Composition for anti-inflammation, skin whitening and regeneration comprising mixture of hempseed oil, Artemisia annua extract and Centella asiatica extract as effective component

Abstract

The present invention relates to a composition for anti-inflammatory, skin whitening, and skin regeneration comprising a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract as an active ingredient. Since the mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract of the present invention has excellent effects in significantly improving anti-inflammatory activity, melanin production inhibitory activity, and skin cell regeneration efficacy compared to hemp seed oil, Artemisia annua, and Centella asiatica extracts alone, it can be usefully used as a functional cosmetic or health functional food for anti-inflammatory, skin whitening, and skin regeneration, or as a treatment for skin pigmentation disorders.

Description

Technology Field

[0001] The present invention relates to a composition for anti-inflammatory, skin whitening, and skin regeneration comprising a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract as active ingredients. Background Technology

[0002] The skin is the body tissue directly exposed to the external environment and acts as a barrier between the body's interior and exterior. It protects the organism from the surrounding environment by defending against the intrusion of microorganisms and external pollutants, including chemicals and ultraviolet rays. Furthermore, skin color is determined by melanin, hemoglobin, and carotene; among these, melanin performs protective functions such as absorbing ultraviolet rays and acting as a free radical scavenger, in addition to determining skin color. However, if melanin is excessively produced due to external environmental changes such as excessive exposure to ultraviolet rays, air pollution, or stress, it causes pigmentation within the skin, leading to skin darkening (melanism) or the appearance of melasma and freckles. Skin darkening occurs through the reaction of skin cells to internal and external factors, with exposure to ultraviolet rays being a representative cause. In other words, when skin is exposed to ultraviolet (UV) rays, tyrosinase is activated. Through an oxidation process in which this tyrosinase acts on tyrosine present in skin tissue to produce DOPA and dopaquinone, a polymer called melanin is synthesized within melanosomes inside melanocytes, which are skin pigment cells. This melanin is then transferred to keratinocytes, the skin's keratin-forming cells, and reaches the skin surface through the keratinization process to protect the skin from UV rays. Therefore, melanin is an indispensable UV defense agent for the human body and also serves as an effective free radical scavenger, eliminating various radicals that modify biological components such as proteins, lipids, and nucleic acids. However, if melanin is synthesized excessively locally, or if the skin's physiological functions decline due to skin lesions or aging, melanin is deposited on the skin surface, causing melasma, freckles, and various forms of pigmentation.As mentioned above, with the elucidation of the causes and mechanisms of skin darkening, methods are generally used in the manufacture of whitening cosmetics to incorporate substances that inhibit the activity of tyrosinase, an enzyme involved in the skin darkening process, or to reduce melanin production by inhibiting certain reactions during the melanin generation process. Commonly known whitening ingredients include substances that inhibit tyrosinase enzyme activity, such as kojic acid and arbutin, as well as hydroquinone, L-ascorbic acid, their derivatives, and various plant extracts. By inhibiting the synthesis of melanin pigment, these ingredients can not only brighten the skin tone and achieve skin whitening, but also improve skin hyperpigmentation conditions such as melasma or freckles caused by ultraviolet rays, hormones, or genetics. However, there are problems such as limitations on usage amounts due to safety concerns like irritation and redness upon skin application, or the insufficient effectiveness that makes it impossible to expect substantial results. Therefore, there is an urgent need for the development of a substance that is safe for the body, has stable active ingredients, and, above all, has excellent whitening effects.

[0003] Meanwhile, when the skin is damaged, an inflammatory response is followed by the promotion of proliferation of various cells, and these processes continue from two days to three weeks after the damage. During this period, the induction of fibroblast proliferation and the stimulation of collagen production fill the gaps in the damaged skin, while the reconstruction of new cells and keratin components occurs at the wound site.

[0004] A closer look at the skin wound healing process reveals that when the skin is injured, mast cells in the dermis secrete histamine, causing blood vessels to dilate, and inflammatory cells gather in these dilated vessels. Among these gathered inflammatory cells, white blood cells secrete cytokines, which activate fibroblasts responsible for maintaining dermal elasticity, thereby promoting the secretion of substances such as collagen and mucopolysaccharides. Subsequently, the epidermis torn by the injury activates genes related to cell proliferation and migration, ultimately leading to the regeneration of the epidermis.

