A microneedle patch containing oriental medicine extracts as active ingredients

Abstract

The present invention relates to a microneedle patch comprising, as an active ingredient, an extract of *Yangtaejinheungbokgo*, a composite material composed of *Guri-ttae* root, *Sukjihwang*, *Ginseng*, *Dioscorea japonica*, and *Ophiopogon japonicus*. Since the extract exhibits excellent antioxidant and anti-inflammatory effects, when used as a composition for a microneedle patch, it can be efficiently delivered into the skin without loss of active ingredients, thereby providing excellent skin protection.

Description

Technology Field

[0001] The present invention relates to a microneedle patch containing a complex extract of herbal medicinal materials as an active ingredient, and more specifically, to a microneedle patch containing an extract of *Yangtaejinheungbokgo*, a complex material composed of *Guri-ttae* root, *Sukjihwang*, *Ginseng*, *Dioscorea japonica*, and *Ophiopogon japonicus*, as an active ingredient. Background Technology

[0002] Recently, consumer awareness of skin health has changed due to COVID-19, and interest in skincare has increased more than in makeup. Additionally, the number of consumers concerned about skin troubles caused by fine dust and harmful substances is continuously increasing.

[0003] Meanwhile, interest in self-beauty care at home to protect the skin barrier from external stimuli is also steadily increasing.

[0004] Reflecting these consumer needs, research is continuously being conducted on products that are easy to use at home without the help of external experts and have high skin delivery efficiency.

[0005] Microneedles, which are currently undergoing active development, are a type of transdermal drug delivery system (TDDS) capable of directly injecting active ingredients into the epidermis or dermis. This enables the efficient delivery of beneficial ingredients into the body compared to conventional cosmetics and is receiving keen attention from the relevant industry as a next-generation innovative bio-platform.

[0006] Based on this, the inventor of the present invention aimed to develop a functional herbal microneedle patch product capable of maximizing the delivery of active ingredients from a complex extract derived from the herbal material Yangtaejinheungbokgo, using proprietary microneedle manufacturing technology.

[0007] In particular, while many herbal materials have been used in the past for skin improvement effects, there have been no previously introduced cases of using a complex mixture of Angelica dahurica root (Bai Zhi), Rehmannia glutinosa, ginseng, Dioscorea japonica, and Ophiopogon japonicus as a composition for a microneedle patch, as in the present invention. The problem to be solved

[0008] The objective of the present invention is to provide a system for effectively delivering a pharmaceutical composition for treating skin diseases using an extract of *Yangtaejinheungbokgo* into the skin. means of solving the problem

[0009] To achieve the above objective, the present invention is characterized by providing a natural herbal composition comprising, as an active ingredient, an extract of a composite material composed of Angelica dahurica root, Rehmannia glutinosa, ginseng, Dioscorea japonica, and Ophiopogon japonicus.

[0010] According to one embodiment of the present invention, the composite material is characterized by being a mixture of copper root, Rehmannia glutinosa, ginseng, Dioscorea japonica, and Ophiopogon japonicus in a mass ratio of 1:1:1:1:1.

[0011] According to one embodiment of the present invention, the extract is characterized as being a hot water extract or a 70% ethanol extract.

[0012] According to one embodiment of the present invention, the composition is characterized by having antioxidant activity.

[0013] According to one embodiment of the present invention, the composition is characterized by having the ability to inhibit melanin production.

[0014] According to one embodiment of the present invention, the composition is characterized by inhibiting the activity of elastase.

[0015] According to another embodiment of the present invention, a microneedle patch comprising the above herbal composition can be provided.

[0016] In addition, the material forming the microneedle patch is characterized by comprising hyaluronic acid, trehalose, glycerin, sodium alginate, and purified water.

[0017] In addition, the microneedle patch is characterized by being manufactured by extruding a mixture of 10 parts by weight of the herbal composition of claim 1, 8 parts by weight of hyaluronic acid, 2 parts by weight of trehalose, 1 part by weight of glycerin, and 0.5 parts by weight of sodium alginate into a microneedle mold, relative to 100 parts by weight of purified water.

