A number of tyrosinase inhibitors from both natural and synthetic sources have been identified. However, the definition of “tyrosinase inhibitor” is sometimes misleading: many authors use that terminology in reference to melanogenesis inhibitors, whose action mainly resides in some interference in melanin formation, regardless of any direct inhibitor/enzyme interaction. Many putative inhibitors are examined in the presence of tyrosine or dopa as the enzyme substrate, and activity is assessed in terms of dopachrome formation. Thus, experimental observation of the inhibition of tyrosinase activity can be accomplished by one of following:
- Reducing agents causing chemical reduction of dopaquinone such as ascorbic acid, which is used as a melanogenesis inhibitor because of its capacity to reduce back o-dopaquinone to dopa, thus avoiding dopachrome and melanin formations.
- o-Dopaquinone scavenger such as most thio-containing compounds, which are well-known melanogenesis inhibitors and react with dopaquinone to form colorless products. The melanogenetic process is therefore slowed until all the scavenger is consumed, and then it goes at its original rate.
- Alternative enzyme substrates such as some phenolic compounds, whose quinoid reaction products absorb in a spectral range different from that of dopachrome. When these phenolics show a good affinity for the enzyme, dopachrome formation is prevented, and they could be mistakenly classified as inhibitors.
- Nonspecific enzyme inactivators such as acids or bases, which non-specifically denature the enzyme, thus inhibiting its activity.
- Specific tyrosinase inactivators such as mechanism-based inhibitors, which are also called suicide substrates. These inhibitors can be catalyzed by tyrosinase and form covalent bond with the enzyme, thus irreversibly inactivating the enzyme during catalytic reaction. They inhibit tyrosinase activity by inducing the enzyme catalyzing “suicide reaction.”
- Specific tyrosinase inhibitors such as most compounds discussed in this review. The compounds reversibly bind to tyrosinase and reduce its catalytic capacity.
Among the six types of compounds described above, only specific tyrosinase inactivators (5) and inhibitors (6) are regarded as “true inhibitors,” which actually bind to the enzyme and inhibit its activity. General speaking, the mistaken inhibitors exhibit only weak inhibitory activity due to their reactive and consumable properties toward the enzyme or the quinone products. Although some tyrosinase inhibitors exhibited multifunctional activities, the compounds, which are well-known reductive agents, dopaquinone scavengers, or tyrosinase substrates and do not obviously belong to the true inhibitors, are not suitable for comparison with the true inhibitors and are ignored in the present article.
Usually, “true inhibitors” are classified into four types, including competitive inhibitors, uncompetitive inhibitors, mixed type (competitive/uncompetitive) inhibitors, and non-competitive inhibitors (). A competitive inhibitor is a substance that combines with a free enzyme in a manner that prevents substrate binding. That is, the inhibitor and the substrate are mutually exclusive, often because of true competition for the same site. A competitive inhibitor might be a copper chelator, non-metabolizable analogs, or derivatives of the true substrate. In contrast, an uncompetitive inhibitor can bind only to the enzyme-substrate complex. A mixed (competitive and uncompetitive mixed) type inhibitor can bind not only with a free enzyme but also with the enzyme-substrate complex. For most mixed-type inhibitors, their equilibrium binding constants for the free enzyme and the enzyme-substrate complex, respectively, are different. However, a special case among the mixed inhibitors is the non-competitive inhibitors, which bind to a free enzyme and an enzyme-substrate complex with the same equilibrium constant. In addition to the inhibitory mechanism, inhibitory strength is the primary criterion of an inhibitor. Inhibitor strength is usually expressed as the inhibitory IC50 value, which is the concentration of an inhibitor needed to inhibit half of the enzyme activity in the tested condition. However, for the tyrosinase inhibitors in the literature, the IC50 values are incomparable due to the varied assay conditions, including different substrate concentrations, varied incubation time, and different batches of commercial tyrosinase. Fortunately, in most studies conducted to discover new tyrosinase inhibitors, a well-known tyrosinase inhibitor such as kojic acid is often used as a positive standard at the same time. Hence, in order to compare the inhibitors described in different literature in a more practical manner, a relative inhibitory activity (RA), which is calculated by dividing the IC50 value of kojic acid with that of a newly found inhibitor in the same report, is used to express and compare the inhibitory strength of an inhibitor with others in this review.
Scheme 1. Action mechanism of reversible inhibitors. E, S, I, and P are the enzyme, substrate, inhibitor, and product, respectively; ES is the enzyme-substrate complex, and EI and ESI are the enzyme-inhibitor and enzyme-substrate-inhibitor complexes, respectively. (more ...)
Kojic acid (), the most intensively studied inhibitor of tyrosinase, is a fungal metabolite currently used as a cosmetic skin-whitening agent and as a food additive for preventing enzymatic browning [25
]. Kojic acid shows a competitive inhibitory effect on monophenolase activity and a mixed inhibitory effect on the diphenolase activity of mushroom tyrosinase. The ability of kojic acid to chelate copper at the active site of the enzyme may well explain the observed competitive inhibitory effect. In addition, kojic acid is reported to be a slow-binding inhibitor of the diphenolase activity of tyrosinase [26
]. This means that the active form of tyrosinase, generated in the catalytic cycle in the presence of the substrate, is required before binding of the inhibitor to the enzyme can occur. Other slow-binding inhibitors of tyrosinase are the very potent inhibitor tropolone [27
] and the substrate analog l
] (). Strikingly, these slow-binding inhibitors of tyrosinase all contain an α-hydroxyketone group. Kojic acid together with tropolone and l
-mimosine are often used as the positive control in the literature for comparing the inhibitory strength of the finding inhibitors.
Figure 3. Chemical structures of selected tyrosinase inhibitors belonging to some standard ones (a), flavonoids (b–j) or N-benzylbenzamides analogs (k). RAa and RAb are the relative diphenolase and monophenolase inhibitory activity, respectively, against (more ...)
In addition to the standard tyrosinase inhibitors, a huge number of new inhibitors, especially for those discovered in the last five years, are collected in this review and classified into five major classes, including polyphenols, benzaldehyde and benzoate derivatives, long-chain lipids and steroids, other natural or synthetic inhibitors, and irreversible inactivators based on either the chemical structures or the inhibitory mechanism.
Polyphenols represent a diverse group of compounds containing multiple phenolic functionalities and are widely distributed in nature. Polyphenols are also the largest groups in tyrosinase inhibitors until now. Since several polyphenols are accepted as substrates by tyrosinase, it depends on the presence and position of additional subsistent whether a polyphenol may act as an inhibitor. Flavonoids are among the most numerous and best-studied polyphenols, that is, benzo-γ-pyrone derivatives consisting of phenolic and pyrene rings. Widely distributed in the leaves, seeds, bark, and flowers of plants, more than 4,000 flavonoids have been identified to date. In plants, these compounds give protection against UV radiation, pathogens, and herbivores [29
]. They are also responsible for the characteristic red and blue colors of berries, wines, and certain vegetables. Flavonoids may be subdivided into seven major groups, including flavones, flavonols, flavanones, flavanols, isoflavonoids, chalcones, and catechin. Different classes of flavonoids are distinguished by additional oxygen-heterocyclic rings, by positional differences of the B ring, and by hydroxyl, methyl, isoprenoid, and methoxy groups distributed in different patterns about the rings. The structure of flavonoids is also in principle compatible with the roles of both substrates and (presumably competitive) inhibitors of tyrosinase. Detailed studies have shown that some flavonoids are in fact rather potent inhibitors and discussed in this section. In addition to flavonoids, other polyphenols, which were also identified as tyrosinase inhibitors, contain stilbenes and coumarin derivatives.
