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.     

Cardioprotective properties of raw and cooked eggplant (Solanum melongena L)

Although eggplants are known to be part of a healthy diet, the effects of this fruit on cardioprotection are not known. The present study examined the role of raw and grilled eggplants on cardioprotection using an isolated perfusion heart model. The animals were fed freeze-dried products of either raw or grilled eggplants for 30 days. After 30 days, isolated working hearts were subjected to 30 min ischemia followed by 2 h of reperfusion. Left ventricular function was monitored, and myocardial infarct size and cardiomyocyte apoptosis were assessed. To determine the antioxidant function of eggplants, their DPPH scavenging ability were determined, and polyphenolic components, especially nasunin content, were determined. The chemical composition of raw and grilled eggplants were determined in order to examine whether grilling was associated with major changes in their composition. The results of this study demonstrated eggplants as containing potent cardioprotective compounds judging by their ability to increase left ventricular function, and reduce myocardial infarct size and cardiomyocyte apoptosis. However, there was no difference in cardioprotective ability between the raw and grilled products. The antioxidant vitamins, including vitamin A, vitamin C and β-carotene, were lower and some of the polyphenolic components, especially nasunin content, were higher in grilled eggplants, but they were unable to demonstrate better cardioprotective properties compared to the raw fruit.     

Effects of Grilling on Total Polyphenol Content and Antioxidant Capacity of Eggplant (Solanum melongena L.)

Cooking can change the polyphenol contents of eggplant. This study elucidated the effects of grilling on total polyphenol content (TPC), antioxidant capacity, and the inner structures of eggplant. After identical hollowing, cylindrical eggplant samples were prepared and were then grilled until their center temperatures (CT) respectively reached 50, 65, 75, 85, and 95 °C. Chemical assays and observations of the inner structures clarified that TPC and 1, 1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity decreased as CT increased when CT was below 65 °C. Results also showed that TPC and DPPH radical scavenging activity increased as CT increased when CT was between 65 °C and 95 °C. For CT 65 °C, the samples retained polyphenol oxidase (PPO) activity up to 40% of the raw state activity. The 3 grilled eggplant models, chlorogenic acid, chlorogenic acid—sugar and chlorogenic acid—amino acid model, yielded results showing that phenol functional groups on chlorogenic acid were thermally stable and that phenol functional groups on chlorogenic acid reacted neither with sugar nor with amino acids. Results show that PPO activity is a primary reason for the decrease of the 2 indices. Optical microscopic and scanning electron microscopic observations revealed collapsed cells and inter‐tissue cracks around the surface area for CT 85 and 95 °C. Scanning electron microscopic observations clarified that intercellular bonds for CT 85 and 95 °C became thinner than those for CT 75 °C around the middle area. The phenomena explained above are reasons for the increase of TPC and DPPH radical scavenging activity.     

Photo-induced changes in the total chlorogenic acid content of potato (Solanum tuberosum) Tubers

The exposure of potato tubers to light resulted in a significant cultivar-dependent increase in chlorogenic acid content. The magnitude of the observed increase resulting from 48 h light exposure was significantly correlated with the initial chlorogenic acid content of the tubers and also with the previously reported photo-induced increases in total glycoalkaloids. Time-course studies indicated that the greatest rate of increase occurred between 8 and 16 h continual exposure. Prolonged storage under light revealed that chlorogenic acid values in some cultivars reached a maximum value after 48 h, whilst in others these continued to increase almost linearly until the end of the experiment 5 days later.     

Evaluating eggplant (Solanum melongena L) genotypes for bioactive properties: A chemometric approach