[0018] In addition, the viscosity of the above mixture is characterized to be in the range of 800 to 1,200 cps. Effects of the invention

[0019] The present invention relates to a skin protection composition containing, as an active ingredient, a composition extracted from a composite material of Angelica dahurica root (Bai Zhi), Rehmannia glutinosa, ginseng, Dioscorea japonica, and Ophiopogon japonicus, known as Yangtaejinheungbokgo. It has been confirmed to have excellent antioxidant and anti-inflammatory effects, and can be used as a skin protection composition containing the natural product as an active ingredient by effectively delivering it transdermally using a microneedle patch. Brief explanation of the drawing

[0020] Figure 1 is the ABTS test result of the Yangtaejinheungbokgo extract of the present invention. Figure 2 is the DPPH test result of the Yangtaejinheungbokgo extract of the present invention. Figure 3 is the result of the Elastase inhibition Assay test of the Yangtaejinheungbokgo extract of the present invention. Figure 4 is the result of the Collagenase inhibition Assay test of the Yangtaejinheungbokgo extract of the present invention. Figure 5 shows the results of tyrosinase activity inhibition by the Yangtaejinheungbokgo extract of the present invention. Figures 6 and 7 are the results of the MTT Assay test of the Yangtaejinheungbokgo extract of the present invention. Figures 8 and 9 are the results of a test on the ROS generation inhibition ability of the Yangtaejinheungbokgo extract of the present invention. Figures 10 and 11 are the results of a test on the ability of the Yangtaejinheungbokgo extract of the present invention to inhibit nitric oxide production. Figures 12 and 13 are the results of measuring protein expression through western blot of the Yangtaejinheungbokgo extract of the present invention. Specific details for implementing the invention

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. Furthermore, the nomenclature used herein is well known and commonly used in the art.

[0022] In this invention, the term "extract" encompasses all substances obtained by extracting components from natural products, regardless of the extraction method or the type of component. For example, it is a broad concept that includes substances obtained by extracting solvent-soluble components from natural products using water or organic solvents, as well as substances obtained by extracting only specific components of natural products, such as oils.

[0023] First, the herbal composition included in the microneedle patch of the present invention comprises a composition extracted from a powder mixed with Angelica dahurica root (Baekji), Rehmannia glutinosa, ginseng, Dioscorea japonica, and Ophiopogon japonicus, which are the main components of Yangtaejinheungbokgo, an herbal prescription known for skin beauty.

[0024] Angelica dahurica is a perennial herb belonging to the Apiaceae family. It is distributed in Northeast Asia and grows mainly on the edges of mountains and valleys. It is about 1 to 1.5 meters tall, and has many fibrous roots growing from its fleshy rhizome.

[0025] The leaves branch out into multiple lobes and are oval-shaped, measuring 5 to 10 cm in length. White flowers bloom from June to August, and oval-shaped fruits ripen around October. In traditional Korean medicine, the root of the Angelica dahurica is called Baekji and is used as a sedative, analgesic, and hemostatic agent.

[0026] In addition, Angelica dahurica contains a component called imperatorin, which has a powerful anti-inflammatory effect. It is known to be beneficial for various skin diseases and pruritus, and is effective against acne, itching, suppuration, freckles, and hives.

[0027] In addition, white peony root contains a component called frangenidin, which shrinks pores and makes the skin radiant.

[0028] Sukjihwang is a medicinal herb made by steaming and drying the root of Rehmannia glutinosa, which belongs to the Scrophulariaceae family. If the root is used as is, it is called Saengjihwang; if it is dried, it is called Geonjihwang; and if it is soaked in alcohol and then steamed, it is called Sukjihwang.

[0029] Rehmannia glutinosa is rich in carotene, which eliminates free radicals that accelerate aging and performs antioxidant functions that promote cell regeneration.

[0030] Korean ginseng is a perennial plant belonging to the Araliaceae family that grows mainly in the Korean Peninsula, Manchuria, and the Primorsky Krai region, and contains components such as saponin, vitamin A, vitamin B, vitamin C, and inorganic salts.

[0031] Ginseng helps improve blood circulation by expanding the skin's capillaries, makes the skin radiant, adds elasticity, and inhibits wrinkles.

