JP2017052912A - Peptide heteropolysaccharide, manufacturing method therefor, and complement third component activator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material capable of more effectively conducting activation of a complement third component, a manufacturing method of the material and a complement third component activator containing the material as an active component.SOLUTION: There are provided peptide heteropolysaccharide obtained by removing low molecular weight material with molecular weight of 10,000 or less from an extract obtained by hot water extraction of a cell wall crushed article of Chlorella, fractionating the resulting crude polysaccharide and purifying the same, a manufacturing method therefor and a complement third component activator containing the peptide heteropolysaccharide as an active component.SELECTED DRAWING: None

Description

  The present invention relates to a peptide heteropolysaccharide obtained from chlorella, a method for producing the same, and a complement third component activator comprising the peptide heteropolysaccharide as an active ingredient.

  The most primitive of the defense mechanisms is a series of defense mechanisms that begin with phagocytosis represented by macrophages and activation of complement.

  Complements and macrophages treat the invasion of exogenous foreign substances including pathogenic microorganisms, self-derived foreign components, waste products, excess products, waste activated products, etc., and homeostasis of the living body (homeostasis) It plays a very important role in maintaining

  Complement activation pathways centering on the third component of complement (C3) act on cellular factors such as lymphocytes, macrophages, erythrocytes, platelets, granular leukocytes, and mast cells. Plays an important role. In particular, complement plays a variety of roles in immune defense such as immune hemolysis, lysis, phagocytosis, opsonization, chemotaxis, immune cell lysis, and complement-dependent cell lysis. Furthermore, complement, which is a main factor of humoral immunity, is also involved in cellular immunity.

  JP-A-2015-44753 discloses the activation of a third complement component extracted from a chlorella cell wall disruption product with hot water and containing a polysaccharide having a molecular weight of 10,000 or more as an active ingredient.

JP 2015-44753 A

  An object of the present invention is to provide a substance capable of more effectively activating the complement third component, a method for producing the substance, and a complement third component activator containing the substance as an active ingredient. .

  The peptide heteropolysaccharide of the present invention, the production method thereof, and the complement third component activator can be expressed as follows.

(1) A peptide heteropolysaccharide having the following physicochemical properties (a) to (e):
(a) Average molecular weight: 1 million
(b) Specific rotation: [α] _D −11.6 (measurement temperature 25 ° C.)
(c) Nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%
(d) Sugar composition (molar ratio): Gal: GIc: Man: Xyl: Rha: Ara: Fru = 32: 25: 13: 8: 7: 5: 3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100%

  (2) The peptide heteropolysaccharide according to the above (1), which is derived from a hot water extract of a cell wall fragment of Chlorella pyrenoidosa.

(3) A low molecular weight material having a molecular weight of 10,000 or less is removed from an extract obtained by hot water extraction of chlorella cell wall crushed material to obtain a liquid from which a low molecular weight material having a molecular weight of 10,000 or less has been removed;
Anhydrous ethanol is added to this solution to obtain a crude polysaccharide as a precipitate,
The obtained crude polysaccharide is dissolved in water, subjected to ion exchange chromatography, and the obtained fraction is further subjected to gel filtration to purify the peptide heteropolysaccharide described in (1) above. A method for producing a peptide heteropolysaccharide.

  (4) The method for producing a peptide heteropolysaccharide according to the above (3), wherein the hot water extraction is performed with hot water at 95 to 100 ° C.

  (5) The method for producing a peptide heteropolysaccharide according to (3) or (4) above, wherein the chlorella is chlorella pyrenoidosa.

  (6) A complement third component activator comprising the peptide heteropolysaccharide according to (1) or (2) as an active ingredient.

  A complement third component activator comprising the peptide heteropolysaccharide obtained by the production method according to any one of (2) to (4) as an active ingredient.

  (7) The complement third component activator according to (6) above, wherein the complement activation pathway is the complement second pathway.

  (8) The complement third component activator according to (6) or (7), which is an orally administered agent.

  The complement third component activator comprising the peptide heteropolysaccharide of the present invention, the peptide heteropolysaccharide obtained by the production method of the present invention, and the peptide heteropolysaccharide of the present invention as an active ingredient is a complement third component ( C3) can be activated effectively, and macrophages can be activated effectively.

