Compound having immunomodulatory activity

Abstract

To provide a novel compound exhibiting superior immunomodulatory activity.SOLUTION: A compound comprising a rhamnogalacturonan (RG-I) part and a xylogalacturonan (XGA) part, the compound having a molecular weight of 10000 or less, the XGA part comprising at least one xylose constituent unit.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel compounds with immunomodulatory properties. [Background technology]

[0002] Immune responses can be broadly divided into innate immunity and adaptive immunity. Innate immunity is the immune system we are born with, and phagocytes such as neutrophils, macrophages, and dendritic cells are representative of the cellular factors responsible for its defense mechanism. These cells recognize, rapidly ingest, and eliminate foreign substances such as microorganisms with the help of humoral factors such as complement and lectins. Furthermore, innate immunity not only plays an important role in early defense against infection, but also in the subsequent early inductive response and differentiation of later adaptive immunity, i.e., it plays an important role in directing the balance between cellular and humoral immunity.

[0003] Cellular immunity, centered on cytotoxic T cells (also known as killer T cells) and macrophages, primarily functions to defend against intracellular infections such as viral infections and tumors caused by them. On the other hand, humoral immunity, by controlling the class switching of IgG1 and IgE antibodies in B cells, primarily functions to defend against extracellularly growing microorganisms such as protozoa, fungi, parasites, mycoplasma, pneumococcus, and Escherichia coli.

[0004] It has recently become clear that cellular immunity and humoral immunity can be likened to a seesaw balance, and that if the balance is disrupted by some cause (endogenous or exogenous) and the body leans toward cellular immunity, it can lead to autoimmune diseases such as insulin-dependent diabetes and rheumatoid arthritis, while if the body leans toward humoral immunity, it can lead to the development of various allergic diseases and cancer. In other words, maintaining a good balance of immune function is essential for maintaining homeostasis in the body.

[0005] Food and beverage ingredients that regulate the immune system to a normal balance come from fungi such as mushrooms, yeast, lactic acid bacteria, seaweed, herbs, etc. These are collectively called biological response modifiers (BRMs) because of their regulatory effect.

[0006] Immunomodulatory substances have also been found in blackcurrants, a fruit. For example, it has been reported that a polysaccharide-based fraction from blackcurrant juice activates macrophages in vitro and exhibits a strong antitumor effect in vivo in mouse antitumor tests. Such blackcurrant polysaccharide fractions are known as cassis polysaccharides (CAPS).

[0007] Furthermore, it has been reported that a polysaccharide-containing composition having an average molecular weight of approximately 10,000 to 40,000 and having excellent physiological activities such as immunomodulatory activity can be obtained by partially digesting a polysaccharide-containing fraction isolated from black currant juice with β-galactosidase (Patent Document 1). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2007 / 125823 Summary of the Invention

[0009] The present inventors have found that novel compounds with excellent immunomodulatory properties can be obtained by subjecting black currant juice to a specific treatment, and the present invention is based on this finding.

[0010] Thus, the present invention provides novel compounds with excellent immunomodulatory properties.

[0011] That is, the present invention provides the following inventions. (1) A compound consisting of a rhamnogalacturonan (RG-I) portion and a xylogalacturonan (XGA) portion, having a molecular weight of 10,000 or less, and the XGA portion containing at least one xylose building block. (2) The compound according to (1) above, wherein the XGA moiety contains 2 to 7 consecutive xylose building blocks. (3) The compound according to (1) above, having a molecular weight of 2,500 to 10,000. (4) The compound according to (1) above, which contains, as structural units, rhamnose structural units, arabinose structural units, galactose structural units, xylose structural units, galacturonic acid structural units, and glucuronic acid structural units. (5) An immunomodulator comprising the compound according to any one of (1) to (4). (6) The immunomodulator according to (5) above, which has immunostimulatory activity. (7) A composition for immunomodulation, comprising the compound according to any one of (1) to (4). (8) The composition described in (7) above, which has immunostimulatory activity. (9) The composition according to (7) above, which is a food or drink. (10) A method for producing a black currant-derived polysaccharide-containing composition, comprising partially digesting black currant juice or a polysaccharide-containing fraction isolated from black currant juice with pectinase until the average molecular weight of the polysaccharides is 10,000 or less. (11) A polysaccharide-containing composition obtained by the method described in (10) above. (12) An immunomodulator comprising the polysaccharide-containing composition described in (11) above. (13) The immunomodulator according to (12) above, which has immunostimulatory activity. (14) A composition for immunomodulation, comprising the polysaccharide-containing composition according to (11) above. (15) The composition according to (14) above, which has immunostimulatory activity. (16) The composition according to (14) above, which is a food or drink.