[0032] Sanyak, also known as Cham-ma, is a tropical vine and a perennial herb belonging to the Dioscoreaceae family; its scientific name is Dioscorea japonica. Sanyak contains phytomucin, a sticky mucilaginous component that is the main component of glycoproteins. It helps maintain skin health and provides excellent moisturizing and elasticity effects, as well as the ability to detoxify and soothe toxins within the skin.

[0033] Ophiopogon japonicus contains components such as saponins and beta-sitosterol, playing a role in improving and managing problematic and aging skin.

[0034] When the tuberous roots of Ophiopogon japonicus are dried, they turn a translucent pale yellow color and are known to have excellent effects in stopping coughs and phlegm, supporting lung function, and boosting vitality. In traditional Korean medicine, it is used as a tonic, expectorant, antitussive, and cardiotonic. Additionally, Ophiopogon japonicus is effective for skin whitening and moisturizing, and as it keeps the skin hydrated, it is a medicinal herb utilized for dry skin.

[0036] The present invention will be described in detail below through examples and the like.

[0038] <Example 1. Preparation of Ethanol Extract of Yangtaejinheungbokgo>

[0039] The materials for Yangtaejinheungbokgo purchased through an online market—Chinese angelica root, Rehmannia glutinosa, ginseng, Dioscorea japonica, and Ophiopogon japonicus—are each washed 5 to 6 times in distilled water and dried at 45℃ for 3 to 4 days.

[0040] Next, the dried Angelica root, Rehmannia glutinosa, ginseng, Dioscorea japonica, and Ophiopogon japonicus are mixed in a mass ratio of 1:1:1:1:1 and then ground. 500g of distilled water is added per 100g of the ground mixed powder to make a mixture, and the mixture is heated in a water bath for at least 5 hours until the water evaporates and the weight of the remaining liquid is 100g.

[0041] The mixture heated in a water bath is placed in a round flask equipped with a reflux condenser, 500 ml of ethanol with a volume concentration of 70% is added, and the mixture is heated three times for about 8 to 9 hours each in a 90°C water bath, followed by extraction and filtration to obtain an ethanol extract of Yangtaejinheungbokgo.

[0042] Here, 70% volume concentration ethanol refers to ethanol composed of ethanol and purified water in a volume ratio of 7:3, and is referred to as '70% ethanol' below.

[0044] <Example 2. Preparation of Hot Water Extract of Yangtaejinheungbokgo>

[0045] A mixed powder ground in the same manner as in Example 1 is obtained. 1 L of water is added per 50 g of the mixed powder, and then heated at 95°C for 2 hours, followed by extraction and filtration, to obtain a hot water extract of Yangtaejinheungbokgo.

[0047] <Example 3. Preparation of a Microneedle Patch Containing Yangtaejinheungbokgo Extract>

[0048] A mixed solution is prepared by mixing 10 parts by weight of a 70% ethanol extract or hot water extract of Yangtaejinheungbokgo, 8 parts by weight of hyaluronic acid, 2 parts by weight of trehalose, 1 part by weight of glycerin, and 0.5 parts by weight of sodium alginate relative to 100 parts by weight of purified water, and then stirring at room temperature for 10 minutes.

[0049] At this time, the preferred content of the Yangtaejinheungbokgo extract in the above mixed solution is preferably 5 to 15 parts by weight per 100 parts by weight of purified water, and if the extract is 5 parts by weight or less, the amount of active ingredient included in the microneedle patch is small, so a useful effect on the skin cannot be obtained.

[0050] On the other hand, if the above extract is included in an amount of 15 parts by weight or more, there is a risk of cytotoxicity.

[0051] In addition, it is desirable to maintain the viscosity of the above-mentioned mixed solution at 800 to 1,200 cps. This is because if the viscosity is 800 cps or lower, the mixed solution penetrates well into the tip grooves within the microneedle mold, but the composition may flow out of the mold during the shaking step for removing bubbles, resulting in a loss of the active ingredient.

[0052] On the other hand, if the viscosity of the above mixed solution is greater than 1,200 cps, not only is it difficult to remove air bubbles within the composition, but it also does not penetrate into small tip grooves within the mold, making it difficult to produce microneedles and thus making it impossible to create a microneedle patch that facilitates the delivery of active ingredients.

