Comparative assessment of polyphenol stability, bioactivity, and digestive availability in purified compounds and fruit extracts from four black chokeberry cultivars

Abstract

This study evaluated the stability and biological activity of polyphenols in purified polyphenolic extract (IPE) and fruit matrix extract (FME) from four black chokeberry cultivars during in vitro digestion. Fifteen polyphenolic compounds were identified in both extracts by UPLC-PDA-MS/MS, with similar qualitative profiles but quantitative differences. This is the first comparative analysis of IPE and FME from four cultivars—Nero, Viking, and the newly characterized Aron and Hugin. The aim was to assess their antioxidant, anti-inflammatory, antimicrobial, and prebiotic properties across the gastric, intestinal, and absorptive phases. Multivariate statistical analysis (PCA/HCA) was used to evaluate digestion- and cultivar-specific patterns. Anthocyanins dominated the polyphenol profile (79%), while flavonols represented 6%. The highest total polyphenol content was found in the FME of cv. Nero (38.9 mg/g dry matter (d.m.)). Despite containing 2.3 times fewer polyphenols, IPE showed superior bioactivity, including 1.4–3.2 times higher antioxidant potential (FRAP, OH·), up to 6.7-fold stronger inhibition of LOX, and 3–11 times higher bioaccessibility and bioavailability indices across polyphenol classes. These enhancements were likely due to enrichment in more stable phenolic acids and flavonols and the removal of interfering matrix components. Simulated digestion resulted in a 20–126% increase in polyphenol content during the gastric and intestinal stages in IPE, followed by ~ 60% degradation post-absorption, whereas FME showed a 49–98% loss throughout digestion. IPE also exhibited higher bioavailability indices for antioxidant and anti-inflammatory activities. Moreover, the Viking cultivar demonstrated antimicrobial activity against Candida albicans, Escherichia coli, Listeria monocytogenes, and Yersinia enterocolitica. These findings highlight the functional potential of purified polyphenol extracts from black chokeberry and their relevance for the development of stable and bioavailable nutraceutical ingredients.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-13104-1.

Introduction

Polyphenols are widely recognized for their broad-spectrum health benefits, particularly their antioxidant, anti-inflammatory, and cardioprotective properties, as extensively documented in both preclinical and clinical studies^1–4. However, their bioavailability and stability during digestion remain critical factors determining their efficacy in functional foods^5,6. The digestive stability of polyphenols is influenced by various factors, including pH, enzymatic degradation, and interactions with dietary components, leading to significant structural modifications that may alter their bioactivity^2,7–9. The gastrointestinal tract presents a challenging environment where polyphenols undergo degradation through oxidation, enzymatic hydrolysis, and microbial metabolism¹⁰. Black chokeberry (Aronia melanocarpa) is a fruit native to North America and increasingly cultivated in Europe due to its high content of bioactive compounds. It is particularly rich in polyphenols, including anthocyanins (mainly cyanidin-3-O-galactoside), proanthocyanidins, flavonols, and phenolic acids, which contribute to its potent antioxidant potential^15–18. The health benefits of black chokeberry have been demonstrated in numerous in vitro and in vivo studies, showing antioxidant, anti-inflammatory, antidiabetic, antihypertensive, and antimicrobial effects^19–22. Due to its dense phytochemical profile and therapeutic potential, black chokeberry is considered a promising candidate for functional food and nutraceutical applications. Moreover, its high anthocyanin content and unique polyphenol composition make it an excellent model for studying the stability and bioactivity of fruit-derived polyphenols under digestive conditions. Despite its potential, comparative studies evaluating the gastrointestinal behavior of different chokeberry cultivars and extract forms remain scarce. Previous studies have reported that polyphenol-rich fruit extracts exhibit varying degrees of stability, with independent polyphenol extracts (IPE) often demonstrating higher resistance to digestive degradation than fruit matrix extracts (FME), likely due to reduced interactions with macromolecules such as fibers, proteins, and pectins present in the food matrix. In this context, we hypothesize that polyphenol degradation during digestion differs between IPE and FME, with IPE maintaining greater stability due to the absence of interfering compounds such as dietary fibers, proteins, or pectins, which are known to bind polyphenols and reduce their release and activity^2,7,8,11,12. However, beyond the matrix effect, the extraction method itself can selectively enrich or deplete specific subclasses of polyphenols, potentially altering their structural stability and bioactivity during digestion^13,23. For example, Kim et al.¹⁰ demonstrated that purified anthocyanin fractions from Aronia melanocarpa exhibited different degradation kinetics and bioavailability compared to whole fruit or juice, while Sánchez-Velázquez et al.⁴ observed that purification enhanced the retention of polyphenols after gastrointestinal digestion in berries. Despite such evidence, comprehensive comparative studies systematically evaluating both IPE and FME from the same plant source, especially across different cultivars, remain scarce.

Despite the well-documented benefits of polyphenols, most research has focused on their in vitro antioxidant capacity rather than their stability under digestive conditions. The present study aims to fill this gap by evaluating the gastrointestinal stability of polyphenols from selected fruit matrices. This approach provides new insights into the potential application of polyphenol-rich fruit extracts in functional food development. Unlike previous research centered on clinical applications, this study emphasizes the importance of maintaining polyphenol stability in food products, improving their dietary intake and effectiveness^2,3,10,13,14. Understanding how different extraction methods affect polyphenol stability may aid in optimizing food formulations that retain bioactive properties throughout digestion.

In this context, we hypothesize that polyphenol degradation during digestion differs between IPE and FME, with IPE maintaining greater stability due to the absence of interfering compounds from the fruit matrix. The study primarily employs an in vitro simulated digestion model, allowing for a controlled assessment of degradation pathways and bioaccessibility. Focusing on functional food applications, this research provides a foundation for developing polyphenol-enriched dietary products that maximize health benefits while ensuring stability in real-world scenarios. We hypothesized that the IPE, despite its reduced anthocyanin content, would exhibit enhanced bioactivity due to a higher relative proportion of stable polyphenol classes such as phenolic acids and flavonols, and minimized matrix interactions that otherwise hinder compound release, solubility, and enzymatic accessibility during digestion. This assumption is supported by our unpublished pilot data and earlier studies (e.g., Kim et al.¹⁰), which indicate that removal of insoluble matrix components—such as dietary fiber and polysaccharides—significantly improves polyphenol retention, stability, and bioactivity under gastrointestinal conditions.

Therefore, this study aimed to evaluate the stability and bioactivity of polyphenolic compounds in both isolated polyphenolic extracts (IPE) and fruit matrix extracts (FME) from four black chokeberry cultivars (Hugin, Viking, Nero, and Aron—two of which, Hugin and Aron, are analyzed for the first time in this context) during their passage through simulated gastrointestinal stages: gastric digestion (GD), intestinal digestion (GID), and absorption (AD). To date, no studies have evaluated the polyphenol composition, digestive stability, or biological activity of cv. Aron, thus its inclusion provides novel insights into cultivar-dependent variability and supports a broader understanding of genotype-specific functional properties. While previous studies have explored the digestion of chokeberry-derived polyphenols, none to date have conducted a direct, multi-cultivar comparison of IPE versus FME, nor have they linked this with detailed in vitro biological activities (antioxidant, anti-inflammatory, antimicrobial, and prebiotic effects). By combining polyphenol profiling with activity assessment and potentially bioavailability indices, this study provides novel insights into both the matrix-dependent stability and the cultivar-specific nutraceutical potential of black chokeberry, which is essential for optimizing its application in functional foods.

Results and discussion

Stability of phenolic compounds during passage through a simulated digestive tract of four cultivars in different matrices

