Composition for anti-oxidation or immunosuppression comprising compounds derived from Ilex rotunda extracts

Abstract

The present invention relates to a composition for suppressing an immune response comprising, as an active ingredient, one or more compounds selected from the group consisting of zebeiresinol, syringaresinol, and coniferaldehyde compounds. The said compounds have effects such as inhibiting IL-2 production, strongly inhibiting the phosphorylation levels of ERK, JNK, and p38 proteins whose expression levels increased after CD3 / CD28 stimulation to inactivate the MAPK signaling pathway, inhibiting the phosphorylation of p65 NF-κB to block the process of NF-κB moving from the cytoplasm to the nucleus, and reducing the phosphorylation level of Kαβ NF-κB to prevent the degradation of IKαβ, and can be usefully used as a composition for preventing or treating immune diseases.

Description

Technology Field

[0001] The present invention relates to an antioxidant or immunosuppressive composition comprising a compound derived from the Chinese holly tree as an active ingredient, and more specifically, to a composition for suppressing an immune response comprising one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Coniferaldehyde, which are compounds derived from the methylene chloride fraction of the Chinese holly tree leaf and branch extract, and to an antioxidant composition comprising one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Medioresinol as an active ingredient. Background Technology

[0002] Free radicals are highly reactive due to the presence of unpaired electrons within their molecular structure and readily react with other molecules to achieve stability. They belong to the category of reactive oxygen species (ROS). These radicals and peroxynitrite are known to have positive and negative effects on the immune system [1]. Free radicals play a crucial regulatory role in intracellular signaling processes and contribute to host defense mechanisms, such as eliminating pathogens through respiratory bursts or regulating the activation of immune cells through the regulation of redox reactions [2,3]. In these processes, reactive oxygen species (ROS) can damage cellular components and induce the destruction and repair of cellular components. Low or moderate levels of reactive oxygen species (ROS) activate various immune signaling pathways, regulating the production of inflammatory cytokines, and amplifying the immune response by bringing more immune cells to the site of injury or infection [4]. However, excessive free radicals and ROS can cause DNA mutations, lipid peroxidation, and protein damage, leading to cellular dysfunction and aging. Furthermore, excessive production of ROS is associated with inflammatory diseases and impaired immune function, and is known to contribute to the development of cardiovascular diseases, neurodegenerative diseases, cancer, and autoimmune diseases [5,6]. Therefore, it is important to properly regulate the production of ROS. Maintaining a balance between oxidative stress and antioxidant defense is essential for maintaining health.

[0003] IL-2 is a cytokine essential for regulating immune responses, playing a particularly important role in regulating the activation and proliferation of T cells. When IL-2 signaling is activated, T cells develop into short-lived terminally differentiated effector cells, stimulate CD8+ T cells to produce important cytotoxic effector molecules and cytokines, and can ultimately promote maturation into memory T cells [7]. Additionally, low concentrations of IL-2 signaling contribute to maintaining the memory phenotype in activated CD8+ T cells and can help CD4+ T cells form a similar memory phenotype or follicular helper-like characteristics. However, because overactivation or dysregulation of IL-2 can have negative effects in inflammatory diseases [8], immunosuppression, and autoimmune diseases, it is very important to regulate the balance of IL-2 [9].

[0004] When IL-2 binds to the receptor, a conformational change occurs in the receptor structure, and various signaling pathways, including the MAPK pathway, are activated. When the IL-2 receptor is activated, a family of kinases such as Ras, Raf, MEK, ERK, and MAPK is sequentially activated, and as this process proceeds, specific transcription factors are phosphorylated, move to the nucleus, and become involved in gene expression and cell growth regulation [10,11].

[0005] Phosphorylation of MAPK kinases was induced by a co-stimulator, but the active compounds (1, 2, 7) showed an effect of inhibiting the downstream signaling of these kinases by blocking the phosphorylation of ERK / JNK / p38 MAPK. That is, these compounds can contribute to regulating autoimmune responses or preventing excessive inflammation by interfering with the ability of MAPK kinases to function in immune cell signaling processes.

[0006] Phenolic compounds are the most abundant and heterogeneous group of secondary metabolites in plants, playing an important role in protecting plants from disease and environmental ultraviolet radiation

[12] . These compounds exhibit antioxidant

[13] and anti-inflammatory

[14] effects. According to prior studies, phenolic compounds have been reported to have a significant impact on immune responses by interacting with key molecular targets related to inflammation and immune cell function through various signaling pathways [15,16].

[0007] The inventor of this invention is a barn tree ( Ilex rotunda While conducting research on ), various effects such as antioxidant and anti-inflammatory properties were discovered in the extracts and fractions of the leaves and branches of *Ilex crenata*, and this was filed as Patent Application No. 10-2024-0019755. Subsequently, while conducting continuous research, including searching for phenolic compounds using FBMN-GNPS (feature-based molecular networking and Global Natural Products Social Molecular Networking) analysis on the extracts and fractions of *Ilex crenata* leaves and branches, it was confirmed that the content of phenolic compounds such as coniferaldehyde and sinapaldehyde was relatively high in the MC fraction of the *Ilex crenata* leaf and branch extracts, thereby completing the present invention. Prior art literature

[0008] [1] Tumilaar, S.G.; Hardianto, A.; Dohi, H.; Kurnia, D. A comprehensive review of free radicals, oxidative stress, and antioxidants: Overview, clinical applications, global perspectives, future directions, and mechanisms of antioxidant activity of flavonoid compounds. Journal of Chemistry 2024, 2024, 5594386.[2] Pacher, P.; Beckman, J.S.; Liaudet, L. Nitric oxide and peroxynitrite in health and disease. Physiological Reviews 2007, 87, 315-424[3] Genestra, M. Oxyl radicals, redox-sensitive signalling cascades and antioxidants. Cellular Signalling 2007, 19, 1807-1819.[4] Mittal, M.; Siddiqui, M.R.; Tran, K.; Reddy, S.P.; Malik, A.B. Reactive oxygen species in inflammation and tissue injury. Antioxidants & Redox Signaling 2013, 20, 1126-1167[5] Maria, A.P.; Elena, P.; Gerardo Alvarez de, C.; Manuel, A.d.P. Dietary antioxidants: Immunity and host defense. Current Topics in Medicinal Chemistry 2011, 11, 1752-1766[6] Shields, H.J.; Traa, A.; Van Raamsdonk, J.M.Beneficial and detrimental effects of reactive oxygen species on lifespan: A comprehensive review of comparative and experimental studies. Frontiers in Cell and Developmental Biology 2021, 9, 628157.[7] Ross, S.H.; Cantrell, D.A. Signaling and function of interleukin-2 in T lymphocytes. Annual Review of Immunology 2018, 36, 411-433,[8] Lykhopiy, V.; Malviya, V.; Humblet-Baron, S.; Schlenner, S.M. IL-2 immunotherapy for targeting regulatory T cells in autoimmunity. Genes & Immunity 2023, 24, 248-262[9] Orozco Valencia, A.; Camargo Knirsch, M.; Suavinho Ferro, E.; Antonio Stephano, M. Interleukin-2 as immunotherapeutic in the autoimmune diseases. International Immunopharmacology 2020, 81, 106296

[10] Xu, W.; Yan, M.; Lu, L.; Sun, L.; Theze, J.; Zheng, Z.; Liu, X. The p38 MAPK pathway is involved in the IL-2 induction of TNF-βgene via the EBS element. Biochemical and Biophysical Research Communications 2001, 289, 979-986,

[11] Koike, T.; Yamagishi, H.; Hatanaka, Y.; Fukushima, A.; Chang, J.-w.; Xia, Y.; Fields, M.; Chandler, P.; Iwashima, M. A novel ERK-dependent signaling process that regulates interleukin-2 expression in a late phase of T cell activation*. Journal of Biological Chemistry 2003, 278, 15685-15692

[12] Scalbert, A.; Manach, C.; Morand, C.; Rιmιsy, C.; Jimιnez, L. Dietary polyphenols and the prevention of diseases. Critical Reviews in Food Science and Nutrition 2005, 45, 287-306.

[13] Matsumura, Y.; Kitabatake, M.; Kayano, S.-i.; Ito, T. Dietary phenolic compounds: their health benefits and association with the gut microbiota. Antioxidants 2023, 12, 880.

[14] Liu, W.; Cui, X.; Zhong, Y.; Ma, R.; Liu, B.; Xia, Y. Phenolic metabolites as therapeutic in inflammation and neoplasms: Molecular pathways explaining their efficacy. Pharmacological Research 2023, 193, 106812.

[15] Ding, S.; Jiang, H.; Fang, J. Regulation of immune function by polyphenols. Journal of Immunology Research 2018, 2018, 1264074.

[16] Mamun, M.A.; Rakib, A.; Mandal, M.; Kumar, S.; Singla, B.; Singh, U.P. Polyphenols: Role in modulating immune function and obesity. Biomolecules 2024, 14, 221.

[17] Bhol, N.K.; Bhanjadeo, M.M.; Singh, A.K.; Dash, U.C.; Ojha, R.R.; Majhi, S.; Duttaroy, A.K.; Jena, A.B. The interplay between cytokines, inflammation, and antioxidants: mechanistic insights and therapeutic potentials of various antioxidants and anti-cytokine compounds. Biomedicine & Pharmacotherapy 2024, 178, 117177.

[18] Yang, Y.; Bazhin, A.V.; Werner, J.; Karakhanova, S. Reactive oxygen species in the immune system. International Reviews of Immunology 2013, 32, 249-270.

[19] Le, D.D.; Jang, Y.S.; Truong, V.; Dinh, T.; Dang, T.; Yu, S.; Lee, M. Anti-inflammatory effects and metabolomic analysis of Ilex rotunda extracted by supercritical fluid extraction. International Journal of Molecular Sciences 2024, 25, 11965. 해결하려는 과제

[0009] One objective of the present invention is to provide a pharmaceutical composition for inhibiting an immune response comprising, as an active ingredient, one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Coniferaldehyde.

[0010] Another objective of the present invention is to provide a food composition for inhibiting an immune response comprising, as an active ingredient, one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Coniferaldehyde.

[0011] Another objective of the present invention is to provide a food composition for antioxidant or immune response inhibition comprising one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Medioresinol as active ingredients. means of solving the problem

[0012] In one embodiment, the present invention provides a pharmaceutical composition for inhibiting an immune response comprising one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Coniferaldehyde as active ingredients.

[0013] In the present invention, Zhebeiresinol is a compound having the structure of the following chemical formula 1, a compound separated from the methanol and water subfraction of the methylene chloride fraction of a Chinese elm branch extract, and is a compound represented as Compound 1 in this specification.

[0014] [Chemical Formula 1]

[0015]

[0016] In the present invention, syringaresinol is a compound having the structure of the following chemical formula 2, a compound separated from the methanol and water subfraction of the methylene chloride fraction of a Chinese elm branch extract, and is a compound represented as compound 2 in this specification.

[0017] [Chemical Formula 2]

[0018]

[0019] In the present invention, coniferaldehyde is a compound having the structure of the following chemical formula 7, a compound separated from the methanol and water subfraction of the methylene chloride fraction of the Ilex crenata leaf extract, and is a compound represented as compound 7 in this specification.

