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Seasonal changes of natural antioxidant content in the leaves of Hungarian forest trees
Industrial Crops and Products 98 (2017) 53–59
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Seasonal changes of natural antioxidant content in the leaves of Hungarian forest trees Esztella Tálos-Nebehaj ∗ , Tamás Hofmann, Levente Albert University of West-Hungary, Faculty of Forestry, Institute of Chemistry, Bajcsy-Zsilinszky Street 4, 9400 Sopron, Hungary
a r t i c l e
i n f o
Article history: Received 10 August 2016 Received in revised form 5 January 2017 Accepted 9 January 2017 Keywords: Forest trees Leaf Polyphenols Antioxidant capacity Seasonal variation
a b s t r a c t Despite the numerous investigations on the phenolic composition and antioxidant properties of different plant tissues, there are only a few studies about forest tree leaves, moreover, the monitoring of seasonal changes is an underexplored area. The present investigation aimed to provide a comparative analysis of the antioxidant properties of the leaves from 12 selected Hungarian coniferous and deciduous forest tree species, during the vegetation period. From the methanolic extracts, the total phenol, flavonoid and flavan-3-ol contents were determined, as well as the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ferric reducing ability of plasma (FRAP) antioxidant capacities. The investigated species revealed diverse seasonal tendencies in their chemical parameters, including seasonal extremities. According to results, polyphenols had a determinant role in the antioxidant efficiency of the leaf extracts. The evaluation of the overall antioxidant power was accomplished by a scoring system. The species with the highest antioxidant capacity values (scores) were: European hornbeam (Carpinus betulus L.) > sweet chestnut (Castanea sativa Mill.) > Turkey oak (Quercus cerris L.) > downy oak (Quercus pubescens Willd.) > pedunculate oak (Quercus robur L.). The leaves of the listed species could be future sources of food antioxidants and nutritional supplements. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Antioxidant compounds are found in numerous plant materials, such as fruits, cereal crops, vegetables, seeds, nuts, bark, roots, spices, herbs and leaves. The consumption of dietary plant antioxidants has been strongly associated with a decrease in the amount of free radicals and reactive oxygen species in the human body, thus, having a positive impact (anticancer, anti-inflammatory, antidiabetic, etc.) on health (Thériault et al., 2006). Despite the numerous investigation on the phenolic composition and antioxidant properties of fruits, vegetables and herbs (Campanella et al., 2003; Lim et al., 2007), there are limited studies on the antioxidant composition of forest tree leaves, with only certain species investigated in detail. Olive leaves were found to have beneficial health effects due to critical amounts of simple phenols, flavonoids and secoiridoids (Talhaoui et al., 2015). Katsube et al. (2009) evidenced five flavonol glycosides as well as quercetin 3-(6-malonylglucoside) were the most prominent antioxidant polyphenols in the leaf extracts of white mulberry
∗ Corresponding author. E-mail addresses: [email protected] (E. Tálos-Nebehaj), [email protected] (T. Hofmann), [email protected] (L. Albert). http://dx.doi.org/10.1016/j.indcrop.2017.01.011 0926-6690/© 2017 Elsevier B.V. All rights reserved.
(Morus alba L.) and concluded that the mulberry leaf could be a promising dietary source of antioxidants, such as quercetin. In silver birch (Betula pendula Roth) leaves, quercetin-glycosides, chlorogenic acid, and p-coumaric acid components were identified: antioxidant polyphenols which enable the potential innovative use of these leaf extracts in skin whitening products (Germanò et al., 2012). The leaves of Chinese crab apple (Malus hupehensis Pamp.), which are used as a common green tea, contained substantial amounts of flavonoids (Liu et al., 2015). In addition to polyphenols, terpene trilactones and ginkgolide derivatives are responsible for many of the dietary and health benefits of ginkgo (Ginkgo biloba L.) leaves (Pereira et al., 2015). The leaves of sweet chestnut (Castanea sativa Mill.) have been used in traditional folk medicine to treat coughing, diarrhea and rheumatic diseases. The main active compounds were identified as polyphenols (Díaz-Reinoso et al., 2011). The extracts of the needles and of the cones of Juniperus sibirica Burgsdorf. were found to exhibit potent antioxidant and anti-inflammatory effects. These effects were explained by high concentrations of luteolin-7-O-glucoside, apigenin-7-O-glucoside and rutin, yet authors pointed out that phenols are not the only compounds which are responsible for anti-inflammatory potency of the extracts (Lesjak et al., 2011). This study aimed to provide a comparative analysis of the antioxidant properties of the leaves from 12 common Hungarian
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forest tree species, namely European beech (Fagus sylvatica L.) (E. beech), European hornbeam (Carpinus betulus L.) (E. hornbeam), downy oak (Quercus pubescens Willd.), sweet chestnut (Castanea sativa Mill.), black locust (Robinia pseudoacacia L.), Norway maple (Acer platanoides L.), Turkey oak (Quercus cerris L.), pedunculate oak (Quercus robur L.), sessile oak (Quercus petraea Liebl.), poplar (Populus x euramericana Dode), Scots pine (Pinus sylvestris L.) and black pine (Pinus nigra J. F. Arnold). No specific applications for the leaves of these species have yet been identified, apart from sweet chestnut. However, the leaves of these species are forestry byproducts that represent a substantial amount of renewable biomass and could contain important amounts of antioxidant compounds. Considering forestry by-products of the investigated species, the bark of European beech (Fagus sylvatica L.) for example was found recently to contain significant amounts of extractable polyphenols with excellent antioxidant properties (Hofmann et al., 2015). The antioxidant capacity of the leaf extracts was measured by DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2 -azino-bis(3ethylbenzothiazoline-6-sulphonic acid)) and FRAP (ferric reducing ability of plasma) assays. The total polyphenol (TPC), total flavonoid (TFC) and total flavan-3-ol (TF3L) contents were also determined to identify the chemical class of the phenolic compounds responsible for the antioxidant effects. Additionally, the seasonal dynamics of the abovementioned chemical parameters were measured. The species/extracts which had the best antioxidant parameters, and thus could be a potential source of antioxidants for foods, nutrition supplements and in healthcare and medical products, or industrial material protecting products were identified.
2. Materials and methods 2.1. Chemicals and reagents Double distilled water was prepared for the extractions, using conventional distillation equipment. Methanol (HPLC grade) was obtained from VWR International (Budapest, Hungary). (+)-Catechin, quercetin, ascorbic acid, 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), ABTS, potassium persulfate, DPPH, 2,4,6-tripyridyl-S-triazine (TPTZ), iron(III)-chloride, p-dimethylaminocinnamaldehyde (DMACA), acetic acid, sodium acetate, potassium acetate, hydrochloric acid, sulfuric acid, sodium carbonate, potassium hydrogen phosphate and potassium-dihydrogen-phosphate were obtained from Sigma-Aldrich (Budapest, Hungary). Folin-Ciocâlteu reagent was purchased from Merck (Darmstadt, Germany).
2.2. Sample collection and extraction Leaf samples were harvested in the Botanic Garden of the University of West-Hungary, Sopron, between May and September 2014, on 5 occasions (15th–22nd of each month). The test species were as mentioned earlier E. beech, E. hornbeam, downy oak, sweet chestnut, black locust, Norway maple, Turkey oak, pedunculate oak, sessile oak, poplar, Scots pine and black pine. From each species, one healthy tree was investigated by taking 15 shade and 15 sun leaves at each sampling occasion. Leaves were combined and treated with microwave irradiation (1 min at 700 W in a household microwave oven) to ensure the inactivation of polyphenol oxidizing enzymes (Wang et al., 2008; Makk et al., 2013; Nebehaj et al., 2013). After, the leaf samples were ground and extraction was immediately performed, as follows: leaf samples (0.2 g leaf powder) were extracted with 20 mL methanol:water 80:20 v/v, for 20 min in an ultrasonic bath (Elma Transsonic T570, Elma Schmidbauer GmbH, Singen, Germany) at room temperature.
Extracts were filtered (0.45 m cellulose acetate syringe filters), prior to analysis.
2.3. Antioxidant assays A U-1500 spectrophotometer (Hitachi Ltd., Tokyo, Japan) was used. All measurements were done at least in triplicate. Trolox, (+)-catechin and rutin were used as the reference compounds. The ABTS assay was determined at 734 nm, based on the method of Stratil et al. (2007), using Trolox as standard and a 10 min reaction time. The results were expressed as mg equivalents of Trolox/g dry leaf units (mg TE/g dw.). The FRAP assay was performed as described by Benzie and Strain (1996), using ascorbic acid as standard. Results were expressed as mg equivalents of ascorbic acid/g dry leaf units (mg AAE/g dw.). The DPPH antiradical activity of the leaf extracts was determined using a slightly modified method of Sharma and Bhat (2009). Methanol (2090 L), methanolic DPPH solution (900 L, 2 × 10−4 M) and 10 L of the extract were mixed. After incubation in the dark at room temperature for 30 min, the decrease in absorbance was measured at 515 nm. Results were calculated as IC50 (50% inhibition concentration) and expressed as g extractives/mL assay (g/mL) units, representing the amount of extractives that react with 50% of the added DPPH• radicals in the total volume of the assay (3000 L) under the conditions used.
2.4. Determination of the TPC, TFC and TF3L levels The determination of TPC was achieved using the FolinCiocâlteu assay at 760 nm and quercetin as the standard (Singleton and Rossi, 1965). The results were expressed as mg equivalents of quercetin/g dry leaf units (mg QE/g dw.). The TFC was determined according to Kalita et al. (2013) at 415 nm, using quercetin as the standard and the results were expressed as mg equivalents of quercetin/g dry leaf units (mg QE/g dw.). The determination of TF3L was performed using the DMACA assay (Treutter, 1989): this method provides a highly specific reaction with catechin-type compounds and with their proanthocyanidin polymers (Treutter, 1989). The assay was performed as follows: 2350 L of methanol was mixed with 100 L DMACA-reagent (2 m/v% DMACA dissolved in methanol: 6N H2 SO4 50:50 v/v solution) and 20 L sample. The reaction was performed at room temperature for 20 min under minimal light conditions. Then, the absorbance was measured at 640 nm. (+)-Catechin was used as the reference compound and the results were expressed as mg equivalents of (+)-catechin/g dry leaf units (mg CE/g dw.). All measurements were done in triplicate by using a U-1500 type spectrophotometer (Hitachi Ltd., Tokyo, Japan).
2.5. Total extractives The extracts (5 mL) were evaporated to dryness at 70 ◦ C in a laboratory oven and the remaining solids were weighed. The total extractive content was expressed as mg extractives/mL extract unit. Results were used to calculate the DPPH IC50 values.
2.6. Statistics Analysis of variance (ANOVA) was performed using Statistica 11 (StatSoft Inc., Tulsa, USA) software, with post hoc Tukey’s HSD test to compare the respective chemical parameters of the leaf extracts. Prior to ANOVA, the values were tested for normal distribution and homogeneity of variances using Bartlett’s Chi-square test.
