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Chapter I Introduction Page 1 Introduction Tyrosinases are ubiquitously present in all life forms. These enzymes play versatile roles in biological functions along with heterogeneity Halaouli et al, 2006. The characteristic feature of tyrosinase is the presence of antiferromagnetically coupled copper ions as CuA and CuB that are coordinated by six histidine residues located in a ‘four ?-helix bundle’. It is the type 3 copper centre found commonly in tyrosinases, catecholoxidases and haemocyanins. It is also found in multi-copper oxidases Claus et al, 2006. The copper present in the active site of tyrosinases catalyzes two different reactions by binding with oxygen. Atmospheric oxygen is utilized by tyrosinase to convert monophenols into diphenols by monophenolase action (cresolase activity) and further conversion of diphenols into o-quinones by diphenolase action (catecholase activity) Claus and Decker, 2006; Kong et al, 2006; Seetharam and Saville, 2002.

These quinones formed are highly reactive which undergoes nonenzymatic polymerization and results into melanin. The copper-ion valency and the binding of molecular oxygen decide the existence of active site in three different intermediate states as deoxy CuI-CuI, oxy CuII-O2-CuII and met CuII-CuII. The exact mechanism is not clear completely still it is assumed that diphenolase action of tyrosimase follows Michaelis-Menten Kinetics while monophenolase action shows a phase of latency Halaouli et al, 2006.

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Tyrosinases participate in wound healing and primary immune responses in plants, sponges and verterbrates. They are also involved in parasite encapsulation and sclerotization in insects Kim et al, 2005. In mammals, tyrosinases are present in melanocytes, where they functions in synthesis of melanin by the process of melanogenesis.

Melanin functions as photoprotection, photoconductivity, thermoregulation and chelation of metal ions Wan et al, 2008. It is the pigment responsible for the pigmentation of hair, skin, retina, feathers, scales while in the internal structures of brain and inner ear Matuszak et al, 2006. It protects skin from harmful UV rays and avoids skin diseases. The unusual distribution of melanin pigments leads to several diseases. Albinism is a genetic abnormality caused due to deficiency in melanin biosynthesis, which results into hypopigmentation of the skin, hair, and eyes. Hypopigmentation in the skin is associated withChapter I Introduction Page 2 sensitivity to UV radiation that leads into skin cancer. However, abnormal accumulation of melanin pigments leads to hyperpigmentation like freckles, spots, melasma, senile lentigines and Kim et al, 2002. Tyrosinase is also responsible for the production of neuro-melanins but excessive productions of dopaquinone by oxidation of dopamine results in neuronal damage related to Parkinson’s disease (PD) Selinheimo et al, 2007.

In this disease (PD) dopamine producing neurons in the basal gangalia of the brain are destroyed progressively. Effects like rigidity (muscles resistant to movement), akinesia (inability to initiate movement), bradykinesia (slowness of movement), and rest tremor are shown by the patient due to loss of neurotransmitter dopamine (Bhatnagar and Andy, 1995; Brodal, 1998; Lang and Lozano, 1998 sush). Colour, appearance, texture, flavor and nutritional value are the important factors considered by consumers Zeng et al, 2008. In fruits, vegetables and crustaceans, tyrosinase causes undesirable browning during peeling, brushing, crushing procedures and post harvest handling which leads to poor aesthetic nature and lowers the market value Kim et al, 2002; Kim et al, 2005. There are two reactions possibly responsible for the browning of food products as enzymatic and non-enzymatic reactions.

Non-enzymatic reactions include reactions between amino acids, peptides, proteins with sugars and vitamin C called as Millard’s reaction. Tyrosinase plays key role in enzymatic browning of food results into destruction of essential amino acids and decrease in digestability as well as inhibition of proteolytic and glycolytic enzymes Loizzo et al, 2012. The enzymatic pathway mainly depends upon concentration of tyrosinase, phenolic substrates, availability of oxygen, pH, temperature, etc.

Various strategies have been employed to inhibit the enzymatic browning reaction. Many chemical methods were planned including both natural and synthetic compounds for inhibition of tyrosinase. Sulfites, cysteine, sodium chloride, oxalic acid, nitric oxide, critric acid, ascorbic acid and 4-hexylresorcinol were used as antibrowning agents. Sulfites were successful antibrowning agents but due to its allergic reactions among individuals, FDA has banned it for safety purpose Saisung et al, 2009. Hence, there is need of development and screening of tyrosinase inhibitors and thus there is an increasing demand of tyrosinase in medical, cosmetic, agriculture and food industry ChengChapter I Introduction Page 3 et al, 2002. A large number of tyrosinase inhibitors including flavanoids, kojic acid, annisaldehyde, cinnamaldehyde, arbutin and hydroquinones have been reported till date Kim et al, 2005; Uchida et al, 2014.

Surface plasmon resonance (SPR) technique is an optical method for measuring the refractive index of very thin layer of material immobilized on a metal gold surface. SPR is a new biophysical method. In 1990, Biocore was to release the first commercial instrumentJonsson, U.; Fagerstam, L.; Ivarsson, B.; Johnsson, B.

; Karlsson, R.; Lundh, K.; Löfås, S.; Persson, B.; Roos, H.; Rönnber, I.

; et al. Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. BioTechniques 1991, 11, 620–627. It is the powerful and versatile spectroscopic method for determining the biomolecular interactions including ligand-receptor coupling, antibody-antigen coupling, protein-DNA interactions, Patil et al, 2014; Surwase et al, 2015. This technique can be facilitates binding analysis from small molecules to complex mixtures, lipid vesicles, viruses, bacteria, and eukaryotic cells Jason-Moller, L.

; Murphy, M.; Bruno, J. Overview of Biacore systems and their applications. Curr.

