KR20210074807A - Inflammatin alleviating composition comprising an extract of white opium-free poppy as an effective ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, a cosmetic composition, or a health functional food composition containing a Papaver nudicaule extract as an active component. The Papaver nudicaule extract of the present invention has excellent inhibitory activity on the expression of cytokines and inflammatory mediators, thereby being useful as a functional natural material for inhibiting, relieving or alleviating inflammation.

Description

Inflammation alleviating composition comprising an extract of white opium-free poppy as an effective ingredient}

The present invention relates to a pharmaceutical composition, a cosmetic composition, and a health functional food composition for inhibiting, alleviating or improving inflammation containing an extract of Papaver nudicaule (white) as an active ingredient.

Corn poppy, which has been used for ornamental purposes such as roadside landscape construction, is also called flower poppy and is known as a safe plant without narcotic substances.

It has been found that the leaves of poppy contain substances that are effective in anti-cancer, anti-inflammatory, anti-pain, as well as antioxidant. According to recent studies, useful physiologically active substances such as chelidonin, which suppresses cancer cells and relieves spasms in poppy leaves, protopin, which is known to be effective in anticancer, anti-inflammatory, and arthritis, and cryptophine, which is used as a hypnotic or analgesic, etc. reported to exist.

However, in natural plants such as poppy, the types and contents of physiologically active substances produced by plants vary greatly depending on the variety and growth period, so it is necessary to conduct research in consideration of these characteristics in order to develop them as industrially valuable materials. However, there is a limitation in that conventional studies have been focused on the identification of specific physiological activities of extracts. In addition, an optimized extraction method for extracting specific physiologically active substances present in corn poppy has not yet been established.

It is an object of the present invention to provide a composition having an excellent effect of inhibiting, alleviating, or improving inflammation comprising a white flower poppy extract as an active ingredient.

Another object of the present invention is to provide an optimal extraction method for producing a white flower poppy extract with high industrial utility value as an anti-inflammatory material.

As a result of intensive research to solve the above problems, surprisingly, the present invention confirmed that the extract obtained from white flower poppy collected at a specific growth period exhibits excellent anti-inflammatory and alleviation-improving activity, and completed the present invention.

Accordingly, the present invention provides a composition for inhibiting, alleviating, or improving inflammation comprising, as an active ingredient, an extract obtained from white flower poppy collected at 75 to 105 days from the time of germination.

In one embodiment, the composition may be a pharmaceutical composition.

Also, in one embodiment, the composition may be a cosmetic composition.

Also, in one embodiment, the composition may be a health functional food composition.

The white flower poppy extract according to the present invention is an extract extracted from white flower poppy collected on the 75th to 105th day from the germination time, and it exhibits excellent anti-inflammatory effect and is safe due to low cytotoxicity, so that it is safe for use in pharmaceuticals, cosmetics, health functional foods, etc. It has great industrial utility value in that it can be used in a variety of ways.

1 is a papaver nudicaule of different flower colors ( Papaver nudicaule ) This is a photo showing 5 varieties and 1 variety of Papaver fauriei with different species.
Figure 2 is a papaver nudicaule of 5 varieties separated by flower color and papaver fauriei of one variety ( Papaver fauriei ) It is a diagram showing the result of checking through MTT analysis whether the extract obtained from the sample (leaf) collected by dividing by growth period (about 60 days after germination, about 90 days after germination) shows cytotoxicity.
Figure 3 divides five varieties of papaver nudicaule and one variety of papaver fauriei separated by flower color by growth period (about 60 days after germination, about 90 days) It is a diagram showing the result of confirming whether the extract obtained from the collected sample (leaf) exhibits Nitric Oxide (NO) inhibitory activity.
4 is a view showing the results of confirming the PEG2 expression inhibitory activity of the white flower poppy extract (NW90) of the present invention.
5 is a view showing the results of confirming the COX2, NOS2 expression inhibitory activity of white flower poppy extract (NW90) according to the present invention.
6 is a result of analyzing the effect of white flower poppy extract (NW90) according to the present invention on the mRNA expression level of cytokines (IL-6, IL-1β, TNF-α) through RT-PCR (FIG. 6a); The result of analyzing the effect of white flower poppy extract (NW90) according to the present invention on cytokine (IL-6, IL-1β, TNF-α) protein production through a multiplex assay (FIG. 6b) is shown It is a drawing.
7 is a view showing the results of analyzing the effect of a white flower poppy extract (NW90) according to the present invention on NF-κB luciferase activity.
8 is a view showing the results of analyzing the expression and phosphorylation of p65, IκBα and STAT3 according to phosphorylation of NF-κB protein by Western blot analysis after treatment with white flower poppy extract (NW90) according to the present invention.
9 is a view showing the results of quantitative analysis of the expression of p65, p-p65, IκBα, p-IκBα, STAT3, and p-STAT3 after treatment with white flower poppy extract (NW90) according to the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily carry out the present invention. The embodiments of the present invention are provided in order to more completely explain the present invention to those of ordinary skill in the art. Accordingly, the embodiment of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

In the present specification, when a part "includes" a certain component, this means that other components may be further included, rather than excluding other components, unless otherwise stated.

As used herein, the term “about” refers to at most 15%, preferably 10%, more preferably 5%, relative to the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. means a level, value, number, frequency, percentage, dimension, size, mass, weight or length that varies within the error range of

The present invention relates to a white flower poppy extract, preferably an extract of a white flower poppy or a part thereof collected at a specific time from the germination point, specifically, a white flower poppy or a part thereof collected when more than 60 days and less than 120 days from the germination point, in ethanol or It provides a composition for inhibiting, alleviating or improving inflammation containing the extract obtained by extraction with ethyl acetate as an active ingredient.