[0005] Meanwhile, hemp is an annual plant belonging to the genus Cannabis of the family Cannabis, and its scientific name is Cannabis sativa It is a plant that has been cultivated as a fiber crop in Korea and China, and in the West, has been utilized as a medicinal plant for its terpenoid-based component, cannabinoids. Hemp seeds are round and dark brown, with a diameter of approximately 3 to 5 mm. The oil contained in hemp seeds consists of unsaturated fatty acids that help lower bad cholesterol levels and improve blood circulation, thereby aiding in the prevention of cardiovascular diseases such as arteriosclerosis. Additionally, it is known to be rich in fiber, which aids digestion and is effective in preventing constipation.

[0006] Artemisia annua ( Artemisia annua Artemisia annua is an annual herb of the Asteraceae family that grows mainly in China but is cultivated in various regions of Korea. Artemisia annua has traditionally been used to treat fever, colds, malaria, infantile convulsions, indigestion, and dysentery, and is known as the source plant for the antimalarial drug artemisinin.

[0007] Centella asiatica Centella asiatica It is a perennial plant belonging to the Apiaceae family. It grows commonly in the mountains and fields of the southern islands of the Korean Peninsula and is also known as tiger grass. The main stem spreads sideways, and there are two degenerate, scale-like leaves near the nodes where roots grow. In India, it is used for medicinal purposes, but it is known to be toxic if consumed in large quantities.

[0008] Regarding technologies related to skin whitening and regeneration, Korean Registered Patent No. 2077708 discloses a 'composition for skin whitening and skin regeneration containing a mixture of fermented extracts of Poria cocos, Coix seed extract, and Stellaria media extract using Hericium erinaceus mycelium as active ingredients,' and Korean Registered Patent No. 1697614 discloses a 'composition for antioxidant activity, whitening, and wrinkle improvement containing Paeonia lactiflora, Atractylodes macrocephala, Angelica dahurica, and Tribulus terrestris, and a functional cosmetic containing the same,' but there has not yet been any disclosure of the present invention's 'composition for anti-inflammatory, skin whitening, and skin regeneration containing a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract as active ingredients.' The problem to be solved

[0009] This invention was derived from the above-mentioned requirements and was devised as a result of the research of the project "Development and Commercialization of Functional Cosmetic Products Utilizing Hemp Seed Oil and Oilseed Meal Processing By-product Resources."

[0010] The present invention provides a composition for anti-inflammatory, skin whitening, and skin regeneration comprising a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract as an active ingredient, and the present invention was completed by confirming that the mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract of the present invention has the effect of significantly improving anti-inflammatory activity, melanin production inhibitory activity, and skin cell regeneration efficacy compared to hemp seed oil, Artemisia annua extract alone, and Centella asiatica extract alone. means of solving the problem

[0011] To achieve the above objective, the present invention comprises hemp seed oil, Artemisia annua ( Artemisia annua ) extract and Centella asiatica ( Centella asiatica A cosmetic composition for anti-inflammatory, skin whitening, and skin regeneration is provided, comprising a mixture of ) extracts as an active ingredient.

[0012] In addition, the present invention relates to hemp seed oil, Artemisia annua ( Artemisia annua) extract and Centella asiatica ( Centella asiatica The present invention provides a health functional food composition for anti-inflammatory, skin whitening, and skin regeneration containing a mixture of extracts as an active ingredient.

[0013] In addition, the present invention relates to hemp seed oil, Artemisia annua ( Artemisia annua ) extract and Centella asiatica ( Centella asiatica The present invention provides a pharmaceutical composition for the prevention or treatment of hypermelanosis of pigmentation, comprising a mixture of extracts as an active ingredient. Effects of the invention

[0014] The present invention relates to a composition for anti-inflammatory, skin whitening, and skin regeneration comprising a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract as an active ingredient. The mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract of the present invention has the effect of significantly improving anti-inflammatory activity, melanin production inhibitory activity, and skin cell regeneration efficacy compared to hemp seed oil, Artemisia annua extract alone, and Centella asiatica extract alone. Brief explanation of the drawing

