JP6757012B1 - Bisel containing a compound having a rigid chemical structure and an external composition containing it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for external use which is safe for a human body. SOLUTION: The bicelle contains 50% by mass or more of anionic phospholipid, and the bicelle is a compound having a stabilizing energy of 3 to 25 kcal / mol or less (excluding paclitaxel) or a glycoside thereof. Contains 3. [Selection diagram] FIG. 11

Description

Compounds such as resveratrol, piceid, pterostilbene, or fullerenes have a conjugated aromatic ring chemical structure. Since these compounds exhibit antioxidant properties in vivo due to this aromatic ring, they can be applied to skin care materials such as sunscreens by utilizing their functions. However, since the above molecules are hydrophobic molecules, they aggregate in water, and when these compounds are applied to bioapplicable materials, they need to be dispersed in water (Patent Documents 1 to 4 and Non-Patent Documents). 1-3). Studies have been conducted on bicelle, which is a lipid-lipid aggregate having a disk-like structure (Patent Document 4).

JP-A-2002-308728 Special Table 2016-506925 International Publication No. 2006/011633 Japanese Unexamined Patent Publication No. 2011-089946 International Publication No. 2017/090740

Ruivo, J. et al. et al. Brazilian Jiu-Jitsu. Pharm. Sci. 2015, 51,499-5137. Peng, R.M. -M. et al. Biofactors 2018, 44, 5-15. Boshi, S.M. et al. Euro. J. Med. Chem. 2003, 38, 913-923. Barbosa-Barros, L .; et al. Small 2012,8,807-818 Barbosa-Barros, L .; et al. Langmuir 2008, 24, 5700-5706.

Phospholipids are bio-derived molecules whose biocompatibility is guaranteed. In addition, since phospholipid is a surfactant registered as a cosmetic ingredient, it is a promising material as a dispersant used when producing nanoparticles by dispersing hydrophobic molecules. Among such phospholipids, neutral phospholipids tend to form large self-assemblies in water. As a method for producing the aggregate of the phospholipid, there is a method called the Hungham method (Patent Document 5 and Non-Patent Document 4 or 5).

However, the above-mentioned Hungham method is a method using an organic solvent, and there is a concern that it may cause toxicity to the human body. Therefore, an object of the present invention is to provide a composition for external use that is safe for the human body.

As a result of diligent research to solve the above problems, the present inventors have found that a compound having a stabilizing energy of 3 to 25 kcal / mol or less can be contained in a bicelle containing 50% or more of anionic phospholipids. .. Then, they found that the particle size of the bicelle was as small as 10 nm or more and 5 μm or less. It has been found that a compound having a stabilizing energy of 3 to 25 kcal / mol or less contained therein can be delivered not only to the stratum corneum but also to the epidermis and dermis tissue by using such a small particle size bicelle.

It should be noted that some of the present inventors have confirmed that bicelle, which is a disk-shaped lipid aggregate containing a single type of anionic phospholipid as a main component, has a skin penetrating ability. Has also succeeded in containing paclitaxel.

The present invention has been completed based on such findings, and broadly includes the inventions shown below.

Item 1 A bicelle containing a compound having an anionic phospholipid of 50% by mass or more and a stabilizing energy of 3 to 25 kcal / mol or less (excluding paclitaxel) or a glycoside thereof.

Item 2 The bicelle according to Item 1, wherein the average particle size of the bicelle is 10 nm or more and 5 μm or less.

Item 3 The bicelle according to Item 1 or 2 above, wherein the anionic phospholipid is a glycerophospholipid.

Item 4 The bicelle according to Item 3 above, wherein the glycerophospholipid is represented by the following formula (1);

(In the formula, R ¹ is a linear or branched chain alkyl group having 10 to 36 carbon atoms, or a linear or branched chain alkenyl group, both identically or independently.
R ² is a nonionic or anionic group. )

Claim 5 wherein R ¹ is the same or independently, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, heneicosyl , Docosyl group, tridecylic group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group, hentolycontyl group, dotriacyl group, tritoriacontyl group, tetratriacontyl group, pentadecanoic acid Item 4. The bicelle according to Item 4, which is selected from the group consisting of a chill group, a hexatridecanoic acid group, and an alkenyl group having 1 to 5 double bonds attached to these groups.

Item 6 The bicell according to Item 4 above, wherein R ² is selected from the groups represented by the following formulas (2-1) to (2-4);

Item 7. The bicelle according to any one of Items 1 to 6 above, wherein the anionic phospholipid is at least one selected from the group consisting of DPPG, DSPG, and DPPA.

Item 8. The bicelle according to any one of Items 1 to 7, wherein the compound is at least one selected from the group consisting of stilbenoids, flavonoids, indomethacin compounds, and fullerene compounds.

Item 9 The above item, wherein the stilbene is at least one selected from the group consisting of resveratrol, piceatannol, oxyresveratrol, rhapontigenin, isolapontigenin, pterostilbene, gnetol, pinosylvin, and pinostilbene. Bisel according to 8.

Item 10 The flavonoids are kenferol, anthocyanidin, anthocyanin, flabanone, naringenin, flavan, catechin, epicatechin, epicatechin gallate epigalocatechin, epigalocatechin gallate, flavon, flavon derivative, flavonol, quercetin, isoflavoneoid, isoflavone, isoflavone. The bicell according to item 8 above, which is at least one selected from the group consisting of isoflavone diols, neoflavonoids, biflavonoids, and aurones.

Item 11 The indomethacin compound is at least one selected from the group consisting of indomethacin, indomethacin farnesyl, acemetacin, indomethacin morpholinylamide, pravadrine, and 1-pentyl-3- (4-methoxybenzoyl) indole. Bisel described in.

Item 12 The fullerene compound is C60 fullerene, C70 fullerene, C76 fullerene, C78 fullerene, C84 fullerene, C60-SAM, 60 pyrrolidinetris-acid, polyhydroxylated C60, C60 ethyl nipecotate, Bis [60] PCBM, [60]. Item 8. The bicelle according to item 8 above, which is at least one selected from the group consisting of PCBM, [70] PCBM, [84] PCBM, [60] PCB-C4, [60] PCB-C8, and [60] ThCBM. ..

