- Email: [email protected]
Tyrosinase Inhibitory Activity of Total Triterpenes and Poricoic Acid A Isolated from Poria cocos
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328
321
Available online at SciVarse ScienceDirect
Chinese Herbal Medicines (CHM) ISSN 1674-6384
Journal homepage: www.tiprpress.com
E-mail: [email protected]
Original article
Tyrosinase Inhibitory Activity of Total Triterpenes and Poricoic Acid A Isolated from Poria cocos Shu-hua Hu1, Gao Zhou1, You-wei Wang1, 2* 1. Institute of TCM and Natural Products, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China 2. MOE Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University, Wuhan 430072, China
ARTICLE INFO
ABSTRACT
Article history
Objective With the improvement of people’s living standards, people’s requests for
Received: June 12, 2017 Revised: June 22, 2017 Accepted: July 20, 2017 Available online: October 10, 2017
DOI: 10.1016/S1674-6384(17)60111-4
beauty are increasing. Skin whitening and lightening have become the pursuit of many
women, and whitening and removing freckles have become the focus of scientific research. At present, widely used whitening agents, such as kojic acid, vitamin C, and
its derivatives, have shortcomings such as poor stability and retarded effect. Therefore,
safer and more effective whitening products from herbs are urgently needed. To
explore the possibility of triterpenes as whitening active substance, the effects of total triterpenes of Poria (TTP) and poricoic acid A (PAA) on mushroom tyrosinase activities
and B16 cells were investigated, and their mechanisms on mushroom tyrosinase were also studied. Methods
Using arbutin and nicotinamide as reference substances, we
determinated the inhibitory effects of TTP and PAA on mushroom tyrosinase and tyrosinase in B16 cells and then studied the inhibitory mechanism on mushroom tyrosinase. Results
TTP and PAA exhibited good inhibitory effects on the activities of
monophenolase and diphenolase in mushroom tyrosinase, as well as a certain inhibitory effect on tyrosinase in B16 cells. Conclusion TTP and PAA are potential whitening active ingredients.
Key words
mushroom tyrosinase; Poria cocos; poricoic acid A; triterpenes; whitening activity
© 2017 published by TIPR Press. All rights reserved.
1. Introduction The ultimate skincare goal of many women is to solve the problems of age spots, chloasma, and other skin problems, thereby eventually achieving the effect of whitening. Therefore, the development of safe and efficient whitening products, as well as those that remove freckles, has received extensive concern. Widely used whitening ingredients are arbutin, kojic acid and its derivatives, vitamin C and its derivatives, *
Corresponding author: Wang YW
endothelin antagonists, flavonoids and their derivatives, stilbene compounds and their derivatives, polysaccharides, and organic acids (Zhang et al, 2008). However, single chemical composition for whitening has a variety of drawbacks, such as poor stability and retarded effect. By contrast, Chinese herbal medicine, compared with Western medicine, has the features of safety, gentleness and slight side effects; Thus, the addition of herbal whitening ingredients into whitening products has become popular in recent years (Maack and Pegard, 2016).
Tel: +86-27-6875 9323 Fax: +86-27-6875 9010 E-mail: [email protected]
Fund: Project of National Twelve-Five Year Research Program of China (2011BAI06B03-2 and 2012BAI29B03)
322
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328
The color of human skin is mainly based on the content and distribution of melanin. Its formation is regulated to a variety of enzymes and hormones, and tyrosinase plays a key role in the synthesis of melanin (Wang et al, 2006). Tyrosine is the substrate of the first step in all enzymatic reactions, and it forms L-DOPA through the catalysis of tyrosinase, which requires participation of oxygen (Kamal et al, 2017). Therefore, whitening can be attained by scavenging oxygen free radicals and inhibiting tyrosinase activity (Zhang et al, 2008). Poria, one of the most important well-known traditional Chinese medicines, is the dried sclerotium of Poria cocos (Schw.) Wolf, which has traditionally been used as diuretic and sedative agent (Lee et al, 2017a). Its major chemical components are polysaccharides and triterpenoids (Wang et al, 2015). Previous studies have shown that triterpenes possessed antitumor (Gao et al, 2016), anti-convulsant (Zhang et al, 2009), anti-inflammatory (Lee et al, 2017b), and anti-oxidative properties (Cheng et al, 2013). Some triterpenic acids also strengthen human immunity (Fan et al, 2009), and they are widely recognized as important active ingredients of Poria. Triterpenoids can be divided into four types according to chemical structure (Wang et al, 2015), namely, lanosta-8-ene type, lanosta-7,9(11)-diene type, 3,4-seco-lanosta-8-ene type (Akihisa et al, 2007) and 3,4-seco-lanosta-7,9(11)-diene type. Poricoic acid A (PAA) (Hu et al, 2006), the main representative compound of 3,4-seco-lanosta-7,9(11)-diene type, has the highest content in this structure. PAA exhibits good water solubility because of its double carbonyl structure. Modern pharmacology initially proved that triterpenes of the surface layer of Poria demonstrated good biological activities such as antibacterial, anti-inflammatory, antioxidative effects and inhibition of tyrosinase activity (Shen et al, 2009). However, the use of PAA for whitening has not been reported. This study aimed to further prove the skin whitening effect of Poria triterpene and provide a theoretical and scientific basis of the use of triterpenes from the surface layer of Poria for skin care. In this paper, we used triterpenoids as the main research object, and experiments were conducted on mice B16 melanoma cells. In addition, the inhibition on catalytic activity of mushroom tyrosine and its mechanism of action were analyzed. Thus, results of this study may demonstrate whether triterpenoids from the surface layer of Poria can be used as skin whitening products.
2. Materials and methods 2.1 Chemicals and reagents B16F1 melanoma cells were purchased from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences. The reagent 2,20-azinobis(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) was procured from Fluka (USA). 1,1-Diphenyl2-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and L-DOPA were purchased from Sigma Aldrich
Chemicals Co. (USA). PAA was prepared in our laboratory. Trysinase (T3824-25ku) was purchased from Sigma-Aldrich Chemicals Co. (USA).
2.2 Extraction and preparation of Poria triterpenoid compounds Poria peel herbs were purchased from Jinzhai county, Anhui province, China. The samples were sieved and extracted twice with methanol at 70 °C for 2 h at a material to liquid ratio of 1:20 (Shen et al, 2009). After combining the extracting solution, the solvent was distilled and dried to obtain the crude extract of total triterpenes of Poria (TTP). A part of the total extract was added to the aqueous solution and then extracted with petroleum ether, chloroform and ethyl acetate individually. The mixture was concentrated to obtain the chloroform part of TTP. PAA was prepared in our laboratory.
2.3 Quantitative analysis on PAA in TTP A small amount of TTP was dissolved in methanol and filtered over a 0.45 nm organic membrane filter. The HPLC system was set under the following conditions: Shimadzu LC−20AT, Agilent TC-C18 (2) (150 mm × 4.6 mm, 5 µm); mobile phase A: methanol-0.1% formic acid aqueous solution, mobile phase B: acetonitrile; flow rate of 1 mL/min; column temperature of 25 °C; injection volume of 20 μL. PAA, used as a standard, was detected and quantitated in TTP.
2.4 Scavenging effect of DPPH and ABTS radicals by TTP The scavenging activity of DPPH radicals was measured. An aliquot of 2.7 mL of 0.2 mmol/K DPPH in ethanol and 0.3 mL TTP in ethanol at various concentration were mixed. Each mixture was shaken vigorously and allowed to reach a steady state in dark for 30 min. The absorbance of the mixture was determined at 517 nm with arbutin, BHT and vitamin C as reference substances. Radical scavenging activity was calculated as follows: [(A1 − A2/A2) / A1] × 100 Where A1 is the absorbance of pure DPPH and A2 is the absorbance of DPPH in the presence of different samples.
The scavenging ability of ABTS radicals was determined by the following method. ABTS+ solution was prepared to 2.08 mL with 7.6 mg ABTS+ and 1.4 mg potassium persulfate after incubation at room temperature in dark for 24 h. The solution was then diluted with 80% ethanol to obtain an absorbance of 0.700 ± 0.005 at 734 nm. The ABTS+ solution (0.9 mL) was thoroughly mixed with 0.1 mL test sample (TTP). The reaction mixture was allowed to set at 30 °C for 30 min and then added to the 96-well plate. The absorbance at 734 nm was recorded with arbutin, BHT and vitamin C as reference substances. Radical scavenging activity was calculated as follows. [(A1 − A2/A2)/A1] × 100 Where A1 is the absorbance of pure ABTS and A2 is the absorbance of ABTS in the presence of different samples.