[0012] According to the present invention, novel compounds having excellent immunomodulatory properties are provided. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 is a graph showing the results of adding black currant-derived polysaccharides to RAW264.7 cells and measuring the levels of IL-6, TNF-α, and iNOS mRNA expression in the cells (NC: untreated cells; flow-through fraction: black currant-derived polysaccharide flow-through fraction from anion chromatography; 0.5 M NaCl fraction: 0.5 M NaCl elution fraction; **p<0.01 vs. NC). [Figure 2] Figure 2 is a graph showing the results of adding black currant polysaccharides to RAW264.7 cells and measuring the concentrations of IL-6 and TNF-α in the culture medium (NC: untreated cells; 0.5 M NaCl fraction: black currant polysaccharide 0.5 M NaCl elution fraction; 0.5 M NaCl fraction enzyme treatment: 0.5 M NaCl elution fraction pectinase enzyme treatment fraction; **p<0.01 vs NC). [Figure 3] Figure 3 is a graph showing the results of adding black currant-derived polysaccharides to RAW264.7 cells and measuring the mRNA expression levels of IL-6, TNF-α, and iNOS in the cells (NC: untreated cells; 0.5 M NaCl fraction: black currant-derived polysaccharide 0.5 M NaCl elution fraction; 0.5 M NaCl fraction enzyme treatment: 0.5 M NaCl elution fraction pectinase enzyme treatment fraction; **p<0.01 vs NC). [Figure 4] Figure 4 is a graph showing the results of adding fraction 1-b, purified from black currant-derived polysaccharides, to RAW264.7 cells at concentrations of 25 to 200 μg / mL and measuring the NO (nitric oxide) concentration in the culture medium (NC: untreated cells; PC: LPS-added cells; *p<0.05 vs. NC, **p<0.01 vs. NC). [Figure 5] Figure 5 is a graph showing the results of adding fraction 1-b, purified from black currant polysaccharides, to RAW264.7 cells at concentrations of 25 to 200 μg / mL and measuring the concentrations of IL-6 and TNF-α in the culture medium (NC: untreated cells; *p<0.05 vs. NC, **p<0.01 vs. NC). [Figure 6]Figure 6 is a graph showing the results of adding fraction 1-b, purified from black currant-derived polysaccharides, to RAW264.7 cells at concentrations of 25 to 200 μg / mL and measuring the mRNA expression levels of IL-6, TNF-α, and iNOS in the cells (NC: untreated cells; *p<0.05 vs. NC, **p<0.01 vs. NC). Specific Description of the Invention

[0014] According to the present invention, novel compounds having excellent immunomodulatory properties are provided. The compounds of the present invention are particularly advantageous in that they have strong immunostimulatory activity.

[0015] The compound according to the first aspect of the present invention is a compound comprising a rhamnogalacturonan (RG-I) moiety and a xylogalacturonan (XGA) moiety. This compound has a molecular weight of 10,000 or less, preferably 2,500 to 10,000, and more preferably 5,000 to 10,000. This compound contains at least one xylose building block in the XGA moiety, preferably two or more, and more preferably two to seven xylose building blocks. Furthermore, when the XGA moiety contains two or more xylose building blocks, these xylose building blocks can be consecutive.

