KR101828204B1 - Anti-atopic composition using the extract of cornus officinalis - Google Patents

Abstract

Provided is an anti-atopic composition derived from natural ingredients with outstanding effects of preventing and alleviating atopy. To this end, the anti-atopic composition contains a corni extract, a wild brier extract, an Oriental arbor vitae extract, a Lespedeza bicolor Turcz. extract, and a chestnut leaf extract as active ingredients.

Description

[0001] The present invention relates to an anti-atopic composition using an extract of corn oil,

The present invention relates to an anti-atopic composition, and more particularly to a composition for anti-atopic use using a saponin extract.

The cause of atopic dermatitis is not only hereditary factors but also various kinds such as food, stress, and stimuli caused by external environment, and itchy skin is caused by dry skin at the onset of atopic dermatitis, Causing skin lesions. Although there are various products that treat such atopic dermatitis, there is a growing concern about the safety of chemical products. Recently, the necessity of products containing natural substances has increased without using coloring matters, artificial flavors, surfactants and steroids.

Natural materials mainly use forest products such as mushrooms, sap, herbs, nuts, and fruit, which are produced or harvested in forests. These forest products have been recently used for product development such as food safety. Accordingly, the inventors of the present invention have completed the present invention while developing products having the effect of preventing and reducing atopy of new formulations based on domestic natural materials.

An object of the present invention is to provide a composition for natural atypical atopic dermatitis which is excellent in atopy prevention and abatement effect.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object, an anti-atopic composition according to an embodiment of the present invention comprises an extract of Cornus sylvestris, Bryce's extract, Antrodia camphorata, Cryptomeria japonica extract, and Curcuma longa extract as active ingredients.

The above extract can be obtained by extracting an extraction target with an ethanol solution.

The composition can be obtained by first mixing and extracting the object to be extracted.

Corn seed oil, briquette, lateral bark, sycamore and perennials to be extracted can be formulated in the following weight ratio.

(Sansui): (brioche + cobwebs): (sycamore + yobu) = (1 ~ 2): 1: 1

The details of other embodiments are included in the detailed description.

According to the anti-atopic composition according to the present invention, it is made of a natural material and has no side effects, and has an excellent effect in prevention and reduction of atopy.

Figs. 1 and 2 show chromatograms obtained by analyzing the components of the corn oil extract and the briar extract by LC / MS / MS analysis, respectively.
FIG. 3 is a view sequentially showing extraction steps of the anti-atopic composition according to one embodiment of the present invention.
FIG. 4 is a graph showing the concentration of IFN-y in serum by ELISA for each experimental group.
5 is a graph showing the concentration of IL-4 in serum by ELISA for each experimental group.
FIG. 6 shows photographs of skin lesions induced by DNCB for each experimental group.
7 is a scoring of the skin pathology of the skin.
FIG. 8 is a graph showing the thickness of epithelium stained with H & E in the dorsal skin of each experimental group.
FIG. 9 shows the results of confirming the infiltration of mast cells by staining mouse or other skin tissues with toluidine blue.

The present invention will be described more specifically based on examples and experimental examples. However, these examples and experimental examples are provided only for the understanding of the present invention, and the scope of the present invention is not limited by these examples.

Example  1. Preparation of sample

<1-1> Composition of raw materials

A base material used in the present invention, Cornus officinalis (Cornus officinalis: fruit - were used, except seeds) (A), anti-eczema activity and antioxidant Hagi trees (Lespedeza bicolor for active reinforcement) and yulyeop (Castanea Active reinforcement material Set 1 (C) with crenata leaf (chestnut leaf) and active reinforcement material with bacterium Rosa multiflora and Platycladus orientalis in order to further enhance anti-inflammation / antioxidant and antimicrobial activity (B) were mixed.

<1-2> Extraction of sample

The sample was extracted with 70% ethanol (EtOH) solution. 70% ethanol was added at a rate of 10 times (w / v) per 100 g of the pulverized sample, and the mixture was extracted with a reflux condenser at 80 ° C for 4 hours. Then, the extract was filtered with a filter paper (Whatman, NJ, USA). The filtrate was concentrated under reduced pressure at 70 캜 for 1 hour in a vacuum concentrator, solidified and stored frozen at -70 캜. The solidified raw material was filtered through a filter (0.45 um) before use in the test.

<1-3> LC / MS analysis

The LC / MS / MS analysis was performed to determine the physiological activity of the extracts from the ethanol extracts of corn oil, berry, and berry, and to utilize them for the extraction process. The extract was concentrated under reduced pressure to remove the solvent, and the residue was dissolved in methanol and analyzed by LC / MS / MS (Accela HPLC, LTQ-Velocity trap mass spectrometer, Thermo Fisher Scientific, San Jose, CA, USA).

