Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Determination of some biochemical properties of polyphenol oxidase from Emir grape (Vitis vinifera L. cv. Emir)

Polyphenol oxidase (PPO) was extracted from Emir grapes grown in Turkey and its characteristics in terms of pH and temperature optima, thermal inactivation, kinetic parameters and potency of some PPO inhibitors were studied. The optimum pH and temperature for grape PPO were found to be 4.2 and 25 °C respectively using catechol as substrate. K_m and V_max values were found to be 25.1 ± 2.72 mmol L⁻¹ and 0.925 ± 0.04 OD₄₁₀ min⁻¹ respectively. Of the inhibitors tested, the most potent was sodium metabisulfite, followed by ascorbic acid. The thermal inactivation curve was biphasic. Activation energy (E_a) and Z values were calculated as 251.4 kJ mol⁻¹ (r² = 0.996) and 8.92 °C (r² = 0.993) respectively. Copyright © 2006 Society of Chemical Industry     

Partial characterization of polyphenoloxidase from a hybridized wheat (Triticum aestivum L.)

Polyphenol oxidase was extracted and partially purified from wheat leaves by a procedure that included ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures led to 35.21-fold purification with 17.65% recovery. Optimum pH, temperature, and ionic strength were determined with six substrates. Some kinetic properties of the enzyme such as V _max, K _M, and k _cat were calculated for the substrates. The k _cat/K _M values of the PPO for catechol, catechin, pyrogallol, l-dopa, dopamine, and 4-methyl catechol were 31408, 31167, 28404, 15378, 4865, and 4967 mM/min, respectively. The best substrate of wheat PPO was found to be catechol. The native molecular weight of the PPO was estimated to be 243 kDa based on its mobility in gel filtration column. The inhibitory effects of glutathione, sodium azide, ascorbic acid, oxalic acid, l-cysteine, and thiourea on the reaction catalyzed by the enzyme were tested, and I ₅₀ values were estimated to be 8.0 mM, 10.12 mM, 11.18 mM, 77.33 mM, 183 mM, and 413 mM, and K _i constants were also calculated as 0.416 ± 0.244 mM, 0.317 ± 0.208 mM, 0.820 ± 0.111 mM, 13.80 ± 1.179 mM, 14.10 ± 5.069 mM, and 130 ± 62.45 mM, respectively, by means of Lineweaver–Burk graphs. The most effective inhibitor was glutathione. Glutathione, sodium azide, oxalic acid, and thiourea were competitive inhibitors, whereas ascorbic acid and l-cysteine were also noncompetitive inhibitors.     

Oregano: chemical analysis and evaluation of its antimalarial, antioxidant, and cytotoxic activities

Abstract: GC‐FID and GC‐MS analysis of essential oil from oregano leaves (Origanum compactum) resulted in the identification of 46 compounds, representing more than 98% of the total composition. Carvacrol was the predominant compound (36.46%), followed by thymol (29.74%) and p‐cymene (24.31%). Serial extractions with petroleum ether, ethyl acetate, ethanol, and water were performed on aerials parts of Origanum compactum. In these extracts, different chemical families were characterized: polyphenols (gallic acid equivalent 21.2 to 858.3 g/kg), tannins (catechin equivalent 12.4 to 510.3 g/kg), anthocyanins (cyanidin equivalent 0.38 to 5.63 mg/kg), and flavonoids (quercetin equivalent 14.5 to 54.7 g/kg). The samples (essential oil and extracts) were subjected to a screening for antioxidant (DPPH and ABTS assays) and antimalarial activities and against human breast cancer cells. The essential oil showed a higher antioxidant activity with an IC₅₀= 2 ± 0.1 mg/L. Among the extracts, the aqueous extract had the highest antioxidant activity with an IC₅₀= 4.8 ± 0.2 mg/L (DPPH assay). Concerning antimalarial activity, Origanum compactum essential oil and ethyl acetate extract showed the best results with an IC₅₀ of 34 and 33 mg/mL, respectively. In addition, ethyl acetate extract (30 mg/L) and ethanol extract (56 mg/L) showed activity against human breast cancer cells (MCF7). The oregano essential oil was considered to be nontoxic.     

