TWI734032B - Use of tartary buckwheat husk extracts for improving mitochondrial activity - Google Patents

Abstract

The present invention provides use of a Tartary buckwheat husk extract for manufacture of a composition for improving mitochondrial activity. The Tartary buckwheat husk is preferably prepared by extraction of Tartary buckwheat husk using water as solvents. This extract promotes energy production, and therefore prevents functional deterioration and maintains health in a subject.

Description

Use of Tartary Buckwheat Seed Coat Extract to Improve Mitochondrial Activity

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 enhancing mitochondrial activity.

With the increase in 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 become more and more important. Aging can be roughly defined as the decline of physiological functions over time, which is associated with many epidemic diseases, such as cardiovascular diseases, cancer, metabolic diseases, neurodegenerative diseases and so on. The aging process is complicated and affected by many factors, including diet, exercise, mental state, and genetic factors. At the molecular or cellular level, possible internal mechanisms include shortening of telomeres at the ends of chromosomes, gene mutations, dysregulation of gene expression, constant loss of protein, and decreased mitochondrial function. In addition, free radicals (such as reactive oxygen species) generated by physical or chemical factors in the surrounding environment of the cell can damage the chromosomal DNA, protein, and lipid biofilms of the cell, thereby destroying the function of the cell.

In view of the above factors that cause aging, anti-aging can start from many aspects, including changing diet and exercise habits, maintaining a happy mood, reducing exposure to environmental factors that induce the formation of free radicals (such as ultraviolet light), and promoting the operation of cell protection mechanisms ( For example, the expression of genes that promote longevity). Among them, the method of promoting cell protection mechanism is in the development stage. Although preliminary research results show that it has quite anti-aging potential, its exact effect needs to be further confirmed. Therefore, it is really necessary to develop a novel composition that can promote cell protection mechanisms and provide a new direction for slowing the decline of cell function and maintaining cell health.

For this reason, one purpose of the present invention is to provide a use of a Tartary Buckwheat ( Fagopyrumtataricum ) Seed Coat Extract for preparing a composition that enhances mitochondrial activity, promotes anti-aging gene expression, and inhibits protein saccharification, wherein the Tartary Buckwheat Seed Coat Extract The material is obtained by extracting a tartary buckwheat seed coat with a solvent.

In an 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 an 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 chaperonin containing TCP1 complex (CCT) protein subunit, which is selected from CCT2, CCT5, CCT6A, CCT7, CCT8, and A group composed of any combination.

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 regulation 2 homolog 1 (silent mating type information regulation 2 homolog 1 or sirtuin 1, SIRT1) or a fork head in cells irradiated by blue light. Box protein O3 (forkhead box class O 3, FOXO3).

The tartary buckwheat seed coat extract of the present invention can enhance the activity of mitochondria and promote the expression of multiple anti-aging-related genes 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 , Human fibroblasts and human peripheral blood mononuclear cell anti-aging related gene group gene expression, and can reduce the production of glycated protein, and ultimately achieve the effect of preventing the decline of individual functions and maintaining health. Therefore, the present invention provides the use of a tartary buckwheat seed coat extract for preparing a composition for enhancing mitochondrial activity, promoting anti-aging gene expression, 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 a body by oral administration, skin application, etc.

The following will further illustrate the embodiments of the present invention in conjunction with the drawings. The following examples are used to illustrate the inventive features and applications of the present invention, rather than to limit the scope of the present invention. Anyone familiar with the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the protection scope of the present invention shall be subject to those defined by the appended patent scope.

Figure 1 shows the percentage of cells with normal mitochondrial membrane potential of human umbilical cord 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 The relative performance.

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

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

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

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

The present invention provides a use of a tartary buckwheat seed coat extract for preparing a composition for enhancing mitochondrial activity, promoting anti-aging gene expression, 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, and 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 treatment of the tartary buckwheat seed coat extract 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 analysis of gene expression, the tartary buckwheat seed coat extract promotes the gene expression of anti-aging-related gene groups in umbilical cord vein endothelial cells, human retinal cells, human fibroblasts, and human peripheral blood mononuclear cells. The proteins encoded by these anti-aging genes include the protein subunit of the companion protein T complex (CCT), phosphatase and tensin homologue inducing kinase 1 (PINK1), silent information regulator 2 homologous protein 1 (SIRT1) and Forkhead box protein O3 (FOXO3).

