Use of plant juice to increase the expression of skin anti-aging genes and / or regulate the NAD+ concentration of skin cells

Abstract

The present invention provides a plant juice for increasing the expression of skin anti-aging genes and / or regulating the NAD+ concentration of skin cells, which can comprehensively increase the anti-aging activity of the skin and promote the reversal of the skin to a youthful and energetic state. The plant juice of the present invention is composed of Tianshan Saussurea ovata juice, longan shell juice and buckwheat juice.

Description

[0001] This application is a divisional application of the invention patent application with Chinese national application number 202011310462.5, application date November 20, 2020, and invention name "Use of plant juice to increase the expression of anti-aging genes and / or increase GSH content". Technical Field

[0002] The present invention relates to the use of a plant juice for increasing the expression of skin anti-aging genes and / or regulating the NAD+ concentration of skin cells, and in particular to the use of a plant juice composed of Tianshan Saussurea scabra juice, longan shell juice and buckwheat juice for preparing a composition for increasing the expression of skin TCP1-containing folded protein genes, the expression of autophagy-related genes and the expression of mitochondrial serine / threonine protein kinase genes, and / or regulating the NAD+ concentration of skin cells. Background Art

[0003] In biology and medicine, aging refers to the phenomenon that the physiological state of an organism deteriorates over time. Aging will cause the functional decline or weakening of tissues or organs in various parts of the body, making the health function of the organism worse, and finally leading to the death of the organism. Modern people live under great pressure, and the accumulation of long-term stress will accelerate the aging of body functions and also lead to many chronic diseases.

[0004] In addition, with the rise of health awareness and the advancement of medical technology, life expectancy has increased, but functional degradation caused by aging still increases with age. Therefore, in addition to the treatment and prevention of diseases, the prevention of aging is also one of the important issues in an aging society.

[0005] Although many genes and related mechanisms related to aging have been studied, there are still no effective drugs or methods to delay aging. Most of them can only recommend eating more foods with antioxidant activity, maintaining proper exercise, keeping a normal schedule and a happy mood.

[0006] In summary, in order to conveniently and effectively achieve the effect of delaying aging, it is necessary to develop a composition that can effectively increase the expression of anti-aging genes and antioxidant capacity. Summary of the invention

[0007] Therefore, an object of the present invention is to provide a plant juice for preparing a composition for increasing the expression of skin TCP1-containing folding protein (CCT) gene, autophagy-related gene (Atg) gene and mitochondrial serine / threonine protein kinase (Pink1) gene, and the plant juice is composed of Tianshan Saussurea involucrata juice, longan shell juice and buckwheat juice.

[0008] Another object of the present invention is to provide a plant juice for preparing a composition for regulating the NAD+ concentration of skin cells, and the plant juice is composed of Tianshan Snow Lotus juice, longan shell juice and buckwheat juice.

[0009] In one embodiment of the present invention, the CCT gene is a Chaperonin containing TCP1 subunit 2 (CCT2) gene, a Chaperonin containing TCP1 subunit 5 (CCT5) gene, a Chaperonin containing TCP1 subunit 6A (CCT6A) gene, a Chaperonin containing TCP1 subunit 7 (CCT7) gene, a Chaperonin containing TCP1 subunit 8 (CCT8) gene, or a combination thereof; and the Atg gene is an autophagy-related gene 1 (Atg1) gene, an autophagy-related gene 8 (Atg8) gene, or a combination thereof.

[0010] In yet another embodiment of the present invention, the plant juice has the ability to increase the anti-aging activity of the skin.

[0011] In another embodiment of the present invention, the plant juice is obtained by extracting with water.

[0012] In yet another embodiment of the present invention, the concentration of the plant juice is at least 0.5% (v / v).

[0013] In another embodiment of the present invention, the plant juice is prepared by mixing the Tianshan Saussurea involucrata juice, the longan shell juice, and the buckwheat juice in a ratio of 2.4-3.6:129.6-194.4:128.8-193.2.

[0014] The plant juice of the present invention is composed of Tianshan Saussurea juice, longan shell juice and buckwheat juice; the Tianshan Saussurea juice, the longan shell juice and the buckwheat juice can all effectively increase the expression of genes related to mitochondrial or cellular anti-aging in skin cells. The combination of the three juices can also directly and effectively increase the expression of skin CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Atg1 gene, Atg8 gene and Pink1 gene, which can comprehensively improve the anti-aging activity of the skin, and the plant juice of the present invention can simultaneously regulate the NAD+ concentration of skin cells; the plant juice of the present invention can effectively promote the reversal of the body back to a youthful and energetic state. Therefore, the plant juice of the present invention can be used for the preparation of a composition for skin anti-aging, wherein the composition is a medicine, a food or a skin care product, which can be given to an individual by oral administration, skin application, etc.

[0015] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments, but is not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a histogram showing the effect of the Tianshan Saussurea Juice according to one embodiment of the present invention on increasing the expression levels of the CCT2 gene, the CCT5 gene, and the CCT6A gene in blood cells. *p value <0.05.