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Table 1 ABTSa antioxidant capacity of the leaves (mean ± standard deviation)b . ABTS (mg TE/g dw.)c Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
119.45 ± 6.64 a bc 329.78 ± 23.89 c h 323.63 ± 16.00 c h 136.04 ± 3.53 cd cd 166.48 ± 4.54 a e 158.35 ± 6.29 b de 257.14 ± 5.44 bc g 199.69 ± 5.42 d f 193.16 ± 7.36 b f 92.82 ± 1.67 a a 104.18 ± 2.95 a ab 99.53 ± 2.78 a ab
156.82 ± 4.82 b a 315.18 ± 3.21 bc e 304.78 ± 12.05 bc e 147.81 ± 2.19 d a 187.28 ± 4.57 bc b 235.75 ± 2.0 e c 274.81 ± 4.20 c d 197.52 ± 6.85 cd b 182.68 ± 2.39 b b 159.29 ± 3.75 d a 154.65 ± 0.52 d a 159.14 ± 2.24 d a
132.28 ± 11.60 a bc 280.83 ± 4.57 b f 199.10 ± 5.00 a e 111.96 ± 1.84 b a 187.34 ± 2.96 bc e 142.86 ± 2.47 a cd 189.82 ± 4.05 a e 125.65 ± 1.05 a b 155.39 ± 3.18 a d 125.81 ± 1.48 b b 141.21 ± 3.23 c c 133.58 ± 2.06 c bc
115.54 ± 3.55 a bc 293.92 ± 14.53 bc h 279.51 ± 13.34 b h 75.26 ± 8.23 a a 182.16 ± 4.52 b ef 208.51 ± 4.04 d fg 235.45 ± 22.96 bc g 140.73± 3.56 b cd 162.63 ± 2.59 a de 137.93 ± 6.32 c cd 105.26 ± 2.92 a b 121.69 ± 8.00 b bc
155.05 ± 11.46 b bc 236.20 ± 5.68 a f 275.78 ± 13.10 b g 126.97 ± 4.88 c a 194.96 ± 3.58 c de 179.62 ± 6.60 c cd 220.87 ± 22.59 ab ef 185.72 ± 5.44 c d 214.41 ± 2.35 c ef 130.53 ± 2.02 bc ab 122.70 ± 6.89 b a 108.37 ± 2.68 a a
a b c
2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). Different letters indicate significant differences at p < 0.05, between months (superscript) and species (subscript). mg equivalents of Trolox/g dry leaf units.
3. Results and discussion 3.1. Seasonal variations in the antioxidant capacity Measuring the antioxidant capacity of biological samples is complicated due to various influencing factors (e.g. the type of radicals used, sample matrix, solvent composition, pH and sensitivity of the test compounds (Balogh et al., 2010)), thus multiple methods are generally used. In this study, three assays (ABTS, FRAP, DPPH) were used to determine the antioxidant properties of the leaf extracts. Table 1 summarizes the results of the ABTS antioxidant capacity measurements. The highest values were observed in May and June for most of the species. The exceptions were Norway maple (September, 194.96 mg TE/g dw.) and sessile oak (September, 214.41 mg TE/g dw.). Sati et al. (2013) also reported a maximum ABTS during the fall, for Ginkgo biloba leaves. During the growing season, a typical July minimum was observed for sweet chestnut and all the oak species. Overall, E. hornbeam (May, 329.78 mg TE/g dw.), sweet chestnut (May, 323.63 mg TE/g dw.) and Turkey oak (June, 274.81 mg TE/g dw.) leaf extracts showed the strongest reducing power against the ABTS.+ radical ion. E. beech, Scots pine, black pine, poplar, and black locust had the lowest antioxidant capacities (115.54, 104.18, 99.53, 92.82 and 75.26 mg TE/g dw., respectively). It was difficult to compare these results with similar data found in the literature because the execution of the ABTS assay (and also of the other assays) often involves various approaches among researchers. Some researchers base their evaluation on the absorbance value read at the end of the reaction, which is then compared with the respective value of a given standard compound
(Razali et al., 2012). Others, use the absorbance at the start and at the end of the reaction to calculate the scavenging capacity (Fu et al., 2015). Furthermore, the standard compound used in the assay and the expressed units of the results are often inconsistent between researchers, so that a comparison of literature measurements is often not feasible. Antioxidant capacity can be related to either gram of extract (Uysal et al., 2016) or dry weight of the sample. According to Razali et al. (2012) the ABTS antioxidant capacity of the methanolic extracts of tamarind (Tamarandus indica) leaves was 1.65 mmol TE/g dw. This value was higher than the best performing samples (May extracts of E. hornbeam and sweet chestnut) of the current study (recalculated values: 1.39 and 1.22 mmol TE/g dw, respectively). Teleszko and Wojdyło (2015) determined the ABTS antioxidant capacity in the leaves of apple (Malus domestica) (35.94 mmol TE/100 g dw) and quince (Cydonia oblonga) (116.49 mmol TE/100 g dw). For comparative purposes, the recalculated value for E. hornbeam, and sweet chestnut was 138.7 and 122.4 mmol TE/100 g dw, respectively, indicating that leaves of these two forest trees had superior ABTS antioxidant capacity than Malus domestica and Cydonia oblonga. Table 2 summarizes the results of the FRAP antioxidant capacity determination. Similar to the ABTS method, significant differences between the months were observed for many species. Generally, the highest values were detected in August and September. The exceptions were black pine (July, 45.74 mg AAE/g dw.), Scots pine (June, 25.81 mg AAE/g dw.) and Turkey oak (May, 106.86 mg AAE/g dw.). According to the literature, other species, such as Ginkgo biloba, can also have maximum FRAP values during the initial (spring) growing season (Sati et al., 2013). The species with the highest FRAP antioxidant capacity were E. hornbeam (August, 106.24 mg AAE/g dw.), Turkey oak (May,
Table 2 FRAPa antioxidant capacity of the leaves (mean ± standard deviation)b . FRAP (mg AAE/g dw.)c . Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
30.26 ± 1.60 a bc 78.79 ± 0.39 ab g 80.24 ± 1.94 c g 34.93 ± 2.20 b c 46.19 ± 0.63 a d 48.93 ± 2.20 a d 106.86 ± 2.73 c h 60.34 ± 3.20 c e 68.59 ± 4.20 b f 21.68 ± 2.19 a a 18.10 ± 0.62 a a 24.25 ± 1.15 a ab
40.13 ± 0.71 c c 77.89 ± 1.21 a g 71.71 ± 3.37 b f 32.20 ± 0.49 b b 47.85 ± 2.54 a d 80.38 ± 2.58 c g 70.39 ± 1.94 a f 51.21 ± 1.82 b d 58.64 ± 2.11 a e 40.33 ± 3.12 b c 25.81 ± 0.86 d a 38.51 ± 0.79 b c
36.40 ± 0.53 b b 84.04 ± 2.67 b h 62.85 ± 2.57 a f 40.55 ± 2.63 c bcd 50.05 ± 1.82 a e 67.03 ± 2.12 b fg 69.23 ± 2.28 a g 43.09 ± 2.93 a cd 64.21 ± 2.52 ab fg 38.59 ± 1.27b bc 19.96 ± 0.33 b a 45.74 ± 1.80 d de
36.71 ± 0.63 b c 106.24 ± 3.10 d i 83.86 ± 2.28 c g 11.59 ± 0.72 a a 64.75 ± 1.37 b f 96.18 ± 1.83 d h 92.60 ± 2.87 b a 48.78 ± 0.65 b e 67.55 ± 1.43 b f 45.19 ± 0.92 c de 17.18 ± 0.66 a b 42.13 ± 0.78 c d
53.10 ± 1.53 d c 92.08 ± 1.21 c gh 93.28 ± 0.39 d h 51.41 ± 1.87 d c 69.27 ± 3.45 b d 79.56 ± 3.46 c e 87.61 ± 1.54 b fg 82.77 ± 6.37 d ef 85.92 ± 1.79 c f 47.88 ± 1.51 c bc 22.19 ± 1.10 c a 43.61 ± 1.74 cd b
a b c
Ferric reducing ability of plasma. Different letters indicate significant differences at p < 0.05, between months (superscript) and species (subscript). mg equivalents of ascorbic acid/g dry leaf units.
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Table 3 DPPHa antioxidant capacity of the leaves (mean ± standard deviation)b . DPPH (IC50 )c (g extractives/mL assay). Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
10.47 ± 1.25 a ab 6.87 ± 0.39 a a 11.90 ± 4.21 a ab 11.63 ± 1.73 a ab 6.59 ± 0.81 ab a 8.16 ± 1.49 a a 6.02 ± 0.93 a a 10.86 ± 2.42 a ab 9.16 ± 2.06 bc ab 29.98 ± 2.69 a c 30.42 ± 3.69 a c 17.10 ± 0.88 ab b
18.18 ± 1.92 c c 6.37 ± 1.90 a a 9.17 ± 2.98 a ab 18.16 ± 0.35 bc c 8.21 ± 0.66 b a 7.82 ± 0.35 a a 7.58 ± 0.35 ab a 10.34 ± 2.68 a ab 14.53 ± 1.38 d bc 24.40 ± 1.61 a d 38.04 ± 2.32 a e 17.62 ± 1.82 ab c
13.36 ± 0.63 ab cd 5.51 ± 0.85 a a 10.53 ± 2.16 a bc 10.19 ± 0.70 a bc 7.32 ± 0.44 ab ab 8.05 ± 0.38 a ab 7.21 ± 0.47 ab ab 10.35 ± 0.59 a bc 7.73 ± 0.67 ab ab 26.57 ± 1.58 a e 38.73 ± 2.19 a f 14.79 ± 0.47 ab d
15.23 ± 1.04 bc e 4.63 ± 0.88 a a 7.05 ± 0.66 a abc 22.79 ± 4.50 c f 6.36 ± 0.31 a ab 7.49 ± 0.78 a abc 6.20 ± 0.47 a ab 10.67 ±1.20 a bcd 11.74 ± 0.22 cd cde 15.70 ± 0.14 b de 42.38 ± 0.00 a g 14.03 ± 1.46 a de
11.35 ± 0.73 a cde 4.69 ± 0.28 a a 9.02 ± 0.30 a bcd 12.51 ± 1.23 ab de 6.30 ± 0.46 a ab 8.41 ± 0.43 a abcd 8.38 ± 0.89 b abc 11.11 ± 1.81 a cde 5.02 ± 0.40 a ab 13.97 ± 2.11 b e 37.83 ± 2.99 a g 18.87 ± 1.53 b f
a b c
2,2-diphenyl-1-picrylhydrazyl. Different letters indicate significant differences at p < 0.02, between months (superscript) and species (subscript). 50% inhibition concentration.