Protoc. Protein Sci. 2006, 19, doi:10.1002/0471140864.

ps1913s45. A SPR-based instrument allows the analysis of biomolecular interaction in real time with no labeling requirements and can measure mass changes down to 10pg/mm2. Applications shown include kinetic measurements (ka, kd), binding site analysis and concentration determination, screening of drug molecules, specificity analysis Fagerstam et al, 1992; Pattanaik et al, 2005. In present study, various natural sources were screened for potent tyrosinase activity. As per the screening study yam (Amorphophallus paeoniifolius) tyrosinase was isolated and showed potent tyrosinase activity. Yam tyrosinase was purified and characterized. The purified enzyme was then subjected to SPR studies.

The SPR technique was employed to determine the affinity of various small molecules inhibitors using surface immobilized yam tyrosinase (Amorphophallus paeoniifolius). Changes in refractive index indicate binding of the inhibitors at different concentrations were monitored by SPR. The sensor detected these inhibitor molecules and their corresponding binding affinity constants were generated after the analysis. These results were also confirmed by spectroscopic inhibition assay. This data will help to study the other proteins which undergo conformational changes on binding of inhibitor molecules.Chapter I Introduction Page 4 Further, another approach regarding effect of combination of inhibitors on yam tyrosinase was studied using SPR. The results were confirmed by spectroscopic inhibition assays. The data obtained from fluoroscence spectra during inhibition of tyrosinase by combination of inhibitors also supported the strategy.

The kinetics, binding affinity data was obtained through SPR studies which can be helpful in understanding the mechanism of tyrosinase.Chapter II Review of Literature Page 5 2. Review of Literature Tyrosianse is a binuclear copper containing enzyme present universally in all living organisms.

The enzyme catalyzes the hydroxylation of monophenols into diphenols which further oxidizes into quinones Claus and Decker, 2006; Kong et al, 2000; Jeon et al, 2006. These quinones are highly reactive compounds those polymerases to form melanin. Tyrosinase is the key enzyme responsible for synthesis of melanin by process of melanogenesis D’Mello et al, 2016; Rescigno et al, 2002. Melanins are the pigments responsible for pigmentation of skin, hair, eye-lens and substantia nigra and locus ceruleus of the brain Bazelton et al, 1967; Nicolaus, 1968. They have biological functions of diverse colouration Solano et al, 2014, absorption of UV radiations Slominski, 2015 and electron transfer properties Gan et al, 1976 and prevents sun induced skin injuries Jeon et al, 2005. 2.1.

Role of tyrosinase Plants, fruits, vegetables, fungi Tyrosinase protects plants from insects and microbial attacks by forming impervicious scab of melanin against further attack Whitaker et al, 1995. Tyrosinase plays defence related roles in plants like tomato, potato, nicotiana, watermelon, etc. When wounded tissues exposed to air, it gets oxidized and gives to browning reactions. Tyrosinases are responsible for this undesirable browning during post harvesting, handling and processecing of fruits and vegetables Gelder et al, 1997; Bourvellec et al, 2004. Enzymatic browning has been studied in various plants like aubergine Dogan et al, 2002, Origanum Dogan et al, 2005, apricot Arslan et al, 1998, Thymus species Dogan et al, 2003; 2005, SalVia Gundogmaz, 2003, spinach Golbeck, 1981, and tea leaves Zawistowski, 1988.

Browning is mainly of two types enzymatic browning and non-enzymatic browning. Enzymes like tyrosinase, polyphenol oxidases generally contribute to enzymatic browning while, nonenzymatic browning is due to reactions between sugars and amino acids called as Millard’s reaction Loizzo et al, 2012.This enzymatic browning results in negative effect on colour, taste, texture, flavor and nutritional value Holderbaum et al, 2010. This may reduce the market value of agricultural products. Browning of mushrooms (Agaricus bisporus) has been reported due to the action of tyrosinase Jolivet et al, 1998. In fungi, tyrosinase mainly contributes to the functions likeChapter II Review of Literature Page 6 defence against stress of different radiations, dehydration, extreme temperatures as well as helps in fungal cell wall resistance against hydrolytic enzymes without cellular lysis Beel et al, 1986.

Fungal tyrosinases are also involved in defence and virulence mechanisms Soler et al, 1997; E. S. Jacobson, 2000, formation and stability of spores Mayer and Harel, 1979. Invertebrates, insects In insects, tyrosinase is involved in sclerotization of exoskeleton and protection of insects from other organisms by encapsulating them in melanin Claus and Decker, 2006. Insects and other arthropods require tyrosinase for melanin formation which is used for exoskeletal pigmentation, cuticular hardening, wound healing and innate immune responses Sugumaran and Barek, 2016.

In many insects, sclerotization and cuticle colouration are associated with each other,which may be due to melanin or quinonoid end products of sclerotizing catechols or both Sugumaran et al, 2010; Andersen et al, 2010. Blackening of insect’s blood due to the attack of parasitic fungus was observed and tyrosinase was considered to be the reason of blackening of blood Alfred S. Sussman, 1949. Mammals, humans In mammals, tyrosinases are responsible for the synthesis of melanin by the process of melanogenesis in melanocytes Videira et al, 2013, Shimizu et al, 2002. Melanins are heteropolymers produced by spontaneous polymerization of quinones generated by action of tyrosinases. It also may be formed due to the chemical oxidation of phenolic compounds like L-tyrosine, L-DOPA, catechols, etc. H.S.

Mason, 1948; G.Prota, 1980; H.S,Raper, 1928.

Melanin is the pigments found throughout the nature. It is found in skin, hair, eye-retina, irides, feathers, scales and internal structures of brain and inner ear Bazelton et al, 1967; Matuszak et al, 2006. Melanin is classified as eumelanins, pheomelanins and allomelanins. Eumelanins are brown-black polymers synthesized from tyrosine and pheomelanins are reddish brown polymers generated from tyrosine and cystein whereas allomelanins are dark brown-black polymers produced from tetrahydroxynaphthalene. Land et al, 2004; F.

