JP2022184634A - Anti-aging agent and production method of the same - Google Patents

Abstract

To provide an anti-aging agent showing an excellent action such as a mitochondrial activating action, and to provide a production method of the same.SOLUTION: An anti-aging agent contains polysaccharides derived from algae of the genus Arthospira as the active ingredient. A production method of the anti-aging agent includes a step of extracting polysaccharides from algae of the genus Arthospira using a phenol aqueous solution or a trichloroacetic acid aqueous solution.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to an anti-aging agent exhibiting excellent effects such as mitochondrial activating action, and a method for producing the same.

The average life expectancy in Japan has increased significantly in the latter half of the 20th century, making Japan one of the world's leading longevity countries. On the other hand, problems due to aging are becoming more alive. For example, age-related aging phenotypes include cognitive decline, muscle weakness, osteoporosis, increased visceral fat, reduced cardiovascular function, and insomnia.

The aging process was once thought to be so complex that intervention was impossible. However, in recent years, scientific advances have revealed that aging is one of the cell-biological processes that can be intervened. BACKGROUND ART Anti-aging techniques are being studied to reduce the probability of developing age-related diseases such as cancer and cancer, and to maintain the quality of life (QOL: Quality Of Life) while aiming for healthy longevity.

By the way, algae called Spirulina is a type of blue-green algae, and is one of the oldest plants that was born on the earth more than 3 billion years ago. For this reason, it has recently been used as a nutritional supplement for modern people who tend to have an unbalanced diet.

For example, Patent Document 1 shows that Spirulina itself has a hydroxyl radical scavenging ability, and describes that it is effective in preventing and treating hepatitis. In addition, Patent Document 2 discloses a cosmetic containing phycocyanin, which is a blue pigment protein extracted from spirulina, and is said to exhibit anti-aging action, whitening action, and the like.

JP 2009-256230 A JP 2020-183356 A

As described above, Spirulina is being considered for use as an active ingredient in preventive agents, therapeutic agents, and the like, in addition to dietary supplements. However, for the anti-aging action and whitening action described in Patent Document 2, for example, there is only subjective qualitative evaluation such as whether the application of a cosmetic containing a protein derived from spirulina makes the skin feel elastic or white. not done.
Accordingly, an object of the present invention is to provide an anti-aging agent exhibiting excellent effects such as mitochondrial activating action, and a method for producing the same.

The present inventors have made intensive studies to solve the above problems. As a result, the inventors have found that polysaccharides extracted from Arthrospira algae exhibit objectively excellent anti-aging effects, and have completed the present invention.
The present invention is shown below.

[1] An anti-aging agent characterized by containing a polysaccharide derived from Arthrospira algae as an active ingredient.
[2] The anti-aging agent according to [1] above, wherein the Arthrospira algae is Arthrospira platensis.
[3] The anti-aging agent according to [1] or [2] above, which exhibits a mitochondrial activation action.
[4] The anti-aging agent according to any one of [1] to [3], which exhibits antioxidant action.
[5] The anti-aging agent according to any one of [1] to [4], which exhibits collagen biosynthesis-enhancing action.
[6] A method for producing an anti-aging agent, comprising:
A method comprising a step of extracting polysaccharides from Arthrospira algae using an aqueous phenol solution or an aqueous trichloroacetic acid solution.
[7] The method according to [6] above, wherein Arthrospira platensis is used as the Arthrospira algae.

The polysaccharide derived from Arthrospira algae according to the present invention has effects such as activation of mitochondria, antioxidant action, enhancement of biosynthesis of collagen and molecular chaperones involved in folding of the endoplasmic reticulum, enhancement of collagen biosynthesis, etc. Objective effective and excellent anti-aging action. In addition, the polysaccharide according to the present invention is derived from Spirulina (Arthrospira algae), which itself is used as a nutritional supplement, or by a method similar to the method for extracting endotoxin from Gram-negative bacteria. It has been experimentally proven to have low cytotoxicity even though it is extracted from Therefore, the present invention is industrially extremely superior as a technology relating to an excellent anti-aging agent.

