KR100840391B1 - A pharmaceutical agent for ameliorating symptoms of atopic dermatitis containing refining wood vinegar - Google Patents

Abstract

The present invention relates to a medicament for improving atopic dermatitis symptoms containing purified wood vinegar. According to the present invention, it comprises 2 to 4% by weight of acetic acid, 0.05 to 0.15% by weight of formic acid, 0.05 to 0.15% by weight of propionic acid, 1 to 3% by weight of wormwood components, 0.01 to 1% by weight of sodium acetate and the remaining amount of purified water Provided is a medicament for improving atopic dermatitis symptoms comprising purified wood vinegar as an active ingredient. Purified wood vinegar of the present invention has excellent bactericidal activity, excellent antimicrobial and anti-pruritic properties, no harmful substances, no toxicity, no odor, and particularly effective in improving atopic dermatitis symptoms.

Crude wood liquor, wood vinegar, phosphoric acid, hydrogen peroxide, acetic acid, peracetic acid, methanol, formic acid

Description

A pharmaceutical agent for ameliorating symptoms of atopic dermatitis containing refining wood vinegar}

1 shows a synthetic route diagram of the purified wood vinegar of the present invention,

Figure 2a is a photograph showing the antimicrobial effect of the purified wood vinegar of the present invention,

Figure 2b is a photograph showing the antimicrobial effect of ethanol as a comparative example,

3 is a graph showing the change over time of plasma IgE levels in NC / Nga mice after administration of the test substance,

4 is a graph showing DNCB-induced enhancement of IgE levels in NC / Nga mice after test substance administration.

5 is a graph showing changes in plasma IgE levels after application of test substances in mice with DNCB-induced atopic dermatitis,

6 is a graph showing the ratio of the plasma IgG2a concentration to the IgG1 concentration after applying the test substance,

7 is a graph showing the ratio of IFNγ level to IL-4 level in culture supernatants of splenic T cells stimulated with anti-CD3 polyclonal activator after test substance application.

8 is a graph showing the reduction ratio of the scratching frequency for 30 minutes before and after application of the test substance,

9 is a graph showing the number of skin mast cells after application of the test substance, and

10 is a graph showing COX-1 expression in the skin of DNCB-sensitized / challenged mice after test substance application.

The present invention relates to a drug for improving the symptoms of atopic dermatitis containing purified wood vinegar, and more particularly, atopy dermatitis containing tableted wood vinegar with the pH of which is removed by using wormwood powder and pH adjusted using sodium acetate. It relates to a drug for improving symptoms.

Crude vinegar is a liquid obtained by aging and refining a liquid obtained by cooling smoke generated during carbonization of wood for a long time. The main component is acetic acid, and various organic acids such as furfuryl acid; It is a mixed composition containing alcohols, such as methanol and ethanol.

In other words, when wood is heated to 300-400 ℃ in a place where there is little air, such as charcoal kiln, carbonization occurs and white smoke is generated. The liquid obtained by cooling the smoke generated at this time is crude wood vinegar, and the crude wood vinegar contains harmful substances such as tar, methanol, phenol and benzopyrene.

Wood vinegar has excellent ability to remove free radicals in cells, and it is known to be effective for various diseases such as atopic skin, allergy and acne, diabetes, constipation, indigestion, athlete's foot, otitis media, cancer, etc. Or drinking together.

However, as described above, crude wood vinegar contains harmful ingredients such as tar, phenol, methanol, cresol, benzopyrene and the like, and its application is limited due to the intrinsic charcoal. Therefore, there is a need for purification and deodorization of crude wood vinegar to make it suitable for application directly to the skin or for use as an additive in food, beverages and cosmetics.

For example, Korean Patent No. 212472 discloses a method of distilling at 95-105 ° C. and removing methanol and phenol from crude wood vinegar using activated carbon.

Korean Patent Application No. 2001-61278 discloses a method for producing a drinking wood vinegar by aging and distilling using non-leaflets, but it is inefficient to require long-term aging of 6 months or more.

Korean Patent Application No. 2001-9198 discloses a reduced pressure distillation purification method of wood vinegar comprising distilling at a very low pressure of -40 to -7 atm, but it requires a considerable cost to maintain the low pressure as described above. There is a problem.

In addition, wood vinegar is widely used for the treatment or improvement of atopic dermatitis, but currently used wood vinegar is burnt and the acidity (about pH 2.2 ~ 2.8) of wood vinegar is irritating to sensitive skin such as children and women. There is a problem that can be.

Accordingly, an object of the present invention is to provide a drug for improving atopic dermatitis symptoms containing purified wood vinegar solution from which harmful components and charcoal are removed.

According to the present invention, there is provided a medicament for improving atopic dermatitis symptoms containing purified wood vinegar.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

Crude wood vinegar obtained by cooling the smoke generated during the carbonization of wood contains carbon and harmful substances from carbonization. Therefore, in the present invention, by using a catalyst, effective organic acid is formed from the acid and alcohol components contained in a large amount of wood vinegar, which has excellent bactericidal activity and uses purified wood vinegar from which harmful substances and coal are removed.

The crude wood vinegar is mostly composed of various acids including acetic acid, perpriic acid and the like, and alcohols such as methanol and ethanol. In the present invention, crude wood vinegar containing various acids and alcohols is used as a starting material.

According to the present invention, among the various components in the crude wood vinegar, acetic acid and meol are converted into effective organic acids, which are peracetic acid and formic acid, respectively, and the crude wood vinegar is purified by the adsorption power of formic acid. 1 shows a synthetic route diagram of the crude wood vinegar purification method of the present invention.

The crude wood acetate is reacted with hydrogen peroxide so that acetic acid and methanol in the crude wood component are oxidized to an effective organic acid such as peracetic acid and formic acid, and is reacted at 30 to 70 ° C, preferably 50 to 70 ° C, in the presence of phosphoric acid. Hydrogen peroxide solution is used as oxidant and phosphoric acid as catalyst.

When crude wood vinegar is reacted with hydrogen peroxide solution in the presence of phosphoric acid at 30-70 ° C., acetic acid in wood vinegar is oxidized to peracetic acid and methanol is formic acid as shown in Chemical Formula 2 below. In the oxidation reaction, a small amount of furfuric acid contained in crude wood vinegar is converted to furfural acetic acid, phenol to carbolic acetic acid and ethanol to acetic acid, respectively. Furyl acetic acid and carboacetic acid are present in traces, removed in a subsequent distillation step and acetic acid is oxidized to peracetic acid.

As shown in Scheme 1, acetic acid is oxidized to peracetic acid and methanol is formic acid as shown in Scheme 2 below. In the oxidation reaction, a small amount of furfuric acid contained in crude wood vinegar is converted to furfural acetic acid, phenol to carbolic acetic acid and ethanol to acetic acid, respectively. Furyl acetic acid and carboacetic acid are present in traces, removed in a subsequent distillation step and acetic acid is oxidized to peracetic acid.