Characterization of Sultaniye grape (Vitis vinifera L. cv. Sultana) polyphenol oxidase

Polyphenol oxidase (PPO) was extracted from Sultaniye grapes grown in Turkey, and its characteristics in terms of pH and temperature optima, thermal inactivation, kinetic parameters and potency of some PPO inhibitors were studied. Optimum pH and temperature for grape PPO were found to be 3.4 and 30 °C, using catechol as substrate. K_m and V_max values were found to be 44.5 ± 5.47 mm and 0.695 ± 0.0353 OD₄₁₀ min^?1, respectively. Four inhibitors were tested in this study and the most potent inhibitor was sodium metabisulphite, followed by ascorbic acid. From the thermal inactivation studies in the range of 65–80 °C, the half‐life values of the enzyme ranged between 2.6 and 49.5 min. Activation energy (E_a) and Z values were calculated to be 208.5 kJ mol^?1 (r² = 0.9544) and 10.95 °C (r² = 0.9517), respectively.     

Purification and enzymatic characteristics of a novel polyphenol oxidase from lotus seed (Nelumbo nucifera Gaertn.)

A polyphenol oxidase (PPO) from lotus seed was purified by the procedures including ammonium sulphate precipitation and affinity chromatography. The apparent molecular mass was 38.6 kDa by SDS‐PAGE. Kinetic studies showed that the K_m and V_max values for catechol were 6.04 mm and 416.67 U, respectively. The PPO performed optimal activity in 20 °C and pH 7.0. The enzymatic activity could be mainly maintained up to 50 °C and pH 4.0–7.0. The activity could be inhibited by various inhibitors including thiourea, urea, sodium hydrogen sulphite, EDTA·2Na, SDS, citric acid, guanidine hydrochloride, ascorbic acid, sodium sulphite and sodium thiosulphate. The metal ions Ba²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺ and Zn²⁺ could inhibit the activity of PPO, while Cu²⁺ performed obvious enhancement. The enzymatic properties of PPO could probably provide practical application in inhibiting the PPO activity and preventing enzymatic browning in the process of picking, transportation, processing and storage of fresh lotus seeds.     

Structural features,antioxidant and tyrosinase inhibitory activities of proanthocyanidins in leaves of two tea cultivars

In this study the structural features, antioxidant, and tyrosinase inhibitory activities of proanthocyanidins in leaves of two tea cultivars, namely Huangjingui and Qilan, were investigated. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thiolysis-high performance liquid chromatography-electrospray ionization mass spectrometry analyses confirmed that these proanthocyanidins were built up a mixture of procyanidins, propelargonidins, and prodelphinidins, with the predominance of procyanidins. The proanthocyanidins in leaves of Huangjingui and Qilan cultivars exhibited potent antioxidant activities with IC₅₀ values of 87.36 ± 0.83 and 97.33 ± 0.61 µg/mL in 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, 43.57 ± 0.25 and 55.01 ± 0.22 µg/mL in 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay, and with ferric reducing antioxidant power values of 4.83 ± 0.03 and 3.97 ± 0.02 mmol ascorbic acid equivalent/g in ferric reducing antioxidant power assay. It was also found that these proanthocyanidins had strong inhibition on tyrosinase activity with IC₅₀ values of 54.8 ± 1.27 and 20.0 ± 0.89 µg/mL for Huangjingui and Qilan cultivars, respectively. The results indicated that the proanthocyanidins in leaves of two tea cultivars could be considered as natural antioxidants and tyrosinase inhibitors applied in pharmaceutical and cosmetic industries in the future.     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

Chemical Composition and Anticancer and Antioxidant Activities of Schinus Molle L. and Schinus Terebinthifolius Raddi Berries Essential Oils

