NOVEL SEA ALGAE-DERIVED ALKYL-AGARBIOSIDE, PREPARATION METHOD THEREFOR, OR USE THEREOF

Abstract

The present invention identifies moisturizing and physiological activities of agarobiose and provides a novel substance alkyl-agarobioside for maintaining moisturization activity of agarbiose and securing both pH and temperature stability, and a preparation method therefor. According to the present invention, alkyl-agarobioside can be produced in a cost-efficient manner as a moisturizing material for foods, medicinal products, and cosmetic products.

Claims

1. A compound represented by Chemical Formula 1 below, a derivative thereof, or a salt thereof: ##STR00003## in Chemical Formula 1, R is a C.sub.1-5 alkyl group.

2. The compound of claim 1, wherein R is an ethyl group.

3. A method of producing a compound represented by Chemical Formula 1 below, the method comprising: treating agar or agarose with a strong acid and an alkanol; neutralizing a product from the treating process; and separating and purifying a compound represented by Chemical Formula 1 below from the neutralized product: ##STR00004## in Chemical Formula 1, R is a C.sub.1-5 alkyl group.

4. The method of claim 3, wherein the strong acid is one or more of hydrochloric acid, sulfuric acid, and nitric acid.

5. The method of claim 3, wherein 1 or 20% (w/v) of agar or agarose is treated with the strong acid at a concentration of 10 to 100 mM.

6. The method of claim 3, wherein the strong acid and the alkanol are treated sequentially or simultaneously, regardless of order.

7. The method of claim 3, wherein the alkanol is ethanol, and R is an ethyl group.

8. A moisturizing composition comprising a compound represented by Chemical Formula 1 below, a derivative thereof, or a salt thereof: ##STR00005## in Chemical Formula 1, R is a C.sub.1-5 alkyl group.

9. A moisturizing cosmetic composition comprising a compound represented by Chemical Formula 1 below, a derivative thereof, or a salt thereof: ##STR00006## in Chemical Formula 1, R is H or a C.sub.1-5 alkyl group.

10. The composition of claim 8, wherein the alkyl group is an ethyl group.

11. The composition of claim 9, wherein the alkyl group is an ethyl group.

Description

DESCRIPTION OF DRAWINGS

[0038] FIG. 1 illustrates the results of testing the HAS2 expression effect of AB in HaCaT cells, which are human skin cells, and the pH stability of AB: (A) cytotoxicity according to AB concentration, (B) HAS2 expression level in HaCaT cells according to AB concentration, and (C) pH stability of AB.

[0039] FIG. 2 illustrates the results of measuring the produced ethyl-agarobioside according to the sulfuric acid concentration: (A) the produced ethyl-agarobioside concentration according to the sulfuric acid concentration, and (B) HPLC results of the produced ethyl-agarobioside according to the sulfuric acid concentration.

[0040] FIG. 3 illustrates the HPLC results of the produced ethyl-agarobioside according to the type of strong acid.

[0041] FIG. 4 illustrates the results of measuring ethyl-agarobioside purified using size exclusion chromatograph with G-10 resin: (A) the product obtained by decomposing agarose with sulfuric acid and ethanol, and (B) the result of separation and purification using size exclusion chromatography with G-10 resin.

[0042] FIG. 5 illustrates the LC-HRMS and 2D-HSQC NMR analyses results of the separated and purified ethyl-agarobioside: (A) LC-HRMS analysis results of separated and purified ethyl-agarobioside, and (B) 2D-HSQC NMR analysis results of separated and purified ethyl-agarobioside.

[0043] FIG. 6 illustrates the results of confirming the HAS2 expression effect of ethyl-agarobioside in HaCaT cells, which are human skin cells, and the pH stability of ethyl-agarobioside: (A) cytotoxicity according to ethyl-agarobioside concentration, (B) HAS2 expression level in HaCaT cells according to ethyl-agarobioside concentration, and (C) pH stability of ethyl-agarobioside.

[0044] FIG. 7 illustrates the results of comparing HAS2 expression levels when HaCaT cells, which are human skin cells, are treated with 100 ?g/mL of L-AHG, AB, and ethyl-agarobioside.

[0045] FIG. 8 illustrates the results of comparing the temperature stability of AB and ethyl-agarobioside.

[0046] FIG. 9 illustrates the comparison of concentration changes of AB and ethyl-agarobioside by temperature to confirm the temperature stability of ethyl-agarobioside: (A) 4? ? C., (B) 30? C., and (C) 45? C.

MODES OF THE INVENTION

[0047] Hereinafter, the present invention will be described in detail by examples. However, the following examples are given for the purpose of illustration only, and the present invention is not limited to the examples described below.

[Example 1] Moisturizing Effect of AB on HaCaT Cells (Human Skin Cells)

[0048] AB was produced from sea algae according to the related art (Korean Registered Patent No. 10-1864800), and the produced AB was confirmed to have a moisturizing effect in HaCaT cells, which are human skin cells, and pH stability (FIG. 1).

