Compositions comprising carrot extract or fractions thereof and uses thereof

Abstract

The carrot extract of the present invention or a fraction thereof, in particular cis-4-hydroxycinnamyl 4-hydroxybenzoate derived therefrom, has anti-arthritic, anti-inflammatory, antioxidant, anti-aging, or anticancer effects and can be effectively used in pharmaceutical compositions and / or compositions for health functional foods.

Description

Technology Field

[0001] The present invention relates to the anti-arthritis, anti-inflammatory, antioxidant, anti-aging, or anticancer effects of carrot extract or fractions thereof. Background Technology

[0003] carrot; Daucus carota subsp. sativus It is a biennial plant of the Apiaceae family, order Apiaceae, class Dicotyledon. It is also called red carrot and grows to a height of about 1m. A variety similar to the carrots commonly cultivated today was improved in France and was widely distributed throughout Europe by the 13th century.

[0004] Carrots contain various types of vitamins, including B vitamins, vitamin C, and vitamin K. In addition, they contain a substance called beta-carotene, which is converted into vitamin A in the human body, and are known to be rich in sugar and iron.

[0005] Natural products are less toxic than synthetic compounds and possess a proven track record of efficacy and safety with relatively few side effects through extensive clinical experience; consequently, they tend to require less time, cost, and have a lower probability of failure compared to conventional new drug development processes. Furthermore, alongside the rapid increase in disease prevalence driven by factors such as increased life expectancy, changes in lifestyle patterns, and climate change, consumer interest in natural products is also growing, leading to an annual growth in the related market size. Prior art literature

[0007] KR 10-2022-0185484 (2022-12-27) The problem to be solved

[0008] As a result of diligent efforts to provide a component derived from a natural product that can be utilized in new drug development, the inventors confirmed that a component derived from a carrot extract or a fraction thereof has anti-arthritis, anti-inflammatory, antioxidant, anti-aging, or anticancer effects, and completed the present invention.

[0009] Accordingly, the object of the present invention is to provide anti-arthritic, anti-inflammatory, antioxidant, anti-aging, or anticancer effects of carrot extract or fractions thereof. means of solving the problem

[0011] The present invention provides an anti-aging composition comprising a compound derived from a fraction of carrot extract.

[0012] 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.

[0013] 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.

[0014] According to a preferred embodiment of the present invention, the compound is cis-4-hydroxycinnamyl 4-hydroxybenzoate.

[0015] According to a preferred embodiment of the present invention, the anti-aging is an anti-aging of one or more selected from the group consisting of chondrocytes, skin cells, and senescent tumor cells.

[0016] 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.

[0017] In addition, the present invention provides an anti-inflammatory composition comprising a compound derived from a fraction of carrot extract.

[0018] In addition, the present invention provides an antioxidant composition comprising a compound derived from a fraction of carrot extract.

[0019] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a compound derived from a fraction of carrot extract.

[0020] According to a preferred embodiment of the present invention, the inflammatory disease is one or more selected from the group consisting of asthma, dermatitis, arthritis, cancer, and inflammatory bowel disease.

[0021] In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases comprising a compound derived from a fraction of carrot extract.

[0022] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a compound derived from a fraction of carrot extract.

[0023] According to a preferred embodiment of the present invention, the cancer is aging cancer.

[0024] According to a preferred embodiment of the present invention, the cancer is one or more selected from the group consisting of breast cancer, stomach cancer, skin cancer, colorectal cancer, liver cancer, bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer, and thyroid cancer.

[0025] In addition, the present invention provides a health functional food composition for preventing or improving cancer comprising a compound derived from a fraction of carrot extract.

[0026] In addition, the present invention provides an anticancer adjuvant comprising a compound derived from a fraction of carrot extract.

[0027] In addition, the present invention comprises i) a step of preparing a methanol extract of carrots;

[0028] ii) a step of preparing an ethyl acetate fraction of the methanol extract;

[0029] iii) a step of purifying the above fraction by silica gel column chromatography;

[0030] iv) a step of purifying the above purified product by flash chromatography; and

[0031] v) A step of purifying the above purified product using HPLC (High performance liquid chromatography);

[0032] A method for preparing a compound derived from a carrot fraction containing

[0033] According to a preferred embodiment of the present invention, the compound is a compound for anti-arthritis, anti-inflammation, anti-aging, antioxidant, and anticancer purposes. Effects of the invention

[0035] The carrot extract or fraction thereof of the present invention has anti-arthritis, anti-inflammatory, antioxidant, anti-aging, or anticancer effects and can be effectively used in pharmaceutical compositions and / or compositions for health functional foods. Brief explanation of the drawing