[0053] In order to maintain the viscosity of the mixed solution within a certain range, the optimally selected amounts of hyaluronic acid, trehalose, glycerin, and sodium alginate are 8 parts by weight of hyaluronic acid, 2 parts by weight of trehalose, 1 part by weight of glycerin, and 0.5 parts by weight of sodium alginate per 100 parts by weight of purified water.

[0054] In addition, viscosity can be further controlled by varying the degree of binding of the hyaluronic acid molecules used.

[0055] Next, the above mixed solution is degassed for 10 minutes using a degasser to remove bubbles.

[0056] Then, 240 µl of the above solution, from which air bubbles have been removed, is dispensed onto the microneedle mold.

[0057] After pressurizing the microneedle mold from which the mixture has been discharged using a vacuum device under a pressure of 0.06 MPa for 30 minutes, the mold is placed on a shaker and then rotated at 3,300 rpm for 2 minutes to remove residual bubbles on the back surface.

[0058] Finally, the mold containing the air-blanked microneedles is placed in a 45°C dryer and dried for 45 minutes, and then the microneedles are separated from the mold using an adhesive to complete the microneedle patch containing herbal materials.

[0060] <Experimental Example 1. ABTS Radical Assay Test>

[0061] The antioxidant activity of the 2-2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)) radical was measured according to the Rice-Evans (1974) method. ABTS reacts with potassium persulfate to produce the ABTS+ radical, which exhibits a characteristic blue-green color. When a sample is added, the characteristic blue-green color is decolorized, and the antioxidant capacity can be measured by measuring the absorbance.

[0062] 7 mM 2,2-azino-bis and 2.4 mM potassium persulfate were mixed in equal proportions, and the mixture was left to react for 12 to 24 hours in a shaded area before proceeding with the experiment.

[0063] Samples were prepared by diluting them to concentrations of 10, 100, and 1000 μg / mL, and Ascorbic Acid (Vitamin C) was used as a positive control.

[0064] Afterward, 100 µl of the solution and 100 µl of each extract were added to a 96-well plate and reacted at 25°C for 7 minutes, after which the absorbance was measured at a wavelength of 734 nm and expressed as the inhibition rate (%).

[0065] The hot water extract and 70% ethanol extract of Yangtaejinheungbokgo were measured at concentrations of 10, 100, and 1000 μg / mL, and the results are shown in Figure 1.

[0066] According to Figure 1, the hot water extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo W) showed radical scavenging activity of 16.3±0.9% at 10㎍ / mL, 64.5±1.4% at 100㎍ / mL, and 97.0±2.8% at 1000㎍ / mL.

[0067] The 70% ethanol extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo E) showed radical scavenging activity of 20.0±1.4% at 10㎍ / mL, 55.0±0.4% at 100㎍ / mL, and 92.0±1.9% at 1000㎍ / mL.

[0068] From these results, the antioxidant effect of the test group, which is the test substance in the ABTS radical measurement test, was confirmed at different concentrations (0.001%, 0.01%, 0.1%).

[0070] <Experimental Example 2. DPPH Radical Test>

[0071] The scavenging activity against 2-2-Diphenyl-1-picrylhydrazyl (DPPH) radical was measured according to the method of Blois (1958). DPPH is a relatively stable free radical and is widely used to measure antioxidant activity.

[0072] The aforementioned free radicals cause chemical damage to human DNA, proteins, and metabolites, and it is known that such damage is a cause of aging; antioxidant capacity can be measured by measuring their absorbance.

[0073] First, DPPH was prepared by dissolving it in 99% ethanol to a concentration of 0.45 mM, and samples were prepared by diluting them to concentrations of 10, 100, and 1000 μg / mL, respectively, and Ascorbic Acid (Vitamin C) was used as a positive control.

[0074] Afterwards, 120 µl of each sample and 60 µl of DPPH were added to a 96-well plate, reacted for 15 minutes in the dark at room temperature, and the scavenging activity was measured using an ELISA reader at a wavelength of 517 nm.

[0075] The hot water extract and 70% ethanol extract of Yangtaejinheungbokgo were measured at concentrations of 10, 100, and 1000 μg / mL, respectively, and the results are shown in Figure 2.

[0076] According to Figure 2, the hot water extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo W) showed radical scavenging activity of 14.0±0.6% at 10㎍ / mL, 26.0±2.5% at 100㎍ / mL, and 63.0±1.3% at 1000㎍ / mL.

