Chamaecyparis obtusa leaf fermentation product fermented with ganoderma applanatum, and use thereof

Abstract

In the present invention, a Chamaecyparis obtusa leaf fermentation product fermented with ganoderma applanatum is prepared, and antioxidant and anti-inflammatory effects thereof are identified. The Chamaecyparis obtusa leaf fermentation product fermented with ganoderma applanatum, according to the present invention, does not exhibit cytotoxicity, exhibits an antioxidant effect, effectively inhibits signaling pathways and the expression of inflammatory cytokines and signal molecules inducing the expression thereof, and inhibits the production of nitric oxide caused by inflammation, and the expression of factors involved therein, thereby exhibiting excellent anti-inflammatory effects, and thus can be effectively applied to uses such as prevention, alleviation or treatment of inflammatory diseases.

Description

Fermented cypress leaf extract fermented with Ganoderma lucidum and its uses

[0001] The present invention relates to a fermented cypress leaf product fermented with *Jannabibullocho* and its anti-inflammatory use.

[0002] Inflammatory responses occur when tissues (cells) are damaged or infected by external infectious agents (bacteria, fungi, viruses, and various types of allergens), involving various inflammatory mediators and immune cells in local blood vessels and body fluids. This results in a series of complex physiological reactions, such as enzyme activation, secretion of inflammatory mediators, fluid infiltration, cell migration, and tissue destruction, as well as external symptoms like erythema, edema, fever, and pain. In normal individuals, the inflammatory response serves to restore biological functions by eliminating external infectious agents and regenerating damaged tissues; however, if antigens are not eliminated or if the inflammatory response becomes excessive or persistent due to internal substances, it instead becomes a major pathological phenomenon of the disease (hypersensitivity disorders, chronic inflammation) and acts as an impediment during treatment processes such as blood transfusion, drug administration, and organ transplantation.

[0003] The actions of factors involved in the inflammatory response are described as follows.

[0004] The complement system is a key humoral factor that activates and amplifies inflammation in the early stages of the immune response. The active proteins (anaphylatoxins; C3a, C4a, C5a) and the membrane attack complex (MAC) generated during the complement activation process are associated with various inflammatory diseases, such as rheumatoid arthritis, systemic lupus erythematosus, adult respiratory disease syndrome, and Alzheimer's dementia, and are a direct cause of hyperacute rejection in organ transplants. ICAM-1 is a representative protein of the group of cell adhesion molecules expressed on the surface of endothelial cells. Under normal conditions, it is expressed at very low levels; however, when stimulated by inflammatory mediators such as cytokines—including TNF-α, interferon gamma (IFN-γ), and interleukin-1β—its expression level increases rapidly. It plays a role in attaching inflammatory cells, such as monocytes and lymphocytes, migrating in the bloodstream and facilitating their migration to inflamed tissues.

[0005] Nitric oxide (NO) is produced along with L-citrulline after L-arginine is oxidized by nitric oxide synthase (NOS). NO acts on the vascular system as a mediator involved in vasodilation, platelet adhesion and aggregation, neurotransmission, digestive tract motility, and penile erection. It is produced not only in inflammatory cells but also in non-immune cells, providing a defense against microbial infections. Meanwhile, inducible-NOS (hereinafter iNOS), one of the NOS involved in NO production, is expressed independently of calcium or calmodulin in response to stimulation by lipopolysaccharide (hereinafter LPS) and cytokines (such as IFNγ and TNFα). Since cyclooxygenase-2 (hereinafter COX-2) is also activated by such stimulation, leading to the production of prostaglandins (hereinafter PGs), which are inflammatory mediators, iNOS expression and COX-2 expression are closely related, and the generated NO also influences COX-2 expression. In macrophages, NO production is selectively induced by the expression of iNOS, and because this result also triggers the activation of other inflammatory responses, NO can be considered an important factor in inflammatory diseases.

[0006] The expression of such various inflammatory mediators activates the phagocytosis of foreign factors and participates in inflammatory responses through the activation of various inflammatory signaling networks, including transcription factors such as NF-κB (nuclear factor-kappaB) and MAPK (mitogen-activated protein kinase). Therefore, substances capable of inhibiting and regulating their production can be utilized as resources for treating or preventing inflammatory diseases.

[0007] Meanwhile, the Japanese cypress (*Chamaecyparis obtusa*) is an evergreen tree native to Japan that grows to a height of about 40 m and a diameter of about 2 m. Its branches spread horizontally, forming a conical shape. The bark is reddish-brown, splits vertically, and peels off. The leaves are scale-like, overlapping in layers, thick in texture, and have blunt tips. In Korea, it is also widely distributed as an evergreen coniferous tree planted in Jeju Island and the southern regions.

[0008] While Hinoki cypress is known to be a natural product with various beneficial properties, further research is needed regarding Hinoki cypress-derived substances that exhibit significant anti-inflammatory effects without toxicity.

[0009] The objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases.

[0010] In addition, another objective of the present invention is to provide a composition for an external medical product for the prevention or improvement of inflammatory diseases.

[0011] In addition, another objective of the present invention is to provide a cosmetic composition for preventing or improving inflammatory diseases.

[0012] In addition, another objective of the present invention is to provide a food composition for preventing or improving inflammatory diseases.

[0013] In addition, another objective of the present invention is to provide a health functional food composition for the prevention or improvement of inflammatory diseases.

[0014] In addition, another objective of the present invention is to provide an anti-inflammatory composition.

[0015] In addition, another objective of the present invention is to provide a method for producing a fermented cypress leaf product fermented with *Jannabibullocho*.

[0016] Another objective of the present invention is to provide a method for preventing or treating inflammatory diseases.

[0017] Another objective of the present invention is to provide a method for preventing, alleviating, or treating inflammation.

[0018] Another objective of the present invention is to provide a method for increasing the antioxidant capacity of an individual.

[0019] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0020] In addition, to achieve the above other objectives, the present invention provides an external pharmaceutical composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0021] In addition, to achieve the above-mentioned other objective, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0022] In addition, to achieve the above-mentioned other objective, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0023] In addition, to achieve the above-mentioned other objective, the present invention provides a health functional food composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0024] In addition, to achieve the above-mentioned other objective, the present invention provides an anti-inflammatory composition comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0025] In addition, to achieve the above other objective, the present invention provides a method for preparing a fermented cypress leaf product fermented with *Jannabis bullocho*, comprising the following steps: 1) treating the cypress with a solvent and extracting at 20 to 30°C for 12 to 48 hours; and 2) inoculating the extract with *Jannabis bullocho* and fermenting at 100 to 300 rpm for 12 hours to 4 weeks.

[0026] In addition, to achieve the above-mentioned other objective, the present invention provides a method for preventing or treating inflammatory diseases, comprising the step of administering to an individual a fermented product obtained by fermenting a Chamaecyparis obtusa (Siebold & Zucc.) Endl. extract with Ganoderma applanatum.

