TW201916886A - Use of Eutrema japonica leaf extracts for manufacture of composition for suppressing MITF gene expression - Google Patents

Abstract

The present invention provides uses of a Eutrema japonica leaf extract for manufacture of composition for suppressing MITF gene expression. The Eutrema japonica leaf extract is prepared by extraction of Eutrema japonica leaves using water, alcohols, or mixtures of water and alcohols as solvents. The Eutrema japonica leaf extract can reduce melanogenesis via suppressing MITF gene expression in skin melanocytes, and thus contributes to reducing skin darkness and fading brown spots and sunspots.

Description

   Use of wasabi leaf extract for preparing composition for inhibiting MITF gene expression   

The present invention relates to the use of a wasabi leaf extract, in particular to the use of a wasabi leaf extract to prepare a composition for inhibiting MITF gene expression.

Wasabi scientific name is Eutrema japonica , also known as "mandrill", is a plant belonging to the genus Crassulaceae. Wasabi has a strong pungent taste, but its spicyness is different from pepper. The hotness of pepper stimulates the tongue, while the spicy taste of wasabi stimulates the sinuses. Wasabi is commonly grown beside valleys and rivers in Japan. Other varieties include Wasabi koreana and Wasabi tetsuigi ; the two types of wasabi commonly sold in the market are Wasabi japonica Daruma and Wasabi japonica Mazuma.

Wasabi leaves are edible and have the spicy taste of wasabi stems. The method of cooking wasabi is marinated with salt and vinegar sauce overnight to make a wasabi salad, cooked with soy sauce and boiled water to serve as a side dish for rice and wine, or as a sushi roll, and can even be dipped into starch and fried into tempura Luo.

Microphthalamia-associated transcription factor (MITF) is a transcription factor required for normal melanocyte development in the skin. It can regulate the gene expression of several other proteins involved in melanin formation, such as tyrosinase ), Tyrosinase related protein-1 (TYRP1), and dopachrome tautomerase (DCT) and other genes.

To achieve the goal of deep skin whitening, it is necessary to develop a novel composition that is safe and effective in inhibiting the growth of melanocytes.

For this reason, an object of the present invention is to provide a use of a leaf extract of wasabi ( Eutrema japonica ) for preparing a composition for inhibiting the expression of a small eye malformation-related transcription factor (MITF) gene, wherein the wasabi leaf extract is a solvent It is obtained by extracting a wasabi leaf.

In one embodiment of the present invention, the solvent is water, alcohols, or an alcohol-water mixture, and the liquid-to-solid ratio of the solvent to the wasabi leaf is 5-20: 1 ~ 5, and the extraction system is at 50 ° C-100 ° C And the wasabi leaf extract is further obtained through a filtering step and a concentration step.

In one embodiment of the present invention, the wasabi leaf extract is a water wasabi leaf extract having a concentration of at least 0.5 mg / mL.

The wasabi leaf extract obtained by using water, alcohol, or a mixture of alcohol and water as a solvent can reduce the formation of melanin by inhibiting the gene expression of MITF in skin melanocytes, and finally achieve the effect of whitening the skin, thus helping to improve the skin. Dull skin and fade dark spots and sun spots. Therefore, the wasabi leaf extract of the present invention can be used to prepare a composition that inhibits MITF gene expression and inhibits melanin formation. The composition can be in the form of a cream, emulsion, dispersion, foam, gel, ointment, powder, patch, or mask Present and can be made into a pharmaceutical, skincare, or cosmetic product for topical application to the skin.

The embodiments of the present invention will be further described below with reference to the drawings. The examples listed below are intended to clarify the features and applications of the invention, but not to limit the scope of the invention. Anyone skilled in the art will not depart from it. Within the spirit and scope of the present invention, some modifications and retouching can be done. Therefore, the protection scope of the present invention shall be determined by the scope of the attached patent application.

Figure 1 shows the relative expression of MITF gene in melanoma cells treated with wasabi leaf extract of the present invention.