Chemical extract

Ultra-performance liquid chromatography identified 15 polyphenolic compounds in both extract types. The analysis was performed on four black chokeberry cultivars (purified polyphenolic extract (IPE) and fruit matrix extract (FME)) (Figure S1; Table 1). The polyphenols belonged to 3 classes of compounds, i.e., anthocyanins (ANC), phenolic acids (PA), and flavonoids (FL)^6,10. Among the anthocyanins, four cyanidin derivatives were identified, all showing the characteristic fragment ion m/z = 287, combined with glucoside, galactoside, arabinoside, or xyloside moieties⁶. In turn, Phenolic acids included chlorogenic and neochlorogenic acid (m/z = 353), and caffeic acid (m/z = 179), the latter detected as a diglucoside conjugate (2 × 162 Da). Flavonoids were represented by two kaempferol derivatives (m/z = 285) and six quercetin derivatives (m/z = 301), characterized by neutral losses corresponding to rhamnose (− 146 Da), pentose (− 132 Da), hexose (− 162 Da), and deoxyhexose (− 308 Da = 146 + 162 Da)^10,15. The total polyphenol content (PC) averaged 36 mg/g d.m., where the highest content was recorded in cv. Nero and cv. Hugin showed the highest content in both IPE and FME. The sum of PC, as well as individual compound classes, was significantly (p < 0.05) (Table S1) influenced by cultivars and isolation methods of obtaining the polyphenol mixture, as well as the in vitro digestion and absorption process itself^7,8. ANCs were the dominant group in the tested products, regardless of the type of extract, accounting for an average of 79% of all polyphenols¹⁰, and differences in content did not affect the change in their percentage. This pattern suggests that polyphenols in FME were initially more abundant—2.3 times higher than in IPE—possibly due to the less selective extraction process. However, this also implies that FME compounds may be more susceptible to degradation during digestion, especially in the absence of protective concentration or purification, as seen in IPE. In FME, the lowest content was observed in cv. Hugin and the highest in cv. Nero. A detailed ANC profile obtained that cyanidin 3-O-glucoside was the dominant compound, accounting for 62% on average. Kim et al.¹⁰ detected an ANC content of approximately 25 mg/g in the black chokeberry cv. Viking, which was similar to our observations; however, in that study, cyanidin 3-O-galactoside was dominant, accounting for 80% of all anthocyanins, and cyanidin 3-O-xyloside was not recorded in the present study. They also indicated that the purified black chokeberry extract contained about 12 times fewer individual ANCs compared to the fruit. A similar observation was noted in this report; however, there were only 3 times fewer ANCs in the IPE. The differences were mainly influenced by the extraction method of anthocyanins. Although the total anthocyanin content in IPE was approximately three times lower than in FME, this can be explained by partial loss of highly water-soluble anthocyanins during the ion-exchange purification process. Ion-exchange purification tends to retain less polar phenolics, such as phenolic acids and flavonols, while allowing more hydrophilic anthocyanins to elute during the desalting step. As a result, IPE showed enrichment in other polyphenolic classes, contributing to its superior bioactivity despite lower anthocyanin content. Moreover, despite the lower content of anthocyanins after isolation, the concentration of other polyphenolic compounds was higher in IPE. This may contribute to the observed higher antioxidant activity, as discussed below. Another group of compounds was PAs, accounting for an average of 16% of all polyphenols⁶, regardless of the product analyzed. The lowest content was recorded in the cv. Viking and the highest in the cv. Nero in botch extracts. The last group of compounds was FLs, constituting an average of 6% of all polyphenols⁶. The total FL content ranged from 4.9 (Viking) to 5.7 mg/g d.m. (Nero), and the results were similar to the IPE extract. The dominant compounds identified among the flavonols were kaempferol 3-O-rhamnoside-7-O-pentoside and quercetin 3-O-galactoside-rhamnoside, which accounted for an average of 19% and 22% in both products tested.

Table 1.

Identified polyphenolic compounds in black chokeberry extracts using UPLC-PDA-MS/MS.

No	Identified compounds	RT	[M-H]-	Fragment ion	UV–Vis λmax
		(min)	(m/z)	(m/z)	(nm)
1	Cyanidin 3-O-glucoside*	2.84	449	287	279, 514
2	Cyanidin 3-O-galactoside*	3.13	449	287	279, 516
3	Cyanidin 3-O-arabinoside*	3.36	419	287	279, 516
4	Cyanidin 3-O-xyloside*	3.61	419	287	279, 516
5	Neochlorogenic acid	2.48	353	191	299sh, 324
6	Chlorogenic acid	3.05	353	191	299sh, 327
7	Caffeic acid di-glucoside	3.45	503	341, 179	299sh, 324
8	Kaempferol 3-O-rutinoside	3.58	593	285	264, 345
9	Quercetin 3-O-sophoroside	3.90	625	301	255, 355
10	Kaempferol 3-O-rhamnoside-7-O-pentoside	4.18	563	431, 285	264, 355
11	Quercetin 3-O-glucoside-pentoside	4.38	595	301	255, 355
12	Quercetin 3-O-rutinoside	4.48	609	301	255, 355
13	Quercetin 3-O-galactoside-rhamnoside	4.54	609	301	255, 355
14	Quercetin 3-O-glucoside	4.62	463	301	255, 355
15	Quercetin 3-O-galactoside	4.86	463	301	255, 355

*Compounds identified in positive ionization (the rest in negative).

The different classes of polyphenols in the IPE and FME of four black chokeberry cultivars were subjected to a simulated gastrointestinal tract in vitro at the gastric digestion (GD), intestinal digestion (GID), and absorption (AD) stages (Fig. 1; Table S1 and S2). Due to its complexity, dynamics, and multi-stage nature, the digestion process is challenging. However, it provides an effective alternative for assessing digestibility and potentially bioavailability in animals or humans¹⁶.

Fig. 1.

The content of polyphenolic compounds before and after in vitro digestion in four cultivars of IPE and FME.

The gastric digestion

The GD stage showed an increase in PC from 14% in the cv. Aron to 47% in the cvs. Hugin and Viking, while in the cv. Nero, there was a 7% decrease in IPE. Meanwhile, in FME, there was an average decrease of 49% in polyphenols. Yu et al.¹⁴ also confirmed the decreasing trend of isolated polyphenols and freeze-dried black chokeberry fruit. In the case of ANC during digestion in the simulated digestive tract, there was as much as 13% (cv. Aron) to 43% (cvs. Viking and Hugin) higher content of ANC in IPE and 35% (cv.Viking) to 59% (cv. Aron) significant reduction of these compounds in FME. The higher content of ANC and thus stability was particularly influenced by cyanidin 3-O-glucoside and cyanidin 3-O-arabinoside, whose content also increased on average by 49% and 36%, respectively. During GD, the content of cyanidin 3-O-galactoside and cyanidin 3-O-xyloside decreased on average by 38% and 35%, respectively, relative to the sample extract for IPE. This may be related to the reduced stability of these glycosides under acidic conditions, though further mechanistic studies are required to confirm this. The individual cyanidins in FME were significantly (p > 0.05) reduced despite the low pH (Table S1-S3). This degradation pattern may be explained by differences in chemical stability among cyanidin derivatives, as the glycosylation type and position can influence resistance to acid hydrolysis. Cyanidin 3-O-galactoside and 3-O-xyloside may be more susceptible to degradation due to less stable glycosidic linkages or lower solubility. Moreover, interactions with residual enzymatic activity or oxidation in solution can also contribute to partial breakdown, especially in the absence of protective co-pigments or matrix structures. After the GD stage, PA content was, on average, 1.4 times higher for IPE, with the highest stability and retention of these compounds at 73% in the cv. Hugin. Additionally, high PA retention was indicated in all identified compounds for IPE, with chlorogenic acid having the highest retention from 11 to 87%. An average of 58% PA degradation was observed in the FME. The GD stage equalized the FL concentration in the extracts tested and thus influenced a higher degradation of these compounds in FME at levels ranging from 37% (cv. Viking) to 73% (cv. Aron). While in IPE, retention of these compounds was recorded at levels ranging from 16% (cv. Aron) to 76% (cv. Hugin). The best stability and retention during gastric conditions were achieved by kaempferol 3-O-rutinoside, indicating a 30% (cv .Nero) to 150% (cv. Hugin) higher content in IPE, while in FME, it showed the lowest degradation, ranging from 19% (cv. Viking) to 44% (cv. Nero). In contrast, quercetin 3-O-galactoside-rhamnoside obtained retention ranging from 33 to 96%. Tych et al.¹⁷ confirmed a 22% decrease in ANC concentration after GD of fresh blackcurrant juice and a decrease of about 90% after GID and AD. Yu et al.¹⁴ also confirmed ANC degradation from black chokeberry fruit, as in the case of FME. Besides, the GD step was preceded by the action of simulated oral saliva, which may have had the effect of increasing the potentially availability of the analyzed compounds, as it plays an important role in the solubilization of polyphenols as described by Ginsburg et al.¹⁸ In the acidic environment of the stomach, the breakdown of flavonoid oligomers into smaller units is most common¹⁹. As reported by Gayoso et al.²⁰, most polyphenols show high stability after digestion with saliva and in the stomach, which may also be related to the protective potential of acidic pH on the degradation of these compounds²¹. Bermúdez-Soto et al.¹³ showed that digestion at the GD stage (pH 3.5) did not significantly alter the main polyphenols in black chokeberry juice compared to the GDI stage. This was in line, and the results of this are in agreement with the previous one in the case of FME, while in IPE, there was an increase mainly in ANC. The increase in ANC may be due to the acidic pH (approx. 2.0) in the gastric phase, which stabilizes anthocyanins in their flavylium cation form—an effect well documented in previous studies^13,21. In contrast, deglycosylation under the influence of the enzymes of the above-mentioned form unstable genins, which are then cleaved into other phenolic derivatives. The metabolites of this process can most often be the resulting protocatechuic acid, gallic acid, caffeic acid, and vanillic acid^4,13, which, however, were not identified in our study. Besides, caffeic acid, vanillic acid, or gallic acid can be formed in spontaneous cleavage under the influence of other processes such as single hydration, dehydration, carboxylation, or decarboxylation^4,22. Because these ANCs, in particular, are localized in the vacuoles of fruit skin plant cells. The stomach is an excellent extractor, so these compounds are efficiently released⁴, while depending on the food matrix, they may undergo complexation, which could probably have occurred in the FME analysis. In the case of FL, the absence of significant changes at the GD stage has been reported¹³.

