JP2008120707A - Immunopotentiating substance, and pharmaceutical composition, food, feed and cosmetic each containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new immunopotentiating substance derived from Ascophyllum nodosum, and to provide a pharmaceutical composition, food, feed and a cosmetic each containing the same. <P>SOLUTION: The new immunopotentiating substance, which is extracted from Ascophyllum nodosum as a kind of brown algae, comprises, as an active ingredient, ascophyllan as a sulfated polysaccharide with D-glucuronic acid as the main constituent other than L-fucose and D-xylose, and has immunopotentiating activities including G-CSF production-inducing ability, TNF-α production-inducing ability and apoptosis-inducing ability. A pharmaceutical composition, food, feed and a cosmetic each containing the immunopotentiating substance and having immunopotentiating activities are also provided, respectively. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to an immunostimulatory substance containing ascophyllan extracted from Ascophyllum nodosum as an active ingredient, and a pharmaceutical composition, food, feed and cosmetics containing the same.

In recent years, the relationship between abnormal immune function and disease has attracted attention. For example, side effects of anticancer drugs and reduced immune function due to HIV infection cause so-called opportunistic infections, and patients may die. Although cancer accounts for about one-third of the causes of death in Japanese people, recent studies have shown that abnormal immune functions increase the risk of onset. Furthermore, the possibility that a decrease in immune function due to stress in daily life may cause illness is being clarified.
Under such circumstances, research and development relating to pharmaceuticals, foods, and health foods having immunostimulatory activity have been actively conducted from the viewpoint of disease prevention and treatment.

In recent years, natural products, especially seaweed-derived immunostimulatory substances, have been attracting attention in response to the recent increase in safety orientation for pharmaceuticals and foods.
For example, Patent Document 1 discloses that an aqueous solvent extract fraction of seaweed belonging to the genus Ogonori has a macrophage activating effect. Patent Document 2 discloses that an enzyme-treated acidic polysaccharide extracted from seaweed belonging to red algae has a macrophage activating action. Patent Document 3 discloses that a porphyran enzyme degradation product has the ability to induce the production of activated interleukin 12. Patent Document 4 discloses that porphyran, which is a water-soluble fraction derived from Amanori, improves IgA production ability and serum IgA concentration of mesenteric lymph node lymphocytes. Patent Document 5 discloses that an alginic acid oligomer, which is an alginic acid degradation product, acts on human terminal blood leukocytes to induce the production of tumor necrosis factor.
Patent Document 6 discloses 4,7,10,13-hexadecatetraenoic acid, 6,9,12,15-octadecatetraenoic acid, 5,8,11, which are unsaturated fatty acids derived from seaweed. It is disclosed that 14,17-eicosapentaenoic acid and 9,12,15-octadecatrienoic acid activate eosinophils.

Ascophilum nodsum, a kind of brown algae, is a large seaweed that grows up to a length of 2 m and grows over the coast of Northern Europe. It has been used as a feed for livestock and a raw material for alginic acid. In recent years, it has also been used as a raw material for health foods (see Non-Patent Document 1).

Japanese Patent Laid-Open No. 5-139988 JP-A-6-256208 JP 2002-193828 A JP 2005-126429 A JP 2005-145895 A Japanese Patent Laid-Open No. 2005-23028 A. T.A. Critchley, Ohno, “Seaweed Resources of the world”, published by International Cooperation Agency, 1998, p. 205-209

Ascofilum nodosum is known to contain various sulfated polysaccharides including fucoidan. Among these, for fucoidan, although health functions such as immunostimulatory activity have attracted attention, there has been little biochemical evaluation of other sulfated polysaccharides.
The present invention has been made in view of such a problem, and an object thereof is to provide a novel immunostimulatory substance derived from Ascofilam nodosum, and a pharmaceutical composition, food, feed and cosmetics containing the same.