The invention provides a use of wasabi leaf extract for preparing a composition for inhibiting MITF gene expression. The wasabi leaf extract of the present invention is obtained by extracting a wasabi leaf with a solvent, wherein the solvent is water, alcohols, or a mixture of alcohol and water, and the liquid-solid ratio of the solvent to the wasabi leaf is 5-20: 1 ~ 5, and the extraction is performed at 50 ° C to 100 ° C. By analyzing the expression amount of genes related to melanin formation, the wasabi leaf extract of the present invention was confirmed to reduce the expression amount of MITF gene in melanoma cells.

definition

The values used in this article are approximate. All experimental data are shown in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

Materials and Methods material

Buy DMEM medium (Dulbecco's modified Eagle's medium) from Gibco, product number 12100-038; fetal bovine serum (FBS), product number 10438-026; penicillin / streptomycin, product number 15140 -122; phosphate buffered saline (PBS solution), product number 14200-075; lysis buffer, containing 0.1mM phenylmethylsulfonyl fluoride (PMSF) and 1% polyethylene glycol octylphenyl ether (Triton X-100) in 0.1M phosphate buffer (pH 6.8).

Cell culture

The following examples use mouse skin melanoma cell line B16F10 (ATCC CRL-6475) purchased from the American Type Culture Collection (ATCC) for in vitro experiments. B16F10 cells were cultured at 37 ° C and 5% carbon dioxide in a DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin, hereinafter referred to as cell culture medium.

Gene expression analysis

In the present invention, the quantitative polymerase chain reaction (qPCR) is used to determine the expression of genes related to melanin formation in cells. The steps are briefly described as follows:

1. RNA is extracted from the cells using a RNA Extraction Kit (Geneaid).

2. Use the reverse transcriptase SuperScript® III Reverse Transcriptase (Invitrogen) to reverse transcribe RNA (2000ng) into complementary DNA (cDNA).

3. The qPCR reaction was performed using the Step One Plus (ABI) and qPCR kits (KAPA SYBR FAST qPCR Kits (2x), KAPA Biosystems). The primer used in the qPCR reaction includes a forward primer (5'-GCCTCCAAGCCTCCGATAAG-3 ', SEQ ID NO: 1) and a reverse primer (5'-GCACTCTCTGTTGCATGAACT-3', SEQ ID) directed to the MITF gene as a target gene. NO: 2), and forward primer (5'-CATGTACGTTGCTATCCAGGC-3 ', SEQ ID NO: 3) and reverse primer (5'-CTCCTTAATGTCACGCACGAT-3') directed against the actin β (ACTB) gene as an internal control , SEQ ID NO: 4). The reaction conditions were that the unwinding temperature was set to 95 ° C, the sticking temperature was set to 60 ° C, the extension temperature was set to 65 ° C, and the reaction was performed for 40 cycles.

4. Analyze the melting curve obtained from the aforementioned qPCR reaction.

5. Determine the relative expression of MITF gene by 2- ^{△△ CT} method. The method is the threshold cycle (C _T) ACTB gene as an internal control of the reference cycle threshold gene, calculated by the following formula relative fold change: C gene of △ C _T = the experimental group or the control group _T - internal control C _T

△△ C _T = test group △ C _T - △ C _T of the control group

Multiple change = 2- ^{△△ Ct average}

6. Use the STDEV function in Excel software to calculate the standard deviation of the relative expression of each gene, and use the one-tailed Student's T test (TTEST) to calculate the statistical difference.

Example 1 Preparation of Wasabi Leaf Extract

This example illustrates the method for preparing wasabi leaf extract of the present invention. First, the wasabi leaves are washed and dried, and the wasabi leaves are coarsely crushed by a pulverizer. Secondly, crude wasabi leaves were extracted with water, alcohols, or a mixture of alcohol and water as a solvent. The solvent is preferably water, and the solvent and the coarse wasabi leaves are uniformly mixed according to a liquid-to-solid ratio of 5-20: 1-5, and the extraction temperature is between 50 ° C and 100 ° C, preferably between 75 ° C and 95 ° C. In this embodiment, the extraction time is 0.5 to 3 hours.

