TWI779251B - Use of a plant extraction for enhancing the gene expression of cct, atg, nadsyn, mrps5, sod3, and tert and/or increase the amount of gsh - Google Patents

Abstract

The present invention provides a use of a plant extraction for enhancing the gene expression of CCT, Atg, NADSYN, MRPS5, SOD3, and TERT and/or increase the amount of GSH. The plant extraction can comprehensively improve the anti-aging activity of the body and/or antioxidant activity of a subject to promote the body to a youthful and energetic state. The plant extraction of the present invention is composed of a day of a Peruvian ground apple extract, a Longan shell extract, a Common buckwheat extract, and any combination thereof.

Description

The use of plant extracts for increasing the expression of CCT gene, Atg gene, NADSYN gene, MRPS5 gene, SOD3 gene, and TERT gene and/or increasing the content of GSH

The present invention relates to the use of a plant extract for increasing the expression of CCT gene, Atg gene, NADSYN gene, MRPS5 gene, SOD3 gene, and TERT gene and increasing the GSH content, especially a kind of extract from Snow Lotus of Tiantian Mountain , a longan shell extract, a buckwheat extract, and a plant extract composed of any combination thereof are used to prepare CCT2 gene, CCT7 gene, CCT8 gene, Atg1 gene, Atg8 gene, NADSYN gene, MRPS5 gene, SOD3 gene, And TERT gene expression level, and/or the use of the composition for increasing the content of GSH.

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 tissues or organs in various parts of the body, making the health of the organism worse. Ultimately, it leads to the death of organisms. Modern people live under high pressure. The accumulation of long-term stress will accelerate the aging of body functions and lead to many chronic diseases.

In addition, with the rise of health care awareness and the advancement of medical technology, the average life expectancy increases, but the functional degradation caused by aging is still intensified with age. Therefore, in addition to the treatment and prevention of diseases, the prevention of aging is also an important aspect of the aging society. one of the important subjects.

Although many genes and related mechanisms related to aging have been studied, there are still no drugs or methods that can be effectively applied to delay aging. Proper exercise and maintain a normal work and rest and a happy mood.

To sum up, in order to achieve the anti-aging effect conveniently and effectively, it is necessary to develop a composition that can effectively increase the expression level of anti-aging genes and the antioxidant capacity.

For this reason, one object of the present invention is to provide a kind of plant extract to be used for preparing and promoting the folder protein (Chaperonin containing TCP1, CCT ) gene containing TCP1, autophagy-related gene (Autophagy-related gene, Atg ) gene, glutamine Acid-dependent tobacco amide adenine dinucleotide synthetase (Glutamine-dependent NAD ⁺ synthetase, NADSYN ) gene, mitochondrial ribosomal protein 5 (mitochondrial ribosomal protein S5, MRPS5 ) gene, superoxide dismutase 3 ( Superoxide dismutase 3, SOD3 ) gene, and telomerase reverse transcriptase (Telomerase reverse transcriptase, TERT ) gene expression composition; wherein the plant extract is composed of a snow lotus extract, a longan shell extract, A buckwheat extract and any combination thereof.

Another object of the present invention is to provide a plant extract for the preparation of a composition for increasing glutathione (Glutathione, GSH) content, wherein the plant extract is composed of Tianshan Snow Lotus extract, a longan shell extract , a buckwheat extract, and any combination thereof.

In one embodiment of the present invention, the CCT gene is a chaperonin containing TCP1 subunit 2 (CCT2) gene, a chaperonin containing TCP1 subunit 7 ( CCT7 ) gene, and a chaperonin containing TCP1 subunit 7 ( CCT7 ) gene. gene, the folding protein (Chaperonin containing TCP1 subunit 8, CCT8 ) gene containing the 8th unit of TCP1, and any combination thereof; and the Atg gene is an autophagy-related gene 1 (Autophagy-related gene 1, Atg1 ) gene, autophagy Phagocytosis-related gene 8 (Autophagy-related gene 8, Atg8 ) gene or its combination.

In yet another embodiment of the present invention, the plant extract increases anti-aging activity.

In yet another embodiment of the present invention, the plant extract modulates NAD ⁺ concentration, and/or telomere length of a cell.

In yet another embodiment of the present invention, the plant extract increases antioxidant activity.

In yet another embodiment of the present invention, the plant extract is extracted with a solvent, and the solvent is water, alcohol, or a mixture of alcohol and water.

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

In another embodiment of the present invention, the plant extract is composed of the Tianshan Snow Lotus extract, the longan shell extract, and the buckwheat extract in a ratio of 2.4-3.6:129.6-194.4:128.8-193.2.

