Ken-ichi Nihei

Molecular design of antibrowning agents: antioxidative tyrosinase inhibitors

Abstract Dodecyl (C12) gallate (3,4,5-trihydroxybenzoate) was found to inhibit the oxidation of l -3,4-dihydroxyphenylalanine ( l -DOPA) catalyzed by tyrosinase. This inhibition is a slow and reversible reaction with residual enzyme activity. The inhibition kinetics analyzed by Lineweaver–Burk plots revealed that dodecyl (lauryl) gallate is an uncompetitive inhibitor (KIS=0.636 mM). Gallic acid, the parent compound of dodecyl gallate, acts as a reducing agent for o-quinone produced by the enzymic oxidation.

Identification of oxidation product of arbutin in mushroom tyrosinase assay system

In order to elucidate whitening mechanisms of arbutin (hydroquinone-O-beta-D-glucopyranoside), its effects on mushroom tyrosinase were analyzed by spectrophotometric, polarographic, and HPLC experiments. It was found that as soon as catalytic amounts of L-DOPA become available as a cofactor, arbutin acts as a monophenol substrate. A significant enzymatic product was identified as 3,4-dihydroxyphenyl-O-beta-D-glucopyranoside by NMR and MS experiments.

Non-antibiotic antibacterial activity of dodecyl gallate

Dodecyl (C(12)) gallate (3,4,5-trihydroxybenzoate) (1) was found to possess antibacterial activity specifically against Gram-positive bacteria, in addition to its potent antioxidant activity. The time-kill curve study indicates that this amphipathic gallate exhibits bactericidal activity against methicillin resistant Staphylococcus aureus (MRSA) strains. Dodecyl (lauryl) gallate inhibited oxygen consumption in whole cells and oxidation of NADH in membrane preparation. The antibacterial activity of this gallate comes in part from its ability to inhibit the membrane respiratory chain. As far as alkyl gallates are concerned, their antimicrobial spectra and potency depend in part on the hydrophobic portion of the molecule.

Tyrosinase Inhibitors from Galls of Rhus javanica Leaves and Their Effects on Insects

Abstract As a defense mechanism of the leaves of Rhus javanica (Anacardiaceae) against the aphid Melaphis chinensis (Aphididae) attack, tannic acid is rapidly accumulated and forms galls along the midrib of the leaves resulting in a unique natural medicine Gallae Rhois. Tannic acid was found to inhibit the oxidation of ʟ-3,4-dihydroxyphenylalanine (ʟ-DOPA) catalyzed by tyrosinase (EC 1.14.18.1) with an IC50 of 22 μᴍ. The aphid would detoxify the ingested toxic tannic acid to relatively nontoxic gallic acid, whereas the non-adapted pink bollworm Pectinophora gossypiella larvae are sensitive to the ingested tannic acid.

Tyrosinase inhibition kinetics of anisic acid.

Abstract Anisic acid (p-methoxybenzoic acid) was characterized as a tyrosinase inhibitor from aniseed, a common food spice. It inhibited the oxidation of l-3,4-dihydroxyphenylalanine (ʟ- DOPA) catalyzed by tyrosinase with an IC50 of 0.60 mᴍ. The inhibition of tyrosinase by anisic acid is a reversible reaction with residual enzyme activity. This phenolic acid was found to be a classical noncompetitive inhibitor and the inhibition constant KI was obtained as 0.603 mᴍ. Anisic acid also inhibited the hydroxylation of ʟ-tyrosine catalyzed by tyrosinase. The lag phase caused by the monophenolase activity was lengthened and the steady-state activity of the enzyme was decreased by anisic acid.

