KR102088329B1 - Pharmaceutical composition for prevention and treatment of pruritus comprising Isodon serra extract as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, health functional food or a cosmetic composition for preventing or treating pruritus or itching containing an Isodon serra extract as an active component. The Isodon serra extract provided in one aspect of the present invention excellently suppresses the expression of IL31, which is a factor involved in itching, and inhibits the secretion of chemokine involved in IL31, thereby having a very excellent effect of alleviating pruritus. When using a cosmetic composition containing the Isodon serra extract as the active component, there are effects of increasing a water content, reducing the amount of transepidermal water loss, reducing skin keratin, alleviating skin itching symptoms, etc. Thus, the present invention can significantly contribute to related industries such as cosmetics that can alleviate pruritus.

Description

Pharmaceutical composition for prevention and treatment of pruritus comprising Isodon serra extract as active ingredient}

It relates to a pharmaceutical composition for preventing or treating containing a self-propelled extract as an active ingredient.

Pruritus, also known as itching, refers to an unpleasant skin sensation that causes the urge to scratch or rub the skin. Such pruritus is a symptom that anyone can easily experience and encounter. Recently, people who complain of pruritus are increasing.

Pruritus is caused by weak stimulation of the skin nerves, skin diseases such as xeroderma, pathogen infestation and insect bites, atopic dermatitis, contact dermatitis, hives, and chronic liver disease, It is a common symptom in systemic diseases such as chronic kidney failure, Hodgkin's disease, diabetes (diabetes mellitus), obstructive biliary disease, and uremia. Frequently, pruritus is caused by external environmental stimuli, but it can also occur without external stimuli.

Pruritus occurs in keratinocytes of the epidermis and is transmitted to non-myelinated C nerve fibers (non-myelinated nerve fibers, non-myelinated nerve fibers, unmyelinated amyelinoned nerve fibers). When a number of neurotransmitters and receptors related to pruritus or pain are expressed, it is recognized by the afferent C nerve fibers under the epidermis (afferent amylinoned nerve fibers, afferent amyelinoned nerve fibers), and the 'external spinal cord. It is transmitted to the thalamus and sensory cortex of the brain via the sagittal pathway ', causing pruritus.

It can be classified into four types: pruritoceptive itch, neurogenic itch, neuropathic itch, and psychogenic itch. Receptive pruritus occurs due to damage to the skin barrier or inflammation of the skin, and neurotic pruritus occurs as a result of neurochemical activity through mediators acting on the central nervous system without nerve damage and primary skin lesions. Neuropathic pruritus refers to secondary pruritus according to mental illness.

Histamine is a representative medium for pruritus. This substance is in mast cells, and upon specific stimulation, it releases histamine to swell and itch the skin. Antihistamines inhibit this histamine, have little effect on chronic pruritus, have an effect on acute pruritus, and have recently been reported to have serious side effects.

Recently, the problem of antihistamine has emerged, and interest in a new form of pruritus mechanism not related to the antihistamine pruritus mechanism has increased and is currently being actively studied. In particular, specific neuropathy causing pruritus has been studied. Among them, research on the action and regulation of the inflammatory response affecting chronic pruritus is ongoing. It has been reported that gastrin-releasing peptide receptor (GRPR) acts as a specific neurotransmitter pathway in pruritus in the spinal cord, and IL31 secreted from T cells is also increasing interest as a new mediator for itching.

IL31 is mainly produced in T lymphocytes, and IL31 is known to act directly on receptors in skin nerve fibers, causing pruritus. It is also known that IL31 plays a role in skin barrier disorders. In the mouse atopic dermatitis model, it was found that when the skin lesion occurred, the frequency of scratching was significantly reduced when anti-IL31 antibody was administered. This supported IL31 as an important vehicle for itching.

On the other hand, chemokine (Chemokine) has been reported as a factor regulating IL31 in other studies. Chemokines are responsible for bringing specific cells to specific sites. That is, when a specific chemokine is expressed, various types of cells collected by the chemokine move toward the cell expressing the chemokine. It is known to act as a kind of aggregate signal. Chemokines include CCL17 / TARC (T-cell recovery and trigger) (Exp Dermatol. 2004 Apr; 13 (4): 265-71), RANTES (eosinophil and T cell migration / activation) (OPINION Volume 22, Issue 2, p83 -87, february 01, 2001). These substances are also produced in keratinocytes. In particular, it is highly expressed in the skin of patients with atopic dermatitis or psoriasis.

IL31, unlike other itching triggers, does not affect inflammation-mediated immune cells and acts only on keratinocytes and sensory nerves and is involved in itching.

