KR20240073365A - Composition for improving inflammation or endotoxemia comprising cherry tree extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for improving inflammation or endotoxemia containing cherry tree extract as an active ingredient, which not only exhibits an improving effect on inflammation or endotoxemia by binding to the TLR4/MD2 complex, but also uses natural materials. It relates to a composition for improving inflammation or endotoxemia that can reduce side effects in the human body.

Description

Composition for improving inflammation or endotoxemia comprising cherry tree extract as an active ingredient}

The present invention relates to a composition for improving inflammation or endotoxemia containing cherry tree extract as an active ingredient. More specifically, it relates to a composition for improving inflammation and endotoxemia that can not only show an improvement effect on inflammation and endotoxemia by binding to the TLR4/MD2 complex, but also reduce side effects in the human body using natural materials. .

Inflammation is the body's main protective response, which involves activation of immune system processes. The inflammatory response is a highly regulated, self-limiting process aimed at identifying and destroying invading pathogens and restoring normal tissue structure and function (Conti et al. 2004, Freire and Van Dyke 2013).

However, excessive inflammatory responses are recognized as a major cause of chronic inflammation, such as cardiovascular disease, rheumatoid arthritis, inflammatory bowel disease, Alzheimer's disease, and even cancer (Amin et al., 1999, Freire and Van Dyke 2013).

Specifically, when macrophages are excessively activated by inflammatory stimulants, including the Gram-negative bacterial endotoxin lipopolysaccharides (LPS), the cells produce nitric oxide (NO) and prostaglandin E2 (PGE). ₂ ) production of inflammatory mediators and several signaling pathways such as nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling. It induces the production of inflammatory cytokines along with its activation, and excessive production of the above inflammatory mediators and cytokines is known to cause the pathogenesis of many inflammatory diseases (McDaniel et al., 1996, Muralidharan and Mandrekar, 2013) .

Meanwhile, another important factor in inflammation is the production of reactive oxygen species (ROS) and oxidative stress (Brune et al. 2013, Mills and O'Neill 2016). ROS overproduced by activated macrophages play an important role in the generation of inflammation and are known to be involved in the production of inflammatory mediators in LPS-stimulated macrophages (Haddad and Land 2002).

As a result, inhibiting the production of inflammatory factors by blocking macrophage activity could be used as a potential therapeutic approach to alleviate the progression of inflammatory and oxidative disorders.

Meanwhile, existing anti-inflammatory drugs that can treat these inflammatory diseases are largely divided into steroidal and non-steroidal anti-inflammatory drugs, and most synthetic anti-inflammatory drugs have problems with side effects in the body. Accordingly, there is a need to develop new anti-inflammatory agents derived from natural products that are highly effective in treating inflammation and have no side effects.

Accordingly, the present applicant has confirmed the mechanism by which an active ingredient derived from the natural product cherry tree binds to the TLR4/MD2 complex, and intends to use this mechanism as a composition that exhibits an anti-inflammatory effect and an improvement effect on endotoxemia.

KR 10-2127282 B1

The purpose of the present invention is to provide a composition for improving inflammation and endotoxemia containing cherry tree extract as an active ingredient.

Another object of the present invention is to provide a composition that binds to the TLR4/MD2 complex and exhibits an improvement effect on inflammation and endotoxemia.

Another object of the present invention is to provide a food composition containing the above composition.

Another object of the present invention is to provide a pharmaceutical composition containing the above composition.

In order to achieve the above object, a composition for improving inflammation according to an embodiment of the present invention contains cherry tree extract as an active ingredient.

The composition is TLR4/MD2 It binds to the complex and shows an improvement effect on inflammation.

The composition is excellent in downregulating iNOS and COX-2 expression and inhibiting the release of NO and PGE ₂ .

A composition for improving endotoxemia according to another embodiment of the present invention contains cherry tree extract as an active ingredient.

The composition exhibits an improving effect on endotoxemia by binding to the TLR4/MD2 complex.

A food composition for improving inflammation or endotoxemia according to another embodiment of the present invention includes the composition.

A pharmaceutical composition for improving inflammation or endotoxemia according to another embodiment of the present invention includes the composition.

Hereinafter, the present invention will be described in more detail.

The term 'extract' used in this specification has a meaning commonly used in the art as a crude extract, but in a broad sense also includes fractions obtained by additional fractionation of the extract. In other words, plant extracts include not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process. For example, fractions obtained by passing the extract through an ultrafiltration membrane with a certain molecular weight cut-off value, separation by various chromatographs (designed for separation according to size, charge, hydrophobicity, or affinity), etc. Fractions obtained through various purification methods are also included in the extract of the present invention.

The extract used in the present invention can be prepared in powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying.

As used herein, the term “prophylaxis” refers to blocking the appearance of a disease or symptoms associated with a disease in an asymptomatic subject at risk of developing the disease, for example, by exposure to the cause of the disease.

The term “treatment” used in the present invention refers to any action that improves or beneficially changes the symptoms of a specific disease by administering the composition of the present invention.

As used herein, the term “improvement” refers to any action that reduces at least the severity of a parameter, such as a symptom, related to the condition being treated.

As used herein, the term "comprising an active ingredient" refers to an amount sufficient to effect a desired biological effect, such as beneficial results, including but not limited to preventing, reducing, alleviating or eliminating signs or symptoms of a disease or disorder. It means that it contains, and this amount is called the “effective amount.” Accordingly, the total amount of each active ingredient in the composition or method is sufficient to produce significant individual benefit. Accordingly, the effective amount will vary depending on the circumstances in which it is administered. An effective amount may be administered in one or more prophylactic or therapeutic administrations.