[0020] [Chemical Formula 7]

[0021]

[0022] The above-mentioned zebeiresinol, syringaresinol, and coniferaldehyde compounds can be used by isolating them from the methylene chloride fraction of the leaves or branches of the Chinese holly as described above, but they can also be used by methods such as synthesis by chemical synthesis, purchasing commercially available compounds, isolation from extracts of various plants including the Chinese holly and their fractions, or production by microorganisms that produce the compounds or microorganisms transformed to produce the compounds.

[0023] The above-mentioned zebeiresinol, syringaresinol, and coniferaldehyde compounds may exist in the form of pharmaceutically acceptable salts. As said salts, acid addition salts formed by pharmaceutically acceptable free acids are useful. The term "pharmaceutically acceptable salt" in this invention refers to any organic or inorganic addition salt of said compounds at a concentration having an active effect that is relatively non-toxic and harmless to the patient, such that side effects caused by said salt do not impair the beneficial efficacy of said compounds of this invention.

[0024] Acid addition salts are prepared by conventional methods, for example, by dissolving a compound in an excess amount of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent, for example, methanol, ethanol, acetone, or acetonitrile. An equal molar amount of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water may be heated, and then the mixture may be dried by evaporation or the precipitated salt may be filtered by suction.

[0025] At this time, organic acids and inorganic acids may be used as free acids. Inorganic acids may include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc., and organic acids may include methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, hydroiodic acid, etc., but are not limited to these.

[0026] In addition, pharmaceutically acceptable metal salts can be prepared using a base. Alkali metal salts or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium, or calcium salts as the metal salts, but is not limited to these. In addition, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0027] Pharmaceutically acceptable salts of the compounds of the present invention comprise salts of acidic or basic groups that may be present in said compounds, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium, and potassium salts of hydroxyl groups, and other pharmaceutically acceptable salts of amino groups include hydrobromides, sulfates, hydrogen sulfates, phosphates, hydrogen phosphates, dihydrogen phosphates, acetates, succinates, citrates, tartrates, lactates, mandelates, methanesulfonates (mesylates), and p-toluenesulfonates (tosylates), and may be prepared through methods for preparing salts known in the art.

[0028] As a salt of the compound of the present invention, any salt of a compound that is pharmaceutically acceptable and exhibits pharmacological activity equivalent to that of the compound, such as inhibiting reactive oxygen species, inhibiting IL production, or inhibiting the MAPK signaling pathway or NF-κB signaling pathway of T cells, thereby exhibiting antioxidant activity and immunosuppressive activity in vivo, may be used without limitation.

[0029] In addition, the compound according to the present invention comprises, without limitation, its pharmaceutically acceptable salts as well as solvates such as possible hydrates that can be prepared therefrom and all possible stereoisomers. The solvates and stereoisomers of the compound may be prepared from the compound represented by the compound using methods known in the art.

[0030] Furthermore, the compound according to the present invention may be prepared in a crystalline or amorphous form, and if prepared in a crystalline form, it may be optionally hydrated or solvated. The present invention may include not only stoichiometric hydrates of the compound but also compounds containing varying amounts of water. The solvates of the compound according to the present invention include both stoichiometric solvates and non-stoichiometric solvates.

[0031] In the present invention, “immunosuppression” or “suppression of immune response” refers to suppressing an immune response that is not beneficial to the body, induced by external or self antigens.

[0032] One or more compounds selected from the group consisting of zebeiresinol, syringaresinol, and coniferaldehyde compounds of the present invention may be used to suppress immune rejection reactions that may occur during the transplantation of any cell, tissue, and organ. For example, they may be used to suppress immune rejection reactions during the transplantation of skin, blood, cornea, liver, lung, intestine, pancreas, heart, kidney, bone marrow, stem cells, and progenitor cells, and preferably, they may be used to suppress immune rejection reactions that may occur during skin transplantation, bone marrow transplantation, stem cell transplantation, blood transfusion, and organ transplantation.

[0033] Accordingly, the composition of the present invention can be usefully used for the treatment, improvement, and prevention of transplant rejection and / or graft-versus-host disease (GVHD) by suppressing the immune response to transplanted organs, tissues, or cells.

[0034] In this specification, "treatment" and "improvement" mean stopping or delaying the progression of a disease when used on an object exhibiting symptoms of onset, and "prevention" means stopping or delaying the signs of onset when used on an object that does not exhibit symptoms of onset but is at high risk of such onset.

[0035] In addition, the composition of the present invention can also be usefully used for the treatment and prevention of various immune hypersensitivity diseases caused by immune hypersensitivity reactions, preferably allergic diseases, autoimmune diseases, and inflammatory diseases.

[0036] The above allergic diseases may include, but are not limited to, bronchial asthma, allergic rhinitis, atopic dermatitis, urticaria (angioedema, rash, etc.), anaphylactic reaction, allergic conjunctivitis, seasonal allergy, drug allergy, food allergy, etc.

[0037] The above-mentioned autoimmune diseases may include, but are not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), systemic sclerosis, autoimmune hemolytic anemia, insulin-dependent diabetes mellitus (IDDM), type 1 diabetes, Graves' disease, Graves' hyperthyroidism, Kikuchi disease, hemophagocytic lymphohistiocytosis, adult-onset Still's disease, Behcet's disease, IgG4-associated disease, psoriasis, etc.

[0038] The above-mentioned inflammatory diseases include inflammatory skin diseases such as asthma, eczema, psoriasis, allergy, rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, psoriasis, acne, atopic rhinitis, allergic dermatitis, and seborrheic dermatitis; inflammatory bowel diseases such as osteoarthritis, gastritis, gout, gouty arthritis, ulcer, chronic bronchitis, acute lung injury, pulmonary inflammation, airway hyperresponsiveness, Crohn's disease, and ulcerative colitis; acute or chronic inflammatory diseases such as ankylosing spondylitis, sepsis, septic shock, vasculitis, and bursitis; osteoporosis; cancer including solid tumors; Alzheimer's disease; atherosclerosis; viral infections including Epstein-Barr virus, cytomegalovirus, herpes simplex virus, HIV, and influenza virus; It includes, but is not limited to, ataxia telangiectasia.

[0039] In one specific example, DPPH and ABTS radical scavenging activities were tested on nine compounds isolated from fractions of Ilex rotunda leaf and branch extracts. As a result, the compounds of the present invention, zebeiresinol (compound 1), syringaresinol (compound 2), and medioresinol (compound 4), exhibited superior DPPH and ABTS radical scavenging activities compared to other compounds (see Figures 4 and 5).

[0040] In another specific embodiment, the inhibitory effect of nine compounds isolated from fractions of Ilex rotunda leaf and branch extracts on the production of reactive oxygen species (ROS) in RAW264.7 macrophages stimulated with LPS (Lipopolysaccharide) was tested. As a result, the compounds of the present invention, zebeiresinol (compound 1), syringaresinol (compound 2), and medioresinol (compound 4), showed a strong inhibitory effect on the production of reactive oxygen species compared to other compounds without cytotoxicity (see Fig. 6).

[0041] Excessive and prolonged production of reactive oxygen species (ROS) is known to have a negative impact on the activation and differentiation processes of T cells. ROS can impair immune function and cause DNA damage, protein oxidation, lipid peroxidation, mitochondrial dysfunction, and reduced immune response. These negative effects can lead to immune cell dysfunction, chronic inflammation, autoimmune diseases, and cancer

[17] . Therefore, maintaining a balance between ROS production and the antioxidant defense system is essential for maintaining optimal immune function, and strategies to reduce oxidative stress can help support immune health

[18] .

[0042] Accordingly, the zebeiresinol and syringaresinol compounds of the present invention can be useful as active ingredients of the immunosuppressive composition according to the present invention due to their inhibitory effect on the generation of reactive oxygen species and their antioxidant effect. In addition, medioresinol, which has excellent antioxidant activity, can also be used as an additional ingredient of the immunosuppressive composition according to the present invention.

[0043] The above-mentioned medioresinol is a compound having the structure of the following chemical formula 4, a compound isolated from the methanol and water subfraction of the methylene chloride fraction of the Ilex chinensis branch extract, and is a compound represented as Compound 4 in this specification. The above-mentioned medioresinol compound may also be used in the form of a pharmaceutically acceptable salt, etc., and may be utilized by methods such as chemical synthesis, purchasing a commercially available compound, isolation from extracts of various plants including Ilex chinensis and fractions thereof, or production by microorganisms that produce the compound or microorganisms transformed to produce said compound.

[0044] [Chemical Formula 4]

[0045]

[0046] Meanwhile, compounds such as Zebeiresinol, Syringaresinol, and Medioresinol, which exhibit excellent inhibitory effects on the generation of free radicals and antioxidant effects, may also be used as active ingredients in antioxidant compositions. The above-mentioned antioxidant composition will be described in more detail below.

[0047] IL-2 is a cytokine essential for regulating immune responses, playing a particularly important role in regulating the activation and proliferation of T cells. When IL-2 signaling is activated, T cells develop into short-lived terminally differentiated effector cells, stimulate CD8+ T cells to produce important cytotoxic effector molecules and cytokines, and can ultimately promote maturation into memory T cells [7]. Additionally, low concentrations of IL-2 signaling contribute to maintaining the memory phenotype in activated CD8+ T cells and can help form a similar memory phenotype or follicular helper-like characteristics in CD4+ T cells. However, because overactivation or dysregulation of IL-2 can have negative effects in inflammatory diseases [8], immunosuppression, and autoimmune diseases, it is very important to regulate the balance of IL-2 [9].

[0048] In one specific embodiment, the compounds of the present invention, zebeiresinol (compound 1), syringaresinol (compound 2), and coniferaldehyde (compound 7), inhibited IL-2 production in Jurkat T cells that promoted IL-2 production via CD3 / CD28 induction with a higher inhibition rate compared to other compounds (see FIG. 8).

[0049] In another specific embodiment, when IL-2 binds to a receptor, a conformational change occurs in the receptor structure, and various signaling pathways, including the MAPK pathway, are activated. When the IL-2 receptor is activated, a family of kinases such as Ras, Raf, MEK, ERK, and MAPK is sequentially activated, and as this process proceeds, specific transcription factors are phosphorylated, move to the nucleus, and become involved in gene expression and cell growth regulation. Accordingly, the correlation between the compounds of the present invention—zebeiresinol (compound 1), syringaresinol (compound 2), and coniferaldehyde (compound 7)—and MAPK-specific proteins, including ERK, JNK, and p38, was investigated. As a result, in Jurkat T cells treated with CD3 / CD28, the compound of the present invention strongly inhibited the phosphorylation levels of ERK, JNK, and p38 proteins, whose expression levels increased after CD3 / CD28 stimulation, thereby exhibiting the effect of inactivating the MAPK signaling pathway (see Fig. 9).

