Anti-inflammatory agents and anti-inflammatory compositions, and methods for producing the same.

Abstract

To provide an anti-inflammatory agent and an anti-inflammatory composition each of which comprises at least one distillate selected from the group consisting of pine distillate, yew distillate and cedar distillate, which were traditionally discarded as by-products, as an active ingredient, and is derived from plants, less irritative to the skin, and popular among users, and methods for producing them.SOLUTION: An anti-inflammatory agent comprises at least one distillate selected from the group consisting of pine distillate, yew distillate and cedar distillate as an active ingredient.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to anti-inflammatory agents, compositions for anti-inflammation, and methods for producing them.

Background Art

[0002] In normal skin, the outermost stratum corneum functions as a barrier, playing a role in retaining skin moisture and preventing the invasion of foreign substances such as bacteria and harmful substances from the outside world. When the skin barrier function decreases due to some reason, the skin's moisture retention becomes insufficient, and foreign substances from the outside world easily invade the skin, which is considered to be one of the causes of skin inflammation. Skin inflammation, to varying degrees, is a problem that many people have. Inflammation accelerates skin aging, causing roughness, wrinkles, dullness, etc. in the skin, and is also known to cause a decrease in the skin barrier function (a decrease in skin moisture content, dryness), hypersensitivity, etc. Therefore, in recent years, substances having an anti-inflammatory effect are required not only as active ingredients of pharmaceuticals but also in the fields of quasi-drugs, cosmetics, etc.

[0003] Picea glehnii is an evergreen coniferous tree of the genus Picea in the Pinaceae family and is a representative afforestation tree species in Hokkaido. It has been clarified that the essential oil obtained by steam distillation of the branches and leaves of Picea glehnii has various functions such as antibacterial action and relaxation action.

[0004] Non-Patent Document 1 discloses that Picea glehnii essential oil exhibits antibacterial action against food poisoning-causing bacteria such as Staphylococcus aureus and Escherichia coli. Further, Non-Patent Document 2 discloses that Picea glehnii essential oil exhibits antibacterial action against acne bacteria that cause acne.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] However, while Non-Patent Documents 1 and 2 disclose the antibacterial activity of essential oil obtained by steam distillation of Sakhalin fir, they do not mention the effects of the distillate, which is a byproduct of steam distillation. Furthermore, substances with anti-inflammatory effects are still needed in the fields of pharmaceuticals such as topical skin preparations, as well as quasi-drugs and cosmetics, and the development of new anti-inflammatory agents and compositions is awaited.

[0007] The present invention has been made in view of the above circumstances, and aims to provide an anti-inflammatory agent and anti-inflammatory composition that are plant-derived, less irritating to the skin, and preferred by users, using at least one distillate selected from the group consisting of pine distillate, yew distillate, and cedar distillate, which were conventionally discarded as by-products, as an active ingredient, as well as methods for producing the same. [Means for solving the problem]

[0008] To achieve the above objective, the anti-inflammatory agent according to the first aspect of the present invention is: The active ingredient is a distillate selected from the group consisting of pine distillate, yew distillate, and cedar distillate.

[0009] For example, the pine tree is selected from the group consisting of Sakhalin spruce, Abies sachalinensis, Japanese larch, and Japanese stone pine.

[0010] For example, the pine tree is a Sakhalin spruce.

[0011] For example, anti-inflammatory drugs are topical medications applied to the skin.

[0012] The anti-inflammatory composition according to a second aspect of the present invention is: The active ingredient is a distillate selected from the group consisting of pine distillate, yew distillate, and cedar distillate.

[0013] For example, the pine tree is selected from the group consisting of Sakhalin spruce, Abies sachalinensis, Japanese larch, and Japanese stone pine.

[0014] For example, the pine tree is a Sakhalin spruce.

[0015] For example, an anti-inflammatory composition is a composition for topical application to the skin.

[0016] For example, an anti-inflammatory composition is a cosmetic composition.

[0017] A method for producing an anti-inflammatory agent according to a third aspect of the present invention is: The process includes obtaining a distillate by distilling a plant selected from the group consisting of pine, yew, and cedar.