Item 13. The bicelle according to any one of Items 1 to 12, wherein the glycoside is a monosaccharide glycoside or a polysaccharide glycoside.

Item 14. The bicelle according to any one of Items 1 to 13 above, wherein the compound or a glycoside thereof is any one of resveratrol, pterostilbene, piceid, kaempferol, indomethacin, or C60 fullerene.

Item 15 An external composition containing the bicelle according to any one of Items 1 to 14 above.

Item 16. The bicelle according to Item 15, wherein the external composition is a cosmetic composition.

Item 17. The bicelle according to Item 15, wherein the external composition is a pharmaceutical composition.

Item 18 The method for producing a bicelle according to any one of Items 1 to 15 above, which comprises the following steps (1) and (2);
(1) A step of dispersing a mixture of an anionic phospholipid and a compound having a stabilizing energy of 3 to 25 kcal / mol or less in an aqueous dispersion medium under conditions below the phase transition temperature of the anionic phospholipid, and (2) A step of heating the aqueous dispersion obtained in the above step (1) to a temperature equal to or higher than the phase transition temperature of the anionic phospholipid.

Since the bicelle of the present invention can contain a compound having a stabilizing energy of 3 to 25 kcal / mol or less, it is used as an external composition (cosmetic composition or pharmaceutical composition). Since the particle size of the bicelle of the present invention is as small as 10 nm or more and 5 μm or less, it is used as an external composition. Since the bicelle of the present invention exerts the effect of penetrating into the skin, it is used as an external composition.

FIG. 1 is a diagram illustrating a manufacturing process of the bicelle of the present invention and the result thereof. FIG. 2 is a photographic image of the bicelle of the present invention. a is a resveratrol-containing bicelle containing DPPC as a main component or a bicelle containing DPPG as a main component. b is a bicell containing piceid and containing DPPC as a main component or a bicell containing DPPG as a main component. c is a bicell containing pterostilbene and containing DPPC as a main component or a bicell containing DPPG as a main component. Reference numeral d is a bicell containing fullerene and containing DPPC as a main component or a bicell containing DPPG as a main component. e is a bicell containing kaempferol and containing DPPC as a main component or a bicell containing DPPG as a main component. reference numeral f is a bicelle containing indomethacin and containing DPPC as a main component or a bicelle containing DPPG as a main component. g is a resveratrol-containing bicelle containing DSPG as a main component or a bicelle containing DPPA as a main component. FIG. 3 is a diagram showing the results of ³¹ P NMR analysis of the bicelle of the present invention. A is a bicell containing resveratrol and containing DPPG as a main component. B is a bicelle containing piceid and containing DPPG as a main component. The horizontal axis in the figure is δ (ppm). FIG. 4 is a TEM image of the bicelle of the present invention. A is a TEM image of a bicell containing resveratrol and containing DPPG as a main component (bar is 1 μM). B is a TEM image of a bicelle containing piceid and containing DPPG as a main component (the bar is 500 nM, and the inside of the dotted line is the bicelle). FIG. 5 is a diagram showing the results of SAXS analysis of resveratrol-containing and DPPG-based bicelle of the present invention. The vertical axis in the figure is the intensity. The horizontal axis in the figure is q (nm ^-1 ). FIG. 6 is a diagram showing an absorption spectrum of the bicelle of the present invention. A is a bicell containing resveratrol and containing DPPG as a main component. B is a bicelle containing piceid and containing DPPG as a main component. The vertical axis in the figure is the absorbance. The horizontal axis in the figure is the wavelength (nm). FIG. 7 is a diagram showing the results of laser diffraction of the bicelle of the present invention. A is a bicell containing resveratrol and containing DPPG as a main component. B is a bicelle containing piceid and containing DPPG as a main component. C is a bicelle containing fullerene and containing DPPG as a main component. D is a bicell containing kaempferol and containing DPPG as a main component. E is a bicelle containing indomethacin and containing DPPG as a main component. The vertical axis in the figure is the volume fraction (%). The horizontal axis in the figure is the particle size (μm). FIG. 8 is a diagram showing the result of the particle size of the bicelle of the present invention after ultrasonic treatment. A is a bicell containing resveratrol and containing DPPG as a main component. B is a bicelle containing piceid and containing DPPG as a main component. The vertical axis in the figure is a fraction (%). The horizontal axis in the figure is the particle size (nm). FIG. 9 is a diagram showing the results of a subcutaneous penetration experiment of the bicelle of the present invention. A is a bicell containing resveratrol and containing DPPG as a main component. B is a bicelle containing piceid and containing DPPG as a main component. C is a bicell containing fullerene and containing DPPG as a main component. The bar in the figure is 100 μm. FIG. 10 is a graph quantifying the results shown in FIG. The vertical axis in the graph is the amount of permeation into the tissue or the amount of permeation through Franz cell (μmol / cm ² ). FIG. 11 is a schematic view of the bicelle of the present invention.

Hereinafter, the present invention will be described in detail. As used herein, the term "contains" or "contains" includes the meanings of "consisting of" and "consisting of". In addition, the description of the numerical values of ◯ to Δ in this specification means that it is ◯ or more and Δ or less. And, "mass" in this specification can be read as "weight".

<Buysell>
As shown in Patent Document 4 and the like, the bicelle is a lipid aggregate having a disk-like (disk-like) structure. Such bicelles tend to disperse in an aqueous solvent, like liposomes, which are other lipid aggregates. The disk-shaped structure that characterizes such a bicell can be specified by SAXS (Small Angle X-ray Scattering Method). Specifically, the structure in the dispersion can be specified by the slope (q- ² ) of the graph obtained by the SAXS analysis. For example, if the inclination is "-4", it can be specified as a spherical structure, if it is "-1", it can be specified as a rod-shaped structure, and if it is "-2", it can be specified as a disk-shaped structure.

The bicelle of the present invention is a bicelle containing 50% by mass or more of anionic phospholipid, and the bicelle contains a specific compound or a glycoside thereof. The specific compound is a compound having a stabilizing energy of 3 to 25 kcal / mol or less. Paclitaxel is clearly excluded from the specific compounds contained in the bicelle of the present invention.

