JP2009227618A - Immunostimulant derived from kjellmaniella crassifolia miyabe and method of extracting viscous polysaccharide derived from kjellmaniella crassifolia miyabe - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunostimulant having, as an active ingredient, a viscous polysaccharide selected from viscous polysaccharides derived from Kjellimaniella crassifolia Miyabe under predetermined conditions and a method of extracting a viscous polysaccharide derived from Kjellimaniella crassifolia Miyabe. <P>SOLUTION: The immunostimulant has a viscous polysaccharide extracted from Kjellimaniella crassifolia Miyabe whose aqueous solvent solution has concentration of more than 0.05% and less than 0.3% as an active ingredient. The process for producing an immunostimulant is a method of extracting a viscous polysaccharide derived from Kjellimaniella crassifolia Miyabe having a pulverization step of pulverizing Kjellimaniella crassifolia Miyabe, an extraction step of mixing the pulverized Kjellimaniella crassifolia Miyabe with an aqueous solvent and shaking to obtain an extract solution of a viscous polysaccharide, and a salt removal step of removing salts from the viscous polysaccharide. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an immunostimulant containing as an active ingredient a viscous polysaccharide obtained by extraction from gagome (Kjellmaniella crassifolia Miyabe), and an extraction method of gagome-derived viscous polysaccharide. .

  Gagome (Kjellmaniella crassifolia Miyabe) is an algae that grows on rocky reefs with a depth of 7 to 25 m, mainly in the limited areas such as Hakodate on the Hokkaido coast, the east coast of the Oshima Peninsula, Matsumae Kojima, and Cape Oma in the northeastern Tohoku region. It is a perennial algae with a body length of 2 m and a width of 30 cm or more. A large and small wrinkle is formed on the entire surface of the leaf-like part, and the viscous polysaccharide that causes stickiness is secreted in large quantities from the alga. This viscous polysaccharide is composed of fucoidan, laminaran, alginic acid and the like, and it has recently been clarified that this viscous polysaccharide is useful for health, and has attracted attention as a raw material for health foods and an active ingredient of drugs.

  For example, WO97-026896 discloses an apoptosis inducer using fucoidan derived from gagome (Patent Document 1). JP-A-2005-231998 discloses a therapeutic agent for dialysis amyloidosis using fucoidan derived from Gagome (Patent Document 2).

  On the other hand, an immunostimulant using a polysaccharide extracted from seaweed as an active ingredient has been proposed. For example, Japanese Patent Application Laid-Open No. 6-256208 discloses an immunostimulant containing, as an active ingredient, an acid polysaccharide extracted from red algae that has been reduced in viscosity by the action of β-agarase (Patent Document 3). JP-A-2007-230980 discloses an immunostimulant prepared by preparing a polysaccharide extract from Mekabu and centrifuging the polysaccharide extract to extract a precipitate fraction (Patent Document 4). .

WO97-026896 JP-A-2005-231998 JP-A-6-256208 JP 2007-230980 A

  None of the inventions disclosed in Patent Document 1 and Patent Document 2 relate to immunostimulation. The invention disclosed in Patent Document 3 does not relate to an immunostimulant derived from Gagome (Kjellmaniella crassifolia Miyabe), but relates to an immunostimulant in which the viscosity of a viscous polysaccharide is reduced by enzyme treatment. Furthermore, the invention disclosed in Patent Document 4 also relates to an immunostimulant using a precipitate fraction obtained by preparing and centrifuging a polysaccharide extract instead of a gagome-derived immunostimulant.

  On the other hand, the inventors of the present application have conducted extensive studies on the immunostimulatory effect by extracting viscous polysaccharides from gagome, quantifying the production of various cytokines and antibodies, and measuring the YAC-1 tumor cytotoxic activity. As a result, it was found that the immunostimulatory effect varies depending on the extraction conditions, concentration, viscosity, light intensity of reflected light, etc., and the present invention has been completed.

  That is, the present invention has an object to provide an immunostimulant having an active ingredient selected from among gagome-derived viscous polysaccharides according to predetermined conditions, and a method for extracting gagome-derived viscous polysaccharides. .

  A first aspect of the immunostimulant according to the present invention is a viscous polysaccharide extracted from gagome (Kjellmaniella crassifolia Miyabe), and the concentration of the aqueous solvent solution is higher than 0.05% and lower than 0.3% It contains a viscous polysaccharide as an active ingredient.