[0018] A method for producing an anti-inflammatory composition according to the fourth aspect of the present invention is: The process includes obtaining a distillate by distilling a plant selected from the group consisting of pine, yew, and cedar. [Effects of the Invention]

[0019] According to the present invention, it is possible to provide an anti-inflammatory agent and anti-inflammatory composition that are plant-derived, less irritating to the skin, and preferred by users, using at least one distillate selected from the group consisting of pine distillate, yew distillate, and cedar distillate, which were conventionally discarded as by-products, as an active ingredient, as well as methods for producing the same. [Brief explanation of the drawing]

[0020] [Figure 1] It is a schematic diagram for measuring the anti-inflammatory effect and cell metabolic activity of the Abies firma distillate in this example. [Figure 2] (a) is a graph of the component analysis of the Abies firma essential oil in the comparative example, and (b) is a graph of the component analysis of the Abies firma distillate in this example. [Figure 3] (a) is a graph showing the NO production rate of the Abies firma distillate and the Abies firma essential oil in the comparative example in this example, and (b) is a graph showing the cell metabolic activity of the Abies firma distillate and the Abies firma essential oil in the comparative example in this example. [Figure 4] (a) is a graph showing the NO production rate of camphor and bornyl acetate in the Abies firma distillate, and (b) is a graph showing the cell metabolic activity of camphor and bornyl acetate in the Abies firma distillate. [Figure 5] (a) is a graph showing the NO production rate for each molecular weight fraction of the Abies firma distillate in this example, and (b) is a graph showing the cell metabolic activity for each molecular weight fraction of the Abies firma distillate in this example. [Figure 6] (a) is a graph showing the NO production rate of the distillate derived from pine in this example, and (b) is a graph showing the cell metabolic activity of the distillate derived from pine in this example. [Figure 7] (a) is a graph showing the NO production rate of the distillates derived from Japanese red pine, Japanese larch and Japanese cedar in this example, and (b) is a graph showing the cell metabolic activity of the distillates derived from Japanese red pine, Japanese larch and Japanese cedar in this example.

Mode for Carrying Out the Invention

[0021] (1. Anti-inflammatory agent) The anti-inflammatory agent according to the present invention contains, as an active ingredient, a distillate selected from at least one of the group consisting of pine distillate, Japanese larch distillate and Japanese cedar distillate.

[0022] The pine trees used in this invention can be any tree belonging to the Pinaceae family without any particular restrictions. For example, trees belonging to the genus Picea of ​​the Pinaceae family include Sakhalin spruce and spruce. Trees belonging to the genus Abies of the Pinaceae family include Sakhalin fir, Abies firma, Japanese fir, Abies mariesii, Abies veitchii, Japanese fir, Balsam fir, Three-leaved fir, White fir, Amabilis fir, Japanese fir, California red fir, Grand fir, Noble fir, etc. Trees belonging to the genus Pinus of the Pinaceae family include Japanese red pine, Japanese pine, Eastern white pine, and Japanese stone pine. Trees belonging to the genus Larix of the Pinaceae family include Japanese larch, etc. Trees belonging to the genus Hemlock of the Pinaceae family include Hemlock, etc. Two or more different types of pine may be mixed and used.

[0023] The pine used in the present invention is preferably at least one species selected from the group consisting of Sakhalin spruce, Abies sachalinensis, Japanese larch, and Japanese stone pine, and more preferably Sakhalin spruce is used.

[0024] The Japanese yew (scientific name: Taxus cuspidata) used in this invention can be any tree belonging to the genus Taxus in the family Taxaceae without any particular restrictions. In this invention, Japanese yew also includes ver. Cuspidata (f. luteobaccata: Yellow yew) and ver. Nana (Japanese yew) (Aurescens: Golden yew).

[0025] The Japanese cedar (scientific name: Cryptomeria japonica) used in this invention can be any tree belonging to the genus Cryptomeria in the subfamily Cupressoideae of the family Cupressaceae, without any particular restrictions.

[0026] Regarding the parts of pine, yew, and cedar to be used, any part of the pine, yew, and cedar can be used, including branches, leaves, trunks, roots, flowers, and fruits, but preferably, the branches and leaves of the pine, yew, and cedar are used.

