JP2004315520A - Autoimmunity potentiating agent, method for producing the same, and cosmetic by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new autoimmunity potentiating agent having such specific characteristics as not to eliminate its bonding properties to saccharides even when heat-treated, to be excellent in saccharide chain recognition selectivity, and to have autoimmunity potentiating activity, such as cellular immunological competence potentiating activity. <P>SOLUTION: The autoimmunity potentiating agent contains a liquid extract extracted from a Gracilaria species of red algae with an aqueous salt solution as an active ingredient. A method for producing the agent comprises extracting the Gracilaria species of the red algae with the aqueous salt solution to obtain an extract solution, conducting first-step salting-out by adding ammonium sulfate to the obtained extract solution until its final concentration reaches a saturation concentration of 20-40%, removing impurities which are precipitated by the salting-out, conducting second-step salting-out by further adding the ammonium sulfate to the extract solution until its final concentration reaches a saturation concentration of 60-80%, recovering a crude active fraction from the extract solution as a precipitate, separating a liquid extract exhibiting the cellular immunological competence potentiating activity by dissolving the precipitate with a suitable solvent, and collecting the liquid extract. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention uses a red alga (Gracilaria sp.) Of the genus Ogonori as a raw material, which is characterized in that sugar binding is not lost by heat treatment, recognition sugar chain selectivity is excellent, and it has autoimmune enhancement activity such as activation of cellular immunity. The present invention relates to a novel autoimmune enhancer having specific properties, a method for producing the same, and a cosmetic using the same.

  Hemagglutinin exhibits a specific behavior with respect to the erythrocytes of each animal, and thus is widely used as a test reagent or a separation material in medical, pharmaceutical, biochemical fields and the like. This hemagglutinin is roughly classified into those derived from animals and those derived from plants.However, in view of availability in large quantities and ease of processing, plant-derived hemagglutinins have attracted attention in practical use. I have.

  Heretofore, as hemagglutinins derived from these plants, terrestrial plants have been derived from concanavalin A (Con A) from jack bean or wheat germ lectin (WGA) from wheat (see Non-Patent Document 1). Known as plant-derived ones are GVAI from Ogonori (Graciliaria verrucosa), Hypnin A, B, C and D from Hypnea japonica (see Non-Patent Document 2).

However, those derived from terrestrial plants can obtain a sample with high agglutinating activity relatively easily, but the hemagglutinating activity is also inhibited by simple sugars such as monosaccharides and disaccharides. It has the disadvantage of low chain selectivity. Marine plant-derived products are considered to have high recognition sugar chain selectivity because hemagglutination activity is not inhibited by monosaccharides or disaccharides and is inhibited by glycoproteins such as fetuin and asialofetuin. It has the drawback that it is difficult to obtain a highly active preparation. Both have the drawback that the aggregation activity cannot be controlled due to the change in ionic strength.
Further, in general, hemagglutinin has a disadvantage in that its heat treatment at 100 ° C. loses its sugar chain binding ability.

  Among the biological activities of hemagglutinin on cells, a breakthrough is the reaction with lymphocytes. Culture of lymphocytes with very low concentrations of hemagglutinin causes the lymphocytes to proliferate and divide. The effect of triggering the lymphocytes in the stationary phase to a state in which they grow and proliferate is called mitogen stimulation, and is an important phenomenon that is the key to the immune response of a living body to a foreign substance (antigen). The mitogen stimulating function is one of the cellular immunity stimulating functions and serves as an indicator of the innate immunity enhancing activity of hemagglutinin.

  Hemagglutinins mainly used as mitogens include concanavalin A (Con A), kidney bean lectin pi (PHA-P), kidney bean lectin L (PHA-L), and pokeweed lectin (PWM), etc. The cells are cultured for 48 to 72 hours and assayed by measuring the rate of increase in labeled thymidine incorporated into DNA.

  Hemagglutinin with mitogenic potential can activate most of the activatable lymphocytes, regardless of the antigen specificity of the cells, making it easier to pursue and study changes due to cell proliferation. is there. It has also been clarified that hemagglutinin induces T lymphocytes to induce cytotoxic activity. Since the induced cytotoxicity of T cells is non-specific for antigen, it is exerted on various normal cells and malignant cells.

  As described above, mitogen activation by hemagglutinin is a means for determining the immunity of patients with various illnesses including AIDS because of easy and simple use. It is also used for investigating various immunosuppressive and immunotherapy effects. More recently, it has also attracted attention as a lymphocyte mitogen in LAK therapy, a new treatment for cancer.