Inflammation is a highly regulated self-defense mechanism to detect and destroy invading pathogens and restore normal tissue structure and function. Once pathogens invade, blood monocytes rapidly migrate to the site of infection and fully differentiate into tissue macrophages. It's rough. Macrophages then release proinflammatory molecules, including interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α), nitric oxide (NO), and prostaglandin E ₂ (PGE ₂ ), triggering an immediate inflammatory response. It starts with However, excessive inflammatory responses are recognized as a major cause of chronic inflammation and a variety of chronic inflammations, including vascular disease, rheumatoid arthritis, inflammatory bowel disease, and cancer. Accordingly, many natural compounds that inhibit the induction of inflammatory cytokines and mediators have been developed to treat inflammatory diseases.

Meanwhile, lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, strongly induces macrophages and neutrophils to produce proinflammatory cytokines and mediators. LPS micelles are detected by soluble LPS binding protein and CD14 and delivered to the Toll-like receptor 4 (TLR4) and myeloid differentiation factor (MD2) complex.

Specifically, when LPS binds to the TLR4/MD2 complex, the intracellular Toll-IL-1 receptor (TIR) domain recruits key adapter molecules, including myeloid differentiation primary response 88 (MyD88), which stimulates inflammatory responses. Additionally, MyD88 promotes the activity of IL-1 receptor-related kinase 4 (IRAK4), which phosphorylates the IκB kinase (IKK) complex and activates nuclear factor-κB (NF-κB). The NF-κB mainly consists of two protein subunits, p50 and p65, and binds to the inhibitory protein of IκB and exists in the cytoplasm in an inactive form.

Subsequently, when exposed to pro-inflammatory stimuli such as LPS, IKK is rapidly phosphorylated and IκB releases NF-κB subunits such as p50 and p65 and translocates to the nucleus, ultimately producing iNOS, COX-2, IL-12, and TNF-α. Activates transcription of inflammatory genes, including: Accordingly, various antagonists targeting TLR4 and NF-κB are being developed as treatments for LPS-induced inflammatory diseases.

Accordingly, the present applicant confirmed that the active ingredient derived from cherry trees, a natural material, is effective in suppressing LPS-induced inflammation and endotoxemia, and through additional research, the active ingredient was confirmed to be effective in suppressing LPS-induced TLR4/MD2. By inhibiting the signaling pathway, it was confirmed that the anti-inflammatory effect and the endotoxemia improvement effect were exhibited, and a composition containing the same was developed.

In order to achieve the above object, a composition for improving inflammation according to an embodiment of the present invention contains cherry tree extract as an active ingredient.

Meanwhile, a composition for improving endotoxemia according to another embodiment of the present invention contains cherry tree extract as an active ingredient.

The cherry tree (Prunus serrulata Lindl. f. serrulata spontanea (Maxim.) Chin S. Chang) is a deciduous tree of the Rosaceae family of the Rosaceae family of dicotyledonous plants and is widely grown in mountainous areas. It reaches a height of 20m, the bark peels off from the side, is black and purplish in color, and the small branches are hairless. The leaves are alternate, egg-shaped or egg-shaped with a sharply pointed end, and the bottom is round or has a wide sharp base and is 6 to 12 cm long. There are needle-like double teeth on the edge of the leaf. It has no hair and is reddish-brown or greenish-brown at first, but when it fully grows, it becomes dark green on the front and light green with a slightly off-white color on the back. The petiole is 2 to 3 cm long and has 2 to 4 nectaries. Flowers bloom in pink or white from April to May, and 2 to 5 flowers grow in corymbs or racemes. There are bracts on the peduncle, and the peduncle, calyx tube, and style are hairless. The fruit is round and ripens from red to black between June and July and is called a cherry. Cherry trees are sometimes difficult to distinguish from Prunus leveilleana, but the difference is that the bottom of the teeth are wide and do not resemble needles, and all those that grow in northeastern China are given the scientific name of Prunus leveilleana. In China, the phosphorus from the stone fruit is used medicinally, and in the private sector, the inner skin of the cherry tree is used as a cough medicine. Distributed in Korea, China, and Japan.

Various pharmacological activities by active ingredients extracted from the cherry tree have been reported.

The composition of the present invention contains the cherry tree extract as an active ingredient and has excellent effects on improving inflammation and endotoxemia.

Specifically, the active ingredient can exhibit an anti-inflammatory effect in macrophages stimulated with LPS and has the characteristic of inducing an anti-endotoxemia effect in LPS microinjected zebrafish larvae.

The composition is TLR4/MD2 It binds to the complex and shows an improvement effect on inflammation.

Furthermore, the composition TLR4/MD2 By binding to the complex, it shows an improving effect on endotoxemia.

The composition is excellent in downregulating iNOS and COX-2 expression and inhibiting the release of NO and PGE ₂ .

Specifically, the composition may not only show an improvement effect on inflammation through a down-regulating effect on iNOS and COX-2 expression and an inhibitory effect on the release of NO and PGE ₂ , but also may show an improvement effect on endotoxemia. will be.

More specifically, the cherry tree extract included as an active ingredient in the composition of the present invention reduces the production of nitric oxide (NO) and prostaglandin E ₂ (PGE ₂ ) induced by LPS, and cyclooxygenase-2 (COX-2). ) and can inhibit the expression of inducible NO synthase. Furthermore, it inhibits the production of pro-inflammatory cytokines, including interleukin-12 (IL-12) and tumor necrosis factor-α, in LPS-stimulated macrophages while inhibiting nuclear translocation of nuclear factor-κB. It was confirmed that it does, and through this, it can exhibit anti-inflammatory effects and anti-endototoxemia effects.

More specifically, the cherry tree extract prevents LPS from binding to myeloid differentiation factor (MD2) and Toll-like receptor 4 (TLR4), and consequently inhibits TLR4/MD2-mediated inflammatory response.

That is, the cherry tree extract included as an active ingredient in the composition of the present invention binds directly to the TLR4/MD2 complex and prevents the binding of LPS to TLR4/MD2, thereby improving LPS-induced inflammation and endotoxemia. will be.

