Artículo científico

 

Effect of storage temperature on enzyme activity and antioxidant capacity in Salvia officinalis L. shoots

 

Efecto de la temperatura de almacenamiento en la actividad enzimática y capacidad antioxidante en brotes de Salvia officinalis L.

 

María de la Luz Romero-Tejeda; María Teresa Martínez-Damián*; Juan Enrique Rodríguez-Pérez

 

Departamento de Fitotecnia, Universidad Autónoma Chapingo. Carretera México-Texcoco km 38.5, Chapingo, México, C.P. 56230, MÉXICO. Correo-e: teremd13@gmail.com, tel.: (595) 95 21 616, ext.: 6163 *(Autor para correspondencia).

 

Received: January 19, 2015.
Accepted: September 23, 2015.

 

Abstract

The aim of this study was to evaluate the effect of low temperatures on antioxidant activity and enzyme activity in sage (Salvia officinalis L.). To do this, terminal buds of this plant, previously packed in polyethylene bags at 0, 6 and 23 °C (control), were stored for 21 days. Every three days, total phenolic content (TP), antioxidant capacity (AC), and the activity of the enzymes catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and polyphenol oxidase (PPO) were evaluated. In the 0 and 6 °C treatments, TP content increased from 4.52 to 5.07 mg·kg^-1 FW up to 12 days after storage (das). A 6 °C, AC had the highest values at 12 das with 53.75 to 88.57 mg VCEAC·g^-1 FW. SOD activity decreased considerably at 9 das in the cooling treatments, falling from 5.2 to 3.2 U·mg^-1 P. On the other hand, low storage temperatures increased CAT activity, relative to the control, although during storage it was on the decrease. Cooling decreased POD and PFO activity relative to the control, but at 12 das the highest activity of these enzymes in the three treatments was observed. The cooling decreased total phenolic content, antioxidant capacity and the enzyme activity of POD, and increased the activity of PFO, CAT and SOD.

Keywords: cooling, catalase, superoxide dismutase, peroxidase, polyphenol oxidase.

 

Resumen

El presente estudio tuvo como objetivo evaluar el efecto de las bajas temperaturas sobre la capacidad antioxidante y actividad enzimática en salvia (Salvia officinalis L.). Para ello, se almacenaron brotes terminales de esta planta, previamente empacados en bolsas de polietileno en 0, 6 y 23 °C (testigo), durante 21 días. Cada tres días, se evaluó el contenido de fenoles totales (FT), capacidad antioxidante (CA), y la actividad de las enzimas catalasa (CAT), superóxido dismutasa (SOD), peroxidasa (POD) y polifenoloxidasa (PFO). En los tratamientos de 0 y 6 °C se incrementó el contenido de FT de 4.52 a 5.07 mg·kg^-1 PF hasta los 12 días de almacenamiento (dda). A 6 °C la CA tuvo los mayores valores a los 12 dda con 53.75 a 88.57 mg AAEVC·g^-1 PF. La actividad de la SOD disminuyó considerablemente a los 9 dda en los tratamientos de refrigeración de 5.2 a 3.2 U·mg^-1 de P. Por otra parte, bajas temperaturas de almacenamiento aumentaron la actividad de la CAT, con respecto al testigo, aunque durante el almacenamiento fue en decremento. La refrigeración disminuyó la actividad de la POD y PFO con respecto al testigo, no obstante, a los 12 dda se observó la mayor actividad de estas enzimas en los tres tratamientos. La refrigeración disminuyó el contenido de fenoles totales, capacidad antioxidante y la actividad enzimática de la POD, y aumentó la actividad de la PFO, CAT y SOD.

Palabras clave: refrigeración, catalasa, superóxido dismutasa, peroxidasa, polifenol oxidasa.

 

INTRODUCTION

Research on natural antioxidants and enzyme activity in plants has increased considerably in recent years, partly because synthetics are volatile, readily degrade at high temperatures and produce negative effects on health (Rodríguez-Vaquero, Tomassini-Serravalle, Manca-DeNadra, & Strasser-De Saad, 2010). For this reason, some herbs, among which the most notable are basil (Ocimum basilicum L.), peppermint (Mentha x piperita L.), oregano (Origanum vulgare) and thyme (Thymus vulgaris), have been studied in order to evaluate the enzymes related to the biosynthesis of different metabolites that have antioxidant capacity in plant tissues (Nickavar, Alinaghi, & Kamalinejad, 2008).

