SA-β-gal expression inhibitor and autophagy inducer

Abstract

To provide an SA-β-gal expression inhibitor having excellent SA-β-gal expression inhibitory action and high safety, an autophagy inducer having excellent autophagy inducing action and high safety, and an anti-aging composition having excellent anti-aging action and high safety.SOLUTION: The present invention provides an SA-β-gal expression inhibitor or an autophagy inducer comprising extract from the germ of Nelumbo nucifera Gaertn., and an anti-aging composition comprising at least one of the SA-β-gal expression inhibitor and the autophagy inducer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an SA-β-gal expression inhibitor, an autophagy inducer, and an anti-aging composition. [Background technology]

[0002] Due to the recent increase in consumer health awareness, there has been a great deal of research and development into superior functional ingredients derived from natural products that can be used in cosmetics, food and beverages, etc.

[0003] For example, lotus germ extract, which has estrogenic activity, collagen production promoting activity, and fibroblast proliferation activity, has been proposed as an alternative to conventional moisturizing agents (see, for example, Patent Document 1).

[0004] Furthermore, neferine, a component isolated from lotus embryos, has been reported to have the effect of inducing autophagy (see, for example, Non-Patent Document 1).

[0005] Furthermore, a mitophagy activator containing lotus flower extract as an active ingredient has been proposed (see, for example, Patent Document 2).

[0006] As mentioned above, various studies have been conducted to date. However, there remains a strong demand for new materials that have excellent functionality and are highly safe, and therefore can be widely used as ingredients in cosmetics, foods and beverages, research reagents, and the like, and there is currently a need for their rapid development. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2002-029980 [Patent Document 2] Japanese Patent Application Laid-Open No. 2017-178920 [Non-patent literature]

[0008] [Non-Patent Document 1] J.-S. Yoon et al., Phytomedicine, 20(2013), 1013-1022 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to solve the above-mentioned conventional problems and achieve the following object: Namely, the present invention aims to provide an SA-β-gal (senescence-associated beta-galactosidase) expression inhibitor that has excellent SA-β-gal expression inhibitory activity and is highly safe. Another object of the present invention is to provide an autophagy inducer that has excellent autophagy-inducing activity and is highly safe. Another object of the present invention is to provide an anti-aging composition that has an excellent anti-aging effect and is highly safe. [Means for solving the problem]

[0010] As a result of extensive research conducted by the inventors to solve the above problems, they discovered that lotus embryo extract has excellent SA-β-gal expression inhibitory effects and autophagy induction effects, and that the SA-β-gal expression inhibitory effect is dependent on the autophagy induction effect, and thus completed the present invention.

[0011] The present invention is based on the above findings of the present inventors, and the means for solving the above problems are as follows: <1> The SA-β-gal expression inhibitor is characterized by containing lotus embryo extract. <2> An autophagy inducer characterized by containing lotus germ extract. <3> The aforementioned <1> The SA-β-gal expression inhibitor according to claim 1, <2> The anti-aging composition is characterized by containing at least one of the autophagy inducers described above. [Effects of the Invention]

[0012] The SA-β-gal expression inhibitor of the present invention can solve the above-mentioned problems of the prior art and achieve the above-mentioned objectives, and can provide an SA-β-gal expression inhibitor that has excellent SA-β-gal expression inhibitory activity and is highly safe. The autophagy inducer of the present invention can solve the above-mentioned conventional problems and achieve the above-mentioned object, and can provide an autophagy inducer that has excellent autophagy-inducing activity and is highly safe. According to the anti-aging composition of the present invention, the above-mentioned conventional problems can be solved, the above-mentioned objects can be achieved, and an anti-aging composition having excellent anti-aging effects and high safety can be provided. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of staining the cells after culture in Test Example 1, which were fixed with a fixative. [Figure 2] FIG. 2 shows the results of staining the cells after culture in Test Example 3, which were fixed with a fixative. [Figure 3A] FIG. 3A is a diagram 1 showing the results of the autophagy induction test in Test Example 4. [Figure 3B] FIG. 3B is FIG. 2 showing the results of the autophagy induction test in Test Example 4. [Figure 4A] FIG. 4A shows the results of measuring the expression of ATG7 and the like in Test Example 5. [Figure 4B] FIG. 4B shows the results of staining the cells after culture in Test Example 5, which were fixed with a fixative. DETAILED DESCRIPTION OF THE INVENTION

[0014] (SA-β-gal expression inhibitor and autophagy inducer) The SA-β-gal expression inhibitor and autophagy inducer of the present invention contain lotus embryo extract as an active ingredient, and may further contain other ingredients as needed.