[0027] Pine distillate is a distillate obtained by distilling pine, and is not pine essential oil (pine oil). Pine essential oil and pine distillate can be obtained by distilling pine. Normally, pine essential oil is used in cosmetics, fragrances, etc., and pine distillate is treated as a by-product and is not utilized. However, in this invention, pine distillate, which was conventionally discarded, is used. Examples of distillation methods include steam distillation, atmospheric distillation, low-pressure distillation, reduced-pressure distillation, molecular distillation, simple distillation, rectification, continuous distillation, and batch distillation, but steam distillation is preferred. More specifically, a steam distillation method can be described as a method in which pine is placed in a distillation vessel, water is added and heated to generate vapor from the pine components, and the vapor is passed through a cooling pipe or the like to condense the vapor and obtain pine essential oil (upper layer) and pine distillate (lower layer).

[0028] Yew distillate is a distillate obtained by distilling yew wood in the same manner as described above. Similarly, cedar distillate is a distillate obtained by distilling cedar wood in the same manner as described above. Details of the distillation methods for each are the same as described above.

[0029] The method for producing pine distillate (steam distillation) will be explained using Sakhalin spruce as an example. The branches and leaves of the Sakhalin spruce are cut with scissors and crushed, then a predetermined amount of water is added and placed in a known steam distillation apparatus. By heating for several hours, vapor is generated from the components of the Sakhalin spruce, and this vapor is condensed by passing it through a cooling pipe or the like to obtain pine essential oil (upper layer) and pine distillate (lower layer). Of these, the pine distillate can be separated by a known method to obtain Sakhalin spruce distillate.

[0030] The anti-inflammatory agent according to the present invention contains as an active ingredient a distillate selected from the group consisting of pine distillate, yew distillate, and cedar distillate. The anti-inflammatory agent according to the present invention may contain, for example, only pine distillate, a mixture of two distillates, pine distillate and yew distillate, or a mixture of three distillates, pine distillate, and cedar distillate.

[0031] The anti-inflammatory agent according to the present invention is applied for the prevention and treatment of inflammation, inflammatory diseases, and various symptoms caused thereby, but is particularly suitable for skin inflammation. Examples of skin inflammation include allergic dermatitis, atopic dermatitis, contact dermatitis (rash), psoriasis, pemphigus vulgaris, and various other skin inflammatory diseases associated with rough or dry skin. In this specification, "prevention" of a disease includes suppressing or delaying the onset of the disease and suppressing its recurrence, and "treatment" of a disease includes not only completely curing the disease but also alleviating symptoms and suppressing its progression.

[0032] The anti-inflammatory agent according to the present invention is administered to mammals that require anti-inflammatory therapeutic or preventive effects, such as rodents including mice, rats, hamsters, and guinea pigs; primates including humans, chimpanzees, and rhesus monkeys; livestock including pigs, cattle, goats, horses, and sheep; and companion animals including dogs and cats. The preferred target is humans.

[0033] The method of administering the anti-inflammatory agent according to the present invention can be appropriately selected from topical, oral, local, intravenous, intraperitoneal, intradermal, sublingual, and other methods. The dosage form may also be arbitrary, and an appropriate drug delivery system (DDS) may be used. These dosage forms are manufactured by formulating the active ingredient by conventional methods. Furthermore, various pharmaceutically acceptable formulation substances can be added as needed for the formulation. The formulation substances can be appropriately selected depending on the dosage form of the formulation, and examples include buffering agents, surfactants, stabilizers, preservatives, excipients, diluents, additives, disintegrants, binders, coatings, lubricants, slicks, flavoring agents, sweeteners, solubilizers, and the like.

[0034] The anti-inflammatory agent according to the present invention is preferably used as a topical skin preparation, applied directly to the skin. The anti-inflammatory agent of the present invention can be mixed with a base or carrier commonly used in pharmaceuticals, quasi-drugs, and cosmetics, and optionally with additives commonly used in pharmaceuticals, quasi-drugs, and cosmetics (e.g., surfactants, stabilizers, antioxidants, colorants, pearlescent agents, dispersants, chelating agents, pH adjusters, preservatives, thickeners, irritation reducers, etc.) according to conventional methods, and emulsified or solubilized as necessary to form various topical skin preparations. In addition to pine distillate, yew distillate, or cedar distillate, which are essential components of the present invention, the present invention may also contain optional components (for example, other anti-inflammatory agents, cooling agents, disinfectants, vitamins, organic acids, moisturizing components, polyhydric alcohols, scrubbing agents, UV absorbing components, UV scattering components, astringent components, peptides or their derivatives, amino acids or their derivatives, cleansing components, keratin softening components, cell activating components, anti-aging components, blood circulation promoting components, whitening components, powders, etc.) as long as they do not impair the effects of the present invention.

