Impact on the Health-Promoting Potential of Cranberries for Food Applications Through Soilless Cultivation Practices in Piemonte Region (Italy): A Sustainable Opportunity for Nutraceutical Production

Abstract

Cranberry (Vaccinium macrocarpon Aiton), a traditional berry crop cultivated in North America, is appreciated for its high amounts of bioactive compounds and polyphenols. The exploration of its cultivation in different geographic areas may support crop diversification and sustainable production of fruits and derived products rich in health-promoting molecules. The present research evaluated the antioxidant capacity, phytochemical profile, and nutritive composition of the ‘Pilgrim’ cranberry cultivar grown in soilless conditions in Northwestern Italy (Bra, Piemonte Region), compared to a reference sample from North America (Canada). Physical–chemical parameters such as weight, fruit size, titratable acidity, and total soluble solids were considered. Additionally, anthocyanins, total phenolics, antioxidant capacity, and proanthocyanidins (PACs) were evaluated using spectrophotometric protocols. Chromatographic techniques (HPLC-MS/MS and HPLC-DAD) were used for detailed profiling of phenolic acids, flavonoids, vitamin C, sugars, organic acids, and PAC types (A- and B-type dimers and trimers). The results highlighted that Italian-grown cranberry fruits, although smaller, showed significantly higher levels of PACs (+61%), anthocyanins (+58%), total polyphenolic compounds (+48%), and antioxidant capacity than North American ones. This may be due to the inhibition of fruit growth by elevated temperatures, resulting in a better synthesis of antioxidants and bioactive compounds. This study may promote the cultivation of cranberries in different climatic regions, as a complementary strategy to international imports, and improve the production of new food applications with a high content of health-promoting molecules. Additionally, the production of antioxidants in plants under challenging conditions may potentially stimulate further studies to address climate change and investigate crop diversification.

1. Introduction

Cranberry (Vaccinium macrocarpon Aiton) is a perennial, evergreen shrub that belongs to the Ericaceae family. It is native to North America and is also known as “big cranberry” to distinguish it from “small cranberry” (Vaccinium oxycoccos L.) that is cultivated in Europe [1]. North America and Canada produce 96.4% of cranberries in the world, especially in Massachusetts, New Jersey, Wisconsin, Oregon, Washington, Quebec, and British Columbia. Europe produces only 0.2% of total cranberries in Germany, Netherlands (Terschelling Island), Belarus, Latvia, Lithuania, Poland, and Russia [2,3]. The relatively small area of production is linked to the environmental requirements that cranberries need. This species grows in temperate climates, in acid soils, with a pH from 4.0 to 5.0, with a sandy loamy texture, and a high content of organic matter, from 3 to 15% [2,4]. Also, flowering occurs only after 1.000–2.500 h of vernalization, which is a significantly longer period compared to other fruit trees of temperate climate [4,5]. Historically, cranberries are cultivated in bogs in North America, formed after the last Ice Age. These bogs are characterised by different layers: a hardpan that allows the flooding of the field, which is necessary to harvest fruits, followed by layers rich in sand and organic matter [2].

Cranberry plants have horizontal stolons, up to 2 m long, with shoots growing upright and producing from two to seven pale pink flowers. However, only one to three berries are produced for fruiting stems every year due to insufficient pollination. Flowering starts at the lower part of the shoots and proceeds upwards from mid-June to July. Fruit ripening occurs from August to October according to the cultivar. Cranberry shape can vary from round to oval, and colour can be dark red or purple [1,2,3].

The interest in cranberries is linked to their uses as an ingredient in food and as a raw material to produce extracts for dietary supplements. Cranberries contain a variety of bioactive chemical compounds, including phenolic acids (e.g., caffeic acid and hydroxycinnamic acids), flavonoids (such as quercetin and myricetin), and anthocyanins [6,7]. Additionally, cranberries contain resveratrol, a stilbene compound [8]. The most represented tannins are proanthocyanidins (PACs), which are oligomers or polymers of flavan-3-ol units (e.g., catechin and epicatechin), linked by C4–C8 or C4–C6 (B-type) bonds and by an additional C2-O-C7 for PACs with A-type interflavan bonds. PACs are also found in other food sources like berries, grape seeds, tea leaves, and chocolate [9]. PACs degree of polymerisation can vary, leading to a range of molecular sizes [10]. Cranberries also contain glucose (3.4–4.7 g 100 g⁻¹) and fructose (0.4–0.7 g 100 g⁻¹), vitamin C (10.0–20.7 mg 100 g⁻¹), vitamins B and E, and a large amount of potassium and nitrogen (72.51 and 42.1 mg 100 g⁻¹, respectively) [1,3,11,12,13].