[0017] Figure 2 This is a histogram showing the effect of longan shell juice in one embodiment of the present invention on increasing the expression of CCT5 gene, CCT6A gene, CCT8 gene, Pink1 gene, Atg1 gene, Atg8 gene, and FOXO3 gene in blood cells. *p value < 0.05; **p < 0.01; ***p < 0.001.

[0018] Figure 3 This is a histogram showing the effect of buckwheat juice in increasing the expression of CCT2 gene and CCT5 gene in blood cells according to one embodiment of the present invention. *p value <0.05.

[0019] Figure 4 This is a histogram showing the effect of the Tianshan Saussurea Juice in one embodiment of the present invention on increasing the expression levels of CCT6A gene, CCT7 gene, Atg1 gene, Atg8 gene, SIRT1 gene, and FOXO3 gene in skin cells. *p value <0.05; **p <0.01.

[0020] Figure 5This is a histogram showing the effect of longan shell juice in one embodiment of the present invention on increasing the expression of CCT2 gene, CCT5 gene, CCT7 gene, Pink1 gene, Atg1 gene, Atg8 gene, SIRT1 gene, and FOXO3 gene in skin cells. *p value < 0.05; **p < 0.01; ***p < 0.001.

[0021] Figure 6 This is a histogram showing the effect of buckwheat juice in increasing the expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, and Pink1 gene in skin cells according to one embodiment of the present invention. ***p value <0.001.

[0022] Figure 7 This is a histogram of the effect of the Tianshan Saussurea Juice of one embodiment of the present invention on increasing the expression of CCT2 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Atg1 gene, and SIRT1 gene in cardiovascular cells. *p value <0.05; **p <0.01; ***p <0.001.

[0023] Figure 8 This is a histogram showing the effect of longan shell juice in one embodiment of the present invention on increasing the expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Atg1 gene, SIRT1 gene, and FOXO3 gene in cardiovascular cells. *p value < 0.05; **p < 0.01; ***p < 0.001.

[0024] Fig. 9 This is a histogram showing the effect of buckwheat juice in one embodiment of the present invention on increasing the expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Atg1 gene, SIRT1 gene, and FOXO3 gene in cardiovascular cells. *p value < 0.05; **p < 0.01; ***p < 0.001.

[0025] Fig.10 This is a histogram of the effect of longan shell juice and buckwheat juice on increasing the expression of CCTs gene, Pink1 gene, Atg8 gene, SIRT1 gene, or FOXO3 gene in retinal cells according to one embodiment of the present invention. **p<0.01; ***p<0.001.

[0026] Fig.11 This is a histogram showing the effect of the plant juice composition according to one embodiment of the present invention on increasing the expression of CCTs genes. *p<0.05; ***p<0.001.

[0027] Fig.12The histogram is a graph showing the effect of the plant juice composition according to one embodiment of the present invention on increasing the expression of Atg1 gene and Atg8 gene. *p<0.05; ***p<0.001.

[0028] Fig.13 This is a histogram showing the effect of the plant juice composition according to one embodiment of the present invention on increasing the expression levels of the NADSYN gene, the MRPS5 gene, and the SOD3 gene. *p<0.05; ***p<0.001.

[0029] Fig.14 This is a histogram showing the effect of the plant juice composition according to one embodiment of the present invention on increasing the expression of the TERT gene. ***p<0.001.

[0030] Fig.15 The histogram is a graph showing the effect of the plant juice composition according to one embodiment of the present invention on increasing the GSH content. **p<0.01; ***p<0.001. DETAILED DESCRIPTION

[0031] The numerical values ​​used herein are approximate values, and all experimental data are expressed within a range of 20%, preferably within a range of 10%, and most preferably within a range of 5%.

[0032] Statistical analysis was performed using Excel software. Data were expressed as mean ± standard deviation (SD), and differences between individuals were analyzed using student's t-test.

[0033] Smallanthus sonchifolius is a perennial herb of the genus Smallanthus in the family Compositae. It is also known as Peruvian ground apple. Its underground tubers are edible and have the effects of cooling and relieving fever, lowering blood lipids, and helping bowel movements.

[0034] Longan (Euphoria longana Lam.) is an evergreen tree plant of the genus Euphoria in the family Sapindaceae. Its English name is Longan. The root of longan has the effect of clearing away meridians; the leaves have the effect of clearing away heat and detoxifying; the core has the effect of stopping bleeding and relieving pain; the fruit is sweet and has the effect of replenishing qi and blood and calming the nerves; the peel (i.e. the longan shell) has the effect of clearing away heat and cooling blood.

[0035] Buckwheat (Fagopyrum esculentum Moench.) is an annual herbaceous plant of the genus Fagopyrum in the family Polygonaceae. It is also known as triangular wheat and its English name is Common buckwheat. Buckwheat stems and leaves have the effects of stopping bleeding and lowering blood pressure; the seeds have the effects of reducing swelling, detoxifying and reducing fever.

[0036] As used herein, the term "Xuanshuilian juice" means the extract obtained by extracting the callus tissue of Xuanshuilian and a solvent at a ratio of 1:1-5 (w / w) for a specific time and temperature.

[0037] As used herein, the term "longan shell juice" means the juice obtained by extracting longan shell and solvent in a ratio of 1:5-10 (w / w) for a specific time and temperature.

