Anti-aging composition containing unripe citrus unshiu peel extract

Abstract

The present invention relates to an anti-aging composition containing an unripe Citrus unshiu peel extract. The composition containing an unripe Citrus unshiu peel extract, of the present invention, has excellent free radical scavenging ability, inhibits skin cell damage caused by ultraviolet rays (photoaging inhibition), and has the effect of inhibiting the expression of MMPs, which are collagen-degrading enzymes, and thus can be effectively used as a pharmaceutical composition, a cosmetic composition, and a functional food composition, for anti-aging.

Description

Anti-aging composition containing green tangerine peel extract

[0001] The present invention relates to an anti-aging composition containing an extract of green tangerine peel, and more specifically, to a pharmaceutical composition, a cosmetic composition and a functional food composition having an antioxidant effect, an anti-collagen decomposition effect and an anti-photoaging effect, which contain an ethanol extract of green tangerine peel as an active ingredient.

[0002] Human skin undergoes various physicochemical changes during the aging process. These changes are broadly categorized into intrinsic aging and photoaging, and research on these factors has been actively conducted. Free radicals (or reactive oxygen species) can be activated by UV rays, stress, disease, environmental factors, injury, and aging. If this condition worsens, it destroys the body's antioxidant defenses, damaging cells and tissues, accelerating the onset of adult diseases and aging. In other words, lipids, proteins, polysaccharides, and nucleic acids, the major constituents of the skin, become oxidized, destroying skin cells and tissues and ultimately contributing to skin aging.

[0003] Therefore, the skin can recover quickly and maintain health by eliminating free radicals generated during the body's metabolic process, ultraviolet irradiation, and inflammatory reactions to protect the cell membrane, and by regenerating damaged cells through active metabolism to proliferate cells.

[0004] Within the human body, reactive oxygen species (ROS) are constantly generated through biochemical reactions that provide energy. These are largely eliminated by the body's self-defense mechanisms. However, if these ROS are not properly eliminated, resulting in oxidative stress, these ROS can cause oxidative damage to cellular components such as lipids, proteins, carbohydrates, and DNA, as well as alter enzyme activity. This can lead to brain diseases such as stroke and Parkinson's disease, as well as adult diseases such as heart disease, ischemia, and arteriosclerosis, as well as various diseases such as cancer.

[0005] In addition, reactive oxygen species not only damage biological tissues, but also are known to affect gene expression, cell adhesion, metabolism, cell cycle, and apoptosis by altering various signal transduction processes. Signal transduction induced by reactive oxygen species is known to include signal transduction mediated by mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), phosphatidylinositol 3-kinase (PI3K), p53, and β-catenin / Wnt. Recently, various studies have been conducted on antioxidant and anti-aging substances that protect the human body from damage caused by reactive oxygen species. In particular, plants are known to contain a large amount of antioxidant and anti-aging ingredients that can remove reactive oxygen species. Many studies have been reported on antioxidant and anti-aging substances such as vitamins, phenolic compounds, and carotenoids obtained from plants such as fruits, vegetables, tea, and herbs.

[0006] Aging is not only affected by free radicals, but also by enzymes called matrix metalloproteinases (MMPs), which degrade collagen. While the synthesis and degradation of extracellular matrix components like collagen are regulated within the body, as we age, collagen synthesis declines and the expression of matrix metalloproteinases (MMPs), enzymes that degrade collagen, is accelerated, leading to decreased skin elasticity and the formation of wrinkles. Furthermore, these degrading enzymes can be activated by UV exposure.

[0007] As a prior art related to such skin anti-aging, Korean Patent Publication No. 2021-0055622 discloses a cosmetic composition for preventing skin aging and improving skin wrinkles containing a Viburnum stellatomentosum extract, and Korean Patent Registration No. 2420544 discloses a composition for preventing skin aging containing a rose petal extract as an active ingredient.

[0008] Therefore, the technical problem to be solved in the present invention is to provide a composition having an antioxidant effect, an anti-collagen decomposition effect, and an anti-photoaging effect, including a natural substance.

[0009] In order to solve the above-mentioned technical problem, the present invention provides an anti-aging composition characterized by containing a green tangerine peel extract as an effective ingredient.

