Hyaluronic acid production promoter and method for producing same

Abstract

To provide a new hyaluronic acid production accelerating agent and a method for producing the hyaluronic acid production accelerating agent.SOLUTION: A hyaluronic acid production accelerating agent comprises a cypress essential oil derived from a trunk material of cypress as an active ingredient. The cypress essential oil contains at least α-pinene, δ-cadinene, α-cadinol, and T-murolol. In the GC-MS analysis of the cypress essential oil, the peak area ratio of α-pinene is at least 20% of the total, and the sum of the peak area ratios of δ-cadinene, α-cadinol, and T-murolol is at least 25% of the total.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a hyaluronic acid production promoter using cypress essential oil and a method for producing the same. [Background technology]

[0002] Hyaluronic acid (HA), a type of mucopolysaccharide, is distributed in the body in the skin, joints, vitreous body, and other areas. Hyaluronic acid has the ability to retain moisture, which has been reported to moisturize the skin and prevent dryness and wrinkles. In recent years, ingredients that promote hyaluronic acid production have been attracting attention in the cosmetics industry. In particular, there is a growing demand for functional ingredients derived from natural materials, and much research is being conducted on this topic.

[0003] For example, it has been reported that extracts obtained from the seeds of Cucurbitaceae plants have the effect of promoting hyaluronic acid production (Patent Document 1).It has also been reported that plant essences obtained from frankincense (Boswellia genus, Burseraceae) and sandalwood (Saltaceae) have the effect of promoting hyaluronic acid production (Patent Document 2).

[0004] Hinoki cypress is a coniferous tree that grows abundantly in Japan, and its wood is widely used for building materials, pulp, paper products, etc. However, a large amount of unused cypress wood is also generated, such as thinned wood and parts that cannot be used for products. In recent years, efforts have been made to make effective use of unused wood, and for example, cypress essential oil is distributed as a fragrance with a refreshing scent.

[0005] However, at present, cypress essential oil is mainly used as a fragrance, and there have been few reports on its functionality. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-273815 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-23437 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a novel hyaluronic acid production promoter and a method for producing the same. [Means for solving the problem]

[0008] The hyaluronic acid production promoter of the present invention is characterized by containing cypress essential oil derived from the trunk wood of cypress as an active ingredient.

[0009] The hyaluronic acid production promoter of the present invention contains cypress essential oil as an active ingredient, and the cypress essential oil is characterized by containing at least α-pinene, δ-cadinene, α-cadinol, and T-muurolol.

[0010] In GC-MS analysis (gas chromatography-mass spectrometry) of the above cypress essential oil, the peak area ratio of the above α-pinene is 20% or more of the total, and the sum of the peak area ratios of the above δ-cadinene, the above α-cadinol and the above T-muurolol is 25% or more of the total.

[0011] The cypress essential oil further contains α-muurolol, and is characterized in that in GC-MS analysis of the cypress essential oil, the total peak area ratio of the α-pinene, δ-cadinene, α-cadinol, T-muurolol, and α-muurolol is 60% or more of the total.

[0012] Hinoki essential oil is further characterized as containing α-muurolene, terpinyl acetate, γ-cadinene, δ-cadinol, β-elemene, bornyl acetate, α-terpineol, and citronellol.

[0013] It is characterized by being a cosmetic material.

[0014] The method for producing the hyaluronic acid production promoter is characterized in that the cypress essential oil is obtained by steam distillation of cypress trunk wood. [Effects of the Invention]

[0015] The hyaluronic acid production promoter of the present invention contains cypress essential oil derived from cypress trunk wood as an active ingredient, and as will be shown in the examples below, it exhibits excellent hyaluronic acid production promoting effects. For example, when used as a cosmetic ingredient, it is expected to increase the skin's moisturizing power and prevent dryness and wrinkles.

