Understanding the Molecular Mechanism of Phytoconstituents As

Saudi Journal of Medical and Pharmaceutical Sciences Abbreviated Key Title: Saudi J Med Pharm Sci ISSN 2413-4929 (Print) |ISSN 2413-4910 (Online) Scholars Middle East Publishers, Dubai, United Arab Emirates Journal homepage: https://saudijournals.com

Review Article

Understanding the Molecular Mechanism of Phytoconstituents as Tyrosinase Inhibitors for Treatment of Hyperpigmentation Lata Kothapalli1*, Pooja Sawant2, AshaThomas1, Ravindra Wavhale1, Komal Bhosale2

1Faculty, Department of Pharmaceutical Chemistry, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Gaikwad Haraibhau Vinayan Rd, Sant Tukaram Nagar, Pimpri Colony, Pimpri-Chinchwad, Maharashtra 411018, India 2Research Scholar, Department of Pharmaceutical Quality Assurance, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Gaikwad Haraibhau Vinayan Rd, Sant Tukaram Nagar, Pimpri Colony, Pimpri-Chinchwad, Maharashtra 411018, India

DOI: 10.36348/sjmps.2021.v07i02.010 | Received: 08.01.2021 | Accepted: 20.01.2021 | Published: 27.02.2021

*Corresponding author: Lata Kothapalli

Abstract

Skin Pigmentation is a phenomenon in which the accumulation of melanin occurs due to various factors and leads to skin tanning and other skin disorders. Now a day’s human beings are more conscious about quality of life and appearance. With this fascination towards various skin whitening or glowing agents is gaining more adherences. While synthetic chemicals approved as skin whitening agents have got the due recognition in market, the possible adverse effects on sensitive skin creates a question regarding their use. The natural plant extracts available as tyrosinase inhibitors are mostly phenolic entities. In this review the inhibitory effect of phenolic compound and flavonoids on tyrosinase enzyme using molecular simulations is studied. The insilico study of flavonoids helps to predict the mechanism of inhibition as well as identification of potential natural tyrosinase inhibitors. This review is oriented to understand the tyrosinase inhibitory activity of an array of phenolic compounds with emphasis on flavonoids to explain how position of the substituents and their interaction with the enzyme can be correlated for the efficacy against the tyrosinase activity. Keywords: Flavanoids, Tyrosinase inhibitor, Hyperpigmentation, Molecular docking. Copyright © 2021 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY-NC 4.0) which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited.

NTRODUCTION condition which affects any part of skin where pigment I cells stop functioning and lead to progressive Skin hyperpigmentation is a major cause of appearance of patches or discolouration of skin colour concern and is caused by sun exposure, pregnancy, [9, 10]. Ephelides (freckles) are dark brown spot on hormonal contraception, family history [1]. With a high skin, caused due to exposure to sun rays or also as prevalence in the Indian population. Melanin level are genetic makeup, over production of melanin and twice the concentration of normal skin and contributes irregular distribution of melanin lead to irregular to darker skin phenotypes, where the pigment is patches on skin. Melasma (chloasma or mask of expressed in melanocytes present in the basal layer of pregnancy) is predominant in women because of the epidermal layer [2, 3]. Melanosome degradation hormonal changes during pregnancy [11]. rates vary in darker skin when compared with lighter skin types as studied by Vashi and Kundu, 2013 [4]. In the melanogenesis process, Tyrosinase Also the melanin content and dispersion pattern of enzyme presents a potential target for inhibition as it is melanosomegives the required protection against UV a rate limiting enzyme [12]. The enzyme also is radiation[5].The hyper pigmentation is phenomenon in expressed in fruits and vegetables besides the fungi due which the accumulation of melanin will leads to several to which the browning is seen [13]. skin disorders. Among the pigmentary disorders lentigines and melasma, postinflammatory For the same, huge demand for novel, safe hyperpigmentation, and dark eye circles are most andpotent tyrosinase inhibitors is increasing and has common [6]. Liver spot (senile lentigo) is skin disorder application in food and cosmetic industry [14]. The skin in which small pigmented spot on skin having diameter whitening agents are also used in scars, acne to make of 1mm which is mainly observed in people above the the skin uniform in colour. As there is increase in age of 40years. Excessive exposure to ultraviolet demand for tyrosinase inhibitors, various tools for radiation expresses the melanocytes and formation of screening of compounds, invitro and invivo assay and melanin pigment in keratinocytes [7,8]. Vitiligo is a Citation: Lata Kothapalli et al (2021). Understanding the Molecular Mechanism of Phytoconstituents as Tyrosinase Inhibitors for 135 Treatment of Hyperpigmentation. Saudi J Med Pharm Sci, 7(2): 135-144.