It has been reported that IL31 is only involved in pruritus, not inflammation. Rats injected with IL31 significantly increased the frequency of scratching, whereas rats with IL31 or anti-IL31 receptors that nullified previously injected mice significantly reduced the frequency of scratching. However, the degree of inflammation showed little difference. (Ayako Takamori et al. IL-31 is crucial for induction of pruritus, but not inflammation, in contact hypersensitivity.Scientific reports, (2018))

Therefore, IL-31 has emerged as an effective factor in the development of treatments for pruritus that does not cause inflammation, such as itching of neuropathy or neuropathy. (Stephan Weidinger et al. Atopic dermatitis.Nature review, (2018) 4: 1)

Treatments for pruritus developed to date include topical or oral treatments such as moisturizers, topical steroids, anesthetics, antihistamines, antidepressants, and anti-opioid agents. However, they have been reported to exhibit significant side effects-resistant symptoms, insomnia, constipation, drowsiness, elevated prolactin, etc. Accordingly, there is an urgent need for the development of a new treatment for pruritus, which does not have any of the side effects mentioned above and has excellent relief or treatment effect.

The present inventors inhibit the expression of IL31, a factor that is involved in itching, and inhibit the secretion of chemokine involved in IL31, reduce the amount of transepidermal water loss, reduce skin keratin, and relieve skin itching symptoms It was confirmed that it has an excellent effect on pruritus.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating pruritus, a health functional food composition for preventing or improving pruritus, and a cosmetic composition for alleviating or improving pruritus, which contains an Isodon serra extract as an active ingredient. will be.

In order to achieve the above object,

An object in one aspect of the present invention is to provide a pharmaceutical composition for preventing or treating pruritus containing an extract of Isodon serra as an active ingredient.

An object in another aspect of the present invention is to provide a health functional food composition for preventing or improving pruritus containing an extract of Isodon serra as an active ingredient.

An aspect of the present invention provides a cosmetic composition for alleviating or improving pruritus containing an extract of Isodon serra as an active ingredient.

The pharmaceutical composition comprising the self-propelling extract provided as an active ingredient in the present invention excellently suppresses the expression of IL31, a factor involved in itching, and suppresses the secretion of chemokines involved in IL31, so it is very effective in improving pruritus. It has an excellent effect, and the use of a cosmetic composition containing the self-propellant extract as an active ingredient has an excellent effect in increasing the moisture content, reducing the amount of transepidermal water loss, reducing skin keratin and alleviating skin itching symptoms.

Figure 1 shows the results of measuring the cytotoxicity in the cell line EL4 cells of the self-propelled grass extract prepared in Example 1, it can be seen that there is no cytotoxicity even at a high concentration of self-propelled grass extract.
Figure 2 shows the results of measuring the cytotoxicity in the cell line HaCaT cells of the self-propelled extract prepared in Example 1, it can be seen that there is no cytotoxicity even at a high concentration of self-propelled extract.
Figure 3 shows the results of confirming the regulation of the secretion of Rantes and Tarc among the chemokine (Chemokine) in the keratinocyte cell line extract prepared in Example 1, it was confirmed that the oleander extract extract inhibits the secretion of chemokine in a concentration-dependent manner You can.
Figure 4 shows the results confirming the effect of the self-propellant extract prepared in Example 1 on the IL31 gene promoter, it can be seen that self-propelled extract extract inhibits the electron-promoting activity of IL31 in a concentration-dependent manner.
Figure 5 shows the results confirming the effect of the self-propelled extract prepared in Example 1 on IL31 gene regulation in T cells, it can be confirmed that the self-propelled extract extract inhibits the expression of IL31 gene in a concentration-dependent manner.
FIG. 6 shows the results confirming the effect of the self-propelled extract prepared in Example 1 on the regulation of IL31 protein in T cells, and it can be confirmed that the self-propelled extract extract inhibits IL31 protein expression.
7 is a graph showing the results of confirming the change in skin moisture content (AU) before, after 2 weeks of use, and after 4 weeks of use of the self-propelled cream prepared in Example 4, and the self-propelled cream is used for skin moisture content It can be seen that it is effective in increasing.
8 is a graph showing the results of confirming the moisture loss amount (g / m ² h) of the transdermal epithelium before, 2 weeks after and 4 weeks after use of the self-propelled cream prepared in Example 4. It can be seen that the cream is effective in reducing the amount of water loss in the epidermis.
9 is a graph showing the results of confirming the change in skin keratin (%) before, after 2 weeks of use, and after 4 weeks of use of the self-propelled cream prepared in Example 4, and the self-propelled cream was used to reduce skin exfoliation. You can see that it works.
Figure 10 shows the results of taking a change in skin keratin before and after use, 2 weeks after use, and 4 weeks after use of the self-propelled cream prepared in Example 4 with Visiosacan VC98, self-propelled cream is effective in reducing skin exfoliation You can confirm that there is.
11 is a graph showing the results of confirming the degree of skin itching improvement (cm) before, after 2 weeks of use, and after 4 weeks of use of the self-propelled cream prepared in Example 4, and the self-propelled cream improves skin itching You can see that it works.

Hereinafter, the present invention will be described in detail.

Isodon serra is often confused with Isodon japonica . However, the medicinal herb, also called Yeonmyeongcho, has been studied a lot, unlike the self-propelled herb, and the efficacy of anti-cancer, immune system, hair growth, etc. has been reported. According to a paper published by Hebei Medical University in China, four of the 27 components of the self-propellant pool analyzed by chromatography and the 24 components of the self-propelled pool (Ponicidin, Enmenol, Lasiodonin, Sodoponin) It is a plant that has a distinctly distinct composition (Chromatographia 2010, 72, August (No. 3/4); RSC Adv., 2019, 9, 1403-1418). In addition, the length of the calyx, petals, and stamens are different, so they can be distinguished by appearance.