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328
323
2.5 Effects of TTP and PAA on activity of mushroom tyrosinase
absence and presence of 25 μg/mL TTP and PAA were recorded by the UV−2600 Spectrophotometer (Espin and Wichers, 2001).
The activity of monophenolase enzyme in mushroom tyrosinase was spectrophotometrically determined using L-tyrosine as substrate following a previously described method (Huang et al, 2013) with slight modification. Approximately 60 μL of each sample solution with various concentration (125–2000 μg/mL) and 180 μL of 1 mmol/L L-tyrosine solution were added into 300 μL of 0.2 mmol/L phosphate buffer (pH 6.8). After incubation of the reaction mixture at 25 °C for 20 min, 60 μL mushroom tyrosinase (250 units/mL) was added to an assay mixture (total volume of 600 μL). After incubation of the reaction mixture at 25 °C for 10 min, the absorbance of mixture was determined at 475 nm in a microplate reader. The inhibitory rate of tyrosinase activity was calculated by the following equation.
2.8 Fluorescence quenching reaction
sample, and Aa is absorbance at 475 nm with enzyme and test
The fluorescence intensities were recorded using an F–4600 Spectrofluorophotometer (Shimadzu, Japan) with an excitation wavelength (ex) of 290 nm and excitation and emission slit widths of 5 nm (Wang et al, 2016). Therefore, 250 U/mL tyrosinase was chosen as the concentration for fluorescence quenching experiments. Serial dilutions of sample solutions (250–1000 μg/mL) were prepared in DMSO. For each data point, 0.4 mL of the appropriate sample solution was added to 1.6 mL tyrosinase solution. The final concentration of samples ranged from 50 to 200 μg/mL. The change in the intensity of emission fluorescence was measured within 1 min when the samples were added to tyrosinase solution.
sample.
2.9 Cell viability analysis with TTP and PAA
Inhibitory rate = 1 − Aa/Ab Where Ab is absorbance at 475 nm with enzyme and without test
The activity of diphenolase enzyme in mushroom tyrosinase using L-DOPA as substrate was spectrophotometrically determined following a previously described method (Yu et al, 2011) with slight modification. About 50 μL of each sample solution with different concentration (125– 2000 μg/mL) and 150 μL of 2.5 mmol/L L-DOPA solution were added to 320 μL of 0.2 mmol/L phosphate buffer (pH 6.8). After incubation of the reaction mixture at 25 °C for 20 min, 60 μL mushroom tyrosinase (250 units/mL) was added to an assay mixture (total volume of 600 μL). The absorbance of the mixture was determined at 475 nm in a microplate reader without delay. The inhibitory rate of tyrosinase activity was calculated by the following equation: Inhibitory rate = 1 − Aa/Ab Where Ab is absorbance at 475 nm with enzyme and without test sample and Aa is absorbance at 475 nm with enzyme and test sample.
2.6 Effect of TTP and PAA on spectra obtained from oxidation of L‐tyrosine and L‐DOPA by mushroom tyrosinase The samples were tested by the methods described in 2.5 by a UV−2600 Spectrophotometer (Shimadzu, Japan) in real time. The spectra of products obtained during the oxidation of L-tyrosine and L-DOPA by mushroom tyrosinase in the range of 200–600 nm were recorded at different reaction time (Jiménez-Atiénzar et al, 2004). The sample concentration was 250 µg/mL, the final concentration in the L-tyrosine system was 25 µg/mL, and the final concentration in the L-DOPA system was 20.83 µg/mL.
2.7 Effect of TTP and PAA on spectra of obtained product during oxidation of L‐DOPA by NaIO4 L-DOPA (20 μg/mL) was oxidized by 20 μg/mL sodium periodate (NaIO4). The different spectra obtained in the
B16F1 melanoma cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin G, and 100 μg/mL streptomycin. Cells were instantly incubated at 37 °C in atmosphere of 5% CO2. The cell viability of TTP and PAA was assessed by MTT assay. In brief, 1 × 104 B16F1 cells were seeded and made to adhere to each well that contained 100 μL culture medium in 96-well plate. After 24 h, the culture medium was aspirated and added to 100 μL of the culture medium with or without TTP and PAA. After incubation for 48 h, the culture medium was aspirated and washed with PBS. A total of 20 μL MTT was added to each well, and the plate was incubated for 4 h at 37 °C. Subsequently, MTT solution was aspirated and quickly added into each well along with 100 μL DMSO for dissolving the blue formazan reactant. The optical density of each well was measured at 570 nm with the help of μQuant Microplate Spectrophotometer (Tecan, Switzerland) (Mei et al, 2011).
2.10 Cellular tyrosinase activity following TTP and PAA treatment B16F1 cells (1 × 105) were seeded and made to adhere to wells of a six-well plate at 37 °C in 5% CO2 for 24 h. The culture medium was removed and the cells were treated in the presence or absence of TTP and PAA for 48 h. The cells were then collected into trypsin-EDTA solution and immediately centrifuged at 5000 r/m for 5 min to obtain cell pellets. The cell pellets were mixed with lysis buffer (1% Triton X-100, 0.1 mmol/L PBS with pH of 6.8 and phenylmethylsulfonyl fluoride). The supernatant (80 μL) and L-DOPA (20 μL) were mixed in a 96-well plate and incubated at room temperature. After 10 min of incubation, the optical densities were determined at 475 nm using μQuant Microplate Spectrophotometer (Ye et al, 2010).
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328
3. Results and discussion 3.1 Quantitative analysis on PAA in TTP As shown in Figure 1, the peak marked 1 was PAA. The standard curve of PAA was y = 30940x + 78956, r2 = 0.9994 and the linear range was 12.8–256.0 μg/mL as previously established. y represents the peak area of triterpenes, whereas x represents the different concentration. The content of PAA in TTP was (37.57 ± 7.5709) mg/g.
mushroom tyrosinase. At this concentration, TPP and arbutin were not significantly different in inhibiting monophenolase activity, meanwhile they were significantly different from PAA and nicotinamide, but TTP, arbutin and PAA were all significantly stronger than other positive control (i.e., nicotinamide). 80
Inhibitory rate / %
324
ab 40
10
Figure 1
20
30 t / min
40
50
The scavenging effects of DPPH and ABTS radicals by TTP were shown in Table 1. TTP exerted a certain clearance effect on DPPH and ABTS free radicals. The IC50 values of clearing DPPH and ABTS free radicals were (2892.19 ± 923.10) and (180.75 ± 10.61) μg/mL, respectively. Anti-oxidant activity of triterpenes from the peels of P. cocos including removing the superoxide anion free radicals(O2−) and hydroxyl free radicals (OH) has been reported in some reference (Cheng et al, 2013). Thereinto, the IC50 values were also close to our results. Free radical scavenging activity or IC50 values of TTP
( x ± s ,n = 3) IC50 values / (μg·mL−1) DPPH
ABTS
TTP
2892.19 ± 923.10a
180.75 ± 10.61a
BHT
22.87 ± 2.54b
1.91 ± 0.22b
Arbutin Vc
PAA
Arbutin
Nicotinamide
Inhibitory effect of TTP and PAA on mushroom
tyrosinase for catalysis of L-Tyr arbutin and nicotinamide as positive controls, same as Figure 3 a−c refer to a significant difference among groups p < 0.05
3.2 Scavenging effect of TTP on DPPH and ABTS radicals
Samples
TTP
HPLC of total triterpenes isolated from P. cocos 1: poricoic acid A
Table 1
d
20
Figure 2 0
ac
60
0
1
a
c
1.65 ± 0.06bc
8.05 ± 0.61d
2.15 ± 0.13bd
76.35 ± 7.12
BHT: butylated hydroxytoluene; Vc: ascorbic acid a−d: significant difference between groups, p < 0.05
3.3 Effects of TTP and PAA on activity of monophenolase enzyme in mushroom tyrosinase Effects of TTP and PAA on tyrosinase monophenolase were shown in Figure 2. We selected two common whitening active components of arbutin and nicotinamide as positive controls. At 200 μg/mL, PAA and TTP had significant inhibitory effects on the activity of monophenolase enzyme in
3.4 Effects of TTP and PAA on activity of diphenolase enzyme in mushroom tyrosinase The relationship between the concentration of TTP and PAA with tyrosinase activity was shown in Figure 3. We also selected arbutin and nicotinamide as the positive controls. The positive control agents, as well as TTP and PAA, could significantly inhibit the activity of diphenolase in mushroom tyrosinase and showed a dose-effect relationship. Furthermore, the inhibitory effect was enhanced with increasing concentration. At the five concentrations selected, the inhibitory activity of PAA on diphenolase in mushroom tyrosinase at low concentration was the weakest. However, as the dose increased, the inhibitory activity was increased rapidly. We found that PAA had the best inhibitory effect when its concentration reached 166.67 μg/mL. The inhibitory effect of PAA was better than those of nicotinamide and arbutin. Its inhibitory rate was 45.24% at 166.67 μg/mL. Furthermore, the inhibitory effect of TTP was better than that of arbutin in the range of 10.42–166.67 μg/mL.