[0016] According to a preferred embodiment of the present invention, the compound according to the first aspect of the present invention comprises, as building blocks, rhamnose building blocks, arabinose building blocks, galactose building blocks, xylose building blocks, galacturonic acid building blocks, and glucuronic acid building blocks. According to another preferred embodiment of the present invention, the compound according to the first aspect of the present invention is derived from black currant (e.g., black currant juice).

[0017] The compound according to the second aspect of the present invention contains rhamnose, arabinose, galactose, xylose, galacturonic acid, and glucuronic acid building blocks. The linkages of these building blocks in this compound are analyzed as follows: T-Ara (4%), 5-Ara (26%), T-Xyl (2%), 4-Xyl (26%), 2,4-Xyl (4%), 2-Rha (3%), T-Gal (2%), and 3-Gal (5%). Here, "T-" refers to the non-reducing terminal sugar residue linked only via C(O)-1.

[0018] According to a preferred embodiment of the present invention, the compound according to the second aspect of the present invention has a molecular weight of 10,000 or less, preferably 2,500 to 10,000, more preferably 5,000 to 10,000.

[0019] According to another preferred embodiment of the present invention, the compound according to the second aspect of the present invention comprises a rhamnogalacturonan (RG-I) portion and a xylogalacturonan (XGA) portion, and more preferably, the XGA portion contains at least one xylose building block, more preferably two or more, and even more preferably 2 to 7 xylose building blocks. Furthermore, when the XGA portion contains two or more xylose building blocks, these xylose building blocks can be consecutive. According to another preferred embodiment of the present invention, the compound according to the first aspect of the present invention is derived from black currant (e.g., black currant juice).

[0020] According to the present invention, there is provided a method for producing a polysaccharide-containing composition derived from black currant, which method comprises partially digesting black currant juice or a polysaccharide-containing fraction isolated from black currant juice with pectinase until the average molecular weight of the polysaccharides is reduced to not more than 10,000. By this method, a polysaccharide-containing composition containing the compound of the present invention can be obtained.

[0021] A polysaccharide-containing fraction isolated from blackcurrant juice can be obtained by subjecting the blackcurrant juice to a process for concentrating polysaccharides, such as dialysis or anion chromatography. Dialysis can be performed using a dialysis membrane with an appropriate molecular weight cutoff (e.g., 14,000), and the supernatant liquid remaining inside the dialysis membrane can be collected as the polysaccharide-containing fraction. Anion chromatography involves fractionating the polysaccharide-containing liquid using an appropriate column, separating it into a passed fraction and a fraction eluted by passing an NaCl solution (e.g., 0.5 M NaCl solution), and collecting the fraction eluted by the NaCl solution as the polysaccharide-containing fraction.

[0022] The amount of pectinase to be added can be determined appropriately taking into consideration the concentration of black currant juice or the polysaccharide-containing fraction isolated from black currant juice in the reaction solution, pH, reaction temperature, reaction time, the organism of origin and degree of purification of the pectinase, etc. If the amount added is too small, the reaction may take too long, and in some cases, the enzyme may be inactivated midway, making it impossible to obtain a degradation product with the desired molecular weight. Conversely, if the amount added is too large, the reaction may proceed too rapidly, making it difficult to stop the reaction at the appropriate time. Those skilled in the art will be able to determine the amount of pectinase to be added taking these factors into consideration.

[0023] Pectinases derived from various organisms can be used, and are not particularly limited, but for example, those derived from Aspergillus niger can be preferably used. Other examples of preferred pectinases include pectinases used for clarification of fruit juice and peeling of agricultural products such as fruits and vegetables. Commercially available pectinases can also be used, and for example, pectinase (derived from Aspergillus niger) from Tokyo Chemical Industry Co., Ltd. can be preferably used.