[0010] Preferably, the composition may be a pharmaceutical, cosmetic or functional food composition.

[0011] Preferably, the green tangerine peel extract is characterized in that it is obtained by extracting green tangerine peel using 60 to 80% ethanol at 20 to 30°C for 40 to 90 minutes.

[0012] Preferably, the anti-aging agent is characterized by being an anti-oxidant, preventing collagen decomposition, or preventing photoaging.

[0013] In this way, the composition containing the green tangerine peel extract of the present invention has excellent free radical scavenging ability, inhibits damage to skin cells caused by ultraviolet rays (inhibits photoaging), and has an effect of inhibiting the expression of MMPs, a collagen-decomposing enzyme, and thus can be usefully used as an anti-aging pharmaceutical composition, a cosmetic composition, and a functional food composition.

[0014] The following drawings attached to this specification illustrate preferred embodiments of the present invention and, together with the contents of the invention described above, serve to further understand the technical idea of ​​the present invention, and therefore, the present invention should not be interpreted as being limited to matters described in such drawings.

[0015] Figure 1 shows the results of measuring the DPPH radical scavenging ability of citrus peel extract.

[0016] Figure 2 shows the results of DPPH radical scavenging activity measurement according to concentration of green tangerine peel extract C13.

[0017] Figure 3 shows the results of measuring the superoxide anion radical scavenging ability of green tangerine peel extract C13 according to concentration.

[0018] Figure 4 shows the results of measuring the hydroxyl radical scavenging ability of green tangerine peel extract C13 according to concentration.

[0019] Figure 5 shows the results of measuring the intracellular ROS scavenging ability of citrus peel extract.

[0020] Figure 6 shows the results of confirming the cytotoxicity of citrus peel extract.

[0021] Figure 7 shows the results of confirming the cytotoxicity of green tangerine peel extract C13 according to concentration.

[0022] Figure 8 shows the results of the concentration-dependent scavenging ability of green tangerine peel extract C13 for intracellular ROS induced by H2O2 or UVB.

[0023] Figure 9 shows the results of the scavenging ability of green tangerine peel extract C13 against UVB-induced intracellular ROS.

[0024] Figure 10 is a fluorescence photograph measuring the scavenging ability of green tangerine peel extract C13 against UVB-induced intracellular ROS.

[0025] Figure 11 shows the results of measuring the inhibitory effect of green tangerine peel extract C13 on changes in intracellular calcium levels induced by UVB.

[0026] Figure 12 is a fluorescence photograph measuring the inhibitory effect of green tangerine peel extract C13 on changes in intracellular calcium levels induced by UVB.

[0027] Figure 13 shows the results of measuring the inhibitory effect of green tangerine peel extract C13 on UVB-induced MMP-1 activity.

[0028] Figure 14 shows the results of Western blotting analysis of green tangerine peel extract C13 on UVB-induced MMPs protein expression.

[0029] Figure 15 shows the results of Western blotting analysis of green tangerine peel extract C13 on the expression of phospho-JNK, a signaling protein induced by UVB.

[0030] Figure 16 shows the results of Western blotting analysis of green tangerine peel extract C13 on the expression of UVB-induced signaling proteins c-Fos and phospho-c-Jun.

[0031] Figure 17 shows the UV absorption behavior of green tangerine peel extract C13 at UV wavelength.

[0032] Figure 18 shows the protective effect of green tangerine peel extract C13 on UVB-induced cell growth and morphological changes.

[0033] Figure 19 shows the inhibitory effect of green tangerine peel extract C13 on UVB-induced β-Galactosidase enzyme activity related to cell aging.

[0034] Figure 20 shows the inhibitory effect of green tangerine peel extract C13 on the expression of phospho-H2AX protein, a DNA damage marker induced by UVB.

[0035] Figure 21 shows the inhibitory effect of green tangerine peel extract C13 on UVB-induced aging or signal-related protein (P16, CyclinD1, CDK4, P21, Cyclin E, CDK2, phospho P53, P53) expression.

[0036] The present invention will be described in more detail below.

[0037] The present invention provides an anti-aging composition characterized by containing a geramon peel extract as an active ingredient.

