Monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids, preparation methods and uses thereof

Abstract

The present invention relates to an enzyme for preparing a hydroxy derivative of a polyunsaturated fatty acid, a method for preparing a hydroxy derivative of a polyunsaturated fatty acid using the enzyme, and an SPMs composite composition containing the hydroxy derivative of a polyunsaturated fatty acid. The enzyme can prepare the hydroxy derivative of a polyunsaturated fatty acid in a single reaction, and therefore, can be very effectively used for in vitro preparation. In addition, the SPMs composite composition containing the hydroxy derivative of a polyunsaturated fatty acid promotes collagen production, inhibits the production of NO and the expression of TNF-α and IL-6, and increases the expression of filaggrin and loricrin, thereby being effectively used to prevent or alleviate skin aging or wrinkles, enhance skin barrier, or prevent, alleviate or treat skin diseases including atopic dermatitis.

Description

Technical Field

[0001] The invention relates to a method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids, and to use of a composite composition containing the derivatives of polyunsaturated fatty acids in relieving skin wrinkles, relieving skin inflammation or enhancing skin barrier. Background Art

[0002] Since the inflammatory response plays an important role as the body's primary defense against pathogen infection and various toxic substances, but at the same time, it serves as a potential factor causing various chronic diseases, the inflammatory response is a physiological phenomenon that needs to be strictly controlled by complex regulatory mechanisms in the body.

[0003] Prostaglandins or leukotrienes are derivatives of arachidonic acid (C20: 4n-6) corresponding to omega-6 polyunsaturated fatty acids, and are a representative inflammatory response initiation signal substance, and induce an imbalance in the inflammatory response, which can cause various chronic diseases in the body. Generally, it is believed that the inflammatory response naturally terminates when the inflammatory response initiation signal substance is not produced, and various steroidal or nonsteroidal anti-inflammatory therapeutic agents have been developed to inhibit the production of this substance. However, in recent years, the termination mechanism of the inflammatory response is also carried out through a complex regulatory process, in which it is believed that hydroxy derivatives of omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA, C22: 6n-3) and eicosapentaenoic acid (EPA, C20: 5n-3) can act as signal substances that play a role in the termination mechanism of the inflammatory response. Those hydroxy derivatives that act as signal substances for the termination mechanism of the inflammatory response are called specific pro-resolution mediators (SPMs; resolvins, protectins, and phagocytins). It has been determined that these hydroxy derivatives exhibit superior effects over existing anti-inflammatory therapeutics such as corticosteroids and the nonsteroidal anti-inflammatory drug (NSAID) aspirin.

[0004] In addition, unlike conventional steroidal or nonsteroidal anti-inflammatory therapeutic agents that mainly induce the production of inflammatory response-initiating signal substances, SPM has the advantage of showing efficacy at all stages of inflammatory response. Therefore, for various diseases such as various chronic inflammatory diseases (various chronic inflammatory diseases such as blood vessels, myocardial infarction, stroke, dementia, osteoarthritis, lung diseases including asthma, gastroenteritis, periodontitis, dry eye and fatty liver), infectious sepsis, burns, wound recovery, pain, cancer and diabetes, research is actively being conducted on whether there is a therapeutic effect using SPM.

[0005] On the other hand, SPM, which is an inflammatory response termination signal substance in the body, has a structure in which two or three hydroxyl groups are inserted into omega-3 polyunsaturated fatty acids, and it is known that a very small amount of SPM is produced by the continuous action of two or more enzymes. Therefore, in order to prepare SPM sufficient for the development of the efficacy and therapeutic agent of the inflammatory response termination signal substance, efforts have been made to prepare it by an organic synthesis process (see Non-Patent Document 1), but there is a limitation that the process is very complicated.

[0006] Therefore, there is a continuing need to develop a technology capable of efficiently preparing monohydroxy or dihydroxy derivatives using omega-3 polyunsaturated fatty acids through an in vitro enzymatic reaction.

[0007] On the other hand, the skin is an important organ that protects the human body from harmful external environmental influences and is the largest immune organ. The stratum corneum that constitutes the outermost layer of the skin has resistance to physical and chemical damage and prevents microorganisms and harmful chemicals from invading the body. In addition, keratinocytes, Langerhans cells, fibroblasts, mast cells, dendritic cells, and capillary endothelial cells form a complex immune system, thereby protecting the human body through immune response when antigens invade from the outside.

[0008] The skin is constantly exposed to external irritants (ultraviolet rays, chemicals, heat, physical friction), and recently, new irritants such as fine dust and blue light are also increasing. In addition, internal factors such as mental stress, sleep disorders, nutritional imbalances and medication are known to cause stress on the skin.

[0009] According to recent studies, inflammatory responses are generated due to skin irritation, and the results of the studies have revealed that such inflammatory responses cause skin aging. Franceschi first used the term "inflammaging" in 2000 to explain the correlation between inflammation and aging, and the results of the studies have continuously revealed that chronic inflammation is the cause of aging or aging-related diseases such as diabetes, arteriosclerosis, and dementia.

[0010] Proinflammatory cytokines secreted during the onset of inflammation promote the expression of matrix metalloproteinases (MMPs). It is known that the increase in MMPs is directly related to skin aging, and it promotes aging such as wrinkles and loss of elasticity by decomposing collagen, which accounts for 70% of the skin's composition.

[0011] In general, substances used to inhibit skin aging include: enzymes such as superoxide dismutase for removing active oxygen; antioxidants such as vitamin C and tocopherol; peptides related to botulinum toxin; and vitamin A derivatives such as retinol. Among them, vitamin A derivatives have excellent collagen synthesis ability and are most effective in removing wrinkles, but they have side effects such as being unstable to light or heat, causing skin irritation, and unstable content in the formula.

[0012] Atopic dermatitis is a disease caused by abnormal genetic, environmental and immune responses and skin barrier disruption, and is a chronic inflammatory skin disease that causes dryness, itching and erythematous eczema. Recently, the prevalence is increasing worldwide due to environmental changes, dietary habits and environmental pollution, and interest in solutions is also increasing. However, since the cause is unknown, it is difficult to cure and mostly relies on symptomatic treatment.

[0013] On the other hand, it is believed that hydroxylated derivatives of docosahexaenoic acid (DHA, C22: 6n-3) and eicosapentaenoic acid (EPA, C20: 5n-3) can act as signal substances that act on the termination mechanism of inflammatory responses, and those substances that act as signal substances of the termination mechanism of inflammatory responses are named SPM (specific pro-resolving mediators; resolvins, protectins, and phagocytic factors).

[0014] Recently, although research on the use of SPM for the treatment of various chronic inflammatory diseases (e.g., blood vessels, myocardial infarction, stroke, dementia, osteoarthritis, asthma and other lung diseases, gastroenteritis, periodontitis, dry eye and fatty liver, etc.), infectious sepsis, burns, wound recovery, pain, cancer and diabetes has been actively conducted, research and experiments on the effects against skin aging, skin barrier enhancement and atopic dermatitis are still insufficient. Summary of the invention

[0015] Technical issues

[0016] One aspect of the present invention is to provide an enzyme capable of preparing monohydroxy or dihydroxy derivatives from polyunsaturated fatty acids, and a method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vivo or in vitro using the enzyme.

[0017] Another aspect of the present invention is to provide a cosmetic composition or a food composition that improves skin conditions by preventing or alleviating skin wrinkles, preventing or alleviating skin aging, preventing or alleviating skin inflammation, regenerating skin, and enhancing skin barrier.

[0018] Another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating one or more skin diseases selected from skin wounds, skin scars, dermatitis, atopic dermatitis, pruritus, eczematous skin diseases, dry eczema, tinea pedis, erythema, urticaria, psoriasis, weak rash, acne and alopecia.

[0019] Another aspect of the present invention is to provide a method for improving skin conditions, wherein the method comprises administering to a subject an effective amount of a complex composition comprising a hydroxy derivative of a polyunsaturated fatty acid as an active ingredient.

[0020] Another aspect of the present invention is to provide a method for preventing or treating skin diseases, wherein the method comprises administering to a subject a complex composition comprising a hydroxy derivative of a polyunsaturated fatty acid as an active ingredient.

[0021] Another aspect of the present invention is to provide a use of a complex composition comprising a hydroxy derivative of a polyunsaturated fatty acid as an active ingredient in the production of a medicament for preventing or treating skin diseases.

[0022] Technical Solution

[0023] One aspect of the present invention provides an enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids, wherein the amino acid sequence of the enzyme has at least 90% homology to SEQ ID NO:1 or SEQ ID NO:2.

[0024] Another aspect of the present invention provides a method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vitro, wherein the method comprises reacting the enzyme for preparing the monohydroxy or dihydroxy derivatives with polyunsaturated fatty acids.

[0025] Another aspect of the present invention provides a method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vivo, wherein the method comprises: culturing a transformant in the presence of polyunsaturated fatty acids, in which a gene encoding the enzyme for preparing the monohydroxy or dihydroxy derivatives is transformed; and isolating the monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids from the cultured culture.

[0026] Another aspect of the present invention provides a cosmetic composition for improving skin conditions, wherein the cosmetic composition comprises 17-hydroxydocosahexaenoic acid (17-HDHA), Resolvin D5 and Protectin DX as active ingredients.

[0027] Another aspect of the present invention provides a food composition for improving skin conditions, wherein the food composition comprises 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX as active ingredients.

[0028] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases, wherein the pharmaceutical composition comprises 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX as active ingredients.

[0029] Another aspect of the present invention provides a method for improving skin conditions, wherein the method comprises administering an effective amount of a complex composition comprising 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX as active ingredients to a subject in need of improving skin conditions.

[0030] Another aspect of the present invention provides a method for preventing or treating skin diseases, wherein the method comprises administering a complex composition comprising 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX as active ingredients to a subject in need of treatment for the skin disease.

[0031] Another aspect of the present invention provides use of a complex composition comprising 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX as active ingredients in preventing or treating skin diseases.

[0032] Beneficial Effects

[0033] According to the present invention, the enzyme can prepare monohydroxy or dihydroxy derivatives in a single reaction by using polyunsaturated fatty acids as substrates, and can be very effectively used for the in vitro preparation of monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids.

[0034] The SPMs composite composition containing the docosahexaenoic acid hydroxy derivative of the present invention increases collagen synthesis, repairs damaged skin barrier, and reduces transepidermal water loss, thereby inhibiting skin aging or alleviating skin wrinkles.