Many flavonols have been isolated from plants, and some were identified as tyrosinase inhibitors. The inhibitory mode of flavonol inhibitors is usually competitive inhibition for the oxidation of l
-dopa by tyrosinase and the 3-hydroxy-4-keto moiety of the flavonol structure acts as the key role in copper chelation [30
]. In terms of inhibitor strength, the discovered flavonol inhibitors are ranking as follows: quercetin (5,7,3′,4′-tetrahydroxyflavonol) > myricetin (5,7,3′,4′,5′-pentahydroxy-flavonol) > kaempferol (5,7,4′-trihydroxyflavonol) > galangin (5,7-dihydroxyflavonol) >> morin, buddlenoid A, buddlenoid B [32
]. In addition, the flavonol glycosides of quercetin or kaempferol were found to be less active than their corresponding aglycones [34
]. Recently, 6-hydroxykaempferol was synthesized and confirmed to possess two times more activity than kaempferol [35
]. Although many flavonols have been identified as tyrosinase inhibitors, all the flavonol inhibitors listed above are very weak inhibitors; the most active flavonol, quercetin (), showed only 20% of the inhibitory strength of kojic acid toward diphenolase activity of mushroom tyrosinase. Hence, it is obvious that these flavonol inhibitors have little potential in applications of skin whitening or food antibrowning.
Flavones, flavanones, and flavanols
Citrus peel as a by-product of the citrus juice industry contains a large amount of flavonoids. Some of the flavonoids were identified as tyrosinase inhibitors, including nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone), naringin (5,7,4′-trihydroxyflavanone), and neohesperidin (5,7,3′-trihydroxy-4′-methoxyflavone). However, the inhibitory strength of the three inhibitors was found to be poorly active toward mushroom tyrosinase compared with kojic acid [36
]. Although no potent tyrosinase inhibitors have been isolated from citrus fruit until now, the ethanolic extract of citrus fruit exhibited in vitro
inhibitory effects on melanogenesis in melanoma cells and in vivo
prevention against UVB-induced pigmentation of dorsal skin in brown guinea pigs. The melanogenesis inhibitory activity of citrus crude extracts was found to be mainly attributed to the antioxidant activity of neohesperidin in citrus fruit.
In addition to citrus extracts, the extracts from Morus
species, which has been well-known as a polyphenol-rich plant and used as a non-toxic natural therapeutic agent, also have high potential in applications as skin-whitening agents due to many potent tyrosinase inhibitors being isolated from different parts of the plant. Mulberroside F (moracin M-6,3′-di-O
-glucopyranoside) purified from the leaves of the plant showed the antidiphenolase activity of mushroom tyrosinase to be 4.5-fold higher than that of kojic acid and exhibited an inhibitory effect on melanin formation within melanoma cells [38
]. Norartocarpetin (5,7,2′,4′-tetrahydroxyflavone, ), 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 μM) [39
]. The flavone was also demonstrated to be a slow-binding inhibitor just like kojic acid and tropolone. In addition to the leaves and stem of the plant, the roots of the Morus
species were also found to contain many very potent tyrosinase inhibitors, including oxyresveratrol [40
], norartocarpetin, and streppogenin (5,7,2′,4′-tetrahydroxy-flavavone, ) [41
]. Oxyresveratrol is a hydroxystilbene with trans
configuration, and its inhibitory property will be discussed in the later “silbenes
” section. The chemical structure of streppogenin (a flavanone) is very similar to that of norartocarpetin (a flavone) with the same four substituted hydroxyl groups. It is reasonable that streppogenin possesses an inhibitory property similar to that of norartocarpetin including similarly inhibitory strength on monophenolase activity (IC50
= 0.57 and 0.47 μM, respectively) and the same slow-binding and competitive inhibition mode (KI
= 0.7 and 0.6 μM, respectively) against mushroom tyrosinase. Moreover, the two potent inhibitors, together with another two analogs, dihydromorin (5,7,2′,4′-tetrahydroxyflavanol, ) and artocarpetin (5,2′,4′-trihydroxy-7-methoxyflavone, ), were recently isolated from the wood of Artocarpus heterophyllus
It is very interesting to compare the inhibitory strength toward monophenolase activity of mushroom tyrosinase among the four analogs described above. First, it was found that methoxylation of the hydroxyl group at the C7 position of the flavone skeleton revealed a 100-fold decrease in inhibitory activity by comparing the inhibitory strength between artocarpetin and norartocarpetin. In fact, for most tyrosinase inhibitors, glycosylation and methoxylation of the specific hydroxyl group on the aromatic ring would heavily affect exerting their inhibitory activity. Similar results were also found that forty-five flavonoids glycosides were recently isolated from Marrubium
species, and all exhibited very low tyrosinase inhibitory activity [44
]. The reasons for causing the decreased inhibitory activity by glycosylation and methoxylation of flavonoids include the loss of the specific hydroxyl group, which plays a key role in showing the inhibition and formation of the hydrophilic and stereohindrance effects of the bulky glycoside moiety, which prevent the inhibitor from fitting into the active site of the enzyme. Second, it has been mentioned that norartocarpetin and streppogenin have very similar inhibitory activity and mode toward mushroom tyrosinase. In contrast, dihydromorin, which contains an extra 3-hydroxy group and hence belongs to the flavanol class of flavonoids, exhibited a 20-fold decrease in inhibitory activity compared with that of streppogenin and norartocarpetin. As described in the “flavonol
” section, it has been demonstrated that the 3-hydroxy-4-keto moiety in the flavonol inhibitors acts as the key role in copper chelation, and loss of the group will completely abolish inhibitory activity [31
]. Based on the mechanism, it is difficult to explain the lowered inhibitory capacity of dihydromorin, which contains the 3-hydroxy group and thus forms the 3-hydroxy-4-keto moiety in its structure. Although the 3-hydroxy-4-keto moiety of dihydromorin could form the copper chelating capacity, it seems that the extra 3-hydroxy group in dihydromorin had more drawbacks on tyrosinase inhibition. According to this, the criteria for tyrosinase inhibition by the flavonoids become an interesting issue. Especially, the effects of both the numbers and the positions of hydroxyl groups attached to the flavonoids skeletons on the inhibitory activity of mushroom tyrosinase are the main concern. Kim et al
. recently systematically tested a large number of flavonoids with different numbers and positions of hydroxyl groups using the fluorescence quenching spectroscopic method [45
]. Both the tyrosinase inhibitory activities and copper chelating capacities of the flavonoids were evaluated in the work. From the results, the authors concluded that the enzyme tyrosinase is primarily quenched by the hydroxyl groups of A and B rings on the ether side of the flavonoids (C6 to C8 and C2’ to C4’). According to the finding, the extra 3-hydroxyl group of dihydromorin would indeed reduce its inhibitory activity compared to streppogenin due to the 3-hydroxyl group making dihydromorin more difficult to form a binding complex with the enzyme. Their result could also explain that most flavonols or flavanols inhibitors exhibited only weak to moderate inhibitory strength.