[0027] In addition, to achieve the above-mentioned other objective, the present invention provides a method for preventing, alleviating, or treating inflammation, comprising the step of administering to an individual a fermented product obtained by fermenting a Chamaecyparis obtusa (Siebold & Zucc.) Endl. extract with Ganoderma applanatum.

[0028] In addition, to achieve the above-mentioned other objective, the present invention provides a method for increasing the antioxidant capacity of an individual, comprising the step of administering to the individual a fermented product obtained by fermenting a Chamaecyparis obtusa (Siebold & Zucc.) Endl. extract with Ganoderma applanatum.

[0029] The fermented cypress leaf extract fermented with *Jannabibullocho* according to the present invention does not exhibit cytotoxicity and shows antioxidant effects. It effectively inhibits inflammatory cytokines and the expression of signaling molecules that induce their expression, as well as signal transduction pathways, and suppresses the production of nitric oxide due to inflammation and the expression of factors involved therein, thereby exhibiting excellent anti-inflammatory effects. Consequently, it can be effectively applied for purposes such as the prevention, improvement, or treatment of inflammatory diseases.

[0030] Figure 1 shows the results of confirming changes in composition due to fermentation. Figure 1(a) shows the results of confirming the composition of Hinoki cypress leaf extract (70COL), and Figure 1(b) shows the results of confirming the composition of a fermented product (70COLGA) obtained by fermenting Hinoki cypress leaf extract with Ganoderma lucidum. In Figure 1 above, ns indicates not significant, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Student's t-test).

[0031] Figure 2 shows the results of determining the phenol and flavonoid content of 70COL and 70COLGA. Figure 2(a) shows the results of determining the total phenol content, and Figure 2(b) shows the results of determining the total flavonoid content. In Figure 2, ns indicates insignificance, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Student's t-test).

[0032] Figure 3 shows the results confirming the antioxidant activity of 70COL and 70COLGA. Figure 3(a) shows the results confirming DPPH radical scavenging activity, and Figure 3(b) shows ABTS + This is the result of confirming radical scavenging ability. In Figure 3 above, ns indicates insignificance, * is p < 0.05, ** is p < 0.01, and *** is p < 0.001 (Student's t-test).

[0033] Figure 4 shows the results of treating cells with various concentrations of 70COL or 70COLGA and confirming their cell viability. In Figure 4, ns indicates insignificance, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Student's t-test).

[0034] Figure 5 shows the results of treating cells with 70 COLGA to induce inflammation and confirming their morphological changes.

[0035] Figure 6 shows the results of treating inflammation-induced cells with 70COLGA and confirming its anti-inflammatory effect. Figure 6(a) shows the results of confirming nitric oxide production, Figure 6(b) shows the results of confirming the expression levels of iNOS and COX-2 proteins, Figure 6(c) shows the results of confirming the iNOS mRNA expression level, and Figure 6(d) shows the results of confirming the COX-1 mRNA expression level. In Figure 6, *** p < 0.001 (Student's t-test).

[0036] Figure 7 shows the results of treating inflammation-induced cells with 70COLGA and confirming the expression of inflammatory factor mRNA and the activation of inflammation-related signaling pathways. Figures 7(a) and 7(b) show the results of confirming the expression levels of proteins involved in inflammation-related signaling pathways, Figure 7(d) shows the results of confirming IL-1β mRNA expression, Figure 7(c) shows the results of confirming IL-6 mRNA expression, and Figure 7(f) shows the results of confirming TNF-α mRNA expression. In Figure 7, *** p < 0.001 (Student's t-test).

[0037] Figure 8 shows the results of treating inflammation-induced cells with 70COLGA and confirming the inhibitory effect on inflammatory molecules. Figure 8(a) visualizes the heatmap of mRNA expression differences between the control group (CON) and the LPS-treated group of RAW264.7 cells based on the GSE76572 dataset. Figure 8(b) shows the results of cytokine array analysis of the supernatant of the culture medium in which inflammation was induced by pre-treating with 70COLGA and then treating with LPS. Figure 8(c) is a graph quantifying the results of Figure 8(b). The red line indicates that expression decreased by more than half compared to the 200 ng / ml LPS-treated group. Figure 8(d) is a Venn diagram showing 27 cytokine genes upregulated in the GSE6572 dataset and 8 cytokines downregulated identified in Figure 8(b).

[0038] Figure 9 shows the results confirming that treating inflammation-induced cells with 70COLGA directly inhibits the STAT signaling pathway. Figure 9(a) illustrates the steps of the process for obtaining the conditioning medium (LPS-CM). Figure 9(b) shows the results of confirming relative protein expression after treating RAW264.7 cells with 70COLGA for 2 hours and then treating them with LPS-CM containing 70COLGA for 30 minutes.

[0039] The present invention will be described in more detail below.

[0040] The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0041] In the present invention, the above-mentioned *Ganoderma applanatum* is a mushroom belonging to the genus *Ganoderma* of the family Ganodermataceae, order Polyporales, phylum Basidiomycota, and is also known as the *Ganoderma applanatum*. It is a perennial mushroom that grows year-round, primarily on dead stumps or stems of deciduous trees, or on wounds on living trees. It occurs singly or in clusters and forms white rot fungi. It is known to possess various effects, including antitumor, antiviral, antibacterial, and antidiabetic effects.

[0042] The term "extract" as used in this invention refers to an active ingredient isolated from a natural product, that is, a substance exhibiting the desired activity.

[0043] In the present invention, the cypress extract may be extracted from one or more parts selected from the group consisting of seeds, fruits, flowers, stems, leaves, roots, and bark, and preferably from leaves. In addition, the cypress leaves may be wild, cultivated, or commercially available without limitation. They may be used as they are, used after pretreatment, used in a pretreated state, or used in powdered form. The pretreatment may be a drying process, and any known drying method may be applied without limitation. Although not limited thereto, it may be, for example, vacuum distillation, hot air drying, freeze-drying, or spray drying.

[0044] In the present invention, the cypress extract may be used without limitation as long as it is a solvent capable of extracting the useful components of the cypress leaves having an anti-inflammatory effect identified in the present invention. Preferably, any one solvent selected from the group consisting of water, C1 to C6 alcohols, and mixtures thereof may be used, and more preferably, ethanol may be used. Even more preferably, 30 to 90% (v / v) ethanol may be used, and even more preferably, 70% (v / v) ethanol may be used, but is not limited thereto.

[0045] In addition, conventional methods in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction, may be used as the extraction method for the cypress extract of the present invention. Although the immersion extraction method was used in the present invention, it is not limited thereto. The method for obtaining the cypress extract of the present invention may include all methods that can be modified by a person with ordinary knowledge in this art field.

[0046] In addition, the cypress leaf extract of the present invention may be obtained by vacuum concentration using vacuum vacuum or vacuum rotary evaporation, but is not limited thereto. Furthermore, after filtration and / or concentration, it may be dried under reduced pressure, vacuum dried, boiling dried, spray dried, or freeze-dried, but is not limited thereto.