The self-propelled grass is a perennial plant of the dicotyledonous plant, the honeydew family, and blooms in purple in September-October and grows to about 1 m. Self-propelled grass is known to help in the treatment of jaundice, cholecystitis, diarrhea, shigellosis, furuncle, and distortion pain in oriental medicine, but relieve or improve pruritus. Has not been studied.

The self-propellant extract provided in one aspect of the present invention does not show cytotoxicity even in high concentration extracts in lymphocyte EL4 cells (Experimental Example 1, FIG. 1), and did not show cytotoxicity despite high concentrations in the skin keratinocyte cell line HaCaT cells. (Experimental Example 2, Figure 2). In an experiment to prove the effect of alleviating or improving pruritus of the self-propelling plant extract, it was confirmed that it inhibits the secretion of chemokine that regulates IL31, a factor involved in pruritus (Experimental Example 3, FIG. 3), and promotes transcription of IL31. Was excellently inhibited (Experimental Example 4, FIG. 4), as well as effectively inhibiting IL31 gene expression (Experimental Example 4, FIG. 5), and was confirmed to suppress IL31 protein excellently (Experimental Example 6, FIG. 6). .

As an example of a cosmetic composition containing an extract of self-propelled grass provided as an active ingredient in one aspect of the present invention, the cream increases the moisture content through a panel test of 23 adults with dry skin or medium dry skin (Experimental Example 7 , FIG. 7), decrease in transepidermal water loss (Experimental Example 8, FIG. 8), reduction in skin keratinization (Experimental Example 9, FIG. 10), improvement of skin itching (Experimental Example 11, FIG. 11), effectiveness and preference (Experimental Example 11) And 12) In the experiment, a significant effect could be confirmed.

One aspect of the present invention provides a pharmaceutical composition for prevention or treatment of pruritus containing an extract of Isodon serra as an active ingredient.

At this time, the extract may be extracted with water, a lower alcohol of C1 to C2, or a mixed solvent thereof. If it is an extraction solvent that may contain the self-propelled grass as an active ingredient and may indicate the purpose of alleviating or improving pruritus of the self-propelled grass Without being limited thereto, water, alcohol, ethyl acetate, glycerin, ethylene glycol, propylene glycol, butylene glycol, and mixed solvents thereof (for example, hydrous alcohol, hydrous glycerin, hydrous ethylene glycol, hydrous propylene glycol, hydrous butylene) Glycol).

The composition inhibits the expression of IL31 (Interleukin 31), a factor that causes itching.

The composition may be to inhibit the secretion of chemokine (Chemokine), the chemokine may be Rantes (Regulated on Activation, Normal T Cell Expressed and Secreted) or Tarc (Thymus and activation-regulated chemokine), which modulates IL31 If it is an element, it is not limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive is starch, gelatinized starch, microcrystalline cellulose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opary, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, stearic acid Aluminum, calcium stearate, white sugar, dextrose, sorbitol and talc may be used, but are not limited thereto.

The pharmaceutical composition of the present invention may be administered orally or parenterally according to a desired method, and when administered parenterally, it is used for external or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. It is preferable to select an injection method.

In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Another aspect of the present invention provides a health functional food composition for preventing or improving pruritus containing an extract of Isodon serra as an active ingredient.

The mixed amount of the self-propelling extract of the health functional food composition according to the present invention may be appropriately determined according to its purpose of use (for prevention or improvement). The form and type of health functional food are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids and pills. The health functional food may include various flavors, sweeteners or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include tau martin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. Further, the natural carbohydrate may be monosaccharide, disaccharide, polysaccharide, oligosaccharide and sugar alcohol.

The health functional food of the present invention, in addition to the additional ingredients described above, nutrients, vitamins, electrolytes, flavoring agents, colorants, pectans and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives , Glycerin, alcohol, and the like. These ingredients can be used independently or in combination. The proportion of the additive may be selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Another aspect of the present invention provides a cosmetic composition for alleviating or improving pruritus containing an extract of Isodon serra as an active ingredient.

At this time, the cosmetic composition may be prepared in any formulation that is conventionally prepared in the art, for example, cream, emulsion, lotion, pack, foundation, hair cosmetic and the like. Specifically, the cosmetic composition of the present invention is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, Contains formulations of pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser.

Hereinafter, examples and experimental examples of the present invention will be specifically illustrated and described below. However, the Examples and Experimental Examples described below are merely illustrative of a part of the present invention, but are not limited thereto.

<Example 1> Preparation of self-propelled grass extract

930 g of shaded self-propelled grass was extracted with 100% methanol in a volume 10 times the amount of the sample at room temperature to obtain an extract. Subsequently, the extract was filtered, and the extracts obtained by repeating these extraction and filtration processes were collected and concentrated under reduced pressure at 50 ° C (EYELA N-1200B). Freeze-dried the filtrate obtained by concentration under reduced pressure to obtain 27.59 g of self-propelled grass extract. Obtained. This extract was dissolved in 100% dimethyl sulfoxide (DMSO) and used in the experiment.

