Composition of Phenolic Compounds, Cyanogenic Glycosides, Organic Acids and Sugars in Fruits of Black Cherry ( Prunus serotina Ehrh.)

Compounds, Glycosides, Abstract: The forest understory is rich in fruit-bearing trees and shrubs. They include both native species and invasive alien plant species (IAPS). One of the most spreading IAPS is black cherry ( Prunus serotina E.). Problems with the invasiveness of P. serotina is well known in many European countries. However, there are very few studies related to this IAPS. This article aims to ﬁll the gap in research on the bioactive compounds of black cherry fruits and thus to start a discussion on the potential use of this species on an industrial or semi-industrial scale, which may lead to the reduction of this species presence in forests. Fruits were collected in a forest of Northern Poland. Contents of sugars, phenolic compounds as well as cyanogenic glycosides were determined. Pphenolic compounds and cyanogenic glycoside were assayed using HPLC-DAD coupled with MS n . Sugars and organic acids were determined with the use of HPLC-DAD. Fruits of P. serotina can be considered as rich in anthocyanins, even at a lower level of total phenolics than in their plantation-grown couterparts, fruits of black cherry are still a good source of dietary phenolics of natural, forest-grown origin. When stones are not crushed during processing the level of GCC is within a safe, admissible range.


Introduction
One of the fruit-producing plants growing abundantly in European forests is the black cherry, Prunus serotina (Ehrh.). This is an invasive alien plant species (IAPS) for many European countries, such as e.g., Germany, the Netherlands, France, Hungary, Italy, Norway and Belgium [1][2][3][4][5][6][7]. As an invasive species P. serotina has spread to areas previously occupied by the European bird cherry (Prunus padus Mill.). Fruits of black cherry are globe-shaped and very dark [8]. Their dark color suggests a high level of anthocyanins which are well known for their colorant effect as well as antioxidant and health-promoting properties [9][10][11]. Results of a study [12] show a correlation between the color and content of phytochemicals. The darker the fruit, the higher the content of phenolics and other phytochemicals. Several studies have been carried out on phenolic compounds of black cherry [13][14][15][16]. These compounds play an important role in the antioxidant activity of fruits and various fruit-based products. If the high content of phenolics, including anthocyanins, is confirmed it may indicate considerable potential for the use of P. serotina fruits in the food industry as a natural colorant or, thanks to their antioxidant activity, as a functional additive.
Prunus serotina was introduced in Poland in 1813 to fertilize weak soils. It did not take long for it to move from weak soils to fertile ones, where it spread rapidly and widely. Since its introduction black cherry has become a common understory species [17]. Now it is treated as an invasive plant species. Researchers from many European countries are now focused on determining factors that limit or promote the spread of black cherry [18][19][20]. Very few studies have been conducted on the characterization of chemical constituents in black cherry fruits growing in Poland [21].
The aim of this study was to fill the gap in research on the compounds in fruits of the genus Prunus by analyzing fruits of P. serotina, which contain many valuable bioactive substances such as sugars, organic acids and phenolic compounds as well as cyanogenic glycosides, which can be toxic to humans.

Plant Materials
Fruits of Prunus serotina for this study were collected from the area of the Dabrowa Forest District of the Polish State Forests (location: latitude 53 • 29 45.74 N, longitude 18 • 31 0.72 E, 79 m altitude). Fully ripe fruits were collected on 20 August 2019. The collected fully ripe fruits were immediately frozen and stored at −20 • C until further analysis.

Preparation of Phenolic and Cyanogenic Glycoside Extracts
In total, 2 g of fresh fruits with intact stones were placed in a 12 mL screw-top tube with 5 mL of the extraction solution (60% MeOH (aq) and 3% of formic acid). The share of methanol with water in the extraction solution and the mass of added fruit material was experimentally optimized by testing samples using HPLC. The solution concentration was optimized based on chromatography peaks. Extraction was conducted in an ice-cooled ultrasonic bath. After 45 min the extraction screw-top tube with extracts were centrifuged for 9 min, 12.857 g (Eppendorf centrifuge with rotor F-34-6-38) at 4 • C and then filtered through a polyamide 0.2 µm filter (Macherey-Nagel, Duren, Germany) into HPLC vials. Vials with the samples were stored in the freezer at −20 • C until further analyses.
The fruit extract was prepared in 10 repetitions.

Preparation of Organic Acid and Sugar Extracts
For the extraction of sugars and organic acids the flesh of fruit was separated from stones. The flesh (3 g) of destoned fresh fruits was homogenized with 25 mL of double distilled water. The homogenized mixture was left for 30 min at room temperature with frequent stirring at 250 rpm (Unimax 1010, Heidolph, Schwabach, Germany). Samples were then centrifuged for 9 min at 12.857 g (Eppendorf centrifuge with rotor F-34-6-38). Then the extract was filtered through a 0.2 µm cellulose filter (Macherey-Nagel) into HPLC vials and stored at −20 • C until further analysis.
The fruit extract was prepared in 10 repetitions.

