KR20220046736A - A composition comprising Chrysanthemum zawadskii and Cudrania tricuspidata Bureau having anti-inflammation activity - Google Patents

Abstract

The present invention relates to a composition containing a complex extract of Chrysanthemum zawadskii and Cudrania tricuspidata as an active component. Specifically, the present invention relates to an anti-inflammatory composition which contains a complex extract of Chrysanthemum zawadskii and Cudrania tricuspidata as an active component. The composition which contains the complex extract of Chrysanthemum zawadskii and Cudrania tricuspidata as an active component of the present invention has a significantly increased anti-inflammatory effect than single extracts of Chrysanthemum zawadskii flowers or Cudrania tricuspidata fruits alone, and also has a superior anti-inflammatory effect than hot water extracts thereof, so it is possible to be widely used as the anti-inflammatory composition.

Description

{A composition comprising Chrysanthemum zawadskii and Cudrania tricuspidata Bureau having anti-inflammation activity}

The present invention relates to a composition comprising a gujeolcho and cujippong complex extract as an active ingredient, and specifically, to an anti-inflammatory composition comprising a gujeolcho and cujippong complex extract as an active ingredient.

In modern times, along with economic development, the form of diet changes rapidly, the aging of the population and many changes in diseases caused by the extension of life expectancy. It has been widely used as a treatment for diseases or as a prescription for herbal supplements, but in recent years, it is being developed as a material for various functional foods considering health and nutrition in terms of preventive medicine.

Gujeolcho (Chrysanthemum zawadskii) is a perennial dicotyledonous plant belonging to the Asteraceae family. Gujeolcho is known to grow wild in mountains and highlands throughout the country, and to grow in China, Russia, Mongolia, and Japan. The stems and leaves of Gujeolcho are dried and used as a herbal medicine for menstruation, cold uterus, infertility, gynecological diseases, and gastrointestinal diseases since ancient times.

As an example, Korean Patent Registration No. 10-2132655 discloses the antioxidant efficacy of a composition containing the gujeolcho extract, but the synergistic effect of the gujeolcho and kkujippong complex extract on the anti-inflammatory effect was first identified by the present inventors.

In addition, Cuji mulberry is a small tall tree belonging to the Morus family, and its shape resembles a mulberry tree, so it has been given the name 'curdling mulberry tree'. it is known Cudrania tree, which has recently been spotlighted as its efficacy has been scientifically revealed, is a natural organic agricultural product known as a fruit that is not harmed by pests and diseases even without pesticides due to the immunity of the plant itself as our unique plant resource. It is so precious that it has been said that fresh fish enjoyed it, and it is known to have antioxidant, anti-diabetic, cold prevention, anti-cancer, anti-aging effects, etc.

As an example, Korean Patent No. 10-1773249 discloses a method for producing a cucurbita health food in which leaf and fruit extracts of Cuji mulberry are mixed, but the present inventors was first identified by

On the other hand, in the human body, macrophages respond to pathogens to induce inflammation such as tumor necrotic factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), etc. Produce factors, synthesize inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to synthesize nitric oxide (NO) and prostaglandin E2, PGE2 ) is created. Physiologically, NO plays a variety of roles, including eliminating bacteria and tumors, regulating blood pressure, and mediating neurotransmission. However, when an inflammatory response occurs, the expression of iNOS in related cells increases and a large amount of NO is generated, and excessively generated NO causes tissue damage, gene mutation, nerve damage, etc., and increases vascular permeability, resulting in edema. promotes an inflammatory response. In order to reduce the inflammatory response, various studies on novel anti-inflammatory substances are being conducted.

Under this background, the present inventors have made intensive research efforts to develop a natural product-derived material having a new anti-inflammatory effect, and confirmed the anti-inflammatory synergistic effect in the gujeolcho and cujippong complex extract, completing the present invention.

One object of the present invention is to provide an anti-inflammatory composition comprising a complex extract of gujeolcho and kkujippong as an active ingredient.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object provides an anti-inflammatory composition comprising a complex extract of gujeolcho and kkujippong as an active ingredient.

Specifically, the gujeolcho extract may be fresh flower, dried flower, or petal stem extract, preferably, raw flower or dried flower, and more specifically, the gujeolcho extract may be comprised of 80 to 99.99% by weight relative to the total weight. However, the present invention is not limited thereto.

