PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF CRATAEGUS MONOGYNA JACQ. AND CRATAEGUS MACROCARPA HEGETSCHW. LEAVES

Phenolic contents of methanolic extracts prepared from leaves and fruits of Crataegus monogyna Jacq. and Crataegus macrocarpa Hegetschw. were determined. The antioxidant capacity was assessed by DPPH, ABTS and FRAP assay. The results showed that leaves were richer in the content of phenols (59.23 91.91 GAE g-1), flavonoids (2.38 4.08 mg QE g-1 and 5.24 8.9 mg RE g-1) , phenolic acids (33.40 68.98 CAE g-1) and proanthocyanidins (26.15 48.60 CE g-1) while in fruits anthocyanins dominateted (0.43 0.80 CG g-1). Leaves also had higher antioxidant capacity than fruits for both species. Generelly, C. monogyna fruits had higher content of anthocyanins. Total phenols, phenolic acids and proanthocyanidins were highly corelated with DPPH (r2 = 0.8703 0.9618), ABTS (r2 = 0.7833 0.9443) and FRAP (r2 = 0.903 0.9695) assay. The results suggests that these compounds were the major contributors to the antioxidant capacity in leaves and fruits extracts of both species. Higher contents of bioactive compounds and higher antioxidant capacity were determined for C. x macrocarpa samples. Therefore, C. x macrocarpa leaves and fruits are valuble source of antioxidant polyphenols with high potential for use in preparation of different natural health products.


INTRODUCTION -Uvod
Natural oxidants from fruits and vegetables have been studed for severel decedes in order to isolate compounds which can prevent or decrease different patological conditions assosiated with oxidative stress (cancer, heart deasese, atherosleroses, neuridegenerative desorders, diabiites, aging) (MANDEL SEİFİRED ET AL., 2007;TEMPLE, 2000). Especially important group of natural antioxidants are phenols and studies showed their protective role against heart deases and cancer due to high antioxidant activity. The antioxidant activity of phenols is based on their redox properties and ability to scavenge wide range of reactive radical Genus Crataegus in Bosnia and Herzegovina have been studied by several researches (BECK, 1927;MALY, 1919MALY, , 1940FUKAREK 1974;JANJIĆ, 1998;BAŠIĆ, 2004, CHRISTENSEN AND JANJIĆ, 2006. One of the most abundant species of genus Crataegus in Bosnia flora is Crataegus monogyna Jacq with very wide ecological amplitude (BAŠIĆ, 2004). Since interspecies breedings are common in Crataegus this study included investigations on C. x macrocapa a hybride between C. laevigata x C. rhypidophylla. Presence of this hybride is well documented in Europe for a long time, and today is known under name C. x macrocapa Hegetschw which was introduced for the first time by HRABĔTOVÁ-UHROVÁ (1969). This hybride specie is recently registreted in flora of Bosnia and Herzegovina (BAŠİĆ, 2004).
Investigations of Crataegus species in terms of chemical composition and antioxidant activity have been started recently by our research group and they were focused mainly on C. monogyna and C. rhypidophylla from native populations around Sarajevo. According to our best knowledge this is the first paper on phenolic content and antioxidant activity of C. monogyna and C. x macrocarpa from Zenica region.
In this work we investigated phenolic contents and antioxidant activity of C. monogyna and C. x macrocarpa leaf and fruit methanolic extracts. Obtained extracts were used in spectrophotometric determinations of total phenols, flavonoids, phenolic acids, monomeric anthocyanins and proanthocyanidins. Antioxidant activity for all extracts was investigated with three methods: DPPH, ABTS and FRAP using Trolox as a standard for expression of final results. Correlations between antioxidant capacities and different phenolic compounds were also investigated.

MATERIAL AND METHODS -Materijal i metode
Plant material -Biljni materijal C. monogyna and C. x macrocarpa leaf and fruit samples were collected in October 2014. in Zenica region at locality of Smetovi. Two samples per species were collected from wider area and samples were identified by Prof Bašić, a plant taxonomist. Voucher specimens were deposited at the Herbarium of the Department of Ecology at Faculty of Forestry. The plant material was air-dried at room temperature and powdered before analysis.
Butanol was obtained from Merck Chemical Suppliers (Germany). Potassium chloride and ferrous ammonium sulfate were sourced from Kemika Zagreb (Croatia). All other chemicals and solvents were of analytical grade.