Separation of the materials was carried out using a solvent A (0.1% formic acid in water) and a solvent B (0.1% formic acid in acetonitrile) in a UPLC-BEH C18 column (1.7 μm, 150 × 2.1 mm id, Acquity, Waters, Milford, Mass. ) At a flow rate of 400 uL / min. Here, the solvent conditions were as shown in Table 1 below, and the column temperature was 36 ° C., and the absorbance at 180-800 nm was measured.

Time (min) Solvent A Solvent B 0 95 5 One 95 5 20 30 70 24 0 100 27 0 100

MS information was collected at 100-1,500 m / z for both positive ion mode ([M + H] ⁺ ) and negative ion mode ([MH] ^- ). The capillary temperature was adjusted to 275 ° C and the source voltage was adjusted to 5 kV. Peak densities and alignments of chromatograms were obtained using SIEVE software (Thermo Fisher Sciencfic, Waltham, Mass., USA). The exact analysis of cool quality was based on the MS mass spectra, UV / visible spectra, MS / MS patterns and various databases.

Example  2. Mixing of raw materials

The raw materials were mixed to optimize the raw materials. When the mixture of A, B and C is a mixture of A, B and C when the mixture of the bark extract and the perennial extract is referred to as "C", the mixture of A, B and C is referred to as " Samples of the composite according to the method are shown in Table 2 below. In order to optimize the extraction of each raw material and simplify the extraction process, optimization was performed for mixing before extraction and after extraction. For example, in the case of 'A + B', in case of 'pre-extraction mixing', the mixture of acid water, briquette and dorsal is firstly extracted, and in case of 'extraction after mixing', a mixture of acid water extract, Can be understood. Hereinafter, the raw materials in which the five plant extracts are mixed is referred to as 'C2RTLC'.

Samples No combination A, B, C Combination after extract A + B, A + C, B + C, A + B + C Combination before extract A + B, A + C, B + C, A + B + C

In order to optimize the mixing ratio of each complex, experiments were carried out by varying the blending ratios of the respective extracts as shown in Table 3 below. (A = extract of Cornus officianalis ; B = combined extracts of Rosa multiflora and Thuja orientalis ; C = combined extracts of Lespedeza bicolor and Castanea crenata leaf). On the other hand, the brieblose extract and the lateral bark extract constituting B can be mixed with each other at various ratios, but preferably they can be composed at the same ratio. Culm tree extract and cotyledon extract constituting C can be mixed with each other at various ratios, but they can preferably be composed at the same ratio.

Compound Compound ratio ABC-1 A: B: C = 4: 1: 1 ABC-2 A: B: C = 4: 2: 1 ABC-3 A: B: C = 4: 1: 2 ABC-4 A: B: C = 4: 2: 2 ABC-5 A: B: C = 4: 2: 2

The aim of this study was to evaluate the antioxidative, anti - inflammatory and anti - degranulation activities of extracts and mixing ratios.

Example  3. Antioxidant activity measurement

&Lt; 3- 1> Electron Difficulty  Measure

The electron donating ability (EDA) was measured by modifying the Blois method. 50 μL of 0.2 mM DPPH (1,1-diphenyl-2-picrylhydrazyl) was added to 100 μL of each sample solution. After stirring for 10 minutes, absorbance was measured at 517 nm. As shown in the following formula 1, the electron donating ability is represented by the absorbance decreasing rate of the addition solution of the sample solution and the non-addition solution.

[Formula 1]

Electron donating ability (%) = (absorbance of the sample addition group) / (absorbance of the no-addition group) x 100

<3-2> ABTS Radical Scatters

The ABTS + radical cation scavenging activity was measured by dissolving 7.4 mM 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and 2.4 mM potassiom persulfate in tertiary distilled water at a ratio of 1: And reacted at room temperature for 15 hours or longer to form ABTS +. The absorbance of ABTS + solution was 0.700 ± 0.005 before dilution with ethanol at 734 nm. 10 μL of methanol-soluble extract was added to 200 μL of the above reaction solution, reacted at room temperature for 6 minutes, and absorbance was measured at 734 nm (VersaMax microplate reader; Molecular Devices). Trolox equivalent antioxidant capacity (TEAC) was calculated by the following equation 2 and calculated by IC ₅₀ .

[Formula 2]

ABTS radical scavenging ability (%) = [(absorbance of untreated sample) - (absorbance of sample added sample)] / (absorbance of untreated sample) × 100

Example  4. Anti-inflammatory efficacy verification

<4-1> Cytotoxicity

The MTT assay was performed to determine the concentration range for the NO assay. RAW264.7 cells (1 × 10 ⁵ cells / mL) were cultured overnight in a 96-well culture plate with 100 μL of DMEM medium, and cultured for 24 hours at various concentrations. 10 μL of 5 mg / mL MTT solution was added to each well, followed by incubation for 3 hours to induce the reduction reaction. 100 μL of DMSO solution was added to completely dissolve the violet formazan crystal. The degree of color development was measured at 570 nm using a microplate reader, and the cytotoxicity was calculated for the relative cell viability of the drug-treated group based on 100% survival of the untreated group only.