FIG. 1 is a graph showing JC-1 fluorescence intensity, which is an indicator of activation of mitochondria in senescent cells by Spirulina polysaccharide according to the present invention. FIG. 2 is a graph showing temporal changes in gene expression of mitochondrial superoxide dismutase 2 (SOD2) in senescent cells cultured in a medium containing the Spirulina polysaccharide according to the present invention. FIG. 3 is an immunoblotting photograph showing temporal changes in the amount of mitochondrial superoxide dismutase 2 (SOD2) biosynthesis in senescent cells cultured in a medium containing the Spirulina polysaccharide according to the present invention. FIG. 4 is a graph showing the oxidative activity of senescent cells cultured in a medium containing Spirulina polysaccharide according to the present invention. FIG. 5 is a graph showing the amount of collagen produced by senescent cells cultured in a medium containing the Spirulina polysaccharide according to the present invention. Fig. 6 (1) shows the production capacity of SOD2 in senescent cells transformed with control siRNA or siRNA against mitochondrial superoxide dismutase 2 (SOD2), with or without treatment with the Spirulina polysaccharide according to the present invention. FIG. 6(2) is a graph showing the amount of collagen produced. FIG. 7 (1) is a graph showing the relative gene expression level of mitochondrial superoxide dismutase 2 (SOD2) in senescent cells cultured in a medium containing spirulina polysaccharide (SPC) or lipopolysaccharide (LPS) according to the present invention. FIG. 7(2) is a graph showing relative gene expression levels of cytokine IL-6. FIG. 8 (1) is a graph showing the relative gene expression levels of the endoplasmic reticulum molecule chaperone HSP47 in senescent cells cultured in a medium containing the Spirulina polysaccharide according to the present invention, and FIG. FIG. 2 is a graph showing relative gene expression levels of chaperone GRP78. FIG. FIG. 9 is a graph showing the results of a cytotoxicity test of Spirulina polysaccharides according to the present invention.

The anti-aging agent according to the present invention contains a polysaccharide derived from Arthrospira algae as an active ingredient.

Arthrospira algae are spiral-shaped algae that live in freshwater areas and are 5 to 8 μm wide and 300 to 500 μm long.

Algae of the genus Arthrospira that can be used in the present invention are not particularly limited as long as they contain polysaccharides exhibiting an anti-aging effect. , Arthrospira geitleri, Arthrospira siamese, Spirulina major, Spirulina subsalsa, etc., preferably Arthrospira alxima platensis and/or Arthrospira platensis • Platensis is more preferred.

A polysaccharide is a polymer in which monosaccharide molecules are linked by glycosidic bonds. In the present invention, as will be described later, the active ingredient is extracted from Arthrospira algae by a method similar to the method for extracting endotoxin from Gram-negative bacteria. structure, but not lipid A, which is responsible for endotoxin toxicity. On the other hand, the polysaccharide according to the present invention may have a polysaccharide structure as well as structures such as peptides, amino acids, phosphate groups, and hydrocarbon groups.

The inventors' experiments have found that the polysaccharide according to the present invention activates mitochondria. Mitochondria are responsible for the metabolism of carbohydrates, proteins, lipids, and the like, and produce ATP and heat, which are energy substances. Therefore, the polysaccharide according to the present invention contributes to the supply of energy for maintaining constant life activity through activation of mitochondria, and also 4-aminobutyric acid (GABA) that brings mental stability. It may also stimulate the secretion of insulin, which lowers blood sugar levels.

In addition, the experimental findings of the present inventors have revealed that the polysaccharide according to the present invention promotes gene expression and biosynthesis of mitochondrial antioxidant enzymes. Active oxygen in the body originally has an important function of protecting the body by attacking bacteria and viruses that have invaded the body, but excessive active oxygen attacks normal cells and is caused by active oxygen. A vicious cycle may occur in which the lipid peroxides that have been oxidized further generate active oxygen. Since antioxidant enzymes decompose excess active oxygen and render it harmless, for example, the polysaccharide according to the present invention activates mitochondria through the activation of mitochondrial antioxidant enzymes and maintains health. and contribute to the maintenance of skin condition.

Furthermore, the present inventors have experimentally demonstrated that the polysaccharide according to the present invention promotes collagen biosynthesis. Collagen is a fibrous protein that constitutes the skin, tendon, cartilage, etc. With aging, the enzyme that produces collagen from its precursor decreases, and the amount of collagen biosynthesis decreases, causing wrinkles and wrinkles. It is known to lead to a decrease in skin tension. Therefore, it is conceivable that the polysaccharide according to the present invention improves skin conditions such as the occurrence of wrinkles due to aging and the reduction of skin tension.