The plant extract of the present invention is composed of Tianshan Snow Lotus extract, a longan shell extract, a buckwheat extract, and any combination thereof; the Tianshan Snow Lotus extract, the longan shell extract, and the buckwheat extract are all Can effectively increase the expression of genes related to mitochondrial or cell anti-aging in blood cells, skin cells, cardiovascular cells, and/or retinal cells, and the three extracts have a significant anti-aging effect on cardiovascular cells . The combination of these three extracts 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 extract of the present invention can increase the content of GSH at the same time, so as to comprehensively enhance the antioxidant activity in the body; the plant extract of the present invention can effectively promote the body to return to a youthful and energetic state. Therefore, the plant extract 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 an individual by oral administration or skin application.

Fig. 1 is a bar graph showing the effect of Saussurea tianshanensis extract on enhancing the expression of CCT2 gene, CCT5 gene, and CCT6A gene in blood cells according to one embodiment of the present invention. *p-value <0.05.

Fig. 2 is a bar graph showing the effect of longan shell extract on enhancing the expression of CCT5 gene, CCT6A gene, CCT8 gene, Pink1 gene, Atg1 gene, Atg8 gene, and FOXO3 gene in hemocytes according to an embodiment of the present invention. *p-value<0.05;**p<0.01;***p<0.001.

Fig. 3 is a bar graph showing the effect of buckwheat extract on increasing the expression levels of CCT2 gene and CCT5 gene in hemocytes according to an embodiment of the present invention. *p-value <0.05.

Fig. 4 is a bar graph showing the effect of Saussurea tianshanensis extract on enhancing the expression of CCT6A gene, CCT7 gene, Atg1 gene, Atg8 gene, SIRT1 gene, and FOXO3 gene in skin cells according to one embodiment of the present invention. *p-value<0.05;**p<0.01.

Figure 5 is a strip showing the effect of the longan shell extract on enhancing the expression of CCT2 gene, CCT5 gene, CCT7 gene, Pink1 gene, Atg1 gene, Atg8 gene, SIRT1 gene, and FOXO3 gene in skin cells according to an embodiment of the present invention picture. *p-value<0.05;**p<0.01;***p<0.001.

Fig. 6 is a bar graph showing the effect of buckwheat extract on enhancing the expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, and Pink1 gene in skin cells according to an embodiment of the present invention. ***p-value <0.001.

7 is a bar graph showing the effect of Saussurea tianshanensis extract on enhancing the expression of CCT2 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Atg1 gene, and SIRT1 gene in cardiovascular cells according to one embodiment of the present invention. *p-value<0.05;**p<0.01;***p<0.001.

Figure 8 is a longan shell extract of one embodiment of the present invention to enhance the expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Atg1 gene, SIRT1 gene, and FOXO3 gene expression in cardiovascular cells. bar chart. *p-value<0.05;**p<0.01;***p<0.001.

Figure 9 shows the effect of buckwheat extract in one embodiment of the present invention on enhancing 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 bar chart. *p-value<0.05;**p<0.01;***p<0.001.

Figure 10 is a bar graph showing the effect of longan shell extract and buckwheat extract on enhancing the expression of CCTs gene, Pink1 gene, Atg8 gene, SIRT1 gene, or FOXO3 gene in retinal cells according to an embodiment of the present invention. **p<0.01;***p<0.001.

Fig. 11 is a bar graph showing the effect of a composition of plant extracts according to an embodiment of the present invention on enhancing the gene expression of CCTs . *p<0.05;***p<0.001.

Fig. 12 is a bar graph showing the effect of the composition of the plant extract according to one embodiment of the present invention on enhancing the expression of Atg1 gene and Atg8 gene. *p<0.05;***p<0.001.

Fig. 13 is a bar graph showing the effects of the composition of plant extracts according to one embodiment of the present invention on enhancing the expression levels of NADSYN gene, MRPS5 gene, and SOD3 gene. *p<0.05;***p<0.001.

Fig. 14 is a bar graph showing the effect of the composition of plant extracts according to one embodiment of the present invention on enhancing the expression level of TERT gene. ***p<0.001.

Fig. 15 is a bar graph showing the effect of increasing the GSH content of the composition of plant extracts according to one embodiment of the present invention. **p<0.01; ***p<0.001.

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

Statistical analysis was performed using Excel software. The data are expressed as mean ± standard deviation (SD), and the differences between them are analyzed by student's t -test.

Tianshan Snow Lotus ( Smallanthus sonchifolius ) is a perennial herbaceous plant of the genus Smallanthus ( Smallanthus ) in the family Compositae , also known as Chrysanthemum potato, and its English name is Peruvian ground apple. Aids in defecation.

Longan ( Euphoria longana Lam. ) is an evergreen tree plant of Sapindaceae ( Sapindaceae ) longan genus ( Euphoria ). The English name is Longan. The fruit is sweet and has the effect of nourishing qi and blood and calming the nerves; the peel (that is, longan shell) has the effect of clearing away heat and cooling blood.

