Skin quality improving agent containing perilla extract

Abstract

To provide a skin texture improver containing a red perilla extract as a component that induces an action more clearly compared with the conventional art, and topical preparations, cosmetics, and food containing the skin texture improver.SOLUTION: A skin texture improver contains a red perilla extract as a component for inducing at least one action selected from acne-prevention, antiallergic, anti-wrinkle, and melanogenesis inhibitory actions. There are also provided topical preparations, cosmetics, and food containing the skin texture improver.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to improving skin quality containing perilla extract. In the agent It relates thereto.

Background Art

[0002] Living a healthy daily life has always been people's wish, unchanged from the past. Especially in modern society where the average life expectancy continues to increase, it can be said that it is desirable to live a lively daily life even as one ages.

[0003] In order to live such a healthy life, for example, daily intake of foods containing components that can prevent or alleviate diseases is effective.

[0004] In this regard, perilla has long been widely known as a familiar food ingredient to the Japanese people and has continued to be used as a traditional Chinese medicine with a history of relieving colds and food poisoning.

[0005] In recent years, perilla has been attracting attention as a food ingredient containing components that cause disease prevention and improvement effects, and for example, usage methods as disclosed in Patent Document 1 and Patent Document 2 are known.

[0006] Patent Document 1 discloses an agent for preventing or improving nocturia. This preventive and improving agent uses rosmarinic acid or its salt contained in the extract of perilla as an active ingredient and reduces the number of times of waking up for urination during sleep. Therefore, according to such a preventive and improving agent, it is said that it can reduce the sense of anxiety regarding urination during sleep and support healthy sleep.

[0007] In addition, Patent Document 2 discloses an adiponectin secretion promoter. The secretion promoter is an extract of a Lamiaceae plant and is characterized by containing at least one or more components selected from rosmarinic acid, luteolin, apigenin, or chrysoeriol. And according to the secretion promoter, it is said that it is possible to prevent or improve the symptoms of lifestyle-related diseases such as diabetes, arteriosclerosis, hypertension, hyperlipidemia, obesity, and metabolic syndrome, and it is possible to provide an adiponectin secretion promoter that is derived from natural components and has high safety even when administered for a long time.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] As described above, shiso has excellent effects in preventing or improving diseases.

[0010] By the way, as people get older, the decline becomes more noticeable externally. In particular, spots and wrinkles may increase with age, and it is normal for the skin condition to change significantly compared to when one was young.

[0011] In order to have a young and beautiful appearance even as one ages, it is preferable to routinely ingest external preparations, cosmetics, or foods containing components that can induce an anti-wrinkle effect for preventing and improving wrinkles and a melanogenesis inhibitory effect for preventing and improving spots and freckles.

[0012] Of course, red perilla is also used for beauty purposes in health foods and cosmetics. However, the health foods and cosmetics proposed so far may not always have a clear significance in containing the perilla extract. That is, many of the perilla extracts contained in health foods and cosmetics are only vaguely described as "good for the body" or "good for the skin", and are provided to users without a clear purpose. As a result, users may not be able to guess exactly how the contained components of the perilla extract exert their effects.

[0013] The present invention has been made in view of such circumstances, and provides a skin quality improving agent containing a perilla extract as a component that causes a clearer action compared to the prior art. The present invention also provides an external preparation, a cosmetic, and a food containing the above skin quality improving agent.

Means for Solving the Problems

[0014] The skin quality improving agent according to the present invention Red perilla ethanol extract without rosmarinic acid and As an ingredient that induces the acne prevention effect is characterized by containing the same.

[0015] In addition, the For preventing acne external preparation, For preventing acne cosmetic and Acne prevention function food according to the present invention are made to contain the above skin quality improving agent.

Effects of the Invention

[0019] According to the skin quality improving agent of the present invention, Red perilla ethanol extract without rosmarinic acid and As an ingredient that induces the acne prevention effect is characterized by containing the same, so that a skin quality improving agent containing a perilla extract can be provided under a clearer purpose.

[0020] In addition, according to the For preventing acne external preparation or For preventing acne cosmetic, Acne prevention function food of the present invention, Red perilla ethanol extract without rosmarinic acidto As an ingredient that induces the acne prevention effect Since it was decided to include the above-mentioned skin quality improving agent containing As an ingredient that induces the acne prevention effect , it is possible to improve the skin quality For preventing acne external preparations and For preventing acne cosmetics, Acne prevention function foods can be provided.

Brief Description of Drawings

[0024]

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Mode for Carrying Out the Invention

[0025] The present invention relates to a skin quality improving agent containing a perilla extract and its use, and also provides external preparations, cosmetics, foods, etc. containing the skin quality improving agent.

[0026] In particular, the skin quality improving agent according to the present embodiment is characterized in that it contains a perilla extract as an inducing component of at least one action selected from prevention of acne occurrence, anti-allergy, anti-wrinkle, and suppression of melanin production.

[0027] Prevention of acne occurrence means suppressing the growth of bacteria that cause acne. Such a function can be confirmed, for example, in the lipase inhibitory activity test and the anti-acne bacteria activity test described later. That is, the skin quality improving agent containing the perilla extract according to the present invention, when used topically in the form of an external preparation or a cosmetic and / or orally in the form of a food (hereinafter referred to as the topical / oral use mode), can increase the lipase inhibitory activity or suppress the growth of acne bacteria, and thus can be said to have an acne prevention effect.

[0028] Anti-allergy means suppressing itching and swelling associated with allergic symptoms. Such a function can be confirmed, for example, by measuring the β-hexosaminidase activity as in the anti-allergy activity test described later. That is, the skin quality improving agent containing the perilla extract according to the present invention can be said to have an anti-allergy effect if it suppresses the β-hexosaminidase activity when used in the topical / oral use mode.