  Further, the immunostimulant according to the present invention has a value of relative viscosity of the aqueous solvent solution of more than 2.5 and less than 25.8 when the value of water viscosity at 20 ° C. is 1. Also good.

  The second aspect of the immunostimulant according to the present invention is a viscous polysaccharide extracted from gagome, wherein the viscosity of the aqueous solvent solution is larger than 2.5 centipoise and smaller than 25.8 centipoise. As an active ingredient.

  In the immunostimulant according to the present invention, the viscous polysaccharide may be extracted with an aqueous solvent having a temperature of less than 30 ° C., and may be extracted under conditions where the pH of the viscous polysaccharide is 5 or more and less than 8.

  A third aspect of the immunostimulant according to the present invention is a viscous polysaccharide extracted from gagome, and the light intensity of 0.1% alginate aqueous solution measured with the excitation wavelength and the fluorescence wavelength of the fluorometer as 350 nm. When the value of 100 is 100, a viscous polysaccharide having a light intensity value of 600 or more of the aqueous solvent solution is contained as an active ingredient.

  In the immunostimulant according to the present invention, the viscous polysaccharide may be processed to remove salts.

  On the other hand, the method for extracting viscous polysaccharides according to the present invention is a method for extracting viscous polysaccharides derived from gagome, comprising a crushing step of crushing gagome, and mixing and shaking the crushed gagome and an aqueous solvent. It has the extraction process which obtains the extract of viscous polysaccharide, and the salt removal process which removes salts from viscous polysaccharide.

  According to the present invention, among the polysaccharides derived from Gagome (Kjellmaniella crassifolia Miyabe), the active ingredient is selected from the polysaccharides selected according to predetermined conditions, and the viscosity enhancer derived from Gagome has an effective immunostimulatory action. A method for extracting polysaccharides can be provided.

  Hereinafter, the extraction method of the gagome (Kjellmaniella crassifolia Miyabe) origin immunostimulant and gagome origin viscous polysaccharide which concern on this invention is demonstrated using embodiment. First, the Gagome-derived immunostimulator according to the present invention will be described.

  The immunostimulant according to the present invention uses, as an active ingredient, a viscous polysaccharide extracted from gagome selected according to a predetermined condition. The “viscous polysaccharide” in the present invention refers to a sticky substance extracted from gagome using a water-soluble solvent, and a product obtained by treating the extracted sticky substance, and is a kind of polysaccharides such as alginic acid, laminaran, and sulfuric acid. The main component is a polysaccharide such as fucoidan, which is a kind of polysaccharide. In addition, the term “treatment” as used herein generally refers to changing the properties by processing, but refers to an aspect that does not impair the immunostimulatory action, such as heat treatment, cooling treatment, freezing treatment, acid / alkali treatment, It refers to a mode of crosslinking treatment, solid-liquid separation treatment, stirring treatment, drying treatment, desalting treatment, etc. that does not impair the immunostimulatory action.

  In the present invention, “immunostimulation” can be replaced with “immunity stimulation”, “immunity enhancement”, “immunity enhancement”, “immunity activity”, “immunity activation”, “immunity enhancement”, or “immunity enhancement”. The term “immune enhancer” is used interchangeably with “immunostimulatory agent”, “immune enhancer”, “immune enhancer”, “immunoactive agent”, or “immune enhancer”. Therefore, “immunostimulatory effect” can also be expressed as “immunostimulatory effect”, for example.

  In this embodiment, the dried gagome is crushed with a food cutter, and a mixture prepared by mixing it with distilled water 25 times the weight of the crushed gagome is extracted by shaking for 24 hours at 20 ° C. The supernatant obtained in this way is used as a viscous polysaccharide.

  Here, in the present embodiment, the viscous polysaccharide is extracted from the dried gagome, but the `` gagome '' in the present invention is not limited thereto, and may be a raw one that has been stored immediately after harvesting, It may be once frozen and stored. The size of the crushing is not particularly limited. For example, a shredded size (a size of about 10 mm to 20 mm), a crushing size (a size of about 5 mm to 10 mm), a fine crushing size (a size of 1 mm to 5 mm), a powder It can be a size (size of 1 mm or less). In the present embodiment, it is preferable to use fine pulverization of 1 mm to 3 mm.

