KR101144117B1 - Method for Preparing the Extract of Marc of Actinidia polygama and Composition for Preventing and Treating Inflammation or Allergy Comprising the Same - Google Patents

Abstract

The present invention relates to a method for preparing the extract of Caesar meal, and to a composition for preventing and treating inflammation or allergies containing the same, more specifically, while maintaining the physiologically active component of the foreseed cabbage peculiar strong flavor and taste removed It relates to a method for preparing the extract and a composition for preventing and treating inflammation or allergy containing the same. Method of producing a extract of Actinidia polygama gourd according to the present invention comprises the steps of: (a) supercritical extraction with a supercritical fluid extraction device; (b) recovering the sea breeze remaining after the supercritical extraction; (c) extracting the leek gourd with a solvent selected from the group consisting of distilled water, ethanol (edible alcohol) and mixtures thereof; And (d) recovering the extracted Caesar meal.

Weeping, weeping gourd, supercritical extraction, inflammation, allergy

Description

Method for preparing the extract of Marc of Actinidia polygama and Composition for Preventing and Treating Inflammation or Allergy Comprising the Same}

The present invention relates to a method for preparing the extract of Caesar meal, and to a composition for preventing and treating inflammation or allergies containing the same, more specifically, while maintaining the physiologically active component of the foreseed cabbage peculiar strong flavor and taste removed It relates to a method for preparing the extract and a composition for preventing and treating inflammation or allergy containing the same.

Inflammation is the defense of biological tissue against local injuries. In other words, in response to various harmful stressors, the biodefense reaction to remove the injury caused by the stimulus and restore the original state is an inflammatory response. Inflammatory stimuli include infection or chemical or physical stimuli. Bioconstituents involved in the inflammatory response include low-molecular or high-molecular chemicals such as free radicals, proteins, sugars, and lipids, and plasma, blood cells, blood vessels, and connective tissue.

The inflammatory process is usually divided into two types, which can be divided into acute and chronic. Acute inflammation is a short-term reaction within a few days. Plasma components and blood cells are associated with the removal of foreign bodies with microcirculation. Chronic inflammation has a long duration and is accompanied by tissue proliferation.

Allergic diseases are most often caused by chemical mediators (primarily histamine, prostaglandins, leukotrienes, TNFα, cytokines, etc.) that are released from granules of mast cells in tissues activated by antigen-antibody responses. Since the use of allergy therapeutic drugs currently used remains symptomatic, there is an urgent need for the development of more fundamental therapeutic drugs.

Key mediators that induce inflammation and allergic diseases include prostaglandindes, leukotriens, platelet activators (PAFs), phospholipase A2, and cyclooxygenase ) And lipoxygenase to form the precursor arachidonic acid. In addition, enzymes related to nitric oxide synthase (NOS) biosynthesis, an enzyme that generates nitric oxide (NO), are also known to play an important role in mediating the inflammatory response. NOS, an enzyme that produces NO from arginine (L-Arginine), is also a major goal in blocking inflammation. Recent studies have shown that there are several types of NOS, such as brain NOS (brain NOS) in the brain, neuronal NOS in the nervous system, and endothelial NOS in the vascular system. It is expressed at a certain level, and a small amount of NO produced by them plays an important role in maintaining normal homeostasis, such as inducing neurotransmission or vasodilation, whereas various inflammatory cytokines and lipopolysaccharides (lipopolyssacharide) NO, which is rapidly produced by iNOS (induced NOS) induced by external stimulants such as LPS), is known to cause cytotoxicity and various inflammatory reactions, and chronic inflammation is associated with increased iNOS activity. (Miller MJ et al., Mediators of inflammation, 4, pp 387-396, 1995: Appleton L. et al., Adv. Pharmacol., 35, pp 27-28, 1996).

In order to treat the above inflammatory diseases, various types of inflammatory disease treatments have been developed. The inflammatory disease therapeutic substances reported to date are induced by tissue cells damaged by acute inflammation, cells involved in inflammation, or chemotactic factor. It is a drug that inhibits the production of eicosanoids from the cell membrane of the white blood cells. Allergy agents are also drugs that inhibit degranulation, liberation of histamine receptors, inhibition of leukotriene production, and leukotriene receptor block, which are released during antigen-antibody reactions.

Korean Patent Application No. 92-11752 discloses anti-inflammatory, anti-allergic and antirheumatic agents of biflavonoids obtained from Lonicera japonica , and Korean Patent Registration No. 100744 discloses biflavonoids obtained from ginkgo leaves. Anti-inflammatory, anti-allergic and antirheumatic agents containing

On the other hand, Actinidia polygama is a fruit of the deciduous vines of the deciduous vines belonging to the genus Tami ( Mihuri ) and (Actinidiaceae), and the components thereof are sucrose, mucus, starch, protein, tannin, organic acid and vitamin C It is known to contain vitamin A, vitamin P, etc., and the effects so far known include the treatment of jigal, haeban fever and urinary stones. Metabolism, Younglimsa, p386-387, 1998).