Buckwheat ( Fagopyrum esculentum Moench. ) is an annual herbaceous plant of the genus Fagopyrum in the Polygonaceae family. , detoxification, antipyretic effect.

As used herein, the term "Tianshan Saussurea extract" means that the callus of Tianshan Saussurea and solvent are extracted at a ratio of 1:1-5 (w/w) for a specific time and temperature.

As used herein, the term "longan shell extract" means that longan shell and solvent are extracted at a ratio of 1:5-10 (w/w) for a specific time and temperature.

As used herein, the term "buckwheat extract" means that buckwheat seed coat and solvent are extracted at a ratio of 1:10-15 (w/w) for a specific time and temperature.

As used herein, the term "composition of plant extracts" means to combine the aforementioned Saussurea tianshanensis extract, the aforementioned longan shell extract, and the aforementioned buckwheat extract at a ratio of 2.4-3.6:129.6-194.4:128.8- The composition obtained by mixing in a ratio of 193.2.

The present invention provides the use of a plant extract for preparing an anti-aging composition. The plant extract of the present invention is obtained from a snow lotus extract, a longan shell extract, a buckwheat extract, and any combination thereof. It can be used to increase the expression of genes related to anti-aging, can comprehensively enhance anti-aging activity, and can increase the content of GSH at the same time, and can comprehensively enhance the antioxidant activity of people, so that the body can return to youthful vitality status.

At the same time, the anti-aging composition of the present invention may also include an effective amount of plant extract and a pharmaceutically acceptable carrier, and the composition is a medicine, a food or a skin care product.

The detailed extraction methods of the Tianshan Snow Lotus Extract, Longan Shell Extract, and Buckwheat Extract of the present invention will be described in detail below; Efficacy test of anti-aging gene expression in vascular cells and/or retinal cells; and efficacy test of enhancing anti-aging gene expression and increasing GSH content with the composition of the plant extract of the present invention. To prove that the plant extracts of the present invention can effectively and comprehensively improve anti-aging and anti-oxidation, so that the body can return to a youthful and energetic state.

Embodiment 1 The preparation method of the Tianshan Snow Lotus Extract of the present invention

In one embodiment of the present invention, first, the callus of Snow Lotus Tianshan is mixed with water, alcohol, or an extraction solvent of a mixture of alcohol and water, the extraction solvent is preferably water, and mixed with a weight ratio of 1:1-5, after homogenization After extracting in a solvent at 50-100°C for 0.5-2 hours, cool the crude extract to room temperature, and filter the crude extract with a 400mesh filter to obtain a filtrate, wherein the extraction temperature is preferably 70-100 ℃. Finally, after the filtrate is concentrated under reduced pressure at 45-70° C., the Snow Lotus extract of the present invention is obtained.

Embodiment 2 The preparation method of the longan shell extract of the present invention

In one embodiment of the present invention, the longan shell tissue is first mixed with water, alcohol, or an extraction solvent of a mixture of alcohol and water. The extraction solvent is preferably water, mixed at a weight ratio of 1:5-10, and mixed in the solvent after homogenization After extraction at 50-100°C for 0.5-2 hours, the crude extract is cooled to room temperature and 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 longan shell extract of the present invention.

Embodiment 3 The preparation method of the buckwheat extract of the present invention

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

Example 4 The efficacy of the Tianshan snow lotus extract, longan shell extract, and buckwheat extract of the present invention in enhancing the expression of systemic anti-aging genes

One embodiment of the present invention uses human peripheral blood mononuclear cells (Human peripheral blood mononuclear cells, PBMC) to test whether the Tianshan Snow Lotus Extract, Longan Shell Extract, and Buckwheat Extract of the present invention can enhance the expression of systemic anti-aging genes efficacy test. Wherein, the human peripheral blood mononuclear cell line was purchased from the American Type Culture Collection, numbered ATCC® PCS- ^{800-011™ ,} and cultured in X-VIVO ^™ 10 Serum-free hematopoietic cell medium (purchased from Lonza, Switzerland, No. BE02-055Q).

