Novel compound derived from carrot, derivative thereof, use thereof, and preparation method therefor

Abstract

A carrot extract or a fraction thereof of the present invention, particularly cis-4-hydroxycinnamyl 4-hydroxybenzoate derived therefrom, has anti-arthritis, anti-inflammatory, antioxidant, anti-aging, or anticancer effects, and thus can be effectively used in a pharmaceutical composition, a functional health food composition, or a treatment method. In addition, the present invention relates to 3-(4-Hydroxyphenyl)propyl 4-hydroxybenzoate derived from a novel carrot-derived compound cis-4-hydroxycinnamyl 4-hydroxybenzoate, and an effective method for preparing same.

Description

Novel compounds derived from carrots, derivatives thereof, uses thereof, and methods for preparing the same

[0001] This application claims the benefit of Korean Patent Application Nos. 10-2024-0025242 (2024-02-21), 10-2024-0025243 (2024-02-21), and 10-2024-0025244 (2024-02-21), the entire contents of which are incorporated herein by reference.

[0002]

[0003] The present invention relates to the anti-arthritic, anti-inflammatory, antioxidant, anti-aging or anti-cancer effects of carrot extract or fractions thereof.

[0004] The present invention relates to a novel compound derived from carrot, cis-4-hydroxycinnamyl 4-hydroxybenzoate, and a method for producing the same.

[0005] The present invention relates to 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate derived from a novel compound derived from carrots and a method for producing the same.

[0006]

[0007] The present invention was completed with the support of the Ministry of Education of the Republic of Korea under Project No. 2023RIS-009 (1345370817) and Project No. 2020R1A6C101A188 (1345346392).

[0008]

[0009] The carrot (Daucus carota subsp. sativus) is a biennial herb of the Apiaceae family in the Umbelliferous order of dicotyledons. Also known as red carrots, it grows to about 1 m tall. A variety similar to the carrot commonly cultivated today was bred in France and widely distributed throughout Europe by the 13th century.

[0010] Carrots contain a variety of vitamins, including the B vitamins, vitamin C, and vitamin K. They also contain beta-carotene, which the body converts to vitamin A, and are known to be rich in sugar and iron.

[0011] Natural products are less toxic than synthetic compounds, and their long-term clinical experience demonstrates relatively fewer side effects, efficacy, and safety. Therefore, compared to conventional new drug development, they tend to require less time, cost, and have a lower failure rate. Furthermore, with the rapid increase in morbidity due to increased life expectancy, changing lifestyles, and climate change, consumer interest in natural products is also growing, leading to annual growth in the related market.

[0012]

[0013] [Prior Art Literature]

[0014] [Patent Document]

[0015] (Patent Document 1) KR 10-2022-0185484 (2022-12-27)

[0016] (Patent Document 2) KR 10-2015-0085752 (2015-06-17)

[0017]

[0018] The present inventors have made efforts to provide a natural product-derived ingredient that can be utilized in the development of new drugs, and as a result, have confirmed that a carrot extract or a component derived from a fraction thereof has anti-arthritis, anti-inflammatory, antioxidant, anti-aging, or anti-cancer effects, thereby completing the present invention.

[0019] Accordingly, the object of the present invention is to provide anti-arthritic, anti-inflammatory, antioxidant, anti-aging or anti-cancer effects of carrot extract or fractions thereof.

[0020] In addition, an object of the present invention is to provide a novel carrot-derived compound, cis-4-hydroxycinnamyl 4-hydroxybenzoate, and a method for producing the same.

[0021] In addition, an object of the present invention is to provide 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate derived from a novel compound derived from carrots and a method for producing the same.

[0022]

[0023] The present invention provides an anti-aging composition comprising a compound derived from a fraction of a carrot extract.

[0024] According to a preferred embodiment of the present invention, the carrot extract is extracted using water, an organic solvent, or a mixture thereof as a solvent.

[0025] According to a preferred embodiment of the present invention, the fraction is obtained by fractionating a carrot extract using hexane, chloroform, ethyl acetate, butanol, water or a mixture thereof as a solvent.

[0026] According to a preferred embodiment of the present invention, the compound is cis-4-hydroxycinnamyl 4-hydroxybenzoate.

[0027] According to a preferred embodiment of the present invention, the anti-aging is anti-aging against at least one selected from the group consisting of cartilage cells, skin cells, and senescent tumor cells.

[0028] According to a preferred embodiment of the present invention, the composition is a cosmetic composition, a food composition, a quasi-drug composition or a pharmaceutical composition.

[0029] Additionally, the present invention provides an anti-inflammatory composition comprising a compound derived from a fraction of a carrot extract.

[0030] In addition, the present invention provides an antioxidant composition comprising a compound derived from a fraction of a carrot extract.

[0031] In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising a compound derived from a fraction of a carrot extract.

[0032] According to a preferred embodiment of the present invention, the inflammatory disease is at least one selected from the group consisting of asthma, dermatitis, arthritis, cancer, and inflammatory bowel disease.

[0033] In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases, which comprises a compound derived from a fraction of a carrot extract.

[0034] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a compound derived from a fraction of a carrot extract.

[0035] According to a preferred embodiment of the present invention, the cancer is a senescent cancer.

[0036] According to a preferred embodiment of the present invention, the cancer is at least one selected from the group consisting of breast cancer, stomach cancer, skin cancer, colon cancer, liver cancer, bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer, and thyroid cancer.

[0037] In addition, the present invention provides a health functional food composition for preventing or improving cancer comprising a compound derived from a fraction of a carrot extract.

[0038] In addition, the present invention provides an anticancer adjuvant comprising a compound derived from a fraction of a carrot extract.

[0039] In addition, the present invention comprises the steps of i) preparing a methanol extract of carrot;

[0040] ii) a step of preparing an ethyl acetate fraction of the methanol extract;

[0041] iii) a step of purifying the above fraction by silica gel column chromatography;

[0042] iv) a step of purifying the above purified product by flash chromatography; and

[0043] v) A step of purifying the above purified product using HPLC (High performance liquid chromatography);

[0044] A method for producing a compound derived from a carrot fraction including .

[0045] According to a preferred embodiment of the present invention, the compound is an anti-arthritis, anti-inflammatory, anti-aging, antioxidant and anti-cancer compound.

[0046] The present invention also provides a method for treating an inflammatory disease, comprising administering to a subject in need thereof a compound derived from a fraction of a carrot extract.

[0047] The present invention also provides a method for treating cancer, comprising administering to a subject in need thereof a compound derived from a fraction of a carrot extract.

[0048] The present invention provides a compound represented by the following [chemical formula 11], an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0049] [Chemical Formula 11]

[0050]

[0051] (In the above chemical formula 11, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

[0052] According to a preferred embodiment of the present invention, R is hydrogen, hydroxy or methoxy.

[0053] According to a preferred embodiment of the present invention, the compound is cis-4-hydroxycinnamyl 4-hydroxybenzoate.

[0054] In addition, the present invention comprises the steps of: i) producing a compound represented by [chemical formula 4] by performing an acetylation reaction on a compound represented by [chemical formula 3];

[0055] [Chemical Formula 3]

[0056]

[0057] [Chemical Formula 4]

[0058]

[0059] ii) a step of producing a compound represented by [chemical formula 6] by subjecting the above-mentioned compound to a Sonogashira coupling reaction;

[0060] [Chemical Formula 6]

[0061]

[0062] iii) A step of producing a compound represented by [chemical formula 7] by partially hydrogenating the compound produced above;

[0063] [Chemical Formula 7]

[0064]

[0065] iv) a step of producing a compound represented by [chemical formula 10] by performing an esterification reaction on the above-mentioned compound; and

[0066] [Chemical Formula 10]

[0067]

[0068] v) A step of producing a compound represented by [Chemical Formula 11] by subjecting the above-mentioned compound to a deacetylation reaction;

[0069] [Chemical Formula 11]

[0070]

[0071] (In the above chemical formula 11, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

[0072] A method for producing a compound represented by [chemical formula 11], an isomer thereof, or a pharmaceutically acceptable salt thereof is provided.

[0073] According to a preferred embodiment of the present invention, the acetylation reaction of step i) is performed using acetic anhydride (Ac₂O, Acetic Anhydride), TEA (Triethylamine) and CH₂Cl₂ (Dichloromethane).

[0074] According to a preferred embodiment of the present invention, the Sonogashira reaction of step ii) is performed using prop-2-yn-1-ol represented by [chemical formula 5], Pd(PPh₃)₂Cl₂, CuI, TEA and DMF.

[0075] [Chemical Formula 5]

[0076]

[0077] According to a preferred embodiment of the present invention, the partial hydrogenation reaction of step iii) is performed using H₂, Lindlar's catalyst and MeOH.

[0078] According to a preferred embodiment of the present invention, the esterification reaction of step iv) is performed using 4-chlorocarbonylphenyl) acetate represented by [chemical formula 9], DMAP, TEA and CH₂Cl₂.

[0079] [Chemical Formula 9]

[0080]

[0081] According to a preferred embodiment of the present invention, the 4-chlorocarbonylphenyl) acetate is prepared through an acyl chloride synthesis reaction of a compound represented by [chemical formula 8].

[0082] [Chemical Formula 8]

[0083]

[0084] According to a preferred embodiment of the present invention, the deacetylation reaction of step v) is performed using acetic acid; hydrazine and DMF.

[0085] The present invention provides a compound represented by the following [chemical formula 14], an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0086] [Chemical Formula 14]

[0087]

[0088] (In the above chemical formula 14, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

[0089] According to a preferred embodiment of the present invention, R is hydrogen, hydroxy or methoxy.

[0090] According to a preferred embodiment of the present invention, the compound is 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate.

[0091] In addition, the present invention comprises the steps of: i) producing a compound represented by [Chemical Formula 13] by subjecting a compound represented by [Chemical Formula 12] to a nucleophilic substitution reaction; and

[0092] [Chemical Formula 12]

[0093]

[0094] [Chemical Formula 13]

[0095]

[0096] ii) A step of producing a compound represented by [Chemical Formula 14] by subjecting the above-mentioned compound to a nucleophilic substitution reaction;

[0097] [Chemical Formula 14]

[0098]

[0099] (In the above chemical formula 14, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

[0100] A method for producing a compound represented by [Chemical Formula 14], an isomer thereof, or a pharmaceutically acceptable salt thereof is provided.

[0101] According to a preferred embodiment of the present invention, the nucleophilic substitution reaction of step i) is performed using HBr.

[0102] According to a preferred embodiment of the present invention, the nucleophilic substitution reaction of step ii) is performed using 4-hydroxybenzoic acid, DBU and CH₃CN.

[0103]

[0104] The carrot extract of the present invention or a fraction thereof, particularly cis-4-hydroxycinnamyl 4-hydroxybenzoate derived therefrom, has anti-arthritis, anti-inflammatory, antioxidant, anti-aging or anti-cancer effects and can be effectively used in pharmaceutical compositions and / or health functional food compositions.

[0105] “Improvement” or “treatment” of the present invention may mean any action that improves or benefits a parameter, for example, the degree of a symptom, associated with arthritis, inflammation, oxidation, aging or cancer, by the carrot extract or fraction thereof of the present invention.

[0106] The “senescent cancer” of the present invention may refer to a cancer (tumor) cell that does not undergo cell division due to various causes, which is a senescent tumor cell.

[0107] The “isomers” of the present invention may include, in particular, optical isomers (essentially pure enantiomers, essentially pure diastereomers, or mixtures thereof), as well as conformation isomers (isomers that differ only in the angle of one or more chemical bonds), position isomers (tautomers, etc.), or geometric isomers (cis-trans isomers, etc.).

[0108] The “pharmaceutically acceptable salt” of the present invention refers to a salt according to one aspect of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. The salt is formed (1) with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, (2) an acid addition salt formed with an organic acid such as tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, or muconic acid; or (3) a salt formed when an acidic proton present in the parent compound is substituted.

[0109]

[0110] Therefore, the present invention can provide an anti-aging composition comprising a compound derived from a fraction of a carrot extract.

[0111] According to a preferred embodiment of the present invention, the carrot extract may be extracted using water, an organic solvent, or a mixture thereof. Preferably, the carrot extract is extracted using water, a C1 to C4 lower alcohol, or a mixture thereof, and most preferably, methanol.

[0112] According to a preferred embodiment of the present invention, the fraction may be a fraction obtained by fractionating a carrot extract using hexane, chloroform, ethyl acetate, butanol, water, or a mixture thereof as a solvent. Preferably, the fraction may be a fraction obtained by fractionating ethyl acetate, butanol, water, or a mixture thereof as a solvent, and most preferably, a fraction obtained by fractionating ethyl acetate as a solvent.

[0113] According to a preferred embodiment of the present invention, the compound may be cis-4-hydroxycinnamyl 4-hydroxybenzoate. The compound may have a structure represented by the following [Chemical Formula 1].

[0114] [Chemical Formula 1]

[0115]

[0116] According to a preferred embodiment of the present invention, the anti-aging may be anti-aging against at least one selected from the group consisting of cartilage cells, skin cells, and senescent tumor cells.

[0117] According to a preferred embodiment of the present invention, the composition may be a cosmetic composition, a food composition, a quasi-drug composition, or a pharmaceutical composition.

