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Functionality study of santalin as tyrosinase inhibitor: A potential depigmentation agent
Accepted Manuscript Title: Functionality study of santalin as tyrosinase inhibitor: a potential depigmentation agent Author: Hridya Hemachandran Anantharaman Amrita Sankari Mohan Mohan Gopalakrishnan Thirumal Kumar Dakshinamurthy George Priya Doss Ramamoorthy Siva PII: DOI: Reference:
S0141-8130(16)30099-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.01.098 BIOMAC 5788
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
30-11-2015 21-1-2016 26-1-2016
Please cite this article as: Hridya Hemachandran, Anantharaman Amrita, Sankari Mohan, Mohan Gopalakrishnan, Thirumal Kumar Dakshinamurthy, George Priya Doss, Ramamoorthy Siva, Functionality study of santalin as tyrosinase inhibitor: a potential depigmentation agent, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.01.098 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Functionality study of santalin as tyrosinase inhibitor: a potential depigmentation agent Hridya Hemachandrana, Anantharaman Amritaa, Sankari Mohana, Mohan Gopalakrishnanb, Thirumal Kumar Dakshinamurthya, George Priya Dossa and Ramamoorthy Sivaa* [email protected] a
School of Bio Sciences and Technology, VIT University, Vellore – 632014, Tamil Nadu, India
b
School of Advanced Sciences, VIT University, Vellore – 632014, Tamil Nadu, India
*
Corresponding author. Tel.: (+91) 9443448905; Fax: (+91) 416 2243092.
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Graphical abstract
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Abstract Excessive melanin production leads to hyperpigmentation disorders which results in distressing aesthetic values. Though there are some synthetic depigmentation agents available it has been reported to possess cytotoxic and mutagenic effects. Hence there is a need for the development of safe and non toxic natural tyrosinase inhibitors. Here we report the role of santalin, the chief constituent of Pterocarpus santalinus in inhibition of tyrosinase and melanin synthesis. Santalin inhibited tyrosinase activity dose dependently. Inhibitory kinetic studies revealed mixed type of inhibition with reversible mechanism. Santalin was found to interact with the fluorophore amino acid residue of tyrosinase. Analysis of circular dichroism spectra showed the binding of santalin to tyrosinase which induced the loss of secondary helical structure. Molecular docking result suggested that santalin interact with the catalytic core of tyrosinase through strong hydrogen and hydrophobic bonding. The results of in vitro studies showed santalin inhibited melanogenesis through down regulation of MITF, tyrosinase, TRP-1 and TRP-2 without any cytotoxic effects towards B16F0 melanoma cells. Therefore, our results suggested that santalin possesses antityrosinase activity, which could be utilized as a safe depigmentation agent in the cosmetic field for the treatment of hyperpigmentation disorder.
Keywords: hyperpigmentation; santalin; tyrosinase
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1. Introduction Melanin, a biopolymer synthesized in melanosomes primarily determines the skin colour [1]. Melanosomes contain three melanogenic specific enzyme such as tyrosinase, tyrosinase related protein 1 (TRP-1) and tyrosinase related protein 2 (TRP-2) also known as dopachrome tautomerase (Dct), which was mainly regulated by microphthalmia-associated transcription factor (MITF) [1-3]. MITF is a critical transcription factor which regulates the expression of tyrosinase, TRP-1 and TRP-2 in melanosomes by binding to the consensus motif of M box [4-7]. Tyrosinase (EC 1.14.18.1) is a copper-containing key enzyme of melanogenesis which catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and further the oxidation of DOPA to DOPA quinone [8-10]. TRP-2 catalyzes the conversion of dopachrome into 5, 6-dihydroxyindole or 5,6-dihydroxyindole-2-carbixylic acid (DHICA). TRP-1 oxidizes 5,6- DHICA into eumelanin which was responsible for the skin pigmentation [11]. Melanin acts as a photo-protectant of skin against harmful effect of UV radiation. However, excessive accumulation of melanin leads to hyperpigmentation disorders such as lentigo, nevus, freckles, age spots, chloasma, melanoma, solar lentigo, ephelides, melasma, and postinflammatory state, which causes serious aesthetic problems [12-17]. Hyperpigmentation disorder was mainly caused by exposing skin to UV light, ageing, drug reaction, pregnancy, endocrine disorders, sex hormones treatment and chronic inflammation [18, 19]. It can be prevented by avoiding skin towards UV exposure or topical application of depigmentation agents [20]. Most of the commercial depigmentation agents target tyrosinase activity such as kojic acid, hydroquinone and retinoids has been reported to possess cytotoxic and mutagenic effects [18, 2123]. Hence, there is a need for the development of safe and non toxic natural tyrosinase inhibitors. 4
In folk medicine, Pterocarpus santalinus was widely used for fair skin complexion [24]. Santalin is a natural red constituent present in the bark of P. santalinus, been used as histological stain. Apart from this, it has been used in the pharmaceutical and food manufacturing products [25-27]. The presence of two hydroxyl group in santalin indicates the involvement towards inhibitory mechanism of tyrosinase activity [28-30]. As not much information is available on the pharmacological studies of santalin, we have explored the role of santalin as a tyrosinase inhibitor and its mode of inhibition by diphenolase activity and kinetic study. Further using fluorescence spectroscopy, circular dichroism and computational docking methods here we report the interaction mechanism of santalin towards tyrosinase. Through in vitro studies, the effect and molecular mechanism of santalin on melanogenesis process was evaluated in B16F0 melanoma cell lines. We believe the results obtained from our study could be used as a template for the discovery of tyrosinase inhibitors in various application fields such as cosmetics, pharmaceutical and food industries. 2. Materials and methods 2.1 Chemicals Tyrosinase was purchased from Sigma (St. Louis, MO, USA), 3, 4-Dihydroxyphenylalanine (LDOPA), dimethyl-sulfoxide (DMSO), Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal bovine serum (FBS), antibiotics (Penicillin and streptomycin), Trypsin-EDTA, 3- (4, 5dimetylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were obtained from Himedia, India. All other reagents used were of analytical grade. 2.2 Isolation of santalin Santalin was isolated from the dried heart wood of P. santalinus as per the protocol of Tennakone et al., [31]. The obtained santalin was characterized by UV-Visible spectroscopy,
