KR20110134015A - Stabilizing method for glabridin - Google Patents

Abstract

PURPOSE: A method for stabilizing glabridin is provided to prevent oxidative decomposition of glabridin and denaturation and discoloration of a cosmetic product. CONSTITUTION: A method for stabilizing glabridin comprises: a step of dissolving chitosan in acid to prepare a solution 1; a step of dissolving glabridin in a solvent to prepare a solution 2; a step of mixing the solutions 1 and 2. The acid is acetic acid, lactic acid, formic acid, formic acid, hydrochloric acid, or nitric acid. The solvent is alcohol, ethyl acetate, or acetone. The chitosan is N-acylated chitosan such as N-propionyl chitosan, N-butytyl chitosan, or N-hexanoyl chitosan.

Description

Stabilizing Method for Glabridin

The present invention relates to a method of stabilizing glabridin. More specifically, the present invention relates to a technique for preventing oxidative degradation of glabridine by synthesizing nanoparticles by ion-bonding glabridin with chitosan.

Licorice (Glycyrrhiza uralensis Fisch, Glycyrrhiza glabra L.) is a perennial herbaceous plant belonging to Leguminosae, whose roots and stems are used for medicinal and edible food. In particular, licorice is a medicinal plant that is used as an antidote, antitussive expectorant, laxative, peptic ulcer and gastric ulcer in oriental medicine and folk medicine.In addition, it works to relieve pain caused by rapid tension of muscles or tissues, weight gain, white blood cell There is an increase, diuretic, anti-inflammatory effect. Therefore, in order to use the medicinal effect licorice is used as a herbal medicine or licorice extract has been attempted to use in food, tobacco, cosmetics.

Licorice extract is composed of Glycyrrhizin, Glycyrrhetic acid, Licoricidin, Hispaglabridin A, Hispaglabridin B, and Glabridine (Glycyrrhizin). Glabridin), 4-O-methylglabridine

 (4-O-methylglabridin), isoprenylchalcone derivative, Isoliquiritigenin, Formononetin and the like. Glycyrrhizin in licorice extract has anti-inflammatory, detoxification, anti-ulcer, and hydrolyzate hormonal effects, so that conventional methods for extracting and purifying only glycidazine are known. The method of extracting glycyrrhizin from licorice is a method of precipitating impurities by separating soda silicate and aluminum sulfate and then adjusting the pH to 4.5 to 6.5 when licorice root is extracted with water or alkaline aqueous solution. A method of combining or sequentially extracting an methanol extraction process, an anion exchange resin decolorization process, and an ethanol recrystallization process is known. As such, a variety of methods for extracting glycyrrhizin from licorice are known, and the glycyrrhizin extracted therefrom is of high purity.

Glabridine, the main component of the licorice extract, is known to have an effect of inhibiting oxidation of low density phospholipids (Low Density Lipoprotein) harmful to the human body (Belinky, P., A., Aviram, M., Fuhrman, B., Rosenblat, M., and Vaya, J., 'The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation', Atherosclerosis, Vol. 137, 49, (1998)). Low-density phospholipids are responsible for delivering cholesterol throughout the body. Oxidation occurs when excess low-density phospholipids are present in the body, which can cause atherosclerosis, diabetes, and adult disease. In addition, glabridine is one of the hydrophobic substances having an important effect on the skin, and has an excellent effect on skin whitening, and there are an increasing number of examples of producing functional cosmetics having a whitening effect from the raw material (Korean Patent Publication 1993- 21190).

However, glablidin components in oil-soluble licorice extract have oxidative degradation from environmental factors such as light, heat, and oxygen, and thus easily change or discolor to cosmetic whitening materials.

Accordingly, the present inventors have come up with a method of making nanoparticles by ion-bonding with biodegradable chitosan in order to prevent oxidative degradation of the globdine from environmental factors and to prevent it from being easily changed or discolored into a whitening material of cosmetics.

