Biotechnology and Bioprocess Engineering 20: 814-823 (2015) DOI 10.1007/s12257-014-0867-x

RESEARCH PAPER

Active Compounds from Schisandra chinensis Exhibiting Tyrosinase Activity and Melanin Content Inhibition in B16 Melanoma Cells Zheng-Fei Yan, Jian Guo, Feng-Hua Tian, Xin-Xin Mao, Yu Li, and Chang-Tian Li

Received: 19 December 2014 / Accepted: 11 May 2015 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract Schisandra chinensis has been used as traditional medicine. The structures of isolate active compounds (schisandrin B, deoxyschisandrin, schisandrin C) from S. chinensis were characterized by physical and spectroscopic analyses. Active compounds were tested for their potential to act as anti-melanogenesis or skin-whitening agents by their abilities to inhibit tyrosinase activity in the cell-free mushroom tyrosinase assay and cellular tyrosinase derived from B16 melanoma cells. The tyrosinase inhibitory activity was correlated to the inhibition of melanin productions in α-MSH-stimulated and unstimulated B16 cells. Cellular tyrosinase kinetics were analyzed and showed by LineweaverBurk plot. Schisandrin B was minimally cytotoxic (cell viability: 88.99% at 0.75 µM) and the IC50 value for suppression of mushroom tyrosinase activity was estimated as 0.6 µM. Zymography analysis demonstrated schisandrin B’s concentration-dependent effects and the kinetic analysis indicated schisandrin B’s noncompetitive-inhibitory action. Keywords: schisandrin B, B16 melanoma cells, tyrosinase activity, melanin content

1. Introduction Schisandra chinensis, a well-known traditional Chinese medicine, grows in the most eastern parts of Russia, southern Jian Guo, Feng-Hua Tian, Xin-Xin Mao, Yu Li, Chang-Tian Li* Engineering Research Center of Edible and Medicinal Fungi, Ministry of Education, Jilin Agricultural University, Changchun 130-118, China Tel: +86-431-84533310; Fax: +86-431-84532989 E-mail: [email protected] Zheng-Fei Yan Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, Yongin 446-701, Korea

Sakhalin, northeastern China, Korea, and Japan. It has been officially listed in the Chinese Pharmacopoeia. S. chinensis has been used traditionally in folk medicine for the hepatoprotective, antioxidant and membrane stabilizing properties [1-4], the cisplatin-induced DNA damage in brain tissue [5]. Melanin is the main component determining the color of skin and up to 10% of cells in the innermost layer of the epidermis produce melanin pigments [6]. The major role of melanin is to protect the skin from damaging effects of ultraviolet radiation [7]. Melanin biosynthesis is a wellknown physiological response of human skin upon exposure to ultraviolet light and other stimuli. Melanogenesis is regulated by enzymes such as tyrosinase [8]. The inhibition of tyrosinase is the most common approach to achieve skin whiteness as it is the key enzyme that catalyzes the ratelimiting step of melanin biosynthesis [9]. Tyrosinase plays a crucial role in the initial step of melanin synthesis by catalyzing the oxidation of L-tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to dopaquinone. Oxidative polymerization of several dopaquinone derivatives gives rise to melanin [10-13]. Thus substances that inhibit tyrosinase may be useful ingredients to be incorporated into cosmetic preparations. Previous studies have shown that antioxidants may reduce hyperpigmentation and support skin health [14]. S. chinensis’s antioxidant property might reduce hyperpigmentation [15]. A study was undertaken to investigate if S. chinensis possesses any anti-melanogenesis effects with a view of its possible use as a treatment for hyperpigmentation and its use as a skin-whitening agent in cosmetics. In this study, the active compounds were isolated and purified by using silica gel column chromatography and preparative high performance liquid chromatography (PHPLC). The ability of the active compounds to act as a skin-whitening agent

Active Compounds from Schisandra chinensis Exhibiting Tyrosinase Activity and Melanin Content Inhibition in B16 Melanoma…

was tested by its ability to inhibit tyrosinase. The rate limiting enzyme in melanogenesis, initially using a cellfree mushroom tyrosinase system. The ability to inhibit cellular tyrosinase derived from melanin producing B16 melanoma cells as well as its ability to inhibit melanogenesis were also tested. Kojic acid that is well known to be an inhibitor of tyrosinase and melanogenesis [16] was used as a positive control.

