ANTIOXIDANT CAPACITY AND PHENOLIC CONTENT OF FRAXINUS ORNUS L. AND FRAXINUS PENNSYLVANICA MARSCH. LEAVES AND BARK EXTRACTS

Total contents of phenols, flavonoids, phenolic acids and proanthocyanidins have been determined in methanolic extracts of Fraxinus ornus L. and Fraxinus pennsylvanica Marsch. leaves and branches bark. DPPH and FRAP assay were used in the determination of antioxidant capacity. F. ornus leaves had higher contents of flavonoids (13.08 mg RE g DW) and proanthocyanidins (7.29 mg LCE g DW) while the bark had higher contents of phenols (56.47 mg GAE g DW), phenolic acids (14.32 mg CAE g DW) and coumarins (94.81 mg CE g DW). F. pennsylvanica leaves were richer in contents of phenols (25.73 mg GQE g DW), flavonoids (2.87 mg CE g DW and 5.13 mg RE g DW), phenolic acids (14.60 mg CAE g DW) and coumarins (20.01 mg CE g DW) while the bark had more proanthocyanidins (6.88 mg CE g DW). F. ornus leaves had lower contents of phenolic acids (11.09 mg CAE g DW) than F. pennsylvanica leaves. Also, F. pennsylvanica bark had higher contents of flavonoids (1.25 mg RE g DW) and proanthocyanidins (6.88 mg CE g DW) than F. ornus bark. Antioxidant capacity for both species was higher for the leaves than the bark. Generally, F. ornus had better antioxidant capacity than F. pennsylvanica. Very high correlations were found between antioxidant capacity and phenols (r= 0.9361-0.9805), flavonoids (r= 0.9358-0.9876) and coumarins (r= 0.9661-0.9938) in leaves. In bark, correlations were found for phenols (r= 0.97440.9796) and coumarins (r= 0.9757-0.9911).

Potassium chloride and ferrous ammonium sulphate were obtained from Kemika Zagreb (Croatia) and butanol from Merck Chemical Suppliers (Germany).All other chemicals and solvents were of analytical grade.

Sample extracts preparation -Priprema ekstrakata uzoraka
Leaf and bark extracts were prepared by ultrasound extraction.Leaf or bark (0.5 g per sample) was extracted twice with 12 ml of 80% aqueous methanol for 30 minute at controlled temperature (40 o C).Obtained supernatants from each step were combined and the final volume was adjusted to 25 ml with extraction solvent.The extracts were kept at -20 o C until use.

Determination of total phenols -Određivanje ukupnih fenola
Determination of total phenols (TP) was done with Folin-Ciocalteu reagent with some modifications (SINGLETON ET AL., 1974).After incubation for 30 minutes (40 o C), absorbance of the blue product was measured at 765 nm.Gallic acid was used as a standard in preparation of a calibration curve.Final results are expressed as mg of gallic acid equivalents per gram of dry sample (mg GAE g -1 ).

Determination of total flavonoids -Određivanje ukupnih flavonoida
Total flavonoids were determined by methods described by ORDONEZ ET AL. (2006) and QUETTIER ET AL. (2000) with aluminium chloride.Quercetin and rutin were used as standards for the expression of the results.Absorbance of the samples was measured at 420 nm (quercetin) and 415 nm (rutin) one hour after incubation at room temperature.Sample blanks were also included in each procedure in order to correct absorbance of the sample.Results for total flavonoids (TFq and TFr) are expressed as mg equivalents of quercetin /rutin per gram of dry sample (mg QE g -1 and mg RE g -1 ).

Determination of total phenolic acids -Određivanje ukupnih fenolnih kiselina
Arnow`s method (GAWLIC-DZIKI, 2012) with some modifications was used for determination of total phenolic acids (TPHA).Briefly, one milliliter of previously diluted sample was mixed with 5 mL of water, 1 mL HCl (0.5 M), 1 mL of Arnow`s reagent (10 g Na 2 MoO 4 and 10 g NaNO 2 dissolved in 100 mL of distillated water) and 1 mL of NaOH (1M).Final volume was made up to 10 mL with distillated water.Caffeic acid was used as a standard to prepare calibration curve and absorbance was measured at 490 nm.The results are expressed as caffeic acid equivalents per gram of dry sample (mg CAE g -1 ).