Scheme 1

CH ₃ COOH + H ₂ O _2- > CH ₃ COOOH + H ₂ O

Scheme 2

CH ₃ OH + H ₂ O _2- > HCOOH + 2H ₂ O

The addition of hydrogen peroxide (H ₂ O ₂ ) to the resulting carboxylic acid provides the effect of supplying active hydrogen (H ₂ ) with a strong reducing effect and oxygen (O ₂ ) with a high electronegativity, Forming a dimer of a stable hydrogen bond such as (dimer) exhibits a high bactericidal power of medicinal activity and reduces the active oxygen by the active hydrogen to reduce side effects.

[Formula 1]

Acetic Dimer Structure

[Formula 2]

Formic Acid Dimer Structure

[Formula 3]

Propionate Dimer Structure

Peracetic acid has excellent removal ability and inhibitory activity against fungi and molds, and formic acid has excellent insecticidal properties. Therefore, according to the purification method of the present invention, the wood vinegar solution obtained by oxidizing acetic acid to peracetic acid and methanol to formic acid has excellent sterilization, insecticide and Antifungal effect shows antibacterial effect.

In addition, formic acid (HCOOH) is an acid that acts to aggregate rubbery substances and organic proteins, such as tar, phenol, cresol, benzopyrene, etc. contained in crude wood vinegar by formic acid formed by the reaction of Scheme 2. Impurities and the like containing harmful components are aggregated into a carbonized suspension, and the aggregated carbonized suspension is easily removed by filtration. As the carbonized suspension is coagulated by the formic acid, the coal is also simultaneously removed.

The oxidation reaction using hydrogen peroxide solution of acetic acid and methanol is performed at about 30-70 degreeC, Preferably it is 50-70 degreeC. The reaction takes a long time when the reaction temperature is less than 30 ℃, and if it exceeds about 70 ℃ low boiling alcohol and organic solvents are volatilized is not preferred.

In addition, the oxidation reaction is performed for about 8-12 hours. When the reaction time is less than 8 hours, acetic acid and methanol are not sufficiently oxidized with peracetic acid and formic acid, and when the reaction is carried out for about 12 hours, the oxidation reaction according to Chemical Formulas 1 and 2 proceeds sufficiently and the reaction does not proceed any further. Reacting over time is uneconomical.

After the oxidation reaction, the mixture is left for about 36 to 40 hours, and the carbonized suspension contained in the crude wood vinegar is agglomerated by formic acid during the time of standing. If the standing time is less than 36 hours, the carbonized suspension is not sufficiently agglomerated, and if it exceeds 40 hours, the carbonized suspension is sufficiently agglomerated by being left for about 36 to 40 hours.

Hydrogen peroxide used as oxidant in the process of the present invention can be used in stoichiometric amounts to measure the content of acetic acid and methanol in the wood vinegar and they can be sufficiently converted to peracetic acid and formic acid according to Schemes 1 and 2. However, in the actual reaction, the reaction may be added in a somewhat excessive amount, for example, about 5-10% by weight, so that the reaction proceeds sufficiently, but is not limited thereto. For example, 2.5 to 4.0 g of hydrogen peroxide per 100 g of crude wood vinegar. Water can be added.

Phosphoric acid as a catalyst may be used in a small amount, but is not limited thereto. For example, 0.1 to 2.5 g of phosphoric acid may be used per 100 g of crude wood vinegar. If less than 0.1g of phosphoric acid is used, the reaction efficiency is lowered, and if it is used more than 2.5g, the efficiency of reaction is not economically improved.

The aggregate of the carbonized suspension formed by aggregation by formic acid is filtered off as a solid. That is, tar, phenol, cresol, benzopyrene, etc., which are harmful components in wood vinegar, are not only removed in the form of aggregates, but also in the coal.

The highly purified high-purity wood vinegar from which impurities are removed is obtained by first distilling the wood vinegar solution from which the carbonized suspension is removed at about 90-120 ° C. for about 3.5-4.5 hours. The primary distillation is distilled at about 90-120 ° C. for about 3.5-4.5 hours, considering the boiling point of peracetic acid and the boiling point of formic acid. Preferably the distillation is carried out until the yield reaches about 60%. By distillation, peracetic acid, formic acid, and the like are volatilized, and it is cooled and taken to obtain purified wood vinegar. Furfural acetic acid and carboacetic acid produced during the oxidation reaction have a higher boiling point than distillation temperature and are thus removed without distillation.

On the other hand, after removal of the carbonized suspension formed by leaving the oxidation reaction, by adding an adsorbent to further remove the remaining impurities and / or unknown toxic substances such as heavy metals in the treated wood vinegar to remove the impurities by additional adsorption. Can be. As the adsorbent, any material capable of adsorbing and removing impurities and / or heavy metals may be used. However, without limitation, charcoal and / or activated carbon powder may be used in an amount of 3 to 5% by volume based on the volume of wood vinegar. . If the content is less than 3% by volume, it is insufficient for sufficient adsorption, and if it exceeds 5% by volume, impurities and / or heavy metals are not only sufficiently adsorbed, but also undesirable in view of the subsequent filtration and removal of the adsorbent.

The adsorbent is added and left for about 20-30 hours to allow the adsorbent to sufficiently adsorb impurities and / or heavy metals, and then the adsorbent is removed by filtration under reduced pressure. If the leaving time is less than 20 hours, impurities are not sufficiently adsorbed and removed by the adsorbent, and even if it exceeds 30 hours, the adsorption is not increased any more and it is uneconomical.

After the first distillation, the dried mugwort powder is added to the distillate in an amount of about 1-3 wt%, preferably about 2 wt%, flavored at 70 to 90 ° C. for 3.5 to 4.5 hours, and then further distilled. It may further include. At this time, the mugwort powder added acts as a flavoring agent and significantly reduces the ethanol of wood vinegar. If the added amount of mugwort powder is less than 1% by weight, the flavoring effect is insignificant, and if it exceeds 3% by weight, it is not preferable because a too thick mugwort may cause rejection. If the flavoring condition is out of the above range, no suitable mugwort flavor can be provided. In addition, the conditions for performing such further distillation are not limited thereto, but it is preferably performed at about 90-120 ° C. for about 3.5-4.5 hours. Preferably the further distillation is carried out until the yield reaches about 80%.

In addition, the method for purifying crude wood vinegar of the present invention includes adjusting the pH of the secondary distillate to 3-3.5. If the pH of the secondary distillate is less than 3, sodium acetate is added to adjust the pH. In addition, if the pH exceeds 3.5, the pH can be adjusted by adding zinc sulfate. More preferably the pH of the secondary distillate is adjusted to 3.3-3.5. As such, by adjusting the pH of the purified wood vinegar to 3-3.5, acetic acid, formic acid and propionic acid can be maintained without being denatured, respectively.

