Method for preparing 3,6-anhydro-L-galactose, and use thereof

Abstract

The present invention relates to a method for preparing 3,6-anhydro-L-galactose, and use thereof. More specifically, 3,6-anhydro-L-galactose, which is a monosaccharide constituting agar, is produced in a high yield through chemical and enzymatic methods, and the physiological activities thereof such as whitening, moisturizing, antioxidant, antiinflammatory activities and the like are displayed, thereby enabling industrial use thereof.

Description

BACKGROUND

1. Field of the Invention

The present invention relates to 3,6-anhydro-L-galactose prepared through chemical and enzymatic methods and industrial use thereof using physiological efficacy.

2. Discussion of Related Art

Agar (or agarose) which is a main polysaccharide constituting a red alga is a polymer in which two monosaccharides, 3,6-anhydro-L-galactose and D-galactose, are alternately bound via α-1,3 linkage and β-1,4 linkage. It has been widely known in that an agarooligosaccharide produced by non-specifically hydrolyzing agarose using a chemical catalyst such as hydrochloric acid, sulfuric acid, and acetic acid exhibits excellent physiological activities such as antioxidant, antiinflammatory, anticancer, whitening, and antiallergic activities. Due to a variety of such physiological activities, agarooligosaccharides and neoagarobiose that are disaccharides have been widely used as functional materials in the fields of food and beauty industries.

As one of monosaccharides constituting the agarooligosaccharides, 3,6-anhydro-L-galactose has problems in that it is produced as a monosaccharide with a very low yield through conventional chemical treatments, and is easily degraded to excessive extents since it has an unstable reducing end and is easily converted into hydroxymethylfurfural in the presence of a high-concentration strong acid under high-temperature reaction conditions (Jol et al (1999) Anal Biochem. 268, 213-222, Kim et al (2010) Bull Korean Soc. 31(2) 511-514). Also, although the hydrolysis using chemical treatment has an advantage in that it is readily applicable for the purpose of commercialization due to low production cost and simple treatment conditions, it has a problem in that a desired product is produced with a very low yield when specific linkages are broken to obtain the product since the chemical treatment non-specifically breaks the linkages (Chen et al (2005) Food Technol Biotechnol. 43(1) 29-36). Further, enzymes specifically breaking linkages in 3,6-anhydro-L-galactose to produce monosaccharides have been reported in recent years, but studies remain to be done to separate, purify, and quantify only 3,6-anhydro-L-galactose from a reaction product.

SUMMARY OF THE INVENTION

Therefore, the present invention is directed to a method for preparing 3,6-anhydro-L-galactose with a high yield through enzymatic degradation while minimizing over-degradation effects caused by chemical treatment.

Also, the present invention is directed to industrial use thereof to investigate the physiological activities of 3,6-anhydro-L-galactose.

According to an aspect of the present invention, there is provided a method for preparing 3,6-anhydro-L-galactose, which includes preparing an agarooligosaccharide by allowing agarose to react with a weak acid at a concentration of 0.5 to 60% (w/v) at a temperature of 40 to 150° C. and a rotary speed of 100 to 200 rpm for 30 minutes to 6 hours, and allowing the agarooligosaccharide to react with an agarose-degrading enzyme and an α-neoagarobiose hydrolase at a temperature of 20 to 40° C. and a rotary speed of 0 to 200 rpm for 30 minutes to 7 days.

In this case, the agarose-degrading enzyme may be an enzyme that breaks a β-1,4-glycosidic linkage between D-galactose and 3,6-anhydro-L-galactose of agarose. More specifically, the agarose-degrading enzyme may be represented by an amino acid sequence set forth in SEQ ID NO: 1.

The neoagarobiose hydrolase may be represented by an amino acid sequence set forth in SEQ ID NO: 3.

According to one exemplary embodiment, the agarose-degrading enzyme and the neoagarobiose hydrolase may be derived from Saccharophagus degradans (S. degradans) 2-40^T.

The method for preparing 3,6-anhydro-L-galactose according to one exemplary embodiment may further include separating and purifying the 3,6-anhydro-L-galactose by sequentially performing adsorption chromatography and gel-permeation chromatography on the 3,6-anhydro-L-galactose.

According to another aspect of the present invention, there is provided a cosmetic composition for skin whitening or moisturizing including 3,6-anhydro-L-galactose.

According to still another aspect of the present invention, there is provided cosmetic use of 3,6-anhydro-L-galactose for skin whitening or moisturizing.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a skin pigmentation disease including 3,6-anhydro-L-galactose.

According to still another aspect of the present invention, there is provided use of the 3,6-anhydro-L-galactose for preparing the pharmaceutical composition for preventing or treating a skin pigmentation disease.

According to still another aspect of the present invention, there is provided a cosmetic or medical method for whitening or moisturizing a skin of a mammal, preferably, a human, which requires skin whitening or moisturizing. Here, the cosmetic or medical method includes administering an effective amount of 3,6-anhydro-L-galactose, preferably, a pharmaceutical or cosmetic composition including 3,6-anhydro-L-galactose.

According to still another aspect of the present invention, there is provided a cosmetic or medical method for treating a condition, disease, and/or lesion of the skin associated with regulation of pigmentation. Here, the method includes applying a pharmaceutical or cosmetic composition including 3,6-anhydro-L-galactose onto the skin, and the condition, disease, and/or lesion may be locally advanced due to an increase in synthesis of a melanin pigment, and may be at least one selected from the group consisting of chloasma, freckles, lentigo, nevi, pigmentation caused by use of drugs, post-inflammatory pigmentation, and hyperpigmentation occurring on dermatitis.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating an inflammatory disease, which includes 3,6-anhydro-L-galactose.

According to still another aspect of the present invention, there is provided a method for treating an inflammatory disease in an animal. Here, the method includes administering the composition for preventing or treating an inflammatory disease, which includes a pharmaceutically effective amount of 3,6-anhydro-L-galactose, to a subject.

According to yet another aspect of the present invention, there is provided use of 3,6-anhydro-L-galactose to prepare a pharmaceutical composition for preventing or treating an inflammatory disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating a procedure of preparing 3,6-anhydro-L-galactose from agarose according to one exemplary embodiment of the present invention. In FIG. 1, DP represents a degree of polymerization of a polysaccharide.

