WO2015041498A1 - Immune enhancement or anticancer composition containing neoagarooligosaccharide - Google Patents

Abstract

One embodiment of the present invention provides an immune enhancement or anticancer composition containing a neoagarooligosaccharide or a neoagarooligosaccharide mixture as an active ingredient. A neoagarooligosaccharide, which is an active ingredient of the composition according to the present invention, has very remarkable immune enhancement and anticancer effects. Accordingly, the composition according to the present invention can be used as a drug or a nutraceutical for enhancing immunity or preventing, alleviating or treating cancer. In addition, since the active ingredient of the composition according to the present invention can be obtained through the enzymatic reaction of a substrate such as agar or agarose, which is readily available, and DagA, which is Streptomyces coelicolor-derived β-agarase, the active ingredient can be produced at relatively low cost and can be readily taken by anyone due to the absence of side effects.

Description

An immunostimulating or anticancer composition comprising neoagarooligosaccharide

The present invention relates to a novel use of a neoagarooligosaccharide or neoagarooligosaccharide mixture, and more particularly to a neoagarooligosaccharide or neoagarooligosaccharide mixture for enhancing immune function or anticancer use.

Agar is a representative algae-derived polysaccharide widely used for food additives, medicines, cosmetics, livestock feed and industrial raw materials for a long time. In Korea, agar is a relatively abundant fishery resource with annual production of about 2,000 ~ 5,000 tons Lt; / RTI > However, in actual use, only a part of the total production amount is processed as a simple raw material and used as cheap raw materials, and the rest of the total amount is neglected. Therefore, there is a great demand for research on the development of new applications of agar and the improvement of added value.

Agar is composed mostly of polysaccharides except for small amounts of protein, ash and fat. The polysaccharides constituting the agar include agarose, a neutral polysaccharide, and agaropectin, an acidic polysaccharide. Agarose is an agarobiose in which D-galactose and 3,6-anhydro-L-galactose are combined in a β-1,4 form, , And agarobiose has a straight-chain structure in which α-1,3 bonds are repeatedly connected to each other, and gelation power is strong. On the other hand, agaropectin, like agarose, has agarobiose as a unit, but contains an acidic group such as a sulfate group and the like and has a weak gelling power.

Among them, agarose is degraded into neoagarobiose via neoagarotetraose by β-agarase acting on β-1,4 bond, and then α-1 Galactose and 3,6-anhydro-L-galactose by α-agarase, which acts on the β-3 linkage. In addition, the agarose is degraded to a neoagarobiose by dilute acid or alpha-agarase. In general, neoagarooligosaccharides are obtained by hydrolyzing agar or agarose with beta-agarase, such as neoagarobiose, neoagarotetraose, neoagarohexaose ( neoagarohexaose, neoagarooctaose, and the like. In addition, agarooligosaccharides can be obtained by hydrolyzing agar or agarose with a dilute acid or alpha-agarase, such as Aeoagarobiose, Agarotetraose, Agarohexaose, , Agarooctaose, and the like. The neoagarooligosaccharide has 3,6-anhydro-L-galactose as a non-reducing end, while the agarooligosaccharide has 3-anhydro-L-galactose as a non- , And these structural differences cause different physiological activities.

Streptomyces coelicolor A3 (2), which is actinomycetes, is known to produce an agarase that degrades agar in the form of extracellular (secreted out of cells) (Stanier et al., 1942, J. Mol. Bacteriol .; Hodgson and Chater, 1981, J. Gen. Microbiol.), The agarase is encoded by the DagA gene. The DagA gene is a beta-agarase gene whose function is the only known in actinomycetes, and thus plays an important role in the study of agarase production through actinomycetes. In particular, Streptomyces sericola is the most widely used strain in the molecular biology of actinomycetes. In 2002, the sequence of chromosomal DNA was analyzed by the Sanger center in England (Bantley et al., 2002, Nature).

Regarding the preparation or use of neo-agarooligosaccharides, Korea Patent No. 10-0794593 discloses a method of producing Thalassomonas sp. SL-5 (Thalassomonas sp. SL-5) having agar resolving ability, KCCM 10790P, Discloses a method for producing at least one neoagarooligosaccharide selected from the group consisting of neoagarobiose, neoagarotetraose and neoagarohexaose using agarase. Korean Patent Publication No. 10-1072503 also discloses that a strain of the genus Escherichia coli SL-12 KCCM 10945P (Glaciecola sp. SL-12 KCCM 10945P) having an agar-resolving ability and a β-agarase (β- agarase is used to produce at least one neoagarooligosaccharide selected from the group consisting of neoagarobiose, neoagarotetraose and neoagarohexaose. Korean Patent No. 10-1303839 discloses beta-agarase isolated from a strain of Pseudoalteromonas sp., And a method of using neoagarotetraose and neoagarohexaose A method of producing at least one neoagarooligosaccharide selected from the group consisting of Korean Patent Registration No. 10-1295659 discloses beta-agarase isolated from Saccharophagus sp. Strain and neoagarotetraose and neoagarohexaose using the same. ) In the presence of a reducing agent. Korean Patent Registration No. 10-1212106 discloses that beta-agarase isolated from a Saccharophagus sp. Strain is selected from the group consisting of agar, neoagarotetraose, and neoagarohexose. A method of producing neoagarobiose by reacting with a substrate or more is disclosed. Korean Patent Registration No. 10-1206006 also discloses that the flameoovirga sp. Mbrc-1 KCCM 11151P strain having the agarase-degrading activity, mbrc-1 strain mbrc-1, and the beta-agarase (β -agarase is reacted with agar to produce at least one neoagarooligosaccharide selected from the group consisting of neoagarobiose, neoagarotetraose and neoagarohexaose. Korean Patent Registration No. 10-1302655 discloses a neoagarotetraose which is characterized by reacting agarase derived from Streptomyces coelicolor with agarose or agar. (neoagarotetraose) and neoagarohexaose are disclosed. Korean Patent Registration No. 10-1190078 discloses a nucleotide sequence of SEQ ID NO: 7 comprising a promoter of a trypsin gene (sprT) derived from Streptomyces griseus and a coding region of a signal peptide. A DNA fragment displayed; And a DNA fragment represented by the nucleotide sequence of SEQ ID NO: 2 from which a signal peptide code has been removed from a Streptomyces coelicolor-derived beta-agarase gene (dagA), wherein the DNA fragment is capable of transforming a prokaryote A beta-agarase recombinant expression vector and a method for producing beta-agarase using the recombinant expression vector. Korean Patent Laid-Open Publication No. 10-2014-0060045 also discloses an enzymatic production method of neoagarobiose or neoagarotetraose using a novel beta-agarase-producing gene. Korean Patent Laid-Open Publication No. 10-2009-0044987 discloses a skin whitening composition comprising neoagarotetraose as an active ingredient. Korean Patent Laid-Open Publication No. 10-2013-0085017 discloses a pharmaceutical composition for preventing or treating skin pigmentation diseases comprising 3,6-anhydro-L-galactose, A cosmetic composition for skin whitening or moisturizing comprising 3,6-anhydro-L-galactose, 3,6-anhydro-L-galactose, -galactose), and the like are disclosed. As described above, the prior art is mainly concerned with a novel strain comprising a beta-agarase as a gene, a method of producing a beta-agarase through a novel strain or a recombinant strain, or a method of producing beta-agarase from agar, And a variety of physiological functions of neoagarooligosaccharides prepared by using neoagarooligosaccharides, in particular beta-agarase, have hardly been studied.

