Lemon verbena (Lippia citrodora) essential oil effects on antioxidant capacity and phytochemical content of raspberry (Rubus ulmifolius subsp. sanctus)

Scientia Horticulturae 248 (2019) 297–304

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Lemon verbena (Lippia citrodora) essential oil effects on antioxidant capacity and phytochemical content of raspberry (Rubus ulmifolius subsp. sanctus)

T

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Shirin Rahmanzadeh Ishkeh, Mohammadreza Asghari, Habib Shirzad , Abolfazl Alirezalu, Ghader Ghasemi Department of Horticultural Sciences, Faculty of Agriculture, Urmia University, Urmia, P.O. Box: 165-5715944931, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Antioxidant activity Raspberry Phenolic compounds PAL enzyme activity Essential oil Shelf life

The aim of this study was to evaluate the effects of Lemon verbena essential oil on antioxidant and enzyme activity and phytochemical contents of raspberry. Lemon verbena essential oil at concentrations of 0, 250, 500, and 750 μl l−1 were used as a fruit edible coating with antioxidant and fungicide properties. The results showed that, in most parameters, there were statistically significant differences between the treatments and control fruits. The findings revealed that the antioxidant activity measured by DPPH method was increased in most treatments during storage and the maximum amount was found in the treated fruits with 500 μl l−1 EO after nine days (85.63%). Also, flavonoid compounds showed some changes similar to antioxidant activity and the maximum amount was 3.97 mg Qu ml-1 extract. The PAL enzyme activity in most of the essential oil concentrations had an upward trend and its activity level changed from 51.17 to 130.78 μmol trans-cinnamic acid min−1. Our results indicated that the use of essential oil coating was effective in enhancing phytochemical contents and to providing a longer storage life with acceptable external and internal qualities in raspberry fruit.

1. Introduction

nitric oxide, jasmonates, salicylates, polyamines, brassinostroiedes, and especially essential oils and extraction of plants (Asghari and Soleimani Aghdam, 2010; Asghari and Zahedipour, 2016). Among various coatings, essential oil has been tested in berries. As secondary metabolites, essential oils are found in aromatic plants which are volatile, natural, and complex compounds. However, they play an important role in protecting the plants via their antibacterial, antiviral, antifungal, and insecticidal functions (Bakkali et al., 2008). EOs1 are secondary metabolites of plants that increase durability in horticulture crops by their antimicrobial and antifungal activities. This ability of EOs is a natural process for reducing the postharvest loss and it is a safe for consumption by humans, which it has led to increase in the intend of different people who use EOs mainly as potential alternative antimicrobials (Vergis et al., 2015). The genus of Lippia is one of the 200 species belonging to the family of Verbenaceae that mainly is distributed in America and Africa (Pascuala et al., 2001). Typically, L. citriodora is used to make taste in food preparations and flavoring beverages (Shahhoseini et al., 2013). L. citriodora is known as an aromatic medicinal plant which has antiviral properties due to containing essential oils and phenolic compounds (flavonoids) (Pascuala et al., 2001). In addition, it has been reported that L. citriodora embraces antispasmodic, antipyretic,

Rubus ulmifolius subsp. sanctus is a member of family Rosaceae and its sub-family is Rosoideae, a bramble native to some parts of Asia and Europe, and it is popular as holy bramble. Berry fruits contain abundant phenol and important pigments of anthocyanin as well as a good source of natural antioxidant vitamins A, B, and C. In addition, it is suitable for treatment of several diseases, particularly diabetes (Kanegusuku et al., 2007). Berries, including strawberries, blueberries, and raspberries, are considered as an important group of commercial fruits. One of the most important shortcomings in postharvest of berries is their susceptibility to water loss, softening, mechanical injuries, and especially postharvest pathogens (such as Botrytis cinerea, Rhizopus sp, Penicillium expansum and Aspergillus niger) (Reddy et al., 2000). Techniques used for controlling pathogens, particularly the use of synthetic fungicides, have led to an increased fungal resistance (Yu et al., 2014) and, at the same time reduced the water loss and soften the use of modified atmospheres with high CO2, cooling, heat and osmotic treatments, irradiation, and edible coatings (Velickova et al., 2013). Edible coatings include gamma-aminobutyric acid (GABA), DL-β-aminobutyric acid (BABA), chitosan,

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Corresponding author. E-mail address: [email protected] (H. Shirzad). 1 Essential oils. https://doi.org/10.1016/j.scienta.2018.12.040 Received 1 October 2018; Received in revised form 11 December 2018; Accepted 21 December 2018 Available online 12 January 2019 0304-4238/ © 2018 Elsevier B.V. All rights reserved.

