JP2018162217A5 - - Google Patents

Description

Peyer's board activator

The present invention relates to a sesame seed bulk material, an aqueous solvent extract of sesame seed sesame oil squeezed meal, and a small intestinal Peyer's patch activator containing a polysaccharide obtained from a proteolytic enzyme digestion solution of the sesame seed aqueous solvent extract as an active ingredient. Regarding

Peyer's patches are highly functionalized lymphoid follicle tissues present in the upper intestinal tract, including antigen-specific and non-specific secretory IgA-producing plasmablasts, antigen-specific and non-specific effectors, and regulatory T and B. It has become one of the induced tissues that is an important starting point for the induction of lymphocytes, antigen-specific Th17 lymphocytes and the like. Peyer's patches play an important role in the immune mechanism involved in the body's defense by the local mucosal immune system called the intestinal immune system and the systemic immune system, and the immune mechanism involved in the tolerance of innocuous antigens such as food antigens and self-antigens. It is known to carry. For example, lymphocytes in Peyer's patches are constantly recruited to other local mucosal immune system and systemic immune system accessory tissues and hematopoietic tissues by the homing phenomenon, and have the function of transmitting immune information and regulating these biological mechanisms.

One of the Peyer's patch immune function-regulating activities is the production of bone marrow cell growth-promoting factors (eg, cytokines) in Peyer's patches. As such bone marrow cell growth promoting factors, for example, IL-6, IFN-γ, and IL-10, which are cytokines produced in Peyer's patches, are known (Non-patent document 1 and Non-patent document 2).

On the other hand, up to now, a polysaccharide obtained from the rhizome of the Asteraceae Atractylodes lancea DC., and a polysaccharide obtained from the above-ground part of the legume Astragalus mongholicus Bunge and amothae (Anemarrhea) of the family Liliaceae. It has been reported that a polysaccharide obtained from rhizome has Peyer's patch immune function regulating activity (Non-Patent Documents 2 and 3).

Functional components derived from the seeds of sesame, which is an annual herb of the sesame family Sesamum (Sesamum indicum), are known to have several sesamin mainly, but components having Peyer's patch immune function regulating activity, for example, A component having an action of enhancing the production of a cytokine having a bone marrow cell proliferation-promoting activity in Peyer's patches (hereinafter, this action is also referred to as "Peyer's plate activating action") has not been known so far.

T. Hong, T.; Matsumoto, H.M. Kiyohara and H.M. Yamada: Enhanced production of hematopoietic growth factors through T cell activation in Peyer's patches by oral administration of Kampo (Japanese herbal) medicines, 'Juzen-taiho-to', Phytomedicine, 5, pp. 353-360 (1998) Hiroaki Kiyohara, Toshiake Matuzaki, Haruki Yamada: Intestinal Peyer's patch, 3rd year, 3rd year, 13th year, 3rd year, 13th year Hiroaki Kiyohara, Toshiake Matsuzaki, Tsuka Matsumoto, Takayuki Nagai, Haruki Yamada: Yakugaku Zasshi, Vol. 128-Vol.

Artificial regulation of Peyer's patch immune function will be beneficial because it will be possible to control the immune system related to systemic biological defense and immune tolerance through the intestinal immune system. As described above, several substances having Peyer's patch immune function regulating activity have been known so far. However, it cannot be said that there is a sufficient repertoire to meet the diverse needs of consumers. In view of the above circumstances, it is an object of the present invention to provide a novel Peyer's patch activator having an action of promoting the production of cytokines having a bone marrow cell growth promoting activity in Peyer's patches.

The present inventors have found that sesame seed bulk material and an aqueous solvent extract of sesame seed sesame oil squeezed meal, and a heteroglycan purified from a proteolytic enzyme digestion solution of an aqueous sesame seed aqueous solvent extract, in Peyer's patches, bone marrow cells They have found that they have an action of enhancing the production of cytokines having a growth promoting activity, and completed the present invention.

That is, the present invention provides a polysaccharide composition containing a polysaccharide obtained from sesame seeds, and a Peyer's patch activator containing the polysaccharide composition as an active ingredient. The polysaccharide composition of the present invention has at least an action of enhancing the production of cytokines (eg, IL-2, IFN-γ, IL-5) having bone marrow cell growth promoting activity in Peyer's patches. Therefore, by regulating the above cytokine production in Peyer's patches, it is possible to exert the Peyer's patch immune function regulating activity. Since sesame has been used as a food since ancient times, it is possible to provide a safer Peyer's patch activator.

Therefore, the present invention relates to a polysaccharide composition containing a polysaccharide obtained from sesame seeds, and a Peyer's patch activator containing the polysaccharide composition as an active ingredient.

In the present specification, the “sesame seed” is not particularly limited as long as it is sesame applied to food, and those subjected to roasting treatment (roasted sesame seed) and non-roasted (non-roasted sesame seed) ). Examples of the sesame seed include, but are not limited to, white sesame (thin sesame seed, thick sesame seed), tea sesame, gold sesame, and black sesame. Further, the polysaccharide of the present invention may be a polysaccharide obtained by using squeezed lees remaining after squeezing sesame oil from sesame seeds (herein referred to as “sesame oil squeezed lees”) instead of sesame seeds. Sesame oil squeezed cake is either a cake obtained by squeezing sesame oil from roasted sesame (roasted sesame oil cake) or a cake obtained by squeezing sesame oil from non-roasted sesame (non-roasted sesame oil cake). It may be.

The polysaccharide in the present invention has a heteroglycan as a main component. The polysaccharide derived from roasted sesame seeds has a peak molecular weight in the range of 10,000 to 100,000, preferably 12,000 to 90,000, 14,000 to 80,000, or 16,000 to. It is within the range of 61,000. The polysaccharide derived from non-roasted sesame seeds has a peak molecular weight in the range of 500,000 to 1,600,000.

Preferably, in the polysaccharide of the present invention, the proportion of arabinose in all constituent sugars is 10 to 50%, 11 to 48%, 12 to 45%, or 13 to 44%, and the proportion of rhamnose is 5 to 20%. 6-18%, 7-15%, or 9-13%, xylose proportion is 5-25%, 6-20%, or 7-19%, and mannose proportion is 0.5-15%. , 1 to 13%, or 2 to 11%, the proportion of galactose is 1 to 25%, 2 to 23%, 3 to 20%, or 5 to 17%, and the proportion of galacturonic acid is 1 to 60%. , 1.5 to 55%, or 2 to 53%, and the proportion of glucuronic acid is 0.5 to 8%, 1 to 5%, or 1 to 4%.

Further, preferably, in the polysaccharide of the present invention, the proportion of non-reducing terminal arabinose is 1 to 20%, 1.5 to 18%, or 2 to 14%, and the proportion of 4 or 5 linked arabinose is 5 to 30. %, 7 to 28%, or 9 to 25%.

Further, the polysaccharide in the present invention may contain a sugar chain that binds to arabinogalactan protein (AGP). That is, the polysaccharide in the present invention is exo-β-D-(1→3)-galactanase (for example, derived from dolicerase) and/or endo-β-D-(1→4)-galactanase (for example, It may be a polysaccharide in which the oligosaccharide is released by enzymatic digestion using Aspergillus niger (derived from Aspergillus niger) and the reactivity with β-D-glucosyl-yalibu antigen is reduced. In the present specification, "reactivity with β-D-glucosyl-yalib antigen is reduced by enzymatic digestion with galactanase" means that the polysaccharide is β-D-glucosyl-yalib antigen before enzymatic digestion with galactanase. It means that the reactivity of the polysaccharide with the β-D-glucosyl-yalibu antigen after enzymatic digestion with galactanase is low compared to the reactivity. The degree of decrease in reactivity is not particularly limited, but may be, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. May be. Further, the degree of decrease in reactivity due to exo-β-D-(1→3)-galactanase and the degree of decrease in reactivity due to endo-β-D-(1→4)-galactanase are the same. There is no need, and for example, a polysaccharide whose reactivity is recognized to be reduced only by enzymatic digestion of one side is also included in the polysaccharides of the present invention whose reactivity with β-D-glucosyl-yalibu antigen is reduced by enzymatic digestion.