  Hemagglutinin (hemagglutinin composed of protein is generally called lectin) has the ability to specifically recognize and bind to sugar chains. This property is that when administered directly to the living body or transdermally to the skin as a mitogen, it recognizes cell surface sugar chains and can bind to cells, so mitogens that do not have sugar chain binding ability (eg, lipopolysaccharide, etc.) Compared with mitogens, it is considered that they can function more effectively as mitogens by binding to sugar chains on the cell surface and accessing the cell surface.

  However, these hemagglutinins having mitogenic ability are mainly composed of proteins, and lose their sugar binding ability when subjected to heat treatment at a high temperature (about 100 ° C.) or left at 40-50 ° C. for a long time. Combination with other reagents at the time of administration and combination with other components when used as a cream or ointment for application to the skin are inevitably limited. Therefore, there is a need for a hemagglutinin having a mitogenic ability capable of retaining a sugar chain binding ability even after heat treatment, and the present inventors have previously conducted a method for producing highly active hemagglutinin from Ogonori red algae. It was proposed (Patent Document 1). However, it has not been known that the highly active hemagglutinin has a physiological activity such as an autoimmune enhancement effect.

"Journal of Bacteriol.", 1936, Vol. 32, pp. 227-237 (claims and others) "Bultan of Japanese Society of Scientific Fisheries (Bul. Jap. Soc. Sci. Fishe.)", 1981, Vol. 47, pp. 1079-1084 (Claims and Others) ) JP-A-7-278004 (Claims and others)

  Under such circumstances, the present invention has a specificity such that the sugar binding is not lost by heat treatment, the recognition sugar chain selectivity is excellent, and the autoimmune activity such as activation of cellular immunity is exhibited. The purpose of the present invention is to provide a novel autoimmune enhancer having properties.

  The present inventors have conducted various studies on hemagglutinin derived from plants, particularly from marine plants, and as a result, hemagglutinin extracted from specific species of red algae (Graciliaria sp.) Under specific conditions is: In addition to high aggregation activity and high recognition sugar chain selectivity, the aggregation activity is controlled by ionic strength, sugar binding is not lost by heat treatment, excellent recognition sugar chain selectivity, and self- The present inventors have found that they have an immunopotentiating activity, and have accomplished the present invention based on this finding.

That is, the present invention provides an autoimmune enhancer containing a liquid extract from a salt aqueous solution of Gracilaria sp. As an active ingredient, and extracts a red alga of Gracilaria sp. With an aqueous salt solution. Ammonium sulfate was first added to the extracted solution to a final concentration of 20 to 40% to carry out a salting-out in the first stage to remove precipitated contaminants. Ammonium sulfate is added until the concentration reaches 80% saturation, salting out is performed in the second stage, the crude active fraction is separated as a precipitate, and the precipitate is dissolved in an appropriate solvent to obtain a liquid crude active fraction. Further, if necessary, the contaminating proteins are removed by heat treatment at 100 ° C. for 1 to 10 minutes, and then the molecular weight is reduced to 100,100 by gel filtration chromatography. The present invention provides a method for producing an autoimmune enhancer having cell immunity stimulating activity, and a cosmetic using the same by separating and purifying the fractions obtained by fractionating 00 or more fractions by chromatography. is there.
The saturation concentration of the above ammonium sulfate is described in “Green, AA & Hughes, WL” (1955) “Methods in Enzymology, Vol. Pages 67-90 "[Table of Relationship between Addition Amount of Crystalline Ammonium Sulfate and Concentration (% Saturation)].