[0035] In addition, the SPMs composite composition of the present invention has significantly improved effects of inhibiting the expression of TNF-α and IL-6, increasing the expression of filaggrin and loricrin, and anti-oxidative effects, thereby enhancing the skin barrier and effectively preventing or treating various skin diseases including atopic dermatitis.

[0036] However, the effects of the present invention are not limited to those mentioned above, and other effects not mentioned herein will be clearly understood by those skilled in the art from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The results of confirming the overexpression of the protein having the amino acid sequence represented by SEQ ID NO: 1 (A) and the protein having the amino acid sequence represented by SEQ ID NO: 2 (B) by SDS-PAGE are shown;

[0038] Figure 2 shows the results of normal phase HPLC analysis of a reaction product obtained by reacting a protein having an amino acid sequence represented by SEQ ID NO: 1 with docosahexaenoic acid (DHA);

[0039] Figure 3 shows the results of normal phase HPLC analysis of a reaction product obtained by reacting a protein having an amino acid sequence represented by SEQ ID NO: 2 with docosahexaenoic acid (DHA);

[0040] Figures 4a to 4d The results of analyzing the homology of the amino acid sequences of human 5LOX (AAA36183), human 12LOX (AAA51533), human 15LOX (AAA36183), soybean 15LOX (AAA33986), potato LOX (AAB81595) and red algae PhLOX2 (AGN54275), which are lipoxygenases having similar activities, are shown for a protein (K1) having an amino acid sequence represented by SEQ ID NO: 1 and a protein (K2) having an amino acid sequence represented by SEQ ID NO: 2;

[0041] Figure 5 The results of confirming the cytotoxicity of hydroxy derivatives produced by a protein having an amino acid sequence represented by SEQ ID NO: 1 (SPM 1) and a hydroxy derivative produced by a protein having an amino acid sequence represented by SEQ ID NO: 2 (SPM 2) with respect to human dermal fibroblasts are shown;

[0042] Figure 6 The results of confirming the cytotoxicity of hydroxy derivatives produced by a protein having an amino acid sequence represented by SEQ ID NO: 1 (SPM 1) and a hydroxy derivative produced by a protein having an amino acid sequence represented by SEQ ID NO: 2 (SPM 2) with respect to human keratinocytes are shown;

[0043] Figure 7 and Figure 8The TNF-α expression inhibitory activity of the hydroxy derivative produced by the protein having the amino acid sequence represented by SEQ ID NO: 1 (SPM 1) and the hydroxy derivative produced by the protein having the amino acid sequence represented by SEQ ID NO: 2 (SPM 2) was confirmed for human keratinocytes ( Figure 7 ) and IL-6 expression inhibitory activity ( Figure 8 ) results;

[0044] Fig. 9 The results of confirming the collagen production enhancing activity of the hydroxy derivative produced by the protein having the amino acid sequence represented by SEQ ID NO: 1 (SPM 1) and the hydroxy derivative produced by the protein having the amino acid sequence represented by SEQ ID NO: 2 (SPM 2) for human dermal fibroblasts are shown;

[0045] Fig.10 The results of confirming the collagenase production inhibitory activity of the hydroxy derivative produced by the protein having the amino acid sequence represented by SEQ ID NO: 1 (SPM 1) and the hydroxy derivative produced by the protein having the amino acid sequence represented by SEQ ID NO: 2 (SPM 2) for human dermal fibroblasts are shown;

[0046] FIG. 11 shows the results of comparing the IL-6 expression inhibitory activity of a single component and a SPMs composite composition on human keratinocytes: Fig.11a The IL-6 expression inhibitory activity according to the concentration of each of 17S-HDHA, protectin DX, and resolvin D5 is shown; Fig.11b The IL-6 expression inhibitory activity according to the concentrations of the SPMs composite composition 1 to the SPMs composite composition 3 according to the present invention is shown; Fig.11c The synergistic IL-6 expression inhibitory effect of the SPMs composite composition of the present invention compared with the single component is shown [untreated control group: a group not irradiated with UVB, Con(-): a group not subjected to any treatment after UVB irradiation, Con(+)HC: a positive control group treated with hydrocortisone after UVB irradiation];

[0047] FIG. 12 shows the results of confirming the procollagen synthesis effects of single components and SPMs composite compositions on human dermal fibroblasts: Fig.12a The effect of procollagen synthesis according to the concentration of each of 17S-HDHA, protectin DX and resolvin D5 is shown; Figure 12b The procollagen synthesis effects of the concentrations of the SPMs composite composition 1 to the SPMs composite composition 3 according to the present invention are shown; Fig.12cThe synergistic procollagen synthesis effect of the SPMs composite composition of the present invention compared with the single component of SPM [untreated control group: untreated group, Con(+)RP: positive control group treated with retinyl palmitate] is shown;

[0048] Fig.13 The results of confirming the effect of the SPMs composite composition of the present invention on improving the ability to inhibit the expression of collagenase (MMP-1) in human dermal fibroblasts [untreated control group: a group not irradiated with UVB, control group: a group not subjected to any treatment after UVB irradiation, retinoic acid: a positive control group treated with retinoic acid after UVB irradiation] are shown;

[0049] Fig.14 The results of confirming the effect of the SPMs composite composition of the present invention on improving the ability to suppress TNF-α expression in human keratinocytes are shown [untreated control group: a group not irradiated with UVB, control group: a group not subjected to any treatment after UVB irradiation, hydrocortisone: a positive control group treated with hydrocortisone after UVB irradiation];

[0050] Fig.15 The results of confirming the NO production reducing effect of the SPMs composite composition of the present invention are shown [untreated control group: a group not irradiated with UVB, control group: a group not subjected to any treatment after UVB irradiation, hydrocortisone: a positive control group treated with hydrocortisone after UVB irradiation, Vit.C: a positive control group treated with vitamin C after UVB irradiation];

[0051] Fig.16 The results of confirming the effect of the SPMs composite composition of the present invention on reducing lipid peroxide production in response to stimulation induced by fine dust are shown [untreated control group: a group not treated with fine dust, control group: a group not subjected to any treatment after irradiation induced by fine dust, hydrocortisone: a positive control group treated with hydrocortisone after irradiation induced by fine dust, Vit. C: a positive control group treated with vitamin C after irradiation induced by fine dust];

[0052] FIG. 17 shows the results of confirming the skin barrier improvement effect of the SPMs composite composition of the present invention by the expression level of the core protein constituting the skin barrier: Fig.17a Results showing an increase in filaggrin expression were confirmed. Fig.17b Results confirming increased expression of loricrin are shown [untreated control group: a group not subjected to any treatment, hyaluronic acid: a positive control group treated with hyaluronic acid];

[0053] FIG. 18 shows the results of confirming the skin barrier improvement effect of the SPMs composite composition of the present invention through human efficacy evaluation: Fig.18a The change rate (%) of the skin barrier index is shown. Fig.18b Skin barrier recovery rate (%) is shown;

[0054] FIG. 19 shows the results of confirming the improvement effect of the SPMs composite composition of the present invention on atopic dermatitis through human efficacy evaluation: Fig.19a The change rate (%) of skin moisture in the damaged area is shown. Fig.19b The change rate of transepidermal water loss (%) at the injured site is shown. Fig.19c The change rate of the skin pruritus index (5) at the lesion site is shown;

[0055] FIG. 20 shows the results of confirming the effects of the SPMs composite composition of the present invention on alleviating eye wrinkles and improving skin moisture through human efficacy evaluation: Fig.20a The change rate (%) of eye wrinkles is shown. Fig.20b Shows the moisture content of the skin around the eyes. Fig.20c The improvement rate (%) of moisture of the skin around the eyes is shown. DETAILED DESCRIPTION

[0056] Hereinafter, the present invention will be described in detail.

[0057] 1. Enzymes for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids

[0058] One aspect of the present invention provides an enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids.

[0059] In addition, another aspect of the present invention provides a nucleic acid molecule encoding the enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids.

[0060] The enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids of the present invention comprises an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and may be a variant or fragment of amino acids having a different sequence by deletion, insertion, substitution or combination of amino acid residues within the range that does not affect the function of the enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids. Amino acid exchange at the level of proteins and peptides is known in the relevant art without completely changing the activity of the enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids. In some cases, the amino acid exchange may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. Therefore, the present invention includes proteins having an amino acid sequence substantially identical to that of a protein comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and variants or active fragments thereof. Substantially identical proteins refer to proteins having at least 90%, preferably at least 93%, and most preferably at least 95% homology with the amino acid sequence, but are not limited thereto, and proteins having at least 90% homology with the amino acid sequence and having the same enzyme activity are included within the scope of the present invention.

[0061] The gene of the enzyme for preparing the monohydroxy or dihydroxy derivative of polyunsaturated fatty acids is preferably composed of the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. However, the enzyme encoding the monohydroxy or dihydroxy derivative of polyunsaturated fatty acids of the present invention and its variant or active fragment may have various modifications in the coding region within the scope of not changing the amino acid sequence of the enzyme expressed by the coding region or its variant or active fragment. Even in the part outside the coding region, various mutations may be present within the scope that does not affect the expression of the gene, and such mutant genes are also included in the scope of the present invention. Therefore, the present invention includes a gene consisting of a nucleotide sequence substantially identical to the nucleic acid molecule of SEQ ID NO: 3 or SEQ ID NO: 4 and a fragment of the gene. A gene consisting of a substantially identical nucleotide sequence refers to a gene having a sequence homology of 80% or more, preferably 90% or more, and most preferably 95% or more, but is not limited thereto, and a gene having a sequence homology of 80% or more and having the same enzyme activity as the encoded protein is included in the present invention. In this way, the gene of the enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids of the present invention can be mutated by replacing, deleting and inserting at least one nucleotide or a combination thereof, as long as the gene encodes a protein with the same activity as the gene, and these mutants are also included in the scope of the present invention.

[0062] The amino acid sequence represented by SEQ ID NO: 1 contained in the enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids is preferably encoded by a gene consisting of a nucleotide sequence represented by SEQ ID NO: 3, and the amino acid sequence represented by SEQ ID NO: 2 is preferably encoded by a gene consisting of a nucleotide sequence represented by SEQ ID NO: 4, but the present invention is not limited thereto. The amino acid sequence may also be encoded by a gene consisting of another nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, as long as the amino acid sequence can encode the protein of the present invention having the same amino acid sequence. These nucleotide sequences may be single-stranded or double-stranded, and may be DNA molecules or RNA molecules.

[0063] In one specific example of the present invention, the present inventors have isolated and purified a protein having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, thereby discovering the function of the protein having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 (see Figure 1 As a result of studying its activity, the present inventors confirmed that the protein having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 has the activity of an enzyme capable of producing a monohydroxy derivative, or a dihydroxy derivative in which two hydroxy groups are introduced into a polyunsaturated fatty acid (see Figure 2 and Figure 3 ).

