KR102892533B1 - Composition for Anti-inflammation Using an Extract of Zanthoxylum piperitum and Minerals of Desalinized Lave Seawater - Google Patents

Abstract

The present invention discloses an anti-inflammatory composition using a Zinnia japonica extract and desalted lava seawater minerals, which suppresses NO production in a concentration-dependent manner and suppresses the production of inflammatory cytokines (TNF-α, IL-6, IL-1β) in a mouse macrophage cell line (RAW 264.7 cells) stimulated with LPS (lipopolysaccharide) without exhibiting any particular cytotoxicity.

Description

{Composition for Anti-inflammation Using an Extract of Zanthoxylum piperitum and Minerals of Desalinized Lave Seawater}

The present invention relates to an anti-inflammatory composition using an extract of Zanthoxylum piperitum and demineralized lava seawater minerals.

Inflammation is a localized defense response of the body in response to pathological conditions caused by physical trauma, harmful chemicals, infections by bacteria, fungi, or viruses, or irritants among the body's metabolic products. Inflammation is triggered by various inflammatory mediators produced by damaged tissue and migrating cells. During an inflammatory response, plasma accumulates at the site of inflammation, diluting the toxins released by bacteria. This increases blood flow and produces symptoms such as erythema, pain, swelling, and fever. Normally, the body uses the inflammatory response to neutralize or eliminate pathogens and regenerate damaged tissues, restoring normal structure and function. However, failure to do so can lead to disease states such as chronic inflammation.

Recent advances in molecular biology have led to extensive research on inflammatory responses at the molecular level.

Although various biochemical phenomena are involved in the inflammatory response, macrophages in particular are known to play an important role in the inflammatory response by producing inflammatory cytokines such as nitric oxide (NO) and IL-1β, TNF-α, and IL-6 in response to chemical stimuli (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) in the brain, nNOS (neuronal NOS) in the nervous system, and eNOS (endothelial NOS) in the vascular endothelium are always expressed at a certain level in the body, and the small amount of nitric oxide (NO) produced by them 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. In contrast, iNOS (induced NOS), whose expression is induced by certain stimuli, produces excessive NO, and the nitric oxide produced excessively by iNOS reacts with superoxide to form peroxynitrite, which acts as a strong oxidant and damages cells, thereby being 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).

Meanwhile, cyclooxygenase (COX) has the function of COX and hydroperoxidase (HOX) activity, and synthesizes intermediates PGG ₂ and PGH ₂ from arachidonic acid, and produces PGE ₂ , PGF ₂ , PGD ₂ , prostacyclin, and thromboxane A ₂ (TxA2) from these compounds. There are two isoforms of COX. COX-1 is constantly expressed in most tissues and synthesizes prostaglandins (PGs) necessary for cell protection, whereas COX-2 is rapidly induced in response to inflammation and plays an important role in causing inflammation by producing PGE _2, etc. (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).

Recent studies have reported that macrophage activation is related to mitogen-activated protein kinases (MAPKs) (KSBB J. 2015, 30:182-190). MAPKs are serine-threonine protein kinases that are activated by various cell proliferation factors, carcinogenic promoters, etc., and mainly include extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p-38. When macrophages are stimulated by LPS, they are rapidly activated and induce the production of inflammatory cytokines (KSBB J. 2015, 30:182-190; Microbiol. Biotechnol. Lett. 2016, 44(4), 479-.487).

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 P. N. Rao et al., Pharmaceuticals., 3:1530-1549, 2010). Therefore, active research is being conducted to find new drugs with long-lasting effects from natural substances that have anti-inflammatory activity and fewer side effects.

The present invention discloses the anti-inflammatory activity of a pine tree extract and demineralized lava seawater minerals.

The purpose of the present invention is to provide an anti-inflammatory composition using a pine bark extract and demineralized lava seawater minerals.

Other or specific purposes of the present invention will be presented below.

The present invention was completed by confirming that, as confirmed in the examples and experimental examples below, a complex of a pine bark extract and desalinated lava seawater minerals inhibits NO production in a concentration-dependent manner in a mouse macrophage cell line (RAW 264.7 cells) stimulated with LPS (lipopolysaccharide) without showing any particular cytotoxicity, and inhibits the production of inflammatory cytokines (TNF-α, IL-6, IL-1β).

Considering the above, the present invention can be understood as an anti-inflammatory composition comprising a pine tree extract and a desalinated lava seawater mineral complex as effective ingredients in one aspect.