Determination of total coumarins -Određivanje ukupnih kumarina
Method described by OSORIO AND MARTINS ( 2004) was used to determine contents of coumarins in leaf and bark samples.To 0.5 ml of diluted plant extract, 2 ml distilled water and 0.5 ml of lead acetate (5%, w/v) solution were added in a test tube and after shaking volume was made up to 10 ml with distilled water. 2 ml of this solution was taken in another test tube and 8 ml of 0.1 M (v/v) hydrochloric acid solution was added.The solution was kept for 30 minutes at room temperature and absorbance was recorded at 320 nm.The total coumarin content was expressed as mg of coumarin equivalents per gram of dry sample (mg CE g -1 ).

Determination of total proanthocyanidins -Određivanje ukupnih proantocijanidina
Butanol/HCl assay described by HAGERMAN (2000B) was used for determination of total proanthocyanidins (TPA).Before and after heating of the samples in boiling water-bath (60 minutes), absorbance of the sample was read at 550 nm.The results were expressed as mg of leucocyanidin equivalents per gram of dry sample (LCE g -1 ) assuming that the specific absorbance of leucocyanidin was 460.
Absorbance was read at 517 nm after incubation in the dark for 30 minutes.Results were expressed as vitamin C equivalents according to obtained equation:  = 17.787 ×  + 1.2817Where I (%) = [(A DPPH -A ext )/A DPPH ] x100 presents percentage inhibition of DPPH radical and c is a concentration of ascorbic acid standards expressed in mgdm -3 .All measurements were performed in triplicate.The mean values of results were expressed as vitamin C equivalents per gram of dry sample (AAE g -1 ).

FRAP assay -FRAP esej
BENZIE AND STRAIN (1996) method was used in the determination of ferric reducing antioxidant power (FRAP).The method is based on reduction of ferric tripyridiyltriazine (Fe(III)-TPTZ) to blue coloured ferrous trypyridyltriazine (Fe(II)-TPTZ) which is monitored spectrophotometrically at 593 nm.Acetate buffer (300 mM, pH 3.6), TPTZ (10 mM in 40 mM HCl acid) and FeCl 3 (20 mM) were mixed in the ratio 10:1:1 to prepare FRAP reagent and wormed at 37 o C before use.The reagent (1.9 mL) was mixed with 0.1 mL of the extracts and leaved in the dark for 30 minute before measurements.Absorbance of the coloured product was measured at 593 nm against the blank which contained 0.1 ml of water instead of the extract.A standard curve was made with Fe(II) sulphate and the results were expressed as mmol Fe(II) equivalents per gram of dry sample (mmol Fe(II) g -1 ).

Statistical analysis-Statistička analiza
All measurements were carried out in triplicate and obtained results are expressed as means.Regression analysis was used in order to investigate correlation between analysed active compounds and antioxidant capacity.