3.5 Effects of TTP and PAA on spectra obtained from oxidation of L‐tyrosine by mushroom tyrosinase The tyrosinase-catalyzed oxidation of the L-tyrosine reaction spectrum was shown in Figure 4A. The absorbance at 475 nm during the catalytic oxidation of tyrosine increased, and the curves from the bottom to the top indicated the change in the absorption spectrum of the oxidation product from 0 to 20 min. Figures 4B and 4C showed the spectra of the tyrosinase-catalyzed oxidation of L-tyrosine in the presence of TTP and PAA. Compared with Figure 4A, the absorbance at 475 nm was significantly attenuated in the presence of TTP and PAA. This result was consistent with monophenolase inhibitory activity.
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328 50
3.6 Effects of TTP and PAA on spectra obtained from oxidation of L‐DOPA by mushroom tyrosinase
Inhibitory rate / %
40 30 20
TTP PAA Arbutin Nicotinamide
10 0
Figure 3
0
50 100 150 Concentration / (μg·mL−1)
Inhibitory effect of TTP and PAA on mushroom
tyrosinase for catalysis of L-DOPA
0.9
0.6
0.6
400
500
600
0.151 260 300 2.3 2.0
D Absorption
1.5 1.0 0.5 0.0 260 300 Figure 4
400
500
600
400 500 Wavelength / nm
0.6
0.3
2.3 2.0
E
1.5 1.0 0.5 0.0 260 300
400 500 Wavelength / nm
C
0.151 600 260 300
Absorption
260 300
2.3 2.0 Absorption
0.9
B
0.3
0.3 0.151
Figure 4D showed the UV-vis spectra of L-DOPA catalyzed by tyrosinase. The absorbance at 475 nm during catalytic oxidation of L-DOPA increased. Curves from the bottom to the top indicated the absorption spectra of the oxidation product at 0–10 min after the reaction time. Figures 4E and 4F showed the spectra of L-DOPA catalyzed by tyrosinase in the presence of TTP and PAA, respectively. Compared with Figure 4D, the absorbance at 475 nm in the presence of TTP and PAA was significantly attenuated. The order of decreasing absorption peak was TTP > PAA, which was consistent with inhibitory activity of diphenolase.
Absorption
A
Absorption
Absorption
0.9
325
600
400
500
600
400
500
600
F
1.5 1.0 0.5 0.0 260 300
Consecutive spectra obtained during oxidation of L-Tyr (A−C) and L-DOPA (D−F) by mushroom tyrosinase
A: L-tyrosine+ enzyme; B: 25 μg·mL−1 TTP + L-tyrosine + enzyme; C: 25 μg·mL−1 PAA + L-tyrosine + enzyme; D: L-DOPA + enzyme; E: 20.83 μg·mL−1 TTP + L-DOPA + enzyme; F: 20.83 μg·mL−1 PAA + L-DOPA + enzyme
3.7 Interaction of TTP and PAA with o‐quinones We tested the production of dopaquinone with NaIO4 in the presence and absence of TTP and PAA to determine whether TTP and PAA are directly oxidized with dopaquinone to produce diphenols. Figures 5A and 5B showed that no absorption peak occurred at 475 nm when L-DOPA was alone, and the absorption peak appeared at 475 nm after NaIO4 was added. The absorption peak at 475 nm was significantly reduced when the sample of TTP and PAA were added into the solution of L-DOPA and NaIO4, as shown in Figures 5A and 5B. Curves from the bottom to top in Figures 5D and 5E showed the changes in the dopaquinone uptake spectrum of the oxidized product at 0–4 min after adding PAA and TTP into the mixture solution with L-DOPA and NaIO4, respectively. Figure 5C showed that the intensity of the absorption peak of the mixture solution with L-DOPA
and NaIO4 at 475 nm increased with time. Thus, the oxidation reaction occurred in TTP, PAA and dopaquinone, which was oxidized with L-DOPA by NaIO4.
3.8 Effect on emission of fluorescence in mushroom tyrosinase Tryptophan fluorescence has been frequently examined among the intrinsic aromatic fluorophores in tyrosinase molecules to obtain information about conformational changes. The effects of TTP and PAA on the fluorescence emission of tyrosinase at an excitation wavelength of 290 nm were shown in Figure 6. Tyrosinase exhibited the largest emission fluorescence at 366 nm. Figure 6A showed that TTP could change the fluorescence emission spectrum of tyrosinase at 50–200 μg/mL. The intensity of the fluorescence peak decreased, and the maximum wavelength of emission
326
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328 A
2.0 Absorption
2.5
L-DOPA L-DOPA + PAA L-DOPA + NaIO4 + PAA L-DOPA + NaIO4
1.5 1.0
1.0 0.5
300
400
500
300
4 min
0.5
300
400
500
600
0.5 0 min
200
600
E
0.0
0 min
0.0
500
1.5 Absorption
1.0
200
400
D
1.5
Figure 5
0.0 200 Wavelength / nm
600
1.5 Absorption
Absorption
200
C
2.0
L-DOPA L-DOPA + TTP L-DOPA + NaIO4 + TTP L-DOPA + NaIO4
1.5
0.5 0.0
B
2.0 Absorption
2.5
0 min 4 min
0.0
4 min
300
0.5
400 500 Wavelength / nm
600
200
300
400
500
600
Scanning spectra for 20 μg/mL L-DOPA+20 μg/mL NaIO4 + 25 μg/mL PAA (A), 25 μg/mL TTP (B), consecutive spectra of
absorption obtained during oxidation of L-DOPA by NaIO4 in 4 min (C), consecutive spectra of absorption obtained during the oxidation of L-DOPA + 25 μg·mL−1 PAA by NaIO4 in 4 min (D) and consecutive spectra of absorption obtained during oxidation of L-DOPA + 25 μg·mL−1 TTP by NaIO4 in 4 min (E) A
Control 50 μg·mL−1 TTP 100 μg·mL−1 TTP 200 μg·mL−1 TTP
200
100
0 350 Figure 6
400
300 Fluorescence intensity
Fluorescence intensity
300
B
Control 50 μg·mL−1 PAA 100 μg·mL−1 PAA 200 μg·mL−1 PAA
200
100
0 450 Wavelength / nm
350
400
450
Effect of TTP (A) and PAA (B) on emission spectra of mushroom tyrosinase
fluorescence showed a redshift from 366 nm to 377 nm. The results showed that TTP had a fluorescence quenching effect on tyrosinase, and an obvious relationship occurred with the concentration of samples. The redshifts increased with concentration. Thus, TTP influenced emission fluorescence quenching. In addition, TPP possibly played a role by changing the hydrophobic environment of the amino acid residues in tyrosinase. Figure 6B showed that PAA had a minimal effect on the fluorescence emission of tyrosinase at 50–200 μg/mL.
