Influence of Extraction Methods on Polyphenol Profile and Antiradical Activity of Hops

Abstract

Hop polyphenols contribute to beer stability and possess a wide range of biological activities; however, information on how extraction conditions influence their recovery and antioxidant potential remains limited. This study aimed to compare hot-water extraction, simulating wort boiling, with 80% aqueous acetone extraction as initial sample preparation steps for assessing the polyphenol content and antiradical activity of hops. Sample sets of four Czech hop cultivars from two contrasting harvest years were examined. Group-type polyphenols, total polyphenols, anthocyanogens, flavanoids and UHPLC–HRMS flavonoid profiles were evaluated. Antiradical activity was evaluated by the DPPH assay. Extraction solvent markedly affected both the composition and antiradical properties of hop extracts. Aqueous acetone provided substantially higher yields of anthocyanogens and prenylflavonoids and consistently higher antiradical activity, particularly in cultivars with elevated α-acid content. In contrast, hot-water extracts yielded slightly higher total polyphenols but significantly lower anthocyanogens and reduced antioxidant capacity. Correlation analyses showed that antiradical activity in water extracts was strongly driven by polyphenol levels, whereas in acetone extracts it was influenced by additional constituents, including bitter acids. Extraction solvent critically determines the polyphenolic profile and antioxidant behavior of hop extracts. Aqueous acetone proved more effective for isolating bioactive polyphenol fractions relevant to antiradical performance.

1. Introduction

Hops (Humulus lupulus L.) have been used for centuries—likely for millennia—primarily as a raw material for brewing, although numerous health-promoting effects of hop constituents are well documented. Hop cones contain a wide spectrum of natural compounds that differ markedly in chemical structure and physicochemical properties. In addition to resins (bitter acids) and essential oils, hop polyphenols represent a technologically and biologically important group. Various biological activities of hop polyphenols have been described, including antioxidant capacity, free-radical scavenging, and metal chelation [1,2,3]. These activities are related to several protective effects that are relevant to the prevention of chronic diseases, particularly cancer and cardiovascular disorders. Some polyphenolic compounds—especially prenylflavonoids and proanthocyanidins—also exhibit antimicrobial properties [4,5].

Historically, interest in hop polyphenols within brewing science has been linked mainly to their role in colloidal stability, haze formation during beer production, and the development of non-biological turbidity during storage [6,7]. Their radical-scavenging and metal-chelating activities also contribute to the sensory stability of beer [8,9,10,11], which remains a major challenge in brewing technology. Furthermore, several simple and polymeric polyphenols, as well as their oxidation products, affect beer bitterness and astringency [12,13].

Most hop polyphenols are localized in the bract and strig of the cone, whereas prenylflavonoids are secreted by lupulin glands together with resins and essential oils [14]. Hop polyphenols are commonly classified into flavonols, flavan-3-ols (catechins), phenolic acids (benzoic- and cinnamic-acid derivatives), and additional phenolic compounds such as prenylflavonoids and stilbenoids [1].

Hops contain approximately 2–7% total polyphenols. Approximately 20% of the total hop polyphenols consist of low-molecular-weight substances or monomer substances such as the phenolic carboxylic acids as well as the flavonoids and their glycosides, which form the majority of this faction. Hop flavonoids consist mainly of catechins and their polymers, proanthocyanidins, quercetin and kaempferol [14].

The major component of the prenylflavonoids in fresh hops is the chalcone xanthohumol (0.2–1.7% of hop cone weight). A smaller amount of desmethyl-xanthohumol is also found. During the brewing process, these prenylchalcones are largely converted into their isomeric flavanones, iso-xanthohumol and prenylnaringenins, respectively. Xanthohumol is considered a powerful antioxidant, while 8-prenylnaringenin has been identified as the most active plant phytoestrogen [3,14].

The hop polyphenols solubility in wort and beer is not the same for all compounds. The hydrophilic groups of substances such as hydroxybenzoic, hydroxycinnamic acids, flavan-3-ols and proanthocyanidins have a high solubility. The flavonoids have an intermediate solubility, while prenylflavonoids are more difficult to bring into solution [2,3,14].

A wide range of analytical methods has been developed for the isolation and characterization of polyphenols in plant matrices, including hops. Group-type analytical methods based on characteristic reactivity of polyphenol classes are incorporated in brewing analytics and include assays for total polyphenols, flavonoids, anthocyanogens, and tannoids. For a more detailed description of individual polyphenolic groups, high-performance liquid chromatography coupled with mass spectrometry [15,16,17] or coulometric detection [18,19,20] is routinely employed.