FIG. 2 shows the thin-layer chromatography (TLC) results obtained in a procedure of preparing and purifying 3,6-anhydro-L-galactose from agarose. In the processing step, Lane 1 represents agarose, Lane 2 represents an acetic acid hydrolysate, Lane 3 represents an Aga50D reaction product, Lane 4 represents an SdNABH reaction product, Lane 5 represents 3,6-anhydro-L-galactose separated by silica gel chromatography, Lane 6 represents 3,6-anhydro-L-galactose purified by biogel P2 chromatography, AHG in the Y axis represents 3,6-anhydrogalactose, and NAB represents neoagarobiose.

FIG. 3 shows the results of silica gel chromatography-TLC which is the first step of purifying 3,6-anhydro-L-galactose. In FIG. 3, AHG represents 3,6-anhydro-galactose.

FIG. 4A shows GC/MS total ion chromatograms of a D-form 3,6-anhydro-galactose standard material;

FIG. 4B shows 3,6-anhydro-L-galactose purified according to one exemplary embodiment of the present invention.

FIG. 5 shows the ¹H NMR analysis results of 3,6-anhydro-L-galactose separated and purified according to one exemplary embodiment of the present invention.

FIG. 6 shows the 2D-HSQC NMR analysis results of 3,6-anhydro-L-galactose separated and purified according to one exemplary embodiment of the present invention.

FIG. 7 shows the whitening activities of 3,6-anhydro-L-galactose, neoagarobiose, D-galactose, and 3,6-anhydro-D-galactose. In FIG. 7, α-MSH represents an α-melanocyte-stimulating hormone, NAB represents neoagarobiose, and D-AHG and L-AHG represent D- and L-forms of 3,6-anhydrogalactose, respectively.

FIG. 8 shows the antioxidant activities of 3,6-anhydro-L-galactose, neoagarobiose, D-galactose, and 3,6-anhydro-D-galactose.

FIG. 9A shows the results of tyrosinase expression in the cells exposed to α-MSH after treatment according to concentrations of L-AHG

FIG. 9B shows a graph obtained by plotting the results of FIG. 9A as relative intensity of tyrosinase to β-actin.

FIG. 10A shows the results of TRP-1 expression in the cells exposed to α-MSH after treatment according to concentrations of L-AHG

FIG. 10B shows a graph obtained by plotting the results of FIG. 10A as relative intensity of TRP-1 to β-actin.

FIG. 11 shows effects of L-AHG on in vitro tyrosinase activities.

FIG. 12A shows an expression level of HAS2 after treatment with 100 μg/mL of L-AHG according to the elapse of time.

FIG. 12B shows an expression level of HAS2 after treatment according to concentrations of L-AHG

FIG. 13A shows the ERK phosphorylation results after treatment with 100 μg/mL of L-AHG according to the elapse of time.

FIG. 13B shows the ERK phosphorylation results after treatment according to concentrations of L-AHG

FIG. 14A shows the AKT phosphorylation results after treatment with 100 μg/mL of L-AHG according to the elapse of time.

FIG. 14B shows the AKT phosphorylation results after treatment according to concentrations of L-AHG

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, exemplary embodiments of the present invention will be described in further detail.

The present invention is directed to a method for preparing 3,6-anhydro-L-galactose, which includes preparing an agarooligosaccharide by allowing agarose to react with a weak acid at a concentration of 0.5 to 60% (w/v) at a temperature of 40 to 150° C. and a rotary speed of 100 to 200 rpm for 30 minutes to 6 hours, and allowing the agarooligosaccharide to react with an agarose-degrading enzyme and an α-neoagarobiose hydrolase at a temperature of 20 to 40° C. and a rotary speed of 0 to 200 rpm for 30 minutes to 7 days.

The method for preparing 3,6-anhydro-L-galactose according to one exemplary embodiment of the present invention is characterized in that agarose is hydrolyzed under mild conditions using a weak acid, an agarooligosaccharide separated from the acid hydrolysate is allowed to react with an exo-type agarose-degrading enzyme producing disaccharides to produce a neoagarobiose, and monosaccharides, galactose and 3,6-anhydro-L-galactose, are produced through additional reaction with an α-neoagarobiose hydrolase.

The 3,6-anhydro-L-galactose may have a structure represented by the following Formula 1:

The method of the present invention will be described in further detail with reference to the procedure of preparing 3,6-anhydro-L-galactose as shown in FIG. 1.

The first step is to prepare an agarooligosaccharide by treating agarose with a weak acid.

The weak acid may be at least one selected from the group consisting of acetic acid, formic acid, succinic acid, citric acid, malic acid, maleic acid, and oxalic acid, which may be used alone or in combination.

The weak acid may be used at a concentration of 0.5 to 60% (w/v) in consideration of the unit production cost of and separation of salts produced after neutralization of weak acid. More specifically, the weak acid may be used at a concentration of 20 to 40% (w/v).

The reaction of the weak acid with the agarose may be performed at a temperature of 40 to 150° C. and a rotary speed of 100 to 200 rpm for 30 minutes to 6 hours. Within these ranges, formation of over-degradation products of agarose by the weak acid may be minimized.

The reaction product obtained after the reaction is an agarooligosaccharide which may be washed to remove the over-degradation product with the residual weak acid, and dried so that the agarooligosaccharide can be obtained in a powdery form.

As the washing solvent, a lower alcohol having 1 to 6 carbon atoms may be used, but the present invention is not limited thereto.

The second step is to prepare 3,6-anhydro-L-galactose by enzymatic degradation of the agarooligosaccharide. Here, the enzymatic degradation is performed by producing a disaccharide, neoagarobiose, by treating the agarooligosaccharide with an exo-type agarose-degrading enzyme and degrading the neoagarobiose into D-galactose and 3,6-anhydro-L-galactose by treating the neoagarobiose with an α-neoagarobiose hydrolase.

The enzymatic reaction may be performed at a temperature of 20 to 40° C. and a rotary speed of 0 to 200 rpm for 30 minutes to 7 days.

