The aim of the present study is to preliminarily investigate the antimelanogenesis effect of
Medicinal mushrooms had an established history of being used in nutritionally functional food as well as traditional oriental therapies. Traditional medicines derived from medicinal mushrooms were increasingly being used to treat a wide variety of clinical conditions, with relatively little knowledge of their modes of action. The mushroom
In this report, we describe the differential extraction of dried and powdered
Mushroom tyrosinase (EC1.14.18.1), Dimethyl sulfoxide (DMSO), L-tyrosine (L-Tyr), L-3, 4-dihydroxyphenylalanine (L-DOPA), and
Powdered
The Shimadzu LC-20AT series high performance liquid chromatography system was equipped with a diode array detector (DAD). Analysis was carried out using an Inertsil ODS-SP column (250 mm × 4.6 mm i.d., 5
Enzymatic assay was performed for screening active fractions according to the procedure of Chen and Liu [
For active fractions that inhibited mushroom tyrosinase by above method described at 0, 4, 6, 8, 10, and 12
The B16 melanoma cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Hyclone’s Modified RPMI-1640’s Medium (Hyclone, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum, 1% Penicillin-Streptomycin Solution, and 100x (Beyotime Institute of Biotechnology, China) in culture flasks in a CO2 incubator with a humidified atmosphere containing 5% CO2 in air at 37°C. The cell culture medium was changed every 2-3 days and subcultured by trypsinisation after beginning to adhere and grow for 3 days. The cells were seeded at the appropriate cell density by using BD Accuri C6 (BD, USA) into wells of cell culture plates for further experiments.
To determine the safety of the various compounds, after treatment with the test compounds cell viability was determined by using MTT colorimetric assay and cell apoptosis rate by using AnnexinV-FITC apoptosis analysis kit (Tianjin Sungene Biotech Co., Ltd). 1 × 106 cells were added to individual wells of a 24-well plate. After 24 h incubation, test compounds or kojic acid (100, 200, 400
Cellular tyrosinase activity and melanin content were measured using a previously described method [
For melanin content, the cells were treated with test compounds or kojic acid in the presence or absence of
The cells were treated with test compounds as described above for the determination of tyrosinase activity. Each well of a 96-well plate contained 40
Due to the colour interference of the extract, the tyrosinase inhibitory effect of original extracts from
Screening of tyrosinase inhibitors with using Tyr as the substrate, concentrations of fraction were 10
The number of compounds in fraction a-d (Fa-d) was analyzed by HPLC, respectively. In the HPLC chromatogram, there were two, two, one, one major peaks (Fa-a, Fa-b, Fb-a, Fb-b, Fc-a, Fd-a) in different fractions. Compound Fa-a, Fa-b, Fb-a, Fb-b, Fc-a, Fd-a were further obtained by PHPLC. The structural information of six compounds was obtained using NMR. All spectral data were consistent with the data of known betulin (Fa-a), trametenolic acid (Fa-b), inotodiol (Fb-a), lanoserol (Fb-b) nicotinie acid (Fc-a), and 3
Chemical structure of betulin (Fa-a), trametenolic acid (Fa-b), inotodiol (Fb-a), lanoserol (Fb-b), nicotinie acid (Fc-a), and 3
l3C-NMR (300 MHZ, DMSO) spectral data of compounds.