3.9 Cell viability analysis on TTP and PAA Drug cytotoxicity is of chief importance when the drug is used either as a medicine or as a cosmetic agent. Therefore, the cell safety of TTP and PAA was examined in B16F1 melanoma with the help of MTT assay. The effects of TTP and PAA on cell inhibition were shown in Figure 7A. In
B16F1 melanoma cells, TTP with high concentration (12.5–100 μg/mL) was associated with cell viability of 58%–86%. At a low concentration (6.25 μg/mL), TTP did not show obvious cytotoxicity when the cell viability was higher than 90%. In addition, PAA with high concentration (12.5–100 μg/mL) was associated with cell viability of 27%–77%. At a low concentration (6.25 μg/mL), PAA did not show obvious cytotoxicity when the cell viability was higher than 90%. The IC50 values of the cell viability of TTP and PAA were 118.26 and 48.01 µg/mL, respectively. These results indicated that a concentration of 0.78–12.5 μg/mL was necessary to evaluate their cellular tyrosinase activity using B16F1 cells.
3.10 Cellular tyrosinase activity following TTP and PAA treatment Melanogenesis due to melanin overproduction is highly
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328 80
60
A
B
50
60
Inhibitory rate / %
Inhibitory rate / %
70
327
50 40 30 TTP
20
0 0 Figure 7
20
40
60
80
30 TTP
20
PAA Arbutin
10
PAA
10
40
0 100 120 0 Concentration / (μg·mL−1)
5
10
15
Inhibitory effect of TTP and PAA on proliferation (A) and tyrosinase activity (B) in mouse B16 cells Arbutin was used as positive control.
dependent on the activation of cellular tyrosinase (Huang et al, 2012). To elucidate the inhibitory effect on melanogenesis of triterpenes, the cellular tyrosinase activity in TTP- and PAA-treated B16F1 cells was investigated. The effects of TTP and PAA on tyrosinase of B16 cells were shown in Figure 7B, and arbutin was selected as positive control. The effects of TTP and PAA on the inhibition of tyrosinase activity in cells increased with rising concentration of 12.5 μg/mL. The inhibitory rates of TTP and PAA were 40.86% and 39.53%, respectively.
4. Conclusion In this paper, we used triterpenoids as the main research object to exploit safer and more effective whitening products from herbs. TTP and PAA exerted a highly effective inhibitory effect on either mushroom tyrosinase or cell tyrosinase. Ultraviolet spectrophotometry and fluorophotometry revealed the possible mechanisms of TTP and PAA on mushroom tyrosinase. TTP and PAA played a key role mainly by combining with dopaquinone formed by the oxidation of DOPA and by changing the hydrophobic environment of the amino acid residues in tyrosinase. In addition, TTP significantly influenced the fluorescence quenching of tyrosinase, whereas PAA only slightly affected the fluorescence emission of tyrosinase. In other words, TTP could change the conformation of mushroom tyrosinase, but PAA could not. Therefore, other triterpenes in TTP may play the same role in inhibiting tyrosinase activity. P. cocos triterpenoids are potential whitening active ingredients, but more in-depth studies are needed to develop them into whitening products. Conflict of interest statement The authors declare no conflict of interest. References Akihisa T, Nakamura Y, Tokuda H, Uchiyama E, Suzuki T, Kimura Y, 2007. Triterpene acids from Poria cocos and their anti-tumorpromoting effects. J Nat Prod 70(6): 948-953.
Ali S, Naaz I, 2015. Current challenges in understanding the story of skin pigmentation-bridging the morpho-anatomical and functional aspects of mammalian melanocytes. Muscle Cell and Tissue. InTech Open House, Europe: 262-285. Cheng SM, Gui Y, Shen S, Huang W, 2013. The antioxidant activity of triterpenes in the peels of Poria cocos. Med Plant 4(8): 38-41. Espin JC, Wichers HJ 2001. Effect of captopril on mushroom tyrosinase activity in vitro. Biochim Biophys Acta 1544(1-2): 289-300. Fan Z, Z XF, Wang BC, Liu HY, Li CY, Liu ZH, Zhang GW, Lu H, Chi C, Wang F, 2009. Pachymic acid, a novel compound for anti-rejection: Effect in rats following cardiac allograft transplantation. Chin Med J 122(23): 2898-2902. Gao Y, Yan H, Jin R, Lei P, 2016. Antiepileptic activity of total triterpenes isolated from Poria cocos is mediated by suppression of aspartic and glutamic acids in the brain. Pharm Biol 54(11): 2528-2535. Hu B, Yang YP, Ye Y, 2006. Chemical constituents from Poria cocos. Chin Tradit Herb Drugs 37(5): 655-658. Huang XW, Feng YC, Huang Y, Li HL, 2013. Potential cosmetic application of essential oil extracted from Litsea cubeba fruits from China. J Essent Oil Res 25(2): 112-119. Huang HC, Wang HF, Yi KH, Chang LZ, Chang TM, 2012. The dual anti-melanogenic and antioxidant activities of the essential oil extracted from the leaves of acorus macrospadiceus (Yamamoto) F. N. Wei et Y. K. Li. Evid Based Complement Alternat Med. Vol. 2012, Art. ID: 781280, 10pages. Jiménez-Atiénzar M, Cabanes J, Gandıa-Herrero F, Garcıa-Carmona F, 2004. Kinetic analysis of catechin oxidation by polyphenol oxidase at neutral pH. Biochem Biophys Res Commun 319(3): 902-910. Kamal UZ, Sharique AA, Ayesha SA, 2017. Melanogenic effect of purified mushroom tyrosinase on B16F10 Melanocytes: A phase contrast and immunofluorescence microscopic study. J Microsco Ultrastruct 5(2): 82-89. Lee RS, Lee S, Moon E, Park HJ, Kim KH, 2017a. Bioactivity-guided isolation of anti-inflammatory triterpenoids from the sclerotia of Poria cocos using LPS-stimulated Raw264.7 cells. Bioorg Chem 70: 94-99. Lee S, Lee D, Lee SO, Ryu JY, Choi SZ, Kang KS, Kim KH, 2017b. Anti-inflammatory activity of the sclerotia of edible fungus, Poria cocos Wolf and their active lanostane triterpenoids. J Funct Foods 32: 27-36. Maack A, Pegard A, 2016. Populus nigra (Salicaceae) absolute rich in phenolic acids, phenylpropanoïds and flavonoids as a new potent
328
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328
tyrosinase inhibitor. Fitoterapia 111: 95-101. Mei X, Wu YY, Mao X, Tu YY, 2011. Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols. Environ Pollut 159(1): 164-168. Shen S, Li FJ, Mei GM, Huang W, 2009. Study on content determination and antibacterial effects of triterpenes from sclerotia of Poria cocos. Food Science 30(1): 95-98. Wang KH, Lin RD, Hsu FL, Huang YH, Chang HC, Huang CY, Lee MH, 2006. Cosmetic applications of selected traditional Chinese herbal medicines. J Ethnopharmacol 106(3): 353-359. Wang W, Dong H, Yan R, Wang ZM, 2015. Comparative study of lanostane-type triterpene acids in different parts of Poria cocos (Schw.) Wolf by UHPLC-Fourier transform MS and UHPLCtriple quadruple MS. J Pharm Biomed Anal 102(5): 203-214.
Wang XY, Li MZ, He Y, Wang QH, 2016. Study on the interaction of cinnamic acid and azelaic acid to tyrosinase by fluorescence quenching method. J Tradit Chin Med 21(12): 5348-5351. Ye Y, Chou GX, Mu DD, Wang H, Chu JH, Leung AK, Fong WF, Yu ZL, 2010. Screening of Chinese herbal medicines for antityrosinase activity in a cell free system and B16 cells. J Ethnopharmacol 129(3): 387-390. Yu LL, Hu WC, Ding G, Li RT, Wei JH, Zou ZM, Wang MH, 2011. Gusanlungionosides A−D, potential tyrosinase inhibitors from Arcangelisia gusanlung. J Nat Prod 74(5): 1009-1014. Zhang JY, Fang YY, Wu XQ, Zhang Y, 2008. Status of skin-whitening cosmetics and its future development. Fine Chem 25(1): 72-75. Zhang QQ, Wang MZ，Wang HK, Wang XF, Gu YT, 2009. The influence on hippo camp amino acid content of the total triterpene of Poria in rats. Chin Pharmacol B 25(2): 279-280.