The term flower poppy is commonly used as a term to refer to a poppy, and it also refers to a poppy that can be used for horticulture or road flowers, except for those that are legally prohibited from cultivation and distribution.

Most of the landscaping poppies, which are often seen as road flowers, are called 'flower poppies'.

In the National Standard Plant List, the scientific name of 'poppy' is recorded as Papaver nudicaule. The flower poppy is called Iceland poppy in English, but unlike the flower name, it is not native to Iceland, and mainly inhabits Siberia, North America, Central Asia, northern China, and Mongolia. It is believed that the flower was given the name 'Iceland' because it mainly grows in cold climates.

In the wild state, poppies produce mainly yellow and white flowers. Through breeding, poppy has various flower colors such as yellow, yellow, crimson, and pink in addition to the wild flower color, and has become a flower often planted for landscaping and horticulture.

In the present specification, the varieties of 'flower poppy' are divided into white, yellow, yellow, scalet, pink, etc. based on flower color.

In the present specification, 'white flower poppy' refers to a flower poppy showing a white flower color.

In the present specification, the white flower poppy used as a raw material of the extract may be the whole or part of a flower poppy exhibiting a white flower color, such as a flower, a stem, a leaf, a root, an outpost, or a mixture thereof, but preferably a white flower poppy. Leaves, stems, or mixtures of said leaves and stems may be used. In the case of a mixture of leaves and stems, both with and without flower buds can be used, but when flower buds are formed, more components necessary for flowering are generated than components exhibiting desired anti-inflammatory activity in the extract of the present invention. , it is preferable in terms of activity and ease of handling to use those before buds are formed.

In the present specification, unless otherwise defined, white-flowered poppy or a portion thereof is more than about 60 days and less than 120 days, preferably 75 days to 105 days, more preferably 80 days to 100 days, most preferably 85 days after germination. It means that it is collected from days to 95 days.

With respect to the collection period of the above-mentioned white-flowered poppy, when it is collected more than about 60 days and less than about 120 days after germination, the total amount of biosynthesized alkaloids in the white-flowered poppy may be insufficient, and the type and biosynthetic amount of active substances (eg, after germination) Berberine, Chelidonine, Allocrytopine, Protopine, Dihydrosanguinarine, DL-Demethylcoclaurine, Tetrahydroberberine, Noscapine, Tetrahydropapaverine, etc.; data not shown), which peaks at about 90 days, and/or activity may decrease, so The desired level of anti-inflammatory effect may not be achieved.

In the present specification, the term 'extract' is a concept that includes all those obtained by separating a certain active ingredient from a natural product with an appropriate solvent, and the extract used in the present invention includes all extracts and purified substances obtained in each step of extraction or purification, and their dilutions. , a concept that includes both concentrates and dried products.

In one embodiment, the extract may be obtained by extraction, separation and fractionation from nature using a method known in the art for extraction, separation and fractionation, and may be extracted according to various extraction or fractionation solvents and extraction methods. have.

In one embodiment, the extract includes all of a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract of white flower poppy or a part thereof, and among them, it is preferable that the extract is a polar solvent-soluble extract. Furthermore, the polar solvent-soluble extract may be water or an organic solvent, for example, as the solvent, one or more solvents selected from the group consisting of water, C1-C4 lower alcohols, and mixed solvents thereof.

For example, ethanol or an aqueous ethanol solution may be used as the extraction solvent, and specifically, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or less, 80 or less, 85 or less, 90 or less% (v/v) may be an aqueous ethanol solution of, preferably 50 or more and 90 or less% (v/v) ethanol aqueous solution, and most preferably 1000% (v/v) ethanol aqueous solution, but is not limited thereto .

In another example, ethyl ether may be used as an extraction solvent, and ethyl ether may be used alone or in combination with ammonia. The concentration of ammonia may be changed as needed, but it may be preferable to use it at a concentration of about 10% (v/v).

In one embodiment, the extraction temperature is preferably 25 to 100 °C, but is not limited thereto.

In one embodiment, the extraction method may include a hot water extraction method, a cold extraction method, a reflux cooling extraction method, a solvent extraction method, a steam distillation method, an ultrasonic extraction method, an elution method, a compression method, etc., but is not limited thereto.

In one embodiment, the extract may be obtained by using water or an organic solvent and then cooled, heated and filtered at an appropriate temperature (eg, room temperature) by a conventional method known in the art to obtain a liquid extract, or further The solvent may be evaporated, spray dried or lyophilized. In addition, the extract extracted as described above may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying.

In one embodiment, the extract of white flower poppy or a portion thereof of the present invention is subjected to various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, and the like. It can also be obtained as a fraction that is further purified using a graph.

In the present specification, the content of the extract (unit: % by weight) of the white flower poppy or a portion thereof of the present invention is 0.000001 or more, 0.000005 or more, 0.00001 or more, 0.00005 or more, 0.0001 or more, 0.0005 or more, 0.001 or more, 0.005 or more with respect to the entire composition. , 0.01 or more, 0.05 or more, 0.1 or more, 0.5 or more, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less by weight, preferably may be 0.000001 to 10% by weight, more preferably 0.000001 to 1% by weight, but is not limited thereto.

The extract of white flower poppy or a part thereof of the present invention may exhibit excellent anti-inflammatory activity.

Specifically, the extract of white flower poppy or a portion thereof of the present invention can inhibit cytokine secretion by regulating the transcriptional activity of NF-κB, STAT-3, or both.