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Fourier Transform Infrared (FTIR), and Nuclear Magnetic Resonance (NMR) analysis, which was compared with the published data (Fig. A.1-4) [31]. 2.3 Anti-tyrosinase activity and kinetic analysis Anti-tyrosinase assay was performed as per the modified protocol of Xie et al [32]. Briefly the assay used 3ml of reaction mixture containing 2.8 ml of L-DOPA in 0.1 M phosphate buffer (pH 6.8), 0.1 ml of tyrosinase (700 units/ml) and santalin with varying concentration (5, 10, 25, 50 100 mM) respectively and the absorbance was measured immediately at 475 nm in Systronics AU-2701 UV–Visible double beam spectrophotometer for 5 min. The % of relative enzymatic activity was calculated using below equation. Relative enzymatic activity (%) = [OD1/ OD2] × 100
(1)
Where OD1 is the slope of reaction kinetics equation obtained from reaction with inhibitor; OD2 is the slope of reaction kinetics equation obtained from reaction without the inhibitor The IC50 value of santalin was calculated through linear fitting. The kinetic reaction mixture consisted of different concentrations of L-DOPA (2–10 mM) as a substrate and tyrosinase (700 units/ ml) in 0.1M phosphate buffer (pH 6.8). Different concentrations of santalin (5, 10, 25, 50 mM) were added to the reaction mixture and the change in the absorbance per minute was recorded at 475 nm. For mixed type of inhibition, The Lineweaver burk equation in double reciprocal form can be written as : 1
1
(2)
∝
Secondary plots were constructed from the following equation 6
(3)
∝
(4)
Where v is the reaction velocity; Km is the Michaleis constant; Vm is the maximal velocity; I is the concentration of inhibitor; [S] is the concentration of substrate; Ki is the constant of the inhibitor binding with free enzyme and αKi is the constant of the inhibitor binding to the enzyme substrate complex The Lineweaver-Burk plot was applied to determine the inhibition mode. The inhibition constant (Ki and αKi) was determined by the secondary plots of the apparent slope or intercept versus different concentrations of santalin (equation 2-4). 2.4 Intrinsic fluorescence binding and circular dichroism The intrinsic fluorescence quenching of tyrosinase by santalin was recorded using Hitachi F7000 spectrofluorophotometer. The fluorescence intensities of constant concentration of tyrosinase (0.77 µM) in 0.1 M phosphate buffer (pH 6.8), was recorded initially at excitation wavelength of 290 nm. With the increasing concentrations of santalin (5, 10, 25, 50 and 100 mM) was added and the change in fluorescence intensity was measured. Fluorescence quenching was described by the Stern-Volmer equation [29]. F0/ F= 1 + kq τ0 [Q] = 1 + KSV [Q]
(5)
Where F0 and F are the fluorescence intensities before and after the addition of the quencher, respectively, kq is the bimolecular quenching constant, τ0 is the lifetime of the fluorophore in the absence of the quencher, [Q] is the concentration of the quencher, and KSV is the Stern-Volmer quenching constant. Hence, the above equation is applied to determine the KSV using linear 7
regression of a plot of F0/F against [Q]. A linear Stern-Volmer plot is generally indicative of a single class of fluorophores, all equally accessible to the quencher. The conformational change in the secondary structure of tyrosinase in phosphate buffer pH 6.8 was recorded using CD spectroscopy (Jasco J-715 spectropolarimeter) with wavelength range 200-250 nm at room temperature under constant nitrogen flush. All spectra were accumulated for three times with a bandwidth of 1.0 nm. The CD measurement was recorded for tyrosinase (10 µg/ ml) initially to which increasing concentrations of santalin was added and the spectral changes were monitored. The secondary structural content was analyzed from Jasco Secondary Structure estimation software version 1. 2.5 Molecular docking studies The protein structure of tyrosinase (PDB ID- 2Y9W) was obtained from the protein data bank (http://www.rcsb.org/pdb/). The canonical SMILES of santalin (ligand) were obtained from PubChem database (http://pubchem.ncbi.nlm.nih.gov/). The Canonical SMILES was converted to
3D
structure
using
online
CORINA
server
(https://www.molecular-
networks.com/online_demos/corina_demo). These two structures were energy minimized using swiss PDB Viewer and further subjected to docking studies. ArgusLab 4.0.1 was used for our docking studies [33, 34]. Protein structure was loaded into the ArgusLab 4.0.1 and hydrogen atoms were added to the molecule. The grid box was fixed around the binding sites. Next, the ligand was introduced into the ArgusLab and hydrogen bonds were added to it. The residues of ligand were converted into ligand group and docking was allowed to execute using AScoring function. Flexible docking was performed in order to achieve docking scores from non-rotatable bonds. Torsions were created and obtained poses during every rotation [35]. Ten independent runs were initiated for each complex. The best pose was selected based on the least AScore 8
calculated by ArgusLab. This least energy expresses the highest binding affinity. The repetitive docking (3 trails) was performed in order to find the best binding affinity. 2.6 Cell culture B16F0 melanoma cell line was purchased from National Centre for Cell Science, (NCCS, Pune, India). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum, penicillin and streptomycin (100 units/ml) in culture flasks in a 5% CO2 incubator at 37ᵒC. 2.7 Cell viability assay Cells were seeded at a density of 3x103 cells in each well of a 96 well plate and incubated overnight at 37oC. The cells were treated for 48 h with the different concentrations santalin (5, 10, 25, 50, 100, 200 mM). After 48 h treatment, the cells were treated with 10 µl MTT (5 mg/ml) for 4 h. The formed formazan blue precipitates were dissolved in 100 µl DMSO and the absorbance was measured at 570 nm in a Biotek ELX-800 microplate reader. Values were expressed as percentage of control. 2.8 Determination of cellular tyrosinase activity and melanin content For cellular tyrosinase activity, cells were seeded in a 24 well plate at a density of 5x104 cells per well. Plates were incubated for overnight at 37oC for cell adherence. Cells were then treated with the different concentrations of santalin (5, 10, 25, 50, 100, 200 mM) for 48 h. The treated cells were lysed with ice cold 0.1 M PBS buffer (pH 6.8) containing 1% triton X-100. The lysate was centrifuged at 12,000 rpm for 15 min. The resulting supernatant was assayed for cellular tyrosinase activity and the pellet was assessed for melanin content [36]. 150 µl of the supernatant was added to 150 µl of 2.5 mM L-DOPA and then incubated for 20 min at 37ᵒC. The absorbance was read at 475 nm using a microplate reader (Biotek ELX-800 microplate reader) for 9