[0050] In another specific embodiment, IL-2 binds to the IL-2 receptor on T cells to activate various intracellular signaling pathways, thereby activating several kinases to phosphorylate the intracellular domain of the receptor. NF-κB is one of the major downstream signaling pathways activated by this phosphorylation, playing a crucial role in promoting T cell survival and proliferation and regulating immune responses. When the p65 subunit is activated along the NF-κB pathway via IL-2 signaling, it translocates to the nucleus to promote gene expression related to immune response regulation, cell survival, and proliferation. This pathway is essential for regulating T cell function throughout the immune response as well as maintaining immune tolerance. Furthermore, the interaction between IL-2 signaling and the NF-κB pathway plays a critical role in the proliferation and survival of immune cells, as well as in regulating inflammation and maintaining immune homeostasis. Accordingly, to confirm the mechanism of the inhibitory effect of the compounds of the present invention—zebeiresinol (compound 1), syringaresinol (compound 2), and coniferaldehyde (compound 7)—on IL-2 production, the inhibitory effect of phosphorylating p65 NF-κB and IKαβ in the signaling pathway was tested. As a result of the test, the compounds of the present invention inhibited the phosphorylation of p65 NF-κB, thereby blocking the process of NF-κB moving from the cytoplasm to the nucleus (see Fig. 10). In addition, the compounds of the present invention reduced the phosphorylation level of IKαβ NF-κB, thereby preventing the degradation of IKαβ (see Fig. 10). Through this, there is a possibility of inhibiting the differentiation of inflammatory T cell subtypes (Th1, Th17), which can serve as an important mechanism for regulating inflammatory responses.

[0051] In another specific embodiment, in silico molecular docking analysis was performed to determine how the compounds of the present invention—zebeiresinol (compound 1), syringaresinol (compound 2), and coniferaldehyde (compound 7)—interact with specific amino acids within the binding pocket of a protein. As a result, the compounds of the present invention were docked at the same position as the native ligand, exhibited significant binding affinity, and showed a binding free energy (ΔG) similar to that of the redocked native ligand (see FIGS. 11 to 17).

[0052] Accordingly, one or more compounds selected from the group consisting of zebeiresinol, syringaresinol, and coniferaldehyde compounds of the present invention can be usefully used as a composition for suppressing immune responses, and furthermore, can be widely applied to the treatment of immune diseases such as transplant rejection, graft-versus-host disease, allergic diseases, autoimmune diseases, and inflammatory diseases that require suppression of immune responses.

[0053] In the present invention, the "pharmaceutical composition" refers to one prepared for the purpose of preventing or treating a disease, and can be formulated into various forms according to conventional methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and can be formulated into parenteral formulations using diluents or excipients such as lubricants, humectants, flavoring agents, emulsifiers, suspending agents, preservatives, and surfactants. Additionally, it can be formulated into external preparations, suppositories, topical skin preparations, and sterile injectable solutions.

[0054] In addition, depending on each formulation, the formulation may be manufactured by additionally including a pharmaceutically acceptable carrier, such as a buffer, analgesic, solubilizing agent, isotonic agent, stabilizer, base, etc., which are known in the art. The term "pharmaceutically acceptable carrier" as used in the present invention may refer to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the injected active ingredient. The types of carriers usable in the present invention are not particularly limited, and any carrier that is commonly used and pharmaceutically acceptable in the relevant technical field may be used. Non-limiting examples of said carriers include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used alone or in a mixture of two or more. The said carrier may be a non-naturally occurring carrier.

[0055] The composition of the present invention may be administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The effective dose level may be determined based on factors including the patient's health condition, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. Specifically, the dose is generally 0.01 mg to 5000 mg per day per 1 kg of body weight of the subject being administered, and may be divided into one or several doses per day at regular intervals at the discretion of a physician or pharmacist, but is not limited thereto.

[0056] The immunosuppressive composition of the present invention, comprising one or more compounds selected from the group consisting of zebeiresinol, syringaresinol, and coniferaldehyde compounds as active ingredients, can be used together with other previously known immunosuppressants.

[0057] In addition, the pharmaceutical composition of the present invention may be used alone, in combination with other pharmaceutically active compounds exhibiting an immunosuppressive effect, or in a suitable combination. Examples of such other pharmaceutically active compounds include, but are not limited to, cyclosporine A, rapamycin, tacrolimus, sacrolimus, methotrexate, azathioprine, mycophenolate mofetil, or various steroid preparations.

[0058] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without causing side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0059] In the present invention, "administration" means introducing the pharmaceutical composition of the present invention to an individual by any appropriate method, and the route of administration of the composition of the present invention may be administered through various oral or parenteral routes as long as it can reach the target tissue.

[0060] The method of administration of the pharmaceutical composition according to the present invention is not particularly limited and may follow methods commonly used in the art. As a non-limiting example of the method of administration, the composition may be administered orally or parenterally.

[0061] The pharmaceutical composition according to the present invention can be produced in various formulations depending on the intended method of administration.

[0062] The frequency of administration of the composition of the present invention is not particularly limited thereto, but it may be administered once a day or divided into several doses.

[0064] In another aspect, the present invention provides a food composition for preventing or improving an immune hypersensitivity reaction comprising, as an active ingredient, one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Coniferaldehyde.

[0065] The above-mentioned hypersensitivity reaction refers to an immune response that is not beneficial to the body, triggered by external or self-antigens, and may include, for example, immune rejection reactions upon transplantation of skin, blood, cornea, liver, lung, intestine, pancreas, heart, kidney, bone marrow, stem cells, and progenitor cells, graft-versus-host disease, allergic diseases, autoimmune diseases, inflammatory diseases, etc.

[0066] In addition, the present invention provides an antioxidant food composition comprising one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Medioresinol as active ingredients.

[0067] The compound included as an active ingredient in the above food composition and its antioxidant and preventive or improving effects on immune hypersensitivity reactions are as described above.

[0068] When the composition of the present invention is used as a food additive, the compound may be added as is or used in combination with other foods or food ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment), and may additionally include food-grade food additives that are food-gradely acceptable. Since the composition of the present invention is a compound derived from natural products and has almost no cytotoxicity, there are no issues regarding stability, so there are no significant restrictions on the mixing amount.

[0069] The food composition of the present invention may include all foods in the conventional sense and may be used interchangeably with terms known in the art, such as functional foods and health functional foods.

[0070] The term "functional food" in the present invention refers to a food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body, and "functionality" means consuming for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body.

[0071] In addition, the term "health functional food" in the present invention refers to a food manufactured or processed by methods such as extraction, concentration, purification, or mixing, using a specific component as a raw material or a specific component contained in a food raw material for the purpose of health supplementation, and refers to a food designed and processed to sufficiently exert biological regulatory functions on the body, such as biological defense, regulation of biological rhythms, and prevention and recovery of disease, through the said component, and the composition for the health food can perform functions related to the prevention of disease and recovery of disease.

[0072] The food composition of the present invention may be prepared by mixing a known additive with other suitable auxiliary ingredients that may be contained in food, according to the choice of a person skilled in the art. Examples of foods to which the composition may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may be prepared by adding the extract according to the present invention to juices, teas, jellies, and juices prepared using the extract as the main ingredient.

[0073] In addition, the foods to which the present invention can be applied may include all foods, such as special nutritional foods (e.g., infant formula, baby food, etc.), processed meat products, fish products, tofu products, jelly products, noodles (e.g., ramen, noodles, etc.), health supplements, seasoning foods (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery products (e.g., snacks), dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (e.g., fruit and vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (e.g., ramen soup, etc.).

[0074] When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavorings, or natural carbohydrates as additional ingredients, as in conventional beverages. In addition to the above, the health functional food composition of the present invention may contain various nutritional supplements, vitamins, electrolytes, flavorings, colorings, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, it may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages.

[0076] In another aspect, the present invention provides an antioxidant cosmetic composition comprising, as an active ingredient, one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Medioresinol.

[0077] The compound included as an active ingredient in the above cosmetic composition and its antioxidant effect are as described above.

[0078] The above cosmetic composition may additionally include ingredients commonly used in cosmetic compositions in addition to one or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Medioresinol as active ingredients. For example, it may include conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0079] The above cosmetic composition may be prepared in any formulation conventionally manufactured in the art, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, it may be prepared in the form of a nourishing cream, astringent lotion, softening lotion, lotion, essence, nourishing gel, or massage cream.

[0080] In the case where the formulation of the above cosmetic composition is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0081] In the case where the formulation of the above cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

[0082] When the formulation of the above cosmetic composition is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan.

[0083] In the case where the formulation of the above cosmetic composition is a suspension, liquid diluents such as water, ethanol, or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth may be used as carrier components.

[0084] In the case where the formulation of the above cosmetic composition is a cleansing product containing a surfactant, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0085] One or more compounds selected from the group consisting of Zhebeiresinol, Syringaresinol, and Medioresinol may be included in an amount of 0.0001 to 50 weight%, more preferably 0.001 to 30 weight%, and even more preferably 0.001 to 10 weight% based on the total weight of the cosmetic composition. At this time, if the content of the compound is less than 0.0001 weight%, an antioxidant effect cannot be effectively obtained, and if it exceeds 50 weight%, the effect may be inefficient as it is not proportional to the increase in content, and there is a problem that the stability of the formulation is not ensured. Effects of the invention

[0086] The zebeiresinol, syringaresinol, and coniferaldehyde compounds according to the present invention exhibit excellent antioxidant activity and have effects such as inhibiting IL-2 production, strongly inhibiting the phosphorylation levels of ERK, JNK, and p38 proteins whose expression levels increased after CD3 / CD28 stimulation to inactivate the MAPK signaling pathway, inhibiting the phosphorylation of p65 NF-κB to block the process of NF-κB moving from the cytoplasm to the nucleus, and reducing the phosphorylation level of IKαβ NF-κB to prevent IKαβ degradation. Therefore, as active ingredients of compositions for suppressing immune responses, they can effectively prevent or treat immune diseases selected from autoimmune diseases, transplant rejection, allergic diseases, and inflammatory diseases. In addition, the zebeiresinol, syringaresinol, and medioresinol compounds according to the present invention exhibit excellent antioxidant activity and can be usefully utilized as active ingredients in antioxidant compositions; furthermore, since they effectively inhibit the generation of reactive oxygen species that negatively affect the activation and differentiation processes of T cells, they can be used as active ingredients in compositions for suppressing immune responses. Brief explanation of the drawing