SUMMARY OF THE INVENTION The present invention has been made under the conventional technical background, and it is an object of the present invention to provide various physiological uses of neoagarooligosaccharide or neoagarooligosaccharide mixture.

The present inventor intends to develop a method for effectively utilizing agar, which is an abundant fishery resource. Since agar is a polysaccharide, various saccharide-derived compounds that exhibit physiological activity during the degradation process of agar can be produced by appropriately utilizing a glycosidase Attention was paid to possibility. Actinomycetes are microorganisms which produce useful physiologically active substances such as antibiotics. Since these physiologically active substances contain various sugar-related compounds, actinomycetes include strains having an enzyme capable of decomposing or transforming agar to convert them into useful sugar compounds I thought there would be. Accordingly, the present inventors have searched for useful enzymes from actinomycetes and applied them to the enzymatic reaction of agar to produce useful physiologically active substances.

Recently, physiological activity studies using medicinal plants or edible plants have been actively conducted. Especially, polysaccharides derived from natural materials have shown a great variety of physiological activities, and have been attracting much attention in the field of functional foods and alternative medicine materials. For example, it has been known that mushroom-derived polysaccharides have antitumor activity by resuscitation or enhancement of immune function, and related research is also proceeding variously. The antitumor activity by the enhancement of the immune function is greatly attracted to unlike the anticancer agent which shows the conventional cytotoxicity because it restores or enhances the decreased immune function of the host to a considerable level and exerts the anticancer effect.

The present inventors determined that the sugar compound produced through the enzymatic reaction of Actinomycetes, especially Streptomyces sericola-derived glucosease, exhibits such immune function enhancement and antitumor activity. In order to examine its efficacy, And in vivo. As a result, it was confirmed that the enzyme reaction product obtained through the reaction of DagA enzyme of actinomycetes Streptomyces sericola with agar or agarose has immune function enhancement and anticancer effect.

In order to solve the object of the present invention, an example of the present invention is a composition comprising a neoagarooligosaccharide or a neoagarooligosaccharide mixture as an active ingredient, wherein the neoagarooligosaccharide is neoagarobiose, neoagarobiose, Wherein the neoagarooligosaccharide is any one selected from the group consisting of neoagarotaxin, neoagarotaxin, neoagarotetraose, neoagarohexaose and neoagarooctaose, wherein the neoagarooligosaccharide mixture is selected from the group consisting of neoagarobiose, The composition for immunomodulating or anticancer cancer according to claim 1, wherein the composition comprises at least two selected from the group consisting of neoagarotetraose, neoagarohexaose, and neoagarooctaose. to provide. The neoagaroligosaccharide mixture preferably contains neoagarobiose, neoagarotetraose, and neoagarohexaose, and the neoagarooligosaccharide mixture preferably contains neoagarooligosaccharide mixture based on the total weight of the neoagarooligosaccharide mixture , It is more preferable that it contains 10% by weight or less of neoagarobiose, 50 to 70% by weight of neoagarotetraose and 20 to 50% by weight of neoagarohexaose . The neoagarooligosaccharide mixture may also be an enzyme reaction product of a substrate selected from agar or agarose with DagA, a Streptomyces coelicolor- derived beta-agarase, or a purified product thereof .

In order to solve the object of the present invention, another example of the present invention is an enzyme reaction product of a substrate selected from agar or agarose with DagA, a beta-agarase derived from Streptomyces coelicolor Or a purified product thereof as an active ingredient, wherein the enzyme reaction product or the purified product is selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose and neoagarose A neoagarooligosaccharide, and a neoagarooctaose. The present invention also provides a composition for enhancing immunity or an anti-cancer composition, which comprises the neoagarooligosaccharide. At this time, the content of neoagarooligosaccharide in the enzyme reaction product or the purified product is preferably 45 to 85% by weight based on the total weight of the enzyme reaction product or purified product. The enzymatic reaction product or the purified product may also contain up to 10% neoagarobiose, 50 to 70% neoagarotetraose and 20 to 50% by weight, based on the total weight of the neoagarooligosaccharide, It is preferred that it contains neoagarohexaose in weight percent. In addition, the enzyme reaction is preferably carried out at a temperature of 35 to 45 ° C and a pH of 6 to 8. In addition, the enzyme reaction is preferably performed by adding DagA at a concentration of 2 to 250 unit / ml to an agar or agarose solution of 0.5 to 5% (w / v). In addition, the DagA preferably comprises the 31st to 309th amino acids of SEQ ID NO: 2.

The neoagarooligosaccharide, which is an active ingredient of the composition according to the present invention, has excellent immunosuppressive and anticancer effects. Therefore, the composition according to the present invention can be used as a medicine or a functional food for enhancing immunity or for preventing, improving or treating cancer. Further, the active ingredient of the composition according to the present invention can be obtained by an enzymatic reaction between a readily available substrate such as Agar or Agarose and DagA, a beta-agarase derived from Streptomyces coelicolor It can be produced at a relatively low cost and has no side effects, so that it can be easily ingested by anyone.

Figure 1 is a cleavage map of the pUWL201pw vector used for cloning the DagA gene in the present invention.

Fig. 2 is a cleavage map of a recombinant vector into which the DagA gene is introduced into the pUWL201pw vector.

3 is a graph showing the results of HPLC-ELSD analysis of the partially purified DagA enzyme reaction product obtained in Example 3 of the present invention.

FIG. 4 shows the expression levels of cytokines (TNF-α, IL-1β, IL-6, and IL-12p70) in the mouse macrophage cell line Raw264.7 cells treated with the partially purified DagA enzyme reaction product obtained in Example 3 To the control (LPS treatment).

FIG. 5 shows the expression levels of TNF-.alpha. In the mouse macrophage cell line Raw264.7 cells treated with the partially purified DagA enzyme reaction product obtained in Example 3 with the DagA enzymatic reaction product components (DP2, DP4, DP6 (LPS, AO treatment), and the standard sample treatment group (? -Glucan treatment).

6 is a graph comparing the amount of cytokine (TNF-α, IL-6) expressed in THP-1 cells treated with the partially purified DagA enzyme product obtained in Example 3 with the control (LPS treatment) It is a graph.

FIG. 7 shows the expression levels of cytokines (TNF-α, IL-1β, IL-6, IL-10 and IL-12p70) upon treatment of the partially purified DagA enzymatic reaction products obtained in Example 3 on dendritic cells derived from mice To the control (LPS treatment).

FIG. 8 shows the expression levels of cytokines (TNF-α, IL-1β, IL-6, IL-10 and IL-12p70) upon treatment of the partially purified DagA enzymatic reaction products obtained in Example 3 on dendritic cells derived from mice (DP2, DP4, and DP6 treated) of the DagA enzyme reaction products.

9 shows the expression levels of surface protein factors (CD80, CD86, MHC class I, and MHC class II), which are dendritic cell activation indicators, in the mouse Dendritic cells treated with the partially purified DagA enzyme reaction product obtained in Example 3, (LPS treatment).

