KR102028472B1 - Sweet-tasting protein Brazzein having anti-inflammatory activity - Google Patents

Abstract

The present invention relates to brazein and / or multiple variants thereof having anti-inflammatory activity, more specifically, brazein and / or multiple variants thereof exhibits an anti-inflammatory effect through the NO production inhibitory activity, anti-inflammatory pharmaceutical composition, It can be usefully used as an anti-inflammatory cosmetic composition and health functional food.

Description

Sweet-tasting protein Brazzein having anti-inflammatory activity

The present invention relates to sweet protein brazein and multiple variants thereof having anti-inflammatory activity.

Bra Jane Serpentine native African dipeulran de la Alhambra The Ana (Pentadiplandra brazzeana A sweet protein found in the fruit of Baillon). It has a sweetness of about 500-2000 times the weight of sugar. Brazein is composed of 54 amino acid residues and has a molecular weight of 6.5 kDa, the smallest of sweet proteins. There are two types of brazein, Major and Minor. Major form consists of 54 amino acid residues located at the N -terminus of pyroglutamic acid (pGlu), whereas Minor form is composed of 53 amino acids without pGlu at the N -terminus. Consists of. Among them, the minor form ( des -pGlu1-brazzein) is known to be twice as sweet as the major form. In addition, brazein has high solubility and has four disulfide bonds, resulting in high thermal and pH stability.

In order to increase the applicability and commercial value of sweet protein brazein, it is necessary to study additional bioactivity. Research into the physiological activity of various proteins and peptides has long been made. Defensins and cathelicidins are representative antimicrobial and antifungal peptides, and proteins such as casein, beta-lactoglobulin, transferrin, lactoferrin and corn protein, zein, are known to have antioxidant effects. So far, little research has been conducted on the biological activities of sweet proteins such as brazein, thaumatin, and monellin.

Therefore, in the present invention, it was intended to explore the various physiological activities of the sweet protein brazein and its multiple variants to obtain the results, and if the efficacy is found, it is expected to be useful as a functional food additive.

In order to determine the actual efficacy, the anti-inflammatory activity of the wild type ( des -pGlu1-brazzein) [patent document 1] and multiple variants was investigated.

Domestic patent registration # 1239669 Domestic Patent Registration No. 981087 Domestic Patent Registration No. 1416892

Thus, the present inventors have completed the present invention by identifying anti-inflammatory activity in certain brazeins and multiple variants thereof.

It is therefore an object of the present invention to provide brazein and / or multiple variants thereof having anti-inflammatory activity.

As a means for solving the above problems, the present invention provides anti-inflammatory pharmaceutical composition and anti-inflammatory cosmetic composition comprising a brazein and / or multiple variants thereof.

In addition, as another means for solving the above problems, the present invention provides a health food comprising brazein and / or multiple variants thereof.

Since the brazein and / or multiple variants thereof of the present invention exhibit an anti-inflammatory effect through NO production inhibitory activity, it can be usefully used as an anti-inflammatory pharmaceutical composition, anti-inflammatory cosmetic composition and health functional food.

1 shows the results of SDS-polyacrylamide gel electrophoresis analysis of purified recombinant brazein [lane M, molecular weight marker (polypeptide marker); Lane 1, purified recombinant brazein wild type ( des- pGlu1-brazzein), lane 2, purified recombinant 3M (H30R_E35D_E40A) variant; Lane 3, purified recombinant K4R-3M (K4R_H30R_E35D_E40A) variant samples were dissolved by 16.5% Tris-tricin gel].
Figure 2 shows homologous proteins with primary and tertiary structures of brazein measured by the BLAST and VAST programs [(A) Brazein (2LY5_A) using T-Coffee and BOXSHADE 3.21 program, defensin-like protein 195 Multiple alignments of [Arabidopsis thaliana] (XP_018437331.1) and putative trypsin inhibitor 4 [Arabidopsis thaliana] (CAG15211.1); Identical residues are shaded in black and similar residues are shaded in grey; (B) tertiary structure of a protein having high structural homology with brazein. (a) Brazzein; (b) Aesculus hippocastanum antimicrobial protein 1; (c) Drosomycin; (d) Scorpion alpha-toxin OD1; (e) Tityus serrulatus neurotoxin; (f) Petunia hybrida defensin 1 PhD1].
Figure 3 shows the cytotoxic effect of brazein and its multiple variants.
4 shows anti-inflammatory activity of brazein, 3M variant and K4R-3M variant against murine macrophage RAW 264.7 cells.