[0015] Figure 1 shows the results of confirming cell viability when (A) macrophages (RAW264.7), (B) mouse melanoma cells (B16F10), and (C) human skin fibroblasts (HS68) were treated with hemp seed oil, Artemisia annua, Centella asiatica extracts alone, and a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract of the present invention at different concentrations, respectively. CON represents the untreated control group. Figure 2 shows the results of confirming the inhibitory effect on nitric oxide (NO) production when (A) hemp seed oil, (B) Artemisia annua extract, (C) Centella asiatica extract, and (D) a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract were treated to macrophages (RAW264.7) at different concentrations. CON is the untreated control group, LPS is the group treated with LPS (1 μg / ml), a nitric oxide synthesis inducer, alone, and RE is the positive control group treated with LPS and resveratrol (10 μg / ml). ### indicates that the amount of nitric oxide produced in the group treated with LPS alone increased statistically significantly compared to the untreated control group, with p<0.001. *** indicates that the amount of nitric oxide produced in the positive control group and the test group decreased statistically significantly compared to the group treated with LPS alone, with p<0.001. Figure 3 shows the results of confirming the inhibitory effect on melanin production when mouse melanoma cells (B16F10) were treated with (A) hemp seed oil, (B) Artemisia annua extract, (C) Centella asiatica extract, and (D) a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract at different concentrations. CON is the untreated control group, α-MSH is the group treated with α-MSH (100 nM), a melanin synthesis inducer, alone, and Arbutin is the positive control group treated with α-MSH and Arbutin (100 μg / ml). ### indicates that the amount of melanin produced in the group treated with α-MSH alone increased statistically significantly compared to the untreated control group, with p<0.001. *** indicates that the amount of melanin produced in the positive control group and the test group decreased statistically significantly compared to the group treated with α-MSH alone, with p<0.001. Figure 4 shows the results of confirming the cell regeneration effect when human skin fibroblasts (HS68) were treated with (A) hemp seed oil, (B) Artemisia annua extract, (C) Centella asiatica extract, and (D) a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract at different concentrations. CON is the untreated control group, and UV-B is UV-B (100 mJ / cm²) for inducing cell stress stimulation. 2 ) is the group treated with UV-B alone, and Retinoic acid is the positive control group treated with UV-B and Retinoic acid (0.03 μg / ml). ### indicates that the cell regeneration rate of the group treated with UV-B alone was statistically significantly reduced compared to the untreated control group, with p<0.001. *, *** indicate that the cell regeneration rate of the positive control group and the test group was statistically significantly increased compared to the group treated with UV-B alone, with * being p<0.05 and *** being p<0.001. Specific details for implementing the invention

[0016] To achieve the objective of the present invention, the present invention comprises hemp seed oil, Artemisia annua ( Artemisia annua ) extract and Centella asiatica ( Centella asiatica A cosmetic composition for anti-inflammatory, skin whitening, and skin regeneration is provided, comprising a mixture of ) extracts as an active ingredient.

[0017] The mixing ratio of the hemp seed oil, Artemisia annua extract, and Centella asiatica extract is preferably 1 to 10:1 to 10:1 to 10 by weight, more preferably 1 to 2:1 to 2:1 to 2 by weight, and even more preferably 1:1:1 by weight, but is not limited thereto.

[0018] The above Artemisia annua extract and Centella asiatica extract may be prepared by a method comprising the following steps, but are not limited thereto:

[0019] (1) A step of extracting each of Artemisia annua or Centella asiatica by adding an extraction solvent;

[0020] (2) A step of filtering the extract of step (1); and

[0021] (3) A step of preparing an extract by drying the filtered extract of step (2).

[0022] In step (1) above, the extraction solvent is preferably selected from water, C1-C4 lower alcohols, or a mixture thereof, more preferably water or ethanol, and even more preferably ethanol, but is not limited thereto. In the above manufacturing method, the extraction method may utilize any conventional method known in the art, such as hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. It is preferable to extract by adding the extraction solvent in an amount of 1 to 20 times the weight of each herbal medicine. The extraction temperature is preferably 20 to 100°C, but is not limited thereto. In addition, the extraction time is preferably 1 to 10 hours, and more preferably 2 to 6 hours, but is not limited thereto. In step (3) above, drying is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze-drying, and more preferably by reduced pressure drying, but is not limited thereto.

