KR20190111554A - Compositon for Prevention or Treatment of Allergic Skin Disease comprising Tribulus terrestris L - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating allergic skin diseases, which contains a Tribulus terrestris extract as an active component. Since the Tribulus terrestris extract inhibits the activity of eosinophils and mast cells causing allergic inflammatory reactions and inhibits the secretion of inflammatory cytokines such as TNF-α and IL-4, it is possible to be usefully used for treating or preventing allergic skin diseases and being able to alleviate side effects of steroids.

Description

Pharmaceutical composition for the prevention or treatment of allergic skin disease comprising the extract of white chicken as an active ingredient {Compositon for Prevention or Treatment of Allergic Skin Disease comprising Tribulus terrestris L}

The present invention relates to a pharmaceutical composition for preventing or treating allergic skin diseases, including the extract of white chicken as an active ingredient.

Skin plays a very important role as a barrier to protect individuals from the outside. The barrier function is to protect against various stimuli (chemicals, air pollutants, dry environment, ultraviolet rays, etc.) from the outside and to prevent excessive release of moisture through the skin. When atopic dermatitis develops, the barrier function of the skin may be impaired and psychological and physical discomfort may be caused in the affected patients.

Atopic dermatitis (AD) is a chronic skin disease that occurs as a result of the production of IgE antibodies. It is severe and itching and other allergic diseases such as hives, metal allergies, asthma, rhinitis. Atopic dermatitis is one of the major diseases that have developed worldwide since the 1970s, especially in developed countries. In Korea, about 20% of elementary and middle school students suffer from atopic dermatitis.

In patients with atopic dermatitis, transepidermal water loss (TEWL) is severe and the skin barrier is disrupted, resulting in an increase in calcium ion concentration and cytokine secretion in epidermis due to changes in the filagrin and intercellular lipid composition. It is known. In addition, the vicious cycle is repeated because the decrease in skin barrier function increases the influx of external allergens, thereby increasing the chance of inducing an immune response mediated by mast cells.

Atopic dermatitis has developed various therapeutic agents because the cause of the disease is not clear, but the fundamental treatment is a difficult disease, and the treatment of atopic dermatitis mainly by using corticosteroids (steroids), antihistamines and immunosuppressants, mainly ointments and oral medicines Injectables are used. However, in the case of steroid preparations, the symptoms may be temporarily improved by suppressing the immune response, but the side effects of atopic symptoms may become more severe due to the reaction that occurs when the ointment or drug is stopped after treatment. Eventually, the steroid is not treated with weak steroids, so the use of strong steroids, the resulting harm is very serious symptoms.

On the other hand, the white girl is also called namgasae, jjiljiji, tired, the scientific name Tribulus terrestris L. Fruit is used medicinally, the fruit is divided into five, each piece has two pointed projections, the epidermis is keratin. If you are dizzy with high blood pressure, have severe headaches, and are depressed, take it to reduce blood pressure, and treat eye diseases such as redness of eyes, tears, loss of vision, and cloudiness of the cornea.

 1. J Invest Dermatol. 2002; 119; 433-439

One object of the present invention is to provide a pharmaceutical composition for preventing or treating allergic skin diseases, including the extract of white chicken as an active ingredient.

Another object of the present invention is to provide an external composition for skin reduction or improvement of side effects caused by steroid treatment for allergic skin diseases, including the white powder extract as an active ingredient.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating allergic skin diseases, which comprises whitening extract as an active ingredient.

As used herein, the term 'prevention' means that the pharmaceutical composition of the present invention is applied to allergic skin diseases so as to prevent or block an allergic reaction so that an allergic disease does not occur in advance.

As used herein, the term 'treatment' includes the cure of skin diseases as well as the partial cure, improvement and alleviation of symptoms as a result of applying the pharmaceutical composition of the present invention to allergic skin diseases.

According to one embodiment of the present invention, the white chalk extract may be prepared according to conventional methods known in the art. Specifically, after drying and pulverizing the white nitric acid, it can be prepared using a conventional solvent under the conditions of the usual temperature and pressure.

The solvent may be selected from the group consisting of distilled water, n-hexane, 1,3-butylene glycol, alcohol having 1 to 4 carbon atoms, ethyl acetate, chloroform, diethyl ether, dichloromethane, acetone, and mixtures thereof. It is preferable that it is distilled water or C1-C4 alcohol. Appropriate amount of the extractant is 0.5 to 20 times the dry weight of white nitrogen, more preferably 5 to 15 times.

