KR102337965B1 - Composition for Anti-inflammation Using an Extract of Lemon Peel and a Root Extract of Sophora flavescens - Google Patents

Abstract

The present invention discloses an anti-inflammatory composition using a lemon peel extract and ginseng root extract, which have concentration-dependently inhibiting NO production in LPS (lipopolysaccharide)-stimulated mouse macrophage cell lines (RAW 264.7 cells).

Description

Composition for Anti-inflammation Using an Extract of Lemon Peel and a Root Extract of Sophora flavescens

The present invention relates to an anti-inflammatory composition using a lemon ( Citrus limon ) peel extract and Sophora flavescens root extract.

Inflammation is a local defense reaction of the living body that appears in response to a pathological condition caused by physical trauma, harmful chemicals, infection by bacteria, fungi, viruses, or irritants in in vivo metabolites. Inflammation is triggered by various inflammatory mediators produced from damaged tissues and migrating cells. In the case of an inflammatory reaction, plasma is accumulated in the inflammatory site to dilute the toxins secreted by bacteria, blood flow increases, and symptoms such as erythema, pain, edema, and fever are accompanied. In a normal case, the living body neutralizes or removes the onset factor through an inflammatory response and regenerates damaged tissue to restore normal structure and function, but otherwise, it may progress to a disease state such as chronic inflammation.

With the recent development of molecular biology, many studies have been made on the inflammatory response at the molecular level.

Although various biochemical phenomena are involved in the inflammatory response, macrophages in particular produce nitric oxide (NO) and inflammatory cytokines such as IL-1β, TNF-α, and IL-6 by chemical stimuli. It is known to play an important role (Ito T., et al., Curr Drug Traget Inflamm Allergy, 2(3):257-265, 2003).

Nitric oxide is synthesized from L-arginine by the action of nitric oxide synthase (NOS), and NOS exists in several isoforms. bNOS (brain NOS) present in the brain, nNOS (neuronal NOS) present in the nervous system, and eNOS (endothelial NOS) present in the vascular endothelial system are always expressed at a certain level in the body, and monoxide produced in small amounts by these Nitrogen (NO) plays an important role in maintaining homeostasis in the human body by performing various physiological reactions related to blood pressure regulation, neurotransmission, learning, and memory. On the other hand, iNOS (induced NOS) whose expression is induced by a certain stimulus produces excessive NO, and the nitric oxide produced excessively by iNOS reacts with superoxide to form peroxynitrite and It acts as a powerful oxidizing agent and by damaging cells, it is involved in various pathological processes, including inflammation and cancer (Gupta SC et al., Exp Biol Med., 236:658-671, 2011; Riehemann et al., FEBS). Lett., 442:89-94, 1999; Stamler et al., Science, 258:1898-1902, 1992).

On the other hand, cyclooxygenase (COX) has the function of COX and hydroperoxidase (HOX) activity and _{synthesizes intermediates PGG 2} and PGH ₂ from arachidonic acid, and these compounds PGE ₂ , PGF ₂ , PGD ₂ , prostacyclin and thromboxane A ₂ (TxA2), in which there are two isoforms of COX. COX-1 is always expressed in most tissues and synthesizes prostaglandins (PGs) necessary for cytoprotective action, whereas COX-2 is rapidly induced during _{an inflammatory response to generate PGE 2,} etc. to cause an inflammatory response. plays an important role (Weisz A., Biochem. J., 316:209-215, 1996; (Miller MJ et al., Mediators of inflammation, 4:387-396, 1995: Appleton L. et al., Adv) (Pharmacol., 35:27-28, 1996).

Non-steroidal anti-inflammatory drugs (NSAIDS), which are currently widely used as anti-inflammatory agents, are known to cause serious side effects such as gastrointestinal disorders, liver disorders, and renal disorders (Rainsford KD., Subcell biochem., 42:3 -27, 2007; Guruprasad P. Aithal., Rheumatology., 7:139-150, 2011; Praveen PN Rao et al., Pharmaceuticals., 3:1530-1549, 2010). Therefore, research is being actively conducted to find a new drug that has an anti-inflammatory activity and has a continuous effect from a natural substance with few side effects.

The present invention discloses the anti-inflammatory activity of extracts from lemon peel and ginseng root.

An object of the present invention is to provide an anti-inflammatory composition using a lemon peel extract and a ginseng root extract.

Other objects or specific objects of the present invention will be set forth below.

As confirmed in the Examples and Experimental Examples below, the present invention stimulates each of the lemon peel extract and ginseng root extract, particularly the lemon peel ethyl acetate fraction and the ginseng root 70% ethanol extract, and their complexes with LPS (lipopolysaccharide). This was completed by confirming that the concentration-dependent inhibition of NO production in the mouse macrophage cell line (RAW 264.7 cells).

