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JFBC12728

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J Food Biochem. 2018;e12728. wileyonlinelibrary.com/journal/jfbc | 1 of 9https://doi.org/10.1111/jfbc.12728

© 2018 Wiley Periodicals, Inc.

Received:19July2018  |  Revised:13October2018  |  Accepted:24October2018DOI:10.1111/jfbc.12728

F U L L A R T I C L E

Effect of chrysin omega-3 and 6 fatty acid esters on mushroom tyrosinase activity, stability, and structure

Zohreh Jamali1 | Gholam Reza Rezaei Behbahani2 | Karim Zare1 | Nematollah Gheibi3

1Department of Chemistry, Science and Research Branch, Islamic Azad University, Tehran, Iran2Department of Chemistry, Imam Khomeini International University, Qazvin, Iran3Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

CorrespondenceNematollah Gheibi, Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran.Email: gheibi_n@yahoo.com

Funding informationQazvin University of Medical Sciences; Islamic Azad University

AbstractThe estreification of chrysin with α‐Linolenic acid (complex I) and linoleic acid (com‐plex II) poly unsaturated fatty acids resulted to design of new mushroom tyrosinase (MT) inhibitors. Thermodynamic parameters of enzymes, including the melting point (Tm)and∆G values, were obtained from thermal and chemical denaturation curves. Complexes I and II showed a competitive inhibitory effect on MT with Ki values of 0.45and0.29mM,respectively.TheTmvalueswerecalculatedas328.6,322.4,and318Kandthe∆Gvaluesas62.8,52.9,and47.1KJmol−1for the enzyme alone and its interaction with complexes I and II, respectively. Intrinsic and extrinsic fluorescence techniques showed structural instability of the enzyme in concomitance with a de‐crease in the regular secondary structure acquired using CD spectrometry. This data clearly prove that the new derivatives show a stronger inhibitory effect than the separate compounds. Molecular docking analysis showed that the best possible in‐teractionconditionwasachievedforchrysinwithn‐6.

Practical applicationsMT is a suitable model in medicine for the investigation of melanogenesis, skin disor‐ders, and hyperpigmentation because of its accessibility and close structural similar‐ity to mammalian tyrosinase. In recent years, the designing of tyrosinase inhibitors from natural substances for prevention of hyperpigmentation in medicine, skin cos‐metics, and undesired browning in agriculture and food industry has risen sharply. Many of the pharmaceutical products based on the use of flavonoids and poly un‐saturated acids as natural compounds or on their semi‐synthetic derivatives have been interested for investigations because of their usefulness in many pathological conditions such as inflammation, cancer, and skin disorders. The limitation of the flavonoids applications are low bioavailability, permeability, and solubility for the cells.Inthisstudy,conjugationofchrysinwithn‐3andn‐6fattyacidsresultedinastronger inhibitors of MT with a synergic inhibitory effect on its activity.

K E Y W O R D S

chrysin, fatty acid, inhibition, molecular docking, mushroom tyrosinase

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1  | INTRODUC TION

Despitetheextensiveresearchontyrosinase(E.C.1.14.18.1),awide‐spread enzyme with high functional capabilities, important issues re‐maintobeaddressed(Eisnhofeetal.,2003;StrackandSchliemann,2001). The first stage of melanogenesis is L‐tyrosine hydroxylated to L‐DOPA. This primary step inmelanin synthesis is followed byoxidation of the catecholamine to O‐quinine (Cooksey, Garratt,Land, & Ramsden, 1998). Irregular tyrosinase activity results in med‐ical disorders such as melanoma, skin hyperpigmentation, albinism, piebaldism, and unwanted browning of fruit, vegetables, beverages, and seafood products along with several other important biological phenomena(Lozano,2006).

