Detection and identification of polyphenoloxidase substrates in apple and pear skins

doi:10.1016/0003-9861(55)90337-4

Archives of Biochemistry and Biophysics

Volume 56, Issue 1, May 1955, Pages 97-102

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Abstract

A paper chromatographic technique for the detection and identification of substances susceptible to darkening with polyphenoloxidase is described. Color formation occurs with the susceptible substances on the paper after it is sprayed with a polyphenoloxidase enzyme from apples. The principal browning substrate on chromatograms of the skin extract from Grimes Golden and Golden Delicious apple and Bartlett pear was l-epicatechin. Bartlett pear contained also smaller amounts of d-catechin, another browning substrate.

References (16)

R.L. Mayer et al.
Nordstrom, C. G., and Swain, T.J. Chem. Soc.1953...
A.E. Bradfield et al.
Biochim. et Biophys. Acta
(1950)
E.A.H. Roberts et al.
Biochem. J
(1953)
Bradfield, A. E., and Penney, M., J. Chem. Soc.1948,...
Bate-Smith, E. C., and Swain, T., Chemistry & Industry1953,...
Williams, A. H., Chemistry & Industry1953,...
T. Nakabayashi
J. Agr. Chem. Soc. Japan
(1953)

There are more references available in the full text version of this article.

Cited by (16)

