Inhibitory effects of salicylic acid family compounds on the diphenolase activity of mushroom tyrosinase

Mushroom tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, inhibitory effects on the diphenolase activity of 10 compounds of the salicylic acid-family on the diphenolase activity of mushroom tyrosinase have been studied. The results show that some of these compounds behave as reversible inhibitors. Salicylic acid is a competitive inhibitor while 4-methoxysalicylic acid is non-competitive, 5-methoxysalicylic acid is mixed-I type and 4-methylsalicylic acid and 5-methylsalicylic acid are mixed-II type. The inhibition constants of these five compounds were evaluated. The inhibition strength follows the order: 4-methylsalicylic acid > 5-methylsalicylic acid > 4-methoxysalicylic acid > salicylic acid > 5-methoxysalicylic acid. Models of the interaction between the enzyme and the inhibitors are further discussed and compared.     

Inhibitory effects of α-cyano-4-hydroxycinnamic acid on the activity of mushroom tyrosinase

The effects of α-cyano-4-hydroxycinnamic acid (HCCA) on the activity of mushroom tyrosinase have been studied. Results showed that HCCA could inhibit both the monophenolase activity and diphenolase activity of mushroom tyrosinase. For the monophenolase activity, the lag phase was obviously lengthened, and the steady-state activity of the enzyme decreased sharply. When the concentration of HCCA reached to 80 μM, the lag time was lengthened from 20 s to 150 s and the steady-state activity was lost by about 75%. The IC₅₀ value was estimated to be 48 μM. For the diphenolase activity, the inhibitory effect of HCCA was also dose-dependent and the IC₅₀ value was estimated to be 2.17 mM. The kinetic analyses showed that the inhibition of HCCA on the diphenolase activity was reversible and competitive with the inhibition constants (K_I) determined to be 1.24 mM.     

Inhibitory effects of 4-chlorosalicylic acid on mushroom tyrosinase and its antimicrobial activities

The inhibitory effects of 4-chlorosalicylic acid on the activity of mushroom tyrosinase have been investigated. The results showed that 4-chlorosalicylic acid could strongly inhibit both monophenolase activity and diphenolase activity. The IC₅₀ values were estimated as 1.89 mM and 1.10 mM for monophenolase and diphenolase activities, respectively. For the monophenolase activity, 4-chlorosalicylic acid could not only lengthen the lag time, but also decrease the steady-state rate. For the diphenolase activity, kinetic analyses showed that the inhibition by 4-chlorosalicylic acid was reversible and its mechanism was mixed-II type, which is different from salicylic acid. The inhibition constants (K_I and K_IS) were determined to be 1.51 mM and 0.82 mM, respectively. Furthermore, the antibacterial activity against Escherichia coli, Bacillus subtilis and Staphyloccocus aureus and antifungal activity against Aspergillus niger and Candida boidinii were investigated. The results showed that 4-chlorosalicylic acid was the most effective against E. coli with the MIC of 250 μg/ml and with the MBC of 500 μg/ml.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Antityrosinase and antimicrobial activities of 2-phenylethanol, 2-phenylacetaldehyde and 2-phenylacetic acid

The inhibition kinetics of 2-phenylethanol, 2-phenylacetaldehyde and 2-phenylacetic acid on the enzyme activity of mushroom tyrosinase have been investigated. The results showed that these aromatic compounds can lead to reversible inhibition of the enzyme; furthermore, 2-phenylacetaldehyde and 2-phenylacetic acid are uncompetitive inhibitors and 2-phenylethanol is a mixed-type inhibitor. The inhibition constants have been determined and the inhibiting ability was: 2-phenylacetaldehyde > 2-phenylacetic acid > 2-phenylethanol, indicating that the functional group on the benzene ring group played an important role in the inhibition of the enzyme. In addition, 2-phenylacetic acid and 2-phenylethanol were found to have effective antibacterial activities, and 2-phenylacetic acid was more effective against Escherichia coli and Ralstonia solanacearum than 2-phenylethanol, but 2-phenylacetaldehyde lacked of antibacterial activity.     

