PHENOLIC CONTENT AND ANTIOXIDANT CAPACITY OF VISCUM ALBUM LEAVES AND TWIGS FROM VARIOUS HOST TREES

Phenolic compounds content and antioxidant capacity from Viscum album ssp. album Beck. leaves and twigs extracts were determined. Common mistletoe was collected from four hosts (Crataegus monogyna, Malus domestica, Prunus cerasifera, and Populus x canadensis.). Folin-Ciocalteu method, AlCl3 method, method with Arnow reagent, and acid-butanol assay were applied for determination of total phenols, flavonoids, phenolic acids and proanthocyanidins respectively. DPPH, ABTS and FRAP methods were applied in the determination of antioxidant capacity of the extracts. Total phenols were in the range 7.16-11.70 mg GAE/g DW, flavonoids 2.115-4.668 mg RE/g DW, phenolic acids 0.811-1.789 mg caffeic CAE/g DW, and proanthocyanidins 0.71-2.62 mg LCE/g DW. Content of phenols and flavonoids was higher in the leaves than the twigs. The highest antioxidant capacity in leaves (50.8665.93 μmol Trolox equivalents/g DW) was determined for mistletoe collected from Crataegus monogyna while the highest capacity in twigs had mistletoe collected from Malus domestica (67.28-81.72 μmol Trolox equivalents/g DW). Also, high correlation has been noticed between total phenols, flavonoids, phenolic acids and antioxidant capacity for V. album leaves. For twigs, high correlation was obtained between phenols and antioxidant capacity, and in some moderate extends for proanthocyanidins.

Polysaccharides, phenylpropanes, lecitins, viscotoxins, alkaloids, flavonoids, caffeic and other acids appear to be the main bioactive compounds in V. album (ERGUN and DELIORMAN, 1995). The main antioxidants are flavonoids and phenolic acids although phytochemical composition mainly depends on a host tree (LUCZKIEWICH et al, 2001). Estimatation of antioxidant capacity of the plant extracts can be carried out with different methods: TEAC (Trolox equivalent capacity), FRAP (ferric-reducing ability), TRAP (total radical trapping capacity, and ORAC (oxygen radical absorbance capacity) (WU et al, 2004).
The aim of this work was to determine total content of different phenolic compounds: phenols, flavonoids, phenolic acids and proanthocyanidins in V. album ssp. album leaves and twigs collected from hawthorn, cherry plum, apple and Canadian poplar trees. Antioxidant activity of the extracts was determined with DPPH, ABTS and FRAP methods with Trolox as a standard. Correlations between investigated compounds and antioxidant capacity of the extracts obtained from leaves and twigs are reported. According to our best knowledge, these are the first results on the investigated species from selected host trees.

Plant material -Biljni materijal
Leaves and twigs were collected from four different hosts in area of Sarajevo in November 2015. Plant material for leaves (l) and twigs (t) was marked as follows: VAC (Crataegus monogyna), VAM (Malus domestica), VAPR (Prunus cerasifera), and VAPO (Populus x canadensis). The plant material was dried in a ventilated place at room temperature and stored in paper bags until use. Voucher specimens of the plants were deposited at herbarium of Department of Forest Ecology at Faculty of Forestry University of Sarajevo.

Chemicals and reagents -Hemikalije i reagensi
All chemicals used in this study were highest purity grade obtained from Sigma-Aldrich Chemical Company (Germany).

Sample extracts preparation -Priprema ekstrakata uzoraka
Powdered dry sample of leaves or twigs (0.5 g) was extracted with 80% aqueous methanol (2 x 12 mL) during 30 minutes in an ultrasonic bath (Elmasonic S 60H). The mixture was centrifuged for 15 min at 3000 rpm (Centric 322 B, Techtnica). Obtained supernatants were combined, filtrated and volume of the extract was adjusted with extraction mixture up to 25 mL. Extracts were stored at -20 o C until analysis.

