KR20120070682A - Synthesis and method for preparing astragalin derivative and the biochemical characterization of the thereof - Google Patents

Abstract

PURPOSE: A composition containing an astragalin derivative is provided to suppress MMP-1 expression and to suppress melanogenesis. CONSTITUTION: An astragalin derivative is denoted by chemical formula 1. The derivative is prepared by adding glycosyltransferase to a solution containing 0.01-1.0 M of sucrose such that the final active concentration is 0.1-100 U/ml, adding astragalin in a reactor at 15-70deg. C. The glycosyltransferase is Leuconostoc mesenteroides-derived dextran sucrase. A composition for enhancing antioxidant activity contains the derivative as an active ingredient. A composition for skin whitening contains the derivative as an active ingredient. The content of the derivative is 0.001-20 wt%.

Description

Astragalin derivatives and compositions containing them {Synthesis and Method for preparing astragalin derivative and the biochemical characterization of the groom}

The present invention relates to an astragalin derivative and a composition containing the same, and more particularly, to astragalin glucoside and astragalin isomaltoligosaccharide and a composition for improving skin containing the same.

Astragalin (Kaempferol-3-D-O-glucoside), a type of flavonoid substance in which glucose is bound to kaempferol, is one of flavonols and is distributed in the leaves and flowers of various species of plants. Astragalin has excellent effects such as antioxidant and anti-inflammatory, and also has a function of inhibiting histamine secretion through basophils to prevent or treat atopic diseases (Oral administration of persimmon leaf extract ameliorates skin symptoms and transepidermal water loss in atopic dermatitis model mice, NC / Nga, M. Matsumoto et al. British Journal of Dermatology (2002) 146: p221-227).

However, most of the astragalins currently used are in the form of extracts, and since the actual amount of astragalin is very small, it is difficult to express practical effects, and in terms of economics for the purification and manufacturing of a high content of astragalin. There are many difficult issues.

Therefore, the present inventors prepared a novel astragalin glucoside, astragalin isomaltoligosaccharide as a derivative of astragalin, a glycoside of flavonoid camphorol, and confirmed a surprising effect on skin improvement of the derivative of astragalin, and the present invention. Was completed.

It is an object of the present invention to provide a novel derivative of Astragalin and its preparation method.

Another object of the present invention is to provide a composition containing a derivative of the novel Astragalin.

In order to achieve the above object, the present invention provides a novel derivative of Astragalin and its preparation method.

The present invention provides a composition containing a derivative of the novel Astragalin.

The novel astragalin derivatives of the present invention are characterized in that they are astragalin glucoside and astragalin isomaltooligosaccharide.

More specifically, the novel astragalin derivatives of the present invention are characterized as astragalin alpha-di-glucoside and astragalin alpha-di-isomaltooligosaccharide in which glucose is linked to astragalin by alpha bonds.

The novel astragalin derivative of the present invention is characterized in that astragalin glucoside and astragalolin isomalto oligosaccharides are synthesized by using astragalin as a receptor and dextransucrase successively transferring glucose of sucrose to the receptor. do.

The novel derivative of Astragalin of the present invention is characterized by having antioxidant activity, skin whitening and wrinkle improvement.

Hereinafter, the present invention will be described in detail.

The present invention provides an astragaline derivative represented by the following formula (1).

The astragalin derivative comprises the steps of: 1) transferring the glucosyl group of sucrose to astragalin, a receptor, to produce astragalin glucoside; 2) sequential transfer of the glucosyl group of sucrose to the produced astragalin glucoside to produce astragalin isomaltoligosaccharide; And 3) isolating and purifying the receptor reactant; Characterized in that it is prepared to include.

In the present invention, the astragalin derivative is prepared by adding a sugar transfer enzyme to a mixture solution of sucrose and astragalin, and then reacting. More specifically, the astragalin derivative is a sugar transfer enzyme in a solution containing sucrose at a concentration of 0.01 to 1.0 M. After the final active concentration of the reactor is added to 0.1 to 100 U / ㎖, it is characterized in that it is prepared by adding astragalin to a final concentration of 0.01 to 0.5 M in a reactor at 15 to 70 ℃, and then reacted.

