KR20190089309A - Method for producing esculetin using acid hydrolysis - Google Patents

Abstract

The present invention relates to a method for producing esculetin using acid hydrolysis. More specifically, the present invention can hydrolyze esculin by treating acid to a Fraxinus rhynchophylla extract containing a large amount of esculin, thereby producing a large amount of esculetin. Thus, it is possible to efficiently produce esculetin which is good for skin care in a rapid and convenient way, compared to the conventional production method.

Description

TECHNICAL FIELD The present invention relates to a method for producing escululin by acid hydrolysis,

The present invention relates to a method for producing escululin using an acid hydrolysis method.

Glucoside is a compound in which a monosaccharide has a cyclic hemi-acetal structure and a hydroxyl group is formed by loss of water and condensation between hydroxyl groups of other alcohols or phenolic compounds . A natural product having a hydroxyl group is present in the form of a glycoside bond in the form of a glycoside bond in a sugar in the plant, and the non-glycoside form separated from the sugar is called an aglycone.

Esculetin is an aglycone form of esculin glycoside and esculin is a derivative of escululin extracted from ash tree with glucopyranoside. Esculin is a component of general medicines and cosmetics, and is used for the purpose of strengthening capillary vessels and functioning as an anti-inflammatory drug, similar in function to vitamin P. In addition, bile aesculin agar treated with iron citrate and bile acid salts was found to have a strong affinity for escululin, escululin, and ascorbic acid, which were hydrolyzed and hydrolyzed by Escherichia escululatin and glucose by enterococci, Is reacted with iron citrate to form blackish brown or black color and is used for identification and analysis of bacteria. This principle is based on the fact that fluorescence is lost when the esculoline, which exhibits fluorescence at 360 nm of the spectrophotometer, is hydrolyzed.

In addition, aglycon, which is an unglycosylated compound, is very advantageous in terms of bioavailability compared to glycosides. There are two methods for converting esculin into escululin, and there is an acid hydrolysis method and an enzyme hydrolysis method using? -Glucosidase. Among them, the acid hydrolysis method is a method in which hydrochloric acid or sulfuric acid is added to a compound and hydrolysis is carried out at a temperature of 100 ° C for a few minutes to 2 hours, whereby the aglycon can be produced in the shortest time.

Recently, a lot of studies have been conducted to inhibit the production of melanin excess in relation to whitening materials. As a known inhibitor of tyrosinase, hydroquinone, arbutin, and kojic acid. However, only a limited amount of ingredients are used due to problems such as skin safety and product formulation stability. Accordingly, the development of natural materials, which reduce melanin synthesis and have no side effects, is attracting attention. Wrinkle-improving materials have also not actually developed substances with superior efficacy in clinical trials.

Therefore, in this study, a method of mass production of esculetin, an aglycone form of esculin, was established through the hydrolysis of esculin glycosides, which are abundant in ash trees, Escululin, which has better whitening and wrinkle-reducing effects, can be efficiently produced in a simple and rapid manner.

Korean Patent No. 1324654 discloses a cosmetic composition for improving wrinkles and anti-aging comprising escululin as an active ingredient. Korean Patent No. 1602630 discloses a method for obtaining an isoflavone aglycon from an isoflavone glycoside Lt; / RTI > However, the production method of escululin using the acid hydrolysis method of the present invention has not yet been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and the inventors of the present invention have found that the extracts of ash tree were prepared by extracting conditions according to extraction conditions, Confirming that esculetin can be mass-produced by hydrolyzing esculin by treating an ash extract in which leucine is present, thereby completing the present invention.

In order to solve the above-mentioned problems, the present invention provides a method for extracting ashulchin, comprising the steps of: 1) obtaining an extract of ash tree containing esculin by adding an extraction solvent to the shell of Fraxinus rhynchophylla containing esculin; 2) hydrolyzing esculin by treating the ash extract containing the esculin with an acid; And 3) separating and purifying escululin from the esculin hydrolyzate. The present invention also provides a method for producing escululin.

According to the present invention, it is possible to mass-produce escululin by hydrolyzing esculin by treating an ash extract with a large amount of esculin, and it is possible to mass-produce escululin, It is possible to efficiently produce choletin.

