JP7699334B2 - Skin quality improving agent containing red shiso extract - Google Patents

Description

The present invention relates to a skin quality improving agent containing a red perilla extract.

Living a healthy daily life has always been a desire of people. In particular, in today's society where the average life expectancy continues to increase, it is desirable to be able to live a vibrant daily life even as we get older.

In order to live such a healthy lifestyle, for example, it is effective to regularly consume foods that contain ingredients that can prevent or alleviate disease.

In this regard, red shiso has been widely known as a familiar food ingredient to Japanese people since ancient times, and continues to be used as a traditional Chinese medicine to relieve chills and food poisoning.

In recent years, red shiso has also been attracting attention as a food ingredient that contains ingredients that have disease prevention and improvement effects, and methods of using it are known, such as those disclosed in Patent Documents 1 and 2.

Patent Document 1 discloses an agent for preventing or improving nocturia. This preventive and improving agent contains rosmarinic acid or its salt, which is contained in red shiso extract, as an active ingredient, and reduces the number of times the patient wakes up to urinate during sleep. It is therefore said that such a preventive and improving agent can reduce anxiety about urination during sleep and support healthy sleep.

Patent Document 2 also discloses an adiponectin secretion promoter. The secretion promoter is an extract of a Lamiaceae plant, and is characterized by containing at least one component selected from rosmarinic acid, luteolin, avigenin, and chrysoeriol. The secretion promoter can prevent or improve symptoms of lifestyle-related diseases such as diabetes, arteriosclerosis, high blood pressure, hyperlipidemia, obesity, and metabolic syndrome, and is said to provide an adiponectin secretion promoter derived from natural ingredients that is highly safe even when administered over the long term.

JP 2019-206519 A JP 2007-106718 A

As mentioned above, red shiso has excellent effects in preventing and improving illness.

As people age, the deterioration of their appearance becomes more noticeable. In particular, age can increase the appearance of age spots and wrinkles, and the condition of the skin usually changes significantly compared to when we were younger.

In order to maintain a youthful and beautiful appearance even as we age, it is preferable to take topical preparations, cosmetics, or foods on a daily basis that contain ingredients that have anti-wrinkle effects to prevent and improve wrinkles, or ingredients that can suppress melanin production to prevent and improve age spots and freckles.

Of course, red shiso is also used for beauty purposes in health foods and cosmetics. However, the significance of including red shiso extract in health foods and cosmetics proposed up until now has not always been clear. In other words, the red shiso extract contained in health foods and cosmetics is often vaguely described as being "good for the body" or "good for the skin," and is provided to users without a clearly stated purpose, so users may not know what specific effects the ingredients in the red shiso extract are providing.

The present invention was made in consideration of these circumstances, and provides a skin quality improving agent that contains red shiso extract as an inducing component with a clearer effect than conventional agents. The present invention also provides external preparations, cosmetics, and foods that contain the skin quality improving agent.

The skin quality improving agent of the present invention is characterized by containing an ethanol extract of red shiso that does not contain rosmarinic acid as an ingredient that induces acne formation prevention effect .

In addition, the topical agent for preventing acne occurrence , the cosmetic composition for preventing acne occurrence , and the functional food for preventing acne occurrence according to the present invention contain the above-mentioned skin quality improving agent.

According to the skin quality improving agent of the present invention, since it is characterized by containing an ethanol extract of red shiso that does not contain rosmarinic acid as an ingredient that induces the acne occurrence prevention effect , it is possible to provide a skin quality improving agent containing red shiso extract with a more specific purpose.

Furthermore, according to the topical preparation for preventing the occurrence of acne , cosmetic preparation for preventing the occurrence of acne, and functional food for preventing the occurrence of acne of the present invention, since they contain the above-mentioned skin quality improving agent which contains an ethanol extract of red shiso that does not contain rosmarinic acid as an ingredient that induces the acne occurrence prevention effect , it is possible to provide a topical preparation for preventing the occurrence of acne , cosmetic preparation for preventing the occurrence of acne , and functional food for preventing the occurrence of acne that can improve skin quality.

FIG. 2 is an explanatory diagram showing the test results of red shiso extract in HPLC analysis. FIG. 2 is an explanatory diagram showing the test results of the water extract in HPLC analysis. FIG. 1 is an explanatory diagram showing the test results of an ethanol extract in HPLC analysis. FIG. 1 is a diagram comparing area values of ELSD detected peaks in HPLC analysis. FIG. 1 is an explanatory diagram showing the test results of TLC analysis. FIG. 1 is an explanatory diagram showing the test results of TLC analysis. FIG. 1 is an explanatory diagram showing the test results of GC/MS analysis. FIG. 1 is an explanatory diagram showing the test results of GC/MS analysis. FIG. 1 is an explanatory diagram showing the test results of GC/MS analysis. 10 is a bar graph showing the results of the test of FIG. FIG. 1 is an explanatory diagram showing the test results of a lipase inhibitory activity test. FIG. 1 is an explanatory diagram showing the test results of an anti-allergy test. FIG. 1 is an explanatory diagram showing the test results of a hyaluronic acid production evaluation test. FIG. 1 is an explanatory diagram showing the test results of a collagen production evaluation test. FIG. 1 is an explanatory diagram showing the test results of a melanin production evaluation test. FIG. 1 is an explanatory diagram showing the test results of an anti-acne bacteria activity test. FIG. 1 is an explanatory diagram showing the test results of an elastase inhibitory activity evaluation test.

