CN112823782B - Use of plant juice for increasing expression level of anti-aging gene in cardiovascular cells - Google Patents
Use of plant juice for increasing expression level of anti-aging gene in cardiovascular cells Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides an application of plant juice for improving the expression quantity of anti-aging genes in cardiovascular cells, which can comprehensively improve the anti-aging activity and promote reversion to a youthful and active state. The plant juice of the invention is composed of a saussurea involucrata (Smallanthus sonchifolius) callus juice, a longan shell juice and a buckwheat juice.
Description
Technical Field
The invention relates to application of plant juice in improving expression quantity of CCT genes, atg genes, NADSYN genes, MRPS5 genes, SOD3 genes and TERT genes and improving GSH content, in particular to application of plant juice consisting of saussurea involucrata juice, longan shell juice, buckwheat juice and any combination thereof in preparing a composition for improving expression quantity of CCT2 genes, CCT7 genes, CCT8 genes, atg1 genes, atg8 genes, NADSYN genes, MRPS5 genes, SOD3 genes and TERT genes and/or improving GSH content.
Background
In biology and medicine, aging (aging) refers to a phenomenon that the physiological state of an organism deteriorates with time, and aging causes the deterioration of the functions or functions of tissues or organs of each part of the organism, so that the health function of the organism is deteriorated, and finally the organism dies, the living pressure of modern people is high, the long-term pressure accumulation accelerates the aging of the body functions, and a plurality of chronic diseases are also derived.
In addition, with the improvement of health consciousness and medical technology, life span of people is prolonged, but functional degradation caused by aging is still increased with age, so that prevention of aging is one of important subjects of the advanced society in addition to treatment and prevention of diseases.
Although many genes and related mechanisms related to aging have been studied, there are no drugs or methods available for delaying aging, and most of them only suggest the intake of foods with antioxidant active substances from the diet, and maintain proper exercise and normal work and pleasure.
In view of the above, it is necessary to develop a composition capable of effectively improving the expression level of anti-aging genes and the antioxidant ability in order to achieve the effect of delaying aging conveniently and effectively.
Disclosure of Invention
Accordingly, an object of the present invention is to provide a use of a plant juice for preparing a composition for enhancing expression amounts of a folding protein (Chaperonin containing TCP, CCT) gene containing TCP1, an Autophagy-related gene (Atg) gene, a glutamate-dependent tobacco adenine dinucleotide synthetase (nad+sylase, NADSYN) gene, a mitochondrial ribosomal protein 5 (mitochondrial ribosomal protein S, MRPS 5) gene, a superoxide dismutase 3 (Superoxide dismutase 3, sod 3) gene, and a telomerase reverse transcriptase (Telomerase reverse transcriptase, TERT) gene; wherein the plant juice is composed of herba Saussureae Involueratae juice, arillus longan juice, semen Fagopyri Esculenti juice, and any combination thereof.
Another object of the present invention is to provide the use of a plant juice for preparing a composition for elevating Glutathione (GSH) content, wherein the plant juice is composed of saussurea involucrata juice, a longan shell juice, a buckwheat juice, and any combination thereof.
In one embodiment of the present invention, the CCT gene is a folded protein (Chaperonin containing TCP t2, CCT 2) gene comprising the 2 nd subunit of TCP1, a folded protein (Chaperonin containing TCP t7, CCT 7) gene comprising the 7 th subunit of TCP1, a folded protein (Chaperonin containing TCP t8, CCT 8) gene comprising the 8 th subunit of TCP1, and any combination thereof; and the Atg gene is Autophagy-related gene 1 (Atg 1) gene, autophagy-related gene 8 (Atg 8) gene or a combination thereof.
In yet another embodiment of the present invention, the plant juice is to increase anti-aging activity.
In yet another embodiment of the invention, the plant juice is a regulatory NAD+ concentration, and/or telomere length, of a cell.
In yet another embodiment of the present invention, the plant juice is to increase antioxidant activity.
In yet another embodiment of the present invention, the plant juice is extracted with a solvent, which is water, alcohol, or an alcohol-water mixture.
In yet another embodiment of the present invention, the plant juice is at a concentration of at least 0.5% (v/v).
In another embodiment of the present invention, the plant juice is composed of the saussurea involucrata juice, the longan shell juice, and the buckwheat juice mixed in a ratio of 2.4-3.6:129.6-194.4:128.8-193.2.
The plant juice of the invention is composed of saussurea involucrata juice, longan shell juice, buckwheat juice and any combination thereof; the saussurea involucrata juice, the longan shell juice and the buckwheat juice can effectively improve the expression quantity of genes related to mitochondria or cell aging resistance in blood cells, skin cells, cardiovascular cells and/or retina cells, and the three juices have obvious aging resistance effects on the cardiovascular cells. The combination of the three juices can also directly and effectively improve the expression levels of CCT2 genes, CCT7 genes, CCT8 genes, atg1 genes, atg8 genes, NADSYN genes, MRPS5 genes, SOD3 genes and TERT genes, and can comprehensively improve the anti-aging activity, and the plant juice can simultaneously improve the GSH content so as to comprehensively improve the in-vivo antioxidant activity; the plant juice can effectively promote the reversion in vivo to the youthful and active state. Thus, the plant juice of the present invention can be used for preparing an anti-aging composition, wherein the composition is a pharmaceutical product, a food product or a maintenance product, which can be administered to a subject by oral administration, skin application, etc.
