TWI774335B - Use of tartary buckwheat husk extracts for reducing retinal damage caused by blue light - Google Patents

Abstract

The present invention provides use of a Tartary buckwheat husk extract for manufacture of a composition for enhancing expression of anti-aging genes. The Tartary buckwheat husk is preferably prepared by extraction of Tartary buckwheat husk using water as solvents. This extract reduces glycated protein formation, and therefore prevents functional deterioration and maintains health in a subject.

Description

Buckwheat seed coat extract for preventing retinal damage due to blue light exposure way

The present invention relates to the health-care use of a plant extract, in particular to the use of a tartary buckwheat seed coat extract for preparing and promoting the expression of anti-aging genes.

With the extension of the average age of the population, having a healthy and high-quality elderly life has become one of the focuses of public attention, and scientific research on aging has also received more and more attention. Aging can be broadly defined as the decline in physiology over time, which is associated with many prevalent diseases such as cardiovascular disease, cancer, metabolic disease, neurodegenerative disease, and the like. The aging process is complex and affected by many factors, including diet, exercise, psychological state, and genetic factors. At the molecular or cellular level, possible internal mechanisms include shortening of telomeres at the ends of chromosomes, genetic mutations, deregulated gene expression, loss of protein invariance, and decreased mitochondrial function. In addition, free radicals (such as reactive oxygen species) generated by physical or chemical factors in the surrounding environment of cells can damage the chromosomal deoxyribonucleic acid (DNA), proteins, and lipid biofilms of cells, thereby disrupting cell function.

In view of the above-mentioned factors that cause aging, anti-aging can be achieved in many ways, including changing diet and exercise habits, maintaining a good mood, reducing exposure to environmental factors that induce free radical formation (such as UV light), and promoting the operation of cell protective mechanisms ( such as the expression of genes that promote longevity). Among them, methods to promote cytoprotective mechanisms are in the developmental stage, although preliminary studies have The results show that it has anti-aging potential, but its exact efficacy has yet to be further confirmed. Therefore, it is necessary to develop a novel composition that can promote cytoprotective mechanisms and provide a new direction for slowing down cell function decline and maintaining cell health.

Therefore, an object of the present invention is to provide the use of a buckwheat ( Fagopyrum tataricum ) seed coat extract for preparing a composition for enhancing mitochondrial activity, promoting the expression of anti-aging genes, and inhibiting protein saccharification, wherein the tartary buckwheat seed coat extract The system was obtained by extracting a tartary buckwheat seed coat with a solvent.

In one embodiment of the present invention, the weight ratio of the solvent to the tartary buckwheat seed coat ranges from 20:1 to 1:1, and the extraction is performed at 50°C to 100°C.

In one embodiment of the present invention, the solvent is water, and the concentration of the tartary buckwheat seed coat extract is 0.125 mg/mL to 0.25 mg/mL, preferably 0.125 mg/mL.

In one embodiment of the present invention, the anti-aging gene encodes a protein subunit of chaperonin containing TCP1 complex (CCT), which is selected from the group consisting of CCT2, CCT5, CCT6A, CCT7, CCT8, and any combination of them.

In one embodiment of the present invention, the anti-aging gene encodes a phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1).

In an embodiment of the present invention, the anti-aging gene encodes a silent mating type information 2 homologous protein 1 in cells irradiated by blue light regulation 2 homolog 1 or sirtuin 1, SIRT1) or a forkhead box class O3 (forkhead box class O 3, FOXO3).

The tartary buckwheat seed coat extract of the present invention can enhance mitochondrial activity and promote the expression of various genes related to anti-aging in several human cells, such as activating the mitochondrial activity of vascular endothelial cells, and enhancing umbilical cord vein endothelial cells and human retinal cells. , The gene expression of anti-aging related gene groups in human fibroblasts and human peripheral blood mononuclear cells, and can reduce the production of glycated proteins, and ultimately achieve the effect of preventing individual functional decline and maintaining health. Therefore, the present invention provides the use of buckwheat seed coat extract for preparing a composition for enhancing mitochondrial activity, promoting expression of anti-aging genes, and inhibiting protein glycation. The composition can be powder, granule, solution, colloid or paste, and can be made into food, drink, medicine, or nutritional supplement, and administered to an individual by oral administration, skin application and the like.