[0037] Figure 1 illustrates the step of obtaining a novel compound derived from a fraction of the carrot extract of the present invention. Figure 2 shows the purification of a compound 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 the expression of inflammation and was analyzed by TLC. Figures 3a and 3b show the results of purifying compounds derived from silica fraction #4 using flash chromatography eluted with MeOH (50-100%) and analyzing them via MPLC and TLC. Figure 4A shows the results of analyzing a purified sample of carrot extract using an HPLC chromatogram, Figure 4B shows the UV absorption spectrum of the HPLC-UV peak appearing at 14.343 minutes in the chromatogram, and Figure 4C shows the results of analyzing the HPLC-purified sample using TLC and developing it with chloroform and methanol (10:1). Spots were detected using UV light. Figure 5a shows the isolated compound. 1 Shows the H-NMR spectrum. Figure 5b shows the isolated compound. 13 It shows the C-NMR spectrum. Figure 6a shows the isolated compound. 1 H- 1It shows the H COSY spectrum. Figure 6b shows the HMQC spectrum of the isolated compound. Figure 7a shows the HMBC spectrum of the isolated compound. Figure 7b shows the HMBC correlation of the isolated compounds. Figure 8a shows the adducts generated after deconvolution as an analysis for the identification of metabolite estimation by Progenesis QI using ESI (+) lysophospholipid PC (16:0 / 0:0) as a representative example. Figure 8b shows the isotopic similarity and fragment ion assignments that match the chromatogram and database as an analysis for the identification of metabolite estimates by Progenesis QI using ESI (+) lysophospholipid PC(16:0 / 0:0) as a representative example. Figure 8c shows the analysis for the identification of metabolite estimates 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 carrots and standards, and B shows the UPLC-MRM MS / MS spectrum of Jeju carrots and standards. Figure 9a shows the adducts generated after deconvolution as an analysis for the estimation identification of metabolites by Progenesis QI using ESI (-) caffeic acid as a representative example. Figure 9b shows the assignment of fragment ions that match isotopic similarity and chromatograms and databases as an analysis for the presumptive identification of metabolites by Progenesis QI using ESI (-) caffeic acid as a representative example. Figure 9c shows an analysis for the estimation identification of metabolites in an in-house database by Progenesis QI using ESI (+) falcarindiol as a representative example. Figure 10a shows the cell survival effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate and the inhibitory effect on catabolic factor expression induced by pro-inflammatory cytokines in primary cultured articular chondrocytes. Figure 10b shows the results of a quantitative analysis of the inhibitory effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate on catabolic factor expression induced by pro-inflammatory cytokines in primary cultured articular chondrocytes. Figure 11 confirms the anti-aging effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) on chondrogenic cells. A shows an experimental overview 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 (brightfield) images. Figure 12 shows the results of quantifying aging-related gene expression when chondrochronous cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) (A). β-actin was used as a loading control. B shows the effect of Doxo-induced aging on IL-6 release, and C shows the histogram of the cell count analysis of uPAR (CD87) in C20A4. Figure 13 shows representative images of ROX green staining of aged and normal C20A4 cells treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) (A). B shows the results of mitochondrial superoxide (MitoSOX; red) staining of aged and normal C20A4 cells. Figure 14 shows the results of Western blot analysis of inflammation-related proteins when chondrogenic cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) (A). B shows the analysis of mitochondrial respiration in C20A4 cells (Seahorse). Differences in bioenergetic parameters in the areas under the curves corresponding to the stages of basal respiration, discontinuous respiration, maximal respiration, and spare respiration capacity are displayed in the histogram. Figure 15 shows the anti-inflammatory and antioxidant activities of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) in RAW 264.7 cells. A is the result of the MTS analysis for the antiproliferative effect after treating RAW 264.7 cells with increasing HA concentrations for 24 hours, and B shows the antioxidant activity as TEAC and FRAP during treatment. HA was treated at a concentration of 100 µM. Figure 16 shows the antioxidant activity of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) in RAW 264.7 cells. A indicates NO production in RAW 264.7 cells (3 × 10⁶ in a 6-well plate). 6This indicates the reduction caused by HA in (dog cells / plate). 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 concentration of HA and then stimulated with LPS (1 μg / mL) for 1 day. iNOS mRNA levels were examined by reverse transcription polymerase chain reaction. C shows the inhibitory effect of HA on iNOS protein levels. Protein lysates were isolated from cells with or without the indicated concentration of HA and then treated with LPS (1 μg / mL) for 24 hours. 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 either untreated or pretreated with HA (10, 20, and 30 µM) followed by stimulation with LPS (1 µg / mL) for 1 day. Total RNA was isolated from RAW 264.7 cells. mRNA levels of IL-1β and TNF-α were determined using reverse transcription polymerase chain reaction. B shows IL-1β, and TNF-α production in the culture medium was quantified using an enzyme-linked immunosorbent assay kit. 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 treating macrophages with 30 μM HA for 1 day before treating them 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. 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 confirmed using the CellROX Green staining assay. Macrophages were treated with 50 µM HA and NAC (1 mM) for 1 hour before treatment with LPS (1 µg / mL) for 1 hour. Figure 20a shows the effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) on carrageenan-induced foot edema. A shows a representative appearance of a mouse foot, B shows the relative thickness of the foot edema, and C shows a comparison of DPPH radical scavenging activity and catalase (CAT) activity in mouse liver samples. Figure 20b shows the effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) on carrageenan-induced foot edema. A shows the Western blotting results of 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 polymerase chain reaction. Figure 21 shows the effects of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) and HA+doxorubicin on the cell viability and SA-β-galactosidase activity of breast cancer cells. A shows the results of performing a cell proliferation assay in breast cancer cells with 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) and 100 nM doxorubicin after treating cells with HA or HA+doxorubicin. B shows representative images of β-galactosidase activity against H2O2-induced aging in control and HA-treated cells. HA was treated at 20, 40 μM (MDA-MB-231) and 15, 30 μM (MCF-7). Figures 22a and 22b show the results of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) inhibiting cellular senescence-related mRNA and protein levels. The gene expression levels of the SASP factor in cellular senescent cells were measured by real-time PCR and Western blotting analysis. Figure 23a shows that cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) inhibited 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. Figure 23b shows that cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) inhibited 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. Figure 23c shows that cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) inhibited cellular senescence-related genes and increased apoptotic activity. qPCR analysis showed telomere shortening (20-30%) in senescent breast cancer cells, which means that it was restored by HA treatment. Figure 24 shows the anti-aging efficacy of cis-4-hydroxycinnamyl 4-hydroxybenzoate (HA) in UVB-induced aged skin cells. Specific details for implementing the invention

[0038] The present invention will be described in more detail below.

[0040] The carrot extract of the present invention or a fraction thereof, in particular cis-4-hydroxycinnamyl 4-hydroxybenzoate derived therefrom, has anti-arthritic, anti-inflammatory, antioxidant, anti-aging, or anticancer effects and can be effectively used in pharmaceutical compositions and / or compositions for health functional foods.

[0041] The “improvement” or “treatment” of the present invention may mean any act that causes parameters related to arthritis, inflammation, oxidation, aging, or cancer, such as the degree of symptoms, to be improved or beneficial due to the carrot extract or fractions thereof of the present invention.

[0042] The “aging cancer” of the present invention refers to aging tumor cells, which may mean cancer (tumor) cells that do not undergo cell division due to various causes.

[0044] Accordingly, the present invention can provide an anti-aging composition comprising a compound derived from a fraction of carrot extract.

[0045] According to a preferred embodiment of the present invention, the carrot extract may be extracted using water, an organic solvent, or a mixture thereof as a solvent. Preferably, it is extracted using water, a C1 to C4 lower alcohol, or a mixture thereof as a solvent, and most preferably, it is extracted using methanol as a solvent.

[0046] According to a preferred embodiment of the present invention, the fraction may be 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 obtained by fractionating using ethyl acetate, butanol, water, or a mixture thereof as a solvent, and most preferably, it is obtained by fractionating using ethyl acetate as a solvent.

[0047] According to a preferred embodiment of the present invention, the compound may be cis-4-hydroxycinnamyl 4-hydroxybenzoate. The compound may have the structure of [Chemical Formula 1] below.