Korean Patent Registration No. 0511550 discloses a pharmaceutical composition for the treatment and prevention of allergic diseases and non-allergic inflammatory diseases, which contains the wormwood extract, which is extracted from the conventional organic solvent extraction method. Because of retaining the aroma and taste, there was a problem that is not suitable for processed products or medicines for people sensitive to the aroma and taste, such as patients or children.

Thus, the present inventors have made a diligent effort to solve the above problems, as a result, when extracting the sea bream gourd which removes the fragrance component exhibiting the unique fragrance of the sea bream under specific conditions, the bioactive component of the sea squid is The present invention was completed by confirming that the extract of Cauliflower Park, which has been removed from the strong aroma and taste peculiar to Camembert, was maintained.

It is an object of the present invention to provide a method of preparing a Cauliflower gourd extract, which has been removed the strong aroma and taste unique to Caucasus while maintaining the bioactive component of Cabbage.

It is another object of the present invention to provide a composition for preventing or treating inflammation and allergy, which contains the extract of Cauliflower, which has been removed the strong aroma and taste peculiar to Caucasus.

In order to achieve the above object, the present invention (a) using a supercritical fluid extraction device at a temperature of 35 ~ 55 ℃, pressure (psi) of 1500 ~ 6000 and CO ₂ flow rate of 1 ~ 3mL / min 1 Supercritical extraction for ˜2 hours; (b) recovering the sea breeze remaining after the supercritical extraction; (c) immersing the sea squirt gourd in a solvent selected from the group consisting of distilled water, ethanol, and mixtures thereof, followed by extraction by heating at a temperature of 70-100 ° C. for 30 minutes to 90 minutes; And (d) said gaedarae recovering foil extract extraction; the production method of the include, and recovering gaedarae foil extract gaedarae wherein the fat of the total components gaedarae is less than 1 wt% (Actinidia polygama) foil extract to provide.

The present invention also provides a pharmaceutical composition for treating inflammation or allergy containing the extract of Actinidia polygama gourd prepared by the above method.

The present invention also provides a functional food for preventing inflammation or allergy containing the extract of Actinidia polygama gourd prepared by the above method.

When using the manufacturing method of the extract according to the present invention, while maintaining the various biologically active components of the foreseed, the strong aroma and taste peculiar to the foreseed can be easily and effectively removed, produced by this May be usefully used in the manufacture of functional foods or therapeutic pharmaceutical compositions for those who are sensitive to the aroma and taste of dogs, such as patients or children.

In the present invention, when using a supercritical fluid extracting device, it was intended to confirm whether the components showing the strong aroma and taste peculiar to the litter can be easily and effectively removed while maintaining various bioactive components of the litterworm.

In one embodiment of the present invention, after extracting the seaweed persimmon with a supercritical fluid extracting device, and recovering the remaining seared gourd gourd was extracted by using a solvent to prepare a seaweed gourd extract. As a result of confirming the components of the extract of Cabbage Fructus, it was confirmed that the components exhibiting the aroma and taste peculiar to Cabbage Fever decreased and that the components such as protein, ash, carbohydrate, free sugar and the like were maintained.

Accordingly, the present invention in one aspect, (a) supercritical extraction with the supercritical fluid extraction device; (b) recovering the sea breeze remaining after the supercritical extraction; (c) extracting the sea squid with a solvent selected from the group consisting of distilled water, ethanol and mixtures thereof; And (d) relates to a method for producing a dog extract ( actinidia polygama ) gourd extract comprising the step of recovering the extracted extract.

The corpus larva is also called a lapper tree, and grows under deep mountain trees or in valleys. The stem is about 5m long, white inside the stem, and the twigs have light brown hairs when they are young, and rarely burly-like hairs. Its leaves are alternately membranous, membranous, broad oval or oval, with sharp tips, with brown hairs on the veins, and fine jagged teeth. Generally, from June to July, 3 ~ 10 white flowers with a diameter of 1.5cm hang on the upper leaf axilla. Forsythia fruit is long oval with berry and ripens yellow and hangs down from September to October.

In the present invention, it is preferable to use the fruit of the litter, and the dried fruit of the litter is also referred to as "mokcheon".

In general, the supercritical fluid refers to a supercritical fluid, which is a fluid in a special situation under high temperature and high pressure by applying a critical pressure and a critical temperature to a material such as carbon dioxide, water, alcohol, helium, and the like. This supercritical fluid has a value similar to the density of the liquid, the viscosity is close to the gas and its diffusion coefficient is about 100 times larger than the liquid. Therefore, when the supercritical fluid is penetrated into the extraction target and reacted with the desired contents, the contents of high purity can be extracted.

The supercritical extraction is performed for 1 to 2 hours at a temperature of 35 ~ 55 ℃, pressure (psi) of 1500 ~ 6000psi. And CO ₂ flow rate of 1 ~ 3mL / min. In the supercritical extraction, the temperature, pressure, and time have a complex effect on each other, but when the respective conditions are out of range, the extraction yield is lowered or does not increase any more, which is economically undesirable. Considering the temperature, pressure, and extraction time, it is preferable to extract for about 1 hour at about 45 ℃, 4500psi.