The embodiments of the present invention will be further described below in conjunction with the drawings. The following examples are used to illustrate the inventive features and applications of the present invention, but not to limit the scope of the present invention. Within the spirit and scope of the present invention, some changes and modifications can be made, so the protection scope of the present invention should be determined by the scope of the appended patent application.

Figure 1 shows the percentage of cells with normal mitochondrial membrane potential in human umbilical vein endothelial cells treated with or without tartary buckwheat seed coat extract for 24 hours.

Figure 2 shows the CCT2, CCT5, CCT6A, CCT7, CCT8, PINK1, ATG8, SIRT1 and FOXO3 genes of human retinal pigment epithelial cells irradiated with blue light after 48 hours of treatment with or without tartary buckwheat seed coat extract relative to control cells relative performance.

Figure 3 shows the relative expression levels of CCT2, CCT5, CCT6A, CCT7, CCT8, and PINK1 genes in human dermal fibroblasts treated with or without buckwheat seed coat extract for 48 hours relative to control cells.

Figure 4 shows the relative expression levels of CCT2 and CCT5 genes in human peripheral blood mononuclear cells treated with or without tartary buckwheat seed coat extract for 48 hours relative to cells in the control group.

Figure 5 shows the relative expression of CCT2, CCT5, CCT6A, CCT7, CCT8, and PINK1 genes in human umbilical vein endothelial cells treated with or without buckwheat seed coat extract for 48 hours relative to control cells.

Figure 6 shows the inhibitory effect of different concentrations of tartary buckwheat seed coat extract on collagen glycation.

The present invention provides the use of a tartary buckwheat seed coat extract for preparing a composition for enhancing mitochondrial activity, promoting expression of anti-aging genes, and inhibiting protein saccharification. The tartary buckwheat seed coat extract of the present invention is obtained by extracting a tartary buckwheat seed coat with a solvent, wherein the solvent is preferably water, the weight ratio of the solvent to the tartary buckwheat seed coat is 20:1 to 1:1, and the The extraction is carried out at 50°C to 100°C. The tartary buckwheat seed coat extract treatment can enhance the mitochondrial activity of human cells and reduce the production of collagen glycation end products through the following examples. In addition, based on the results of quantitative gene expression analysis, the tartary buckwheat seed coat extract promotes the gene expression of anti-aging related gene groups in umbilical vein endothelial cells, human retinal cells, human fibroblasts and human peripheral blood mononuclear cells. The proteins encoded by these anti-aging genes include the chaperone T complex (CCT). Protein subunits, phosphatases and tensin homolog-inducible kinase 1 (PINK1), silent information regulator 2 homolog 1 (SIRT1) and forkhead box protein O3 (FOXO3).

definition

Numerical values used herein are approximations and all experimental data are expressed within 20%, preferably within 10%, and most preferably within 5%.

The term "anti-aging gene" herein generally refers to the gene whose existence is related to longevity of the organism or the function of its protein product can maintain the normal function of cells. The functions of the protein encoded by the anti-aging gene include assisting the folding of other proteins, enabling the normal functioning of mitochondria, and regulating the gene expression of proteins involved in nutrient metabolism or cellular stress response.

