Inhibition of α-Amylase, α-Glucosidase, Pancreatic Lipase, 15-Lipooxygenase and Acetylcholinesterase Modulated by Polyphenolic Compounds, Organic Acids, and Carbohydrates of Prunus domestica Fruit

This work aimed to establish the content of phenolic compounds, carbohydrates, and organic acids and to determine their potential to inactivate α-amylase, α-glucosidase, pancreatic lipase, 15-lipoxygenase (15-LOX), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE), and antioxidant activity (ABTSo+ and FRAP) in 43 Prunus domestica cultivars. We identified 20 phenolic compounds, including, in the order of abundance, polymeric procyanidins, flavan-3-ols, phenolic acids, flavonols, and anthocyanins. The total content of phenolic compounds varied depending on the cultivar and ranged from 343.75 to 1419 mg/100 g d.w. The cultivars of Ś2, Ś11, and Ś16 accumulated the greatest amounts of polyphenols, while in cvs. Ś42, Ś35, and Ś20 polyphenols were the least abundant. The highest antioxidant potential of 7.71 (ABTSo+) and 13.28 (FRAP) mmoL Trolox/100 g d.w. was confirmed for cv. Ś11. P. domestica fruits showed inhibitory activity toward α-amylase (2.63–61.53), α-glucosidase (0.19–24.07), pancreatic lipase (0.50–8.20), and lipoxygenase (15-LOX; 4.19–32.67), expressed as IC50 (mg/mL). The anti-AChE effect was stronger than the anti-BuChE one. Cv. Ś3 did not inhibit AChE activity, while cv. Ś35 did not inhibit BuChE. Thanks to the abundance of biologically active compounds, P. domestica offers several health-promoting benefits and may prevent many diseases. For these reasons, they are worth introducing into a daily diet.


Introduction
Over 400 species in the Prunus genus belong to the Rosacae family, but only 89 are listed in the Genetic Resources Information System [1]. The main representatives of the Prunus genus are plums, cherries, peaches, apricots, and almonds [2]. Plum (Prunus L.) fruits have gained importance in recent years, as they constitute a considerable share of fruit production in Poland and Europe. The fruits are valued for sensory reasons (taste and smell) and technological properties (attractive and desirable products). The most commonly grown cultivars are European plum (Prunus domestica L.) and Japanese plum (Prunus salicina) [1]. P. domestica fruits come in different skin and flesh colours, which, depending on the cultivar, range from dark purple-red, red to yellow or yellowish-green.
The fruits can be divided into early ('Królowa Wiktoria', 'Kirka'), moderately early ('Renkloda', 'Węgierka'), and late ('Anna Spath', 'Stanley') [3]. A wide variety of P. domestica cultivars, including late-ripening ones, considerably prolongs their availability on the market, even until October, in the climatic conditions of Europe. Therefore, P. domestica fruits are the second, after peaches and nectarines, the most often produced stone fruits worldwide. Their cultivation area is 2,700,000 ha, and their total annual production is about fruit) was mixed with distilled water, exposed to ultrasounds (Sonic 6D; Polsonic, Warsaw, Poland) for 15 min, heated at 90-100 • C for 30 min, and finally centrifuged (MPW-55; Warsaw, Poland) at 12,000× g for 10 min at 4 • C. The supernatant (2.5 mL) was injected into a Sep-Pak C-18 cartridge (1 g, Millipore Waters, Milford, MA, USA) and eluted with H 2 O into Eppendorf tubes. Before analysis, the extract was filtered through a hydrophilic PTFE membrane (0.20 µm; Millex Simplicity filters; Merck, Germany). The organic acids were analyzed on Polymex IEX H column (8 µm, 250 × 8 mm, Watrex; Prague, Czech Republic) using isocratic elution with 0.9 M sulfuric acid in H 2 O for 20 min. The carbohydrates were analyzed on Alltech ® Prevail TM Carbohydrate ES HPLC Column-W 250 × 4.6 mm, 5 µm (Columbia, MD, USA) using isocratic elution with 70% acetonitrile in H 2 O for 20 min. The results were expressed in g per 100 g of d.w.

Analysis of Biological Activity
All analyses were made using a multi-mode microplate reader SynergyTM H1 (BioTek, Winooski, VT, USA) in three repetitions. The antioxidant activity of ABTS o+ and FRAP was expressed in mmoL Trolox per 100 g. Other results were expressed as the sample capable of reducing the enzyme activity by 50% (IC 50 ) in mg/mL.