The specific anionic phospholipid contained in the bicelle of the present invention exerts the effect of the present invention, and the entire molecule is negatively charged in the pH range of 6 to 8 (particularly preferably pH 7). As long as it is a lipid, it is not particularly limited. For example, sphingolipids or glycerophospholipids can be mentioned. Glycerophospholipids are preferred in view of more effectively exerting the effects of the present invention or being easily produced or available. It should be noted that the fact that the entire molecule is negatively charged can be easily confirmed by titration of the isoelectric point of the target phospholipid.

Such specific glycerophospholipids are not particularly limited as long as the effects of the present invention are exhibited. For example, a glycerophospholipid represented by the following formula (1) can be mentioned.

R ¹ in the above formula (1) is a linear or branched chain alkyl group having 10 to 36 carbon atoms, or a linear or branched chain alkenyl group, both identically or independently. The preferable lower limit of the carbon number of the R ¹ is 11, 12, 13, or 14, and the preferable upper limit of the carbon number of the R ¹ is 35, 34, 33, 32, 31, 30, 29, 28. , 27, or 26. The specific number of double bonds contained in the alkenyl group is not particularly limited as long as the effects of the present invention are exhibited. For example, it can be 1, 2, 3, 4, or 5.

The specific R ¹ in the above formula (1) is not particularly limited as long as the effect of the present invention is exhibited. For example, nonyl group, decyl group, undecylic group, dodecyl group, tridecylic group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecil group, icosyl group, henicosyl group, docosyl group, tridecylic group, tetracosyl group, Pentacosylic group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group, hentolycontyl group, dotriacylic group, tritoriacontyl group, tetratriacontyl group, pentatriacontyl group, hexatriacontyl group, or Examples thereof include alkenyl groups in which 1 to 5 double bonds are provided in these groups.

R ² in the above formula (1) is a nonionic group or an anionic group. The specific nonionic group or anionic group is not particularly limited as long as the effect of the present invention is exhibited. For example, the groups represented by the following formulas (2-1) to (2-4) can be mentioned.

In the case of (2-1) above, the anionic phospholipid is phosphatidic acid having the same or different fatty acids, and in the case of (2-2), phosphatidylglycerol having the same or different fatty acids, (2-3). In the case of phosphatidylserine having the same or different fatty acids, and in the case of (2-4), phosphatidylinositol having the same or different fatty acids.

The compound represented by the above formula (1) can also be a compound of an aspect such as a metal salt such as a sodium salt or a potassium salt, or an ammonium salt.

Preferred embodiments of the anionic phospholipid contained in the bicelle of the present invention include, for example, dipalmitoylphosphatidylglycerol (DPPG; phase transition temperature 41 ° C.), distearoylphosphatidylglycerol (DSPG; phase transition temperature 55 ° C.), dipalmitoylphosphatidyl. Serine (DPPS; phase transition temperature 54 ° C.) or dipalmitoylphosphatidylate (DPPA; phase transition temperature 65 ° C.) can be mentioned. Among these, DPPG is the most preferable anionic phospholipid contained in the bicelle of the present invention.

The bicelle of the present invention contains 50% by mass or more of the anionic phospholipid with respect to 100 parts by mass of the bicelle. That is, the lipid component constituting the bicelle of the present invention is mainly composed of anionic phospholipids. The preferable content of the anionic phospholipid is 80 parts by mass or more, more preferably 90 parts by mass or more, and most preferably 95 parts by mass or more with respect to 100 parts by mass of bicelle. The anionic phospholipid contained in such a bicelle of the present invention may be contained alone or may contain two or more kinds of anionic phospholipids.

In addition to the above anionic phospholipids, the bicelle of the present invention contains a specific compound having a stabilizing energy of 3 to 25 kcal / mol. A compound having a value of about 4 to 21 kcal / mol is preferable. Such specific compounds are known as compounds having a rigid chemical structure. Specifically, the stabilizing energy of a compound having such a rigid chemical structure is, for example, J.I. AM. CHEM. SOC. 2002, 124, 10887-93, Acc. Chem. Res. 2013, 46, 1029-38, or Coll. Czechoslovak Chem. Commun. It can be calculated by referring to the description of 2006, 71, 443 to 531 and the like.

The specific compound contained in the bicelle of the present invention is not particularly limited as long as the effects of the present invention are exhibited. Specific examples thereof include stilbenoids, flavonoids, indomethacin compounds, fullerene compounds and the like.

The stilbenoid is a compound having a stilbene skeleton and is also called a stilbene compound. The stilbenoid is not particularly limited as long as the effect of the present invention is exhibited. Specific examples thereof include resveratrol, piceatannol, oxyresveratrol, rhapontigenin, isolapontigenin, pterostilbene, gnetol, pinosylvin, pinostilbene and the like.

Considering that resveratrol, which is a typical example of the above stilbenoid, is a compound having two benzene rings and one benzene ring is 2 to 3 kcal / mol, its stabilizing energy is 4 to 6 kcal / mol. It can be calculated as mol. Therefore, it can be determined that the stilbenoid has a stabilizing energy of about 4 to 6 kcal / mol.

The flavonoid is a compound having a flavon skeleton and is not particularly limited as long as the effect of the present invention is exhibited. Specifically, kenferol, anthocyanidin, anthocyanin, flavanone, naringenin, flavan, catechin, epicatechin, epicatechin gallate epigalocatechin, epigalocatechin gallate, flavon, flavone derivative, flavonol, quercetin, isoflavone, isoflavone, isoflavone. , Isoflavone diol, neoflavanoid, biflavonoid, auron and the like.

Considering that kaempferol, which is a typical example of the flavonoid, is a compound having a flavone skeleton containing two benzene rings, its stabilization energy can be calculated as 4 to 6 kcal / mol. Therefore, it can be determined that the flavonoid has a stabilizing energy of about 4 to 6 kcal / mol.

The indomethacin compound is a compound having an indole skeleton, and is not particularly limited as long as the effects of the present invention are exhibited. Specific examples thereof include indomethacin, indometacin farnesyl, acemetasin, indomethacin morpholinylamide, pravadrine, and 1-pentyl-3- (4-methoxybenzoyl) indole.

Considering that indomethacin, which is a typical example of the above indomethacin compound, is a compound having an indole skeleton containing a benzene ring, its stabilization energy can be calculated to be 6 to 9 kcal / mol. Therefore, it can be determined that the indomethacin compound has a stabilizing energy of about 6 to 9 kcal / mol.

