TWI736855B - Use of passiflora flower extracts for enhancing mitochondrial activity and anti-aging gene expression in skin cells - Google Patents

Abstract

The present invention provides use of a Passiflora flower extract for manufacture of composition for enhancing mitochondrial activity or anti-aging gene expression in skin cells. The passion flower extract not only helps to increase cellular energy production, but also enhances the expression of multiple anti-aging genes, such as PINK1, ATG1, ATG8, SIRT1, FOXO3, TERT and TERC, that are associated with renewal of mitochondria, cellular stress response, and telomere elongation. Therefore, said extract can maintain skin cell vitality and skin youth.

Description

The use of passion flower extract to promote mitochondrial activity of skin cells and the expression of anti-aging genes

The present invention relates to the health care use of a passionflower extract, in particular to the use of a passionflower extract to prepare a composition that promotes the mitochondrial activity of skin cells or the expression of anti-aging genes.

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 complexity of the above-mentioned factors that cause aging, delaying the aging of the body and even various cells can be done in many ways, 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 strengthening The operation of cell protection mechanisms (such as promoting the expression of anti-aging genes). Among them, the methods to promote cell protection mechanisms are in the development stage, and what kind of methods can strengthen the cell protection mechanisms is still under study. 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 object of the present invention is to provide a use of a Passiflora (Passiflora spp.) flower extract for preparing a composition for promoting mitochondrial activity of skin cells, wherein the Passiflora spp. is extracted with a solvent Obtained from a passiflora flower.

In an embodiment of the present invention, the passionflower extract is extracted with water, alcohols, or a mixture of alcohol and water as a solvent.

In one embodiment of the present invention, the passionflower extract inhibits the gene expression of adenosine monophosphate ribose polymerase (also known as poly(ADP-ribose) polymerase, PARP), and the Adenosine polyphosphate ribose polymerase includes polyadenosine diphosphate ribose polymerase 1 (PARP1) and polyadenosine diphosphate ribose polymerase 2 (PARP2). It is known that PARP is related to mitochondrial dysfunction, so Passiflora extract may, but is not limited to, achieve the effect of enhancing mitochondrial activity by inhibiting PARP gene expression.

Another object of the present invention is to provide a use of a passionflower extract for preparing a composition for promoting anti-aging gene expression of skin cells, wherein the passionflower extract is obtained by extracting a passionflower flower with a solvent.

In an embodiment of the present invention, the anti-aging gene encodes an autophagy-related protein (ATG), including autophagy-related protein 1 (ATG1) and autophagy-related protein 8 (ATG8).

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 forkhead box protein O3 (forkhead box class protein O3). O 3, FOXO3).

In an embodiment of the present invention, the anti-aging gene encodes telomerase reverse transcriptase (TERT), telomerase RNA component (TERC), or a combination thereof. Therefore, the passion flower extract can promote the synthesis of telomerase in skin cells.

In an embodiment of the present invention, the concentration of the Passiflora extract used to promote the mitochondrial activity of skin cells or the expression of anti-aging genes is 0.125 mg/mL to 0.25 mg/mL.

Based on the results of in vitro cell experiments and gene expression analysis, the present invention reveals that Passiflora extract enhances the mitochondrial activity of skin cells and the performance of a variety of anti-aging genes. In view of the fact that the maintenance of mitochondrial activity is extremely important for cell viability, and the aforementioned anti-aging genes are involved in aging mitochondrial renewal, cellular stress response (that is, a variety of molecular mechanisms that help cells reduce damage in adversity), and telomere extension and other beneficial cell rejuvenation In the event, passion flower extract has the potential to maintain the vitality of skin cells and the youthful state of the individual's skin. Therefore, the present invention provides the use of Passiflora extract for preparing a composition that promotes mitochondrial activity of skin cells or anti-aging gene expression. The composition can be in the form of powder, granules, liquid, gel or paste, and can be made into food, drink, medicine, reagent or nutritional supplement, and administered to a body by oral administration, skin application and other methods.