[0023] In the present invention, the skin whitening effect is intended to be an effect of improving or preventing dark skin, melasma, freckles, and dark circles caused by various factors such as exposure to ultraviolet rays, changes in hormone balance, and genetic programming; an effect of making the skin transparent and beautiful, or maintaining transparent and beautiful skin; and an effect of reducing dullness of the skin to increase radiance and firmness. Generally, it is known that dark skin, melasma, freckles, and dark circles occur when melanocytes are stimulated by irritation from ultraviolet rays or changes in hormone balance, and the melanin pigment biosynthesized therefrom is deposited in the skin. Therefore, if the production of melanin can be suppressed, it is possible to prevent and improve dark skin, melasma, freckles, and dark circles.

[0024] In a cosmetic composition according to one embodiment of the present invention, the cosmetic composition may have any one formulation selected from skin, skin softener, skin toner, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, eye cream, moisture cream, hand cream, essence, nourishing essence, pack, cleansing foam, cleansing water, cleansing cream, body lotion, body cleanser, soap, and powder, but is not limited thereto. A cosmetic composition made of each of these formulations may contain various bases and additives that are necessary and appropriate for the formulation of the formulation, and the types and amounts of these ingredients can be easily selected by a person skilled in the art.

[0025] In the case where the formulation of the cosmetic composition of the present invention is a paste, cream, or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0026] When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane-butane, or dimethyl ether.

[0027] When the formulation of the cosmetic composition of the present invention is a solution or an emulsion, a solvent, a solvating agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan.

[0028] In the case where the formulation of the cosmetic composition of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be used as carrier components.

[0029] In the case where the formulation of the cosmetic composition of the present invention is a cleansing product containing a surfactant, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0030] In addition, the present invention relates to hemp seed oil, Artemisia annua ( Artemisia annua ) extract and Centella asiatica ( Centella asiatica The present invention provides a health functional food composition for anti-inflammatory, skin whitening, and skin regeneration containing a mixture of extracts as an active ingredient.

[0031] The above-mentioned health functional food has the advantage of providing superior effects when consumed in the form of an inner beauty food. The above-mentioned inner beauty refers to a food referred to as "edible cosmetics or beauty food," which is a food that changes the skin constitution to a healthy state by absorbing various skin-beneficial ingredients into the body. Just as one chooses cosmetics suited to their skin type, one can select and consume an inner beauty food tailored to their individual needs by considering their skin condition and lifestyle. More preferably, when a cosmetic containing the above-mentioned cosmetic composition is combined with an inner beauty food containing the above-mentioned active ingredient, the anti-inflammatory, skin whitening, and regenerative effects are significantly higher compared to using the cosmetic alone, thereby providing the advantage of achieving more effective anti-inflammatory, skin whitening, and regenerative effects.

[0032] The health functional food composition of the present invention may be prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages, but is not limited thereto.

[0033] When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The active ingredient may be used appropriately according to its intended use (prevention or improvement). Generally, when manufacturing food or beverages, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, relative to the raw material. However, in the case of long-term consumption for health purposes, the above amount may be less than the above range, and since there are no issues regarding safety, the active ingredient may be used in an amount greater than the above range.

[0034] There are no special restrictions on the types of the above-mentioned health functional foods. Examples of foods to which the above-mentioned health functional food composition may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, and vitamin complexes, and include all health foods in the conventional sense.

[0035] In addition, the health functional food composition of the present invention may be manufactured into a food, particularly a functional food. The functional food of the present invention may include ingredients that are typically added. For example, it includes proteins, carbohydrates, fats, nutrients, and flavoring agents. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredients. The natural carbohydrate is preferably a monosaccharide (e.g., glucose, fructose, etc.), a disaccharide (e.g., maltose, sucrose, etc.), an oligosaccharide, a polysaccharide (e.g., dextrin, cyclodextrin, etc.), or a sugar alcohol (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., taumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

[0036] In addition to the above health functional food composition, it may further contain various nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Although the proportion of these added ingredients is not of great importance, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health functional food composition of the present invention.

