JP5608792B2 - Nikuzuku processed product and manufacturing method thereof - Google Patents

Description

  The present invention relates to a processed product of nutmeg, a method for producing the same, a cooling sensation agent and a TRPM8 activator using the processed product of nutmeg.

  Nikuzuku and processed products thereof (for example, nutmeg oil) are useful compounds that have been used for gastrointestinal drugs, headache drugs, spices and the like since ancient times.

On the other hand, for the purpose of imparting a refreshing sensation during and after use, various products such as cosmetics, hair care products, toiletry products, bathing agents, pharmaceuticals and the like are often blended with cooling sensation substances. Menthol is widely used as a sensitive substance.
It is thought that menthol imparts a cooling sensation by direct action of menthol on sensory nerve endings present in the skin and mucous membrane tissues, and studies have been conducted on mechanisms for imparting a sensation of cooling.

  First, it was discovered that among sensory neurons in rats, neurons that generate inward ionic currents in response to weak cooling stimuli exhibit similar responsiveness to menthol (Non-Patent Document 1). This discovery revealed that the sensation of menthol caused the cold feeling caused by an inward ionic current.

  As a receptor responsive to menthol and cold stimulation, CMR-1 (cold and menthol sensitive receptor) was identified from trigeminal neurons (Non-patent Document 2). This receptor is called TRPM8 (Non-Patent Document 3), and this receptor, which is an excitable ion channel belonging to the TRP ion channel family, is considered to cause the aforementioned ionic current.

  From these reports, it is clear that menthol is bound to TRPM8 present in the sensory nerve and an inward current is generated, thereby causing a cooling sensation due to menthol.

REID, G. & FLONTA, M.M. L. (2002), Neurosci. Lett. , 324, p164-168 MCKEMY, D.M. D. , NEUHAUSSER, W.M. M.M. & JULUS, D. (2002), Nature, 416, p52-56. PEIER, A.M. M.M. , MOQRICH, A.M. , HERGARDEN, A. C. , REEVE, A. J. et al. , ANDERSSON, D.M. A. , STORY, G .; M.M. , EARLEY, T. J. et al. DRAGONI, I. , MCINTYRE, P.M. , BEVAN, S. & PATAPOUTIAN, A. (2002), Cell, 108, p705-715. BEHRENDT, H.C. -J. , GERMANN, T .; , GILLEN, C.I. , HATT, H. & JOSTOCK, R. (2004), Br. J. et al. Pharmacol. 141, p737-745

However, when menthol is used in a large amount, the amount of use may be limited because of irritation to the skin and mucous membranes and peculiar unpleasant irritating odor.
In addition to menthol, linalool, geraniol, hydroxycitronellal and the like are known to activate TRPM8 (Non-Patent Document 4), but the refreshing feeling exhibited by the compounds described in Non-Patent Document 4 is not necessarily the same. It was not enough.

  The present invention relates to a processed plant product having an excellent TRPM8 activation action, a method for producing the same, a cooling sensation agent and a TRPM8 activator using the processed plant product.

  The present inventors treated the nutmeg or extract thereof with alcohol or an aqueous solution of alcohol of 25% (v / v) or more and one or more selected from protonic acid and H-type cation exchange resin at the same time, The present invention was completed by finding that a processed product having a superior TRPM8 activation action can be obtained.

That is, the present invention is a method for producing a processed product of nutmeg, wherein the nutmeg or extract thereof is selected from alcohol or an aqueous solution of alcohol of 25% (v / v) or more, a protonic acid and an H-type cation exchange resin. The present invention provides a production method comprising a step of simultaneously treating with one or more kinds.
The present invention also provides a processed product obtained by the above-described production method.
Furthermore, this invention provides the cooling sensation agent which uses the said processed product of a nutmoku as an active ingredient.
Furthermore, this invention provides the TRPM8 activator which uses the said processed product of a nutmeg as an active ingredient.
Furthermore, the present invention provides a cosmetic containing the above-described processed product.
Furthermore, this invention provides the composition for oral cavity containing the said nutmeg processed material.

According to the production method of the present invention, it is possible to produce a processed product having a superior TRPM8 activation action.
In addition, the processed product of the nutmeg of the present invention has an excellent TRPM8 activation action, and by using this, a good cooling feeling can be imparted.

It is a figure which shows the cool feeling of the mouthwash of Example 13.

  In the method for producing a processed product of nutmeg of the present invention, the nutmeg or extract thereof is made of alcohol or an aqueous alcohol solution of 25% (v / v) or more, and one or more selected from protonic acid and H-type cation exchange resin. It is characterized by including the process processed simultaneously (Hereinafter, the processed product obtained with the manufacturing method of this invention is also only called a processed product).