Another flavanol, taxifolin (5,7,3′,4′-tetrahydroxyflavanol, ) isolated from the sprout of Polygonum hydropiper
, showed equal inhibitory activity of kojic acid toward monophenolase activity of mushroom tyrosinase [46
]. In an advance study, it is interesting to find that taxifolin would effectively inhibit both the tyrosinase activity of the living cells and cellular melanogenesis as effectively as arbutin, despite its effects on increasing the tyrosinase protein level [47
]. In addition to flavonoid monomers, one flavone-flavanone dimmer was isolated from seashore plants, Garcinia subelliptica
, and revealed to be 3.6-fold more active than kojic acid against the monophenolase activity of mushroom tyrosinase [48
The extracts from the roots and seeds of Glycyrrhiza
species (Leguminoseae) have long been regarded as an effective constituent for skin-whitening agents in East Asian countries. The melanogenesis inhibitory activity of the extracts mainly comes from the isoflavonoids in the plant. Two isoflavans were purified from the roots of the plant and identified as potent tyrosinase inhibitors. Glabridine () was the first confirmed inhibitor with 15 times activity of kojic acid and exhibited higher depigmenting activity than that of arbutin [49
]. In contrast, glabridine’s analog, glabrene, was found to be 100-fold less active than glabridine [50
]. Kinetics study showed that glabridin and glabrene inhibited the enzyme with non-competitive and uncompetitive modes, respectively. Glyasperin C () was recently isolated from the same part of the plant and shown to be two times more active than glabridin [51
]. Although glyasperin C is the most active inhibitor, glabridin had the best melanogenesis inhibitory activity among them.
On the other hand, we isolated several isoflavones derivatives from soybean koji fermented with Aspergillus oryzae
and demonstrated three hydroxyisoflavones including 6-hydroxydaidzein (6,7,4′-trihydroxyisoflavone), 8-hydroxydaidzein (7,8,4′-trihydroxyisoflavone), and 8-hydroxygenistein (5,7,8,4′-tetrahydroxyisoflavone) as potent tyrosinase inhibitors () [52
]. Among them, 6-hydroxydaidzein with 6-fold more than kojic acid acts competitively on the L-tyrosine binding site of the enzyme while the other two 8-hyroxyisoflavones irreversibly inactivate the enzyme and belong to the suicide substrates of tyrosinase (discussed in the “irreversible inactivators
” section) [54
]. It is interesting to note that the position and number of hydroxyl groups in the A ring of the isoflavone structure can strongly affect both the inhibitory strength and the inhibitory mode of the isoflavones to mushroom tyrosinase. For example, an isoflavone with hydroxyl groups at both the C6 and C7 positions in the A ring (6,7,4′-trihydroxyisoflavone) would increase more than 10 times both the inhibitory activity (judged by IC50
values) and affinity to the enzyme (judged by Michaelis constants) compared to that of isoflavones either with only one hydroxyl group at the C7 position or without any hydroxyl group in the A ring. Alternatively, the hydroxyl groups at both the C7 and C8 positions (7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone) would completely change the inhibitory mode of the isoflavones from the reversible competitive to the irreversible suicide form. The elucidation of the detailed mechanism of the effects of the hydroxyl groups in the A ring in the isoflavones structure on their inhibitory activity toward tyrosinase needs further study.
In addition to the isoflavonoids from Glycyrrhiza
plants, Lee et al.
recently identified other natural isoflavonoids as potent tyrosinase inhibitors. Haginin A (2′,3′-dimethoxy-7,4′-dihdroxyisoflav-3-ene, ) isolated from the branch of Lespedeza cyrtobotrya
was shown to be 10-fold more active than kojic acid against monophenolase activity of mushroom tyrosinase with a non-competitive mode [55
]. The compound significantly inhibited melanin synthesis in melanoma cells and decreased UV-induced skin pigmentation in brown guinea pigs. Furthermore, haginin A presented remarkable inhibition on the body pigmentation in the zebrafish model system. Dalbergioidin (5,7,2′,4′-tetrahyroxyisoflavan) was also isolated from L. cyrtobotrya
and non-competitively inhibited the monophenolase activity of mushroom tyrosinase [56
]. Fifty percent of melanin biosynthesis in melanoma cells was inhibited by 27 μM of dalbergioidin, and 80% of cell viability was maintained in this concentration. The third potent inhibitor discovered by the authors is calycosin (4′-methoxy-7,4′-dihydroxyisoflavone, ), which showed slighter higher monophenolase inhibitory activity toward mushroom tyrosinase than that of kojic acid. The compound contains two mechanisms to reduce melanogenesis in melanoma cells, including inhibiting tyrosinase activity and reducing the expression of tyrosinase [57
Chalcones consist of two aromatic rings in trans
configuration, separated by three carbon atoms, of which two are connected by a double bond and the third is a carbonyl group. Some natural prenylated chalcones showed potent tyrosinase inhibitory activity. Three chalcones derivatives, including licuraside, isoliquiritin, and licochalcone A were isolated from the roots of the Glycyrrhiza
species and competitively inhibited the monophenolase activity of mushroom tyrosinase. Among them, licochalcone A () was 5.4-fold more active than kojic acid [58
]. Another prenylated chalcone, kuraridin (), was isolated from the plant Sophora flavescens
and identified as a potent tyrosinase inhibitor, which was 34 times activity of kojic acid against monophenolase activity of mushroom tyrosinase [59
]. Following, its hydroxyl analog, kuraridinol (), was found to be 18.4-fold more active compared to kojic acid [60
]. More interesting, the two prenylated chalcones exhibited significantly more activity than those of their corresponding flavanone analogs. Kuraridin (a chalcone) is 10-fold more active than kurarinone (a flavanone) 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. Recently, 2,4,2′,4′-tetrahydroxy-3-(3-methyl-2-butenyl)-chalcone (TMBC, ), which was isolated from the stems of Morus nigra
, was proved to be 26-fold more potent than kojic acid in diphenolase inhibitory activity of mushroom tyrosinase [61
]. The kinetic study showed that the compound is competitive to the L-dopa binding site of the enzyme with a KI
value of 1 to 1.5 μM. Moreover, 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.