[0087] Figure 1 shows the results of analyzing the total phenolic content of the total extract and its fractions of *Ilex crenata* branches (T: total extract, H: hexane fraction, MC: methylene chloride fraction, E: ethanol fraction, B: butanol fraction). Figure 2 shows the results of analyzing the total phenolic content of the total extract and its fractions of *Ilex crenata* leaves (T: total extract, H: hexane fraction, MC: methylene chloride fraction, E: ethanol fraction, B: butanol fraction). Figure 3 shows a feature-based molecular network generated from the total extract (T, pink) and various fractions (Bu (B, green), water (W, yellow), ethyl acetate (E, purple), n-hexane (H, cyan), MC (MC, orange)) of each of the leaves and branches of the Chinese holly tree. Figure 4 shows compounds isolated from Ilex crenata leaves and branches. 1 inside 9 This figure shows the DPPH radical scavenging activity. Each experiment was repeated three times, and data are expressed as mean ± standard deviation (mean ± SD). p < 0.05, ** p < 0.01, shows a significant difference compared to the positive control group (ascorbic acid, AA) or the LPS-alone treatment group (LPS). Figure 5 shows compounds isolated from Ilex crenata leaves and branches. 1 inside 9 This figure shows the ABTS radical scavenging activity. Each experiment was repeated three times, and data are expressed as mean ± standard deviation (mean ± SD). p < 0.05, ** p < 0.01, shows a significant difference compared to the positive control group (ascorbic acid, AA) or the LPS-alone treatment group (LPS). Figure 6 shows compounds isolated from the leaves and branches of the Chinese holly tree. 1 inside 9This figure shows the inhibitory activity of on ROS production in LPS-induced RAW264.7 cells. Each experiment was repeated three times, and data are expressed as mean ± standard deviation (mean ± SD). p < 0.05, ** p < 0.01, shows a significant difference compared to the positive control group (ascorbic acid, AA) or the LPS-alone treatment group (LPS). Figure 7 shows compounds isolated from CD3 / CD28-activated Jurkat T cells ( 1 - 9 This figure shows the cytotoxicity results of ). #p < 0.05 indicates a significant difference compared to the untreated group (CTL). *p < 0.05, **p < 0.01 indicate a significant inhibitory effect in CD3 / CD28-activated cells compared to the group not treated with the sample. Fig. 8 shows compounds isolated from CD3 / CD28-activated Jurkat T cells ( 1 - 9 This figure shows the inhibitory effect of ) on IL-2 production. #p < 0.05 indicates a significant difference compared to the untreated group (CTL). *p < 0.05, **p < 0.01 indicate a significant inhibitory effect in CD3 / CD28-activated cells compared to the group not treated with the sample. Figure 9 shows compounds isolated from Ilex crenata leaves and branches in Jurkat T cells ( 1 , 2 , and 7 This figure shows the inhibitory effect of CD3 / CD28 stimulation on the MAPK signaling pathway. #p < 0.05 vs. untreated group (CTL). Significant differences were observed at the *p < 0.05 and **p < 0.01 levels compared to the control group in CD3 / CD28 stimulation. Figure 10 shows compounds isolated from Ilex crenata leaves and branches in Jurkat T cells ( 1 , 2 , and 7This figure illustrates the inhibitory effect on the NF-κB signaling pathway induced by CD3 / CD28 stimulation. Experimental data are expressed as the mean ± standard deviation (Mean ± SD) of three individual experiments. Significant differences were observed at the #p < 0.05 level when compared to the control group, and significant differences were observed at the *p < 0.05 and **p < 0.01 levels when compared to the CD3 / CD28 stimulation group. Figure 11 shows a compound 1 (Blue), compound 2 (Magenta), Hwahipmul 7 This figure shows the molecular docking interaction between the ERK protein (MAPK, PDB ID: 6NBS) and KJ4 (green), and KJ4 (cyan, N-{3-[(2Z,4S)-1-(2-{2-(2-amino-1H-imidazol-1-yl)ethylamino}ethyl)-3-(3-cyclohexylpropyl)-2-iminoimidazolidin-4-yl]propyl}guanidine). Figure 12 shows the compound docked with the ERK protein (PDB ID: 6NBS). 1 (Blue), compound 2 (Magenta), compound 7 This is a figure showing the interactions of amino acids with (green), and KJ4 (cyan, N-{3-[(2Z,4S)-1-(2-{2-(2-amino-1H-imidazol-1-yl)ethylamino}ethyl)-3-(3-cyclohexylpropyl)-2-iminoimidazolidin-4-yl]propyl}guanidine)). Figure 13 shows a compound 1 (Blue), compound 2 (Magenta), Hwahipmul 7This is a figure showing the molecular docking interaction between JNK protein (PDB ID: 1PMN) and KJ4 (green), and KJ4 (cyan, N-{3-[(2Z,4S)-1-(2-{2-(2-amino-1H-imidazol-1-yl)ethylamino}ethyl)-3-(3-cyclohexylpropyl)-2-iminoimidazolidin-4-yl]propyl}guanidine). Figure 14 shows a compound docked with the JNK protein (PDB ID: 1PMN). 1 (Blue), compound 2 (Magenta), compound 7 This is a figure showing the interactions of amino acids with (green), and KJ4 (cyan, N-{3-[(2Z,4S)-1-(2-{2-(2-amino-1H-imidazol-1-yl)ethylamino}ethyl)-3-(3-cyclohexylpropyl)-2-iminoimidazolidin-4-yl]propyl}guanidine)). Figure 15 shows a compound 1 (Blue), compound 2 (Magenta), Hwahipmul 7 This figure shows the molecular docking interaction between p38 protein (PDB ID: 1KV2) and KJ4 (green), and KJ4 (cyan, N-{3-[(2Z,4S)-1-(2-{2-(2-amino-1H-imidazol-1-yl)ethylamino}ethyl)-3-(3-cyclohexylpropyl)-2-iminoimidazolidin-4-yl]propyl}guanidine). Figure 16 shows a compound docked with the p38 protein (PDB ID: 1KV2). 1 (Blue), compound 2 (Magenta), compound 7This is a figure showing the interactions of amino acids with (green), and KJ4 (cyan, N-{3-[(2Z,4S)-1-(2-{2-(2-amino-1H-imidazol-1-yl)ethylamino}ethyl)-3-(3-cyclohexylpropyl)-2-iminoimidazolidin-4-yl]propyl}guanidine)). Figure 17 shows the compound docked with the p65 protein (PDB ID: 1VKX). 1 (Blue), compound 2 (Magenta), compound 7 This is a figure showing the interactions of amino acids with (green), and KJ4 (cyan, N-{3-[(2Z,4S)-1-(2-{2-(2-amino-1H-imidazol-1-yl)ethylamino}ethyl)-3-(3-cyclohexylpropyl)-2-iminoimidazolidin-4-yl]propyl}guanidine)). Specific details for implementing the invention

[0088] Hereinafter, the present invention will be described in detail with reference to examples and the like to aid in understanding the invention. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention should not be interpreted as being limited to the following embodiments. The embodiments of the present invention are provided to more completely explain the invention to those with average knowledge in the art.

[0090] Example 1: Preparation of Ilex rotunda leaf and branch extracts and fractions

[0091] Leaves and branches of *Ilex crenata* growing wild in the Jeonnam region were collected, cold-air dried, and then ground into powder. Each of these *Ilex crenata* leaf and branch powders was pretreated and extracted using a supercritical fluid extraction system following the same procedure. First, the temperature was set to 50°C before being injected into the equipment. After the temperature of the equipment stabilized, the samples were introduced via a high-pressure pump, and CO₂ was subsequently injected to increase the pressure until it reached 400 bar. The valve was adjusted to ensure that the pressure changed linearly, maintaining a constant internal pressure. Ethanol (300 mL) was used as a co-solvent and added at a rate of 5 mL over 120 minutes. The resulting solution was then concentrated under vacuum to obtain 200 g of residue. This residue was suspended in distilled water and fractionated using solvents with increasing polarity from n-hexane to n-butanol (n-BuOH). Through this, extracts and fractions of H(n-hexane), MC(methylene chloride), E(EtOAc), B(n-BuOH), and W(water) were obtained and stored in a refrigerator at 4℃ for subsequent use.

[0093] Example 2: Analysis of Total Phenol Content (TPC) of Ilex crenata Leaf or Branch Extracts and Fractions

[0094] Extracts and fractions of either the leaves or branches of *Ilex crenata* were dissolved in 2% DMSO to prepare a 10,000 μg / mL stock solution, which was then diluted tenfold with distilled water. Subsequently, 1 mL of distilled water was added to 100 μL of each sample solution. To this, 100 μL of Folin-Ciocalteu's phenol reagent was added. After 6 minutes, 1 mL of 7% sodium carbonate (Na2CO3) was added to each mixture. In the final step, all solutions were thoroughly mixed and reacted in a dark room for 90 minutes. Finally, absorbance was measured at 750 nm using a spectrophotometer. Gallic acid was used as the standard solution, and experiments were repeated three times at each concentration range of 7.8125–500 μg / mL. Based on this, a calibration curve was constructed including a regression equation of Y = 0.0031x - 0.0179, and the coefficient of determination ( r 2 The value was found to be 0.9965, showing very high linearity. Total phenolic content was expressed in milligrams of gallic acid (mg GAE / g DE) per 1g of dry extract.

[0095] As a result, the total phenolic content of the extracts from the branches of *Ilex crenata* ranged from 3.11 ± 0.01 to 7.86 ± 0.01 mg GAE / g DE (Figure 1). Among the various fractions, the MC fraction showed the highest phenolic content at 7.86 ± 0.01 mg GAE / g DE. This was followed by the extract (7.41 ± 0.05 mg GAE / g DE), fraction E (6.42 ± 0.01 mg GAE / g DE), fraction B (5.93 ± 0.01 mg GAE / g DE), and fraction H (3.11 ± 0.01 mg GAE / g DE).

[0096] Meanwhile, the total phenolic content of the extract of *Ilex crenata* leaves ranged from 4.38 ± 0.00 to 6.36 ± 0.00 mg GAE / g DE (Fig. 2). The MC fraction showed the highest value at 6.36 ± 0.00 mg GAE / g DE. This was followed by the extract (5.86 ± 0.00 mg GAE / g DE), E fraction (5.61 ± 0.00 mg GAE / g DE), Bu fraction (5.11 ± 0.01 mg GAE / g DE), and H fraction (4.38 ± 0.00 mg GAE / g DE).

[0097] Based on these results, the MC fraction (leaves and stems) with a high total phenolic content was selected as the primary material for the isolation of active ingredients.

[0098] Meanwhile, in the experiments mentioned above and below, all experimental data were expressed as the mean ± standard deviation (SD) of the results from three repeated experiments. For statistical analysis, one-way ANOVA and Duncan's multiple range test were performed using GraphPad Prism software (version 8.0, GraphPad Software, Inc., San Diego, CA, USA) to test for differences between treatments. Statistically significant differences * p < 0.05 and ** p was set to < 0.01.

[0100] Example 3: Molecular network and bioactive basis of Ilex crenata ( I. rotunda Separation of compound (1-9) from )

[0101] In the above Example 2, the Chinese holly ( Ilex rotundaThe methylene chloride (MC) fraction of the leaf and branch extracts of *Ilex crenata* showed a high total phenolic content, and a previous study

[19] confirmed that it exhibited high levels of triterpenoid compounds and showed a strong inhibitory effect on IL-2 production in CD3 / CD28-stimulated Jurkat T cells. Accordingly, using the UHPLC-Orbitrap-MS system, *Ilex crenata* ( I. rotunda HR-ESI-MS / MS data of the total extract and fractions obtained from the leaves and stems were generated.

[0102] Data processing was performed using the mzMine program for the analysis and annotation of secondary metabolites of the extracts, and the distribution patterns of the compounds were analyzed through the FBMN-GNPS web platform. The metabolite identification process was based on ion precursor data dereplication, mass isotopic pattern analysis, fragmentation, and spectral matching with public databases, and was optimized using the GNPS open web tool.

[0103] Among the annotated phenol compounds in the FBMN network (Fig. 3), the precursor ion peak from the FBMN data of the Chinese elm stem sample at node 1278 ( m / z 179.0703 [M+H] + ) was detected. The compound in question is m / z 148.0977 [M+H-CH3O] + and m / z 119.0897 [M+H-CH3O-CHO] + It generated fragment ions, and when these fragmentation patterns were compared with publicly available data, they were identified as coniferaldehyde.

[0104] also, m / z209.0808 [M+H] + Another compound with a precursor ion peak was identified, and this compound is related to water molecules and methyl group reduction, respectively. m / z 191.0709 [M+H-OH] + and m / z 177.0608 [M+H-OH-CH3] + It produced fragment ions. Based on this, this compound was identified as sinapaldehyde.