In this specification, the term "Zopi extract" means an extract obtained by leaching Zopi leaves, stems, fruits, peels, pulps or mixtures thereof with water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof, an extract obtained by using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract, and any method such as cold steeping, reflux, heating, ultrasonic radiation, or supercritical extraction may be applied as the extraction method, taking into consideration the polarity of the active substance, the degree of extraction, and the degree of preservation. In the case of a fractionated extract, it means a fraction obtained by suspending the extract in a specific solvent and then mixing and allowing it to settle with a solvent of different polarity, and a fraction obtained by adsorbing the crude extract on a column filled with silica gel or the like and using a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent has been removed by a method such as freeze-drying, vacuum drying, hot air drying, or spray drying. Preferably, it means an extract obtained using water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof as an extraction solvent, and more preferably, it means an extract obtained using a mixed solvent of water and an alcohol having 1 to 4 carbon atoms as an extraction solvent.

Also, in this specification, “lava seawater” has the same meaning as “salt groundwater existing in the eastern region of Jeju Island” as defined and described in Korean Patent No. 0853244 (Application No. 10-2008-0027861), entitled “Method for producing mineral composition and mineral composition obtained by the method.” Specifically, 'the eastern region of Jeju Island' refers to Gujwa-eup, Seongsan-eup, and Pyoseon-myeon in terms of administrative districts of Jeju Island, and 'salt groundwater' refers to groundwater containing a certain amount of salt or more, and specifically, it can be understood to include both low-salt groundwater (electrical conductivity of 1,700 ㎲/cm to 17,350 ㎲/cm) and saline groundwater (electrical conductivity of 17,350 ㎲/cm or more) according to the criteria for classifying Jeju Island groundwater based on the salt concentration used in the thesis of Go Gi-won et al. (Go Gi-won et al., Study on the Hydrogeology of the Eastern Region of Jeju Island (Ⅱ), 2003, Kyungwon University; considered as a part of this specification), but preferably, saline groundwater with an electrical conductivity of 17,350 ㎲/cm or more or a salt concentration of 30‰ (permilliage) or more (normally, the salt concentration of seawater is 32 to 35‰) It can be understood to mean.

Also, in this specification, "demineralized lava seawater mineral" can be understood as something obtained by desalinating lava seawater containing salt and minerals, wherein the desalination can be performed by any method known in the art, such as reverse osmosis or electrodialysis. The desalinated lava seawater can be dehydrated into a liquid concentrate or a powder concentrate.

Also, in this specification, “active ingredient” means an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself.

Also, in this specification, “anti-inflammatory” means improving (alleviating symptoms), treating, inhibiting or delaying the onset of an inflammatory disease as defined below.

In addition, in this specification, the above-mentioned "inflammatory disease" is defined as a pathological symptom caused by an inflammatory response that is specific to a local or systemic biological defense response to external physical or chemical stimuli, infection by external infectious agents such as bacteria, fungi, viruses, and various allergens, or autoimmunity. This inflammatory response involves a series of complex physiological reactions such as activation of various inflammatory mediators and enzymes related to immune cells (e.g., iNOS, COX-2, etc.), secretion of inflammatory mediators (e.g., secretion of NO, TNF-α, IL-6, etc.), fluid infiltration, cell migration, tissue destruction, etc., and is externally manifested by symptoms such as erythema, pain, edema, fever, and decline or loss of specific bodily functions. Since the above-mentioned inflammatory disease may be acute, chronic, ulcerative, allergic, or necrotic, as long as any disease is included in the definition of the above-mentioned inflammatory disease, it does not matter whether it is acute, chronic, ulcerative, allergic, or necrotic. Specifically, the inflammatory diseases may include 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 infections (e.g., hepatitis C), bacterial infections, fungal infections, burns, wounds resulting from surgical or dental work, prostaglandin E hyperactivity syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, and the like.

The anti-inflammatory composition of the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit improvement activity for the inflammatory disease intended to be treated, depending on the specific use, formulation, etc., and the typical effective amount will be determined within the range of 0.001 wt % to 15 wt % based on the total weight of the composition. Here, the "effective amount" refers to the amount of the active ingredient that can exhibit the intended medical/pharmacological effect, such as improvement, treatment, or inhibition/delay of the onset of an inflammatory disease, when the composition of the present invention is administered to a mammal, preferably a human, which is the subject of application, during the administration period recommended by a medical professional. Such an effective amount can be experimentally determined within the normal ability of a person skilled in the art.