From the naturally found chalcones listed above, it seems that the 4-resorcinol moiety (2,4-dihydroxyl groups in the aromatic ring) in the chalcone structure is the key substituted group in exerting potent inhibitory activity. Some simple 4-alkylresorcinols were proved to exhibit strong tyrosinase inhibitory activity [62
]. To evaluate the structure-activity relationship between the numbers and the positions of hydroxyl groups in the chalcone skeleton and the inhibitory activity toward mushroom tyrosinase, the inhibitory activity against mushroom tyrosinase of many chemically synthesized hydroxychalcones was examined. In an early study, Nerya et al
. reported that the 4-hydroxyl group in the B ring of the chalcone skeleton is the major factor affecting inhibitory potency, because it results in a molecular skeleton closely similar to that of L-tyrosine [64
]. Following, they 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 [65
]. Therefore, they concluded that 4-resorcinol moiety in the chalcone skeleton plays the key role in exhibiting inhibitory potency. Similarly, Jun et al.
recently chemically synthesized a series of hydroxychalcones and determined the tyrosinase inhibitory activity [66
]. The results were also consistent with that by Nerya et al
. Moreover, they found that 2,4,2′,4′,6′-pentahydroxychalcone () is 5-fold stronger than 2,4,2′,4′-tetrahydroxychalcone. The inhibitory mode of the compound is also competitive to the L-tyrosine binding site of the enzyme with a KI
value of 3.1 μM. On the other hand, as seen in the description in the earlier “flavone
” section, 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 contain 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.
In similar research with N
-benzylbenzamide as the structure skeleton, which is analogous to that of chalcone but with an amide moiety (not the alkyl double bond) connecting between the two aromatic rings, the inhibitory activities of N
-benzylbenzamide derivatives () were found to rank by 3,5,2′,4′-tetrahydroxyl > 2,4,2′,4′-tetrahydroxyl > 3,5,4′-trihydroxyl > 2,4,4′-trihydroxyl substitutions [67
]. Accordingly, it was concluded that the 4-resorcinol in the B ring has more effect than in the A ring on exerting potent inhibitory activity, while the 5-resorcinol in the A ring is preferred to the 4-resorcinol. Very interesting, the structural criterion for exerting potent tyrosinase inhibitory activity in the chalcone-like structure, i.e., the 4-resorcinol moiety in the B ring and the 5-resorcinol in the A ring, is partially consistent with that in the flavone structure studied by the previous fluorescence quenching spectroscopic method, i.e., the hydroxyl groups of A and B rings on the ether side of the flavones (C6 to C8 and C2’ to C4’) [45
]. In addition, Khatib et al
. recently used computer modeling to dock the enzyme crystallographic structure by some 4-resorcinol derivatives with different ester chains and compared the experimental inhibitory IC50
values with the calculated free energy and docking energy [68
]. The authors’ results emphasize again the importance of the 4-resorcinol skeleton in exerting potent tyrosinase inhibitory activity and the extent to which a lipophilic moiety, combined with the resorcinol skeleton, can contribute to this activity. The additional lipophilic groups affect both the inhibition potency, as well as the ability of the tyrosine to compete with the inhibitors. Such a lipophilic unit, which contains a minor bulky group, was a preferred inhibitor over a long chain or highly bulky functional moiety, as it may interact with the enzyme hydrophobic pocket and augment binding affinity. The conclusion could explain the potent inhibitory activity of prenylated chalcone with 4-resorcinol moiety such as kuraridin and kuraridinol described above.
A stilbene consists of an ethene double bond substituted with a benzyl ring on both carbon atoms of the double bond. Oxyresveratrol (2,4,3′,5′-tetrahydroxy-trans
-stilbene, ), which was initially isolated from Morus alba
, exhibited 32-fold more inhibitory activity than that of kojic acid [40
]. The inhibitor acts non-competitively on both monophenolase and diphenolase activity of mushroom tyrosinase. Based on the potent inhibitory capacity, oxyresveratrol reduced pigmentation in melanoma cells. The melanogenesis inhibitory mechanism of the compound was proved to directly inhibit enzyme activity but not affect gene expression [69
]. It is worth noting that oxyresveratrol also contains the structure of the 4-resorcinol moiety in the B ring and the 5-resorcinol moiety in the A ring as described in the former “chalcone
” section. In fact, oxyresveratrol is 50-fold more active than its analog, resveratrol (2,3′,5′-trihydroxy-trans
-stilbene), which loses the 4-resorcinol moiety in its structure. Taken together with all these findings, the key role of the structural criterion (the 4-resorcinol moiety in the B ring and the 5-resorcinol moiety in the A ring) of an inhibitor in tyrosinase inhibition has been validated in flavones, chalcones, and stilbenes, and this criterion becomes a golden rule for further design of a potent tyrosinase inhibitor.
Chemical structures of selected tyrosinase inhibitors belonging to stilbenes (a), bibenzyl derivatives (b), coumarins (c), and benzaldehyde derivatives (d). RAa and RAb have the same meanings as those of .
Following oxyresveratrol, another three hydroxystilbenes were also purified and identified as potent tyrosinase inhibitors. Chloroporin (4-geranyl-3,5,2′,4′-tetrahydroxy-trans
-stilbene, ) was purified from the heartwood of Chlorophora excelsa
and displayed 14.8-fold inhibitory activity of kojic acid against diphenolase of mushroom tyrosinase [70
]. Gnetol (2,6,3′,5′-tetrahydroxy-trans
-stilbene, ), isolated from the roots of Gnetum gnemon
, exhibited 30-fold more diphenolase inhibitory activity of murine tyrosinase than that of kojic acid [71
]. Recently, piceatannol (3,5,3′,4′-tetrahydroxy-trans
-stilbene, ), isolated from grapes and red wine, showed 32.7-fold antimonophenolase activity of kojic acid toward mushroom tyrosinase [72
]. The hydroxyl positions of the structure of piceatannol, which contains o
-(3′,4′-)dihydroxy groups, are significantly different from other naturally found hydroxystilbenes. The o
-dihydroxy groups play an important role in antioxidant activity (free radical scavenging). In fact, piceatannol has strong suppressive activity in reactive species generation and enhances the reduced/oxidized glutathione ratio in cells. Hence, the inhibitory mechanism of piceatannol on tyrosinase monophenolase activity may be exerted by both direct tyrosinase inhibition and quinone products scavenging, and is not the same with that of oxyresveratrol. However, a detailed kinetic study of the inhibition by piceatannol is lacking.
Aside from naturally occurring hydroxystilbenes, a number of synthetic stilbene derivatives are also good tyrosinase inhibitors. To examine the structure-activity relationship, a variety of hydroxystilbene compounds were synthesized and assayed for their tyrosinase inhibitory activity. The role of the double bond between the two aromatic rings of the stilbene skeleton in the tyrosinase inhibition has been discussed in these studies. By comparing the diphenolase inhibitory activity of murine tyrosinase by cis
- and trans
-isomers of 3,3′-dihydroxystilbene, it was concluded that the trans
-olefin structure of the parent stilbene skeleton is essential for inhibition [73
]. Indeed, all the natural hydroxystilbenes possessing potent antityrosinase activity are trans
-isomers. However, recently, Song et al.