[0118] The cosmetic composition of the present invention contains a compound derived from a fraction of a carrot extract as an active ingredient and can be manufactured in the form of a basic cosmetic composition (a lotion, a skin protectant, a cream, an essence, a cleansing foam and a cleansing water such as a face pack, a body oil, a full-body cleanser), a color cosmetic composition (a foundation, a lipstick, a mascara, a makeup base), a hair product composition (a shampoo, a rinse, a scalp cleanser, a hair conditioner, a hair gel), and a soap, etc., together with a dermatologically acceptable excipient.

[0119] The above excipients are not limited thereto, but may include, for example, skin softeners, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners, and solvents. In addition, fragrances, pigments, bactericides, antioxidants, preservatives, and moisturizers may be additionally included, and thickeners, inorganic salts, synthetic polymers, and the like may be included for the purpose of improving physical properties. For example, when preparing a cleanser and soap using the cosmetic composition of the present invention, the cleanser and soap may be easily prepared by adding a compound derived from a fraction of the carrot extract to a conventional cleanser and soap base. When preparing a cream, the cream may be prepared by adding a compound derived from a fraction of the carrot extract or a salt thereof to a general oil-in-water (O / W) cream base. To this, synthetic or natural materials such as fragrances, chelating agents, pigments, antioxidants, preservatives, and proteins, minerals, and vitamins for the purpose of improving physical properties may be additionally added.

[0120] The content of the compound derived from the fraction of carrot extract contained in the cosmetic composition of the present invention is not limited thereto, but is preferably 0.001 to 10 wt%, and more preferably 0.01 to 5 wt%, based on the total weight of the entire composition. If the content is less than 0.001 wt%, the desired skin regeneration or wound treatment / improvement effect cannot be expected, and if it exceeds 10 wt%, there may be difficulties in safety or formulation manufacturing.

[0121]

[0122] The food composition according to the present invention can be manufactured in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible plant oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.), and the like. In addition, nutritional supplements include, but are not limited to, capsules, tablets, pills, and the like, and the like, and the like, and the like, the like, may be manufactured by adding a compound derived from a fraction of the carrot extract of the present invention. In addition, the food is not limited thereto, but for example, the fraction of the carrot extract of the present invention itself can be manufactured into the form of tea, juice, and drink, and consumed by being liquefied, granulated, encapsulated, and powdered so that it can be consumed (health drink). In addition, in order to use the compound derived from the fraction of the carrot extract of the present invention in the form of a food additive, it can be manufactured and used in the form of a powder or concentrate. In addition, the compound derived from the fraction of the carrot extract of the present invention can be manufactured in the form of a composition by mixing it with a known active ingredient known to have a skin regeneration or wound improvement effect.

[0123] When the compound derived from the fraction of the carrot extract of the present invention is used as a health beverage, the health beverage composition may contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage. The natural carbohydrates mentioned above may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetener may be a natural sweetener such as thaumatin and stevia extract; or a synthetic sweetener such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

[0124] In addition, the compound derived from the fraction of the carrot extract of the present invention can be contained as an effective ingredient of a food composition for skin regeneration or wound improvement, and the amount thereof is not particularly limited to an amount effective to achieve skin regeneration or wound improvement action, but is preferably 0.01 to 100 wt% based on the total weight of the entire composition. The food composition of the present invention can be prepared by mixing the compound derived from the fraction of the carrot extract with other active ingredients known to be effective for skin regeneration or wound improvement.

[0125] In addition to the above, the food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, the food of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination.

[0126]

[0127] The term "quasi-drug" in the present invention refers to products that have a milder effect than pharmaceutical products among products used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases of humans or animals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are products excluding products used for pharmaceutical purposes, and include products used for treating or preventing diseases of humans or animals, products that have a mild effect on the human body or do not act directly, etc.

[0128] When using a compound derived from a fraction of the carrot extract of the present invention as an over-the-counter drug additive, the composition may be added as is or used in combination with other over-the-counter drug ingredients, and may be used appropriately according to conventional methods. The amount of active ingredients mixed may be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment).

[0129]

[0130] The pharmaceutical composition of the present invention may be administered orally or parenterally in various dosage forms. When formulating the composition, it may be prepared using one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, or surfactants, diluents, or excipients.

[0131] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing one or more compounds with at least one excipient, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose or gelatin. For example, tablets or sugar-coated tablets can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture.

[0132] In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, or preservatives may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants in some cases, and anticoagulants, flavoring agents, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, packaging agents, pigments, and preservatives may be additionally included.

[0133] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, or suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0134] The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be formulated in the form of an external skin injection; an intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, epidural, or intracerebrovascular injection; or a transdermal injection; according to a method known in the art.

[0135] In the case of the above injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for the injection include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. In addition, the injection may in most cases additionally include isotonic agents such as sugars or sodium chloride.

[0136] Transdermal administration includes forms such as ointments, creams, lotions, gels, topical solutions, pastes, liniments, and aerosols. Transdermal administration, as described above, refers to topically administering a pharmaceutical composition to the skin, thereby delivering an effective amount of the active ingredient contained in the pharmaceutical composition into the skin.

[0137] The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. That is, the total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered as a fractionated treatment protocol in which multiple doses are administered over a long period of time. It is important to consider all of the above factors and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0138] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0139] The 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.

[0140] The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing a compound derived from a fraction of a carrot extract as an active ingredient. When the pharmaceutical composition for skin regeneration or wound treatment of the present invention is used as an external preparation for skin, it may additionally contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in external preparations for skin, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic active agents, lipophilic active agents, or lipid vesicles. In addition, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

[0141] When the compound derived from the fraction of the carrot extract of the present invention is provided as a topical agent for skin, it may be in the form of, but is not limited to, an ointment, a patch, a gel, a cream, or a spray.

[0142]

[0143] In addition, the present invention can provide an anti-inflammatory composition comprising a compound derived from a fraction of a carrot extract.

[0144] Since the compound derived from the fraction of the above carrot extract is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0145] According to a preferred embodiment of the present invention, the composition may be a cosmetic composition, a food composition, a quasi-drug composition, or a pharmaceutical composition.

[0146] Since the above cosmetic composition, food composition, quasi-drug composition or pharmaceutical composition is the same as the concept used in the above anti-aging composition, the description is replaced with the description thereof.

[0147]

[0148] In addition, the present invention can provide an antioxidant composition comprising a compound derived from a fraction of a carrot extract.

[0149] Since the compound derived from the fraction of the above carrot extract is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0150] According to a preferred embodiment of the present invention, the composition may be a cosmetic composition, a food composition, a quasi-drug composition, or a pharmaceutical composition.

[0151] Since the above cosmetic composition, food composition, quasi-drug composition or pharmaceutical composition is the same as the concept used in the above anti-aging composition, the description is replaced with the description thereof.

[0152]

[0153] In addition, the present invention can provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising a compound derived from a fraction of a carrot extract.

[0154] Since the compound derived from the fraction of the above carrot extract is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0155] According to a preferred embodiment of the present invention, the inflammatory disease may be at least one selected from the group consisting of asthma, dermatitis, arthritis, cancer, and inflammatory bowel disease.

[0156] Since the above pharmaceutical composition is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0157]

[0158] In addition, the present invention can provide a health functional food composition for preventing or improving inflammatory diseases, which comprises a compound derived from a fraction of a carrot extract.

[0159] Since the compound derived from the fraction of the above carrot extract is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0160] According to a preferred embodiment of the present invention, the inflammatory disease may be at least one selected from the group consisting of asthma, dermatitis, arthritis, cancer, and inflammatory bowel disease.

[0161] Since the above health functional food composition is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0162]

[0163] In addition, the present invention can provide a pharmaceutical composition for preventing or treating cancer comprising a compound derived from a fraction of a carrot extract.

[0164] Since the compound derived from the fraction of the above carrot extract is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0165] According to a preferred embodiment of the present invention, the cancer may be a senescent cancer.

[0166] According to a preferred embodiment of the present invention, the cancer may be at least one selected from the group consisting of breast cancer, stomach cancer, skin cancer, colon cancer, liver cancer, bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer, and thyroid cancer.

[0167] Since the above pharmaceutical composition is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0168]

[0169] In addition, the present invention can provide a health functional food composition for preventing or improving cancer, which comprises a compound derived from a fraction of a carrot extract.

[0170] Since the compound derived from the fraction of the above carrot extract is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0171] According to a preferred embodiment of the present invention, the cancer may be a senescent cancer.

[0172] According to a preferred embodiment of the present invention, the cancer may be at least one selected from the group consisting of breast cancer, stomach cancer, skin cancer, colon cancer, liver cancer, bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer, and thyroid cancer.

[0173] Since the above health functional food composition is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0174]

[0175] In addition, the present invention can provide an anticancer adjuvant comprising a compound derived from a fraction of a carrot extract.

[0176] Since the compound derived from the fraction of the above carrot extract is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0177] According to a preferred embodiment of the present invention, the cancer may be a senescent cancer.

[0178] According to a preferred embodiment of the present invention, the cancer may be at least one selected from the group consisting of breast cancer, stomach cancer, skin cancer, colon cancer, liver cancer, bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer, and thyroid cancer.

[0179] The anticancer adjuvant of the present invention refers to any form that enhances the anticancer effect of an anticancer agent or suppresses or ameliorates the side effects of the agent. The anticancer adjuvant of the present invention can be administered in combination with various types of anticancer agents or anticancer adjuvants. When administered in combination, even at lower doses than conventional anticancer agents, the anticancer agent can exhibit an equivalent level of anticancer therapeutic effect, thereby enabling safer anticancer treatment.

[0180] The above-mentioned anticancer adjuvant may be administered via any conventional route as long as it can reach the target tissue. The anticancer adjuvant of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, orally, intrapulmonary, or rectally, depending on the intended purpose, but is not limited thereto. Furthermore, the anticancer adjuvant may be administered via any device capable of transporting the active substance to target cells.

[0181] The anticancer adjuvant of the present invention can be preferably formulated as an anticancer adjuvant by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Carriers, excipients or diluents that can be included in the anticancer treatment adjuvant of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0182] The anticancer adjuvant of the present invention may be a formulation for oral or parenteral administration, and the description of the formulation is replaced with the description of the formulation of the pharmaceutical composition.

[0183]

[0184] In addition, the present invention comprises the steps of i) preparing a methanol extract of carrot;

[0185] ii) a step of preparing an ethyl acetate fraction of the methanol extract;

[0186] iii) a step of purifying the above fraction by silica gel column chromatography;

[0187] iv) a step of purifying the above purified product by flash chromatography; and

[0188] v) A step of purifying the above purified product using HPLC (High performance liquid chromatography);

[0189] A method for producing a compound derived from a carrot fraction including .

[0190] According to a preferred embodiment of the present invention, the compound may be cis-4-hydroxycinnamyl 4-hydroxybenzoate. The compound may have a structure represented by the following [Chemical Formula 1].

[0191] [Chemical Formula 1]

[0192]

[0193] According to a preferred embodiment of the present invention, the compound may be an anti-arthritis, anti-inflammatory, anti-aging, antioxidant and anti-cancer compound.

[0194] The above anti-aging may be anti-aging against at least one selected from the group consisting of cartilage cells, skin cells, and senescent tumor cells.

[0195]

[0196] The present invention also provides a method for treating an inflammatory disease, comprising administering to a subject in need thereof a compound derived from a fraction of a carrot extract.

[0197] Since the compound derived from the fraction of the above carrot extract is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0198] According to a preferred embodiment of the present invention, the inflammatory disease may be at least one selected from the group consisting of asthma, dermatitis, arthritis, cancer, and inflammatory bowel disease.

[0199]

[0200] The present invention also provides a method for treating cancer, comprising administering to a subject in need thereof a compound derived from a fraction of a carrot extract.

[0201] Since the compound derived from the fraction of the above carrot extract is identical to the concept used in the above anti-aging composition, the description is replaced with that description.

[0202] According to a preferred embodiment of the present invention, the cancer may be at least one selected from the group consisting of breast cancer, stomach cancer, skin cancer, colon cancer, liver cancer, bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer, and thyroid cancer.

[0203]

[0204] The present invention can provide a compound represented by the following [chemical formula 11], an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0205] [Chemical Formula 11]

[0206]

[0207] (In the above chemical formula 11, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

[0208] According to a preferred embodiment of the present invention, R may be hydrogen, hydroxy or methoxy.

[0209] According to a preferred embodiment of the present invention, the compound may be cis-4-hydroxycinnamyl 4-hydroxybenzoate.

[0210] The above cis-4-hydroxycinnamyl 4-hydroxybenzoate may also be named (Z)-3-(4-hydroxyphenyl)allyl 4-hydroxybenzoate and may have a structure of the following [chemical formula 1].

[0211] [Chemical Formula 1]

[0212]

[0213]

[0214] In addition, the present invention comprises the steps of: i) producing a compound represented by [chemical formula 4] by performing an acetylation reaction on a compound represented by [chemical formula 3];

[0215] [Chemical Formula 3]

[0216]

[0217] [Chemical Formula 4]

[0218]

[0219] ii) a step of producing a compound represented by [chemical formula 6] by subjecting the above-mentioned compound to a Sonogashira coupling reaction;

[0220] [Chemical Formula 6]

[0221]

[0222] iii) A step of producing a compound represented by [chemical formula 7] by partially hydrogenating the compound produced above;

[0223] [Chemical Formula 7]

[0224]

[0225] iv) a step of producing a compound represented by [chemical formula 10] by performing an esterification reaction on the above-mentioned compound; and

[0226] [Chemical Formula 10]

[0227]

[0228] v) A step of producing a compound represented by [Chemical Formula 11] by performing a deacetylation reaction on the above-mentioned compound;

[0229] [Chemical Formula 11]

[0230]

[0231] (In the above chemical formula 11, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

[0232] A method for producing a compound represented by [chemical formula 11], an isomer thereof, or a pharmaceutically acceptable salt thereof can be provided.