[0038] The composition containing the green tangerine peel extract of the present invention has excellent free radical scavenging ability, inhibits damage to skin cells caused by ultraviolet rays (inhibits photoaging), and has the effect of inhibiting the expression of MMPs, a collagen-decomposing enzyme.

[0039] The active ingredient used in the present invention, unripe tangerines, contain more than twice as many flavonoids and citric acid as fully ripe tangerines. They are rich in antioxidants and have anti-inflammatory and skin-hydrating properties. These tangerines are grown exclusively in summer for the purpose of utilizing their functional properties. While their exteriors are unripe and bluish, they are richer in functional ingredients than fully ripe tangerines.

[0040] The term "extract" of the present invention means a preparation obtained by squeezing raw materials with an appropriate extractant and evaporating the extractant to concentrate them, and is not limited thereto, but may be an extract obtained by extraction treatment, a diluted or concentrated extract, a dried product obtained by drying the extract, a controlled product, or a purified product thereof.

[0041] The extracts used in the present invention can be extracted using each conventional extraction solvent known in the art, and the extracts can be used in liquid form or after being concentrated and / or dried. At this time, the extraction solvent can be obtained using, for example, (a) water, (b) anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) a mixed solvent of the lower alcohols and water, (d) acetone, (e) ethyl acetate, (f) chloroform, or (g) 1,3-butylene glycol as an extraction solvent. Preferably, extraction is performed using methanol, ethanol, or butane. Since the degree of extraction and loss of the active ingredient of the extract may differ depending on the extraction solvent, an appropriate extraction solvent should be selected and used. The above extraction method is not particularly limited, and includes, for example, hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and pressing.

[0042] In addition, the extract can be obtained through a conventional purification process in addition to the extraction method using the extraction solvent. For example, the extract can be obtained through fractions obtained through various additional purification methods, such as separation using an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatographies (designed for separation according to size, charge, hydrophobicity, or affinity).

[0043] According to an embodiment of the present invention, a natural plant extract may be obtained by using an extraction method such as stirring extraction, hot water extraction, cold immersion extraction, reflux cooling extraction or ultrasonic extraction, using water, an alcohol having 1 to 5 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, or a mixed solvent of water and alcohol, in an amount of about 1 to 20 times, preferably about 2 to 10 times, the weight of a green tangerine peel sample, and then extracting the obtained extract at preferably 20 to 100°C, and then filtering, concentrating under reduced pressure or drying the obtained extract.

[0044] Specifically, the green tangerine peel extract is characterized in that it is obtained by drying green tangerine peel and then extracting it at 20 to 30°C for 40 to 90 minutes using water, an organic solvent, or a mixture thereof, preferably 60 to 80% ethanol, as an extraction solvent.

[0045] According to one embodiment of the present invention, the green tangerine peel extract is characterized in that it is included in an amount of 0.5 to 10 wt% based on the total weight of the anti-aging composition. In this case, if the green tangerine peel extract is included in an amount less than 0.5 wt%, it is difficult to obtain an anti-aging effect, and if the content of the green tangerine peel extract exceeds 10.0 wt%, the effect is not proportional to the amount used, which is uneconomical.

[0046] In the present invention, “anti-aging” may be for anti-oxidation, prevention of collagen decomposition, or prevention of photoaging, but is not particularly limited thereto.

[0047] According to one embodiment of the present invention, the composition may be a pharmaceutical, cosmetic or functional food composition.

[0048] The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable auxiliary agents in addition to the above-mentioned effective ingredient, and the auxiliary agents can include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents.

[0049] The above pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described effective ingredients for administration.

[0050] The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracheacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0051] In the composition formulated as a liquid solution, acceptable pharmaceutical carriers include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0052] Furthermore, it can be preferably formulated according to the ingredients using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, as an appropriate method in the relevant field.

[0053] The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and oral administration is preferred.

[0054] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and a generally skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0055] According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g / kg.

[0056] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the method can be performed. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0057] In the present invention, "cosmetic composition" means a product used on the human body to cleanse and beautify the human body, add attractiveness, brighten the appearance, or maintain or improve the health of the skin and hair, and has a mild effect on the human body.