Determination of total phenols -Određivanje ukupnih fenola
Folin-Ciocalteu method is used for the determination of total phenols content (SINGELTON et al, 1974). Diluted sample was mixed with 7.9 mL of distillated water and 0.5 mL of Folin-Ciocalteu reagent was added. After 5 minutes, 20% Na 2 CO 3 (1.5 mL) was added in the reaction mixture, and it was left to stand for 30 minutes in a water bath at 40 o C. Blank was prepared by using distillated water instead extract. Absorbance of the colored product was measured at 765 nm and a calibration curve was prepared with gallic acid as a standard. Final results are expressed as mg gallic acid equivalents (GAE) per gram of dry sample (DW). All spectrophotometric measurements were done with Shimadzu UV-mini 1240 spectrophotometer.

Determination of total flavonoids -Određivanje ukupnih flavonoida
Determination of total flavonoids was done with AlCl 3 method (QUETTIER et al, 2000), with rutin as a standard. Shortly, equal volumes of extracts and reagent are mixed and left to stand at room temperature for 1 hour, and absorbance of the colored product was measured at 415 nm against the blank. Sample blank was also used in the analysis for the correction of sample absorbance. Final results are expressed as mg rutin equivalents (RE) per gram of dry sample.

Determination of total phenolic acids -Određivanje ukupnih fenolnih kiselina
Arnow reagent was used for the quantification of total phenolic acids as described by GAWLIC-DZIKI, (2012). Diluted sample (1 mL) was mixed with distilled water (5 mL), HCl (1 mL, 0.5 M), Arnow reagent (1 mL) and NaOH (1 mL, 1 M). A calibration curve was established with caffeic acid as a standard. Absorbance was measured at 490 nm, and the results are expressed as mg caffeic acid equivalents (CAE) per gram of dry sample.

Determination of total proanthocyanidins -Određivanje ukupnih proantocijanidina
Butanol-HCl assay was used for the determination of proanthocyanidins as described by HAGERMAN (2000). Diluted extract (0.5 mL) was mixed with butanol-HCl reagent (3.0 mL, butanol-HCl 95:5 v/v) and ferric reagent (0.1 mL, 2% ferric ammonium sulphate in 2 M HCl). Samples were heated at boiling water bath for 60 minutes. Absorbance was measure before and after heating at 550 nm against blank. Results are expressed as mg leucocyanidin equivalents (LCE) using specific absorbance of leucocyanidin 460.

Determination of antioxidant activity -Određivanje antioksidacijske aktivnosti DPPH method -DPPH metoda
Methods of BRAND-WILLIAMS et al (1995) andTHAIPONG et al (2006) were used for determination of antioxidant activity of the extracts. Dilution of the extracts was done with methanol. DPPH stock solution (0.094 M) was prepared in methanol on a daily basis and diluted to absorbance of 1.1±0.02 at 515 nm before use. Aliquots of extracts (0.1 mL) were mixed with 1.9 mL of DPPH solution and left at room temperature in the dark for 30 minutes. Standard solutions of Trolox were used for preparation of a calibration curve. Final results are expresses as µmol of Trolox equivalents (TE) per gram of dry sample.

ABTS method -ABTS metoda
The method of RE et al (1999) modified by THAIPONG et al. (2006) was used in ABTS assay. Stock solutions of ABTS (7 mM) and potassium persulfate (2.45 mM) were mixed in equal volumes and left to stand in the dark for 12-16 hours prior use. Freshly prepared ABTS + solution was diluted with methanol to absorbance of 1.1 ±0.02 units at 734 nm. Plant extracts (0.1 mL) were allowed to react with 1.9 mL of working ABTS + solution for 6 minutes after that the reduction in absorbance was measured. Standard solutions of Trolox were used to prepare a calibration curve, and the results are expressed as µmol of Trolox (TE) per gram of dry sample.

