The results of this study will provide new knowledge and useful information about the content of phenolic compounds in some selected non-traditional plants leaves and the antioxidant activity of their extracts, which will give a wide range of possibilities to employing these plants as the sources of phenolic compounds.

Objects of this study were leaves of 12 species of non-traditional plants, namely, Amelanchier alnifolia (Nutt.) Nutt. ex M. Roem., Aronia mitschurinii A.K. Skvortsov & Maitul., Castanea sativa Mill., Chaenomeles japonica (Thunb.) Lindl., Cornus mas L., Diospyros kaki L., Diospyros lotus L., Diospyros virginiana L., Lycium barbarum L., Lycium сhinense Mill., Pseudocydonia sinensis (Thouin) C.K. Schneid., Ziziphus jujuba Mill. (Figure 1). The raw materials were collected in the 2018 August on the experimental collection of the Institute of Biological Conservation and Biosafety, the Slovak University of Agricultural in Nitra.

The collected samples were packed in cotton bags and air-dried for several days. The air-dried samples were finely powdered using laboratory blender and kept in zip-locked bags until further analysis. Duplicate specimens were collected for the herbarium preparation.

All the chemicals used were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA) and CentralChem (Slovakia).

The dry non-traditional plants leaves were used for the detection of total phenolic content and total flavonoid content. An amount of 0.25 g of each sample was extracted with 20 mL of 80% ethanol for 24 h. Then, the sample in 80% ethanol was centrifuged at 4605 RCF (Rotofix 32 A, Hettich, Germany) for 10 min and the supernatant was used for measurement with the DPPH and molybdenum reducing antioxidant power methods.

The total polyphenol content (TPC) was measured by the method of Singleton and Rossi (1965) using the Folin-Ciocalteu reagent. A quantity of 0.1 mL of each sample was mixed with 0.1 mL of the Folin-Ciocalteu reagent, 1 mL of 20% (w/v) sodium carbonate and 8.8 mL of distilled water. After 30 min in darkness, the absorbance at 700 nm was measured with the spectrophotometer Jenway (6405 UV/Vis, England). Gallic acid (25 – 300 mg.L⁻¹; R² = 0.998) was used as the standard. The results were expressed in mg.g^-1 DM gallic acid equivalent.

The total flavonoid content (TFC) was determined by the modified method described by Shafii et al. (2017). An aliquot of 0.5 mL of the sample was mixed with 0.1mL of 10% (w/v) ethanolic solution of aluminum chloride, 0.1 mL of 1 M potassium acetate and 4.3 mL of distilled water. After 30 min in darkness, the absorbance at 415 nm was measured using the spectrophotometer Jenway (6405 UV/Vis, England). Quercetin (1 – 400 mg/L; R2 = 0.9977) was used as the standard. The results were expressed in mg.g^-1 DM quercetin equivalent.

Total phenolic acid (TPA) content was determined using the method of Farmakopea Polska (1999). A 0.5 mL of sample extract was mixed with 0.5 mL of 0.5 M hydrochloric acid, 0.5 mL Arnova reagent (10% NaNO₂+10% Na₂MoO₄), 0.5 mL of 1 M sodium hydroxide (w/v) and 0.5 mL of water. Absorbance at 490 nm was measured using the spectrophotometer Jenway (6405 UV/Vis, England). Caffeic acid (1 – 200 mg/L, R² = 0.999) was used as a standard and the results were expressed in mg.g^-1 DM caffeic acid equivalents.

Basic statistical analyses were performed using PAST 2.17. Data were analyzed with ANOVA test and differences between means compared through the Tukey-Kramer test (α = 0.05). The variability of all these parameters was evaluated using descriptive statistics. Correlation coefficients were calculated by CORR analysis.

Phenolic compounds have been proven to have a particularly strong antioxidant effect (Scalbert et al., 2005; Pandey and Rizvi, 2009), which is closely related to the anti-inflammatory (Pastore et al., 2009), strong antimicrobial (Cushnie and Lamb, 2005), antiviral (Chávez et al., 2006), and anticancer (Kandaswami et al., 2005) effect. Based on a large amount of scientific data proving the beneficial effect of phenolic compounds in humans, it is appropriate to perform estimation of these compounds content of leaves extracts of some selected non-traditional plants.

The amount of total phenolic acid content varied with the plant species (Figure 2). Total phenolic acids were ranged from 3.51 (Aronia mitschurinii) to 24.67 (Chaenomeles japonica) mg CAE.g^-1 DM.

Comparing the works of literature, Barreira et al. (2010) reported a total phenol content of 228.37 – 522.98 mg.g^-1 of Castanea sativa. According to Lavola, Karjalainen and Julkunen-Tiitto (2012), total phenolic acid content for Amelanchier alnifolia cultivars was from 22.78 to 26.75 mg.g^-1 DW. In this case, chlorogenic acids had maximal values (17.55 – 20.16 mg.g^-1). Also, this study showed that that among investigated organs of A. alnifolia cultivars leaves had the most content phenolic acids as well as other phenolic compounds. In our study leaves, extracts of this species showed less content of phenolic acids. The total phenolic contents of water extracts of Cornus mas was 341.09 mg.g^-1 (Stankovic et al., 2014). Methanol extracts of Ziziphus jujuba had total phenolic content 68.10 mg.g^-1 (Al-Saeedi et al., 2016). The total amount of phenolic compounds in the methanol extracts of Chaenomeles japonica leaves varied from 12.94 to 64.79 mg GAE.100g^-1 (Urbanaviciute et al., 2019). The total phenolic contents have been investigated in other plant leaf extracts, including Mangifera indica L. (65 mg.g^-1), Anacardium occidentale L. (58.57 mg.g^-1), Cymbopogon citratus (DC.) Stapf (28.30 mg.g^-1), Carica papaya L. (21.80 mg.g^-1) (Iyawe and Azih, 2011), Euphorbia spp. (19.10 – 20.30 mg.g^-1) (Gapuz and Besagas, 2018), and Azadirachta indica A. Juss. (14.43 mg.g^-1) (Iyawe and Azih, 2011).

