KR101871922B1 - Composition for promoting mature melanocyte migration comprising chemokine, detection kit for skin-active ingredients comprising chemokine gene and the method for detecting skin-active ingredients by using chemokine gene - Google Patents

Abstract

The present invention relates to a composition containing chemokine and promoting adult melanocyte cell migration in skin or hair, and more particularly, to a composition for promoting the migration of adult melanocyte cells containing a chemokine gene and used for the treatment of hypochromic diseases Characterized in that the hypochromic disease is vitiligo or white moth.
The present invention also relates to a kit for screening a skin active substance and a method for searching for a skin active substance using the kit. More particularly, the present invention relates to a kit for screening a skin active substance using a search kit comprising a chemokine gene, The present invention relates to a kit for searching for a skin active substance such as a substance for treating a whitening substance and a low pigment disease, and a method for searching for a skin active substance using the same.

Description

[0001] The present invention relates to a composition for promoting the migration of adult melanocytes containing a chemokine, a kit for screening a skin active substance using a chemokine gene, and a method for searching for a skin active substance using the kit. ingredients comprising chemokine gene and the method for detecting < RTI ID = 0.0 > chemokine &

The present invention relates to a composition containing chemokine and promoting adult melanocyte cell migration in skin or hair, and more particularly, to a composition for promoting the migration of adult melanocyte cells containing a chemokine gene and used for the treatment of hypochromic diseases Characterized in that the hypochromic disease is vitiligo or white moth.

The present invention also relates to a kit for screening a skin active substance and a method for searching for a skin active substance using the kit. More particularly, the present invention relates to a kit for screening a skin active substance using a search kit comprising a chemokine gene, The present invention relates to a kit for searching for a skin active substance such as a substance for treating a whitening substance and a low pigment disease, and a method for searching for a skin active substance using the same.

Cell migration is one of the physiological activities of normal cells. It occurs in the development and differentiation of tissues, migration due to activation of immune cells, formation of new blood vessels, regeneration of damaged tissues, and the like. If the migration of these cells is improperly controlled, it is possible to prevent developmental diseases such as brain neurological diseases due to abnormality of neural network, inflammatory diseases due to abnormal immune cell activation such as abnormal rheumatoid arthritis, Delay of wound healing caused by inhibition, cancer metastasis, etc.

Melanocytes develop from the precursor neural crest cells and develop into melanoblasts. Melanocyte progenitor cells are differentiated into adult melanocytes and finally distributed in basal epidermis, hair follicle, iris, etc. and maintain a constant number. The migration of melanocyte progenitor cells is well known, and various signal transduction mechanisms and transcription factors have been identified. However, the phenomenon of adult melanocyte migration has been rarely studied. Recently, however, there is a growing need for studies on the migration of melanocytes, including abnormal activation of skin melanocytes as well as diseases such as white rot and vitiligo.

Acne and other inflammatory sites are caused by chemokines secreted by macrophages and various immune cells migrate and accumulate in the affected area. Recently, the phenomenon that the movement of melanin cells in the mouse is abnormally promoted by the exposure of UVB and the like, and the melanocytes localized in the hair follicles migrate to the epidermis and are distributed in the literature.

It is reported that the pigmentation of the basement membrane between epidermis and dermis is a typical pigmentation disease. The melanocytes migrate to the dermis due to the collapse of the basement membrane, which is likely to cause pigmentation diseases such as stain and blackness. Therefore, inhibition of melanocyte migration to the dermis due to basement membrane collapse can prevent intractable pigmentation diseases such as stinging.

Vitiligo is a disease characterized by low pigmentation of skin and hair. It occurs in all races and occurs in 0.5% of the world. There is no clear pathogenesis of a decrease in the number of melanocytes. Therefore, a definite treatment for vitiligo has not been developed. Local corticosteroid application, phototherapy, photochemotherapy, etc. have been used to increase pigmentation in patients with vitiligo. Such UV light treatment aims to induce the proliferation of melanocytes and the migration of melanocytes to the affected part. Melanin progenitor cells in the outer root sheath of the hair follicle proliferate by UV light and the increased melanin progenitor cells migrate to the surrounding skin and the skin tone of the lesion is similar to that of the normal area . Therefore, the chemokine of the present invention which induces the migration of adult melanocytes can be used for hypochromic diseases such as vitiligo and white moth.