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 relative amount (%) of JC-1 aggregates in human skin fibroblasts treated with or without Passiflora extract for 24 hours.

Figure 2 shows the relative expression levels of PARP1 and PARP2 genes of human skin fibroblasts after 48 hours of treatment with or without Passiflora extract, relative to the control group cells.

Figure 3 shows the relative expression levels of PINK1, ATG1 , ATG8, SIRT1, FOXO3, TERT , and TERC genes of human skin fibroblasts treated with or without Passiflora extract for 48 hours relative to control cells.

The present invention provides a use of Passiflora extract for preparing a composition for promoting mitochondrial activity of skin cells or anti-aging gene expression. The Passiflora extract of the present invention is obtained by extracting a Passiflora flower with a solvent, wherein the solvent can be water, alcohol, or a mixture of alcohol and water, preferably water. The following examples show that the treatment of the passionflower extract can enhance the mitochondrial activity of human skin cells and inhibit the gene expression of various poly ADP ribose polymerases (PARP; such as PARP1 and PARP2) related to mitochondrial dysfunction . In addition, the passion flower extract promotes anti-inflammatory effects in human skin cells. The performance of aging genes. The product of the aforementioned anti-aging gene expression can be ribonucleic acid (RNA) or protein, including but not limited to: phosphatase and tensin homologue inducing kinase 1 (PINK1), various autophagy-related proteins (ATG; such as ATG1 and ATG8) , Silencing information regulator 2 homologous protein 1 (SIRT1), forkhead box protein O3 (FOXO3), and telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) involved in the telomerase reaction.

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 "mitochondrial activity" in this article refers to the effectiveness of mitochondrial operations, including energy production, material metabolism, and the removal efficiency of reactive oxygen species. The assessment of mitochondrial activity can be accomplished according to the biochemical, molecular biology and cytological techniques known in the technical field of the present invention, such as the measurement of mitochondrial membrane potential described herein.

The "anti-aging genes" used herein generally refer to genes whose existence is related to the longevity of organisms or whose RNA or protein products can maintain normal cell function. The functions of the protein encoded by the anti-aging gene include promoting the normal operation of mitochondria, assisting in the folding of other proteins, promoting the synthesis of telomerase, and regulating the gene expression of proteins involved in nutrient metabolism or cellular stress response.

Materials and Methods Material

Gibco Eagle's minimum essential medium (MEM), Gibco fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin (Gibco Eagle's minimum essential medium, FBS) containing Earle's balanced salt solution (EBSS) Penicillin/streptomycin) and phosphate buffered saline (Gibco phosphate buffered saline, PBS) were purchased from Thermo Fischer Scientific.

Cell culture

The following examples used human skin fibroblast CCD-966SK (BCRC 60153) purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute. CCD-966SK cells are cultured in MEM medium (containing EBSS) at 37°C and 5% carbon dioxide, which is supplemented with 10% fetal bovine blood Clear, 1mM sodium pyruvate, 0.01M non-essential amino acid, and 1% penicillin-streptomycin, hereinafter referred to as MEM cell culture medium.

Preparation of Passiflora Flower Extract

Herein called passion flower extract with a solvent-based plant Passiflora a flower (Passiflora spp.) (Referred to as passion flower, for example, passion fruit (Passiflora edulis), Passiflora foetida (Passiflora foetida) Flowers ) Is obtained by extraction. The solvent may be water, alcohols, or a mixture of alcohol and water, preferably water. In one embodiment, the passiflora flower is washed and dried before extraction, and then coarsely crushed using a homogenizer. Thereafter, the solvent and the homogenous passiflora flower are uniformly mixed in a stirring manner for 0.5 to 3 hours to obtain a passiflora extract; the weight ratio of the solvent to the homogenous passiflora flower is in the range of 20:1 to 2 :1, preferably 5:1; extraction temperature is 40°C to 75°C. The Passiflora extract used in the following examples was purchased from Shaanxi Jiahe Biotechnology Co., Ltd., and was prepared in a similar manner to the foregoing steps.