The invention will now be described in more detail with reference to the drawings and specific examples, which are not intended to limit the invention thereto.
Drawings
FIG. 1 is a histogram showing the effect of the saussurea involucrata juice of increasing the expression level of CCT2 gene, CCT5 gene, and CCT6A gene in blood cells according to an embodiment of the present invention. * p value <0.05.
FIG. 2 is a histogram showing the effect of increasing the expression level of CCT5 gene, CCT6A gene, CCT8 gene, pink1 gene, atg8 gene, and FOXO3 gene in blood cells using longan shell juice according to an embodiment of the present invention. * p value <0.05; * P <0.01; * P <0.001.
FIG. 3 is a histogram showing the effect of the buckwheat juice on increasing the expression level of CCT2 genes and CCT5 genes in blood cells according to an embodiment of the present invention. * p value <0.05.
FIG. 4 is a histogram showing the effect of the saussurea involucrata juice 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.
FIG. 5 is a histogram showing the effect of improving expression levels of CCT2 gene, CCT5 gene, CCT7 gene, pink1 gene, atg8 gene, SIRT1 gene, and FOXO3 gene in skin cells using longan peel juice according to an embodiment of the present invention. * p value <0.05; * P <0.01; * P <0.001.
FIG. 6 is a histogram showing the effect of increasing the expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, and Pink1 gene in skin cells using the juice of buckwheat according to an embodiment of the present invention. * P value <0.001.
FIG. 7 is a histogram showing the effect of the saussurea involucrata juice of the present invention on increasing the expression levels 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.
FIG. 8 is a histogram showing the effect of the juice of longan seed on improving the expression level of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, atg1 gene, SIRT1 gene and FOXO3 gene in cardiovascular cells according to an embodiment of the present invention. * p value <0.05; * P <0.01; * P <0.001.
FIG. 9 is a histogram showing the effect of improving the expression level of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, pink1 gene, atg1 gene, SIRT1 gene, and FOXO3 gene in cardiovascular cells according to an embodiment of the present invention. * p value <0.05; * P <0.01; * P <0.001.
FIG. 10 is a histogram showing the effect of the longan seed juice and the buckwheat seed juice on increasing the expression level 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 histogram showing the effect of the composition of plant juice on improving the expression level of CCTS genes according to an embodiment of the present invention. * p <0.05; * P <0.001.
FIG. 12 is a histogram showing the effect of the composition of plant juice on improving the expression level of the Atg1 gene and the Atg8 gene according to an embodiment of the present invention. * p <0.05; * P <0.001.
FIG. 13 is a histogram showing the effect of improving the expression levels of NADSYN gene, MRPS5 gene and SOD3 gene by using the plant juice composition according to the embodiment of the present invention. * p <0.05; * P <0.001.
FIG. 14 is a histogram showing the effect of a composition of plant juice on enhancing the expression level of TERT gene according to an embodiment of the invention. * P <0.001.
Fig. 15 is a histogram of the effect of a composition of plant juice on elevating GSH content according to an embodiment of the present invention. * P <0.01; * P <0.001.
Detailed Description
The values used herein are approximations, and all experimental data are presented in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.
Statistical analysis was performed using Excel software. Data are expressed as mean.+ -. Standard Deviation (SD), and the differences between these are analyzed by student's t-test (student's s t-test).
Saussurea involucrata (Smallanthus sonchifolius) is a perennial herb of the genus Smallanthus (Smallanthus) of the family Compositae (Compositae), also known as Smallanthus, with the name Peruvian ground apple, and a large underground root tuber, and has effects of cooling, relieving fever, reducing blood lipid, and facilitating defecation.
Longan (Euphoria longana lam.) is a plant of the genus Euphoria (Euphoria) of the family Sapindaceae, the English name being Longan, and the root of Longan has effects of dredging meridian; the leaves have the effects of clearing heat and detoxicating; the fruit core has hemostatic and analgesic effects; the fruit is sweet in taste and has the effects of tonifying qi and blood and soothing nerves; the pericarp (namely longan shell) has the effects of clearing heat and cooling blood.
Semen Fagopyri Esculenti (Fagopyrum esculentum Moench.) is annual herbaceous plant of Fagopyrum (Fagopyrum) of Polygonaceae, also called Trigonella Foenum Graecum, and has hemostatic and blood pressure lowering effects; the seeds have the effects of detumescence, detoxification and fever abatement.
As used herein, the term "saussurea involucrata juice" means that the callus tissue of saussurea involucrata is extracted with a solvent in a ratio of 1:1-5 (w/w) for a specified time and temperature.
As used herein, the term "longan seed juice" means that longan seed 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 juice" means that the seed coat of buckwheat and the solvent are extracted at a ratio of 1:10-15 (w/w) for a specific time and temperature.