[0048] [Chemical Formula 1]

[0049]

[0050] According to a preferred embodiment of the present invention, the anti-aging may be an anti-aging effect on one or more selected from the group consisting of chondrocytes, skin cells, and senescent tumor cells.

[0051] 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.

[0052] The cosmetic composition of the present invention contains a compound derived from a fraction of carrot extract as an active ingredient and can be prepared in the form of a basic cosmetic composition (toner, skin protectant, cream, essence, facial cleanser such as cleansing foam and cleansing water, pack, body oil, body cleanser), a color cosmetic composition (foundation, lipstick, mascara, makeup base), a hair product composition (shampoo, rinse, scalp cleanser, hair conditioner, hair gel), and soap, together with dermatologically acceptable excipients.

[0053] The above excipients are not limited thereto but may include, for example, skin softeners, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners, and solvents. Additionally, fragrances, colorants, disinfectants, antioxidants, preservatives, and moisturizers may be additionally included, and thickeners, inorganic salts, synthetic polymer materials, etc. may be included for the purpose of improving physical properties. For example, when preparing facial cleansers and soaps using the cosmetic composition of the present invention, they can be easily prepared by adding a compound derived from the fraction of the carrot extract to a conventional facial cleanser or soap base. When preparing a cream, it can be prepared by adding a compound derived from the fraction of the carrot extract or a salt thereof to a general oil-in-water (O / W) cream base. In addition to this, fragrances, chelating agents, colorants, antioxidants, preservatives, etc., and synthetic or natural materials such as proteins, minerals, and vitamins for the purpose of improving physical properties may be additionally added.

[0054] 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 weight% with respect to the total weight of the composition, and more preferably 0.01 to 5 weight%. If the content is less than 0.001 weight%, the desired skin regeneration or wound treatment / improvement effect cannot be expected, and if it exceeds 10 weight%, there may be difficulties in safety or formulation preparation.

[0056] The food composition according to the present invention can be prepared in various forms according to conventional methods known in the art. General foods may be prepared by adding a fraction of the carrot extract of the present invention to beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and its processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, malt syrup, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., soybean paste, soy sauce, sauces, etc.). Additionally, nutritional supplements may be prepared by adding a compound derived from the fraction of the carrot extract of the present invention to capsules, tablets, pills, etc., although not limited thereto. In addition, although not limited to this, the fraction of the carrot extract of the present invention itself may be consumed by liquefying, granulating, encapsulating, and powdering it so that it can be prepared in the form of tea, juice, and drink (health beverage). Furthermore, to use the compound derived from the fraction of the carrot extract of the present invention as a food additive, it may be prepared and used in the form of a powder or a concentrate. Additionally, the compound derived from the fraction of the carrot extract of the present invention may be prepared in the form of a composition by mixing it with a known active ingredient known to have skin regeneration or wound improvement effects.

[0057] When a compound derived from a fraction of the carrot extract of the present invention is used as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The aforementioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract; or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrates 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.

[0058] In addition, the compound derived from the fraction of the carrot extract of the present invention may be included as an active ingredient in a food composition for skin regeneration or wound improvement. The amount is not specifically limited to an amount effective for achieving skin regeneration or wound improvement, but it is preferable to be 0.01 to 100 weight% with respect to the total weight of the composition. The food composition of the present invention may 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 wounds.

[0059] In addition to the above, the food of the present invention may contain various nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. Furthermore, the food of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. These ingredients may be used independently or in combination.

[0061] The term "quasi-drug" in the present invention refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases of humans or animals, and having a milder effect than pharmaceuticals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are defined as articles excluding those used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases of humans or animals, and products that have a mild effect on the human body or do not act directly on it.

[0062] When a compound derived from a fraction of the carrot extract of the present invention is used as an additive to a quasi-drug, the composition may be added as is or used together with other quasi-drug ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

[0064] The pharmaceutical composition of the present invention may be in various oral or parenteral formulations. When formulating the composition, it may be prepared using one or more buffers (e.g., saline solution or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

[0065] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient with one or more compounds, for example, 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 hydroxypropylmethylcellulose, or gelatin, etc. For example, tablets or sugar-coated tablets can be obtained by combining an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and processing it into a granular mixture.

[0066] In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, or syrups, and may contain various excipients, such as humectants, sweeteners, flavorings, or preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants, and may additionally include anticoagulants, flavorings, emulsifiers, solubilizers, dispersants, flavorings, antioxidants, packaging agents, pigments, and preservatives.

[0067] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, or suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cocoa paste, laurin paste, glycerol, gelatin, etc. may be used as bases for suppositories.

[0068] The composition of the present invention may be administered orally or parenterally, and when administered parenterally, it may be formulated according to methods known in the art in the form of an external application for the skin; an injectable for injection into the abdominal cavity, rectum, vein, muscle, subcutaneous, intrauterine dura mater or cerebral blood vessel; or a transdermal application.

[0069] The above-mentioned injectable must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Suitable carriers for the injectable may include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils, as solvents or dispersion media. More preferably, suitable carriers may include Hanks' solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the injectable from microbial contamination, various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. Additionally, the injectable may, in most cases, further include isotonic agents such as sugars or sodium chloride.

[0070] Transdermal administration methods include forms such as ointments, creams, lotions, gels, topical solutions, pastes, liniments, and aerosols. In the above, transdermal administration means administering a pharmaceutical composition topically to the skin so that an effective amount of active ingredients contained in the pharmaceutical composition are delivered into the skin.

[0071] 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, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. That is, the total effective amount of the composition of the present invention may be administered to the patient as a single dose, or administered via a fractionated treatment protocol involving long-term administration of multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all the aforementioned factors, and this can be easily determined by a person skilled in the art.

[0072] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's body weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease.

[0073] 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.

[0074] The pharmaceutical composition of the present invention may also be provided in the form of a topical formulation comprising a compound derived from a fraction of carrot extract as an active ingredient. When the pharmaceutical composition of the present invention for skin regeneration or wound treatment is used as a topical application for the skin, it may additionally contain adjuvants commonly used in the field of dermatology, such as fatty substances, organic solvents, solvents, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, non-ionic emulsifiers, fillers, metal ion chelating agents, chelating agents, preservatives, vitamins, blockers, humectants, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators, or lipid vesicles. Furthermore, said ingredients may be introduced in amounts commonly used in the field of dermatology.

[0075] When a compound derived from a fraction of the carrot extract of the present invention is provided as an external skin preparation, it may be in the form of an ointment, patch, gel, cream, or spray, but is not limited thereto.