<Example 2> Preparation of self-propelled grass extract

930 g of shaded self-propelled grass was extracted with 100% ethanol in a volume 10 times the amount of the sample at room temperature to obtain an extract. Subsequently, the extract was filtered, and the extracts obtained by repeating these extraction and filtration processes were collected and concentrated under reduced pressure at 50 ° C (EYELA N-1200B). The extract filtrate concentrated under reduced pressure was freeze-dried to obtain 27.59 g of a self-propelling extract. This extract was dissolved in 100% dimethyl sulfoxide (DMSO) and used in the experiment.

<Example 3> Preparation of self-propelled butylene glycol extract

930 g of shaded self-propelled grass was extracted with 100% butylene glycol in a volume 10 times the amount of the sample at room temperature to obtain an extract. Subsequently, the extract was filtered, and the extracts obtained by repeating these extraction and filtration processes were collected and concentrated under reduced pressure at 50 ° C (EYELA N-1200B). The extract filtrate concentrated under reduced pressure was freeze-dried to obtain 27.59 g of a self-propelling extract. This extract was dissolved in 100% dimethyl sulfoxide (DMSO) or 40% butylene glycol and used in the experiment.

<Experimental Example 1> Cytotoxicity measurement in cell line EL4 cells

In order to evaluate the cytotoxicity of the self-propellant extract prepared in Example 1, a WST assay was performed. EL4 cells (T lymphocytes) used in the present invention are 10% fetal bovine serum (FBS) and antibiotic-antimycotics (PSF: 100 units / ml sodium penicillin G, 100 μg / ml streptomycin, and 250 ng / ml amphotericin B) in RPMI (Rosewell Park Memorial Institue) 1640 medium, dispensed to ⁴ ⅹ 10 ⁵ cells / ml per well in a 24 well plate, and diluted samples (10, 20 μg / ml) for 24 hours Treatment. After 24 hours, WST-1 reagent was added to each well, and after 2 hours, absorbance was measured at 450 nm by ELISA. The results are shown in FIG. 1.

As a result, as shown in FIG. 1, the self-propelled extract showed a survival rate of about 90% even at high concentration, and thus it was confirmed that there was no cytotoxicity.

<Experimental Example 2> Measurement of cytotoxicity in skin keratinocyte cell line HaCaT cells

In order to evaluate the cytotoxicity of the self-propellant extract prepared in Example 1, an MTT assay was performed. Cytotoxicity was tested by MTT [3- (4,5-dimethylthiazol-2-yl) -2, 5-diphenyl-tetrazolium bromide] method. MTT assay is a method to investigate the proliferation or toxicity of cells through the measurement of formazan produced by the intracellular mitochondrial dehydrogenase, 10% FBS, 1% HaCaT used in the present invention The cells were passaged in DMEM (Dulbecco's Modified Eagle's Medium) medium containing antibiotic-antimycotics (antibiotics-antimycotics). After dispensing at ² plate 10 ⁵ cells / ml per well in a 6 well plate, it was cultured for 24 hours in a 37 ° C CO incubator. Afterwards, 10 and 20 μg / mL of the self-propelling extract dissolved in 100% DMSO in HaCaT cells were pre-treated for 6 hours and treated with IFN-γ (10 ng / mL) and TNF-α (10 ng / mL). Then, after 24 hours, the culture solution was discarded, and a culture solution containing MTT (Sigma-Aldrich, MO, USA) (0.5 mg / mL) was added and 1 mL per well was cultured for 3 hours. After the cultivation was completed, the culture solution was discarded, washed three times with PBS, DMSO was added 2 mL per well, allowed to stand at room temperature for 5 minutes, and the absorbance of the dissolved formazan MTT was measured at 570 nm with an ELISA reader. The results are shown in FIG. 2.

As a result, as shown in FIG. 2, the self-propelled extract showed a survival rate of about 90% even at high concentration, and thus it was confirmed that there was no cytotoxicity.

<Experimental Example 3> Confirmation of chemokine secretion regulation in keratinocyte line (ELISA)

An enzyme-linked immunosorbent assay (ELISA) analysis was performed to confirm the control of chemokine secretion for the self-producing oleander extract prepared in Example 1. The chemokines confirmed expression in Example 4 are Tarc (Thymus and activation-regulated chemokine) involved in the recovery and trigger of T-cells, and Rantes (Regulated on Activation, Normal T Cell Expressed) involved in the movement and activation of eosinophils and T cells. and Secreted). It is known that such chemokines are expressed in large amounts in the skin of psoriasis patients. HaCaT used in the present invention was cultured in DMEM medium containing 10% FBS, 1% antibiotic-antimycotics, and the like. After dispensing in a 6 well plate at 2/10 ⁵ cells / ml per well, the cells were cultured for 24 hours in a 37 ° C, 5% CO₂ incubator. Afterwards, 10 and 20 μg / mL of the self-propelled extracts dissolved in 100% DMSO in HaCaT cells were pre-treated for 6 hours and treated with IFN-γ (10 ng / mL) and TNF-α (10 ng / mL). Then, after 24 hours, the supernatant of the cells was collected and tested. The cytokine of Tarc and Rantes was measured using an ELISA kit sold by abcam. The results are shown in FIG. 3.