An object of the present invention is to prevent oxidative degradation of glabridine by ionically binding glabridine to chitosan, which is easily unstable from environmental factors. More specifically, an object of the present invention is to prevent oxidative degradation of glabridine by synthesizing nanoparticles by ionically bonding a hydroxyl group, which is a functional group of glabridine, with an amine group of chitosan.

In order to achieve the object of the present invention as described above, the present invention

(a) dissolving chitosan in an acid;

(b) dissolving glabridine in the solvent;

(c) mixing the solutions (a) and (b);

It provides a method for stabilizing glabridine comprising a.

The acid of step (a) is preferably acetic acid, lactic acid, formic acid, hydrochloric acid or nitric acid.

The solvent of step (b) is preferably alcohol, ethyl acetate or acetone.

In the mixing of the step (c), the ratio of the solution (a) to the solution (b) is preferably 5: 5 to 8: 2.

The chitosan of step (a) may include N-acylated chitosan.

N-acylated chitosan is preferably N-propionyl Chitosan, N-butyryl Chitosan or N-hexanoyl Chitosan.

The present invention also provides a chitosan-glabridine complex or an N-acylated chitosan-glabridine complex synthesized by the above method.

The N-acylated chitosan-glabridine complex may be an N-propionic chitosan-glybridine complex, an N-butanoic chitosan-glabridine complex or an N-hexa It is preferred that it is a N-hexanoic chitosan-glabridine complex.

According to the present invention, resorcinol present in the globridine molecule is oxidized to prevent radicals and oxidative decomposition of glabridine, thereby preventing easily changing or discoloring the cosmetic whitening material. can do.

1 is a chemical structural formula of glabridine. The spherical part represents the chemical formula of resorcinol.
2 is a chemical structural formula of chitosan.
3 is a chemical structural formula of chitosan and N-acylated chitosan.
4 is a chemical structural formula of ionic bonds between glabridine and chitosan.
5 is a chemical structural formula of ionically bonding glabridine and acylated chitosan.
6 is an FT-IR spectrum of a chitosan sample. (a) chitosan (b) N-propionyl chitosan (c) N-butyryl chitosan (d) N-hexanoyl chitosan. FT-IR confirmed that acyl group was attached to chitosan.
7 is a ¹ H-NMR spectrum of a chitosan sample in 2 wt% CD3COOD / D2O solvent.
FIG. 8 is FT-IR spectra of chitosan, glabridin, chitosan-glabridin complex and N-acylated chitosan. FIG. (A) chitosan from above; (b) N-acylated chitosans; (c) glabridine; (d) chitosan-glabridine complex; (e) N-propionyl Chitosan-Glabridin complex; (f) N-butyryl Chitosan-Glabridin complex; (g) N N-hexanoyl Chitosan-Glabridin complex.
FIG. 9 is a TEM image of chitosan-glabridine composite nanoparticles. (A) and (C) are 5: 5 ratios of chitosan solution to glabridine solution, and (B) and (D) are chitosan solution to article The ratio of the labridine solution is 8: 2. A and B are 10K times larger and C and D are 150K times larger.
FIG. 10 is a particle size distribution of particles of chitosan-glabridine complex nanoparticles.
FIG. 11 is a particle size distribution of particles of chitosan-glabridine complex nanoparticles. FIG.
12 is a UV spectrum of methanol extraction from each sample and appropriate control.
13 shows the encapsulation efficiency. This was measured to see what percentage it attached when complexed with chitosan and N-acylated chitosan.
14 shows Loading Capacity. This is to find out how many g of glybridine is attached per g of chitosan in the chitosan-glibridine complex.
15 shows the UV radioactivity sample quantitatively by HPLC. This is to know about the stability of glabridine upon UV irradiation.
Figure 16 shows G: glabridine, C: chitosan-glybridine complex, P: N-propionyl chitosan-glybridine complex, B: N-butyryl chitosan-globridine complex, H: N-hexanoyl Thermal stability of the chitosan-globridine complex. Thermal stability with temperature conditions (refrigerated, 25 degrees, 40 degrees).
FIG. 17 shows light exposure (●), cooling (○), 25 ° C. (▼) and 40 ° C. (Δ): L ^* (white 100, black 0), a ^* (+ red, -green), and b ^* It shows the change of color according to (+ yellow, -blue). (G: glabridine solution, N: chitosan absent, CH: complex solution)
18 shows 1. in the absence of glabridine, 2. in the absence of chitosan, 3. chitosan-glybridine complex solution, 4. N-propionyl chitosan-glybridine complex solution, 5. N-butyryl chitosan- Glabridine complex solution, 6. N-hexanoyl chitosan-glabridine complex solution 1 month later change is shown.

