JP2011074058A - Reverse vesicle made of biosurfactant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide new reverse vesicles that can be formed in oily substances of great many kinds by simple operation and are suitable to be blended into cosmetics, drugs or the like because of excellent transdermal absorption. <P>SOLUTION: A sugar-type biosurfactant compound is agitated in a liquid oily substance to form the reverse vesicles. The stability of the reverse vesicles is increased by adding water to the liquid oily substance, which enables use of various oily substances. The formed reverse vesicles can be used as a compounding agent for cosmetics or drugs having a wide range of functions, by adding an agent to the water, liquid oily substance or both of them. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an inverted vesicle formed with a sugar-type biosurfactant as a main component.

It is known that the human stratum corneum is formed by laminating intercellular lipids in layers (lamella structure), and exhibits an important function for a barrier function and a moisturizing function. For external preparations for skin such as creams and emulsions, it is required to improve such functions and improve the condition of the skin as an excellent preparation. From the viewpoint of enhancing the above functions, it is desirable in terms of permeability and moisturizing effect to provide a preparation having the same lamellar structure as that of the skin.
Lipids or amphiphiles that have both hydrophilic and hydrophobic groups in the molecule are oriented in the order of (continuous phase) hydrophilic group-hydrophobic group; hydrophobic group-hydrophilic group (inner side) in an aqueous solution that is a continuous phase. An endoplasmic reticulum (capsule) composed of a bilayer lamellar structure is called a “vesicle”. A preparation utilizing vesicles is considered to have a higher function in terms of permeability, moisturizing effect and the like because the structure is similar to the cell structure. Compared to micelles and emulsions, it has a large interfacial (surface) area through a bilayer membrane and is excellent in mass transfer and transport, so it is also effective for drug and food applications such as drug delivery system (DDS) carriers. Is big.

However, vesicles in which the continuous phase is not water but oil (nonpolar solvent), that is, “reverse vesicles (inverted vesicles)” ((continuous phase side) hydrophobic group-hydrophilic group; hydrophilic group-hydrophobic group (inner side) in this order. There are very few reports on vesicles composed of oriented bilayers, and there are no examples of their application. This is because, in the first place, the materials that can form reverse vesicles are limited, the conditions (oil type, temperature) that can be formed are very narrow, and the manufacturing method is complicated.
Reverse vesicles were first reported in 1991 by Kunieda et al. (Non-Patent Document 1), but there were also limited reports after that, and reports were made with nonionic synthetic surfactants whose hydrophilic groups were polyoxyethylene type. (Non-Patent Documents 2 to 4). In the case of natural materials, phospholipid (lecithin) -based systems (Non-Patent Documents 5 and 6) and glycolipid-based sucrose fatty acid monoester systems (Non-Patent Documents 7 to 9) have also been reported. However, in many cases, the type of oily substance that becomes a continuous phase is limited, and the addition of a third component such as an oily substance other than the continuous phase or another surfactant is required. Recently, with the aim of producing stable reverse vesicles, reports have been made on selection and combination of raw materials and improvement of the mixing method (Patent Documents 1 and 2), but the conditions are very limited. Development of a method for easily forming reverse vesicles in a wide range of oily substances is strongly required in practice.

JP 2009-62365 A JP 2009-62386

H. Kunieda et.al., J. Am. Chem. Soc., 113, 1051-1052 (1991) H. Kunieda et.al., Langmuir, 7, 1915-1919 (1991) H. Kunieda et.al., J. Colloid Interfac. Sci., 150, 277-280 (1992) H. Kunieda et.al., Langmuir, 15, 3118-3122 (1999) H. Kunieda et.al., Adv. Mater., 4, 291-293 (1992) S-H. Tung et.al., J. Am. Chem. Soc., 130, 8813-8817 (2008) H. Kunieda et.al., J. Phys. Chem., 97, 9525-9531 (1993) H. Kunieda et.al., J. Colloid Interfac. Sci., 156, 446-453 (1993) H. Mollee et.al., J. Pharm. Sci., 89, 930-939 (2000)

  An object of the present invention is to provide a reverse vesicle by a simple operation using a single component in a wide variety of oily substances.

The present inventors have developed biosurfactants (amphiphiles produced by microorganisms) having excellent liquid crystal forming ability, such as easily forming various molecular aggregates by self-assembly in solution based on their unique molecular structure. We have found that reverse vesicles can be easily formed in a wide range of liquid oily substances by using this material.
That is, the present invention is as follows.