106.86 mg AAE/g dw.) and sweet chestnut (September, 93.28 mg AAE/g dw.). The leaf extracts of coniferous trees as well as of black locust (August, 11.59 mg AAE/g dw.) exhibited the weakest FRAP antioxidant power. The DPPH radical scavenging activity was characterized by the IC50 value (50% inhibition concentration), with low IC50 indicating high antioxidant capacities. According to Table 3, no significant differences were indicated between IC50 values and the month for E. hornbeam, sweet chestnut, downy oak, pedunculate oak and Scots pine. A marked decrease of the IC50 levels during the growing season was only observed for sessile oak and poplar, while for the remaining species, a fluctuation or slight increase in the IC50 levels was evident. Overall, E. hornbeam (August, 4.63 g/mL) and sessile oak (September, 5.02 g/mL) showed the best (lowest) IC50 values, while the highest values were determined for Scots pine throughout the entire growing season (30.42–42.38 g/mL). In Eucommia ulmoides Oliver leaf, which is a traditional medicine and functional food in China, Zhang et al. (2013) found the best DPPH IC50 values were obtained from the August extracts. Considering reference compounds, it was concluded that E. hornbeam leaf extracts have medium to high antioxidant power compared with respective DPPH IC50 (Trolox: 4.29 g/mL, (+)-catechin: 7.40 g/mL, rutin: 13.94 g/mL), FRAP (Trolox: 550 mg AAE/g, (+)-catechin: 524 mg AAE/g, rutin: 295 mg AAE/g) and ABTS ((+)-catechin: 3514 mg TE/g, rutin: 1760 mg TE/g) values. Seasonal tendencies of the FRAP, ABTS and DPPH antioxidant capacities of leaf extracts were found to be markedly different due to the selective variation of these methods. Results of the individual assays should be summarized to obtain a comprehensive measure of the overall antioxidant power of the leaf extracts. Moreover, the seasonal changes between various types of antioxi-
dant compounds, mainly polyphenols, also need to be performed. Such information can be used to determine which compounds or types of compounds are responsible for the antioxidant power of the extracts. For this purpose, the total polyphenol contents of the extracts were assayed and compared with their respective antioxidant capacity values. 3.2. Seasonal variation of phenolic compounds 3.2.1. Seasonal variation of the TPC Phenolic compounds contribute directly to the antioxidant power of leaf extracts (Awika et al., 2003), hence the TPC of the extract was determined (Table 4). For the majority of the species, the TPC increased significantly from May to August/September with some slight fluctuations in between. However, this tendency was not observed in Turkey oak, sweet chestnut and Scots pine, which had maximum TPC values in May/June. There have been numerous investigations on the seasonal TPC variations of tree leaves, which describe a general increasing tendency from spring to fall. In Moringa oleifera Lam. the TPC was lowest in the newly opened leaves and values increased gradually with the maturation of the leaves (Iqbal and Bhanger, 2006). Pirvu et al. (2013) also reported an increasing TPC of E. beech leaves during the vegetation period, with overall maximum values in September. The TPC of the methanolic leaf extracts of Gingko biloba L. was higher in the fall than in spring (Sati et al., 2013). In the two evergreen species, the TPC maximum was observed in June for Scots pine (42.56 mg QE/g dw.) and in July for black pine (53.80 mg QE/g dw.). Pines differ from the other test species as pine needles remain on the trees throughout the entire year and do not fall at regular intervals. The lifetime of pine needles can span from a few months to several decades, thus determining their degree of
Table 4 Total phenol content of the leaves (mean ± standard deviation)a . Total phenol content (mg QE/g dw.)b . Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
37.82 ± 1.01 a b 78.81 ± 0.59 a f 71.30 ± 3.53 ab e 29.30 ± 0.99 b a 46.63 ± 2.80 a c 37.52 ± 0.54 a b 78.99 ± 3.35 c f 59.85 ± 2.27 b d 56.47 ± 1.34 a d 29.03 ± 0.29 a a 27.12 ± 0.78 a a 27.37 ± 0.97 a a
56.83 ± 0.99 c d 93.08 ± 3.29 b h 78.52 ± 1.13 b g 29.73 ± 0.73 b a 57.37 ± 1.98 b d 71.68 ± 2.32 c fg 65.82 ± 1.59 ab ef 58.46 ± 2.50 b de 51.23 ± 3.89 a cd 53.63 ± 3.24 b cd 42.56 ± 1.62 b b 46.61 ± 4.23 b bc
48.12 ± 1.28 b bc 105.93 ± 5.57 c h 62.51 ± 1.59 a de 43.25 ± 0.21 c ab 80.21 ± 1.4 e g 63.80 ± 3.31 bc e 65.89 ± 1.54 ab ef 48.32 ± 4.82 a bc 59.15 ± 4.06 a de 73.73 ± 3.05 d fg 37.47 ± 3.9 b a 53.80 ± 1.56 b cd
47.38 ± 3.14 b b 94.27 ± 5.38 b g 76.32 ± 5.25 b f 19.89 ± 2.89 a a 65.88 ± 2.83 c def 92.85 ± 3.67 d g 73.73 ± 5.81 bc ef 49.36 ± 4.21 a bc 53.70 ± 2.14 a bcd 61.96 ± 2,74 c cde 24.63 ± 3.98 a a 45.39 ± 5.51 b b
57.71 ± 2.59 c bc 80.81 ± 3.05 a e 75.49 ± 4.67 b de 49.63 ± 1.84 d b 73.73 ± 1.26 d de 60.31 ± 5.73 b c 59.69 ± 2.52 a c 72.21 ± 0.46 c d 70.01 ± 2.42 b d 57.61 ± 0.58 bc bc 36.07 ± 1.74 b a 35.78 ± 2.30 a a
a b
Different letters indicate significant differences at p < 0.05, between months (superscript) and species (subscript). mg equivalents of quercetin/g dry leaf units.
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Table 5 Total flavonoid content of the leaves (mean ± standard deviation)a . Total flavonoid content (mg QE/g dw.)b . Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
4.34 ± 0.07 c cd 11.05 ± 0.52 b i 5.72 ± 0.28 a fg 4.65 ± 0.23 c cde 5.33 ± 0.46 a efg 3.80 ± 0.31 a c 5.99 ± 0.21 b g 5.44 ± 0.40 ab efg 4.85 ± 0.41 b def 8.40 ± 0.39 a h 1.90 ± 0.04 a b 0.82 ± 0.04 a a
4.15 ± 0.10 c de 11.03 ± 0.75 b h 4.73 ± 0.53 a ef 4.43 ± 0.26 bc ef 5.33 ± 0.19 a fg 3.26 ± 0.07 a bcd 4.03 ± 0.24 a cde 5.71 ± 0.28 b g 3.18 ± 0.30 a bc 15.10 ± 0.09 d i 2.57 ± 0.09 c b 1.07 ± 0.08 ab a
2.80 ± 0.14 b bc 10.03 ± 0.68 b g 4.66 ± 0.38 a e 4.27 ± 0.24 bc de 6.06 ± 0.15 b f 3.71 ± 0.36 a cde 3.76 ± 0.52 a cde 4.59 ± 1.02 ab e 3.19 ± 0.19 a bcd 12.82 ± 0.37 c h 2.30 ± 0.09 b b 0.93 ± 0.17 ab a
2.23 ± 0.07 a b 12.84 ± 0.28 c h 5.15 ± 0.09 a f 2.60 ± 0.16 a bc 6.74 ± 0.20 b g 3.75 ± 0.39 a d 4.52 ± 0.24 a e 4.09 ± 0.19 a de 2.96 ± 0.05 a c 12.78 ± 0.16 c h 2.36 ± 0.03 b b 1.46 ± 0.06 c a
2.70 ± 0.09 b bc 8.42 ± 0.26 a g 5.16 ± 0.37 a e 4.00 ± 0.11 b d 6.47 ± 0.23 b f 3.25 ± 0.47 a cd 3.93 ± 0.23 a d 5.54 ± 0.43 ab e 3.54 ± 0.35 a d 10.22 ± 0.05 b h 2.09 ± 0.09 a b 1.12 ± 0.07 b a
a b
Different letters indicate significant differences at p < 0.05, between months (superscript) and species (subscript). mg equivalents of quercetin/g dry leaf units.
maturity is complicated (Ewers and Schmid, 1981) and these type of measurements have not been carried out. Overall, black locust had the lowest TPC (August, 19.89 mg QE/g dw.) while the highest levels were measured in E. hornbeam (July, 105.93 mg QE/g dw.), downy oak (August, 92.85 mg QE/g dw.), Turkey oak (May, 78.99 mg QE/g dw.) and sweet chestnut (June, 78.52 mg QE/g dw.). Pedunculate oak, sessile oak, E. beech, poplar and the conifers were characterized by mediocre TPC (27.12–73.73 mg QE/g dw.).
3.2.2. Seasonal variation of the TFC The characteristics of the seasonal TFC variations (Table 5) differed significantly from the seasonal TPC variation. The TFC values decreased from May to August/September in E. beech, Turkey oak, sessile oak and black locust, while a seasonal increase in the TFC was measured in E. hornbeam, Norway maple, poplar and in the pines. The seasonal tendency determined for E. beech corresponds with the previous results of Pirvu et al. (2013). According to the results, the seasonal TFC variation in the leaves of forest trees is a characteristic of the investigated species (similar to the previously discussed ABTS, FRAP, DPPH and TPC seasonal tendencies). In August, a seasonal minimum was observed for black locust (2.60 mg QE/g dw.) and pedunculate oak (4.09 mg QE/g dw.) while in E. hornbeam (12.84 mg QE/g dw.) and Norway maple (6.74 mg QE/g dw.) and black pine (1.46 mg QE/g dw.) a seasonal maximum was determined in the same month. A seasonal maximum in May was measured for E. beech (4.34 mg QE/g dw.), sweet chestnut (5.72 mg QE/g dw.), black locust (4.65 mg QE/g dw.), downy oak (3.80 mg QE/g dw.), Turkey oak (5.99 mg QE/g dw.) and sessile oak (4.85 mg QE/g dw.), similar to that found in the leaves of Eucommia ulmoides Oliver (Zhang et al., 2013). It should also be noted that seasonal TFC variation can also depend
on the gender: Zhang et al. (2016) determined that the seasonal TFC (March–September) of female individuals of Pistacia chinensis Bunge tended to increase, while the male individuals tended to decrease. Overall, the highest TFC was found in poplar (June, 15.10 mg QE/g dw.) and in E. hornbeam (August, 12.84 mg QE/g dw.), while the lowest TFCs were measured in the coniferous species (0.82–2.57 mg QE/g dw.).