Thirty four genotypes of eggplant and its wild relatives were evaluated for their total phenolics, flavonoid content and antioxidant capacity. Free radical scavenging was evaluated using four in-vitro assays, viz. FRAP (Ferric reducing antioxidant power), CUPRAC (cupric reducing antioxidant capacity), TEAC (Trolox equivalent antioxidant capacity) and DPPH (2, 2-diphenylpicrylhydrazyl). Total phenolics in eggplant showed a wide variation, ranging from 22.62 to 234.46 mg gallic acid equivalents (GAE)/100 g fw (244.28 to 2990.64 mg GAE/100 g dw). With the exception of Solanum aethiopicum and its accessions (Ac-1, Ac-2 and Ac-3), all wild relatives had significantly (p < 0.05) higher total phenolics and flavonoid content than cultivated ones. Total antioxidant capacity (FRAP) in wild genotypes ranged from 1.13 to 8.04 μmol Trolox (TE)/g. The hierarchy in decreasing order was Solanum khasianum > Solanum torvum > Solanum sisymbriifolium > Solanum incanum > Solanum integrifolium > S. aethiopicum. The antioxidant capacity correlated high with total phenolics in all assays. In cultivated group, JBR-99 and RCMBL-3 possessed high antioxidant capacity than the rest. Principal component analysis (PCA) and Hierarchical cluster analysis (HCA) revealed distinct similarity between JBR-99 (green), S. sisymbriifolium, S. khasianum and S. torvum. Overall results indicate that the wild species of S. torvum, S. incanum and S. sisymbriifolium are potential candidates for improving the functional quality of cultivated eggplant.     

Cultural Methods and Mineral Content of Eggplant (Solanum melongena) Fruit

Mineral content in edible portions of vegetables may be affected by cultural methods. This study was conducted to determine if fertiliser rate and irrigation regime affected mineral (N, NO₂, NO₃, P, PO₄, K, SO₄, Ca, Fe, Na, Mg, and Mn) of eggplant ( Solanum melongena L, cv Black Bell) fruit. The experiment on a Bernow fine-loamy, siliceous, thermic Glossic Paleudalf soil at Lane (OK, USA) with three fertiliser rates and three irrigation regimes. The data were analysed as a split-plot with harvests as the sub-plot. All P was accounted for by PO₄. Levels of NO₂ and NO₃ comprised <0·001% of the N content. Harvest number affected only N, P, K, SO₄ and Mg levels in both years, and Na in one year. For many of the minerals the highest levels in fruit were not consistent over harvests in both years. When there was less precipitation, and fertiliser was applied at the recommended rate, fewer irrigations per week were necessary to increase levels of N, P and K in fruit. The interaction of irrigation and fertiliser can affect mineral content of fruit and should be considered when fertiliser recommendations are made.     

Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l-cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K⁺, other metal ions such as Zn²⁺, Cu²⁺, Fe³⁺, Ca²⁺, Fe²⁺ and Na⁺ accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent K_m and V_max values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho-diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.     

Polyphenols content and antioxidant capacity of eggplant pulp

The influence of organic and conventional farming practices on the phenolic content in eggplant samples belonging to two cultivars, Blackbell (American eggplant) and Millionaire (Japanese eggplant) grown under similar environmental conditions was evaluated. Phytochemical investigation of the eggplant extracts showed that N-caffeoylputrescine, 5-caffeoylquinic acid, and 3-acetyl-5-caffeoylquinic acid made up the bulk of total eggplant phenolics. In addition, this is the first report on identification of trace quantities of three additional flavonols, namely, quercetin-3-glucoside, quercetin-3-rhamnoside, and myricetin-3-galactoside in freeze-dried eggplant pulp. The phenolic content of the conventionally grown Blackbell variety was marginally higher or equal to those grown organically. However, the phenolic content of the Millionaire organic variety was much greater than the conventionally grown samples. Eggplant extract inhibition of cupric-ion mediated oxidation of low-density lipoproteins was highly correlated with the content of 5-caffeolquinic acid (R² = 0.9124), the most abundant phenolic acid identified.     

Effects of gibberellic acid and chlorocholine chloride on tuberisation and growth of potato (Solanum tuberosum L)

Treatment of potato plants with gibberellic acid (GA₃) enhanced both shoot and stolon growth and dry weight but delayed tuber initiation and decreased tuber yield. Conversely, treatment with chlorocholine chloride (CCC) growth retardant reduced shoot and stolon growth and dry weight but promoted tuberisation. Chlorophyll a and b contents were both increased appreciably with CCC. In the tubers, CCC increased starch content by 11% compared to untreated control, whereas GA₃ decreased starch content by about 13%. A very high reducing sugar content in the stem of GA₃-treated crop indicated active hydrolysis of sucrose coming from the leaves, leading to its reduced supply to the tubers because of further possible sucrose hydrolysis while passing through the long stolons. However, in the CCC treated crop, the higher chlorophyll content of the leaves with reduced stolon length will promote efficient sucrose supply to the tubers. © 1998 Society of Chemical Industry.     

