VARIETAL DEPENDENCE OF CHEMOPROTECTIVE SUBSTANCES IN FRESH AND FROZEN SPINACH ( SPINACIA OLERACEA L . )

Spinach (Spinacia oleracea L.) is an important source of bioactive compounds. It is commonly consumed fresh or frozen products. Spinach is rich sources of polyphenols, it is a good source of vitamin C and has potential beneficial properties for human health. This study provides some knowledge about content of total polyphenols, and antioxidant activity in selected varieties of fresh and frozen spinach samples. Four spinach cultivars ('Boa', 'Hudson', 'Chica', 'Trombone') were analysed. The content of the total polyphenols (TPC) was determined by the Folin-Ciocalteu reagent (FCR). Antioxidant activity (AA) was measured using a compound DPPH (2,2-diphenyl-1-picrylhydrazyl). The content of total polyhenols in fresh samples of spinach ranged from 975 ±97.15 mg.kg to 1493 ±50.42 mg.kg and values of antioxidant activity were in interval from 77.55 ±0.34% to 82.57 ±0.83%. The highest level of TP content in fresh spinach was recorded in variety 'Hudson' (1493 mg.kg) and the lowest in variety 'Chica' (975 mg.kg). Between these varieties statistically significant difference in the content of total polyphenols was found. The highest value of antioxidant activity in fresh spinach was recorded in variety 'Trombone' (82.57%) and the lowest in variety 'Boa' (78.59%). This difference was also statistically significant. The highest level of TP content in frozen spinach samples was found in variety 'Hudson' (1749 mg.kg) and the lowest in variety 'Chica' (855 mg.kg). The values of antioxidant activity in frozen spinach samples were in range from 45.86 ±7.84% to 79.67 ±0.88%. The highest value of antioxidant activity in frozen spinach was found in variety 'Hudson' (79.67%) and the lowest in variety 'Chica' (45.86%).


INTRODUCTION
Spinach (Spinacia oleracea L.) belongs to the Amaranthaceous family with relatively short growing season.It is native to South-West Asia and cultivated through the world as vegetables (Rao et al., 2015), it is an important dietary vegetable and a common raw material in the food processing industry.
Spinach is not a popular plants in Europe, despite its high content of health-promoting substances.It is the most important leafy vegetable commonly consumed fresh or as canned or frozen products (Koh et al., 2012).It is widely known for their nutraceutical value, but there is relatively little information about its polyphenols content.Polyphenols are divided into two main groups, flavonoids and phenolic acid (Tapiero et al., 2002).Phenolic acids and their derivatives exhibit effects of primary antioxidants.Their activity depends on the number of hydroxyl groups in the molecule (Musilová et al., 2013).It is known that phenolic compounds are involved in maintenance of redox status of cell and its response to cold, UV radiation and the effect of pathogenes (Lukaszewicz et al., 2004).Spinach is rich source of polyphenols often associated with beneficial health effects.Their content depends on various factors, such as cultivar or cultivation conditions.These compounds possess an aromatic ring bearing one or more hydroxyl groups and their structures may range from that of a simple phenolic molecule to that of a complex high-molecular weight polymer (Balasundram et al Raw vegetables are subjected to some form of preservation in order to make them available for later consumption.Freezing is considered an effective technique for preserving of total polyphenol content, ascorbic acid and antioxidant activity in all vegetables. Freezing of fruits and vegetables is generally regarded as superior to other food preservation techniques such as canning and dehydration, with respect to retention in sensory attributes and nutritive properties.Freezing is often employed to maintain fresh-like characteristics with minimal loss of nutrients such as vitamins and antioxidant contents (Prochaska et al., 2000).
The aim of this study was to evaluate the content of total polyphenols and antioxidant activity of chosen varieties of fresh and frozen spinach.

MATERIAL AND METHODOLOGY Characteristic of varieties
̓ Boa ̓ is excellent as early spring and also late autumn cultivation.It has a large dark green leaves, suitable for industrial processing but also for fresh market.̓ Hudson ̓ is early variety of spinach.The variety has characteristic smooth leaves.̓ Hudson ̓ variety tends to run out into flower.̓ Chica ̓ is a slow-growing variety.The variety has a bright green smooth leaves.̓ Trombone ̓ is characterized by smooth leaves, very profitable for industrial processing.

Plant samples
Four spinach cultivars ('Boa', 'Hudson', 'Chica', 'Trombone') were obtained from a local producer in area Vinica, Slovak Republic.All cultivars were cultivated conventionally under he same condition.Altitude of this area is in the range from 164 to 400 meter above the sea level.Climatic conditions: this area belongs to the warm climatic area, regional middle dry.The annual mean temperature is 9.1 C, during growing season is 15 C.Annual mean rainfalls is 640 mm.
Samples of selected varieties of spinach were washed with distilled water several times, gently dried and part immediately stored in plastic bags at -18 C.Samples of spinach were homogenized (50 g) in 100 mL 80% ethanol 12 h at 250 rpm.Extracts were then filtered through filter paper (130 g.m -2 ; Filtrak Brandt GmbH, Thermalbad Wiesenbad, Germany) and kept at 8 C for further analysis.The experiment was based on four replications.

