Enhancement of the antioxidant defense system of post-harvested ‘Daw’ longan fruit by chlorine dioxide fumigation

Scientia Horticulturae 178 (2014) 138–144

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Enhancement of the antioxidant defense system of post-harvested ‘Daw’ longan fruit by chlorine dioxide fumigation Warunee Chomkitichai a , Bualuang Faiyue b , Pornchai Rachtanapun c , Jamnong Uthaibutra a,d , Kobkiat Saengnil a,d,∗ a

Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Department of Biology, Mahidol Wittayanusorn School, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand c Division of Packaging Technology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50200, Thailand d Postharvest Technology Research Institute, Chiang Mai University/Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok 10140, Thailand b

a r t i c l e

i n f o

Article history: Received 19 June 2014 Received in revised form 14 August 2014 Accepted 18 August 2014 Keywords: Enzymatic antioxidant Antioxidant capacity Chlorine dioxide Browning Longan fruit

a b s t r a c t The response of the antioxidant defense system to chlorine dioxide (ClO2 ) of post-harvested ‘Daw’ longan fruit was determined. Fresh fruit was fumigated with 10 mg/L ClO2 for 10 min and then packed into cardboard boxes and stored at 25 ± 1 ◦ C with 82 ± 5% RH for 7 days. Changes in free radicals elimination systems were examined every day. The activities of enzymatic antioxidants including superoxide dismutase, catalase and ascorbate peroxidase in non-fumigated fruit (control) increased and reached the peak on day 2 and decreased thereafter. The contents of non-enzymatic antioxidant such as total phenolic compounds, ascorbic acid (ASA), total glutathione, ␣-tocopherol and total antioxidant capacities (TAC) decreased continuously throughout storage time. These decreases coincided with the onset of pericarp browning. ClO2 fumigation enhanced the antioxidant defense system of longan fruit during storage which was associated with less pericarp browning. The enzymatic antioxidant activities, nonenzymatic antioxidant contents and TAC showed significantly higher levels in the ClO2 treated fruit than those in the control throughout the storage period. It was concluded that enhanced antioxidant defense system by ClO2 alleviates browning in ‘Daw’ longan during storage. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The antioxidant defense system is an important mechanism for plant protection against oxidative damage from free radicals especially reactive oxygen species (ROS) such as superoxide radical (O2 •− ), hydrogen peroxide (H2 O2 ), hydroxyl radical (OH• ), perhydroxyl radical (HO2 • ), singlet oxygen (1 O2 ), and peroxyl radical (ROO• ). The antioxidant defense system is composed of enzymatic and non-enzymatic components. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), peroxidase (POD), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) are important enzymatic components. Nonenzymatic antioxidants include phenolic compounds, ascorbic acid (ASA), glutathione, carotenoids and ␣-tocopherol (Blokhina et al., 2003; Gill and Tuteja, 2010; Sharma et al., 2012).

∗ Corresponding author. Tel.: +66 53 943346; fax: +66 53 892259. E-mail address: kobkiat [email protected] (K. Saengnil). http://dx.doi.org/10.1016/j.scienta.2014.08.016 0304-4238/© 2014 Elsevier B.V. All rights reserved.

SOD converts O2 •− to O2 and H2 O2 and then H2 O2 is scavenged by CAT, APX, GPX and/or POD (Mittler, 2002; Gill and Tuteja, 2010; Sharma et al., 2012). Non-enzymatic antioxidants scavenge ROS by donating electrons or hydrogen atoms (Blokhina et al., 2003; Gill and Tuteja, 2010; Sharma et al., 2012). Phenolic compounds have a strong capacity to donate electrons or hydrogen atoms. They can directly scavenge molecules of ROS, and inhibit lipid peroxidation by trapping the lipid alkoxyl radical. Moreover, they are oxidized by polyphenol oxidase (PPO) and POD and act in the H2 O2 scavenging system (Sharma et al., 2012). Ascorbic acid can directly scavenge O2 •− , OH• and 1 O2 and reduce H2 O2 to water via APX reaction. It can regenerate ␣-tocopherol from the tocopheroxyl radical (Blokhina et al., 2003; Gill and Tuteja, 2010; Sharma et al., 2012). Glutathione is a potential scavenger of 1 O2 , O2 •− , H2 O2 and most dangerous ROS like OH• . It also has a key role in the antioxidant defense system by regenerating ascorbic acid via the ascorbate–glutathione cycle (ASA-GSH cycle). ␣-Tocopherols are involved in scavenging 1 O2 , OH• (Munné-Bosch, 2005) and prevents the lipid peroxidation by scavenging lipid peroxide radicals (Blokhina et al., 2003; MunnéBosch, 2005; Gill and Tuteja, 2010; Sharma et al., 2012).