Solano, 2014; Plonka and Grabacka, 2006. Melanin bears physicochemical properties like UV rays absorbtion, cation exchangers, drug carriers, semiconductors, X-ray and ? –ray absorbtion,etc Saran et al,1976; Bell and Wheeler, 1986, della-Cioppa et al, 1990; Hill, 1992; Hoti and Balaraman, 1993; KrolChapter II Review of Literature Page 7 and Liebler, 1998; Rozanowska et al, 1999; Wang et al, 2000; Nofsinger et al, 2002; Ortonne, 2002. The abnormal synthesis of melanin leads to the skin problems.

Increased melanin synthesis causes hyperpigmentation while, decreased melananin synthesis results into hypopigmentation Baxter and Pavan, 2013. There are many inherited pigmentary disordersresulting into increased melanocyte density (e.g. freckles) or decreased melanocyte density (e.g. vitiligo) Yamaguchi et al, 2007.

Albinism, melasma, age spots, post inflammatory, lentigo, leukoplakia, moles are common skin problems due to abnormal synthesis of melanin by action of tyrosinase Park et al, 2010; Alam et al, 2017. Many reports on neromelanins have also been mentioned describing the significance of melanin and indirectly tyrosinase in brain aging process. Neuromelanin is a complex polymer pigment found in catecholaminergic neurons of the human brain Dzierzega-Lecznar et al, 2004. It get acuumulated with the aging in the substantia nigra region of human brains and plays very important role in Parkinson’s disease (PD) M. D’Ischia and G.Prota, 1997; Double et al, 1999; Zecca et al, 2001; Greggio et al, 2005. In brain, tyrosinase oxidizes excess amount of DOPA and L-DOPA present in cytoplasm.

This prevents self-oxidation of DOPA and maintains its optimum levels. Tyrosinase also shows catecholamine synthesizing activity in absence of tyrosine hydroxylase. Hence, this dual role of tyrosinase enables its potent application in synthesis of DOPA, therefore can be used in treatment of Parkinson’s disease Asanuma et al, 2003. 2.

2. Structure and active centre of tyrosinase A pair of copper ions present in the active site of tyrosinase is co-ordinated by six histidine residues Park et al, 2003. This is the peculiar characteristics of tyrosinases, catecholoxidases and haemocyanins. All of them share same structural features: (i) valency and conformational change during oxygen binding Longa et al, 1996, (ii) spectroscopic and magnetic properties Himmelwright et al, 1980 (iii) homologies in primary sequences Gelder et al, 1997, (iv) catechol oxidase exhibit monophenolase activity on 4-hidroxyanisole Espin et al, 1998, (v) hemocyanins carrying oxygen shows catecholase activity after in vitro treatments of sodium dodecyl sulphate or protease Decker and Rimke, 1998; Jaenicke and Decker, 2004.Chapter II Review of Literature Page 8 In 2006, Matoba et al determined the crystal structure of tyrosinase in a complex with ORF378 at 1.2–1.8 Å resolution, by the multiple isomorphous replacement method including the anomalous scattering effect. As per study, tyrosinase shows helicle structure with the core of the enzyme formed by four helix bundles as ?2, ?3, ?6, and ?7 helices.

The copper ions are present at the bottom of the large concavity which acts as active site for substrate binding. The active site comprised of hydrophobic residues. Along with ?-helices, tyrosinase also possesses few ? sheets.

Another report on the X-ray absorption near edge structure (XANES) spectra of mushroom tyrosinase in oxy and deoxy forms. The study revealed same conformational changes in the active sites from oxy to deoxy form in both mushroom tyrosinase and mollusc hemocyanins Longa et al, 1996. The amino acid residue adjacent to the copper ions is highly conserved among tyrosinase, catecholoxidases and mollusc haemocyanins Halaouli et al, 2006.

The crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 2.0–2.3 Å. The study shows two monomeric units in asymmetric unit, associated in a homomeric structure with dimensions 45 Å× 25 Å × 80 Å. Both copper ions were coordinated by three His residues, which project out from the four surrounding helices (?2, ?3, ?7, and ?8) and by one loop. CuA is coordinated by His42 (?2), His69 (?3), and His60, located on a large loop intervening between these two helices.

Whereas, the second copper ion CuB is coordinated by His204, His208 (both on ?7), and His231 (?8) Sendovski et al, 2011. Crystal structure of (Agaricus bisporus) mushroom tyrosinase was identified to be tetramer at the molecular level. The structure has two H subunits of ?392 residues and two L subunits ?150 residues Ismaya et al, 2011.Chapter II Review of Literature Page 9 Fig.

1.Overview of tyrosinase from Bacillus megaterium (TyrBm) structure. Copper atoms in active site are presented in brown. Adapted from Sendovski et al, 2011 (http://www.pymol.org/) 2.3.

Mechanism of action Many researchers have studied tyrosinases and recently, it has been considered as model in many reviews for biological oxidation catalysis Que and Tolman, 2008; Rosenzweig and Sazinsky, 2006. In 1983, Lerch et al proposed a reaction mechanism of tyrosinase which describes the common catalytic site for two different reactions showing three different forms of enzyme as met, oxy and deoxy. Depending upon the availability of molecular oxygen and oxidation states of copper ions the enzyme changes its form Olivares and Solano, 2009. The copper pair present at active site of enzyme binds to atomospheric oxygen so as to exhibit two different activities: (1) the ortho-hydroxylation of monophenols by cresolase activity and (2) the oxidation of o-diphenols to o-diquinones by catecholase activity Claus and Decker, 2006; Decker et al, 2000 (fig.2).Chapter II Review of Literature Page 10 Fig.

