JP4540318B2 - Cytokine production promoter - Google Patents

Description

The present invention relates to a novel polysaccharide derived from cassis fruit juice, a cytokine production promoter containing the polysaccharide as an active ingredient, and a health food.

In recent years, consumers' awareness of health has increased. On the other hand, the modern society under recession is flooded with factors that damage the immune system, such as irregular lifestyles, dietary bias, and mental stress. Conversely, when immunity is activated, various effects such as suppression of carcinogenesis, anticancer activity, anti-infection, anti-allergy, and recovery of physical condition rhythm / maintenance can be expected. Many types of cells are involved in the immune mechanism, but the role of leukocytes is particularly large. Among them, macrophages are ubiquitous in all animals and are an important type of white blood cell that is involved in all stages of the immune response, particularly in the early stages. In recent years, the function of leukocytes has been elucidated at the substance level, and it has been found that the function of leukocytes and the intercellular phase action are borne by trace proteins (cytokines) secreted by leukocytes.

There are many types of cytokines, and tumor necrosis factor (TNF) and interleukins (IL) are attracting attention. Among them, inflammatory cytokines such as TNF-α, IL-1β, and IL-12p70 have been reported to be released mainly from macrophages and eventually show anticancer and antiinfective effects. Yes. Research has also been conducted on methods for producing these cytokines using genetic engineering techniques and using them as drugs, but on the other hand, by using natural materials and their extracts to increase the amount of certain cytokines in the body. Material search and examination for enhancing the immunity and resistance of the host are also widely performed (see Non-Patent Documents 1 and 2). However, most of the active ingredients contained in conventional immunostimulants and health foods are derived from bacteria and fungi. Moreover, in general, there are many things that are difficult to obtain, expensive, or have a safety problem or poor image when used in food.

An Immunomodulatory Polysaccharide-Rich Substance from the Fruit Juice of Morinda citrifolia (Noni) with Antitumour Activity (Phytotherapy Research 13,380-387 (1999)) Immunopharmacological activity of Echinacea preparations following simulated digestion on murine macrophages and human peripheral blood mononuclear cells (J. Leukocyte Biology 68 (10), 503-510 (2000))

The present invention finds a substance that promotes the production of cytokines such as TNF-α and certain types of interleukins in the body in daily foods, and uses the substance or a purified extract to safely An object of the present invention is to provide a health food that is highly resistant and has an excellent immunostimulatory effect.

In order to solve the above-mentioned problems, the present inventors searched for an immunostimulatory substance in foods that are routinely provided for food, particularly fruits. More specifically, by using mouse peritoneal macrophages, the fruit juice of various fruits and the cytokine production promoting activity of the fruit skin extract were measured. It has been found that it has significantly superior cytokine production promoting activity compared to other fruits, and that the active substances are two types of polysaccharides (polysaccharide A and polysaccharide B), thereby completing the present invention. It was.

That is, the present invention provides the following novel polysaccharide derived from Cassis fruit juice, a cytokine production promoter and a health food containing the polysaccharide as an active ingredient.
1. Polysaccharide A having the following properties (1) to (5).
(1) The average molecular weight is about 45,000,
(2) As a constituent sugar, at least glucose, galactose and arabinose are included, and the weight composition ratio thereof is 21:64:15;
(3) Easily soluble in water and an aqueous ethanol solution of about 35% (v / v) or less,
(4) Insoluble in 70% (v / v) or higher ethanol aqueous solution,
(5) Insoluble in 65% saturated or higher ammonium sulfate aqueous solution.
2. Said polysaccharide A of 1 obtained from a cassis fruit juice.

3. Polysaccharide B having the following properties (1) to (5).
(1) The average molecular weight is about 430,000,
(2) As a constituent sugar, at least glucose, galactose and arabinose are included, and their weight composition ratio is 41: 50: 9,
(3) Easily soluble in water,
(4) About 35% (v / v) or more insoluble in ethanol aqueous solution,
(5) Insoluble in 65% saturated or higher ammonium sulfate aqueous solution.
4). 4. The polysaccharide B described in 3 above, obtained from cassis juice.
5). A cytokine production promoter comprising the polysaccharide A described in 1 or 2 and the polysaccharide B described in 3 or 4 as active ingredients.
6). A health food comprising the polysaccharide A described in 1 or 2 and the polysaccharide B described in 3 or 4 as active ingredients.