dopachrome formation. Melanin content was determined by suspending the pellet obtained with 1 N sodium hydroxide containing 20% DMSO and then incubated for 1 h at 80◦C .The absorbance was read at 490 nm. 2.9 Gene expression studies: 2.9.1 Total RNA isolation and Reverse transcriptase-polymerase chain reaction Effect of santalin on melanogenic specific gene expression (MITF, Tyrosinase, TRP-1 and TRP2) was evaluated. Cells were cultured overnight in 24 well plate at a density of 5 x 104 cells per well. Cells were treated with different concentrations of santalin (50, 100 and 200 mM) for 48 h. Total cellular RNA was isolated using RNA isolation kit (Genei, Merck) according to the manufacturer’s instructions. Total RNA (2 µg/reaction) was converted into cDNA using oligo DT reverse transcriptase primer. The oligonucleotide gene specific primers used for PCR (Eppendorf, Germany), were given in Table 2. The PCR reaction was performed for 30 cycles at 94ᵒC for 30 s, 55ᵒC for 30 s and 72ᵒC for 30 s. The amplified products were subjected to 1% agarose gel containing Ethidium bromide and visualized in Image Quant 300 Gel Imaging System. 3. Results and discussion 3.1 Effect of santalin on tyrosinase activity and kinetics analysis The anti tyrosinase activity of santalin was assessed. (Fig 1) shows the inhibitory effect of santalin towards tyrosinase activity in a dose dependent manner. The IC50 value of santalin was 15.21 ± 0.326 mM respectively. The result shows that santalin has tyrosinase inhibition action in a dose dependent manner. Kojic acid is a well known tyrosinase inhibitor which is commercially used with an IC50 value of 0.014 mM [21]. Kojic acid has been reported for chronic, cytotoxic and mutagenic effect on humans [18]. Even though santalin exhibit less significant anti 10
tyrosinase activity than that of kojic acid, the ability of santalin as a tyrosinase inhibitor is well understood through this study. The kinetic inhibition of tyrosinase by santalin was determined through the Lineweaver burk plot as shown in (Fig 2). With the increasing concentrations of santalin, tyrosinase activity was inhibited in a mixed type manner, which was indicated through change in both Km and Vmax values [37]. It implies that santalin bound to both tyrosinase enzyme and tyrosinase-DOPA complex. Both the secondary replots showed linear fitting. The inhibition constant value Ki and Kα was determined by equation 2-4. The Ki and Kα value was 3.514 mM and 1.24 mM respectively, which indicates that santalin inhibits tyrosinase through reversible mechanism [38]. Moreover, the value of Kα was lower than Ki which indicates that the inhibitor santalin had a stronger binding affinity towards enzyme substrate complex than the free enzyme [39]. Overall the datas obtained fit very well to the equations 2-4 and the predicted (Scheme 1). In summary, inhibition kinetic analysis result showed that santalin inhibited tyrosinase in a mixed type manner through reversible mechanism and also it is evident that santalin binds with the active site of tyrosinase as well as tyrosinase-DOPA complex and inhibit the activity. 3.2 Effect of santalin on tyrosinase conformational changes The binding affinity between protein and ligand was effectively investigated by intrinsic fluorescence quenching studies [40]. Fluorescence emission spectrum of tyrosinase was found to be 340 nm and it was exhibited by the fluorophore amino acid residues such as tyrosine, tryptophan and phenylalanine [29, 38]. Upon increasing concentrations of santalin, the intrinsic fluorescence of tyrosinase was quenched which revealed that santalin interacts with the fluorophore amino acid residues (Fig 3 a). The quenching of intrinsic fluorescence of tyrosinase by santalin shows the interaction with the enzyme [29, 40]. Moreover, the strong binding affinity 11
exists between santalin and tyrosinase through the presence of hydroxyl group in santalin [29, 36]. The binding constant (Ksv) was determined through linear slope of Stern-Volmer plot and found to be 1.27x10-2 mM. These results showed that the quenching of intrinsic fluorescence and change in the band intensity of tyrosinase by santalin exhibited probabilistic interaction of the fluorophore amino acid residues with santalin. The change in the secondary structure of tyrosinase upon interaction with the santalin was investigated by circular dichroism. The tyrosinase exhibits two characteristic negative peak at 209 nm and 222 nm, which indicates the n-π* transfer of peptide bond in the α-helical structure of tyrosinase [38]. As shown in the Fig. 3b decreased peak intensity was observed at 209 nm without any peak shift. The secondary structure content was calculated by Jasco Secondary Structure estimation software version 1 and the content is given in (Table 1). The loss of αhelical structure and breakage of hydrogen bonding network was indicated through decreased band intensity in CD spectrum upon interaction of main polypeptide chain of tyrosinase with ligand (Table 1). Anantharaman et al., 2014 reported that the conformational change of helical structure in tyrosinase is indicated by unfolding and exposure of hydrophobic regions. Thus santalin induce conformational change by the loss of α-helix and bound to the main polypeptide chain of tyrosinase enzyme respectively [38]. 3.3 Molecular docking studies The in silico docking studies confirmed the interaction between tyrosinase and santalin which was shown in Fig 4. The binding energy was -8.322 kcal/mol and this indicates strong interaction of santalin towards the active site residues of tyrosinase. The hydrogen bonding was formed between the hydroxyl groups of santalin with PHE-292, HIS-263 residues of tyrosinase. The
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molecular docking studies were in concordance with the fluorescence quenching study through the binding of PHE-292 residue with santalin. Apart from this, santalin interacts with the active site amino acid residues such as HIS-296, HIS-295, ASN-260, PHE-264, THR-261, HIS-259, MET-257, HIS-61, CYS-83, HIS-85, PHE-90, VAL-248 and HIS-244. Santalin does not involve in the interaction with the copper ion of tyrosinase, which indicates that santalin prevented the substrate entry by binding to the active site. The hydrophobic interaction was exhibited between santalin and tyrosinase, which has affected the conformational flexibility and leads to the inhibition of catalytic activity of tyrosinase [36, 41]. Through our result of in silico docking studies, it’s further proved that santalin inhibited tyrosinase activity through hydrogen bonding and hydrophobic interaction with the active site residues of tyrosinase. 3.4 Cell viability assay The effect of santalin towards the cell viability of B16F0 cell line was assessed by MTT assay. As shown in Fig. 5a, santalin showed no cytotoxic effects towards B16F0 up to the concentration of 200 mM. The viability of cells was maintained above 80% for 48 h without any change in the cell morphology. For development of an effective skin whitening agent it must be devoid of undesirable cytotoxic effects [42]. Thus, the MTT assay results demonstrated that santalin does not affect the viability of B16F0 cell line. 3.5 Effect of santalin on cellular tyrosinase activity and melanin content The cellular tyrosinase activity and melanin synthesis of B16F0 treated cells with different concentrations of santalin was shown in Fig 5b and 5c. Santalin has significantly inhibited tyrosinase activity and melanin synthesis in a dose dependent manner without any change in B16F0 cell viability. These results were in concordance with the kinetic inhibition analysis, CD