The nutmeg is Myristica fragrance Hout. Means. As the nutmeg, seeds, maces, leaves, fruits and the like are used, and seeds and maces are preferable from the viewpoint of activating TRPM8. In addition, you may use these parts individually by 1 type or in combination of 2 or more types.
The nutmeg can be used as it is or after being dried and then cut and pulverized to an appropriate size.

  In the present invention, the extraction of nutmeg is a method of impregnating the above-mentioned nutmeg into a solvent at room temperature or under heating, a solvent extraction method performed using an extraction device such as a Soxhlet extractor, and carbon dioxide gas etc. in a supercritical state A supercritical extraction method to be performed, a pressing method to obtain an extract by pressing, or the like can be used. Alternatively, extraction may be performed again using a solvent or the like from a residue obtained by steam distillation or microwave steam distillation.

  Moreover, although it may distill after extracting by the above methods, it is preferable to use a distillation residue from a viewpoint of TRPM8 activation effect | action in this case. The distillation method may be a known method such as molecular distillation, steam distillation, microwave distillation or the like. Moreover, such distillation operation can be performed under normal pressure or reduced pressure, and the distillation temperature is usually 30 to 150 ° C, preferably 60 to 130 ° C.

When an extraction solvent is used for the extraction, either a polar solvent or a nonpolar solvent may be used as the extraction solvent. For example, water; alcohols such as methanol, ethanol, propanol, butanol and cyclohexanol (for example, alcohols used in the treatment described later); polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; acetic acid Esters such as methyl and ethyl acetate; linear or cyclic ethers such as diethyl ether and tetrahydrofuran; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane and cyclohexane; aromatic hydrocarbons such as benzene and toluene In addition to halogenated hydrocarbons such as dichloromethane, chloroform and dichloroethane, supercritical carbon dioxide, fats and oils, waxes, oils and the like can be mentioned. You may use these individually by 1 type or in combination of 2 or more types.
Among these, a mixed solution of water and a polar solvent and a polar solvent are preferable, and a water / alcohol mixed solution and alcohols are more preferable. As will be described later, the extraction solvent may contain a protonic acid or the like.
Moreover, when using the liquid mixture of the said water and a polar solvent, the density | concentration of the polar solvent becomes like this. Preferably it is 25% (v / v) or more, More preferably, it is 35% (v / v) or more, More preferably, it is 50%. (V / v) or more, more preferably 70% (v / v) or more, more preferably 80% (v / v) or more, more preferably 90% (v / v) or more, preferably 99 .9% (v / v) or less. Specifically, the concentration of the polar solvent in the mixed solution is preferably 25 to 99.9% (v / v), more preferably 35 to 99.9% (v / v), and still more preferably. Is 50 to 99.9% (v / v), more preferably 70 to 99.9% (v / v), still more preferably 80 to 99.9% (v / v), Preferably it is 90 to 99.9% (v / v).

  The amount of the extraction solvent used is not particularly limited, but is usually 1 part by mass or more, preferably 3 parts by mass or more, more preferably 5 parts by mass or more, and usually 50 parts by mass with respect to 1 part by mass of the nutmeg. Part or less, preferably 30 parts by weight or less, more preferably 25 parts by weight or less, and still more preferably 15 parts by weight or less. Specifically, the use amount of the extraction solvent is preferably 1 to 50 parts by mass, more preferably 3 to 30 parts by mass, and further preferably 5 to 25 parts by mass with respect to 1 part by mass of the nutmeg. is there.

  The extraction temperature is usually 3 ° C. or higher, preferably 10 ° C. or higher, more preferably 20 ° C. or higher, and is usually 100 ° C. or lower, preferably 60 ° C. or lower, more preferably 45 ° C. or lower. Specifically, extraction temperature becomes like this. Preferably it is 3-100 degreeC, More preferably, it is 10-60 degreeC, More preferably, it is 20-45 degreeC.

The extraction time is usually 1 hour or longer, and usually 14 days or shorter, preferably 7 days or shorter, more preferably 5 days or shorter. Specifically, the extraction time is preferably 1 hour to 14 days, more preferably 1 hour to 7 days, and even more preferably 1 hour to 5 days.
Moreover, the pressure at the time of extraction is usually normal pressure, but can also be high pressure.