[0035] The dosage and frequency of administration of the anti-inflammatory agent according to the present invention can be appropriately determined by those skilled in the art, depending on the type of inflammation, the patient's health condition, age, weight, route of administration, form of administration, etc., so that an effective amount is administered to the patient.

[0036] (2. Anti-inflammatory composition) The anti-inflammatory composition according to the present invention contains as an active ingredient a distillate selected from the group consisting of pine distillate, yew distillate, and cedar distillate. Details regarding pine, yew, cedar, distillate, etc., are the same as described above.

[0037] The pine trees used in this invention can be any tree belonging to the Pinaceae family without particular restrictions, but examples include the same types of pine as described above. Preferably, at least one species is selected from the group consisting of Sakhalin spruce, Abies sachalinensis, Japanese larch, and Japanese stone pine, and more preferably, Sakhalin spruce is used. The same applies to the Japanese yew and Japanese cedar used in this invention as described above. The parts of each plant used are the same as described above.

[0038] The anti-inflammatory composition according to the present invention is applied for the prevention and treatment of inflammation, inflammatory diseases, and various symptoms caused thereby, but is particularly suitable for skin inflammation, as described above. The target of application, dosage, and frequency of administration are the same as described above.

[0039] The method of administering the anti-inflammatory agent according to the present invention is optional, as described above, but preferably, it is used by applying it directly to the skin as a topical skin composition. Details of the formulation of the topical skin composition are as described above.

[0040] The anti-inflammatory composition according to the present invention can be used, for example, as a cosmetic composition to prevent or improve skin roughness, skin stiffness, stratum corneum disorder, decreased barrier function, dry skin, decreased skin moisture content, hypersensitivity, skin redness, uneven skin tone, fine wrinkles, dullness, loss of skin texture, loss of firmness, pigmentation, skin tone dullness, and rough skin associated with skin inflammation.

[0041] The cosmetic composition of the present invention can be used, for example, as skincare products (lotions, emulsions, serums, creams, etc.); makeup products; makeup bases; sunscreen lotions, sunscreen creams; cleansing agents such as body soaps and facial washes; hair care products such as shampoos, conditioners, dry shampoos, scalp care products, hair mists, hair growth products, and hair restoration products; cleansing agents, cleansing lotions; deodorants, antiperspirants; bath additives; ointments; liquids; turbidants; emulsifiers (emulsifiers and creams); gels; liniments; lotions; poultices; mists, aerosols; powders, granules, tablets (including effervescent tablets); and solid soaps. It may also be used by impregnating a nonwoven fabric with the composition. The cosmetic composition of the present invention is effective against symptoms such as inflammatory aging, and may be used as a cosmetic composition for anti-aging purposes. It may also be used as a cosmetic composition for sensitive skin, temporary skin irritation due to seasonal or environmental changes, changes in physical condition, and so-called "unstable skin."

[0042] (3. Method for producing anti-inflammatory agents or anti-inflammatory compositions) The present invention provides a method for producing an anti-inflammatory agent or anti-inflammatory composition, comprising the step of obtaining a distillate by distilling a plant selected from the group consisting of pine, yew, and cedar. Details of the anti-inflammatory agent and anti-inflammatory composition are as described above.

[0043] The pine used in this invention can be any tree belonging to the Pinaceae family without any particular restrictions, but examples include the same types of pine as described above. Preferably, at least one species is selected from the group consisting of Sakhalin spruce, Abies sachalinensis, Japanese larch, and Japanese stone pine, and more preferably, Sakhalin spruce is used.

[0044] The Japanese yew (scientific name: Taxus cuspidata) used in this invention can be any tree belonging to the genus Taxus in the family Taxaceae without any particular restrictions. In this invention, Japanese yew also includes ver. Cuspidata (f. luteobaccata: Yellow yew) and ver. Nana (Japanese yew) (Aurescens: Golden yew).

