JP2012171949A - Expression enhancer of melatonin receptor and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an expression enhancer of melatonin receptor capable of sufficiently enhancing the melatonin receptor expression.SOLUTION: The expression enhancer of melatonin receptor includes an extract of at least one kind of plants selected from the group consisting of clove, eucalyptus, thyme and lavender as an active component.

Description

  The present invention relates to a melatonin receptor expression enhancer and a method for producing the same.

  Melatonin, which is a hormone secreted from the pineal gland of the brain at night, is known to adjust the internal rhythm by acting on the supraoptic nucleus and induce natural sleep (Non-patent Document 1). Recent studies have revealed that melatonin has not only an effect of adjusting the internal rhythm but also an effect of reducing oxidative stress, which is a cause of aging and disease (Non-patent Document 2). ing. Furthermore, it has been clarified that melatonin also has a physiological action that promotes recovery of the barrier function of the skin (Patent Document 1).

  On the other hand, expression of type I melatonin receptor has been recognized in a wide range of tissues including dermal fibroblasts and epidermal keratinocytes (Non-patent Documents 3 to 6).

  Regarding the physiological action of melatonin, the involvement of melatonin receptors has also been suggested. Melatonin is understood as an important physiologically active substance that contributes to the maintenance of skin function through acting on the melatonin receptor to promote the restoration of barrier function as well as the protective effect against oxidative stress in skin tissue .

  However, it is known that melatonin in blood decreases with aging (Non-patent Document 7). Therefore, it is considered extremely difficult to continuously receive antioxidant stress due to melatonin or maintenance of skin function in the skin tissue. In order to overcome this problem, cosmetics and external preparations containing melatonin have been studied for the purpose of maintaining the function of the skin by supplying melatonin from the outside (for example, Patent Documents 2 to 4).

JP 2004-175687 A Japanese Patent Laid-Open No. 61-221104 JP 2002-326922 A JP 2004-107261 A

J Clin Sleep Med. 3, S17-23 (2007) Toxicology. 278, 55-67 (2010) (abstract) Cell Mol Life Sci. 59, 1706-13 (2002) Prog Neurobiol. 85, 335-53 (2008) Biomed Pharmacother. 60, 97-108 (2006) Exp Dermatol. 17, 713-30 (2008) J Pineal Res. 3, 379-88 (1986) (abstract)

  However, in the method of maintaining the skin function by applying melatonin itself to the skin, the function of the skin is significantly increased in concentration over the blood level (10 pM to 1 nM) normally maintained by humans. The effect to protect is only recognized (patent document 1). From the viewpoint of the composition of general external preparations, this is an extremely high concentration. When melatonin is used as an internal preparation, it is known that there are some acute side effects such as sleep disturbance and abdominal pain due to excessive intake. There are concerns that undesired side effects may be caused. In addition, since an antioxidant such as melatonin has high reactivity, it is susceptible to chemical changes such as oxidation and decomposition during storage. Therefore, it is difficult to maintain an effective amount of melatonin for radical scavenging in vivo.

  By the way, it is considered that when the expression of melatonin receptor is enhanced, the sensitivity to melatonin under physiological conditions in the skin is sensitized. Therefore, it is sufficiently expected that melatonin's physiological actions such as protection against oxidative stress and restoration of epidermal barrier function can be enhanced without administering melatonin itself.

  Then, an object of this invention is to provide the melatonin receptor expression enhancer which can fully enhance the expression of a melatonin receptor.

  As a result of diligent search and evaluation of plants in nature, the present inventor has found that a crustaceae plant (Futomoceae family plant) and a eucalyptus plant (Futomoceae family eucalyptus plant), and a prickly plant (Lamiaceae) The present inventors have found that extracts of Lamiaceae plants such as plants) and lavenders (Lamidaceae Lavandula plants) have an action of enhancing the expression of melatonin receptors.

  That is, the present invention provides a melatonin receptor expression enhancer containing, as an active ingredient, an extract of at least one plant selected from the group consisting of clove, eucalyptus, ginger and lavender.

  According to the present invention, it is possible to sufficiently enhance the expression of melatonin receptors on the cell membrane by using the extract of the specific plant.

  In another aspect, the present invention relates to a melatonin receptor expression enhancer containing an extract, comprising the step of extracting the extract from at least one plant selected from the group consisting of clove, eucalyptus, periwinkle and lavender. It relates to the manufacturing method.