The present inventors have found that ascofilan extracted from ascophyllum dossum has various immunostimulatory activities, and have completed the present invention.
The immunostimulatory substance according to the first invention that meets the above object contains ascofilan extracted from ascophyllum dossum as an active ingredient. Therefore, the immunostimulatory substance according to the first invention has an immunostimulatory activity derived from ascophyrane.
Ascophyllan is a kind of unique sulfated polysaccharide contained in ascophyllum dosam. Although its complete structure has not been elucidated, the electrophoretic pattern difference, the structure of its partial hydrolyzate Other seaweed-derived sulfated polysaccharides such as fucoidan in that it is 3-OD-xylosyl-L-fucose, and that D-xylose and D-glucuronic acid are included in addition to L-fucose as constituent sugars A distinction is made (see, for example, Larsen B, Proc. Int. Seaweed Symp. 5th, p. 287-294, 1965).
In the present invention, “ascofilan” refers to any sulfuric acid extracted from ascophyllumnodosa by the method described later and containing D-glucuronic acid as a main component in addition to L-fucose and D-xylose. Means a modified polysaccharide. In the present invention, “immunostimulatory activity” includes not only activation of immunocompetent cells but also apoptosis-inducing activity against tumor cells and the like.

In the immunostimulatory substance according to the first invention, the ascofilan may have a sulfate group content of 18 to 22% by mass and a uronic acid content of 18 to 22% by mass.
Here, the “sulfuric acid group content” means a sulfuric acid group content measured by a turbidimetric method using barium sulfate described later, and “uronic acid content” means a uronic acid content measured by a carbazole sulfuric acid method described later. Say.

The immunostimulatory substance according to the first invention may have G-CSF (Granulocyte Colony Stimulating Factor) production inducing ability.
G-CSF acts on granulocyte stem cells in the bone marrow to promote differentiation, promotes outflow of produced granulocytes from the bone marrow, and has an action of enhancing the function of granulocytes.
Currently, G-CSF, M-CSF (macrophage production stimulating factor), etc. are administered to leukopenia seen during the treatment of anticancer drugs, and clinical effects are achieved. Leukopenia during infection, after cancer chemotherapy and regeneration The effect of G-CSF in leukopenia during aplastic anemia has been studied.

The immunostimulatory substance according to the first invention may have a TNF-α (Tumor necrosis factor) production inducing ability.
TNF-α is a cytokine that induces hemorrhagic necrosis at the tumor site, and has a protective ability in various infectious diseases. A substance that induces the production of TNF-α activates the immune system in the living body and is expected to have an immunostimulatory effect.
TNF-α is a cytokine called death ligand having apoptosis-inducing ability together with lymphotoxin, Fas ligand, and TRAIL. When TNF-α binds to the receptor, an initiator caspase is assembled to the death domain in the cell region via an adapter molecule, and apoptosis is caused by activating the caspase cascade. From this, TNF-α and apoptosis are very closely related.

The immunostimulatory substance according to the first invention may have apoptosis inducing ability.
Apoptosis is a physiological cell death caused by normal cell replacement in a living body, and is distinguished from necrosis (necrosis) which is a pathological cell death. Since cancer cells are unable to control the growth of cells and cells that should originally die no longer die and cause abnormal growth, the use of a substance having an ability to induce apoptosis is one method for suppressing the growth of cancer cells. It is being considered.

The pharmaceutical composition which concerns on 2nd invention contains the immunostimulatory substance which concerns on 1st invention, and has immunostimulatory activity.

The food according to the third invention includes the immunostimulatory substance according to the first invention and has immunostimulatory activity.

The feed according to the fourth invention includes the immunostimulatory substance according to the first invention and has immunostimulatory activity.

The cosmetic according to the fifth invention includes the immunostimulatory substance according to the first invention and has immunostimulatory activity.

The immunostimulatory substance which concerns on 1st invention contains the ascofilan extracted from the ascophyllum dosum which has the track record utilized also as a foodstuff raw material as an active ingredient. Therefore, it can be safely used as a raw material for pharmaceutical compositions, foods, feeds, and cosmetics having an immunostimulatory activity derived from Ascofilan.

In the immunostimulatory substance according to the first invention, when the sulfate group content of ascofilan is 18 to 22% by mass and the uronic acid content is 18 to 22% by mass, the immunostimulatory activity is particularly high. A substance can be obtained.

When the immunostimulatory substance according to the first invention has the ability to induce G-CSF production, such as promotion of granulocyte production in the bone marrow and release into the blood, enhancement of granulocyte function, etc. An immunostimulatory substance having an effect can be obtained.

When the immunostimulatory substance which concerns on 1st invention has TNF- (alpha) production induction ability, the immunostimulatory substance which has effects, such as an improvement of the protective ability with respect to an infectious disease, can be obtained.