The enzymatic reaction will be described in further detail, as follows.

First, an enzyme breaking a β-1,4-glycosidic linkage between D-galactose and 3,6-anhydro-L-galactose of agarose (hereinafter referred to as ‘Aga50D’) may be used as the agarose-degrading enzyme degrading the agarooligosaccharide to produce the disaccharide, neoagarobiose.

The agarose-degrading enzyme may include an amino acid sequence set forth in SEQ ID NO: 1, and amino acid sequences having sequence identities of 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with respect to the amino acid sequence.

In the present invention, a polypeptide having a sequence identity of certain percentage (for example, 80%, 85%, 90%, 95%, or 99%) with respect to another sequence means that two sequences has the same amino acid residues at any percentage upon sequence comparison when the sequences are aligned to each other. The alignment and percentage homology or identity may be determined using any proper software program known in the related art, for example, those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al., (eds) 1987 Supplement 30 section 7.7.18). A preferred program includes GCG Pileup programs, for example FASTA (Pearson, et al., 1988 Proc. Natl. Acad. Sci. USA 85: 2444-2448), and BLAST (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al., 1997 NAR25: 3389-3402). Another preferred alignment program is ALIGN Plus (Scientific and Educational Software, PA), which preferably uses basic parameters. Still another preferred sequence software program which may be used herein is a TFASTA Data Searching program available for Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison Wis.).

In this specification, the terms “protein” and “polypeptide” may be used interchangeably. In this specification, one-letter or three-letter codes for amino acid residues are typically used herein.

The enzyme may be derived from S. degradans, but the present invention is not limited thereto.

The enzyme may be transcribed and translated through a coding gene, that is, a DNA fragment which is associated with production of a polypeptide including regions upstream and downstream of a coding region of the enzyme and an intervening sequence between individual coding fragments. For example, the enzyme may have a sequence set forth in SEQ ID NO: 2, but the present invention is not limited thereto.

The agarose-degrading enzyme according to one exemplary embodiment of the present invention may be separated and purified from a supernatant of the S. degradans culture broth, and may be produced and separated in the strains other than S. degradans using a genetic recombination technique or an artificial chemical synthesis method.

When the recombination technique is used, factors used to facilitate expression of typical recombinant proteins, for example, antibiotic-resistant genes, and reporter proteins or peptides usable for affinity column chromatography may be used herein. Such a technique falls within a category which can be easily carried out by those skilled in the art to which the present invention belongs. Also, a supernatant of the S. degradans culture broth may be used instead of the agarose-degrading enzyme according to one exemplary embodiment of the present invention, or a supernatant of a culture broth of an edible strain, for example, a transformed yeast strain obtained by transforming a yeast strain, may be used instead of the agarose-degrading enzyme.

In the present invention, the term “recombination” used in connection with cells, nucleic acids, proteins, or vectors means that the cells, nucleic acids, proteins, or vectors are modified by introduction of heterogeneous nucleic acids or proteins or alteration of innate nucleic acids or proteins, or that the cells are derived from such modified cells. That is, the recombinant cells, for example, express genes which are not found in the cells in an original non-recombinant form, or express original genes which are expressed abnormally upon expression or not expressed at all.

In this specification, the term “nucleic acid” encompasses all kinds of single- or double-stranded DNAs, RNAs, and chemical variants thereof. The terms “nucleic acid” and “polynucleotide” may be used interchangeably herein. Since the genetic codes are degenerated, one or more codons may be used to encode one certain amino acid, and the present invention covers polynucleotides encoding certain amino acid sequences.

The term “introduction” used to insert a nucleic acid sequence into cells refers to “transfection,” “transformation,” or “transduction,” and encompasses reference to integration of a nucleic acid sequence into eucaryotic or procaryotic cells. In this case, the nucleic acid sequence is integrated into the genome (for example, a chromosome, a plasmid, a chromatophore, or mitochondrial DNA) of a cell, and converted into an autonomous replicon or expressed temporally.

The reaction of the agarooligosaccharide with the agarose-degrading enzyme may be performed at a temperature of 20 to 40° C. and a rotary speed of 0 to 200 rpm for 30 minutes to 7 days. More specifically, such a reaction may be performed at a temperature of 25 to 35° C. and a rotary speed of 100 to 150 rpm for 1 to 4 days.

When the agarooligosaccharide is in a powdery form, the agarooligosaccharide may be dissolved in a conventional buffer solution to be used, but the present invention is not limited thereto.

Next, the neoagarobiose hydrolase that can degrade the neoagarobiose produced through the enzymatic reaction into D-galactose and 3,6-anhydro-L-galactose may be a protein having an amino acid sequence set forth in SEQ ID NO: 3 and a mutant protein having at least one substitution, deletion, translocation, and addition in the enzyme. Proteins having the neoagarobiose-hydrolytic activities are also included in a category of the enzymes according to one exemplary embodiment of the present invention, and, preferably, has an amino acid sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with respect to the amino acid sequence set forth in SEQ ID NO: 3 (hereinafter referred to as ‘SdNABH’).

The enzyme may be derived from S. degradans 2-40, but the present invention is not limited thereto.

The enzyme may be transcribed and translated through a coding gene, that is, a DNA fragment which is associated with production of a polypeptide including regions upstream and downstream of a coding region of the enzyme and an intervening sequence between individual coding fragments. For example, the enzyme may have a sequence set forth in SEQ ID NO: 4, but the present invention is not limited thereto.

The neoagarobiose hydrolase may be separated and purified from a supernatant of the S. degradans culture broth, and may be produced and separated in the strains other than S. degradans using a genetic recombination technique or an artificial chemical synthesis method.

When the recombination technique is used, factors used to facilitate expression of typical recombinant proteins, for example, antibiotic-resistant genes, and reporter proteins or peptides usable for affinity column chromatography may be used herein. Such a technique falls within a category which can be easily carried out by those skilled in the art to which the present invention belongs. Also, a supernatant of the S. degradans culture broth may be used instead of the neoagarobiose hydrolase according to one exemplary embodiment of the present invention, or a supernatant of a culture broth of an edible strain, for example, a transformed yeast strain obtained by transforming a yeast strain, may be used instead of the neoagarobiose hydrolase.