[0024] In pectinase treatment, the peaks of low-molecular-weight saccharides (monosaccharides to oligosaccharides) with molecular weights of approximately 1000 or less are ignored, and the enzymatic reaction is terminated when the peak of the polysaccharides (those with molecular weights exceeding 1000) reaches an average molecular weight of 10,000 or less (preferably 2,500 to 10,000, more preferably 5,000 to 10,000). This allows the production of a composition containing the target polysaccharide. In this case, the timing of reaction termination can be determined by, for example, sampling portions of the reaction solution over time after the start of the reaction and analyzing the molecular weight by gel filtration using high-performance liquid chromatography (HPLC). A differential refractive index detector, which can primarily detect only sugars (monosaccharides, disaccharides, oligosaccharides, and polysaccharides), is recommended as the detector for high-performance liquid chromatography (HPLC). The enzymatic reaction can be carried out near the optimal temperature for the enzyme (e.g., 37°C). Because the resulting polysaccharides are highly thermostable, the enzymatic reaction can also be terminated by boiling the mixture for several minutes, e.g., 5 to 10 minutes, to inactivate the enzyme.

[0025] After the enzyme is inactivated, the polysaccharide-containing composition can be obtained by purification as needed. The purification method is not particularly limited; for example, the small amount of insoluble precipitate produced by boiling can be removed by centrifugation or other means. Furthermore, fractionation can be performed by column chromatography to obtain several fractions, and the most active fraction can be selected. Fractionation can also be performed using other purification techniques, such as paper chromatography or thin layer chromatography. Furthermore, these fractionation techniques, including column chromatography, may be performed multiple times, or multiple techniques may be used in combination. Furthermore, if necessary, the polysaccharide-containing composition can be obtained as a dry product by lyophilization. This polysaccharide-containing composition contains the compound of the present invention, and the concentration of the compound of the present invention can be increased by the above-mentioned purification.

[0026] The polysaccharide-containing composition produced according to the above method has been demonstrated to have excellent immunomodulatory activity (particularly immunostimulatory activity) in the Examples described below ("Fraction 1-b" in the Examples). Therefore, this polysaccharide-containing composition also constitutes one aspect of the present invention. Hereinafter, the compound of the present invention and this polysaccharide-containing composition may be collectively referred to as the "active ingredient of the present invention."

[0027] The active ingredient of the present invention has immunomodulatory ability (particularly immunostimulatory activity). Thus, the present invention provides an immunomodulator comprising the active ingredient of the present invention, and a composition for immunomodulation comprising the active ingredient of the present invention. The present invention also provides the active ingredient of the present invention for use in immunomodulation, and the use of the active ingredient of the present invention in immunomodulation. Furthermore, the present invention provides a method for modulating immunity in a subject, the method comprising having the subject ingest (e.g., orally ingest) an effective amount of the active ingredient of the present invention. The immunomodulation is preferably immunostimulation.

[0028] The active ingredient of the present invention can be used as an immunomodulator containing the active ingredient. The immunomodulator may be a molded product of the active ingredient itself, or a molded mixture of the active ingredient and various other ingredients. Examples of molding methods include spray-drying or freeze-drying the active ingredient or a mixture of the active ingredient and various other ingredients to remove moisture, physically mixing the resulting dry powder with an excipient, and granulating it to form a powder or granular composition. Examples of such materials include sweeteners, colorants, storage stabilizers, antioxidants, acidulants, flavorings, bittering agents, spices, and seasonings. Examples of excipients that can be used include lactose, crystalline cellulose, starch (dextrin), sucrose fatty acid esters, and glycerin fatty acid esters.

[0029] Furthermore, the active ingredient of the present invention can be used as a composition for immunomodulation (immunomodulatory composition) containing the active ingredient. The immunomodulatory composition is a mixture of the active ingredient of the present invention with various ingredients, and may be formed into a shape after mixing, if necessary. Examples of such ingredients that can be used include sweeteners, colorants, storage stabilizers, antioxidants, acidulants, flavorings, bittering agents, spices, and seasonings.