In addition, when the CO ₂ flow rate is less than 1mL / min, there is a problem that the extraction yield is greatly lowered and the extraction time takes a lot, and if it exceeds 3mL / min, there is a problem that the extraction yield does not increase any more.

When the litterworm is extracted with a supercritical fluid extraction device, fat-soluble components associated with the unique flavor and taste of the litterworm are extracted. After the extraction, the remaining cabbage gourd may be used for extracting the bioactive of the cabbage.

In the present invention, the solvent extraction may be immersed in a solvent for the seaweed foil, left to stand at room temperature for 24 hours or extracted for 1 hour at 70 ~ 100 ℃.

Distilled water, ethanol or a mixture thereof may be used as the solvent. The temperature of the solvent extraction may vary depending on the solvent used but is preferably 70 ~ 100 ℃. For example, if the ethanol content is high 70 ~ 80 ℃, if the distilled water content is high, it is preferable to extract at 90 ~ 100 ℃. In the present invention, the ethanol is preferably used edible alcohol.

Gaedarae according to the invention (Actinidia polygama ) The method for producing a gourd extract is characterized in that it further comprises the step of recovering the antler gourd extract, then filtration and lyophilization of the recovered antler gourd extract. The filtration may be performed using a filter paper, and lyophilization may be performed in the same manner as in the conventional method of removing water by sublimating ice by freezing the decocted gourd gourd extract and decompressing (減壓).

On the other hand, when extracting the supercritical fluid of the cortex, it was predicted that the extract of the cortex pertussis would have anti-inflammatory and anti-allergic effects, since the bioactive component remained in the cortex, if the bioactive component was not extracted.

In another embodiment of the present invention, as a result of the anti-inflammatory and anti-allergic activity of the extract of Caesar gourd extract, it was confirmed that the extract of Cabbage gourd was excellent in anti-inflammatory and anti-allergic activity.

Therefore, in another aspect, the present invention relates to a pharmaceutical composition for treating inflammation or allergy containing the extract of Actinidia polygama gourd prepared by the above method.

The pharmaceutical composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of oral dosage forms, such as powders, granules, tablets, capsules, syrups, external preparations, suppositories, and injections according to conventional methods.

The pharmaceutical compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

The present invention also provides a method of making a spear ( Acinidia) polygama ) relates to functional foods for the prevention of inflammation or allergies containing gourd extract.

The functional food may include the extract of Corsagea Park and food acceptable food additives.

The functional food may preferably be one selected from tablets, granules, soft capsules, liquid formulations, beverages, and teas, wherein the perilla extract is added to the total weight of the food composition so that it may be absorbed by the human body to express inflammation or allergy prevention. It is preferable to contain 0.1 weight% or more.

Actinidia at this time polygama ) gourd extract is reduced in the smell of peculiar odor component, there is no objection to ingestion, the fat containing these scent may be less than 1% of the total content of the lag.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Supercritical Extraction and Recovery of Persimmon Foil

Using the supercritical fluid extractor (SFT-100XW, Supercritical fluid technologies, Inc., USA) was extracted under the conditions of Table 1. That is, 40 g of the sample was placed in an extraction tank of 100 mL, extracted in a dynamic mode of CO ₂ flow rate of 2 mL / min at a constant temperature and pressure extraction conditions (Table 1), and the extract and the remaining catnip were recovered. The yield of the extract is shown in Table 2 below.

division Extraction conditions Temperature (℃) 35, 45, 55 Pressure (psi) 1500, 3000, 4500, 6000 Hour (hr) 1, 2

Temperature
(℃) pressure
(psi) time 1 hr (%) 2 hr (%) 35 ℃ 1500 0.15 ± 0.02 0.31 ± 0.07 3000 0.38 ± 0.01 0.52 ± 0.02 4500 0.50 ± 0.03 0.55 ± 0.09 6000 0.41 ± 0.01 0.53 ± 0.02 45 ° C 1500 0.09 ± 0.01 0.18 ± 0.04 3000 0.34 ± 0.06 0.48 ± 0.04 4500 0.65 ± 0.03 0.72 ± 0.04 6000 0.45 ± 0.02 0.51 ± 0.03 55 ° C 1500 0.02 ± 0.00 0.07 ± 0.01 3000 0.31 ± 0.03 0.49 ± 0.02 4500 0.36 ± 0.01 0.76 ± 0.11 6000 0.32 ± 0.02 0.52 ± 0.02

As shown in Table 2, the supercritical extraction yields of the litterworms were increased with increasing pressure at the same temperature, and decreased with increasing temperature at the same pressure. Also, the same temperature and pressure increased as the extraction time increased. Especially, when the pressure increased from 3000 psi to 4500 psi at 45 and 55 ℃, the yield tended to increase by about 2 times, but at 45 ℃ and 4500 psi, it was 0.72 when extracted for 2 hours from 0.65 ± 0.03% for 1 hour. Extraction yield did not increase significantly with increasing time to ± 0.04%. In consideration of temperature, pressure, and extraction time, it is determined that extraction for 1 hour at 45 ° C. and 4500 psi is most preferable.