[0016] The hyaluronic acid production promoter of the present invention contains cypress essential oil as an active ingredient, and the cypress essential oil contains at least α-pinene, δ-cadinene, α-cadinol, and T-muurolol.In addition, in GC-MS analysis, the peak area ratio of α-pinene is 20% or more of the total, and the total peak area ratio of δ-cadinene, α-cadinol, and T-muurolol is 25% or more of the total, and as a result, it exhibits excellent hyaluronic acid production promoting effect.

[0017] The above-mentioned cypress essential oil is obtained by steam distillation of the trunk wood of cypress, so that the desired components can be extracted more efficiently in terms of the hyaluronic acid production promoting effect than, for example, extracts obtained by solvent extraction, and the obtained cypress essential oil shows excellent hyaluronic acid production promoting effect. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the results of GC-MS analysis of cypress essential oil. [Figure 2] FIG. 1 shows a detected image of hyaluronic acid in cypress essential oil. [Figure 3] FIG. 1 shows the amount of hyaluronic acid produced in cypress essential oil (stem). [Figure 4] FIG. 1 shows the amount of hyaluronic acid produced in cypress essential oil (stem, branches, and leaves). [Figure 5] FIG. 1 shows the amount of hyaluronic acid produced by cypress essential oil (stem) and other essential oils (stem). [Figure 6] FIG. 1 shows the amount of hyaluronic acid produced in cypress essential oil (stem) and other oils. [Figure 7] FIG. 1 is a graph showing the amount of hyaluronic acid produced depending on the extraction method. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present inventors have carried out intensive research to find new functions of cypress essential oil from the viewpoint of effectively utilizing unused cypress wood, and have found that the cypress essential oil derived from cypress trunk wood has excellent hyaluronic acid production promoting effect, and further, have found that the cypress essential oil obtained by steam distillation has excellent hyaluronic acid production promoting effect.The present invention is based on this finding.

[0020] The hyaluronic acid production promoter of the present invention contains cypress essential oil derived from cypress trunk wood as an active ingredient. Cypress (scientific name: Chamaecyparis obtusa) is a coniferous tree of the genus Chamaecyparis and the family Cupressaceae. The origin of cypress is not particularly limited, and cypress from Japan or Korea can be used, but it is preferable to use cypress from Japan in terms of quality control, etc. The cypress trunk wood used as the raw material for cypress essential oil of the present invention is the xylem (heartwood) part, for example, the log part that has been felled and the branches and leaves cut off.

[0021] The cypress essential oil that stems from is a mixture of many kinds of compounds, and has a refreshing and clean fragrance.Here, comparing the cypress essential oil that stems from and the cypress essential oil that stems from leaves, the cypress essential oil that stems from contains a lot of compounds with low volatility (high boiling point), and the cypress essential oil that stems from leaves contains a lot of compounds with high volatility (low boiling point).In the present invention, the compound that stems from the cypress essential oil that stems from exerts the effect of promoting hyaluronic acid production.

[0022] The cypress essential oil used in the present invention mainly contains monoterpenes, sesquiterpenes, and sesquiterpene alcohols, such as α-pinene, δ-cadinene, α-cadinol, and T-muurolol.

[0023] α-Pinene is a monoterpene known as a fragrance component. Cypress essential oil derived from trunk wood may contain β-pinene, a structural isomer of α-pinene, but its content is extremely small compared to α-pinene. δ-cadinene is a sesquiterpene, and is a structural isomer of the α-, β-, and γ-forms, which differ in the position of the double bond. Cypress essential oil derived from trunk wood may contain α-, β-, and γ-forms in addition to the δ-form. For example, among the four structural isomers, the δ-form is detected as the peak with the largest peak area in GC-MS analysis. α-Cadinol and T-Murolol are sesquiterpene alcohols.

[0024] The cypress essential oil used in the present invention may further contain other components such as muurolene, β-elemene, terpinyl acetate, and d-limonene.