3.2.3. Seasonal variation of the TF3L Results of the TF3L measurements are summarized in Table 6. Similar to the previous parameters investigated in this study, the TF3L of leaf extracts followed diversified seasonal tendencies. A characteristic increase from spring to fall was evidenced for sweet chestnut, downy oak and E. beech. For the remaining species, a maximum value was observed at discrete months. A July maximum was determined for black locust, Norway maple, black pine and E. hornbeam. Downy oak and Turkey oak had an August maximum, while pedunculate oak, poplar and Scots pine had seasonal maximum TF3L levels in June. The highest TF3L was measured in E. beech (September, 24.05 mg CE/g dw.), black pine (July, 18.89 mg CE/g dw.), Norway maple (July, 18.69 mg CE/g dw.), black locust (July, 16.45 mg CE/g dw.), and pedunculate oak (June, 15.55 mg CE/g dw.). Scots pine, sessile oak and downy oak had moderate values (0.52–7.71 mg CE/g dw.), while the lowest TF3L content was measured in E. hornbeam, sweet chestnut and Turkey oak (0.29–4.90 mg CE/g dw.). The measured values for E. beech, are comparable to previous results obtained for the yellow and green leaves of E. beech, wherein the TF3L content varied between 30–100 mg CE/g dw., depending on the year of the study and the health state (yellow/green) of the leaves (Sen et al., 2013). Feucht et al. (1994) concluded that flavan-
Table 6 Flavan-3-ol content of the leaves (mean ± standard deviation)a . Flavan-3-ol content (mg CE/g dw.)b . Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
12.41 ± 0.40 a hi 0.47 ± 0.02 a b 0.29 ± 0.00 a a 11.66 ± 0.46 bc h 7.27 ± 0.11 a fg 0.52 ± 0.01 a c 1.38 ± 0.04 a d 13.28 ± 1.10 b i 6.66 ± 1.20 d f 4.17 ± 0.15 a e 4.27 ± 0.18 b e 7.86 ± 0.27 a g
22.57 ± 1.04 c i 0.73 ± 0.01 b a 1.36 ± 0.02 c b 12.81 ± 0.44 c h 10.87 ± 0.80 b h 3.65 ± 0.01 b e 2.08 ± 0.01 b c 15.55 ± 1.03 c f 2.50 ± 0.06 ab d 11.38 ± 0.24 d h 7.71 ± 0.1 e g 13.07 ± 0.55 c h
22.97 ± 0.65 c k 1.82 ± 0.0 e b 1.13 ± 0.01 b a 16.45 ± 0.99 d i 18.69 ± 0.75 c j 4.35 ± 0.04 c e 2.27 ± 0.04 b c 14.63 ± 0.11 bc h 3.44 ± 0.12 c d 7.84 ± 0.08 b g 5.66 ± 0.33 d f 18.89 ± 0.29 d j
15.45 ± 0.61 b h 1.66 ± 0.01 d a 3.43 ± 0.06 d cd 3.27 ± 0.19 a c 11.55 ± 0.57 b g 5.85 ± 0.0 e e 4.87 ± 0.10 d e 9.67 ± 0.67 a f 2.08 ± 0.11 a b 8.27 ± 0.19 b f 3.64 ± 0.10 a d 11.76 ± 0.61 b g
24.05 ± 0.10 c g 1.10 ± 0.05 c a 4.90 ± 0.1 e c 10.94 ± 0.18 b e 11.16 ± 0.61 b e 5.15 ± 0.04 d c 2.73 ± 0.10 c b 13.30 ± 0.46 b f 2.90 ± 0.07 bc b 9.56 ± 0.42 c d 5.04 ± 0.12 c c 9.13 ± 0.20 a d
a b
Different letters indicate significant differences at p < 0.02, between months (superscript) and species (subscript). mg equivalents of (+)-catechin/g dry leaf units.
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Fig. 1. Seasonal variations of the total phenol content and antioxidant capacities in European hornbeam leaves. 1: mg equivalents of quercetin/g dry leaf units. 2: Ferric reducing ability of plasma. 3: mg equivalents of ascorbic acid/g dry leaf units. 4: 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). 5: mg equivalents of Trolox/g dry leaf units. Table 7 Overall antioxidant power of the leaf extracts evaluated by the scoring system. Combined antioxidant values. Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
1.21 2.65 2.50 1.30 1.67 1.62 2.68 1.84 1.94 0.50 0.50 0.90
1.26 2.59 2.41 1.14 1.73 2.27 2.32 1.74 1.65 1.11 0.58 1.27
1.25 2.54 1.87 1.30 1.77 1.76 1.99 1.38 1.79 0.90 0.44 1.32
1.14 2.85 2.50 0.52 1.93 2.34 2.44 1.49 1.74 1.31 0.18 1.25
1.57 2.48 2.53 1.41 2.03 2.02 2.27 2.01 2.32 1.35 0.42 1.09
3-ols play a major role in the plant antioxidant system and in the defense reactions of the E. beech leaves. 3.3. Evaluation of the antioxidant assays The ABTS, DPPH and FRAP methods show a diverse selectivity to the various types of antioxidant compounds (Müller et al., 2011), thus a summarized evaluation of these methods is needed to obtain a comprehensive measure of the overall antioxidant efficiency of the leaf extracts. This was achieved by a scoring system, assigning 0 points to the weakest values and 1 to the best values within each antioxidant capacity method, using linear approximation between lowest and highest values. In the ABTS and FRAP methods, the lowest value was scored 0 and the highest value was scored 1. For the DPPH values, the opposite scoring system was used because the lowest IC50 value (score: 1) represent the highest antioxidant capacity, and the highest IC50 (score: 0) represent the weakest antioxidant power. The DPPH, FRAP and ABTS scores for each sample were summed up to obtain a measure of their overall antioxidant efficiency. Results of the overall antioxidant power of the samples are summarized in Table 7. The differences between values are demonstrated with the aid of gray shading. According to Table 7, the species with the overall best antioxidant power were E. hornbeam, sweet chestnut, Turkey oak and downy oak. The leaves of the most abundant Hungarian forest tree species (pedunculate oak, sessile oak, E. beech and black locust) were characterized by medium to low antioxidant power, while poplar and the coniferous trees had the weakest antioxidant capacity. By
comparing the TPC values (Table 4) with the scores in Table 7 it was noticed that both the highest TPC and scores occurred in respective samples of E. hornbeam, sweet chestnut, downy oak and Turkey oak. It was established that in E. hornbeam, sweet chestnut and downy oak, the polyphenols are primarily responsible for the antioxidant properties of the leaf extracts. Regarding E. hornbeam, which was the overall best-performing species in this study, its leaves had high TFL (Table 5) and relatively low TF3L (Table 6) levels. Thus, flavonoid-type compounds, as well as other polyphenols, without the flavan-3-ol structure (e.g. tannins, phenolic acids), are responsible for the excellent antioxidant power of E. hornbeam leaves. Nevertheless, antioxidant properties and phenolic concentrations followed diverse seasonal tendencies (Fig. 1). It was assumed that the optimum antioxidant properties of E. hornbeam leaves were between July and August. Similar tendencies can be suggested for sweet chestnut and downy oak, although their TFC is lower and their TF3L somewhat higher compared with E. hornbeam. The biosynthesis of phenolic antioxidants depends on environmental factors (e.g. light, temperature, water availability, mineral nutrition) and also on genetic factors, which can consequently affect the antioxidant properties of the leaves. The seasonal variation of leaves’ polyphenol and overall antioxidant content is also closely related to the plant’s adaptation to biotic and abiotic environmental factors (Nour et al., 2014). Each species has its highest antioxidant values in distinct months; hence, the harvesting time of the leaves should be carefully selected when the future use of the leaves is being considered. 4. Conclusions In this work the detailed comparative analysis of the antioxidant properties of the leaves from 12 selected Hungarian coniferous and deciduous forest tree species were targeted. Sample collection was carried out between May and September which enabled to track seasonal changes of the antioxidant properties and of the major groups of polyphenolic compounds, too. Results indicated that seasonal variations of the measured chemical parameters in the leaves are highly dependent on the wood species. Each species has its highest antioxidant values in distinct months; hence, the harvesting time of the leaves should be carefully selected when the future use of the leaves is being considered. According to the present work the leaf extracts of E. hornbeam, sweet chestnut and downy oak exhibited the highest antioxidant
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capacities. E. hornbeam leaf extracts have stronger antioxidant properties and higher concentrations of polyphenolic compounds than sweet chestnut, which has been used as a traditional medicine for various ailments due to the high antioxidant concentration of its leaves (Díaz-Reinoso et al., 2011). Despite the superior antioxidant properties of E. hornbeam leaf extracts, its physiological effects and possible beneficial impacts on human health have not yet been investigated thoroughly; only limited data is found in the literature (Müller et al., 2011). The high-performance liquid chromatographic separation and mass spectrometric identification of the polyphenolic antioxidants in the leaves of E. hornbeam has already been carried out by Hofmann et al. (2016) in an earlier study. Altogether 171 compounds, including phenolic acids, flavonoid glycosides, tannins, catechins and procyanidins have been characterized and identified. According to the HPLC-PDA peak areas, the most abundant polyphenolic compounds in the August leaf extracts were chlorogenic acid, ellagic acid, ellagitannins, myricetin-, luteolin-, quercetin- and apigenin glycosides. The authors concluded that further research is needed for the elucidation of the compounds which are primarily responsible for the exceptional antioxidant properties of E. hornbeam leaves. The leaves of forest trees are a renewable biomass, which is an abundant source of antioxidants that could be used for nutritional purposes (e.g. food additives, food preservatives), healthcare products and medical applications. The leaves of the species with promising antioxidant properties and polyphenol contents (E. hornbeam, sweet chestnut and downy oak) should be tested in the future to determine their antibacterial, antiproliferative and antiviral effects and to unveil their potential as a source of antioxidants in food and healthcare products. Acknowledgement This research was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and financed by the VKSZ 12-1-2013-0034 Agrárklíma. 2 project. References Awika, J.M., Rooney, L.W., Wu, X., Prior, R.L., Zevallos, L.C., 2003. Screening methods to measure antioxidant activity of sorghum (Sorghum bicolor) and sorghum products. J. Agric. Food Chem. 51, 6657–6662. Balogh, E., Heged"us, A., Stefanovits-Bányai, É., 2010. Application and correlation among antioxidant and antiradical assays for characterizing antioxidant capacity of berries. Sci. Hortic. 125, 332–336. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 70–76. Campanella, L., Bonanni, A., Favero, G., Tomassetti, M., 2003. Determination of antioxidant properties of aromatic herbs, olives and fresh fruit using an enzymatic sensor. Anal. Bioanal. Chem. 375, 1011–1016. Díaz-Reinoso, B., Moure, A., Domínguez, H., Parajó, J.C., 2011. Membrane concentration of antioxidants from Castanea sativa leaves aqueous extracts. Chem. Eng. J. 175, 95–102. Ewers, F.W., Schmid, R., 1981. Longevity of needle fascicles of Pinus longaeva (Bristlecone Pine) and other North American pines. Oecologia 51, 107–115. Feucht, W., Treutter, D., Christ, E., 1994. Accumulation of flavanols in yellowing beech leaves from forest decline sites. Tree Physiol. 14, 403–412. Fu, R., Zhang, Y., Guo, Y., Chen, F., 2015. Chemical composition, antioxidant and antimicrobial activity of Chinese tallow tree leaves. Ind. Crops Prod. 76, 374–377. Germanò, M.P., Cacciola, F., Donato, P., Dugo, P., Certo, G., D’Angelo, V., Mondello, L., Rapisarda, A., 2012. Betula pendula leaves: polyphenolic characterization and potencial innovative use in skin whitening products. Fitoterapia 83, 877–882. Hofmann, T., Nebehaj, E., Albert, L., 2015. The high-performance liquid chromatography/multistage electrospray mass spectrometric investigation and extraction optimization of beech (Fagus sylvatica L.) bark polyphenols. Ind. Crops Prod. 1393, 96–105. Hofmann, T., Nebehaj, E., Albert, L., 2016. Antioxidant properties and detailed polyphenol profiling of European hornbeam (Carpinus betulus L.) leaves by multiple antioxidant capacity assays and high-performance liquid
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Seasonal changes of natural antioxidant content in the leaves of Hungarian forest trees Esztella Tálos-Nebehaj ∗ , Tamás Hofmann, Levente Albert University of West-Hungary, Faculty of Forestry, Institute of Chemistry, Bajcsy-Zsilinszky Street 4, 9400 Sopron, Hungary
a r t i c l e
i n f o
Article history: Received 10 August 2016 Received in revised form 5 January 2017 Accepted 9 January 2017 Keywords: Forest trees Leaf Polyphenols Antioxidant capacity Seasonal variation
a b s t r a c t Despite the numerous investigations on the phenolic composition and antioxidant properties of different plant tissues, there are only a few studies about forest tree leaves, moreover, the monitoring of seasonal changes is an underexplored area. The present investigation aimed to provide a comparative analysis of the antioxidant properties of the leaves from 12 selected Hungarian coniferous and deciduous forest tree species, during the vegetation period. From the methanolic extracts, the total phenol, flavonoid and flavan-3-ol contents were determined, as well as the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ferric reducing ability of plasma (FRAP) antioxidant capacities. The investigated species revealed diverse seasonal tendencies in their chemical parameters, including seasonal extremities. According to results, polyphenols had a determinant role in the antioxidant efficiency of the leaf extracts. The evaluation of the overall antioxidant power was accomplished by a scoring system. The species with the highest antioxidant capacity values (scores) were: European hornbeam (Carpinus betulus L.) > sweet chestnut (Castanea sativa Mill.) > Turkey oak (Quercus cerris L.) > downy oak (Quercus pubescens Willd.) > pedunculate oak (Quercus robur L.). The leaves of the listed species could be future sources of food antioxidants and nutritional supplements. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Antioxidant compounds are found in numerous plant materials, such as fruits, cereal crops, vegetables, seeds, nuts, bark, roots, spices, herbs and leaves. The consumption of dietary plant antioxidants has been strongly associated with a decrease in the amount of free radicals and reactive oxygen species in the human body, thus, having a positive impact (anticancer, anti-inflammatory, antidiabetic, etc.) on health (Thériault et al., 2006). Despite the numerous investigation on the phenolic composition and antioxidant properties of fruits, vegetables and herbs (Campanella et al., 2003; Lim et al., 2007), there are limited studies on the antioxidant composition of forest tree leaves, with only certain species investigated in detail. Olive leaves were found to have beneficial health effects due to critical amounts of simple phenols, flavonoids and secoiridoids (Talhaoui et al., 2015). Katsube et al. (2009) evidenced five flavonol glycosides as well as quercetin 3-(6-malonylglucoside) were the most prominent antioxidant polyphenols in the leaf extracts of white mulberry
∗ Corresponding author. E-mail addresses: [email protected] (E. Tálos-Nebehaj), [email protected] (T. Hofmann), [email protected] (L. Albert). http://dx.doi.org/10.1016/j.indcrop.2017.01.011 0926-6690/© 2017 Elsevier B.V. All rights reserved.
