THE CONTENT OF TOTAL POLYPHENOLS, ASCORBIC ACID AND ANTIOXIDANT ACTIVITY IN SELECTED VARIETIES OF QUINCE (CYDONIA OBLONGA MILL.)

Quince fruit (Cydonia oblonga Miller) is an important source of bioactive compounds, especially of polyphenolic compounds, phenolic acids, flavonoids also of minerals and vitamins. This compounds exhibit health promoting properties including antibacterial, anti-inflammatory, anticancer, antidiabetic and cardioprotective properties. Quine fruit have a high therapeutic value, can be used as good sources of antioxidants. This study provides some knowledge about content of total polyphenols, ascorbic acid and antioxidant activity in selected varieties of quince fruit samples. Four quince fruit cultivars (Semenáč, Konstantinopler Apfelquitte, Cydora Robusta, Mammut) were analysed. The content of the total polyphenols (TPC) was determined by the Folin-Ciocalteu reagent (FCR) at 765 nm using spectrophotometer. Ascorbic acid (AsA) content was determined using standard HPLC gradient method. Antioxidant activity (AA) was measures using a compound DPPH ̇(2.2-diphenyl-1-picrylhydrazyl). The content of (TPC) in fresh samples of quince fruit ranged from 661 ±11.60 mg.kg to 1044 ±11.03 mg.kg and content of AsA were in interval from 151 ±0.58 mg.kg to 215 ±0.75 mg.kg. The values of antioxidant activity in quince fruit samples were in range from 26.90 ±0.61% to 49.14 ±0.38%. Statistically significant highest content TPC, AsA and AA was recorded in cultivar Konstaninopler Apfelquitte and statistically lowest content was recorded in cultivar Semenáč. The content of TPC, AsA and AA beside the variety may be affected by many factors also climatic conditions and the agrochemical composition of the soil.


INTRODUCTION
Quince fruit (Cydonia oblonga Miller) is a fruit of the Rosaceae family. It is one of the oldest crops, originates in the warmer areas of Sout-west Asia and has spread to Europe. It was a popular fruit species in Ancient Roma as well. The main production areas are Iraq, France and Portugal.
Quince fruit (Cydonia oblonga Miller) is mostly consumed in processed form. Because of its astringency, bitterness and firmness, it belongs to less popular species of the core fruit. However, when ripe, it is very demanded fruit for processing of marmalades, cakes and aromatic distillates as well.
The scientific investigations showed that quince fruit contains high quantities of bioactive substances, which are effective in prevention of different types of cancer and heart diseases and has beneficial effects on the human health is the major phenolic compound in quince. Organic acids, identified in quince fruit, are citric, ascorbic, malic, shikimic and fumaric (Silva et al., 2002). Monosaccharides, situated in quince fruit, include: rhamnose, mannose, glucose and galactose (Hopur et al., 2011). The nutritional value of quince fruit is high. It is also considered as a good source of vitamins and minerals such as K, Ca, Mg, Fe, Cu, Zn, and Mn (Al-Snafi, 2016). Wani et al. (2013) also considers quince to be a good source of bioactive substances like vitamin (A, C, E, riboflavin, folic acid), carotenoids, and flavonoids (isorhamnetin, quercetin, myricetin) and their glucoside compounds.
The aim of the present study was to determine the content of polyphenols, ascorbic acid and antioxidant activity in selected varieties of quince fruit.

Characteristic of varieties
Semenáčthe oldest and unbred cultivar. The fruits are similar to apples or pears, golden-yellow in colour.
Konstantinopler Apfelquittebred cultivar, is among the best tasting and oldest cultivars. Fruits are very aromatic.
Cydora Robustais among the most recent bred cultivars. The fruits are big, similar to the pears, bright yellow in colour. Mammut -It is very profitable cultivar. Fruits are big and pear shaped, bright yellow in colour.

Plant samples
Four quince fruit cultivars (Semenáč, Konstantinopler Apfelquitte, Cydora Robusta, Mammut) were obtained from a local producer in area Banka, Slovak Republic. All cultivars were cultivated conventionally under the same condition.
Samples of quince fruit 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.
The compound 2.2-diphenyl-1-pikrylhydrazyl (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.

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
Quince (Cydonia oblonga Miller) is regarded as an important source of beneficial effective bioactive compounds, which have a positive effect on human health.       Table 1. The content of total polyphenols in quince fruit samples ranged from 661 ±11.60 mg.kg -1 to 1044 ±11.03 mg.kg -1 . The highest level of total polyphenols content was found in cv. Konstantinopler Apfelquitte and the statistically significant lowest content of total polyphenols was recorded in cv. Semenáč. Based on the measured values of total polyphenols content, cultivars of quince can be classified as follows: Konstantinopler Apfelquitte (1044 mg.kg -1 ) Mammut (819 mg.kg -1 ) Cydoria Robusta (799 mg.kg -1 ) Semenáč (661 mg.kg -1 ). Many studies have dealt with the content of polyphenols in the parts of quince. The research results vary depending on the cultivars and also climatic and agrochemical conditions. Our results are in correspondence with the results of referred, that total polyphenol content in fresh quince was in interval from 412.3 to 975.10 mg.kg -1 . It follows from the above, that the quince ranks among the significant and easily available sources of the polyphenols substances.
Another significant parameter, that was monitored, was the content of ascorbic acid in selected varieties of quince. The highest level of ascorbic acid content was found in cv. Konstantinopler Apfelquitte, (215 ±0.75 mg.kg -1 ) and the statistically significant lowest content of ascorbic acid was recorded in cv. Mammut (151 ±0.58 mg.kg -1 ). compared antioxidant activity in peel, flesh extracts and oven-dried fruits and determine following values: 87%, 23.20% and 83.35%, respectively. In this study we have found positive correlation between the content of total polyphenols and antioxidant activity (r = 0.967, r = 0.815, r = 0.911, r = 0.922). Results are shown in Figure 1, Figure  2, Figure 3 and Figure 4. These results are in good accordance with Drogoudi et al. (2008), who reported a positive correlation between total antioxidant activity and total phenolic content in apple flesh and peel (r = 0.914 and r = 0.977, respectively). Silva et al. (2008) also observed a positive relationship between the content of polyphenolic coumpounds and antioxidant activity.

CONCLUSION
Despite the fact, that Quince is among the less popular fruit, this study indicates, that it is a rich source of compounds with interesting biological effects. The Quince is mainly valued for its high content of polyphenols (flavonols, flavanols, proanthocyanidins and many more), which protect the body against many diseases of civilization. The highest content of polyphenols was determined in breeding cultivar Konstantinopler Apfelquitte. The content of bioactive compounds may be affected by many factors, e.g. cultivars, agrochemical composition of the soil and climatic conditions. In the future, the information determined in this study, should be used for development of new functional foods with the addition of quince.