The polysaccharide of the present invention can be obtained, for example, by the following method. First, after crushing the cassis fruit and separating the solids such as the peels into fruit juice, the pH is adjusted to 7.2 to 7.4 using aqueous ammonia, aqueous sodium hydroxide, aqueous potassium hydroxide, etc., and the resulting insoluble matter is centrifuged. Remove by separation to obtain a supernatant. Cassis used as a raw material is not particularly limited in its production area and time, and commercially available fruit juice may be used without adjusting the pH, and may be applied to the following steps.

  Separation and purification of a further active ingredient from the obtained supernatant can be performed according to a method for purifying polysaccharides from ordinary natural products. First, for example, cation exchange columns such as Amberlite IR 120B H AG (manufactured by Organo), CM Sepharose FF (Pharmacia), AG 50W-X2 (manufactured by Biorad), and Amberlite IRA 410 OH AG ( Various ionic substances are removed by sequentially passing through anion exchange columns such as Organo), DEAE Sepharose FF (Pharmacia), AG 1-X2 (BioRad). Note that either the cation exchange treatment or the anion exchange treatment may be performed first, and the resin may be used in a mixed bed. Next, the solution is passed through a reverse phase column such as SEP-PAK (C18) (manufactured by Waters) to remove the polyphenols to obtain an extract. Furthermore, if necessary, drying treatment such as freeze drying and spray drying can be performed to obtain a dried product containing the two polysaccharides A and B of the present invention. The two polysaccharides of the present invention can be obtained by further purifying the dried product or the extract before drying by the following ethanol fractionation method.

  In a state where the polarity is increased by adding a small amount of a salt solution such as sodium chloride or potassium chloride to the extract containing the two polysaccharides of the present invention, ethanol is added to obtain a concentration of about 35% (v / v), and centrifugation is performed. By performing the separation, polysaccharide B can be obtained in the precipitate fraction. From the supernatant fraction, polysaccharide A can be obtained by removing low-molecular sugars such as monosaccharides, disaccharides and oligosaccharides by dialysis and ultrafiltration.

Alternatively, in a state where the polarity is increased, first, ethanol is added so as to be 70% (v / v) or more, and the mixture is centrifuged to co-precipitate polysaccharide A and polysaccharide B, and the resulting precipitate is added to an appropriate aqueous solution. After suspending in (5), ethanol is added to give a concentration of about 35% (v / v) and the mixture is centrifuged to obtain polysaccharide A in the supernatant fraction and polysaccharide B in the precipitate fraction. In order to coprecipitate polysaccharide A and polysaccharide B, ammonium sulfate may be added so as to be 65% saturated or higher.

The physicochemical properties of the two types of polysaccharides thus obtained are as follows.
1. Physicochemical properties of polysaccharide A (1) Molecular weight The average molecular weight measured using high performance liquid chromatography is about 45,000.
(2) Sugar composition After hydrolysis with hydrochloric acid, the sugar composition measured using high performance liquid chromatography contains at least glucose, galactose and arabinose as constituent sugars, and the weight composition ratio thereof is 21:64:15.
(3) It is easily soluble in soluble water and an aqueous ethanol solution of about 35% (v / v) or less, and insoluble in an aqueous ethanol solution of 70% (v / v) or more. Some are insoluble in aqueous ethanol solutions up to about 35% (v / v) to 70% (v / v). Insoluble in 65% saturated or higher ammonium sulfate aqueous solution.

(4) Adsorption characteristics in various chromatography The constituent polysaccharide of polysaccharide A did not bind to the cation exchange resin and the anion exchange resin, but partly bound to concanavalin A-Sepharose resin (Pharmacia).
(5) Infrared absorption spectrum The infrared absorption spectrum of polysaccharide A is shown in FIG. Polysaccharide A dissolved in pure water showed a high absorption pattern as a whole in the vicinity of 950 to 1200 cm ⁻¹ .
(6) UV-visible absorption spectrum The UV-visible absorption spectrum of polysaccharide A is shown in FIG.
(7) Specific rotation The specific rotation of polysaccharide A is [α] D = + 123 (c 0.25, H 2 O).

(8) Heat stability In the state where polysaccharide A and polysaccharide B coexist, heat treatment at 100 ° C. for 10 minutes did not deactivate cytokine production promoting activity against macrophages.
(9) Color reaction The color reaction by the phenol-sulfuric acid method is positive.
(10) Color of the substance After dialysis with pure water, the product freeze-dried is light brown.

