EFFECT OF ACETOHYDROXAMIC ACID (AHA) AND SALICYLHYDROXAMIC ACID (SHAM) ON THE OXIDATION OF o-DIHYDROXY- AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

Acetohydroxamic acid (AHA) and salicylhydroxamic acid (SHAM) each inhibited the rate of oxidation of different o-dihydroxy- and trihydroxyphenols by tyrosinase when assayed spectrophotometrically or polarographically. SHAM was a much more effective inhibitor than AHA. Spectral changes occurring during the oxidation of different o-dihydroxyphenols by tyrosinase in the presence of AHA or SHAM were different than the spectral changes occurring in their absence. AHA and SHAM also had an effect on the spectrum of the final product(s) formed when different o-dihydroxyphenols were oxidized by the enzyme, suggesting that AHA and SHAM conjugate with the o-quinones formed. A lack of an effect of AHA and SHAM on the spectrum of product(s) formed when trihydroxyphenols were oxidized by tyrosinase suggest that AHA and SHAM do not conjugate with the o-quinones derived from trihydroxyphenols.     

EFFECT OF KOJIC ACID ON THE OXIDATION OF TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE1

Kojic acid [5-hydroxy-2-(hydroxymethyl)-4-pyrone] inhibited effectively the rate of pigment formation during the oxidation of pyrogallol, 2, 3,4-THAP (2, 3,4-trihydroxyacetophenone) and 2, 4,5-THBP (2, 4,5-trihydroxybutyrophenone) by tyrosinase. On the other hand, kojic acid had a synergistic effect on the rate of methyl gallate and n-propyl gallate oxidation to pigmented product(s) (λ_max= 360 nm and λ_max= 380 nm, respectively). However, kojic acid inhibited effectively the rate of oxygen uptake when each of the above trihydroxyphenols was oxidized by tyrosinase. These results suggest that kojic acid inhibits tyrosinase per se (probably due to its ability to bind copper at the active site of the enzyme) and that it exerts only an apparent stimulatory effect during the formation of pigmented product (s) from methyl gallate and n-propyl gallate. Proof for the latter was obtained by a time-course experiment of kojic acid addition and examination of the spectra of pigmented product(s) formed in the absence versus presence of kojic acid, which suggested that the o-quinone of n-propyl gallate and the o-quinone of methyl gallate can each convert kojic acid to a yellow product(s) absorbing at the 360–380 nm region.     

EFFECT OF HYDROXYLAMINE, p-AMINOBENZOIC ACID AND p-AMINOSALICYLIC ACID ON THE OXIDATION OF O-DIHYDROXY- AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

The effect of hydroxylamine (NH₂OH), p-aminobenzoic acid (PABA) and p-aminosalicylic acid (PASA) on the spectrum of the final product (s) formed when o-dihydroxy- and trihydroxyphenols were oxidized by tyrosinase was examined. New pigmented product(s), probably oximes, were formed by the interaction of NH₂OH with the o-quinones of 4-methyl catechol, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylpropionic acid (3,4-DPPA) but not with the o-quinones of catechol or protocatechuic acid. Interaction of PABA or PASA with the o-quinones of catechol, 4-methyl catechol, protocatechuic acid, DOPAC and 3,4-DPPA also yielded pigmented oximes. The interaction of the o-quinones of trihydroxyphenols with NH₂OH, PABA or PASA had little effect on the spectrum of the final product (s), suggesting that oximes are not formed in these reactions.     

EFFECT OF MALTOL ON THE OXIDATION OF o-DIHYDROXYPHENOLS BY MUSHROOM TYROSINASE AND BY SODIUM PERIODATE1

Maltol (3-hydroxy-2-methyl-4H-pyran-4-one) inhibits the rate of oxidation of different o-dihydroxyphenols by tyrosinase when assayed spectrophotometrically, but not when assayed polarographically. The spectral changes occurring during the oxidation of different o-dihydroxyphenols by tyrosinase (or by sodium periodate) in the absence or presence of maltol were different, suggesting that maltol conjugates with the o-quinones formed. Maltol does not inhibit tyrosinase activity per se but only gives an apparent inhibition probably due to its ability to conjugate with o-quinones.     

