JP6178162B2 - Macrophage activator containing mucin treated with β-galactosidase - Google Patents

Description

  The present invention provides a composition comprising food-derived mucins treated with β-galactosidase for activating macrophages.

  It has been shown that macrophages play a central role in the immune response in the body and attack bacteria / viruses and cancer cells that have entered the body. Today, macrophage activation therapy that activates macrophages in the body and treats infections, cancers, and the like has been developed and attracts attention.

  The present inventors have previously treated vitamin D-binding protein (hereinafter referred to as “DBP”), which is a glycoprotein present in human serum, with β-galactosidase, and if necessary, further treated with sialidase, It has been found and reported that macrophage activation can be imparted to DBP by modifying the sugar chain of DBP (Patent Document 1).

  However, there is still a need in the art for a means that can activate macrophages simply and efficiently.

  Among glycoproteins, high molecular glycoproteins consisting of 1 to several tens of sugar chains periodically linked by O-glycosidic bonds to peptide chains with a simple repeating structure are called “mucins”. Collectively. Mucin exists naturally as a component of mucus in animals and plants, and in the biological system, it exhibits antibacterial effects as an extracellular matrix in addition to physical actions such as moisturizing, protecting, and lubricating cellular tissues, and suppresses infections such as viruses. It is known to perform various important functions.

WO2012 / 029954

  An object of this invention is to provide the new means which can activate a macrophage.

  As a result of intensive studies to solve the above problems, the present inventors have found that a food-derived mucin can be treated with β-galactosidase to impart a macrophage activating action to the mucin, thereby completing the present invention. It came.

That is, the present invention has the following features.
[1] A composition containing a food-derived mucin, wherein the mucin is treated with β-galactosidase.
[2] The composition of [1], wherein the food is a plant.
[3] A method for producing a composition containing mucin treated with β-galactosidase, comprising treating mucin derived from food with β-galactosidase.
[4] The method according to [3], wherein the food is a plant.
[5] A macrophage activator comprising a food-derived mucin, wherein the mucin is treated with β-galactosidase.
[6] The macrophage activator according to [5], wherein the food is a plant.

  ADVANTAGE OF THE INVENTION According to this invention, the composition which has the effect | action which activates a macrophage from food-derived mucin can be provided. The composition of the present invention is a composition that can be obtained from mucins derived from foods rich in dietary experience, and can be taken, used, or administered on a daily basis for the purpose of activating macrophages. It is a highly composition.

FIG. 1 (A) is a photograph showing the results of CBB staining. Sample applied to each lane: (M) marker, (lane 1) mucin extract, (lane 2) processed mucin extract. 1 (B) and (C) are photographic diagrams showing the results of Western blotting using the HPA lectin of Example 2. FIG. (B) Sample applied to each lane: (lane 1) mucin extract, (lane 2) processed mucin extract. (C) Applied sample: (lane 3) purified processed mucin extract. FIG. 2 shows the phagocytic activity of macrophages treated with GcMAF, mucin extract, or processed mucin extract. FIG. 3 shows the phagocytic activity of macrophages derived from mice administered with purified processed mucin extract.

1. Mucin treated with β-galactosidase “Mucin” is a generic term for macromolecular glycoproteins in which an innumerable sugar chain is bound to a core protein called apomucin through O-glycosidic bonds (TE Maureen & D. Kurt: Introduction to Glycobiology, Chemical Doujin (2005)). More specifically, mucin has a core protein having a repeating structure consisting of 10 to 80 residues rich in serine and / or threonine, and N-acetyl at the reducing end of the sugar chain is added to the hydroxyl group of serine and / or threonine. Galactosamine (hereinafter referred to as “GalNAc”) has a structure in which α-O-glycoside bonds are bonded at a high frequency. The sugar chain may include N-acetylglucosamine, galactose, fucose, sialic acid and the like linked to GalNAc.

  In the present invention, the “mucin” is not particularly limited as long as it has the above-mentioned structure, but a mucin derived from a natural product (so-called food) that can be edible is preferable from the viewpoint of availability and safety. Foods containing mucin are known, for example, potatoes (sweet potatoes, yams, yams (Tsukuneimo, Yamatoimo), taros, etc.), lotus root, okra, moroheiya, ashitaba, tsurumurasaki, aloe and other plants, spider webs, seaweeds, Nameko, jellyfish and the like.