Inhibitory effects of the water extracts of Lavendula sp. on mushroom tyrosinase activity

This study aimed to evaluate the inhibition properties of six lavender species, including Lavendula angustifolia, Lavandula angustifolia “Vera”, Lavendula X allardii, Lavendula stoechas, Lavendula viridis and Lavendula X heterophylla, toward the activity of mushroom tyrosinase. When using l-3,4-dihydroxyphenylalanine (l-Dopa) as the substrate for mushroom tyrosinase, the water extracts of leaves and stems from L. stoechas and L. angustifolia “Vera” showed strong inhibitory effects against the activity of mushroom tyrosinase (70% and 66.4% inhibition, respectively). Oven-drying the leaves and stems or free-drying the water extracts significantly decreased the inhibitory abilities of the water extracts from all lavender species. The water extract from L. stoechas decreased the V_max values when using l-Dopa, catechol and 3,4-dihydroxyphenylacetic acid (DHPAA) as the substrates. It increased the value of K_m when l-Dopa and catechol were the substrates but it decreased the K_m when DHPAA was used. It behaved as a mixed-type inhibitor toward mushroom tyrosinase.     

Inhibition of aldehyde dehydrogenase enzyme by Durian (Durio zibethinus Murray) fruit extract

The scientific basis of the adverse, or at times lethal, effect of ingesting durian (Durio zibethinus Murray) while imbibing alcohol has not been established. Symptoms are reminiscent of the disulfiram–ethanol reaction (DER) arising from the inhibition of aldehyde dehydrogenase (ALDH). Cognizant of the inhibitory effect of sulphur compounds like disulfiram on ALDH and the rich sulphur content of durian, the influence of durian fruit extract on the ALDH-mediated oxidative metabolism of acetaldehyde was investigated. We report a dose-dependent inhibition of yeast ALDH (yALDH), at most 70% at 0.33 ppm (mg extract/l assay mix), by dichloromethane:pentane extracts. Sulphur-rich TLC fruit extract fractions that eluted farthest from the origin effected the greatest inhibitory action. yALDH assay using diethyl disulfide as internal standard further supports the role of durian’s sulfury constituents in the fruit’s ALDH-inhibiting property. Insight into the etiology of DER-like symptoms felt upon simultaneous durian and alcohol consumption is hereby presented.     

Cytotoxic effects of geranyl flavonoid derivatives from the fruit of Artocarpus communis in SK-Hep-1 human hepatocellular carcinoma cells

Breadfruit (Artocarpus communis) is cultivated in tropical and subtropical regions as a traditional starch crop. This study investigated the cytotoxic activity of geranyl flavonoid derivatives isolated from the fruit of A. communis. Two new geranyl flavonoid derivatives, arcommunol A and arcommunol B, together with four known compounds, were isolated from the fruit of A. communis. The effects of these compounds on the viability of HepG2, Hep3B, PLC5 and SK-Hep-1 cells were investigated. Arcommunol A showed the highest inhibitory activity with 2.05 μM IC₅₀ in SK-Hep-1 cells. Treatment with arcommunol A changed the ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members and subsequently induced the activation of apoptosis-inducing factor, apoptotic protease activating factor-1, caspase-9 and caspase-3, which may result in a cleavage of poly(ADP-ribose) polymerase. These findings provide critical information regarding the chemopreventive potential of arcommunol A from the fruit of A. communis.     

Glycosylation,amino acid analysis and kinetic properties of a major Kunitz-type trypsin inhibitor from Acacia victoriae Bentham seeds

An Acacia victoriae trypsin inhibitor (AvTI) was purified from the seeds of prickly wattle (A. victoriae Bentham) by salt precipitation, ion-exchange and gel filtration chromatography, and its degree of glycosylation, amino acid composition, and kinetic properties were determined. Gel electrophoresis revealed at least four glycoprotein bands in the crude extract, salt-precipitated and ion-exchange protein fractions, while the purified AvTI showed only one band and a degree of glycosylation of 2.06%. Glutamate (13.3%), aspartate (10.3%), leucine (7.62%) and lysine (7.01%) were the major amino acids in AvTI while the contents of sulphur-containing amino acids, cysteine (1.38%) and methionine (0.75%), as well as of tryptophan (1.17%) were low. Its dissociation constant (K_i) for the inhibition of bovine trypsin was found to be 1.06 × 10⁻⁸ M, indicating a high affinity between AvTI and this enzyme, and its role as a competitive inhibitor was confirmed by a double reciprocal plot. These results complement our earlier studies which indicated the presence of three isoforms of this Kunitz-type trypsin inhibitor in prickly wattle seed.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