(11) Enzyme treatment stability Among the constituent polysaccharides of polysaccharide A, the fraction that binds to concanavalin A-sepharose is endo-β-Galactosidase having an endo-type galactosidase activity (Escherichia freundii: manufactured by Seikagaku Corporation) And β-Glucanase FBP (Bacillus subtilis: manufactured by Nippon Biocon) are not decomposed.
(12) ¹³ C-nuclear magnetic resonance spectrum (125 MHz)
FIG. 5 shows the ¹³ C-nuclear magnetic resonance spectrum of polysaccharide A measured in heavy water using acetone as an internal standard.
(13) ¹ H-nuclear magnetic resonance spectrum (500 MHz)
The ¹ H-nuclear magnetic resonance spectrum of polysaccharide A measured in heavy water is shown in FIG.

2. Physicochemical properties of polysaccharide B (1) Molecular weight The average molecular weight measured using high performance liquid chromatography is about 430,000.
(2) Sugar composition After hydrolysis with hydrochloric acid, the sugar composition measured using high performance liquid chromatography contains at least glucose, galactose and arabinose as constituent sugars, and the weight composition ratio thereof is 41: 50: 9.
(3) It is easily soluble in soluble water and insoluble in ethanol aqueous solution of about 35% (v / v) or more. Some ethanol is insoluble at ethanol concentrations from 10% (v / v) to about 35% (v / v). Insoluble in 65% saturated or higher ammonium sulfate aqueous solution.

(4) Adsorption characteristics in various chromatography The constituent polysaccharide of polysaccharide B did not bind to the cation exchange resin and the anion exchange resin and did not bind to the concanavalin A-sepharose resin.
(5) Infrared absorption spectrum The infrared absorption spectrum of polysaccharide B is shown in FIG. Polysaccharide B dissolved in pure water showed a particularly high absorption pattern around 1040 cm ⁻¹ .
(6) UV-visible absorption spectrum The UV-visible absorption spectrum of polysaccharide B is shown in FIG.
(7) Specific rotation The specific rotation of polysaccharide B is [α] D = + 177 (c 0.25, H 2 O).

(8) Heat stability In the state where polysaccharide A and polysaccharide B coexist, heat treatment at 100 ° C. for 10 minutes did not deactivate cytokine production promoting activity against macrophages.
(9) Color reaction The color reaction by the phenol-sulfuric acid method is positive.
(10) Color of the substance After dialysis with pure water, the product freeze-dried is light brown.

Polysaccharide A and polysaccharide B thus obtained can be used for health foods because they have cytokine production promoting activity. In order to use polysaccharide A and polysaccharide B for health foods, those in any purification stage can be used. For example, those in various purification stages can be used in various foods such as soft drinks, juices, teas, and teas. By adding to alcoholic beverages, candy, various processed foods, etc., a health food that promotes cytokine production and activates the immune mechanism can be obtained.

The polysaccharide obtained by the present invention and an extract containing the same are useful as a cytokine production promoter, an immunostimulant, or a functional food and a health food to which the substance or ingredient is added, and are used on a daily basis. Since it is an extract from the fruit eaten by the worm, it is extremely low in toxicity and does not have any adverse effects on the human body even in long-term continuous use.

  EXAMPLES Next, although an Example is described and this invention is demonstrated in detail, this invention is not limited to a following example.

Example 1: Cytokine production promoting effect of various fruit juices, pericarp extracts and herbal extracts Passion fruit, Sikhwasha, Peach, Watermelon, Prunes, Figs, Kiwi, Apricot, Guava, Papaya, Cassis (English name: Blackcurrant), La France, red raspberry, red plum, mango, cranberry, lychee, ume, loquat, orange, grapefruit, yuzu, lemon, amla, natsumikan, lime, lime, blueberry, acerola, pineapple, pomegranate, ruby grapefruit, banana, apple, Blood orange, melon, grape juice (cabernet sauvignon) and grape juice (sauvignon blanc), orange peel, banana peel, lemon peel, grapefruit peel and yuzu peel peels, Furthermore, the extract of herbs (Echinacare) was tested for the effect of promoting production of cytokines (TNF-α, IL-1β, IL-12p70) using a mouse peritoneal macrophage system.

(Preparation of test sample)
The fruit juice was diluted twice with deionized water, adjusted to pH 7.2 to 7.4 with a 1N aqueous sodium hydroxide solution, further added with deionized water, and finally a 4-fold diluted solution was prepared. This was centrifuged (15,000 rpm × 5 minutes), and the resulting supernatant was used as a test sample. For the peel, an equal amount of deionized water was added and sufficiently stirred and crushed with a mixer. After adjusting the pH to 7.2 to 7.4, deionized water was further added to finally prepare a 4-fold diluted solution. This was centrifuged (1,5000 rpm × 5 minutes), and the resulting supernatant was used as a test sample.