<Experimental Example 1> Comparison of Components

The general components, free sugars, free amino acids, fragrances, etc. of the gourd (leaves gourd) remaining after supercritical extraction at 45 ° C, 4500 psi, and 1 hour were compared. In the case of fat (Table 3), the fat decreased significantly from 1.17% to 0.48%, and it can be seen that the fat, which is the fat-soluble component of the seaweed, is extracted by supercritical extraction. The value is shown. In addition, free amino acids (Table 4) and free sugars (Table 5) showed almost the same values of fortune and fortune persimmon, indicating that supercritical extraction did not affect free sugar and free amino acid values of fortune. After extracting the fragrance components (lipophilic essential oils) of forage and perilla gourd using a continuous steam distillation extractor, analyzed by GC (Table 6), the total fragrance content of perilla at 59.7 ± 14.4 mg / kg It was found to be greatly reduced to 5.8 mg / kg, and it was found that the indigenous odor component was significantly removed by the supercritical extraction.

Item Dog sputum (%) Jacktail Park (%) moisture 4.67 4.44 Fat 1.17 0.48 protein 14.81 14.98 Ash 6.95 7.00 carbohydrate 72.40 73.10

amino acid Thigh (μg / mg) Forth Shrimp (μg / mg) Cys 0.55 0.60 ASP 0.52 0.51 GLU 0.24 0.24 ASN 1.72 1.75 SER 0.23 0.23 GLN 0.81 0.82 GLY 0.05 0.05 HIS 0.08 0.07 ARG 3.55 3.61 THR 0.10 0.11 ALA 0.97 0.97 PRO 0.18 0.18 TYR 0.20 0.23 VAL 0.19 0.20 MET 0.07 0.06 Cys2 0.04 0.05 ILE 0.08 0.08 LEU 0.06 0.06 PHE 0.30 0.31 TRP 0.46 0.52 LYS 0.33 0.33 Total 10.73 10.97

Glass sugar Dog sputum (%) Jacktail Park (%) Fructose 1.44 1.57 Glucose 1.76 1.62 Sucrose 0.84 0.67

A dog Corkscrew Night Total Aroma Content
(mg / kg) 99.7 ± 14.4 58.0 ± 5.8

< Example  2> A dog  Night Hydrothermal  And preparation of alcohol extracts

40 g of Cauliflower gourd, recovered at 45 ° C., 4500 psi and 1 hour of Example 1, was extracted with distilled water and 75% ethanol (edible alcohol). In the case of distilled water, distilled water corresponding to 20 times the weight of litter gourd and litter gourd was put into the flask and extracted at 90 ° C. for 1 hour. For 75% ethanol (edible alcohol), the weight of litter gourd and litter gourd 75% ethanol (edible alcohol) corresponding to the pear was put into the flask, and then extracted at 80 ° C. for 1 hour. The extract was filtered and then lyophilized.

<Comparative Example 1> Preparation of hot water and alcohol extract

Except for using the sea squirrel instead of the sea squirrel gourd was extracted using distilled water and 75% ethanol (edible alcohol) in the same manner as in Example 2.

Experimental Example 2 Comparison of Hydrothermal and Alcoholic Extracts

The yields of the hot water extract and the alcoholic extract of the extract of Example 2 and Comparative Example 1 were confirmed, the total flavonoid and total polyphenol content was analyzed by GC, the results are shown in Table 7.

extract Yield (%) Total flavonoids
(ppm / catechin) Total polyphenols
(ppm / gallic acid) A dog Hot water extract 31.1 ± 0.5 21.2 ± 0.5 85.0 ± 0.6 Alcohol extract 21.8 ± 1.5 26.8 ± 0.8 95.6 ± 1.2 Corkscrew Night Hot water extract 33.6 ± 1.4 22.3 ± 0.3 85.9 ± 1.0 Alcohol extract 21.3 ± 0.4 27.3 ± 0.6 95.9 ± 1.9

As shown in Table 7, hot water extracts (31.1 ± 0.5 ~ 33.6 ± 1.4%) showed higher yields than alcoholic extracts (21.3 ± 0.4 ~ 21.8 ± 1.5%), but there was almost no difference between seaweed and seaweed meal. It was.

The total flavonoids and total polyphenol contents were higher in alcoholic extracts than hot water extracts, but there was little difference between total flavonoids and total polyphenol contents in litter and perilla gourd.

Therefore, it was confirmed that the supercritical extraction did not have any effect on the yield or the extraction of flavonoids and polyphenols, which are the main functional components, when hot water or ethanol was extracted by supercritical extraction.