321
Available online at SciVarse ScienceDirect
Chinese Herbal Medicines (CHM) ISSN 1674-6384
Journal homepage: www.tiprpress.com
E-mail: [email protected]
Original article
Tyrosinase Inhibitory Activity of Total Triterpenes and Poricoic Acid A Isolated from Poria cocos Shu-hua Hu1, Gao Zhou1, You-wei Wang1, 2* 1. Institute of TCM and Natural Products, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China 2. MOE Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University, Wuhan 430072, China
ARTICLE INFO
ABSTRACT
Article history
Objective With the improvement of people’s living standards, people’s requests for
Received: June 12, 2017 Revised: June 22, 2017 Accepted: July 20, 2017 Available online: October 10, 2017
DOI: 10.1016/S1674-6384(17)60111-4
beauty are increasing. Skin whitening and lightening have become the pursuit of many
women, and whitening and removing freckles have become the focus of scientific research. At present, widely used whitening agents, such as kojic acid, vitamin C, and
its derivatives, have shortcomings such as poor stability and retarded effect. Therefore,
safer and more effective whitening products from herbs are urgently needed. To
explore the possibility of triterpenes as whitening active substance, the effects of total triterpenes of Poria (TTP) and poricoic acid A (PAA) on mushroom tyrosinase activities
and B16 cells were investigated, and their mechanisms on mushroom tyrosinase were also studied. Methods
Using arbutin and nicotinamide as reference substances, we
determinated the inhibitory effects of TTP and PAA on mushroom tyrosinase and tyrosinase in B16 cells and then studied the inhibitory mechanism on mushroom tyrosinase. Results
TTP and PAA exhibited good inhibitory effects on the activities of
monophenolase and diphenolase in mushroom tyrosinase, as well as a certain inhibitory effect on tyrosinase in B16 cells. Conclusion TTP and PAA are potential whitening active ingredients.
Key words
mushroom tyrosinase; Poria cocos; poricoic acid A; triterpenes; whitening activity
© 2017 published by TIPR Press. All rights reserved.
1. Introduction The ultimate skincare goal of many women is to solve the problems of age spots, chloasma, and other skin problems, thereby eventually achieving the effect of whitening. Therefore, the development of safe and efficient whitening products, as well as those that remove freckles, has received extensive concern. Widely used whitening ingredients are arbutin, kojic acid and its derivatives, vitamin C and its derivatives, *
Corresponding author: Wang YW
endothelin antagonists, flavonoids and their derivatives, stilbene compounds and their derivatives, polysaccharides, and organic acids (Zhang et al, 2008). However, single chemical composition for whitening has a variety of drawbacks, such as poor stability and retarded effect. By contrast, Chinese herbal medicine, compared with Western medicine, has the features of safety, gentleness and slight side effects; Thus, the addition of herbal whitening ingredients into whitening products has become popular in recent years (Maack and Pegard, 2016).
Tel: +86-27-6875 9323 Fax: +86-27-6875 9010 E-mail: [email protected]
Fund: Project of National Twelve-Five Year Research Program of China (2011BAI06B03-2 and 2012BAI29B03)
322
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328
The color of human skin is mainly based on the content and distribution of melanin. Its formation is regulated to a variety of enzymes and hormones, and tyrosinase plays a key role in the synthesis of melanin (Wang et al, 2006). Tyrosine is the substrate of the first step in all enzymatic reactions, and it forms L-DOPA through the catalysis of tyrosinase, which requires participation of oxygen (Kamal et al, 2017). Therefore, whitening can be attained by scavenging oxygen free radicals and inhibiting tyrosinase activity (Zhang et al, 2008). Poria, one of the most important well-known traditional Chinese medicines, is the dried sclerotium of Poria cocos (Schw.) Wolf, which has traditionally been used as diuretic and sedative agent (Lee et al, 2017a). Its major chemical components are polysaccharides and triterpenoids (Wang et al, 2015). Previous studies have shown that triterpenes possessed antitumor (Gao et al, 2016), anti-convulsant (Zhang et al, 2009), anti-inflammatory (Lee et al, 2017b), and anti-oxidative properties (Cheng et al, 2013). Some triterpenic acids also strengthen human immunity (Fan et al, 2009), and they are widely recognized as important active ingredients of Poria. Triterpenoids can be divided into four types according to chemical structure (Wang et al, 2015), namely, lanosta-8-ene type, lanosta-7,9(11)-diene type, 3,4-seco-lanosta-8-ene type (Akihisa et al, 2007) and 3,4-seco-lanosta-7,9(11)-diene type. Poricoic acid A (PAA) (Hu et al, 2006), the main representative compound of 3,4-seco-lanosta-7,9(11)-diene type, has the highest content in this structure. PAA exhibits good water solubility because of its double carbonyl structure. Modern pharmacology initially proved that triterpenes of the surface layer of Poria demonstrated good biological activities such as antibacterial, anti-inflammatory, antioxidative effects and inhibition of tyrosinase activity (Shen et al, 2009). However, the use of PAA for whitening has not been reported. This study aimed to further prove the skin whitening effect of Poria triterpene and provide a theoretical and scientific basis of the use of triterpenes from the surface layer of Poria for skin care. In this paper, we used triterpenoids as the main research object, and experiments were conducted on mice B16 melanoma cells. In addition, the inhibition on catalytic activity of mushroom tyrosine and its mechanism of action were analyzed. Thus, results of this study may demonstrate whether triterpenoids from the surface layer of Poria can be used as skin whitening products.
2. Materials and methods 2.1 Chemicals and reagents B16F1 melanoma cells were purchased from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences. The reagent 2,20-azinobis(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) was procured from Fluka (USA). 1,1-Diphenyl2-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and L-DOPA were purchased from Sigma Aldrich
Chemicals Co. (USA). PAA was prepared in our laboratory. Trysinase (T3824-25ku) was purchased from Sigma-Aldrich Chemicals Co. (USA).
2.2 Extraction and preparation of Poria triterpenoid compounds Poria peel herbs were purchased from Jinzhai county, Anhui province, China. The samples were sieved and extracted twice with methanol at 70 °C for 2 h at a material to liquid ratio of 1:20 (Shen et al, 2009). After combining the extracting solution, the solvent was distilled and dried to obtain the crude extract of total triterpenes of Poria (TTP). A part of the total extract was added to the aqueous solution and then extracted with petroleum ether, chloroform and ethyl acetate individually. The mixture was concentrated to obtain the chloroform part of TTP. PAA was prepared in our laboratory.
2.3 Quantitative analysis on PAA in TTP A small amount of TTP was dissolved in methanol and filtered over a 0.45 nm organic membrane filter. The HPLC system was set under the following conditions: Shimadzu LC−20AT, Agilent TC-C18 (2) (150 mm × 4.6 mm, 5 µm); mobile phase A: methanol-0.1% formic acid aqueous solution, mobile phase B: acetonitrile; flow rate of 1 mL/min; column temperature of 25 °C; injection volume of 20 μL. PAA, used as a standard, was detected and quantitated in TTP.
2.4 Scavenging effect of DPPH and ABTS radicals by TTP The scavenging activity of DPPH radicals was measured. An aliquot of 2.7 mL of 0.2 mmol/K DPPH in ethanol and 0.3 mL TTP in ethanol at various concentration were mixed. Each mixture was shaken vigorously and allowed to reach a steady state in dark for 30 min. The absorbance of the mixture was determined at 517 nm with arbutin, BHT and vitamin C as reference substances. Radical scavenging activity was calculated as follows: [(A1 − A2/A2) / A1] × 100 Where A1 is the absorbance of pure DPPH and A2 is the absorbance of DPPH in the presence of different samples.
The scavenging ability of ABTS radicals was determined by the following method. ABTS+ solution was prepared to 2.08 mL with 7.6 mg ABTS+ and 1.4 mg potassium persulfate after incubation at room temperature in dark for 24 h. The solution was then diluted with 80% ethanol to obtain an absorbance of 0.700 ± 0.005 at 734 nm. The ABTS+ solution (0.9 mL) was thoroughly mixed with 0.1 mL test sample (TTP). The reaction mixture was allowed to set at 30 °C for 30 min and then added to the 96-well plate. The absorbance at 734 nm was recorded with arbutin, BHT and vitamin C as reference substances. Radical scavenging activity was calculated as follows. [(A1 − A2/A2)/A1] × 100 Where A1 is the absorbance of pure ABTS and A2 is the absorbance of ABTS in the presence of different samples.