[0029] Anti-wrinkle means preventing skin dryness or suppressing the decrease in the components constituting the dermis. Such functions can be confirmed, for example, in the collagen production evaluation test and hyaluronic acid production evaluation test described later. That is, if the skin quality improving agent containing the perilla extract according to the present invention promotes the production of collagen or hyaluronic acid when used in external or oral use modes, it can be said to have an anti-wrinkle effect.

[0030] Melanin production suppression means suppressing the excessive production of melanin by melanocytes. Such functions can be confirmed, for example, in the melanin production evaluation test described later. That is, if the skin quality improving agent containing the perilla extract according to the present invention can suppress the production of melanin when used in external or oral use modes, it can be said to have a melanin production suppressing effect.

[0031] Perilla is a plant known as Perilla frutescens var. frutescens form. purpurea belonging to the genus Perilla of the family Lamiaceae, and is a plant widely and commonly cultivated in Japan.

[0032] The perilla extract contained in the skin quality improving agent according to the present embodiment is extracted using the whole herb of perilla or one or more of leaves, flowers, fruits, stems, and roots as raw materials.

[0033] Extraction is a process of obtaining an extract by pressing or a process of leaching components into an extraction solvent.

[0034] The extraction solvent for obtaining the extract is not particularly limited, and for example, water or ethanol can be used. The water extract is extracted from perilla leaves using water at a predetermined temperature as the extraction solvent. Also, the ethanol extract is extracted from perilla leaves using ethanol at a predetermined temperature as the extraction solvent. The extraction temperature using these extraction solvents is not particularly limited, and for example, it can be set to 4°C to 100°C, limitedly 10°C to 50°C, and more limitedly about 15°C to 30°C.

[0035] The method of extraction treatment when obtaining water extracts or ethanol extracts is not particularly limited. As an example, there is a method of containing an extraction solvent and a dried product of perilla leaves in a predetermined container and performing shaking extraction.

[0036] Also, the extract after extraction may be directly contained in the skin quality improving agent according to this embodiment, or it can also be dried and then used as a perilla extract.

[0037] The drying method of the extract is not particularly limited and can be appropriately selected according to the extraction scale and accuracy. For the drying of the water extract, for example, freeze-drying can be adopted. For the drying of the ethanol extract, for example, drying by a rotary evaporator or drying in a desiccator can also be adopted. When drying with a rotary evaporator, the drying conditions can be appropriately adjusted according to the purpose and situation, such as under heating and reduced pressure or with or without boiling as necessary.

[0038] The water or ethanol extract of perilla extracted in this way can be used as it is as the skin quality improving agent according to this embodiment regardless of the state such as solid (powder) or liquid, and auxiliary components can also be added as necessary.

[0039] Here, the auxiliary components include, for example, components for suppressing the loss of function over time of components that cause the action of improving skin quality, components necessary for shaping the dosage form, components for adjusting the fragrance, and other necessary components.

[0040] More specifically, if the component that causes the action of improving skin quality loses its function over time due to oxidation, an antioxidant for suppressing this belongs to the auxiliary components. Also, if it loses its function due to denaturation, a predetermined anti-denaturing agent belongs to the auxiliary components.

[0041] In addition, auxiliary components may be added as necessary when processing into external preparations, cosmetics, foods, reagents, etc., such as excipients and fragrances.

[0042] Here, the external preparation is something that is used by applying, sticking, or spraying on the skin or mucous membranes, and it is a concept that includes pharmaceuticals and quasi-drugs, as well as things not classified as cosmetics or foods. External preparations include ointments, creams, external liquid preparations, eye drops, nasal drops, suppositories, patches, inhalants, sublingual preparations, and the like.

[0043] Cosmetics include, in addition to so-called makeup cosmetics, basic cosmetics, hair tonics, perfumes, toothpastes, shampoos, rinses, soaps and detergents used for body washing, bath preparations, and other toiletry products, as well as medicinal cosmetics that claim preventive effects and the like.

[0044] Moreover, examples of makeup cosmetics include foundation, eyebrow pencils, mascara, eyeshadow, eyeliner, lipstick, gloss, blusher, powder, and nail polish. Examples of basic cosmetics include lotion, emulsion, facial cleanser, cleansing product, beauty essence, and cream. Note that lotion also includes liquid preparations (spray liquids) that are sprayed on the face and hand skin like a refreshing mist.

[0045] Functional foods are a concept that encompasses all foods that are said to contribute to maintaining and promoting health without containing pharmaceutical ingredients. For example, in addition to dietary supplements, health supplements, and nutritional regulatory foods, they also include general foods such as so-called supplements, as well as health functional foods such as foods for specified health use, nutritional functional foods, and foods with function claims.

[0046] Note that the descriptions of the above-mentioned external preparations, cosmetics, and functional foods are examples of articles corresponding to these for the purpose of understanding the present invention, and the interpretation of each term is not limited to these listed articles. However, when the applicant of this application rights the present application, it does not prevent the present invention from being limited to these articles.

[0047] Moreover, the skin quality improving agent according to the present embodiment can also be used as an experimental reagent or a clinical test drug.

[0048] Thus, by using the perilla extract as a component that induces each of the above-described actions, it is possible to provide a skin quality improving agent that can be ingested in daily life, an external preparation containing the improving agent, a cosmetic, and a functional food.

[0049] Hereinafter, with reference to the process of examining whether the skin quality improving agent according to the present embodiment induces acne prevention, anti-allergy, anti-wrinkle, or melanin production inhibitory action, an explanation will be given.