2 Monophenolase and diphenolase activities of tyrosinases. Claus and Decker, 2006 The type 3 copper protein exihibits different forms during catalysis of reaction as: The deoxy form Cu(I)-Cu(I) which is a reduced species, after binding with oxygen gives the oxy formCu(II)-O22–Cu(II).In the oxy form, molecular oxygen is bound as peroxide in a µ-?2:?2 (peroxodicopper II intermediate) side on bridging mode, which destabilizes the O–O bond and activates it. The met form Cu(II)-Cu(II) is assumed to be a resting enzymatic form, where Cu(II)ions are usually bridged to a small ligands, such as a water molecule or hydroxide ionZekeri et al, 2013.

In the absence of any substrate more than 85% of the enzyme is in the met state, which can be considered as the resting phase of the enzyme. According to current assumptions, both, the met and the oxy-state of tyrosinase are responsible for diphenolase activity, whereas the oxy state contributes to monophenolase activity Claus et al, 2006. The peroxodicopper II intermediate or bis-µ-oxodicopper III intermediate are formed during catalysis of reaction. These intermediates have characteristic EPR and resonance Raman spectra Soloman et al, 1996; Decker and Tuczek, 2000; Siegbahn and Wirstam, 2001. 2.

3.1 Monophenolase activity of tyrosinase It is the first step in melanization pathway by o-hydroxylation of monophenols into diphenols (fig.3).

The monophenolase activity of tyrosinase is conjunctioned with diphenolase activity due to the competitive inhibition pattern among monophenolase and diphenolaseChapter II Review of Literature Page 11 substrates Mason et al, 1956. The monophenolase reaction shows characteristic lag phase before the hydroxylation step has reached Cabanes et al, 1987. Fig.3 Diphenolase and Monophenolase catalytic cycles of tyrosinase. Adopted from Wilcox et al, 1985; Garcaí-Borron and Solano, 2002 2.3.2 Diphenolase activity of tyrosinase The diphenolase activity involves the oxidation of two molecules of o-diphenols to two molecules of o-quinones with a concomitant 4 e- reduction of O2, generating two molecules of water Sanchez-Ferrer,et al, 1994. If non-cyclizable diphenol like 4-methyl catchol is used, then both accumulation of quinones and decrease in oxygen concentration are linear and no lag period is observedSanchez-Ferrer et al, 1998.

In case of cyclizable diphenol like L-DOPA, quinone production is pH dependent. The quinone production and decrease in oxygen concentration is linear at pH 7 whereas at pH myricetin (5,7,3′,4′,5′- pentahydroxy-flavonol) ; kaempferol (5,7,4′-trihydroxyflavonol) > galangin (5,7- dihydroxyflavonol) > morin, buddlenoid A, buddlenoid B Xie et al, 2003; Matsuda et al, 1995. Recently, 6-hydroxykaempferol was synthesized and observed to possess two times more tyrosinase inhibition activity than kaempferol Gao et al, 2007. All these flavanols are comparably less potent inhibitors than kojic acid, which is the most potent tyrosinase inhibitor. Hence, flavanols have very least application in skin whitening and anti-browning.

Some flavanoids like nobiletin, naringin, and neohesperidin were poor inhibitors of mushroom tyrosinase Zhang et al, 2007; Itoh et al, 2009. The extracts of plant parts of Morus species possess wide range of applications in skin whitening. The compound like mulberroside F was purified from the leaves of the plant exhibit the antidiphenolase activity of mushroom tyrosinase which is 4.5-fold higher than that of kojic acid and showed inhibitory effect on melanin formation within melanoma cells Lee et al, 2002.

Norartocarpetin isolated from the stem bark of the plant, was found to be 10.4-fold more active than kojic acid against monophenolase activity of mushroom tyrosinase with a competitive inhibition mode (KI = 1.35 ?M) Ryu et al, 2008.

The flavones seem to be slow binding tyrosinase inhibitors like kojic acid and tropolone. Along with leaves and stem of the plants, roots were also contributed with many potent tyrosinase inhibitors including oxyresveratrol Shin et al, 1998, norartocarpetin, and streppogenin Jeong et al, 2009. Taxifolin was isolated from the sprout of Polygonum hydropiper, showed equal inhibitory activity of kojic acid toward monophenolase activity of mushroom tyrosinase Miyazawa and Tamura, 2007. On the other hand flavone-flavanone dimmer was isolated from seashore plants Garcinia subelliptica and proved to be 3.6-fold more active than kojic acid towards the monophenolase activity of mushroom tyrosinase Masuda et al, 2005.

In East Asian countries, the extracts of roots and seeds of Glycyrrhiza species (Leguminoseae) bears isoflavanoids which were being used as an effective ingredient in skin whitening agent. Two of the potent tyrosinase inhibitors glabridine and glyasperin C were isolated from same plant. Glabridine showed tyrosinase inhibition with 15 times activity of kojic acid and exhibited higher depigmenting activity than that of arbutin Yokota et al, 1998. But glabridine analogs, glabrene were 100 times less active than glabridine Nerya et al, 2003. BothChapter II Review of Literature Page 22 glabridine and gabrene inhibited tyrosinase by non-competitive and uncompetitive modes respectively. Whereas, glyasperin C was found to be two times more active than glabridine Kim et al, 2005.

Several different derivatives of isoflavanoids were isolated from soyabean koji when fermented with Aspergillus oryzae and displayed three hydroxyisoflavones such as 6-hydroxydaidzein (6,7,4’trihydroxyisoflavone), 8-hydroxydaidzein (7,8,4′-trihydroxyisoflavone), and 8-hydroxygenistein (5,7,8,4′-tetrahydroxyisoflavone) as potent tyrosinase inhibitors Chang et al, 2005; Chang et al, 2007. Among these, 6-hydroxydaidzein with 6-fold more than kojic acid show competitive mode of action on the L-tyrosine while the other two 8-hyroxyisoflavones irreversibly inactivate the enzyme and belong to the suicide substrates of tyrosinase T. S. Chang, 2007. Haginin A isolated from the branch of Lespedeza cyrtobotrya was shown to be 10-fold more active than kojic acid towards monophenolase activity of mushroom tyrosinase in a non-competitive mode Kim et al, 2008.