In one embodiment, the cytokine may be IL-1β or IL-6, but is not limited thereto.

The extract of white flower poppy or a part thereof of the present invention can suppress the expression of inflammation-related mediators by regulating the expression of NOS2 (nitric oxide synthase 2), COX2 (Cytochrome c oxidase subunit 2), or both.

In one embodiment, the inflammation-related mediator may be nitric oxide (NO) or prostaglandin E2 (PGE2), but is not limited thereto.

The extract of white flower poppy or a part thereof of the present invention can effectively inhibit the expression of inflammation-related mediators such as NOS2 and COX2 by endotoxin of lipopolysaccharide (LPS) of Gram-negative bacteria, which is an inflow source, and NF-κB and STAT3 are involved in the inflammatory signaling pathway, resulting in the release of p65, p-p65, IκBα, p-IκBα and p-STAT3 Since phosphorylation can be inhibited, gram-negative bacteria such as Salmonella, Shigella, Meningitis, etc. Abdominal pain, diarrhea, and both, or inflammation accompanied by inflammation caused by endotoxin of lipopolysaccharide (LPS) present in the outer membrane It may be useful as an active ingredient of a pharmaceutical composition for inhibiting, alleviating, or ameliorating pneumonia accompanying pneumonia.

In one aspect, the present invention provides an extract of white flower poppy or a part thereof, preferably more than 60 days but less than 120 days, preferably 75 days to 105 days from the time of germination, white flower poppy or its leaves and/or Inhibition of inflammation comprising, as an active ingredient, an extract of a stem, more preferably an extract obtained by extracting the white flower poppy or its leaves and/or stems collected after 75 to 105 days from the time of germination with ethanol or ethyl acetate; A pharmaceutical composition for alleviation or improvement is provided.

As used herein, the term 'administration' refers to providing a composition comprising an extract of a predetermined white flower poppy or a portion thereof to an individual by any suitable method, and the composition may be administered in a pharmaceutically effective amount. .

As used herein, the term 'pharmaceutically effective amount' means an amount sufficient to inhibit, alleviate or ameliorate inflammation or disease or abnormal symptoms accompanying inflammation, and the effective dose level is determined by the type and severity of inflammation, the age of the subject. , sex, sensitivity to drugs, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.

In one example, the daily dose of the composition comprising the extract of the white flower poppy or a part thereof according to the present invention is 0.1 mg/kg/day to 5000 mg/kg/day, more specifically 50 mg/kg/day to 500 mg It can be, but is not limited to, /kg/day.

In one embodiment, the composition may be administered as a separate therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or concurrently with a conventional therapeutic agent. In addition, the composition may be administered single or multiple. It is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects by comprehensively considering the above factors, which can be determined by those skilled in the art without particular difficulty.

In one embodiment, the composition may be administered to a subject by various routes. Examples of routes of administration include, but are not limited to, oral, rectal, intravenous, intramuscular, or subcutaneous administration.

In one embodiment, the composition can be used alone or in combination with methods using diet, physical therapy, and biological response modifiers for the inhibition, alleviation or improvement of inflammation itself or inflammation accompanying disease or abnormal symptoms. can

In another aspect, the present invention provides an extract of white flower poppy or a part thereof, preferably an extract of white flower poppy or its leaves and/or stems collected after 75 to 105 days from the time of germination, more preferably from the time of germination It provides a cosmetic composition for inhibiting, alleviating, or improving skin inflammation, comprising an extract obtained by extracting white flower poppy or its leaves and/or stems collected after 75 to 105 days with ethanol or ethyl acetate.

In the present specification, the 'cosmetic composition' is a concept that includes all things that make the appearance of humans or animals beautiful, and the composition of the present invention suppresses adverse effects on the skin caused by physiological phenomena or external stimuli according to the circadian rhythm. Or a concept that refers to an object that makes the appearance of an animal beautiful, but is not limited thereto.

In one embodiment, the cosmetic composition further contains conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, etc. commonly used in cosmetic compositions in addition to the extract of white flower poppy or a part thereof of the present invention. may be included. For example, the cosmetic composition may further include auxiliary components such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, and allantoin. .

In one embodiment, since the cosmetic composition is basically applied to the skin, it may be prepared in any formulation conventionally prepared with reference to a cosmetic composition in the art. For example, it can be prepared in the form of a paste, gel, cream, powder, spray, solution, emulsion, suspension, surfactant-containing cleansing, lotion, soap, oil, foundation, and the like formulations.

In one embodiment, 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, Zinc oxide and the like may be included.

In one embodiment, when the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be included as a carrier component, and in particular, in the case of a spray, additional chloro propellants such as fluorohydrocarbons, propane/butane, dimethyl ether, and the like.

In one embodiment, when the formulation of the present invention is a solution or emulsion, a solvent, a solubilizer, an emulsifier, etc. may be included 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 esters, polyethylene glycol, fatty acid esters of sorbitan, and the like may be included.

In one embodiment, when the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol, propylene glycol; suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tracanth, and the like may be included.

In one embodiment, when the formulation of the present invention is a surfactant-containing cleansing agent, as carrier ingredients, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, ethoxylated glycerol fatty acid ester, and the like.

In another aspect, the present invention provides an extract of white flower poppy or a part thereof, preferably an extract of white flower poppy or its leaves and/or stems collected after 75 to 105 days from the time of germination, more preferably the time of germination It provides a health functional food composition for inhibiting, alleviating or improving inflammation comprising an extract obtained by extracting white flower poppy or its leaves and/or stems with ethanol or ethyl acetate as an active ingredient when 75 to 105 days have elapsed from do.

