Here we examine the effects of extracts of
Melanin plays a key role in photoprotection and imparts skin color. It is well documented that overproduction and excessive accumulation of melanin leads to various human skin disorders, such as melasma, freckles, age spots, and malignant melanomas [
The following chemicals were procured from Sigma-Aldrich, St. Louis, MO, USA: ascorbic acid, 1,1-diphenyl,2-picryl hydrazyl (DPPH), gallic acid, vanillin, (+)-catechin, sulfuric acid, sodium dodecyl sulphate (SDS), sulfuric acid (H2SO4), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, potassium ferricyanide (K3[Fe(CN)6]), ferrous sulphate (FeSO4), ferric chloride (FeCl3), sodium carbonate (Na2CO3), sodium nitrite (NaNO2), sodium hydroxide (NaOH), aluminum chloride (AlCl3), copper(II) chloride (CuCl2), iron(II) chloride (FeCl2), ethanolic neocuproine, Folin–Ciocalteu’s phenol reagent, 2,2′-bipyridyl, ethylenediaminetetraacetic acid (EDTA), ammonium acetate, dimethyl sulfoxide (DMSO), and propidium iodide (PI). Potassium persulfate (Junsei, Japan), HPLC grade methanol, and ethanol (J.T Baker, U.S.A) were the other chemicals used.
RPMI 1640 medium was purchased from Thermo SCIENTIFIC, DMEM from Gendepot, and the cell counting Kit-8 (CCK-8) from Dojindo Laboratories.
Freeze-dried plum pulp was purchased from Suncheon N Plum Ltd. (Suncheon City, Republic of Korea) and extracted. Control is cultured media, PC1% (freeze-dried plum powder 1%+cultured media) and PP (
Catechins and proanthocyanidins reactive to vanillin were analyzed using the vanillin method of Richard and William (1978) [
The total flavonoid content was evaluated using the method of Thomas et al. (2012) [
The total phenolic content method of Thomas et al. (2012) [
Antioxidant activity was studied using the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) method as described by Blois (1958) [
The metal chelating ability of extracts were predicted according to the method of Dinis et al. (1994) [
The reducing power of Cu2+ was studied using the reducing ability method described by Apak et al. (2006) [
Melanoma B16F0 cells (CRL-6322) were obtained from ATCC (Manassas, VA, U.S.A.) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin G, and 100
Cell viability was determined using the cell counting Kit-8 (CCK-8) assay. Melanoma B16F0 cells were suspended in Dulbecco’s Modified Eagle Medium (DMEM) at a density of 1×105 cells/mL; 100
To determine the amount of melanin produced, 1×106 B16F0 cells were plated per well and exposed to 1000
Tyrosinase activity was estimated by measuring the rate of oxidation of 3, 4-dihydroxy-L-phenyl-alanine (L-DOPA). About 1×106 cells/well of the B160 cells were exposed to 1000
Differences in the data between groups are presented as the mean ± S.D. of three replicates. Statistical differences were analyzed using the Student’s t-test. Probability values less than 0.05 are considered to be significant (P values
The total phenolic compounds, flavonoid content, and condensed tannin content of extracts of the cultured
The chemical composition of cultured
Type of Sample | Polyphenols (GAE) | Tannins (CE) | Flavonoids (QE) |
---|---|---|---|
Control | 2.120 ± 0.002 | 12.667 ± 0.002 | 1.286 ± 0.001 |
1% PP | 2.887 ± 0.002 | 13.222 ± 0.001 | 2.476 ± 0.001 |
PC | 2.212 ± 0.004 | 17.667 ± 0.001 | 2.714 ± 0.002 |
0.1% PPE | 2.964 ± 0.001 | 14.889 ± 0.001 | 2.952 ± 0.001 |
0.3% PPE | 3.323 ± 0.005 | 15.444 ± 0.001 | 2.714 ± 0.001 |
1% PPE | 3.878 ± 0.003 | 18.222 ± 0.001 | 3.429 ± 0.001 |
3% PPE | 4.701 ± 0.006 | 21.000 ± 0.003 | 5.333 ± 0.001 |
10% PPE | 4.546 ± 0.006 | 18.779 ± 0.002 | 6.048 ± 0.001 |
Standard deviations (SD) did not exceed 5%, nd: not detected.
