Inhibition Of Polyphenol Oxidase Research Articles

In vitro and in vivo inhibition of Hass avocado polyphenol oxidase enzymatic browning by paeonol, β-cyclodextrin, and paeonol:β-cyclodextrin inclusion complex

Polyphenol oxidase (PPO) was extracted from Hass avocados and its physicochemical properties were analyzed. The optimum pH and temperature of the enzyme were pH 7.5 and 20°C. This PPO showed a high thermal stability, since 26% of the initial activity was retained by the enzyme after heating at 60°C for 40min. Inhibition studies were performed using different chemical reagents, and the order in the inhibition efficiency was paeonol>4-hydroxybenzaldehyde>β-cyclodextrin (β-CD). The first two inhibitors presented a non-competitive mechanism while the inhibition by β-CD results from a mixed type mechanism. Since the aqueous solubility of paeonol (a natural compound) is very low, the inclusion complex between this drug and β-CD was obtained in solution and solid state. The stoichiometry of the paeonol:β-CD complex was 1:1 and its ΔG° of formation was-26kJ/mol. The complexation of paeonol by β-CD not only enhances the aqueous solubility and thermal stability of the drug, but also improves the invitro inhibition efficiency against PPO. Colorimetric analysis on avocados pulp (invivo) showed that the inclusion complex does not increase the inhibitory effect of paeonol, remaining practically unchanged. However, the formulation of paeonol:β-CD inclusion complex allows employing this compound as PPO inhibitor in aqueous solutions.

Ornithine decarboxylase is involved in methyl jasmonate-regulated postharvest quality retention in button mushrooms (Agaricus bisporus).

In the present study, we investigated the role of ornithine decarboxylase (ODC) in the methyl jasmonate (MeJA)-regulated postharvest quality maintenance of Agaricus bisporus (J. E. Kange) Imbach button mushrooms by pretreating mushrooms with a specific irreversible inhibitor called α-difluoromethylornithine (DFMO) before exposure to MeJA vapor. Mushrooms were treated with 0 or 100 µmol L-1 MeJA or a combination of 120 µmol L-1 DFMO and 100 µmol L-1 MeJA, respectively, before storage at 4 °C for 21 days. Treatment with MeJA alone induced the increase in ODC activity whereas this effect was greatly suppressed by pretreatment with DFMO. α-Difluoromethylornithine strongly attenuated the effect of MeJA on decreasing cap opening, slowing the decline rate of soluble protein and total sugar, and accumulating total phenolics and flavonoids. α-Difluoromethylornithine pretreatment also counteracted the ability of MeJA to inhibit polyphenol oxidase and lipoxygenase activities, and malondialdehyde production, and to stimulate superoxide dismutase and catalase activities. It also largely downregulated MeJA-induced accumulation of free putrescine (Put). These results reveal that ODC is involved in MeJA-regulated postharvest quality retention of button mushrooms, and this involvement is likely to be associated with Put levels. © 2018 Society of Chemical Industry.

Inhibition of Polyphenol Oxidase by Quercetin Derivatives in Grape Skin

Polyphenol oxidase (PPO) inhibitors were isolated from grape skin and identified as quercetin derivatives. Among the inhibitors isolated, quercetin-3-glucoside (Q3Glc) was the most potent. Kinetic analysis of the inhibitory activity toward PPO was carried out with authentic Q3Glc using catechol as substrate. The inhibition was reversible, the inhibitory mechanism obtained from Lineweaver-Burk plots indicated that Q3Glc is a competitive inhibitor, and the inhibitory constant (Ki) was 84.6 µM.

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

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

Inhibitory effect of chemical and natural anti-browning agents on polyphenol oxidase from ginger (Zingiber officinale Roscoe).