[0105] These two phenolic compounds were detected in the FBMN network obtained from leaves and stems via the same fragmentation pathway, showing the highest content particularly in the MC fraction. Based on these results, additional separation processes were carried out on the MC fractions of leaves and stems.

[0106] The MC fraction of the Chinese elm branch is RP-MPLC (YMC Triart ODS C 18 It was eluted using a gradient solvent system consisting of methanol and water (containing 0.1% formic acid) (from 35% B to 100% B, for 150 minutes, flow rate 5.0 mL / min) with a 250 x 20 mm, 5.5 μm filter, and UV detection was performed at 200, 216, and 260 nm to obtain a total of 14 fractions (MC_A-MC_N).

[0107] The subfraction MC_L (259.6 mg) is Triart C 18 UV detection at 254 nm was performed using a column (10 × 250 mm, 5 μm) and prep HPLC at a flow rate of 3.0 mL / min, and elution was performed using water (containing 0.1% formic acid, A) and acetonitrile (B) as a gradient solvent system [0 min (48% B) - 10 min (60% B) - 50 min (61% B) - 80 min (100% B)] to obtain two peaks ( t R 17 minutes and t R I got 50 minutes. t RThe peak at 50 minutes is from prep TLC (preparative thin layer chromatography, silica gel 60 F) using methylene chloride (MC) and methanol (M) in a 15:1 ratio. 254 Compounds further purified through Merck 1 I obtained.

[0108] Subfraction MC_M was eluted using a Biotage Sfar column with ethyl acetate and n-hexane in a gradient solvent system (110 min, flow rate 5 mL / min, UV detection: 205 and 225 nm) from 15% to 100% to obtain a total of 7 fractions (MC_M1–MC_M7). MC_M5 (34.8 mg) was prepared by thin-layer chromatography (silica gel 60 RP-18 F 254S Using , Merck), acetone (A) and distilled water (DW) in a 6:1 ratio as the mobile phase, the compound 2 and 4 I obtained.

[0109] The MC fractions of *Ilex crenata* leaves were eluted using a Biotage Sfar C18 400g column with methanol and water in a gradient solvent system (30% to 100%, 100 min, flow rate 100 mL / min, UV detection: 200 and 254 nm) to obtain a total of 23 fractions (MC1–MC23). Subfraction MC4 (37.6 mg) was YMC-Triart C 18 The compound was processed by prep HPLC using a column (10 × 250 mm, 5 μm) and eluted with an H2O-CH3CN gradient (30:70-0:100) (flow rate 3.0 mL / min, detection wavelength 313 nm). 3 ( t R 45 minutes), compound 5 ( t R 62 minutes), compound 6 ( t R 49 minutes), compound 7 ( t RI got 59 minutes.

[0110] In addition, fraction B of the Chinese holly branch is YMC Triart C 18 UV detection at 202 nm was performed using prep HPLC with a column (10 × 250 mm, 5 μm) at a flow rate of 3.0 mL / min, and the compound was eluted using a gradient solvent system from 0 min (15% B) to 88 min (100% B). 8 ( t R 55 minutes) and compounds 9 ( t R I got 45 minutes.

[0112] Example 4: Chinese holly ( I. rotunda ) Determination of the structure of compounds (1-9) isolated from leaf and stem extracts

[0113] As described in Example 3 above, a total of 9 compounds ( 1-9 ) were separated using various chromatographic techniques. The structures of these compounds were determined by interpreting the fragmentation patterns of mass spectrometry data and comparing them with spectroscopic data reported in the literature.

[0114] separated compounds 1 inside 9 The spectroscopic data and structural formula are as follows.

[0115] compound 1 It separated into a white amorphous powder. HR-ESI-MS analysis results showed that the deprotonated ion peak m / z 279.0875 [MH] (Calculated value: C 14 H 15 It was identified at O6, 279.0869. Compound 1 of 1 H NMR spectrum analysis results showed that two symmetric protons in the meta-substituted benzene ring δ HIt appeared at 6.67 (2H, s, H-2, 6), and two methoxy (-OCH3) groups δ H It was detected at 3.85(6H, s, 3-OCH3, 5-OCH3). The presence of a furofuran backbone was confirmed through COSY spectral analysis by verifying the interrelationships between H-7 / H-8 / H-9 and H-7' / H-8' / H-9'. As a result of comparing mass and spectroscopic data with the literature, the compound 1 It was identified as Zhebeiresinol. Compound 1 The structural formula and spectroscopic data are as follows.

[0116] [Chemical Formula 1]

[0117]

[0118] Zhebeiresinol ( 1 ): White amorphous powder; HR-ESI-MS: m / z 279.0875 [MH] - (calc. for C 14 H 15 O6, 279.0869); 1 H NMR (600 MHz, CD3OD): 3.15-3.22 (1H, m, H-1'), 4.67 (1H, d, J = 8.4 Hz, H-2'), 4.31 (1H, t, J = 8.7 Hz, H-4'), 3.54-3.57 (1H, td, J = 3.4, 8.8 Hz, H-5'), 4.09 (1H, dd, J = 3.4, 9.2 Hz, H-7'), 4.55 (1H, dd, J = 6.8, 9.6 Hz, H-8'), 6.67 (2H, s, H-2), 3.85 (3H, s, 3-OCH3, 5-OCH3), 6.09 (1H, brs, 4-OH), 6.67 (2H, brss, H-2, 6); 13C NMR (150 MHz, CD3OD): 131.0 (C-1), 103.0 (C-2), 148.7 (C-3), 134.1 (C-4), 148.7 (C-5), 103.0 (C-6), 46.2 (C-1'), 85.9 (C-2'), 69.9 (C-4'), 48.7 (C-5'), 178.1 (C-6'), 70.4 (C-8'), 56.1 (3, 5-OCH3).

[0120] compound 2 It separated into a white amorphous powder. In HR-ESI-MS analysis, the deprotonation ion peak was m / z 417.1552 [MH] (calculated value: C 22 H 25 It was detected in O8, 417.1549). 1 H and 13 3C NMR analysis results show a symmetric structure, and proton peaks in the two meta-substituted benzene rings δ H It appeared at 6.67 (4H, s, H-2, 6, 2', 6'), and four methoxy groups (-OCH3) were respectively δ H It was detected at 3.84 (12H, s, 3-OCH3, 5-OCH3, 3'-OCH3, 5'-OCH3). In COSY spectral analysis, the furofuran backbone was confirmed through the intercorrelation between H-7 / H-8 / H-9 and H-7' / H-8' / H-9'. In HMBC analysis δ H 6.66(H-2, H-6) and δ C A correlation was observed between 87.6(C-7, C-7'), elucidating the substructure. In addition, δ H The HMBC cross-peak of the methoxy group detected at 3.84 δ C The presence of 3-OCH3, 5-OCH3, 3'-OCH3, and 5'-OCH3 was confirmed by identification at 149.4 (oxygenated quaternary carbon). Based on these results, the compound2 It was identified as Syringaresinol. Compound 2 The structural formula and spectroscopic data are as follows.

[0121] [Chemical Formula 2]

[0122]

[0123] Syringaresinol 2 ): White amorphous powder; HR-ESI-MS: m / z 417.1552 [MH] - (calc. for C 22 H 25 O8, 417.1549); 1 H NMR (600 MHz, CD3OD): 4.72 (2H, d, J = 4.0 Hz, H-7), 4.26 (2H, dd, J = 6.9, 9.0 Hz, H-9a), 3.88 (2H, dd, J = 3.8, 9.3 Hz, H-9b), 4.72 (2H, d, J = 4.0 Hz, H-7'), 4.26 (2H, dd, J = 6.9, 9.0 Hz, H-9'a), 3.88 (2H, dd, J = 3.8, 9.3 Hz, H-9'b), 6.66 (4H, s, H-2, H-6), 3.14 (2H, m, H-8), 6.66 (4H, s, H-2', H-6'), 3.14 (2H, m, H-8'), 3.84 (12H, s, 3-OCH3, 5-OCH3, 3'-OCH3, 5'-OCH3); 13C NMR (150 MHz, CD3OD): 133.2 (C-1), 104.6 (C-2), 149.4 (C-3), 136.3 (C-4), 149.4 (C-5), 104.6 (C-6), 87.6 (C-7), 55.5 (C-8), 72.8 (C-9), 133.2 (C-1'), 104.6 (C-2'), 149.4 (C-3'), 136.3 (C-4'), 149.4 (C-5'), 149.4 (C-5'), 104.6 (C-6'), 87.6 (C-7'), 55.5 (C-8'), 72.7 (C-9'), 56.8 (3, 5-OCH3, 3', 5'-OCH3).

[0125] compound 3 It was separated into a white amorphous powder. HR-ESI-MS analysis results showed that the deprotonated ion peak m / z 357.1341 [MH] (Calculated value: C 20 H 21 It was detected in O6, 357.1338). 1 H and 13 C NMR analysis results, compound 2 It showed a structure similar to but with two reduced methoxy groups. 1 In the H NMR spectrum, two ABX spin systems δ H 6.95(2H, d, J = 2.0 Hz, H-2, 2'), 6.81(2H, dd, J = 1.9, 8.1 Hz, H-5, 5'), 6.77(2H, d, J = 8.1 Hz, H-6, 6'). Signals associated with the furofuran skeleton are δ H 4.71(2H, d, J = 4.4 Hz, H-7, 7'), 4.24(2H, dd, JIt was detected at 6.9, 9.1 Hz, H-9a, 9'a), 3.85 (2H, m, H-9b, 9'b), and 3.15 (2H, m, H-8, 8'). δ H Two methoxy groups (3-OCH3, 3'-OCH3) were detected at 3.85, which were identified as (+)-pinoresinol upon comparison with existing literature. 3 The structural formula and spectroscopic data are as follows.

[0126] [Chemical Formula 3]

[0127]

[0128] Pinoresinol ((+)-Pinoresinol) ( 3 ): White amorphous powder; HR-ESI-MS: m / z 357.1341 [MH] - (calc. for C 20 H 21 O6, 357.1338); 1 H NMR (600 MHz, CD3OD): 6.95 (2H, d, J = 2.0 Hz, H-2), 6.81 (2H, dd, J = 1.9, 8.1 Hz, H-5), 6.77 (2H, d, J = 8.1 Hz, H-6), 4.71 (2H, d, J = 4.4 Hz, H-7), 4.24 (2H, dd, J = 6.9, 9.1 Hz, H-9), 6.95 (2H, d, J = 2.0 Hz, H-2'), 6.81 (2H, dd, J = 1.9, 8.1 Hz, H-5'), 6.77 (2H, d, J = 8.1 Hz, H-6'), 4.71 (2H, d, J = 4.4 Hz, H-7'), 4.24 (2H, dd, J= 6.9, 9.1 Hz, H-9'), 3.15 (2H, m, H-8), 3.15 (2H, m, H-8'), 3.85 (6H, s, 3, 3'-OCH3); 13 C NMR (150 MHz, CD3OD): 133.8 (C-1), 108.6 (C-2), 149.2 (C-3), 147.4 (C-4), 113.8 (C-5), 116.1 (C-6), 87.5 (C-7), 55.4 (C-8), 72.6 (C-9), 133.8 (C-1'), 108.6 (C-2'), 149.2 (C-3'), 147.4 (C-4'), 113.8 (C-5'), 116.1 (C-6'), 87.5 (C-7'), 55.4 (C-8'), 72.6 (C-9'), 56.4 (3, 3'-OCH3).