First, 1×10 ⁵ PBMC cells were cultured in a 6-well culture dish containing 2 mL of the above-mentioned cell culture medium, and cultured at 37°C for 16-18 hours, and then the cells were divided into the following four groups: (1) Add only the cell culture medium The control group, (2) the experimental group adding 0.5% (v/v) of the Saussurea chinensis extract of the present invention, (3) the experimental group adding 0.25% (v/v) of the longan shell extract of the present invention, and ( 4) Add 0.125% (v/v) of the buckwheat extract of the present invention to the experimental group, and act on the cells of these groups for 24 hours or 48 hours at 37°C respectively, and test the expression of the target gene in the PBMC cells of each group. Performance; Wherein, the Tianshan Snow Lotus extract, the longan shell extract, and the buckwheat extract use water as the extraction solvent. First, the PBMC cells were recovered with cell lysate (RB buffer, purchased from Geanaid Company, Taiwan, Cat No. RBD300), and then collected separately using the RNA extraction kit (purchased from Geneaid Company, Taiwan, Cat No. RBD300) The RNA in the four groups of cells was then reverse-transcriptase (purchased from Invitrogene, the U.S., No. ^18080-051 ) using 2000 ng of extracted RNA as a template, and reversed with the combined primers and reverse transcriptase in Table 1. Transcription, to produce the cDNA products corresponding to the mRNA of these genes, then use ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific Company, the United States), and KAPA SYBR FAST (purchased from Sigma Company, the United States, No. 38220000000) The four sets of reverse-transcribed products were subjected to quantitative real-time polymerase chain reaction (qPCR) test with the combined primers in Table 1. The conditions were 95°C for 1 second and 60°C for 20 seconds. 40 loops. For quantifying the 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 Expression, 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 Equation 2 ^{- △△Ct} , where △C _T = C _{T comparison group or experimental group target gene / control group Target gene} -C _{T GAPDH} (glyceraldehyde-3-phosphate dehydrogenase, Glyceraldehyde-3-phosphate dehydrogenase); △△C _T = C _{T comparison group or experimental group target gene} -C _{T control group target gene} ; The fold change of each gene is 2 ^-△△Ct . Then, use Excel software to determine whether the coefficient of variation and whether there is a statistically significant difference (*p value<0.05; **p value<0.01; ***p value<0.001).

The CCT gene is a type of chaperonin, whose main function is to correct misfolded proteins and send the proteins that cannot be successfully repaired to the proteasome for hydrolysis. Previous studies have pointed out that if this protein regulates The failure of the reuse system will lead to the accumulation of misfolded proteins in cells, which will cause the decline of cell function and accelerate the aging and death of cells, so it is considered to be related to the regulation of aging in individuals.

The Pink1 gene is a serine/threonine protein kinase located in mitochondria. Its function is to protect mitochondria from failure during cellular stress. Mutations in this gene are known to be associated with Parkinson's disease Related, and previous studies have pointed out that the excessive expression of this gene can increase the lifespan of mice, so it is also considered to be related to the regulation of aging in individuals.

The Parkin gene is an enzyme that exists in the ubiquitin-proteasome system (Ubiquitin-proteasome system) and acts as a regulator of protein breakdown. It is known that the mutation of this gene is related to Parkinson's disease, and previous studies have also pointed out that the increase of this gene The expression level of can delay the aging effect of cells, so it is considered to be related to the aging effect of cells.

The Atg gene is an autophagy-related gene, which is involved in the degradation of waste in cells and autophagy in the cycle. 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 of cells.

Previous studies have pointed out that compared with old mice, young mice contain more NAD ⁺ in their bodies; and increasing the concentration of NAD ⁺ in old mice can make their physiological conditions younger, so NAD ⁺ is It is considered to be related to delaying the aging of individuals, and the research results have shown that SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, UBL-5 gene, and SOD3 gene are related to the regulation of NAD ⁺ .

The experimental results of the Snow Lotus extract of the present invention to enhance the expression of anti-aging genes in blood cells are shown in FIG. 1 . After being treated with the Saussurea tianshanensis extract of the present invention for 48 hours, compared with the control group, the expression levels of CCT2 gene, CCT5 gene, and CCT6A gene in PBMC cells can be increased by about 1.2-1.4 times, and the expression levels of other genes are similar to those of the control group. There was little difference in the control group (results not shown), but there was a significant difference in the expression of CCT6A gene. The results show that the Saussurea tianshanensis extract of the present invention can effectively increase the expression levels of CCT2 gene, CCT5 gene, and CCT6A gene in PBMC cells, and can effectively improve the systemic anti-aging ability.

The experimental results of the longan shell extract of the present invention increasing the expression of anti-aging genes in blood cells are shown in FIG. 2 . After being treated with the longan shell extract of the present invention for 24 hours, compared with the control group, the expression levels 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, and the expression of other genes had little difference with the control group (results not shown). This result shows that the longan shell extract 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 improve the ability of systemic anti-aging .

The experimental results of the buckwheat extract of the present invention increasing the expression of anti-aging genes in blood cells are shown in FIG. 3 . After being treated with the buckwheat extract of the present invention for 48 hours, compared with the control group, the expression levels of CCT2 gene and CCT5 gene in PBMC cells can be increased by about 1.2 times, and the expression levels of other genes are not significantly different from the control group (results not shown shown), in which the expression of CCT2 gene was significantly different. The results show that the buckwheat extract of the present invention can effectively increase the expression levels of CCT2 gene and CCT5 gene in PBMC cells, and can effectively improve the systemic anti-aging ability.