The reaction of the neoagarobiose with the neoagarobiose hydrolase may be performed at a temperature of 20 to 40° C. and a rotary speed of 0 to 200 rpm for 30 minutes to 7 days. More specifically, such a reaction may be performed at a temperature of 25 to 35° C. and a rotary speed of 100 to 150 rpm for 1 to 4 days.

Also, the method for preparing 3,6-anhydro-L-galactose according to one exemplary embodiment of the present invention may further include separating and purifying only 3,6-anhydro-L-galactose from the degradation products of neoagarobiose.

The 3,6-anhydro-L-galactose may be separated and purified with a high purity of approximately 96% by sequentially performing silica gel chromatography that is adsorption chromatography and biogel P2 chromatography that is gel-permeation chromatography.

The purified product is subjected to ¹H-NMR and 2D-heteronuclear single quantum coherence (HSQC) NMR analysis, and ¹H ppm and ¹³C ppm of previously reported 3,6-anhydro-L-galactose may be compared to identify the structure of the purified product.

In the preparation method according to one exemplary embodiment of the present invention, 76 g of the agarooligosaccharide may be produced from 100 g of agarose through chemical hydrolysis, and 37.55 g of the neoagarobiose and 15.21 g of the 3,6-anhydro-L-galactose may be produced using the Aga50D and SdNABH enzymes, respectively. Also, 3.98 g of pure 3,6-anhydro-L-galactose may be purified through a purification procedure using two kinds of chromatographies.

The present invention is also directed to a cosmetic composition for skin whitening or moisturizing including 3,6-anhydro-L-galactose.

Further, the present invention is directed to cosmetic use of 3,6-anhydro-L-galactose for skin whitening or moisturizing.

The 3,6-anhydro-L-galactose has higher whitening activities than arbutin that has been most widely known as a conventional whitening material, and has an effect of inhibiting the expression of tyrosinase, TRP-1, and the like, which are associated with promoting synthesis of melanin, in a concentration-dependent manner.

According to one exemplary embodiment of the present invention, an in vitro tyrosinase activity test shows that the 3,6-anhydro-L-galactose exhibits a whitening effect by inhibiting expression of the enzyme rather than inhibiting the tyrosinase activities.

Also, the 3,6-anhydro-L-galactose has an effect of promoting the expression of a HAS2 protein that is a moisturizing marker associated with the synthesis of hyaluronic acid.

According to one exemplary embodiment of the present invention, the expression of the HAS2 protein may be regulated by various inflammatory signal transduction pathways such as ERK, and AKT. Here, the 3,6-anhydro-L-galactose may increase phosphorylation of ERK and AKT, and the highly phosphorylated ERK and AKT may increase the expression of the HAS2 protein.

The 3,6-anhydro-L-galactose may be used without limitation as long as it may be synthesized, or may be prepared using the method for preparing 3,6-anhydro-L-galactose according to one exemplary embodiment of the present invention.

Based on an effective amount, a cosmetic preparation or cosmetics including the 3,6-anhydro-L-galactose itself or a mixture obtained by mixing a cosmetologically acceptable carrier with the 3,6-anhydro-L-galactose may be provided. In this case, the cosmetic preparation or cosmetics may be prepared into typical emulsified and solubilized formulations. In the emulsified formulation, the cosmetics may include a lotion, a cream, an essence, and the like, and, in the solubilized formulation, the cosmetics may be a toner. Proper formulations of the cosmetics may, for example, be provided in the form of a solution, a gel, a solid or pasty anhydrous product, an emulsion obtained by dispersing an oily phase in a water phase, a suspension, a microemulsion, a microcapsule, a microgranulocyte, or an ionic (liposomal) or non-ionic vesicle dispersing agent, or may also be provided in the form of a cream, a toner, a lotion, a powder, an ointment, a spray, or a conceal stick.

Also, the formulations of the cosmetics may be prepared in the form of a foam or an aerosol composition further including a compressed propellant.

In this case, the method of preparing cosmetics in the above-described form, and the carrier are apparent to those skilled in the art, and thus the specific description of the method of preparing cosmetics is omitted for clarity.

In addition to the 3,6-anhydro-L-galactose according to one exemplary embodiment of the present invention, the cosmetics may include supplemental agents typically used in the field of cosmetology, such as a fat material, an organic solvent, a dissolving agent, a concentrating agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizing agent, a foaming agent, an aromatic, a surfactant, water, an ionic or non-ionic emulsifying agent, a filler, a metal ion sequestering and chelating agent, a preservative, a vitamin, a blocking agent, a moisturizing agent, an essential oil, a dye, a pigment, a hydrophilic or lipophilic activating agent, a lipid vesicle, or any other components typically used in cosmetics.

When the 3,6-anhydro-L-galactose is used for foods or as a food additive, the 3,6-anhydro-L-galactose itself may be added, or may be used in connection with other foods or food components. In this case, the 3,6-anhydro-L-galactose may be properly used according to conventional methods. The amount of the mixed active component may be determined according to a purpose of use (prophylaxis, health, or therapeutic treatment). In general, the composition according to one exemplary embodiment of the present invention may be added at a content of 15 wt % or less, preferably 10 wt % or less, based on the total amount of the active component, upon preparation of foods. However, when the 3,6-anhydro-L-galactose is taken in for a long period of time for the purpose of health and hygiene or the purpose of regulation of health, the amount of the mixed active component may be lower than or equal to the above-described content. When the mixed active component has no problems regarding the safety, the active component may also be used within this content range or less.

The present invention is also directed to a pharmaceutical composition for preventing or treating a skin pigmentation disease, which includes 3,6-anhydro-L-galactose.

Also, the present invention is directed to use of the 3,6-anhydro-L-galactose for preparing the pharmaceutical composition for preventing or treating a skin pigmentation disease.

The pharmaceutical composition according to one exemplary embodiment of the present invention may further include a suitable carrier, excipient, and diluent which are typically used to prepare pharmaceutical compositions.