<4-2> Measurement of inhibitory activity of NO (nitric oxide)

The amount of NO produced from RAW264.7 cells was measured using the Griess reagent reaction method in the form of NO ^{2 -} present in the cell culture medium. That is, RAW264.7 cells were pretreated with LPS (1 ug / mL) for 1 hour and cultured for 24 hours at various concentrations of the drug. The cell culture medium was collected, and the same amount of Griess reagent (0.1% NED / 1% sulfanilamide in 5% H ₃ PO ₄ ) was added to 100 uL of the culture solution. The degree of color development was measured at 540 nm absorbance of a microplate reader. 100% activity was defined as the secretion amount of the group treated only with LPS, and the relative amount (% of control) was calculated.

Example  5. Anti-atopic  Active measurement

&Lt; 5-1 > hexosaminidase  release Inhibition  evaluation

RBL-2H3 cells were cultured for 24 h at a density of 2 × 10 ⁵ cells / well in a 24-well plate and sensitized with DNP-specific IgE (10 μg / well) for 18 h. The grown medium was replaced with Tyrodes' assay buffer (119 mM NaCl, 5 mM KCl, 5.6 mM Glucose, 0.4 mM MgCl 2, 25 mM PIPES, 40 mM NaOH, 1 mM CaCl 2, 0.1% BSA, pH 7.2). Then, Ig E sensitized cells were cultured for 10 minutes, and the extracts were treated with various concentrations (0-0.3 mg / mL) and incubated for 30 minutes. After treatment, the cells were stimulated with 20 uL of DNP-HSA (10 ug / mL) for 30 minutes and then placed at 4 for 10 minutes to stop the reaction. Cells were centrifuged for 5 min at 12000 × g for 5 min. The supernatant was then equilibrated with 200 μL of substrate solution (4.8 mM p-nitrophenyl N-acetyl-beta- D-glucosamine) and incubated at 37 for 1 h . The enzyme reaction was stopped by adding 400 uL of 0.1 M NaCO3 / NaHCO3 and the reacted product was measured at 405 nm. In order to determine the total amount of β-hexosaminidase, the remaining cells were dissolved in DDW, and the absorbance was measured in the same manner as in determining the activity of the supernatant.

Example  6. DNCB  Preliminary tests for establishing an inducible atopic mouse model

In order to evaluate the efficacy of the test substance on atopic dermatitis, an atopic mouse model was established. This model refers to "Atopic dermatitis" (Biopharmaceutical Bureau, Korea Food and Drug Administration, 2016) guideline for the efficacy test of herbal medicine and herbal medicine.

&Lt; 6-1 >

Specific pathogen free male BALB / c mice (Orient) at 5 weeks of age were purchased and aseptically administered. During the experimental period, the temperature of the breeding room was maintained at 21-23 ℃, the humidity was around 60%, and the photoperiod and cancer cycle were adjusted to 12 hours. Prior to this experiment, three mice were assigned to each of the groups shown in Table 4 below in order to establish a DNCB inducible atopic mouse model, and a preliminary experiment was conducted once. Each group had a purge period of one week.

Group Treatment Normal control (Con) Saline solution Negative control (NC) DNCB-induced atopic dermatitis with no treatment Positive control (PC) DNCB-induced atopic dermatitis with 0.1% of Tacrolimus treatment

<6-2> DNCB  Manufacturing and Atopy Induction

In this experiment, DNCB (Sigma, St, Louis, MO) solution was diluted to 2.5% and 1% in 3: 1 mixture of acetone and olive oil. After 1 week of adaptation to the environment, the back of the test animal was epilated from the neck to the pelvis the day before application of the test substance, and left to stand for 24 hours to heal the wound of the epilation. After applying the 2.5% DNCB solution to the skin of the neck of the mice, 150 μL was applied to the back area for 4 days after inducing the primary immunity reaction to induce atopic dermatitis. In order to prevent errors caused by natural healing, Week.

<6-3> Sample collection

Two weeks after the start of the test, the mice were fasted for 12 hours and anesthetized with CO2. One ml of blood was collected from the heart using a syringe and subjected to CBC at 100 μL, and the remaining 900 μL was centrifuged at 4 to 18,000 rpm for 10 minutes The plasma was separated and stored at -70.

Skin tissue such as in the area then after sacrificing the mouse by cervical dislocation cardiac blood collection 1 × ² cm 2 Collected by cutting to size and, 1 × 1 cm ² Were used to measure histopathology and cytokines.

<6-4> Measurement of blood cell count in blood

The blood obtained from cardiac puncture was placed in an EDTA tube and a common blood cell (CBC) was performed using an automatic full digiital cell counter (Cergy-Pontoise, France).