, concluded the opposite: they found that the diphenolase inhibitory capacity on mushroom tyrosinase by cis
-3,5-dihydroxystilbene was stronger than that by its corresponding trans
]. The reasons to explain the obtainted opposite results are not clear. On the other hand, a gnetol analog, dihydrognetol, which loses the double bond between the two aromatic rings, exerted a greatly lowered inhibitory effect on diphenolase activity of murine tyrosinase [73
], and it was concluded that the role of the double bond in the stilbene skeleton for tyrosinase inhibition is critical. In contrast, an oxyresveratrol analog, dihydrooxyresveratrol (), which also loses the double bond between the two aromatic rings, showed 8-fold more diphenolase inhibitory activity toward mushroom tyrosinase than its analog, oxyresveratrol [75
]. The authors suggested that the higher tyrosinase inhibitory activity of dihydrooxyresveratrol was probably due to its bibenzyl structure, which gave more flexibility and thus allowed the phenolic groups to interact with the enzyme more effectively. Similar results were also found by both Oozeki et al.
] and Vielhaber et al
]. The former authors synthesized another bibenzyl analog but with 2,4,2′,4′-tetrahydroxyl substitution groups () and identified the compound to be 20-fold more active than kojic acid while the latter authors found that 4-(1-phenylethyl)1,3-benzenediol () inhibited mushroom tyrosinase 22 times more effective than kojic acid. These results implied again that the double bond in the stilbene structure is not essential. Despite the undefined inhibitory mechanism of hydroxystilbenes, one Korean research group led by Chung and Suh recently successfully synthesized a new isostere of oxyresveratrol, HNB [4-(6-hydroxy-2-naphthyl)-1,3-bezendiol, ], which exhibited 546-fold more inhibitory activity toward monophenolase of mushroom tyrosinase than that of kojic acid [78
]. HNB is also the strongest tyrosinase inhibitor published until now. The kinetic study demonstrated that HNB is a competitive inhibitor with a KI
value of 4.78 to 6.21 nM. Due to the potent tyrosinase inhibitory activity, it is reasoned that HNB suppressed cellular tyrosinase activity and total melanin content to 27% and 35%, respectively, in melanoma cells at 100 μM concentration, where cells maintained 87% viability [79
Coumarins are lactones of phenylpropanoid acid with an H
-benzopyranone nucleus. Among the coumarin-type tyrosinase inhibitors, aloesin () is the famous one, which is a natural hydroxycoumarin glucoside isolated from Aloe vera
. Aloesin performed more inhibitory activity toward crude murine tyrosinase than mushroom tyrosinase and has been recently used in topically applied cosmetics due to its natural source and multifunctional activity in skin care [80
]. A coumarin analog, esculetin, was isolated from the seeds of Euphorbia lathyris
and showed one-quarter of the antityrosinase activity of kojic acid [82
]. However, the compound was recently demonstrated to be a substrate of mushroom tyrosinase [83
]. Another coumarin analog, 9-hydroxy-4-methoxypsoralen (), was isolated from Angelica dahurica
and exhibited six times more tyrosinase inhibitory activity than that of kojic acid [84
]. Recently, a new coumarin derivative, 8′-epi-cleomiscosin A (), was isolated from the aerial parts of Rhododendron collettianum
and showed 12.8-fold diphenolase inhibitory activity of kojic acid toward mushroom tyrosinase [85
]. Interestingly, another purified compound, cleomiscosin A, had a structure highly similar to that of 8′-epi-cleomiscosin A but exhibited a 14-fold decrease in tyrosinase inhibitory activity compared to that of 8′-epi-cleomiscosin A. The only difference between these two compounds is that the α-proton at the position 8′ for the former, where the β-proton at the same position for the latter. A change in stereochemistry of a single proton that drastically changes the inhibitory activity of the compound is rarely found in the reviewed literature.
3.2. Benzaldehyde and Benzoate Derivatives
In the past decade, a large number of benzaldehyde and benzoate derivatives have been isolated from plants and identified as tyrosinase inhibitors, including benzoic acid, benzaldehyde, anisic acid, anisaldehyde, cinnamic acid, and methoxycinnamic acid from the roots of Pulsatilla cernua
], 4-substituted benzaldehydes from cumin [87
], 2-hydroxy-4-methoxybenzaldehyde from roots of Mondia whitei
-coumaric acid from the leaves of Panax ginseng
], hydroxycinnamoyl derivatives from green coffee beans [90
], and vanillic acid and its derivatives from black rice bran [91
]. The aldehyde group is known to react with biologically important nucleophilic groups such as sulfhydryl, amino, and hydroxyl groups. The tyrosinase inhibitory mechanism of benzaldehyde-type inhibitors comes from their ability to form a Schiff base with a primary amino group in the enzyme [92
]. In contrast, benzoate inhibits tyrosinase by a copper chelating mechanism and belongs to a typical HA-type acid tyrosinase inhibitor, whose inhibitory mechanism involves the interaction between the non-ionized form of the inhibitor and the copper in the active site of the enzyme [94
]. In terms of inhibitory strength, all the naturally occurring benzaldehyde and benzoate derivatives listed above showed only weak-to-moderate tyrosinase inhibitory activity while none are stronger than kojic acid. Recently, flourobenzaldehydes [95
], methyl trans
], salicylic acid [97
], hydroxybenzaldehydes [98
], and 4-β-d
] were also proved to be weak tyrosinase inhibitors with one to two orders of magnitude of lower antityrosinase activity than that of kojic acid. The most potent natural inhibitor of benzaldehyde type was not identified in plants but in a fungus. Protocatechualdehyde () was isolated from the fruiting body of Phellinus linteus
and exhibited 7.8-fold more tyrosinase inhibitory activity than that of kojic acid [100
]. It is worth noting that its analog, protocatechuic aldehyde, with two methoxyl groups replacing the two hydroxyl groups showed one order of magnetite of lower activity than that of protocatechualdehyde [101
]. Moreover, another analog, protocatechuic acid isolated from black rice bran with a benzoate skeleton, showed another one order of magnetite of lower activity [91
]. Thus, it is indicated that both the o
-dihydroxyl group and the aldehyde group of the protocatechualdehyde structure play important roles in exerting its inhibition activity. On the other hand, to improve the inhibitory strength of the benzaldehyde-type inhibitors, some derivatives were chemically synthesized and assayed to determine their inhibitory activity. 4-Vinylbenzaldehyde [102
], 4-alkylbenzaldehyde [103
], 2-hydroxy-4-isopropyl-benzaldehyde [105
-ethyloxime () [106
], and 4-butyl-benzaldehyde thiosemicarbazone [107
] were successfully synthesized and showed 35-, 100-, 350-, 1500-, and 2000-fold, respectively, more potent activity than that of the precursor benzaldehyde. However, advanced data for cell cytotoxicity and cellular melanogenesis inhibition are lacking for these improved benzaldehyde derivative inhibitors.