W. Chomkitichai et al. / Scientia Horticulturae 178 (2014) 138–144

Decline in the antioxidant defense system contributes to fruit browning after harvesting such as lychee (Sun et al., 2010, 2011; Yi et al., 2010; Duan et al., 2011), rambutan (Shao et al., 2012), loquat (Abbasi et al., 2013) and chestnut (You et al., 2012). It leads to free radical accumulation, resulting in oxidative damage in both cellular and organelle membranes. The phenolic compounds leaked from damaged vacuoles are oxidized and changed to a complex brown polymer, catalyzed by PPO and POD. This oxidative damage increases browning. The relationship between the antioxidant defense system and browning during storage of longan fruit has been reported. Duan et al. (2007) reported that the decrease in CAT activity, APX activity, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and reducing power in ‘Shixia’ longan fruit is associated with pericarp browning during storage at 28 ◦ C for 6 days. Cheng et al. (2009) reported that DPPH radical scavenging activity of ‘Shixia’ longan fruit decreased throughout 6 days of storage at 28 ◦ C with an increase in pericarp browning. Chlorine dioxide (ClO2 ) is a new chemical used to reduce longan browning. Saengnil et al. (2014) reported that ClO2 gas maintained the quality of longan fruit cv. Daw during storage at 25 ◦ C for 7 days. They found that fumigating with 10 mg/L ClO2 for 10 min was the most effective treatment to reduce pericarp browning by decreasing PPO and POD activities. It also reduced the levels of ROS and membrane damage in longan fruit. The levels of O2 •− , H2 O2 and OH• , lipoxygenase (LOX) activity, conjugated diene, malondialdehyde (MDA) contents and electrolyte leakage (EL) rate were significantly lower in the fumigated group than those in the control group (Chomkitichai et al., 2014). The present study investigated the changes in antioxidant defense system during storage of ‘Daw’ longan fruit and the effect of ClO2 on enhancing the antioxidant defense system in longan fruit.

139

extract for CAT (EC 1.11.1.6) and APX (EC 1.11.1.11) assays. For the SOD (EC 1.15.1.1) assay, the enzyme extract was dialyzed against 10 mM potassium phosphate buffer (pH 7.8) using regenerated cellulose tubular membrane (Cellu•Sep® , USA) overnight at 4◦ C. The dialyzed extract was centrifuged at 15,000 × g for 20 min at 4 ◦ C. The supernatant was used as an enzyme extract for SOD assay. SOD activity was assayed in a 2 mL reaction mixture containing 0.2 mL of 500 mM potassium phosphate buffer (pH 7.8), 0.2 mL of 0.1 mM cytochrome C from horse heart, 0.2 mL of 1 mM xanthine dissolved in 10 mM NaOH, 0.04 mL of xanthine oxidase, 1.32 mL of distilled water and 0.04 mL of enzyme extract. The rate of reduction of cytochrome C was measured by the increase in absorbance at 550 nm. The SOD activity was expressed as Unit mg−1 protein. One unit SOD activity is defined as the amount of enzyme that inhibits the rate of cytochrome c reduction by half under specific conditions. CAT activity was assayed in a 2 mL reaction mixture comprising 1.9 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 25 mM H2 O2 and 0.1 mL of enzyme extract. The subsequent decomposition of H2 O2 was observed at 240 nm (E = 0.0394 mM−1 cm−1 ). The CAT activity was expressed as ␮mol H2 O2 decomposition g−1 protein min−1 . APX activity was assayed in a 2 mL reaction mixture containing 0.5 mL of 100 mM potassium phosphate buffer (pH 7.0), 0.5 mL of 1 mM ascorbic acid, 0.5 mL of 0.4 mM EDTA, 0.02 mL of 10 mM H2 O2 , 0.38 mL of distilled water and 0.1 mL of enzyme extract. The subsequent decrease in ascorbic acid was observed at 290 nm (E = 2.8 mM−1 cm−1 ). The APX activity was expressed as ␮mol ASA decomposition mg−1 protein min−1 . Protein content was determined according to Lowry et al. (1951) using bovine serum albumin as the standard. 2.3. Determination of non-enzymatic antioxidant contents

2. Materials and methods 2.1. Plant materials and ClO2 treatment Mature longan (Dimocarpus longan Lour. cv. Daw) fruit was harvested from a commercial orchard in Lamphun province, Thailand and transported to the Postharvest Physiology and Technology Research Laboratory at Chiang Mai University within 2 h. The fruit without defect was individually selected for uniformity of shape, color and size. They were divided into two groups of 480 fruits each for a non-fumigated (control) group and a fumigated group with 10 mg/L ClO2 for 10 min. After fumigation, the fruit was ventilated for 30 min in order to remove gas residues. They were then packed into cardboard boxes (20 (L) × 14 (W) × 7 (H) cm) with 20 fruits in each box and were subdivided into 3 subgroups of 8 boxes each before storing at 25 ± 1 ◦ C with relative humidity 82 ± 5% for 7 days. One box from each subgroup was randomly sampled every day to determine the activities of antioxidant enzymes (SOD, CAT and APX), the amount of non-enzymatic antioxidants (total phenolic compound (TPC), ASA, total glutathione, ␣-tocopherol), total antioxidant capacities (TAC) and pericarp browning. 2.2. Extraction and assay of enzymatic antioxidant activities Enzyme extraction and assays were performed according to the method of Sunohara and Matsumoto (2004) with some modifications. Longan pericarp (1 g) was sliced and homogenized at 4 ◦ C for 30 s in 10 mL of 25 mM potassium phosphate buffer (pH 7.8) containing 0.4 mM EDTA, 1 mM ascorbic acid and 2% polyvinyl polypyrrolidone (PVPP). The homogenate was centrifuged at 15,000 × g for 20 min at 4 ◦ C and the supernatant was filtered through filter paper (Whatman® No.1, England). The filtrate was used as an enzyme