[0233] According to a preferred embodiment of the present invention, the acetylation reaction of step i) may be performed using acetic anhydride (Ac₂O, Acetic Anhydride), TEA (Triethylamine) and CH₂Cl₂ (Dichloromethane).

[0234] According to a preferred embodiment of the present invention, the Sonogashira reaction of step ii) may be performed using prop-2-yn-1-ol represented by [chemical formula 5], Pd(PPh₃)₂Cl₂, CuI, TEA and DMF.

[0235] [Chemical Formula 5]

[0236]

[0237] According to a preferred embodiment of the present invention, the partial hydrogenation reaction of step iii) may be performed using H₂, Lindlar's catalyst and MeOH.

[0238] According to a preferred embodiment of the present invention, the esterification reaction of step iv) may be performed using 4-chlorocarbonylphenyl) acetate represented by [chemical formula 9], DMAP, TEA and CH22Cl2.

[0239] [Chemical Formula 9]

[0240]

[0241] According to a preferred embodiment of the present invention, the 4-chlorocarbonylphenyl) acetate may be prepared through an acyl chloride synthesis reaction of a compound represented by [chemical formula 8].

[0242] [Chemical Formula 8]

[0243]

[0244] According to a preferred embodiment of the present invention, the deacetylation reaction of step v) may be performed using acetic acid; hydrazine and DMF.

[0245] According to a preferred embodiment of the present invention, R may be hydrogen, hydroxy or methoxy.

[0246] According to a preferred embodiment of the present invention, the compound may be cis-4-hydroxycinnamyl 4-hydroxybenzoate.

[0247] The above cis-4-hydroxycinnamyl 4-hydroxybenzoate may also be named (Z)-3-(4-hydroxyphenyl)allyl 4-hydroxybenzoate and may have a structure of the following [chemical formula 1].

[0248] [Chemical Formula 1]

[0249]

[0250]

[0251] In addition, the present invention can provide a compound represented by the following [chemical formula 14], an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0252] [Chemical Formula 14]

[0253]

[0254] (In the above chemical formula 14, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

[0255] According to a preferred embodiment of the present invention, R may be hydrogen, hydroxy or methoxy.

[0256] According to a preferred embodiment of the present invention, the compound may be 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate.

[0257] The above 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate may have a structure of the following [chemical formula 2].

[0258] [Chemical Formula 2]

[0259]

[0260]

[0261] In addition, the present invention comprises the steps of: i) producing a compound represented by [Chemical Formula 13] by subjecting a compound represented by [Chemical Formula 12] to a nucleophilic substitution reaction; and

[0262] [Chemical Formula 12]

[0263]

[0264] [Chemical Formula 13]

[0265]

[0266] ii) A step of producing a compound represented by [Chemical Formula 14] by subjecting the above-mentioned compound to a nucleophilic substitution reaction;

[0267] [Chemical Formula 14]

[0268]

[0269] (In the above chemical formula 14, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

[0270] A method for producing a compound represented by [Chemical Formula 14], an isomer thereof, or a pharmaceutically acceptable salt thereof can be provided.

[0271] According to a preferred embodiment of the present invention, the nucleophilic substitution reaction of step i) may be performed using HBr.

[0272] According to a preferred embodiment of the present invention, the nucleophilic substitution reaction of step ii) may be performed using 4-hydroxybenzoic acid, DBU and CH₃CN.

[0273] According to a preferred embodiment of the present invention, R may be hydrogen, hydroxy or methoxy.

[0274] According to a preferred embodiment of the present invention, the compound may be 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate.

[0275] The above 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate may have a structure of the following [chemical formula 2].

[0276] [Chemical Formula 2]

[0277]

[0278]

[0279] The carrot extract of the present invention or a fraction thereof has anti-arthritis, anti-inflammatory, antioxidant, anti-aging, or anti-cancer effects and can be effectively used in pharmaceutical compositions, health functional food compositions, or therapeutic methods. In addition, the present invention can provide a novel carrot-derived compound, cis-4-hydroxycinnamyl 4-hydroxybenzoate, 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate derived therefrom, and an effective method for preparing the same.

[0280]

[0281] Figure 1 shows a step for obtaining a novel compound derived from a fraction of the carrot extract of the present invention.

[0282] Figure 2 shows the purification of compounds derived from carrot extract using SiO2 gel chromatography eluted with CHCl3: MeOH (10:1). Fraction #4 was obtained and its activity was confirmed. Fraction #4 inhibited inflammation and was analyzed by TLC.

[0283] Figures 3a and 3b show the results of purification of compounds derived from silica fraction #4 using flash chromatography eluted with MeOH (50-100%), and MPLC and TLC analyses thereof.

[0284] Figure 4 A shows the results of HPLC chromatography analysis of a purified sample of carrot extract, B shows the UV absorption spectrum of the HPLC-UV peak appearing at 14.343 minutes in the chromatogram, and C shows the results of TLC analysis of the HPLC-purified sample and development with chloroform and methanol (10:1). Spots were detected with UV light.

[0285] Figure 5a shows the separated compound. 1 Shows the H-NMR spectrum.

[0286] Figure 5b shows the separated compound. 13 Shows the C-NMR spectrum.

[0287] Figure 6a shows the separated compound. 1 H- 1 H shows the COSY spectrum.

[0288] Figure 6b shows the HMQC spectrum of the separated compound.

[0289] Figure 7a shows the HMBC spectrum of the isolated compound.

[0290] Figure 7b shows the HMBC correlation of the separated compounds.

[0291] Figure 8a shows the adducts generated after deconvolution as an analysis for the identification of metabolites by Progenesis QI using ESI (+) lysophospholipid PC(16:0 / 0:0) as a representative example.

[0292] Figure 8b shows the analysis for putative metabolite identification by Progenesis QI using ESI (+) lysophospholipid PC(16:0 / 0:0) as a representative example, showing isotopic similarity and fragment ion assignments matching the chromatogram and database.

[0293] Figure 8c shows the analysis for the putative identification of metabolites by Progenesis QI using ESI (+) lysophospholipid PC(16:0 / 0:0) as a representative example. A shows the UPLC-MRM chromatogram of cis-4-hydroxycinnamyl 4-hydroxybenzoate in Jeju carrot and standard, and B shows the UPLC-MRM MS / MS spectrum in Jeju carrot and standard.

[0294] Figure 9a shows the adducts generated after deconvolution as an analysis for the putative identification of metabolites by Progenesis QI using ESI (-) caffeic acid as a representative example.

[0295] Figure 9b shows the analysis for putative identification of metabolites by Progenesis QI using ESI (-) caffeic acid as a representative example, showing isotopic similarity and assignment of fragment ions matching the chromatogram and database.

[0296] Figure 9c shows an analysis for putative identification of metabolites in an in-house database by Progenesis QI using ESI (+) Falcarindiol as a representative example.

[0297] Figure 10a shows the cell survival effect and inhibitory effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate on the expression of catabolic factors induced by proinflammatory cytokines in primary cultured articular chondrocytes.

[0298] Figure 10b shows the results of a quantitative analysis of the inhibitory effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate on the expression of catabolic factors induced by proinflammatory cytokines in primary cultured articular chondrocytes.

[0299] Figure 11 shows the anti-aging effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) on senescent cartilage cells. A shows the experimental outline of the in vitro study, B shows the results of cell viability analysis of C20A4 senescent cells or normal cells using WST analysis, and C shows phase contrast and SA-β-gal (bright-field) images.

[0300] Figure 12 shows the quantification of senescence-related gene expression in chondrocytes treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) (A). β-Actin served as a loading control. B shows the effect of Doxo-induced senescence on IL-6 release, and C shows a histogram of the cell count analysis of uPAR (CD87) in C20A4 cells.

[0301] Figure 13 shows representative images of ROX green staining in senescent and normal C20A4 cells treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) (A). B shows the results of mitochondrial superoxide dismutase (MitoSOX; red) staining in senescent and normal C20A4 cells.

[0302] Figure 14 shows the results of Western blot analysis of inflammation-related proteins in chondrocytes treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) (A). Figure B shows mitochondrial respiration analysis (Seahorse) in C20A4 cells. The differences in bioenergetic parameters, represented by the areas under the curves corresponding to the stages of basal respiration, discontinuous respiration, maximal respiration, and respiratory capacity reserve, are displayed in the histogram.

[0303] Figure 15 shows the anti-inflammatory and antioxidant activities of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) in RAW 264.7 cells. A shows the results of an MTS assay for the antiproliferative effect after treating RAW 264.7 cells with increasing concentrations of HA for 24 hours, and B shows the antioxidant activity as TEAC and FRAP during the treatment. HA was treated at a concentration of 100 μM.

[0304] Figure 16 shows the antioxidant activity of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) in RAW 264.7 cells. A shows NO production in RAW 264.7 cells (3 × 10 in 6-well plates). 6 (Cells / plate) shows a decrease in HA-induced iNOS. Cells were pretreated with HA before treatment with LPS (1 μg / mL) for 1 day. B shows the inhibitory effect of HA on iNOS mRNA levels. Total RNA was isolated from RAW 264.7 macrophages with the indicated concentrations of HA and then stimulated with LPS (1 μg / mL) for 1 day. The mRNA levels of iNOS were examined by reverse transcription-quantitative polymerase chain reaction. C shows the inhibitory effect of HA on the protein levels of iNOS. Protein lysates were isolated from cells in the presence or absence of the indicated concentrations of HA and then treated with LPS (1 μg / mL) for 24 h.

[0305] Figure 17 shows the effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) on LPS-induced IL-1β and TNF-α mRNA expression in RAW 264.7 cells (A). RAW 264.7 cells were left untreated or pretreated with HA (10, 20, and 30 μM) and then stimulated with LPS (1 μg / mL) for 1 day. Total RNA was isolated from RAW264.7 cells. The mRNA levels of IL-1 and TNF-α were determined using reverse transcription-quantitative polymerase chain reaction. (B) IL-1β and TNF-α production in the culture medium were quantified using an enzyme-linked immunosorbent assay kit.

[0306] Figure 18 shows that cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) inhibits LPS-induced IL-1β, TNF-α production and nuclear translocation of NF-kB (p65) in RAW264.7 cells. A shows the results of macrophages treated with 30 μM HA for 1 day before treatment with LPS (1 μg / mL) for 1 hour, B shows the effect of HA on LPS-stimulated MAPK activation, and C shows the effect of HA on LPS-stimulated ROS accumulation and HA-induced increase in Nrf2 and HO-1 protein levels in RAW 264.7 cells.

[0307] Figure 19 shows the effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) on LPS-stimulated reactive oxygen species accumulation in RAW264.7 macrophages. The effect of HA on reactive oxygen species accumulation was determined using the CellROX Green staining assay. Macrophages were treated with 50 μM HA and NAC (1 mM) for 1 h prior to treatment with LPS (1 μg / mL) for 1 h.

[0308] Figure 20a shows the effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) on carrageenan-induced paw edema. A represents a representative appearance of a mouse paw, B represents the relative paw edema thickness, and C represents a comparison of DPPH radical scavenging activity and catalase (CAT) activity in mouse liver samples.

[0309] Figure 20b shows the effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) on carrageenan-induced paw edema. A shows Western blotting results for TNF-α, IL-1β, and IL-6, and B shows gene expression in HA-treated cells. mRNA levels of TNF-α, IL-1β, and IL-6 were measured by reverse transcription-quantitative polymerase chain reaction.

[0310] Figure 21 shows the effects of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) and HA+doxorubicin on cell viability and SA-β-galactosidase activity in breast cancer cells. A shows the results of a cell proliferation assay performed on breast cancer cells treated with HA or HA+doxorubicin, followed by the addition of HA (0, 20, 40, 60, 80, 100, 120, 150, 200 μM) and HA (0, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500 μM) together with 100 nM doxorubicin. B shows representative images of β-galactosidase activity in response to H2O2-induced senescence in control and HA-treated cells. HA was treated at 20 and 40 μM (MDA-MB-231) and 15 and 30 μM (MCF-7).

[0311] Figures 22a and 22b show the results of suppression of mRNA and protein levels associated with cellular senescence by cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA). Gene expression levels of SASP factors in cellular senescent cells were measured by real-time PCR and Western blotting analysis.

[0312] Figure 23a shows that cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) suppressed cellular senescence-related genes and increased apoptotic activity. The levels of each protein were examined by immunoblot analysis using antibodies. β-Actin was used as a control.

[0313] Figure 23b shows that cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) suppressed cellular senescence-related genes and increased apoptotic activity. The apoptotic cell population (green) increased under the influence of HA. Cells were stained with annexin V-FITC and PI.

[0314] Figure 23c shows that cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) suppressed cellular senescence-related genes and increased apoptotic activity. qPCR analysis revealed telomere shortening (20-30%) in senescent breast cancer cells, which was restored by HA treatment.

[0315] Figure 24 shows the anti-aging effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) in UVB-induced aged skin cells.