The skin is a precisely regulated immune system that secretes a variety of cytokines, chemokines, and other inflammatory mediators. Chemokine is an immune response mediator with a small size of 8-14 kD. It has selective chemotaxis to surrounding cells and has the function of inducing cell migration and integration into target organs. While cytokines are already well known to play an important role in skin physiology such as atopy and contact dermatitis, chemokines are not well known for their role in skin physiology.

Accordingly, the present inventors discovered that a specific chemokine affects the migration of adult melanocytes, and found that chemokine can be used for promoting melanocyte migration of skin or hair using the same. Accordingly, an object of the present invention is to provide a composition containing chemokine to promote the migration of adult melanocytes in skin or hair.

Furthermore, the present inventors have developed a kit containing chemokine to identify skin-active substances such as keratin-forming cells, fibroblasts, melanocytes and the like by confirming the expression of chemokines in skin cells, The present invention has been completed. Accordingly, another object of the present invention is to provide a search kit for screening a skin active substance through expression of a chemokine gene in skin cells, and a method for searching for a skin active substance using the same.

In order to accomplish the above object, the present invention provides a composition containing chemokine and promoting adult melanocyte cell migration. More particularly, the present invention relates to a composition for promoting the migration of adult melanocytes in skin or hair by containing chemokine, wherein the chemokine promotes the migration of adult melanocytes, preferably CTACK, I-TAC, Lptn, PARC, SDF1, TARC , RANTES, IP10, and Eotaxin. The composition can be used for the treatment of diseases characterized by low pigmentation of the skin such as vitiligo and white moth.

The present invention also relates to a method for detecting the expression level of chemokine in skin cells, and thereby screening a substance capable of regulating chemokine expression in skin cells, thereby searching for a skin active substance. The chemokine promotes the migration of adult melanocytes, and is preferably at least one selected from the group consisting of CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10, and Eotaxin. More preferably, one or more selected from the group consisting of CTACK, I-TAC, Lptn, PARC, SDF1, and TARC can be used.

More particularly, the present invention relates to a method for screening a whitening substance by screening a substance that reduces the expression of chemokine in the skin cells. The whitening substance may include whitening substances that lighten the skin tone by reducing melanocyte cell migration, whitening substances that improve skin pigmentation such as stain, blackness, and whitening substances that improve pigmentation after inflammation such as acne.

The present invention also relates to a method for screening a substance for treating low-pigment disease by screening a substance that promotes expression of chemokine in the skin cells. The hypochromic disease refers to a disease that exhibits hypochromia in skin and hair, and is preferably vitiligo or white moth.

A search kit of the invention capable of detecting the expression level of chemokine in skin cells comprises a chemokine-specific antibody, a secondary antibody, a chromogenic label, and a skin cell, wherein the expression level of chemokine is regulated in the skin cell By screening the substance, the skin active substance can be searched. The chemokine promotes the migration of adult melanocytes, and is preferably at least one selected from the group consisting of CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10, and Eotaxin.

The chemokine-specific antibody may be, but is not limited to, a capture antibody contained in a commercial kit for CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10 and Eotaxin. Preferably, CTACK capture antibody (MAB3761, R & D systems), I-TAC capture antibody (MAB672, R & D systems), Lptn capture antibody (MAB6951, R & D systems), PARC capture antibody (MAB364, R & D systems), RANTES capture antibody (MAB678, R & D systems), IP10 capture antibody (MAB266, R & D systems) and Eotaxin capture antibody (MAB364, R & D systems) Can be used.

The secondary antibody may be CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10, and Eotaxin-specific detection antibody. (BAF376, R & D systems), an I-TAC detection antibody (BAF672, R & D systems), Lptn detection antibody , R & D systems), TARC detection antibody (BAF364, R & D systems), RANTES detection antibody (BAF278, R & D systems), IP10 detection antibody (BAF266, R & D systems) and Eotaxin detection antibody (BAF320, R & D systems).

Streptavidin-HRP (Streptavidin-HRP) contained in a commercial kit for CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10 and Eotaxin may be used as the label for color development. But is not limited thereto. Preferably, streptavidin-conjugated to horseradish-peroxidase conjugated with horseradish peroxidase is used.