[0050] 〔1. Preparation of Evaluation Samples〕 Leaves were collected from perilla cultivated in a perilla field in Ashiya-cho, Tōgō-gun, Fukuoka Prefecture, and three types of evaluation samples were prepared. That is, the evaluation samples are a perilla extract obtained by squeezing the leaves of perilla, a perilla extract residue obtained by vacuum-drying the leaves of perilla after extracting the perilla extract, and a perilla dried product obtained by drying the leaves of perilla at 60°C.

[0051] Furthermore, the perilla extract was used as it was in each test, and the perilla extract residue and the perilla dried product were used after being pulverized with a mill and then subjected to an extraction treatment.

[0052] For the extraction treatment of the perilla extract residue, 1 g of pulverized leaves was separately collected in two Erlenmeyer flasks, 100 mL of water was dispensed into one flask, and 100 mL of ethanol was dispensed into the other flask.

[0053] Then, after sealing, both flasks were attached to a shaking extractor, and shaking extraction treatment was performed at 200 rpm for 24 hours at room temperature.

[0054] After the shaking extraction treatment, the contents of the water extraction flask were subjected to centrifugation at 3000 rpm for 10 minutes to obtain a supernatant, which was then subjected to freeze-drying treatment. The obtained freeze-dried product was used as the water extract of the perilla extract residue.

[0055] Also, the ethanol extraction flask after the shaking extraction treatment was filtered through No. 2 filter paper, and the contents were concentrated and dried using a rotary evaporator. The obtained dried product was used as the ethanol extract of the perilla extract residue.

[0056] In addition, for the extraction treatment of the perilla frutescens dried product, the same treatment as the preparation method of the extract of the perilla frutescens extract residue was performed to obtain the water extract and ethanol extract of the perilla frutescens dried product.

[0057] [2. HPLC analysis, TLC analysis] Next, in order to obtain the component profiles of the perilla frutescens extract, the water extract and ethanol extract of the perilla frutescens extract residue, and the water extract and ethanol extract of the perilla frutescens dried product, component analysis by high performance liquid chromatography (HPLC) and thin layer chromatography (TCL) was carried out.

[0058] In the HPLC analysis of this test, the evaluation samples were pretreated to prepare HPLC samples. The HPLC samples were those in which the water extract was dissolved in the initial eluent (10% methanol) at 10 mg / mL and passed through a 0.2 μm filter, the ethanol extract was dissolved in methanol at 10 μg / mL and passed through a 0.2 μm filter, and the perilla frutescens extract was directly passed through a 0.2 μm filter.

[0059] The analysis conditions for the HPLC analysis are as shown in Table 1.

Table 1

[0060] Regarding the TLC analysis, three types of developing solvents were tested using a TLC aluminum plate (Hexane:Ethyl acetate = 50:50, Ethyl acetate = 100, Ethyl acetate:MeOH = 50:50).

[0061] The results of the HPLC analysis will be explained based on Figures 1 to 4. Figures 1 to 3 show the chromatograms of the HPLC analysis. Figure 1 shows the results of the perilla extract, Figure 2(a) shows the results of the water extract of the perilla extract residue, Figure 2(b) shows the results of the water extract of the dried perilla, Figure 3(a) shows the results of the ethanol extract of the perilla extract residue, and Figure 3(b) shows the results of the ethanol extract of the dried perilla. And Figure 4 is a table and graph showing the peak area values detected by ELSD in the water extract and ethanol extract.

[0062] As shown in Figure 1, a peak was observed near RT = 12.5 minutes at UV (280 nm) in the chromatogram of the perilla extract, but no peak was observed by ELSD, and the details were unclear in this analysis result.

[0063] Next, the results of the water extract of the perilla extract residue and the water extract of the dried perilla are compared. As shown in Figures 2(a) and 2(b), at 280 nm, multiple peaks were confirmed in the water extract of the perilla extract residue up to around RT = 10 minutes, while the dried perilla had fewer peaks. Also, in the ELSD results shown in Figure 4, a single peak was observed near RT = 1.5 minutes in both cases.

[0064] Furthermore, the results of the ethanol extract of the perilla extract residue and the ethanol extract of the dried perilla are compared. As shown in Figures 3(a) and 3(b), a large number of peaks were confirmed in both. In particular, the ethanol extract of the dried perilla is presumed to contain a large amount of components because of the large peak height. Also, in the ELSD results shown in Figure 4, three peaks regarded as the main components were confirmed near RT = 23 - 25 minutes.

[0065] From the above results, it became clear that in the perilla extract residue and the dried perilla, the ethanol extract contains more kinds of components and a larger amount of those components than the water extract.

[0066] The results of the TLC analysis will be described based on Fig. 5. Note that Fig. 5(a) shows the results using Hexane:Ethyl acetate = 50:50 as the developing solvent, Fig. 5(b) shows the results using Ethyl acetate = 100 as the developing solvent, and Fig. 5(c) shows the results using Ethyl acetate:MeOH = 70:30 as the developing solvent. In Fig. 5, the symbol 3 represents the result of the water extract of the perilla extract residue, the symbol 4 represents the result of the ethanol extract of the perilla extract residue, the symbol 5 represents the result of the water extract of the dried perilla, and the symbol 6 represents the result of the ethanol extract of the dried perilla, respectively.

[0067] As shown in Fig. 5, even when the developing solvent was changed for testing, there were no significant differences in the peak positions in the ethanol extracts of the perilla extract residue and the dried perilla, although there were differences in the shades of the peaks.

[0068] Next, it was confirmed whether rosmarinic acid and luteolin were contained as components in the ethanol extract of the dried perilla by TLC test. The results will be described with reference to Fig. 6.