[0316] Figure 25 shows the overall reaction scheme for preparing cis-4-hydroxycinnamyl 4-hydroxybenzoate or (Z)-3-(4-hydroxyphenyl)allyl 4-hydroxybenzoate of the present invention.

[0317] Figure 26 shows the results of LC-MS analysis demonstrating the completion of the reaction in [Reaction Scheme 2]. The upper chromatogram was measured at a wavelength of 220 nm, and the lower chromatogram was measured at a wavelength of 254 nm, showing that the same major peak was detected at both wavelengths. The major single peak showed strong signals at 1.036 min (retention time, RT) and 1.037 min (RT), suggesting that the target compound was isolated with high purity. Additionally, a small amount of peak presumed to be a trace impurity or intermediate was detected in the front region (approximately 0.3 to 1.0 min), but the area was relatively insignificant compared to the area of ​​the main peak. These LC-MS results demonstrate that the synthesized compound was obtained with high purity and that the reaction was completed.

[0318] Figure 27 shows the results of LC-MS analysis demonstrating the completion of the reaction in [Scheme 2]. A specific molecular ion peak was detected on the chromatogram, indicating the successful synthesis of the target compound. The major peak in the analyzed mass spectrum matched the expected molecular weight and was used to confirm the presence of impurities or unreacted substances. These results are useful for assessing the purity of the synthesized compound, and the major signals identified through LC-MS analysis are judged to be consistent with the structural characteristics of the compound.

[0319] Figure 28 shows the LC-MS analysis results demonstrating the completion of the reaction in [Scheme 2]. The upper and lower mass spectra show major molecular ion peaks, which are consistent with the expected molecular weight of the synthesized compound. In particular, strong ion signals at 262.9 and 303.9 m / z were detected, which are interpreted as key evidence supporting the presence of the target compound. Additionally, the analysis results can be utilized to confirm the presence of impurities, and the detected ion pattern serves as an important criterion for evaluating the efficiency of the synthetic process. These LC-MS data serve as evidence that the reaction was successful and that the target compound was identified as the main product.

[0320] Figure 29 shows that the reaction of [Reaction Scheme 3] is completed. 1 Shows the results of H-NMR analysis. 1 The H-NMR spectrum clearly showed various chemical shift (δ, ppm) values ​​that can confirm the structure of the synthesized compound. The aromatic proton (Ar-H) signal was observed as a complex multiple peak in the δ 7.12–7.50 ppm region, which corresponds to the hydrogen signal of the benzene ring. In addition, the signals related to allyl or alkoxy protons (-CH=CH-, -OCH3, etc.) were identified as two major peaks around δ 4.28–5.57 ppm, which were consistent with the predicted compound structure. The signals related to methylene (-CH2-) and methyl (-CH3) groups were detected as two separate peaks in the δ 2.00–3.00 ppm region, which provides evidence supporting the presence of the corresponding functional groups. Additionally, characteristic chemical shifts associated with the ester functional group (-COOCH3) were detected at δ4.00–4.50 ppm, suggesting that the ester bond within the synthesized compound was maintained. These analytical results are in good agreement with the predicted structure of the target compound, confirming the successful synthesis.

[0321] Figure 30 shows the results of H NMR analysis showing that the reaction of [Reaction Scheme 4] was completed. In the spectrum, various chemical shift (δ, ppm) signals reflecting the structural features of the synthesized compound were clearly observed. Aromatic protons (Ar-H) appear as multiple peaks in the δ 7.12–7.28 ppm region, which are interpreted as signals supporting the presence of a benzene ring. In addition, peaks related to allyl (-CH=CH-) and alkoxy (-OCH3) protons appear at δ 4.20–4.93 ppm, which shows a result consistent with the allylic structure of the target compound. Signals corresponding to methylene (-CH2-) and methyl (-CH3) groups were observed as strong peaks at δ 2.00–2.50 ppm, which are consistent with the expected structure. In particular, characteristic signals reflecting the ester (-COOCH3) functional group were identified at δ4.00–4.50 ppm, indicating that the synthesized compound maintained the target structure. Overall, the analytical results demonstrate that the synthesis was successful according to [Scheme 4].

[0322] Figure 31 shows the results of 2D NMR analysis showing that the reaction of [Reaction Scheme 4] was completed. The corresponding spectrum is 1 H- 1H correlations, which provide important information for confirming the interactions between protons in the target compound. The peaks appearing along the diagonal represent individual proton signals, and the correlation peaks appearing at intersections off the diagonal suggest that specific protons are adjacent to each other and forming spin-spin couplings (J couplings). In particular, the correlation peaks observed at δ6.44 ppm and δ5.81 ppm, and δ5.81 ppm and δ4.21 ppm support the connection between the aromatic protons and the allyl or alkoxy protons in the compound. This serves as important evidence to confirm that the synthesized structure matches the target compound. In addition, the compound structure and numbered proton configuration information presented on the right side of the spectrum provide useful criteria for interpreting the 2D NMR data, and the results of this analysis can be used as decisive evidence to prove the successful synthesis of the target compound.

[0323] Figure 32 shows the results of 2D NMR analysis showing that the reaction of [Reaction Scheme 4] was completed. This spectrum 1 H- 1Analysis of H correlations provides information on the bonding between protons in the synthesized compound, and serves as important experimental evidence for confirming the structure of the target compound. Diagonal peaks in the spectrum represent the chemical shifts of individual protons, and cross-peaks outside the diagonal indicate J-coupling between protons that are correlated with each other. In particular, correlation peaks such as δ7.26 ppm and δ6.44 ppm, δ6.44 ppm and δ5.81 ppm, and δ5.81 ppm and δ4.21 ppm demonstrate interactions between protons in the aromatic ring and allyl or alkoxy protons, indicating that the synthesized compound matches the expected structure. In addition, matching the positions of each proton with the structural formula of the compound presented on the right can help interpret the 2D NMR data. These results demonstrate the successful synthesis of the target compound.

[0324] Figure 33 shows the results of 2D NMR analysis showing that the reaction of [Reaction Scheme 4] was completed. This analysis 1 H and 13 By confirming the correlation between C, it is used to clearly identify the carbon-hydrogen bond of the target compound. In particular, the signals at δ7.12 ppm to 122.11 ppm, δ7.26 ppm to 130.47 ppm, δ6.44 ppm to 128.54 ppm, and δ5.81 ppm to 134.26 ppm indicate bonds between the carbons of the aromatic ring and the corresponding hydrogens, which shows a result consistent with the expected compound structure. In addition, strong signals were observed at δ4.21 ppm and 58.04 ppm, suggesting carbon-hydrogen bonds of the alkoxy or allyl group. The correspondence of the NMR peaks can be confirmed with the molecular structure presented on the right, which verifies the successful synthesis of the target compound.

[0325] Figure 34 shows the results of H NMR analysis showing that the reaction of [Reaction Scheme 5] was completed. In this spectrum, hydrogen signals of the aromatic ring were identified in the range of δ7.00–8.00 ppm, which shows characteristic chemical shifts corresponding to the benzene ring of the compound. In addition, signals of allyl and alkoxy functional groups were detected at δ5.68–6.84 ppm, which are consistent with the expected structure. Signals of methylene (-CH2-) and methyl (-CH3) groups appeared at δ2.00–2.50 ppm, suggesting the presence of an ester functional group.

[0326] Figure 35 shows the results of LC-MS analysis demonstrating the completion of the reaction in [Scheme 6]. A single major peak (approximately 0.95–0.96 min) was detected at wavelengths of 220 nm and 254 nm in the upper and lower chromatograms, which serves as an important indicator of the identity and purity of the target compound. The signal intensity demonstrated high sensitivity, and peaks of impurities or unreacted substances were relatively low or barely detectable. This confirmed the successful completion of the reaction and the high purity of the synthesized compound.

[0327] Figure 36 shows the results of LC-MS analysis demonstrating the completion of the reaction in [Scheme 6]. In the total ion chromatogram (TIC), a major peak was detected with strong signal intensity at approximately 1.00 min, providing clear evidence for the presence of the target compound. Additionally, some faint peaks, presumed to be reaction intermediates or unreacted reagents, were detected, but with relatively low intensities. This confirms that the reaction primarily proceeded toward the formation of the target compound, ensuring high yield and purity during the synthetic process.

[0328] Figure 37 shows the results of LC-MS analysis demonstrating the completion of the reaction in [Scheme 6]. In the mass spectrometry spectrum, the main molecular ion peak shows a strong signal at m / z 293, indicating that it matches the molecular weight of the target compound. In addition, fragment ion peaks such as m / z 273.2 and 133.1 were observed, showing a pattern consistent with the main structural features of the compound. These results strongly suggest that the reaction proceeded in the desired direction and that the synthesized compound had the targeted molecular weight and structure.

[0329] Figure 38 is an analysis result for cis-4-hydroxycinnamyl 4-hydroxybenzoate of the present invention. 1 The H-NMR spectrum is the result of structural analysis of a compound presumed to be a p-hydroxycinnamic acid derivative. Hydroxy signals appear at approximately 9.4-11 ppm, aryl hydrogens of the benzene ring at 6-8 ppm, olefinic or allyl protons at 4-6 ppm, and methylene or methyl signals at 2-3 ppm. Additionally, solvent peaks of DMSO-d6 and TMS are observed near 2.50 ppm and 0 ppm, respectively. Overall, the analyzed data are consistent with the predicted structure of the compound.

[0330] Figure 39 shows the results of LC-MS analysis of the compound cis-4-hydroxycinnamyl 4-hydroxybenzoate of the present invention, measured at two different wavelengths (220 nm and 254 nm) using a PDA (Photodiode Array) detector. In each chromatogram, a major peak was observed at approximately 1.85 minutes (retention time, RT), which is expected to be the main component of the analyte. In addition, small amounts of other components were detected at times such as 0.53, 0.79, and 1.04 minutes, but since the peak areas were relatively small, they are presumed to be impurities or other trace components. The RT values ​​of the major peaks at the two wavelengths were similar, and the absorbance intensity was detected to be relatively higher at 254 nm.

[0331] Figure 40 shows the LC-MS analysis results for cis-4-hydroxycinnamyl 4-hydroxybenzoate of the present invention. The analysis results were presented in the form of a TIC (Total Ion Chromatogram) using a mass spectrometry (MS) chromatogram, and a main peak was observed at about 1.85 minutes (retention time, RT), indicating the main component of the compound to be analyzed.

[0332] Figure 41 is an LC-MS analysis result for cis-4-hydroxycinnamyl 4-hydroxybenzoate of the present invention, showing the mass spectrum of the compound, and the molecular formula C through the molecular ion peak (m / z 279.2) and fragmentation pattern. 16 H 14O4, a compound with an exact molecular weight of 270.09 Da, was identified. The retention time was detected as 1.847 minutes, suggesting that the synthesized compound elutes stably within a specific time period. Major fragment ions (m / z 133.3, 205.2, 279.2, etc.) were detected in the spectrum, which appear to reflect the structural characteristics of the compound. The analysis results confirmed that the target compound is consistent with a cinnamic acid derivative series compound.

[0333] Figure 42 shows the HPLC analysis results for cis-4-hydroxycinnamyl 4-hydroxybenzoate of the present invention. The analysis was performed at two wavelengths, 220 nm and 254 nm, using a PDA (Photodiode Array) detector, and the main peaks in the chromatogram were detected at retention times (Rt) of 2.576 and 2.577 minutes. This confirms that the compound has a constant Rt and is detected as a single peak, indicating that it has been separated with high purity. Additionally, no separate peaks suspected to be impurities were detected in the chromatogram, confirming that the purity of the material is excellent. This demonstrates that the cis-4-hydroxycinnamyl 4-hydroxybenzoate of the present invention has been successfully synthesized and purified, and that its purity and separation characteristics can be clearly verified through HPLC analysis.

[0334] Figure 43 shows the overall reaction scheme for preparing 3-(4-Hydroxyphenyl)propyl 4-hydroxybenzoate.

[0335] Figure 44 shows that the reaction of [Reaction Scheme 7] is completed. 1 The results of H NMR analysis are shown. The analysis was performed in CDCl3 solvent, and the molecular formula of the target compound is C9H11 BrO, molecular weight was confirmed to be 215 g / mol. In the spectrum, aromatic protons (δ6.17 ~ 7.28 ppm), characteristic signals of alkoxy (-OH) functional groups (δ4.61 ppm), benzyl and methylene (-CH₂-) signals (δ2.00 ~ 3.39 ppm) were observed, indicating that the synthesized compound was consistent with the target structure. In particular, peaks related to bromoalkyl (-CH₂Br) groups appeared at δ3.3 ~ 3.9 ppm, confirming that the reaction proceeded in the desired direction.

[0336] Figure 45 is a diagram of 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate of the present invention. 1 The results of H-NMR analysis are shown. The measurements were performed in DMSO-d6 solvent using a Bruker Avance Neo 400 MHz instrument, and the molecular formula of the compound (C 16 H 16 O4) and molecular weight (272 g / mol) were confirmed. In the spectrum, the main peaks were observed in the multiplets hydrogen signals of the aromatic ring (δ 6.5 ~ 8.0 ppm), singlet peaks of the hydroxyl (-OH) functional group (approximately δ 9.4 ppm and δ 10.1 ppm), allyl and methylene (-CH₂-) regions (δ 2.0 ~ 3.0 ppm), and hydrogen signals of alkoxy and benzyl positions (δ 3.3 ~ 4.2 ppm). This pattern was consistent with the expected structure of the target compound.