In the present specification, the term 'health functional food' refers to a food group or food composition that has added value to act and express the function of the food for a specific purpose by using physical, biochemical, or bioengineering methods, etc. It is a food that has been designed and processed to sufficiently express the body control functions related to disease prevention and recovery, etc., and should be harmless to the human body when taken for a long time.

In one embodiment, the health functional food may include a nutraceutical acceptable food supplement additive, and may further include an appropriate carrier, excipient and diluent commonly used in the manufacture of health functional food.

In one embodiment, when the health functional food composition is used as a food additive, the composition may be added as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

In one embodiment, examples of the health functional food include dairy products, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, etc., so as to act and express the function of the food in a conventional sense for a specific purpose. It includes all foods designed to sufficiently express the body control functions related to the biological defense rhythm control, disease prevention and recovery, etc. of the food group or food composition with added value.

In one embodiment, the health functional food composition comprises various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, It may further include glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like, and in addition, the health functional food composition of the present invention may further include fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. . These components may be used independently or in combination.

Overlapping contents are arbitrarily omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification should be understood to have the meanings commonly used in the art to which the present invention pertains.

Hereinafter, examples and formulations will be described in detail for better understanding of the present invention. However, the following Examples and Formulation Examples merely illustrate the content of the present invention, and the scope of the present invention is not limited to the following Examples and Formulation Examples. Examples and formulation examples of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

material

As shown in Figure 1, papaver nudicaule with different flower colors ( Papaver nudicaule ) The leaf/stem part of one variety of Papaver fauriei , which is different from 5 varieties and species itself, was collected at about 30 days, about 60 days, and about 90 days from the germination time and used as samples. . Although there is a slight deviation in the sampling timing of each sample, for convenience of description, NW (30), NW (60), and NW (90) are indicated as follows.

Varieties name and collection time

* DAT: about 30, 60, 90 days after germination

[Reference Example] Establishment of optimal extraction method for extract samples

Selection of optimal extraction solvent

Five different solvents (50% methanol, 80% methanol, 100% methanol, 100% methanol, ethyl ether + 10% ammonia solution) were used to identify the solvent from which the desired active ingredient could be extracted most efficiently. Thus, extraction was carried out.

5, 10, and 20 mg of leaf samples collected by growth period from 6 prepared varieties are placed in an Eppendorf tube with a capacity of 2 ml, 1 mL of each of the 5 solvents is added, vortexed for 30 seconds, and then at room temperature for 30 minutes Extraction was performed using ultrasound.

When ethyl ether was used as an extraction solvent, concentration was carried out immediately after extraction because of strong volatility, and re-dissolved in 100% methanol. Then, the extract was filtered using a syringe filter having a pore size of 0.2 μm, and the obtained filtrate was placed in a 1 ml vial for analysis.

To confirm the degree of optimization of the extraction solvent, protopine components present in all samples were analyzed and confirmed, and the results are summarized below.

Regions of product ions of protopene

Protopine component was detected in all samples, and the extraction efficiency of the extraction solvent was compared using the region of the protopene peak.

The area value of the product ion (Q3) was ethyl ether + 10% ammonia > 100% ethanol > 80% ethanol > 100% methanol > 50% methanol in the order, and the extraction method using ethyl ether was the best in terms of extraction efficiency. Confirmed.

However, in the case of ethyl ether, the extraction method using 100% ethanol, which had the second highest efficiency, was confirmed as the optimal sample extraction method because it was difficult to handle due to its strong volatility and difficult pipetting during pretreatment.

In the following experimental examples, samples extracted by an extraction method using 100% ethanol were used.

[Experimental Example 1] Cytotoxicity analysis using MTT assay

To compare the cytotoxicity of each extract by variety/growth period, cell viability was measured by MTT assay. The MTT assay measures the degree of reduction of MTT tetrazolium to MTT formazan[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] by dehydrogenase action in mitochondria of living cells. As a test method, a cell line (RAW264.7 mouse macrophage cell line) was used in this experimental example.

A cell line (RAW264.7 mouse macrophage cell line) was added to DMEM medium (PAN-biotech) with 10% fetal bovine serum (BioProjects International) and 1% antibiotic/antimycotic (Corning) and cultured at _{37°C, 5% CO 2 in an incubator.} prepared (in the case of experimental examples using the following RAW264.7 cell line, all were prepared in the same manner).

The prepared RAW264.7 macrophage cell line was dispensed in a 96-well plate at 10 ⁴ cells/well and cultured overnight. On the next day of culture, extracts of each variety were treated 1 hour prior to each concentration up to 500 μg/ml, and then treated with LPS at 100 ng/ml for 24 hours. After 24 hours, 20 μl of MTT reagent (Sigma Aldrich) of 2 mg/ml was added to each well, and then incubated at 37° C. for 3 hours. After DMSO was added, absorbance was measured at 540 nm using a VersaMax Pro Microplate Reader (Molecular Devices, California, USA). The measurement results were analyzed using SoftMax Pro Software.

As shown in Figure 2, the cytotoxicity of each ethanol extract obtained from 6 varieties harvested by growth period (60 days, 90 days after germination) was confirmed through MTT assay, NY60, NW60, NW90, NP60, NP90, NS60, FW60 and FW90 showed less than 10% cytotoxicity at the highest treatment concentration (500 μg/ml), confirming that there was no significant level of cytotoxicity.