Mitochondrial activity analysis

In order to evaluate the mitochondrial activity of cells, flow cytometer (Beckman Coulter Life Sciences) and mitochondrial membrane potential detection kit (BD ^TM MitoScreen) were used to measure the changes in mitochondrial membrane potential. The steps are briefly described. as follows. According to the manufacturer's instructions, rinse the cells to be tested with PBS solution, 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 a PBS solution and centrifuged again (400 xg, 5 minutes). Mix 100μL of JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) operating solution with the precipitated cells in the dark for 15 minutes for fluorescent labeling. Wash the cells twice with washing solution and centrifugation step (400 xg, 5 minutes). Finally, the cells were resuspended in a PBS solution containing 2% FBS, and a flow cytometer was used to detect the relative amount of JC-1 aggregates indicating the mitochondrial membrane potential (excitation wavelength is about 490nm, detection wavelength is about 595nm ). The greater the amount of JC-1 aggregates, the greater the potential difference in the inner mitochondrial membrane, which is regarded as the higher the mitochondrial activity.

Gene expression analysis

Based on the quantitative polymerase chain reaction (qPCR) to determine the mitochondrial activity and anti-aging gene expression 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. Secondly, use the qPCR kit (KAPA CYBRFAST qPCR Kit (2X); KAPA Biosystems) and the target genes listed in Table 1 below and the glyceraldehyde 3- The primer pair of phosphate dehydrogenase (Glyceraldehyde 3-phosphate dehydrogenase, GAPDH) gene is qpCR performed on the aforementioned cDNA in a PCR reaction machine (Step One Plus 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 value}

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 Passionflower extract enhances the mitochondrial activity of skin cells

Mitochondria are organelles that supply cell energy and participate in cell redox constant and nutrient metabolism. Their normal operation is essential to maintain cell viability and proliferation. In order to explore the influence of passion flower extract on mitochondrial function of skin cells, this example uses flow cytometry to evaluate the changes in mitochondrial activity of human skin fibroblasts CCD-966SK after treatment with passion flower extract . First, CCD-966SK cells ^{were seeded in a 6-well plate at 1×10 5} cells/well, and each well contained 2 mL of MEM cell culture medium. After culturing the cells at 37°C for 24 hours, remove the cell culture medium and treat the cells with 1 mL of MEM cell culture medium containing 0.125 mg/mL Passiflora extract (experimental group), or treat the cells only with MEM cell culture medium as a control Group. The cells of each group were cultured at 37°C for 24 hours and then used for mitochondrial activity analysis. The fluorescent dye JC-1 was used to indicate the mitochondrial membrane potential and activity.

Figure 1 shows the relative amount (%) of JC-1 aggregates in the aforementioned groups of CCD-966SK cells. The higher the value, the higher the average mitochondrial activity of the cells; ^*** means p<0.001 compared to the control group . According to Figure 1, compared with the control group, the administration of 0.125% Passiflora extract significantly increased the relative amount of JC-1 aggregates of CCD-966SK cells by about 40%, indicating that Passiflora extract can enhance the mitochondria of skin cells Activity, so that skin cells have enough vitality to perform specific physiological functions or proliferate to replace old cells. Therefore, Passiflora extract is beneficial to maintain the youthful state of individual human skin.

Example 2 Passionflower extract enhances cellular anti-aging power

In order to explore the influence of passion flower extract on the anti-aging ability of skin cells, qPCR was used to determine the poly ADP ribose polymerase 1 and 2 (PARP1 and PARP2) of human skin fibroblasts CCD-966SK after treatment with passion flower extract. , Phosphatase and tensin homologue inducible kinase 1 (PINK1), autotrophic related proteins 1 and 8 (ATG1 and ATG8), silencing information regulator 2 homologous protein 1 (SIRT1), forkhead box protein O3 (FOXO3) Changes in gene expression of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC). In short, CCD-966SK cells ^{were seeded in a 6-well plate at 1.5×10 5} cells/well, and each well contained 2 mL of MEM cell culture medium. After culturing the cells at 37°C for 24 hours, remove the cell culture medium and treat the cells with 1 mL of MEM cell culture medium containing 0.125 mg/mL or 0.25 mg/mL passionflower extract (experimental group), or only MEM cell culture medium Treat the cells as a control group. The aforementioned cells were cultured at 37°C for 48 hours and then used for qPCR analysis.