Cytoprotective and antioxidant activity of free,conjugated and insoluble-bound phenolic acids from swallow root (Decalepis hamiltonii)

Swallow root (Decalepis hamiltonii) was extracted for free (SRFP), conjugated (SRCP) and insoluble-bound phenolic acids (SRIBP), and evaluated for cytoprotectivity, 1,1,diphenyl-2-picrylhydrazyl (DPPH) scavenging ability, reducing power and protection to DNA damage. In addition, the constituent phenolic acids in the extracts were also analysed. Results indicated a total phenol content of 20.72, 7.97 and 11.52 mg gallic acid equivalents (GAE)/g for SRFP, SRCP and SRIBP extracts, respectively. At 0.12 μg/mL concentration SRCP showed 87% cytoprotection (on NIH 3T3 cells) compared to SRFP (47%) and SRIBP (65%). DPPH radical scavenging activity indicated an IC₅₀ of 0.046, 0.06 and 0.128 μg/mL for SRCP, SRIBP and SRFP, respectively. Also, SRCP showed higher reducing power and DNA protectivity (80%). HPLC analysis of phenolic acid extracts showed the presence of hydroxybenzoate and cinnamate derivatives. Among the phenolics identified gallic, gentisic, protocatechuic and p-coumaric acids were the major contributors to antioxidant activity.     

Effect on lamb meat quality of including thyme (Thymus zygis ssp. gracilis) leaves in ewes’ diet

The aim of this study was to investigate the effect of including thyme leaves (TL) in the diet of pregnant sheep on the sensorial characteristics, bacterial spoilage and oxidative stability of lamb meat stored in modified atmosphere (70% O₂:30% CO₂). For this, thirty-six sheep were randomly assigned to three groups: control (basal diet), T₁ (3.7% thyme leaves), T₂ (7.5% thyme leaves). Meat spoilage (TV, PSY, MY, ENT, and LA), TBARS, CIELAB coordinates, metmyoglobin and the sensory characteristics of fresh lamb meat were analyzed on days 0, 7, 14 and 21. The presence of antioxidant compounds in the diet containing TL delayed (P < 0.05) colour deterioration, lipid oxidation and bacterial counts, while at the same time imparting a better appearance to the fresh lamb meat. In general, this effect was more pronounced at the higher level of TL (7.5%). High Pearson’s correlation coefficients were found between the sensory attributes, CIELAB coordinates and TBARS.     

Chemical composition,antioxidant and antimicrobial activities of basil (Ocimum basilicum) essential oils depends on seasonal variations

Chemical composition, antioxidant and antimicrobial activities of the essential oils from aerial parts of basil (Ocimum basilicum L.) as affected by four seasonal, namely summer, autumn, winter and spring growing variation were investigated. The hydro-distilled essential oils content ranged from 0.5% to 0.8%, the maximum amounts were observed in winter while minimum in summer. The essential oils consisted of linalool as the most abundant component (56.7–60.6%), followed by epi-α-cadinol (8.6–11.4%), α-bergamotene (7.4–9.2%) and γ-cadinene (3.2–5.4%). Samples collected in winter were found to be richer in oxygenated monoterpenes (68.9%), while those of summer were higher in sesquiterpene hydrocarbons (24.3%). The contents of most of the chemical constituents varied significantly (p < 0.05) with different seasons. The essential oils investigated, exhibited good antioxidant activity as measurements by DPPH free radical-scavenging ability, bleaching β-carotene in linoleic acid system and inhibition of linoleic acid oxidation. Evaluation of antimicrobial activity of the essential oils and linalool, the most abundant component, against bacterial strains: Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pasteurella multocida and pathogenic fungi Aspergillus niger, Mucor mucedo, Fusarium solani, Botryodiplodia theobromae, Rhizopus solani was assessed by disc diffusion method and measurement of determination of minimum inhibitory concentration. The results of antimicrobial assays indicated that all the tested microorganisms were affected. Both the antioxidant and antimicrobial activities of the oils varied significantly (p < 0.05), as seasons changed.     