(1) A reverse vesicle comprising a self-assembled bilayer membrane of a sugar-type biosurfactant compound.
(2) The reverse vesicle according to (1), wherein the sugar-type biosurfactant compound is mannosyl alditol lipid represented by the following general formula (1).
[In the above formula (1), R ¹ represents a linear or branched saturated or unsaturated aliphatic acyl group having ² to 24 carbon atoms, and R ² represents hydrogen or an acetyl group. R ³ represents hydrogen or a linear or branched saturated or unsaturated aliphatic acyl group having 2 to 24 carbon atoms. Two R ² and R ³ may be the same or different. n is an integer of 1-4. ]
(3) The reverse vesicle according to (2) above, wherein the compound represented by the general formula (1) has a structure described in the following formula (2).
(In the formula, R represents an aliphatic acyl group having 4 to 20 carbon atoms which may be the same or different.)
(4) The reverse vesicle according to any one of (1) to (3) above, which contains a drug.
(5) The method of forming a reverse vesicle according to (1) above, wherein the sugar-type biosurfactant compound described in the general formula (1) is stirred in a nonpolar liquid oily substance to which water has been added.
(6) The method for producing a reverse vesicle according to (5) above, wherein a water-soluble drug is contained in water and / or an oil-soluble drug is contained in a nonpolar liquid oily substance.

Biosurfactant is an amphiphile produced by microorganisms using various types of biomass as raw materials, and has excellent compatibility with the environment and living organisms. Therefore, it should be converted into a reverse vesicle and used in various pharmaceutical materials such as cosmetics and topical skin preparations. Is very useful in terms of safety compared to existing synthetic surfactant systems. In addition, biosurfactant exhibits high surface activity at a very low concentration as compared with general-purpose synthetic surfactants, and also has unique self-organization characteristics and various physiological functions. Therefore, compared to existing compounds, the same or better effect can be expected when used in a small amount, and biofunctionality utilizing various physiological activities can be imparted to the reverse vesicles. The effect is expected to be extremely high.
Furthermore, by using the present invention, reverse vesicles can be formed with a single component in a very wide variety of solvents that have not been obtained in the past. The type of oil can be selected according to the application, and a wide variety of products such as creams, emulsions and ointments can be provided.

4 is a photograph showing the result of microscopic observation of the reverse vesicle prepared in Example 1. FIG. FIG. 3 is a diagram showing the results of small-angle X-ray scattering (SAXS) measurement of an inverted vesicle prepared in Example 1. It is the photograph which showed the result of the fluorescence observation by the confocal laser microscope of the reverse vesicle which includes the calcein aqueous solution prepared in Example 23.

[Biosurfactant]
Currently, biosurfactants (surfactants produced by microorganisms) are classified into five groups: glycolipids, acyl peptides, phospholipids, fatty acids, and polymer compounds. Among them, glycolipid surfactants are most well studied because they are excellent in productivity and can be mass-produced, and are expected to have high physiological activity derived from sugar chains. Substances have been reported.
Including Pseudomonas aeruginosa (Pseudomonas aeruginosa), mainly rhamnolipid such that Pseudomonas (Pseudomonas) bacteria of the genus produce, sophorolipid acids including Sutamerera Bonbikora (Starmerella bombicola), which is mainly Candida (Candida) yeast of the genus production, Corynebacterium (Corynebacterium) or the genus Rhodococcus (Rodococcus) genus trehalose lipid compounds that bacteria production, including, Ustilago Meidisu (Ustilago maydis) and Cryptococcus Humicola (Cryptococcus humicola) cellobiose lipid acids to produce bacteria, including such as, Pseudozyma ( Pseudozyma) genus mannosyl alditol lipid acids that yeast to production, such as, such as oligosaccharides lipid compounds that Tsukamurella (Tsukamurella) genus yeast to production, Baiosa typical glycolipid system Is Fakutanto.

