JP6144997B2 - Complement third component activator - Google Patents

Description

  The present invention relates to a complement third component activator comprising a specific chlorella extract 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 exogenous foreign substances including pathogenic microorganisms, self-derived foreign components, waste products, excess products, waste activated products, etc. It plays a very important role in maintaining.

  So far, chlorella extract is known to induce Th1 (type 1 helper T cell) activity and promote production of IFN-γ (interferon gamma) and IL-12 (interleukin 12) ( Hasegawa T, Ito K, et al .: Oral administration of hot water of extracts of chlorella vulgaris reduces IgE production against milk case in mice. Int J Immunopathol pharmacol 21 (5): 311-323 1999).

  As one of its active ingredients, two polysaccharides mainly composed of galactose or rhamnose were isolated from Chlorella pyrenoidosa extract to inhibit the growth of lung cancer cells (A549 human lung adenocarcinoma cells). (Sheng J, Yu F, et al .: Preparation, identification and their antitumor activities in vitro of polysaccharides from Chlorella pyrenoidosa. Food Chem 105: 533-539 2007).

  However, it is not known about the activation of the third component of complement by the chlorella extract.

Hasegawa T, Ito K, et al .: Oral administration of hot water of extracts of chlorella vulgaris reduces IgE production against milk casein in mice. Int J Immunopathol pharmacol 21 (5): 311-323 1999 Sheng J, Yu F, et al .: Preparation, identification and their antitumor activities in vitro of polysaccharides from Chlorella pyrenoidosa. Food Chem 105: 533-539 2007

  It is an object of the present invention to provide a complement third component activator comprising a specific chlorella extract as an active ingredient.

  The complement third component activator of the present invention can be expressed as follows.

  (1) A complement third component activator comprising, as an active ingredient, a polysaccharide having a molecular weight of 10,000 or more, extracted from a chlorella cell wall fragment with hot water.

  (2) The complement third component activator according to (1), wherein the complement activation pathway is the complement second pathway.

  (3) The complement third component activator according to (1) or (2), wherein the polysaccharide is extracted from a chlorella cell wall fragment with hot water at 95 to 100 ° C.

  (4) The complement third component activator according to any one of (1) to (4), wherein the chlorella is chlorella pyrenoid.

  (5) The complement third component activator according to any one of (1) to (5), which is an orally administered agent.

  The complement third component activator comprising as an active ingredient a polysaccharide having a molecular weight of 10,000 or more extracted from the chlorella cell wall crushed material of the present invention with hot water effectively comprises the complement third component (C3). Can be activated.

Cross immunoelectrophoresis pattern of complement third component (C3)

  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, Chlorella regularis and the like. Most suitable for the present invention is Chlorella pyrenoidosa.

  The chlorella cell wall crushed material used for the production of the complement third component activator 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.

  The polysaccharide having a molecular weight of 10,000 or more, which is an active ingredient of the complement third component activator of the present invention, can be obtained, for example, by filtering a hot water extract from a chlorella cell wall fragment.

  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.

  The form of oral administration in the complement third component activator of the present invention is not particularly limited. For example, it can be a powder, a tablet, a hard capsule, or a soft capsule.

  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, for example, 5 mg to 50 mg in the content of polysaccharide having a molecular weight of 10,000 or more extracted from the chlorella cell wall crushed material as an active ingredient with hot water. / Day is preferred.

  The complement third component activator of the present invention has a molecular weight of 1 extracted with hot water from a chlorella cell wall crushed material, which is an active ingredient, in a food or beverage composition that constitutes, for example, a nutritional food, a nutritional supplement, or a beverage liquid It can also be set as the form containing 10,000 or more polysaccharides, and can also contain the various components which do not impair the effect of this invention.

  A test was conducted on the third component activation effect of a polysaccharide extracted from a chlorella cell wall fragment with hot water and having a molecular weight of 10,000 or more.

  1. Manufacture of test substances

  A dry powder of Chlorella pyrenoidosa (hereinafter, also simply referred to as “chlorella”) having a disrupted cell wall 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. The cells were cooled below, dried under vacuum, and then pulverized to obtain a cell wall-crushed chlorella / pyrenoidosa dry powder as a test substance.

  The obtained cell wall-disrupted chlorella / pyrenoid 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 Diafilter G-10T (manufactured by Bioengineering Co., Ltd .: molecular weight cut off 10,000) to obtain a first filtration residue.