Sample extracts preparation -Priprema ekstrakata uzoraka
Leaf and fruit samples (0.5 g each) were extracted twice in the extraction solvent containing 80% methanol (12 mL) with ultrasound bath, (Elmecs, Italy). Each extraction step was performed at room temperate for 30 minutes. Obtained supernatants for each sample were combined and collected in a volumetric flask and volume adjusted to 25 mL with extraction solvent. The extracts were kept at -20 o C until analysis.

Determination of total phenols -Određivanje ukupnih fenola
Procedure with Folin-Ciocalteu method described by SINGLETON ET AL. (1974) was used for the determination of total phenols (TP). The absorbance of the colored product was measured at 765 nm. Appropriate calibration curve was prepared with gallic acid as standard, and 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
Colorimetric method with AlCl 3 given by CHRIST AND MULLER (1960) and ABDENNACER ET AL. (2015) was used for the determination of total flavonoids. Briefly, sample aliquot (0.5 mL) was mixed with 1.5 mL methanol and 0.1 mL CH 3 COONa (1M). Six minutes later, 0.1 mL AlCl 3 (10%) was added and dilution was made up to 5 mL with water. The solution was kept at room temperature for 30 minutes after that absorbance was measured at 430 nm against blank. Sample blanks were also included. Standard solutions of rutin and quercetin were used to prepare calibration curves. Final 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
Total phenolic acids (TPHA) were quantified with Arnov method described by GAWLIC-DZIKI (2012) with some modifications. One mililitre of appropriately diluted sample was mixed with 5 mL of water, 1 mL HCl (0.5 M), 1 mL of Arnov`s reagent (10 g Na 2 MoO 4 and 10 g NaNO 2 dissolved in 100 mL of distillated water), 1 mL of NaOH (1M) and the volume was made up to 10 mL with distillated water. Calibration curve was established with standard solutions of caffeic acid and apsorbance was measured at 490 nm. Solvent instead of extract was used as a blank. The results are expressed as caffeic acid equivalents per gram of dry sample (mg CAE g -1 ).

Determination of monomeric anthocyanins -Određivanje monomernih antocijanina
In the determination of total monomeric anthocyanins (TMA), pH differential method by LEE  was used Extracts were diluted in the ratio 1:10 and apsorbances were measured at 520 and 700 nm at room temperature after 15 min. The content of total monomeric anthocyanins was expressed in mg of cyanidin-3-glucoside equivalents (CGE) per gram of dry fruits. A molar extinction coefficient of cyanidin-3-O-glucoside of 26900 l mol -1cm -1 and molar weight (MW) (449.2 g mol -1 ) were used for calculations.

Determination of total proanthocyanidins -Određivanje ukupnih proantocijanidina
Total proanthocyanidins (TPA) were determined with butanol/HCl assay (HAGERMAN, 2002). The method was based on their acid hydrolysis to anthocyanidins and color formation with the added reagent which is monitored spectrophotometrically. Absorbance of the sample was read at 550 nm before and after heating of the samples at 95 o C for 40 minutes. Butanol/HCl mixture was used as a blank. The results were expressed as mg of cyanidin chloride equivalents (CE) per gram of dry fruit.
Determination of antioxidant capacity -Određivanje antioksidacijskog kapaciteta DPPH assay -DPPH esej DPPH assay was done according to BRAND-WILLIAMS ET AL. (1995) and THAIPONG . The method is based on the ability of standard and extracts to scavenge stable DPPH radical which leads to its decolonization and formation of yellow non-radical form. Stock solution of DPPH in methanol (0.094 M) was freshly prepared and diluted with methanol to absorbance of 1.1 ±0.02 at 515 nm. After that, 100 µL of previously diluted extracts was mixed with 1.9 mL of working DPPH solutions and kept in the dark for 30 minutes before measurements. Calibration curve was prepared with standard solutions of Trolox and the results are express in terms of Trolox equivalent antioxidant capacity (TEAC) as mmol Trolox equivalents per gram of dry sample weight.
ABTS assay -ABTS esej ABTS assay was done according TO REE  with some modification given by THAIPONG . Basically it is a decolorisation assay which can be applied to lipophilic and hydrophilic antioxidants. In this assay radical monocation of 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS •+ ) is reduced with standard or extract. Stock solutions of ABTS (7 mM) and potassium persulphate (2.45 mM) were prepared in water and kept in the dark for 16 hours. Equal volumes of the stock solutions are mixed and diluted to absorbance of 1.1 ±0.02 at 734 nm to prepare ABTS radical cation (ABTS •+ ) solution. Freshly prepared solution was used for each assay. Working solution of ABTS •+ (1.9 mL) was mixed with 100 µL of previously diluted extracts and after 6 minutes the reduction in apsorbance was measured at 734 nm. Calibration curve was prepared with standard solutions of Trolox and the results are express in terms of Trolox equivalent antioxidant capacity (TEAC) as mmol Trolox equivalents per gram of dry sample weight.