Molecular design of multifunctional antibacterial agents against methicillin resistant Staphylococcus aureus (MRSA)

Antibacterial activity of a series of alkyl gallates (3,4,5-trihydroxybenzoates) against Gram-positive bacteria, especially methicillin resistant Staphylococcus aureus (MRSA) strains was evaluated. Gram-positive bacteria are all susceptible to alkyl gallates. Dodecyl gallate was the most effective against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 microg/mL (74 microM). The time-kill curve study showed that dodecyl gallate was bactericidal against this MRSA strain at any growth stage. This activity was observed even in the chloramphenicol-treated cells, but the rate of decrease of cell number was slower than that in the exponentially growing cells. The bactericidal activity of medium-chain alkyl gallates was noted in combination with their ability to disrupt the native membrane-associated function nonspecifically as surface-active agents (surfactants) and to inhibit the respiratory electron transport. Subsequently, the same series of alkyl protocatechuates (3,4-dihydroxybenzoates) were studied and the results obtained are similar to those found for alkyl gallates. The length of the alkyl chain is not a major contributor but is related to the activity.

Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase

5-Pentadecatrienylresorcinol, isolated from cashew nuts and commonly known as cardol (C₁₅:₃), prevented the generation of superoxide radicals catalysed by xanthine oxidase without the inhibition of uric acid formation. The inhibition kinetics did not follow the Michelis-Menten equation, but instead followed the Hill equation. Cardol (C₁₀:₀) also inhibited superoxide anion generation, but resorcinol and cardol (C₅:₀) did not inhibit superoxide anion generation. The related compounds 3,5-dihydroxyphenyl alkanoates and alkyl 2,4-dihydroxybenzoates, had more than a C9 chain, cooperatively inhibited but alkyl 3,5-dihydroxybenzoates, regardless of their alkyl chain length, did not inhibit the superoxide anion generation. These results suggested that specific inhibitors for superoxide anion generation catalysed by xanthine oxidase consisted of an electron-rich resorcinol group and an alkyl chain having longer than C9 chain.

Antifungal Cyclodepsipeptides, W493 A and B, from Fusarium sp.: Isolation and Structural Determination

W493 A and B, which showed strong antifungal activity, were isolated from a culture broth of Fusarium sp. The structure of W493 B was determined to be that of a cyclodepsipeptide, cyclo(3S,4R-HMTA-D-allo-Thr-L-Ala-D-Ala-L-Gln-D-Tyr-L-Ile) (1) by MS and NMR data, an amino acid analysis, and synthesis of the component. HMTA represents 3-hydroxy-4-methyltetradecanoic acid. W493 A (2) had a similar structure, except that L-Ile in 1 was replaced by L-Val. The absolute configuration of each amino acid was determined by chiral HPLC, and the sequence of the components was determined by HMBC experiments. The sequence of the two alanines was determined to be a L-Ala-D-Ala by a chiral HPLC analysis of the peptide fragment containing only one Ala residue. The absolute configuration of HMTA obtained from the hydrolysis of W493 B was determined to be 3S and 4R by comparing with four isomers prepared by enantioselective synthesis via Sharpless asymmetric epoxidation.

Synthesis and evaluation of bibenzyl glycosides as potent tyrosinase inhibitors

Bibenzyl glycosides 1-6 were synthesized from 2,4-dihydoxybenzaldehyde and xylose, glucose, cellobiose or maltose. The key steps in the synthesis were the Wittig reaction and trichloroacetimidate glycosylation. Tests for tyrosinase inhibitory activity showed that all were significantly active, indicating that they are unique hydrophilic tyrosinase inhibitors. Bibenzyl xyloside 2 is a particularly potent inhibitor (IC(50) = 0.43 μM, 17 times higher than that of kojic acid). These results suggest that the hydrophilic cavity of tyrosinase might accommodate the bulky carbohydrate on the bibenzyl scaffold.

Molecular design of potent tyrosinase inhibitors having the bibenzyl skeleton.

In order to develop water soluble tyrosinase inhibitors, bibenzyl xyloside 1 isolated from Chlorophytum arundinaceum (liliaceae), and its derivatives 2 and 3 were synthesized by using Wittig reaction and trichloroimidate glycosylation procedure as key steps. Xylosides 1-3 showed potent tyrosinase inhibitory activity with IC(50)s of 1.6, 0.43, and 0.73 microM, respectively, although each NMR data of synthetic bibenzyls was not identical to that of naturally occurring xyloside 1.