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distilled water, EO was mixed by Tween 80 (0.5%, v/v) due to their oily nature and their insignificant solubility in water. Then, 60 g fruits per unit experiment were dipped into the essential oil emulsion at a desired concentration for three minutes. Then, the fruits were put into the sterilized containers and covered with parafilm to prevent air. Finally, all fruits were transferred to a cold storage with a temperature of 4 ± 1 °C and relative humidity of 90–95%. Before measuring the traits such as total phenolic content, total flavonoid content, total anthocyanin content, antioxidant activity (DPPH and FRAP) and enzymatic activity (PAL and guaiacol peroxidase), fruits were washed twice with distilled water.

sedative, and digestive properties (Pascuala et al., 2001). Previous research has shown that essential oil of its leaves exhibit antimicrobial activity. Neral, 1,8-cineole, geraniol, and limonene are the most important compounds of L. citrodora and they are monoterpens naturally. Because of high percentage of L. citrodora’s essential oil, neral, it is believed to have high antioxidant and fungicidal properties. Nevertheless, the antifungal activity of neral against several postharvest pathogens has been well documented in in-vitro trials (Neri et al., 2009). Postharvest treatment of fruits with Neral showed a low degree of efficacy in controlling the blue mold and brown rot (Neri et al., 2009), however it failed to control lenticel rot (Neri et al., 2009). Due to the susceptibility of raspberry fruit to all types of postharvest damages and, as a cause for its low storage life, for the necessity of application of postharvest treatments, especially safe and natural treatments such as essential oils, is undeniable. Therefore, the objectives of this study was to evaluate the effects of various concentrations of the essential oil extracted from L. citriodora on raspberries during postharvest cold storage and observation of phytochemical contents, enzymatic activities and increasing the shelf life of raspberries. Moreover, the study aimed to get a better understanding of the effects of essential oil to maintain the valuble compounds of the fruits for human health".

2.4. Chemical analysis At first, a number of fruits were squeezed with a hand press (Schieber et al., 2001), and the juice was used to assay three factors, namely TA2, TSS3, and pH. TSS was determined using a refractometer (AZ 8601). TA was evaluated by diluting each 2 ml aliquot of raspberry juice in 20 ml of distilled water and titrated to pH 8.2 using 0.1 molars NaOH. It should be noted that pH was measured by pH meter (pHMeter CG 824). 2.5. Panel test

2. Materials and methods Panel test was performed by 10 panelists and, average points for each test were reported. Based on the panelists’ report, the samples were selected which were odor, flavor, and marketable. Score 1 was given to the highest quality and score 10 for the lowest quality. Measurements were repeated three times on days 3, 6, and 9 after the storage. Overall quality (percentage of decaying, shrinking and adverse effects in fruit surface) was identified as the fruit marketability index and evaluated by 10 trained panelists using a 1–10 scale. In that scale, different scores were assigned to various qualities including 1-2 = excellent (no decay, shrink, or any other adverse effects on fruit surface), 3-4 = good (up to 5% surface affected), 5-6 = acceptable (5–20% surface affected), 7-8 = bad (20–50% surface affected), and 910 = unacceptable (> 50% surface affected). Results were shown in terms of overall quality indices.