As a result, as shown in FIG. 3, it was confirmed that the self-propelled extract reduces the amount of Rantes and Tarc in a concentration-dependent manner in the HaCaT keratinocyte culture medium. Specifically, when 10 μg / mL was treated with the self-propelled extract, Rantes expression was almost half lower than that of the control group, and when treated with 20 μg / mL, it was reduced by about 66% or more. When treated with 20 μg / mL of oleander extract, Tarc expression was also reduced by more than about 40% compared to the control group. Therefore, it can be seen that the self-propelling extract according to Example 1 of the present invention is effective in reducing dry skin or pruritus by reducing the expression of chemokines such as Rantes and Tarc.

<Experimental Example 4> Check the effect on IL31 gene promoter

EL4 cells were adjusted to ⁴ 가지고 10 ⁵ cells / ml per well in a 24 well plate with RPMI 1640 medium containing 5% FBS, 0.5 μg of plasmid with a luciferase attached IL promoter region, pRRL, and Fugene (Promega) was mixed in RPMI medium and transfected for 6 hours, followed by pretreatment of diluted samples (10, 20 μg / mL) for 1 hour and treatment with PMA (50 μg / mL) and Ionomycin (1 μM). After incubation for 24 hours. After that, the cells were collected and suspended in 1ⅹ Passive Lysis Buffer (E194A, Promega, Madison, WI) for 15 minutes and then mixed with a Luminometer (Cetro LB 960, Berthold, using Dual Luciferase Kit (E1910, Promega, Madison, WI)). Luciferase activity was measured with Geremany), and transfection efficiency was corrected with pRL luciferase activity. The results are shown in FIG. 4.

As a result, as shown in FIG. 4, it was confirmed that when the IL31 promoter plasmid was introduced into EL4 T cells, the luciferase activity increased compared to the control group that did not. So, in the state in which the IL31 promoter plasmid was introduced into EL4 cells, the self-propelling extract was added and the promoter activity was confirmed. As a result, it was confirmed that the self-propellant extract inhibits the transcription-promoting activity of IL31 in a concentration-dependent manner. Specifically, it can be seen that when the oleander extract is treated with 20 μg / mL, the activity of the promoter is reduced by about 60%. Therefore, it can be seen that the self-propellant extract effectively inhibits the activity of luciferase in a concentration-dependent manner, thereby suppressing the transcriptional promoting activity of IL31, and thus is effective for dry skin or pruritus.

<Experimental Example 5> Effect on the regulation of IL31 gene in T cells

EL4 (4ⅹ10 ⁵ cells / ml) cells were cultured for 24 hours at 37 ° C and 5% CO₂ in a 100 mm dish with RPMI medium containing 10% FBS, and then diluted samples were added to the medium for 1 hour and pretreated and PMA (50 μg / mL) and Ionomycin (1 μM) and incubated for another hour. RNA was sufficiently lysed using TRI reagent (Invitrogen, Carlsbad, CA, USA), and then chloroform was added to extract RNA and precipitated using isopropanol. The RNA precipitate was washed with 70% ethanol, dried in air, and then dissolved in nuclease-free water (AM9937, Ambion, Austin, TX), heated at 55 ° C for 10 minutes, and heat treated at 70 ° C for 5 minutes to generate RNA. It was made to exist in a single strand state. Total RNA was quantified using NanoDrop (ND-1000, Dae Myung Science, Seoul, Korea) and diluted to 1 μg / μL, followed by Reverse Transcription System (Cat.No.A3500; Promega, Madison, WI, USA) CDNA was made. iQTM SYBR® Green Supermix (# 170-8880, Bio-Rad, Hercules, CA), target gene-specific primers (SEQ ID NO: IL31 sense; SEQ ID NO: 2: IL31 antisense; SEQ ID NO: 3: β-actin sense; sequence No. 4: β-actin antisense) was used to amplify the target gene MiniOpticonTM Real-time PCR Detection system (CFB-3120, Bio-Rad, Hercules, CA). Real-time PCR conditions were denatured at 95 ° C for 20 seconds, followed by 40 cycles of 95 ° C for 20 seconds (denaturation), 56 ° C for 20 seconds (annealing), and 72 ° C for 30 seconds (elongation). Then, the final step was performed at 95 ° C for 1 minute and at 55 ° C for 1 minute. It was analyzed using MJ Opticon Monitor software (Opticon Monitor 3.1.32, Bio-rad, Hercules, CA). The results are shown in FIG. 5.

As a result, as shown in FIG. 5, it was confirmed that the self-propelled extract inhibits IL31 gene expression in the EL4 T cell line. Specifically, it was confirmed that the IL31 mRNA level was suppressed to about 40% even when only the 10 μg / mL was treated with the self-propelled extract. Therefore, it was confirmed that the self-propelled herb extract effectively suppresses IL31 gene expression, and thus has an excellent effect on pruritus.