The biological effects of many plant species depend on flavonoids; therefore, studies on the variation in their content are important and relevant (Liaudanskas et al., 2014).

In the present work, Lycium barbarum, Ziziphus jujuba and Diospyros lotus leaf extract showed the highest amount of flavonoids (54, 61, 54.35 and 54.21 mg QE.g^-1 DM, respectively) (Figure 3). The least value of flavonoids determined in extracts of Pseudocydonia sinensis (22.47 mg QE.g^-1 DM).

According to Barreira et al. (2010), total flavonoid contents Castanea sativa 73.31-90.39 mg.g^-1. Water extracts of Cornus mas leaves had total flavonoid contents 22.18 mg.g^-1 (Stankovic et al., 2014). Aronia mitschurinii leaves extracts in another study showed the content of flavonoids 103.6 – 163.7 mg CE.g^-1 DW (Thi and Hwang, 2014). Also, Shahin et al. (2019) found that the total content of flavonoids in dried leaves of A. melanocarpa was 96.16 mg.mL^-1. It should be noted that most studies about antioxidant parameters of Aronia species concerning of berries. The value of this parameter for Ziziphus jujuba methanol extracts was 90.28 mg.g^-1 (Al-Saeedi et al., 2016) but ethanol extract of this species in our study was less. A study by Aryal et al. (2019) showed the flavonoid content in methanol leaves extracts eight selected wild vegetables from Nepal ranged from 37.86 to 66.1 mg QE.g^-1.

Natural plant antioxidants include main classes such as phenolic compounds, vitamins, carotenoids, etc. Phenolic compounds are a large group of antioxidants that can have structures from simple molecules to polyphenols (like flavonoids). Moreover, polyphenol compounds characterized by numerous activities that can be useful in human life such as antimicrobial, antifungal, anti-inflammatory, etc. (Lourenço, Moldão-Martins and Alves, 2019).

The concentrations of total polyphenols content in non-traditional plant leaves are presented in Figure 4.

The highest total polyphenol content was obtained from Lycium barbarum and Castanea sativa (95.84 and 80.58 mg GAE.g^-1 DW, respectively). In contrast, the lowest polyphenol content (40.24 and 38.02 mg GAE.g^-1 DW, respectively) was obtained from Diospyros kaki and Ziziphus jujuba leaves.

Thi and Hwang (2014) found that Aronia mitschurinii total polyphenol content varied from 139.3 to 250.8 mg GAE.g^-1 DW showed higher results than in the present study. The result of another study represented by Shahin et al. (2019) related to Aronia melanocarpa demonstrated that the total polyphenol content of dried leaves was 765.63 mg GAE.g^-1. As reported Męczarska et al. (2017), leaves of Amelanchier alnifolia demonstrated the total content of polyphenols 185.23 mg GAE.g^-1 DW that was 3 times less comparing with our result.

After studying the total phenolic compounds content it is important to examine and assess the antioxidant activity in the extracts of non-traditional plant species leaves. The results obtained during studies will be useful for the assessment and standardization of the quality of plant raw materials and will allow predicting an antioxidant effect of leaves non-traditional plants species.

The DPPH radical scavenging activities of non-traditional plant leaves at different stages of growth are shown in Figure 5. All tested samples exhibited DPPH• radical scavenging activities with values from 6.92 (Aronia mitschurinii) to 9.0 (Cornus mas) mg TEAC.g^-1 DW.

Serteser et al. (2009) reported that the methanolic extracts of the Cornus mas fruits showed EC50 (mg/ml) (DPPH reduction) values as 0.71.

The DPPH radical scavenging activity of the distilled water and 80% ethanol extracts of samples Aronia mitschurinii increased in a concentration-dependent manner (12.5~100 μg.mL^-1) (Thi and Hwang, 2014). Ziziphus spina-christi (L.) Desf. ethanolic extracts exhibited good radical scavenging activity, with IC50 (the extract concentration providing 50% of inhibition) values of 54.3 μg.mL^-1, respectively (Khaleel et al., 2016). In the leaves of wild-growing plants of Nepal, the DPPH radical scavenging potency with a minimum IC50 value was recorded from 9.89 to 45.68 mg.mL^-1 (Aryal et al., 2019).

Antioxidant activity by molybdenum reducing antioxidant power method ranged from 109.43 (Aronia mitschurinii) to 322.95 (Cornus mas) mg TEAC.g^-1 DM (Figure 6).

Comparison analyses of obtained data with results of other reviews often difficult to use in a similar study because of using different methods of determination of antioxidant parameters, especially it is concerning standards that used and units.

The Pearson correlation coefficients between antioxidant activities and TPC, TPA, and TFC were depicted in Figure 7.

The result revealed the existence of a positive and negative correlation between tested antioxidant assays. TPC was significantly correlated with DPPH and phosphomolybdate assays (r = 0.609 – 0.718, p <0.05). The considerable correlation was noted in TPA and DPPH assay (r = 0.611).