Specifically, the composition of the present invention contains cherry tree extract as an active ingredient, and the cherry tree extract is a compound represented by the following formula (1):

[Formula 1]

here,

n is an integer from 0 to 3,

L ₁ is a single bond or a substituted or unsubstituted alkylene group having 1 to 10 carbon atoms,

Ar ₁ is a substituted or unsubstituted cycloalkyl group with 3 to 30 carbon atoms, a substituted or unsubstituted aryl group with 5 to 30 carbon atoms, a substituted or unsubstituted heteroaryl group with 2 to 30 carbon atoms, or a substituted or unsubstituted heteroaryl group with 3 to 30 carbon atoms. selected from the group consisting of an arylalkyl group and a substituted or unsubstituted aryloxy group having 6 to 30 carbon atoms,

R ₁ is hydrogen, deuterium, halogen, cyano group, hydroxy group, substituted or unsubstituted alkyl group with 1 to 30 carbon atoms, substituted or unsubstituted alkenyl group with 2 to 30 carbon atoms, substituted or unsubstituted alkynyl group with 2 to 24 carbon atoms, Substituted or unsubstituted heteroalkyl group having 2 to 30 carbon atoms, substituted or unsubstituted aralkyl group having 6 to 30 carbon atoms, substituted or unsubstituted aryl group having 5 to 30 carbon atoms, substituted or unsubstituted heteroaryl group having 2 to 30 carbon atoms, substituted or an unsubstituted heteroarylalkyl group with 3 to 30 carbon atoms, an alkoxy group with 1 to 30 carbon atoms, a substituted or unsubstituted cycloalkyl group with 3 to 20 carbon atoms, a substituted or unsubstituted heterocycloalkyl group with 3 to 20 carbon atoms, or a substituted or unsubstituted carbon number. It is selected from the group consisting of 3 to 20 cycloalkenyl groups, substituted or unsubstituted heteroaralkyl groups with 3 to 30 carbon atoms, and substituted or unsubstituted heteroalkenyl groups with 1 to 20 carbon atoms,

The substituents of L ₁ , Ar ₁ and R ₁ are hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, alkyl group with 1 to 30 carbon atoms, alkenyl group with 2 to 30 carbon atoms, alkynyl group with 2 to 24 carbon atoms, Heteroalkyl group with 2 to 30 carbon atoms, aralkyl group with 6 to 30 carbon atoms, cycloalkyl group with 3 to 20 carbon atoms, heterocycloalkyl group with 3 to 20 carbon atoms, aryl group with 5 to 30 carbon atoms, heteroaryl group with 2 to 30 carbon atoms, A substituent selected from the group consisting of a heteroarylalkyl group with 3 to 30 carbon atoms, an alkoxy group with 1 to 30 carbon atoms, an alkylsilyl group with 1 to 30 carbon atoms, an arylsilyl group with 6 to 30 carbon atoms, and an aryloxy group with 6 to 30 carbon atoms. It is substituted, and when substituted with a plurality of substituents, they are the same or different from each other.

Preferably, the compound represented by Formula 1 is a compound represented by Formula 2 below:

[Formula 2]

here,

n is an integer from 0 to 3,

R ₁ is hydrogen, deuterium, halogen, cyano group, hydroxy group, substituted or unsubstituted alkyl group with 1 to 30 carbon atoms, substituted or unsubstituted alkenyl group with 2 to 30 carbon atoms, substituted or unsubstituted alkynyl group with 2 to 24 carbon atoms, It is selected from the group consisting of a substituted or unsubstituted aryl group having 5 to 30 carbon atoms, an alkoxy group having 1 to 30 carbon atoms, and a substituted or unsubstituted cycloalkyl group having 3 to 20 carbon atoms,

The substituent for R ₁ is hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, alkyl group with 1 to 30 carbon atoms, alkenyl group with 2 to 30 carbon atoms, alkynyl group with 2 to 24 carbon atoms, hetero group with 2 to 30 carbon atoms. It is substituted with a substituent selected from the group consisting of an alkyl group, an aralkyl group with 6 to 30 carbon atoms, an aryl group with 5 to 30 carbon atoms, and an alkoxy group with 1 to 30 carbon atoms. When substituted with a plurality of substituents, they are the same or different from each other.

That is, the composition of the present invention contains the compound represented by Formula 1, which is an active ingredient extracted from cherry trees, as an active ingredient, and the compound represented by Formula 1 is capable of binding to the TLR4/MD2 complex, through which LPS By preventing binding to the complex, it can consequently exhibit an anti-inflammatory effect on the inflammatory response derived from LPS and an improvement effect on endotoxemia.

More preferably, the compound represented by Formula 2 is a compound represented by Formula 3 below:

[Formula 3]

A food composition for improving inflammation or endotoxemia according to another embodiment of the present invention includes the composition.

The food composition according to the present invention may be provided in the form of powder, granules, tablets, capsules, syrup or beverage, and may be used with other foods or food additives, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on its purpose of use, such as prevention, health, or therapeutic treatment.

As a specific example, the above food composition can be used to produce processed food that can enhance the improvement effect on inflammation and endotoxemia. These processed foods include, for example, snacks, beverages, alcoholic beverages, fermented foods, canned foods, processed milk foods, processed meat foods, noodles, etc. Confectionery includes biscuits, pies, cakes, bread, candy, jellies, gum, cereals, etc. Beverages include drinking water, carbonated beverages, functional ionic beverages, functional ionic beverages, juices (e.g., apple, pear, grape, aloe, tangerine, peach, carrot, tomato juice, etc.), Sikhye, etc. Alcoholic beverages include sake, whiskey, soju, beer, liquor, and fruit wine. Fermented foods include soy sauce, soybean paste, and red pepper paste. Canned food includes canned seafood (e.g., canned tuna, mackerel, saury, canned conch, etc.), canned livestock products (canned beef, pork, chicken, turkey, etc.), and canned agricultural products (canned corn, peaches, canned file apple, etc.). Milk processed foods include cheese, butter, yogurt, etc. Processed meat products include pork cutlet, beef cutlet, chicken cutlet, and sausage. Includes sweet and sour pork, nuggets, neobiani, etc. Includes noodles such as raw noodles in sealed packaging. In addition, the composition can be used in retort foods, soups, etc.