FIG. 5 is an antigen-antibody cross-immunoelectrophoresis pattern for dextran. FIG. It is an antigen antibody cross immunoelectrophoresis pattern about ATSO derived from Kawaratake. It is an antigen antibody cross immunoelectrophoresis pattern about FA-1a.

  (1) The chlorella in the present invention is a unicellular green algae belonging to the genus Chlorella, and examples thereof include Chlorella pyrenoidosa, Chlorella ellipsoidea, Chlorella vulgaris, and Chlorella regularis. Most suitable for the present invention is Chlorella pyrenoidosa.

  (2) A cell wall disrupted product of chlorella (preferably chlorella pyrenoidosa) that can be used for production of the peptide heteropolysaccharide of the present invention can be obtained, for example, as follows. That is, first, a chlorella powder / water suspension having a chlorella concentration of 10 to 25% by weight is adjusted to 10 ° C. or lower. Next, this suspension is fed into a continuous wet pulverizer as described below, and pulverized so that the slurry immediately after crushing becomes 40 ° C. or lower. Subsequently, the chlorella slurry thus obtained is immediately cooled to 10 ° C. or lower, so that a chlorella having a crushed cell wall can be obtained without causing quality deterioration.

  In the continuous wet pulverizer, a large number of glass beads having a diameter of 0.5 to 1.5 mm are enclosed in a closed cylinder having a cooling mantle. The capacity of the glass beads is 80 to 85% of the capacity of the sealed cylinder, and the glass beads are mixed and rotated with the inflowing liquid to grind the substance in the inflowing liquid.

  The chlorella in which the cell wall is crushed in this way can be used as it is, but it may be used after an appropriate treatment such as pulverization after vacuum drying.

  (3) The peptide heteropolysaccharide of the present invention can be produced, for example, as follows.

  Chlorella (preferably Chlorella pyrenoidosa) cell wall crushed material (for example, vacuum-dried cell wall crushed material) and hot water extraction (for example, extraction with 95-100 ° C. hot water. Extraction time is 2 for example. Time, but not limited to this), and the extract is left as a supernatant after extraction, for example.

  The low molecular weight material having a molecular weight of 10,000 or less is removed from the obtained extract (preferably the supernatant obtained by centrifugation), for example, by diafiltration or ultrafiltration, and the low molecular weight material having a molecular weight of 10,000 or less is removed. To obtain a liquid.

  To this solution is added absolute ethanol (for example, 3 times the volume), and crude polysaccharide (FA) is obtained as a precipitate (for example, a centrifuged precipitate). The crude polysaccharide (FA) is preferably obtained by washing the precipitate with absolute ethanol, then washing with anhydrous ether, and drying (preferably vacuum drying).

The obtained crude polysaccharide (FA) is dissolved in water, ion-exchange chromatography [eg, using DEAE-Cellulose (Cl ⁻ ) manufactured by Wako Pure Chemical Industries, Ltd.] and necessary gradient elution [eg, (gradient elution with 0 → 1.0M NaCl)] to obtain a fraction (FA-1).

  By purifying FA-1 by gel filtration, for example, gel filtration using Sephadex G-50 manufactured by Wako Pure Chemical Industries, Ltd. and necessary gradient elution [for example, (gradient elution with 0 → 2.0 FA-1a, that is, the peptide heteropolysaccharide of the present invention can be obtained by purification with M NaCl)].

(4) The peptide heteropolysaccharide of the present invention has the following physicochemical properties (a) to (e). Preferably, it is a peptide heteropolysaccharide derived from a hot water extract of a cell wall fragment of Chlorella pyrenoidosa.
(a) Average molecular weight: 1 million
(b) Specific rotation: [α] _D −11.6 (measurement temperature 25 ° C.)
(c) Nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%
(d) Sugar composition (molar ratio): Gal: GIc: Man: Xyl: Rha: Ara: Fru = 32: 25: 13: 8: 7: 5: 3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100%

  (5) The complement third component activator of the present invention comprises the peptide heteropolysaccharide described in (4) above as an active ingredient. Alternatively, the peptide heteropolysaccharide obtained by the production method described in (3) above is used as an active ingredient.