Abstract: Essential oils were obtained by steam distillation from berries of Schinus molle L. and Schinus terebinthifolius Raddi originating from southern of Tunisia and analyzed by GC-FID and GC-MS. Among 57 and 62 compounds (%[mg/100 g dry matter]) identified in these oils, the main were α-phellandrene (46.52%[1256.15] and 34.38%[859.60]), β-phellandrene (20.81%[561.74] and 10.61%[265.15]), α-terpineol (8.38%[226.26] and 5.60%[140.03]), α-pinene (4.34%[117.29] and 6.49%[162.25]), β-pinene (4.96%[133.81] and 3.09%[77.30]) and p-cymene (2.49%[67.28] and 7.34%[183.40]), respectively. A marked quantity of γ-cadinene (18.04%[451.05]) was also identified in the S. terebinthifolius essential oil whereas only traces (0.07%[1.81]) were detected in the essential oil of S. molle. The in vitro antioxidant and antiradical scavenging properties of the investigated essential oils were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. Essential oil of S. terebinthifolius expressed stronger antioxidant activity in the ABTS assay, with an IC₅₀ of 24 ± 0.8 mg/L, compared to S. molle (IC₅₀= 257 ± 10.3 mg/L). Essential oils were also evaluated for their anticancer activities against human breast cancer cells (MCF-7). S. terebinthifolius essential oil was more effective against tested cell lines (IC₅₀= 47 ± 9 mg/L) than that from S. molle (IC₅₀= 54 ± 10 mg/L). Suggestions on relationships between chemical composition and biological activities are outlined.     

Some properties of polyphenol oxidase from lily

A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first‐order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the V_max/K_m ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l ‐cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning.     

Antioxidant and semicarbazide‐sensitive amine oxidase inhibitory activities of alginic acid hydroxamates

The commercial polysaccharides of alginic acid (medium (3500 cps, 2% solution) and low (250 cps, 2% solution) viscosities) were esterified with acidic methanol (1 mmol L^?1 HCl) at 4 °C with gentle stirring for 5 days to obtain methyl esters of medium‐viscosity alginic acid (ME‐MVA) and low‐viscosity alginic acid (ME‐LVA). These ME‐MVA and ME‐LVA were reacted with alkaline hydroxylamine to obtain medium‐viscosity alginic acid hydroxamates (MVA‐NHOH) and LVA‐NHOH. The percentages of hydroxamic acid content in MVA‐NHOH and LVA‐NHOH were calculated as 25% and 20%, respectively. The hydroxamate derivatives of alginic acid were used to test the antioxidant and semicarbazide‐sensitive amine oxidase (SSAO) inhibitory activities in comparison with original materials (MVA and LVA). The half‐inhibition concentrations, IC₅₀, of scavenging activity against 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) were 24.5 and 29.8 µg mL^?1 for MVA‐NHOH and LVA‐NHOH, respectively. However, few scavenging activities of the MVA and LVA were found at the same concentrations. The IC₅₀ of the positive control of butylated hydroxytoluene was 5 µg mL^?1. The scavenging activity of DPPH radical was pH‐dependent, and the optimal pH for both of MVA‐NHOH and LVA‐NHOH was the Tris‐HCl buffer (pH 7.9). Using electron spin resonance (ESR) to detect the activity of scavenging hydroxyl radicals, both alginic acid hydroxamates showed dose‐dependent scavenging activities, and the IC₅₀ was 90 and 92 µg mL^?1, respectively, for MVA‐NHOH and LVA‐NHOH. Both alginic acid hydroxamates also exhibited protection against hydroxyl radical‐mediated DNA damage. Both MVA‐NHOH and LVA‐NHOH showed dose‐dependent inhibitory activities against bovine SSAO (2.53 units); the IC₅₀ was 0.16 and 0.09 µg mL^?1, respectively, for MVA‐NHOH and LVA‐NHOH, compared with 3.81 µg mL^?1 of semicarbazide (positive controls). Amine oxidase activity staining also revealed that both MVA‐NHOH and LVA‐NHOH exhibited SSAO inhibitory activities. Both MVA‐NHOH and LVA‐NHOH showed mixed non‐competitive inhibition against bovine SSAO. It was found that the V′_max value was reduced and the K′_m value was either increased (added MVA‐NHOH, 0.05 µg mL^?1) or reduced (added LVA‐NHOH, 0.11 µg mL^?1) in the presence of alginic acid hydroxamate. Copyright © 2006 Society of Chemical Industry     