In the present invention, the sugar transfer enzyme is Leuconostoc ( Leuconostoc) mesenteroides ) is characterized in that the dextran sucrase derived from strains.

More specifically, to the solution containing 100 mM sucrose, dextran sucrase derived from Leuconostoc mesenteroides species strain was added so that the reactor final active concentration was 0.5 U / mL, This was reacted by adding astragalin as a receptor to a final concentration of 50 mM in a reactor at 20 ° C. to prepare an astragalin derivative.

In the present invention, the astragalin derivative prepared by the above production method is characterized in that at least one selected from the following compounds.

The compound is Camperol-3-O-beta-di-glucopyranoside-6-O-alpha-di-glucopyranoside (Kaempferol-3-O-β-D-Glucopyranoside-6-O-α- D-Glucopyranoside); Camperol-3-O-beta-di-glucopyranoside-3-O-alpha-di-glucopyranoside (Kaempferol-3-O-β-D-Glucopyranoside-3-O-α-D-Glucopyranoside ); And camphorol-3-O-beta-di-isomaltooligosaccharide; and at least one member selected from the group consisting of all compounds having new structures.

The present invention provides a composition for enhancing antioxidant activity, containing the astragalin derivative as an active ingredient.

The present invention provides a composition for skin whitening containing the astragalin derivative as an active ingredient.

The present invention provides a composition for improving skin wrinkles containing the astragalin derivative as an active ingredient.

Astragalin derivatives according to the present invention have superior ability to inhibit the expression of intracellular collagen degrading enzyme MMP-1, enhance antioxidant activity and inhibit melanin production, compared to astragalin present in the associated plant, thereby improving skin whitening and wrinkles, and preventing aging. Excellent effect Such effects are specified in the examples below.

The astragalin derivatives each contain 0.001 to 20% by weight based on the total weight of the composition. When the astragalin derivatives are contained in an amount less than 0.001% by weight, it is difficult to expect a substantial effect of the composition, and the content is greater than 20% by weight. In this case, it affects the stability of the formulation and the product, and it is not economical because the synergistic effect of the overdose is small.

The components included in the composition of the present invention may further include components conventionally used in cosmetic compositions in addition to the astragalin derivative as an active ingredient, such as conventional auxiliaries such as stabilizers, solubilizers, vitamins, pigments and flavorings, and And a carrier.

Cosmetics prepared from the composition containing the astragalin derivative of the present invention as an active ingredient can be prepared in the form of a general emulsion formulation and solubilized formulation. Cosmetics of the emulsified formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations are flexible cosmetics. In addition to cosmetics containing the astragalin derivative of the present invention, by containing a dermatologically acceptable medium or base may be prepared in the form of adjuvants that can be used topically or systemically applied commonly used in the field of dermatology.

Suitable cosmetic formulations include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionics (liposomes), nonionics obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase. It may be provided in the form of a vesicle dispersant, in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the composition of the present invention is in addition to astragalin derivatives in addition to fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water Commonly used in ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic or dermatology field, such as any other ingredient. And the above ingredients may be introduced in amounts generally used in the field of dermatology.

Products to which the composition of the present invention can be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, cleansing agents, face washes, soaps, treatments, cosmetics, etc. There is this.

Specific formulations of the composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, soap , Shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, loose powders, eye shadows and the like.

The method of preparing the astragalin derivative of the present invention is not only an advantage of efficiently producing a large amount of astragalin derivative using an enzyme, but also the astragalin derivative prepared by the method is superior to the astragalin present in the associated plant. It has the ability to inhibit the expression of collagen degrading enzyme MMP-1, enhance antioxidant activity and inhibit melanin production, and contribute to functional cosmetic materials with excellent effect on skin whitening and wrinkle improvement and anti-aging.