Sample preparation, particularly extraction, is a critical step that strongly influences analytical outcomes. Although the literature provides extensive information on solvents and extraction techniques for polyphenols from various botanical sources, surprisingly little attention has been given to how extraction conditions affect the polyphenol content and antiradical activity of hops [21]. Owing to the broad polarity range of hop constituents, solvent selection becomes especially important. Ethanol, a polar solvent that dissolves a wide array of hop components, is frequently used for polyphenol extraction; however, polyphenols remain a minor fraction of such extracts [21]. Other organic solvents—methanol, acetone, ethyl acetate, propanol, dimethylformamide, and their mixtures—are also commonly applied [22]. Numerous studies have shown that pure, anhydrous organic solvents are not optimal for polyphenol extraction, and therefore aqueous mixtures are often preferred [23]. Supercritical or liquid carbon dioxide extraction is widely used in both industrial and laboratory settings to obtain hop resins and essential oils [24], but due to their polarity, polyphenols are extracted only to a minimal extent [14].

For brewing research purposes, we have established a hot-water extraction procedure that simulates wort boiling [25]. This procedure was later codified in a slightly modified form in the European Brewery Convention (EBC) collection of analytical methods for determining the total polyphenol content of hops and hop pellets (EBC Method 7.14) [26]. Hot-water extract preparation is also common in other plant matrices such as tea [27]. More recently, we extracted polyphenols from hops using 80% aqueous acetone [28]. Acetone is a polar aprotic solvent that is often used to isolate a wide range of polar and less polar substances from food matrices. For instance, Taylor et al. [29] extracted hop polyphenols with 70% aqueous acetone.

Antiradical activity is an important criterion in the evaluation of plant polyphenol extracts. The methods used are primarily based either on the ability of the antioxidants present to scavenge free radicals (ABTS, DPPH, ORAC, PCL) or on their reducing potential (FRAP, voltammetry, HPLC-ECD) [30]. Methods based on radical deactivation can be divided into assays that primarily involve hydrogen atom transfer (HAT) and those based on single-electron transfer (SET). The results differ mainly in reaction kinetics and the occurrence of side processes; in real systems, HAT and SET take place simultaneously, and their relative contribution depends on the structure of the antioxidants and the pH of the environment [31]. Consequently, the results of determination and evaluation of the antiradical properties of plant matrices (including hops) as well as beverages (including beer) differ significantly depending on the method used [32,33,34]. The antiradical activity of hop extracts is commonly determined using established methods; the DPPH assay is very widely applied [16,35,36,37], although other methods, such as ORAC, have also been used [33].

Beer is generally considered to be a source of a number of substances that offer health benefits, including vitamins, minerals, dietary fiber, and, last but not least, polyphenols. Twenty to thirty percent of the polyphenols in beer come from hops, which contain substances that are absent in malt (prenylflavonoids) or present in negligible amounts (flavonol glycosides) [2,3,14,34]. Since many of these biologically active substances are lost during the brewing process [34], considerable attention is paid to hops as a primary source of health-beneficial substances [2,3,4].

Reliable knowledge of the polyphenol content and antiradical activity of hops is valuable when selecting raw materials and processing strategies aimed at improving the sensory stability of beer [8,9,10,11]. The aim of the present study was to compare hot-water extraction and aqueous acetone extraction of hops as initial sample preparation steps for determining polyphenol content and antioxidant activity.

2. Materials and Methods

2.1. Hop Samples

Samples of four Czech hop varieties—Saaz (SAZ), Sládek (SLD), Premiant (PRE), and Agnus (AGN)—from two harvests (designated A and B) that differed markedly in their α-acids content were analyzed. For each combination of variety and harvest, five samples of freshly harvested and industrially dried hops were examined. The samples were randomly selected from a set of approximately 300 harvest samples originating from all growing regions. Samples of freshly harvested and dried hops aliquots from the sample collection gathered by the laboratory of Chmelařství, Cooperative Žatec, were stored at a temperature of 0–2 °C until analysis.

The antiradical activity of polyphenolic fractions was further investigated using samples of “polyphenol pellets” P1 (SAZ) and P2 (AGN). These pellets were a by-product of the production of P60 hop pellets, i.e., material containing hop polyphenols except for prenylflavonoids and only bitter acid residues. Hop samples as well as pellet samples were kindly provided by Chmelařství, Cooperative Žatec (Žatec, Czech Republic). The antiradical activity of acids and iso-α-acids was tested using pure preparations of these compounds kindly supplied by the Hop Research Institute, Žatec (Žatec, Czech Republic).

2.2. Extraction of Polyphenolic Compounds

The study compared two contrasting methods of extracting polyphenolic compounds. Hot-water extraction is an intensive process, but it can cause changes in the extracted compounds due to oxidative and thermal stress. Cold aqueous acetone extraction is a gentle process for obtaining even less water-soluble compounds. Both tested methods were used in parallel to extract and analyze hop samples, which had first been finely milled.

2.2.1. Hot-Water Extraction

A 1.0 g portion of hop dry matter was suspended in 250 mL of water and boiled for 30 min under reflux. After cooling, the solid fraction was separated by centrifugation (15 min, 6000 rpm). This procedure has been used at our laboratory since 2002 [25] and is practically identical to the preparation of an extract for the determination of total polyphenols in hops according to the Analytica of the European Brewery Convention, method 7.14 [26].