It inhibited melanin synthesis in melanoma cells and decreased UV induced skin pigmentation in brown guinea pigs. In the zebrafish model system too, haginin A showed remarkable inhibition in the body pigmentation. Another isoflavanoid, dalbergioidin was also isolated from L. cyrtobotrya and non-competitively inhibited the monophenolase activity of mushroom tyrosinase Baek et al, 2008. The third potent inhibitor discovered by the authors is calycosin, which showed slightly higher monophenolase inhibitory activity against mushroom tyrosinase than that of kojic acid. It bears two mechanisms to reduce melanogenesis in melanoma cells, including inhibiting tyrosinase activity and reducing the expression of tyrosinase Kim et al, 2009.

Another group of flavanoids, chalcones also proved to be potent tyrosinase inhibitors. The chalcones derivatives like licuraside, isoliquiritin, and licochalcone A were isolated from the roots of the Glycyrrhiza species and competitively inhibited the monophenolase activity of mushroom tyrosinase. Licochalcone A showed 5.

4-fold more inhibition than kojic acid Fu et al, 2005. The prenylated chalcone, kuraridin was isolated from the plant Sophora flavescens and identified as a potent tyrosinase inhibitor. It was 34 times active than kojic acid against monophenolase activity of mushroom tyrosinase Kim et al, 2003. Its hydroxyl analog, kuraridinol was found to be 18.4-fold more active compared to kojic acid Hyun et al, 2008. The two prenylated chalcones displayed significantly more activity than those of their corresponding flavanone analogs.

Kuraridin (a chalcone) is 10-fold more active than kurarinone (a flavanone)Chapter II Review of Literature Page 23 while kuraridinol (a chalcone) is also 10-fold more active than kurarinol (a flavavone). This emphasizes that tyrosinase inhibitors with a chalcone structure have enhanced potential than flavanone. Recently, 2,4,2′,4′-tetrahydroxy-3-(3-methyl-2-butenyl)chalcone (TMBC) was isolated from the stems of Morus nigra, exhibited 26-fold more potent than kojic acid in diphenolase inhibitory activity of mushroom tyrosinase Zhang et al, 2009. The kinetic study showed that the compound is competitive to the L-dopa binding site of tyrosinase with a KI value of 1 to 1.5 µM. Even, the melanin content of melanoma cells was reduced to 31% by 30 µM of TMBC treatment.

The inhibitory effect of TMBC on melanogenesis was attributed to the direct inhibition of tyrosinase activity, rather than suppression of tyrosinase gene expression. The 4-resorcinol moiety (2,4dihydroxyl groups in the aromatic ring) in the chalcone structure is responsible for potent inhibitory activity. Few simple 4-alkylresorcinols were exhibited strong tyrosinase inhibitory activity Shimizu et al, 2000; Chen et al, 2004. Later on it was found that 2,4,2′,4′-tetrahydroxychalcone possesses the most potent monophenolase inhibitory activity compared with 3,4,2′,4′-tetrahydroxychalcone and 2,4,3′,4′-tetrahydroxychalcone Khatib et al, 2005.

Therefore, it was concluded that 4-resorcinol moiety in the chalcone skeleton plays the prime role in exhibiting inhibitory potency. In 2007, Jun et al. chemically synthesized a series of hydroxychalcones and determined the tyrosinase inhibitory activity Jun et al, 2007. As discussed earlier, it is interesting to note that the identified potent tyrosinase inhibitors with a flavone, flavanone, or flavonol skeleton such as artocarpetin, norartocarpetin, and streppogenin all possess 4-resorcinol in the B ring.

Thus, the 4-resorcinol moiety plays an important role in the inhibition of tyrosinase activity not only in chalcones but also in other flavonoid structures. Stilbenes are other polyphenols also showed potent tyrosinase inhibitory activity. Oxyresveratrol is a stilbene which was isolated from Morus alba, exhibited 32-fold more inhibitory activity than that of kojic acid Shin et al, 1998. It inhibits both monophenolase and diphenolase activity of mushroom tyrosinase non-competitively.

The mechanism of the compound in inhibition of melanogenesis proved that it directly inhibits enzyme activity but does not affect gene expression Kim et al, 2002. Apart from oxyresveratrol, other three hydroxystilbenes were also purified and identified as potent tyrosinase inhibitors. Chloroporin was isolated from the heartwood of Chlorophora excelsa and gives 14.8-fold inhibitory activity than kojic acid towards diphenolase activity of mushroom tyrosinase Kuniyoshi et al, 2003.Chapter II Review of Literature Page 24 Gnetol was extracted from the roots of Gnetum gnemon, exhibited 30-fold more diphenolase inhibitory activity of murine tyrosinase than that of kojic acid Ohguchi et al, 2003. Recently, piceatannol was isolated from grapes and red wine, gave 32.7-fold inhibitory activity for monophenolase than kojic acid toward mushroom tyrosinase Yokozawa and Kim, 2007.

Coumarins also proved to be strong tyrosinase inhibitors. Aloesin is a natural hydroxycoumarin glucoside isolated from Aloe vera. Aloesin shows more inhibition of crude murine tyrosinase than mushroom tyrosinase and has been recently used in cosmetics Jones et al, 2002; Choi et al, 2002.