In order to achieve the object of the present invention as described above, the present invention provides a technique for preventing the oxidative decomposition of glabridine by synthesizing nanoparticles by ion-bonding the active ingredient glabridine with chitosan. This will be described in detail below.

In one embodiment of the present invention, chitosan, which is ion-bonded with glabridine, is distributed on the shells of shellfish such as shrimp and crabs, the epidermis of fungi, and the cell walls of fungi. N-acetyl glucosamine is a compound in which the residues are linearly bound to β-1,4 by 2,000-3,000 residues or more. In addition, chitosan has a unique structure, which is the only natural animal dietary fiber on earth, which is positively charged in dissolution state, unlike most conventional plant fiber (dietary fiber) that is negatively charged. Due to the natural structure of such a specific structure, it is a highly active natural material that is highly compatible with living organisms and has been put to practical use in various applications such as polymer coagulants, cosmetic additives, cholesterol lowering effects, food additives, medical sutures, anticancer agents, and wound healing agents.

In one embodiment of the present invention, the amine group attached to the second carbon of chitosan (see FIG. 2) is reacted, and chitosan may act as an anion molecule.

In addition, in one embodiment of the present invention, the acylated chitosan may be ionically bonded to glabridine. Acylated chitosan can cause an acyl reaction by reacting chitosan with a carboxylic acid derivative or an acid anhydride. As the chain length of the carboxylic acid increases, hydrophobicity increases, which may affect the stabilization of glabridine.

In one embodiment of the present invention, the active ingredient glabridine (see Fig. 1) is known to have an effect of inhibiting the oxidation of low density phospholipids (Low Density Lipoprotein) harmful to the human body (Belinky, P., A. , Aviram, M., Fuhrman, B., Rosenblat, M., and Vaya, J., 'The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation', Atherosclerosis, Vol. 137, 49, (1998 )). In addition, glabridine is one of the hydrophobic substances having an important effect on the skin, and has an excellent effect on skin whitening, and can be used as a raw material to produce a functional cosmetic having a whitening effect.

However, glabridine is easily oxidized and decomposed by environmental factors, resulting in browning and odor changes. This is because glabridine is a polyphenol, and when resorcinol in a molecule is oxidized by environmental factors, it forms quinones to form radicals to decompose glabridine.

Therefore, in one embodiment of the present invention, the resorcinol of glabridine is oxidized through the ionic bond of two hydroxyl groups (see FIG. 1) of resorcinol having activity in the molecule of glabridine. By stabilizing the glabridine, the glabridine can act as a cationic molecule.

In one embodiment of the present invention

(a) dissolving chitosan in an acid;

(b) dissolving glabridine in the solvent;

(c) mixing the solutions (a) and (b);

It provides a method for stabilizing glabridine comprising a.

Chitosan has limited solvent dissolving due to the hard crystal structure resulting from the formation of hydrogen bonds between amino and hydroxyl groups. In one embodiment of the present invention, an acid is preferable as the solvent in which the chitosan is dissolved, and non-limiting examples include organic acids such as acetic acid, lactic acid and formic acid, and inorganic acids such as weak hydrochloric acid and nitric acid. Acetic acid is more preferred.