The intestinal digestion

The GID stage indicated an additional increase in PC only for IPE, with the lowest increase in the cv. Nero (75%) and the highest in the cv. Aron (124%). This apparent increase may result from partial breakdown of the plant matrix and improved solubilization of polyphenols that were previously bound or entrapped, thus becoming more detectable by the assay after enzymatic and pH-driven release. The FME extract showed an additional decrease of 27% (cv. Hugin) to 49% (cv. Viking), and there was a 33% increase in the cv. Aron. Additionally, IPE at this stage contained, on average, 2.7 times more polyphenolic compounds compared to FME. Chen et al.²³ similarly detected a 26% increase in PC in freeze-dried wild blackberries after GID as in IPE, which may have been influenced by controlled environmental conditions, i.e., light and oxygen access, which were not controlled in the present study. IPE samples showed a higher concentration of polyphenols after the intestinal digestion stage, suggesting potential protective effects. Most literature indicates that polyphenol degradation increases in alkaline environments, which may explain the observed trend²⁴. Sánchez-Velázquez et al.⁴ also reported an approximately 70% decrease in polyphenols from blackberry after GDI, where a similar trend was observed in FME. The decrease may be due to the interaction of FME matrix polyphenols with other food components. In contrast, the content of color compounds at the GID stage was 2.2 times higher in IPE compared to FME. Furthermore, a 17% and 12% increase in ANCs was recorded at this stage in the IPE of the cvs. Viking and Hugin, while an 8% and 32% reduction in their content relative to the GD stage was indicated in the cvs. Aron and Nero. In FME, a significantly high degradation of ANC was recorded, ranging from 74% (cv. Hugin) to 87% (cv. Viking). Furthermore, the lowest degradation of cyanidin was recorded when combined with xyloside at a level of 5%, while the same compound in FME showed 14 times higher degradation. The lowest stability was recorded for cyanidin 3-O-glucoside at 24% for IPE and 65% degradation for FME. Sánchez-Velázquez et al.⁴ and Kim et al.¹⁰ also showed that GID conditions affected approximately 75% ANC loss in the blackberry freeze-drying and approximately 37% in the purified black chokeberry compounds^4,10. Kim et al.¹⁰ detected that the greater loss was cyanidin 3-O-galactoside than 3-O-glucoside, which was also confirmed in this report. The authors suggest that the cyanidin hexosidic residue and the glucosidic grouping may interact to increase ANC stability. The decrease in ANC concentration at the GID stage was explained by the effect of a change in pH from acidic to basic, which acts to alter the structure of these compounds. It leads to the ring disruption of aglycones and the formation of brown, polymeric, insoluble compounds²⁴. Besides, the release of colored compounds at different stages of digestion into the matrix, such as bolus, chyme, or chyle from plant tissue, is gradual, where the tissue structure is affected by different factors such as a change in redox potential, anaerobic atmosphere, pH, and digestive enzymes⁴. The calculated bioaccessibility index for anthocyanins reached 0.3 for cv. Hugin in IPE. These values suggest better potential release from the IPE matrix than from FME. High bioaccessibility above 1 was indicated by cyanidin 3-O-glucoside and 3-O-arabinoside. Peixoto et al.²⁵, in a paper on the effect of GID on blueberry ANC potentially bioavailability, demonstrated that malvidin derivatives, due to the presence of methylated functional groups, are more stable after digestion than delphinidin or cyanidin derivatives with hydroxyl functional groups. They also indicate that the more OCH₃ groups, the more resistant the compound is to digestion conditions, which increases its bioavailability in the intestine. This is explained by the size of the structure of the molecules studied, which promotes steric hindrance by showing protective properties against degradation or structural change of compounds to an undetectable form. Similar observations were noted for the potentially bioavailable cyanidin fruit extract, which was also low. However, cyanidins were highly bioavailable for compounds isolated on resin. The stability of ANC during digestion in an in vitro system is influenced by various factors, such as the number of acids attached to the glycoside or the number of sugars attached to the aglycone and their nature. Glycosylation also improves the stability and solubility of ANC compared to aglycones. Monoglucosides showed higher stability than diglucosides of ANC, while non-acylated compounds showed lower stability than compounds with an aromatic acyl group^4,16,24,26. Because the FME showed a higher ANC content than the IPE, a saturation of salivary α-amylase by ANC may have occurred, producing a form of carbinol pseudosaccharide that can convert to colorless chalcone, all of which may lead to a lack of subsequent enzymatic activity, deglycosylation, and chemical degradation^4,27, and consequently to lower concentrations of these compounds after gastrointestinal digestion. Additionally, increasing the pH from the gastric to the intestinal environment may convert the flavylium cation into other structures. These include colorless pseudobases (as observed in the oral cavity), a purple chinoid base, or yellow chalcone. Such transitions may result in the complete or partial degradation of ANC^13,14. In vivo studies conducted on pigs fed raspberry meal reported a 40% to 80% recovery of ANC, which was largely dependent on the sugar substituent. The lowest recovery was for cyanidin 3-O-glucoside, around 2%, probably due to degradation, as its metabolites were not reported in urine²⁸. A study by Bermúdez-Soto et al.¹³ obtained as much as 57% recovery of this compound in digested black chokeberry juice, while, in contrast, recovery was 102% in IPE and 19% in FME. The differences can be explained, among other things, by the matrix tested and the in vivo conditions, the results of which often differ from those performed in vitro. At the GID stage, high PA retention of both total content and individual compounds was also indicated, while this content in FME was 5.1 times lower compared to IPE. Additionally, the IPE mixture products indicated a high potential bioavailability of 5.3 (cv. Nero) to 9.7 (cv. Hugin) and were 11 times higher than FME. The exceptions in FME were neochlorogenic acid (cv. Aron) and caffeic acid diglucoside (in all cultivars), whose bioavailability index was above 1. PAs are counted among the main metabolites of FLs, or ANC, and other metabolized compounds, and are completely degraded or pre-absorbed (AD) by the intestinal epithelial wall⁴. In our study, we noted an increase in PA, particularly after GID in IPE, although in FME, an increase in acids was seen with an increase in cyanidin metabolism. Neochlorogenic acid is the main metabolite of delphinidins²⁷, although our study suggests that it may also be a metabolite of cyanidins. Bermúdez-Soto et al.¹³ in their study obtained a 28% and 55% decrease in PA after GID of chokeberries and blueberries, which may indicate its isomerization or degradation. They also noted an increase in chlorogenic acid in favor of a loss of neochlorogenic acid in digested black chokeberry juice and indicated that there was a slight conversion to neochlorogenic acid during incubation of pure chlorogenic acid, although stability was higher¹³. This suggests that the dietary matrix should not be neglected and that the action of pancreatic juice conditions may affect the isomerization of the two acids, and there may be polyphenol oxidase-mediated degradation of mainly neochlorogenic acid. In contrast, Sánchez-Velázquez et al.⁴ indicated the complete degradation of this acid after blackberry digestion. Celep et al.²⁹ also indicated a 43% loss of chlorogenic acid during the GID of blackberry. In the case ofFLs, the GID stage influenced an average increase of 5.8 and 3.4-fold compared to GD in the FME and IPE groups, respectively, while the highest retention was observed in the cv. Aron. Despite the higher content of the individual FLs of the kaempferol and quercetin family, no cyanidin 3-O-arabinoside and cyanidin 3-O-xyloside were recorded in the cvs. Viking and Aron after this digestion step (Table 1, S1). In addition, the analyzed products were particularly potentially bioavailable, and their index was between 2.7 (cv. Nero) and 5.7 (cv. Hugin) for IPE products and 3.2 (cv. Viking) and 4.2 (cv. Aron) for FME. In addition, all identified individual compounds indicated high release rates after GID, the exceptions being quercetin 3-O-galactoside and kaempferol 3-O-rhamnoside-7-O-pentoside cvs. Nero and Hugin for FME. The high stability of FL after the GID process was also confirmed in an earlier study in an in vitro system of black chokeberry juice¹³. In contrast, onion and apple digestion affected the loss of 50 to 75% quercetin and 10% quercetin 3-O-glucoside only after GID, while no significant changes were observed at the GD stage³⁰. Furthermore, it was shown that the pure quercetin 3-glucoside compound was significantly more stable (97% recovery) compared to the compound digested in the black chokeberry juice matrix (70% recovery). Similar observations were made for quercetin contained in onion compared to its pure form³⁰. It has been suggested that interactions with matrix components may affect the stability of individual compounds during digestion¹³. Moreover, if quercetin is oxidized, it may convert to ortho-quinone and transient adducts, which can influence its bioavailability and absorption¹³. Furthermore, polyphenols are sensitive to alkaline intestinal conditions. They can undergo partial metabolism in the duodenum into other polyphenolic forms. However, pure IPE forms appear more stable than polyphenols encapsulated in FME matrices. These findings underscore the importance of selecting stable glycosylated forms (e.g., cyanidin 3-O-arabinoside, kaempferol derivatives) and using purified polyphenol systems like IPE to enhance retention through digestion. This knowledge directly supports the development of functional food products with improved antioxidant stability and bioactive delivery.

