JP6444133B2 - Anti-inflammatory polypeptide and anti-inflammatory composition containing the same - Google Patents

Description

  The present invention relates to an anti-inflammatory polypeptide and an anti-inflammatory composition comprising the polypeptide.

  In Japan, where there are few natural resources, it has long been recognized that the effective use of unused natural resources is an issue. In particular, in Japan, which has a land surrounded by the ocean, the search and use of useful marine resources has important significance.

  Among marine resources, seaweed that grows quickly and can be easily collected in the near sea has been effectively used in various fields as a raw material for bioethanol or as a raw material for various bioactive components such as fucoxanthin.

  Red algae (red algae) is mentioned as an example of the seaweed inhabiting the Japanese coast and the near sea. Red algae are seaweeds that exhibit a purple to red color as a whole, including pigments involved in photosynthesis, such as chlorophyll, phycocyanin, or phycoerythrin, as photosynthesis pigments, and are reported to exist in about five to six thousand species.

  Red algae have long been used as a food ingredient. For example, red algae belonging to the order of Coleoptera (Maca, etc.), Ogonori (Aegonori, etc.), or Species (Aegonori, etc.) as a raw material for Tokoroten and agar, It's being used.

  Red algae are also used as or as a source of various physiologically active substances. In particular, for an aqueous extract of Dulse (English name Dulse, scientific name Palmaria palmata), which is a kind of algae of the red alga Dulceae, the immunosuppressive action (Patent Document 1), the antitumor action (Patent Document 2), the anti-tumor action It is known to exhibit physiological activities such as radical action (Patent Document 3), cell activation action (Patent Document 4), and melanin production inhibitory action (Patent Document 5).

  The present inventors have found that a hydrolyzate obtained by successively digesting dalcosic phycobiliprotein with pepsin and trypsin has an anti-inflammatory action such as significantly suppressing carrageenin-induced edema. (Non-Patent Document 1).

Lee Daei et al., 67th Annual Meeting of the Japan Society of Nutrition and Food, Lecture No. 3L-03a, Japan Society of Nutrition and Food, April 30, 2013, page 217

JP-A-6-298661 JP-A-1-66126 Japanese Patent Publication No. 5-50483 JP-A-8-259443 Japanese Patent Laid-Open No. 10-330219

  An object of the present invention is to provide a novel anti-inflammatory polypeptide derived from phycobiliproteins contained in marine resources, red algae, and an anti-inflammatory composition containing the same.

  In the process of studying the anti-inflammatory action of a hydrolyzate derived from phycobiliprotein, the present inventors have found that an oligopeptide having a specific amino acid sequence has an anti-inflammatory action, and the following inventions Was completed.

(1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which 1 to 3 amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 are substituted or deleted, or an amino acid shown in SEQ ID NO: 1. A polypeptide comprising an amino acid sequence having 1 to 3 amino acid residues added to the sequence and having anti-inflammatory activity.
(2) An anti-inflammatory agent comprising at least one of the polypeptides according to (1) as an active ingredient.
(3) An anti-inflammatory composition comprising at least one of the polypeptides according to (1).
(4) The composition according to (3), wherein the composition is an algal thermolysin degradation product containing phycoerythrin.
(5) The composition according to (4), wherein the algae is red algae.
(6) The composition according to (5), wherein the red algae is duls.

  The polypeptide of the present invention and the anti-inflammatory composition containing the polypeptide are a degradation product of a polypeptide derived from a natural marine resource algae, in particular, a dietary red algae dals or thermolysin, It can be used as a polypeptide having excellent properties or an anti-inflammatory composition.