[0064] 2. Expression vector and transformant of enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids

[0065] Another aspect of the present invention provides a recombinant expression vector and a transformant introduced with the recombinant expression vector, wherein the recombinant expression vector comprises a gene of the enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids of the present invention.

[0066] The recombinant expression vector of the present invention comprises the gene of the enzyme for preparing the monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids.

[0067] The expression vector includes a plasmid vector, a cosmid vector, a phage vector, a viral vector, etc., but is not limited thereto.

[0068] The recombinant expression vector can be appropriately combined with expression regulatory sequences such as promoters, terminators, enhancers, etc., or sequences for secretion according to the purpose and the type of host cells, so as to prepare the enzyme of the present invention for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids.

[0069] The expression vector may further include a selectable marker for selecting host cells into which the vector has been introduced and, in the case of a replicable expression vector, an origin of replication.

[0070] In addition, the recombinant expression vector may contain a sequence for promoting the purification of the expressed protein, specifically, it may be connected to a gene encoding a tag for separation and purification, so that the gene encoding the enzyme for preparing the monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids of the present invention is operable. At this time, the tag for separation and purification may be used alone, such as GST, poly-Arg, FLAG, histidine tag (His-tag), c-myc, etc., or may be used by sequentially connecting two or more tags.

[0071] The gene of the enzyme of the monohydroxyl or dihydroxy derivative of the described coding for the preparation of polyunsaturated fatty acids can be cloned by the restriction enzyme cleavage site.When using the gene of the encoding protein cutting enzyme recognition site in the vector, this gene is connected with the gene frame (in frame) of the enzyme of the monohydroxyl or dihydroxy derivative for the preparation of polyunsaturated fatty acids.When obtaining enzyme then by the protein lyase cracking, the enzyme of the original type of the monohydroxyl or dihydroxy derivative for the preparation of polyunsaturated fatty acids can be prepared.

[0072] In a specific example of the present invention, the gene of the enzyme for preparing the monohydroxy or dihydroxy derivative of polyunsaturated fatty acid of the present invention (the gene comprising the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4) is inserted into the plasmid vector pET28a (+) to prepare a recombinant cloning vector. In addition to pET28a (+) used in the preparation of the cloning vector, various vectors for prokaryotic cells or eukaryotic cells (such as pPIC and pPICZ, etc.) are known, and therefore, in addition to the above vectors, various expression vectors can be used according to the purpose of expression.

[0073] The recombinant expression vector of the present invention comprises the gene of the enzyme for producing the monohydroxy or dihydroxy derivative of polyunsaturated fatty acid, and therefore, can be effectively used as a vector capable of producing the gene of the enzyme.

[0074] In addition, the transformant of the present invention is introduced with the recombinant expression vector comprising the gene encoding the enzyme for producing the monohydroxy or dihydroxy derivative of polyunsaturated fatty acid.

[0075] According to the expression purpose, the recombinant expression vector according to the present invention is transformed into any suitable host cell selected from bacteria, yeast, Escherichia coli, fungi, plant cells and animal cells to prepare a transformant. For example, the host cell can be Escherichia coli (Escherichia coli BL21 (DE3), DH5a, etc.), yeast cells (Saccharomyces, Pichia, etc.), etc. At this time, those skilled in the art can easily select appropriate culture methods and culture medium conditions, etc. according to the type of host cells from techniques known in the art.

[0076] As a method for introducing a recombinant expression vector for preparing the transformant of the present invention, a known technique, ie, a heat shock method, an electric shock method, etc. can be used.

[0077] In a specific example of the present invention, a transformant is prepared by using Escherichia coli as a host cell and transforming a recombinant expression vector containing a gene encoding an enzyme for preparing a monohydroxy or dihydroxy derivative of a polyunsaturated fatty acid of the present invention into Escherichia coli.

[0078] Since the protein expressed by the transformant is a protein of a novel sequence having the activity of an enzyme for producing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids, the transformant is cultured on a large scale to express the gene, thereby facilitating large-scale production of the enzyme for producing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids.

[0079] 3. Method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vitro

[0080] Another aspect of the present invention provides a method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vitro.

[0081] The method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vitro of the present invention comprises: reacting a polyunsaturated fatty acid with an enzyme for preparing monohydroxy or dihydroxy derivatives described in the item "1. Enzyme for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids". The polyunsaturated fatty acid is a fatty acid containing three or more double bonds between carbons, specifically omega-3 fatty acids, more specifically docosahexaenoic acid or eicosapentaenoic acid, but is not limited thereto. As described above, when the polyunsaturated fatty acid is used as a substrate for an enzyme for preparing monohydroxy or dihydroxy derivatives, the substrate contains multiple double bonds, and hydroxyl groups can be introduced into the double bonds to achieve the preparation of monohydroxy or dihydroxy derivatives.

[0082] The method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vitro of the present invention further comprises: recovering the monohydroxy or dihydroxy derivatives from the reaction product of the enzyme and the polyunsaturated fatty acid.

[0083] The recovery can be achieved by carrying out methods commonly performed in the art, such as centrifugation and filtration, and can be achieved by additionally carrying out a purification process in a conventional manner. For example, the purification process can be carried out alone, or in combination with techniques such as solvent extraction, dialysis, gel filtration, ion exchange, and chromatography such as reverse phase column chromatography.

[0084] The reaction of the polyunsaturated fatty acid with the enzyme for preparing the monohydroxy or dihydroxy derivative can be carried out in the temperature range of 10° C. to 40° C., preferably in the temperature range of 15° C. to 35° C., but is not limited thereto. In addition, the reaction can be carried out in the range of pH 4 to pH 10, preferably in the range of pH 7 to pH 9, but is not limited thereto. When the reaction is carried out in the above temperature and pH range, the activity of the enzyme for preparing the monohydroxy or dihydroxy derivative is maximized, thereby more efficiently preparing the monohydroxy or dihydroxy derivative.

[0085] 4. Method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vivo

[0086] Another aspect of the present invention provides a method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vivo.

[0087] The method for preparing the monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vivo of the present invention comprises: culturing in the presence of polyunsaturated fatty acids in the presence of 2. Enzymes for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids Expression vectors and transformants ” and isolating the monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids from the cultured culture.

[0088] In a transformant introduced with a recombinant expression vector containing a gene encoding the enzyme for preparing a monohydroxy or dihydroxy derivative, the enzyme for preparing a monohydroxy or dihydroxy derivative can be expressed. The enzyme for preparing a monohydroxy or dihydroxy derivative present in the transformant is referred to as " 1. Monomers used to prepare polyunsaturated fatty acids Hydroxyl or dihydroxy derivatives of enzymes " item, and its detailed description will be omitted.

[0089] In the transformant, the enzyme for preparing the monohydroxy or dihydroxy derivative is expressed. Therefore, by using it, the transformant is cultured in a medium containing a polyunsaturated fatty acid as a substrate to prepare the monohydroxy or dihydroxy derivative without requiring a separate process of isolating the enzyme.

[0090] The culture of the transformant can be carried out according to appropriate culture medium and culture conditions known in the art. Those skilled in the art can easily adjust and use culture medium and culture conditions according to the type of transformant to be selected. The culture method can include batch, continuous, batch feed or a combination thereof.

[0091] The culture medium may contain various carbon sources, nitrogen sources and trace element components.

[0092] The carbon source may include, for example, sugars such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose; animal and vegetable oils such as soybean oil, sunflower oil, castor oil and coconut oil; fatty acids such as palmitic acid, stearic acid and linoleic acid; alcohols such as glycerol and ethanol; organic acids such as acetic acid; or combinations thereof. Glucose may be used as a carbon source for culturing. The nitrogen source may include an organic nitrogen source such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL) and soy milk; an inorganic nitrogen source such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate; or combinations thereof. As a source of phosphorus, the culture medium may include, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and their corresponding sodium salts; and metal salts such as magnesium sulfate or ferrous sulfate.

[0093] In addition, the medium may contain amino acids, vitamins, appropriate precursors, etc. The medium or individual components may be added to the medium in a batch or continuous type.

[0094] In addition, during the culture process, the generation of bubbles can be suppressed by using an antifoaming agent such as polyethylene glycol fatty acid ester.

[0095] Composition for improving skin condition or preventing or treating skin diseases

[0096] In another aspect, another aspect of the present invention provides a composition for improving skin conditions or preventing or treating skin diseases.

[0097] In a specific embodiment, the present invention provides a composition for improving skin conditions, comprising 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX as active ingredients.

[0098] The 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX are lipoxygenase (LOX) metabolites from the omega-3 fatty acid, docosahexaenoic acid (DHA), and are specific pro-resolvative mediators (SPMs).

[0099] The 17-hydroxydocosahexaenoic acid (17-HDHA) is (±)17-hydroxy(dihydroxy)-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid and is represented by the structure of Formula 1 below.

[0100] [Formula 1]

[0101]

[0102] The resolvin D5 (RvD5) is 7S,17S-dihydroxy-4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid and is represented by the structure of Formula 2 below.

[0103] [Formula 2]

[0104]

[0105] The protectin DX (PDX) is 10S, 17S-dihydroxy-4Z, 7Z, 11E, 13Z, 15E, 19Z-docosahexaenoic acid and is an isomer of protein D1, and is represented by the structure of Formula 3 below.

[0106] [Formula 3]

[0107]

[0108] The 17-HDHA, resolvin D5 and protectin DX may be biosynthesized, chemically synthesized or commercially purchased by treating docosahexaenoic acid with the enzyme of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention, but is not limited thereto.

[0109] In one specific example of the present invention, as a result of treating human dermal fibroblasts with a composite composition comprising 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX, it can be confirmed that the expression of inflammatory factors increased by ultraviolet irradiation is suppressed and procollagen synthesis is increased. In particular, it can be confirmed that the composite composition of the present invention synergistically induces collagen synthesis effect and inflammation inhibition effect compared with a single compound. In addition, in human application experiments, it can be confirmed that the composite composition of the present invention relieves dry skin and itching caused by atopic dermatitis by restoring the damaged skin barrier and reducing transepidermal water loss.

[0110] Therefore, the composition of the present invention can be used to improve skin conditions.

[0111] As used herein, the term "skin condition" may be skin aging, spots, freckles, skin wounds, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, rash or acne due to ultraviolet rays.