On the other hand, the white gum extract includes not only the extract by the solvent extraction method described above, but also an extract that has undergone a conventional purification process. Fractions obtained through various additional purification methods, such as, for example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). It may also be included in the Baeknyeo extract of the present invention. In addition, the white itch extract may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying.

Baekilyeo extract prepared by the above-described method is a natural ingredient, there is no irritation to the human body can be used for the prevention and treatment of allergic dermatitis.

According to one embodiment of the invention, the composition may be to inhibit the activity of eosinophils and mast cells causing allergic inflammatory reactions.

As used herein, the term 'eosinophils' is a type of granulocyte, and is known to be involved in cell-mediated immune responses mainly in parasitic infections or allergic diseases. It has a high-affinity IgE receptor (FcεR I) bound to IgE antibody on the cell surface. When antigen binds to IgE, cross-linking occurs and FcεR I beneath it gathers and releases immune response mediators by degranulation. .

As used herein, the term 'mast cells' is a kind of immune cell that induces an allergic reaction and has a receptor to which an IgE antibody can bind to a membrane surface. When allergen enters the human body and binds to IgE, the mast cells that bind to IgE are activated, and the activated mast cells are degranulated and are neurotransmitters such as histamine and beta-hexosaminidase. -hexosaminidase) is released to the outside causing allergic reactions.

According to one embodiment of the present invention, the white matter extract suppresses an increase in the number of eosinophils and mast cells and reduces the degranulation of mast cells induced by IgE stimulation, thereby suppressing an allergic inflammatory response.

In addition, the white chlorophyll extract inhibits the expression of inflammatory cytokines such as TFN-α and IL-4 in the transcriptional stage, thereby suppressing an allergic inflammatory response induced by activation of eosinophils and mast cells.

Steroid preparations commonly used in the treatment of allergic skin diseases, such as hydrocortisone, can cause side effects such as weight loss and increased liver toxicity. On the other hand, the white cheese extract of the present invention can be usefully used for preventing or treating allergic skin diseases because it effectively inhibits allergic reactions without the above side effects.

The pharmaceutical composition comprising the white chlorophyll extract according to the present invention may be used alone or in combination with methods using surgery, hormonal therapy and drug therapy for the prevention and treatment of allergic skin diseases.

According to one embodiment of the present invention, when using a combination of hydrocortisone and the pharmaceutical composition compared to the treatment of hydrocortisone alone, since the use of significantly lower concentrations of hydrocortisone, side effects of steroids are reduced and allergic Can effectively treat skin diseases.

According to one embodiment of the invention, the allergic skin disease may be selected from the group consisting of atopic dermatitis, psoriasis, contact allergy and urticaria.

On the other hand, the pharmaceutical composition for preventing or treating allergic skin diseases may further include a pharmaceutically acceptable carrier in addition to containing the white baekryeum extract as an essential ingredient. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and that, when administered to a human, typically does not cause allergic or similar reactions, such as gastrointestinal disorders, dizziness, and the like. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, and parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. And the like may further comprise stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). The pharmaceutical composition according to the present invention may be formulated in a suitable form according to methods known in the art together with the pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention may be prepared in various parenteral or oral administration forms according to known methods. As representative of formulations for parenteral administration, isotonic aqueous solutions or suspensions are preferred for injectable formulations. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

Pharmaceutical compositions formulated in such a manner can be administered in a effective amount via several routes, including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, an 'effective amount' refers to an amount that exhibits a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected depending on the route of administration, subject to administration, age, gender weight, individual difference and disease state.

Another aspect of the present invention provides a skin external preparation composition for reducing or ameliorating side effects caused by steroid treatment for allergic skin diseases, including an extract of white chicken as an active ingredient.

Since the external preparation composition for skin is using the same white extract as the pharmaceutical composition for preventing or treating allergic skin diseases including the above-described white extract, the common content between the two is described in order to avoid excessive complexity of the present specification. Omit.

Side effects caused by steroid treatment for allergic skin diseases may include weight loss, vasodilation, osteoporosis, increased concentrations of glutamate-oxaloacetate transminase (GOT) and glutamate-pyruvate transminase (GPT).

According to one embodiment of the present invention, the topical skin composition may inhibit weight loss due to steroid treatment, and may suppress an increase in the concentration of GOT and GPT (IU / L). If the liver is damaged by an inflammatory response, the enzymes in the hepatocytes can flow into the blood, increasing the concentration of GOT and GPT.

The external composition for skin preparations preferably contains 0.001 to 30% by weight, preferably 0.01 to 20% by weight, based on the total weight of the composition. If it is less than 0.001%, it may be difficult to expect the efficacy of the white cheese extract, and if it exceeds 30% by weight may cause problems in the stability of the formulation.