[0038] As used herein, the term "buckwheat juice" means the juice obtained by extracting buckwheat seed coat and solvent at a ratio of 1:10-15 (w / w) for a specific time and temperature.

[0039] As used herein, the term "composition of plant juices" means a composition obtained by mixing the aforementioned Saussurea involucrata juice, the aforementioned Longan shell juice, and the aforementioned Buckwheat juice in a ratio of 2.4-3.6:129.6-194.4:128.8-193.2.

[0040] The present invention provides a use of a plant juice for preparing an anti-aging composition. The plant juice of the present invention is obtained from a snow lotus juice, a longan shell juice, a buckwheat juice, and any combination thereof. The plant juice can be used to increase the expression of genes related to anti-aging, can comprehensively increase the anti-aging activity, and can simultaneously increase the content of GSH, can comprehensively increase the antioxidant activity of humans, so as to reverse the body back to a youthful and energetic state.

[0041] Meanwhile, the anti-aging composition of the present invention may also include an effective amount of plant juice and a pharmaceutically acceptable carrier. The composition is a medicine, a food or a skin care product.

[0042] The following will describe in detail the detailed extraction methods of the Saussurea involucrata juice, Longan shell juice, and Buckwheat juice of the present invention; the efficacy tests of the Saussurea involucrata juice, Longan shell juice, and Buckwheat juice of the present invention in increasing the expression of anti-aging genes in the whole body, skin cells, cardiovascular cells, and / or retinal cells; and the efficacy tests of the plant juice composition of the present invention in increasing the expression of anti-aging genes and increasing the GSH content. This verifies that the plant juice of the present invention can effectively and comprehensively increase anti-aging and antioxidant properties, so as to reverse the body back to a youthful and energetic state.

[0043] Example 1 Preparation method of Tianshan Saussurea ovata juice of the present invention

[0044] In one embodiment of the present invention, the callus tissue of Saussurea involucrata is first mixed with water, alcohol, or an alcohol-water mixture as an extraction solvent, preferably water, at a weight ratio of 1:1-5, homogenized, and then extracted in the solvent at 50-100° C. for 0.5-2 hours, the crude juice is cooled to room temperature, and the crude juice is filtered through a 400-mesh filter to obtain a filtrate, wherein the extraction temperature is preferably 70-100° C. Finally, the filtrate is concentrated under reduced pressure at 45-70° C. to obtain the Saussurea involucrata juice of the present invention.

[0045] Embodiment 2 Preparation method of longan shell juice of the present invention

[0046] In one embodiment of the present invention, firstly, the longan shell tissue is mixed with water, alcohol, or an alcohol-water mixture leaching solvent, preferably water, at a weight ratio of 1:5-10, homogenized, and then leached in the solvent at 50-100° C. for 0.5-2 hours, and then the crude juice is cooled to room temperature and filtered through a 400-mesh filter to obtain a filtrate, wherein the leaching temperature is preferably 70-100° C. Finally, the filtrate is concentrated under reduced pressure at 45-70° C. to obtain the longan shell juice of the present invention.

[0047] Example 3 Preparation method of buckwheat juice of the present invention

[0048] In one embodiment of the present invention, first, the seed coat of buckwheat is mixed with a leaching solvent of water, alcohol, or a mixture of alcohol and water, preferably water, at a weight ratio of 1:10-15, and after homogenization, the mixture is leached in the solvent at 50-100° C. for 0.5-2 hours, and then the crude juice is cooled to room temperature and filtered through a 400-mesh filter to obtain a filtrate, wherein the leaching temperature is preferably 70-100° C. Finally, the filtrate is concentrated under reduced pressure at 45-70° C. to obtain the buckwheat juice of the present invention.

[0049] Example 4 The efficacy of the Tianshan Saussurea Juice, Longan Shell Juice, and Buckwheat Juice of the present invention in increasing the expression of systemic anti-aging genes

[0050] One embodiment of the present invention uses human peripheral blood mononuclear cells (PBMC) to test the efficacy of the Tianshan Saussurea Juice, Longan Shell Juice, and Buckwheat Juice of the present invention in increasing the expression of systemic anti-aging genes. The human peripheral blood mononuclear cells were purchased from the American Type Culture Collection, numbered PCS-800-011 TM , and cultivated in X-VIVOTM 10 Serum-free hematopoietic cell medium (purchased from Lonza, Switzerland, number BE02-055Q).