Cranberry juice or extract in tablets are already used to promote urinary tract well-being, alleviate inflammation status and urinary infection occurrences, support digestive health, and reduce oxidative damage [14]. Americans are the first producers and consumers with a 1 kg year⁻¹ per capita of cranberries, almost entirely in juice form. 95% of cranberries in the United States are processed into juice or as dietary supplements [15]. According to FAOSTAT and USDA analysis, the United States produced from 20 to 25 t ha⁻¹ of cranberries, referring to the last six years (2017–2022) with a value of 0.6$ kg⁻¹. These results are promising to promote the production and consumption of cranberries in other areas of the world. Among the various cultivars of Vaccinium macrocarpon, the one presented in this study is the ‘Pilgrim’ (Prolific × McFarlin). ‘Pilgrim’ is derived from a breeding project developed at the University of Wisconsin in 1929, aiming to develop more productive fruits with larger diameter, better colour, and higher concentrations of bioactive metabolites [4,11]. Abeywickrama, et al. [16] and Narwojsz, et al. [13] studied the physical characteristics, antioxidant activity, and phenolic profile of cranberry cultivars, including ‘Pilgrim’ harvested in Poland and Canada. Results show that physical characteristics, contents of bioactive compounds, and their health-promoting activities vary according to climate conditions, agronomic methods, and storage conditions. So, it is important to study the results of the cultivation of cranberries in new areas and with different agronomic approaches and environmental stimuli. Currently, no data are available for cranberry plants grown under soilless conditions in a Mediterranean climate.

This exploratory study analysed the phytochemical composition of Vaccinium macrocarpon cv ‘Pilgrim’ cultivated in North-West Italy in a soilless cultivation, comparing its characteristics with those of the American-cultivated ones. The comparison between Canadian commercially obtained and Italian locally grown cranberries was aimed as a qualitative approach to provide a qualitative framework to evaluate the phytochemical and quality results, rather than as a direct experimental assessment because of the different cultivation system. The aim was to preliminarily evaluate whether the environmental (latitude, temperature, sunlight exposure) and agronomic factors affect the content of the most valuable secondary metabolites for use as dietary supplements. However, this constitutes only an initial preliminary investigation, and future studies are necessary to guarantee the consistency, stability, and reliability of the outcomes and confirm them after a minimum of three crop cycles (or three harvests). The main aim was to define and identify key parameters for future studies and experiments rather than to establish definitive biochemical or agronomic conclusions. In any case, this preliminary study may facilitate the expansion of crop diversification in regions where cranberry cultivation is not traditionally practised. It fosters direct interaction between farmers, who are interested in new, sustainable and economically interesting productions, and end-consumers, who are interested in health-promoting food applications.

2. Materials and Methods

2.1. Plant Materials

Two-year-old cranberry plants, cv ‘Pilgrim’, were cultivated in Bra (Cuneo, Italy) during the season 2022 in a soilless cultivation (Italian cranberry—IC). In April, 84 plants were transplanted in 50 × 20 × 20 cm pots filled with peat and perlite (80:20), placing two plants in each pot. The pots were placed on a ground drainage channel at 1.5 m apart between rows under a plastic tunnel with an extension of 6 × 10 m. The plants were fertigated three times/day with a nutritive solution containing 0.16, 0.91, and 0.41 g L⁻¹ of NPK, respectively, and 0.20 g L⁻¹ of S. The substrate was kept at around 30% of volumetric water content and 150 µs cm⁻¹ electrical conductivity (pH = 4.0–5.0). In the first week of November, fruits were harvested from all the plants and pooled, then they were stored at 4 °C and 95% R.H. for a few days until the extraction processes (Autumn 2023). A comparative sample of fresh cranberry (cv ‘Pilgrim’) (North American cranberry—AC) harvested at the end of October 2023 from bogs in Columbia Britannica (Canada) was provided by Abel Nutraceuticals (Turin, Italy) and stored at 4 °C and 95% R.H. for a few days until extraction (Autumn 2023). Canadian cranberries were only used here as a reference material to provide an indicative comparison of selected biochemical properties and physicochemical parameters. The comparison intended to provide an exploratory context to evaluate the quality traits and phytochemical properties of V. macrocarpon grown under soilless conditions in Piemonte Region (Italy). The fruits were manually collected from the cranberry plants in accordance with specific qualitative criteria (such as total soluble solids, firmness, and colour), based on previous literature and the expertise of researchers and berry growers. Indeed, in their unripe state, cranberry fruits are characterised by a range in colour from green to pale red and firm texture, while ripe cranberries show a consistent deep red hue and are softer than unripe ones. Analyses started in January 2023.

Analyses were performed by the Department of Agricultural, Forestry and Food Sciences—University of Turin (DISAFA), both on cranberries cultivated in Bra (Italian cranberry, IC) and North America (Canadian cranberry, CC).

2.2. Qualitative Analysis

Average fruit weight (g) and size (mm) were evaluated on 30 fruits with a Mettler PM460 Delta Range Electronic Balance (Mettler, Greifensee, Switzerland) and with a digital calliper (Traceable Digital Calliper-6″, VWR International, Milano, Italy). Total soluble solids (TSS, °Brix) were determined on different biological and analytical repetitions (N = 3) for Italian and American cranberries with a digital refractometer DBR35 (Tsingtao Unicom-Optics Instruments, Laixi, China). pH (TitroMatic 2S, Crison, SAVATEC, Torino, Italy) and titratable acidity (TA, meq L⁻¹) were defined on different biological repetitions, both for Italian and American berries. 10 mL of cranberry pulp juice was mixed with 90 mL of distilled water and titrated with a solution of NaOH (0.2 mol L⁻¹). Dry matter analyses were performed with a moisture analyser (ALGS60, Dini Argeo, Fiorano Modenese, Italy—0.001 g resolution) and equipped with a halogen heating source. The system was operated at 110 °C for the necessary time to reach a stable weight measurement for at least 5 min.