FRAP method -FRAP metoda
Ferric reducing antioxidant power (FRAP) was determined by method of BENZIE and STRAIN (1999). FRAP reagent was prepared by mixing 300 mM acetate buffer, pH= 3.6; 10 mM TPTZ (2,4,6-tripiridil-s-triazine) in 40 mM HCl acid and 20 mM FeCl 3 in the ratio 10:1:1. Obtained solution was heated at 37 o C for 30 minutes in a water bath. Plant extracts (0.1 mL) were mixed with 1.9 mL of working FRAP solution and left in the dark for additional 30 minutes. Absorbance of the formed blue complex was measured at 593 nm against a blank. Standard solutions of Trolox were used to prepare a calibration curve, and the results are expressed as µmol of Trolox (TE) per gram of dry sample.
(2011a) who found that leaves extracts are richer in the content of phenolic compounds than the twigs extracts. Mistletoe leaves hosted by VAC, VAM and VAPR had higher content of phenols, flavonoids and phenolic acids than the twigs. However, twigs of mistletoe from VAPO were richer in the content of total phenolic acids than their leaves. The highest content of total phenols (11.70 mg GAE/g DW), total flavonoids (4.668 mg RE/g DW) and total phenolic acids (1.789 mg CAE/g DW) was found in mistletoe leaves from VAC, while the lowest levels of phenols (7.32 mg GAE/g DW), flavonoids (3.576 mg RE/g DW) and phenolic acids (0.811 mg CAE/g DW) was determined in mistletoe leaves from VAPO. The highest content of proanthocyanidins was found in mistletoe leaves from VAPR (1.42 LCE/g DW) while the lowest content was in mistletoe leaves from VAPO (0.71 LCE/g DW). The highest content of flavonoids (2.707 mg RE/g DW) and phenolic acids (1.138 mg CAE/g DW) had the mistletoe twigs extracts hosted by C. monogyna (VAC), content of proanthocyanidins (2.62 LCE/g DW) was the highest with mistletoe from VAPR and total phenols (9.92 mg GAE/g DW) were the highest for mistletoe from VAM. These results are lower than the results obtained for mistletoe collected from Robinia pseudoacacia obtained in our previous investigation (TAHIROVIĆ and BAŠIĆ, 2017). Also, V. album from R. pseudoacacia L. had higher content of flavonoids than other investigated hosts (ORHAN et al 2014). It is also found that mistletoe from Malus domestica (17.48 mg %) is richer in phenolic acids content than Populus nigra (12.34 mg %) (LUCZKIEWIEZ et al 2001), which is in agreement with the results in this study. The lowest content of phenols (7.16 mg GAE/g DW) and flavonoids (2.115 mg RE/g DW) was found in mistletoe twigs collected from VAPR. In mistletoe twigs collected from VAPO content of phenolic acid was 1.045 mg CAE/g DW and for mistletoe from VAM had the lowest content of proanthocyanidins (1.31 LCE/g DW). Influence of the host tree may have a key role in the phenolic composition of mistletoe leaves or twigs (VICAŞ et al 2011A;ORHAN et al 2014). As it was observed by VICAŞ et al (2011B) differences in phenolic contents highly depends on harvesting seasons and generally they are higher for samples harvested in spring than in autumn. The same authors found that leaves of mistletoe are richer in phenolic contents (total phenols and phenolic acids) than the twigs. (VICAŞ et al, 2011B).