[0015] Although the details of the substance contained in the lotus germ extract that exerts the SA-β-gal expression inhibitory effect are unknown, it was not previously known that the lotus germ extract has such excellent effects and is useful as an SA-β-gal expression inhibitor, and this is a new discovery by the inventors.

[0016] As mentioned above, it is known that neferine, a component contained in lotus germ, has autophagy-inducing properties. However, as will be shown in the Examples section below, lotus germ extract has a stronger autophagy-inducing effect than neferine. Although the details of why the lotus germ extract exerts such a stronger autophagy-inducing effect than neferine are unknown, the fact that the lotus germ extract has such excellent properties and is useful as an autophagy inducer (sometimes referred to as an "autophagy activator") was previously unknown and is a new discovery made by the present inventors. Note that, as will be shown in the Examples section below, the lotus germ extract has autophagy-inducing properties but does not have mitophagy-inducing properties.

[0017] <Lotus germ extract> The lotus (scientific name: Nelumbo nucifera Gaertn.) is a perennial aquatic plant belonging to the genus Nelumbo in the family Nymphaeaceae, native to tropical Asia. In Japan, the rhizome of the lotus is eaten as lotus root, and it is a highly safe plant. Lotus is distributed in southeastern Europe, northern Australia, eastern Asia, etc., and is cultivated in ponds, rice paddies, moats, etc., and can be easily obtained from these areas.

[0018] The lotus germ extract may be prepared from the germ used as the extraction raw material, or a commercially available product may be used.

[0019] The lotus plant component used as the raw material for the lotus extract is the lotus germ, which is a green, rod-shaped embryo found inside the lotus seed. The shape, structure, and size of the germ used as the extraction raw material are not particularly limited and can be appropriately selected depending on the purpose.

[0020] The method for preparing the lotus extract raw material is not particularly limited and can be selected appropriately depending on the purpose. For example, the method may involve drying the embryo and then pulverizing it either directly or using a crusher. The dried material can be subjected to solvent extraction either directly or after pulverization. The drying can be performed in the sun or using a commonly used dryer.

[0021] The lotus germ extract can be easily obtained by a method generally used for plant extraction. The form of the lotus germ extract is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include the extract itself, a diluted extract, a concentrated extract, a dried product thereof, a roughly purified product thereof, and a purified product thereof.

[0022] The extraction method is not particularly limited and can be selected appropriately depending on the purpose, and examples include a method of extracting at room temperature or under reflux using any extraction device, and more specifically, a method of adding the lotus embryos, which are the extraction raw material, to a treatment tank filled with an extraction solvent, stirring appropriately as necessary, and leaving the mixture to stand for, for example, 30 minutes to 4 hours to elute the soluble components, and then filtering to remove the extraction residue, thereby obtaining an extract.The extract may be further dried after distilling off the extraction solvent. Alternatively, the extract may be used as a raw material for extraction after pre-treatment such as degreasing with a non-polar solvent such as hexane. Pre-treatment such as degreasing allows for efficient extraction with a polar solvent.

[0023] The conditions for extracting lotus embryos (extraction time and extraction temperature), the extraction solvent, and the amount of extraction solvent used are not particularly limited and can be appropriately selected depending on the purpose.

[0024] The extraction solvent is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include water, a hydrophilic solvent, and a mixed solvent of water and a hydrophilic solvent.

[0025] The water is not particularly limited and can be appropriately selected depending on the purpose. Examples include pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, and water obtained by various treatments. Examples of treatments for water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, etc. Water that can be used as the extraction solvent also includes purified water, hot water, ion-exchanged water, saline, phosphate buffer, phosphate-buffered saline, etc. The water may be used alone or in combination of two or more types.

[0026] The hydrophilic solvent is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include lower alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones, such as acetone and methyl ethyl ketone; and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin. These may be used alone or in combination of two or more.

[0027] The amount of the hydrophilic solvent used relative to the water in the mixed solvent is not particularly limited and can be appropriately selected depending on the purpose. However, when a lower alcohol is used, it is preferable to add 1 to 90 parts by volume relative to 10 parts by volume of water, when a lower aliphatic ketone is used, it is preferable to add 1 to 40 parts by volume relative to 10 parts by volume of water, and when a polyhydric alcohol is used, it is preferable to add 1 to 90 parts by volume relative to 10 parts by volume of water.