EFFECT OF BENZOIC ACID AND SOME OF ITS DERIVATIVES ON THE RATE OF DL-DOPA OXIDATION BY MUSHROOM TYROSINASE1

Vanillic acid and salicylic acid inhibited the rate of dihydroxyphenylalanine (DL-DOPA) oxidation to dopachrome (γ_max=475 nm) by tyrosinase at all concentrations tested. Benzoic acid and p-hydroxybenzoic acid (PHBA), at relatively low concentrations, slightly stimulated the rate of DL-DOPA oxidation, whereas at higher concentrations each inhibited the reaction. p-Hydroxybenzoic acid methyl ester (PHBAME), at relatively low concentrations, had a pronounced synergistic effect on the reaction, whereas at relatively high concentrations it inhibited the rate of DL-DOPA oxidation. The synergistic effect of 1.6–6.6 mM PHBAME on the rate of DL-DOPA oxidation to dopachrome was found to be only an apparent effect due to the ability of PHBAME to be hydroxylated very slowly by tyrosinase to a yellow pigmented product(s) with DL-DOPA serving as a reductant (AH₂) for the hydroxylation reaction, thus hastening the conversion of PHBAME to pigmented product(s). Vanillic acid, salicylic acid, benzoic acid and PHBA could not be hydroxylated by tyrosinase.     

EFFECT OF 2,3,4-TRIHYDROXYACETOPHENONE (2,3,4-THAP) AND 2,4,5-TRIHYDROXYBUTYROPHENONE (2,4,5-THBP) ON THE RATE OF DL-DOPA OXIDATION BY MUSHROOM TYROSINASE1

Various concentrations of 2,3,4-trihydroxyacetophenone (2,3,4-THAP) and 2,4,5-trihydroxybutyrophenone (2,4,5-THBP) have a synergistic effect on the rate of DL-3,4-dihydroxyphenylalanine (DL-DOPA) oxidation by mushroom tyrosinase as measured by absorbance at 475 nm. The synergism results from the ability of dopaquinone to nonenzymatically oxidize 2,3,4-THAP and 2,4,5-THBP to the corresponding o-quinone. Moreover, various concentrations of 2,3,4-THAP and 2,4,5-THBP prevent the enzymatic conversion of DL-DOPA to DOPA-melanin, probably due to the ability of these trihydroxyphenols to interact nonenzymatically with dopaquinone.     

EFFECT OF KOJIC ACID ON THE HYDROXYLATION OF L-TYROSINE AND TYRAMINE BY MUSHROOM TYROSINASE1

Kojic acid inhibits effectively the rate of L-tyrosine and tyramine hydroxylation by mushroom tyrosinase. It also affects the spectrum of product (s) formed, this being due to the ability of dopaquinone and dopamine-o-quinone to oxidize kojic acid to a yellow product(s). Kojic acid prevents the conversion of these o-quinones to their corresponding melanins. An insoluble yellow product(s) is formed when tyramine, but not L-tyrosine, is incubated with tyrosinase for 20 h in the presence of excess kojic acid.     

p-HYDROXYPHENYLACETIC ACID AND-3,4-DIHYDROXYPHENYLACETIC ACID AS SUBSTRATES FOR MUSHROOM TYROSINASE

The hydroxylation of p-hydroxyphenylacetic acid (PHPAC) and the oxidation of 3,4-dihydroxyphenylacetic acid (DOPAC) by mushroom tyrosinase are illustrated. DOPAC quinone (4-carboxy-methyl-o-benzoquinone (λ_max= 400±10 nm) is the initial pigmented product formed when DOPAC is oxidized by the enzyme. DOPAC quinone is very unstable, and, once formed, is converted rapidly to further oxidation product(s).
The relationships between the rate of p-dihydroxyphenylacetic acid hydroxylation and 3,4-dihydroxyphenylacetic acid oxidation as a function of various concentrations of each substrate and of mushroom tyrosinase, are described.
The effect of the addition of various chemicals that can potentially conjugate otherwise affect DOPAC quinone was studied The Km value of 3,4-dihydroxyphenylacetic acid for mushroom tyrosinase was estimated to be 4.0 mM.     