FIG. 10 shows the results of analysis of the cell death using Annexin V-FITC and propidium iodide (PI) when the partially purified DagA enzyme reaction product obtained in Example 3 was treated with mouse derived dendritic cells, .

Fig. 11 is a graph showing the effect of the partially purified DagA enzyme reaction product obtained in Example 3 on the dendritic cells derived from WT mouse, TLR2 - / - mouse, TLR4 - / - mouse and TLR9 - IL-12p70, IL-1 ?, IL-10, and IL-12p70) compared to the control (LPS treatment).

Fig. 12 shows the results of the treatment of the partially purified DagA enzyme reaction product obtained in Example 3 with dendritic cells derived from WT mouse, TLR2 - / - mouse, TLR4 - / - mouse and TLR9 - / - (CD80, CD86) expressing cells were compared with the control group (LPS treatment).

FIG. 13 shows the result of confirming the signal transmission mechanism by western blotting after treating the partially purified DagA enzyme reaction product obtained in Example 3 to dendritic cells derived from WT mouse and TLR4 - / - mice for a predetermined time to be.

14 shows the effect of inhibiting the growth of cancer cells when administered with the partially purified DagA enzyme reaction product obtained in Example 3 after xenotransplantation of 4T-1 breast cancer cells into Balb / c mice, compared with the control group (administered with physiological saline) It is a graph.

15 shows the anticancer effect of the partially purified DagA enzyme reaction product obtained in Example 3 on the skin cancer cell animal model (B16F1 melanoma tumor model) with the control (PBS treatment) or the standard test group (? -Glucan treatment) FIG.

16 is a graph showing the survival rate of Salmonella injected into Balb / c mouse and injected with the partially purified DagA enzyme reaction product obtained in Example 3 in the control group (PBS treatment, AO treatment) and the standard test group (beta-glucan treatment) .

FIG. 17 is a graph comparing the expression level of activated NK cells in peritoneal cells when injected with DP6 purified in Example 5 in a peritoneal cavity of C57bl / 6 mice, compared with a control (PBS solution injection).

18 is a graph showing the amount of activated cells when the partially purified DagA enzyme reaction product obtained in Example 3 was treated with NK cells isolated from spleen of C57bl / 6 mouse and immune cells other than NK cells.

FIG. 19 shows the results of treatment of the partially purified DagA enzyme reaction product obtained in Example 3 on dendritic cells and the resultant supernatant was treated with immune cells other than NK cells and NK cells isolated from the spleen of C57bl / 6 mice, Lt; / RTI >

4 to 19, "NAO" represents the partially purified DagA enzyme reaction product obtained in Example 3 of the present invention, "LPS" represents lipopolysaccharide, "CON" represents no treatment, and "AO" represents ≪ / RTI >

Hereinafter, terms used in the present invention will be described.

As used herein, the terms " pharmaceutically acceptable " and " pharmaceutically acceptable " are intended to mean not significantly irritating the organism and not interfering with the biological activity and properties of the administered active substance.

As used herein, the term " prophylactic " means any act that inhibits the symptoms of a particular disease or delays the progress of the disease upon administration of the composition of the present invention.

The term " improvement " as used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.

The term " treatment " as used herein refers to any action that improves or alleviates the symptoms of a particular disease upon administration of the composition of the present invention.

The term " administering " as used herein is meant to provide any desired composition of the invention to an individual by any suitable method. The term " individual " means any animal such as a human, a monkey, a dog, a goat, a pig, or a mouse having a disease in which symptoms of a specific disease can be improved by administering the composition of the present invention.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The composition for immunomodulating or anticancer cancer according to an embodiment of the present invention includes neoagarooligosaccharide or neoagarooligosaccharide mixture as an active ingredient. The neoagaroligosaccharide may be any one selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose, and neoagarooctaose. The neoagarooligosaccharide may be any one selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose and neoagarooctaose. , And it is more preferable that it is selected from neoagarotetraose or neoagarohexaose, but it is not limited thereto. The neoagarooligosaccharide mixture may also be selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose, and neoagarooctaose. The present invention is not particularly limited as long as it includes species or more. In view of the synergistic effect of the constituents, neoagarobio, neoagarotetraose, neoagarohexaose and neoagarooctaose neoagarooctaose, and more preferably at least three selected from the group consisting of neoagarobiose, neoagarotetraose, and neoagarohexaose. The neoagarooligosaccharide mixture may also contain up to 10% neoagarobiose, 50 to 70% neoagarotetraose, and 20 to 50% neoagarotrophin, based on the total weight of the neoagarooligosaccharide mixture. It is preferred to include neoagarohexaose by weight and neoagaroboose by weight of 1 to 5% neoagarobiose, 50 to 70% neoagarotetraose and 25 to 45% It is more preferred to include neoagarohexaose in weight percent. The neoagarooligosaccharide mixture may also be an enzyme reaction product of a substrate selected from agar or agarose with DagA, a Streptomyces coelicolor- derived beta-agarase, or a purified product thereof . A method of obtaining a neoagarooligosaccharide mixture through an enzyme reaction between a substrate and DagA is described in detail later.

Another embodiment of the present invention provides an immune enhancing composition or anticancer composition comprising an enzyme reaction between a substrate selected from agar or agarose and DagA, a beta-agarase derived from Streptomyces coelicolor Product or purified product thereof as an active ingredient. Wherein the enzyme reaction product or the purified product is selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose, and neoagarooctaose And is not particularly limited as long as it contains one or more neoagarooligosaccharides. In view of the synergistic effect of the components, neoagarobiose, neoagarotetraose, neoagarohexaose, And neoagarooctaose, and more preferably at least three kinds of neoagarooligosaccharides. The term " neoagarooctaose " The content of neoagarooligosaccharide in the enzyme reaction product or the purified product is preferably 45 to 85% by weight, more preferably 50 to 80% by weight, based on the total weight of the enzyme reaction product or purified product, But is not necessarily limited to. The enzymatic reaction product or the purified product may also contain up to 10% neoagarobiose, 50 to 70% neoagarotetraose and 20 to 50% by weight, based on the total weight of the neoagarooligosaccharide, It is preferred to include neoagarohexaose by weight and neoagaroboose by weight of 1 to 5% neoagarobiose, 50 to 70% neoagarotetraose and 25 to 45% It is more preferred to include neoagarohexaose in weight percent. In addition, the enzyme reaction is preferably carried out at a temperature of 35 to 45 ° C and a pH of 6 to 8. In addition, the enzyme reaction may be carried out by adding DagA to a 0.5 to 5% (w / v) agar or agarose solution at a concentration of 2 to 250 unit / ml, preferably 10 to 150 unit / ml, more preferably 10 To 100 units / ml. At this time, 3.9 ml of a 50 mM PBS solution (pH 7) in which agarose was dissolved at a concentration of 0.2% (w / v) was reacted at 40 ° C for 5 minutes (reaction solution: 4 ml) (Prepared by dissolving 6.5 g of dinitrosalicylic acid, 325 ml of 2M NaOH and 45 ml of glycerol in 1 liter of distilled water), boiled for 10 minutes, cooled and measured for absorbance at 540 nm. Absorbance at 540 nm 0.001. ≪ / RTI >