[0130] compound 4 It was separated into a white amorphous powder. In HR-ESI-MS analysis, the deprotonated ion peak m / z 387.1447 [MH] (Calculated value: C 21 H 23 It was detected in O7, 387.1444). 1 ABX spin system in 1H NMR spectrum δ H 6.92(1H, d, J = 2.0 Hz, H-2'), 6.79(1H, dd, J = 2.0, 8.0 Hz, H-6'), 6.74(1H, d, J = 8.1 Hz, H-5') appeared. Symmetric protons in two meta-substituted benzene rings δ H It was identified at 6.63(2H, s, H-2, 6), and three methoxy groups (-OCH3) δ H 3.83(6H, s, 3-OCH3, 5-OCH3) and δ H The compound was detected at 3.84 (3H, s, 3'-OCH3). 2It was confirmed that the structure has one reduced methoxy group compared to (syringaresinol). 1 H and 13 As the C NMR data is consistent with existing literature, this compound was identified as (+)-medioresinol. Compound 4 The structural formula and spectroscopic data are as follows.

[0131] [Chemical Formula 4]

[0132]

[0133] Medioresinol ((+)-Medioresinol) ( 4 ): White amorphous powder; HR-ESI-MS: m / z 387.1447 [MH] - (calc. for C 21 H 23 O7, 387.1444); 1 H NMR (600 MHz, CD3OD): 4.69 (d, J = 4.3 Hz, H-7), 6.92 (d, J = 2.0 Hz, H-2'), 6.74 (d, J = 8.1 Hz, H-5'), 6.79 (dd, J = 2.0, 8.0 Hz, H-6'), 4.69 (d, J = 4.3 Hz, H-7'), 6.63 (s, H-2, 6), 3.12 (m, H-8), 3.83 (m, H-9a), 4.23 (m, H-9b), 3.12 (m, H-8'), 3.83 (m, H-9'a), 4.23 (m, H-9'b), 3.83 (s, 3, 5-OCH3), 3.84 (s, 3'-OCH3); 13C NMR (150 MHz, CD3OD): 131.2 (C-1), 103.4 (C-2), 149.3 (C-3), 135.1 (C-4), 149.3 (C-5), 103.4 (C-6), 87.1 (C-7), 54.9 (C-8), 72.8 (C-9), 131.2 (C-1'), 110.6 (C-2'), 147.2 (C-3'), 135.1 (C-4'), 115.7 (C-5'), 119.8 (C-6'), 87.1 (C-7'), 54.9 (C-8'), 72.8 (C-9'), 56.2 (3, 5-OCH3), 56.1 (3'-OCH3).

[0135] compound 5 It was separated into a white amorphous powder. In HR-ESI-MS analysis, the deprotonated ion peak m / z 213.1139 [MH] (Calculated value: C 11 H 17 It was detected in O4, 213.1127). 1 In the H NMR spectrum, olefinic hydrogens with double bonds (olefinic protons, δ H 5.75, 1H, s, H-7) and oxygenated methylene group (oxymethine, δ H 4.21, 1H, quint, J = 3.5 Hz, H-3) was confirmed. Two methylene groups δ H It was detected at 2.42, 1.99, 1.99, and 1.78, and three methyl groups (-CH3) were, respectively δ H It was confirmed at 1.76, 1.47, and 1.28. 13 Eleven carbon signals were detected in C NMR analysis, and carbonyl carbon ( δ C 171.7, C-8), two double-bonded carbons ( δ C 185.4, C-1 and 113.8, C-7), two oxygenated carbons ( δ C 88.7, C-2 and 67.3, C-4), two methylene carbons ( δ C 47.9, C-5 and 46.5, C-3), three methyl carbons ( δ C 31.0, 27.4, 26.6) were confirmed. In HMBC analysis, 2-CH3 and 6-CH3 were bonded to C-1, forming a double bond (D 1(7) It was confirmed that ) is formed. In addition, HMBC analysis confirmed that it has an 8-COOH structure, as H-7 showed cross-peaks with C-2, C-6, and C-8. Accordingly, the compound 5 It was identified as (Z)-2-(2,4-dihydroxy-2,6,6-trimethylcyclohexylidene) acetic acid. Compound 5 The structural formula and spectroscopic data are as follows.

[0136] [Chemical Formula 5]

[0137]

[0138] ( Z )-2-(2,4-dihydroxy-2,6,6-trimethylcyclohexylidene) acetic acid(( Z )-2-(2,4-Dihydroxy-2,6,6-trimethylcyclohexylidene) acetic acid) ( 5 ): White amorphous powder; HR-ESI-MS: m / z 213.1139 [MH] - (calc. for C 11 H 17 O4, 213.1127); 1 H NMR (600 MHz, CD3OD): 2.42 (dt, J = 2.6, 13.3 Hz, H-3a), 1.78 (dd, J = 3.3, 14.2 Hz, H-3b), 4.21 (quint, J = 3.5 Hz, H-4), 1.99 (ddd,J = 2.2, 3.1, 14.4 Hz, H-5a), 1.99 (dt, J = 3.7, 14.4 Hz, H-5b), 5.75 (s, H-7), 1.47 (s, 6-CH3), 1.28 (s, 6-CH3), 1.76 (s, 2-CH3); 13 C NMR (150 MHz, CD3OD): 185.4 (C-1), 88.7 (C-2), 46.5 (C-3), 67.3 (C-4), 47.9 (C-5), 36.8 (C-6), 113.3 (C-7), 171.7 (C-8), 27.4 (6-CH3), 31.0 (6-CH3), 26.6 (2-CH3).

[0140] compound 6 It was separated as a white amorphous powder. Mass spectrometry and spectroscopic data were similar to Compound 5, but a difference was confirmed at the C-3 position. Therefore, Compound 6 silver compounds 5 It was identified as the epimer of and was identified as rel-(Z)-2-(2,4-dihydroxy-2,6,6-trimethylcyclohexylidene) acetic acid. Compound 6 The structural formula and spectroscopic data are as follows.

[0141] [Chemical Formula 6]

[0142]

[0143] rel-( Z )-2-(2,4-dihydroxy-2,6,6-trimethylcyclohexylidene) acetic acid (Rel-( Z )-2-(2,4-dihydroxy-2,6,6-trimethylcyclohexylidene) acetic acid ( 6 ): White amorphous powder; 1 H NMR (600 MHz, CD3OD): 2.42 (dt, J = 2.6, 13.3 Hz, H-3a), 1.78 (dd, J= 3.3, 14.2 Hz, H-3b), 4.21 (quint, J = 3.5 Hz, H-4), 1.99 (ddd, J = 2.2, 3.1, 14.4 Hz, H-5a), 1.99 (dt, J = 3.7, 14.4 Hz, H-5b), 5.75 (s, H-7), 1.47 (s, 6-CH3), 1.28 (s, 6-CH3), 1.76 (s, 2-CH3).

[0145] compound 7 It separated into a white amorphous powder. In HR-ESI-MS analysis, the deprotonated ion peak m / z 177.0563 [MH] (Calculated value: C 10 It was detected in H9O3, 177.0552). 1 Characteristic signal of the aldehyde group (-CHO) in the H NMR spectrum ( δ H 9.46, 1H, d, J = 8.1 Hz, H-3') was confirmed. Two trans-olefinic protons were each δ H 7.54(1H, d, J = 15.4 Hz, H-1') and 6.52(1H, dd, J The ABX spin system was confirmed at = 8.1, 15.4 Hz, H-2'). δ H 7.1313 (1H, d, J = 2.3 Hz, H-2), 7.10(1H, dd, J = 2.6, 8.2 Hz, H-6), 6.69(1H, d, J = 8.4 Hz, H-5) was detected. 13 Carbonyl carbon (in C NMR) δ C 191.9, C-3') and 5 unsaturated carbons ( δ C112.3(C-2), 116.7(C-5), 125.1(C-6), 156.2(C-1'), and 127.6(C-2') were identified. Through comparison with existing literature, this compound was identified as coniferaldehyde. Compound 7 The structural formula and spectroscopic data are as follows.

[0146] [Chemical Formula 7]

[0147]

[0148] Coniferaldehyde 7 ): White amorphous powder; HR-ESI-MS: m / z 177.0563 [MH] - (calc. for C 10 H9O3, 177.0552); 1 H NMR (600 MHz, CD3OD): 7.13 (1H, d, J = 2.3 Hz, H-2), 6.69 (1H, d, J = 8.4 Hz, H-5), 7.10 (1H, dd, J = 2.6, 8.2 Hz, H-6), 7.54 (1H, d, J = 15.4 Hz, H-1'), 6.52 (1H, dd, J = 8.1, 15.4 Hz, H-2'), 9.46 (1H, d, J = 8.1 Hz, H-3'), 3.85 (3H, s, 3-OCH3); 13 C NMR (150 MHz, CD3OD): 126.7 (C-1), 112.3 (C-2), 151.7 (C-3), 149.5 (C-4), 116.7 (C-5), 125.1 (C-6), 156.2 (C-1'), 127.6 (C-2'), 196.1 (C-3').

[0150] compound 8 It was separated into amorphous powder. In HR-ESI-MS analysis, the deprotonated ion peak m / zIt was detected at 121.0299 [MH] (calculated value: C7H5O2, 121.0290). 1 Aldehyde group in H NMR spectrum ( δ H 8.55(1H, s, H-7)) and A2B2 spin system( δ H 7.95((2H, d, J = 9.0 Hz, H-2, 6), 6.45(2H, d, J = 9.0 Hz, H-3, 5)) was confirmed, 13 The compound was identified as 4-hydroxybenzaldehyde through 3C NMR analysis. 8 The structural formula and spectroscopic data are as follows.

[0151] [Chemical Formula 8]

[0152]

[0153] 4-Hydroxybenzaldehyde 8 ): White amorphous powder; HR-ESI-MS: m / z 121.0299 [MH] - (calc. for C7H5O2, 121.0290); 1 H NMR (600 MHz, CD3OD): 7.64 (2H, d, J = 9.2 Hz, H-2, H-6), 6.81 (2H, d, J = 9.3 Hz, H-3, H-5); 13 C NMR (150 MHz, CD3OD): 131.2 (C-1), 132.8 (C-2), 116.2 (C-3), 165.3 (C-4), 116.2 (C-5), 132.8 (C-6), 191.9 (CHO).

[0155] compound 9 is a compound 8It had a structure similar to, and was in a form where the aldehyde group (-CHO) was substituted with a carboxyl group (-COOH). In HR-ESI-MS analysis, the deprotonator ion peak m / z It was detected at 137.0246 [MH] (calculated value: C7H5O3, 137.0239). 1 A2B2 spin system in H NMR spectrum ( δ H 7.88(2H, d, J = 8.9 Hz, H-2, 6), δ H 6.82(2H, d, J = 8.9 Hz, H-3, 5)) was confirmed, 13 Carbonyl carbon (in C NMR) δ C 170.1, C-7), 2 quaternary carbons ( δ C 163.4 (C-4) and 131.1 (C-1)) and 4 methine carbons ( δ C 133.0 (C-2,6) and 116.0 (C-3,5)) were detected. As a result of comparison with existing literature, this compound was identified as 4-hydroxybenzaldehyde. Compound 9 The structural formula and spectroscopic data are as follows.