Example 5 The efficacy of Tianshan snow lotus extract, longan shell extract, and buckwheat extract of the present invention in enhancing the expression of anti-aging genes in skin cells

One embodiment of the present invention uses human skin fibroblast (Human skin fibroblast) CCD-966SK cells to test the efficacy of the Tianshan snow lotus extract, longan shell extract, and buckwheat extract of the present invention to enhance the expression of anti-aging genes in skin cells test. Wherein, the CCD-966SK cell line was purchased from the American Type Culture Collection (ATCC, U.S.), the number is ATCC ^® CRL-1881, and the cell line was cultured in 10% fetal bovine serum (Fetal Bovine Serum) and 90 The culture solution of Minimum Essential Medium (MEM) (purchased from Gibco, U.S.) containing 1 mM sodium pyruvate (Sodium pyruvate, purchased from Gibco, U.S.), and 1% penicillin-streptomycin (penicillin/streptomycin ).

First, ^1.5x105 CCD-966SK cells were cultured in a 6-well culture dish containing 2 mL of the above-mentioned culture solution in each well, and cultured at 37°C for 16-18 hours, and then the cells were divided into the following four groups: (1) Add only cells The control group of the culture medium, (2) the experimental group adding 0.5% (v/v) of the Tianshan Snow Lotus extract of the present invention, (3) the experimental group of adding 0.5% (v/v) of the longan shell extract of the present invention, And (4) add 0.125% (v/v) of the buckwheat extract of the present invention to the experimental group, and after the cells of these groups acted at 37°C for 24 hours or 48 hours, test the CCD-966SK cells in each group The expression level of the target gene; wherein, the extract of Snow Lotus Tianshan, the extract of longan husk, and the extract of buckwheat use water as the extraction solvent. Then, after the four groups of CCD-966SK cells were recovered with cell lysate, the CCT2 gene, CCT5 gene, CCT6A gene, CCT7 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 mRNA expression, and then use Excel software to determine the coefficient of variation and whether it is statistically Significantly different (*p-value<0.05;**p-value<0.01;***p-value<0.001).

The experimental results of the Snow Lotus extract of the present invention to enhance the expression of anti-aging genes in skin cells are shown in FIG. 4 . After being treated with the Saussurea tianshanensis extract of the present invention for 24 hours, compared with the control group, the expression levels 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 levels of SIRT1 gene and FOXO3 gene were about 1.05 times, and the expression levels of other genes were not significantly different from those of the control group (results not shown). The results show that the Saussurea tianshanensis extract of the present invention can effectively increase the expression levels of CCT6A gene, CCT7 gene, Atg1 gene, Atg8 gene, SIRT1 gene, and FOXO3 gene in CCD-966SK cells, and can effectively improve the anti-aging ability of skin cells.

The experimental results of the longan shell extract of the present invention increasing the expression of anti-aging genes in skin cells are shown in FIG. 5 . After being treated with the longan shell extract of the present invention for 24 hours, compared with the control group, the levels 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. The expression levels ranged from 1.1 to 1.9 times, and the expression levels of other genes were not significantly different from those in the control group (results not shown). Among them, the expression levels of the Pink1 gene, SIRT1 gene, and FOXO3 gene were significantly different. This result shows that the longan shell extract of the present invention can effectively increase the expression levels 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 improve skin The ability of cells to resist aging.

The experimental results of the buckwheat extract of the present invention increasing the expression of anti-aging genes in skin cells are shown in FIG. 6 . After being treated with the buckwheat extract of the present invention for 48 hours, compared with the control group, the expression levels 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, CCT5 The expression levels of genes and Pink1 gene were about 1.2 times, and the expression levels of other genes were not significantly different from those of the control group (results not shown). The results show that the buckwheat extract of the present invention can effectively increase the expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, and Pink1 gene in CCD-966SK cells, and can effectively improve the anti-aging ability of skin cells.

Example 6 The effect of Tianshan snow lotus extract, longan shell extract, and buckwheat extract of the present invention on enhancing the expression of anti-aging genes in cardiovascular cells

One embodiment of the present invention uses human umbilical vein/vascular endothelial cells (Human umbilical vein/vascular endothelium cell, HUVEC) to test that the Tianshan snow lotus extract, longan shell extract, and buckwheat extract of the present invention can enhance the anti-aging in cardiovascular cells Efficacy test of gene expression. Wherein, the HUVEC cell line was purchased from Taiwan Biological Resource Conservation and Research Center, the number is BCRC No.H-UV001ATCC ^® CRL- ^1730TM , and the cell line was cultured in a culture medium containing 10 mL of 50x LSGS (purchased from Gibco, the United States). Liquid Medium 200 (purchased from Thermo, the United States, No. M200500), which contains 1% penicillin-streptomycin (penicillin/streptomycin).