Figure 2 shows the relative expression levels of PARP1 and PARP2 genes of CCD-966SK cells treated with 0.125mg/mL Passiflora extract for 48 hours relative to control group cells. According to Figure 2, the treatment of Passiflora extract inhibits the gene expression of multiple poly ADP ribose polymerase (PARP) in CCD-966SK cells. In view of previous studies that the inhibition of this enzyme activity can improve mitochondrial activity, this result suggests that Passiflora extract can increase mitochondrial activity, thereby improving cell energy production and combating aging.

Figure 3 shows the relative expression levels of PINK1, ATG1, ATG8, SIRT1, FOXO3, TERT , and TERC genes of CCD-966SK cells treated with 0.25mg/mL Passiflora extract for 48 hours relative to control group cells ^{; *} , ^{* *} , and ^*** indicate p<0.05, p<0.01, and p<0.001 compared to the control group, respectively. According to Figure 3, compared with the control group, administration of Passiflora extract to CCD-966SK cells significantly improved the expression of PINK1, ATG1 , and ATG8 genes related to aging mitochondrial turnover, and promoted SIRT1, which is related to cellular stress response. And the performance of FOXO3 gene. In addition, Passiflora extract also enhances the expression of TERT and TERC genes involved in the telomerase reaction to prolong telomeres. This result shows that the extract of Passionflower flower enhances the products of various anti-aging genes in skin cells, thereby helping to maintain the vitality of skin cells and delay skin aging.

In summary, Passiflora extract can enhance the mitochondrial activity of skin cells and the performance of a variety of anti-aging genes, thereby maintaining the vitality of skin cells and the youthful state of the individual's skin. Therefore, the present invention provides the use of Passiflora extract for preparing a composition that promotes mitochondrial activity of skin cells or anti-aging gene expression. The composition can be in the form of powder, granules, liquid, gel or paste, and can be made into foods, drinks, nutritional supplements, or medicines, and can be administered to a body by oral administration, skin application and other methods.

<110> Dajiang Biomedical Co., Ltd.

<120> Passionflower extract is used to promote mitochondrial activity of skin cells and the expression of anti-aging genes

<130> 107B0594-I1

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<170> PatentIn version 3.5

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Claims (5)

A passiflora extract is used to prepare a composition for promoting mitochondrial activity of skin cells, wherein the passiflora extract is obtained by extracting a passiflora flower with a solvent, and the concentration of the passiflora extract is From 0.125mg/mL to 0.25mg/mL. The use described in item 1 of the scope of patent application, wherein the passiflora extract inhibits the gene expression of adenosine monophosphate ribose polymerase (PARP), and the adenosine polyphosphate ribose polymerase comprises poly diphosphate Adenosine ribose polymerase 1 and polyadenosine ribose polymerase 2. The use as described in item 1 of the scope of patent application, wherein the passion flower extract increases phosphatase and tensin homologue inducing kinase 1 (PINK1), autotrophic related protein 1 (ATG1), or autotrophic related protein 8 The performance of genes. A passiflora flower extract is used to prepare a composition that promotes skin cell anti-aging gene expression by extending telomeres, wherein the passiflora flower extract is obtained by extracting a passiflora flower with a solvent, and wherein the anti-aging The aging gene is a protein of telomerase reverse transcriptase (TERT) or telomerase ribonucleic acid component (TERC), wherein the concentration of the passionflower extract is 0.125 mg/mL to 0.25 mg/mL. Such as the application described in item 1 or item 4 of the scope of patent application, wherein the solvent is water, alcohol, or a mixture of alcohol and water.

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