The fullerene compound is not particularly limited as long as the effect of the present invention is exhibited. Specifically, C60 fullerene, C70 fullerene, C76 fullerene, C78 fullerene, C84 fullerene, C60-SAM, 60 pyrrolidinetris-acid, polyhydroxylated C60, C60 ethyl nipecotate, Bis [60] PCBM, [60] PCBM. , [70] PCBM, [84] PCBM, [60] PCB-C4, [60] PCB-C8, [60] ThCBM and the like.

Considering that C60 fullerene, which is a typical example of the above fullerene compound, is a compound having seven benzene rings conjugated at the contact interface, its stabilization energy can be calculated as 14 to 21 kcal / mol. Therefore, it can be determined that the fullerene compound has a stabilizing energy of about 14 to 21 kcal / mol.

The bicelle of the present invention can contain glycosides of the above-mentioned specific compounds. The glycoside is a compound in which the above-mentioned specific compound and a monosaccharide or a polysaccharide are glycosidic bonded.

The specific glycosidic bond in the glycoside is not particularly limited as long as the effect of the present invention is exhibited. For example, O-glycosidic bond, N-glycosidic bond, C-glycosidic bond and the like can be mentioned. In view of the simple production of the glycoside, it is preferable to use an O-glycosidic bond.

The specific mode of anomeric binding in the glycoside is not particularly limited as long as the effect of the present invention is exhibited. For example, it can be an α bond or a β bond. In view of the stability of the glycoside, β-bond is preferable.

The monosaccharide is not particularly limited as long as the effect of the present invention is exhibited. Specific examples thereof include monosaccharides of aldose, ketose, or deoxy sugar, which can have any structure of xylose ring, oxetose ring, pyranose ring, furanose ring, septanose ring, or octanose ring structure. it can. Among such monosaccharides, glucose, maltose, or galactose is preferable, and glucose is used, in view of the fact that the glycoside can be easily produced or the glycoside can be obtained at low cost. Is the most preferable. The monosaccharide may be an oxidized product such as uronic acid, aldonic acid, or aldaric acid, or a reduced product such as alditol.

The above-mentioned polysaccharide is not particularly limited as long as the effect of the present invention is exhibited. Specifically, a polysaccharide in which two or more of the above monosaccharides are glycosidic bonded to each other can be mentioned. The monosaccharide can be similar to a monosaccharide that glycosidic bonds to the specific compound described above. The specific number of monosaccharides bound to the above-mentioned polysaccharide is not limited as long as the effect of the present invention is exhibited. For example, 3 pieces, 4 pieces, 5 pieces, 6 pieces, 7 pieces, 8 pieces, 9 pieces, 10 pieces, 11 pieces, 12 pieces, 13 pieces, 14 pieces, or 15 pieces can be mentioned.

The specific glycosidic bond of two or more monosaccharides in the above-mentioned polysaccharide is not particularly limited as long as the effect of the present invention is exhibited. For example, O-glycosidic bond, N-glycosidic bond, C-glycosidic bond and the like can be mentioned. The specific glycosidic bonds of such two or more monosaccharides may be the same or different. Considering that the glycosides can be easily produced or that the glycosides can be obtained at low cost, it is preferable that all the glycosides have the same glycoside bond, and above all, it is preferable that all the glycosides have an O-glycosidic bond.

The specific mode of anomeric binding of two or more monosaccharides in the above-mentioned polysaccharide is not particularly limited as long as the effect of the present invention is exhibited. For example, it can be an α bond or a β bond. The specific anomeric binding modes of the two or more monosaccharides may be the same or different. Considering that the glycosides can be easily produced or that the glycosides can be obtained at low cost, it is preferable that all the glycosides have the same anomeric bond, and among them, all of them are preferably α-bonds.

In the above-mentioned polysaccharide, the binding mode between specific carbon numbers of two or more monosaccharides is not particularly limited as long as the effect of the present invention is exhibited. For example, (1-1) glycosidic bond, (1-2) glycosidic bond, (1-3) glycosidic bond, (1-4) glycosidic bond, (1-6) glycosidic bond mode and the like can be mentioned. The binding mode between the specific carbon numbers of such two or more monosaccharides may be the same or different. Considering that the glycoside can be easily produced or that the glycoside can be obtained at low cost, it is preferable to use the same bonding mode between all carbon numbers, and among them, all (1-4) glycosides. It is preferably a bond.

Among the above-mentioned specific compounds having a stabilizing energy of 3 to 25 kcal / mol or less, resveratrol, pterostilbene, piceid, kaempferol, indomethacin, or C60 fullerene is preferable.

The specific compound or glycoside thereof contained in the bicelle of the present invention can be substituted or modified with various labels for the purpose of confirming the behavior of the bicelle. The specific label is not particularly limited as long as the effect of the present invention is exhibited. For example, radioactive isotopes such as deuterium, ¹³ C, or ³² P; fluorescent dyes such as nitrobenzoxadiazole (NBD) groups, luciferin, rhodamine, fluorescein, or derivatives thereof; or GFP, KFP, RFP, or Fluorescent proteins such as YFP can be mentioned.

The bicelle of the present invention may contain one or more of the above-mentioned specific compounds or glycosides thereof. The content of the specific compound or glycoside thereof in the bicelle of the present invention is not particularly limited as long as the effect of the present invention is exhibited. Specifically, it can be usually about 0.01 parts by mass to 40 parts by mass with respect to 100 parts by mass of the bicelle of the present invention. It is preferably about 0.01 parts by mass to 30 parts by mass.

The bicelle of the present invention has a disk-like structure. More specifically, as shown in FIG. 11, it has two flat portions (1) and a bent portion (2), and the major axis of the flat portion can be defined as the size of the bicelle of the present invention. it can. The size of such a bicell can be measured, for example, by an electron microscope image represented by TEM or the like, as shown in FIG. The specific size of the bicelle of the present invention is not particularly limited as long as the effect of the present invention is exhibited. Specifically, it is about 10 nm or more and 5 μm or less, preferably about 20 nm or more and 3 μm or less. More preferably, it is about 20 nm or more and 2 μm or less.