[0112] Skin aging manifests itself in symptoms such as decreased skin elasticity, reduced radiance, wrinkle generation, reduced regeneration or severe dryness, and includes both intrinsic and extrinsic aging caused by the passage of time or external environment. Specifically, skin aging may be photoaging, which refers to skin damage that occurs when the skin is repeatedly or chronically exposed to ultraviolet rays.

[0113] In addition, wrinkles refer to wrinkles that occur in all body parts including the face, and include both static wrinkles and dynamic wrinkles.

[0114] As used herein, the term "improving skin condition" is a comprehensive concept including prevention or alleviation of skin wrinkles, prevention or alleviation of skin aging, prevention or alleviation of skin inflammation, proliferation and regeneration of skin cells, shrinking or reducing pores, improving skin barrier function, alleviating skin irritation, anti-oxidation and enhancing collagen synthesis.

[0115] As used herein, "comprising as an active ingredient" means comprising an effective amount to the extent that skin improvement effects can be exhibited, for example, wrinkle relief, inflammation relief, skin regeneration, atopic dermatitis relief, or skin barrier enhancement and improvement.

[0116] According to one embodiment of the present invention, the composition can promote the synthesis of collagen in cells or inhibit the decomposition of collagen by inhibiting the expression or activity of matrix metalloproteinase (MMP). The composition can, for example, inhibit the decomposition of extracellular matrix by inhibiting the expression or activity of MMP-1 that decomposes type 1 collagen.

[0117] According to one embodiment of the present invention, the composition may have antioxidant activity. The composition may inhibit the formation of lipid peroxides. According to another embodiment of the present invention, the composition may inhibit inflammatory factors. The composition may inhibit NO, TNF-α and IL-6 as inflammatory initiators.

[0118] TNF-α (tumor necrosis factor-a) and IL-6 (interleukin-6) are representative inflammation-inducing cytokines. TNF-α is a proinflammatory cytokine produced by lymphocytes activated by lipopolysaccharide (LPS), which is an endotoxin in the cell membrane of Gram-negative bacteria, and IL-6 is secreted by macrophages and T cells to stimulate immune responses to promote inflammation.

[0119] The composite composition of the present invention comprising 17-HDHA, resolvin D5 and protectin DX can achieve excellent anti-inflammatory effects by inhibiting the expression of inflammatory cytokines TNF-α and IL-6, and has excellent antioxidant effects of inhibiting the production of lipid peroxides due to oxidative stress occurring in the skin, thereby improving the skin's resistance to external stimuli.

[0120] According to yet another embodiment of the present invention, the composition can be used to enhance the skin barrier.

[0121] The stratum corneum is the outermost layer of the epidermis of the skin barrier, mainly composed of anucleated flat keratinocytes. The multilayer lipid membrane formed by intercellular lipids such as ceramide, cholesterol and fatty acids acts as a protective barrier to prevent the evaporation of water in the skin, and the intercellular lipids are synthesized by the keratinocytes of the skin barrier maintained by the division and differentiation process of normal epidermal cells. In these intercellular lipids, omega hydroxyceramides are chemically covalently linked to the involucrin as the protein of the outer layer of keratinocytes to form a corneocyte lipid envelope (CLE), so that the intercellular lipids are physically stabilized in the form of multilayer lipid membranes to act, thereby enhancing barrier function.

[0122] The composition of the present invention is delivered to the stratum corneum by skin application and promotes the differentiation of keratinocytes, thereby producing an effect of increasing the thickness of the epidermis. In addition, the composition has an excellent effect of repairing the damage of the skin barrier, and thus, can be effectively used to treat and prevent skin diseases caused by skin barrier damage. Skin diseases caused by skin barrier damage include, but are not limited to, atopic dermatitis, dry skin disease, psoriasis, ichthyosis, acne, etc.

[0123] In addition, the skin barrier enhancement effect was determined by increasing the expression of filaggrin and loricrin. Filaggrin is one of several structural proteins expressed by keratinocytes in the differentiation stage and is involved in the differentiation from the basal layer of the epidermis to the stratum corneum. It is used as a primary indicator of skin moisturizing and skin membrane function because it also forms the main component of the natural moisture factor (NMF) necessary for maintaining moisture in skin tissue. Due to the significant increase in the gene expression of filaggrin or loricrin, the composite composition comprising 17-HDHA, resolvin D5 and protectin DX according to the present invention has excellent skin barrier protection, enhancement and improvement functions.

[0124] Due to the complex synergistic effects of 17-HDHA, resolvin D5 and protectin DX included as active ingredients, the composite composition of the present invention comprising 17-HDHA, resolvin D5 and protectin DX can achieve significantly improved IL-6 expression inhibition effect and procollagen synthesis effect, especially compared with using the same concentration of each of these compounds (Figures 11 and 12).

[0125] In one embodiment of the present invention, the complex comprises 17-HDHA, resolvin D5 and protectin DX in a weight ratio of 3 to 85:10 to 60:5 to 50, for example, a weight ratio of 3:10:5, a weight ratio of 5:60:50, a weight ratio of 85:10:5, but is not limited thereto. In one example of the present invention, a composite composition comprising 17-HDHA, resolvin D5 and protectin DX in a weight ratio of 3:47:50, 25:56:19 and 84:10:6 was used to confirm the skin improvement effect.

[0126] On the other hand, the composition for improving skin condition may be used as a cosmetic composition, a pharmaceutical composition or a food composition.

[0127] Therefore, in one aspect, the present invention provides a cosmetic composition for improving skin conditions, comprising 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX as active ingredients.

[0128] The complex composition comprising 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX and the contents related to improvement of skin condition are the same as described above.

[0129] When the composite composition of the present invention is used as a cosmetic composition, in addition to 17-HDHA, resolvin D5 and protectin DX, it also contains ingredients commonly used in cosmetic compositions, for example, sulfating agents, stabilizers, solubilizers, vitamins, conventional adjuvants such as pigments and fragrances, and carriers.

[0130] The cosmetic composition of the present invention can be prepared as any formula usually prepared in this area. The cosmetic composition can be formulated as, for example, solution, suspension (anhydrous and aqueous), anhydrous product (oils and glycols), emulsion, paste, gel, mask, dressing, powder, frost, lotion, powder, soap, surfactant-containing cleansing agent, oil, foundation, liquid foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, the cosmetic composition can be prepared as formula such as toner (skin), essence toner (milk emulsion), essence cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, dressing, spray or powder. At this point, the easy accessibility of the cosmetic composition can be further improved, and the chronic recurrence of atopic dermatitis can be prevented by implementing a moisturizing effect.

[0131] When the cosmetic composition is formulated as a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicon dioxide, talc, zinc oxide and the like may be used as carrier components.

[0132] When the cosmetic composition is formulated as a powder or a spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and specifically, when the formulation is a spray, a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether may be included.

[0133] When the dosage form of the cosmetic composition is a solution or an emulsion, a solvent, a solubilizer or an emulsifier may be used as a carrier component, and examples thereof include water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester.

[0134] When the formula of the cosmetic composition is a suspension, the following can be used as carrier components: liquid diluents such as water, ethanol and propylene glycol; suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters and polyoxyethylene sorbitan esters; microcrystalline cellulose; aluminum metahydroxide; bentonite; agar; tragacanth gum, etc.

[0135] When the formula of the cosmetic composition is a surfactant-containing cleanser, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyl taurate, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glyceryl fatty acid esters can be used as carrier components.

[0136] When the cosmetic composition is in the form of soap, it can be prepared by including skin moisturizers, emulsifiers, water softeners, etc. as additives. As a base material for soap, vegetable oils such as coconut oil, palm oil, soybean oil, olive oil, palm kernel oil, jojoba oil, etc. can be used; or animal fats such as tallow, lard, sirloin, fish oil, etc. can be used. As a moisturizer, glycerol, erythritol (detipritol), propylene glycol, butylene glycol, hexylene glycol, isopropyl myristate, aloe, sorbitol, etc. can be used. As an emulsifier, natural oils, waxes, hydrocarbons, etc. can be used, and as a water softener, EDTA tetrasodium, etc. can be used, but are not limited thereto.

[0137] The soap may further contain antibacterial agents, foam suppressants, solvents, corrosion inhibitors, fragrances, colorants, chelating agents, preservatives, etc. as additives.

[0138] When the composition of the present invention is used as a cosmetic composition, 17-HDHA, resolvin D5 and protectin DX in the composition may be contained at a concentration of 30 μM or more, specifically 35 μM or more, more specifically 40 μM or more. However, this varies depending on the form in which the cosmetic composition is prepared and its specific application site (face or hand) or application dose, and is therefore not limited thereto.

[0139] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases, wherein the pharmaceutical composition comprises 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX as active ingredients.

[0140] The skin disease may be skin wounds, skin scars, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, athlete's foot, erythema, urticaria, psoriasis, rash, acne or hair loss.

[0141] The complex composition comprising 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX and the contents related to skin diseases are the same as described above.

[0142] When the composition is used as a pharmaceutical composition, the composition may further include a pharmaceutically acceptable carrier or additive in addition to 17-HDHA, resolvin D5 and protectin DX.

[0143] "Pharmaceutically acceptable" means that it is not toxic beyond the tolerance of the intended subject without inhibiting the activity of the active ingredient. "Carrier" is defined as a compound that facilitates the incorporation of a compound into cells or tissues.

[0144] The 17-HDHA, resolvin D5 and protectin DX of the present invention can be administered alone or in combination with any convenient carrier, etc., and such dosage form can be a single dose or a repeated dose preparation. The pharmaceutical composition can be a solid preparation or a liquid preparation. Solid preparations include powders, granules, tablets, capsules and suppositories, but are not limited thereto. Solid preparations may include carriers, flavor enhancers, binders, preservatives, disintegrants, lubricants, fillers, etc., but are not limited thereto. Liquid preparations include solutions such as water and propylene glycol solutions, suspensions and emulsions, but are not limited thereto, and can be prepared by adding appropriate colorants, flavoring agents, stabilizers, thickeners, etc. For example, powders can be prepared by simply mixing appropriate pharmaceutically acceptable carriers such as lactose, starch, microcrystalline cellulose, etc. with 17-HDHA, resolvin D5 and protectin DX of the present invention as active ingredients. Granules can be prepared by mixing 17-HDHA, resolvin D5 and protectin DX of the present invention, a suitable pharmaceutically acceptable carrier and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropylcellulose, and then using a wet granulation method using a solvent such as water, ethanol and isopropanol, or a dry granulation method using a compression force. In addition, tablets can be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then tableting the mixture using a tablet press.