In addition, the method for producing a processed product of nutmeg of the present invention comprises a method of extracting a nutmeg or an extract thereof (hereinafter referred to as “nikuzuku or an extract thereof”), an alcohol or an aqueous alcohol solution of 25% (v / v) or more (hereinafter referred to as “nikuzuku”). 1 or more selected from proton acids and H-type cation exchange resins (hereinafter referred to as proton acids and H-type cation exchange resins) 1 or more of them are also treated with a protonic acid, etc.), but this means coexistence of a nutmeg or the like, an alcohol or the like with a protonic acid or the like, and the order of addition of each component does not matter. . Further, such processing may be performed as the above-mentioned extraction of nutmeg, or may be performed after extraction of nutmeg.
Specific examples of the treatment include (1) a method for performing the above extraction using an extraction solvent containing an alcohol or the like and a protonic acid, or (2) an extraction solvent containing an alcohol or the like but not containing a protonic acid or the like. And then extracting the extract with a protonic acid or the like, and (3) extracting the nutmeg extract obtained by the above extraction using a suitable extraction solvent with an alcohol or the like and a protonic acid or the like. The method of processing at the same time, for example, by coexisting with the.
In the above method (1), the obtained nutmeg extract may be distilled and the distillation residue may be used as a nutmeg processed product. In the method (3), the extract of the nutmeg obtained by the above extraction using an appropriate extraction solvent may be further distilled, and the distillation residue may be simultaneously treated with alcohol or the like and protonic acid or the like.

Alcohol or a 25% (v / v) or higher alcohol aqueous solution is used for the treatment included in the production method of the present invention. As alcohol used for such a process, monohydric alcohol is preferable. Moreover, the carbon number is preferably 1 to 10, more preferably 1 to 6, and further preferably 2 to 6 from the viewpoint of the TRPM8 activation action. For example, methanol, ethanol, propanol, butanol, cyclohexanol and the like can be mentioned, and these one kind may be used alone or in combination of two or more kinds.
The concentration of the aqueous alcohol solution is preferably 35% (v / v) or more, more preferably 50% (v / v) or more, and still more preferably 70% (v / v), from the viewpoint of TRPM8 activation. More preferably, it is 80% (v / v) or more, more preferably 90% (v / v) or more, and preferably 99.9% (v / v) or less. Specifically, the concentration of the aqueous alcohol solution is preferably 35 to 99.9% (v / v), more preferably 50 to 99.9% (v / v), and still more preferably 70 to 99. 0.9% (v / v), more preferably 80 to 99.9% (v / v), and still more preferably 90 to 99.9% (v / v). In addition, when using alcohol aqueous solution, if the density | concentration is less than 25% (v / v), the TRPM8 activation effect | action of a processed product will become inadequate.

  The amount of alcohol or the like used is not particularly limited, but is usually 0.1 parts by mass or more, preferably 1 part by mass or more, more preferably 2 parts by mass or more, and usually 1000 parts by mass with respect to 1 part by mass such as scratches. It is not more than part by mass, preferably not more than 500 parts by mass, more preferably not more than 200 parts by mass. Specifically, the use amount of alcohol or the like is preferably 0.1 to 1000 parts by mass, more preferably 1 to 500 parts by mass, and still more preferably 2 to 200 parts per 1 part by mass of scabs or the like. Part by mass.

  Further, at least one selected from protonic acid and H-type cation exchange resin is used for the treatment. Examples of the protonic acid used for such treatment include organic acids and inorganic acids. You may use these individually by 1 type or in combination of 2 or more types.

Examples of the organic acid include citric acid, acetic acid, p-toluenesulfonic acid, and the like.
Moreover, as said inorganic acid, a sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid etc. are mentioned, for example.

  Examples of the H-type cation exchange resin include Amberlist (Dow Chemical), DOWEX (Dow Chemical), DIAION (Mitsubishi Chemical), and the like. The treatment with the H-type cation exchange resin may be the addition of the H-type cation exchange resin or the liquid passing through a column filled with the H-type cation exchange resin.

  Among the above-mentioned protonic acids and H-type cation exchange resins, strong acidic protonic acids and strong acidic H-type cation exchange resins are preferable from the viewpoint of TRPM8 activation action.

  The amount of protonic acid and the like is not particularly limited, but is usually 0.001 parts by mass or more, preferably 0.01 parts by mass or more, and usually 50 parts by mass or less, relative to 1 part by mass such as scratches. Preferably it is 25 mass parts or less, More preferably, it is 10 mass parts or less, More preferably, it is 5 mass parts or less. Specifically, the use amount of protonic acid or the like is preferably 0.01 to 50 parts by mass, more preferably 0.01 to 10 parts by mass, and still more preferably, with respect to 1 part by mass such as nutmeg. 0.01 to 5 parts by mass.

  The treatment temperature is usually 3 ° C. or higher, preferably 15 ° C. or higher, more preferably 25 ° C. or higher, and is usually 100 ° C. or lower, preferably 70 ° C. or lower, more preferably 60 ° C. or lower. Specifically, processing temperature becomes like this. Preferably it is 3-100 degreeC, More preferably, it is 15-70 degreeC, More preferably, it is 25-60 degreeC.

