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Screening method of whitening substance using mitochondrial dynamics and kit using same

A screening method, mitochondrial technology, applied in the direction of biochemical equipment and methods, scientific instruments, microbial measurement/inspection, etc., can solve a lot of cost and effort, does not show the inhibitory effect of human skin hyperpigmentation, melanin production and dispersion Increase and other issues

Inactive Publication Date: 2019-03-12
AMOREPACIFIC CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there is a tendency to make its symptoms worse due to ultraviolet rays, melanin production and dispersion will continue to increase in the absence of ultraviolet rays
Therefore, in the case of whitening substances developed based on the existing ultraviolet irradiation method, there are many cases where the human skin hyperpigmentation inhibitory effect is not actually shown clinically
In addition, in order to study skin hyperpigmentation, it requires a lot of cost and effort to use after biopsy of the skin of the transitional part of the pigmentation of the test subject. Not only is it not easy to obtain human skin tissue, but it is also difficult to obtain human skin tissue unlike ultraviolet irradiation models. Animal Models of Skin Hyperpigmentation

Method used

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  • Screening method of whitening substance using mitochondrial dynamics and kit using same
  • Screening method of whitening substance using mitochondrial dynamics and kit using same
  • Screening method of whitening substance using mitochondrial dynamics and kit using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Induction of Mitochondrial Fusion Using Drugs

[0052] mouse melanoma B16F1 cells (purchased from ATCC), 1.6×10 4 Cell / ml concentration, placed in 12-well plate. After 24 hours, they were treated with 10 μM and 15 μM Mdivi-1 (Enzo Life Sciences) and 200 nM α-MSH (positive control group for melanin induction). After the fusion was induced by treating Mdivi1, observed with confocal laser microscope, it can be observed as figure 2 A shows the fusion of mitochondria. Cells were harvested after 48 hours and lysed with 1N NaOH at 100°C for 30 minutes. Afterwards, the cells were collected, and the total amount of melanin was observed with the naked eye, which was compared with figure 2 B shows the same. Measure the absorbance (SpectraMax190 microplate reader, Molecular Instruments Corporation of America, the U.S.) at 470nm afterwards. figure 2 The total amount of melanin measured by quantifying the results of B is shown in the graph of Fig. 2C. Also, b...

Embodiment 2

[0053] Example 2: Induction of Mitochondrial Fusion Using DRP1 siRNA

[0054] Mouse melanoma B16F1 cells, 1.6×10 4 Cells / ml concentration, placed on a 12-well plate. 24 hours later, DRP1 siRNA #1100 pmol was transfected using liposome reagent (Invitrogen). After treatment with DRP1siRNA to induce fusion, observe with a laser confocal microscope (LSM510, Carl Zeiss (Carl Zeiss), USA), and it can be observed that image 3 A shows the fusion of mitochondria. Cells were harvested after 72 hours and lysed with 1N NaOH at 100°C for 30 minutes. Afterwards, the cells were collected, and the total amount of melanin was observed with the naked eye, which was compared with image 3 B shows the same. Then the absorbance was measured at 470nm, and the figure 2 The results of B are quantified to measure the total amount of melanin, using image 3 The graph of C shows. Also, by Western blotting image 3 B shows the amount of the melanogenesis-regulating protein inside the cell afte...

Embodiment 3

[0057] Example 3: Induction of Mitochondrial Fission Using Drugs

[0058] Mouse melanoma B16F1 cells, 1.6×10 4 Cells / ml concentration, placed on a 12-well plate. After 24 hours, the cells were treated with 2.5 μM and 5 μM CCCP (Sigma) and 200 nM α-MSH. At this time, CCCP was treated under the condition that treatment of α-MSH induces melanin biosynthesis. In addition, after treatment with CCCP alone, after inducing division, observe with a laser confocal microscope (LSM510, Carl Zeiss, USA), it can be observed as Figure 4 A shows the division of mitochondria. After 48 hours, the cells were harvested and lysed at 100° C. for 30 minutes with 1 N NaOH. Afterwards, the cells were collected, and the total amount of melanin was observed with the naked eye, which was compared with Figure 4 B shows the same. Measure the absorbance (SpectraMax190 microplate reader, Molecular Instruments Corporation of America, the U.S.) at 470nm afterwards. Figure 4 The results of B are quant...

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Abstract

The invention relates to a method for screening a whitening substance by means of the detection of mitochondrial dynamics, and a kit using the method. More specially, the invention relates to a method for screening a whitening substance, by reducing the activity of mitochondria, the dynamics is blocked, then a candidate substance is used to process the mitochondria, whether the candidate substance has a whitening effect or not is evaluated by whether the dynamics of the mitochondria is improved or not, and by means of the characteristic of the mitochondria, the kit can screen whitening substance simply.

Description

technical field [0001] The present invention relates to a method for screening whitening substances by measuring mitochondrial kinetics and a kit using the same. In more detail, it relates to a screening method for a whitening substance that evaluates whether the candidate substance has a whitening effect by measuring whether the mitochondrial kinetics increases or not after treatment with a candidate substance after reducing the activity of the mitochondrial stereotype to hinder the kinetics, And a kit that can easily screen whitening substances by utilizing the characteristics of such mitochondria. Background technique [0002] In the past, the following methods have been used for the development of new whitening substances. The candidate substances are directly treated with melanocytes, which are melanocytes, to confirm whether they inhibit melanin production, or the melanocytes are irradiated with ultraviolet rays to confirm whether the candidate substances inhibit melan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02
CPCC12Q1/025G01N33/5079
Inventor 罗勇柱张希景朴录贤李海光
Owner AMOREPACIFIC CORP
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