Considering the above, the anti-inflammatory composition of the present invention is characterized in that it contains a lemon peel extract, a ginseng root extract, or a mixture thereof as an active ingredient.

In particular, in the present invention, the lemon peel extract is preferably an ethyl acetate fraction obtained from the ethyl acetate layer when the water and ethanol mixed solvent extract of lemon peel is fractionated with water and ethyl acetate, and the Korean ginseng root extract is a mixed solvent extract of water and ethanol. It is preferable to be

In addition, as used herein, the term "active ingredient" refers to a component that alone exhibits the desired activity or can exhibit activity together with a carrier that has no activity by itself.

In addition, as used herein, "anti-inflammatory" is meant to include alleviation (relief of symptoms), treatment, and suppression or delay of the onset of an inflammatory disease defined below.

In addition, as used herein, the term "inflammatory disease" refers to an inflammatory reaction specified as a local or systemic biological defense reaction against external physical or chemical stimuli or external infectious agents such as bacteria, fungi, viruses, and various allergens, or autoimmunity. It can be defined as the pathological symptom that causes This inflammatory response involves the activation of various inflammatory mediators and immune cell-related enzymes (eg, iNOS, COX-2, etc.), secretion of inflammatory mediators (eg, secretion of NO, TNF-α, IL-6, etc.), infiltration of body fluids, It is accompanied by a series of complex physiological reactions such as cell migration and tissue destruction, and is externally manifested by symptoms such as erythema, pain, edema, fever, and deterioration or loss of specific body functions. Since the inflammatory disease can be acute, chronic, ulcerative, allergic or necrotic, whether it is acute, chronic, ulcerative, allergic or Regardless of whether it is necrotic or not. Specifically, the inflammatory disease includes asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis , irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, myofascial disease, viral infection (such as hepatitis C infection), bacterial infection, fungal infection, burns, surgical or dental wounds, pro Stagladin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, etc. will be included

The anti-inflammatory composition of the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit the inflammatory disease improvement activity intended for treatment, etc. depending on the specific use, formulation, etc., but a typical effective amount is the composition It will be determined within the range of 0.001 wt % to 15 wt % based on the total weight. As used herein, the term "effective amount" refers to when the composition of the present invention is administered to a mammal, preferably a human subject to which it is applied, during the administration period as suggested by a medical professional, etc., to improve, treat, or inhibit the onset of such pathological symptoms as inflammatory diseases. / Refers to the amount of active ingredient that can produce the intended medical and pharmacological effects, such as delay. Such an effective amount can be determined empirically within the ordinary ability of one of ordinary skill in the art.

In addition to the active ingredient, the anti-inflammatory composition of the present invention is administered through addition of similar activities such as anti-allergic activity, skin protection activity (suppression of skin damage caused by UV rays, skin moisturizing, etc.) In order to enhance the convenience of ingestion, any compound or natural extract that has already been verified for safety in the art and known to have the corresponding activity may be further included.

Such compounds or extracts include compounds or extracts, drugs listed in compendial documents such as pharmacopeias of each country (“Korea Pharmacopoeia” in Korea) and health functional food regulations of each country (“Health Functional Food Standards and Specifications” announced by the Ministry of Food and Drug Safety in Korea). Each country's laws governing the manufacture and sale of compounds or extracts and health functional foods that have been approved for items in accordance with the laws of each country (the "Pharmaceuticals Act" in Korea) governing the manufacture and sale of '), compounds or extracts whose functionality has been individually recognized are included. For example, MSM (dimethylsulfonylmethane) with 'arthritis improvement' function, N-acetylglucosamine with 'arthritis improvement' function and 'skin moisturizing' function, etc. according to the Korean Health Functional Foods Code, according to the Korea 「Health Functional Food Act」 Enterococcus faecalis heat-treated dry powder, which has been individually recognized for its functionality as ‘alleviating hypersensitivity immune response’, complexes such as guava leaf extract, Actinidia extract, leaf extract, picaopreto powder, etc., PLAG (1-palmitoyl-2-linoleoyl- 3-acetyl-rac-glycerol), L. sakei Probio 65, which has been individually recognized for its functionality as 'improving sensitive skin conditions', oils and fats containing gamma-linolenic acid, L. plantarum CJLP133, a lactic acid bacterium derived from fruits and vegetables, and probiotics ATP, etc. It would be an extract.

One or more of these compounds or natural extracts may be included in the anti-inflammatory composition of the present invention together with the active ingredient.