[0037] In addition, the present invention relates to hemp seed oil, Artemisia annua ( Artemisia annua ) extract and Centella asiatica ( Centella asiatica The present invention provides a pharmaceutical composition for the prevention or treatment of hypermelanosis of pigmentation, comprising a mixture of extracts as an active ingredient.

[0038] The term "hyperpigmentation" as used herein means becoming darker or blacker than other areas due to an excessive increase in melanin in specific parts of the skin or nails. The hyperpigmentation conditions include, but are not limited to, freckles, senile spots, liver spots, melasma, brown or black spots, solar pigmentation spots, cyanic melasma, hyperpigmentation after drug use; hyperpigmentation after inflammation caused by abrasions, burns, or dermatitis; or gravidic chloasma.

[0039] The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient, and may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, topical preparations, suppositories, and sterile injectable solutions according to conventional methods, but is not limited thereto.

[0040] Carriers, excipients, and diluents that may be included in the above pharmaceutical composition include 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.

[0041] When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0042] 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, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. with the active ingredient. In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0043] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and 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, cacao oil, laurin oil, glycerogelatin, etc. may be used as bases for suppositories.

[0044] The appropriate dosage of the pharmaceutical composition according to the present invention can be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity. The concentration of the active ingredient included in the composition of the present invention can be determined by considering the therapeutic purpose, the patient's condition, the duration of need, etc., and is not limited to a specific range of concentrations.

[0046] The present invention will be described in detail below by way of embodiments. However, the following embodiments are merely illustrative of the present invention, and the content of the present invention is not limited to the following embodiments.

[0048] Example 1. Preparation of a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract

[0049] Hemp seed oil was extracted using a cold-pressing method considering oxidative stability, functionality, and sensory characteristics. Oil was extracted by applying a pressure of 650 MPa to shelled hemp seeds for 30 minutes without a roasting process, and then filtered to obtain cold-pressed hemp seed oil.

[0050] Artemisia annua ( Artemisia annua ) and Centella asiatica( Centella asiatica The above-ground parts excluding the roots were used. For each of Artemisia annua and Centella asiatica, 20 times the volume of 70% (w / v) ethanol solvent was added, homogenized, and shaken for 3 hours at room temperature for extraction. Subsequently, only the supernatant was collected, and the remaining residue was extracted once more using the same method. The supernatant was then concentrated under reduced pressure, filtered through a 0.45 µm syringe filter, and vacuum dried to obtain the single extracts. The mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract was prepared by mixing hemp seed oil, Artemisia annua extract, and Centella asiatica single extracts in a weight ratio of 1:1:1.

[0052] Example 2. Confirmation of cell viability of a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract

[0053] Cell viability of hemp seed oil, Artemisia annua extract, and Centella asiatica extract, as well as a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract, was determined in mouse macrophages (RAW 264.7), mouse melanoma cells (B16F10), and human skin fibroblasts (HS68). RAW264.7, B16F10, and HS68 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin (P / S) at a temperature of 37°C and under 5% CO2 conditions.

[0054] Cytotoxicity was confirmed by MTT (3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) quantitative analysis. 1 × 10⁶ cells were used per well. 5The cells were dispensed into 96-well plates at a density of [number], and treated with hemp seed oil; Artemisia annua extract alone; Centella asiatica extract alone; and a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract at various concentrations, followed by incubation for 24 hours. Subsequently, MTT solution was added and reacted for 3 hours to confirm the formation of purple formazan within the cells. After completely removing the solution from each well and dissolving the formazan crystals in DMSO, cell viability was measured at a wavelength of 570 nm.

[0055] As a result, as disclosed in Figure 1, cell viability was maintained up to a concentration of 200 μg / ml for the Artemisia annua extract alone, and cell viability was maintained up to a concentration of 100 μg / ml for hemp seed oil; Centella asiatica extract alone; and a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract, so that each extract was used at a concentration that was not toxic in subsequent experiments.

[0057] Example 3. Anti-inflammatory effect of a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract

[0058] To confirm the anti-inflammatory effects of hemp seed oil, Artemisia annua, and Centella asiatica extracts, changes in nitric oxide (NO) production were measured for hemp seed oil, Artemisia annua extract alone, Centella asiatica extract alone, and a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract.