  The melatonin receptor expression enhancer obtained by the production method according to the present invention can sufficiently enhance the expression of melatonin receptor on the cell membrane.

  In the manufacturing method which concerns on this invention, it is preferable to extract an extract from the said plant at 10-30 degreeC.

  The extraction solvent is preferably selected from alcohol, water and a mixed solvent thereof. The plant is preferably clove or eucalyptus.

  According to the present invention, the expression of melatonin receptor can be sufficiently enhanced. By enhancing the expression of the melatonin receptor, the physiological action of melatonin can be enhanced without necessarily requiring the administration of melatonin itself. Skin is roughly divided into epidermis and dermis because of its structure. Since the decrease in the ability of fibroblasts responsible for maintaining the function of the dermis to protect against oxidative stress leads to a decrease in skin elasticity and the formation of wrinkles, it is considered that oxidative stress protection by melatonin is important in the dermal fibroblasts. From such a point of view, a natural product material that enhances the expression of type I melatonin receptor was searched using fibroblasts.

It is a graph which shows the influence with respect to the expression level of a type I melatonin receptor of the plant extract extracted with the water-containing ethanol evaluated using the normal human skin fibroblast. It is a graph which shows the influence with respect to the expression level of a type I melatonin receptor of the plant extract extracted with the hot water evaluated using the normal human skin fibroblast. It is a graph which shows the influence with respect to the expression level of a type I melatonin receptor of the clove extract extracted with the water-containing ethanol evaluated using the normal human epidermal keratinocytes.

  Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

  The melatonin receptor expression enhancer according to the present embodiment contains, as an active ingredient, an extract of at least one plant selected from the group consisting of clove, eucalyptus, periwinkle and lavender, and these extracts themselves are melatonin as they are. It can also be used as a receptor expression enhancer. The active ingredients of the melatonin expression enhancer according to this embodiment are preferably clove and eucalyptus, more preferably clove.

  The extract may be extracted from any part of the plant body. In the case of clove, the above-ground part is preferable, and the flower bud part is more preferable. In the case of eucalyptus, the above-ground part is preferable, and the foliage part is more preferable. In the case of Tachy musk, the above-ground part is preferable, and the foliage part is more preferable. In the case of lavender, the above-ground part is preferable, and the flower bud part is more preferable. The plant may be used as it is after collection, or may be used after drying treatment such as sun drying and shade drying. Preferably, those powdered after drying are used.

  The plant to be extracted may be in any form, but is preferably a cut piece or a pulverized powder from the viewpoint of improving the extraction efficiency. The method and means for solvent extraction are not limited. The plant extract may be the extract itself obtained by solvent extraction, or the concentrate or dilution obtained by concentrating or diluting the extract or distilling off the solvent in the extract. It may be a dry product or a granule.

  The method for preparing the extract is not particularly limited. For example, the extract is extracted by immersing the plant in various appropriate extraction solvents at a low temperature, room temperature, or warming.

  The extraction solvent is not particularly limited. The extraction solvent may be an organic solvent or water. Examples of the organic solvent include lower alcohols such as methanol, ethanol, n-propanol, isopropanol, and t-butanol, liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol, ketones such as acetone, and acetic acid. An organic solvent such as an ester such as ethyl ester can be used. These extraction solvents are used singly or in combination of two or more. In the present embodiment, alcohol, water and a mixed solvent thereof are preferable. The alcohol is preferably ethanol and / or methanol. A mixed solvent of water and ethanol (hydrous ethanol) is particularly preferable. By using hydrous ethanol as the extraction solvent, the solvent removal operation after extraction tends to be easy. The ethanol concentration in the hydrous ethanol is preferably 0 to 100% by volume, more preferably 90% by volume.

  The amount of the extraction solvent is not particularly limited, but is, for example, about 20 mL per 1 g of dried plant.

  When the extraction solvent contains an alcohol, the extraction temperature is preferably 10 to 30 ° C, more preferably 20 to 25 ° C. The extraction time is not necessarily limited because it varies depending on the part of the plant, whether or not it is dried, and the type and amount of the extraction solvent, but is preferably 1 to 5 days, more preferably 2 to 4 days.

  When the extraction solvent is water, the extraction temperature is preferably 50 to 100 ° C, more preferably 60 to 90 ° C. The extraction time is preferably 1 hour to 5 hours, more preferably 2 hours to 4 hours.