Lata Kothapalli et al., Saudi J Med Pharm Sci, Feb, 2021; 7(2): 135-144 design of synthetic inhibitors are undergoing rapid The importance of structural features of development. Various microbial species express polyphenols in treating hyperpigmentation using tyrosinase enzyme like Streptomyces glaucescens, molecular modelling supported by enzyme inhibition Agaricus bisporus and Neurospora crassa. assay can help in development of analogs using the Mushroomtyrosinase enzyme obtained from Agaricus phenolics as lead. A systematic search was performed bisporusiswidely used for screeningtyrosinaseinhibitory on PubMed, Scopus, Embase, Medline, databases using activity by various small molecules and can be proven the words ―tyrosinase inhibitor, phytoconstituents viainsilico study. The structural features of Mushroom docking, skin lightening agents, hyperpigmentation and tyrosinase have highest similarity with human data was collected over the last 20 years. tyrosinase compared to other tyrosinase enzyme sources [15] and the one studied by Ismaya et al., 2011 as Melanin Biosynthesis crystal structure composed of tetramer with the co - Melanin is a Greek word i.e. melanos means crystallised ligand tropolone as inhibitor has been dark. Chemically melanin is a blend of various widely used for understanding the binding interaction biopolymersobtained from serially oxidised derivatives with various inhibitors (PDB code 2Y9X) [16]. Another of tyrosine [19]. Skin, hairs, and eye (ciliary body and crystal structures of tyrosinase used for the study of choroid) with parts of the mammalian cerebrum contain inhibitors is a well resolved dimer from Bacillus melanin, which additionally spread in theliver, the gaps megaterium [17]. The computational drug design tools in layer of serous membrane and amphibian organs for molecular modelling can be an important tool for [20]. Tyrosinase present in melanocytes helps in understanding the anti tyrosinase activity of compounds synthesis of pigment melanin and stored in using the lead from natural resources [18]. melanosomes. Melanocyte produces two kinds of pigment that is eumelanin and pheomelanin by Rapor- This review is oriented to understand the Mason Pathway [21]. tyrosinase inhibitory activity of an array of phenolic compounds with emphasis on flavonoids to explain Biosynthesis of melanin begins with L- how position of the substituents and their interaction tyrosine which is oxidised to L-3, 4- with the enzyme can be correlated for the efficacy dihydroxyphenylalanine and later to dopaquinone with against the tyrosinase activity. the assistance of enzyme tyrosinase [22].

Fig-1: Melanin biosynthesis

As seen in Fig-1, Dopaquinone by cyclization glutathionyldopa or cysteinyl dopa with the help of form leukodopachrome and further by oxidation to glutathione or cysteine and these are further converted dopachrome and Indole-2 carboxylic acid - 5, 6- to pheomelanin (red-yellow) by forming benzothiazine quinone(DHI). In presence of enzyme Tyrosine Related as intermediate [24]. Protein-1 (TRP-1) dopaquinone is converted into dihydroxyindole-2-carboxylic acid (DHICA) and Microphthalmia associated transcription factor oxidized into dihydroxyindole (DHI) [23]. Further DHI (MITF) controls the melanin biosynthesis and control by oxidation with tyrosinase enzyme converts into the gene expressions of TRP-1, TRP-2, and tyrosinase. indolequinone(IQ). By polymerization of DHICA, IQ and ICAQ result in the formation of eumelanin (black- brown). On the opposite side, dopaquinone modifies to