(Morus alba L.) and concluded that the mulberry leaf could be a promising dietary source of antioxidants, such as quercetin. In silver birch (Betula pendula Roth) leaves, quercetin-glycosides, chlorogenic acid, and p-coumaric acid components were identified: antioxidant polyphenols which enable the potential innovative use of these leaf extracts in skin whitening products (Germanò et al., 2012). The leaves of Chinese crab apple (Malus hupehensis Pamp.), which are used as a common green tea, contained substantial amounts of flavonoids (Liu et al., 2015). In addition to polyphenols, terpene trilactones and ginkgolide derivatives are responsible for many of the dietary and health benefits of ginkgo (Ginkgo biloba L.) leaves (Pereira et al., 2015). The leaves of sweet chestnut (Castanea sativa Mill.) have been used in traditional folk medicine to treat coughing, diarrhea and rheumatic diseases. The main active compounds were identified as polyphenols (Díaz-Reinoso et al., 2011). The extracts of the needles and of the cones of Juniperus sibirica Burgsdorf. were found to exhibit potent antioxidant and anti-inflammatory effects. These effects were explained by high concentrations of luteolin-7-O-glucoside, apigenin-7-O-glucoside and rutin, yet authors pointed out that phenols are not the only compounds which are responsible for anti-inflammatory potency of the extracts (Lesjak et al., 2011). This study aimed to provide a comparative analysis of the antioxidant properties of the leaves from 12 common Hungarian
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forest tree species, namely European beech (Fagus sylvatica L.) (E. beech), European hornbeam (Carpinus betulus L.) (E. hornbeam), downy oak (Quercus pubescens Willd.), sweet chestnut (Castanea sativa Mill.), black locust (Robinia pseudoacacia L.), Norway maple (Acer platanoides L.), Turkey oak (Quercus cerris L.), pedunculate oak (Quercus robur L.), sessile oak (Quercus petraea Liebl.), poplar (Populus x euramericana Dode), Scots pine (Pinus sylvestris L.) and black pine (Pinus nigra J. F. Arnold). No specific applications for the leaves of these species have yet been identified, apart from sweet chestnut. However, the leaves of these species are forestry byproducts that represent a substantial amount of renewable biomass and could contain important amounts of antioxidant compounds. Considering forestry by-products of the investigated species, the bark of European beech (Fagus sylvatica L.) for example was found recently to contain significant amounts of extractable polyphenols with excellent antioxidant properties (Hofmann et al., 2015). The antioxidant capacity of the leaf extracts was measured by DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2 -azino-bis(3ethylbenzothiazoline-6-sulphonic acid)) and FRAP (ferric reducing ability of plasma) assays. The total polyphenol (TPC), total flavonoid (TFC) and total flavan-3-ol (TF3L) contents were also determined to identify the chemical class of the phenolic compounds responsible for the antioxidant effects. Additionally, the seasonal dynamics of the abovementioned chemical parameters were measured. The species/extracts which had the best antioxidant parameters, and thus could be a potential source of antioxidants for foods, nutrition supplements and in healthcare and medical products, or industrial material protecting products were identified.
2. Materials and methods 2.1. Chemicals and reagents Double distilled water was prepared for the extractions, using conventional distillation equipment. Methanol (HPLC grade) was obtained from VWR International (Budapest, Hungary). (+)-Catechin, quercetin, ascorbic acid, 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), ABTS, potassium persulfate, DPPH, 2,4,6-tripyridyl-S-triazine (TPTZ), iron(III)-chloride, p-dimethylaminocinnamaldehyde (DMACA), acetic acid, sodium acetate, potassium acetate, hydrochloric acid, sulfuric acid, sodium carbonate, potassium hydrogen phosphate and potassium-dihydrogen-phosphate were obtained from Sigma-Aldrich (Budapest, Hungary). Folin-Ciocâlteu reagent was purchased from Merck (Darmstadt, Germany).
2.2. Sample collection and extraction Leaf samples were harvested in the Botanic Garden of the University of West-Hungary, Sopron, between May and September 2014, on 5 occasions (15th–22nd of each month). The test species were as mentioned earlier E. beech, E. hornbeam, downy oak, sweet chestnut, black locust, Norway maple, Turkey oak, pedunculate oak, sessile oak, poplar, Scots pine and black pine. From each species, one healthy tree was investigated by taking 15 shade and 15 sun leaves at each sampling occasion. Leaves were combined and treated with microwave irradiation (1 min at 700 W in a household microwave oven) to ensure the inactivation of polyphenol oxidizing enzymes (Wang et al., 2008; Makk et al., 2013; Nebehaj et al., 2013). After, the leaf samples were ground and extraction was immediately performed, as follows: leaf samples (0.2 g leaf powder) were extracted with 20 mL methanol:water 80:20 v/v, for 20 min in an ultrasonic bath (Elma Transsonic T570, Elma Schmidbauer GmbH, Singen, Germany) at room temperature.
Extracts were filtered (0.45 m cellulose acetate syringe filters), prior to analysis.
2.3. Antioxidant assays A U-1500 spectrophotometer (Hitachi Ltd., Tokyo, Japan) was used. All measurements were done at least in triplicate. Trolox, (+)-catechin and rutin were used as the reference compounds. The ABTS assay was determined at 734 nm, based on the method of Stratil et al. (2007), using Trolox as standard and a 10 min reaction time. The results were expressed as mg equivalents of Trolox/g dry leaf units (mg TE/g dw.). The FRAP assay was performed as described by Benzie and Strain (1996), using ascorbic acid as standard. Results were expressed as mg equivalents of ascorbic acid/g dry leaf units (mg AAE/g dw.). The DPPH antiradical activity of the leaf extracts was determined using a slightly modified method of Sharma and Bhat (2009). Methanol (2090 L), methanolic DPPH solution (900 L, 2 × 10−4 M) and 10 L of the extract were mixed. After incubation in the dark at room temperature for 30 min, the decrease in absorbance was measured at 515 nm. Results were calculated as IC50 (50% inhibition concentration) and expressed as g extractives/mL assay (g/mL) units, representing the amount of extractives that react with 50% of the added DPPH• radicals in the total volume of the assay (3000 L) under the conditions used.
2.4. Determination of the TPC, TFC and TF3L levels The determination of TPC was achieved using the FolinCiocâlteu assay at 760 nm and quercetin as the standard (Singleton and Rossi, 1965). The results were expressed as mg equivalents of quercetin/g dry leaf units (mg QE/g dw.). The TFC was determined according to Kalita et al. (2013) at 415 nm, using quercetin as the standard and the results were expressed as mg equivalents of quercetin/g dry leaf units (mg QE/g dw.). The determination of TF3L was performed using the DMACA assay (Treutter, 1989): this method provides a highly specific reaction with catechin-type compounds and with their proanthocyanidin polymers (Treutter, 1989). The assay was performed as follows: 2350 L of methanol was mixed with 100 L DMACA-reagent (2 m/v% DMACA dissolved in methanol: 6N H2 SO4 50:50 v/v solution) and 20 L sample. The reaction was performed at room temperature for 20 min under minimal light conditions. Then, the absorbance was measured at 640 nm. (+)-Catechin was used as the reference compound and the results were expressed as mg equivalents of (+)-catechin/g dry leaf units (mg CE/g dw.). All measurements were done in triplicate by using a U-1500 type spectrophotometer (Hitachi Ltd., Tokyo, Japan).
2.5. Total extractives The extracts (5 mL) were evaporated to dryness at 70 ◦ C in a laboratory oven and the remaining solids were weighed. The total extractive content was expressed as mg extractives/mL extract unit. Results were used to calculate the DPPH IC50 values.
2.6. Statistics Analysis of variance (ANOVA) was performed using Statistica 11 (StatSoft Inc., Tulsa, USA) software, with post hoc Tukey’s HSD test to compare the respective chemical parameters of the leaf extracts. Prior to ANOVA, the values were tested for normal distribution and homogeneity of variances using Bartlett’s Chi-square test.