Melanoma is a prevalent and lethal cancer. Reduction of tyrosi‐nase activity has been linked to the development of skin abnormali‐ties and targeting this enzyme could be beneficial for the prevention andtreatmentofmelanoma(Funayamaetal.,1995).Mushroomty‐rosinase (MT) from Agaricus bisporus is a good model for the design of inhibitors, structural analysis and other physicochemical studies on this keyenzyme (Gheibi et al.,2005;Haet al.,2011).Becauseof its accessibility and close structural similarity to mammalian ty‐rosinase, it is a suitable model for these investigations (Gheibi, Taherkhani,Ahmadi,Haghbeen,&Ilghari,2015).Ithasbroadappli‐cations in cosmetics, medicine, agriculture, and the food industry, which makes tyrosinase inhibition useful for better understanding of its mechanism of action (Cabanes, Chazarra, & Garic‐Carmona, 1994).Tyrosinase inhibitorssuchaskojicacid,hydroquinone,elec‐tron‐rich phenols, arbutin, and azelaic acid have been evaluated for their capacity to prevent overproduction of melanin (Chang et al., 2007;Haetal.,2007).

Unsaturated fatty acids have anti‐cancer effects in the human body through their anti‐oxidant activity (Arendse, 2014; Asghari,Chegini,Amini,&Gheibi,2016;Zajdel,Wilczok,&Tarkowski,2015).They are divided into two groups according to the number of double bonds as polyunsaturated fatty acids (PUFAs) and monounsaturated fatty acids (Das, 2006; Hussain, Schmitt, Loeffler, &Gonzalez deAguilar,2013).Bothgroupshavecrucialfeaturesthatpreventmalig‐nancy(Girao,Ruck,Cantrill,&Davidson,1985;Ljungblad,Johnsen,Wickström,Kogner,&Gleissman,2015).

PUFAscomprisethen‐3andn‐6familiesandcanconveyanti‐canceractivityinvitroandinvivo(Arendse,2014;Das,1991;Zajdeletal.,2015).Researchhasrevealedthatnaturalflavonoidssuchas5,7‐dihydroxy flavone (chrysin),acopper‐chelating inhibitor,caninhibit tyrosinase much more effectively in a reversible manner (Rho et al., 2010). Other flavonoids (cupferron, hexylresorcinol,dodecylresorcinol, and alkylbenzaldehydes) have been reported to competitivelyinhibitL‐DOPAoxidationbyMT(Qiu,Chen,Wang,Huang,&Song,2005).Studiesonthesetwoimportantbiologicalcompounds have suggested the conjugation of both compounds in new design complexes and their assessment on MT. In the present study, the inhibitoryeffectsof complexes I (chrysin+n‐3) and II(chrysin+n‐6)on thekinetics, structureandstabilityofMTwasevaluated to obtain a better understanding of the enzymatic mech‐anism of action. The results clearly demonstrate that the tested complexes can inhibit the diphenolase activity of MT.

2  | MATERIAL S AND METHODS

2.1 | Materials

A. bisporus tyrosinase(specificactivity3,600units/mg),L‐DOPA,chry‐sin, isopropanol, PUFAs (α‐linolenic acid (ALA) and linoleic acid (LA))

and 1‐anilino‐8‐naphthalene sulfonate (ANS) were purchased from Sigma‐Aldrich (USA). Isopropanol was used to make all stock solutions for the substrates and inhibitors. Biological complexes I and II were synthetized using the Fisher method with esterification of chrysin as the flavonoid and LA and ALA as the PUFAs, respectively (Figure 1). Phosphate‐buffered saline (PBS; Na2HPO4andNaH2PO4;10mM;pH

6.8)wasusedthroughoutandthecorrespondingsaltswereobtainedfromMerck(Germany).Allexperimentswerecarriedoutat25°C.

2.2 | Experimental analysis of MT

Kinetic analysis of MT inhibition | 2.2.1

The diphenolase activity of MT was measured by the oxidation‐ureactionofL‐DOPAtoDOPA‐quinoneat475nm(dopachromeaccmulationwavelength)at25°Cusingthemolarabsorptioncoefficient

F I G U R E 1   The structure of Chrysin—Linoleic acid (complex I) (A); Chrysin—Alpha Linoleic acid (complex II) (B)

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of 3,700M–1 cm–1 (Gheibi et al., 2016; Haghbeen et al., 2010).Absorption measurements were accomplished utilizing a UV‐2100 spectrophotometer (Rayleigh).