Amaranth Sprouts: A Potential Health Promoting and Nutritive Natural Food

Amaranth sprouts are an edible food with good nutritional qualities and potential biological activities of their proteins. The chemical composition, angiotensin converting enzyme inhibitory activity and antioxidant activity of the sprouts were determined. Sprouts showed a protein content similar to the seeds’ on a dry basis (16%) and a high fiber content (17%). Amaranth sprout proteins presented a capacity to inhibit angiotensin converting enzyme activity similar to other plant proteins (IC₅₀ = 0.9 ± 0.6 mg/mL). This capacity increased after in vitro gastrointestinal digestion (IC₅₀ = 0.26 ± 0.07 mg/mL). Besides other non protein molecules, the amaranth sprout proteins also presented ABTS^+. scavenging activity (TEAC = 0.32 ± 0.05 μmol/mg) that increased after in vitro gastrointestinal digestion (TEAC = 0.72 ± 0.08 μmol/mg) and oxygen radical antioxidant capacity. According to these results amaranth sprouts are a nutritive food with potential health promoting properties.     

Phenolic Compound Identification and Antioxidant Capacity of Alperujo Extracts from Region del Maule,Chile

This study was carried out to determinate phenolic, flavonoid content, and antioxidant capacity in methanolic extract from three Alperujo varieties. Alperujo Barnea showed the highest concentration of phenols and flavonoid. The greater hydroxytyrosol content was obtained in the same extract (4.93 ± 0.37 µg/mg extract), whereas the greater tyrosol content (0.23 ± 0.012 µg/mg extract) was found in Arbequina extract. These results were correlated with the greatest radical scavenging and the highest inhibition of lipoperoxidation process observed in Barnea extract (IC₅₀ of 27.9 ± 1.04 µg/mL; IC₅₀ 22.8 ± 3.5 µg/mL, respectively). In spite of differences, alperujo extracts exhibited notable antioxidant capacities.     

Variability and the genotypic effect on antioxidant activity,total phenolics,carotenoids and ascorbic acid content in seventeen natural population of Seabuckthorn (Hippophae rhamnoides L.) from trans-Himalaya

Seventeen natural population of Seabuckthorn (SBT), which comprised 187 plants from trans-Himalaya, were studied to find out variability and genotypic effect on total phenolic content (TPC), total antioxidant capacity (TAC), ascorbic acid and carotenoids content in fruit pulp. The fruits were found to be rich in TPC ranging from 964 to 10,704 mg gallic acid equivalent/100 g. The free radical-scavenging activity in terms of inhibitory concentration (IC₅₀) ranged from 0.7 to 9.1 mg/ml and ferric reducing antioxidant potential (FRAP) from 180 to 1355 FeSO₄·7H₂O μg/ml. The ascorbic acid and carotenoids content ranged from 56 to 3909 mg/100 g and 0.1–14.4 mg/100 g, respectively. A variation of 1–11 fold in TPC, 1–14 folds in IC₅₀ by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and 1–8 fold in ferric reducing antioxidant potential, 1–70 fold in ascorbic acid content and 1–206 fold in carotenoid content among the examined fruit across 17 populations underlines the important role played by genetic background and the geographical location for determining the health promoting compounds. Significant correlation was observed between TPC, IC₅₀, FRAP, carotenoids, ascorbic acid, fruit lightness (L*) and plant height. Among the 20 morphological traits studied, fruit colour and plant height showed positive correlation with the health promoting compounds.     