  In the present invention, “mucin” can be used in the form of an extract extracted from the food using a solvent or a dried product thereof. Any extraction solvent may be used as long as it can extract mucin from food. For example, water, alcohol (preferably ethanol), a mixed solution thereof, or the like can be used. Preferably, water is used as the extraction solvent. The dried extract can be obtained by subjecting the obtained extract to known drying means such as freeze drying, spray drying, blow drying, and vacuum drying.

  In the present invention, “β-galactosidase” may be derived from any organism, and those derived from bovine (bovine liver), Escherichia coli, Aspergillus, Penicillium, and the like can be used. In addition, a recombinant enzyme obtained by recombinant expression in a suitable host cell using a nucleic acid encoding β-galactosidase can also be used. β-galactosidase can take any form such as a crudely purified form, a purified form, and an immobilized form. Crude forms include, for example, processed products (eg, extracts, lyophilized products, etc.) from cells, tissues, or cell cultures. The immobilized form means a state in which it is immobilized on an appropriate solid phase. Examples of such a solid phase material include, but are not limited to, cellulose derivatives such as cellulose and nitrocellulose, sepharose, agarose, metal, glass, ceramic, resin, and the like. The shape and material of the solid phase are not particularly limited.

  The treatment of mucin with β-galactosidase can be performed by reacting mucin with β-galactosidase in an aqueous solvent. The reaction only needs to be able to remove / release mucin, more specifically, galactose bound to GalNAc in the sugar chain. 20-60 ° C., preferably 35-42 ° C., of mucin and β-galactosidase in an aqueous solvent More preferably, it can be carried out by reacting at 37.5 ° C. for 0.5 to 5 hours, preferably about 1 to 3 hours. The reaction pH is from pH 5 to pH 11, preferably in the neutral range, and may be carried out by a batch method or a continuous method. As the aqueous solvent, water or a phosphate buffer (such as a sodium phosphate buffer) can be used. The reaction can be stopped by inactivating the enzyme by heat treatment (at 60 to 70 ° C. for 5 to 15 minutes).

  The obtained mucin treated with β-galactosidase may be included in a suitable aqueous solvent to form a liquid, or may be subjected to a known drying means to form a dried product.

  The mucin treated with the extract and / or the β-galactosidase can be further purified. For purification, methods commonly used for purification of mucin can be used. For example, ammonium sulfate salting out, precipitation separation with an organic solvent (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectric point chromatography, gel Filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using substrates or antibodies, chromatography such as reverse phase column chromatography, filtration treatment such as microfiltration, ultrafiltration, reverse osmosis filtration, etc. Can be purified using one or more in combination. It is sufficient that at least a protein having a molecular weight of preferably about 45-80 kDa, more preferably about 56-80 kDa can be recovered by purification.

  In the composition or macrophage activator of the present invention, an amount appropriately selected from the range of 0.0001 mg to 100 mg can be included in the amount of protein of mucin treated with β-galactosidase. The content includes the form, usage, and dose of the composition or macrophage activator, and the age, weight, severity of disease, etc. of the human (patient) or animal in need of the composition or macrophage activator It can vary depending on the factors.

2. Other components In addition to mucin treated with β-galactosidase, the composition or macrophage activator of the present invention contains other components that are acceptable in the final form such as foods, drinks, animal feeds, pharmaceuticals, and cosmetics. Further can be included. Examples of such components include excipients, binders, disintegrants, lubricants, diluents, solubilizers, suspending agents, isotonic agents, pH adjusters, buffers, and stabilizers. , Colorant, flavoring agent, flavoring agent, antioxidant, thickener, preservative, surfactant, chelating agent, emulsifier, fruit juice, sweetener, acidulant, salt, flavor, vitamins, seasoning, spice, One or more components selected from oils, ultraviolet absorbers, alcohols, various skin nutrients, and the like can be appropriately selected and included depending on the final form of foods, drinks, animal feeds, pharmaceuticals, cosmetics, and the like.

3. Composition and use thereof The composition of the present invention can be used as a food / beverage composition, an animal feed composition, a cosmetic composition, or a pharmaceutical composition having a macrophage activating action.