It is a graph which shows the mouse | mouth carrageenin edema inhibitory effect of the thermolysin decomposition product of the phycobiliprotein derived from dulls. In the figure, the broken line represents the control group, the solid line represents the thermolysin degradation product administration group, and the dotted line represents the indomethacin administration group. TNF-α from a panel of mouse macrophage-like cells (Raw 264.7 cells, stimulated with bacterial lipopolysaccharide (LPS)) obtained by adding trifluoroacetic acid to a thermolysin degradation product of a phycobiliprotein derived from Dulse (panel) It is a graph which shows the production inhibitory effect of the upper) and IL-6 (bottom panel). *** is p <0.001, and all indicate the test results with the additive-free section (Control) by the Student-T test. It is a spectrum when the precipitate obtained by adding trifluoroacetic acid to the thermolysin degradation product of the phycobiliprotein derived from dulls is analyzed by MALDI-TOF MASS. It is a graph which shows the production inhibitory action of TNF- (alpha) (upper panel) and IL-6 (lower panel) of chemically synthesized polypeptide (DDP83-92). * Is p <0.05, ** is p <0.01, and both show the results of the test with the additive-free section (0) by the Student-T test. Degradation product of Dulse phycobiliproteins digested with various proteolytic enzymes shows TNF-α (upper panel) and IL-6 (lower panel) production inhibitory action from Raw264.7 cells (stimulated with LPS) It is a graph.

  The anti-inflammatory polypeptide which is one embodiment of the invention disclosed in the present application is a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or 1 to 3 amino acid residues of the amino acid sequence represented by SEQ ID NO: 1. It is a polypeptide having an anti-inflammatory activity, comprising a substituted or deleted amino acid sequence or an amino acid sequence in which 1 to 3 amino acid residues are added to the amino acid sequence shown in SEQ ID NO: 1.

  The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is a polypeptide consisting of the partial amino acid sequence from the 83rd to the 92nd of the phycoerythrin β chain, which is a subunit constituting the phycobiliprotein. Hereinafter, this polypeptide is represented as DDP83-92.

  DDP83-92 is a 10-residue, which was found from a degradation product obtained by digesting phycobiliprotein contained in a water extract fraction of Dulse with a thermolysin (thermolysin, EC 3.4.24.27). It is a relatively short polypeptide.

  DDP83-92 exhibits anti-inflammatory effects such as suppression of nitric oxide (NO) or TNF-α production from macrophages, or suppression of carrageenan-induced edema, as shown in part in later examples. Thus, DDP83-92 can be used as an anti-inflammatory agent. The anti-inflammatory effect in the present invention can suppress at least one of production of nitric oxide (NO), TNF-α or interleukin 6 (IL-6) from macrophages, or carrageenan-induced edema. Defined as an action.

  The present application also provides a modified form of DDP83-92, such as an amino acid sequence in which 1 to 3 amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 are substituted or deleted, or an amino acid sequence shown in SEQ ID NO: 1. A polypeptide comprising an amino acid sequence to which three amino acid residues are added and having anti-inflammatory activity is also provided. Hereinafter, such a polypeptide is referred to as a DDP83-92 modified polypeptide.

  A preferred example of the DDP83-92 modified polypeptide is a polypeptide in which one to three amino acid residues shown in SEQ ID NO: 1 are so-called conservatively substituted. Examples of such polypeptides include glutamic acid in the amino acid sequence shown in SEQ ID NO: 1 aspartic acid, aspartic acid in glutamic acid or asparagine, arginine in lysine, glycine in alanine, leucine in isoleucine or valine, And / or modified polypeptides in which isoleucine is replaced by leucine or valine, respectively. In addition, a modified polypeptide in which about 1 residue each of the N-terminal and / or C-terminal of DDP83-92 is deleted, and one to several arbitrary amino acid residues at the N-terminal and / or C-terminal of DDP83-92 Examples of DDP83-92 modified polypeptides include polypeptides with added functional groups, functional amino acid sequences such as histidine tags, and polypeptides with added amino acid sequences that can constitute fusion proteins with arbitrary proteins. it can.

  The present application provides a thermolysin degradation product of algae containing phycoerythrin, particularly red algae containing phycoerythrin, as an anti-inflammatory composition.

  As shown in the following examples, the anti-inflammatory effect of Duls thermolysin degradation product itself is similar to DDP83-92. DDP83-92 is the 83rd to 92nd phycoerythrin β chain, which is a subunit constituting phycobiliprotein, found from a degradation product obtained by digesting phycobiliprotein contained in the aqueous extract of Dulse with thermolysin. It is a polypeptide consisting of the partial amino acid sequence.