  Moreover, in this embodiment, although the viscous polysaccharide is extracted using distilled water 25 times the weight of the crushed gagome, the type and amount of the solvent used for the extraction of the viscous polysaccharide is an aqueous solvent. It is not particularly limited as long as it is an amount capable of extracting the viscous polysaccharide, and can be appropriately selected. As an aqueous solvent in this invention, water or an aqueous solvent can be mentioned, for example, Water is preferable. Examples of water include distilled water, ion-exchanged water from which metal ions and the like have been removed, pure water, water obtained by sterilizing these, and the like. Examples of the aqueous solvent include alcohols, acids and bases.

  Next, in the present invention, among the extracted viscous polysaccharides, those having a predetermined concentration, relative viscosity, viscosity, extraction temperature, extraction pH, or reflected light intensity when used as an aqueous solvent solution, Select as an active ingredient of an immunostimulator.

  In the present invention, a viscous polysaccharide having a concentration higher than 0.05% and lower than 0.3% in the case of an aqueous solvent solution is used as an active ingredient of the immunostimulator according to the present invention. Here, the “concentration” in the present invention is the concentration of an aqueous solvent solution of a viscous polysaccharide. That is, the concentration in the present invention includes not only the sugar concentration but also the concentration of a polysaccharide contained in the viscous polysaccharide and an arbitrary mixture selected from sulfate groups, reducing ends, ash, and the like. The concentration in this embodiment is the sugar concentration of an aqueous solution of a viscous polysaccharide. In this embodiment, the concentration of sugar in the aqueous solution is 0.07% to 0.29%. Sugars.

  The sugar concentration can be expressed as w / w% or w / v% based on the weight of the sugar and the weight or volume of the aqueous solution as necessary. The sugar concentration can be represented by Brix (w / w%), which is a ratio of the soluble solid content expressed based on the refractive index of the sugar liquid. The sugar concentration can be determined by measuring the absorbance of the colored compound by the phenol sulfate method, the anthrone sulfate method, etc., and both monosaccharides and polysaccharides can be converted into any sugar. Can be quantified {Basic Biochemical Experimental Method Vol. 5 p118 (2000)}. In the present embodiment, the sugar concentration is measured using fucoidan as a standard sugar by the phenol sulfate method {Hodge, JE and Hofreiter, B. T., Method in Carbohydrate Chemistry, 1338 (1962)}.

  The viscous polysaccharide in the present invention can also be selected by the value of the relative viscosity of the aqueous solvent solution having a correlation with the concentration of the aqueous solvent solution. That is, in the present invention, the relative viscosity of the aqueous solvent solution when the concentration of the aqueous solvent solution is higher than 0.05% and lower than 0.3% and the value of water viscosity at 20 ° C. is 1. A substance having a value larger than 2.5 and smaller than 25.8 can be used as an active ingredient of the immunostimulant according to the present invention. In the present embodiment, a more suitable viscous polysaccharide having a sugar concentration of 0.07% to 0.29% and a relative viscosity value of 5.8 to 20.3 is used. It is said.

  In the present invention, the relative viscosity of the aqueous solution of the viscous polysaccharide is set to 1 at a water viscosity at 20 ° C., but the method for measuring the relative viscosity is not particularly limited, and a desired viscometer or relative viscometer is used. It can measure suitably by using. In the present embodiment, the relative viscosity is measured using an Ostwald relative viscometer having an inner diameter of 0.75 mm and 1.5 mm under the condition of 20 ° C.

  Moreover, since the viscosity of water at 20 ° C. is 1 centipoise (cP), the viscosity polysaccharide in the present invention has a viscosity of 2.5 centipoise larger than 25.8 centipoise when used as an aqueous solvent solution. What has viscosity can be used as an active ingredient of the immunostimulant according to the present invention. In addition, in this embodiment, the thing whose viscosity of aqueous solution is 5.8 cP-20.3 cP is made into the suitable viscous polysaccharide.

  Further, in the present invention, a viscous polysaccharide extracted with an aqueous solvent under a condition of less than 30 ° C. and / or a viscous polysaccharide extracted with an aqueous solvent under a condition of pH of 5 or more and less than 8 is selected. Can do. It has been clarified by the inventors that a polysaccharide having a high viscosity can be obtained by extraction under conditions of less than 30 ° C. and / or a pH of 5 or more and less than 8. In the present embodiment, a viscous polysaccharide extracted under conditions of 4 ° C. to 20 ° C. and / or a pH of 5.8 to 7.6 is used as a suitable viscous polysaccharide, and 20 ° C. And / or a viscous polysaccharide extracted under the condition of pH 6.3 to 7.4 is used as a more suitable viscous polysaccharide.