[0069] Note that Fig. 6(a) shows the results using Hexane:Ethyl acetate = 50:50 as the developing solvent, Fig. 6(b) shows the results using Ethyl acetate = 100 as the developing solvent, and Fig. 6(c) shows the results using Ethyl acetate:MeOH = 70:30 as the developing solvent. In Fig. 6, the symbol 6 represents the result of the ethanol extract of the dried perilla, the symbol Flav represents the result of developing the detection limit concentration of luteolin as a positive control, and the symbol Ros represents the result of developing the detection limit concentration of rosmarinic acid as a positive control, respectively.

[0070] From Fig. 6(b), it was revealed that the luteolin in the ethanol extract of the dried perilla was below the detection limit concentration. Also, from Fig. 6(c), it was revealed that the rosmarinic acid in the ethanol extract of the dried perilla was below the detection limit concentration.

[0071] [3. GC / MS Analysis] Next, in order to obtain the profiles of the volatile components contained in perilla extract, perilla extract residue, or dried perilla, collection of volatile components by solid-phase microextraction (SPME) and component analysis by gas chromatography / mass spectrometer (GC / MS) were carried out.

[0072] In this test, 5 mL of perilla extract was directly sealed into a 20 mL vial for SPME. For perilla extract residue and dried perilla, 5 g of the milled samples were sealed into a 20 mL vial for SPME. Before capping each vial, the gas phase was replaced with nitrogen gas.

[0073] After replacement, using an SPME fiber (100 μm polydimethylsiloxane), perilla extract was collected at room temperature for 1 minute, and perilla extract residue and dried perilla were collected at room temperature for 30 minutes.

[0074] After collection, each vial was analyzed by GC / MS under the conditions shown in Table 2.

Table 2

[0075] The results of the GC / MS analysis will be described based on Figures 7 to 10. Note that Figure 7(a) shows the results of the GC / MS analysis of perilla extract. Figure 7(b) shows the results of the GC / MS analysis of perilla extract residue. Figure 7(c) shows the results of the GC / MS analysis of dried perilla. Figure 8 is a chromatogram in which the vertical axis of the results of the GC / MS analysis of perilla extract residue shown in Figure 7(b) is adjusted to the same dimension as the results of dried perilla shown in Figure 7(c). Figure 9 is a list of the components identified by GC / MS analysis, and Figure 10 is a bar graph showing the peak areas of the identified components.

[0076] As shown in Fig. 7(a), Fig. 9, and Fig. 10, it was revealed that the perilla extract contains benzaldehyde (around RT = 7 minutes) and perillaldehyde (around RT = 16 minutes) as the main components. Perillaldehyde is an aroma component contained in perilla and accounts for about 50% of the essential oil of perilla. In addition, D-limonene, eucalyptol (cineole), linalool, perillyl alcohol, etc. were also detected in the perilla extract.

[0077] From Fig. 7(b), Fig. 7(c), Fig. 9, and Fig. 10, it was possible to confirm a larger number of peaks in the perilla extract residue and the perilla dried product compared to the perilla extract. That is, it became clear that the perilla extract residue and the perilla dried product contain more kinds of volatile components than the perilla extract.

[0078] Also, from Fig. 7(c), Fig. 8, Fig. 9, and Fig. 10, it can be seen that the perilla extract residue contains various compounds. On the other hand, although the perilla dried product has fewer types of components compared to the perilla extract residue, it was revealed that their respective contents tend to be high.

[0079] [4. Lipase Inhibition Activity Test] Lipase is known as an enzyme that decomposes fat. It is also known that such lipase is secreted by Propionibacterium acnes, which is the cause of acne, and decomposes sebum as its own nutrition. That is, Propionibacterium acnes grows while decomposing sebum and secretes components related to the inflammation that causes acne. In other words, if the action of lipase is inhibited, it becomes possible to prevent the occurrence of acne by suppressing the growth of Propionibacterium acnes and the secretion of inflammatory components.

[0080] In this test, 25 μL of each sample was mixed with 50 μL of a 0.1 mM 4-methylumbeliferyl oleate (4-MUO) solution ( / 13 mM Tris-HCl, 150 mM NaCl, 1.3 mM CaCl2 buffer). 25 μL of pancreatic lipase at 150 μg / mL was dissolved in the same buffer as above and incubated at 25 °C for 30 minutes.

[0081] After incubation, 100 μL of 0.1 M citrate buffer (pH 4.2) was added to stop the reaction. Then, the fluorescence intensity (Em: 355, Ex: 460 nm) of 4-methylumbelliferone (4-MU) released by the action of lipase on 4-MUO was measured to evaluate the effect on lipase activity.

[0082] The final concentrations of the samples in this test were 12.5, 37.5, 75, and 150 ppm for perilla extract, and 1.5625, 12.5, 25, 100, 400, and 800 μg / mL for the water extract and ethanol extract of perilla extract residue, as well as the water extract and ethanol extract of perilla dry matter.

[0083] This test was also conducted with n = 4, and orlistat (Orl) was used as a positive control.

[0084] Figure 11(a) is a graph showing the lipase inhibitory activity of perilla extract. Figure 11(b) is a graph showing the lipase inhibitory activity of the water extract and ethanol extract of perilla extract residue. Figure 11(c) is a graph showing the lipase inhibitory activity of the water extract and ethanol extract of perilla dry matter.

[0085] In this test, as shown in Figure 11(a), regarding the perilla extract, when confirmation was carried out at sample concentrations of 12.5, 37.5, 75, and 150 ppm, lipase inhibitory activity of approximately 5% to approximately 10% was observed at all concentrations. Also, in the perilla extract, it was not confirmed that the lipase inhibitory activity increased in a concentration-dependent manner.