2. Materials and Methods 2.1. Reagents Mushroom tyrosinase (EC1.14.18.1), dimethyl sulfoxide (DMSO), L-tyrosine (L-Tyr), and L-3,4-dihydroxyphenylalanine (L-DOPA) were purchased from Sigma (St. Louis, MO, USA). kojic acid was purchased from the National Institute for the Control of Pharmaceutical and Bioproducts (Beijing, China). Absolute benzene, acetone, ethanol, and methanol (HPLC grade) were obtained from Merck (Darmstadt, FR, Germany). Column chromatography was carried out over silica gel (200 mesh, Merck) and reversed silica gel was carried out with silica gel (400 mesh, Merck). Preparative High Performance Liquid Chromatograph (PHPLC) was run on a JASCO PU-1586 instrument equipped with a differential refractometer (RI 1531). Fractions obtained from column chromatograph were monitored by TLC (silica gel 60 F254). The water used was distilled. 2.2. Extraction and isolation Dried fruits of S. chinensis were ground into powder (500 g), and extracted with methanol employing an automatic reflux apparatus for 1 h at 50oC. The methanol solution was evaporated under reduced pressure and the resulting dark brown residue (19.5 g) was then suspended in distilled water and partitioned with n-hexane. The n-hexane solution was evaporated under reduced pressure and the resulting dark yellow residue (9.0 g) was subjected to silica gel (0.2 kg) column chromatography. Elution of the column with benzene-acetone (98:2, V/V) obtained residues: A (Fr. No. 1-20, 0.5 g), B (Fr. No. 21-37, 1 g), and C (Fr. No. 3854, 0.7 g). Residue B was rechromatographed by reversed silica gel (400 mesh, 30g) column chromatography using methanol-water (12:1, V/V) to give compound I (19.5 g) from fraction No. 19-37. Residue C was rechromatographed on reversed silica gel (200 mesh, 10 g) using methanol-water (12:1) to afford compounds II and III mixture (90.3 mg), which was separated with PHPLC [ODS, MeOH-H2O (90:10)] to give compounds II (10 g) and III (10.2 g), respectively. Compound structure was confirmed by its 1H and 13C NMR spectral data. The compounds were redissolved with deionized water and prepared to a suitable

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concentration for screening active compound. The crystal habit and melting point were determinate [17,18]. 2.3. Determination of the concentration of compounds in dried fruits of Schisandra chinensis by HPLC and method validation Concentrations of three compounds were determined by HPLC a column Inertsil ODS-SP (250 mm × 4.6 mm i.d., 5 µm) (LC-20AT, Shimadzu, Japan) with the photodiode array detector (SPD-M20A). A linear gradient elution of eluents A (methanol) and B (water) was used for separation. The elution program was optimized and conducted as follows: a linear gradient of 0% B (0 ~ 5 min), 2% B (5 ~ 6 min), 3% B (6 ~ 7 min), 4% B (7 ~ 10 min), 3% B (10 ~ 15 min), 2% B (15 ~ 20 min), 1% B (20 ~ 25 min), 0% B (25 ~ 30 min). The peaks were recorded using PDA absorbance at 224 nm and the solvent flow rate was 0.5 mL/min and the oven temperature was set at 30°C. The reference compounds (schisandrin B, deoxyschisandrin, schisandrin C) and samples were weighed and dissolved in methanol. The solutions (2 mL) were filtered through a 0.45 µm membrane filter prior to HPLC analysis. For all experiments, volumes of 10 µL of the reference compounds (10 concentration gradient) and isolable compounds were injected. The compound calibration curves were obtain by abscissa for concentration; ordinate for absorbance. The intra-day and inter-day precision for sample extracts was performed on day one and next four consecutive day [19]. The relative standard deviation (RSD) ranges of the retention time and peak area of the reference peak in the sample extracts were obtained for intra-day and inter-day analysis. The recovery test was performed using the method of standard addition [20]. Using rutin as a target in the sample extracts, the sample extracts was spiked with the high, intermediate and low levels of standard solution. The recovery was calculated by comparing the determined amount of those standards with the added amount originally. The stability was assessed by analyzing the same sample solution at 0, 2, 4, 8, 12, and 24 h after extraction. The RSD ranges of the retention time and peak area of the reference peak in the sample extracts was also determined. 2.4. Mushroom tyrosinase activity analysis as phytochemical screening For screening active compounds, mushroom tyrosinase was reconstituted in 50 mM Na2HPO4-NaH2PO4 buffer (pH 6.8) at 1,000 U/mL and stored at −20°C prior to use. There action mixture consisted of 2.5 mM L-Tyr, 500 U/mL mushroom tyrosinase, and various concentrations (0, 0.75, 1.5, and 3 µM) of the active compounds or kojic acid. After incubation for 30 min at 37oC, the absorbance was measured at 475 nm by a Beckman TU-1810 spectro-