[0077] The 70% ethanol extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo E) showed radical scavenging activity of 27.0±2.5% at 10㎍ / mL, 37.0±0.9% at 100㎍ / mL, and 84.0±1.5% at 1000㎍ / mL.

[0078] From these results, the antioxidant effect of the test group, which is the test substance in the DPPH radical measurement test, was confirmed at different concentrations (0.001%, 0.01%, 0.1%).

[0080] <Experimental Example 3. Elastase Inhibition Assay>

[0081] Elastase inhibitory activity was tested based on the method of Cannell et al. (1988). Elastin is a very important protein for maintaining skin elasticity within the dermis and is present in the skin's connective tissue, playing a role in tissue elasticity and flexibility.

[0082] Elastase is an enzyme that breaks down elastin and is one of the causes of skin aging, such as wrinkles and sagging skin.

[0083] The experimental method involved using N-syccinyl-(L-Ala)-p-nitroanailide to measure the amount of p-nitroanilide produced from the substrate at 405 nm for 25 minutes at 36°C using ELISA.

[0084] The sample was diluted to a concentration of 1,000 µg / mL, 40 µl of the sample solution was taken, and 40 µl of a solution of porcine pancreas elastase (0.5 unit / mL) dissolved in 50 mM Tris-HCl buffer (pH 8.6) was added. Then, N-succinyl-(L-Ala)3-p-nitroanailide dissolved in 50 mM Tris buffer (pH 8.6) was added as a substrate, and the reaction was allowed to proceed for 20 minutes before the results were measured. Adenosine was used as a positive control.

[0085] The hot water extract and 70% ethanol extract of Yangtaejinheungbokgo were measured at concentrations of 10, 100, and 1000 μg / mL, and the results are shown in Figure 3.

[0086] As shown in Figure 3, which shows the results of the elastase activity inhibition experiment at each concentration, the hot water extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo W) showed radical scavenging activity of 2.0±1.7% at 10㎍ / mL, 17.0±1.2% at 100㎍ / mL, and 33.0±0.6% at 1000㎍ / mL.

[0087] The 70% ethanol extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo E) showed radical scavenging activity of 18.0±1.8% at 10㎍ / mL, 29.0±0.8% at 100㎍ / mL, and 55.0±0.3% at 1000㎍ / mL.

[0088] From these results, the test group, which is the test substance in the Elastase inhibition measurement test, confirmed the elastase inhibitory effect at different concentrations (0.001%, 0.01%, 0.1%).

[0090] <Experimental Example 4. Collagenase inhibition Assay>

[0091] Collagen is a major matrix protein produced by skin fibroblasts that forms most of the organic material of the skin, bones, and teeth, and makes up more than 90% of the dermis layer, protecting and maintaining the skin.

[0092] Collagenase is an enzyme that breaks down this collagen, and it is one of the causes of wrinkles, loss of elasticity, and skin sagging due to the destruction of connective tissue that maintains skin elasticity.

[0093] To confirm the skin aging inhibitory effect, collagenase inhibitory activity was measured as follows. Specifically, for the reaction group, 4 mM CaCl2 was added to 0.1 M Tris-HCl buffer (pH 7.5). 0.15 ml of collagenase (0.2 mg / ml) was added to a mixture of 0.25 ml of substrate solution containing dissolved 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (0.3 mg / ml) and 0.1 ml of sample solution, and the mixture was left at room temperature for 20 minutes. Then, 0.5 ml of 6% citric acid was added to stop the reaction, and 1.5 ml of ethyl acetate was added, and the absorbance of the supernatant was measured at 320 nm.

[0094] Collagenase inhibitory activity was expressed as the rate of decrease in absorbance between the sample solution addition group and the control group.

[0095] The hot water extract and 70% ethanol extract of Yangtaejinheungbokgo were measured at concentrations of 10, 100, and 1000 μg / mL, and the results are shown in Figure 4.

[0096] Figure 4, which shows the results of measuring collagenase inhibitory activity at each concentration, shows that the hot water extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo W) exhibited radical scavenging activity of 16.0±0.1% at 10㎍ / mL, 20.0±0.4% at 100㎍ / mL, and 30.0±0.3% at 1000㎍ / mL.