[0030] The active ingredient of the present invention can exert its effects, for example, by oral ingestion. Therefore, the immunomodulator and immunomodulatory composition of the present invention can be prepared to be suitable for oral ingestion. The amount of intake of the active ingredient of the present invention can be determined based on, for example, the user's health condition, age, weight, etc., and is not particularly limited, but a typical range is, for example, 0.1 g to 100 g per day. Ingestion may be once a day or in multiple divided doses. Therefore, the immunomodulator and immunomodulatory composition of the present invention can contain the active ingredient of the present invention so that the determined daily intake can be easily taken.

[0031] Furthermore, when the active ingredient of the present invention is orally ingested, the immunomodulatory composition of the present invention can be formulated as a food or drink, which is used to regulate immunity, particularly to activate immunity.

[0032] The food and drink of the present invention can be prepared by incorporating the active ingredient of the present invention into the food and drink. The food and drink may be in any form, such as a solid food, a semi-liquid food such as a creamy food, a gel food, or a beverage, or may be in the form of a powder, granules, capsule, tablet, liquid, or the like. When incorporating the active ingredient of the present invention into the food and drink, other ingredients can also be incorporated together. Examples of such other ingredients include glucose, fructose, sucrose, maltose, sorbitol, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, casein, gelatin, pectin, agar, amino acids, colorants, flavorings, preservatives, etc.

[0033] Specific examples of the food and drink of the present invention include soft drinks, juices, jams, confectioneries, dairy products, and the like.

[0034] The subjects to which the active ingredient, immunomodulator, immunomodulatory composition, and immunomodulatory food and drink of the present invention are to be ingested are not limited to humans, but are also applicable to pets such as dogs and cats, and all non-human animals. Accordingly, the food and drink of the present invention also includes animal feed.

[0035] When the compound having immune function, which is the active ingredient of the present invention, is provided as a food, it can be provided as is or can be incorporated into a food. The food provided in this manner is a food containing an effective amount of the compound having immune function. As used herein, "containing an effective amount" of the compound having immune function refers to a content such that the compound having immune function is ingested within the range described below when ingested in the amount normally consumed in a particular food. Furthermore, the term "food" is used to include health foods, functional foods, dietary supplements, supplements, health-promoting foods (e.g., foods for specified health uses, foods with nutrient functions, foods with functional claims), and foods for special dietary uses (e.g., foods for infants, foods for pregnant women, foods for the sick).

[0036] Because the compositions and compounds having immune function of the present invention maintain, enhance, and / or improve immune function, they can be provided by being incorporated into foods consumed daily or as supplements. In this case, the compositions, immunomodulators, and compounds having immune function of the present invention can be provided in unit packaging forms in which the amount to be ingested per meal is predetermined. Examples of unit packaging forms per meal include packs, wrappings, cans, bottles, etc., in which a fixed amount is specified. To better exert the various effects of the compounds having immune function of the present invention, the intake amount per meal can be determined according to the intake amount per serving of the compounds having immune function, as described below. The foods of the present invention may be provided with instructions regarding the intake amount displayed on the packaging or with a document or the like containing such instructions.

[0037] The food of the present invention may be labeled to indicate that it has the effect of maintaining, enhancing, and / or improving immune function. In this case, in order to make the labeling easier for consumers to understand, the food of the present invention may be labeled with some or all of the following: "sustains, enhances, improves, improves, strengthens, supports, maintains, activates immune function; reduces the risk of catching a cold; reduces the risk of infection with viruses such as influenza virus, norovirus, or rotavirus." It goes without saying that in the present invention, "maintaining, enhancing, and / or improving immune function" is used in a sense that includes the above-mentioned labels.

[0038] The food or beverage of the present invention may be a non-alcoholic beverage. Since the non-alcoholic beverage of the present invention has an alcohol content of less than one alcoholic content, it is not included in the category of alcoholic beverages defined as "drinks with an alcohol content of one or more alcoholic contents" under the Liquor Tax Act of Japan. The non-alcoholic beverage of the present invention includes not only so-called "non-alcoholic beverages" but also "soft drinks."