[0058] In addition to the active ingredient, the cosmetic composition of the present invention may further contain components commonly added to cosmetic compositions, such as conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0059] The cosmetic composition of the present invention can be manufactured into any formulation commonly manufactured in the art, and for example, can be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, it can be manufactured into the formulation of an ampoule, a nourishing cream, an astringent toner, an emollient toner, a lotion, an essence, a nourishing gel, or a massage cream.

[0060] When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0061] When the formulation of the present invention is a powder or spray, toluene, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0062] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0063] When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth gum, etc. can be used as a carrier component.

[0064] When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0065] The cosmetic composition of the present invention may further contain adjuvants commonly used in the fields of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, film-forming agents, water, ionic or nonionic emulsifiers, fillers, metal ion sequestering and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients commonly used in cosmetics. In addition, the above ingredients may be introduced in amounts commonly used in the field of dermatology.

[0066] The cosmetic composition of the present invention may be applied alone or in combination, or may be applied in combination with other cosmetic compositions other than those of the present invention. Furthermore, the cosmetic composition of the present invention may be used according to conventional methods, and the frequency of application may vary depending on the user's skin condition or preference.

[0067] In one embodiment of the present invention, the food composition may be in the form of a tablet, capsule, powder, granule, liquid, pill, solution, syrup, juice, suspension, emulsion, or drop, etc. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an orally acceptable, non-toxic, inert carrier such as ethanol, glycerol, water, etc. In addition, if desired or necessary, suitable binders, lubricants, disintegrants, and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracheacanth, or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0068] In addition, the food composition of the present invention can be used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, tea, beverages, meat, chocolate, jellies, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0069] In addition, the food composition may contain, in addition to the effective ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0070] The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, stick liquids, pills, etc.

[0071] In the present invention, health functional food refers to a food manufactured and processed using raw materials or ingredients with functionality useful to the human body according to the Health Functional Food Act, and is meant to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological action.

[0072] The health functional food of the present invention may contain conventional food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0073] The items listed in the above food additive code include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0074] For example, health functional foods in tablet form can be made by mixing active ingredients with excipients, binders, disintegrants, and other additives, granulating them using conventional methods, adding lubricants, etc., and then compression molding them, or by directly compression molding the mixture. Furthermore, the above-mentioned health functional foods in tablet form can also contain a binder, etc., as needed.

[0075] Among health functional foods in capsule form, hard capsules can be manufactured by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by filling a mixture of the extract with additives such as excipients into a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.

[0076] A health functional food in the form of a ring can be prepared by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc., and molding the mixture using a conventionally known method. If necessary, the mixture can be coated with white sugar or another coating agent, or the surface can be coated with a substance such as starch or talc.

[0077] A health functional food in granular form can be manufactured into a granular form by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc. using a conventionally known method, and may contain a flavoring agent, a flavoring agent, etc., as needed.

[0078] In the manufacture of the composition of the present invention, in addition to the effective ingredient as a food composition, various flavoring agents and natural carbohydrates may be contained as additional ingredients, as in a food composition commonly used, and physiologically acceptable adjuvants may be used, and the adjuvants may include excipients, sweeteners, coating agents, bulking agents, lubricants, binders, or flavoring agents.

[0079] The food composition of the present invention can be used as a functional food or added to various foods. Examples of foods to which the composition of the present invention can be added include chocolate, beverages, foods, snacks, noodles, gum, candy, and health supplements.

[0080] The health functional food of the present invention may include commonly used food additives, and the food additives are judged according to the specifications and standards in accordance with the general provisions and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety.

[0081] In this way, the composition containing the green tangerine peel extract of the present invention has excellent free radical scavenging ability, inhibits damage to skin cells caused by ultraviolet rays (inhibits photoaging), and has an effect of inhibiting the expression of MMPs, a collagen-decomposing enzyme, and thus can be usefully used as an anti-aging pharmaceutical composition, a cosmetic composition, and a functional food composition.

[0082]

[0083] Hereinafter, the present invention will be described in more detail through examples. These examples are intended merely to illustrate the present invention, and therefore, the scope of the present invention is not to be construed as being limited by these examples.