[0077] In addition, the present invention can provide an anti-inflammatory composition comprising a compound derived from a fraction of carrot extract.

[0078] Since the compound derived from the fraction of the carrot extract is identical to the concept used in the anti-aging composition, the description is replaced by the description thereof.

[0079] 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.

[0080] 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 by the description thereof.

[0082] In addition, the present invention can provide an antioxidant composition comprising a compound derived from a fraction of carrot extract.

[0083] Since the compound derived from the fraction of the carrot extract is identical to the concept used in the anti-aging composition, the description is replaced by the description thereof.

[0084] 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.

[0085] 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 by the description thereof.

[0087] In addition, the present invention may provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a compound derived from a fraction of carrot extract.

[0088] Since the compound derived from the fraction of the carrot extract is identical to the concept used in the anti-aging composition, the description is replaced by the description thereof.

[0089] According to a preferred embodiment of the present invention, the inflammatory disease may be one or more selected from the group consisting of asthma, dermatitis, arthritis, cancer, and inflammatory bowel disease.

[0090] Since the above pharmaceutical composition is identical to the concept used in the above anti-aging composition, the description is replaced by the description thereof.

[0092] In addition, the present invention may provide a health functional food composition for preventing or improving inflammatory diseases comprising a compound derived from a fraction of carrot extract.

[0093] Since the compound derived from the fraction of the carrot extract is identical to the concept used in the anti-aging composition, the description is replaced by the description thereof.

[0094] According to a preferred embodiment of the present invention, the inflammatory disease may be one or more selected from the group consisting of asthma, dermatitis, arthritis, cancer, and inflammatory bowel disease.

[0095] Since the above-mentioned health functional food composition is identical to the concept used in the above-mentioned anti-aging composition, the description is replaced by the description thereof.

[0097] In addition, the present invention may provide a pharmaceutical composition for the prevention or treatment of cancer comprising a compound derived from a fraction of carrot extract.

[0098] Since the compound derived from the fraction of the carrot extract is identical to the concept used in the anti-aging composition, the description is replaced by the description thereof.

[0099] According to a preferred embodiment of the present invention, the cancer may be aging cancer.

[0100] According to a preferred embodiment of the present invention, the cancer is breast cancer, stomach cancer, skin cancer, colorectal cancer, liver cancer, It may be one or more selected from the group consisting of bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer and thyroid cancer.

[0101] Since the above pharmaceutical composition is identical to the concept used in the above anti-aging composition, the description is replaced by the description thereof.

[0103] In addition, the present invention may provide a health functional food composition for cancer prevention or improvement comprising a compound derived from a fraction of carrot extract.

[0104] Since the compound derived from the fraction of the carrot extract is identical to the concept used in the anti-aging composition, the description is replaced by the description thereof.

[0105] According to a preferred embodiment of the present invention, the cancer may be aging cancer.

[0106] According to a preferred embodiment of the present invention, the cancer is breast cancer, stomach cancer, skin cancer, colorectal cancer, liver cancer, It may be one or more selected from the group consisting of bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer and thyroid cancer.

[0107] Since the above health functional food composition is the same as the concept used in the above anti-aging composition, the description is replaced by the description thereof.

[0109] In addition, the present invention may provide an anticancer adjuvant comprising a compound derived from a fraction of carrot extract.

[0110] Since the compound derived from the fraction of the carrot extract is identical to the concept used in the anti-aging composition, the description is replaced by the description thereof.

[0111] According to a preferred embodiment of the present invention, the cancer may be aging cancer.

[0112] According to a preferred embodiment of the present invention, the cancer is breast cancer, stomach cancer, skin cancer, colorectal cancer, liver cancer, It may be one or more selected from the group consisting of bile duct cancer, lung cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer and thyroid cancer.

[0113] The anticancer adjuvant of the present invention refers to any form intended to enhance the anticancer effect of an anticancer drug or to suppress or improve the side effects of an anticancer drug. The anticancer adjuvant of the present invention may be administered in combination with various types of anticancer drugs or anticancer adjuvants; when administered in combination, an equivalent level of anticancer therapeutic effect can be achieved even when the anticancer drug is administered at a lower dose than the conventional dosage, thereby enabling safer anticancer treatment.

[0114] The above-mentioned anticancer adjuvant may be administered via any general route as long as it can reach the target tissue. Depending on the purpose, the anticancer adjuvant of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, or orally, intrapulmonaryly, or rectally, but is not limited thereto. Additionally, the above-mentioned anticancer adjuvant may be administered by any device capable of delivering the active substance to the target cells.

[0115] The anticancer adjuvant of the present invention may preferably be formulated as an anticancer adjuvant by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Carriers, excipients, or diluents that may 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, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0116] The anticancer adjuvant of the present invention may be a formulation for oral or parenteral administration, and the description of the formulation is replaced by the description of the formulation of the pharmaceutical composition above.

[0118] In addition, the present invention comprises i) a step of preparing a methanol extract of carrots;

[0119] ii) a step of preparing an ethyl acetate fraction of the methanol extract;

[0120] iii) a step of purifying the above fraction by silica gel column chromatography;

[0121] iv) a step of purifying the above purified product by flash chromatography; and

[0122] v) A step of purifying the above purified product using HPLC (High performance liquid chromatography);

[0123] A method for preparing a compound derived from a carrot fraction containing can be provided.

[0124] According to a preferred embodiment of the present invention, the compound may be cis-4-hydroxycinnamyl 4-hydroxybenzoate. The compound may have the structure of [Chemical Formula 1] below.

[0125] [Chemical Formula 1]

[0126]

[0127] According to a preferred embodiment of the present invention, the compound may be an anti-arthritis, anti-inflammatory, anti-aging, antioxidant, and anticancer compound.

[0128] The above anti-aging may be an anti-aging effect on any one or more selected from the group consisting of cartilage cells, skin cells, and senescent tumor cells.

[0130] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0132] Isolation of cis-4-hydroxycinnamyl 4-hydroxybenzoate, a novel compound, from carrots

[0133] <1-1> Preparation of Carrot Methanol Extract

[0134] After crushing 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, mixed with water, and the methanol was evaporated.

[0136] <1-2> Preparation of Ethyl Acetate Fraction

[0137] 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 and then solubilized with methanol for use in the following examples.