In addition, the kkujippong extract may be a kkujippong fruit extract, preferably 0.1 to 20% by weight relative to the total weight may be included.

In addition, the composition may have an additional antioxidant effect.

In the present invention, the solvent of the extract is preferably ethanol, but is not limited thereto.

As used herein, the term “Gujeolcho (Chrysanthemum zawadskii)” is a perennial plant belonging to the Asteraceae family, and is wild throughout Korea. In the folklore, it has been mainly used as a therapeutic effect for gynecological diseases such as poor circulation, dysmenorrhea, and menstrual irregularities.

In the present invention, for the anti-inflammatory effect of the gujeolcho extract, preferably fresh or dried gujeolcho may be used.

As used herein, the term “Cudrania tricuspidata Bureau” is a small tall tree belonging to the Morus family, and has the name 'Cudrania tricuspidata Bureau' due to its resemblance to a mulberry tree. , hypertension, and gynecological diseases are known to have been used.

It is a deciduous tree of the Morus family widely distributed in East Asia including China and Japan, including Korea, and is known to have various pharmacological effects. As a natural organic agricultural product known as a fruit that is not harmed by pesticides because of its immunity, Cudrania mulberry tree is so precious that it has been said that freshers enjoyed it in oriental medicine since ancient times. It is known that it has an excellent effect in treating various diseases because of its efficacy, but its anti-inflammatory effect was first identified by the present inventors.

In the present invention, for the anti-inflammatory effect of the cujippong, it may preferably be one using the cujippong fruit.

As used herein, the term “complex extract” refers to an extract in which two or more extracts are combined into one.

In the present invention, the extract may be a combination of gujeolcho and kkujippong extract, or may be extracted by mixing the gujeolcho and kkujippong extract, but is not limited thereto.

As used herein, the term “extract” refers to a substance obtained by extracting a certain substance, specifically, an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, and the extract It includes extracts of all formulations that can be formed using the extract itself and the extract, such as a crude or purified product or a mixture thereof.

The extraction method is not particularly limited, and extraction may be performed according to a method commonly used in the art. Non-limiting examples of the extraction method include a solvent extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, and these may be performed alone or in combination of two or more methods.

The type of the extraction solvent used for extraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, and these may be used alone or in combination of one or more. Specifically, the extraction solvent may be ethanol.

As used herein, the term “active ingredient” refers to a component capable of exhibiting the desired activity alone or in combination with a carrier having no activity by itself.

As used herein, the term “anti-inflammatory” refers to the action of inhibiting inflammation, and the regulation of the inflammatory response is known to be very complex, which is known to enhance the in vivo repair system and reduce damage. However, if the inflammatory response continues due to repeated tissue damage or regeneration, ROS and RNS are overproduced in inflammation-related cells, resulting in permanent gene modification. As such, ROS and RNS are deeply related to the inflammatory response that regulates the actions of various cells in vivo. During the inflammatory process, large amounts of proinflammatory cytokines, nitric oxide (NO), and prostaglandin E2 (PGE2) are released from inducible nitric oxide synthase (iNOS) and cyclooxygenase. Produced by (cyclooxygenase-2, COX-2).

In one embodiment of the present invention, by extracting gujeolcho by solvent (Fig. 1), the antioxidant efficacy of ethanolic extract of gujeolcho through DPPH and ABTS assays of each solvent was confirmed (Fig. 2), and cytotoxicity evaluation was performed. (FIG. 3), after confirming the antioxidant efficacy and anti-inflammatory effect (FIG. 4), extracting cujippong fruit for each solvent in the same way (FIG. 5), and confirming the antioxidant efficacy of each cujibbong extract through DPPH and ABTS assays ( 6), cytotoxicity evaluation was performed for each cujippong extract through the MTT assay (FIG. 7), to confirm the anti-inflammatory effect (FIG. 8), and to confirm the synergistic effect of the complex extract mixed with gujeolcho and cujippong, The cytotoxicity evaluation of gujeolcho raw flower and cujippong complex extract was performed (Fig. 9), and the anti-inflammatory efficacy of gujeolcho raw flower and cujippong complex extract was confirmed through inflammation-mediated gene expression measurement (Fig. And the cytotoxicity evaluation of the cujippong complex extract was conducted (FIG. 12), and the anti-inflammatory efficacy of the dried gujeolcho dry and cujippong complex extract was confirmed through the measurement of inflammatory-mediated gene expression (FIGS. 13 to 14).

