HK1261670B - Polysaccharide having innate immunity stimulating activity and innate immunity stimulating agent or food and drink comprising thereof - Google Patents

Description

Polysaccharides with natural immune activation effects and natural immune activators or foods and beverages containing the same

Technical Field

The present invention relates to polysaccharides having an innate immunity activating effect, and innate immunity activating agents or foods and beverages containing the polysaccharides.

Background Art

Higher vertebrates like humans possess two immune systems: innate and acquired immunity. These two mechanisms work together to protect against infection. Meanwhile, many other organisms, such as insects, lack acquired immunity and rely solely on innate immunity to protect against infection.

Innate immunity is a common infection defense mechanism shared by all organisms. Being nonspecific, it is characterized by rapid response and effective function against a wide range of infectious agents. In higher vertebrates, such as humans, nonspecific innate immunity is considered more important than specific immunity against infectious agents, from the perspectives of early-stage infection resistance, prevention of cancer and lifestyle-related diseases, and tissue repair.

Therefore, innate immunity is an important host factor in the body's defense mechanism in the early stages of infectious diseases, and activation of innate immunity is believed to be effective in the prevention and treatment of infectious diseases. Furthermore, the development of anti-cancer drugs has become increasingly important in recent years, and the ability to activate innate immunity, exemplified by the anti-tumor activity of polysaccharides from mushrooms, has garnered attention.

The inventors of the present invention have previously discovered that injecting innate immunity-activating substances, such as fungal β-glucan or bacterial peptidoglycan, into silkworm muscle specimens induces paralytic peptides, which act as insect cytokines, activating innate immunity and causing muscle contraction (Patent Document 1, Non-Patent Documents 1-3). Furthermore, they have established a simple system for searching for innate immunity-activating substances, using changes in body length due to muscle contraction as an indicator, and have discovered innate immunity-activating abilities in polysaccharides extracted from, for example, green tea (Non-Patent Documents 4 and 5).

It is known empirically that vegetables are important for the intake of nutrients necessary for maintaining human health. On the other hand, they are also used as traditional medicinal herbs worldwide and are believed to contain substances that activate natural immunity. However, the substances that are considered to have the effect of activating natural immune function have not been clearly known so far.

The inventors of the present invention have discovered that broccoli extracts exhibit innate immune-stimulating effects (Patent Document 1). However, the specific components in broccoli that are directly involved in this innate immune-stimulating effect have not yet been determined. Consequently, it is also unclear which part (chemical structure) of this component (compound) may have this innate immune-stimulating effect.

Therefore, there are few attempts to use biologically derived extracts (compounds) as innate immune activators by breaking down the chemical bonds of these extracts, extracting only the active component (chemical structure). In other words, there are few attempts to prepare (synthesize) innate immune activators with chemical structures effective for innate immune activation using biologically derived extracts (compounds) as raw materials.

Prior art literature

Patent Literature

Patent Document 1: International Publication No. 2008/126905

Non-patent literature

Non-patent document 1: Activation of the silkworm cytokine by bacterial and fungal cell wall components via a reactive oxygen species-triggeredmechanism. Ishii K, Hamamoto H, Kamimura M, Sekimizu K.J Biol Chem. 2008, 283(4), 2185-91.

Non-patent document 2: Insect cytokine paralytic peptide (PP) induces cellular and hummoral immune responses in the silkworm Bombyx mori. Ishii K, Hamamoto H, Kamimura M, Nakamura Y, Noda H, Imamura K, Mita K, Sekimizu K.J Biol Chem. 2010, 285(37), 28635-42.

Non-patent document 3: Porphyromonas gingivalis peptidoglycans induce excessive activation of the innate immune system in silkworm larvae. Ishii K, Hamamoto H, Imamura K, Adachi T, Shoji M, Nakayama K, Sekimizu K.J Biol Chem. 2010, 285(43), 33338-47.

Non-patent document 4: Purification of innate immunostimulant from green teausing a silkworm muscle contraction assay. Dhital S, Hamamoto H, Urai M, Ishii K, Sekimizu K. Drug Discoveries&Therapeutics. 2011, 5(1), 18-15.