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photometer as previously described [21]. For active compounds that inhibited mushroom tyrosinase, the extent of inhibition was here expressed as the amount of samples needed to inhibit 50% of enzyme activity (IC50) by above method described, and then they were investigated the effects on cellular tyrosinase activity, melanin content, cytotoxicity test of B16 melanoma cells. 2.5. Cell culture and treatment The B16 melanoma cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The B16 melanoma cells were initially grown in co-culture with Hyclone’s Modified RPMI1640’s Medium (Hyclone, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum, 1% Penicillin-Streptomycin Solution, 100 × (Beyotime Institute of Biotechnology, China) in 96-well plates in a CO2 incubator with a humidified atmosphere containing 5% CO2 in air at 37°C. The cells culture medium was replaced three times a week and then cell were sub-cultured by trypsinisation after growing 3 days. The cells were seeded at the appropriate numbers counted with BD Accuri C6 (BD, USA) into wells of cell culture plates for further experiments. 2.6. Determination of cellular tyrosinase activity and melanin content in active compounds To investigate the effect of active compounds on cellular tyrosinase activity and melanin content, 1 × 106 B16 melanoma cells were seeded into each well of a 6-well tissue culture plates and allowed to attach overnight. The cells were exposed to various concentrations (0, 0.75, 1.5, and 3 µM) of the pure compounds or kojic acid for 72 h in the presence or absence of 200 nM α-MSH. Tyrosinase activity was estimated by measuring the rate of oxidation of L-DOPA [22]. Cells (1 × 106) were suspended in 50 µL cold M/15 phosphate buffer, pH 6.8, containing 1% (w/v) Triton X-100. After vortexing to lyse the cells, the extract was clarified by centrifugation at 10,000 × g for 5 min. LDOPA (3 mM) was prepared in phosphate buffer as above without Triton X-100 (assay buffer). Samples (40 µL) of cell lysate were added to the wells of a 96-well plate and the assay was started by the addition of 100 µL L-DOPA solution at 30°C. Control wells contained 40 µL lysis buffer or boiled cell lysate. Absorbance at 475 nm was read every minute for at least 20 min at 30°C. One unit of tyrosinase activity was arbitarily defined as a rate of increase of 1 absorbance unit per h per 106 cells in the initial linear region of a plot of absorbance against time [23]. There was no increase in absorbance in the control wells. Tyrosinase activity in the protein was calculated by the following formula [24]:

Biotechnology and Bioprocess Engineering 20: 814-823 (2015)