When using the food composition of the present invention as a food additive, the food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The active ingredient can be used appropriately depending on its purpose of use (prevention or improvement).

At this time, the amount of the extract in the food or beverage can be added at about 0.01 to 15% by weight of the total weight of the food, and the health drink composition can be added at a rate of about 0.01 to 10g based on 100 ml, but is not limited thereto. .

When the food composition of the present invention is a beverage composition, other than containing the extract as an essential ingredient in the ratio indicated, there are no particular restrictions on other ingredients, and various flavoring agents or natural carbohydrates may be contained as additional ingredients like ordinary beverages. You can. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used, but are not limited to these. The ratio of the natural carbohydrate may generally range from about 1 to 20 g per 100 ml of the composition of the present invention, but may not be limited thereto.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salt, organic acid, protective colloidal thickener, Ph adjuster, stabilizer, preservative, glycerin, alcohol, carbonating agent used in carbonated beverages, etc., but may not be limited thereto.

In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks, and these ingredients can be used independently or in combination. The proportion of these additives is not critical, but may be selected in the range of about 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto.

A pharmaceutical composition for improving inflammation or endotoxemia according to another embodiment of the present invention includes the composition.

The pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterilized injection solutions according to conventional methods.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose ( It can be prepared by mixing sucrose, lactose, gelatin, etc.

Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and hydrogen phosphate. Calcium, lactose, mannitol, taffy, 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, white sugar, dextrose, sorbitol, and talc may be used.

Additionally, the dosage of the pharmaceutical composition according to the present invention may be increased or decreased depending on the route of administration, severity of disease, gender, weight, age, etc. Accordingly, the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" in the present invention refers to the amount used to treat or prevent a disease with a reasonable benefit/risk ratio applicable to medical treatment or prevention. It refers to a sufficient amount, and the effective dose level is determined by the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the administration time, route of administration, and excretion rate of the composition of the present invention used. Factors including the duration of time, drugs used in combination or concurrently with the compositions of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered alone or in combination with a known immunotherapeutic agent. It is important to consider all of the above factors and administer the amount that will achieve the maximum effect with the minimum amount without side effects.

According to the present invention, a composition for improving inflammation and endotoxemia containing cherry tree extract as an active ingredient, the active ingredient binds to the TLR4/MD2 complex, thereby showing an excellent improvement effect on inflammation and endotoxemia, and using natural materials. This has the effect of minimizing side effects.

Figure 1 shows the results of confirming the cytotoxicity of a composition for improving inflammation and endotoxemia according to an embodiment of the present invention.
Figure 2 shows the results of confirming the effect of downregulating iNOS and COX-2 expression and inhibiting the release of NO and PGE ₂ of a composition for improving inflammation and endotoxemia according to an embodiment of the present invention.
Figure 3 shows the results of confirming the effect of reducing the production of IL-12 and TNF-α of the composition for improving inflammation and endotoxemia according to an embodiment of the present invention.
Figure 4 shows the results of confirming the down-regulating effect on the nuclear translocation of NF-κB of a composition for improving inflammation and endotoxemia according to an embodiment of the present invention.
Figure 5 shows the results of confirming the interaction between a composition for improving inflammation and endotoxemia and the TLR4/MD2 complex according to an embodiment of the present invention.
Figure 6 shows the results of confirming the anti-endotoxemia effect of a composition for improving inflammation and endotoxemia according to an embodiment of the present invention.
Figure 7 shows the results of confirming the effect of a composition for improving inflammation and endotoxemia according to an embodiment of the present invention on down-regulating pro-inflammatory genes and weakening the recruitment of macrophages and neutrophils.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement it. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

Manufacturing example

[Material preparation and experimental method]

Reagents and Antibodies

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), trypsin-ethylenediamine tetraacetic acid (EDTA), and antibiotic mixture were purchased from WelGENE (Daegu, Korea). LPS extracted from E. coli O55:B5, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), methylene blue, 4′ 6-diamidine-2′ -phenylindole dihydrochloride (DAPI) , and Alexa Fluor 647-conjugated secondary antibody were purchased from Sigma-aldrich (USA). Antibodies against iNOS (sc-7271), COX-2 (sc-19999), MyD88 (sc-74532), p50 (sc-8414), p65 (sc-8008), β-actin (sc-69879), nucleolin ( sc-13057), and peroxidase-labeled anti-mouse antibody (sc-16102) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Anti-phospho (p)-IRAK4 antibody was purchased from Invitrogen (Waltham, MA, USA), and peroxidase-labeled anti-rabbit antibody was purchased from Koma Biotechnology (Seoul, Republic of Korea).

Meanwhile, the cherry tree (Prunus serrulata Lindl. f. serrulata spontanea (Maxim.) Chin S. Chang) extract was purchased from the Freshwater Biological Resources Bank under resource number FBCC-EP1345, and all other chemicals were purchased from Sigma-Aldrich.

Meanwhile, as a result of analyzing the cherry tree extract using a ¹ H-NMR analyzer, it was confirmed to be a compound (Pi) represented by the following formula 3:

[Formula 3]

Cell culture and viability experiments

RAW 264.7 macrophages were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 5% FBS at 37°C in a 5% CO ₂ humidified incubator. For relative cell viability analysis, RAW 264.7 macrophages were seeded in a 24-well plate at a density of 1Х10 ⁵ cells/mL, and 2 hours before treatment with 300 ng/mL LPS, the compound (Pi) represented by Formula 3 above (0 to 40 μM). Then, colorimetric MTT analysis was performed after 24 hours of incubation. Specifically, cells were treated with MTT solution (0.5 mg/mL) for 30 min at 37°C, dark purple formazan was dissolved in dimethyl sulfoxide, and microplate reader (Thermo Fisher Scientific, Rockford, IL, USA) was used. The absorbance was measured at 570 nm.

flow cytometry

RAW 264.7 macrophages were seeded at a density of 1Х10 ⁵ cells/mL, treated with compound (Pi) (0 to 40 μM) represented by Formula 3 above for 2 hours, and then treated with 300 ng/mL of LPS for 24 hours. To estimate viable cell numbers and dead cell populations, harvested cells were washed with cold phosphate-buffered saline (PBS) and stained with the Muse Cell Count and Viability Kit (Luminex, Austin, Texas, USA) for 5 min. Viable cell numbers and dead cell populations were determined using the Muse Cell Analyzer (Luminex).