Non-patent document 5: Evaluation of innate immune stimulating activity ofpolysaccharides using a silkworm (Bombyx mori) muscle contraction assay. T. Fujiyuki, H. Hamamoto, K. Ishii, M. Urai, K. Kataoka, T. Takeda, S. Shibata and K. Sekimizu. Drug Discoveries&Therapeutics, 6(2), 88-93, 2012.

Summary of the Invention

Problems to be solved by the invention

The present invention has been made in view of the above-mentioned background art, and an object of the present invention is to provide a novel substance having an innate immunity activating effect.

Solutions to the Problem

To address the above-mentioned issues, the inventors of the present invention conducted extensive research and, using a method they established to measure silkworm muscle contraction activity, purified and structurally elucidated an innate immune-activating substance from broccoli extract. Furthermore, they investigated the structure essential for its activity.

As a result, by purifying broccoli extracts, they discovered that a substance with a natural immune-activating effect exists among polysaccharides whose structure was previously unknown.

Furthermore, the inventors have discovered that polygalacturonic acid chains, which are constituent components of the polysaccharides, play an important role in activating innate immunity, leading to the completion of the present invention.

That is, the present invention provides a polysaccharide having an innate immune activating effect, characterized in that, as constituent sugars, it contains 25 to 50 mol parts of galacturonic acid, 15 to 50 mol parts of galactose, 0 to 7 mol parts of glucose, 0 to 30 mol parts of arabinose, 0 to 6 mol parts of xylose, and 3 to 15 mol parts of rhamnose; and as a main chain, it contains a polygalacturonic acid chain having galacturonic acid with an α-1,4 bond.

Furthermore, an innate immunity activator containing the above-mentioned polysaccharide as an active ingredient is provided.

Furthermore, a food or beverage containing the above-mentioned polysaccharide is provided.

Effects of the Invention

According to the present invention, a polysaccharide having a novel chemical structure and an innate immunity activating effect can be provided.

Furthermore, it is possible to provide an innate immunity activator containing the polysaccharide as an active ingredient, and a food or beverage containing the polysaccharide and having an innate immunity activating effect.

Furthermore, the polysaccharides of the present invention are extremely safe and have no side effects. Furthermore, they can be easily prepared into various dosage forms and added to food and beverages. Therefore, they can be used as safe natural immune activators or functional foods to effectively activate natural immunity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the results of measuring the specific activity (C value) when the DEAE-cellulose adsorbed fraction was injected into silkworms.

FIG. 2( a ) is a graph showing the results of HPLC analysis of a standard sample, and FIG. 2( b ) is a graph showing the hydrolyzate of the DEAE-cellulose adsorption fraction.

FIG3( a ) is a 500 MHz ¹ H NMR spectrum of the DEAE-cellulose adsorption fraction, and FIG3( b ) is a 125 MHz ¹³ C NMR spectrum of the DEAE-cellulose adsorption fraction.

FIG. 4 is a graph showing the results of methylation analysis of sugar alcohol acetate derivatives in the DEAE-cellulose adsorption fraction.

FIG5(a) is a 500 MHz ¹ H NMR spectrum of the oxalic acid hydrolyzate, and FIG5(b) is a 125 MHz ¹³ C NMR spectrum of the oxalic acid hydrolyzate.

FIG. 6 is a graph showing the results of fractionation of a pectinase-treated product by gel filtration chromatography.

FIG. 7 is a schematic diagram showing an example of the chemical structure of the polysaccharide and the processed product of the polysaccharide according to the present invention.

Specific embodiments of the invention

The present invention will be described below. However, the present invention is not limited to the following specific embodiments and can be arbitrarily modified within the technical scope of the present invention.

Polysaccharides

The polysaccharide of the present invention has an innate immune activation effect, characterized in that:

The sugar composition comprises 25 to 50 mole parts of galacturonic acid, 15 to 50 mole parts of galactose, 0 to 7 mole parts of glucose, 0 to 30 mole parts of arabinose, 0 to 6 mole parts of xylose, and 3 to 15 mole parts of rhamnose.

As a main chain, it contains a polygalacturonic acid chain having galacturonic acid with α-1,4 linkage.