The pharmaceutical composition according to one exemplary embodiment of the present invention may be formulated in the form of external preparations such as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, syrup, and an aerosol, and in the form of a sterile injectable solution according to conventional methods. Preferably, the pharmaceutical composition may be used in the form of a cream, a gel, a patch, a spraying agent, an ointment, a plaster, a lotion, a liniment, a paste, or a cataplasma.

The carrier, excipient, and diluent that may be included in the pharmaceutical composition according to one exemplary embodiment of the present invention may include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, the pharmaceutical composition may be prepared using a diluent or excipient widely used in the related art, such as a filler, an extending agent, a binding agent, a wetting agent, a disintegrating agent, a surfactant, and the like.

A solid preparation for oral administration may include a tablet, a pill, a powder, a granule, a capsule, and the like. Such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, or gelatin, with the above-described compound. In addition to the simple excipients, lubricants such as magnesium stearate and talc may also be used. A liquid preparation for oral administration may include a suspension, a liquid for internal use, an emulsion, syrup, and the like. In addition to the widely used simple diluents such as water and liquid paraffin, the liquid preparation may include various excipients, for example, a wetting agent, a sweetening agent, an aromatic, a preservative, and the like. A preparation for parenteral administration may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, a suppository, and the like. A vegetable oil such as propylene glycol, polyethylene glycol, or olive oil, or an injectable ester such as ethyl oleate may be used as the non-aqueous solvent and the suspension. Witepsol, Macrogol, Tween 61, cocoa butter, laurin butter, glycerogelatin, and the like may be used as base of the suppository.

The pharmaceutical composition according to one exemplary embodiment of the present invention may be a formulation for external use in the skin which can be applied to the skin. In this case, the pharmaceutical composition may be prepared in the form of a liquid for external use, such as a cream, a gel, a patch, a spraying agent, an ointment, a plaster, a lotion, a liniment, a paste, or a cataplasma, but the present invention is not limited thereto.

A desirable dose of the pharmaceutical composition according to one exemplary embodiment of the present invention may vary according to the conditions and body weight of a patient, the severity of a disease, the type of a drug, and the route and duration of administration, but may be properly chosen by those skilled in the related art. However, the composition according to one exemplary embodiment of the present invention may be administered daily at a dose of 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, in order to show a desirable medicinal effect. The liquid for external use may be administered once or several times a day. The dosage is not intended to limit the scope of the present invention at any aspects.

Also, the present invention is directed to a cosmetic or medical method for whitening or moisturizing a skin of a mammal, preferably, a human, which requires skin whitening or moisturizing. Here, the cosmetic or medical method includes administering an effective amount of 3,6-anhydro-L-galactose, preferably, a pharmaceutical or cosmetic composition including 3,6-anhydro-L-galactose.

Likewise, the present invention is directed to a cosmetic or medical method for treating a condition, disease, and/or lesion of the skin associated with regulation of pigmentation. Here, the method includes applying a pharmaceutical or cosmetic composition including 3,6-anhydro-L-galactose onto the skin, and the condition, disease, and/or lesion may be locally advanced due to an increase in synthesis of a melanin pigment, and may be at least one selected from the group consisting of chloasma, freckles, lentigo, nevi, pigmentation caused by use of drugs, post-inflammatory pigmentation, and hyperpigmentation occurring on dermatitis.

The application frequency of the pharmaceutical or cosmetic composition including 3,6-anhydro-L-galactose may vary according to the demands of individual subjects. For example, the application frequency is proposed to be ten times a month to a day, preferably four times a week to a day, more preferably three times a week to a day, and the most preferably one or twice a day.

Also, the present invention is directed to use of the 3,6-anhydro-L-galactose for preparing a pharmaceutical or cosmetic composition for treating, caring, and/or dressing the skin, preferably the skin of a face, a neck, a neckline, a hand, an armpit, a groin, an elbow, and/or a knee, and more preferably a local site of a face, a neck, and/or a hand.

Further, the present invention is directed to a pharmaceutical composition for preventing or treating an inflammatory disease, which includes 3,6-anhydro-L-galactose.

Also, the present invention is directed to a method of treating an inflammatory disease in an animal, which includes administering the composition for preventing or treating an inflammatory disease, which includes a pharmaceutically effective amount of 3,6-anhydro-L-galactose, to a subject.

Furthermore, the present invention is directed to use of the 3,6-anhydro-L-galactose for preparing a pharmaceutical composition for preventing or treating an inflammatory disease.

The 3,6-anhydro-L-galactose has an antioxidant activity to inhibit generation of nitrite (NO₂), and thus may be used in the pharmaceutical composition for preventing or treating an inflammatory disease since the 3,6-anhydro-L-galactose has the highest antioxidant activity, compared to D-form 3,6-anhydro galactose, and another disaccharide and monosaccharide, such as neoagarobiose and galactose, which constitutes a sea alga (see FIG. 8).

Since the pharmaceutical composition used in the method of treating an inflammatory disease, and the method of administration are described above, the overlapping description of the pharmaceutical composition and the method of administration are omitted for clarity. Meanwhile, the subject to which the pharmaceutical composition for preventing or treating an inflammatory disease may be administered may include all kinds of animals. For example, the subject may be an animal except a human being, including a dog, a cat, a mouse, and the like.

The effective amount of the active component in the pharmaceutical composition means an amount required to treat a disease. Therefore, the effective amount of the active component may be adjusted according to various factors including the kind of a disease, the severity of a disease, the kinds and contents of the active component and other components included in a composition, the kind of a formulation, the age, body weight, general physical condition, gender, and diet of a patient, the time and route of administration, the secretion rate of the composition, the duration of treatment, and drugs used together.

Also, the term “treating” or “treatment” means relieving symptoms, temporally or permanently removing the causes of the symptoms, or alleviating the onset and the progress of, or preventing the illness, disorder, or disease, but the present invention is not limited thereto.

Hereinafter, the present invention will be described in further detail with reference to the following preferred Examples. However, it should be understood that the following Examples are given by way of illustration of the present invention only, and are not intended to limit the scope of the present invention.