  The treatment time is usually 0.1 hour or longer, preferably 1 hour or longer, more preferably 2 hours or longer, and is usually 5 days or shorter, preferably 3 days or shorter, more preferably 2 days or shorter. Specifically, the treatment time is preferably 0.1 hour to 5 days, more preferably 1 hour to 3 days, and further preferably 2 hours to 2 days.

In addition, the obtained processed product of the nutmeg can be diluted, concentrated, freeze-dried, etc., and then prepared and used as a powder or paste as necessary.
In addition, as a solvent used for the dilution of the said processed product, the alcohol used for the said process, water, and these liquid mixture are mentioned. Examples of the means for concentrating and purifying the above processed product include filtration, activated carbon treatment, and liquid-liquid distribution of the processed product.

And the nutmeg processed product of this invention has the outstanding TRPM8 activation effect | action, as shown in the postscript Example, although it is derived from a natural product.
Therefore, the processed processed product of the present invention can be used as it is, or together with various preparation carriers, preservatives and the like as a cooling sensation agent and a TRPM8 activator (hereinafter also referred to as a cooling sensation agent).

Next, aspects of the cooling sensation agent and TRPM8 activator of the present invention will be described.
The cooling sensation agent itself may be a medicine for humans or animals, a cosmetic, a food or drink, a composition for oral cavity, a pet food, other luxury goods (such as tobacco), etc. The material which provides the cooling sensation added to may be sufficient.

The medicament can be administered in any dosage form. The administration forms are roughly classified into parenteral administration such as transmucosal and transdermal and oral administration.
Moreover, the dosage form of the said pharmaceutical is not specifically limited. Examples of pharmaceutical dosage forms for parenteral administration include, for example, liquid preparations, gel preparations, cream preparations, ointments, poultices, aerosols, lotions, foundations, and other skin external preparations, eye drops, nasal drops, etc. Is mentioned.
On the other hand, pharmaceutical dosage forms for oral administration include, for example, tablets, capsules, granules, powders, powders, pills, dragees, liquids for internal use, suspensions, syrups and the like.
Moreover, the said pharmaceutical may contain other medicinal components, such as an anti-inflammatory analgesic, a bactericidal disinfectant, an astringent, and antibiotics. In addition, you may use these individually by 1 type or in combination of 2 or more types.

Moreover, the form of the said cosmetics, food-drinks, and composition for oral cavity is not specifically limited.
Cosmetics include, for example, skin external preparations (insect repellent spray, etc.), cleaning agents, lotions, emulsions, skin creams, foundations, lipsticks, scalp cosmetics, hair cosmetics (shampoos, hair tonics, etc.), bath preparations, sheets Product. In addition, the cosmetics are processed oils, ceramides, pseudoceramides, sterols, humectants, antioxidants, ultraviolet absorbers, whitening agents, alcohols, chelating agents, pH adjusters, An antiseptic etc. may be included. In addition, you may use these individually by 1 type or in combination of 2 or more types.
Examples of the food and drink include candy, gum, tablet, capsule, and drinking water.
Examples of oral compositions include dentifrices, mouthwashes, and gingival massage creams.

  In addition, the content of the processed product of nutmeg is not particularly limited, but in the case of a pharmaceutical product, food and drink, oral composition or pet food for oral administration, preferably 0.1 ppm from the viewpoint of TRPM8 activation action. More preferably, it is 1 ppm or more, more preferably 10 ppm or more, further preferably 50 ppm or more, preferably 10,000 ppm or less, more preferably 5000 ppm or less, still more preferably 2500 ppm or less, still more preferably 1000 ppm or less. Specifically, the content of the processed product in the above case is preferably 0.1 to 10,000 ppm, more preferably 1 to 10,000 ppm, and still more preferably, from the viewpoint of the TRPM8 activation action. It is 10-5000 ppm, More preferably, it is 50-2500 ppm.

  In the case of pharmaceuticals, cosmetics or other luxury products for parenteral administration, the content of the processed processed product is preferably 0.001% by mass or more, more preferably from the viewpoint of TRPM8 activation action. It is 0.01 mass% or more, Preferably it is 10 mass% or less. Specifically, the content of the processed product in the above case is preferably 0.001 to 10% by mass, more preferably 0.01 to 10% by mass from the viewpoint of the TRPM8 activation action. It is.

  In relation to the above-described embodiment, the present invention further discloses the following manufacturing method and the like.

  <1> A method for producing a processed product of nutmeg, wherein at least one selected from an alcohol or an aqueous alcohol solution of 25% (v / v) or more, a protonic acid, and an H-type cation exchange resin. The manufacturing method characterized by including the process processed simultaneously.