Isolation of total RNA and RT-PCR experiments

Total RNA was extracted from RAW 264.7 macrophages using the Easy-BLUE Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam-si, Gyeonggi-do). RNA was reverse transcribed using moloney murine leukemia virus (MMLV) reverse transcriptase (Bioneer, Daejeon, Korea), and the synthesized cDNA was amplified using EzWay Neo Taq PCR MasterMix (Koma Biotechnology). Meanwhile, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control to evaluate the relative expression of iNOS, COX-2, IL-12, and TNF-α.

Western blotting

Total cell extracts were prepared from RAW 264.7 macrophages using radioimmunoprecipitation assay lysis buffer (iNtRON Biotechnology) with protease inhibitor cocktail (Sigma-Aldrich). Specifically, lysis buffer was added to cells on ice for 30 minutes, and at the same time, cytoplasmic and nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagent (Pierce, Rockford, IL, USA). Lysates were centrifuged at 15,000 g for 10 min at 4°C, and protein concentration was quantified using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Afterwards, samples were stored at -80°C or used immediately for Western blotting. Meanwhile, equal amounts of proteins were separated on SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific). Afterwards, each protein was detected using a chemiluminescent substrate (Thermo Fisher Scientific).

NO analysis

RAW 264.7 macrophages (1x10 ⁵ cells/mL) were seeded in a 24-well plate. Cells were pretreated with compound (Pi) (0-20 μM) represented by Formula 3 above for 2 hours and then stimulated with 300 ng/mL LPS for 24 hours. NO levels in culture supernatants were determined using the Griess reagent assay. Specifically, the supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride) and incubated in the dark at 25°C for 15 minutes. Absorbance was measured at 540 nm using a microplate reader (Thermo Fisher Scientific), and a standard curve of sodium nitrite was used to determine nitrite concentration.

ELISA analysis

ELISA analysis was performed to quantify secretion levels of PGE ₂ (Cayman Chemicals, Ann Arbor, MI, USA), IL-12 (R&D Systems, Minneapolis, MN, USA), and TNF-α (R&D Systems). RAW264.7 macrophages were pretreated with compound (Pi) (0-20 μM) represented by Formula 3 above for 4 hours and 2 hours before adding 300 ng/mL of LPS. Then, the supernatant was collected and the levels of PGE ₂ , IL-12, and TNF-α were measured using ELISA.

Immunofluorescence staining experiment

RAW 264.7 macrophages were cultured at a density of ^1x10 cells/mL on 3% gelatin-coated coverslips, treated with 20 μM compound (Pi) represented by Formula 3 above for 2 hours, and then stimulated with 300 ng/mL LPS for 1 hour. did. Afterwards, the cells were fixed with 4% paraformaldehyde for 10 minutes at 37°C and permeabilized with PBST for 10 minutes at 25°C. Cells were then washed with ice-cold PBST for 5 min, blocked with 10% donkey serum for 1 h, and incubated with anti-p65 antibody (200 μg/mL, 1:100 dilution in 10% donkey serum) at 4°C. Incubated overnight. Afterwards, cells were washed with ice-cold PBST and incubated with conjugated secondary antibodies at 25°C for 2 hours. Afterwards, cells were counterstained with the nuclear stain DAPI (300 nM) for 10 minutes. The coverslip was mounted on a glass slide using aqueous mounting medium (DAKO, Carpinteria, CA, USA), and immunofluorescence was visualized using the CELENA S Digital Imaging System (Logos Biosystems, Anyang, Gyeonggi-do).

Molecular docking

Crystal structures of human TLR4/MD2 complex [Protein Database Bank (PDB) ID: 3FX1] and mouse TLR4/MD2 complex (PDB ID: 3VQ2) were obtained from RCSB PDB, and then all non-amino acids were removed. The chemical structure of compound (Pi) represented by Formula 3 was obtained through PubChem. Meanwhile, molecular docking was calculated using Mcule and interaction modes were visualized using UCSF Chimera. 2D diagram interactions were visualized using BIOVIA Discovery Studio Visualizer.

Maintenance of zebrafish

Experiments using zebrafish were approved by the Animal Care and Use Committee of Jeju National University (Jeju Special Self-Governing Province, Republic of Korea) and were performed according to the approved guidelines (approval number 2022-0021). Embryos were collected through natural spawning of zebrafish and placed in embryo medium supplemented with 1% methylene blue [(NaCl-34.8g, KCl-1.6g, _CaCl2-5.8g , _MgCl2-9.78g , containing double distilled water, pH 7.2]). ) were cultured at 28°C.

Experiment to confirm heart rate, abnormality and mortality of LPS microinjected Zebrafish larvae

At 3 days after fertilization, zebrafish larvae (dpf, n = 20 per group) were anesthetized using 0.002% tricaine, and the same volume of PBS (2 nL) was injected into untreated controls. Thereafter, the larvae were transferred to 3 mL embryo medium in the presence or absence of compound (Pi) (0-20 μM) represented by Formula 3 above. After 24 h, heart rate per minute was manually calculated at 4 dpf, and in parallel experiments, mortality and abnormalities were measured at 5 dpf using a stereomicroscope (Olympus, Tokyo, Japan).