  Examples of the pathway by which the third complement activator of the present invention activates complement include the complement second pathway.

  The complement third component activator of the present invention can be applied in various other dosage forms that can be usually employed as a complement third component activator in addition to oral administration.

There is no particular limitation on the form of oral administration in the complement third component activator of the present invention,
For example, it can be a powder, a tablet, a hard capsule, a soft capsule, a solution dissolved in water, or another solution.

  In forming various forms, various excipients, binders, disintegrants, lubricants, coating agents, coloring agents, flavoring agents, flavoring agents, plasticizers, and the like can be appropriately used.

  Examples of excipients include sugars (lactose, sucrose, glucose, mannitol), starch (potato, wheat, corn), minerals (calcium carbonate, calcium sulfate, sodium bicarbonate, sodium chloride), crystalline cellulose, plant powder ( Licorice powder, gentian powder) and the like.

  Examples of binders include starch paste, gum arabic, gelatin, sodium alginate, meth / recellulose (MC), ethylcellulose (EC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC). , Carboxymethylcellulose (CMC) and the like.

  Examples of the disintegrant include starch, agar, gelatin powder, crystalline cellulose, CMC / Na, CMC / Ca, calcium carbonate, sodium hydrogen carbonate, sodium alginate and the like.

  Examples of lubricants include magnesium stearate, talc, hydrogenated vegetable oil, macrogol, silicone oil and the like.

  Examples of coating agents include sugar coating (sucrose, HPC, shellac), glue (gelatin, glycerin, sorbitol), film coating [hydroxypropyl methylcellulose (HPMC), EC, HPC, PVP], enteric coating [hydroxyprovir Methyl cellulose phthalate (HPMCP), cellulose acetate phthalate (CAP)] and the like.

  Examples of the colorant include water-soluble food dyes and lake dyes. Examples of the corrigent include lactose, sucrose, glucose, mannitol) and the like. Examples of flavoring agents include aromatic essential oils) and light blocking agents (titanium oxide). Examples of the plasticizer include phthalic acid esters, vegetable oils, polyethylene glycol) and the like.

  In addition, the human dose of the complement third component activator of the present invention is preferably, for example, about 3 mg to 250 mg / day in terms of the content of the peptide heteropolysaccharide as an active ingredient.

  A test was performed for the effect of activating the third component of complement of peptide heteropolysaccharide.

  1. Manufacture of test substances

  (1) A dried powder (hereinafter also simply referred to as “chlorella”) of Chlorella pyrenoidosa with the cell wall crushed was produced as follows.

  A large number of glass beads of 0.5 to 1.5 mm in diameter with a capacity of 80 to 85% of the capacity of the sealed cylinder are enclosed in a sealed cylinder having a cooling jacket, and the glass beads are mixed and rotated with the inflowing liquid. In a continuous wet pulverizer (trade name: DYNOMILL [KD type, manufactured by WAB, Inc.) that crushes substances in the inflowing liquid, the concentration of chlorella pyrenoids adjusted to 10 ° C. or lower is 10 to 25% by weight. The chlorella / pyrenoid powder / water suspension was fed and pulverized so that the slurry immediately after crushing was 40 ° C. or lower, and then the chlorella / pyrenoid slurry thus obtained was immediately cooled to 10 ° C. By cooling to the following, vacuum drying, and pulverization, a cell wall-crushed chlorella pyrenoidosa dry powder was obtained.

  (2) The obtained cell wall disrupted chlorella pyrenoidosa dry powder was suspended in water and then boiled at 95 to 100 ° C. for 2 hours.

  After cooling this to room temperature, the supernatant was allowed to stand still and ultrafiltered using a diafilter G-10T (manufactured by Nippon Vacuum Engineering Co., Ltd .: molecular weight cut off 10,000) to obtain a first filtration residue.