Through this, the antioxidant, cytotoxicity, and anti-inflammatory effects of single extracts for each solvent of fresh flowers, dried flowers, dried petals of Gujeolcho and dried cucurbita fruit were confirmed. , the optimal mixing ratio of the complex extract was confirmed.

Another aspect of the present invention provides an anti-inflammatory pharmaceutical composition comprising the extract as an active ingredient.

In this case, the terms “extract”, “active ingredient”, and “anti-inflammatory” are the same as described above.

As used herein, the term “pharmaceutical composition” may further include a pharmaceutically acceptable carrier, excipient or diluent, and the carrier may include a non-naturally occurring carrier.

Specifically, "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the organism and does not impair the preventive or therapeutic activity and properties of the composition of the present invention for preventing or treating hyperpigmentation disease symptoms. Examples of acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol. And one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed.

In addition, the pharmaceutically acceptable carrier of the present invention may include a non-natural carrier.

More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid (Poly Lactic Acid), poly-L-lactic acid (poly-L-lactic acid), mineral oil, and the like.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. The carrier may include various amorphous carriers, microspheres, nanofibers, and the like.

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term, “pharmaceutically effective amount” is sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. means the amount, and the effective dose level is the severity of the disease, the drug activity, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and the rate of excretion Treatment period , may be determined according to factors including drugs used in combination with or concurrently with the composition of the present invention and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a known therapeutic agent for pterygium. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects.

Another aspect of the present invention provides an anti-inflammatory food composition comprising the extract as an active ingredient.

In this case, the terms “extract”, “active ingredient”, and “anti-inflammatory” are the same as described above.

When the composition of the present invention is used as a food additive, the compound may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment), and may further include a food pharmaceutically acceptable supplementary additive. Since the composition of the present invention uses a compound synthesized from a natural product-derived compound as an active ingredient, there is no problem in terms of stability, so there is no significant limitation on the mixing amount.

As used herein, the term “food composition” may include any food in a conventional sense, and may be used interchangeably with terms known in the art, such as functional food and health functional food.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition comprising the compound of the present invention as an active ingredient can be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the compound according to the present invention as a component to juice, tea, jelly, juice, and the like.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (eg, formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplements , Seasoned foods (eg soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, sweets (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.) ), beverages (eg, fruit, vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

As used herein, the term “health functional food” refers to a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and 'functionality' refers to the It refers to obtaining useful effects for health purposes such as regulating nutrients with respect to structure and function or physiological actions. On the other hand, health food means food that has an active health maintenance or promotion effect compared to general food, and health supplement means food for the purpose of health supplementation. In some cases, the terms health functional food, health food and health supplement food can be mixed.

More specifically, it refers to food with high medical and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutritional supply, and the food includes tablets, capsules, powders, granules, It may be prepared in various forms such as liquid, pills, and the like.

The food composition of the present invention may further include a food pharmaceutically acceptable carrier.

There is no particular limitation on the type of food to which the composition of the present invention can be added, for example, various beverages, gum, tea, vitamin complexes, and health supplements. Other ingredients that do not interfere with the prevention or improvement effect of obesity may be added to the food composition, and the type thereof is not particularly limited.

For example, it may contain various herbal extracts, food-logically acceptable food supplements or natural carbohydrates as additional ingredients, such as conventional food.

In addition, the composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu). In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene ( BHT), etc.), colorant (tar pigment, etc.), color developer (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweetener (dulcin, cyclmate, saccharin, sodium, etc.) , flavorings (vanillin, lactones, etc.), bulking agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam suppressants, solvents, food additives such as improving agents ) can be added. The additive is selected according to the type of food and used in an appropriate amount.

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, or natural carbohydrates as additional ingredients, as in a conventional beverage. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin , alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage.

The health functional food of the present invention can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, there are no side effects that may occur when taking the drug for a long time by using food as a raw material, and it can be excellent in portability.