Figure 1 shows the TLC analysis of the astragalin derivative prepared by the production method of the present invention,
(1: 100 mM sucrose, 2: 100 mM glucose, 3: 100 mM fructose, 4: dextran sucrose + sucrose reaction solution, 5: dextran sucrose + sucrose + astragalin reaction solution, 6: ultraviolet ray) Astragalin receptor reaction solution identified in the region (254 nm)
Figure 2 shows the results of the HMBC analysis of the structure of the astragalin glucoside (Ast-G1; n = 0) and astragallin isomaltooligosaccharide (Ast-IMO; 1≤n≤7) prepared by the method of the present invention ( Where the arrow indicates the correlation of the bond between carbon and hydrogen based on HMBC data),
Figure 3 shows the results of the HMBC analysis of the astragalin glucoside (Ast-G1 ') derivative prepared by the method of the present invention (where the arrow means the correlation of the bond between carbon and hydrogen based on the HMBC data).

Hereinafter, preferred preparation examples and experimental examples, etc. are provided to help the understanding of the present invention. However, the following Preparation Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Preparation Example 1 Preparation of Dextran Sucrase Enzyme Liquid

As the dextran sucrase used in the present invention, an enzyme obtained from a strain of Leuconostoc mesenteroides was used. Leuconostoc mesenteroides B-512FMCM, used to obtain dextransucrase (EC.2.4.1.5), was used to vacuum Leuconostoc mesenteroides B-512FMC. A bacterium obtained by using ultraviolet light constitutively produces dextran sucrose. Leuconostoc mesenteroides B-512FMCM was cultured using LM medium containing 2% (w / v) glucose as a carbon source and the growth temperature was maintained at 28 ° C.

Example 1

1) sugar transferase reaction

Synthesis reaction conditions of astragalin glucoside and astragalin isomaltooligosaccharide were 50 mM astragalin receptor reaction solution containing 0.5 U / ml dextran sucrose, 100 mM sucrose, and 10% DMSO prepared in Preparation Example 1. The reaction was carried out at 28 ° C. until all of the sucrose in the reactor was consumed (about 3 hours) and the reaction was stopped by boiling the reaction mixture for 10 minutes. One unit of enzyme used is expressed in μmol of fructose liberated from sucrose per ml of enzyme per minute.

Production of the receptor reaction product and confirmation of complete reaction of sucrose were carried out on a TLC plate (Merck K6F, Darmstadt, Germany) after taking 1 μl of the reaction solution, followed by nitromethane: normal propanol: water = 2: 5: 1.5 (v / v / v) or acetonitrile: water = 85: 15 (v / v) using a solvent prepared, and the separated carbohydrate component of the thin layer chromatography (TLC) plate 0.5% (w / v) α -Mukerjea R. et al. Simplified and improved methylation analysis of saccharides using a modified procedure and thin-layer chromatography, Carbohydrate Research, 1996 , 292, 11-20).

2) Astragaline Glucosides Astragaline Isomaltooligosaccharide  refine

Astragalin glucoside and astragallin isomaltooligosaccharide produced in 1) can be separated and purified by conventional methods. The present invention administers a sample to a column chromatography filled with Sephadex LH-20 (GE Healthcare Biosciences AB), a gel filtration chromatography (Gel filtration Chromatograph), and fractional elution with ethanol (0-100%) The degree of sugar separation was confirmed using TLC. Purified synthetic products were collected by size, lyophilized and prepared in powder form.

3) refined Astragaline  Receptor Reaction products  Structure determination

After purification by size using Sephadex LH-20 column chromatography, the molecular weight of each material was analyzed using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF). To this end, the purified reaction product (1mg / 1mL) is diluted with distilled water and mixed with 2,5-dihydroxybenzoic acid (dehyeroxy-benzoic acid, 1mg / 1mL) in a volume of 1: 1. 1 μl of the mixed solution was added to a stainless steel matrix and then slowly dried at room temperature.