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spectrum, intrinsic fluorescence quenching and molecular docking studies, which signifies that santalin act as a potent depigmentation agent. 3.6 Effect of santalin on gene expression of MITF, tyrosinase and tyrosinase related protein The melanogenesis process was mainly regulated by tyrosinase gene family namely MITF, tyrosinase, TRP-1 and TRP-2 [43]. In our present study, santalin has down regulated the mRNA expression level of MITF, tyrosinase and tyrosinase related protein (TRP-1 and TRP-2) in a dose dependent manner as shown in the Fig 5d. In hyperpigmentation disorders, the level of tyrosinase, TRP-1 and TRP-2 were over expressed [1, 43 and 44]. The down-regulation of MITF gene leads to the repression of tyrosinase related genes [45, 46]. These result suggested that santalin inhibited melanin formation by down regulating MITF gene and that subsequently leads to the inhibition of tyrosinase and tyrosinase related protein activity. 4. Conclusion: This study shows the inhibitory mechanism of santalin towards tyrosinase activity and melanogenesis process, which was evaluated through multi-spectroscopic technique, molecular docking and in vitro studies in B16F0 cell lines. Santalin inhibited tyrosinase activity in a dose dependent manner. Kinetic analysis revealed that santalin inhibited tyrosinase by mixed type inhibition with reversible mechanism. Intrinsic fluorescence binding revealed the binding affinity of santalin towards tyrosinase. CD spectrum showed change in secondary structure of tyrosinase upon interaction of santalin. Molecular docking studies demonstrated that santalin interacts towards the active site residue of tyrosinase through hydrophobic and hydrogen bonding In in vitro studies, the inhibition of melanin synthesis in B16F0 melanoma cells by santalin through down-regulation of MITF, tyrosinase, TRP-1 and TRP-2. Our investigation concluded the utilization of santalin as a depigmentation agent, which could contribute for the replacement of 14
toxic tyrosinase inhibitors and also for the development of novel compound. This research work has suggested that santalin may be used in cosmetic product with further in vivo studies. 5. Acknowldegment: The authors desire to express thank to the management of the VIT University for their constant support. We thank the staff at VIT- Sophisticated Instrumentation Facility Laboratory, School of Advanced Sciences, Chemistry Division, for fluorescent spectroscopy. We wish to thank Dr. Asit Ranjan Ghosh, Dr. G. Jayaraman, S. Prathiba, J. Miriam and Dr. V. Pragasam, , School of Bio Sciences and Technology, VIT University for cell culture lab, CD spectrum and microplate reader facility
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Figure Captions
Fig 1. Inhibitory effect of santalin on tyrosinase activity. Data are expressed as Mean ± SD.
23
Fig 2. Inhibition kinetics of santalin: The Lineweaver–Burk plot exhibited the inhibition kinetics of different concentration of santalin 0 (■), 5 (●), 10 (▲), 25 (♦) and 50 mM (◄) on tyrosinase enzyme. Inset secondary replot of slope versus different concentrations of santalin and Y intercept versus different concentrations of santalin. Data are expressed as Mean ± SD.
24
Fig 3. (a) Spectral changes on intrinsic fluorescence of tyrosinase upon increasing concentration of santalin. Inset plot of (I0/I) versus different concentrations of santalin, where I0 is the fluorescence intensity of tyrosinase and I is the fluorescence intensity of tyrosinase with santalin. Santalin concentration was 10-120 mM. (b) Circular dichroism spectral changes of secondary structure tyrosinase upon santalin interaction.
25
Fig 4. Molecular docking studies of santalin interacting with tyrosinase.
26
Fig 5. Effect of santalin on B16F0 melanoma cell lines for 48 h on (a) cell viability (b) cellular tyrosinase and (c) melanin content. Data are expressed as Mean ± SD. (d) RT-PCR B16F0 cells were treated with santalin in 50, 100 and 200 mM for 48 hours. mRNA expression of melanogenic genes namely MITF, tyrosinase, TRP-1, and TRP-2. GAPDH was used as internal control.
27
E+S + I Ki
EI + S
Km
ES + I
E+P
αKi
EIS
EI + P
Scheme 1: Santalin interacts with enzyme and enzyme substrate complex. E - enzyme tyrosinase; S - substrate L-DOPA; I - inhibitor santalin; P- product dopachrome; Ki -inhibitor dissociation constant; α - modifying factor
28
Tables Table 1: CD spectral secondary structural changes of tyrosinase with increase concentration of santalin at pH 6.8 Santalin (mM) 0 5 10 25 50 100
α-Helix (%) 28.5 28.1 27.5 26.8 26.3 25.4
β- Sheet (%) 16.9 20.4 19.3 17.3 15.4 8.7
29
β-Turn (%) 20.5 18.7 19.5 21.5 21.1 24.8
Random coil (%) 34.0 32.8 33.7 34.4 37.2 41.0
Table 2: Primers used for melanogenic gene expression study Primers MITF Tyrosinase TRP-1 TRP-2 GAPDH
Forward Sequence 5’-AGTACAGGAGCTGGAGATG-3’ 5’-GGCCAGCTTTCAGGCAGAGGT-3’ 5’-GGCCTCTGAGGTTCTTTAAT-3’ 5’-ATGAGAAACTGCCAACCTTA-3’ 5’-GTGAAGGTCGGTGTGAACG-3’
30
Reverse Sequence 5’-GTGAGATCCAGAGTTGTC-3’ 5’-TGGTGCTTCATGGGCAAA-3’ 5’-AATGACAAATTGAGGGTGAG-3’ 5’-AGGAGTGAGGCCAAGTTATGA-3’ 5’-CTCGCTCCTGGAAGATGGTG-3’
S0141-8130(16)30099-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.01.098 BIOMAC 5788
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
30-11-2015 21-1-2016 26-1-2016
Please cite this article as: Hridya Hemachandran, Anantharaman Amrita, Sankari Mohan, Mohan Gopalakrishnan, Thirumal Kumar Dakshinamurthy, George Priya Doss, Ramamoorthy Siva, Functionality study of santalin as tyrosinase inhibitor: a potential depigmentation agent, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.01.098 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Functionality study of santalin as tyrosinase inhibitor: a potential depigmentation agent Hridya Hemachandrana, Anantharaman Amritaa, Sankari Mohana, Mohan Gopalakrishnanb, Thirumal Kumar Dakshinamurthya, George Priya Dossa and Ramamoorthy Sivaa* [email protected] a
School of Bio Sciences and Technology, VIT University, Vellore – 632014, Tamil Nadu, India
b
School of Advanced Sciences, VIT University, Vellore – 632014, Tamil Nadu, India
*
Corresponding author. Tel.: (+91) 9443448905; Fax: (+91) 416 2243092.
