The Influence of Locality on Phenolic Profile and Antioxidant Capacity of Bud Extracts

Gemmotherapy represents the most recent therapeutic technique that uses the properties of extracts from fresh meristematic plant tissues, mainly buds and sprouts, by macerating them in ethanol and glycerol. The harvesting time and the location can significantly affect the chemical composition of the buds. Therefore, this work aimed to point out the possible variability in the phenolic content and the antioxidant potential of extracts prepared from commonly grown trees in the Czech Republic. Extracts from buds collected during autumn and spring in three different localities were analysed using UHPLC-MS (ultra-high-pressure liquid chromatography) for the phenols profile. Five tests assays were used for the evaluation of the extract antioxidant potential. The sampling time positively affected the content of total phenols, flavonoids, and phenolic acids. The increased levels of total phenols and flavonoids in localities with high and medium pollution may be the result of the higher levels of NO and SO2, the main air pollutants. However, surprisingly, the content of phenolic acid showed the highest values in the area with the lowest pollution. The results of antioxidant tests did not completely correlate with the levels of phenolic metabolites, which may be due to the involvement of other active molecules (e.g., ascorbate, tocopherol, or proline) in the antioxidant machinery.


Introduction
Phenolic compounds represent common secondary metabolites in vascular plants. They exhibit great structural diversity and play an important role in the defence responses against various environmental stimuli, such as ultraviolet radiation, heavy metal pollutions, or plant-insect interactions. They are present in all plant organs and are therefore an integral part of the human diet. Polyphenols have drawn increasing attention due to their wide distribution in food, beverages, phytotherapeutics, and potent antioxidant properties [1].
Antioxidants are compounds that can delay, inhibit, or prevent the oxidation of oxidizable materials by scavenging free radicals and diminishing oxidative stress. The imbalance between the excess of reactive oxygen/nitrogen species and endogenous antioxidants leads to oxidative stress, and subsequently to the development of chronic degenerative diseases, such as cancer, atherosclerosis, diabetes mellitus, rheumatism, cardiovascular diseases, or inflammatory injury [1,2].
In recent years, the demand for traditional and alternative medicine products that use the power of substances naturally contained in plants has increased. One such phytotherapeutic method is gemmotherapy, which uses more biologically active substances in the embryonic parts of plants rather than the grown parts of plants. Here, the extracts are obtained from fresh buds and other meristematic tissues (young sprouts, leaves, or The extraction method followed the protocol for bud preparation detailed in the European Pharmacopoeia [12]. The mother extract solution was prepared macerating one part fresh buds and 20 parts solution containing 42% v/v ethanol and 25.5% v/v glycerol. After four weeks of cold maceration (in a dark place at laboratory temperature), extracts were filtered (Whatman Filter Paper, London, UK) and diluted with the same solution at a ratio of 1:10 and used for the following analyses.

Total Phenolic, Tannins, and Flavonoids Content
The content of total phenols was determined spectrophotometrically (Cintra 101, Dandenong, Australia) using the Folin-Ciocalteu method with gallic acid as a standard. Briefly, 30 µL of the sample extract, 470 µL of distilled water, 975 µL 2% (w/v) sodium carbonate, and 25 µL of Folin-Ciocalteu reagent solution were incubated for 60 min at 45 °C. After cooling, the absorbance was read at 750 nm [13].
The aluminium chloride method, with quercetin as a standard, was used for the determination of the total flavonoids level [13]. The sample extract (500 µL) was mixed with 500 µL of 2% aluminum chloride (w/v, diluted with methanol) and then incubated at room temperature for 60 min. The absorbance was read at 420 nm.