position | l3C-NMR data | |||||
---|---|---|---|---|---|---|
Fa-a | Fa-b | Fb-a | Fb-b | Fc-a | Fd-a | |
1 | 40.9(t) | 35.6(t) | 35.5(t) | 36.1(t) | 35.6(t) | |
2 | 27.3(t) | 28.8(t) | 27.8(t) | 28.3(t) | 154.0(d) | 27.8(t) |
3 | 78.1(d) | 77.2(d) | 78.9(d) | 78.9(d) | 90.1(s) | 79.8(d) |
4 | 39.3(s) | 39.0(s) | 38.8(s) | 39.3(s) | 137.4(d) | 38.9(s) |
5 | 55.9(d) | 50.5(d) | 50.42(d) | 50.8(d) | 124.5(d) | 50.4(d) |
6 | 18.8(t) | 18.4(t) | 19.1(t) | 18.6(t) | 150.9(d) | 19.43(d) |
7 | 34.9(t) | 28.0(t) | 12.6(t) | 26.7(t) | 167.0(d) | 25.6(t) |
8 | 41.2(d) | 134.8(s) | 134.6(s) | 134.4(s) | 134.6(s) | |
9 | 50.8(d) | 143.7(s) | 134.2(s) | 134.8(s) | 134.2(s) | |
10 | 37.6(s) | 37.0(s) | 37.0(s) | 37.4(s) | 37.0(s) | |
11 | 21.1(t) | 21.1(t) | 20.9(t) | 21.4(t) | 21.0(t) | |
12 | 25.7(t) | 30.6(t) | 29.1(t) | 31.4(t) | 29.7(t) | |
13 | 37.5d(d) | 44.2(s) | 44.8(s) | 44.9(s) | 44.8(s) | |
14 | 43.0(s) | 49.4(s) | 49.5(s) | 50.2(s) | 49.4(s) | |
15 | 27.6(t) | 32.8(t) | 31.0(t) | 31.0(t) | 30.9(t) | |
16 | 30.0(t) | 27.0(t) | 31.0(t) | 28.6(t) | 30.9(t) | |
17 | 48.6(s) | 48.1(d) | 47.2(d) | 50.8(d) | 47.4(d) | |
18 | 48.4(d) | 16.0(q) | 15.6(q) | 15.7(q) | 15.7(q) | |
19 | 49.1(d) | 19.4(q) | 18.3(q) | 19.5(q) | 19.1(q) | |
20 | 151.3(s) | 47.3(d) | 41.7(d) | 36.7(d) | 42.9(d) | |
21 | 29.7(t) | 177.4(s) | 12.5(q) | 19.1(q) | 12.5(q) | |
22 | 34.2(t) | 27.0(t) | 73.3(d) | 36.7(t) | 74.6(d) | |
23 | 28.0(q) | 26.1(t) | 27.2(t) | 25.7(t) | 27.2(t) | |
24 | 15.3(q) | 124.3(d) | 121.4(d) | 125.2(d) | 41.1(t) | |
25 | 16.0(q) | 131.5(s) | 134.9(d) | 131.3(d) | 70.8(s) | |
26 | 16.1(q) | 25.7(q) | 26.5(q) | 26.1(q) | 30.0(q) | |
27 | 15.3(q) | 17.9(q) | 18.2(q) | 17.9(q) | 18.0(q) | |
28 | 10.4(t) | 28.5(q) | 28.0(q) | 28.4(q) | 15.4(q) | |
29 | 109.6(q) | 15.5(q) | 15.3(q) | 15.7(q) | 27.9(q) | |
30 | 19.1(t) | 24.5(q) | 24.2(q) | 24.2(q) | 24.4(q) |
Fa-a and Fa-b appeared to have some cytotoxic and apoptotic rates, a more highly cytotoxic and apoptotic rates (Fc-a), and a less cytotoxic and apoptotic rates (Fb-a, Fb-a, Fd-a) and also could be showed in Table
Effects of test compounds and kojic acid on cell viability and apoptosis rate in B16 melanoma cells. Control groups (from wells without test material or kojic acid) were set as 100% for cell viability and set as 0% for apoptosis rate. Experimental groups were expressed as a percentage of controls (mean ± SD).