Food browning is undesirable as it causes deterioration in food quality and appearance. This phenomenon was related to polyphenol oxidase (PPO), which catalyzes conversion of phenolic compounds into o-quinones. The present work evaluated the use of chemical and natural anti-browning agents to prevent the browning of ginger PPO. Sodium metabisulfite (5mM) is a better chemical inhibitor compared to l-cysteine and sodium chloride as 55.00% of ginger PPO inhibition was achieved. The percentage of inhibition increased as the concentration of anti-browning agents increases. The addition of heated onion, chili pepper and pineapple extracts exhibited a stronger inhibitory effect on ginger PPO than unheated extracts. Heated chili pepper extract was the best natural inhibitor found in this study and it inhibited the ginger PPO (47.97%) mixed-competitively. Natural anti-browning agents have potential to be used to control the browning of ginger as well as other vegetables and fruits.

Chitosan Coating: A Postharvest Treatment to Delay Oxidative Stress in Loquat Fruits during Cold Storage

Loquat is a non-climacteric fruit consumed fresh for its essential nutrients and phytochemical compounds. In this study, the effects of chitosan coating (1% w/v) on changes in the enzymatic antioxidant and membrane damage in three loquat selections (CREAFRC-S18; CREAFRC-S35 and CREAFRC-S36) and three loquat cultivars (Golden Nugget, Algerie and Nespolone rosso di Trabia) stored at 7 °C over 21 days were evaluated. Chitosan treatment enhanced the activities of superoxide dismutase, catalase and ascorbate peroxidase. Moreover, this treatment inhibited polyphenol oxidase and guaiacol peroxidase activities, extending the storage life of loquat. Chitosan also preserved membrane integrity by inhibiting lipoxygenase activity and malondialdehyde accumulation. Principal component analysis provided a global view of the responses of both loquat selections and cultivars to the postharvest chitosan coating and storage temperature. These findings suggest that chitosan treatment could be a valid tool for improving the activity of antioxidant enzymes, preserving the enzymatic browning of loquat fruits.

Development of carbazole-bearing pyridopyrimidine-substituted urea/thiourea as polyphenol oxidase inhibitors: synthesis, biochemistry, and theoretical studies

Polyphenol oxidase (Tyrosinase, PPO) has received considerable attention, since it is the key enzyme in melanin biosynthesis. In this study, we investigated prepared novel carbazole-containing pyridopyrimidine-substituted with urea and thiourea derivatives and their PPO activities on the diphenolase activity of banana tyrosinase. The structures of the compounds synthesized were confirmed by 1 H NMR, 13 C NMR, FTIR and elemental analysis. PPO enzyme was purified from banana on an affinity gel comprised of Sepharose 4B-L-tyrosine-p-amino benzoic acid. For evaluating the enzyme activity, the synthesised compounds were subjected to tyrosinase inhibition assay using catechol as substrate. While some of the compounds (6, 7, 8f, 8h, 8i, 8j) showed enzyme inhibitor effect, some of them (8a, 8b, 8c, 8d, 8e, 8g, 8k) activated the PPO enzyme activity. Gaussian software was used for the molecular calculations to explain the results for the prepared compounds.

Effect of ascorbic acid postharvest treatment on enzymatic browning, phenolics and antioxidant capacity of stored mung bean sprouts

Enzymatic browning limits the postharvest life of minimally processed foods, thus the study selected the optimal inhibitors of polyphenol oxidase (PPO) and evaluated their effect on enzymatic browning, phenolics and antioxidant capacity of stored mung bean sprouts. The sprouts treated with 2mM and 20mM ascorbic acid had a lowered PPO activity; compared to the control by 51% and 60%, respectively. The inhibition was reflected in a significant decrease in enzymatic browning. The sprouts treated with 20mM ascorbic acid had 22% and 23% higher phenolic content after 3 and 7days of storage, respectively. Both storage and ascorbic acid treatment increased potential bioaccessibility of phenolics. Generally, there was no effect of the treatments on the antioxidant capacity; however, a significant increase in the reducing potential was determined for the sprouts washed with 20mM ascorbic acid. In conclusion, ascorbic acid treatments may improve consumer quality of stored sprouts.