Further, the size of the bicelle of the present invention can be an average particle diameter measured by a laser diffraction method, a dynamic light scattering (DLS) method, or the like. Based on the numerical values measured by these methods, the particle size (particle size at which the cumulative frequency is 50% in the frequency distribution of volume fraction or fraction) can be calculated. The particle size of the bicelle of the present invention is about 10 nm or more and 5 μm or less. It is preferably about 20 nm or more and 3 μm or less. More preferably, it is about 20 nm or more and 2 μm or less.

As shown in FIG. 11, all of the above-mentioned specific compounds or glycosides (3) thereof are contained in a fully encapsulated manner inside the lipid aggregate which is the basic structure of bicelle. Alternatively, the compound or a part of a glycoside thereof may be contained in an incorporated manner.

The thickness of the bicelle of the present invention corresponds to the region between the two flat portions (1). The specific thickness of the bicelle of the present invention is not particularly limited as long as the effect of the present invention is exhibited. Specifically, it can be about 2 to 6 nm.

The bisels of the present invention include Na ⁺ , K ⁺ , NH ⁴⁺ , Li ⁺ , Mg ²⁺ , Ca ²⁺ , tetramethylammonium (Me ₄ N ⁺ ), tetraethylammonium (Et ₄ N ⁺ ), tetrapropylammonium (Pr ₄ N). It can also have a counter ion such as ⁺ ), tetrabutylammonium (Bu ₄ N ⁺ ), or trishydroxymethylaminomethane ion (Tris).

For the purpose of confirming the behavior of the bicelle of the present invention, the anionic phospholipid contained in the bicelle can be replaced or modified with various labels. The specific label is not particularly limited as long as the effect of the present invention is exhibited. For example, it can be the same as the label described in the specific compound contained in the bicelle of the present invention or its glycoside.

Since the bicelle of the present invention can contain the above-mentioned specific compound, it can be expected to be used as an external composition (cosmetic composition or pharmaceutical composition). Further, the particle size of the bicelle of the present invention is as small as about 10 nm or more and 5 μm or less, and it is expected to exert an effect of penetrating into the skin, so that it can be used as an external composition.

<External composition>
Since the particle size of the bicelle of the present invention of the present invention is as small as about 10 nm or more and 5 μm or less, this can be effectively used as an external composition. Therefore, the external composition of the present invention contains the above-mentioned bicelle of the present invention. Examples of such an external composition include a pharmaceutical composition or a cosmetic composition.

<Cosmetic composition>
The cosmetic composition, which is one aspect of the external composition of the present invention, also includes quasi-drugs such as medicated cosmetics. The content of the bicelle of the present invention in such a cosmetic composition is not particularly limited. For example, it can be usually set in a numerical range larger than 0 parts by mass with respect to 100 parts by mass of the cosmetic composition, and the upper limit thereof is about 5 parts by mass, about 10 parts by mass, about 15 parts by mass, and so on. It can be about 20 parts by mass, about 25 parts by mass, or about 30 parts by mass.

The specific embodiment of the cosmetic composition is not particularly limited as long as the effects of the present invention are exhibited. For example, make-up cosmetics such as foundation, cheek red, or white powder; basic cosmetics such as conditioner, milky lotion, skin cream, lotion, oil, or pack; skin cleanser such as face wash, cleansing, or body soap; shampoo , Rinse, conditioner, hair conditioner, hair restorer, and other hair cosmetics; bath salts, bath tablets, bath liquids, and other bathing agents, massage agents, and cleaning agents.

The shape of the cosmetic composition is not particularly limited as long as the effects of the present invention are exhibited. For example, lotion-like (liquid), mousse-like, gel-like, jelly-like, emulsion-like, suspension-like, cream-like, ointment-like, sheet-like, aerosol-like, spray-like and the like can be mentioned. By forming the cosmetic composition into such a shape, it can be applied not only to the skin but also to the scalp.

The specific amount of the cosmetic composition applied is not particularly limited as long as the effects of the present invention are exhibited. The amount of the cosmetic composition provided can be appropriately set based on the gender and age of the person to be applied, the application form of the cosmetic composition, the degree of desired effect, etc., and the book in the cosmetic composition. In terms of the amount of the above-mentioned specific compound contained in the bicelle of the present invention, the amount can be, for example, about 1 to ⁵ mg with respect to 1 cm ^{2 of the} skin or scalp.

<Pharmaceutical composition>
The specific content of the bicelle of the present invention in the pharmaceutical composition which is one aspect of the external composition of the present invention is not particularly limited as long as the effect of the present invention is exhibited. For example, it can be usually set in a numerical range larger than 0 parts by mass with respect to 100 parts by mass of the pharmaceutical composition, and the upper limit thereof is about 5 parts by mass, about 10 parts by mass, about 15 parts by mass, 20 parts. It can be about parts by mass, about 25 parts by mass, or about 30 parts by mass.

The specific dosage form of the pharmaceutical composition is not particularly limited as long as the effects of the present invention are exhibited. For example, aerosols, liquids, extracts, (eye) ointments, transdermal preparations, suspensions, emulsions, lotions, patches, tinctures, poultices, which are mainly used externally. Dosage forms such as aromatic water agents, liniment agents, flow extracts, lotions and the like can be mentioned.

The pharmaceutical composition can be produced by blending the bicelle of the present invention with a pharmacologically acceptable and acceptable known carrier or additive in order to obtain the above-mentioned topical dosage form. The specific carrier or additive is not particularly limited as long as the effect of the present invention is exhibited. For example, any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, medium, delivery system, emulsifier, tablet degrading substance, absorbent, preservative, surfactant, colorant, fragrance, etc. Can be mentioned.

The specific dose of the pharmaceutical composition is not particularly limited as long as the effects of the present invention are exhibited. The amount of the pharmaceutical composition provided can be appropriately set based on the gender and age of the target person, the application form of the pharmaceutical composition, the degree of desired effect, etc., and the bicelle of the present invention in the pharmaceutical composition can be appropriately set. In terms of the amount of the above-mentioned specific compound contained in, the amount can be, for example, about 1 to 5 mg / cm ² per day for a 60 kg human adult.