[0051] First, 1×10 5 PBMC cells were cultured in a 6-well culture dish containing 2 mL of the cell culture medium at 37° C. for 16-18 hours. Then, the cells were divided into the following four groups: (1) a control group to which only the cell culture medium was added, (2) an experimental group to which 0.5% (v / v) of the Saussurea involucrata juice of the present invention was added, (3) an experimental group to which 0.25% (v / v) of the Longan shell juice of the present invention was added, and (4) an experimental group to which 0.125% (v / v) of the Buckwheat juice of the present invention was added. After the cells of the groups were exposed to 37° C. for 24 hours or 48 hours, the expression level of the target gene in the PBMC cells of each group was tested. The Saussurea involucrata juice, the Longan shell juice, and the Buckwheat juice were extracted with water as the solvent. First, PBMC cells were recovered with cell lysis buffer (RBbuffer, purchased from Geneaid, Taiwan, China, Cat No. RBD300), and RNA extraction reagent kit (purchased from Geneaid, Taiwan, China, Cat No. RBD300) was used to collect RNA in the four groups of cells. III reverse transcriptase (purchased from Invitrogene, USA, number 18080-051) was used with 2000 ng of extracted RNA as template, and reverse transcription was performed with the combination of primers and reverse transcriptase in Table 1 to generate cDNA products corresponding to the mRNA of these genes. Then, the four sets of reverse transcription products were subjected to quantitative real-time polymerase chain reaction (qPCR) test with the combination of primers in Table 1 using ABI StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, USA) and KAPA SYBR FAST (purchased from Sigma, USA, number 38220000000), respectively, under the conditions of 95°C for 1 second, 60°C for 20 seconds, and a total of 40 cycles. It is used to quantify the expression of mRNA of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, and UBL-5 gene, wherein the quantitative value is obtained by the threshold cycle number (Ct), and the relative amount of mRNA of the target gene is derived from Equation 2 -ΔΔCt, where ΔC T =C T比较组或实验组目标基因 / 控制组目标基因 -C T GAPDH (Glyceraldehyde-3-phosphate dehydrogenase); ΔΔCT=C T比较组或实验组目标基因 -C T控制组目标基因 ; The fold change of each gene in each group is 2 -ΔΔCt Then, Excel software was used to determine whether the coefficient of variation was statistically significant (*p value < 0.05; **p value < 0.01; ***p value < 0.001).

[0052] Table 1. Combination primers for quantitative real-time reverse transcription polymerase chain reaction

[0053]

[0054]

[0055] The CCT gene is a type of chaperonin, whose main function is to correct misfolded proteins and send proteins that cannot be successfully repaired to the protease complex (Proteasome) for hydrolysis. Previous studies have pointed out that if this protein-regulated reuse system fails, misfolded proteins will accumulate in cells, causing cell function decline and accelerating cell aging and death. Therefore, it is considered to be related to the regulation of individual aging.

[0056] The Pink1 gene is a serine / threonine protein kinase located in the mitochondria. Its function is to protect mitochondria from malfunctioning during cellular stress. Mutations in this gene are known to be associated with Parkinson's disease, and previous studies have shown that excessive expression of this gene can increase the lifespan of mice. Therefore, it is also believed to be related to the regulation of individual aging.

[0057] The Parkin gene is an enzyme present in the ubiquitin-proteasome system and acts as a regulator of protein degradation. Mutations in this gene are known to be associated with Parkinson's disease, and previous studies have also shown that increasing the expression of this gene can delay cell aging. Therefore, it is believed to be related to cell aging.

[0058] The Atg gene is an autophagy-related gene that is involved in the degradation and recycling of waste in cells. Previous studies have shown that excessive expression of this gene can extend the lifespan of mice, so it is considered to be related to cell aging.

[0059] Previous studies have pointed out that young mice contain more NAD+ than old mice; and increasing the NAD+ concentration in old mice can make their physiological condition younger. Therefore, NAD+ is considered to be related to delaying individual aging. Research results show that the SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, UBL-5 gene, and SOD3 gene are related to regulating NAD+.

[0060] The experimental results of the present invention on the effect of the Tianshan Saussurea Juice on increasing the expression of anti-aging genes in blood cells are as follows: Figure 1 As shown. After 48 hours of action of the Tianshan Saussurea juice of the present invention, compared with the control group, the expression of CCT2 gene, CCT5 gene, and CCT6A gene in PBMC cells can be increased by about 1.2-1.4 times, and the expression of other genes is not much different from that of the control group (results not shown), among which the expression of CCT6A gene has a significant difference. This result shows that the Tianshan Saussurea juice of the present invention can effectively increase the expression of CCT2 gene, CCT5 gene, and CCT6A gene in PBMC cells, and can effectively enhance the systemic anti-aging ability.

[0061] The experimental results of the present invention on the longan shell juice increasing the expression of anti-aging genes in blood cells are as follows: Figure 2 As shown. After 24 hours of action of the longan shell juice of the present invention, compared with the control group, the expression of CCT5 gene, CCT6A gene, CCT8 gene, Pink1 gene, Atg1 gene, Atg8 gene, and FOXO3 gene in PBMC cells can be significantly increased by about 1.3-20 times, while the expression of other genes is not much different from that of the control group (results not shown). This result shows that the longan shell juice of the present invention can effectively increase the expression of CCT5 gene, CCT6A gene, CCT8 gene, Pink1 gene, Atg1 gene, Atg8 gene, and FOXO3 gene in PBMC cells, and can effectively enhance the systemic anti-aging ability.

[0062] The experimental results of the buckwheat juice of the present invention on increasing the expression of anti-aging genes in blood cells are as follows: Figure 3 As shown. After 48 hours of the buckwheat juice of the present invention, compared with the control group, the expression of CCT2 gene and CCT5 gene in PBMC cells can be increased by about 1.2 times, and the expression of other genes is not much different from that of the control group (results not shown), among which the expression of CCT2 gene has a significant difference. This result shows that the buckwheat juice of the present invention can effectively increase the expression of CCT2 gene and CCT5 gene in PBMC cells, and can effectively enhance the systemic anti-aging ability.