Lata Kothapalli et al., Saudi J Med Pharm Sci, Feb, 2021; 7(2): 135-144 The upregulation of MITF activity leads to an Tyrosinase inhibition mechanism: increase in the synthesis of melanin and downregulation Tyrosinase enzyme inhibition occurs in the of MITF activity leads to a decrease in the synthesis of same way as all enzymes react with inhibitors based on melanin [25]. two different mechanism namely suicide inactivators and true inhibitors.Suicide inactivators are specific Role of tyrosinase enzyme tyrosinaseinactivators where in the substrate brings Enzymes are activated by metal ions as about conformational changes with the help of solvent cofactors. Enzyme Tyrosinase is also a metalloenzyme molecule and alters the tertiary and quaternary with two copper ions and ismembrane-bound type- structures of tyrosinase in presence of inhibitor leading 3glycoprotein. X-Ray Crystallographic structure of to suicide inactivation [29]. Land et al., D-tyrosinase is tyrosinase obtained from PDB databaseshows active an example of irreversible inhibitor of the enzyme and site with a huge vacant space surrounded with six stop the melanin formation provoked by UV irradiation histidine residues attached with copper ion which or α-MSH [30]. True Inhibitors compete with the makes the structure of enzyme highly flexible. Active substrate for binding in active site of the enzyme and site of enzyme has a substrate binding pocket which can are the competitive inhibitors E.g. Morin, Hesperitin orient based on theflexibility and position to fit the [31]. Noncompetitive inhibitors like substrate [26, 27]. The measurement of cell size and 7,8,4trihydroxyflavoneand Mixed (competitive and count of melanosomes helps in understanding the uncompetitive) Inhibitor such ascinnamic acid, thymol regulation of melanin formationat the cellular level analogues [32] have multiple binding in active pocket while expression of enzymes involved in melanin of enzyme or in allosteric site of the enzyme–substrate synthesis is driven by signaling pathways [28]. complex while derivatives like Barbarin are Uncompetitive Inhibitor. Some examples of flavonoids are as mentioned in Table-1.

Table-1: Different Flavanoids with the type of inhibition Classes of Biological source Chemical constituent Mechanism of IC50 value flavonoids tyrosinase inhibition Flavones and Heart wood of 7,8,4trihydroxyflavone Noncompetitive 10.31±0.41µM dihydroflavone Artocapusaltilis Hydroxyflavone Heart wood of Baicalein Mixed 0.11µM. Artocapusaltilis Flavanoles Cudraniacolchinchinens Morin Competitive& 8.13 µM reversible Flavanones Citrus fruit Hesperetin Competitive& 11.25μM. reversible Flavanolsand Extract of stem and fruit Epicatechin, Mixed and 23.6 µM flavan-3,4-diols of Clausenalansium Gallocatechin, competitive inhibition 7.0 µM Epigallocatechin Curcuminoids extract of Curcuma Curcumin, Competitive 267.3 ± 6.1µM xanthorrhiza demethoxycurcumin and xanthorrizol

Polyphenols as Tyrosinaseinhibitors for treatment of also studied the inhibitors for overcoming the adverse hyperpigmentation and toxic effects especially when used for food storage Living organisms develop their own protective and medicinal applications. Besides this an ever pathways to fight against the adverse surroundings. One increasing demand around the globe is towards skin such pathway beneficial against excessive exposure to glowing and skin whitening products [35]. The harmful ultraviolet radiation is the Melanin pathway understanding of the structural features of the (Skin tanning) and the control on the melanin pathway phytoconstituents will help in developing more stable is done by the innumerable sources of tyrosinase and efficient derivatives for the treatment. The inhibitors [33]. Various scientists have studied computational drug design tools like structure based tyrosinase inhibitors from natural sources like plant, drug design where in molecularmodelling can be an fungi and marine life to understand their structural important tool for understanding the antityrosinase features to understand the structure-activity relationship activity of compounds using the lead from natural (SAR) and further modify the existing compounds to resources. overcome issues related to stability and efficacy [34]. Natural products have gained more attention in Polyphenols cosmetics as compared to synthesized compound Plant polyphenols make an amalgam of specially the plant concentrates being studied to compounds with multiple phenolic functionalities. discover new lead compounds. A number of researchers These compounds are secondary metabolites formedby