When the immunostimulatory substance according to the first invention has apoptosis-inducing ability, an immunostimulatory substance having effects such as removal of cells that have caused carcinogenic mutations can be obtained.

Since the pharmaceutical composition which concerns on 2nd invention contains the immunostimulatory substance which concerns on 1st invention, and has immunostimulatory activity, it can be used as an active ingredient of the therapeutic agent of an immune disease.

Since the food which concerns on 3rd invention contains the immunostimulatory substance which concerns on 1st invention, it can be used as a functional food which performs immunostimulatory activity.

Since the feed which concerns on 4th invention contains the immunostimulatory substance which concerns on 1st invention, it can be used as a functional feed which has immunostimulatory activity.

Since the cosmetic according to the fifth invention contains the immunostimulatory substance according to the first invention, it can be used as a functional cosmetic having immunostimulatory activity.

Subsequently, an embodiment of the present invention will be described to provide an understanding of the present invention.
The immunostimulatory substance which concerns on one embodiment of this invention contains ascofilan extracted from ascophyllum dossum which is 1 type of brown algae as an active ingredient.

As an ascophyllum dosam which is an extraction raw material, the one from any production area can be used. In addition, since raw algal bodies have a high water content, it is preferable to use a dried algal body from which salt has been removed by washing with water as a raw material for extraction.
The average composition of Ascophyramnodosa dry powder used as an extraction raw material is 5 to 10% protein, 45 to 60% carbohydrate, 17 to 20% ash, 2 to 4% lipid, and 10 to 12% moisture. . In addition, as a pretreatment, the ascophyramnodosa dry powder may be treated with an acid aqueous solution to remove acid-soluble components.

Extraction of ascofilan from the dry powder of ascofilum nodosum is performed using 10 to 100 times (mass ratio) water of the dry powder of ascofilam nodosum. When the water to which Ascophyramnodosa dry powder is added is stirred at room temperature for 12 to 24 hours, Ascofilan is extracted into water together with alginic acid and the like. If necessary, water may be newly added, and extraction may be further performed at 100 ° C. for 12 to 24 hours. The crude extract thus obtained contains other polysaccharides, proteins, carbohydrates, lipids, ash, minerals, pigments, polyphenols, etc., but none of them has any harm to humans and animals. It can be added to food and feed as it is.

In order to obtain high-purity Ascofilan, purification can be performed by the method described below. Alginic acid contained in the extract can be removed by adding hydrochloric acid or the like to lower the pH of the extract and filtering the produced precipitate. Alternatively, alginic acid lyase may be added to selectively hydrolyze only alginic acid, and the hydrolyzate of alginic acid may be removed by dialysis. When ethanol is added to the extract, the resulting precipitate is dissolved in water, desalted by dialysis, and lyophilized to obtain almost pure ascofilan. If further purification is required, reprecipitation may be performed from water-ethanol.

Ascofilan, which contains a sulfate group and a D-glucuronic acid group and is a polyanion, may form a salt with any cation that does not inhibit water solubility and immunostimulatory activity. Examples of such cations include sodium ions, potassium ions, magnesium ions, calcium ions, ammonium ions, and the like.

Ascofilan that can be used as an active ingredient of an immunostimulatory substance is any sulfated polysaccharide extracted from ascophyllumnodsum and containing D-glucuronic acid as a main component in addition to L-fucose and D-xylose There is no particular limitation on the molecular weight or the like. The sulfate group content and the uronic acid content are not particularly limited, but those having a sulfate group content of 18 to 22% by weight measured by a turbidimetric method using barium sulfate are preferred, and uron measured by a carbazole sulfate method Those having an acid content of 18 to 22% by weight are preferred.

Ascofilan can be used as a pharmaceutical composition by mixing with a carrier or the like. Examples of the dosage form of the pharmaceutical composition for humans or animals include oral, rectal, parenteral (for example, intravenous administration, intramuscular administration, subcutaneous administration, etc.), and the dosage is the formulation of the pharmaceutical composition. However, in the case of humans, the active ingredient generally contained in the formulation is difficult to determine and to be determined uniquely depending on the administration method, purpose of use, and age, weight, and symptoms of the subject to be applied. The amount is preferably 0.1 to 2000 mg / kg per day for an adult. Of course, since the dosage varies depending on various conditions, an amount smaller than the above dosage may be sufficient or may be necessary beyond the range.