Absorption

The AD stage of the PC in both products tested influenced their loss, with that in FME being 92% (cv. Nero) to 97% (cv. Aron) compared to GID. In the case of IPE, it was indicated that only 21% (cv. Nero) to 14% (cv. Aron) of the polyphenols were absorbed relative to the entire digestion process. The average content of the tested compounds in IPE was nine times higher than in FME. The study indicates that compounds from IPE were more stable throughout the digestion and AD process compared to FME. Furthermore, on average, the cv. Viking (16.4 mg/g d.m.) had the lowest compound content and stability in IPE and the cv. Nero (21.2 mg/g d.m.) had the highest, while there was no significant difference in FME. Additionally, PC was highly bioaccessible in IPE, where it was highest in the cv. Hugin (2.8), while in FME, the index value was 3.2 times lower, indicating poor bioaccessibility. After the AD stage, individual ANC content was 11% in IPE, about 2.9 times higher than in FME. The mechanism of AD of natural pigments is not well understood. At the same time, it has been demonstrated from available human and animal in vivo studies that the AD of ANC can occur both in the small intestine, partially in the oral cavity, and in the gastric mucosa via active transport in the form of glycosides²². Talavera et al.³¹ noted that the AD of ANC occurred at the gastric stage. Hence, the high stability of these compounds after digestion under gastric conditions may result, suggesting that the metabolites present in gastric fluid under in vivo conditions may belong to ANC¹⁰. The intestinal microflora is equally important for the mechanism of AD and the metabolism of color compounds^24,32. Regardless of the samples tested, the potentially bioavailability index was below 1, indicating low bioaccessibility of ANC. There was also a high content of PA after AD, at 17% to 23% relative to the GID of the IPE stage, with a 16-fold higher content compared to FME. The post-AD potentially bioavailability index averaged 0.2 in the IPE product, indicating very low potentially bioavailability, in addition to being 3.1 times higher compared to FME. The exceptions were the chlorogenic acids for the cvs. Viking and Aron, as this index was 1.6 and 0.8, respectively. In contrast, the AD of the FL stage showed an 18.6-fold difference in the content of these compounds in favor of the IPE mixture. The digestion process also led to a qualitative change; quercetin 3-O-galactoside degraded completely, quercetin 3-O-sophoroside degraded only in FME, and cyanidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-xyloside were not recorded in the cvs. Viking and Aron of FME. Additionally, the potentially bioavailability index of FL after AD averaged 0.2 and was, on average, 9.1 times higher in IPE compared to FME. Furthermore, kaempferol 3-O-rutinoside and 3-O-rhamnoside-7-O-pentoside, whose potentially bioavailability indices averaged 0.8 and 0.6, and quercetin 3-O-galactoside-rhamnoside 1.0 (cv. Viking) for IPE, deserve special mention. The chemical properties of FLs, such as molecular weight, hydrophobicity, solubility, and isomer configuration, show a significant influence on their stability during digestion³³. In turn, the stability of FLs in the gastric environment depends, among other things, on the physicochemical properties of the compounds or the interaction with the compound mixture, enzymes, or dietary components³. The gut microflora, which has the ability to metabolize macromolecular compounds into simple compounds, plays a major role in the digestion and AD of polyphenols and their respective classes, as compounds above 450 g/mol cannot be bioavailable. Therefore, their metabolites are more often identified in body fluids³⁴. Moreover, digestive conditions are mainly responsible for the biotransformation of polyphenols, and factors such as gender, age, health status, matrix components, or gut microflora determine the profiling of polyphenol intake. The study indicates that the choice of cultivar for further research and the design of health-promoting foods is important; however, it was IPE that proved to be the more valuable material for use in both the food and pharmaceutical industries, as the stability of these compounds was found to be significantly higher.

To the best of our knowledge, this is one of the first comparative studies to evaluate absorption-stage behavior and potential bioavailability indices of polyphenols across both IPE and FME forms in four black chokeberry cultivars, highlighting cultivar- and matrix-specific effects and supporting the rationale for future encapsulation strategies and clinical validation.

Biological and antimicrobial activity in an in vitro model

Black chokeberry contains numerous polyphenolic compounds that exhibit strong antioxidant activity through various mechanisms of action. We used a combination of in vitro digestion and biological assays to assess the antioxidant, anti-inflammatory, and antimicrobial activities of polyphenols from IPE and FME forms. The non-enzymatic antioxidant activity (AA) was assessed using anti-radical assay (ABTS), ferric reducing antioxidant power (FRAP), metal-chelating power (CHP), hydroxyl radical removal activity (OH·), and inhibition of linoleic acid peroxidation (LP) (Fig. 2) before and after digestion in an in vitro model of intact IPE and FME. In turn, enzymatic AA was measured by catalase assay (CAT) and xanthine oxidase assay (XOI), and inflammatory activity was tested by lipoxygenase activity assay (LOX) (Fig. 3) before and after digestion in the in vitro model of purified polyphenolic extract (IPE) and fruit matrix extract (FME). Both the antioxidant and inflammatory activity were significantly influenced by the cultivars and the analysed product, as well as the in vitro digestion process. Significant differences in AA were observed depending on the cultivar, the extract type (IPE vs. FME), and the phase of the simulated gastrointestinal digestion.

Fig. 2.

The non-enzymatic antioxidant activity before and after in vitro digestion in four cultivars of IPE and FME.

Fig. 3.

The enzymatic antioxidant activity before and after in vitro digestion in four cultivars of IPE and FME.

Non-enzymatic antioxidant activity

The ABTS activity in undigested extract was averaged 3,260.7 mg TE/g d.m. in FME, with the highest value observed in the cv. Viking and the lowest in cv. Nero and Hugin were directly proportional to the PC content. Similar observations were detected by Sidor et al.³⁵ This value was 1.2 times higher compared to IPE, which the initial lower PC content could have also influenced. The higher initial ABTS activity in FME likely reflects the synergistic effect of polyphenols with other native matrix components, such as sugars or organic acids. However, after digestion, IPE outperformed due to the greater chemical stability of purified polyphenols and the absence of interfering macromolecules, which may otherwise reduce recovery during gastrointestinal transformation. The GD stage influenced an average sevenfold higher ABTS in IPE compared to FME; furthermore, the activity value decreased by approximately 40% in IPE and up to 92% in FME. At the GID stage, an increase in activity was noted from 67% (cv. Aron) to 97% (cv. Hugin) in FME, whereas in IPE, this increase was on average 11%, although the ABTS value was still 4 times higher in this extract, likely influenced by the polyphenol concentration. On the other hand, the potential bioavailability was close to a value of 1 in IPE, while in FME, it was, on average, 5 times lower. At the AD stage, the difference in ABTS values was 3.4-fold in favor of IPE, and the bioavailability value was 0.5, which was 2 times higher than in FME. For clarity, bioavailability indices are interpreted as follows: values > 1 indicate enhanced bioavailability after digestion, values around 1 suggest moderate retention of active compounds, while values < 1 indicate degradation or reduced availability⁴⁴. Our observations were confirmed in the study by Sánchez-Velázquez et al.⁴, where a decrease in ABTS radical inhibition during digestion was also indicated, with the highest degree of inhibition noted after GID. The decrease in ABTS activity coincided with polyphenol content losses. It is possible that digestion conditions—particularly pH—did not directly affect ABTS solubility but rather influenced compound stability⁴. However, Gião et al.³⁶ noted up to a 70% decrease in ABTS activity after GD of commercial raspberries and a slight increase after GID, as in the case of IPE from black chokeberry. Meanwhile, Yu et al.¹⁴ observed a growing decrease in AA of black chokeberry fruits evaluated by the DPPH assay depending on the digestion stage, similar to the evaluation of FME, suggesting the influence of the raw material matrix on the final results.

The activity of OH· was 8,007.9 mg TE/g d.m., with the highest observed in the cv. Nero and the lowest in cvs. Viking and Hugin for FME. Similar observations were detected by Sidor et al.³⁶ This value was 1.2 times higher in FME than in IPE. On the other hand, at the GD stage, a 9% increase in activity was noted in IPE and a 39% decrease in FME, with the difference between the powders being 1.6-fold. At the GID stage, a significant increase in OH· activity was observed, averaging 300% in IPE and 100% in FME, which was also noticeable in the case of phenolic acids and flavonols, especially in IPE. Furthermore, the difference in activity between the powders was 3.2-fold in favor of IPE. Particularly high OH· activity is shown by high potential bioavailability ranging from 3.8 (cv. Viking) to 4.9 (cv. Nero) in IPE and between 0.9 (cvs. Aron and Nero) and 1.6 (cv. Hugin) in FME. Meanwhile, AD resulted in an average 89% and 92% decrease in activity in both products, which was directly proportional to the content of polyphenolic compounds. The bioavailability at this stage was comparable for the tested varieties and products, averaging 0.1. The markedly higher OH· scavenging activity in IPE after digestion suggests better stability and sustained radical-neutralizing potential, likely due to the concentration of low-molecular-weight flavonoids and phenolic acids less prone to degradation. This indicates stronger protective potential against hydroxyl-radical-induced cellular damage compared to FME. The antioxidant activity against the hydroxyl radical OH· was approximately 1,293 μmol GAE/g d.m. extract, similar to the activity assessed for black elderberry and blackberry. However, no activity is mentioned after the simulated in vitro digestion³⁶.

The inhibition of LP in the sample extract averaged 447.7 mg/g d.m. in IPE and was 2.2 times higher compared to FME, with the highest value observed in cv. Viking and the lowest in the cv. Nero. On the other hand, the GD stage resulted in an increase in LP ranging from 5% (cv. Viking) to 350% (cv. Nero) in IPE, while LP in FME decreased from 62% (cv. Viking) to 93% (cv. Hugin). At the GID stage, a significant increase in LP was also noted, averaging 206% in IPE and 240% in FME. FME exhibited LP values 26 times lower than IPE. Meanwhile, potential bioavailability was particularly high in IPE, indicating values ranging from 2.4 (cv. Viking) to 15.4 (cv. Nero). On the other hand, the AD stage detected a significant decrease in LP, averaging 90% and 98%, respectively, in IPE and FME. The bioavailability was at a level of 0.1. The ability to inhibit LP is mainly attributed to cyanidin 3-O-glucoside, quercetin 3-O-glucoside, and 3-O-galactoside³⁷. According to reports by Achimowicz et al.³⁸, no correlation was found between apple polyphenols and LP inhibition, as also indicated in this study, but only in sample extracts. This indicator changed and showed a strong correlation after absorption, especially with IPE for the ANC, FL, and PC classes, and a moderate correlation with PA. A strong correlation was obtained only with PA and FL at the GID stage and with FL at the GD stage. To the best of our knowledge, the impact of LP inhibition on polyphenolic compounds in black chokeberry during digestion has not been analyzed. Furthermore, polyphenols can directly act as reactive oxygen species (ROS) in the lipid peroxidation chain reaction by neutralizing free radicals, providing electrons to them, and inhibiting the chain reaction¹⁵.