[0025] The above-mentioned components of the cypress essential oil used in the present invention can be defined by their peak areas in GC-MS analysis. In the GC-MS analysis of the cypress essential oil, the peak area ratio of α-pinene is, for example, 20% to 50%, or may be 20% to 40%, relative to the total. The peak area ratio of δ-cadinene is, for example, 5% to 20%, or may be 10% to 20%, relative to the total. The peak area ratio of α-cadinol is, for example, 5% to 20%, or may be 10% to 20%, relative to the total. The peak area ratio of T-muurolol is, for example, 2% to 10%, or may be 5% to 10%, relative to the total.

[0026] In GC-MS analysis, the peak area ratio of α-pinene is preferably 20% or more relative to the total, and the sum of the peak area ratios of δ-cadinene, α-cadinol, and T-muurolol is preferably 25% or more relative to the total. Furthermore, the sum of the peak area ratios of α-pinene, δ-cadinene, α-cadinol, T-muurolol, and α-muurolol is preferably 60% or more relative to the total. The conditions for GC-MS analysis are shown in the Examples below.

[0027] On the other hand, the cypress essential oil used in the present invention may not contain sabinene, elemol, beyerene, and fenchyl acetate, or may contain only trace amounts. For example, in GC-MS analysis, the peak area ratio of each of these may be, for example, less than 3% (including 0%) or even less than 1% (including 0%) of the total. These are components that are contained in large amounts in cypress essential oil derived from, for example, branches and leaves.

[0028] As described above, the cypress essential oil used in the hyaluronic acid production promoter of the present invention is derived from trunk wood and also contains specific components. Because it contains the desired components, it exhibits excellent hyaluronic acid production promoting effects, unlike, for example, cypress essential oil derived from branches and leaves.

[0029] The raw material for the cypress essential oil used in the present invention is cypress trunk wood. For example, logs crushed into chips can be used as the trunk wood. In particular, using unused wood allows for effective use of resources.

[0030] The cypress essential oil used in the present invention can be obtained by subjecting cypress trunk wood to steam distillation or solvent extraction. Steam distillation involves generating heated steam from the bottom of a still containing raw materials, vaporizing the components in the raw materials together with the steam, and then cooling the mixture to collect the liquid essential oil. Solvents used in solvent extraction include ethanol, ethyl acetate, diethyl ether, normal hexane, and methanol. It is preferable to use cypress essential oil obtained by steam distillation, as it can efficiently extract the desired components for promoting hyaluronic acid production. For example, the amount of steam injected per hour during steam distillation is 15 to 40 parts by mass per 100 parts by mass of raw materials.

[0031] The hyaluronic acid production promoter of the present invention can be used as it is (100%), and can also be used as the cypress essential oil derived from the stem material of cypress, and can be mixed with any component in this cypress essential oil.In addition, the form of hyaluronic acid production promoter is not limited to liquid, and can also be prepared in gel or solid form.

[0032] The hyaluronic acid production promoter of the present invention is expected to prevent or improve skin aging, wrinkles, etc. by promoting hyaluronic acid production, and is particularly useful as a material to be incorporated into cosmetics or topical medicines.Such cosmetics or topical medicines can be incorporated with various optional components generally used in the relevant fields.Optional components can include, for example, various oils and fats, surfactants, viscosity agents, oil-soluble active ingredients, water-soluble active ingredients, powders, various plant extracts, etc.The form of the above-mentioned cosmetics or topical medicines can be any form such as cream, emulsion, ointment, lotion, pack cosmetics, facial cleansing cosmetics, cleansing cosmetics, spray, gel, etc., depending on the purpose. [Example]

[0033] (1) Production of Hinoki essential oil (trunk, branches and leaves) As raw materials, the trunk wood of Japanese cypress and the branches and leaves of Japanese cypress produced in Japan were used respectively. The said trunk wood and branches and leaves were used in a state of being crushed into chips. Regarding the trunk wood, 950 kg of raw material was subjected to steam distillation (steam amount: 280 kg / h) to obtain 7.8 kg of Japanese cypress essential oil (trunk). Regarding the branches and leaves, 40 kg of raw material was subjected to steam distillation (steam amount: 7 L / h) to obtain 380 g of Japanese cypress essential oil (branches and leaves).