[0145] According to the disease to be treated and the condition of the subject, the 17-HDHA, resolvin D5 and protectin DX of the present invention can be administered by oral dosage, injection (e.g., intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection and implant), inhalation, nasal administration, vaginal administration, rectal administration, sublingual administration, transdermal administration, topical administration, etc., but are not limited thereto. The derivatives can be formulated into appropriate dosage unit preparations containing pharmaceutically acceptable carriers, additives and excipients, which are conventionally used according to the route of administration and are non-toxic.

[0146] The pharmaceutical composition of the present invention can be administered at a daily dose of about 0.0001 mg / kg to about 10 g / kg and about 0.001 mg / kg to about 1 g / kg. However, the dosage can vary according to the degree of purification of the mixture, the patient's condition (age, sex, body weight, etc.) and the severity of the condition for treatment. As needed, for convenience, the total daily dose can be divided in one day and administered multiple times.

[0147] When the composition of the present invention is used as a pharmaceutical composition, 17-HDHA, resolvin D5, and protectin DX in the composition may be contained at a concentration of 30 μM or more, specifically 35 μM or more, more specifically 40 μM or more.

[0148] Another aspect of the present invention provides a food composition for improving skin conditions, wherein the food composition comprises 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX as active ingredients.

[0149] The complex composition comprising 17-hydroxydocosahexaenoic acid (17-HDHA), resolvin D5 and protectin DX and the contents related to the improvement of skin condition are the same as described above.

[0150] When the composition is used as a food composition, the composition may contain acceptable food supplement additives, and may further contain appropriate carriers, excipients and diluents commonly used in food preparation.

[0151] In the present invention, food refers to a natural product or processed product containing one or more nutrients, specifically, refers to a state that can be directly eaten through a specific processing process, and as a conventional meaning, is used as all of various foods, functional foods, beverages, food additives and beverage additives. Examples of food include various foods, beverages, soft candies, teas, vitamin complexes, functional foods, etc. In addition, the food of the present invention includes: special nutritional foods (such as formula foods, baby foods, etc.), processed meat products, fish products, tofu, mud (muk), noodles (such as ramen, noodles, etc.), health supplement foods, seasoning foods (such as soy sauce, bean paste, chili sauce, mixed sauce, etc.), sauces, candies (such as snacks), dairy products (such as fermented milk, cheese, etc.), other processed foods, kimchi, salted foods (various kimchi, pickles, etc.), beverages (such as fruit beverages, vegetable beverages, soy milk, fermented beverages, ice cream, etc.), natural seasonings (such as ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages and other health supplements, but are not limited to this. The functional food, beverage, food additive or beverage additive can be prepared by a conventional preparation method.

[0152] The term "functional food" refers to a food that is designed and processed to fully express the human regulatory functions on the human body regarding biodefense rhythm control, disease prevention and recovery, etc., with respect to a food group or food composition, which has an added value of acting and expressing the functions of the corresponding food for a specific purpose by using physical, biochemical and biotechnological methods, etc., and specifically, may be a health functional food.

[0153] The term "healthy functional food" used in the present invention refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills by using raw materials or ingredients with functionality useful to the human body. Here, "function" refers to adjusting nutrients to the structure and function of the human body, or refers to obtaining useful effects for health applications such as physiological effects. The health functional food of the present invention can be prepared by methods commonly used in the art, and can be prepared by adding raw materials and ingredients commonly used in the art in the preparation. In addition, the formula of the health functional food can also be prepared without restriction, as long as the formula is considered to be a health functional food. The food composition of the present invention can be prepared as various types of formulas, and unlike conventional drugs, the food composition has the advantage that, by using food as a raw material, there are no side effects that occur when taking drugs for a long time, and it has excellent portability, but the health functional food of the present invention can be considered as a supplement for enhancing the skin improvement effect.

[0154] In addition, the functional food may include nutritionally acceptable food supplement additives, and may also include appropriate carriers, excipients and diluents commonly used in the preparation of functional foods.

[0155] In addition, in the food composition, 17-HDHA, resolvin D5, and protectin DX in the composition may be contained at a concentration of 30 μM or more, specifically 35 μM or more, and more specifically 40 μM or more.

[0156] In addition to the active ingredients, the food composition of the present invention may also include sweeteners, flavoring agents, physiologically active ingredients, minerals, etc. Sweeteners can be used in an amount that gives food an appropriate sweet taste, and can be natural or synthetic. Specifically, when using natural sweeteners, examples of natural sweeteners can include sugar sweeteners, such as starch syrup dry powder, honey, sucrose, fructose, lactose and maltose. Flavoring agents can be used to improve taste or flavor, and both natural flavoring agents and synthetic flavoring agents can be used. Specifically, natural flavoring agents are used. In the case of using natural flavoring agents, in addition to seasoning, the purpose of nutritional enhancement can be combined. Natural flavoring agents can be obtained from apples, lemons, citrus, grapes, strawberries, peaches, etc., or from green tea leaves, Polygonatum multiflorum, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. In addition, flavoring agents obtained from ginseng (red ginseng), bamboo shoots, aloe and ginkgo can be used. Natural flavoring agents can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents can be used, and synthetic flavoring agents can use esters, alcohols, aldehydes, terpenes, etc. As physiologically active ingredients, catechins such as catechin, epicatechin, epigallocatechin and epigallocatechin and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin can be used. As minerals, calcium, magnesium, chromium, cobalt, copper, fluorine, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc and the like can be used.

[0157] In addition, in addition to sweeteners, the food composition of the present invention may also include preservatives, emulsifiers, acidulants, thickeners, etc., as required. These preservatives, emulsifiers, etc. are preferably added and used in very trace amounts, as long as the application to be added can be realized. When expressed in numbers, trace amounts refer to the gross weight based on the food composition, the scope of about 0.0005 weight % to about 0.5 weight %. Available preservatives may include sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, ethylenediaminetetraacetic acid (EDTA), etc. Available emulsifiers may include gum arabic, carboxymethyl cellulose, xanthan gum, pectin, etc. Available acidulants may include citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. These acidulants may be added so that the food composition has appropriate acidity, so as to be used for the purpose of inhibiting the proliferation of microorganisms in addition to the purpose of enhancing taste. As thickeners that can be used, there are suspending agents, settling aids, gel forming agents, swelling agents and the like.

[0158] Hereinafter, the present invention will be described in detail by way of examples.

[0159] However, the following embodiments are specific illustrations of the present invention, and the contents of the present invention are not limited to the following embodiments.

[0160] [Example 1]

[0161] Preparation of a protein comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2

[0162] [1-1] Preparation of vectors for protein expression

[0163] Bioneer Co., Ltd was requested to synthesize the nucleotide sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4 encoding the respective amino acids of SEQ ID NO: 1 and SEQ ID NO: 2. Using the respective synthetic nucleotide sequences as templates, preliminary denaturation was performed at 94° C. for 5 minutes using primer pairs of SEQ ID NO: 5 to SEQ ID NO: 8, and then PCR was performed by repeating a cycle of reaction at 94° C. for 30 seconds, reaction at 61° C. for 30 seconds, and reaction at 72° C. for 2 minutes 20 times to amplify the respective genes of SEQ ID NO: 3 and SEQ ID NO: 4.

[0164] [Table 1]

[0165]

[0166] The PCR products containing the nucleotide sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4 amplified as described above were respectively inserted into the plasmid vector pET28a (+) (Novagen, USA) to prepare a recombinant expression vector. It was confirmed by nucleotide sequence analysis (Solgent) that the nucleotide sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4 were properly inserted.

[0167] [1-2] Protein expression and purification

[0168] The recombinant expression vector prepared in Example [1-1] was transformed into Escherichia coli BL21 (DE3), and each transformant was inoculated into 3 ml of LB medium and seed cultured at 37°C until the absorbance at 600 nm became 2.0. Then, main culture was performed by adding the medium cultured with seeds to 500 ml of LB medium. When the absorbance at 600 nm became 0.6, IPTG (isopropyl-1-thio-β-D-galactoside) was added to a final concentration of 1 mM to induce overexpression of a protein having an amino acid sequence represented by SEQ ID NO: 1 and a protein having an amino acid sequence represented by SEQ ID NO: 2. During the induction of overexpression as described above, the culture temperature was maintained at 37°C before the addition of IPTG, and the culture temperature was reduced to 20°C after the addition of IPTG.

[0169] The culture medium of the transformant induced to overexpression is centrifuged to separate the supernatant, and the cells of the transformant are lysed from the pellet from which the supernatant is separated to obtain a cell lysate of the transformant.

[0170] As a result of SDS-PAGE of 100 μl of the cell lysate obtained as described above, Figure 1 As shown in , it can be confirmed that a protein having an amino acid sequence represented by SEQ ID NO: 1 and a size of about 96.9 KDa ( Figure 1 A) and a protein having an amino acid sequence represented by SEQ ID NO: 2 having a size of about 96.8 KDa ( Figure 1 B) is overexpressed.

[0171] Regarding the cell lysate in which overexpression of the protein having the amino acid sequence represented by SEQ ID NO: 1 and the protein having the amino acid sequence represented by SEQ ID NO: 2 was confirmed as described above, the protein having the amino acid sequence represented by SEQ ID NO: 1 and the protein having the amino acid sequence represented by SEQ ID NO: 2 were separated and purified using Ni-NTA adsorption chromatography.

[0172] [Example 2]

[0173] Confirm protein activity - confirm the activity of mono- or di-hydroxy derivative preparations

[0174] For the protein having the amino acid sequence represented by SEQ ID NO: 1 and the protein having the amino acid sequence represented by SEQ ID NO: 2 purified and separated in Example [1-2], 6 KU or 10 KU of each protein and 100 μM of docosahexaenoic acid (DHA) were reacted at pH 7 and room temperature for 30 minutes, the reaction product was reduced by adding 1 M sodium borohydride so that the final concentration became 50 mM, and then the reaction was terminated by adding 5 μl / ml of acetic acid.

[0175] After the reaction product was purified using a solid phase column (SPE, C18 500 mg), the type of compound in the reaction product was analyzed using normal phase HPLC.

[0176] Specifically, normal phase HPLC analysis was carried out as follows: 20 ml of a mobile phase consisting of 95% n-heptane, 5% isopropanol, 0.1% acetic acid and 0.1% 2,2-dimethoxypropane was developed on a supelcosil LC-Diol column (Supelco, 25 cm×3 mm, 5 μm) at a flow rate of 0.5 ml / min for 40 minutes, and finally a diode array detector (DAD) was used to detect the compounds in the reaction product.