Preferably, the polysaccharide obtained from sesame seeds of the present invention has a heteroglycan derived from roasted sesame seeds as a main component, has a peak molecular weight in the range of 61,000 to 16,000, and accounts for all constituent sugars. The arabinose proportion is 13-44%, the rhamnose proportion is 9-13%, the xylose proportion is 7-19%, the mannose proportion is 1-11%, the galactose proportion is 5-17%, and the galacturonic acid proportion is 2 to 53%, and the proportion of glucuronic acid is 1 to 4%, and the proportion of non-reducing terminal arabinose is 2 to 14%, and the proportion of arabinose having 4 or 5 bonds is 9 to 25%, which is derived from Irpex lacteus. Enzymatic digestion with exo-β-D-(1→3)-galactanase and Aspergillus niger-derived endo-β-D-(1→4)-galactanase liberates oligosaccharides. Polysaccharide having reduced reactivity with β-D-glucosyl-yalib antigen; and a non-roasted sesame seed-derived heteroglycan as a main component, with a peak molecular weight in the range of 600,000 to 1,600,000 And a polysaccharide showing.

In the present specification, the “molecular weight distribution curve” means a distribution curve obtained when the molecular weight of a test sample (eg, sesame polysaccharide fraction of sesame extract) is analyzed by high performance gel filtration chromatography (HPSEC). .. Regarding the molecular weight, for example, a calibration curve of molecular weight vs. retention time coefficient (Kav) is prepared from the retention time of standard polysaccharide (pullulan P-800, 400, 200, 100, 50, 20, 10 and 5; Showa Denko) in HPSEC. It can be calculated from the retention time of the test sample. The HPSEC condition can be set as follows, for example.
Column: Asahi-pak GS520HQ and Asahi-pak GS320HQ (each 0.75 i.d.×30 cm) (Showa Denko) connected column Liquid sending device: JASCO PV-980 (JASCO)
Detector: Shodex RI SE-62 (Showa Denko) (sensitivity: x2)
Eluent: 0.2 M NaCl (0.8 mL/min)

In the present specification, the “peak molecular weight” means the molecular weight corresponding to the peak top of the peak in the above-mentioned “molecular weight distribution curve”.

In one aspect, the polysaccharide composition of the present invention comprises an aqueous solvent extract from sesame seeds or sesame oil cake. In addition, the polysaccharide composition of the present invention may include an enzymatic digestion product of an aqueous solvent extract of sesame seeds or sesame oil squeezed meal with a proteolytic enzyme.

Moreover, in another aspect, the polysaccharide composition of the present invention comprises an ethanol-precipitated fraction of sesame seeds or sesame oil cake. The ethanol-precipitated fraction is preferably an ethanol-precipitated fraction of an aqueous solvent extract from sesame seeds or sesame oil cake, or an enzymatic digestion product of an aqueous solvent extract from sesame seeds or sesame oil cake with a proteolytic enzyme. Is an ethanol-precipitated fraction of. Further, the polysaccharide composition of the present invention may contain an enzymatic digestion product of an ethanol-precipitated fraction of an aqueous solvent extract of sesame seeds or sesame oil squeezed meal with a proteolytic enzyme.

In yet another aspect, the polysaccharide composition of the present invention is an aqueous solvent extract of sesame seeds or sesame oil squeezed meal, an ethanol-precipitated fraction of the aqueous solvent extract, or an enzymatic digestion product thereof with a proteolytic enzyme, and dialysis and And/or a fraction obtained by removing low molecular weight substances and proteins by membrane treatment. Alternatively, the polysaccharide composition of the present invention may contain the above-mentioned fraction from which neutral polysaccharide has been removed. The fraction obtained by removing low molecular weight substances and proteins by dialysis and/or membrane treatment was passed through a column packed with an anion exchange resin as a carrier and adsorbed on the anion exchange resin. An elution fraction obtained by eluting the component with an elution solvent having a low to high ionic strength, or a fraction obtained by further subjecting it to gel filtration is preferable.

For example, the polysaccharide composition of the present invention, for the polysaccharide fraction derived from roasted sesame seeds, the ethanol precipitation fraction is passed through a column packed with an anion exchange resin equilibrated with water as a carrier, Each elution fraction obtained by eluting the component adsorbed on the anion exchange resin with 0.08M NaCl and then with 0.21M NaCl and then with 0.30M NaCl, or the obtained elution fraction Is an eluate obtained by further passing through a gel filtration column having a molecular weight cut off of 2×10 ³ to 4×10 ⁵ Da, wherein in the analysis by gel filtration HPLC, the polysaccharide component in the above-mentioned peak molecular weight range is It is preferable to include a fraction corresponding to the elution volume to be eluted. The polysaccharide composition of the present invention is a polysaccharide fraction derived from a non-roasted sesame seed, and the ethanol precipitation fraction is passed through a column packed with an anion exchange resin equilibrated with water as a carrier, Fractions obtained by eluting the components adsorbed on the anion exchange resin with 0.152M NaCl, or the obtained elution fractions were further fractionated by gel filtration with a molecular weight of 2×10 ³ to 4×10 ⁵ Da. In the analysis by gel filtration HPLC of the eluate obtained by passing through the column, it is preferable to contain a fraction corresponding to the elution volume in which the polysaccharide component having the above-mentioned peak molecular weight is eluted.

Preferably, the polysaccharide composition of the present invention is an aqueous solvent extract from sesame seeds and/or sesame oil pomace of sesame seeds, or a raw material containing a proteolytic enzyme digestion solution of an aqueous solvent extract of sesame seed raw material. A composition containing a fraction obtained by ethanol precipitation and removing a low molecular weight substance from the obtained precipitate by dialysis and/or membrane treatment. Sesame seeds that have been roasted may be used, or those that have not been roasted may be used. In addition, the polysaccharide composition of the present invention may contain a substance in which the protein content is further lowered by performing digestion with an additional proteolytic enzyme after ethanol precipitation, followed by dialysis and/or ultrafiltration.

Since the polysaccharide composition of the present invention has a Peyer's patch activating action, a pharmaceutical composition, in particular, a Peyer's patch activator, an intestinal immunity enhancer, or a disease to be treated or prevented by activation of the Peyer's patch, or It can be used as a therapeutic or prophylactic agent for disorders. That is, in one aspect, the present invention relates to a pharmaceutical composition or a Peyer's patch activator containing the polysaccharide composition of the present invention as an active ingredient. The Peyer's patch activator of the present invention may contain sesame seeds or sesame oil mash itself as an active ingredient. In addition, it can be used for prevention and treatment of diseases involving IFN-γ, IL-2 and IL-5 through the intestinal tract immunity enhancing action. The Peyer's patch activator of the present invention can also be used as a food composition, food additive or feed for activating Peyer's patches. Diseases or disorders treated or prevented by activation of Peyer's patches can include infectious diseases, inflammatory diseases, autoimmune diseases, or allergies. Whether a certain disease or disorder is a disease or disorder treated or prevented by activation of Peyer's patches is exacerbated by, for example, suppression of Spi-B gene expression, or a disease improved by administration of RANKL. Or a disorder (Kanaya T et al., Nat Immunol. 2012 Jun 17; 13(8):729-36.), it can be confirmed as a disease or disorder to be treated or prevented by activation of Peyer's patches. ..

As used herein, the term “Peyer's patch activation” means to enhance the immunoregulatory function in Peyer's patches, and the proliferation and activity of CD8-positive T cells (killer T cells) and CD4-positive T cells (helper T cells). Activation, proliferation, differentiation and activation of NK cells, activation of monocytes/macrophages, antiviral effect, enhancement of cytotoxic activity of NK cells, CTLs and macrophages, promotion of expression of MHC class II molecules, Th2 cells Inhibition of IgE antibody production by suppression and/or B cell development/differentiation promoting action.

The pharmaceutical composition of the present invention can be prepared, for example, as a pharmaceutical composition for parenteral administration in the form of intravenous administration, drip, etc., or a pharmaceutical composition for oral administration. The pharmaceutical composition of the present invention may contain a pharmacologically acceptable carrier (additive for formulation). Those skilled in the art can appropriately select the type of the pharmaceutical additive used for producing the pharmaceutical composition, the ratio of the pharmaceutical additive to the active ingredient, or the method for producing the pharmaceutical composition, depending on the form of the composition. Is.

Since the pharmaceutical composition of the present invention has a Peyer's patch activating action, for example, an intestinal immunity enhancer, an intestinal immunomodulator, an intestinal infection improving agent, an anti-inflammatory agent, an antiallergic agent, a viral infection improving agent, It can also be used as a bacterial infection improving agent, a diabetes improving agent, an obesity improving agent, a mood disorder improving agent or a preventive agent for those diseases.

Moreover, since the polysaccharide composition of the present invention has a Peyer's patch activating effect, it can be used as a food composition or a feed. The shape of the pharmaceutical composition, Peyer's patch activator, food composition, and feed of the present invention is not particularly limited, and may be liquid or powder, or may be a solid preparation or a liquid preparation using a commonly used carrier for preparation. Good. The Peyer's patch activator thus obtained can be stored in liquid or powder form. The storage is preferably refrigerated, especially when it is liquid.