Polyphenol Oxidase from Apple (Malus domestica Borkh. cv Bramley's Seedling): Purification Strategies and Characterization

ABSTRACT Polyphenol oxidase (PPO) was isolated from Bramley's Seedling apples with 75.7‐fold purification and 26.5% recovery by ammonium sulfate precipitation, phenyl sepharose chromatography, ion exchange chromatography, and hydroxyapatite chromatography. Molecular weight was estimated to be about 45 kDa by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS PAGE). Optimum PPO activity was at pH 6.5 and greater than 50% activity was retained during storage for 72 h at pH 5.5 to 6.5. Optimum temperature for activity was 30 °C and the enzyme had residual activity of greater than 50% during storage for 72 h at 20 °C to 30 °C and for 24 h at 40 °C to 50 °C. Of the substrates tested, activity was greatest with 4‐methylcatechol followed by catechol, pyrogallol, and (?)epicatechin. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid.     

Purification and properties of polyphenol oxidase from oil bean (Pentaclethra macrophylla Benth) seeds

Polyphenol oxidase extracted from oil bean (Pentaclethra macrophylla) seeds was purified 165-fold over the crude enzyme extract. The apparent molecular weight of the enzyme by gel filtration was 110.8 k ± 9.0 k while SDS-PAGE indicated a single species of molecular weight 28.0 k. A copper content of 1.9 mg g^?1 corresponds to one copper atom for each of the four subunits. The purified enzyme oxidised pyrogallol, catechol, 4-methylcatechol and L-dihydroxyphenylalanine but had low activity towards tyrosine. p-Cresol, caffeic acid and cholorogenic acid were not oxidised. Thio-compounds were strong inhibitors of the enzyme. The phenolic compounds tyrosine, resorcinol and orcinol inhibited catechol oxidation but became oxidised in the process.     

Steam blanching effect on polyphenoloxidase,peroxidase and colour of mango (Mangifera indica L.) slices

The heat stability of peroxidase (POD) and polyphenoloxidase (PPO) was investigated in mango (Mangifera indica L.) slices, and the relative colour was studied after different steam blanching times. There was complete inactivation after 5 min for POD and 7 min for PPO. Steam blanching of 3 min gave residual activity of 2.85% and 8.33% for PPO and POD, respectively, and when compared with samples blanched for 5 min had no effect on colour over 20 days of storage. Correlation was found between activities of PPO, POD and colour change over 20 days. After 7 min steam blanching the browning index was stable but less than at 3 and 5 min because non-enzymic browning had occurred. This research suggests that yellowness (b) and lightness (L) values contribute positively to the browning index (BI), compared to redness (a).     

Free radical scavenging, inhibition of polyphenoloxidase activity and copper chelating properties of model Maillard systems

Antioxidant properties of Maillard reaction products (MRP) obtained from aqueous mixtures of glucose (0.8 mol/l) with proline, glycine, arginine, lysine, cysteine or glutathione (0.5 mol/l) heated at 103 °C for 1-92 h were investigated using different in vitro tests. Free radical scavenging activity was determined by measuring their reactivity towards DPPH° and lipidic radicals produced by AAPH. Contrary to glucose-proline and glucose-glycine MRP, glucose-lysine and glucose-arginine MRP displayed high scavenging activities. The activity of glucose-lysine MRP peaked after 14 h of heating while the activity of glucose-arginine constantly increased during the whole heat treatment. The high scavenging capacity observed towards DPPH° for glucose-cysteine mixtures could be attributed to the sulfhydryl group of cysteine.The inhibitory effect of MRP on activity of two copper-oxidoreductases, apple polyphenoloxidase and mushroom tyrosinase, assessed by polarography, showed that thiol-derived MRP were the most potent inhibitors, even at very low levels in the reaction medium. Conversely, the other MRP were only slightly efficient at high levels. Unheated mixtures containing thiol compounds exhibited a potent copper chelating ability, as efficient as EDTA, when determined by the tetramethylmurexide (TMM) test. After heating, these mixtures lost part of their chelating efficiency, but it remained higher than that of the other MRP, suggesting that the sulfhydryl group also played a role in the copper chelating properties.     