[0039] The type of non-alcoholic beverage is not particularly limited as long as the effects of the present invention are achieved, and examples include non-alcoholic beverages that have the taste or flavor of various alcoholic beverages such as various soft drinks, beer, wine, sparkling wine, sangria, cocktails, chuhai, sake, shochu, plum wine, etc. In one embodiment, the non-alcoholic beverage is a non-alcoholic wine-flavored beverage. [Example]

[0040] The present invention will be described below with reference to specific embodiments, but it will be understood that the present invention is not limited to these embodiments, and that various changes and modifications therein can be made by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

[0041] Test Example 1: Immunostimulatory effect of blackcurrant polysaccharides on macrophage-like cells In this example, the immunostimulatory effect of polysaccharides isolated from black currant was evaluated using mouse-derived macrophage-like cells (RAW264.7).

[0042] (1) Production of blackcurrant-derived polysaccharides Blackcurrant juice was dialyzed using a dialysis membrane with a molecular weight cutoff (MWCO) of 14,000. The supernatant was then centrifuged. The supernatant was then fractionated using an anion chromatograph (DEAE-Sephadex® A-25 (Merck)) to separate it into a flow-through fraction and a 0.5 M NaCl elution fraction (0.5 M NaCl elution fraction).

[0043] The collected 0.5M NaCl eluted fraction was treated with pectinase (derived from Aspergillus niger, Tokyo Chemical Industry Co., Ltd.) (389 units) for 6 hours (37°C) to obtain the 0.5M NaCl eluted fraction, pectinase-treated polysaccharide. The 0.5M NaCl eluted fraction, pectinase-treated polysaccharide, was then loaded onto Bio-Gel P-2 Gel (Bio-Rad) and fractionated by column chromatography to obtain fraction 1-b. "Unit" represents the enzyme titer, with 1 unit being the titer required to liberate 1.0 μmol of galacturonic acid from polygalacturonan in 1 minute at 37°C and pH 4.0.

[0044] The results of the analysis of the constituent sugars of the polysaccharide in fraction 1-b are shown in Table 1. As shown in Table 1, this polysaccharide contains rhamnose, arabinose, galactose, xylose, galacturonic acid, and glucuronic acid residues in specific proportions. [Table 1]

[0045] Furthermore, the linkage modes detected for polysaccharides in fraction 1-b were T-Ara (4%), 5-Ara (26%), T-Xyl (2%), 4-Xyl (26%), 2,4-Xyl (4%), 2-Rha (3%), T-Gal (2%), and 3-Gal (5%), where "T-" refers to the non-reducing terminal sugar residue linked exclusively via C(O)-1.

[0046] These results suggest that the polysaccharide in fraction 1-b has a molecular weight of less than 10,000 and is a compound consisting of rhamnogalacturonan (RG-I) and xylogalacturonan (XGA) moieties.

[0047] (2) Measurement of immunostimulatory activity RAW264.7 cells, 5.0 x 10 5 The concentration of cells / ml was adjusted, and 500 μL of each was seeded onto a 24-well plate and pre-cultured for 24 hours (37°C, 5% CO2). DMEM medium containing 10% FBS was used for the culture. The prepared flow-through fraction and 0.5M NaCl elution fraction were then added at a concentration of 100 μg / mL, and the culture was continued for 24 hours. Changes in mRNA expression of cytokines involved in immunostimulatory activity were then measured using real-time RT-PCR.

[0048] Next, black currant-derived polysaccharides (0.5M NaCl eluted fraction, 0.5M NaCl eluted fraction pectinase enzyme-treated fraction, and fraction 1-b) were added at a concentration of 200 μg / mL and cultured for 48 hours. After that, the concentrations of cytokines involved in immunostimulatory activity in the cell culture medium were measured using ELISA, and changes in the mRNA expression levels of the same cytokines were measured using real-time RT-PCR.