[0139] <1-3> Silica Gel Column Chromatography

[0140] After concentrating the methanol fraction, it was loaded onto a column (30×450 mm) packed with silica gel 60 resin (MERCK, Darmstadt, Germany), and five fractions (fractions 1, 2, 3, 4, and 5) were obtained by silica gel chromatography using chloroform:methanol (10:1, v / v) as the elution solvent. As a result of investigating the anti-inflammatory activity of the five fractions, fraction 4 showed an anti-inflammatory effect and was concentrated to be used as a sample for secondary fractionation. [Figure 2] is a diagram showing the evaluation of samples fractions 1 to 5 separated by silica gel column chromatography using TLC (Thin layer chromatography). After concentrating the sample fraction 4 showing anti-inflammatory activity, it was used in the next step <1-4>.

[0142] <1-4> Flash Chromatography

[0143] Fraction sample No. 4 obtained in the above Examples <1-3> was loaded into a flash chromatograph and flash chromatography was performed using methanol as the elution solvent. Flash chromatography analysis was performed using a Biotage Isolera Spektra One MPLC (Biotage, uppsala, Sweden). Through this, the anticancer and anti-inflammatory activities of fraction sample No. 1 were evaluated, and [Figure 3] shows the results of evaluation using MPLC and TLC (Thin layer chromatography) on fractions 1, 2, and 3.

[0145] <1-5> HPLC (High performance liquid chromatography)

[0146] Substance No. 1 obtained through the flash chromatography above 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 substances were sieved through a 0.2 μm filter. The injection volume was 0.5 mL, the flow rate was 5 mL / min, and the column temperature at 254 and 365 nm was room temperature. The mobile phase consisted of water (solvent A) and methanol (solvent B). For gradient elution, solvent B was initially set to 50% and increased to 100% at 23 minutes. The peak of the final separated and purified active compound was indicated at 14.343 minutes. [Figure 4] is a diagram showing the HPLC results and the final separated fractions evaluated using TLC (Thin layer chromatography).

[0148] Identification of the novel compound cis-4-hydroxycinnamyl 4-hydroxybenzoate

[0149] <2-1> Analysis of Carrot-Derived Natural Products

[0150] A putative identification analysis of metabolites using Progenesis QI was performed using the natural product cis-4-hydroxycinnamyl 4-hydroxybenzoate isolated from the carrot methanol extract through the above <Example 1>.

[0151] Specifically, in the metabolite analysis workflow using Progenesis QI, Lysophospholipid PC (16:0 / 0:0) was used as a representative example in ESI(+) mode. First, the adductors generated after deconvolution were identified, and the reliability of candidate substances was evaluated by analyzing isotopic similarity and chromatograms. Additionally, the structures of the fragment ions generated through MS / MS analysis were assigned by comparing them with a database, and specific compounds were identified by comparing the UPLC-MRM chromatograms of the carrot extract and the standard substances. Finally, it was confirmed whether the substance detected in the carrot was identical to the standard substances by comparing MS / MS spectra (Figs. 5a–8c).

[0152] In addition, to analyze the structure of the finally isolated and purified active compound, the structure was identified through NMR (nuclear magnetic resonance) analysis, and the molecular weight was measured using an ESI-Mass spectrometer and confirmed to be 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).

[0153] [Chemical Formula 1]

[0154]

[0156] <2-2> Metabolite Analysis of Natural Products Derived from Jeju Carrots and Identification of Novel Compounds

[0157] A novel compound, 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate (Chemical Formula 2), was synthesized, which can exhibit activity similar to that of the natural product cis-4-hydroxycinnamyl 4-hydroxybenzoate isolated from carrot methanol extract.

[0158] [Chemical Formula 2]

[0159]

[0161] In the metabolite analysis workflow using Progenesis QI, metabolites were identified using caffeic acid in ESI(-) mode and falcarindiol in ESI(+) mode as representative examples. First, the adducts generated after deconvolution were identified, and reliability was evaluated by analyzing isotopic similarity and chromatograms. Subsequently, structures were assigned by comparing MS / MS fragment ions with a database. In addition, for falcarindiol, an in-house database was utilized to enhance analytical reliability. Subsequently, Nuclear Magnetic Resonance (NMR) 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 was 272 g / mol. Through this, it was finally confirmed that the novel compound purified from Jeju carrots in this study is 3-(4-hydroxyphenyl)propyl 4-hydroxybenzoate (Figs. 9a–9c).

[0163] Anti-arthritic effect of carrot extract or fractions thereof

[0164] <3-1> Analysis of MTT cell viability

[0165] 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) assays were performed 24 hours after treatment. Briefly, primary cultured chondrocytes (5×10⁶) 4) was seeded into a 96-well plate. After 24 hours of incubation, cis-4-hydroxycinnamyl 4-hydroxybenzoate (0–20 μM) was added to the cells. MTT solution was added to each well for 4 hours at the final incubation time. After adding 100 μL of dimethyl sulfoxide solution, optical density was recorded using a microplate reader (570 nm).

[0166] When mouse primary chondrocytes were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate (0, 5, 10, 20 uM), no toxicity was observed in the chondrocytes.

[0168] <3-2> Efficacy Verification via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

[0169] Chondrocytes were isolated from the femoral condyle and tibial plateau of 4-day-old mice (n = 8) by digesting cartilage tissue with DMEM supplemented with 0.2% collagenase (Sigma). Passage "0" (P0) primary chondrocytes (3 x 10⁶ 530mm 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) were treated for 24 hours in a 5% CO2 incubator at 37°C. Then, chondrocytes were 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 the TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). The quality and concentration of RNA were evaluated 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 evaluated using the SYBR premixed Extaq reagent (Takara Bio, Mountain View, CA, USA), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal control. It was confirmed that carrot samples (0, 5, 10, 20 uM) inhibit the transcription of catabolic factors (Mmp3, Mmp13, Adamts 4, Adamts 5) induced by inflammatory cytokines IL-1β (1 ng / ml), TNF-α (10 ng / ml), and IL-6 (100 ng / ml). It was also confirmed that they inhibit the transcription of catabolic factors (Mmp3, Mmp13, Adamts 4, Adamts 5) induced by LPS (10 ng / ml).In addition, quantitative analysis of transcription factors also showed that IL-1β inhibited the expression of Mmp3 and Mmp13, and TNF-α inhibited the expression of Mmp3, Mmp13, Adamts4, and Adamts5 (Fig. 10a, Fig. 10b).

[0171] <3-3> Analysis of WST cell viability using chondrocytes

[0172] Anti-aging activity was confirmed by treating human chondrocyte cell line C20A4 with cis-4-hydroxycinnamyl 4-hydroxybenzoate.