[0045] The Japanese cedar (scientific name: Cryptomeria japonica) used in this invention can be any tree belonging to the genus Cryptomeria in the subfamily Cupressoideae of the family Cupressaceae, without any particular restrictions.

[0046] Regarding the parts of pine, yew, and cedar to be used, any part of the pine, yew, and cedar can be used, including branches, leaves, trunks, roots, flowers, and fruits, but preferably, the branches and leaves of the pine, yew, and cedar are used.

[0047] In the present invention, examples of distillation methods include steam distillation, atmospheric distillation, low-pressure distillation, reduced-pressure distillation, molecular distillation, simple distillation, rectification, continuous distillation, and batch distillation, but steam distillation is preferred. For example, when pine is used, pine essential oil and pine distillate can be obtained by distilling the pine, but in the present invention, the essential oil is not used, and the water-soluble pine distillate is used as an anti-inflammatory agent or anti-inflammatory composition (similarly, yew essential oil or cedar essential oil is not used, and yew distillate or cedar distillate is used). More specifically as a steam distillation method, for example, pine is placed in a distillation pot, water is added and heated to generate vapor from the pine components, and the vapor is passed through a cooling pipe or the like to condense the vapor and obtain pine essential oil (upper layer) and pine distillate (lower layer).

[0048] The manufacturing method of the present invention will be explained using a method using Sakhalin spruce (steam distillation) as an example. The branches and leaves of the Sakhalin spruce are cut with scissors and crushed, then a predetermined amount of water is added and placed in a known steam distillation apparatus. By heating for several hours, vapor is generated from the components of the Sakhalin spruce, and the vapor is condensed by passing it through a cooling pipe or the like to obtain essential oil (upper layer) and distillate (lower layer). Of these, the distillate can be separated by a known method to obtain Sakhalin spruce distillate, which is used as an anti-inflammatory agent or anti-inflammatory composition.

[0049] Furthermore, the present invention includes anti-inflammatory agents or anti-inflammatory compositions obtained by the above-described manufacturing method.

[0050] (4. Summary) As described above, the anti-inflammatory agent and anti-inflammatory composition according to the present invention contain as an active ingredient at least one distillate selected from the group consisting of pine distillate, yew distillate, and cedar distillate, which were conventionally discarded as by-products, and therefore can be produced at low cost. Since these distillates are plant-derived, unlike chemical substances, they are expected to have reduced side effects and cause less skin irritation. Furthermore, in light of the recent increase in interest in topical preparations and cosmetics using natural materials, they can be suitably used by users of topical preparations and cosmetics. [Examples]

[0051] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples.

[0052] (Example 1) The anti-inflammatory effects of the distillate of Picea jezoensis were investigated.

[0053] (sample) (1) Branches and leaves of Picea jezoensis Leaves from branches of Japanese red spruce trees planted in Ebetsu City, Hokkaido were collected, the branches and leaves were shredded with scissors, and then crushed using a crush mill (IFM-C20G, Iwatani).

[0054] (2) Purification of essential oils and distillates Using a steam distillation apparatus (Pure Stiller, Yellow River), 1.2 L of tap water was added to 600 g of Sakhalin fir and distilled for 2 hours, yielding approximately 2-3 mL of Sakhalin fir essential oil and approximately 400 mL of Sakhalin fir distillate.

[0055] (3) Preparation of essential oil inclusion complexes 0.2 g of Sakhalin fir essential oil was solubilized in 20 mL of a 2% hydroxyethyl-β-cyclodextrin (2OH-CD) (323-84242, Wako) solution to form a 1% essential oil inclusion complex. 2OH-CD is a cyclodextrin with numerous hydroxyl groups introduced into it, and forming an inclusion complex allows for the water-soluble conversion of essential oils.

[0056] (4) Vortex of distillate In the case of the Sakhalin fir distillate, since it was expected that the lipophilic aroma components would be dispersed, the undiluted sample bottle was shaken well immediately before dilution, and the mixture was also thoroughly stirred with a vortex mixer each time it was diluted with the culture medium.