The coumarin analog, 9-hydroxy-4- methoxypsoralen was isolated from Angelica dahurica and displays six times more tyrosinase inhibitory activity than that of kojic acid Piao et al, 2004. Recently, a new coumarin derivative, 8′-epi-cleomiscosin A was extracted from the aerial parts of Rhododendron collettianum and exhibits 12.8-fold diphenolase inhibitory activity than that of kojic acid towards mushroom tyrosinase Ahmad et al, 2004. Since last decade, many benzaldehye and benzoate derivatives have been used as strong tyrosinase inhibitors such as benzoic acid, benzaldehyde, anisic acid, anisaldehyde, cinnamic acid, and methoxycinnamic acid from the roots of Pulsatilla cernua Lee, 2002. The other derivatives were also gained like 4-substituted benzaldehydes from cumin Jimenez et al, 2001, 2-hydroxy-4-methoxybenzaldehyde from roots of Mondia whitei Kubo and Kinst-Hori, 1999, p-coumaric acid from the leaves of Panax ginseng Lim et al, 1999, hydroxycinnamoyl derivatives from green coffee beans Iwai et al, 2004, and vanillic acid and its derivatives from black rice bran Miyazawa, 2003. All of these inhibitors show low to moderate inhibiton of tyrosinase as compared tgo kojic acid. The tyrosinase inhibitory mechanism of benzaldehyde-type inhibitors is due to formation of a Schiff base with a primary amino group in the enzyme Kubo and Kinst-Hori, 1998; 1999. Whereas, benzoate inhibits tyrosinase by chelating copper and belonging to a typical HA-type acid tyrosinase inhibitor, its inhibitory mechanism involves the interaction between the non-ionized form of the inhibitor and the copper in the active site of the enzyme Conrad et al, 1994.

Protocatechualdehyde was isolated from the fruiting body of Phellinus linteus and showed 7.8 fold more anti-tyrosinase activity than that of kojic acid Kang et al, 2004. Its analogue like protocatechuic aldehyde, with two methoxyl groups replacing the two hydroxyl groups gives one order of magnetude of lower activity than that of protocatechualdehyde No et al, 2004. Another analog, protocatechuic acid isolated from black rice bran with a benzoate skeleton, showed lowerChapter II Review of Literature Page 25 activity Miyazawa et al, 2003. To improve the inhibitory strength of the benzaldehyde-type inhibitors, some derivatives were chemically synthesized and their tyrosinase inhibitory activities were determined.

4-Vinylbenzaldehyde with 35 fold Song et al, 2005, 4-alkylbenzaldehyde with 100 fold Xue et al, 2008; Chen et al, 2003, 2-hydroxy-4-isopropylbenzaldehyde with 350 fold Nihei et al, 2004, 3,4-dihydroxybenzaldehyde-O-ethyloxime Lev and Bertram, 2001 and 4-butylbenzaldehyde thiosemicarbazone with 1500 and 2000 fold respectively Xeu et al, 2007 were more potent than that of the precursor benzaldehyde . Gallic acid was isolated and identified as a tyrosinase inhibitor from many plants. The ester derivatives of gallic acid and its inhibitory mechanism was well studied Kubo et al,2000; 2003.

It was found that gallic acid inhibited diphenolase activity of mushroom tyrosinase with a IC50 value of 4500 µM, which is much lower than that of kojic acid. It was found that gallic acid is very toxic to melanoma cells with cytotoxicity as compared with hydroquinone Kang et al, 2003. Recently, several lipids were purified from natural sources and showed anti- tyrosinase activity. A triacylglycerol, trilinolein was isolated from sake lees, (byproducts of sake production) and proved to be as potent as kojic acid for inhibition of diphenolase activity of mushroom tyrosinase Jeon et al, 2006. A glycosphingolipid, soyacerebroside I was isolated from the leaves of Guioa villosa and inhibited monophenolase and diphenolase activitivities of mushroom tyrosinase with half-activity of kojic acid Maqid et al, 2008 while another glycosphinogolipid, cerebroside B, from Phellinus linteus showed no inhibition against the tyrosinase Kang et al, 2004. Three steroids were isolated from the aerial parts of Trifolium balansae giving higher diphenolase inhibitory activity toward mushroom tyrosinase than that of kojic acid Sabudak et al, 2006. Among them, stigmast-5-ene-3?,26-diol showed 7-fold more inhibition.

A long-chain ester, 2?(2S)-hydroxyl-7(E)-tritriacontenoate was isolated from Amberboa ramose . It inhibited diphenolase activity of mushroom tyrosinase 12.3-fold more than kojic acid Khan et al, 2005. A triterpenoid, 3?,21,22,23-tetrahydroxycycloart-24(31),25(26)-diene had an extremely potent inhibition (12.6-fold) against diphenolase activity of mushroom tyrosinase than kojic acid Khan et al, 2006.

At the same time, they also purified some triterpenoid glycosides from the roots of Astragalus taschkendicus. However, theChapter II Review of Literature Page 26 triterpenoid glycosides showed only inhibitory activity same as that of kojic acid against mushroom tyrosinase Khan et al, 2006. Furthermore, Choudhary and Khan purified nine pentacyclic triterpenes from the aerial part of the plant Rhododendron collettianum, and all showed potent diphenolase inhibition of mushroom tyrosinase.

arjunilic acid was the strongest tyrosinase inhibitor among them which was 16.7-fold inhibitory active of kojic acid Ullah et al, 2007. Along with triterpenoids, the authors also purified many diterpenoids from the aerial parts of Aconitum leave Shaheen et al, 2005. However, most of the diterpenoids did not inhibit tyrosinase activity except lappaconitine hydrobromide, revealed activity similar to that of kojic acid Sultankhodzhaev et al, 2005. As like diterpenoids, few monoterpenoids were also seemed to be strong tyrosinase inhibitors. Only a monoterpenoid, crocusatin-K, isolated from the petals of Crocus sativus, displayed inhibitory activity equal to that of kojic acid Li et al, 2004. Many inhibitors from natural and synthetic sources were screened out showing potent tyrosinase activity. Recently, an anthraquinone, physcion (1,8-dihydroxy-2-methoxy-3-methylanthraquinone) was found to show similar anti-tyrosinase activity like that of kojic acid Leu et al, 2008.

