Inhibitory potential of 4-hexylresorcinol against α-glucosidase and non-enzymatic glycation: Activity and mechanism.

Inhibition of α-glucosidase as well as non-enzymatic glycation is thought as an effective method for treating type-2 diabetes mellitus. In this study, we investigated the inhibitory potential and mechanism of 4-hexylresorcinol against α-glucosidase and non-enzymatic glycation by using multispectroscopic analyses and molecular docking. The results of enzyme kinetics showed that 4-hexylresorcinol reversibly inhibited α-glucosidase activity in a noncompetitive way. Fluorescence quenching then revealed that it increased the hydrophobicity of α-glucosidase and changed the conformation of the enzyme by forming the α-glucosidase-hexylresorcinol complex. Thermodynamic analysis and molecular docking further demonstrated that the inhibition of 4-hexylresorcinol on the α-glucosidase was mainly dependent on hydrogen bond and hydrophobic interaction. Moreover, the 4-hexylresorcinol moderately inhibited the formation of fructosamine, and strongly suppressed the generation of α-dicarbonyl compounds and advanced glycation end products (AGEs). The interaction between 4-hexylresorcinol and bovine serum albumin was mainly driven by hydrophobic interaction. This study showed a novel inhibitor of α-glucosidase as well as non-enzymatic glycation, and provided a drug candidate for the prevention and treatment of type-2 diabetes.

Rapid autographic method for detection of enzymatic browning inhibitors based on enzyme immobilization.

This work describes a TLC-coupled bioautographic assay suitable for the separation and detection of apple polyphenol oxidase (PPO) inhibitors from natural extracts. PPO was immobilised in agar containing l-DOPA as substrate and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) to enhance colour development. The inhibition was detected as white spots on reddish background. Minimum amount of PPO inhibitors detected was 0.0125 µg of 4-hexylresorcinol, 0.025 µg of ascorbic acid, 0.5 µg of cysteine and 1 µg of kojic acid. The assay was compatible with normal and reverse phase TLC systems and allows detecting compounds that directly had action on the enzyme as well as agents that could convert quinones back to their reduced form. The chromatographic run evidenced the different nature of enzymatic browning inhibitory compounds from garlic and onion extracts. Using natural enzymes will provide a fast and cheap alternative for target specific exploration of natural enzymatic inhibitors.

Determination of 4-Hexylresorcinol in Shrimp Samples by Solid Phase Extraction Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry.

A method for the rapid determination of 4-hexylresorcinol (4-HR) residue in shrimp by solid phase extraction (SPE) ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established. 4-HR was extracted twice with methanol, and the extract was formulated into methanol-water solution (1:1). After being cleaned up and concentrated by a PRIME HLB solid phase extraction column, the sample was analyzed by UPLC-MS/MS and quantitatively determined by an external standard method. The separation was performed with a gradient system consisting of water and acetonitrile as the mobile phase. Monitoring was performed by electrospray ionization (ESI) in negative ion mode using multiple ion reaction monitoring (MRM). Good linearity was obtained in the concentration range of 1.0⁻100.0 µg/L, with correlation coefficients larger than 0.999. The limit of detection (LOD) was 0.25 µg/kg and the limit of quantification (LOQ) was 0.80 µg/kg. The average recoveries of 4-HR at spiked concentrations of 2.40, 6.40 and 16 µg/kg ranged from 81.35% to 94.68% with the relative standard deviations (n = 6) from 3.57% to 6.86%. The results showed that the method is simple, fast, sensitive, reliable, and reproducible; thus, it could be used as a rapid confirmation and quantitative analysis method of 4-HR residue in aquatic products.

Accelerated biodegradation of silk sutures through matrix metalloproteinase activation by incorporating 4-hexylresorcinol.