Purified wood vinegar of the present invention purified as described above is 2 to 4% by weight of acetic acid, 0.05 to 0.15% by weight formic acid, 0.05 to 0.15% by weight propionic acid, 1-3% by weight of wormwood components, 0.01-1% by weight of sodium acetate and purified water remaining Amount is made. In addition, the vinegar solution may contain a trace amount of zinc sulfate. For example, zinc sulfate may be included at 0.01-1% by weight.

Hazardous substances purified by the above method and the ethanol is removed from the ethanol can be diluted and drinkable, can be directly applied to the skin, etc., can be used as an additive in food, drinks and other cosmetics. Therefore, it is expected to be used in various fields. For example, woody liquor purified by the method according to the invention in food, beverages and cosmetics can be added in an amount of up to 5% by weight. Purified wood vinegar of the present invention may be added in a suitable amount in consideration of the action, effect, etc. required for plants, beverages and cosmetics. However, since the food additive is currently regulated by the maximum amount of 5% by weight, purified wood vinegar can be added to less than 5% by weight in food, beverages and cosmetics. Wood vinegar can also be used as a main ingredient in the synthesis of pharmaceuticals.

The wood vinegar purified according to the present invention can be effectively used especially for improving atopic dermatitis symptoms. When the wood vinegar is used as a drug for improving atopic dermatitis symptoms, the wood vinegar is preferably contained in 1 to 5% by weight or less of the total weight of the drug. In addition to the wood vinegar, it may further comprise a pharmaceutically acceptable carrier generally known. Pharmaceutically acceptable carriers that can be used are conventional in the pharmaceutical art, for example in the case of topical administration agents, bases, excipients, lubricants, preservatives and the like. A typical single dose of the drug is preferably 1,000 to 30000 mg / kg when administered transdermally. However, the dosage of this drug may vary depending on the weight, age, sex and severity of the patient.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are illustrative of the present invention, which does not limit the present invention.

Example  One

To the round flask was added 600 g of crude wood vinegar and 2 g of 80% phosphoric acid. Thereafter, a cooling condenser device was installed in the flask, and 24 g of 30% hydrogen peroxide solution was slowly added to the crude wood vinegar and heated to 70 ° C. while stirring at 110 rpm to react for 8 hours. Thereafter, the resulting aggregates were left to stand at room temperature for 40 hours, filtered, and then heated and distilled at 90-120 ° C. for 4 hours in a distiller to obtain 410 g of purified wood vinegar (about 68% yield).

The physical properties of the purified wood vinegar and the presence of toxic substances were evaluated.

(1) physical properties

Purified wood vinegar was tested by the method according to the standard and quality Article 9 quality standards and quality test standards of the National Forest Research Institute Notice 2004-04 (2004.6.22), the results are shown in Table 1 below.

TABLE 1

Bome weight (g / ml) Acid amount (%)  pH Melt Tar (%) Refractive Index (% Brix) color 0.8 3.7 2.5 0.01 2.6 Transparent pale yellow

(2) Contains toxic substance

The content of toxic substances in the wood vinegar purified by GC-ECD method was measured (Korea Testing and Research Institute). As a result, α-BHC, β-BHC, γ-BHC, δ-BHC, op-DDT, pp-DDT, op-DDD, pp-DDD, op-DDE, pp-DDE, aldrin, Dieldrin and Endrin were not detected.

Example  2

600 g of crude wood vinegar was added to the round flask and 1 g of 83% phosphoric acid was added thereto. Thereafter, a cooling condenser was installed in the flask, and 20 g of 30% hydrogen peroxide solution was slowly added to the crude wood vinegar, and the mixture was heated at 60 ° C. with stirring at 100 rpm to react for 6 hours. Thereafter, the heating was stopped and left for 40 hours to allow the carbonized suspension to aggregate, and then the aggregated carbonized suspension was filtered off.

Charcoal powder 2g was added to the wood vinegar from which the carbonized suspension was removed, and the mixture was left at room temperature for 24 hours, followed by filtration under reduced pressure at 9.0 mbar to remove charcoal powder. After filtration, the mixture was heated and distilled at 90-120 ° C. for about 4 hours to obtain 390 g (yield 63%) of purified wood vinegar.

Antimicrobial properties, physical properties and the presence of harmful substances of the obtained wood vinegar was measured by the following method.

(1) antibacterial

Antimicrobial activity was determined by the Halo Test according to KS K 0692.

That is, in the antimicrobial test, spore suspension was prepared from Trichophyton rubrum bacteria stored in Sabouraud Dextrose Agar, and then rod culture of 200 µl of spore suspension was carried out, and a sterile disc was placed in the center of the medium. Then, 50 µl of purified wood vinegar and 50 µl of 70% ethanol as a control sample were dropped, and then cultured at 24-26 ° C. for 7 days, and then the diameter of the inhibition zone was measured. Was evaluated. The photograph showing the restraint band when using the purified wood vinegar in the present Example is shown in FIG. 1A, and the photograph showing the restraint band formed when ethanol is used is shown in FIG. As can be seen in Figure 1a, in the case of purified wood vinegar in the present embodiment, a wide antibacterial inhibitory band is formed from the center, while the alcohol is used in Figure 1b almost no antimicrobial inhibitors are formed, from which the excellent antimicrobial activity Able to know.

(2) Physical properties and content of harmful substances

The acidity and solid content of wood vinegar were tested in accordance with the National Forest Research Institute Notice 2004-04 Specification and Quality Article 9 Quality Standards and Quality Test Criteria. Other harmful substances content was determined in accordance with the Korean Food Additives Code. The test was conducted by the Environmental Testing Institute, and the results are shown in Table 2 below.

TABLE 2

Test Items Test result Acidity (%) 2.9 Solid content (%) 0.03 Lead (mg / kg) Not detected Cadmium (mg / kg) Not detected Iron (mg / kg) Not detected Tar color Not detected Methyl alcohol (mg / kg) 743 Phenolic (%) Not detected Salicylic Acid (mg / kg) Not detected

Example  3

500 g of crude wood vinegar was added to the round flask and 2 g of 83% phosphoric acid was added thereto.

Thereafter, a cooling condenser was installed in the flask, and 15 g of 30% hydrogen peroxide solution was slowly added to the crude wood vinegar and heated to 30 ° C. while stirring at 100 rpm to react for 10 hours. Thereafter, the heating was stopped and left for 35 hours to allow the carbonized suspension to aggregate, and then the aggregated carbonized suspension was filtered off.

5 g of activated carbon powder was added to the wood vinegar from which the carbonized suspension was removed, and the activated carbon powder was removed by filtration under reduced pressure at 9.0 mbar for 25 hours. After filtration, heat distillation was performed at 90-120 ° C. for about 3 hours to obtain 300 g of purified wood vinegar (60% yield).

After the first distillation, the dried mugwort powder was added to the distillate in an amount of 2% by weight, and the second distillation was performed at 80 ° C. for 4 hours. The pH of the second distilled distillate was adjusted to 3 to 3.5 using sodium acetate or zinc sulfate to obtain 240 g of purified wood vinegar (80% yield). The inventors named the purified wood vinegar according to the present invention thus obtained as 'vinesalline'.