The TPC content was determined by the Folin-Ciocalteu method according to the procedure of Singleton and Rossi (1965) with some modifications. Longan pericarp (1 g) was sliced and homogenized in 10 mL of 80% ethanol for 30 s at 4 ◦ C. The homogenate was centrifuged at 16,000 × g for 20 min at 4 ◦ C. Then, 0.4 mL of the supernatant was mixed with 2 mL of 10% Folin-Ciocalteu’s phenol reagent for 8 min before 1.6 mL of 7.5% sodium carbonate was added. The mixture was placed at room temperature for 2 h. The absorbance was measured with a visible spectrophotometer at 765 nm. The TPC was determined from the linear equation of a standard curve prepared with gallic acid (GA) and expressed as mg GA g−1 FW. ASA content was determined according to the methods of Deepa et al. (2006) and AOAC (1990) with some modifications. Longan pericarp (1 g) was sliced and homogenized in 10 mL of 3% (w/v) metaphosphoric acid for 30 s at 4 ◦ C. The homogenate was centrifuged at 3,000 × g for 20 min at 4 ◦ C and 2 mL of the crude extract was added to 5 mL of 3% metaphosphoric acid and then titrated with 0.1 mM 2,6-dichloroindophenol (DPIP) to the end point. The ASA content was expressed as mg AsA g−1 FW. Total glutathione content was determined according to the method of Gronwald et al. (1987) with some modifications. Longan pericarp (1 g) was sliced and homogenized in 12 mL of 5% trichloroacetic acid (TCA) for 30 s at 4 ◦ C. The homogenate was centrifuged at 12,000 × g for 10 min at 4 ◦ C. The supernatant was diluted (1:1) with 0.5 M potassium phosphate buffer (pH 8.0). This dilution was further diluted (1:9) in 0.1 M potassium phosphate buffer (pH 8.0). The reaction medium consisted of 2.2 mL of 0.1 M potassium phosphate buffer (pH 7.5) containing 5 mM EDTA and 0.2 mL of 1 mM NADPH. The NADPH used was previously dissolved in 0.1 M potassium phosphate buffer (pH 7.5) containing 5 mM EDTA and 0.2 mL (1 unit) of glutathione reductase (GR) prepared in 0.1 M potassium phosphate buffer (pH 7.5). These components

140

W. Chomkitichai et al. / Scientia Horticulturae 178 (2014) 138–144

were equilibrated in test tube at 25 ◦ C for 2 min. The reaction was initiated by adding 0.2 mL of 6 mM 5-5 -dithiobis (2-nitrobenzoic acid) (DTNB) prepared in 0.1 M potassium phosphate buffer (pH 7.5) and 0.2 mL of diluted supernatant. The change in absorbance of the mixture was measured by UV/VIS spectrophotometer at 412 nm every 1 min for 5 min. Total glutathione content was determined from the linear equation of a standard curve prepared with reduced glutathione (GSH) and expressed as ␮g GSH g−1 FW. ␣-Tocopherol was determined according to the method of Contreras-Guzmán and Strong (1982) with some modifications. Longan pericarp (1 g) was sliced and immersed in 20 mL of absolute ethanol for 30 min in a water bath at 85 ◦ C. The solution was allowed to cool and then filtered into a separating funnel. Twenty milliliters of heptane was added and the solution was shaken for 5 min. Then, 20 mL of 1.25% sodium sulphate was added and the solution was shaken again for 2 min and allowed to separate into layers. The upper layer was separated to eliminate the interfering substances by the slight saponification method. The mixture containing 7 mL of upper layer solution, 5 mL of 5% L-ASA and 5 mL of 50% potassium hydroxide was shaken for 2.5 min and allowed to separate into layers. Five milliliters of the upper layer solution was mixed with 10 ml of 80% ethanol, shaken for 1 min and allowed to separate into layers. The upper layer was used to determine total tocopherols by a reaction with cupric ions and complexed with 2,2 -biquinoline (cuproine). A volume of 0.5 mL of ␣-tocopherol in absolute ethanol was processed in the same way as the sample and used as a standard. The ␣-tocopherol content was expressed as ␮g ␣-tocopherol g−1 FW. 2.4. Determination of total antioxidant capacities (TAC) Longan pericarp (1 g) was sliced and homogenized in 10 mL of 80% methanol for 30 s at 4 ◦ C. The homogenate was centrifuged at 16,000 × g for 20 min at 4 ◦ C. The supernatant was collected as a sample solution to determine the TAC by using 2,2 -Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and DPPH radical scavenging and ferric reducing antioxidant power (FRAP) assays. ABTS radical scavenging activity was determined according to the method of Huang et al. (2005) with some modifications. A volume of 0.02 mL of sample solution was mixed with 2 mL of ABTS•+ solution which was prepared from 2 mL of 7 mM ABTS added to 3 mL of 2.45 mM of potassium persulfate. The mixture was kept in the dark at 5 ◦ C for 16 h to give a dark blue solution before being diluted with 80% ethanol until the absorbance of ABTS•+ dilution was 7.0 at 734 nm. The mixture was placed at room temperature for 10 min. The absorbance was measured with a visible spectrophotometer at 734 nm. ABTS radical scavenging activity was compared to that of Trolox, a water-soluble vitamin E analogue. The TAC was determined from the linear equation of a standard curve prepared with Trolox and expressed as ␮mol Trolox g−1 FW. DPPH radical scavenging activity was determined according to the method of Mun’im et al. (2003) with some modifications. A volume of 0.1 mL of sample solution was mixed with 0.4 mL of 0.3 M acetate buffer (pH 5.5) and 2.5 mL of 0.12 mM DPPH• with 100% methanol. The mixture was placed in a dark at room temperature (25 ± 1 ◦ C) for 30 min. The absorbance was measured with a visible spectrophotometer at 517 nm. DPPH radical scavenging activity was determined from the linear equation of a standard curve prepared with Trolox and expressed as ␮mol Trolox g−1 FW. The FRAP assay was determined according to the methods of Benzie and Strain (1996, 1999) with some modifications. A volume of 0.3 mL of sample solution was mixed with 2.7 mL of FRAP solution which was freshly prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6- tripyridy-s-triazine (TPTZ) in 40 mM hydrochloric acid (HCl) and 20 mM ferric chloride hexahydrate (FeCl3 ·6H2 O) in the ratio of 10: 1: 1 and was kept in the dark at room temperature