In the case of sage, belonging to the family Labiatae, it is used in gastronomy to flavor dishes and is beneficial to health, as it is considered antidiabetic, antidiarrheal, anti-inflammatory and antiseptic; it is also used in the food, cosmetic and liquor industries (Melero, Moré, & Cristóbal, 2009).

In studies related to antioxidants and enzymes in plants, a key role is the reduction of oxygen (O₂) to water, which occurs sequentially. In this sense, if the O₂ molecule only receives one to three electrons, reactive oxygen species (ROS) are formed. These, in turn, incorporate into the hydrogen peroxide molecule, which has similar chemical properties to those of superoxide and can easily form the highly reactive hydroxyl radicals (Quirós-Sauceda, Palafox, Robles-Sánchez, & González-Aguilar, 2011). ROS play an important role in plant signaling, since they control processes such as growth, development, senescence, response to both biotic and abiotic environmental stimuli and programmed cell death (Bailey-Serres & Mitler, 2006).

Some abiotic changes, such as low and high temperatures, excess irradiation, drought, salinity, injury, anoxia and natural contaminants, modify cellular balance and increase the generation of reactive oxygen species; therefore, the response of a plant tissue to produce these species may result not only from the existence and function of different proteins, but also from the presence or absence of antioxidants in the cell. Reactive oxygen species are closely related to the delay or inhibition of the oxidation of lipids and other molecules, accompanied by the progressive deterioration of plants. It is also linked to the rapid cell growth that causes heart disease and cancer (Rodríguez, Valdés, & Alemán, 2006); this is why the consumption of foods high in antioxidants coupled with other health care-related alternatives can prevent these alterations (Erdemoglu, Turan, Cakõcõ, Sener, & Aydin, 2006).

Generally plants with high antioxidant capacity contain more antioxidants, most of which are phenolic compounds (Connor, Luby, Hancock, Berkheimer, & Hanson, 2002) with the presence of hydroxyl groups, a situation that also occurs in aromatic plants. Specifically in the case of sage, it has been found to contain a high content of polyphenolic compounds that contribute to its strong antioxidant capacity (Bichra, El-Modafar, El-Abbassi, Bouamama, & Benkhalti, 2013).

The antioxidant activity of phenolic compounds is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen extinguishers (Pizzale, Bortolomeazzi, Vichi, Beregger, & Lanfranco, 2005). Therefore, the content of antioxidants is an increasingly important parameter with respect to their role in human health and vegetable quality; therefore, it is of great interest to evaluate their changes during postharvest storage (Ayala, Wang, Wang, & González-Aguilar, 2004).

In this regard, the antioxidant defense systems in plants involve a non-enzymatic system, which promotes the synthesis of many secondary metabolites (phenolic acids, flavonoids, tannins and vitamins) in the phenylpropanoid pathway (Yanishlieva, Marinova, & Pokorny, 2006), and an antioxidant system based on enzyme activity that is located in several cellular compartments, which include the enzymes superoxide dismutase, which converts superoxide to H₂O₂, peroxidase, which converts H₂O₂ to water, and catalase, which eliminates H₂O₂ (Oueslati, Karray-Bouraoui, Attia, Rabhi, Ksouri, & Lachal, 2010).

Added to this, the enzyme activity in fruits and vegetables has served as an indicator of intrinsic metabolic disorders, manifested as external physiological disorders (Pérez, Vargas, Díaz, & Tellez, 1999). This makes it desirable to quantify the activity of various enzymes such as CAT and SOD, which catalyze reactions that lower ROS concentrations, and in some plants they are increased due to the effect of cold during storage (Aquino & Mercado, 2004; Sala & Lafuente, 2000), and peroxidase, which participates in the construction and lignification of the cell wall, the biosynthesis of ethylene and hydrogen peroxide, the regulation of auxin levels, and protection against tissue damage and infection by pathogenic microorganisms. However, postharvest studies of enzymatic and antioxidant behavior in sage plants stored in refrigeration are scarce. Therefore, the aim of the present study was to evaluate the effect of three temperatures (0, 6 and 23 °C) on the antioxidant capacity and activity of the enzymes superoxide dismutase, catalase, peroxidase and polyphenol oxidase during seven storage times in sage terminal buds.