2.2.2. Cold Acetone Extraction

A 1.0 g portion of sample dry matter was suspended in 250 mL of an acetone:water mixture (80:20, v/v) and extracted at a laboratory temperature of 20–22 °C for 20 min on a magnetic stirrer. The extraction duration was optimized in preliminary tests to achieve maximal polyphenol yield. Solids were separated by centrifugation (15 min, 6000 rpm).

2.3. Analysis of Polyphenols and Antiradical Activity

2.3.1. Bitter Acids

The contents of α-acids and β-acids in hop cones and extracts and iso-α-acids in extracts were determined using EBC methods 7.7 and 7.8 [26].

2.3.2. Group-Type Polyphenols

Three group-type analytical methods were applied:

• Total Polyphenols (TPs; EBC 7.14) [26]: Polyphenols react with ferric ions (ferric ammonium citrate) in an alkaline medium to form a red complex measured spectrophotometrically at 600 nm.

• Anthocyanogens (ANTs; MEBAK 2.16.2) [38]: In acidic conditions, leucoanthocyanidins form red oxonium salts detected at 550 nm.

• Flavanoids (FLAs; EBC 9.12) [26]: Flavan-3-ols (catechins, proanthocyanidins) react with p-dimethylcinnamaldehyde in acidic medium to form a green chromophore measured at 640 nm.

2.3.3. UHPLC–HRMS Analysis of Selected Polyphenols

Detailed profiling of key flavonoids—flavanols (catechin, epicatechin, and their glycosides) and flavonols (quercetin, kaempferol, and their glycosides)—was performed using a previously published QuEChERS extraction followed by UHPLC–Q-Exactive LC–MS/MS quantification [39]. Flavonoids were extracted from a mixture of hop extract (10 mL) and acetonitrile (10 mL), after addition of a mixture of salts (4 g MgSO₄ and 1 g NaCl) followed by centrifugation (4500 rpm for 7 min). One milliliter of the acetonitrile layer was first evaporated to dryness and subsequently dissolved in 1 ml of methanol:water (1:1, v/v). The LC-HR/MS assay was performed on an XSelect HSS T3 chromatography column (2.5 μm, 2.1 × 100 mm, Waters, Milford, MA, USA) with a C18 reverse phase, and the analytes were separated by gradient elution of water and acetonitrile, both acidified by the addition of 0.1% formic acid. The column temperature was 40 °C and the injection volume was 3 μL. The data were recorded by a full-scan scanning mass spectrometer over a mass range of 120 to 900 m/z. The content of individual flavonoids in extracts was quantified using an external calibration curve constructed in the range of 10 to 200 μL/L for all analytes of interest.

2.3.4. Antiradical Activity (DPPH Assay)

The antiradical activity was evaluated using the previously published DPPH method [25]. A spectrophotometric assay was applied, and the following parameters were derived using measurement program created for this analysis in a spectrophotometer.

• ARA1 (%): Antiradical activity after 1 min (rapidly reacting antioxidants).

• ARA2 (%): Antiradical activity after 10 min (total reducing species).

• ARP (%): Integrated DPPH reduction over the 0–10 min interval.

2.3.5. Statistical Analysis

Excel was used for statistical analysis and graphical representation of the experimental data. The agglomerative hierarchical clustering (AHC) of the flavonoid profile was carried out using XLSTAT 2021.4.1.

3. Results and Discussion

The study compared two contrasting methods of polyphenol extraction. The first method involved hot-water extraction, which simulated wort boiling or tea infusion conditions, and potentially induced changes in the extracted substances, polyphenols and others (bitter acids) as a result of oxidative and thermal stress. In contrast, the second method, cold aqueous acetone, is in principle a gentle and effective procedure with a high yield of various polyphenol groups, as reported in the literature [3,14]. Regarding hops, lipophilic substances, i.e., bitter acids, are extracted to a greater extent, which some authors attribute to strong antiradical activity [36].

Our initial focus when discussing the results was on polyphenolic substances, followed by an examination of the antiradical activity of the substances extracted by both methods in relation to their spectrum. When discussing the relationship between the antiradical activity of extracts and the bitter acid content of raw hops, we consider the known behavior of these substances in various solvents, their thermal conversion, and the results of a correlation analysis of data on polyphenols and ARA.

3.1. Polyphenols

A range of aroma and bitter varieties, as well as varieties with different genetic backgrounds, were represented by four Czech hop cultivars: Saaz, Sládek, Premiant and Agnus. Saaz belongs to the European Saaz-type group, Sládek and Premiant are classified within the European Fuggle–Saaz group, whereas Agnus represents cultivars of the American-type pedigree [40]. These cultivars differ markedly in both the quantity and composition of their polyphenolic constituents [11,28].