(25 ± 1 ◦ C) for 30 min. The absorbance was measured with a visible spectrophotometer at 593 nm. FRAP was determined from the linear equation of a standard curve prepared with ferrous sulphate (FeSO4 ) and expressed as ␮mol Fe2+ g−1 FW. 2.5. Assssment of pericarp browning Pericarp browning was estimated using the browning index (BI) as described by Jiang and Li (2001). Browning was assessed visually by measuring the extent of the brown area on each fruit surface using the following scales; 1 = no browning (excellent quality), 2 = slight browning, 3 = less than 25% browning of the total surface, 4 = 25–50% browning, and 5 = more than 50% browning (poor quality). The browning index was calculated as  (browning scale × percentage of fruit in each class). Fruit having a browning index above 3.0 were considered as unacceptable for visual marketing quality. 2.6. Statistical analysis The experiments were arranged in a completely randomized design with three replications. Data were analyzed by one-way analysis of variance (ANOVA) using statistical packages for the social science (SPSS) version 17.0. Least significant differences (LSD) were calculated to compare significant effects at P = 0.05. 3. Results 3.1. Enzymatic antioxidant activities The activities of SOD, CAT and APX in the control fruit increased and reached the highest peak on Day 2 and then decreased gradually from day 3 to day 7 (Fig. 1). The activities of SOD, CAT and APX in the fruit treated with ClO2 significantly increased with a similar pattern as the control (Fig. 1). Fumigation with ClO2 increased the activities of SOD, CAT and APX by 35%, 133% and 40%, respectively, on day 2 as compared to the control (Fig. 1). The average increase in enzyme activities was 70%, 135% and 209% for SOD, CAT and APX, respectively, during 7 days of storage. 3.2. Non-enzymatic antioxidant contents The content of all non-enzymatic antioxidants in the untreated control fruit decreased with storage time (Fig. 2). TPC and ␣tocopherol continuously decreased throughout storage (Figs. 2A and D). Ascorbic acid and total glutathione rapidly decreased during the first 2 days of storage and decreased continuously thereafter (Figs. 2B and C). ClO2 fumigation significantly delayed the decrease of non-enzymatic antioxidant contents during storage. As a result, non-enzymatic antioxidant contents in ClO2 fumigated fruit were significantly higher than those in the control fruit (Fig. 2). The amounts of TPC, ascorbic acid, total glutathione and ␣-tocopherol in fumigated fruit were 39%, 66%, 66% and 29%, respectively higher than those in the control fruit, throughout storage. 3.3. Total antioxidant capacities (TAC) TAC in the untreated control fruit gradually decreased throughout storage (Fig. 3). Changes in TAC analyzed by ABTS, DPPH and FRAP assays in the untreated control fruit showed a similar pattern, although ABTS assay had higher TAC than DPPH and FRAP assays (Fig. 3). ClO2 fumigation significantly delayed the decrease in TAC throughout storage (Fig. 3). TAC in the fumigated fruit analyzed by ABTS, DPPH and FRAP assays were 48%, 43% and 33%, respectively higher than those in the control fruit during storage.

W. Chomkitichai et al. / Scientia Horticulturae 178 (2014) 138–144

141

Fig. 1. Changes in activities of SOD (A), CAT (B) and APX (C) of ‘Daw’ longan fruit during storage at 25 ± 1 ◦ C for 7 days. Vertical bars with the same letters between the control and treatment in each storage time indicate non-significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

3.4. Pericarp browning Longan pericarp browning of ClO2 untreated fruit occurred rapidly and became unacceptable on Day 1 of storage with a browning index of more than 3 (Fig. 4). ClO2 fumigation significantly delayed and reduced pericarp browning with browning index below 3 for 5 days (Fig. 4). 4. Discussion Under normal conditions, ROS is continuously generated in plants as byproducts of aerobic metabolism. At low levels of ROS, there is an appropriate balance between production and scavenging of ROS by various cellular enzymatic and non-enzymatic mechanisms. Under stress conditions the levels of ROS exceeded the defense mechanisms causing adverse effects such as oxidative damage of biological molecules, membrane damage, loss of cell structure and function leading to browning in plants. Browning of ‘Daw’ longan pericarp rapidly increased within 1 day of storage at 25 ◦ C (Fig. 4). This agree with Khunpon et al. (2011)