In a specific embodiment, the anti-inflammatory composition of the present invention can be regarded as a food composition.

The food composition of the present invention can be prepared in any form, for example, beverages such as tea, juice, carbonated drinks, and ion drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean sweets, bread, confectionery, noodles, etc. Foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and health functional food preparations such as bars can be manufactured.

In addition, the food composition of the present invention may have any product classification in terms of legal and functional classification as long as it conforms to the enforcement laws at the time of manufacture and distribution. For example, it is a health functional food according to Korea's "Health Functional Food Act", or confectionery, beans, tea, and beverages according to each food type in the Food Ordinance of Korea "Food Sanitation Act" (Ministry of Food and Drug Safety Notification "Food Standards and Specifications") , special purpose food, and the like.

The food composition of the present invention may contain food additives in addition to the active ingredients thereof. Food additives can be generally understood as substances that are added and mixed or infiltrated into food in manufacturing, processing, or preserving food. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Ordinance in accordance with the laws of each country that regulates the manufacture and distribution of food (“Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Specifications” announced by the Ministry of Food and Drug Safety), food additives are classified into chemically synthetic products, natural additives, and mixed preparations in terms of ingredients. It is divided into agents, preservatives, emulsifiers, acidulants, thickeners, etc.

The sweetener is used to impart appropriate sweetness to food, and both natural and synthetic ones may be used in the composition of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents may be used to improve taste or aroma, and both natural and synthetic ones may be used. Preferably, it is a case where a natural thing is used. In the case of using a natural product, the purpose of nutritional enhancement in addition to flavor may be concurrently used. The natural flavoring agent may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or obtained from green tea leaves, horseradish leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo can be used. The natural flavoring agent may be a liquid concentrate or a solid extract. Optionally, a synthetic flavoring agent may be used, and the synthetic flavoring agent may be an ester, an alcohol, an aldehyde, a terpene, or the like.

As a preservative, sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin, etc. are mentioned, and acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. can be used as an acidulant. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As a thickening agent, a suspending agent, a settling agent, a gel-forming agent, a bulking agent, etc. can be used.

The food composition of the present invention may contain, in addition to the food additives described above, physiologically active substances or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutrition and guaranteed stability as food additives.

Examples of such physiologically active substances include catechins contained in green tea and the like, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, and the like. Minerals include calcium preparations such as calcium citrate, magnesium stearate Magnesium preparations, such as iron preparations, such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, etc. are mentioned.

In the food composition of the present invention, the food additives as described above may be included in an appropriate amount to achieve the purpose of the addition according to the product type.

In relation to other food additives that may be included in the food composition of the present invention, reference may be made to the Food Ordinance of each country or the Food Additives Code.

The composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment.

The pharmaceutical composition of the present invention may be prepared as an oral dosage form or a parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of the combination of two or more routes is a case in which two or more formulations of drugs according to the route of administration are combined. For example, one drug is first administered by an intravenous route and the other drug is secondarily administered by a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country including the "Korea Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers according to methods known in the art together with a suitable carrier It can be prepared in a formulation such as Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol etc. are mentioned. In the case of formulation activity, an appropriate binder, lubricant, disintegrant, colorant, diluent, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrist, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and oleic acid as lubricant. sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc., and the disintegrating agent is starch, methyl cellulose , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, and the like.

When the pharmaceutical composition of the present invention is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or isotonic solution such as 5% dextrose may be used. . When formulated for transdermal administration, it can be formulated in the form of an ointment, a cream, a lotion, a gel, an external solution, a pasta agent, a liniment agent, an air roll, and the like. In the case of nasal inhalants, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, and the like. witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like can be used.

Specific formulations of pharmaceutical compositions are known in the art, and reference may be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). This document is considered a part of this specification.

A preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, severity of the patient, and the route of administration. It can be in the range /kg. Administration may be performed once or divided into several times a day. These dosages should not be construed as limiting the scope of the invention in any respect.

In another specific embodiment, the composition of the present invention can be identified as a cosmetic composition. When the composition of the present invention is identified as a cosmetic composition, its use may be understood as a use for suppressing inflammatory skin troubles, alleviating inflammatory skin irritation, and the like.

Even when the composition of the present invention is identified as a cosmetic composition, the cosmetic composition can be classified into any product in terms of its use or by law, and specifically functional cosmetics, non-functional products having uses such as improvement of skin troubles and improvement of atopic dermatitis It may be general cosmetics and the like. In terms of product form, it may take any product form, and specifically, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax It can take the form of products such as foundation and spray. In a specific product form, it may be in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

The cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the active ingredient, for example, conventional adjuvants such as stabilizers, solubilizers, surfactants, vitamins, pigments and fragrances, and carriers.