2.3. Reagents

The following products were purchased from VWR (Milan, Italy): acetonitrile Chromanorm (ACN), ammonium acetate (AA), 1-propanol Reactapur (1-PrOH), hydrochloric (HA), sulphuric (SA), and orthophosphoric (OA) acid Chromanorm. Merck (Darmstadt, Germany) provided ethanol Emparta (EtOH), formic acid Emparta (FA), and methanol Emparta (MeOH). 4-dimethylaminocinnamaldehyde (DMAC) was purchased from TCI Europe (Zwijndrecht, Belgium). Milli-Q system to produce ultrapure water, Millipore Corporation (Billerica, MA, USA). Proanthocyanidin A2 dimer (PAC A2) >98% was purchased by Extrasynthese (Lyon, France).

2.4. Extraction of Bioactive Compounds

Different methods were used to extract phytochemicals and nutritional compounds according to Donno, et al. [17]. The detailed description of the extraction methods is reported in the Supplementary Materials (Table S1). Three biological replications with three analytical repetitions (both for IC and CC) were prepared for the following analysis. Each biological replicate was derived from a pool of 30 fruits from multiple independently grown plants (i.e., several pots) to maintain biological independence between replicates.

2.5. Total Anthocyanin Content

Total Anthocyanin Content (TAC) was evaluated by pH-differential method according to previous studies [17,18] and expressed as cyanidin-3-O-glucoside per 100 g of fresh weight (mgC3G 100 g⁻¹ FW).

2.6. Total Polyphenolic Content

Total Polyphenolic Compounds (TPC) were defined according to the Folin–Ciocalteu method [19] and expressed as mg of gallic acid equivalent (GAE) per 100 g of fresh weight (FW).

2.7. Ferric Reducing Antioxidant Power

The Ferric Reducing Antioxidant Power test [20] was used to evaluate antioxidant capacity (AOC). Results were expressed as millimoles of ferrous iron (Fe²⁺) equivalents per kg (solid food) of FW. These analyses were carried out on three biological replications with three analytic repetitions, both for Italian and American cranberries.

2.8. Total PACs Analysis

The analyses of total soluble PACs (TPAC) were performed with the Modified DMAC/A2 Method as published in the American Herbal Pharmacopoeia [21] relative to cranberry fruit. The assay is based on the reaction of PACs with 4-dimethylaminocinnamaldehyde, an aromatic aldehyde, yielding a green chromophore whose intensity can be measured at 640 nm. For the analysis, 20 g of cranberry fruits were ground in a pestle and mortar. 100 mg of the resulting pulp were weighed and extracted in 10 mL of 0.4 N H₂SO₄ methanolic solution. The sample was vortexed for 30 s and extracted in a sonic bath for 30 min. After 5 min of centrifugation at 5000 rpm, the liquid fraction was collected and diluted 20-fold in the 0.4 N H₂SO₄ methanolic solution. 200 μL of the diluted sample solution were added to 1 mL of a 1 mg mL⁻¹ DMAC 0.4 N H₂SO₄ methanolic solution. The 640 nm absorbance was measured after 4 min with a Cary-60 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The quantitation was performed by external standard calibration using PAC A2 (5, 10, 15, 20, 25 and 30 ppm).

2.9. Phytochemicals and Nutritional Compounds

The detailed description of the HPLC methods (Table S2) is reported in the Supplementary Materials. HPLC analyses were carried out on an Agilent 1200 HPLC—UV-Vis Diode Array Detector (Agilent Technologies, Santa Clara, CA, USA). Cinnamic acids, flavonols, benzoic acids, catechins, vitamin C, organic acids, and sugars were analysed to define the nutritional properties of cranberries [17]. In particular, polyphenolic molecules are selected as health-promoting markers because they play a role in protecting human bodies from the negative actions of free radicals, and they have been correlated to lower rates of chronic issues such as cancer and heart disease. Including a large variety of polyphenol-rich foods, such as cranberries, in the diet may contribute to overall well-being and health [22]. Six HPLC methods (A–F) were performed using either Kinetex C18 (methods A–E) or Sphereclone NH₂ (method F) columns. Several mobile phases were utilized in gradient/isocratic elution programs, and different wavelengths were optimized for the detection of each specific analyte group. Methods A–E targeted phenolic acids (λ = 330 and 280 nm), flavonols and catechins (λ = 330 and 280 nm, respectively), monoterpenes (λ = 250 nm), organic acids (λ = 214 nm), and vitamin C (λ = 261 nm for ascorbic acid and 348 nm for dehydroascorbic acid), while Method F was used for sugar analysis (λ = 267 and 286 nm). Elution programs ranged between gradient and isocratic modes depending on compound class, with different flow rates varying from 0.5 to 1.5 mL·min⁻¹. All the details on the calibration and validation parameters of the compounds considered are reported in the Supplementary Materials (Table S3).

2.10. Anthocyanins Profiling

An aliquot of about 2 g of pulp was weighed and extracted in a sonic bath in 10 mL of 2% HCl acidified methanol for 30 min at room temperature. The solution was centrifuged to precipitate the residues of plant material. The semi-quantitative analysis of anthocyanins was led with an HPLC-PDA apparatus, an LC-40 HPLC system from Shimadzu (Milan, Italy) consisting of an HPG binary pump and an SPD40 PDA spectrophotometric detector. Shimadzu GIST-HP C18 (3 × 150 mm, 3 μm) was used as the analytical separation column, and the mobile phases were prepared as listed: A: 99.5:0.5 (v/v) water:concentrated phosphoric acid; B: 50:48.5:1:0.5 (v/v) water:acetonitrile:glacial acetic acid:concentrated phosphoric acid. The gradient was set as follows: t = 0 min, B% = 10; t = 1 min, B% = 10; t = 38 min, B% = 40; t = 42 min, B% = 75. The column reconditioning was performed for the next 6 min; column flow was kept at 0.25 mL min⁻¹, and the injection volume was 5 μL.