However, extracts NO60 and NO90 exhibited cytotoxicity of up to about 15 to 20% at the highest treatment concentration (500 μg/ml), and NS90 and NY90 exhibited cytotoxicity of up to about 25 to 28%. Therefore, it can be seen that there is a significant difference in the cytotoxicity of the extract depending on the growth period and variety.

[Experimental Example 2] Comparison of NO (Nitric Oxide) inhibitory activity by breed according to the growth period of each breed of dog poppy

In order to compare the anti-inflammatory effects of the extracts of each variety according to the growth period, the amount of NO (Nitric Oxide) production was measured.

The prepared RAW264.7 macrophage cell line was treated with ethanol extracts of each variety at a concentration of 125 to 500 μg/ml for 1 hour, and then treated with LPS at 100 ng/ml and cultured for 24 hours. The culture medium was harvested and mixed with Griess reagent (equivalent volume to 1% (w/v) sulfanilamide and 0.1% (w/v) naphthylethyleneamine-HCl in 5% (v/v) phosphoric acid) at room temperature was reacted for 10 minutes. After completion of the reaction, absorbance was measured at 570 nm using a Microplate reader (Molecular Devices, California, USA).

As shown in Figure 3, from the Papa Bell press dicarboxylic ulre (Papaver nudicaule) 5 dog breeds and Papa Bell par We Avon (Papaver fauriei) 1 dog breed harvested by growing period (about 60 days after germination, 90 days) As a result of the analysis of the inhibitory ability of Nitric Oxide (NO) expression, which is a representative inflammatory index, when inflammation is induced with LPS by treatment concentration of each ethanol extract obtained, NO expression was suppressed in all extracts.

However, concentration-dependent NO inhibitory activity was not observed in NO90 and NS90. In NY and NW, the NO inhibitory activity increased depending on the growth period (about 60 days after germination, about 90 days after germination) and the treatment concentration.

Combining the results of the NO inhibitory activity analysis, it was found that the extract with the highest NO inhibitory activity and excellent anti-inflammatory activity was NW90. Accordingly, considering the growth rate, state change (eg, bud generation, flowering), and the type and amount of biosynthetic substances based on NW60 and NW90, more than 60 days and less than 120 days, more precisely 90 days It can be expected that the NW extract obtained at the growth period of ±15 days (ie, 75 to 105 days) will exhibit the same or similar level of anti-inflammatory activity as that of NW90.

[Experimental Example 3] Confirmation of PGE2 inhibitory effect of NW90 through ELISA assay

The effect on PGE2 expression of the NW90 extract, which showed the highest NO inhibitory activity, was confirmed by ELISA assay.

The RAW264.7 macrophage line was seeded in a 6-well plate at 10 ⁶ cells/well and cultured overnight. On the next day of culture, the NW90 extract was treated for 1 hour at each concentration up to 500 μg/ml, then 100 ng/ml of LPS was treated and cultured for 24 hours. After 24 hours, 1 ml of the cell culture solution was removed with a pipette and transferred to an e-tube to prepare a sample.

In the R&D ELISA kit (R&D Systems, MN, USA), 50 μl of each sample was placed in a microplate coated with IL-1beta and reacted at room temperature for 2 hours. 400 μl of washing buffer was added and washed 5 times. 100 μl of the conjugated antibody was added and reacted at room temperature for 2 hours. 400 μl of washing buffer was added and washed 5 times. 100 μl of the substrate solution was added and reacted at room temperature for 30 minutes. 100 μl of Stop solution was added at a time and carefully shaken. Values were measured at a wavelength of 540 nm using a VersaMax Pro Microplate Reader (Molecular Devices) equipment.

As shown in FIG. 4 , the effect of NW90 on the secretion of PGE2, an inflammatory mediator, during LPS treatment in RAW264.7 cell line was confirmed through an ELISA assay. As a result, the NW90 extract was concentration-dependently PGE2 from a treatment concentration of 250 μg/ml. has been shown to inhibit production.

[Experimental Example 4] Confirmation of the inhibitory effect of NW90 on COX2 and NO production through Western blot analysis

To determine the effect of NW90 on the expression of COX2, a cell signaling material involved in PGE2 expression, and NOS2, an enzyme involved in NO production, a Western blot assay was performed.

The RAW264.7 macrophage line was aliquoted into ^{3 X 10 6 cells in a 100 mm dish and cultured overnight.} The next day, the NW90 extract was treated 1 hour before each concentration up to 500 μg/ml, and then treated with LPS at 100 ng/ml for 24 hours. After 24 hours, the cells were washed with PBS (1XPBS, PBST (0.1% Tween20) buffer), and then protein lysis buffer (10X Protein lysis buffer (Cell signaling Technology), 1 mM PMSF, PIC (Sigma-aldrich)) was used. to decompose the protein. After quantifying the protein of the sample using the Bio-rad protein assay (Bio-rad lab, California, USA) by the Bradford method, 20 μg of each sample was prepared in the same amount of the protein sample.

An 8% SDS-PAGE gel was prepared and the gel was mounted in a Western blot tank of a Western blot electrophoresis device Mini-PROTEAN 3 multi-casting chamber (Bio-rad lab, California, USA), and then the development buffer was filled. . After the protein marker (ELPIS bio) and the sample were loaded on the gel, the tank and the power were connected by matching +/-, and developed at 90V for 2 hours. Proteins separated by SDS-PAGE were transferred to an NC membrane (PALL corp, NY, USA) (transfer step).