The present invention relates to a skin quality improving agent containing red shiso extract and its use, and also provides topical preparations, cosmetics, foods, etc. that contain the skin quality improving agent.

In particular, the skin quality improving agent according to this embodiment is characterized in that it contains red shiso extract as an ingredient that induces at least one of the effects selected from the prevention of acne occurrence, anti-allergy, anti-wrinkle, and inhibition of melanin production.

Preventing acne occurrence means inhibiting the proliferation of bacteria that cause acne. Such a function can be confirmed, for example, by the lipase inhibitory activity test and the anti-acne bacteria activity test described below. In other words, the skin quality improving agent containing the red shiso extract according to the present invention can be said to have an acne occurrence prevention effect if it is used externally in the form of an external preparation or cosmetic and/or orally in the form of a food (hereinafter referred to as external/oral use mode) to increase lipase inhibitory activity and inhibit the proliferation of acne bacteria.

Anti-allergic means suppressing itching and swelling associated with allergic symptoms. Such a function can be confirmed, for example, by measuring β-hexosaminidase activity as in the anti-allergic activity test described below. In other words, the skin quality improving agent containing the red shiso extract of the present invention can be said to have an anti-allergic effect if it suppresses β-hexosaminidase activity when used externally or orally.

Anti-wrinkle means preventing dry skin and inhibiting the decrease of components that make up the dermis. Such functions can be confirmed, for example, by the collagen production evaluation test and hyaluronic acid production evaluation test described below. In other words, the skin quality improving agent containing the red shiso extract of the present invention can be said to have an anti-wrinkle effect if it promotes collagen production or hyaluronic acid production when used topically or orally.

Inhibition of melanin production means suppressing the excessive production of melanin by melanocytes. Such a function can be confirmed, for example, by the melanin production evaluation test described below. In other words, if the skin quality improving agent containing the red shiso extract of the present invention can suppress melanin production when used topically or orally, it can be said to have a melanin production inhibitory effect.

Red shiso is a plant known as Perilla frutescens var. frutescens form. purpurea, which belongs to the Lamiaceae family and the Lamiaceae genus, and is a plant that is commonly cultivated widely in Japan.

The red shiso extract contained in the skin quality improving agent according to this embodiment is an extract made from the whole plant of red shiso or one or more of the leaves, flowers, fruits, stems, and roots.

Extraction is the process of obtaining an extract by pressing or infusing ingredients into an extraction solvent.

The extraction solvent for obtaining the extract is not particularly limited, but for example, water or ethanol can be used. The water extract is extracted from red shiso leaves using water at a specific temperature as the extraction solvent. The ethanol extract is extracted from red shiso leaves using ethanol at a specific temperature as the extraction solvent. The extraction temperature using these extraction solvents is not particularly limited, but can be, for example, 4°C to 100°C, more specifically 10°C to 50°C, and even more specifically 15°C to 30°C.

The extraction method for obtaining a water extract or an ethanol extract is not particularly limited, but one example is a method in which an extraction solvent and dried red shiso leaves are placed in a specified container and shaken for extraction.

The extracted liquid may be directly contained in the skin quality improving agent of this embodiment, but it can also be dried to make a red shiso extract.

The method for drying the extract is not particularly limited and can be selected appropriately depending on the scale and precision of the extraction. For example, freeze-drying can be used to dry the water extract, and drying using a rotary evaporator or a desiccator can be used to dry the ethanol extract. When drying using a rotary evaporator, the drying conditions can be adjusted as necessary depending on the purpose and circumstances, such as heating and reducing pressure, or whether or not boiling is performed.

The water or ethanol extract of red shiso thus extracted can be used as is as a skin quality improving agent according to this embodiment, regardless of whether it is in a solid (powder) or liquid state, but auxiliary ingredients can also be added as necessary.

Here, auxiliary ingredients include, for example, ingredients that suppress the loss of function over time of ingredients that cause the effect of improving skin quality, ingredients necessary for adjusting the dosage form, ingredients for adjusting the flavor, and other necessary ingredients.

More specifically, if an ingredient that brings about an effect of improving skin quality loses its function over time due to oxidation, an antioxidant that inhibits this corresponds to an auxiliary ingredient. Also, if the ingredient loses its function due to denaturation, a specific denaturation inhibitor corresponds to an auxiliary ingredient.

In addition, auxiliary ingredients such as excipients and fragrances may be added as necessary when processing into topical agents, cosmetics, foods, reagents, etc.

Topical preparations here are used by applying, sticking, or spraying onto the skin or mucous membranes, and include medicines and quasi-drugs, as well as products that are not classified as cosmetics or food. Topical preparations include ointments, creams, liquid preparations for external use, eye drops, nasal drops, suppositories, patches, inhalants, sublingual preparations, etc.

Cosmetics include not only so-called makeup cosmetics, but also basic skin care products, hair tonics, perfumes, toothpaste, shampoos, conditioners, soaps and detergents used for body washing, bath additives and other toiletry products, as well as medicated cosmetics that claim to have preventive effects, etc.

Makeup cosmetics include, for example, foundation, eyebrow pencil, mascara, eye shadow, eyeliner, lipstick, gloss, blush, face powder, and nail polish, while basic cosmetics include, for example, lotion, milky lotion, facial cleanser, cleanser, serum, and cream. Lotion also includes liquids (spray liquids) that are sprayed onto the face or hands, such as refresh mist.

Functional foods are a concept that encompasses all foods that do not contain pharmaceutical ingredients and are believed to contribute to maintaining and improving health, and include, for example, nutritional supplements, health supplements, and nutritionally adjusted foods, as well as general foods such as so-called supplements, and health functional foods such as foods for specified health uses, foods with nutritional functions, and foods with functional claims.