[0057] (5) Molecular weight fraction of the distillate The distillate was fractionated by ultrafiltration (Amicon Ultra, Merck) to obtain three fractions with molecular weights of [less than 3,000 Da], [3,000 to 10,000 Da], and [greater than 10,000 Da].

[0058] (Measurement of anti-inflammatory activity in macrophage cells) To assess the effect of suppressing nitric oxide (NO) release by macrophage cells activated by lipopolysaccharide (LPS) (indicated as "CN" in Figures 3-7), the native oxide of NO, NO2 ions, was colorimetrically quantified by a diazo coupling reaction using Griess reagent (Figure 1) (Reiji Aoki. (2007). Simple evaluation of NO production using macrophages. The Japan Society for Food Science and Technology. Manuals for the Evaluation of Liquid Functions of Foods, Vol. II. 118-123). In addition, cellular metabolic activity (toxicity) was measured using the formazan reagent WST-1 (Figure 1).

[0059] (1) Cell subculture Mouse macrophage-like cell line RAW264 (RCB0535, RIKEN BRC) was cultured in a CO2 incubator (MCO-175, Sanyo) using a culture medium consisting of 10% inanimate fetal bovine serum (FBS) (10437-028, Thermo) and 1% penicillin-streptomycin (168-23191, Wako) in D-MEM medium (D-5790, Wako). When the growth rate in a 75 cm2 culture flask reached 80-90%, the cells were subcultured every 2-3 days.

[0060] (2) Preparation of reagents (2-1)1mg / mL LPS stock solution 25 mg of LPS (127-05141, Wako) derived from E. coli was dissolved in 25 mL of water, dispensed into 1 mL 1.5 mL tubes, and stored at -30°C. Immediately before use, it was diluted 1,000-fold with culture medium.

[0061] (2-2) 20 mmol / L quercetin solution 60.4 mg of quercetin (10005169, Cayman) was dissolved in 1 mL of dimethyl sulfoxide (DMSO), 9 mL of ethanol was added and mixed, then dispensed into 2 mL tubes in 1 mL portions and stored at -30°C. Immediately before use, the solution was diluted 100-fold with culture medium (in Figures 3-7, "PC: Positive Control").

[0062] (2-3) 0.25 mmol / L NO2 ion standard stock solution 0.862 g of NaNO2 (199-02565, Wako) was dissolved in distilled water to a volume of 50 mL, placed in a light-shielding bottle, and stored at 4°C. Before use, serial dilution was performed using distilled water to create a standard solution dilution series ranging from 25 to 100 μmol / L.

[0063] (2-4) Griess reagent 0.1% naphthylethylenediamine dihydrochloride (NED) solution: 200 mg of NED (147-00763, Wako) was dissolved in 200 mL of water. 1% sulfanilamide (SUL) (191-04502, Wako) solution: 2 g of SUL was dissolved in 40 mL of 21.25% H3PO4 (a 4-fold dilution of the 85% stock solution), and the total volume was diluted to 200 mL with distilled water. Both NED and SUL solutions were stored at 4°C, and Griess reagent was prepared by mixing equal volumes just before use.

[0064] (3) Measurement of anti-inflammatory (NO production suppression) effect (Figure 1) 10 μL of 1 μg / mL LPS was dispensed into all wells of a 96-well cell culture plate (92096, TPP) using a multichannel micropipette. Then, 50 μL each of 200 μmol / L quercetin or a 10- to 40-fold dilution (2.5-10%) of the sample (Sakhalin fir distillate or Sakhalin fir essential oil) in culture medium was dispensed as a positive control (n=6). Next, 2 × 10⁻⁶6 50 μL of cell suspension, adjusted to cells / mL (using a cell counter, BMS), was dispensed into each well and incubated at 37°C for 24 hours using a CO2 incubator. 80 μL of the culture supernatant was transferred to another flat-bottomed 96-well plate (655101, Greiner). Additionally, 80 μL of a diluted series of NO2 ion standard solutions was dispensed into the unused wells of the same plate. 80 μL of Griess reagent was dispensed into all wells and left at room temperature for 20 minutes. Absorbance at 550 nm was measured using a microplate reader (Emax, Molecular Devices), and the amount of NO produced by the standard solution was determined from the calibration curve. The relative NO production rate of each sample well compared to the control (LPS added only) was calculated to evaluate the NO production inhibitory effect.