These are excellent in the ability to form liquid crystals composed of bimolecular films, and are expected to have high suitability for vesicle-based preparations. In particular, mannosyl alditol lipid is desirable for forming reverse vesicles because it can form liquid crystals in a very wide range of concentrations and temperatures.
The chemical structure of mannosyl alditol lipid is represented by the general formula (1), and mannosyl alditol lipid (hereinafter referred to as MAL) is obtained by culturing MAL-producing microorganism, and 4-O-β-D-mannopyranosyl-alditol. Is the basic structure.
[In the above formula (1), R ¹ is an aliphatic acyl group having 2 to 24 carbon atoms, and includes a linear or branched saturated or unsaturated aliphatic acyl group. R ² represents hydrogen or an acetyl group. R ³ is hydrogen or a C2-C24 aliphatic acyl group, and includes a linear or branched saturated or unsaturated aliphatic acyl group. Two R ² and R ³ may be the same or different. n is an integer of 1-4. ]

  Here, alditol is a sugar alcohol in which both ends of a linear saccharide are reduced, and glycerin having 3 carbon atoms (n = 1), erythritol having 4 carbon atoms, and threitol (n = 2). Examples thereof include arabinitol having 5 carbon atoms, xylitol, ribitol (n = 3), allitol having 6 carbon atoms, sorbitol, mannitol, iditol, galactitol, and taritol (n = 4).

In addition, the type of the aliphatic acyl group of the substituents R ¹ and R ³ of the general formula (1) and the number of carbons thereof basically depend on the fatty acid in the fats and oils contained in the MAL production medium. The number of carbons varies depending on the degree of assimilation of fatty acids of the MAL-producing bacteria used. Therefore, each MAL obtained is usually in the form of a mixture of compounds in which the fatty acid residue portion of the substituent R is different.

Among the mannosyl alditol lipids, the mannosyl erythritol lipid represented by n = 2 in the formula (1) is most excellent in productivity, so that it is desirable to use it.
Mannosyl erythritol lipid (hereinafter referred to as MEL) used in the present invention is obtained by culturing a MEL-producing microorganism and is represented by n = 2 in the above formula (1). As typical examples of MEL, chemical structures of MEL-A, MEL-B, MEL-C, MEL-D, single-chain MEL, and three-chain MEL-A are shown below.

In these formulas, R ¹ is the same group as the above formula (1), and Ac represents an acetyl group.

These representative homologues can be selectively produced depending on the production microorganisms and the difference in raw materials. By way of example, the Pseudozyma Antakutika (Pseudozyma antarctica), etc. is MEL-A, the Pseudozyma tsukubaensis (Pseudozyma tsukubaensis) such that MEL-B, etc. Pseudozyma Guraminikora (Pseudozyma graminicola) to produce MEL-C as a main product It is known that each homologue can be selectively obtained by culturing them. Moreover, MEL-D can be manufactured by hydrolyzing only an acetyl group from these homologues. Furthermore, single-chain MEL is obtained by limiting the main raw material to hydrophilic substrates such as carbohydrates, and three-chain MEL is selectively added by adding a large amount of hydrophobic substrate to control the culture time. Is obtained.

In addition, there are optical isomers in which the erythritol moiety is inverted (Formula 4).
Many common MEL producing microorganism is producing MEL left conformation, Pseudozyma tsukubaensis (Pseudozyma tsukubaensis), Pseudozyma crassa (Pseudozyma crassa) are known to produce MEL right conformation .
The above homologues are known to exhibit different physical properties, but they can be easily isolated by a normal separation and purification process such as silica gel column chromatography.

[Formation and use of reverse vesicles]
Reverse vesicles can be formed by performing a method of forming vesicles in a normal aqueous system in various liquid oily substances.
The liquid oily substance is preferably nonpolar, and examples thereof include linear, unsaturated and cyclic hydrocarbons having 5 or more carbon atoms, but nonpolar silicone oils such as dimethylsilicone oil or cyclic dimethylsilicone oil may be used. It was confirmed that reverse vesicles were easily formed in these liquid oily substances.
Various known methods can be used in forming the reverse vesicle of the present invention, but it is generally preferable to use a thin film method (bangham method). In this method, a biosurfactant sample is dissolved in a volatile solvent such as chloroform or acetone and weighed in a container, and the solvent is removed by drying under reduced pressure to form a biosurfactant thin film on the inner surface of the container. Next, various non-polar liquid oily substances that become a continuous phase are added, and the lamella phase is dispersed in the oily substance by stirring or shaking with a vortex mixer or ultrasonic waves to form a reverse vesicle. The