The above explanations of topical agents, cosmetics, and functional foods are merely examples of equivalent products to aid in the understanding of the present invention, and the interpretation of each term is not limited to these listed products. However, this does not preclude the applicant from limiting the present invention to these products when granting rights to this application.

The skin quality improving agent of this embodiment can also be used as an experimental reagent or clinical test drug.

In this way, by using red shiso extract as an ingredient that induces each of the above-mentioned effects, it is possible to provide a skin quality improving agent that can be ingested in daily life, as well as topical agents, cosmetics, and functional foods that contain the same improving agent.

The skin quality improving agent of this embodiment will be described below with reference to the process of investigating whether it has the effect of preventing acne, preventing allergies, preventing wrinkles, or inhibiting melanin production.

1. Preparation of evaluation samples
Leaves were collected from red perilla grown in a red perilla field in Ashiya-machi, Onga-gun, Fukuoka Prefecture, and three types of evaluation samples were prepared. That is, the evaluation samples were a red perilla extract obtained by squeezing red perilla leaves, a red perilla extract residue obtained by vacuum-drying red perilla leaves after extraction of the red perilla extract, and a red perilla dried product obtained by drying red perilla leaves at 60°C.

Furthermore, the red shiso extract was used as is in each test, while the red shiso extract residue and dried red shiso were ground in a mill and then extracted before use.

To extract the red shiso extract residue, 1 g of crushed leaves was placed in each of two Erlenmeyer flasks, 100 mL of water was poured into one flask, and 100 mL of ethanol was poured into the other flask.

Then, both flasks were sealed and attached to a shaking extractor, and the shaking extraction process was carried out at room temperature at 200 rpm for 24 hours.

After the shaking extraction process was completed, the flask contents of the water extract were centrifuged at 3000 rpm for 10 minutes, and the supernatant was obtained and freeze-dried. The resulting freeze-dried material was used as the water extract of red shiso extract residue.

After the shaking extraction process, the contents of the ethanol extraction flask were filtered through No. 2 filter paper and concentrated and dried using a rotary evaporator. The resulting dried material was used as the ethanol extract of red shiso extract residue.

The extraction process for the dried red shiso was carried out in the same manner as for preparing the extract from the red shiso extract residue, to obtain a water extract and an ethanol extract from the dried red shiso.

[2. HPLC analysis, TLC analysis]
Next, in order to obtain component profiles of the red shiso extract, the water extract and ethanol extract of the red shiso extract residue, and the water extract and ethanol extract of dried red shiso, component analysis was performed using high-performance liquid chromatography (HPLC) and thin-layer chromatography (TCL).

For the HPLC analysis in this test, the evaluation samples were pretreated to prepare HPLC samples. The HPLC samples were prepared by dissolving the water extract at 10 mg/mL in the initial eluent (10% methanol) and passing it through a 0.2 μm filter, dissolving the ethanol extract at 10 μg/mL in methanol and passing it through a 0.2 μm filter, and passing the red shiso extract directly through a 0.2 μm filter.

The analytical conditions for the HPLC analysis are as shown in Table 1.

For TLC analysis, three different developing solvents were used (Hexane:Ethyl acetate = 50:50, Ethyl acetate = 100, Ethyl acetate:MeOH = 50:50) using TLC aluminum plates.

The results of the HPLC analysis are explained based on Figures 1 to 4. Figures 1 to 3 show chromatograms of the HPLC analysis. Figure 1 shows the results of the red shiso extract, Figure 2(a) shows the results of the water extract of the red shiso extract residue, Figure 2(b) shows the results of the water extract of dried red shiso, Figure 3(a) shows the results of the ethanol extract of the red shiso extract residue, and Figure 3(b) shows the results of the ethanol extract of dried red shiso. Figure 4 is a table and graph showing the area values of the peaks detected by ELSD in the water extract and ethanol extract.

As shown in Figure 1, the chromatogram of red shiso extract showed a peak at around RT = 12.5 minutes using UV (280 nm), but no peak was observed using ELSD, and the details of the analysis results were unclear.

Next, the results of the water extract of red shiso extract residue and the water extract of dried red shiso were compared. As shown in Figures 2(a) and 2(b), at 280 nm, the water extract of red shiso extract residue showed multiple peaks up to around RT=10 minutes, whereas dried red shiso showed fewer peaks. In addition, in the ELSD results shown in Figure 4, a single peak was observed in both cases around RT=1.5 minutes.

Furthermore, the results of the ethanol extract of the red shiso extract residue and the ethanol extract of dried red shiso were compared. As shown in Figures 3(a) and 3(b), numerous peaks were confirmed in both. In particular, the ethanol extract of dried red shiso has large peak heights, suggesting that it contains a large amount of ingredients. Furthermore, in the ELSD results shown in Figure 4, three peaks that are thought to represent the main components were confirmed around RT = 23 to 25 minutes.

These results show that in red shiso extract residue and dried red shiso, the ethanol extract contains a greater variety of components than the water extract, and the amounts of these components are greater.

The results of the TLC analysis are explained based on Figure 5. Figure 5(a) shows the results when hexane: ethyl acetate = 50: 50 was used as the developing solvent, Figure 5(b) shows the results when ethyl acetate = 100 was used as the developing solvent, and Figure 5(c) shows the results when ethyl acetate: MeOH = 70: 30 was used as the developing solvent. In Figure 5, the symbol 3 shows the results of the water extract of the red shiso extract residue, the symbol 4 shows the results of the ethanol extract of the red shiso extract residue, the symbol 5 shows the results of the water extract of the dried red shiso, and the symbol 6 shows the results of the ethanol extract of the dried red shiso.