When prepared as an orally administered preparation, it can be prepared in the form of tablets, granules, powders, capsules, coatings, liquids, suspensions, etc. It can be prepared in the form of drops, suppositories and the like. Any known method can be used for formulation. For example, ascofilan and a pharmaceutically acceptable carrier or diluent, stabilizer, and other desired additives can be blended to form the desired dosage form described above.

The food containing the immunostimulatory substance can take any form commonly used in foods or health foods, such as those prepared by ascofilan as food, added to other foods, capsules, tablets, etc. .
When ingested or administered in foods, mix with excipients, extenders, binders, thickeners, emulsifiers, colorants, flavors, food additives, seasonings, etc. Accordingly, it can be formed into a powder, granule, tablet or the like. Moreover, it can be ingested by preparing a food as a functional food having immunostimulatory activity by appropriately mixing in a food material and preparing a food.

The feed containing the immunostimulatory substance can take various forms such as those prepared as is, as well as those prepared in the feed.
When mixed in feed and administered to animals such as livestock, it can be mixed in advance with feed ingredients to prepare a feed with functionality. It can also be added to the feed for administration. That is, an immunostimulatory substance containing ascofilan as an active ingredient can be safely and immunized by adding it to livestock such as pigs, chickens, cows, horses and sheep, and feed such as fish and pets (dogs, cats and birds). It can be used as a functional feed having activation activity.

Cosmetics containing an immunostimulatory substance can take various forms such as those obtained by blending Ascofilan in a lotion or cream.
In order to mix and administer to cosmetics etc., it can mix with liquid or creamy cosmetics suitably, and can prepare as functional cosmetics etc. In that case, solubilizers, stabilizers, emulsifiers and the like well known in the preparation of cosmetics and the like can be used.

Next, examples carried out for confirming the effects of the present invention will be described.
Example 1 Extraction of Ascofilan Extraction of Ascofilan was performed using the method of Larsen et al. (Acta Chemica Scandinavica, vol. 20, p.219-230, 1966).
20 g of Ascophyramnodosa dry powder was placed in 500 mL of 0.2N hydrochloric acid and stirred overnight. The solid was collected by filtration, 0.2N hydrochloric acid was added again and stirred for 4 hours, and then the solid was collected by filtration. The obtained solid was placed in 1000 mL of distilled water and stirred, and then neutralized with NaOH solution. Then, it stirred at 20 degreeC for 20 hours, and isolate | separated into the extract and solid substance by filtration. The obtained solid was again put into 1000 mL of distilled water and extracted at 100 ° C. for 2 hours with stirring. After extraction, it was separated into an extract and a solid by filtration. Two extracts were mixed and adjusted to pH 1.3 with 0.2N hydrochloric acid. After standing overnight, the precipitated alginic acid was removed by filtration. The filtrate was neutralized with a NaOH solution and then concentrated using an evaporator. The resulting concentrated solution was dialyzed to remove salts generated by neutralization.
After desalting, the solution was concentrated to 100 mL, and 50 mL of 0.13 M phosphate buffer solution (pH 7.5) was added. Sodium chloride and then alginic acid lyase were added to this solution to 3% w / v, and the mixture was reacted at 30 ° C. for 20 hours to decompose the remaining alginic acid. The enzymatic reaction was stopped by heat inactivation, and the alginic acid decomposition product was removed by dialysis.
To the aqueous solution thus obtained, 30 mL of 1M sodium chloride aqueous solution and 180 mL of ethanol were added dropwise and allowed to stand overnight. Thereafter, the precipitate and the supernatant were separated by centrifugation. The obtained precipitate was dissolved in distilled water so as to be 1% (w / v), and 1/5 volume of 0.8 M sodium chloride aqueous solution and 6/5 volume of ethanol were added dropwise. After standing overnight, it was separated into a precipitate and a supernatant by centrifugation. The obtained precipitate was dissolved in water, dialyzed and lyophilized to obtain 0.85 g of Ascofilan.

