KR102249038B1 - Composition for Improving, Preventing or Treating Stress-Related Alopecia Comprising Catechin 7-O-β-D-apiofuranoside As Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for alleviating, preventing or treating stress-related alopecia, containing a catechin 7-O-β-D-apiofuranoside compound as an active component. The compound of the present invention inhibits apoptosis caused by oxidative stress in dermal papilla cells, and thus can be used as an active material for treatment, prevention, or alleviation of stress-related alopecia.

Description

Composition for Improving, Preventing or Treating Stress-Related Alopecia Comprising Catechin 7-O-β -D-apiofuranoside As Active Ingredient}

The present invention relates to a composition for improving, preventing or treating stress-related alopecia comprising catechin 7-O-β-D-apiofuranoside as an active ingredient.

Hair loss does not have a pathological disorder problem compared to other pathological diseases, but it affects the appearance and greatly affects the person who suffers hair loss as a social and psychological problem. In the past, hair loss has been recognized as an aging phenomenon, but recently, it appears due to various factors such as stress, nutritional imbalance, changes in social activities, diseases, childbirth, changes in diet, irregular life, and excessive use of chemical hairdressing agents along with genetic factors. have. The types of hair loss can be broadly classified into male-type hair loss, female-type hair loss, circular hair loss, postpartum hair loss, hair loss due to seborrheic scalp infection, and hair loss due to stress.

Hair loss due to stress appears in the form of alopecia areata. Alopecia areata is a disease in which hair falls from part or all of the body. Often, bald spots about the size of a few coins occur on the scalp, which can be caused by psychological stress, and there are cases in which the hairs on the scalp, the hairs of the beard or the entire body such as the eyebrows are all gone, leading to permanent hair loss.

In addition, hair loss due to stress may appear in the form of telogen hair loss, and telogen hair loss is hair loss that occurs when the normal growth cycle of the hair enters the telogen. Human hair grows and disappears repeatedly in a 3-6 year cycle, but hair that has grown for 3-6 years degenerates over 3-4 weeks, and then folds into a resting period where the hair has been removed for about 3 to 5 months. Lift. Usually, the hair that enters the telogen is about 10% (about 10,000 strands) of the total hair, and this type of hair loss is called telogen hair loss when the hair enters the telogen.

IGF-1 promotes the proliferation of epithelial cells in culture, increases the length of hair follicle tissue, acts as a factor that promotes hair growth, and is an important growth factor involved in the regulation of hair growth (Itami et al., 1995). ; Hibino & Nishiyama, 2004).

IGF-1 growth factor is reported to play an important role in the mechanism of action that causes testosterone to act on the hair and causes androgenetic alopecia by increasing the expression of male hormones (Boo-seop Shin, 2014). Prevention, improvement of dementia and depression, prevention of lifestyle-related diseases such as diabetes or high blood pressure, increase of immunity and increase of bone density, help to prevent skin aging, and are used for hair loss treatment by inducing the transformation from the resting state of the hair to the growing state (Pyeonjuri , 2012).

When TGF-β1 inducing degenerative development was injected into the skin on the back of the mouse, the number of keratinocytes decreased by proliferation of hair follicles, and the number of TUNEL cells was increased in TGF-β1 administered mice compared to the control group, and TGF-β type II receptor (TGFRIl) And dual visualization of TUNEL reactivity.It was found that colocalization of the apoptotic nucleus and TGFRII of the degenerative hair follicles was found, so that the TGF-βRII agonist and antagonist could be provided as a useful treatment for human hair growth diseases based on early or delayed degenerative alopecia and hirsutism. (Foitzik et al., 2000).

Shin et al. (2013) reported that androgenic alopecia is closely related to TGF-β1 secretion. Androgen receptor-transformed murine bladder papillary cells (DP-6) showed increased ROS production after androgen treatment, and TGF-β1 secretion was increased by androgen treatment in DP-6, whereas androgen-induced TGF-β1 was It has been reported that the induction of androgen-induced TGF-β1 is mediated by ROS of hair follicle DPCs, confirming that it is significantly inhibited by N-acetyl cysteine, suggesting the use of antioxidant therapy for the treatment of androgenetic alopecia.

Regarding the effect of growth factors on hair growth, growth factors such as epidermal growth factor (EGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) were tested through several experiments. It has already been found that it is involved in hair growth by acting on a specific place in the hair follicle (Tsuboi, 1997). In 1994, the Hervert research team found that mice deficient in the fibroblast growth factor (FGF)-5 gene had an abnormally prolonged anagen phase, resulting in relatively long hair.FGF-7, IGF-1, and HGF were responsible for hair growth. It was reported as a paracrine growth factor that promotes (Hervert et al., 1994).