In the pharmaceutical composition of the present invention, the sesame polysaccharide of the present invention may be contained as the only active ingredient, or may be contained together with other active ingredients.

According to the present invention, a novel Peyer's patch activator is provided. According to the present invention, an agent containing a polysaccharide obtained from sesame seeds as an active ingredient is administered to a mammal (for example, orally) to give, for example, a cytokine in Peyer's patches (for example, a bone-associated cell growth promoting factor). IL-2 and the like) can be produced. Thereby, for example, it is possible to enhance the immune mechanism related to biological defense and immune tolerance through the enhancement of intestinal immunity.

Further, the Peyer's patch activator of the present invention is derived from sesame seeds that humans have eaten since ancient times, so that it does not harm the health of humans or industrial animals such as livestock or poultry that humans eat. , Safe.

It is a graph which shows the result of the Peyer's board activation action test in Test Example 1. It is a graph which shows the result of the Peyer's board activation action test in Test Example 1. It is a graph which shows the result of the Peyer's board activation action test in Test Example 1. 3 is a graph showing an elution pattern in column chromatography of heteroglycan (fraction A1) performed in Production Example 1. 3 is a graph showing an elution pattern in column chromatography of heteroglycan (fraction B1) performed in Production Example 1. It is a graph which shows the elution pattern in the column chromatography of the heteroglycan (fraction C1) performed in Production Example 1. 7 is a graph showing an elution pattern in column chromatography of heteroglycan (fraction D1) performed in Production Example 2. 3 is an HPSEC chart showing the molecular weight distribution of the heteroglycan fraction in Test Example 2. 9 is an HPSEC chart showing the molecular weight distribution of the heteroglycan fraction (fraction D1) obtained from the non-roasted sesame seed sesame oil squeezed meal in Test Example 2. 3 is a graph showing the results of Peyer's patch activation action test of the heteroglycan fraction in Example 1. 3 is a graph showing the results of Peyer's patch activation action test of the heteroglycan fraction in Example 1. 5 is a graph showing the results of Peyer's patch activation test by oral administration to BALB/c mice in Example 2.

[Method for producing polysaccharide]

<Sesame seeds>
Sesame seeds are roughly classified into four types, white sesame seeds, tea sesame seeds, black sesame seeds, and gold sesame seeds, depending on the color of the seed coat, but any type of sesame seeds can be used in the present invention. The origin of sesame is Africa, but it is now cultivated all over the world. Main producing areas are Myanmar, India, China, Mexico, Paraguay, Bolivia, etc. in addition to Africa, and sesame seeds from any producing area can be used in the present invention.

<Roast>
Roasting is the heating of sesame seeds for the purpose of enhancing the flavor and flavor by producing the color and aroma peculiar to sesame oil. The temperature of the seeds to be heated is generally 160 to 210° C., but the temperature is not limited to this.

<Sesame oil cake>
The squeezed lees remaining after squeezing sesame oil from sesame seeds (sesame oil squeezed lees) are obtained by the following method. A squeezing method and an extraction method are used to produce sesame oil. The former is a method of producing sesame oil by pressing raw sesame or roasted sesame seeds. On the other hand, the extraction method is a method in which the oil content of the seeds is extracted by treating the sesame seeds with an organic solvent such as hexane. Since the sesame oil squeezed cake obtained by any of these methods also contains the Peyer's patch activated polysaccharide, the sesame oil squeezed cake in the present specification can be produced by any production method.

<Aqueous solvent extract>
The aqueous solvent extract of roasted or non-roasted sesame or sesame oil cake can be extracted with water at room temperature or cold temperature. More preferably, roasted or non-roasted sesame or sesame oil squeezed lees can be more efficiently obtained by infusing in open water until the amount becomes about half. Therefore, the aqueous solvent extract in the present specification is a cold water or hot water extract. An insoluble solid component can be removed by filtering the decoction using filter paper or the like, if necessary. In addition, if necessary, it may be further concentrated under reduced pressure before use.

<Proteolytic enzyme treatment>
The proteolytic enzyme treatment of the aqueous solvent extract or ethanol-precipitated fraction of the present specification is carried out by adding a proteolytic enzyme (for example, actinase E) in a buffer solution (pH 8.0) at 37°C for 1 to 7 days. It can be performed by incubating. If necessary, the sample after the proteolytic enzyme treatment may be further heated in a boiling water bath for 5 minutes, then rapidly cooled in ice water, and dialyzed with purified water (3 to 17 days) for purification. ..

<Ethanol precipitation fraction>
The ethanol-precipitated fraction of the present invention can be obtained by ethanol-precipitating an aqueous solvent extract of roasted or non-roasted sesame seeds or sesame oil cake, or a proteolytic enzyme treatment solution thereof. The obtained precipitate may be further purified by removing low molecular weight substances by dialysis and/or membrane treatment, if necessary. The ethanol-precipitated fraction may be treated with the above-mentioned proteolytic enzyme and, if necessary, purified by dialysis and/or membrane treatment to further reduce the protein content. The yield of the ethanol-precipitated fraction is usually about 5% by mass based on the sesame oil squeezed lees, for example, when the sesame oil squeezed lees of sesame seeds are used. In addition to polysaccharides, the ethanol-precipitated fraction contains components such as polyphenols such as lignin and salts.

<Dialysis/Membrane treatment>
For dialysis, a dialysis membrane generally used for desalting can be used. For the membrane treatment, a UF membrane used for desalting and removing monosaccharides can be used.

<Polysaccharide obtained from roasted or non-roasted sesame seeds or sesame oil cake>
A polysaccharide obtained from roasted or non-roasted sesame seeds or sesame oil cake (hereinafter referred to as "sesame polysaccharide") is obtained by producing (purifying) by the following method.
For the production of sesame polysaccharide, an aqueous solvent extract of sesame seeds or sesame oil squeezed lees, a proteolytic treatment solution thereof, or an ethanol precipitate fraction thereof can be used, but the ethanol precipitate fraction is preferably used. Therefore, the method for purifying sesame polysaccharides will be described below by taking the case of using the ethanol-precipitated fraction as an example.

The ethanol precipitation fraction obtained by the above method is passed through a column packed with an anion exchange resin as a carrier to adsorb the components. The anion exchange resin is preferably a weak ion exchange resin. For example, a resin having a DEAE group can be used. DEAE-Sepharose Fast Flow (FF) etc. can be mentioned as a specific example of the column provided with such a carrier. These resin carriers are preferably equilibrated with water. For example, in the case of DEAE-Sepharose FF, the DEAE group is activated by immersing it in 2M sodium chloride (the chloride ion of the counter ion is bound to the tertiary amino group covalently bonded to Sepharose, that is, the Cl ⁻ type is formed). Then, the remaining sodium chloride is removed by washing with water, and then suspended in water (equilibrated with water).

Next, the components adsorbed on the carrier are eluted by letting an elution solvent pass through so that the ionic strength gradually increases. As the elution solvent, it is preferable to use an aqueous solution in which an anion salt such as NaCl, NH ₄ HCO ₃ or HCOONH ₄ is dissolved.

Sesame polysaccharide can be obtained as a heteroglycan fraction from the elution fraction obtained by elution with a low to high ionic strength elution solvent. As the low ionic strength elution solvent, for example, when Cl ⁻ is used as the anion, an elution solvent having a concentration of the anion of 50 to 100 mM can be used, and an elution solvent of 70 mM to 80 mM is more preferable. .. Further, as the medium ionic strength elution solvent, an elution solvent having a concentration of the anion of 150 mM to 250 mM can be used, and an elution solvent of 150 mM to 210 mM is more preferable. Further, as the high ionic strength elution solvent, an elution solvent having a concentration of the anion of 250 mM to 350 mM can be used, and an elution solvent of 290 mM to 310 mM is more preferable. For example, a heteroglycan fraction containing sesame polysaccharides derived from roasted sesame seeds is eluted with an elution fraction (fraction A ) obtained by eluting with an elution solvent of 70 mM to 80 mM and an elution solvent of 190 mM to 210 mM. The elution fraction (fraction B ) thus obtained and the elution fraction (fraction C ) obtained by elution with an elution solvent of 290 mM to 310 mM may be purified and then used in combination. it can. As the sesame polysaccharide fraction derived from non-roasted sesame seeds, a fraction (fraction D ) obtained by an elution method with an elution solvent of 140 to 160 mM can be used.