[0337] Figure 46 is a diagram of 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate of the present invention. 13 The results of C-NMR analysis are shown. The measurements were performed in DMSO-d6 solvent using a Bruker Avance Neo 100 MHz instrument, and the molecular formula of the compound (C16 H 16 O4) and molecular weight (272 g / mol) were confirmed. In the spectrum, the main carbon peaks were observed in the carbon signals of the aromatic ring (δ115 ~ 160 ppm), the ester carbonyl (C=O) carbon peak (approximately δ166 ppm), and the allyl and propyl carbon regions (δ30 ~ 70 ppm).

[0338] Figure 47 shows the HPLC analysis results of 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate of the present invention. The analysis was performed at wavelengths of 214.4 nm and 254.4 nm using a diode array detector (DAD), and the same single peak was detected at 3.133 minutes (retention time, RT) in each chromatogram, confirming the high purity of the compound. The signal intensities were measured to be approximately 1750 mAU at 214.4 nm and approximately 2000 mAU at 254.4 nm, indicating that the analyzed compound exhibited strong absorbance at those wavelengths. Considering that no impurities or additional peaks were detected in the chromatograms, this means that the compound was separated and purified with high purity according to the HPLC analysis standards. These HPLC results demonstrate the chemical purity and consistency of 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate synthesized in the present invention.

[0339] Figure 48 shows the LCMS analysis results of 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate of the present invention. The analysis was performed at wavelengths of 214.4 nm and 254.4 nm using a diode array detector (DAD), and a major single peak was commonly detected at 1.148 minutes (retention time, RT) in both chromatograms, confirming the high purity of the analyzed compound and whether it was a single component. In addition, a strong ion signal was detected at the same RT (1.148 minutes) in the TIC (Total Ion Chromatogram), and a major peak of -174 m / z appeared, showing a pattern consistent with the expected molecular weight of the compound. These LC-MS analysis results demonstrated the purity, molecular weight, and quantitative analysis of the 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate synthesized in the present invention. Therefore, this substance exists as a single component, suggesting that impurities generated during the synthesis were minimized.

[0340] Figure 49 shows the LC-MS analysis results of 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate of the present invention. The analysis was performed in MM-ES+APCI (Negative Mode), and the main molecular ion peaks (m / z 271.1 and 272.1) were detected in the measured spectrum, confirming that they match the expected molecular weight (MW = 272 g / mol) of the present compound. In addition, the signals at m / z 173.0 and 385.0 represent the main fragment pattern of the compound, and are interpreted as specific ion fragments generated when specific bonds within the molecule are broken during the ionization process. These LC-MS analysis results are important basis for verifying the molecular weight and chemical purity of the compound synthesized in the present invention, and suggest that material identification can be performed more clearly through a unique mass spectrometry pattern.

[0341] Figure 50 shows the HMBC analysis results of 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate of the present invention. The HMBC spectrum shows the long-range neighbor coupling (J 2 and J 3The correlation between the carbons of the aromatic ring (δ115-160 ppm) and the carbons of the propyl (-CH2-CH2-CH3) group (δ20-50 ppm) is clearly observed. In addition, long-range interactions with the carbonyl carbon of the hydroxyl (-OH) and ester bonds (C=O, δ165-170 ppm) were detected, which plays an important role in confirming that the structure of the target compound matches. The spectral analysis can verify that the molecular structure of the synthesized compound matches the expected chemical environment. These results support that the intramolecular bonds of the synthesized compound match the expected chemical environment, and verify that the target material of the present invention was successfully synthesized.

[0342]

[0343] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0344]

[0345] [Example 1]

[0346] Isolation of a novel compound, cis-4-hydroxycinnamyl 4-hydroxybenzoate, from carrots.

[0347] <1-1> Preparation of carrot methanol extract

[0348] After crushing the Jeju carrots, methanol extraction was performed. 7.0 L of methanol was added to 1 kg of crushed carrots to obtain a methanol extract, which was then concentrated, water was added, mixed, and the methanol was evaporated.

[0349]

[0350] <1-2> Preparation of ethyl acetate fraction

[0351] An ethyl acetate extract was obtained by adding an equal amount of ethyl acetate (EA) to the water fraction from which methanol had been evaporated. The EA extract was concentrated, solubilized with methanol, and used in the examples below.

[0352]

[0353] <1-3> Silica gel column chromatography

[0354] The methanol fraction was concentrated and then loaded onto a column (30×450 mm) packed with silica gel 60 (silica gel 60, 0.035-0.2 mm) resin (MERCK, Darmstadt, Germany) and subjected to silica gel chromatography using chloroform:methanol (10:1, v / v) as an eluting solvent to obtain five fractions (fractions 1, 2, 3, 4, and 5). The anti-inflammatory activity of the five fractions was examined, and fraction 4 showed an anti-inflammatory effect, which was concentrated and used as a sample for secondary fractionation. [Figure 2] is a diagram showing the evaluation of fractions 1 to 5 separated by silica gel column chromatography using TLC (Thin layer chromatography). Fraction 4 sample showing anti-inflammatory activity was concentrated and used in the next step <1-4>.

[0355]

[0356] <1-4> Flash Chromatography

[0357] The fraction 4 sample obtained in the above Example <1-3> was loaded into a flash chromatography and flash chromatography was performed using methanol as the eluting solvent. Flash chromatography analysis was performed using Biotage Isolera Spektra One MPLC (Biotage, Uppsala, Sweden). The anticancer and anti-inflammatory activities of fraction 1 of the fractionated sample were evaluated, and [Figure 3] is a diagram evaluating fractions 1, 2, and 3 using MPLC and TLC (Thin Layer Chromatography).

[0358]

[0359] <1-5> HPLC (High performance liquid chromatography)

[0360] The substance 1 obtained through the above flash chromatography was injected into preparatory high-performance liquid chromatography (prep HPLC). HPLC analysis was performed using an Agilent HPLC (Agilent, Santa Clara California, USA). HPLC separation was performed using an ODS (10 × 250 mm) C18 column, and the separated substance was sieved through a 0.2 μm filter. At this time, the injection volume was 0.5 mL, the flow rate was 5 mL / min, the column temperature was room temperature at 254 and 365 nm, and the mobile phase consisted of water (solvent A) and methanol (solvent B). In the case of gradient elution, solvent B was initially set to 50% and increased to 100% at 23 min. The peak of the final isolated and purified effective compound appeared at 14.343 min. [Figure 4] is a diagram showing the HPLC results and the final separated fractions evaluated using TLC (Thin layer chromatography).

[0361]

[0362] [Example 2]

[0363] Identification of a novel compound, cis-4-hydroxycinnamyl 4-hydroxybenzoate.

[0364] <2-1> Analysis of natural products derived from carrots

[0365] Using the natural product cis-4-hydroxycinnamyl 4-hydroxybenzoate isolated from the carrot methanol extract through the above <Example 1>, a presumptive identification analysis of metabolites using Progenesis QI was performed.

[0366] Specifically, in the metabolomic analysis workflow using Progenesis QI, using Lysophospholipid PC (16:0 / 0:0) as a representative example in ESI(+) mode, adducts generated after deconvolution were first identified, and isotopic similarity and chromatograms were analyzed to evaluate the reliability of candidate compounds. In addition, fragment ions generated through MS / MS analysis were compared with a database to assign structures, and UPLC-MRM chromatograms of carrot extracts and standards were compared to identify specific compounds. Finally, MS / MS spectra were compared to confirm whether the compounds detected in carrots were identical to the standards (Figs. 5a to 8c).

[0367] In addition, in order to analyze the structure of the finally separated and purified effective compound, the structure was identified through NMR (nuclear magnetic resonance) analysis, and the molecular weight was measured using an ESI-Mass spectrometer to confirm that the molecular weight was 270 g / mol. Through this, it was confirmed that one of the novel compounds purified from the Jeju carrot of the present invention is cis-4-hydroxycinnamyl 4-hydroxybenzoate (chemical formula 1).

[0368] [Chemical Formula 1]

[0369]

[0370]

[0371] <2-2> Metabolomic analysis of natural products derived from Jeju carrots and identification of new compounds

[0372] A novel compound, 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate (chemical formula 2), which can exhibit activity similar to that of cis-4-hydroxycinnamyl 4-hydroxybenzoate, a natural product isolated from carrot methanol extract, was synthesized.

[0373] [Chemical Formula 2]

[0374]

[0375]

[0376] In the metabolite analysis workflow using Progenesis QI, caffeic acid was used in ESI(-) mode, and falcarindiol was used in ESI(+) mode to identify metabolites. First, adducts generated after deconvolution were identified, and isotopic similarity and chromatogram analysis were used to assess reliability. Then, the structure was assigned by comparing MS / MS fragment ions with a database. In addition, for falcarindiol, an in-house database was used to increase the analysis reliability. Subsequently, NMR (Nuclear Magnetic Resonance) analysis was performed to identify the structure of the synthesized compound, and the molecular weight was measured using an ESI-Mass spectrometer, confirming that the compound had a molecular weight of 272 g / mol. Through this, the novel compound purified from Jeju carrot in this study was finally confirmed to be 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate (Figs. 9a–9c).

[0377]

[0378] [Example 3]

[0379] Anti-arthritic effect of carrot extract or its fraction

[0380] <3-1> MTT cell viability analysis

[0381] Primary cultured chondrocytes were exposed to various concentrations of cis-4-hydroxycinnamyl 4-hydroxybenzoate (0–20 μM). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed 24 h after treatment. Briefly, primary cultured chondrocytes (5 × 10 4) were seeded in 96-well plates. After 24 h of incubation, cis-4-hydroxycinnamyl 4-hydroxybenzoate (0–20 μM) was added to the cells. MTT solution was added to each well for 4 h at the final incubation time. After adding 100 μL of dimethyl sulfoxide solution, the optical density was recorded using a microplate reader (570 nm).

[0382] When cis-4-hydroxycinnamyl 4-hydroxybenzoate was treated (0, 5, 10, 20 uM) to mouse primary chondrocytes, no toxicity was observed to the chondrocytes.

[0383]

[0384] <3-2> Efficacy testing through reverse transcription-polymerase chain reaction (RT-PCR) analysis

[0385] Chondrocytes were isolated from the femoral and tibial plateaus of 4-day-old mice (n = 8) by digesting the cartilage tissue with DMEM supplemented with 0.2% collagenase (Sigma). Passage “0” (P0) primary chondrocytes (3 × 10 5 / 30 mm culture dishes) were maintained as a monolayer in DMEM (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum and antibiotics (100 units / mL penicillin G and 100 μg / mL streptomycin; Gibco, Waltham, MA, USA) for 24 h in a 5% CO2 incubator at 37°C. Chondrocytes were then exposed to various concentrations of cis-4-hydroxycinnamyl 4-hydroxybenzoate (0–20 μM) in the absence or presence of IL-1β (1 ng / mL), IL-6 (100 ng / mL), TNF-α (10 ng / mL), or LPS (10 ng / mL). Total RNA was extracted from primary cultured chondrocytes using TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). RNA quality and concentration were assessed using a NanoDrop™2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA was reverse transcribed, and the resulting cDNA was amplified by PCR. mRNA expression was measured using SYBR premixed Extaq reagent (Takara Bio, Mountain View, CA, USA), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal control. Carrot samples (0, 5, 10, 20 uM) were confirmed to inhibit the transcription of catabolic factors (Mmp3, Mmp13, Adamts 4, Adamts5) induced by inflammatory cytokines IL-1b (1 ng / ml), TNF-a (10 ng / ml), and IL-6 (100 ng / ml). In addition, it was confirmed to inhibit the transcription of catabolic factors (Mmp3, Mmp13, Adamts 4, Adamts5) induced by LPS (10 ng / ml).In addition, in quantitative analysis of transcription factors, the expression of Mmp3 and Mmp13 was inhibited by IL-1b, and the expression of Mmp3, Mmp13, Adamts4, and Adamts5 was inhibited by TNF-a (Fig. 10a, Fig. 10b).

[0386]

[0387] <3-3> Analysis of WST cell viability using senescent cartilage cells

[0388] The anti-aging activity of cis-4-hydroxycinnamyl 4-hydroxybenzoate was confirmed by treating the human chondrocyte cell line C20A4.

[0389] Specifically, to create senescent cells from cartilage cell lines, they were cultured for a total of 6 days, and a low concentration of 25 nM doxorubicin was used as a drug to create senescent cells.

[0390] As a result, cis-4-hydroxycinnamyl 4-hydroxybenzoate was found to be inactive in normal chondrocytes, whereas it was active in aged chondrocytes. In other words, it was confirmed that cis-4-hydroxycinnamyl 4-hydroxybenzoate inhibits the survival of aged cartilage cells (Fig. 11 AB).

[0391]

[0392] <3-4> SA-β-galactosidase staining

[0393] The effect was confirmed using SA-β-galactosidase staining, a well-defined biomarker for cellular senescence. After creating a senescent cell line using the same method as in Example <3-3> above, staining was performed using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, #9860). As a result, it was confirmed that the SA-β-galactosidase staining area was reduced in the area treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate compared to the control group.