(+) sponge-3M paper (Whatman 3MM paper; Thermo Fisher Scientific)-NC membrane-SDS PAGE gel-3M paper-sponge After mounting the cassette stacked in the (-) direction in the Western blot tank, fill the transfer buffer, Run at 100V for 2 hours. The membrane was placed in 5% skim milk buffer and reacted at room temperature for 1 hour (blocking step).

Dilute each primary antibody [Cell Signaling Technology (COX2, Danvers, MA, USA) and NOS2 (Santa Cruz, CA, USA)] to a dilution ratio of 1:100 to 500 in 5% Bovogen biologicals, Australia (BSA) buffer After that, it was reacted overnight at 4°C (rocking step). The membrane was washed repeatedly with PBST buffer 3 times for 10 minutes each.

After diluting the secondary antibody (Jackson ImmunoResearch Laboratories, Inc, PA, USA) in 5% skim milk (Difco Skim milk (Becton Dickinson, NJ, USA)) at a dilution ratio of 1:5000 to 10000, at room temperature for 1 hour reacted. The membrane was washed repeatedly with PBST buffer 3 times for 10 minutes each. After the ECL solution (DoGEN Bio) was reacted with the membrane for 1 minute, the band was analyzed by photosensitizing in a dark room using Kodak GBX developer/Fixer (FIG. 5a).

Expressions of COX2 and NOS2 were normalized to β-actin protein expression levels and quantified by densitometry analysis for each band using ImageJ software ( FIGS. 5b and 5c ). Data are presented as mean±SD (standard deviation) from at least 3 independent experiments. (*P<0.05, **P<0.01, and ***P<0.001 indicate a statistically significant difference compared to the group treated with LPS alone)

As shown in Figures 5a to 5c, the NW90 extract confirmed the effect on the expression of COX2, a cell signaling material involved in PGE2 expression, and NOS2, an enzyme involved in NO production, the NW90 extract inhibited the expression of COX2 and NOX2 proteins appeared to do This led to the conclusion that NW90 could effectively induce inhibition of PGE2 and NO production.

[Experimental Example 5] Analysis of the effect of NW90 extract on the production of inflammatory cytokines

(Experimental Example 5-1) Analysis of the effect of NW90 extract on cytokine RNA expression level

The effect of NW90 on the mRNA expression level of Cytokines (IL-6, IL-1β, TNF-α) was analyzed by RT-PCR (qPCR).

The RAW264.7 macrophage line was seeded in a 6-well plate at 10 ⁶ cells/well and cultured overnight. The next day, the NW90 extract was treated 1 hour before each concentration up to 500 μg/ml, and then treated with LPS at 100 ng/ml for 24 hours. After 24 hours, total RNA was isolated from cells using TRI Reagent solution (Ambion, Waltham, MA, USA). RNA was quantified by nano drop. cDNA was synthesized using 2 μg of quantified RNA and PrimeScript first strand cDNA synthesis kit (Takara Korea Biomedical Inc.). A mixture was prepared using the synthesized cDNA and the following forward/reverse primer, SYBR, and nuclease-free water (cDNA 100 ng, forward primer/reverse primer, each 10 μM 0.8 μl, SYBR 10 μl nuclease -Filled with free water, to a final volume of 20ul). PCR was performed using StepOne Plus realtime equipment (Waltham, Massachusetts, USA) (Realtime PCR conditions: 95°C - 2min, 95°C-10s, 60°C-10s, 72°C-20s-40 cycle).

Primer sequence information

As shown in FIG. 6a , NW90 affected the transcription of IL-6, IL-1β, and TNFα, thereby reducing the mRNA expression level of cytokines increased by LPS treatment.

(Experimental Example 5-2) Analysis of the effect of NW90 extract on cytokine protein production

The effect of the NW90 extract on the production of cytokines (IL-6, IL-1β, TNF-α) was analyzed through a multiplex assay.

The prepared RAW264.7 macrophage cell line ^{was aliquoted in a 6-} well plate at 10 6 cells/well and cultured overnight. The next day, the poppy extract was pretreated for 1 hour at each concentration up to 500 μg/ml, and then treated with LPS at 100 ng/ml for 24 hours. After 24 hours, 1 ml of the cell culture solution was pipetted and collected in an e-tube to prepare a sample. Using the cytokine beads in the Magnetic R&D Multi-Analyte kit (R&D Systems, MN, USA), 50 ul of beads and 50 ul of sample were reacted overnight at 4°C (shaking). It was washed three or more times using a wash buffer. Biotin antibody was added 50 ul, and reacted (shaking) at room temperature for 1 hour. It was washed three or more times using a wash buffer. 50 μl of Streptavidin-PE was added, followed by shaking at room temperature for 30 minutes. It was washed three or more times using a wash buffer. After adding 100 μl of wash buffer and reacting for 2 minutes, cytokines were measured using Luminex 200 instrument (Luminex 200 instrument/xPONENT3.1 software (Texas, USA)).

As shown in FIG. 6b , the NW90 extract exhibited the activity of inhibiting the protein production of IL-6 and IL-1β increased by LPS. In particular, IL-1β production was more efficiently inhibited than IL-6. However, NW90 had no effect on TNF-α protein production.

[Experimental Example 6] Analysis of the effect of NW90 extract on the signaling pathway of transcription factors

The effect of NW90 extract on p65, IκBα, and STAT3 of NF-κB, a transcription factor acting on inflammatory signaling by LPS, was analyzed.