FIG. 1 is a graph showing the results of the hydrolysis of esculin by treating an acid to hydrolyzate of ash tree according to an embodiment of the present invention, and then treating esculetin extracted from the esculin with B16 melanoma cells at different concentrations. Melanin content in the blood. Nor is a normal group with no treatment, Con is a control group treated with α-MSH (α-melanocyte stimulating hormone), and arbutin is a positive control group.
FIG. 2 is a graph showing the results of hydrolysis of esculin by treatment of acid with hot water extract of ash tree according to an embodiment of the present invention, and esculetin isolated therefrom is treated with B16 melanoma cells in concentration- And the effect of increasing the procollagen type-I biosynthesis performance. Nor is a normal group that did not do anything, and Con UV-B (ultraviolet-B) and EGCG (Epigallocatechin gallate) are positive controls.

In order to accomplish the object of the present invention, the present invention provides: 1) an ash tree containing esculin ( Fraxinus rhynchophylla ) to obtain an ash tree extract containing esculin;

2) hydrolyzing esculin by treating the ash extract containing the esculin with an acid; And

And 3) separating and purifying escululin from the esculin hydrolyzate. The present invention also provides a method for producing escululin.

In the method for producing escululin of the present invention, the esculin and escululin are each represented by the following general formulas (1) and (2).

[Chemical Formula 1]

(2)

The ash tree extract may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, preferably extracted with water or ethanol, more preferably water But the present invention is not limited thereto.

The ash tree extract may be prepared by adding 2 to 4 parts by weight of water to 1 part by weight of the ash tree bark, and then extracting the mixture at 75 to 95 ° C for 80 to 100 minutes, And adding 3 parts by weight of water to 1 part by weight of water, followed by extraction at 90 캜 for 90 minutes. However, the present invention is not limited thereto.

The hydrolysis method of esculin may include hydrolyzing esculin at 90 DEG C for 90 minutes by treating 10 parts by weight of hydrochloric acid (HCl) with respect to 1 part by weight of the asurein containing esculin, It is not limited.

The separation and purification of escululin in step 3) above may be performed by separating and purifying escululin from esculin hydrolyzate using column chromatography using ODS (octadecylsilica) gel, but is not limited thereto.

In addition, the escululin may have a skin whitening and wrinkle-reducing effect, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Materials and methods

1. Preparation of extract, fractionation and separation of active substance

Extracts of ash bark were prepared according to extraction conditions (extraction solvent and extraction temperature), and their yield and tyrosinase inhibitory activity were measured and compared. Specifically, 3 parts by weight of water and ethanol (50% and 70%, v / v) were added to 1 part by weight of the ash bark, respectively, and then an extract was prepared for each extraction temperature (50, 70 and 90 ° C) , And then concentrated at 40 DEG C or lower using a rotary vacuum evaporator. Then, the extraction yield and skin whitening efficacy of ash tree extracts prepared according to the extraction conditions were confirmed and optimal extraction conditions were established. 10 parts by weight of hydrochloric acid (1N HCl) was added to 1 part by weight of the ash tree extract prepared under the optimum extraction conditions, and the mixture was hydrolyzed at 90 ° C for 90 minutes. ODS (octadecylsilica) gel . The eluent was H ₂ O-MeOH solvent system using a stepwise gradient mode.

In addition, bioassay-guided isolation using the skin whitening and wrinkle-reducing activity as an activity index was performed by HPLC analysis of the fractions in which activity was concentrated, Active substances were separated using a filler such as LH-20, MCI, Diaion HP-20, and ODS gel.

2. Determination of structure of active substance

The bioassay-guided isolation using the skin whitening and wrinkle-removing activity as an activity index was tested for purity by TLC and HPLC analysis for pure compounds separated by activity as an index, and ¹ H -, ¹³ C-, and 2D-NMR such as COZY, NOESY (or ROESY), HMBC and HMQC were measured to identify the structure of the compound. In addition, data necessary for obtaining structural information of a compound by using UV, IR, MS and the like were collected, and absolute arrangement of the active material was performed through various chemical methods.