First, ^1.5x105 HUVEC cells were cultured in a 6-well culture dish containing 2 mL of the above-mentioned culture medium per well, and cultured at 37°C for 16-18 hours, and then the cells were divided into the following four groups: (1) Add only cell culture medium The control group, (2) the experimental group adding 0.5% (v/v) of the Saussurea tianshanensis extract of the present invention, (3) the experimental group adding 0.5% (v/v) of the longan shell extract of the present invention, and ( 4) Add 0.125% (v/v) of the buckwheat extract of the present invention to the experimental group, and react the cells of these groups at 37°C for 48 hours, then test the expression of the target gene in each group of HUVEC cells; Wherein, the Snow Lotus extract, the longan shell extract, and the buckwheat extract use water as the extraction solvent. Next, after the cells of the four groups of HUVEC cells were recovered with cell lysate, the CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, and Pink1 gene in the four groups of HUVEC cells were quantified by the steps described in Example 4. , Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, and UBL-5 gene mRNA expression, and then use 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).

The experimental results of the Saussurea tianshanensis extract of the present invention to enhance the expression of anti-aging genes in cardiovascular cells are shown in FIG. 7 . After being treated with the Saussurea tianshanensis extract of the present invention, compared with the control group, the expression levels 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, and the expression of other genes had little difference with the control group (results not shown). This result shows that the Saussurea tianshanensis extract 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 improve the anti-aging ability of cardiovascular cells. ability.

The experimental results of the longan shell extract of the present invention increasing the expression of anti-aging genes in cardiovascular cells are shown in FIG. 8 . After being treated with the longan shell extract of the present invention, compared with the control group, the expression levels 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 was about 1.8 times, and the expression of other genes was not significantly different from the control group (results not shown). This result shows that the longan shell extract 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 expression of cardiovascular cell Anti-aging ability.

The experimental results of the buckwheat extract of the present invention increasing the expression of anti-aging genes in cardiovascular cells are shown in FIG. 9 . After being treated with the buckwheat extract of the present invention, compared with the control group, the expression levels of CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Atg1 gene, SIRT1 gene, and FOXO3 gene in HUVEC cells can be significantly improved by about 2-4 times, the expression of CCT2 gene was about 1.1 times, and the expression of other genes was not significantly different from the control group (results not shown). The results show that the buckwheat extract of the present invention can effectively increase the expression levels 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 heart rate. Ability of vascular cells to resist aging.

Example 7 The effect of the longan shell extract and buckwheat extract of the present invention on enhancing the expression of anti-aging genes in retinal cells

One embodiment of the present invention uses human retinal pigment epithelial (Human retinal pigment epithelial) ARPE-19 cells to test whether the extracts of Saussurea chinensis, longan shell extract, and buckwheat extract of the present invention can enhance the expression of anti-aging genes in skin cells efficacy test. Wherein, the ARPE-19 cell line was purchased from ATCC Company (U.S.), numbered CRL-2302, and the cell line was cultured in DMEM/F12 culture medium containing 10% fetal bovine serum and 90%, wherein the culture medium contained 1:1 ratio of DMEM medium (Dulbecco's Modified Eagle Medium, purchased from Gibco, USA, 12100-046) and Han's F12 medium (Ham's F12 Nutrient Mixture, purchased from Gibco, USA, 12500-026), and containing 0.5 mM sodium pyruvate (sodium pyruvate) and 15 mM 4-hydroxyethyl piperazineethanesulfonic acid (hydroxyethyl piperazineethanesulfonic acid, HEPES) buffer solution.

First, ^1.5x105 ARPE-19 cells were cultured in a 6-well culture dish containing 2 mL of the above-mentioned culture solution in each well, and cultured at 37°C for 16-18 hours, and then the cells were divided into the following five groups: (1) Add only cells A blank control group with culture medium and no blue light irradiation, (2) a positive control group with only cell culture medium and blue light irradiation, (3) an experimental group with 0.5% (v/v) Snow Lotus extract of the present invention added, ( 4) The experimental group adding 0.5% (v/v) of the longan shell extract of the present invention, and (5) the experimental group adding 0.5% (v/v) of the buckwheat extract of the present invention; wherein (2)-( 5) Groups of cell lines were first placed in a blue light box and irradiated with blue light at room temperature for 15 minutes, and then treated with samples at 37°C for 48 hours to test the expression of target genes in each group of ARPE-19 cells; among them, The longan shell extract and the buckwheat extract use water as the extraction solvent. Next, after the five groups of ARPE-19 cells were recovered with cell lysate, the steps described in Example 4 were used to quantify the CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, and CCT8 gene in the five groups of ARPE-19 cells. Gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, and UBL-5 gene mRNA expression, and then use Excel software to determine the coefficient of variation and whether it is statistically significant Difference (*p-value<0.05;**p-value<0.01;***p-value<0.001).