  Cyanobacteria and red algae are known to have a protein supercomplex composed of a core and a rod called phycobilisome for photosynthesis. The phycobilisome includes a chromoprotein called phycobiliprotein in which phycobilin and a specific apoprotein are covalently bound, and a linker protein that links the chromoprotein. In phycobilisomes, the transmission of light energy in photosynthesis is performed through allophycocyanin, phycocyanin, phycoerythrin, and the like, which are a plurality of phycobiliproteins having different photochemical properties. Phycoerythrin is a red-fluorescent multi-subunit protein having a phycoerythrin β chain as a constituent element, forms a phycobilisome, and is the red source of red algae.

  Therefore, it is expected that not only Darus but also algae containing phycoerythrin, particularly a red seaweed thermolysin degradation product containing phycoerythrin, can be used as an anti-inflammatory composition.

  The algae usable in the present invention are algae containing phycobiliprotein phycoerythrin, and are preferably red algae having a high content. Examples of such red algae include Acrocaetium, Chinolemo, Oxenaceae, Cypridina, Erythropertis, Euphorbiaceae, Kakraito, Sangomo, Dulce, Proboscis, Rhodogorgon, Benimara, Examples include red seaweeds belonging to the order of the order of the scorpionid, the scallop, the cynomolgus, the scorpiona, the scorpiona, the scorpiona, the crabs and the saccharid. A preferred red algae is a red algae belonging to the order of Dulse, and a particularly preferred red algae is a dull.

  The thermolysin digestion of algae may be performed on the raw algae that have been refined, or on the water extraction fraction obtained by immersing the dried and micronized algae in an appropriate amount of water. Solvents and adjuvants may be used but are not essential. The thermolysin digestion may be performed under the recommended reaction conditions using thermolysin commercially available as a reagent or as a bulk product, and no special processing conditions are required. What is necessary is just to use the usage-amount of thermolysin suitably in the range of 0.1 to 1% weight with respect to the dry weight (gram) of algal protein. The treatment time may be about 30 minutes to 6 hours, preferably 1 to 3 hours.

  DDP83-92 does not lose its anti-inflammatory effect even when placed at 65-85 ° C, or even 100 ° C, which is the preferred temperature range for thermolysin. Such high temperature resistance of DDP83-92 can provide the advantage that algal thermolysin digestion can be carried out at high temperatures and contamination contamination can be prevented.

  DDP 83-92 is a general salt salt, chromatographic, and other ordinary methods that can be commonly performed by those skilled in the art from a decomposition product obtained by digesting a phycobiliprotein contained in the water extract fraction of the red algae, particularly the dulls, with thermolysin. The method can be used to purify or isolate in part or in high purity.

  DDP83-92 can also be produced by various chemical methods represented by the t-Boc method. Furthermore, it can also be produced by a genetic engineering method using a nucleic acid encoding the amino acid sequence of DDP83-92. Similarly, DDP83-92 modified polypeptides can also be produced by chemical and / or genetic engineering methods. Production of such polypeptides by chemical methods or genetic engineering methods may be performed using general methods well known or well known to those skilled in the art.

  In a preferred embodiment of the polypeptide or anti-inflammatory composition provided by the present application, when the anti-inflammatory composition is a thermolysin degradation product, the effective amount is 0.1 mg / kg to 1 kg body weight of the administered individual. 500 mg, preferably 1 mg to 300 mg, more preferably 5 mg to 100 mg, which can be administered in one or more divided doses. In addition, the effective amount of the anti-inflammatory composition other than the thermolysin degradation product and the effective amount of the polypeptide are appropriately determined by those skilled in the art by referring to the polypeptide amount contained in the thermolysin degradation product.

  The polypeptide or anti-inflammatory composition provided by the present application may be processed into a liquid or solid form appropriately diluted with purified water or other solvent or diluent. Further, the polypeptide or anti-inflammatory composition provided by this application forms or formulates a pharmaceutical composition with any pharmaceutically acceptable excipient, carrier or other component known to those of skill in the art. can do. Such pharmaceutical compositions and preparations are also included in the anti-inflammatory composition of the present invention.