The anti-aging agent according to the present invention contains a polysaccharide derived from Arthrospira algae as an active ingredient. The active ingredient refers to a component that exerts an anti-aging effect among the components contained in the anti-aging agent according to the present invention. Contains polysaccharides. Specifically, although not particularly limited, for example, the proportion of polysaccharides in the anti-aging agent according to the present invention can be 10% by mass or more and 100% by mass or less. Moreover, when the anti-aging agent according to the present invention is an external preparation, the proportion of polysaccharides can be 0.1% by mass or more and 10% by mass or less.

The administration frequency and dosage of the anti-aging agent according to the present invention may be appropriately adjusted according to the age, sex, condition, etc. of the subject, and the amount that can exhibit anti-aging effects is administered to the subject. For example, the dosage of the anti-aging agent per day can be 10 mg/kg body weight or more and 1 g/kg body weight or less. Moreover, when the anti-aging agent according to the present invention is an external preparation, the amount of the anti-aging agent to be applied per day may be 0.1 mg or more and 10 mg or less. The number of times of administration and the number of times of application per day are not particularly limited.

The anti-aging agent according to the present invention can be administered not only to humans but also to animals other than humans. Examples of animals to be administered include domestic animals such as horses, cattle, pigs, sheep, goats, camels, and llamas; sports animals such as racehorses; pets such as dogs and cats; experiments using mice, rats, guinea pigs, rabbits, etc. Animals; poultry such as chickens, ducks, turkeys and ostriches.

The dosage form of the anti-aging agent according to the present invention is not particularly limited. For example, it may be a polysaccharide itself, a composition combined with other ingredients, a solution or suspension thereof may be The dosage form of the anti-aging agent according to the present invention is not particularly limited, and examples thereof include tablets, powders, capsules, sugar-coated tablets, granules, liquids, external preparations and the like. Pharmaceutically acceptable additives may be used in the anti-aging agent according to the present invention according to the dosage form. Such additives include, for example, excipients, disintegrants, lubricants, binders, antioxidants, colorants, sweeteners, aggregation inhibitors, preservatives, active ingredient dissolution aids, stabilizers, and the like. can be mentioned.

The polysaccharide, which is the active ingredient of the anti-aging agent according to the present invention, can be extracted from Arthrospira algae by the same method as the method for extracting endotoxin (lipopolysaccharide) from Gram-negative bacteria. Such methods include the Westphal method and the TCA extraction method.

The Westphal method is a method of dissociating membrane components using an aqueous phenol solution and extracting polysaccharides into the aqueous phase. As the raw material, the Arthrospira algae, its culture solution may be used, but from the viewpoint of purification, it is preferable to use the Arthrospira algae itself or its dried body. For example, an aqueous phenol solution is added to an aqueous dispersion of Arthrospira algae. At this time, the cells are destroyed by heating. The heating temperature may be adjusted as appropriate, and may be, for example, 50°C or higher and lower than 100°C. At this time, the ratio of phenol to the total of water and phenol can be, for example, 30% by mass or more and 60% by mass or less. In addition, since water and phenol are not completely miscible and may separate into two layers, vigorous stirring is preferred. The stirring time can be, for example, 5 minutes or more and 30 minutes or less.

The TCA extraction method is a method of extracting polysaccharides from Arthrospira algae using trichloroacetic acid (TCA). For example, cold trichloroacetic acid is added to a cold dispersion of Arthrospira algae, and the mixture is shaken for about 1 hour to 10 hours to extract polysaccharides. At this time, the concentration of TCA with respect to the entire extraction solvent can be, for example, 15% by mass or more and 30% by mass or less. This method is a relatively mild extraction method.

After extraction, the polysaccharide may be purified by a general method. Purification methods include, for example, liquid separation, dialysis, concentration, centrifugation, freeze-drying and the like.

As shown in the following examples, the polysaccharide according to the present invention has an effect of activating mitochondria, an antioxidant effect, an effect of enhancing the biosynthesis of collagen and molecular chaperones involved in the folding of the endoplasmic reticulum, an effect of enhancing the biosynthesis of collagen, etc. , shows an objective and excellent anti-aging action. In addition, as shown in the following examples, the polysaccharide according to the present invention is derived from Spirulina (Arthrospira algae), which itself is used as a nutritional supplement, and is extracted from Gram-negative bacteria. Although it is extracted by the same method as the method, it has low cytotoxicity. Therefore, the anti-aging agent according to the present invention can be constantly ingested as a health food having an excellent anti-aging effect.