Analysis of Antioxidant Activities of ABTS o+ and FRAP
Antioxidant properties were assessed using the ABTS o+ method, which determines the ability to reduce the ABTS o+ cation radical, and the FRAP method, in which Fe 3+ is reduced to Fe 2+ . The samples for the analysis were prepared as described by Wojdyło et al. [11]. The fruit powder (about 0.5 g) was mixed with 5 mL of methanol:H 2 O:HCl (79:20:1; v/v/v), exposed to ultrasounds at 20 • C for 15 min and left for 24 h at 4 • C. Then, the extract was again exposed to ultrasounds for 15 min and centrifuged for 15 min at 15,000× g. In triplicate, all measurements were performed using a PC UV-2401 spectrophotometer (Shimadzu, Kyoto, Japan). The antioxidant activity of ABTS o+ and FRAP was expressed in mmol Trolox per 100 g d.w.

Inhibition of α-Amylase, α-Glucosidase, and Pancreatic Lipase
The inhibitory effect on the activity of α-amylase and α-glucosidase (antidiabetic activity), and pancreatic lipase (antiobesity activity) of P. domestica fruits was determined according to the procedure described before by Wojdyło et al. [15,16]. The inhibition of α-amylase activity by the P. domestica extracts was evaluated using the ability of α-amylase to hydrolyze α-1,4-glycosidic bonds. The hydrolysis causes gradual cleavage of starch chains and results in a color reaction of iodine with KJ. Depending on the degree of starch degradation, the colour is dark blue to violet after incubation at 37 • C and shows maximum absorption at 600 nm. Inhibition of α-glucosidase activity by the P. domestica extracts was evaluated based on the interaction of α-glucosidase with PNPG (4-nitrophenylα-D-glucopyranose). This reaction in an alkaline environment yields glucose and p-αnitrophenol (PNG). The latter is yellow and shows a maximum absorbance of 405 nm. The stronger the enzyme inhibition capacity of the tested extracts, the less p-α-nitrophenol is released from PNPG due to enzymatic hydrolysis.
Reference samples and positive control were prepared with a buffer instead of the enzymes and acarbose.
Inhibition of pancreatic lipase by the P. domestica extracts was evaluated based on the enzyme activity mediating the formation of p-nitrophenol from p-nitrophenol acetate at 37 • C. The reaction product shows maximum absorbance at 400 nm. Reference samples and positive control were prepared with a buffer instead of the enzyme and orlistat.

Inhibition of 15-Lipoxygenase
Inhibition of 15-lipoxygenase activity was determined as described by Wojdyło et al. [17]. The inhibitory properties of the P. domestica extracts were assessed based on the formation of conjugated double bonds in linoleic acid hydroperoxide during the reaction carried Antioxidants 2023, 12, 1380 5 of 24 out at 37 • C for 20 min. The product showed a maximum absorbance of 210 nm [15]. In the reference samples, the enzyme was replaced with Tris-HCl buffer. The results were expressed as IC 50 values.
2.6.4. Inhibition of Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) Inhibition of cholinesterase was assessed using the acetylcholinesterase (AChE) and butylcholinesterase (BuChE) methods described before by Wojdyło et al. [17]. The reaction mixture consisted of a sample of P. domestica extract, Tris-HCl buffer (pH 8.0), acetylthiocholine iodide or S-butylthiocholine iodide and 5,5 -dithiobis (2-nitrobenzoic acid). After incubation at 37 • C for 10 min, AChE or BuChE solution was added. The absorbance was measured after 15 min at 412 nm. The results were expressed as IC 50 (mg/mL). All assays were performed in triplicate with a PC UV-2401 spectrophotometer (Shimadzu, Kyoto, Japan).

Statistical Analysis
The statistical analysis was performed with the Statistica package, version 15.03 (StatSoft, Kraków, Poland). Significant differences (p ≤ 0.05) between mean responses were assessed by one-way ANOVA with the Duncan test. Principal component analysis (PCA) was performed using XLSTAT Statistical Software for Microsoft Excel 2017 (Microsoft Corp., Redmond, WA, USA).