[0047] In the present invention, the fermented product may be obtained through the following steps, but is not limited thereto: 1) treating cypress wood with a solvent and extracting for 12 to 48 hours; and 2) inoculating the extract with *Ganoderma lucidum* and fermenting for 12 hours to 4 weeks.

[0048] In the above step 1), the extraction may be performed at 20 to 30°C, but is not limited thereto. Preferably, it may be performed at 20 to 25°C, and more preferably at 21 to 23°C, but is not limited thereto.

[0049] In addition, the above extraction may preferably be performed for 12 to 36 hours, and preferably for 24 hours, but is not limited thereto.

[0050] In addition, in step 2) above, the fermentation may be performed at 100 to 300 rpm at 10 to 40°C, but is not limited thereto. Preferably, it may be performed at 100 to 200 rpm at 20 to 30°C, and more preferably at 180 rpm at 25°C, but is not limited thereto.

[0051] In addition, the above fermentation may preferably be carried out for 1 to 4 weeks, but is not limited thereto. Preferably, it may be carried out for 2 to 4 weeks, and more preferably for 3 weeks, but is not limited thereto.

[0052] In addition, if necessary, filtration and / or concentration and / or drying methods known in the art may be additionally used after the above fermentation. The dried and concentrated extracts and fermented products used in the present invention refer to those dried or concentrated by methods known as above.

[0053] The above filtration process may be carried out using known filtration methods, but is not limited thereto; for example, filtration using filter paper, a filter mesh, or a microfilter, centrifugation, and a separatory funnel may be used. The above concentration process may be carried out using known concentration methods, but is not limited thereto; for example, concentration may be carried out using rotary vacuum, precipitation concentration, evaporation concentration, vacuum concentration, ultrafiltration, reverse osmosis, and centrifugation.

[0054] The above drying process may be carried out by known drying methods, but is not limited thereto; for example, it may be freeze-drying, spray drying, or hot-air drying.

[0055] The present invention has the technical feature of deriving a composition with significant efficacy by changing the composition of unfermented cypress extract through bioconversion via fermentation.

[0056] More specifically, the fermented product obtained by fermenting the cypress extract of the present invention with *Jannabibulrocho* is characterized by an increase in quercitrin content compared to before fermentation. Furthermore, the fermented product obtained by fermenting the cypress extract of the present invention with *Jannabibulrocho* is characterized by an increase in the content of one or more selected from the group consisting of phenol and flavonoid compared to before fermentation. Accordingly, the fermented product obtained by fermenting the cypress extract of the present invention with *Jannabibulrocho* is characterized by possessing an antioxidant effect.

[0057] Through this, it was confirmed that the fermented product obtained by fermenting the cypress extract of the present invention with *Jannabibullocho* alleviates the toxicity of the cypress extract while simultaneously exhibiting excellent antioxidant effects.

[0058] In addition, the fermented product obtained by fermenting the cypress extract of the present invention with the monkey's ear plant is characterized by inhibiting the production of nitric oxide induced by inflammation. Furthermore, the fermented product obtained by fermenting the cypress extract of the present invention with the monkey's ear plant is characterized by inhibiting the expression of one or more mRNAs or proteins selected from the group consisting of iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) induced by inflammation.

[0059] Furthermore, the fermented product obtained by fermenting the Hinoki cypress extract of the present invention with *Ganoderma lucidum* is characterized by inhibiting the expression of one or more factors selected from the group consisting of IL-27, MIP-2, GM-CSF, G-CSF, IL-1β, and IL-6 induced by inflammation. In addition, the fermented product obtained by fermenting the Hinoki cypress extract of the present invention with *Ganoderma lucidum* is characterized by directly inhibiting the STAT signaling pathway, thereby exhibiting a significant anti-inflammatory effect.

[0060] In the present invention, the inflammatory disease may be one or more selected from the group consisting of arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleuritis, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, allergy, atopy, periodontitis, gingivitis, otitis media, pharyngitis, arthritis, rheumatoid arthritis, tendinitis, tenosynovitis, degenerative neuroinflammation, and acute to chronic inflammatory diseases, but is not limited thereto.

[0061] In the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, spread, and recurrence of an inflammatory disease by administering a pharmaceutical composition according to the present invention, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of an inflammatory disease by administering a composition of the present invention. A person skilled in the art to which the present invention pertains would be able to determine the precise criteria for diseases to which the composition of the present invention is effective, and to judge the degree of improvement, enhancement, and treatment, by referring to materials provided by organizations such as the Korean Medical Association.

[0062] In the present invention, the "therapeutically effective amount" used in combination with the active ingredient refers to an amount effective for preventing or treating a certain inflammatory disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, and the patient's condition. Therefore, when used in the human body, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments.

[0063] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally 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 status, the type of inflammatory disease, the cause and severity of the inflammatory disease, the drug's activity, sensitivity to the drug, the method of administration, the time of administration, the route of administration and elimination rate, the duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0064] The pharmaceutical composition of the present invention may include a carrier, a diluent, an excipient, or a combination of two or more of these commonly used in biological preparations. As used in the present invention, the term "pharmaceutical acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and may be used, for example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, by utilizing appropriate methods in this field, it can be preferably formulated according to each disease or component.

[0065] In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, topical preparations, suppositories, sterile injectable solutions, and sprays, but is not limited thereto. The term "administration" as used in the present invention means providing a specific substance to an individual or patient by any appropriate method, and may be administered non-parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally as an injectable formulation) or orally depending on the intended method, and the dosage may vary depending on the patient's body weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease, etc. Liquid formulations for oral administration of the composition of the present invention include suspensions, oral liquids, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. The pharmaceutical composition of the present invention may also be administered by any device capable of delivering the active substance to target cells. Preferred modes of administration and preparations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip infusions, etc. Injectable preparations can be manufactured using aqueous solvents such as physiological saline solution and Ringer's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin), etc., and may include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0066] The term "individual" as used in the present invention refers to any animal including monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, including humans, that has developed or may develop the inflammatory disease, and the diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the individual.

[0067] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0068] The pharmaceutical composition of the present invention may also be provided in the form of an external preparation containing, as an active ingredient, a fermented product obtained by fermenting the above-mentioned cypress extract with *Ganoderma lucidum*. When the pharmaceutical composition of the present invention for the prevention or treatment of inflammatory diseases is used as an external skin preparation, it may additionally contain adjuvants commonly used in the field of dermatology, such as fatty substances, organic solvents, solvents, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, non-ionic emulsifiers, fillers, metal ion chelating agents, chelating agents, preservatives, vitamins, blockers, humectants, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators, or lipid vesicles. Furthermore, said ingredients may be introduced in amounts commonly used in the field of dermatology.

[0069] When the pharmaceutical composition of the present invention for the prevention or treatment of inflammatory diseases is provided as a topical application to the skin, it may be in the form of an ointment, patch, gel, cream, or spray, but is not limited thereto.

