KR20100079586A - Composition for anti atopy-dermatitis containing cirsium japonicum var. ussuriense extract - Google Patents

Abstract

PURPOSE: A composition containing cirsium japonicum var. ussuriense extract for anti-atopic dermatitis is provided to relieve skin rash caused by atopy without side effects. CONSTITUTION: A composition for anti-atopic dermatitis contains 0.01-20 weight% of cirsium japonicum var. ussuriense extract as an active ingredient. The cirsium japonicum var. ussuriense extract is 60-100% of C1-C3 alcohol extract of cirsium japonicum var. ussuriense root. An external use skin formulation contains the composition. The cirsium japonicum var. ussuriense extract is obtained by pulverizing or crushing cirsium japonicum var. ussuriense and adding 60-100% of C1-C3 low alcohol solution. The low alcohol is methanol, ethanol, propanol, or isopropanol.

Description

Composition for anti-Atopy-Dermatitis containing cirsium japonicum var. ussuriense extract}

The present invention relates to an anti-atopic dermatitis composition, and more particularly, to an anti-atopic dermatitis composition that can effectively relieve skin rashes caused by atopic dermatitis by containing thistle extract as an active ingredient.

Atopy dermatitis is a disease with severe pruritus caused by abnormalities in the stratum corneum, the outermost skin barrier due to genetic, environmental, and immunological causes. Atopic dermatitis is a complex entanglement of a variety of causes, repeated relief and recurrence. Allergic diseases caused by atopy predisposition include allergic dermatitis, allergic rhinitis, asthma, allergic conjunctivitis, and atopic urticaria.

Atopic dermatitis suffers from a significant number of people, with 0.5 to 1 percent of the population and 5 to 10 percent of children suffering from atopic dermatitis. 50% of patients heal within two stones, but 25% go to adolescence, and the remaining 25% will continue without atopic dermatitis even in adults.

The cause of this atopic dermatitis is not well known, but it has been found to be mainly related to genetic factors and immune system deficiency. A congenital immunodeficiency patient with severe atopic dermatitis was successfully implanted with a bone marrow and corrected for defects in immune function.Atopic skin rash completely disappeared, or a patient without atopic dermatitis was transplanted from a person with atopic dermatitis. Atopic dermatitis is not a structural disease of the skin but an immune disease caused by bone marrow-derived immune cells. In addition, dry skin, skin itch characteristics, infections caused by bacteria, viruses, and fungi, emotional factors, and environmental factors appear to be combined with each other. Most allergic reactions are mediated by mast cells, and chemical carriers, including histamine, prostaglandins, and tumor necrosis factor (TNF), are the causative agents of various clinical symptoms that can occur early in the allergic reaction.

The main symptoms of atopic dermatitis are severe itching, dry skin, rashes, rashes, crusts and scaly skin. Above all, itching is characterized by severe itching.

Because atopic dermatitis cannot be fundamentally repaired, atopic dermatitis is a disease that is controlled by proper treatment and avoiding the triggers rather than aiming at cure. Prescriptions for atopic dermatitis are mainly drug therapy such as steroids, antihistamines and antibiotics. Steroids (adrenal corticosteroids) have anti-inflammatory and immunosuppressive effects and have excellent effects, but when applied for a long time, side effects such as skin weakness, systemic hormonal symptoms, and addictive effects may occur. Antihistamines prevent the release of histamine from mast cells and reduce itchy symptoms, but may be used as a temporary measure, such as insomnia, anxiety, and loss of appetite.

Therefore, there is a need for a new concept of anti-atopic composition that is effective against atopic dermatitis and has no side effects.