Content of Carbohydrates and Organic Acids
Organic acids and carbohydrates contribute significantly to the sensory desirability of fruits, conferring their pleasant taste and aroma. The sugar-to-organic acid ratio is an important quality indicator. The higher the ratio, the more attractive the fruits and the products of their processing [18]. The composition and content of sugars and organic acids in fruit depend mainly on the cultivar. However, environmental factors and growing conditions may also affect their total content [19]. From the technological perspective, acids affect the gelling properties of pectin. They are also less susceptible to changes during storage and processing than other fruit components, such as flavor and aroma compounds or pigments [18].
Carbohydrates content in P. domestica cultivars was highly variable and ranged from 15.51 to 5.49 g/100 g d.w. (Table 1). The main saccharides identified were sucrose and fructose, followed by glucose and sorbitol. Another study showed the dominance of fructose and glucose in the total carbohydrate pool of Prunus domestica L. fruit, while sucrose and sorbitol were detected at low amounts [18]. The highest total carbohydrates content was identified in the cultivarsŚ25,Ś30,Ś26,Ś11,Ś38,Ś40, andŚ43 (>13 g/100 g d.w.), and the lowest in cvs.Ś32,Ś17,Ś35,Ś27,Ś42,Ś29,Ś15, andŚ21 (<8 g/100 g d.w.). Wu et al. [20] analyzed fruits of various Prunus persica L. Batsch cultivars and found sucrose to be the most abundant carbohydrate in all cultivars. Also, all the cultivars had a higher content of fructose than glucose.
Depending on the cultivar, P. domestica fruits are much richer in sugars than cherries (16.33 and 9.09 mg/100 g, respectively). A comparison of P. domestica fruits with apple [21] showed the same or higher content of sugars in P. domestica. Finally, P. domestica fruits contained less sugar than peaches [22].
The content of organic acids in the analyzed P. domestica cultivars varied and ranged from 4.33 to 1.34 g/100 g d.w. (Table 1). The most common organic acid was malic acid, quinic, and oxalic acid. For the other acids, the order of abundance was as follows: succinic, formic, and citric acid, and they were detected only in trace amounts.
The fruits most popular among consumers should have a ten times high sugar to organic acid ratio [24].