  After the extract is filtered, the solvent in the extract is removed by an evaporator, and then the residue suspended in water is dried by a method such as freeze-drying to obtain an extract. The resulting extract is often in powder form.

  The melatonin receptor expression enhancer according to this embodiment can be used for both external and internal uses. Considering that the expression of the type I melatonin receptor is enhanced, for example, when it is intended to more effectively promote the recovery of the epidermal barrier function, the composition for external use with the skin is easy to contact and approach the skin or its target site More preferably, an enhancer is used. Examples of the composition for external use of skin include cosmetics such as face-wash cream, lotion and emulsion. The amount of the melatonin receptor expression enhancer is preferably 0.0001 to 10% by mass, more preferably 0.001 to 1% by mass, based on the total amount of the external composition for skin.

  The composition for external use of skin is a surfactant, fats and oils, polyhydric alcohol, lower alcohol, thickener, UV absorber, UV scattering agent, preservative, antioxidant, chelating agent, pH adjuster (potassium hydroxide, Citric acid and the like), fragrances, pigments, and other components such as water may be included.

  As the surfactant, polyoxyethylene (hereinafter abbreviated as POE-) octyldodecyl alcohol and POE-branched alkyl ethers such as POE-2-decyltetradecyl alcohol, POE such as POE-oleyl alcohol ether and POE-cetyl alcohol ether -POE-sorbitan such as alkyl ether, sorbitan monooleate, sorbitan esters such as sorbitan monoisostearate and sorbitan monolaurate, POE-sorbitan monooleate, POE-sorbitan monoisostearate and POE-sorbitan monolaurate Glycerol fatty acid esters such as esters, glyceryl monooleate, glyceryl monostearate and glyceryl monomyristate, POE-glyceryl monooleate, POE- POE-cured castor oil fatty acid ester such as POE-glycerin fatty acid ester such as lyseryl monostearate and POE-glyceryl monomyristate, POE-dihydrocholesterol ester, POE-hardened castor oil and POE-hardened castor oil isostearate, POE-alkyl aryl ethers such as POE-octylphenol ether, glycerol monostearate (such as self-emulsifying glyceryl monostearate and lipophilic glyceryl monostearate), glycerol esters such as glycerol monoisostearate and glycerol monomyristate, POE-glycerol ethers such as POE-glycerol monoisostearate and POE-glycerol monomyristate, diglyceryl monostearate, decaglyce Polyglycerin fatty acid esters such as rudecastearate, decaglyceryl decaisostearate and diglyceryl diisostearate, and nonionic surfactants such as sucrose fatty acid ester, myristic acid, stearic acid, palmitic acid, behenic acid , Salts of higher fatty acids such as isostearic acid and oleic acid with potassium, sodium, diethanolamine, triethanolamine and amino acids, the above alkali salts of ether carboxylic acids, salts of N-acyl amino acids, N-acyl sarconates, and Anionic surfactants such as higher alkyl sulfonates, cationic surfactants such as alkylamine salts, polyamines, amino alcohol fatty acid organic silicone oils and alkyl quaternary ammonium salts, and lecithin, betaine derivatives (coconut oil fats) Amphoteric surfactants such as acid amidopropyl betaine).

  Fats and oils include castor oil, olive oil, cacao oil, coconut oil, palm oil, tree wax, jojoba oil, grape seed oil, avocado oil and other animal oils such as mink oil and egg yolk oil, beeswax, whale Waxes such as wax, lanolin, carnauba wax and candelilla wax, hydrocarbons such as liquid paraffin, squalene, microcrystalline wax, ceresin wax, paraffin wax and petrolatum, lauric acid, myristic acid, stearic acid, oleic acid, isostearic acid And natural and synthetic fatty acids such as behenic acid, natural and higher alcohols such as cetanol, stearyl alcohol, hexyl decanol, octyl decanol, oleyl alcohol and lauryl alcohol, and isopropyl myristate and isopropyl palmitate Octyldodecyl myristate, octyldodecyl oleate, esters such as myristyl lactate and cholesterol oleate.

  Examples of the polyhydric alcohol include ethylene glycol, polyethylene glycol, propylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, dipropylene glycol, glycerin, diglycerin, triglycerin and tetraglycerin, and other polyglycerin, and Examples thereof include sugar alcohols such as glucose, maltose, maltose, sucrose, fructose, xylitolose, sorbitol (sorbitol), maltotriose, threitol, and erythritol.