  In a pharmaceutical composition or formulation comprising a polypeptide or anti-inflammatory composition provided by the present application and an appropriate solvent, excipient, carrier and / or adjuvant, etc., an anti-inflammatory effect is exhibited. It is preferable to contain a sufficient amount of the polypeptide or anti-inflammatory composition. Such an amount can be appropriately determined by those skilled in the art according to the administration method, patient symptoms, age and the like. For example, it is preferable that 0.1 to 50% by weight, preferably 1 to 10% by weight of the thermolysin degradation product is contained per weight of the composition.

  Pharmaceutically acceptable excipients, carriers and other components are known or well known to those skilled in the art, and those skilled in the art will be able to describe within the scope of normal performance, for example, in the 16th revision Japanese Pharmacopoeia and other standards. The selected components can be used as appropriate according to the form of the preparation.

  Examples of excipients, carriers or adjuvants include, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch , Carboxymethylcellulose calcium, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, soft anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan Fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polyso Bate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water or the like.

  A pharmaceutical composition or formulation comprising a polypeptide or anti-inflammatory composition provided by the present application can take any dosage form. Examples of dosage forms include tablets, capsules, granules, fine granules, oral solutions, powders and syrups, suppositories, ointments, creams, gels, patches, sprays, lotions, etc. And external preparations, liquid preparations, suspensions, injections and the like. These formulations can be prepared by those skilled in the art according to conventional methods.

  A pharmaceutical composition or formulation comprising a polypeptide or anti-inflammatory composition provided by this application may be administered by any route known to those skilled in the art, for example, oral administration, parenteral administration such as a topical skin preparation, and intrarectal. It can be administered by administration. Such a pharmaceutical composition or preparation may be produced using a general method known or well known to those skilled in the art.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, these examples are intended to help understanding of the present invention, and the technical scope of the present invention is not limited thereto. .

<Example 1>
1) Preparation of Dullus Thermolysin Degradation Product Dulse leaf bodies were washed with running water, freeze-dried, and further pulverized. 100 mL (liter) of distilled water was added to 10 g (gram) of fine powder, and water extraction was performed with stirring at 5 ° C. for 30 minutes. The supernatant was collected by centrifugation, 70% ammonium sulfate fractionation was performed three times to collect the precipitate, and the salt was removed by dialysis to obtain a crude phycobiliprotein fraction.

  1% (w / w) of thermolysin (Wako Pure Chemical Industries, 9200 PU / mg, Bacillus thermoproteolyticus Roko) from 50 mL of crude phycobiliprotein fraction (10 mg protein / mL, pH adjusted to 8.0) And after incubation at 70 ° C. for 180 minutes, the thermolysin was inactivated by heating at 95-98 ° C. for 15 minutes. This was centrifuged at 20,000 × g for 30 minutes, and the supernatant was recovered to obtain a thermolysin degradation product.

2) Confirmation of anti-inflammatory action The volume and thickness of the right hind footpad of 6-week-old male ICR mice (n = 7) were measured with a plethysmometer (MK-101P, manufactured by Muromachi Kikai), and 0 hours Was measured. One hour after oral administration (300 mg protein / kg body weight) of the thermolysin degradation product prepared above, 40 μL of 1% carrageenan dissolved in physiological saline was injected into the right hind limbs subcutaneously, and administered every hour for 5 hours. Until then, the volume and thickness of the right hind footpad were measured. In addition, ICR mice administered with 10 mg / kg of indomethacin instead of thermolysin degradation products were prepared as positive control.

  A control in which only a thermolysin degradation product and an equal volume of distilled water are orally administered is prepared, and the carrageenin edema rate due to the thermolysin degradation product or indomethacin when the average value is 100% is shown in FIG.

  As a result of the above test, it was confirmed that the thermolysin degradation product can suppress the exacerbation of carrageenan edema (increase in edema rate) by about 40% relative to the control.

<Example 2>
10 mg of the thermolysin degradation product (referred to as Total in FIG. 2) prepared in Example 1) was added to a 0.1% trifluoroacetic acid (TFA) solution at 10 mg / mL and stirred at room temperature for 10 minutes. Thereafter, the mixture was centrifuged at 20,000 × g for 10 minutes to recover the supernatant and the precipitate. The supernatant was lyophilized as it was to obtain a supernatant fraction. For precipitation, 1 mL of distilled water containing 0.1% TFA was added and dissolved and allowed to stand still by sonication at room temperature. The finally obtained precipitate fraction (referred to as precipitate in FIG. 2) and the supernatant fraction (referred to as supernatant in FIG. 2) were lyophilized.