Hereinafter, a method for obtaining an active ingredient of the composition according to the present invention through an enzyme reaction between a substrate and DagA will be described. In the present invention, DagA derived from Streptomyces coelicolor means a protein having an amino acid sequence of between 31 and 309 of SEQ ID NO: 2, and is a protein having an amino acid sequence selected from the group consisting of Streptomyces coelicolor , Such as by incorporating a labeling amino acid or by altering the amino acid sequence for heterologous expression in order to make the purification more favorable, within the scope of not altering to completely different or losing agarase activity, Proteins. When DagA is originally translated from the beta-agarase gene of Streptomyces sericola, it has 309 amino acids of SEQ ID NO: 2 and is produced with a molecular weight of about 35 kDa, and the N-terminal 30 amino acid signal peptide is cleaved Lt; / RTI > (approximately 32 kDa). DagA of Streptomyces sericola The gene may be represented by the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 1 is the nucleotide sequence of the gene present in the genome of Streptomyces sericola A3 (2) and is named "SCO3471" on NCBI (US National Bioinformation Center) database. As confirmed by in vitro experiments, the transcription of the DagA gene is regulated by four or five other promoters that are recognized by at least three other holoenzymes of the RNA polymerase. DagA Transcriptional phase analysis of the gene revealed that the transcription of DagA was initiated at the 32nd, 77th, 125th and 220th bases of the coding sequence.

Although DagA used in the present invention can be produced through cultivation of Streptomyces lividans, which is a production strain of DagA, it is preferable to use an expression system of Streptomyces lividans, a heterologous strain, in order to increase production efficiency Do. DagA can be produced by inserting the DagA gene into a stactobacterium vector to prepare a recombinant vector, transforming the streptomyces ribidus with a recombinant vector, and culturing the transformant. In this case, the recombinant vector is preferably constructed such that the transcription of the DagA gene can be regulated by the actinomycete-derived promoter. Since there are various promoters such as sgtR promoter (sgtRp), ermE promoter (ermEp), and tipA promoter (tipAp), actinomycetes -derived promoters can be selected and used when preparing recombinant vectors. Several kinds of vectors constructed so that transcription can be regulated by these promoters have been developed. When SCO3471 is cloned into this vector, a recombinant vector having a structure in which transcription is regulated by actinomycetes-derived promoter can be produced. The transformant can be prepared by transforming the host strain with a recombinant vector in which the DagA gene is cloned. Since a variety of transformation methods are available depending on the host strain, an appropriate method can be selected and used. For example, when a Streptomyces ribidus is used as a host strain, a transformation method based on PEG (polyethylene glycol) can be used. A DagA producing strain such as a transformant can be produced in a liquid medium to produce DagA, and a culture solution can be obtained, and a high purity DagA can be produced using a conventional protein purification method such as an ultrafiltration method. At this time, if agar or agarose is included in the liquid medium, DagA can be produced more efficiently.

When the DagA thus produced is added to a substrate solution such as agar or agarose and reacted at a specific temperature and pH, an enzyme reaction product used as an active ingredient in the composition according to the present invention can be obtained. Further, the enzyme reaction product can be partially purified by ultrafiltration or passed through gel filtration chromatography to obtain a purified product of the enzyme reaction product.

The composition for enhancing immunity according to the present invention can be used to increase the immunity of a person having a weakened immunity or to improve the immunity balance of a person having a weakened immune balance. In addition, the anticancer composition according to the present invention can be used to prevent, ameliorate, or treat cancer of an individual. In addition, the composition according to the present invention may be formulated into pharmaceutical compositions, food additives, food compositions (especially functional foods) or feed additives depending on the intended use or aspects, and the content of the active ingredients in the composition may also be a specific form , And can be adjusted in various ranges depending on the intended use or aspect.

When the immunoconjugate composition or anti-cancer composition according to the present invention is formulated into a pharmaceutical composition, the content of the active ingredient in the pharmaceutical composition is not limited to a great extent, and may be, for example, 0.1 to 99% by weight, May be 0.5 to 50 wt%, more preferably 1 to 30 wt%. In addition, the pharmaceutical composition of the present invention may further comprise, in addition to the active ingredient, an additive such as a pharmaceutically acceptable carrier, excipient or diluent. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention may further contain one or more known active ingredients having immunosuppressive or anticancer effects in addition to neoagarooligosaccharide. The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration by a conventional method, and can be formulated into a pharmaceutical composition such as a filler, an extender, a binder, a wetting agent, a disintegrant, Diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, Sucrose, Lactose, Gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention may be administered orally or parenterally to a mammal including a human according to a desired method. Examples of the parenteral administration method include external dermal application, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, Intravenous injection or intra-thoracic injection. The dosage of the pharmaceutical composition of the present invention is not limited as long as it is a pharmacologically effective amount and is not limited as long as it depends on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, Varies. The typical daily dose of the pharmaceutical composition of the present invention is not particularly limited, and is preferably 0.1 to 1000 mg / kg, more preferably 1 to 500 mg / kg, based on, for example, And may be administered once or several times a day.

When the immunoconjugate composition or anticancer composition according to the present invention is formulated into a food composition, the content of the active ingredient in the food composition is not limited to a great extent and is preferably 0.01 to 50% by weight, 0.1 to 25% by weight, more preferably 0.5 to 10% by weight. The food composition of the present invention may be in the form of a pill, a powder, a granule, an infusion, a tablet, a capsule, or a liquid preparation. Examples of the food include meat, sausage, bread, chocolate, candy, snack, Other noodles, gums, dairy products including ice cream, various soups, drinks, tea, functional water, drinks, alcoholic beverages and vitamin complexes. The food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient in addition to the active ingredient. In addition, the food composition of the present invention can be used as a food composition containing various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Natural flavors such as tau Martin and stevia extract, and synthetic flavors such as saccharin and aspartame may be used as the flavor.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

Example 1: Production of DagA enzyme

Polymerase chain reaction (PCR) was performed using the chromosomal DNA of Streptomyces sericola A3 (2) as a template and using the following primers and Ex-Taq (TAKARA) polymerase. As a result, amplified DagA A fragment of the gene fragment (a signal peptide of DagA and a fragment of 947 bp in length encoded with the completed peptide, corresponding to a part of the sequence of the primer connected to both ends of the nucleotide sequence of SEQ ID NO: 1) was obtained.

Primers used for amplification of the DagA gene fragment

Asm-F (Forward Primer): 5'-GACATATGGTGGTCAACCGACGTGATC-3 '(NdeI) (SEQ ID NO: 3)

Asm-R (Reverse Primer): 5'-GGTGGATCCCTACACGGCCTGATACG-3 '(BamHI) (SEQ ID NO: 4)

Then, the DagA gene was cloned into the pUWL201pw vector of FIG. 1 using the restriction enzyme site (NdeI / BamHI) of the amplified DagA gene fragment to prepare the recombinant vector of FIG. In the recombinant vector of Fig. 2, transcription of the DagA gene is regulated by the ermE promoter. Thereafter, a recombinant strain of Streptomyces lividans TK24 was transformed with the recombinant vector of FIG. 2 to prepare a recombinant strain, and the recombinant strain was used for the production of DagA.