LPS Macrophage and Neutrophil Staining in Microinjected Zebrafish

LPS (2nL, 0.5 mg/mL) was microinjected into the yolk sac of 3 dpf zebrafish larvae and then treated with the compound represented by Formula 3 (0-20 μM). After 24 h (4 dpf), whole larvae were fixed with paraformaldehyde for 2 h at room temperature and washed with PBS. Afterwards, larval macrophages were stained with 5 μg/mL neutral red solution for 6 hours. Meanwhile, in parallel experiments, larval neutrophils were stained with Sudan Black as previously described. Afterwards, larvae were washed with 70% ethanol and gradually rehydrated. Meanwhile, the recruitment of macrophages and neutrophils to the inflammation site was observed using a stereomicroscope (Olympus).

Isolation of Total Zebrafish mRNA and RT-PCR

Zebrafish larvae at 3 dpf were microinjected with LPS (2 nL, 0.5 mg/mL) into the egg yolk and then treated with compound (Pi) (0-20 μM) represented by Formula 3 above. Meanwhile, total RNA was extracted at 4 dpf using the Easy-BLUE Total RNA Extraction Kit (iNtRON Biotechnology). RNA was reverse transcribed using MMLV reverse transcriptase (Bioneer), and cDNA was amplified using primers. β-Actin was used as a loading control to evaluate the relative expression of iNOS, COX-2α, IL-12, and TNF-α.

Statistical analysis

RT-PCR and Western blot images were visualized using ImageQuant LAS 500 (GE Healthcare Bio-Science AB, Uppsala, Sweden) and ImageJ 1.50i (National Institute of Health, Manassas, VA, USA; www.imagej.net). It was quantified using . All data represent the average of at least three independent experiments and are expressed as mean ± standard error (SE). Statistical analysis was performed using Student's t-test and analysis of variance with Bonferroni correction using SigmaPlot version 12.0 (Systat Software, San Jose, CA, USA, www.sysstatsofware.com). Statistical significance was set at ^* , p < 0.05, ^** , p < 0.05 and ^*** , ^&&& and ^### , p < 0.001.

[Experiment result]

Cytotoxicity results of compound (Pi) represented by Formula 3 above

MTT analysis and flow cytometry were performed to determine the effect of the compound (Pi) represented by Formula 3 above on the viability of RAW 264.7 macrophages. The compound (Pi) represented by Formula 3 at a concentration of less than 20 μM did not show a significant decrease in relative cell viability, but the compound (Pi) represented by Formula 3 at a concentration of 40 μM decreased the survival rate regardless of the presence or absence of LPS. (Figure 1A).

Meanwhile, a clear decrease in relative cell viability was confirmed in LPS treatment alone. Morphological changes in RAW 264.7 macrophages were not observed under the condition of the compound (Pi) represented by Formula 3 at a concentration of less than 20 μM (Figure 1B). However, some floating and contracted RAW 264.7 macrophages could be observed under the condition of 40 μM of the compound (Pi) represented by Formula 3 above.

Flow cytometry was performed to accurately measure the effect of the compound (Pi) represented by Formula 3 above on cell viability (Figure 1C). As shown in Figure 1D, the compound (Pi) represented by Formula 3 slightly reduced the total number of viable cells regardless of the presence of LPS, and in the case of treatment with LPS alone, the number of viable cells was significantly reduced compared to untreated cells. It was adjusted downward.

It was confirmed that the compound (Pi) represented by Formula 3 reduced the number of viable cells in a concentration-dependent manner, but did not induce significant cell death at a concentration of less than 20 μM (Figure 1E). However, it was confirmed that 40 μM of the compound (Pi) represented by Formula 3 significantly induced cell death. Through the above experiment, it was confirmed that the compound (Pi) represented by Formula 3 at a concentration of 20 μM or less was not toxic to RAW 264.7 macrophages.

Effect of downregulation of iNOS and COX-2 expression and NO and PGE ₂ Confirm the emission suppression effect of

To evaluate the anti-inflammatory effect of the compound (Pi) represented by Formula 3 in LPS-stimulated RAW 264.7 macrophages, the levels of NO and PGE ₂ released into the culture medium were examined using Griess reagent analysis and ELISA, respectively. .

RAW 264.7 macrophages not treated with LPS were confirmed to spontaneously release low levels of NO (1.4 ± 0.1 μM). However, it was confirmed that treatment with LPS alone significantly improved the NO production level (25.8 ± 0.7 μM, Figure 2A).

Meanwhile, treatment with the compound (Pi) represented by Formula 3 was confirmed to reduce LPS-induced NO release in a concentration-dependent manner (15.4 ± 1.4 μM, 9.6 ± 0.3 μM, and 2.8 ± 2.0 at 5, 10, and 20 μM, respectively) μM). Additionally, referring to Figure 2B, stimulation of RAW 264.7 macrophages with LPS increased PGE ₂ production (2279.1 ± 50.2 pg/mL) compared to LPS-untreated RAW 264.7 macrophages (543.1 ± 5.6 pg/mL). Confirmed.

It was confirmed that pretreatment with the compound (Pi) represented by Formula 3 significantly attenuated LPS-stimulated PGE ₂ production in a concentration-dependent manner (1796.4 ± 12.0 pg/mL, 1505.9 ± 7.4 at 5, 10, and 20 μM, respectively) pg/mL and 1403.4 ± 38.1 pg/mL).

Meanwhile, it was tested whether the compound (Pi) represented by Formula 3 affects LPS-induced iNOS and COX-2 expression at the transcription and translation levels. According to RT-PCR analysis, LPS treatment showed a significant increase in iNOS and COX-2 expression, but pretreatment with the compound (Pi) represented by Formula 3 attenuated this increase in expression in a concentration-dependent manner. This was confirmed (Figure 2C). Meanwhile, consistent with the RT-PCR data, it was confirmed that the compound (Pi) represented by Formula 3 significantly inhibited LPS-induced iNOS and COX-2 expression at the translation level (Figure 2D).

Taking the above results together, it can be confirmed that the compound (Pi) represented by Formula 3 inhibits LPS-stimulated NO and PGE ₂ release and the expression of iNOS and COX-2.