RAW264.7 cells cultured in DMEM medium containing 10% fetal bovine serum were seeded in a 96-well plate at 2.0 × 10 ⁵ cells / well and incubated for 2 hours. After changing the medium, the measurement sample and LPS (final concentration 2.5 ng / mL) were immediately added and cultured for 24 hours (37 ° C., 5% CO ₂ ). Thereafter, the amounts of TNF-α and IL-6 in the culture medium were measured by ELISA. In the measurement of TNF-α, an anti-mouse TNF-α monoclonal antibody and a biotin-labeled TNF-α antibody were used for the primary antibody and the secondary antibody, respectively. In the measurement of IL-6, an anti-mouse IL-6 monoclonal antibody and a biotin-labeled IL-6 antibody were used for the primary antibody and the secondary antibody, respectively. Both were quantified by an absorption method using an HRP-labeled anti-biotin antibody and a TMB solution. The result is shown in FIG. The numbers in the figure are the amount of each sample added (μg / mL).

  As shown in FIG. 2, a stronger anti-inflammatory effect was confirmed in the precipitate fraction compared to the supernatant fraction.

<Example 3> Identification and activity evaluation of DDP83-92 1) MALDI-TOF MASS analysis The substances contained in the precipitate fraction prepared in Example 2 were mixed with MALDI-TOF MS (Applied Biosystem, AB4700, matrix was α -CHCA). The obtained spectrum is shown in FIG.

  When Mw = 1247.7 was selected as the main peak on the spectrum, and its structure was further analyzed in detail by the Collision Induced Dissociation (CID) -High energy collation method, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 It was confirmed that. When this amino acid sequence was searched for DNA Data Bank of Japan as a query, it was confirmed that the amino acid sequence matches the partial amino acid sequence from the 83rd to the 92nd position of the phycoerythrin β chain.

2) Polypeptide synthesis Using a peptide synthesizer (model MultiSynTech, SyroII), a polypeptide (DDP83-92) having a purity of 95% or more consisting of a partial amino acid sequence from the 83rd to the 92nd amino acid sequence of the phycoerythrin β chain is obtained. Chemically synthesized. The ability of chemically synthesized DDP83-92 to inhibit TNF-α production and IL-6 production was measured by the method described in Example 2. The result is shown in FIG.

  As shown in FIG. 4, synthesized DDP83-92 suppressed both TNF-α and IL-6 production, and was confirmed to have an anti-inflammatory effect.

<Test example>
In place of the thermolysin of Example 1 1), using the enzymes shown in Table 1 below (all commercially available products), digesting the dallus crude phycobiliprotein at the optimum temperature and pH of each enzyme, A degradation product by each enzyme was prepared. The amount of enzyme was unified to 1% of the crude phycobiliprotein. The anti-inflammatory action of each degradation product was confirmed by measuring the ability to suppress TNF-α production and IL-6 production. The result is shown in FIG. Note that pepsin / trypsin represents a degradation product obtained by continuous digestion of pepsin and trypsin.

  As shown in FIG. 5, it was confirmed that the thermolysin degradation product showed the strongest anti-inflammatory effect as compared with the degradation products by other enzymes.

  The polypeptide of the present invention and the anti-inflammatory composition containing the polypeptide are a degradation product of a polypeptide derived from a natural marine resource algae, in particular, a dietary red algae dals or thermolysin, It can be used as a polypeptide having excellent properties or an anti-inflammatory composition.

Claims (6)

A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted or deleted, or one amino acid in the amino acid sequence shown in SEQ ID NO: 1 A polypeptide comprising an amino acid sequence to which residues are added and having anti-inflammatory activity.   An anti-inflammatory agent comprising at least one of the polypeptides according to claim 1 as an active ingredient. An anti-inflammatory composition comprising at least one of the polypeptides according to claim 1.   The composition according to claim 3, wherein the composition is an algal thermolysin degradation product containing phycoerythrin.   The composition according to claim 4, wherein the algae is red algae.   The composition according to claim 5, wherein the red algae is duls.

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