Lata Kothapalli et al., Saudi J Med Pharm Sci, Feb, 2021; 7(2): 135-144 most higher plants and contribute for Flavonoids severalbiologicalactivities. Nguyen et al., studied the Flavonoids are a huge class of compound methanolic extract of heartwood of Artocapusalti. among natural products and are utilized broadly as lead liscontaining about 13phenolic compounds against the compounds or drugs. Looking into the biosynthetic tyrosinase inhibitory activity wherein it was seen that pathway for the flavonoids, it is seen that chalcone curcumin, desmethoxycurcumin and p-coumaric acid initiates the synthesis of flavonoids as reviewed by were more potent than kojic acid [36]. Kim D et al., Falcone et al., 2012 [38]. Polysubstituted derivatives 2006 further carried out molecular modeling of the p- such as 2,4,3’ ,4’-hydroxychalcone and 2,4,2’,4’- coumaric acid and phenyl thiourea(PTU) as standard hydroxychalcone present IC50 of 0.2 and 0.02M, depigmenting agent used in understanding the blocking respectively against the monophenolase enzyme as of pigmentation process in zebrafish demonstrated by Khatib et al., 2005 [39]. Further few embryo.Thebinding interaction of Chain A tyrosinase more studies on the hydroxyl substituted derivatives enzyme (PDB ID = 3NQ1).with standard PTU and p- were done by Shimizu et al., 2000 [40] using coumaric acid was analysed and compared with the phytoconstituents and synthetic analogs and Ohguchi et kojic acid using the PyRx,a software of Autodock 4. al., 2003 [41] explained importance of number and The binding interaction of p-coumaric acid was place of phenolic hydroxyl group on the flavonoids different from those seen with the standard. Kojic acid impact the inhibition of tyrosinaseenzyme. Kim D et al., interacted with Pro201, Arg209 via Pi-Alkyl 2006 correlated the Stern-Volmer quenching constants interactions while Gly200 showed Carbon-Hydrogen values with the IC 50 values of about 25 flavonoids bond. Interaction with p-coumaric acid with dock score when tested against the Tyrosinaseenzyme [42]. The of -6.00kcal/mol involved five amino acid residues number of phenolic hydroxyl on the two aromatic rings namely H-bonding with Gln242, His245, His279 and (Fig-2) of flavonoid structure, can significantly impact Pi-sigma interaction with Ile243. It was seen that PTU inhibition of tyrosinase enzyme. The hydroxyl groups binding pattern did not overlap with kojic acid site. For of A and B rings separated by the etherical bond of the PTU, π-σ interaction were seen with Val218 and while C (pyran) ring mainlycontributein the tyrosinase Gly216with H-bonding, Ala221showed π-Alkyl inhibitory activity. The experimental finding were interaction, HIS208 showed Pi-Pi stacked interaction confirmed with molecular docking studies. For the with binding score of -5.53 kcal/mol. With this same,homology modeling was used to prepare modeling study it was concluded that p-coumaric acid mushroom tyrosinasewith crystal structure (PDB code, and PTU show different binding pattern within the 1BT3) of Catechol oxidase using the template Octopus active site of the enzyme. The experimental study with hemocyanin (PDB code, 1JS8).The docking result on zebrafish model and molecular docking correlated that prepared tyrosinase enzyme showed that the catechol p-coumaric acid can be an important lead molecule to group and copper ions complex well thereby inhibiting inhibit dermal pigmentation [37]. enzyme activity. Further activity is lost or decreases with additional hydroxyl groups which add to steric hindrance.

Fig-2: General structure of flavonoids

Lata Kothapalli et al., Saudi J Med Pharm Sci, Feb, 2021; 7(2): 135-144 Flavones and Dihydroflavone 7,8,4´‐ trihydroxyflavone. When taking varied The derivative of flavones, that is 7, 8,4’ - concentration of substrate and inhibitor, it was observed trihydroxyflavone which occur extensively in petals and that the enzyme inhibition with Ki 9.50±0.40µM is foliage of the plant and having widespread reversible and non‐ competitive. It inhibits both pharmacological activity. Shang et al., 2018 carried out diphenolase and monophenolase activity in a reversible experiments to correlate the mechanism for tyrosinase and non-competitive manner with IC50 value of inhibition with the molecular docking simulation for 10.31±0.41µM and 12.95 µM respectively [43].

