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.     

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 p-alkylbenzoic acids on the activity of polyphenol oxidase from potato (Solanum tuberosum)

In order to prevent potato from browning during preservation and processing, the effects of p-alkylbenzoic acids (p-propylbenzoic acid, p-butylbenzoic acid, p-pentylbenzoic acid, p-hexylbenzoic acid, p-heptylbenzoic acid, p-octylbenzoic acid) on the activity of potato polyphenol oxidase (PPO) have been studied. The PPO was extracted with PBS (pH 6.8), fractionated with ammonium sulphate, concentrated, and purified with Sephadex G-100 (1.8 × 100 cm) filtration chromatography. The active fractions were pooled and the PPO activity was determined to be 79.83 U/mg. The results showed that p-alkylbenzoic acids could strongly inhibit the activity of potato PPO. The values of IC₅₀ of these six inhibitors were determined to be 0.213, 0.180, 0.152, 0.106, 0.075 and 0.047 mM, respectively. Kinetic analyses showed that p-alkylbenzoic acids used in this experiment were reversible and noncompetitive inhibitors to the enzyme. The inhibitory effects were potentiated with increasing lengths of the hydrocarbon chains, indicating that the inhibitory efficiency on the enzyme was influenced by the steric effect of the substituted groups.     

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.     

Chemical composition,antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone

We investigate in this work the chemical composition by GC–EIMS, the antibacterial and the cytotoxic activities of Tunisian Nigella sativa essential oil and its bioactive compound, thymoquinone, were tested against various clinical cariogenic bacteria (n = 30). Eighty-four compounds were identified in the essential oil. The major one was p-cymene (49.48%) whereas thymoquinone represented only 0.79%. The essential oil (2.43 mg/disc) containing only 3.35 μg of thymoquinone showed pronounced dose dependant antibacterial activity against Streptococcus mitis, Streptococcus mutans, Streptococcus constellatus and Gemella haemolysans (15.5 ± 0.707 mm). However, pure thymoquinone compound (150 μg/disk) was active against all the studied strains especially S. mutans and S. mitis (24.5 ± 0.71 and 22 ± 1.41 mm inhibition zones, respectively).     

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.     

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.     

Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-β-GlcNAc) hydrolysis was five; the optimum temperature was 50 °C; the K_m was 0.18 mM, V_max was 37.6 μmol min⁻¹ mg⁻¹ and activation energy (E_a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30–45 °C for 40 min, but its activity decreased significantly when the temperature exceeded 50 °C. Heavy metal ions, Ag⁺ and Hg²⁺, at a concentration of 0.25 mM and Zn²⁺ and Cu²⁺, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The β-NAHA had only one active site for binding both pNP-β-GlcNAc and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-β-GalNAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon–Webb plots.     

Screening and identification of the antibacterial bioactive compounds from Lonicera japonica Thunb. leaves

Our aim was to screen for antibacterial bioactive compounds from Lonicera japonica leaves. Staphylococcus aureus and Escherichia coli were used as the indicator bacteria. Bacteriostatic assay-guided extraction and stepwise partitioning of the samples yielded five compounds of interest. Antimicrobial activities of the compounds were determined using a disk diffusion assay. Extracts, fractions, and compounds from L. japonica leaves possessed considerable antibacterial activities against the tested bacterial strains and the most active fraction was attributed to J3B2, which primarily contained 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid. Meanwhile, five bacteriostatic constituents were isolated (3-O-caffeoylquinic acid, secoxyloganin, luteoloside, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid), among which, secoxyloganin was isolated for the first time from leaves. The antibacterial activity of the compounds was in the order of 3,5-bis-O-caffeoyl quinic acid, 4,5-bis-O-caffeoylquinic acid, luteoloside > 3-O-caffeoylquinic acid > secoxyloganin. Our results suggested that the phenolic compounds might significantly contribute to antibacterial activity and were the most responsible for the bacteriostatic activity of L. japonica leaves.     

Rapid,simple and sensitive determination of the apparent formation constants of trans-resveratrol complexes with natural cyclodextrins in aqueous medium using HPLC

The study of the complexation of trans-resveratrol with natural cyclodextrins (CDs) in aqueous medium under different physico-chemical conditions of pH or temperature is essential if this antioxidant compound is to be used successfully in the food industry as ingredient of functional foods, due its poor stability, bioavailability and solubility. In this paper, a rapid, simple and sensitive determination of the apparent formation constant of trans-resveratrol/CD complexes by HPLC in aqueous medium has been investigated for first time. It can be observed that trans-resveratrol forms a 1:1 complex with α-, β- and γ-CD. The highest value of the apparent formation constant (K_F = 1922 ± 89 M⁻¹) was found for β-CD and a strong dependence of K_F on pH can be seen in the region where the trans-resveratrol begins the deprotonation of their hydroxyl groups. Moreover, an increase in the system’s temperature produced a decrease in the values of K_F. Finally, to gain information on the mechanism of the trans-resveratrol affinity for CD, the thermodynamic parameters of the complexation were obtained.     