FRAP assay -FRAP esej
Ferric reducing antioxidant power (FRAP) was measured according to BENZIE AND STRAIN (1996) method. The method is based on reduction of ferric tripyridiyltriazine (Fe(III)-TPTZ) to ferrous trypyridyltriazine (Fe(II)-TPTZ) by sample extracts. As a result of reagent reduction, a blue product is formed which can be monitored spectrophotometrically. Briefly, FRAP reagent was prepared by mixing 300 mM acetate buffer, pH 3.6; 10 mM TPTZ in 40 mM HCl acid and 20 mM FeCl 3 in the ratio 10:1:1. The fresh working solution was wormed at 37 o C before using. This reagent (1.9 mL) was mixed with 0.1 mL of the extracts and leaved in the dark for 30 minute before measurements. Apsorbance of the colored product was measured at 593 nm against the blank which contained 0.1 ml of methanol instead of the extract. A standard curve was made with Trolox and the results were expressed as mmol Trolox equivalents per gram of dry sample weight.

Statistical analysis-Statistička analiza
All measurements were carried out in triplicate and obtained results are expressed as mean±SD. Correlation between investigated active compounds and antioxidant activity was established by regression analysis.

RESULTS AND DISCUSSION-Rezultati i diskusija
Phenolic compounds isolated from plant materials represent a reach source of natural antioxidants which receive much attention during last years. Flavonoids (flavonols, flavones and anthocyanins) show good antioxidant properties capable to scavenge free radicals (RICE-EVANS AND MULLER, 1997). Anthocyanins used as natural colorant becomes important due to their antioxidant and antibacterial properties (NAZ ET AL., 2007). Also, hydroxycinammic acids represent important class of phenolic compounds since they act as antioxidants in plant protection (CHEN AND HO, 1997). They are usually found at higher concentrations in plants (MANACH  and some of them such as caffeic acid can inhibite formation of mutagenic compounds in humans (OLTHOF ET AL., 2001).
In this study we investigated contents of a range of bioactive compounds (phenols, flavonoids, phenolic acids, anthocyanins and proanthocyanidins) of C. monogyna and C. x macrocarpa leaf and fruit methanolic extracts. Antioxidant capacity of the extracts was also examined with three methods: DPPH, ABTS and FRAP using Trolox as a standard. The results for quantitative contents of bioactive compounds for two investigated Crataegus species are presented in Table 1.  Leaf and fruit extracts of C. monogyna were rich in phenolics, phenolic acids and proanthocyanidins. The average values in leaves were: phenols 61.98 mg GAE g -1 DW, phenolic acids 36.41 mg CAE g -1 DW, and proanthocyanidins 30.74 mg CE g -1 DW. In fruits, avarage values were for phenols 28.19 mg GAE g -1 DW, , phenolic acids 16.05 mg CAE g -1 DW, and proanthocyanidins 13.06 mg CE g -1 DW . Avarage flavonoid content was 2.95 QE g -1 and 6.47 RE g -1 DW for leaves and for fruits 0.55 QE g -1 and 1.24 RE g -1 DW (Table 1). According to the results of several studies given by EDWARDS ET AL. (2012) for C. monogyna fruits total phenols were in the range 9.1-17.8 mg g -1 and 16.42-57.07 mg g -1 ; total flavonoids 4.46-147 mg g -1 , , and total proanthocyanidins 19.29. mg g -1 . Also, leaves contained bioactive compounds in the following order: proanthocyanidins 32.83-53.48 mg g -1 and flavonoids 24.95-28.60 mg g -1 . Results obtained in this work for flavonoids and proanthocyanidins in leaves and fruits are lower than the above mentioned which can be explained by different ecological conditions (climate, type of soil, exposore to the light) (BAHRİ-SAHLOUL ET AL., 2009 A; LİU ET AL., 2005; BAHORUN ET AL., 1994).