The external preparation composition for skin containing the white chlorophyll extract of the present invention is used for the purpose of reducing or improving side effects caused by steroid treatment, and is not particularly limited in the formulation thereof. For example, it may be a cosmetic composition having a soft cosmetic lotion, a nourishing lotion, a cream, a pack, a gel, or a skin adhesive type formulation. It may also be a transdermal dosage form such as an ointment, gel, cream, patch or spray.

The ointment, gel, and cream may further contain a carrier component in addition to the extract of white chicken as an active substance. For example, it may contain animal and vegetable fats, wax paraffins, starches, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide or mixtures of these substances, the components being introduced in amounts generally used. Can be.

The spray agent may contain lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder as carrier components, and additionally typically contain a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether, and the like. can do. The components can be introduced in amounts generally used.

The method of administering the external preparation composition for skin may be generally applied to the affected part, and a preferred method of administration may be selected according to the dosage form. In addition, the external preparation composition may be administered in combination with a steroid for the purpose of treating allergic skin diseases.

According to one embodiment of the present invention, the white blood extract inhibits the activity of eosinophils and mast cells causing allergic inflammatory reactions, and inhibits the secretion of inflammatory cytokines such as TFN-α and IL-4, thereby allergic skin diseases. It can be usefully used for therapeutic and prophylactic purposes, and can be used for reducing or improving side effects of steroids.

Figure 1 shows the results confirming the degranulation inhibitory effect of the extract of white baekryum in eosinophilic mast cells.
Figure 2 shows the results confirming the inhibitory effect of the inflammatory allergic reaction of the white blood extract in eosinophilic mast cells.
Figure 3 schematically shows a process for producing atopic dermatitis mouse according to an embodiment of the present invention.
Figure 4 shows the results of measuring the transdermal moisture loss after applying the white vaginal extract to atopic dermatitis mice.
Figure 5 shows the results of confirming the degree of skin lesions after the application of the white cheese extract to atopic dermatitis mice.
Figure 6 shows the results confirmed histological changes and mast cell infiltration in the skin tissue of atopic dermatitis mice to which the white blood extract was applied.
Figure 7 shows the results of confirming the change in the number of eosinophils and mast cells in the skin tissue of atopic dermatitis mice to which the white blood extract was applied.
Figure 8 shows the results of confirming the degree of T cell infiltration and the activity of the ion channel protein in the skin tissue of atopic dermatitis mice coated with white chicken extract.
Figure 9 shows the results of measuring the body weight and GOT / GPT levels in atopic dermatitis mice to which the white gum extract was applied.

Hereinafter, one or more embodiments will be described in more detail with reference to Examples. However, these examples are provided to illustrate one or more embodiments illustratively and the scope of the present invention is not limited to these examples.

Example  One: White girl  Preparation of Extract

The dry bag niece (The fruits of Tribulus terrestris L, Tribuli Fructus, Leguminosae; Hereinafter referred to as TF) to obtain a powder. 1,000 ml of 30% ethanol was added to 100 g of white nitric acid powder, stirred for 3 hours, and the extract solution was filtered to prepare a white nitric acid 30% ethanol extract (hereinafter referred to as TF extract).

Example  2: White girl  Evaluation of allergic reaction inhibitory effect of extract in vitro )

2-1: eosinophils Degranulation ( degranulation ) Inhibitory efficacy measurement

Rat basophilic leukemia cell lines (RB-2H3), which are eosinophilic mast cells, were cultured in 96-well plates and cultured with DNP-IgE (HNP) and human serum albumin (HSA) to induce degranulation of eosinophils. Treated and incubated again for 16 hours. Next, the TF extract was treated with concentrations of 0, 0.1, 0.2 and 0.5 mg / ml, or incubated for 3 hours by treatment with 30 μM / ml of DSCG (disodium crompglycate; Cromolyn) known as an anti-histamine. After incubation, DNP-BSA was added and left at 37 ° C. for 1 hour, and 0.05 M sodium bicarbonate buffer (pH 10.0) was added to terminate the reaction. The absorbance was measured at 450 nm to calculate the secretion amount of beta-hexosaminidase, the cells were stained with toluidine blue, and the degree of degranulation of eosinophils was observed under a microscope.

As a result, as shown in Figure 1 TF extract was found to reduce the secretion of the degranulase beta- hexosaminidase concentration-dependently at a concentration without cytotoxicity. In particular, when TF extract 0.5 mg / ㎖ treated with beta- hexosaminidase secretion was reduced to a similar level as the DSCG treatment. In addition, it was confirmed that inducing the mast cell stabilization by inhibiting the degranulation of eosinophils. In FIG. 1A, a means a significant value compared to a normal group, b means a significant value compared to an IgE control group, and ** indicates p <0.01 and * indicates p <0.05. Arrows in FIG. 1B refer to cells in which degranulation has occurred.