[0049] The mRNA expression levels of IL-6, TNF-α, and iNOS were elevated in cells treated with the 0.5M NaCl fraction compared to untreated cells (NC) (Figure 1). In contrast, no changes were observed in cells treated with the flow-through fraction. This indicates that the active ingredient is contained in the 0.5M NaCl fraction. Next, the concentrations of IL-6 and TNF-α in the medium were elevated in cells treated with the 0.5M NaCl fraction compared to untreated cells (NC) (Figure 2). Furthermore, the secretion levels of these proteins were elevated in cells treated with pectinase-treated polysaccharides from the 0.5M NaCl fraction or with fraction 1-b. Furthermore, the mRNA expression levels of IL-6, TNF-α, and iNOS were significantly elevated, particularly in cells treated with fraction 1-b (Figure 3). These results suggest that fraction 1-b contains polysaccharides that exhibit potent immunostimulatory effects.

[0050] Next, RAW264.7 cells were cultured at a cell count of 5.0 × 10 5 The cells were adjusted to a concentration of 100 cells / ml and seeded in 500 μL aliquots onto 24-well plates for 24 hours at 37°C and 5% CO2. Cultures were performed in DMEM medium containing 10% FBS. Fraction 1-b, prepared from blackcurrant extract polysaccharides, was then added at concentrations of 25, 50, 100, and 200 μg / ml and cultured for 48 hours. These cells were compared with cells treated with Escherichia coli lipopolysaccharide (LPS) at 0.1 μg / ml as a positive control, which increases inflammation-related factors. Immunostimulatory activity was assessed by measuring nitric oxide (NO) levels in the cell culture medium using the Griess assay, measuring the levels of immunostimulatory cytokines in the cell culture medium using ELISA, and measuring changes in mRNA expression of these cytokines using real-time RT-PCR.

[0051] Fraction 1-b was added to RAW264.7 cells at concentrations of 25-200 μg / mL, and the NO concentration in the medium was measured, revealing a concentration-dependent increase (Figure 4). Furthermore, the concentrations of IL-6 and TNF-α in the medium were measured by ELISA, revealing a similar concentration-dependent increase (Figure 5). Furthermore, an increase in mRNA expression was also observed (Figure 6). These results demonstrate that fraction 1-b, a polysaccharide obtained by enzymatically treating and fractionating black currant polysaccharide, possesses potent immunostimulatory activity.

Claims

1. A compound consisting of a rhamnogalacturonan (RG-I) portion and a xylogalacturonan (XGA) portion, the compound having a molecular weight of 10,000 or less, and the XGA portion containing at least one xylose building block.

2. 2. The compound of claim 1, wherein the XGA moiety comprises 2 to 7 consecutive xylose building blocks.

3. The compound according to claim 1, having a molecular weight of 2,500 to 10,000.

4. 2. The compound according to claim 1, comprising as building blocks rhamnose building blocks, arabinose building blocks, galactose building blocks, xylose building blocks, galacturonic acid building blocks, and glucuronic acid building blocks.

5. An immunomodulatory agent comprising a compound according to any one of claims 1 to 4.

6. The immunomodulator according to claim 5 , which has immunostimulatory activity.

7. A composition for immunomodulation comprising a compound according to any one of claims 1 to 4.

8. The composition of claim 7 having immunostimulatory activity.

9. The composition according to claim 7, which is a food or drink.

10. A method for producing a black currant-derived polysaccharide-containing composition, comprising partially digesting black currant juice or a polysaccharide-containing fraction isolated from black currant juice with pectinase until the average molecular weight of the polysaccharides is 10,000 or less.

11. A polysaccharide-containing composition obtainable by the method of claim 10.

12. An immunomodulator comprising the polysaccharide-containing composition of claim 11.

13. The immunomodulator according to claim 12, having immunostimulatory activity.

14. A composition for immunomodulation comprising the polysaccharide-containing composition of claim 11.

15. The composition of claim 14 having immunostimulatory activity.

16. The composition according to claim 14, which is a food or drink.

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