[0394]

[0395] <3-5> RT-qPCR

[0396] To compare aging-related gene expression, messenger RNA expression was compared using a real-time PCR system. C20A4 cells were seeded in 6-well plates and cultured for 1 day. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate for 24 hours. Total RNA was purified using the TaKaRa MiniBEST RNA Extraction Kit (TaKaRa, Kyoto, Japan) according to the manufacturer's protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using RNA-direct™SYBR® Green Realtime qPCR Master Mix (Enzynomics, Daejeon, Korea). The RT-qPCR mixture contained 10 μL of SYBR Green Realtime qPCR Master Mix, 1 μL of enzyme, 1 μL of template RNA (100 ng / μL), 1 μL of specific primer-F (10 pmol / μL), 1 μL of specific primer-R (10 pmol / μL), and 6 μL of nuclease-free H2O. Primers used in RT-PCR were purchased from Bioneer (Daejeon, Korea).

[0397] As a result, it was confirmed that the expression of aging-related genes tended to decrease compared to the control group when treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate. In addition, when cytokine changes were confirmed, changes in IL-1b, IL-6, and MMP9 were confirmed (Fig. 12 A).

[0398]

[0399] <3-6> Histogram analysis for uPAR (CD87)

[0400] uPAR, a factor involved in cell motility and survival, may be affected by microenvironmental changes associated with aging. To analyze uPAR (CD87) expression in C20A4 cells, cells were stained using FACS (BD, Flow cytometry) and fluorescence signals were measured. Cells were stained with a uPAR antibody and expression levels were quantitatively assessed using FACS. The analysis results showed that uPAR expression tended to increase in aged cells and was reduced by treatment with cis-4-hydroxycinnamyl 4-hydroxybenzoate (Fig. 12B).

[0401]

[0402] <3-7> Analysis of ROS production and mitochondrial ROS fluorescence expression using fluorescence microscopy

[0403] To evaluate the effects of drug A in aged C20A4 cells, ROX Green and MitoSOX staining were performed. After treating cells with cis-4-hydroxycinnamyl 4-hydroxybenzoate, ROX Green was used to detect reactive oxygen species (ROS) production, and MitoSOX was used to measure mitochondrial oxidative stress. After fluorescent staining, changes were observed using a fluorescence microscope.

[0404] As a result, ROS and mitochondrial stress levels were significantly reduced in the cis-4-hydroxycinnamyl 4-hydroxybenzoate treatment group (Fig. 13).

[0405]

[0406] <3-8> Immunoblot analysis

[0407] After harvesting, cells were lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to an Immobilin-FL polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After blocking with Odyssey® Blocking Buffer (LI-COR, Lincoln, NE, USA) for 1 hour, the blots were incubated with primary antibodies overnight at 4°C. After washing, the blots were incubated for 60 minutes with IRDye 680- and IRDye 800-labeled secondary antibodies diluted in Odyssey Blocking Buffer containing 0.2% Tween-20 / 0.01% sodium dodecyl sulfate. Protein bands were detected using an Odyssey CLx imaging machine (LI-COR).

[0408] In the present invention, we used Western blot analysis to confirm changes in the expression of p65, pERK, ERK, pSTAT3, p-p38, and LC3 proteins. The experiment was conducted using β-actin as an internal control, and protein expression was compared relatively. The results showed significant changes in the protein expression of p65, pERK, and LC3. The increased expression of p65 and pERK suggests the activation of specific signaling pathways, which may be related to the regulation of inflammatory responses and intracellular signaling. Conversely, the change in LC3 expression suggests the possibility of being related to the regulation of the autophagy process. These results provide evidence that HA influences specific signaling pathways and autophagy mechanisms (Figure 14A).

[0409]

[0410] <3-9> Analysis of intracellular mitochondrial metabolism

[0411] The Seahorse kit was used to analyze intracellular mitochondrial metabolism. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured. After culturing the cells under appropriate conditions, mitochondrial respiration and glycolysis activity were assessed using the Seahorse analyzer.

[0412] As a result, significant changes in OCR and ECAR were observed under specific conditions affecting mitochondrial function and energy metabolism. This suggests that mitochondrial metabolism can be regulated according to cellular status and environmental changes.

[0413]

[0414] [Example 4]

[0415] Antioxidant and anti-inflammatory effects of carrot extract or its fractions

[0416] <4-1> Cell lines and culture conditions

[0417] RAW 264.7 macrophage cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Macrophage cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin / streptomycin (Hyclone). Cells were cultured at 37°C in a 5% CO2 atmosphere.

[0418]

[0419] <4-2> Cell proliferation

[0420] RAW 264.7 cells (3×10 6(Cells / plate) were seeded in 96-well plates and cultured for 1 day. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate for 1 day. Cell viability was tested using CellTiter 96 AQueous One Solution (Promega, Madison, WI, USA). After mixing the culture medium and aqueous solution in a 5:1 ratio, 100 μL was added to a 96-well plate and incubated at 37°C for 2 hours. The absorbance was estimated at OD490 using a Molecular Devices (SPECTRAMAX ID5) reader.

[0421]

[0422] <4-3> Iron reducing antioxidant power (FRAP) analysis

[0423] The FRAP working solution was prepared as a solution (300 mM sodium acetate buffer + 10 mM TPTZ + 20 mM FeCl3 = 10:1:1). After adding 150 μl of the FRAP working solution and 50 μl of the extract, the reaction was carried out at 37°C for 30 min. The absorbance was measured at 593 nm using a microplate reader. A standard curve was created based on the value of FeSO4. As a result, it was confirmed that the solution had excellent iron-reducing antioxidant activity, similar to that of FeSO4 used as a standard substance (Fig. 15B).

[0424]

[0425] <4-4> Trolox Equivalent Antioxidant Capacity (TEAC) Analysis

[0426] Free radical scavenging activity was assessed using the Trolox Equivalent Antioxidant Capacity (TEAC). The ABTS solution was mixed with 2,2-azino-bis-(3-ethylbenzo-thiazoline-6-sulfonic acid) and 2.45 mM potassium persulfate. They were reacted for 18 h. Afterwards, 180 μl of the ABTS solution and 20 μl of cis-4-hydroxycinnamyl 4-hydroxybenzoate were mixed and reacted for 5 min. After the reaction, the samples were measured at 734 nm. A standard curve was created by adding Trolox.

[0427] As a result, radical cations were generated by the antioxidant substance cis-4-hydroxycinnamyl 4-hydroxybenzoate, resulting in a blue-green discoloration. For comparison, the results of Trolox and ascorbic acid were also presented as positive controls.

[0428] As a result, it was confirmed that the antioxidant inhibition effect was superior to that of the two substances used as positive controls (Figure 15 B).

[0429]

[0430] <4-5> NO Analysis

[0431] Nitric oxide (NO) is a biological mediator produced by lipopolysaccharide (LPS)-stimulated macrophages. The inhibitory effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (C4H4) on LPS (lipopolysaccharide from E. coli O111:B4; InvivoGen, San Diego, CA, USA)-induced NO production in RAW 264.7 cells was evaluated.

[0432] Specifically, RAW 264.7 cells (3×10 6(Cells / plate) were seeded in 96-well plates and cultured for 1 day. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate for 24 h and LPS (1 μg / mL) for 1 day. NO was assessed in the supernatant culture medium using the NO Plus Detection Kit (iNtRON Biotechnology, Gyeonggi, Korea). 100 μl of culture medium or nitrite standard was pre-induced by adding 50 μL of N1 buffer (sulfanilamide in buffer) to each well of a 96-well plate. The 96-well plate was then incubated for 20 min, after which the mixture was reacted with 50 μL of N2 buffer (naphthyl-ethylenediamine in buffer). After incubating the mixture for 10 min, NO formation was measured as absorbance values ​​at OD560 nm using Molecular Devices (SPECTRAMAX ID5).

[0433] As a result, LPS stimulation induced an increase in NO concentration, whereas the cis-4-hydroxycinnamyl 4-hydroxybenzoate-treated sample group showed a concentration-dependent decrease compared to the LPS-treated control group. Therefore, NO production was induced by LPS, and cis-4-hydroxycinnamyl 4-hydroxybenzoate appeared to reduce LPS-induced NO production (Fig. 16 A).

[0434]

[0435] <4-6> RT-qPCR

[0436] RAW 264.7 cells (3×10 6(Cells / plate) were seeded in 96-well plates and cultured for 1 day. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate (C4H4) together with LPS (1 μg / mL) for 24 h. Total RNA was purified using the TaKaRa MiniBEST RNA Extraction Kit (TaKaRa, Kyoto, Japan) according to the manufacturer's protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using RNA-direct™SYBR® Green Realtime qPCR Master Mix (Enzynomics, Daejeon, Korea). The RT-qPCR mixture contained 10 μL of SYBR Green Realtime qPCR Master Mix, 1 μL of enzyme, 1 μL of template RNA (100 ng / μL), 1 μL of specific primer-F (10 pmol / μL), 1 μL of specific primer-R (10 pmol / μL), and 6 μL of nuclease-free H2O. Primers used in RT-PCR were purchased from Bioneer (Daejeon, Korea).

[0437] As a result, as shown in B of [Fig. 16], LPS-stimulated RAW 264.7 cells showed increased iNOS protein expression, whereas cis-4-hydroxycinnamyl 4-hydroxybenzoate significantly suppressed it. In addition, analysis of the transcription level of iNOS in RAW264.7 cells revealed that LPS increased the iNOS transcript level, whereas cis-4-hydroxycinnamyl 4-hydroxybenzoate decreased the increased transcript levels of iNOS, TNF-a, and IL-1b (Fig. 16 A, Fig. 17 A).

[0438]

[0439] <4-7> ELISA

[0440] The secretion of IL-1β and TNF-α was quantified using an enzyme-linked immunosorbent assay (ELISA) kit from BioLegend. After collecting cell culture supernatants, ELISA was performed according to the manufacturer's protocol, and absorbance was measured at 450 nm.

[0441] As a result, while the LPS-treated group showed a high secretion amount, the production of IL-1β and TNF-α tended to decrease in a concentration-dependent manner according to HA treatment. This suggests that HA regulates the expression of inflammatory cytokines (Fig. 17 B).

[0442]

[0443] <4-8> Immunoblot analysis

[0444] Cells were harvested and lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to an Immobilin-FL polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After blocking with Odyssey® Blocking Buffer (LI-COR, Lincoln, NE, USA) for 1 hour, the blots were incubated with primary antibodies overnight at 4°C. After washing, the blots were incubated for 60 minutes with IRDye 680- and IRDye 800-labeled secondary antibodies diluted in Odyssey blocking buffer containing 0.2% Tween-20 / 0.01% sodium dodecyl sulfate. Protein bands were detected using an Odyssey CLx imaging instrument (LI-COR). Primary antibodies against iNOS were obtained from Cell Signaling Technology (Beverly, MA, USA). β-actin was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

[0445] As a result, as shown in C of [Fig. 16], LPS-stimulated RAW 264.7 cells showed increased iNOS protein expression, whereas cis-4-hydroxycinnamyl 4-hydroxybenzoate inhibited NO production through downregulation of iNOS. While the levels of pERK1 / 2 and pJNK were increased by LPS stimulation, they were decreased compared to the LPS-stimulated control cells when treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate. Therefore, it was confirmed that the LPS-induced inflammatory response of RAW 264.7 cells was suppressed by reducing the levels of pERK and pJNK and inhibiting MAPK signaling. Therefore, it was confirmed that the above compound is associated with the MAPK kinase pathway and that the drug action occurs through the above signaling system (Fig. 18 A, B).

[0446] In [Figure 18] C, the effect of inducing protein expression of HO-1 and NRF2 was analyzed. The Nrf-2 (nuclear factor erythroid-2 like 2; Nfe2I2) and heme oxygenase 1 (HO-1) signaling axis plays a role in protecting multiple organs by reducing oxidative stress in tissues and animal models (multiorgan protector). Therefore, Western blot analysis was performed to evaluate whether cis-4-hydroxycinnamyl 4-hydroxybenzoate could induce the Nrf2 / HO-1 signaling axis. Cis-4-hydroxycinnamyl 4-hydroxybenzoate confirmed protein changes in HO-1 and Nrf2 and changed the expression of Keap1 protein after 3 hours of treatment. Accordingly, it was confirmed that cis-4-hydroxycinnamyl 4-hydroxybenzoate induced the protein levels of HO-1 and Nrf2 (Fig. 18 C).

[0447]

[0448] <4-9> Analysis of ROS expression using fluorescence microscopy

[0449] The effect of HA on LPS-induced ROS (reactive oxygen species) accumulation in RAW264.7 macrophages was evaluated. ROS accumulation was measured by measuring intracellular ROS concentration using Invitrogen CellROX® Green Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Cells were pretreated with HA (50 μM) and NAC (1 mM) for 1 h, followed by treatment with LPS (1 μg / mL) for 1 h. Cells were then stained with CellROX green dye for 10 min at 37°C. After washing with 1X PBS, the stained ROS were visualized using an automated microscope (Cytation5, Agilent, VT, USA). After fluorescent staining, ROS signals were observed using a fluorescence microscope.

[0450] As a result, ROS accumulation tended to decrease in the HA-treated group (Fig. 19). Therefore, it was confirmed that cis-4-hydroxycinnamyl 4-hydroxybenzoate can exert a potent effect as a ROS inhibitor against LPS in RAW 264.7 macrophages.