1
Graphical abstract
2
Abstract Excessive melanin production leads to hyperpigmentation disorders which results in distressing aesthetic values. Though there are some synthetic depigmentation agents available it has been reported to possess cytotoxic and mutagenic effects. Hence there is a need for the development of safe and non toxic natural tyrosinase inhibitors. Here we report the role of santalin, the chief constituent of Pterocarpus santalinus in inhibition of tyrosinase and melanin synthesis. Santalin inhibited tyrosinase activity dose dependently. Inhibitory kinetic studies revealed mixed type of inhibition with reversible mechanism. Santalin was found to interact with the fluorophore amino acid residue of tyrosinase. Analysis of circular dichroism spectra showed the binding of santalin to tyrosinase which induced the loss of secondary helical structure. Molecular docking result suggested that santalin interact with the catalytic core of tyrosinase through strong hydrogen and hydrophobic bonding. The results of in vitro studies showed santalin inhibited melanogenesis through down regulation of MITF, tyrosinase, TRP-1 and TRP-2 without any cytotoxic effects towards B16F0 melanoma cells. Therefore, our results suggested that santalin possesses antityrosinase activity, which could be utilized as a safe depigmentation agent in the cosmetic field for the treatment of hyperpigmentation disorder.
Keywords: hyperpigmentation; santalin; tyrosinase
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1. Introduction Melanin, a biopolymer synthesized in melanosomes primarily determines the skin colour [1]. Melanosomes contain three melanogenic specific enzyme such as tyrosinase, tyrosinase related protein 1 (TRP-1) and tyrosinase related protein 2 (TRP-2) also known as dopachrome tautomerase (Dct), which was mainly regulated by microphthalmia-associated transcription factor (MITF) [1-3]. MITF is a critical transcription factor which regulates the expression of tyrosinase, TRP-1 and TRP-2 in melanosomes by binding to the consensus motif of M box [4-7]. Tyrosinase (EC 1.14.18.1) is a copper-containing key enzyme of melanogenesis which catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and further the oxidation of DOPA to DOPA quinone [8-10]. TRP-2 catalyzes the conversion of dopachrome into 5, 6-dihydroxyindole or 5,6-dihydroxyindole-2-carbixylic acid (DHICA). TRP-1 oxidizes 5,6- DHICA into eumelanin which was responsible for the skin pigmentation [11]. Melanin acts as a photo-protectant of skin against harmful effect of UV radiation. However, excessive accumulation of melanin leads to hyperpigmentation disorders such as lentigo, nevus, freckles, age spots, chloasma, melanoma, solar lentigo, ephelides, melasma, and postinflammatory state, which causes serious aesthetic problems [12-17]. Hyperpigmentation disorder was mainly caused by exposing skin to UV light, ageing, drug reaction, pregnancy, endocrine disorders, sex hormones treatment and chronic inflammation [18, 19]. It can be prevented by avoiding skin towards UV exposure or topical application of depigmentation agents [20]. Most of the commercial depigmentation agents target tyrosinase activity such as kojic acid, hydroquinone and retinoids has been reported to possess cytotoxic and mutagenic effects [18, 2123]. Hence, there is a need for the development of safe and non toxic natural tyrosinase inhibitors. 4
In folk medicine, Pterocarpus santalinus was widely used for fair skin complexion [24]. Santalin is a natural red constituent present in the bark of P. santalinus, been used as histological stain. Apart from this, it has been used in the pharmaceutical and food manufacturing products [25-27]. The presence of two hydroxyl group in santalin indicates the involvement towards inhibitory mechanism of tyrosinase activity [28-30]. As not much information is available on the pharmacological studies of santalin, we have explored the role of santalin as a tyrosinase inhibitor and its mode of inhibition by diphenolase activity and kinetic study. Further using fluorescence spectroscopy, circular dichroism and computational docking methods here we report the interaction mechanism of santalin towards tyrosinase. Through in vitro studies, the effect and molecular mechanism of santalin on melanogenesis process was evaluated in B16F0 melanoma cell lines. We believe the results obtained from our study could be used as a template for the discovery of tyrosinase inhibitors in various application fields such as cosmetics, pharmaceutical and food industries. 2. Materials and methods 2.1 Chemicals Tyrosinase was purchased from Sigma (St. Louis, MO, USA), 3, 4-Dihydroxyphenylalanine (LDOPA), dimethyl-sulfoxide (DMSO), Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal bovine serum (FBS), antibiotics (Penicillin and streptomycin), Trypsin-EDTA, 3- (4, 5dimetylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were obtained from Himedia, India. All other reagents used were of analytical grade. 2.2 Isolation of santalin Santalin was isolated from the dried heart wood of P. santalinus as per the protocol of Tennakone et al., [31]. The obtained santalin was characterized by UV-Visible spectroscopy,
5
Fourier Transform Infrared (FTIR), and Nuclear Magnetic Resonance (NMR) analysis, which was compared with the published data (Fig. A.1-4) [31]. 2.3 Anti-tyrosinase activity and kinetic analysis Anti-tyrosinase assay was performed as per the modified protocol of Xie et al [32]. Briefly the assay used 3ml of reaction mixture containing 2.8 ml of L-DOPA in 0.1 M phosphate buffer (pH 6.8), 0.1 ml of tyrosinase (700 units/ml) and santalin with varying concentration (5, 10, 25, 50 100 mM) respectively and the absorbance was measured immediately at 475 nm in Systronics AU-2701 UV–Visible double beam spectrophotometer for 5 min. The % of relative enzymatic activity was calculated using below equation. Relative enzymatic activity (%) = [OD1/ OD2] × 100
(1)