Antioxidant properties of different cultivars of Portulaca oleracea

Methanolic extracts of six cultivars of Portulaca oleracea were analyzed for their total phenol content (TPC) using the Folin–Ciocalteu method. The antioxidant activity was measured using the 1,1-diphenyl-2-picrylhydrazyl, ferric-reducing antioxidant power (FRAP) and β-carotene bleaching (BCB) assays. The iodine titration method was used to determine the ascorbic acid content (AAC). The TPC of the cultivars of P. oleracea ranged from 127 ± 13 to 478 ± 45 mg GAE/100 g of fresh weight of plant. There was good correlation between the TPC value and its AEAC, IC₅₀ and FRAP values (r² > 0.9) for all the cultivars. The AAC for the cultivars ranged from 38.5 ± 0.6 to 73.0 ± 17.5 mg/100 g. The TPC value of the common variety PO1, was the lowest compared to the ornamental cultivars (PO2–PO6). The BCB assay showed that all cultivars were capable of inhibiting lipid peroxidation and the inhibition power did not correlate with TPC value.     

Evaluation of antioxidant activity of aqueous extract of some selected nutraceutical herbs

The objectives of this study were to examine the antioxidant activities and free radical scavenging effects of extracts of aqueous leaves of Psidium guajava L. (PE), Camellia sinensis (GABA tea; CE), Toona sinensis Roem. (TE) and Rosemarinus officinalis L. (RE). Among the four extracts, PE exhibited the strongest efficiency and showed over 50% scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals at the concentration of 100 μg/mL. The reducing power of four nutraceutical herbs was in the order of PE > RE > CE > TE. The antioxidant activities of nutraceutical herbs were evaluated in a liposomes oxidation system promoted by Fe³⁺/ascorbic acid/H₂O₂. PE still showed the strongest antioxidant activity and exhibited over 95% inhibition at concentration of 50 μg/mL. The antioxidant activity of TE was still lower than that of other herbal plants; however, it also displayed 89% inhibition at concentration of 250 μg/mL. RE exhibited well inhibitory effects on the UVB-induced oxidation of erythrocyte ghosts at lower concentration (100 μg/mL). However, the protection of PE on the UVB-induced oxidation was significantly raised with increasing the concentrations and reached 95.4% inhibitory effects at concentration of 500 μg/mL. These results show that the tested herbal tea, especially PE could be considered as a natural antioxidant source.     

Chemical,biochemical and electrochemical assays to evaluate phytochemicals and antioxidant activity of wild plants

Plants are a source of compounds that may be used as pharmacologically active products. Cytisus multiflorus, Filipendula ulmaria and Sambucus nigra have been used as important medicinal plants in the Iberian Peninsula for many years, and are claimed to have various health benefits. Herein, the phytochemical composition and antioxidant activity of the mentioned wild medicinal plants were evaluated in vitro, based on chemical, biochemical and electrochemical methods. F. ulmaria was found to be richest in antioxidant phytochemicals, such as phenolics (228 mg GAE/g DW), flavonoids (62 mg CE/g DW), ascorbic acid (2700 μg/g DW) and tocopherols (497 μg/g DW). The antioxidant activity was found to vary in the order: F. ulmaria > S. nigra > C. multiflorus, irrespective of the analysis method. Electrochemical methods have proven to be rapid and inexpensive techniques to characterise the antioxidant activity of plant extracts.     

Antioxidant and antibacterial activities of exopolysaccharides from Bifidobacterium bifidum WBIN03 and Lactobacillus plantarum R315

The objective of this study was to investigate the antioxidant and antibacterial activities of exopolysaccharide (EPS) from Bifidobacterium bifidum WBIN03 (B-EPS) and Lactobacillus plantarum R315 (L-EPS). The 1,1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging, hydroxyl radical-scavenging, and superoxide radical-scavenging abilities were measured to evaluate antioxidant activity. Inhibition of erythrocyte hemolysis and lipid peroxidation was also measured. Both B-EPS and L-EPS had strong scavenging ability against DPPH and superoxide radicals at high concentration. The inhibitory effect of B-EPS on erythrocyte hemolysis was stronger than that of L-EPS in a concentration range from 0.30 to 1.00 mg/mL, whereas the hydroxyl scavenging ability of L-EPS (39.15 ± 0.58%) was significantly higher than that of 0.15 mg/mL ascorbic acid (24.33 ± 1.17%) and B-EPS (17.89 ± 3.30%) at 0.10 mg/mL. The inhibition of lipid peroxidation of 0.50 mg/mL B-EPS and L-EPS was 13.48 ± 1.74% and 12.43 ± 0.51%, respectively, values lower than that of ascorbic acid at the same concentration (23.20 ± 1.41%). Furthermore, all these abilities were enhanced in a concentration-dependent manner. Agar diffusion assay showed that both EPS exhibited antibacterial activities against tested pathogens such as Cronobacter sakazakii, Escherichia coli, Listeria monocytogenes, Staphyloccocus aureus, Candida albicans, Bacillus cereus, Salmonella typhimurium, and Shigella sonnei at 300 μg/mL. In conclusion, both EPS have antimicrobial and antioxidant activities and could have applications in the food industry.     

Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.)

Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical characteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K_M and V_max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min⁻¹ for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min⁻¹ for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a temperature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione ≈ ascorbic acid > l-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

Quality assessment of wild European eel (Anguilla anguilla) stored in ice

The quality assessment of wild European eel (Anguilla anguilla) stored in ice and in boxes without ice (3 ± 1 °C) was investigated by the sensory analysis, levels of nucleotide breakdown products and biogenic amines for up to 19 days. Sensory analysis was assessed using the Tasmanian Food Research Unit Scheme. K and related values (K_i, G, P, H and F_r) were used as freshness indicators. Linear regressions (r²) obtained from K, K_i, G, P, H and F_r were 0.95, 0.96, 0.83, 0.96, 0.99 and 0.96, respectively, for eel stored in ice whereas, for eel kept in boxes without ice, the values were 0.86, 0.86, 0.96, 0.91, 0.98 and 0.86, respectively. When eel stored in ice and in boxes without ice were considered at the limit of acceptability by assessors at ∼12–14 days and ∼5–7 days, respectively, the average K, K_i and P values were ∼70–85%, H values were ∼60% and F_r values were ∼10% for both storage conditions. The level of histamine exceeded the legal limit (5 mg/100 g fish) in eel stored without ice after 6–7 days and, in ice, after 13–14 days of storage, at which time eels were rejected by the sensory panel. The concentrations of biogenic amines were higher in eel stored in boxes without ice than in eel kept in ice. The levels of histamine in the muscle of eel kept in boxes without ice and in ice increased to the maximum levels of 17.9 mg/100 g on day 12 and 12.6 mg/100 g on day 19, respectively.     

Kinetics of browning onset in white wines: influence of principal redox-active polyphenols and impact on the reducing capacity

The browning capacity of white wines was studied, employing an accelerated test and samples produced and stored under identical conditions. Browning was approached from a kinetic point of view and efforts were focussed on the investigation of plausible correlations with major redox-active polyphenols, including substances with an o-diphenol feature, such as gallic acid, caftaric acid, 2-S-glutathionylcaftaric acid (GRP), caffeic acid, catechin, and epicatechin. Over a period of ten days, browning development was shown to obey zero-order kinetics from the third day of the treatment, and browning rate constants (k) varied from 15.3 to 74.5 × 10⁻³ day⁻¹. Regression analysis between k values and concentration of individual phenolics provided strong evidence that epicatechin is the principal browning agent (r² = 0.8033, P < 0.01). Furthermore, the monitoring of the reducing power (P_R), throughout treatments, indicated that increases in browning are accompanied by a commensurate decline in the reducing ability, raising concerns about the impact of browning reactions on the in vitro antioxidant properties of white wines.     

Biochemical characteristics of Trametes multicolor pyranose oxidase and Aspergillus niger glucose oxidase and implications for their functionality in wheat flour dough

Similar to glucose oxidase (GO), pyranose oxidase (P₂O) may well have desired functionalities in some food applications in general, particularly breadmaking. As its name implies, P₂O oxidises a variety of monosaccharides. P₂O purified from a culture of Trametes multicolor (P₂O-Tm) had high affinity towards d-glucose (K_M = 3.1 mM) and lower affinity to other monosaccharides. GO from Aspergillusniger (GO-An) had a K_M value of 225 mM towards glucose, which points to a significant difference in glucose affinity between the two enzymes. Furthermore, P₂O-Tm had higher affinity towards O₂ (K_M = 0.46 mM) than GO-An (K_M = 2.9 mM). Dehydroascorbic acid did not accept electrons in the reactions catalysed by P₂O-Tm and GO-An. For the same activity towards glucose in saturating conditions, the rate of ferulic acid oxidation in a model system and of thiol oxidation in a wheat flour extract were higher with P₂O-Tm, than with GO-An. The demonstrated differences in properties and functional features between P₂O-Tm and GO-An allow prediction of differences in functional behaviour of the enzymes, in food applications.     

Evaluation of antioxidant,antibacterial and anti-tyrosinase activities of four Macaranga species

The methanolic fresh leaf extracts of Macaranga gigantea, Macaranga pruinosa, Macaranga tanarius and Macaranga triloba were screened for their antioxidant properties (AOP), tyrosinase inhibition and antibacterial activities. Total phenolic content (TPC), 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, ferric-ion reducing power (FRAP), ferrous-ion chelating (FIC) and lipid peroxidation inhibition (LPI) activities were used to evaluate the AOP. Modified 3,4-dihydroxy-L-phenylalanine (L-DOPA) method was used to determine tyrosinase inhibition activity, whereas antibacterial activity was determined using the disc-diffusion technique. TPC screening of the same species from different collection sites showed no significant difference between sites. M.triloba showed the highest ascorbic acid equivalent antioxidant activity (AEAC), FRAP and LPI values. M. tanarius, which showed the lowest TPC, AEAC, FRAP and LPI activities, exhibited the best FIC activity. M. pruinosa showed the best tyrosinase inhibition activity, whereas M. triloba showed the best antibacterial activity against Gram-positive bacteria species, with minimal inhibition dosage (MID) values as low as 10 μg/disc.