[0097] The 70% ethanol extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo E) showed collagenase inhibition of 16.0±0.3% at 10㎍ / mL, 29.0±0.8% at 100㎍ / mL, and 63.0±0.3% at 1000㎍ / mL.

[0098] From these results, the test group, which is the test substance in the collagenase inhibition measurement test, was found to have a collagenase inhibitory effect at different concentrations (0.001%, 0.01%, 0.1%).

[0100] <Experimental Example 5. Tyrosinase Inhibitory Activity>

[0101] Tyrosinase is an enzyme involved in the most critical initial rate-determining step of the melanin biosynthesis pathway in the human body; therefore, experiments to measure tyrosinase inhibitory activity were conducted to determine whether inhibiting this enzyme would result in the inhibition of melanin production.

[0102] The effect of the extract on the inhibition of intracellular tyrosinase activity was measured using the method of Martinez-Esparza et al. B16F10 cells at 1.5×10 6 After dispensing into cell culture dishes at a concentration of cells / dish, the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours for attachment and stabilization.

[0103] Next, the extracts were treated at different concentrations in a 37℃, 5% CO2 incubator and cultured for 48 hours. After culture, the cells were suspended in 1 ml of Protein Extraction solution, and then the cells were vortexed and centrifuged at 1,000 rpm for 5 minutes. The supernatant obtained was used as the enzyme solution for measuring activity.

[0104] 100 µl of the substrate L-DOPA (2 mg / ml) was added to a 96-well plate to measure tyrosinase activity, 40 µl of enzyme solution was added, and the reaction was carried out for 1 hour at 37°C in a 5% CO2 incubator, after which the activity was measured at 405 nm using a Microplate Reader.

[0105] As shown in Figure 5, which examines the tyrosinase inhibitory activity at each concentration, the hot water extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo W) showed radical scavenging activity of 5.1±3.0% at 10㎍ / mL, 22.2±2.1% at 100㎍ / mL, and 76.0±0.5% at 1,000㎍ / mL.

[0106] The 70% ethanol extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo E) showed scavenging activity of 17.1±0.8% at 10㎍ / mL, 42.1±0.1% at 100㎍ / mL, and 86.4±0.1% at 1,000㎍ / mL.

[0107] From these results, the test group, which is the test substance in the Tyrosinane inhibitory activity measurement test, showed Tyrosinase inhibitory activity effects at different concentrations (0.001%, 0.01%, 0.1%).

[0109] <Experimental Example 6. MTT Assay>

[0110] Cytotoxicity was measured using the MTT assay. 3.0 × 10⁴ per well in a 96-well plate 4Cells were seeded and cultured for 1 day at 37°C and 5% CO2.

[0111] After collecting the supernatant following incubation, 180p of DMEM medium and 20p of the extract were added, and the mixture was cultured again for 2 days. After collecting the supernatant following incubation, 100p of MTT solution was added, and crystallization was carried out for 4 hours.

[0112] After removing the MTT solution, the crystallized MTT was dissolved again by treatment with 100p DMSO, and the amount of crystallized MTT was measured using 560nm absorbance. The ability to inhibit nitric oxide formation was confirmed.

[0113] Figures 6 and 7 show the results of cytotoxicity evaluations after treating with hot water and ethanol extracts of Yangtaejinheungbokgo at concentrations of 25, 50, 100, and 200 μg / mL. As the cell viability was considered to be 100% for the control group, it was confirmed in Figure 6 that the cell viability of the test group was 80% or higher.

[0114] The hot water extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo W) showed 91% cell viability at 100 µg / mL. The 70% ethanol extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo E) showed 89% cell viability at 50 µg / mL.

[0116] Accordingly, NO, iNox, COX-2, and ROS tests were conducted at concentrations within 50 μg / mL.

[0118] <Experimental Example 7. Inhibitory Ability of Reactive Oxygen Species (ROS) Generation>

[0119] Intracellular ROS production inhibitory activity was measured using an intracellular ROS assay kit (Cell Biolabs, San Diego, CA, USA). 5×10⁴ 4RAW 264.7 cells were seeded into the cell / well and cultured to attach the cells to the well, after which the medium was changed to serum-free DMEM.