  Thickeners include sodium alginate, xanthan gum, aluminum oxalate, quince seed extract, tragacanth gum, starch, collagen and sodium hyaluronate and other natural polymeric substances, methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, soluble starch and cationized Examples include semi-synthetic polymer materials such as cellulose, and synthetic polymer materials such as carboxyvinyl polymer and polyvinyl alcohol.

  Examples of ultraviolet absorbers include paraaminobenzoic acid, isopropyl paramethoxycinnamate, butylmethoxybenzoylmethane, glyceryl-mono-2-ethylhexanoyl-di-paramethoxybenzophenone, digalloyl trioleate, 2-2'-dihydroxy- 4-methoxybenzophenone, ethyl-4-bishydroxypropylaminobenzoate, 2-ethylhexyl-2-cyano-3,3′-diphenyl acrylate, ethyl hexyl paramethoxycinnamate, 2-ethylhexyl salicylate, glyceryl paraaminobenzoate, homomethyl salicylate , Methyl orthoaminobenzoate, 2-hydroxy-4-methoxybenzophenone, amyl-para-dimethylaminobenzoate, 2-phenylbenzimidazole-5-sulfonic acid Beauty hydroxy-4-methoxybenzophenone-5-sulfonic acid and the like.

  Examples of preservatives include benzoate, salicylate, sorbate, dehydroacetate, p-hydroxybenzoate, 2,4,4′-trichloro-2′-hydroxydiphenyl ether, 3,4,4′-trichlorocarbani Examples include lido, benzalkonium chloride, hinokitiol, resorcin, and ethanol.

  Examples of the antioxidant include tocopherol, ascorbic acid, butylhydroxyanisole, dibutylhydroxytoluene, nordihydroguaiaretic acid, propyl gallate, and the like.

  Examples of chelating agents include sodium edetate and sodium citrate.

  Among these additive components, by enhancing the stability or transdermal absorbability of the melatonin receptor expression enhancer according to the present embodiment, the effect of further improving the effectiveness of the external composition for skin according to the present embodiment. Some have The dosage form of the external composition for skin is arbitrary, and any of a soluble system, an emulsion system, a powder dispersion system, and the like may be used. The composition for external use of skin can be widely used for basic cosmetics such as lotion, milky lotion, cream and pack, as well as makeup cab cosmetics such as foundation.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Production Example 1: Preparation of clove extract (hydrous ethanol extraction)
Extraction was carried out by adding 100 mL of water-containing ethanol containing 90% by volume of ethanol to dried and shredded flower buds of clove (5 g) and allowing to stand at 25 ° C. for 3 days. Thereafter, the extraction solvent (hydrous ethanol) was removed from the extract collected by filtration with an evaporator. The residue was once suspended in water, water was further removed by freeze-drying, and a clove extract extracted with hydrous ethanol was obtained.
(Hot water extraction)
100 mL of water was added to the dried chopped flower buds (5 g), and extraction was performed for 3 hours while heating to 85 ° C. Thereafter, water was removed from the extract collected by filtration by freeze-drying to obtain a clove extract extracted with hot water.

Production Example 2: Preparation of Eucalyptus Extract Eucalyptus extract extracted with water-containing ethanol or hot water was obtained in the same manner as in Production Example 1 from dried and chopped eucalyptus leaves (5 g).

Manufacture example 3: Preparation of an extract of periwinkle from a dried and minced leaf of periwinkle (5 g) and extracted with water-containing ethanol in the same manner as the water-containing ethanol extraction of production example 1 Got.

Production Example 4: Preparation of Lavender Extract A lavender extract extracted with hydrous ethanol was obtained from dried chopped lavender flowers (5 g) in the same manner as the hydrous ethanol extraction of Production Example 1. .

Test Example 1: Evaluation of expression level of type I melatonin receptor using human normal skin fibroblasts A substance having an action of enhancing expression of type I melatonin receptor (type I melatonin receptor expression enhancing action) It has been reported to increase the amount of type I melatonin receptors in cells. Therefore, by comparing the expression level of the type I melatonin receptor on the cells with the control group according to the Western blotting method widely used as a method for comparing the expression level at the protein level, the type I melatonin receptor The effect of enhancing the expression of was confirmed.