In addition, after adjusting the hot water and ethanol extracts of sea bream and coriander gourd to 1% concentration, the sensory characteristics were derived through the flavor description test on the extracts of 10 trained sensory inspectors. Was carried out and the results are shown in Table 8. In addition, the sensory test was conducted to evaluate the sensory preferences of the extracts by the 9-point symbol scale method (1 point: very dislike, 9 points: very liking) about color, aroma, taste, body feeling, and overall acceptability. The results are shown in Table 9.

A dog Corkscrew gourd Hot water extract Alcohol extract Hot water extract Alcohol extract bitter 6.0 ± 0.9 8.7 ± 0.5 5.2 ± 1.3 8.1 ± 0.8 Astringent 4.9 ± 0.8 7.4 ± 0.9 4.7 ± 1.2 7.3 ± 1.0 sweetness 3.3 ± 1.6 2.4 ± 1.5 3.3 ± 2.0 2.6 ± 1.5 Sweet taste 4.2 ± 1.2 3.4 ± 1.0 3.9 ± 1.8 2.9 ± 1.3 Herbal Medicine 5.4 ± 1.1 5.9 ± 1.7 4.6 ± 0.9 5.2 ± 1.8 Dirt smell 5.0 ± 1.4 5.1 ± 1.8 4.2 ± 1.5 4.5 ± 1.2 Peppermint 2.7 ± 1.4 4.3 ± 1.2 2.6 ± 0.7 3.6 ± 1.1

As shown in Table 8, through the flavor description test, a total of seven sensory characteristics, including taste characteristics such as bitterness, astringent taste, sweetness, and sweet taste, and aroma characteristics such as herbal medicine fragrance, earthy smell and peppermint flavor, were derived. It was also found that alcohol extracts had strong strengths such as astringent taste, astringent taste, and medicinal herb extract. In addition, the characteristics of both hot-water extracts and alcohol extracts were significantly reduced in the case of forked yam prepared by supercritical extraction.

A dog Corkscrew gourd Hot water extract Alcohol extract Hot water extract Alcohol extract color 6.7 ± 0.5 4.2 ± 1.0 6.7 ± 0.9 4.3 ± 1.7 incense 5.8 ± 0.8 5.3 ± 2.0 6.4 ± 1.3 5.9 ± 1.5 flavor 5.7 ± 1.0 3.2 ± 1.0 6.2 ± 1.7 3.9 ± 0.8 Body 5.6 ± 0.5 4.0 ± 1.5 5.6 ± 1.6 4.3 ± 1.3 Comprehensive likelihood 5.8 ± 1.1 3.5 ± 1.1 6.3 ± 1.1 4.4 ± 1.1

As shown in Table 9, the results of sensory evaluation on color, aroma, taste, body feeling, and comprehensive preference in 9-point symbol scale method were used to evaluate the sensory preferences of hot water and alcohol extracts of forage and perilla gourd. Compared to the alcoholic extracts, the degree of acceptability of hot water extracts was higher in all items than in alcoholic extracts.

< Example  3> A dog  Preparation of alcoholic extract of gourd

1 kg of Cauliflower gourd collected at 45 ° C., 4500 psi, and 1 hour condition of Example 1 was repeatedly extracted by immersion in 10 times (w / v) of 80% ethanol (edible alcohol) for 24 hours at room temperature. The extract was filtered twice using filter paper (Whatman No. 3, England), and the supernatant was concentrated at 55 ° C. with a rotary vacuum evaporator (EYELA UNI TRAP UT-1000, JAPAN), followed by freeze-drying to obtain 190 g of the fungus extract. It was.

<Comparative Example 2> Preparation of Caesar extract

1 kg of dried Cauliflower fruits were purchased, washed with water and dried. For 24 hours at room temperature, forty-four (1 kg) fruit was immersed in 10-fold (w / v) of 70% ethanol (edible alcohol) and extracted three times in total. The extract was filtered twice using filter paper (Whatman No. 3, England), and the supernatant was concentrated at 55 ° C. with a rotary vacuum evaporator (EYELA UNI TRAP UT-1000, JAPAN) and lyophilized to obtain 306.9 g of Ganoderma lucidum extract. It was.

Experimental Example 3 Anti-inflammatory Effects of Extracts

1. Cell line culture

RAW 264.7 cell line, a murine macrophage cell line, was distributed from Korea Cell Line Bank (KCLB, Seoul, Korea), and DMEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotics (penicillin / streptomycin). Subsequently, passages were performed 2-3 times a week in a 37 ° C. incubator with 5% CO ₂ .

2. Measurement of Cytotoxicity and Nitric Oxide (NO) Production

MTT (3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazoliumbromide) assay for measuring the cytotoxicity of the extract of Cauliflower Park, extracted from Example 3 and the extract of Cauliflower, extracted from Comparative Example 2 Was measured. RAW 264.7 cells 1 × 10 ⁵ cells / well were aliquoted into 96 well plates and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours. After culturing the cells with serum free medium, LPS (100 ng / mL) and the samples were treated for 24 hours and incubated for 24 hours, 10 μL of 5 mg / mL MTT solution was added to each well and incubated in the incubator for 4 hours. After incubation, the supernatant was removed and 100 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 550 nm using a microplate reader. Cytotoxicity was expressed as a percentage of the absorbance of the control group. It was.