Alternatively, an anion exchanger is used to first elute with water to remove the neutral components, and then with an elution solvent having an ionic strength of 310 mM or more to elute, so that the sesame seeds are collected as an acidic fraction. Heteroglycan fractions containing polysaccharides can also be prepared.

The obtained elution fraction is dialyzed using a dialysis membrane (for example, Visking tube, MWCO 12,000 to 14,000) as needed, and then the dialysis internal solution is freeze-dried before the next step. You may use.

In addition, the sesame polysaccharide of the present invention may be obtained as a heteroglycan fraction obtained by fractionating the above-mentioned elution fraction by molecular weight using a gel filtration column, if necessary. For example, with respect to the fraction A , a gel having a fraction molecular weight of 2×10 ³ to 4×10 ⁵ Da is used, and a peak showing a peak molecular weight of 61,000 to 26,000 is flowed out by gel filtration HPLC analysis. Fractions (fraction A 1) may be obtained. In addition, as for the fraction B, a fraction in which a peak showing a peak molecular weight of 33,000 was analyzed by gel filtration HPLC using a gel filtration column having a molecular weight cutoff of 2×10 ³ to 4×10 ⁵ Da (fraction B 1) may be recovered. For the fraction C , for example, a gel filtration column having a molecular weight cutoff of 2×10 ³ to 4×10 ⁵ Da is used, and a fraction in which a peak having a peak molecular weight of 220,000 is analyzed by gel filtration HPLC. The fraction (fraction C 1) in which the peak having a peak molecular weight of 16,000 is eluted may be recovered. Alternatively, an elution pattern was prepared based on sugar, uronic acid, and UV absorption at 280 nm for the elution fraction during gel filtration, and the molecular weight fractions of the above-mentioned fractions A and B (fractions A 1 and Fraction B 1) may be collected. Further, the molecular weight fraction (fraction C 1) of the above-mentioned fraction C may be collected by the same operation. Further, regarding the fraction D , for example, a peak showing a peak molecular weight of 2,000,000 to 300,000 by gel filtration HPLC analysis using a gel filtration column having a molecular weight cutoff of 2×10 ³ to 4×10 ⁵ Da. The fraction (flow-out limit elution fraction, fraction D 1) may be collected.

As the gel filtration column having a molecular weight cut-off of 2×10 ³ to 4×10 ⁵ Da, for example, Sephacryl S-300, Superose 12 and the like can be used.

As the sesame polysaccharide of the present invention, each fraction obtained by gel filtration from fraction A, fraction B, fraction C, and fraction D (fraction A 1, fraction B 1, fraction C) 1 and fraction D 1) can be used individually or in combination. The obtained heteroglycan fraction may be dialyzed by a conventional method and then freeze-dried.

[Pharmaceutical composition, Peyer's patch activator, food composition, feed]
When the pharmaceutical composition, Peyer's patch activator, food composition, and feed of the present invention are solid preparations, for example, sesame polysaccharide of the present invention as an active ingredient, or sesame seeds or sesame oil squeezed dextrin, corn starch or defatted rice bran May be mixed and prepared. Methods for formulation of these formulations are known.

The administration timing of the pharmaceutical composition/Peyer's patch activator of the present invention is not particularly limited. The dose of the Peyer's patch activator of the present invention is not particularly limited because it varies depending on the formulation form, the type of target human or non-human animal, health condition, age and degree of growth. For example, it is possible to administer 1 to 1,000 mg/kg body weight per day in terms of the above-mentioned polysaccharide as the active ingredient.

The dosage form of the pharmaceutical composition/Peyer's patch activator of the present invention is not particularly limited, but it can be administered by oral, enteral and nasal methods. It can also be administered in a form that allows easy storage in the oral cavity (including chewing gum and the like).

The subject to be administered is not particularly limited as long as it is a mammal, and includes, for example, humans, dogs, cats, rabbits, rats, mice, cows, pigs, sheep, goats, horses, and preferably humans.

In one aspect, the present invention relates to the use of the sesame polysaccharide of the present invention for producing a Peyer's patch activator or a therapeutic or prophylactic agent for a disease treated or prevented by activation of Peyer's patches. In another aspect, the present invention is used as a sesame polysaccharide of the present invention for activation of Peyer's patches, or as a medicine (preferably a therapeutic or prophylactic agent for a disease treated or prevented by activation of Peyer's patches). To the sesame polysaccharide of the present invention. In yet another aspect, the invention is treated or prevented by a method of activating Peyer's patches or activation of Peyer's patches comprising administering to a subject in need thereof an effective amount of the sesame polysaccharide of the invention. The present invention relates to a method for treating or preventing a disease. Alternatively, the invention relates to the use of the sesame polysaccharides of the invention for activating Peyer's patches.

Hereinafter, the present invention will be described more specifically based on Examples. However, the present invention is not limited to these examples. All documents cited throughout this application are incorporated into this application as they are by reference.

<Statistical test>
All results in the examples are mean±S.D. in in vitro experiments. D. , In vivo test means ±S. E. It showed with. The statistically significant difference between the control and test samples was tested by Fisher's PLSD after the ANOVA test.

[Test Example 1: Peyer's patch activation test by ethanol precipitation fraction of sesame seed-derived aqueous solvent extract]
(1) Preparation of ethanol precipitation fraction from roasted sesame seeds 100 mL of purified water of white sesame seeds (thin sesame seeds, dark sesame seeds) and black sesame seeds sesame oil meal (each 10 g) treated under different roasting conditions It was brewed with an open flame until about half the amount was used. The infusion was subjected to suction filtration using a glass fiber filter paper, concentrated under reduced pressure to 50 mL, 4 volumes of ethanol (200 mL) was added with stirring, and the mixture was stirred overnight at room temperature. The obtained precipitate was separated by centrifugation (6,000 rpm, 4°C, 30 minutes), and then dialyzed against purified water for 7 days. An insoluble matter in the obtained dialyzed solution was removed by centrifugation (6,000 rpm, 4°C, 30 minutes), and then freeze-dried to obtain an ethanol precipitate fraction. 0.42 g (yield: 4.2%) from thin sesame seeds, 0.38 g (yield: 3.8%) from thick sesame seeds, and 0.4 g (yield: 4.0) from black sesame seeds. %) ethanol precipitated fractions were obtained.

(2) Preparation of ethanol precipitation fraction from non-roasted sesame seeds Purified water (2L) was added to non-roasted white sesame seed sesame oil squeezed lees (100 g), and the mixture was decocted with an open flame until the volume became half. The residue was removed by suction filtration. With respect to the residue, a decoction was prepared again using purified water (1 L) by the same method, and then combined with the above decoction, and concentrated under reduced pressure to 500 mL. To this infusion, four volumes of ethanol was added with stirring, and the mixture was stirred overnight at room temperature. The resulting precipitate was separated by centrifugation (6,000 rpm, 4°C, 30 minutes), and the obtained precipitate was dialyzed against running water and purified water (7 days), and then the insoluble matter in the non-dialysable fraction was centrifuged. It was removed by separation (6,000 rpm, 4°C, 30 minutes), and freeze-dried to obtain an ethanol precipitate fraction (yield: 2.82 g, yield: 2.8%).

(3) Proteolytic enzyme digestion of ethanol-precipitated fraction from non-roasted sesame seeds Ethanol-precipitated fraction (103 mg) from the non-roasted sesame seed sesame oil cake obtained in (2) above was added to 10 mM potassium chloride and The enzyme was dissolved in a 50 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 0.05% sodium azide, 5 mg of a protein-degrading enzyme (Actinase E, Kaken Pharmaceutical Co., Ltd.) was added, and the mixture was incubated at 37°C for 3 days. Digested. The digested liquid was heated in a boiling water bath for 5 minutes, then rapidly cooled in ice water, and dialyzed with purified water (7 days). The actinase E-treated ethanol-precipitated fraction (yield: 32.7 mg, yield: 31.5%) was obtained by freeze-drying the non-dialysable fraction.

(4) Preparation of sugar chain decomposition ethanol precipitation fraction Ethanol precipitation fraction (20 mg) prepared from sesame oil squeezed lees of roasted sesame seeds (thin sesame seeds) obtained in (1) above was added to a 50 mM acetate buffer solution (pH 4). 0.5, 12 mL), the acetate buffer (4 mL) containing 100 mM sodium periodate was added, and the mixture was stirred at 4° C. in the dark for 96 hours to perform periodate oxidation. Ethylene glycol (0.4 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour to decompose excess sodium periodate. Then, the reaction mixture was dialyzed against purified water for 2 days, and the dialyzed solution was concentrated under reduced pressure to about half the volume. Furthermore, sodium borohydride (250 mg) was added, and the mixture was stirred in the room for 12 hours, and then acetic acid was added dropwise to neutralize. Further, this reaction solution was dialyzed with purified water for 7 days, and the dialyzed solution was freeze-dried to obtain an ethanol-precipitated fraction for glycolysis. (Yield: 11.4 mg, yield: 56.5%).