Jiang et al. (1995) found that TGF-β1 among the endocrine factors affecting hair growth and hair loss inhibits the growth of hair at the anagen stage, causing it to enter the catagen stage faster than normal, inducing hair loss, and IGF. Growth factors such as -1, TGF-β2, EGF, and FGF promote hair growth and prevent degeneration of hair cells, thereby preventing hair loss.

In a study by Hyun-wook Na et al. (2006), it is reported that hair growth has a more intimate relationship with blood circulation, while insulin, a hormone, and IGF-1 and EGF, which are hormones, promote hair growth and affect hair follicle growth. Has become.

Minoxidil was developed as a vasodilator for the treatment of hypertension in the early 1970s, but was used as a hair growth promoting agent as hirsutism was reported as a side effect. Although the mechanism for the hair growth effect is not accurately identified, it has been reported that hair growth is induced by increasing nutrient supply due to vasodilation. However, minoxidil is not effective in the front of the scalp, which has already become bald, and if it is not steadily applied to the scalp twice a day for 6-12 months, the effect cannot be seen. Side effects of minoxidil include edema, tachycardia, local peeling, dermatitis, soreness at the site of administration, erythema, peeling of the skin, itchiness, dryness, skin dropping, and contact dermatitis, and it has been reported that hirsutism can be induced. In addition, since minoxidil affects the hair cycle, there may be a phenomenon in which hair loss becomes more severe due to an increase in shedding during the resting period, and there is a shortcoming in that the period of returning to the state before treatment is short when the dialysis is stopped.

The patent documents and references mentioned in this specification are incorporated herein by reference to the same extent as if each document was individually and clearly specified by reference.

Korean Patent Registration No. 10-2016002 Korean Patent Registration No. 10-1659516 Republic of Korea Patent Publication No. 10-2013-0123626

The present inventors have made research efforts to develop a natural product-derived anti-hair loss agent that is safer and has no side effects. As a result, the catechin 7-O-β-D-apiofuranoside (catechin 7-O-β-D-apiofuranoside) compound isolated as a single compound from the extract of the supercritical extract of Elm plant The present invention was completed by experimentally demonstrating that it effectively inhibits apoptosis caused by oxidative stress of, and confirming that the compound can be developed as a therapeutic agent for stress-related alopecia.

Accordingly, an object of the present invention is to provide a composition for improving, preventing or treating stress alopecia.

Other objects and technical features of the present invention are presented in more detail by the following detailed description, claims, and drawings.

According to one aspect of the present invention, the present invention provides a catechin 7-O-β-D-apiofuranoside (catechin 7-O-β-D-apiofuranoside) compound as an active ingredient for preventing stress-related alopecia, It provides a composition for improvement or treatment.

Catechin 7-O-β-D-apiofuranoside, an active ingredient of the composition of the present invention, is a catechin glycoside compound represented by the following formula (1).

[Formula 1]

Catechin 7-O-β-D-apiofuranoside, the active ingredient of the composition of the present invention, can be used isolated from natural products, but chemically synthesized ones are also extracted. It is obvious to a person skilled in the art that it exhibits the same effect as that.

The catechin 7-O-β-D-apiofuranoside compound of the present invention is contained in a large amount in the solvent extract of the supercritical extract of the elm plant, which is a natural product extract, from this extract by a conventional single compound separation method. You can get it.

In the present invention, the supercritical extract residue of the Elm genus refers to a residue left after extracting the Elm genus plant by the supercritical extraction method.

The Elm genus (Ulmus) plant used in the production of the supercritical extract of the present invention includes a plant of the Rosaceae elm family, for example, Ulmus davidiana, Elm tree (Ulmus davidiana var. japonica), and lump elm. Trees (Ulmus davidiana var.japonica for.Suberosa), Ulmus parvifolia, Ulmus pumila, Ulmus laciniata, and Ulmus macrocarpa. Most representatively, the elm tree (Ulmus davidiana), the elm tree (Ulmus davidiana var. japonica) can be used.

The elm plant used for supercritical extraction in the present invention is the whole of the elm plant; Alternatively, a part of an Elm genus plant selected from the group consisting of stems, roots, leaves, flowers, fruits, and seeds may be used. Preferably, a stem, branch or root of an Elm genus plant may be used. In the present invention, before the supercritical extraction, the whole or part of the elm plant may be dried and/or pulverized.