Inhibitory effect of coumarin derivatives on apple (cv. Idared) polyphenol oxidase

Inhibitory effect of 32 coumarin derivatives (20 Schiff bases, 5 thiosemicarbazides, 5 thiazolidinones, and their precursors, 7- hydroxy-4-methylcoumarin and 4-methylcoumarin- 7-yl hydrazine carboxylate) on partially purified apple polyphenol oxidase was investigated. Thirteen coumarin derivatives inhibited polyphenol oxidase (5 Schiff bases, 5 thiosemicarbazides, 1 thiazolidinone, 4-methyl- 7-hydroxycoumarin and 4-methylcoumarin-7-yl hydrazine carboxylate), while 19 derivatives showed no effect on enzyme activity. The most effective inhibitors were thiosemicarbazides, with 4-methyl-1-(2-(4-methyl-2-oxo-2H-chromen- 7-yloxy)acetyl) thiosemicarbazide (compound C23) being the most prominent inhibitor (IC50 = 10.45 µM). The importance of thiosemicarbazide moiety as crucial structure element for strong apple PPO inhibition was confirmed by its cyclisation to thiazolidinone bearing the same substituents as corresponding thiosemicarbazide. Capture of the sulphur atom of thiosemicarbazide group within tiazolidinone ring caused significant loss of inhibitory effect against apple PPO.

Effect of catechin and its derivatives on inhibition of polyphenoloxidase and melanosis of Pacific white shrimp

This study aimed to investigate the effect of tea catechin (C) and 4 of its derivatives on the Pacific white shrimp PPO inhibition and melanosis during refrigerated storage. Epigallocatechin gallate (EGCG) exhibited the highest inhibition towards PPO, followed by C. Inhibitory activity of all compounds tested was in a dose dependent manner (0.1-2.0mM). Based on activity staining, EGCG most effectively inhibited PPO. For inhibition kinetic studies, C and epicatechin (EC) showed uncompetitive type, whereas epicatechin gallate (ECG), epigallocatechin (EGC) and EGCG exhibited mixed type inhibition. When whole shrimps were treated with EGCG solution at various concentrations (0.25-0.75%), those treated with 0.5 or 0.75% EGCG had lower melanosis scores throughout storage for 10days at 4°C, compared with the control and the 1.25% sodium metabisulfite treated samples (P<0.05). Therefore, EGCG could be used as a potential inhibitor for melanosis in raw Pacific white shrimp during refrigerated storage.

Ratoon rice generated from primed parent plants exhibit enhanced herbivore resistance.

Rice ratooning is practiced in many rice-growing countries for achieving increased rice production with limited labour input. Here, we report that attack by insect herbivores, or treatment with a defense signaling compound in parent plants, can prime anti-herbivore defense responses in subsequent ratoon plants. We compared the defense responses of rice ratoons generated from parent plants that had been either infested by Cnaphalocrocis medinalis (rice leaffolder, LF) caterpillars or treated with methyl jasmonate (MeJA) during vegetative growth, with ratoons generated from control parent plants. Ratoon plants generated from parents receiving prior LF infestation or MeJA treatment exhibited higher jasmonic acid (JA) levels, as well as elevated levels of transcripts of defense-related genes associated with JA signaling. In addition, elevated activities of peroxidase, polyphenol oxidase and trypsin protease inhibitor were observed, as well as enhanced resistance towards subsequent LF infestation. Pre-priming of ratoon defense responses was significantly reduced in plants where expression of OsAOS (allene oxide synthase, involved in JA biosynthesis) or OsCOI1 (CORONATINE INSENSITIVE1, involved in JA perception) was inhibited by RNA interference. Our results indicate that herbivore exposure or MeJA treatment in rice parent plants enhances anti-herbivore resistance in subsequently generated ratoons through priming of JA-mediated defenses.