[0120] After culturing the cells for 24 hours, each sample was treated at different concentrations and cultured for another 24 hours at 37°C and 5% CO2. After washing three times with PBS (pH 7.4), 100 µl of 1X DCFH-DA was added to the medium and cultured for 1 hour at 37°C and 5% CO2, followed by washing three times again with PBS.

[0121] After adding 100 µl of Lysis buffer and mixing, fluorescence was measured at excitation 485 nm and emission 535 nm using a microplate reader (Perkin-Elmer Inc., Waltham, MA, USA) and expressed as the relative ROS generation inhibition rate compared to the LPS-alone treatment group.

[0122] The ROS production inhibitory activity of solvent-specific extracts was confirmed in RAW 264.7 macrophages treated with H2O2.

[0123] As a result of measuring the amount of ROS generated by pre-treating the extracts for 1 hour and stimulating them with H2O2, it was confirmed that ROS generation was inhibited as the concentration of the hot water and 70% ethanol extracts of Yangtaejinheungbokgo increased, and both extracts showed excellent inhibitory effects.

[0124] The control group, which was treated with H2O2, showed ROS expression nearly 10 times higher than the group not treated with H2O2. According to the results of treating with Yangtaejinheungbokgo W (hot water extract) at concentrations of 25 and 50 μg / mL, it was confirmed that as the concentration increased, the amount of ROS expression was inhibited by 12.1% and 24.7%, respectively.

[0125] As a result of treating with the 70% ethanol extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo E) at concentrations of 25 and 50 μg / mL, it was confirmed that ROS expression was inhibited by 18.5% and 54.5% as the concentration increased (see Figs. 8 and 9).

[0127] <Experimental Example 8. Nitric oxide (NO) generation inhibition ability>

[0128] Nitric oxide (NO) was measured by determining the amount of NO in the cell supernatant using nitrite and nitrate (Green et al., 1982). Cell line RAW 264.7 was seeded into a 96-well plate at a density of 1 × 10⁴ cells / well and cultured in an incubator at 37°C under 5% CO₂ conditions for 24 hours.

[0129] Subsequently, the culture medium was replaced with serum-free DMEM medium, and lipopolysaccharide (LPS) was added to induce an inflammatory response. Next, LPS was added to a concentration of 1 ppm / well, and after treating each sample solution at different concentrations, the samples were incubated in an incubator at 37°C under 5% CO2 conditions for 24 hours.

[0130] After that, 100 µl of cell culture medium was transferred to a new 96-well plate, 100 µl of Griess reagent was added, and the mixture was reacted at room temperature for 5 minutes. Then, the absorbance was measured at 540 nm and the results were calculated.

[0131] To confirm the ability to inhibit nitric oxide production in Raw 264.7 cells, the cells were treated at concentrations of 25 and 50 μg / mL to demonstrate the ability to inhibit nitric oxide production.

[0132] When the lipopolysaccharide treatment group was considered as 100%, the nitric oxide formation inhibitory activity in Raw 264.7 was 83.0% for the hot water extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo W) at a concentration of 25 μg / mL and 73.0% for the 70% ethanol extract (Yangtaejinheungbokgo E) at a concentration of 25 μg / mL (see Fig. 10).

[0133] The control group, treated with LPS, showed nearly five times higher NO expression compared to the LPS-untreated group. When treated with Yangtaejinheungbokgo W (hot water extract) at concentrations of 25 and 50 μg / mL, it was confirmed that NO expression was inhibited by 17.0% and 35.0%, respectively, as the concentration increased.

[0134] When the 70% ethanol extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo E) was treated at concentrations of 25 and 50 μg / mL, it was confirmed that as the concentration increased, NO expression was inhibited by 27.0% and 51.0%, respectively.

[0136] <Experimental Example 9. Confirmation of iNOS and COX-2 Activity>

[0137] To confirm the activity of iNOS and COX-2, 1 × 10⁶ cells of cell line Raw 264.7 were placed in a 100 mm tissue culture dish. 6 After seeding cells into cells / well, the cells were stabilized by culturing for 24 hours.

[0138] After removing the medium, LPS was treated at a concentration of 1 μg / ml for 2 hours, then cultured in medium treated with different concentrations of the extract for 24 to 48 hours, after which the medium was removed again and washed twice with PBS.