 

MATERIALS AND METHODS

Plant material

The Salvia officinalis commercial variety Extrakta, grown in the spring-summer of 2012 with export quality, provided by the Glezte S. P. R. de R. L. company, located in Axochiapan, Morelos, Mexico, was used. After harvesting sage terminal buds of 20 cm in length, they were precooled for 24 h at 5 °C; subsequently, 250 g of sage were packed in 500-g, low-density, transparent, 40 x 60 cm polyethylene bags, with six holes of 0.5 cm in diameter per side, which remained closed during storage.

 

Experiment location and evaluated treatments

The material was moved to the Fruit Physiology and Multipurpose Laboratories at the Universidad Autónoma Chapingo. The packed sage was stored for 21 days at temperatures of 0 to 6 °C, in a cold-storage room and at 23 °C (control treatment).

 

Experimental unit and design

The experimental unit was a package with 250 g of sage. The experimental design used in each sampling was completely randomized with four replications, in each of which four analytical determinations were performed.

 

Evaluated variables

At each temperature level, four packages were transferred to a freezer (-20 °C) for seven different times: 3, 6, 9, 12, 15, 18 and 21 days of storage.

The following were recorded in each sampling: total phenolic concentration (TP), antioxidant capacity (AC), and enzyme activity of superoxide dismutase (SOD), peroxidase (POD) and polyphenol oxidase (POF). Catalase (CAT) activity was determined from acetone extract and the activity of the other enzymes by acetone powder. The results of the variables are reported based on fresh weight.

 

Extract preparation

Acetone powder was made from the frozen samples (Alia-Tejacal, Colinas-León, Martínez-Damián, & Soto-Hernández, 2005); for this purpose, 80 mL of frozen acetone (-15 °C) were added to 15 g of leaves (without petiole), homogenized in a blender for 25 s and vacuum filtered. After homogenizing and filtering twelve times, the acetone extract was kept under refrigeration (4 ± 2 °C) and the solid was allowed to dry for 15 min at room temperature (23 ± 2 °C), obtaining the acetone powder, which was weighed and stored in a freezer (-20 °C). The weight of the acetone powder was determined according to the ratio: fresh weight of macerated leaves/dry weight of powder.

 

Total phenolic concentration

Phenols were quantified by the Folin-Ciocalteu method described by Waterman and Mole (1994). First, 7.9 mL of deionized water and 0.5 mL of Folin and Ciocalteu reagent (2N) were added to 0.1 mL of acetone extract; the mixture was vigorously stirred and 1.5 mL of sodium carbonate solution (20 %) were added. Then it was allowed to stand for 2 h in darkness; then the samples were read at 760 nm in a spectrophotometer. By means of a standard curve of tannic acid, the results were reported in mg of tannic acid equivalent expressed as fresh weight (mg TAE·kg^-1 FW).

 

Antioxidant capacity

The antioxidant capacity was determined according to the 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method (Ozgen, Reese, Tulio, Scheerens, & Miller, 2006). An ABTS solution (7 mM) with potassium persulfate (2.45 mM) was kept in darkness for 24 h at room temperature; then the solution was diluted with phosphate buffer (0.1 M, pH 7.4) until obtaining an absorbance value of 0.700 nm ± 0.02 in a spectrophotometer (GENESYS™-10UV) calibrated at 734 nm.

The assay of the samples was performed with 2.9 mL of ABTS solution and 0.1 mL of ethanol extract from acetone powder (0.05 g of acetone powder in 5 mL of ethanol, homogenized with 24 h rest); after 2 h, reading was performed at 734 nm. Quantification was done by calibration curve with ascorbic acid, and the values were reported in mg of vitamin C equivalent antioxidant capacity per g of sample expressed in fresh weight (mg VCEAC·g^-1 FW).