Fig. 2. Changes in the contents of TPC (A), ascorbic acid (B), total glutathione (C) and ␣-tocopherol (D) of ‘Daw’ longan fruit during storage at 25 ± 1 ◦ C for 7 days. Vertical bars with the same letters between the control and treatment in each storage time indicate non-significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

and Saengnil et al. (2014) who also found that pericarp browning in longan fruit at 25 ◦ C started quickly in 1 day. This pericarp browning in longan was primarily attributed to the oxidation of phenolic compounds activated by PPO and POD (Khunpon et al.,

142

W. Chomkitichai et al. / Scientia Horticulturae 178 (2014) 138–144

Fig. 4. Change in pericarp browning of ‘Daw’ longan fruit during storage at 25 ± 1 ◦ C for 7 days. Vertical bars with the same letters between the control and treatment in each storage time indicate non-significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

Fig. 3. Changes in TAC by ABTS (A), DPPH (B), and FRAP (C) assays of ‘Daw’ longan fruit during storage at 25 ± 1 ◦ C for 7 days. Vertical bars with the same letters between the control and treatment in each storage time indicate non-significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

2011; Saengnil et al., 2014). The enzyme-catalyzed browning reaction was caused by the over accumulation of ROS in the pericarp (Chomkitichai et al., 2014). However, stress-induced ROS accumulation is counteracted by enzymatic and non-enzymatic systems (Gill and Tuteja, 2010; Sharma et al., 2012). Fig. 1 showed that the activities of SOD, CAT and APX in longan pericarp initially increased during early storage of oxidative stress after harvest and then decreased thereafter. Environmental stresses during fruit storage, such as water and nutrition deficits, drought, wounds and aging have been reported to stimulate the production and accumulation of ROS (Gill and Tuteja, 2010; Sharma et al., 2012). ROS at low levels produced at early stage of stress is a signal for upregulation of defense system against the environmental stress (Apel and Hirt, 2004; Bhattacharjee, 2012; Sharma et al., 2012) It is possible that the accumulation of ROS in longan pericarp at the beginning of storage (Chomkitichai et al., 2014) activated SOD, CAT and APX activities in first two days of storage (Fig. 1). This indicates that

these enzymes have important roles in scavenging ROS and balancing between the ROS production and antioxidants in longan during storage. CAT showed the most increasing activity among the three antioxidant enzymes (Fig. 1). High activity of CAT might be due to a high level of H2 O2 , a substrate of CAT, generated in longan pericarp during storage (Chomkitichai et al., 2014). Although H2 O2 is also a substrate for APX, this reaction requires ascorbic acid; thus the activity of APX was lower than that of CAT. However, the results showed that prolonged periods of storage caused a decrease in the activities of these three enzymes (Fig. 1). This shows that the ability to scavenge ROS by the enzyme was weakened by oxidative stress due to the over production of ROS such as O2 •− , H2 O2 and OH• during storage and inducing oxidative damage (Chomkitichai et al., 2014). Our results are consistent with Sun et al. (2010) and Duan et al. (2011) who reported that SOD activity declined concomitant with the over production of ROS in lychee fruit throughout the storage time. The increase in activities of SOD, CAT and APX of ‘Daw’ longan fruit during early storage and decreased thereafter are consistent with the results found in ‘Deltastar’ cucumber fruit (Yang et al., 2011), in ‘shixia’ longan (Duan et al., 2007) and in ‘Feizixiao’ lychee fruit (Sun et al., 2010) for SOD, and in ‘Feizixiao’ lychee fruit for APX (Sun et al., 2011). Plants also have non-enzymatic antioxidants which scavenge excessive ROS. They scavenge the ROS by donor electron or hydrogen atom to ROS (Blokhina et al., 2003; Gill and Tuteja, 2010; Sharma et al., 2012). Our results showed that the contents of TPC, ASA, total glutathione and ␣-tocopherol gradually decreased throughout storage (Fig. 2). The levels of ascorbic acid and total glutathione rapidly decreased in early storage (Figs. 2B and C). This indicates that ascorbic acid and glutathione had played more important roles as non-enzymatic antioxidants than TPC and ␣tocopherol which eliminate ROS in longan fruit. TPC can react with O2 •− , H2 O2 , OH• and lipid alkoxyl radicals and functions directly as a free radical scavenger (Sharma et al., 2012). ROS scavenging by APX also requires ascorbic acid and glutathione for the ascorbateglutathione cycle (ASA-GSH cycle) (Arora et al., 2002; Mittler, 2002; Apel and Hirt, 2004; Gill and Tuteja, 2010). ASA is also used to regenerate ␣-tocopherol from the tocopheroxyl radical (Blokhina et al., 2003; Gill and Tuteja, 2010). It is possible that total glutathione (Fig. 2C) and ␣-tocopherol (Fig. 2D) are involved in ROS scavenging resulting in a decrease in ascorbic acid during storage of longan fruit (Fig. 2B), leading to a decrease in APX activity (Fig. 1C). The decrease of non-enzymatic antioxidant during storage of ‘Daw’ longan fruit is consistent with Cheng et al. (2009) who reported that TPC content decreased during storage of post-harvested ‘Shixia’