Where OD1 is the slope of reaction kinetics equation obtained from reaction with inhibitor; OD2 is the slope of reaction kinetics equation obtained from reaction without the inhibitor The IC50 value of santalin was calculated through linear fitting. The kinetic reaction mixture consisted of different concentrations of L-DOPA (2–10 mM) as a substrate and tyrosinase (700 units/ ml) in 0.1M phosphate buffer (pH 6.8). Different concentrations of santalin (5, 10, 25, 50 mM) were added to the reaction mixture and the change in the absorbance per minute was recorded at 475 nm. For mixed type of inhibition, The Lineweaver burk equation in double reciprocal form can be written as : 1
1
(2)
∝
Secondary plots were constructed from the following equation 6
(3)
∝
(4)
Where v is the reaction velocity; Km is the Michaleis constant; Vm is the maximal velocity; I is the concentration of inhibitor; [S] is the concentration of substrate; Ki is the constant of the inhibitor binding with free enzyme and αKi is the constant of the inhibitor binding to the enzyme substrate complex The Lineweaver-Burk plot was applied to determine the inhibition mode. The inhibition constant (Ki and αKi) was determined by the secondary plots of the apparent slope or intercept versus different concentrations of santalin (equation 2-4). 2.4 Intrinsic fluorescence binding and circular dichroism The intrinsic fluorescence quenching of tyrosinase by santalin was recorded using Hitachi F7000 spectrofluorophotometer. The fluorescence intensities of constant concentration of tyrosinase (0.77 µM) in 0.1 M phosphate buffer (pH 6.8), was recorded initially at excitation wavelength of 290 nm. With the increasing concentrations of santalin (5, 10, 25, 50 and 100 mM) was added and the change in fluorescence intensity was measured. Fluorescence quenching was described by the Stern-Volmer equation [29]. F0/ F= 1 + kq τ0 [Q] = 1 + KSV [Q]
(5)
Where F0 and F are the fluorescence intensities before and after the addition of the quencher, respectively, kq is the bimolecular quenching constant, τ0 is the lifetime of the fluorophore in the absence of the quencher, [Q] is the concentration of the quencher, and KSV is the Stern-Volmer quenching constant. Hence, the above equation is applied to determine the KSV using linear 7
regression of a plot of F0/F against [Q]. A linear Stern-Volmer plot is generally indicative of a single class of fluorophores, all equally accessible to the quencher. The conformational change in the secondary structure of tyrosinase in phosphate buffer pH 6.8 was recorded using CD spectroscopy (Jasco J-715 spectropolarimeter) with wavelength range 200-250 nm at room temperature under constant nitrogen flush. All spectra were accumulated for three times with a bandwidth of 1.0 nm. The CD measurement was recorded for tyrosinase (10 µg/ ml) initially to which increasing concentrations of santalin was added and the spectral changes were monitored. The secondary structural content was analyzed from Jasco Secondary Structure estimation software version 1. 2.5 Molecular docking studies The protein structure of tyrosinase (PDB ID- 2Y9W) was obtained from the protein data bank (http://www.rcsb.org/pdb/). The canonical SMILES of santalin (ligand) were obtained from PubChem database (http://pubchem.ncbi.nlm.nih.gov/). The Canonical SMILES was converted to
3D
structure
using
online
CORINA
server
(https://www.molecular-
networks.com/online_demos/corina_demo). These two structures were energy minimized using swiss PDB Viewer and further subjected to docking studies. ArgusLab 4.0.1 was used for our docking studies [33, 34]. Protein structure was loaded into the ArgusLab 4.0.1 and hydrogen atoms were added to the molecule. The grid box was fixed around the binding sites. Next, the ligand was introduced into the ArgusLab and hydrogen bonds were added to it. The residues of ligand were converted into ligand group and docking was allowed to execute using AScoring function. Flexible docking was performed in order to achieve docking scores from non-rotatable bonds. Torsions were created and obtained poses during every rotation [35]. Ten independent runs were initiated for each complex. The best pose was selected based on the least AScore 8
calculated by ArgusLab. This least energy expresses the highest binding affinity. The repetitive docking (3 trails) was performed in order to find the best binding affinity. 2.6 Cell culture B16F0 melanoma cell line was purchased from National Centre for Cell Science, (NCCS, Pune, India). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum, penicillin and streptomycin (100 units/ml) in culture flasks in a 5% CO2 incubator at 37ᵒC. 2.7 Cell viability assay Cells were seeded at a density of 3x103 cells in each well of a 96 well plate and incubated overnight at 37oC. The cells were treated for 48 h with the different concentrations santalin (5, 10, 25, 50, 100, 200 mM). After 48 h treatment, the cells were treated with 10 µl MTT (5 mg/ml) for 4 h. The formed formazan blue precipitates were dissolved in 100 µl DMSO and the absorbance was measured at 570 nm in a Biotek ELX-800 microplate reader. Values were expressed as percentage of control. 2.8 Determination of cellular tyrosinase activity and melanin content For cellular tyrosinase activity, cells were seeded in a 24 well plate at a density of 5x104 cells per well. Plates were incubated for overnight at 37oC for cell adherence. Cells were then treated with the different concentrations of santalin (5, 10, 25, 50, 100, 200 mM) for 48 h. The treated cells were lysed with ice cold 0.1 M PBS buffer (pH 6.8) containing 1% triton X-100. The lysate was centrifuged at 12,000 rpm for 15 min. The resulting supernatant was assayed for cellular tyrosinase activity and the pellet was assessed for melanin content [36]. 150 µl of the supernatant was added to 150 µl of 2.5 mM L-DOPA and then incubated for 20 min at 37ᵒC. The absorbance was read at 475 nm using a microplate reader (Biotek ELX-800 microplate reader) for 9