In the present invention, the supercritical extraction method refers to a method of extracting flavors, pigments, oils, or functional substances contained in natural products without denaturation by using a fluid in a supercritical state.

The fluid in the supercritical state refers to a fluid in a state in which gas and liquid cannot be distinguished beyond a limit point of a constant high temperature and high pressure at a supercritical point. The supercritical fluid may be, for example, supercritical carbon dioxide. The supercritical carbon dioxide is carbon dioxide in a state exceeding a critical temperature (31° C.) and a critical pressure (7.5 MPa), and is a fluid having both gas and liquid properties.

The supercritical extraction method in the present invention includes, for example, a pressure change method in which a mixture of a supercritical fluid and a solute is decompressed and expanded at a temperature equal to the extraction temperature to reduce the dissolving power of the supercritical fluid and the solute is separated; Alternatively, the temperature of the supercritical fluid may be increased to decrease the dissolving power, and thus, the supercritical fluid may be separated from the solute, but the method is not limited thereto.

In the present invention, a co-solvent may be used with the supercritical fluid, and the co-solvent includes a polar solvent commonly used in the art, and preferably alcohol, water, ethylene glycol, polyethylene glycol, and propylene It can be used by selecting from the group consisting of carbonate, formic acid, acetic acid, acetonitrile, chlorodifluoromethane, or mixtures thereof.

In the present invention, the container for supercritical extraction may be controlled in temperature and pressure, and it is not particularly limited as long as it is a container configured to contact the extraction raw material and the supercritical fluid.

In the present invention, the pressure in the supercritical extraction may be in the range of 100-600 bar, the temperature in the supercritical extraction may be in the range of 10-100°C, and the flow rate of the supercritical fluid and the auxiliary solvent in the supercritical extraction may be 10 May be -100 g/min. Those of ordinary skill in the art may use the pressure, temperature, fluid and co-solvent flow rates in the supercritical extraction to be appropriately adjusted within the above range.

The supercritical extract of the elm plant obtained after performing the supercritical extraction method is subjected to extraction using a solvent to obtain an extract. Preferably, the solvent extraction is performed through a process of contacting the supercritical extract foil of elm with an extraction solvent. Before the solvent extraction step, the supercritical extract foil of the elm tree may be dried and/or pulverized. In the present invention, the extraction solvent may preferably use an alcohol, and the alcohol is an anhydrous or water-containing lower alcohol having 1-4 carbon atoms, such as methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, and normal -Butanol is mentioned.

In the present invention, a fraction can be obtained by performing an additional fractionation on the solvent extract of the supercritical extract of the elm tree.

The fractionation is carried out by contacting the fractionation solvent with the extract, similarly to extraction. The solvent used for the fractionation is (a) water, (b) alcohol, (c) a mixed solvent of the lower alcohol and water, (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl Acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether. Preferably, it may be a fraction obtained using ethyl acetate.

From the fraction, catechin 7-O-β-D-apiofuranoside can be separated using a single compound separation method known in the art such as chromatography. A method of separating and purifying a single compound that can be used in the present invention may use, for example, a method of thin layer chromatography (TLC) or high performance liquid chromatography (HPLC).

As demonstrated in one specific example of the present specification, catechin 7-O-β-D-apiofuranoside, the active compound of the present invention, is apoptosis in dermal papilla cells (HFDPC) due to oxidative stress. By inhibiting this, it has the effect of preventing, treating, and improving stress-related hair loss.

As used herein, the term “including as an active ingredient” includes an amount sufficient to achieve the efficacy of the catechin 7-O-β-D-apiofuranoside compound of the present invention to prevent, ameliorate, or treat stress-related alopecia. Means to do.

In the present specification, the term “hair loss” refers to a state in which there is no hair in a region where hair should normally exist due to the loss of abnormal hair, and the term “alopecia” refers to a condition caused by the aforementioned hair loss.

In the present invention, "stress alopecia" refers to alopecia caused by psychological or physical stress, and includes stress-related alopecia areata and stress telogen alopecia.

In the present invention, “stress alopecia” includes stress alopecia occurring in animals other than humans as well as stress alopecia in humans.

The composition of the present invention may be provided in the form of a pharmaceutical composition for preventing or treating stress-related alopecia, and the pharmaceutical composition may include a pharmaceutically acceptable carrier.