<6-5> Immunoglobulin  E (Ig E) measurement

On the sacrifice day, the blood obtained from cardiac puncture was centrifuged at 4 to 1,800 rpm for 10 minutes, and plasma was separated and stored at -70. Immunoglobulin E was measured by ELISA kit (Shibayagi, Japan). The analytical method was performed according to the manufacturer's instructions, and the method is as follows. The plate coated with antibody is washed three times, and 50 μL of Ig E standard solution and sample are dispensed. Reaction is carried out at room temperature for 2 hours. After washing again, 50 uL of biotin-conjugated anti-IgE antibody was dispensed, reacted for 2 hours, and washed. HRP-avidin was reacted at room temperature for 1 hour, washed with chromogenic substrate solution, incubated for 20 minutes, stoppered, and absorbance was measured at 450 nm using a tunable microplate reader (Molecular device, US) .

<6-6> Measurement of Histamine Change in Serum

On the day of sacrifice, the blood obtained from cardiac puncture was centrifuged at 4 to 1,800 rpm for 10 min. After the plasma was separated and stored at -70, an ELISA kit (IBL HAMBURG, Germany) was used to measure histamine changes Respectively.

&Lt; 6-7 > Cytokine ( Cytokine )

Immediately after sacrifice, 1 × 1 cm ² of dorsal skin tissue was sampled, pulverized in 5 ml of phosphate buffer containing protease inhibitor, and centrifuged at 20,000 rpm for 30 minutes. The amount of cytokine was quantified by using the supernatant in IL-1β (BIOSOURCE, Europe SA) and IFN-γ (KOMA BIOTECH, Korea) ELISA.

<6-8> Visual pathology , Weighing and Histopathology

On the day of sacrifice, the skin parenchyma was measured and the weight of the skin tissue of 1 × 1 cm ² size was measured. The histopathology of mouse skin was commissioned to the Department of Veterinary Pathology, Chungnam National University.

<6-9> DNCB  Establishment of an inducible atopic mouse model

For the establishment of the DNCB inducible atopic mouse model, a preliminary experiment was conducted by setting the control group (Con), DNCB induction group (negative control group), DNCB induction group + 0.1% tacrolimus treatment group (positive control group: PC) . In this preliminary experiment, pathological observations of dietary intake, body weight change, blood analysis (leukocyte, eosinophil, basophil and IgE change), skin gross lesion, and skin lesion were evaluated by DNCB for atopic dermatitis It was confirmed to be significant as the efficacy evaluation item.

Example  7. Development of Prototype

In order to develop a prototype of the plant extract (C2RTLC) of the present invention as a raw material (hereinafter referred to as "PT-C2RTLC"), a cream preparation with tracer vaseline was designed.

<7-1> C2RTLC  Establish extraction process and selection of indicator material

We used the C2RTLC extract and its base to control the quality of the prototype to be prepared and to establish the extraction process and select the indicator material in order to obtain consistent efficacy in the future.

<7-2> C2RTLC  Solubility assessment

The solubility of C2RTLC was evaluated to design a cream with optimal emissivity. Solubility was measured by using purified water, ethanol, acetonitrile, methanol and the like as a solvent in order to select an appropriate test solution in the release test. Excess C2RTLC was added to the test solution. After stirring for 12 hours, the test solution was filtered, and the solubility of C2RTLC was evaluated with an ultraviolet absorption spectrophotometer.

<7-3> C2RTLC  Manufacture of cream formulations containing

To prepare a cream containing C2RTLC, C2RTLC was dissolved at 65 ° C. The water phase was mixed with white vaseline (Sigma) and sufficiently stirred at 65 ° C using a homogenizer. Stirring was continued until cooling to 30 캜 and solidification was carried out to prepare a W / O cream.

Example  8. Acute Oral Toxicity Assessment of Prototype

This test was conducted to determine the pattern and intensity of the toxicity observed in the Acute Toxic Class Method (ATC method, OECD TG 423) of the acute oral toxicity test for SD female rats against the prototype (PT-C2RTLC) . An autopsy (PT-C2RTLC) was performed at a dose of 2,000 mg / kg bw in the stomach for 14 days after the intramuscular injection, and the autopsy was carried out after the lethal rate, weight change, clinical symptoms and observation period for the test substance.

<8-1> Preparation of test substances

This test substance was used as it was without any preparation. On the day of the test, the weight of the test substance was weighed to prepare 2000 mg / 20 ml and then used according to the dosage. After the test, the remaining test material was discarded.

<8-2> Test system

- Strains and species: NTac: Sprague-Dawley Rat, SPF

- Supplier: Orient Co., Ltd.

- sex, number and age of animals (when available): 6 females (5 weeks old, 156.8 ± 5 g)

(5 weeks of age, 157 ± 1.0 g), Step 2: 3 females (5 weeks of age, 156.7 ± 7.6 g)

<8-3> Breeding environment

The environment of this experiment was set up at environmental temperature of 22 ± 3, relative humidity of 50 ± 20%, ventilation frequency of 10-15 times / hour, illumination period of 12 hours, illumination of 150-300 Lux, veterinary pharmacology physiology laboratory .