<Experimental Example 6> Effect on the regulation of IL31 protein in T cells

EL4 (4ⅹ10 ⁵ cells / ml) cells were cultured for 24 hours at 37 ° C and 5% CO₂ in a 100 mm dish with RPMI medium containing 10% FBS and 1% AA, and then diluted samples were added to the medium and pretreated for 1 hour. And treated with PMA (50 ug / ml) and Ionomycin (1 μM) and incubated for another hour. After that, the supernatant was discarded, the cells were collected, washed 2-3 times with PBS, and then the cells were lysed using a lysis buffer, and the protein was quantified, and 20 μg was electrophoresed at 100 V for 2 hours using SDS-PAGE. The isolated protein was transferred to a PVDF membrane (membrane, ISEQ15150, Millipore, Bedford. MA) for 1 hour at 100 V, and then placed in a blocking buffer (5% Albumin from Bovine Serum in PBS containing 0.1% Tween-20 (TBST)) at room temperature. Incubated for 1 hour. The primary antibody was then diluted with TBST 3% Albumin from Bovine Serum (BSA (A9647, Sigma, St Louis, WI)) and reacted with membrane for 4 hours at 4 ° C. After washing the membrane (Membrane, ISEQ 1S150, Millipore, Bedford, MA) three times with TBST for 5 minutes, HRP (horseradish peroxidase) -binding secondary antibody with TBST at 3% BSA at a rate of 1: 1,000 to 1: 2,000 The mixture was diluted and reacted for 2 hours at room temperature with a membrane. After washing TBST 3 times for 5 minutes, the Western blotting substrate (WEST-ZOL PLUS (16021, Intron)) was treated to confirm the protein expression level in LAS-4000 (Fuji Film Corp., Japan). The results are shown in FIG. 6.

As a result, as shown in FIG. 6, it was confirmed that the self-propelled extract inhibits IL31 protein expression in the EL4 T cell line.

<Example 4> Preparation of cream for improving pruritus symptoms

4-1. Preparation of cream for improving pruritus symptoms including self-propelling extract (Example 4)

Using the self-propelled butylene glycol extract prepared in Example 3, a cream cosmetic (Example 4) containing self-propelled grass as an active ingredient was prepared with the composition shown in Table 1 below.

ingredient Content (% by weight) Self-propellant extract 10 glycerin 15 Caprylic / Capric / Mystic / Stearic Triglyceride 2 Diisostearyl maleate 2 Cetyl ethyl hexanoate 2 Polyglyceryl methyl glucose distearate 2 Glyceryl stearate 1.5 Polyglyceryl stearate 1.5 Cetearyl Alcohol One 1,2-hexanediol One Herb extract 0.36 Ammonium acryloyl dimethyl taurate / VP copolymer 0.5 Pentylene glycol 0.5 Caprylyl Glycol 0.2 Xanthan gum 0.1 Carbomer 0.08 Tromethamine 0.06 Hydrogenated lecithin 0.3 Polyglyceryl behenate a very small amount Disodium ID 0.01 Tocopherol a very small amount Purified water Balance

4-2. Preparation of Comparative Example Cream (Comparative Example)

In Example 4, a cream having the same ingredient composition and weight ratio was prepared, except that it was prepared by adding more purified water instead of adding the self-propellant extract from the component of the cream for improving pruritus symptoms (Table 1).

The experiments of Experimental Examples 7 to 13 below selected 23 adults (17 dry skin, 6 dry skin) who had itching due to dry skin (moisture content of 29 or less) among adult women aged 29 to 50 years, Twice a day, in the morning and evening, take an appropriate amount in the cream phase and apply it evenly over the itchy areas. In addition, during the human application test period, the use of cosmetics (eg, functional cosmetics such as whitening, wrinkle improvement, etc.) containing active ingredients that may affect the test results was prohibited in addition to the cream of Example 4.

<Experimental Example 7> Moisture content measurement (Corneometer CM825)

The moisture content was measured using Corneometer CM825 (Courage + Khazaka electronic GmbH, Germany), and the same itch was measured before and after the creams of Example 4 and Comparative Example. The measurement was performed three times through the sensor by contacting the corneometer probe to the skin, and the average value was used as the evaluation data for the skin moisture content (moisturizing amount). Corneometer can measure the moisture content by measuring the capacitance of the probe contact, and the moisture content and capacitance tend to be proportional to each other, so the higher the moisture in the skin, the higher the measured value. The unit is an arbitrary unit (A.U.), which is a unit constant, and A.U. As the value increases, it means that it is effective in improving the moisture content. The changes in moisture content by using the cream of Example 4 are shown in Tables 2 and 7 below. As a result of confirming the change in the moisture content after using the cream of Example 4, compared to before use, it was significantly increased (p <0.025) 2 weeks after use and 4 weeks after use. On the other hand, when the cream of the comparative example was used, the water content did not increase significantly after 2 weeks of use and 4 weeks after use.