In the present specification, the term “prevention” refers to the suppression of the occurrence of symptoms due to hair loss or complications of hair loss, and the term “treatment” means reduction or elimination of symptoms due to already occurring hair loss or symptoms due to hair loss complications Means.

Pharmaceutically acceptable carriers in the composition of the present invention are commonly used at the time of formulation, and are lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.

The daily dosage of the pharmaceutical composition of the present invention is 0.001-10,000 mg/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

According to another aspect of the present invention, there is provided a functional cosmetic composition for preventing or improving stress-related alopecia comprising catechin 7-O-β-D-apiofuranoside as an active ingredient.

In the present specification, the term “improvement” means alleviation or alleviation of symptoms due to hair loss or symptoms due to complications of hair loss.

The functional cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing It may be formulated as a cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. In more detail, it may be prepared in the form of a flexible lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. I can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

The ingredients included in the functional cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to the active ingredient and the carrier ingredient, and include conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. Can include.

The catechin 7-O-β-D-apiofuranoside compound of the present invention inhibits apoptosis due to oxidative stress in dermal papilla cells, thereby being an active substance for the treatment, prevention or improvement of stress-related alopecia. Can be used.

Figure 1a is a process flow diagram summarizing the separation process of the compound of the present invention.
Figure 1b shows the chromatographic results confirming catechin 7-O-β-D-apiofuranoside in the ethanol extract of the supercritical extract of elm branches.
Figure 1c shows the chromatographic results confirming the catechin 7-O-β-D-apiofuranoside in the fraction fractionated with ethyl acetate for the ethanol extract of the supercritical extract of elm branches.
Figure 1d is a result of confirming the catechin 7-O-β-D-appiofuranoside compound obtained by separating the fraction fractionated with ethyl acetate for the ethanol extract of the supercritical extract of elm branches by Prep-LC.
Figure 2a is a TOF-MS analysis result of the catechin 7-O-β-D- apiofuranoside compound.
2B is 13C-NMR data of a catechin 7-O-β-D-apiofuranoside compound.
2C is 1H-NMR data of a catechin 7-O-β-D-apiofuranoside compound.
Figure 2d is the result of structural analysis of the catechin 7-O-β-D-apiofuranoside compound.
3 is a catechin 7-O-β-D-apiofuranoside compound inhibits the expression of Bax protein in dermal papilla cells treated with hydrogen peroxide, increases Bcl-2 protein expression, and inhibits PARP-1 protein expression. It is an experimental result showing that.
FIG. 4A is a graph showing the effect of catechin 7-O-β-D-apiofuranoside in promoting the expression of IGF, which is helpful for hair growth, in H2O2 treated dermal papilla cells in a concentration-dependent manner. *P<0.001 vs normal group. **P<0.005 vs H2O2 control group.
FIG. 4B is a graph showing the effect of catechin 7-O-β-D-apiofuranoside in inhibiting the expression of TGF-β1 in a concentration-dependent manner in H2O2 treated dermal papilla cells. *P<0.001 vs normal group. **P<0.005 vs H2O2 control group.

The specific examples described in the present specification below are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that variations and other uses of the invention do not depart from the scope of the invention described in the claims of this specification.

Example

1. Experimental materials and methods

(1) Isolation of Catechin 7-O-β-D-Apiofuranoside Compound

In this experiment, supercritical extraction of Ulmus branches was performed using a supercritical fluid extraction production equipment (SCFE-P400, Ilsin Autoclave, Daejeon, Korea).

Elm branches were purchased at Seoul Yangnyeong Market, foreign substances were removed, washed, and then shaded to be used as an experimental material. 100 kg of the dried sample was pulverized so as to pass through a crushing net of 200 mesh, and the temperature of the pulverized elm branch was adjusted to 50° C. to maintain the temperature. When the temperature is stabilized, insert an elm branch sample and _{maintain the CO 2} gas at isostatic pressure, and then adjust the control valve through a line using a high-pressure pump until the experimental pressure condition reaches 400 bar. And injected. After reaching the set pressure, ethanol (alcohol: edible alcohol) was added to the bottom of the extraction tank at 0.5~0.6L per minute for a total of 60~80L for 160 minutes, and the pressure was set to remove the residual ethanol remaining in the sample. The extraction was completed by flowing _{CO 2} using a high-pressure pump at over temperature for 60 to 120 minutes.