As used herein, the term "plant juice composition" means a composition obtained by mixing the saussurea involucrata juice, the longan shell juice, and the buckwheat juice at a ratio of 2.4-3.6:129.6-194.4:128.8-193.2.
The plant juice is obtained from saussurea involucrata juice, longan shell juice, buckwheat juice and any combination thereof, can be used for improving the expression level of genes related to ageing resistance, can comprehensively improve the ageing resistance activity, can simultaneously improve the GSH content, can comprehensively improve the antioxidant activity of a person, and can enable the person to return to a youth active state in vivo.
Also, the present invention is useful for preparing anti-aging compositions, which may also comprise an effective amount of plant juice and a pharmaceutically acceptable carrier, as a pharmaceutical, a food or a maintenance product.
The detailed extraction method of the saussurea involucrata juice, the longan shell juice, and the buckwheat juice of the present invention will be described in detail below; the invention relates to an efficacy test for improving the expression level of anti-aging genes in systemic, skin cells, cardiovascular cells and/or retina cells by using saussurea involucrata juice, longan shell juice and buckwheat juice; and efficacy tests for improving expression level of anti-aging genes and improving GSH content of the plant juice composition. So as to prove that the plant juice can effectively and comprehensively promote the aging resistance and the oxidation resistance of the plant juice, and enable the plant juice to return to the youthful and active state in vivo.
EXAMPLE 1 preparation method of the present invention of Tianshan saussurea involucrata juice
In one embodiment of the invention, the callus of saussurea involucrata is first mixed with a leaching solvent, preferably water, in a weight ratio of 1:1-5, which is preferably water, and after homogenization, leaching in the solvent at 50-100 ℃ for 0.5-2 hours, the crude juice is cooled to room temperature and filtered through a 400mesh sieve to obtain a filtrate, wherein the leaching temperature is preferably 70-100 ℃. Finally, the filtrate is decompressed and concentrated at 45-70 ℃ to obtain the saussurea involucrata juice.
EXAMPLE 2 preparation method of longan Shell juice of the invention
In one embodiment of the invention, the longan shell tissue is first mixed with a leaching solvent, preferably water, of water, alcohol or alcohol-water mixture in a weight ratio of 1:5-10, homogenized, leached in the solvent at 50-100 ℃ for 0.5-2 hours, the crude juice is cooled to room temperature and filtered through a 400mesh sieve to obtain a filtrate, wherein the leaching temperature is preferably 70-100 ℃. Finally, the filtrate is decompressed and concentrated at 45-70 ℃ to obtain the longan shell juice.
Example 3 preparation method of buckwheat juice of the present invention
In one embodiment of the invention, the buckwheat seed hulls are first mixed with a leaching solvent, preferably water, in a weight ratio of 1:10-15, of water, homogenized, leached in a solvent at 50-100 ℃ for 0.5-2 hours, the crude juice is cooled to room temperature and filtered through a 400mesh screen to obtain a filtrate, wherein the leaching temperature is preferably 70-100 ℃. Finally, the filtrate is decompressed and concentrated at 45-70 ℃ to obtain the buckwheat juice.
Example 4 efficacy of the Tianshan herba Saussureae Involueratae juice, arillus longan juice and semen Fagopyri Esculenti juice of the invention in improving systemic anti-aging Gene expression
One embodiment of the present invention uses human peripheral blood mononuclear cells (Human peripheral blood mononuclear cell, PBMC) to test the efficacy test of the saussurea involucrata juice, longan shell juice, and buckwheat juice of the present invention to promote the expression of systemic anti-aging genes. Wherein the human peripheral blood mononuclear cells are obtained from the American type culture Collection under the accession number
PCS-800-011 TM And is cultured in X-VIVO TM 10Serum-free hematopoietic cell medium (available from Lonza, switzerland under the number BE 02-055Q).