  Next, the viscous polysaccharide in the present invention can be selected according to the light intensity of the reflected light of the aqueous solvent solution. The viscous polysaccharide in the present invention is a case where a plurality of polysaccharides having high viscosity are entangled with each other to form a complex having a higher-order structure, and this higher-order structure complex has a certain size and density. The inventors believe that an immunostimulatory effect is exhibited. The value of the light intensity of the reflected light is proportional to the concentration of the aqueous solvent solution in a certain range, but it has been clarified by the inventors that the value of the light intensity decreases when the concentration is too high. This indicates that the higher-order structure complex having a certain size and density formed at the certain concentration has an immunostimulatory effect. According to the inventors, the size and density of the existing higher-order structure complex greatly affect the reflection of light, and when the sugar concentration exceeds a certain level, the size of the higher-order structure complex becomes too large. For this reason, it is thought that the reflection of light is not performed well.

  In this embodiment, the light intensity value of the aqueous solution is measured by setting the excitation wavelength and the fluorescence wavelength of the fluorometer to 350 nm, and the light intensity value of the alginate aqueous solution 0.1% is 100. A viscous polysaccharide having a light intensity value of about 600 to 900 is a suitable viscous polysaccharide, and a viscous polysaccharide having a light intensity value of about 710 to 900 of the aqueous solution is a more suitable viscous polysaccharide. Sugars.

  Furthermore, the viscous polysaccharide in this invention can select the thing by which the process which removes salts is carried out. The “salts” in the present invention include not only so-called sodium chloride but also inorganic salts such as carbonates, sulfates, magnesium salts, calcium salts, minerals (minerals), and trace elements. In the present invention, the “removing process” includes not only an act of removing all but also an act of removing at least a part thereof.

  In the present invention, the treatment for removing salts is not particularly limited. For example, dialysis or electrodialysis using a dialysis membrane, ultrafiltration membrane, desalting column, suction filtration or ultrafiltration, or ion exchange resin. Any physical or chemical method / means known to those skilled in the art can be mentioned. In this embodiment, the process which removes salts is made | formed by the dialysis using a cellulose tube dialysis membrane. It is believed by the inventors that when salts are removed by dialysis, the charge balance changes and the molecular structure expands, resulting in the formation of a giant structure and increased viscosity.

Next, the extraction method of the gagome-derived viscous polysaccharide according to the present invention will be described. The method for extracting a viscous polysaccharide derived from Gagome according to the present invention comprises:
(i) Crushing process for crushing gagome
(ii) Extraction step of obtaining an extract of viscous polysaccharide by mixing pulverized gagome and aqueous solvent and shaking
(iii) A salt removal step of removing salts from the viscous polysaccharide The steps (i) to (iii) are included.

  The method for extracting a gagome-derived viscous polysaccharide according to the present invention may have other optional steps as long as it has the above steps (i) to (iii), for example, a heating step or a cooling step. You may have arbitrary processes well-known to those skilled in the art, such as a process, a freezing process, an acid / alkali addition process, a bridge | crosslinking process, a solid-liquid separation process, a stirring process, a drying process, a desalting process. In this embodiment, a food cutter is used. However, the “pulverization” in the present invention is not limited to this, and any means known to those skilled in the art can be used.

  Here, “shaking” in the present invention generally means sieving, but is not limited to this and includes the meaning of “stirring”. That is, in the present invention, a desired shaking device can be used, and a stirring device such as a magnetic stirrer or a mechanical stirrer can also be used. In the present embodiment, shaking is performed using a constant temperature shaking incubator.

  Hereinafter, the Example of the extraction method of the immunostimulant which concerns on this invention, and the viscous polysaccharide derived from Gagome is described. Note that the scope of the present invention is not limited to the features shown by these examples.

<Examination of extraction conditions>
In order to extract highly viscous viscous polysaccharides from Gagome (Kjellmaniella crassifolia Miyabe), the relationship between the concentration and viscosity of viscous polysaccharides was examined while examining extraction temperature, time, and pH.