[0028] Among the above extraction solvents, 50% by volume of ethanol is preferred because it is easier to obtain a lotus embryo extract having a more excellent effect.

[0029] The temperature of the extraction solvent is not particularly limited and can be appropriately selected depending on the purpose, but it is preferably used at a temperature between room temperature and the boiling point of the solvent.

[0030] The obtained lotus germ extract may be subjected to treatments such as dilution, concentration, drying, purification, etc. according to conventional methods to obtain a diluted product, concentrate, dried product, roughly purified product, purified product, etc. of the lotus germ extract.

[0031] The purification method for the lotus embryo extract is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, etc. Purification by such a purification method can increase the concentration of active ingredients and remove unnecessary substances.

[0032] The lotus embryo extract thus obtained can be used as an SA-β-gal expression inhibitor or autophagy inducer as is, but the concentrate and dried product are preferred for ease of use. When obtaining the dried product, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.

[0033] The amount of lotus germ extract contained in the SA-β-gal expression inhibitor or autophagy inducer is not particularly limited and can be appropriately adjusted depending on the physiological activity of the extract, etc. The SA-β-gal expression inhibitor or autophagy inducer may consist solely of the lotus germ extract.

[0034] <Other ingredients> The other components are not particularly limited and can be selected appropriately depending on the form of use of the SA-β-gal expression inhibitor or autophagy inducer, and examples thereof include excipients, moisture-proofing agents, preservatives, strengthening agents, thickeners, emulsifiers, antioxidants, sweeteners, acidulants, seasonings, coloring agents, fragrances, whitening agents, moisturizers, oily components, UV absorbers, surfactants, thickeners, alcohols, powder components, coloring agents, aqueous components, water, skin nutrients, etc. These may be used alone or in combination of two or more.

[0035] The content of the other components in the SA-β-gal expression inhibitor or autophagy inducer is not particularly limited and can be selected appropriately depending on the purpose.

[0036] <Application> The uses of the SA-β-gal expression inhibitors or autophagy inducers are not particularly limited and can be appropriately selected depending on the purpose, and examples include cosmetics, pharmaceuticals, quasi-drugs, and foods and beverages. The SA-β-gal expression inhibitor or autophagy inducer has excellent SA-β-gal expression inhibitory or autophagy inducing effects and is highly safe, and therefore can be suitably used, for example, as an active ingredient in an anti-aging composition.

[0037] The SA-β-gal expression inhibitors or autophagy inducers of the present invention are preferably applied to humans, but can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as their respective effects are achieved.

[0038] The method of use of the SA-β-gal expression inhibitor or autophagy inducer is not particularly limited and can be appropriately selected depending on the purpose, and examples include topical, oral, and parenteral administration.

[0039] The dosage form of the SA-β-gal expression inhibitor or autophagy inducer is not particularly limited and can be selected appropriately depending on the purpose. Examples include topical preparations such as lotion, emulsion, cream, ointment, beauty serum, lotion, pack, jelly, lip balm, lipstick, foundation, bath additives, soap, body soap, astringent, hair tonic, hair lotion, hair cream, hair liquid, pomade, shampoo, rinse, and conditioner; oral preparations such as tablets, powders, capsules, granules, extracts, and syrup; and parenteral preparations such as injections, drips, and suppositories. The method for producing each of the above dosage forms of the SA-β-gal expression inhibitor or autophagy inducer is not particularly limited, and any known method can be appropriately selected.

[0040] The method of use, such as the amount and duration of use, of the SA-β-gal expression inhibitor or autophagy inducer is not particularly limited and can be appropriately selected depending on the purpose.

[0041] Furthermore, the SA-β-gal expression inhibitors or autophagy inducers of the present invention can also be used as reagents for research into the mechanism of action of SA-β-gal expression inhibitory action or autophagy induction action.

[0042] As described above, the SA-β-gal expression inhibitor of the present invention has excellent SA-β-gal expression inhibitory activity. Therefore, the present invention also relates to a method for inhibiting SA-β-gal expression, which comprises administering the aforementioned SA-β-gal expression inhibitor to an individual.

[0043] Furthermore, as described above, the autophagy inducer of the present invention has excellent autophagy-inducing activity. Therefore, the present invention also relates to a method for inducing autophagy, which comprises administering the autophagy inducer to an individual.