Confirmation of the effect of reducing the production of IL-12 and TNF-α induced by LPS

The potential effect of the compound (Pi) represented by Formula 3 on the production of pro-inflammatory cytokines, including IL-12 and TNF-α, in LPS-stimulated RAW 264.7 macrophages was confirmed.

Referring to Figures 3A and 3B, IL-12 and TNF-α were weakly expressed in cells not treated with LPS (235.5 ± 4.5 pg/mL and 101.7 ± 0.4 pg/mL, respectively), but were expressed in cells treated with LPS for 24 hours. Afterwards, it was confirmed that the release amounts of IL-12 (1206.0 ± 4.9 pg/mL) and TNF-α (1563.1 ± 44.9 pg/mL) were significantly increased.

Meanwhile, pretreatment with the compound (Pi) represented by Formula 3 inhibited the release of LPS-induced IL-12 and TNF-α in a concentration-dependent manner, and the compound (Pi) represented by Formula 3 at the highest concentration (20 μM) was Its expression was attenuated maximally (879.7 ± 5.4 pg/mL IL-12 and 627.7 ± 3.2 pg/mL TNF-α).

To confirm whether the compound (Pi) represented by Formula 3 down-regulates the expression of IL-12 and TNF-α induced by LPS, RT-PCR analysis was performed 6 hours after LPS treatment. RT-PCR data in RAW 264.7 macrophages stimulated with LPS confirmed that the compound (Pi) represented by Formula 3 reduced the expression of IL-12 and TNF-α in a concentration-dependent manner (Figure 3C).

Considering the above results, it can be said that the compound (Pi) represented by Formula 3 downregulates the release of LPS-stimulated IL-12 and TNF-α by suppressing gene expression.

Confirmation of down-regulation effect on nuclear translocation of NF-κB

Since NF-κB is a major transcription factor that regulates the expression of pro-inflammatory mediators and cytokines, it was investigated whether the compound (Pi) represented by Formula 3 downregulates the nuclear translocation of NF-κB in LPS-treated RAW 264.7 macrophages. An experiment was conducted to determine whether or not.

As shown in Figure 4A, LPS significantly increased the expression of p50 and p65 in the nucleus, but the compound (Pi) represented by Formula 3 down-regulated the expression. In addition, as shown in immunostaining of p65 in LPS-treated RAW 264.7 macrophages, the compound (Pi) represented by Formula 3 inhibited the nuclear translocation of p65 induced by LPS (Figure 4B).

Considering the above results, it can be said that the compound (Pi) represented by Formula 3 inhibits the expression of pro-inflammatory mediators and cytokines by inhibiting the nuclear translocation of NF-κB.

Compound (Pi) represented by Formula 3 above and confirmation of binding between TLR4/MD2 complexes.

LPS binding to the membrane TLR4/MD2 complex stimulates the NF-κB signaling pathway, leading to high expression of proinflammatory genes including iNOS, COX-2, IL-12, and TNF-α. Based on this, the binding effect of the compound (Pi) represented by Formula 3 of the present invention on the TLR4/MD2 complex was confirmed.

Specifically, through molecular docking simulation, four binding sites for the compound (Pi) represented by Formula 3 were revealed where it binds to the human TLR4/MD2 complex (PDB ID: 3FXI). The strongest binding activity (Pose 1) did not form traditional hydrogen bonds, but was found to have a binding score of -5.5 (Figure 5A and Table 1).

Table 1 below shows the compound (Pi) and TLR4/MD2 represented by Formula 3 above. It shows docking poses, scores, hydrogen bond interaction, and hydrogen bond distances between complexes (PDB ID: 3FXI).

Receptor Docking pose Docking score Binding AA
(H-bond) H-bond distance ( ) TLR4/MD2(3FXI) One -5.5 - - 2 -4.9 SER438(BOG) 2.902 3 -4.8 - - 4 -4.6 SER438(BOG) 3.076 AGR460(BO) 1.947

(A.A., amino acid; H-bond; hydrogen bond.)

It was confirmed that the compound (Pi) represented by Formula 3 in pose 1 blocked the binding by interfering with the hydrophobic pocket of MD2 where LPS binds. At the same time, a hydrogen bond is formed between ARG90 of MD2 and GLU439 of TLR4, showing that the compound (Pi) represented by Formula 3 induces a rare interaction between MD2 and TLR4 (Figure 5A, right). Additionally, the 2D interaction diagram shows that the compound (Pi) represented by Formula 3 above forms a specific carbon hydrogen bond with LYS89 in MD2 and forms ð-alkyl or alkyl and van der Waals interactions with the TLR4/MD2 complex. is giving (Figure 5B).

In pose 2, the compound (Pi) represented by Formula 3 formed a hydrogen bond with SER483 of TLR4 at a distance of 2.902 Å, and the docking score was -4.9 (Table 1).

The 2D interaction diagram shows that the compound (Pi) represented by Formula 3 above forms a hydrogen bond with GLN436 and SER438 of TLR4 and ð-alkyl or alkyl interactions in pose 2 (PRO88 of MD2, ARG460 and ALA462 of TLR4) It was confirmed that

In Pose 3, the compound (Pi) represented by Formula 3 showed a docking score of -4.8 (Table 1), but no clear traditional hydrogen bond was observed and two ð-alkyl or alkyl interactions (PRO88 and Only ARG460) of TLR4 was confirmed.

Meanwhile, it can be seen that the compound (Pi) represented by Formula 3 in pose 4 formed two hydrogen bonds with SER438 and AGR460 of TLR4 at distances of 3.076 Å and 1.947 Å, respectively (Table 1).

It was confirmed that the 2D interaction diagram of pose 4 shows hydrogen bonding with ARG460 of TLR4 and ð-alkyl or alkyl interactions with PRO88 of MD2 and ALA462 of TLR4. In addition, through molecular docking, it was confirmed that the compound (Pi) represented by Formula 3 interacts with the mouse TLR4/MD2 complex (PDB ID: 3VQ2) through many non-covalent bonds, but no hydrogen bonds were found.