More specifically, the present invention provides a search kit for screening a substance that reduces the expression of chemokine in the skin cells, thereby searching for a whitening substance. The whitening substance may include whitening substances that lighten the skin tone by reducing melanocyte cell migration, whitening substances that improve skin pigmentation such as stain, blackness, and whitening substances that improve pigmentation after inflammation such as acne.

The present invention also provides a search kit for screening a substance for promoting the expression of chemokines in the skin cells, thereby searching for a substance for treating hypochromic diseases. The hypochromic disease refers to a disease that exhibits hypochromia in skin and hair, and is preferably vitiligo or white moth.

In one embodiment of the present invention, the skin cells are selected from the group consisting of NHEK (normal human epidermal keratinocytes), NHF (normal human fibroblasts), melanocytes (NHEM), human epidermal- And a cell co-cultured with human keratinocyte and melanocyte, but the present invention is not limited thereto.

In addition, the present invention provides a method for screening a skin cancer cell, comprising the steps of: treating a skin cell with a candidate substance and confirming expression of the chemokine using the search kit; And a method for separating and purifying a substance that reduces chemokine expression, thereby searching for a skin active substance.

The composition according to the present invention promotes the movement of melanocytes in the skin and hair, and thus can be applied to the affected part in hypochromic diseases such as vitiligo and white moth, thereby exhibiting symptom relief and therapeutic effects.

In addition, the skin active substance search kit according to the present invention can quickly and easily screen skin active substances such as a substance for treating a whitening substance and a hypochromic disease through the expression of a chemokine gene in skin cells.

1 is a graph showing the results of confirming cell migration in human melanocytes treated with chemokine.
2 is a graph showing the results of confirming cell migration in human keratinocytes treated with chemokine.
FIG. 3 is a graph showing the results of confirming the amount of RANTES expression in human keratinocytes and human melanocytes treated with kojic acid.
4 is a graph showing the results of confirming the amount of RANTES expression in human epidermal-derived immune cells treated with ginsenoside F1.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. It is to be understood that the scope of the present invention is not limited to these embodiments and test examples, and that variations, substitutions, and insertions conventionally known in the art And this is also included in the scope of the present invention.

 [ Reference example  1] Preparation of melanocytes

Human melanocytes purchased from Cascade (Normal Human Epidermal Melanocyte-Moderately pigmented, product number C-102-5C) were seeded on a 100 cm 2 plate in a number of 6 x 10 ⁵ , and the basic medium was HMGS (product number S- 254 medium (product number M-254-500, Gibco) containing Gibco) was used and incubated in a 5% CO ₂ incubator at 37 ° C. When the density reached about 70-80%, subculture was performed, and melanocytes were provided in subsequent experiments.

[ Reference example  2] Preparation of keratinocyte

 NHE-Neo, Pooled (Neonatal Normal Human Epidermal Keratinocytes, Pooled, Cat. No. 00192906, Lonza) provided by Lonza was used for human keratinocyte (Normal Human Epidermal Keratinocyte). KGM-Gold Bullet Kit (product number 192060, Lonza) can be used as the primary medium. The dish was coated with 0.1% gelatin to maintain the proliferation of keratinocytes attached to the basement membrane.

Human keratinocytes were seeded at 6 × 10 ⁵ cells in 100 cm 2 plates and cultured in a basic medium at 37 ° C. in a 5% CO ₂ incubator. Subculture was performed at a density of about 70-80%, and keratinocytes were provided in subsequent experiments.