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Table 1 ABTSa antioxidant capacity of the leaves (mean ± standard deviation)b . ABTS (mg TE/g dw.)c Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
119.45 ± 6.64 a bc 329.78 ± 23.89 c h 323.63 ± 16.00 c h 136.04 ± 3.53 cd cd 166.48 ± 4.54 a e 158.35 ± 6.29 b de 257.14 ± 5.44 bc g 199.69 ± 5.42 d f 193.16 ± 7.36 b f 92.82 ± 1.67 a a 104.18 ± 2.95 a ab 99.53 ± 2.78 a ab
156.82 ± 4.82 b a 315.18 ± 3.21 bc e 304.78 ± 12.05 bc e 147.81 ± 2.19 d a 187.28 ± 4.57 bc b 235.75 ± 2.0 e c 274.81 ± 4.20 c d 197.52 ± 6.85 cd b 182.68 ± 2.39 b b 159.29 ± 3.75 d a 154.65 ± 0.52 d a 159.14 ± 2.24 d a
132.28 ± 11.60 a bc 280.83 ± 4.57 b f 199.10 ± 5.00 a e 111.96 ± 1.84 b a 187.34 ± 2.96 bc e 142.86 ± 2.47 a cd 189.82 ± 4.05 a e 125.65 ± 1.05 a b 155.39 ± 3.18 a d 125.81 ± 1.48 b b 141.21 ± 3.23 c c 133.58 ± 2.06 c bc
115.54 ± 3.55 a bc 293.92 ± 14.53 bc h 279.51 ± 13.34 b h 75.26 ± 8.23 a a 182.16 ± 4.52 b ef 208.51 ± 4.04 d fg 235.45 ± 22.96 bc g 140.73± 3.56 b cd 162.63 ± 2.59 a de 137.93 ± 6.32 c cd 105.26 ± 2.92 a b 121.69 ± 8.00 b bc
155.05 ± 11.46 b bc 236.20 ± 5.68 a f 275.78 ± 13.10 b g 126.97 ± 4.88 c a 194.96 ± 3.58 c de 179.62 ± 6.60 c cd 220.87 ± 22.59 ab ef 185.72 ± 5.44 c d 214.41 ± 2.35 c ef 130.53 ± 2.02 bc ab 122.70 ± 6.89 b a 108.37 ± 2.68 a a
a b c
2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). Different letters indicate significant differences at p < 0.05, between months (superscript) and species (subscript). mg equivalents of Trolox/g dry leaf units.
3. Results and discussion 3.1. Seasonal variations in the antioxidant capacity Measuring the antioxidant capacity of biological samples is complicated due to various influencing factors (e.g. the type of radicals used, sample matrix, solvent composition, pH and sensitivity of the test compounds (Balogh et al., 2010)), thus multiple methods are generally used. In this study, three assays (ABTS, FRAP, DPPH) were used to determine the antioxidant properties of the leaf extracts. Table 1 summarizes the results of the ABTS antioxidant capacity measurements. The highest values were observed in May and June for most of the species. The exceptions were Norway maple (September, 194.96 mg TE/g dw.) and sessile oak (September, 214.41 mg TE/g dw.). Sati et al. (2013) also reported a maximum ABTS during the fall, for Ginkgo biloba leaves. During the growing season, a typical July minimum was observed for sweet chestnut and all the oak species. Overall, E. hornbeam (May, 329.78 mg TE/g dw.), sweet chestnut (May, 323.63 mg TE/g dw.) and Turkey oak (June, 274.81 mg TE/g dw.) leaf extracts showed the strongest reducing power against the ABTS.+ radical ion. E. beech, Scots pine, black pine, poplar, and black locust had the lowest antioxidant capacities (115.54, 104.18, 99.53, 92.82 and 75.26 mg TE/g dw., respectively). It was difficult to compare these results with similar data found in the literature because the execution of the ABTS assay (and also of the other assays) often involves various approaches among researchers. Some researchers base their evaluation on the absorbance value read at the end of the reaction, which is then compared with the respective value of a given standard compound
(Razali et al., 2012). Others, use the absorbance at the start and at the end of the reaction to calculate the scavenging capacity (Fu et al., 2015). Furthermore, the standard compound used in the assay and the expressed units of the results are often inconsistent between researchers, so that a comparison of literature measurements is often not feasible. Antioxidant capacity can be related to either gram of extract (Uysal et al., 2016) or dry weight of the sample. According to Razali et al. (2012) the ABTS antioxidant capacity of the methanolic extracts of tamarind (Tamarandus indica) leaves was 1.65 mmol TE/g dw. This value was higher than the best performing samples (May extracts of E. hornbeam and sweet chestnut) of the current study (recalculated values: 1.39 and 1.22 mmol TE/g dw, respectively). Teleszko and Wojdyło (2015) determined the ABTS antioxidant capacity in the leaves of apple (Malus domestica) (35.94 mmol TE/100 g dw) and quince (Cydonia oblonga) (116.49 mmol TE/100 g dw). For comparative purposes, the recalculated value for E. hornbeam, and sweet chestnut was 138.7 and 122.4 mmol TE/100 g dw, respectively, indicating that leaves of these two forest trees had superior ABTS antioxidant capacity than Malus domestica and Cydonia oblonga. Table 2 summarizes the results of the FRAP antioxidant capacity determination. Similar to the ABTS method, significant differences between the months were observed for many species. Generally, the highest values were detected in August and September. The exceptions were black pine (July, 45.74 mg AAE/g dw.), Scots pine (June, 25.81 mg AAE/g dw.) and Turkey oak (May, 106.86 mg AAE/g dw.). According to the literature, other species, such as Ginkgo biloba, can also have maximum FRAP values during the initial (spring) growing season (Sati et al., 2013). The species with the highest FRAP antioxidant capacity were E. hornbeam (August, 106.24 mg AAE/g dw.), Turkey oak (May,
Table 2 FRAPa antioxidant capacity of the leaves (mean ± standard deviation)b . FRAP (mg AAE/g dw.)c . Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
30.26 ± 1.60 a bc 78.79 ± 0.39 ab g 80.24 ± 1.94 c g 34.93 ± 2.20 b c 46.19 ± 0.63 a d 48.93 ± 2.20 a d 106.86 ± 2.73 c h 60.34 ± 3.20 c e 68.59 ± 4.20 b f 21.68 ± 2.19 a a 18.10 ± 0.62 a a 24.25 ± 1.15 a ab
40.13 ± 0.71 c c 77.89 ± 1.21 a g 71.71 ± 3.37 b f 32.20 ± 0.49 b b 47.85 ± 2.54 a d 80.38 ± 2.58 c g 70.39 ± 1.94 a f 51.21 ± 1.82 b d 58.64 ± 2.11 a e 40.33 ± 3.12 b c 25.81 ± 0.86 d a 38.51 ± 0.79 b c
36.40 ± 0.53 b b 84.04 ± 2.67 b h 62.85 ± 2.57 a f 40.55 ± 2.63 c bcd 50.05 ± 1.82 a e 67.03 ± 2.12 b fg 69.23 ± 2.28 a g 43.09 ± 2.93 a cd 64.21 ± 2.52 ab fg 38.59 ± 1.27b bc 19.96 ± 0.33 b a 45.74 ± 1.80 d de
36.71 ± 0.63 b c 106.24 ± 3.10 d i 83.86 ± 2.28 c g 11.59 ± 0.72 a a 64.75 ± 1.37 b f 96.18 ± 1.83 d h 92.60 ± 2.87 b a 48.78 ± 0.65 b e 67.55 ± 1.43 b f 45.19 ± 0.92 c de 17.18 ± 0.66 a b 42.13 ± 0.78 c d
53.10 ± 1.53 d c 92.08 ± 1.21 c gh 93.28 ± 0.39 d h 51.41 ± 1.87 d c 69.27 ± 3.45 b d 79.56 ± 3.46 c e 87.61 ± 1.54 b fg 82.77 ± 6.37 d ef 85.92 ± 1.79 c f 47.88 ± 1.51 c bc 22.19 ± 1.10 c a 43.61 ± 1.74 cd b
a b c
Ferric reducing ability of plasma. Different letters indicate significant differences at p < 0.05, between months (superscript) and species (subscript). mg equivalents of ascorbic acid/g dry leaf units.
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Table 3 DPPHa antioxidant capacity of the leaves (mean ± standard deviation)b . DPPH (IC50 )c (g extractives/mL assay). Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
10.47 ± 1.25 a ab 6.87 ± 0.39 a a 11.90 ± 4.21 a ab 11.63 ± 1.73 a ab 6.59 ± 0.81 ab a 8.16 ± 1.49 a a 6.02 ± 0.93 a a 10.86 ± 2.42 a ab 9.16 ± 2.06 bc ab 29.98 ± 2.69 a c 30.42 ± 3.69 a c 17.10 ± 0.88 ab b
18.18 ± 1.92 c c 6.37 ± 1.90 a a 9.17 ± 2.98 a ab 18.16 ± 0.35 bc c 8.21 ± 0.66 b a 7.82 ± 0.35 a a 7.58 ± 0.35 ab a 10.34 ± 2.68 a ab 14.53 ± 1.38 d bc 24.40 ± 1.61 a d 38.04 ± 2.32 a e 17.62 ± 1.82 ab c
13.36 ± 0.63 ab cd 5.51 ± 0.85 a a 10.53 ± 2.16 a bc 10.19 ± 0.70 a bc 7.32 ± 0.44 ab ab 8.05 ± 0.38 a ab 7.21 ± 0.47 ab ab 10.35 ± 0.59 a bc 7.73 ± 0.67 ab ab 26.57 ± 1.58 a e 38.73 ± 2.19 a f 14.79 ± 0.47 ab d
15.23 ± 1.04 bc e 4.63 ± 0.88 a a 7.05 ± 0.66 a abc 22.79 ± 4.50 c f 6.36 ± 0.31 a ab 7.49 ± 0.78 a abc 6.20 ± 0.47 a ab 10.67 ±1.20 a bcd 11.74 ± 0.22 cd cde 15.70 ± 0.14 b de 42.38 ± 0.00 a g 14.03 ± 1.46 a de
11.35 ± 0.73 a cde 4.69 ± 0.28 a a 9.02 ± 0.30 a bcd 12.51 ± 1.23 ab de 6.30 ± 0.46 a ab 8.41 ± 0.43 a abcd 8.38 ± 0.89 b abc 11.11 ± 1.81 a cde 5.02 ± 0.40 a ab 13.97 ± 2.11 b e 37.83 ± 2.99 a g 18.87 ± 1.53 b f
a b c
2,2-diphenyl-1-picrylhydrazyl. Different letters indicate significant differences at p < 0.02, between months (superscript) and species (subscript). 50% inhibition concentration.