First, 1X 105 PBMC cells were cultured in a 6-well culture dish containing 2mL of the above cell culture solution, and cultured at 37℃for 16-18 hours,the cells were then divided into the following four groups: (1) a control group to which only the cell culture solution 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 seed 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, and after allowing the cells of these groups to act at 37℃for 24 hours or 48 hours, respectively, the expression level of the target gene in the cells of each group of PBMC was tested; wherein the saussurea involucrata juice, the longan shell juice and the buckwheat juice are leaching solvents. First, after PBMC cells were collected as a cell lysate (RB buffer, available from Geanid Corp., taiwan, cat No. RBD 300), RNA was collected from each of the four groups of cells using an RNA extraction kit (available from Genaid Corp., taiwan, cat No. RBD 300), followed by the use of
III reverse transcriptase (available from Invitrogene, U.S. Pat. No. 18080-051) reverse transcription was performed using 2000ng of leached RNA as a template and the combined primers and reverse transcriptase of Table 1 to generate cDNA products corresponding to mRNA of these genes, and the four sets of reverse transcribed products were then subjected to quantitative real-Time polymerase chain reaction (Quantitative real-Time polymerase chain reaction, qPCR) using ABI StepOnePlusTM Real-Time PCR systems (Thermo Fisher Scientific, U.S. Pat. No. 3) and KAPA SYBR FAST (available from Sigma, U.S. Pat. No. 38220000000) with the combined primers of Table 1, respectively, at 95℃for 1 second and 60℃for 20 seconds for a total of 40 cycles. For quantifying the expression level 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 a threshold cycle number (Ct) and the relative amount of mRNA of the target gene is derived from equation 2 -ΔΔCt Wherein DeltaC T ＝C T comparison group or experimental group target gene/control group target gene -C T GAPDH (Glyceraldehyde-3-phosphate dehydrogenase, glycal dehyde-3-phosphate dehydrogenase); ΔΔct=c T comparison group or experimental group target gene -C T control group target gene The method comprises the steps of carrying out a first treatment on the surface of the The fold change of each gene in each group is 2 -ΔΔCt . Then, excel software is used to determine whether the coefficient of variation and the statistically significant difference (p-value)<0.05; * P value<0.01; * P value<0.001)。
Table 1, combination primer for quantitative real-time reverse transcription polymerase chain reaction
CCT gene is a kind of folded protein (Chaperonin), and has the main functions of correcting misfolded proteins and feeding proteins that cannot be successfully repaired into a proteolytic enzyme complex (proteome) for hydrolysis, and previous studies have indicated that if the re-use system of this protein regulation fails, misfolded proteins will accumulate in cells and cause cell function to decline and accelerate aging and death of cells, and thus are considered to be relevant for aging regulation of individuals.
The Pink1 gene is a mitochondrial-located Serine/threonine protein kinase (Serine/threonine protein kinase) whose function is to protect mitochondria from failure during cellular stress, mutations in which are known to be associated with Parkinson's disease, whereas previous studies have pointed to more that overexpression of this gene can increase the longevity of mice and thus is also believed to be associated with aging regulation in individuals.
The Parkin gene is an enzyme present in the Ubiquitin-protease system (Ubiquitin-proteasome system) and acts as a regulator of proteolysis, mutations in this gene are known to be associated with parkinson's disease, and previous studies have also indicated that increasing the expression of this gene can delay the aging effect of cells and are therefore believed to be associated with the aging effect of cells.
The Atg gene is an autophagy-related gene involved in degradation of wastes in cells and circulating autophagy, and previous studies indicate that excessive expression of the gene can prolong the life of mice and thus is considered to be related to aging of cells.
Previous studies have indicated that young mice contain more nad+ in vivo than older mice; and increasing the concentration of NAD+ in aged mice can make their physiological conditions younger, so NAD+ is considered to be related to delaying aging of individuals, and research results show that SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, UBL-5 gene and SOD3 gene are related to NAD+ regulation.
The experimental results of increasing the expression level of anti-aging genes in blood cells from saussurea involucrata juice of the present invention are shown in FIG. 1. Compared with the control group, the saussurea involucrata juice has the advantages that the expression amounts of CCT2 genes, CCT5 genes and CCT6A genes in PBMC cells can be improved by about 1.2-1.4 times after 48 hours of the saussurea involucrata juice is acted, and the expression amounts of other genes are not different from the control group (the result is not shown), wherein the expression amounts of the CCT6A genes are obviously different. The result shows that the saussurea involucrata juice can effectively improve the expression amount of CCT2 genes, CCT5 genes and CCT6A genes in PBMC cells and can effectively improve the systemic anti-aging capacity.
The experimental results of increasing expression level of anti-aging gene in blood cells by longan shell juice of the present invention are shown in FIG. 2. After 24 hours of action of the longan shell juice, compared with a control group, the expression amounts of CCT5 gene, CCT6A gene, CCT8 gene, pink1 gene, atg8 gene and FOXO3 gene in PBMC cells can be obviously improved, the expression amounts of the genes are about 1.3-20 times different, and the expression amounts of other genes are not greatly different from the control group (the result is not shown). The result shows that the longan shell juice can effectively improve the expression quantity of CCT5 genes, CCT6A genes, CCT8 genes, pink1 genes, atg8 genes and FOXO3 genes in PBMC cells, and can effectively improve the systemic anti-aging capacity.
The experimental results of increasing expression level of anti-aging gene in blood cells from buckwheat juice of the present invention are shown in FIG. 3. After the buckwheat juice of the invention is acted for 48 hours, compared with a control group, the expression quantity of CCT2 genes and CCT5 genes in PBMC cells can be improved by about 1.2 times, and the expression quantity of other genes is not greatly different from that of the control group (the result is not shown), wherein the expression quantity of the CCT2 genes is obviously different. The results show that the buckwheat juice can effectively improve the expression level of CCT2 genes and CCT5 genes in PBMC cells and can effectively improve the systemic anti-aging capacity.
EXAMPLE 5 efficacy of the Tianshan herba Saussureae Involueratae juice, arillus longan juice and semen Fagopyri Esculenti juice of the invention in improving expression level of anti-aging Gene in skin cells
One embodiment of the present invention uses human dermal fibroblasts (Human skin fibroblast) CCD-966SK cells to test the efficacy of the saussurea involucrata juice, longan shell juice, and buckwheat juice of the present invention to increase the expression level of anti-aging genes in skin cells. Wherein the CCD-966SK cell line is available from the American type culture Collection (ATCC, USA) under the number
CRL-1881 and the cell line was cultured in a medium (available from Gibco, USA) containing 10% fetal bovine serum (Fetal Bovine Serum) and 90% Minimum Essential Medium (MEM) containing 1mM Sodium pyruvate (available from Gibco, USA) and 1% penicillin-streptomycin.