OD475 of sample Tyrosinase activity (%) = --------------------------------------× 100 OD475 of control For melanin determination, 1 × 106 cells were solubilised in 100 µL 1 M NaOH and diluted with 400 µL distilled water. Absorbance at 475 nm was compared with a standard curve of synthetic melanin (Sigma) prepared in a final NaOH concentration of 0.2 M. 2.7. Kinetic analysis of tyrosinase activity inhibition analysis by active compounds The cells were treated with active compounds as described above for the determination of tyrosinase activity. Each well of a 96-well plate contained 40 µg of lysate protein, 0.1 M PBS (pH 6.8), and various concentrations of LDOPA (0.25, 0.3, 0.5, 0.6, and 1 mM) and active compounds (0, 2.25, 1.2, 0.6, and 0.18 µM). After incubation at 37oC for 30 min, the absorbance was measured at 475 nm. The apparent inhibition constants for active compounds and inhibition type were calculated using Lineweaver-Burk plot [25]. The Lineweaver-Burk plot was widely used to determine important terms in enzyme kinetics, such as Km and Vmax. The plot provided a useful graphical method for analysis of the Michaelis-Menten equation [26,27]: Vmax [S] V = ------------------Km + [ S ] Taking the reciprocal gives Lineweaver-Burk plot: Km + [ S ] Km 1 1- = ------------------1--- = ---------- ------- + ---------V Vmax [S] Vmax [S] Vmax Where V is the reaction velocity (the reaction rate), Km is the Michaelis-Menten constant, Vmax is the maximum reaction velocity, and [S] is the substrate concentration. The y-intercept of such a graph was equivalent to the inverse of Vmax; the X-intercept of the graph represents −1/Km. It also gave a quick, visual impression of the different forms of enzyme inhibition. The inhibition constant was generated from the slope of the apparent Kmax/Vmax or 1/Vmax versus the concentrations of active compounds. 2.8. Cytotoxicity tests of active compound in cell culture The in vitro cytotoxicity of active compound was tested on B16 melanoma cells. The active compounds and kojic acid were dissolved in ethanol at 0, 0.75, 1.5, and 3 µM. The cells were treated as described above for the determination of tyrosinase activity. Cells at the exponential growth phase were harvested from the culture plates by trypsination and centrifuging at 180 g for 3 min, and then resuspended in fresh medium at 1 × 106 cells/mL. The cell suspension was dispensed into a 96-well plates at 100 µL/well and incubated

Active Compounds from Schisandra chinensis Exhibiting Tyrosinase Activity and Melanin Content Inhibition in B16 Melanoma…

in a humidified atmosphere with 5% CO2 at 37oC for 24 h, and then treated with active compounds and kojic acid, respectively. Cell proliferation in the plates was determined with the MTT assay as described in detail elsewhere [28]. Cell proliferation activity was expressed as the percentage of MTT count of extract-treated cells relative to that of the control (% of control) [24]. OD492 of sample Cell viability rate % = --------------------------------------× 100 OD492 of control

3. Results 3.1. Identification of compounds by spectral data Compounds were further purified on PHPLC. The structural information of three compounds was obtained using NMR. All spectral data were consistent with the data of schisandrin B (compound I), deoxyschisandrin (compound II), schisandrin C (compound III) [29-32]. Three compounds were known (schisandrin B for white square crystal, mp 104 ~ 106oC; deoxyschisandrin for white columnar crystal, mp 114 ~ 115oC; schisandrin C for white needle crystal, mp 122 ~ 123oC). Chemical structures of compounds were shown in Fig. 1 as well as NMR data of compounds in Tables 1, 2, and 3, respectively. 3.2. Compounds concentration in S. chinensis HPLC provided an efficient tool for sensitive and selective determination of compounds. The correlation coefficient of the three reference compounds calibration curve was very satisfactory, R = 0.998 in the studied concentration range (0.1 ~ 10 µg/mL). Table 4 summarized the calibration curve parameters and detection limits. Precision was determined by analyzing three same sample extracts. The sample was

Fig. 1. Chemical structures of schisandrin B (Compound I), deoxyschisandrin (Compound II), schisandrin C (Compound III).

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extracted in duplicate. Intra-day analysis was assessed by replicate injections (five times) of sample. Inter-day analysis was also assessed by replicate injections (four times) of second sample in 4 consecutive day. Mean deoxyschisandrin concentration in this sample was determined to be 19.75 ± 0.071 µg/g for intra-day analysis, 19.90 ± 0.05 µg/g for inter-day analysis and RSD < 3.0%. For instrument sensitivity and stability, it was assessed by analyzing the same sample solution at 0, 2, 4, 8, 12, and 24 h. The concentrations of deoxyschisandrin were determined from six analysis were nearly identical. The consistent results obtained over the two-week period of sample processing and analysis also supported this correlations. Using rutin as a target in the samples, the samples were spiked with the high, intermediate and low levels of standard solution in order to determine the recovery of rutin. The native rutin signals from the original extract analyses were subtracted from the native rutin signals of the spiked samples and the resulting data were used to determine the rutin recoveries. The recovery rate was determined to be 94.4% and RSD < 3.0%. This suggested that the extraction method, HPLC analysis was suitable for determining compounds levels. The compounds found in examined Table 1. 1H and13C NMR spectral data of compound I in CDCl3 1 Position H NMR dataa H-4 6.54 (s) 2.58 (dd, J = 10.1,6.7 Hz) H2-6 2.49 (d, J = 10.1 Hz) H-7 1.81(m), H-8 1.90 (m), 2.23 (dd, J = 13.3,9.4 Hz) H2-9 2.03 (d, J = 13.3 Hz) H-11 6.47 (s) 0.72 (s) CH3-17 0.97 (d, J = 7.0 Hz) CH3-18 OCH3-1 3.53 (s) OCH3-2 3.81 (s) OCH3-3 3.88 (s) OCH3-14 3.95 (s) -OCH2O- 5.94 (s)