The breeding box is made of stainless steel mesh lid with polycarbonate breeding box (270x500x200mm). Three animals were housed during the refinement period. Betachip irradiated with gamma rays was used as the rug.

Feeds were fed free of animal feed rats and water was fed free of ultraviolet sterilized filtered water (R / O water).

<8-4> Test method

The composition of the test group is shown in Table 5 below.

Test step Dose
(mg / kg bw) castle Animal number
(Two) Death First-line treatment group 2,000 female 1-3 (3) none Second-tier administration group 2,000 female 1-3 (3) none

In this study, the limit dose of 2,000 mg / kg bw was set as the initial dose and the next dose was administered after observing the death for up to 48 hours.

Since it is a product to be applied to humans, first oral toxicity was administered, and the administration frequency was once / once a day. The dose (mg / kg) was calculated based on the weight on the day of administration. After 12-16 hours of fasting (except for the negative water) the day before administration, the prepared test substance was forcibly administered once in the stomach using oral administration sonde. Animals were fed to the test animals 4 hours after administration.

The test was conducted according to the Acute Toxic Class Method (ATC method, OECD TG 423) of the Acute Oral Toxicity Test (RCCA Notice 2013-21, 2013.6.28, Attachment 12 " (II), 2008. 11, Korea Food and Drug Administration).

The dose of the initial test was set to 2000 mg / kg bw in the first step, and the test substance was administered to three females. After observation of death or absence of vomiting for 48 hours after administration, all three animals survived and were re-administered 2000 mg / kg bw to 3 other females. In the second stage 2000 mg / kg bw dose, all three mice survived for 48 hours, so the next dose level was not administered.

<8-5> Observation items

a) Observation of general symptoms

All animals were intensively observed from 30 minutes to 4 hours after administration, and general symptoms were observed until 1 day / day 14 days. The mortality and clinical symptoms were recorded on an individual basis.

b) Weight measurement

All animals were measured immediately before dosing (on the day of dosing), on days 3, 6, 9, 10 and 14 after dosing.

c) Autopsy

At the end of the experiment, all surviving animals were euthanized by CO ₂ gas aspiration method, and the organ was visually examined at the end of the test.

d) lethal dose (LD ₅₀ )

Half lethal dose was calculated by referring to this test step flow chart of toxicity grade method.

Example  9. Evaluation of efficacy of prototype using atopic mouse model

<9-1> Experimental animals

Specific pathogen free male BALB / c mice (Orient) at 5 weeks of age were purchased and aseptically administered. During the experimental period, the temperature of the breeding room was maintained at 21-23, the humidity around 60%, and the photoperiod and cancer cycle were adjusted to 12 hours. The experimental group was divided into five groups as shown in Table 6 below.

Group Treatment Normal control (Con) Without atopy and treatment Negative control (NC) DNCB treatment + vehicle (Vaseline) 2.5% TG DNCB treatment + 2.5% PT-C2RTLC ointment treated 5% TG DNCB treatment + 5% PT-C2RTLC ointment treated Postive control (PC) DNCB treatment + betamethasone cream treated

That is, a normal control (Con) not coated with 1-chloro-2,4-dinitro-benzene (DNCB), a negative control (NC) coated with DNCB and a DNCB were applied, (PT-C2RTLC) treatment group (TG), DNCB and betamethasone-treated positive control (PC). Each group had a purge period of one week.

<9-2> NCB  Manufacturing and Atopy Induction

In this experiment, DNCB (Sigma, St, Louis, MO) solution was diluted to 1% and 0.2% in 3: 1 mixture of acetone and olive oil. After 1 week of adaptation to the environment, the back of the test animal was epilated from the neck to the pelvis the day before application of the test substance, and left to stand for 24 hours to heal the wound of the epilation. After applying the 2.5% DNCB solution to the skin of the mouse neck, 4 days after inducing the primary immunity, 150uL was applied to the back area to induce atopic dermatitis. In order to prevent errors caused by natural healing, 2 times for 2 weeks or the like

<9-3> Measurement of blood cell count in blood

Blood collected through cardiac puncture was placed in an EDTA tube and a common blood cell was measured using an automated hematology analyzer (URIT medical electronic, China).

&Lt; 9-4 > Cytokine )

Immediately after sacrifice, 1 × 1 cm ² of dorsal skin tissue was sampled, pulverized in 5 ml of phosphate buffer containing protease inhibitor, and centrifuged at 20,000 rpm for 30 minutes. The amount of cytokine was quantified by using the supernatant in IL-1β (BIOSOURCE, Europe SA) and IFN-γ (KOMA BIOTECH, Korea) ELISA.

On the day of sacrifice, the blood obtained through cardiac puncture was centrifuged at 4 ° C at 1,800 rpm for 10 minutes to separate plasma and stored at -70 ° C. The amount of cytokine was determined by ELISA using IFN-γ, IL-2, IL-4, IL-5, IL-6 and IL-13 (KOMA BIOTECH, Korea).