[0086] As shown in Fig. 11(b), regarding the water extract of the perilla extract residue, when confirmed at sample concentrations of 1.5625, 12.5, 25, 100, 400, and 800 μg / mL, high lipase inhibitory activity was observed in a concentration-dependent manner at concentrations of 12.5 μg / mL or higher. In contrast, no lipase inhibitory activity was observed at 1.5625 μg / mL.

[0087] Also, regarding the ethanol extract of the perilla extract residue, when confirmed at sample concentrations of 1.5625, 12.5, 25, 100, 400, and 800 μg / mL, high lipase inhibitory activity was observed in a concentration-dependent manner at concentrations of 12.5 μg / mL or higher. In contrast, no lipase inhibitory activity was observed at 1.5625 μg / mL.

[0088] From these results, when comparing the experimental results of the water extract and the ethanol extract of the perilla extract residue, it was revealed that both showed a lipase inhibitory activity in a concentration-dependent manner, but the ethanol extract showed a particularly high effect.

[0089] As shown in Fig. 11(c), regarding the water extract of the dried perilla, when confirmed at sample concentrations of 1.5625, 12.5, 25, 100, 400, and 800 μg / mL, lipase inhibitory activity was confirmed at all concentrations, and moreover, it was found that the effect became stronger in a concentration-dependent manner.

[0090] Also, regarding the ethanol extract of the dried perilla, when confirmed at sample concentrations of 1.5625, 12.5, 25, 100, 400, and 800 μg / mL, high lipase inhibitory activity was observed in a concentration-dependent manner at concentrations of 12.5 μg / mL or higher. In contrast, no lipase inhibitory activity was observed at 1.5625 μg / mL.

[0091] From these results, when comparing the experimental results of the water extract and the ethanol extract of the dried perilla, it was revealed that both showed a lipase inhibitory activity in a concentration-dependent manner, but at concentrations of 12.5 μg / mL or higher, the ethanol extract showed a higher effect than the water extract.

[0092] Therefore, it was revealed that the water extract and ethanol extract of the perilla extract residue, as well as the water extract and ethanol extract of the dried perilla, exhibited high lipase inhibitory activity in a concentration-dependent manner at a concentration of 12.5 μg / mL or higher. In addition, when calculating the IC50 value (50% inhibitory concentration) for the perilla extract and the dried perilla, as shown in Table 3, it was revealed that the ethanol extract had extremely strong lipase inhibitory activity in both cases.

Table 3

[0093] 〔5. Anti-allergy test〕 Allergy is one of the causes of symptoms such as skin itching, redness, papules, and small blisters. Allergy develops when immune cells called basophils release granules containing β-hexosaminidase. That is, by suppressing the release of granules containing β-hexosaminidase, these symptoms caused by allergy can be suppressed.

[0094] In this test, samples were added to rat basophilic leukemia cells (RBL-2H3) as model cells for basophils, and the amount of β-hexosaminidase in the cell supernatant was measured to determine the amount of granule release, that is, the effect on the onset of allergy. In this test, the water extract and ethanol extract of the perilla extract residue, as well as the water extract and ethanol extract of the dried perilla, were used as samples.

[0095] Specifically, rat basophilic leukemia cells (RBL-2H3) were prepared at a concentration of 5×10 5 cells / mL and seeded at 100 μL in a 96-well plate. Two such plates were prepared, one for measuring β-hexosaminidase activity and the other for the MTT test, and pre-cultured in a CO2 incubator (37 °C, 5% CO2) for 40 hours.

[0096] After pre-culture, the medium was removed and the wells were washed with Tyrode's buffer. 100 μL of Tyrode's buffer (or EMEM medium) containing 0.5 μL of each diluted sample was added to each well. After culturing at 37 °C in 5% CO2 for 1 hour, for the plate used to measure β-hexosaminidase activity, the supernatant was discarded, the wells were washed, and 100 μL of A23187 (in Tyrode's buffer) was added. After culturing in an incubator for 1 hour, 50 μL of the supernatant from each well was aliquoted and transferred to a new 96-well plate.

[0097] To measure β-hexosaminidase activity, 50 μL of p-nitrophenyl-N-acetyl-beta-glucosaminide solution was added as a substrate to each well of the new plate where 50 μL aliquots of the supernatant had been taken. After shielding from light with aluminum foil, the reaction was allowed to proceed at room temperature for 1 - 3 hours. 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.

[0098] For the MTT assay, for the plates to which each diluted sample had been added and 1 hour had elapsed, first, the medium in each well was removed, and 100 μL of MTT staining solution (5 mg / ml in medium) was added to each well, and static culture was carried out at 5% CO2 and 37 °C for 4 hours. Then, the medium was removed, 1 mL of hydrochloric acid - isopropanol solution was added to each well, shielded from light, and left at room temperature for 4 hours. Thereafter, the absorbance at 570 nm was measured using a microplate reader and used as an index of cell viability.

[0099] Also, this test was conducted with n = 3, and quercetin was used as a positive control.

[0100] Figure 12 is a graph showing the cell viability and β-hexosaminidase activity of the water extract and ethanol extract of the perilla frutescens extract residue, as well as the water extract and ethanol extract of the dried perilla frutescens.