[0063] Example 5 The efficacy of the Tianshan Saussurea Juice, Longan Shell Juice, and Buckwheat Juice of the present invention in increasing the expression of anti-aging genes in skin cells

[0064] In one embodiment of the present invention, human skin fibroblast CCD-966SK cells were used to test the efficacy of the Tianshan Saussurea Juice, Longan Shell Juice, and Buckwheat Juice of the present invention in increasing the expression of anti-aging genes in skin cells. The CCD-966SK cell line was purchased from the American Type Culture Collection (ATCC, USA) and is numbered CRL-1881, and the cell line was cultured in a culture medium containing 10% fetal bovine serum (Fetal Bovine Serum) and 90% Minimum Essential Medium (MEM) (purchased from Gibco, the United States), which contained 1 mM sodium pyruvate (Sodium pyruvate, purchased from Gibco, the United States), and 1% penicillin / streptomycin.

[0065] First, 1.5×10 5 CCD-966SK cells were cultured in a 6-well culture plate containing 2 mL of the above culture medium in each well, and cultured at 37° C. for 16-18 hours. Then, the cells were divided into the following four groups: (1) a control group to which only the cell culture medium was added, (2) an experimental group to which 0.5% (v / v) of the Saussurea involucrata juice of the present invention was added, (3) an experimental group to which 0.5% (v / v) of the Longan shell juice of the present invention was added, and (4) an experimental group to which 0.125% (v / v) of the Buckwheat juice of the present invention was added. After the cells of these groups were exposed to 37° C. for 24 hours or 48 hours, the expression level of the target gene in each group of CCD-966SK cells was tested. The Saussurea involucrata juice, the Longan shell juice, and the Buckwheat juice were extracted with water as the extraction solvent. Next, the four groups of CCD-966SK cells were recovered with cell lysis buffer, and the mRNA expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, and UBL-5 gene in the four groups of CCD-966SK cells were quantified according to the steps described in Example 4, and the coefficient of variation and whether there was a statistically significant difference were determined using Excel software (*p value <0.05; **p value <0.01; ***p value <0.001).

[0066] The experimental results of the present invention on the effect of the Tianshan Saussurea Juice on the expression of anti-aging genes in skin cells are as follows: Figure 4As shown. After 24 hours of action of the Tianshan Saussurea juice of the present invention, compared with the control group, the expression of CCT6A gene, CCT7 gene, Atg1 gene, and Atg8 gene in CCD-966SK cells can be significantly increased by about 1.1-2.2 times, the expression of SIRT1 gene and FOXO3 gene is about 1.05 times, and the expression of other genes is not much different from the control group (results not shown). This result shows that the Tianshan Saussurea juice of the present invention can effectively increase the expression of CCT6A gene, CCT7 gene, Atg1 gene, Atg8 gene, SIRT1 gene, and FOXO3 gene in CCD-966SK cells, and can effectively enhance the anti-aging ability of skin cells.

[0067] The experimental results of the present invention on the longan shell juice increasing the expression of anti-aging genes in skin cells are as follows: Figure 5 As shown. After 24 hours of action of the longan shell juice of the present invention, compared with the control group, the expression of CCT2 gene, CCT5 gene, CCT7 gene, Pink1 gene, Atg1 gene, Atg8 gene, SIRT1 gene, and FOXO3 gene in CCD-966SK cells can be increased by about 1.1-1.9 times, and the expression of other genes is not much different from that of the control group (results not shown), among which the expression of Pink1 gene, SIRT1 gene, and FOXO3 gene has significant differences. This result shows that the longan shell juice of the present invention can effectively increase the expression of CCT2 gene, CCT5 gene, CCT7 gene, Pink1 gene, Atg1 gene, Atg8 gene, SIRT1 gene, and FOXO3 gene in CCD-966SK cells, and can effectively enhance the anti-aging ability of skin cells.

[0068] The experimental results of the buckwheat juice of the present invention on increasing the expression of anti-aging genes in skin cells are as follows: Figure 6 As shown. After 48 hours of the buckwheat juice of the present invention, compared with the control group, the expression of CCT2 gene, CCT6A gene, CCT7 gene, and CCT8 gene in CCD-966SK cells can be significantly increased by about 1.2-2.9 times, the expression of CCT5 gene and Pink1 gene is about 1.2 times, and the expression of other genes is not much different from the control group (results not shown). This result shows that the buckwheat juice of the present invention can effectively increase the expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, and Pink1 gene in CCD-966SK cells, and can effectively enhance the anti-aging ability of skin cells.

[0069] Example 6 The efficacy of the Tianshan Saussurea Juice, Longan Shell Juice, and Buckwheat Juice of the present invention in increasing the expression of anti-aging genes in cardiovascular cells

[0070] One embodiment of the present invention uses human umbilical vein / vascular endothelium cells (HUVEC) to test the efficacy of the Tianshan Saussurea Juice, Longan Shell Juice, and Buckwheat Juice of the present invention in increasing the expression of anti-aging genes in cardiovascular cells. The HUVEC cell line was purchased from the Bioresource Conservation and Research Center, Taiwan, China, with the number BCRC No. CRL-1730 TM The cell line was cultured in 10 mL of 50x LSGS (purchased from Gibco, USA) in a culture medium Medium 200 (purchased from Thermo, USA, No. M200500) containing 1% penicillin / streptomycin.