The RAW264.7 macrophage line was seeded in a 6-well plate at 3 X 10 ⁵ cells/well and cultured overnight. After replacing the cell culture medium with a serum-free medium, a transfection mixture was prepared. In 200 μl of serum-free medium, 1.5 μg of Luc plasmid DNA (pSTAT3-Luc, NF-kb-Luc) and 4 μl of lipofectamine (Transfection agent lipofectamine 2000 (Invitrogen, California, USA)) were added and mixed by pipetting. , and reacted for 20 minutes. After the mixed solution was treated with the cells, the reaction was carried out for at least 7 hours. After 7 hours, the medium was replaced with 10% FBS, and the next day, the transfected cells were re-dispensed in a ^{24-well plate at 3 X 10 5 cells/well and cultured overnight.} The next day, the NW90 extract was pretreated for 1 hour at each concentration up to 500 μg/ml, and then reacted with 100 ng/ml LPS for 3 hours (NF-kb)/24h (pSTAT3). After the reaction time, the cell culture solution was drained and the cells were washed with PBS buffer. Cells were lysed by treating the cells with the cell lysis buffer of the Luciferase assay kit (Promega, Madison, WI, USA). 100 μl of Luciferase reagent and 20 μl of cell lysis buffer were added to a 96-well plate for luminometer and mixed, and then the luminescence value was measured using a LuBi Microplate Luminometer (MicroDigital Co).

As shown in FIG. 7 , after treatment with NW90 extract at 250 μg/ml and 500 μg/mlg, luciferase activity was measured after 3 hours ( FIG. 7a ) and 24 hours ( FIG. 7b ). As a result, the NW90 extract was LPS It has been shown to transmit the activity of transcription factors that act on inflammatory signaling by

Further, expression and phosphorylation of p65, IκBα and STAT3 were analyzed in RAW264.7 cells treated as described above using Western blot assay. In this case, β-actin was used as an internal control. As a result, as shown in FIG. 8, phosphorylation of NF-kB protein was inhibited in a concentration-dependent manner according to NW90 treatment.

Expressions of p65, p-p65, IκBα, p-IκBα, STAT3, and p-STAT3 were normalized to β-actin protein expression levels and quantified by densitometry analysis for each band using ImageJ software ( Fig. 9). Data are presented as mean±SD (standard deviation) from at least 3 independent experiments. (*P<0.05, **P<0.01, and ***P<0.001 indicate statistically significant differences compared to the group treated with LPS alone). As shown in FIG. 9 , it can be seen that the result of quantifying the expression levels of p65, p-p65, IκBα, p-IκBα and p-STAT3 is overall decreased, from which NW90 is NF-κB p65 , it was found to effectively regulate phosphorylation of IκBα and STAT3. Through these results, the NW90 extract of the present invention can exhibit excellent anti-inflammatory activity by effectively inhibiting the transcriptional activity of NF-κB and STAT3, thereby participating in the inflammatory signaling pathways in which they are involved, and thus as a natural anti-inflammatory material. It can be concluded that the useful value is large.

Hereinafter, an example of a formulation using the extract according to the present invention will be described, but it is intended to be used as a means to describe the present invention in detail, not to limit it.

<Formulation Example 1> Preparation of pharmaceutical composition

1-1. ointment preparation

ingredient content (wt%) White flower poppy extract of the present invention One glycerin 8 butylene glycol 4 liquid paraffin 15 beta glucan 7 Carbomer 0.1 Caprylic/Capric Triglycerides 3 squalane One cetearyl glucoside 1.5 Sorbitan Stearate 0.4 cetearyl alcohol One beeswax 3 Preservatives, Colors, Flavors appropriate amount Purified water to 100 (remaining amount)

After mixing according to the component composition, an ointment can be prepared according to a conventional ointment preparation method. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust them appropriately.

1-2. tablet manufacturing

ingredient Content (mg) White flower poppy extract of the present invention 100 corn starch 100 lactose 100 magnesium stearate 2

After mixing according to the above component composition, tablets can be prepared by tableting according to a conventional method for manufacturing tablets. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust them appropriately.

1-3. Capsule manufacturing

ingredient Content (mg) White flower poppy extract of the present invention 10 crystalline cellulose 3 lactose 14.8 magnesium stearate 0.2

After mixing according to the component composition, the capsule can be prepared by filling the gelatin capsule. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust them appropriately.

1-4. powder manufacturing

ingredient Content (mg) White flower poppy extract of the present invention 20 lactose 100 talt 10

After mixing according to the component composition, it is possible to prepare a powder by filling in an airtight cloth. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust them appropriately.

<Formulation Example 2> Preparation of cosmetic composition

2-1. Milk lotion manufacturing

ingredient content (%) White flower poppy extract of the present invention 0.1 glycerin 5.1 propylene glycol 4.2 Tocopheryl Acetate 3.0 liquid paraffin 4.6 triethanolamine 1.0 squalane 3.1 Macadamia Nut Oil 2.5 Polysorbate-60 1.6 Propylparaben 0.6 Carboxyvinyl Polymer 1.5 incense 0.1 antiseptic 0.1 Purified water to 100 (remaining amount)

Milk lotion can be prepared by a conventional method using the mixture having the above composition. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust them appropriately.

2-2. Nutrient cream manufacturing

ingredient content (%) White flower poppy extract of the present invention 0.5 glycerin 4.0 vaseline 3.5 triethanolamine 2.1 liquid paraffin 5.3 squalane 3.0 beeswax 2.6 Tocopheryl Acetate 5.4 Polysorbate-60 3.2 carboxyl vinyl polymer 1.0 Sorbitan sesquioleate 3.1 incense 0.1 antiseptic 0.1 Purified water to 100 (remaining amount)

A nutritional cream can be prepared by a conventional method using the mixture having the above composition. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust appropriately.