[0177] Results, such as Figure 2 and Figure 3 As shown in , it can be confirmed that both the protein having the amino acid sequence represented by SEQ ID NO: 1 and the protein having the amino acid sequence represented by SEQ ID NO: 2 can prepare 17S-monohydroxy-DHA, 7S, 17S-dihydroxy-DHA (resolvin D5) and 10S, 17S-dihydroxy-DHA (protectin DX) from DHA. In each case, it can be confirmed that the contents of the above three monohydroxy or dihydroxy derivatives are respectively present in the ratios described in the following Table 2.

[0178] [Table 2]

[0179]

[0180] [Example 3]

[0181] Protein amino acid sequence analysis

[0182] For the protein having the amino acid sequence represented by SEQ ID NO: 1 and the protein having the amino acid sequence represented by SEQ ID NO: 2, homology analysis and systematic analysis of the amino acid sequences compared with human 5LOX (AAA36183), human 12LOX (AAA51533), human 15LOX (AAA36183), soybean 15LOX (AAA33986), potato LOX (AAB81595) and red algae PhLOX2 (AGN54275) were performed.

[0183] Results, such as Figures 4a to 4d As shown in, it can be confirmed that the protein having the amino acid sequence represented by SEQ ID NO: 1 and the protein having the amino acid sequence represented by SEQ ID NO: 2 were found to show very low sequence homology with human 5LOX (AAA36183), human 12LOX (AAA51533), human 15LOX (AAA36183), soybean 15LOX (AAA33986), potato LOX (AAB81595) and red algae PhLOX2 (AGN54275).

[0184] In addition, if Figures 4a to 4d As can be understood, it can be confirmed that both the protein having the amino acid sequence represented by SEQ ID NO: 1 and the protein having the amino acid sequence represented by SEQ ID NO: 2 have an N-terminal region for linking to membrane lipids and show high similarity in the C-terminal region. Specifically, it was confirmed that residues that bind to iron ions, such as His, His, His and Asn / Ser, and the terminal amino acid, exist, and Ala, which is closely related to the hydroxylation performance of the enzyme, exists.

[0185] In addition, as a result of analyzing the secondary structures of the protein having the amino acid sequence represented by SEQ ID NO: 1 and the protein having the amino acid sequence represented by SEQ ID NO: 2, it was confirmed that α-helical structure, extended chain and random coil were present in the proportions shown in Table 3 below, respectively.

[0186] [Table 3]

[0187]

[0188] [Example 4]

[0189] Confirmation of the effects of the reaction products prepared by the enzymatic activity of the protein of the present invention - inhibition of skin aging and alleviation of wrinkles Pattern

[0190] As in Example 2 above, the product obtained by reacting docosahexaenoic acid with the protein having the amino acid sequence represented by SEQ ID NO: 1 of the present invention (SPM 1), and the product obtained by reacting docosahexaenoic acid with the protein having the amino acid sequence represented by SEQ ID NO: 2 (SPM 2) were treated with human dermal fibroblasts to confirm the cytotoxicity, MMP-1 expression and procollagen synthesis effects, and were treated with human keratinocytes to confirm the changes in cytotoxicity, TNF-α expression and IL-6 expression.

[0191] [4-1] Confirmation of cytotoxicity

[0192] Human dermal fibroblasts and human keratinocytes were treated with SPM 1 and SPM 2 at concentrations of 0.001 ppm, 0.01 ppm, 0.1 ppm, and 1 ppm, respectively, to confirm cell viability.

[0193] Results, such as Figure 5 and Figure 6 As shown in , both reaction products (SPM 1, SPM 2) showed more than 90% cell activity at all concentrations, from which it can be understood that the products prepared from DHA by the protein of the present invention have no cytotoxicity.

[0194] [4-2] Confirmation of the improvement effect of TNF-α and IL-6 expression inhibitory activity

[0195] Human keratinocytes were cultured at 4 × 10 4 The cells were plated at a concentration of 10 cells / well in a 48-well plate and cultured at 5% CO2 and 37°C for 24 hours, then the medium was removed and the cells were starved with DMEM serum-free medium for 24 hours.

[0196] SPM 1 and SPM 2 were diluted to 1 ppm with DMEM (2% FBS), and then sequentially diluted to 0.1 ppm, 0.01 ppm, and 0.001 ppm, and then cultured as above and treated with human keratinocytes from which the medium had been removed for 1 hour. Then, for TNF-α or IL-6 expression, the medium was exchanged with 250 μl of DPBS (WelGENE), and 160 mJ / cm 2UVB irradiation, then treated with SPM 1 and SPM 2 again and cultured for 24 hours. In addition, after collecting the culture medium, the amount of TNF-α was measured using Human TNF-α DuoSet ELISA (R&D system), and the amount of IL-6 was measured using Human IL-6 DuoSet ELISA (R&D system). The cells adhered to the bottom were washed with DPBS, dissolved with 1N NaOH, and the amount of protein was measured by BCA analysis to measure the amount of TNF-α or IL-6 synthesized by each given protein. At this point, the effects of two reaction products SPM 1 and SPM 2 were confirmed by using the situation of not doing treatment as a negative control group and the situation of being treated with 10ppm of dexamethasone as a positive control group.

[0197] First, in the case of TNF-α, Figure 7 As shown in , the expression of TNF-α was increased by about 2 times by irradiation with UVB. This increased expression of TNF-α was reduced by about 35% by treatment with 10 ppm of dexamethasone, by about 24% by treatment with 1 ppm of SPM 1, and by about 56% by treatment with 1 ppm of SPM 2. The expression inhibition of TNF-α showed a trend that depended on the treatment concentrations of SPM 1 and SPM2.

[0198] Next, in the case of IL-6, Figure 8 As shown in , the expression of IL-6 increased by about 1.6 times by irradiation with UVB. This increased expression of IL-6 was reduced by about 88% by treatment with 10 ppm of dexamethasone, by about 35% by treatment with 1 ppm of SPM 1, and by about 92% by treatment with 1 ppm of SPM 2. The expression inhibition of IL-6 also showed a trend that depended on the treatment concentrations of SPM 1 and SPM 2.

[0199] From the above results, it can be understood that all products prepared from DHA by the protein of the present invention have the effect of stopping inflammation.

[0200] [4-3] Confirmation of the effect of improving procollagen synthesis ability

[0201] Collagen is a major component of the extracellular matrix and is synthesized in the form of a precursor called procollagen. It is known that when polymerization occurs, procollagen breaks and separates from the collagen molecule. By measuring the amount of procollagen, the extent of collagen biosynthesis in the cell can be estimated.

[0202] Human dermal fibroblasts were treated with SPM 1 and SPM 2 at concentrations of 0.01ppm, 0.1ppm and 1ppm, respectively, and cultured for 24 hours, then the cultured cell culture medium was collected, and the amount of procollagen was measured using a procollagen type I c-peptide (PIP) EIA kit (TAKARA, MK101). At this point, the effects of the two reaction products SPM 1 and SPM 2 were confirmed by using a negative control group with no treatment and a positive control group with 26.2ppm of retinyl palmitate.

[0203] Results, such as Fig. 9 As shown in , it was confirmed that both reaction products (SPM 1 and SPM 2) had a greater amount of procollagen than the negative control group that was not subjected to any treatment. Specifically, in the case of SPM 2, at a concentration of 0.1 ppm or more, a greater amount of procollagen was confirmed compared to the positive control group (26.2 ppm of retinyl palmitate). From this, it can be understood that all products prepared from DHA by the protein of the present invention improve the ability to synthesize procollagen.

[0204] [4-4] Confirmation of the effect of improving the ability to inhibit collagenase (MMP-1) expression

[0205] MMPs (matrix metalloproteinases) are enzymes with proteolytic activity, and by decomposing proteins constituting the extracellular matrix, they play an important role in the binding of cells and proteins constituting the extracellular matrix. There are 28 types of MMPs, among which MMP-1 is an enzyme that decomposes collagen. In this example, the expression level of MMP-1 in cells was confirmed.

[0206] After culturing human dermal fibroblasts for 24 hours, the medium was replaced with 250 μl / well DPBS (WelGENE) and 100 mJ / cm 2 Then, SPM 1 and SPM 2 were treated with DMEM (2% FBS) to dilute to concentrations of 0.01ppm, 0.1ppm and 1ppm, respectively, and cultured for 48 hours. After collecting the cultured cell culture medium, the amount of MMP-1 was measured using a humantotal MMP-1 ELISA kit (R&D systems, DY901). At this point, the effects of the two reaction products SPM 1 and SPM 2 were confirmed by using a negative control group with no treatment and a positive control group with 6ppm of retinoic acid treatment.

[0207] Results, such as Fig.10As shown in , the expression of MMP-1 was increased by about 2 times by irradiation of UVB. This increased expression of MMP-1 was reduced by about 70% by treatment with 6 ppm of retinoic acid, by about 17.2% and 80.1% by treatment with 0.1 ppm and 1 ppm of SPM 1, respectively, by about 11.4% by treatment with 0.01 ppm of SPM 2, by about 26.1% by treatment with 0.1 ppm of SPM 2, and by about 63.3% by treatment with 1 ppm of SPM 2. The expression inhibition of MMP-1 also showed a trend that depended on the treatment concentrations of SPM 1 and SPM 2.

[0208] [Example 5]

[0209] Preparation of SPMs composite compositions and preparation of single compounds

[0210] [5-1] A complex composition of 17-HDHA, resolvin D5 and protectin DX (hereinafter referred to as "SPMs complex composition")

[0211] Lipoxygenase having an amino acid sequence represented by SEQ ID NO: 1 was added to docosahexaenoic acid (DHA) at different concentrations and reacted at pH 7 and room temperature for 30 minutes. The reaction product was reduced by adding 1M sodium borohydride so that the final concentration became 50mM, and then the reaction was terminated by adding 5μl / ml of acetic acid. After the reaction product was purified using a solid phase column (SPE, C18500mg), the type of compound in the reaction product was analyzed using normal phase HPLC.

[0212] As a result, it was confirmed that 17S-HDHA, resolvin D5 (7S, 17S-diHDHA) and protectin DX (10S, 17S-diHDHA) were prepared from DHA. Thus, complexes 1 to 3 were prepared, which were SPMs complex compositions in a mixed state at the ratios described in Table 4 below.

[0213] [Table 4]

[0214] Complex 1 Complex 2 Complex 3 17S-HDHA 3 25 84 Resolvin D5 47 56 10 Protectin DX 50 19 6

[0215] [5-2] Isolation and purification of 17-HDHA, resolvin D5 and protectin DX

[0216] 17S-HDHA, resolvin D5 (7S, 17S-diHDHA) and protectin DX (10S, 17S-diHDHA) were used by separating and purifying each product of the SPMs complex composition from the above Example [5-1] by preparative HPLC equipped with a C18 column or a diol column.