Another aspect of the present invention provides an anti-inflammatory cosmetic composition comprising the extract as an active ingredient.

In this case, the terms “extract”, “active ingredient”, and “anti-inflammatory” are the same as described above.

As used herein, the term “cosmetic composition” may be prepared in the form of general emulsified formulations and solubilized formulations. The emulsified formulation includes nutrient lotion, cream, essence, and the like, and the solubilized formulation includes softening lotion and the like. Suitable formulations include, but are not limited to, for example, solutions, gels, solid or kneaded dry products, emulsions obtained by dispersing an oily phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), ionic It may be in the form of a vesicle dispersant, cream, skin, lotion, powder, ointment, spray or in the form of a cone stick. In addition, it may be in the form of a foam or an aerosol composition further containing a compressed propellant.

The cosmetic composition may additionally contain fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metals Commonly used adjuvants such as sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetic compositions may contain.

Another aspect of the present invention provides an anti-inflammatory use of a composition comprising the extract as an active ingredient.

In this case, the terms “extract”, “active ingredient”, and “anti-inflammatory” are the same as described above.

The composition comprising the gujeolcho flower and Cujipon fruit complex extract of the present invention as an active ingredient has a significantly increased anti-inflammatory effect than the gujeolcho flower or Cujipon fruit alone extract, and also has a superior anti-inflammatory effect than its hot water extract. , it will be widely used as an anti-inflammatory composition.

1 is a schematic diagram showing the extraction method of Gujeolcho extract.
Figure 2A is a graph showing the antioxidant efficacy of the ethanol extract of Gujeolcho through DPPH and ABTS assays.
Figure 2B is a graph showing the antioxidant efficacy of gujeolcho hot water extract through DPPH and ABTS assays.
Figure 3A is a graph showing the cytotoxicity evaluation results of ethanol extract from Guulcho.
3B is a graph showing the cytotoxicity evaluation results of Guulcho dry ethanol extract.
3C is a graph showing the cytotoxicity evaluation results of ethanol extracts from dried petals of Gujeolcho.
Figure 4A is a graph showing the antioxidant efficacy of the ethanol extract of Gujeolcho.
Figure 4B is a photograph showing the anti-inflammatory efficacy of the ethanol extract of Gujeolcho through the measurement of inflammatory-mediated gene expression.
Figure 5 is a schematic diagram showing the extraction method of kkujippong extract.
Figure 6 is a graph showing the antioxidant efficacy of each kkujippong extract through the DPPH and ABTS assay.
7 is a graph showing the cytotoxicity evaluation results for each kkujippong extract through the MTT assay.
Figure 8 is a photograph showing the anti-inflammatory effect of each kkujippong extract.
Figure 9 is a graph showing the cytotoxicity evaluation results of gujeolcho raw flower and kkujippong complex extract.
10 is a photograph showing the anti-inflammatory efficacy of the gujeolcho raw flower and kkujippong complex extract through the measurement of inflammatory-mediated gene expression.
11 is a graph showing the anti-inflammatory efficacy of gujeolcho raw flower and kkujippong complex extract through inflammation-mediated gene expression measurement.
Figure 12 is a graph showing the cytotoxicity evaluation results of the dried gujeolcho dry and kkujippong complex extract.
Figure 13 is a photograph showing the anti-inflammatory efficacy of the gujeolcho dried dry and kkujippong complex extract through the measurement of inflammatory-mediated gene expression.
14 is a graph showing the anti-inflammatory efficacy of the gujeolcho dry dry and kkujippong complex extract through the measurement of inflammatory-mediated gene expression.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples only.

Example 1. Gujeolcho sample preparation

Fresh flowers of Gujeolcho, dried flowers (dried flowers) of Gujeolcho, and dried petals were pulverized to prepare extract samples using ethanol or water.

More specifically, after grinding raw or dried Gujeolcho and dissolving it in 99% ethanol, ultrasonic waves were applied at 60°C at 40kHz and 300W for 2 hours, followed by extraction for 2 hours to prepare an ethanol extract.

In addition, the hot water extract was prepared by grinding raw or dried Gujeolcho, applying a pressure of 0.1 MPa in water at 121° C., and extracting it for 1 hour ( FIG. 1 ).