2.11. A-Type and B-Type PACs Analysis

The relative quantity of soluble A- and B-type PACs in their dimeric and trimeric forms was determined by an HPLC-MRM approach; the relative ratio of A- and B-type PACs peak area was used to calculate their concentration with respect to their total content obtained by DMAC assay. The pulp of cranberry samples (1 g) was extracted in 10 mL of 0.1% v/v formic acid methanolic solution. The obtained solution was then sonicated for 30 min at RT and centrifuged to precipitate the insoluble plant material. The analysis was performed with an LC-MS8045 MS coupled to an LC-40 HPLC system Shimadzu (Milan, Italy). Chromatographic separation was performed on a Raptor Biphenyl (2.1 × 100 mm, 2.7 μm) Restek (Milan, Italy) column using A: 0.1% v/v formic acid in water and B: 0.1% v/v formic acid in methanol. Gradient was set as follows: t = 0 min, B% = 5; t = 24 min, B% = 100; t = 27 min, B% = 100. The column reconditioning was performed for the next 8 min. The mass spectrometer was operated in negative ion mode with the following ion transitions for PACs molecule identification: A-type PAC dimer: Q1 = 575.2 m/z; Q2 285 m/z; CE: 30.0 v, B-type PAC dimer: Q1 = 577.2 m/z; Q2 425 m/z; CE: 15 v; A-type PAC trimer: Q1 = 863.3 m/z; Q2 711.5 m/z; CE: 15.0 v, B-type PAC trimer: Q1 = 856.2 m/z; Q2 289 m/z; CE: 36.0 v. To get rid of the fragmentation efficiency bias typical of triple quadrupole-based instrumentation, PACs relative quantitation was performed using the SIM signal area for each molecular ion of Q1.

2.12. Statistical Analysis

The results were expressed as mean values and their standard deviation (±SD). Analysis of t-Student (SPSS 22.0 Software) was used to define significant differences (p < 0.05, N = 3) between American and Italian cranberries.

3. Results and Discussion

3.1. Fruit Quality Analysis

Cranberries cultivated in Northern Italy (IC) resulted in being significantly different (p < 0.05) from Canadian (CC) ones in terms of dimensions and weight (Figure 1). IC was smaller and lighter than CC, as shown in

Table 1. Physical and qualitative features of Italian and Canadian cranberries.

Feature	Italian Cranberry (IC)	Canadian Cranberry (CC)
Weight (g)	0.763 ± 0.361 a	1.771 ± 0.532 b
Dry matter (g 100 g−1)	12.5 ± 0.10 a	11.7± 0.12 b
Width (mm)	10.52 ± 1.91 a	14.47 ± 1.79 b
Length (mm)	11.59 ± 2.53 a	16.25 ± 2.36 b
TSS (°Brix)	10.3 ± 0.1 a	8.0 ± 0.7 b
pH (pH units)	2.54 ± 0.04 a	2.51 ± 0.01 a
TA (meq L−1)	159 ± 1.4 a	170 ± 5.7 a

Data are expressed as mean value ± standard deviation (N = 3). Values in the same line with different letters indicate significant differences (p < 0.05).

. IC showed a circular shape if compared to CC, which presented an oval shape.

TSS were significantly different (p < 0.05) between the two samples, with higher content and less variability of IC than CC, in accordance with Oszmiański, et al. [12] (10.00 ± 0.02 °Brix). pH and titratable acidity were not significantly different (p < 0.05) between IC and CC (Table 1), and the data were in accordance with other studies [1]. IC showed an average dimension lower than data reported in the literature for cv ‘Pilgrim’ cultivated in Poland (width 15.60 ± 1.84, length 19.04 ± 2.41) [13].

The differences in fruit size observed in IC compared with the Canadian reference (CC) may be due to several climatic, environmental, and physiological factors. In particular, reduced variation in diurnal temperature and lower accumulated chilling hours (about 600–900 in Bra and about 1100–1300 in Victoria, estimated by “0.0–7.2 °C Model”) may have influenced fruit set and flowering induction and, consequentially, maturation and development of fruits grown in North-Western Italy [23,24]. Moreover, the higher temperatures typical of summer in the Italian experimental site may have contributed to plant dormancy, moderated photosynthetic activity, and/or partial growth inhibition in this period. These factors may have limited the availability of carbohydrates for fruit enlargement.

Canada is one of the biggest producers of cranberries and has similar climatic conditions to the Piemonte Region, classified as Cfa according to the Köppen–Geiger climate classification [25], but some factors may be important to consider in order to understand the observed differences in fruit size and quality parameters. In 2022, Bra in Piemonte Region (Italy) recorded higher precipitation (about 1100 mm) and mean annual temperature (13.0 °C) than Victoria (about 970 mm, 10.2 °C) in British Columbia (Canada). Moreover, seasonal contrasts were higher in Bra, with temperature values of 30.1 °C in July and −1.5 °C in January, versus Victoria (23.0 °C and −0.5 °C, respectively). Peak sunshine was observed in July for both locations (about 11.9 h/day in Bra and about 11.2 h/day in Victoria), while minimum values occurred in November (Bra) and January (Victoria). Snowfall remained minimal in both cities, though slightly more frequent in Victoria (about 30–35 cm/year).