The autoimmune enhancer of the present invention can be prepared, for example, by adding ammonium sulfate to an extract obtained by extracting an aqueous salt solution from a red alga of the genus Gracilaria sp. , And the precipitated contaminants are removed. Then, ammonium sulfate is added to the extract to a final concentration of about 60 to 80% saturation, and the second step of salting out is performed to obtain a crude active fraction. Is recovered as a precipitate, and the liquid crude active fraction obtained by dissolving the precipitate with an appropriate solution, and if necessary, heat treatment at 100 ° C. for 1 to 10 minutes to remove contaminating proteins, and then gel The fraction having a molecular weight of 100,000 or more is separated by filtration chromatography, and the components are further separated by chromatography. It is a liquid obtained by collecting.
The salt aqueous solution used at this time is, for example, selected from physiological saline, phosphate buffer, Tris-HCl buffer or sodium chloride, potassium chloride, zinc sulfate, zinc chloride, 2-mercaptoethanol and dithiothreitol. There is a liquid to which at least one is added, particularly a phosphate buffer, a Tris-HCl buffer or a liquid to which at least one selected from sodium chloride, potassium chloride, zinc sulfate and 2-mercaptoethanol is added. .
The liquid obtained above contains hemagglutinin containing sugar as a main component. As such a saccharide, those in which the proportion of galactose in the monosaccharide constituting the saccharide is 70 to 100%, particularly 90 to 100% are preferable. When the above liquid is used as the autoimmune enhancer of the present invention, it may contain, in addition to sugar, a protein having a protein mass of 0.4 or less with respect to the mass of sugar.
The quantification of sugar is performed by the phenol sulfate method using galactose as a standard sample, and the quantification of protein is performed by the Lowry method using bovine serum albumin as a standard sample.

As a raw material at this time, red algae of the genus Ogonori are used, and especially Ogonori (Graciliaria verrucosa), Tsurusilamo (Graciliaria chorda), and subspecies thereof are preferable. In the present invention, the red alga of the genus Ogonori (Gracilaria sp.) Is defined as (1) a seaweed classified as a seaweed of the genus Ogonori (Graciliaria sp.), Or (2) a seaweed of Gracilaria psi. Or (3) Gracilariopsis sp. Including seaweeds classified in the past.
For example, among seaweeds from Japan, the red alga of the genus Ogonori (Graciliaria sp.) Is described in Non-Patent Document "New Japanese Seaweed Journal of Japanese Seaweeds, by Tadao Yoshida, published by Uchida Lao Tsuruho, 1998," Gracilariales: Gracilariales: It includes seaweeds classified as Gracilariaceae.
These red algae are also present in the cold sea but particularly in the warm sea, and are distributed in almost all coastal areas in Japan, and are used for agar augmentation and sashimi clams.

  In order to suitably produce the autoimmune enhancer of the present invention, (a) an extraction step of a water-soluble fraction, (b) a fractionation step of a crude active fraction, and if necessary, (C) sequentially performing the agglutinin purification step.

  Each of the above steps will be described in more detail. First, in the step (a), a salt-containing aqueous solution such as a physiological saline, a phosphate buffer, a Tris-HCl buffer or sodium chloride is added to the starting red algae. A solution containing at least one selected from potassium chloride, zinc sulfate, zinc chloride, 2-mercaptoethanol and dithiothreitol, preferably a phosphate buffer, a Tris-HCl buffer or sodium chloride, potassium chloride, After adding and homogenizing a liquid to which at least one selected from zinc sulfate and 2-mercaptoethanol is added, centrifugation is performed to obtain a crude extract as a supernatant.

  Next, in the step (b), ammonium sulfate is added to the extract obtained in the step (a) until a final concentration of 20 to 40% saturation is reached, and a first-stage salting-out is carried out. Is removed by centrifugation. By this operation, impurities such as dyes are removed as a precipitate fraction. Next, ammonium sulfate was added to the supernatant obtained by the centrifugal separation treatment until a final concentration of 60 to 80% saturation was reached, followed by salting-out in the second step. The fraction is redissolved in a buffer such as a phosphate buffer containing sodium chloride and, if desired, purified by dialysis against a buffer such as a phosphate buffer containing sodium chloride to obtain a crude active fraction. This crude active fraction can be used as it is as the autoimmune enhancer of the present invention.

  This crude active fraction can be further subjected to step (c) if desired. In the step (c), the crude active fraction obtained in the step (b) is heat-treated at 100 ° C. for 1 to 10 minutes to remove precipitated contaminating proteins, and then subjected to gel filtration chromatography to obtain a molecular weight of 100,000 or more. The fractions are fractionated, and the components are further separated by chromatography to obtain purified hemagglutinin. In this case, it is advantageous to use ion exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography or a combination thereof as the chromatography used in the final step.

  Here, the fraction having a molecular weight of 100,000 or more refers to a fraction corresponding to a molecular weight of 100,000 or more as a result of calculating the molecular weight of the eluted fraction using a globular protein as a standard molecular weight substance in gel filtration chromatography. Say.