The present invention relates to new activity, namely anti-inflammatory activity, of brazein and / or multiple variants thereof.

The brazein means a brazein subtype (minor form), and consists of the amino acid sequence of SEQ ID NO: 1.

The brazein multiple variant refers to a variant that is secondary to quaternary of the braze minor (part) type (SEQ ID NO: 1). For example, H30R_E35D_E40A (Domestic Registration No. 981087), fourth order, as a tertiary variant. The variant may be K4R_H30R_E35D_E40A (Domestic Patent No. 1416892).

The tertiary variant H30R_E35D_E40A is a histidine residue, the thirtieth amino acid of the brazein subtype protein, as an arginine residue, and a thirty-third amino acid glutamic acid residue as an aspartic acid residue, 40 The first glutamic acid residue is a brazein variant substituted with an alanine residue, and may be represented by SEQ ID NO: 2.

In the fourth variant K4R_H30R_E35D_E40A, the lysine residue, which is the fourth amino acid of the brazein subtype protein, is replaced with an arginine residue, and the thirtieth amino acid histidine residue is replaced with an arginine residue. A variant in which the 35th amino acid glutamic acid residue is substituted with an aspartic acid residue and the 40th glutamic acid residue is substituted with an alanine residue, may be represented by SEQ ID NO: 3 .

The brazein and / or multiple variants thereof of the present invention confirmed the anti-inflammatory effect through NO production inhibitory activity.

As such, the present invention provides an anti-inflammatory pharmaceutical composition comprising at least one selected from the group consisting of brazein and multiple variants thereof as an active ingredient.

In one embodiment of the invention, the brazein has an amino acid sequence represented by SEQ ID NO: 1, the multiple variant has an amino acid sequence represented by SEQ ID NO: 2 or 3.

The term "inflammation" as used herein refers to the defense response of biological tissues against local injury to the body. In other words, the inflammatory response is a biodefense reaction that attempts to recover the original state by removing the injury caused by the stimulus in response to various harmful stressors. Inflammatory stimuli include infection or chemical or physical stimuli. Bioconstituents involved in the inflammatory response include low-molecular or high-molecular chemicals such as free radicals, proteins, sugars, lipids, plasma, blood cells, blood vessels, and connective tissue. The process of inflammation is usually divided into two types, which can be divided into acute and chronic inflammation. Acute inflammation is a short-term reaction within a few days. Plasma components and blood cells are associated with the removal of foreign bodies by posting a microcirculatory system. Chronic inflammation has a long duration, and tissue growth is seen.

As used herein, "anti-inflammatory" means preventing or treating (or ameliorating) an inflammatory disease.

The inflammatory disease includes, but is not limited to, at least one selected from the group consisting of asthma, periodontitis, dermatitis, stomatitis, peritonitis, gastritis, enteritis, arthritis, nephritis, prostatitis, hepatitis, pelvic inflammation, vasculitis and degenerative diseases.

As used herein, the term "prevention" means any action that delays the development of an inflammatory disease by administration of a composition of the present invention.

As used herein, the terms "treatment" and "improvement" refer to any action in which the symptoms of the disease improve or benefit altered by administration of the composition of the present invention.

As used herein, the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner.

