JP5608793B1 - Cooling sensation agent, TRPM8 activator, cosmetic and oral composition - Google Patents

Abstract

The present invention provides a cooling sensation agent, an TRPM8 activator, a cosmetic, and an oral composition having an excellent TRPM8 activation action.
A cooling sensation agent comprising a distillation residue of a nutmeg extract as an active ingredient.
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Description

  The present invention relates to a cooling sensation agent, a TRPM8 activator, a cosmetic, and an oral composition.

  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 cooling agent, an TRPM8 activator, a cosmetic, and an oral composition having an excellent TRPM8 activation effect.

  The present inventors have found that a distillation residue of a nutmeg extract has an excellent TRPM8 activating action, and completed the present invention.

That is, this invention provides the cooling agent which uses the distillation residue of a nutmeg extract as an active ingredient.
Moreover, this invention provides the TRPM8 activator which uses the distillation residue of a nutmeg extract as an active ingredient.
Furthermore, this invention provides the cosmetics containing the distillation residue of a nutmeg extract.
Furthermore, this invention provides the composition for oral cavity containing the distillation residue of a nutmeg extract.

  The cooling sensation agent and TRPM8 activator of the present invention have an excellent TRPM8 activating action, and by using this, a good cooling sensation can be imparted.

  The present invention uses a distillation residue of a nutmeg extract. Such a 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, the nutmeg extract may use these parts independently, and may use it in combination of 2 or more types. Further, it may be extracted as it is, or may be extracted after being dried and cut and pulverized to an appropriate size.

Moreover, as said nutmeg extract, you may use a commercially available thing, but you may use what extracted the nutmeg according to the conventional method. Such extraction includes a method of impregnating the above-mentioned nutmeg with a solvent at room temperature or under heating, a solvent extraction method performed using an extraction device such as a Soxhlet extractor, and a supercritical extraction performed using carbon dioxide gas or the like in a supercritical state. It can be carried out using a method, a squeezing method for obtaining an extract by squeezing. Alternatively, extraction may be performed again using a solvent or the like from a residue obtained by steam distillation or microwave steam distillation.
Among the above extraction methods, when obtaining an extract to be used for the distillation residue, the supercritical extraction method is preferable from the viewpoint of activating TRPM8. The supercritical extraction method is a method in which a supercritical fluid is brought into contact with a nutmeg, specifically, a substance such as carbon dioxide is brought into a supercritical state (supercritical fluid), and this is extracted in a state where it can be extracted. And the like, and the like.

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; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; Linear or cyclic ethers such as ether and tetrahydrofuran; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane and cyclohexane; aromatic hydrocarbons such as benzene and toluene; halogens such as dichloromethane, chloroform and dichloroethane In addition to activated hydrocarbons, 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, when obtaining the extract used for the distillation residue, from the viewpoint of TRPM8 activation action, a mixed solution of water and a polar solvent, a polar solvent, and supercritical carbon dioxide are preferable, and a water / alcohol mixed solution, alcohol More preferred is supercritical carbon dioxide, and even more preferred is supercritical carbon dioxide.
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.

Distillation of the extract obtained as described above may be carried out by a known method such as molecular distillation, steam distillation or microwave distillation, and the distillation conditions, number of stages and apparatus are not particularly limited.
Further, such a distillation operation can be performed under normal pressure or reduced pressure, and the distillation pressure is preferably 0.05 Pa or more, more preferably 0.1 Pa or more, and preferably 1000 Pa or less, more preferably Is 200 Pa or less, more preferably 30 Pa or less. Specifically, it is preferably 0.05 to 1000 Pa, more preferably 0.1 to 200 Pa, and still more preferably 0.1 to 30 Pa.
The distillation temperature is preferably 30 ° C. or higher, more preferably 45 ° C. or higher, further preferably 60 ° C. or higher, preferably 150 ° C. or lower, more preferably 140 ° C. or lower, and still more preferably 130 ° C. or lower. Specifically, distillation temperature becomes like this. Preferably it is 30-150 degreeC, More preferably, it is 45-140 degreeC, More preferably, it is 60-130 degreeC.
Moreover, you may use the residue obtained by distilling the distillation residue of the nutmeg extract obtained as mentioned above once or more (preferably 1-3 times) as a distillation residue. By using such a distillation residue, the TRPM8 activation action is improved.