Materials and Methods cell culture

The following examples use human umbilical vein endothelial cells (human umbilical vein endothelial cells) purchased from the American Type Culture Collection (ATCC) or the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute. vein endothelial cells, HUVEC, BCRC H-UV001 or ATCC CRL-1730), human retinal pigment epithelial cells ARPE-19 (ATCC CRL-2302), and human skin fibroblast (human skin fibroblast) CCD-966SK (BCRC 60153) . Human peripheral mononuclear mononuclear cells (PBMC) are isolated from donor blood. All cells were cultured at 37°C, 5% carbon dioxide. HUVEC cells were cultured in M200 medium (Medium 200; Thermo Fischer Scientific) supplemented with 10% low serum growth supplement (LSGS; Thermo Fischer Scientific). Hereinafter referred to as M200 cell culture medium. ARPE-19 cells were cultured in DMEM/F12 medium in a 1:1 volume ratio mixed with DMEM medium (Dulbecco's modified Eagle's medium; Thermo Fischer Scientific) and Han's F12 medium (Ham's F12 medium; Thermo Fischer Scientific), supplemented with 0.5 mM acetone sodium, 15mM HEPES buffer solution, and 10% fetal bovine serum (FBS), hereinafter referred to as DMEM/F12 cell culture medium. CCD-966SK cells were cultured in minimal basal medium (Minimum Essential Medium (MEM); Thermo Fischer Scientific) supplemented with 10% FBS and 1% penicillin-streptomycin, hereinafter referred to as MEM cell culture medium. PBMC cells were cultured in RPMI medium (RPMI medium 1640; Thermo Fischer Scientific) supplemented with 10% FBS and 1% penicillin-streptomycin, hereinafter referred to as RPMI cell medium.

Mitochondrial activity assay

In order to evaluate the mitochondrial activity of cells, flow cytometer (BD Accuri) and mitochondrial membrane potential detection kit (BD ^TM MitoScreen) were used to determine the proportion of cell populations with changes in mitochondrial membrane potential. A brief description is as follows. Cells to be tested were rinsed with phosphate buffered saline (PBS; Thermo Fischer Scientific) according to the manufacturer's instructions, then collected into 1.5 mL microcentrifuge tubes and centrifuged (400×g, 5 min). After removing the supernatant, the cells were resuspended in PBS solution and centrifuged again (400xg, 5 minutes). The precipitated cells were uniformly mixed with 100 μL of JC-1 operating solution for 15 minutes in the dark for fluorescent labeling, and then the cells were washed twice with washing solution and centrifugation (400×g, 5 minutes). Finally, the cells were resuspended in PBS containing 2% FBS, and the percentage of cells with normal mitochondrial membrane potential was counted using a flow cytometer.

Gene expression analysis

Based on quantitative polymerase chain reaction (quantitative polymerase chain reaction, referred to as qPCR) to determine the expression of anti-aging genes in cells, the steps are briefly described as follows. According to the manufacturer's instructions, RNA was isolated from the cells using an RNA Extraction Kit (Geneaid), and 2000 ng of RNA was reverse transcribed into cDNA with the reverse transcriptase SuperScript® III Reverse Transcriptase (Invitrogen) at 37°C. Afterwards, using the primer pair of the target gene and the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal control (Table 1), using the qPCR kit (KAPA CYBR FAST qPCR Kit (2X) ; KAPA Biosystems) in a PCR reactor (StepOnePlus Real-Time PCR system; Applied Biosystems) to perform qPCR on the aforementioned cDNA to obtain a melting curve.

Finally, the relative expression amount of the target gene was determined using the 2 ^-ΔΔCT method. In this method, the cycle threshold ( _CT ) of the GAPDH gene is used as the cycle threshold of the reference gene of the internal control, and the relative fold change is calculated according to the following formula: △ _CT = the _CT of the target gene of the experimental group or the control group - the C of the internal control _T △△C _T = △C _{T of the experimental group - △C T fold} change of the control group = 2 ^{- △△Ct mean}

The Department of Statistical Analysis used the STDEV function in the Excel software to calculate the standard deviation of the relative expression levels of each gene, and calculated the statistical differences with the one-tailed Student's t-test (TTEST).

Example 1 Preparation of buckwheat seed coat extract

First, the husk of the whole grain of bitter buckwheat, that is, the seed coat of bitter buckwheat, is washed and dried, and pulverized using a pulverizer. The ground buckwheat seed coat can be selectively filtered with a 10 mesh (mesh) screen. Secondly, the ground tartary buckwheat seed coat was extracted with water as a solvent. The weight ratio of the solvent mixed with the ground buckwheat seed coat is in the range of 20:1 to 1:1. The extraction temperature is between 50°C to 100°C, preferably 80°C to 90°C. The buckwheat seed coat extracts in the following Examples 2-4 were all extracted with water, and the extraction time was 0.5 to 3 hours.