The mass spectrum was analyzed using MALDI-TOF-MS (Voyager DE-STR; Appled Bio Systems, USA), at which time 65 kV acceleration voltage was used in positive mode. From the MALDI-TOF mass spectrometry, the purified reaction product was found to increase the molecular weight as much as the molecular weight of glucose from which water molecules were separated by one molecule of glucose into astragalin.

Structural analysis of the reaction product using NMR dissolved 2 to 3 mg of the purified reaction product in 250 μl of D ₂ O, and then placed in an NMR (3 mm diameter) tube, using a VNMRS spectrometer (Varian, Palo Alto, CA). And ¹ H NMR with 600 MHz equipment, ¹³ C NMR with 150 MHz equipment was analyzed.

The binding sites of astragalin and sugar are homozygous correlation correlation (COSY) that connects hydrogen to each other, heteronuclear single quantum coherence (HSQC) that connects hydrogen and carbon, and hydrogen to carbon, but is connected by multiple bonds. Analysis was performed using heteronuclear multiple bond correlation (HMBC) (FIG. 2 and FIG. 3).

(The values in parentheses are the pairing constant J value (Hz), and o means overlap with other peaks.)

As a result of analysis by NMR of Table 1, it was possible to isolate and confirm three novel astragalin derivatives represented by the following compounds. That is, astragalin is kaempferol-3-O-β-D-Glucopyranoside, and astragalin glucoside is camphorol-3-O-beta-di-glu Copyranoside-6-O-alpha-di-glucopyranoside (Kaempferol-3-O-β-D-Glucopyranoside-6-O-α-D-Glucopyranoside) and camphorol-3-O-beta-di It was confirmed that it is isomaltoligosaccharide, and another astragalin glucoside was camphorol-3-O-beta-di-glucopyranoside-3-O-alpha-di-glucopyranoside (Kaempferol-3-O- β-D-Glucopyranoside-3-O-α-D-Glucopyranoside).

[ Test Example  One]

1) Evaluation of wrinkle improvement efficacy Matrix metalloproteinase - 1 (MMP-1) inhibition  activation)

Collagen is a major protein constituting the dermis of the skin and plays a role in maintaining skin structure and elasticity. Collagen is known to show a decrease in production with increasing age and to increase the decomposition to induce depression of the dermal dermal layer to produce wrinkles of the skin. In addition, the degradation of collagen is increased due to the expression of MMP such as collagenase by UV, which causes photoaging, and wrinkles appear. Therefore, by measuring the ability to inhibit MMP-1 expression in the cell can be evaluated the effect of the skin wrinkle improvement material.

Dulbecco's containing penicillin (100 units / ml), streptomycin (100 ug / ml), 10% fetal bovine serum (FBS) after inoculation of human fibroblasts (primary cell line) or similar fibroblasts to the bottom of the culture dish. Modified essential medium (DMEM) was added and incubated in an incubator containing 37 ° C. and 5% CO ₂ . Fibroblasts are aliquoted into 2 × 10 ⁴ cells per well in a 24-well plate, incubated for 24 hours in cell culture conditions, discarded, and starvated for 12 hours in a medium without FBS. Discard the medium, wash with PBS and irradiate or irradiate 10 J / cm 2 with UVA. Add sample solution and fresh medium and incubate for 48 hours. Cultures were taken and subjected to the MMP-1 ELISA method (R & D Systems Elisa, Minneapolis, MN, USA) according to the R & D Systems ELISA Protocol.

As can be seen in Table 2, the production of MMP-1, a collagen glucoside and astragalin isomaltoligosaccharide synthesized using a sugar transfer enzyme according to the preparation method of the present invention, is inhibited from that of collagen decomposing enzyme MMP-1. It was confirmed that the effect was improved.

2) evaluation of antioxidant activity DCF - DA assay )

A method for measuring the ability to remove free radicals induced by treating a fibroblast or a keratinocyte cell line (HaCaT) with a sample, astragalin, a standard material, and astragalin glucoside separated and purified in Example 1, The antioxidant activity of astragallin isomaltooligosaccharide was measured.