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328
323
2.5 Effects of TTP and PAA on activity of mushroom tyrosinase
absence and presence of 25 μg/mL TTP and PAA were recorded by the UV−2600 Spectrophotometer (Espin and Wichers, 2001).
The activity of monophenolase enzyme in mushroom tyrosinase was spectrophotometrically determined using L-tyrosine as substrate following a previously described method (Huang et al, 2013) with slight modification. Approximately 60 μL of each sample solution with various concentration (125–2000 μg/mL) and 180 μL of 1 mmol/L L-tyrosine solution were added into 300 μL of 0.2 mmol/L phosphate buffer (pH 6.8). After incubation of the reaction mixture at 25 °C for 20 min, 60 μL mushroom tyrosinase (250 units/mL) was added to an assay mixture (total volume of 600 μL). After incubation of the reaction mixture at 25 °C for 10 min, the absorbance of mixture was determined at 475 nm in a microplate reader. The inhibitory rate of tyrosinase activity was calculated by the following equation.
2.8 Fluorescence quenching reaction
sample, and Aa is absorbance at 475 nm with enzyme and test
The fluorescence intensities were recorded using an F–4600 Spectrofluorophotometer (Shimadzu, Japan) with an excitation wavelength (ex) of 290 nm and excitation and emission slit widths of 5 nm (Wang et al, 2016). Therefore, 250 U/mL tyrosinase was chosen as the concentration for fluorescence quenching experiments. Serial dilutions of sample solutions (250–1000 μg/mL) were prepared in DMSO. For each data point, 0.4 mL of the appropriate sample solution was added to 1.6 mL tyrosinase solution. The final concentration of samples ranged from 50 to 200 μg/mL. The change in the intensity of emission fluorescence was measured within 1 min when the samples were added to tyrosinase solution.
sample.
2.9 Cell viability analysis with TTP and PAA
Inhibitory rate = 1 − Aa/Ab Where Ab is absorbance at 475 nm with enzyme and without test
The activity of diphenolase enzyme in mushroom tyrosinase using L-DOPA as substrate was spectrophotometrically determined following a previously described method (Yu et al, 2011) with slight modification. About 50 μL of each sample solution with different concentration (125– 2000 μg/mL) and 150 μL of 2.5 mmol/L L-DOPA solution were added to 320 μL of 0.2 mmol/L phosphate buffer (pH 6.8). After incubation of the reaction mixture at 25 °C for 20 min, 60 μL mushroom tyrosinase (250 units/mL) was added to an assay mixture (total volume of 600 μL). The absorbance of the mixture was determined at 475 nm in a microplate reader without delay. The inhibitory rate of tyrosinase activity was calculated by the following equation: Inhibitory rate = 1 − Aa/Ab Where Ab is absorbance at 475 nm with enzyme and without test sample and Aa is absorbance at 475 nm with enzyme and test sample.
2.6 Effect of TTP and PAA on spectra obtained from oxidation of L‐tyrosine and L‐DOPA by mushroom tyrosinase The samples were tested by the methods described in 2.5 by a UV−2600 Spectrophotometer (Shimadzu, Japan) in real time. The spectra of products obtained during the oxidation of L-tyrosine and L-DOPA by mushroom tyrosinase in the range of 200–600 nm were recorded at different reaction time (Jiménez-Atiénzar et al, 2004). The sample concentration was 250 µg/mL, the final concentration in the L-tyrosine system was 25 µg/mL, and the final concentration in the L-DOPA system was 20.83 µg/mL.
2.7 Effect of TTP and PAA on spectra of obtained product during oxidation of L‐DOPA by NaIO4 L-DOPA (20 μg/mL) was oxidized by 20 μg/mL sodium periodate (NaIO4). The different spectra obtained in the
B16F1 melanoma cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin G, and 100 μg/mL streptomycin. Cells were instantly incubated at 37 °C in atmosphere of 5% CO2. The cell viability of TTP and PAA was assessed by MTT assay. In brief, 1 × 104 B16F1 cells were seeded and made to adhere to each well that contained 100 μL culture medium in 96-well plate. After 24 h, the culture medium was aspirated and added to 100 μL of the culture medium with or without TTP and PAA. After incubation for 48 h, the culture medium was aspirated and washed with PBS. A total of 20 μL MTT was added to each well, and the plate was incubated for 4 h at 37 °C. Subsequently, MTT solution was aspirated and quickly added into each well along with 100 μL DMSO for dissolving the blue formazan reactant. The optical density of each well was measured at 570 nm with the help of μQuant Microplate Spectrophotometer (Tecan, Switzerland) (Mei et al, 2011).
2.10 Cellular tyrosinase activity following TTP and PAA treatment B16F1 cells (1 × 105) were seeded and made to adhere to wells of a six-well plate at 37 °C in 5% CO2 for 24 h. The culture medium was removed and the cells were treated in the presence or absence of TTP and PAA for 48 h. The cells were then collected into trypsin-EDTA solution and immediately centrifuged at 5000 r/m for 5 min to obtain cell pellets. The cell pellets were mixed with lysis buffer (1% Triton X-100, 0.1 mmol/L PBS with pH of 6.8 and phenylmethylsulfonyl fluoride). The supernatant (80 μL) and L-DOPA (20 μL) were mixed in a 96-well plate and incubated at room temperature. After 10 min of incubation, the optical densities were determined at 475 nm using μQuant Microplate Spectrophotometer (Ye et al, 2010).
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328
3. Results and discussion 3.1 Quantitative analysis on PAA in TTP As shown in Figure 1, the peak marked 1 was PAA. The standard curve of PAA was y = 30940x + 78956, r2 = 0.9994 and the linear range was 12.8–256.0 μg/mL as previously established. y represents the peak area of triterpenes, whereas x represents the different concentration. The content of PAA in TTP was (37.57 ± 7.5709) mg/g.
mushroom tyrosinase. At this concentration, TPP and arbutin were not significantly different in inhibiting monophenolase activity, meanwhile they were significantly different from PAA and nicotinamide, but TTP, arbutin and PAA were all significantly stronger than other positive control (i.e., nicotinamide). 80
Inhibitory rate / %
324
ab 40
10
Figure 1
20
30 t / min
40
50
The scavenging effects of DPPH and ABTS radicals by TTP were shown in Table 1. TTP exerted a certain clearance effect on DPPH and ABTS free radicals. The IC50 values of clearing DPPH and ABTS free radicals were (2892.19 ± 923.10) and (180.75 ± 10.61) μg/mL, respectively. Anti-oxidant activity of triterpenes from the peels of P. cocos including removing the superoxide anion free radicals(O2−) and hydroxyl free radicals (OH) has been reported in some reference (Cheng et al, 2013). Thereinto, the IC50 values were also close to our results. Free radical scavenging activity or IC50 values of TTP
( x ± s ,n = 3) IC50 values / (μg·mL−1) DPPH
ABTS
TTP
2892.19 ± 923.10a
180.75 ± 10.61a
BHT
22.87 ± 2.54b
1.91 ± 0.22b
Arbutin Vc
PAA
Arbutin
Nicotinamide
Inhibitory effect of TTP and PAA on mushroom
tyrosinase for catalysis of L-Tyr arbutin and nicotinamide as positive controls, same as Figure 3 a−c refer to a significant difference among groups p < 0.05
3.2 Scavenging effect of TTP on DPPH and ABTS radicals
Samples
TTP
HPLC of total triterpenes isolated from P. cocos 1: poricoic acid A
Table 1
d
20
Figure 2 0
ac
60
0
1
a
c
1.65 ± 0.06bc
8.05 ± 0.61d
2.15 ± 0.13bd
76.35 ± 7.12
BHT: butylated hydroxytoluene; Vc: ascorbic acid a−d: significant difference between groups, p < 0.05
3.3 Effects of TTP and PAA on activity of monophenolase enzyme in mushroom tyrosinase Effects of TTP and PAA on tyrosinase monophenolase were shown in Figure 2. We selected two common whitening active components of arbutin and nicotinamide as positive controls. At 200 μg/mL, PAA and TTP had significant inhibitory effects on the activity of monophenolase enzyme in
3.4 Effects of TTP and PAA on activity of diphenolase enzyme in mushroom tyrosinase The relationship between the concentration of TTP and PAA with tyrosinase activity was shown in Figure 3. We also selected arbutin and nicotinamide as the positive controls. The positive control agents, as well as TTP and PAA, could significantly inhibit the activity of diphenolase in mushroom tyrosinase and showed a dose-effect relationship. Furthermore, the inhibitory effect was enhanced with increasing concentration. At the five concentrations selected, the inhibitory activity of PAA on diphenolase in mushroom tyrosinase at low concentration was the weakest. However, as the dose increased, the inhibitory activity was increased rapidly. We found that PAA had the best inhibitory effect when its concentration reached 166.67 μg/mL. The inhibitory effect of PAA was better than those of nicotinamide and arbutin. Its inhibitory rate was 45.24% at 166.67 μg/mL. Furthermore, the inhibitory effect of TTP was better than that of arbutin in the range of 10.42–166.67 μg/mL.