[0084]

[0085] <Example 1> Preparation of green tangerine peel extract

[0086] 20 g of citrus peel powder was extracted at 25°C for 1 hour with 200 L of 70% ethanol. Each peel extract extracted according to the citrus species shown in Table 1 below was designated C1 to C14. The extract was concentrated using a vacuum concentrator, and then the extract powder was obtained using a freeze dryer and stored at -70°C until use in the experiment.

[0087]

[0088]

[0089]

[0090] <Test Example 1> Measurement of DPPH radical scavenging activity of green tangerine peel extract

[0091] The DPPH radical scavenging activity was confirmed by measuring the absorbance at 520 nm using a microreader after treating the green tangerine peel ethanol extract (50 μg / ml, 100 μg / ml) with a DPPH radical solution and reacting for 3 hours at room temperature. The superoxide anion and hydroxyl radical scavenging activity were measured using an ESR spectrometer. After treating the green tangerine peel ethanol extract (50 μg / ml) with a 3 M DMPO and xanthin / xanthine oxidase system or a 0.3 M DMPO and FeSO4+H2O2 system, the superoxide anion scavenging activity or hydroxyl radical scavenging activity was measured using an ESR spectrometer. N-acetyl cysteine ​​(NAC) was used as an antioxidant and a positive control for ROS removal.

[0092] Figure 1 shows the results of DPPH radical scavenging activity measurements of citrus peel extracts. As shown here, when the activity of 14 types of citrus peel extracts was measured at concentrations of 50 μg / ml and 100 μg / ml, C4, C6, C7, C8, and C13 peels showed concentration-dependent scavenging activity. In particular, green tangerine peel extract C13 showed the highest scavenging activity at 20% and 31%.

[0093] Figure 2 shows the results of DPPH radical scavenging activity measurement according to concentration of green tangerine peel extract C13. As can be seen here, the DPPH radical scavenging activity of green tangerine peel extract C13 and the radical scavenging activity through ESR technique showed that green tangerine peel extract C13 showed a concentration-dependent increase in DPPH radical scavenging activity from 20 μg / ml, but the increase decreased from 50 μg / ml.

[0094] Figure 3 shows the results of measuring the superoxide anion radical scavenging ability of green tangerine peel extract C13 at various concentrations. As seen here, the superoxide anion radical scavenging ability of green tangerine peel extract C13 (50 μg / ml) was measured and the superoxide anion radical index was reduced from 8.6 to 5.6.

[0095] Figure 4 shows the results of measuring the hydroxyl radical scavenging activity of green tangerine peel extract C13 at various concentrations. As seen here, the hydroxyl radical scavenging activity of green tangerine peel extract C13 (50 μg / ml) was measured and the hydroxyl radical index was reduced from 112.5 to 68.6.

[0096]

[0097] <Test Example 2> Measurement of the scavenging ability of citrus peel extracts to scavenge intracellular reactive oxygen species (ROS).

[0098] Human normal skin epidermal cells (HaCaT) were maintained in DMEM medium containing 10% FBS and 1% antibiotics in a 37°C, 5% CO2 incubator, and these cells were used in all experiments.

[0099] Cells were pretreated with citrus peel extract (50 μg / ml, 100 μg / ml) and treated with 1 mM H2O2 or 30 mJ / cm 2 After treatment with medium-wave ultraviolet (UVB) light, the cells were treated with DCF-DA fluorescence reagent, and the fluorescence intensity was measured using a fluorescence spectrophotometer to confirm the production of intracellular ROS.

[0100] Figure 5 shows the results of measuring the intracellular ROS scavenging activity of citrus peel extracts. As shown here, when the H2O2-induced intracellular ROS scavenging activity was measured at concentrations of 50 μg / ml and 100 μg / ml of 14 types of citrus peel extracts, C1, C2, C3, C4, C6, C7, C8, C11, C12, C13, and C14 peels showed relatively high scavenging activity.

[0101]

[0102] <Test Example 3> Measurement of cytotoxicity of green tangerine peel extract

[0103] Cells were seeded and cultured for 16 hours, then treated with citrus peel extract (50 μg / ml, 100 μg / ml) and incubated for another 24 hours in a 37°C incubator. MTT reagent was added and the reaction was performed at 37°C for 4 hours. After collecting the supernatant, formazan crystals were dissolved in DMSO solution and the absorbance at 540 nm was measured using a microreader.