[0065] (4) Measurement of cellular metabolic activity (toxicity) (Figure 1) To evaluate the cytotoxicity of the samples, the metabolic activity of cells was measured using a formazan reagent. (4-1)5.5mmol / L WST-1 solution 100 mg of WST-1 (346-06454, Dojindo) was dissolved in 27.9 mL of phosphate-buffered saline (PBS) solution (166-23555, Wako), and then dispensed into 15 mL centrifuge tubes in 0.9 mL portions and stored at -30°C. (4-2)2mmol / L 1-Methoxy PMS solution 1-Methoxy PMS (345-04001, Dojindo) 67.2 mg was dissolved in 100 mL of water and stored at 4°C. (4-3) WST-1 Reagent Immediately before use, 0.9 mL of 5.5 mol / L WST-1 solution was mixed with 0.1 mL of 1-Methoxy PMS solution and 10 mL of PBS. (4-4) WST-1 assay After removing the culture supernatant, 100 μL of WST-1 reagent was added to the cells in the wells, the wells were covered, and the incubator was left standing on an aluminum block at 37°C for 1 hour. Using a microplate reader, the absorbance at 450 / 650 nm was measured, and the cellular metabolic activity of each sample was determined for both the control group and the LPS-added sample.

[0066] (Measurement of aroma components by gas chromatography) The hexane-diluted essential oils were measured using flame ionization detection gas chromatography (FID-GC, main unit GC353B, autosampler ASI240) under the following conditions with an Inert Cap WAX-HT separation column (0.25 mm × 30 m × 0.5 μm, GL Sciences). For the aroma component standards, the peak height ratio with the internal standard ethyl octanoate (all from Tokyo Chemical Industry Co., Ltd.) was calculated to quantify the aroma component concentration in each sample.

[0067] [Table 1]

[0068] (result) (GC measurement results) Figures 2(a) and 2(b) show the results of aroma component measurements of the Sakhalin fir essential oil and Sakhalin fir distillate obtained as described above. In Figure 2, the horizontal axis of the graph shows the lot number of the Sakhalin fir essential oil and Sakhalin fir distillate. The aroma component of the Sakhalin fir essential oil was approximately 13 mg / mL, with bornyl acetate accounting for about half, followed by α-pinene, camphene, limonene, and camphor in that order (Figure 2(a)). On the other hand, the Sakhalin fir distillate also contained aroma components in small amounts, approximately 0.05 mg / mL, with camphor (approximately 100 μg / mL) accounting for the majority, and a small amount of bornyl acetate (approximately 20 μg / mL) also present (Figure 2(b)). This indicates that the distillate of Sakhalin fir also contains lipophilic aromatic components (camphor and bornyl acetate), albeit in small amounts of about 0.05 mg / mL, which is about 1 / 200th the amount found in Sakhalin fir essential oil.

[0069] (Anti-inflammatory (NO production suppression) effect) (1) Essential oil inclusion complex and distillate Figures 3(a) and 3(b) show the results of measuring the anti-inflammatory effects and cellular metabolic activity of the Sakhalin fir essential oil inclusion complex (2OH-CD inclusion complex) and Sakhalin fir distillate. The 2OH-CD inclusion complex was prepared as an aqueous solution using cyclodextrin. Both the Sakhalin fir essential oil 2OH-CD inclusion complex (comparative example) and the Sakhalin fir distillate (example) showed concentration-dependent anti-inflammatory effects, but the Sakhalin fir distillate showed a stronger anti-inflammatory effect than the Sakhalin fir essential oil (Figure 3(a)). No cytotoxicity was observed in either case (Figure 3(b)). Therefore, it was demonstrated that Sakhalin fir distillate possesses anti-inflammatory properties.