Example 2: Composition analysis of ascofilan (1) Analysis of sugar composition The analysis of the sugar composition in ascofilan was performed by the method of Anno et al. (Biosci. Biotechnol. Biochem. Vol. 63, p.1353-1359, 1999). Used.
To the reaction test tube, 10 μL of an ascofilan aqueous solution and 10 μL of an 8M aqueous trifluoroacetic acid solution were added. After sealing, the mixture was stirred with a vortex mixer, and the solution was collected at the bottom of the test tube by centrifugation. After hydrolysis at 100 ° C. for 3 hours, the solution was cooled with air, and the solution was collected by centrifugation at the bottom of the test tube. After removing the lid and evaporating the solution to dryness, 40 μL of 2-propanol was added and stirred. The solvent was evaporated again, and 40 μL of pyridine / methanol (10/90, v / v) was added to the reaction test tube containing the dried sample. After stirring, 10 μL of acetic anhydride was further added and stirred. In order to prevent contamination of the solution, the test tube was covered with aluminum foil and left at room temperature for 30 minutes. Thereafter, the solution was evaporated to dryness, and 10 μL of pure water and 40 μL of ABEE (4-aminobenzoic acid ethyl ester) reagent (Seikagaku Corporation catalog No. 400871) were added and sealed. After stirring, the solution was centrifuged and collected at the bottom of the test tube, and reacted at 80 ° C. for 60 minutes. After air cooling, the solution was centrifuged and collected at the bottom of the test tube. The lid was removed, and 200 μL of pure water and 200 μL of chloroform were added. After sealing, the mixture was vigorously stirred and centrifuged. The aqueous layer (upper layer) was collected and subjected to HPLC (high performance liquid chromatography) analysis.

The conditions for HPLC analysis are as follows.
column:
Honen Pack C18 (Seikagaku Corporation catalog No. 80000445, column length 75 mm x inner diameter 4.6 mm)
Eluent:
A 0.2M potassium borate buffer (pH 8.9) / acetonitrile (93/7, v / v)
B 0.02% trifluoroacetic acid / acetonitrile (50/50, v / v)
Elution was performed using eluent A for elution time 0 to 50 minutes, eluent B for 50 to 55 minutes, and eluent A for 55 to 75 minutes.
Flow rate: 1 mL / min
Detection method: UV (305 nm)

The results of the sugar composition analysis of ascofilan are shown in Table 1. In Table 1, “Gal”, “Man”, “Glc”, “Xyl”, and “Fuc” represent D-galactose, D-mannose, D-glucose, D-xylose, and L-fucose, respectively. The sugar composition was described as a molar ratio to L-fucose.

According to the literature (Larsen B, Proc. Int. Seaweed Symp. 5th, p.287-294, 1965), the molar ratio of L-fucose and D-xylose contained in ascofilan is 1: 1.1. Although reported, similar results were obtained in this compositional analysis.
In addition, the sugar composition analysis of Okinawa mozuku-derived fucoidan and gagomecomb-derived fucoidan was also conducted. These fucoidans are polysaccharides (fucan) containing sulfated L-fucose as a main component and are clearly different from ascofilan. Has a composition.

(2) Determination of sulfate group content The sulfate group content was determined using the method of Dodgson et al. (Turbidimetric method using barium sulfate) (Biochem. J., Vol. 84, p.106-110, 1962). I went.
5 mg of Ascofilan powder was collected in a test tube and 2 mL of 1N hydrochloric acid was added. Under a nitrogen atmosphere, the test tube was sealed and hydrolyzed at 110 ° C. for 5 hours. After opening the test tube, 0.2 mL of the hydrolysis solution was collected and mixed with 3.8 mL of a 3% aqueous trichloroacetic acid solution. Furthermore, BaCl ₂ - sample solution prepared by adding gelatin solution 1 mL, and BaCl ₂ a blank solution prepared by adding the gelatin solution without 15 minutes standing, using an absorption photometer measures the absorbance of 360 nm, the sample The difference in absorbance between the solution and the blank solution was determined. As a result of calculating the content of sulfate groups contained in ascofilan based on a calibration curve prepared using an aqueous potassium sulfate solution having a known concentration, a quantitative value of 18.9% was obtained.