(1) Examination of temperature and time First, the dried gagome was finely pulverized with a food cutter, cut into pieces of 1 mm to 3 mm, and suspended in 25 times weight of distilled water. The suspension was shaken using a constant temperature shake incubator (TAITEC) at 100 rpm / min for 1 to 24 hours under a temperature condition of 4 to 100 ° C., and the viscosity of the supernatant was adjusted. It was measured. The extraction temperature was performed in 8 steps of 4, 10, 20, 30, 40, 60, 80, and 100 ° C. Viscosity was measured 6 times each in a 20 ° C. bath using an Ostwald viscometer (tube inner diameter 0.75 mm or 1.5 mm; Shibata Chemical), and the average value of the relative viscosity with respect to water was calculated. The result is shown in FIG. 1 (a), and a graph of this is shown in FIG. 1 (b).

  As shown in FIGS. 1 (a) and 1 (b), it is clear that the viscosity of the supernatant of the viscous polysaccharide extract becomes the highest when extraction is performed for 12 to 24 hours at a temperature of 20 ° C. became.

(2) Examination of pH Next, dried gagome finely pulverized to a size of 1 to 3 mm using a food cutter is suspended in 25 times weight of distilled water, and then 0.1 N HCl aqueous solution or 0.1 N NaOH. The pH was adjusted to 3.8-9.4 by adding an aqueous solution. This pH-adjusted suspension was shaken at 100 rpm / min for 24 hours at a temperature of 20 ° C. using a constant temperature shaking incubator (TAITEC) to extract viscous polysaccharides, and the viscosity of the supernatant was measured. The measurement and calculation of the viscosity were performed in the same manner as (1). The sugar concentration in the supernatant was measured using the phenol sulfate method {Hodge, JE and Hofreiter, BT, Method in Carbohydrate Chemistry, 1338 (1962)}. The results are shown in FIG. 2 (a), and the relationship between pH and relative viscosity is shown in FIG. 2 (b).

  As shown in FIGS. 2 (a) and 2 (b), it has been clarified that the viscosity of the supernatant of the viscous polysaccharide extract is highest when extraction is performed under the condition of pH 7.1. .

(3) Relationship between sugar concentration and viscosity Contained using an extract of viscous polysaccharide extracted under the conditions of pH 5.8 at a temperature below 30 ° C. in the same manner as in (1) and (2) above. The sugar concentration (%) of the viscous polysaccharide was measured and the relationship with the relative viscosity was examined. The result is shown in FIG.

  As shown in FIG. 3, when the sugar concentration of the extract was 0.07% to 0.29%, the relative viscosity was a value of 5.8 to 20.3. And it became clear that the relative viscosity tends to increase as the sugar concentration increases.

<Component analysis of viscous polysaccharide extract>
A dried gagome finely pulverized to a size of 1 to 3 mm using a food cutter is suspended in 25-fold weight of distilled water, and then kept at a temperature of 20 ° C. for 24 hours at 100 rpm / min. The viscous polysaccharides were extracted by shaking using, and the filtrate and gagome residue were separated into solid and liquid by suction filtration. The filtrate was further centrifuged at 1500 rpm for 20 minutes to remove the precipitate and obtain a supernatant. 25 mL of the obtained supernatant was applied to a cellulose tube dialysis membrane (planar width: 32 mm, diameter: 20 mm, wall thickness: 0.0305 mm, permeation molecular weight: about 14,000 or less; Sanko Junyaku Co., Ltd.) The dialysis treatment was carried out with stirring. The dialysis treatment was performed until 24 hours had passed after changing the external solution 3 hours, 6 hours, and 12 hours after the start of dialysis. The solution after dialysis treatment was freeze-dried, sterilized distilled water was added, and the mixture was allowed to stand overnight and dissolved to obtain a viscous polysaccharide extract (GE). On the other hand, the solution after the dialysis treatment was heated at 60 ° C. for 1 hour, then lyophilized, and sterilized distilled water was added and allowed to stand overnight for dissolution to obtain a viscosity reducing solution (GH).

  The viscosity of the solution after the dialysis treatment (extract), the total amount of sugar contained in the lyophilized solution (lyophilized product), the amount of each polysaccharide, the sulfate group, the reducing end, and Quantification with ash was performed. The viscosity and total sugar amount were the same as in Example 1. The amount of each polysaccharide was determined by the method of Nishide et al. (Nishide, E .; Yoshihara, M .; Kato, T .; Hakone, T .; Kamata, Y .; Anzai, H .; Uchida, N. Fractionation of laminaran and fucoidan by DEAE-sephadex column chromatography. Bull. Coll. Agr. & Vet. Med., Nihon Univ. 1994, 51, 103-107), respectively, followed by phenol sulfate method {Hodge, JE and Hofreiter, BT , Method in Carbohydrate Chemistry, 1338 (1962)}. Furthermore, the sulfate group was quantified by the rosin acid method (Terho, TT; Hartiala, K. Method for determination of the sulfate content of glycosaminoglycans. Anal Biochem. 1971, 41, 471-476.) Somgyi, M. Note on sugar determination. J Biol Chem. 1952, 195, 19-23. The ash content was determined by the direct ashing method (550 ° C ashing method). . The result is shown in FIG.