At this time, the concentration of the biosurfactant sample is preferably 0.0001 to 50% by mass, preferably 0.001 to 20%, and more preferably 0.01 to 10%.
Further, at this time, it is possible to form a reverse vesicle without adding other components, but when water is added, the reverse vesicle can be formed more stably. When adding water, it is preferable to contain 0.0001-5 mass% by a total composition ratio, and it is preferable to contain 0.001-3%, and also 0.01-1%.
Furthermore, in order to separate the liquid oily substance and the reverse vesicle of the present invention by filtration, drying, etc., when more stability is required, when forming the reverse vesicle, further, a nonionic surfactant, Alternatively, known stabilization means such as addition of a stabilizer such as cholesterol may be applied.

  The film constituting the inverted vesicle of the present invention has a lamellar structure, and has a structure in which one or more self-assembled bilayer films are stacked as a structural unit, and is usually obtained in a mixture of these stacked states. It is done. The bimolecular film has a structure in which two monomolecular films are laminated, and the hydrophobic group (aliphatic acyl group) and the hydrophilic group (hydroxyl group) of the biosurfactant are hydrophobic groups from the outside-hydrophilic group :: hydrophilic group- Hydrophobic groups are oriented in order. Further, when two or more bimolecular films are laminated, the hydrophobic groups of the bimolecular film are aligned adjacently to form a laminated state.

On the other hand, the inside of the inverted vesicle is hollow, and the hollow space contains an oily substance used as a continuous phase when forming the inverted vesicle. Moreover, the added water can be contained in the hydrophilic group orientation portion of the self-assembled bilayer film. Therefore, when the reverse vesicle of the present invention contains a drug, the oily substance and / or water used in the above-described reverse vesicle formation may contain the drug. The reverse vesicle of the present invention includes an oil A soluble drug, a water-soluble drug, or both can be included.
That is, the drug that can be contained in the reverse vesicle of the present invention is not particularly limited, and examples thereof include anti-inflammatory agents, bactericides, astringents, preservatives, moisturizers, various fat-soluble physiologically active substances (hormonal agents, vitamins). Etc., cell activators, etc.).

  The reverse vesicle of the present invention can be blended in a cosmetic base material or a pharmaceutical base material itself or in a state of being present in the liquid oily substance. Since the reverse vesicle of the present invention has a lamellar structure similar to that of skin, it has good affinity with skin and excellent percutaneous absorption.

In the present invention, the formation of reverse vesicles usually means that the solution is observed under a microscope under polarized light according to the method of observing liquid crystal, and the self-assembly formed in the solution is not a reverse micelle. Can be confirmed from the anisotropic pattern of the structure, which is confirmed by having anisotropy. More specifically, it can be confirmed by seeing the Maltese cloth. In order to analyze in more detail, the bilayer structure (lamella structure) is confirmed by X-ray crystal structure analysis, and the structure is observed in a capsule (hollow) shape by an electron microscope.
The present invention will be described more specifically with reference to the following examples. However, these are merely examples, and the present invention is not limited thereto.

[Example 1]
Mannosyl erythritol lipid-D (hereinafter, MEL-D) was selected as a biosurfactant sample used for the formation of reverse vesicles. The molecular structure is shown in the following chemical formula (5).
Here, in the formula, R represents an aliphatic acyl group having 4 to 20 carbon atoms which may be the same or different, and the composition ratio of R of the sample used this time is as shown in Table 1.

(Reverse vesicle preparation method)
Prepare a 1% MEL-D chloroform / methanol (50/50 vol) solution, weigh 0.5 mL into a test tube, concentrate under reduced pressure using a rotary evaporator, and dry under reduced pressure to completely remove the solvent. A thin film of MEL-D was formed on the tube wall surface. Next, 6 mL of decane and 20 μL of water were added, and the solution was sufficiently stirred using a vortex mixer and ultrasonic shaking to obtain a cloudy solution.

(Confirmation of reverse vesicle formation)
The obtained solution was observed with a microscope, and a spherical assembly was observed by differential interference observation. This assembly was observed as a Maltese cross under crossed Nicols by polarization observation, confirming the formation of a reverse vesicle ( FIG. 1). Furthermore, after a lapse of time, a lower layer portion (precipitate) formed by sedimentation of the reverse vesicle was collected, and a result of small-angle X-ray scattering (SAXS) measurement (FIG. 2), a lamella at a ratio of 1: 2: 3. A peak indicating the structure was detected. This result also confirmed that the biosurfactant sample formed a lamellar structure in decane, which supported the formation of inverted vesicles by dispersion.