 

Enzyme activity of superoxide dismutase (Enzyme Commission [EC] number 1.15.1.1, SOD)

Extraction of SOD was made from 0.05 g of acetone powder, to which 5 mL of Tris-HCl buffer (0.1 M, pH 7.8) were added and homogenized for 20 s; it was centrifuged at 12,000 x g at 4 °C for 30 min and the supernatant was stored. For the enzyme assay, the methodology proposed by Beyer and Fridovich (1987) was used; 27 mL of phosphate buffer (0.05 M, pH 7.8) containing 0.1 mM of EDTA (pH 7.8), 1.5 mL of L-methionine (30 mg·mL^-1), 1 mL of nitro blue tetrazolium (1.41 mg·mL^-1) and 0.75 mL of Triton X-100 at 1 % were mixed. Then 0.5 mL of the supernatant and 0.03 mL of riboflavin (4.4 mg·100 mL^-1) were added to this reaction mixture; the mixture was stirred and illuminated for seven minutes with fluorescent light, and then the samples were read in absorbance at 560 nm. The enzyme activity was reported as units of enzyme activity per mg of protein (U·mg^-1 P), where a U of superoxide dismutase is equal to the amount of supernatant that photo-inhibits 50 % of the formation of nitro blue tetrazolium formazan (Giannopolities & Ries, 1977).

The increase in absorbance due to the formation of nitro blue tetrazolium formazan per unit of time equals the rate of reaction, and the absorbance in the absence or presence of various amounts of superoxide dismutase was used to determine the number of units per ml of superoxide dismutase in the solution (Stauffer, 1989).

Enzyme activity of catalase (EC number 1.11.1.6, CAT)

 

CAT was extracted from 0.05 g of acetone powder and then homogenized with 5 mL of Tris-HCl (0.1 M, pH 8.5) containing 1 % polyvinylpyrrolidone (PVP). The mixture was centrifuged at 12,000 x g for 40 min at 4 °C, and the supernatant was retained. Catalase activity was determined using the method described by Blackwell, Murray, and Lea (1990), for which, in a quartz cell, 3 mL of Tris-HCl buffer (10 mM, pH 8.5) and 0.1 mL of 88 % H₂O₂ were placed. The reaction was initiated by adding 0.1 mL of the supernatant. Finally, the change in absorbance of the samples at 240 nm was observed, by recording readings at 20, 60 and 180 s. The enzyme activity was reported as units of enzyme activity per mg of protein (U·mg^-1 P), where a U is equal to the decomposition of 1 μmol·min^-1 of H₂O₂.

 

Enzyme activity of peroxidase (EC number 1.11.1.7, POD)

POD was extracted from 0.05 g of acetone powder that was homogenized for 10 s, with 5 mL of Tris-HCl (0.1 M, pH 7.1) with 1 % PVP; the mixture was centrifuged at 12,000 x g at 4 °C for 40 min, and the supernatant was saved. For this test, the Flurkey and Jen method (1978) was applied; 2.45 mL of Tris-HCl buffer (0.1 M, pH 7.1), 0.25 mL of guaiacol (0.1 M), 0.1 mL of H₂O₂ at 0.25 % and 0.2 mL of supernatant were mixed. In this mixture, with a total volume of 3 mL, the change in absorbance was determined at 470 nm and sample readings were made at 30, 120 and 180 s. The enzyme activity was reported as units of enzyme activity per mg of protein (U·mg^-1 P), where U is equal to the formation of 1 μmol of tetraguaiacol per minute.

 

Enzyme activity of polyphenol oxidase (EC number 1.14.18.1, PFO)

PFO was extracted with the same procedure used with peroxidase. Polyphenol oxidase enzyme activity was assessed by the method proposed by Laminkanra (1995); 3 mL of catechol (60 mM) dissolved in Tris-HCl buffer (0.1 M, pH 7.1) and 0.2 mL of supernatant were used. With this mixture, the change in absorbance of the samples at 420 nm was evaluated, by recording readings at 20, 60 and 120 s. The enzyme activity was reported as units of enzyme activity per mg of protein (U·mg^-1 P), where U is equal to the formation of 1 μmol of o-benzoquinone per minute.