[0044] (Anti-aging composition) The anti-aging composition of the present invention contains at least one of the SA-β-gal expression inhibitor and autophagy inducer of the present invention, and further contains other components as needed.

[0045] <SA-β-gal expression inhibitor and autophagy inducer> The SA-β-gal expression inhibitor and autophagy inducer are the SA-β-gal expression inhibitor and autophagy inducer of the present invention described above.

[0046] The content of the SA-β-gal expression inhibitor and autophagy inducer in the anti-aging composition is not particularly limited and can be appropriately adjusted according to the form of the anti-aging composition and the physiological activity of the lotus germ extract, etc. However, in terms of the lotus germ extract, 0.0001% to 20% by mass is preferable, and 0.0001% to 10% by mass is more preferable. The anti-aging composition may consist only of the SA-β-gal expression inhibitor or the autophagy inducer.

[0047] <Other components> The other components in the anti-aging composition are not particularly limited and can be appropriately selected according to the usage form of the anti-aging composition. For example, those similar to the other components described in the items of the above SA-β-gal expression inhibitor and autophagy inducer can be mentioned. These may be used alone or in combination of two or more.

[0048] The content of the other components in the anti-aging composition is not particularly limited and can be appropriately selected according to the purpose.

[0049] <Aspect> The aspect of the anti-aging composition is not particularly limited and can be appropriately selected according to the purpose. For example, cosmetics, pharmaceuticals, quasi-drugs, food and drink products, etc. can be mentioned. The anti-aging composition of the present invention can be used on a daily basis, and the lotus germ extract, which is the active ingredient, can extremely effectively exert various physiologically active effects, including anti-aging effects.

[0050] The anti-aging composition of the present invention is preferably applied to humans, but can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as the respective functional effects are exerted.

[0051] The method of use of the anti-aging composition of the present invention is not particularly limited and can be appropriately selected depending on the purpose. Examples of the method of use include external application, oral administration, and parenteral administration.

[0052] Examples of the oral composition include the above-mentioned orally administered agents and foods and beverages. Here, foods and beverages refer to those that are unlikely to be harmful to human health and are taken orally or by administration through the digestive tract in normal social life, and are not limited to administrative classifications such as foods, medicines, and quasi-drugs. Therefore, the foods and beverages refer to a wide range of foods and beverages that are taken orally, including general foods, health foods (functional foods and beverages), health functional foods (foods for specified health uses, foods with nutrient functions, and foods with functional claims), quasi-drugs, and pharmaceuticals.

[0053] The type of oral composition is not particularly limited and can be appropriately selected depending on the purpose. Examples of the oral composition include beverages such as tea drinks, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, alcoholic drinks, coffee drinks, and coffee-containing soft drinks (including concentrated liquids and powders for adjusting these beverages); frozen desserts such as ice cream, ice sorbet, and shaved ice; noodles such as soba noodles, udon noodles, vermicelli, gyoza wrappers, shumai wrappers, Chinese noodles, and instant noodles; confectioneries such as candy, candy, gum, chocolate, candy tablets, snacks, biscuits, jelly, jam, cream, baked goods, and bread; and seafood such as crab, salmon, clams, tuna, sardines, shrimp, bonito, mackerel, whale, oysters, saury, squid, ark shells, scallops, abalone, sea urchin, salmon roe, and tokobushi sea bream. ; Processed seafood and livestock foods such as kamaboko, ham, and sausage; Dairy products such as processed milk and fermented milk; Oils and fats and oil-processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; Condiments such as sauces and dressings; Retort pouch foods such as curry, stew, oyakodon, porridge, rice porridge, Chinese rice bowl, katsudon, tendon, unadon, hayashi rice, oden, mabo dolph, beef bowl, meat sauce, egg soup, omelet rice, gyoza, shumai, hamburger steak, and meatballs; Side dishes such as salads and pickles; Health, beauty, and nutritional supplements in various forms; Pharmaceuticals and quasi-drugs such as tablets, granules, capsules, drinks, lozenges, and mouthwash; Oral fresheners used in the mouth such as mouth fresheners and anti-halitosis agents, and toothpaste.

[0054] Examples of the composition other than oral use include the above-mentioned parenteral administration agent and external use agent.More specifically, for example, skin lotion, emulsion, cream, ointment, beauty serum, lotion, gel, beauty oil, pack, jelly, lip balm, lipstick, foundation, bath additive, soap, body soap, astringent, hair tonic, hair lotion, hair cream, hair liquid, pomade, shampoo, rinse, conditioner, etc. can be used as the external use agent for skin.