First, 1.5x105 CCD-966SK cells were cultured in a 6-well culture dish containing 2mL of the above culture solution per well, cultured at 37℃for 16-18 hours, and then the cells were divided into the following four groups: (1) a control group to which only the cell culture solution 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 seed 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, and after allowing the cells of these groups to act at 37℃for 24 hours or 48 hours, the expression level of the target gene in each group of CCD-966SK cells was tested; wherein the saussurea involucrata juice, the longan shell juice and the buckwheat juice use water as leaching solvent. Then, after recovering the cells from the four sets of CCD-966SK cells with cell lysates, the mRNA expression amounts 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 sets of CCD-966SK cells were quantified by the procedure described in example 4, and then the variation coefficient and statistically significant difference were determined by Excel software (p value <0.05 p value <0.01 p value < 0.001).
The experimental results of the present invention of the saussurea involucrata juice to increase the expression level of anti-aging genes in skin cells are shown in FIG. 4. Compared with the control group, the invention can obviously improve the expression amounts of CCT6A gene, CCT7 gene, atg1 gene and Atg8 gene in CCD-966SK cells by about 1.1-2.2 times, the expression amounts of SIRT1 gene and FOXO3 gene by about 1.05 times, and the expression amounts of other genes are not greatly different from the control group (the result is not shown). The result shows that the saussurea involucrata juice can effectively improve the expression amounts of CCT6A genes, CCT7 genes, atg1 genes, atg8 genes, SIRT1 genes and FOXO3 genes in CCD-966SK cells, and can effectively improve the anti-aging capacity of skin cells.
The experimental results of the longan seed husk juice of the present invention for increasing the expression level of anti-aging gene in skin cells are shown in FIG. 5. After the longan shell juice is acted for 24 hours, compared with a control group, the expression amounts of CCT2 genes, CCT5 genes, CCT7 genes, pink1 genes, atg8 genes, SIRT1 genes and FOXO3 genes in CCD-966SK cells can be improved to be about 1.1-1.9 times different, and the expression amounts of other genes are not greatly different from the control group (the result is not shown), wherein the expression amounts of the Pink1 genes, the SIRT1 genes and the FOXO3 genes are obviously different. The result shows that the longan shell juice can effectively improve the expression quantity of CCT2 genes, CCT5 genes, CCT7 genes, pink1 genes, atg8 genes, SIRT1 genes and FOXO3 genes in CCD-966SK cells, and can effectively improve the anti-aging capacity of skin cells.
The experimental results of the expression level of anti-aging gene in skin cells of the buckwheat juice of the present invention are shown in FIG. 6. After the buckwheat juice of the invention is acted for 48 hours, compared with a control group, the expression amounts of CCT2 genes, CCT6A genes, CCT7 genes and CCT8 genes in CCD-966SK cells can be obviously improved by about 1.2-2.9 times, the expression amounts of CCT5 genes and Pink1 genes are about 1.2 times, and the expression amounts of other genes are not greatly different from the control group (the result is not shown). The results show that the buckwheat juice can effectively improve the expression level of CCT2 genes, CCT5 genes, CCT6A genes, CCT7 genes, CCT8 genes and Pink1 genes in CCD-966SK cells, and can effectively improve the anti-aging capacity of skin cells.
EXAMPLE 6 efficacy of the Tianshan herba Saussureae Involueratae juice, arillus longan juice and semen Fagopyri Esculenti juice of the invention in improving expression level of anti-aging Gene 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 the efficacy of the saussurea involucrata juice, longan shell juice, and buckwheat juice of the present invention to increase expression of anti-aging genes in cardiovascular cells. Wherein the HUVEC cell line is purchased from Taiwan biological resource conservation and research center of China, and is numbered BCRC No. H-UV001
CRL-1730 TM And the cell line was cultured in Medium 200 (available from Thermo, U.S. under the number M200500) containing 10mL of 50 XLSGS (available from Gibco, U.S.) and 1% penicillin-streptomycin.
First, 1.5x105 HUVEC cells were cultured in 6-well plates containing 2mL of the above culture solution per well, and cultured at 37℃for 16-18 hours, followed by dividing the cells into four groups: (1) a control group to which only the cell culture solution 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 seed 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, and after allowing the cells of these groups to act at 37℃for 48 hours, the expression level of the target gene in each group of HUVEC cells was tested; wherein the saussurea involucrata juice, the longan shell juice and the buckwheat juice use water as leaching solvent. Next, after recovering the cells from the four HUVEC cells with the cell lysate, the 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 mRNA expression amounts in the four HUVEC cells were quantified by the procedure described in example 4, and then the Excel software was used to determine whether the coefficient of variation was statistically significantly different from that of <0.05 × p <0.01 × p <0.001 ×.