Another anthraquinone, 1,5dihydroxy-7-methoxy-3-methylanthraquinone with hydroxyl and methoxyl groups at different positions exhibited 72-fold more antityrosinase activity compared with those of physcion Devkota et al, 2007. Therefore, it reveals structure-activity relationship between the functional groups attached to the anthraquinone skeleton and the antityrosinase activity. In addition, many lignans were isolated from the roots of Vitex negundo showed higher tyrosinase inhibitory activity than kojic acid. The most active lignan from the plant was (+)-lyoniresinol with 5.2-fold higher activity than that of kojic acid Azhar-Ul-Haq et al, 2006. Along with inhibitors from plant sources some inhibitors from marine environment also showed production of substances responsible for inhibition of tyrosinase. dieckol is a phloroglucinol derivative was isolated from a marine brown alga, Ecklonia stolonifera and showed three times more activity than that of kojic acid Kang et al, 2004.

A marine-derived fungus Myrothecium sp. was found to contain 6-n-pentyl-?-pyrone proved to be a potent tyrosinase inhibitor Li et al, 2005. Recently, another tyrosinase inhibitor was purified from a marine bacteria, Trichoderma viride strain H1-7, and showed competitive inhibitionChapter II Review of Literature Page 27 against monophenolase activity of mushroom tyrosinase by binding to a copper active site of the enzyme Tsuchiya et al, 2008. Many synthetic tyrosinase inhibitors have also been synthesized and used as antityrosinase agents. N-Phenylthiourea (PTU) is a well-known tyrosinase inhibitor belonging to the type-3 copper protein group. It inhibits diphenolase activity such that the sulfur atom of the compound binds to both copper ions in the active site of the enzyme and blocks enzyme activity Gerdemann et al, 2002. Various analogs of N-phenylthiourea were synthesized and it was noticed that theses analogs showed more inhibitory activity against diphenolase activity of mushroom tyrosinase than that of N-phenylthiourea Criton and Mellay-Hamon, 2008.

In addition, an analog of cupferron, which is a well-known metal chelating agent was synthesized by Shiino et al. cupferron. It inhibited competitively both monophenolase and diphenolase activity of mushroom tyrosinase Xie et al, 2003, and later on it was found that N-substituted-N-nitrosohydroxylamines inhibited mushroom tyrosinase by interacting with the copper ions at the active site of the enzyme Shiino et al, 2001. Furthermore, new tyrosinase inhibitors were developed by chemically synthetic methods included as sildenafil Khan et al, 2005, oxadiazole Khan et al, 2005, oxazolones Khan et al, 2006, and tetraketones types Khan et al, 2006. Another group of authors demonstrated that 1,3 selenazol-4-one derivatives Koketsu et al, 2002, selenourea derivatives Ha et al, 2005, and selenium-containing carbohydrates Ahn et al, 2006 has inhibitory activity similar to that of kojic acid against diphenolase activity of mushroom tyrosinase. Many people synthesized some simple phenyl and biphenyl compounds and used as potent tyrosinase inhibitors. 4,4′-Dihyldroxybiphenyl showed monophenolase inhibitory activity of mushroom with a competitive inhibition mode Kim et al, 2005 whereas its glucoside derivatives isolated from the fruit of Pyracantha fortuneana displayed low inhibitory activity Dai et al, 2006.

In addition, S-phenyl N-phenylthiocarbamate Lee et al, 2005 and 4-(2′,4′-dihydroxyphenyl)(E)-3-buten-2-one Kuo et al, 2005 were recently found to be more potent inhibitors than kojic acid toward diphenolase activity of mushroom tyrosinase. However, though a huge numbers of synthetic inhibitors were successful used in tyrosinase inhibition, few have been used in melanogenesis inhibiting activity in cells or skin models.Chapter II Review of Literature Page 28 2.

7. Surface plasmon resonance (SPR) biosensors Surface plasmon resonance (SPR) technique is an optical method for measuring the refractive index of very thin layer of material immobilized on a metal gold surface. It is an indespensible tool used for label free and molecular binding interactions. In earlier days, SPR biosensors were bearing stratified medium model with dicrete molecules approximated with a uniform thin film Yu et al, 2014. Whereas since last decade, it is the powerful and versatile spectroscopic method for determining the biomolecular interactions including protein:ligand, protein:protein, protein:DNA, protein:membrane binding, ligand-receptor coupling, antibody-antigen coupling Patil et al, 2014; Surwase et al, 2015.

A SPR-based instrument allows the analysis of biomolecular interaction in real time with no labeling requirements and can measure mass changes down to 10pg/mm2. Applications shown include kinetic measurements (ka, kd), binding site analysis and concentration determination, screening of drug molecules, specificity analysis Fagerstam et al, 1992; Pattanaik, 2005; Liedberg et al, 1993; Rich and Myszka, 2000; 2001; 2002. 2.

7.1. Principle of Surface plasmon resonance The SPR-based biosensors use an optical method to measure the change in refractive index near a sensor surface. For detection of an interaction, one molecule is immobilised onto the sensor surface while the other molecule (the analyte) is injected in aqueous solution over the sensor surface. When the analyte binds to the immobilised molecule on the sensor surface, the mass at the surface increase, and thereby the refractive index increases. This change in refractive index is measured in real time, and plotted as response or resonance units (RU) versus time (a sensorgram). The sensorgram provides essentially two kinds of information: (1) the rate of interaction (association, dissociation or both), which provides information on kinetic rate constants and analyte concentration; and (2) the binding level which can provide information on affinity constants and analyte concentration The principle of SPR, however, only occurs when the light’s wavevector component parallel to the metal surface matches that of the surface plasmon polaritons (SPP). This happens at specific angles of incidence, appearing like a drop in the reflectivity of incident light 17,18.