[0451]

[0452] <4-10> Mouse experiment related to foot edema (inflammation)

[0453] The effect of HA was evaluated in a carrageenan-induced paw edema model. [Fig. 20a] A shows representative changes in mouse paws according to HA treatment, and [Fig. 20a] B compares the relative paw edema thickness. [Fig. 20a] C analyzes DPPH radical scavenging activity and catalase (CAT) activity in mouse liver samples. [Fig. 20b] A confirms TNF-α, IL-1β, and IL-6 protein expression using Western blotting, and [Fig. 20b] B analyzes gene expression changes in HA-treated cells. The mRNA levels of TNF-α, IL-1β, and IL-6 were measured using reverse transcription-quantitative PCR, and the effect of HA on the inflammatory response was evaluated.

[0454] As a result, the thickness of foot edema was significantly reduced in the HA treatment group, and the protein and mRNA expression of inflammatory cytokines also tended to decrease.

[0455]

[0456] [Example 5]

[0457] Anticancer (anti-aging) effects of carrot extract or its fractions

[0458] <5-1> Cell proliferation

[0459] MCF-7, MDA-MB-231 breast cancer cells (1.5 × 10 6(Cells / plate) were seeded in 96-well plates and cultured for 1 day. They were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate and doxorubicin, which is used for senescence, for 1 day. Cell viability was tested using CellTiter 96 AQueous One Solution (Promega, Madison, WI, USA). After mixing the culture medium and aqueous solution in a 5:1 ratio, 100 μL was added to a 96-well plate and incubated at 37°C for 2 hours. The absorbance was estimated at OD490 using a Molecular Devices (SPECTRAMAX ID5) reader. As a result, it was evaluated as non-toxic at the concentration used in the experiment (Fig. 21A).

[0460] The anti-aging activity of cis-4-hydroxycinnamyl 4-hydroxybenzoate was confirmed by treating human breast cancer cell lines MCF-7 and MDA-MD-231. To create senescent cells from breast cancer cell lines, the cells were cultured for a total of 6 days, and a low concentration of 50 nM doxorubicin was used as a drug to create senescent cells. While cis-4-hydroxycinnamyl 4-hydroxybenzoate was inactive in normal breast cancer cells, its activity was confirmed in aged senescent breast cancer cells. As a result, cis-4-hydroxycinnamyl 4-hydroxybenzoate inhibited the survival of senescent breast cancer cells (Fig. 21A).

[0461]

[0462] <5-2> SA-β-galactosidase staining

[0463] The effect was confirmed using SA-β-galactosidase staining, a well-defined biomarker for cellular senescence. After creating senescent cancer cell lines using the same method as in Example <5-1>, staining was performed using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, #9860).

[0464] As a result, it was confirmed that the SA-β-galactosidase staining area was reduced in the area treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate compared to the control group.

[0465]

[0466] <5-3> RT-qPCR

[0467] MDA-MB-231 cells (5×10 5Cells / plate) were seeded in 6-well plates and cultured with doxorubicin for 6 days. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate together with DOX (50 nM) for 2 days. Total RNA was purified using the TaKaRa MiniBEST RNA Extraction Kit (TaKaRa, Kyoto, Japan) according to the manufacturer's protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using RNA-direct™SYBR® Green Realtime qPCR Master Mix (Enzynomics, Daejeon, Korea). The RT-qPCR mixture contained 10 μL of SYBR Green Realtime qPCR Master Mix, 1 μL of enzyme, 1 μL of template RNA (50 ng / μL), 1 μL of specific primer-F (10 pmol / μL), 1 μL of specific primer-R (10 pmol / μL), and 6 μL of nuclease-free H2O. Primers used in RT-PCR were purchased from Bioneer (Daejeon, Korea).

[0468] We tested the effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate on elevated levels of p21, p53, and several senescence-associated genes at the transcriptional level in breast cancer cells using RT-qPCR.

[0469] As a result, as shown in [Figure 22a], doxorubicin treatment increased p21 and p53 gene expression in aged breast cancer cells, whereas cis-4-hydroxycinnamyl 4-hydroxybenzoate suppressed this expression. Therefore, we confirmed that cis-4-hydroxycinnamyl 4-hydroxybenzoate induces apoptosis of senescent cancer cells through downregulation of p21, p53, and several senescence-related genes.

[0470]

[0471] <5-4> Immunoblot analysis

[0472] Normal and senescent cells were harvested and lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to an Immobilin-FL polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After blocking with Odyssey® Blocking Buffer (LI-COR, Lincoln, NE, USA) for 1 hour, the blots were incubated with primary antibodies overnight at 4°C. After washing, the blots were incubated for 60 minutes with IRDye 680- and IRDye 800-labeled secondary antibodies diluted in Odyssey Blocking Buffer containing 0.2% Tween-20 / 0.01% sodium dodecyl sulfate. Protein bands were detected using an Odyssey CLx imaging machine (LI-COR).

[0473] As shown in [Figure 22b], the expression of pp53, p21, and p16 was significantly increased in senescent cells compared to normal cells. This suggests the activation of cell cycle inhibitory proteins during the aging process, demonstrating the enhancement of senescence-related signaling pathways. In contrast, β-actin expression remained constant compared to the control group, serving as an appropriate internal control for the experiment. The present invention suggests that changes in protein expression associated with aging are regulated through specific signaling pathways.

[0474] Furthermore, [Figure 23a] showed that HA dose-dependently decreased the protein levels of YAP1, mTOR, PD-L1, PD-L2, p-AMPK, and GPX4. In particular, the decrease in YAP1 and mTOR suggests that HA may affect cell growth and metabolic regulation, while the simultaneous decrease in PD-L1 and PD-L2 suggests that HA may more broadly modulate the effects of immune evasion mechanisms. The decrease in p-AMPK demonstrates the effects of HA on energy homeostasis and metabolic regulatory pathways, and the decrease in GPX4 suggests the possibility that cytoprotective mechanisms related to lipid peroxidation regulation may be suppressed. This may imply that HA may also be involved in novel cell death pathways such as ferroptosis (iron-dependent apoptosis).

[0475] Taken together, these results suggest that HA may influence cell growth, metabolism, immune regulation, and antioxidant mechanisms by modulating specific signaling pathways in senescent cells, and may contribute to the removal of senescent cells by activating cell death pathways such as ferroptosis.

[0476]

[0477] <5-5> Apoptosis assay

[0478] Apoptosis assay was performed using flow cytometry, using the Accuri™ C6 Plus Flow Cytometer and FITC Annexin V Apoptosis Detection Kit I from BD Biosciences. After treating cells with HA, Annexin V-FITC and PI staining were performed to analyze the degree of apoptosis.

[0479] As a result, the proportion of Annexin V-positive (Early Apoptosis) cells significantly increased in the HA-treated group, suggesting that HA has the effect of promoting early apoptosis. On the other hand, the proportion of PI-positive (Necrosis) cells tended to decrease, confirming the possibility that HA may influence the induction of programmed cell death rather than late apoptosis (Fig. 23b).

[0480]

[0481] <5-6> Telomeres

[0482] In this study, we analyzed telomere length using qPCR and confirmed the effect of HA in aged breast cancer cells. To measure telomere length, qPCR was performed using specific primers, and relative changes were compared with the telomere length of a negative control group. The experimental results showed that telomere length significantly decreased by 20-30% in aged breast cancer cells, suggesting that telomere shortening occurs during cellular aging. However, after HA treatment, telomere length tended to increase again, suggesting that HA may affect telomere maintenance in aged breast cancer cells. These results suggest that HA not only regulates aging-related signaling pathways but also plays a role in restoring telomere stability.

[0483]

[0484] [Example 6]

[0485] Evaluation of anti-aging efficacy in human skin cells

[0486] We aimed to evaluate the antiaging efficacy of cis-4-hydroxycinnamyl 4-hydroxybenzoate in UVB-induced aged skin cells.

[0487] After culturing HaCaT cells, bright-field microscopy was performed using an Agilent Cytation 5 instrument to evaluate morphological changes following UVB irradiation and HA treatment. The experiment was conducted by setting up a control group, a UVB-treated group, and a UVB and HA (1, 2, 5, 10, and 20 μM) treatment group.

[0488] Microscopic analysis revealed that, compared to the control group, UVB-treated cells exhibited increased cell size, morphological changes, and senescence (Fig. 24). In the HA-treated group, a gradual effect was observed starting from 5 μM, and at concentrations of 10 μM and 20 μM, selective killing of senescent cells was observed, while non-senescent cells were not significantly affected at the same concentration. These results suggest that HA exhibits an anti-aging effect by selectively killing senescent cells, and at high concentrations, it exerts its effect on senescent cells, promoting their removal.

[0489]

[0490] [Example 7]

[0491] Preparation of cis-4-hydroxycinnamyl 4-hydroxybenzoate

[0492] <7-1> Preparation of 4-(chlorocarbonyl)phenyl acetate

[0493] [Reaction Formula 1]

[0494]

[0495] A solution of 4-acetoxybenzoic acid (formula 8) (1.50 g, 8.33 mmol) was added to CH2Cl2 (15 mL), and (COCl)2 (1.06 g, 8.33 mmol, 728 μL) and dimethyl formamide (DMF) (60.8 mg, 832 μmol, 64.0 μL) were added. The mixture was stirred at 25 °C for 1 h. TLC (Thin Layer Chromatography; petroleum ether / EtOAc = 3 / 1, Rf = 0.4) showed that the reaction was completed. The reaction mixture was concentrated with the filtrate to yield 4-(chlorocarbonyl)phenyl acetate (formula 9) (1.70 g, crude) as a white solid.

[0496]

[0497] <7-2> Manufacturing of 4-iodophenyl acetate

[0498] [Reaction Formula 2]

[0499]

[0500] To a solution of 4-iodophenol (3) (30.0 g, 136 mmol) in CH2Cl2 (300 mL), TEA (Triethanolamine; 41.3 g, 409 mmol, 56.9 mL) and Ac2O (13.9 g, 136 mmol, 12.8 mL) were added. The mixture was stirred at 20°C for 2 h. LC-MS (Liquid chromatography-mass spectrometry; EB13070-11-P1A) showed that the reaction was complete (Figs. 26-28). The reaction mixture was diluted with water (300 mL) and extracted with EtOAc (300 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated to obtain 4-iodophenyl acetate (4) (36.0 g, crude) as a white oil. LCMS: EB13070-11, m / z = 262.9 (M+H)+, Rt = 1.036 min.

[0501]

[0502] <7-3> Preparation of 4-(3-hydroxyprop-1-yn-1-yl)phenyl acetate

[0503] [Reaction Formula 3]

[0504]

[0505] To a solution of 4-iodophenyl acetate (4) (25.0 g, 55.9 mmol) in DMF (250 mL) were added TEA (16.9 g, 167 mmol, 23.3 mL), CuI (1.07 g, 5.59 mmol), Pd(PPh3)2Cl2 (3.93 g, 5.59 mmol), and prop-2-yn-1-ol (5) (3.39 g, 60.4 mmol, 3.57 mL), followed by degassing and purging with N2 three times. The mixture was stirred at 40 °C under N2 atmosphere for 2 h. TLC (petroleum ether / EtOAc = 3 / 1, Rf = 0.3) showed the reaction was completed. The reaction mixture was diluted with water (200 mL) and extracted with EtOAc (200 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated to obtain a residue, which was purified by column chromatography (SiO2, petroleum ether / EtOAc = 100 / 1~3 / 1) to obtain 4-(3-hydroxyprop-1-yn-1-yl)phenyl acetate (chemical formula 6) (4.80 g, yield 22.8%) as a white solid. This was confirmed by H NMR (EB13070-12-P1D) (Fig. 29).

[0506] 1 H NMR: EB13070-12-P1D (400 MHz, DMSO-d6)

[0507] δ 7.38-7.52 (m, 2H), 7.05-7.18 (m, 2H), 5.34 (t, J = 6.0 Hz, 1H), 4.29 (d, J = 6.0 Hz, 2H), 2.27 (s, 3H)

[0508]

[0509] <7-4> Preparation of (Z)-4-(3-hydroxyprop-1-en-1-yl)phenyl acetate

[0510] [Reaction Formula 4]

[0511]

[0512] To a solution of 4-(3-hydroxyprop-1-yn-1-yl)phenyl acetate (6) (2.96 g, 7.89 mmol) in MeOH (15 mL) was added LINDLAR CATALYST (1.95 g, 5% purity). The mixture was stirred at 20°C under H2 (15 Psi) for 5 h. H NMR (EB13070-16-P1D1) showed the reaction was complete (Figs. 30–33). The mixture was filtered through Celatem and washed with MeOH (100 mL). The filtrate was concentrated under reduced pressure to obtain (Z)-4-(3-hydroxyprop-1-en-1-yl)phenyl acetate (chemical formula 7) (1.50 g, yield 98.9%) as a white solid.