Fig-3

For 7,8,4-trihydroxyflavone (Fig-3) binding to inhibition [45]. Baicalein Fig-3 shows inhibition of tyrosinase enzyme is through conformational tyrosinase with the conformational changes in the changes.The mushroom tyrosinase( PDB 2Y9X) active structure of enzyme. Along with tyrosinase inhibition site has twosets of three histidineresidues namely Set A baicalein shows multiple activities such as antioxidant (His61, His85 and His94) and Set B with His259, properties and is safe molecule [46]. Nihong Guo et al., His263 and His294 that couple with copper ions. 7,8,4 estimated the active site for binding interaction is Trihydroxy flavone stations itself in the active site of present at the bottom of the cavity of tyrosinase. From catalytic domain surrounded by Tryptophan amino acid docking studies carried out using the same tyrosinase and is explained by the binding affinity between protein enzyme (PDB: 2Y9X), intermolecular interaction and ligand. Lowest binding free energy is ranked as confirmed the binding between baicalein and tyrosinase best confirmation, 7,8,4trihydroxy flavone showed the which effectively produced docking score - binding score -7.4kcal/mol which represents binding 6.20kcal/mol for binding conformation. Several interactions. The oxygen of carbonyl group of Ring C adjacent residues are His85, Val 248, Phe264, Met280, acts as HBA for Hydrogen bond interactionwhile 7-OH Ser282, Val283; Ala286 helps to form hydrophobic group is HBD in ring A with His 244 and Met280 interaction with benzene ring and increases stability of residues with a bond length of 3.34A0and 3.07A0 binding interaction. Phenolic hydroxyl group(C-7) of respectively. The van der Waal force and hydrophobic baicalein and carbamoyl oxygen atom of Met 280 interaction were seen between carbamoyl group of residue form hydrogen bonding which stabilizes the Asn260 and phenyl ring of 7,8,4 trihydroxyflavone complex. C-7 hydroxy group was considered very respectively, which is seen to help in improving essential for enhancing its inhibitory activity against stability of binding interaction. tyrosinase.

Another flavone Baicalein is chemically 5,6,7- Vander waals interaction are seen between the trihydroxyflavone which is obtained from roots and carbamoyl group of Asn260 and baicalein. It also aerial parts of Chrysanthemum morifolium, involves pi –pi interaction with His85 and Phe264 and Artemisiaannua and Oroxylumindicum (Indian trumpet pi-σ interaction with residues Val283 [47, 48]. flower) [44]. Baicalein exhibit inhibition at a later stage of oxidation diphenolase enzymewith mixed type of

Lata Kothapalli et al., Saudi J Med Pharm Sci, Feb, 2021; 7(2): 135-144 Flavanols various fruits and its different parts namely pericarp of Morin is a flavones analog (3,5,7,2,4- Kiwi fruit (Actinidiasinensis),.Avocado fruit pentahydroxyflavone) (Fig-3) isolated from Cudrania (Perseaamericana) [58] and fruit seed of Chinese colchinchinensis and Cudrania hawthorn (Crataeguspinnatifida B) [59]. tricuspidata (family:Moraceae) is tyrosinase inhibitor an Proanthocyanidins extracted from fruit pericarp of essential bioactive flavonoid compound studied by Clausenalansium (Lour.) Skeels showed inhibitory Zheng et al., 2011 [49]. It is widely present in onion, activity in concentration dependent manner withIC50 guava leaves and seaweeds and displaywide spectrum values calculated to be 23.6 ± 1.2 for monophenolase a of activities against inflammatory mediators and nd 7.0 ± 0.2 μgmL−1 against diphenolase. In addition, antioxidant activities [50, 51]. Morin is a reversible and the proanthocyanidins also exhibited cell proliferation competitive inhibitor and shows conformational inhibition with suppression of melanin production changes in the enzyme (IC50 value 8.13 µM) [52]. evaluated on B16 mouse melanoma cells. RP HPLC- ESI-MS chromatographic analysis of the treated Another study done by Wang, Y et al., proanthocyanidins thiolytic products, prodelphinidins explained the inhibitory action of Morin using the (PD) were in greater proportion to procyanidins (PC) circular dichroism spectral study along with docking and accounts for the enhanced activity against carried out using PDB code 2Y9X. The study explained tyrosinase at every step of melanogenesis. that morin when interact with tyrosinase induces a rearrangement followed by some conformational R Flavan-3-ol Proanthocyanidines changes in the enzyme. Morin binds in hydrophobic H Epicatechin Procyanidin cavity surrounded by amino acid residues His85, His94, OH Epigallocatechin Prodelphindin His244, His259, Asn260, His263, Phe264, Met280, Gly281, Val283 and His296. Hydrogen bonding is On taking up the molecular docking studies mainly between His85 and His 244 and C-3 and C-5 using the MOE (molecular operation environment, hydroxyl group of morin respectively. Thetyrosinase 2010) study, Tyrosinase enzyme obtained from inhibition displayed by morin is due to the fitting of Streptomyces castaneo globisporus (oxy-form) bound morin into the active site of catalyticdomain which does with copper and stabilized with a caddie protein (PDB not interfere with L-DOPA, however Morin shows code 1WX2)was selected as the protein model and was conformational changes in tyrosinase [53]. refined to remove the caddie protein. The hydroxyl