The activity of CHP was 1.2 times higher in FME, with the highest observed in the cv. Aron and the lowest in cv. Viking, while in IPE, the cv. Nero exhibited higher CHP activity. Similar observations were indicated by Sidor et al.³⁶. The higher activity in FME in the sample extract was associated with higher PC in these extracts. At the GD stage, an average decrease of 50% and 86% in CHP activity was noted, respectively, in IPE and FME. Conversely, at the GID stage, observations were reversed, and a significant increase in activity was noted, which was also shown in PA and FL, ranging from 7.7 (cv. Viking) to 9.9 (cv. Nero) for FME and from 13.5 (cv. Hugin) to 18.8 (cv. Viking) for IPE, which was on average 1.5 times lower. The potential bioavailability index was particularly high in IPE, averaging 4.4, and in FME 2.3. At the AD stage, approximately 41% and 31% of CHP activity were noted, respectively, in IPE and FME, and the bioavailability index was comparable in both products, averaging 0.4. Stanisavljević et al.³⁹ noted that the digestion process of black chokeberry juice and pomace contributed to an increase in CHP activity, similar to the results obtained in this study. It was suggested that peptides from matrix proteins are released during digestion in both juice and pomace, which influences the increase in CHP activity. In the present study, a similar mechanism may be involved, as matrix-bound proteins in FME could release peptides during digestion, potentially enhancing metal-chelating capacity alongside polyphenols. They also pointed out that post-digestion juice exhibited lower CHP than pomace, which was explained by the polyphenols in the juice inhibiting matrix protein digestion, probably due to interactions and the formation of stable complexes between these components³⁹. Additionally, it was reported that chlorogenic acid or rutin exhibits the ability to inhibit the formation of glycation products, which is related to CHP’s ability¹⁵.

The average FRAP activity was 621.3 mg TE/g d.m. in FME, with the highest activity observed in the cv. Hugin and the lowest in cv. Viking, the value of which was 1.4 times lower than in IPE. The GD stage resulted in an increase in FRAP values only in IPE, similar to ANC, from 1 to 35% for cvs. Viking and Nero. Meanwhile, the FRAP activity in FME was, on average, 11 times lower. Conditions at the GID stage led to a decrease in activity by an average of 16% and 18%, respectively, in IPE and FME. Despite the decrease in activity, only IPE indicated high potential bioavailability, ranging from 1.6 (cv. Viking) to 2.1 (cv. Nero). The greater stability and higher FRAP activity observed in IPE may result from the purification process, which concentrates specific polyphenol classes—particularly flavonols and phenolic acids—known for strong reducing capacity. This enrichment enhances not only the chemical stability of these compounds during digestion but also their potential to maintain redox homeostasis in vivo by donating electrons and preventing oxidative cellular injury. AD also led to a reduction in FRAP activity by an average of 73% for both products compared to GID. Furthermore, these extracts did not indicate high bioavailability. The conducted reducing power test assessing blackberry at various stages of digestion detected a significant decrease in its power, especially during digestion in the small intestine, which is attributed to the degradation of polyphenolic compounds⁴. It is also suggested through hydrogen atom transfer mechanisms that the method mimics the modulation of redox tone by the ability to accept polyphenols both in serum and tissues⁴⁰. However, Corrêa et al.⁴¹ demonstrated that the reducing power of digested grape pomace increased especially after GID, similar to our observations, only in the mixture of isolated compounds from black chokeberry. The increase in AA may be the result of the release of polyphenols and their metabolites⁴¹. On the other hand, Świeca et al.⁴² noted that the activity of CHP and LPO after simulated in vitro digestion decreased in fortified bread; a similar trend was noted in FME. IPE detected an increase in these activities. Thus, the food matrix and interactions between the matrix and polyphenols will mainly be responsible for this activity.

Kim et al.¹⁰ indicate an increase in AA assessed by the DPPH method from the intestinal to the colonic fraction, explaining this by the influence of metabolites formed from the degradation of ANC. Meanwhile, the increase in AA after digestion in pomegranate juice was explained by the influence of lactic acid bacteria on the polyphenol composition⁴³. Therefore, the decrease or increase in AA, depending on the test used during GD or GID conditions, and even AD, may be mainly associated with structural rearrangement of polyphenolic compounds due to greater stability or sensitivity to solution pH. The formation of complexes with the food matrix may also affect AA by reducing it^16,19. Furthermore, to our knowledge, this is the first document describing the effect of digestion at different stages on the stability and activity of black chokeberry compounds, especially those isolated.

These results collectively indicate that IPE offers superior bioaccessibility, functional activity, and stability across digestion stages, positioning it as a preferred formulation for nutraceutical development.

Enzymatic antioxidant activity

To our knowledge, this is one of the first in vitro studies assessing the effect of simulated digestion on both enzymatic antioxidant and anti-inflammatory activity of purified black chokeberry polyphenols compared to matrix-bound forms across multiple cultivars. Catalase (CAT) and xanthine oxidase (XOI) are key enzymes involved in oxidative stress regulation. CAT decomposes hydrogen peroxide into water and oxygen, thus protecting cells from oxidative damage, while XOI contributes to ROS production during purine metabolism⁴⁴. In CAT, the value was recorded at an average of 0.4 mUnits/g d.m. in IPE and was, on average, 1.4 times higher than in FME, with the highest inhibition activity shown in the cv. Nero. The GD process resulted in a decrease in CAT activity by an average of 39% and 45%, respectively, in IPE and FME. There was also a 2.3-fold difference between the powders in favour of IPE. Meanwhile, the GID stage led to an increase in inhibition of CAT activity from 58% (cv. Hugin) to 72% (cv. Nero) in IPE, with no change in activity observed for cvs. Aron and Viking, while in FME, the increase in inhibition ranged from 50% (cvs. Aron and Nero) to 73% (cv. Viking). At this stage, the potential bioavailability was also determined, with a high value indicated in the Aron (FME) and Hugin (IPE) varieties. In vitro AD led to a decrease in inhibition activity by 63% and 84%, respectively, in IPE and FME, with 2.6 times higher ability to inhibit CAT activity observed in IPE. In previously published animal studies, IPE from chokeberry was shown to protect the parotid gland in rats under low to moderate cadmium exposure conditions⁴⁴. Polyphenolic compounds from the extract improved the enzymatic barrier of CAT and protected against oxidative lipid damage in in vivo studies⁴⁴, which was confirmed by the increase in CAT activity after black chokeberry digestion, especially after GID, depending on the cultivars in this study. This suggests a protective effect of black chokeberry polyphenols against disruption of the antioxidant balance and the parotid gland. Furthermore, black chokeberry extract affected a higher level of antioxidant enzymes, CAT in pancreatic β-cells, protecting them from H₂O₂ and glucose toxicity⁴⁵.

The inhibition of XOI ranged between 256.9 and 248.2 U/g d.m., respectively, in the cvs. Aron and Viking in FME were, on average, comparable to IPE. The GD stage led to a decrease in enzymatic activity inhibition by an average of 50% and 22%, respectively, in IPE and FME. The GID stage resulted in a significant increase in XOI activity inhibition, ranging from 102% (cv. Nero) to 128% (cv. Aron) in IPE and from 44% (cv. Aron) to 85% (cv. Hugin) in FME. Moreover, the potential bioavailability at this digestion stage was particularly high in IPE, with an average value of 1.1, which was 3.2 times higher than in FME. However, the AD stage, regardless of the cultivars and product, led to a decrease in XOI activity inhibition by an average of 72%, with a bioavailability of 0.3 on average. An isolated mixture of polyphenols from black chokeberry cv. Moskow indicated moderate inhibition activity of 26% at a concentration of 42 μg/mL⁴⁶. On the other hand, the ethanolic extract of black chokeberry cvs. Moscow, Hugin, and Nero showed XOI inhibitory activity at levels of 25.5, 34.5, and 23.7% at a concentration of 83.3 ug/mL, suggesting that the cultivars did not determine the ability to inhibit XOI⁴⁷, similar to this study, both before and after the in vitro digestion process.