3. Measurement of Melanin Biosynthesis Inhibitory Activity

Measurement of melanin biosynthesis inhibition from B16 melanonma cells was performed according to the method of Hosoi et al. (1985, Cancer Res., 45 (4), 1474-1478). The melanoma cell line cultured with the DMEM medium was divided into 2 × 10 ⁶ cells / dish in a culture dish of 100 mm. After culturing for 24 hours, 2 ml of the sample was prepared by concentration, and 2 days later, phosphoric acid And washed with buffer (pH 7.4). Cells were then desensitized with 0.25 M trypsin-EDTA solution and harvested cells were treated with 1 ml of 5% TCA (trichloroacetic acid) per 1 × 10 ⁶ cells. Then, after centrifuging twice at 2500 rpm, the separated melanin was washed with phosphate buffer, and then 1 ml of ether: ethanol (1: 3) was added, followed by centrifugation twice, washing with 1 ml of ether and drying. The dried melanin was reacted with 1N NaOH at 56 ° C for 1 hour, and the absorbance was measured at 470 nm using a spectrophotometer. Inhibition of melanin biosynthesis was expressed by the absorbance reduction ratio of the sample solution and the non-added sample.

4. Procollagen  Type-I biosynthesis measurement

Collagen is synthesized in the form of a precursor, procollagen. Procollagen contains a peptide sequence called a propeptide at the amino terminus and at the carboxyl terminus. The function of the propeptide helps to fold the procollagen molecule in the endoplasmic reticulum and, at the same time, cleaves and separates from the collagen molecule when the collagen polymerization takes place. Therefore, by measuring the amount of the separated propeptide, the degree of collagen biosynthesis in the cell can be measured. Thus, the skin melanoma cells (B16 melanonma cells) were inoculated in a 12-well plate at a concentration of 5 x 10 ⁴ cells / well, samples were added to each well, and the cells were cultured in a CO ₂ incubator for 48 hours. The amount of the pro peptide was measured using a procollagen type-I peptide (PIP).

5. Measurement of tyrosinase activity

The activity of tyrosinase was determined by modifying the method of Choi et al. B16F10 melanoma cells were inoculated into 6 wells to be 5 x 10 ⁴ cells and cultured. After 24 hours, the samples were treated for 48 hours in each well, washed twice with PBS, and then lysed in lysis buffer (1 % triton X-100, 0.1 M sodium phosphate buffer and 50 mM PMSF, pH 6.8). Cells were disrupted on ice and centrifuged, and only the supernatant was collected and used as the enzyme solution. The substrate was prepared by dissolving 2 mg / ml of L-DOPA in 0.1 M sodium phosphate buffer (pH 6.8), adding 40 μl of the enzyme solution to 160 μl of the substrate, heating the mixture at 37 ° C for 1 hour, DOPA chrome) was measured at 490 nm and calculated using the following equation.

(%) = (1-absorbance of sample / absorbance of control) x 100

Example  1. Acid hydrolysis through ash tree Heat number  Identification of active substance changes in extracts

The extraction yields and tyrosinase inhibitory activities of the extracts prepared according to the extraction conditions (extraction solvent, extraction time and extraction temperature) on the ash bark were measured and as a result, water was used as an extraction solvent as shown in Table 1 below Extraction yield and tyrosinase inhibition activity of the 7th hot - water extract prepared at 90 ℃ were the best. The changes of active substances in the extracts were confirmed by acid hydrolysis method. As a result, it was confirmed that the content of escululin increased proportionally with the disappearance of esculin, the main component of ash tree, in the hydrolyzate of the ash extract.

The absolute content of esculin and escululin present in the 7th hot water extract having the best extraction yield and tyrosinase inhibitory activity among the above extracts was measured by HPLC analysis. As a result, as shown in Table 2 Esculetin, which is the aglycone form of esculin, was measured in an amount of 0.7 mg / g in the hydrolyzate of the ash tree before the acid hydrolysis method, and 80.9 mg / g after the acid hydrolysis method Not only did the content increase. In the tyrosinase-inhibiting activity, tyrosinase inhibitory activity was significantly increased when the acid hydrolysis method was treated more than the 7th hot-water extract (Table 3).

In addition, active substance separation was carried out by using column chromatography using an ODS (octadecylsilica) gel for an ash tree hydrothermal extract (No. 7) having excellent tyrosinase inhibitory activity. As the eluent, a water-methanol solvent system Step wise gradient mode was used. As a result, fr. 81-100, 3.5 mg of pure substance E-1, fr. At 133-145, 35.1 mg of E-2, a pure substance, was isolated. The chemical shifts were measured using ¹ H and ¹³ C using NMR (Varian 600 MHz) for E-1 (6.16 min) and E-2 (8.68 min) The chemical shift value was compared with its reference and the structure of the compound was determined as E-1 as esculin and E-2 as esculetin.