Differences in suberin content and composition between two varieties of potatoes (Solanum tuberosum) and effect of post-harvest storage to the composition

The waterproof defence barrier of the potato (Solanum tuberosum) tuber periderm consists of the suberized cells of phellem. The distinct polyaliphatic and polyaromatic domains of suberin have separate roles in the development of resistance to infections. The aliphatic suberin composition and changes in the amounts of peel and extractive free suberized membrane i.e. raw suberin were studied in two potato varieties, Nikola and Asterix, during post-harvest storage for one year. The amount of peel increased from 2.33 to 4.80 g/kg in yellow-skinned Nikola and from 3.50 to 5.54 g/kg in red-skinned Asterix. The raw suberin fraction accounted for 258.1 ± 16.6 mg/g in the peel of Nikola and 250.7 ± 36.3 mg/g in the peel of Asterix on average during the storage period. In addition to increase in suberin up to 6 months, the microscopic images of the peel and raw suberin indicated that the other components contributed to the peel mass increase. The CHCl₃-soluble suberin monomer fraction after methanolysis accounted for about 20% of the raw suberin in both varieties, indicating a constant ratio of suberin polyaliphatic and polyaromatic domain over the storage period. α,ω-Diacids, fatty acids and aromatic compounds were more abundant in Asterix, while ω-hydroxyacids and fatty alcohols were more abundant in Nikola (all p = 0.000). Small changes in the aliphatic monomer composition within each variety were seen during storage.     

Profiling the chlorogenic acids of sweet potato (Ipomoea batatas) from China

The leaves, stem and root of sweet potato cultivated in China have been analysed qualitatively for chlorogenic acids by structure-diagnostic LC–MS³. Chlorogenic acids were not detected in the root. Caffeoylquinic acids were quantitatively the major subgroup of chlorogenic acids detected in the stem and the only subgroup detected in the leaves. This subgroup was dominated by 5-caffeoylquinic acid. The stem also contained three feruloylquinic acids, 3,5- and 4,5-dicaffeoylquinic acid, and small amounts of at least four caffeoyl-feruloylquinic acids. This is the first report of feruloylquinic and caffeoyl-feruloylquinic acids from sweet potato. Two chemically unrelated and coeluting substances with the same molecular mass (M_r = 530) extracted from the Chinese sweet potato interfered with the characterisation of the caffeoyl-feruloylquinic acids. At least five caffeoyl-feruloylquinic acids were detected in the peel of sweet potato cultivated in Tanzania that lacked these interfering substances.     

Inhibitory effects of Chrysanthemum species extracts on formation of advanced glycation end products

The corolla of Chrysanthemum species (C.morifolium R. and C.indicum L.) has long been used to treat eye and inflammatory disease. However, little is known about the antiglycation properties of Chrysanthemum species. Our study sought to characterise their activity against the formation of advanced glycation end products (AGEs) in glycation model reactions. In BSA/glucose (fructose) systems, both Chrysanthemum species strongly inhibited the formation of AGEs and N^ε-(carboxymethyl)lysine (CML). C.morifolium R., not C.indicum L., also acted to inhibit the formation of fluorescent AGEs, including pentosidine. This difference correlated with the values of polyphenol and flavonoid components. We characterised the active components in these plants by liquid chromatography-diode array detector-atmospheric pressure chemical ionisation/mass spectrometry, which showed that C. morifolium R. contains large amounts of chlorogenic acid, flavonoid glucoside varieties, and apigenin, while C.indicum L. contains large amounts of caffeic acid, luteolin, and kaempferol. Our findings raise hopes for the successful treatment of pathogenesis in conditions associated with diabetic complications and aging.     