For example, the polysaccharide according to the present invention can be used as an anti-aging agent in general foods and drinks. The food and drink to which the polysaccharide according to the present invention is added is not particularly limited, but for example, milk drinks, soft drinks, sports drinks, nutritional drinks, beauty drinks, liquid nutritional supplements; chewing gums, chocolates, candies, jellies, cakes Confectionery such as , biscuits and crackers; Frozen desserts such as ice cream and frozen desserts; Noodles such as udon noodles, Chinese noodles, spaghetti and instant noodles; Examples include bread, ham, rice porridge, cooked rice, soup, various retort foods, and various frozen foods. The food and drink products containing the polysaccharide according to the present invention are so-called health foods, supplements, functional foods, functionally labeled foods, nutritional supplements, foods for specified health uses, foods with nutrient function claims, nursing foods, smile care foods, chewing and It can be used for applications such as swallowing aids, concentrated liquid foods, and foods for sick people.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples, and can be modified appropriately within the scope that can conform to the gist of the above and later descriptions. It is of course possible to implement them, and all of them are included in the technical scope of the present invention.

Example 1: Preparation of Spirulina Extract by the Westphal Method Spirulina pacifica was first selected in 1984 by Cyanotech from strains of the edible Spirulina (Arthrospira (Spirulina) platensis).
Dried Spirulina pacifica cells (10 g) were washed with acetone and dispersed in distilled water (55 mL), followed by addition of 90% phenol water (45 mL) at 68° C. with vigorous stirring. After continuing stirring for 120 minutes, the mixture was filtered and the two layers of filtrate were separated. The remaining phenol was removed from the resulting aqueous phase by dialysis. Specifically, the filtrate was placed in a dialysis membrane (“RC dialysis tube” manufactured by Spectrum), immersed in water (2 L), and held at 4° C. for 48 hours to remove residual phenol. It was then lyophilized.
The resulting crude product (0.1 g) was dissolved in water (10 mL), subjected to ultracentrifugation at 100,000 g for 6 hours to remove solids, and the supernatant was lyophilized to obtain a jelly. A deposit was obtained. A Westphal fraction was obtained from the resulting jelly deposit.
The resulting extract was analyzed by mass spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 1000-20,000. As a reference for analysis, E. coli 055:B5 LPS (Smooth type, manufactured by Sigma-Aldrich) was used.
Although lipopolysaccharides are generally extracted from Gram-negative bacteria according to the Westphal method, the following experiments revealed that the extract according to the present invention was derived from Spirulina, and lipopolysaccharides, which are endotoxins, Since it was found to exhibit properties different from those of polysaccharides, and it is thought that it does not contain lipid A, which is the cause of the toxicity of lipopolysaccharides, the resulting extract is hereinafter referred to as "Spirulina polysaccharides".

Test Example 1: Mitochondrial Activity Test (1) Mitochondrial Activity Test Using a medium containing 10% FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin in MEMα medium, human dermal fibroblasts (NB1RGB cells) were Senescent cells were obtained by culturing for 89 days.
The Spirulina polysaccharide produced in Example 1 was added to the medium at a rate of 150 μg/mL, and the human skin fibroblast senescent cells were further cultured at 37° C. for 24 hours. For comparison, culture was carried out in the same manner except that Spirulina polysaccharide was not added.
Mitochondrial activity was then measured by JC-1 (Dojindo Molecular Technologies) staining using a mitochondrial membrane potential detection kit (“JC-1 MitoMP Detection kit” Dojindo) according to the manufacturer's protocol. Fluorescence was measured with a fluorescence microplate reader ("Infinite M200" TECAN) using 535 nm/590 nm and 485 nm/535 nm filter pairs.
When the mitochondrial activity is high, the membrane potential difference is maintained, and the low-molecular-weight fluorescent dye JC-1 aggregates to emit fluorescence with a longer wavelength. becomes a monomer and emits fluorescence with a shorter wavelength. Thus, mitochondrial activity can be measured as the ratio of the longer wavelength fluorescence measured at 535 nm/590 nm and the lower wavelength fluorescence measured at 485 nm/535 nm. The results are shown in FIG. 1 as a ratio of Spirulina polysaccharide-treated examples to controls. In FIG. 1, "*" indicates significant difference at p<0.05 by Tukey's test followed by two-way analysis of variance.
As the results shown in FIG. 1 , it was revealed that mitochondria were significantly activated in senescent cells treated with Spirulina polysaccharide as compared to untreated senescent cells.