To suitably produce the autoimmune enhancer of the present invention, 0.1 ml of a purified hemagglutinin having a mitogenic ability is added to a TSK gel G3000 PWXL column, subjected to gel filtration chromatography, and subjected to gel filtration chromatography. Collect elution fractions from the column in 0.1 ml increments. At this time, thyroglobulin (molecular weight 669,000), ferritin (molecular weight 440,000), bovine serum albumin (molecular weight 67,000), and ovalbumin (molecular weight 43,000) are used as standard molecular weight substances. As a result, it was found that the peak of the peak having the agglutinating activity, which indicates the fraction in which the hemagglutinin having the mitogenic ability was eluted (the fraction of the hemagglutinating activity), corresponds to a molecular weight of 5.64 × 10 ⁵ .

The autoimmune enhancer of the present invention thus obtained is further characterized by the following.
(1) has the property of agglutinating sheep erythrocytes treated with pronase, and this agglutinating activity is not inhibited by monosaccharides or disaccharides, but is inhibited by fetuin or asialofetuin;
(2) that the agglutinating activity on rabbit erythrocytes changes with ionic strength;
(3) elution in a fraction corresponding to a molecular weight of 100,000 or more in gel filtration chromatography using a globular protein as a standard molecular weight substance;
(4) having a cellular immunity stimulating activity;
(5) having sugar chain binding activity even after heat treatment at 100 ° C. for 10 minutes;
(6) having the activity of transforming human lymphocytes;
(7) To promote the incorporation of tritiated thymidine into cell nuclei.

  The autoimmune enhancer of the present invention is a novel one derived from red algae, whose aggregation activity can be controlled by ionic strength, has excellent recognition sugar chain selectivity, and has autoimmune activity such as activation of cellular immunity. At 100 ° C. for 10 minutes.

  Next, the best mode for carrying out the present invention will be described with reference to examples, but the present invention is not limited to these examples.

(A) Extraction step of water-soluble fraction Tsurusilamo (from Yoshinogawa estuary, Tokushima prefecture) was washed with a 0.15 M aqueous sodium chloride solution and then dried in the sun to obtain a dried product. To 100 g of the dried product, 700 ml of 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride was added and homogenized, and the homogenized solution was left at 4 ° C. for 6 hours, centrifuged, and the supernatant was obtained. A crude extract was obtained.

(B) Separation Step of Crude Active Fraction Next, ammonium sulfate was added to the crude extract so as to give a solution having a final concentration of 35% saturation, and the first step of salting out was performed. After the addition of ammonium sulfate was completed, the mixture was left at 4 ° C. for 1 hour, and the formed precipitate was removed by centrifugation. By this operation, impurities such as dyes were removed as a precipitate fraction. Next, ammonium sulfate was added to the supernatant obtained by centrifugation so that the solution had a final concentration of 70% saturation. After completion of the addition, the mixture was allowed to stand at 4 ° C. overnight. The resulting precipitate was separated by centrifugation. The obtained precipitate fraction was redissolved in 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride to obtain a crude active fraction. The hemagglutinating activity of the obtained crude active fraction on rabbit erythrocytes was 256 units, the specific activity was 3372.9 units / mg protein, and the activity recovery was 62.4%. Here, the unit of the agglutinating activity was defined as the reciprocal of the maximum dilution ratio of the sample from which the agglutinating activity could be detected. Table 5 shows the results.

(C) Purification step of agglutinin Next, the crude active fraction thus obtained was heat-treated at 100 ° C for 10 minutes, centrifuged to remove insoluble contaminating proteins, and then subjected to gel filtration chromatography for molecular weight analysis. Fractions of 100,000 or more were fractionated and separated by ion exchange chromatography using TSKgelDEAE-5PW to obtain a purified sample. The minimum protein concentration showing hemagglutination activity on rabbit erythrocytes of the obtained purified sample was 0.8763 μg / ml. From the above results, it can be seen that the use of the autoimmune enhancer of the present invention allows red algae-derived haemagglutinin to be effectively obtained while maintaining its activity.

  When the purified sample was tested for ionic strength dependence of agglutinating activity on rabbit erythrocytes, the agglutinating activity at a concentration of 0.15 M sodium chloride was 2048 units, while the agglutinating activity at a concentration of 0.4 M sodium chloride was 8 units. Met. Table 6 shows the results.

  The agglutinating activity of the purified sample after heat treatment at 100 ° C. for 10 minutes was 2048 units, and no disappearance of the agglutinating activity by the heat treatment was observed. Since the hemagglutinin agglutinin activity is one of the indicators of the hemagglutinin's sugar-binding activity, the above results show that the autoimmune enhancer of the present invention has a stable heat-binding activity to hemagglutinin against heat. I understand what it is.