<9-5> Visual pathology , Weighing and Histopathology

Based on the following items on the day of sacrifice, the visual evaluation result was expressed as the total sum after evaluating each of the following items, assigning the scores.

(1) excoriation / erosion, (2) scarring / dryness, (3) edema, (4) excoriation / erosion, 0 (none) , 1 (mild), 2 (moderate), and 3 (severe). Additionally, the weight of the size 1 × 1 cm ² tissue was measured. Histopathologic analysis of the resected mouse skin was also performed.

Reference example  1. Statistical Analysis

All data analyzes were performed using Statistical AnalysisSystem (SAS, Ver. 8.01). Results were expressed as mean and standard deviation (SD). The significance of the mean between the experimental groups was verified at the p <0.05 level by the ANOVA test.

Experimental Example  1. Sample extraction

Figs. 1 and 2 show chromatograms obtained by analyzing the components of the corn oil extract and the briar extract by LC / MS / MS analysis, respectively. In both types of extracts, more diverse peaks were detected in the positive ion mode than in the negative ion mode.

Loganin, Cornuside and Naringenin-7-O-β-D-glucoside were detected in the ethanol extract of corn oil. Loganin is known as a plant metabolite and is presented as an indicator component of marine oil that is registered as a standard herbal medicine by the Food and Drug Safety Agency. Cornuside and Naringenin-7-O-β-D-glucoside have been shown to have various physiological activities. Among them, anti-inflammatory activity and antimicrobial activity directly related to anti-atopic activity were also confirmed. Therefore, various anti-atopy physiologically active ingredients are contained in the ethanol extract of corn oil.

Gallic acid, Glucopyranosyl methyl gallate and Methyl gallate were detected in the negative ion mode of the berry ethanol extract and flavonoid compounds such as Ellagic acid, Quercetin and Kaempferol were detected in positive ion mode. Therefore, it can be seen that the antioxidant activity of the briar extract is excellent.

Experimental Example  2. Optimization of raw materials

In this specification, 'A' represents the extract of the base material (except for the fruit juice seeds), 'B' represents the extract of the active reinforcing material Set 2 (briefer, lateral), 'C' represents the active reinforcing material Set 1 , And yellows).

<2-1> Antioxidant activity evaluation

Table 7 below shows the electron donating ability of various samples. As shown in Table 7, when the three samples were mixed (A + B + C), they were excellent in terms of efficacy, and the extracts after mixing were 10 times more effective (DPPH assay) Respectively. Therefore, in order to increase the antioxidant activity, it is preferable to mix the three samples (A + B + C) and to mix them after extraction.

Sample Combination / extract IC ₅₀ (mg / mL) A No combination 0.338 B No combination 0.345 C No combination 0.105 A + B Combination after extract 0.098 A + C Combination after extract 0.032 B + C Combination after extract 0.035 A + B + C Combination after extract 0.056 A + B Combination before extract 0.085 A + C Combination before extract 0.064 B + C Combination before extract 0.010 A + B + C Combination before extract 0.005

<2-2> Evaluation of anti-inflammatory activity

Table 8 below shows the NO inhibiting activity of various samples. Referring to Table 8, when the three samples were mixed, LPS-induced NO production was inhibited in a concentration-dependent manner. When comparing the mixture before extraction with the mixture after extraction, the effect of mixing before extraction was confirmed to be about 3 times. Therefore, it was confirmed that the anti - inflammatory activity of the pre - extraction mixture was superior to that of the post - extraction mixture.

Some of the two samples were found to have excellent anti - inflammatory activity. However, considering both antioxidant activity and anti - inflammatory activity, the pre - extraction method is more effective than the other three methods.

Sample Combination / extract IC ₅₀ (mg / mL) A + B Combination after extract 8 A + C Combination after extract 758 B + C Combination after extract 2.9 A + B + C Combination after extract 512 A + B Combination before extract 3.2 A + C Combination before extract 221 B + C Combination before extract 665 A + B + C Combination before extract 152

<2-3> Anti-atopic  Activity evaluation

Table 9 below shows the inhibition of β-hexosaminidase release. The activity of anti-atopy is indirectly confirmed by confirming the inhibition of β-hexosaminidase secretion in mast cells. In the case of mixed samples, inhibition pattern of β-hexoxaminidase secretion was observed only in the mixture of A + B and A + B + C. In case of mixing three samples, the effect was more than doubled. When the mixtures were compared before and after extraction, the pre - extraction mixing was somewhat higher than the post - extraction mixing.

Sample Combination / extract IC ₅₀ (mg / mL) A + B Combination after extract 2140 A + C Combination after extract > 3000 B + C Combination after extract > 3000 A + B + C Combination after extract 73.21 A + B Combination before extract 136.8 A + C Combination before extract > 3000 B + C Combination before extract > 3000 A + B + C Combination before extract 51.56

Experimental Example  3. Optimization of mixing ratio

The mixing ratio of the combined extract (A + B + C) using the three samples mentioned in Table 3 was optimized. After mixing, antioxidant, anti-inflammatory and anti-atopic activities were measured.