[0513] 1 H NMR: EB13070-16-P1D1 (400 MHz, DMSO-d6)

[0514] δ 7.27 (d, J = 8.4 Hz, 2H), 7.09-7.14 (m, 2H), 6.44 (d, J = 12.0 Hz, 1H), 5.73-5.85 (m, 1H), 4.92 (t, J = 5.2 Hz, 1H), 4.18-4.25 (m, 2H), 2.26(s, 3H)

[0515]

[0516] <7-5> Preparation of (Z)-3-(4-acetoxyphenyl)allyl 4-acetoxybenzoate

[0517] [Reaction Formula 5]

[0518]

[0519] To a solution of (Z)-4-(3-hydroxyprop-1-en-1-yl)phenyl acetate (Formula 7) (1.40 g, 7.28 mmol) in CH2Cl2 (20 mL), TEA (2.21 g, 21.8 mmol, 3.04 mL) and DMAP (4-Dimethylaminopyridine; 88.9 mg, 728 μmol) were added at 0°C, followed by the addition of (4-chlorocarbonylphenyl) acetate (Formula 9) (1.45 g, 7.28 mmol). The mixture was stirred at 0°C for 1.5 h. The reaction was completed according to TLC (petroleum ether / EtOAc = 3 / 1, Rf = 0.5). The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated to obtain a residue, which was purified by column chromatography (SiO2, petroleum ether / EtOAc = 100 / 1~3 / 1) to obtain (Z)-3-(4-acetoxyphenyl)allyl 4-acetoxybenzoate ((Z)-3-(4-acetoxyphenyl)allyl 4-acetoxybenzoate, chemical formula 10) (3.00 g, yield 76.3%) as a white solid (Figure 34).

[0520] 1 H NMR: EB13070-21-P1D (400 MHz, DMSO-d6)

[0521] δ 8.02 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 7.14-7.21 (m, 2H), 6.73 (d, J = 12.0 Hz, 1H), 5.83-6.00 (m, 1H), 5.06-5.08 (m, 2H), 2.29 (d, J = 8.4 Hz, 6H)

[0522]

[0523] <7-6> Preparation of (Z)-3-(4-hydroxyphenyl)allyl 4-hydroxybenzoate

[0524] [Reaction Formula 6]

[0525]

[0526] To a solution of (Z)-3-(4-acetoxyphenyl)allyl 4-acetoxybenzoate ((Z)-3-(4-acetoxyphenyl)allyl 4-acetoxybenzoate, chemical formula 10) (1.70 g, 4.80 mmol) was added DMF (17 mL), and acetic acid; hydrazine (1.99 g, 21.5 mmol) was added. The mixture was stirred at 0°C for 2 hours. LC-MS (EB13070-25-P1A) showed that the reaction was completed (Figs. 35-37). The reaction mixture was concentrated to obtain a residue by filtrating the filtrate, which was purified by prep-SFC (P1: Rt = 1.606; P2: Rt = 1.472; P3: Rt = 1..354; P4: Rt = 1.274) (C column: DAICEL CHIRALPAK AD (250 mm * 50 mm, 10 um); mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 40%, isocratic elution mode) to obtain (Z)-3-(4-hydroxyphenyl)allyl 4-hydroxybenzoate ((Z)-3-(4-hydroxyphenyl)allyl 4-hydroxybenzoate, chemical formula 1) (350 mg, yield 26.9%, purity 100%) as a white solid (Figs. 38-42).

[0527] LCMS: EB13070-25-P1A, m / z = 293.0 (M+Na)+, Rt = 0.956 min.

[0528] 1 H NMR: EB13070-25-P1D2 (400MHz, DMSO-d6)

[0529] δ 9.95-10.76 (m, 1H), 9.28-9.92 (m, 1H), 7.83 (d, J = 8.8Hz, 2H), 7.14 (d, J = 8.4Hz, 2H), 6.75-6.89 (m, 4H), 6.57 (d, J = 12.0Hz, 1H), 5.67-5.75 (m, 1H), 4.96-4.98 (m, 2H) LCMS: EB13070-25-P1B, m / z = 293.0 (M+Na)+, Rt = 1.849 min HPLC: EB13070-25-P1A3

[0530]

[0531] [Experimental Method]

[0532] 1 H NMR spectra were recorded on a Bruker Avance Neo 400 MHz and a Bruker Fourier 300 MHz, with TMS used as an internal standard.

[0533] LCMS was performed on an Agilent LC / MSD 1200 series (column: ODS 2000 (50 × 4.6 mm, 5 μm)) quadrupole mass spectrometer under the following conditions: ES(+) or (-) ionization mode; T=30°C; flow rate=1.5 mL / min; detection wavelength: 214 nm, 254 nm.

[0534]

[0535] [Example 8]

[0536] Preparation of 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate

[0537] <8-1> Synthesis of 4-(3-bromopropyl)phenol

[0538] [Reaction Formula 7]

[0539]

[0540] A solution of 4-(3-hydroxypropyl)phenol (chemical formula 12) (800 mg, 5.27 mmol) in aqueous HBr (15 mL) was stirred at 90°C for 4 h. The reaction mixture was diluted with water (50 mL), extracted with dichloromethane (DCM) (3 × 50 mL), and the organic phases were combined. The mixture was dried over Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM / MeOH = 50:1) to obtain 4-(3-bromopropyl)phenol (chemical formula 13) (786 mg, yield 69.4%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6): δ7.07 (d, J = 8.4Hz, 2H), 6.76 (d, J = 8.8Hz, 2H), 4.61 (s, 1H), 3.38 (t, J = 6.4Hz, 2H), 2.71 (t, J = 7.2Hz, 2H), 2.16-2.09(m, 2H) (Figure 44).

[0541]

[0542] <8-2> Synthesis of 3-(4-Hydroxyphenyl)propyl 4-hydroxybenzoate

[0543] [Reaction Formula 8]

[0544]

[0545] 4-(3-bromopropyl)phenol (4-(3-bromopropyl)phenol, chemical formula 13) (786 mg, 4-hydroxybenzoic acid) (764 mg, 5.53 mmol) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) (562 mg, 3.69 mmol) were added to DMF (10 mL), and the reaction mixture was stirred at 80°C for 16 h. The reaction was quenched with water (50 mL), extracted with DCM (Dichloromethane) (3 x 50 mL), the organic phases were combined, dried over Na2SO4, and concentrated in vacuo. The crude product was purified by HPLC to give 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate (chemical formula 2) (370 mg, yield 37.8%) was obtained as a white solid (Fig. 45-50).

[0546] 1 H-NMR (400 MHz, DMSO-d6): δ9.53(s, 2H), 7.81(d, J = 8.8Hz, 2H), 7.01(d, J = 8.4Hz, 2H), 6.85(d, J = 8.8Hz, 2H), 6.68(d, J = 8.4Hz, 2H), 4.17 (t, J = 6.4 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H), 1.97-1.86 (m, 2H). 13 C-NMR (100 MHz, DMSO-d6): δ 166.08, 162.60, 155.92, 131.87, 131.65, 129.62, 120.82, 115.84, 115.60, 63.88, 31.19, 30.68. LCMS (mobile phase: 95% water (0.02% NH4Ac) in 5% CH3CN to 5% water (0.02% NH4Ac) in 95% CH3CN for 6.5 min, and finally 0.5 min at these conditions), purity: 99.62%, Rt = 3.133 min; MS Calcd.: 272.1; MS Found: 271.1 (MH)-.

[0547]

[0548] The carrot extract or fraction thereof of the present invention has anti-arthritis, anti-inflammatory, anti-oxidant, anti-aging or anti-cancer effects, and thus can be effectively used in pharmaceutical compositions, health functional food compositions or therapeutic methods, and thus has industrial applicability. In addition, the present invention provides a novel carrot-derived compound, cis-4-hydroxycinnamyl 4-hydroxybenzoate, and 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate derived therefrom, and a method for preparing the same, and thus has industrial applicability.

Claims

1. An anti-aging composition comprising a compound derived from a fraction of carrot extract.

2. A composition according to claim 1, characterized in that the carrot extract is extracted using water, an organic solvent, or a mixture thereof as a solvent.

3. In the first paragraph, the fraction is a composition characterized in that the fraction is obtained by fractionating a carrot extract using hexane, chloroform, ethyl acetate, butanol, water or a mixture thereof as a solvent.

4. A composition according to claim 1, characterized in that the compound is cis-4-hydroxycinnamyl 4-hydroxybenzoate.

5. A composition according to claim 1, characterized in that the anti-aging is anti-aging against at least one selected from the group consisting of cartilage cells, skin cells, and senescent tumor cells.

6. A composition according to claim 1, characterized in that the composition is a cosmetic composition, a food composition, a quasi-drug composition, or a pharmaceutical composition.

7. An anti-inflammatory composition comprising a compound derived from a fraction of carrot extract.

8. An antioxidant composition comprising a compound derived from a fraction of carrot extract.

9. A pharmaceutical composition for preventing or treating inflammatory diseases comprising a compound derived from a fraction of carrot extract.

10. A composition according to claim 9, wherein the inflammatory disease is at least one selected from the group consisting of asthma, dermatitis, arthritis, cancer, and inflammatory bowel disease.

11. A health functional food composition for preventing or improving inflammatory diseases, comprising a compound derived from a fraction of carrot extract.

12. A pharmaceutical composition for preventing or treating cancer comprising a compound derived from a fraction of carrot extract.

13. A composition according to claim 12, characterized in that the cancer is senescent cancer.

14. A composition according to claim 12, wherein the cancer is at least one selected from the group consisting of breast cancer, stomach cancer, skin cancer, colon cancer, liver cancer, bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer, and thyroid cancer.

15. A health functional food composition for preventing or improving cancer comprising a compound derived from a fraction of carrot extract.

16. An anticancer adjuvant comprising a compound derived from a fraction of carrot extract. 17.i) Step of preparing a methanol extract of carrot; ii) a step of preparing an ethyl acetate fraction of the methanol extract; iii) a step of purifying the above fraction by silica gel column chromatography; iv) a step of purifying the above purified product by flash chromatography; and v) A step of purifying the above purified product using HPLC (High performance liquid chromatography); A method for producing a compound derived from a carrot fraction comprising:

18. A manufacturing method according to claim 17, wherein the compound is an anti-arthritis, anti-inflammatory, anti-aging, antioxidant and anti-cancer compound.

19. A method for treating an inflammatory disease, comprising administering to a subject in need thereof a compound derived from a fraction of a carrot extract.

20. A method for treating cancer, comprising administering to a subject in need thereof a compound derived from a fraction of a carrot extract.

21. A compound represented by the following [chemical formula 11], an isomer thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula 11] (In the above chemical formula 11, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

22. A compound, an isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that in claim 21, R is hydrogen, hydroxy, or methoxy.

23. A compound, an isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that the compound in claim 21 is cis-4-hydroxycinnamyl 4-hydroxybenzoate. 24.i) A step of producing a compound represented by [Chemical Formula 4] by performing an acetylation reaction on a compound represented by [Chemical Formula 3]; [Chemical Formula 3] [Chemical Formula 4] ii) a step of producing a compound represented by [chemical formula 6] by subjecting the above-mentioned compound to a Sonogashira coupling reaction; [Chemical Formula 6] iii) A step of producing a compound represented by [chemical formula 7] by partially hydrogenating the compound produced above; [Chemical Formula 7] iv) a step of producing a compound represented by [chemical formula 10] by performing an esterification reaction on the above-mentioned compound; and [Chemical Formula 10] v) A step of producing a compound represented by [Chemical Formula 11] by performing a deacetylation reaction on the above-mentioned compound; [Chemical Formula 11] (In the above chemical formula 11, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro) A method for producing a compound represented by [chemical formula 11], an isomer thereof, or a pharmaceutically acceptable salt thereof.

25. A manufacturing method according to claim 24, characterized in that the acetylation reaction of step i) is performed using acetic anhydride (Ac₂O, Acetic Anhydride), TEA (Triethylamine) and CH₂Cl₂ (Dichloromethane).

26. A manufacturing method characterized in that, in the 24th paragraph, the Sonogashira reaction of step ii) is performed using prop-2-yn-1-ol represented by [chemical formula 5], Pd(PPh₃)₂Cl₂, CuI, TEA and DMF. [Chemical Formula 5] 27. A manufacturing method according to claim 24, characterized in that the partial hydrogenation reaction of step iii) is performed using H₂, Lindlar's catalyst and MeOH.

28. A manufacturing method characterized in that in the 24th paragraph, the esterification reaction of step iv) is performed using 4-chlorocarbonylphenyl) acetate represented by [chemical formula 9], DMAP, TEA and CH₂Cl₂. [Chemical Formula 9] 29. A manufacturing method according to claim 28, wherein the 4-chlorocarbonylphenyl) acetate is manufactured through an acyl chloride synthesis reaction of a compound represented by [chemical formula 8]. [Chemical Formula 8] 30. A manufacturing method characterized in that in the 24th paragraph, the deacetylation reaction of step v) is performed using acetic acid; hydrazine and DMF.

31. A compound represented by the following [chemical formula 14], an isomer thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula 14] (In the above chemical formula 14, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro).

32. A compound, an isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that in claim 31, R is hydrogen, hydroxy, or methoxy.

33. A compound, an isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that the compound in claim 31 is 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate. 34.i) A step of producing a compound represented by [Chemical Formula 13] by performing a nucleophilic substitution reaction on a compound represented by [Chemical Formula 12]; and [Chemical Formula 12] [Chemical Formula 13] ii) A step of producing a compound represented by [Chemical Formula 14] by subjecting the above-mentioned compound to a nucleophilic substitution reaction; [Chemical Formula 14] (In the above chemical formula 14, R is hydrogen, hydroxy, methoxy, C1 to C4 straight or branched alkyl, halogen, or nitro) A method for producing a compound represented by [chemical formula 14], an isomer thereof, or a pharmaceutically acceptable salt thereof.

35. A manufacturing method according to claim 34, characterized in that the nucleophilic substitution reaction of step i) is performed using HBr.

36. A manufacturing method according to claim 34, characterized in that the nucleophilic substitution reaction of step ii) is performed using 4-hydroxybenzoic acid, DBU and CH₃CN.

Images