[0059] 1 × 10⁶ RAW264.7 cells per well 6The samples were dispensed into 96-well plates at a density of [number] and cultured for 24 hours, after which the medium was replaced with serum-free DMEM. At this time, 10 µg / ml of resveratrol (positive control); hemp seed oil; Artemisia annua extract alone; Centella asiatica extract alone; and a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract were each pretreated simultaneously and cultured for 1 hour, followed by treatment with 1 µg / ml of LPS (lipopolysaccharide) and culture for 24 hours. Subsequently, the degree of nitrite accumulation in the culture medium was measured using Griess reagent. The supernatant of the culture medium was mixed with Griess reagent for 10 minutes, and the absorbance was measured at a wavelength of 540 nm; the amount of nitrite in the culture medium was determined by referring to the sodium nitrite (NaNO2) standard graph. The amount of nitric oxide (NO) produced was expressed as a percentage (%) compared to the LPS-alone treatment group.

[0060] As a result, as disclosed in Fig. 2, the amount of nitric oxide (NO) produced in the LPS-only treatment group was significantly increased compared to the untreated control group (CON), but the amount of nitric oxide produced in the hemp seed oil; Artemisia annua extract alone; Centella asiatica extract alone; and the mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract was statistically significantly reduced compared to the LPS-only treatment group. In particular, it was confirmed that the nitric oxide production inhibitory effect was significantly increased in the group treated with the mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract of the present invention (Fig. 2D) compared to the groups treated with the single extracts (Figs. 2A, 2B, and 2C).

[0062] Example 4. Skin whitening effect of a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract

[0063] To confirm the whitening effect of hemp seed oil, Artemisia annua, and Centella asiatica extracts, the inhibitory effect on melanin production by hemp seed oil, Artemisia annua extract alone, Centella asiatica extract alone, and a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract was confirmed.

[0064] 5×10 B16F10 cells per well 4 The cells were seeded into 12-well plates at a density of [number] and cultured for 24 hours. Subsequently, melanin synthesis was induced by treating each well with 100 nM α-MSH (melanocyte-stimulating hormone). At this time, 100 µg / ml arbutin (positive control); hemp seed oil; Artemisia annua extract alone; Centella asiatica extract alone; and a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract were each treated simultaneously and cultured for 48 hours. Afterward, the medium was removed and the cells were washed twice with PBS (phosphatate buffer saline). Lysis buffer (0.1 M sodium phosphate buffer containing 1% Triton X-100 and 0.2 mM PMSF) was added to each well, and the cells were lysed by incubating at -70°C for 1 hour. Subsequently, the cells were left at room temperature for about 30 minutes, and the lysed cells were collected and centrifuged at 12,000 rpm for 15 minutes at 4°C. To the pellet obtained from centrifugation, 200 µl of 1N NaOH solution containing 10% (v / v) DMSO (dimethyl sulfoxide) was added and reacted at 80°C for 1 hour, after which the absorbance was measured at 405 nm. The amount of melanin produced was expressed as a percentage (%) relative to the α-MSH-alone treatment group.

[0065] As a result, as disclosed in Fig. 3, the amount of melanin produced in the α-MSH-only treatment group was significantly increased compared to the untreated control group (CON), but the amount of melanin produced was statistically significantly decreased in the hemp seed oil; Artemisia annua-only extract; Centella asiatica-only extract; and the mixture of hemp seed oil, Artemisia annua-only extract, and Centella asiatica-only extract compared to the α-MSH-only treatment group. In particular, it was confirmed that the melanin production inhibitory effect was significantly increased in the mixture of hemp seed oil, Artemisia annua-only extract, and Centella asiatica-only extract of the present invention (Fig. 3D) compared to the single extract treatment groups (Figs. 3A, 3B, and 3C).

[0067] Example 5. Skin regeneration effect of a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract

[0068] To confirm the skin regeneration effects of hemp seed oil, Artemisia annua, and Centella asiatica extracts, the skin cell regeneration effects of hemp seed oil, Artemisia annua extract alone, Centella asiatica extract alone, and a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract were confirmed.