(5) Preparation of Delignified Ethanol Precipitate Fraction An ethanol precipitate fraction (20 mg) prepared from sesame oil cake of roasted sesame seeds (thin sesame seeds) obtained in (1) above was added to a 4% acetic acid aqueous solution (20 mL). ), chlorous acid (100 mg) was added, and the mixture was stirred in a water bath at 70°C until the color tone of the solution changed (about 10 minutes). The reaction solution was neutralized with 3M sodium hydroxide under ice cooling, and then this reaction solution was dialyzed with purified water for 4 days, and the non-dialysable fraction was lyophilized to remove the delignified ethanol-precipitated fraction. (Yield: 12.7 mg, Yield: 63.5%).

(6) Preparation and Culture of Mouse Peyer's Patch Cells C3H/HeJ mice were euthanized with isoflurane (escaine inhalation anesthetic, Mylan Pharmaceutical Co., Ltd.), and Peyer's patches were excised from the small intestine using ophthalmic scissors. This Peyer's patch is placed in a sterilized petri dish containing RPMI1640 medium (2 mL) containing ice-cooled 5% fetal bovine serum (FBS) and crushed on a stainless steel mesh (200 mesh) using a rubber rubber portion of the inner cylinder of a disposable syringe of 5 mL. This released Peyer's patch cells. The suspension of free cells was transferred to a 50 mL Falcon tube and vortexed briefly. Then, the cell suspension was filtered through a stainless mesh (150 mesh), centrifuged (1,500 rpm, 4° C., 7 minutes), and the medium was decanted to obtain Peyer's patch cells. The cells thus obtained were washed by repeating the same operation four times using FBS-containing RPMI1640 medium (10 mL), and then filtered with a stainless mesh (200 mesh). The cell number was counted using a cell counter using this cell suspension (20 μL), and then a Peyer's patch cell suspension of 1-2×10 ⁶ cells/mL was prepared using FBS-containing RPMI1640 medium.

The resulting Peyer's patch cell suspension (180 μL/well) and ethanol-precipitated fraction solution (polysaccharide sample solution) (20 μL/well, final concentration of ethanol-precipitated fraction: -100 μg/mL) were placed in a 96-well culture plate (3072). , FALCON), and cultured at 37° C. for 2 to 6 days under 5% CO ₂ -95% air. The culture supernatant was transferred to another 96-well culture plate and stored at -20°C until use. The culture supernatant obtained by adding water for injection (20 μL/well) instead of the ethanol-precipitated fraction solution and culturing was used as a control.

(7) Preparation of mouse bone marrow cells C3H/HeJ mice (7-week-old, female) were euthanized with isoflurane, the femurs were excised, and FBS-containing RPMI1640 medium (5 mL was used using a 5 mL syringe equipped with a 23 G needle. The bone marrow cells were collected by pushing out from the femur. The obtained bone marrow cells were dispersed by a vortex mixer, filtered with a stainless mesh (200 mesh), and then centrifuged (1,200 rpm, 4° C., 7 minutes) to collect bone marrow cells. The same operation was repeated 3 times, and after washing the cells, the bone marrow cells were suspended in RPMI1640 medium containing FBS (10 mL), the number of cells was counted with a cell counter, and the bone marrow cell suspension (5 x 10 ⁵ cells/mL) was prepared.

(8) Peyer's patch activating action test In a 96-well culture plate, Peyer's patch cell culture supernatant (50 μL/well), bone marrow cell suspension (5×10 ⁵ cells/mL, 100 μL/well), and FBS-containing RPMI1640 medium (50 μL/well) was added, and the mixture was cultured at 37° C. under 5% CO ₂ -95% air for 6 days. Alamar Blue (20 μL/well, Biosource) was added to the cultivated bone marrow cell culture suspension, and after culturing at 37° C. for 6 to 24 hours in 5% CO ₂ -95% air, the amount of the generated fluorescent substance was measured. Measurement was performed with a reader (Infinite M200, Tecan, excitation wavelength: 544 nm, measurement wavelength: 590 nm), and the number of proliferating bone marrow cells as the obtained relative fluorescence intensity was taken as the amount of myeloid cell growth promoting factor.

(9) Results FIG. 1 shows the results of the Peyer's patch activation test of ethanol-precipitated fractions prepared from sesame oil squeezed meal of roasted sesame seeds. The amount of myeloid cell growth promoting factor was shown as the activation effect of Peyer's patches. The Peyer's patch activating effect was observed in the ethanol-precipitated fractions derived from the sesame oil squeezed lees of white sesame seeds (thin sesame seeds and thick sesame seeds) treated under different roasting conditions. In addition, the ethanol-precipitated fraction prepared from sesame oil squeezed lees of non-roasted black sesame seeds also had the same Peyer's patch activation effect as the ethanol-precipitated fraction derived from the roasted white sesame seeds (Fig. 1).

Furthermore, the ethanol-precipitated fraction prepared from sesame oil squeezed lees of white sesame which has not been subjected to roasting treatment was confirmed to have a Peyer's patch activating effect by reducing the protein content with a proteolytic enzyme (actinase E), No significant Peyer's patch activating effect was observed in the ethanol-precipitated fraction not treated with actinase E (Fig. 2).

On the other hand, in the ethanol-precipitated fraction prepared from sesame oil squeezed lees of roasted thin-mouth sesame, its sugar chain was decomposed by periodate oxidation (“glycolysis” in FIG. 3). The activity was drastically reduced to a level not significantly different from the control, as compared with the fraction (FIG. 3). Further, the activity of the same ethanol-precipitated fraction that was delignified by chlorite treatment was lower than that of the untreated ethanol-precipitated fraction, but significant activity was observed (in FIG. 3, "delignification"). ). As a result, it was suggested that the polysaccharide-containing component of the ethanol-precipitated fraction in the sesame seed-derived extract contained a substance having a Peyer's patch activating effect.

[Production Example 1: Preparation of heteroglycan fraction from roasted sesame seeds]
(1) Preparation of Ethanol Precipitated Fraction 200 g of sesame oil squeezed lees of roasted sesame seeds (thin sesame seeds) were brewed with purified water (4 L) until the liquid volume became about half. The residue was again decocted with the same amount of purified water, the extracts were combined and separated by centrifugation (6,000 rpm, 4° C., 30 minutes), suction filtered, and the filtrates were combined and concentrated under reduced pressure to 500 mL. .. To this extract, 4-fold amount of ethanol was added with stirring, and the mixture was stirred at room temperature overnight. The generated precipitate was separated by centrifugation (6,000 rpm, 4°C, 30 minutes), redissolved in water, and then dialyzed with tap water for 4 days and then with purified water for 7 days. The obtained dialyzed solution was centrifuged (6,000 rpm, 4°C, 30 minutes) to remove insoluble matter, and then freeze-dried to obtain an ethanol-precipitated fraction (yield: 10.52 g , Yield: 5.3%).

(2) Preparation of Heteroglycan Fraction A thin-mouth sesame ethanol precipitation fraction (2.0 g) was dissolved in 300 mL of purified water and then added to a DEAE-Sepharose FF (Cl ⁻ type) column (5.0×38.5 cm). The extract was washed with purified water to remove the neutral polysaccharide fraction. The adsorbed fractions were 0.0776 M saline (1.7 L, fraction A), 0.2067 M saline (1.4 L, fraction B), and then 0.3035 M saline (0.9 L, fraction C). Were sequentially eluted with to obtain an adsorption fraction. Each fraction was dialyzed using a dialysis membrane (rejection limit: 12,000-14,000), and the dialyzed solution was freeze-dried to give fraction A (yield: 135.8 mg, yield: 6.79%). , Fraction B (yield: 437.23 mg, yield: 21.9%) and Fraction C (yield: 174.75 mg, yield: 8.7%) were obtained.

(3) Purification of heteroglycan The obtained fraction A was fractionated by Sephacryl S-300 (2.6 id×90 cm) equilibrated with 0.2 M NaCl solution, and "fraction A1" in FIG. The fraction indicated by was collected as the main fraction (yield: 0.11 g, yield: 0.5%). Further, the fraction B obtained in (2) was fractionated by Sephacryl S-300 (2.6 id×90 cm) equilibrated with a 0.2 M NaCl solution, and the fraction B was designated as “fraction B1” in FIG. The indicated fraction was collected as the main fraction (yield: 0.14 g, yield: 6.9%). Similarly, the fraction C obtained in (2) was fractionated by Sephacryl S-300 (2.6 id×90 cm) equilibrated with a 0.2 M NaCl solution, and “fraction C1” in FIG. The fraction shown by was collected as the main fraction excluding the Vo elution fraction (yield: 0.13 g, yield: 6.6%).