Anti-inflammatory activity

Inhibition of lipoxygenase (LOX) is directly associated with anti-inflammatory potential, as LOX contributes to the formation of pro-inflammatory mediators and reactive oxygen species. The inhibition of LOX in the sample extract was comparable in both products and averaged 1.2 mUnit/g d.m. This value after gastric digestion (GD) decreased on average by 79% and 94%, respectively, in purified polyphenolic extract (IPE) and fruit matrix extract (FME), with LOX inhibition in IPE being 2.7 times higher than in FME. Subsequently, the intestinal digestion (GID) stage also resulted in a decrease in LOX activity inhibition by an average of 36% and 69%, respectively, in IPE and FME. However, the cv. Hugin was an exception, showing a 16% increase in inhibition activity in IPE and only a 34% decrease in inhibition activity in FME. Moreover, LOX inhibition activity at this stage was 6.7 times higher in IPE than in FME. At this stage, particularly low potential bioavailability was also noted, amounting to 0.2 in IPE but higher by 8 times than in FME, on the other hand, the AD stage, compared to enzymatic and non-enzymatic methods, led to a significant increase in LOX activity inhibition by an average of 163% and 569%, respectively, in IPE and FME. A particularly high bioavailability was also noted at an average level of 6.7 in FME, which was 2.7 times higher than in IPE. Xiong et al.¹⁶, in studies on the digestion of berry pomace, detected that the anti-inflammatory activity after digestion depends on the composition of polyphenolic compounds, and additional protection in the form of encapsulation protects the compounds, enhancing the anti-inflammatory activity¹⁶. Extracts of pure polyphenolic compounds from black chokeberry cv. Moscow showed high inhibition activity of 15-LOX with an IC₅₀ of 57 (ug/mL), indicating that ANC and proanthocyanidins exhibited high inhibition activity of 15-LOX⁴⁷. On the other hand, the ethanolic extract of black chokeberry cvs. Moscow, Hugin, and Nero showed inhibitory activity of 15-LOX at levels of 4, 10.9, and 8.9% at a concentration of 83.3 ug/mL⁴⁷. Similarly, slight differences between varieties were noted in this study, although these differences changed after the digestion process. The authors also point out that the inhibition activity of 15-LOX may depend not only on the content of ANC but also on other polyphenolic compounds⁴⁷.

Both prooxidative enzymes, lipoxygenase (LOX) and xanthine oxidase (XOI), contribute to the formation of ROS, which promote oxidative stress in vascular cells. Inhibiting LOX and XOI may therefore reduce inflammation and oxidative damage⁴⁶. Additionally, black chokeberry extracts after simulated GD indicate a moderate LOX/XOI correlation with polyphenols, whereas this index changes at the AD stage, indicating a strong correlation. Therefore, the conducted research indicates that LOX inhibitors are highly bioavailable and XOI inhibitors are highly bioaccessible, especially in the case of IPE, suggesting that they may act at the in vivo level as effective inhibitors, although this requires further confirmation.

Among all tested parameters, the highest potential bioavailability was observed in the inhibition of lipid peroxidation (up to 15.4), hydroxyl radical scavenging (up to 4.9), and metal-chelating power (up to 4.4), particularly in IPE after the intestinal digestion stage, while FME generally showed lower indices across all assays.

Antimicrobial and prebiotic activity

Antimicrobial and prebiotic activity was measured in sample extracts. The cultivars and the analysed product significantly influenced the microbiological activity.

Antibacterial activity was not observed by fruit matrix extract (FME), probably due to the presence of components such as sugars that promote the growth of the tested microorganisms (Table S3, S4). However, in the case of purified polyphenolic extract (IPE) devoid of fibrous substances, high activity was obtained depending on the analyzed cultivars of black chokeberry. The cv. Viking showed antimicrobial activity against Candida albicans, Escherichia coli, Listeria innocua, Listeria monocytogenes, Yersinia enterocolitica, with the largest clear zone in mm observed for L. monocytogenes (13.5/15 mm) > E. coli > C. albicons, L. innocua, Y. enterocolitica. The cv. Aron showed inhibitory activity against L. innocum and L. monocytogenes, also with a high clear zone at 13/15 mm. Meanwhile, the cv. Nero and cv. Hugin varieties presented inhibitory activity against E. coli, L. innocua, and L. monocytogenes, with the largest clear zone observed for inhibition against Y. enterocolitica (13/14 mm and 14/15 mm) and the smallest against E. coli (10.5/11 mm). The minimum inhibitory concentration (MIC) for all tested varieties against strains C. albicans, E. coli, L. innocua, L. monocytogenes, and Y. enterocolitica was 15 mg/mL of polyphenolic extract. In studies conducted by Daoutidou et al.⁵, black chokeberry extracts at a concentration of 62.5 mg/mL showed inhibitory activity against E. coli, S. aureus, S. enterica with clear zones of 8-, 9-, and 12-mm⁵. On the other hand, Liepina et al.⁴⁸ noted antimicrobial activity against Bacillus cereus, Staphylococcus aureus, and Pseudomonas aeruginosa but did not indicate inhibitory activity against E. coli. They also found that Gram-positive bacteria showed higher sensitivity to black chokeberry extract, possibly due to the presence of anthocyanins, than Gram-negative bacteria, due to differences in cell wall structure⁴⁸. This antimicrobial effect is largely attributed to the high content of anthocyanins and proanthocyanidins, which are known to inhibit bacterial growth. The potent antimicrobial properties of these compounds suggest their potential application in controlling pathogenic bacteria in the gastrointestinal tract, thus contributing to improved gut health⁴⁹.

Regarding the variation in antimicrobial activity across different chokeberry cultivars, this can be linked to differences in polyphenol profiles and total polyphenolic content. The Viking cultivar exhibited the highest antimicrobial activity, potentially due to its elevated levels of anthocyanins, phenolic acids, and flavonols (Table S1), which may act synergistically to enhance antimicrobial activity^10,14. Studies have suggested that anthocyanins, particularly cyanidin 3-O-glucoside, exert strong antibacterial properties by altering bacterial membrane permeability and interfering with quorum-sensing mechanisms²⁵. Furthermore, flavonols such as quercetin derivatives can inhibit bacterial DNA gyrase and ATP synthesis, thereby reducing bacterial viability^4,32.

To quantitatively validate these relationships, a correlation analysis was conducted between antimicrobial activity and the key polyphenolic compounds present in IPE and FME (Fig. 4). Results indicate a strong positive correlation (r > 0.85, p < 0.05) of antimicrobial activity (inhibition zone diameter, MIC) with anthocyanin content, particularly cyanidin derivatives, and a moderate correlation (r = 0.65–0.80, p < 0.05) with chlorogenic acid levels. These findings further confirm that specific phenolic compounds play a crucial role in determining antimicrobial potency.

Fig. 4.

Analysis of Pearson correlation in chemical extract. Explanation: CE, chemical extract; PC, polyphenolic compounds; ANC, anthocyanins; FL, Flavonols; PA, phenolic acids; XO, xanthine oxidase assay; CAT, catalase assay; CHP, chelate iron ions; OH·, hydroxyl radical scavenging; LP, linoleic acid peroxidation; A1, VIKING;A2, ARON; A3, NERO; A4, HUGIN; B1, VIKING; B2, ARON; B3, NERO; B4, HUGIN; (A) pure polyphenols; (B) fruit powder.

It is important to clarify that antimicrobial activity refers to the inhibition of pathogenic microorganisms (measured by inhibition zones and MIC), whereas prebiotic potential is assessed by the ability to stimulate the growth of beneficial gut bacteria. Thus, prebiotic activity was not observed for FME; however, IPE exhibited both strong antimicrobial effects and notable prebiotic potential, particularly by promoting the growth of Bifidobacterium longum^50–53 (Table S5). This dual functionality underscores the critical role of polyphenols in modulating the gut microbiome and supporting overall gut health. The most substantial inhibitory effect was observed in the cv. Aron, with a clear zone of 26 mm, while the cv. Nero demonstrated a narrower clear zone of 19 mm. Additionally, the MIC of IPE required to inhibit the growth of prebiotic bacteria, irrespective of the cultivar, was 15 mg/mL. These findings suggest that polyphenols can effectively modulate gut flora, enhancing both antimicrobial defenses and the growth of beneficial bacteria.

However, the observed differences in antimicrobial activity between FME and purified polyphenolic extracts IPE can be attributed to the matrix effect and polyphenol composition. Polyphenols within the fruit matrix are often bound to fibers, proteins, and other macromolecules, which may limit their bioavailability and antimicrobial efficacy. In contrast, IPE consists of purified, more readily available polyphenols, allowing for direct interaction with microbial cells, thereby enhancing their antimicrobial potency. Previous studies have demonstrated that isolated phenolic compounds exhibit superior antimicrobial activity compared to those embedded in the plant matrix, as they can more effectively penetrate bacterial cell walls and disrupt cellular functions^{13,24,53–55}.

PCA and HCA

In order to visualize the obtained results, two statistical methods were applied: hierarchical cluster analysis (HCA) for the sample extract results depending on the cultivars and principal component analysis (PCA) to assess the relationship between polyphenolic compounds results and in vitro health-promoting activity before and after simulated in vitro digestion (Fig. 5). The HCA method indicated 4 main clusters, where the first cluster grouped FME cv. Aron, which exhibited the highest metal-chelating power (CHP) and xanthine oxidase assay (XOI) activity, the second cluster comprised FME cvs. Hugin and Nero were characterized by the highest levels of phenolic acid (PA) and flavonols (FL) and high ferric reducing antioxidant power (FRAP), catalase assay (CAT), and lipoxygenase activity assay (LOX) activity; the third cluster included fruit matrix extract (FME) cv. Viking had the lowest individual polyphenol values, while the fourth cluster consisted of IPE cvs. Aron, Viking, Nero, Hugin, whose initial compound content was approximately 3 times lower compared to FME. Figure 5 presents data for "Biplots-PCA”. PCA analysis indicates that the first two components explained 80.9% of the variables, where Dim1 accounted for 71.9% and Dim2 for 9%. FME was clearly grouped in the "Biplots-PCA" plot, which was consistent with the HCA analysis. "Biplots-PCA" indicates a high positive correlation between XO, LOX, and CHP activity, and a moderate correlation with hydroxyl radical removal activity (OH·) after gastric digestion (GD) with all classes of polyphenolic compounds in sample extracts, and the tested activities evaluated at the absorption digestion (AD) stage also showed strong positive correlations with identified compounds after absorption. Simulated compound classes in Fig. 5 also indicate that digestion in the in vitro system contributed significantly to greater stability and quantity of tested IPEs of all varieties compared to sample extracts, which predominated in FME. These extracts also exhibited high activity, especially in CHP, OH·, XO, and LOX tests. Meanwhile, during the GD stage, concentrations in the tested products were equalized, indicating significant degradation of these compounds in FME. Kim et al.¹⁰, using PCA analysis, showed distinct clustering of extracts from mulberry and black chokeberry, correlations between simulated black chokeberry extracts with cyanidin 3-O-galactoside, and the occurrence of p-coumaric acid, protocatechuic acid, and its hexoside with the colon and rectum fraction.