<Manufacturing method of Bisel>
The bicelle of the present invention can be produced by the following steps (1) and (2);
(1) A step of dispersing a mixture of an anionic phospholipid and a compound having a stabilizing energy of 3 to 25 kcal / mol or less in an aqueous dispersion medium under conditions below the phase transition temperature of the anionic phospholipid. , And (2) a step of heating the aqueous dispersion obtained in the above step (1) to a temperature equal to or higher than the phase transition temperature of the anionic phospholipid.

Process 1
In step 1, a mixture of an anionic phospholipid and a compound having a stabilizing energy of 3 to 25 kcal / mol or less is dispersed in an aqueous dispersion medium under conditions below the phase transition temperature of the anionic phospholipid. ..

The anionic phospholipid used in step 1 can be the same as that described in <Bisell> above.

The specific amount of the anionic phospholipid to be dispersed in the aqueous dispersion medium in step 1 is not particularly limited as long as the effect of the present invention is exhibited. For example, the concentration of the anionic phospholipid in the aqueous dispersion medium can be usually about 2 to 10% by mass, preferably about 3 to 7% by mass, and more preferably about 5% by mass.

The compound (specific compound) having a stabilizing energy of 3 to 25 kcal / mol or less used in step 1 can be the same as that described in <Bisell> above. The amount of the specific compound dispersed in the aqueous dispersion medium in step 1 is not particularly limited as long as the effects of the present invention are exhibited. Specifically, the concentration of the specific compound in the aqueous dispersion medium can be, for example, about 0.01 to 40% by mass, preferably about 0.1 to 30% by mass.

The specific aqueous dispersion medium used in step 1 is not particularly limited as long as the effects of the present invention are exhibited. For example, deionized water, distilled water, water such as pure water; or phosphate buffer, citrate phosphate buffer, Tris-HCl buffer, Tris-EDTA buffer, or Tris-EDTA buffer, which can be adjusted to pH 6-8. A buffer solution such as a HEPES buffer solution can be mentioned.

In step 1, the temperature at which the mixture of the anionic phospholipid and the specific compound is dispersed in the aqueous dispersion medium is a temperature lower than the phase transition temperature of the anionic phospholipid. The temperature below the specific phase transition temperature is not particularly limited. For example, room temperature can be mentioned. The term "room temperature" as used herein means a temperature at which the reaction system is neither heated nor cooled from the outside, and can vary depending on the season, altitude, or atmospheric pressure. Although it cannot be determined, for example, a temperature of about 1 to 35 ° C. can be mentioned.

The specific means for dispersing the mixture of the anionic phospholipid and the specific compound in the aqueous dispersion medium is not particularly limited as long as the effect of the present invention is exhibited. For example, a means for simply mixing the two, or a means for subjecting the two to ultrasonic treatment after mixing the two can be mentioned.

Process 2
In step 2, the aqueous dispersion prepared in step 1 is heated to a temperature equal to or higher than the phase transition temperature of the anionic phospholipid.

In step 2, when two or more kinds of the anionic phospholipids are used, the heating temperature is set to be equal to or higher than the phase transition temperature of the anionic phospholipid having the highest phase transition temperature. The specific heating temperature in step 2 is not particularly limited as long as the effect of the present invention is exhibited. For example, a temperature of about 2 ° C. or higher and 30 ° C. or lower can be mentioned from the phase transition temperature of the anionic phospholipid.

The specific heating means in the step 2 is not particularly limited as long as it exhibits the effect of the present invention and the temperature of the entire aqueous dispersion becomes higher than the desired temperature. For example, a method of immersing the dispersion liquid in a container in a hot water bath to warm it, a method of irradiating microwaves with a microwave oven, and the like can be mentioned.

The specific heating time in step 2 can be appropriately set by the heating means, and is not particularly limited as long as the effect of the present invention is exhibited. For example, it can be about 0.1 to 60 minutes.

The method for producing a bicelle of the present invention may be provided with step 3 shown below, if necessary.

Process 3
In step 3, the aqueous dispersion heated in step 2 is cooled to a temperature lower than the phase transition temperature of the anionic phospholipid.

The specific cooling temperature in step 3 is not particularly limited as long as the effect of the present invention is exhibited. For example, a temperature of about 2 ° C. or lower than the phase transition temperature of the anionic phospholipid can be mentioned. The temperature is preferably about 15 ° C. or lower.

The specific cooling means in the step 3 is not particularly limited as long as it exhibits the effect of the present invention and the temperature of the entire aqueous dispersion becomes lower than the desired temperature. For example, a means for allowing the aqueous dispersion to stand at room temperature or a means for immersing the aqueous dispersion in an ice bath can be mentioned.

The specific cooling time in the step 3 can be appropriately set by the cooling means, and is not particularly limited as long as the effect of the present invention is exhibited. For example, it can be about 0.1 to 60 minutes.

Examples are shown below for explaining the present invention in more detail. Needless to say, the present invention is not limited to the inventions described in the following examples.

<Test Example 1>
Production of resveratrol-containing bicelle (DPPG) An attempt was made to produce resveratrol-containing bicelle (DPPG) by the procedure shown in FIG. Here, the main component of the lipid constituting the bicelle is dipalmitoylphosphatidylglycerol (DPPG). Specifically, 5% by weight DPPG and 1 mM resveratrol powder were mixed at room temperature, and then the mixed solution was subjected to ultrasonic treatment for 15 minutes to disperse both. The dispersion was heated to 60 ° C. and then cooled to room temperature. A photographic image of the obtained dispersion is shown in FIG.

As shown in FIG. 1, a transparent dispersion was obtained. Therefore, since a substance that is uniformly dispersed in water was obtained, it was suggested that the substance is a bicelle containing DPPG as a main lipid constituent, and that the bicelle contains resveratrol.

<Test Example 2>
Production of Bicell (DPPG, DSPG, or DPPA) Containing Various Compounds An attempt was made to produce bicell containing various compounds by the same method as in Test Example 1 above. Specifically, instead of resveratrol used in Test Example 1, 1 mM piceid (IUPAC name: 2- [3-Hydroxy-5-[(E) -2- (4-hydroxyphenyl) ethenyl] phenoxy] The production of bicelle was attempted in the same manner as in Test Example 1 except that -6- (hydroxymethyl) oxane-3,4,5-triol, 1 mM pterostilbene, and 0.1 mM fullerene were used.