(3) Determination of uronic acid content The uronic acid content was determined using the carbazole sulfate method improved by T. Bitter and HN Muir.
0.5 mL of Ascofilan aqueous solution was collected in a test tube, and 2.5 mL of sulfuric acid reagent was added dropwise. The mixed solution was stirred well and then heated in a boiling water bath for 10 minutes. After cooling to room temperature, 0.1 mL of carbazole reagent was added and mixed, and further heated in a boiling water bath for 15 minutes. After cooling to room temperature, the absorbance at 530 nm was measured. As a result of calculating the uronic acid content contained in ascofilan based on a calibration curve prepared using a glucuronic acid aqueous solution having a known concentration, a quantitative value of 20.5% was obtained.

Example 3: Measurement of G-CSF production inducing ability of Ascofilan to RAW264.7 cells (1) As a cultured macrophage cell of RAW264.7 cells, mouse monocyte-derived cell line RAW264.7 (ATCC number TIB-71) was used. did. As a culture medium for RAW264.7 cells, D-MEM medium containing 10% FBS (fetal bovine serum) supplemented with penicillin (100 units / mL) and streptomycin (100 μg / mL) was used. The passage operation was performed according to a conventional method. First, the medium in the flask was removed, the cells were detached using a 0.25% trypsin / EDTA solution, and the trypsin reaction was stopped by adding a culture medium. Centrifugation was performed at 1000 rpm for 1 minute, the supernatant was removed, and a growth medium was added so that the cell concentration was 1 × 10 ⁵ cells / mL. The cell culture solution thus obtained was cultured in a 25 cm ² flask. The culture was performed in an incubator under conditions of 5% CO ₂ and 37 ° C.

(2) Addition of Ascofilan to RAW264.7 cells RAW264.7 cells cultured in a 25 cm ² flask were collected by the method described in (1), and the number of cells per well was 2 × 10 6 on a 96-well microplate. ^The seeds were sowed to ⁴ pieces. After culturing for 1 day under conditions of 5% CO ₂ and 37 ° C. and confirming that the cells were attached to the bottom surface of the well, the medium was removed and the Ascofilan solution (125, 250, 500 dissolved in the culture medium) was removed. , 1000 μg / mL). Furthermore, the cell supernatant was extract | collected after culture | cultivation for 1 day, and the production amount of G-CSF was measured.

(3) Evaluation of the ability of Ascofilan to induce G-CSF production G-CSF was measured by ELISA (enzyme-linked immunosorbent assay). 100 μL of a primary antibody (Monoclonal Anti-Mouse G-CSF Antibody) diluted with PBS (phosphate buffered saline) was added to a 96-well plate for ELISA. After standing at room temperature for 24 hours, it was washed 5 times with a washing solution (PBS solution containing 0.025% Tween 20 (registered trademark)), and a blocking solution (PBS solution containing 1% BSA (bovine serum albumin)) was added to one well. 200 μL was added per unit. After standing at room temperature for 24 hours, washed 5 times with a washing solution, diluted with PBS to 0-2000 pg / mL (Recombinant Mouse G-CSF), RAW264.7 cell culture solution supplemented with ascofilan solution 100 μL of the diluted diluted liquid was added per well and allowed to stand at room temperature for 2 hours. After washing 5 times with a washing solution, a secondary antibody (Biotinylated Anti-Mouse G-CSF Antibody) diluted with a blocking solution was added and allowed to stand at room temperature for 2 hours. Washed 5 times with washing solution, added 100 μL of enzyme-antibody solution diluted with blocking solution (Streptavidin conjugated to horseradish-peroxidase) per well, allowed to stand at room temperature for 20 minutes, washed 5 times with washing solution, then TMB A substrate solution in which (3,3′5,5′-tetramethylbenzidine) peroxidase substrate and peroxidase substrate solution B (KPL, USA) were mixed at a ratio of 1: 1 was added per well, and allowed to stand at room temperature for 30 minutes. 50 μL of 1N phosphoric acid aqueous solution was added as a reaction terminator and stirred well, and the absorbance (A = 450 nm) was measured with a microplate reader.

A graph showing the relationship between the amount of Ascofilan added and the amount of G-CSF produced from R264.7 cells is shown in FIG. It can be seen that the amount of G-CSF produced from RAW264.7 cells is increased depending on the concentration of ascofilan.