Physicochemical changes of ‘Phulae’ pineapple fruit treated with short-term anoxia during ambient storage

The effects of short-term anoxia exposure for 16h on physicochemical changes of 'Phulae' pineapple fruit stored at ambient temperature (25±2°C) were investigated. The respiratory rate of the fruit was induced by the anoxia treatment. However, it retarded the increase in moisture loss and maintained both flesh and pulp colour by inhibiting polyphenol oxidase (PPO) activity of the both tissues. The anoxia exposure delayed the increase in total sugar content and enhanced total ascorbic acid content during storage. The half-cut pineapple fruit showed that the anoxia exposure completely inhibited internal transparency of the flesh tissue adjacent to core during the storage. In conclusion, the short-term anoxia exposure for 16h maintained postharvest quality, retarded physiological disorder and enhanced nutritional values of the pineapple fruit stored at ambient temperature (25±2°C).

Fall Armyworm-Associated Gut Bacteria Modulate Plant Defense Responses.

Mechanical damage caused by insect feeding along with components present in insect saliva and oral secretions are known to induce jasmonic acid-mediated defense responses in plants. This study investigated the effects of bacteria from oral secretions of the fall armyworm Spodoptera frugiperda on herbivore-induced defenses in tomato and maize plants. Using culture-dependent methods, we identified seven different bacterial isolates belonging to the family Enterobacteriacea from the oral secretions of field-collected caterpillars. Two isolates, Pantoea ananatis and Enterobacteriaceae-1, downregulated the activity of the plant defensive proteins polyphenol oxidase and trypsin proteinase inhibitors (trypsin PI) but upregulated peroxidase (POX) activity in tomato. A Raoultella sp. and a Klebsiella sp. downregulated POX but upregulated trypsin PI in this plant species. Conversely, all of these bacterial isolates upregulated the expression of the herbivore-induced maize proteinase inhibitor (mpi) gene in maize. Plant treatment with P. ananatis and Enterobacteriaceae-1 enhanced caterpillar growth on tomato but diminished their growth on maize plants. Our results highlight the importance of herbivore-associated microbes and their ability to mediate insect plant interactions differently in host plants fed on by the same herbivore.

Characterization of polyphenol oxidase from blueberry (Vaccinium corymbosum L.)

Polyphenol oxidase (PPO) was extracted and characterized from high-bush blueberries. PPO showed an optimum activity at pH 6.1–6.3 and 35°C, with the enzyme showing significant activity over a wide temperature range (25–60°C). Catechol was the most readily oxidized substrate followed by 4-methylcatechol, DL-DOPA, and dopamine. Blueberry PPO showed a Km of 15mM and Vmax of 2.57 ΔA420nm/min×10−1, determined with catechol. PPO was completely inactivated in 20min at 85°C, however, after 30minat 75°C it showed about 10% residual activity. Thermal treatment at 55 and 65°C for 30min resulted in the partial inactivation of PPO. Ascorbic acid, sodium diethyldithiocarbamic acid, L-cysteine, and sodium metabisulfite were effective inhibitors of PPO at 1.0mM. Benzoic acid and cinnamic acid series inhibitors showed relatively weak inhibition of PPO (21.8–27.6%), even at as high as 2.0mM concentration.

Chemical elicitors improve the shelf life of phalsa (Grewia asiaticaL.) by inducing antioxidants and controlling microbes