Since glabridine is a nonpolar substance, non-limiting examples of the solvent in which glabridine is dissolved are preferably alcohol, ethyl acetate, acetone.

When the solution in which the chitosan is dissolved and the solution in which the glabridine is dissolved are mixed, two hydroxyl groups (see FIG. 1) and the amine group of the chitosan in the glabridine may form nanoparticles through ionic bonds. Therefore, resorcinol of glabridine can be prevented from being oxidized to stabilize the glabridine.

The mixing preferably has a ratio of the solution in which chitosan is dissolved to the solution in which glabridine is dissolved is 5: 5 to 8: 2. More preferably, the size of the nanoparticles is small and evenly distributed at the time of mixing at a ratio of 8: 2 (see FIG. 9).

In addition, in one embodiment of the present invention, the N-acylated chitosan may be ion-bonded with glabridine to stabilize the glabridine. N-acylated chitosan is not limited thereto, but is preferably N-propionyl Chitosan, N-butyryl Chitosan, N-hexanoyl Chitosan Do. As the carbon number of the alkyl group increases, the hydrophobicity of the chitosan increases, and the stability of the glabridine upon UV irradiation increases with the glabridine (see FIG. 15).

In addition, one embodiment of the present invention provides a chitosan-glybridine complex or N-acylated chitosan-glybridine complex synthesized by the method of stabilizing the glabridine. The complex may include nanoparticles.

The N-acylated chitosan-glybridine complex is not limited thereto, but is not limited to N-propionyl Chitosan-glybridine complex, N-butyryl Chitosan-glybridine Complex, N-hexanoyl Chitosan-glybridine complex.

Hereinafter, the present invention will be described in detail through examples. The following examples are only illustrative of the present invention, and the scope of the present invention is not limited thereby.

< Example >

measuring equipment

Nanoparticle size and distribution were measured using a particle size analyzer (Particle Analyzer, Model LM10, NTA1.3 analytical software, NanoSight Ltd, UK). (Model LM10, NTA1.3 analytical software, NanoSight Ltd, UK). Scanning profiles were obtained using UV / Visible Spectrophotometer (S-3150, SCINCO, S.Korea), and IR-Spectrum was measured by FT-IR Spectrometer (FT-IR Spectrometer, FTIR-8400S, Shimadzu, Japan) 400-4000 cm ¹ Recorded in the range. The synthesized nano-composites were observed using transmission electron microscopy (JEM1010, JEOL, Japan). HPLC (305 system, Gilson, France) was used to analyze the amount of glabridine and HPLC columns (C ₁₈ , 5 μm, 150 × 4.6 mm) were purchased from Grace Davison Discovery Sciences (USA).

 material ( Materials )

Low molecular chitosan (Fluka, Switzerland) has a deacetylation degree of 78%, propionic anhydride, butanoic anhydride, hexanoic anhydride to synthesize N-acylated chitosan. ) (Sigma-Aldrich, USA) was used. Glabridine (Bioland, S.Korea) was supplied from Bioland, and the reagents were acetic acid (Sigma-Aldrich, USA), methanol (JT Baker, USA), acetone and ether. Chitosan (Mw 16 X 104, Regen) was pulverized chitosan in flake form by grinding it finely for 5 days in a ball mill to form a powder. Chitosan in powder form was divided into sieves, of which chitosan powder was used. N-acylated chitosan synthesis includes acetic acid (additional purification, Aldrich), methanol, propionic anhydride (97%, Aldrich), butanoic anhydride (98%, Aldrich), hexanoic anhydride (99%, Aldrich), palmitic acid Anhydride (90%, Aldrich) was used and acetone and diethyl ether were used as wash solution.