[0104] Figure 6 shows the results of examining the cytotoxicity of citrus peel extracts. As seen here, while 50 μg / ml concentrations generally exhibited no toxicity, 100 μg / ml concentrations did not exhibit significant toxicity.

[0105] Next, the cytotoxicity of green tangerine peel extract C13 according to concentration was confirmed using the MTT method, and significant cytotoxicity was observed starting from a concentration of 90 μg / ml. Figure 7 shows the results of confirming the cytotoxicity of green tangerine peel extract C13 according to concentration.

[0106]

[0107] <Test Example 4> Antioxidant effect of green tangerine peel extract on intracellular ROS induced by UVB irradiation

[0108] The scavenging effect of C13, a peel extract of green tangerine, on intracellular ROS induced by H2O2 or UVB was measured.

[0109] Figure 8 shows the results of the concentration-dependent scavenging activity of green tangerine peel extract C13 against intracellular ROS induced by H2O2 or UVB. As seen here, the H2O2 group showed a significantly high scavenging activity in a concentration-dependent manner starting from 50 μg / ml (29.5%), and the UVB group showed a concentration-dependent scavenging activity starting from 10 μg / ml (12.8%).

[0110] In addition, the scavenging effect of green tangerine peel extract C13 on UVB-induced intracellular ROS was confirmed through flow cytometry after cell staining with DCF-DA fluorescent reagent.

[0111] Figure 9 shows the results of the scavenging activity of green tangerine peel extract C13 against UVB-induced intracellular ROS. As shown here, the scavenging activity of green tangerine peel extract C13 (50 μg / ml) was confirmed, reducing the ROS fluorescence intensity value from 205.5 to 110.8.

[0112] In addition, the effect of green tangerine peel extract C13 on intracellular ROS induced by UVB irradiation was confirmed through confocal microscopy after cell staining with DCF-DA fluorescent reagent.

[0113] Figure 10 is a fluorescence image measuring the scavenging ability of green tangerine peel extract C13 against UVB-induced intracellular ROS. As shown here, green tangerine peel extract C13 (50 μg / ml) significantly reduced the green fluorescence emitted by UVB irradiation.

[0114]

[0115] <Test Example 5> Inhibitory effect of green tangerine peel extract on changes in intracellular calcium levels induced by UVB irradiation

[0116] The cells were pretreated with 50 μg / ml of green tangerine peel extract C13 and 30 mJ / cm 2 After UVB irradiation, the cells were treated with Fluo-4 fluorescent reagent, and the intracellular calcium level was measured by measuring fluorescence intensity using a flow cytometer or confocal microscope.

[0117] Figure 11 shows the results of measuring the inhibitory effect of green tangerine peel extract C13 on changes in intracellular calcium levels induced by UVB. As shown here, the inhibitory effect of green tangerine peel extract C13 (50 μg / ml) was confirmed, and it was confirmed that the fluorescence intensity value decreased from 206.9 to 155.8.

[0118] After cell staining with Fluo-4 fluorescent reagent, the effect of green tangerine peel extract C13 (50 μg / ml) on UVB-irradiated intracellular calcium levels was confirmed using a confocal microscope.

[0119] Figure 12 is a fluorescent photograph measuring the inhibitory effect of green tangerine peel extract C13 on UVB-induced changes in intracellular calcium levels. As shown here, the inhibitory effect of green tangerine peel extract C13 (50 μg / ml) was confirmed, showing that it significantly reduced the green fluorescence emitted by UVB irradiation.

[0120]

[0121] <Test Example 6> Measurement of the activity and expression of MMPs and signaling proteins induced by UVB irradiation of green tangerine peel extract

[0122] After collecting cells, they were washed with PBS, and proteins were extracted with lysis buffer (RIPA buffer, protease inhibitor), and centrifuged at 15,000× RPM for 10 minutes to isolate the proteins. Then, equal amounts of protein and sample buffer were mixed and heated at 95℃ for 5 minutes to induce protein denaturation, and then the proteins were developed by SDS-PAGE electrophoresis. After electrophoresis, the SDS-PAGE was transferred to nitrocellulose membranes, and then diluted primary antibodies against aging-related proteins (MMP-1, MMP-2, MMP-9, phospho-SEK, phospho-JNK, phospho-c-Jun, c-Fos) were reacted, and secondary antibodies against the primary antibodies were reacted with anti-rabbit IgG conjugated horse radish peroxidase or anti-mouse IgG conjugated horse radish peroxidase, and then developed into X-ray film using an ECL kit.