Climate data showed that the climate is similar in the considered areas except for hotter and colder temperatures in summer/winter months in Columbia Britannica that guarantee a greater accumulation of chill hours that cranberries need (Figure 2).

Table 2. Comparative summary of climatic conditions in Piemonte (Italy) and Columbia Britannica (Canada) in the period 1994–2024.

Parameter	Bra, Piemonte, Italy	Victoria, Columbia Britannica, Canada
Mean Annual Temperature	About 12.4 °C	About 9.9 °C
Mean Annual Precipitation	About 1052 mm	About 961 mm
Warmest Month	July (about 29.1 °C)	July (about 22.1 °C)
Coldest Month	January (about −0.5 °C)	January (about 3.4 °C)
Peak Sunshine	July (about 11.6 h/day)	July (about 11 h/day)
Lowest Sunshine	November (about 3.5 h/day)	January (about 2.5 h/day)
Snowfall	Occasional	Minimal (about 33 cm/year)

shows a comparison of climate data between the two locations (period 1994–2024), showing that 2022 values were higher than the average of the last 30 years. The Italian climate of 2022 was abnormal if compared to the average from 1991 to 2020, with the hottest temperature (+1.12 °C) and lower precipitation (−21%) (ISPRA). Hot temperature and less water during the growing season of cranberries may reduce their dimensions. Further data, which can support the previous statement, is found in the dry matter, significantly higher in IC (12.5%) than in CC (11.7%). Indeed, the difference in dry matter between IC and CC samples may be correlated to environmental factors (e.g., lower relative humidity, suboptimal irrigation efficiency, higher temperatures, etc.), which may have limited fruit growth and increased transpiration rates. For this reason, the observed differences in the phytochemical may result from differences in environmental stress and fruit water content as well as intrinsic metabolic differences. Previous studies reported that transient water limitations and/or moderately elevated temperatures may stimulate the phenolic and anthocyanin biosynthesis in Vaccinium species as an effect of an adaptive response [26,27]. Even if no physiological stress parameters were directly measured in this research, the high temperatures recorded in the experimental area in the summer period may have induced a similar metabolic response, contributing to the higher phenolic and anthocyanin amounts observed in the IC cranberries.

In addition, the soilless cultivation system, useful to ensure phytosanitary conditions and control availability of nutrients, may have limited water relations and root expansion, reducing the berry expansion. The optimization of environmental conditions and irrigation control may be a key purpose in future trials to improve fruit uniformity and size. The adaptation of this species to local conditions may have also played a role in fruit growing, as V. macrocarpon may require several seasons to reach a better fruit size and productivity under Mediterranean climatic conditions.

A smaller size of cranberries may represent a limitation for commercial purposes (i.e., fresh consumption or material for food supplements), but the high levels of nutraceutical compounds and high antioxidant capacity detected in the IC samples highlighted their significant health-promoting potential. Future studies will be carried out to optimize agronomic and environmental parameters to improve fruit size without reducing healthy quality and properties.

3.2. Total Phenolic, Anthocyanin and Proanthocyanidin Content and Antioxidant Activity

Phenolics are a group of natural molecules found in many plant materials and foods that have been shown to have several health-positive effects. These bioactive compounds present many antibacterial, antioxidant, and anticancer properties [28]. However, this study referred to the presence of bioactive molecules with recognized antioxidant capacity and to health-promoting evidence already reported in the scientific literature. The observed phytochemical composition was discussed in terms of its nutraceutical and antioxidant characteristics, without attributing specific health or physiological effects.

Analyses on total phenolics, anthocyanin content, proanthocyanidins, and antioxidant capacity showed that IC has a higher concentration with significant statistical differences (p < 0.05) compared to CC (

Table 3. Total polyphenolic, anthocyanin and proanthocyanidin contents and antioxidant capacity of Italian and Canadian cranberries.

		Italian Cranberry (IC)	Canadian Cranberry (CC)
TPC	Total polyphenolic content (mgGAE 100 g−1 FW)	299.13 ± 22.02 a	201.73 ± 12.09 b
TAC	Total anthocyanin content (mgC3G 100 g−1 FW)	112.05 ± 25.50 a	70.72 ± 25.72 b
TPAC	Total PACs content (mg PAC A2 100 g−1 FW)	1156.05 ± 79.87 a	717.85 ± 30.89 b
AOC	Antioxidant capacity (mmol Fe2+ kg−1 FW)	35.51 ± 1.12 a	32.86 ± 1.39 b

Data are expressed as mean value ± standard deviation (N = 3). Values in the same line with different letters indicate significant differences (p < 0.05). FW: fresh weight of fruit material; GAE: gallic acid equivalent; C3G: cyanidin-3-O-glucoside.