Two contrasting harvest years were included: Harvest A exhibited higher α-acid content and lower polyphenol levels, whereas Harvest B showed lower α-acids and elevated polyphenol levels (Table 1). Both α-acid and polyphenol contents, which are key quality parameters of hops, may vary widely among years in response to climatic conditions [16,28,41]; under unfavorable conditions, polyphenol content increases as part of the plant’s response to environmental stressors [42]. Group-type polyphenols were quantified using standardized brewing analytics.

Table 1.

Comparison of hot-water and acetone extracts of four hop varieties from two harvests.

(a) Polyphenols content
		Water			Acetone
	Alpha	TPs	ANTs	FLAs	TPs	ANTs	FLAs
Variety	% w	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g
	Harvest A
SAZ	4.58 ± 0.91	44.75 ± 3.80	30.24 ± 2.39	8.09 ± 0.70	38.06 ± 3.13	38.71 ± 2.47	8.85 ± 1.38
SLD	8.28 ± 0.42	29.05 ± 6.29	12.60 ± 2.63	3.85 ± 1.00	23.06 ± 4.17	29.14 ± 5.89	4.26 ± 1.16
PRE	10.12 ± 0.07	28.30 ± 3.61	16.32 ± 6.29	3.92 ± 0.43	24.06 ± 1.48	30.19 ± 3.16	4.34 ± 0.26
AGN	10.70 ± 0.72	30.80 ± 3.20	11.90 ± 0.73	3.53 ± 0.39	26.88 ± 3.07	34.30 ± 1.65	5.35 ± 0.95
	Harvest B
SAZ	2.90 ± 0.55	70.20 ± 10.03	34.19 ± 2.04	13.33 ± 1.59	56.13 ± 6.81	49.18 ± 6.36	11.75 ± 1.81
SLD	4.74 ± 1.27	32.53 ± 3.98	20.43 ± 2.56	6.07 ± 1.13	28.80 ± 3.53	28.48 ± 2.62	6.17 ± 0.67
PRE	5.10 ± 1.19	42.00 ± 6.14	26.45 ± 4.75	8.53 ± 1.45	34.27 ± 5.03	35.97 ± 5.22	8.25 ± 1.55
AGN	11.90 ± 1.18	40.06 ± 5.45	26.56 ± 8.59	7.01 ± 0.91	31.80 ± 4.72	39.43 ± 7.39	8.01 ± 0.92
(b) Antiradical activity
		Water			Acetone
		ARA1	ARA2	ARP	ARA1	ARA2	ARP
Variety		%/g	%/g	%/g	%/g	%/g	%/g
		Harvest A
SAZ		7.36 ± 0.80	14.9 ± 1.71	11.05 ± 1.24	8.83 ± 0.87	17.19 ± 0.78	12.81 ± 0.72
SLD		5.12 ± 0.89	9.28 ± 1.74	7.05 ± 1.31	9.38 ± 1.00	14.99 ± 14.99	11.81 ± 1.38
PRE		5.89 ± 0.69	10.3 ± 1.33	7.91 ± 1.00	10.34 ± 0.56	16.14 ± 0.76	12.82 ± 0.65
AGN		5.71 ± 0.74	10.58 ± 1.29	8.06 ± 0.97	11.47 ± 0.48	17.87 ± 0.73	14.35 ± 0.42
		Harvest B
SAZ		12.26 ± 1.30	21.34 ± 1.94	17.08 ± 1.62	12.36 ± 1.03	22.02 ± 1.57	17.49 ± 1.29
SLD		8.17 ± 1.73	12.21 ± 2.10	10.13 ± 1.82	10.00 ± 0.78	16.46 ± 0.88	13.30 ± 0.75
PRE		10.90 ± 1.82	17.33 ± 2.84	14.12 ± 2.34	12.87 ± 1.13	21.16 ± 1.75	17.08 ± 1.44
AGN		10.37 ± 1.76	16.53 ± 2.46	13.40 ± 2.10	20.55 ± 2.22	25.48 ± 1.44	22.60 ± 1.45

Alpha—α-acids in hops; TPs—Total polyphenols; ANTs—Anthocyanogens; FLAs—Flavanoids; SAZ—Saaz; SLD—Sládek; PRE—Premiant; AGN—Agnus; ARA1—Antiradical activity DPPH, reaction 1 min; ARA2—Antiradical activity DPPH, reaction 10 min; ARP—Antiradical potential DPPH.

Across both extraction methods, the highest values of total polyphenols (TPs), anthocyanogens (ANTs), and flavonoids (FLAs) were consistently observed in Saaz, while the remaining three cultivars did not differ significantly from one another (Table 1). Harvest year was a major source of variation: TP, ANT, and antiradical activity (ARA) values were generally higher in Harvest B, especially in Saaz, whereas α-acid levels were significantly lower. Location-specific factors—soil and climate conditions, weather patterns, and plant age—also contributed to variability in phenolic profiles. All of these factors significantly influence the phenolic composition of hops [28,43]. Thus, the genotype and harvesting conditions jointly determine the phenolic composition and biological activity of hops. The choice of extraction medium can accentuate or suppress certain differences.