[0101] As shown in Fig. 12, regarding the aqueous extract of the perilla extract residue, when confirmation was carried out at sample concentrations of 10, 100, and 400 μg / mL, it was revealed that the β-hexosaminidase activity was promoted in a concentration-dependent manner. Also, regarding the ethanol extract of the perilla extract residue, when confirmation was carried out at the same sample concentrations, it was found that at concentrations of 100 μg / mL or less, the β-hexosaminidase activity was inhibited in a concentration-dependent manner, but at 400 μg / mL, the β-hexosaminidase activity was promoted.

[0102] Thus, it was clarified that the aqueous extract and ethanol extract of the perilla extract residue at sample concentrations of 100 μg / mL or less showed opposite properties of "promotion" and "inhibition" with respect to the β-hexosaminidase activity, and that the ethanol extract showed the property of "promoting" the β-hexosaminidase activity at a sample concentration of 400 μg / mL.

[0103] Also, regarding the aqueous extract of the dried perilla, when confirmation was carried out at sample concentrations of 10, 100, and 400 μg / mL, it was revealed that the β-hexosaminidase activity was promoted to the same extent at all concentrations. Also, regarding the ethanol extract of the dried perilla, when confirmation was carried out at the same sample concentrations, it was found that the β-hexosaminidase activity was inhibited in a concentration-dependent manner and the cell viability was increased.

[0104] Thus, in the perilla extract residue and the dried perilla, inhibition of the β-hexosaminidase activity was observed only in the ethanol extract. That is, it is considered that the hydrophobic components of perilla act as components that induce an anti-allergic effect.

[0105] 〔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 maintaining the structure of the skin and is important for keeping the skin looking young and preventing wrinkles.

[0106] Collagen is produced by fibroblasts present in the dermis of the skin. If the production of collagen by fibroblasts can be promoted, youthful skin can be maintained. Therefore, components that promote the production of collagen by fibroblasts are considered useful as anti-aging cosmetic materials.

[0107] Hyaluronic acid is mainly produced by fibroblasts, which are the main cells of the skin dermis, and other connective tissue cells. It is a main component of the extracellular matrix and is involved in various cell-cell interactions. In addition, it is present in synovial fluid at high concentrations and is responsible for normal water retention and lubrication of joints.

[0108] In this test, the ability to produce collagen and hyaluronic acid by adding the water extract and ethanol extract of the perilla frutescens dry matter according to this embodiment to the fibroblast culture system was measured, and an evaluation was made on the skin water retention effect in the perilla frutescens extract residue and the extract of the perilla frutescens dry matter. As this test method, adult skin fibroblasts (NHDF-Ad) were used as model cells. 1.0×10 4 cells / well were seeded in a microplate and pre-cultured in a CO2 incubator (37 °C, 5% CO2) for 24 hours.

[0109] After pre-culture, the medium was removed, and fresh medium and each diluted sample diluted to a predetermined concentration were added, and the cells were cultured again in a CO2 incubator for 72 hours. The amounts of collagen and hyaluronic acid produced in the culture solution were measured using a commercially available ELISA kit. Similarly, an MTT test was performed using the same cells.

[0110] Figure 13 shows the hyaluronic acid production ratio by the ethanol extract of the perilla frutescens dry matter relative to the control.

[0111] In this test, no hyaluronic acid production effect was observed for the water extract of the perilla frutescens dry matter.

[0112] On the other hand, as shown in Fig. 13, regarding the ethanol extract of the perilla frutescens dry product, when confirmed at sample concentrations of 10 and 100 μg / mL, an increase in hyaluronic acid production was observed in a concentration-dependent manner.

[0113] Fig. 14 shows the collagen production ratio by the ethanol extract of the perilla frutescens dry product relative to the control.

[0114] In this test, regarding the water extract of the perilla frutescens dry product, no collagen production effect was observed.

[0115] On the other hand, as shown in Fig. 14, regarding the ethanol extract of the perilla frutescens dry product, when confirmed at a sample concentration of 100 μg / mL, an increase in collagen production was observed at a sample concentration of 100 μg / mL.

[0116] From these results, it was suggested that the ethanol extract of the perilla frutescens dry product contains components that induce the promotion of hyaluronic acid and collagen production.

[0117] [7. Melanin Production Evaluation Test] Melanin is a general term for substances formed by the polymerization of phenolic substances into pigments. Melanin is produced by melanin pigment cells (melanocytes) inside the skin and protects cells from damage by ultraviolet rays. However, excessive ultraviolet rays also cause excessive production of melanin and are a cause of the formation of freckles. That is, if the perilla frutescens extract can suppress the production of melanin, it can be said that it can be used as a skin quality improver having the effect of improving freckles.

[0118] In this test, using the mouse melanoma cell line (B16), the effect of sample addition on melanin production ability was evaluated. Also, as the evaluation samples, ethanol extracts (0.1, 1, 10 ng / mL) of the perilla frutescens dry product were used.

[0119] Mouse melanoma cell lines were pre-cultured in 75 cm flasks with DMEM (low glucose, containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) until confluent. 2 Subsequently, they were seeded into 24-well plates at a concentration of 1.0×10 5 cells / mL and cultured overnight in a CO2 incubator (37°C, 5% CO2).

[0120] After overnight culture, each evaluation sample was added and cultured in a CO2 incubator for 48 hours. 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.

[0121] After 48 hours of culture, the 24-well plates from which the cell supernatant had been removed were washed with PBS, and the cells were detached with a 0.25% trypsin-EDTA solution, and the cells were collected from each well. After centrifugation and washing operations, the cells were transferred to 1.5 mL tubes, centrifuged again, the supernatant was removed, 120 μL of 1M NaOH solution was added to each, and the reaction was carried out 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 with a plate reader.

[0122] Figure 15 is a graph showing the melanin production rate of the ethanol extract of the dried perilla leaves. As shown in Figure 15, it was revealed that when the concentration of the ethanol extract was 1 ng / mL or 10 ng / mL, there was a tendency to decrease the melanin production rate.