Total polyphenol content (TPC) determination
Total polyphenol content was determined by the method of Lachman et al., (2003) and expressed as mg of gallic acid equivalent per kg fresh mater.Gallic acid is usually used as a standard unit for phenolic content because a wide spectrum of phenolic compounds.The Folin-Ciocalteau phenol reagent was added to a volumetric flask containing 100 mL of extracts.The content was mixed and 5 mL of a sodium carbonate solution (20%) was added after 3 min.The volume was adjusted to 50 mL by adding of distilled water.After 2 hours, the samples were centrifuged for 10 min.and the absorbance was measured at 765 nm of wave length against blank.The concentration of polyphenols was calculated from a standard curve plotted with known concentration of gallic acid.

Antioxidant activity (AA) determination
Antioxidant activity was measured by the Brand-Williams et al., (1995) method-using a compound DPPH˙ (2,2-diphenyl-1-picrylhydrazyl).2,2-diphenyl-1-picrylhydrazyl (DPPH) was pipetted to cuvette (3.9 mL) then the value of absorbance which corresponded to the initial concentration of DPPH˙ solution in time Ao was written.Then 0.1 mL of the followed solution was added and then the dependence A = f (t) was immediately started to measure.The absorbance of 1, 5 and 10 minutes at 515.6 nm in the spectrophotometer Shimadzu UV/VIS -1240 was mixed and measured.The percentage of inhibition reflects how antioxidant compound are able to remove DPPH˙ radical at the given time.Formula: Inhibition (%) = (Ao -At / Ao) x 100

Statistical analysis
Results were statistically evaluated by the Analysis of Variance (ANOVA -Multiple Range Tests, Method: 95.0 percent LSD) using statistical software STATGRAPHICS (Centurion XVI.I, USA).

RESULTS AND DISCUSSION
Spinach (Spinacia oleracea L.) contains a range of phytochemicals such as polyphenols (flavonols, flavones), vitamins and minerals, which significantly contribute to their antioxidant activity.Freezing of spinach influenced the content of total polyphenols and antioxidant activity in spinach, however freezing helps to preserve fruit and vegetables through the slowing of senescence.Both studied parametersthe content of total polyphenols and antioxidant activity in selected varieties of fresh and frozen spinach are described in Table 1.
The content of total polyphenols in fresh spinach samples ranged from 975 ±97.15 mg.kg -1 to 1493 ±50.42 mg.kg -1 .The highest level of total polyphenol content was found in variety of Hudson and statistically significant lowest content of total polyphenols was recorded in variety of Chica.Based on the measured values of total polyphenols content varieties of spinach can be classified as follows: 'Hudson' (1493 mg.kg -1 ) >'Boa' (1481 mg.kg -1 ) >'Trombone' (1459 mg.kg -1 ) >'Chica' (975 mg.kg -1 ).Another indicator that has been evaluated and compared was the value of antioxidant activity in varieties of spinach.The values of antioxidant activity in fresh spinach samples were similar and ranged from 78.59 ±1.68% to 82.57±0.83%.Based on the measured values of antioxidant activity in fresh spinach samples cultivars can be classified as follows: 'Trombone' >'Chica' >'Hudson' >'Boa '. Turkmen et al., (2005) found the value of antioxidant activity in spinach in range from 67 to 85%.Statistically significant lowest level of antioxidant activity was recorded in variety 'Boa' (78.59%), on the other hand there was measured the highest level of total polyphenol content.These gained results indicate a fact that phenolic compounds may not be necessarily able to reflect antioxidant activity.Mainly structure of present phenolic substances is very important for antioxidant properties.Zhu et al., (2001) reported that the phenolic compounds with hydroxy groups in the position ortho (1,2), have a stronger antioxidant properties than phenolic compounds which have a hydroxy groups at other position.Wojdylo et al., (2007) also referred that the differences in the structure and substitution influenced the phenoxyl radical stability and thereby the antioxidant properties of the phenolic compounds.
In the present work the content of total polyphenols and antioxidant activity in frozen spinach samples after 3 months of freezing was detected.In the frozen spinach samples (except a variety 'Chica') slight increase of total polyphenol content was recorded.The highest level of total polyphenol content in frozen spinach samples was found in variety 'Hudson' (1749 mg.kg -1 ) and the lowest in variety 'Chica' (855 mg.kg -1 ).The greatest increase of total polyphenol content after 3 months of freezing was found in variety 'Hudson' (17.15%).The total polyphenol content is changed among other things by processing methods (Hegedűsová et al., 2015).Ganbaatar et al., (2015) also reported a decline of some polyphenols after boiling or after the fermentation.Some researchers reported an increase of total polyphenol content after freezing the spinach  cv.Boa (after freezing)

Figure 1
Figure 1Relationship between TPC and AA in fresh spinach in cv.'Chica'.

Figure 2
Figure 2Relationship between TPC and AA in fresh spinach in cv.'Trombone'.

Figure 3
Figure 3Relationship between TPC and AA in frozen spinach in cv.'Hudson'.

Figure 4
Figure 4Relationship between TPC and AA in frozen spinach in cv.'Boa'.

Bunea et al., 2008).
In green vegetables such as spinach only the green chlorophylls are seen by the consumer because they mask the bright colours of carotenoids (
a-c values with different letters mean significant differences (p 0.05) among selected varieties, values TPC and AA are expressed as arithmetic mean No. 1/2015