<3-1> Evaluation of antioxidant activity

Table 10 below shows the measurement of electron donating ability according to the mixing ratio. In particular, the IC ₅₀ of ABC-4 (A: B: C = 4: 2: 2) and ABC-5 (A: B: C = 4: 2: 2) were 211.9 and 169.1 ug / Antioxidant activity was found to be up to 2.5 times higher.

Compound Compound ratio IC ₅₀ (ug / mL) ABC-1 A: B: C = 4: 1: 1 538.9 ABC-2 A: B: C = 4: 2: 1 401.9 ABC-3 A: B: C = 4: 1: 2 381.2 ABC-4 A: B: C = 4: 2: 2 211.9 ABC-5 A: B: C = 4: 2: 2 169.1

<3-2> Evaluation of anti-inflammatory activity

Table 11 below shows the NO inhibitory activity measured according to the mixing ratio. In particular, the IC ₅₀ of ABC-4 (A: B: C = 4: 2: 2) and ABC-5 (A: B: C = 4: 2: 2) were induced by LPS at 823 and 176 ug / It was confirmed that the production of NO was effectively suppressed.

Compound Compound ratio IC ₅₀ (ug / mL) ABC-1 A: B: C = 4: 1: 1 > 1000 ABC-2 A: B: C = 4: 2: 1 > 1000 ABC-3 A: B: C = 4: 1: 2 > 1000 ABC-4 A: B: C = 4: 2: 2 823.5 ABC-5 A: B: C = 4: 2: 2 172.6

<3-3> Anti-atopic  Activity evaluation

Table 12 below shows the inhibition of β-hexosaminidase release according to mixing ratio. In particular, ABC-4 (A: B: C = 4: 2: 2) was found to be three times more potent than ABC-5 (A: B: C = 4: 2: 2) .

Compound Compound ratio IC ₅₀ (ug / mL) ABC-1 A: B: C = 4: 1: 1 722 ABC-2 A: B: C = 4: 2: 1 > 3000 ABC-3 A: B: C = 4: 1: 2 987.3 ABC-4 A: B: C = 4: 2: 2 95.25 ABC-5 A: B: C = 4: 2: 2 225.6

<3-4> Sintering

As a result of the optimization of the mixing method and the mixing ratio through the above-mentioned experiments, it was confirmed that 'pre-extraction mixing' is preferable considering antioxidative activity, anti-inflammatory activity and anti-atopic activity. The mixing ratio was (1 to 2): 1: 1 (1 to 2) for the raw material (A), the briquette and the lateral bark (active reinforcement materials Set-2 and B) , Preferably 2: 1: 1. Here, the brieblose extract and the progeny extract constituting B may be mixed with each other at various ratios, but they may preferably be composed at the same ratio. Culm tree extract and cotyledon extract constituting C can be mixed with each other at various ratios, but they can preferably be composed at the same ratio.

Through the above experiments, an effective extraction process including extraction method, extraction time, concentration process, and solidification process for prototype production was established. FIG. 3 is a view sequentially showing extraction steps of the anti-atopic composition according to one embodiment of the present invention.

Experimental Example  4. Acute Oral Toxicity Assessment of Prototype

The Acute Toxic Class method test was performed on the SD female rats to determine the toxicity and toxicity of the protozoal when given once orally in the stomach. SD rats were treated with the test substance at a dose of 2,000 mg / kg bw at a single oral dose of 1, 2, and 14 days. After 14 days, death, And the following results were obtained.

a) The prototype was administered to three female rats at a dose of 2,000 mg / kg bw (step 1), and 2,000 mg / kg bw was again administered to three female rats (stage 2) No deaths were observed in all.

b) No specific clinical symptoms were observed from the administration to the end of the observation period at both 2000 mg / kg bw in the first and second phase groups.

c) No weight change associated with the test substance was observed after the prototype administration.

d) After the observation period, gross examination by autopsy was performed on the surviving animals of the 2,000 mg / kg bw group, and no abnormality was observed in all individuals.

e) The approximate lethal dose (LD ₅₀₎ of the prototype was found to be 2000 to 5000 mg / kg bw.

From these experimental results, the prototype was identified as a safe substance as GHS Category 5 (LD ₅₀ cut-off value: 2000 mg / kg bw).

Experimental Example  5. Evaluation of efficacy of prototype using atopic mouse model

The effect of improving the atopic dermatitis of the prototype (PT-C2RTLC) was evaluated for the five experimental groups shown in Table 6 above.

<5-1> Blood analysis (CBC analysis)

Table 13 below shows the blood leukocyte counts of atopic blood cells measured in mice of five experimental groups. The number of white blood cells (WBCs) was significantly reduced in the control group (NC) compared to the control group (PT-C2RTLC) (2.5% TG and 5% TG). This was similar to that of the positive control group (PC) and the normal control group (Con), which were treated with betamethasone.