<Example 1> Hydrolysis of Agarose Using Acetic Acid

Agarose that is a representative polysaccharide constituting a sea alga was hydrolyzed with acetic acid. 5% (w/v) of agarose was reacted with 3M acetic acid at 80° C. for 70 minutes and dried to remove acetic acid. Also, acetic acid which remained after drying through a washing procedure using ethanol, and over-degradation products that were able to be produced upon hydrolysis were removed to produce an agarooligosaccharide in the form of a pure powder (FIG. 2).

<Example 2> Production of 3,6-Anhydro-L-Galactose Using Aga50D and SdNABH

To degrade the acid hydrolysate produced in Example 1 into monosaccharides, D-galactose and 3,6-anhydro-L-galactose, the acid hydrolysate was reacted with an exo-type disaccharide-producing enzyme, Aga50D, (see Korean Patent Publication No. 2010-0040438) to produce neoagarobiose as a reaction product.

When the reaction with Aga50D was completed, the Aga50D reaction product was reacted with an SdNABH enzyme (see Korean Patent Publication No. 2010-0108241) in order to produce the monosaccharides, D-galactose and 3,6-anhydro-L-galactose, from the neoagarobiose. The enzymatic reaction conditions were as follows: 5% (w/v) of agarooligosaccharide was dissolved in 100 mL of a 50 mM Tris-HCl buffer solution (pH 7.4), and reacted at 30° C. and 150 rpm for 3 days. The amounts of the enzymes used upon the enzymatic reaction were 10 mg and 2.5 mg for Aga50D and SdNABH, respectively (FIG. 2).

<Example 3> Separation and Purification Using Silica Gel Chromatography and Biogel P2 Chromatography

Chromatography was performed to separate and purify only 3,6-anhydro-L-galactose from the reaction products produced in Examples 1 and 2. The reaction products were adsorbed onto celite to form a sample in the form of powder, and subjected to silica gel chromatography that was adsorption chromatography. A solvent obtained by mixing chloroform, methanol, and water at a ratio of 78:20:2 (v/v/v) was used as a mobile phase, and the total volume of the solvent as the mobile phase was 3 L. The volume of one fraction was 20 mL, and the sample composed of a total of 150 fractions was analyzed through TLC. Among these, the fractions containing 3,6-anhydro-L-galactose were collected. Since the biogel P2 chromatography to be further performed separated materials according to the molecular weights, the fractions containing D-galactose having a molecular weight similar to 3,6-anhydro-L-galactose among the fractions containing 3,6-anhydro-L-galactose were excluded (FIG. 3).

Only 3,6-anhydro-L-galactose was able to be purified from the disaccharides and the agarooligosaccharide having a low degree of polymerization with high purity through biogel P2 chromatography. The mobile phase used herein was water, and the volume of one fraction was 2 mL. Among the respective fractions, only the fractions showing spots of 3,6-anhydro-L-galactose on TLC were collected to obtain high-purity 3,6-anhydro-L-galactose (FIG. 2).

As shown in FIG. 2, 76 g of the agarooligosaccharide was produced from 100 g of agarose through chemical hydrolysis in Examples 1 to 3, and 37.55 g of neoagarobiose and 15.21 g of 3,6-anhydro-L-galactose were produced using the Aga50D and SdNABH enzymes, respectively. Also, 3.98 g of the pure 3,6-anhydro-L-galactose was purified through two chromatographies.

Also as shown in FIG. 3, no saccharides were observed in fractions 0.25-067 of elution volume (L), the sugars other than 3,6-anhydro-L-galactose and galactose were observed in fractions 0.77-1.77 (a sample subjected to biogel P2 chromatography after the concentration), and galactose and other sugars were observed in fractions 1.87-2.67.

<Example 4> Qualification and Quantification of 3,6-Anhydro-L-Galactose Using GC/MS Analysis

The high-purity 3,6-anhydro-L-galactose produced in Example 3 was quantified through GC/MS analysis. A derivatization procedure for GC/MS analysis was as follows. The purified sample was dried in a speed-vac, added to 50 μl of 2% (w/v) O-methylhydroxylamine hydrochloride in pyridine, and then reacted at 75° C. for 30 minutes. Then, 80 μl of N-methyl-N-(trimethylsilyl)trifluoroacetamide was added, and reacted at 40° C. and 150 rpm for 30 minutes. The equipment conditions for GC/MS analysis were as follows. A column used for analysis was a DB5-MS capillary column. In the case of the GC column temperature conditions, first, the sample was maintained at a temperature of 100° C. for 3.5 minutes, heated to 160° C., and maintained for 20 minutes. Thereafter, the sample was heated to 200° C., maintained for 15 minutes, finally heated to 280° C., and maintained for 5 minutes. 1 μl of the sample was analyzed at a split ratio of 9.6 (FIG. 4B).

<Example 5> Identification of Structure of 3,6-Anhydro-L-Galactose Through ¹H-NMR and 2D HSQC NMR Analyses

NMR analyses were performed to identify the chemical structure of the high-purity 3,6-anhydro-L-galactose produced in Examples 1 to 3. 2 mg of a 3,6-anhydro-L-galactose sample was dissolved in D₂O, and 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid was added as the internal standard to calculate the chemical shift. It was confirmed that the chemical structure was identified by comparing the chemical shifts in ¹H-NMR and 2D HSQC NMR to the resulting values of the previously reported documents (Sugano et al (1994) J Bacteriol. 176(22) 6812-6818, Miller et al (1982) Aust J Chem. 35(4) 853-856)(FIGS. 5 and 6).

<Example 6> Examination of Whitening and Antioxidant Activities of Separated and Purified 3,6-Anhydro-L-Galactose

To determine the whitening activities of the high-purity 3,6-anhydro-L-galactose produced in Examples 1 to 3, the melanoma cells, B16F10, were incubated. Before treatment with α-melanocyte-stimulating hormone that was a hormone promoting the melanin production, the melanoma cells were treated with each of arbutin most widely used as a whitening agent, saccharides (neoagarobiose, D-galactose, and 3,6-anhydro-D-galactose) constituting a red alga, and 3,6-anhydro-L-galactose at concentration of 1, 10, and 100 μg/mL, and the resulting reaction mixture was reacted with the α-melanocyte-stimulating hormone. After the melanoma cells were incubated together for 4 days, the optical densities of the melanoma cells were measured at 475 nm to determine the content of melanin.