2,3-DIHYDROXYBENZOIC ACID AS A SUBSTRATE FOR MUSHROOM TYROSINASE1,2

When 2, 3-dihydroxybenzoic acid (2, 3-DBA) is acted upon by mushroom tyrosinase, a yellow intermediate, 2, 3-DBA-o-quinone, characterized by a peak at 415 nm, is the first product detected. 2, 3-DBA-o-quinone gives rise to a “final blue product” (λmax = 230, 410, 620 nm), and to “soluble oxidation product(s)” (λmax = 275–280, 350–360 nm). Kinetic data (assayed spectrophotometrically and polarographically) obtained when different concentrations of 2, 3-DBA were oxidized by a fixed amount of mushroom tyrosinase, deviated from classic Michaelis-Menten kinetics. Reduction of the “final blue product” with ascorbate resulted in the loss of the blue chromophore at 620 nm and the concomitant appearance of a “yellowish reduced final product.” The “yellowish reduced final product” could be reoxidized with either mushroom tyrosinase or with NaIO4 to the “final blue product,” indicating that the latter has carbonylic quinonoid groups in ortho position to each other.     

EFFECT OF KOJIC ACID ON THE OXIDATION OF o-DIHYDROXYPHENOLS BY MUSHROOM TYROSINASE1

Kojic acid is a very effective inhibitor of mushroom tyrosinase as judged by its effect on the rate of pigmented products formation and on the rate of oxygen uptake when different o-dihydroxyphenols are oxidized by the enzyme. In addition to the ability of kojic acid to inhibit the enzyme per se, the data show that kojic acid can change the spectrum of some pigmented products formed in its absence, probably due to the ability of some o-quinones formed enzymatically to oxidize kojic acid to a yellow product(s). These findings call for caution in the use of kojic acid as an inhibitor of enzymatic browning in tissues and processed foods that are not yellow originally.     

p-HYDROXYPHENYLPROPIONIC ACID (PHPPA) AND 3,4-DIHYDROXYPHENYLPROPIONIC ACID (3,4-DPPA) AS SUBSTRATES FOR MUSHROOM TYROSINASE

p-Hydroxyphenylpropionic acid (PHPPA) and 3,4- dihydroxyphenylpropionic acid (3,4-DPPA) serve as substrates for tyrosinase. The Km value of 3,4-DPPA for tyrosinase is 1.3 mM. The yellow o-quinone of 3,4-DPPA (4-carboxyethyl-o-benzoquinone) (λ_max= 400nm), is detected initially and it is then converted to a red product(s) (λ_max= 480±10 nm), the o-quinone of 6,7-dihydroxy 3-dihydrocumarin (dihydroesculetin). When the concentration of the latter is relatively high, it polymerizes to a final brown product(s), characterized by an ill-defined spectrum.
H₂O₂ shortens the lag period of PHPPA hydroxylation, hastens the conversion of the yellow o-quinone of 3,4-DPPA to the red o-quinone of dihydroesculetin, and prevents the polymerization of the latter to the final brown product(s).
The relatively unstable o-quinone of 3,4-DPPA interacts with amines such as hydroxylamine (NH₂OH), p-aminosalicylic acid (PASA) and p-aminobenzoic acid (PABA), forming relatively stable final product(s) characterized by different spectra from those formed in their absence.
Acetohydroxamic acid (AHA) and salicylhydroxamic acid (SHAM) each has an effect on the spectrum of product(s) obtained when 3,4-DPPA is oxidized by tyrosinase, indicating that these hydroxamic acids derivatives interact with the o-quinone of 3,4-DPPA. The spectrum of the final product(s) was also different when 3,4-DPPA was oxidized by tyrosinase in the presence of benzenesulfinic acid than in its absence, suggesting the formation of a stable phenylsulfonyl derivative.     