[0139] 100 µl of Complete mini 1tab was added to 10 ml of Radio-immunoprecipitationassay (RIPA) buffer to dissolve the solution, and the mixture was centrifuged at 4°C at 13,200 rpm for 20 minutes.

[0140] The supernatant obtained by centrifugation was quantified using a BCA protein assay kit, and 20 µl of protein was separated by electrophoresis on a 10% acrylamide gel.

[0141] The isolated proteins were transferred to a polyvinylidenefluoride (PVDF) membrane using a transfer device (BIORAD) and then incubated in blocking buffer (5% skim milk in TBST) at room temperature for 1 hour.

[0142] The primary antibodies against iNOS, COX-2, and β-actin were diluted and incubated overnight at 4°C, then washed three times with tris-buffered saline and tween 20 (TBST) at 10-minute intervals.

[0143] Anti-rabbit secondary antibodies for iNOS and COX-2 and anti-mouse secondary antibodies for β-actin were used, diluted 1:1,000, and incubated at room temperature for 2 hours. After washing three times with TBST, bands were confirmed and quantified using a LAS 4,000 instrument.

[0144] Inflammatory responses were induced in macrophages (RAW 264.7) using LPS, and experiments were conducted to investigate the effects of iNOS and COX-2, which are inflammation-causing genes, on mRNA by treating with hot water extract and 70% ethanol extract of Yangtaejinheungbokgo at concentrations of 25 and 50 μg / mL.

[0145] As a result, compared to the LPS-induced Control group, when treated with Yangtaejinheungbokgo W (hot water extract) at concentrations of 25 and 50 μg / mL, iNOS showed inhibition rates of 33.5% and 47.8%, respectively, and COX-2 showed inhibition rates of 22.82% and 53.24%, respectively, as the concentration increased, and the expression level showed a tendency to decrease in a concentration-dependent manner.

[0146] When the 70% ethanol extract of Yangtaejinheungbokgo (Yangtaejinheungbokgo E) was treated at concentrations of 25 and 50 μg / mL, as the concentration increased, each iNOS showed inhibition rates of 34.73% and 55.31%, and COX-2 showed inhibition rates of 33.23% and 61.63%, and the expression level showed a tendency to decrease in a concentration-dependent manner.

[0147] From these experimental results, it was confirmed that mRNA expression was inhibited as the concentrations of the hot water and 70% ethanol extracts of Yangtaejinheungbokgo increased, and the anti-inflammatory activity was verified through the inhibition of mRNA expression of the inflammatory inducers iNOS and COX-2. Both types of extracts exhibited excellent inhibitory effects. (See Figs. 12 and 13)

[0149] Although the present invention has been described above with reference to one embodiment, those skilled in the art will understand that various modifications and changes can be made to the invention without departing from the spirit and scope of the invention as described in the following claims.

Claims

Claim 1 A microneedle patch for skin whitening or anti-inflammatory skin, characterized by comprising a mixture of hyaluronic acid, trehalose, glycerin, sodium alginate, and purified water, wherein the extract is included in an amount of 5 to 15 parts by weight per 100 parts by weight of purified water, and the viscosity of the mixture is 800 to 1,200 cps. Claim 2 A microneedle patch for skin whitening or skin anti-inflammation according to claim 1, characterized in that the mixture comprises 10 parts by weight of the extract, 8 parts by weight of hyaluronic acid, 2 parts by weight of trehalose, 1 part by weight of glycerin, and 0.5 parts by weight of sodium alginate per 100 parts by weight of purified water. Claim 3 A microneedle patch for skin whitening or skin anti-inflammation according to claim 1, characterized in that the mixture is discharged at 240 µL into a microneedle mold after degassing, pressurized for 30 minutes under a pressure of 0.06 MPa, rotated at 3,300 rpm for 2 minutes to remove bubbles, and then dried at 45°C for 45 minutes to form microneedles. Claim 4 A microneedle patch for skin whitening or skin anti-inflammatory according to claim 1, characterized in that the microneedle patch inhibits tyrosinase activity, inhibits ROS generation, inhibits nitric oxide (NO) generation, and inhibits iNOS expression or COX-2 expression. Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete

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