Thistle is a perennial herbaceous plant belonging to the Asteraceae family, which is used for young sterling silver food and mature one for medicinal purposes. Thistle has a significant hemostatic effect and is used for urine bleeding, fecal bleeding, nosebleed, uterine bleeding and traumatic bleeding. In particular, pulmonary tuberculosis, hepatic blood, while removing expectoration, expectoration and chest pain, hemorrhagic blood, acute infectious hepatitis has an antibacterial effect and lowers blood pressure. In the private sector, alcohol is applied to the roots to treat neuralgia and back pain. However, it has not been reported that thistle is effective in treating atopic dermatitis by effectively inhibiting skin rash caused by atopic dermatitis.

The present invention aims to solve the problems of the prior art and to provide a novel anti-atopic dermatitis composition which is excellent in atopic dermatitis and does not have side effects of the conventional therapeutic agent.

The present invention for achieving the above object relates to an anti-atopic dermatitis composition containing thistle extract as an active ingredient.

Thistle extract of the present invention effectively inhibited the secretion of TNF-α induced by PMA / A23187 in HMC-1 cell culture, and showed no cytotoxicity at experimental concentrations.

Thistle extract of the present invention was applied to the atopic dermatitis-induced NC / Nga mice and observed the effect, papules and rinseol / peel was not significantly different from the control group, but erythema and taeyanghwa with pruritic action significantly reduced the skin It was confirmed that it is effective for the treatment of the rash. In the hydrocortisone treatment group used as a positive control, all the items showed therapeutic effects such as papule, rinseol / cuff, erythema, and thyroiditis, but there was no side effect of hair growth on the hydrocortisone-coated area. On the other hand, in the case of thistle extract, none of the short-term side effects visible to the naked eye was observed.

Serum cytokine, a factor related to atopic dermatitis, showed that levels of IL-4, IL-5, and IL-13 and histamine release were statistically insignificant for some levels, but all decreased, compared to hydrocortisone. The reduction effect was better. In the results of optical micrographs of the back skin tissue, the infiltration of thickened epidermis and mast cells according to the induction of atopic dermatitis was effectively suppressed, and when the thistle extract of the present invention or the conventional therapeutic hydrocortisone was plated, the epidermis was thinned again and the degree of infiltration of mast cells. Markedly reduced.

The content of thistle extract in the composition is preferably 0.01 to 20% by weight. If the content of thistle extract is too low, you can not expect a therapeutic effect. Thistle itself has not been used for the treatment of atopic dermatitis, but it has been used for a long time as a folk remedy for other purposes. Since the toxicity has not been reported, the extract itself can be used for the treatment of atopic dermatitis, but it is more easily absorbed. In order to use economically and effectively in the form, it is more preferable to mix with other carriers or excipients so that the content of thistle extract is within 20% by weight. The dosage of the active ingredient according to the present invention may be appropriately selected depending on the absorbency of the active ingredient in the formulation, the age, sex and condition of the patient, the severity of the disease to be treated and the like.

Thistle extract of the present invention may be prepared by pulverizing or grinding thistle finely, 60 to 100% extracted using a lower alcohol aqueous solution of C1 ~ C3 and then concentrated under reduced pressure. As the lower alcohol, methanol, ethanol, propanol and isopropanol may be used. If the content of alcohol is too low, the extraction of the active ingredient may not be effective. The thistle may be prepared using all the parts of the thistle, but may be mainly used only the root part. The aqueous alcohol solution used for extraction is preferably used 2-6 times (L / kg) compared to thistle. If the amount of the aqueous alcohol solution is too small, the extraction is not efficient, too much usage is advantageous in terms of extraction efficiency, but it is not economical because not only a lot of reagents used but also takes a long time to concentrate.

It is preferable that the anti-atopic dermatitis composition of the present invention is formulated into a formulation for unit administration or several administrations and used as an external preparation for skin directly smearing on the site where skin rash appears. For example, it may be a cosmetic composition having a softening lotion, nourishing lotion, massage cream, cleansing cream, nourishing cream, pack, gel or adhesive formulation, and also a lotion, ointment, gel, cream, patch or spray formulation. It may be a pharmaceutical preparation having. In addition, in the external composition of each formulation, other components than the thistle extract described above may be appropriately selected and blended by those skilled in the art without difficulty according to the formulation or purpose of use of other external preparations.