Pharmaceutical compositions comprising the brazein of the present invention and / or multiple variants thereof as an active ingredient are useful for anti-inflammatory and the combination formulations of the present invention are conventional non-toxic pharmaceutically acceptable carriers, adjuvants and carriers for these purposes. orally (subcutaneously, intravenously, intramuscularly, intrasternally, or infusion), or parenterally, or rectally, in the form of a dosage unit formulation comprising a vehicle).

The pharmaceutical compositions according to the invention may further comprise inert ingredients, including pharmaceutically acceptable carriers, diluents, fillers, solubilizers or emulsifiers and salts well known in the art, and according to conventional methods powders, granules, Oral formulations such as tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions. For example, tablets used in combination therapy may be formulated according to conventional methods using solid carriers well known in the art.

"Pharmaceutically acceptable carrier" is a term that refers to a composition, in particular ingredients other than the active substance of the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like, and diluents such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. Or excipients.

Oral solid preparations include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one starch, calcium carbonate, sucrose or lactose, gelatin and the like. Excipients and lubricants such as magnesium stearate, talc, and the like, and oral liquid preparations include suspending agents, liquid solutions, emulsions, syrups, and the like, and diluents such as water, liquid paraffin, wetting agents, sweeteners, fragrances, It may include, but is not limited to, preservatives.

Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ethyl. Injectable esters, such as oleate, and the like, and suppository bases include, but are not limited to, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like. .

In one embodiment of the present invention, the brazein and / or multiple variants thereof of the present invention may be included in 0.00001 to 30% by weight relative to the total weight of the composition.

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

In another embodiment of the present invention, the pharmaceutical composition of the present invention may be an external preparation composition for skin. The dosage of the pharmaceutical composition according to the present invention is a conventional external preparation, preferably applied in the morning and evening in an appropriate amount depending on the affected area once to several times a day.

The external skin pharmaceutical composition of the present invention is a skin external agent having an effect of protecting the skin from active oxygen species, and includes a cream, gel, patch, spray, ointment, warning agent, lotion agent, liniment agent, pasta agent or cataplasma agent. It may be prepared and used as a pharmaceutical composition in the form of external preparations for skin, but is not limited thereto.

In addition, the present invention provides an anti-inflammatory cosmetic composition comprising at least one selected from the group consisting of brazein and multiple variants thereof as an active ingredient.

In one embodiment of the invention, the brazein has an amino acid sequence represented by SEQ ID NO: 1, the multiple variant has an amino acid sequence represented by SEQ ID NO: 2 or 3.

When the composition of the present invention is made of a cosmetic composition, the composition of the present invention may include not only the above-described brazein and / or variants thereof, but also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, Conventional adjuvants such as solubilizers, vitamins, pigments and flavorings, and carriers.

In addition, the composition of the present invention can be used in addition to the above-described brazein and / or its multiple variants in combination with the antioxidants that have been conventionally used so long as it does not impair the skin protective effect by reacting with the brazein and / or its multiple variants. It may be. Examples of the antioxidants that can be used include water-soluble and oil-soluble vitamins.

The water-soluble vitamins may be any compound as long as they can be incorporated into cosmetics, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, and the like. And salts thereof (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-sodium phosphate salt, ascorbic acid-2-magnesium phosphate salt, etc.) can also be used in the present invention. Included in

The oil-soluble vitamin may be any compound that can be formulated in cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3 and vitamin E (d1-α-tocopherol, d-α-tocopherol, d-δ-tocopherol) And derivatives thereof (ascorbic palmitate, ascorbic stearate, ascorbic acid dipalmitine, dl-α-tocopherol acetate, dl-α-tocopherol vitamin E, DL-pantothenyl alcohol, D, and the like). Pantothenyl alcohol, pantothenyl ethyl ether, etc.) are also included in the oil-soluble vitamins used in the present invention.

Such vitamins can be obtained by conventional methods such as microbial transformation, purification from microorganism culture, enzyme or chemical synthesis.

Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, soaps, treatments, and essences. Etc.

Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.