[0217] [Example 6]

[0218] Confirmation of the synergistic IL-6 expression inhibitory effect by the SPMs complex composition

[0219] To confirm the synergistic anti-inflammatory relief effect of the SPMs complex composition, HaCaT cells used as human keratinocytes were treated with complex compositions of 17-HDHA, resolvin D5, and protectin DX (complexes 1 to 3) and individual SPM substances to confirm changes in IL-6 expression.

[0220] [6-1] Experimental methods

[0221] Specifically, human keratinocytes were first cultured at 4 × 10 4 The cells were plated in a 48-well plate at a concentration of 10 cells / well and cultured at 5% CO 2 and 37° C. for 24 hours. The medium was then removed and the cells were starved with DMEM serum-free medium for 24 hours.

[0222] The SPMs composite composition was diluted to 0.5 μg / ml, 1 μg / ml, 2 μg / ml and 5 μg / ml with DMEM (FBS 2%), and then cultured as above and treated with human keratinocytes from which the medium was removed for 1 hour. Then, for IL-6 expression, the medium was exchanged with 250 μl of DPBS (WelGENE) and 160 mJ / cm 2 The IL-6 expression inhibition of SPMs composite compositions of the present invention and each single component was confirmed by using the UVB irradiation of 10 μ m hydrocortisone as Con (-) and the situation of processing with 10 μ m hydrocortisone as Con (+) after UVB irradiation.

[0223] [6-2] Confirmation of the IL-6 inhibitory effect of SPM alone

[0224] The results are shown in Table 5 and Fig.11a As shown in, the expression of IL-6 was increased by irradiating human keratinocytes with UVB. When treated with 2 μg / ml of 17S-HDHA, protectin DX and resolvin D5, respectively, this increased expression of IL-6 was reduced by 10.8%, 11.8% and 0.5% compared to Con(-), and showed a trend dependent on the treatment concentration of SPM.

[0225] From this, we can understand that 17-HDHA, protectin DX, and resolvin D5 all have inflammation-relieving effects.

[0226] [Table 5]

[0227]

[0228] [6-3] Confirmation of IL-6 inhibitory effect of SPMs complex composition

[0229] In addition, as shown in Table 3 and Figure 1 As shown in B, the expression of IL-6 was increased by irradiating the SPMs composite composition with UVB. When treated with 2 μg / ml of the SPMs composite composition of the present invention (Composition 1, Composition 2, and Composition 3), the increased expression of IL-6 was reduced by 18%, 33.8%, and 29.5%, respectively, compared with Con(-), and showed a trend dependent on the treatment concentration of SPM.

[0230] Therefore, it can be understood that the SPMs composite compositions of the present invention all have excellent inflammation alleviating effects.

[0231] [Table 6]

[0232]

[0233] [6-4] Confirmation of the synergistic IL-6 inhibitory effect of SPMs complex composition

[0234] As shown in Table 7 and Fig.11c As shown in the results, when treated with 2 μg / ml of the composite composition, it was confirmed that the expression of IL-6 was reduced by 18% in composition 1, 33.8% in composition 2, and 29.5% in composition 3 compared to Con(-). When treated with the same concentration of SPM alone, it was confirmed that the effect of reducing the expression of IL-6 was increased compared to that.

[0235] [Table 7]

[0236]

[0237] Thus, it can be understood that the SPMs composite composition of the present invention has a significantly increased inflammation relief effect compared to a single component. In addition, the above results show that due to the synergistic effect of 17S-HDHA, PDX and RvD5, when they are used as a composite composition instead of using them alone, the amount used is reduced. Thus, it can be understood that potential side effects that may occur when SPM is used in excess can be prevented.

[0238] [Example 7]

[0239] Confirmation of the synergistic procollagen synthesis effect by SPMs composite composition

[0240] Collagen is a major component of the extracellular matrix and is synthesized in the form of a precursor called procollagen. It is known that procollagen breaks and separates from collagen molecules when polymerization occurs. By measuring the amount of procollagen, the extent of collagen biosynthesis in cells can be estimated.

[0241] [7-1] Experimental methods

[0242] Human dermal fibroblasts were treated with SPM single materials and their composite compositions (compositions 1 to 3) at concentrations of 0.1 μg / ml, 1 μg / ml, 2 μg / ml and 5 μg / ml, respectively, and cultured for 24 hours, and then the cultured cell culture medium was collected and the amount of procollagen was measured using a procollagen type I c-peptide (PIP) EIA kit (TAKARA, MK101). At this point, the effects of SPM single components and their composite compositions (compositions 1 to 3) were confirmed by using the untreated case as an untreated control group and the case treated with 50 μM of retinyl palmitate as Con (+).

[0243] [7-2] Confirmation of the procollagen synthesis effect of SPM alone

[0244] As shown in Table 8 and Fig.12a As shown in , 17-HDHA, Protectin DX, and Resolvin D5 were all confirmed to have more procollagen than the untreated control group. Specifically, Protectin DX and Resolvin D5 were confirmed to have more procollagen than the positive control group (50 μM retinyl palmitate) at concentrations of 1 μg / ml or more.

[0245] From this, it can be understood that 17-HDHA, protectin DX, and resolvin D5 all improve the ability to synthesize procollagen.

[0246] [Table 8]

[0247]

[0248] [7-3] Confirmation of the procollagen synthesis effect of SPMs composite composition

[0249] In addition, in the case of SPMs composite compositions, as shown in Tables 9 and Figure 12b As shown in , all compositions 1 to 3 having different mixing ratios were confirmed to have procollagen in an amount 2 to 3 times or more of that of the untreated control group. Specifically, in the SPMs composite composition, at a concentration of 0.1 μg / ml or more, more procollagen was confirmed in all compositions 1 to 3 than in Con (+) treated with 50 μM retinyl palmitate.

[0250] Therefore, it can be understood that all the SPMs composite compositions of the present invention significantly improve the procollagen synthesis ability.

[0251] [Table 9]

[0252]

[0253] [7-4] Confirmation of the synergistic procollagen synthesis effect of SPMs composite composition

[0254] As shown in Table 10 and Fig.12c As shown in , it was confirmed that all SPMs composite compositions (Composition 1, Composition 2, and Composition 3) had a greater amount of procollagen than that of 17-HDHA, Protectin Dx, or Resolvin D5 as a single component.

[0255] [Table 10]

[0256]

[0257] Thus, it can be understood that the SPMs composite composition of the present invention has a significantly improved procollagen synthesis ability compared to a single component. In addition, the above results show that due to the synergistic effect of 17S-HDHA, PDX and RvD5, when they are used as a composite composition instead of using them alone, the amount used can be reduced. Thus, it can be understood that potential side effects that may occur when SPM is used in excess can be prevented.

[0258] [Example 8]

[0259] Confirmation of the improvement effect of the ability to inhibit collagenase (MMP-1) expression by the SPMs composite composition

[0260] MMPs (matrix metalloproteinases) are enzymes with proteolytic activity, and by decomposing proteins constituting the extracellular matrix, they play an important role in the binding of cells and proteins constituting the extracellular matrix. There are 28 types of MMPs, among which MMP-1 is an enzyme that decomposes collagen. In this example, the expression level of MMP-1 in cells was confirmed.

[0261] Specifically, after culturing human dermal fibroblasts for 24 hours, the medium was replaced with 250 μl / well of DPBS (WelGENE) and 100 mJ / cm 2Then, composition 2 was treated with DMEM (2% FBS) to dilute to concentrations of 0.1ppm, 1ppm, 2ppm and 5ppm, respectively, and cultured for 48 hours. After collecting the cultured cell culture medium, the amount of MMP-1 was measured using a human total MMP-1 ELISA kit (R&D systems, DY901). At this point, the effect of composition 2 was confirmed by using a negative control group without treatment and a positive control group with 20μM retinoic acid.

[0262] Results, such as Fig.13 As shown in, the expression of MMP-1 was increased by about 2 times by irradiation with UVB. This increased expression of MMP-1 was reduced by about 70% by treatment with 20 μM retinoic acid, by about 11.4% by treatment with 0.01 ppm, by about 26.1% by treatment with 0.1 ppm, and by about 63.3% by treatment with 1 ppm of the SPMs composite composition. The expression inhibition of MMP-1 also showed a trend that depended on the treatment concentration of the SPMs composite composition.

[0263] Therefore, it can be understood that the SPMs composite composition of the present invention has an excellent effect of alleviating skin wrinkles.

[0264] [Example 9]

[0265] Confirmation of the effect of improving the ability to suppress TNF-α expression by the SPMs composite composition

[0266] TNF-α is a representative inflammatory cytokine, and it is known that when its expression level increases, inflammatory skin diseases are aggravated. TNF-α is a tumor necrosis factor, which is an important factor that plays the most central role in many inflammatory responses, and the regulation of TNF-α expression plays an important role in inflammatory diseases. Therefore, in order to confirm the inflammation-relieving effect of the SPMs composite composition of the present invention, the expression pattern of TNF-α was confirmed.

[0267] Specifically, human keratinocytes were cultured at 4 × 10 4 The cells were plated at a concentration of 10 cells / well in a 48-well plate and cultured at 5% CO 2 and 37° C. for 24 hours, then the medium was removed and the cells were starved with DMEM serum-free medium for 24 hours.

[0268] Composition 2 was diluted to 0.5 μg / ml, 1 μg / ml, 2 μg / ml and 5 μg / ml in DMEM (2% FBS), and then cultured as above and treated with human keratinocytes from which the medium was removed for 1 hour. Then, for TNF-α expression, the medium was exchanged with 250 μl of DPBS (WelGENE) and 160 mJ / cm 2UVB irradiation, then treated with SPMs again and cultured for 24 hours. In addition, after collecting the culture medium, the amount of TNF-α was measured respectively using Human TNF-αDuoSet ELISA (R&D system). The cells adhered to the bottom were washed with DPBS, dissolved with 1N NaOH, and the amount of protein was measured by BCA analysis to measure the amount of TNF-α synthesized by each given protein. At this point, the effect of the SPMs composite composition was confirmed by using the situation of not doing treatment as a negative control group and the situation of treating with 10 μM hydrocortisone as a positive control group.

[0269] As a result, in the case of TNF-α, Fig.14 As shown in , the expression of TNF-α was increased by about 5 times by irradiation with UVB. This increased expression of TNF-α was reduced by about 52% by treatment with 10 μM hydrocortisone and by about 49% by treatment with 1 ppm of the SPMs composite composition. The TNF-α expression inhibition showed a trend that depended on the treatment concentration of the SPMs composite composition.