3.5 Effects of TTP and PAA on spectra obtained from oxidation of L‐tyrosine by mushroom tyrosinase The tyrosinase-catalyzed oxidation of the L-tyrosine reaction spectrum was shown in Figure 4A. The absorbance at 475 nm during the catalytic oxidation of tyrosine increased, and the curves from the bottom to the top indicated the change in the absorption spectrum of the oxidation product from 0 to 20 min. Figures 4B and 4C showed the spectra of the tyrosinase-catalyzed oxidation of L-tyrosine in the presence of TTP and PAA. Compared with Figure 4A, the absorbance at 475 nm was significantly attenuated in the presence of TTP and PAA. This result was consistent with monophenolase inhibitory activity.
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328 50
3.6 Effects of TTP and PAA on spectra obtained from oxidation of L‐DOPA by mushroom tyrosinase
Inhibitory rate / %
40 30 20
TTP PAA Arbutin Nicotinamide
10 0
Figure 3
0
50 100 150 Concentration / (μg·mL−1)
Inhibitory effect of TTP and PAA on mushroom
tyrosinase for catalysis of L-DOPA
0.9
0.6
0.6
400
500
600
0.151 260 300 2.3 2.0
D Absorption
1.5 1.0 0.5 0.0 260 300 Figure 4
400
500
600
400 500 Wavelength / nm
0.6
0.3
2.3 2.0
E
1.5 1.0 0.5 0.0 260 300
400 500 Wavelength / nm
C
0.151 600 260 300
Absorption
260 300
2.3 2.0 Absorption
0.9
B
0.3
0.3 0.151
Figure 4D showed the UV-vis spectra of L-DOPA catalyzed by tyrosinase. The absorbance at 475 nm during catalytic oxidation of L-DOPA increased. Curves from the bottom to the top indicated the absorption spectra of the oxidation product at 0–10 min after the reaction time. Figures 4E and 4F showed the spectra of L-DOPA catalyzed by tyrosinase in the presence of TTP and PAA, respectively. Compared with Figure 4D, the absorbance at 475 nm in the presence of TTP and PAA was significantly attenuated. The order of decreasing absorption peak was TTP > PAA, which was consistent with inhibitory activity of diphenolase.
Absorption
A
Absorption
Absorption
0.9
325
600
400
500
600
400
500
600
F
1.5 1.0 0.5 0.0 260 300
Consecutive spectra obtained during oxidation of L-Tyr (A−C) and L-DOPA (D−F) by mushroom tyrosinase
A: L-tyrosine+ enzyme; B: 25 μg·mL−1 TTP + L-tyrosine + enzyme; C: 25 μg·mL−1 PAA + L-tyrosine + enzyme; D: L-DOPA + enzyme; E: 20.83 μg·mL−1 TTP + L-DOPA + enzyme; F: 20.83 μg·mL−1 PAA + L-DOPA + enzyme
3.7 Interaction of TTP and PAA with o‐quinones We tested the production of dopaquinone with NaIO4 in the presence and absence of TTP and PAA to determine whether TTP and PAA are directly oxidized with dopaquinone to produce diphenols. Figures 5A and 5B showed that no absorption peak occurred at 475 nm when L-DOPA was alone, and the absorption peak appeared at 475 nm after NaIO4 was added. The absorption peak at 475 nm was significantly reduced when the sample of TTP and PAA were added into the solution of L-DOPA and NaIO4, as shown in Figures 5A and 5B. Curves from the bottom to top in Figures 5D and 5E showed the changes in the dopaquinone uptake spectrum of the oxidized product at 0–4 min after adding PAA and TTP into the mixture solution with L-DOPA and NaIO4, respectively. Figure 5C showed that the intensity of the absorption peak of the mixture solution with L-DOPA
and NaIO4 at 475 nm increased with time. Thus, the oxidation reaction occurred in TTP, PAA and dopaquinone, which was oxidized with L-DOPA by NaIO4.
3.8 Effect on emission of fluorescence in mushroom tyrosinase Tryptophan fluorescence has been frequently examined among the intrinsic aromatic fluorophores in tyrosinase molecules to obtain information about conformational changes. The effects of TTP and PAA on the fluorescence emission of tyrosinase at an excitation wavelength of 290 nm were shown in Figure 6. Tyrosinase exhibited the largest emission fluorescence at 366 nm. Figure 6A showed that TTP could change the fluorescence emission spectrum of tyrosinase at 50–200 μg/mL. The intensity of the fluorescence peak decreased, and the maximum wavelength of emission
326
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328 A
2.0 Absorption
2.5
L-DOPA L-DOPA + PAA L-DOPA + NaIO4 + PAA L-DOPA + NaIO4
1.5 1.0
1.0 0.5
300
400
500
300
4 min
0.5
300
400
500
600
0.5 0 min
200
600
E
0.0
0 min
0.0
500
1.5 Absorption
1.0
200
400
D
1.5
Figure 5
0.0 200 Wavelength / nm
600
1.5 Absorption
Absorption
200
C
2.0
L-DOPA L-DOPA + TTP L-DOPA + NaIO4 + TTP L-DOPA + NaIO4
1.5
0.5 0.0
B
2.0 Absorption
2.5
0 min 4 min
0.0
4 min
300
0.5
400 500 Wavelength / nm
600
200
300
400
500
600
Scanning spectra for 20 μg/mL L-DOPA+20 μg/mL NaIO4 + 25 μg/mL PAA (A), 25 μg/mL TTP (B), consecutive spectra of
absorption obtained during oxidation of L-DOPA by NaIO4 in 4 min (C), consecutive spectra of absorption obtained during the oxidation of L-DOPA + 25 μg·mL−1 PAA by NaIO4 in 4 min (D) and consecutive spectra of absorption obtained during oxidation of L-DOPA + 25 μg·mL−1 TTP by NaIO4 in 4 min (E) A
Control 50 μg·mL−1 TTP 100 μg·mL−1 TTP 200 μg·mL−1 TTP
200
100
0 350 Figure 6
400
300 Fluorescence intensity
Fluorescence intensity
300
B
Control 50 μg·mL−1 PAA 100 μg·mL−1 PAA 200 μg·mL−1 PAA
200
100
0 450 Wavelength / nm
350
400
450
Effect of TTP (A) and PAA (B) on emission spectra of mushroom tyrosinase
fluorescence showed a redshift from 366 nm to 377 nm. The results showed that TTP had a fluorescence quenching effect on tyrosinase, and an obvious relationship occurred with the concentration of samples. The redshifts increased with concentration. Thus, TTP influenced emission fluorescence quenching. In addition, TPP possibly played a role by changing the hydrophobic environment of the amino acid residues in tyrosinase. Figure 6B showed that PAA had a minimal effect on the fluorescence emission of tyrosinase at 50–200 μg/mL.