(2) mRNA expression analysis In the same manner as in Test Example 1 (1), human skin fibroblast senescent cells were further cultured at 37° C. for 24 hours using a medium supplemented with spirulina polysaccharide at a rate of 150 μg/mL. Samples were collected 0, 3, 6, 12, and 24 hours after the start of culture, and a high-efficiency real-time PCR master mix ("THUNDERBIRD ^(R) Next SYBR ^(R) qPCR Mix" manufactured by Toyobo) was used to extract mitochondria. gene expression of superoxide dismutase 2 (SOD2), an antioxidant enzyme in Total RNA was normalized in each reaction using β-actin complementary DNA as an internal standard. The results are shown in FIG. In FIG. 2, "*" indicates significant difference at p<0.05 by Tukey's test followed by two-way analysis of variance.
As shown in FIG. 2, it was revealed that the Spirulina polysaccharide according to the present invention promoted the expression of the mitochondrial antioxidant enzyme SOD2 gene and the production of SOD2 mRNA.

(3) Analysis of Amount of SOD2 Biosynthesis The amount of SOD2 biosynthesis was analyzed using an anti-SOD2 antibody (manufactured by Cell Signaling Technology) and anti-β-actin (manufactured by Sigma).
Specifically, a protein solution was prepared from the sample obtained in the same manner as in Test Example 1 (2), subjected to polyacrylamide gel electrophoresis, transferred to a membrane, and the anti-SOD2 antibody product was multiplied by 1000. A β-actin antibody was diluted 10,000 times, the membrane was immersed, incubated at 4° C. for 18 hours, and then washed. Next, HRP-labeled anti-rabbit and anti-mouse secondary antibody products (manufactured by Promega) were diluted 5000-fold, the membrane was immersed, incubated at room temperature for 1 hour, and then washed. Next, the membrane was immersed in a luminol reaction solution, incubated at room temperature for 5 to 10 minutes until luminescence occurred, and then exposed to an X-ray film for development.
The fluorescence intensity of the SOD2 band was corrected with the fluorescence intensity of the β-actin band in each sample. The results are shown in FIG. "Intensity" in FIG. 3 indicates the correction value.
As shown in FIG. 3, the Spirulina polysaccharide according to the present invention was found to increase the amount of mitochondrial antioxidant protein SOD2 biosynthesis over time.

(4) Antioxidant activity measurement: (Dojindo: SOD assay kit)
Human skin fibroblast senescent cells were further cultured at 37° C. for 24 hours in a medium supplemented with 150 μg/mL of spirulina polysaccharide in the same manner as in Test Example 1 (1) to obtain a sample. For comparison, samples were separately obtained by culturing in the same manner except that Spirulina polysaccharide was not added.
The antioxidant activity of the obtained sample was evaluated using an antioxidant capacity measurement kit (“SOD Assay Kit-WST” manufactured by Dojindo Molecular Technologies) according to the manufacturer's protocol. Fluorescence was measured with a fluorescence microplate reader (“Infinite M200” manufactured by TECAN) equipped with a 380 nm/485 nm filter pair. The results are shown in FIG. In FIG. 4, "*" indicates a significant difference at p<0.05 by Tukey's test following two-way analysis of variance.
As the results shown in FIG. 4, the ROS scavenging ability in the control cells was 27%, but the ROS scavenging ability increased to 47% in the cells treated with the Spirulina polysaccharide according to the present invention. It was found that the antioxidant activity was significantly increased in the cells treated with Spirulina polysaccharide.

The above experimental results demonstrate that the Spirulina polysaccharide according to the present invention activates the mitochondria of senescent cells, and particularly promotes the expression of mitochondrial antioxidant enzymes.