The diphenolase reaction of MT was carried out in 10 mM phos‐phatebuffer(pH6.8)at25°Cwith40unitsofMTanddifferentcon‐centrationsofL‐DOPA(0.1,0.5,1,1.5,2,and2.5mM)assubstrateswith and without the presence of inhibitors I and II (0, 0.01, 0.02, and0.04mM).Theincubationtimeoftheinhibitorsandenzymewas3minandallexperimentswererepeatedatleastthreetimes.Thedi‐phenolase activity of MT was analyzed using the Michaelis–Menten equation, followed by the double reciprocal plots of Lineweaver–Burk to obtain the Km, Vmax, and Ki values. The IC50 values of inhibi‐tors were calculated by Equation (1).

2.2.2 | Secondary structure measurements with CD

A spectropolarimeter (Aviv;Model 215;USA)was applied forCDmeasurement in a 0.1cm length and 0.3ml quartz cell. The CDspectraofthe40unitMTalonewas incubated(3min)withvarious

concentrationsofcomplexI(20,40,and60µM)andcomplexII(20,40,and60µM)andtheresultswererecordedinthefarUVregion(190–260nm).Theα‐helix, β‐sheet, turn, and random coils were ana‐lyzed in the far UV‐CD spectra using CD deconvolution software.

2.2.3 | Internal fluorescence and thermal analysis of MT

The fluorescence intensity of MT and complexes I and II was meas‐ured using a Cary Eclipse model 100‐Bio spectrofluorimeter. For in‐ternal fluorescence, an excitation wavelength of 280 nm was used and theemission spectrawere collected at300–420nm.TheMTconcentrationwas fixedat0.2mg/ml for interactionwith50,100,200, 300, or 400µMof complex I and 100, 200, 300, 400, 500,or 600µM of complex II. The thermal denaturation curves weredeveloped by measuring the fluorescence intensity at an excitation wavelengthof280nmandemissionwavelengthof350nmas thetemperatureincreasedfrom278to368K.

2.2.4 | External ANS fluorescence spectroscopy

Astock solutionof40mMANSwasprepared in thedistilled anddeionizedwaterwhichwasexcitedat350nmandtheemissionspec‐tra of the solutions are produced at 400–600nm in 50µMANS.The emission spectra were recorded in the presence of ANS alone (50µM),ANSandMT,ANS,MT,and200µMofcomplexesIandII.TheMTconcentrationwas0.2mg/mlin10mMPBSbufferatapHof6.8and25°Casobtainedusingaspectrofluorimeter(CaryEclipsemodel 100‐Bio).

2.3 | Theoretical analysis

2.3.1 | Protein preparation

Thecrystalstructureoftyrosinaseat3.25ÅwasdownloadedfromtheProteinDataBank(PDBID:5M6B).Residuesmissingfromthecrystallographic structure filewere nos. 462‐469,whichwere re‐stored using MODELLER software and the multiple‐template ap‐proach (Fiser, Do, & Sali, 2000). Autodock Tools (Sanner, 1999) was used to add the tyrosinase after determination of the Gasteiger charges of the polar hydrogen atoms (Gasteiger & Marsili, 1978).

2.3.2 | Optimization of protein homology by molecular dynamics simulation

In order to optimize the side chain orientation for protein balanc‐ing and determining the correct configuration of the atoms, molec‐ulardynamics simulationwithaCHARMM27force fieldwasused(Bjelkmar,Larsson,Cuendet,Hess,&Lindahl,2010).Afterapplyingperiodicboundaryconditions, theTIP3Pwater‐solvated formatofthe protein was placed in a cubic box with a box edge of 1 nm on the surface atoms. By replacing Na+, the total charge of the system was neutralized and the type of PME electrostatic interaction was determined. Minimization of the potential energy was performed for 50pstoreachanenergylevelof1,000kJ/molandfreethesystemfrom high‐energy interactions and steric clashes. In the next step, thesystemwasbalancedataconstanttemperatureof310Kandaconstant pressure of 1bar for 50ps.Molecular dynamics calcula‐tionswereperformedfor5nsusingGROMACSsoftware(VanDijk,Wassenaar,&Bonvin,2012).

2.3.3 | Ligand preparation

Themolecularstructureofchrysin,chrysin+n‐3,andchrysin+n‐6were drawn using Gaussian View. Their potential energy minimiza‐tionwascalculatedusingthePM3methodinGauss09wsoftwareandtransformedto3Dstructures(Almodarresiyehetal.,2014).