 N- Acylation  Chitosan synthesis ( Preparation of  N- acylated chitosan )

1 g of chitosan was dissolved in 20 ml of 20% acetic acid and the fully dissolved chitosan / acetic acid solution was diluted in 100 ml methanol. Each propionic anhydride, butyric anhydride, and hexanoic anhydride are added at a molar equivalent per glucosamine residue, followed by methanol. ) Into 100 ml. 0.4 molar equivalent of propionic anhydride, butyric anhydride, hexanoic anhydride / 1 glucosamine residue solution was added to the chitosan / acetic acid solution. The solution was allowed to react to room temperature overnight and the solutions gelled during the reaction. 200 ml of acetone (aceton) was added thereto, and the mixture was shaken hard to break the gel. The gel mixed in acetone was centrifuged at 3,000 rpm for 10 minutes to remove only the solution and washed. In this way, each washed three times with acetone and ethyl ether, and the washed gel was dried in vacuo to give N-acylated chitosan in powder form (see Figure 3).

 Chitosan and N- Acylated  Chitosan Glabridine  Nanoparticle Manufacturing

Chitosan and synthesized N-acylated chitosan samples were completely dissolved in 1% acetic acid at a concentration of 0.1% for one day. The glabridine powder sample was completely dissolved in methanol at a concentration of 1%, and the chitosan and N-acylated chitosan solutions were added to the glabridine solution and mixed. The two solutions were mixed and analyzed using a vortex.

N- Acylated  Chitosan Analysis

Chitosan and synthesized N-propionyl Chitosan, N-butyryl Chitosan, N-hexanoyl Chitosan powders were subjected to FT-IR and ¹ H-NMR. Synthesis and substitution rate (Table 2) were examined. 5 mg of each chitosan NMR sample was measured on a 300 MHz NMR spectrometer using 0.5 ml of 2 wt% CD3COOD / D2O as solvent. (See FIGS. 6 and 7) Through the H-NMR, it was possible to confirm whether acto groups were synthesized and how many acyl groups were attached to chitosan.

Table 1 below shows the chemical shift of N- acylated chitosan, and Table 2 shows the substitution rate.

Dsubs (%) were found by applying integral values obtained from NMR data to the above equation.

Nanoparticle Complex ( Nanoparticle complex ) analysis

FT-IR was used to determine whether chitosan and each N-acylated chitosan and glabridine complex solution were synthesized. Each nanoparticle composite solution was lyophilized to measure the sample by FT-IR, and the sponge-like sample was mixed with KBr powder to make a disc (see FIG. 8). The FT-IR data of each of the N-acylated chitosan-glybridine complexes shows that the glybridine is present in the complex because it has the same peak as glabridine in the fingerprint area below 1500 cm-1. In addition, it was confirmed that there is also chitosan as the OH peak of chitosan broadly appearing in the 3000cm-1 band (see FIG. 8). The enlarged IR peak shows a semi doublet peak at 1590 cm −1, which is a result of the electrostatic interaction of the amine group of chitosan and the hydroxyl group of glabridine. These results confirm that chitosan and glabridine are present in the complex and can be confirmed to bind by electrostatic interaction (see FIG. 8).

Nanoparticle structure ( Nanoparticle morphology )

TEM was used to confirm the shape of the nanoparticles made of chitosan and glabridine. Nanoparticle samples made of chitosan solution and glabridine solution in a ratio of 5: 5 and 8: 2 were placed on a grid and measured at a magnification of 10K and 150K (see FIG. 9). It can be seen that the chitosan solution and the glabridine solution are smaller and evenly distributed in the size of the 8: 2 ratio than the 5: 5 ratio.

Nanoparticle size ( Nanoparticle you ) Measure

The size of the nanoparticles made of chitosan and each N -acylated chitosan and glabridine was measured using a particle analyzer. Each nanoparticle complex solution was measured by diluting 1,000 times to measure the size due to the large number of particles (see FIGS. 10 and 11).

In nanoparticles Glabridine  Confirm

UV spectroscopy was performed to determine whether the nanoparticles had glabridine. In order to measure UV spectroscopy, each nanoparticle composite solution was lyophilized, extracted with methanol, a solvent in which only glabdine was dissolved, but not chitosan, and measured by UV spectroscopy at 280 nm (see FIG. 12).