  As shown in FIG. 4, it was shown that GE contains fucoidan, laminaran, and alginic acid as polysaccharides. In addition, the relative viscosity of GH was much lower than that of GE, and it was revealed that the viscosity of the extract was lowered by heat treatment at 60 ° C. for 1 hour.

《Study of immunostimulatory effect using mice》
The immunostimulatory action of viscous polysaccharides extracted from gagome using mice was investigated.
First, mice were divided into groups using 6-week-old male C57BL / 6Cr (Sankyo Lab Service). A total of 7 groups, ie, a control group, GE10, GE30, GE100, GH10, GH30, and GH100, were set as test composition groups, and 5 mice were used for each group. The GE obtained in Example 2 was prepared to 1, 3, 10 mg / mL with sterilized distilled water, and these aqueous solutions were administered as test substances to three groups of GE10, GE30, and GE100. On the other hand, GH obtained in Example 2 was prepared to 1, 3, 10 mg / mL with sterilized distilled water, and these aqueous solutions were administered as test substances to three groups of GH10, GH30, and GH100. Sterile distilled water was administered to the control group.

  In the test method, the day of grouping was defined as day 0, and the test substance or sterilized distilled water was orally administered daily for 14 days each time using a sonde once a day from the next day. Body weight was measured daily during the administration period. In addition, feces were collected on the 14th day of the last administration day, and the mice were dissected on the 15th day after the last administration day. For dissection, the mice were first sacrificed with carbon dioxide gas, and after blood was collected from the posterior vena cava, the spleen was removed. Animal experiments were conducted in accordance with “Regulations for Animal Experiments at Hokkaido University”.

  Using the data and samples obtained above, the immunostimulatory effect was examined. In the data analysis, an average value and a standard deviation in each group were calculated based on the obtained numerical values. The statistical analysis between each group performed the T test using Excel (trademark; Microsoft), and evaluated the effect of the to-be-tested substance by comparing the average value with a control group. Significance levels are 5% and 1%, and are indicated by “*” and “**”, respectively.

(1) Body weight Changes in body weight of each group during the test period are shown in FIGS. 5 (a) to (g). On the 14th day of the last day of administration, the average value of the body weight of each group is calculated and data analysis is performed. It was. The result is shown in FIG. 6A, and a graph of this is shown in FIG. 6B. As shown in FIGS. 5 (a) to (g), there was no difference in body weight among the individual groups in all groups. In addition, as shown in FIGS. 6A and 6B, no significant difference was observed in all of the GE or GH administration groups as compared to the control group.

(2) Spleen weight
The weight of the extracted spleen was measured and data analysis was performed. The result is shown in FIG. 7A, and a graph of this is shown in FIG. 7B.

  As a result, compared with the control group, the spleen weight value was significantly higher in GE30 (p <0.01), and it was revealed that the spleen was enlarged.

(3) Amount of cytokine and antibody produced by spleen cells The amount of cytokine and immunoglobulin produced from the extracted spleen cells of the spleen was quantified by ELISA. The measurement method is to prepare a cell suspension (2.8 × 10 ⁶ cells / mL) from the extracted spleen, dispense it to a culture plate, and then add concanavalin A (ConA) to a final concentration of 1 μg / mL. Then, under the conditions of a culture temperature of 37 ° C. and a CO ₂ concentration of 5%, the culture supernatant produced from the spleen cells was obtained by culturing the cytokine for 3 days and the immunoglobulin for 5 days, respectively. Then, cytokine (IFN-γ, IL-12, IL-18, IL-6) concentration and immunoglobulin (IgA, IgM) concentration in the culture supernatant were measured by sandwich enzyme immunoassay (sandwich ELISA). The results are shown in FIGS.

  As a result, compared with the control group, all values of IFN-γ, IL-12, IL-18, IL-6, IgA, and IgM were significantly higher in GE30 (p <0.05). Have been shown to promote the production of cytokines and antibodies.