In addition to the effective ingredient, the composition of the present invention may additionally include any compound or natural extract that has already been verified as safe and known to have the corresponding activity in the art in order to enhance or reinforce the anti-inflammatory effect or to improve the convenience of taking or ingesting through the addition of similar activities such as anti-allergic activity, skin protection activity (inhibition of skin damage caused by ultraviolet rays, skin moisturizing, etc.).

These compounds or extracts include compounds or extracts listed in the pharmacopoeia of each country (in Korea, the “Korean Pharmacopoeia”), the code of health functional foods of each country (in Korea, the “Standards and Specifications for Health Functional Foods,” a notice issued by the Ministry of Food and Drug Safety), compounds or extracts approved for use in accordance with the laws of each country regulating the manufacture and sale of pharmaceuticals (in Korea, the “Pharmaceutical Affairs Act”), and compounds or extracts individually recognized for their functionality in accordance with the laws of each country regulating the manufacture and sale of health functional foods (in Korea, the “Act on Health Functional Foods”). For example, MSM (dimethylsulfonylmethane) with the 'arthritis improvement' functionality according to the Korean Health Functional Food Codex, N-acetylglucosamine with the 'arthritis improvement' and 'skin moisturizing' functionality, and compounds such as heat-treated dried powder of Enterococcus faecalis, guava leaf extract, perilla extract, leaf extract, pico preto powder, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), etc., which are individually recognized for their functionality as 'alleviating hypersensitive immune response' according to the Korean Health Functional Food Act, and L. sakei Probio 65, gamma-linolenic acid-containing oil, L. plantarum CJLP133, a lactic acid bacterium derived from vegetables, and probiotics ATP, etc., which are individually recognized for their functionality as 'improving sensitive skin conditions', would be such compounds or extracts.

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.

The anti-inflammatory composition of the present invention, in a specific embodiment, can be understood as a food composition. In this case, the use of the present invention can be understood as suppressing an inflammatory response.

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

In addition, the food composition of the present invention may be classified into any product category, as long as it complies with the applicable laws and regulations at the time of manufacturing and distribution, in terms of legal and functional classification. For example, it may be a health functional food according to the Korean "Health Functional Food Act," or a confectionery, legume, tea, beverage, special-purpose food, etc. according to each food type according to the food code of the Korean "Food Sanitation Act" (MFDS Notice "Food Standards and Specifications").

The food composition of the present invention may contain food additives in addition to its effective ingredients. Food additives can generally be understood as substances added to, mixed with, or infiltrated into food during the manufacturing, processing, or preservation of food. Since they are consumed daily and over a long period of time with food, their safety must be guaranteed. The Food Additive Codex, which is based on the laws of each country that regulate the manufacturing and distribution of food (in Korea, the “Food Sanitation Act”), provides limited regulations on food additives with guaranteed safety in terms of ingredients or functions. The Korean Food Additive Codex (Ministry of Food and Drug Safety Notice “Food Additive Standards and Specifications”) classifies food additives into chemically synthesized products, natural additives, and mixed preparations in terms of ingredients. These food additives are classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, and thickeners in terms of functions.

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

Flavoring agents can be used to enhance taste or aroma, and both natural and synthetic flavors can be used. Natural flavorings are preferred. When using natural flavorings, they can also serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents can be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or from green tea leaves, Polygonum multiflorum, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. Also, flavoring agents obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo biloba, etc. can be used. Natural flavoring agents can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents can be used, such as esters, alcohols, aldehydes, and terpenes.

Preservatives that can be used include sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc., and emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc., and acidulants that can be used include citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. In addition to the purpose of enhancing taste, acidulants can be added to ensure that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms.

As thickeners, suspending agents, sedimentation agents, gelling agents, and bulking agents can be used.

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

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, and dibenzoylthiamine, and examples of minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferrous citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

The food composition of the present invention may include the aforementioned food additives in an appropriate amount that can achieve the purpose of addition depending on the product type.

With regard to other food additives that may be included in the food composition of the present invention, reference may be made to the food codes or food additive codes of each country.

The composition of the present invention may be considered as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared as an oral or parenteral formulation 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 appropriate route, including the topical route, the oral route, the intravenous route, the intramuscular route, and direct absorption through mucosal tissues, and a combination of two or more routes may be used. An example of a combination of two or more routes is a case where two or more drug formulations according to the route of administration are combined, for example, one drug is administered intravenously first, and the other drug is administered topically second.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specific examples can be found in the pharmacopoeias of each country, including the Korean Pharmacopoeia.