[0071] First, 1.5×10 5 HUVEC cells were cultured in a 6-well culture plate containing 2 mL of the above culture medium in each well, and cultured at 37° C. for 16-18 hours. Then, the cells were divided into the following four groups: (1) a control group to which only the cell culture medium was added, (2) an experimental group to which 0.5% (v / v) of the Saussurea involucrata juice of the present invention was added, (3) an experimental group to which 0.5% (v / v) of the Longan shell juice of the present invention was added, and (4) an experimental group to which 0.125% (v / v) of the Buckwheat juice of the present invention was added. After the cells of the groups were treated at 37° C. for 48 hours, the expression level of the target gene in the HUVEC cells of each group was tested. The Saussurea involucrata juice, the Longan shell juice, and the Buckwheat juice were extracted with water as the extraction solvent. Next, the four groups of HUVEC cells were recovered with cell lysis buffer, and the mRNA expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, and UBL-5 gene in the four groups of HUVEC cells were quantified according to the steps described in Example 4, and the coefficient of variation and whether there was a statistically significant difference were determined using Excel software (*p value <0.05; **p value <0.01; ***p value <0.001).

[0072] The experimental results of the present invention on the effect of the Tianshan Saussurea Juice on the expression of anti-aging genes in cardiovascular cells are as follows: Figure 7As shown. After the Tianshan Saussurea juice of the present invention is acted upon, compared with the control group, the expression of CCT2 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Atg1 gene, and SIRT1 gene in HUVEC cells can be significantly increased by about 1.6-4.4 times, while the expression of other genes is not much different from that of the control group (results not shown). This result shows that the Tianshan Saussurea juice of the present invention can effectively increase the expression of CCT2 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Atg1 gene, and SIRT1 gene in HUVEC cells, and can effectively enhance the anti-aging ability of cardiovascular cells.

[0073] The experimental results of the present invention on the longan shell juice increasing the expression of anti-aging genes in cardiovascular cells are as follows: Figure 8 As shown. After the longan shell juice of the present invention is applied, compared with the control group, the expression of CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Atg1 gene, SIRT1 gene, and FOXO3 gene in HUVEC cells can be significantly increased by about 1.8-4.3 times, the expression of CCT2 gene is about 1.8 times, and the expression of other genes is not much different from the control group (results not shown). This result shows that the longan shell juice of the present invention can effectively increase the expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Atg1 gene, SIRT1 gene, and FOXO3 gene in HUVEC cells, and can effectively enhance the anti-aging ability of cardiovascular cells.

[0074] The experimental results of the buckwheat juice of the present invention on increasing the expression of anti-aging genes in cardiovascular cells are as follows Fig. 9 As shown. After the buckwheat juice of the present invention is applied, compared with the control group, the expression of CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Atg1 gene, SIRT1 gene, and FOXO3 gene in HUVEC cells can be significantly increased by about 2-4 times, the expression of CCT2 gene is about 1.1 times, and the expression of other genes is not much different from the control group (results not shown). This result shows that the buckwheat juice of the present invention can effectively increase the expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Atg1 gene, SIRT1 gene, and FOXO3 gene in HUVEC cells, and can effectively enhance the anti-aging ability of cardiovascular cells.

[0075] Example 7 The effect of the longan shell juice and buckwheat juice of the present invention on increasing the expression of anti-aging genes in retinal cells

[0076] In one embodiment of the present invention, human retinal pigment epithelial cells (ARPE-19) were used to test the efficacy of the inositolium juice, longan shell juice, and buckwheat juice of the present invention in increasing the expression of anti-aging genes in skin cells. The ARPE-19 cell line was purchased from ATCC (USA) with a serial number of CRL-2302, and was cultured in a medium containing 10% fetal bovine serum and 90% DMEM / F12 culture medium, wherein the culture medium contained a 1:1 ratio of DMEM culture medium (Dulbecco's Modified Eagle Medium, purchased from Gibco, USA, 12100-046) and Ham's F12 culture medium (Ham's F12 Nutrient Mixture, purchased from Gibco, USA, 12500-026), and contained 0.5 mM sodium pyruvate and 15 mM 4-hydroxyethyl piperazineethanesulfonic acid (HEPES) buffer solution.

[0077] First, place the 1.5x10 5 Each ARPE-19 cell was cultured in a 6-well culture plate containing 2 mL of the culture medium in each well, and cultured at 37° C. for 16-18 hours. The cells were then divided into the following five groups: (1) a blank control group in which only the cell culture medium was added and no blue light was irradiated, (2) a positive control group in which only the cell culture medium was added and blue light was irradiated, (3) an experimental group in which 0.5% (v / v) of the Tianshan Saussurea scabra juice of the present invention was added, (4) an experimental group in which 0.5% (v / v) of the longan shell juice of the present invention was added, and (5) an experimental group in which 0.5% (v / v) of the buckwheat juice of the present invention was added. The cell lines of groups (2) to (5) were first placed in a blue light box and irradiated with blue light at room temperature for 15 minutes, and then the samples were respectively exposed to blue light at 37° C. for 48 hours, and the expression level of the target gene in each group of ARPE-19 cells was tested. The longan shell juice and the buckwheat juice were extracted with water as the extraction solvent. Next, the five groups of ARPE-19 cells were recovered with cell lysis buffer, and the mRNA expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, and UBL-5 gene in the five groups of ARPE-19 cells were quantified according to the steps described in Example 4, and the coefficient of variation and whether there was a statistically significant difference were determined using Excel software (*p value <0.05; **p value <0.01; ***p value <0.001).