[0069] 1 × 10⁶ HS68 cells per well 5 The cells were dispensed into a 24-well plate at a density of [number] and incubated for 24 hours; afterward, the medium was removed and 500 µl of PBS was added. Subsequently, UV-B was applied at 100 mJ / cm². 2 Stress stimulation was induced in the cells by treating them for 30 seconds under the specified conditions. Subsequently, 0.03 μg / ml of retinoic acid (positive control); hemp seed oil; Artemisia annua extract alone; Centella asiatica extract alone; and a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract were treated simultaneously, respectively, and cultured for 48 hours. Afterward, MTT solution was added and reacted for 3 hours to confirm the formation of purple formazan within the cells. The solution in each well was completely removed, and the formazan crystals were dissolved in DMSO; subsequently, the cell regeneration rate was measured at a wavelength of 570 nm. The cell regeneration rate was expressed as a percentage (%) relative to the untreated control group.

[0070] As a result, as disclosed in Figure 4, the cell regeneration rate in the UV-B-only treatment group was significantly reduced compared to the untreated control group (CON), but the cell regeneration rate in the group treated with a mixture of Centella asiatica extract alone, hemp seed oil, Artemisia annua extract, and Centella asiatica extract was statistically significantly increased compared to the UV-B-only treatment group. Meanwhile, the group treated with hemp seed oil and Artemisia annua extract alone did not show efficacy in skin cell regeneration, but the cell regeneration effect in the group treated with a mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract of the present invention was significantly increased compared to the UV-B-only treatment group, confirming the synergistic effect of the mixture of hemp seed oil, Artemisia annua extract, and Centella asiatica extract of the present invention.

Claims

Claim 1 Hemp seed oil, Artemisia annua ( Artemisia annua ) extract and Centella asiatica ( Centella asiatica A cosmetic composition for anti-inflammatory, skin whitening, and skin regeneration comprising a mixture of ) extracts as an active ingredient. Claim 2 A cosmetic composition for anti-inflammatory, skin whitening, and skin regeneration according to claim 1, characterized in that the mixing ratio of the hemp seed oil, Artemisia annua extract, and Centella asiatica extract is a weight ratio of 1 to 10:1 to 10:1 to 10. Claim 3 A cosmetic composition for anti-inflammatory, skin whitening, and skin regeneration according to claim 1, wherein the extraction solvent of the Artemisia annua and Centella asiatica extracts is water, a C1-C4 lower alcohol, or a mixture thereof, respectively. Claim 4 A cosmetic composition for anti-inflammatory, skin whitening, and skin regeneration according to claim 1, characterized in that the cosmetic composition is prepared in any one formulation selected from skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, eye cream, moisture cream, hand cream, essence, nourishing essence, pack, cleansing foam, cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap, and powder. Claim 5 Hemp seed oil, Artemisia annua ( Artemisia annua ) extract and Centella asiatica ( Centella asiatica A health functional food composition for anti-inflammatory, skin whitening, and skin regeneration comprising a mixture of ) extracts as an active ingredient. Claim 6 A health functional food composition for anti-inflammatory, skin whitening, and skin regeneration according to claim 5, characterized in that the health functional food is an inner beauty food. Claim 7 A health functional food composition for anti-inflammatory, skin whitening, and skin regeneration according to claim 5, characterized in that the composition is prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages. Claim 8 Hemp seed oil, Artemisia annua ( Artemisia annua ) extract and Centella asiatica ( Centella asiatica A pharmaceutical composition for the prevention or treatment of hypermelanosis of pigmentation, comprising a mixture of ) extracts as an active ingredient. Claim 9 A pharmaceutical composition for the prevention or treatment of a melanin hyperpigmentation disease according to claim 8, characterized in that the melanin hyperpigmentation disease is freckles, senile spots, liver spots, melasma, brown or black spots, solar pigmentation spots, cyanic melasma, hyperpigmentation after drug use; hyperpigmentation after inflammation caused by abrasions, burns or dermatitis; or gravidic chloasma. Claim 10 A pharmaceutical composition for the prevention or treatment of hypermelanosis, characterized in that, in claim 8, the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. Claim 11 A pharmaceutical composition for the prevention or treatment of hypermelanosis, characterized in that, in addition to the active ingredient in claim 8, it further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

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