Determination of Phenolic Acid
The contents of phenolic acids were determined by UHPLC on Zorbax RRHD Eclipse plus C18 column (2.1 × 50 mm, 1.8 µm) (Agilent) with a 6470 Series Triple Quadrupole mass spectrometer (Agilent) (electrospray ionization in negative ion mode) as a detector.  The extraction method followed the protocol for bud preparation detailed in the European Pharmacopoeia [12]. The mother extract solution was prepared macerating one part fresh buds and 20 parts solution containing 42% v/v ethanol and 25.5% v/v glycerol. After four weeks of cold maceration (in a dark place at laboratory temperature), extracts were filtered (Whatman Filter Paper, London, UK) and diluted with the same solution at a ratio of 1:10 and used for the following analyses.

Total Phenolic, Tannins, and Flavonoids Content
The content of total phenols was determined spectrophotometrically (Cintra 101, Dandenong, Australia) using the Folin-Ciocalteu method with gallic acid as a standard. Briefly, 30 µL of the sample extract, 470 µL of distilled water, 975 µL 2% (w/v) sodium carbonate, and 25 µL of Folin-Ciocalteu reagent solution were incubated for 60 min at 45 • C. After cooling, the absorbance was read at 750 nm [13].
The aluminium chloride method, with quercetin as a standard, was used for the determination of the total flavonoids level [13]. The sample extract (500 µL) was mixed with 500 µL of 2% aluminum chloride (w/v, diluted with methanol) and then incubated at room temperature for 60 min. The absorbance was read at 420 nm.

Determination of Phenolic Acid
The contents of phenolic acids were determined by UHPLC on Zorbax RRHD Eclipse plus C18 column (2.1 × 50 mm, 1.8 µm) (Agilent) with a 6470 Series Triple Quadrupole mass spectrometer (Agilent) (electrospray ionization in negative ion mode) as a detector. Eluents: (A) 0.05% formic acid in water and (B) 0.05% formic acid in acetonitrile were used in the following gradient program  [14]. A representative chromatogram is shown in Figure 2.

DPPH Free Radical Scavenging Assay
Bud extracts were assayed by the discoloration of a solution of DPPH· as previously reported [15] with some modifications. The mixture of 60 µM methanolic solution of DPPH· (1.5 mL) and the sample extract (10 µL) was left in the dark for 30 min and evaluated spectrophotometrically at 517 nm. The percentage of DPPH· scavenging effect was calculated using the formula: where Asample is the absorption of the solution with extract, and Acontrol is the absorbance of the solution without extract.

ABTS Radical Decoloration Assay
The ABTS + assay was performed by bleaching the cationic radical ABTS + as described by Soto et al. [16]. A solution of 7 mM ABTS + and 2.5 mM potassium persulfate was left to stabilize from 12 to 16 h in the dark at room temperature before use. Afterward, the solution was diluted with methanol (80%, v/v) until an initial absorbance of 0.70 ± 0.02 was obtained at 734 nm. The sample extract (10 µL) was mixed with 300 µL of prepared working ABTS + solution in a 96-well plate and incubated for 6 min in the dark at room temperature. The absorbance was measured at 734 nm in a microplate reader (SPARK ® Tecan Trading AG, Männedorf City, Switzerland). The scavenging effect was calculated using the formula: where Asample is the absorption of the solution with extract, and Acontrol is the absorbance of the solution without extract.  Bud extracts were assayed by the discoloration of a solution of DPPH· as previously reported [15] with some modifications. The mixture of 60 µM methanolic solution of DPPH· (1.5 mL) and the sample extract (10 µL) was left in the dark for 30 min and evaluated spectrophotometrically at 517 nm. The percentage of DPPH· scavenging effect was calculated using the formula: where A sample is the absorption of the solution with extract, and A control is the absorbance of the solution without extract.

ABTS Radical Decoloration Assay
The ABTS + assay was performed by bleaching the cationic radical ABTS + as described by Soto et al. [16]. A solution of 7 mM ABTS + and 2.5 mM potassium persulfate was left to stabilize from 12 to 16 h in the dark at room temperature before use. Afterward, the solution was diluted with methanol (80%, v/v) until an initial absorbance of 0.70 ± 0.02 was obtained at 734 nm. The sample extract (10 µL) was mixed with 300 µL of prepared working ABTS + solution in a 96-well plate and incubated for 6 min in the dark at room temperature. The absorbance was measured at 734 nm in a microplate reader (SPARK ® Tecan Trading AG, Männedorf City, Switzerland). The scavenging effect was calculated using the formula: where A sample is the absorption of the solution with extract, and A control is the absorbance of the solution without extract.