2-3. massage cream manufacturing

ingredient content (%) White flower poppy extract of the present invention 0.5 glycerin 4.0 vaseline 3.5 triethanolamine 0.5 liquid paraffin 4.0 squalane 3.0 beeswax 2.1 Tocopheryl Acetate 0.1 Polysorbate-60 2.4 carboxyl vinyl polymer 1.0 Sorbitan sesquioleate 2.3 incense 0.1 antiseptic 0.1 Purified water to 100 (remaining amount)

A massage cream can be prepared by a conventional method using the mixture having the above composition. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust appropriately.

2-4. Manufacture of body cleanser for cleansing and soothing the skin

ingredient content (g) White flower poppy extract of the present invention One Anionic surfactant 18 nonionic surfactant 5 glycerin 7 sodium chloride 3 Natural Olive Liquid Soap 15 Spices 0.1 water to 100 (remaining amount)

By using the mixture having the above composition, a body cleanser for cleansing and soothing the skin can be prepared by a conventional method. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust appropriately.

2-5. Soft lotion manufacturing

ingredient content (%) White flower poppy extract of the present invention 0.1 1,3-butylene glycol 5.2 oleyl alcohol 1.5 ethanol 3.2 Polysorbate-20 3.2 Benzophenone-9 2.0 Carboxyvinyl Polymer 1.0 glycerin 3.5 incense 0.01 antiseptic 0.01 Purified water remaining amount

A softening lotion can be prepared by a conventional method using the mixture having the above composition. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust appropriately.

<Formulation Example 3> Preparation of health functional food composition

3-1. Functional health food manufacturing

ingredient Content (mg) White flower poppy extract of the present invention 100 vitamin A acetate 7.0 vitamin E 1.0 vitamin B1 0.13 vitamin B2 0.15 vitamin B6 0.5 vitamin B12 0.2 vitamin C 10 biotin 1.0 Nicotinamide 1.7 folic acid 5.0 Calcium Pantothenate 0.5 ferrous sulfate 1.75 zinc oxide 0.82 magnesium carbonate 2.53 monobasic potassium phosphate 1.5 Dibasic Calcium Phosphate 5.5 Potassium Citrate 9.0 calcium carbonate 100 magnesium chloride 24.8

After mixing the above ingredients, it is possible to prepare a health functional food by formulating it in the form of granules (pills). In particular, the mixing ratio of the vitamin component and the mineral component may be appropriately changed according to the type of health food, preference, and the like. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust appropriately.

3-2. Functional health drink manufacturing

ingredient content (g) White flower poppy extract of the present invention 0.1 vitamin A 0.2 vitamin B1 0.25 vitamin B2 0.3 vitamin C 15 Vitamin E (powder) 10 iron lactate 19.75 zinc oxide 3.5 Nicotinamide 3.5 incense 0.1 Purified water to 100 (remaining amount)

According to a conventional functional health beverage manufacturing method, the above ingredients are mixed, and then stirred and heated at about 85° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2L container, sealed and sterilized, and then stored in a refrigerator for health Beverages can be prepared. In this case, the mixing ratio of the components may be changed according to the preference of the demand class, the demand region, and the like. A person skilled in the art may further add other ingredients with reference to the above formulating table, or may adjust appropriately.

Claims (15)

A composition for inhibiting or alleviating inflammation containing an extract of Papaver nudicaule (white) as an active ingredient. The composition for inhibiting or alleviating inflammation according to claim 1, wherein the white flower poppy extract is obtained from white flower poppy or a part thereof collected at a period of more than 60 to less than 120 days after germination. According to claim 1, NF-κB, STAT-3, or inhibiting cytokine secretion by regulating the transcriptional activity of both, the composition for inhibiting or alleviating inflammation. The composition of claim 3, wherein the cytokine is IL-1β or IL-6. According to claim 1, NOS2, COX2, or by regulating the expression of both, inhibiting the expression of inflammation-related mediators, the composition for inhibiting or alleviating inflammation. The composition for inhibiting or alleviating inflammation according to claim 5, wherein the inflammation-related mediator is NO or PGE2. The composition for inhibiting or alleviating inflammation according to claim 1, wherein the white flower poppy extract is obtained by extracting white flower poppy or a portion thereof collected at 75 to 105 days after germination with an aqueous ethanol solution or an ethyl ether solution. The composition for inhibiting or alleviating inflammation according to claim 1, which is a pharmaceutical composition or a cosmetic composition. According to claim 1, Abdominal pain accompanied by inflammation caused by Gram-negative bacteria, diarrhea, or both, or inhibiting, alleviating, or ameliorating pneumonia accompanying inflammation, the composition for inhibiting or alleviating inflammation. A health functional food composition for inhibiting, alleviating or improving inflammation, comprising an extract of Papaver nudicaule (white) as an active ingredient. The health functional food composition according to claim 10, wherein the white flower poppy extract is obtained from white flower poppy or a part thereof collected at a period of more than 60 to less than 120 days after germination. The health functional food composition according to claim 11, wherein the white flower poppy extract is obtained by extracting white flower poppy or a part thereof collected from 75 to 105 days after germination with an aqueous ethanol solution or an ethyl ether solution. The health functional food composition according to claim 10, wherein the white flower poppy extract suppresses the expression of inflammation-related mediators by regulating the expression of NOS2, COX2, or both. The health functional food composition according to claim 13, wherein the inflammation-related mediator is NO or PGE2. The health functional food composition according to claim 10, which is formulated as a liquid or pill.

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