definition

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

The so-called "anti-aging genes" in this article generally refers to genes whose existence is related to the longevity of organisms or whose protein products can maintain the normal function of cells. The functions of the protein encoded by the anti-aging gene include assisting in the folding of other proteins, enabling the normal operation 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 cells) purchased from American Type Culture Collection (ATCC) or Bioresource Collection and Research Center (BCRC). 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 CCD-966SK (BCRC 60153) . Human peripheral mononuclear mononuclear cells (PBMC) are isolated from donor blood. All cells were cultured at 37°C and 5% carbon dioxide. HUVEC cells are 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 medium. ARPE-19 cells were cultured in DMEM/F12 medium (Dulbecco's modified Eagle's medium; Thermo Fischer Scientific) and Han's F12 medium (Ham's F12 medium; Thermo Fischer Scientific) in a 1:1 volume ratio mixed with DMEM/F12 medium, and 0.5mM acetone was added to it Sodium, 15mMHEPES buffer solution, and 10% fetal bovine serum (FBS), hereinafter referred to as DMEM/F12 cell culture medium. CCD-966SK cells are cultured in a minimum 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 culture medium.

Mitochondrial activity analysis

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

Gene expression analysis

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

Finally, use the 2- ^△△CT method to determine the relative expression of the target gene. The method is the threshold cycle (C _T) GAPDH gene as an internal control of the reference cycle threshold gene, calculated by the following formula relative fold change: C gene of △ C _T = the experimental group or the control group _T - internal control C _T

△△ C _T = test group △ C _T - △ C _T of the control group

Multiple change = 2- ^{△△Ct average}

The statistical analysis department uses the STDEV function in Excel software to calculate the standard deviation of the relative expression of each gene, and uses the one-tailed Student’s T test (TTEST) to calculate the statistical difference.

Example 1 Preparation of Tartary Buckwheat Seed Coat Extract

First, wash and dry the whole tartary buckwheat husk, which is the seed coat of tartary buckwheat, and crush it with a grinder. The crushed tartary buckwheat seed coat can be selectively filtered with a 10 mesh screen. Secondly, the crushed tartary buckwheat seed coat is extracted with water as a solvent. The weight ratio of the solvent and the pulverized tartary buckwheat seed coat is in the range of 20:1 to 1:1. The extraction temperature is between 50°C and 100°C, preferably between 80°C and 90°C. The extracts of tartary buckwheat seed coats in the following Examples 2-4 are all extracted with water, and the extraction time is 0.5 to 3 hours.

After the tartary buckwheat seed coat extract obtained by the above extraction step is cooled to room temperature, it can be centrifuged at 5000 rpm for 10 minutes, and then filtered with a 400 mesh filter to remove residual solids. 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 concentrated tartary buckwheat seed coat extract can be dried to remove the solvent by a drying method such as freeze drying and spray drying, 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 operation is essential for maintaining cell viability and proliferation. In order to explore the effect of the extract of tartary buckwheat seed coat on mitochondrial function, in this example, flow cytometry was used to evaluate the changes in the mitochondrial activity of human umbilical vein endothelial cells (HUVEC) after treatment with the extract of tartary buckwheat seed coat. First, HUVEC cells ^{were seeded at 1×10 5} cells/well in each well of a 6-well plate containing 2mLM200 cell culture medium, and cultured at 37°C for 24 hours. Thereafter, the 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 only M200 medium was used to treat the cells as a control group. Cells of each group were cultured at 37°C for 24 hours and then used for mitochondrial activity analysis.

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

Example 3 Expression of anti-aging genes of Tartary Buckwheat Seed Coat Extract 3.1 Retinal pigment epithelial cells

In order to explore the effect of tartary buckwheat seed coat extract on the expression of aging-related genes, qPCR was used to determine the associated protein T complex (CCT) subunit, phosphatase and phosphatase of human retinal pigment epithelial cells ARPE-19 after being treated with tartary buckwheat seed coat extract. Genes for tensin homologue-inducing kinase 1 (PINK1), autophagy-related protein 8 (ATG8), silencing information regulator 2 homologous protein 1 (SIRT1) and forkhead box protein O3 (FOXO3) Performance changes. In short, cells ^{were seeded into each well of a 6-well plate containing 2 mL of cell culture medium at 1×10 5} cells/well, and after culturing at 37° C. for 24 hours, the cell culture medium was removed and the cells were washed with PBS solution. Thereafter, 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 and received blue light (wavelength approximately) at room temperature. (425-475nm) irradiation for 15 minutes. The cell line in the blue light irradiation 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 the control group was treated with DMEM/F12 medium without tartary buckwheat seed coat extract but not Irradiated by blue light. Finally, the cells of each group were collected for qPCR analysis.