As shown in Figure 5, even when different developing solvents were used, there were differences in the intensity of the peaks in the ethanol extract of the red shiso extract residue and dried red shiso, but no significant differences were observed in the peak positions.

Next, a TLC test was used to confirm whether rosmarinic acid and luteolin were contained in the ethanol extract of dried shiso. The results are explained with reference to Figure 6.

Figure 6(a) shows the results when the developing solvent was hexane: ethyl acetate = 50: 50, Figure 6(b) shows the results when the developing solvent was ethyl acetate = 100, and Figure 6(c) shows the results when the developing solvent was ethyl acetate: MeOH = 70: 30. In Figure 6, the symbol 6 indicates the results of an ethanol extract of dried red shiso, the symbol Flav indicates the results when the detection limit concentration of luteolin was used as a positive control, and the symbol Ros indicates the results when the detection limit concentration of rosmarinic acid was used as a positive control.

Figure 6(b) shows that the luteolin concentration in the ethanol extract of dried red shiso is below the detection limit. Figure 6(c) shows that the rosmarinic acid concentration in the ethanol extract of dried red shiso is below the detection limit.

[3. GC/MS analysis]
Next, in order to obtain a profile of the volatile components contained in the red shiso extract, red shiso extract residue, and dried red shiso, volatile components were collected by solid-phase microextraction (SPME) and the components were analyzed by gas chromatography/mass spectrometry (GC/MS).

In this test, 5 mL of red shiso extract was placed directly into a 20 mL SPME vial. The red shiso extract residue and dried red shiso were ground in a mill and placed into a 20 mL SPME vial at 5 g. The gas phase of each vial was replaced with nitrogen gas before capping.

After the replacement, the red shiso extract was collected using an SPME fiber (100 μm polydimethylsiloxane) at room temperature for 1 minute, and the red shiso extract residue and dried red shiso were collected at room temperature for 30 minutes.

After collection, each vial was analyzed by GC/MS under the conditions shown in Table 2.

The results of the GC/MS analysis are explained with reference to Figures 7 to 10. Figure 7(a) shows the results of GC/MS analysis of red shiso extract. Figure 7(b) shows the results of GC/MS analysis of red shiso extract residue. Figure 7(c) shows the results of GC/MS analysis of dried red shiso. Figure 8 is a chromatogram in which the vertical axis of the results of GC/MS analysis of red shiso extract residue shown in Figure 7(b) is adjusted to the same dimensions as the results of dried red shiso shown in Figure 7(c). Figure 9 is a list of components identified by GC/MS analysis, and Figure 10 is a bar graph showing the peak areas of the identified components.

As shown in Figure 7 (a) and Figures 9 and 10, it was revealed that red shiso extract contains benzaldehyde (RT = around 7 minutes) and perillaldehyde (RT = around 16 minutes) as its main components. Perillaldehyde is an aroma component contained in shiso, and is said to account for approximately 50% of shiso essential oil. In addition, D-limonene, eucalyptol (cineole), linalool, perill alcohol, etc. were also detected in the red shiso extract.

Figures 7(b) and 7(c) as well as Figures 9 and 10 show that the red shiso extract residue and dried red shiso contain a greater number of peaks than the red shiso extract. In other words, it was revealed that the red shiso extract residue and dried red shiso contain a greater variety of volatile components than the red shiso extract.

In addition, Figures 7(c) and 8, as well as Figures 9 and 10, show that red shiso extract residue contains a wide variety of compounds. On the other hand, it was revealed that although dried red shiso contains fewer types of components compared to red shiso extract residue, the content of each component tends to be higher.

[4. Lipase inhibitory activity test]
Lipase is known as an enzyme that breaks down fat. It is known that acne bacteria, which are believed to cause acne, also secrete lipase, which breaks down sebum using lipase to feed themselves. In other words, acne bacteria grow while breaking down sebum, and secrete components involved in inflammation that causes acne. In other words, if the action of lipase is inhibited, it is possible to prevent the occurrence of acne by suppressing the growth of acne bacteria and the secretion of inflammatory components.

In this test, 25 μL of each sample was mixed with 50 μL of 0.1 mM 4-methylumbeliferyl oleate (4-MUO) (13 mM Tris-HCl, 150 mM NaCl, 1.3 mM _CaCl2 buffer solution), and 25 μL of 150 μg/mL pancreatic lipase was dissolved in the same buffer as above, and incubated at 25°C for 30 minutes.

After incubation, 100 μL of 0.1 M citrate buffer (pH 4.2) was added to stop the reaction. The fluorescence intensity (Em: 355, Ex: 460 nm) of 4-methylumbelliferone (4-MU), which was liberated by the action of lipase on 4-MUO, was then measured to evaluate its effect on lipase activity.

The final concentrations of the samples in this test were 12.5, 37.5, 75, and 150 ppm for red shiso extract, and 1.5625, 12.5, 25, 100, 400, and 800 μg/mL for the water extract and ethanol extract of red shiso extract residue, and the water extract and ethanol extract of dried red shiso.

This study was also conducted with n=4, and orlistat (Orl) was used as a positive control.

Figure 11(a) is a graph showing the lipase inhibitory activity of red shiso extract. Figure 11(b) is a graph showing the lipase inhibitory activity of the water extract and ethanol extract of red shiso extract residue. Figure 11(c) is a graph showing the lipase inhibitory activity of the water extract and ethanol extract of dried red shiso.