According to a preferred embodiment of the present invention, the content of brazein and / or multiple variants thereof of the present invention is 0.00001% to 30% by weight, preferably 0.5-20% by weight, more preferably, based on the total weight of the composition. Is 1.0-10% by weight. If the content of the brazein and / or multiple variants thereof is less than 0.00001% by weight, the anti-inflammatory effect is greatly reduced, and if it exceeds 30% by weight, skin irritation may occur, and formulation problems may occur.

The food or health functional food of the present invention may be prepared and processed in a form including, but not limited to, tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing or improving inflammatory symptoms. The term "health functional food" used in the present invention refers to a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the Health Functional Food Act No. 6767, regarding the structure and function of the human body It means the ingestion for the purpose of obtaining a beneficial effect for health use such as nutrient control or physiological action.

The health functional food of the present invention may include a conventional food additive, and the suitability as the "food additive" is applicable in accordance with the General Regulations of the Food Additives Code and General Test Methods, etc. approved by the Food and Drug Administration, unless otherwise specified. Judge according to the standards and standards for the item.

Examples of the items listed in the "Food Additive Revolution" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as color pigments, licorice extracts, crystalline cellulose, high color pigments, guar gum, Mixed preparations, such as a sodium L- glutamate preparation, an addition of an alkali, a preservative, and a tar coloring agent, etc. are mentioned.

For example, a dietary supplement in tablet form granulates a mixture of brazein and / or its multiple variants with excipients, binders, disintegrants, and other additives in a conventional manner, followed by compression molding with lubricants, etc. Alternatively, the mixture may be compression molded directly. In addition, the health functional food in the form of tablets may contain a mating agent and the like, if necessary, may be coated with a suitable coating agent.

Hard capsules of the health functional foods in the form of capsules may be prepared by filling a mixture of additives such as excipients or mixtures of granules or avoided granules with conventional hard capsules, and soft capsules with brazein and And / or multiple variants thereof may be prepared by filling a mixture with an additive such as an excipient into a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.

The dietary supplement in cyclic form may be prepared by molding a mixture of brazein and / or multiple variants thereof with excipients, binders, disintegrants, etc. in a suitable manner, and may be coated with sucrose or other suitable coating agent, or starch, if necessary. It may also be celebrity with talc or a suitable substance.

The health functional food in the form of granules may be prepared by granulating a mixture of brazein and / or its multiple variants with excipients, binders, disintegrants, etc. in a suitable manner, and may contain a flavoring agent, a coagulant, etc. as necessary. .

Terminology definitions for the excipients, binders, disintegrants, glidants, copulation agents, flavoring agents, etc. of the present invention are those described in literature known in the art and include those having the same or similar functions. Sacrament, Korean College of Pharmacy, 5 revised edition, p33-48, 1989).

Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited to the examples given below. In addition, the brazein wild type and its multiple variants used in this example are described in detail for the expression, purification and production method in Patent Documents 1-3.

EXAMPLE

1. Materials and Experimental Methods

1.1. Chemicals and Reagents

CM-Sepharose resin was obtained from GE Healthcare (Little Chalfont, UK) and Coomassie Brilliant Blue R-250 for protein staining from Fluka (St. Louis, MO, USA). Polypeptide SDS-PAGE molecular weight standards were purchased from Bio-Rad (Hercules, CA, USA). 2,2'-diphenyl-1-picrylhydrazyl (2,2'-Diphenyl-1-picrylhydrazyl, DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid ( 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, ABTS), L-ascorbic acid, celecoxib, anti-DNP IgE, DNP-BSA, grease reagent, fumaric acid ke Ketotifen fumarate, lipopolysaccharides, NG-methyl-L-arginine acetate salt (L-NMMA), p-nitrophenyl N-acetyl-β-D-glucosamine, p-nitrophenyl α-D- Glucopyranoside, and norfloxacin were purchased from Sigma-Aldrich (St. Louis, Mo., USA) DMSO and MTT formazan were obtained by Duchefa Biochemie (Amsterdam, The Netherlands).