The docking scores were found to be -7.2, -6.6, -6.3, and -6.2 in each pose.

Meanwhile, we investigated whether cherry tree extract binds to the TLR4/MD2 complex and regulates the expression of intracellular signaling molecules MyD88 and IRAK4.

As shown in Figure 5, it can be seen that the compound (Pi) represented by Formula 3 inhibits the LPS-induced increase in MyD88 expression and IRAK4 phosphorylation in a concentration-dependent manner.

Taking the above results together, it can be seen that the compound (Pi) represented by Formula 3 binds to the LPS binding site between LPS and the TLR4/MD2 complex, thereby preventing LPS from binding to the TLR4/MD2 complex.

Confirmation of mortality reduction effect of LPS microinjected Zebrafish larvae

To evaluate the anti-endotoxemia effect of the compound (Pi) represented by Formula 3, LPS was microinjected into zebrafish larvae at 3 dpf with or without the compound (Pi) represented by Formula 3, and heart rate was measured. , mortality and abnormalities were monitored.

Referring to Figure 6A, after 24 hours (at 4 dpf), there was a significant increase in heart rate (144.8 ± 0.9 heartbeat/min) for LPS microinjected larvae compared to PBS microinjected larvae (179.6 ± 2.3 heartbeat/min, Figure 6A). showed a decrease.

Meanwhile, when the concentration of the compound (Pi) represented by Formula 3 was increased, the decreased heart rate gradually recovered to almost normal level at 20 μM (174.8 ± 1.9 heart beats/min).

Referring to Table 2 below, it can be seen that when LPS was microinjected, 45% mortality and 45% abnormalities were induced in zebrafish larvae after 48 hours (at 5 dpf), and 10% of zebrafish larvae survived. there is.

Specifically, Table 2 below confirms the effect of the compound (Pi) represented by Formula 3 on the mortality and abnormalities of zebrafish larvae caused by LPS microinjection.

Group Phenotype (%)/48 h (n = 20) Normal Death Abnormal Untreated 100 0 0 Pi at 20 μM 100 0 0 LPS 10 45 45 5 μM of Pi + LPS 30 10 60 10 μM of Pi + LPS 70 0 30 20 μM of Pi + LPS 90 0 10

In contrast, it can be seen that the compound (Pi) represented by Formula 3 gradually reduces mortality and abnormalities in LPS microinjected zebrafish larvae.

The compound (Pi) represented by Formula 3 at a concentration of 5 μM reduced the mortality rate by 10%, while the mortality rate slightly increased to 60%.

Meanwhile, it was confirmed that treatment with 10 μM or more of the compound (Pi) represented by Formula 3 completely blocks the mortality caused by LPS microinjection and strongly suppresses abnormalities (30% and 10% at 10 μM and 20 μM, respectively).

In particular, LPS microinjected zebrafish showed 45% abnormalities at 5 dpf ( Figure 6B ), including cell division (5%), yolk necrosis (5%), yolk edema (5%), and yolk fragmentation (10%). , swollen pericardium (15%), and head deformity (5%, Figure 6C, left).

Meanwhile, in zebrafish larvae treated with 20 μM of the compound (Pi) represented by Formula 3, abnormalities induced by LPS microinjection were strongly blocked, and 5% cell division and 5% pericardial edema were observed ( Figure 6C, right).

Taking the above results together, it can be seen that the compound (Pi) represented by Formula 3 shows an improving effect on LPS-induced endotoxemia in zebrafish larvae.

Confirmed effect of down-regulation of pro-inflammatory genes and weakening of recruitment of macrophages and neutrophils

The effect of the compound (Pi) represented by Formula 3 on LPS-induced macrophage and neutrophil infiltration of zebrafish larvae was confirmed through neutral red and Sudan black staining, respectively.

As shown in Figure 7A, neutral red staining showed a predominantly increased number of macrophages in yolk sacs microinjected with LPS compared to larvae microinjected with LPS.

However, when treated with the compound (Pi) represented by Formula 3, the accumulation of macrophages at the site of inflammation was significantly reduced in a concentration-dependent manner. In addition, in zebrafish microinjected with PBS, a large number of neutrophils stained with Sudan black were observed in the post-hematopoietic region (PBI), but it was confirmed that microinjection of LPS into the yolk sac clearly reduced neutrophils in PBI (Figure 7B), which is It would be said to indicate that neutrophils have migrated from PBI to the site of infection.

Meanwhile, in LPS microinjected zebrafish larvae, the compound (Pi) represented by Formula 3 significantly reduced neutrophil recruitment in a concentration-dependent manner. In addition, in the case of LPS microinjected larvae at 4 dpf, the expression of pro-inflammatory genes including iNOS, COX-2α, IL-12, and TNF-α was confirmed to be high (Figure 7C), while the Compound (Pi) was confirmed to reduce gene expression in LPS microinjected zebrafish larvae in a concentration-dependent manner.

Considering the above results, it can be seen that the compound (Pi) represented by Formula 3 strongly suppresses endotoxemia by suppressing the recruitment of macrophages and neutrophils along with downregulating the expression of pro-inflammatory genes.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. falls within the scope of rights.

Claims (7)

Contains cherry tree extract as an active ingredient
Composition for improving inflammation. According to paragraph 1,
The composition is TLR4/MD2 It binds to a complex and shows an improvement effect on inflammation.
Composition for improving inflammation. According to paragraph 1,
The composition has an excellent effect of downregulating iNOS and COX-2 expression and suppressing the release of NO and PGE _2.
Composition for improving inflammation. Contains cherry tree extract as an active ingredient
Composition for improving endotoxemia. According to clause 4,
The composition binds to the TLR4/MD2 complex and exhibits an improving effect on endotoxemia.
Composition for improving endotoxemia. Comprising a composition according to any one of claims 1 to 5
Food composition for improving inflammation or endotoxemia. Comprising the composition according to any one of claims 1 to 5.
Pharmaceutical composition for improving inflammation or endotoxemia.