Elsevier

Phytochemistry

Volume 65, Issue 10, May 2004, Pages 1389-1395
Phytochemistry

Chalcones as potent tyrosinase inhibitors: the effect of hydroxyl positions and numbers

https://doi.org/10.1016/j.phytochem.2004.04.016Get rights and content

Abstract

The inhibition of tyrosinase is one of the major strategies to treat hyperpigmentation. Various limitations are associated with many of these inhibitors, such as high cytotoxicity, poor skin penetration and low stability in formulations. In continuation of our previous study [J. Agric. Food Chem. 51 (2003) 1201], showing that isoliquiritigenin chalcone (ILC) is a potent tyrosinase inhibitor, the present study aims to characterize the chalcone family as new tyrosinase inhibitors, and demonstrate their potential whitening potency. Nine mono-, di-, tri- and tetrahydroxychalcones were tested as inhibitors of tyrosinase mono- and diphenolase activities, showing that the most important factor in their efficacy is the location of the hydroxyl groups on both aromatic rings, with a significant preference to a 4-substituted B ring, rather than a substituted A ring. Neither the number of hydroxyls nor the presence of a catechol moiety on ring B correlated with increasing tyrosinase inhibition potency. 4-Hydroxychalcone (4-HC), ILC and Butein inhibited tyrosinase and shortened the lag period of enzyme monophenolase activity from about 490 min (control) to 30 min (ILC). As pigmentation also results from auto-oxidation, the antioxidant activity of 4-HC, ILC and Butein, were tested. Results showed that chalcones are also potent antioxidants, with Butein the most potent. We may conclude that chalcones are potentially potent new depigmentation agents, with their double effect of reduction and antioxidant activity. A deeper understanding of the relation between their structures to their potency will contribute to designing the optimal agents.

Chalcones with non-, mono-, di-, tri-, or tetra-substituted hydroxyl groups were examined as new family of tyrosinase inhibitors, demonstrating their potential whitening potency.

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Introduction

Tyrosinase (monophenol monooxygenase, E:C:1.14.18.1), also known as polyphenol oxidase (Whitaker, 1995), is a copper-containing enzyme widely distributed in nature. It catalyzes two reactions involving molecular oxygen in the melanin biosynthesis pathway: the hydroxylation of monophenols to o-phenols (monophenolase activity), and the oxidation of the o-phenols to o-quinones (diphenolase activity). These quinones are highly reactive and tend to polymerize spontaneously to form brown pigments of high molecular weight (melanins), which determine the color of mammalian skin and hair (Seo et al., 2003). Quinones can also react with amino acids and proteins and thus enhance the development of brown color.

Various dermatological disorders, such as melasama, age spots and sites of actinic damage, arise from the accumulation of an excessive level of epidermal pigmentation. Tyrosinase inhibitors have become increasingly important in medication (Seo et al., 2003) and in cosmetics (Maeda and Fukuda, 1991) to prevent hyperpigmentation, by inhibiting enzymatic oxidation. A number of naturally occurring tyrosinase inhibitors have been described, the majority consisting of a phenol structure or of metal chelating agents (Mayer, 1987; Mayer and Harel, 1979; Passi and Nazzaro-Porro, 1981; Pifferi et al., 1974). Some of these inhibitors suffer from number of limitations, such as low activity, high toxicity, and insufficient penetrative ability. Other potentially active agents, such as kojic acid and arbutin, have not yet been demonstrated as clinically efficient (Briganti et al., 2003). Hydroquinone, a widely used skin lightening agent, is a compound considered to be cytotoxic to melanocytes, and hence potentially mitogenic (Briganti et al., 2003; Dooley, 1997; Frenk, 1995; Hermanns et al., 2000). A possible way to overcome some of these limitations could be the use of a combination of compounds in a mixture, which may act as whitening agents through different mechanisms and in a complementary way, such as combining tyrosinase inhibitors with efficient antioxidants, together with antiperoxidases.

Chalcones are a group of compounds widely present in higher plants (Star and Mabry, 1971; Stevens et al., 2000), which may combine these various activities. They contain two aromatic rings with an unsaturated chain. Many biological activities have been attributed to this group, such as anticancer (Satyanarayana and Rao, 1993; Shibata, 1994), anti-inflammatory, antipyretic and analgesic (Satyanarayana and Rao, 1993), cytotoxic in vitro (Dhar, 1981), bactericidal, insecticidal, anti-fungal (Satyanarayana and Rao, 1993), antioxidant (Ruby et al., 1995; Vaya et al., 1997) and phytoestrogenic activities (Maggiolini et al., 2002; Tamir et al., 2001). In our previous work (Vaya et al., 1997), two chalcones were isolated from licorice root, isoliquiritigenin (2,4,4-OH chalcone, ILC) and isoprenylchalcone (IPC), the former presenting antioxidant and phytoestrogenic activities (Tamir et al., 2001) and the latter not active in the tests carried out. The aim of the present study was to explore the potential of chalcones as whitening agents: this group of compounds has not been studied before in this context, despite their structural similarity to t-stilbene, a well-known tyrosinase inhibitor. The inhibitory activity of a series of chalcones was set against their structure and their antioxidant potency (which can contribute to prevent pigmentation resulting from non-enzymatic oxidation). Such a comparison may lead to the design of new whitening agents which overcome some of the limitations associated with existing depigmentation compounds.

Section snippets

Inhibition of tyrosinase activity by chalcones

The inhibitory effect of tyrosinase by nine chalcones was examined at both enzyme activity stages: the hydroxylation of tyrosine to L-dopa, and the oxidation of the L-dopa to L-dopa quinone. A summary of the results obtained, shown in Table 1, demonstrates that when either no hydroxyl group is attached to the chalcone skeleton, or when one or two hydroxyls are present only on ring A of the chalcone moiety, the compounds are practically inactive. Thus, chalcone and 2-HC did not inhibit

Discussion

In the present study, the relation between the structure of nine different chalcones was compared to their tyrosinase inhibition activity, showing that the position of the hydroxyl groups attached to the A and B aromatic rings is of major importance, while hydroxylation on ring B contributes markedly more to inhibition than when it is on ring A. Butein, an effective tyrosinase inhibitor, was also able to delay linoleic acid auto-oxidation, as shown by conjugated diene (CD) formation.

The

Chemicals and reagents

Tyrosinase (EC1.14.18.1, Sigma Product T7755, with an activity of 6680 units/mg), Butein and trolox (a synthetic, hydrophilic derivate of vitamin E), were purchased from Sigma. The chalcones, 4-hydroxychalcone (4-HC), 4-hydroxychalcone (4-HC), 2-hydroxychalcone (2-HC), 2,4-dihydroxychalcone (2,4-HC), 4,2-dihydroxychalcone (4,2-HC), 4,2,4-trihydroxychalcone (ILC) and 2,4,4-trihydroxychalcone-3,3-di-isoprenyl chalcone (IPC) were purchased from Indofine Chemical Co.

Tyrosinase assay

Potassium

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