The polysaccharide of the present invention must contain 25 to 50 molar parts of galacturonic acid, 15 to 50 molar parts of galactose, 0 to 7 molar parts of glucose, 0 to 30 molar parts of arabinose, 0 to 6 molar parts of xylose, and 3 to 15 molar parts of rhamnose as constituent sugars.

It preferably contains 25 to 45 parts by mole of galacturonic acid, 20 to 50 parts by mole of galactose, 1 to 7 parts by mole of glucose, 0 to 20 parts by mole of arabinose, 0 to 6 parts by mole of xylose, and 4 to 14 parts by mole of rhamnose as constituent sugars.

More preferably, it contains 25 to 40 parts by mole of galacturonic acid, 30 to 50 parts by mole of galactose, 3 to 7 parts by mole of glucose, 0 to 10 parts by mole of arabinose, 0 to 5 parts by mole of xylose, and 5 to 13 parts by mole of rhamnose as constituent sugars.

It is particularly preferred to contain 30 to 40 parts by mole of galacturonic acid, 35 to 45 parts by mole of galactose, 5 to 6 parts by mole of glucose, 0 to 5 parts by mole of arabinose, 0 to 5 parts by mole of xylose, and 6 to 12 parts by mole of rhamnose as constituent sugars.

The polysaccharide of the present invention may contain monosaccharides other than the above-mentioned monosaccharides within a range not impairing the effects of the present invention.

The polysaccharide of the present invention contains a polygalacturonic acid chain having galacturonic acid with an α-1,4 bond as a main chain. The polygalacturonic acid chain may contain monosaccharides other than galacturonic acid as long as the effects of the present invention are not impaired.

Examples of the "monosaccharide other than galacturonic acid" include rhamnose.

The bonding ratio of galacturonic acid (unit) in the "polygalacturonic acid chain" is preferably 20 mol% to 99 mol%, more preferably 30 mol% to 97 mol%, and particularly preferably 40 mol% to 95 mol%, relative to the entire "polygalacturonic acid chain".

If the ratio of galacturonic acid (units) in the main chain "polygalacturonic acid chain" is too low, the natural immunity activating effect of the polysaccharide may be reduced. On the other hand, if it is too high, it is difficult to obtain the polysaccharide of the present invention.

The polysaccharide of the present invention does not contain methyl galacturonate, making it a polysaccharide with a novel structure, unlike pectin. Furthermore, the polysaccharide of the present invention is a novel substance, as it is derived from a chemical structure that has a natural immune-stimulating effect and is extracted from a natural substance.

The polysaccharides of the present invention were discovered by purifying broccoli extract. Broccoli extract is composed of multiple components. It is not easy to identify the components that have natural immune activation effects.

The present invention was achieved by repeatedly measuring the innate immune-stimulating effects of the various components of the broccoli extract using a simple and ethically sound method for measuring silkworm muscle contraction activity. Furthermore, chemical structures (units) were extracted from these components and, using the same method for measuring silkworm muscle contraction activity, identified chemical structures (units) with innate immune-stimulating effects from a large number of these chemical structures (units), ultimately leading to the present invention.

The polysaccharide of the present invention may be a polysaccharide derived from a natural substance obtained by purification, decomposition, etc., a polysaccharide obtained by chemically modifying a natural substance as a raw material, or a polysaccharide obtained by complete synthesis.

＜Natural Immune Activator＞

The innate immunity activator of the present invention is characterized by containing the above-mentioned polysaccharides as an active ingredient.

The polysaccharide contained as an active ingredient in the innate immunity activator of the present invention may be a natural polysaccharide or a synthetic polysaccharide.

The content of the polysaccharide as an active ingredient in the innate immunity activator of the present invention relative to the total innate immunity activator is not particularly limited and can be appropriately determined depending on the intended purpose. The total amount of the polysaccharide, based on 100 parts by mass of the total innate immunity activator, is preferably 0.01 to 100 parts by mass, more preferably 0.1 to 99 parts by mass, particularly preferably 1 to 95 parts by mass, and even more preferably 10 to 90 parts by mass.