Normal human adult skin-derived fibroblasts (normal human skin fibroblasts: NHDF) were used in the test. Normal human skin fibroblasts were suspended in Dulbecco's modified Eagle medium (GIBCO) containing 10% by volume fetal bovine serum (GIBCO) and seeded in a 100 mm culture dish. Cultivation was carried out at 37 ° C. in a CO ₂ incubator (95 vol% air / 5 vol% carbon dioxide).

After culturing until the cells became confluent, the medium was removed from the petri dish and replaced with a sample-added medium containing the clove extract extracted with hydrous ethanol obtained in Production Example 1. That is, the medium cultured as described above was removed from the petri dish, Dulbecco's modified Eagle medium containing 1% by volume fetal calf serum added so that the clove extract had a concentration of 20 μg / mL was placed in the petri dish and cultured. . The culture was performed in a CO ₂ incubator (95% by volume air, 5% by volume carbon dioxide) at 37 ° C. for 24 hours.

The cells cultured for 24 hours in this manner were washed with PBS (phosphate buffered saline) and collected with a scraper. After centrifugation at 1000 rpm for 5 minutes with a centrifuge, the supernatant was removed. RIPA Lysis Buffer (registered trademark; manufactured by Millipore), final concentration of 1 mM NaF, final concentration of 5 mM Na ₃ VO ₄ , final concentration of 10 μM MG132 reagent (Calbiochem), and protease inhibitor (trade name: Complete protease inhibitor) (Registered trademark; manufactured by Roche) was added to a concentration of 1 × according to the attached instruction to prepare a cell lysate. The cells were suspended in 50 μL of this cell lysate, and the cells were lysed by pipetting. Thereafter, the cells were sufficiently lysed by sonication, and the amount of protein in the sample in which the cells were lysed was quantified by Pierce BCA Protein Assay Kit (registered trademark: Thermo Scientific). Based on the obtained value, a sample was prepared so as to have a concentration of 30 μg / 10 μL, and SDS (sodium dodecyl sulfate) sample buffer (50 mM Tris-HCl (pH 6.8), 2 volumes) % SDS, 6 volume% β-mercaptoethanol, 10 volume% glycerol, and appropriate amount of bromophenol blue) were added and boiled at 100 ° C. for 5 minutes. The sample (protein sample) thus obtained was subjected to Western blotting.

  Next, a gel for electrophoresis was prepared according to the following procedure. First, 30 mass% acrylamide solution (6 mL), 1.5 M Tris-HCl (pH 8.8) solution (3.8 mL), milli-Q water (5 mL), 10 mass% SDS (150 μL), APS (ammonium persulfate, Ammonium persulfate (125 μL), TEMED (tetramethylethylenediamine, tetramethylethylenediamine) (12 μL) were added to a 50 ml centrifuge tube, stirred well, poured into a 100 mm × 80 mm glass plate for gel preparation, and subjected to a polymerization reaction. A separation gel was formed. In the formed separation gel, the concentration of acrylamide is 12%. Subsequently, 30% by mass acrylamide solution (0.75 mL), 0.5 M Tris-HCl (pH 6.8) solution (1.9 mL), milli-Q water (4.8 mL), 10% by mass SDS (75 μL), APS (62.5 μL) and TEMED (6 μL) were added to each 50 mL centrifuge tube and stirred well, then overlaid on the separation gel, combed, and allowed to stand at room temperature for 20 minutes to polymerize the concentration gel. Reacted. Thus, a gel for electrophoresis to be finally used was obtained.

  The prepared gel was attached to an electrophoresis apparatus (manufactured by Nippon Aido), and a protein sample of cells cultured in a medium containing a clove extract prepared by the above method was added to one lane so that the amount of protein was 30 μg. Electrophoresis was performed for approximately 1 and a half hours. Thereafter, the protein separated in the gel was transferred to a polyvinylidene fluoride membrane (PVDF membrane) (Millipore) using iBLOT (registered trademark; manufactured by Invitrogen). The protein derived from horse serum is adsorbed to the PVDF membrane by immersing the PVDF membrane, on which the protein has been transferred, in PBST (PBS + 0.05% by volume Tween20) solution containing 10% by volume horse serum (GIBCO) at room temperature for 2 hours. And blocking was performed.