[0173] Specifically, to induce senescent cells of the chondrocyte cell line, the cells were cultured for a total of 6 days, and a low concentration of 25 nM of doxorubicin was used as the drug to induce senescence.

[0174] As a result, cis-4-hydroxycinnamyl 4-hydroxybenzoate showed no activity in normal chondrocytes, whereas activity was confirmed in aged chondrocytes. In other words, it was confirmed that cis-4-hydroxycinnamyl 4-hydroxybenzoate inhibits the survival of chondrogenic cells (Fig. 11 AB).

[0176] <3-4> SA-β-galactosidase staining

[0177] The effect was confirmed using SA-β-galactosidase staining, a well-defined biomarker for cellular senescence. After preparing senescent cell lines 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 region treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate compared to the control group.

[0179] <3-5> RT-qPCR

[0180] To compare the expression of aging-related genes, messenger RNA expression was compared using a real-time gene amplifier. C20A4 cells were seeded into 6-well plates and cultured for 1 day. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate for 24 hours. Total RNA was purified according to the manufacturer's protocol using the TaKaRa MiniBEST RNA Extraction Kit (TaKaRa, Kyoto, Japan). 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. The primers used for RT-PCR were purchased from Bionia (Daejeon, Korea).

[0181] 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 examined, changes in IL-1b, IL-6, and MMP9 were observed (Fig. 12 A).

[0183] <3-6> Histogram Analysis of uPAR(CD87)

[0184] uPAR is a factor involved in cell motility and survival, and can be affected by changes in the microenvironment associated with aging. To analyze uPAR (CD87) expression in C20A4 cells, cells were stained and fluorescence signals were measured using FACS (BD, Flow Cytometry). Cells were stained with uPAR antibodies, and expression levels were quantitatively evaluated via FACS. The analysis results showed that uPAR expression tended to increase in aged cells, and decreased when treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate (Fig. 12 B).

[0186] <3-7> Analysis of ROS Generation and Mitochondrial ROS Fluorescence Expression via Fluorescence Microscopy

[0187] ROX Green and MitoSOX staining were performed to evaluate the effects of drug A on aged C20A4 cells. After treating cells with cis-4-hydroxycinnamyl 4-hydroxybenzoate, ROX Green was used to check for the production of ROS (reactive oxygen species), and MitoSOX was used to measure oxidative stress within mitochondria. Changes were observed using a fluorescence microscope after fluorescence staining.

[0188] As a result, ROS and mitochondrial stress levels were significantly reduced in the cis-4-hydroxycinnamyl 4-hydroxybenzoate-treated group (Fig. 13).

[0190] <3-8> Immunoblot Analysis

[0191] After harvesting the cells, they 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 then electrotransferred onto 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 overnight with the primary antibody at 4°C. After washing, the blots were incubated for 60 minutes with IRDye 680- and IRDye 800-labeled secondary antibodies diluted with 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).

[0192] In this invention, changes in the expression of proteins p65, pERK, ERK, pSTAT3, p-p38, and LC3 were confirmed using Western blot analysis. The experiment was conducted using β-actin as an internal control, and protein expression was compared relatively. As a result of the experiment, the protein expression of p65, pERK, and LC3 changed significantly. The increased expression of p65 and pERK suggests the activation of specific signaling pathways, which may be related to inflammatory responses and intracellular signaling. On the other hand, changes in LC3 expression suggest 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 (Fig. 14 A).

[0194] <3-9> Analysis of Intracellular Mitochondria Metabolism

[0195] The Seahorse kit was used to analyze intracellular mitochondrial metabolism. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured through experiments. After culturing cells under appropriate conditions, mitochondrial respiration and glycolysis activities were evaluated using the Seahorse analyzer.

[0196] 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 by changes in cellular state and the environment.

[0198] Antioxidant and anti-inflammatory effects of carrot extract or fractions thereof

[0199] <4-1> Cell Line and Culture Conditions

[0200] RAW 264.7 macrophage cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Macrophage cells were cultured in high-glucose Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin / streptomycin (Hyclone). Cells were cultured at 37°C under a 5% CO2 atmosphere.

[0202] <4-2> Cell Proliferation

[0203] RAW 264.7 cells (3×10⁶ 6Canine cells / plates were seeded into 96-well plates and cultured for 1 day. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate for 1 day. Cell viability was assessed 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. Absorbance was estimated at OD490 using a Molecular Devices (SPECTRAMAX ID5) reader.

[0205] <4-3> Analysis of Iron-Reducing Antioxidant Capacity (FRAP)

[0206] 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 minutes. The absorbance was measured at 593 nm using a microplate reader. A standard curve was constructed based on the FeSO4 value. As a result, it was confirmed that it possesses excellent iron-reducing antioxidant activity similar to that of FeSO4, which was used as a standard (Fig. 15 B).

[0208] <4-4> Analysis of Trolox Equivalent Antioxidant Capacity (TEAC)

[0209] Free radical scavenging activity was investigated using the Trolox Equivalent Antioxidant Dose (TEAC). For the reaction with the ABTS solution, 2,2-azino-bis-(3-ethylbenzo-thiazolin-6-sulfonic acid) and 2.45 mM potassium persulfate were mixed. They were reacted for 18 hours. Subsequently, 180 µl of the ABTS solution was mixed with 20 µl of cis-4-hydroxycinnamyl 4-hydroxybenzoate and reacted for 5 minutes. After the reaction, samples were measured at 734 nm. A standard curve was constructed by adding Trolox.

[0210] As a result, radical cations were generated by the antioxidants of cis-4-hydroxycinnamyl 4-hydroxybenzoate, and the color changed to blue-green. In addition, the results of Trolox and Ascorbic acid were presented as positive controls for comparison.

[0211] As a result, superior antioxidant inhibitory efficacy compared to the two substances used as positive controls was confirmed (Fig. 15 B).

[0213] <4-5> NO Analysis

[0214] Nitric oxide (NO) is a biological mediator produced by LPS-stimulated macrophages. We intended to evaluate the inhibitory effect of cis-4-hydroxycinnamyl 4-hydroxybenzoate on NO production induced by LPS (lipopolysaccharide from E. coli O111:B4; InvivoGen, San Diego, CA, USA) in RAW 264.7 cells.