[0049] Cytotoxicity tests were performed using the MTT(3-4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. HaCaT cells were cultured in an animal cell incubator at 37? C. and 5% CO.sub.2 in a laboratory using Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v/v) FBS, 100 U/mL penicillin, and 100 g/mL streptomycin as antibiotics. The produced AB was dissolved in DMSO and added to the culture medium at 0-100 g/mL, and cells were cultured in the medium for 24 hours. The cells were treated with an MTT solution (5 mg/mL) and cultured for another 4 hours, and then an amount of the produced formazan as a blue crystal, was measured by measuring absorbance at 595 nm using an ELISA reader. Toxicity to cells was expressed as a percentage of the average absorbance value of each control group. To investigate the cytotoxicity of AB in HaCaT keratinocytes, when AB was treated at a concentration of 0-100 g/mL, the degree of change in cell proliferation was calculated compared to control groups treated with only DMSO. The results of Example 1 are shown in A of FIG. 1.

[0050] B of FIG. 1 illustrates the presence or absence of a cytotoxic effect when HaCaT keratinocytes are treated with AB according to an example of the present invention. Cell viability was measured to be 100% or more at 100 g/mL AB. Therefore, it was confirmed that there is no cytotoxicity below 100 g/mL AB.

[0051] Hyaluronan (HA) is a glycosaminoglycan composed of D-glucuronic acid and N-acetyl-D-glucosamine. Due to its ability to retain large amounts of water, HA plays an important role in regulating hydration and osmotic pressure. AH is synthesized in cell membranes by HAS1, HAS2, and HAS3, and HAS2 in particular appears in human normal tissues. In previous studies, it was found that a genetic defect of HAS2 causes fetal lethality in a mouse model and shows reduced HAS2 gene expression in the epidermis and dermis of adult human skin. Therefore, increasing HAS2 expression may be a great strategy to maintain skin homeostasis. To determine the AB induction time and AB concentration for HAS2 expression, Western blot analysis was performed. Cells used for the analysis were HaCaT cells, which were cultured at 37? C. in a 5% CO.sub.2 atmosphere using DMEM with 10% FBS and penicillin/streptomycin. Cells (1?10.sup.5) were cultured in 6-cm dishes for 24 hours and then starved in serum-free medium for another 24 hours to remove the FBS effect on the activation of the kinase. Then, AB was treated at a certain time and concentration. Cells were lysed with a lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na.sub.2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ?-glycerophosphate, 1 mM Na.sub.3VO.sub.4, 1 g/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail tablet]. The protein concentration was measured using a dye-conjugated protein assay kit (Bio-Rad Laboratories Inc.) according to the manufacturer's instructions. Dissolved proteins (20-40 ?g) were subjected to 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane by electrophoresis (Millipore Corp., Bedford, MA, USA). After blotting, the membrane was blocked with 5% skim milk for 2 hours and incubated overnight at 4? C. with a primary antibody (goat anti-mouse IgG-HRP). Subsequently, the membrane was incubated with a secondary antibody (goat anti-rabbit IgG HRP-conjugated secondary antibody), and the protein bound to the antibody was detected using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). Representative values of two independent experiments were used as data. As shown in B of FIG. 1, AB increased HAS2 expression 3 hours after treatment. In addition, AB increased HAS2 expression in a concentration-dependent manner. The moisturizing activity of AB was confirmed from the result.

[Example 2] pH Stability of AB

[0052] The pH stability of AB was assessed (C of FIG. 1). For a pH stability experiment, the degree of denaturation in each sample (AB) over time was measured at 25? C. under conditions of pH 3 (20 mM citrate), pH 7 (20 mM Tris-HCl), and pH 9 (20 mM Tris-HCl). As shown in C of FIG. 1, the sample was stable over time at pH 3 and pH 7, but it was found that the sample was completely denatured within 6 weeks at pH 9. Since the buffer and AB peaks were not separated at pH 3 using HPLC, AB was quantified through GC/MS analysis. The derivatization process for GC/MS analysis is as follows. After drying a purified sample with a speed vac, 10 ?L of 40 mg/mL O-methylhydroxylamine hydrochloride in pyridine was added and reacted at 30? ? C. and 200 rpm for 90 minutes. Then, 45 ?L of N-methyl-N-(trimethylsilyl)trifluoroacetamide was added and reacted at 37? C. and 200 rpm for 30 minutes. Instrument conditions for GC/MS analysis are as follows. A DB5-MS capillary column was used in the analysis, and the GC column temperature condition was maintained at 50? C. for 1 minute and then raised to 280? C. and maintained for 5 minutes. 1 ?L of sample was analyzed in a splitless mode. Each concentration of agarose at pH 7 and pH 9 was analyzed using HPLC. HPX-87H was used as a column, and the sample was analyzed at a column temperature of 65? C. and a flow rate of 0.5 mL/min. 0.005 M sulfuric acid was used as a mobile phase.