Antioxidant capacity-antioksidacijski kapacitet
Antioxidant capacity of mistletoe leaves and twigs extracts was investigated by DPPH, ABTS and FRAP methods. The results are presented in Table 2. In mistletoe leaves, antioxidant capacity varied from 25.57 to 50.86 µmol Trolox/g DW for DPPH, from 39.98 to 57.67 µmol Trolox/g DW for ABTS, and from 40.28 to 65.93 µmol Trolox/g DW for FRAP method. Generally, antioxidant capacity decreased in the order: FRAP>ABTS>DPPH. Antioxidant capacity of V. album has been investigated by different researches (ONAY-UCAR et al, 2006;OLUWASEUN and GANIYU, 2007;VICAŞ et al, 2008VICAŞ et al, , 2009PAPUC et al, 2010;SHARMA and BHAT, 2010;ROMAN et al 2010). As it was presented by VICAŞ et al (2008VICAŞ et al ( , 2009 extracts of V. album harvested in autumn were richer in phenolics as antioxidant with ferric reducing ability. These differences were explained by different environmental factors. Antioxidant capacity of mistletoe leaves extracts from VAC was higher than the corresponding twigs extracts. Interestingly, antioxidant capacity of mistletoe leaves extracts collected from VAPO was lower than corresponding twigs extracts. This can be explained by higher contents of phenols and phenolic acids found in mistletoe twigs from VAPO than in leaves which may contribute to higher values of antioxidant capacity in twigs. Antioxidant capacity for leaves decreased in the following order: VAC>VAM>VAPR>VAPO for all three methods. The highest antioxidant capacity was determined in mistletoe leaves from VAC while the lowest values were found for mistletoe leaves from VAPO. These results are in agreement with other investigators who found that antioxidant capacity differs depending on the host trees and harvesting time (ONAY-UCAR et al, 2006;OLUWASEUN and GANIYU, 2007;VICAŞ et al, 2009).
Antioxidant capacity for twigs varied from 37.14 to 44.63 µmol Trolox/g DW for DPPH, from 39.79 to 50.62 µmol Trolox/g DW for ABTS and from 43.33 to 57.13 µmol Trolox/g DW for FRAP (Table 2). Antioxidant capacity for twigs decreased in the following order: VAM>VAC>VAPO>VAPR and for leaves in the order VAM>VAPO=VAC>VAPR for DPPH and FRAP methods. The highest values of antioxidant capacity were determined for mistletoe twigs from VAM and the lowest for twigs from VAPR. VICAŞ et al (2009B) found that methanol extracts of V. album from M. domestica had the highest antioxidant capacity (0.14 mg/g fw). Results obtained for antioxidant capacity in this study are lower compared to the results for mistletoe leaves and twigs extracts collected from Robina pseudoacacia (ONAY-UCAR et al, 2006;TAHIROVIĆ and BAŠIĆ, 2017).
Correlations between antioxidant capacity and bioactive compounds in leaves and twigs are given in Table 3. For leaves, correlation coefficients were in range r 2 =0.9293-0.9818 for phenols; r 2 =0.8787-0.9117 for flavonoids and for phenolic acids r 2 =0.7302-0.8593. Moderate correlations were found between phenolic acids and antioxidant capacity (r 2 =0.5082-0.5458) which suggest that these compounds also contribute to the antioxidant capacity of the extracts but in some less extend. TP-total phenols, TF-total flavonoids, TPA -total phenolic acids, TPC-total proanthocyanidins TP-ukupni fenoli, TF-ukupni flavonoidi, TPA-ukupne fenolne kiseline, TPC-ukupni proantocijanidini In case of twigs, correlations were in range of 0.8348-0.9211 for phenols and 0.6919-0.8075 for proanthocyanidins. The lowest correlation coefficients were found for flavonoids r 2 =0.5918-0.7724 in leaves and for twigs r 2 =0.688-0.873. Other investigators found that phenolics and flavonoids may contribute mostly to antioxidant capacity of mistletoe extracts (PAPUC et al, 2010;ORHAN et al, 2014;PIETRZAK et al, 2014). No correlation was observed between other investigated compounds and antioxidant capacity.

CONCLUSIONS -Zaključci
In all investigated samples, flavonoids are presented in higher amounts in the leaves than the twigs. Mistletoe from Crataegus monogyna was the richest in the content of flavonoids and phenolic acids. Mistletoe from Populus x canadensis had the lowest content of phenols, flavonoids and phenolic acids in leaves. In twigs, the lowest content of phenols and flavonoids was found for mistletoe from Prunus cerasifera, phenolic acids from Populus x canadensis and proanthocyanidins from Malus domestica.