The total phenolic content in 3% PPE was 4.701 ± 0.006
Higher levels of phenolics and flavonoids were confirmed in 3% PPE extracts as compared to other concentrations of PPE. Additionally, the active ingredients that aid antioxidation through fermentation were also confirmed to be higher in 3% PPE. Condensed tannin was detected only after fermentation and, thus, was thought to be formed through the metabolism or the fermenting microbes. These results indicate that flavonoids of natural products increase the antioxidant activities, such as the ability to donate electrons, in proportion to the content of phenolic materials (Table
Figure
(a) The DPPH radical scavenging activity of extracts of
By the electron-donating ability assay, we found that 3% PPE has a 33.38% activity at 1000
Figure
(a) The Cu2+ reducing ability of extracts of
In the Fenton oxidation reaction, H2O2 and Fe2+ form an OH radical intermediate that bonds with organic compounds. To measure the reducing power in this oxidation reaction, we measured the reducing powers of Fe (Fe2+) and Cu (Cu2+), as well as the antioxidant activities of chelating reactions, which inhibit the formation of the Fe2+- ferrozine complex.
The FRAP assay is based on the principle that, at low pH, the ferric tripyridyl triazine (Fe3+- TPTZ) complex is reduced to ferrous tripyridyl triazine (Fe2+- TPTZ) by a reducing agent. In the 3% PPE extract, the FRAP value was determined to be 0.338 ± 0.010 (OD) at 1000
When measuring the reducing power of ferrous-ferricyanide (Fe3+) stabilizing free radicals by donating hydrogen to the ferric-ferricyanide, the 3% PPE extract and control were 0.482 ± 0.061 (OD) and 0.119 ± 0.011 (OD), respectively. Additionally, the reducing power of Cu2+ was greater in 10% PPE (0.349 ± 0.012) than in the control (0.216 ± 0.002), a pattern similar to that observed for phenolic contents and radical scavenging abilities.
The chelating activities of 0.1% PPE and control at 1000
We investigated whether different concentrations of the extract induce apoptosis in B16F0 cells. As shown in Figure
(a) Cytotoxicity of extract on mouse B16 melanoma cells. B16F0 cell line was treated with extracts for 24 hours and cell cytotoxicity was determined by CCK-8 assay. Culture supernatants were removed, and cell counting Kit-8 (CCK-8) was added. (b) Inhibitory effects of extract on the activity of tyrosinase. The lysates of B16F0 melanoma cells containing tyrosinase were incubated with DOPA for 1 h. Tyrosinase activity was measured as described in the Material and Methods. (c) Inhibitory effects of extract on the melanin synthesis in B16F0 melanoma cells. The cells were cultured in the presence of the extracts at concentration of 1000
Next, we evaluated the effects of the extracts on melanin synthesis in B16F0 cells, with an aim to evaluate potent antiwhitening properties. This was compared to the Prunus extract, which is known to exert an antiwhitening effect. Arbutin, a well-known inhibitor of melanin synthesis in B16F0 cells, was used as the positive control. As presented in Figure
Tyrosinase is a well-known major regulator enzyme involved in melanin synthesis. Numerous inhibitors of melanin synthesis reduce melanogenesis by directly inhibiting the tyrosinase activity. The effect of the obtained extract on tyrosinase activity was assessed to tentatively evaluate their antimelanogenic properties since we identified that obtained extracts inhibited melanin synthesis. Likewise, arbutin, a well-known tyrosinase inhibitor, was used as a positive control. We further compared this with the Prunus extract, which is known to have an antiwhitening effect. We observed that exposure to the fermented extracts resulted in increased tyrosinase inhibition activities in the B16 melanoma cells (31%, 33%, 59%, 45%, and 37%) with increasing content of Prunus in the medium, as compared to cells treated with only Prunus (Figure
In conclusion, we summarize the effects of extracts of the cultured
The data used to support the findings of this study are available from the corresponding author upon request.
The authors have declared that there are no conflicts of interest.
This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (316009-5).