Fig. 5.

Analysis of main components: (A) agglomerative hierarchical cluster analysis (HCA) of distance between sample extracts in 4 black chokeberry cultivars (Aron, Nero, Hugin, Viking) of IPE and FME; (B) PCA-biplom. Explanations: 1, Viking IPE; 2, Aron IPE; 3, Nero IPE; 4, Hugin IPE; 5, Viking FME; 6, Aron FME; 7, Nero FME; 8, Hugin FME; CE, sample extract; GD, gastric digestion; GI, intestinal digestion; A, absorption, PA, phenolic acids; ANC, anthocyanins; FL, flavonols.

Overall, despite the 2.3-fold lower total polyphenol content in IPE compared to FME, the purified extract exhibited significantly superior biological activity, including 1.4–3.2 times higher antioxidant potential (e.g., FRAP, OH·, LP), stronger inhibition of LOX and XOI enzymes (up to 6.7-fold for LOX), and 3–11 times higher bioaccessibility and potential bioavailability indices across all polyphenol classes (ANC, PA, FL), underscoring the effectiveness of polyphenol purification in enhancing functional properties.

Material and methods

Plant material and preparation of the extract

Plant materials of the black chokeberry (Aronia melanocarpa Nutt.) cultivars (cvs. Hugin, Viking, Nero, Aron). The ZUT plantation was used in the study. The Aron cultivar is a relatively new variety introduced to Poland and, to the best of our knowledge, has not yet been analyzed in terms of its polyphenolic profile or digestive stability, which adds novelty to the current research. Its inclusion allows for a broader understanding of cultivar-dependent variation in polyphenol stability and activity. The Aron cultivar is a relatively new variety introduced in Poland and, to the best of our knowledge, has not yet been analyzed for its polyphenol profile or digestive stability. Its inclusion adds novelty and expands understanding of cultivar-dependent differences in polyphenol behavior. The fruits were harvested at full maturity, which was determined based on their deep black coloration, firm texture, and total soluble solids (TSS) content. The harvest took place in September 2023, ensuring optimal ripeness for polyphenol content analysis. This addition enhances the reproducibility of the study. This addition enhances the reproducibility of the study. The fruit matrix extract (FME) was obtained through a two-step extraction process, where crushed black chokeberry was treated with 70% ethanol containing 1% formic acid, assisted by ultrasound (30 min, 30 °C, Sonic 10, Polsonic, Warsaw, Poland). The extract was then centrifuged (10 min at 10,000 × g; Eppendorf, Hamburg, Germany), and the resulting supernatant was concentrated using a rotary evaporator (R-215 Rotavapor System, Buchi, Flawil, Switzerland). For the purified extract (isolated polyphenolic extract; IPE), the crude extract was passed through a resin (Amberlite XAD-16, Brenntag, Poland) preconditioned with methanol and equilibrated with water. After the extract was loaded onto the column, it was washed with water, and the purified fraction was concentrated again using the evaporator. The crude and purified extracts were freeze-dried (ALPHA 1–2 LD plus, Osterode, Germany) and refrigerated until further use.

A schematic overview of the in vitro digestion and absorption model used in this study is provided in Figure S2.

Digestion in an in vitro system

In vitro digestion was assessed at the gastric, intestinal, and absorption stages and was performed as described by Lachowicz et al.^7,8 and Gawlik-Dziki et al.⁵⁴ The simulated digestion experiment was conducted for each sample in three independent replicates across three stages: (i) The first stage was halted at the gastric digestion phase, and the resulting fraction was subjected to further analyses; (ii) The second stage involved completing the full gastric-intestinal digestion, which was stopped at the intestinal digestion phase, and the obtained extract was analyzed; (iii) The third stage included completing the full gastric-intestinal digestion, followed by absorption simulation. The fraction was halted post-absorption and subsequently analyzed.

A simulated saliva solution was prepared by dissolving 2.4 g Na₂HPO₄, 0.2 g KH2PO4, and 8.0 g NaCl in 1 L of distilled water. The solution was adjusted to pH 6.7, and α-amylase (EC 3.2.1.1.) was added to obtain 200 U per mL of enzyme activity. For gastric digestion, 300 U/mL of pepsin (from porcine stomach mucosa; EC 3.4.23.1) was prepared in 0.03 mol/L HCl, pH 1.2. Further, simulated intestinal juice was prepared by dissolving 0.05 g of pancreatin and 0.3 g of bile extract in 35 mL of 0.1 mol/L NaHCO₃. The samples were subjected to simulated gastrointestinal digestion as follows: 100 mg of a powdered sample was homogenized in a Stomacher laboratory blender for 1 min to simulate mastication in the presence of 5 mL of simulated salivary fluid; subsequently, the samples were shaken for 10 min at 37 °C. The samples were adjusted to pH 1.2; 5 mL of simulated gastric fluid was added next. The samples were shaken for 120 min at 37 °C. After digestion with the gastric fluid, the samples were adjusted to pH 6, and then 5 mL of a mixture of the bile extract and pancreatin (activity equivalent to 4 × USP) was added. The extracts were adjusted to pH 7, and finally, 5 mL of 120 mmol/L NaCl and 5 mL of mmol/L KCl were added to each sample. Once prepared, the samples were submitted to in vitro digestion for 120 min at 37 °C in the dark. The samples were centrifuged, and the supernatants were used for further analysis. After the in vitro digestion (AD), the samples were centrifuged for 30 min at 6800 × g at 4 °C, and the supernatants were discarded for analysis. The centrifuged supernatant was subjected to ultrafiltration using a 3-kDa molecular weight limit membrane to obtain potential AD⁵¹. After that, the enzyme activity was stopped using methanol (1:1 ratio). Theoretical approach: the bioaccessibility⁷ was calculated as RE per GE, where GE = amount of components/activity after simulated gastro or intestinal digestion, and RE = amount of components/activity after chemical extraction (raw extract); the bioavailability was calculated as AE per GE, where, GE = amount of components/activity after simulated intestinal digestion, and AE = amount of components/activity after absorption. Besides, the Aron variety is new in Poland and has not been analyzed yet.

Polyphenolic compounds

Chemical extraction: The extraction procedure was adapted from the protocol described by Lachowicz et al.⁸ and Lachowicz et al.⁵² with slight modifications to improve recovery efficiency from different matrices. Briefly, for polyphenols, a sample (100 mg) was extracted with 10 mL of a mixture containing UPLC-grade methanol (30%) and acetic acid (1% of reagent). The extraction was performed twice by incubation for 20 min under sonication (Sonic 6D, Polsonic, Warsaw, Poland) and with occasional shaking. Next, the slurry was centrifuged at 19,000 g for 10 min, and the supernatant was filtered through a hydrophilic PTFE 0.20 μm membrane (Millex Simplicity Filter, Merck, Darmstadt, Germany) and used for analysis. Polyphenols determination by UPLC-PDA-MS/MS system (Waters Acquity, Milford, MA, USA): the runs were monitored at the following wavelengths: phenolic acids at 320 nm, flavonols at 360 nm, anthocyanins at 520 nm, and flavan-3-ols at 280 nm. Individual polyphenols were separated using a UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters Corporation, Milford, MA) at 30 °C. The samples (10 μL) were injected, and the elution was completed in 15 min with a sequence of linear gradients and isocratic flow rates of 0.45 mL/min. The mobile phase comprised solvent A (2.0% formic acid, v/v) and solvent B (100% acetonitrile). The program began with isocratic elution with 99% solvent A (0–1 min), and then a linear gradient was used until 12 min, lowering solvent A to 0%; from 12.5 to 13.5 min, the gradient returned to the initial composition (99% A), and then it was held constant to re-equilibrate the column. To quantitatively determine the content of selected compounds, the following standards were used: cyanidin-3-O-galactoside, chlorogenic acid, quercetin, and rutin. The PDA spectra were measured over the 200–600 nm wavelength range in steps of 2 nm. The experiment was exhibited in triplicate, and consequently, data (results) were obtained. The results were expressed as mg/g of dry matter (d.m.). The method was validated for parameters such as linearity, accuracy (relative error, RE), limit of detection (LOD), limit of quantification (LOQ), and precision (relative standard deviation, RSD). External standard calibration lines were generated by three repeated injections of standard solutions at seven concentration levels (0.05, 0.1, 0.25, 0.5, 1, 2.5, and 5 mg/L) at 1 day. A plot of peak area with respect to the corresponding concentration was used to demonstrate linearity. The linear regression equation and correlation coefficient were calculated by weighted (1/ × 2) least-squares linear regression analysis. Linearity was considered to be acceptable when correlation coefficients were R² ≤ 0.9998. The LOD was determined as a signal-to-noise ratio (S/N) of 3:1, and the LOQ was determined as a S/N of > 10. An acceptable RE within 20% and the intra- and inter-day variations were determined using relative standard deviation (RSD) values, which were < 3.5% for all the analysed compounds.