After the tartary buckwheat seed coat extract obtained in the above extraction step is cooled to room temperature, it can be centrifuged at 5000 rpm for 10 minutes, and then filtered through a 400-mesh filter to remove residual solids thing. The filtered tartary buckwheat seed coat extract can be further concentrated under reduced pressure at 45°C to 70°C to obtain a concentrated product. In order to obtain a solid tartary buckwheat seed coat extract, the aforementioned concentrated tartary buckwheat seed coat extract can be subjected to drying methods such as freeze drying and spray drying to remove the solvent, thereby obtaining a dried product of the tartary buckwheat seed coat extract.

Example 2 Tartary buckwheat seed coat extract enhances mitochondrial activity

Mitochondria are organelles that supply energy to cells, and their normal functioning is critical to maintaining cell viability and proliferation. In order to investigate the effect of tartary buckwheat seed coat extract on mitochondrial function, flow cytometry was used to evaluate the changes in mitochondrial activity of human umbilical vein endothelial cells (HUVEC) treated with tartary buckwheat seed coat extract. First, HUVEC cells were seeded at 1×10 ⁵ cells/well in each well of a 6-well plate containing 2 mL of M200 cell culture medium, and cultured at 37° C. for 24 hours. Thereafter, cells were treated with 1 mL of M200 medium containing 0.25 mg/mL or 0.125 mg/mL tartary buckwheat seed coat extract (experimental group), or cells were treated with M200 medium alone as a control group. Cells in each group were cultured at 37°C for 24 hours and used for mitochondrial activity analysis.

Figure 1 shows the percentage of mitochondrial membrane potential in the HUVEC cells of the aforementioned groups with normal values. The higher the value, the more cells with normal function mitochondria; ^* means p<0.05 compared to the control group, ^{* **} indicates p<0.001 compared to the control group. According to Figure 1, administration of 0.125mg/mL tartary buckwheat seed coat extract significantly increased cells with normal functioning mitochondria, while administration of 0.25mg/mL tartary buckwheat seed coat extract resulted in a smaller number of cells compared to cells in the control group. increase, this result indicates that buckwheat seed coat extract can increase the average energy production of cells, so that cells have the ability to perform specific physiological functions or proliferate to replace old cells, so it is beneficial to maintain the health status of animals or human individuals.

Example 3 Expression of anti-aging genes of buckwheat seed coat extract

3.1 Retinal Pigment Epithelial Cells

To investigate the effect of tartary buckwheat seed coat extract on the expression of anti-aging-related genes, qPCR was used to determine the chaperone protein T complex (CCT) subunit, phosphatase and phosphatase levels of ARPE-19 in human retinal pigment epithelial cells treated with tartary buckwheat seed coat extract. Genes of tensin homolog-inducible kinase 1 (PINK1), autophagy-related protein 8 (ATG8), silent information regulator 2 homolog 1 (SIRT1) and forkhead box O3 (FOXO3) Performance changes. Briefly, cells were seeded at ¹ x 105 cells/well in each well of a 6-well plate containing 2 mL of cell culture medium, and after 24 hours of incubation at 37°C, the cell culture medium was removed and cells were washed with PBS solution. After that, the cells were treated with 1 mL of DMEM/F12 cell culture medium containing 0.5% (w/v) tartary buckwheat seed coat extract for 48 hours, and then the 6-well culture plate was placed in a blue light box to receive blue light (wavelength approx. 425-475nm) for 15 minutes. The cell line as a blue light irradiated group was treated with DMEM/F12 medium without tartary buckwheat seed coat extract and irradiated with blue light for 15 minutes; the cell line as a control group was treated with DMEM/F12 medium without tartary buckwheat seed coat extract but not treated with blue light. irradiated by blue light. Finally, each group of cells was collected for qPCR analysis.

Figure 2 shows the relative expression levels of CCT2, CCT5, CCT6A, CCT7, CCT8, PINK1, ATG8, SIRT1, and FOXO3 genes in the aforementioned three groups of ARPE-19 cells relative to the control group cells; ^* and ^** represent the comparison with blue light irradiation, respectively Groups were p<0.05 and p<0.01. According to Figure 2, blue light exposure reduced the expression of most anti-aging genes compared to the control group. However, compared with the blue light irradiation group, the treatment of tartary buckwheat seed coat extract significantly enhanced the gene expression of the CCT2, CCT5, and CCT6A subunits of the chaperone T complex that assists in protein folding in ARPE-19 cells, and the significant increase was associated with longevity. The gene expression of SIRT1 and FOXO3, and slightly increased the gene expression of PINK and ATG8 related to mitochondrial activity. This result indicated that buckwheat seed coat extract can promote the synthesis of various anti-aging genes in retinal cells exposed to blue light, and thus has the potential to reduce the damage or death of the cells due to blue light exposure.