[0055] The method for producing the anti-aging composition is not particularly limited and can be appropriately selected depending on the form in which the anti-aging composition is to be used.

[0056] The amount of the anti-aging composition used, the period of use, etc. are not particularly limited and can be appropriately selected depending on the purpose.

[0057] The anti-aging effect of the lotus germ extract is exerted, for example, by suppressing SA-β-gal expression and inducing autophagy. Therefore, the anti-aging composition preferably has at least one of the effects of suppressing SA-β-gal expression and inducing autophagy. Furthermore, the anti-aging composition preferably has the effect of suppressing SA-β-gal expression via the induction of autophagy excluding mitophagy. Note that the anti-aging effect of the lotus germ extract may include anti-aging effects other than those exerted based on the above-mentioned effects.

[0058] The anti-aging composition of the present invention can also be used as a reagent for research into the mechanism of anti-aging action.

[0059] As described above, the anti-aging composition of the present invention has an excellent anti-aging effect. Therefore, the present invention also relates to an anti-aging method, which comprises administering the anti-aging composition to an individual. [Example]

[0060] Hereinafter, production examples, test examples and formulation examples of the present invention will be explained, but the present invention is not limited to these production examples, test examples and formulation examples.

[0061] (Production Example 1) The lotus germ extract was prepared as follows. 100 g of dried water chestnut embryos were added with 1,500 mL of 50% by volume ethanol, and reflux extraction was performed at 80 - 90 °C for 2 hours, followed by filtration while hot. 0.2% by mass of activated carbon was added to the volume of the obtained extract, stirred at room temperature for 2 hours, and then filtered. The obtained extract was dried to obtain 18 g of water chestnut embryo extract (powder).

[0062] (Comparative Production Example 1) In Production Example 1, 13 g of water chestnut flower extract (powder) was obtained in the same manner as in Production Example 1, except that 100 g of dried water chestnut embryos was replaced with 100 g of dried water chestnut flowers.

[0063] (Test Example 1: SA-β-gal Expression Inhibition Test - 1) Using the above water chestnut embryo extract as a test sample, the inhibitory effect on SA-β-gal (senescence-associated beta-galactosidase) expression was tested by the following test method.

[0064] <Preparation of Human Skin Fibroblast Aging Cells> Using a medium prepared by mixing 10% FBS, 100 μg / mL penicillin, and 100 μg / mL streptomycin in MEMα medium, human skin fibroblast cells (NB1RGB cells) were cultured for 80 - 89 days to obtain aging cells.

[0065] <SA-β-gal Activity> To the culture solution containing the above human skin fibroblast aging cells, the water chestnut embryo extract was added to a concentration of 0 (control), 10, 50, or 100 μg / mL, and the human skin fibroblast aging cells were further cultured at 37 °C for 72 hours. Thereafter, using the Senescence Detection Kit (abcam), according to the manufacturer's protocol, the cultured cells were fixed with a fixing solution and stained. Cells with SA-β-gal activity (aging cells) were stained blue. The results are shown in Figure 1.

[0066] In addition, using a 96-well cellular senescence assay (CELL BIOLABS), the SA-β-gal activity of the cultured cells was measured according to the manufacturer's protocol. With the SA-β-gal activity in the control set as 100%, the SA-β-gal activity when the lotus germ extract was added (50 μg / mL) was calculated. The results are shown in Table 1.

[0067]

Table 1

[0068] From the above results, it was confirmed that the lotus germ extract has the effect of reducing the SA-β-gal activity specific to senescent cells. Therefore, it was also confirmed that the lotus germ extract has an anti-aging effect.

[0069] (Test Example 2: SA-β-gal expression inhibition test - 2) Using the above lotus germ extract as a test sample, it was tested whether the lotus germ extract directly inhibits the activity of SA-β-gal by the following test method.

[0070] <Preparation of human skin fibroblast senescent cells> Human skin fibroblast senescent cells were prepared in the same manner as in Test Example 1.

[0071] <SA-β-gal activity> The above-prepared human skin fibroblast senescent cells were cultured at 37°C for 24 hours. Thereafter, using a 96-well cellular senescence assay (CELL BIOLABS), the cultured cells were lysed according to the manufacturer's protocol. Then, the lotus germ extract was added to the lysate at 0 (control), 50, or 100 μg / mL, and the SA-β-gal activity was measured. With the SA-β-gal activity in the control set as 100%, the SA-β-gal activity when the lotus germ extract at each formulation amount was added to the cell lysate after cell culture was calculated. The results are shown in Table 2.