The protein extracted from the plant, with which the activity of SOD, CAT, POD and POF was reported, was determined by the method described by Bradford (1976), for which 0.05 g of acetone powder were homogenized for 10 s with 5 mL of Tris-HCl (0.1 M, pH 7.1), which had 1 % PVP. This was centrifuged at 12,000 x g for 40 min at 4 °C. Then 0.2 mL of the supernatant was taken and 5 mL of coomassie blue solution were added to it. It was stirred and after 12 min the absorbance at 595 nm was recorded. Quantification was done using a calibration curve with bovine albumin.

 

Statistical analysis

In each of the seven samplings, analysis of variance and Tukey's test (P ≤ 0.05) were performed using Statistical Analysis System, ver. 9.0 software (SAS, 2002).

 

RESULTS AND DISCUSSION

Total phenolic concentration

In relation to total phenolic content, it was observed that at 3, 6 and 15 days of storage, the treatments at 0 and 6 °C showed statistically (P ≤ 0.05) similar values ranging from 4.2 to 4.4 mg TAE·kg^-1 FW; however, the latter was higher than the control value of TAE 4.1 mg·kg^-1 FW, which presented the lowest content of phenols in sage shoots (Table 1). This behavior may be associated with increased antioxidant capacity, since phenols in conjunction with enzymes such as superoxide dismutase and catalase are the first line of defense against oxidative stress induced by low temperatures, keeping the integrity of the cell membrane stable (González, Wang, & Buta, 2011). On the other hand, at 9 and 12 das the three evaluated temperatures were statistically (P ≤ 0.05) different, with the highest amount of phenols occurring at 6 °C, with values of between 4.45 and 4.47 mg TAE·kg^-1 FW. These results are similar to those reported by Martínez-Damián, Cruz-Álvarez, Colinas-León, Rodríguez-Pérez, and Ramírez-Ramírez (2013), in mint (Mentha x piperita L.) stored for 15 days, where total phenolic content was greater at 6 and 10 °C, compared to the control. In this sense, Kevers, Falkowski, Tabart, Defraigne, Dommes, and Pincemail (2007) indicate that the stability of phenolic compounds depends on factors such as the refrigeration temperature, relative humidity and oxygen availabilty; however, products such as tomatillo (Phisalis ixocarpa Brot.), table grapes (Vitis vinifera) and broccoli (Brassica oleracea italica) have showed a slight increase in phenolic compounds during the first days of storage. At 18 and 21 das, the highest values statistically (P ≤ 0.05) for total phenolic content were obtained from plants stored at 6 °C. This behavior was probably because the storage period affected the total concentration of phenols, which under these conditions had an intermediate oxidation state with greater radical-scavenging activity, which promoted the enzymatic oxidation (Kirca & Arslan, 2008).

 

Antioxidant capacity

Between 3 to 9 das the highest antioxidant capacity in sage terminal buds was observed at 23 °C. From 12 das the lowest values were reported under the 0 °C condition (Table 1). In this regard it has been noted that antioxidant capacity and total phenolic content increase when products are handled at high temperatures, results observed by Kevers et al. (2007) in asparagus (Asparagus officinalis L.), spinach (Spinacia oleracea) and celery (Apium graveolens), stored at 20 °C for 22, 19 and 8 days, respectively. By contrast, other fruits and green leafy vegetables when stored at ±4°C such as flamboyant (Delonix regia), curry plant (Helichrysum thianschanicum) leaves and katuk (Sauropus androgynus) (Mathiventhan & Sivakanesan, 2013), or at ± 5 °C like lettuce (Lactuca sativa), and at 10-12 °C in the case of cucumber (Cucumis sativus) and tomato (Phisalis ixocarpa Brot.) (Kevers et al., 2007), where phenol and antioxidant activity levels remained low because the low temperatures slow the metabolic processes of plants (Mathiventhan & Sivakanesan, 2013).