1) Toxicity test of single oral administration in rats

Toxicity test of single oral administration was performed on rats using the purified wood vinegar solution (hereinafter referred to as vinesarin) according to the present invention obtained in Example 3. The test was conducted by the Institute for Safety Evaluation, affiliated with the Korea Research Institute of Chemical Technology, and the test method was conducted in accordance with Korean Food and Drug Administration Notification No. 1999-61, 'Toxicity Test Standards for Drugs.'

In order to investigate the toxicity of acute oral administration of test substance, mortality, general symptoms, weight change and autopsy findings of 15 days were observed after single oral administration of 5 male and female doses of 0 and 20 ml / kg to SD rats. . The test results are as follows.

A) No dead animals were observed in males, but only one in 20ml / kg female group.

B) In general symptoms, ptosis was observed in 20ml / kg group of male animals, spontaneous exercise deterioration, ptosis and flow in female 20ml / kg group.

C) In the case of body weight change, no change due to the test substance was observed in all the male and female test groups.

Autopsy Results Gross necropsy findings due to the test substance were not observed in all test groups.

2) Toxicity test of single transdermal administration using rats

Toxicology of single transdermal administration was performed on rats using vineserin. The test was conducted by the Institute for Safety Evaluation, affiliated with the Korea Research Institute of Chemical Technology, and the test method was conducted in accordance with Korean Food and Drug Administration Notification No. 1999-61, 'Toxicity Test Standards for Drugs.'

In order to investigate the acute dermal toxicity of the test substance, 15 days of mortality, general symptoms, weight change and necropsy were observed after single transdermal administration with 5 male and female doses of 0 and 2,000 mg / kg in 5 male and female rats. The test results are as follows.

A) Death animals were not observed in all male and female test groups.

B) In the general symptom, flow was observed in the male and female 2,000 mg / kg group.

C) In the case of body weight change, no change due to the test substance was observed in all the male and female test groups.

Autopsy Results Gross necropsy findings due to the test substance were not observed in all test groups.

3) Skin irritation test using rabbit

Skin irritation tests were performed on rabbits using vineserin. The test was conducted by the Institute for Safety Evaluation, affiliated with the Korea Research Institute of Chemical Technology, and the test method was conducted in accordance with Korean Food and Drug Administration Notification No. 1999-61, 'Toxicity Test Standards for Drugs.'

To investigate the skin irritation of the test substance in New Zealand White male rabbits, the test substance was applied to the abrasion and non-abrasion areas for 24 hours, and then mortality, general symptoms, weight change and local irritation up to 72 hours were evaluated. It was. The results of the test are summarized as follows.

A) No dead animals due to test substance application were observed during the test period.

B) As a result of general symptoms, abnormal findings due to the application of the test substance were not observed.

C) As a result of weight measurement, no weight change due to application of the test substance was observed.

D) As a result of observing the local irritation of the abrasion and non-abrasive areas after finishing the application of the test substance, no abnormal findings were observed by applying the test substance. Therefore, the primary irritation index of this test substance was '0' and was evaluated as non-irritant.

From the above results, it was determined that the skin application of the wood vinegar obtained in Example 3 in New Zealand White rabbits did not cause any irritation to the abrasion and non-abrasive areas. Therefore, vineserin is considered to be a non-irritating substance.

4) Eye irritation test using rabbit

The ocular mucosa stimulation test was performed on rabbits using vineserin. The test was conducted by the Institute for Safety Evaluation, affiliated with the Korea Research Institute of Chemical Technology, and the test method was conducted in accordance with Korean Food and Drug Administration Notification No. 1999-61, 'Toxicity Test Standards for Drugs.'

In order to investigate ocular irritation of the test substance in New Zealand White male rabbits, the mortality, general symptoms, weight change and local irritation were evaluated for 7 days after treatment with 0.1 ml of test substance. The test results are summarized as follows.

A) No dead animals due to test substance application were observed during the test period.

B) As a result of general symptoms, abnormal findings due to the application of the test substance were not observed.

C) As a result of weight measurement, no weight change due to application of the test substance was observed.

D) As a result of local irritation, conjunctival abnormalities were observed up to 4 days after application, but no abnormalities were observed in cornea and iris. Therefore, the acute ocular irritation index of the test substance was evaluated as '4.0' in the non-eye group and '6.0' in the eye group.

In conclusion, ophthalmic mucosa application of the wood vinegar obtained in Example 3 in New Zealand White male rabbits causes mild abnormalities in the conjunctiva but does not show any abnormalities in corneas and irises. Vinesarin is believed to be a mild-irritant.

5) Skin sensitization test using guinea pig

Skin sensitization tests were performed on guinea pigs using vineserin. The test was conducted by the Institute for Safety Evaluation, affiliated with the Korea Research Institute of Chemical Technology, and the test method was conducted in accordance with Korean Food and Drug Administration Notification No. 1999-61, 'Toxicity Test Standards for Drugs.'

In order to investigate skin sensitization, T1 group (test substance sensitization group, 10 animals), V1 group (5 sensitizers for excipients for test substance, 5), T2 group (positive control) using 30 Hartley male guinea pigs Guinea pig maximization tests were performed, consisting of four groups: substance sensitization group (10) and V2 group (sensitization of excipients for positive control, 5). Two sensitizations (first sensitization: intradermal administration, second sensitization: closed patch) were performed on the back of the animal at weekly intervals. The skin reaction was observed.

Throughout the observation period, no general symptoms or body weight changes presumed to be due to administration of the test substance were observed. At 48 hours, erythema and edema of the test substance applied were 0.0% and the sensitization rate was 0%, which was significantly lower than that of the positive control skin and 1.6 and 100% sensitization rate, and the excipient control group (mean skin response: 0.0, A very weak skin sensitization was observed, with the same percentage of sensitization. Similarly, the 72-hour observation showed that the average skin response was 0.3 and the sensitization rate was 30%.

The above results suggest that skin sensitization of vineserin in Hartley guinea pig is very weak (grade 1).

Example  4

Efficacy test for atopic dermatitis using NC / Nga mouse

I. Purpose

The purpose of this study was to select a disease model using NC / Nga mice known to induce atopy as a test system and to evaluate the efficacy of Vinesarin for atopic dermatitis. Was carried out.

II. Test Methods

1. Experimental Animal

Four-week-old NC / Nga mouse males were purchased from Charles River, Japan, and used for testing from five weeks of age after the acclimation period. Mice were bred in air-uncontrolled conventional rooms to induce spontaneous atopic dermatitis, similar to atopic dermatitis in humans, and drinking non-sterile tap water. Irradiated sterilized diets (Halan Teklad Irradiated Rodent diets) were used to feed the mice before feeding them to a non-sterile condition, and the cages were reared without the filter top. All of these breeding conditions were to establish the dirty condition (dirty condition) that can optimize the expression of atopic dermatitis in the breeding room.