Gallic acid (3,4,5-trihydroxybenzoate) has been isolated and identified as a tyrosinase inhibitor from many plants, and its inhibitory mechanism together with those of its ester derivatives has been well studied by Kubo et al.
]. They found that gallic acid inhibited diphenolase activity of mushroom tyrosinase with a IC50
value of 4500 μM, which is 100-fold lower than that of kojic acid. In addition, gallic acid itself and its short alkyl chain esters (<C10) were oxidized by mushroom tyrosinase as substrates, but the long-chain alkyl chain esters (>C10) inhibited the enzyme without being oxidized. Gallic acid was also found to be very toxic to melanoma cells with cytotoxicity comparable to that of hydroquinone [111
]. Recently, Nithitanakool et al.
isolated both gallic acid and its methyl derivative from seed kernels of Mangifera indica
, and determined that both compounds were poor inhibitors against diphenolase activity of mushroom tyrosinase with 300- and 30-fold, respectively, lower activity than that of kojic acid [112
]. These results were consistent with previous studies. In spite of the poor inhibitory activity of gallic acid itself, some compounds with gallate moiety were found to inhibit mushroom tyrosinase more effectively. Some flavonoids with gallate moiety bonded to the 3-hydroxyl position, including GCG [(+)-gallocatechin-3-O
-gallate] and EGCG [(−)-epigallocatechin-3-O
-gallate], were isolated from green tea leaves and showed stronger inhibitory activity [113
]. Similarly, three tyrosyl gallates were synthesized and showed stronger inhibitory activity [114
]. In recent times, 1,2,3,4,6-pentagalloylglucopyranose isolated from the seed kernels of M. indica
] and the roots of Paeonia suffruticosa
], respectively, was found to be 16-fold more active than gallic acid and inhibited monophenolase activity of mushroom tyrosinase with a non-competitive mode.
3.3. Long-chain Lipids and Steroids
Recently, several lipids were purified from natural sources and exhibited tyrosinase inhibitory activity. A triacylglycerol, trilinolein (), was isolated from sake lees, which are byproducts of sake production, and proved to be as potent as kojic acid for inhibition of diphenolase activity of mushroom tyrosinase [116
]. Based on the findings, including inability of copper chelating, lack of free radical scavenging, and kinetically non-competitive inhibition of the inhibitor, the inhibitory mechanism was proposed by binding of the compound to some site of the tyrosinase, except the catalytic site. A glycosphingolipid, soyacerebroside I, which is composed of a sphingoid base skeleton, an amide aliphatic long-chain fatty acid, and a β-glucopyranose moiety, was isolated from the leaves of Guioa villosa
and found to inhibit monophenolase and diphenolase activity of mushroom tyrosinase with half-activity of kojic acid [117
] while another glycosphinogolipid, cerebroside B, from Phellinus linteus
showed no inhibitory activity against the enzyme [100
]. In addition, trans
geranic acid from Cymbopogon citrates
(lemongrass) was recently identified as a tyrosinase inhibitor but with only one-tenth of the inhibitory activity of kojic acid [118
Chemical structures of selected tyrosinase inhibitors belonging to long-chain lipids (a) or steroids (b). RAa and RAb have the same meanings as those of .
In addition to long-chain lipids, some steroids were also determined to be tyrosinase inhibitors. Many studies in this field were contributed by the research group of Choudhary and Khan. Three steroids isolated by these authors from the aerial parts of Trifolium balansae
showed higher diphenolase inhibitory activity toward mushroom tyrosinase than that of kojic acid [119
]. Among the steroids, stigmast-5-ene-3β,26-diol () was 7-fold more active. The authors also found that a long-chain ester, 2β(2S
)-tritriacontenoate (), from Amberboa ramose
exhibited 12.3-fold more inhibition against the diphenolase activity of mushroom tyrosinase compared to the standard kojic acid [120
]. On the other hand, the derivative containing a d
-galactopyranosyl moiety at C2 was found to lose one order of magnitude on its tyrosinase inhibitory activity. Like most cases of tyrosinase inhibitors, the presence of the bulky and hydrophilic d
-galactopyranosyl moiety interferes with the entrance of the molecule into the active site of the enzyme, thus reducing its inhibitory activity. From the same plant, eight cycloartane triterpenoids were isolated at the same time. A triterpenoid, 3β,21,22,23-tetrahydroxycycloart-24(31),25(26)-diene (), exhibited an extremely potent inhibition (12.6-fold) against diphenolase activity of mushroom tyrosinase when compared with kojic acid [121
]. The same research group also purified some triterpenoid glycosides from the roots of Astragalus taschkendicus
. However, the triterpenoid glycosides showed only inhibitory activity equal to that of kojic acid against mushroom tyrosinase [122
]. In advance, Choudhary and Khan purified nine pentacyclic triterpenes from the aerial part of the plant Rhododendron collettianum
, and all the triterpenes showed potent diphenolase inhibitory activity of mushroom tyrosinase. Among them, arjunilic acid () was the strongest tyrosinase inhibitor, which was 16.7-fold inhibitory activity of kojic acid [123
]. In addition to triterpenoids, the authors also purified many diterpenoids from the aerial parts of Aconitum leave
]. However, most diterpenoids did not inhibit tyrosinase activity. Only one compound, lappaconitine hydrobromide, revealed activity similar to that of kojic acid [125
]. Similar to the diterpenoids, few monoterpenoids were found to be strong tyrosinase inhibitors; only a monoterpenoid, crocusatin-K, isolated from the petals of Crocus sativus
, was proved to display inhibitory activity equal to that of kojic acid [126
]. More recently, Wu et al
. isolated four new sesquiterpenes from the leaves and stems of Chloranthus henryi
] and two new sesquiterpenes dimmers from the leaves of Chloranthus tianmushanensis
], and found that these sesquiterpenes showed either no or weak tyrosinase inhibitory activity. In addition to the original steroids from plants, two 11α-hydroxylated steroid metabolites were isolated from the fungus Cunninghamella elegans
cultivations feeding with 17α-ethynyl- or 17α-ethylsteroids () and showed 2.9- and 9.8-fold higher tyrosinase inhibitory activity, respectively, than that of kojic acid [129
]. Regarding the difficult permeability of skin, these hydrophobic steroid or long-chain lipid inhibitors seems to have superior potential in the development as skin-whitening agents due to their easier cellular permeability. However, all these lipid inhibitors are lack of cellular or clinical assays for determining their depigmentation activity and remain less utilized by the cosmetics industry until now.