[0078] The experimental results of the present invention on the longan shell juice and buckwheat juice increasing the expression of anti-aging genes in retinal cells are as follows: Fig.10 As shown. The expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Atg8 gene, SIRT1 gene, and FOXO3 gene in the positive control group cells are lower than those in the blank control group, indicating that irradiation with blue light can indeed reduce the expression level of anti-aging genes in retinal pigment epithelial cells; and after being treated with the longan shell juice of the present invention, compared with the control group, the expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene in ARPE-19 cells can be increased. , Atg1 gene, SIRT1 gene, and FOXO3 gene expression levels ranged from 1.1 to 1.7 times, and the expression levels of other genes were not much different from those of the control group (results not shown); after being treated with the buckwheat juice of the present invention, the expression levels of CCT2 gene, CCT5 gene, CCT6A gene, Atg1 gene, and FOXO3 gene were increased by 1.2 to 1.5 times, and the expression levels of other genes were not much different from those of the control group (results not shown); after being treated with the Tianshan Saussurea Juice of the present invention, the expression levels of these genes were not much different from those of the control group (results not shown). These results show that the longan shell juice and buckwheat juice of the present invention can effectively increase the expression levels of CCTs gene, Pink1 gene, Atg8 gene, SIRT1 gene, or FOXO3 gene in ARPE-19 cells, and can effectively enhance the anti-aging ability of retinal cells.

[0079] Example 8 Preparation method of the combination of plant juices of the present invention

[0080] In one embodiment of the present invention, the Tianshan Saussurea juice of the present invention, the longan shell juice of the present invention, and the buckwheat juice of the present invention are mixed in a weight ratio of 2.4-3.6:129.6-194.4:128.8-193.2 to obtain the plant juice combination of the present invention.

[0081] Example 9 The combination of plant juices of the present invention enhances the anti-aging activity

[0082] One embodiment of the present invention is to test the efficacy of the combination of plant juices of the present invention in enhancing anti-aging activity. First, 7 adults aged 30-50 years or older were recruited as subjects. They took 2 capsules (each capsule containing 2.4-3.6 mg of the Tianshan Saussurea juice of the present invention, 129.6-194.4 mg of the longan shell juice of the present invention, and 128.8-193.2 mg of the buckwheat juice of the present invention) of the combination of plant juices of the present invention every day. At the 0th week, 2nd week and 4th week of administration of the plant juice, blood samples of each subject were collected for anti-aging gene expression analysis and biochemical protein analysis. Among them, in the combination of plant juices of the present invention used in this embodiment, the Tianshan Saussurea juice, the longan shell juice and the buckwheat juice were all extracted juices using water as the solvent.

[0083] The analysis of the expression of anti-aging genes was performed by first separating peripheral blood mononuclear cells from the blood of the test subjects with lymphocyte separation fluid, and then quantifying the mRNA expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, UBL-5 gene, SOD3 gene, TERT gene, and TERC gene in the peripheral blood mononuclear cells using the steps described in Example 4 and the combined primers in Tables 1 and 2, and then using Excel software 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).

[0084] Table 2. Primer combinations for quantitative real-time reverse transcription polymerase chain reaction

[0085]

[0086] The biochemical protein analysis was conducted by using an intracellular GSH assay kit (purchased from abcam, number ab112132) to analyze the glutathione (GSH) content in blood cells. The blood of the subject was adjusted to 106 cells / mL with PBS, and then stained with the intracellular GSH assay kit at room temperature for 15 minutes. After washing and re-dissolving with PBS, the blood was placed on a flow cytometer (Accuri C6 Plus, BD) to detect fluorescence in the FL1 channel (Ex / Em=490 / 525nm), and the fluorescence signal was used as the glutathione content in the blood of the subject.

[0087] Telomeres are repetitive DNA sequences at the ends of biological chromosomes. Their main function is to maintain the integrity of chromosomes and control the cell division cycle. Each time DNA is replicated, the telomeres shorten. Once the telomeres are exhausted, the cells will undergo apoptosis. Therefore, the length of telomeres is related to the age of cells. Among them, the TERT gene and TERC gene are known to be related to regulating the length of telomeres.

[0088] Biochemical protein analysis is to analyze the content of glutathione (GSH) in the blood of the subjects. Glutathione, also known as glutathione, is an important antioxidant enzyme in the body. It has a higher concentration in the liver, so it is also an important detoxifier. Glutathione is also an important auxiliary component of metabolism in the body, so it is related to maintaining the stability and smoothness of various physiological metabolisms in the body. Many studies have pointed out that glutathione in the body will decrease with aging, stress, etc. If the amount of glutathione in the body is insufficient, it may increase the risk of cardiovascular, liver and neurodegenerative diseases.