[0156] [Chemical Formula 9]

[0157]

[0158] p -hydroxy-benzoic acid( p -Hydroxy-benzoic acid) ( 9 ): White amorphous powder; HR-ESI-MS: m / z 137.0246 [MH] - (calc. for C7H5O3, 137.0239); 1 H NMR (600 MHz, CD3OD): 7.92 (2H, d, J = 9.34 Hz, H-2, H-6), 6.43 (2H, d,J = 9.35 Hz, H-3, H-5); 13 C NMR (150 MHz, CD3OD): 121.2 (C-1), 132.3 (C-2), 115.7 (C-3), 164.2 (C-4), 115.7 (C-5), 132.3 (C-6), 171.2 (COOH).

[0160] Example 5: Antioxidant effect of compound (1-9)

[0161] 5-1. DPPH and ABTS Radical Scavenging Activity

[0162] Antioxidant activity was evaluated using the DPPH (Sigma-Aldrich, St. Louis, MO, USA) radical scavenging ability of each sample. 100 μL of a 200 μM DPPH solution diluted in ethanol and 100 μL of the sample (solutions diluted in ethanol at concentrations of 10 and 100 μg / mL) were added to each well. A control group was constructed using ethanol instead of the sample under the same conditions. After thoroughly mixing each well, the mixture was reacted at room temperature in a dark room for 30 minutes, and the absorbance was measured at 517 nm using a microreader (Epoch, Biotek Instruments, Winooski, VT, USA).

[0163] Looking at the DPPH radical scavenging activity, the compound at a concentration of 40 μM 1 class 2 They exhibited strong DPPH radical scavenging activities of 50.01 ± 1.13% and 70.45 ± 1.06%, respectively. 4 It exhibited an intermediate level of DPPH radical scavenging activity of 37.36 ± 0.60%. Even at concentrations of 10 and 20 μM, the compound 2 The compounds maintained high DPPH scavenging activities of 63.11 ± 0.76% and 68.42 ± 0.32%, respectively. 1 class 4Each showed DPPH scavenging activity ranging from 28.32 ± 0.66% to 41.63 ± 0.13%, while other compounds showed negligible activity (Fig. 4).

[0164] In addition, to prepare the ABTS (Sigma-Aldrich, Co.) solution, 2,2′-azobis(2-aminopropane) dihydrochloride (7 mM) and 2.45 mM ABTS were mixed and reacted at 4°C for 16 hours. For the experiment, 50 μL of the sample and 100 μL of the ABTS solution were mixed in a 96-well plate, reacted at room temperature for 20 minutes, and the absorbance was measured at 734 nm.

[0165] As a result, the compound 1 , 2 and 4 It significantly reduced ABTS radicals under tested conditions. At a concentration of 40 μM, the compound 1 class 2 They exhibited ABTS radical scavenging activities of 52.33 ± 0.30% and 40.94 ± 0.30%, respectively. 4 It showed a relatively low scavenging activity of 16.04 ± 0.38%. Even at concentrations of 10 and 20 μM, the compound 1 class 2 They exhibited scavenging activities ranging from 17.15 ± 0.04% to 33.81 ± 0.52%, respectively. The other compounds showed negligible or almost no ABTS radical scavenging activity (Fig. 5).

[0166] Additionally, active compound ( 1 , 2 , 4 DPPH and ABTS radical scavenging activity experiments were performed on ) at various concentrations ranging from 0 to 80 μM to EC 50 The value was determined. As a result, the EC for DPPH scavenging activity 50 Compounds as value 2 The lowest EC was 9.72 ± 1.99 μM. 50 It indicated the value, and next, the compound1 This EC of 39.16 ± 1.04 μM 50 value, compound 4 GA's EC is 70.73 ± 9.21 μM 50 The values ​​were shown (Table 1)

[0167] In addition, EC regarding ABTS scavenging activity 50 value, compound 1 This is the lowest EC of 33.23 ± 0.66 μM 50 It indicated the value, and next, the compound 2 GA has an EC of 42.90 ± 2.12 μM 50 Value, EC of compound 4 at 57.62 ± 6.00 μM 50 The values ​​were shown (Table 1).

[0168] compound EC 50 (μM) DPPH ABTS 1 39.16 ± 1.04 33.23 ± 0.66 2 9.72 ± 1.99 42.90 ± 2.12 4 70.73 ± 9.21 57.62 ± 6.00 Ascorbic acid (AA) 4.22 ± 0.17 24.83 ± 0.27

[0169] 5-2. Inhibitory Effect of Reactive Oxygen Species (ROS) on LPS-Induced RAW264.7 Cells

[0170] To further evaluate the antioxidant effects of the isolated compounds, LPS (Lipopolysaccharide)-induced ROS production was measured in RAW264.7 macrophages.

[0171] 5-2-1. RAW264.7 Cell Culture and Cell Viability Analysis

[0172] RAW264.7 cells were obtained from the Korean Cell Lines Bank (Seoul, South Korea). Cells were cultured in a humid environment (5% CO2, 37℃) using DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% heat-inactivated FBS, streptomycin sulfate (100 μg / mL), and penicillin (100 IU / mL).

[0173] Cells were seeded into a 96-well plate and pre-cultured for 20 hours; subsequently, each compound was treated for 1 hour, followed by stimulation with LPS (1 μg / mL) for 20 hours. Then, the compound ( 1-9After treating for 2 hours, additional stimulation with LPS (100 ng / mL) was performed for 20 hours.

[0174] Cultured cells were treated with MTT solution at 37°C for 4 hours for the MTT experiment, after which the supernatant was removed and 100 μL of DMSO (dimethyl sulfoxide, Sigma Aldrich, Saint Louis, MO, USA) was added. After 5 minutes, the absorbance of the formazan crystals was measured at 570 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA).

[0175] 5-2-2. Measurement of Reactive Oxygen Species (ROS) Generation

[0176] Each compound ( 1-9 ) was treated at concentrations of 10, 20, and 40 μM, and cells were placed in a 6-well plate at a rate of 1 × 10⁶ per well. 4 After inoculating at a cell density, the cells were cultured for 20 hours. Subsequently, after removing all the cell culture medium, 10 μM DCFH-DA (dichlorodihydrofluorescein diacetate) diluted to a ratio of 1 / 1000 was added to serum-free medium. After reacting in a dark room at 37°C for 20 minutes, the cells were washed three times with PBS.

[0177] Cells were observed using an inverted fluorescence microscope, and fluorescence intensity was measured at 485 nm (excitation) and 530 nm (emission) using a fluorescence microplate reader.

[0178] 5-2-3. Experimental Results

[0179] As a result of the MTT cytotoxicity evaluation, none of the compounds affected the viability of RAW264.7 cells at concentrations of 40 μM or lower (Fig. 7). It was confirmed that LPS treatment increased ROS production in RAW264.7 macrophages, while compound treatment decreased ROS production. At a concentration of 40 μM, the compound 1 , 5 , 8 , 9 It inhibited ROS production by 63.52 ± 4.12%, 64.97 ± 6.15%, 65.69 ± 2.31%, and 71.54 ± 0.54%, respectively (Fig. 6). At a concentration of 20 μM, the compound 1 , 6 , 8 This significantly reduced LPS-induced ROS production. Even at a concentration of 10 μM, the compound 1 and 5-9 It significantly reduced ROS production compared to the control group (cells treated only with LPS). In RAW264.7 cells, none of the compounds showed toxicity under experimental conditions (Fig. 7).

[0181] Example 5: Inhibitory effect of CD3 / CD28-induced IL-2 production in activated Jurkat T cells

[0182] Jurkat T cells were cultured in RPMI medium (Gibco-RBL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS, AusGeneX, Santa Clara, CA) and PenStrep (Gibco-RBL), and cultured in a humidified incubator maintained at 37°C, 5% CO2, and 95% air. Jurkat T cells (1 × 10⁶ 6) were treated with CD3 / CD28 (7 μg / mL and 2 μg / mL, respectively) for 24 hours. For anti-CD3 / CD28 stimulation, cells were cultured in culture dishes coated with anti-CD3 antibodies, and then stimulation was induced by adding anti-CD28 antibodies (2 μg / mL). For superantigen stimulation, Jurkat T cells were co-cultured with Raji B cells treated with Staphylococcus enterotoxin E (1 μg / mL). For pre-treatment and post-treatment experiments, various concentrations of compounds were applied 60 minutes before and 30 minutes after CD3 / CD28 stimulation of Jurkat T cells. Subsequently, the supernatant of the cell culture medium was collected, and the concentration of IL-2 was measured using the Duoset Human IL-2 ELISA kit (R&D Systems) according to the manufacturer's instructions.

[0183] First, all isolated compounds did not affect the viability of Jurkat T cells at concentrations of 40 μM or less (Fig. 7), and thus were deemed suitable for evaluating the inhibitory effect on CD3 / CD28-induced IL-2 production.

[0184] IL-2 secretion significantly increased compared to the untreated group through CD3 / CD28 co-stimulation, but the isolated compounds showed a tendency to inhibit IL-2 production in a concentration-dependent manner. In particular, the compound 1 class 2 It significantly inhibited co-stimulation-induced IL-2 production in Jurkat cells at all concentrations. At a concentration of 40 μM, the compound 2 It inhibited IL-2 production by approximately 99.5% compared to co-stimulation without the compound. 1 The compound inhibited IL-2 production with a high inhibition rate of 97.17%. 7It significantly inhibited IL-2 production with an inhibition rate of 76.54%. Compound 4 , 5 , 8 , and 9 It showed moderate IL-2 inhibitory effects with inhibition rates of 64.16%, 51.61%, 62.35%, and 59.10%, respectively.

[0185] Compound at a concentration of 20 μM 2 It strongly inhibited IL-2 production with an inhibition rate of 96.54%, and the compound 1 The compound significantly inhibited IL-2 production with an inhibition rate of 88.58%. 4 , 7 , and 8 ...inhibited IL-2 production with inhibition rates of 54.66%, 67.54%, and 56.76%, respectively. At a concentration of 10 μM, the compound 1 and 2 It strongly inhibited IL-2 production with inhibition rates of 83.85% and 85.86%. Compound 7 It significantly inhibited IL-2 production with an inhibition rate of 60.70%. Other compounds ( 3 and 6 The inhibitory effect of ) on CD3 / CD28-induced IL-2 production was negligible (Fig. 8).

[0187] Example 6: Inhibitory effect of Compounds 1, 2, and 7 on CD3 / CD28-induced MAPK signaling pathway

[0188] To evaluate the correlation between the active compound and MAPK-specific proteins including ERK, JNK, and p38, protein expression levels were measured using Western blotting.

[0189] Specifically, Jurkat T cells cultured as in Example 6 were stimulated with CD3 / CD28 for 2 hours and lysed using PRO-PREPTM (Intron Biotechnology, Seoul, Republic of Korea).