Figure 2 shows the relative expression levels of the CCT2, CCT5, CCT6A, CCT7, CCT8, PINK1, ATG8, SIRT1, and FOXO3 genes of the aforementioned three groups of ARPE-19 cells relative to the control group cells; ^* and ^** respectively indicate the relative expression levels of blue light irradiation The group was p<0.05 and p<0.01. According to Figure 2, compared with the control group, blue light irradiation will reduce the performance of most anti-aging genes. However, compared with the blue light irradiation group, the treatment of tartary buckwheat seed coat extract significantly improved the gene expression of the CCT2, CCT5, and CCT6A subunits of the accompanying protein T complex that assisted in protein folding in ARPE-19 cells, and at the same time, it significantly increased and 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 indicates that the seed coat extract of tartary buckwheat can promote the synthesis of various anti-aging genes in the retinal cells under blue light irradiation, and therefore has the potential to reduce the damage or death of the cells due to blue light irradiation.

3.2 Skin Fibroblasts

Using qPCR to determine the changes in CCT protein subunits and PINK1 gene expression of human skin fibroblasts CCD-966SK treated with tartary buckwheat seed coat extract. In short, cells ^{were seeded into each well of a 6-well plate containing 2 mL of cell culture medium at 1×10 5} cells/well, and after culturing at 37° C. for 24 hours, the cell culture medium was removed and the cells were washed with PBS solution. Thereafter, the cells were treated with 1 mL of MEM medium containing 0.125 mg/mL tartary buckwheat seed coat extract (experimental group), or the cells were treated with MEM medium only as a control group. The aforementioned two groups of cells were cultured at 37°C for 48 hours and then used for qPCR analysis.

Figure 3 shows the relative expression levels of the CCT2, CCT5, CCT6A, CCT7, CCT8, and PINK1 genes of the two groups of CCD-966SK cells compared to the control group; ^*** means p<0.001 compared to the control group. According to Figure 3, the treatment of the seed coat extract of tartary buckwheat significantly improved the gene expression of the CCT2, CCT6A, CCT7, and CCT8 subunits of the chaperone protein T complex in CCD-966SK cells, while slightly increasing the expression of the CCT5 and PINK1 genes. This result indicates that the extract of tartary buckwheat seed coat 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 the protein subunits of CCT of human peripheral blood mononuclear cells (PBMC) treated with extracts of tartary buckwheat seed coat were determined by qPCR. In short, cells ^{were seeded into each well of a 6-well plate containing 2 mL of cell culture medium at 1×10 5} cells/well, and after culturing at 37° C. for 24 hours, the cell culture medium was removed and the cells were washed with PBS solution. Thereafter, the cells were treated with 1 mL of RPMI medium containing 0.125 mg/mL tartary buckwheat seed coat extract (experimental group), or the cells were treated with only RPMI medium as a control group. The aforementioned two groups of cells were cultured at 37°C for 48 hours and then used for qPCR analysis.

Figure 4 shows the relative expression levels of the CCT2 and CCT5 genes of the two groups of PBMC cells relative to the control group; ^* and ^*** indicate p<0.05 and p<0.001 compared to the control group, respectively. According to Fig. 4, the treatment of the seed coat extract of tartary buckwheat significantly improved the gene expression of the 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 blood monocytes (such as T cells, B cells, and monocytes).

3.4 Vascular endothelial cells

QPCR was used to determine the changes in CCT protein subunit and PINK1 gene expression of human umbilical cord vein endothelial cells (HUVEC) after treatment with tartary buckwheat seed coat extract. In short, cells ^{were seeded into each well of a 6-well plate containing 2 mL of cell culture medium at 1×10 5} cells/well, and after culturing at 37° C. for 24 hours, the cell culture medium was removed and the 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 only M200 medium was used as a control group. The aforementioned two groups of cells were cultured at 37°C for 48 hours and then used for qPCR analysis.