3.4. Other Natural and Synthetic Inhibitors
Other inhibitors from natural sources
Anthraquinones from different plant sources have been widely used since ancient times due to their laxative and cathartic properties. In addition, this class of compounds has shown a wide variety of pharmacological activities, such as antiinflammatory, wound healing, analgesic, antipyretic, antimicrobial, and antitumor activities. Recently, an anthraquinone, physcion (1,8-dihydroxy-2-methoxy-3-methylanthraquinone, ), was found to show similar tyrosinase inhibitory activity with that of kojic acid [130
]. Interestingly, another anthraquinone, 1,5-dihydroxy-7-methoxy-3-methylanthraquinone (), with hydroxyl and methoxyl groups at different positions compared with those of physcion exhibited a 72-fold increase on antityrosinase activity [131
]. Therefore, it is worthy to systematically evaluate the structure-activity relationship between the functional groups attached to the anthraquinone skeleton and the antityrosinase activity. In addition, many lignans isolated from the roots of Vitex negundo
showed higher tyrosinase inhibitory activity than kojic acid, while the most active lignan from the plant was (+)-lyoniresinol (), whose activity was 5.2-fold higher than that of kojic acid [132
]. In addition to the inhibitors from plants, marine beings inhabit virtually any environment in the sea, and they have been shown to produce novel substances with utilities in fine chemicals, drug, and cosmetics products, including tyrosinase inhibitors. One phloroglucinol derivative, dieckol, was isolated from a marine brown alga, Ecklonia stolonifera
, and displayed three times more activity than that of kojic acid [133
]. In addition, a marine-derived fungus Myrothecium sp
. was found to contain 6-n
-pentyl-α-pyrone (), which was a potent tyrosinase inhibitor with 9.6-fold inhibitory activity of kojic acid [134
]. Recently, another tyrosinase inhibitor was purified from a marine bacteria, Trichoderma viride
strain H1-7, and showed competitive inhibition toward monophenolase activity of mushroom tyrosinase through binding to a copper active site of the enzyme [135
Chemical structures of other tyrosinase inhibitors from natural (a) or synthetic (b) sources. RAa and RAb have the same meanings as those of .
Other inhibitors from synthetic sources
For smaller molecules, it is easier to rationally design the structures of the molecules by a chemically synthetic method. Improving the tyrosinase inhibitory activity of a known inhibitor by properly changing the substitute groups on its structure is expected. N
-Phenylthiourea (PTU, ) is a well-known inhibitor for inhibiting diphenolase enzyme that belongs to the type-3 copper protein group. The sulfur atom of the compound binds to both copper ions in the active site of the enzyme and blocks enzyme activity [136
]. Criton and Le Mellay-Hamon synthesized a series of analogs of N
-phenylthiourea and put into assays of the inhibitory activity of the analogs against diphenolase activity of mushroom tyrosinase. They found that when the amino group and the sulfur moieties of N
-phenylthiourea were replaced by N
-hydroxylamine and oxygen, respectively, the resulting compound () exhibited six times more activity than that of N
]. In another study, the same authors synthesized N
-(phenylalkyl)cinnamides derived from the coupling cinnamic acid with phenylalkylamines and found that two synthetic compounds showed higher inhibitory activity than that of kojic acid [138
]. Similarly, Kang et al
. designed a series of compounds by combining the structures of two putative tyrosinase inhibitors, kojic acid and caffeic acid, to form new inhibitors, which showed antidiphenolase activity equal to that of kojic acid toward mushroom tyrosinase but enhanced depigmentation activity in melanoma cells [139
]. In addition, Shiino et al
. synthesized analogs of cupferron, which is a well-known metal chelating agent and inhibits competitively both monophenolase and diphenolase activity of mushroom tyrosinase [140
], and found that N
-nitrosohydroxylamines inhibited mushroom tyrosinase by interacting with the copper ions at the active site of the enzyme [141
]. As removal of nitroso or hydroxyl moiety completely diminished the inhibitory activity, both functional groups were suggested to be essential for chelating activity. Among the derivatives, the authors found that N
-nitrosohydroxylamines showed the best inhibitory activity, which is comparable with that of kojic acid [142
]. In advance, they recently reported that these N
-nitrosohydroxylamines inhibited mushroom tyrosinase in a pH-dependent manner due to the non-ionized, electrically neutral form of the compounds [143
]. Another research group led by Khan also did a lot of successful work for developing new tyrosinase inhibitors by chemically synthetic methods. The discovered new types of inhibitors included sildenafil [144
], oxadiazole [145
], oxazolones [146
], and tetraketones types [147
] (). The strongest inhibitor of each type list above was between 7- and 14-fold stronger than kojic acid. On the other hand, Kim et al
. focused on developing new type inhibitors containing a selenium atom, which is an essential biological trace element and an integral component of several enzymes. The use of selenium as a nutritional supplement has been popularized recently due to its potential roles as an antioxidant and an anticancer agent. The authors demonstrated that some 1,3-selenazol-4-one derivatives [148
], selenourea derivatives [149
], and selenium-containing carbohydrates [150
] possessed inhibitory activity similar to that of kojic acid against diphenolase activity of mushroom tyrosinase. However, these selenium-containing derivatives were applied in melanogenesis inhibition assays in melanoma cells; most compounds showed both cytotoxicity and depigmenting effects and had restrictions in applications to cosmetics or foods.
Some simple phenyl and biphenyl compounds were also synthesized and identified as potent tyrosinase inhibitors. 4,4′-Dihyldroxybiphenyl () showed 12-fold more monophenolase inhibitory activity of mushroom tyrosinase than that of kojic acid with a competitive inhibition mode [151
] while its glucoside derivatives from the fruit of Pyracantha fortuneana
displayed only low inhibitory activity [152
]. In addition to directly inhibiting tyrosinase activity, 4,4′-dihyldroxybiphenyl was also found to suppress several cellular key parameters in the melanogenic pathway by downregulating the cAMP-dependent protein kinase K signaling pathway and decreasing gene expression of microphthalmia transcription factor, which in turn suppressed tyrosinase expression [153
]. In addition, S
-phenylthiocarbamate () [154
] and 4-(2′,4′-dihydroxyphenyl)-(E
] were recently found to be 44-fold and 6-fold, respectively, more active than kojic acid toward diphenolase activity of mushroom tyrosinase. However, although huge numbers of synthetic inhibitors were successful in inhibiting tyrosinase activity, few have been confirmed in melanogenesis inhibiting activity in cells or skin models.
3.5. Irreversible Inactivators
In contrast to the huge number of reversible inhibitors has been identified, rarely irreversible inhibitors of tyrosinase were found until now. These irreversible inhibitors, which are also called specific inactivators, can form irreversibly covalent bond with the target enzyme and then inactivate it. However, the tyrosinase irreversible inhibitors are different from non-specifically irreversible enzyme inactivators such as acids or bases, which destroy all protein structures. Instead, they are generally specific for tyrosinase and do not inactivate all proteins; they work by specifically altering the active site of the enzyme. The substrate reaction kinetics method described by Tsou has been widely used in studies of inactivation of various enzymes by different types of inactivation or inhibitions [156
]. As shown in , irreversible inhibitors form a reversible non-covalent complex with the enzyme (EI or ESI), and this then reacts to produce the covalently modified “dead-end complex” Ei
. The rate at which Ei
is formed is called the inactivation rate or kinact
. It is worth noting that irreversible inhibitors display time-dependent inhibition, and their potency therefore cannot be characterized by an IC50
value. This is because the amount of active enzyme in a given concentration of irreversible inhibitor will be different depending on how long the inhibitor is pre-incubated with the enzyme.