W. Chomkitichai et al. / Scientia Horticulturae 178 (2014) 138–144

longan fruit. Similar results were reported for ‘Chinese’ lychee and ‘Huaizhi’ lychee fruit during storage (Yi et al., 2010; Duan et al., 2011). In ‘Feizixiao’ lychee fruit, ASA, glutathione and TPC contents decreased during oxidative stress storage (Sun et al., 2010, 2011). You et al. (2012) reported that TPC and ascorbic acid contents decreased during storage of ‘Guilin’ water chestnut slices. TAC by ABTS, DPPH and FRAP assays decreased steadily throughout storage (Fig. 3) as a result of the decrease in both enzymatic antioxidant activity (Fig. 1) and non-enzymatic antioxidant content (Fig. 2). ABTS radical scavenging assay (Fig. 3A) was showed higher TAC than that of DPPH (Fig. 3B) and FRAP (Fig. 3C) and was more similar in capacity to DPPH than FRAP. The continuous decrease in ABTS and DPPH radicals scavenging indicates that ROS scavenging capacity in longan pericarp deteriorates with storage time. The decrease in FRAP also indicates that electron donation capacity or reducing power of longan pericarp gradually lost efficacy. Reducing power and free radical scavenging activity in plants including ABTS and DPPH radicals, superoxide radical and hydroxyl radical are measured from non-enzymatic antioxidant activity (Duan et al., 2007, 2011). TAC and non-enzyme antioxidant contents have similar decreasing trends (Figs. 2 and 3). The relationship between TAC and non-enzymatic antioxidants (r = 0.896–0.982) was higher than that of TAC and enzymatic antioxidants (r = 0.596–0.919) (Table 1). This indicates that high levels of TCA from both non-enzymatic and enzymatic antioxidants correlated with oxidative stress in longan pericarp. The decrease in enzymatic antioxidant activity (Fig. 1), nonenzymatic antioxidant content (Fig. 2) and TAC (Fig. 3) coincided with the onset of longan pericarp browning (Fig. 4). These decreases are consistent with the increases in ROS levels, including O2 •− , H2 O2 and OH• in longan pericarp (Chomkitichai et al., 2014). The over production and accumulation of ROS cause membrane damage in both cellular and organelle membranes, as measured by the electrolyte leakage (EL) rate, lypoxygenase (LOX) activity and the contents of conjugated diene and malondialdehyde (MDA) (Chomkitichai et al., 2014). The decline in the antioxidant defense system led to the development of pericarp browning during storage of longan fruit. It was found that antioxidant defense system was significantly and negatively correlated with BI (r = 0.520–0.900) during storage (Table 1). BI highly correlated with non-enzymatic antioxidant content (r = 0.879–0.900) and TAC (r = 0.862–0.900) and poorly correlated with enzymatic antioxidant activity (r = 0.520–0.757) (Table 1). The relationship between high activity of antioxidant defense system and reduced browning in plants has been reported in ‘Shixia’ longan (Duan et al., 2007; Cheng et al., 2009), ‘Feizixiao’ lychee (Sun et al., 2010, 2011), Chinese lychee (Yi et al., 2010) ‘Guilin’ Chinese water chestnut (You et al.,

143

2012), rambutan (Shao et al., 2012), ‘Longyan Mei’ Japanese apricot (Shi et al., 2013) and ‘Sufid’ loquat (Abbasi et al., 2013) ClO2 fumigation enhanced enzymatic antioxidants of SOD, CAT and APX during storage of ‘Daw’ longan fruit (Fig. 1). Their levels were consistently higher in ClO2 fumigated fruit than those in the control fruit. A statistically significant difference between the fumigated and control fruit was observed for all the three enzymes indicating a link between increased activities of the antioxidant enzymes and decreased levels of ROS and supports that enzymatic antioxidant was increased by ClO2 . These findings are in agreement with earlier observation by Chomkitichai et al. (2014) who hypothesized that reducing ROS levels (O2 •− , H2 O2 and OH• ) might be due to the enhancing enzymatic antioxidant activities stimulated by ClO2 fumigation during storage of longan fruit. These results are also consistent with the study by Wu et al. (2014) who reported that ClO2 enhanced the activities of SOD, CAT and POD during oxidative stress storage of ‘Xiaobai’ apricot at room temperature for 10 days. It is possible that ClO2 might activate the expression of SOD, CAT and APX genes which increased their antioxidative enzyme activities. Similarly, gene expression and activities of SOD, CAT and APX were induced by O3 and salicylic acid (SA) in aspen and sweet cherry (Noormets et al., 2000; Xu and Tian, 2008). ClO2 fumigation maintained a higher content of non-enzymatic antioxidant, viz. TPC, ASA, glutathione and ␣-tocopherol during storage of longan fruit (Fig. 2). ClO2 might promote the biosynthesis of phenolic compounds to relieve the oxidative stress and triggers the acceleration of the antioxidant defense system which is linked to the shikimic acid pathway, leading to the production phenolic of compounds. It is probable that the increase in glutathione by ClO2 contributed to the regeneration of ASA, via the ascorbate–glutathione cycle, causing an increase in ASA content. This increase increases the generation of ␣-tocopherol from the tocopheroxyl radical. Higher TAC by ABTS, DPPH and FRAP assays enhances the antioxidant defense system by ClO2 in longan pericarp. This increase is caused by the increases in enzymatic and non-enzymatic antioxidants during storage of longan fruit by ClO2. An enhanced antioxidant defense system by ClO2 is closely correlated with reduced pericarp browning (Table 1). An activated antioxidant defense system reduces oxidative damage from ROS and retards the reaction between PPO and POD with phenolic compounds leading to a reduction in pericarp browning. Chomkitichai et al. (2014) reported that ClO2 reduced ROS levels and maintained membrane integrity of ‘Daw’ longan fruit and consequently reduced pericarp browning. Reduction in pericarp browning of ‘Daw’ longan fruit by ClO2 resulted from reduced PPO and POD activities by oxidizing amino acids such as histidine and cysteine at the active sites of the enzymes and inhibiting the enzymes to