In order to measure the inhibition of NO production of the samples, 100 μL of each sample extract and LPS-treated culture were taken in 96 well plates for nitrite measurement. After adding the same amount of Griess reagent and reacting for 10 minutes, the absorbance was measured at 540 nm using a microplate reader. The concentration of nitrite was calculated by comparing with a standard straight line obtained using sodium nitrite (NaNO ₂ ), and the calculated value was expressed as mean ± SEM value of the experiment data repeated three times.

Inflammatory reactions produce a variety of proinflammatory mediators, including prostaglandin, produced by nitric oxide (NO) and cyclooxygenase-2 (COX-2), which are produced by inducible nitric oxide synthase (iNOS). E ₂ (PGE ₂ ), and the like. These inflammatory factors activate the nuclear factor-kB (NF-kB), a transcription factor of the inflammatory response, resulting in excess NO and PGE ₂ , resulting in inflammation. Inflammatory factors include tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which are inflammatory cytokines.

Sample Concentration
(μg / mL) Cell survival rate (%) NO inhibition rate (%) ^* EtOH ext. 10 95.32 ± 5.06 4.61 ± 0.91 100 102.73 ± 0.28 19.00 ± 2.34 Marc ext. 10 96.18 ± 4.23 36.63 ± 4.00 100 98.17 ± 1.71 70.58 ± 3.35

As shown in Table 10, scavenging of EtOH ext. And Marc ext. For NO, which is one of the reactive nitrogen species and known to be important agents of the inflammatory response. As a result of observing the effect, LPS induction of NO production was about 45 μM in the control. On the other hand, when each extract was treated with 10 and 100 μg / mL, the NO production of EtOH ext. (EtOH ext.) Was 42.93 and 36.45 μM, respectively, indicating that NO scavenging activity was insignificant. .) Showed 28.97 and 13.45 μM, respectively, showing good NO scavenging activity of about 36% and 70% at each concentration. Therefore, the extract of the supercritical extracts showed better NO scavenging ability compared to alcohol extracts. In addition, the cell survival rate of more than 95% at these concentrations showed that the anti-inflammatory effect of the extract was not related to the decrease in cell viability.

3. PGE ₂  And TNF-α production

To measure the amount of PGE _{2 in} cell culture, a commercial competitive enzyme immunoassay kit was purchased from Cayman chemical (Michigan, USA) and tested. PGE _{2 was} measured using a cultured barley extract and extracts treated in LPS (100ng / mL) in Example 3. 50 μL of the culture solution was loaded on 96 well plates coated with goat anti-mouse, and 50 μL of the primary antibody solution and 50 μL of the PGE ₂ conjugate were added thereto and then overnight at 4 ° C. After washing 4 times with Washing Buffer and treating the substrate solution by 200 μL for 5 to 60 minutes, the absorbance was measured at 405 nm after treatment with 50 μL of stop solution. In addition, Enzyme-Liked Immunosorbent Assay (ELISA) was performed to measure the amount of cytokines in the cell culture. In addition, TNF-α was measured using a cultured barley extract and extracts treated in LPS (100ng / mL) in Example 2. After diluting the culture to an appropriate concentration, 50 μL was added to a 96 well plate coated with cytokine and overnight at 4 ° C. After washing three times with washing buffer, the reaction was treated with 100 μL of streptavidine-HRP solution at room temperature for 1 hour and then washed three times with washing buffer again. After treatment with 100 μL of di (2-ethylhexyl) -2,4,5-trimethoxybenzalmalonate (TMB) substrate for 5-30 minutes, the absorbance was measured at 450 nm after treatment with 100 μL of stop solution.

As a result of measuring the effect of the extract of Ganoderma lucidum on the production of PGE ₂ and TNF-α, the production of PGE ₂ was increased by about 20-fold when 100 ng / mL LPS was treated in RAW 264.7 cells. When the Cauliflower gourd extract was treated with 10 and 100 μg / mL, respectively, the PGE ₂ content was about 740 and 500 pg / mL, and the Cauliflower gourd extract showed more than 90% inhibition of PGE ₂ production. In addition, TNF-α was effectively induced during LPS treatment, and when treated with Cauliflower extract, it had no effect at the concentration of 10 μg / mL, but produced TNF-α at the concentration of 100 μg / mL. Reduced by more than% (FIGS. 1 and 2).