division Example 4 (A.U.) Comparative Example (A.U.) Before use 25.055 26.156 2 weeks after use 37.719 29.897 4 weeks after use 43.288 31.364

<Experimental Example 8> Measurement of transepidermal water loss (Vapometer)

For the measurement of transepidermal water loss, the same itching site was measured before and after using the cream prepared in Example 4 using a Vapometer (Delfin Technologoes Ltd., Finland). The amount of transepidermal water loss that predicts the skin's ability to retain water and is an indicator of skin water barrier functioin indicates the amount of water lost through the skin (g / m ² h). Was used as the evaluation data for transepidermal water loss. The lower the moisture loss value, the more effective it is to improve the transepidermal moisture loss. The results of measuring the transepidermal water loss after using the cream of Example 4 are shown in Tables 3 and 8 below. As a result of confirming the change in the amount of transepidermal water loss after using the cream of Example 4, compared with the use of the cream of Example 4, it was significantly reduced (p <0.025) after 2 weeks of use and 4 weeks after use than when using the cream of the comparative example.

division Example 4 (g / m ² h) Comparative Example (g / m ² h) Before use 15.480 14.964 2 weeks after use 13.323 14.169 4 weeks after use 11.694 13.698

<Experiment 9> Skin exfoliation measurement (Tape stripping, Visioscan VC98)

Skin exfoliation was measured and analyzed by using the Visioscan VC98 (Courage + Khazaka electronic GmbH, Germany) to collect and exfoliate the same itchy areas before and after using the test product. For measurement, the keratin was collected using a special film, D-Squame Scan 850A Instrument (Cuderm, USA), and then an image was taken and analyzed with Visiosacan VC98, and the measured value DI (Desquamation Index,%) was used as skin keratin evaluation data. Did. D.I. It was confirmed that as the value decreases, it is effective in improving skin keratin. Table 4 and FIG. 9 show the results of confirming the change in skin keratin after using the cream of Example 4. In addition, a photograph obtained from two test subjects among keratin photographs taken with Visiosacan VC98 was selected and shown in FIG. 10. As a result of confirming the change in skin keratin after using the cream prepared in Example 4, compared with before, it was significantly reduced (p <0.025) 2 weeks after use and 4 weeks after use than when using the cream of the comparative example. After 4 weeks of use, it was confirmed that skin keratin decreased to about 60% compared to before use. In addition, as can be seen in Figure 10, it can be seen that the keratin of the test subject was significantly reduced by using the self-propelled cream of Example 4 provided in one aspect of the present invention.

division Example 4 (%) Comparative Example (%) Before use 10.370 10.168 2 weeks after use 7.398 9.897 4 weeks after use 6.618 9.165

<Experiment 10> Evaluation of skin itching improvement (VAS, Visual Analog Scale Assessment)

In the evaluation of the degree of itching of the skin, the test subject was directly marked on the 10 cm line from 0 (no itching) to 10 (no itching) and the length of the test subject was measured. . The degree of itching improvement was evaluated by comparing the lengths of line segments indicating the degree of itching before and after the cream of Example 4. The results of confirming the degree of skin itching improvement after using the cream of Example 4 are shown in Tables 5 and 11 below. As a result of confirming the change in the degree of skin itching (VAS) after using the cream of Example 4, it was significantly reduced (p <0.025) after 2 weeks of use and 4 weeks after use as compared to before use. After 4 weeks of use, it was found that itching was improved by more than half compared to before use.

division Example 4 (cm) Comparative Example (cm) Before use 5.757 5.697 2 weeks after use 3.778 5.345 4 weeks after use 2.226 4.645

<Experimental Example 11> Effectiveness Assessment Survey (Global Assessment)

After using the cream prepared in Example 4, to answer the questionnaire directly in 5 steps: very good (4), good (3), normal (2), bad (1), very bad (0) After that, the percentage of the number of test subjects for each answer was obtained to determine whether the cream of Example 4 was effective. Example 4 In the survey conducted on the degree of effect of alleviating itching and improving skin exfoliation due to drying of the skin after using the cream, the percentage of the number of subjects for the mean, standard deviation, and response was determined. The results are shown in Table 6 below. As a result of the questionnaire evaluation, 100.000% of the test subjects rated more than normal for all items of alleviation of itching caused by dry skin and improvement of keratin. Specifically, more than 90% of the subjects selected a very good or good item for alleviating itchiness due to dry skin, and about 87% of the test subjects selected a very good or good item for improving skin keratin.