[0034] (2) GC-MS analysis of Japanese cypress essential oil The Japanese cypress essential oil (trunk) and Japanese cypress essential oil (branches and leaves) obtained above were subjected to GC-MS analysis under the following conditions. <GC conditions> Equipment used: QP2010 (manufactured by Shimadzu Corporation) Column used: InertCap Pure-WAX (0.25 mm I.D. × 30 m, df = 0.25 μm, manufactured by GL Sciences) Temperature rising condition: 50°C (hold for 3 min) to 250°C at 5.0°C / min (hold for 15 min) Carrier gas: He Injection volume: 1 μl Split ratio: 1:100 Ionization method: Electron ionization method (EI method) Ionization voltage: 70 eV Ion source temperature: 200°C Interface temperature: 230°C Detection mass range: 29 - 450 m / z

[0035] The chart showing the obtained total ion chromatogram (TIC) relatively is shown in Fig. 1. Fig. 1(a) shows the relative TIC chart of Japanese cypress essential oil (trunk), and Fig. 1(b) shows the relative TIC chart of Japanese cypress essential oil (branches and leaves). Also, in Table 1, 10 components are listed in descending order of peak area ratio (%) from each TIC chart, and their peak area ratio (%) and retention time (min) are shown. The total of the peak area ratios of each 10 components shown in Table 1 was 80% or more for the whole in each case.

[0036] [Table 1]

[0037] As shown in Figure 1 and Table 1, the components contained in cypress essential oil (stem) and cypress essential oil (branch and leaf) were found to differ significantly. In cypress essential oil (stem), α-pinene was the most abundant peak area ratio, exceeding 30%, followed by δ-cadinene and α-cadinol, each exceeding 10%. The total peak area ratio of these components was over 50% of the total. Furthermore, cypress essential oil (stem) also contained t-muurolol and α-muurolol. The total peak area ratio of these five components, including the above three, was over 60% of the total. Cypress essential oil (stem) also contained relatively high amounts of other components, such as α-muurolene, terpinyl acetate, γ-cadinene, δ-cadinol, and β-elemene. Although not listed in Table 1, cypress essential oil also contained bornyl acetate, α-terpineol, and citronellol. On the other hand, sabinene, elemol, beyerene, and fenchyl acetate were not detected. Note that when the value was below the detection limit (for example, the peak area ratio was less than 0.01%), it was considered as not detected.

[0038] In the cypress essential oil (branches and leaves), sabinene had the highest peak area percentage, followed by terpinyl acetate and elemol. The total area percentage of each of these peaks was over 40% of the total. α-cadinol, α-muurolol, α-muurolene, γ-cadinene, δ-cadinol, and β-elemene were not detected. Furthermore, in the cypress essential oil (branches and leaves), several high-height peaks were detected, particularly between retention times of 10 and 16 minutes. The total area percentage of each peak detected during this period (sabinene, β-myrcene, d-limonene, etc.) was over 30% of the total. In contrast, in the cypress essential oil (stem), few characteristic peaks were observed between retention times of 10 and 16 minutes, and the total area percentage of each peak detected during this period (d-limonene, etc.) was less than 5% of the total.

[0039] (3) Evaluation test of hyaluronic acid production promotion effect The following cells were treated with the above-obtained cypress essential oil to evaluate its effect of promoting hyaluronic acid production. Cells used: Human dermal fibroblast cell line NB1RGB Culture medium used: MEMα (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with L-glutamine, penicillin, and streptomycin, and 10% FBS (fetal bovine serum) was used.