According to the composition containing the thistle extract of the present invention as described above, it is possible to effectively suppress skin rashes caused by atopic dermatitis, and has been used for a long time in folk remedies for conventional medicines such as steroid drugs and antihistamines. It can be used safely without any side effects.

The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

Example

Example 1 Preparation of Thistle Extract

500 g of thistle roots were ground using a blender and placed in a 3 L Erlenmeyer flask, followed by 2 L of 80% (v / v) EtOH and extracted at room temperature for 10 hours. The extract was filtered and the residue was put back into a 3 L Erlenmeyer flask and filtered again with 2 L of 80% (v / v) EtOH. The first extraction filtrate and the second extraction filtrate were combined and concentrated under reduced pressure with a rotary concentrator at 40 ° C. or lower to obtain an EtOH extract.

Example 2 Effect of Thistle Extract on Atopic Therapy in invitro

1) Effect on TNF-α Secretion in Cell Culture Solution

To confirm the atopic therapeutic effect of thistle extract in vitro, HMC-1 cell monoculture (M), HMC-1 cell and PMA / A23187 coculture (P), and HMC-1 cell, PMA / A23187 and thistle EtOH Extracts were cocultured. PMA (phorbol myristate acetate) and A23187 were used at concentrations of 50 nM and 1 μM, respectively. As a control, cycloclosporin (C) was co-cultured with HMC and PMA / A23187 at a concentration of 10 nM and compared with the effect of thistle extract. Thistle extract concentrations of 200, 100, 50 ㎍ / ml was used. Cultivation of HMC-1 cells was performed at 37 ° C. for 8 hours while maintaining a constant CO ₂ concentration of 5% in IMEM medium not containing 10% FBS.

Cell culture solution of each culture group was centrifuged at 1500 rpm for 5 minutes to collect the cell supernatant, and the secretion amount of TNF-α was measured by ELISA. ELISA was measured according to the manufacturer's method (BioSource, U.S.A). Cytokine secretion by the natural product extract was calculated by the following formula and the results are shown in FIG.

As can be seen in Figure 1, thistle extract was concentration-dependently inhibited the secretion of TNF-α, showing the best inhibitory effect at 200㎍ / ml was superior to cyclosporin. No effect was found at concentrations below 50 μg / ml.

2) cytotoxicity

After incubating HMC-1 cells by the same method as in 1), 50 μl of cold 50% TCA (trichloroacetic acid) is slowly added from above. After sufficient fixation for 1 hour, and after the fixation was completed, the mixture was washed 5 times or more with distilled water, and 100 μl of 0.4% SRB (sulforhodamine B) solution was added thereto, followed by dyeing at room temperature for 30 minutes or more. After dyeing, washed 5 times with 1% acetic acid and dried well. The SRB dye was dissolved well with 150 μl 10 mM unbuffered Tris solution and the absorbance was measured in the range of 540 nm with a microplate reader for 96 well plates. Absorbance represents the total amount of protein present in each well, which is proportional to the number of viable cells remaining in that well. The average OD540 value in the test group was calculated as a percentage of the average OD540 value in the control group to obtain cell viability of the test group, and the results are shown in FIG. 2. Thistle extract showed no cytotoxicity up to a concentration of 200 μg / ml.

Example 3 Effect of Thistle Extract on Atopic Skin Rash in in-vivo

1) Induction of Atopic Skin Rash in NC / Nga Mice

The 7-week-old NC / Nga mice were acclimated for 1 week and then challenged with the 18-week-old NC / Nga mice, which had previously developed atopic dermatitis, in the same space for 2 weeks. After anesthesia with chloral hydrate (10%) as an anesthetic agent, the ear and dorsal neck area was cleaned with hair removal agent and left for 24 hours to heal fine wounds of the skin. And atopic dermatitis caused formulation Biostir mite antigen cream (BMAC containing 0.5% Tween 20 and mite antigen; a central laboratory animals, Dermatophagoides farinae crude extract (mite antigen , lyophilized) 2 times 100mg / per · mouse concentration at 1 week 3 Apply evenly over the back and neck area during the day, disrupting the skin layer by spraying 4% SDS solution 2-3 hours before application.