The experimental results of the longan shell extract and buckwheat extract of the present invention in enhancing the expression of anti-aging genes in retinal cells are shown in FIG. 10 . 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 cells of the positive control group were directly lower than those of the blank control group, showing that the exposure to blue light can indeed reduce retinal pigment The expression of anti-aging genes in epithelial cells; after the action of the longan shell extract of the present invention, compared with the control group, it can increase the CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene in ARPE-19 cells gene, Atg1 gene, SIRT1 gene, and FOXO3 gene expression amount up to 1.1-1.7 times, and other gene expression amount is not much different from the control group (result not shown); after the buckwheat extract of the present invention acts, then can Increase the expression levels of CCT2 gene, CCT5 gene, CCT6A gene, Atg1 gene, and FOXO3 gene by 1.2-1.5 times, and the expression levels of other genes have little difference with the control group (results not shown); After the effect of the drugs, the expression levels of these genes were not significantly different from those of the control group (results not shown). These results show that the longan shell extract and buckwheat extract of the present invention can effectively increase the expression of CCTs gene, Pink1 gene, Atg8 gene, SIRT1 gene, or FOXO3 gene in ARPE-19 cells, and can effectively improve the anti-aging effect of retinal cells. ability.

Example 8 The preparation method of the combination of plant extracts of the present invention

In one embodiment of the present invention, the Tianshan Snow Lotus extract of the present invention, the longan shell extract of the present invention, and the buckwheat extract of the present invention are mixed in a weight ratio of 2.4-3.6:129.6-194.4:128.8-193.2 , which is the combination of plant extracts of the present invention.

Example 9 The combination of plant extracts of the present invention enhances the anti-aging effect

One embodiment of the present invention is to test the efficacy of the combination of plant extracts of the present invention in enhancing anti-aging activity. Firstly, 7 adults over 30-50 years old are recruited as subjects, and they take 2 capsules (each capsule contains 2.4 - 3.6mg of Saussurea tianshanensis extract of the present invention, 129.6-194.4mg of longan shell extract of the present invention, 128.8-193.2mg of buckwheat extract of the present invention) the combination of the plant extract of the present invention, in the administration of the plant extract At week 0, week 2, and week 4, blood samples from each subject were collected for Perform anti-aging gene expression analysis and biochemical protein analysis; among the combination of plant extracts of the present invention used in this embodiment, the Saussurea tianshanensis extract, the longan shell extract, and the buckwheat extract are all made of water as a solvent Extracts.

The anti-aging gene expression analysis is to first separate the peripheral blood mononuclear cells in the subject's blood with the lymphocyte separation solution, and then use the steps described in Example 4 and the combined primers in Table 1 and Table 2, quantification 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, The mRNA expression levels of UBL-5 gene, SOD3 gene, TERT gene, and TERC gene, and then use 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).

Among them, the biochemical protein analysis system used the intracellular GSH assay kit (Intracellular GSH assay kit, purchased from abcam, number ab112132) to analyze the content of glutathione (GSH) in the blood cells, in which the subject’s blood was collected as PBS was adjusted to 106cells/mL, and then the intracellular GSH assay reagent kit was used for staining at room temperature for 15 minutes, washed and redissolved with PBS, and placed on a flow cytometer (Accuri C6 Plus, BD). , the fluorescence is detected as FL1 channel (Ex/Em=490/525nm), and the fluorescence signal is used as the content of glutathione in the subject's blood.

The telomere system is the DNA repeating sequence at the end of the biological chromosome. Its main function is to maintain the integrity of the chromosome and control the cell division cycle. Every time the DNA is replicated, the telomere will be shortened. Once the telomere is exhausted, the cell will go to Apoptosis, so the length of telomere is related to the age of cells, and it is also known that TERT gene and TERC gene are related to regulating the length of telomere.

The biochemical protein analysis system analyzes the content of glutathione (GSH) in the blood of the subjects. Among them, glutathione, also known as glutathione, is an important antioxidant enzyme in the body. It has a high concentration in the liver, so it is also an important antidote, and 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.

The combination of plant extracts of the present invention improves the expression of CCTs genes as shown in Figure 11; the results of improving the expression of Atg1 and Atg8 genes are shown in Figure 12; the results of improving the expression of NADSYN gene, MRPS5 gene and SOD3 gene The result is shown in Figure 13; the result of increasing the expression level of TERT gene is shown in Figure 14; the result of increasing the GSH content in blood is shown in Figure 15.