2.1. Fruit Raspberry fruits normally grow in Khandaracy region (Longitude: 45° 07’ 09’’, Latitude: 37° 19’ 16’’ and 2392 m above sea level) and are in the form of shrubs, near the wild river, Barandoschay, Urmia, Iran. Fruits were harvested at commercial maturity stage with similar color, shape, and size, also fruits with no defects were selected. In this study, the process of harvesting was conducted in onset morning, and then the fruits were transferred into cold storage at the laboratory of Horticulture Department, Urmia University. Cold storage temperature was 4 ± 1 °C and 60 g fruits were used for each experimental unit. 2.2. Essential oil isolation In order to extract L. citriodora essential oil, the dried leaves (30 g; in the room temperature and darkness) were used for water distillation using a Clevenger apparatus for three hours. EO was kept in a vial at 4 °C and darkness conditions before analysis. Gas chromatography–mass spectrometry (GC–MS) analyses were performed on a Thermo Finnigan capillary gas chromatograph directly joined to the mass spectrometer system (model Trace GC/Trace MS Plus system). A non-polar fused silica capillary column (HP-5 ms; 30 m × 0.250 mm i.d., film thickness 0.25 μm) was used. The GC Oven temperature was held at 40 °C for 2 min and then was increased to 160 °C with the temperature rate of 3 °C/min and finally increased to 280 °C at a rate of 5 °C/min for 2 min. Injector temperature: 280 °C, ion source: 200 °C and interface temperature: 260 °C. The carrier gas was helium at a flow rate of 1 ml/min, and ionization energy was 70 eV. 1 μl of EO is solved in diethyl ether (2 μl of the oil in 2 ml solvent) was injected via a split injector (1:20). Retention indices (RI) were calculated based on the injected n-alkenes (C6–C24) with same experimental conditions. Compounds were identified by comparison of their RI with those reported in the literature (Adams et al., 2007) and mass spectra of compounds were obtained using X-Calibur (2.07) with its available libraries. The percentages of compounds were calculated by the area normalization method, without considering the response factors.

2.6. Fungal decay measurement This index was visually inspected on days 3, 6 and 9 after storage at a temperature of 4 °C. All fruits were classified in ten scores by the extent of decay: 1-2 = excellent (no decay), 3-4 = good (up to 5% decay), 5-6 = acceptable (5–20% decay), 7-8 = bad (20–50% decay), and 9-10 = unacceptable (> 50% decay). At this stage, the index was used by 10 panelists. Based on their ratings, the average was calculated. Through this method, the researchers rank the samples by scores. 2.7. Total phenolics content (TPC) To assay total phenol content, colorimetric determination by FolinCiocalteu phenol reagent was used by brief modifications (Slinkard and Singleton, 1977). The juice was centrifuged at 11,000 × g for 15 min at 4 °C. Then, 30 μl concentered extract of fruit juice was mixed with 180 μl of distilled water. Later, 1200 μl of 10% folin was added to the solution and was kept for 5–10 min. Then, 960 μl of 7.5% sodium carbonate (Na2CO3) was added to this solution and placed in darkness for 30 min. Finally, using gallic acid as a standard, the color change of the extracts was calorimetrically determined at a wavelength of 760 nm by UV–vis spectrophotometer. The data are expressed as milligram gallic acid equivalent per ml fruit extract (mg GAE/ml fruit juice).

2.3. The process of applying the treatments After preparing different concentrations of EO (0, 250, 500 and 750 μl l−1) of L. citrodora, due to its lipophilic nature and insolubility in

2 3

298

Titratable Acidity. Total Soluble Solids.

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Dickerson et al. (1984). A 30 μl sample of the extract enzyme was incubated in 0.5 ml of 0.1 M borate buffer with pH 8.8 containing 0.5 ml of 12 mM L-phenylalanine for 30 min at 30 °C. The optical density (O.D) was recorded at 290 nm wavelength and the amount of trans-cinnamic acid was calculated using its extinction coefficient of 9630 M−1. Enzymatic activity was reported as μmol trans-cinnamic acid min−1.

2.8. Total flavonoid content (TFC) TFC was determined using a colorimetric assay (Shin et al., 2007). At first fifty μl of fruit juice was mixed with 150 μl 5% sodium nitrite (NaNO2) and was kept at room temperature for 5 min. Next, it was mixed with 300 μl of 10% aluminium chloride (AlCl3) and was kept for 5–10 min. Then, 1 ml of 1 mol l−1 sodium hydroxide was added to the solution and was brought to 5 ml volume with adding distilled water. The absorption mixture was read in 380 nm wavelength. The results are expressed as quercetin equivalents using a standard curve prepared from authentic quercetin. The TFC amount of fruit juice is reported according to mg quercetin equivalent per ml fruit juice (mg Qu/ml fruit juice).

2.12. Guaiacol-peroxidase activity (GPX) Guaiacol-peroxidase activity was assayed according to Roy et al. (1996). The reaction mixture consisted of 200 μl centrifuged fruit juice with 200 μl guaiacol solution (22 mM) and100 μl H2O2 (100 mM). The final mixture included 1 ml of the final volume with 125 mM potassium–phosphate buffer (pH = 7.0). Tetraguaiacol concentration in the reaction mixture was measured spectrophotometrically using UV–vis at 470 nm (ε = 26.6 mM−1 cm−1).