Silk suture material is primarily composed of silk fibroin and regarded as a non-resorbable material. It is slowly degraded by proteolysis when it is implanted into the body. 4-Hexylresorcinol (4HR) is a well-known antiseptic. In this study, the biodegradability of 4HR-incorporated silk sutures were compared to that of untreated silk sutures and polyglactin 910 sutures, a commercially available resorbable suture. 4HR-incorporated silk sutures exhibited anti-microbial properties. Matrix metalloproteinase (MMP) can digest a wide spectrum of proteins. 4HR increased MMP-2, -3, and -9 expression in RAW264.7 cells. MMP-2, -3, and -9 were able to digest not only silk fibroin but also silk sutures. Consequently, 59.5% of the 4HR-incorporated silk suture material remained at 11 weeks after grafting, which was similar to that of polyglactin 910 degradation (56.4% remained). The residual amount of bare silk suture material at 11 weeks after grafting was 91.5%. The expression levels of MMP-2, -3 and -9 were high in the 4HR-incorporated silk suture-implanted site 12 weeks after implantation. In conclusion, 4HR-treated silk sutures exhibited anti-microbial properties and a similar level of bio-degradation to polyglactin 910 sutures and induced higher expression of MMP-2, -3, and -9 in macrophages.

Cobalt(II) complexation with small biomolecules as studied by 57Co emission Mössbauer spectroscopy.

In the emission (57Co) variant of Mössbauer spectroscopy (EMS), the 57Co radionuclide (with a half-life of 9months) is used that undergoes a nuclear decay 57Co→57Fe via electron capture followed by the emission of a γ-quantum, the energy of which is modified by the chemical state and the close coordination environment of the parent 57Co atom. While EMS has been used largely in materials science and nuclear chemistry, its high sensitivity can also be of great advantage in revealing fine structural features and for speciation analysis of biological complexes, whenever the 57Co2+ cation can be used directly as the coordinating metal or as a substitute for native cobalt or other metal ions. As such EMS applications are yet rare, in order to reliably interpret emission spectra of sophisticated 57Co2+-doped biosystems, model EMS studies of simple cobalt biocomplexes are necessary. In this work, EMS spectroscopic data are analysed and discussed for 57Co2+ complexes with a range of small biomolecules of different structures, including 4-n-hexylresorcinol, homoserine lactone and a few amino acids (spectra measured in rapidly frozen dilute aqueous solutions or in the dried state at T=80K). The EMS data obtained are discussed with regard to the available literature data related to the coordination modes of the biocomplexes under study.

Development and validation of an analytical method for the determination of 4-hexylresorcinol in food.

This study presents a method validation for extraction and quantitative analysis of 4-hexylresorcinol residues in shrimp and crab meat using HPLC-FLD. We were focused on the collaboratively analysis of each shrimp and crab meat samples, and developed LC-MS/MS method for the correct confirmation of the identity of compound. Validation parameters; selectivity, linearity, LOD, LOQ, accuracy, precision, and measurement of uncertainty were attained. The measurement of uncertainty was based on the precision study, data related to the performance of the analytical process and quantification of 4-hexylresorcinol. For HPLC-FLD analysis, the recoveries of 4-hexylresorcinol from spiked samples at levels of 0.2-10.0 ppm ranged from 92.54% to 97.67% with RSDs between 0.07% and 1.88%. According to these results, the method has been proven to be appropriate for extraction and determination of 4-hexylresorcinol, and can be used to maintain the safety of shrimp and crab products containing 4-hexylresorcinol residues.

Tyrosinase-Catalyzed Hydroxylation of 4-Hexylresorcinol, an Antibrowning and Depigmenting Agent: A Kinetic Study.

4-Hexylresorcinol (HR) is a compound used in the food and cosmetic industries as an antibrowning and lightening agent. Its use is mainly attributed to its inhibitory effect on the enzyme tyrosinase. However, the enzyme hydroxylates HR to an o-diphenol, which it then oxidizes to an o-quinone, which rapidly isomerizes to p-quinone. For tyrosinase to act in this way, the Eox form (oxy-tyrosinase) must be present in the reaction medium, which can be brought about by (a) hydrogen peroxide, (b) ascorbic acid, or (c) catalytic concentrations of o-diphenol and a reductant (NADH) to maintain it constant. This work demonstrates that HR is a substrate of tyrosinase and proposes a mechanism for its action. Its kinetic characterization provides a catalytic constant of 0.85 ± 0.04 s(-1) and a Michaelis constant of 60.31 ± 6.73 µM.

Silk fibroin and 4-hexylresorcinol incorporation membrane for guided bone regeneration.