Reaction mechanism of irreversible inhibitors. E and Ei are the enzyme and the inactivated enzyme, respectively; S, I, and P are the substrate, inhibitor, and product, respectively; ES, EI and ESI are the intermediates.
Captopril (), an antihypertensive drug [(2S
-proline], forms both a copper-captopril complex and a disulfide bond between captopril and cysteine-rich domains at the active site of tyrosinase [157
]. Accordingly, the drug is able to prevent melanin formation by irreversibly inhibiting monophenolase and diphenolase activity of mushroom tyrosinase in a non-competitive and competitive manner, respectively, as well as by scavenging the generated o
-quinones to form a colorless conjugate. The kinetic study showed that the value of the rate inactivation constant kinact
was 1.98 × 10−4
is also known as an inactivator of several copper-containing enzymes, such as dopamine β-monooxygenase [158
] and mushroom tyrosinase [159
]. It was proposed that high concentrations of H2
could oxidize a methionine residue in position 374 of the mushroom tyrosinase active site to methionine sulfoxide, and this event would inactivate the enzyme [160
]. Chen et al.
discovered two other irreversible inhibitors of tyrosinase, including cetylpyridinium chloride [161
] and 3,5-dihydroxyphenyl decanoate [162
] (). The fluorescence spectroscopic results demonstrated that cetylpyridinium chloride can bind to the tyrosinase molecule and induce enzyme conformational changes, which undergo a slow irreversible inactivation of the enzyme. The kinetic study determined the values of the inactivation constants of the inhibitor toward the free enzyme and enzyme-substrate complex to be 3.957 and 6.078 × 10−3
, respectively. On the other hand, the inhibition mechanism of 3,5-dihydroxyphenyl decanoate was irreversible and competitive/uncompetitive mixed type inhibition with varied inactivation constants ranging from 1.93 to 7.912 × 10−3
. Recently, p
-hydroxybenzyl alcohol showed binding capability of mushroom tyrosinase and irreversibly inhibited the enzyme [163
]. It was demonstrated that p
-hydroxybenzyl alcohol () exhibited an inhibitory effect on melanogenesis in melanoma cells at non-cytotoxic concentrations. The suppression of melanin synthesis by the compound was attributed to its behavior in directly inhibiting cellular tyrosinase activity without effects on the expression level of tyrosinase mRNA. In another study, it is very interesting to find that hen egg white lysozyme (HEWL) inhibited mushroom tyrosinase with a reversibly and irreversibly mixed inhibition mechanism [164
]. The authors suggested that HEWL binds to glycoside linkage in tyrosinase and induces its conformational change. Some of the bound HEWL continues to non-specifically cleavage glycosidic linkages and induces irreversible inhibition while the other portion of the HEWL-tyrosinase complex restored the diphenolase activity of tyrosinase after depletion of HEWL.
Chemical structures of irreversible tyrosinase inhibitors.
Among the irreversible inhibitors, suicide substrates belong to a special class. It is known that tyrosinase could be irreversibly inhibited by its o
-diphenol substrates, such as l
-dopa and catechol [165
]. These substrates were also named suicide substrates or mechanism-based inhibitors. The mechanism of the suicide substrate has been extensively studied by Waley [166
], who proposed a simple branched reaction pathway as , in which an intermediate Y may give either an active enzyme and product, or an inactive enzyme. In the scheme, the intermediate Y has a choice of reaction, governed by the partition ratio r, where r = k+3
. The r value is called the molar proportion for inactivation, i.e., the number of molecules of inhibitors required to completely inactivate one molecule of the enzyme, and may be determined by plotting the fractional activity remaining against the ratio of the initial concentration of inhibitor to that of enzyme. The intercept on the abscissa is 1 + r in the plot, when r > 1 [167
]. As in general irreversible inhibitors, the inhibitory strength of a suicide substrate is also not determined by an IC50
value but expressed by its r value, where a smaller r value of a suicide substrate means fewer inhibitor molecules are needed to inactivate all enzyme activity and being more powerful inhibition.
Action mechanism for suicide substrate. E and Ei are the enzyme and inactivated enzyme, respectively; P is the product; X is the first intermediate, and Y is another intermediate.
In addition to the kinetic reaction mechanism, Land et al.
recently proposed a model to explain the detailed molecular reaction mechanism of the suicide inactivation of tyrosinase based on the chemical structures of substrates and crystallographic tyrosinase [168
]. In their model, the inactivation of tyrosinase during catechol oxidation is due to the catechol being capable of an alternative “cresolase” (monophenolase) presentation (). Oxygen addition to the catechol ring by cresolase activity generates an intermediate product that can undergo deprotonation and reductive elimination. This deprotonation leads to the inactivation of the enzyme by formation of a copper atom, which does not bind to the histidine ligands. According to the model, suicide inactivation of tyrosinase depends on anomalous catechol oxidation and the consequence of the formation of zero-valency copper. Their model can explain well the experimental observation that half of the copper is lost from the active site during catechol inactivation. Moreover, the authors in advance used hplc/mass to identify the hydroquinone product in the suicide inactivation of mushroom tyrosinase with 4-methylcatechol as a substrate [169
]. Very interestingly, tyrosinase has the following two unique properties: (1) Tyrosinase is able to catalyze oxidations of both monophenols (cresolase activity) and diphenols (catecholase activity) to o
-quinones, and (2) tyrosinase exhibits suicide inactivation during the oxidation of catechols. In the Land cresolase-dependent model, the two properties are neatly connected, and suicide inactivation is the result of catecholic substrates being processed by the cresolase route.
Figure 8. Molecular reaction mechanism of suicide inactivation of tyrosinase by the oxidation of an o-diphenol substrate. The curly arrows shows the effect of deprotonation leading to the reduction of copper from bivalent to zero-valent form, elimination of an (more ...)
Although both the kinetic and the molecular reaction mechanism of suicide inactivation of tyrosinase have been well studied, potent suicide substrates have rarely been discovered. Recently, we found that two isoflavones, 7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone (), were potent and unique suicide substrates of mushroom tyrosinase with low partition ratios, low Michaelis constants, and high maximal inactivation rate constants [54
]. The partition ratios between molecules of suicide substrate in the formation of the product and in the inactivation of the enzyme were determined to be 81.7 and 35.5, for 7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxy-isoflavone, respectively. In the reviewed literature, 5,7,8,4′-tetrahydroxyisoflavone is the most potent suicide substrate of mushroom tyrosinase until now and has high potential in application as a skin-whitening agent. This compound was in advance creamed in the proper condition [170
] and showed higher depigmenting activity than that of ascorbic acid 2-glucoside (AA2G) in an in vivo
assay conducted with 60 volunteers by our laboratory (unpublished data).