Introduction. Phalsa (Grewia asiatica L.) fruit has limited marketability due to its high degree of perishability, which leads to extensive postharvest losses. In view of the short postharvest shelf life and perishable nature of the fruit, this study aimed to determine the efficacy of salicylic acid (SA) or sodium benzoate (SB) alone or in combination with calcium chloride (CaCl2 ) treatments for improving the shelf life and quality of phalsa fruit. Materials and methods. Phalsa fruits dipped for 15 min in solutions of T1 = SA 2 mM, T2 = SA 2 mM + CaCl2 1%, T3 = SB 0.1% and T4 = SB 0.1% + CaCl2 1% were stored at two different temperatures, low temperature (10 ± 1°C) and room temperature (25 ± 1°C), while the untreated fruit served as control.Results and discussion. The treatments of 0.1% sodium benzoate (T3) and salicylic acid 2 mM + calcium chloride 1% (T2) were found to be effective in enhancing antioxidants and bioactive compounds such as ascorbic acid, total anthocyanins, etc. These treatments also increased total phenolics inhibited polyphenol oxidase activity and reduced the microbial load in phalsa fruit stored at low as well as room temperature. Conclusion. The treatment of 0.1% sodium benzoate could increase the shelf life of phalsa fruit to 14 days in low temperature storage conditions as compared with only 7 days in the control.

Green tea extract as an anti-browning agent for cloudy apple juice.

Enzymatic browning of fruits and vegetables and their products is an important factor worsening their quality. The influence of five green tea extracts at the concentrations of 1 g L-1 , 2 g L-1 and 3 g L-1 on polyphenol oxidase (PPO) activity in fresh cloudy apple juice was investigated. Moreover, PPO inhibition by tea extract and colour stability of juice during short-time refrigerated storage was studied. The changes of juice colour during storage was expressed as the total colour differences (ΔE*), browning index (BI), yellowness index (YI), and the absorbance at 420 nm (A420 ). All extracts inhibited PPO activity in fresh apple juice in concentration-dependent manner. PPO activity in pure apple juice decreased by 7% after 48 h, whereas PPO activity in samples with 1 g L-1 , 2 g L-1 and 3 g L-1 tea extract decreased by 53%, 74%, and 96%, respectively. Browning of apple juice during storage decreased with increased concentration of green tea extract. After 48 h, extract at 1 g L-1 , 2 g L-1 and 3 g L-1 inhibited browning of juice expressed as BI by 48%, 60%, and 86%, respectively, comparing to pure apple juice. Green tea extract may be an effective anti-browning agent for short-time stored cloudy apple juices. © 2016 Society of Chemical Industry.

Shelf-life extension of Pacific white shrimp using algae extracts during refrigerated storage.

Shrimp is a low-fat, high-protein aquatic product, and is susceptible to spoilage during storage. To establish an effective method for the quality control of Pacific white shrimp, the effects of polyphenols (PP) and polysaccharides (PS) from Porphyra yezoensis on the quality of Pacific white shrimp were assessed during refrigerated storage. Pacific white shrimp samples were treated with 5 g L-1 polyphenols, and 8 g L-1 polysaccharides, then stored at 4 ± 1 °C for 8 days. All samples were subjected to measurement of total viable count (TVC), pH, total volatile basic nitrogen (TVB-N), K-value, thiobarbituric acid (TBA), polyphenol oxidase (PPO) activity, and were also assessed by sensory evaluation. The results showed that PP, PS, and the mixture of polyphenols and polysaccharides (PP+PS) could inhibit the increase of total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA) and K-value, and reduce total viable count (TVC) compared with the control group. PP could also inhibit polyphenol oxidase (PPO) activity. Sensory evaluation proved the efficacy of PP and PS by maintaining the overall quality of Pacific white shrimp during refrigerated storage. Moreover, PP+PS could extend the shelf-life of shrimp by 3-4 days compared with the control group. PP+PS could more effectively maintain quality and extend shelf-life during refrigerated storage. © 2016 Society of Chemical Industry.

Partial purification and characterization of polyphenol oxidase from Chinese parsley (Coriandrum sativum).