  The precipitate in the standing state is again boiled with water at 95 to 100 ° C. for 2 hours, cooled to room temperature, and then the supernatant is left to stand in a diafilter G-10T (manufactured by Nippon Vacuum Technology Co., Ltd.). : A fractional molecular weight of 10000) to obtain a second filtration residue, and the first filtration residue and the second filtration residue were mixed.

  The mixture was allowed to stand at 3 ° C. for 12 hours, and then centrifuged at 10,000 rpm for 20 minutes at room temperature to separate into a supernatant and a precipitate.

  The supernatant is collected and ultrafiltered at 3 ° C. using Diafilter G-10T (manufactured by Nippon Vacuum Engineering Co., Ltd .: molecular weight cut off 10,000) to obtain an unfiltered fraction (MW 10,000 or more) and a filtered fraction. (MW 10,000 or less).

  To this non-filtered fraction, 3 times the volume of absolute ethanol was added and centrifuged at room temperature at 10,000 rpm to separate into supernatant and precipitate.

  The precipitate was washed 3 times with absolute ethanol and then once with anhydrous ether. Then, it vacuum-dried at 40 degreeC and obtained FA (crude polysaccharide).

FA is dissolved in water and subjected to ion exchange chromatography [using DEAE-Cellulose (Cl ⁻ ) manufactured by Wako Pure Chemical Industries, Ltd.] and gradient elution [(gradient elution with 0 → 1.0 M NaCl)]. -1, FA-2, and FA-3.

  Fraction FA-1 was purified by gel filtration [using Sephadex G-50 manufactured by Wako Pure Chemical Industries, Ltd.] and gradient elution (gradient elution with 0 → 2.0 M NaCl) to obtain FA-1a. .

  The yields of crude polysaccharide FA and peptide heteropolysaccharide FA-1a of the present invention from 400 g of chlorella pyrenoids dried powder were 15.63 g and 253.27 mg, respectively, as a vacuum lyophilized powder.

  As a result of analyzing FA-1a as described below, it was found to be a peptide heteropolysaccharide having the following physicochemical properties (a) to (e).

  (i) Analysis of sugar composition

  Total sugars were analyzed by the phenol-sulfuric acid method (colorimetric method).

  Analysis of the constituent sugar composition was performed as follows.

  The sample FA-1a (2 mg) was dissolved in dimethyl sulfoxide (DMSO, 0.5 mL), and sodium hydroxide (20 mg) and methyl iodide (0.2 mL) were added to the solution.

  After stirring this, extraction with chloroform (1.5 mL) was carried out, followed by drying under reduced pressure to obtain a methylated polysaccharide.

  On the other hand, 80% formic acid (1 mL) was added and vacuum-dried, and then methylated polysaccharide was obtained with 2N trifluoroacetic acid (1 mL).

  To this, 80% formic acid (1 mL) was added and vacuum-dried, and then the methylated polysaccharide was hydrolyzed with 2N trifluoroacetic acid (1 mL) and vacuum-dried to obtain a partially methylated polysaccharide.

  A small amount of distilled water and sodium borohydride (5 mg) were added to the partially methylated polysaccharide, and after standing overnight, sodium borohydride was decomposed with acetic acid.

  Further, boric acid was removed to obtain partially methylated alditol. To this was added an acetic anhydride / pyridine (1: 1) mixture (1 mL), and the mixture was heated for 4 hours and then cooled to dryness. Subsequently, chloroform (2 mL) and distilled water (1 mL) were added and mixed, the chloroform layer was recovered, anhydrous sodium sulfate (1.5 g) was added, and dehydration treatment was performed, followed by drying under reduced pressure.

  This was dissolved in 200 μL of distilled water, and 30 μL of the solution was subjected to high performance liquid chromatography (HPLC) analysis (Shimadzu LC-9A type) to identify and quantify the constituent sugars.

  (ii) Determination of total nitrogen

  It was measured by the semi-micronardal method.

  (iii) Protein quantification

  Protein quantification was performed by the Lowry method.

  (iv) Amino acid analysis

  A sample (1 mg) was sealed with 6N hydrochloric acid (1 mL), subjected to hydrolysis treatment at 110 ° C. for 20 hours, and then subjected to a Hitachi 835 amino acid automatic analyzer.