Compounds | Cell viability | Apoptosis rate | ||||
---|---|---|---|---|---|---|
100 |
200 |
400 |
100 |
200 |
400 | |
Control | 100 | 100 | 100 | 0 | 0 | 0 |
Fa-a | 88.31 ± 3.21* | 75.13 ± 2.65* | 65.32 ± 4.41 | 9.01 ± 2.21 | 12.21 ± 3.03 | 15.31 ± 2.90 |
Fa-b | 96.45 ± 3.08* | 85.32 ± 4.13* | 70.02 ± 2.05 | 5.11 ± 4.01 | 6.51 ± 3.31 | 10.09 ± 4.31 |
Fb-a | 99.34 ± 1.53* | 99.43 ± 1.41 | 98.24 ± 1.35 | 3.91 ± 3.27 | 5.10 ± 2.61* | 6.07 ± 3.53** |
Fb-b | 99.98 ± 2.56** | 96.26 ± 2.61 | 94.56 ± 3.03 | 0.31 ± 4.37*** | 2.62 ± 3.33*** | 5.12 ± 2.35* |
Fc-a | 73.53 ± 3.11** | 45.31 ± 2.56 | 40.51 ± 2.12 | 10.12 ± 1.14 | 25.44 ± 1.53*** | 30.51 ± 1.11*** |
Fd-a | 96.70 ± 1.01 | 97.12 ± 1.12 | 85.03 ± 1.51 | 1.21 ± 2.31 | 2.15 ± 3.92 | 7.34 ± 3.28 |
Kojic acid | 92.43 ± 4.41*** | 86.08 ± 2.12** | 70.91 ± 3.12** | 9.45 ± 1.15 | 12.36 ± 1.19 | 15.05 ± 2.21 |
The B16 cells line was used because they produce melanin and contain tyrosinase which is associated with melanogenesis under
Effects of test compounds and kojic acid on cellular tyrosinase activity in B16 melanoma cells. Data are expressed as a percentage of control which was set at 100%. Each column represents the mean ± SD of three independent experiments.
Effects of test compounds and kojic acid on cellular tyrosinase activity in a-MSH-stimulated B16 melanoma cells compared with kojic acid. The cells were incubated with 100
Effects of test compounds and kojic acid on cellular melanin content in B16 melanoma cells. The control readings were set as 100%. Data are expressed as a percentage of control which was set at 100%. Each column represents the mean ± SD of three independent experiments.
Effects of test compounds and kojic acid on cellular melanin content in a-MSH-stimulated B16 melanoma cells compared with kojic acid. The cells were incubated with 100
Fa-a and Fa-b significantly and Fc-a slightly reduced the tyrosinase activity, Fb-a and Fb-b significantly increased the tyrosinase activity of B16 melanoma cells, and Fd-a had a little or no inhibition effect on the tyrosinase activity. So Fa-a and Fa-b were investigated to examine their mechanism of action. We performed an enzyme kinetics study of Fa-a and Fa-b in B16 melanoma cells based tyrosinase assays with various concentrations of the L-DOPA substrate. A Lineweaver-Burk plot of the data was shown in Figures
(a) Inhibitory effects of Fa-a on tyrosinase activity in B16 melanoma cells. Lineweaver-Burk plots in the absence (control) or in the presence of Fa-a with L-DOPA as the substrate are shown. Concentrations of Fa-a for the curve 1–5 at 0, 25, 50, 100, and 180
(a) Inhibitory effects of Fa-b on tyrosinase activity in B16 melanoma cells. Lineweaver-Burk plots in the absence (control) or in the presence of Fa-b with L-DOPA as the substrate are shown. Concentrations of Fa-b for the curve 1–5 at 0, 25, 50, 100, and 180
Six compounds isolated from
Dimethyl sulfoxide
L-tyrosine
L-3,4-dihydroxyphenylalanine
Diode array detector
Preparative high performance liquid chromatography
High performance liquid chromatography
50% inhibiting concentration
50% accelerating concentration.
The authors declare that there is no conflict of interests. The authors declare that they have no financial and personal relationships with other people or organizations that can inappropriately influence our work; there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the position presented in this paper.
Zheng-Fei Yan developed the algorithm. Zheng-Fei Yan and Yang Yang carried out most of the analyses. Feng-Hua Tian and Xin-Xin Mao participated in the design of the study and helped in algorithm development. Zheng-Fei Yan drafted the paper. Yu Li and Chang-Tian Li conceived and coordinated the study. All authors have read and approved the final paper.
This work was supported by Jilin Province Science and Technology Agency-Jilin Province Innovative Drug Incubation Base Project (no. 2011ZX09401-305-46).