2.3.4 | Molecular docking

The process of molecular docking of the ligands to tyrosinase was performedusingAutodockVinasoftware(Trott&Olson,2010).Alldocking calculations were carried out using the optimization algo‐rithm for repetitive local search assuming that the protein is inflex‐ibleandtheligandflexible.Agridboxof14×18×16Åpointswasdefined and placed in an active site region of the enzyme. The grid distancewassetat1,000Åandotherparameterswereconsideredas the default. After docking, the best conformation with the low‐est binding energy was selected. The hydrogen and hydrophobic interactions of the tyrosinase‐ligand complex and the length of the hydrogen bonds were analyzed using Lig‐Plot software (Wallace,Laskowski,&Thornton,1995).

(1)IC50=Ki

([

S]

Km

+1

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2.4 | Results and Discussion

2.4.1 | Esterification analysis by FTIR

Esterification was performed using the Fischer method in which carboxylic acid reacts with alcohols or phenol groups to form es‐ters(Ahkong,Fisher,Tampion,&Lucy,1973).Thisisanendergonic(endothermic) reversible reaction with a high activation energy bar‐rierintheabsenceofacatalyst(HCl).Intheforwarddirection,itiscalled an esterification reaction because it produces an ester. In this study, an ester bound formed between ortho state of the chrysin and each PUFA (LA and ALA) (Figure 1A,B). The FTIR data showed theshiftingof theC=Oabsorptionbond from1,709 to1,739cm−1 (Figure2A).Theseresultsindicatedthat–COOHfunctionalgroupofPUFAs has been convertedto–COOC–andconfirmedtheformation

ofcomplexesI and II (Figure 2B).

2.4.2 | Complexes I and II competitive inhibition of diphenolase activity of MT

The inhibition kinetics of complexes I and II on the diphenolase ac‐tivity of MT were evaluated and the kinetic characteristics were ac‐quired from Lineweaver–Burk plots. This is demonstrated by the fact that an increase in the concentration of inhibitor resulted in a set of lineshavingdifferentslopescrossingattheY‐intercept(Figure3A,B).

The magnitudes of Ki and IC50 were obtained from secondary

plotsofLineweaver–Burk(insetsofFigure3A,B)andEquation(1),re‐spectively. The enzyme competitive type of inhibition was obtained

at Ki=0.45,IC50=0.83mM,andKi = 0.29, IC50=0.53mM,forcom‐

plexes I and II, respectively. The type of inhibition proposed the co‐ordination of these compounds with the copper clusters of enzyme active sites. The lower Ki of complexes I and II in comparison with chrysin (Ki = 0.79 and IC50 =1.45mM) and LA and ALA (Ki =0.53;IC50 =0.97mM and Ki=0.34; IC50 = 0.63 mM, respectively; data

not published) confirm that their esterification produced stronger inhibitorsofMT(Gheibietal.,2016,2014).

A variety of flavonoids with inhibitory effects on tyrosinase have been recently isolated from different natural sources. Flavonoid derivatives are reported to be able to reversibly hinder the binding of L‐DOPA to tyrosinase active sites (Xie, Chen,Huang,Wang,&Zhang,2003).Kineticsdatafrompreviousstudiesclearlyshowthatflavonoid derivatives reversibly block the catecholase activity of MT with inhibitory potencies in the following order: chrysin < querce‐tin < naringin < Gallic acid. Nonetheless, these structurally similar substances inhibit enzyme activity through different mechanisms (Gheibietal.,2016).

Studies have shown that saturated and unsaturated fatty acids have inhibitory effects on tyrosinase activity. Changes in activity with an increase in unsaturated fatty acids may increase degradation of tyrosinase by a proteasome complex, whereas an increase in satu‐ratedfattyacidshastheoppositeeffect(Gheibi,Saboury,Haghbeen,Rajaei,&Pahlevan, 2009; 2015;Gheibi et al., 2015;Ibiebele,Nagle,

Bain,&Webb,2012).Thus,bychangingthecomposition of the fatty acids in the cell, it may be possible to con‐trol abnormalities occurring during melanin production, including its

F I G U R E 2   FTIR spectra comparison before (A) and after (B) esterification

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overproduction (Jiménez, Chazarra, Escribano, Cabanes, & García‐Carmona, 2001).