  (v) Measurement of average molecular weight

  The average molecular weight was measured by gel filtration (Sephadex G-100, Wako Pure Chemical Industries).

  As the polysaccharide molecular weight markers for gel filtration, α-1,6-linked linear polysaccharide pullulan kit Shodex standard P-82 (Showa Denko) and standard dextran (Sigma-Aldrich) were used.

  (vi) Measurement of specific rotation

The circular rotation spectrophotometer (DIP-360 type) was used to measure the specific rotation, and the measurement temperature was 25 ° C.
(a) Average molecular weight: 1 million
(b) Specific rotation: [α] _D −11.6 (measurement temperature 25 ° C.)
(c) Nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%
(d) Sugar composition (molar ratio): Gal: GIc: Man: Xyl: Rha: Ara: Fru = 32: 25: 13: 8: 7: 5: 3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100%

  2. Measurement of complement activity by cross immunoelectrophoresis

  (1) Material

  From a centrifuge tube with a lid (2 mL) into which 0.2 mL of human fresh autologous serum was dispensed, the test substance FA-1a 200 μg, the negative control dextran (molecular weight 228,000; manufactured by Hanai Chemicals) 200 μg, the positive control derived from Kawaratake 200 μg of ATSO was added, and these were used as test sera.

  As a positive control, ATSO derived from Kawaratake is one for every three linear glucose residues with β-1,3 glucoside bonds, and one molecule of glucose has β-1,6 glucoside bonds. It is a polysaccharide that branches through and exhibits antitumor activity and complement C3 activity. FA-1a which is a peptide heteropolysaccharide of the present invention is a substance different from ATSO having a β (1,3) (1,6) glucan structure.

  For the possibility of mixing polysaccharides with antigen specificity, that is, lipopolysaccharides formed by binding lipid A of 0 antigen (endotoxin endotoxin) with FA-1a, the Limulus test was conducted. Negative due to negative performance.

  (2) Measurement method

  Complement activity of each test serum was determined by antigen-antibody crossed immunoelectrophoresis [Crossed C3 antigen-antibody crossed by addition of ATSO, Shiro Shimura, Toshiro Shiomi, Hitoshi Ito, Chisuke Naruse ATSO Measurement of residual C3 activity by electrophoresis image History of medicine 1977 103 20-22, [Shimura K, Ito H, Hibasami H Screening of host-mediated antitumor polysaccharides by crossed immunoelectrophorphoresis using fresh human serum Jpn. J. Pharmacol 1983 33 403 -408]).

  Electrophoresis was performed under the condition of Tricine veronal buffer (TVB) (pH 8.6, μ = 0.05). As a support, 1.2% agarose-TVB prepared by dissolving agarose A-45 (Nacalai Tex) to 1.2% with TVB was used.

  Anti-human serum was prepared by immunizing rabbits with Freund's complete adjuvant (Difco Lab.) With human C3 (Gene Tex. Inc.). The anti-human serum diluted 10-fold with 1.2% agarose-TVB was poured on a 76 × 76 mm glass slide and solidified, and this was used as an imunoplate.

  Separately, after electrophoresis of 1.2% agarose-TVB (6-7mm width) containing 5 μL of test serum on a slide glass, this was placed on an immunoplate containing 10% of anti-human serum, and two-dimensionally. Development electrophoresis was performed. The electrophoresis conditions were 1.2 mA / cm for 5 hours.

  (3) Measurement results

  The two-dimensional antigen-antibody cross-immunoelectrophoresis patterns performed using human fresh autologous serum are shown in FIGS. 1 to 3, respectively. 1 to 3, the right side is an anode (+), and the left side is a cathode (-).

As shown in FIGS. 1 to 3, dextran (negative control), ATSO (positive control), and FA-1a show almost no fluctuation in peaks of Tr (transferrin) and α ₂ -M (macroglobulin). There was no.

  In dextran, which is a negative control, C3 (native C3) appeared upright along the inner side of the (−) side of Tr and formed a skirt on the (+) side (FIG. 1).