After supercritical extraction of elm branches as described above, 100 kg of supercritical extract sample of elm tree obtained was dried in the shade, extracted once at room temperature with 60% ethanol, and filtered. The extract was concentrated under reduced pressure and freeze-dried to obtain a final 4.81 kg.

1 kg of ethanol extract (“USCFR”) of the obtained elm supercritical extract was quantified, and ethyl acetate solvent fractionation was performed using a separatory funnel to obtain 185.2 g of an ethyl acetate solvent fraction (“USCFREA”).

Using Prep-LC (Waters, USA) from the obtained ethyl acetate solvent fraction (“USCFREA”), and when using silica gel resin, use Chloroform:Methanol:Water (70:30:4) as a mobile phase solvent. And, when using a C18 column resin, a single compound was purified using MeOH:Water (0-100%) mobile phase conditions (see FIGS. 1A to 1D).

(2) Analysis of indicator compounds in extracts using TOF-MS

A high-resolution time-of-flight mass spectrometer (TOF-MS) using a high vacuum and high sensitivity detector through TMP. The catechin 7-O-β-D-Apiofuranno, the main component in the ethanol extract of elm branch supercritical gourd using a direct probe. The molecular weight and molecular structure of the side were confirmed. Sample information and analysis conditions are described in Table 1 below.

The analysis results of TOF-MS are shown in Fig. 2A, 13C-NMR data are shown in Fig. 2B, and 1H-NMR data are shown in Fig. 2C. The results of structural analysis of the catechin 7-O-β-D-apiofuranoside compound of the present invention are shown in FIG. 2D.

(3) cell culture

Hair follicle dermal papilla cells (HFDPC) used in this example are cells that exhibit the characteristics of hair growth and maintenance, and were purchased and used from PromoCell (Heidelberg, Germany). HDFPC cells were inoculated in a papilla cell-only medium recommended by the supplier (PromoCell, Heidelberg, Germany or Cell biologics Inc, IL, USA) and subcultured for 24 to 48 hours under conditions of _{37°C and 5% CO 2 and used in the experiment.}

(4) Inhibitory effect of intracellular apoptosis

It was tested whether the catechin 7-O-β-D-apiofuranoside compound could inhibit apoptosis due to oxidative stress in dermal papilla cells. In this experiment, the inhibitory effect of oxidative stress on dermal papilla cells (HDFPC) induced by _{H 2} O _{2 was measured through the MTT experiment.} HDFPC cells were dispensed into a 96-well plate at 5×10 ⁵ cells/mL, and _{600 μM of H 2} O ₂ and samples were simultaneously treated for 4 hours after 12 hours. The HDFPC cells in this state were treated with MTT stock solution to a final concentration of 0.2 mg/ml, left at 37° C. for 4 hours, and then purple-stained formazan was dissolved in DMSO, and the absorbance was measured at 570 nm. Cell viability was expressed as a% value for the control.

(5) Western blot analysis

After treating HDFPC cells with H2O2 and samples, cells were recovered at each time point, and the cells were lysed in a cell lysis buffer (RIPA buffer). The sample was centrifuged at 13,000 g for about 10-15 minutes, and then only the supernatant was taken. The protein concentration of the supernatant was measured with a BCA protein assay kit (Bio-Rad, CA, USA) and then heated at 95° C. for 10 minutes. The prepared sample was subjected to electrophoresis on an SDS-polyacrylamide gel, transferred to a polyvinylidinedifluoride membrane (PVDF, Bio-rad, CA, USA), and then blocked with 5% BSA, and reacted with the presented specific antibody. And after reacting the HRP-bound secondary antibody (Santacruz, CA, USA), it was detected with the ECL system (Bio-Rad, MO, USA). Santacruz antibody (CA, USA) was used as the primary antibody against Bax-1, Bcl-2, PARP-1, and β-actin.

(6) ELISA analysis

An ELISA kit was purchased and used to perform an immunochemical quantitative analysis related to hair growth promotion and hair loss inhibition. TGF-β and IGF-1 kit were purchased and used from R&D systems (CA, USA). The use of each kit was tested according to the supplier's manual, and the result was derived by converting the amount of antigen (pg or μg) per volume (ml) of cell lysate into a concentration unit.

(7) statistical processing

All experiments were repeated three times to present the mean value and standard deviation. In addition, using SPSS (Statistical Package for Social Science, ver. 18, IBM, Chicago IL. USA), the significance of the mean values of the experimental group and the control group at the P<0.05 level was verified with Student's t-test.