Flavanones groupspresent in B ring of the proanthocyanidins Hesperetin is chemically (3,5,7-trihydroxy-4- interact withthe two copper ions, and was further methoxyflavanone) Fig-3) is abundantly present in correlated by fluorescence quenching. Epicatechin citrus fruits and the medicinal herbs of family Rutaceae, shows binding interaction of copper with His85, His61 Rosaceae, Leguminosae [54, 55]. Hesperidin is and Phe264 residues, and other interaction with His263, theglycoside of hesperitinand has been reported to show Met280, Val248. Epigallocatechin shows interaction of anti-inflammatory, anti-carcinogenic, anti-hypertensive, copper ion with Asn260 and Phe264 and shows anti-atherogenic, antiviral and antioxidant activity [56]. hydrogen binding interaction with His244 residue. Gallocatechin shows interaction with Phe264, His85, Yue-Xiu Si et al., 2012 evaluated the Val283, His263 residues [60]. tyrosinase inhibitory mechanism with the help of kinetic analysis and computational simulation with Curcuminoids hesperitin as the inhibitor and kojic acid as standard. It Curcumin, demethoxycurcumin and was shown that hesperitin showed a competitive xanthorrizol (Fig-3) obtained from inhibition with IC50 value 11.25 ± 1.73 mM (n = 3). ethanolic/methanolicextract of Curcuma longa and Hesperetin enter the active site of the enzyme by Curcuma xanthorrhiza, exhibitedanti tyrosinase effect competing with L-dopa. Docking was carried out using wherein IC50 value estimated were206.7± 9.2 µg/ml and AutoDock4.2, where the binding score for hesperiin 267.3 ± 6.1 µg/mlamong the 35 plants analysedby was −5.67 kcal/mol while kojic acid had binding energy Batubara et al., 2010 [61]. Further studies were carried −3.91 kcal/mol. Hesperetin binds into the active site of out by Lee et al., to depict the inhibitory effect of enzyme and reversibly chelate with copper bound to the curcumin against thesignal pathway stimulated by three Histidine residues (His61, His85, and His259)and αMSH and expression of enzymes like tyrosinase, modify the hydrophobic surfaces. Hydrogen bonding TRP1/TRP2 along with regulating proteins were interactions are also seen between 5-OH and 7-OH of analysed in B16F10 cells [62]. The work carried out by substrate with Met280, His61, His85, His259 residues. Mustarichie et al., wherein the docking simulation Computational simulations carried out by Si YX et al., results of curcumin,demethoxycurcumin, xanthorrizol 2012 also confirmed similar binding pattern [57]. and Kojic Acid was carried out using AutoDock wherein it was seen that Hydrogen bonding and Flavanols and flavan-3,4-diols vanderwaal forces of interaction occur with all three Wei-Ming Chai et al., 2014 studied actives. All the three compounds Curcumin, proanthocyanidins (flavan-3-ol) (Fig-3) present in

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