<2> including a step of extracting nutmeg using an extraction solvent containing alcohol or an aqueous solution of alcohol of 25% (v / v) or higher and one or more selected from protonic acids and H-type cation exchange resins. A method for producing a processed product of a nutmeg product.
<3> Scratch was extracted using an extraction solvent containing alcohol or an aqueous solution of alcohol of 25% (v / v) or more but not including one or more selected from protonic acids and H-type cation exchange resins. A method for producing a processed product of a nutmeg product, comprising a step of treating the extract with one or more selected from a protonic acid and an H-type cation exchange resin.
<4> Extracting the nutmeg using an extraction solvent, and the resulting extract is one or more selected from alcohol or 25% (v / v) aqueous alcohol solution, protonic acid and H-type cation exchange resin The manufacturing method of the processed product which is characterized by including the process processed by these.
<5> Any of the above <1> to <4>, wherein an alcohol having 1 to 10 carbon atoms, more preferably an alcohol having 1 to 6 carbon atoms, and still more preferably an alcohol having 2 to 6 carbon atoms is used as the alcohol. Manufacturing method.
<6> The concentration of the aqueous alcohol solution is preferably 35% (v / v) or more, more preferably 50% (v / v) or more, still more preferably 70% (v / v) or more, and even more preferably 80% ( v / v) or more, more preferably 90% (v / v) or more, and preferably 99.9% (v / v) or less, the production method according to any one of <1> to <5> above .
<7> Alcohol or 25% (v / v) or more of the alcohol aqueous solution is used in an amount of preferably 0.1 parts by mass or more, more preferably 1 part by mass or more, based on 1 part by mass of the nutmeg or extract thereof. More preferably, it is 2 parts by mass or more, preferably 1000 parts by mass or less, more preferably 500 parts by mass or less, and still more preferably 200 parts by mass or less, in any one of the above <1> to <6> .
<8> The method according to any one of <1> to <7>, wherein at least one selected from organic acids and inorganic acids is used as the protonic acid.
The manufacturing method in any one of said <1>-<8> using 1 or more types chosen from <9> strong acidic protonic acid and strong acidic H type cation exchange resin.
<10> The amount of one or more selected from protonic acids and H-type cation exchange resins is preferably 0.001 parts by mass or more, more preferably 0.01 based on 1 part by mass of nikuzu or its extract. The production method according to any one of the above items <1> to <9>, which is not less than 50 parts by mass, preferably not more than 50 parts by mass, more preferably not more than 25 parts by mass, and still more preferably not more than 10 parts by mass.
<11> The treatment temperature is preferably 3 ° C. or higher, more preferably 15 ° C. or higher, further preferably 25 ° C. or higher, preferably 100 ° C. or lower, more preferably 70 ° C. or lower, still more preferably 60 ° C. or lower. Any one of <1> to <10> above.
<12> The treatment time is preferably 0.1 hour or longer, more preferably 1 hour or longer, still more preferably 2 hours or longer, preferably 5 days or shorter, more preferably 3 days or shorter, still more preferably 2 The production method according to any one of <1> to <11>, wherein the production time is not more than one day.
<13> Scratched processed product obtained by the production method according to any one of <1> to <12>.
<14> A cooling sensation agent comprising the processed product of <13> above as an active ingredient.
<15> A TRPM8 activator comprising the processed processed product of <13> above as an active ingredient.
<16> A cosmetic comprising the processed product of <13> above.
<17> A composition for oral cavity containing the processed product of <13> above.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

Preparation Example 1 Ethanol-treated extract 50 mL of 99.5 vol% ethanol was added to 5 g of seeds of Myrista fragrance Hout., Soaked at room temperature for 2 days, extracted, filtered, and crude extract (acid-untreated) Product). The evaporation residue of the obtained crude extract was 1.05 w / v%.

Example 1 Extract treated with ethanol / hydrochloric acid To 5 mL of the crude extract obtained in Preparation Example 1 (solid content 52.5 mg), hydrochloric acid (1.1 M aqueous solution, 0.05 mL) was added and mixed so that the whole became uniform. After that, the mixture was allowed to stand at 60 ° C. for 2 days to obtain an extract treated with ethanol and hydrochloric acid (evaporation residue 1.05 w / v%).

Example 2 Ethanol / strongly acidic cation exchange resin-treated extract To the crude extract 5 mL (solid content 52.5 mg) obtained in Preparation Example 1, a strongly acidic cation exchange resin (Amberlyst 15JW, manufactured by Dow Chemical Co., Ltd.) Was added and stirred, and then allowed to stand at 60 ° C. for 2 days. By filtering the resin in this extract, an extract (evaporation residue 1.05 w / v%) treated with ethanol and a strongly acidic cation exchange resin was obtained as a filtrate.