[0070] (2) Camphor and bornyl acetate standards Figures 4(a) and 4(b) show the results of measuring the anti-inflammatory effect and cellular metabolic activity of spruce distillate to which camphor and bornyl acetate, which were also present in the original spruce distillate, were added. Standard samples of camphor and bornyl acetate, corresponding to their respective concentration levels, were added to the spruce distillate and measured, but no clear anti-inflammatory effect was observed (Figure 4(a)). In the case of spruce essential oil, bornyl acetate accounts for approximately half of the aroma components, and camphor is a trace component. However, in spruce distillate, camphor is more water-soluble and therefore considered to be the main component. Therefore, it was expected that these essential oil aroma components were involved in the anti-inflammatory effect of spruce distillate. Standard solutions of camphor and bornyl acetate were dispersed in water or spruce distillate and measured, but the expected anti-inflammatory effect was not observed. This suggests that components other than camphor and bornyl acetate are involved in the anti-inflammatory effect of spruce distillate.

[0071] (3) Molecular weight fraction of the distillate Figures 5(a) and 5(b) show the results of measuring the anti-inflammatory effect and cellular metabolic activity of the molecular weight fraction of the Sakhalin fir distillate. Amicon Ultra was used for molecular weight fractionation of the Sakhalin fir distillate. In the comparison of the molecular weight fractions of the Sakhalin fir distillate, the strongest anti-inflammatory effect was observed in the fraction with a molecular weight of 3,000 Da or less (Figure 5(a)). This suggests the presence of a water-soluble, low-molecular-weight anti-inflammatory substance with surfactant properties in the Sakhalin fir distillate, distinct from the essential oil components.

[0072] (Example 2) The anti-inflammatory effects of various pine distillates were investigated.

[0073] Similar to Example 1, distillates of Sakhalin fir, Abies sachalinensis, and Japanese larch were prepared, and their anti-inflammatory (NO production inhibition) activity and cellular metabolic activity (toxicity) were measured in the same manner (Figure 1). For Abies sachalinensis and Japanese larch, branches and leaves of Abies sachalinensis and Japanese larch planted in Ebetsu City, Hokkaido were collected, and distillates of Abies sachalinensis and Japanese larch were prepared in the same manner as in Example 1.

[0074] The results are shown in Figure 6. Each pine distillate showed a concentration-dependent anti-inflammatory effect (Figure 6(a)). The distillate of Sakhalin spruce showed the highest anti-inflammatory effect. Furthermore, no cytotoxicity was observed with any of the pine distillates (Figure 6(b)). This indicates that the distillates of Sakhalin spruce, Abies sachalinensis, and Larch possess anti-inflammatory properties.

[0075] (Example 3) The anti-inflammatory effects of distillates from Japanese stone pine, Japanese yew, and Japanese cedar were investigated.

[0076] In the same manner as in Example 1, distillates of Japanese stone pine, Japanese yew, and Japanese cedar were prepared, and their anti-inflammatory (NO production inhibition) activity and cellular metabolic activity (toxicity) were measured in the same manner (Figure 1). For Japanese stone pine, Japanese yew, and Japanese cedar, branches and leaves of Japanese stone pine, Japanese yew, and Japanese cedar planted in Ebetsu City, Hokkaido were collected, and distillates of Japanese stone pine, Japanese yew, and Japanese cedar were prepared in the same manner as in Example 1.

[0077] The results are shown in Figure 7. Each distillate showed a concentration-dependent anti-inflammatory effect (Figure 7(a)). Similar to the Sakhalin fir, Abies sachalinensis, and Japanese larch distillates that showed anti-inflammatory effects in Examples 1 and 2, the Japanese stone pine distillate also showed a high anti-inflammatory effect. Furthermore, similar to the various pine distillates, the Japanese yew and Japanese cedar distillates also showed high anti-inflammatory effects. In addition, no cytotoxicity was observed in any of the distillates (Figure 7(b)). From this, it was shown that the distillates of Japanese stone pine, Japanese yew, and Japanese cedar have anti-inflammatory effects.

[0078] Based on the above, the strong anti-inflammatory effects of the pine, yew, and cedar distillates in this example were confirmed. Since no cytotoxicity was observed with each of the distillates in this example, it was demonstrated that effective anti-inflammatory agents and compositions with minimal skin irritation can be obtained. Applications in topical skin preparations for medical use, quasi-drugs, cosmetic compositions, etc., are expected.

Claims

1. NO production inhibitor containing Japanese red spruce distillate as the active ingredient.

2. For anti-inflammatory purposes, The NO production inhibitor according to feature 1.

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