Table 1 Pearson correlation coefficients of enzymatic and non-enzymatic antioxidants, total antioxidant capacity and browning of longan fruits during storage at 25 ± 1 ◦ C. Trait

SOD CAT APX TPC Ascorbic Glutathione Tocopherol ABTS DPPH FRAP BI * **

r value SOD

CAT

APX

TPC

Ascorbic

Glutathione

Tocopherol

ABTS

DPPH

FRAP

BI

1 0.887** 0.873** 0.894** 0.894** 0.838** 0.913** 0.919** 0.903** 0.844** −0.757**

1 0.862** 0.723** 0.668** 0.594* 0.669** 0.741** 0.728** 0.645** −0.598*

1 0.676** 0.656** 0.577* 0.698** 0.703** 0.716** 0.596** −0.520*

1 0.913** 0.917** 0.979** 0.961** 0.982** 0.974** −0.881**

1 0.968** 0.936** 0.950** 0.919** 0.896** −0.883**

1 0.951** 0.937** 0.926** 0.924** −0.900**

1 0.971** 0.981** 0.974** −0.879**

1 0.977** 0.940** −0.900**

1 0.967** −0.898**

1 −0.862**

1

Significant at P < 0.05 probability level. Significant at P < 0.01 probability level.

144

W. Chomkitichai et al. / Scientia Horticulturae 178 (2014) 138–144

catalyze the browning reaction (Du et al., 2009; Chen et al., 2010; Wu et al., 2011; Saengnil et al., 2014). In conclusion, deterioration of the antioxidant defense system during storage of ‘Daw’ longan fruit causes in pericarp browning. The enhancement of antioxidant defense system induced by ClO2 , alleviated pericarp browning. Acknowledgements This study was financially supported by the Office of the Higher Education Commission, Bangkok, Thailand; Uttaradit Rajabhat University, Uttaradit, Thailand; Faculty of Science and the Graduate School, Chiang Mai University, Chiang Mai, Thailand. References Abbasi, N.A., Akhtar, A., Hussain, A., Ali, I., 2013. Effect of anti-browning agents on quality changes of loquat [Eriobotraya japonica (Thunb.) Lindley] fruit after harvest. Pak. J. Bot. 45, 1391–1396. AOAC, 1990. Official Methods of Analysis, 12th ed. Association of Official Agricultural Chemists, Washington, D.C. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. Arora, A., Sairam, R.K., Srivastava, G.C., 2002. Oxidative stress and antioxidative system in plant. Curr. Sci. 82, 1227–1238. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: the FRAP assay. Anal. Biochem. 239, 70–76. Benzie, I.F.F., Strain, J.J., 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant capacity of biological fluids and modified version for stimultaneous measurement of total antioxidant power an ascorbic acid concentration. Methods Enzymol. 299, 15–27. Bhattacharjee, S., 2012. The language of reactive oxygen species signaling in plants. J. Bot. 2012, 1–22. Blokhina, O., Virolainen, E., Fagerstedt, K.V., 2003. Antioxidant, oxidative damage and oxygen deprivation stress: a review. Ann. Bot. 91, 179–194. Chen, Z., Zhu, C., Zhang, Y., Niu, D., Du, J., 2010. Effects of aqueous chlorine dioxide treatment on enzymatic browning and shelf-life of fresh-cut asparagus lettuce (Lactuca sativa L.). Postharvest Biol. Technol. 58, 232–238. Cheng, G., Jiang, Y., Duan, X., Macnish, A., You, Y., Li, Y., 2009. Effect of oxygen concentration on the biochemical and chemical changes of stored longan fruit. J. Food Quality 32, 2–17. Chomkitichai, W., Chumyam, A., Rachtanapun, P., Uthaibutra, J., Saengnil, K., 2014. Reduction of reactive oxygen species production and membrane damage during storage of ‘Daw’ longan fruit by chlorine dioxide. Sci. Hortic. 170, 143–149. Contreras-Guzmán, E., Strong III, F.C., 1982. Determination of total tocopherols in grain, grain products, and commercial oils, with saponification, and by a new reaction with curic ion. J. Agr. Food Chem. 30, 1109–1112. Deepa, N., Kaur, C., Singh, B., Kapoor, H.C., 2006. Antioxidant activity in some red sweet pepper cultivars. J. Food Comp. Anal. 19, 572–578. Du, J., Fu, Y., Wang, N., 2009. Effects of aqueous chlorine dioxide treatment on browning of fresh-cut lotus root. LWT-Food Sci. Technol. 42, 654–659. Duan, X., Su, X., You, Y., Qu, H., Li, Y., Jiang, Y., 2007. Effect of nitric oxide on pericarp browning of harvested longan fruit in relation to phenolic metabolism. Food Chem. 104, 571–576. Duan, X., Liu, T., Zhang, D., Su, X., Lin, H., Jiang, Y., 2011. Effect of pure oxygen atmosphere on antioxidant enzyme and antioxidant activity of harvested litchi fruit during storage. Food Res. Int. 44, 1905–1911.

Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930. Gronwald, J.W., Fuerst, E.P., Eberlein, C.V., Egli, M.A., 1987. Effect of herbicide antidotes on glutathione content and glutathione S-transferase activity of sorghum shoots. Pestic. Biochem. Physiol. 29, 66–76. Huang, D., Ou, B., Prior, R.L., 2005. The Chemistry behind antioxidant capacity assays. J. Agri. Food Chem. 53, 1841–1856. Jiang, Y.M., Li, Y., 2001. Effects of chitosan coating on postharvest life and quality of longan fruit. Food Chem. 73, 139–143. Khunpon, B., Uthaibutra, J., Faiyue, B., Saengnil, K., 2011. Reduction of enzymatic browning of harvested ‘Daw’longan exocarp by sodium chlorite. ScienceAsia 37, 234–239. Lowry, O.H., Rosebrough, N.J., Far, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410. Munné-Bosch, S., 2005. The role of ␣-tocopherol in plant stress tolerance. J. Plant Physiol. 162, 743–748. Mun’im, A., Negishi, O., Ozawa, T., 2003. Antioxidant compounds from Crotalaria sessiliflora. Biosci. Biotechnol. Biochem. 67, 410–414. Noormets, A., Podila, G.K., Karnosky, D.F., 2000. Rapid response of antioxidant enzymes to O3 -induced oxidative stress in Populus tremuloides clones varying in O3 tolerance. Forest Genet. 7, 335–338. Saengnil, K., Chumyam, A., Faiyue, B., Uthaibutra, J., 2014. Use of chlorine dioxide fumigation to alleviate enzymatic browning of harvested ‘Daw’ longan pericarp during storage under ambient conditions. Postharvest Biol. Technol. 91, 49–56. Shao, Y., Xie, J., Chen, P., Li, W., 2012. Changes in some chemical components and in the physiology of rambutan fruit (Nephelium lappaceum L.) as affected by storage temperature and packing material. Fruits 68, 15–24. Sharma, P., Jha, A.B., Dubey, R.S., Pessarakli, M., 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J. Bot. 2012, 1–26. Shi, T., Li, Z., Zhang, Z., Zhang, C., Gao, Z., 2013. Effect of 1-methylcyclopropene (1MCP) treatment on antioxidant enzymes of postharvest Japanese apricot. Afr. J. Biotechnol. 12, 689–694. Singleton, V.L., Rossi, J.R., 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–157. Sun, D., Liang, G., Xie, J., Lei, X., Mo, Y., 2010. Improved preservation effects of lychee fruit by combining chitosan coating with ascorbic acid treatment during postharvest storage. Afr. J. Biotechnol. 9, 3272–3279. Sun, J., You, X., Li, L., Peng, H., Su, W., Li, C., He, Q., Liao, F., 2011. Effects of a phospholipase D inhibitor on postharvest enzymatic browning and oxidative stress of litchi fruit. Postharvest Biol. Technol. 62, 288–294. Sunohara, Y., Matsumoto, H., 2004. Oxidative injury induced by the herbicide quinclorac on Echinochloa oryzicola Vasing. and the involvement of antioxidative ability in its highly selective action in grass species. Plant Sci. 167, 597–606. Wu, B., Li, X., Hu, H., Lui, A., Chen, W., 2011. Effect of chlorine dioxide on the control of postharvest diseases and quality of lychee fruit. Afr. J. Biotechnol. 10, 6030–6039. Wu, B., Guo, Q., Wang, G., Peng, X., Wang, J., Che, F., 2014. Effects of different postharvest treatments on the physiology and quality of ‘Xiaobai’ apricots at room temperature. J. Food Sci. Technol., In Press. Xu, X., Tian, S., 2008. Salicylic acid alleviated pathogen-induced oxidative stress in harvested sweet cherry fruit. Postharvest Biol. Technol. 49, 379–385. Yang, S., Chen, Y., Feng, L., Yang, E., Su, X., Jiang, Y., 2011. Effect of methyl jasmonate on pericarp browning of postharvest lychees. J. Food Process. Preserv. 35, 417–422. Yi, C., Jiang, Y., Shi, J., Qu, H., Xue, S., Duan, X., Shi, J., Prasad, N.K., 2010. ATPregulation of antioxidant properties and phenolics in litchi fruit during browning and pathogen infection process. Food Chem. 118, 42–47. You, Y., Jiang, Y., Sun, J., Liu, H., Song, L., Duan, X., 2012. Effects of short-term anoxia treatment on browning of fresh-cut Chinese water chestnut in relation to antioxidant activity. Food Chem. 118, 1191–1196.