Example 3 Ethanol / citric acid-treated extract To 5 mL of the crude extract obtained in Preparation Example 1 (solid content 52.5 mg), citric acid (960 mg) was added and stirred, and then allowed to stand at 60 ° C. for 2 days. . Thereafter, ethanol was removed by nitrogen flow, and hexane (2.5 mL) and saturated aqueous sodium hydrogen carbonate solution (2.5 mL) were added. After discarding the aqueous phase, the hexane phase was concentrated and dissolved again in ethanol (5 mL) to obtain an extract treated with ethanol and citric acid (evaporation residue 0.60 w / v%).

Example 4 n-Butanol / hydrochloric acid treated extract 5 mL of the crude extract obtained in Preparation Example 1 (solid content 52.5 mg) was concentrated and then dissolved in n-butanol (5 mL), and hydrochloric acid (1.1 M aqueous solution, 0 .05 mL) was added. After stirring, the mixture was allowed to stand at 60 ° C. for 2 days to obtain an extract treated with n-butanol and hydrochloric acid (evaporation residue 0.85 w / v%).

Example 5 n-Butanol / strongly acidic cation exchange resin-treated extract After concentration of the crude extract 5 mL (solid content 52.5 mg) obtained in Preparation Example 1, it was dissolved in n-butanol (5 mL) 52.5 mg of ion exchange resin (Amberlyst 15JW, manufactured by Dow Chemical Co., Ltd.) was added and stirred, and then allowed to stand at 60 ° C. for 2 days. By filtering the resin in this extract, an extract (evaporation residue 0.78 w / v%) treated with n-butanol and a strongly acidic cation exchange resin was obtained as a filtrate.

Example 6 Ethanol / strongly acidic cation exchange resin treated extract 8 mL of 99.5 vol% ethanol was added to 8 g of a mace of Myristica fragrance Hout., Extracted by immersion for 1 day at room temperature, and then filtered. A crude extract (evaporation residue 1.17 w / v%) was obtained. To this was added 936 mg of strongly acidic cation exchange resin (Amberlyst 15JW, manufactured by Dow Chemical Co.), and the mixture was stirred and allowed to stand at 60 ° C. for 2 days. By filtering the resin in this extract, an extract (evaporation residue 1.17 w / v%) treated with ethanol and a strongly acidic cation exchange resin was obtained as a filtrate.

Example 7 Extract of ethanol / strongly acidic cation exchange resin treated extract 400 mL of 99.5 vol% ethanol was added to 20 g of leaves of Myristica fragrance Hout. And extracted by immersion for 4 days at room temperature, followed by filtration. A crude extract (evaporation residue 0.32 w / v%) was obtained. To this was added 640 mg of strongly acidic cation exchange resin (Amberlyst 15JW, manufactured by Dow Chemical Co.), and the mixture was stirred and allowed to stand at 60 ° C. for 2 days. By filtering the resin in the extract, an extract (evaporation residue 0.32 w / v%) treated with ethanol and a strongly acidic cation exchange resin was obtained as a filtrate.

Example 8 Treated product of ethanol / strongly acidic cation exchange resin In the same manner as in Preparation Example 1, 100 mL of 99.5 vol% ethanol was added to 10 g of seeds of Myristica fragrance Hout. And soaked at room temperature for 2 days. And then filtered to obtain a crude extract (evaporation residue: 1.05 w / v%). This solution was passed through a column packed with 5 g of a strongly acidic cation exchange resin (Amberlyst 15JW, manufactured by Dow Chemical) at 50 ° C. for 2 days to obtain an extract treated with ethanol and a strongly acidic cation exchange resin. It was.
Next, the extract was concentrated by hydrolysis and then dissolved again in 100 mL of ethanol. Activated carbon was added thereto, and the mixture was allowed to stand at room temperature for 2 days, and then the activated carbon was filtered off. Next, the filtrate was concentrated under reduced pressure to obtain a nutmeg extract (141 mg) treated with ethanol and a strongly acidic cation exchange resin.

Comparative Example 1 DMSO / hydrochloric acid treated extract 5 mL (52.5 mg solid content) of the crude extract obtained in Preparation Example 1 was concentrated to remove ethanol, and then dissolved in DMSO (5 mL), and dissolved in hydrochloric acid (1.1 M aqueous solution). 0.05 mL) was added. After stirring, the mixture was allowed to stand at 60 ° C. for 2 days to obtain an extract treated with DMSO and hydrochloric acid (evaporation residue 1.05 w / v%).

Preparation Example 2 Ethanol-treated product As in Preparation Example 1, 100 mL of 99.5 vol% ethanol was added to 10 g of seeds of Myristica fragrance Hout., Soaked at room temperature for 2 days, extracted, and then filtered. A crude extract (evaporation residue 1.05 w / v%) was obtained. This extract was concentrated by hydrolysis to obtain a nutmeg extract (835 mg) treated with ethanol.