(4) YAC-1 tumor cytotoxic activity Natural killer (NK) cells were isolated from the excised spleen, and tumor cytotoxic activity using YAC-1 as a target cell was measured as NK activity. That is, after isolating NK cells from the extracted spleen, it was prepared to a concentration of 6.25 × 10 ⁶ cells / mL, and this was used as effector cells (E). On the other hand, YAC-1 cells (Dainippon Sumitomo Pharma Co., Ltd.) were prepared to a concentration of 2.5 × 10 ⁵ cells / mL, 5 × 10 ⁵ cells / mL, 1 × 10 ⁶ cells / mL, and these were designated as target cells (T). did. A 96-well plate mixed and seeded at a cell number ratio of E: T of 100: 1, 50: 1, 25: 1 was cultured at 37 ° C. (5% CO ₂ ) for 22 hours. The 96-well plate was stained with WST-1 reagent (Takara Bio) for 2 hours, and then the absorbance value at 450 nm was measured using a microplate reader (Corona). In addition, the damaging activity with respect to YAC-1 cell was calculated | required by following Formula.
(Following formula)
% (Cytotoxic activity) = [1-{(absorbance value of ET co-culture−absorbance value of E culture) / (absorbance value of T culture)}] × 100
Each data analysis is performed based on the obtained numerical values, and the results are shown in FIGS. 9 (a) to 9 (c), respectively, and graphs of the results are shown in FIGS. 10 (a) to 10 (c).

  As a result, in the case of an E: T ratio of 100: 1, a significant increase in NK cell activity was observed in GE30 compared to the control group (p <0.05).

<Viscosity measurement>
The viscosity of each test substance (administration solution) administered in Example 3 was measured. The viscosity was measured 6 times by the method used in Example 1. Individual relative viscosities in each group are shown in FIGS. 11 (a) to (f), and an average value of six measurement data is shown in FIG. 12.

  As a result, it has been clarified that the value of relative viscosity increases as the concentration increases in both GE and GH.

<Light intensity measurement>
(1) Measurement of administration liquid In order to investigate the characteristics of the structure of the test substance (administration liquid) administered in Example 3, the light intensity of the reflected light was measured using a fluorometer. The test substance administered in Example 3 was used as a measurement sample, purified alginic acid (Sigma) prepared to 1,3,10 mg / mL, and carboxymethylcellulose (CMC; Kanto Chemical) prepared to 10 mg / mL A control sample was used. CMC was dissolved in sterilized distilled water, and purified alginic acid was dissolved in a 12% aqueous sodium carbonate solution. 0.6 mL of each sample was subjected to a fluorometer, and the light intensity of the reaction light was measured by setting both the fluorescence wavelength and the excitation wavelength to 350 nm. After the measurement, each light intensity was calculated with the light intensity of the 0.1% aqueous solution of alginic acid as 100. The result is shown in FIG. 13A, and a graph of this is shown in FIG. 13B.

  As shown in FIGS. 13A and 13B, a significantly high light intensity was detected in GE30. In Example 3 above, a significant immunostimulatory effect was observed for GE30, suggesting that 3 mg / mL GE has a specific structure that enhances immunostimulation. Further, the light intensity of GH30 was reduced by 36.3% as compared with GE30. This indicates that the viscosity of GH30 is lower than that of GE30 in Example 4, and it is suggested that the higher order structure of the sugar molecule is destroyed by the heat treatment. The reason why the light intensity in 10 mg / mL alginic acid shows a high value is considered to be that a large cluster is formed by cross-linking due to the presence of the metal ion salt at the center of the molecular structure.

(2) Examination of GE concentration Samples prepared with GE in sterile distilled water to 1, 2, 3, 5, 10 mg / mL are designated as GE10, GE20, GE30, GE50, and GE100, respectively, as in (1) above. The light intensity of the reflected light was measured. Sample preparation and measurement were each performed 4 times. The result is shown in FIG. 14 (a), and a graph of this is shown in FIG. 14 (b).

  As a result, it was found that the light intensity at GE30, GE50, and GE100 showed a value of 600 or more, and that at GE30 and GE50, the light intensity showed a high value of 700 or more.

  According to the present Example as described above, the viscous polysaccharide derived from Gagome has a specific viscosity or a reflectance by a specific measurement, so that cytokine production, antibody production, and YAC-1 tumor cell damage in spleen cells It is suggested that the activity is promoted and acts on the immune response as shown in FIG.