Specifically, the recombinant strain was inoculated into an R2YE liquid medium containing agar of 0.3% (w / v) and pre-cultured for about 60 hours at 28 ° C and 120 rpm. After the pre-culture, the cultivation was carried out for about 60 hours. The culture solution obtained through the present culture was centrifuged to remove cells, and the supernatant was separated and purified by filtration through an ultrafiltration membrane (5 kDa cut-off membrane). The enzyme concentrate which did not pass through the filtration membrane was freeze-dried to solidify it, and the solidified DagA enzyme was stored and used in the subsequent experiments.

Example 2: Measurement of agarase activity of DagA enzyme

The agarase activity of the DagA enzyme obtained in Example 1 was measured by the method of reducing equivalence (DNS method). 100 μl of the DagA enzyme solution prepared by dissolving the DagA enzyme obtained in Example 1 in PBS (phosphate buffered saline) at a concentration of 10 mg / ml and 50 ml of 50 mM PBS solution in which the agarose was dissolved at a concentration of 0.2% (w / v) (manufactured by dissolving 6.5 g of dinitrosalicylic acid, 325 ml of 2M NaOH and 45 ml of glycerol in 1 l of distilled water) was added to the reaction mixture, followed by addition of 10 ml of 10 The mixture was boiled for minutes, then allowed to cool, and the absorbance was measured at 540 nm. 1 U (Unit) of the enzyme was defined as an activity having an absorbance at 540 nm of 0.001.

Example 3: Preparation of DagA Enzyme Reaction Products

1 L of a 20 mM Tris-HCl solution in which agar was dissolved at a concentration of 1.5% (w / v) was prepared, heated at 100 캜 for about 10 minutes, and then cooled to 40 캜. To this was added a 125 U / Was treated to 20,000 U based on the amount of the whole sample and reacted for about 24 hours. Then, the enzyme reaction product was centrifuged to remove the undegraded agar, the supernatant was recovered, and the enzyme reaction product was confirmed by TLC (thin layer chromatography). The recovered supernatant was partially purified by filtration through an ultrafiltration membrane (5KDa cut-off membrane). The DagA enzyme reaction product passed through the filter membrane was lyophilized and solidified. The solidified DagA enzyme reaction product was stored and used in the subsequent experiments. The above procedure was repeated to obtain partially purified enzyme reaction products corresponding to 4 batches.

Example 4: Determination of neoagarooligosaccharide composition of DagA enzyme reaction product by HPLC-ELSD analysis

The neo-agarooligosaccharide composition of the enzyme reaction product obtained in Example 3 was confirmed by HPLC-ELSD analysis. HPLC-ELSD analysis conditions are as follows.

* Column: NH ₂ P-50 4E multimode column (250 mm x 4.6 mm)

* Mobile phase: a mixed solution of acetonitrile and water (acetonitrile: water in a volume ratio of 65:35

FIG. 3 is a graph showing the results of HPLC-ELSD analysis of the partially purified DagA enzyme reaction product obtained in Example 3. FIG. The four peak graphs shown in Figure 3 correspond to the partially purified enzyme reaction products obtained in each of the 4 batches. In addition, the area (%) in the table in Fig. 3 represents the area of the peak corresponding to each neoagaroligosaccharide as a percentage of the total peak area and can be interpreted in the same sense as% by weight in the art. As shown in FIG. 3, the DagA enzymatic reaction products are mainly composed of neoagarobiose (DP2), neoagarotetraose (DP4), and neoagarohexaose neoagarohexaose, hereinafter referred to as " DP6 "). The content of neoagarooligosaccharide in the DagA enzyme reaction product was found to be about 65 5% by weight based on the total weight of solids (including components not detected in the HPLC-ELSD assay) The above content can be selected in various ranges. For example, the content of neoagarooligosaccharide in the agA enzyme reaction product may be 65 ± 20% by weight based on the total weight of the solids. The DP2, DP4 and DP6 contents in the DagA enzyme reaction products were 2 to 4 wt%, 65 to 70 wt% and 25 to 30 wt%, respectively, based on the total weight of the neoagarooligosaccharide, Depending on the enzyme reaction conditions, the content can be selected from a wide range. For example, the content of DP2, DP4, and DP6 in the DagA enzyme reaction product may be 0 to 10 wt%, 50 to 70 wt%, and 20 to 50 wt%, respectively, based on the total weight of the neoagarooligosaccharide.

Example 5 Purification of DagA Enzyme Reaction Products Using Gel Filtration Chromatography

The partially purified enzyme reaction product obtained in Example 3 was purified by gel permeation chromatography (GPC; BioGel P-2 gel, Biorad, Cat. No. 150-4115) And purified and separated into neoagarohexaose (DP6), neoagarotetraose (DP4) and neoagarobiose (DP2). The purified products were then lyophilized to solidify, and the solidified DagA enzyme reaction purified product was stored and used in subsequent experiments. The purity of the purified products, DP2, DP4 and DP6, was found to be about 85% (w / w) by TLC and HPLC.

Example 6: Test for confirming the immunological activity and anticancer efficacy of DagA enzyme reaction product

6-1. Determination of cytokine expression level in immune cells

Raw264.7 cells and mouse mononuclear cell line, THP-1 cells, were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and used for experiments. The cells were cultured in RPMI 1640 (Hyclone Laboratories Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum and 1% streptomycin / penicillin (Gibco BRL, Grand Island, Cells were cultured in an incubator at 37 ° C and 5% CO ₂ .

Raw 264.7 cells were seeded at about 5 × 10 ⁴ cells / well in a 24-well culture plate and cultured for about 24 hours. Then, the partially purified DagA enzyme reaction product obtained in Example 3 Hereinafter referred to as " NAO ") was added at various concentrations ranging from 0.005 to 12.5 mg / ml, and cultured in an incubator having a temperature condition of 37 ° C and 5% CO ₂ for 24 hours. As a positive control, LPS (lipopolysaccharide) was added at a concentration of 50ng / ml instead of the DagA enzyme reaction product, and the cells were cultured under the same conditions. Negative control cells were cultured in the same conditions without any treatment with macrophages. After incubation, the supernatant was collected and the amount of secreted TNF-α, IL-1β (interleukin-1 beta), IL-6 (interleukin-6) and IL-12p70 (interleukin-12p70) Enzyme-Linked ImmunoSorbent Assay (ELISA). FIG. 4 shows the expression levels of cytokines (TNF-α, IL-1β, IL-6, and IL-12p70) in the mouse macrophage cell line Raw264.7 cells treated with the partially purified DagA enzyme reaction product obtained in Example 3 To the control (LPS treatment). As shown in FIG. 4, the expression levels of TNF-α, IL-1β, IL-6 and IL-12p70 were increased in a dose-dependent manner as the amount of NAO treated increased. These results indicate that NAO exhibits immune function enhancing effects.