In order to examine the mitogenic activity of the purified preparation, a human lymphocyte blastogenesis test was performed. The lymphocyte blastogenesis test is often used to measure and compare the DNA synthesizing ability of peripheral blood lymphocytes of patients and healthy persons. This response is believed to be indicative of a general cellular immune response capacity. As a measuring method, there are a method of counting the number of cells in which chromosomes have appeared in a fixed stained sample, a method of morphological observation, and the like.In this example, a method of measuring the incorporation of ³ H-thymidine into the cell nucleus was performed. Was. The experiment was performed using lymphocytes from samples of three healthy persons.

As a culture solution, an aqueous solution prepared by dissolving 1.05 g of RPMI 1640, 0.2 g of NaHCO ₃ , 10,000 Unit of penicillin, 10 mg of streptomycin, and 10 ml of fetal calf serum in 100 ml of pure water was prepared. And stored in a small bottle at -20 ° C.

  As a comparative mitogen, common bean lectin was dissolved in a culture solution to adjust the concentration to 10 to 50 μg / ml. Dispensed into sterile small test tubes, sealed and stored at -20 ° C.

Lymphocytes were separated as follows. That is, lymphocytes were separated from heparin-added blood by the Ficoll-Conray method, and washed three times with CMF-PBS (pH 7.0). The separated lymphocytes were suspended in 1 ml of the culture solution, and the number of lymphocytes was calculated. Then, a lymphocyte suspension adjusted to 5 × 10 ⁵ cells / ml with a culture solution was obtained.

Culture of lymphocytes was performed as follows. That is, 200 μl of the lymphocyte suspension was dispensed into each well of the microplate. Next, as a mitogen solution, a purified preparation, a comparative mitogen as a positive control, and a phosphate buffer (PES) as a negative control were dispensed in an amount of 20 μl to each well. Then, the cells were cultured for 3 days in a humidified state at 37 ° C. in air at a CO ₂ concentration of 5%. Eight hours before the end of the culture, ³ H-thymidine was dispensed into each well so that the final concentration in the culture solution was 1 μCi / ml.

  The activity was measured as follows. That is, cells were collected on a glass fiber filter while harvesting the inside of the wells with a saline solution using Labo-MASH, and the cells were continuously aspirated to wash the cells on the filter (about 20 seconds in physiological saline for about 1 second). .5 ml). Next, the cell-fixed portion on the glass filter was peeled off and placed in a counting vial. After sufficiently drying, 5 ml of toluene scintillator (0.1 g of POPO + 5 g / l toluene of PPO) as a liquid scintillator was dispensed into each vial using a dispenser, and counted with a scintillation counter. The results are shown in Table 1 as the average of three measurements per sample.

  From this table, it can be seen that the autoimmune enhancer of the present invention shows higher mitogenic activity than conventionally known hemagglutinin derived from land plants.

(A) Extraction step of water-soluble fraction Tsurusilamo (from Yoshinogawa estuary, Tokushima prefecture) 500 g of wet mass was washed with 0.15 M aqueous sodium chloride solution and then frozen at -30 ° C. A 0.5 M tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.2) containing 30 mM potassium chloride, 3 μM zinc sulfate, and 5 mM 2-mercaptoethanol was used as an extraction buffer, and finely ground frozen seaweed (Trushiramo) was used. After adding 800 ml of extraction buffer to the mixture and homogenizing the mixture, the homogenized solution was allowed to stand at 4 ° C. for 6 hours and centrifuged to obtain a supernatant crude extract.

(B) Separation Step of Crude Active Fraction Next, ammonium sulfate was added to this crude extract so that a final concentration of a 35% by mass saturated solution was obtained, and the first stage of salting out was performed. After the addition of ammonium sulfate was completed, the mixture was left at 4 ° C. for 1 hour, and the formed precipitate was removed by centrifugation. By this operation, impurities such as dyes were removed as a precipitate fraction. Next, ammonium sulfate was added to the supernatant obtained by centrifugation so as to obtain a 70% saturated solution at the final concentration. After completion of the addition, the mixture was allowed to stand at 4 ° C. overnight, and the formed precipitate was separated by centrifugation. The fractionated precipitate fraction was redissolved in 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride, and then dialyzed against 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride. Thus, a crude active fraction was obtained. The hemagglutination activity of the obtained crude active fraction on rabbit erythrocytes was 256 units. Here, the unit of the agglutinating activity was defined as the reciprocal of the maximum dilution ratio of the sample from which the agglutinating activity could be detected.