). TPC and TAC in IC were 48% and 58% higher than in CC, respectively; a similar result is reflected in the TPAC value in IC, which is 61% higher than in CC. If compared to the literature, TPAC values obtained in this study are slightly higher [29] since their investigation ranged in results between 700 and 1400 mg 100 g⁻¹ of fresh weight (FW). The present results, furthermore, showed a higher level of polyphenolic and anthocyanin compounds than the results reported in the literature. Borowska, et al. [11] and Narwojsz, et al. [13] studied phenolic compounds of five cultivars of cranberries cultivated in Poland. Cultivar ‘Pilgrim’ only presented 163.4 mgGAE 100 g⁻¹ FW of TPC and 60.0 mgC3G 100 g⁻¹ FW of TAC. Vanden Heuvel and Autio [30] also underlined that high temperatures during the bloom and fruit-set stages of cranberries increase anthocyanin content. Hot temperatures during the preharvest of cranberries increase the storage of total phenolic compounds. Therefore, the high temperatures of summer 2022 may have caused a greater accumulation of these compounds in IC.

These trends were also consistent with studies from northern Europe. For example, in Latvia, extensive research on introduced and locally cultivars reported high amounts of health-promoting agents, with total anthocyanin content commonly ranging between 8 and 10 mg/g DW and a substantial proanthocyanidin accumulation, highlighting a good health-positive potential under Baltic pedoclimatic conditions [31]. Some studies carried out in Lithuania showed that the anthocyanin qualitative was represented by peonidin-3-galactoside/-arabinoside and cyanidin-3-galactoside/-arabinoside [32].

The evaluation of the antioxidant capacity of a plant material or substance is a very complex process involving several units of measurement and methods, causing a hard comparison of results with previous studies [12]. In this research, antioxidant capacity also showed significant statistical differences (p < 0.05) between IC (35.51 ± 1.12 mmol Fe²⁺ kg⁻¹ FW) and CC (32.86 ± 1.39 mmol Fe²⁺ kg⁻¹ FW), with FRAP values similar to previous research [3,12]. Some studies highlighted that FRAP values may be mainly influenced by maturity stage, further confirming and supporting the key role of phenology and environment in defining cranberry quality in European contexts [33], in particular in the northern European climate.

In this study, the FRAP assay was selected as a screening method for antioxidant capacity evaluation due to its fast results, low cost, and simplicity. This test may underestimate the real biological antioxidant capacity, but it is effective in evaluating the potential of a molecule or a substance as an inhibitor of specific target-molecule oxidation [34]. It is necessary to highlight that the antioxidant action of bioactive compounds is not limited to scavenging free radicals, but it also includes other mechanisms (e.g., redox cell signalling and modulation of gene expression). The FRAP assay provides an index value for ordering and comparing several materials in terms of their antioxidant properties. In any case, it should be recommended to use different methods to ensure reliable results because of the large range of antioxidants present in biological samples [35].

The results confirmed the positive potential of cranberries cultivated under soilless conditions as health-promoting food or material for food supplements. The fruits showed high levels of total phenolics, which are very important to significantly contribute to the V. macrocarpon antioxidant capacity in accordance with previously reported studies for cranberries cultivated in North America [4,36], showing the consistency of their health-promoting value in different growing systems. This research also reinforced the role of cranberries as an excellent dietary source of phenolics and other health-promoting compounds, supporting their potential use in food supplements, functional food applications, and healthy formulations.

3.3. Bioactive Compounds and Nutritional Substances

Bioactive compounds were considered to define IC nutraceutical properties. Different phenols and vitamin C were studied, according to their antioxidant capacity and benefits on human health [1,9]. Results have been reported in

Table 4. Content of bioactive compounds and nutritional substances in Italian (IC) and Canadian (CC) cranberries.

Bioactive Class	Compound	Italian Cranberry (IC)	Canadian Cranberry (CC)
Cinnamic acids	Caffeic acid	<0.10	<0.10
	Chlorogenic acid	0.99 ± 0.39	<0.31
	Coumaric acid	0.91 ± 0.11	<0.97
	Ferulic acid	3.75 ± 1.31 a	6.95 ± 7.72 a
Flavonols	Hyperoside	<0.34	<0.34
	Isoquercitrin	<0.03	<0.03
	Quercetin	<0.41	<0.41
	Quercitrin	<0.55	<0.55
	Rutin	<0.29	<0.29
Benzoic acids	Ellagic acid	0.49 ± 0.44 a	0.35 ± 0.12 b
	Gallic acid	<0.15	<0.04
Catechins	Catechin	2.13 ± 1.62 a	5.12 ± 0.24 b
	Epicatechin	13.82 ± 3.50 a	14.74 ± 1.24 a
Vitamin C	Ascorbic acid	5.27 ± 0.15 a	5.54 ± 0.16 b
	Dehydroascorbic acid	10.92 ± 4.89 a	19.04 ± 3.27 b
Organic acids	Citric acid	<1.88	<1.88
	Malic acid	50.50 ± 6.65 a	64.01 ± 23.80 a
	Oxalic acid	47.89 ± 4.70 a	43.70 ± 5.36 a
	Quinic acid	<2.61	<2.61
	Succinic acid	<0.71	<0.71
	Tartaric acid	49.43 ± 5.85 a	30.73 ± 7.01 b
Anthocyanins (relative to TAC)	Cyanidin-3-O-Gal	32.01± 1.98 a	20.14 ± 1.29 b
	Cyanidin-3-O-Glu	1.91± 0.15 a	0.52 ± 0.09 b
	Cyanidin-3-O-A	17.73 ± 0.07 a	12.45 ± 0.36 b
	Peonidin-3-O-Gal	40.1 ± 1.13 a	26.19 ± 0.89 b
	Peonidin-3-O-Glu	4.86 ± 0.29 a	2.11 ± 0.1 b
	Peonidin-3-O-A	15.44 ± 0.78 a	9.31 ± 0.76 b
Proanthocyanidins (relative to TPAC)	A-type dimer	283.2 ± 7.18 a	250.4 ± 0.15 a
	A-type trimer	352 ± 3.5 a	180.5 ± 0.05 b
	B-type dimer	487.9 ± 3.39 a	269.6 ± 16.6 b
	B-type trimer	32.9 ± 0.28 a	17.4 ± 0.1 b
Sugars	Fructose	0.31 ± 0.03 a	0.19 ± 0.03 b
	Glucose	3.24 ± 0.85 a	7.00 ± 0.10 b
	Sucrose	1.95 ± 0.54 a	0.77 ± 0.09 b