3.2 Dermal fibroblasts

The expression changes of CCT protein subunit and PINK1 gene in human dermal fibroblasts CCD-966SK treated with tartary buckwheat seed coat extract were determined by qPCR. Briefly, cells were seeded at ¹ x 105 cells/well in each well of a 6-well dish containing 2 mL of cell culture medium, after 24 hours of incubation at 37°C, the cell culture medium was removed and cells were washed with PBS solution. Thereafter, cells were treated with 1 mL of MEM medium containing 0.125 mg/mL tartary buckwheat seed coat extract (experimental group), or cells were treated with MEM medium alone as a control group. The aforementioned two groups of cells were used for qPCR analysis after culturing at 37°C for 48 hours.

Figure 3 shows the relative expression levels of CCT2, CCT5, CCT6A, CCT7, CCT8, and PINK1 genes in the aforementioned two groups of CCD-966SK cells relative to the control group cells; ^*** means p<0.001 compared to the control group. According to Figure 3, the treatment of tartary buckwheat seed coat extract significantly increased the gene expression of CCT2, CCT6A, CCT7, and CCT8 subunits of the chaperone T complex in CCD-966SK cells, while slightly increasing the expression of CCT5 and PINK1 genes. This result indicates that buckwheat seed coat extract is beneficial to maintain the vitality of skin cells and therefore has the potential to delay skin aging.

3.3 Peripheral blood mononuclear cells

The gene expression changes of CCT protein subunits in human peripheral blood mononuclear cells (PBMC) treated with tartary buckwheat seed coat extract were determined by qPCR. Briefly, cells were seeded at ¹ x 105 cells/well in each well of a 6-well dish containing 2 mL of cell culture medium, after 24 hours of incubation at 37°C, the cell culture medium was removed and cells were washed with PBS solution. Thereafter, cells were treated with 1 mL of RPMI medium containing 0.125 mg/mL tartary buckwheat seed coat extract (experimental group), or cells were treated with RPMI medium alone as a control group. The aforementioned two groups of cells were used for qPCR analysis after culturing at 37°C for 48 hours.

Figure 4 shows the relative expression levels of CCT2 and CCT5 genes in PBMC cells of the aforementioned two groups relative to cells in the control group; ^* and ^*** represent p<0.05 and p<0.001 compared to the control group, respectively. According to Figure 4, the treatment of tartary buckwheat seed coat extract significantly enhanced the gene expression of CCT2 and CCT5 subunits of the chaperone T complex in CCD-966SK cells. This result indicates that the buckwheat seed coat extract contributes to the normal function of monocytes (eg, T cells, B cells, and monocytes) in the blood.

3.4 Vascular endothelial cells

The expression changes of CCT protein subunit and PINK1 gene in human umbilical vein endothelial cells (HUVEC) treated with buckwheat seed coat extract were determined by qPCR. Briefly, cells were seeded at ¹ x 105 cells/well in each well of a 6-well dish containing 2 mL of cell culture medium, after 24 hours of incubation at 37°C, the cell culture medium was removed and cells were washed with PBS solution. Thereafter, the cells were treated with 1 mL of M200 medium containing 0.125 mg/mL tartary buckwheat seed coat extract (experimental group), or the cells were treated with M200 medium alone as a control group. The aforementioned two groups of cells were used for qPCR analysis after culturing at 37°C for 48 hours.