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

When the formulation 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. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc. may be used.

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

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 as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like may be used.

The cosmetic composition of the present invention may be prepared according to a method for preparing a cosmetic composition conventionally performed in the art, except that it contains an active ingredient exhibiting anti-inflammatory activity.

As described above, according to the present invention, it is possible to provide an anti-inflammatory composition using a lemon peel extract and a ginseng root extract.

The anti-inflammatory composition of the present invention may be commercialized as food, cosmetics, drugs, etc. for use, such as improvement of inflammatory diseases, and alleviation of inflammatory skin irritation.

1 and 2 are cytotoxicity evaluation results of lemon peel extract and Korean ginseng root extract.
3 and 4 are results of evaluation of the NO production inhibitory activity of the lemon peel extract and the ginseng root extract.
5 is an evaluation result of NO production inhibitory activity of a complex of lemon peel ethyl acetate fraction and 70% ethanol extract of ginseng root.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example> Anti-inflammatory activity test of lemon peel extract and ginseng root extract

1. Preparation of Samples

(1) Preparation of hot water extract

The dried Jeju organic lemon ( Citrus limon ) peel and old ginseng ( Sophora flavescens ) root were each crushed, added with tertiary distilled water, and stirred at 60 ° C., heating mantel for 8 hours. The leached sample was subjected to a filtrate using a reduced pressure filtration device, and the same procedure was repeated three times. The filtrate was concentrated on a water bath with a rotary vacuum concentrator under reduced pressure to obtain each extract. The yield of each sample was 34.04% of lemon peel and 13.52% of ginseng root.

(2) Preparation of 70% ethanol extract

The dried Jeju organic lemon peel and ginseng root were each crushed, and 70% ethanol was added thereto and stirred at room temperature for 24 hours. The leached sample was subjected to a filtrate using a reduced pressure filtration device, and the same procedure was repeated three times. The filtrate was concentrated on a water bath with a rotary vacuum concentrator under reduced pressure to obtain each extract. The yield of each sample was 36.47% of lemon peel and 14.26% of ginseng root.

(3) Preparation of ethyl acetate fraction

Jeju organic lemon peel and 70% ethanol freeze-dried product of Korean ginseng root are placed in a separatory funnel, distilled water and 99% ethyl acetate are added as a solvent in a 1:1 volume ratio, extracted for 2 hours at room temperature while shaking every 30 minutes, and separated by stagnation. A fraction of the organic layer was obtained, and the same amount of ethyl acetate was again treated with the remaining aqueous layer in the same manner, and this was repeated three times. The ethyl acetate layer thus obtained was filtered using a reduced pressure filtration device, and the filtrate was concentrated on a water bath with a vacuum rotary vacuum concentrator under reduced pressure to obtain each extract. The yield of each sample was 2.72% of lemon peel and 9.72% of ginseng root.

2. Experimental method

2.1 RAW264.7 Cell Culture

Murine macrophage cell line RAW264.7 cells used for anti-inflammatory experiments were purchased through the Korea Cell Line Bank (Koeran Cell Line Bank). Cells were cultured in Dulbecco's Modified Eagle medium (DMEM) medium containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) _{in an incubator at 37 °C and 5% CO 2} conditions, and passaged once every 2 days. Culture was carried out.

2.2 Cytotoxicity assessment

In order to examine the effect of the sample on cell survival, an MTT experiment was performed.

In the MTT experiment, 3-(4,5-dimethylthiaxo-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a yellow water-soluble substrate, was converted to purple insoluble formazan by the dehydrogenase action of mitochondria in living cells. It is a test method using the ability of mitochondria to reduce

RAW 264.7 cells were cultured in DMEM medium containing 1% penicillin and 10% FBS for 24 hours. 7 × 10 ⁴ cells/well in each well were dispensed in a 24 well plate, and after incubation for 48 hours, samples were treated by concentration. After removing the supernatant, the MTT reagent was diluted with DMEM medium (0.4 mg/mL) to a concentration of 400 μL in each well and treated for 4 hours. Then, the supernatant was removed, DMSO was added to formazan crystals, dissolved at room temperature for 1 hour, transferred to a 96 well plate, and absorbance was measured at 570 nm using a microplate reader (Tecan, Mannedorf, Swizerland). It was calculated as a percentage compared to the sample untreated group.