The innate immunity activator of the present invention may contain "other ingredients" in addition to the above-mentioned polysaccharides as active ingredients.

The "other components" are not particularly limited and can be appropriately selected according to the intended purpose within a range not impairing the effects of the present invention. Examples thereof include pharmaceutically acceptable carriers.

Such a carrier is not particularly limited and can be appropriately selected according to, for example, the dosage form described below. The content of the "other components" in the innate immunity activator is also not particularly limited and can be appropriately selected according to the intended purpose.

The dosage form of the innate immunity activator of the present invention is not particularly limited and can be appropriately selected according to the desired administration method as described below.

Specific examples include oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquids (internal liquids, syrups, elixirs, etc.), injections (solvents, suspensions, etc.), ointments, patches, gels, creams, external powders, sprays, inhalation sprays, etc.

The oral solid preparation can be prepared by, for example, adding an excipient to the active ingredient and, if necessary, further adding additives such as a binder, a disintegrant, a lubricant, a colorant, and a flavoring agent according to a conventional method.

Examples of the excipients include lactose, saccharose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid.

Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinyl pyrrolidone.

Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose.

Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol.

Examples of the colorant include titanium oxide and iron oxide.

Examples of the flavoring/odor correcting agent include white sugar, orange peel, citric acid, and tartaric acid.

The oral liquid preparation can be prepared by adding additives such as flavoring/smell correctives, buffers, stabilizers, etc. to the active ingredients, for example, and using conventional methods.

Examples of the flavoring/smell corrective include white sugar, orange peel, citric acid, tartaric acid, etc. Examples of the buffer include sodium citrate, etc. Examples of the stabilizer include gum tragacanth, gum arabic, gelatin, etc.

The injections can be prepared by adding a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. to the active ingredients, and by conventional methods for subcutaneous, intramuscular, or intravenous injections.

Examples of the pH adjuster and buffer include sodium citrate, sodium acetate, and sodium phosphate. Examples of the stabilizer include sodium metabisulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

The ointment can be prepared by, for example, adding a known base, stabilizer, wetting agent, preservative, etc. to the above-mentioned active ingredient and mixing them by a conventional method.

Examples of the base include liquid paraffin, white petrolatum, white wax, octyldodecanol, and paraffin. Examples of the preservative include methylparaben, ethylparaben, and propylparaben.

The patch can be produced, for example, by applying a cream, gel, paste, or the like, such as the above-mentioned ointment, onto a known support by conventional methods. Examples of the support include woven fabrics, non-woven fabrics, films of soft vinyl chloride, polyethylene, polyurethane, and the like, and foam sheets made of cotton, staple fibers, or chemical fibers.

The innate immunity activator of the present invention can be used, for example, by administering to individuals who need to activate the innate immune mechanism (e.g., individuals who need to maintain health or recover from fatigue; individuals who need to prevent or treat cancer or lifestyle diseases; individuals infected with bacteria, fungi, viruses, etc.).

Animals to which the innate immunity activator of the present invention can be administered are not particularly limited, and examples thereof include humans, mice, rats, monkeys, horses, livestock such as cattle, pigs, sheep, and chickens, and pets such as dogs and cats.

The method of administering the innate immunity activator is not particularly limited and can be appropriately selected depending on the dosage form of the innate immunity activator. Examples include oral administration, intraperitoneal administration, injection into the blood, and intestinal injection.

In addition, the dosage of the above-mentioned natural immunity activator is not particularly limited and can be appropriately selected based on the age, weight, degree of desired effect, etc. of the individual to be administered. For example, the daily dosage for adults is preferably 1 mg to 30 g, more preferably 10 mg to 10 g, and particularly preferably 100 mg to 3 g, based on the total amount of the above-mentioned polysaccharide as an active ingredient.

Furthermore, the administration time of the innate immunity activator is not particularly limited and can be appropriately selected depending on the purpose. For example, the agent can be administered prophylactically or therapeutically.

＜Food and Beverage＞

The food and drink of the present invention is characterized by containing the aforementioned polysaccharide or the aforementioned innate immunity activator of the present invention.

The food and drink of the present invention has an effect of activating natural immunity.