In this test, as shown in Figure 11 (a), red perilla extract was tested at sample concentrations of 12.5, 37.5, 75, and 150 ppm, and lipase inhibitory activity of approximately 5% to approximately 10% was observed at all concentrations. Furthermore, it was not confirmed that red perilla extract showed a concentration-dependent increase in lipase inhibitory activity.

As shown in Figure 11 (b), when the aqueous extract of red shiso extract residue was examined at sample concentrations of 1.5625, 12.5, 25, 100, 400, and 800 μg/mL, a concentration-dependent high lipase inhibitory activity was observed at concentrations of 12.5 μg/mL or higher. In contrast, no lipase inhibitory activity was observed at 1.5625 μg/mL.

In addition, when the ethanol extract of red shiso extract residue was examined at sample concentrations of 1.5625, 12.5, 25, 100, 400, and 800 μg/mL, a concentration-dependent high lipase inhibitory activity was observed at concentrations of 12.5 μg/mL or higher. In contrast, no lipase inhibitory activity was observed at 1.5625 μg/mL.

These results show that when comparing the experimental results of the aqueous extract and ethanol extract of red shiso extract residue, both show concentration-dependent lipase inhibitory activity, but the ethanol extract shows a particularly strong effect.

As shown in Figure 11 (c), when the aqueous extract of dried red shiso was examined at sample concentrations of 1.5625, 12.5, 25, 100, 400, and 800 μg/mL, lipase inhibitory activity was confirmed at all concentrations, and the effect was found to be concentration-dependent.

In addition, when the ethanol extract of dried red shiso was examined at sample concentrations of 1.5625, 12.5, 25, 100, 400, and 800 μg/mL, a concentration-dependent high lipase inhibitory activity was observed at concentrations of 12.5 μg/mL or higher. In contrast, no lipase inhibitory activity was observed at 1.5625 μg/mL.

From these results, it was revealed that when comparing the experimental results of the water extract and ethanol extract of dried red shiso, both showed concentration-dependent lipase inhibitory activity, but at concentrations of 12.5 μg/mL or higher, the ethanol extract showed a stronger effect than the water extract.

Therefore, it was revealed that the aqueous and ethanolic extracts of the red perilla extract residue and the aqueous and ethanolic extracts of the dried red perilla exhibited high lipase inhibitory activity in a concentration-dependent manner at concentrations of 12.5 μg/mL or more. Furthermore, when the IC50 values (50% inhibitory concentration) were calculated for the red perilla extract and the dried red perilla, it was revealed that the ethanolic extract had extremely strong lipase inhibitory activity, as shown in Table 3.

5. Anti-allergy Testing
Allergies are one of the causes of symptoms such as itching, redness, papules, and small blisters on the skin. Allergies are caused by immune cells called basophils releasing granules containing β-hexosaminidase. In other words, these allergic symptoms can be suppressed by inhibiting the release of granules containing β-hexosaminidase.

In this test, samples were added to rat basophilic leukemia cells (RBL-2H3) as a model cell for basophils, and the amount of granules released, i.e., the effect on allergy onset, was measured by measuring the amount of β-hexosaminidase in the cell supernatant. In this test, water and ethanol extracts of red shiso extract residue, as well as water and ethanol extracts of dried red shiso were used as samples.

Specifically, rat basophilic leukemia cells (RBL-2H3) were prepared at a concentration of 5 x ¹⁰⁵ cells/mL and seeded in a 96-well plate at 100 μL. Two such plates were prepared, one for measuring β-hexosaminidase activity and the other for the MTT test, and pre-cultured for 40 hours in a _CO2 incubator (37°C, 5% _CO2 ).

After pre-incubation, the medium was removed and the wells were washed with Tyrode's buffer, and 100 μL of Turode's buffer (or EMEM medium) containing 0.5 μL of each diluted sample was added to each well. After 1 hour of incubation at ₃₇ °C and 5% CO2, the supernatant was discarded from the plate for measuring β-hexosaminidase activity, the wells were washed, and 100 μL of A23187 (in Tyrode's buffer) was added. After 1 hour of incubation in an incubator, 50 μL of the supernatant from each well was taken and transferred to a new 96-well plate.

To measure β-hexosaminidase activity, 50 μL of p-Nitrophenyl-N-acetyl-β-glucosaminide solution was added as a substrate to each well of a new plate from which 50 μL of the supernatant had been removed, and the reaction was carried out at room temperature for 1 to 3 hours while shielding the wells from light with aluminum foil. After the reaction, 100 μL of reaction stop solution was added to all wells to stop the enzyme reaction, and the absorbance at 405 nm was measured using a plate reader to measure β-hexosaminidase activity.

For the MTT test, the medium was removed from each well of the plate one hour after the addition of each diluted sample, 100 μL of MTT staining solution (5 mg/ml in medium) was added, and the plate was incubated at 5% CO ₂ and 37°C for four hours. The medium was then removed, and 1 mL of hydrochloric acid-isopropanol solution was added to each well. The plate was then shielded from light and left at room temperature for four hours. The absorbance at 570 nm was then measured using a microplate reader, and used as an index of cell viability.

This study was also conducted with n=3, and quercetin was used as a positive control.

Figure 12 is a graph showing the cell viability and β-hexosaminidase activity of the water extract and ethanol extract of red shiso extract residue, and the water extract and ethanol extract of dried red shiso.