2.3 NO (Nitrite) Generation Evaluation

RAW 264.7 cells were seeded in a 24-well plate at 2.0×10 ⁵ cells/mL and cultured at 37° C., 5% CO ₂ condition for 24 hours. The next sample (100㎍/ml) and LPS 1㎍/ml were simultaneously treated and cultured for 24 hours, the supernatant of the culture solution was recovered, and the same amount of griess reagent (1% sulfanilamide + 0.1% naphthylendiamine dihydrochloride) in 100 μl of the supernatant. , 1:1, Sigma, MO, USA) was mixed and reacted at room temperature for 10 minutes, and absorbance was measured at 540 nm using an ELISA Microplate Reader. The result was calculated as a percentage compared to the sample untreated group (LPS alone treated group).

2.4 Statistical processing

All experimental results using student's t-test were statistically (*) when p -values < 0.05, (**) when p -values < 0.01, and (***) when p -values < 0.001. marked as significant. Each result is presented as the mean ± SD of three independent experiments.

3. Experimental results

3.1 Cytotoxicity evaluation results

The cytotoxicity evaluation results of lemon peel extract (JLP) and Korean ginseng root extract (JSR) are shown in FIGS. 1 and 2 . In Figure 1, A is a lemon peel hot water extract (JLP-W), B is a lemon peel 70% ethanol extract (JLP-70% EtOH), C is a lemon peel ethyl acetate fraction (JLP-EtOAc fr) cytotoxicity evaluation results, , In FIG. 2, A is a hot ginseng root extract (JLP-W), B is a high ginseng root 70% ethanol extract (JLP-70% EtOH), and C is a cytotoxicity evaluation result of a high ginseng root ethyl acetate fraction (JLP-EtOAc fr) to be.

As a result of cytotoxicity evaluation, lemon peel hot water extract (JLP-W) was 87% at a concentration of 1,250 μg/mL, lemon peel 70% ethanol extract (JLP-70% EtOH) was 89% at a concentration of 1,000 μg/mL, and lemon peel ethyl The acetate fraction (JLP-EtOAc fr) showed 98% viability at 50 μg/mL ( FIG. 1 ).

In addition, hot ginseng root extract (JSR-W) is 102% at 50 μg/mL concentration, high ginseng root 70% ethanol extract (JSR-70% EtOH) is 500 μg/mL 76%, old ginseng root ethyl acetate fraction (JSR-EtOAc fr) showed a survival rate of 75% at a concentration of 20 μg/mL.

In the subsequent experiment, the experiment was carried out using a concentration with a cell viability of 90% or more.

3.2 NO production inhibitory activity evaluation result

Lemon peel extract (JLP) and Korean ginseng root extract (JSR) and a complex of these extracts (lemon peel ethyl acetate fraction and Korean ginseng root 70% ethanol extract (JLS complex, 3:7 weight ratio, lemon peel ethyl acetate) The fraction is 3 weight), the NO production inhibitory activity evaluation results are shown in FIGS. 3 to 5, respectively.

Referring to Figure 3, lemon peel hot water extract (JLP-W) and lemon peel 70% ethanol extract (JLP-70% EtOH) do not show NO production inhibitory activity, but lemon peel ethyl acetate fraction (JLP-EtOAc fr ) showed NO production inhibitory activity in a concentration-dependent manner. Nagosam root 70% ethanol extract (JSR-70% EtOH) clearly showed NO production inhibitory activity.

Referring to FIG. 5 , the complex of lemon peel ethyl acetate fraction and 70% ethanol extract of Korean ginseng root showed significantly more NO production inhibitory activity than each of the lemon peel ethyl acetate fraction and 70% ethanol extract of Korean ginseng root in a concentration-dependent manner. (showing 52.31% NO production inhibitory activity at 400 μg/mL). Ammonium 1-pyrrolidinedithiocarbamate (APDTC) was used as a positive control.

Claims (5)

(i) an ethyl acetate fraction extract of lemon peel and (ii) a mixture of water and ethanol mixed solvent extract of ginseng root as an active ingredient,
The lemon peel extract is an anti-inflammatory composition, characterized in that it is an ethyl acetate fraction obtained from the ethyl acetate layer when the water and ethanol mixed solvent extract of the lemon peel is fractionated with water and ethyl acetate.
According to claim 1,
The water and ethanol mixed solvent extract of the lemon peel is a 70% ethanol extract of the lemon peel,
The composition, characterized in that the water and ethanol mixed solvent extract of the old ginseng root is a 70% ethanol extract of the old ginseng root.
3. The method of claim 1 or 2,
The composition is a pharmaceutical composition, characterized in that the composition.
3. The method of claim 1 or 2,
The composition is a food composition, characterized in that the composition.
3. The method of claim 1 or 2,
The composition is a cosmetic composition, characterized in that the composition.

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