[0040] [Fluorescence detection of hyaluronic acid] This was done according to the following procedure. 1. A 13 mm diameter cover glass was placed on the bottom of a 24-well plate (TPP), and NB1RGB cells were seeded on top of it at 2500 cells / well (medium volume: 500 μL). 2. To allow the cells to settle, they were cultured for 24 hours in an incubator set at 37°C and 5% CO2. 3. Each sample was prepared in a DMSO:ethanol = 9:1 solution to give concentrations of 20, 40, and 80 mg / mL, and then diluted 1 / 1000 with medium to give treatments of 20, 40, and 80 μg / mL. 4. Treatment of each sample was initiated by replacing the medium with the prepared medium. 5. 72 or 144 hours after treatment, the medium was removed and the cells were washed once with phosphate buffered saline (PBS). 6.4% paraformaldehyde (PFA) / PBS was added and the cells were fixed for 10 minutes at room temperature in the dark. The 7.4% PFA / PBS was removed, and the sections were washed three times with PBS. 8. 0.1% Triton X-100 / PBS was added, and the cell membrane was permeabilized at room temperature for 10 minutes. 9. The 0.1% Triton X-100 / PBS was removed, and the plate was washed once with PBS. 3% bovine serum albumin (BSA) / PBS was added, and the plate was blocked at 37°C for 1 hour. A Biotin-HABP (Hokudo Co., Ltd.) solution diluted 200-fold with 10.1% bovine serum albumin (BSA) / PBS was dropped onto the cover glass and incubated at 4°C for 16 to 20 hours. 11. Washed with 0.05% Tween 20 / PBS three times for 5 minutes. An FITC avidin solution diluted 200-fold with 12.1% BSA / PBS was dropped onto the cover glass and incubated at 37°C for 45 minutes. 13. Washed with 0.05% Tween 20 / PBS three times for 5 minutes. 14. Hoechst 33342 solution diluted 10,000 times with PBS was added and the nuclei were stained at room temperature for 5 minutes. 15. The sections were washed once with PBS and once with water, and then mounted on slides with 2.5% DABCO / 90% glycerol / 1×PBS. 16. Photographs were taken using a fluorescence microscope (Olympus Corporation, CKX53).

[0041] [Image quantification] This was done according to the following procedure. Ten images of each sample taken at 1.200x magnification were stacked using Image J software (Ver. 1.52u), and the same threshold was set, after which the intensity density of all images was measured. 2. The quantitative values ​​obtained were divided by the number of cells present in each image, and the values ​​were used to compare the amount of HA production between samples. 3. Dunnett's method was used for multiple testing.

[0042] In the above evaluation test, TGFβ1 (1 ng / mL) was used as a positive control for promoting hyaluronic acid production.

[0043] The evaluation results of the promotion of hyaluronic acid production by each test sample are shown in Figures 2 and 3. Figure 2 shows an image of the fluorescence detection of hyaluronic acid after 72 hours of treatment with TGFβ1 and cypress essential oil (stem), with the white area in the image representing hyaluronic acid.

[0044] Figure 3 shows the quantitative results of the fluorescence detection of hyaluronic acid after 72 or 144 hours of treatment with TGFβ1 and cypress essential oil (stem). Specifically, the fluorescence detection amount for the untreated (control) is set to 1, and the fluorescence detection amounts for the other test samples are shown as relative values. As shown in Figure 3, cypress essential oil (stem) showed a concentration-dependent effect of promoting hyaluronic acid production. Furthermore, the hyaluronic acid production-promoting effect of cypress essential oil (stem) also increased in a treatment time-dependent manner.

[0045] Next, Figure 4 shows the quantitative results of fluorescent detection of hyaluronic acid after 72 hours of treatment with TGFβ1, cypress essential oil (stem), and cypress essential oil (branch and leaf). As shown in Figure 4, cypress essential oil (stem) showed a concentration-dependent effect of promoting hyaluronic acid production, whereas cypress essential oil (branch and leaf) did not show any effect of promoting hyaluronic acid production.

[0046] Next, we compared the hyaluronic acid production promoting effect when treated with other essential oils (stem) and cypress essential oil (stem). Following the procedure described above, each sample was added to the culture medium at various concentrations, treated for 72 hours, and the degree of hyaluronic acid production promotion was compared. All essential oils used were obtained by steam distillation from the stem (xylem). The results are shown in Figure 5.