[0089] The results of the combination of plant juices of the present invention in increasing the expression of CCTs genes are as follows: Fig.11 As shown; the results of increasing the expression of Atg1 gene and Atg8 gene are as follows Fig.12 As shown; the results of increasing the expression of NADSYN gene, MRPS5 gene, and SOD3 gene are as follows Fig.13 As shown; the results of increasing the expression of TERT gene are as follows Fig.14 The results of increasing the GSH content in the blood are shown as follows Fig.15 shown.

[0090] After taking the combination of the plant juice of the present invention for 2 weeks or 4 weeks, the expression levels of the CCT2 gene and the CCT7 gene in the subjects were significantly higher than before the test, ranging from about 2.4 to 3.9 times, and the expression level of the CCT8 gene was about 1.8 and 1.3 times higher than before the test, respectively; the expression level of the Atg1 gene was significantly higher than about 2.8 and 3.8 times, respectively, and the Atg8 gene was about 1.4 and 2.5 times higher than before the test, respectively; the expression levels of the NADSYN gene and the MRPS5 gene were significantly higher than about 3.9 to 15 times, respectively, and the expression level of the SOD3 gene was about 1.04 and 1.2 times higher than before the test, respectively, and the expression levels of other genes were not much different from those of the control group (results not shown); and after taking the combination of the plant juice of the present invention for 4 weeks, the expression level of the TERT gene was also significantly higher than about 1.5 times before the test, and the expression levels of other genes were not much different from those of the control group (results not shown). These results show that the combination of plant juices of the present invention can effectively increase the expression of CCT2 gene, CCT7 gene, CCT8 gene, Atg1 gene, Atg8 gene, NADSYN gene, MRPS5 gene, SOD3 gene, and TERT gene, and can effectively and comprehensively enhance the anti-aging activity.

[0091] After taking the combination of plant juices of the present invention for 2 weeks or 4 weeks, the GSH content in the blood was significantly higher than that before the test by about 1.16 times and 1.4 times. The results show that the combination of plant juices of the present invention can effectively increase the GSH content in the blood and can effectively and comprehensively improve the antioxidant capacity.

[0092] In summary, the plant juice of the present invention is composed of one Tianshan Saussurea juice, one Longan shell juice, one Buckwheat juice, and any combination thereof; the Tianshan Saussurea juice, the Longan shell juice, and the Buckwheat juice can all effectively increase the expression of genes related to mitochondrial or cellular anti-aging in blood cells, skin cells, cardiovascular cells, and / or retinal cells, and the three juices have significant anti-aging effects on cardiovascular cells. The combination of the three juices can also directly and effectively increase the expression of CCT2 gene, CCT7 gene, CCT8 gene, Atg1 gene, Atg8 gene, NADSYN gene, MRPS5 gene, SOD3 gene, and TERT gene, and can comprehensively improve the anti-aging activity, and the plant juice of the present invention can simultaneously increase the content of GSH to comprehensively improve the antioxidant activity; the plant juice of the present invention can effectively promote the body to revert back to a youthful and energetic state. Therefore, the plant juice of the present invention can be used to prepare an anti-aging composition, wherein the composition is a medicine, a food or a skin care product, and can be administered to a subject by oral administration, skin application, etc.

[0093] Of course, the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, technicians familiar with the field may make various corresponding changes and deformations based on the present invention, but these corresponding changes and deformations should all fall within the scope of protection of the claims of the present invention.

Claims

1. A use of plant juice, characterized in that: The plant juice is used to prepare a composition for increasing the expression of skin TCP1-containing folding protein (CCT) gene, autophagy-related gene (Atg) gene and mitochondrial serine / threonine protein kinase (Pink1) gene and / or regulating the NAD+ concentration of skin cells, and the plant juice is composed of Tianshan snow lotus juice, longan shell juice and buckwheat juice, wherein the plant juice is obtained by extracting with water, and the Tianshan snow lotus juice is prepared from the callus tissue of Tianshan snow lotus Smallanthus sonchifolius.

2. The use according to claim 1, characterized in that The CCT gene is a folding protein (CCT2) gene containing the 2nd subunit of TCP1, a folding protein (CCT5) gene containing the 5th subunit of TCP1, a folding protein (CCT6A) gene containing the 6A subunit of TCP1, a folding protein (CCT7) gene containing the 7th subunit of TCP1, a folding protein (CCT8) gene containing the 8th subunit of TCP1, or a combination thereof.

3. The use according to claim 1, characterized in that The Atg gene is the autophagy-related gene 1 (Atg1) gene, the autophagy-related gene 8 (Atg8) gene, or a combination thereof.

4. The use according to claim 1, characterized in that The plant juice has the ability to increase the anti-aging activity of the skin.

5. The use according to claim 1, characterized in that: The concentration of the plant juice is at least 0.5% (v / v).

6. The use according to claim 1, characterized in that: The plant juice is prepared by mixing the Tianshan Saussurea involucrata juice, the longan shell juice and the buckwheat juice in a ratio of 2.4-3.6:129.6-194.4:128.8-193.2.

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