EFFECT OF SALICYLHYDROXAMIC ACID (SHAM) ON DL-DOPA OXIDATION BY MUSHROOM TYROSINASE AND BY NaIO4

Salicylhydroxamic acid (SHAM) inhibits very effectively the rate of DL‐DOPA oxidation by mushroom tyrosinase. SHAM also affects the spectrum of the initial produces) formed when DL‐DOPA is oxidized by mushroom tyrosinase or by NaIO4. Moreover, at certain concentrations, SHAM prevents the polymerization of dopaquinone formed enzymaticatty or nonenzymatically probably due to a chemical interaction between dopaquinone and SHAM.     

SYNERGISM EXERTED BY 4-TERT-BUTYLCATECHOL AND TERT-BUTYLCATECHOL QUINONE ON THE RATE OF DL-DOPA OXIDATION BY MUSHROOM TYROSINASE1

4-tert-butylcatechol (t-BC), at concentrations ranging from 0.033 to 16.6 mM, has a pronounced synergistic effect on the rate of DL-DOPA oxidation to material absorbing at 475 nm (dopachrome) by mushroom tyrosinase. This phenomenon was found to be due to the ability of t-BC-quinone (formed by the oxidation of t-BC by mushroom tyrosinase or by sodium periodate) to oxidize DL-DOPA nonenzymatically.     

文冠果壳皂苷提取物抑制酪氨酸酶活性的研究

分别采用70％的乙醇溶液、蒸馏水2种浸提液提取文冠果壳中的皂苷,并测定其含量以确定最佳提取溶剂.通过对酪氨酸酶催化L-多巴氧化速率的测定研究了文冠果壳粉的乙醇提取液对体外酪氨酸酶活性的抑制作用.结果表明,皂苷粗提液对酪氨酸酶的抑制率与浓度呈非线性变化,随着浓度的增加,抑制剂对酪氨酸酶活性的抑制率先增加后趋于平稳.当皂苷质量浓度为0.36 mg/mL,抑制率可达到64.6％.通过酶抑制作用的Lineweaver-Burk图,分析结果显示,文冠果壳皂苷粗提液对酪氨酸酶的抑制作用类型为非竞争性抑制.上述研究为进一步开发文冠果壳中的美白成分提供了依据,同时又实现了废物利用,提高了农产品的经济价值.     

Inhibitory Effects of ‘Enokitake’ Mushroom Extracts on Polyphenol Oxidase and Prevention of Apple Browning

‘Enokitake’ mushroom (Flammulina velutipes) extracts were prepared by three different solvents: 70 mL/100 mL acetone, 70 mL/100 mL ethanol as well as hot water. Effects of the extracts on mushroom tyrosinase activity and browning of apple were investigated. Mushroom tyrosinase activities assayed spectrophotometrically and by oxygen uptake were found to be inhibited significantly by all three extracts. Lyophilized ‘enokitake’ fruit body powder inhibited the browning of fresh apple. Immersing sliced apple into the acetone extract was found to prevent effectively the browning development. Freshly squeezed apple juice with added hot-water extract showed the concentration-dependent inhibition of browning. No browning was observed on the apple juice when mixed with the extract containing 1.0 g wet ‘enokitake’/mL in a mixture. Under the same conditions, no significant changes in color in terms of a* and b* values of the mixture were observed for up to 6 h. These observations suggested that ‘enokitake’ mushroom contained certain compounds which inhibited mushroom tyrosinase activity and the development of browning induced by the catalytic oxidation due to polyphenol oxidase.     

KOJIC ACID CONVERSION TO A YELLOW PRODUCT(S) BY THE HEMOGLOBLIN/H2O2 SYSTEM1

Kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one; also named 5-hydroxy-2-(hydroxymethyl)-γ-pyrone) in the presence of hydrogen peroxide, but not in its absence, can be oxidized by hemoglobin (Hb) to a yellow product(s). The yellow product(s) formed is characterized by a peak at 370–380 nm and is fluorescent. The relationship between the rate of oxidation of kojic acid and various concentrations of hemoglobin and of H₂O₂ is described. The changes with time in the spectrum of product(s) obtained when kojic acid is acted upon by the Hb/H₂O₂ system and the relationship between the various concentrations of hemoglobin, of kojic acid and of H₂O₂ on the spectrum of the “final yellow product(s)” are shown.     