One week after BMAC application, symptoms such as erythema, papules, rinsing (dandruff), skin (scab), and thyroiditis began to appear as in atopic dermatitis in humans. It was confirmed that the skin rash was maintained until after the week Fig. 3 is a photograph showing a photograph of the raw line 2 weeks after the start of BMAC application (12 weeks old) and 2 weeks after the completion of the application (15 weeks old) with the normal group.

2) Skin smear of thistle extract

The milk thistle extract prepared in 1) was mixed with a base (a water-soluble ointment, Sama Pharmaceutical Co., Ltd.) to a concentration of 10% (w / w) to prepare a milk thistle extract ointment. BMAC was applied for 2 weeks by the method of 1), and the ointment was applied 0.2 g / mouse to the 12-week-old NC / Nga mice that induced atopic dermatitis for 3 weeks with a cotton swab on the rashes between 11 am and 12 am daily. Spread evenly. In order to more easily understand the experimental procedure for the NC / Nga mice are shown in the graph in FIG. The last positive control drug was a 1% hydrocortinsone (HC) ointment. Each experimental group used 10 NC / Nga mice.

3) sensory evaluation

Sensory evaluation was visually performed at 15 weeks of age after the application of the ointment for 3 weeks in 2), and the photograph of the result is shown in FIG.

FIG. 5A is a photograph immediately before smearing and applying for 3 weeks to ointment prepared with thistle extract in NC / Nga mice induced with atopic dermatitis according to the method of 2). Papules and scarring / dryness of the back skin were not different from those of the control group, but erythema / hemorrhage and excoriation / erosion were significantly reduced according to the smear of thistle extract. .

The smear of 1% hydrocortison used as a positive control almost eliminated erythema, papules, linseol / skin, and thyroiditis with pruritus.However, due to the side effects, the hydrocortison 1% -coated area did not grow hair compared to other experimental groups. .

4) Measurement of cytokine and histamine release in serum

Cytokines and histamine release in serum collected from NC / Nga 15-week-old mice subjected to sensory evaluation in 3) were measured by ELISA, and the results are shown in FIG. 6. In Figure 6, None is a normal mouse group, BMAC is a group of mice induced only atopic dermatitis by BMAC, Hydrocortisone is a group of mice coated with 1% hydrocortisone as a positive control, thistle is a mouse group smeared with 10% thistle extract In the following graphs, the same meanings are indicated unless otherwise stated.

In atopic patients, TH2 cells, which mainly secrete IL-4, IL-5, and IL-13, were increased in the subgroup of T helper cells compared to TH1 cells. Therefore, the degree of relaxation of atopy can be estimated by measuring the amount of such cytokines.

FIG. 6A shows IL-4 and IFN-γ levels in serum. IL-15 levels after the end of the 15-week experiment were higher than those of the control group (BMAC) in which atopic dermatitis was induced compared to the normal group (none). Markedly increased. Thistle extract decreased IL-4 levels slightly, whereas hydrocortisone increased IL-4 levels slightly but was not significant.

IFN-γ serum levels were increased in both the control and experimental groups compared to the normal group. Recent studies have reported that IFN-γ increases in microbial toxins due to infections such as skin barrier damage when atopic diseases become chronic (J Allergy Clin Immunol. 2000; 105 (5): 860-76). This may be due to the increase of serum IFN-γ in BMAC-induced atopy-induced animal models, and hydrocortisone significantly increased IFN-γ levels. Thistle also showed a slight increase in IFN-γ levels compared to the control, but was not significant.