Further, a bicell production was attempted in the same manner as in Test Example 1 except that DSPG (distearoylphosphatidylglycerol) and DPPA (dipalmitoylphosphatidylate) powder having the same concentration were used instead of DPPG. Then, as a comparative example, production of bicelle was attempted in the same manner as in Test Example 1 except that DPPC (dipalmitoylphosphatidylcholine) having the same concentration, which was not an anionic phospholipid, was used. These results (photographic images) are shown in FIG.

FIG. 2a shows a bicell containing resveratrol-containing DPPC as a main lipid component or a bicell containing DPPG as a main lipid component. It was revealed that when DPPC was used, the obtained dispersion became cloudy, unlike the case where DPPG was used. From these results, it was clarified that the use of DPPC, which is not an anionic phospholipid, agglomerates having a large particle size were formed, suggesting that resveratrol-containing bicelle was not produced. Similarly, when piceid of FIG. 2 b, pterostilbene of c, fullerene of d, kaempferol of e, and indomethacin of f are used, if DPPC is the main lipid component, DPPG is the main lipid component. It was clarified that the obtained dispersion became cloudy, unlike the case where it was used as a component.

Further, as shown in g of FIG. 2, DSPG or DPPA, which is an anionic phospholipid, is the main lipid constituent component (the compound contained therein is the same resveratrol as in FIGS. 1 and 2a. ), A transparent dispersion that does not become cloudy was obtained.

From the above results, by the method shown in Test Example 1, a rigid structure such as resveratrol, piceid, and fullerene was added to a bicelle containing anionic liposomes such as DPPG, DSPG, and DPPA as the main lipid constituents. It was suggested that the compound to be contained could be contained.

<Test Example 3>
Resveratrol-containing bicelle and piceid-containing bicelle ³¹ P NMR measurement By the same method as in Test Example 1, 5 wt% DPPG and 0.2 mM resveratrol were used as raw materials, and 5 wt% DPPG and 0. An attempt was made to produce bicelle using 2 mM piceid as a raw material. The obtained dispersion was subjected to 31P NMR measurement. The measuring instrument used was (Bruker, Ascend ^TM- 600). The results are shown in FIGS. 3A (resveratrol) and B (piceid).

As shown in FIGS. 3A and 3B, peaks in the region where the δ value was negative, which is peculiar to the bicell structure, were observed in both. Therefore, it was clarified that the bicell containing resveratrol and piceid was produced by the method shown in Test Example 1.

<Test Example 4>
Resveratrol-containing bicelle and piceid-containing bicelle using the same method as in TEM imaging test example 1, using 5% by weight DPPG and 10 mM resveratrol as raw materials, and 5% by weight DPPG and 10 mM piceid. Bisel was manufactured as a raw material. A TEM image of the bicelle produced in this way was taken. The equipment used for photography is (JEOL, JEM-1230). The results are shown in FIGS. 3A (resveratrol-containing bicelle) and B (piceid-containing bicelle).

As shown in FIGS. 4A and 4B, a disk region as shown by the dotted line was observed, so it is clear that a disk-shaped bicelle containing resveratrol and piceid is produced by the method shown in Test Example 1. It became.

<Test Example 5>
SAXS Analysis Results of Resveratrol-Containing Bisel Bisel was produced using 0.5% by weight of DPPG and 0.1 mM resveratrol as raw materials in the same manner as in Test Example 1. The SAXS analysis of the bicelle produced in this way was performed. The instrument used for the analysis is (SPring8 beamline BL45XU). The result is shown in FIG.

The slope of the graph (q- ² ) obtained by SAXS analysis allows us to understand the structure of the particles in the dispersion. For example, if the inclination is "-4", it is a spherical structure, if it is "-1", it is a rod-like structure, and if it is "-2", it is a disk-like structure. ..

From the results shown in FIG. 5, it can be read that the slope (q- ² ) is " ^-2 ". Therefore, only DPPG containing resveratrol obtained by the method shown in Test Example 1 is a lipid component. It was clarified that the bicelle has a disk-like structure.

<Test Example 6>
Absorption spectrum analysis results of resveratrol-containing bicelle and piceid-containing bicelle Using 5% by weight DPPG and 1 mM resveratrol as raw materials, and 5% by weight DPPG and 1 mM piceid in the same manner as in Test Example 1. And was used as a raw material to manufacture Bisel. The absorption spectra of resveratrol and piceid in water were analyzed together with the resveratrol and piceid-free bicelle produced in this manner. The equipment used for the analysis is (JASCO, V-760, UV / VIS / NIR spectrum meter). The results are shown in FIGS. 6A and 6B.

Since resveratrol and piceid were insoluble in water, a peak near 300 nm specific to the stilbene structure was not observed, but resveratrol and piceid were contained in bicelle containing DPPG as a main lipid component. As a result, it was dispersed in water, and a peak near 300 nm, which is specific to the stilbene structure, was observed.

From these results, it was clarified that resveratrol and piceid are contained in the bicelle containing DPPG as a main lipid component obtained by the method shown in Test Example 1.

<Test Example 7>
Particle size measurement of resveratrol, piceid, fullerene, kenferol, or indomethacin-containing bicelle In the same manner as in Test Example 1, 5% by weight DPPG or DPPC and 1 mM resveratrol, piceid, fullerene, kenferol, Alternatively, bicell was produced using fullerene as a raw material. Resveratrol, piceid, and fullerene were subjected to laser diffraction together with each of the bicelles thus produced, and the particle size of each was measured. The instrument used for the measurement is (Horiba, LA-960 laser diffraction particle size analyzer). The results are shown in FIGS. 7A (resveratrol-containing bicelle), B (piceid-containing bicelle), C (fullerene-containing bicelle) D (kaempferol-containing bicelle), and E (indomethacin-containing bicelle).

From the results shown in FIGS. 7A to 7C, when resveratrol, piceid, and fullerene are contained in bicelle containing DPPC, which is not an anionic phospholipid, as a main lipid component, the average particle size before the inclusion is almost the same or slightly. Although the average particle size becomes smaller, when resveratrol, piceid, fullerene, kenferol, and indomethacin are contained in bicelle containing DPPG, which is an anionic phospholipid, as the main lipid component, DPPC becomes the main lipid component. It has been clarified that the average particle size can be remarkably reduced as compared with the case where these compounds are contained in the bicelle. A bicelle having such a small particle size is effectively used as an external composition.