In addition, DP2, DP4, DP6 and NAO, which are constituents of the DagA enzyme reaction product, were treated with mouse macrophage cell line Raw264.7 cells at a concentration of 500 mu g / ml, respectively, and then the expression amount of TNF-alpha was measured. As a control, LPS was treated at a concentration of 100 ng / ml or agarooligosaccharide (AO; Takara Bio Inc., JP) was treated at a concentration of 500 μg / ml, and β- Ml and then compared the expression levels of TNF-α. FIG. 5 shows the expression levels of TNF-.alpha. In the mouse macrophage cell line Raw264.7 cells treated with the partially purified DagA enzyme reaction product obtained in Example 3 with the DagA enzymatic reaction product components (DP2, DP4, DP6 (LPS, AO treatment), and the standard sample treatment group (? -Glucan treatment). As shown in FIG. 5, the expression level of TNF-α was the highest in the NAO treatment group. In particular, the NAO-treated group showed a higher expression of TNF-α than the same amount of DP2, DP4, DP6 treated group, and this result is considered to be a synergistic effect by the combination of various neoagarooligosaccharides.

In addition, THP-1 cells, another mononuclear cell line, were divided into 3 × 10 ⁴ cells / well in a 96-well plate, treated with PMA (phorbol myristate acetate), and cultured for 24 hours to induce differentiation into macrophages. The differentiated THP-1 cells were treated with NAO at concentrations of 0.1 mg / ml, 0.5 mg / ml and 1 mg / ml, respectively, and incubated for 24 hours in an incubator at 37 ° C and 5% CO ₂ . As a positive control, LPS (lipopolysaccharide) was added at a concentration of 100 ng / ml or 500 ng / ml instead of the DagA enzyme reaction product and cultured under the same conditions. After the culture was completed, the supernatant was collected and the amount of secreted TNF-α and IL-6 (interleukin-6) was measured by ELISA (enzyme-linked immunosorbent assay). 6 is a graph comparing the amount of cytokine (TNF-α, IL-6) expressed in the human macrophage cell line THP-1 cells treated with the partially purified DagA enzyme reaction product obtained in Example 3 with the control (LPS treatment) It is a graph. As shown in FIG. 6, the expression levels of TNF-α and IL-6 were increased in THP-1 cells as well as in the case of Raw264.7 cells, depending on the concentration of NAO treated. From these results, it can be seen that NAO shows immunity enhancement effect in various animals.

6-2. Identification of immune activity in dendritic cells

6-2-1. Confirming the enhancement of immune function in dendritic cells

The effect of DagA enzyme reaction products on immune function was confirmed by using dendritic cells, which are responsible for the mammalian immune system and capture the antigens and present them on the surface and are known to have the best ability to support the activities of other immune cells.

Dendritic cells from mouse bone marrow mononuclear cells were cultured for about 6 days before use in the experiment. In culture medium was used that the addition of 10% FBS, 1% streptomycin / penicillin and 20ng / ㎖ GM-CSF (granulocyte -macrophage stimulating factor) in RPMI 1640, with the temperature conditions and 5% CO ₂ conditions of 37 ℃ Cells were cultured in an incubator.

Dendritic cells were plated at approximately 2 × 10 ⁵ cells / well in a 12-well culture plate. To each well of the culture plate, NAO was added at a concentration of 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / / Ml and 2 mg / ml, and cultured in an incubator at 37 ° C and 5% CO ₂ concentration for 24 hours. The expression level of surface protein (CD80, CD86, MHC class I, MHC class II) was then confirmed.

In addition, the dendritic cells were treated with NAO at a concentration of 0.025 mg / ml, 0.1 mg / ml, 0.25 mg / ml and 1 and 2.5 mg / ml respectively and then cultured in the presence of TNF-α, IL- IL-10, and IL-12p70.

In addition, the dendritic cells were treated with the NAO and the NAO constituents DP2, DP4, and DP6 at a concentration of 2 mg / ml, respectively, and then cultured with TNF-α, IL-1β, IL-6, IL- Were compared.

For comparison with NAO, LPS (lipopolysaccharide) was added at a concentration of 50 ng / ml instead of NAO as a positive control group, cultured under the same conditions, and negative control cells were cultured under the same conditions without any treatment on dendritic cells.

FIG. 7 shows the expression levels of cytokines (TNF-α, IL-1β, IL-6, IL-10 and IL-12p70) upon treatment of the partially purified DagA enzymatic reaction products obtained in Example 3 on dendritic cells derived from mice To the control (LPS treatment). 8 is a graph showing the effect of the partially purified DagA enzyme reaction product obtained in Example 3 on the dendritic cells derived from mice in the presence of cytokines (TNF- ?, IL-1 ?, IL-6, IL-10 and IL-12p70) (DP2, DP4, and DP6 treated) of the DagA enzyme reaction products. 9 shows the expression levels of surface protein factors (CD80, CD86, MHC class I and MHC class II), which are indicators of dendritic cell activation, upon treatment of the partially purified DagA enzyme reaction products obtained in Example 3 on dendritic cells derived from mice To the control (LPS treatment). 10 shows the results of analysis of the cell death using Annexin V-FITC and propidium iodide (PI) when the partially purified DagA enzyme reaction product obtained in Example 3 was treated with dendritic cells derived from mice, Processing).

As shown in FIG. 7, as the concentration of NAO treated with dendritic cells increased, the expression level of cytokine was increased. As shown in FIG. 8, the dendritic cells were treated with DP2, DP4, DP6 In the case of treatment with the same amount, the amount of cytokine expression was the most increased in the NAO treatment group, and the expression amount of the surface protein factor was also increased depending on the treatment concentration of NAO as shown in FIG. These results indicate that NAO shows immunity - enhancing effect and that the combination of various neo - agarooligosaccharides maximizes the enhancement of immune function. In addition, as shown in Fig. 10, even when the dendritic cells were treated with NAO at a concentration of 2.5 mg / ml, apoptosis did not occur at all (in the graph of Fig. 10, the upper right region corresponds to the apoptotic zone, The greater the distribution of dots in the area, the more likely it is that cell death has occurred.

6-2-2. Comparison of Immune Function Enhancement Effect in TLR2 - / -, TLR4 - / -, TLR9 - / - Mouse Derived Dendritic Cells

NAO was treated at a concentration of 2.5 mg / ml in each of the dendritic cells obtained from WT (Wild Type) mice, TLR2 - / - mice, TLR4 - / - mice and TLR9 - / - mice, ₂ concentration for 24 hours, and the expression level of surface protein (CD80, CD86) was confirmed.

Each of the dendritic cells was treated with NAO at a concentration of 0.5 mg / ml and 2.5 mg / ml, cultured in an incubator at 37 ° C and 5% CO ₂ for 24 hours, -1β, IL-10, and IL-12p70.

For comparison with NAO, LPS (lipopolysaccharide) was added at a concentration of 50 ng / ml instead of NAO as a positive control group, cultured under the same conditions, and negative control cells were cultured under the same conditions without any treatment on dendritic cells.