On the other hand, in ATSO and FA-1a, the peak of native C3 decreased, and the converted C3 (converted C3) shifted to the (+) side with a trimodality (the letters in FIGS. 2 and 3 are attached). No arrow). Comparing the migration patterns of dextran, ATSO and FA-1a, the ratio of the height of the stable α ₂ -M peak to the peak of converted C3 was the lowest for dextran, and ATSO and FA-1a were almost the same. It was.

  From the above results, it was confirmed that FA-1a also acts as a complement third component (C3) activator like ATSO.

  As the complement activation pathway, the pathway that is activated in the order of C1-> C4-> C2-> C3-> C5-> C6-> C7-> C8-> C9 called the classical pathway (C1assical pathway) and the alternative pathway (alternative) There is a known pathway that activates C3 and later (C3 → C5 → C6 → C7 → C8 → C9) without activating C1, C4, and C2 (pathway).

C1 of the first complement component is a complex in which three components C1q, C1r and C1s are bound via Ca ²⁺ . Ca ²⁺ chelating reagent ethylene glycol bis (β-aminoethyl ether) N, N, N ′, N′-tetraacetic acid (EGTA) In the presence of 10 mM, each of FA-1a and ATSO was electrophoresed similarly. A migration pattern was observed.

  From this, it was clarified that FA-1a and ATSO both act on the alternative pathway of the complement and activate complement C3.

  Note that the filtration fraction (MW 10,000 or less) composed of a polysaccharide having a molecular weight of less than 10,000 exhibits the same migration pattern as that of dextran, which is a negative control, (Japanese Patent Application No. 2013-44553). Publication).

  3. Measurement of in vivo complement C3 activity

  (1) Laboratory animals and rearing conditions

  6 ICR / Slc female mice of appropriate age (manufactured by SLC Japan) are raised in a barrier system environment at a temperature of 23 ± 2 ° C., a relative humidity of 55 ± 5%, and a light / dark cycle of 12 hours (starting at 8 o'clock). Feed (CLEA CE-7, manufactured by CLEA Japan) and tap water were collected freely.

  After 7 days of preliminary breeding, 5 mice were grouped in plastic cages as groups of 10 mice that had no abnormality observed in general symptom observation and urinalysis, and used for the test.

  (2) Measurement method

  For each group of mice, FA-1a (50 mg / kg) and ATSO (150 mg / kg) as test substances and dextran (250 mg / kg) as control substances were forced twice daily for 7 days by gastric catheter. Orally.

  Two hours after the final administration, intraperitoneal macrophages were collected and the number of cells was measured as follows.

  After blood removal from the mice under ether anesthesia, 3 mL of phosphate buffered saline (PBS) was injected into the abdominal cavity with a syringe. After gently rubbing the abdomen, the skin of the abdomen was peeled off, the peritoneum was grasped with tweezers, a small cut was made with scissors, and all of the peritoneal exudate cell suspension was collected in a centrifuge tube with a Komagome pipette and immediately stored in ice water.

  The collected peritoneal exudate cell suspension was placed in a centrifuge tube and centrifuged (225 × g, 3 minutes). The supernatant was discarded, and 3 mL of ice-cold PBS was added to resuspend the peritoneal exudate cells. The same operation was performed 3 times to wash the cells.

After discarding the final supernatant, add 2 mL of serum-free medium (cosmedium 001, manufactured by Cosmo Bio), float the cells with a pipette, transfer to a Petri dish (3.5 × 10 mm), and in a CO ₂ incubator at 37 ° C. Incubated for 2 hours.

  A portion of this peritoneal exudate cell suspension was taken up in melanger, diluted with Turk solution, and the number of cells was counted with a hemocytometer.

  A part of the collected intraperitoneal macrophages was suspended in Eagle's medium (manufactured by Nissui) and attached to a cover slip. Cells that did not adhere to the coverslip were washed off with PBS and immediately fixed with ethanol. The coverslip was then reacted with anti-mouse C3F (ab ') 2, washed thoroughly, and then reacted with fluorescein isothiocyanate (FITC) -labeled anti-rabbit immunoglobulin G (IgG) to obtain the complement receptor for macrophages ( The presence of C3 cleavage antigen binding to (CR) was observed with a fluorescence microscope.