Data are expressed as mean value ± standard deviation (N = 3) and as mg 100 g⁻¹ fresh weight; anthocyanins and proanthocyanidins concentrations are expressed further in terms of area % relative to the total (in brackets), and sugars are expressed as g 100 g⁻¹ fresh weight. Values in the same line with different letters indicate significant differences (p < 0.05).

. In particular, the evaluation of the phenolic content is very important for predicting the health-promoting effects of preparations based on cranberries and assessing the fruit quality [31].

Both IC and CC resulted in being rich in catechins, in particular, epicatechin (about 13–15 mg 100 g⁻¹ FW). These compounds have an important role in human health according to their antioxidant and anti-inflammatory activity, and prevention of neurodegenerative disease, cancer, and cardiovascular disease [37]. Ferulic acid has been the most present cinnamic acid in both IC and CC. This molecule has superior antioxidant capacities and has low toxicity. It has several useful properties for human health, such as antioxidant, anticancer, antidiabetic, and anti-inflammatory properties [38]. Chlorogenic and coumaric acids were detected only in IC at low concentrations (0.91–0.99 mg 100 g⁻¹ FW), whereas in CC their amounts were below the limit of quantification (LOQ). As well as chlorogenic and coumaric acids, other bioactive molecules, reported in Table 4, were lower than the limit of detection (LOD) or LOQ of the used analytical methods. Similarly, caffeic acid was not quantified in both IC and CC samples (<0.10 mg 100 g⁻¹ FW). The most abundant derivative of benzoic acid was ellagic acid; it has a significantly higher concentration in IC (p < 0.05), whereas gallic acid has not been detected by HPLC-DAD. Phenolic acids also have antioxidant activity that prevents damage to human cells [39]. Flavonols were identified in trace amounts both in IC and CC due to their low concentration. Other studies reported higher values of flavonols (about 20–40 mg 100 g⁻¹ FW), but it may depend on genetic and environmental factors [9]. Moreover, the levels of flavonols may also be strongly influenced by different ripening periods, highlighting the importance of harvest time as reported by Šedbarė, et al. [31] in several cultivars such as ‘Red Star’, ‘Howes’, and ‘Pilgrim’. Several studies from Poland, Latvia, and Lithuania, confirmed the present results, highlighting that European (in particular, northern) cultivation can yield cranberries with phenolic fingerprinting and antioxidant capacity comparable to values reported in North America, while size traits and some specific compounds depend on genotype, ripening dynamics, and local climate [40].

Vitamin C was analysed as ascorbic acid and its oxidised form (dehydroascorbic acid). Both are biologically active [41]. Dehydroascorbic acid resulted in higher than ascorbic acid levels both in IC and CC, as already reported in other studies [1]. Another study reported that cv ‘Pilgrim’ is characterised by a low content of ascorbic acid compared to other cultivars, 16.0 mg 100 g⁻¹ FW vs. 28.5 mg 100 g⁻¹ FW of ‘Stevens’ [1]. Viskelis, et al. [36] also underlined that the amount of ascorbic acid varies according to the ripening stage, cultivation techniques, and exposure of cranberries to high temperatures. As reported in previous studies, vitamin C plays an important role in human health, developing connective tissues, activating vitamin B and folic acid, reducing oxidative damage, and reducing cholesterol levels. Fruits like citrus, strawberries, blueberries, peppers, and tomatoes are the main sources of vitamin C in the human diet [41]; according to the present study, cranberries could also be an important source of this essential compound.

Organic acids were also quantified since they play an important role in human health. For example, they may provide metabolism regulation and energy to protect the myocardial and immune systems in humans [42]. Table 4 shows that IC was rich in malic acid (50.50 ± 6.65 mg 100 g⁻¹ FW), followed by tartaric (49.43 ± 5.85 mg 100 g⁻¹ FW) and oxalic acid (47.89 ± 4.70 mg 100 g⁻¹ FW). A similar trend was also observed for CC, but with a higher concentration of malic acid and a lower concentration of oxalic and tartaric acid. Otherwise, citric, quinic, and succinic acid signals were below LOD.

Regarding the concentration of sugars, both samples showed a higher concentration of glucose, followed by fructose and sucrose. The glucose concentration was significantly higher in CC with respect to IC, whereas the fructose and sucrose amounts were significantly higher in IC in comparison to CC (Table 4). The amounts of these molecules were similar in comparison to another study [12], even if some conditions, such as different stages of ripeness, cultivars, and storage conditions, may influence their concentrations [3].