HPLC-DAD MS n Analysis of Phenolic Compounds and Cyanogenic Glycosides
Detection of phenolic compounds was performed in the Thermo Scientific Dionex HPLC system with a diode array detector (Thermo Scientific, San Jose, CA, USA) using the Chromeleon workstation software. The method of analysis was previously described [22]. Compounds were analyzed at three wavelengths of 280, 350 and 530 nm. The used column was Gemini C18 (150 × 4.6 mm 3 µm; Phenomenex, Torrance, CA, USA). The mobile phases consisted of phase A: 3% acetonitrile/0.1% formic acid/96.9% double-distilled water; phase B: 3% water/0.1% formic acid/96.9% acetonitrile. The injected extract volume was 20 µL, flow rate was 0.6 mL/min, column temperature 25 • C.
Phenolics were identified using a mass spectrometer (LTQ XL Linear Ion Trap Mass Spectrometer, Thermo Fisher Scientific, USA) with electrospray ionization (ESI) in the negative and positive ion mode (for anthocyanins). The scanning range was between m/z 110 and 1700. The capillary temperature was 320 • C, the sheath gas and auxiliary gas were 20 and 8 units, the source voltage was 4 kV for negative ionization and 0.1 kV for positive ionization. The normalized collision energy was set between 20% and 35%. The injection volume was 10 µL, flow rate was 0.6 mL/min. For compounds lacking standards, quantification was carried out with a chemically similar standard. Thus, feruloylquinic acids were quantified in equivalents of ferulic acid, p-coumaroylquinic acids in equivalents of p-coumaric acid, caffeoylquinic acids in equivalents of chlorogenic acid, caffeic acid hexoside in eqv. of caffeic acid, all procyanidins in eqv. of procyanidin B1, kaempferol glycosides in eqv. of kaempferol-3-glucoside, isorhamnetin glycosides in eqv. of isorhamnetin-3-glucoside and cyanidin derivatives in eqv. of cyanidin-3-galactoside. All available standards were dissolved in methanol to obtain stock solution of 1 mg/mL, which was stored at −80 • C until analyzed. Calibration curves were constructed using six standard solutions containing 0.05, 0.01, 0.025, 0.005, 0.001 and 0.0001 mg/mL of each standard. Concentrations of identified compounds in fruits were expressed in mg/kg of fresh weight.

HPLC-MS n Analysis of Cyanogenic Glycosides
The presence of cyanogenic glycosides was confirmed on a TSQ Quantum Access Max quadrupole mass spectrometer (MS). The MS instrument was operated using an (ESI) source in the positive ion mode. All instrument parameters and analytical conditions were described in [23]. Prunasin and amygdalin were analyzed in the selected reaction monitoring (SRM) mode, which provides excellent sensitivity for the quantification of target compounds, and additionally by comparison with the SRM data of both standards. Contents were expressed in mg per g fruit.

Analysis of Organic Acids and Sugars by HPLC-DAD
Analysis of organic acids and sugars was conducted using HPLC (Thermo Scientific, San Jose, CA, USA). The methods were previously described [24]. For organic acids the Rezex ROA column was used (300 × 7.8 mm) (Phenomenex). For sugars the Rezex RCMmonosaccharide column was used (300 × 7.8 mm) (Phenomenex). Both columns were heated at 65 • C. The mobile phase for organic acids was 4 mM sulfuric and for sugars it was double-distilled water. Detection was performed by an ultraviolet detector (UV) set to 210 nm for organic acids and a refractive index (RI) detector for sugars. Quantification was performed with the help of the standard calibration curve with the known concentration. Calibration curves of sugars were composed using standard solutions containing 0.50, 1.00, 2.50 and 5.00 mg/mL of each standard. Where calibration curves of organic acids were constructed using standard solutions containing 0.05, 0.10, 0.20, 0.50 mg/mL of each standard, the contents were expressed in g/kg FW fruits.

Statistical Evaluation
Obtained data were analyzed with the StatGraphics Plus 4.0 software (Manugistics, Rockville, MD, USA). The results are presented in Tables 1-3 as mean values ± standard deviation.