Purification and characterization of polyphenol oxidase (PPO) from Chinese parsley (Coriandrum sativum) were achieved. Crude PPO exhibited an enzyme activity of 1,952.24 EU/mL. PPO was partially purified up to 6.52x with a 10.89% yield using gel filtration chromatography. Maximal PPO activity was found at 35°C, pH 8.0 for 4-methylcatechol and at 40°C, pH 7.0 for catechol. PPO showed a higher affinity towards 4-methylcatechol, but a higher thermal stability when reacting with catechol. LCysteine was a better inhibitor than citric acid for reducing PPO activity at concentrations of 1 and 3mM in the presence of either substrate. Two 46 kDa isoenzymes were identified using SDS-PAGE. Isolation and characterization of Chinese parsley serves as a guideline for prediction of enzyme behavior leading to effective prevention of enzymatic browning during processing and storage, including inhibition and inactivation of PPO.

Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants.

Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) has been performed using cotyledonary node explants (CNs), which release phenolics upon excision that are detrimental to the viability of Agrobacterium tumefaciens and result in low transformation frequency. Twelve low molecular weight phenolic compounds and salicylic acid were identified in the exudates released upon excision during the preparation of cotyledonary nodes by reverse phase high-performance liquid chromatography (RP-HPLC). Zone inhibition assays performed with the explant exudates released at periodic intervals after excision showed the inhibition of A. tumefaciens. Agroinoculation of freshly excised cotyledonary nodes of chickpea showed 98-99% inhibition of colony forming units (cfu). Osmium tetraoxide fixation of excised tissues showed enhanced accumulation of phenolics in the sub-epidermal regions causing enzymatic browning, affecting the viability and performance of A. tumefaciens for T-DNA delivery. The periodic analysis of exudates released from excised CNs showed enhanced levels of gallic acid (0.2945 ± 0.014μg/g), chlorogenic acid (0.0978 ± 0.0046μg/g), and quercetin (0.0971 ± 0.0046μg/g) fresh weight, which were detrimental to A. tumefaciens. Quantitative assays and the elution profile showed the maximum leaching of phenolics, flavonoids, and salicylic acid immediately after the excision of explants and continued till 4 to 8h post-excision. Pre-treatment of excised explants with inhibitors of polyphenol oxidase like L-cysteine, DTT, and sodium thiosulfate before co-cultivation showed the recovery of A. tumefaciens cfu, decreased the accumulation of phenolics, and improved transformation frequency. Our results show the hypersensitive response of excision stress for the expression of defense response-related genes and synthesis of metabolites in grain legume chickpea against pathogen infestation including Agrobacterium.

High pressure thermal processing of pears: Effect on endogenous enzyme activity and related quality attributes

Abstract The effect of high pressure-thermal (HPT) processing (600 MPa, 20–100 °C) on the activity of pear enzymes and related quality attributes was investigated. HPT processing at 20 °C for 5 min resulted in 32%, 74% and 51% residual activities of polyphenol oxidase (PPO), peroxidase (POD) and pectin methylesterase (PME), respectively. Increasing processing temperature to 40 and 60 °C reduced the level of PPO and POD inactivation, with the maximum residual activities of 64% and 123%, respectively observed after 3-min treatments at 40 and 60 °C. Overall, HPT at 20 to 60 °C had minimal effect on quality, although enzymatic browning was observed upon air exposure. HPT at 80 to 100 °C caused almost complete inactivation of PPO and POD with 90% and 92% inactivation respectively after 3-min processing at 100 °C, which reduced browning upon air exposure. Nevertheless, the lowest texture retention of 22% was observed under this condition. Industrial relevance The study examined the effects of combined high pressure thermal processing on quality related pear enzymes and related instrumental quality attributes such as colour and texture. The study enabled identification of processing regimes for enzyme inactivation and quality retention. The excellent quality retention following HPP at 20 to 40 °C makes this condition suitable for ‘fresh-like’ small portion products for immediate consumption after unpacking that do not require complete PPO and POD inactivation. On the other hand, the almost complete inactivation of oxidative enzymes PPO and POD at 100 °C makes this condition more appropriate for the production of bulk products for food service applications or pureed ingredients for baby food, or pear pieces for yoghurt, that require PPO inhibition but not necessarily high firmness retention.