Fig. 11 is a graph showing the effect of the partially purified DagA enzyme reaction product obtained in Example 3 on the dendritic cells derived from WT mouse, TLR2 - / - mouse, TLR4 - / - mouse and TLR9 - IL-12p70, IL-1 ?, IL-10, and IL-12p70) compared to the control (LPS treatment). 12 shows the results of the partially purified DagA enzyme reaction products obtained in Example 3 on dendritic cells derived from WT mice, TLR2 - / - mice, TLR4 - / - mice and TLR9 - / - mice, Surface protein factor (CD80, CD86) expression level in comparison with the control group (LPS treatment). As shown in FIG. 11, when NAO was treated with dendritic cells derived from WT mice, TLR2 - / - mice and TLR9 - / - mice, the amount of cytokine expression was increased, but the dendritic cells derived from TLR4 - / - mice were treated with NAO , The expression level of cytokines was reduced in a similar manner to that of the control (LPS treatment). In addition, as shown in FIG. 12, when NAO was treated with dendritic cells derived from WT mice, TLR2 - / - mice and TLR9 - / - mice, the amount of surface protein factor was increased, but the dendritic cells derived from TLR4 - / - , The expression level of the surface protein factor was reduced in a manner similar to that of the control (LPS treatment). At this time, LPS is known as a substance via TLR4 (Toll-like receptor-4). For example, binding of LPS to TRL4 promotes intracellular signaling and produces large amounts of cytokines.

6-2-3. Confirmation of immunity enhancement signaling mechanism in dendritic cells derived from TLR4 - / - mice

The dendritic cells obtained from TLR4 - / - mice were treated with NAO at a concentration of 1 mg / ml for 0, 10, 20, 30, and 60 minutes, and western blotting was performed on proteins obtained from each sample The phosphorylation of ERK, JNK, p38, AKT and p65 was analyzed.

FIG. 13 shows the result of confirming the signal transmission mechanism by western blotting after treating the partially purified DagA enzyme reaction product obtained in Example 3 to dendritic cells derived from WT mouse and TLR4 - / - mice for a predetermined time to be. As shown in FIG. 13, treatment of NAO with WT mouse-derived dendritic cells reduced phosphorylation of ERK, p38, JNK and AKT, which are lower signaling mechanisms of TLR4, and decreased amount of p65 in the cell matrix, In the case of dendritic cells, there was no difference in the degree of phosphorylation by NAO treatment.

6-3. Confirmation of Breast Cancer Cell Growth Inhibitory Effect

A 6-week-old Balb / c female mouse fat pad was injected with 2 × 10 ⁵ cells of mouse breast cancer cell line 4T-1. From the 2nd day of tumor formation after the injection of the breast cancer cell line, physiological saline was administered to the control group and NAO was administered to the experimental group daily at a dose of 500 mg / kg or 1000 mg / kg. The size of the tumor was measured daily from 5 days after the injection of the breast cancer cell line. 14 shows the effect of inhibiting the growth of cancer cells when administered with the partially purified DagA enzyme reaction product obtained in Example 3 after xenotransplantation of 4T-1 breast cancer cells into Balb / c mice, compared with the control group (administered with physiological saline) It is a graph. As shown in FIG. 14, NAO inhibited the growth of breast cancer cells in a concentration-dependent manner.

6-4. Confirmation of skin cancer cell growth inhibitory effect

6-week-old C57bl / 6 mice were divided into 10 mice per group, and PBS (phosphate buffered saline) solution was administered to the control group and NAO or β-glucan (quizgenbiotech) The size of the tumor was observed over time. Specifically, mice were injected intraperitoneally with NAO at a dose of 500 mg / kg or β-glucan at a dose of 25 mg / kg every other day for 9 times, and the drug was administered intraperitoneally At the time of injection, B16F1 melanoma tumor cells were subcutaneously transplanted into mice at a dose of 4 × 10 ⁴ cells. Tumor size was measured from 10 days after transplantation of cancer cells. 15 shows the anticancer effect of the partially purified DagA enzyme reaction product obtained in Example 3 on the skin cancer cell animal model (B16F1 melanoma tumor model) with the control (PBS treatment) or the standard test group (? -Glucan treatment) FIG. As shown in Fig. 15, tumor size in the NAO-treated group was greatly reduced compared to the control (PBS treatment) and standard sample treatment group (β-glucan treatment).

6-5. Graft-versus-host mortality assay

Balb / c mice were divided into 5 groups of 8 mice per group, and salmonella injection and drug injection were performed in mice, and survival rates were calculated. Specifically, PBS (phosphate buffered saline) solution was intraperitoneally injected once a day for 3 days, salmonella was injected, and PBS solution was intraperitoneally injected once every morning for 7 days. In group 2, β-glucan (quizgen biotech), a standard sample, was intraperitoneally injected once daily for 3 days at a dose of 50 ㎎ / ㎏, then salmonella was injected and β-glucan was added at a dose of 50 ㎎ / Daily intraperitoneal injection was performed once a day. In group 3, agarooligosaccharide (AO; Takara Bio Inc., JP) was intraperitoneally injected once daily for 3 days at a dose of 50 mg / kg, then salmonella was injected and again agarooligosaccharide was added at 50 mg / Kg daily for 7 days. In group 4, NAO was intraperitoneally injected once daily for 3 days at a dose of 50 ㎎ / ㎏, then salmonella was injected, and NAO was intraperitoneally injected once a day for 7 days at a dose of 50 ㎎ / ㎏. In group 5, NAO was intraperitoneally injected once daily for 5 days at a dose of 50 ㎎ / ㎏, then salmonella was injected, and NAO was intraperitoneally injected once every morning for 7 days at a dose of 50 ㎎ / ㎏. 16 is a graph showing the survival rate of Salmonella injected into Balb / c mouse and injected with the partially purified DagA enzyme reaction product obtained in Example 3 in the control group (PBS treatment, AO treatment) and the standard test group (beta-glucan treatment) . As shown in FIG. 16, the number of salmonella was decreased in the NAO-treated group, especially in the group injected with NAO for 5 days before the salmonella injection. In other words, NAO has the effect of extending the life span of laboratory animals infected with pathogens.

6-6. Identification of NK cell activation ability in peritoneal cells

6-6-1. Identification of NK cell activation ability of DP6 in peritoneal cells

6 to 8-week-old C57bl / 6 mice were divided into 3 groups, and PBS (phosphate buffered saline) solution was injected into mouse abdominal cavity of control group. DP6 obtained by purifying in Example 5 was injected into mouse abdominal cavity of experimental group at 500 mg / kg By volume. Twenty-four hours after the drug injection, 10 ml of PBS (phosphate buffered saline) solution was injected into the mouse abdominal cavity of the control group and the experimental group by the syringe, and the abdominal cells were obtained. Thereafter, 2 × 10 ⁶ cells of the peritoneal cavity were fluorescently stained with NK1.1 and CD69, and the number of NK1.1 + / CD69 + cells was analyzed by flow cytometry.

FIG. 17 is a graph comparing the expression level of activated NK cells in peritoneal cells when injected with DP6 purified in Example 5 in a peritoneal cavity of C57bl / 6 mice, compared with a control (PBS solution injection). As shown in FIG. 17, in the DP6-treated group, the ratio of CD69 + cells, which is an activation index of NK cells in the peritoneal cells, was about 9.5%, which is about four times higher than that of the control group. In other words, when peritoneal injection of DP6 was performed, the number of activated NK cells capable of attacking cancer cells was increased.