In addition, the prepared extract was dried and then the yield was measured (Table 1).

Through this, Guulcho extracts for each solvent were prepared, and then, the ethanol extract was dissolved in DMSO and the hot water extract was dissolved in water and used in the experiment.

Example 2. Antioxidant activity analysis of each Gujeolcho sample

The antioxidant capacity of each sample of Gujeolcho prepared above was analyzed through DPPH and ABTS assays. Specifically, the DPPH assay is an assay that confirms the radical scavenging ability by measuring DPPH, a purple compound, when a structural change occurs by reacting DPPH with a substance with antioxidant activity, which does not absorb light at 520 nm. The assay reacts with a phenolic compound to form a cation, and when a change in color appears, it is an assay to measure antioxidant activity at 600 to 750 nm.

As a result, as can be seen in FIG. 2 , in the case of the ethanol extract of Gujeolcho raw flower, there was a radical reduction effect of 50% at 200μg/mL, and in the case of the ethanol extract of Gujeolcho dried petal stem, there was a 70% radical reduction effect at 500μg/mL was confirmed.

On the other hand, there was no radical reduction effect in the dried ethanol extract of Guulcho.

In addition, in the case of the hot water extract, it was confirmed that the radical reduction effect of 2000 μg/mL of Gujeolcho fresh flower extract was confirmed, and it was confirmed that the radical effect was insufficient in the hot water extract of dried and dried petal stems.

Through this, it was confirmed that the antioxidant activity was shown in the ethanol extract of Gujeolcho, while the antioxidant activity was low in the hot water extract of Guulcho.

Example 3. Cytotoxicity analysis of each Guulcho sample

The cytotoxicity of the selected Guulcho ethanol extract was confirmed.

More specifically, the skin cell line HaCaT cells and macrophage RAW 264.7 cells were treated with 1, 10, and 100 ug/ml of ethanol extracts of fresh, dried, and dried petal stems of Guulcho, followed by MTT 24 and 48 hours later. The assay was performed.

As a result, as can be seen in FIG. 3 , it was confirmed that some cytotoxicity appeared in the dried ethanol extract of Guulcho, but in the ethanol extract of fresh flower/dried petal stems of Guulcho Cho, after 48 hours of treating the cells with the extract, the cell viability was generally It was confirmed that there was no cytotoxicity through the high one.

Through this, it was confirmed that the low cytotoxicity of the ethanol extract of Gujeolcho.

Example 4. Analysis of antioxidant and anti-inflammatory efficacy by sample of Gujeolcho

The cellular antioxidant efficacy of the selected Guulcho ethanol extract was confirmed.

Specifically, an assay using DCF-DA was performed. When DCF-DA, a chemically reduced form of cell-permeable fluorescein, reacts with ROS generated in the cell, the acetic acid group is cleaved and converted into DCF with high fluorescence. By measuring the fluorescence expression level in Ex 492, Em 527 using a microplate reader, the amount of ROS generated in the cell was confirmed.

More specifically, 100 μg/mL of fresh, dried, and dried petal stems of Gujeolcho were treated, stimulated with LPS for 24 hours, and treated for 30 minutes by replacing the medium with DCF-DA added, and then cells were harvested. Thus, changes in ROS generation were observed.

As a result, as can be seen in FIG. 4A, it was confirmed that all Guulcho ethanol extracts significantly reduced ROS generated by LPS stimulation than other ethanol extracts.

In addition, the cellular anti-inflammatory efficacy of the ethanol extract of Guulcho selected above was confirmed. Gujeolcho fresh flowers 10, 100, 500, μg/mL, Gugucho dried flowers 1, 10, 100, μg/mL, and Gujeolcho dried petals 1, 10, and 100 μg/mL were treated.

Specifically, after RNA was extracted from the cells, cDNA was synthesized, and changes in gene expression of iNOS, COX-2, IL-6, and β-actin were confirmed through PCR.

As a result, as can be seen in FIG. 4B, the expression of the inflammation-mediated genes was reduced in a concentration-dependent manner in all ethanol extracts of Gujeolcho.

Through this, it was confirmed that the ethanol extract of Gujeolcho has antioxidant and anti-inflammatory effects.