  Although the form of the composition of this invention is not specifically limited, In one aspect | mode, it can be set as the liquid, solid form, or semisolid composition suitable for oral ingestion or oral administration. Solid or semi-solid compositions include, for example, pharmaceuticals having forms such as granules, capsules, tablets, fine granules, caplets, powders, pills, health foods, nutritional functional foods, dietary supplements ( Supplements), nursing foods, liquid foods, nutritionally adjusted foods, nutritional supplements, etc. A solid or semi-solid composition is prepared by mixing mucin treated with β-galactosidase with an excipient such as an excipient, a bulking agent, a binder, a disintegrant, a lubricant thickener, etc. It can be molded and manufactured according to the law. The liquid composition is provided, for example, as a liquid food (for example, a beverage such as juice, mineral beverage, sports drink) or a liquid pharmaceutical for oral administration. The liquid composition is a composition based on water, and can be produced by mixing mucin treated with β-galactosidase with water.

  In another embodiment, the composition of the present invention may be a liquid or semi-solid composition suitable for parenteral administration (eg, transdermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc.). it can. The composition in such a form is provided as a cosmetic composition in the form of a lotion, milky lotion, cream or the like, or a pharmaceutical in the form of an injection, an ointment or the like. These compositions can be produced according to a conventional method by mixing mucin treated with β-galactosidase together with auxiliary agents such as suspending agents, surfactants, emulsifiers, thickeners and the like together with water.

  The composition of the present invention has an action of activating macrophages, and can activate macrophages in humans or animals that have been ingested or administered the composition. In the present invention, “macrophage activation” means that macrophage phagocytic ability (particularly phagocytic ability via Fc receptor), active oxygen production ability and antigen presenting action are enhanced. The intake or dose of the composition is the amount of mucin protein treated with β-galactosidase per kg of body weight per intake or administration, and an appropriate amount selected from the range of 0.0001 mg to 100 mg. Or it can be administered. The amount can vary depending on the form of the composition, usage, dosage, and factors such as the age, weight, severity of disease of the human (patient) or animal in need of the composition.

  Treating or ameliorating a disease or disorder known or potentially treatable by macrophage activation by ingesting or administering the composition of the present invention, or against the disease or disorder Resistance can be enhanced. Examples of such diseases and disorders include wound healing, allergic diseases, autoimmune diseases, side effects due to therapeutic agents, cancer, and the like.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[Example 1] Preparation of mucin extract and processed mucin extract (preparation of extract)
50 g of Naruto gold from Tokushima was peeled off, and 450 ml of distilled water was added and ground with a mixer (ZOJIRUSHI BM-RE08-HA) for 180 seconds. After stirring at room temperature for 5 hours, the water extraction filtered through filter paper was centrifuged at 4 ° C. and 3000 rpm for 10 minutes (TOMY CX-200 TA-4), and the supernatant was recovered. The collected liquid was dispensed 100 ml each on a dialysis membrane clipped on one side, the other side was also clipped, floated and floated in 4 L of ion exchange water for dialysis. 120 minutes after the start of dialysis, ion-exchanged water was replaced with fresh one and dialyzed overnight to obtain about 400 ml of mucin extract.

(Protein quantification)
Protein quantification in the mucin extract was performed using the bicinchoninic acid (BCA) protein measurement kit (PIERCE, Reagent A [Lot NO.HH106101, PROD # 23223], and Reagent B [Lot NO. CE49183, PROD # 23224] (Takara Bio Inc.) A 10 mM phosphate buffer solution of pH 7.0 was added to the mucin extract, diluted 25-fold and 50-fold, and 25 μl each was applied to a 96-well plate, separately from bovine serum albumin (Albumin from Bovine Serum from Sigma-Aldrich) COH) was adjusted to 0, 0.1, 0.25, 0.5, 1.0, 2.0 mg / ml by adding 10 mM phosphate buffer at pH 7.0, and 25 μl each was applied to the 96-well plate. 1 ml: mixed at a ratio of 20 μl of solution B) was applied 200 μl at a time, reacted at 37 ° C. for 30 minutes, and the absorbance was measured at 570 nm.
As a result, the amount of protein in the mucin extract was 0.906 μg / μl.

(Preparation of processed mucin extract)
37.5 by adding 28.3 μl of β-galactosidase (WAKO: dissolved in 100 mM sodium phosphate buffer pH 7.0 to 2000 U / ml) to 50 ml of mucin extract (protein amount 0.906 μg / μl) Incubated for 3 hours at ° C. Thereafter, the enzyme was inactivated by heat treatment at 60 ° C. for 10 minutes to obtain a processed mucin extract treated with β-galactosidase.