(C) Purification step of agglutinin Next, the crude active fraction thus obtained was heat-treated at 100 ° C. for 1 minute, centrifuged to remove insoluble contaminating proteins, and then subjected to gel filtration chromatography for molecular weight of 100,000 or more. Was fractionated and separated by ion exchange chromatography using TSKgelDEAE-5PW to obtain a purified sample. The hemagglutination activity of the purified sample thus obtained on rabbit erythrocytes was 2048 units. The above results indicate that the autoimmune enhancer of the present invention can be obtained while maintaining its activity.

  When the ionic strength dependence of the agglutinating activity on rabbit erythrocytes of the purified sample was examined, the agglutinating activity at a concentration of 0.15 M sodium chloride was 2048 units, whereas the agglutinating activity at a concentration of 0.4 M sodium chloride was not. 8 units.

  When the purified sample was subjected to a heat treatment at 100 ° C. for 10 minutes, the agglutinating activity was measured.

  Mitogenic activity was measured for the crude active fraction and the purified preparation. A human lymphocyte blastogenesis test was performed.

Next, a human lymphocyte blastogenesis test based on incorporation of ³ H-thymidine was performed to measure the mitogenic activity of the crude active fraction and the purified sample. In this case, materials necessary for all cell culture, for example, microplate, cell harvester, glass fiber filter, counting vial, ³ H-thymidine, toluene scintillator (POPO 0.1 g + PPO 5 g / liter toluene), preparation of liquid scintillation counter and All operations using these were performed aseptically.

Next, as a culture solution, an aqueous solution prepared by dissolving 1.05 g of RPMI 1640, 0.2 g of NaHCO ₃ , 10,000 Unit of penicillin, 10 mg of streptomycin, and 10 ml of fetal bovine serum in 100 ml of pure water was prepared. According to the amount used, the mixture was packed in a small bottle, sealed and stored at -20 ° C. In this state, storage was possible for two months. They were used up when used and freeze-thaw was not repeated.

Lymphocytes were separated from heparinized blood by the Ficoll-Conley method. Next, after washing three times with CMF-PBS (pH 7.0), the cells were suspended in 1 ml of a culture solution, and the number of lymphocytes was calculated. Then, it was adjusted to 5 × 10 ⁵ cells / ml with a culture solution.

  Lymphocyte culture was performed by dispensing 200 μl of the lymphocyte suspension into each well of the microplate.

Then, the microplate containing the lymphocytes was placed in a clean booth. The experiment was performed by three experimental plots. A control group A, which was left in a clean booth for 30 minutes without irradiation with ultraviolet rays, was set as a group A. An experimental plot in which the lymphocytes in the microplate were irradiated with ultraviolet rays from above for 30 minutes was designated as experimental plot B. An experimental plot in which the lymphocytes in the microplate were irradiated with ultraviolet rays from above for 16 hours was designated as experimental plot C. Ultraviolet irradiation was performed as follows. The microplate is placed in a chromatoview portable dark box (Funakoshi Co., Ltd.), and ultraviolet light of a long wavelength (365 nm) is emitted from a 6-watt handy type UV lamp UVL-56 type black ray lamp (Funakoshi Co., Ltd.) attached to the upper part of the dark box. Irradiated. At this time, the UV intensity at 365 nm was measured by connecting a MODEL UVX-36 sensor (Funakoshi Co., Ltd.) to a digital UVX RADIOMETER UV intensity meter (Funakoshi Co., Ltd.). The ultraviolet intensity at the position of the microplate was 0.63 mW / cm ² .

Next, as a mitogen solution, 20 μl of a crude active fraction, a purified sample, and a phosphate buffer (PES) were dispensed to each well in each well. As the crude active fraction, a diluted solution (10-fold dilution to 320-fold dilution) diluted with a buffer solution was prepared and used for the experiment. The amount of ³ H-thymidine incorporation (cpm) in the crude active fraction was determined by multiplying the measured value in the diluent by the dilution factor and calculating the value converted to the undiluted solution. As the purified sample, a diluent (10-fold to 320-fold) diluted with a buffer solution was prepared, and the diluted solution was used for the experiment. The amount of incorporation of ³ H-thymidine in the purified sample (cpm) was determined by multiplying the measured value of the diluent by the dilution factor and calculating the value converted to the undiluted solution.
Then, the cells were cultured in the air containing 5% CO ₂ at 37 ° C. in a wet state for 3 days. Eight hours before the end of the culture, ³ H-thymidine was dispensed into each well so that the final concentration per culture solution was 1 μCi / ml.