Identification and Quantification of Polyphenolic Compounds in P. domestica Fruits
Polyphenols are phenolic compounds synthesized in plants as by-products or secondary metabolites. They provide significant health benefits in human nutrition. For example, they reduce the risk of developing chronic diseases, and have antioxidant, antiinflammatory, anticancer, antiallergic, antihypertensive, and antiviral properties [25]. In the fruits of the investigated P. domestica cultivars, LC-MS Qtof analysis confirmed the presence of 19 phenolic compounds belonging to four groups: phenolic acids (neochlorogenic, cryptochlorogenic, chlorogenic, 3-caffeoylshikimic, and 3-feruloylquinic acid), flavonols   The content of phenolic compounds in P. domestica fruits has been studied by some researchers [18,26], but these studies were limited to several or single cultivars different from those analyzed in our work. In this study, we quantified the concentration of anthocyanins, phenolic acids, flavan-3-ols, and flavonols ( Table 3). The average content of polyphenols ranged from 343.75 to 1419.14 mg/100 g d.w. These values indicated significant differences between the cultivars regarding the total content of polyphenols. The highest total content of polyphenolic compounds was determined for cvs.Ś2,Ś9,Ś11,Ś12,Ś16,Ś17,Ś18,Ś19, S21,Ś29,Ś33 andŚ43 (>1000 mg/100 g d.w.), and the lowest for cvs.Ś7,Ś20,Ś35, andŚ42 (<500 mg/100 g d.w.).
Flavan-3-ols were the main group of polyphenols (16.70-83.79%). Phenolic acids constituted next the main group of polyphenolic compounds, accounting for 10.47% to 69.08% of the total phenolic compounds. The other most abundant groups were flavonols (0.82% to 16.08%). Regardless of the cultivar, the least abundant group were anthocyanins (0.35% to 6.63%). The main reason for significant differences in the content of polyphenolic compounds was the cultivar, and the other factors were related to the method of cultivation, climatic conditions of a growing season, and the degree of fruit maturity [27].
Phenolic acids usually occur in plants in a bound form of esters and glycosides. They are extremely important for plants, as they actively defend the living tissues against injuries, infections, or insolation. Phenolic acids are characterized by antioxidant activity. They regulate seed germination and plant growth. Esterified derivatives of caffeic acid that is, neochlorogenic and chlorogenic acids, have strong antioxidant, antimutagenic, and anticancer properties and regulate carbohydrate metabolism by lowering the level of glucose in the human body [30]. Research conducted by Navarro-Orcajada et al. [30] revealed anti-inflammatory, hepatoprotective, antimicrobial, cardioprotective, and neuroprotective effects, and Zhao et al. [28] also reported their antihypertensive properties.
Flavonols are the most common biologically active compounds in plants and have several beneficial properties. The flavonols in the investigated P. domestica cultivars ranged from 7.6 to 128.77 mg/100 g d.w. (Table 2). Their highest total content was determined for cvs.Ś32,Ś18,Ś39,Ś31, andŚ37 (>75 mg/100 g d.w.), and the lowest for cvs.Ś28,Ś27, S24,Ś21,Ś8,Ś42,Ś4 (<25 mg/100 g d.w.). In the analyzed P. domestica fruit samples, Quercetin, a compound commonly found in plants, shows many biological activities [33]. It scavenges free radicals, increases the concentration of glutathione, reduces lipid peroxidation, and thus limits oxidative stress. Thanks to these properties, quercetin may reduce the risk of neurodegenerative and cardiovascular diseases [34]. It is also known for its antibacterial, anticancer, and antiangiogenic activity [35], and it plays an important role in eliminating mycotoxins, thus protecting plant cells from damage [36]. A study by Sharma et al. [37] showed that quercetin-3-O-rutinoside can considerably protect the digestive tract from damage caused by gamma radiation. The authors confirmed the interaction of quercetin-3-O-rutinoside with essential antioxidant and anti-inflammatory proteins. The interaction of all tested antioxidant proteins (heme oxygenase-1, glutathione S-transferase, glutamate-cysteine ligase catalytic subunit, and thioredoxin reductase 1) significantly increased in the presence of quercetin-3-Orutinoside [37]. Quercetin-3-O-rutinoside was a potent antioxidant, as it effectively quenched free radicals and efficiently chelated iron ions [38].
Anthocyanins are flavonoids commonly found in fruits and vegetables. Their presence in fruits is manifested by the red, blue, or purple color. These compounds exert strong antioxidant activity and play an important health-promoting role. They also protect the plants against abiotic and biotic stresses [39]. Anthocyanins were the least abundant polyphenolic compounds in the investigated fruits, with a maximum amount of up to 8.49%, depending on the cultivar. Their content in the investigated P. domestica cultivars ranged from 3.24 to 53.14 mg/100 g d.w. ( Table 2). As all fruits of the P. domestica cultivars had light flesh, all detected anthocyanins accumulated in the fruit skin. According to Michalska et al. [40], the content of anthocyanins, responsible mainly for P. domestica fruit color, ranged from 18 to 170 mg/100 g d.w. The analyzed P. domestica samples contained four anthocyanins, with dominant cyanidin-3-O-rutinoside (51.03%). Their highest content was determined in cvs.Ś18,Ś30,Ś41,Ś32, andŚ16 (>20 mg/100 g d.w.), and the lowest in cvs.Ś28,Ś24,Ś42,Ś27,Ś15,Ś21,Ś23, andŚ7 (<5 mg/100 g d.w.). Another identified anthocyanin was cyanidin-3-O-galactoside, which accounted for 16.28% of all anthocyanins. Its highest content was found in cvs.Ś32,Ś39, andŚ31 (>9 mg/100 g d.w.), and the lowest in cvs.Ś21,Ś9,Ś15,Ś27,Ś13,Ś38,Ś22,Ś8 (<1 mg/100 g d.w.). The remaining anthocyanins accounted for 14.95% of their total pool. However, their content did not exceed 10 mg/100 g. These data are in line with the reports of Tomić et al. [18], who found cyanidin-3-O-glucoside and -3-O-rutinoside to be the two most common anthocyanins in P. domestica fruit.