[0123] Figure 13 shows the results of measuring the inhibitory effect of green tangerine peel extract C13 on UVB-induced MMP-1 activity. As shown here, the inhibitory effect of green tangerine peel extract C13 (50 μg / ml) was confirmed, reducing the fluorescence intensity index from 1.275 to 1.159.

[0124] Figure 14 shows the results of Western blotting analysis of green tangerine peel extract C13 on UVB-induced MMPs protein expression. As shown here, Western blot analysis confirmed that UVB-induced expression of MMP-1, MMP-2, and MMP-9 proteins was significantly inhibited by treatment with green tangerine peel extract C13.

[0125] Figure 15 shows the results of Western blotting analysis of green tangerine peel extract C13 on the expression of phospho-JNK, a signaling protein induced by UVB.

[0126] Figure 16 shows the results of Western blotting analysis of green tangerine peel extract C13 on the expression of UVB-induced signaling proteins c-Fos and phospho-c-Jun.

[0127] As shown in Figures 15 and 16, it was confirmed that the expression of signaling proteins induced by UVB irradiation was significantly inhibited by treatment with green tangerine peel extract C13.

[0128]

[0129] <Test Example 7> Measurement of UV absorption of green tangerine peel extract

[0130] 100% ethanol was used as a blank, and the green tangerine peel extract C13 was diluted to 50 μg / ml using 100% ethanol as a solvent, and the UV absorption was measured at a UV wavelength of 200 nm-400 nm using a spectrophotometer.

[0131] Figure 17 shows the UV absorption of green tangerine peel extract C13 at UV wavelengths. As can be seen here, green tangerine peel extract C13 clearly exhibits absorption at UVB wavelengths.

[0132]

[0133] <Test Example 8> Measurement of the protective effect of green tangerine peel extract on cell growth and morphological changes induced by UVB irradiation

[0134] Human normal skin epidermal cells (HaCaT) were seeded in a 60 mm culture dish and normal cell morphology was confirmed through a phase contrast microscope after 16 hours. After pretreatment with green tangerine peel extract C13 for 1 hour, 30 mJ / cm 2 The changed cell morphology was confirmed 24 hours after UVB irradiation.

[0135] Figure 18 shows the protective effect of green tangerine peel extract C13 on UVB-induced cell growth and morphological changes. As shown here, when UVB-induced cell morphological changes were observed using a phase-contrast microscope, cell growth was inhibited and apoptosis was observed, but a clear recovery pattern was observed after treatment with green tangerine peel extract C13.

[0136]

[0137] <Test Example 9> Measurement of the inhibitory effect of green tangerine peel extract on β-Galactosidase enzyme activity related to cell aging induced by UVB irradiation

[0138] Cells were seeded in 6-well culture dishes, and 16 hours later, they were pretreated with green tangerine peel extract C13 for 1 hour and treated with 30 mJ / cm 2 UVB was irradiated. After 24 hours, the cells were treated with β-Galactosidase staining reagent for 30 minutes, harvested, and measured for fluorescence using a flow cytometer or confocal microscope.

[0139] Figure 19 shows the inhibitory effect of green tangerine peel extract C13 on UVB-induced cellular senescence-related β-galactosidase enzyme activity. As shown here, β-galactosidase enzyme activity was measured using a flow cytometer and confocal microscope after SA-β-gal staining, and it was confirmed that high β-galactosidase activity was observed in UVB-induced cells, but was significantly reduced after treatment with green tangerine peel extract C13.