[ Reference example  3] Preparation of skin-derived immune cells

CD3 ⁺ skin epidermal T cells (CD3 ⁺ epidermal T cells) are obtained from human skin tissue. Skin tissues were prepared by dissolving dermal and epidermal cells in a basal medium (2 mM L-glutamine, 100 U / ml penicillin, 100 [mu] l) containing 2 units / ml Dispase II solution (product number 4942078001, Roche) ml / ml streptomycin and inactivated 10% fetal calf serum, product no. 12-115F, Lonza) at 4 ° C for 12 hours. After separating only the epidermal layer using sterile forceps, add 2 units / ml of Dispase II solution to the skin layer and incubate at room temperature for 10 minutes. Add 10 mM EDTA (product number E6758, Sigma) to inactivate the enzyme. To obtain a suspension with lymphocytes, use a filter (cell strainer 100 mm; product number REF352360, BD falcon) to obtain a suspension. Cells were isolated using a centrifugal separator (Fincoll-Paque gradient centrifugation, Hitopagne-1077, product no. 1077-1, Sigma), and then 50 units / ml of interleukin 2 (IL-2, ) At 37 ° C for 24 hours. To isolate T cells, T cells were obtained by culturing the cultured cells and the basic medium in a 100 mm culture dish coated with CD3 antibody (anti-CD3 antibody, product number MBA100, R & D systems) for 6 days.

 [ Reference example  4] Human keratinocytes and human melanocytes Co-culture

Human melanocytes (Normal Human Epidermal Melanocyte-Moderately pigmented, product number C-102-5C, Cascade) and human keratinocytes (Neonatal Normal Human Epidermal Keratinocytes, Pooled, product number 00192906, Lonza) Specifically, 1.25 x 10 ⁵ human keratinocytes cultured in Reference Examples 1 and 2 and 2.5 x 10 ⁴ human melanocytes were mixed on a 6-well plate to prepare KGM-Gold Bullet Kit (product number 5% CO ₂ incubator at 37 ° C in a 1: 1 mixture of 254 medium (product number M-254-500, Gibco) containing L-glutamine, It is cultured for 24 hours and then used for experiments.

[ Test Example  One] To chemokine  Of Melanocytes

In order to determine whether melanocytes were induced by chemokines, human melanocytes were treated with chemokines secreted from skin cells as well as immune cells. FK506 (Sigma Aldrich, product No. F4679) was used as a control. The chemokine was purchased from the R & D system and the product numbers were CTACK (product number 376-CT), Fracktalkin (product number 362-CX), MCP1 (product number 279-MC), MCP4 (product number 327- (Product number: 271-BME), PARC (product number 394-PA), RANTES (product number 278-RN), IP10 (product number 266-IP), Eotaxin -IT), MIG (product number 392-MG), MIP1a (product number 270-LD), SDF1 (product number 350-NS), TARC (Product number 281-CP) and MDC (product number 336-MD) were used.

The human melanocytes cultured in Reference Example 1 were cultured in a basic medium (254medium, product number M-254-500; Gibco) without HMGS for 4 hours at 37 ° C in a 5% CO ₂ incubator. The cultured human melanocytes were separated using trypsin / EDTA, cultured in a basic medium containing 0.1% BSA at 37 ° C for 30 minutes, and the tube containing the cells was gently patched every 10 minutes.

The basal medium containing 0.1% BSA supplemented with 200 .mu.g / ml of the above chemokine CTACK, Fracktalkin, MCP1, MCP4, MIP1b, PARC, RANTES, IP10, Eotaxin, I-TAC, MIG, MIP1a, SDF1, TARC, Lptn, MCP2, , And 27 [mu] l of the solution was dispensed into the lower chamber of the Boyden chamber. Melanocytes were treated with 1 [mu] M of FK506 as a positive control.

Place the glazed portion of the membrane down on the bottom chamber, and assemble the gasket and top chamber in order. The cultured human melanocytes were divided into 104 cells / 56 μl into the upper chamber, and the assembled Boyden chamber was cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. Dye the membrane using H & E staining (Hematoxylin and Eosin staining), wipe the unmoved cells with a cotton swab, place the shiny side on a glass plate to touch the glass. The number of transferred cells was counted by taking 6 pictures per each well, and the movement of melanocytes was calculated according to Equation 1 below.

As shown in FIG. 1, CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10 and Eotaxin among the chemokines used in the experiments caused migration of melanocytes 1.5 times more than the control group. However, eight other chemokines did not induce melanocyte migration. From this, it can be confirmed that not all chemokines secreted from the skin migrate melanocytes nonspecifically, but only specific chemokines induce the migration of melanocytes.

[ Test Example  2] To chemokine  Of keratinocytes

In order to confirm whether or not the chemokine of the present invention induces migration of skin cells other than melanocytes, the migration of keratinocytes, which are major constituent cells of skin epidermis in which melanocytes are present, by chemokines was examined.