Example 5. Cujippong sample preparation

An extract sample using ethanol or water of kkujippong was prepared.

More specifically, after pulverizing kkujippong fruit and dissolving it in 99% ethanol, ultrasonic wave was applied at 60 ℃ at 40 kHz, 300W and extracted for 2 hours, to prepare an ethanol extract.

In addition, the hot water extract was prepared by applying a pressure of 0.1 MPa in water at room temperature or 121 °C (warmed), and extracting for 1 hour (FIG. 5).

In addition, the prepared extract was dried and then the yield was measured (Table 2).

Through this, each solvent kkujippong extract was prepared, and then, the ethanol extract was dissolved in DMSO, and the water extract was dissolved in water and used in the experiment.

Example 6. Analysis of antioxidant activity for each sample of Cujippong

The antioxidant capacity of each sample prepared in the same manner as in Example 2 was analyzed through DPPH and ABTS assays.

Specifically, kkujippong ethanol extract was treated with 0.6, 1.1, 2.8, 5.6, 35, and 566 μg / mL, kkujippong room temperature or warm water extract was treated with 1.25, 25, 50, 100, 200, and 500 μg / mL Thus, DPPH assay and ABTS assay were performed.

As a result, as can be seen in Figure 6, kkujippong ethanol extract showed a free radical scavenging ability from 0.6 μg / mL, it was confirmed that close to 90% at 566 μg / mL. On the other hand, it was confirmed that there was no antioxidant effect in the room temperature and warm water extracts.

Example 7. Cytotoxicity analysis for each Kujippong sample

The cytotoxicity of the selected kkujippong ethanol extract was confirmed above.

Specifically, in the same manner as in Example 3, it was confirmed by MTT assay in HaCaT cells, a skin cell line, and RAW 264.7 cells, a macrophage cell line.

As a result, as can be seen in Figure 7, it was confirmed that the overall cytotoxicity does not appear in all kkujippong extracts.

Through this, it was confirmed that the low cytotoxicity of the kkujippong extract.

Example 8. Analysis of the anti-inflammatory efficacy of each Kujippong sample

The cellular anti-inflammatory effect of the selected kkujippong ethanol extract was confirmed above.

Specifically, after RNA was extracted from the cells in the same manner as in Example 4, cDNA was synthesized, and changes in gene expression of iNOS, COX-2, IL-6, and β-actin were confirmed through PCR.

More specifically, kkujippong ethanol extract was treated with 0.56, 5.6, and 55.6 μg/mL, room temperature and warm water extract was treated with 1, 10, and 50 μg/mL, and LPS was treated with 100ng/mL to induce an inflammatory response. .

As a result, as can be seen in Figure 8, kkujippong ethanol extract was shown to reduce the expression of inflammation-mediated genes from 0.56 μg / mL. On the other hand, it was confirmed that there is no anti-inflammatory effect in the water extract of Cujippong (room temperature/warm temperature).

Through this, it was confirmed that the Cujippong ethanol extract has significantly superior anti-inflammatory and antioxidant effects than the water extract.

Example 9. Analysis of cytotoxicity and anti-inflammatory efficacy of gujeolcho raw flower and kkujippong complex extract

Since the excellent antioxidant and anti-inflammatory effects of the ethanol extract of gujeolcho raw flower and kkujippong ethanol extract were confirmed through the above examples, the cytotoxicity and anti-inflammatory synergistic efficacy of the complex extract mixed with the extract was confirmed.

Specifically, after treatment with a mixture of Gujeolcho raw flower ethanol extract 1, 10 μg/mL and Kujippong ethanol extract 0.05 and 0.1 μg/mL, respectively, intracellular toxicity of RAW264.7 was confirmed through MTT assay.

As a result, as can be seen in Figure 9, it was confirmed that the overall high cell viability appears at various concentrations of gujeolcho raw flower and kkujippong complex extract.

Through this, it was confirmed that the complex extract of Gujeolcho Saenghwa and Cujippong did not have cytotoxicity.

In addition, the expression changes of the inflammation-mediated genes (iNOS, COX-2, and IL-6) induced by LPS were confirmed by treating the gujeolcho raw flower and kkujippong complex extract.