As a result, it was revealed that the 3,6-anhydro-L-galactose showed very excellent whitening activities, compared to the other treated groups, and, in fact, that the 3,6-anhydro-L-galactose showed higher whitening activities than arbutin which is known as a functional material showing whitening activities (FIG. 7).

<Example 7> Antioxidant Activities of Separated and Purified 3,6-Anhydro-L-Galactose

To determine the antioxidant activities of the high-purity 3,6-anhydro-L-galactose produced in Examples 1 to 3, the concentration of nitrite (NO₂) in an RAW264.7 cell culture broth was measured by a method using a Griess reaction. The RAW264.7 cells were treated with a lipopolysaccharide at a concentration of 2 μg/mL, and simultaneously treated with each of neoagarobiose, galactose, 3,6-anhydro-D-galactose, and 3,6-anhydro-L-galactose at concentrations of 50, 100, and 200 μg/mL. Thereafter, the RAW264.7 cells were treated together to determine the antioxidant activities. After the RAW264.7 cells were incubated together for 24 hours, the optical densities of the RAW264.7 cells were measured at 540 nm to determine the antioxidant activities.

As a result, it was revealed that the 3,6-anhydro-L-galactose showed very excellent antioxidant activities, compared to the other treated groups, and that the 3,6-anhydro-L-galactose showed higher antioxidant activities than the previously reported 3,6-anhydro-D-galactose (FIG. 8).

<Example 8> Experiment of Whitening Effect of 3,6-Anhydro-L-Galactose in Human Epidermal Melanocytes

Melanin is a natural pigment found to give colors to the skin and hair. The known main function of melanin is to protect the skin from DNA damage caused by irradiation of absorbed and scattered ultraviolet rays. However, the formation or abnormal distribution of excessive melanin may cause irregular skin hyperpigmentations such as chloasma, freckles, senile lentigos, and the like. The previous studies showed that the ultraviolet irradiation increases levels of an α-melanocyte-stimulating hormone (α-MSH) and ACTH, and a related receptor thereof, that is, a melanocortin 1 receptor (MC1R) to promote the expression of melanin biosynthesis enzymes including tyrosinase, and tyrosinase-related proteins (TRPs). A copper-containing glycoprotein, tyrosinase, is a rate limiting enzyme which is important for synthesis of melanin in certain cell organs, that is, melanosomes produced only in melanocytes. Therefore, a decrease in tyrosinase expression was considered to be a good strategy for inhibiting the pigmentation of the skin.

Also, TRP-1 is structurally associated with tyrosinase, has an amino acid homology of approximately 40%, and is present in the melanosomes such as tyrosinase. The previous studies reported that the mutations of TRP-1 resulted in pale skin or hair color, which was observed in a certain type of oculocutaneous albinism. Also, it was proposed that TRP-1 played a role in melanin biosynthesis since the inhibition of TRP-1 expression was associated with the hypopigmentation.

Therefore, effects of L-AHG on tyrosinase and TRP-1 expression induced by α-MSH were examined in order to evaluate whether L-AHG had the probability of showing the whitening activities on the skin. Properly pigmented human epidermal melanocytes (HEMs, Cascade Biologics (Oregon, USA)) derived from the neonatal epidermis were used as the cells for this purpose. The cells were cultured at a temperature of 37° C. under a moisturizing environment containing 5% CO₂. The culture medium for HEMs was Medium 254 (Cascade Biologics (Oregon, USA)) supplemented with HMGS.

HEMs (1×10⁵) were cultured in a 6-cm dish for 24 hours. Before the HEMs were exposed to 100 nM α-MSH (Sigma-Aldrich, St. Louis, Mo., USA) for several days, the cells were treated with 3,6-anhydro-L-galactose (L-AHG) at concentrations of 0, 25, 50, and 100 μg/mL for an hour. Then, the cells were lysed in a lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 g/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail tablet]. The concentration of the proteins was measured according to the manufacturer's manual using a dye-conjugated protein assay kit (Bio-Rad Laboratories Inc. Hercules, Calif., USA). 10% SDS-PAGE was performed on the lysed proteins (20 to 40 μg), and the lysed proteins were transferred to a polyvinylidene fluoride (PVDF) membrane through electrophoresis (Millipore Corp., Bedford, Mass., USA). After blotting, the membrane was blocked with 5% skim milk (MB cell, Los Angeles, Calif., USA) for 2 hours, and incubated with a primary antibody (goat anti-mouse IgG-HRP) overnight at 4° C. Thereafter, the membrane was incubated with a secondary antibody (a goat anti-rabbit IgG HRP-conjugated secondary antibody), and the antibody-bound proteins were detected using a chemifluorescence detection kit (Amersham Pharmacia Biotech, Piscataway, N.J.). As the control, an expression level of β-actin was measured. The data represented the values obtained from the independent experiments performed in duplicate.

The in vitro tyrosinase analyses were performed using a slight modification of the reported method (Ishihara Y et al., The journal of antibiotics (Tokyo) 1991; 44:25-32; An SM, Koh J S, Boo Y C, Phytotherapy Research 2010; 24:1175-1180). In brief, 250 μl of a 0.1M potassium phosphate buffer (pH 6.8) was put into a 24-well plate, and 25 μl of a mushroom-derived tyrosinase (2,000 units/mL), 25 μl of a sample, and 225 μl of distilled water were put into each well. The plate was incubated at 25° C. for 10 minutes, and 25 μl of 1 mM L-tyrosine used as a substrate was added to the wells. Then, the plate was incubated at 25° C. for 10 minutes, and the optical density of each well measured at 475 nm using a microplate reader (Sunrise-Basic Tecan, Austria). Each of the measured values was represented by a variation (%) with respect to the control. L-tyrosine was used as the substrate, and arbutin was used as the positive control. The results were represented by the relative tyrosinase activities to the non-treated control.

As shown in FIGS. 9A and 9B, it was revealed that the tyrosinase expression induced by α-MSH significantly decreased in the HEMs when the HEMs were treated with 100 μg/mL of L-AHG, compared to the tyrosinase expression induced by α-MSH.