The content of the polysaccharide or the innate immunity activator in the food and drink containing the above-mentioned polysaccharide or the above-mentioned innate immunity activator (hereinafter sometimes referred to as "the food and drink of the present invention") is not particularly limited and can be appropriately selected according to the purpose or the form (type) of the food and drink. When the food and drink as a whole is set to 100 parts by mass, the total amount of the above-mentioned innate immunity activator is preferably 0.001 to 100 parts by mass, more preferably 0.01 to 100 parts by mass, and particularly preferably 0.1 to 100 parts by mass.

In addition, any one of the polysaccharides or innate immunity activators can be used alone or in combination of two or more. When two or more are used in combination, the content ratio of each substance in the above-mentioned food and beverage is not particularly limited and can be appropriately selected according to the purpose.

The food and drink of the present invention may contain "other ingredients" in addition to the above-mentioned polysaccharides or the above-mentioned innate immunity activator of the present invention.

As above-mentioned " other components " in the food and drink product of the present invention with such natural immunity activation, there is no particular restriction, can suitably select according to purpose within the scope that does not damage effect of the present invention, can enumerate for example various food raw materials etc.In addition, the content of " other components " is not particularly restricted, can suitably select according to purpose.

There is no particular restriction on the types of the above-mentioned food and beverages, and they can be appropriately selected according to the purpose. Examples include snacks such as jelly, candy, chocolate, and biscuits; palatable beverages such as green tea, black tea, coffee, and cold drinks; dairy products such as fermented milk, yogurt, and ice cream; processed vegetable and fruit products such as vegetable drinks, fruit drinks, and jams; liquid foods such as soups; processed cereal products such as breads and noodles; various seasonings, etc.

There are no particular restrictions on the method for producing these foods and drinks, and they can be appropriately produced according to, for example, conventional methods for producing various foods and drinks.

In addition, above-mentioned food and drink products for example can be made into oral solids such as tablets, granules, capsules or oral liquids such as liquid for internal use, syrups and manufacture.The manufacture method of above-mentioned oral solids, oral liquids is not particularly limited, can suitably be selected according to purpose, for example, can be manufactured according to the manufacture method of oral solids, oral liquids of above-mentioned medicament.

These foods and drinks are considered to be particularly useful as functional foods, health foods, and the like aimed at activating the natural immune system.

When the polysaccharide or natural immunity activator of the present invention is used in the manufacture of food and beverages, the manufacturing method can be carried out using methods well known to those skilled in the art. As long as a person skilled in the art can appropriately combine the following steps to produce the target food and beverage, the steps include: a step of mixing the polysaccharide of the present invention with other ingredients, a forming step, a sterilizing step, a fermentation step, a firing step, a drying step, a cooling step, a granulation step, a packaging step, etc.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples, Comparative Examples, and Test Examples. However, the present invention is not limited to these Examples and the like unless departing from the gist of the present invention.

＜Hot water extraction＞

After cutting various vegetables, add water and steam them (121°C, 20 minutes). After cooling naturally, centrifuge (8000 rpm, 10 minutes) and use the supernatant as the "hot water extract".

<Silkworm muscle contraction activity assay>

After dissolving the sample in buffer, 100 μL was injected into a silkworm muscle specimen, and muscle contraction was measured. If the value (specific activity or C value (Contraction value)) obtained by dividing the difference in muscle length before and after injection by the length before injection was 0.15 or higher, the specimen was judged to have the ability to activate innate immunity.

Purification of the DEAE-cellulose column adsorption fraction

A hot water extract of broccoli was subjected to ethanol precipitation. The hot water extraction method followed the method described in Patent Document 1. The resulting precipitate was dissolved in MilliQ water, dialyzed against MilliQ water, and then freeze-dried. This was subjected to DEAE-cellulose column chromatography, and the amount of reducing sugars in each fraction was measured using the phenol-sulfuric acid method. The eluted sugar peak was collected, dialyzed, and then freeze-dried.