[Example 3] Production of Ethyl-Agarobioside from Red Algae (Agarose)

[0053] Agarose, which is a representative polysaccharide constituting sea algae, was decomposed using strong acids such as sulfuric acid, hydrochloric acid, and nitric acid. Ethyl-agarobioside was produced through a one-pot reaction of 2% (w/v) agarose with 12.5 mM sulfuric acid and 100 mL of ethanol at 70? C. overnight (about 18 hours). The sulfuric acid was neutralized and removed using tertiary distilled water and calcium hydroxide (Ca(OH).sub.2) in order to use the ethyl-agarobioside. In addition, to determine the concentration of sulfuric acid that can produce a large amount of ethyl-agarobioside, experiments were conducted with 3.125 mM, 6.25 mM, 12.5 mM, 25 mM, 50 mM, and 100 mM of sulfuric acid, and the produced ethyl-agarobioside was quantified, and it was confirmed that the greatest amount of ethyl-agarobioside was produced with 12.5 mM sulfuric acid (FIG. 2). In addition, it was confirmed that when agarose was treated with hydrochloric acid (12.5 mM) or nitric acid (12.5 mM, 25 mM) in a 20 mL reaction volume under the same experimental conditions as sulfuric acid above, the greatest amount of ethyl-agarobioside was produced in each case (FIG. 3).

[Example 4] Separation and Purification of Ethyl-Agarobioside from Fermentation Products Using Size-Exclusion Chromatography

[0054] Size-exclusion chromatography was used to separate and purify ethyl-agarobioside produced in Example 3. Sephadex G-10 (GE Healthcare) was used as a resin, and distilled water was used as a mobile phase (FIG. 4).

[Example 5] Identification of Molecular Weight and Structure of Ethyl-Agarobioside Through LC-HRMS and 2D HSQC NMR Analyses

[0055] LC-HRMS and 2D HSQC NMR analyses were conducted to identify the molecular weight and chemical structure of ethyl-agarobioside produced in Examples 3 and 4 (FIG. 5). LC-HRMS analysis results showed that the molecular weight of ethyl-agarobioside was 352.14 when agarose was converted to ethyl-agarobioside. Excluding a hydrogen ion, the actual molecular weight measured became 351. 2 mg of an ethyl-agarobioside sample was dissolved in D.sub.2O, and 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid was used as an internal standard to calculate a chemical shift. Chemical shifts in 2D HSQC NMR analysis were compared to previously reported results in documents to identify the chemical structure (Anatoly I. Usov et al (1980) Biopolymers, 19: 977-990; Cyrille Rochas (1986) Carbohydrate Research, 148: 199-207; E. Murano (1992) Carbohydrate Polymers. 18: 171-178).

[Example 6] Experiment for Moisturizing Effect of Ethyl-Agarobioside in HaCaT Cells (Human Skin Cells)

[0056] The experiment was performed in the same manner as Example 1, and it was confirmed that ethyl-agarobioside also exhibits moisturizing activity without cytotoxicity in HaCaT cells, which are human skin cells (A of FIG. 6 and B of FIG. 6).

[Example 7] pH Stability of Ethyl-Agarobioside

[0057] The experiment was performed in the same manner as Example 2, and it was confirmed that the stability of ethyl-agarobioside was maintained at pH 3, pH 7, and pH 9. The ethyl-agarobioside concentration was measured using GC-MS and HPLC (C of FIG. 6).

[Example 8] Experiment for Comparing HAS2 Expression of L-AHG, AB, and Ethyl-Agarobioside in HaCaT Cells (Human Skin Cells)

[0058] The experiment was performed in the same manner as Example 1. In a previous study, it was reported that L-AHG (Korean Registered Patent No. 10-1525298) has moisturizing activity in HaCaT cells, which are human skin cells, and in the present invention, the effects on the regulation of HAS2 expression in HaCaT cells, which are human skin cells, when AB and ethyl-agarobioside were treated at the same concentration were compared with L-AHG. It was confirmed that, compared to L-AHG showing a moisturizing effect, HAS2 expression was greatly increased when treated with AB and ethyl-agarobioside, so it was expected that the moisturizing effect of AB and ethyl-agarobioside would be better (FIG. 7).

[Example 9] Temperature Stability of AB and Ethyl-Agarobioside

[0059] The temperature stability of agarobiose was tested. The experimental method measured the denaturation/decomposition degrees of AB and ethyl-agarobioside over time at 4? C., 30? C., and 45? C. As shown in FIG. 8, it was confirmed that AB maintained stability for 15 weeks at 4? C. and 30? C., but at 45? C., stability slowly decreased. On the other hand, it was confirmed that ethyl-agarobioside maintained stability for 15 weeks at 4? C. and 30? C., but ethyl-agarobioside was less stable than AB after 6 weeks at 45? C. AB and ethyl-agarobioside were quantified using HPLC. As a result of analyzing reactant components of ethyl-agarobioside at 45? C. using HPLC, it was confirmed that AB, which was not present initially, was produced, and as the temperature increased, ethyl-agarobioside was converted to AB and was present along with AB. From these results, it was determined that not only the functionality of the cosmetic composition including ethyl-agarobioside may not be affected because ethyl-agarobioside is converted to AB, exhibiting the same moisturizing activity as ethyl-agarobioside, at high temperatures above 45? C., but also high-temperature stability may be secured.