NC 2.5% TG 5% TG PC Con WBC 8.85 ± 1.34 5.2 ± 1.13 4.75 ± 1.63 4.35 ± 1.2 3.25 ± 0.64 Lymphocyte 0.65 0.35 1.15 + 0.07 0.3 ± 0.14 0.55 + 0.07 0.1 ± 0.00 Granulocyte 4.55 + 1.48 2.7 ± 0.07 2.3 ± 1.13 2.85 0.35 2.9 ± 0.56

<5-2> Measurement of serum immunocytocyte change

FIG. 4 is a graph showing the concentration of IFN-y in serum by ELISA for each experimental group. Referring to FIG. 4, the concentration of IFN-y was significantly higher in the NC group than in the Con group. In contrast, the level of IFN-γ was lower in the group treated with 2.5% or 5% prototype (PT-C2RTLC) (ie, 2.5% TG and 5% TG) and betamethasone Respectively. In particular, the level of IFN-γ in the 2.5% TG group was similar to that in the PC group, and the level of IFN-γ in the 5% TG group was similar to that of the Con group.

5 is a graph showing the concentration of IL-4 in serum by ELISA for each experimental group. IL-4 is a typical cytokine associated with Th2 cell response in combination with IL-5, IL-6, and IL-13 and stimulates B-cell to participate in IgE production, mucin secretion, mast cell and eosinophil activity. Serum levels of IL-4 were the highest in NC group and significantly lower in Con group, 2.5% TG group, 5% TG group and PC group.

<5-3> Visual pathology  And weighing

The skin condition was checked by visual inspection once every 6 days and scored as 0-3 according to the item of skin symptom. FIG. 6 shows photographs of skin lesions induced by DNCB for each experimental group. Immediately after applying DNCB to the skin, major clinical signs and symptoms began to appear. Repeated DNCB treatment showed moderate erythema, hemorrhage, and occasional edema in the posterior period after skin dryness. The Con group had normal skin findings, covering the majority of the back skin with full hair growth. On the contrary, in the NC group, the scalp was partially formed, and the hair did not grow completely. In the case of the PC group, the hair was almost grown without the skin. DNCB-induced skin lesions were improved in a dose-dependent manner in the anti-atopic composition administration group of the present invention. In particular, in 5% PT-C2RTLC administration group (5% TG) Growth of hair was confirmed.

7 is a scoring of the skin pathology of the skin. Particularly, in the PC group after the administration, 2.5% or 5% TG group showed improvement of the skin symptom from the 12th day to 6.63, 5.89, and 5.25, respectively, Respectively. In particular, the 5% TG group was found to improve DNCB - induced skin symptoms at a level similar to that of betamethasone, a steroidal drug used as a dermatitis treatment.

<5-4> Skin Histopathology

In order to confirm the effect of the anti-atopic composition or prototype (PT-C2RTLC) of the present invention on DNCB induced atopic dermatitis in mice, a part of the skin after sacrifice at the end of the experiment was collected and subjected to H & E (hematoxylin & eosin) Respectively. FIG. 8 is a graph showing the thickness of epithelium stained with H & E in the dorsal skin of each experimental group. Referring to FIG. 8, in the skin histopathological findings, the thickness of skin tissue of the negative control group (NC) was significantly increased compared to the thickness of the normal control group (Con) (13.26 +/- 0.47 um) due to the growth of pidermis. On the other hand, the thickness of the epidermis in the 2.5% TG group was 51.85 ± 4.53 μm and the thickness of the epidermis in the 5% TG group was 31.68 ± 3.04 μm. The infiltration of inflammatory cells such as leukocytes in the negative control group (NC) was relatively decreased in the PT-C2RTLC-treated group.

On the other hand, mast cells that secrete histamine can be stained with toluidine blue. FIG. 9 shows the results of confirming the infiltration of mast cells by staining mouse or other skin tissues with toluidine blue. Referring to FIG. 9, mast cells were highly infiltrated around the dermis of the negative control group (NC). In contrast, the infiltration of mast cells in the dermis was significantly decreased in the 2.5% TG and 5% TG groups. This was similar to that of the positive control group (PC), which was treated with betamethasone.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, You will understand. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (4)

The present invention relates to an anti-atopic composition comprising as an active ingredient an extract of corn oil, a briebl extract, a horsetail extract, a horsetail extract, a curd leaf extract, and an anti-atopic composition comprising an extract of corn oil, briquette, Atopic composition.
(Sansui): (brioche + cobwebs): (sycamore + yobu) = (1 ~ 2): 1: 1 The anti-atopic composition according to claim 1, wherein the extract is obtained by extracting an extract from an ethanol solution. The anti-atopic composition according to claim 1, wherein the composition is obtained by first mixing and then extracting an object to be extracted. 2. The anti-atopic composition according to claim 1, wherein the briebl extract and the progeny extract are constituted in the same proportion, and the cypress extract and the yeast extract are composed in the same ratio.

Images