Similarly to the results for C. monogyna, the most abundant compounds in of C. x macrocarpa leaf and fruit were phenols, phenolic acids and proanthocyanidins. Average contents in leaves were for phenols 82.44 mg GAE g -1 DW, phenolic acids 57.60 CAE g -1 DW and proanthocyanidins 43.11 mg CE g -1 DW. In fruits, it was determined avarage content of phenols 48.60 mg GAE g -1 DW, phenolic acids 31.72 CAE g -1 DW and proanthocyanidins 18.43 mg CE g -1 DW (Table 1). However, we could not find literature data to compare our results with the results of other investigators.
Generally, leaf extracts of C. monogyna had higher contents of investigated compounds than fruits except monomeric anthocyanins content (0.10 mg CG g -1 DW in leaves and 0.62 mg CG g -1 DW in fruits). ). This is in agreement with data given BY EDWARDS ET AL. (2012) for content of anthocyanins (0.15-0.58 mg g -1 ). Similar observations are found for C. x macrocarpa leaf (0.21 mg CG g -1 DW) and fruit extracts (0.55 mg CG g -1 DW). Also, several studies concerning different plants confirmed that level of anthocyanins are higher in fruits than in leaves which is probably connected with coloration role of anthocyanins in fruits (ABDENNACER ET AL., 2015 AND REFERENCES THEREIN). Compering content of investigated compounds between two species we can conclude that C. x macrocarpa leaves are richer in the content of phenols, flavonoids, phenolic acids, proanthocyanidins and anthocyanins while fruits of C. monogyna have only higher content of monomeric anhtocyanins.
Three different assays for the estimation of antioxidant capacity (AC) of plant extracts were used in this work. Since different reaction mechanisms can be involved in evaluation of antioxidant capacity two or more reactions are usually applied. In all cases, Trolox was used as a standard, and the results are expressed as Trolox equivalent antioxidant capacity (TEAC). Generally, the higher DPPH, ABTS and FRAP values point to greater antioxidant activity of the sample. Antioxidant activity of polyphenols is due to redox properties acting as a reducing agents, hydrogen donors and singlet oxygen quenchers (HANRAFI AND HAMRANI, 2010). The results are given in Table 2. Antioxidant capacities determined with DPPH, ABTS and FRAP were higher for C. x macrocarpa leaves and fruits samples than C. monogyna samples. Average values of antioxidant capacity found for C. monogyna leaves were DPPH =0.36 mmol Trolox g -1 DW, ABTS =0.47 mmol Trolox g -1 DW, and FRAP =0.41 mmol Trolox g -1 DW while for C. x macrocarpa leaves were DPPH =0.46 mmol Trolox g -1 DW, ABTS =0.72 mmol Trolox g -1 DW, and FRAP =0.50 mmol Trolox g -1 DW ( Table 2). Fruits of both species had lower antioxidant capacity than leaves. Average values of antioxidant capacity found for C. monogyna fruits were DPPH = 0.11 mmol Trolox g -1 DW, ABTS =0.25 mmol Trolox g -1 DW, FRAP =0.18 mmol Trolox g -1 DW while higher value were determined in C. x macrocarpa fruits DPPH =0.26 mmol Trolox g -1 DW, ABTS =0.51 mmol Trolox g -1 DW, and FRAP =0.32 mmol Trolox g -1 DW ( Table 2). This can be explained with higher contents of all investigated compounds in C. x macrocarpa leaves and fruits than C. monogyna samples. This is in agreement with several studies where it was found that antioxidant capacity of leaf extracts are higher than fruit extracts (ABDENNACER ET AL., 2015 AND REFERENCES THEREIN). We can also conclude that leaves and fruits of both Crataegus species have high antioxidant capacity. Values obtained for antioxidant capacity in this work for C. monogyna fruits are much higher than the values reported by RUIZ-RODRIGEZ ET AL. (2014) (TEAC = 1.54 -7.11 mmol Trolox 100g -1 fresh weight) and EGEA, ET AL. (2010) (TEAC ABTS = 8.43 µmol Trolox g -1 fresh weight). According to RUIZ-RODRIGEZ ET AL. (2014), AC for fresh fruits were in the range of 0.84 -6.12 mmol Trolox100 g -1 for ABTS assay; 0.76 -2.03 mmol Trolox100 g -1 for DPPH assy, and 3.28 -10.99 mmol Trolox100 g -1 for FRAP assay. ÖZYÜREK  found that different variety of C. monogyna leaves in Turkey had TEAC ABTS