<Structural analysis of the DEAE-cellulose column adsorption fraction>

[[1. Monosaccharide composition analysis]]

The DEAE-cellulose column adsorbed fraction was hydrolyzed with trifluoroacetic acid, and the hydrolyzate was labeled with ethyl aminobenzoate and analyzed by HPLC using an ODS column. The same treatment was performed on a sample prepared by mixing standard monosaccharides including L-arabinose (Ara), L-fucose (Fuc), D-galactose (Gal), D-glucose (Glc), D-mannose (Man), L-rhamnose (Rha), D-ribose (Rib), D-xylose (Xyl), D-galacturonic acid (GalA), D-glucuronic acid (GlcA), N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), and N-acetyl-D-mannosamine (ManNAc), and the retention times were compared.

2. NMR analysis

27 mg of the DEAE-cellulose column adsorption fraction was dissolved in heavy water (D ₂ O) at 40°C, and analyzed by ¹ H and ¹³ C NMR, DQF-COSY, HMQC, HMBC, and TOCSY.

3. Methylation Analysis

The DEAE-cellulose column adsorption fraction was methylated and then hydrolyzed with trifluoroacetic acid. The resulting product was acetylated to produce sugar alcohol acetate, which was then analyzed by GCMS.

Selective decomposition of the DEAE-cellulose column adsorption fraction

1. Selective decomposition of arabinose by oxalic acid hydrolysis

The DEAE-cellulose column adsorption fraction was hydrolyzed with oxalic acid, neutralized, dialyzed, and freeze-dried.

[[2. Decomposition of polygalacturonic acid chains by pectinase]]

The DEAE-cellulose column adsorption fraction was treated with pectinase (Sigma). After inactivating the enzyme by heating, the fraction was subjected to Bio-Gel P4 gel filtration column chromatography, and the amount of reducing sugars in each fraction was determined using the phenol-sulfuric acid method. The Bio-Gel P4 gel filtration column used a 1150 nm x 15 mm diameter column, and 0.2 M acetic acid was used as the eluent. The eluted sugar peak was collected and dried under reduced pressure.

Example 1

[Exploration of vegetables containing natural immune-activating substances]

Hot water extracts of 17 vegetables (broccoli, cabbage, carrot, green pepper, monk fruit, spinach, garlic, pumpkin, ginger, cherry tomatoes, white radish, bean sprouts, cilantro, cucumber, eggplant, green onion, and Chinese cabbage) were compared for their natural immune activation abilities using a silkworm muscle contraction activity assay.

[Table 1]

Table 1 shows the specific activity (C value) of hot-water extracts of 17 vegetables using the silkworm muscle contraction activity assay. Specific activity (C value) values of 0.15 or higher were considered to have innate immune activation potential. The results in Table 1 confirm that broccoli hot-water extract has a strong innate immune activation potential.

Example 2

[Purification of natural immune-activating substances from broccoli]

Innate immunity-activating substances were purified from broccoli hot water extract using silkworm muscle contraction activity as an indicator. Table 2 shows the specific activity (C value) results at each purification stage.

[Table 2]

Ethanol precipitation of the hot water extract revealed that activity was recovered in the precipitate (Table 2), indicating that the active substance is a polysaccharide. Subsequent DEAE-cellulose column chromatography revealed a uniform sugar peak eluted along a NaCl gradient, and an increase in specific activity was observed in this fraction (Table 2).

Next, the dosage responsiveness of this fraction was examined, and the results are shown in Figure 1. In Figure 1, the vertical axis represents the specific activity (C value), and the horizontal axis represents the DEAE-cellulose adsorption fraction (μg).

When the activity showing a C value (Contraction value) of 0.15 was defined as 1 unit, a specific activity of 67 units/mg was shown ( FIG1 ). In this example, this fraction was used as a purified fraction and further analysis was attempted.

Example 3

[Structural analysis of the DEAE-cellulose column adsorption fraction]

To clarify the monosaccharide composition of the DEAE-cellulose column adsorption fraction, the trifluoroacetic acid hydrolyzate was analyzed by HPLC. The analysis results are shown in Figure 2. Figure 2a shows the results of the HPLC analysis of the standard sample, and Figure 2b shows the results of the HPLC analysis of the DEAE-cellulose column adsorption fraction.