2. Mouse grouping according to test substance administration condition

1) First experiment

The first experiment was an observation of symptoms of spontaneous atopic dermatitis for 13 weeks (18 weeks after birth). This was based on the results of the authors who developed the first atopic dermatitis expression model using NC / Nga mice (the highest degree of dermatitis at 17 weeks of age, Matsuda et al. 1997).

Each group consisted of 10 animals, which consisted of 1.5%, 3%, and 6% of the test substance, the non-treated group, the non-treated group, the third distilled water applied group, the dilution solvent for the vineserin, and the positive substance applied group. . As a positive substance, pure care ointment (diarrheal drug) and attoclear ointment (dong pharmaceutic drug) were used successively. The following table summarizes the test information by group.

TABLE 3

number process Throughput Cage label Remarks Initial Processing Date One Vinesarin 1.5% 100 μl Group 1 Test group 2005-09-27 2 No treatment Group 2 No treatment 2005-09-27 3 Vinesarin 3.0% 100 μl Group 3 Test group 2005-09-27 4 Positive control group 100 μl (0.1 g) 4th group Positive control group 2005-09-27 5 Solvent treatment 100 μl 5 groups Solvent administration group 2005-09-27 6 Vinesarin 6.0% 100 μl 6 groups Test group 2005-09-27

For the administration of the material, first, the area of the back of the mouse was depilated to a pelvis area of 2 × 4 cm, 100 μl of test substance was applied, and the same amount of test substance was also applied to the left ear. It was applied once a day for 5 days a week, and hair was applied at weekly site.

2) 2nd experiment

In the second experiment, it was judged that spontaneous atopic dermatitis was not expressed in NC / Nga mice as a result of the first experiment, and the immunomechanical atopic composition combining 1% DNCB (2,4-dinitrochlorobenzene) sensitization and 0.2% DNCB challenge was performed. It is an experiment evaluating the efficacy by inducing dermatitis expression and applying test substance (Tomimori et al. 2004). That is, sensitization twice with 1% DNCB or DNCB solvent material (acetone: olive oil = 4: 1 mixture) followed by blood collection after DNCB challenge to confirm double IgE levels with 0.2% DNCB or DNCB solvent material. Then, after applying the test material for 2 weeks, the mice were sacrificed and necropsied.

3. Observe skin symptoms

Atopic dermatitis symptoms such as abscess inflammation, bleeding, eczema, erythema, erosion, and peeling skin were observed twice a week. In addition, in the second experiment, the number of times of scratching the skin or face was measured for 30 minutes each on the day after the completion of the two DNCB challenges and on the day of the application of the test substance.

4. Determination of Antibody Levels in Plasma

Blood was collected by orbital or cardiac collection and the plasma separated from the blood was stored in a -80 ° C freezer until use. Total IgE was quantified by sandwich ELISA method for comparative analysis of IgE levels present in plasma (Accept et al. 2002). This method is briefly described as follows.

Rat anti-mouse IgE capture antibody (BD Pharmingen, San Diego, Calif.) Was added to 2 μg / ml (100 μl per well) in Immulon 2 plate (Dynex, Chantilly, VA), then allowed to stand for 24 hours in refrigeration and then 1% After blocking nonspecific binding with BSA-PBS, diluted experimental plasma and standard IgE (purified mouse IgE mAB isotype standard, BD Pharmingen) were added and allowed to stand for 24 hours in the cold. The next day, biotinylated rat anti-mouse IgE (1 μg / ml 1% BSA-PBS, BD Pharmingen) was added and allowed to stand at room temperature for 2 hours to allow detection antibodies to attach, followed by addition of avidin-peroxidase, followed by another 1 hour room temperature. Political Subsequently, color development was induced by ABTS (Sigma, St. Louis, MO) and then absorbance was measured at 405 nm.

To determine the levels of IgG1 and IgG2a, the isotypes of IgG present in plasma, a sandwich ELISA method using goat anti mouse IgG1 and IgG2a (Serotec, Raleigh, NC) capture antibodies and peroxidase conjugated anti mouse IgG detection antibodies It was.

5. Cytokine Production Analysis

Type 1 cytokines centered on interleukin-4 (IL-4), which induces isotype switching of IgE and IgG1, and type 1 cytokines such as interferon-gamma (IFNγ), which antagonizes the role of these cytokines Comparative analysis of their production capacity is considered as a way to assess the bias of basic cellular immune function (Heo et al. 1996; Snapper and Paul 1987). To this end, spleen or axillary lymph nodes were collected from each mouse, and then a single cell group was prepared. Then, 0.5 × / well in a 24-well-plated plate coated with anti-CD3 mAB (5 µg / well, BD Pharmingen) a day before. 10 ⁶ cells were dispensed and incubated for 48 hours in a CO ₂ incubator. Quantification of IL-4 and IFNγ cytokine levels in cell culture was performed by sandwich ELISA (Heo et al. 1998). The lower detection limit for each cytokine was 4 pg / ml for IL-4 and 100 pg / ml for IFNγ.

6. Plasma Histamine Level Analysis

Plasma concentrations of histamine, secreted from activated mast cells or basophils and recognized as representative markers of allergic expression, were quantified by ELISA (Kim, H. et al. 2005). IBL histamine ELISA kit (IBL Immuno-biological laboratories, Hamburg, Germany) was used to quantify histamine, which is briefly described as follows.

First, 20 µl of experimental plasma, standard plasma, and positive control plasma were mixed with 20 µl of indicator buffer, 4 µl of acylated reagent was added and mixed again, and then 50 µl of this mixture was added to goat anti-histamine against histamine. Place in a 96 well plate with rabbit antiserum antibody attached. Immediately add 50 μl of peroxidase-conjugated histamine and finally 50 μl of rabbit antiserum to histamine and mix for 3 hours in an orbital shaker. After washing the plate and adding TMB (tetramethylbenzidine) chromophore 20 minutes after the absorbance was measured using an ELISA reader (Bio-Tek EL800, Winoski, VT) at 450nm (reference wavelength 610nm).

7. Skin Tissue Cell Changes

7-1. Observation of mast cells through toluidine blue staining in skin tissue

1) Mast Cell Staining Method

Staining of mast cells in skin tissues was performed under staining conditions as shown in the table below.

[Table 4] Staining Conditions of Mast Cells

Dyeing Process Condition fixing 10% buffered formalin solution, (pH7.0) Technique Paraffin, 4-6 μm reagent 0.5% Toluidine Blue Solution Toluidine Blue --- 5g Distilled Water --- 100ml process of dyeing 1) paraffin in xylene 2) Rinse in tap water for 5 minutes. 3) Stain in 0.5% toluidine blue solution. Stain for 5 minutes for mast cell observation. For other tissue observations, stain for 6-20 minutes. 4) Rinse quickly 10 times with 2 steps of 95% ethyl alcohol solution. 5) Dehydrate for 1 min each with 2 steps of anhydrous ethyl alcohol solution. 6) Encapsulated in glycerin- jelly. Dyeing result Mast Cell Granules ----------- Red to Purple Nucleus --------------------------- Blue

2) Calculation of mast cell number

The staining method and the distribution of cells were observed through the above staining method, and then the average value was calculated by counting 10 sections as ideal areas, and all experimental procedures were performed by blind tests.