[0123] 〔8. Anti-acne bacteria activity test〕 Acne bacteria are known to be the cause of acne. Acne bacteria grow using sebum as a nutrient source, and the skin causes inflammation against the growing acne bacteria, thereby causing acne. That is, if the perilla extract can suppress the activity of acne bacteria, it can be said that it can be used as a skin quality improving agent with an acne prevention effect.

[0124] In this test, Propionibacterium acnes was used to examine the effect of suppressing the growth of Propionibacterium acnes by adding samples. In addition, the evaluation samples used were the ethanol extract of the dried perilla frutescens and the ethanol extract. The test was conducted so that the final concentrations of these evaluation samples were 20, 200, 400, and 800 μg / mL, respectively.

[0125] As the test strain, Propionibacterium acnes was used. The bacterial solution (10% glycerin solution) stored in an -80°C deep freezer was added to 5 mL of GAM broth medium and anaerobically cultured at 37°C in an incubator for 48 hours with static placement. The obtained bacterial solution was diluted with GAM broth to prepare a solution with OD630 = 0.1 (10 5 CFU / mL).

[0126] 890 μL of the diluted and prepared GAM medium and 10 μL of the sample solution were mixed, and 135 μL each was dispensed into a 96-well plate (n = 3). To this, 15 μL of the bacterial solution was added to each well, and 15 μL of the medium was added instead of the bacterial solution as a blank, and they were cultured at 37°C for 24 hours. After culturing, the turbidity at 570 nm was measured and used as an index of antibacterial activity.

[0127] Figure 16 shows the results of the anti-Propionibacterium acnes activity test. As shown in Figure 16, the water extract significantly suppresses the survival rate of Propionibacterium acnes at 20 μg / mL compared to the control (water). In addition, the ethanol extract significantly suppresses the survival rate of Propionibacterium acnes at 800 μg / mL compared to the control (DMSO).

[0128] Therefore, it can be said that the perilla frutescens extract can be used as a skin quality improver with an acne prevention effect because it suppresses the growth of Propionibacterium acnes.

[0129] 〔9. Elastase Inhibition Activity Evaluation Test〕 The skin is formed in layers of the epidermis, dermis, and subcutaneous tissue in that order from the outside. Elastin is known as one of the components that make up the dermis, which is 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 stimuli such as aging, ultraviolet rays, reactive oxygen species, and stress. Elastin is decomposed by elastase, and the cross-linked structure becomes brittle. Such decomposition of elastin causes the loss of skin elasticity and the formation of wrinkles. That is, an anti-wrinkle effect can be expected by inhibiting elastase activity.

[0130] In this test, elastase activity was evaluated by measuring the amount of nitroaniline produced using N-Succinyl-Ala-Ala-Ala-p-nitroanilide as a substrate with elastase derived from human skin fibroblasts (NHDF-Ad). In addition, based on the elastase activity, the skin elasticity retention function of the target material was evaluated.

[0131] The samples for evaluating elastase inhibitory activity were the water extract and ethanol extract of the dried perilla leaves. The test was conducted so that the respective sample concentrations were 0.04, 0.4, 4, 40, and 400 μg / mL.

[0132] Human skin fibroblasts were pre-cultured in a φ10 cm dish in a CO2 incubator (37°C, 5% CO2) using Dulbecco's Modified Eagle Medium (DMEM) (high glucose) (containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) until confluent.

[0133] The pre-cultured human skin fibroblasts were washed with phosphate-buffered saline (PBS) and lysed with a cell lysate (0.2 M Tris-HCl (pH 8.0) containing 1 mM PMSF and 0.5% Triton x-100) at 1.0×10 6Resuspended to cells / ml. The suspension was sonicated and centrifuged (13,000 rpm, 4 °C, 15 minutes), and then the supernatant (cell lysate) was collected.

[0134] To 12.5 μL of the collected supernatant, 35.5 μL of 0.2 M Tris-HCl buffer (pH 8.0) and 2 μL of samples at each concentration were mixed 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 with a microplate reader, and the elastase activity was calculated.

[0135] Figure 17 shows the results of the elastase inhibitory activity evaluation test. As shown in Figure 17, the water extract and ethanol extract showed elastase inhibitory activity in a concentration-dependent manner. In particular, the water extract showed an inhibition rate of about 82% at 800 mg / mL, and the ethanol extract showed an inhibition rate of about 58% at 800 mg / mL.

[0136] Therefore, it can be said that the perilla extract can be used as a skin quality improving agent having an anti-wrinkle effect because it inhibits elastase activity.

[0137] As described above, the perilla extract has the effects of preventing acne, anti-allergy, anti-wrinkle, and suppressing melanin production. That is, it can be said that the perilla extract can be used as a component that induces at least one of the effects selected from preventing acne, anti-allergy, anti-wrinkle, and suppressing melanin production.

[0138] Next, an evaluation test by humans was conducted using external preparations, cosmetics, and foods containing a skin quality improving agent containing such a perilla extract.

[0139] 〔10. Human evaluation test of external preparation〕 In this test, 12 subjects, two males and two females each in their 20s, 40s, and 60s, were made to use an external preparation containing the skin quality improving agent according to the present invention for a predetermined period (hereinafter referred to as the "test group"). After the lapse of the period, the evaluation was carried out in five grades on the effects on acne (acne prevention effect), redness and itching (anti-allergic effect), wrinkles (anti-wrinkle effect), and spots (melanin production inhibitory effect).