The magnitude of UV-Visible absorption by material varies with wavelength, and the absorption spectrum according to wavelength has a unique value for each material. Λ max having unique values for each material was identified using a UV-Visible spectrophotometer. Glabridine has an intrinsic λ max at 232 nm and 280 nm. Referring to FIG. 12, it was found that glabridine is present in the chitosan-glabridine complex produced by the experiment.

Encapsulation efficiency  & Loading capacity

The encapsulation efficiency and loading capacity were measured by PVDF membrane filter (membrane) to determine the efficiency of how glabridine adheres to chitosan and N-acylated chitosan in the chitosan-glibridine complex and the N-acylated chitosan-globridine complex. This was measured using a filter) (see Tables 3 and 4). The solution drawn out through the PVDF membrane filter (membrane filter) was called Gout, the resulting cake was filtered out of the PVDF membrane filter and extracted with only glybridine with methanol was referred to as Gin (see FIGS. 13 and 14). Table 3 relates to encapsulation efficiency (EE) and Table 4 relates to Loading Capasity (LC).

EE: Encapsulation Efficiency

G0: total amount of Glabridin

Gout: amount of free Glabirdin

Gin: amount of extract glabridin in chitosan-glabridin complex

LC: Loading Capacity

G0: total amount of Glabridin

Gout: amount of free Glabirdin

Gin: amount of extract glabridin in chitosan-glabridin complex

C0: total amount of chitosan

UV  On exposure Glabridine  Stability measurement

Chitosan-Glabridine complex and N-acylated chitosan-Glabridine complex were irradiated with UV wavelength of 280nm for 0min, 10min and 60min to measure the stability of glabridine following UV exposure. As a 1% glabridine / methanol solution was used. HPLC was used to see the change in glabridine in each sample examined and detected by 280 nm UV (see FIG. 15).

According to temperature Glabridine  Stability measurement

In order to measure the stability of glabridine with temperature, the chitosan-glibridine complex and N-acylated chitosan-glabridine complex were refrigerated for 0 days, 3 days, and 7 days, at 25 ° C and 40 ° C. Measured under conditions. HPLC was used to see the change in glabridine as in the stability experiment of glabridine upon UV exposure (see FIG. 16).

Color difference meter

Each sample was observed for color change using a color difference meter in daylight, chilled, 25 ° and 40 ° conditions (see FIG. 17).

Change of state

The state after one month of each sample was observed (see FIG. 18). Photograph 1 month after A of FIG. 18 is B. FIG. Sample 2 was precipitated and subsided after 1 month with a solution free of chitosan. However, in samples 3-6, the particles were dispersed evenly after one month without sinking.

Although embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (9)

(a) dissolving chitosan in an acid;
(b) dissolving glabridine in the solvent;
(c) mixing the solutions (a) and (b);
Method of stabilizing the glabridine comprising a. The method of claim 1,
The acid of step (a) is acetic acid, lactic acid, formic acid, hydrochloric acid or nitric acid. The method of claim 1,
The solvent of step (b) is characterized in that the alcohol, ethyl acetate or acetone. The method of claim 1,
The mixing of step (c) is characterized in that the ratio of (a) solution to (b) solution is 5: 5 to 8: 2. The method of claim 1,
The chitosan of step (a) is characterized in that the N-acylated chitosan. 6. The method of claim 5,
The N-acylated chitosan is N-propionyl Chitosan, N-butyryl Chitosan or N-hexanoyl Chitosan. Chitosan-globridine complex synthesized by the method of any one of claims 1 to 4. N-acylated chitosan-glybridine complex synthesized by the method of claim 5 or 6. The method of claim 8,
The N-acylated chitosan-glybridine complex is N-propionyl Chitosan-glybridine complex, N-butyryl Chitosan-glybridine complex or N-hexa A complex characterized in that the N-hexanoyl Chitosan-glybridine complex.

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