  The activity was measured as follows. The cells were collected on a glass fiber filter while harvesting the inside of the well with a saline solution using Labo-MASH or the like, and this was continuously aspirated to wash the cells on the filter (for about 20 seconds, about 1. 5 ml). Next, the cell-fixed portion on the glass filter was peeled off and placed in a counting vial. Next, after sufficient drying, 5 ml of a liquid scintillator was dispensed into each vial using a dispenser, and the count was performed with a scintillation counter. An experiment was performed using lymphocytes from three samples (hereinafter, referred to as samples a, b, and c) different from the three used in Example 1. The number of experiments under certain experimental conditions was set to 3 (described in the table as 1, 2, and 3), and the average indicates the average value of three measurements. Table 2 shows the results for the sample a, Table 3 shows the results for the sample b, and Table 4 shows the results for the sample c.

As is clear from the experimental section A in Tables 2 to 4, the autoimmune enhancer of the present invention comprising the crude active fraction obtained in Example 2 and the purified sample was more effective than the negative control in the amount of ³ H-thymidine. Since the incorporation amount of, was 600 times or more and 3400 times or more, respectively, it can be seen that excellent mitogenic activity was exhibited.
In addition, as is clear from the average values of the negative controls in Tables 2 to 4, it can be seen that the amount of ³ H-thymidine incorporation, that is, the immunity, decreases when irradiated with ultraviolet rays. As can be seen, the addition of the autoimmune enhancer of the present invention promotes the incorporation of ³ H-thymidine even when irradiated with ultraviolet light.
From the above results, it was found that the crude active fraction of the autoimmune enhancing component / purified sample was added to human lymphocytes having reduced immunity such as DNA synthesis ability (such as incorporation of ³ H-thymidine) by ultraviolet irradiation treatment. In addition, immunity such as the DNA synthesis ability of the lymphocyte can be enhanced. When the ultraviolet irradiation time is within 30 minutes, the DNA synthesis ability can be increased to the same level as when an autoimmune enhancing component is added to human lymphocytes not irradiated with ultraviolet light. Irradiation with ultraviolet light for 16 hours can increase the DNA synthesis ability to 50% or more of that obtained by adding an autoimmune enhancing component to human lymphocytes not irradiated with ultraviolet light, and a negative control without irradiation with ultraviolet light It can be seen that the incorporation of ³ H-thymidine is much larger than that of.

Comparative Example 1
In the fractionation step of the crude active fraction in Example 1- (b), instead of the fractionation treatment by two-stage salting out by adding ammonium sulfate, fractionation treatment with 50% by mass ethanol [“Phytochemistry”, Vol. 27 , Pages 2063-2067 (1988)], to obtain a crude active fraction in the same manner as in Example 1. The hemagglutinating activity of the crude active fraction on rabbit erythrocytes was 4 units, the specific activity was 53.4 units / mg protein, and the activity recovery was 5.0%. Table 5 shows the results.

Comparative Example 2
According to a conventional method [the method described in “Comparative Biochemistry and Physiology” (Comp. Biochem. Physiol.), Vol. 102B, pp. 445-449 (1992)], it is derived from red algae. Hemagglutinin was obtained. The obtained crude active fraction had a hemagglutination activity of 16 units, a specific activity of 149.5 units / mg protein, and an activity recovery of 19.5%. Table 5 shows the results. The minimum protein concentration of the purified sample showing the hemagglutination activity on rabbit erythrocytes was 32.6 μg / ml, which corresponded to a specific activity of about 1/40 of Example 1.

Comparative Example 3
Purified from red algae according to a conventional method [the method described in “Comparative Biochemistry and Physiology” (Comp. Biochem. Physiol.), Vol. 102B, pp. 445-449 (1992)]. Regarding agglutinin having a molecular weight of 50,000, the ion concentration dependency of the agglutinating activity on rabbit erythrocytes was examined. The aggregation activity at the concentrations of 0.15 M sodium chloride and 0.4 M sodium chloride was both 1024 units, and the ionic strength dependence of the aggregation activity was not observed. Table 6 shows the results.