7-2. Observation of eosinophils, lymphocytes and macrophages in skin tissue

Quantitative changes of eosinophils, lymphocytes and macrophages were observed through conventional hematoxylin & eosin (H & E) staining.

8. Analysis of COX-1 Protein Expression in Skin Tissue

Cyclooxygenase enzyme (COX) is a mediator of the production of prostagladin and thromboxane from arachidonic acid. These substances increase as the inflammation progresses in many organs, including the skin (Roca and FitzGerald, 2002). In this experiment, the expression level of COX-1 enzyme, which is primarily involved in the production of these substances, was compared and analyzed using a combination of Western and chemiluminescent methods. The amount of skin tissue protein used in Western analysis was 30 µg, and the chemoluminescent antibodies were rabbit antimurine COX-1 antibody (Cayman) and peroxidase-conjugated donkey anti-rabbit IgG (Amersham).

9. Statistical Processing

Significant differences in measurements between groups were reviewed using the SigmaPlot statistical program (SPSS, Chicago, USA). First, the normal distribution of the data was verified and then tested for significance by single factor ANOVA and Dunnett's t-test or Kruskal-Wallis ANOVA and Dunn's test. If necessary, the student's t-test or Mann-Whitney test was used as an additional significance test, and the p-value was 0.05 or less. * Indicates that there is a statistically significant difference at the p <0.05 level.

III. Results and Discussion

Changes in Plasma IgE Levels in the First Experiment of Spontaneous Atopic Dermatitis

Blood was collected through orbital blood collection five times from one week after the application of the test substance to one week after the end of the application, and the plasma was separated and the level of IgE in the plasma was measured (FIG. 3). Measuring IgE levels in plasma, as described above, is representative of immunological markers of atopic dermatitis, which is a significant increase in IgE levels, particularly in humans as well as studies using NC / Nga mice (Matsumoto et al. 1999). This has been confirmed in one study (Abdel-Hamid KM 2003).

The characteristics of this test were that there was no significant decrease in IgE levels in the 1.5%, 3%, and 6% treated groups, compared to the untreated or solvent-treated group. Interestingly, although not statistically significant, IgE levels at 7, 11, 13, and 16 weeks of the vineserin 3% -coated group were always lower than those of the untreated or solvent-administered groups. There was a similar trend in plasma at 7, 11, and 16 weeks of the application group. However, symptoms such as redness, bleeding, eczema, erosion, and dermal formation on the skin of the face, ear, and back, reported as skin symptoms accompanying atopic dermatitis in NC / Nga mice, were significantly increased in the untreated or solvent-applied group. Since it was not observed, it is difficult to infer that spontaneous atopic dermatitis was expressed in the NC / Nga mice used in this experiment, and thus, it was judged that it was not appropriate to evaluate the efficacy of suppressing the atopic dermatitis of vineserin (FIG. 3). . Originally, the researchers who developed the model indicated that spontaneous atopic dermatitis was developed when the animals were bred in the air-conditioned Convention Room conditions, that is, the most contaminated conditions (Matsuda et al. 1997). However, the conditions in which the mice were raised were the most contaminated breeding conditions such as drinking general tap water, using no filter for dropping dust, not cleaning the cage during the breeding period, and not replacing the cage. The fact that it was not possible to induce clear atopic dermatitis expression was considered a problem to be discussed with Japanese researchers who published the model. When this problem was raised in the company supplying the experimental animals, it was later recognized that spontaneous atopic dermatitis was difficult to express, unlike the minimum researchers announced in the supplied NC / Nga mice.

2. Changes in IgE Level in Induction-Atopic Dermatitis-Induced Second Experiment Using DNCB

Since it was difficult to determine that spontaneous atopic dermatitis was expressed in the first experiment, induced-atopic dermatitis was induced using DNCB used in allergic contact dermatitis expression model. That is, 7 mice in each group sensitized 1% DNCB, and 3 animals in each group were sensitized twice a week with a mixture of acetone and olive oil (4: 1), a DNCB solvent substance, followed by 1% DNCB sensitization mice. 0.2% DNCB was also challenged twice with DNCB solvent material application group for one week. After the challenge, blood was collected and analyzed for plasma IgE levels to determine whether induced-atopic dermatitis was induced.

As shown in FIG. 4, the IgE level of the DNCB-coated mice was increased by 5-15 times compared to the solvent-coated mice in all groups. In addition, the IgE level was increased by about four times or more than the highest level of IgE (13-week-old time) of the first experiment of inducing spontaneous atopic dermatitis shown in FIG. These results suggest that this model can evaluate the efficacy of inhibiting atopic dermatitis of the testosterone test substance. Therefore, the test sample was applied to each group for the next two weeks as in the first experiment.

Next, 1.5%, 3%, 6% of test substance, or sterile tertiary distilled water, which is a diluting solvent, was applied to the mice induced with DNCB-induced atopic dermatitis in the first experiment, or positive. Plasma IgE levels were analyzed by blood collection after two weeks of material application or no treatment. As shown in FIG. 5, the IgE level was significantly decreased in the 1.5% of the nonsarinin, 3%, the sterile tertiary distilled water, and the positive material application group (p <0.05) as compared to immediately after the DNCB challenge.

In addition, the IgE level decreased by 0.287 ± 0.169 times after the application of the testosterone test compared to the DNCB challenge immediately after the application of the 3% nonnesine test, followed by the positive control application application group (0.294 ± 0.148) and the solvent substance. The application group (0.379 ± 0.133), the vinesarin 1.5% application group (0.418 ± 0.187), the untreated group (0.480 ± 0.331), and the vinesarin 6% application group (0.562 ± 0.318).

3. Changes in IgG1 Isotype Levels in the Induced-Atopic Dermatitis-Induced Secondary Experiment Using DNCB

It is known that in mice, isotype-switching is induced by IL-4 and IgG2a is induced by IFNγ. Thus, if the IgG1 concentration in the blood is increased relative to the IgG2a concentration, the body's immune system responds to type 2 response. It is mentioned that this predominantly atopy tendency is shown (Abbas et al. 2000). Therefore, in this study, blood was collected through cardiac collection at the time of final sacrificial necropsy, plasma was isolated, and then plasma IgG1 and IgG2a levels were measured, and the ratio of IgG2a concentration divided by IgG1 concentration was calculated.