Extraction optimisation, purification and major antioxidant component of red pigments extracted from Camellia japonica
2011, Food Chemistry
Citation Excerpt :
White needle crystals (MeOH); HR-ESI-MS: m/z 313.0684 [M + Na]+, calculated for C15H14O6Na 313.0688; 1H NMR (400 MHz, DMSO): δ 4.73 (1H, s, H-2), 4.00 (1H, s, H-3), 2.48 (1H, dd, J = 16.2, 3.5, H-4A), 2.68 (1H, dd, J = 16.2, 4.5, H-4B), 5.71 (1H, d, J = 2.2, H-6), 5.89 (1H, d, J = 2.2, H-8), 6.89 (1H, d, J = 2.8, H-2′), 6.68 (1H, dd, J = 8.0, 2.8, H-6′), 6.64 (1H, d, J = 8.0, H-5′), 9.09 (1H, s, -OH), 8.88 (1H, s), 8.78 (1H, s, -OH), 8.70 (1H, s, -OH), 4.64 (1H, s, 3-OH); 13C NMR (100 MHz, DMSO): 78.5 (C-2), 65.3 (C-3), 28.6 (C-4), 98.9 (C-4a), 156.9 (C-5), 95.5 (C-6), 156.6 (C-7), 94.5 (C-8), 156.2 (C-8a), 131.0 (C-1′), 115.3 (C-2′), 144.9 (C-3′), 144.8 (C-4′), 115.2 (C-5′), 118.4 (C-6′). (−)-Epicatechin belongs to the flavone, was present in other fruits, such as pear skins (Siegelman, 1955), mango kernels (Arogba, 2000) and apple peels (Alonso-Salces et al., 2004). It exhibited a wide spectrum of pharmacological properties such as chlorinating activity (Kirchner, Flemmig, Furtmüller, Obinger, & Arnhold, 2010), antimutagenic activity (Geetha, Garg, Chopra, & Kaur, 2004), antioxidant effects (Cuevas et al., 2009), and inhibition of NADPH oxidase (Steffen, Schewe, & Sies, 2007).
The optimised extraction conditions of red pigments (RP) from Camellia japonica obtained with an orthogonal design L₉(3⁴) were solid/liquid ratio, temperature, pH and extraction time as 1/10, 60 °C, 1.5 and 4 h, respectively. The RP were then purified by the macroporous resin method, which showed the resin LX-68 was appropriate for purifying the pigments from C. japonica. The antioxidant activities of these pigments were also investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical in vitro model systems. The DPPH scavenging activity of pigment extract was comparable to that of standard butylated hydroxyanisole (BHA). The IC₅₀ values of the RP and BHA were 4.55 and 4.17 μg ml⁻¹, respectively. The pigments showed higher hydroxyl radical-scavenging activities than that of mannitol at the same concentration. Following activity-oriented separation, (−)-epicatechin was isolated as an active principle, which exhibited excellent DPPH free radical scavenging activities with IC₅₀ 5.08 μg ml⁻¹.
Identification of (-)-epicatechin as the direct substrate for polyphenol oxidase from longan fruit pericarp
2008, LWT
Citation Excerpt :
In combination with the data of [α]D, 1H NMR, 13C NMR and ESI-MS, the direct substrate for PPO from pericarp tissues of longan fruit was identified as (−)-epicatechin. ( −)-Epicatechin has also been identified from other fruit tissues, such as apple peel (Alonso-Salces et al., 2004), mango kernel (Arogba, 2000), pear skin (Siegelman, 1955; Veberic et al., 2005) and apricot fruit (Radi, Mahrouz, Jaouad, & Amiot, 2004). Furthermore, the contents of (−)-epicatechin of fruit pericarp tissues of longan fruit were determined by HPLC.
Postharvest browning of longan fruit results in a short life and a reduced commercial value. The experiments were conducted to separate, then purify and finally identify the polyphenol oxidase (PPO) substrates that cause longan fruit to brown. PPO and its substrates were, respectively, extracted from longan fruit pericarp tissues. The substrate for longan PPO was separated and purified using polyamide column chromatography, Sephadex LH-20 column chromatography and silica gel column chromatography, respectively. The substrate was further identified by 0.5% FeCl₃ solution and enzymatic reaction with longan PPO. On the bases of UV, ¹H NMR, ¹³C NMR, and ESI-MS data, the direct substrate for the PPO from pericarp tissues of longan fruit was identified to be (−)-epicatechin. Furthermore, the contents of (−)-epicatechin of pericarp tissues of longan fruit of two major cultivars were determined by high performance liquid chromatography (HPLC). The HPLC analysis exhibited that the contents of (−)-epicatechin of fruit pericarp of ‘Shixia’ and ‘Chuliang’ were 0.26 and 0.56 mg/g on fresh weight (FW) basis at harvest and 0.15 and 0.09 mg/g FW after 3 days of storage. The more rapid decrease in the (−)-epicatechin content of ‘Chuliang’ was due to the oxidization catalyzed by PPO, which was in agreement with the higher browning index.
Identification of (-)-epicatechin as the direct substrate for polyphenol oxidase isolated from litchi pericarp
2006, Food Research International
Postharvest browning of litchi fruit results in a short life and a reduced commercial value. The experiments were conducted to separate, purify and identify the polyphenol oxidase (PPO) substrates that cause litchi fruit to brown. PPO and its substrates were, respectively, extracted from fruit pericarp tissues. The substrates for litchi PPO were separated and purified using polyamide column chromatography, silica gel column chromatography and preparative thin layer chromatography. The substrate was further identified by 0.5% FeCl₃ solution and enzymatic reaction with litchi PPO. On the basis of UV, ¹H NMR, ¹³C NMR, and ESI-MS data, the direct substrate for the PPO from litchi fruit pericarp tissues was identified to be (−)-epicatechin.
O-diphenol oxidase activity, phenolic content and colour of New Zealand wheats, flours and milling streams
1990, Journal of Cereal Science
Phenolic content, o-diphenol oxidase activity and bread crumb colour have been compared for flour streams from a local mill and for flours and wholemeals from six New Zealand wheat cultivars. Phenolic content and o-diphenol oxidase activity varied significantly betwen milling flour streams and between cultivars. Flour and bread crumb colour correlated significantly with phenolic content and o-diphenol oxidase activity in the milling flour streams. A similar relationship was observed also between o-diphenol oxidase activity and the colour of flour and bread prepared from the different cultivars. These observations suggest an involvement of o-diphenol oxidase and phenolics in the pigmentation of both wheat flour and bread.
Polyphenoloxidase from apple, partial purification and some properties
1989, Phytochemistry
The optimum conditions for polyphenoloxidase (PPO) extraction from apple fruit were obtained with a buffer solution above pH 7 containing 15 mM ascorbic acid and 0.5% of Triton X100. In 12 cultivars grown in France, the PPO activity ranged from 3.1 to 0.62 in the cortex and from 3 to 0.3 mkat/kg in the peel. For both parts, the cultivar Red Delicious exhibited the highest activity, whereas the lowest activity was in Elstar. In the Red Delicious apples, the PPO activity steadily declined during the eight weeks around the commercial date of harvest, the total decrease being close to 30%. PPO from Red Delicious cortex has been purified 120-fold, with a total yield close to 40%, by ammonium sulphate precipitation and hydrophobic chromatography on Phenyl Sepharose CL4B. The pIs, obtained by isoelectrofocusing were at pH 4.5 and 4.8 for two major bands and near neutral for a minor one. The M_r determined by gel filtration was unique (ca46 000). The optimum pH of activity was between 4.5 and 5 for 4-methylcatechol, chlorogenic acid and (+) catechin. The K_m values were almost independent of pH on the acid side of the pH optimum and were in the range of 5 mM for the three substrates studied. At pH 4 which is close to the pH of apple vacuoles, chlorogenic acid is a better substrate for apple PPO than (+) catechin.
Erwinia salicis in cricket bat willows: peroxidase, polyphenoloxidase, β-glucosidase, pectinolytic and cellulolytic enzyme activity in diseased wood
1978, Physiological Plant Pathology
Erwinia salicis produced peroxidase, β-glucosidase, cellulase and pectinolytic enzymes in artificial media, but not polyphenoloxidase. The pectic enzyme produced by E. salicis (N.C.P.P.B. isolate Preece 14) was a constitutive rans-eliminase and using the thiobarbituric acid procedure was shown to have a pH optimum of 9·8 and to be Ca²⁺ dependent. In healthy, uninoculated cricket bat willow wood extracts, peroxidase, polyphenoloxidase, β-glucosidase and cellulase activity was detected, but not pectinolytic enzymes. Wood with the watermark disease sympton following infection by E. salicis shows marked increase in peroxidase, β-glucosidase and cellulase and slight increase in polyphenoloxidase and polygalacturonate trans-eliminase.

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Detection and identification of polyphenoloxidase substrates in apple and pear skins

Abstract

Biochim. et Biophys. Acta

Biochem. J

J. Agr. Chem. Soc. Japan