Inflammation is one of the defense responses of biological tissues to external stimuli. Clinically, symptoms include redness, fever, swelling, pain, and dysfunction, and various mediators such as cytokines, PGE ₂ , lysosomal enzymes, and free radicals are involved. Doing. After the treatment of Caesar gourd extract on the RAW 264.7 cell line, LPS treatment induced inflammation and measured the production of inflammatory factors NO, PGE ₂ and the production of proinflammatory cytokines TNF-α. It has been shown to effectively reduce the induced inflammatory factors, which may be effectively applied to the prevention and treatment of atherosclerosis and chronic inflammatory diseases such as hypertension, cancer, diabetes and arthritis.

Experimental Example 4 Anti-allergic Effect of Extract

1. Cell culture and cytotoxicity measurement

Cytotoxic activity of rat-derived mast cells (RBL-2H3) was measured by MTT method. The cell line RBL-2H3 cells used in this experiment were tested by subcultured through the pre-cultured through the Korea Cell Line Bank (Seoul, Korea). The medium used for cell culture was used after culturing for 2-3 days in 5% CO ₂ incubator at 37 ° C using MEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotics (penicillin / streptomycin). . The cytotoxicity of the sample was measured after 12 hours treatment of the extract of Example 3, which was extracted from Example 2 and the extract of Comparative Example 2, on cells activated with IgE and antigen, and the concentration of 10-400 ㎍ / mL was measured. It was not toxic to mast cell lines (FIG. 3).

2. Mast cell protective effect

Allergic reactions are largely divided into four types (I-IV), and allergic diseases such as atopic dermatitis, allergic rhinitis and bronchial asthma belong to type I allergic reactions. Mast cells play an important role in this type I allergic reaction and are widely distributed in connective tissues in vivo such as skin, respiratory tract, blood vessels, and brain. When mast cells are activated pharmacologically or non-immunologically, degranulation is induced, and histamine stored in intracellular granules is released, resulting in allergic symptoms. The degranulation inhibitory activity of the sample was measured by measuring the activity of β-hexosaminidase, which is released together with histamine and is widely used as an indicator of degranulation in RBL-2H3 cells.

2-1. Determination of β-Hexosaminidase Release Inhibition

The mast cell line RBL-2H3 was sensitized in 1 ml (2 x 10 ⁵ cells, 24 well plates) with anti-DNP IgE (0.45 μg / ml) for 16 hours and before activation with DNP-BSA (10 μg / mL) 30 20 ml of concentration-specific samples were treated at 37 ° C. for minutes. After completion of the reaction, 25 μl of the supernatant obtained by centrifugation at 400 × g for 10 minutes at 4 ° C. was reacted with 25 μl of substrate p-nitrophenyl-N-acetyl-bD-glucosaminide at 37 ° C. for 1 hour, followed by stop solution (0.1 M NaCO / NaHCO, pH 10) 200 μl was added to stop and the absorbance was measured at 405 nm to calculate the amount of b-hexosaminidase.

As shown in FIG. 4, the activity of β-hexosaminidase released from the cells treated at the concentrations of 0.01, 0.1, and 0.4 mg / mL extracted from Comparative Example 2 was 87.9%, 45.6%, and 27.4%, respectively, compared to the control group. In the case of alcoholic extracts of Caesar meal extracted in Example 3, 73.2%, 54.3%, 42.5% showed a decrease in concentration-dependent.

2-2. Protective effect against degranulation by Compound 48/80

In order to confirm the degranulation inhibitory effect of the extract of Cauliflower park extract extracted from Example 3 and the extract of Cauliflower extract from Comparative Example 2, 1 mL (2 x 10 ⁵ cells, 24 well plate) of RBL-2H3 was observed. ) for 4 hours after the incubation, 200 ㎕ in incubation buffer containing 1 mM Ca ^{2 +} 10 bungan after incubation and put into a 0.1% DMSO solution or sample 25 ㎕ per each concentration was reacted at 37 ℃ 10 minutes. After the reaction, 25 µl of a compound 48/80 solution was added and reacted for 20 and 40 minutes. After the reaction, the cells were observed under an inverted microscope (LEICA DMIRE2, Germany) under a magnification of 600 times the shape of the mast cell line on an optical microscope. The total degree of degranulation was confirmed under 200 times magnification, and reproducibility was confirmed by three experiments. Normal mast cell lines have a circular or oval shape with a clear cell profile and a smooth surface, whereas the cells of the granules activated by compound 48/80 treatment are unclear and small in size, with intracellular granules It could be seen protruding or scattered around the cell (FIG. 5). When only the sample was treated without compound 48/80, degranulation did not occur and the cell morphology did not change, and the effects of protecting the cells from degranulation were observed at the concentration of 400 ㎍ / mL. In the concentration of 200 ㎍ / mL, 20 minutes treatment had a protective effect (Data not shown), but after 40 minutes it was confirmed that most of the protective effect disappeared (Fig. 5).