6-6-2. Identification of spleen-derived immune cell activation ability of C57bl / 6 mouse (direct immunity enhancement efficacy confirmation)

(Monocyte, macrophage, dendritic cell, helper T cell, and cytotoxic T cell) other than NK cells and NK cells were isolated from the spleen of C57bl / 6 mice using MACS beads. Then, 2 × 10 ⁵ NK cells Each of the non-NK cells was treated with NAO at a concentration of 0.1 mg / ml, 0.5 mg / ml and 2.5 mg / ml, and cultured in an incubator at 37 ° C and 5% CO ₂ for 24 hours. The cultured cells were collected and fluorescently stained with NK1.1 and CD69, and the number of CD69 + cells was analyzed by flow cytometry. 18 is a graph showing the amount of activated cells when the partially purified DagA enzyme reaction product obtained in Example 3 was treated with NK cells isolated from spleen of C57bl / 6 mouse and immune cells other than NK cells. As shown in FIG. 18, the number of activated NK cells and activated non-NK cells increased depending on the concentration of NAO.

6-6-3. Identification of immune cell activation ability of NAO treated with dendritic cells (indirect immunity enhancement)

After the dendritic cells from mouse bone marrow mononuclear cells, 6 days incubation, the NAO here each 0.1 ㎎ / ㎖, 0.5 ㎎ / ㎖, 2.5 ㎎ / ㎖ treatment at a concentration of, and the temperature conditions of 37 ℃ and 5% CO ₂ And cultured for 24 hours in an incubator under the condition of concentration. (Monocyte, macrophage, dendritic cell, helper T cell, and cytotoxic T cell) other than NK cells and NK cells were isolated from the spleen of C57bl / 6 mice using MACS beads. Then, 2 × 10 ⁵ NK cells Each of the non-NK cells was treated with a supernatant obtained from a dendritic cell culture and cultured in an incubator at 37 ° C and 5% CO ₂ concentration for 24 hours. The cultured cells were collected and fluorescently stained with NK1.1 and CD69, and the number of CD69 + cells was analyzed by flow cytometry. FIG. 19 shows the results of treatment of the partially purified DagA enzyme reaction product obtained in Example 3 on dendritic cells and the resultant supernatant was treated with immune cells other than NK cells and NK cells isolated from the spleen of C57bl / 6 mice, Lt; / RTI > As shown in FIG. 19, the number of activated NK cells and the number of activated non-NK cells were increased depending on the concentration of NAO treated with dendritic cells.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the scope of protection of the present invention is not limited to the specific embodiments but should be construed as including all embodiments belonging to the claims attached hereto.

Claims (20)

1. A composition comprising a neoagarooligosaccharide or neoagarooligosaccharide mixture as an active ingredient,

   The neoagarooligosaccharide is any one selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose and neoagarooctaose, and the neoagarooligosaccharide is any one selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose and neoagarooctaose,

   The neoagarooligosaccharide mixture may be at least two or more selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose and neoagarooctaose. Wherein the composition for immunity enhancement comprises the compound of formula (I).
2. The composition for immunomodulation according to claim 1, wherein the neoagarooligosaccharide mixture comprises neoagarobiose, neoagarotetraose and neoagarohexaose.
3. 4. The method of claim 3, wherein the neoagarooligosaccharide mixture comprises less than 10% neoagarobiose, 50-70% neoagarotetraose, and mixtures thereof, based on the total weight of the neoagarooligosaccharide mixture. And 20 to 50% by weight of neoagarohexaose.
4. The method of claim 1, wherein the neoagarooligosaccharide mixture is an enzyme reaction product of a substrate selected from agar or agarose with DagA, a beta-agarase derived from Streptomyces coelicolor , Wherein the composition is a purified product.
5. A composition comprising an enzyme reaction product of a substrate selected from agar or agarose and DagA as a beta-agarase derived from Streptomyces coelicolor or a purified product thereof as an active ingredient,

   The enzyme reaction product or a purified product thereof may be one or more selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose, and neoagarooctaose. Or more neo-agarooligosaccharide.
6. 6. The composition according to claim 5, wherein the content of neoagarooligosaccharide in the enzyme reaction product or the purified product thereof is 45 to 85% by weight based on the total weight of the enzyme reaction product or purified product thereof.
7. 6. The method of claim 5, wherein the enzymatic reaction product or a purified product thereof comprises no more than 10% neoagarobiose, 50-70% neoagarotetraose based on the total weight of neoagarooligosaccharide, ) And 20 to 50% by weight of neoagarohexaose.
8. The immunomodulating composition according to claim 5, wherein the enzyme reaction is carried out at a temperature of 35 to 45 ° C and a pH of 6 to 8.
9. [Claim 6] The composition for immunity enhancement according to claim 5, wherein the enzyme reaction is performed by adding DagA at a concentration of 2 to 250 unit / ml to a 0.5 to 5% (w / v) agar or agarose solution.
10. [Claim 6] The immunomodulating composition according to claim 5, wherein the DagA comprises the 31st to 309th amino acids of SEQ ID NO: 2.
11. A composition comprising a neoagarooligosaccharide or neoagarooligosaccharide mixture as an active ingredient,

    The neoagarooligosaccharide is any one selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose and neoagarooctaose, and the neoagarooligosaccharide is any one selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose and neoagarooctaose,

    The neoagarooligosaccharide mixture may be at least two or more selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose and neoagarooctaose. Or a pharmaceutically acceptable salt thereof.
12. 12. The anticancer composition according to claim 11, wherein the neoagarooligosaccharide mixture comprises neoagarobiose, neoagarotetraose, and neoagarohexaose.
13. 14. The method of claim 13, wherein the neoagarooligosaccharide mixture comprises less than or equal to 10% neoagarobiose, 50-70% neoagarotetraose based on the total weight of the neoagarooligosaccharide mixture, And 20 to 50% by weight of neoagarohexaose.
14. 12. The method of claim 11, wherein the neoagarooligosaccharide mixture is an enzyme reaction product of a substrate selected from agar or agarose with DagA, a beta-agarase derived from Streptomyces coelicolor , Wherein the composition is a tablet.
15. A composition comprising an enzyme reaction product of a substrate selected from agar or agarose and DagA as a beta-agarase derived from Streptomyces coelicolor , or a purified product thereof as an active ingredient,

    The enzyme reaction product or a purified product thereof may be one or more selected from the group consisting of neoagarobiose, neoagarotetraose, neoagarohexaose, and neoagarooctaose. Or more of neo-agarooligosaccharide.
16. 16. The anticancer composition according to claim 15, wherein the content of neoagarooligosaccharide in the enzyme reaction product or purified product thereof is 45 to 85% by weight based on the total weight of the enzyme reaction product or purified product thereof.
17. 16. The method of claim 15, wherein the enzymatic reaction product or a purified product thereof comprises no more than 10% neoagarobiose, no less than 50% neoagarotetraose, based on the total weight of neoagarooligosaccharide, ) And 20 to 50% by weight of neoagarohexaose.
18. 16. The anticancer composition according to claim 15, wherein the enzyme reaction is carried out at a temperature of 35 to 45 DEG C and a pH of 6 to 8.
19. 16. The anticancer composition according to claim 15, wherein the enzyme reaction is performed by adding DagA at a concentration of 2 to 250 unit / ml to an agar or agarose solution of 0.5 to 5% (w / v).
20. 6. The anticancer composition according to claim 5, wherein the DagA comprises the 31st to 309th amino acids of SEQ ID NO: 2.

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