Example 4: Measurement of TNF-α production inducing ability of Ascofilan to RAW264.7 cells (1) As a cultured macrophage cell of RAW264.7 cells, mouse monocyte-derived cell line RAW264.7 (ATCC number TIB-71) was used. did. As a culture medium for RAW264.7 cells, D-MEM medium containing 10% FBS (fetal bovine serum) supplemented with penicillin (100 units / mL) and streptomycin (100 μg / mL) was used. The passage operation was performed according to a conventional method. First, the medium in the flask was removed, the cells were detached using a 0.25% trypsin / EDTA solution, and the trypsin reaction was stopped by adding a culture medium. Centrifugation was performed at 1000 rpm for 1 minute, the supernatant was removed, and a growth medium was added so that the cell concentration was 1 × 10 ⁵ cells / mL. The cell culture solution thus obtained was cultured in a 25 cm ² flask. The culture was performed in an incubator under conditions of 5% CO ₂ and 37 ° C.

(2) Addition of Ascofilan to RAW264.7 cells RAW264.7 cells cultured in a 25 cm ² flask were collected by the method described in (1), and the number of cells per well was 2 × 10 6 on a 96-well microplate. ^The seeds were sowed to ⁴ pieces. After culturing for 1 day under conditions of 5% CO ₂ and 37 ° C. and confirming that the cells were attached to the bottom of the well, the medium was removed and the Ascofilan solution (62.5, 125 dissolved in the culture medium) was removed. , 250, 500, 1000 μg / mL). Furthermore, the cell supernatant was extract | collected after culture | cultivation for 1 day, and the TNF- (alpha) output was measured by ELISA method.

(3) Measurement of ability to induce TNF-α production of ascofilan TNF-α was measured by ELISA. 100 μL of primary antibody (Anti-Mouse TNF alpha Monoclonal Antibody) diluted with PBS was added to a 96-well plate for ELISA. After standing at room temperature for 24 hours, it was washed 5 times with a washing solution (PBS solution containing 0.025% Tween 20 (registered trademark)), and a blocking solution (PBS solution containing 4% BSA (bovine serum albumin)) was added to one well. 200 μL was added per unit. After leaving still at room temperature for 24 hours, it was washed 5 times with a washing solution. After washing, 100 μL of a standard solution (Recombinant Mouse TNF alpha) diluted with PBS to 0 to 1000 pg / mL and a diluted culture supernatant of RAW264.7 cells supplemented with ascofilan solution were added at room temperature. And left for 2 hours. After washing 5 times with a washing solution, a secondary antibody (Anti-Mouse TNF alpha Polyclonal Antibody) diluted with a blocking solution was added and allowed to stand at room temperature for 2 hours. Washed 5 times with the washing solution, diluted with blocking solution and diluted with blocking solution (Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey) 100 μL per well and allowed to stand at room temperature for 30 minutes. After washing twice, 100 μL of a substrate solution in which TMB (3,3′5,5′-tetramethylbenzidine) peroxidase substrate and peroxidase substrate solution B (KPL, USA) were mixed at a ratio of 1: 1 was added to each well at room temperature. Let stand for a minute. 50 μL of 1N phosphoric acid aqueous solution was added as a reaction terminator and stirred well, and the absorbance (A = 450 nm) was measured with a microplate reader.

A graph showing the relationship between the amount of Ascofilan added and the amount of TNF-α produced from RAW264.7 cells is shown in FIG. It can be seen that the amount of TNF-α produced from RAW264.7 cells increases depending on the concentration of ascofilan.

Example 5: Evaluation of apoptosis-inducing ability of ascofilan (1) Evaluation of apoptosis-inducing ability Human histiocytic lymphoma U937 cells (ATCC number CRL1593.2) were used as cultured cells. The culture was performed using RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% FBS to which penicillin (100 units / mL) and streptomycin (100 μg / mL) were added.
The number of cells in the cell solution entering the logarithmic growth phase was adjusted to 4.0 × 10 ⁵ cells / mL, and 0.9 mL of each cell solution was dispensed into a 24-well plate. Thereafter, Ascofilan dissolved in the culture medium was added to a final concentration of 10, 50, 100, 250 μg / mL. The culture was performed at 37 ° C. in the presence of 5% CO ₂ for 4 days. Cell viability was determined by trypan blue cell staining.