Later, at 15 das, statistical differences (P ≤ 0.05) among the three temperatures used were presented; at 23 °C, antioxidant capacity was higher compared with 0 and 6 °C. Finally at 18 and 21 days, in storage at 6 °C the highest AC values were observed. In these last two assessment dates, the control plants did not have adequate quality to be evaluated. Patthamakanokporn, Puwastien, Nitithamyong, and Sirichakwal (2008) stated that antioxidant capacity depends on total phenolic content; however, antioxidant activity is not limited to phenolic compounds and can be affected by the presence of other antioxidant secondary metabolites such as volatile oils, carotenoids and vitamins (Javanmardi et al., 2003), a situation that agrees with this work at 0 and 6 °C between 6 and 12 das.

 

Enzyme activity of superoxide dismutase

In plants, external factors such as temperature, humidity, light, pathogens and injury induce oxidative stress due to an imbalance in the production of reactive oxygen species. Detoxification of reactive oxygen species in plants depends on the enzyme superoxide dismutase (Huang, Xia, Hu, Lu, and Wang, 2007). This study found that at 12 and 15 das with 0 °C, higher superoxide dismutase activity occurred than at 23 °C (P ≤ 0.05); at 6 and 9 das, this activity was the same at 0 and 6 °C, and higher (P ≤ 0.05) than that at 23 °C. At 3, 18 and 21 das, SOD activity was statistically equal at all storage temperature levels (Table 1).

Subsequently, at 12 and 15 das, the highest enzyme activity of SOD was obtained from samples stored under refrigeration, a situation that prevailed until the last sampling where the highest activity occurred in leaves refrigerated at 0 °C. This can occur because low temperatures confer protection against possible damage to the cell membrane. This is consistent with Cuadra-Crespo and del Amor (2010) and Oliveira, Rufino, Moura, Cavalcanti, Alves, and Miranda (2011), who in evaluating the activity of SOD in sweet peppers (Capsicum annuum var. Annuum) and acerola (Malpighia emarginata), stored at low temperatures and high relative humidity, observed an increase in its activity without damage to their appearance. These results are also consistent with those reported by Martínez-Damián et al. (2013), who stored mint for 15 days and found that at 6 and 10 °C the activity of superoxide dismutase was higher (P ≤ 0.05) (with values between 26.2 and 20.7 U·mg^-1 P, respectively), compared with mint kept at 20 °C (17.89 U·mg^-1 P).

With regard to these results, plant resistance to low temperatures is associated with the joint activity of the enzymes superoxide dismutase and catalase, more than to other physiological factors (Polata, Wiliniska, Bryjak, & Polakovic, 2009), since they are the first line of cellular defense against reactive oxygen species (Ferreira-Silva, Voigt, Silva, Maia, Aragão, & Silveira, 2012). On the other hand, the effect of low-temperature storage is associated with a significant increase in superoxide dismutase activity and decreased senescence, so if this enzyme is active, it may slow the damage caused by plant aging (Balois-Morales, Colinas-León, Peña-Valdivia, Chávez-Franco, & Alia-Tejecal, 2008).

 

Enzyme activity of catalase

Catalase activity (Table 2) showed no differences (α = 0.05) at 3 das, suggesting the stability of the enzyme at the beginning of the experiment. Subsequently and until 15 das, the recorded values of the refrigerated plants (0 and 6 °C) were higher (P ≤ 0.05) than those of the control (23 °C), where conservation at 0 °C was greater, particularly at 15 and 18 das, since in the last evaluations the refrigerated leaves had the same behavior. This increase in catalase activity probably occurred because this enzyme is involved in the defense mechanism against cold stress (Polata et al., 2009).

These results are consistent with those of Duarte-Baquero, Castro-Rivera, and Narváez-Cuenca (2005), who by storing yellow pitaya (Acanthocereus pitajaya) observed an increase in catalase activity at the lowest storage temperature tested (1,300 U·mg^-1 P), unlike what was reported by Balois-Morales et al. (2008) in pithaya (Hylocereus undatus) fruit, where catalase activity showed no association with temperature or cold storage time; moreover, inhibition of catalase activity was partially or completely reversed with storage at 22 ± 1 °C. Also, Oliveira et al. (2011) observed in acerola that catalase activity was minimal, regardless of storage temperature (with values between 0.7 and 0.9 μmolH₂O₂·mg^-1min^-1 P).