In Figure 2, 1 represents D-glucuronic acid, 2 represents D-galacturonic acid, 3 represents D-galactose, 4 represents D-mannose, 5 represents D-glucose, 6 represents L-arabinose, 7 represents D-ribose, 8 represents N-acetyl-D-mannosamine, 9 represents D-xylose, 10 represents N-acetyl-D-glucosamine, 11 represents L-fucose, 12 represents L-rhamnose, and 13 represents N-acetyl-D-galactosamine.

The results in Figure 2 show that galacturonic acid (GalA), galactose (Gal), glucose (Glc), arabinose (Ara) and rhamnose (Rha) were detected, with a molar ratio of GalA:Gal:Glc:Ara:Rha=12.4:4.9:1.0:7.3:1.2.

Next, analysis of the sugar chain structure was attempted using NMR analysis. The results are shown in FIG3 .

The results of 1D NMR analysis showed that no characteristic signals of proteins and lipids were detected, but a characteristic spectrum of carbohydrates was shown (Figure 3).

Next, various 2D NMR analyses were performed, and the detected signals were assigned and summarized in Table 3.

[Table 3]

The signal intensity of GalA (galacturonic acid), the main component, was strong, confirming the formation of polygalacturonic acid chains with α-1,4 linkages. Regarding Ara (arabinose), the second most abundant component, some signals could be assigned, with chemical shift values indicating the formation of α-1,5 linkages. However, the signal intensities of other smaller sugar residues detected in the monosaccharide composition analysis were weak, making it impossible to assign all the signals.

Therefore, we attempted to determine the sugar linkage positions using methylation analysis. The results are shown in Figure 4. In Figure 4, 1 represents T-Araf, 2 represents T-Rhap, 3 represents 3-Araf, 4 represents 5-Araf, 5 represents T-Glcp, 6 represents T-Galp, 7 represents 3,5-Araf, 8 represents 2,4-Rhap, 9 represents 4-GalpA, 10 represents 4-Galp, 11 represents 3-Galp, 12 represents 6-Galp, and 13 represents 3,6-Galp.

The results in Figure 4 show the detection of 13 partially methylated sugar alcohol acetate peaks. The sugar species and bond positions were determined from the retention time and MS fragmentation pattern of each peak and are summarized in Table 4.

In Table 4, for example, "T-Araf" indicates non-reducing terminal arabinose, "3-Galp" indicates 3-linked galactose, and "3,5-Araf" indicates 3,5-linked arabinose.

[Table 4]

peak Bonding mol% 1 T-Araf 5.7 2 T-Rhap 1.3 3 3-Araf 1.0 4 5-Araf 16 5 T-Glcp 3.7 6 T-Galp 3.6 .7 3,5-Araf 4.4 8 2,4-Rhap 3.2 9 4-GalpA 46 10 4-Galp 5.1 11 3-Galp 3.7 12 6-Galp 1.5 13 3,6-Galp 4.5

Methylation analysis revealed 1→4-linked galacturonic acid (GalA) and 1→5-linked arabinose (Ara) as the main components, supporting the NMR analysis results. Furthermore, the linkage positions of other smaller sugar residues detected in the monosaccharide composition analysis were also determined.

Example 4

[Structure of the DEAE-cellulose column adsorption fraction required for innate immune activation]

To determine the structure of the DEAE-cellulose column adsorption fraction required for innate immunity activation, structurally modified products were prepared by selectively decomposing the main components, arabinose and galacturonic acid. Their innate immunity activation ability was evaluated using a silkworm muscle contraction activity assay. The results are shown in Table 5.

[Table 5]

First, we attempted to selectively decompose and remove arabinose (Ara) by oxalic acid hydrolysis. Analysis of the monosaccharide composition revealed a significant reduction in the Ara content (Table 5).

Furthermore, the oxalic acid hydrolyzate was dissolved in deuterated water (D ₂ O) at 70° C. and subjected to NMR analysis. The results are shown in FIG5 .

The results of 1D NMR analysis showed that the arabinose signal disappeared (Figure 5). In addition, the results of silkworm muscle contraction activity measurement of this sample (oxalic acid hydrolyzate) showed that the innate immune activation ability was lost (Table 5).