a

Measured at 400 MHz, J value in Hz. Measured at 100 MHz, d in ppm.

b

Position C-1 C-2

13

C NMR datab 151.5(s) 140(s)

C-3 C-4 C-5

151.6(s) 110.6 (d) 134.1(s)

C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 OCH3-1 OCH3-2 OCH3-3 OCH3-14 -OCH2O-

39.1 (t) 33.4 (d) 40.7 (d) 335.5 (t) 137.8 (s) 102.9 (d) 148.6 (s) 134.5 (s) 140.0 (s) 121.3 (s) 123.2 (s) 12.8 (q) 21.5 (q) 60.9 (q) 60.5 (q) 59.6 (q) 55.81(q) 100.7 (t)

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Biotechnology and Bioprocess Engineering 20: 814-823 (2015)

Table 2. 1H and13C NMR spectral data of compound II in CDCl3 Position H-4 H2-6 H-7 H-8 H2-9 H-11 CH3-17 CH3-18 OCH3-1 OCH3-2 OCH3-3 OCH3-12 OCH3-13 OCH3-14

1

H NMR dataa 66.55 (s) 2.59 (dd, J =13.6,7.2) 2.49 (d, J = 10.1) 1.80 (m) 1.89 (m) 2.27 (dd, J = 13.5, 9.0) 2.02 (brd, J = 13.5) 6.54 (s) 0.72 (s) 0.97 (d) 3.59 (s) 3.87 (s) 3.85 (s) 3.90 (s) 3.83 (s) 3.91 (s)

Position C-1 C-2

C NMR datab 152.5 (s) 140.1 (s)

Table 3. 1H and 13C NMR spectral data of compound III in CDCl3

13

C-3 C-4 C-5

152,8 (s) 107.1 (d) 139.2 (s)

C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 OCH3-1 OCH3-2 OCH3-3 OCH3-12 OCH3-13 OCH3-14

35.5 (t) 40.7 (d) 33.7 (d) 39.1 (t) 133.9 (s) 110.3 (d) 151.5 (s) 139.6 (s) 151.3 (s) 123.3 (s) 122.2 (s) 12.6 (q) 21.9 (q) 61.0 (q) 60.6 (q) 55.9 (q) 55.9 (q) 60.6 (q) 61.0 (q)

Position

1

H NMR dataa

H-4 H2-6

13

Position

66.49 (s) C-1 2.44 (d, J = 13.6) C-2 2.54 (dd, J = 13.6, 7.2) H-7 1.76 (m) C-3 H-8 1.86 (m) C-4 2.00 (d, J = 12.8) C-5 H2-9 2.22 (d, J = 112.8, 7.8) H-11 6.49 (s) C-6 CH3-17 0.97 (d, J = 7.2 Hz) C-7 0.72 (d, J = 7.2) C-8 CH3-18 OCH3-1 3.83 (s) C-9 3.85 (s) C-10 OCH3-14 3-OCH2O-4 5.93 (brd) C-11 C-12 14-OCH2O-15 5.94 (brd) C-13 C-14 C-15 C-16 C-17 C-18 OCH3-1 OCH3-14 3-OCH2O-4 14-OCH2O-15

C NMR datab 141 (s) 134.6 (s)

147.5 (s) 106.0 (d) 132.7 (s) 38.7 (s) 33.5 (d) 40.6 (d) 35.2 (t) 138.1 (s) 103.1 (d) 148.6 (s) 134.3 (s) 140.9 (s) 120.9 (s) 122.l (s) 11.7 (q) 12.4 (q) 59.6 (q) 59.6 (q) 100.7 (t) 100.7 (t)

a

a

Measured at 400 MHz, J value in Hz. Measured at 100 MHz, d in ppm.