RESULTS AND DISCUSSION-Rezultati i diskusija
Contents of phenols, flavonoids, phenolic acids, coumarins and proanthocyanidins were investigated in methanolic extracts of F. ornus and F. pennsylvanica leaf and bark samples.Antioxidant capacity of the extracts was also determined by DPPH and FRAP assay.Ascorbic acid and ferrous sulphate were used as standards, respectively.Obtained results for contents of bioactive compounds are presented in Table 1.Generally, the most abundant compounds in F. ornus leaves are coumarins, phenols and flavonoids and in the bark coumarins, phenols and phenolic acids.The average contents of coumarins, phenols and flavonoids in leaves are 58.27mg CE g -1 DW, 49.90 mg GAE g -1 DW, 13.08 mg RE g -1 DW respectively.In the bark, average values for coumarins, phenols, phenolic acids are 94.81 mg CE g -1 DW, 56.47 mg GAE g -1 DW, and 14.32 mg CAE g -1 DW, respectively.The average contents of flavonoids and proanthocyanidins in the leaves are higher (6.20 mg QE g -1 DW, 13.08 mg RE g -1 DW, 7.29 mg LCE g -1 DW), than their average contents in the bark (0.70 mg QE g -1 DW, 1.09 mg RE g -1 DW, 4.85 mg LCE g -1 DW).Also, average phenolic acid contents in the bark are higher than in the leaves (Table 1).
The most abundant compounds in F. pennsylvanica leaves and bark were phenols, coumarins and phenolic acids.In leaves, their average contents were for phenols 25.73 mg GAE g -1 DW, coumarins 20.01 mg CE g -1 DW, and phenolic acids 14.60 mg CAE g -1 DW.In the bark, average values were for phenols 25.36 mg GAE g - 1 DW, coumarins 15.21 mg CE g -1 DW, and phenolic acids 9.86 mg CAE g -1 DW.Leaves are richer in the contents of all investigated compounds, except proanthocyanidins with their average content of 4.32 mg LCE g -1 DW in leaves compering to the bark with the value of 6.88 mg LCE g -1 DW.We can conclude that F. ornus leaves had higher average contents of phenols, flavonoids, coumarins and proanthocyanidins than F. pennsylvanica leaves.F. ornus bark is richer in the contents of phenols, phenolic acids and coumarins while F. pennsylvanica bark had higher contents of flavonoids and proanthocyanidins.
Literature data concerning quantitative determination of the investigated compounds in F. ornus and F. pennsylvanica leaves and bark are quite limited.It is reported that different Fraxinus species contains phenols, phenolic acids, flavonoids, coumarins, sterols, triterpenes (KOSTOVA AND IOSSIFOVA, 2007 AND REFERENCE THEREIN).Obtained average results for total phenols of F. ornus and F. pennsylvanica leaves were higher compared to the results for F. excelsior and F. angustifolia 1.78-2.505%(IORDAKE ET AL., 2013).According to our previous investigations on F. excelsior and F. angustifolia leaves and bark extracts, we can conclude that F. ornus leaves is a reach source of phenols.Also, F. pennsylvanica had the highest average content of total flavonoids in leaves compared to that for F. excelsior (4.08 mg RE g -1 DW) and F. angustifolia (5.01 mg RE g -1 DW).We found that F. angustifolia leaves were the richest in the contents of phenolic acids (20.30 mg CAE g -1 DW) and proanthocyanidins (7.99 mg LCE g -1 DW).Since the average contents of coumarins for F. excelsior and F. angustifola leaves were 23.58 mg CE g -1 DW and 36.47 mg CE g -1 DW, we can conclude that F. ornus leaves had highest total coumarins content.(TAHIROVIĆ AND BAŠIĆ, 2016).According to KOSTOVA (2011), the levels of coumarins in F. ornus leaves varied from 0.3% up to 4.6% depending on the sampling season.Results obtained in this work for coumarin content in F. ornus leaves are generally higher.NYKOLOV ET AL. (1993) reported that F. ornus bark taken from five different regions in Bulgaria had total coumarin contents in the range of 7.8-9% which is lower than the results obtained in this work for F. ornus bark.These results are also higher in comparison with the results obtained for F. excelsior (37.89 mg CE g -1 DW) and F. angustifolia bark (60.43 mg CE g -1 DW) (TAHIROVIĆ AND BAŠIĆ, 2016).Also, F. pennsylvanica had the highest content of proanthocyanidins and F. ornus highest content of total phenols compared to other investigated species (TAHIROVIĆ AND BAŠIĆ,

Table 1 .
Contents of investigated polyphenolic compounds in extracts of F.ornus and F. pennsylvanica leaves and bark Tabela 1. Sadržaji ispitivanih polifenolnih jedinjenja u ekstraktima F. ornus i F. pennsylvanica lista i kore