[Examples 2 to 14]
Instead of decane as a continuous phase in Example 1, hexane, heptane, octane, dodecane, tetradecane, hexadecane, cyclohexane, 1-octanol, oleic acid, squalane, squalene, silicone oil KF-96A-6cs (dimethyl silicone oil), A similar experiment was conducted using KF-995 (cyclic dimethyl silicone oil) (both manufactured by Shin-Etsu Silicone). The results are summarized in Table 2. The presence or absence of reverse vesicles was determined by observation of maltase cross.
Reverse vesicles were not formed in solvents with polar functional groups such as alcohol (1-octanol) and fatty acids (oleic acid), but other straight hydrocarbon solvents include natural oils (squalene and squalane). In addition, it was confirmed that the reverse vesicle was easily formed even in the silicone oil.

[Example 15]
In Example 2, a similar experiment was performed without adding water (Table 2). As a result, it was confirmed that the reverse vesicle was formed only by the continuous phase (hexane) and the activator (MEL-D) without adding water.

[Examples 16 to 22]
In Example 15, instead of hexane, hexadecane, 1-octanol, oleic acid, squalane, squalene, silicone oil KF-96A-6cs (dimethylsilicone oil), KF-995 (cyclic dimethylsilicone oil) were used as the continuous phase in Example 15. A similar experiment was conducted (Table 2). The formation of reverse vesicles was not observed in alcohols, fatty acids, and hydrocarbons having a large number of carbon atoms, but the formation of reverse vesicles was confirmed even when water was not added.

[Example 23] (Confirmation of structure of reverse vesicle and inclusion of water-soluble substrate between lipid membranes)
When preparing a reverse vesicle in the same manner as in Example 1, instead of 20 μL of water, 6 μL of an aqueous solution (1 mM) of calcein, which is a water-soluble fluorescent indicator, was added, and a reverse vesicle encapsulating the aqueous solution of calcein between the membranes was added. Prepared. As a result of fluorescence observation using a confocal laser microscope, green fluorescence was observed only in the outer shell portion of the spherical assembly, and it was confirmed that the inside was a hollow structure (FIG. 3).
From this result, this aggregate is not a micelle structure having an aqueous phase inside, but the hydrophobic group (aliphatic acyl group) and the hydrophilic group (hydroxyl group) of the biosurfactant from the outside are hydrophobic groups—hydrophilic groups :: hydrophilic groups— It was confirmed that a reverse vesicle was formed in which the outer shell of the aggregate was constructed with a bilayer structure in which hydrophobic groups were oriented in order. In addition, it was confirmed that calcein can be locally encapsulated in the hydrophilic region between the membranes, indicating that a specific hydrophilic drug can be encapsulated in a region having a very small molecular level and a large surface area.

Claims (6)

  A reverse vesicle composed of a self-assembled bilayer membrane of a sugar-type biosurfactant compound. The reverse vesicle according to claim 1, wherein the sugar-type biosurfactant compound is mannosyl alditol lipid represented by the following general formula (1).
[In the above formula (1), R ¹ represents a linear or branched saturated or unsaturated aliphatic acyl group having ² to 24 carbon atoms, and R ² represents hydrogen or an acetyl group. R ³ represents hydrogen or a linear or branched saturated or unsaturated aliphatic acyl group having 2 to 24 carbon atoms. Two R ² and R ³ may be the same or different. n is an integer of 1-4. ] The reverse vesicle according to claim 2, wherein the compound represented by the general formula (1) has a structure represented by the following formula (2).
(In the formula, R represents an aliphatic acyl group having 4 to 20 carbon atoms which may be the same or different.)   The reverse vesicle according to any one of claims 1 to 3, comprising a drug.   The method for forming a reverse vesicle according to claim 1, wherein the sugar-type biosurfactant compound represented by the general formula (1) is stirred in a nonpolar liquid oily substance to which water has been added.   The method for producing a reverse vesicle according to claim 5, wherein a water-soluble drug is contained in water and / or an oil-soluble drug is contained in a nonpolar liquid oily substance.