2.9. Total anthocyanin content assay (TAC) A total anthocyanin content in fruit juice was determined by pH differential method (Wrolstad, 1976). For this purpose, the fruit juice was centrifuged at 11,000 × g for 15 min at 4 °C, and, then, 100 μl of a supernatant was used. The absorption was measured via UV–vis spectrophotometer at 530 and 700 nm wavelengths of buffers containing 1.0 and 4.5 pHs based on A = [(A530 − A700) pH1.0 − (A530 − A700) pH4.5] with molar extinction coefficients of cyanidin-3-O-glucoside for raspberry juice. The results were expressed as mg of cyanidin-3-Oglucoside equivalent to 1 ml−1 of fruit juice.

2.13. Statistical analysis This experiment was carried out based on a completely randomized design (with 3 concentrations of EO and 3 storage durations) with four replications (each replication included 60 g fruits). One-way ANOVA was run for analyzing the data (SAS, version 9.1.3). The Friedman Test based on randomized complete block design was carried out for panel test and decay data analysis. The means of scores were compared using Duncan’s Multiple Range Test. Differences at p < 0.01 were considered significant.

2.10. Antioxidant capacity

3. Results

2.10.1. Antioxidant capacity assay using DPPH measurement Free radical scavenging activity of centrifuged fruit juice was measured based on the principle introduced by Nakajima et al. (2004) with brief changes. 10 μl of the prepared fruit juice was added to 2000 μl of 6 × 10−5 mol l−1 (95% free radical) methanol. The mixture was perfectly mixed and left at a room temperature for 30 min. Then, the absorbance level was measured at 517 nm wavelength using UV–vis spectrophotometer. In order to prepare the control materials, the same method was implemented using 80% methanol that was calculated based on the following formula:

DPPHsc % =

(Abs

control)t = X (Abs

− (Abs sample)t = X control)t = X min

min

min

3.1. Essential oil composition The results of the gas chromatography revealed that the lemon verbena essential oil contains 45 different compositions, major compositions identified include, D-limonene (25.26%), (−)-spathulenol (14.74%), α-curcumene (7.98%), neral (4.16%) and isocaryophyllen (4.12%). All chemical compositions of L. citrodora are shown in Table 1. 3.2. Chemical analysis

× 100 The data analysis showed that the level of titratable acidity changed from 0.84 to 1.42% fruit juice (p < 0.01). Besides, the amount of TSS and pH was influenced by essential oil treatment (p < 0.01). The maximum amount of TSS and pH were observed in ninth day of control fruits and the minimum in the fruits treated with 750 μl l−1 EO on the third-day (Table 2).

Abs control: Absorption rate. Abs sample: Sample absorption rate. 2.10.2. Antioxidant capacity through FRAP assay Antioxidant capacity was measured using FRAP method according to the method described by Asghari and Hsanlooe (2015). In this method, 50 μl centrifuged fruit juice, 3 ml fresh reagent of FRAP [0.3 M acetate buffer pH 3.6, 0.01 M TPTZ (2, 4, 6-tripyridyl-s-triazine) in 0.04 M HCl, and 0.02 M FeCl3 6H2O (10:1:1, v/v/v)] were mixed. The resulted mixture was put in a dark place and hot water bath (37 °C) for 30 min. Next, the absorbance level was measured using a UV–vis spectrophotometer in 593 nm wavelength toward control. The findings were calculated using a standard curve of FeSO4 and expressed as mmol Fe2+ 1000 ml−1 fruit juice.

3.3. Panel test There were significant differences between treatments and control (p < 0.01). The fruits which were treated with 750 μl l−1 EO showed the most desirable taste three days after the storage, while those were in the control group manifested the least desirable taste after nine days (Table 2). These results can be described for coating role of essential oil that increased fruit shelf life after the harvest by acting as a barrier against gases resulting in maintaining the moisture and decreasing oxygen levels in the fruit. In the form of a thin layer, the essential oil covers the surface of fruit and controls gas flow. Thus, respiration and ethylene production decrease and the senility speed are delayed and this causes the maintenance of fruit quality as reported by Sivakumar and Bautista-Banos (2014).