All other chemicals and reagents used were commercially available and top rated.

1.2. Of the primary and tertiary structures of proteins Homology  Analysis structure modelling  And docking simulation

Brazein sequences are retrieved from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). 1 of proteins by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and VAST (http://www.ncbi.nlm.nih.gov/Structure/VAST/vast.shtml) The homology of the primary and tertiary structures was analyzed.

To investigate the interaction between human COX-2 and brazein, structural modeling and docking simulations were performed. Human COX-2 and brazein sequences were retrieved from NCBI. Structural modeling includes the 'SWISS-MODEL EXPASY' program (https://swissmodel.expasy.org/) at Biozentrum's Center for Molecular Life Sciences (Basel University, France) and 'Autodock' at the Scripps Research Institute in the United States. This is done using tools' (http://autodock.scripps.edu/). The docking simulation is the Global Range Molecular Matching (GRAMM-X) program (http://vakser.compbio.ku.edu/resources/gramm) provided by the Computational Biology Center at the University of Kansas. / grammx /). Docking simulation results were analyzed by 'DIMPLOT' provided by the European Institute of Bioinformatics (UK) and 'SEQMOL' (http://biochemlabsolutions.com).

1.3 Preparation of Brazein and Variants

Brazein (New England Biolabs, Ipswich, Mass., USA) was constructed using the expression vector pKLAC2 comprising a Kluyveromyces lactis GG799 strain and an alpha-mate factor signal sequence.

The construction, modification and expression of expression vectors for the production of recombinant brazein (minor form, des-pE1M-brazzein) and / or multiple variants in K. lactis has been described previously (Jo, HJ, Noh, JS, & Kong). , KH (2013) .Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis .; Yun, CR, Kong, JN, Chung, JH, Kim, MC, & Kong, KH (2016) .Improved Secretory Production of the Sweet-Tasting Protein, Brazzein, in Kluyveromyces lactis.Journal of Agricultural and Food Chemistry, 64, 6312-6316.). The expressed recombinant brazein or multiple variants were purified by CM-Sepharose chromatography as previously described (Jo, HJ, Noh, JS, & Kong, KH (2013). Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis.Protein Expression and Purification, 90 , 84-89). Fractions containing purified brazein and / or multiple variants were dialyzed with distilled water and lyophilized. Purity of recombinant brazein and / or multiple variants was confirmed by reverse phase HPLC column chromatography and SDS-polyacrylamide gel electrophoresis (Jo, HJ, Noh, JS, & Kong, KH (2013). Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis.Protein Expression and Purification, 90 , 84-89). Molecular weight markers were polypeptide SDS-PAGE molecular weight standards and Coomassie Blue R-250 was used for protein staining.

1.4. For determining the bioactivity of brazein and variants in vitro   analysis

1.4.1. Anti-inflammatory activity and cytotoxicity

The inhibitory effect on nitric oxide (NO) synthesized from rat macrophage RAW 264.7 cells was investigated in Moro, C., Palacios, I., Lozano, M., D'Arrigo, M., Guillamon, E., Villares, A. , Martinez, JA, & Garcia-Lafuente, A. (2012). Anti-inflammatory activity of methanolic extracts from edible mushrooms in LPS activated RAW 264.7 macrophages. Evaluation was as described above in Food Chemistry, 130 , 350-355. The Griess reagent was used to measure NO production by accumulation of nitrite in the culture supernatant and absorbance at 540 nm. Brazein and multiple variants were each treated at a concentration of 1-15 μM and L-NMMA was used as a positive control. In addition, MTT colorimetric analysis was performed to determine the cytotoxicity of brazein and multiple variants (Moro, C., Palacios, I., Lozano, M., D'Arrigo, M., Guillamon, E., Villares, A., Martinez, JA, & Garcia-Lafuente, A. (2012) .Anti-inflammatory activity of methanolic extracts from edible mushrooms in LPS activated RAW 264.7 macrophages.Food Chemistry, 130 , 350-355).