Figure 5 shows the relative expression levels of the CCT2, CCT5, CCT6A, CCT7, CCT8, and PINK1 genes of the aforementioned two groups of HUVEC cells relative to the control group; ^* and ^** indicate p<0.05 and p<0.01 compared to the control group, respectively . According to Figure 5, the treatment of the seed coat extract of tartary buckwheat significantly improved the gene expression of the CCT2 and CCT5 subunits of the chaperone T complex in HUVEC cells, and also significantly increased the expression of the PINK1 gene. This result indicates that the seed coat extract of tartary buckwheat 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 reaction of protein in the body will lead to the loss of protein function, which will promote the occurrence of aging and related diseases. In order to explore whether tartary buckwheat seed coat extract can inhibit protein glycation, the anti-glycation analysis was used to determine the inhibitory effect of 0.1, 0.5, 1, or 5 mg/mL tartary buckwheat seed coat extract on the glycation reaction of porcine collagen. In short, 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 solution (pH 7.4). In order to carry out the collagen saccharification reaction, a mixture of 0.2 mL collagen solution and 0.2 mL fructose solution was mixed with 0.2 mL samples of tartary buckwheat seed coat extract or deionized water (control group) of various concentrations, and reacted at 50°C for 24 hours. Then aminoguanidine (AG, purchased from Sigma) was added to stop the saccharification reaction. Use a spectrofluorometer (FLx 800, BioTek) to measure the fluorescence intensity (excitation wavelength 360nm, emission wavelength 460nm) of the aforementioned reaction solution (0.1mL) at 0 hours and 24 hours, and calculate the final protein glycation product according to the following formula Generation rate: [(sample fluorescence intensity for _{24 hours} -sample fluorescence intensity for _{0 hours} )/(control group fluorescence intensity for _{24 hours} -control group fluorescence intensity for _{0 hours} )]×100%.

Figure 6 shows the inhibitory effect of different concentrations of tartary buckwheat seed coat extracts on collagen glycation. According to Figure 6, 0.1, 0.5, 1, and 5mg/mL tartary buckwheat seed coat extracts reduce the production of collagen glycation end products by about 8%, 52%, 69%, 81%, respectively, showing that tartary buckwheat seed coat extracts can reduce the body The glycation reaction of protein can delay the progress of aging, such as skin aging.

In summary, Tartary buckwheat seed coat extract can enhance mitochondrial activity and promote the expression of multiple anti-aging-related genes in several human cells, such as activating the mitochondrial activity of vascular endothelial cells, and enhancing umbilical cord vein endothelial cells, Human retinal cells, human fibroblasts, and human peripheral blood mononuclear cells have the gene expression level of anti-aging related gene groups, and can reduce the production of glycated protein, and ultimately achieve the effect of preventing individual function decline and maintaining health. Therefore, the present invention provides the use of a tartary buckwheat seed coat extract for preparing a composition for enhancing mitochondrial activity, promoting anti-aging gene expression, 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 a body by oral administration, skin application, etc.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of tartary buckwheat seed coat extract to enhance mitochondrial activity

<130> N/A

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<170> PatentIn version 3.5

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Claims (5)

The use of a tartary buckwheat seed coat extract for preparing a composition for enhancing the mitochondrial activity of vascular endothelial cells, wherein the tartary buckwheat seed coat extract is obtained by extracting a tartary buckwheat seed coat with water, and the tartary buckwheat seed coat extract enhances encoding phosphoric acid The expression level of enzyme and tensin homologue-induced kinase 1 (PINK1) gene. The use described in item 1 of the scope of patent application, wherein the tartary buckwheat seed coat extract has a concentration of 0.125 mg/mL to 0.25 mg/mL. The use as described in item 1 or item 2 of the scope of the patent application, wherein the composition has a powder, granule, solution, colloid, or paste dosage form. Such as the application described in item 1 or item 2 of the scope of patent application, wherein the weight ratio of the water to the tartary buckwheat seed coat ranges from 20:1 to 1:1. Such as the use described in item 1 or item 2 of the scope of patent application, wherein the extraction is carried out at 50°C to 100°C.

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