SPR biosensing is based on the principle that any change in the dielectric sensing surface causesChapter II Review of Literature Page 29 a shift in the angle of reflectivity, followed by a detector, in order to satisfy the resonance condition as shown in figure (5) Daghestani and Day, 2010. Fig.5.Detection of binding events for SPR 2.7.2. Importance and Applications of Surface plasmon resonance biosensors From over a last decade SPR biosensors are excessively used in a wide range of applications due to its major advantages as fast response, can detect multiple number of analytes at a time and all the analysis are label free and real time based. During 1980s, SPR and related techniques were exploited to study biological and chemical interaction Bernard and Lengeler, 1978; Flanagan and Pantell, 1984; Liedberg et al, 1983.

These sensors are used in examination of biological system including antibody–antigen, ligand–receptor, and protein–nucleic acid interactions Blaesin et al, 1999; Hart et al, 1999. Interactions between DNA–DNA, DNA–protein, lipid–protein, and complex biomolecules can be studied using SPR biosensor-based instruments. It can be used for not only qualitative but also quantitative applications. Qualitative applications consists of ligand fishing Catimel et al, 2000; Guermazi et al, 2000; small molecule and drug screening, molecular assembly, epitope mapping Achen et al, 2000; Vogel etChapter II Review of Literature Page 30 al, 2000 specificity analysis, and small-scale affinity flow purification. As a quantitative tool, SPR biosensors facilitates with reaction kinetics (ka, kd) and affinity constants, equilibrium constants (kD) for molecular interactions, estimation of functionally active concentration, thermodynamics (?Hvant Hoff), stoichiometry, and mechanisms of receptor– ligand interaction Pattanaik, 2005.

SPR technique has been used to determine the kinetics of cholera toxin binding to gangliosides inserted in lipid monolayer Kuziemko et al, 1996l; Terrettaz et al, 1993 and captured vesicles MacKeizie et al, 1997; 2000. In 2000, Cooper and coworkers Cooper et al, 2000 reported a vesicle capture sensor chip used for kinetic analysis of membrane bound receptors. In 2001, Ellson and his team Ellson et al, 2001 studied membrane bound subunits of neutrophils and their role in production of reactive oxygen species by using SPR biosensor along with polymer-supported lipid bi-layers. Ravanat and coworkers had used Biacore system to monitor the specific interaction of platelets with surface bound proteins Ravanat et al, 1998. The speed, automation, and high data resolution of SPR-based biosensors make these instruments ideal as drug discovery tools. The SPR technology has been used for characterization of target molecules and biopharmaceuticals Atwell et al, 1997; Mangold et al, 1999. The screening of compound libraries for binding to target proteins has been possible due to biosensor assays.

With the help of SPR technique, HIV-1 protease inhibitors have been characterized successfully Hamalainen et al, 2000; Markgren et al, 1998. Interaction between thrombin and thrombin inhibitors were also studied using biosensors to evaluate drug target interactions Karlsson et al, 2000. Both high- and low-affinity small molecule interactions can be analysed using SPR Adamczyk et al, 2000. Markgren and coworkers used SPR biosensors in screening of small-molecules Markgren et al, 1998.The assessment of biological processes like adsorption, distribution, metabolism, and excretion (ADME) such as compound’s ability to bind to serum carrier proteins and also permeate across membrane was studied using SPR-based technology Frostell-Karlsson et al, 2000.

The SPR technique was used for the characterization of human protein kinase (PrKX) and its interactions with known inhibitors of cAPK and its regulation by the second messenger cAMP in vitro and in vivo were studied Zimmermann et al, 1999. Further, improvement in SPR instrumentation enabled detection of small molecules, such asChapter II Review of Literature Page 31 drugs (?138 Da) binding to human serum albumin Frostell-Karlsson et al, 2000 and small oligosaccharides (gallicacid; tannic acid ;ascorbic acid. Catechol and pyrogallol showed higher binding affinities towards yam tyrosinase. The significance of SPR is continuous monitoring of label free biomolecular interactions which has great scope in drug discovery. The sensors can be used for detection of small molecules in food and pharmaceutical industries.

This data will help to study the role of tyrosinase in hyperpigmentation which willChapter V Summary and conclusion Page 74 create an avenue for tyrosinase inhibitors. Tyrosinase inhibitors have wide range of applications in cosmetics, medical and food industries. In fourth chapter, a new approach of heterogenous analyte kinetics model was implemented using surface plasmon resonance technique. The study was conducted with inhibitors like kojic acid, crocin and hydroquinone along with substrate L-DOPA with different concentrations were subjected to SPR to determine their individual binding affinities towards yam tyrosinase. At the same time cocktails of inhibitor-inhibitor and inhibitor-substrate with same concentration were used for SPR analysis. The results revealed that cocktails have higher affinities than those of individual inhibitor. KD values in heterogenous mixture were increased than that of individual inhibitors (assuming it prevents oxidation) which resulted into increased binding affinity of both the inhibitors in mixture than individual one.

This method can provide both qualitative and quantitative data. The significance of the present work is that all the experiments were performed on a single immobilized enzyme surface making results more reliable. The fluorescence study also supported the SPR data. The sensors with such strategies can be used in drug discovery and development process.

The study also provides us with a better option of heterogenous mixture of inhibitors in inhibition of an enzyme. It will be helpful in designing inhibition of tyrosinase by using cocktails of two or more inhibitors. It will open the new aspect in the study of inhibition of enzyme.

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