2. Experiment result

(1) Bax protein expression inhibitory effect

Hydrogen peroxide (H2O2) treated to induce apoptotic cytotoxicity of HDFDC cells was confirmed to show a mortality of 50% at a concentration of 600 μM, and this concentration was used in the test. It was confirmed that Bax (Bcl-2-associated X protein) as a representative apoptosis inducing protein in apoptotic cytotoxicity significantly increased the expression of Bax molecules by treatment with H2O2. On the other hand, as a result of pretreatment of the catechin 7-O-β-D-apiofuranoside compound by concentration, it was proved that the expression of Bax was significantly reduced in a concentration-dependent manner. These results suggest that catechin 7-O-β-D-apiofuranoside effectively inhibits dermal papilla cells from apoptosis due to stress.

(3) Bcl-2 protein expression increase effect

In order to evaluate the apoptosis induced by hydrogen peroxide (H2O2) treated to induce apoptotic cytotoxicity of HDFDC in the same way as the Bax expression inhibitory ability evaluation method, 600 μM of H2O2 was treated to the cells. Bcl-2 is a representative protein molecule involved in the inhibition of apoptosis, and it was observed that the treatment of H2O2 to HDFDC significantly reduced the expression of the Bcl-2 molecule. On the other hand, as a result of treatment of the catechin 7-O-β-D-apiofuranoside compound by concentration, it was proved that the expression of Bcl-2 was significantly increased in a concentration-dependent manner. These results suggest that catechin 7-O-β-D-apiofuranoside effectively inhibits dermal papilla cells from apoptosis due to stress.

(4) PARP-1 protein expression inhibitory effect

PARP-1 uses intracellular NAD as a substrate to induce poly-ADP ribosylation of target proteins in the nucleus of cells, thereby activating signaling involved in apoptosis. For this reason, the activation of PARP-1 is used as a leading indicator of apoptosis, and in particular, it is used as a representative marker related to apoptosis. Treatment with 600 μM of hydrogen peroxide (H2O2) significantly increased the expression of PARP-1 in the cells in HDFDC, and this result proves that apoptosis of cells is in progress. On the other hand, as a result of treatment of the catechin 7-O-β-D-apiofuranoside compound by concentration, it was proved that the protein expression level of PARP-1 was significantly suppressed in a concentration-dependent manner. These results suggest that catechin 7-O-β-D-apiofuranoside effectively inhibits dermal papilla cells from apoptosis due to stress.

(5) IGF-1 expression promoting effect and TGF-β1 expression inhibitory effect

In this experiment, the catechin 7-O-β-D-apiofuranoside compound promotes the expression of IGF-1, which is helpful for hair growth (Fig. 4A), and inhibits hair growth and accelerates hair loss at the same time. The inhibitory effect of TGF-β1 (Fig. 4b) was confirmed.

The above experimental results show that catechin 7-O-beta-D-apiofuranoside effectively inhibits apoptosis of dermal papilla cells caused by oxidative stress, resulting in stress. It suggests that it may have an effect on hair loss.

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As described above, specific parts of the present invention have been described in detail, and it is clear that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (9)

A composition for preventing or treating stress-related alopecia comprising a catechin 7-O-β-D-apiofuranoside compound as an active ingredient.
The method according to claim 1, wherein the catechin 7-O-β-D-apiofuranoside compound has an activity of inhibiting apoptosis due to oxidative stress in dermal papilla cells, preventing stress-related alopecia or Therapeutic composition.
The method according to claim 1, wherein the composition is a pharmaceutical composition, a composition for preventing or treating stress alopecia.
The composition for preventing or treating stress alopecia according to claim 1, wherein the stress alopecia is stress alopecia areata or stress telogen alopecia.
The composition for preventing or treating stress alopecia according to claim 1, wherein the stress alopecia is stress alopecia occurring in animals.
Functional cosmetic composition for preventing or improving stress-related alopecia comprising a catechin 7-O-β-D-apiofuranoside compound as an active ingredient.
The method of claim 6, wherein the catechin 7-O-β-D-apiofuranoside compound has an activity of inhibiting apoptosis due to oxidative stress in dermal papilla cells, preventing or improving stress-related alopecia Functional cosmetic composition for use.
The method according to claim 6, wherein the stress alopecia is stress alopecia areata or stress telogen alopecia, functional cosmetic composition for preventing or improving stress alopecia.
The method according to claim 6, wherein the stress alopecia is stress alopecia occurring in animals, functional cosmetic composition for preventing or improving stress alopecia.

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