[0070] In addition, the pharmaceutical composition of the present invention may further include any compound or natural extract known to have safety and anti-inflammatory effects, in addition to the fermented product obtained by fermenting the cypress extract according to the present invention with *Jannabis bullrocho*, to enhance or reinforce the effects of prevention, improvement, or treatment of inflammatory diseases.

[0071] In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the treatment of inflammatory diseases.

[0072] In addition, the present invention provides an external pharmaceutical composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0073] In the present invention, the term "quasi-drug" refers to an article corresponding to one of the following: fibers, rubber products, or similar items used for the purpose of treating, alleviating, managing, or preventing diseases in humans or animals; items similar thereto that are not instruments or machines and have a weak or non-direct effect on the human body; or preparations used for sterilization, insecticidal, and similar purposes to prevent infection. It refers to articles used for the purpose of diagnosing, treating, alleviating, managing, or preventing diseases in humans or animals, excluding those that are not instruments, machines, or devices; and articles used for the purpose of exerting pharmacological effects on the structure and function of humans or animals, excluding those that are not instruments, machines, or devices; and also includes external skin preparations and personal hygiene products.

[0074] When a fermented product obtained by fermenting the cypress extract of the present invention with *Ganoderma lucidum* is included in a quasi-drug for the purpose of preventing or improving inflammatory diseases, the fermented product obtained by fermenting the cypress extract with *Ganoderma lucidum* may be used as is, or it may be used together with other quasi-drug ingredients, or it may be used appropriately according to conventional methods. The mixing amount of the active ingredients may be appropriately determined according to the purpose of use.

[0075] The quasi-drug of the present invention is not particularly limited thereto, but may be manufactured and used in the form of, for example, a cream, lotion, aerosol, shampoo, gel, or pack.

[0076] In the case of creams, ointments, shampoos, gels, or packs, bases such as white petroleum jelly, yellow petroleum jelly, lanolin, bleached beeswax, cetanol, stearyl alcohol, stearic acid, hydrogenated oil, gelled hydrocarbon, polyethylene glycol, liquid paraffin, squalane, etc.; solvents and solubilizing agents such as oleic acid, isopropyl myristate, triisooctanoglycerin, crotamiton, diethyl sebacate, diisopropyl adipoyl adipose, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, vegetable oils, etc.; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, etc.; preservatives such as parahydroxybenzoic acid esters, etc.; moisturizers such as glycerin, propylene glycol, sodium hyaluronate, etc.; Surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, and lecithin; and thickeners such as carboxyvinyl polymers, xanthan gum, carboxymethylcellulose, sodium carboxymethylcellulose salts, hydroxypropylcellulose, and hydroxypropylmethylcellulose.

[0077] In the case of aerosol preparations, bases such as white petroleum jelly, yellow petroleum jelly, lanolin, bleached beeswax, cetanol, stearyl alcohol, stearic acid, hydrogenated oil, gelled hydrocarbon, polyethylene glycol, liquid paraffin, squalane, etc., used in the formulation of ointments, creams, gels, suspensions, emulsions, liquids, and lotions; solvents and solubilizing agents such as oleic acid, isopropyl myristate, diisopropyl adipice, isopropyl sebacate, triisooctanoglycerin, crotamiton, diethyl sebacate, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, and vegetable oils; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylhydroxyanisole; and preservatives such as parahydroxybenzoic acid esters. Humidifiers such as glycerin, propylene glycol, and sodium hyaluronate; surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, and lecithin; thickeners such as carboxyvinyl polymers, xanthan gum, carboxymethylcellulose, sodium carboxymethylcellulose salts, hydroxypropylcellulose, and hydroxypropylmethylcellulose; additionally, various stabilizers, buffers, binders, suspending agents, emulsifiers, fragrances, preservatives, solubilizing agents, and other suitable additives may be incorporated. In addition, stabilizers, preservatives, absorption promoters, pH adjusters, and other suitable additives may be incorporated as needed.

[0078] In the quasi-drug of the present invention, a more specific description is the same as the description of the pharmaceutical composition above.

[0079] In addition, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0080] The ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the fermented product obtained by fermenting a cypress extract with *Jannabis bulrocho* as an active ingredient, and may include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances. The cosmetic composition of the present invention may be prepared in any formulation commonly manufactured in the art, such as emulsions, creams, lotions, packs, foundations, lotions, beauty serums, hair cosmetics, etc.

[0081] Specifically, the cosmetic composition of the present invention includes formulations of skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser.

[0082] In the case where the formulation of the present invention is a paste, cream, or gel, animal fibers, plant fibers, wax, paraffin, starch, tracanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as carrier components.

[0083] In the case where the formulation of the present invention 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.

[0084] In the case where the formulation of the present invention is a solution or emulsion, a solvent, a solvating 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.

[0085] In the case where the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used as carrier components.

[0086] In the case where the formulation of the present invention is a cleansing agent 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, linolenic acid derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0087] The cosmetic composition of the present invention may contain, in addition to the active ingredient, one or more ingredients that help improve skin condition, including an ingredient that exhibits the same or similar anti-inflammatory activity. The above ingredients are hyaluronic acid, butylene glycol, glycerin, amino acid, trehalose, kojic acid and its derivatives, arbutin, ascorbic acid and its derivatives, hydroquinone and its derivatives, resorcinol, 2,7-dinitroindazole, adenosine, retinol, retinyl palmitate, polyethoxylated retinamide, yeast, dipeptide, palmitoyl oligopeptide & palmitoyl tripeptide-7, acetyl Examples include acetyl hexapeptide, epidermal growth factor (EGF), or plant extracts such as tangerine, rice, licorice, shea butter, aloe vera, coconut, olive, and avocado, but are not limited thereto.

[0088] Additionally, the cosmetic composition of the present invention may be provided in a pack. The pack is preferably manufactured in any one formulation selected from a mask pack, a sleeping pack, a cleansing pack, a wash-off pack, and a peel-off pack, but is not limited thereto.

[0089] In addition to the fermented product obtained by fermenting the cypress extract according to the present invention with *Jannabis bulrocho*, the cosmetic composition of the present invention may additionally include any compound or natural extract known to have an anti-inflammatory effect and whose safety has already been verified, in order to enhance and reinforce the anti-inflammatory effect.

[0090] In addition, the cosmetic composition for the prevention or improvement of inflammatory diseases according to the present invention may be used for pets by varying its formulation. For example, it may be manufactured in various forms such as solutions, sol-gels, emulsions, oils, waxes, aerosols, etc., such as pet shampoos and pet rinses, and may be manufactured by adding a neutral detergent that is less irritating to the pet's skin and has excellent moisturizing properties.

[0091] In addition, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0092] In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0093] The food composition according to the present invention includes all forms such as functional food, nutritional supplement, health food, health supplement, and food additives. The food composition of the above types may be formulated in any one form selected from the group consisting of powder, tablet, capsule, pill, and liquid according to conventional methods known in the art, but is not limited thereto. It may be manufactured in various forms using methods known in the art.