[0215] Specifically, RAW 264.7 cells (3×10⁶ 6Canine cells (or plates) were seeded into a 96-well plate and cultured for 1 day. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate along with LPS (1 μg / mL) for 24 hours for 1 day. NO measurements were evaluated in the supernatant culture medium using the NO Plus detection kit (iNtRON Biotechnology, Gyeonggi, Korea). A pre-reaction was induced by adding 50 μL of N1 buffer (sulfanilamide in the buffer) to each well of the 96-well plate with 100 μL of culture medium or nitrite standard. Then, after incubating the 96-well plate for 20 minutes, the mixture was reacted with 50 μL of N2 buffer (naphthyl-ethylenediamine in the buffer). After incubating the mixture for 10 minutes, NO formation was measured by the absorbance value at OD560 nm using Molecular Devices (SPECTRAMAX ID5).

[0216] 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, it was found that NO production is induced by LPS, and cis-4-hydroxycinnamyl 4-hydroxybenzoate reduces LPS-induced NO production (Fig. 16 A).

[0218] <4-6> RT-qPCR

[0219] RAW 264.7 cells (3×10⁶ 6Canine cells (or plates) were seeded into 96-well plates and cultured for 1 day. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate in combination with LPS (1 μg / mL) for 24 hours. Total RNA was purified according to the manufacturer's protocol using the TaKaRa MiniBEST RNA Extraction Kit (TaKaRa, Kyoto, Japan). 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. The primers used for RT-PCR were purchased from Bionia (Daejeon, Korea).

[0220] As a result, as shown in [Fig. 16] B, LPS-stimulated RAW 264.7 cells showed increased iNOS protein expression, whereas cis-4-hydroxycinnamyl 4-hydroxybenzoate highly inhibited it. Additionally, analysis of iNOS transcription levels in RAW264.7 cells revealed that LPS increased iNOS transcript levels, whereas cis-4-hydroxycinnamyl 4-hydroxybenzoate decreased the increased transcript levels of iNOS, TNF-α, and IL-1β (Fig. 16 A, Fig. 17 A).

[0222] <4-7> ELISA

[0223] The secretion amounts of IL-1β and TNF-α were quantified using an ELISA (enzyme-linked immunosorbent assay) kit from BioLegend. After collecting the cell culture supernatant, an ELISA was performed according to the manufacturer's protocol, and the absorbance was measured at 450 nm.

[0224] As a result, while the LPS-treated group showed high secretion levels, the production of IL-1β and TNF-α tended to decrease in a concentration-dependent manner following HA treatment. This suggests that HA regulates the expression of inflammatory cytokines (Fig. 17 B).

[0226] <4-8> Immunoblot Analysis

[0227] 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 onto 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 overnight with the primary antibody at 4°C. After washing, the blots were incubated for 60 minutes with IRDye 680- and IRDye 800-labeled secondary antibodies diluted with 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). The primary antibody against iNOS was obtained from Cell Signaling Technology (Beverly, MA, USA). And β-actin was purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA).

[0228] As a result, as shown in Fig. 16, C, it was confirmed that while iNOS protein expression was increased in LPS-stimulated RAW 264.7 cells, cis-4-hydroxycinnamyl 4-hydroxybenzoate inhibited NO production through the downregulation of iNOS. While the levels of pERK1 / 2 and pJNK were increased by LPS stimulation, they were found to be decreased upon treatment with cis-4-hydroxycinnamyl 4-hydroxybenzoate compared to LPS-stimulated control cells. Therefore, it was confirmed that the inflammatory response in LPS-induced RAW 264.7 cells is suppressed by reducing the levels of pERK and pJNK and inhibiting MAPK signaling. Thus, it was confirmed that the above compounds are associated with the MAPK kinase pathway and that drug action occurs through this signaling system (Fig. 18 A, B).

[0229] In Figure 18, C, the protein expression-inducing effects of HO-1 and NRF2 were analyzed. The Nrf-2 (nuclear factor erythroid-2 like 2; Nfe2I2) and heme oxygenase 1 (HO-1) signaling axis plays a role as a multiorgan protector by reducing oxidative stress in tissue and animal models. Accordingly, 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 changes in HO-1 and Nrf2 proteins and altered the expression of Keap1 protein after 3 hours of treatment. Accordingly, it was confirmed that cis-4-hydroxycinnamyl 4-hydroxybenzoate induces protein levels of HO-1 and Nrf2 (Fig. 18 C).

[0231] <4-9> Analysis of ROS Expression via Fluorescence Microscopy

[0232] The effect of HA on LPS-induced ROS (reactive oxygen species) accumulation in RAW264.7 macrophages was evaluated. ROS accumulation was assessed by measuring intracellular ROS concentrations 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 hour, followed by treatment with LPS (1 μg / mL) for 1 hour. Subsequently, cells were stained with CellROX green dye at 37°C for 10 minutes. After washing with 1X PBS, the stained ROS were visualized using an automated microscope (Cytation5, Agilent, VT, USA). ROS signals were observed using a fluorescence microscope after fluorescent staining.

[0233] 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 an LPS-induced ROS inhibitor in RAW 264.7 macrophages.

[0235] <4-10> Mouse experiment on foot edema (inflammation)

[0236] The effects of HA were evaluated in a carrageenan-induced foot edema model. Figure 20a A shows representative changes in mouse feet following HA treatment, while Figure 20a B compares relative foot edema thickness. Figure 20a C analyzes DPPH radical scavenging activity and catalase (CAT) activity in mouse liver samples. Figure 20b A confirms TNF-α, IL-1β, and IL-6 protein expression using Western blot, and Figure 20b B analyzes changes in gene expression in HA-treated cells. mRNA levels of TNF-α, IL-1β, and IL-6 were measured using reverse transcription quantitative PCR, and the effect of HA on inflammatory responses was evaluated.

[0237] As a result, the thickness of foot edema was significantly reduced in the HA-treated group, and the protein and mRNA expression of inflammatory cytokines also showed a decreasing trend.

[0239] Anticancer (anti-aging) effects of carrot extract or fractions thereof

[0240] <5-1> Cell Proliferation

[0241] MCF-7, MDA-MB-231 breast cancer cells (1.5×10⁶ 6Canine cells (plates) were seeded into 96-well plates and cultured for 1 day. They were treated for 1 day with cis-4-hydroxycinnamyl 4-hydroxybenzoate and doxorubicin, which is used for aging. 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. Absorbance was estimated at OD490 using a Molecular Devices (SPECTRAMAX ID5) reader. As a result, the concentrations used in the experiment were evaluated as non-toxic (Fig. 21 A).