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared in the form of powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier and a method known in the art. Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; cellulosics such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, vegetable oils, ethanol, and glycerol. When formulating, appropriate binders, lubricants, disintegrants, colorants, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch peristalsis, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc., and disintegrants include starch, methyl cellulose, agar, bentonite, xanthan gum, starch, alginic acid, or sodium salts thereof. Other diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine.

When the pharmaceutical composition of the present invention is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalants, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, an aqueous isotonic solution or suspension can be used as a suitable carrier, and specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose can be used. When formulated as a transdermal dosage form, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, paste, liniment, aerosol, etc. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide, and in the case of formulating it as a suppository, the carrier can be witepsol, tween 61, polyethylene glycol, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, or the like.

Specific formulations of pharmaceutical compositions are known in the art and can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

The preferred dosage of the pharmaceutical composition of the present invention may range from 0.001 mg/kg to 10 g/kg per day, preferably from 0.001 mg/kg to 1 g/kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once daily or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

In another specific embodiment, the composition of the present invention can be considered a cosmetic composition. If the anti-inflammatory composition of the present invention is considered a cosmetic composition, its intended uses include suppressing inflammatory skin problems and alleviating inflammatory skin irritation.

Even when the composition of the present invention is identified as a cosmetic composition, the cosmetic composition may be classified into any product category based on its intended use and the law, and specifically, it may be a functional cosmetic for improving skin troubles, improving atopic dermatitis, etc., or a non-functional general cosmetic. It may also be in any product form, and specifically, it may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, etc. In terms of specific product forms, it may be in the form of a flexible toner, a nourishing toner, 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, in addition to its effective ingredient, components commonly used in cosmetic compositions, 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, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

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

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

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

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

The cosmetic composition of the present invention can be manufactured according to a method for manufacturing a cosmetic composition commonly practiced in the art, except that it includes an effective ingredient exhibiting anti-inflammatory activity.

As described above, according to the present invention, an anti-inflammatory composition using a pine bark extract and demineralized lava seawater minerals can be provided.

The composition of the present invention can be commercialized as a food, cosmetic, pharmaceutical, etc. for purposes such as improving inflammatory diseases.

Figure 1 shows the results of cytotoxicity evaluation and NO production inhibition evaluation (NO aasay).
Figures 2 to 4 show the results of evaluation of inhibition of production of inflammatory cytokines (TNF-α, IL-6, IL-1β) through Western blot.

The present invention is described below with reference to examples and experimental examples. However, the scope of the present invention is not limited to these examples and experimental examples.

<Example> Preparation of Chopi tree extract and lava seawater minerals

The dried powder of Zanthoxylum piperitum leaves was added to 20 times 50% ethanol, stirred at room temperature for 24 hours, extracted, filtered, concentrated under reduced pressure, and freeze-dried to prepare a powder extract (ZP).

Desalinated lava seawater minerals are made by removing impurities from lava seawater using a filter paper, desalting it with an electrodialyzer, and then producing desalinated lava seawater mineral water containing only mineral components such as Mg and Ca, and then dehydrating it to produce solid desalinated lava seawater minerals (YSM).

<Experimental Example> Anti-inflammatory activity experiment

<Experimental Example 1> Cytotoxicity Test

RAW 264.7 cells (1.5 × 10 ⁵ cells/mL) were cultured in DMEM medium simultaneously with the sample of the example and 1 μg/mL of Lipopolysaccharide (LPS from Escherichia coli O111:B, Sigma-Aldrich) for 24 hours, and then the culture medium was centrifuged at 3,000 rpm for 5 minutes. LDH (Lactate dehydrogenase) assay was measured using a Non-radioactive cytotoxicity assay kit (PROMEGA). 50 μL of the culture medium obtained by centrifugation and 50 μL of the reconstituted substrate mix were added to a 96-well plate, and after reacting for 30 minutes at room temperature, 50 μL of the stop solution was added, and the absorbance was measured at 490 nm using a microplate reader (Spark, TECAN). The average absorbance value for each sample group was calculated, and cytotoxicity was evaluated by comparing it with the absorbance value of the control group (LDH control, 1:5000). The results of the cytotoxicity evaluation according to the LDH assay are shown in Figure 1. The results are based on three repeated experiments at each concentration.

Referring to Figure 1, the extract of the pine tree, demineralized lava seawater minerals, and their complex did not show any particular cytotoxicity at any treatment concentration.

<Experimental Example 2> Evaluation of NO production inhibition (NO assay)

Raw 264.7 mouse macrophages used in the experiment were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Raw 264.7 mouse macrophages were cultured at 37°C in 5% CO2 conditions.