[0094] For example, as a health food, the fermented product obtained by fermenting the cypress extract according to the present invention with *Jannabis bullhocho* itself can be granulated, encapsulated, or powdered for consumption, or manufactured into the form of tea, juice, or drink for consumption. In addition, the fermented product obtained by fermenting the cypress extract according to the present invention with *Jannabis bullhocho* can be prepared in the form of a composition by mixing it with a known substance or active ingredient known to have preventive, inhibitory, or therapeutic effects on inflammatory diseases.

[0095] In addition, functional foods can be prepared by adding a fermented product obtained by fermenting the cypress extract of the present invention with *Jannabibullocho* to beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and its processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, malt syrup, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., soybean paste, soy sauce, sauces, etc.).

[0096] In addition, the food composition of the present invention may include conventional food additives, and unless otherwise specified, suitability as a "food additive" is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety. Examples of items listed in the "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0097] In the food composition of the present invention, the fermented product obtained by fermenting the cypress extract with *Jannabis bullocho* may preferably be included in an amount of 0.00001 to 50 weight% relative to the food composition. If the content is less than 0.00001 weight%, the effect is negligible, and if it exceeds 50 weight%, the increase in effect relative to the amount used is insignificant and therefore uneconomical.

[0098] In addition, in order to use the fermented product obtained by fermenting the cypress extract of the present invention with the monkey's ear ginseng as a food additive, it can be manufactured and used in the form of tablets, capsules, powder, granules, liquid, pills, etc.

[0099] When the composition of the present invention is prepared as a beverage, it may include various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g, per 100 ml of the composition of the present invention.

[0100] In addition to the above, the composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, 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, the composition of the present invention may include fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0101] In the present invention, “health supplement” or “health functional food” means a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and “functional properties” means consuming for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0102] In addition, the present invention provides an anti-inflammatory composition comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0103] The above anti-inflammatory composition may be one or more selected from the group consisting of pharmaceutical compositions, cosmetic compositions, and food compositions.

[0104] In the present invention, "anti-inflammatory" may be used interchangeably with "inhibition or improvement of inflammation" and may refer to any action that alleviates an inflammatory response.

[0105] In addition, the present invention provides a method for producing a fermented cypress leaf product fermented with *Jannabibulrocho*.

[0106] The above method may comprise: 1) a step of treating cypress wood with a solvent and extracting at 20 to 30°C for 12 to 48 hours; and 2) a step of inoculating the extract with *Jannabibullocho* and fermenting at 100 to 300 rpm for 12 hours to one month.

[0107] In the present invention, the cypress extract can be used without limitation as long as it is a solvent capable of extracting useful components of cypress leaves. Preferably, any one solvent selected from the group consisting of water, C1 to C6 alcohols, and mixtures thereof may be used, and more preferably, ethanol may be used. Even more preferably, 30 to 90% (v / v) ethanol may be used, and even more preferably, 70% (v / v) ethanol may be used, but is not limited thereto.

[0108] In the above step 1), the extraction may be performed at 20 to 30°C, but is not limited thereto. Preferably, it may be performed at 20 to 25°C, and more preferably at 21 to 23°C, but is not limited thereto.

[0109] In addition, the above extraction may preferably be performed for 12 to 36 hours, and preferably for 24 hours, but is not limited thereto.

[0110] In addition, in step 2) above, the fermentation may be performed at 100 to 300 rpm at 10 to 40°C, but is not limited thereto. Preferably, it may be performed at 100 to 200 rpm at 20 to 30°C, and more preferably at 180 rpm at 25°C, but is not limited thereto.

[0111] In addition, the above fermentation may preferably be carried out for 1 to 4 weeks, but is not limited thereto. Preferably, it may be carried out for 2 to 4 weeks, and more preferably for 3 weeks, but is not limited thereto.

[0112] In addition, if necessary, after Step 1 or Step 2 above, filtration and / or concentration and / or drying methods known in the art may be additionally used. The dried and concentrated extracts and fermented products used in the present invention refer to those dried or concentrated by methods known as above.

[0113] The above filtration process may be carried out using known filtration methods, but is not limited thereto; for example, filtration using filter paper, a filter mesh, or a microfilter, centrifugation, and a separatory funnel may be used. The above concentration process may be carried out using known concentration methods, but is not limited thereto; for example, concentration may be carried out using rotary vacuum, precipitation concentration, evaporation concentration, vacuum concentration, ultrafiltration, reverse osmosis, and centrifugation.

[0114] The above drying process may be carried out by known drying methods, but is not limited thereto; for example, it may be freeze-drying, spray drying, or hot-air drying.

[0115] The manufacturing method of the present invention can provide a fermented cypress leaf product fermented with *Jannabibullocho*, which possesses significant efficacy such as antioxidant and anti-inflammatory properties, by changing the composition of an unfermented cypress extract through bioconversion via fermentation.

[0116] In addition, the present invention provides a method for preventing or treating inflammatory diseases, comprising the step of administering to an individual a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

[0117] The present invention also provides a method for preventing, alleviating, or treating inflammation, comprising the step of administering to an individual a fermented product obtained by fermenting a Chamaecyparis obtusa (Siebold & Zucc.) Endl. extract with Ganoderma applanatum.

[0118] In addition, the present invention provides a method for increasing the antioxidant capacity of an individual, comprising the step of administering to the individual a fermented product obtained by fermenting a Chamaecyparis obtusa (Siebold & Zucc.) Endl. extract with Ganoderma applanatum.

[0119] The above "individual" may be administered to an "individual" that requires prevention of inflammation or inflammatory disease, has developed or is likely to develop inflammation or inflammatory disease, or requires an increase in antioxidant capacity, and the above "individual" may refer to any animal, including humans.

[0120]

[0121] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w)% for solid / solid, (w / v)% for solid / liquid, and (v / v)% for liquid / liquid, unless otherwise noted.

[0122] The terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Therefore, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as “comprising” a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0123] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0124] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalent scope interpreted therefrom.

[0125]

[0126] Example 1. Extraction of 70COL and 70COLGA

[0127] Chamaecyparis obtusa (Siebold & Zucc.) Endl. (CO) leaves, COs, were collected in May 2022 in Chukdong-myeon, Sacheon-si, Gyeongsangnam-do, South Korea. A 10-fold excess of 70% ethanol was added to the COs, and extraction was performed at room temperature for 24 hours. This process was repeated twice to prepare the CO leaf 70% ethanol extract (70 COs). The 70 COs was passed through a paper filter (Whatman™ Quantitative Filter Paper; Toyo Kaisha, Tokyo, Japan) and then concentrated using a vacuum rotary evaporator (HS-10SP; Hahnshin S&T, Gimpo, Republic of Korea) at 50 rpm and 40°C. The concentrated extract was freeze-dried using a freeze dryer (Freeze Dryer with Micro Concentrator MCFD; ilShin Biobase, Dongducheon-si, Gyeonggi-do, Republic of Korea).