The amount of reactive oxygen species in the cell was oxidized by the reactive oxygen species produced by UV and treated with 2 ', 7'-Dichlorodihydrofluorescein diacetate (DCF-DA) that exhibits fluorescence and introduced into the cell, and irradiated with UVB 30 mJ / cm 2. . After 2 hours, fluorescence was measured at excitation 485 nm and emission 535 nm. Here, the active oxygen removal ability was expressed as a fold increase in% based on a control group not irradiated with UV, and vitamin C (1 mM) was used as a positive control group.

As a result, as can be seen in Table 2, the antioxidant activity effect of the sugar transition products was confirmed that the antioxidant capacity is improved than the standard astragalin.

3) Whitening Activity Assessment- Intracellular  Melanin production inhibition experiment

Melanocytes are differentiated cells in the basal layer of skin epithelial tissue, which have specialized organs called melanin bodies that synthesize melanin. Melanin is known to play an important role in pigmentation such as blemishes and freckles, as well as protecting skin from UV rays and acting as a scavenger of cytotoxic radicals. In the present invention, the experiment to inhibit intracellular melanin production was performed to evaluate the efficacy as a whitening raw material.

Melan-a (murine melanoma) cells were inoculated at 1 × 10 ⁵ per well in a 12-well plate with RPMI 1640 or equivalent growth medium containing FBS, and then the cells were well maintained at 5% CO ₂ at 37 ° C. Incubate until about 80% adhered to the bottom. After incubation, the medium was removed, the sample was replaced with a medium diluted to a suitable concentration, and then incubated at 5% CO ₂ , 37 ° C for a certain time. The concentration range of the sample was determined by toxicity test. The cells from which the medium was removed were washed with PBS (phosphated buffer saline, pH 7.4) and treated with trypsin to recover the cells. The recovered cells were centrifuged at 5,000 to 10,000 rpm for 10 minutes and then the supernatant was removed to obtain pellets. Cell pellets were dried at 60 ° C., and then 100 μl of 1M NaOH containing 10% DMSO or an appropriate amount of cell lysis buffer was used to obtain intracellular melanin in a 60 ° C. thermostat. The absorbance of the solution was measured at 490 nm using a microplate reader, the protein amount was determined by the Bradford method, and the amount of melanin per predetermined number of cells or the amount of melanin per constant protein was determined and corrected by a negative control group. As a result, as can be seen in Table 2, it was confirmed that the whitening effect of glucose-linked astragalin is superior to that of the standard astragalin.

Claims (9)

Astragalin derivative represented by the following formula (1).

The method of claim 1,
The derivative is an astragalin derivative, characterized in that at least one selected from the following compounds.

3. The method according to claim 1 or 2,
The derivative is an astragalin derivative, characterized in that obtained by the reaction after the addition of the sugar transfer enzyme to the mixed solution of sucrose and astragalin. The method of claim 3, wherein
The derivative is added to the solution containing the sucrose concentration of 0.01 to 1.0 M so that the final active concentration of the reactor is 0.1 to 100 U / ㎖, and then the final concentration of astragaline in the reactor at 15 to 70 ℃ Astragalin derivatives obtained by reacting after addition to 0.01 to 0.5 M. The method of claim 3, wherein
The glycotransferase is an astragaline derivative, characterized in that the dextran sucrase derived from Leuconostoc mesenteroides species strain. Antioxidant activity enhancing composition comprising the astragalin derivative according to claim 1 as an active ingredient. A skin whitening composition comprising the astragalin derivative according to claim 1 as an active ingredient. A composition for improving skin wrinkles comprising the astragalin derivative according to claim 1 as an active ingredient. The method according to any one of claims 6 to 8,
The astragalin derivative is characterized in that it contains 0.001 to 20% by weight relative to the total weight of the composition.

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