  In the present embodiment, a viscous polysaccharide derived from gagome that is effective for enhancing immunostimulation is extracted using water as a solvent, and the appropriate viscosity of the aqueous solution of this viscous polysaccharide and the immunostimulatory action are obtained. Utilization can be expected to develop functional foods with thickness.

In Example 1 (1), it is a figure which shows the examination result of extraction temperature and extraction time. (A) is a chart showing individual relative viscosities measured, and (b) is a graph of (a). In Example 1 (2), it is a figure which shows the examination result of extraction pH. (A) is the chart which shows each relative viscosity measured, and the chart which shows the sugar density | concentration in an extract, (b) is the figure which graphed the relationship between pH of (a) and relative viscosity. In Example 1 (3), it is a figure which shows the relationship between sugar concentration and a viscosity. In Example 2, it is a figure which shows the result of the component analysis of a viscous polysaccharide extract. In Example 3 (1), it is the figure which showed the weight transition of each group. (A)-(g) shows the weight transition of a control group, GE10 group, GE30 group, GE100 group, GH10 group, GH30 group, GH100 group, respectively. In Example 3 (1), it is a figure which shows the average value of the body weight in each group on the last day of administration. (A) is a chart showing measured individual body weights, and (b) is a graph of (a). In Example 3 (3), it is a figure which shows the spleen weight extracted. (A) is a chart showing the measured individual spleen weights, and (b) is a graph of (a). In Example 3 (4), it is a figure which shows the result of having measured the cytokine and the antibody production amount of the spleen cell. (A) to (g) show the production amounts of IFN-γ, IL-12, IL-18, IL-6, IgA, and IgM, respectively. In Example 3 (5), it is a graph which shows the measurement result of YAC-1 tumor cytotoxic activity. (A)-(c) show the measurement results when the ratio of the number of cells of E and T is 25: 1, 50: 1, 100: 1, respectively. In Example 3 (5), it is the figure which graphed the measurement result of YAC-1 tumor cytotoxic activity. (A)-(c) show the measurement results when the ratio of the number of cells of E and T is 25: 1, 50: 1, 100: 1, respectively. In Example 4, it is a figure which shows each numerical value which measured the viscosity. In Example 4, it is a figure which shows the average value computed from each numerical value which measured the viscosity. In Example 5 (1), it is a figure which shows the result of having measured the light intensity. (A) is a chart showing individual measured light intensities, and (b) is a graph of (a). In Example 5 (2), it is a figure which shows the result of having measured the light intensity. (A) is a chart showing individual measured light intensities, and (b) is a graph of (a). It is the figure shown about the immune response considered that the viscous polysaccharide derived from Gagome acts.

Claims (8)

  An immunostimulant comprising a viscous polysaccharide extracted from gagome (Kjellmaniella crassifolia Miyabe) and having an aqueous solvent solution concentration of more than 0.05% and less than 0.3% as an active ingredient.   2. The immunostimulant according to claim 1, wherein the value of the relative viscosity of the aqueous solvent solution when the viscosity value of water at 20 ° C. is 1 is greater than 2.5 and less than 25.8.   An immunostimulant comprising a viscous polysaccharide extracted from Kjellmaniella crassifolia Miyabe and having a viscosity of an aqueous solvent solution larger than 2.5 centipoise and smaller than 25.8 centipoise as an active ingredient.   The immunostimulant according to any one of claims 1 to 3, wherein the viscous polysaccharide is extracted with an aqueous solvent of less than 30 ° C.   4. The immunostimulant extracted according to any one of claims 1 to 3, wherein the viscous polysaccharide is extracted under a condition where the pH of the viscous polysaccharide is 5 or more and less than 8.   In the case of a viscous polysaccharide extracted from Gagome (Kjellmaniella crassifolia Miyabe), the light intensity value of 0.1% alginate aqueous solution measured with an excitation wavelength and a fluorescence wavelength of a fluorometer of 350 nm as 100, An immunostimulant comprising, as an active ingredient, a viscous polysaccharide having a light intensity value of 600 or more in an aqueous solvent solution.   The immunostimulant according to any one of claims 1 to 6, wherein the viscous polysaccharide is subjected to a treatment for removing salts. A method for extracting a viscous polysaccharide from Gagome (Kjellmaniella crassifolia Miyabe),
Crushing step of crushing gagome;
An extraction step of obtaining a viscous polysaccharide extract by mixing and shaking the pulverized gagome and an aqueous solvent;
A method for extracting a gagome-derived viscous polysaccharide comprising a salt removing step of removing salts from the viscous polysaccharide.

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