Figure 5 shows the relative expression levels of CCT2, CCT5, CCT6A, CCT7, CCT8, and PINK1 genes in the aforementioned two groups of HUVEC cells relative to the control group cells; ^* and ^** represent p<0.05 and p<0.01 compared to the control group, respectively . According to Figure 5, the treatment of tartary buckwheat seed coat extract significantly increased the gene expression of CCT2 and CCT5 subunits of the chaperone T complex in HUVEC cells, and also significantly increased the expression of PINK1 gene. This result indicated that buckwheat seed coat extract is beneficial to maintain the viability of vascular endothelial cells, and therefore has the potential to reduce the incidence of vascular-related diseases.

Example 4 Tartary buckwheat seed coat extract inhibits protein glycation

The glycation of proteins in the body can lead to loss of protein function, which in turn promotes aging and related diseases. To investigate whether tartary buckwheat seed coat extract can inhibit protein glycation, the inhibitory effect of 0.1, 0.5, 1, or 5 mg/mL tartary buckwheat seed coat extract on porcine collagen glycation was determined by anti-glycation assay. Briefly, a 60 mg/mL collagen solution (containing 0.06% sodium azide) and a 1.5 M fructose solution were prepared using 200 mM phosphate buffer (pH 7.4). In order to carry out the collagen saccharification reaction, the mixture of 0.2mL collagen solution and 0.2mL fructose solution and 0.2mL each concentration of tartary buckwheat seed coat extract samples or deionized water (control group) were evenly mixed, and reacted at 50 ℃ for 24 hours. Further aminoguanidine (AG, purchased from Sigma) was added to stop the saccharification reaction. Use a spectrofluorometer (spectrofluorometer, FLx 800, BioTek) to measure the fluorescence intensity (excitation wavelength 360 nm, emission wavelength 460 nm) of the aforementioned reaction solution (0.1 mL) at 0 hours and 24 hours, and calculate the final product of protein glycation according to the following formula Generation rate: [(sample fluorescence intensity _{24 hours} -sample fluorescence intensity _{0 hours} )/(control group fluorescence intensity _{24 hours} -control group fluorescence intensity _{0 hours} )]×100%.

Figure 6 shows the inhibitory effect of different concentrations of tartary buckwheat seed coat extract on collagen glycation. According to Figure 6, 0.1, 0.5, 1, and 5 mg/mL tartary buckwheat seed coat extract decreased, respectively Collagen glycation end products are generated at about 8%, 52%, 69%, and 81%, indicating that buckwheat seed coat extract can reduce the glycation reaction of proteins in the body, thereby delaying the progress of aging, such as skin aging.

In conclusion, tartary buckwheat seed coat extract can enhance mitochondrial activity and promote the expression of various genes related to anti-aging in several human cells, such as activating the mitochondrial activity of vascular endothelial cells, and enhancing umbilical vein endothelial cells, The gene expression of anti-aging-related gene groups in human retinal cells, human fibroblasts and human peripheral blood mononuclear cells, and can reduce the production of glycated proteins, and ultimately achieve the effect of preventing individual functional decline and maintaining health. Therefore, the present invention provides the use of buckwheat seed coat extract for preparing a composition for enhancing mitochondrial activity, promoting expression of anti-aging genes, and inhibiting protein glycation. The composition can be powder, granule, solution, colloid or paste, and can be made into food, drink, medicine, or nutritional supplement, and administered to an individual by oral administration, skin application and the like.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of tartary buckwheat seed coat extract to prevent retinal damage due to blue light exposure

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<170> PatentIn version 3.5

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Claims (4)

Use of a tartary buckwheat seed coat extract for preparing a composition for preventing retinal pigment epithelial cells from being damaged by blue light irradiation, wherein the tartary buckwheat seed coat extract can promote the expression of a gene group in retinal pigment epithelial cells under blue light irradiation, the bitter Buckwheat seed coat extract is obtained by extracting a tartary buckwheat seed coat with water; and wherein the gene group is CCT2, CCT5, and CCT6A subunits encoding chaperonin T complex (CCT), as well as SIRT1 gene, FOXO3 gene, PINK gene and At least one of the genes of ATG8. The use as claimed in claim 1, wherein the composition is in the form of powder, granule, solution, colloid, or paste. The use as claimed in claim 1, wherein the weight ratio of the water to the buckwheat seed coat ranges from 20:1 to 1:1. The use as claimed in claim 1, wherein the extraction is carried out at 50°C to 100°C.

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