Moreover, after distilling, concentrating, freeze-drying, etc. the obtained distillation residue, it can also be prepared and used for powder and paste form as needed.
In addition, as a solvent used for dilution of the said distillation residue, alcohol, water, and these liquid mixture are mentioned. Examples of the distillation residue concentration / purification means include distillation residue filtration, activated carbon treatment, and liquid-liquid distribution.

And as shown in the below-mentioned Example, the distillation residue of a nutmeg extract is derived from a natural product and has an excellent TRPM8 activating action, although it can be obtained by a very simple operation. In the present invention, the distillation residue means a residue that remains without being distilled when the nutmeg extract is distilled.
Accordingly, the distillation residue of the nutmeg extract can be used as it is, or together with various pharmaceutical carriers, preservatives, etc. 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, etc. can do. In addition to distillation residues, cosmetics include oils, ceramides, pseudoceramides, sterols, humectants, antioxidants, UV absorbers, whitening agents, alcohols, chelating agents, pH adjusters, preservatives. 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.

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

  In addition, in the case of pharmaceuticals, cosmetics or other luxury products for parenteral administration, the content of the distillation residue is preferably 0.001% by mass or more, more preferably 0.001% from the viewpoint of TRPM8 activation action. It is 01 mass% or more, and preferably 10 mass% or less. Specifically, the content of the distillation residue 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. .

  In relation to the above-described embodiment, the present invention further discloses the following cooling sensation agent and the like.

  <1> A cooling sensation agent comprising a distillation residue of a nutmeg extract as an active ingredient.

<2> A TRPM8 activator comprising a distillation residue of a nutmeg extract as an active ingredient.
<3> Cosmetics containing distillation residue of nutmeg extract.
<4> An oral composition containing a distillation residue of a nutmeg extract.
<5> The cooling sensation agent, TRPM8 activator, cosmetic or oral composition according to any one of <1> to <4>, wherein the nutmeg extract is a nutmeg extract extracted by a supercritical extraction method.
<6> Extract from a nutmeg extract, preferably using a mixture of water and a polar solvent, a solvent selected from polar solvents and supercritical carbon dioxide, more preferably a water / alcohol mixture, alcohols And a cooling sensitizer according to any one of the above <1> to <5>, which is extracted using a solvent selected from supercritical carbon dioxide, and more preferably extracted using supercritical carbon dioxide, TRPM8 Activators, cosmetics or oral compositions.
<7> The concentration of the mixed solution is preferably 25% (v / v) or more, more preferably 35% (v / v) or more, still more preferably 50% (v / v) or more, and further preferably 70% ( v / v) or more, more preferably 80% (v / v) or more, more preferably 90% (v / v) or more, and preferably 99.9% (v / v) or less <6> cooling agent, TRPM8 activator, cosmetic or oral composition.
<8> Distilled residue is distilled at a pressure of 0.05 Pa or more, more preferably 0.1 Pa or more, preferably 1000 Pa or less, more preferably 200 Pa or less, and even more preferably 30 Pa or less. The cooling sensation agent according to any one of <1> to <7>, the TRPM8 activator, the cosmetic, or the composition for oral cavity, which is a residue obtained by
<9> The distillation residue is preferably at a temperature of 30 ° C. or higher, more preferably 45 ° C. or higher, further preferably 60 ° C. or higher, preferably 150 ° C. or lower, more preferably 140 ° C. or lower, still more preferably 130 ° C. or lower. The cooling sensation agent according to any one of <1> to <8>, TRPM8 activator, cosmetic or oral composition, which is a residue obtained by distilling the nutmeg extract.
<10> The cooling sensation agent according to any one of <1> to <9>, the TRPM8 activator, and the cosmetic, wherein the distillation residue is a residue obtained by further distilling the distillation residue of the nutmeg extract once more. Or composition for oral cavity.