[0128] To prepare a 70% EtOH extract (70COLGA) inoculated with mycelium, Ganoderma applanatum (G. applanatum) mycelia (GAM) was cultured in PDB medium for 14 days. The GAM was inoculated into a 70COL solution concentrated to 10% and fermented at 25°C at 180 rpm for 3 weeks. The extract was filtered through a paper filter, freeze-dried using a freeze dryer, and stored at 4°C before use.

[0129]

[0130] Experimental Example 1. Analysis Method

[0131] 1-1. Reagents and Antibodies

[0132] DPPH (2,2-Diphenyl-1-picrylhydrazyl, D9132), ABTS (2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 11557), potassium persulfate (216224), L-ascorbic acid (A0278), tannic acid (403040), quercetin (Q4591), LPS (lipopolysaccharide, L3012), BHA (butylated hydroxyanisole, B1253), and Griess reagent (G4410) were purchased from Sigma Aldrich (St. Louis, MO, USA). Folin-Ciocalteu's reagent (96703S8130) and sodium hydroxide (2020K1192) were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). Sodium carbonate anhydrous (7541-4405) was purchased from Daejung Chemicals & Metals Co., Ltd. (Siheung-si, Games-do, Republic of Korea). Diethylene glycol was purchased from Samchun Chemical (Seoul, Republic of Korea), and MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) reagent (M1415) was purchased from Duchefa Biochemie (Haarlem, The Netherlands).

[0133] In addition, the Anti-COX-2 antibody (#12282), Anti-pY-STAT1 antibody (#8826), Anti-STAT1 antibody (#9172), Anti-pY-STAT3 antibody (#9145), Anti-STAT3 antibody (#30835), Anti-pT / Y-p44 / 42 MAPK antibody (Erk1 / 2) (#9101), Anti-p44 / 42 MAPK antibody (Erk1 / 2) (#4695), Anti-pT / Y-SAPK / JNK antibody (#9251), Anti-SAPK / JNK antibody (#9252), Anti-pT / Y-p38 MAPK antibody (#9211), Anti-p38 MAPK antibody (#9212), Anti-pS-IB antibody (#2859), and Anti-pS-NFB antibody (#3033) are Cell Signaling Technology It was purchased from Danvers, MA, USA. The anti-IB antibody (sc-1643), anti-NFB antibody (sc-8008), and anti-actin antibody (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The HRP (horseradish peroxidase)-tagged anti-rabbit antibody (A21010) was purchased from Abbkine (Wuhan, China). The HRP-tagged anti-mouse antibody (ADI-SAB-100) was purchased from Enzo Life Science (Farmingdale, NY, USA).

[0134]

[0135] 1-2. High-Performance Liquid Chromatography (HPLC) Fingerprint Analysis

[0136] A sample solution was prepared by dissolving 10 mg of the extract in 1 mL of MeOH solvent. The supernatant was then filtered through a 0.22 μm syringe filter. Chemical fingerprint analysis of the extract was performed using an Agilent HPLC 1260 (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was performed using a gilent Eclipse XDB-C18 (4.6 × 250 mm, 5 μm) analytical column, and HPLC analysis was performed under the conditions shown in Table 1 below.

[0137]

[0138]

[0139] 1-3. Analysis of Total Polyphenolic Compound Content

[0140] 50 μL of the extract was mixed with 50 μL of Folin-Ciocalteu reagent. The mixture was reacted at room temperature for 5 minutes, stored in the dark, and then 50 μL of 10% Na2CO3 was added. The reaction mixture was incubated at room temperature for 1 hour. Absorbance was measured at 640 nm using a plate reader (BioTek, Winooski, VT, USA), and the data were expressed as milligrams of tannic acid equivalents (mg TAE / g) per 1 g of sample.

[0141]

[0142] 1-4. Analysis of Total Flavonoid Compound Content

[0143] The extract, diethylene glycol, and sodium hydroxide were mixed in a ratio of 1:10:10. After reacting at 37°C for 1 hour, the absorbance was measured at 420 nm using a plate reader (BioTek). The data were expressed as milligrams of quercetin equivalent per 1 g of sample (mg QE / g).

[0144]

[0145] 1-5. DPPH Radical Scavenging Activity Analysis

[0146] 50 μL of 0.2 mM DPPH solution in 99% EtOH was mixed with 100 μL of extracts at concentrations of 50, 100, 200, and 400 μg / mL. The mixture was then incubated in the dark at room temperature for 30 minutes. Absorbance was measured at 517 nm using a plate reader (BioTek). BHA (Butylated hydroxyanisole) was used as a positive control.

[0147]

[0148] 1-6. ABTS + Radical Scavenging Activity Analysis

[0149] 7.4 mM ABTS and 2.6 mM potassium persulfate were mixed in a 1:1 ratio and reacted for 24 hours in a dark place at room temperature. Before use, ABTS + The solution was diluted with 99% EtOH to obtain an absorbance value of approximately 0.7. Diluted ABTS + The solution and extracts at concentrations of 50, 100, 200, and 400 μg / mL were mixed in a 1:1 ratio and reacted at room temperature in a dark place for 1 minute. Absorbance was measured at 734 nm using a plate reader (BioTek). Ascorbic acid (AA) was used as a positive control.

[0150]

[0151] 1-7. Cell Lines and Culture

[0152] Mouse macrophage cell line RAW264.7 was purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, Capricorn Scientific GmbH, Ebsdorfergrund, Germany) containing 10% heat-inactivated FBS (fetal bovine serum, Capricorn Scientific GmbH) and 1% penicillin / streptomycin (Capricorn Scientific GmbH). Cells were cultured at 37°C in a humidified incubator (PHCbi CO2Incubator, Tokyo, Japan) with 5% CO2 and subcultured every 2 to 3 days.

[0153]

[0154] 1-8. Analysis of Cell Viability

[0155] Cell viability was confirmed via the MTT assay. First, RAW264.7 cells were seeded into a 96-well culture plate and treated with extracts at concentrations of 25, 50, 100, 200, 400, and 800 μg / mL for 24 hours. After treatment, the cells were treated with MTT reagent (5 mg / mL) and incubated for 2 hours. The resulting formazan crystals were dissolved in 100 μL of DMSO, and the absorbance was measured at 570 nm using a plate reader (BioTek).

[0156]

[0157] 1-9. NO (Nitric Oxide) Analysis

[0158] NO levels in the cell culture medium supernatant were measured using Griess reagent. First, RAW264.7 cells were seeded into 6-well culture plates and cultured overnight. After pretreatment with extracts at concentrations of 100 and 200 μg / mL for 2 hours, LPS (200 ng / mL) was added for 16 hours. To measure NO production, 100 μL of the supernatant was reacted with 100 μL of Griess reagent in the dark for 10 minutes. Absorbance was measured at 540 nm using a plate reader (BioTek).