[0190] The Pierce™ Bradford Protein Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used for protein quantification. Extracted protein (20 μg) was separated by SDS-PAGE using 8% and 10% acrylamide gels and then transferred to a PVDF membrane. The primary antibodies used in the experiment included p-p38 (38 kDa), p38 (38 kDa), p-p65 (65 kDa), p65 (65 kDa), Iκαβ (36 kDa), and p-Iκαβ (36 kDa) antibodies purchased from Cell Signaling Technology (Danvers, MA, USA); β42 kDa was supplied by BD Bioscience (San Jose, California, USA); and ERK (44 kDa) and p-ERK (44 kDa) were supplied by Cell Signaling Technology (Beverly, Massachusetts, USA).

[0191] After the protein was transferred to the PVDF membrane, it was blocked with 5% skim milk for 2 hours, and then the primary antibody was diluted in 2.5% skim milk at a ratio of 1:1000 and incubated at 4°C for 18 hours. Subsequently, after washing three times with Tween20 / Tris-buffered saline (T / TBS), the HRP-conjugated secondary antibody was diluted in 5% skim milk at a ratio of 1:2000 and incubated at room temperature for 2 hours. Finally, after washing three times with T / TBS, the expression of the target protein was detected using the Super Signal™ West Femto Maximum Sensitivity Substrate from Thermo Fisher Scientific (Waltham, MA, USA). The results are shown in Figure 9.

[0192] The expression level of p-ERK increased after CD3 / CD28 stimulation. At 40 μM, the compound 2 and 7 silver compounds 1 It reduced the phosphorylation level of ERK more significantly. At 20 μM, the compound 7 silver compounds 1 class 2 It inhibited ERK phosphorylation more strongly.

[0193] The expression level of p-JNK increased after CD3 / CD28 stimulation. As a result of treatment to evaluate the effects of these compounds on p-JNK, the compounds 7 The compound most strongly reduced JNK phosphorylation at 20 μM and 40 μM. 1 class 2 It significantly reduced JNK phosphorylation at 40 μM, and the compound 1 It reduced p-JNK expression levels more vehemently than compound 2. At 10 μM, compound 1 It most strongly inhibited p-JNK expression levels among the three compounds.

[0194] p38 phosphorylation increased after CD3 / CD28 stimulation. After treatment with each compound, p38 phosphorylation was regulated. Compounds at all tested concentrations 1 silver compounds 2 and 7 It inhibited p38 phosphorylation levels more than at 40 μM. 2 is a compound 7 It inhibited p38 phosphorylation levels more. However, at 20 μM, the compound 7 silver compounds 2 It inhibited p38 phosphorylation more strongly. Under the experimental conditions, no compound affected the level of β-expression.

[0196] Example 7: Inhibitory effect of Compounds 1, 2, and 7 on the CD3 / CD28-induced NF-κB signaling pathway

[0197] Regarding the mechanism of the inhibitory effect of the active compound on IL-2 production, phosphorylation of p65 NF-κB and IKαβ in the signaling pathway was investigated using the method described in Example 6 above, and the results are shown in Figure 10.

[0198] After CD3 / CD28 stimulation, the phosphorylation level of p65 increased, indicating activation of the NF-κB pathway. However, after treatment with 40 μM of the active compound, the expression level of p-p65 decreased, and particularly the compound 2 a compound 1 class 7 It reduced p-p65 expression levels more than.

[0199] Under normal conditions, the IκB protein binds to the p65 subunit of NF-κB to form a complex, keeping the NF-κB molecule segregated in the cytoplasm and preventing its translocation to the nucleus and activation of gene expression. When NF-κB is activated, IκB is degraded. Compound 1 , 2 , 7 Since they exhibited an inhibitory effect on p-p65 expression, their influence on the IKαβ pathway was investigated to evaluate their ability to regulate IκB degradation.

[0200] The phosphorylation level of IKαβ increased after CD3 / CD28 stimulation. However, the increase in IKαβ phosphorylation levels decreased after treatment with active compounds, and particularly the compound 2 a compound 1 class 7 They reduced IKαβ phosphorylation more significantly. Under the experimental conditions, these compounds did not affect β-actin expression (Fig. 10).

[0202] Example 8: In silico ( In Silico ) Molecular docking analysis

[0203] To evaluate the binding affinity of an active compound between a target protein In Silico Molecular docking analysis was performed.

[0204] Specifically, the crystal structures of the proteins p65 (PDB ID: 1VKX), ERK (PDB ID: 6NBS), JNK (PDB ID: 1PMN), and p38 MAPK (PDB ID: 1KV2) were downloaded from the RCSB Protein Data Bank (https: / / www.rcsb.org) as of October 25, 2024. Subsequently, each protein was added to MGL Tools 1.5.6 to perform a preprocessing step. After removing water molecules and heteroatoms, the proteins were purified by adding polar hydrogen atoms and Kollman charges, and finally saved in pdbqt format. The 3D structures of the compounds were downloaded in sdf format from PubChem (https: / / pubchem.ncbi.nlm.nih.gov) as of October 26, 2024, and converted to pdbqt format using the Open Babel program. The transformed structure was added to MGL Tools 1.5.6 to impart Gasteiger charges, and the final preparation for docking was completed. Grid box parameters were set using the Pymol program, and the Lamarckian Genetic Algorithm of AutoDock 4.2.6 was applied to search for the optimal binding structure of the ligand. Finally, the docked complex structure was analyzed and visualized using Discovery Studio Visualizer 2021 and the Pymol program.

[0205] 8-1. Results of In Silico Molecular Docking Analysis with ERK Protein

[0206] compound( 1 , 2 , 7) was prepared as a ligand to dock to the ERK protein (Fig. 11). All compounds docked to the same region as the re-docked native ligand, indicating that these compounds docked to the protein's binding pose. According to the data, the compounds 2 The free energy of (ΔG = -6.65 kcal / mol) was lower than that of the co-crystal structure (ΔG = -4.92 kcal / mol). compound 7 It exhibited a binding free energy (ΔG = -4.73 kcal / mol) similar to that of the original ligand, and the compound 1 It showed a higher bond free energy (ΔG = -3.56 kcal / mol).

[0207] These compounds occupied the binding pose of the protein surrounded by interacting amino acids. Compounds 2 and 7 Each has the potential to form a conventional hydrogen bond with the ALA307 residue through interaction with the hydroxyl group of the ligand. In addition, the compound 2 It formed additional hydrogen bonds with the ASP316 amino acid and exhibited hydrophobic interactions with GLU312, TYR315, ASP316, and PRO317 residues. 7 ...exhibited hydrophobic interactions with GLU312, TYR315, and ASP316 residues. 1 It formed hydrogen bonds with TYR129 and exhibited hydrophobic interactions with the amino acids ARG133, GLN313, TYR315, ASP316, and ASP319. Therefore, it was confirmed that the amino acids ALA307, GLN312, TYR314, ASP316, and PRO317 are the major residues of the protein binding posture (Fig. 12).

[0208] 8-2. Results of In Silico Molecular Docking Analysis with JNK Protein

[0209] Similarly, compounds ( 1 , 2 , 7 ) was docked to the same region within the active region of the JNK protein (Fig. 13). Compound 2 It exhibited a bond free energy (ΔG = -12.27 kcal / mol), which was similar to that of the redocked native ligand (ΔG = -12.10 kcal / mol). 1 class 7 In addition, they showed significant binding affinity for proteins. All three compounds were able to form π-sulfur (π-interactions) with the protein's MET146 residue.

[0210] On the other hand, compounds 2 It formed two hydrogen bonds with ASN152 and GLN155 residues and exhibited significant interactions with VAL78, ALA91, LYS93, LEU144, MET149, VAL196, and LEU206 residues within the binding pose. 1 It formed hydrogen bonds with the LYS93 and MET149 amino acids. 7 It exhibited two hydrogen bonds with the ALA91 and LYS93 amino acids. In addition, the compound 1 class 7 It formed interactions with the hydrophobic protein mentioned above (Fig. 14).

[0211] 8-3. Results of In Silico Molecular Docking Analysis with p38 Protein

[0212] compound 1 , 2 , 7It also exhibited significant binding affinity when docked to the p38 protein (Fig. 15). These compounds and their co-crystal structures occupied the same allosteric sites of the protein binding pose. This observation indicates that the docking protocol is appropriate.

[0213] compound 2 It showed a bond free energy (ΔG = -10.58 kcal / mol), which is the compound 1 class 7 It was lower than (ΔG = -6.13 and -5.17 kcal / mol, respectively). Compound 1 It formed two hydrogen bonds with GLU71 and ASP168 residues through hydroxyl and oxygenated bonding. 2 It formed hydrogen bonds with ARG70 and ILE166 residues through interaction with hydroxyl groups. 7 It interacted with histidine (HIS148) and aspartic acid (ASP168) to form hydroxyl and carboxyl bonds (Fig. 16). In addition, these compounds exhibited hydrophobic interactions with amino acids including LYS53, ARG67, LEU75, ILE84, and LEU167.

[0214] 8-4. Results of In Silico Molecular Docking Analysis with p65 Protein

[0215] compound( 1 , 2 , 7 When ) docked to the p65 protein, it exhibited significant binding affinity (Fig. 17). Among them, the compound 7 showed the lowest bond free energy (ΔG = -7.56 kcal / mol), followed by the compound 2 (ΔG= -5.81 kcal / mol) and compound 1It appeared in the order of (ΔG = -4.11 kcal / mol). However, the compound 1 class 7 occupied the same region, and the compound 2 and it was located in a different position from the reference (Fig. 17).

[0216] compound 1 It formed hydrogen bonds with the ARG35 residue through oxygenated interaction. Compound 2 It interacted with the major amino acid residues ARG187, LEU154, and ASN155 by forming hydrogen and oxygen bonds. 7 It formed two hydrogen bonds with the major amino acids ASN155 and ARG187 through hydroxyl groups, and also exhibited hydrophobic interactions through Van der Waals interactions with ARG35.

Claims

Claim 1 A pharmaceutical composition for inhibiting immune responses comprising, as an active ingredient, one or more compounds selected from the group consisting of zebeiresinol, syringaresinol, and coniferaldehyde. Claim 2 A pharmaceutical composition for suppressing an immune response according to claim 1, wherein the composition is intended to suppress an immune rejection response to the transplantation of skin, blood, cornea, liver, lung, intestine, pancreas, heart, kidney, bone marrow, stem cells, or progenitor cells. Claim 3 A pharmaceutical composition for suppressing an immune response according to claim 1, wherein the composition is used for the treatment and prevention of graft-versus-host disease, allergic disease, autoimmune disease or inflammatory disease. Claim 4 A food composition for preventing and improving immune hypersensitivity reactions comprising, as an active ingredient, one or more compounds selected from the group consisting of zebeiresinol, syringaresinol, and coniferaldehyde. Claim 5 In paragraph 4, the above composition is a food composition intended to suppress an immune hypersensitivity reaction to the transplantation of skin, blood, cornea, liver, lung, intestine, pancreas, heart, kidney, bone marrow, stem cells, or progenitor cells. Claim 6 In paragraph 4, the above composition is a food composition that prevents or improves graft-versus-host disease, allergic disease, autoimmune disease or inflammatory disease. Claim 7 An antioxidant food composition comprising one or more compounds selected from the group consisting of zebeiresinol, syringaresinol, and medioresinol as active ingredients. Claim 8 An antioxidant cosmetic composition comprising one or more compounds selected from the group consisting of zebeiresinol, syringaresinol, and medioresinol as active ingredients.

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