Yield and tyrosinase inhibitory activity of ash tree extract No. The extraction solvent (100 ml) Extraction time (min) Extraction temperature (캜) The sample (g) yield(%) Tyrosinase
Inhibitory activity (IC _50,占 퐂 / ml) One H ₂ O 90 50 10 6.7 412.0 + - 21.3 2 50% EtOH 90 50 10 6.1 > 500 3 70% EtOH 90 50 10 5.0 > 500 4 H ₂ O 90 70 10 5.6 332.6 ± 12.1 5 50% EtOH 90 70 10 5.8 > 500 6 70% EtOH 90 70 10 7.5 > 500 7 H ₂ O 90 90 10 10.2 294.3 ± 10.3 8 50% EtOH 90 90 10 9.6 > 500 9 70% EtOH 90 90 10 13.8 > 500

 Comparison of absolute amounts of esculin and escululin before and after hydrolysis of acid hydrolyzate of hydrothermal extract of ash tree sample Absolute content (mg / g) Esculin ( t _R : 6.1 min) S Cool retinoic (t _R: 8.7 min) Ash extraction (No. 7) 192.6 0.7 Acid hydrolysis 2 hours Reactant 0.6 80.9

Comparison of tyrosinase inhibitory activity before and after hydrolysis by treating acid hydrolyzate of ash tree extract Samples (占 퐂 / ml) Tyrosinase inhibitory activity (%) IC ₅₀ ([mu] g / ml) 500 250 125 62.5 31.3 Ash extraction (No.7) 65.6 48.1 23.4 11.1 10.9 294.4 + - 10.3 Acid hydrolysis 2 hours Reactant 87.6 73.5 57.3 43.7 38.7 92.7 ± 7.1

Example  2. Skin whitening and anti-wrinkle efficacy evaluation

(1) Evaluation of tyrosinase inhibitory activity

Compared to arbutin, a positive control for esculin and esculetin, the compounds isolated from acid hydrolysates of the ash extract showing skin whitening efficacy, tyrosinase inhibitory activity . As a result, it was confirmed that the IC ₅₀ value was about 324.7 μM for arbutin, and that esculin had no tyrosinase inhibitory activity while escululin exhibited excellent inhibitory activity at 76.6 μM.

The tyrosinase inhibitory activity of esculin and esculetin compound Tyrosinase inhibitory activity (%) IC ₅₀ ([mu] M) 500 250 125 62.5 31.3 Escululine 89.6 72.4 66.5 50.1 23.1 76.6 ± 5.7 Esculin - - - - - > 500 Arbutin 58.4 43.6 34.8 28.1 9.1 324.7 ± 9.4

(2) Evaluation of melanin synthesis inhibitory activity

Compared to arbutin, a positive control for esculin and esculetin, the compounds isolated from acid hydrolysates of the ash extract exhibiting skin whitening efficacy, B16 melanoma cells The inhibitory activity of melanin synthesis was evaluated. As a result, as shown in Fig. 1, the melanin synthesis inhibitory activity was about 20% when treated with 75 μg / ml of Esculin, whereas about 70% or more of melanin synthesis inhibitory activity was observed with 75 μg / And it was confirmed that melanin synthesis inhibitory effect is superior to that of arbutin, which is a positive control group.

(3) Procollagen  Type-I ( procollagen  type-I) Sum performance  evaluation

The effect on the collagen type-I biosynthesis was evaluated in order to evaluate the skin wrinkle reducing effect on esculin and esculetin which are the compounds separated from the acid hydrolyzate of the ash extract. As a result, as shown in Fig. 2, about 70% of procollagen type-I combination performance was observed at 75 占 퐂 / ml of Esculin treatment whereas about 90% of procolagin Type-I combination performance, and it was confirmed that EGCG, which is a positive control, is superior in the ability to produce procollagen type-I.

Claims (2)

1) an ash containing an esculin ( Fraxinus rhynchophylla ), and extracting the extract at 85 to 95 DEG C to obtain an ash tree extract containing esculin;
2) hydrolyzing esculin by treating the ash extract containing the esculin with an acid; And
3) Separation and purification of escululin from the esculin hydrolyzate. The method for producing escululin according to claim 1, wherein the escululin has skin whitening and wrinkle-reducing effects.

Images