Measured at 400 MHz, J value in Hz. Measured at 100 MHz, d in ppm.

b

b

Table 4. Calibration curves parameters and detection limits for all samples Compounds schisandrin B deoxyschisandrin schisandrin C

Concentration (µg/mL) 0.1 ~ 10 0.1 ~ 10 0.1 ~ 10

Regression equation Y = 912753X + 478.24 Y = 872747X + 429.24 Y = 96567X + 327.24

Table 5. Concentrations of compounds in Schisandra chinensis Compounds schisandrin B deoxyschisandrin schisandrin C

Concentration (µg/g) 3.83 2.17 2.93

Content (%) 0.38 0.21 0.29

samples and content is given in Table 5. 3.3. Determination compounds on mushroom tyrosinase inhibition activity The mushroom tyrosinase inhibition activity of test compounds using L-Tyr as substrate was evaluated for screening active compounds. The results showed that schisandrin B had a strong inhibitory effect on mushroom tyrosinase. Deoxy-

Correlation coefficient 0.998 0.992 0.999

Detection limit (µg/µL) 0.0211 0.0251 0.0111

shisandrin and schisandrin C had a slightly or no inhibitory effect on tyrosinase (Fig. 2). So schisandrin B was next investigated to examine IC50. The inhibitory effects of different concentrations of schisandrin B (0, 2.25, 1.5, 1.2, 0.675, and 0.6 µM) on the oxidation of L-Tyr by the enzyme were studied. The progress curve of the oxidation of L-Tyr without schisandrin B showed an apparent lag period, which allowed the characteristics of monophenolase activity to be observed (Fig. 3A, curve 1). The curve reached a constant rate (the steady-state rate) after the lag period. The rates of lag time and steady-state were estimated by extrapolating the curve to the abscissa. With an increasing schisandrin B concentration, the kinetic course of the oxidation of L-Tyr was shown in Fig. 3A, curves 2-6. All lag time and the steady-state rates were determined, as

Active Compounds from Schisandra chinensis Exhibiting Tyrosinase Activity and Melanin Content Inhibition in B16 Melanoma…

Fig. 2. Effects of compounds and kojic acid on the activity of mushroom tyrosinase. Data are expressed as a percentage of control which was set at 100%. Data from experimental wells were expressed as percentage of control. Each column represents the mean ± SD of five independent experiments. *P < 0.05 versus control group.

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Fig. 4. Effects of 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%. Data from experimental wells were expressed as percentage of control. Each column represents the mean ± SD of five independent experiments. **P < 0.01, *P < 0.05 versus control group.

but the steady-state rate decreased distinctly in a dosedependent manner. The concentration causing 50% loss of enzyme activity (IC50) was determined to be 0.6 µM.

Fig. 3. Inhibitory effects of schisandrin B on the activity of mushroom tyrosinase. (A) Kinetic curves for the oxidation of LTyr by the enzyme. The concentration of inhibitor for curves 1 ~ 6 was 0, 2.25, 1.5, 1.2, 0.675, and 0.6 µM, respectively. (B) Effects of schisandrin B on the lag time of mushroom tyrosinase. (C) Effects of schisandrin B on the steady-state rates of monophenolase.

shown in Figs. 3B and 3C, respectively. The lag time did not change as the concentration of schisandrin B increased,