Hydroxyl (OH) Radical Scavenging Assay
For the generation of the hydroxyl radicals, the deoxyribose method was used. The mixture of the sample extract (10 µL), potassium phosphate buffer (780 µL; 10 mM, pH 7.4), ascorbic acid (10 µL; 0.1 mM), FeCl 3 (50 µL; 0.04 mM), H 2 O 2 (50 µL; 2.13 mM), and deoxyribose (100 µL; 2.8 mM) was incubated in a water bath for 60 min at 37 • C. Then, 1.0 mL of 2.8% TCA (w/v) and 1.0 mL of 1% TBA (w/v) were added and heated in a water Foods 2021, 10, 1608 5 of 13 bath for 15 min at 100 • C. After cooling the absorbance was read at 532 nm. The percentage of deoxyribose degradation was calculated using the formula: where A sample is the absorption of the solution with extract, and A control is the absorbance of the solution without extract [15].
Superoxide radicals were generated by the NADH/PMS system according to the previously described procedure [17]. The sample extract (50 µL) was mixed with 50 µL NADH (166 µM), 150 µL NBT (43 µM), and 50 µL PMS (2.7 µM) in a 96-well plate and incubated for 2 min at room temperature. All components were dissolved in a phosphate buffer (19 mM, pH 7.4). The absorbance was measured at 560 nm. The percentage of scavenging effect was calculated using the formula: where A sample is the absorption of the solution with extract, and A control is the absorbance of the solution without extract.

Nitric Oxide Scavenging Assay (NO)
The activity was determined spectrophotometrically in a 96-well plate reader. The reaction mixtures consisted of the sample extract (100 µL) and 100 µL SNP (20 mM) were preincubated for 60 min at 25 • C under light exposure. Then, 100 µL of Griess reagent was added, and the absorbance was read at 540 nm. The percentage of scavenging effect was calculated using the formula: % Scavenging NO = (A sample /Ac ontrol ) × 100 (5) where A sample is the absorption of the solution with extract, and A control is the absorbance of the solution without extract.

Data Processing
Statistica 10.0 (StatSoft Inc., Tulsa, OK, USA) software was used for statistical analyses. The comparison of differences in the experiment was based on the one-way analysis of variance (ANOVA) and Tukey's test at the significance level p < 0.05. Six individual samples were used for analyses of each parameter.