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

Example 1 Residue obtained by Kugel distillation (simple distillation) of supercritical carbon dioxide extract 15.6 g of supercritical carbon dioxide extract (purchased from PT MITRA AYU ADIPRATAMA) of seeds of Myristica fragrant Hout. The components were separated by two-stage Kugel distillation. The first stage was performed at a reduced pressure of 0.8 mmHg and a temperature of 80 ° C. to obtain a fraction (1) 5.23 g and a distillation residue (1) 7.91 g (loss 2.46 g). The second stage was carried out at a reduced pressure of 0.006 mmHg and a temperature of 120 ° C. From the distillation residue (1), 2.39 g of a fraction (2) and 5.19 g of a distillation residue (2) were obtained (loss 0.33 g). .

Example 2 Residue obtained by Kugel distillation (simple distillation) of EtOH extract 1.6 g of 99.5 vol% ethanol was added to 160 g of seeds of crushed stigma family (Myristica fragrance Hout.) And immersed at room temperature for 3 days. After extraction, a crude extract was prepared by filtration. Subsequently, the crude extract was concentrated with an evaporator to obtain 19.49 g of an EtOH extract, and component separation was performed on 7.59 g of this EtOH extract by two-stage Kugel distillation. The first stage was performed at a reduced pressure of 0.8 mmHg and a temperature of 80 ° C. to obtain 0.09 g of a fraction (3) and 7.05 g of a distillation residue (3) (loss 0.45 g). The second stage was carried out at a reduced pressure of 0.01 mmHg and a temperature of 130 ° C. From the distillation residue (3), 1.31 g of the fraction (4) and 5.62 g of the distillation residue (4) were obtained (loss 0.12 g). .

Example 3 Residue obtained by fractional distillation and molecular distillation of supercritical carbon dioxide extract Supercritical carbon dioxide extract (purchased from PT MITRA AYU ADIPRATAMA) of seeds of Myristica fragrant Hout. Fractionation was carried out under conditions of 0.5 mmHg and a temperature of 80 ° C. to obtain 76.5 kg of a fraction (5) and 151.5 kg of a distillation residue (5) (loss 6 kg). Subsequently, components were separated by distillation in three stages for 132.6 kg of the distillation residue (5). The first stage was performed at a reduced pressure of 0.375 mmHg and a temperature of 60 ° C., and 27.5 kg of a fraction (6) was obtained from the distillation residue (5). The second stage was performed at a reduced pressure of 0.00075 mmHg and a temperature of 80 ° C., and 29.3 kg of a fraction (7) was obtained from the distillation residue of the first stage. The third stage was carried out at a reduced pressure of 0.00075 mmHg and a temperature of 120 ° C. From the distillation residue of the second stage, 16.9 kg of a fraction (8) and 55.3 kg of a distillation residue (6) were obtained (three stages). Loss 3.6 kg).

Test example 1
The above distillation residue (1), (2), (4) and (6), the above fractions (1) and (2), a supercritical carbon dioxide extract purchased from PT MITRA AYU ADIPRATAMA, prepared in Example 2 The obtained EtOH extract was used as a test sample, and the EC ₅₀ value in TRPM8 activation action was measured according to the following procedure.
(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, a test sample (in which each compound shown in Table 1 below is dissolved in a Ringer solution) is added from a dispenser with a built-in FDSS 3000, and the fluorescence intensity The activity of TRPM8 was evaluated based on the change in.

(3) Evaluation of TRPM8 activation TRPM8 activity was obtained by subtracting the autofluorescence component from 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 during F _sub / measurement start of each time point
Each treatment group was evaluated in 2 wells, and the average value was used.

(4) Evaluation results of TRPM8 activation action The TRPM8 activation action of each test sample was evaluated in a final concentration range of 0.1 ppm to 30 ppm, and a capacity dependence curve approximated to Hill's equation was obtained by the least square method. The EC ₅₀ value in the TRPM8 activation action of each test sample calculated from this curve is shown below.

  The fraction (1) and the fraction (2) had no TRPM8 activation action even at a final concentration of 30 ppm.

  As shown in Table 1, the distillation residue obtained in the examples has an excellent TRPM8 activation effect.

Claims (4)

A cooling sensation agent containing only the distillation residue of nutmeg seed extract as an active ingredient. A TRPM8 activator comprising only a distillation residue of a nutmeg seed extract as an active ingredient. An oral composition containing the cooling agent according to claim 1 . An oral composition containing the TRPM8 activator according to claim 2.