in range 0.077-0.330 mmol Trolox g -1 and TEAC FRAP in range 0.064 -0.141 mmol Trolox g -1 which is lower than values obtained in this work (Table 2). Also, very similar values of AC for DPPH i FRAP method were obtained for all investigated samples while AC values for ABTS were higher. They were in the following order: ABTS>FRAP>DPPH. Similar results were obtained for guava fruits extracts which is explained with differences in the ability of antioxidant compounds to reduce DPPH, ASBTS and FRAP reagents (THAIPONG ET AL., 2006). It is also reported that stereoselectivity of the reagents as well as solvent used for extractions can be important factors influencing on scavenging effect of plant extracts (YU ET AL., 2002). Lower results for FRAP assay can be the results of uncomplete reaction of the reagent with flavonoids and phenolic acids (BERKER ET AL., 2007).
Linear regression was used to establish correlation coefficients between contents of bioactive compounds and antioxidant capacities. The obtained results are presented in Table 3. Very high correlations were noticed between DPPH, ABTS and FRAP and contents of phenols, phenolic acids, and proanthocyanidins. Correlation coefficients for phenolic content and DPPH, ABTS, FRAP assay were 0.9618; 0.8949, 0.9695 respectively. Strong correlations showed ABTS (r 2 = 0.9443), FRAP (r 2 =0.9216) and DPPH method (r 2 =0.8703) with phenolic acid content. Also, strong correlations were observed between proanthocyanidins and DPPH (r 2 =0.8953), FRAP (r 2 =0.903) and ABTS (r 2 =0.7833). High correlations were also noticed between antioxidant capacity with DPPH and flavonoid content (r 2 =0.7302 and 0.7301) while other two methods showed insignificant correlations (0.4-0.55). Insignificant correlations were found between antioxidant capacity with all three methods and anthocyanins content.
These results suggest that phenols, phenolic acids and proanthocyanidins are major contributors to the antioxidant capacity as the most abundant compounds in leaves and fruits. Similarly, strong correlations were found for total phenols and proanthocyanidins with ABTS and FRAP in callus extracts (BAHORUN ET AL., 1994), phenols, proanthocyanidins, phenolic acids in fruit extracts (RUIZ-RODRIGUEZ ET AL., 2014), phenols and proanthocyanidins with DPPH and FRAP in fruit extracts (MRAIHI ET AL., 2013).
Interestingly, although flavonoid content expressed as quercetin was much lower compered to rutin equivalents, correlation coefficients for antioxidant capacities and flavonoid contents expressed as quercetin or rutin equivalents were very similar for the same method. This is in agreement with observation of JUNG  that quercetin (flavonol aglicon) has stronger antioxidant activity as a result of presence multiple hydroxyl groups.

CONCLUSION -Zaključak
Leaf and fruit extracts of C. monogyna and C. x macrocarpa were rich inphenolics, phenolic acids and proanthocyanidins.
Leaves of both species are richer in the content of phenols, flavonoids, phenolic acids and proanthocyanidins while fruits of both species are richer in content of monomeric anthocyanins.
C. x macrocarpa leaves had higher contents of all investigated compounds than C. monogyna. On the other hand, C. monogyna fruit had higher content of total monomeric anthocyanins than C. x macrocarpa fruit.
Leaves of both species had higher antioxidant activity than the fruits with all three methods. C. x macrocarpa leaves and fruits had higher antioxidant activity than C. monogyna leaves and fruits.
Our results indicate that phenols including phenolic acids and proanthocyanidins are most probably the major contributors to the antioxidant properties of leaves and fruits extracts. This is supported by high correlation coefficients obtained between different method for antioxidant capacity and content of phenols, phenolic acids and proanthocyanidins in this work.