Purification and characterisation of polyphenol oxidase (PPO) from eggplant (Solanum melongena)

Eggplant (Solanum melongena) is a very rich source of polyphenol oxidase (PPO), which negatively affects its quality upon cutting and postharvest processing due to enzymatic browning. PPO inhibitors, from natural or synthetic sources, are used to tackle this problem. One isoform of PPO was 259-fold purified using standard chromatographic procedures. The PPO was found to be a 112 kDa homodimer. The enzyme showed very low K_m (0.34 mM) and high catalytic efficiency (3.3 × 10⁶) with 4-methyl catechol. The substrate specificity was in the order: 4-methyl catechol > tert-butylcatechol > dihydrocaffeic acid > pyrocatechol. Cysteine hydrochloride, potassium metabilsulphite, ascorbic acid, erythorbic acid, resorcylic acid and kojic acid showed competitive inhibition, whereas, citric acid and sodium azide showed mixed inhibition of PPO activity. Cysteine hydrochloride was found to be an excellent inhibitor with the low inhibitor constant of 1.8 μM.     

杜仲绿原酸的分离纯化及结构鉴定

筛选大孔树脂纯化澄清后的杜仲绿原酸粗提液,并对纯化后的绿原酸进行初步的结构鉴定。采用壳聚糖、活性炭澄清绿原酸的粗提液,选取7 种不同类型的大孔树脂对杜仲绿原酸进行静态吸附解吸实验比较。结果表明,NKA-2 型大孔树脂对绿原酸有较好的选择性,其吸附率和解吸率分别为99.67% 和62.8%。盐酸调整洗脱液的pH 值为3,洗脱液的体积分数为30% 时,洗脱效果最好。澄清后的绿原酸粗提液经乙酸乙酯萃取除去黄酮,再用NKA-2 型树脂进行梯度洗脱,可得到纯度为76.3% 的绿原酸。HPLC-MS 分析表明,绿原酸是咖啡酸和奎尼酸缩合的缩酚酸。     

番石榴多酚氧化酶的部分纯化及其特性研究

番石榴PPO的粗提取液经过80%硫酸铵盐析和DEAE-Toyopearl 650M、CM-Sephadex C-50离子交换柱层析分离后,被纯化了约126倍,回收率为16.13%。该酶迅速地催化焦性没食子酸的酶促氧化反应,而对对苯二酚和绿原酸则完全无催化活性。该酶对焦性没食子酸的Km值为4.6 mmol/L,其最适pH为7.5,pH稳定性范围在pH4.0~11.0,最适温度为50℃,热稳定性相对较高,在≥90℃加热10 min后仍残留约15%的酶活性。该酶的最佳抑制剂是抗坏血酸和NaHSO3,其次是植酸、盐酸-L-半胱氨酸、柠檬酸,Ca2+、Mg2+等金属离子对该PPO也有较强的抑制作用。     

新鲜奶油中黄嘌呤氧化酶的纯化及其酶学性质

采用正丁醇处理、硫酸铵沉淀、凝胶过滤层析和阴离子交换层析等分离技术,从新鲜的稀奶油中纯化得到SDS-PAGE电泳纯的黄嘌呤氧化酶,其最终回收率为19.9%,纯化倍数为82.42,酶比活为8.65U/mg。该黄嘌呤氧化酶的相对分子质量约为280kDa,最适作用温度为40℃,在35℃以下比较稳定;最适pH值是8.5,在pH6.5～8.5时较稳定;Zn2+、Fe2+和K+对黄嘌呤氧化酶活性有促进作用,Ag+、Mn2+、Ca2+和SDS对黄嘌呤氧化酶活性有抑制作用。