[0072]

Table 2

[0073] From the above results, when the loquat germ extract was added to the cell lysate after cell culture, no effect of reducing SA-β-gal activity was confirmed. Therefore, it was considered that the loquat germ extract did not suppress the enzyme activity of SA-β-gal, but rather suppressed the expression of SA-β-gal.

[0074] (Test Example 3: SA-β-gal Expression Inhibition Test - 3) Using the loquat germ extract, the loquat flower extract, and neferine as test samples, the SA-β-gal expression inhibition effect was tested by the following test method.

[0075] <Preparation of Human Skin Fibroblast Senescent Cells> Human skin fibroblast senescent cells were prepared in the same manner as in Test Example 1.

[0076] <SA-β-gal Activity> To the culture solution containing the above-mentioned human skin fibroblast senescent cells, the loquat germ extract was added at 50 μg / mL, the loquat flower extract was added at 50 μg / mL, or neferine was added at 1 μM, and the human skin fibroblast senescent cells were further cultured at 37°C for 72 hours. Also, 0.1% DMSO was added in place of the test sample, and the human skin fibroblast senescent cells cultured at 37°C for 72 hours were used as a control. Thereafter, using the Senescence Detection Kit (abcam), according to the manufacturer's protocol, the cultured cells were fixed with a fixative and stained. The results are shown in Figure 2.

[0077] The SA-β-gal activity of the cultured cells was measured using a 96-well cellular senescence assay (CELL BIOLABS) according to the manufacturer's protocol. The SA-β-gal activity in the control was set at 100%, and the SA-β-gal activity in the presence of each test sample was calculated. The results are shown in Table 3.

[0078] [Table 3]

[0079] These results confirm that lotus flower extract and neferine did not have the effect of suppressing SA-β-gal expression, whereas lotus germ extract did. In other words, it was confirmed that only lotus germ extract had the effect of suppressing SA-β-gal expression.

[0080] (Test Example 4: Autophagy induction test) The lotus embryo extract, the lotus flower extract, and neferine were used as test samples, and the autophagy-inducing activity was tested by the following test method.

[0081] <Preparation of human skin fibroblast senescent cells> Human dermal fibroblast senescent cells were prepared in the same manner as in Test Example 1.

[0082] <Autophagy induction> Using anti-LC3b antibody (Cell Signaling Technology) and anti-β-actin antibody (Sigma), we analyzed the amount of LC3-II biosynthesis (an increase in which autophagy can be determined to be induced). Specifically, the lotus germ extract was added to a culture medium containing the above-mentioned human dermal senescent fibroblasts at concentrations of 0 (control), 5, 10, 25, or 50 μg / mL, and the lotus flower extract was added to a culture medium containing the above-mentioned human dermal senescent fibroblasts at concentrations of 0 (control), 5, 10, 25, or 50 μg / mL, and the human dermal senescent fibroblasts were further cultured at 37°C for 72 hours (FIG. 3A).Also, the lotus germ extract was added to a culture medium containing the above-mentioned human dermal senescent fibroblasts at concentrations of 0 (control), 5, 10, 25, or 50 μg / mL, and neferine was added to a culture medium containing the above-mentioned human dermal senescent fibroblasts at concentrations of 0 (control), 0.1, 0.2, 0.5, or 1 μM, and the human dermal senescent fibroblasts were further cultured at 37°C for 72 hours (FIG. 3B). A protein solution was prepared from the obtained sample and subjected to polyacrylamide gel electrophoresis. The protein was then transferred to a membrane. The anti-LC3b antibody product was diluted 1:1,000, and the anti-β-actin antibody was diluted 1:10,000. The membrane was then immersed in the diluted anti-LC3b antibody product and incubated at 4°C for 18 hours, after which it was washed. The membrane was then immersed in HRP-labeled anti-rabbit and anti-mouse secondary antibody products (Promega) diluted 5:1,000, and incubated at room temperature for 1 hour, after which it was washed. The membrane was then immersed in luminol reaction solution and incubated at room temperature for 5–10 minutes until luminescence was observed. It was then exposed to X-ray film and developed. The LC3-II band intensity was corrected by the β-actin band intensity in each sample, and the relative intensity was calculated by setting the corrected value for the control at 1.0. The results are shown in Figures 3A and 3B. The "Intensity" values ​​in Figures 3A and 3B indicate the relative values.