Biological activity in vitro

The non-enzymatic antioxidant activity (AA) was determined by the antiradical activity of the ABTS method, which was carried out with the method described by Lachowicz et al.⁸ ABTS^·⁺ radical scavenging assay: This assay is based on the ability of antioxidants to quench the ABTS radical cation (ABTS^·⁺), which results in a decrease in absorbance at 734 nm. The ABTS^·⁺ solution was prepared by reacting ABTS with potassium persulfate and incubating in the dark for 12–16 h. The reaction was initiated by mixing the diluted sample with ABTS^·⁺ solution and measuring absorbance after 6 min. Results were expressed as mg Trolox equivalents per g d.m. following Lachowicz et al.⁸

FRAP assay (ferric reducing antioxidant power): The assay measures the reduction of the Fe³⁺-TPTZ complex to Fe²⁺-TPTZ by antioxidants, monitored at 593 nm. The FRAP reagent consisted of TPTZ, ferric chloride, and acetate buffer. The sample was incubated for 10 min at 37 °C, and absorbance was recorded. Results were expressed as mg Trolox/g d.m.⁸.

Hydroxyl radical (·OH) scavenging activity: Assessed based on the ability of the extracts to compete with deoxyribose for hydroxyl radicals generated via the Fenton reaction (Fe²⁺ + H₂O₂ → Fe³⁺ + ·OH). The inhibition of malondialdehyde formation (measured at 532 nm) reflected the scavenging activity. Results were expressed as mg Trolox/g d.m.⁴².

Chelating power of ferrous ions (CHP): Measured by the formation of the ferrozine-Fe²⁺ complex. The presence of chelators in the sample reduces complex formation, decreasing absorbance at 562 nm. EDTA was used as a standard, and results were expressed as mg EDTA equivalents per g d.m.⁴⁴.

Inhibition of linoleic acid peroxidation (LP): Lipid peroxidation was induced in a linoleic acid emulsion, and oxidative degradation was measured at 480 nm using the thiocyanate method. Inhibition indicated antioxidant potential against lipid oxidation. Results were expressed as mg Trolox/g d.m.⁴².

Enzymatic antioxidant activity

Catalase inhibition assay (CAT): This assay evaluates the capacity of the sample to inhibit catalase, which decomposes hydrogen peroxide. The decrease in absorbance at 240 nm reflects residual H₂O₂ concentration. One unit (U) of inhibitory activity was defined as the amount of sample inhibiting 1 U of catalase activity per g d.m.⁴².

Xanthine oxidase inhibition assay (XOI): Assesses the ability to inhibit xanthine oxidase (XO), which catalyzes the oxidation of xanthine to uric acid and generates superoxide radicals. The assay was based on measuring the formation of uric acid at 295 nm. Results were expressed as U or mU inhibitor activity per g d.m., indicating inhibition of enzymatic oxidative processes⁴².

All biological in vitro tests were measured using the BioTek Microplate Reader spectrophotometer (BioTek, Warsaw, Poland).

Anti-inflammatory activity

The LOX inhibitory assay was made based on the method described by Lachowicz et al.⁸ LOX activity was tested in absorbance at 234 nm. One mega unit of inhibitor activity was defined as the activity inhibiting 1 mega unit of enzyme activity. Results were expressed in MUnit per g of d.m.

Anti-microbiological activity

Regeneration of Pathogenic Strains

The regeneration of pathogenic strains (Yersinia enterocolitica ATCC9610, Salmonella enterica KBiMŻ12a, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC15445, Listeria monocytogenes ATCC15313, Listeria innocua ATCC33090, Enterococcus faecalis KBiMŻS72, Escherichia coli ATCC10536, Bacillus cereus ATCC10876, and Candida albicans KBiMŻ11c) was conducted in 2 ml Eppendorf tubes. A single bead containing the bacterial strain was taken from the cryobanks and transferred into 1 ml of the nutrient broth. The cultures were then incubated under aerobic conditions at 37 °C for 24–48 h.

Regeneration of probiotic strains

The probiotic strains were regenerated in 2 ml Eppendorf tubes, following the same procedure as for pathogenic bacteria. Beads with adsorbed bacteria were introduced into 1 ml of MRS broth for Lactobacillus helveticus, Lactobacillus rhamnosus GG, and Lactobacillus salivarius; MRS with L-cysteine for Bifidobacterium animalis and Bifidobacterium longum; and RCM broth for Faecalibacterium prausnitzii. Cultures of Lactobacillus were incubated under microaerophilic conditions, while Bifidobacterium and F. prausnitzii were cultured anaerobically using AnaeroGen®A at 37 °C for 48 h.

Determination of antibacterial activity

Antimicrobial activity was assessed using the well diffusion method. Regenerated indicator strains were swabbed onto Mueller–Hinton agar (OXOID, catalog number CM0405) and solidified in Petri dishes. Wells with a diameter of 10 mm were aseptically cut into the agar using a cork borer, and 100 µl of polyphenol extract solutions at a concentration of 1.5% w/v (60 mg/4 ml in 10% DMSO) were added to the wells. Plates containing anaerobic strains were placed in anaerobic culture containers with AnaeroGen®A sachets. The plates were incubated at 37 °C for 24 h. After incubation, the diameters of inhibition zones (clear zones) were measured in millimeters. Results were recorded using the AES plate reader and analyzed with the EasyCount 2 software, with measurements reported to the nearest 1 mm (SD = 1 mm for all samples).

Determination of MIC

The polyphenol extracts’ minimum inhibitory concentration (MIC) was determined using a series of six two-fold dilutions prepared in U-shaped titration plates, with sterile distilled water as the diluent. Mueller–Hinton agar plates were surface-inoculated with indicator strains using swabs, and 10 µl of each diluted extract was spotted onto the agar. Plates containing anaerobic strains were placed in anaerobic culture containers with AnaeroGen®A sachets and incubated at 37 °C for 24 h. After incubation, the dilution levels with visible clear inhibition zones were recorded. The highest dilution of the extract-producing inhibition zones of at least 3 mm in diameter was used to calculate the MIC value. MIC values were determined for each extract and indicator strain.

Statistics

Statistical analysis, such as one-way ANOVA and median, was analyzed using Statistica 12.5 (StatSoft, Kraków, Poland). One-way ANOVA and Tukey’s multiple range test evaluated significant differences (p < 0.05) between mean values. Prior to performing one-way ANOVA, data normality and homogeneity of variances were assessed using the Shapiro–Wilk and Levene’s tests, respectively. The multivariate analysis was performed by applying principal component analysis (PCA) and agglomerative hierarchical cluster analysis (HCA) by the Ward method (Euclidean distance).

Conclusions

This study demonstrates that black chokeberry polyphenols are highly sensitive to cultivar type, extraction strategy, and simulated gastrointestinal conditions, all of which significantly influence their final concentration, bioaccessibility, and potential bioavailability. A total of fifteen polyphenolic compounds were identified, including four anthocyanins, three phenolic acids, and eight flavonol derivatives. Anthocyanins dominated the polyphenol profile in both extract types—fruit matrix extract (FME) and isolated polyphenolic extract (IPE)—accounting for an average of 79% of total polyphenols, followed by phenolic acids (16%) and flavonols (6%).

Among the studied cultivars, cv. Nero exhibited the highest total polyphenol content (38.9 mg/g d.m.) in FME, whereas cv. Hugin presented the lowest (33.4 mg/g d.m.). On average, IPE contained 2.3 times fewer total polyphenols than FME; however, it showed markedly greater resistance to gastrointestinal degradation. Simulated digestion increased the relative bioaccessibility of polyphenols in IPE by 2.7–9.0 times in the gastric and intestinal phases, while FME experienced cumulative losses of 49%, 62%, and 98% in the gastric, intestinal, and absorptive stages, respectively.

In IPE, the digestion process resulted in a 20% increase in polyphenol content during the gastric phase and up to 126% during the intestinal phase, followed by a 60% loss after simulated absorption. The IPE also exhibited higher bioaccessibility indices for antioxidant mechanisms and enhanced potential bioavailability for anti-inflammatory effects, demonstrating superior functional value compared to FME.

Anthocyanins in IPE showed improved stability under digestive conditions, likely due to reduced interactions with the food matrix and enhanced protection of more stable polyphenol subclasses (e.g., phenolic acids, flavonols). Notably, IPE from cv. Viking showed the strongest antimicrobial activity against five pathogenic strains and stimulated the growth of Bifidobacterium longum, supporting its dual antimicrobial and prebiotic properties.

Overall, the results underscore cultivar-specific and matrix-related differences and highlight IPE as a promising candidate for incorporation into functional foods and nutraceutical formulations. Further research should prioritize encapsulation technologies and in vivo validation to optimize compound stability, targeted delivery, and health outcomes.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Sabina Lachowicz-Wiśniewska: Conceptualization, Methodology, Software, Investigation, Formal analysis, Writing—original draft, Writing—review & editing, Funding acquisition, Project administration. Michał Świeca: Methodology, Writing—review & editing, Ireneusz Kapusta: Methodology, Writing—review & editing, Anna Sip: Methodology, Writing—review & editing, Ireneusz Ochmian: Delivery of plant materials, Writing—review & editing.

Funding

This research was funded by the National Science Centre (NCN), Poland [grant no. 2020/39/D/NZ9/01810].

Data availability

Data is provided within the manuscript and supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This article contains no studies with human or animal subjects.