dopachrome formation. Melanin content was determined by suspending the pellet obtained with 1 N sodium hydroxide containing 20% DMSO and then incubated for 1 h at 80◦C .The absorbance was read at 490 nm. 2.9 Gene expression studies: 2.9.1 Total RNA isolation and Reverse transcriptase-polymerase chain reaction Effect of santalin on melanogenic specific gene expression (MITF, Tyrosinase, TRP-1 and TRP2) was evaluated. Cells were cultured overnight in 24 well plate at a density of 5 x 104 cells per well. Cells were treated with different concentrations of santalin (50, 100 and 200 mM) for 48 h. Total cellular RNA was isolated using RNA isolation kit (Genei, Merck) according to the manufacturer’s instructions. Total RNA (2 µg/reaction) was converted into cDNA using oligo DT reverse transcriptase primer. The oligonucleotide gene specific primers used for PCR (Eppendorf, Germany), were given in Table 2. The PCR reaction was performed for 30 cycles at 94ᵒC for 30 s, 55ᵒC for 30 s and 72ᵒC for 30 s. The amplified products were subjected to 1% agarose gel containing Ethidium bromide and visualized in Image Quant 300 Gel Imaging System. 3. Results and discussion 3.1 Effect of santalin on tyrosinase activity and kinetics analysis The anti tyrosinase activity of santalin was assessed. (Fig 1) shows the inhibitory effect of santalin towards tyrosinase activity in a dose dependent manner. The IC50 value of santalin was 15.21 ± 0.326 mM respectively. The result shows that santalin has tyrosinase inhibition action in a dose dependent manner. Kojic acid is a well known tyrosinase inhibitor which is commercially used with an IC50 value of 0.014 mM [21]. Kojic acid has been reported for chronic, cytotoxic and mutagenic effect on humans [18]. Even though santalin exhibit less significant anti 10
tyrosinase activity than that of kojic acid, the ability of santalin as a tyrosinase inhibitor is well understood through this study. The kinetic inhibition of tyrosinase by santalin was determined through the Lineweaver burk plot as shown in (Fig 2). With the increasing concentrations of santalin, tyrosinase activity was inhibited in a mixed type manner, which was indicated through change in both Km and Vmax values [37]. It implies that santalin bound to both tyrosinase enzyme and tyrosinase-DOPA complex. Both the secondary replots showed linear fitting. The inhibition constant value Ki and Kα was determined by equation 2-4. The Ki and Kα value was 3.514 mM and 1.24 mM respectively, which indicates that santalin inhibits tyrosinase through reversible mechanism [38]. Moreover, the value of Kα was lower than Ki which indicates that the inhibitor santalin had a stronger binding affinity towards enzyme substrate complex than the free enzyme [39]. Overall the datas obtained fit very well to the equations 2-4 and the predicted (Scheme 1). In summary, inhibition kinetic analysis result showed that santalin inhibited tyrosinase in a mixed type manner through reversible mechanism and also it is evident that santalin binds with the active site of tyrosinase as well as tyrosinase-DOPA complex and inhibit the activity. 3.2 Effect of santalin on tyrosinase conformational changes The binding affinity between protein and ligand was effectively investigated by intrinsic fluorescence quenching studies [40]. Fluorescence emission spectrum of tyrosinase was found to be 340 nm and it was exhibited by the fluorophore amino acid residues such as tyrosine, tryptophan and phenylalanine [29, 38]. Upon increasing concentrations of santalin, the intrinsic fluorescence of tyrosinase was quenched which revealed that santalin interacts with the fluorophore amino acid residues (Fig 3 a). The quenching of intrinsic fluorescence of tyrosinase by santalin shows the interaction with the enzyme [29, 40]. Moreover, the strong binding affinity 11
exists between santalin and tyrosinase through the presence of hydroxyl group in santalin [29, 36]. The binding constant (Ksv) was determined through linear slope of Stern-Volmer plot and found to be 1.27x10-2 mM. These results showed that the quenching of intrinsic fluorescence and change in the band intensity of tyrosinase by santalin exhibited probabilistic interaction of the fluorophore amino acid residues with santalin. The change in the secondary structure of tyrosinase upon interaction with the santalin was investigated by circular dichroism. The tyrosinase exhibits two characteristic negative peak at 209 nm and 222 nm, which indicates the n-π* transfer of peptide bond in the α-helical structure of tyrosinase [38]. As shown in the Fig. 3b decreased peak intensity was observed at 209 nm without any peak shift. The secondary structure content was calculated by Jasco Secondary Structure estimation software version 1 and the content is given in (Table 1). The loss of αhelical structure and breakage of hydrogen bonding network was indicated through decreased band intensity in CD spectrum upon interaction of main polypeptide chain of tyrosinase with ligand (Table 1). Anantharaman et al., 2014 reported that the conformational change of helical structure in tyrosinase is indicated by unfolding and exposure of hydrophobic regions. Thus santalin induce conformational change by the loss of α-helix and bound to the main polypeptide chain of tyrosinase enzyme respectively [38]. 3.3 Molecular docking studies The in silico docking studies confirmed the interaction between tyrosinase and santalin which was shown in Fig 4. The binding energy was -8.322 kcal/mol and this indicates strong interaction of santalin towards the active site residues of tyrosinase. The hydrogen bonding was formed between the hydroxyl groups of santalin with PHE-292, HIS-263 residues of tyrosinase. The
12
molecular docking studies were in concordance with the fluorescence quenching study through the binding of PHE-292 residue with santalin. Apart from this, santalin interacts with the active site amino acid residues such as HIS-296, HIS-295, ASN-260, PHE-264, THR-261, HIS-259, MET-257, HIS-61, CYS-83, HIS-85, PHE-90, VAL-248 and HIS-244. Santalin does not involve in the interaction with the copper ion of tyrosinase, which indicates that santalin prevented the substrate entry by binding to the active site. The hydrophobic interaction was exhibited between santalin and tyrosinase, which has affected the conformational flexibility and leads to the inhibition of catalytic activity of tyrosinase [36, 41]. Through our result of in silico docking studies, it’s further proved that santalin inhibited tyrosinase activity through hydrogen bonding and hydrophobic interaction with the active site residues of tyrosinase. 3.4 Cell viability assay The effect of santalin towards the cell viability of B16F0 cell line was assessed by MTT assay. As shown in Fig. 5a, santalin showed no cytotoxic effects towards B16F0 up to the concentration of 200 mM. The viability of cells was maintained above 80% for 48 h without any change in the cell morphology. For development of an effective skin whitening agent it must be devoid of undesirable cytotoxic effects [42]. Thus, the MTT assay results demonstrated that santalin does not affect the viability of B16F0 cell line. 3.5 Effect of santalin on cellular tyrosinase activity and melanin content The cellular tyrosinase activity and melanin synthesis of B16F0 treated cells with different concentrations of santalin was shown in Fig 5b and 5c. Santalin has significantly inhibited tyrosinase activity and melanin synthesis in a dose dependent manner without any change in B16F0 cell viability. These results were in concordance with the kinetic inhibition analysis, CD