[0083] These results confirmed that lotus germ extract has a stronger autophagy-inducing effect than lotus flower extract and neferine. The amount of neferine contained in 50 μg / mL of the lotus germ extract is equivalent to 1 μM of neferine.

[0084] (Test Example 5: Inhibition of autophagy induction and suppression of SA-β-gal expression) The relationship between autophagy induction and SA-β-gal expression was examined using the following test method.

[0085] <sirna> An siRNA targeting the gene encoding the ATG7 protein, essential for the induction of autophagy (hereinafter sometimes referred to as "siATG7"), was prepared with the following target sequence (Stealth RNAi oligonucleocide, manufactured by Invitrogen). A control siRNA (manufactured by Santa Cruz Biotechnology) (hereinafter sometimes referred to as "siControl") was also prepared. -Target sequence- 5'-GGAAACCTTAGAAGCGGACTTAATT-3' (SEQ ID NO: 1)

[0086] <Preparation of human skin fibroblast senescent cells> Human dermal fibroblast senescent cells were prepared in the same manner as in Test Example 1.

[0087] <Test> The siATG7 or siControl and Lipofectamine TM The human dermal fibroblast senescent cells were transfected using RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's instructions. Next, the lotus embryo extract was added to the culture medium containing the above-mentioned human dermal senescent fibroblast cells at a concentration of 0 or 50 μg / mL, and the human dermal senescent fibroblast cells were further cultured at 37°C for 48 hours to obtain samples.

[0088] A protein solution was prepared from the obtained sample and subjected to polyacrylamide gel electrophoresis. The protein was then transferred to a membrane. The membrane was immersed in a 1:1000 dilution of anti-ATG7 antibody (Cell Signaling), a 1:1000 dilution of anti-LC3b antibody product (see Test Example 4), and a 1:10,000 dilution of anti-β-actin antibody (see Test Example 4). The membrane was then incubated at 4°C for 18 hours and then washed. The membrane was then immersed in a 5:1000 dilution of HRP-labeled anti-rabbit and anti-mouse secondary antibody product (Promega), incubated at room temperature for 1 hour, and then washed. The membrane was then immersed in a luminol reaction solution and incubated at room temperature for 5-10 minutes until luminescence occurred, after which it was exposed to X-ray film and developed. The intensities of the ATG7 and LC3-II bands were corrected by the intensity of the β-actin band in each sample, and relative values ​​were calculated when the corrected value for the control was set to 1.0. The results are shown in Figure 4A. The "Intensity" values ​​in Figure 4A indicate the relative values.

[0089] In addition, the cultured cells were fixed with fixative and stained using a Senescence Detection Kit (Abcam) according to the manufacturer's protocol. The results are shown in Figure 4B.

[0090] The SA-β-gal activity of the cultured cells was measured using a 96-well cellular senescence assay (CELL BIOLABS) according to the manufacturer's protocol. The SA-β-gal activity in the control (siControl, lotus embryo extract 0 μg / mL) was set at 100%, and the SA-β-gal activity in the presence of each test sample was calculated. The results are shown in Table 4.

[0091] [Table 4]

[0092] These results suggest that the inhibitory effect of lotus germ extract on SA-β-gal expression is dependent on the induction of autophagy, since inhibition of autophagy induction by siATG7 also inhibits the inhibitory effect of lotus germ extract on SA-β-gal expression.

[0093] (Test Example 6: Mitophagy induction effect test) The lotus embryo extract was used as a test sample to test its mitophagy-inducing activity according to the following test method. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and oligomycin were used as positive controls.

[0094] <Preparation of human skin fibroblast senescent cells> Human dermal fibroblast senescent cells were prepared in the same manner as in Test Example 1.

[0095] <Mitophagy induction> The lotus embryo extract was added to the culture medium containing the above-mentioned human dermal senescent fibroblasts at 100 μg / mL, or FCCP and oligomycin at 1 μM, and the human dermal senescent fibroblasts were further cultured at 37° C. for 24 hours to obtain a sample. A control was also prepared by culturing the cells in the same manner except that the test sample was not added. Next, induction of mitophagy was measured using a mitophagy detection kit (Dojindo) according to the manufacturer's protocol. Observations were performed using an inverted fluorescence microscope (Olympus). The fluorescence intensity of the mitophagy dye was measured, and the relative values ​​calculated with the control fluorescence intensity set to 1.0 are shown in Table 5.