  For herbs, add deionized water so that the extract powder is 4 times diluted in terms of the weight of herbal medicine, stir with a stirrer for 15 minutes, then centrifuge (15,000 rpm x 5 minutes), and the resulting supernatant Was used as a test sample.

(Measurement of cytokine (TNF-α, IL-1β, IL-12p70) production ability)
A mouse (ICR, 8 weeks old, male) was intraperitoneally injected with 2.7 ml of 2.98% thioglycolate medium (Difco). As a result of collecting cells in the abdominal cavity after 4 days, macrophages of about 1 × 10 ⁷ cells / mouse were obtained. PBS ^- (phosphate buffered saline (pH 7.4): Difco) After washing, the final macrophages concentration was suspended in 1% FCS-RPMI1640 so that 1 × 10 ⁶ cells / ml. 200 μl of this was dispensed into a 96-well plate and allowed to stand at 37 ° C. for 2 hours in a CO ₂ incubator. After the wells were washed twice with PBS ⁻ , 180 μl of 5% FCS-RPMI1640 containing antibiotics (penicillin (100 units / ml), streptomycin (100 μg / ml)) was added to each well. 20 μl of the test sample or control was added thereto and cultured for 20 hours. For positive control, LPS (Escherichia coli 0111: manufactured by Funakoshi) prepared with deionized water to 1 μg / ml was used. PBS ^- was used as a negative control. The culture solution is centrifuged (1000 rpm, 10 minutes), and the cytokine concentration in the obtained supernatant is attached by a cytokine ELISA quantification kit (Quantikine mouse TNF-α, IL-1β, IL-12p70: manufactured by R & D Systems). Measured according to the manual.

The results are shown in Table 1-1 and Table 1-2.

  As shown in Table 1-1 and Table 1-2, in the juice, cassis, fig, lychee, loquat and the like showed strong cytokine production promoting ability. Among them, the ability to promote cytokine production in cassis, especially the inducing activity of TNF-α and IL-1β, is well known as an herb that enhances immunity and has been reported to activate mouse macrophages. Higher activity. Furthermore, in the case of cassis, the induction activity of IL-1β was relatively higher than that of other fruits compared with the induction activity of TNF-α.

Example 2: Separation of cytokine production promoting component from cassis (1)
The cassis fruit was crushed and centrifuged (10,000 rpm, 10 minutes) to separate the solids. The pH was adjusted to 7.2 to 7.4 with 1N aqueous sodium hydroxide solution, and the resulting insoluble matter was removed by centrifugation (10,000 rpm, 10 minutes), and the supernatant was collected. The fraction passed through a cation exchange resin (Amberlite IR 120B H AG: Organo) and an anion exchange resin (Amberlite IRA 410 OH AG: Organo) at a flow rate of SV4 was collected. Subsequently, the fraction was passed through a SEP-PAK (C-18) column (Waters) activated with methanol.

Example 3: Separation of cytokine production promoting component from cassis (2)
Sodium chloride was added to the fraction obtained by the SEP-PAK (C-18) column process to a final concentration of 0.1 mole / l. Ethanol was added to this at various concentrations (v / v). The supernatant fraction and the precipitate fraction were separated by centrifugation (15,000 rpm, 10 minutes), and PBS ⁻ was added to the precipitate fraction to suspend the original volume. When the TNF-α-inducing activity of the supernatant fraction and the precipitate fraction at each ethanol concentration was measured, it was clearly separated into two components when the ethanol concentration was 30-40% (v / v) as shown in FIG. did.

Then, the ethanol concentration is 70% (v / v) and so as the precipitate fraction obtained when added PBS ^- suspended in, again ethanol to a concentration of 35% turn (v / v) Added. This was separated into a supernatant fraction and a precipitate fraction by centrifugation, and when examining the cytokine production promoting activity (TNF-α inducing activity) when the supernatant fraction and the precipitate fraction alone were mixed, As shown in FIG. 8, only when two components coexist, strong cytokine production promoting activity appeared.