<Test Example 8>
Particle size measurement of resveratrol, piceid, or fullerene-containing bicelle Using the same method as in Test Example 1, using 5% by weight DPPG and 1 mM resveratrol as raw materials, and 5% by weight DPPG and 1 mM piceid. And was used as a raw material to manufacture bicell. The bicelle thus produced was subjected to ultrasonic treatment for 3 hours, and then the particle size was measured by the DLS method. The instrument used for the measurement is (Malvern, Zetasizer Nano ZSP). The results are shown in FIGS. 8A (resveratrol-containing bicelle) and B (piceid-containing bicelle).

From the results shown in FIGS. 8A and 8B, it was clarified that both the resveratrol-containing bicelle and the piceid-containing bicelle were fine particles of 70 nm or less. Such an effect is an effect obtained by a bicelle having an anionic phospholipid as a main lipid component. For example, in a lipid aggregate containing a neutral anionic phospholipid as a main lipid component, an external preparation is thus used. It is difficult to obtain a small average particle size that can be suitably used as a.

<Test Example 9>
Subcutaneous Penetration Behavior of Resveratrol, Piceid, or Fulleren-Containing Bisel In the same manner as in Test Example 1, 5% by weight of DPPG and 0.1% by weight of NBD-modified resveratrol were used as raw materials. Bicells were made from DPPG and 0.025 wt% FITC-modified piceid, and from 5 wt% NBD-modified DPPG and 0.1 wt% fullerene. Each of the bicells thus produced was administered to rat skin (hairless rat, 8 weeks old, male) set on Franzcel, and a fluorescence photograph was taken 24 hours later, and the result is shown in FIG. In addition, the amount of permeation through each tissue (stratum corneum and epidermis / dermis layer) and Franz cell of rat skin is shown in FIG.

As is clear from the results shown in FIGS. 9 and 10, it was clarified that the resveratrol, piceid, and fullerene-containing bicelle penetrate not only into the stratum corneum of rat skin but also into the epidermis and dermis.

1 Plane part of Bisel 2 Bending part of Bisel 3 Compound or glycoside having stabilizing energy of 3 to 25 kcal / mol (excluding paclitaxel)

Claims (12)

A bicelle containing 50% by mass or more of anionic phospholipid and at least one compound (excluding paclitaxel) selected from the group consisting of stilbenoids, flavonoids, indomethacin compounds, and fullerene compounds, or glycosides thereof. The bicell according to claim 1, wherein the average particle size of the bicell is 10 nm or more and 5 μm or less. The bicelle according to claim 1 or 2, wherein the anionic phospholipid is a glycerophospholipid. The bicelle according to claim 3, wherein the anionic phospholipid is a glycerophospholipid represented by the following formula (1);
(In the formula, R ¹ is the same or independently a linear or branched chain alkyl group having 10 to 36 carbon atoms, or a linear or branched chain alkenyl group. R ² is a nonionic group or an anionic group. Is.) The R ¹ is the same or independently, nonyl group, decyl group, undecylic group, dodecyl group, tridecyl group, tetradecyl group, pentadecanoic group, hexadecyl group, heptadecyl group, octadecyl group, nonadecylic group, icosyl group, henicosyl group, Docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonadecylic group, triacontyl group, hentolycontyl group, dotriacyl group, tritriacontyl group, tetratriacontyl group, pentatriacontyl group The bicell according to claim 4, which is selected from the group consisting of a group, a hexatriacontyl group, and an alkenyl group having 1 to 5 double bonds attached to these groups. The bicell according to claim 4, wherein R ² is selected from the groups represented by the following formulas (2-1) to (2-4);
The bicelle according to any one of claims 1 to 6, wherein the anionic phospholipid is at least one selected from the group consisting of DPPG, DSPG, and DPPA. The bicell according to any one of claims 1 to 7 , which satisfies at least one of the following requirements (i) to (iv);
(I) The stilbeneoid is at least one selected from the group consisting of resveratrol, piceatannol, oxyresveratrol, rhapontigenin, isolapontigenin, pterostilbene, gnetol, pinosylvin, and pinostilbene.
(Ii) The flavonoids are kenferol, anthocyanidin, anthocyanin, flabanone, naringenin, flavan, catechin, epicatechin, epicatechin gallate epigalocatechin, epigalocatechin gallate, flavon, flavon derivative, flavonol, quercetin, isoflavone, isoflavone. , Isoflavone, isoflavone diol, neoflavonoid, biflavonoid, and at least one selected from the group consisting of aurone.
(Iii) The indomethacin compound is at least one selected from the group consisting of indomethacin, indometacin farnesyl, acemetasin, indomethacin morpholinylamide, pravadrine, and 1-pentyl-3- (4-methoxybenzoyl) indole.
(Iv) The fullerene compound is C60 fullerene, C70 fullerene, C76 fullerene, C78 fullerene, C84 fullerene, C60-SAM, 60 pyrrolidinetris-acid, polyhydroxylated C60, C60 ethyl nipecotate, Bis [60] PCBM, [ It is at least one selected from the group consisting of 60] PCBM, [70] PCBM, [84] PCBM, [60] PCB-C4, [60] PCB-C8, and [60] ThCBM. The bicelle according to any one of claims 1 to 8 , wherein the glycoside is a monosaccharide glycoside or a polysaccharide glycoside. The bicelle according to any one of claims 1 to 9, wherein the compound or a glycoside thereof is any one of resveratrol, pterostilbene, piceid, kaempferol, indomethacin, or C60 fullerene. An external composition containing the bicelle according to any one of claims 1 to 10 . The method for producing a bicelle according to any one of claims 1 to 10 , which comprises the following steps (1) and (2);
(1) A mixture of an anionic phospholipid and at least one compound (excluding paclitaxel) selected from the group consisting of a stilbenoid, a flavonoid, an indomethacin compound, and a fullerene compound, or a glycosylation thereof is used as the anionic phospholipid. Under conditions below the phase transition temperature of the lipid, the step of dispersing in the aqueous dispersion medium and (2) heating the aqueous dispersion obtained in the above step (1) to a temperature equal to or higher than the phase transition temperature of the anionic phospholipid. Process.