3.1.1. Effect of Extraction Method on Polyphenolic Composition

Extraction method had a strong influence on extract composition, particularly on ANT and antiradical activity parameters (ARA1, ARA2, ARP). In Harvest A, the differences between extracts for both ANT and ARA were at a higher probability level (p < 1 × 10⁻⁷) than in Harvest B (p < 0.001) (Table 2). In Harvest A, ANT levels in acetone extracts were approximately 40–100% higher than in aqueous extracts, depending on the variety. In Harvest B, the differences between the extracts were approximately 40%. The reason for the differences between harvests could be the composition of ANT in different years, as polyphenols are formed partly as protective substances in response to stressors during the growing season [16,17,28].

Table 2.

Differences between acetone and water extracts in harvests A and B (ANOVA).

	Harvest A			Harvest B
	F	p Value	F crit	F	p Value	F crit
TP	5.2314	0.0275	4.0847	3.4465	0.0712	4.0982
ANT	50.084	1 × 10−8	4.0847	17.731	0.0002	4.0982
FLA	1.7921	0.1882	4.0847	0.0464	0.8306	4.0982
ARA1	84.6438	2 × 10−11	4.08475	10.415	0.0026	4.0982
ARA2	55.79	4 × 10−9	4.0847	13.146	0.0008	4.0982
ARP	67.892	4 × 10−10	4.0847	13.00	0.0009	4.0982

TPs—Total polyphenols; ANTs—Anthocyanogens; FLAs—Flavanoids; ARA1—Antiradical activity DPPH, reaction 1 min; ARA2—Antiradical activity DPPH, reaction 10 min; ARP—Antiradical potential DPPH; F crit—Critical value of the F parameter.

Total polyphenol values were less affected by solvent choice. Acetone extracts showed 15–20% lower TP values than water extracts in both harvests; this difference was significant in Harvest A (p < 0.05) but not in Harvest B. TP comprises the entire spectrum of polyphenols. Some of these polyphenols, along with polysaccharides and proteins, form the structure of hop cone cell walls [14,15,16]. Therefore, it is possible that these structures are partially destroyed during boiling and the polyphenols are released. Flavanoid contents (FLAs) tended to be slightly higher (10–15%) in acetone extracts in Harvest A, but no statistically significant differences were observed, suggesting that these compounds are extracted with similar efficiency by both solvents. FLAs represent simple and short-chain oligomeric flavan-3-ols, readily extractable by water [3,14].

Overall, aqueous acetone was more effective in releasing specific polyphenolic fractions associated with antiradical activity—particularly anthocyanogens and prenylflavonoids—whereas total polyphenol yields were comparable or slightly higher in hot-water extracts.

3.1.2. Polyphenol Groups and Extraction Principles

The finding that acetone-based systems often provide higher yields of selected phenolic classes is consistent with both the hop-specific and general extraction literature. Aqueous mixtures of acetone and methanol typically extract higher amounts of proanthocyanidins, xanthohumol, and other phenolics compared with hot water [44]. Traditionally, aqueous acetone is recommended for proanthocyanidins (anthocyanogens) [17,44]. The lower ANT yield for hot-water extraction is consistent with previous studies. Broader extraction reviews show that polar solvents (or water–solvent mixtures) generally outperform water for many phenolic classes, although optimal solvent choice depends on the target fraction [44]. Medium-polar solvents (ethanol, methanol, acetone) yield higher total polyphenol content and antiradical activity compared with strongly polar (water) and nonpolar solvents [45], with significant differences also found between these medium-polar solvents [46].

However, it should be noted that the outcomes of polyphenol analyses employing limited specific group methods are subject to the profile of polyphenolic substances present in the sample. The determination of total polyphenols (TPs) in beer (hops, wort, sweet wort) by methods codified in the EBC Analytica [26] or MEBAK [38] is based on a method proposed by Jerumanis [47], which relies on the reduction of ferric ions to ferrous ions and multiplication of the color intensity by an empirical coefficient. This method, which has been used in brewing for decades as a rapid and simple approach for both routine process control and research, is, however, similarly to the Folin–Ciocalteu method [48], highly non-specific, as reducing compounds other than phenolic acids and flavonoid polyphenols also react. Polyphenols bearing vicinal OH groups (gallic and chlorogenic acids, catechin, quercetin) are preferentially oxidized [49].

The relatively small difference in TPs between hot-water and acetone extracts reflects the dominance of simple phenolics, phenolic acids, and monomeric flavonoids—compounds that are readily extractable by water. Hot-water extracts may additionally contain polyphenols released through thermal degradation of plant tissues or partial depolymerization of condensed structures [49].