106.86 mg AAE/g dw.) and sweet chestnut (September, 93.28 mg AAE/g dw.). The leaf extracts of coniferous trees as well as of black locust (August, 11.59 mg AAE/g dw.) exhibited the weakest FRAP antioxidant power. The DPPH radical scavenging activity was characterized by the IC50 value (50% inhibition concentration), with low IC50 indicating high antioxidant capacities. According to Table 3, no significant differences were indicated between IC50 values and the month for E. hornbeam, sweet chestnut, downy oak, pedunculate oak and Scots pine. A marked decrease of the IC50 levels during the growing season was only observed for sessile oak and poplar, while for the remaining species, a fluctuation or slight increase in the IC50 levels was evident. Overall, E. hornbeam (August, 4.63 g/mL) and sessile oak (September, 5.02 g/mL) showed the best (lowest) IC50 values, while the highest values were determined for Scots pine throughout the entire growing season (30.42–42.38 g/mL). In Eucommia ulmoides Oliver leaf, which is a traditional medicine and functional food in China, Zhang et al. (2013) found the best DPPH IC50 values were obtained from the August extracts. Considering reference compounds, it was concluded that E. hornbeam leaf extracts have medium to high antioxidant power compared with respective DPPH IC50 (Trolox: 4.29 g/mL, (+)-catechin: 7.40 g/mL, rutin: 13.94 g/mL), FRAP (Trolox: 550 mg AAE/g, (+)-catechin: 524 mg AAE/g, rutin: 295 mg AAE/g) and ABTS ((+)-catechin: 3514 mg TE/g, rutin: 1760 mg TE/g) values. Seasonal tendencies of the FRAP, ABTS and DPPH antioxidant capacities of leaf extracts were found to be markedly different due to the selective variation of these methods. Results of the individual assays should be summarized to obtain a comprehensive measure of the overall antioxidant power of the leaf extracts. Moreover, the seasonal changes between various types of antioxi-
dant compounds, mainly polyphenols, also need to be performed. Such information can be used to determine which compounds or types of compounds are responsible for the antioxidant power of the extracts. For this purpose, the total polyphenol contents of the extracts were assayed and compared with their respective antioxidant capacity values. 3.2. Seasonal variation of phenolic compounds 3.2.1. Seasonal variation of the TPC Phenolic compounds contribute directly to the antioxidant power of leaf extracts (Awika et al., 2003), hence the TPC of the extract was determined (Table 4). For the majority of the species, the TPC increased significantly from May to August/September with some slight fluctuations in between. However, this tendency was not observed in Turkey oak, sweet chestnut and Scots pine, which had maximum TPC values in May/June. There have been numerous investigations on the seasonal TPC variations of tree leaves, which describe a general increasing tendency from spring to fall. In Moringa oleifera Lam. the TPC was lowest in the newly opened leaves and values increased gradually with the maturation of the leaves (Iqbal and Bhanger, 2006). Pirvu et al. (2013) also reported an increasing TPC of E. beech leaves during the vegetation period, with overall maximum values in September. The TPC of the methanolic leaf extracts of Gingko biloba L. was higher in the fall than in spring (Sati et al., 2013). In the two evergreen species, the TPC maximum was observed in June for Scots pine (42.56 mg QE/g dw.) and in July for black pine (53.80 mg QE/g dw.). Pines differ from the other test species as pine needles remain on the trees throughout the entire year and do not fall at regular intervals. The lifetime of pine needles can span from a few months to several decades, thus determining their degree of
Table 4 Total phenol content of the leaves (mean ± standard deviation)a . Total phenol content (mg QE/g dw.)b . Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
37.82 ± 1.01 a b 78.81 ± 0.59 a f 71.30 ± 3.53 ab e 29.30 ± 0.99 b a 46.63 ± 2.80 a c 37.52 ± 0.54 a b 78.99 ± 3.35 c f 59.85 ± 2.27 b d 56.47 ± 1.34 a d 29.03 ± 0.29 a a 27.12 ± 0.78 a a 27.37 ± 0.97 a a
56.83 ± 0.99 c d 93.08 ± 3.29 b h 78.52 ± 1.13 b g 29.73 ± 0.73 b a 57.37 ± 1.98 b d 71.68 ± 2.32 c fg 65.82 ± 1.59 ab ef 58.46 ± 2.50 b de 51.23 ± 3.89 a cd 53.63 ± 3.24 b cd 42.56 ± 1.62 b b 46.61 ± 4.23 b bc
48.12 ± 1.28 b bc 105.93 ± 5.57 c h 62.51 ± 1.59 a de 43.25 ± 0.21 c ab 80.21 ± 1.4 e g 63.80 ± 3.31 bc e 65.89 ± 1.54 ab ef 48.32 ± 4.82 a bc 59.15 ± 4.06 a de 73.73 ± 3.05 d fg 37.47 ± 3.9 b a 53.80 ± 1.56 b cd
47.38 ± 3.14 b b 94.27 ± 5.38 b g 76.32 ± 5.25 b f 19.89 ± 2.89 a a 65.88 ± 2.83 c def 92.85 ± 3.67 d g 73.73 ± 5.81 bc ef 49.36 ± 4.21 a bc 53.70 ± 2.14 a bcd 61.96 ± 2,74 c cde 24.63 ± 3.98 a a 45.39 ± 5.51 b b
57.71 ± 2.59 c bc 80.81 ± 3.05 a e 75.49 ± 4.67 b de 49.63 ± 1.84 d b 73.73 ± 1.26 d de 60.31 ± 5.73 b c 59.69 ± 2.52 a c 72.21 ± 0.46 c d 70.01 ± 2.42 b d 57.61 ± 0.58 bc bc 36.07 ± 1.74 b a 35.78 ± 2.30 a a
a b
Different letters indicate significant differences at p < 0.05, between months (superscript) and species (subscript). mg equivalents of quercetin/g dry leaf units.
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Table 5 Total flavonoid content of the leaves (mean ± standard deviation)a . Total flavonoid content (mg QE/g dw.)b . Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
4.34 ± 0.07 c cd 11.05 ± 0.52 b i 5.72 ± 0.28 a fg 4.65 ± 0.23 c cde 5.33 ± 0.46 a efg 3.80 ± 0.31 a c 5.99 ± 0.21 b g 5.44 ± 0.40 ab efg 4.85 ± 0.41 b def 8.40 ± 0.39 a h 1.90 ± 0.04 a b 0.82 ± 0.04 a a
4.15 ± 0.10 c de 11.03 ± 0.75 b h 4.73 ± 0.53 a ef 4.43 ± 0.26 bc ef 5.33 ± 0.19 a fg 3.26 ± 0.07 a bcd 4.03 ± 0.24 a cde 5.71 ± 0.28 b g 3.18 ± 0.30 a bc 15.10 ± 0.09 d i 2.57 ± 0.09 c b 1.07 ± 0.08 ab a
2.80 ± 0.14 b bc 10.03 ± 0.68 b g 4.66 ± 0.38 a e 4.27 ± 0.24 bc de 6.06 ± 0.15 b f 3.71 ± 0.36 a cde 3.76 ± 0.52 a cde 4.59 ± 1.02 ab e 3.19 ± 0.19 a bcd 12.82 ± 0.37 c h 2.30 ± 0.09 b b 0.93 ± 0.17 ab a
2.23 ± 0.07 a b 12.84 ± 0.28 c h 5.15 ± 0.09 a f 2.60 ± 0.16 a bc 6.74 ± 0.20 b g 3.75 ± 0.39 a d 4.52 ± 0.24 a e 4.09 ± 0.19 a de 2.96 ± 0.05 a c 12.78 ± 0.16 c h 2.36 ± 0.03 b b 1.46 ± 0.06 c a
2.70 ± 0.09 b bc 8.42 ± 0.26 a g 5.16 ± 0.37 a e 4.00 ± 0.11 b d 6.47 ± 0.23 b f 3.25 ± 0.47 a cd 3.93 ± 0.23 a d 5.54 ± 0.43 ab e 3.54 ± 0.35 a d 10.22 ± 0.05 b h 2.09 ± 0.09 a b 1.12 ± 0.07 b a
a b
Different letters indicate significant differences at p < 0.05, between months (superscript) and species (subscript). mg equivalents of quercetin/g dry leaf units.
maturity is complicated (Ewers and Schmid, 1981) and these type of measurements have not been carried out. Overall, black locust had the lowest TPC (August, 19.89 mg QE/g dw.) while the highest levels were measured in E. hornbeam (July, 105.93 mg QE/g dw.), downy oak (August, 92.85 mg QE/g dw.), Turkey oak (May, 78.99 mg QE/g dw.) and sweet chestnut (June, 78.52 mg QE/g dw.). Pedunculate oak, sessile oak, E. beech, poplar and the conifers were characterized by mediocre TPC (27.12–73.73 mg QE/g dw.).
3.2.2. Seasonal variation of the TFC The characteristics of the seasonal TFC variations (Table 5) differed significantly from the seasonal TPC variation. The TFC values decreased from May to August/September in E. beech, Turkey oak, sessile oak and black locust, while a seasonal increase in the TFC was measured in E. hornbeam, Norway maple, poplar and in the pines. The seasonal tendency determined for E. beech corresponds with the previous results of Pirvu et al. (2013). According to the results, the seasonal TFC variation in the leaves of forest trees is a characteristic of the investigated species (similar to the previously discussed ABTS, FRAP, DPPH and TPC seasonal tendencies). In August, a seasonal minimum was observed for black locust (2.60 mg QE/g dw.) and pedunculate oak (4.09 mg QE/g dw.) while in E. hornbeam (12.84 mg QE/g dw.) and Norway maple (6.74 mg QE/g dw.) and black pine (1.46 mg QE/g dw.) a seasonal maximum was determined in the same month. A seasonal maximum in May was measured for E. beech (4.34 mg QE/g dw.), sweet chestnut (5.72 mg QE/g dw.), black locust (4.65 mg QE/g dw.), downy oak (3.80 mg QE/g dw.), Turkey oak (5.99 mg QE/g dw.) and sessile oak (4.85 mg QE/g dw.), similar to that found in the leaves of Eucommia ulmoides Oliver (Zhang et al., 2013). It should also be noted that seasonal TFC variation can also depend
on the gender: Zhang et al. (2016) determined that the seasonal TFC (March–September) of female individuals of Pistacia chinensis Bunge tended to increase, while the male individuals tended to decrease. Overall, the highest TFC was found in poplar (June, 15.10 mg QE/g dw.) and in E. hornbeam (August, 12.84 mg QE/g dw.), while the lowest TFCs were measured in the coniferous species (0.82–2.57 mg QE/g dw.).