3. Measurement of TNF-a Production Inhibition

RBL-2H3 cells were dispensed into 24-well plates (2 × 10 ⁵ cells / well) and incubated for 12 hours. Treated with New Zealand MEM extract extracted from Example 3 and the extract of "Taiwan extract from Comparative Example 2 in concentration (0.01, 0.1, 0.4mg / mL), A23187 (1 μM) and PMA (50 ng) / mL) was treated and incubated for 4 hours, in which Luteolin (5 μM) was used as a control, and after the incubation was completed, the supernatant was separated and stored at -70 ° C and measured using a TNF-α ELISA kit. The amount of TNF-α produced was calculated by measuring the absorbance at 450 nm using an ELISA reader, and as a result of measuring the inhibitory effect on the production of the proinflammatory factor TNF-α, TNF-α was 5.06 pg before cell activation. Activation at / mL significantly increased to 87.14 pg / mL, but when treated with 0.1 mg / mL of alcohol extract and gourd extract, 69.1pg / mL, 68.6pg / mL, similarly inhibited TNF-a production It was confirmed (Fig. 6).

Preparation Example 1 Preparation of Beverage

20% by weight of Cabbage gourd extract powder prepared in Example 2, 0.00075% by weight of vitamin B1 hydrochloride, 0.00075% by weight of sodium vitamin B2 phosphate, 0.00045% by weight of vitamin B6 hydrochloride, 0.00005% by weight of folic acid, 0.003% by weight of nicotinamide, citric acid After mixing by filtering 0.095% by weight, 25.3% by weight of sugar and 54.6% by weight of purified water, the filtered mixture was sterilized and filled for 20 seconds at 90 ℃ or more, and then sterilized for 30 minutes at 85 ℃ to prepare a beverage.

Preparation Example 2 Preparation of Tablet

40 mg of lactose gourd extract powder prepared in Example 2, 100 mg of lactose and 100 mg of starch were mixed and tableted according to a conventional method for preparing tablets.

Preparation Example 3 Preparation of Granules

Granules of 60mesh granules were kneaded together and passed through a granulator, 35% by weight of bark gourd ethanol extract powder prepared in Example 2, 0.3% by weight of vitamin C, 28% by weight of glucose, 28% by weight of dextrin, 8.7% by weight of 70% ethanol Was prepared.

Preparation Example 4 Preparation of Capsule

20 mg, Lactobacillus ethanol extract powder prepared in Example 2, lactose 50 mg, starch 50 mg, talc 2 mg were mixed, and the capsules were prepared by filling gelatin capsules according to a conventional method for preparing capsules.

As described above in detail the specific parts of the present invention, it is apparent to those skilled in the art that such specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

1 is PGE _{2 of the} extract of Cauliflower gourd extract against LPS-stimulated RAW 264.7 cells. It is a graph showing the effect on the production (mean ± SEM value of the experiment data repeated three times, * p <0.05).

Figure 2 is a graph showing the effect on the TNF-α production of Lactobacillus chinensis extract of L. stimulated RAW 264.7 cells (mean ± SEM value, * p <0.05 of three repeated experimental data).

Figure 3 is a graph measuring the cytotoxicity of the extract of Lilium ostrich extract and Lilium pacifist extract on RBL-2H3 cells activated with IgE and antigen (A: Lilium ethanol extract, B: Lilium lip extract.

Figure 4 is a graph measuring the inhibitory ability of β-hexosaminidase release of the Licorice persimmon extract and Licorice persimmon extract on RBL-2H3 cells activated with IgE and DNP-BSA (A: perilla extract, B: perilla persimmon extract ).

Figure 5 is a photograph of the degranulation protection of the Mt. bark extract and the Mt. bark extract for the mast cell line treated with the compound 48/80 solution (A 600, A: barley bark extract, B: barley gourd Alcohol extract, 1,2,3: before treatment with compound 48/80, 4,5,6: after treatment with compound 48/80, 4: untreated 5: 400 μg / mL, 6: 200 μg / mL).

FIG. 6 is a graph measuring the inhibitory ability of TNF-α production of Cauliflower alcohol extract and Cauliflower gourd extract on RBL-2H3 cells stimulated with A23187 (1 μM) and PMA (50 ng / mL). , B: Cortex gourd ethanol extract).

Claims (9)

(a) extracting supernatant for 1 to 2 hours using a supercritical fluid extraction apparatus at a temperature of 35 to 55 ° C., a pressure of 1500 to 6000 (psi) and a CO ₂ flow rate of 1 to 3 mL / min; (b) recovering the sea breeze remaining after the supercritical extraction; (c) immersing the sea squirt gourd in a solvent selected from the group consisting of distilled water, ethanol, and mixtures thereof, followed by extraction by heating at a temperature of 70-100 ° C. for 30 minutes to 90 minutes; And (d) recovering the extracted Cauliflower gourd extract; Recovered cabbage gourd extract is a method of producing a extract of Actinidia polygama gourd, characterized in that less than 1% by weight of fat in the whole component. The method of claim 1, wherein the method further comprises the step of filtration and lyophilization of the recovered Cabbage Fructus extract. delete delete The method of claim 1, wherein the step of extracting with a solvent is immersed in a solvent, soaked in the solvent, and extracted by standing for 24 hours at room temperature. delete delete delete delete

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