[Production Example 2: Preparation of heteroglycan fraction from non-roasted sesame seeds]
(1) Preparation of ethanol precipitation fraction Non-roasted sesame oil squeezed lees (100 g) of white sesame seeds were brewed with purified water (2 L) until the liquid volume became about half, and the extract was concentrated under reduced pressure to 500 mL. Then, 4-fold amount of ethanol (2 L) was added to the extract while stirring, and the mixture was stirred at room temperature overnight. The generated precipitate was separated by centrifugation (6,000 rpm, 4° C., 30 minutes), and dialyzed with purified water for 7 days. The obtained dialysate was concentrated and freeze-dried to obtain an ethanol precipitation fraction (yield: 2.8 g, yield: 2.8%) derived from non-roasted white sesame seed sesame oil squeezed lees.

The ethanol-precipitated fraction (2.82 g) derived from the non-roasted white sesame seed sesame oil squeezed lees was dissolved in 0.05% NaN ₃ /10 mM CaCl ₂ -containing 50 mM Tris-HCl (pH 8.0) (282 mL), Actinase E (141 mg, Kaken Pharmaceutical Co., Ltd.) was added, and enzyme digestion was performed at 37° C. for 3 days. The enzyme reaction solution was neutralized with concentrated hydrochloric acid and then heated in a boiling water bath for 5 minutes to deactivate the enzyme. Further, it was dialyzed with purified water for 7 days and freeze-dried to obtain a deproteinized ethanol precipitate fraction (yield: 751.1 mg, yield: 26.6%).

(2) After dissolving deproteinized ethanol precipitated fraction of the non-roasted from white sesame seeds sesame lees obtained preparation of hetero-glycan fraction (133.52Mg) with purified water 50mL, DEAE-Sepharose FF (Cl - Type) column (2.4×27 cm) to obtain an unadsorbed fraction (yield: 19.0 mg, yield: 14.3%), and then the adsorbed fraction was added with 0.1522 M NaCl (2 L). After elution with water and dialysis, the dialyzed solution was freeze-dried to obtain Fraction D (yield: 82.9 mg, yield: 62.1%).

(3) Purification of heteroglycan Fraction D (82.9 mg) obtained from the deproteinized ethanol-precipitated fraction derived from non-roasted white sesame seeds was equilibrated with 0.2 M NaCl solution Sephacryl S-300 (2. 6 i.d.×90 cm), and a fraction indicated by “fraction D1” in FIG. 7 was obtained as a Vo-eluting fraction which is a main fraction (yield: 40.2 mg, yield: 55). .4%).

[Test Example 2: Analysis of heteroglycan]
(1) Measurement of molecular weight distribution The molecular weight distributions of the heteroglycan fractions obtained in Production Example 1 and Production Example 2 were measured using Asahi-pak GS520HQ and Asahi-pak GS320HQ (each 0.75 id x 30 cm) (Showa era). (High-performance gel filtration chromatography (HPSEC)) using an Asahi-pak GS620HQ (0.75 id×30 cm) coupling column. The molecular weight is a calibration curve of molecular weight vs. retention time coefficient (Kav) from the retention time of standard polysaccharides (pullulan P-1600, P-800, 400, 200, 100, 50, 20, 10 and 5, Showa Denko) in HPSEC. Was calculated and calculated from the retention time of the test sample.
The conditions of HPSEC are as follows.
Liquid sending device: JASCO PV-980 (JASCO Corporation)
Detector; Shodex RI SE-62 (Showa Denko) (sensitivity: x2)
Eluate; 0.2M NaCl (0.8-1.0 mL/min)

(2) Colorimetric determination The total sugar amount was measured using the phenol-H ₂ SO ₄ method, the uronic acid amount was measured using the m-hydroxybiphenyl method, and the protein amount was measured using the Bradford method. As a standard, galactose was used for the phenol-H ₂ SO ₄ method, galacturonic acid was used for the m-hydroxybiphenyl method, and bovine gamma globulin (Bio-Rad) was used for the Bradford method.

(3) Measurement of β-D-(1→3,6)-galactan content β-D-(1→3,6)-galactan content in heteroglycans (fractions A1, B1 and C1) was Holst and Clarke. It was measured using the one-way gel diffusion method using the β-D-glucosyl-yalibu antigen reported by K.K.
An agarose gel (7 mL) containing 1% agarose, 0.15 M sodium chloride, 0.02% sodium azide, and 10 μg/mL β-glucosyl-yalib antigen (Megazyme) was placed on a 7.6×5.1 cm slide glass. After overlaying on top, it was cooled and solidified in a moisture chamber. After making a hole with a diameter of about 2 mm in the gel, an aqueous solution of heteglycan sample (1 mg/mL, 10 μL) was added, and the mixture was incubated overnight at room temperature in a moisture chamber. The gel was dried using filter paper and the diameter of the resulting red settling ring was measured. Acacia arabinogalactan (10 μg, Megazyme) was used as standard β-D-(1→3,6)-galactan, and the same operation was performed to obtain β-D-(1→3,6)-galactan (10 μg). The diameter of the red sedimentation ring formed was measured. The calibration curve was prepared from the square value of the diameter of the red sedimentation ring formed by the standard β-D-(1→3,6)-galactan and the β-D-(1→3,6)-galactan amount (10 μg). The amount of β-D-(1→3,6)-galactan in the test heteroglycan was calculated.

(4) Glycolytic enzyme digestion (exo-β-D-(1→3)-galactanase digestion)
Heteroglycan (fractions A1, B1 or C1) (300 μg) in a 25 mM acetate buffer (pH 4.6) solution (300 μL) together with exo-α-L-arabinofuranosidase (2 μL, Megazyme) together with the method of Trumuraya et al. (Tsumuraya, Y., Mochizuki, N., Hashimoto, Y., Kovak, P., 1990. Purification of an exo-β-(1→3)-D-galactanase of porcelain operase porterus eructus iruce puerus erupterus erupterus erupterus irruis teruis et al. exo-β-D-(1→3)-galactanase (2 μL) purified from dolicerase (Kyowa Hakko Bio) by arabinogalactan-protein. J. Biol. Chem. 265, 7207-7215) at 37°C. The enzyme reaction was performed under the condition of 24 hours. The digest was heat-treated in a boiling water bath for 30 seconds to inactivate the enzyme and stored at -20°C until use.

(Endo-β-D-(1→4)-galactanase digestion)
Endo-β-D-(1→4)-galactanase (2 μL, Megazyme) was added to a 25 mM acetate buffer (pH 4.6) solution (300 μL) of heteroglycan (300 μg), and the conditions were 37° C. and 24 hours. The enzyme reaction was carried out at. The digest was heat-treated in a boiling water bath for 30 seconds to inactivate the enzyme and stored at -20°C until use.

(5) Constituent saccharide analysis Constituent saccharide analysis was performed by the TMS methyl glycoside method.
A standard mixture of monosaccharides (Glc, Gal, GlcA, GalA, Ara, Fuc, Xyl, Man, Rha, 5 μg each) and a test sample (50-100 μg each) were dispensed into a 13 mm screw cap test tube, and further, myo. -Inositol (internal standard: 20 μg) was added. A 1 M HCl-MeOH solution (100 to 300 μL, Wako Pure Chemical Industries, Ltd.) was added to each test tube, and methanolysis (80° C., 15 hours) was performed under sealing. After adding tert-BuOH (5 μL) to the reaction solution and distilling off the solvent under a nitrogen stream (40° C.), Tri-Sil reagent (100 μL, Pierce) was added and the reaction was carried out under sealed condition (80° C., 20 minutes). .. After distilling off the reagent under a nitrogen stream (40° C.), hexane (2 mL) was added to the reaction product and sonicated for several seconds to extract the TMS derivative. The insoluble matter in the extract was removed by centrifugation (2,000 rpm, 4°C, 5 minutes), the solvent was distilled off under a nitrogen stream (40°C), and the obtained hexane solution of the TMS derivative was subjected to gas chromatography. It was analyzed by (GLC). Each monosaccharide derivative was identified by comparison with the retention time of the standard derivative, and the content ratio (mol.%) was determined from the peak area and the response coefficient of each monosaccharide derivative obtained in each experiment to the FID detector. It was calculated.
The conditions of GLC are as follows.
Equipment: HP5890 Series II gas chromatograph (Hewlett Packard)
Column: DB-1 capillary column (0.25 mm id×30 m, liquid film thickness 0.25 μm, J&W Scientific Inc.)
Carrier gas: He (total flow rate: 80 mL/min, column inlet pressure: 21 psi, gas purity: 99.9999%)
Inlet temperature: 250℃
Detector temperature: 280℃
Oven temperature program: 60°C (1 minute), 60°C → 170°C (30°C/min), 170°C → 190°C (1°C/min), 190°C → 300°C (30°C/min), 300°C ( 5 minutes)

(6) Methylation analysis Methylation analysis for sugar binding mode analysis was performed according to a modified method of the Hakomori method and Waeghe method as shown below.