Example 9 Ethanol / strongly acidic cation exchange resin-treated extract 50.0 mg of supercritical carbon dioxide extract (purchased from PT MITRA AYU ADIPRATAMA) of seeds of Myristica fragrance Hout. Dissolved in ethanol (5 mL) did. 50 mg of strongly acidic cation exchange resin (Amberlyst 15JW, manufactured by Dow Chemical Co., Ltd.) was added thereto and stirred, and then allowed to stand at 60 ° C. for 1 day. By filtering the resin in this extract, an extract (evaporation residue: 1.00 w / v%) treated with ethanol and a strongly acidic cation exchange resin was obtained as a filtrate.

Example 10 50% ethanol aqueous solution / strongly acidic cation exchange resin-treated extract 50.0 mg of the same supercritical carbon dioxide extract as in Example 9 was dissolved in 50% ethanol aqueous solution (5 mL). 50 mg of strongly acidic cation exchange resin (Amberlyst 15JW, manufactured by Dow Chemical Co., Ltd.) was added thereto and stirred, and then allowed to stand at 60 ° C. for 1 day. By filtering the resin in the extract, an extract (evaporation residue: 1.00 w / v%) treated with a 50% aqueous ethanol solution and a strongly acidic cation exchange resin was obtained as a filtrate.

Comparative Example 2 Ethanol Solution of Supercritical Carbon Dioxide Extract 50.0 mg of the supercritical carbon dioxide extract similar to Example 9 was dissolved in ethanol (5 mL) to obtain an ethanol solution of the supercritical carbon dioxide extract.

Comparative Example 3 20% Ethanol Aqueous Solution / Strong Acidic Cation Exchange Resin Treated Extract 50.0 mg of the same supercritical carbon dioxide extract as in Example 9 was dissolved in a 20% aqueous ethanol solution (5 mL). 50 mg of strongly acidic cation exchange resin (Amberlyst 15JW, manufactured by Dow Chemical Co., Ltd.) was added thereto and stirred, and then allowed to stand at 60 ° C. for 1 day. By filtering the resin in this extract, an extract (evaporation residue: 1.00 w / v%) treated with a 20% aqueous ethanol solution and a strongly acidic cation exchange resin was obtained as a filtrate.

Example 11 Ethanol / Strongly Acidic Cation Exchange Resin Treated Extract A residue obtained by molecular distillation of a supercritical carbon dioxide extract of seeds of Myristica fragrance Hout. (Purchased from PT MITRA AYU ADIPRATAMA) 50.0 mg of ethanol (5 mL). 50 mg of strongly acidic cation exchange resin (Amberlyst 15JW, manufactured by Dow Chemical Co., Ltd.) was added thereto and stirred, and then allowed to stand at 60 ° C. for 1 day. By filtering the resin in this extract, an extract (evaporation residue: 1.00 w / v%) treated with ethanol and a strongly acidic cation exchange resin was obtained as a filtrate.

Example 12 Extract treated with cyclohexanol / strongly acidic cation exchange resin 50.0 mg of a distillation residue of the same supercritical carbon dioxide extract as in Example 11 was dissolved in cyclohexanol (5 mL). 50 mg of strongly acidic cation exchange resin (Amberlyst 15JW, manufactured by Dow Chemical Co., Ltd.) was added thereto and stirred, and then allowed to stand at 60 ° C. for 1 day. By filtering the resin in this extract, an extract (evaporation residue: 1.00 w / v%) treated with cyclohexanol and a strongly acidic cation exchange resin was obtained as a filtrate.

Test Example 1 Evaluation of TRPM8 Activating Action According to the following procedure, the EC ₅₀ value of the test sample in the TRPM8 activating action was measured.
(1) Production of human TRPM8 stable expression strain To produce the human TRPM8 stable expression HEK293 cell line, the human TRPM8 gene was cloned. The full-length human TRPM8 gene was amplified from human prostate tissue total RNA (manufactured by COSMOBIO) using the RT-PCR method.
The obtained PCR product was cloned into the entry vector pENTR-D / TOPO (Invitrogen), then subcloned into pCDNA3.2-V5 / DEST (Invitrogen), and HEK293 using Lipofectamine 2000 (Invitrogen). Cells were transduced. Transduced cells were selected by growing in DMEM medium containing 450 μg / mL G-418 (Promega). Since HEK293 cells do not express endogenous TRPM8, they can be used as a control for TRPM8 transduced strains.