The much higher ANT (pelargonidin, delphinidin, condensed tannins) values in acetone extracts are attributable both to the greater solubility of these compounds in acetone and to oxidative degradation during hot-water treatment [49].

Only a minor effect of the extraction procedure was observed for the flavanoid group. As “flavanoids” [49], the method determines flavan-3-ols epi/catechin (and oligomers composed of /epi/catechin, /epi/gallocatechin, and /epi/afzelechin structural units with a terminal /epi/catechin molecule) bearing free OH groups at the 3′ and 4′ positions on the B ring. The method is highly specific. Monomeric catechins are relatively well soluble in water [44].

3.1.3. Profiling of Individual Polyphenols

UHPLC profiling revealed strong cultivar-driven patterns in simple flavonoids, i.e., flavanols, flavonols, and their glycosides (Figure 1), consistent with our earlier findings [43]. Extraction method also played a role, with hot-water extracts showing lower total flavanoid levels, particularly due to reduced catechin content (Figure S1). Prenylflavonoids were significantly affected: their total content in acetone extracts was approximately twice that of hot-water extracts (Figure S2). Hot-water extraction leads to the conversion of the major and less polar xanthohumol to more soluble isoxanthohumol and to losses due to sorption onto hop plant material, explaining lower prenylflavonoid levels [14].

Figure 1.

Agglomerative hierarchical clustering (AHC) of flavonoids profile (UHPLC).

3.1.4. Summary of Polyphenol Results

Taken together, hot-water extraction produced slightly higher TPs, whereas it yielded substantially lower ANTs, while both solvents extracted similar amounts of flavanoids. Acetone extraction captured a broader spectrum of phenolic compounds, including those potentially attributed to high antiradical activity.

3.2. Antiradical Activity

Antiradical activity measured by the DPPH assay was significantly higher in acetone extracts in both harvests (Table 2), with the largest differences observed in Harvest A, characterized by higher α-acid and lower polyphenol levels. Cultivar effects were pronounced: the aroma variety Saaz, with the highest polyphenol content, showed the smallest solvent-driven differences, whereas the bitter variety Agnus, with the highest α-acids, showed the greatest increases in acetone extracts (Table 1).

3.2.1. ARA1 as a Marker of Rapidly Reacting Compounds

The most notable solvent effect was seen in ARA1 (1 min reaction time), reflecting rapidly reacting antioxidants. Differences between solvents were most pronounced in Harvest A (Figure S3), suggesting a significant contribution of α-acids to antiradical activity in organic extracts. α-acids have low solubility in water and isomerize during boiling to iso-α-acids [14], which according to Ting et al. [36] do not reduce DPPH. This could explain why hot-water extracts, which are rich in iso-α-acids but poor in α-acids, show lower ARA1 values. The exception was Agnus samples with similar α-acid contents in both harvests and similar differences in ARA1 between water and acetone extracts.

3.2.2. Correlation Patterns

Hot-water extracts showed strong positive correlations (r = 0.73–0.90; p < 0.01) between TP, ANT, FLA, and all ARA metrics, indicating that their antiradical activity is primarily driven by polyphenols.

In acetone extracts, correlations differed: in Harvest A, polyphenols did not correlate significantly with ARA and α-acids content in the original hop sample correlated strongly with ARA1 (r = 0.75, p < 0.01). In Harvest B, some positive correlations were observed but were weaker than in hot-water extracts (Table 3).

Table 3.

Correlation of antiradical activity with polyphenols in acetone and water extracts in harvests A and B.

		Harvest A				Harvest B
		Alpha	TPs	ANTs	FLAs	Alpha	TPs	ANTs	FLAs
Water	ARA1	−0.523 *	0.879 **	0.769 **	0.814 **	−0.232	0.729 **	0.763 **	0.747 **
	ARA2	−0.681 **	0.903 **	0.849 **	0.903 **	−0.297	0.833 **	0.820 **	0.846 **
	ARP	−0.652 **	0.892 **	0.832 **	0.884 **	−0.291	0.822 **	0.816 **	0.837 **
Acetone	ARA1	0.751 **	−0.314	−0.066	−0.317	0.829 **	−0.122	0.226	0.047
	ARA2	0.125	0.386	0.535 *	0.377	0.569 **	0.275	0.559 **	0.462 *
	ARP	0.418	0.137	0.335	0.125	0.703 **	0.112	0.434 *	0.297

* Significant at p = 0.05%; ** significant at p = 0.01%; Alpha—α-acids in hops; TPs—Total polyphenols; ANTs—Anthocyanogens; FLAs—Flavanoids; ARA1—Antiradical activity DPPH, reaction 1 min; ARA2—Antiradical activity DPPH, reaction 10 min; ARP—Antiradical potential DPPH.

These patterns indicate that antiradical activity in acetone extracts is not closely linked to the TP/ANT/FLA group parameters, suggesting that other components, such as bitter acids and prenylflavonoids, substantially contribute to antiradical activity. These components are extracted more effectively in organic solvents [36,50,51].