Test Example 2: Collagen Producing Ability Test (1) Collagen Producing Ability Test In the same manner as in Test Example 1 (1), using a medium supplemented with spirulina polysaccharide at a rate of 150 µg/mL, human dermal fibroblast senescent cells were further added. After culturing at 37° C. for 24 hours, a sample was obtained. For comparison, samples were separately obtained by culturing in the same manner except that Spirulina polysaccharide was not added. A cell lysate was prepared from the obtained sample, and the amount of collagen in the cell layer per 1 μL of the cell lysate containing the same protein concentration was measured using a collagen quantification kit (manufactured by Cosmo Bio), which was determined by the manufacturer. Assessed according to protocol. Fluorescence was measured with a fluorescence microplate reader (“Infinite M200” manufactured by TECAN) equipped with a 380 nm/485 nm filter pair. The results are shown in FIG. In FIG. 5, "*" indicates a significant difference at p<0.05 by Tukey's test following two-way analysis of variance.
As shown in the results shown in FIG. 5, the amount of collagen produced by the control senescent cells cultured without adding Spirulina polysaccharide was 14 μg/mL, but when cultured in the culture solution containing the Spirulina polysaccharide according to the present invention, the amount of collagen produced was 14 μg/mL. It increased to 19 μg/mL in senescent cells. Therefore, it was found that the cells treated with the Spirulina polysaccharide according to the present invention had a significantly increased ability to produce collagen.

(2) Transformation with siRNA siRNA (SEQ ID NO: 1 and SEQ ID NO: 2) or control siRNA (manufactured by Santa Cruz Biotechnology) against the mitochondrial antioxidant protein SOD2 gene and Lipofectamine ^™ RNAiMAX transfection reagent (manufactured by Invitrogen) were used. Human dermal fibroblast senescent cells obtained in the same manner as in Test Example 1 (1) were transformed using according to the manufacturer's instructions.
Next, human skin fibroblast senescent cells were further cultured at 37° C. for 24 hours using a medium supplemented with 150 μg/mL of spirulina polysaccharide in the same manner as in Test Example 1 (1) to obtain a sample. For comparison, samples were separately obtained by culturing in the same manner except that Spirulina polysaccharide was not added.
From the obtained samples, the amount of SOD2 biosynthesis was measured in the same manner as in Test Example 1 (3), and the amount of collagen production was measured in the same manner as in Test Example 2 (1). The amount of SOD2 biosynthesis is shown in FIG. 6(1), and the amount of collagen production is shown in FIG. 6(2). In FIG. 6(2), "SPC" indicates Spirulina polysaccharides, and "*" indicates a significant difference at p<0.05 by Tukey's test following two-way analysis of variance.
As shown in the results shown in FIG. 6, when the biosynthesis of SOD2 is suppressed by siRNA, the effect of increasing the amount of collagen biosynthesis by spirulina polysaccharide (SPC) is also inhibited. was suggested to be dependent on increased expression of SOD2, ie, increased mitochondrial antioxidant capacity.

Test Example 3: Comparison with lipopolysaccharide In the same manner as in Test Example 1 (1), 150 µg/mL of Spirulina polysaccharide or 1 µg/mL of lipopolysaccharide (“Lipopolysaccharide” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added. Human skin fibroblast senescent cells were further cultured at 37° C. for 24 hours using the medium added at the ratio to obtain a sample. For comparison, samples were separately obtained by culturing in the same manner except that Spirulina polysaccharide and lipopolysaccharide were not added. Next, the expression level of SOD2 mRNA or IL-6 mRNA, which is a cytokine, was measured in the same manner as in Test Example 1(2). The results for the SOD2 mRNA expression level are shown in FIG. 7(1), and the results for the IL-6 mRNA expression level are shown in FIG. 7(2). In FIG. 7, "SPC" indicates Spirulina polysaccharide, "LPS" indicates lipopolysaccharide, and "*" indicates a significant difference at p<0.05 by Tukey's test following two-way analysis of variance. indicates that
As shown in FIG. 7, the Spirulina polysaccharide (SPC) according to the present invention strongly promotes the biosynthesis of the mitochondrial antioxidant protein SOD2, while the effect of promoting the biosynthesis of the cytokine IL-6 is weak. . On the other hand, lipopolysaccharide (LPS) promotes the biosynthesis of SOD2 by about 0.4 times that of SPC, which is very weak, but promotes the biosynthesis of IL-6 by about 3 times, which is very strong.
Thus, lipopolysaccharide (LPS) is purified from Gram-negative bacteria by the Westphal method or the like, and is also called endotoxin, and is highly toxic. Although it is purified by such as, it has low toxicity such as inducing IL-6, a cytokine that is involved in the development of inflammation and immune diseases, while it has the effect of strongly promoting the biosynthesis of the mitochondrial antioxidant protein SOD2. show.