As shown in Figure 12, when the aqueous extract of red shiso extract residue was examined at sample concentrations of 10, 100, and 400 μg/mL, it was found to promote β-hexosaminidase activity in a concentration-dependent manner. In addition, when the ethanol extract of red shiso extract residue was examined at the same sample concentrations, it was found to inhibit β-hexosaminidase activity in a concentration-dependent manner at concentrations of 100 μg/mL or less, but to promote β-hexosaminidase activity at 400 μg/mL.

In this way, it was revealed that the aqueous extract and ethanol extract of red shiso extract residue at sample concentrations of 100 μg/mL or less exhibited opposite properties of "stimulating" and "inhibiting" β-hexosaminidase activity, and that the ethanol extract at a sample concentration of 400 μg/mL in turn exhibited the property of "stimulating" β-hexosaminidase activity.

In addition, when the aqueous extract of dried red shiso was tested at sample concentrations of 10, 100, and 400 μg/mL, it was found that it promoted β-hexosaminidase activity to the same extent at all concentrations. In addition, when the ethanol extract of dried red shiso was tested at the same sample concentrations, it was found that it inhibited β-hexosaminidase activity and increased cell viability in a concentration-dependent manner.

Thus, inhibition of β-hexosaminidase activity was observed only in the ethanol extract of red shiso extract residue and dried red shiso. In other words, it is believed that the hydrophobic components of red shiso act as the components that induce the anti-allergic effect.

[6. Collagen and hyaluronic acid production evaluation test]
Collagen is a structural protein that forms connective tissues such as the skin of animals. Collagen contributes to the structure of the skin and is important for keeping the skin youthful and wrinkle-free.

Collagen is produced by fibroblasts present in the dermis of the skin, and if collagen production by fibroblasts can be promoted, youthful skin can be maintained. Therefore, components that promote collagen production in fibroblasts are thought to be useful as anti-aging cosmetic materials.

Hyaluronic acid is also produced mainly by fibroblasts, the main cells in the dermis of the skin, and other cells of connective tissues. It is a major component of the extracellular matrix and is involved in various intercellular interactions. It is also present in high concentrations in synovial fluid, where it is responsible for normal water retention and lubrication of joints.

In this test, the collagen and hyaluronic acid production ability of a blast cell culture system is measured by adding a water extract and an ethanol extract of dried red shiso matter according to this embodiment, and the skin moisture retention effect of red shiso extract residue and an extract of dried red shiso matter is evaluated.
In this test method, adult human dermal fibroblasts (NHDF-Ad) were used as model cells.
The cells were seeded at 1.0×10 ⁴ cells/well onto a microwell plate and pre-cultured for 24 hours in a CO ₂ incubator (37° C., 5% CO ₂ ).

After pre-culture, the medium was removed, and new medium and each diluted sample diluted to a specified concentration were added, and the cells were again cultured in a _CO2 incubator for 72 hours. The amount of collagen and hyaluronic acid produced in the culture medium was measured using a commercially available ELISA kit. Similarly, an MTT test was performed using the same cells.

Figure 13 shows the ratio of hyaluronic acid production by an ethanol extract of dried red shiso to the control.

In this study, the water extract of dried red shiso was not found to have any effect on hyaluronic acid production.

On the other hand, as shown in Figure 13, when the ethanol extract of dried red shiso was examined at sample concentrations of 10 and 100 μg/mL, a concentration-dependent increase in hyaluronic acid production was observed.

Figure 14 shows the collagen production ratio of the ethanol extract of dried red shiso relative to the control.

In this study, the aqueous extract of dried red shiso was not found to have any collagen-producing effect.

On the other hand, as shown in Figure 14, when the ethanol extract of dried red shiso was examined at a sample concentration of 100 μg/mL, an increase in collagen production was observed at the sample concentration of 100 μg/mL.

These results suggest that the ethanol extract of dried red shiso contains components that promote the production of hyaluronic acid and collagen.

[7. Melanin production evaluation test]
Melanin is a general term for pigments that are formed when phenolic substances polymerize. Melanin is produced by melanocytes in the skin and protects cells from damage caused by ultraviolet rays. However, excessive ultraviolet rays can cause excessive melanin production, which can lead to age spots. In other words, if red shiso extract can suppress melanin production, it can be used as a skin quality improvement agent that can improve age spots.

In this study, the effect of sample addition on melanin production was evaluated using a mouse melanoma cell line (B16). The evaluation sample was an ethanol extract (0.1, 1, 10ng/mL) of dried red shiso.

Mouse melanoma cell lines were precultured in 75 ^cm2 flasks using DMEM (low glucose, containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) until they became confluent. Then, they were seeded in 24-well plates at a concentration of 1.0× ¹⁰⁵ cells/mL and cultured overnight in a _CO2 incubator (37℃, 5% _CO2 ).

After overnight incubation, each evaluation sample was added and incubated for 48 hours in a _CO2 incubator. DMSO was used as a control, and arbutin (final concentration 50 μg/mL), which is known to have the property of inhibiting melanin synthesis, was used as a positive control.

After 48 hours of culture, the cell supernatant was removed from the 24-well plate, which was then washed with PBS. The cells were then detached with a 0.25% trypsin-EDTA solution and collected from each well. After centrifugation and washing, the cells were transferred to a 1.5 mL tube and centrifuged again, the supernatant was removed, and 120 μL of 1M NaOH solution was added and reacted at 100°C for 10 minutes. 100 μL of the suspension was transferred to a 96-well plate, and the absorbance at 405 nm was measured using a plate reader.

