, 1.5 times, and 1.8 times that of the control group, respectively. Times, and 1.9 times, and are significantly higher than the control group. This result shows that the Chimonanthus praecox extract of the present invention can effectively increase the expression of CCT2 , CCT5 , CCT6A , CCT7 , and CCT8 genes related to anti-aging and rejuvenation in skin cells, and can prevent or delay skin cell aging , Make the skin firm and shiny.

The experimental results of the Chimonanthus chinensis extract of the present invention in enhancing the expression of Pink1 gene, Parkin gene, and Atg8 gene in human skin fibroblasts are shown in FIG. 4. After human skin fibroblasts were treated with the Chimonanthus praecox extract of the present invention , the expression levels of Pink1 gene, Parkin gene, and Atg8 gene were about 2 times, 2.1 times, and 10 times that of the control group, and were significantly higher than those of the control group. Control group. This result shows that the Chimonanthus chinensis extract of the present invention can effectively increase the expression of Pink1 gene, Parkin gene, and Atg8 gene related to mitochondrial metabolism in skin cells, and can promote the elimination of mutant mitochondria and maintain The normal metabolic function of mitochondria enables skin cells to maintain normal operation and prevent cell senescence.

The experimental results of the Chimonanthus praecox extract of the present invention in enhancing the expression of SIRT1 gene in human skin fibroblasts are shown in FIG. 5. After the human skin fibroblasts were treated with the Chimonanthus praecox extract of the present invention, the expression level of the SIRT1 gene was about 1.2 times that of the control group, and was significantly higher than that of the control group. This result shows that the Chimonanthus chinensis extract of the present invention can effectively increase the expression level of the longevity gene- SIRT1 gene in skin cells, can effectively promote skin repair, prevent the death of skin cells, and prolong the lifespan of skin cells.

The experimental results of the Chimonanthus praecox extract of the present invention in enhancing the expression of NADSYN gene, MRPS5 gene, and Ubl-5 gene in human skin fibroblasts are shown in FIG. 6. After human skin fibroblasts were treated with the Chimonanthus praecox extract of the present invention, the expression levels of NADSYN gene, MRPS5 gene, and Ubl-5 gene were approximately 2 times, 1.6 times, and 1.6 times that of the control group, and were significantly higher In the control group. This result shows that the Chimonanthus chinensis extract of the present invention can effectively increase the expression levels of the NADSYN gene, MRPS5 gene, and Ubl-5 ^{gene that regulate NAD + in} skin cells, enhance the activity of mitochondria, and prolong the activity of skin cells. life.

Example 4 The Chimonanthus chinensis extract of the present invention has anti-glycation activity

In the embodiment of the present invention, an in vitro anti-glycation test tube experiment was used to test the anti-glycation activity of the Chimonanthus praecox extract of the present invention, in which the efficiency of the glycation of collagen (Collagen) produced by inhibiting D-fructose (D-fructose) was performed. Quantitative; Anti-glycation can inhibit non-enzymatic browning to avoid denaturation of functional proteins in the body. First, take 0.2 mL of the Chimonanthus chinensis extract solution of the present invention with a concentration of 1 mg/mL and 0.2 mL of water as the control solution, and add 0.2 mL of 60 mg/mL collagen solution (with 200 mM sodium phosphate Buffer configuration, pH 7.4) and 0.2 mL of 1.5 M D-fructose solution (configured with 200 mM sodium phosphate buffer, pH 7.4) after mixing uniformly, then take 0.1 mL of the mixed solution to excitation light 360nm, radiation Measure the fluorescence value under the light of 460nm, which is the zero point before the reaction, and after incubating 0.45 mL of the mixed solution at 50°C for 24 hours, take out 0.1 mL to measure the fluorescence value, which is the end of the reaction. And with an equal amount of 3mM Aminoguanidine (AG) (configured with 200mM sodium phosphate buffer) to re-dissolve the solvent to the same volume as a positive control group, in which Aminoguanidine (AG) is known to have the effect of inhibiting glycation. Finally, use the following formula to calculate the efficiency of the ability to remove the final glycation product, which represents the anti-glycation activity of each group of samples.

The test results of the anti-glycation activity of the Chimonanthus chinensis extract of the present invention are shown in FIG. 7. After the action of the Chimonanthus chinensis extract of the present invention, it can reduce the production of the final glycation product by up to 44%. This result shows that the Chimonanthus chinensis extract of the present invention has a strong inhibitory activity of saccharification and helps delay chronic diseases. Progress with aging.

In summary, the Chimonanthus praecox extract of the present invention can effectively increase the expression levels of CCT2 , CCT6A , CCT7 , and Atg8 genes related to anti-aging and rejuvenation in peripheral blood mononuclear cells, and can effectively delay systemic cells. The aging of the body can achieve the effect of systemic anti-aging, and enhance the mitochondrial activity of the cells of the whole body, so that the cells of the whole body can maintain the normal operation and prevent the senescence of the cells. At the same time, the Chimonanthus praecox extract of the present invention can also effectively increase the CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg8 gene, SIRT1 gene, related to anti-aging and rejuvenation in skin cells. The expression levels of NADSYN gene, MRPS5 gene, and Ubl-5 gene can effectively delay the aging of skin cells, enhance the activity of mitochondria in skin cells, and maintain the normal metabolic function of mitochondria to promote skin repair, Prevent the death of skin cells and extend the life of skin cells. In addition, the Chimonanthus chinensis extract of the present invention also has potent activity in inhibiting glycation reaction, which helps delay the progression of chronic diseases and aging. Therefore, the Chimonanthus chinensis extract of the present invention can be used to prepare a composition for enhancing the expression of anti-aging-related genes and/or delaying aging, wherein the composition is a medicine, a food or a skin care product. , Can be administered to a body by oral administration, skin application, etc.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of Chimonanthus praecox extract to delay skin cell aging

<130> N/A

<160> 26

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence

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<213> Artificial sequence

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<213> Artificial sequence

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<213> Artificial sequence

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Claims (7)

A use of a Chimonanthus chinensis extract for preparing a composition for delaying skin aging, wherein the composition achieves the purpose of delaying skin aging by enhancing the activity of mitochondria, and wherein the Chimonanthus chinensis extract is used as a solvent It is obtained by extracting Chimonanthus chinensis, and the solvent is water. The use as described in item 1 of the scope of the patent application, wherein the composition is through the elimination of mutated DNA in mitochondria, inhibiting the reduction of mitochondrial biosynthesis function, protecting mitochondria from malfunctioning or assisting during cell stress Decompose abnormal mitochondria in cells to achieve the purpose of delaying skin cell aging. A use of Chimonanthus chinensis extract for preparing a composition for avoiding the denaturation of functional proteins in the body, wherein the composition is used to prevent the denaturation of functional proteins in the body by inhibiting saccharification, and wherein the Chimonanthus chinensis extract is based on It is obtained by extracting a Chimonanthus chinensis with a solvent, and the solvent is water. The use described in item 3 of the scope of patent application, wherein the composition achieves the purpose of delaying skin cell aging by inhibiting non-enzymatic browning to prevent the denaturation of functional proteins in the body. Such as the use described in item 1 or item 3 of the scope of the patent application, wherein the weight ratio of the solvent to the Chimonanthus praecox is 20:1. For the purposes described in item 1 or item 3 of the scope of patent application, the extraction temperature is 50-100°C. Such as the use described in item 1 or item 3 of the scope of patent application, wherein the composition is a medicine, a food or a skin care product.

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