Results
The overall phytochemical profile of the analysed material, phenolic compounds included, is influenced by several factors, such as the genetic origin of the plant, or time and technique of sample collection, and the processing of the material. In addition, environmental conditions (influence of biotic and abiotic factors) significantly affect the qualitative and quantitative composition of plant materials and thus the possible therapeutic potential of medicinal products [1,6]. Therefore, in our study, we analysed the plant material harvested in two time periods (autumn and spring), originating from three localities that differed in the degree of pollution, and compared our results with commercially available preparations.
Based on the results shown in Table 1, it is clear that the time of sampling affected the content of total phenols, flavonoids, and phenolic acids (given as a sum). In all trees, the values were significantly higher in the extracts from the buds collected in the spring. The only exception was in oak, where the sum of phenolic acid decreased depending on the time of harvest. Therefore, only spring extracts were used for further analyses. The changes in metabolite content, with respect to sampling time, may be related to the transition of the buds from the dormant phase to the actively growing phase, where the need for metabolites is different. In birch, the content of hydrolysable tannins (gallotannin and ellagitannins) and flavonoid aglicones decreased by up to 90%, depending on the bud transformation to the adult leaf. The content of phenolic acids (mainly hydroxycinnamic acid derivatives) increased [6]. A similar increase in hydroxycinnamic acids and gallic acid, with respect to the time of harvest, was also observed in four tested Castanea species. The parallel increase in flavonoids, namely quercetin and rutin, and tannins also increased [4]. The work of Varigi et al. [5] pointed out that the effect of the season was considerably greater than that of the genotype, ontogenetic stage, and location. Here, the content of glycosylated flavonoids, epigallocatechin and epicatechin, decreased with the ontogenesis of the blackcurrant bud. Concentration variability, with respect to sampling time, was also observed for other secondary metabolites, for example, the content of terpenes in the essential oil of six blackcurrant cultivars decreased with the disruption of bud dormancy [18].
The monitored trees responded differently to the environmental impact ( Figure 3). For maple and birch, the highest values of total phenols (32.14 and 48.46 mg g −1 FW, respectively) and flavonoids (7.14 and 16.73 mg g −1 FW, respectively) were observed in bud extracts in the slightly contaminated locality HK. In the case of oak, the highest values were in the clean locality J. The commercially available extracts of the monitored trees alone showed significantly lower values compared to our measurements. In the case of phenols, there were no significant differences between N and RA producers, 9.39 and 9.69 mg g −1 FW for maple, 16.83 and 19.21 mg g −1 FW for birch, and 11.75 and 13.48 mg g −1 FW for oak, respectively. The content of flavonoids in the maple extract of the company N (6.72 mg g −1 FW) showed a similar level as the extracts from the localities J and HK. On the contrary, the values in the RA extract (4.92 mg g −1 FW) were comparable with the locality O, and at the same time significantly lower from the above. For birch, the values in N and RA were significantly lower compared to our extracts, 6.36 and 5.63 mg g −1 FW, respectively. For oak, the commercial extracts showed similar values as HK, namely 3.10 mg g −1 FW for N and 3.08 mg g −1 FW for RA. alone showed significantly lower values compared to our measurements. In the case of phenols, there were no significant differences between N and RA producers, 9.39 and 9.69 mg g −1 FW for maple, 16.83 and 19.21 mg g −1 FW for birch, and 11.75 and 13.48 mg g −1 FW for oak, respectively. The content of flavonoids in the maple extract of the company N (6.72 mg g −1 FW) showed a similar level as the extracts from the localities J and HK. On the contrary, the values in the RA extract (4.92 mg g −1 FW) were comparable with the locality O, and at the same time significantly lower from the above. For birch, the values in N and RA were significantly lower compared to our extracts, 6.36 and 5.63 mg g −1 FW, respectively. For oak, the commercial extracts showed similar values as HK, namely 3.10 mg g −1 FW for N and 3.08 mg g −1 FW for RA. Phenolic acids can be divided into two classes: derivatives of hydroxybenzoic acid such as gallic acid, and derivatives of hydroxycinnamic acid (HCA), such as caffeic, chlorogenic, or ferulic acid [1]. Here, we followed the changes of five HCA and six hydroxybenzoic acids. Overall, the highest content of monitored phenolic acids was in birch extracts, with the exception of gallic acid. Here, the highest values were in maple extracts. In maple and oak extracts (Tables 2 and 3), the highest values of all of the monitored HCA were recorded in the clean locality J. In the case of the HK and O sites, the values were similar. In birch ( Table 4) the content of HCA was highest in J (with the exception of chlorogenic acid-locality HK) Table 2. The content of phenolic acids (µg g −1 FW) in bud extracts of Acer pseudoplatanus. Values are expressed as mean ± SDs (n = 5). Values within lines, followed by the same letter(s), are not significantly different according to Tukey's test (p < 0.05).