The objective of this study was to demonstrate that a silk fibroin (SF) and 4-hexylresorcinol (4-HR) incorporation membrane could be used for a guided bone regeneration technique. Fourier transform infrared measurements were obtained to determine change of physical property of SF membrane by 4-HR incorporation. Two peri-implant defects, 3.0 × 5.0 mm (width × length), were prepared on the lateral side of the implant hole in the tibia of New Zealand white rabbits (n = 8). The peri-implant defect was left unfilled in the control group. Silk fibroin + 4-HR membrane was applied to the peri-implant defect in the experimental group. The 8 animals were killed at 8 weeks after implantation. Subsequently, removal torque test and histomorphometric evaluation were done. Fourier transform infrared spectroscopy showed no specific chemical interaction between 4-HR and SF. In the histomorphometric analysis, the mean bone regeneration was 18.3 ± 1.9 mm(2) in the experimental group and 9.3 ± 0.9 mm(2) in the control group (P = 0.004). In conclusion, the SF and 4-HR incorporation membrane successfully regenerated bone in the rabbit tibia peri-implant bone defect model.

A measurement of the unstirred aqueous boundary layer in a Franz diffusion cell.

The unstirred aqueous boundary layer (ABL) thickness was determined for modified Franz diffusion cells using three test compounds--parathion, 4-hexylresorcinol, and 2,4-dinitrochlorobenzene (DNCB). Steady-state fluxes through one, two, and three dialysis membranes in series were achieved in donor-saturated, infinite-dose permeation experiments. These results were then used to calculate the total diffusive resistance for each case, as well as an extrapolated zero membrane value which was used in conjunction with an estimate of the aqueous diffusivities to calculate the ABL thickness. The resulting value (mean ± SE) was 0.070 ± 0.004 cm.

[Effect of a synthetic analogue of bacterial anabiosis autoinducers hexylresorcinol on the stability of membrane structures].

The effect of hexylresorcinol (HR), a chemical analogue of microbial anabiosis autoinducers of the alkylhydroxybenzene (AHB) group, on the stability of biological membranes and monolamellar liposomes formed of egg phosphatidylcholine (ePC) was studied. According to spectrophotometry and electron microscopy studying of HR-loaded liposomes in the presence of a surface-active agent Tween 20, the critical ratio between HR and ePC for liposome preservation was found to be close to equimolar. The trends in HR influence on membrane structural organization and stability confirmed in experiments on liposomes were also reproduced on intact bacterial cells explaining non-species-specific effect of AHBs. The demonstrated high efficiency of AHB biocides may be used in material and equipment protection against biocorrosion.

Solvent effects on the spectroscopic properties of 4-hexylresorcinol.

A study was made on the spectroscopic properties of 4-hexylresorcinol (4HR) in several solvents at room temperature. Absorption and emission spectra were slightly affected by solvent polarity. Stokes' shifts were small (approximately 3000 cm(-1)) and the fluorescence quantum yields varied between 0.05 and 0.68, depending on the solvent. The spectral shifts were correlated with different solvent scales. Multiple regression analysis indicates that both non-specific solute-solvent interactions as well as specific solute-solvent interactions (such as hydrogen bonding) play an important role in the position of the Stokes' shift and on the fluorescence quantum yield in the solvents under study.

Browning prevention by ascorbic acid and 4-hexylresorcinol: different mechanisms of action on polyphenol oxidase in the presence and in the absence of substrates.

We have investigated the mechanism of action of 4-hexylresorcinol (4-HR) and ascorbic acid (AA) on the polyphenol oxidase (PPO) catalyzed oxidation of phenolic substrates. Incubation of PPO with 4-HR diminishes strongly PPO activity. This effect can be erroneously interpreted, due to the high affinity of 4-HR for PPO, as irreversible inactivation of PPO. However, PPO activity can be recovered by dialysis after incubation with 4-HR. 4-hexylresorcinol is a canonical enzyme inhibitor that binds preferentially to the oxy form of PPO. It is a mixed-type inhibitor, because it influences both apparent V(max) (1.26 compared with 0.4 units in the absence and presence of 4-HR, respectively) and K(m) values (0.28 mM compared with 0.97 mM in the absence and in the presence of 4-HR, respectively) of PPO. AA can prevent browning by 2 different mechanisms: In the absence of PPO substrates it inactivates PPO irreversibly, probably through binding to its active site, preferentially in its oxy form. In the presence of PPO substrates, AA reduces PPO oxidized reaction products, which results in a lag phase when measuring PPO activity by monitoring dark product formation but not when monitoring O(2) consumption. The simultaneous use of both 4-HR and AA on PPO results in additive prevention of browning.