2.11. PAL enzymatic activity PAL activity was assayed according to the method used by Karthikeyan et al. (2006) with a brief modification. First, fruits samples (0.5 g) were homogenized in 1.5 ml of ice-cold 0.1 M sodium borate buffer with pH 0.7, 1.4 mM 2-mercaptoethanol, and 0.1 g insoluble polyvinyl-pyrrolidone. The extract was filtered through cheesecloth and was centrifuged in 12,000 × g at 4 °C for 15 min. The supernatant was used as the enzyme source. The PAL activity was considered as the rate of conversion of L-phenylalanine to trans-cinnamic acid, as described in

3.4. Measurement of fungal decay Raspberry fruits are believed to be highly putrefying due to their high metabolic activity, water content, and susceptibility to microbial 299

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oil and the total phenol content of the samples had significant effects on aptness level, up to 1% (p < 0.01) (Fig. 2A). Comparing the average points, the highest amount was observed in the treated fruits by EO (250 μl l−1) on 9th day. In contrast, the lowest TPC was identified in control fruits after 9 days of storage. Moreover, the amount of phenolic compounds in the fruits that were treated with the highest concentration of EO (750 μl l−1) declined over time. However, there isn't a significant difference between samples on days 6 and 9. This can be due to coating effect of EO that leads to a lower exposure of the samples to temperature variations and, consequently, a reduction in the enzymatic activity. But Reducing total phenol content on days 6 and 9 than on day 3 in the EO (750 μl l−1) during storage can be due to the toxic effects of higher EO concentrations on the cells leading to the breakdown of cellular structure due to aging and the enhanced activity of the polyphenol oxidase enzyme (Hussain et al., 2016).

Table 1 Chemical constituents of the essential oil extracted from Lemon verbena (L. citrodora). No.

Compounds

Empirical formula

RI

Percentagea

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

3-Thujene α-Pinene Sabinene 5-Hepten-2-one, 6-methyl( ± )-β-Pinene 4-Carene D-Limonene 3-Carene cis-Sabinenehydrate Linalool Limonene oxide, transLimonene epoxide α-terpineol Neral Geranial γ-Muurolene Copaene β-Bourbonene Germacrene D (Z,E)-Farnesyl acetate (E,E)-Farnesyl acetate (−)-α-Cedrene Isocaryophyllene γ-Cadinene α-cubebene α-Caryophyllene Alloaromadendrene Di-epi-α-cedrene β-Cadinene γ-Cadinene Germacrene D α-Curcumene Isoledene Ylangene α-Cadinene δ-Cadinene Caryophyllene oxide α-Nerolidol (−)-Spathulenol 6-Isopropenyl-4,8a-dimethyl1,2,3,5,6,7,8,8a-octahydronaphthalen-2-ol Cedrol Caryophyllene oxide τ-Cadinol Calarene epoxide Bis(2-ethylhexyl) phthalate

C10H16 C10H16 C10H16 C8H14O C10H16 C10H16 C10H16 C10H16 C10H18O C10H18O C10H16O C10H16O C10H18O C10H16O C10H16O C15H24 C15H24 C15H24 C15H24 C17H28O2 C17H28O2 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H22 C15H24 C15H24 C15H24 C15H24 C15H24O C15H26O C15H24O C15H24O

918 927 955 985 964 1022 1005 991 1064 1082 1134 1133 1144 1235 1264 1466 1347 1339 1461 1812 1812 1407 1387 1494 1340 1422 1430 1385 1518 1495 1451 1461 1373 1347 1533 1503 1517 1533 1572 1714

0.52 2.36 3.2 0.48 0.26 0.49 25.26 0.21 0.48 0.43 0.16 0.23 1.75 4.16 2.72 0.3 2.1 1.32 1.25 0.14 0.33 0.87 4.12 0.36 0.41 0.36 2.2 0.3 0.42 0.47 2.03 7.98 0.57 0.44 0.26 1.29 1.32 1.34 14.74 3.27