When acetone, a polar aprotic solvent, is used, less polar compounds are dissolved to a greater extent; α- and β-bitter acids are also extracted, as their solubility in water is low [52]. The antiradical activity of acetone hop extracts is therefore likely to also involve humulones and lupulones, which are likewise capable of reducing the DPPH radical [36], whereas the antiradical activity of aqueous extracts obtained by boiling during “blank wort boiling” is predominantly associated with polyphenolic compounds, since iso-α-acids do not reduce DPPH [36]. Acetone and aqueous extracts thus differ both in their polyphenol profiles and in their antiradical profiles. Certain compounds included in the anthocyanogen group evidently may exhibit significant antiradical activity [17].

3.2.3. Bitter Acids in Extracts and Their Contribution to ARA

An additional set of samples was analyzed (see Table 4), revealing that hot-water extracts exhibited low concentrations of α-acids (39–83 mg/L) and negligible levels of β-acids (5–12 mg/L), irrespective of their content in hops. The iso-α-acids were found to be present at concentrations ranging from 14 to 31 mg/L, which corresponds to a relatively short 30 min boiling time.

Table 4.

Antiradical activity, polyphenols, and bitter acids in acetone and water extracts.

		Iso-Alpha	Alpha	Beta	TPs	ANTs	FLAs	ARP	ARA1	ARA2
		mg/L	mg/L	mg/L	mg/L	mg/L	mg/L	%	%	%
Water	SAZ	14	39	10	174	90	48	53.8	38.7	67.8
	SLD	25	48	12	67	36	12	25.6	19.8	31.7
	PRE	31	83	11	86	59	22	38.1	30.1	46.7
	AGN	27	59	5	170	118	38	55.4	41.9	68.2
Acetone	SAZ	3	60	90	155	111	42	46.7	34.6	58.2
	SLD	5	90	87	57	48	12	31.0	25.3	37.8
	PRE	5	263	86	92	73	22	41.6	34.2	50.2
	AGN	4	257	96	165	158	40	56.0	45.1	67.5

Alpha—α-acids; Beta—β-acids; TPs—Total polyphenols; ANTs—Anthocyanogens; FLAs—Flavanoids; ARA1—Antiradical activity DPPH, reaction 1 min; ARA2—Antiradical activity DPPH, reaction 10 min; ARP—Antiradical potential DPPH; SAZ—Saaz; SLD—Sládek; PRE—Premiant; AGN—Agnus.

In contrast, acetone extracts contained much higher levels of α-acids (up to 263 mg/L) and β-acids (up to 96 mg/L), corresponding to 75–90% extraction yields (see Table S1), while iso-α-acid levels remained low (3–5 mg/L). These data confirm the solvent-dependent extraction behavior of bitter acids. Iso-α-acids are more polar and more soluble in water, while α-acids and β-acids are more effectively extracted in organic solvents [14,44].

With respect to antiradical activity (ARA1 and ARA2), the aqueous extracts closely reflected the contents of TP, ANT, and FLA, in agreement with the strong correlations observed in the main experiment. In acetone extracts, where TP levels were comparable and ANT contents were 25–30% higher, ARA1 values were approximately 10–25% higher than in the aqueous extracts. However, this increase did not correspond to the concentration of α-acids, which was strongly dependent on the hop cultivar. For the SAZ, characterized by higher TP levels in the aqueous extract, the ARA of the acetone extract was lower, consistent with the main experiment. Thus, the previously inferred significant contribution of α- and β-acids based on correlation analysis (Table 3), including the association between ARA1 and α-acids in acetone extracts, was not confirmed, despite the well-documented antiradical activity of these compounds in the literature [10,36,53].

Concentration-dependent assays of pure α- and iso-α-acids confirmed the findings of Ting et al. [36] regarding the pronounced DPPH scavenging activity of α-acids, whereas iso-α-acids showed no measurable activity (Figure S4). Although pure α-acids showed strong DPPH activity, the expected magnitude of their contribution (~15% increase in ARA at 100 mg/L α-acids) was not observed in real hop extracts, suggesting interactions within the matrix.

3.2.4. Polyphenol Pellets Experiment

To further differentiate the contributions of polyphenols and bitter acids, polyphenol-enriched hop pellets of two contrasting varieties, SAZ and AGN, with only residual bitter acids were examined. Acetone extraction increased both polyphenol content and ARA in both pellets, but the effect was more pronounced in Agnus (Table 5). The similar increase in ANTs in both cultivars and pronounced increase in TPs in AGN (25% rel. vs. 14% rel.) correlated with a 21% rel. increase in ARP, compared with only 5% rel. in SAZ. These results indicate that certain polyphenolic subfractions—particularly less polar or bound phenolics—are major contributors to antiradical activity and are more efficiently extracted by acetone.

Table 5.

Comparison of polyphenol content and antiradical activity in water and acetone extract of bitter acid-free pellets from two hop varieties.