As shown in FIG. 6, the IgG2a / IgG1 ratio was greatest when 3% of vinesarin was applied to mice induced with DNCB-induced atopic dermatitis, while the lowest was applied when 6% of vinesarin was applied. This suggests that mice treated with 3% of vinesarin exhibited a relatively suppressed type 2 response directly related to atopic dermatitis expression compared to mice with other application conditions. It is noteworthy that mice coated with acetone-olive oil mixture, a DNCB solvent instead of DNCB, had a relatively higher IgG2a / IgG1 ratio compared to the DNCB group. It is believed to mean that the induced-atopic dermatitis expression model has been meaningfully applied to the immune system. In other words, it can be inferred that the DNCB sensitization / challenge proceeded to bias the atopic type 2 reaction in which the production of IgG1 was promoted relatively (see Table 5).

TABLE 5 Mean and standard deviation of plasma IgG1 and IgG2a concentrations in NC / Nga mice

DNCB Inspiration / Challenge Application of acetone-olive oil mixture IgG1 (mg / ml) IgG2a (mg / ml) IgG1 (mg / ml) IgG2a (mg / ml) Vinesarin 1.5% 12.1 ± 7.7 * 7.5 ± 1.9 6.4 ± 0.2 10.9 ± 5.2 No treatment 8.9 ± 1.9 7.2 ± 2.5 3.0 ± 1.2 5.1 ± 1.8 Vinesarin 3% 6.2 ± 2.5 6.6 ± 3.0 3.9 ± 2.3 7.5 ± 2.2 Positive control 7.8 ± 2.8 6.4 ± 1.6 5.2 ± 2.0 5.8 ± 1.6 Solvent treatment 6.8 ± 4.0 5.2 ± 0.5 3.3 ± 1.5 4.1 ± 0.9 Vinesarin 6% 10.5 ± 5.4 5.2 ± 0.6 5.0 ± 2.3 4.9 ± 0.8

*: Mean ± standard deviation

4. Changes in cytokine levels in the second trial of induction-atopic dermatitis using DNCB

Above `` 3. T lymphocytes in the subject by analyzing the pattern of cytokines produced after selectively activating T lymphocytes in the spleen or lymph nodes, as described in "Changes in IgG1 Isotype Levels in Induction-Atopic Dermatitis-induced Secondary Experiments Using DNCB". In particular, it is possible to analyze whether type 1 helper T cells are dominant or type 2 helper T cells in the helper T cells (Heo et al. 1998). In other words, if the type-2 helper T cells that secrete IL-4 are superior to the type-1 helper T cells that secrete IFNγ, the immune system centered on T lymphocytes in the body may be said to be atopic (Hoffjan et al. 2004). In this experiment, we determined the relative decrease in the number of type 2 helper T cells by creating a splenocyte single cell group, selectively activating T lymphocytes with anti-CD3 mAb, and dividing the amount of IFNγ generated in each culture by the amount of IL-4. It was.

As shown in FIG. 7, there was no significant difference between groups in mice induced with DNCB-induced atopic dermatitis. However, mice coated with acetone-olive oil mixture, a DNCB solvent instead of DNCB, had a relatively higher IFNγ / IL-4 ratio than the DNCB group, similar to the IgG2a / IgG1 ratio. The induction-atopic dermatitis expression model by DNCB applied by is considered to mean the fact that it has been applied to the immune system. In other words, it can be inferred that type 2 helper T cells secreting IL-4 were more likely to undergo type 2 response by DNCB sensitization / challenge than type 1 helper T cells secreting IFNγ. Can be. In the present study, the DNCB-induced atopic dermatitis-induced mice were not able to confirm the significant differences between the two groups. Or perhaps it was not enough time to affect differentiation into type 1 helper T cells.

In addition, it is noteworthy that the mice coated with acetone-olive oil mixture, DNCB solvent, had a higher type 1 response to inhibit atopy tendency when 3% of vineserin was applied. Mice coated with these acetone-olive oil mixtures did not induce atopic dermatitis expression by DNCB, possibly reflecting the effects of long-term application of test materials, such as vinesarin, applied from the first experiment.

5. Changes in the Scratch Frequency of Mice in Induction-Atopic Dermatitis-induced Secondary Experiments Using DNCB

When atopic dermatitis is expressed in facial, ear, and back skin tissues, itching is caused by histamine secretion. Thus, mice scratch the affected area with their forefoot. Therefore, the frequency of scraping is calculated as an index for the occurrence of atopic dermatitis or allergic contact dermatitis.

In this experiment, the scratching frequency was measured for 30 minutes on the day after DNCB challenge and the day after application of test substance such as vinesarin. It was then calculated as a percentage of how much the scratching frequency decreased after application of the test substance compared to after the DNCB challenge (FIG. 8). Scratching frequency was reduced to 36% when 3% of Vinesarin was applied, showing the largest reduction, which was statistically significant (p <0.05) reduction compared to the no treatment group and the solvent application group (Table 6). .

Table 6 Mean and standard deviation of scratching frequency before and after application of test substance in NC / Nga mice with DNCB-induced atopic dermatitis

After DNCB Challenge After applying test substance % Reduction Vinesarin 1.5% 170 ± 77 * 126 ± 72 78 ± 58 No treatment 155 ± 61 178 ± 94 118 ± 59 Vinesarin 3% 241 ± 107 87 ± 73 36 ± 21 Positive control 209 ± 152 148 ± 91 168 ± 214 Solvent treatment 229 ± 122 243 ± 111 127 ± 74 Vinesarin 6% 259 ± 197 113 ± 48 93 ± 98

*: Mean ± standard deviation

6. Changes in Plasma Histamine Level in Induction-Atopic Dermatitis-Induced Secondary Experiments with DNCB

Histamine is secreted out of cells within 30 minutes of binding of allergen-specific IgE or IgG1 to Fc receptors on the surface of mast cells. For this reason, histamine levels in allergic diseases such as atopic dermatitis are highest when symptoms such as skin rash or hyperemia occur, which is used as an early indicator of the progression of allergic diseases. It is not used as an indicator (Adelman et al. 2002). That is, when the symptoms of atopic dermatitis are progressing, the secretion level is the highest.

As a result (Table?), There was no significant difference in histamine levels between the groups. This may be due to the fact that histamine secretion was out of active period by collecting blood after 2 weeks of application period of test substance after induction-atopic dermatitis caused by DNCB.

TABLE 7 Mean (ng / ml) and standard deviation of plasma histamine levels in NC / Nga mice

After DNCB Challenge After applying test substance Vinesarin 1.5% 235 ± 136 * 246 ± 142 No treatment 117 ± 149 111 ± 164 Vinesarin 3% 197 ± 116 129 ± 111 Positive control 73 ± 104 5 ± 1 Solvent treatment 92 ± 169 145 ± 153 Vinesarin 6% 134 ± 127 43 ± 52

*: Mean ± standard deviation

7. Mast Cells and Other Histopathological Findings in Skin Tissues

Mast cells reside in the mucous membranes and epithelial tissue on the surface of the body and serve to alert the immune system to local trauma and infection. These mast cells are distributed in all the vasculature except the central nervous system and retina, and the initial response of mast cells upon activation releases heparin, an anticoagulant, in addition to the function of releasing inflammatory molecules stored in 50-200 large granules filling the cytoplasm. It is also known as a secretory cell that secretes histamine (5-hydroxytryptamine), which strongly contracts smooth muscle, and increases the permeability of capillaries and vasculature. That is, in allergic dermatitis-induced experiments such as this experiment, the increase in the number of mast cells in the skin tissue and the promotion of degranulation are known to have a correlation with the manifestation of dermatitis symptoms.