2-2: Determination of Inflammatory Allergic Reaction Efficacy

IgE stimulation was induced in RBL-2H3 cells in the same manner as in Example 2-1, and TF extract was incubated with 0, 0.1, 0.2 and 0.5 mg / ml concentrations. After the incubation, the cells were recovered, and RT-PCR (reverse transcriptase-polymerase chain reaction) confirmed the expression changes of inflammatory cytokines TNF-α and Th2 cytokine IL-4.

As a result, as shown in FIG. 2, the expression of TNF-α and IL-4 increased by IgE stimulation, but decreased by TF extract treatment. The experimental results indicate that TF extract can control allergic inflammatory responses due to eosinophil and mast cell activation in the transcriptional stage by inhibiting the gene expression of TNF-α and IL-4.

Example  3: White girl  Evaluation of Atopic Dermatitis Treatment Efficacy in vivo )

3-1: Atopic Dermatitis Mouse Production and White girl  Extract application

SKN-1 hairless mice were used to prepare atopic dermatitis mice (hereinafter referred to as AD mice).

SKN-1 hairless mice were purchased from Raon Bio (Yongin, Republic of Korea), and after one week of adaptation at the experimental animal center (23 ° C, 12 hour contrast cycle, 60 ± 10% humidity and free feeding conditions), Was used. After the adaptation period, 50 μl of 5% oxazolone was applied to the dorsal skin of the mouse once for 8 days. After 8 days, 60 μl of 0.5% oxazolone was applied once every other day, for a total of 9 times up to 24 days.

TF extract alone (hereinafter referred to as' 1% TF treated group '), 1% hydrocortisone (Sigma-Aldrich co., H4001-16, USA; below;' 1% HC) from the 18th day of the experiment Treatment group) alone or a mixture of 1% TF and 0.1% HC (hereinafter referred to as '1% TF + 0.1% HC combination group') was applied to the site where oxazolone was applied 24 to 24 days. It was treated once daily until day. Five mice were used for each experimental group, and mice were sacrificed 24 days after the start of the experiment to evaluate the combined treatment efficacy of TF extract and HC. AD mouse manufacturing process and TF extract and HC treatment process is schematically shown in FIG.

3-2: Determination of Water Loss in AD Mice

In order to confirm the effect of the white vaginal extract on atopic dermatitis, the water content of the back skin of the AD mouse was confirmed by a transepidermal water loss (TEWL) analyzer.

As a result, as shown in Figure 4 it was confirmed that the water loss was reduced in the 1% TF treatment group compared to the control group (AD), the water loss was more effectively suppressed in the 1% TF + 0.1% HC combination treatment group. Could know.

In Figure 4 Nor is normal mouse (normal mouse); AD is a control group that induced atopy by treating oxazolone in hairless mice; 1% TF was experimental group treated with 1% TF extract in AD mice; 1% TF + 0.1% HC refers to the experimental group treated with 1% TF extract and 0.1% HC in AD mice, and 1% HC refers to the experimental group treated with 1% HC in AD mice. In addition, *** means p <0.001 compared with normal mouse (a) or AD mouse (b).

3-3: Determination of the skin symptom score in AD mice

Three blinds were scored by measuring the degree of back skin lesions in AD mice. As a result, as shown in FIG. 5, it was confirmed that the atopy symptom was improved in the 1% TF treatment group compared to the control group (AD), and the atopy symptom was remarkably improved in the 1% TF + 0.1% HC combination treatment group. Could know.

Table 1 describes the degree of lesion according to the score.

score Symptom 0 no symptom One Mild (dryness, scaling) 2 Moderate (dryness, scaling, and erosion) 3 Middle (dryness, scaling, erosion, and excoriation) 4 Severe (dryness, scaling, erosion, excoriation, and hemorrhage)

3-4: of skin from AD mouse Histopathological  Observe changes

After sacrificing the AD mouse prepared in Example 3-1, skin tissue was separated, and the separated skin tissue was fixed with 4% paraformaldehyde. The immobilized skin tissue was embedded in paraffin to produce a paraffin block, and the paraffin block was sectioned to prepare a skin tissue slide. Skin tissue slides were stained with hematoxylin & eosin or toluidine blue according to the manufacturer's instructions for microscopic observation of skin histological changes and mast cell infiltration. In addition, the increase and decrease of the number of eosinophils and mast cells was observed.