All experiments were performed three times and the data presented as mean ± SD.

2. Results and Discussion

2.1. Brazein or Variant Expression and Purification

Recombinant brazein (miner form, des-pE1M-brazzein) and / or multiple variants thereof were produced using yeast K. lactis GG799, which is generally known to be safe. K. lactis cells containing the brazein gene were grown in YPGal medium (pH 5.0) by injecting 2% of the inoculum for 96 hours at 30 ° C for maximum secretion yield of recombinant brazein. Recombinant brazein and / or multiple variants were efficiently produced in active form in the medium in an amount of about 90% of the total secreted protein. Secreted recombinant brazein or multiple variants were applied to a CM-Sepharose column equilibrated with 50 mM sodium acetate at pH 4.0 and eluted with 400 mM NaCl. Purified recombinant brazein or multiple variants of about 90-100 mg / L were obtained and confirmed by SDS-PAGE and HPLC. Purified recombinant brazein or multiple variants appeared in single band with a molecular weight of 6,500 Da (Figure 1) on SDS-PAGE. The elution time of the folded recombinant brazein was 9 ± 0.5 minutes as measured by reversed phase HPLC. Purified recombinant brazein or multiple variants were then used in bioactivity experiments.

2.2. Homology Analysis of Primary and Tertiary Structures of Brazein

Understanding the homology between the primary and tertiary structures of a protein provides insight into biochemical function and evolution. In general, BLAST is used to examine the homology of major structures (amino acid sequences) between peptides and / or proteins. The alignment result of the homologous sequence using the BLAST program is shown in FIG.

The primary structure of brazein was approximately 42-45% homologous to the antimicrobial peptides named defensin and trypsin inhibitors.

In general, proteins function as complex tertiary structures rather than the protein's primary structure. Moreover, the tertiary structure of proteins is more evolutionarily conserved than the primary structure. VAST was used to analyze the similarity of tertiary structure between brazein and protein. As a result of the VAST program, the third structure of brazein is antimicrobial protein I from Aesculus hippocastanum , drosomycin used as an antifungal agent, scorpion alpha-toxin Od1, Very similar to the structure of neurotoxin and plant defensin (FIG. 2B). The tertiary structures of brazein showed about 54-66% similarity to those structures.

2.3. Anti-inflammatory activity of brazein and variants

Macrophages or monocytes stimulate lipopolysaccharide to promote the secretion of interleukin (IL) proteins and pro-inflammatory cytokines such as nitric oxide (NO) (Sharma, JN, Al-Omran, A. , & Parvathy, SS (2007) .Role of nitric oxide in inflammatory diseases.Inflammopharmacology , 15 , 252-259.).

NO is produced from L-arginine by NO synthase, and overproduction of NO induces an acute inflammatory response in humans. To evaluate the anti-inflammatory effects of brazein and its multiple variants, NO production in mouse macrophage RAW 264.7 cells was measured. Prior to NO analysis, cytotoxicity of brazein and / or multiple variants thereof was observed by MTT colorimetric analysis. The MTT assay showed that 1-15 μM brazein and multiple variants thereof did not have a cytotoxic effect on RAW 264.7 cells, and cell viability was about 100% at all concentrations (FIG. 3).

In addition, the anti-inflammatory activity of brazein and / or its multiple variants was determined by NO analysis.

The results shown in FIG. 4 show that NO production from RAW 264.7 cells inhibited 11.83 ± 2.27% in 15μM brazein, 11.62 ± 2.65% in 15μM 3M (H30R_E35D_E40A) and 13.12 ± 1.27% NO production in 15μM K4R-3M (K4R_H30R_E35D_E40A). The inhibitory effect continued to increase at concentrations above 15 μM.