3.2.3. Seasonal variation of the TF3L Results of the TF3L measurements are summarized in Table 6. Similar to the previous parameters investigated in this study, the TF3L of leaf extracts followed diversified seasonal tendencies. A characteristic increase from spring to fall was evidenced for sweet chestnut, downy oak and E. beech. For the remaining species, a maximum value was observed at discrete months. A July maximum was determined for black locust, Norway maple, black pine and E. hornbeam. Downy oak and Turkey oak had an August maximum, while pedunculate oak, poplar and Scots pine had seasonal maximum TF3L levels in June. The highest TF3L was measured in E. beech (September, 24.05 mg CE/g dw.), black pine (July, 18.89 mg CE/g dw.), Norway maple (July, 18.69 mg CE/g dw.), black locust (July, 16.45 mg CE/g dw.), and pedunculate oak (June, 15.55 mg CE/g dw.). Scots pine, sessile oak and downy oak had moderate values (0.52–7.71 mg CE/g dw.), while the lowest TF3L content was measured in E. hornbeam, sweet chestnut and Turkey oak (0.29–4.90 mg CE/g dw.). The measured values for E. beech, are comparable to previous results obtained for the yellow and green leaves of E. beech, wherein the TF3L content varied between 30–100 mg CE/g dw., depending on the year of the study and the health state (yellow/green) of the leaves (Sen et al., 2013). Feucht et al. (1994) concluded that flavan-
Table 6 Flavan-3-ol content of the leaves (mean ± standard deviation)a . Flavan-3-ol content (mg CE/g dw.)b . Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
12.41 ± 0.40 a hi 0.47 ± 0.02 a b 0.29 ± 0.00 a a 11.66 ± 0.46 bc h 7.27 ± 0.11 a fg 0.52 ± 0.01 a c 1.38 ± 0.04 a d 13.28 ± 1.10 b i 6.66 ± 1.20 d f 4.17 ± 0.15 a e 4.27 ± 0.18 b e 7.86 ± 0.27 a g
22.57 ± 1.04 c i 0.73 ± 0.01 b a 1.36 ± 0.02 c b 12.81 ± 0.44 c h 10.87 ± 0.80 b h 3.65 ± 0.01 b e 2.08 ± 0.01 b c 15.55 ± 1.03 c f 2.50 ± 0.06 ab d 11.38 ± 0.24 d h 7.71 ± 0.1 e g 13.07 ± 0.55 c h
22.97 ± 0.65 c k 1.82 ± 0.0 e b 1.13 ± 0.01 b a 16.45 ± 0.99 d i 18.69 ± 0.75 c j 4.35 ± 0.04 c e 2.27 ± 0.04 b c 14.63 ± 0.11 bc h 3.44 ± 0.12 c d 7.84 ± 0.08 b g 5.66 ± 0.33 d f 18.89 ± 0.29 d j
15.45 ± 0.61 b h 1.66 ± 0.01 d a 3.43 ± 0.06 d cd 3.27 ± 0.19 a c 11.55 ± 0.57 b g 5.85 ± 0.0 e e 4.87 ± 0.10 d e 9.67 ± 0.67 a f 2.08 ± 0.11 a b 8.27 ± 0.19 b f 3.64 ± 0.10 a d 11.76 ± 0.61 b g
24.05 ± 0.10 c g 1.10 ± 0.05 c a 4.90 ± 0.1 e c 10.94 ± 0.18 b e 11.16 ± 0.61 b e 5.15 ± 0.04 d c 2.73 ± 0.10 c b 13.30 ± 0.46 b f 2.90 ± 0.07 bc b 9.56 ± 0.42 c d 5.04 ± 0.12 c c 9.13 ± 0.20 a d
a b
Different letters indicate significant differences at p < 0.02, between months (superscript) and species (subscript). mg equivalents of (+)-catechin/g dry leaf units.
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Fig. 1. Seasonal variations of the total phenol content and antioxidant capacities in European hornbeam leaves. 1: mg equivalents of quercetin/g dry leaf units. 2: Ferric reducing ability of plasma. 3: mg equivalents of ascorbic acid/g dry leaf units. 4: 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). 5: mg equivalents of Trolox/g dry leaf units. Table 7 Overall antioxidant power of the leaf extracts evaluated by the scoring system. Combined antioxidant values. Species
May
June
July
August
September
European beech European hornbeam Sweet chestnut Black locust Norway maple Downy oak Turkey oak Pedunculate oak Sessile oak Poplar Scots pine Black pine
1.21 2.65 2.50 1.30 1.67 1.62 2.68 1.84 1.94 0.50 0.50 0.90
1.26 2.59 2.41 1.14 1.73 2.27 2.32 1.74 1.65 1.11 0.58 1.27
1.25 2.54 1.87 1.30 1.77 1.76 1.99 1.38 1.79 0.90 0.44 1.32
1.14 2.85 2.50 0.52 1.93 2.34 2.44 1.49 1.74 1.31 0.18 1.25
1.57 2.48 2.53 1.41 2.03 2.02 2.27 2.01 2.32 1.35 0.42 1.09
3-ols play a major role in the plant antioxidant system and in the defense reactions of the E. beech leaves. 3.3. Evaluation of the antioxidant assays The ABTS, DPPH and FRAP methods show a diverse selectivity to the various types of antioxidant compounds (Müller et al., 2011), thus a summarized evaluation of these methods is needed to obtain a comprehensive measure of the overall antioxidant efficiency of the leaf extracts. This was achieved by a scoring system, assigning 0 points to the weakest values and 1 to the best values within each antioxidant capacity method, using linear approximation between lowest and highest values. In the ABTS and FRAP methods, the lowest value was scored 0 and the highest value was scored 1. For the DPPH values, the opposite scoring system was used because the lowest IC50 value (score: 1) represent the highest antioxidant capacity, and the highest IC50 (score: 0) represent the weakest antioxidant power. The DPPH, FRAP and ABTS scores for each sample were summed up to obtain a measure of their overall antioxidant efficiency. Results of the overall antioxidant power of the samples are summarized in Table 7. The differences between values are demonstrated with the aid of gray shading. According to Table 7, the species with the overall best antioxidant power were E. hornbeam, sweet chestnut, Turkey oak and downy oak. The leaves of the most abundant Hungarian forest tree species (pedunculate oak, sessile oak, E. beech and black locust) were characterized by medium to low antioxidant power, while poplar and the coniferous trees had the weakest antioxidant capacity. By
comparing the TPC values (Table 4) with the scores in Table 7 it was noticed that both the highest TPC and scores occurred in respective samples of E. hornbeam, sweet chestnut, downy oak and Turkey oak. It was established that in E. hornbeam, sweet chestnut and downy oak, the polyphenols are primarily responsible for the antioxidant properties of the leaf extracts. Regarding E. hornbeam, which was the overall best-performing species in this study, its leaves had high TFL (Table 5) and relatively low TF3L (Table 6) levels. Thus, flavonoid-type compounds, as well as other polyphenols, without the flavan-3-ol structure (e.g. tannins, phenolic acids), are responsible for the excellent antioxidant power of E. hornbeam leaves. Nevertheless, antioxidant properties and phenolic concentrations followed diverse seasonal tendencies (Fig. 1). It was assumed that the optimum antioxidant properties of E. hornbeam leaves were between July and August. Similar tendencies can be suggested for sweet chestnut and downy oak, although their TFC is lower and their TF3L somewhat higher compared with E. hornbeam. The biosynthesis of phenolic antioxidants depends on environmental factors (e.g. light, temperature, water availability, mineral nutrition) and also on genetic factors, which can consequently affect the antioxidant properties of the leaves. The seasonal variation of leaves’ polyphenol and overall antioxidant content is also closely related to the plant’s adaptation to biotic and abiotic environmental factors (Nour et al., 2014). Each species has its highest antioxidant values in distinct months; hence, the harvesting time of the leaves should be carefully selected when the future use of the leaves is being considered. 4. Conclusions In this work the detailed comparative analysis of the antioxidant properties of the leaves from 12 selected Hungarian coniferous and deciduous forest tree species were targeted. Sample collection was carried out between May and September which enabled to track seasonal changes of the antioxidant properties and of the major groups of polyphenolic compounds, too. Results indicated that seasonal variations of the measured chemical parameters in the leaves are highly dependent on the wood species. Each species has its highest antioxidant values in distinct months; hence, the harvesting time of the leaves should be carefully selected when the future use of the leaves is being considered. According to the present work the leaf extracts of E. hornbeam, sweet chestnut and downy oak exhibited the highest antioxidant
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capacities. E. hornbeam leaf extracts have stronger antioxidant properties and higher concentrations of polyphenolic compounds than sweet chestnut, which has been used as a traditional medicine for various ailments due to the high antioxidant concentration of its leaves (Díaz-Reinoso et al., 2011). Despite the superior antioxidant properties of E. hornbeam leaf extracts, its physiological effects and possible beneficial impacts on human health have not yet been investigated thoroughly; only limited data is found in the literature (Müller et al., 2011). The high-performance liquid chromatographic separation and mass spectrometric identification of the polyphenolic antioxidants in the leaves of E. hornbeam has already been carried out by Hofmann et al. (2016) in an earlier study. Altogether 171 compounds, including phenolic acids, flavonoid glycosides, tannins, catechins and procyanidins have been characterized and identified. According to the HPLC-PDA peak areas, the most abundant polyphenolic compounds in the August leaf extracts were chlorogenic acid, ellagic acid, ellagitannins, myricetin-, luteolin-, quercetin- and apigenin glycosides. The authors concluded that further research is needed for the elucidation of the compounds which are primarily responsible for the exceptional antioxidant properties of E. hornbeam leaves. The leaves of forest trees are a renewable biomass, which is an abundant source of antioxidants that could be used for nutritional purposes (e.g. food additives, food preservatives), healthcare products and medical applications. The leaves of the species with promising antioxidant properties and polyphenol contents (E. hornbeam, sweet chestnut and downy oak) should be tested in the future to determine their antibacterial, antiproliferative and antiviral effects and to unveil their potential as a source of antioxidants in food and healthcare products. Acknowledgement This research was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and financed by the VKSZ 12-1-2013-0034 Agrárklíma. 2 project. References Awika, J.M., Rooney, L.W., Wu, X., Prior, R.L., Zevallos, L.C., 2003. Screening methods to measure antioxidant activity of sorghum (Sorghum bicolor) and sorghum products. J. Agric. Food Chem. 51, 6657–6662. Balogh, E., Heged"us, A., Stefanovits-Bányai, É., 2010. Application and correlation among antioxidant and antiradical assays for characterizing antioxidant capacity of berries. Sci. Hortic. 125, 332–336. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 70–76. Campanella, L., Bonanni, A., Favero, G., Tomassetti, M., 2003. Determination of antioxidant properties of aromatic herbs, olives and fresh fruit using an enzymatic sensor. Anal. Bioanal. Chem. 375, 1011–1016. Díaz-Reinoso, B., Moure, A., Domínguez, H., Parajó, J.C., 2011. Membrane concentration of antioxidants from Castanea sativa leaves aqueous extracts. Chem. Eng. J. 175, 95–102. Ewers, F.W., Schmid, R., 1981. Longevity of needle fascicles of Pinus longaeva (Bristlecone Pine) and other North American pines. Oecologia 51, 107–115. Feucht, W., Treutter, D., Christ, E., 1994. Accumulation of flavanols in yellowing beech leaves from forest decline sites. Tree Physiol. 14, 403–412. Fu, R., Zhang, Y., Guo, Y., Chen, F., 2015. Chemical composition, antioxidant and antimicrobial activity of Chinese tallow tree leaves. Ind. Crops Prod. 76, 374–377. Germanò, M.P., Cacciola, F., Donato, P., Dugo, P., Certo, G., D’Angelo, V., Mondello, L., Rapisarda, A., 2012. Betula pendula leaves: polyphenolic characterization and potencial innovative use in skin whitening products. Fitoterapia 83, 877–882. Hofmann, T., Nebehaj, E., Albert, L., 2015. The high-performance liquid chromatography/multistage electrospray mass spectrometric investigation and extraction optimization of beech (Fagus sylvatica L.) bark polyphenols. Ind. Crops Prod. 1393, 96–105. Hofmann, T., Nebehaj, E., Albert, L., 2016. Antioxidant properties and detailed polyphenol profiling of European hornbeam (Carpinus betulus L.) leaves by multiple antioxidant capacity assays and high-performance liquid
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