13
spectrum, intrinsic fluorescence quenching and molecular docking studies, which signifies that santalin act as a potent depigmentation agent. 3.6 Effect of santalin on gene expression of MITF, tyrosinase and tyrosinase related protein The melanogenesis process was mainly regulated by tyrosinase gene family namely MITF, tyrosinase, TRP-1 and TRP-2 [43]. In our present study, santalin has down regulated the mRNA expression level of MITF, tyrosinase and tyrosinase related protein (TRP-1 and TRP-2) in a dose dependent manner as shown in the Fig 5d. In hyperpigmentation disorders, the level of tyrosinase, TRP-1 and TRP-2 were over expressed [1, 43 and 44]. The down-regulation of MITF gene leads to the repression of tyrosinase related genes [45, 46]. These result suggested that santalin inhibited melanin formation by down regulating MITF gene and that subsequently leads to the inhibition of tyrosinase and tyrosinase related protein activity. 4. Conclusion: This study shows the inhibitory mechanism of santalin towards tyrosinase activity and melanogenesis process, which was evaluated through multi-spectroscopic technique, molecular docking and in vitro studies in B16F0 cell lines. Santalin inhibited tyrosinase activity in a dose dependent manner. Kinetic analysis revealed that santalin inhibited tyrosinase by mixed type inhibition with reversible mechanism. Intrinsic fluorescence binding revealed the binding affinity of santalin towards tyrosinase. CD spectrum showed change in secondary structure of tyrosinase upon interaction of santalin. Molecular docking studies demonstrated that santalin interacts towards the active site residue of tyrosinase through hydrophobic and hydrogen bonding In in vitro studies, the inhibition of melanin synthesis in B16F0 melanoma cells by santalin through down-regulation of MITF, tyrosinase, TRP-1 and TRP-2. Our investigation concluded the utilization of santalin as a depigmentation agent, which could contribute for the replacement of 14
toxic tyrosinase inhibitors and also for the development of novel compound. This research work has suggested that santalin may be used in cosmetic product with further in vivo studies. 5. Acknowldegment: The authors desire to express thank to the management of the VIT University for their constant support. We thank the staff at VIT- Sophisticated Instrumentation Facility Laboratory, School of Advanced Sciences, Chemistry Division, for fluorescent spectroscopy. We wish to thank Dr. Asit Ranjan Ghosh, Dr. G. Jayaraman, S. Prathiba, J. Miriam and Dr. V. Pragasam, , School of Bio Sciences and Technology, VIT University for cell culture lab, CD spectrum and microplate reader facility
15
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Figure Captions
Fig 1. Inhibitory effect of santalin on tyrosinase activity. Data are expressed as Mean ± SD.
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Fig 2. Inhibition kinetics of santalin: The Lineweaver–Burk plot exhibited the inhibition kinetics of different concentration of santalin 0 (■), 5 (●), 10 (▲), 25 (♦) and 50 mM (◄) on tyrosinase enzyme. Inset secondary replot of slope versus different concentrations of santalin and Y intercept versus different concentrations of santalin. Data are expressed as Mean ± SD.
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Fig 3. (a) Spectral changes on intrinsic fluorescence of tyrosinase upon increasing concentration of santalin. Inset plot of (I0/I) versus different concentrations of santalin, where I0 is the fluorescence intensity of tyrosinase and I is the fluorescence intensity of tyrosinase with santalin. Santalin concentration was 10-120 mM. (b) Circular dichroism spectral changes of secondary structure tyrosinase upon santalin interaction.
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Fig 4. Molecular docking studies of santalin interacting with tyrosinase.
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Fig 5. Effect of santalin on B16F0 melanoma cell lines for 48 h on (a) cell viability (b) cellular tyrosinase and (c) melanin content. Data are expressed as Mean ± SD. (d) RT-PCR B16F0 cells were treated with santalin in 50, 100 and 200 mM for 48 hours. mRNA expression of melanogenic genes namely MITF, tyrosinase, TRP-1, and TRP-2. GAPDH was used as internal control.
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E+S + I Ki
EI + S
Km
ES + I
E+P
αKi
EIS
EI + P
Scheme 1: Santalin interacts with enzyme and enzyme substrate complex. E - enzyme tyrosinase; S - substrate L-DOPA; I - inhibitor santalin; P- product dopachrome; Ki -inhibitor dissociation constant; α - modifying factor
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Tables Table 1: CD spectral secondary structural changes of tyrosinase with increase concentration of santalin at pH 6.8 Santalin (mM) 0 5 10 25 50 100
α-Helix (%) 28.5 28.1 27.5 26.8 26.3 25.4
β- Sheet (%) 16.9 20.4 19.3 17.3 15.4 8.7
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β-Turn (%) 20.5 18.7 19.5 21.5 21.1 24.8
Random coil (%) 34.0 32.8 33.7 34.4 37.2 41.0
Table 2: Primers used for melanogenic gene expression study Primers MITF Tyrosinase TRP-1 TRP-2 GAPDH
Forward Sequence 5’-AGTACAGGAGCTGGAGATG-3’ 5’-GGCCAGCTTTCAGGCAGAGGT-3’ 5’-GGCCTCTGAGGTTCTTTAAT-3’ 5’-ATGAGAAACTGCCAACCTTA-3’ 5’-GTGAAGGTCGGTGTGAACG-3’
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Reverse Sequence 5’-GTGAGATCCAGAGTTGTC-3’ 5’-TGGTGCTTCATGGGCAAA-3’ 5’-AATGACAAATTGAGGGTGAG-3’ 5’-AGGAGTGAGGCCAAGTTATGA-3’ 5’-CTCGCTCCTGGAAGATGGTG-3’