Table 2 shows the ability of ascofilan to induce apoptosis. Apoptosis activity (%) was expressed as a cell growth inhibition rate relative to control cells (no ascofilan added). From this result, it was confirmed that ascofilan has a growth inhibitory action on cancer cells, the action depends on the addition concentration, and the ED ₅₀ value (the amount necessary for the 50% inhibition rate) is also 36.1 μg / It became a very low value with mL.

(2) Detection of DNA fragmentation In order to confirm whether the cell growth inhibitory action of Ascofilan is due to apoptosis, DNA fragmentation was detected by extracting cell DNA and performing electrophoresis. It has been found that fragmentation of nucleosome units is confirmed in apoptotic cells.

As cultured cells, human histiocytic lymphoma U937 cells (ATCC number CRL1593.2) were used. The culture was performed using RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% FBS to which penicillin (100 units / mL) and streptomycin (100 μg / mL) were added.
The number of cells in the cell solution entering the logarithmic growth phase was adjusted to 3.0 × 10 ⁵ cells / mL, and 0.9 mL of each cell solution was dispensed into a 24-well plate. Thereafter, Ascofilan dissolved in the culture medium was added to a final concentration of 1 mg / mL. Culturing was performed at 37 ° C. in the presence of 5% CO ₂ .

The cell solution cultured for 3 days was transferred to a 2 mL tube, centrifuged at 400 G for 5 minutes, and the supernatant was removed. Cell lysis buffer (50 mM Tris-HCl, pH 7.8, 10 mM EDTA · 4Na, 0.5% sodium N-lauroyl sarcosinate) was added and stirred well. 1 μL of RNase solution was added and mixed well, and incubated at 50 ° C. for 30 minutes. Thereafter, 1 μL of proteinase K solution was added and mixed well, and incubated at 50 ° C. for 60 minutes. Water droplets on the wall of the tube were dropped by centrifuging lightly. The sample solution thus obtained was poured into a 2% agarose gel well using a micropipette and subjected to electrophoresis at 100V. After completion of electrophoresis, a polaroid photograph of an agarose gel was taken on a UV transilluminator.

A photograph showing an agarose gel electrophoresis pattern of DNA extracted from U937 cells cultured at 37 ° C. under 5% CO ₂ for 3 days is shown in FIG. Lane 1 is a molecular weight marker (pHY marker), Lane 2 is DNA extracted from U937 cells cultured without addition of Ascofilan, Lane 3 is an electrophoresis pattern of DNA extracted from U937 cells cultured with addition of Ascofilan. Respectively.
From the fact that a ladder-like pattern was observed in lane 3, it was confirmed that the DNA of cells to which Ascofilan had been added was fragmented in units of nucleosomes. In addition, fragmentation could not be confirmed in the DNA of cells to which no ascofilan was added. From this, it was confirmed that the inhibitory action of U937 cell proliferation by Ascofilan is due to apoptosis induction.

It is a graph showing the relationship between the addition amount of Ascofilan and the production amount of G-CSF from RAW264.7 cells. It is a graph showing the relationship between the addition amount of ascofilan and the production amount of TNF-α from RAW264.7 cells. 37 ° C., which is a photograph showing the agarose gel electrophoresis pattern of 5% CO ₂ and below in extracted from 3-day culture were U937 cells DNA.

Claims (9)

An immunostimulatory substance containing ascophyllan extracted from Ascophyllum nodosum as an active ingredient. The immunostimulatory substance according to claim 1, wherein the ascofilan has a sulfate group content of 18 to 22% by mass and a uronic acid content of 18 to 22% by mass. The immunostimulatory substance according to any one of claims 1 and 2, wherein the substance has an ability to induce G-CSF production. The immunostimulatory substance according to any one of claims 1 to 3, wherein the immunostimulatory substance has an ability to induce TNF-α production. The immunostimulatory substance according to any one of claims 1 to 4, wherein the immunostimulatory substance has apoptosis inducing ability. A pharmaceutical composition comprising the immunostimulatory substance according to any one of claims 1 to 5 and having immunostimulatory activity. The foodstuff which contains the immunostimulatory substance of any one of Claims 1-5, and has immunostimulatory activity. A feed comprising the immunostimulatory substance according to any one of claims 1 to 5 and having immunostimulatory activity. A cosmetic comprising the immunostimulatory substance according to any one of claims 1 to 5 and having immunostimulatory activity.