In the above Test Example 1, CTKACK, I-TAC, Lptn, PARC, SDF1 and TARC, which induced the movement of melanocytes, were used, and keratinocytes without any treatment were used as the control group.

Human keratinocytes cultured in Reference Example 2 were cultured in the presence of bovine pituitary extract (BPE), human epidermal growth factor (hEGF), bovine insulin, hydrocortisone, (KGM-Gold ™ BulletKit ™, product number 192060, Lonza) containing Epinephrine and Transferrin at 37 ° C., CO ₂ And incubated with an incubator for 4 hours. The cultured human keratinocytes were separated using trypsin / EDTA, cultured in a basic medium (KGM-Gold ™ BulletKit ™, product number 192060, Lonza) containing 0.1% BSA for 30 minutes at 37 ° C., I gently stroked the tube that contained it.

The chemokine CTACK, I-TAC, Lptn, PARC, SDF1 and TARC were each diluted to 200 μg / ml in a basic medium (KGM-Gold ™ BulletKit ™, product number 192060, Lonza) containing 0.1% BSA, (Boyden chamber) were subdivided into 27 [mu] l each of the lower chambers and keratinized cells which were not treated as the control group were used.

Place the glazed portion of the membrane down on the bottom chamber, and assemble the gasket and top chamber in order. The cultured human keratinocytes were divided into 104 cells / 56 μl in the upper chamber, and then the assembled Bowden chamber was cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Dye the membrane using H & E staining (Hematoxylin and Eosin staining), wipe the unmoved cells with a cotton swab, place the shiny side on a glass plate to touch the glass. FIG. 2 shows the results of calculation of the migration of keratinocytes by the above formula (1) by counting the number of transferred cells by taking pictures in 6 fields per each well.

As shown in FIG. 2, the chemokine CTACK, I-TAC, Lptn, PARC, SDF1, and TARC induced melanocyte migration but did not induce keratinocyte migration. Therefore, it can be seen that when the secretion of the chemokine increases in the skin due to a specific stimulus, it does not induce the migration of keratinocytes, and that the melanocytes are specifically transferred.

[ Example  One] Chemokine  Detection of skin active substance (kojic acid) by expression change

1.25 × 10 ⁵ human keratinocytes and 2.5 × 10 ⁴ human melanocytes co-cultured in Reference Example 4 were suspended in KGM-Gold Bullet Kit (product number 192060, Lonza) and HMGS (product number S-002-5, Gibco (Kojic acid, product number K3125, Sigma Aldrich) 200 mM in a 1: 1 mixture of 254 medium (product number M-254-500, Gibco) , And cultured in a 5% CO ₂ incubator for 48 hours to obtain a supernatant. As a control group, the co-cultured cells were cultured in the same manner for 48 hours except that kojic acid was not treated, and a supernatant was obtained.

100 ml of each of the supernatants obtained above was added to each well of a 96-well plate, and 100 μl of RANTES-specific antibody (MAB678, R & D systems) diluted to 1 μg / ml with PBS was added thereto, Lt; / RTI > for 12 hours. At this time, to obtain the standard curve, the protein concentration of the reference material, recombinant human RANTES was serially diluted to 300, 150, 75, 37.5, 18.75, 9.4, 4.7 ng / ml. After washing with wash buffer (PBS containing 0.05% Tween 20) three times, a blocking solution containing 1% skim milk (PBS containing 10% FBS) was added to prevent nonspecific reaction And cultured at room temperature for 1 hour. The PBS containing the antigen was removed, washed three times with washing buffer, and the remaining solution was completely removed using a paper towel. After washing, 100 μl of sample was added and reacted at room temperature for 2 hours to attach an antigen to a plate attached with an antibody. After removing the sample, each well was washed three times with washing buffer solution. 100 μl of detection antibody (Biotinylated anti-human RANTES) diluted to 20 ng / ml was added and incubated at room temperature for 2 hours. After rinsing twice with washing buffer, streptavidin conjugated to horseradish peroxidase diluted 1: 200 was added for color development, incubated at room temperature for 20 minutes do. Washing is done three times with Washing Buffer Solution, then tap the solution gently with a paper towel to remove any remaining solution. Hydrogen peroxide solution and tetramethylbenzidine were mixed at a ratio of 1: 1 and 100 μl was added to each well. The color reaction was induced and the mixture was incubated at room temperature for 20 minutes. 50 μl of 2N sulfuric acid was added to terminate the color reaction, and the absorbance at 450 nm was measured using a spectrophotometer. The results are shown in FIG.