Inthepresentstudy,chrysinwitheithern‐3orn‐6wasappliedto MT to characterize the inhibitory effects of complexes I (chry‐sin+n‐3) and II (chrysin+n‐6) on catecholase activity of this en‐zyme. In a previous study on carboxylic acids, we emphasized the chelating effects of the carboxylic groups on copper clusters in the MT active sites (Gheibi et al., 2009). The competitive inhibitory ef‐fects exhibited by these complexes suggest that they probably bind to the active sites of the enzyme.

2.4.3 | Thermodynamic parameters of MT in the presence of complexes

Figure4 shows thesigmoidaldenaturationcurvesof theMTwithand without complexes I and II in which the internal fluorescence of MT decreased with a gradual increase in temperature. According to the theory developed by Pace et al., the Gibbs free energy of de‐naturation (ΔG°)wasmeasuredasabenchmarkof conformationalstability of globular protein (Pace, 1992). Denaturation was charac‐terizedinlinewithalterationsintheemissionintensityat350nmanda change was measured in the Gibbs free energy of the denatured protein (ΔG°).Theproteinmeltingpoint (Tm) is the temperature at which a protein reaches half of its two‐state transition during ther‐maldenaturation(Figure4;inset).AnalysisofMTthermaldenatura‐tion curves revealed that, the Tm valueswerecalculatedas328.6,322.4,and318KandtheΔG°valuesas62.8,52.9,and47.1KJmol−1

for the enzyme alone and its interaction with complexes I and II, re‐spectively. The values of ΔG°andTm, as the most important thermo‐dynamic parameters, decreased after incubation of enzyme with the two complexes.

2.4.4 | Change of regular secondary of MT by CD

TheMT far‐UVregion (190–260nm) revealed that theadditionofthe two complexes caused significant changes in the secondary structure of the MT. Figure 5A,B shows changes in the second‐ary structure contents of the enzyme through the CD profiles of the complexes at different concentrations. Table 1 shows that the amountofalpha‐helixandbeta‐sheetsofMTalonewas63.1%and9.1%, respectively. The enzymewith two complexes shows a de‐crease in the sum of the α‐helix and β‐sheet structures with a coin‐cident increase in the random coil values after MT incubation with complexes I and II.

2.4.5 | Internal and external fluorescence changes of MT

The effect of complexes I and II on the tyrosinase structure was examined by the measurement of fluctuations in the intrinsic pro‐teinfluorescence(Figure6A,B).Besides,thesestructuralchangeswere achieved through extrinsic fluorescence with ANS as a tag of the hydrophobic patches (Figure 7). The fluorescence data dem‐onstrates the effect of the complexes on the tertiary structure of MT. The internal fluorescence spectra showed movement of tryptophan residues to hydrophobic regions of the protein. An in‐crease in ANS fluorescence intensity after incubation of MT with the complexes in comparison with ANS alone emphasizes the ef‐fect of the complexes on the exposing of hydrophobic patches to the enzyme surface (Figure 7). The binding of ANS to the hydro‐phobic surface of the protein in an aqueous solution enhanced the fluorescence emission intensity (Sponton, Perez, Carrara, & Santiago,2014).

F I G U R E 3  Lineweaver–Burkplotsofmushroomtyrosinasekineticassayformonophenolasereactions.L‐DOPAwasasubstrate.Thereactionwasperformedin10mMPBS,pH6.8,at25°Cand40μL enzyme, in the presence of different concentrations of (A) Complex I: 0 μM (♦), 10 μM (■), 20 μM (▲),30μM(×);(B)ComplexII:0μM (♦), 10 μM (■), 20 μM (▲),30μM(×),40 μM(Ж).Inset:secondaryplots,theslopeagainstdifferentconcentrationsofinhibitor,whichgivestheinhibitionconstant(−Ki) from the abscissa‐intercepts