[0140] The external preparation used in the test was a body mist containing 400 μg / mL of an ethanol extract of dried perilla leaves. This body mist was used twice a day, morning and evening, for four weeks.

[0141] Also, as a control test, a body mist not containing the skin quality improving agent according to the present invention was used and evaluated under the same conditions (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.

[0142] The results of the human evaluation test of the external preparation are shown. Table 4 shows the results of the test group, and Table 5 shows the results of the control test group.

[0143]

Table 4

Table 5

[0144] From Table 4 and Table 5, it became clear that the skin quality improving agent containing the perilla extract is effective as a component that induces an acne prevention effect, an anti-allergic effect, an anti-wrinkle effect, or a melanin production inhibitory effect when contained in an external preparation.

[0145] 〔11. Human Evaluation Test of Cosmetics〕 In this test, a total of 12 people, two men and two women each in their 20s, 40s, and 60s, were asked to use a cosmetic containing the skin quality improving agent according to the present invention for a predetermined period (hereinafter referred to as the "test group"). After the elapse of the period, the evaluation was carried out in five grades on the effects on acne (acne prevention effect), redness and itching (anti-allergy effect), wrinkles (anti-wrinkle effect), and spots (melanin production inhibitory effect), respectively.

[0146] The cosmetic used in the test was an emulsion containing 400 μg / mL of an ethanol extract of dried perilla leaves. This emulsion was used twice a day, morning and evening, for 4 weeks.

[0147] Also, as a control test, an emulsion not containing the skin quality improving agent according to the present invention was used and evaluated under the same conditions (hereinafter referred to as the "control test group"). The emulsion used in this control test group was an emulsion prepared so as not to contain skin-beautifying ingredients.

[0148] The results of the human evaluation test of the cosmetic are shown. Table 6 shows the results of the test group, and Table 7 shows the results of the control test group.

[0149]

Table 6

Table 7

[0150] From Table 6 and Table 7, it was revealed that the skin quality improving agent containing perilla extract is effective as a component that induces an acne prevention effect, an anti-allergy effect, an anti-wrinkle effect, or a melanin production inhibitory effect when contained in a cosmetic.

[0151] 〔12. Human evaluation test of food〕 In this test, 12 subjects, two men and two women each in their 20s, 40s, and 60s, were given a food containing the skin quality improving agent according to the present invention over a predetermined period (hereinafter referred to as the "test group"). After the passage of the period, the evaluation was carried out in five grades on the effects on acne (acne prevention effect), redness and itching (anti-allergic effect), wrinkles (anti-wrinkle effect), and spots (melanin production inhibitory effect).

[0152] The food for the test was a tablet containing 400 μg / mg of an ethanol extract of dried perilla leaves. This tablet was taken twice after breakfast and dinner for 4 weeks.

[0153] Also, as a control test, a tablet not containing the skin quality improving agent according to the present invention was used and evaluated under the same conditions (hereinafter referred to as the "control test group"). The tablets used in this control test group were commercially available fake drugs that did not contain beauty ingredients.

[0154] The results of the human evaluation test of the food are shown. Table 8 shows the results of the test group, and Table 9 shows the results of the control test group.

[0155]

Table 8

Table 9

[0156] From Tables 8 and 9, it was revealed that the skin quality improving agent containing perilla extract is effective as a component that induces an acne prevention effect, anti-allergic effect, anti-wrinkle effect, or melanin production inhibitory effect when contained in food.

[0157] From the results of such human evaluation tests, it can be said that perilla extract is effective as a component that induces at least one of the effects selected from acne prevention, anti-allergy, anti-wrinkle, and melanin production inhibition when contained in external preparations, cosmetics, or food.

[0158] In addition, from the results of the lipase inhibitory activity test, anti-allergy test, collagen and hyaluronic acid production evaluation test, melanin production evaluation test, anti-acne bacteria activity test, and elastase inhibitory activity evaluation test described above, it was revealed that especially the ethanol extract of perilla frutescens strongly acts on these. Therefore, it can be said that the ethanol extract of perilla frutescens can be used as an active ingredient in external preparations, cosmetics, or foods aimed at at least any one selected from prevention of acne occurrence, anti-allergy, anti-wrinkle, and suppression of melanin production.

[0159] By the way, there is a report on using an external preparation containing luteolin contained in perilla frutescens for the treatment and prevention of acne. As an example of such a report, Japanese Patent Laid-Open No. 2013-203703 is known. Also, Japanese Patent Laid-Open No. 2008-253256 is known as a cosmetic containing rosmarinic acid also contained in perilla frutescens.

[0160] However, as revealed by the HPLC analysis and TLC analysis in this embodiment, the perilla frutescens extract in this embodiment does not contain luteolin and rosmarinic acid. In other words, luteolin and rosmarinic acid are not involved at all in the prevention of acne occurrence, anti-allergy, anti-wrinkle, and suppression of melanin production of the perilla frutescens extract clarified in this embodiment.

[0161] That is, even the perilla frutescens extract obtained from the residue of the leaves of perilla frutescens after extracting luteolin and rosmarinic acid as active ingredients can be used as a component contained in external preparations, cosmetics, or foods aimed at at least any one selected from prevention of acne occurrence, anti-allergy, anti-wrinkle, and suppression of melanin production.

Claims

1. A skin quality improving agent containing, as an ingredient that induces an acne prevention effect, an ethanol extract of perilla frutescens var. acuta that does not contain rosmarinic acid.

2. An external preparation for preventing acne containing the skin quality improving agent according to Claim 1.

3. A cosmetic for preventing acne containing the skin quality improving agent according to Claim 1.

4. A useful functional food for preventing acne containing the skin quality improving agent according to Claim 1.

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