Figure 15 is a graph showing the melanin production rate of an ethanol extract of dried red shiso. As shown in Figure 15, it was revealed that when the concentration of the ethanol extract was 1 ng/mL or 10 ng/mL, it tended to decrease the melanin production rate.

8. Anti-acne bacteria activity test
Propionibacterium acnes is known to be the cause of acne. Propionibacterium acnes grows on sebum as a nutrient source, and the skin causes inflammation to fight against the grown propionate, which causes acne. In other words, if red shiso extract can suppress the activity of propionibacterium acnes, it can be used as a skin quality improvement agent that prevents acne from occurring.

In this test, acne bacteria was used to examine whether the addition of the sample had any effect in inhibiting the proliferation of acne bacteria. The evaluation samples used were an ethanol extract and an ethanol extract of dried red shiso. The final concentrations of these evaluation samples were 20, 200, 400, and 800 μg/mL, respectively.

Propionibacterium acnes was used as the test strain. A bacterial solution (10% glycerin solution) stored in a -80℃ deep freezer was added to 5mL of GAM bouillon medium, and the mixture was incubated anaerobically for 48 hours in a 37℃ incubator. The resulting bacterial solution was diluted with GAM bouillon to adjust the OD630 to 0.1 (10 ⁵ CFU/mL).

890μL of the diluted GAM medium and 10μL of the sample solution were mixed and dispensed into a 96-well plate at 135μL each (n=3). 15μL of bacterial solution was added to each well, and 15μL of medium was added to each well as a blank instead of the bacterial solution as a blank, and the mixture was cultured at 37℃ for 24 hours. After culture, the turbidity at 570nm was measured and used as an index of antibacterial activity.

Figure 16 shows the results of the anti-P. acnes activity test. As shown in Figure 16, the water extract significantly inhibits the survival rate of P. acnes at 20 μg/mL compared to the control (water). In addition, the ethanol extract significantly inhibits the survival rate of P. acnes at 800 μg/mL compared to the control (DMSO).

Therefore, it can be said that red shiso extract can be used as a skin quality improving agent that prevents acne occurrence because it inhibits the proliferation of acne bacteria.

9. Elastase inhibitory activity evaluation test
The skin is layered, consisting of the epidermis, dermis, and subcutaneous tissue, from the outside in. Elastin is known to be one of the components that make up the dermis, which is located inside the epidermis. Elastin is produced by fibroblasts and plays an important role in maintaining the flexibility and elasticity of the skin. In addition, elastase, an elastin-degrading enzyme, is known to be induced by aging, ultraviolet rays, active oxygen, and stress. When elastin is degraded by elastase, its cross-linked structure becomes brittle. Such degradation of elastin causes the skin to lose elasticity and develop wrinkles. In other words, by inhibiting elastase activity, anti-wrinkle effects can be expected.

In this study, elastase derived from human skin fibroblasts (NHDF-Ad) was used to evaluate elastase activity by measuring the amount of nitroaniline produced using N-Succinyl-Ala-Ala-Ala-p-nitroanilide as a substrate. In addition, the skin elasticity-retaining function of the target materials was evaluated based on elastase activity.

The samples used to evaluate elastase inhibitory activity were water extracts and ethanol extracts of dried red shiso. The samples were tested at concentrations of 0.04, 0.4, 4, 40, and 400 μg/mL.

Human dermal fibroblasts were precultured in Dulbecco's Modified Eagle Medium (DMEM) (high glucose) (containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) in a φ10 cm dish in a _CO2 incubator (37°C, 5% _CO2 ) until confluent.

The precultured human dermal fibroblasts were washed with phosphate-buffered saline (PBS) and resuspended in cell lysis solution (0.2 M Tris-HCl (pH 8.0) containing 1 mM PMSF and 0.5% Triton x-100) to a concentration of 1.0 × 10 ⁶ cells/ml. The suspension was sonicated and centrifuged (13,000 rpm, 4°C, 15 min), and the cell lysate was collected.

12.5 μL of the collected supernatant was mixed with 35.5 μL of 0.2 M Tris-HCl buffer (pH 8.0) and 2 μL of each concentration sample, and incubated at 37°C for 15 minutes. Then, 50 μL of 5 mM substrate (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) was added, and incubated at 37°C for 3 hours. The absorbance at 405 nm was measured using a microplate reader, and the elastase activity was calculated.

Figure 17 shows the results of an elastase inhibitory activity evaluation test. As shown in Figure 17, the water extract and ethanol extract exhibited concentration-dependent elastase inhibitory activity. In particular, the water extract showed an inhibition rate of approximately 82% at 800 mg/mL, and the ethanol extract showed an inhibition rate of approximately 58% at 800 mg/mL.

Therefore, it can be said that red shiso extract can be used as a skin quality improving agent with anti-wrinkle effects because it inhibits elastase activity.

As described above, red shiso extract has the effects of preventing acne occurrence, anti-allergy, anti-wrinkle, and suppressing melanin production. In other words, it can be said that red shiso extract can be used as an ingredient that induces at least one of the effects selected from acne occurrence prevention, anti-allergy, anti-wrinkle, and suppressing melanin production.

Next, human evaluation tests were conducted using topical preparations, cosmetics, and foods that contained skin quality improving agents containing red shiso extract.

10. Human evaluation test of topical preparations
In this test, a total of 12 people, two men and two women in their 20s, 40s, and 60s, were asked to use an external preparation containing the skin quality improving agent of the present invention for a specified period of time (hereinafter referred to as the "test group"). After the period had passed, the effects on acne (acne prevention effect), redness and itching (anti-allergic effect), wrinkles (anti-wrinkle effect), and blemishes (melanin production suppression effect) were evaluated on a 5-point scale.