[0159]

[0160] 1-10. Western Blot

[0161] Cells were washed with cold PBS (phosphate-buffered saline), and proteins were extracted using 0.5% Triton X-100 buffer (Sigma Aldrich) containing protease and phosphatase inhibitors (2 mM PMSF (phenylmethanesulfonyl fluoride); 1 mM NaF (sodium fluoride); 2 mM EDTA (ethylenediaminetetraacetic acid); 0.5 mM Na3VO4 (sodium orthovanadate); and 10 μg / mL leupeptin. After incubation on ice for 10 minutes, the lysate was centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was collected. The lysate was separated from an SDS-polyacrylamide gel and transferred to a nitrocellulose (NC) membrane. The membrane was 5% The membranes were immersed in skim milk and blocked at room temperature for 1 hour, then incubated overnight at 4°C with a specific primary antibody (diluted 1:1000). The following day, the membranes were incubated with an HRP-conjugated secondary antibody at room temperature for 1 hour. Subsequently, the membranes were incubated with ECL solution (Bio-Rad, Hercules, CA, USA) for 1 minute. Images were obtained using a Davinch-Chemi Imager™ CAS-400SM (Davinch-K, Seoul, Republic of Korea).

[0162]

[0163] 1-11. RNA Extraction and qPCR (Quantitative Real-Time PCR)

[0164] Total RNA was extracted using RNAiso Plus reagent (Takara, Kusatsu, Shiga, Japan), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer's instructions. qPCR was performed using SYBR Green qPCR Master Mix (Applied Biological Materials, Richmond, BC, Canada), and amplification was achieved using the LightCycler® 96 System (Roche, Basel, Switzerland). PCR was performed under the following conditions: denaturation at 95°C for 15 seconds, followed by 40 cycles of annealing / extension at 60°C for 1 minute. Relative gene expression levels were normalized according to GAPDH expression levels. The sequences of the primers used for qPCR are shown in Table 2 below. The sequences of the primers are denoted as SEQ ID NOs 1 to 12, respectively.

[0165]

[0166]

[0167] 1-12. Cytokine Array

[0168] RAW264.7 cells were pretreated with the extract for 2 hours and treated with LPS (200 ng / mL) for 4 hours. The culture supernatant was collected, and cytokines were measured using the Mouse Cytokine Array Panel kit according to the manufacturer's instructions (ARY006; R&D systems, Minneapolis, MN, USA). Images were acquired using the Davinch-Chemi Imager™ CAS-400SM (Davinch-K), and cytokine blots were quantified using Image J (bundled with 64-bit Java 8).

[0169]

[0170] 1-13. Conditioning Medium of LPS-Induced Macrophages and Heat-Inactivated CM

[0171] RAW264.7 cells were activated by treating with LPS (200 ng / mL) for 4 hours. After washing the LPS-treated medium with PBS, it was replaced with fresh medium and cultured for another 4 hours. Heat-inactivated CM was synthesized by boiling LPS-CM at 100°C for 20 minutes to degrade all secreted cytokines and chemokines, and was used as a negative control.

[0172]

[0173] 1-14. Dataset Analysis

[0174] The dataset provided under access number GSE76563 in the Gene Expression Omnibus (GEO) database (http: / www.ncbi.nlm.nih.gov / geo / (accessed December 5, 2023)) was used. Z-scores for each gene of interest were calculated and displayed using heatmaps created with Microsoft Excel software (Microsoft Office 365, version 2420, Redmond, WA, USA).

[0175]

[0176] 1-15. Statistical Analysis

[0177] Statistical analysis of all data was performed using Microsoft Excel software (Microsoft Office 365, version 2420). All experiments were repeated independently at least three times, and a p-value of less than 0.05 was considered significant.

[0178]

[0179] Example 2. HPLC analysis and antioxidant activity of 70COL and 70COLGA

[0180] It has been reported that the major flavonoids of CO are quercitrin, amentoflavone, and myricetin. Accordingly, HPLC fingerprint analysis results, as shown in Figure 1, revealed that 70COL had a high amentoflavone content (70COL: 26.66%, 70COLGA: 8.84%), and 70COLGA had a high quercitrin content (70COL: 15.08%, 70COLGA: 20.52%). This confirmed that the composition changed through bioconversion. Additionally, as shown in Figure 2, it was confirmed that the total phenol content and total flavonoid content also changed. 70COLGA had higher total phenol and flavonoid content than 70COL.

[0181] Since polyphenols are closely related to antioxidant activity, DPPH analysis and ABTS + Antioxidant activity was confirmed by performing an analysis. As a result, as shown in Figure 3, 70COLGA exhibited significantly superior antioxidant activity compared to 70COL at low concentrations. Since antioxidant activity is associated with anti-inflammatory effects, it was expected that 70COLGA, which has superior antioxidant activity at low concentrations, would have a greater anti-inflammatory effect.

[0182]

[0183] Example 3. Inhibitory effect of 70COLGA on LPS-induced inflammation in RAW264.7 cells

[0184] Prior to confirming anti-inflammatory activity, an MTT assay was performed to evaluate the toxicity of 70COL and 70COLGA. As shown in Figure 4, 70COL exhibited a cell viability of <80% at a concentration of 25 μg / mL, whereas 70COLGA showed a cell viability of nearly 100% even at a maximum concentration of 200 μg / mL. Therefore, it was confirmed that biological conversion to 70COLGA reduces the cytotoxicity of 70COL. Since a cell viability of <80% is considered to indicate cytotoxicity, 70COL was excluded from subsequent experiments. Furthermore, when 70COLGA was treated to RAW264.7 cells with LPS-induced inflammation, it was confirmed that 70COLGA inhibited morphological changes caused by inflammation, as shown in Figure 5. In addition, as shown in Fig. 6, 70COLGA reduced the production of LPS-induced NO, iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2), and significantly inhibited the expression of iNOS and COX-2 mRNA and protein. Therefore, it was confirmed that 70COLGA according to the present invention possesses an anti-inflammatory effect.

[0185]

[0186] Example 4. Inhibitory effect of 70COLGA on inflammatory cytokine production and STAT activation in LPS-induced inflammation of RAW264.7 cells

[0187] LPS-induced macrophage stimulation is known to regulate the expression of inflammatory mediators and inflammatory cytokines through MAPK (mitogen-activated protein kinase), NF-κB (nuclear factor-kappa B) / IκBα (NF-kappa-B inhibitor alpha), and JAK / STAT (Janus kinase-signal transducer and activator transcription factor) signaling.