3.9 Cell viability analysis on TTP and PAA Drug cytotoxicity is of chief importance when the drug is used either as a medicine or as a cosmetic agent. Therefore, the cell safety of TTP and PAA was examined in B16F1 melanoma with the help of MTT assay. The effects of TTP and PAA on cell inhibition were shown in Figure 7A. In
B16F1 melanoma cells, TTP with high concentration (12.5–100 μg/mL) was associated with cell viability of 58%–86%. At a low concentration (6.25 μg/mL), TTP did not show obvious cytotoxicity when the cell viability was higher than 90%. In addition, PAA with high concentration (12.5–100 μg/mL) was associated with cell viability of 27%–77%. At a low concentration (6.25 μg/mL), PAA did not show obvious cytotoxicity when the cell viability was higher than 90%. The IC50 values of the cell viability of TTP and PAA were 118.26 and 48.01 µg/mL, respectively. These results indicated that a concentration of 0.78–12.5 μg/mL was necessary to evaluate their cellular tyrosinase activity using B16F1 cells.
3.10 Cellular tyrosinase activity following TTP and PAA treatment Melanogenesis due to melanin overproduction is highly
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328 80
60
A
B
50
60
Inhibitory rate / %
Inhibitory rate / %
70
327
50 40 30 TTP
20
0 0 Figure 7
20
40
60
80
30 TTP
20
PAA Arbutin
10
PAA
10
40
0 100 120 0 Concentration / (μg·mL−1)
5
10
15
Inhibitory effect of TTP and PAA on proliferation (A) and tyrosinase activity (B) in mouse B16 cells Arbutin was used as positive control.
dependent on the activation of cellular tyrosinase (Huang et al, 2012). To elucidate the inhibitory effect on melanogenesis of triterpenes, the cellular tyrosinase activity in TTP- and PAA-treated B16F1 cells was investigated. The effects of TTP and PAA on tyrosinase of B16 cells were shown in Figure 7B, and arbutin was selected as positive control. The effects of TTP and PAA on the inhibition of tyrosinase activity in cells increased with rising concentration of 12.5 μg/mL. The inhibitory rates of TTP and PAA were 40.86% and 39.53%, respectively.
4. Conclusion In this paper, we used triterpenoids as the main research object to exploit safer and more effective whitening products from herbs. TTP and PAA exerted a highly effective inhibitory effect on either mushroom tyrosinase or cell tyrosinase. Ultraviolet spectrophotometry and fluorophotometry revealed the possible mechanisms of TTP and PAA on mushroom tyrosinase. TTP and PAA played a key role mainly by combining with dopaquinone formed by the oxidation of DOPA and by changing the hydrophobic environment of the amino acid residues in tyrosinase. In addition, TTP significantly influenced the fluorescence quenching of tyrosinase, whereas PAA only slightly affected the fluorescence emission of tyrosinase. In other words, TTP could change the conformation of mushroom tyrosinase, but PAA could not. Therefore, other triterpenes in TTP may play the same role in inhibiting tyrosinase activity. P. cocos triterpenoids are potential whitening active ingredients, but more in-depth studies are needed to develop them into whitening products. Conflict of interest statement The authors declare no conflict of interest. References Akihisa T, Nakamura Y, Tokuda H, Uchiyama E, Suzuki T, Kimura Y, 2007. Triterpene acids from Poria cocos and their anti-tumorpromoting effects. J Nat Prod 70(6): 948-953.
Ali S, Naaz I, 2015. Current challenges in understanding the story of skin pigmentation-bridging the morpho-anatomical and functional aspects of mammalian melanocytes. Muscle Cell and Tissue. InTech Open House, Europe: 262-285. Cheng SM, Gui Y, Shen S, Huang W, 2013. The antioxidant activity of triterpenes in the peels of Poria cocos. Med Plant 4(8): 38-41. Espin JC, Wichers HJ 2001. Effect of captopril on mushroom tyrosinase activity in vitro. Biochim Biophys Acta 1544(1-2): 289-300. Fan Z, Z XF, Wang BC, Liu HY, Li CY, Liu ZH, Zhang GW, Lu H, Chi C, Wang F, 2009. Pachymic acid, a novel compound for anti-rejection: Effect in rats following cardiac allograft transplantation. Chin Med J 122(23): 2898-2902. Gao Y, Yan H, Jin R, Lei P, 2016. Antiepileptic activity of total triterpenes isolated from Poria cocos is mediated by suppression of aspartic and glutamic acids in the brain. Pharm Biol 54(11): 2528-2535. Hu B, Yang YP, Ye Y, 2006. Chemical constituents from Poria cocos. Chin Tradit Herb Drugs 37(5): 655-658. Huang XW, Feng YC, Huang Y, Li HL, 2013. Potential cosmetic application of essential oil extracted from Litsea cubeba fruits from China. J Essent Oil Res 25(2): 112-119. Huang HC, Wang HF, Yi KH, Chang LZ, Chang TM, 2012. The dual anti-melanogenic and antioxidant activities of the essential oil extracted from the leaves of acorus macrospadiceus (Yamamoto) F. N. Wei et Y. K. Li. Evid Based Complement Alternat Med. Vol. 2012, Art. ID: 781280, 10pages. Jiménez-Atiénzar M, Cabanes J, Gandıa-Herrero F, Garcıa-Carmona F, 2004. Kinetic analysis of catechin oxidation by polyphenol oxidase at neutral pH. Biochem Biophys Res Commun 319(3): 902-910. Kamal UZ, Sharique AA, Ayesha SA, 2017. Melanogenic effect of purified mushroom tyrosinase on B16F10 Melanocytes: A phase contrast and immunofluorescence microscopic study. J Microsco Ultrastruct 5(2): 82-89. Lee RS, Lee S, Moon E, Park HJ, Kim KH, 2017a. Bioactivity-guided isolation of anti-inflammatory triterpenoids from the sclerotia of Poria cocos using LPS-stimulated Raw264.7 cells. Bioorg Chem 70: 94-99. Lee S, Lee D, Lee SO, Ryu JY, Choi SZ, Kang KS, Kim KH, 2017b. Anti-inflammatory activity of the sclerotia of edible fungus, Poria cocos Wolf and their active lanostane triterpenoids. J Funct Foods 32: 27-36. Maack A, Pegard A, 2016. Populus nigra (Salicaceae) absolute rich in phenolic acids, phenylpropanoïds and flavonoids as a new potent
328
Hu SH et al. Chinese Herbal Medicines, 2017, 9(4): 321-328
tyrosinase inhibitor. Fitoterapia 111: 95-101. Mei X, Wu YY, Mao X, Tu YY, 2011. Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols. Environ Pollut 159(1): 164-168. Shen S, Li FJ, Mei GM, Huang W, 2009. Study on content determination and antibacterial effects of triterpenes from sclerotia of Poria cocos. Food Science 30(1): 95-98. Wang KH, Lin RD, Hsu FL, Huang YH, Chang HC, Huang CY, Lee MH, 2006. Cosmetic applications of selected traditional Chinese herbal medicines. J Ethnopharmacol 106(3): 353-359. Wang W, Dong H, Yan R, Wang ZM, 2015. Comparative study of lanostane-type triterpene acids in different parts of Poria cocos (Schw.) Wolf by UHPLC-Fourier transform MS and UHPLCtriple quadruple MS. J Pharm Biomed Anal 102(5): 203-214.
Wang XY, Li MZ, He Y, Wang QH, 2016. Study on the interaction of cinnamic acid and azelaic acid to tyrosinase by fluorescence quenching method. J Tradit Chin Med 21(12): 5348-5351. Ye Y, Chou GX, Mu DD, Wang H, Chu JH, Leung AK, Fong WF, Yu ZL, 2010. Screening of Chinese herbal medicines for antityrosinase activity in a cell free system and B16 cells. J Ethnopharmacol 129(3): 387-390. Yu LL, Hu WC, Ding G, Li RT, Wei JH, Zou ZM, Wang MH, 2011. Gusanlungionosides A−D, potential tyrosinase inhibitors from Arcangelisia gusanlung. J Nat Prod 74(5): 1009-1014. Zhang JY, Fang YY, Wu XQ, Zhang Y, 2008. Status of skin-whitening cosmetics and its future development. Fine Chem 25(1): 72-75. Zhang QQ, Wang MZ，Wang HK, Wang XF, Gu YT, 2009. The influence on hippo camp amino acid content of the total triterpene of Poria in rats. Chin Pharmacol B 25(2): 279-280.