C15H26O C15H24O C15H26O C15H24O C24H38O4

1589 1565 1623 1620 2519

0.32 2.11 2.88 1.66 1.56

41 42 43 44 45

3.6. Total flavonoid content (TFC) This study showed that the total flavonoid content in the treated fruits increased during the storage period. The highest was for the treated fruits with 750 μl l−1 EO in ninth day of storage duration and the lowest amount was observed in control fruits after 9th days of storage duration (Fig. 2B). Specifically, flavonoids are known as antioxidants and extremely beneficial to human health. These compounds contain hydroxyl groups that can deactivate free radicals in plants (Pourmorad et al., 2006). 3.7. Total anthocyanin content (TAC) In this study, as an indicator of anthocyanin content of samples, cyanidin 3-glucoside was evaluated and the changes of anthocyanin concentrations were assayed for the treated and control fruits during the storage. As shown in Fig. 2C, the amounts of anthocyanin in treated and control fruits differ significantly (p < 0.01). Anthocyanin content in control raspberries showed a decreasing trend during 9 days of cold storage, while it increased in the treated fruits after 3, 6, and 9 days of cold storage in most cases. The increase in anthocyanins in this study is probably due to the ripening of the fruits, and the increased amounts of sugars and enzymatic activity of phenylalanine amonyaliaz during the storage (Vargas et al., 2006). However, on the harvest day, the control fruits contained less anthocyanin because they have not any coating. It must be considered that changes in the amount of anthocyanin depend on the fruit type and composition index (Pourmorad et al., 2006). 3.8. Antioxidant capacity

a

GC, Gas chromatography; MS, Mass Spectroscopy; RI, retention index determined on HP5-MS column.

3.8.1. Antioxidant capacity measurement using DPPH method The measurement of antioxidant activity using DPPH method showed that its activity level changed from 50.99 % to 85.63 %. Antioxidant activity was decreased in control fruits during storage but the treated fruits showed a high antioxidant activity at the end of storage duration. The maximum and minimum levels of antioxidant activity were found in fruit treated eith 500 μl l−1 EO and in control fruits after nine days, respectively (Fig. 3A).

molds and rots (Hassanpour, 2014). The findings showed that there were statistically significant differences between the treatment and control (p < 0.01) in terms of decay extensionduring cold storage (Fig. 1). The most favorable results were related to the fruits treated with 750 μl l−1 EO, on third day of storage. On the other hand, the unfavorable results belonged to control fruits after 9 days of storage (Table 2). Due to the presence of phenolic compounds such as neral, linalool, and α-terpineol (De Corato et al., 2010), EOs possess fungicidal properties. These compounds play a key role in the resistance of plants against pathogen attacks which, in turn, lead to a slight stress and enable defense mechanisms in plants (Sivakumar and BautistaBanos, 2014).

3.8.2. Antioxidant capacity measurement by FRAP method In this research, the antioxidant activity was also measured through FRAP method and it shows that its amount decreased during storage days. Also the results of this study revealed that the use of essential oil of L. citrodora as an edible coating could reduce the damage caused by reactive oxygen species (ROS) with increasing the antioxidant activity by increasing the essential oil concentration. The utmost antioxidant activity was observed in the fruits treated with 750 μl l−1 EO within the third day with 134.50 mmol Fe2+ 1000 ml−1of the fruit juice. On the other hand, the least amount of antioxidant activity was identified in the control fruits on the sixth day with 82.36 mmol Fe2+ 1000 ml−1 of

3.5. Total phenol content (TPC) The results revealed that TPC in raspberry was affected by the storage duration and different concentration levels of essential oil of L. citrodora. The analyses showed that the interaction between essential 300

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Table 2 Effect of different Lemon verbena (L. citrodora) essential oil treatment on the quality attributes of raspberry (R. ulmifolius subsp. sanctus) at harvest and during storage at 4 ± 1 °C. Treatments

Chemical analysis and Quality attributes

EO μl l−1

Days

TA

Harvest day 0 250 500 750 0 250 500 750 0 250 500 750

0 3 3 3 3 6 6 6 6 9 9 9 9

1.31 1.24 1.37 1.37 1.42 1.25 1.27 1.30 1.35 0.84 0.98 0.99 1.09 **

TSS ± ± ± ± ± ± ± ± ± ± ± ± ±

0.07 0.03b 0.05ab 0.13ab 0.11a 0.11b 0.05b 0.05ab 0.09ab 0.08d 0.03c 0.06c 0.07c

12.06 14.77 13.97 13.67 12.93 14.38 13.27 13.03 13.03 17.33 16.40 16.00 15.60 **

pH ± ± ± ± ± ± ± ± ± ± ± ± ±

0.39 0.30 cd 0.38def 0.62def 0.73f 0.82de 0.33ef 0.81f 0.77f 0.30a 0.46ab 0.31b 0.24bc