Comparative Example 4
25 mg of Con A [manufactured by Wako Pure Chemical Industries, Ltd.] was dissolved in 100 ml of phosphate buffer, and the ionic concentration dependence of rabbit hemagglutination activity was examined. The aggregation activity at both the 0.15 M sodium chloride concentration and the 0.4 M sodium chloride concentration was 64 units, and the ionic strength dependence of the aggregation activity was not observed. Table 6 shows the results.

  No aggregation activity was detected after heat treatment of Con A at 100 ° C. for 10 minutes, and disappearance of the aggregation activity was observed by the heat treatment.

  As is evident from Table 5, the autoimmune enhancer of the present invention comprising the crude active fraction obtained in Example 1 has an agglutinating activity, a specific activity and an activity recovery rate as compared with those of Comparative Examples 1 and 2. In each case, the activity recovery was about 12 times that of Comparative Example 1, about 3 times that of Comparative Example 2, and the specific activity was about 63 times that of Comparative Example 1 and about 23 times that of Comparative Example 2. Table 6 shows that the purified agglutinin of Example 1 is different from those of Comparative Examples 3 and 4, and that the agglutinating activity on rabbit erythrocytes is controlled by the ionic strength.

  The autoimmune enhancer of the present invention is useful as an additive in the fields of clinical treatment, medical treatment, biochemical industry, therapeutic and test materials, and cosmetics.

Claims (16)

  An autoimmune enhancer comprising, as an active ingredient, a liquid extract of a red algae of the genus Ogonori (Graciliaria sp.) With a saline solution.   2. The autoimmune enhancer according to claim 1, wherein the red algae of the genus Ogonori is Ogonori (Gracilia verrucosa) or Tsurusilamo (Graciliaria chorda) or a subspecies thereof.   The liquid extract has the property of agglutinating sheep erythrocytes treated with pronase, and this agglutinating activity is not inhibited by monosaccharides or disaccharides, but is characterized by being inhibited by fetuin or asialofetuin. 3. The autoimmune enhancer according to 2.   The autoimmune enhancer according to any one of claims 1 to 3, wherein the liquid extract is characterized in that the agglutinating activity on rabbit erythrocytes changes depending on the ionic strength.   The autoimmune according to any one of claims 1 to 4, wherein the liquid extract elutes in a fraction corresponding to a molecular weight of 100,000 or more in gel filtration chromatography using a globular protein as a standard molecular weight substance. Enhancer.   The autoimmune enhancer according to any one of claims 1 to 5, wherein the liquid extract has a cellular immunity stimulating activity.   The autoimmune enhancer according to any one of claims 1 to 6, wherein the liquid extract is an extract characterized by having a sugar chain binding activity even after heat treatment at 100 ° C for 10 minutes.   The autoimmune enhancer according to any one of claims 1 to 7, wherein the liquid extract has an activity of transforming human lymphocytes.   The autoimmune enhancer according to any one of claims 1 to 8, wherein the liquid extract promotes the uptake of tritiated thymidine into cell nuclei.   The autoimmune enhancer according to any one of claims 1 to 9, wherein the liquid extract contains sugar and protein, and the mass of the protein is 0.4 or less based on the mass of sugar.   The autoimmune enhancer according to any one of claims 1 to 10, wherein the liquid extract contains 1 to 60% by mass of sulfuric acid.   The red algae of the genus Ogonori are extracted with an aqueous salt solution, and ammonium sulfate is first added to the resulting extract to a final concentration of 20 to 40% to carry out a first-stage salting out to remove precipitated contaminants. After that, ammonium sulfate was added to the extract to a final concentration of 60 to 80% saturation to carry out the second step of salting out. The crude active fraction was collected as a precipitate, and the precipitate was dissolved in an appropriate solvent. A method for producing an autoimmune enhancer, comprising separating and collecting a liquid extract exhibiting a cellular immunity activity activating activity.   The liquid extract was further heat-treated at 100 ° C. for 1 to 10 minutes to remove contaminating proteins, and then fractionated by gel filtration chromatography to fractions having a molecular weight of 100,000 or more, and then separated by chromatography. The method for producing an autoimmune enhancer according to claim 12, which is purified.   14. The method for producing an autoimmune enhancer according to claim 12, wherein the aqueous salt solution is a phosphate buffer containing sodium chloride.   14. The method for producing an autoimmune enhancer according to claim 12 or 13, wherein the aqueous salt solution is a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer containing at least one selected from potassium chloride, zinc sulfate and 2-mercaptoethanol. A cosmetic comprising the autoimmune enhancer according to any one of claims 1 to 11.