  Next, a goat-derived antibody against type I melatonin receptor (Abcam, concentration 0.5 mg / ml, catalog number ab87639) was diluted 1000-fold with a PBS solution containing 5% by volume of horse serum to give a primary antibody reaction. A liquid was prepared. The PVDF membrane was immersed in this, and the primary antibody was reacted at 4 ° C. for 3 days. Thereafter, the PVDF membrane was washed with a PBST solution for 5 minutes three times. An anti-goat immunoglobulin antibody conjugated with HRP (horse radish peroxidase) (Vector Laboratories, concentration 1 mg / ml, catalog number PI-9500) was diluted 50000 times with a PBS solution containing 5% by volume of horse serum. A secondary antibody reaction solution was prepared. This was allowed to react with the secondary antibody by immersing the PVDF membrane at room temperature for 2 hours. Next, the PVDF membrane was washed with PBST solution for 5 minutes four times, and then reacted with a luminescent substrate using an ECL Plus Western Blotting Detection System (registered trademark; manufactured by GE Healthcare). Thereafter, the luminescence intensity reflecting the amount of type I melatonin receptor was detected using LAS4000mini (trade name, manufactured by Fujifilm).

  Hydrous ethanol extracts of plants other than clove (Production Example 1), that is, eucalyptus extract (Production Example 2), ginger extract (Production Example 3) and lavender extract (Production Example 4) are also Similarly, Dulbecco's modified Eagle medium prepared to contain an extract having a final concentration of 20 μg / ml in the amount of dry solids was prepared. Cells were cultured using this medium. After culturing for 24 hours, a protein sample was prepared and used for evaluation together with a protein sample prepared from cells cultured in a medium containing a clove extract.

  On the other hand, a protein sample was prepared in the same manner for cells cultured in a medium containing 1% by volume fetal calf serum to which these plant extracts were not added, and the cells were cultured in a medium containing the plant extract as a control sample. It was used for evaluation together with the protein sample prepared from 1.

  In order to correct the expression of the above-mentioned type I melatonin receptor, the amount of expression of glyceraldehyde 3-phosphate dehydrogenase, which is an internal standard protein, was also monitored. That is, instead of the type I melatonin receptor, a rabbit-derived antibody against glyceraldehyde 3-phosphate dehydrogenase (manufactured by Cell Signaling, catalog number 2118) was diluted 2000 times with a PBS solution containing 5% by volume of horse serum. The PVDF membrane to which the protein sample was transferred by the same method as above was immersed in the primary antibody reaction solution, and the primary antibody reaction was allowed to proceed at room temperature for 1 hour. Thereafter, through the same washing process as described above, HRP-conjugated anti-rabbit immunoglobulin antibody (CELL SIGNALING, Catalog No. 7074) was diluted 2000 times with a PBS solution containing 5% by volume of horse serum to PVDF. The secondary antibody reaction was performed by immersing the membrane at room temperature for 2 hours. Thereafter, a luminescence intensity that reflects the amount of glyceraldehyde 3-phosphate dehydrogenase was detected through a washing process.

  The amount of expression of the type I melatonin receptor in each culture condition was determined using the Scion Image software (trade name, manufactured by Scion), and the luminescence intensity of a band near 39 kDa corresponding to the type I melatonin receptor was determined using the internal standard protein. It was quantified by a method of dividing by the luminescence intensity of a band corresponding to a certain glyceraldehyde triphosphate dehydrogenase. Table 1 shows the ratio (%) of these values when the luminescence intensity is 100% in a protein sample (control sample) obtained from cells cultured without adding a plant extract. Moreover, the increase amount of expression (expression increase amount) represented by the value when the control sample is 1 is shown in FIG.

  As is clear from Table 1 and FIG. 1, the expression level of the type I melatonin receptor in NHDF by the clove extract, eucalyptus extract, perilla extract, or lavender extract extracted with hydrous ethanol is the control sample. As compared with the above, an effect of enhancing the expression of type I melatonin receptor was observed.

  With respect to the clove extract and eucalyptus extract extracted with hot water, the enhancing action on the expression of the type I melatonin receptor was evaluated by the same procedure as that of the water-containing ethanol extract. The results are shown in Table 2 and FIG.

  As is apparent from Table 2 and FIG. 2, the expression level of the type I melatonin receptor in NHDF by the clove extract or eucalyptus extract extracted with hot water is higher than that of the control sample. An effect of enhancing expression was observed. In the case of the clove extract, compared with the hot water extract, the water-containing ethanol extract showed a more remarkable effect of enhancing the expression of the type I melatonin receptor. On the other hand, in the case of the eucalyptus extract, compared with the water-containing ethanol extract, the hot water extract showed a more remarkable effect of enhancing the expression of type I receptor.