Also, it was confirmed that, similar to the decrease in the tyrosinase expression by L-AHG treatment (FIG. 9A), the TRP-1 expression in the HEMs decreased in an L-AHG concentration-dependent manner (FIGS. 10A and 10B).

Next, effects of L-AHG on the tyrosinase activities were examined using a cell-free assay system. Many reports disclosed that the mushroom-derived tyrosinase was primarily used as a model since it was able to be used in the form of a tablet. Arbutin that was a competitive inhibitor of L-tyrosine was used as the positive control, and inhibited the tyrosinase activities in the mushroom (FIG. 11). The results were represented by the relative tyrosinase activities to the non-treated control. The data represented the average values±S.D. obtained from the independent experiments performed in triplicate, and the student's t-test was used for single statistical comparison. A significance value of p<0.05 was used as the standard to the statistical significance. An asterisk represents a significant difference (p<0.05) compared to the non-treated control.

As shown in FIG. 11, it was revealed that L-AHG did induce no significant decrease in the tyrosinase activities in the mushroom, and that arbutin induced a decrease in the tyrosinase activities to 40% at a concentration of 200 μM.

In sum, the results addressed that L-AHG was a novel hypopigmenting agent inhibiting the expression of tyrosinase and TRP-1.

<Example 9> Experiment of Moisturizing Effect of 3,6-Anhydro-L-Galactose in Human Corneous Cells

Hyaluronan (HA) is a glycosaminoglycan composed of D-glucuronic acid and N-acetyl-D-glucosamine. Due to its capability to store a large amount of water, HA functions to play an important role in regulating the water balance and osmotic pressure. HA is synthesized by HASs 1, 2, and 3 in a cell membrane. In particular, HAS2 is found in normal human tissues. The previous studies showed that the genetic defect of HAS2 induced the fatality in the prenatal period in a mouse model, and the HAS2 gene was expressed in the epidermis and the derma of an adult human skin to a decreased level. Therefore, an increase in the HAS2 expression could be a good strategy for maintaining the homeostasis in the skin. To measure an induction time and concentration of L-AHG for HAS2 expression, a western blot assay was performed. The cells used for this purpose were HaCaT cells, and incubated at 37° C. under a 5% CO₂ atmosphere in a Dulbecco's modified Eagle's medium (DMEM, GIBCO Invitrogen, Auckland, NZ) supplemented with 10% FBS and penicillin/streptomycin.

The cells (1×10⁵) were incubated for 24 hours in a 6-cm dish, and fasted in a serum-free medium for another 24 hours to get rid of an effect of FBS on activation of kinases. Thereafter, the cells were treated with 3,6-anhydro-L-galactose (L-AHG) at a predetermined concentration for a predetermined period of time. Then, the cells were lysed in a lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 g/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail tablet]. The concentration of the proteins was measured according to the manufacturer's manual using a dye-conjugated protein assay kit (Bio-Rad Laboratories Inc). 10% SDS-PAGE was performed on the lysed proteins (20 to 40 μg), and the lysed proteins were transferred to a PVDF membrane through electrophoresis (Millipore Corp., Bedford, Mass., USA). After blotting, the membrane was blocked with 5% skim milk for 2 hours, and incubated with a primary antibody (goat anti-mouse IgG-HRP) overnight at 4° C. Thereafter, the membrane was incubated with a secondary antibody (a goat anti-rabbit IgG HRP-conjugated secondary antibody), and the antibody-bound proteins were detected using a chemifluorescence detection kit (Amersham Pharmacia Biotech, Piscataway, N.J.). The data represented the values obtained from the independent experiments performed in duplicate.

As shown in FIGS. 12A and 12B, it was revealed that L-AHG increased the HAS2 expression at 3 hours after the treatment. Also, it was confirmed that L-AHG increased the HAS2 expression in a concentration-dependent manner.

It was reported that the HAS2 expression was regulated by various inflammatory signal transduction pathways such as MAPKs and AKT. Therefore, the effects of L-AHG in the various inflammatory signal transduction pathways were measured.

As shown in FIG. 13A, it was revealed that 100 μg/mL of L-AHG significantly increased the phosphorylation of ERK 3 hours after the treatment. To measure a concentration-dependent effect of L-AHG on the phosphorylation of ERK, the cells were treated with L-AHG at concentrations of 25, 50, and 100 μg/mL. Similar to the HAS2 expression induced by L-AHG (FIG. 13B), it was revealed that the phosphorylation of ERK increased in an L-AHG concentration-dependent manner.

As shown in FIG. 14A, it was revealed that 100 μg/mL of L-AHG significantly increased the phosphorylation of AKT 3 hours after the treatment. To measure a concentration-dependent effect of L-AHG on the phosphorylation of AKT, the cells were treated with L-AHG at concentrations of 25, 50, and 100 μg/mL. And, it was revealed that L-AHG increased the phosphorylation of AKT in a concentration-dependent manner (FIG. 14B).

In summary, the results addressed that the phosphorylation of ERK and AKT induced by L-AHG contributed to an increase in the HAS2 expression by L-AHG

According to exemplary embodiments of the present invention, a monosaccharide, 3,6-anhydro-L-galactose, can be produced with a high yield by preparing an oligosaccharide under a mild chemical treatment condition in which an over-degradation effect according to chemical treatment is minimized and subjecting the oligosaccharide to enzymatic treatment.

Also, according to the present invention, the physiological activities of 3,6-anhydro-L-galactose, such as whitening, moisturizing, antioxidant, and antiinflammatory activities, are first examined, resulting in use in various fields including foods, cosmetics, medicine, and the like. Therefore, the 3,6-anhydro-L-galactose considered to be a by-product or a fermentation inhibitor upon production of red alga-derived bioenergy can be applied to production of higher value-added materials in the field of bioenergy.

According to exemplary embodiments of the present invention, 3,6-anhydro-L-galactose can be used as a whitening agent, a moisturizing agent, an antioxidant, a hypopigmenting agent, or an antiinflammatory agent.

Claims

1. A method for treating inflammatory diseases, comprising administering an effective amount of 3,6-anhydro-L-galactose to a subject in need thereof.

2. The method of claim 1, wherein the subject is a dog, a cat, a mouse, or a human.