[0047] As shown in Figure 5, other kinds of essential oils (stem) hardly show any hyaluronic acid production promoting effect.In addition, 40μg / mL of cypress essential oil and 40μg / mL of sandalwood essential oil treatment show the same hyaluronic acid production promoting effect, but in 80μg / mL treatment, cypress essential oil shows a stronger hyaluronic acid production promoting effect, while sandalwood essential oil shows a weakened hyaluronic acid production promoting effect.Sandalwood essential oil is a plant of the Santalum family, and as described in the above-mentioned Patent Document 2, it is said to show hyaluronic acid production promoting effect, but from the results of this time, it can be said that cypress essential oil has a better hyaluronic acid production promoting effect.

[0048] Next, we compared the hyaluronic acid production promoting effect of treatment with refined oils sold as carrier oils and cypress essential oil (stem). Following the procedure described above, each sample was added to the culture medium at various concentrations, treated for 72 hours, and the degree of hyaluronic acid production promotion was compared. The results are shown in Figure 6.

[0049] As shown in Figure 6, refined olive oil and refined grapeseed oil did not show any hyaluronic acid production promoting effect, but the hyaluronic acid production promoting effect was specifically observed in cypress essential oil (stem).

[0050] Next, an extraction method for cypress trunk wood was investigated. Normal hexane was added to cypress trunk wood (ground into chips) and stirred, after which the organic layer was recovered and the normal hexane was distilled off to obtain a hexane extract. An ethanol extract was also obtained using the same extraction procedure. Following the procedure described above, each sample was added to the medium at various concentrations, treated for 72 hours, and the degree of promotion of hyaluronic acid production was compared. The results are shown in Figure 7.

[0051] As shown in Figure 7, extracts extracted by methods other than steam distillation also showed a concentration-dependent effect of promoting hyaluronic acid production. Among these, the effect of promoting hyaluronic acid production was strongest in the cypress essential oil (stem) obtained by steam distillation. For example, when GC-MS analysis was performed on the hexane extract, almost the same components as those obtained by steam distillation were detected, but the peak area ratio of these components to the total was low, and many other components were also detected. It is thought that the results shown in Figure 7 were due to the compounds that promote hyaluronic acid production being efficiently extracted by steam distillation. [Industrial Applicability]

[0052] The hyaluronic acid production promoter of the present invention contains cypress essential oil derived from cypress trunk material, and therefore has the effect of promoting the production of hyaluronic acid in normal human skin fibroblasts, so it can be expected to be effective in improving the moisturizing power of skin, and preventing dryness, wrinkles, etc.In addition, by expanding the application field of cypress essential oil, it can further effectively utilize unused cypress material, and is expected to be connected to the revitalization of industry through cypress and the contribution to local community by building an environmentally friendly recycling system.

Claims

1. It contains cypress essential oil, a steam distillate of cypress trunk wood, as an active ingredient. The cypress essential oil is a hyaluronic acid production promoter characterized in that it contains at least α-pinene, δ-cadinene, α-cadinol, and T-muurolol.

2. The hyaluronic acid production promoter described in claim 1, characterized in that in GC-MS analysis of the cypress essential oil, the peak area ratio of the α-pinene is 20% or more of the total, and the sum of the peak area ratios of the δ-cadinene, the α-cadinol and the T-muurolol is 25% or more of the total.

3. A hyaluronic acid production promoter as described in Claim 2, characterized in that in GC-MS analysis of the cypress essential oil, the peak area ratios of sabinene, elemol, beyerene, and fenchyl acetate are less than 1% of the total.

4. The hyaluronic acid production promoter according to claim 1 or 2, characterized in that it is a cosmetic material.

5. A method for producing the hyaluronic acid production promoter according to claim 1 or 2, The method for producing a hyaluronic acid production promoter is characterized in that the cypress essential oil is obtained by steam distilling the trunk wood of the cypress, and the amount of steam blown in per hour in the steam distillation method is 15 to 40 parts by mass per 100 parts by mass of raw material.

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