Example 4: Separation of cytokine production promoting components from cassis (3)
The supernatant fraction and the precipitate fraction are washed and concentrated by ultrafiltration (Millipore) with a fractional molecular weight of 10,000 to remove low-molecular substances such as monosaccharides, disaccharides and oligosaccharides, and Protein Assay The protein content and polysaccharide content of each fraction were measured by Kit (Bio-Rad) and phenol-sulfuric acid method. When converted into the concentration before the fractionation operation, that is, in the state of cassis juice, the protein content and sugar content contained in the supernatant fraction were 3 μg / ml and 360 μg / ml, respectively. On the other hand, the protein content and sugar content contained in the precipitate fraction were 39 μg / ml and 4400 μg / ml, respectively. Thus, both fractions were almost entirely occupied by polysaccharides. The polysaccharides contained in the supernatant fraction and the precipitate fraction were designated as polysaccharide A and polysaccharide B, respectively. In this case, the specific rotation of polysaccharide A is [α] D = + 123 (c 0.25, H 2 O), and the specific rotation of polysaccharide B is [α] D = + 177 (c 0.25, H 2 O). )Met.

  Next, the molecular weights of polysaccharide A and polysaccharide B were measured. Measurement was performed by high performance liquid chromatography using a gel filtration column OHpak SB-805 HQ (manufactured by Showa Denko) using dextran (Pharmacia) having various degrees of polymerization as molecular weight markers. As a result, the apparent average molecular weights of polysaccharide A and polysaccharide B were about 45,000 and about 430,000, respectively.

Furthermore, the composition ratio of the constituent sugars was examined. Hydrochloric acid was added to a final concentration of 0.7 N, and the polysaccharide was hydrolyzed by allowing it to stand at 100 ° C. for 12 hours. Glucose, galactose and arabinose were quantified by high performance liquid chromatography using a sugar analysis column SUGAR SC1011 (manufactured by Showa Denko) for the released monosaccharide. As a result, the sugar composition ratios of polysaccharide A and polysaccharide B were 21:64:15 and 41: 50: 9, respectively, by weight.

The chart of the infrared absorption spectrum of the refined polysaccharide A is shown. A vertical axis | shaft shows the absorption difference in each wave number of the aqueous solution of the polysaccharide A and the pure water which is a blank by the light absorbency. The chart of the infrared absorption spectrum of the refined polysaccharide B is shown. The vertical axis represents the absorption difference at each wave number between the aqueous solution of polysaccharide B and pure water as a blank in terms of absorbance. The ultraviolet-visible absorption spectrum of the refined polysaccharide A is shown. The ultraviolet visible absorption spectrum of the refined polysaccharide B is shown.

The ¹³ C-nuclear magnetic resonance spectrum (125 MHz) of the purified polysaccharide A is shown. The ¹ H-nuclear magnetic resonance spectrum (500 MHz) of the purified polysaccharide A is shown. The location situation in each ethanol concentration fraction of the cytokine production promoting component (polysaccharide A and polysaccharide B) in the cassis juice is shown. The location is indicated by the strength of TNF-α-inducing activity. The recovery (synergistic effect) of TNF-α-inducing activity by combining the supernatant fraction (including polysaccharide A) and the precipitate fraction (including polysaccharide B) in the 35% ethanol concentration fraction of Cassis juice is shown.

Claims (6)

Polysaccharide A having the following properties (1) to (5).
(1) The average molecular weight is about 45,000,
(2) As a constituent sugar, at least glucose, galactose and arabinose are included, and the weight composition ratio thereof is 21:64:15;
(3) Easily soluble in water and an aqueous ethanol solution of about 35% (v / v) or less,
(4) Insoluble in 70% (v / v) or higher ethanol aqueous solution,
(5) Insoluble in 65% saturated or higher ammonium sulfate aqueous solution. The polysaccharide A according to claim 1, obtained from cassis fruit juice. Polysaccharide B having the following properties (1) to (5).
(1) The average molecular weight is about 430,000,
(2) As a constituent sugar, at least glucose, galactose and arabinose are included, and their weight composition ratio is 41: 50: 9,
(3) Easily soluble in water,
(4) About 35% (v / v) or more insoluble in ethanol aqueous solution,
(5) Insoluble in 65% saturated or higher ammonium sulfate aqueous solution. The polysaccharide B according to claim 3 obtained from cassis fruit juice. A TNF-α production promoter comprising the polysaccharide A according to claim 1 or 2 and the polysaccharide B according to claim 3 or 4 as active ingredients . An IL-1β production promoter comprising the polysaccharide A according to claim 1 or 2 and the polysaccharide B according to claim 3 or 4 as active ingredients.

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