  The remaining intraperitoneal macrophages were suspended in Eagle's medium, latex beads (1.1 μm, Sigma-Aldrich) were added and mixed well, poured into a petri dish with a cover slip, and incubated at 37 ° C. for 2 hours. After washing with PBS, it was immediately fixed with ethanol, stained with Giemsa, and the phagocytic ability of antibody-independent latex beads was observed under a microscope.

  (3) Results

  The results are shown in Table 1.

The comparison between the dextran-administered group and the FA-1a and ATSO-administered group was performed using Student's t-test, and the ratio difference was tested using χ ² test. Statistical processing was performed by Dunnett's t-test, and less than 5% was determined to be significant (*). Numerical values other than the rate of enhancement of phagocytic ability represent mean values ± standard deviation.

  In dextran-treated mice, 22.5% of fluorescence-positive cells were observed. On the other hand, it increased remarkably to 51.4% in mice treated with FA-1a and 63.7% in mice treated with ATSO.

  In addition, phagocytosis of latex beads was 2.7 times in FA-1a-treated mice and 4.2 times in ATSO-treated mice when the total number taken up by intraperitoneal macrophages of dextran-treated mice was used as a control.

  Oral administration of FA-1a to mice showed an increase in the number of C3 fluorescence-positive cells and the number of intraperitoneal macrophages, as well as an increase in antibody-independent phagocytosis of intraperitoneal macrophages. From these results, it is considered that the C3 cleavage antigen generated by in vivo complement activation binds to CR1 or CR3 receptor of macrophages, and this is triggered to participate in cascade activation of macrophages. In addition, by oral administration of FA-1a, C3b, which is one of C3 Cleavage (C3a, C3b, iC3b) converted from complement C3, binds to the C3b receptor present on the cell membrane of macrophages, and as a result, macrophages It is thought that activation of was caused.

  4). As described above, FA-1a enhances antibody-independent phagocytic ability and promotes the mechanism of foreign body exclusion.Therefore, from the aspect of reducing the immunosuppressive action seen during administration of anticancer drugs and protecting against infection by pathogenic microorganisms, Fa-1a is considered to be advantageous for the defense mechanism, and Fa-1a is also considered to be useful in adjuvant immuno-chemotherapy as a substance that non-specifically enhances or modifies the immune response.

Claims (8)

A peptide heteropolysaccharide having the following physicochemical properties (a) to (e):
(a) Average molecular weight: 1 million
(b) Specific rotation: [α] _D −11.6 (measurement temperature 25 ° C.)
(c) Nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%
(d) Sugar composition (molar ratio): Gal: GIc: Man: Xyl: Rha: Ara: Fru = 32: 25: 13: 8: 7: 5: 3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100%   The peptide heteropolysaccharide according to claim 1, wherein the peptide heteropolysaccharide is derived from a hot water extract of a cell wall disruption of Chlorella pyrenoidosa. Removing a low molecular weight material having a molecular weight of 10,000 or less from an extract obtained by hot water extraction of a chlorella cell wall crushed material to obtain a liquid from which the low molecular weight material having a molecular weight of 10,000 or less has been removed;
Anhydrous ethanol is added to this solution to obtain a crude polysaccharide as a precipitate,
The obtained crude polysaccharide is dissolved in water, subjected to ion exchange chromatography, and the obtained fraction is further subjected to gel filtration to purify the peptide heteropolysaccharide according to claim 1, wherein the peptide heteropolysaccharide is obtained. A method for producing a peptide heteropolysaccharide.   The method for producing a peptide heteropolysaccharide according to claim 3, wherein the hot water extraction is performed with hot water at 95 to 100 ° C.   The method for producing a peptide heteropolysaccharide according to claim 3 or 4, wherein the chlorella is chlorella pyrenoid.   A complement third component activator comprising the peptide heteropolysaccharide according to claim 1 or 2 as an active ingredient.   The complement third component activator according to claim 6, wherein the pathway for activating complement is the complement second pathway.   The complement third component activator according to claim 6 or 7, which is an orally administered agent.