In contrast, IC ₅₀ values for NO synthase inhibition of aspirin, sodium salicylate, piroxicam, indomethacin, ibuprofen and curcumin, known as nonsteroidal anti-inflammatory agents, were 5300 μM, 4200 μM, 750 μM, 100 μM, 80 μM, 6 reported as μM (Brouet, I., & Ohshima , H. (1995). Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochemical and Biophysical Research Communications, 206, 533 -540.) It is believed that brazein and / or multiple variants thereof exhibit higher NO synthase inhibitory activity than the other anti-inflammatory agents except curcumin. These results suggest that brazein and / or multiple variants thereof are not cytotoxic and exhibit anti-inflammatory activity.

In inflammatory processes, together with ROS (Nakagawa, T., & Yokozawa, T. (2002) .Direct scavenging of nitric oxide and superoxide by green tea.Food and Chemical Toxicology, 40 , 1745-1750.) Reactive nitrogen such as nitrogen, NO is produced.

NO is an unstable free radical. Excessive production damages cells and tissues as toxic radicals.

As mentioned above, brazein and / or multiple variants thereof comprise a number of residues reported to have radical scavenging ability and comprise about 43% of the total number of amino acids.

Taken together, we propose that the anti-inflammatory effects of brazein and / or multiple variants thereof are related to the radical scavenging effect based on amino acid composition rather than cytotoxicity against cells.

3. Conclusion

Brazein and / or its multiple variants showed significant anti-inflammatory activity. Based on these results, brazein and / or its multiple variants can be used as functional sweeteners with various physiological activities.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Sweet-tasting protein, Brazzein having anti-inflammatory activity <130> P17U21C0303 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> brazzein minor type <400> 1 Asp Lys Cys Lys Lys Val Tyr Glu Asn Tyr Pro Val Ser Lys Cys Gln   1 5 10 15 Leu Ala Asn Gln Cys Asn Tyr Asp Cys Lys Leu Asp Lys His Ala Arg              20 25 30 Ser Gly Glu Cys Phe Tyr Asp Glu Lys Arg Asn Leu Gln Cys Ile Cys          35 40 45 Asp Tyr Cys Glu Tyr      50 <210> 2 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> brazzein multiple mutated variant H30R_E35D_E40A <400> 2 Asp Lys Cys Lys Lys Val Tyr Glu Asn Tyr Pro Val Ser Lys Cys Gln   1 5 10 15 Leu Ala Asn Gln Cys Asn Tyr Asp Cys Lys Leu Asp Lys Arg Ala Arg              20 25 30 Ser Gly Asp Cys Phe Tyr Asp Ala Lys Arg Asn Leu Gln Cys Ile Cys          35 40 45 Asp Tyr Cys Glu Tyr      50 <210> 3 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> brazzein multiple mutated variant K4R_H30R_E35D_E40A <400> 3 Asp Lys Cys Arg Lys Val Tyr Glu Asn Tyr Pro Val Ser Lys Cys Gln   1 5 10 15 Leu Ala Asn Gln Cys Asn Tyr Asp Cys Lys Leu Asp Lys Arg Ala Arg              20 25 30 Ser Gly Asp Cys Phe Tyr Asp Ala Lys Arg Asn Leu Gln Cys Ile Cys          35 40 45 Asp Tyr Cys Glu Tyr      50

Claims (5)

At least one selected from the group consisting of brazein and multiple variants thereof,
The multiple variant is an anti-inflammatory pharmaceutical composition represented by SEQ ID NO: 2 or SEQ ID NO: 3.
The method of claim 1,
The brazein is an anti-inflammatory pharmaceutical composition represented by SEQ ID NO: 1.
At least one selected from the group consisting of brazein and multiple variants thereof,
The multiple variant is an anti-inflammatory cosmetic composition represented by SEQ ID NO: 2 or SEQ ID NO: 3.
The method of claim 3, wherein
The brazein is an anti-inflammatory cosmetic composition represented by SEQ ID NO: 1.
At least one selected from the group consisting of brazein and multiple variants thereof,
The multiple variant is a health functional food for preventing or improving inflammation represented by SEQ ID NO: 2 or SEQ ID NO: 3.

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