(2) Calcium imaging TRPM8 activity transduced into HEK293 cells was measured by the fluorescent calcium imaging method.
First, cultured TRPM8-expressing cells were seeded (30,000 cells / well) in a 96-well plate (BD Falcon) coated with poly-D-lysine, incubated at 37 ° C. overnight, and then the culture solution was removed. Fluo4-AM solution (manufactured by Dojin Chemical Co., Ltd .; calcium kit II) was added and incubated at 37 ° C. for 30-60 minutes. Thereafter, the 96-well plate was set in a fluorescent plate reader (FDSS3000; manufactured by Hamamatsu Photonics), and the fluorescence image by Fluo4 when excited at an excitation wavelength of 480 nm with the temperature inside the apparatus being set at 32 ° C. Was captured at a detection wavelength of 520 nm.
The measurement is performed every second for a total of 4 minutes, and 15 seconds after the start of the measurement, the test sample shown in Table 1 dissolved in Ringer's solution is added from a dispenser with a built-in FDSS 3000, and the activity of TRPM8 is evaluated by the change in fluorescence intensity. did.

(3) Evaluation of TRPM8 activating action TRPM8 activity was obtained by subtracting the autofluorescent component by the following formula in order to eliminate the influence of autofluorescence by the test sample.
Fluorescence intensity minus autofluorescence (F _sub )
= (Fluorescence intensity of TRPM8 expressing cells at each time point)-{(Fluorescence intensity of HEK293 cells at each time point)-(Fluorescence intensity of HEK293 cells at the start of measurement)}
Moreover, the TRPM8 activation action by each sample was evaluated using the peak of the fluorescence intensity ratio after addition of the test sample. The fluorescence intensity ratio was calculated using the following formula.
Fluorescence intensity ratio = F _sub at each time point / Fluorescence intensity of TRPM8 expressing cells at the start of measurement In each treatment group, evaluation was performed in 2 wells, and the average value was used.

(4) Evaluation result of TRPM8 activation action The TRPM8 activation action of the test sample shown in Table 1 was measured in the range of a final concentration of 0.01 to 10 ppm in terms of dry solid content using the above method. From the measurement results, a capacity dependence curve approximated to Hill's equation was determined using the least square method, and EC ₅₀ values were calculated from this. The EC ₅₀ values in the TRPM8 activation action of the crude extract of Preparation Example 1, Comparative Examples 1 to 3 and Examples 1 to 7 and 9 to 12 were as shown in Table 1.

  As shown in Table 1 above, the processed products of Examples 1 to 7 and 9 to 12 exhibit an excellent TRPM8 activation effect.

Test Example 2 Evaluation of cooling sensation of test sample-containing mouthwash The mouthwashes (test products A, B and C) shown in Table 2 were prepared, and the cooling sensation induction effect was evaluated according to the following procedures and standards. The evaluation results are shown in FIG.

(1) Evaluation procedure A total of 6 persons including 5 male panels and 1 female panel were evaluated for the cool feeling with the mouthwash.
Test product A (Comparative Example 4), Test product B (Comparative Example 5) or Test product C (Example 13) was contained in the mouth for 10 seconds for 30 seconds and discharged. Then, the cool sensation felt in the oral cavity for 30 minutes was evaluated with 0 to 5.0 points (11 step evaluation in 0.5 increments). Each test product was applied with an interval of 30 minutes or more.

(2) Evaluation criteria (cool feeling score)
0 points: Nothing felt 0.5 points 1.0 points: Slightly cool feeling 1.5 points 2.0 points: Feeling cool feeling 2.5 points 3.0 points: Clearly cool feeling 3.5 points 4.0 points: Feeling a strong cool feeling 4.5 points 5.0 points: Feeling a very strong cooling feeling

(3) Evaluation result of cool feeling induction effect As shown in FIG. 1, the mouthwash of Example 13 was the same as the mouthwash of Comparative Example 4 and the mouthwash of Comparative Example 5 (instead of the sample of Example 8). Compared with the sample containing the sample of Preparation Example 2 of concentration), it shows a cool feeling with high strength and durability.

Claims (9)

A method of manufacturing a nutmeg processing products, seeds of nutmeg, a mace or leaf or its these extracts, alcohol or 25% (v / v) or higher alcohol aqueous solution and a protonic acid and H-type cation-exchange resin The manufacturing method characterized by including the process processed simultaneously with 1 or more types chosen from.   The manufacturing method of Claim 1 which uses a C1-C10 alcohol as alcohol.   A processed product obtained by the manufacturing method according to claim 1 or 2. A cooling sensation agent containing only the processed product of the nutmeg product according to claim 3 as an active ingredient. A TRPM8 activator containing only the processed product of nutmeg according to claim 3 as an active ingredient. Cosmetics containing the cooling sensation agent of Claim 4 . The composition for oral cavity containing the cooling agent of Claim 4 . A cosmetic comprising the TRPM8 activator according to claim 5. An oral composition containing the TRPM8 activator according to claim 5.

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