Test Example 2: Evaluation of expression level of type I melatonin receptor using normal human epidermal keratinocytes In the epidermis, keratinocytes are the main constituent cells, Like dermal fibroblasts, it leads to skin aging. In addition, since keratinocytes are considered to be mainly involved in the barrier function of the epidermis, it is important to protect against oxidative stress by melatonin and strengthen the barrier function in epidermal keratinocytes in order to maintain the barrier function. It is thought that. From such a viewpoint, the expression enhancing action of the type I melatonin receptor in the epidermal keratinocytes was evaluated using the clove extract which showed the melatonin receptor expression enhancing action most remarkably among the water-containing ethanol extracts.

  Evaluation of type I melatonin receptor expression enhancing effect on clove extract extracted with normal ethanol using normal human epidermal keratinocytes (NHEK) in the same manner as the test conducted with normal human skin fibroblasts Went. However, instead of Dulbecco's modified Eagle medium, a serum-free liquid medium for proliferation of normal human epidermal keratinocytes (Kurabo, trade name: Humedia-KG2) was used as the medium. The concentration of the clove extract was 5 μg / mL or 10 μg / mL instead of 20 μg / mL. In the medium, insulin necessary for the growth of epidermal keratinocytes, hEGF (human epidermal growth factor), hydrocortisone, and gentamicin and amphotericin B as antibacterial agents were added. The test results are shown in Table 3 and FIG.

  As is apparent from Table 3 and FIG. 3, the expression level of the type I melatonin receptor in NHEK by the clove extract increased depending on the concentration of the clove extract. According to the clove extract, it was confirmed that the epidermal keratinocytes can be expected to contribute to the protective effect against oxidative stress of the skin by melatonin or the promotion of the recovery of the barrier function.

Example 1: Manufacture of face-wash cream Using the type I melatonin receptor expression enhancer obtained in Production Example 1 (clove extract extracted with water-containing ethanol), a face-wash cream having the following formulation was prepared. Each component AC was prescribed | regulated in the following ratio so that the sum total of the component A (Table 4), the component B (Table 5), and the component C (Table 6) might be 100 mass%. First, the mixture of component A was heated and dissolved and maintained at 80 ° C. Subsequently, the mixture of component B separately melted by heating at 80 ° C. was added to the mixture of component A and sufficiently stirred. Then, it cooled, stirring, and added the mixture of the component C at 50 degreeC, and obtained the face-wash cream.

Example 2: Manufacture of lotion The components shown in Table 7 below were formulated so that the total amount was 100% by mass. All ingredients were mixed and stirred at room temperature to obtain a uniform solution, and the pH was adjusted to 5.5 to obtain a lotion.

Example 3: Manufacture of milky lotion Each component DF is prescribed in the following ratio so that the sum total of the component D (Table 8), the component E (Table 9), and the component F (Table 10) may be 100 mass%. did. First, the mixture of component D was heated and dissolved and kept at 80 ° C. Then, separately, the mixture of component E dissolved by heating at 80 ° C. was added to the mixture of component D and sufficiently stirred. Then, it cooled, stirring, and the mixture of the component F was added at 50 degreeC, and the emulsion was obtained.

Claims (7)

  A melatonin receptor expression enhancer comprising, as an active ingredient, an extract of at least one plant selected from the group consisting of clove, eucalyptus, ginger and lavender.   The melatonin receptor expression enhancer according to claim 1, wherein the extract is extracted from the plant with an extraction solvent selected from alcohol, water and a mixed solvent thereof.   The melatonin receptor expression enhancer according to claim 1 or 2, wherein the plant is clove or eucalyptus.   The manufacturing method of the melatonin receptor expression enhancer containing an extract which has the process of extracting an extract from the at least 1 sort (s) of plant selected from the group which consists of a clove, a eucalyptus, a ginger and a lavender.   The manufacturing method of Claim 4 which extracts the said extract from the said plant with the extraction solvent selected from alcohol, water, and these mixed solvents.   The manufacturing method according to claim 4 or 5, wherein the plant is clove or eucalyptus. The manufacturing method as described in any one of Claims 4-6 which extracts the said extract from the said plant at 10-30 degreeC.

Images