The current research represents a preliminary exploration into the phytochemical composition of V. macrocarpon fruits cultivated in the Piemonte Region (North-West of Italy), assessing the identification of the main bioactive secondary metabolites and supporting the diversification of crop cultivation as a new ecological or economic opportunity in non-traditional areas for cranberries. Potted cranberries (Italy) and field-grown cranberries (Canada), two markedly different agronomic systems, were considered. For this reason, several uncontrolled variables, such as differing substrate compositions (peat/perlite vs. field soil), irrigation and fertigation practices, limitations in the root zone due to pot size, and microclimate influences (e.g., heat retention in pots) may be introduced. These factors may potentially influence the synthesis and, then, the accumulation of secondary metabolites, organic acids, and sugars in cranberries. Consequently, the differences observed in phytochemical levels may be attributed not only to climatic variations but also to the aforementioned factors. Therefore, the results highlighted potential trends but further studies under standardized and controlled experimental conditions are necessary to validate these initial findings.

The anthocyanin profile was evaluated by HPLC-PDA. The six main abundant and cranberry-characteristic species have been identified: Cyanidin 3-O-Galactoside, Cyanidin 3-O-Glucoside, Cyanidin 3-O-Arabinoside, Peonidin 3-O-Galactoside, Peonidin 3-O-Glucoside, and Cyanidin 3-O-Arabinoside, as reported in Figure 3.

To obtain their concentration value, relative area abundances were correlated to anthocyanin total content (TAC). The profile of anthocyanins of both IC and CC is consistent with the results published by the American Herbal Pharmacopoeia [21] on V. macrocarpon. Among anthocyanins, the most abundant (%) is Peonidin 3-O-Galactoside (P-3-gal), followed by Cyanidin 3-O-Galactoside; furthermore, in both samples, the relative abundance of those chemicals remains unvaried. Specifically, the area percentages for Cyanidin-3-O-Galactoside were 28.57 ± 1.87 in the IC and 28.48 ± 1.83 in the CC. For Cyanidin-3-O-Glucoside, the values were 1.7 ± 0.13 in the IC and 0.73 ± 0.13 in the CC. Cyanidin-3-O-Arabinoside exhibited values of 15.83 ± 0.06 in the IC and 17.7 ± 0.51 in the CC. Peonidin-3-O-Galactoside displayed values of 35.78 ± 1.01 in the IC and 26.19 ± 0.89 in the CC. For Peonidin-3-O-Glucoside, the values were 4.34 ± 0.26 in the IC and 2.99 ± 0.14 in the CC. Lastly, Peonidin-3-O-Arabinoside showed values of 13.78 ± 0.7 in the IC and 13.17 ± 1.07 in the CC. Literature values report a concentration of (P-3-O-gal) in cranberry fruits of around 180 mg 100 g⁻¹ DW [7]. The present analysis proposes levels of this anthocyanin of 40 mg 100 g⁻¹ FW for IC and 30 mg 100 g⁻¹ FW for CC, which, if transformed for the dry weight, result in 330 and 250 mg 100 g⁻¹, respectively. The relative quantity of PACs in IC and CC was determined on the most soluble species of PACs: dimers and trimers. Their relative concentrations, obtained by the area registered on the ionic chromatogram of HPLC-MS² analysis, were transformed in their concentration value by their correlation with the value of TPAC obtained by DMAC assay. Among the PACS studied, the B-type dimer registers the highest abundance in both samples of IC and CC. The area percentage for the A-type dimer was recorded as 24.5 ± 0.42 in the IC and 34.7 ± 0.64 in the CC. A-type trimer exhibited a value of 30.5 ± 1.06 in the IC and 25.5 ± 0.99 in the CC. For the B-type dimer, the values were 42.2 ± 1.27 in the IC and 37.4 ± 0.42 in the CC. Lastly, the B-type trimer showed values of 2.9 ± 0.21 in the IC and 2.5 ± 0.07 in the CC. A slight variation is present in the relative abundance of PAC-A dimer, whose concentration was enriched in CC. The sum of the relative concentration of type-A PACs in IC registered a value of 55% while in CC was slightly higher (60.2%).

4. Conclusions

This study showed that cranberries (‘Pilgrim’), cultivated soilless in Northern Italy, were rich in antioxidant and bioactive compounds and presented the potential to be valorised in food applications or dietary supplements. Despite Italian cranberries (IC) being smaller than Canadian ones (CC), they presented a higher amount of total polyphenolic, anthocyanin, and proanthocyanidin content and a higher antioxidant capacity. In particular, larger amounts of anthocyanins (cyanidin and peonidin) and proanthocyanidins (A-type and B-type) in IC rather than CC were probably related to the climatic conditions in which IC grew. Probably, high temperatures reduced fruit growth and led to an increased production of antioxidant compounds. These findings highlighted not only the regionality-related differences in the phytochemical profile of the cranberries cultivated in different areas (e.g., Italy vs. North America) but also the potential increased abundance of bioactive compounds in IC. This research represented only a preliminary investigation to assess the potential of V. macrocarpon cultivation under soilless conditions in Piemonte Region (Northwestern Italy) and further studies based on controlled cultivation trials are necessary to confirm these preliminary results. The results of this study are an encouraging factor in promoting the cultivation of cranberries in new areas with varying climates, which could stimulate the biosynthesis of bioactive metabolites, as a complementary strategy for the sourcing of this botanical ingredient. Finally, but not least importantly, the increased production of antioxidants in fruits grown in a cultivation area with climatic conditions characterized by low relative humidity and elevated temperatures, potentially stressing for cranberries, may serve as a starting point for research on crop diversification and tackling climate change.