(Methylation of polysaccharides using sodium methylsulfinylcarbanion)
A test sample (500 μg) was placed in a screw cap test tube (15 id×100 mm), dried under reduced pressure in a desiccator overnight, and then anhydrous dimethyl sulfoxide (dry DMSO, Sigma) was added to the test sample (15 μg) under a nitrogen stream under sealed conditions for 15 minutes. The sample was sonicated, and heated at 50 to 60° C. until the sample was completely dissolved (several hours to 24 hours). Sodium methylsulfinylcarbanion (500 μL) prepared by a conventional method was added to the sample solution, and the mixture was subjected to ultrasonic treatment for 1 hour under a nitrogen stream and then reacted for 3 hours at room temperature. After the reaction, a small amount of the reaction solution (5 to 10 μL) was used, and it was confirmed by triphenylmethane reagent (Wako Pure Chemical Industries, Ltd.) that excess methylsulfinylcarbanion remained. When the methylsulfinylcarbanion sodium was insufficient, additional methylsulfinylcarbanion sodium was added, and the above operation was repeated until an excess amount of methylsulfinylcarbanion sodium remained. After freezing the reaction mixture, CH ₃ I (iodomethane, Yanagishima Pharmaceutical Co., Ltd., special grade, 1 mL) was added, and sonication was performed for 15 minutes under a nitrogen stream under sealed conditions, and the reaction was carried out at room temperature for 4 hours or more. After completion of the reaction, CH ₃ I in the reaction solution was distilled off under reduced pressure, frozen under ice-cooling, and purified water in an amount equal to the total volume of dimethyl sulfoxide and methylsulfinylcarbanion used was added to decompose remaining methylsulfinylcarbanion. Then, the reaction was stopped. Further, saturated sodium thiosulfate (about 250 μL-) was added to the reaction solution until the yellow color disappeared.

(Recovery of fully methylated polysaccharide)
A Sep-pak C18 cartridge (1 mL, Waters Associate Inc.) was washed with distilled ethanol (10 mL×4 times) and then water (2 mL×3 times), and then the above methylation reaction mixture was passed through this cartridge. The methylated polysaccharide was adsorbed on the cartridge. The cartridge was washed with 50% dimethyl sulfoxide (2 mL x 5 times) and then with water (2 mL x 5 times), and the methylated polysaccharide was eluted with distilled ethanol (2 mL x 3 times) and then completely dried by vacuum drying. A methylated polysaccharide was obtained.

(Reduction of carboxyl group of uronic acid in methylated polysaccharide)
The carboxyl methyl ester group of the uronic acid residue in the completely methylated polysaccharide was reduced to a deuterated primary alcohol by the method shown below. That is, the completely methylated polysaccharide sample was dissolved in 95% ethanol (0.21 mL) and tetrahydrofuran (THF, 0.51 mL), sodium deuteride borohydride (NaBD ₄ , 1.8 mg) was added, and the mixture was mixed for 18 hours. The reaction was carried out at room temperature as above and further heated at 70° C. for 1 hour to reduce the carboxymethyl ester group. The reaction solution was neutralized with acetic acid, and the reaction was stopped by adding 7 to 8 drops of acetic acid. After the reaction solution was dried under reduced pressure, in order to remove the generated boric acid, the operation of adding distilled methanol (1 mL) to the reaction product and drying under reduced pressure was repeated at least 4 times. With respect to a 50% dimethyl sulfoxide solution of this reaction mixture, a carboxyl-reduced fully methylated polysaccharide was recovered by the same operation as the method described in the section (Recovery of fully methylated polysaccharide).

(Derivatization and analysis of fully methylated polysaccharide to partially methylated alditol acetate)
The resulting carboxyl-reduced fully methylated polysaccharide is hydrolyzed by heating in a screw cap test tube (15 id×100 mm) with 2M trifluoroacetic acid (TFA, 1 mL) at 121° C. for 1 hour under sealing. went. After completion of the reaction, the reaction solution cooled to room temperature was dried under reduced pressure and further dried under reduced pressure for 30 minutes with a desiccator to remove residual trifluoroacetic acid. The obtained hydrolyzate was dissolved in 95% ethanol (distillation, 1 mL), 25% aqueous ammonia was added 7 to 8 drops to make it alkaline, and then excess sodium borohydride (NaBH ₄ ) was added at room temperature. The reaction was carried out for 4 hours or more. An acetic acid solution was added dropwise to the reaction solution to decompose the remaining sodium borohydride, 7 to 8 drops were further added, and the solvent was evaporated under reduced pressure to dryness. Methanol (1 mL) was added to the reaction product, and the operation of drying under reduced pressure was repeated 4 times to remove the generated boric acid. The reaction product was dried in a desiccator under reduced pressure for 1 hour, acetic anhydride was added, and the mixture was heated at 121° C. for 3 hours in a sealed state to perform acetylation. After leaving the reaction solution to reach room temperature, toluene (1 mL) was added and mixed, and acetic anhydride was removed at 40° C. under an air stream. Water (1 mL) and chloroform (CHCl ₃ , 2 mL) were added to the reaction product, and the mixture was subjected to liquid-liquid distribution, centrifuged (4° C., 2,500 rpm, 5 minutes), and then the upper aqueous layer was removed by suction. Further, the chloroform layer was washed with water (1 mL) about 4 to 5 times, and then the chloroform was distilled off under reduced pressure to obtain a partially methylated alditol acetate derivative. This derivative was analyzed using gas chromatography (GLC) and gas chromatography/mass spectrometry (GLC-MS) under the conditions shown below. The methylated alditol acetate was identified by comparison with the fragment ion of the authentic sample and comparison with the relative retention time for 2,3,4,6-tetra-OMe-1,5-di-OAc-galactitol. The constituent molar ratio (mol.%) of the methylated sugar was determined by the peak area and the response coefficient to the hydrogen flame ion detector (FID).

GLC:
Device: HP5890 SeriesII gas chromatogram (Hewlett Packard)
Capillary column: SP-2380 capillary column (0.25 mm id×30 m, liquid film thickness 0.25 μm, SPELCO/ALDRICH)
Carrier gas: He (total flow rate: 80 mL/min, column inlet pressure: 10 psi, gas purity: 99.9999%)
Inlet temperature: 250℃
Detector: 250℃
Oven temperature: 60°C (1 min), 60°C→150°C (30°C/min), 150°C→250°C (1.5°C/min), 250°C (1 min)

MS:
Mass spectrometer: HP5970B Mass Selective Detector (70 eV, 280° C.)

(7) Results Table 1 shows the chemical properties of the obtained heteroglycan, and Table 2 shows the sugar-binding mode of the heteroglycan. In Table 2, f indicates a furanose type and p indicates a pyranose type. The measurement results of the molecular weight distribution of heteroglycan are shown in FIG. 8 (fractions A1, B1, and C1) and FIG. 9 (fraction D1).