  The precipitate in the stationary state is again boiled with water at 95 to 100 ° C. for 2 hours, cooled to room temperature, and then the supernatant is allowed to stand in a diafilter G-10T (manufactured by Bioengineering Corporation: The second filtration residue was obtained by ultrafiltration using a molecular weight cut-off of 10,000), 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 was collected and ultrafiltered at 3 ° C. using Diafilter G-10T (manufactured by Bioengineering Co., Ltd .: molecular weight cut-off 10000) to obtain a non-filtered fraction (MW 10,000 or more) and a filtered fraction ( MW 10,000 or less: Filter 3) was obtained.

  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, what was vacuum-dried at 40 degreeC was set to Sample F1 (CP). The yield of CP was 2.75%.

  After adding 3 times the volume of absolute ethanol to Filter 3 and concentrating under reduced pressure at 40 ° C., the mixture was centrifuged at 10,000 rpm at room temperature to separate into supernatant and precipitate. The precipitate was washed three times with absolute ethanol, then once with anhydrous ether, and then vacuum-dried at 40 ° C. as Sample F2.

  The obtained non-filtered fraction CP (F1) composed of a polysaccharide having a molecular weight of 10,000 or more and the filtered fraction F2 composed of a polysaccharide having a molecular weight of less than 10,000 were each dissolved in physiological saline and treated at 115 ° C. for 10 minutes. Used after autoclaving.

  2. Measurement of activity of complement third component (C3) by cross immunoelectrophoresis

  Complement third component activity was measured by capturing changes in the immunoelectrophoresis image of the third complement component (C3).

  300 μg of the above CP (non-filtered fraction consisting of polysaccharides with a molecular weight of 10,000 or more) is added to a centrifuge tube with lid (0.2 ml) into which 0.2 ml of serum has been dispensed, 300 μg of dextran as a negative control, and 300 μg of ATSO from Kawaratake as a positive control The migration pattern of native C3 and converted C3 was measured by cross immunoelectrophoresis.

  ATSO is an antitumor glucan in which one molecule of glucose branches via β-1,6 glucoside bonds at a ratio of one linear glucose residue with three β-1,3 glucoside bonds It is.

  As a result, as shown in Fig. 1, ATSO and CP showed a large change in the electrophoretic pattern, showing trimodality and easily moving to the (+) side, whereas the negative control dextran changed to the electrophoretic pattern. No complement activity was observed in classical and alternative pathways.

  The filtrate fraction (F2) having a molecular weight of less than 10,000 also showed the same migration pattern as the negative control.

  From the above results, it was revealed that a polysaccharide having a molecular weight of 10,000 or more extracted from crushed cell wall of chlorella with hot water exerts a complement third component activation effect.

That is, factor B (C3 proactivator, GBG), an activator of the alternative pathway, is activated by factor D (C3 proactivator convertase) to become Bb (C3 activator, GGG). When CP or ATSO was added as shown in FIG. 1, the β ₁ -A peak decreased and the β ₁ -C peak increased.

  Complement activation pathways include pathways that are activated in the order of C1-> C4-> C2-> C3-> C5-> C6-> C7-> C8-> C9 and the alternative pathway (complement second pathway). There is known a pathway for activating C5, C6, C7, C8, and C9 after C3 without activating C1, C4, and C2.

C1 of the first complement component is a complex in which three components C1q, C1r, and C1s are bound via Ca ²⁺ . Therefore, a similar migration pattern was observed even when electrophoresis was performed in the presence of 10 mM of the chelating reagent EGTA [ethylene glycol bis (β-aminoethyl ether) N, N, N ′, N′-tetraacetic acid]. It was revealed that ATSO acted in the alternative pathway and activated complement C3.

  It is presumed that CP is beneficial to the living body from the viewpoint of protection against infection by pathogenic microorganisms.

Claims (5)

A method for producing a complement third component activator comprising as an active ingredient a polysaccharide obtained by filtering a chlorella pyrenoidosa cell wall crushed material extracted with hot water to remove a material having a molecular weight of less than 10,000. The method according to claim 1, wherein the pathway by which the third component activator of complement activates complement is the complement second pathway. The method according to claim 1 or 2, wherein the cell wall disruption product of Chlorella pyrenoidosa and extracts at 95 to 100 ° C. hot water. The method according to any one of claims 1 to 3 , wherein the filtration is ultrafiltration using a filter having a molecular weight cut off of 10,000 . The method according to any one of claims 1 to 4 , wherein the complement third component activator is an oral administration agent.