The experimental results of the present invention of the saussurea involucrata juice to increase the expression level of anti-aging genes in cardiovascular cells are shown in FIG. 7. Compared with the control group, the saussurea involucrata juice has the advantages that the expression amounts of CCT2 genes, CCT6A genes, CCT7 genes, CCT8 genes, pink1 genes, atg1 genes and SIRT1 genes in HUVEC cells can be obviously improved to be about 1.6-4.4 times different, and the expression amounts of other genes are not greatly different from the control group (the result is not shown). The result shows that the Tianshan saussurea involucrata juice can effectively improve the expression amounts of CCT2 genes, CCT6A genes, CCT7 genes, CCT8 genes, pink1 genes, atg1 genes and SIRT1 genes in HUVEC cells and can effectively improve the anti-aging capacity of cardiovascular cells.
The experimental results of the longan seed husk juice of the present invention for increasing the expression level of anti-aging gene in cardiovascular cells are shown in FIG. 8. Compared with the control group, the juice of longan shells of the invention can obviously improve the expression amounts of CCT5 genes, CCT6A genes, CCT7 genes, CCT8 genes, atg1 genes, SIRT1 genes and FOXO3 genes in HUVEC cells to be about 1.8-4.3 times, the expression amount of CCT2 genes is about 1.8 times, and the expression amounts of other genes are not greatly different from the control group (the result is not shown). The result shows that the longan shell juice can effectively improve the expression level of CCT2 genes, CCT5 genes, CCT6A genes, CCT7 genes, CCT8 genes, atg1 genes, SIRT1 genes and FOXO3 genes in HUVEC cells, and can effectively improve the anti-aging capacity of cardiovascular cells.
The experimental results of the present invention for improving the expression level of anti-aging gene in cardiovascular cells using buckwheat juice are shown in FIG. 9. Compared with the control group, the buckwheat juice provided by the invention can obviously improve the expression amounts of CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, pink1 gene, atg1 gene, SIRT1 gene and FOXO3 gene in HUVEC cells by about 2-4 times, the expression amount of CCT2 gene is about 1.1 times, and the expression amounts of other genes are not greatly different from the control group (the result is not shown). The results show that the buckwheat juice can effectively improve the expression level of CCT2 genes, CCT5 genes, CCT6A genes, CCT7 genes, CCT8 genes, pink1 genes, atg1 genes, SIRT1 genes and FOXO3 genes in HUVEC cells, and can effectively improve the anti-aging capacity of cardiovascular cells.
EXAMPLE 7 the longan seed juice and buckwheat juice of the present invention have the effect of improving the expression level of anti-aging genes in retinal cells
One embodiment of the present invention uses human retinal pigment epithelial cells (Human retinal pigment epithelial) ARPE-19 cells to test the efficacy of the saussurea involucrata juice, longan hull juice, and buckwheat juice of the present invention to enhance the expression of anti-aging genes in skin cells. Wherein the ARPE-19 cell line is available from ATCC company (U.S.) under the designation CRL-2302 and is cultured in a DMEM/F12 medium containing 10% fetal bovine serum and 90% DMEM/F12 medium, wherein the medium contains 1:1 ratio of DMEM medium (Dulbecco's Modified Eagle Medium, available from Gibco, U.S. 12100-046) to Hans F12 medium (Ham's F Nutrient Mixture, available from Gibco, U.S. 12500-026) and 0.5mM sodium pyruvate and 15mM 4-hydroxyethylpiperazine ethanesulfonic acid (hydroxyethyl piperazineethanesulfonic acid, HEPES) buffer.
First, 1.5x10 5 The ARPE-19 cells were cultured in 6-well plates containing 2mL of the above culture solution per well, at 37℃for 16-18 hours, and then the cells were divided into the following five groups: (1) a blank control group to which only the cell culture liquid was added and to which no blue light was irradiated, (2) a positive control group to which only the cell culture liquid was added and to which the blue light was irradiated, (3) an experimental group to which 0.5% (v/v) of the saussurea involucrata juice of the present invention was added, (4) an experimental group to which 0.5% (v/v) of the longan shell juice of the present invention was added, and (5) an experimental group to which 0.5% (v/v) of the buckwheat juice of the present invention was added; wherein the cell lines of groups (2) - (5) are first placed in a blue light box at room temperatureAfter irradiating blue light for 15 minutes, respectively using samples to act at 37 ℃ for 48 hours, and testing the expression quantity of target genes in each group of ARPE-19 cells; wherein the longan shell juice and the buckwheat juice use water as leaching solvent. Next, after recovering cells from the five sets of ARPE-19 cells as a cell lysate, the mRNA expression levels of the 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 sets of ARPE-19 cells were quantified by the procedure described in example 4, and the Excel software was used to determine whether the coefficient of variation was statistically significantly different (p-value)<0.05; * P value<0.01; * P value<0.001)。
The experimental results of the longan peel juice and the buckwheat juice of the present invention for improving the expression level of anti-aging genes in retinal cells are shown in FIG. 10. The expression level of the CCT2 gene, the CCT5 gene, the CCT6A gene, the CCT7 gene, the CCT8 gene, the Pink1 gene, the Atg8 gene, the SIRT1 gene and the FOXO3 gene in the positive control group cells is lower than that of the blank control group, so that the expression level of the anti-aging gene in the retinal pigment epithelial cells can be reduced by the irradiation of blue light; compared with the control group, the juice of the longan seed husk can improve the expression amounts of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, pink1 gene, atg1 gene, SIRT1 gene and FOXO3 gene in ARPE-19 cells to be 1.1-1.7 times different, and the expression amounts of other genes are not greatly different from the control group (the result is not shown); after the buckwheat juice of the invention acts, the expression amounts of CCT2 gene, CCT5 gene, CCT6A gene, atg1 gene and FOXO3 gene can be improved by 1.2-1.5 times, and the expression amounts of other genes are not greatly different from those of a control group (the result is not shown); after the saussurea involucrata juice of the invention acts, the expression amounts of the genes are not greatly different from that of the control group (the result is not shown). These results show that the longan shell juice and the buckwheat juice of the invention can effectively improve the expression level of CCTS gene, pink1 gene, atg8 gene, SIRT1 gene or FOXO3 gene in ARPE-19 cells, and can effectively improve the anti-aging capability of retina cells.