Test Example 4: Measurement of expression level of endoplasmic reticulum chaperone Hsp47 is localized in the endoplasmic reticulum and is a molecular chaperone essential for collagen folding. ing. GRP78 is also a molecular chaperone involved in protein folding and assembly in the endoplasmic reticulum. Therefore, the effect of the Spirulina polysaccharide according to the present invention on these molecular chaperones was investigated.
In the same manner as in Test Example 1 (1), human skin fibroblast senescent cells were further cultured at 37° C. for 24 hours using a medium supplemented with spirulina polysaccharide at a ratio of 100 μg/mL or 150 μg/mL to obtain a sample. rice field. For comparison, samples were separately obtained by culturing in the same manner except that Spirulina polysaccharide was not added. Then, the expression level of Hsp47 mRNA or GRP78 mRNA was measured in the same manner as in Test Example 1(2). The results of Hsp47 mRNA expression level are shown in FIG. 8(1), and the results of GRP78 mRNA expression level are shown in FIG. 8(2). In FIG. 8, "SPC" indicates Spirulina polysaccharides, and "*" indicates a significant difference from the control (no SPC addition) at p < 0.05 by Tukey's test following two-way analysis of variance. indicates that there is
As shown in FIG. 8, the Spirulina polysaccharide according to the present invention promotes the expression of molecular chaperone genes involved in the folding of collagen and endoplasmic reticulum, thus promoting the production of various proteins including collagen. It is thought that there are

Test Example 5 Cytotoxicity Test The cytotoxicity of the Spirulina polysaccharide according to the present invention was tested by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method.
Specifically, human skin fibroblast senescent cells were cultured at 37° C. for 24 hours in a medium supplemented with 0 to 150 μg/mL of Spirulina polysaccharide in the same manner as in Test Example 1 (1). Then, an MTT solution was added to the medium so that the MTT concentration in the medium was 1 mg/mL, and the cells were further incubated for 1 to 2 hours. Next, 2-propanol and HCl were added to final concentrations of 50% and 20 mM, respectively, and absorbance at 570 nm was measured using an infinite F50R spectrophotometer (manufactured by TECAN). The results are shown in FIG.
As the results shown in Figure 9, the Spirulina polysaccharide according to the present invention did not show toxicity to cells at any concentration tested.

Test Example 6: Component Analysis Since the extract obtained in Example 1 was obtained by the Westphal method, it was expected to be polysaccharides. Therefore, the amount of saccharides contained in the obtained extract (Spirulina polysaccharides) was measured.
Specifically, using a Total Carbohydrate Assay kit (manufactured by CELL BIOLABS), the saccharide content was determined by the phenol-sulfuric acid method. According to the kit, all saccharides in the sample are hydrolyzed with sulfuric acid to furfural and furfural derivatives, which are further reacted with kit reagents to form dyes. Absorbance is then measured and the measurements are compared to a glucose standard curve to determine the total sugar content. Absorbance was measured with a plate reader ("Infinite F50" manufactured by TECAN) using a 492 nm filter.
As a result, it was found that 66±11% of Spirulina polysaccharides were saccharides in terms of glucose. About 34% of the remainder is polysaccharides that have not been sufficiently decomposed, saccharides that are less reactive than glucose, or peptides, amino acids, phosphate groups, etc. bound to polysaccharides, and most of the extract is polysaccharides. considered to be sugars.

Claims (7)

An anti-aging agent comprising, as an active ingredient, a polysaccharide derived from algae belonging to the genus Arthrospira. 2. The anti-aging agent according to claim 1, wherein the Arthrospira algae is Arthrospira platensis. 3. The anti-aging agent according to claim 1 or 2, which exhibits mitochondrial activation action. The anti-aging agent according to any one of claims 1 to 3, which exhibits antioxidant action. The anti-aging agent according to any one of claims 1 to 4, which exhibits collagen biosynthesis enhancing activity. A method for producing an anti-aging agent, comprising:
A method comprising a step of extracting polysaccharides from Arthrospira algae using an aqueous phenol solution or an aqueous trichloroacetic acid solution. 7. The method according to claim 6, wherein Arthrospira platensis is used as the Arthrospira algae.

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