The topical agent used in the test was a body mist containing 400μg/mL of an ethanol extract of dried red shiso. The subjects were asked to use this body mist twice a day, in the morning and evening, for four weeks.

As a control test, a body mist that does not contain the skin quality improving agent of the present invention was also used under the same conditions and evaluated (hereinafter referred to as the "control test group"). The body mist used by this control test group was a commercially available product that does not contain skin-beautifying ingredients.

The results of human evaluation tests of topical agents are shown below. Table 4 shows the results of the test group, and Table 5 shows the results of the control test group.

Tables 4 and 5 reveal that skin quality improving agents containing red shiso extract are effective as ingredients that prevent acne occurrence, have anti-allergic effects, anti-wrinkle effects, or suppress melanin production when included in topical preparations.

11. Human evaluation test of cosmetics
In this test, a total of 12 people, two men and two women in their 20s, 40s, and 60s, were asked to use a cosmetic containing the skin quality improving agent of the present invention for a specified period of time (hereinafter referred to as the "test group"). After the period had passed, the effects on acne (acne prevention effect), redness and itching (anti-allergic effect), wrinkles (anti-wrinkle effect), and blemishes (melanin production inhibitory effect) were evaluated on a 5-point scale.

The cosmetic product tested was a milky lotion containing 400μg/mL of an ethanol extract of dried red shiso. The subjects were asked to use this lotion twice a day, in the morning and evening, for four weeks.

As a control test, an emulsion that did not contain the skin quality improving agent of the present invention was also used under the same conditions and evaluated (hereinafter referred to as the "control test group"). The emulsion used in this control test group was an emulsion that had been prepared so as not to contain any skin-beautifying ingredients.

The results of human evaluation tests of cosmetics are shown below. Table 6 shows the results of the test group, and Table 7 shows the results of the control test group.

From Tables 6 and 7, it is clear that a skin quality improving agent containing red shiso extract is effective as an ingredient that prevents acne, has anti-allergic effects, anti-wrinkle effects, or inhibits melanin production when added to cosmetics.

12. Human evaluation testing of food
In this test, a total of 12 people, two men and two women in their 20s, 40s, and 60s, were asked to take a food product containing the skin quality improving agent of the present invention for a specified period of time (hereinafter referred to as the "test group"). After the period had passed, the effects on acne (acne prevention effect), redness and itching (anti-allergic effect), wrinkles (anti-wrinkle effect), and blemishes (melanin production suppression effect) were evaluated on a 5-point scale.

The food to be tested was a tablet containing 400μg/mg of an ethanol extract of dried red shiso. The subjects were asked to take this tablet twice a day, once in the morning and once in the evening, after meals, for a period of four weeks.

As a control test, tablets not containing the skin quality improving agent of the present invention were also used under the same conditions and evaluated (hereinafter referred to as the "control test group"). The tablets used in this control test group were commercially available placebos that did not contain any skin-beautifying ingredients.

The results of human evaluation tests of the food are shown below. Table 8 shows the results of the test group, and Table 9 shows the results of the control test group.

From Tables 8 and 9, it is clear that a skin quality improving agent containing red shiso extract is effective as an ingredient that prevents acne, has anti-allergic effects, anti-wrinkle effects, or suppresses melanin production when added to food.

The results of these human evaluation tests indicate that red shiso extract, when added to topical agents, cosmetics, or foods, is effective as an ingredient that induces at least one of the following effects: acne prevention, anti-allergy, anti-wrinkle, and melanin production inhibition.

In addition, the results of the above-mentioned lipase inhibitory activity test, anti-allergy test, collagen/hyaluronic acid production evaluation test, melanin production evaluation test, anti-acne bacteria activity test, and elastase inhibitory activity evaluation test revealed that the ethanol extract of red shiso in particular has a strong effect on these. Therefore, it can be said that the ethanol extract of red shiso can be used as an active ingredient in external preparations, cosmetics, or foods that aim at least one of the following: prevention of acne, anti-allergy, anti-wrinkle, and inhibition of melanin production.

Incidentally, there have been reports of topical preparations containing luteolin, found in shiso, being used to treat and prevent acne. One such report is JP 2013-203703 A. In addition, a cosmetic preparation containing rosmarinic acid, also found in shiso, is known in JP 2008-253256 A.

However, as revealed by the HPLC and TLC analyses, the red shiso extract of this embodiment does not contain luteolin or rosmarinic acid. In other words, luteolin and rosmarinic acid are not involved at all in the acne prevention, anti-allergy, anti-wrinkle, and melanin production inhibition effects of the red shiso extract revealed in this embodiment.

In other words, even a red perilla extract obtained from the residue of red perilla leaves after extracting luteolin and rosmarinic acid as active ingredients can be used as an ingredient in external preparations, cosmetics, or foods for at least one of the following purposes: prevention of acne, anti-allergy, anti-wrinkle, and inhibition of melanin production.

Claims (4)

The skin quality improving agent contains an ethanol extract of red shiso that does not contain rosmarinic acid as an ingredient that prevents acne from occurring . An external preparation for preventing the occurrence of acne, comprising the skin quality improving agent according to claim 1. A cosmetic preparation for preventing the occurrence of acne, comprising the skin quality improving agent according to claim 1. A functional food for preventing acne, comprising the skin quality improving agent according to claim 1.

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