Example 8 method for preparing a combination of plant juice of the invention
In one embodiment of the present invention, the combination of the plant juice of the present invention is obtained by mixing the saussurea involucrata juice of the present invention, the longan shell juice of the present invention, and the buckwheat juice of the present invention in a weight ratio of 2.4-3.6:129.6-194.4:128.8-193.2.
Example 9 efficacy of the plant juice combination of the present invention to enhance anti-aging Activity
In order to test the efficacy of the plant juice composition of the present invention in improving anti-aging activity, 7 adults over 30-50 years old are first recruited and 2 plants of the present invention (each containing 2.4-3.6mg of the saussurea involucrata juice of the present invention, 129.6-194.4mg of the longan shell juice of the present invention, 128.8-193.2mg of the buckwheat juice of the present invention) are taken daily, and blood samples of each subject are collected for anti-aging gene expression analysis and biochemical protein analysis at week 0, week 2 and week 4 of the administration of the plant juice, respectively; in the combination of the plant juice according to the present invention, the saussurea involucrata juice, the longan shell juice, and the buckwheat juice are all leaching juice with water as solvent.
Wherein the anti-aging gene expression level analysis is to separate peripheral blood mononuclear cells in blood of a subject with lymphocyte separation liquid, and then determine the mRNA expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, rank 1 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 by the steps described in example 4 and the combined primers of table 1 and table 2, and determine whether the variation coefficient and the statistically significant difference are present by Excel software (p value <0.05; p value <0.01; p value < 0.001).
TABLE 2 Combined primer for quantitative real-time reverse transcription polymerase chain reaction
The biochemical protein analysis is to analyze the content of Glutathione (GSH) in blood cells by using an intracellular GSH measuring reagent kit (Intracellular GSH assay kit, purchased from abcam, no. ab 112132), wherein the blood of a subject is adjusted to 106cells/mL by using PBS, and then the intracellular GSH measuring reagent kit is dyed for 15 minutes at room temperature, washed and redissolved by using PBS, and then placed on a flow cytometer (Accuri C6 Plus, BD), fluorescence is detected as FL1 channel (Ex/em=490/525 nm), and the fluorescence signal is used as the content of Glutathione in the blood of the subject.
Telomeres are DNA repeats at the ends of a chromosome, the primary function of which is to maintain chromosome integrity and control the cell division cycle, wherein the telomeres are shortened once DNA is replicated, and cells undergo apoptosis once the telomeres are depleted, so that the length of the telomeres is related to the age of the cells, and the TERT gene, and the TERC gene, are known to be related to the length of regulatory telomeres.
Biochemical protein analysis is to analyze the content of Glutathione (GSH) in the blood of a subject. Glutathione, also known as glutathione, is an important antioxidant enzyme in the body and has a high concentration in the liver, and is therefore also an important antidote, and cereal glutathione is also an important auxiliary component of metabolism in the body, and is thus relevant to maintaining the stability and smoothness of various physiological metabolism in the body. Numerous studies have shown that in vivo, there is a decrease in glutathione with aging, stress, etc., and that insufficient amounts of glutathione in vivo may increase the risk of cardiovascular, hepatic and neurodegenerative diseases.
The results of the combination of plant juices of the present invention to enhance the expression level of CCTS gene are shown in FIG. 11; the results of improving the expression level of the Atg1 gene and the Atg8 gene are shown in FIG. 12; the results of increasing expression levels of the NADSYN gene, MRPS5 gene and SOD3 gene are shown in FIG. 13; the results of enhancing TERT gene expression are shown in fig. 14; the results of increasing GSH content in blood are shown in FIG. 15.