As a result, as shown in FIG. 6, it was confirmed that the degree of change in skin tissue was reduced in the 1% TF treatment group, and the 1% TF + 0.1% HC combination treatment group was more effective. In Figure 6, A is the result of staining with hematoxylin & eosin, B is toluidine blue.

In addition, as shown in Figure 7, it was confirmed that the increase in the number of allergic inflammation-induced immune cells eosinophils and mast cells in the 1% TF treatment group was suppressed. In FIG. 7, *** means p <0.001 and * is p <0.05 as compared with normal mouse (a) or AD mouse (b).

3-5: T cell infiltration and ion channel protein expression in AD mouse skin tissue

Since the activity of the ion channel protein Orai-1 is increased when the skin barrier is damaged, it was confirmed whether the change of the activity of Orai-1 occurs by applying the TF extract.

Specifically, paraffin was removed from the skin tissue slide prepared in Example 3-4 using xylene, and hydrated with alcohol. The slides were then washed with PBS, immersed in epitope-retrieval buffer and allowed to react at 95 ° C. for 20 minutes. Next, the slide was immersed in a blocking solution containing 10% goat serum (goat serum) and reacted for 30 minutes at room temperature. The slides were then reacted overnight at 4 ° C. with a CD3 antibody or Orai-antibody diluted with blocking solution. The next day, the biotinylated secondary antibody and the slides were reacted at room temperature for 1 hour and washed with PBS. After washing, the mixture was reacted with 3, 3'-diaminobenzidine tetra hydrochloride (DAB), and counter staining was performed with hematoxylin. After dropping the acid solution on the slide and covering the coverslips, the presence of CD3 + T cells infiltrated using an optical microscope.

As a result, as shown in FIG. 8, in the case of the 1% TF treatment group, the infiltration level of CD3 + positive T cells in the skin epidermis was decreased. In addition, it was found that the expression of Orai-1, an ion channel protein activated during skin barrier damage, was reduced, and the effect was more marked in the 1% TF + 0.1% HC combination group. In FIG. 8, A is stained with a CD3 antibody, and B is stained with an Orai-1 antibody.

3-6: Identification of weight loss inhibitory effects and liver toxicity indicators in AD mice

To determine whether the TF extract can reduce the side effects of HC, the body weight was measured before the sacrifice of the mice, and blood was collected to measure GOT / GPT (glutamic oxaloacetic transaminase / glutamic pyruvic transaminase) levels.

As a result, as shown in FIG. 9, the 1% HC-treated group significantly reduced the weight of the mouse compared to the normal group and the control group, and significantly increased the GOT / GPT level, which is an indicator of liver toxicity. On the other hand, in the 1% TF treatment group or the 1% TF + 0.1% HC combination group, no weight loss effect or GOT / GTP levels could be observed.

The experimental results showed that the 1% HC treatment group had excellent atopic dermatitis improvement effects (moisture loss, symptom improvement, tissue damage suppression, etc.) but could be accompanied by side effects of systemic weakness by causing weight loss and hepatotoxicity. have. However, when treating TF alone or in combination with TF and HC it can be seen that there are almost no side effects.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (9)

Pharmaceutical composition for the prevention or treatment of allergic skin diseases, including white extract as an active ingredient.
The method of claim 1, wherein the white nitrite extract is distilled water, n-hexane, 1,3-butylene glycol, alcohol having 1 to 4 carbon atoms, ethyl acetate, chloroform, diethyl ether, dichloromethane, acetone and mixtures thereof The composition is extracted with a solvent selected from the group consisting of.
The composition of claim 1, wherein the composition inhibits the activity of eosinophils and mast cells causing allergic inflammatory reactions.
The composition of claim 1, wherein the composition inhibits the expression of inflammatory cytokines.
The composition of claim 1, wherein the allergic skin disease is selected from the group consisting of atopic dermatitis, psoriasis, contact allergy and urticaria.
A skin external preparation composition for reducing or improving side effects caused by steroid treatment for allergic skin diseases, which comprises white extract as an active ingredient.
The method according to claim 6, wherein the white nitrite extract is distilled water, n-hexane, 1,3-butylene glycol, alcohol having 1 to 4 carbon atoms, ethyl acetate, chloroform, diethyl ether, dichloromethane, acetone and mixtures thereof The composition is extracted with a solvent selected from the group consisting of.
The composition of claim 6, wherein the allergic skin disease is selected from the group consisting of atopic dermatitis, psoriasis, contact allergies and urticaria.
The composition of claim 6, wherein the composition reduces the concentration of glutamate-oxaloacetate transminase (GOT) or glutamate pyruvate transminase (GPT).

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