Chymotryptic hydrolysates of α-kafirin,the storage protein of sorghum (Sorghum bicolor) exhibited angiotensin converting enzyme inhibitory activity

Kafirin is the main storage protein (prolamin) in sorghum grains. α-Kafirin, the alcohol soluble fraction, was isolated from sorghum flour. Treatment of α-kafirin with chymotrypsin yielded a hydrolysate which on fractionation, using Sephadex G-25 column, yielded four fractions with significant angiotensin converting enzyme (ACE) inhibitory activity in vitro. The IC₅₀ values of these fractions ranged from 1.3 to 24.3 μg/ml. Two of the fractions were found to be competitively inhibiting the enzyme, while two other fractions were non-competitive inhibitors. These results demonstrate that chymotryptic hydrolysates of sorghum prolamin could serve as a good source of peptides with angiotensin I converting enzyme inhibitory activity.     

Dose-effect study on the antioxidant properties of leaves and outer bracts of extracts obtained from Violetto di Toscana artichoke

Artichoke (Cynara scolymus L.) is an edible vegetable largely used in the Mediterranean diet and in folk medicine. The present paper discusses the analysis of the polyphenol content of leaves and outer bracts of Violetto di Toscana artichoke using different extraction procedures with the aim of establishing a correlation between polyphenol subclasses and antioxidant activity measured on human LDL oxidized by copper ions. HPLC/DAD and HPLC/MS analyses revealed that both the matrixes contain identical polyphenol subclasses, with mainly quantitative differences. The antioxidant effect of four artichoke extracts decreases in the following order when the sum of total phenolic compounds was considered: ethanolic extract from leaves (IC₅₀ = 2.92 ± 0.46 μM); ethanolic extract from outer bracts (IC₅₀ = 4.04 ± 0.21 μM); ethyl acetate extract from leaves (IC₅₀ = 4.91 ± 0.11 μM); ethyl acetate extract from outer bracts (IC₅₀ = 10.18 ± 1.6 μM). IC₅₀ were also calculated considering the concentrations of single polyphenol subclasses. In both cases, the potency of antioxidant properties was not related to the amount of total polyphenols or the single subclasses.     

Physicochemical and structural characterisation of protein isolate,globulin and albumin from soapnut seeds (Sapindus mukorossi Gaertn.)

The amino acid (AA) composition and physicochemical and conformational properties of protein isolate (SNPI), globulin (SNG) and albumin (SNA) fractions from soapnut seeds were evaluated. The essential AA of SNG, SNA and SNPI (except sulfur-containing AA) are sufficient for the FAO/WHO suggested requirements for 2–5 year old infants. SNG and SNPI showed similar electrophoresis patterns and AA compositions, the subunit of those proteins consisted of two polypeptides linked by disulfide bonds. In contrast, SNA showed a different AA compositions and SDS–PAGE pattern. Both SNG and SNPI presented a typical U-shape protein solubility (PS)–pH profile, SNA showed a completely different PS–pH profile, especially at pH 2.0–4.0. The near-UV circular dichroism (CD), differential scanning calorimetry (DSC) and tryptophan fluorescence spectra analyses indicated that the flexibility in tertiary conformations decreased in the order: SNA > SNPI > SNG, while soapnut proteins had a similar secondary conformation, with a highly ordered structure (the β-types), as evidenced by far-UV CD spectra.     

Açaí (Euterpe oleraceae) ‘BRS Pará’: A tropical fruit source of antioxidant dietary fiber and high antioxidant capacity oil

This article reports a study of the concentrations of dietary fiber (DF) and antioxidant capacity in fruits (pulp and oil) of a new açaí (Euterpe oleraceae) cultivar—‘BRS-Pará’, with a view to determine the possibility of using it as a source of antioxidants in functional foods or dietary supplements. Results show that ‘BRS-Pará’ açaí fruits has a high content of DF (71% dry matter) and oil (20.82%) as well as a high antioxidant capacity in both defatted matter and oil. ‘BRS-Pará’ Açaí fruits can be considered as an excellent source of antioxidant dietary fiber. Antioxidant capacity of açaí ‘BRS-Pará’ oil by DPPH assay was higher (EC₅₀ = 646.3 g/g DPPH) than extra virgin olive oil (EC₅₀ = 2057.27 g/g DPPH). These features provide açaí ‘BRS-Pará’ fruits with considerable potential for nutritional and health applications.