HPLC Analyses of Phenolic Compounds
To our knowledge this paper is the first report on HPLC-MS analysis of phenolic compounds in Prunus serotina fruits. All identified compounds are listed in Table 1 and the chromatogram shown in Figure 1.  Table 1.
Flavanols are the most abundant group of phenolic compounds in the fruit of black cherry. Their level is 7017 mg per kg of fresh fruits, which indicates that P. serotina fruits are rich sources of these compounds in comparison with other Prunus species: P. padus at 124.14 mg/kg and P. avium at 546 mg/kg [25]. Four dimers and four trimers of procyanidin were identified as well as one tetramer. It is noticeable that fruits of black cherry are very abundant in catechins. The sum of catechin and epicatechin is 3236 mg/kg of fresh fruits, more than the previously reported 1426 mg/kg FW [16]. The analysis of catechins in fruits shows that in Prunus avium it is 62.7 mg/kg of catechins in fruits, while in Prunus cerasus it is only 9.8 mg/kg FW. In that study the most abundant in catechins was apricot (Prunus armeniaca) with 156.0 mg/kg FW [26].
The third group by content in mg/kg are hydroxycinnamic acids. In total they amount to 222 mg/kg FW. The main component in this group is 5-caffeoylquinic acid 1 (chlorogenic acid) at 71 mg/kg FW, followed by vanillic acid hexoside at 58 mg/kg FW. The level of hydroxycinnamic acids in black cherry fruits was lower than in most popular Prunus species. In the work [25] the reported content of hydroxycinnamic acids varied, depending on the species, from 442 to 660 mg/kg FW, which is approximately 2-fold lower than in black cherry.
In the study [32] the total polyphenolic content in sour cherry varied depending on the cultivar from 15,395 to 29,825 mg/kg, i.e., it was higher than the content of total phenolics in our study (11,394 mg/kg), comparing with the work of Vasco [16] at 2433 mg/kg FW the amount of total phenolics determined in our work is more than four times higher. Table 3 shows sugar and organic acid contents in fresh P. serotina fruits. The main sugar in black cherry fruits is glucose; this is also the main sugar in many Prunus species [12,25].

Sugars and Organic Acids
Glucose (111 g/kg) and fructose (95.9 g/kg) are also the main sugars in sweet cherry fruits, varying between cultivars, but in P. avium it is more than two times lower than in black cherry [33]. The third compound was a sugar alcohol, sorbitol (21.9 g/kg FW) and the last was a disaccharide, sucrose, accounting for 6.0 g/kg FW. The concentration of glucose and fructose in sweet cherry is approx. 45 g/kg for glucose and 40 g/kg for fructose, respectively. Black cherry with total sugar contents of 235 g/kg of fresh flesh is similar to the level of sugars in P. avium fruit reported in a study [12], whereas it is 2-fold higher according to the work [33]. The main organic acid was citric acid (28.2 g/kg FFW), which ranked second as a major organic acid in P. padus in a study [34]. The second most abundant organic acid for P. serotina was malic acid at 21.1 g/kg FFW. Shikimic and fumaric acids were detected only in trace amounts. Malic acid is also a common organic acid in P. padus. In the sweet cherry the main organic acid is malic acid, followed by citric and shikimic acids. The total organic acid content is approx. 13 g/kg FW in sweet cherry and approx. 49 g/kg in black cherry [33]. The sugar/acid ratio of 4.79 suggests a sour taste of fruit [25] and a bitter taste due to the high content of flavanols. The sugar/acid ratio of the above-mentioned Prunus avium is 8.15, which indicates a sweet taste of its fruit. Table 4 shows levels of cyanogenic glycosides in fruit of black cherry. Two cyanogenic glycosides (CGG) were identified in the extracts. The main one is amygdalin at 19.24 mg per kilogram of fresh fruits, while the recorded content of prunasin was 11.90 mg/kg FW.

Cyanogenic Glycosides
Amygdalin, also called vitamin B-17, can be found in seeds and other parts of many Prunus species [35]. Among cyanogenic glycosides (CGG), prunasin is present in smaller amounts in the P. serotina fruit extract compared to amygdalin. However, prunasin bioavailability is approx. 50% of the oral dose, while amygdalin is almost impossible to absorb in an unchanged form [36]. The total content of CGG in fruits of black cherry is very low (31 mg/kg) compared to kernels of sweet cherry, which contain 1384 mg/kg [23]. However, if black cherry stones were crushed, the CGG content would be much higher, because they are stored there. Cyanogenic glycosides are known to be toxic by HCN hydrolysis, but their toxicity is lower than theoretical calculations suggest [37]. The level of 20 µg per kg body weight is described (by the European Food Safety Authority) as the maximum safe dose of cyanogenic glycosides [38]. According to the maximum dose of CGG, even 1 kg of processed fruit is not harmful for humans, bearing in mind that the fruit stone must not be crushed or damaged during processing; otherwise, the CGG content will be definitely higher.

Conclusions
The results of this study showed that fruits of this common invasive alien plant species of European forests can be considered rich in anthocyanins, which are known for their antioxidant activity. Although the total phenolic content is lower than in industrially used cherries, black cherry fruits are still a good source of dietary phenolics of natural origin. Wild species with an almond-like aroma are often considered harmful due to their content of cyanogenic glycosides. The study showed that the level of CGG in black cherry fruits is low, even 1 kg of fruits has lower amounts of prunasin and amygdalin than the level considered harmful to humans; nevertheless, it must be remembered that stones may not be crushed.