RAW 264.7 cells were adjusted to 1.5 × 10 ⁵ cells/mL using DMEM medium supplemented with 10% FBS, seeded in 48-well plates, and cultured for 24 hours after being simultaneously treated with the sample of the example and LPS (1 μg/mL). The amount of NO produced was measured in the form of NO 2- present in the cell culture medium using Griess reagent [1% (w/v) sulfanilamide, ^0.1 % (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid]. 100 μL of the cell culture supernatant and 100 μL of Griess reagent were mixed and reacted in 96-well plates for 10 minutes, and the absorbance was measured at 540 nm. The amount of NO produced was compared with sodium nitrite (NaNO ₂ ) as a standard. The results are also shown in Figure 1 as a percentage compared to the untreated group. The results are from three repeated experiments for each concentration.

Demineralized lava seawater mineral alone had no NO production inhibition activity, and the extract of the Chinese juniper tree showed NO production inhibition activity in a concentration-dependent manner. However, the complex of the Chinese juniper tree extract and demineralized lava seawater mineral inhibited NO production more significantly in a concentration-dependent manner than the extract of the Chinese juniper tree.

<Experimental Example 3> Evaluation of inhibitory activity against inflammatory cytokine production using Western blot

RAW 264.7 cells (4 × 10 ⁵ cells/well) were seeded in 6-well and simultaneously treated with the sample of the example and LPS (1 μg/mL) and cultured for 24 hours. After washing 2-3 times with PBS, 200 μL of M-PER lysis buffer (M-PER mammalian protein extraction reagent, ThermoFisher Scientific) was added and incubated at -70°C overnight. The cell lysate was collected and centrifuged at 12,000 rpm and 4°C for 20 minutes to obtain the supernatant excluding cell membrane components. The protein concentration was quantified using a Protein assay kit (Protein assay dye reagent concentrate, Bio-rad) standardized with BSA (bovine serum albumin). 10-30 μg of protein was denatured and separated by 4-12% mini gel SDS-PAGE (poly acrylamide gel electrophoresis) and transferred to a PVDF membrane (iBlot gel transfer, Invitrogen). Membrane blocking was performed in TBST (1X TBS + 0.1% Tween 20) solution containing 5% skim milk (Merck) for 2 hours at room temperature, and then washed four times for 5 minutes each with TBST. The primary antibody to be detected was diluted in TBST, reacted for 2 hours at room temperature, and then washed four times for 5 minutes each with TBST. As a secondary antibody, anti-mouse igG (cell signaling) conjugated to HRP (horse radish peroxidase) was diluted 1:4,000 in TBST, reacted for 1 hour at room temperature, and then washed four times for 5 minutes each with TBST. After reacting the ECL substrate (miracle-star, iNtRON) with the membrane, the expression level of each protein was confirmed using a Western blot imaging system (Fusion solo X, Vilber).

The results are shown in Figures 2 to 4. The desalinated lava seawater minerals themselves generally did not exhibit any activity to inhibit the production of inflammatory cytokines (TNF-α, IL-6, IL-1β), and the extract of the Chinese juniper tree generally exhibited an activity to inhibit the production of inflammatory cytokines in a concentration-dependent manner. However, the complex of the desalinated lava seawater minerals and the extract of the Chinese juniper tree inhibited the production of cytokines more significantly in a concentration-dependent manner than the extract of the Chinese juniper tree.

Claims (6)

An anti-inflammatory food composition comprising a complex of extract of the leaves of the Chinese juniper tree and demineralized lava seawater minerals as active ingredients.
In the first paragraph,
The above-mentioned extract of the pine tree is a mixed solvent extract of water and ethanol of the pine tree leaves,
A composition characterized in that the above desalinated lava seawater mineral is in liquid or powder form.
An anti-inflammatory cosmetic composition comprising a complex of a plant leaf extract and demineralized lava seawater minerals as active ingredients.
In the third paragraph,
The above-mentioned extract of the pine tree is a mixed solvent extract of water and ethanol of the pine tree leaves,
A composition characterized in that the above desalinated lava seawater mineral is in liquid or powder form.
An anti-inflammatory pharmaceutical composition comprising a complex of a plant extract and demineralized lava seawater minerals as active ingredients.
In paragraph 5,
The above-mentioned extract of the pine tree is a mixed solvent extract of water and ethanol of the pine tree leaves,
A composition characterized in that the above desalinated lava seawater mineral is in liquid or powder form.