Antidiabetic and Antiobesity Properties and Inhibition of Lipoxygenase
Human pancreatic α-amylase and intestinal α-glucosidase are responsible for the hydrolysis of carbohydrates into absorbable simple sugars. Inhibition of these enzymes lowers blood glucose levels by limiting the breakdown of polysaccharides into glucose [44]. IC 50 (mg/mL) of α-amylase inhibition in the analyzed P. domestica fruits ranged from 2.63 (Ś34) to 61.53 (Ś41). It was not measured for cvs.Ś1,Ś2,Ś3,Ś8,Ś9,Ś11,Ś12,Ś13,Ś24,Ś28, S33,Ś36, andŚ39. For α-glucosidase, IC 50 oscillated between 0.19 (Ś12) and 24.07 (Ś41), and the parameter was not assessed in cvs.Ś2,Ś6,Ś7,Ś8,Ś9, andŚ43 (Table 4). IC 50 is the concentration of a substance at which 50% of a specific biological or biochemical function is inhibited. Therefore, the lower IC 50 , the smaller the active substance necessary to achieve the desired effect. The inhibition of α-amylase is due to the activity of bioactive plant compounds, such as polyphenolic glycosides, polysaccharides, steroids, and terpenoids [44]. α-Amylase causes postprandial hyperglycemia and increases blood glucose levels, supporting digestion by breaking down polysaccharide molecules into glucose and maltose [45]. De Sales et al. [46] showed that crude extracts and isolated compounds from plant sources could inhibit α-amylase, and flavonoids exhibited the greatest inhibition potential related to the number of hydroxyl groups in their molecules. Of the naturally occurring flavonoid compounds investigated by Kim et al. [47], the most potent inhibitors of α-amylase and α-glucosidase were luteolin, amentoflavone, luteolin 7-O-glucoside, and daidzein. Luteolin at 0.5 mg/mL inhibited α-glucosidase by 36%. Zhang et al. [48], who investigated different cultivars of peaches, found that the fruits inhibited α-glucosidase due to the presence of polyphenolic compounds (chlorogenic acid, neochlorogenic acid, caffeoylquinic acid, 3-O-feruloylquinic acid, catechin, procyanidin C1, procyanidin B1, procyanidin dimer, procyanidin trimer isomer 1, procyanidin trimer isomer 2, procyanidin B2, and prunus inhibitor b). It was also found that the type of phenolic compounds plays an important role in inhibiting α-glucosidase. Inhibition of this enzyme is one of the main strategies for countering the metabolic changes associated with hyperglycemia and type 2 diabetes. Phenolic compounds in fruits and vegetables can affect digestive enzymes involved in the hydrolysis of dietary carbohydrates. In addition, they contribute to the effective prevention of hyperglycemia by limiting lipid absorption [27]. In a study by Nowicka et al. [27], selected peach cultivars showed an inhibitory potential against α-amylase ranging from 1.41 to 4.55 mg/mL, and for α-glucosidase IC 50 ranged from 1.31 mg/mL to 10.51 mg/mL. P. domestica fruits were less effective in inhibiting α-amylase and α-glucosidase. Only a few cultivars (Ś1,Ś5,Ś10) showed inhibitory activity below 0.7 mg/mL (Table 4).
Pancreatic lipase is a key enzyme responsible for the hydrolysis of dietary fats to monoacylglycerols and free fatty acids. This helps reduce overweight and obesity in patients with diabetes by significantly modulating the inhibitory effects of fat absorbed into the bloodstream [49]. In addition, the enzyme is advocated as a weight-lowering agent. P. domestica fruits efficiently inhibited pancreatic lipase, but this ability was cultivardependent (p < 0.05). IC 50 [mg/mL] for pancreatic lipase ranged from 0.5 (Ś35) to 8.2 (Ś1) ( Table 4). The inhibitory potential of pancreatic lipase in peaches examined by Nowicka et al. [27] was between 0.25 and 1.39 mg/mL. Turkiewicz et al. [50] reported that IC 50 for pancreatic lipase in quince fruits ranged from 0.04 to 0.35 mg/mL, depending on the cultivar.
Lipoxygenases (LOXs) are enzymes that catalyze the oxidation of polyunsaturated fatty acids to hydroperoxides [51]. They play an important role in stimulating inflammatory reactions in the human body. Inflammation can be caused by excessive amounts of reactive oxygen species, which stimulate the release of cytokines and subsequent activation of LOX. Studies on the inhibition of lipoxygenases involved in synthesising prostaglandins and leukotrienes were conducted to identify the possibility of preventing conditions such as stroke, cancer, and cardiovascular and neurodegenerative diseases [51]. IC 50 lipoxygenase inhibition in the tested P. domestica fruits ranged from 4.19 (Ś11) to 32.67 (Ś33) ( Table 4)-the cvs.Ś33,Ś17,Ś8,Ś31,Ś15, andŚ37 were characterized by the highest enzyme inhibition capacity (>18.5), while the cvs.Ś11,Ś25,Ś36,Ś43, andŚ6 were the least efficient in this respect (IC 50 < 6). Polyphenols can inhibit lipoxygenase activity by binding to the hydrophobic active site, scavenging lipid radicals, and interacting with the hydrophobic fatty acid substrate [51].