As shown in FIG. 9, the mast cell numbers of the positive control group, the vineserin 6% coated group, and the solvent-administered group were significantly decreased compared with the untreated group, 1.5% of the vineserin, and 3% coated group. In the case of 3% of vinesarin, which was observed to be able to inhibit the expression of atopy in the body in plasma IgE level, IgG2a / IgG1 ratio, IFNγ / IL-4 ratio, scratching frequency, and the like, positive control group, 6% Vinesarin group The exception is that no mast cell count was observed. At present, it is difficult to explain the difference between the results and histopathologic findings in terms of cellular immunity.

In general histopathology, Langerhans cells, basal cells with melanin granules, melanocytes, fibroblasts, and collagen fibers were somewhat different, but most of them were normal. In the 1.5% test substance application group of the low dose group, the hair cycle was shown to be tologen state, and the amount of hair was small in the tissue of the medium dose and the high dose group. The common findings in the test substance application group, including the positive control group, included a large number of coarse wrinkles in the epidermal layer, and a number of fibrotic cells were observed, and the dermal layer was narrower and not uniform in thickness than the normal group. This was observed to be very rough, with inflammatory findings. However, inflammatory changes were observed more significantly in the high dose group, 6% group than the low and medium dose group. On the other hand, lymphocytes were difficult to observe in the positive control group, 3% and 6% of the visarin group, and intermittently observed in some slides of the solvent group. Eosinophils and macrophages were difficult to observe in most experimental groups. These findings indicate that an increase in mast cell number and promotion of degranulation, lymphocyte and macrophage deposition were observed in the skin of human atopic dermatitis symptom in NC / Nga mice (Matsumoto et al., 1997). This suggests that atopic dermatitis-specific histopathological phenomena have been alleviated or disappeared during the two weeks of application of the test or control material following induction of atopic dermatitis using DNCB.

In conclusion, mast cell counts were significantly lower in the 6% Vinesarin group, whereas in the histological findings, inflammatory changes were more severe in the 6% group with higher concentrations. Showed. From the current results, it was observed that DNCB treatment caused atopic dermatitis, but it was very difficult to judge the effect on histopathological aspects of the test substance.

8. Analysis of COX-1 Expression in Skin Tissue

COX-1 is expressed in most cells in the epidermal layer of skin tissues (Lee et al., 2003). Its main function is to produce prostaglandin precursors and is involved in maintaining homeostasis of COX proteins (Crofford, 1997). Therefore, if the test substance inhibits the dermal inflammatory response induced by DNCB application, COX-1 expression was predicted to be somewhat lowered, and the present COX-1 expression was compared and analyzed.

As a result of the analysis, no significant difference was found between the groups (FIG. 10), but the group showed a lower tendency than the other groups in the 1.5%, 3%, and no treatment groups. In particular, no difference was observed between the COX-1 expression level and the test substance application group in the untreated group without any substance applied after the DNCB sensitization / challenge and the third distilled water applied solvent administration group, which is the dilution solvent of the test substance. It is difficult to judge that the application showed a significant effect on the progression of DNCB-induced local dermatitis response. In other words, the Vinesarin test substance has a systemic regulation related to the development of atopic dermatitis, such as the effects on various cellular immunological parameters presented in the results 2-5, while localizing such as inflammatory response. It can be said that the effect is not clear.

IV. conclusion

The main conclusions obtained through the above experimental results are as follows.

1. In the spontaneous atopic dermatitis expression model, the application of 3% of vinesarin for 13 weeks maintained lower IgE levels than the untreated or solvent-coated groups.

2. In the DNCB-induced atopic dermatitis model, plasma IgE levels were significantly lower in the mice treated with 1.5% and 3% of vinesarin for 2 weeks than immediately after atopic dermatitis by DNCB.

3. The highest percentage of plasma IgG2a / IgG1 in the same subject was applied to 3% of vinesarin in mice induced with DNCB-induced atopic dermatitis, which was directly related to the expression of atopic dermatitis compared to mice with other application conditions. It can be regarded as the result that type 2 response is relatively suppressed.

4. In control mice challenged / challenged with acetone-olive oil mixture as a solvent instead of DNCB, 3% of vinesarin suppressed atopic tendency based on the tendency of IL-4 and IFNγ production in T lymphocytes of splenocytes. The type 1 response was observed to be higher than that of the other groups.

5. The frequency of scratching was measured for 30 minutes on the day after DNCB challenge and the day after application of test substance such as vinesarin, and the frequency of scratching was reduced to 36% when 3% of vinesarin was applied. There was a statistically significant decrease.

6. Mast cell count was significantly lower in non-sarin 6% coated group, while general histological findings showed higher inflammatory changes in high concentration 6% coated group. These results were also different from those of the cellular immunological aspects.

7. The expression level of COX-1 protein, which is involved in the progression of local dermatitis reaction, tended to be lower in nonnesine 1.5% and 3% groups than in other groups.

8. In conclusion, it was observed that the combination of various changes in cellular immunological indices related to atopic allergic expression can inhibit the expression of atopic response in the body when applying the purified wood vinegar solution of the present invention.

Water and coal are effectively removed in a short time, resulting in high purity, for example, high purity wood vinegar of more than 99%. In addition, wood vinegar purified by the method of the present invention is excellent in bactericidal activity and does not contain harmful substances, there is no toxicity, does not smell bad, especially effective in improving atopic dermatitis symptoms.

Claims (4)

Oxidation of crude wood vinegar and hydrogen peroxide solution at 30-70 ° C. for 8-12 hours in the presence of phosphoric acid; After the oxidation reaction, leaving for 36 to 40 hours to remove the aggregates formed when left; Primary distillation at 90-120 ° C. for 3.5-4.5 hours; The dried mugwort powder is added in an amount of 1 to 3% by weight based on the first distillate, flavored at 70 to 90 ° C. for 3.5 to 4.5 hours, and then distilled at 90 to 120 ° C. for 3.5 to 4.5 hours. ; And Adjusting the pH to 3 to 3.5 by adding sodium acetate to the secondary distillate Atopic dermatitis symptom improving agent containing wood vinegar purified by the active ingredient. delete According to claim 1, wherein the wood vinegar is a drug, characterized in that contained in 1 to 5% by weight of the total weight of the drug for improving atopic dermatitis symptoms. The pharmaceutical agent of claim 1, further comprising a pharmaceutically acceptable carrier.

Images