After taking the combination of the plant juice of the invention for 2 weeks or 4 weeks, the expression level of CCT2 genes and CCT7 genes in a subject is obviously higher than that before the test by about 2.4-3.9 times, and the expression level of CCT8 genes is respectively higher than that before the test by about 1.8 and 1.3 times; the expression quantity of the Atg1 gene is obviously higher than that of about 2.8 and 3.8 times before the test, and the expression quantity of the Atg8 gene is higher than that of about 1.4 and 2.5 times before the test; the expression levels of NADSYN gene and MRPS5 gene are all obviously higher than that before the test by about 3.9-15 times, the expression level of SOD3 gene is respectively higher than that before the test by about 1.04 and 1.2 times, and the expression levels of other genes are not greatly different from those of a control group (the result is not shown); the expression level of TERT gene was also significantly higher than about 1.5 times before the test after taking the combination of the plant juice of the present invention for 4 weeks, and the expression level of other genes was not much different from that of the control group (results not shown). These results show that the combination of the plant juice of the invention can effectively improve the expression levels 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 improve the anti-aging activity.
After taking the combination of the plant juices of the present invention for 2 or 4 weeks, the GSH content in the blood was significantly higher than about 1.16 and 1.4 times before the test. The results show that the combination of the plant juice can effectively improve the GSH content in blood and can effectively and comprehensively improve the antioxidation capability.
In summary, the plant juice of the present invention is composed of saussurea involucrata juice, longan shell juice, buckwheat juice, and any combination thereof; the saussurea involucrata juice, the longan shell juice and the buckwheat juice can effectively improve the expression quantity of genes related to mitochondria or cell aging resistance in blood cells, skin cells, cardiovascular cells and/or retina cells, and the three juices have obvious aging resistance effects on the cardiovascular cells. The combination of the three juices can also directly and effectively improve the expression levels of CCT2 genes, CCT7 genes, CCT8 genes, atg1 genes, atg8 genes, NADSYN genes, MRPS5 genes, SOD3 genes and TERT genes, and can comprehensively improve the anti-aging activity, and the plant juice can simultaneously improve the GSH content so as to comprehensively improve the anti-oxidation activity; the plant juice of the invention can effectively promote the reversion in vivo to the youthful and active state. Thus, the plant juice of the present invention can be used for preparing an anti-aging composition, wherein the composition is a pharmaceutical product, a food product or a maintenance product, which can be administered to a subject by oral administration, skin application, or the like.
Of course, the present invention is capable of other various embodiments and its several details are capable of modification and variation in light of the present invention by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
[ sequence Listing ]
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<120> use of plant juice for increasing expression level of anti-aging gene and/or increasing GSH content
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Claims (5)
1. The use of plant juice, characterized in that the plant juice is used for preparing a composition for improving the expression level of a folding protein gene containing TCP1, an autophagy related gene, a glutamate dependent tobacco adenine dinucleotide synthetase gene, a mitochondrial ribosomal protein 5 gene, a superoxide dismutase 3 gene and a telomerase reverse transcriptase gene in cardiovascular cells; the plant juice consists of saussurea involucrata (Smallanthus sonchifolius) callus juice, longan shell juice and buckwheat juice; the saussurea involucrata callus juice is obtained by leaching saussurea involucrata callus and a solvent according to the weight ratio of 1:1-5, the longan shell juice is obtained by leaching longan shell and the solvent according to the weight ratio of 1:5-10, and the buckwheat juice is obtained by leaching buckwheat and the solvent according to the weight ratio of 1:10-15; the solvent is water; the plant juice has a concentration of at least 0.5% (v/v); the plant juice is prepared by mixing the saussurea involucrata callus juice, the longan shell juice and the buckwheat juice according to the proportion of 2.4-3.6:129.6-194.4:128.8-193.2.
2. The use according to claim 1, wherein the folding protein gene comprising TCP1 comprises a folding protein gene comprising the 2 nd unit of TCP1, a folding protein gene comprising the 7 th unit of TCP1, a folding protein gene comprising the 8 th unit of TCP1, and any combination thereof.
3. The use according to claim 1, wherein the autophagy-related gene comprises an autophagy-related gene 1 gene, an autophagy-related gene 8 gene, or a combination thereof.
4. The use according to claim 1, wherein the plant juice increases anti-aging activity.
5. The use according to claim 1, wherein the plant juice regulates nad+ concentration and/or telomere length of a cell.
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| CN202310522797.0A CN116440197A (en) | 2019-11-21 | 2020-11-20 | Use of plant juice for increasing expression level of retinal anti-aging gene and/or regulating NAD+ concentration of retinal cells |
| CN202310522799.XA CN116473889A (en) | 2019-11-21 | 2020-11-20 | Use of plant juice for increasing expression level of skin anti-aging gene and/or regulating NAD+ concentration of skin cells |
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| TW108142378A TWI779251B (en) | 2019-11-21 | 2019-11-21 | 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 |
| TW108142378 | 2019-11-21 |
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| CN202310522797.0A Division CN116440197A (en) | 2019-11-21 | 2020-11-20 | Use of plant juice for increasing expression level of retinal anti-aging gene and/or regulating NAD+ concentration of retinal cells |
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| CN202011310462.5A Active CN112823782B (en) | 2019-11-21 | 2020-11-20 | Use of plant juice for increasing expression level of anti-aging gene in cardiovascular cells |
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| TW202120069A (en) | 2021-06-01 |
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