As shown in the results of FIG. 3, it was confirmed that the expression of RANTES was reduced in the case of treatment with kojic acid in the cells co-cultured with human keratinocytes and human melanocytes, as compared with the control group, which was not treated. It is possible to search for whitening substances.

[ Example  2] Chemokine  Skin Active Substances by Change of Expression ( Gin Senocide  F1)

The CD3 ⁺ epidermal-derived culture in Reference Example 3 T cell (CD3 ⁺ epidermal T cell) a CD3 antibody (immobilized anti-CD3 antibody) was fixed on the plate to activate, 200 U / ml interleukin-2 (IL-2) 500ng / ml PHA into the basal medium and treat Ginsoeniside F1 100 mM, a known whitening substance. After culturing for 48 hours in a 5% CO ₂ incubator at 37 ° C, a supernatant was obtained. The control group was a CD3 antibody (immobilized anti-CD3 antibody) and 200 U / ml IL-2 for activation. T cells that had been treated with 500 ng / ml PHA (Phytohaemaglutinin; product number L1668; Sigma) but not treated with Ginsoeniside F1 were used.

100 ml of the supernatant obtained above was added to each well of a 96-well plate and 100 μl of RANTES-specific antibody (MAB678, R & D systems) diluted to 1 mg / ml with PBS was added to the wells at 4 ° C Lt; / RTI > for 12 hours. At this time, to obtain the standard curve, the protein concentration of the reference material, recombinant human RANTES was serially diluted to 300, 150, 75, 37.5, 18.75, 9.4, 4.7 ng / ml. After washing with wash buffer (PBS containing 0.05% Tween 20) three times, a blocking solution containing 1% skim milk (PBS containing 10% FBS) was added to prevent nonspecific reaction And cultured at room temperature for 1 hour. The PBS containing the antigen was removed, washed three times with washing buffer, and the remaining solution was completely removed using a paper towel. After washing, 100 μl of sample was added and reacted at room temperature for 2 hours to attach an antigen to a plate attached with an antibody. After removing the sample, each well was washed three times with washing buffer solution. 100 μl of detection antibody (Biotinylated anti-human RANTES) diluted to 20 ng / ml was added and incubated at room temperature for 2 hours. After rinsing twice with washing buffer, streptavidin conjugated to horseradish peroxidase diluted 1: 200 was added for color development, incubated at room temperature for 20 minutes do. Washing is done three times with Washing Buffer Solution, then tap the solution gently with a paper towel to remove any remaining solution. Hydrogen peroxide solution and tetramethylbenzidine were mixed at a ratio of 1: 1 and 100 μl was added to each well. The color reaction was induced and the mixture was incubated at room temperature for 20 minutes. 50 μl of 2N sulfuric acid was added to terminate the color reaction, and the absorbance was measured at 450 nm using a spectrophotometer. The results are shown in FIG.

As shown in the results of FIG. 4, when Ginsenoside F1 was treated on human epidermal-derived immune cells, the expression of RANTES was decreased as compared with that of the untreated control group. .

Claims (14)

Claims 1. A pharmaceutical composition for improving and treating a low-pigmented disease comprising chemokine as an active ingredient,
The chemokine is CTACK (CC Motif Chemokine Ligand 27)
Wherein said improvement and treatment of said hypochromic disease is by promoting the migration of adult melanocytes to the lesion of hypochromic disease in skin by said chemokine. 2. The pharmaceutical composition according to claim 1, wherein the composition further comprises at least one chemokine selected from the group consisting of PARC (C-C Motif Chemokine Ligand 18) and I-TAC (C-X-C Motif Chemokine Ligand 11). 2. The pharmaceutical composition according to claim 1, wherein the hypochromic disease is gallstone or white moth. delete delete delete delete delete delete delete delete delete delete delete

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