Evaluation of the Phenolic Content,Antiradical, Antioxidant,and Antimicrobial Activity of Different Floral Sources of Honey

Thirty-five honeys were evaluated for total phenolic content by Folin-Ciocalteu method, for potential antioxidant activity using phosphomolibdenum assay and by the 1,1-diphenyl-2-picrylhydrazyl method for antiradical activity. The antimicrobial activity was studied by the agar diffusion method, using 12 bacteria and 2 yeasts. The means of the total phenolic contents of chestnut, honeydew, multifloral, thyme, and astragalus were 47 ± 18, 24.2 ± 0.6, 14 ± 11, 11 ± 6, and 9 ± 7 mg/100 g honey as gallic acid equivalent, respectively. The lowest antioxidant activity was observed in honeydew 70 ± 5 mg ascorbic acid equivalent/g honey while the highest content was observed in astragalus honey 86 ± 16 mg ascorbic acid equivalent/g honey. Correlation between the phenolic content and antioxidant activity were found to be statistically significant. Chestnut honeys (n = 5) exhibited maximum free radical scavenging activity with an average 68 ± 9%. The honey samples showed the highest antimicrobial activity against some microorganisms, especially Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Proteus mirabilis. On the other side, Aeromonas hydrophila, Bacillus subtilis, and E. coli were the most resistant microorganisms. The results revealed that the Turkish honeys studied proved to be a good source of antioxidant and antimicrobial agents that might serve to protect human health.     

Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape

Polyphenol oxidase (PPO) was isolated from Thompson seedless grape (Vitis vinifera ‘Thompson Seedless’), and its biochemical characteristics were studied. The PPO showed activity to catechol and D, L-DOPA, but not towards monophenol l-Tyrosine, diphenols guaiacol and caffeic acid, and triphenols pyrogallic acid and gallic acid. Apparent Michaelis–Menten constant (K _m) and maximum velocity of the reaction (V _max) values were 45.0 ± 0.05 mM and 500.0 ± 15.3 OD_400 nm/min for catechol, and 34.6 ± 0.03 mM and 384.6 ± 11.7 OD_478 nm/min for D, L-DOPA, respectively. The obtained similar specificity values of V _max/K _m ratio of catechol and D, L-DOPA indicated their similar affinity to Thompson seedless PPO. The most effective inhibitor was l-cysteine, followed in decreasing order by ascorbic acid, sodium metabisulfite, EDTA, NaCl, and citric acid. It was discovered that metal ions of Mg²⁺ and Cu²⁺ increased, while Zn²⁺ and K⁺ reduced the PPO activity. Sugars showed inhibition on the PPO activity, with higher effect by sucrose and lower effect by fructose and glucose. Optimum pH and temperature for grape PPO activity were 6.0 and 25 °C with 10 mM catechol as substrate. The enzyme was heat stable between 10 and 25 °C, but showed significant activity loss at temperatures higher than 40 °C and completely inactivation at 70 °C for 10 min. Thermal inactivation of PPO showed a first-order kinetic with an activation energy (E _a) of 146.1 ± 10.8 kJ/mol at pH 6.0.     

Antioxidative Potential of the Polyphenolics of Stephania japonica var. Discolor (Blume) Forman: A Chromatographic (High-Performance Liquid Chromatography) and Spectrophotometric Measure