[0096] [Table 5]

[0097] These results confirmed that lotus embryo extract does not have the effect of inducing mitophagy.

[0098] (Combination example 1) An emulsion having the following composition was prepared by a conventional method. Lotus germ extract (extract produced in Production Example 1) 0.01g Jojoba oil 4.00g 1,3-butylene glycol 3.00g Arbutin 3.00g Polyoxyethylene cetyl ether (20E.O.) 2.50g 2.00g olive oil 2.00g squalane 2.00g of cetanol Glyceryl monostearate 2.00g Polyoxyethylene sorbitan oleate (20E.O.) 2.00g Methyl parahydroxybenzoate 0.15g Stearyl glycyrrhizinate 0.10g Phellodendron Bark Extract 0.10g Dipotassium glycyrrhizinate 0.10g Ginkgo biloba extract 0.10g 0.10g conchiolin Phellodendron bark extract 0.10g Chamomile extract 0.10g · Fragrance 0.05g Purified water, balance (total amount 100g)

[0099] (Combination example 2) A cream having the following composition was prepared by a conventional method. Lotus germ extract (extract produced in Production Example 1) 0.05g Sophora root extract 0.1g Scutellaria root extract 0.1g Liquid paraffin 5.0g 4.0g white beeswax Squalane 10.0g 3.0g of cetanol 2.0g lanolin Stearic acid 1.0g Polyoxyethylene sorbitan oleate (20E.O.) 1.5g Glyceryl monostearate 3.0g Oil-soluble licorice extract 0.1g 1,3-butylene glycol 6.0g Methyl parahydroxybenzoate 1.5g · Fragrance 0.1g Purified water, balance (total amount 100g)

[0100] (Combination example 3) A cosmetic essence having the following composition was prepared by a conventional method. Lotus germ extract (extract produced in Production Example 1) 0.01g Chamomile extract 0.1g 0.3g xanthan gum Hydroxyethylcellulose 0.1g Carboxyvinyl polymer 0.1g 1,3-butylene glycol 4.0g Dipotassium glycyrrhizinate 0.1g 2.0g glycerin 0.25g potassium hydroxide · Fragrance 0.01g Preservative (methyl parahydroxybenzoate) 0.15g 2.0g ethanol Purified water, balance (total amount 100g)

[0101] (Combination example 4) A hair tonic having the following composition was prepared by a conventional method. Lotus germ extract (extract produced in Production Example 1) 0.4g Tocopherol acetate (appropriate amount) Cephalatin 0.002g Isopropylmethylphenol 0.1g Sodium hyaluronate 0.15g 15.0g glycerin Ethanol 15.0g · Appropriate amount of fragrance Chelating agent (sodium edetate) appropriate amount Preservative (hinokitiol) appropriate amount Solubilizer (polyoxyethylene cetyl ether) appropriate amount Purified water, balance (total amount 100g)

[0102] (Combination example 5) A shampoo having the following composition was prepared by a conventional method. Lotus germ extract (extract produced in Production Example 1) 0.5g Marjoram extract 1.0g Plum fruit extract 0.2g Sodium coconut oil fatty acid methyl taurate 10.0g Coconut oil fatty acid amidopropyl betaine 10.0g Sodium polyoxyethylene alkyl ether sulfate 20.0g Coconut oil fatty acid diethanolamide 4.0g Propylene glycol 2.0g · Appropriate amount of fragrance Purified water, balance (total amount 100g)

[0103] (Combination example 6) An oral liquid preparation having the following composition was prepared by a conventional method. <Composition in 1 ampoule (100 mL)> Lotus germ extract 0.3% by mass (Extract produced in Production Example 1) Sorbitol 12.0% by mass Sodium benzoate 0.1% by mass · Fragrance 1.0% by mass Calcium sulfate 0.5% by mass · Purified water remainder

[0104] (Combination example 7) Tablets having the following composition were prepared by a conventional method. Lotus germ extract 5.0mg (Extract produced in Production Example 1) Dolomite 83.4mg (Contains 20% calcium and 10% magnesium) Casein phosphopeptide 16.7mg Vitamin C 33.4mg Maltitol 136.8mg Collagen 12.7mg Sucrose fatty acid esters 12.0mg< / sirna>

Claims

1. An SA-β-gal (sence-associated beta-galactosidase) expression inhibitor characterized by containing lotus germ extract.

2. An autophagy inducer characterized by containing lotus germ extract.

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