[0242] 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 induce senescent cells in the breast cancer cell lines, the cells were cultured for a total of 6 days, and a low concentration of Doxorubicin 50 nM was used as the senescent agent. While cis-4-hydroxycinnamyl 4-hydroxybenzoate showed no activity in normal breast cancer cells, activity was confirmed in senescent breast cancer cells. As a result, cis-4-hydroxycinnamyl 4-hydroxybenzoate inhibited the survival of senescent breast cancer cells (Fig. 21 A).

[0244] <5-2> SA-β-galactosidase staining

[0245] The effect was confirmed using SA-β-galactosidase staining, a well-defined biomarker for cellular senescence. After preparing an senescent cancer cell line using the same method as in Example <5-1> above, staining was performed using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, #9860).

[0246] 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.

[0248] <5-3> RT-qPCR

[0249] MDA-MB-231 cells (5×10⁶ 5Cells / plates were seeded into 6-well plates and cultured with doxorubicin for 6 days. Cells were treated with cis-4-hydroxycinnamyl 4-hydroxybenzoate and DOX (50 nM) for 2 days. Total RNA was purified according to the manufacturer's protocol using the TaKaRa MiniBEST RNA Extraction Kit (TaKaRa, Kyoto, Japan). 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 contains 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. The primers used for RT-PCR were purchased from Bionia (Daejeon, Korea).

[0250] The effects of cis-4-hydroxycinnamyl 4-hydroxybenzoate on elevated levels of p21, p53, and several aging-related genes at the transcriptional level in breast cancer cells were tested using RT-qPCR.

[0251] As a result, as shown in [Figure 22a], the expression of p21 and p53 genes in senescent breast cancer cells was increased by Doxorubicin treatment, whereas cis-4-hydroxycinnamyl 4-hydroxybenzoate inhibited it. Therefore, it was confirmed that cis-4-hydroxycinnamyl 4-hydroxybenzoate induces apoptosis in senescent cancer cells through the downregulation of p21, p53, and several senescence-related genes.

[0253] <5-4> Immunoblot Analysis

[0254] 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 onto 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 overnight with the primary antibody at 4°C. After washing, the blots were incubated for 60 minutes with IRDye 680- and IRDye 800-labeled secondary antibodies diluted with 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).

[0255] As shown in [Fig. 22b], the expression of pp53, p21, and p16 was significantly increased in senescent cells compared to normal cells. This suggests that cell cycle inhibitory proteins were activated during the aging process and indicates that aging-related signaling pathways were strengthened. On the other hand, the expression of β-actin remained constant compared to the control group, effectively serving as an internal control for the experiment. The present invention suggests that changes in protein expression related to aging are regulated through specific signaling pathways.

[0256] In addition, [Figure 23a] showed that HA reduced the protein levels of YAP1, mTOR, PD-L1, PD-L2, p-AMPK, and GPX4 in a concentration-dependent manner. In particular, the reduction in YAP1 and mTOR implies that HA may influence cell growth and metabolic regulation, while the simultaneous reduction in PD-L1 and PD-L2 suggests the possibility that HA more broadly regulates the effects on immune evasion mechanisms. The reduction in p-AMPK demonstrates the effects of HA on energy homeostasis and metabolic regulatory pathways, and the reduction in GPX4 suggests the possibility that cytoprotective mechanisms related to lipid peroxidation regulation are inhibited. This may imply that HA may also be involved in novel apoptotic pathways such as ferroptosis (iron-dependent cell death).

[0257] Taken together, these results suggest that HA may influence cell growth, metabolism, and immune regulation, as well as antioxidant mechanisms, by regulating specific signaling pathways in senescent cells, and may contribute to the removal of senescent cells by activating apoptotic pathways such as ferroptosis.

[0259] <5-5> Apoptosis assay

[0260] An apoptosis assay was performed using flow cytometry, utilizing BD Biosciences' Accuri™ C6 Plus Flow Cytometer and FITC Annexin V Apoptosis Detection Kit I. After treating cells with HA, the degree of apoptosis was analyzed by performing Annexin V-FITC and PI staining.

[0261] As a result, the proportion of Annexin V-positive (Early Apoptosis) cells significantly increased in the HA-treated group, suggesting that HA has an effect of promoting early apoptosis. On the other hand, the proportion of PI-positive (Necrosis) cells showed a decreasing trend, confirming the possibility that HA influences the induction of programmed cell death rather than late apoptosis (Fig. 23b).

[0263] <5-6> Telomere

[0264] In this invention, telomere length was analyzed using qPCR, and the effects of HA on aged breast cancer cells were confirmed. qPCR was performed using specific primers to measure telomere length, and relative changes were compared based on the telomere length of a negative control group. Experimental results showed that telomere length in aged breast cancer cells decreased significantly to the level of 20–30%, suggesting that telomere shortening occurred during the cellular aging process. However, after treatment with HA, telomere length showed a tendency to increase again, indicating the possibility that HA influences telomere maintenance in aged breast cancer cells. These results suggest that HA can play a role in restoring telomere stability as well as regulating aging-related signaling pathways.

[0266] Evaluation of anti-aging efficacy in human skin cells

[0267] We intended to evaluate the anti-aging efficacy of cis-4-hydroxycinnamyl 4-hydroxybenzoate in UVB-induced aged skin cells.

[0268] 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. Experiments were conducted by establishing a control group, a UVB-treated group, and UVB and HA (1, 2, 5, 10, 20 μM) treated groups.

[0269] Microscopic analysis revealed that compared to the control group, UVB-treated cells showed increased cell size, changes in morphology, and progression of aging (Fig. 24). In the group treated with HA, a gradual effect was observed starting from 5 μM, and at concentrations of 10 μM and 20 μM, a selective apoptosis effect was observed in aged cells, while non-aged cells were not significantly affected at the same concentrations. These results suggest that HA exhibits an anti-aging effect by selectively killing aged cells, and at high concentrations, it exerts efficacy against aged cells to promote the removal of aged cells.

Claims

Claim 1 Antioxidant composition comprising a compound derived from a fraction of carrot extract. Claim 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. Claim 3 A composition according to claim 1, wherein the fraction is a carrot extract fractionated using hexane, chloroform, ethyl acetate, butanol, water, or a mixture thereof as a solvent. Claim 4 A composition according to claim 1, characterized in that the compound is cis-4-hydroxycinnamyl 4-hydroxybenzoate. Claim 5 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.

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