Page 9 - H. Cell signaling

Analysis of melanin synthesis inhibitor from Butanol fraction of Ligustrum lucidum Ait

Chae-Rin Kim 1,# , Ji-Eun Park , Ga-Eun Yoo , Hae-Ryeong Lim 1,** and Deug-Chan Lee 1,*
1
1,#
Department of Immunology, College of Biomedical Science, Kangwon National University,
Kangwondaehak-gil, Chuncheon, Gangwondo, 200-701, Korea
*Email: dclee@kangwon.ac.kr, **Email: dimgofud@naver.com
BACKGROUND AIM

Recently, cosmetics industry has expected that bio-cosmetics is a growth engine of future cosmetics industry Ligustrum lucidum Ait is a fruit of Ligustrum japonicus. For many decades, Ligustrum
because people prefer functional-cosmetic and natural cosmetics. As customer expectations about cosmetics are lucidum Ait is used for medical purpose, especially for liver and kidney, in Asia. Also,
gradually increasing, functional cosmetic industry including anti-wrinkle and whiteness is rapidly growing. Skin color
is influenced by skin thickness and amounts of pigment such as hemoglobin, carotene and melanin. Among them, biological effects of Ligustrum lucidum Ait. such as antidiabetic and antioxidant have been
melanin pigment made by melanocyte plays the most important role. The melanin pigment is produced by tyrosine studied. 70% ethanol extract of dried Ligustrum lucidum Ait showed inhibitory effect on
reacting with tyrosinase. Tyrosinase oxidizes tyrosine into DOPA, and DOPA is oxidized into DOPA quinone. The melanin synthesis using mouse melanocyte, B16F10 cells. 70% ethanol extract was
DOPA quinone undergoes auto oxidation and changes to 5,6-dihydroxyindole. Finally, 5,6-dihydroxyindole is fractionated to various solvents (hexane, Dichloromethane, Ethyl acetate, butanol,
converted into dark-brown melanin pigment. Therefore, tyrosinase is the most important enzyme to make melanin aqueous solvent). Butanol fraction produced highest yield and extremely inhibited melanin
production. If it is inhibited, melanin production cannot be proceeded. Ligustrum lucidum Ait is the fruit of Ligustrum
japonicus Thunberg. Due to the benefits for liver and kidney, Ligustrum lucidum Ait has been used to make medicine synthesis than other fractions. In this study, we reveal melanin inhihition effects of
since old days. Studies on Ligustrum lucidum Ait are continuously proceeded, but the effects on melanin inhibition Ligustrum lucidum Ait extract by using B16F10(melanocyte).
have yet to be reported.
METHODS
(1) Extract & fraction : In the preparation of the extract of Ligustrum lucidum Ait, 70% ethanol (1500mL) of an extraction solvent (1500mL) was added to 150g of the pulverized Ligustrum lucidum Ait as follows. And it was prepared
by repeating the extraction three times at room temperature for 24 hours. Based on the extraction yield and efficacy, the 70% ethanol extract was mixed in the same amount in the order of hexane, dichloromethane, ethylacetate,
butanol solvents, that is, from a low polarity solvent to a high solvent, followed by layer separation to obtain each fraction.
(2) B16F10 cell culture : B16F10 cell culture, a malignant melanoma cell line, was used in the experiment while subculture using 60dish according to the method of Yoonetal., (2013). As a medium for cell culture, DMEM (Dulbecco's
Modified Eagle's Medium) containing penicillin (100 UI/mL), streptomycin (100 μg/mL), and 10% FBS (Fetalbovineserum) was used, and the cell culture during the experiment was 5% CO2. , 37 ℃ incubator conditions were tested.
(3) MTT assay (Cell viability assay)
Before analyzing the efficacy of the extract in cells, the cytotoxicity of the extract was assessed by treating the cells with samples of varying concentrations and then evaluating their effects on cell growth, proliferation and
cytotoxicity via MTT assay. The melanoma cell line B16F10 was aliquoted into a 24-well plate at 1x10 cells/well, incubated for 24 hours in a 37 °C incubator containing 5% CO2, and then treated with a new product and cultured for
72 hours. medium. The mg/mL MTT (Triazolblue Tetrazolium Bromide) solution was diluted 1/10 in the medium and incubated for 4 hours, then the culture solution was removed and 600 μL of dimethylsylfoxide (DMSO) solution
was added. 80 μL of each sample culture solution was added to a 96-well plate. Cytotoxicity was measured by measuring absorbance at 550 nm each with a spectrophotometer. Cytotoxicity evaluation was expressed as a
percentage based on the absorbance of the control group using the medium itself according to the method of Chanetal., (2011).
(4) Evaluation of Melanin Synthesis Inhibition Effect of Ligustrum lucidum Ait Extract : B16F10, a malignant melanoma cell line, is dispensed into a 60-dish culture dish by 4x104 cells/dish and incubated for 24 hours in a 37°C
incubator containing 5% CO2. Discard the medium and add the test substance to each desired concentration in a new medium. After dilution and treatment, the cells were cultured for 72 hours. The cultured cell line was washed
with PBS, and 1 mL of PBS was added to scrape the cells with a scrapper and transferred to an e-tube, followed by centrifugation at 7000 rpm for 5 minutes and 30 seconds at 4°C. 1N NaOH and 10% DMSO were added and left to
stand for about 30 minutes in a water bath at 60°C. The supernatant was placed in a 96-wellplate and measured with a 405nm spectrophotometer to measure the inhibition rate of melanin synthesis.
RESULTS
Figure 1 Figure 2
As a result, 70% EtOH extract of
Ligustrum lucidum Ait inhibit the malnin
synthesis. In order to find the melanin
synthesis inhibitor, we proceed fraction
using various solvents (Hexane,
Dichloromethane, Ethyl acetate, Butanol,
water). Among the fractions, butanol
fraction is made highest yield and inhibit
melanin synthesis than other fractions.

Figure 3 Figure 4

Figure 1. (A) The cytotoxicity of Ligustrum lucidum Ait extract B16F10 fibroblast. (B) The inhibitory effect of Ligustrum lucidum Ait extract on Melanin in B16F10 fibroblast
Figure 2. (A) The cytotoxicity of Ligustrum lucidum Ait each solvent extract in B16F10 fibroblast. (B) Activity rate of each solvent from Ligustrum lucidum Ait on Melanin
Figure 3. Fractionation procedure of Ligustrum lucidum Ait
Figure 4. (A) The cytotoxicity of Ligustrum lucidum Ait solvent fractions in B16F10 fibroblast. (B) Activity rate of solvent fractions from Ligustrum lucidum Ait on Melanin
CONCLUSION REFERENCES Contact information

We demonstrated thant 70% EtOH extract 1. Korean J. Oriental Physiology & Pathology
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obtained in nature and this natural doi:10.3390/ijms150916665 **Email: dimgofud@naver.com
material has an advantage over artificial 4. Biol. Pharm. Bull. 36(5) 772–779 (2013) Vol. 36,
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that Ligustrum lucidum Ait has possibility 5. Journal of Life Science 2013 Vol. 23. No. 12.
to become whiteness compound. 1495~1500