3.4. The effects of compounds on cellular tyrosinase activity and melanin content To gain further evidence of compounds involvement in cellular tyrosinase activity and melanogenesis, the effect on cellular tyrosinase activity and melanin content in B16 melanoma cells was tested [23]. Fig. 4 demonstrated that schisandrin B reduced cellular tyrosinase activity in B16 melanoma cells in the absence of α-MSH stimulation. The inhibition was dose-dependent: at 0.75, 1.5, and 3 µM induced significant inhibition on cellular tyrosinase activity by 7.11, 20.52, and 40.01%. Deoxyshisandrin and schisandrin C had a little or no effect on cellular tyrosinase activity. It suggested that they did not showed that exhibiting tyrosinase activity. The effect of compounds on cellular tyrosinase activity in α-MSH-stimulated B16 melanoma cells also was next investigated (Fig. 5). Upon exposure to 0.75 µM α-MSH alone, the cellular tyrosinase activity of B16 melanoma cells was significantly increased, compared to the controls. Schisandrin B significantly reduced the tyrosinase activity of α-MSH-stimulated B16 melanoma cells in a dose-dependent manner, with 10.01% inhibition at 0.75 µM, 20.31% at 1.5 µM and 30.61% at 3 µM. Kojic acid at the same doses reduced tyrosinase activity in a dosedependent manner. While deoxyshisandrin and schisandrin C slightly reduced tyrosinase activity of α-MSH-stimulated

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Fig. 5. Effect of compounds on cellular tyrosinase activity in αMSH-stimulated B16 melanoma cells compared with kojic acid. Baseline cellular tyrosinase activity in control wells not exposed to α-MSH, compounds and kojic acid was set at 100%. Data from experimental wells were expressed as percentage of control. Each column represents the mean ± SD of five independent experiments. ****P < 0.001 versus control group (without α-MSH). ***P < 0.005, **P < 0.01, *P < 0.05 versus α-MSH-treated group.

Biotechnology and Bioprocess Engineering 20: 814-823 (2015)

Fig. 7. Effect of compounds on cellular melanin content in αMSH-stimulated B16 melanoma cells compared with kojic acid. Baseline melanin content in control wells not exposed to α-MSH, compounds and kojic acid was set at 100%. Data from experimental wells were expressed as a percentage of control. Each column represents the mean ± SD of five independent experiments. ***P < 0.005 versus control group (without α-MSH). **P < 0.01, *P < 0.05 versus α-MSH-treated group.

cells. The α-MSH increased the melanin content of B16 melanoma cells alone. schisandrin B and kojic acid also significantly inhibited melanin content in α-MSH-stimulated B16 melanoma cells in a dose-dependent manner, at 0.75, 1.5, and 3 µM induced inhibition by 20.12, 40.15, and 60.24% for schisandrin B and 10.32, 31.64, and 42% for kojic acid, compared to α-MSH-treated group without schisandrin B. Deoxyshisandrin and schisandrin C had a little or no effect on melanin content in α-MSH-stimulated B16 melanoma cells.

Fig. 6. Effects of compounds and kojic acid on cellular melanin content in B16 melanoma cells. Data are expressed as a percentage of control which was set at 100%. Data from experimental wells were expressed as percentage of control. Each column represents the mean ± SD of five independent experiments. **P < 0.01, *P < 0.05 versus control group.

B16 melanoma cells. Fig. 6 showed that schisandrin B reduced cellular melanin content in B16 melanoma cells in the absence of α-MSH stimulation at 0.75, 1.5, and 3 µM induced inhibition by 10.42, 30.05, and 40.14%, respectively. Kojic acid at the same doses also reduced cellular melanin content in a dose-dependent manner. While deoxyshisandrin and schisandrin C slightly reduced cellular melanin content. Fig. 7 showed the similar effect of schisandrin B and kojic acid on melanin content in α-MSH-stimulated B16 melanoma

3.5. Kinetic analysis of tyrosinase activity inhibition by compounds Schisandrin B significantly reduced the tyrosinase activity, deoxyshisandrin and schisandrin C had a slightly or no inhibition effect on the tyrosinase activity. So schisandrin B was next investigated to examine their mechanism of action. We performed an enzyme kinetics study of schisandrin 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 Fig. 8; Schisandrin B acted as a noncompetitive inhibitor with the plots of 1/[v] versus 1/[S] gave a family of straight lines with different slopes, which intersected one another in the X-axis. It decreased the apparent value of Vmax with no effect on Kmax. This indicated that the schisandrin B bound to both free enzyme and enzyme-substrate complex. The inhibition constants for schisandrin B binding with free enzyme, KI, and the enzyme-substrate complex, KIS were the same in value. The inhibition constant was determined

Active Compounds from Schisandra chinensis Exhibiting Tyrosinase Activity and Melanin Content Inhibition in B16 Melanoma…

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Fig. 9. Effects of compounds on cell viability of B16 melanoma cells. Data are expressed as a percentage of control which was set at 100%. Data from experimental wells were expressed as percentage of control. Each column represents the mean ± SD of five independent experiments. ***P < 0.005, **P < 0.01, *P < 0.05 versus control group.