3.23 3.85 3.75 3.67 3.27 3.85 3.73 3.70 3.67 3.97 3.90 3.89 3.89 **

± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 0.06ab 0.01bc 0.12c 0.21d 0.06ab 0.06bc 0.03c 0.03c 0.02a 0.08a 0.05a 0.03a

Panel test

Decay

1.00 4.06 3.80 3.80 3.08 6.85 6.68 7.08 6.43 9.00 8.90 8.85 8.30 **

1.00 4.90 3.50 3.36 3.06 6.36 6.00 6.16 5.06 9.56 8.86 8.70 8.48 **

± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.08gh 0.04hi 0.10hi 0.05i 0.06c-e 0.03d-f 0.63ef 0.02fg 0.00a 0.00ab 0.02bc 0.47b-d

± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.04gh 0.00hi 0.02ij 0.02j 0.06de 0.04bef 1.11ef 0.02fg 0.02a 0.02ab 0.04bc 0.01cd

Data obtained from four replications, mean ± Standard error. 2 2 ChiDecay = 41.70 and ChiPanel test = 41.05. **, * and ns are significant at the 1% and 5% level and non-significant respectively (Duncan’s Multiple Range test).

2016). Given et al. (1988) showed that accumulation of anthocyanins in ripening strawpberry fruit requires high PAL activity. In the present study, essential oil as an edible coating with high activity of the enzyme PAL has increased the accumulation of phenolic compounds. In the other words, the amount of flavonoids and anthocyanins have been correlated with the level of PAL activity (Fig. 4A). 3.10. Guaiacol-peroxidase enzyme activity (GPX) According to Fig. 4B, guaiacol-peroxidase enzyme activity varied based on treatment types and comparatively, its highest amount belonged to 250 μl l−1 EO on day three, while the lowest amount was observed in 500 μl l−1 EO on the ninth day. The results were significant at (p < 0.01). 4. Discussion According to our findings, the amount of acid decreased in the fruits during the storage process, especially in low-temperature rooms. It can be postulated that the acid is broken down into sugar during fruit respiration and its amount decrease gradually. Of course, in some studies, enzymatic activities were observed during fruit juice storage, which can be one cause of acidity reduction in fruits (Vargas et al., 2006). In simple terms, organic acids are consumed by the process of respiration to support the normal activities occurring in a product during storage (Gao et al., 2013). Furthermore, edible coatings can retard the respiration and reduce the enzymatic reactions involved in decomposing organic acids (Amal et al., 2010). As a natural edible coating that is capable of creating a modified atmosphere around the fruit and maintaining CO2 at a higher level, EO reduces respiration and produces ethylene, which in turn decreases the consumption of organic acids (Baldwin et al., 1995). The percentage of soluble solids is one of the important quality indicators in the products. This index can be changed during fruit ripening in the postharvest period by hydrolysis of the polysaccharides and concentrating of the fruit extract as well as a reduction in the associated juice (Vargas et al., 2008). The findings of this study showed that the amount of soluble solids had downward trend when EOs were used and this is consistent with results of Abdolahi et al. (2010). Other important characteristics in determining the quality of fruits are pH of fruit extract which changes into smaller units during respiration, and protein hydrolysis and glycosaccharides decomposition processes (AKhtar et al., 2010) due to the breakdown of carbohydrates and

Fig. 1. Effect of different Lemon verbena (L. citrodora) essential oil treatment on the fungal decay of raspberry (R. ulmifolius subsp. sanctus) fruit stored for 9 days at 4 ± 1 °C. Control refers to untreated raspberries.

the fruit juice (Fig. 3B).

3.9. PAL enzyme activity Our results showed that, in most cases and compared to the control fruits, the amount of PAL enzyme activity in the treated fruits increased during the storage (p < 0.01). Its activity level changed from 51.17 to 130.78 μmol trans-cinnamic acid min−1. High PAL activity is associated with the accumulation of phenolic compounds such as flavonoid in fruits, which is a group of phenolic compounds. It was reported that fruit tissue has several phenolic compounds that have severe antioxidant activity (Asghari and Soleimani Aghadam, 2010; Velickova et al., 2013; Asghari and Hasanlooe, 2015; Asghari and Zahedipour, 301

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Fig. 2. Effect of essential oil treatment (at 0, 250, 500, and 750 μl l−1) on (A) total phenolics content, (B) total flavonoid content and (C) total anthocyanin content of raspberries stored for 9 days at 4 ± 1 °C with 90–95% relative humidity. Control refers to untreated raspberries. The data shown are the mean ± standard error of four replicates. Different letters indicate statistical significance (p < 0.01).