As a result, as can be seen in FIGS. 10 to 11 , when the complex extract was treated than when the ethanol extract of Gujeolcho raw flower and kkujippong ethanol extract was treated alone, the decrease in the expression of inflammation-mediated genes induced by the LPS was further increased. In particular, it was confirmed that the expression of the above genes was most excellently reduced in the groups of Gujjicho fresh flower 1μg/mL + Cuchipon 0.1μg/mL, Gujeolcho fresh flower 10μg/mL + Cujipong 0.1μg/mL group.

Through this, it was confirmed that the excellent synergistic effect on the anti-inflammatory effect of the ethanol extract of Gujeolcho Saenghwa and Cujippong complex was confirmed.

Example 10. Analysis of cytotoxicity and anti-inflammatory efficacy of dried gujeolcho dried and kkujippong complex extract

Following Example 9, the dried ethanol extract of Guulcho 1, 10 μg/mL and kkujippong ethanol extract 0.05 and 0.1 μg/mL were mixed and treated, respectively, and the intracellular toxicity of RAW264.7 was confirmed through MTT assay.

As a result, as can be seen in Figure 12, it was confirmed that the overall high cell viability appears at various concentrations of the dried gujeolcho dried and kkujippong complex extract.

Through this, it was confirmed that the cytotoxicity was somewhat high in the dry ethanol extract of Guulcho, but not cytotoxicity in the dried extract of Gujeolcho and kkujippong complex.

In addition, by treating the dried gujeolcho dry and kkujippong complex extract, it was confirmed that the expression changes of LPS-induced inflammation-mediated genes (iNOS, COX-2, and IL-6).

As a result, as can be seen in FIGS. 13 to 14, when the complex extract is treated than when the dried ethanol extract of Gujeolcho and kkujippong ethanol extract is treated alone, the decrease in the expression of the inflammatory mediator genes induced by the LPS is more increased It was confirmed that, in particular, it was confirmed that the expression of the above genes was reduced the most excellently in the Guulcho dry 10 μg/mL + Kujippong 0.1 μg/mL group.

Through this, it was confirmed that the excellent synergistic effect on the anti-inflammatory effect of the dried gujeolcho dry and cujippong complex ethanol extract.

In other words, in the present invention, the synergistic effect on anti-inflammatory was confirmed by comparing the ethanol extract of Gujeolcho and the ethanol extract of Kujippong fruit and the complex extract.

In addition, there is a time limitation in securing the raw material and there are disadvantages of high cost, but due to the synergistic effect of the dried gujeolcho dried and cujippong fruit complex extract, it was found that it has an anti-inflammatory effect similar to the anti-inflammatory effect of the raw gujeolcho fresh flower and cujippong fruit complex extract. It was confirmed, and it was confirmed that the cytotoxicity which was somewhat high in the ethanol extract of Guulcho dry dry alone was low, and the cytotoxicity was low in the dried Gujeolcho dried and Cuji pony fruit complex extract, confirming the excellent anti-inflammatory synergistic effect and safety.

That is, although the anti-inflammatory efficacy and cytotoxicity of the fresh or dried ethanol extract of Gujeolcho fresh or dried flowers are different from each other, when mixed with the kojipon fruit ethanol extract, it has excellent anti-inflammatory efficacy and high safety without any difference between fresh flowers and dryness. The above time limitation and the disadvantages of cost were solved.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Claims (8)

An anti-inflammatory composition comprising a complex extract of gujeolcho and cucurbita as active ingredients.
The composition for anti-inflammatory according to claim 1, wherein the gujeolcho extract is a fresh or dried extract.
According to claim 1, wherein the gujeolcho extract is contained in 80 to 99.99% by weight based on the total weight, anti-inflammatory composition.
According to claim 1, wherein the kkujippong extract is kkujippong fruit extract will, anti-inflammatory composition.
According to claim 1, wherein the kkujippong extract is comprised of 0.01 to 20% by weight based on the total weight, anti-inflammatory composition.
The composition of claim 1, wherein the extract is an ethanol extract.
The composition for anti-inflammatory according to claim 1, wherein the anti-inflammatory effect is achieved by the ability to reduce iNOS, COX-2, or IL-6 expression.
According to claim 1, wherein the composition will have an additional antioxidant effect, anti-inflammatory composition.

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