Next, polygalacturonic acid was decomposed by pectinase treatment. The degradation products were fractionated by Bio-gel P4 gel filtration column chromatography, and the results are shown in Figure 6. 1 to 4 in Figure 6 correspond to "Peak 1" to "Peak 4" in Table 5, respectively.

As shown in Figure 6, four peaks were detected. Monosaccharide composition analysis of these peaks (Table 5) revealed that GalA, Gal, Glc, Ara, Rha, and Xyl were detected in Peaks 1 and 2. No GalA was detected in Peak 3, while Gal, Glc, Ara, Rha, and Xyl were detected. GalA monomers released by pectinase treatment were detected in Peak 4. The results of silkworm muscle contraction activity assays for each peak (Table 5) showed that the natural immune activation ability decreased with molecular weight reduction, and no activity was detected in Peaks 3 and 4.

<Summary and Examination of Examples>

Using the silkworm muscle contraction activity assay, hot water extracts from 17 vegetables were used to search for innate immunity-activating substances, confirming that broccoli exhibited high activity. Furthermore, this example also demonstrated the purification of a DEAE-cellulose column adsorption fraction from broccoli that exhibited innate immunity-activating properties.

Structural analysis of the DEAE-cellulose column adsorption fraction revealed that it was an acidic polysaccharide containing GalA, Gal, Glc, Ara, and Rha, with a main structure having a polygalacturonic acid chain with α-1,4 linkages as the backbone and arabinan chains with α-1,5 linkages as the side chains.

Based on the above results, it can be considered that the DEAE-cellulose column adsorption fraction is a type of pectin, which is a component of plant cell walls. However, the results of NMR analysis generally showed that the signal of GalA methyl ester contained in pectin was almost undetectable, indicating that the structure is different from the pectin reported to date.

In order to determine the structure of the DEAE-cellulose column adsorption fraction required for the ability to activate natural immunity, the results of selectively decomposing the main components, arabinose and galacturonic acid, were obtained. The polygalacturonic acid chains were decomposed by pectinase treatment, and the ability to activate natural immunity decreased and disappeared along with the molecular weight reduction.

An example of the structure of the polysaccharide of the present invention and the structure of a processed product of the polysaccharide, predicted from the results of the Examples, is schematically shown in FIG7 . The structure of the polysaccharide of the present invention is not limited to the structure shown in the schematic diagram of FIG7 .

While numerous reports have documented the immune-stimulating properties of pectins extracted from various plants, including medicinal herbs, there are few studies investigating the relationship between immune activity and structure. However, some studies have shown that the galactan structure of pectin is essential for its activity.

However, the DEAE-cellulose column adsorption fraction purified in this example showed that the polygalacturonic acid structure was essential for activity, suggesting a mechanism of action different from the immunoactivation ability of pectin reported so far.

Industrial Applicability

According to the present invention, polysaccharides with a high effect of activating natural immune function can be obtained, and thus can be widely used in various food and beverages, tablets, granules, chewable tablets, etc. In addition, the polysaccharides of the present invention are particularly suitable for use as raw materials for functional foods that activate natural immune function in the body, specific health foods, and diets or nursing diets for patients with immunodeficiency problems. In addition, they can also be widely used as raw materials for pharmaceuticals.

Claims (4)

1. A polysaccharide with natural immune-activating properties, wherein, As a constituent sugar, it contains 30–40 moles of galacturonic acid, 35–45 moles of galactose, 5–6 moles of glucose, 0–5 moles of arabinose, 0–5 moles of xylose, and 6–12 moles of rhamnose. As the main chain, it contains polygalacturonic acid chains with α-1,4 bonds in galacturonic acid. The polysaccharides mentioned therein are obtained from broccoli, and The proportion of galacturonic acid units in the polygalacturonic acid chain is from 20 mol% to 99 mol%. 2. The polysaccharide according to claim 1, which is obtained by column chromatography from a hot water extract of broccoli and hydrolysis with oxalic acid. 3. A natural immune activator containing the polysaccharide described in claim 1 or 2 as an active ingredient. 4. A food product containing the polysaccharide as described in claim 1 or 2.