[Example 2] Protein separation by electrophoresis of processed mucin extract and Western blotting using HPA lectin (sample preparation)
The mucin extract (1) and the processed mucin extract (2) prepared by the method described in Example 1 and 500 μl of the mucin extract were ultrafiltered with Amicon Ultra 10K (Lot.R3CA66596) (Merck Millipore). (4 ° C., 13500 rpm, 15 minutes), each sample prepared so that the amount of protein was 2 μg with respect to a solution (purified processed mucin extract) (3) obtained by treating the collected concentrated solution with β-galactosidase (SDS-polyacrylamide) The gel was applied and electrophoresed at 300 V. Subsequently, it was transferred to a PVDF membrane (47 V, 1 hour), washed with TBST buffer (10 minutes × 3 times), and then blocked with 1% BSA (TBST 10 ml: BSA 100 mg). Then, it was washed with TBST buffer (10 minutes × 3 times), and reacted with the primary antibody (HPA lectin diluted 2000 times) at room temperature for 2 hours. After the primary antibody reaction, it was washed with TBST buffer (10 minutes × 3 times) and reacted with the secondary antibody (5000-fold diluted streptavidin) at room temperature for 2 hours. After the secondary antibody reaction, wash with TBST buffer solution (10 minutes x 3 times), apply ECL solution (A solution 1 ml: B solution 1 ml) (GE Healthcare), react at room temperature for 1 minute, and sensitize for 3 minutes And then shot. After shooting, the PVDF membrane was washed with TBST buffer (10 minutes x 3 times), then applied with 1 ml of CBB staining solution (CBB solution 1 ml: 20% acetic acid 1 ml), decolorized with decoloring solution, and dried overnight. .

(result)
FIG. 1 (A) shows the results of SDS-PAGE CBB staining of the mucin extract (1) and the processed mucin extract (2). No band was confirmed in the lane of the mucin extract (1). This suggests that the protein is a high-molecular glycoprotein in which an infinite number of sugar chains are bonded through O-glycoside bonds. In the lane of the processed mucin extract (2), bands were observed near 80, 70, 56, 45 and 26 kDa.

  Fig. 1 (B) shows mucin extract (1) and processed mucin extract (2), and Fig. 1 (C) shows Western blotting of purified processed mucin extract (3) using HPA lectin. . As a result, no HPA lectin-positive band was observed in the lane of the mucin extract (1), but in the lane of the processed mucin extract (2) and the purified processed mucin extract (3), it was around 80, 70 and 56 kDa. HPA lectin positive band was seen.

  These results suggest that an O-type glycoprotein having a GalNAc structure was formed from a huge glycoprotein contained in the mucin extract by cleavage of the sugar chain by β-galactosidase treatment.

  From the above results, it was possible to obtain a HPA lectin-positive glycoprotein by treating the mucin extract and its concentrated solution with β-galactosidase.

[Example 3] Effect of processed mucin extract on macrophage phagocytic activity (in vitro)
The effect of the processed mucin extract prepared by the method described in Example 1 on the phagocytic activity of macrophages was evaluated by the following procedure.

(experimental method)
An intraperitoneal mixed cell was taken out by injecting 10 ml of 10 mM PBS buffer into the abdominal cavity of an 8-week-old ICR mouse (female), and centrifuged at 4 ° C. and 1,000 rpm for 15 minutes. The collected cells were adjusted to 1.0 × 10 ⁶ cells / ml with RPMI1640 medium, and 500 μl each was seeded at 5.0 × 10 ⁵ cells / well on a plate with a cover glass submerged. To the cells, 500 μl of RPMI medium was added and precultured at 37.5 ° C. for 1 hour to fix the macrophages on the cover glass. Thereafter, the supernatant was removed and the adhering macrophage layer was washed, and a fresh RPMI medium was added, followed by culturing at 37 ° C. for 15 hours.