As shown in Table 1 and FIG. 8, the fraction A1 had a peak molecular weight of 41,000 and was distributed in the molecular weight range of 10,000 to 300,000. The fraction B1 peak molecular weight was 33,000, and the molecular weight was distributed in the range of 10,000 to 300,000. Fraction C1 had a peak molecular weight of 16,000 and a molecular weight of 10,000 to 100,000. The ratio of arabinose, rhamnose, xylose, mannose, and galactose in the constituent sugars of the fraction A1 was about 44%, 13%, 7%, 10%, and 16%, respectively. In fraction B1, the proportions of arabinose, rhamnose, xylose, galacturonic acid, and galactose in the constituent sugars were about 37%, 9%, 19%, 15%, and 10%, respectively. The proportions of arabinose, rhamnose, xylose, galacturonic acid, and galactose in the constituent sugars of fraction C1 were about 13%, 10%, 10%, 52%, and 5%, respectively. Of these heteroglycans, fractions B1 and C1 are polysaccharide fractions containing a sugar consisting of a neutral sugar and uronic acid as main components, and fraction A1 is a polysaccharide fraction containing a sugar consisting of a neutral sugar as a main component. Met. As shown in FIGS. 8 and 9, the polysaccharide fraction obtained from the non-roasted white sesame seed sesame oil cake (fraction D1, FIG. 7) is the polysaccharide fraction obtained from the roasted white sesame seed sesame oil cake. The molecular weight is larger than that of (fractions A1, B1 and C1), the peak molecular weight is distributed in the range of 500,000 to 1,600,000, and the molecular weight is distributed in the range of 300,000 to 2,000,000. It was

As shown in Table 2, the heteroglycan (fraction A1) is rich in non-reducing end arabinofuranose at about 13%, 4 or 5 linked arabinose at about 24% and 4 linked mannose at about 21%. There was a feature. Heteroglycan (fraction B1) contains about 14% arabinofuranose at the non-reducing end, about 11% arabinopyranose at the non-reducing end, and about 20% 3, 4 or 3 arabinose having 4 or 5 bonds. It was characterized by containing as much as 11% of 5-branched arabinose. Further, the heteroglycan (fraction C1) contained 4 or 5 linked arabinose in about 9%, 2 linked rhamnose in about 10%, 4 linked mannose in about 9%, and non-reducing terminal galactose in about 11%. It was characterized by containing as much as about 18% of bound galactose and about 18% of 4-bound galacturonic acid. In addition, as shown in Table 1, exo-β-D-(1→3)-galactanase derived from β-D-glucosyl-yalib antigen and endo-β-derived from Aspergillus niger. The β-D-(1→3,6)-galactan structure, which is converted by D-(1→4)-galactanase into a structure that does not react with the yarib antigen, is approximately in fraction A1 and fraction B1, respectively. It had a feature of 29% and 12% inclusion. On the other hand, this structure was not included in the fraction C1.

[Example 1: Peyer's board activation action test]
Using the heteroglycan fractions (fractions A1, B1, C1 and D) prepared in Production Example 1 and Production Example 2, the Peyer's patch activation action test was conducted in the same manner as in Test Example 1. As a positive control, the syrup-polysaccharide fraction (Kiyohara, H. et al., Phytochemistry 71, 280-293.) and the delignified ethanol-precipitated fraction obtained in Test Example 1 (5) were used.

As shown in FIG. 10, the amount of bone marrow cell growth promoting factor was shown as the Peyer's patch activating effect. Heteroglycan fractions (fractions A1, B1 and C1) obtained from roasted white sesame seeds (thin-mouth sesame) sesame oil squeezed lees were all roasted white sesame seeds (thin-mouth sesame) sesame oil squeezed ethanol with lignin removed. It showed a similar or significantly stronger Peyer's patch activating effect than the precipitate fraction. Further, FIG. 11 shows the Peyer's patch activation action test results of the fraction D derived from the non-roasted white sesame seed sesame oil squeezed lees ethanol precipitation fraction. The heteroglycan fraction (fraction D) obtained from the non-roasted white sesame seed sesame oil cake also showed a significant Peyer's patch activating effect.

[Example 2: Peyer's patch activating action by oral administration of sesame seed heteroglycan fraction]
(1) Administration of an ethanol-precipitated fraction derived from roasted white sesame seed sesame oil squeezed lees to mice BALB/c mice (7-week-old, female, Japan SLC) were treated with FTY720 aqueous solution (1.25 mg/kg/day) Oral administration was carried out for 3 days to inhibit the migration of lymphocytes from Peyer's patches. The ethanol-precipitated fraction (50 or 100 mg/kg/sday) derived from the sesame oiled meal of the roasted white sesame seeds (thin sesame seeds) obtained in Production Example 1(1) 3 hours or more after the administration of FTY720 to the above treated mice Was orally administered for 3 days. As a control group, water was orally administered to FTY720-treated mice for the same period. Four days after the start of administration, the mouse was euthanized under isoflurane (Mylan Pharmaceutical) anesthesia, and then Peyer's patches were collected from the small intestine using ophthalmic scissors.

(2) Preparation and culture of mouse Peyer's patch cells The obtained Peyer's patches are placed in a sterile petri dish containing ice-cooled 5% fetal bovine serum (FBS)-containing RPMI1640 medium (2 mL), and placed on a stainless steel mesh (200 mesh) in an amount of 5 mL. Peyer's patch cells were released by crushing using the rubber rubber part of the inner cylinder of the disposable syringe. The cell suspension was transferred to a 50 mL Falcon tube and stirred briefly with a vortex mixer. The cell suspension was filtered through a stainless mesh (150 mesh), centrifuged (1,500 rpm, 4° C., 7 minutes), and the medium was decanted to obtain Peyer's patch cells. The cells were washed by repeating the same operation four times using FBS-containing RPMI1640 medium (10 mL), and then filtered with a stainless mesh (200 mesh). The cell number was counted using a cell counter using this cell suspension (20 μL), and then a Peyer's patch cell suspension of 2×10 ⁶ cells/mL was prepared using FBS-containing RPMI1640 medium.

To a 24-well culture plate (3047, FALCON), the Peyer's patch cell suspension (500 μL/well) and concanavalin A (50 μg/mL, 50 μL, final concentration: 5 μg/mL, Sigma-Aldrich) obtained above were added to T. Under lymphocyte stimulation conditions, the cells were cultured at 37° C. under 5% CO ₂ -95% air for 3 days. The culture supernatant was transferred to another 24-well culture plate and stored at -20°C until use.

(3) Enzyme immunoassay (ELISA)
An anti-mouse cytokine primary antibody (anti-IL-2, IL-5 or IFN-γ antibody, 100 μL/diluted to 1 μg/mL with 50 mM carbonate-bicarbonate buffer (pH 9.6) on an ELISA plate (Immuno-Maxisorp, Nunc). well) was dispensed and incubated overnight at 4°C. This plate was washed three times with 0.05% Tween20-containing phosphate buffered saline (PBST) (300 μL/well), and then PBST containing 1% skim milk (SM) (SM-PBST) (100 μL/well) was used. And incubated at 37°C for 1 hour. The plate was washed 4 times with PBST (300 μL/well), 1% SM-PBST (50 μL/well) was added and preincubated at room temperature for 10 minutes, and then Peyer's plate culture supernatant (50 μL/well) was added. Incubated overnight at 4°C. The plate was washed 3 times with PBST (300 μL/well) and preincubated with 1% SM-PBST (100 μL/well) for 10 minutes at room temperature. Furthermore, a biotin-labeled anti-cytokine secondary antibody (1:1000, 50 μL/well) corresponding to the primary antibody diluted with 1% SM-PBST was added to the plate, and the mixture was incubated at 37° C. for 1 hour, and then PBST (300 μL/well) was used for 3 Washed twice. The plate was preincubated with 1% SM-PBST (100 μL/well) for 10 minutes at room temperature, and then alkaline phosphatase-labeled streptavidin (1:1000, 100 μL/well) diluted with 1% SM-PBST was added to the plate at 37° C. And incubated for 1 hour. After washing the plate 5 times with PBST (300 μL/well), a substrate solution [10% diethanolamine buffer (pH 9.8) solution of disodium p-nitrophenylphosphate (pH 9.8) (1 mg/mL, 150 μL/well) was added, and the temperature was kept at room temperature. Incubated at. The developed yellow color was measured using a microplate reader (Multiskan JX, Thermo Electron Corp.) (measurement wavelength: 405 nm, blank wavelength: 492 nm).

(4) Results FIG. 12 shows that IL-2 from Peyer's patch T lymphocytes by oral administration of an ethanol precipitation fraction, which is a heteroglycan-containing fraction derived from sesame oil squeezed meal of thin-mouth sesame, to FTY720-treated BALB/c mice. Shows the test results for changes in production of IL-5, IL-5 and IFN-γ. Oral administration of the ethanol-precipitated fraction at a dose of 50 or 100 mg/kg/day significantly enhanced the production of Il-2, IL-5 and IFN-γ from Peyer's patch T lymphocytes in a dose-dependent manner. .. IL-2 is involved in proliferation of T lymphocytes and maintenance of regulatory T lymphocytes, IL-5 is involved in differentiation of granulocytes such as neutrophils in hematopoietic system, and IFN-γ is antigen presentation It is known to be involved in enhancement of cell function, enhancement of Th1 lymphocyte reaction, antiviral action and activation of macrophages. From this, it is considered that sesame seed-derived heteroglycan induces T lymphocytes involved in production of these cytokines in Peyer's patches, leading to improvement of pathological conditions in which these cytokines are involved.