KOJIC ACID CONVERSION TO A YELLOW PRODUCT(S) VIA THE HORSERADISH PEROXIDASE/H2O2 SYSTEM1

Horseradish peroxidase in the presence of hydrogen peroxide oxidizes kojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) to a yellow product(s). The yellow product(s) formed has a major absorbance peak at 375 nm and is fluorescent. The relationships between, and effects of, various concentrations of horseradish peroxidase, kojic acid and hydrogen peroxide on the rate of oxidation of kojic acid to the yellow product(s) are described. The observation that the oxidation of kojic acid to the yellow product(s) occurs best in the presence of very low concentrations of hydrogen peroxide, relative to that of kojic acid, suggests that kojic acid is a poor hydrogen donor (AH₂) for horseradish peroxidase.     

Differential Inhibitory Effect of alpha-, beta-, gamma-, and delta-Tocopherols on the Metal-Induced Oxidation of Cholesterol in Unilamellar Phospholipid-Cholesterol Liposomes

ABSTRACT: Cholesterol oxidation products (COPs) are present in biological tissues and in foods. The inhibitory effect of antioxidants, such as tocopherols, on COPs formation has been only partially investigated. The antioxidant effect of dl alpha-, dl beta-, dl gamma-, and dl-delta tocopherol on the metal-induced oxidation of phosphatidylcholine (PC): cholesterol liposomes was assayed. Formation during liposome oxidation of six different COPs was monitored by gas chromatography. dl alpha-, and dl gamma-tocopherol show good inhibitory effect against PC-fatty acid oxidation and also on COPs formation. dl delta-Tocopherol is less effective than the alpha-and gamma-homologous, beta-tocopherol being unable to prevent PC and cholesterol oxidation. dl alpha-, and dl gamma-Tocopherol are more effective to prevent the oxidation of the lateral chain of cholesterol molecule. At the highest tocopherol concentration assayed, dl alpha-tocopherol shows prooxidant effect, enhancing liposomal oxidation and COPs formation. It is concluded that the tocopherols assayed can inhibit cholesterol oxidation but to a different degree.     

Oxidation of Polyphenols and the Effect on In vitro Iron Accessibility in a Model Food System

ABSTRACT: Tannic acid and green tea extract were added to a model food system of wheat bread, before and after incubation with polyphenol oxidase (mushroom tyrosinase), and the effect on In vitro iron accessibility was studied. Tannic acid and green tea extract had a marked inhibitory effect on the In vitro accessibility of iron when added to the model food system. Incubation of the polyphenols with tyrosinase before addition to the model food system significantly increased the accessibility of iron. The results from the study therefore suggest that oxidation of polyphenols may be a promising way to increase the bioavailability of iron in polyphenol containing foods.     

白藜芦醇对酪氨酸酶体外抑制活性的动力学研究

在30℃,pH 6.8的Na2HPO4-NaH2PO4的缓冲体系中,采用酶促动力学方法,研究了白藜芦醇对酪氨酸酶单酚酶和二酚酶的抑制作用。结果表明,白藜芦醇对酪氨酸酶、单酚酶和二酚酶均有抑制作用,对单酚酶抑制活性的IC50值(抑制率达到50%时的白藜芦醇质量浓度)约为5.1mg/mL,对二酚酶抑制活性的IC50值约为5.6 mg/mL。此外,白藜芦醇可延长单酚酶的迟滞效应,8 mg/mL的白藜芦醇能使迟滞时间从22 s延长至62 s,而对二酚酶则无此迟滞作用。Lineweav-er-Burk图分析表明,白藜芦醇对酪氨酸酶的抑制作用为混合型抑制,对游离酶的抑制常数(KI)和对酶-底物络合物的抑制常数(KIS)分别为3.4 mg/mL和35.98 mg/mL。