[0140]

[0141] <Test Example 10> Measurement of the inhibitory effect of green tangerine peel extract on UVB irradiation-induced phospho-H2AX

[0142] Cells were seeded in 4-well chamber slides and pretreated with green tangerine peel extract C13 for 1 hour after 16 hours and treated with 30 mJ / cm 2 After UVB irradiation, the cells were cultured for 24 hours. Cells were fixed with 100% ethanol, permeabilized with 0.5% TritonX 100, blocked with 5% bovine serum, reacted with primary antibody phospho H2AX with 1% bovine serum, and secondary antibody conjugated with FITC with 25% bovine serum in that order, and slides were prepared with a mounting reagent containing DAPI.

[0143] Figure 20 shows the inhibitory effect of green tangerine peel extract C13 on the expression of phospho-H2AX protein, a DNA damage marker induced by UVB. As shown here, the expression of phospho-H2AX protein, a DNA damage marker, was measured and found to be high in UVB-induced cells, but was significantly suppressed after treatment with green tangerine peel extract C13.

[0144]

[0145] <Test Example 11> Measurement of the inhibitory effect of green tangerine peel extract on the expression of aging-related proteins induced by UVB irradiation

[0146] After collecting cells, they were washed with PBS, and proteins were extracted with lysis buffer (RIPA buffer, protease inhibitor), and centrifuged at 15,000× RPM for 10 minutes to isolate the proteins. Equal amounts of protein and sample buffer were mixed and heated at 95℃ for 5 minutes to induce protein denaturation, and then the proteins were developed by SDS-PAGE electrophoresis. After electrophoresis, the SDS-PAGE was transferred to nitrocellulose membranes, and then diluted with primary antibodies against senescence or signaling-related proteins (P16, CyclinD1, CDK4, P21, Cyclin E, CDK2, phospho P53, P53) and reacted, and then secondary antibodies against the primary antibodies were reacted with anti-rabbit IgG conjugated horse radish peroxidase or anti-mouse IgG conjugated horse radish peroxidase, and then developed into X-ray film using an ECL kit.

[0147] Figure 21 shows the inhibitory effect of green tangerine peel extract C13 on the expression of UVB-induced aging or signaling-related proteins (P16, CyclinD1, CDK4, P21, Cyclin E, CDK2, phospho P53, P53). As shown here, the expression of aging-related proteins measured using the Western blot technique showed high expression in UVB-induced cells, but was significantly suppressed after treatment with green tangerine peel extract C13.

[0148]

[0149] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0150]

[0151] In this way, the composition containing the green tangerine peel extract of the present invention has excellent free radical scavenging ability, inhibits damage to skin cells caused by ultraviolet rays (inhibits photoaging), and has an effect of inhibiting the expression of MMPs, a collagen-decomposing enzyme, and thus can be usefully used as an anti-aging pharmaceutical composition, a cosmetic composition, and a functional food composition.

Claims

1. An anti-aging pharmaceutical composition comprising a green tangerine peel extract as an effective ingredient.

2. In paragraph 1, An anti-aging pharmaceutical composition, characterized in that the above-mentioned green tangerine peel extract is obtained by extracting green tangerine peel using 60 to 80% ethanol at 20 to 30°C for 40 to 90 minutes.

3. In paragraph 1, An anti-aging pharmaceutical composition characterized in that the anti-aging agent is an anti-oxidant, an agent for preventing collagen decomposition or an agent for preventing photoaging.

4. An anti-aging cosmetic composition characterized by containing a green tangerine peel extract as an effective ingredient.

5. In paragraph 4, An anti-aging cosmetic composition, characterized in that the above-mentioned green tangerine peel extract is obtained by extracting green tangerine peel using 60 to 80% ethanol at 20 to 30°C for 40 to 90 minutes.

6. In paragraph 4, An anti-aging cosmetic composition characterized in that the anti-aging agent is for anti-oxidation, prevention of collagen decomposition or prevention of photoaging.

7. An anti-aging functional food composition characterized by containing a green tangerine peel extract as an effective ingredient.

8. In paragraph 7, An anti-aging functional food composition, characterized in that the above-mentioned green tangerine peel extract is obtained by extracting green tangerine peel using 60 to 80% ethanol at 20 to 30°C for 40 to 90 minutes.

9. In paragraph 7, An anti-aging functional food composition characterized in that the anti-aging agent is for anti-oxidation, prevention of collagen decomposition or prevention of photoaging.

Images