This study investigated the quantitative phytochemical contents, total phenolics, total flavonoids, total carotenoids, antioxidative capacity, tannin, proanthocyanidins, lycopene, chlorophyll a, and chlorophyll b of the Stephania japonica extract. Comprehensive antioxidative effects of the extract were also investigated. Quantitative assays were conducted through both spectrophotometric and high-performance liquid chromatographic methods. Antioxidative effects were measured through FeCl₃ reducing power, metal chelating power, reducing power, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) scavenging activity, N, N-dimethyl-1,4-diaminobenzene free radical scavenging activity, superoxide radical scavenging activity, and nitric oxide scavenging effect. The contents of total phenolics, total flavonoids, total carotenoids, total antioxidative capacity, tannin, proanthocyanidins, lycopene, chlorophyll a, and chlorophyll b were found to be 47.32 ± 0.75 mg tannic acid equivalent, 61.41 ± 1.58 mg catechin equivalent, 63.29 ± 2.21 mg, 22.85 ± 0.70 mg ascorbic acid equivalent, 76.17 ± 0.97 mg tannic acid equivalent, 94.96 ± 4.49 mg catechin equivalent, 22.19 ± 0.79 µg, 22.19 ± 0.79 µg, 7.52 ± 1.24 mg, and 10.43 ± 2.11 mg, respectively, in 1 g of ethanol extract. A high concentration of epicatechin, p-coumaric acid, and rutin hydrate and moderate concentration of caffeic acid and quercetin was detected in the extract. The IC₅₀ value for ferric reducing power assay, metal chelating assay, reducing power assay, ABTS scavenging assay, N, N-dimethyl-1,4-diaminobenzene scavenging assay, superoxide scavenging assay and nitric oxide (NO) assay were 465.06 ± 7.32 µmol ascorbic acid/g, 1656.52, 270.55, 457.27, 632.74, 217.5, and 464.00 µg/mL, respectively. No beta carotene was detected in the extract. The extract was demonstrated to be a very potential source of antioxidative metabolites.     

Myricetin,an antioxidant flavonol,is a substrate of polyphenol oxidase

The present study demonstrates the antiradical efficiency of myricetin, a flavonol widely distributed in fruits and vegetables, by testing its ability to react with two different free radicals, ABTS^+· and DPPH^·. The polyphenolic nature of myricetin led us to consider the possibility of its oxidation by polyphenol oxidase (PPO). The results reported show that myricetin can be oxidised by PPO extracted and partially purified from broad bean seeds. The reaction was followed by recording spectral changes with time, maximal spectral changes being observed at 372 nm. The presence of two isosbestic points (at 274 and 314 nm) suggested that only one absorbing product was formed. The spectral changes were not observed in the absence of PPO. The oxidation rate varied with the pH, reaching its highest value at pH 5.5. The myricetin oxidation rate increased in the presence of SDS, an activing agent of polyphenol oxidase. Maximal activity was obtained at 1.3 mM SDS. The kinetic parameters were also determined: V _m = 1.35 µM min⁻¹, K _m = 0.3,mM , V _m/ K _m = 4.5 × 10⁻³ min⁻¹. Flavonol oxidation was inhibited by a selective PPO inhibitor such as cinnamic acid (K_I = 1 mM ). The results reported show that myricetin oxidation was strictly dependent on the presence of polyphenol oxidase. © 1999 Society of Chemical Industry     

Characterization of polyphenol oxidase from broccoli (Brassica oleracea var. botrytis italica) florets

Polyphenol oxidase (PPO) from broccoli florets was extracted and purified through (NH₄)₂SO₄ precipitation, ion-exchange and gel filtration chromatography. The molecular weight was estimated to lie between 51.3 and 57 kDa by sodium dodecyl sulphate-polyacrylamide gel electophoresis (SDS-PAGE) and gel filtration. The effects of substrate specificity, pH, and sensitivity to various inhibitors: citric acid, ascorbic acid, sodium sulphate and EDTA (sodium salt of ethylenediaminetetraacetic acid) of partially purified PPO were investigated. Polyphenol oxidase showed the best activity toward catechol (K_M = 12.34 ± 0.057 mM, V_max = 2000 ± 8736 U/ml/min) and 4-methyl catechol (K_M = 21 ± 0.087 mM, V_max = 28.20 ± 0.525 U/ml/min). The optimum pH for broccoli PPO was 5.7 with catechol and 4-methylcatechol as substrates. The most effective inhibitor was sodium sulphate.