After the wasabi leaf extract obtained through the above extraction step is cooled to room temperature, it is filtered through a 400 mesh filter to remove residual solids. The filtered wasabi leaf extract can be further concentrated under reduced pressure at 45 ° C to 70 ° C to obtain a concentrated product. The following examples are performed using concentrated wasabi leaf extracts for cell experiments. In order to obtain a solid wasabi leaf extract, the aforementioned wasabi leaf extract concentrated under reduced pressure may be spray-dried to remove the solvent, thereby obtaining a wasabi leaf extract powder.

Example 2 Expression of wasabi-related genes regulated by wasabi leaf extract

In order to explore the regulation effect of wasabi leaf extract on the melanin formation-related genes of skin melanocytes in this invention, qPCR was used to determine the melanoma formation-related genes of mouse skin melanoma cell line B16F10 after treatment with wasabi leaf extract. Change in performance. First, melanoma cells were seeded at 1.5 × 10 ⁵ cells / well into each well of a 6-well plate containing 2 mL of cell culture medium, and cultured at 37 ° C. for a period of time. Next, cells were treated with 0.5 mg / mL wasabi leaf water extract for 48 hours, and the treated cells were collected for q-PCR. The melanoma cells of the control group were not treated with water extract of wasabi leaves.

The relative expression of the small eye malformation-associated transcription factor (MITF) gene in the above melanoma cells is shown in FIG. 1. The relative expression of the gene in the vertical axis represents the multiple of the RNA expression of the MITF gene in the control group cells. MITF is involved in the differentiation and proliferation of melanocytes, and its gene expression is also positively correlated with melanin formation. According to Fig. 1, compared to the control group, the administration of a water extract of wasabi leaf at a concentration of 0.5 mg / mL to melanoma cells significantly reduced the expression of MITF gene by about 31%. This result shows that the wasabi leaf extract of the present invention can reduce the melanin formation by inhibiting the gene expression of MITF in skin melanocytes.

In summary, the wasabi leaf extract obtained by using water, alcohol, or alcohol-water mixture as a solvent in the present invention can reduce the expression of melanin and pigmentation in the skin by inhibiting the gene expression of MITF in skin melanocytes Skin whitening effect. Therefore, the wasabi leaf extract of the present invention can be used to prepare a composition that inhibits MITF gene expression and inhibits melanin formation. , Gel, ointment, powder, patch, or mask, and may further include a humectant, a whitening agent, a cuticle remover, or any combination thereof.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of wasabi leaf extract for preparing composition for inhibiting MITF gene expression

<130> 106B0410-I1

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Claims (10)

    A wasabi leaf extract is used for preparing a composition for inhibiting the expression of a small eye malformation-related transcription factor (MITF) gene. The wasabi leaf extract is obtained by extracting a wasabi leaf with a solvent.         The use as described in item 1 of the patent application range, wherein the solvent is water, an alcohol, or an alcohol-water mixture.         The use as described in item 1 of the scope of patent application, wherein the liquid-to-solid ratio of the solvent to the wasabi leaf is 5-20: 1-5.         The use described in item 1 of the patent application range, wherein the extraction is performed at 50 ° C to 100 ° C.         The use according to item 1 of the scope of patent application, wherein the wasabi leaf extract is further obtained through a filtering step and a concentration step.         The use according to item 1 of the scope of patent application, wherein the wasabi leaf extract is a water wasabi leaf extract having a concentration of at least 0.5 mg / mL.         The use according to item 1 of the scope of patent application, wherein the wasabi leaf extract is administered topically to the skin.         The use according to item 1 of the scope of patent application, wherein the composition for inhibiting the expression of the MITF gene is a medicine, a skincare product, or a cosmetic.         The use according to item 1 of the scope of the patent application, wherein the composition for inhibiting MITF gene expression is presented in the form of a cream, an emulsion, a dispersion, a foam, a gel, an ointment, a powder, a patch, or a mask.         The use according to item 1 of the scope of the patent application, wherein the composition for inhibiting MITF gene expression further comprises a humectant, a whitening agent, a cuticle remover, or any combination thereof.