transform phenylalanine to trans-cinnamic acids with the help of PAL enzyme as a key element in the phenylpropanoid pathway (PérezBalibrea et al., 2011). In previous studies, the investigation of EO effects on PAL enzyme activity showed that it can play a significant role in enzymatic activities; it reported that the application of tea tree EO on strawberry enhanced the activity of PAL enzyme that was 50% higher than control (Shao et al., 2013). This increase in activity also was observed in our study, and these results were partly consistent with the findings of Shao et al. (2013). Due to their biodegradability, environmental compatibility, cost-effectiveness, and non-toxicity and antifungal, anti-microbial, and antioxidant properties, natural products such as EOs are ideal organic options for use in postharvest technology. Various components of EOs can be effective and useful in controlling microbial growth (Sharma and Tripathi, 2006). Previous observations have concluded that EO treatments enhance antioxidant capacity in berry fruits. For example, strawberries that were treated with eugenol

pectin. The increase in pH of extracts during storage seems to be due to biochemical changes in the fruit such as the decomposition of organic acids into sugars as well as their participation in the respiration cycle (Shin et al., 2007). As edible coatings, EOs reduce gas exchange and prevent decomposition of carbohydrates by reducing the amount of respiration. In addition, the phenolics in the EOs help to reduce respiration and produce ethylene that leads to a decline in metabolic processes and pH maintenance (Baldwin et al., 1995). Also this study demonstrated that EO treatments enhanced the amount of phenolic compounds such as phenolics, flavonoids, anthocyanins, and antioxidant capacity in the fruits. As a new method to control postharvest diseases, the use of plant essential oils is proposed for storage duration of products. Moreover, these compounds increase the antioxidant properties, quality, and shelf life in the fruits (Sellamuthu et al., 2013). In fact, Shikimate-phenylpropanoid-flavonoids pathways are suitable for biosynthesis of phenolicss and flavonoids in plants, catalyzers

Fig. 3. (A) and FRAP (B) method. Effect of essential oil treatments (at 0, 250, 500, and 750 μl l−1) on (A) Antioxidant capacity measured by DPPH and (B) Antioxidant capacity measured by FRAP methods, in raspberries fruit stored for 9 days at 4 ± 1 °C and 90–95% relative humidity. Control refers to untreated raspberries. The data shown are the mean ± standard error of four replicates. Different letters indicate statistical significance (p < 0.01). 302

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Fig. 4. Effect of essential oil treatment (at 0, 250, 500, and 750 μl l−1) on (A) PAL enzyme activity and (B) guaiacol-peroxidase enzyme activity in raspberries stored for 9 days at 4 ± 1 °C and 90–95% relative humidity. Control refers to untreated raspberries. The data shown are the mean ± standard error of four replicates. Different letters indicate statistical significance (p < 0.01).

and thymol showed higher contents of anthocyanin and flavonoids and free radical scavenging capacity (Wang and Xu, 2007). Furthermore, those fruits treated by a high concentration of EO of L. citrodora displayed a reduced decay and an increase in taste. This can be due to the antimicrobial role of D-limonene that is known as generally recognized as safe compound (GRASC) for food preservation and or it can be related to antibacterial activity of isocaryophyllene (Shahhoseini et al., 2013). The findings of the panel tests indicated that the treated fruits had the highest taste and flavor rates and lowest decay index. This is attributable to phenolic compounds of EOs which have antifungal properties and play a key role in the resistance of plants against attacks of pathogen (Asghari and Soleimani Agdam, 2010). Activeness of pathogens engenders a small stress leading to a defensive mechanism in plants. For example, when plants are attacked, it induces the least amount of decay, despite of producing higher levels of tase and flavor (Sivakumar and Bautista-Banos, 2014). Due to the existence of various antimicrobial compounds of EO, one cannot certainly specify the causes of these changes, because several complex antimicrobial mechanisms could presumably activate them (Burt, 2004). As an eadible coating formed on the crop surface, although EOs reduce gas exchange and respiration, however, their antimicrobial properties control fungal decays in fruits such as raspberry and peach (Montero-prado et al., 2011). This property decreases microbial decay and decomposition of crops (Montero-prado et al., 2011).

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