Inhibition of AChE and BuChE Activity
A potential therapeutic strategy in Alzheimer's and Parkinson's diseases is to increase cholinergic levels in the brain by inhibiting the biological activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Therefore, it is important that the diet of people who suffer from these conditions contains potential inhibitors of these enzymes to increase the acetylcholine content in cholinergic synapses and improve nerve conduction [17,49]. Acetylcholinesterase, present in the neuronal synapses of the central nervous system, is a key enzyme in the cholinergic system that terminates the transmission of nerve impulses. Butyrylcholinesterase, as an enzyme associated with glial cells, endothelial cells, neurons, and senile plaques, plays a minor role in the regulation of acetylcholine levels in the brain, but its activity gradually increases in patients with Alzheimer's disease, while the activity of AChE remains unchanged or decreases [27,52]. The source of substances inhibiting the activity of acetylcholinesterase and butyrylcholinesterase are, among others, biologically active compounds found in plants [52]. Molecular docking showed that polyphenols inhibit the activity of AChE and BuChE, offering neuroprotection and improvement of cognitive functions in Alzheimer's and dementia [53]. For this reason, we performed additional experiments to assess P. domestica potential to inhibit AChE and BuChE. Inhibition of AChE, expressed as IC 50 (mg/mL), ranged from 7.62 (Ś19) to 61.82 (Ś36), and it was not assessed in the sampleŚ3 (Table 4). IC 50 for the inhibition of BuChE ranged from 15.60 (Ś29) to 75.73 (Ś36), and the effect was not demonstrated in theŚ35 sample. These results confirmed that P. domestica fruits are not the most effective inhibitors of AChE or BuChE. Raw materials with strong AChE inhibition capacity of over 80% at 0.1 mg/mL include, for example, Rhei radix et rhizome, Polygoni multiflori radix, Salviae miltiorrhiza radix, Radix Paeoniae alba, Radix Paeonie rubra, Chelidonii herba, Corydalis intermediae bulbus, Corydalis intermediae herba, or Corydalis cavae bulbus [52]. In their study on polyphenols in Phyllanthus emblica Linn fruit, Wu et al. [54] reported that myricetin, quercetin, fisetin, and gallic acid were highly efficient in inhibiting AChE. Individual plant compounds were found to exert a specific therapeutic effect, which can be potentiated using the compounds in the right combinations [27]. In comparison with other fruits, that is, peaches (AChE: 4.51-42.90 and for BuChE: 8.85-18.79 mg/mL) [27] and quince (mean inhibition values for AChE 13.24, and BuChE 15.32 mg/mL) [52], we concluded that the analyzed Prunus fruits were less efficient at inhibiting the cholinoesterases.