This indicates that catalase behavior depends on the species, physiological harvest time and storage time, among other factors. The upward trend in catalase at 21 das, compared to 15 das, suggests that in senescent plants the natural production of free radicals in aging tissue promotes an antioxidant response by increasing catalase activity (Duarte-Baquero et al., 2005).

 

Enzyme activity of peroxidase

At 3 das, there were statistical differences (P ≤ 0.05) among the three treatments assayed (Table 3), with the highest peroxidase activity at 0 °C (39.0 U·g^-1 FW). These results are consistent with those of Martínez-Damián et al. (2013), who found that in mint stored for 15 days that peroxidase activity was higher at 10 °C (306.29 U·g^-1 FW) than at 20 °C (162.50 U·g^-1 FW). The increase in peroxidase at low temperatures is considered a consequence of the system's ability to delay senescence, since it is responsible for catalyzing the decomposition of hydrogen peroxide (Balouchi, Peyvast, Ghasemnezhad, & Saadatian, 2011).

From 6 to 15 das, sage shoots stored at 23 °C had the highest peroxidase activity (P ≤ 0.05), increases that may be associated with the generation of oxidative stress caused by the exposure of plant leaves to unfavorable temperature conditions, so a greater release of peroxidase can be generated in the cytoplasm, which increases its activity (Able, Wong, Prasad, & Ohare, 2005).

In terms of 18 to 21 das, POD activity at 0 °C was higher (P ≤ 0.05) than at 6 °C; there was also an increase from the beginning of storage. These results agree with those of Balouchi et al. (2011), who by storing broccoli florets for 40 days observed that in the last days of evaluation the low temperature (0 °C) resulted in a significant increase in POD activity (with values ranging between 5.4 and 7.2 U·g^-1 FW.

 

Enzyme activity of polyphenol oxidase

In this study, at 3 das at 23 °C the highest polyphenol oxidase activity (21.3 U·mg^-1 P) (Table 3) was observed, compared to the treatments at 0 and 6 °C, which had statistically similar activity. At 12 das, similar behavior was presented, although 6 and 23 °C (with values of 27.1 and 26.3 U·mg^-1 P, respectively) were different (P ≤ 0.05) at 0 °C (18.9 U·mg^-1 P). In this regard, Hassanpour, Khavari-Nejad, Nikman, Najafi, and Razavi (2012) note that polyphenol oxidase is only located in plastids and is released to the cytosol due to damage or the senescence process that is related to plant respiration and which accelerates with high temperatures, generating degradation products which increase the concentration of substrates causing dark stains on the leaves (de Almeida, Durigan, Mattiu, Alves, & O'hare, 2006).

The results at 6, 9 and 15 das indicated that the highest polyphenol oxidase activity was obtained in sage stored at 23 °C, an effect similar to that described by Martínez-Damián et al. (2013), who report that polyphenol oxidase activity increases rapidly with high storage temperatures, so there may be a direct association between the enzyme and increased temperature.

At 18 das, a decrease in this enzyme occurred due to the effect of the low temperature, which was significant (P ≤ 0.05) at 21 das, where storage at 0 °C had the lowest polyphenol oxidase activity. This result is consistent with that of López-Blancas, Martínez-Damián, Colinas-León, Bautista-Bañuelos, Martínez-Solís, and Rodríguez-Pérez (2014), who by storing basil at 10 °C observed that polyphenol oxidase activity decreased after a period of 16 storage days, with values ranging from 31.0 to 15.3 U·mg^-1 P. In this sense, when plant tissues are stressed by factors such as cold, there are changes in the respiratory rate, accumulation of phenolic compounds and variations in polyphenol oxidase activity, which increase as a defense against the cold.

 

CONCLUSIONS

Cooling sage decreased total phenolic content and antioxidant capacity, compared to the control; however, the activity of the enzymes superoxide dismutase and catalase increased. In most of the evaluated times (3, 6, 12 and 15 das), the activity of the enzymes peroxide and polyphenol oxidase was lower at low temperatures (0 and 6 °C), compared to the control (23 °C). Sage storage at 0 °C reduced the activity of enzymes linked to oxidative processes, in most of the storage times studied.

 

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