Fig. 8. Lineweaver-Burk plot: (A) for inhibition of schisandrin B on mushroom tyrosinase for the catalysis of L-DOPA at 30°C, pH 6.8. The concentration of schisandrin B for curves 1 ~ 5 was 0, 2.25, 1.2, 0.6, and 0.18 µM, respectively. (B) Secondary plot of the intercept of the straight lines versus concentration of inhibitor, respectively.

from a plot of the vertical intercept (1/Vmax) versus the concentrations of schisandrin B, which was linear as shown in Fig. 5B. The inhibition constant was found to be 2.9 µM. 3.6. Effects compounds on cytotoxicity tests of B16 melanoma cells The effect of compounds on cell viability of B16 melanoma cells was shown in Fig. 9. The results showed that in the cell viability assay, schisandrin B did not have appreciable cytotoxic activity at a dose of 0.75 µM, but reduced viable cells slightly at the higher doses, with 11.01% inhibition at 0.75 µM, 23.31% at 1.5 µM and 31.61% at 3 µM. Deoxyshisandrin, schisandrin C and kojic acid at the same doses similarly cell viability in a dose-dependent manner.

4. Discussion Three compounds had been extracted with solvents of

different polarity and tested for their skin-whitening properties using inhibition of mushroom tyrosinase activity in a cell-free system as screening assays, and then schisandrin B was tested for cellular tyrosinase activity, melanin content, kinetic analysis and cytotoxicity tests in B16 melanoma cells. Our major findings were: schisandrin B showed strongly inhibitory activity but deoxyshisandrin and schisandrin C had no or slight activity on mushroom tyrosinase. Some possible structure-activity relationship could be inferred from tyrosinase inhibitory assay results: (1) Chemical structure of deoxyschisandrin was stable because of symmetric structures and a stable chemical bond (C-O, C-C), it hardly react with other substances. (2) Because of the reaction conditions of the acidity, acidic conditions would hydrolyze the dioxolane moiety. Schisandrin C turned into ionic state (Fig. 10), and then 4 ionic state of schisandrin C was integrated to become a new ring compound with symmetric structures and a stable chemical bond, quickly (Fig. 11). Schisandrin B was able to turn into ionic state, but was not formed new ring compound. Ionic state of schisandrin B might integrate to substrates or tyrosinase, so schisandrin B could inhibited the tyrosinase activity. (3) These results suggested that after functional group (-O-CH2-O-) in the dioxolane moiety was hydrolyzed, it could affect the tyrosinase inhibitory activity, possibly. Schisandrin B inhibited cellular tyrosinase activity as well as melanin content in B16 melanoma cells. The decrease in cellular tyrosinase activity could not be attributed to the smaller number of viable cells present because assays were normalised to use the same quantity of protein from each

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Biotechnology and Bioprocess Engineering 20: 814-823 (2015)

Fig. 10. Formation of the ion state of schisandrin C.

material, we can obtain a large amount of schisandrin B, and that schisandrin B meet two standards for whitening agent: (1) The activity of tyrosinase inhibitory rate was higher, which could significantly reduce the formation of melanin. (2) Safety, non-toxic and non-irritating to human skin.

Acknowledgements This work was supported by grants from the supporting projects of the ministry of science and technology of republic of China (2013BAD16805) and special funds projects of breeding of department of finance of Jilin province(201305).

References

Fig. 11. Forming of new ring compound based on ionic state of schisandrin C.

well. Thus the inhibition of tyrosinase activity was credible [33]. The fact that schisandrin B was also able to inhibit the increase in cellular tyrosinase in α-MSH-stimulated B16 melanoma cells provides further evidence of the direct action of schisandrin B on inhibition of cellular tyrosinase and melanogenesis. However, due to the slightly cytotoxic effects of schisandrin B, they would be used for skin whitening. Content of schisandrin B in S. chinensis is far higher than other plants [34,35]. Using S. chinensis as raw

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