After taking the combination of plant extracts of the present invention for a total of 2 weeks or 4 weeks, the expression levels of CCT2 gene and CCT7 gene in the subject were significantly higher than those before the test by about 2.4-3.9 times, and the expression levels of CCT8 gene were respectively 1.8 and 1.3 times higher than before the test; while the Atg1 gene expression was significantly higher than before the test about 2.8 and 3.8 times, and the Atg8 gene was about 1.4 and 2.5 times higher than before the test; NADSYN gene, and MRPS5 The gene expression levels were all significantly higher than those before the test by about 3.9-15 times, the SOD3 gene expression levels were respectively 1.04 and 1.2 times higher than before the test, and the other gene expression levels were not significantly different from the control group (results not shown shown); and after taking the combination of plant extracts of the present invention for a total of 4 weeks, the expression level of TERT gene was also significantly higher than that before the test by about 1.5 times, and the expression levels of other genes were not significantly different from the control group (results not shown). These results show that the combination of plant extracts of the present invention can effectively enhance 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 Boosts anti-aging activity.

After taking the combination of plant extracts 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 result shows that the combination of plant extracts of the present invention can effectively increase the content of GSH in the blood, and can effectively and comprehensively improve the antioxidant capacity.

In summary, the plant extract of the present invention is composed of Tianshan Snow Lotus extract, a longan shell extract, a buckwheat extract, and any combination thereof; the Tianshan Snow Lotus extract, the longan shell extract, and The buckwheat extracts can effectively increase the expression of genes related to mitochondria or cell anti-aging in blood cells, skin cells, cardiovascular cells, and/or retinal cells, and the three extracts are especially effective for cardiovascular cells Visible anti-aging benefits. The combination of these three extracts 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 immune system. Aging activity, and the plant extract of the present invention can increase the content of GSH at the same time, so as to comprehensively enhance the antioxidant activity; the plant extract of the present invention can effectively promote the body to return to a youthful and energetic state. Therefore, the plant extract 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 an individual by oral administration or skin application.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of plant extracts to increase the expression of CCT gene, Atg gene, NADSYN gene, MRPS5 gene, SOD3 gene, and TERT gene and/or increase the content of GSH

<130> N/A

<160> 36

<170> PatentIn version 3.5

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Claims (8)

A kind of plant extract is used to prepare and promote the folding protein containing TCP1 (Chaperonin containing TCP1, CCT ) gene, autophagy-related gene (Autophagy-related gene, Atg ) gene, glutamine-dependent tobacco amide adenine dinuclear Glutamine-dependent NAD ⁺ synthetase ( NADSYN ) gene, mitochondrial ribosomal protein 5 (mitochondrial ribosomal protein S5, MRPS5 ) gene, and telomerase reverse transcriptase (Telomerase reverse transcriptase, TERT ) gene expression The use of the composition; wherein the plant extract is composed of a buckwheat seed coat extract or a combination of the buckwheat seed coat extract, a snow lotus callus extract and a longan shell extract, wherein the The weight ratio of the Tianshan snow lotus extract, the longan shell extract and the buckwheat extract is 2.4-3.6:129.6-194.4:128.8-193.2, wherein the plant extract is extracted with a solvent, and the solvent is water . The use as described in claim 1, wherein the CCT gene is a chaperonin containing TCP1 subunit 2 ( CCT2 ) gene, a chaperonin containing TCP1 subunit 7, CCT7 ) gene, chaperonin containing TCP1 subunit 8 ( CCT8 ) gene, or any combination thereof. The use as described in claim 1, wherein the Atg gene is an autophagy-related gene 1 (Autophagy-related gene 1, Atg1 ) gene, an autophagy-related gene 8 (Autophagy-related gene 8, Atg8 ) gene or a combination thereof . The use as described in claim 1, wherein the plant extract increases anti-aging activity. The use as claimed in claim 1, wherein the plant extract regulates the NAD ⁺ concentration and/or telomere length of a cell. Use of a plant extract for preparing a composition for increasing glutathione (Glutathione, GSH) content, wherein the plant extract is composed of the callus extract of Snow Lotus It consists or consists of the combination of the Saussurea tianshanensis extract, a buckwheat seed coat extract and a longan shell extract, wherein the combination of the Saussurea sauraceae extract, the longan husk extract and the buckwheat extract The weight ratio is 2.4-3.6:129.6-194.4:128.8-193.2, wherein the plant extract is extracted with a solvent, and the solvent is water. The use as described in claim 6, wherein the plant extract increases antioxidant activity. The use as claimed in claim 1 or 6, wherein the concentration of the plant extract is at least 0.5% (v/v).

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