FIG. 6B shows the levels of IL-5 and IL-13 in serum by experimental group. IL-5 is a cytokine that responds to the activation of eosinophils and can be seen to be significantly increased in the control group compared to the normal group. Staining of hydrocortisone or thistle extract reduced IL-5 levels slightly, but not significantly.

IL-13 is a cytokine produced by the activation of Th2 and mast cells, which was significantly increased in the control group compared to the normal group, but the level was significantly decreased by treatment with hydrocortisone or thistle extract.

6C is a graph showing the amount of histamine released from serum. Degranulation of activated mast cells is known to accelerate histamine release and vascular permeability to accelerate skin inflammation. Serum histamine release levels were markedly increased by BMAC treatment, but not significantly by hydrocortisone or thistle extract, but decreased by control.

These results showed that thistle extract significantly reduced the level of IL-13 and, although not significant, had a moderate effect on the release of IL-4, IL-13 and histamine, which was superior to hydrocortisone. Efficacy was shown.

5) Back skin tissue analysis and analysis of NC / Nga mice

The skin of the dorsal neck was removed from the 15-week-old NC / Nga mice that were terminated in 3) and fixed in formalin for 24 hours in 10% paraform-aldehyde, respectively. The immobilized tissues were formatted in paraffin and blocks were made 5 μm thick. For each block, infiltration of mast cells with hematoxyline / eosin (H & E) staining to identify epidermis, dermis, keratinocytes, neutrophils / eosinophil and other cells and edema and toluidine blue staining to mast cells. Nikon, Japan, x100).

FIG. 7 shows optical micrographs after H & E staining. In the 15-week-old normal mouse skin tissue, epidermis was thinly distributed, whereas atopic dermatitis was induced by BMAC, and the thickness of epidermis was increased to hyperplasia (red arrow). Hyperkeratosis, acanthosis, hypergranulosis, and parakeratosis were significantly increased compared to normal group. When hydrocortisone or thistle extract was applied to the skin rash, epidermis thickness, hyperkeratosis, acanthosis, hypergranulosis and parakeratosis were significantly reduced compared to the control group.

Figure 8 is a photograph of observation of the infiltration of mast cells by optical microscopy with toluidine blue staining. Mast cells were rarely observed in the back skin tissues of normal mice, but the infiltration of mast cells was significantly increased due to the induction of atopic dermatitis. Both hydrocortisone and thistle extracts effectively reduced mast cell infiltration in the back skin tissue of mice with atopic dermatitis.

From this result, it was confirmed that thistle extract has a therapeutic effect on skin rash by reducing epidermis thickness and mast cell invasion by atopic dermatitis.

1 is a graph showing the effect on TNF-α secretion in invitro by thistle extract.

2 is a graph showing that thistle extract does not exhibit cytotoxicity.

Figure 3 is a photograph of normal rats and mice induced atopic dermatitis by BMAC.

4 is a scheme showing an experimental design of an embodiment of the present invention.

Figure 5 is a photograph after treatment with thistle extract or hydrocortisone in atopic dermatitis induced rats.

FIG. 6 is a graph showing various cytokine and histamine release levels in serum when thistle extract was treated in mice induced with atopic dermatitis with the normal group and the comparative group.

Figure 7 is an optical micrograph of H & E stained back skin tissue when thistle extract was treated in atopic dermatitis induced rats.

Figure 8 is an optical micrograph of toluidine blue stained back skin tissue when thistle extract was treated in atopic dermatitis induced rats.

Claims (5)

Anti-atopic dermatitis composition containing thistle extract as an active ingredient. The method of claim 1, The thistle extract is an anti-atopic dermatitis composition, characterized in that the content is 0.01 to 20% by weight. The method of claim 1, The thistle extract is an anti-atopic dermatitis composition, characterized in that the extract of 60 ~ 100% C1 ~ C3 alcohol solution of thistle root. The external preparation for skin containing the anti-atopic dermatitis composition of claim 1. Cosmetics containing the anti-atopic dermatitis composition of Claim 1.

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