[0188] As 70COLGA inhibited LPS-induced inflammation, the mechanism of its anti-inflammatory action was confirmed by investigating the MAPK and NF-κB / IκBα signaling pathways. As a result, as shown in Figures 7(a) and 7(b), 70COGLA failed to prevent the activation of LPS-induced MAPK and NF-κB / IκBα signaling, but as shown in Figure 7(c), it significantly inhibited the activation of STAT1 and STAT3. When macrophages are activated by harmful substances, they secrete inflammatory cytokines, including IL-1β (interleukin-1β), IL-6, and TNF-α (tumor necrosis factor-α), during phagocytosis. To investigate the anti-inflammatory effects of 70COLGA in more detail, LPS-induced IL-1β, IL-6, and TNF-α mRNA expression was examined. As a result, as shown in Figures 7(d) to 7(f), 70COLGA inhibited LPS-induced IL-1β and IL-6 mRNA expression but did not inhibit TNF-α. Through this, it was confirmed that 70COLGA inhibits LPS-induced STAT activation and the expression of inflammatory cytokines IL-1β and IL-6.

[0189]

[0190] Example 5. Inhibitory effect of 70COLGA on inflammatory molecules in LPS-induced inflammation of RAW264.7 cells

[0191] Next, we investigated whether 70COLGA exhibits anti-inflammatory effects by inhibiting inflammatory molecules other than IL-1β and IL-6. First, using the GSE76562 database, we identified upregulated genes in LPS-induced RAW264.7 cells, resulting in the identification of 27 genes as shown in Fig. 8(a). Furthermore, cytokine array analysis revealed that, as shown in Figs. 8(b) to 8(d), the expression of IL-27, MIP-2 (macrophage inflammatory protein-2), GM-CSF (granulocyte-macrophage colony-stimulating factor), and G-CSF (granulocyte colony-stimulating factor) was increased by LPS and decreased by 70COLGA. GM-CSF is a well-known inflammatory cytokine that regulates cytokine responses by increasing the number and activation of immune cells. In addition, IL-27 has been reported to bind to the IL-27 receptor to activate JAK-STAT signaling in macrophages, and to upregulate MIP-2 in the skin of patients with psoriasis, a chronic inflammatory skin disease. Therefore, 70COLGA was confirmed to exhibit anti-inflammatory effects by inhibiting inflammatory molecules such as IL-27, MIP-2, GM-CSF, G-CSF, as well as IL-1β and IL-6.

[0192]

[0193] Example 6. Direct inhibitory effect of 70COLGA on STAT activation in LPS-induced inflammation of RAW264.7 cells

[0194] Since inflammatory cytokines induce STAT activation, we investigated whether 70COLGA directly inhibits STAT activation rather than suppressing the expression of inflammatory cytokines. First, as shown in Fig. 9(a), an equal amount of pro-inflammatory molecule medium was collected as well as LPS-conditioned medium (LPS-CM) obtained from LPS-induced RAW264.7 cells and treated pure RAW264.7 cells. At this time, heat-inactivated LPS-CM was used as a negative control. As a result, as shown in Fig. 9(b), when RAW264.7 cells were treated with LPS-CM, STAT was activated within 30 minutes, but this effect was inhibited by 70COLGA. Therefore, through this, it was confirmed that 70COLGA exhibits a significantly superior anti-inflammatory effect by not only suppressing the expression of inflammatory molecules but also directly inhibiting STAT activation in LPS-induced RAW264.7 cells.

[0195]

[0196] As described above, specific embodiments of the present invention have been described in detail; however, those skilled in the art who understand the spirit of the present invention will be able to easily propose other inventions that are inferior or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the same spirit. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included within the scope of the present invention.

Claims

1. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

2. In Paragraph 1, A pharmaceutical composition in which the above-mentioned cypress is a cypress leaf.

3. In Paragraph 1, A pharmaceutical composition wherein the above cypress extract is extracted with any one solvent selected from the group consisting of water, C1 to C6 alcohols, and mixtures thereof.

4. In Paragraph 1, A pharmaceutical composition in which the above-mentioned fermented product is obtained through the following steps: Step 1) A step of treating cypress wood with a solvent and extracting for 12 to 48 hours; and Step 2) Inoculating the above extract with *Jannabibullocho* and fermenting for 12 hours to 4 weeks.

5. In Paragraph 1, A pharmaceutical composition characterized by the above-mentioned fermented product having an increased quercitrin content compared to before fermentation.

6. In Paragraph 1, A pharmaceutical composition characterized by the above-mentioned fermented product having an increased content of one or more selected from the group consisting of phenol and flavonoid compared to before fermentation.

7. In Paragraph 1, A pharmaceutical composition characterized by the above-mentioned fermented product having an antioxidant effect.

8. In Paragraph 1, A pharmaceutical composition characterized by the above-mentioned fermented product inhibiting the production of nitric oxide induced by inflammation.

9. In Paragraph 1, A pharmaceutical composition characterized by the above fermented product inhibiting the expression of one or more mRNAs or proteins selected from the group consisting of iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) induced by inflammation.

10. In Paragraph 1, A pharmaceutical composition characterized by the above fermented product inhibiting the expression of one or more factors selected from the group consisting of IL-27, MIP-2, GM-CSF, G-CSF, IL-1β, and IL-6 induced by inflammation.

11. In Paragraph 1, A pharmaceutical composition characterized by the above-mentioned fermented product inhibiting the STAT signaling pathway induced by inflammation.

12. In Paragraph 1, A pharmaceutical composition wherein the above-mentioned inflammatory disease is one or more selected from the group consisting of arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleuritis, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, allergy, atopy, periodontitis, gingivitis, otitis media, pharyngitis, arthritis, rheumatoid arthritis, tendinitis, tenosynovitis, degenerative neuroinflammation, and acute to chronic inflammatory diseases.

13. An external pharmaceutical composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

14. A cosmetic composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

15. A food composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

16. A health functional food composition for preventing or improving inflammatory diseases, comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum.

17. An anti-inflammatory composition comprising a fermented product obtained by fermenting an extract of Chamaecyparis obtusa (Siebold & Zucc.) Endl. with Ganoderma applanatum. 18.1 Step) treating the cypress wood with a solvent and extracting at 20 to 30°C for 12 to 48 hours; and Step 2) Inoculating the above extract with *Ganoderma lucidum* and fermenting at 100 to 300 rpm for 12 hours to 4 weeks; comprising a method for preparing a fermented cypress leaf product fermented with *Ganoderma lucidum*.

19. In Paragraph 18, A method of manufacturing in which the above cypress wood solvent is one or more selected from the group consisting of water, C1 to C6 alcohols and mixtures thereof.

20. A method for preventing or treating inflammatory diseases, comprising the step of administering to an individual a fermented product obtained by fermenting a Chamaecyparis obtusa (Siebold & Zucc.) Endl. extract with Ganoderma applanatum.

21. A method for preventing, alleviating, or treating inflammation, comprising the step of administering to an individual a fermented product obtained by fermenting a Chamaecyparis obtusa (Siebold & Zucc.) Endl. extract with Ganoderma applanatum.

22. A method for increasing the antioxidant capacity of an individual, comprising the step of administering to the individual a fermented product obtained by fermenting a Chamaecyparis obtusa (Siebold & Zucc.) Endl. extract with Ganoderma applanatum.

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