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2.4.6 | Molecular docking

Afterdocking,theinteractionofL‐DOPAasasubstratewithcom‐plexes I and II was examined using LigPlot software. The best pos‐sibleconditionachievedwasforchrysin+n‐6.Theenergybindingof the interactions of complexes I and II andMTwas −113.1 and−116.9kcal/mol, respectively. For chrysin+n‐6, Glu474 was in‐volvedinahydrogenbondwithalengthof3.06Åand15residues(Asn254,Asp253,Gly256,Gly259,Val250,His296,His57,Phe279,Ala273,His283,Ala274,Phe87,Glu248,His82,andHis236)wereinvolved in the hydrophobic interaction. For chrysin+n‐3, Ser83wasinvolvedinahydrogenbondwithalengthof2.81Åandwhile20 residues (Phe 87, Asp252, Thr216, Ala273, Thr134, His283,His282,His255,Gly259,Val258,H7q0,Trp132,Phe257,Leu209,Leu138,Cys90,His57,His91,Phe454,andHis251)wereincludedinhydrophobic interaction (Figure 8A,B). Through comparison of these residues and the hydrophobic nature of their interactions, it appears that the four‐helix bundle sheet structure around the active site is the interaction site of these two complexes (Ismaya et al., 2011; Pretzler, Bijelic, & Rompel, 2017).

TA B L E 1   Far‐UV CD analysis of MT structure with and without the complexes

SampleInhibitor concentration (µM) α-helix (%) β-sheet(%) β-turn (%) Random.coil (%)

MT 0 63.1 9.1 15.9 11.9

Complex I 20 47.3 15.9 18.3 18.5

40 41.9 18.3 18.7 21.1

60 38.5 20.1 18.8 22.6

Complex II 20 56.1 11.4 16.8 15.7

40 50.9 13.5 17.4 18.2

60 41.7 17.8 18.8 22.6

F I G U R E 4   (A) Thermal denaturation in the presence of solemushroomtyrosinase0.2mg/mlandwiththeconcentrationsofcomplex I and Complex II. (B) Free energy changes for the thermal denaturation of sole enzyme and with concentrations of complex I and Complex II

F I G U R E 5  UV‐CDspectraregisteredformushroomtyrosinase40 unit (a)andMTinthepresenceofdifferentconcentrationof(A):ComplexIand(B):ComplexII.Enzymesole,20,40,and60μM

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F I G U R E 7   InteractionbetweenmushroomtyrosinaseandANSFluorescenceemissionspectraofANS50µMin10mMPBS,pH6.8,temperature25°Candtheexcitationwavelengthof380nminthepresenceofsoleANS50µM(a),ANSwithmushroomtyrosinase(b),withconcentrationsofcomplexII,10µM(c),and12µM(d),andComplexI:10µM(e),and12µM(f)

F I G U R E 8   Best docked conformations of complex I‐ mushroom tyrosinase (A) and Complex II‐ mushroom tyrosinase (B)

F I G U R E 6   Intrinsic fluorescence emission spectra of mushroom tyrosinase and the enzyme in the presence of different concentration of (A) ComplexI:0(a),50µM(b),100µM(c),200µM(d),300µM(e),and400µM(f);(B)ComplexII:0(a),100µM(b),200µM(c),300µM(d),400µM(e),500µM(f),and600µM(g).Theconcentrationoftheenzymewas0.2mg/ml

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2.5 | Conclusion

Complexes I and II showed competitive inhibition of MT with in‐duced partial unfolding and in‐stability. Moreover, it can conclude thattheyshouldcompetewithL‐DOPAforaccessibilitytotheMTactive sites and induce a competitive type of inhibition. These com‐plexes induced enzyme inhibition having more strength than chrysin, ALA, and LA separately. In line with the kinetic changes, the com‐plexes induced thermodynamic instability and structural changes in MT. The enzyme secondary regular structures switched to a random coil in a concentration‐dependent manner. The molecular docking and experimental results indicate that complex II has better binding potency than complex I.

ACKNOWLEDG MENT

The financial support provided by the Research Council of the Qazvin University of Medical Sciences and Science and Research Branch of Islamic Azad University, is gratefully acknowledged.

CONFLIC T OF INTERE S T

The authors have no conflicts of interest to declare.

ORCID

Nematollah Gheibi https://orcid.org/0000‐0001‐7503‐0894

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How to cite this article:JamaliZ,RezaeiBehbahaniGR,ZareK,GheibiN.Effectofchrysinomega‐3and6fattyacidesterson mushroom tyrosinase activity, stability, and structure. J Food Biochem. 2018;e12728. https://doi.org/10.1111/jfbc.12728

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