	ARP	ARA1	ARA2	ANTs	TPs	ALPHA
	%	%	%	mg/L	mg/L	mg/L
SAZ—water	52.6	41.3	63.5	86.8	327	0.561
SAZ—acetone	55.5	42.6	66.6	153.2	372	0.838
AGN—water	40.4	33	48.3	64.4	228	4.404
AGN—acetone	49	39.2	58.1	112.6	285	8.394

3.2.5. Summary of Antiradical Activity Results

Antiradical activity assessed by the DPPH assay was consistently higher in acetone extracts than in hot-water extracts, with the most pronounced differences observed in Harvest A (lower polyphenols, higher α-acids) and in hop cultivars rich in α-acids. Hot-water extracts showed strong correlations between antiradical activity and group-type polyphenols, indicating that their antiradical capacity is primarily polyphenol-driven. In contrast, acetone extracts exhibited weaker or absent correlations with these polyphenol parameters, suggesting the involvement of additional compounds (bitter acids, prenylflavonoids). Although α- and β-bitter acids are, in principle and in fact, extracted far more efficiently in acetone and pure α-acids displayed strong DPPH scavenging activity, their expected contribution to antiradical activity in real hop extracts was not fully confirmed, likely due to matrix interactions. Although pure α-acids exhibit strong antiradical activity, their effectiveness in hop matrices is limited by concentration, solubility, and probable interactions with polyphenols. Furthermore, specific polyphenolic subfractions that are preferentially extracted by acetone may play a significant role in enhancing antiradical activity. The content of these subfractions varies depending on the variety.

4. Conclusions

This study of four hop cultivars over two harvest years highlights the decisive influence of extraction method on the composition and antioxidant activity of hop extracts. While hot-water extraction yields slightly higher levels of total polyphenols, it provides markedly lower amounts of anthocyanogens and prenylflavonoids and results in substantially reduced antiradical activity. In contrast, aqueous acetone efficiently extracts a broader range of bioactive phenolic compounds—particularly those strongly associated with antiradical performance—and consistently produces extracts with higher DPPH-scavenging activity across cultivars and harvest years.

The aqueous extracts exhibited strong and consistent correlations between grouped polyphenol parameters (TP, ANT, FLA) and antiradical activity, indicating that their antioxidant potential was primarily governed by polyphenol concentration. In contrast, the relationships between polyphenol parameters and ARAs were weaker in acetone extracts, suggesting a substantial contribution of other constituents, particularly bitter acids and prenylflavonoids, extracted more efficiently in this way. Surprisingly, the high antiradical activity determined for pure α-acids was not reflected by corresponding increases in antiradical activity in hop extracts, possibly due to matrix effects. Additionally, an experiment with bitter acid-free hop material revealed that antiradical activity depends on the variety-specific profile of polyphenolic compounds. Thus, aqueous and acetone extracts represent distinct phenolic and antioxidant profiles of hops, which should be considered when interpreting analytical data and evaluating biological activity.

When evaluating the polyphenol profile and antiradical activity of hops, it is necessary to choose an extraction medium in accordance with the objective of the analysis. Aqueous extraction under conditions simulating wort boiling better reflects the behavior of polyphenols in the actual brewing process, while aqueous acetone highlights fractions with high antiradical activity, including anthocyanogens and prenylflavonoids.

The results highlight the selective nature of solvent–matrix interactions in hops and emphasize that assessments of hop antiradical potential must consider extraction conditions. Aqueous acetone emerges as the more suitable extraction medium when the objective is to isolate phenolic fractions relevant to antiradical functionality or to obtain enhanced analytical resolution of hop polyphenols. Future research should focus on detailed profiling of solvent-responsive phenolic subgroups and on elucidating synergistic/antagonistic mechanisms contributing to antiradical activity within hop matrices, including hop bitter acids.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods15040702/s1, Figure S1: UHPLC-HRMS profile of flavonoids in hop extracts using hot water and aqueous acetone; Figure S2: UHPLC-HRMS profile of prenylflavonoids in hop extracts using hot water and aqueous acetone; Figure S3: Comparison of antiradical activity (DPPH-ARA1) of hop extracts obtained with hot water and aqueous acetone in harvests A and B; Figure S4: Relationship between the concentration of pure alpha acid and DPPH antiradical activity. Table S1: Limit concentrations of bitter acids in acetone and water extracts (based on their content in hops).

Author Contributions

A.M. conceived the original idea and led all stages of the work, including study design, methodology, investigation, data curation and analysis, visualization, manuscript writing, and overall project coordination. M.D. contributed by performing HPLC and UHPLC-HRMS analyses, validation, and interpretation. T.V. contributed with formal data analysis and validation. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request. Access is restricted because further, not-yet-published findings could be derived from these data.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Funding Statement

This research was supported by the Ministry of Agriculture of the Czech Republic within the institutional support MZE-RO1923.