[0270] Therefore, it can be understood that the SPMs composite composition of the present invention has an excellent effect of stopping inflammation.

[0271] [Example 10]

[0272] Confirmation of the effect of improving NO generation reduction capability by SPMs composite composition

[0273] Nitric oxide (NO) is a type of reactive oxygen species known to play an important role in inducing inflammation and is a highly reactive biomolecule. NO is a key molecule involved in inflammatory response, is an inflammatory mediator and accelerates inflammatory response, further exacerbating inflammatory response. Therefore, regarding the irritation caused by fine dust, the amount of NO produced was confirmed.

[0274] Specifically, the amount of NO produced by the cells was measured using the Griess Reagent System to measure the concentration of nitrite present in the cell culture medium, and the NO concentration in the culture medium was determined using a standard curve for various concentrations of sodium nitrite.

[0275] Results, such as Fig.15 As shown in , it was confirmed that the production of NO was reduced by 39.2% to 70% in cells treated with 0.1 ppm to 5 ppm of the SPMs composite composition.

[0276] Therefore, it can be understood that the SPMs composite composition of the present invention has the effect of alleviating inflammation.

[0277] [Example 11]

[0278] Confirmation of the improvement effect of the ability to reduce lipid peroxide production by the SPMs composite composition

[0279] Cellular lipid peroxidation is one of the mechanisms of cell damage in animals and plants during aging or disease states, and the extent of cell damage can be determined by measuring total malondialdehyde (MDA) as a product of lipid peroxidation. Therefore, for stimulation caused by fine dust, MDA production by the SPMs composite composition was confirmed.

[0280] Specifically, the amount of total malondialdehyde (MDA) as a lipid peroxidation product was reacted with thiobarbituric acid (TBA), and the MDA-TBA adduct was measured by the TBARS method.

[0281] Results, such as Fig.16 As shown in , it was confirmed that MDA production was reduced by 19.1% to 62.7% in cells treated with 0.1 ppm to 5 ppm of the SPMs composite composition.

[0282] Therefore, it can be understood that the SPMs composite composition of the present invention has the effect of inhibiting skin aging.

[0283] [Example 12]

[0284] Confirmation of the improvement effect of the skin barrier improvement ability of the SPMs composite composition

[0285] It is known that filaggrin, loricrin and involucrin are core proteins that constitute the skin barrier. Among them, filaggrin is a protein produced by the decomposition of profilaggrin, which constitutes the transparent keratin granules (KG) present in the granular cells of the epidermis and plays an important role in the skin barrier by aggregating keratin filaments in keratinocytes. Therefore, an attempt was made to confirm the expression of filaggrin and loricrin by confirming the skin barrier enhancement effect through the sample.

[0286] Human keratinocytes were cultured in DMEM medium under the same conditions as in Example 9. In order to confirm the expression of genes related to skin barrier improvement ability, the cultured cells were treated with the SPMs composite composition of the present invention according to the concentration and cultured, and then RNA was extracted and RT-PCR was performed. The amplified genes were analyzed using the Gel file system and the Image J program.

[0287] Results, such as Fig.17a and Fig.17b As shown in , when treated with 50 ppm of hyaluronic acid, i.e., the positive control group, the expression of filaggrin increased by 52.6%, and the expression of loricrin increased by 97.7%. It can be confirmed that when treated with 0.1 ppm to 5 ppm of the SPMs composite composition of the present invention, filaggrin increased by 31.3% to 52.8%, and loricrin increased by 31.8% to 88.5%.

[0288] Therefore, it can be understood that the SPMs composite composition of the present invention has the effect of strengthening the skin barrier.

[0289] [Example 13]

[0290] Evaluation of the efficacy of SPMs cream in humans

[0291] A cream (cream S) was prepared using the SPMs composite composition with the composition shown in the following Table 11. For comparative experiments, a ceramide cream (cream C) and a base cream (cream B) were also prepared with the following compositions.

[0292] [Table 11]

[0293] Frost S Cream C Cream B SPMs composite composition 2.00 - - Ceramide AC45 - 1.00 - glycerin 5.00 5.00 5.00 1,2-Hexanediol 2.00 2.00 2.00 Oil Moderate Moderate Moderate preservative Moderate Moderate Moderate Purified water Moderate Moderate Moderate

[0294] (Unit: parts by weight)

[0295] [13-1] Confirmation of skin barrier improvement effect of SPMs cream

[0296] The human efficacy evaluation of skin barrier improvement was conducted on 12 women (average age 45 ± 5.54 years old). The forearm was used as the test site, and the physical damage was divided into 3 test sites and a control site (not applied), and applied to 4 sites. Then, Cream S (SPM cream), Cream C (ceramide cream) and Cream B (basic cream) were used twice a day on each designated site for 1 week, and the device was measured compared with the control site (not applied). Measurements and efficacy questionnaires were performed before skin damage, immediately after skin damage, 1 day after using the experimental product, 4 days after, and 7 days after. The skin barrier improvement effect was confirmed by measuring TEWL (transepidermal water loss) using Tewameter TM300.

[0297] Results, such as Fig.18a As shown in , ceramide cream, which is the most widely used skin barrier improvement material, did not show significant improvement compared with base cream, while SPM cream had a statistically significant skin barrier improvement effect compared with the non-application group and base cream (two-tailed test using two groups assuming equal variance, t-test; p < 0.034 after 4 days of use, p < 0.027 after 7 days of use). In addition, as a result of comparing the skin barrier recovery rate (%), it can be confirmed that SPM cream has a relative advantage compared with base cream and ceramide cream, such as Fig.18b As shown in (Wilcoxon signed-rank test; *p≤0.05, **p≤0.01, ***p≤0.001).

[0298] [13-2] Confirmation of the efficacy of SPMs cream in improving atopic dermatitis

[0299] Human efficacy evaluation for relieving itching caused by dryness was conducted on 22 subjects. After measuring skin moisture, 11 subjects were randomly assigned to each experimental product, Cream S and Cream C, by matching the mean values ​​between the groups, and were asked to use each product. Forearms and dry lesions were used as test sites, and device measurements and efficacy questionnaire evaluations were conducted before product use, 2 weeks after product use, and 4 weeks after product use.

[0300] The improvement effect of atopic dermatitis was confirmed by measuring skin moisture using Comeometer CM825, measuring skin moisture loss using Tewameter TM300, and evaluating itching improvement (VAS).

[0301] Confirm skin moisturizing effect

[0302] like Fig.19a As shown in the results, it can be confirmed that ceramide cream, which is the most widely used to relieve the dryness symptoms of atopic dermatitis, showed an increase in skin moisture content compared to before product use, but there was no statistical significance, while SPM cream showed a statistically significant improvement in skin moisture at the lesion site (2 weeks, 4 weeks of use; <0.014, p <0.0115).

[0303] Confirmation of skin barrier improvement effect at damaged areas

[0304] like Fig.19b As shown in the results, also in terms of the rate of change of transepidermal water loss, ceramide cream showed a reduced transepidermal water loss compared with before product use, but there was no statistical significance, while SPM cream showed a statistically significant improvement in transepidermal water loss at the injured site (4 weeks of use; P < 0.01451).

[0305] Confirmed itching relief effect

[0306] like Fig.19c As shown in , in terms of the itch relief effect, it was confirmed that the itch index was statistically significantly improved after using SPM cream and ceramide cream for 2 weeks and 4 weeks.

[0307] Thus, it can be understood that the SPM cream of the present invention exhibits the same level or higher effects of skin moisturizing, improving skin barrier and alleviating itching as ceramide, which is generally used as a functional material for alleviating skin itching and dry itching, preventing moisture loss caused by dry skin diseases such as atopic dermatitis, and improving skin barrier.

[0308] [13-3] Confirmed the effect of SPMs cream in alleviating eye wrinkles and improving skin moisture

[0309] The human efficacy of Cream A and Cream S in alleviating eye wrinkles and improving skin moisture was evaluated on 20 selected test subjects. The eye area was used as the test site, and 10 subjects were instructed to use Cream A and the remaining 10 subjects to use Cream S twice a day for 8 weeks. The effects of alleviating wrinkles and improving skin moisture were confirmed by measuring the test subjects with the device before using the experimental product, after 4 weeks of use, and after 8 weeks of use.

[0310] Specifically, eye wrinkles were measured using the Antera 3D CS, and skin moisture was measured using the Comeometer.

[0311] Confirmed to relieve eye wrinkles

[0312] like Fig.20a As shown in the results, it can be confirmed that the skin wrinkle relief rate (%) using Cream A containing retinol as a functional concern ingredient for wrinkle relief was not statistically significant compared with before product use, while SPM cream showed a significant skin wrinkle relief rate (%) compared with 4 weeks ago and 8 weeks ago (t-test (two groups assuming equal variances, two-tailed test); *, p≤0.05); **, p≤0.01; ***, p≤0.001).

[0313] Confirm eye moisturizing effect

[0314] like Fig.20b and Fig.20c As shown in , the moisture in the periocular skin showed a statistically significant increase when using SPM cream and retinol cream for 8 weeks, while there was no statistically significant difference between SPM cream and retinol cream.

[0315] From this, it can be understood that the SPM cream of the present invention exhibits the effects of increasing the moisture content of the skin around the eyes and alleviating skin wrinkles around the eyes at the same level or higher than retinol, an approved material for wrinkle alleviation.

[0316] As described above, although the preferred embodiments of the present invention have been exemplarily described, the scope of the present invention is not limited to the above specific embodiments, and those skilled in the art can appropriately modify the present invention within the scope of the appended claims.

Claims

1. Use of an enzyme for preparing a monohydroxy or dihydroxy derivative of a polyunsaturated fatty acid, wherein the amino acid sequence of the enzyme is the same as the amino acid sequence represented by SEQ ID NO:

1.

2. The use according to claim 1, wherein The polyunsaturated fatty acid is docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA).

3. A method for preparing monohydroxy or dihydroxy derivatives of polyunsaturated fatty acids in vitro, the method comprising: The enzyme according to claim 1 is reacted with a polyunsaturated fatty acid.

4. The method according to claim 3, further comprising: The monohydroxy or dihydroxy derivative is recovered from the reaction product of the enzyme and the polyunsaturated fatty acid.

5. The method according to claim 3, wherein: The reaction is carried out at 10°C to 40°C and pH 4 to pH 10.

6. The method according to claim 3, wherein: The polyunsaturated fatty acid is docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA).

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