The Elements of Primary Component Analysis
Principal component analysis (PCA) included the following parameters: mean content of sugars, organic acids, phenolic compounds, effects of biological activity (antioxidant [ABTS o+ , FRAP]), inhibition potential of α-amylase, α-glucosidase, pancreatic lipase, lipoxygenase, AChE, and BuChE, and the examined P. domestica cultivars. The PCA model (Figure 1) presents the most important variables and explains the relationships between 43 P. domestica cultivars, allowing for identifying group patterns. The biplot indicates that 65.69% of the total data variance is represented by F1 and F2. Of these two major components, F1 explains 37.54% of the total variance, and F2 explains 28.16%. Cluster 1: Seven cultivars: Ś2, Ś3, Ś6, Ś9, Ś11, Ś19, and Ś27 showed a considerable potential to inhibit α-amylase, α-glucosidase, 15-LOX, and AChE, and high activity of FRAP and ABTS o+ , which was associated with their content of flavan-3-ols and organic acids. The first principal axis showed the strongest correlations with FRAP, ABTS o+ , flavan-3-ol levels and α-amylase and α-glucosidase inhibition levels. The Pearson test confirmed a correlation between flavan-3-ols and: FRAP and ABTS o+ (0.5 and 0.5, respectively), α-amylase, and α-glucosidase (0.4 and 0.4, respectively).
PCA confirmed significant differences in the chemical composition of P. domestica fruit depending on the cultivar. The analysis made it possible to indicate common features of the examined cultivars and to categorize the fruits into those with a higher content of polyphe-nolic compounds and higher biological activity, into more sweet or sour cultivars, and into cultivars characterized by a high and low ability to inhibit α-amylase, α-glucosidase, pancreatic lipase, lipoxygenase, AChE, and BuChE.
We also performed agglomeration and hierarchical clustering to summarize the differences in chemical compounds and biological activity among the P. domestica cultivars. The AHC dendrogram is shown in Figure 2. The binary cluster tree clearly shows the differences between the cultivars. The line at 73% in the graph represents an automatic truncation, showing two homogeneous groups. The dendogram presents two groups. The first is more diverse and consists of 27 P. domestica cultivars. The groups are made up of cultivars that show a large diversity concerning the analyzed compounds, which confirms the relationships shown in the PCA.
PCA confirmed significant differences in the chemical composition of P. domestica fruit depending on the cultivar. The analysis made it possible to indicate common features of the examined cultivars and to categorize the fruits into those with a higher content of polyphenolic compounds and higher biological activity, into more sweet or sour cultivars, and into cultivars characterized by a high and low ability to inhibit α-amylase, αglucosidase, pancreatic lipase, lipoxygenase, AChE, and BuChE.
We also performed agglomeration and hierarchical clustering to summarize the differences in chemical compounds and biological activity among the P. domestica cultivars. The AHC dendrogram is shown in Figure 2. The binary cluster tree clearly shows the differences between the cultivars. The line at 73% in the graph represents an automatic truncation, showing two homogeneous groups. The dendogram presents two groups. The first is more diverse and consists of 27 P. domestica cultivars. The groups are made up of cultivars that show a large diversity concerning the analyzed compounds, which confirms the relationships shown in the PCA.

Conclusions
Our study confirmed significant differences in chemical composition, phenolic compounds content, and biological properties of 43 P. domestica cultivars. We identified 19 phenolic compounds, including procyanidins, belonging to four groups: phenolic acids (neochlorogenic, cryptochlorogenic, chlorogenic, 3-caffeoylshikimic, and 3-feruloylquinic acid), flavonols (quercetin of -pentoside-hexoside, -3-O-galactoside, -3-O-glucoside, -3-Orutinoside, -3-O-arabinoside, 3-O-rhamnoside, 3-O-penthoside-rhamnoside), flavan-3-ols (procyanidin B1 and B3, (+)-catechin), and anthocyanins (cyanidin-3-O-galactoside, -3-Oglucoside, -3-O-rutinoside, and peonidin-3-O-glucoside). P. domestica fruits were confirmed to be a rich source of phenolic compounds, particularly flavan-3-ols (16.70 to 83.79% of total phenolics content) and phenolic acids (10.47 to 69.08%). The cultivars that accumulated the greatest total amounts of biologically active compounds wereŚ16,Ś17,Ś18, andŚ11. The assessment of antioxidant capacity identified cv.Ś11 had the highest ABTS o+ and FRAP activity, which was related to its high content of phenolic compounds, especially flavan-3-ols compounds. The analyzed cultivars more effectively inhibited AChE (IC 50 = 7.62-61.82) than BuChE (IC 50 = 15.60-75.73). P. domestica fruits are a good source of biologically active compounds and provide several health benefits, which make them a desirable element of a daily diet as fresh fruits during the season or some prepared foods such as, i.e., juices, smoothies, dried and others. The fruits of Prunus domestica are rich in flavan-3-ols, which contribute to their ability to inhibit α-amylase, α-glucosidase, and lipoxygenase, and rich in phenolic acid, which contributes to their ability to inhibit pancreatic lipase. For these reasons, they are helpful in the prevention of many noncommunicable diseases, particularly chronic diseases of the cardiovascular system, type 2 diabetes, gastrointestinal diseases, and some cancers. Data Availability Statement: All related data and methods are presented in this paper. Additional inquiries should be addressed to the corresponding authors.