4 3 2 One 0 Average Relieves itching caused by dry skin (%) Example 4 34.783 56.522 8.696 0.000 0.000 3.261 Comparative example 13.564 37.453 48.983 0.000 0.000 2.645 Skin keratin improvement (%) Example 4 30.435 56.522 13.043 0.000 0.000 3.174 Comparative example 10.456 21.190 68.354 0.00 0.00 2.421

<Experimental Example 12> Preference survey evaluation

After using the cream prepared in Example 4, the test subjects were asked to directly answer the questionnaire data. The evaluation items are evaluated in 5 stages: very good (4), good (3), normal (2), bad (1), and very poor (0) for skin moistness, smoothness, spread, absorbency, fragrance, and overall usability. Did. The survey results indicated the mean, standard deviation, and percentage of the number of subjects for answers. The results are shown in Table 7 below. As a result of the questionnaire evaluation, 100.000% of the test subjects evaluated the skin moistness, smoothness, spread, and overall usability as more than normal, and 95.652% of the test subjects rated the absorbency and scent as more than normal. Specifically, 100% of the test subjects selected a very good or good item for moisturizing the skin, and it was confirmed that the self-propelled cream of Example 4 provided in one aspect of the present invention was excellent in moisturizing the skin. , It was found that about 80% of the test subjects selected the items of very good or good for the overall usability, and the overall preference of the self-propelled cream of Example 4 was very good.

4 3 2 One 0 Average Skin moist (%) Example 4 34.783 65.217 0.000 0.000 0.000 3.348 Comparative example 13.992 46.534 39.474 0.000 0.000 2.745 Smoothness (%) Example 4 34.783 56.522 8.696 0.000 0.000 3.261 Comparative example 34.783 56.522 8.696 0.000 0.000 3.261 Spreadability (%) Example 4 26.087 43.478 30.435 0.000 0.000 2.957 Comparative example 34.783 40.213 25.004 0.000 0.000 3.098 Absorbency (%) Example 4 21.739 39.130 34.783 4.348 0.000 2.783 Comparative example 16.546 41.634 36.655 5.165 0.000 2.696 Incense (%) Example 4 8.696 34.783 52.174 4.348 0.000 2.478 Comparative example 13.354 51.534 30.766 4.346 0.000 2.739 Overall usability (%) Example 4 21.739 69.565 8.696 0.000 0.000 3.130 Comparative example 26.564 69.082 4.354 0.000 0.000 3.222

<Experiment 13> Safety evaluation

The safety of the creams prepared in Example 4 and Comparative Examples was obtained by synthesizing the adverse reactions identified for all test subjects who used them and all adverse reactions reported during the test period, and obtaining the incidence of adverse reactions and using it as product safety evaluation data. Did. Example 4 As a result of evaluating the results of the evaluation of the naked eye and the subject questionnaire on the areas of use of the creams and the comparative example creams, the test subjects responded to special skin adverse reactions during the period of using the creams of the examples 4 and comparative examples. There were no reports, and no abnormalities were observed on the physical examination by the dermatologist (erythema, edema, swelling). No symptoms related to skin adverse reactions (itching, pain, burning, stiffness, tingling) were observed in the safety survey results of the subjects.

On the other hand, the self-defense extract according to the present invention can be formulated in various forms depending on the purpose. The following exemplifies several formulation methods in which the self-propellant extract according to the present invention is contained as an active ingredient, and the present invention is not limited thereto.

<Formulation Example 1>

1-1. Manufacture of powder

Self-propelled extract 500 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

Self-propelled extract 500 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

1-3. Manufacture of capsule

Self-propelled extract 500 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

1-4. Preparation of Injectables

Self-propelled extract 500 mg

Appropriate sterile distilled water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, the lemon flavor is appropriately added, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle. Sterilize to prepare the liquid.

<Formulation Example 2> Preparation of cosmetic preparation

2-1. Lotion

Sprout extract 3.00 (%)

L-ascorbic acid-2-magnesium phosphate salt 1.00

Water-soluble collagen (1% solution) 1.00

Sodium citrate0.10

Citric acid 0.05

1,3-butylene glycol 3.00

The whole amount was set to 100 with purified water, and a lotion was prepared at the above mixing ratio.

Above, the present invention has been described in detail through preferred examples and experimental examples, but the scope of the present invention is not limited to the characteristic examples, and should be interpreted by the appended claims. In addition, those skilled in the art should understand that many modifications and variations are possible without departing from the scope of the present invention.

<110> JANG, eun jung <120> Cosmetic composition comprising Isodon serra extract as active          ingredient for alleviating or improving pruritus <130> 2019P-10-004 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL31 sense <400> 1 acaactatag cataaagcag gc 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL31 antisense <400> 2 gattcatcag tatttccagg ca 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actic sense <400> 3 aaggccaacc gtgaaaagat 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> beta-actic antisense <400> 4 gtggtacgac cagaggcata c 21

Claims (7)

Pharmaceutical composition for prevention or treatment of pruritus, which contains Isodon serra extract as an active ingredient.
According to claim 1,
The extract is a pharmaceutical composition, characterized in that extracted with water, C1 to C2 lower alcohol or a mixed solvent thereof.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition characterized in that to inhibit the expression of IL31 (Interleukin 31).
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition characterized by inhibiting the secretion of chemokine (Chemokine).
According to claim 4,
The chemokine is Rantes (Regulated on Activation, Normal T Cell Expressed and Secreted) or Tarc (Thymus and activation-regulated chemokine).
Health functional food composition for prevention or improvement of pruritus, which contains Isodon serra extract as an active ingredient.
Cosmetic composition for relieving or improving pruritus containing Isodon serra extract as an active ingredient.

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