  To this prepared macrophage layer, mucin extract (modified with 100 mM SPB at pH 7.0) or processed mucin extract (1.0 ng, 10 ng or 100 ng in protein amount) or control (note that “control” is the above As a positive control, purified GcMAF (synthesized in the Hori laboratory of the University of Tokushima Sociotechnoscience) 1.0 was used as a positive control. Each ng protein amount was added, and the cells were cultured at 37 ° C. for 3 hours. Subsequently, macrophages were added with 0.5% opsonized SRBC and phagocytosed for 90 minutes, then the macrophages were fixed and stained with Giemsa, and then the phagocytic index (ingestion index) was calculated by counting the phagocytosed SRBC. Macrophage phagocytic activity was evaluated.

(result)
With the control group as the relative value 1.00, the average value (n = 3) of the phagocytic index of the group treated with the processed mucin extract (1.0 ng, 10 ng or 100 ng in terms of protein amount) was 1.89, 1.95, 1.93, respectively. There was a significant increase compared to the control group (see FIG. 2). On the other hand, the average value (n = 3) of the phagocytic index of the group treated with the mucin extract (1.0 ng, 10 ng or 100 ng as the protein amount) was 0.95, 1.02 and 0.91, respectively, which was the same level as the control group. It was.

  As mentioned above, the macrophage phagocytic activity excellent in the processed mucin extract was recognized in vitro.

[Example 4] Effect of purified processed mucin extract on macrophage phagocytic activity (ex vivo)
(Preparation of purified processed mucin extract)
To mucin extract (protein amount 0.906 μg / μl) 22.1 μl prepared by the method described in Example 1, 2.5 μl β-galactosidase (10 mU / μl, Grade III from Bovine Liver, SIGMA, Lot NO. 54H7025, G1875 ) And 100 mM SPB buffer (pH 7.0) was added thereto to make the total volume 200 μl. The resulting mixture was incubated at 37.5 ° C. for 1 hour, and then heated with HOT DRY BATH at 60 ° C. for 10 minutes to inactivate the enzyme. Subsequently, the obtained processed mucin extract was ultrafiltered by the method described in Example 2, and 100 mM SPB was prepared so that the protein amount of the obtained purified processed mucin extract was 6 ng / 500 μl or 60 ng / 500 μl. Adjustment was made using a buffer solution (pH 7.0).

(experimental method)
ICR mice (female) aged 8 weeks were injected intraperitoneally with 6 ng / 500 μl or 60 ng / 500 μl of the prepared purified mucin extract and 500 μl of control, respectively, and left for 3 hours (note that “control” is the above) The supernatant obtained by culturing cells collected from the mouse abdominal cavity in RPMI1640 medium was used.) Subsequently, the intraperitoneal mixed cells were taken out from the abdominal cavity of each mouse by the method described in Example 3, and 500 μl was seeded on a plate with a cover glass submerged at 5.0 × 10 ⁵ cells / well. Add 500 μl of RPMI medium to the cells, pre-incubate for 1 hour at 37.5 ° C., fix macrophages on cover glass, replace with fresh RPMI medium, add 0.5% opsonized SRBC, and phagocytose macrophages for 90 minutes Let Macrophages were fixed and stained with Giemsa, and then the SRBC phagocytosed with a microscope was counted to calculate the ingestion index, and the phagocytic activity of macrophages was evaluated.

(result)
When the control group is expressed as a relative value of 1.00, the average value (n = 3) of the phagocytic index of the group treated with the purified processed mucin extract (6 ng and 60 ng in protein amount) is 1.463 and 1.539, respectively. , Significantly increased compared to the control group (see FIG. 3).

  From this, macrophage phagocytic activity excellent in purified processed mucin extract was observed in vivo.

  ADVANTAGE OF THE INVENTION According to this invention, the highly safe composition which can be ingested, used or administered on a daily basis for the purpose of activating a macrophage can be obtained, and the food-drinks composition which has a macrophage activation effect | action Cosmetic compositions and / or pharmaceutical compositions can be provided.

Claims (6)

A composition for activating macrophages containing food-derived mucin, wherein the mucin is treated with β-galactosidase.   The composition according to claim 1, wherein the food is a plant. A method for producing a composition for activating macrophages containing β-galactosidase-treated mucin, which comprises treating food-derived mucin with β-galactosidase.   The method according to claim 3, wherein the food is a plant.   A macrophage activator comprising a mucin derived from food, wherein the mucin is treated with β-galactosidase.   The macrophage activator according to claim 5, wherein the food is a plant.

Images