Jičín
Hradec Králové Opatovice n.  Phenolic acids can be divided into two classes: derivatives of hydroxybenzoic acid such as gallic acid, and derivatives of hydroxycinnamic acid (HCA), such as caffeic, chlorogenic, or ferulic acid [1]. Here, we followed the changes of five HCA and six hydroxybenzoic acids. Overall, the highest content of monitored phenolic acids was in birch extracts, with the exception of gallic acid. Here, the highest values were in maple extracts. In maple and oak extracts (Tables 2 and 3), the highest values of all of the monitored HCA were recorded in the clean locality J. In the case of the HK and O sites, the values were similar. In birch ( Table 4) the content of HCA was highest in J (with the exception of chlorogenic acid-locality HK)  the highest O 2 − scavenging activity was recorded in birch extracts, specifically in the HK locality. Locations J and O did not differ significantly. In oak and maple, the highest levels were observed in the HK extracts (87% and 90%, respectively). Based on the NO· assay, birch and oak extracts showed the highest activity in locality J; 68% and 79%, respectively. For maple extracts, it was the locality O.
Similar to our results, aqueous and methanol extracts of birch leaves showed high antioxidant activity, more than 80%, measured via the DPPH and ABTS test [27]. However, when compared to the activity of certain phenolic acids, significantly higher concentrations of birch extracts were needed to achieve similar antioxidant activity [28]. The high efficacy of phytotherapeutic extracts was demonstrated in the work of Raiciu et al. [29], where the antioxidant capacity of over 90% birch, Salix, and Ribes extracts was monitored even after considerable dilution of the extract (up to 100-fold).
The different antioxidant activities between tree extracts may be due to the variability of composition, content, and chemical character of various active compounds, and the synergy between them and other natural substances [1]. The study comparing the composition of water bark extracts of alder, pine, and oak reveals the higher contents of phenolic biologically active components (phenols, flavonoids, tannins) and radical scavenging activity were in samples collected in the city with medium pollution from continental climatic zones compared to samples from the clean environment of a coastal natural park [30]. Variability, with respect to genetic structure and environmental condition, was also shown in the Alcea species in the content of total phenolics, flavonoids, anthocyanins, and overall antioxidant activity [31]. A significant effect of climatic zones on the phenolic content and antioxidant potential of the Aloe vera plant was monitored. Extracts of plants from the highland and semi-arid zones of northern India possessed maximum antioxidant potential compared to the tropical zones of southern India [32].
Generally, the radical scavenging activity of phenolic acids depends on the number and position of hydroxyl (−OH) groups and methoxy (−OCH 3 ) substituents in the molecules. Caffeic acid, most often esterified with quinic acid as in chlorogenic acid (both have two −OH groups), gallic acid (three −OH groups), and ferulic acid (one −OH and one −OCH 3 ), showed substantial antioxidant properties to scavenge free radicals [15,33,34]. Thus, higher concentrations of these phenolic acids can be reflected in the antioxidant activity of the extracts. In our samples, the highest concentrations of chlorogenic, caffeic, and ferulic acids were recorded in birch extracts, which, however, was manifested only in the cases of the O 2 − and NO· tests. However, when we focus on the correlation between the content of the mentioned acids and the antioxidant activity within one tree, it is not possible to draw a specific conclusion about the possible influence of the studied locality.
The preparation of gemmotherapeutic extracts is an undemanding method and thus easily accessible to the general public. Weaker botanical knowledge can lead to the easy confusion of individual species, which can also be reflected in the total content of phenolic substances and thus the possible antioxidant potential of the extracts. For example, Meda et al. [35] showed that the extracts prepared from red maple contained higher amounts of total phenols, flavonoids, and anthocyanins than sugar maple, which was also reflected in the higher antioxidant activity measured by the DPPH test. Similar results were recorded in the study of two oak species, where leaf extracts of Q. saliciana showed the maximum inhibition activities in ABTS radical scavenging assays, however, in the DPPH test Q. serrata leaf extracts showed better results [36]. Table 5. Antioxidant activities of bud extracts. Values are expressed as mean ± SDs (n = 5). J, Jičín; HK, Hradec Králové; O, Opatovice n. Labem; N, Naděje; RA, Rabštejnská Apatyka. Different letters show significant differences (p < 0.05) between groups for the same experiment.