Chemicals and materials

Milli-Q® deionized water (Darmstadt, Germany); hexane, methanol, dimethylsulfoxide and acetonitrile were purchased from Bio-Grade® (Durham, USA); hydrochloric acid and sodium bicarbonate from Synth® (Diadema, São Paulo); magnesium sulfate from Êxodo Científica® (Sumaré, São Paulo); acetic acid and magnesium chloride from Dinâmica® (Indaiatuba, São Paulo); Folin–Ciocalteu reagent from Merck® (Darmstadt, Germany); sodium chloride from Neon® (Suzano, São Paulo); monobasic and dibasic potassium phosphate, monobasic and dibasic sodium phosphate, hypoxanthine, ferric chloride hexahydrate, salicylic acid, ethylenediaminetetraacetic acid (EDTA), DPPH·+ (1,1-diphenyl-2-picrylhydrazyl), ABTS·+ [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)], p-nitrophenyl-α-D-glucopyranoside, acetylthiocholine iodide (ATCI), acetylcholinesterase (AChE), 5,5′-dithiobis[2-nitrobenzoic acid] (DTNB), bovine serum albumin fraction V, Trizma hydrochloride solution, xanthohumol, mangiferin, bovine milk xanthine oxidase and α-glucosidase (Saccharomyces cerevisiae) all obtained from Sigma-Aldrich® (Saint Louis, USA); acarbose (Glucobay®) from Bayer® (Leverkusen, Germany); international calibration extract of hop ICE-4 (standard mixture of bitter acids, purity 69.2%) was obtained from Labor Veritas® (Zurich, Switzerland).

Plant material

Commercial pellets of two varieties of hop flowers (Saaz–Hallertau® and Herkules–Barth-Haas Group®) were purchased at a brewery supply store in the city of Fortaleza (Brazil), with more than 2 years of storage until the period of analysis.

Hop cones of the Chinook cultivar (Chinook-1: mature hop/Chinook-2: first bloom) and Cascade (first bloom) were provided by two members of the Brazilian Association of Hop Producers (Aprolúpulo®), from their plantations in the cities Brasília (Federal District) and Monte Alegre (Rio Grande do Norte). The samples were received in vacuum-sealed metallized packages, with humidity between 8 and 12% (w/w). These cones were collected from replicated plants from seedlings imported with certification and registration at the Brazilian Ministry of Agriculture, Livestock and Supply (MAPA), acquired from the Lúpulo Ninkasi® nursery (Teresópolis, Brazil). All samples were kept at 5 °C under cold storage, until the analysis period.

Obtaining hexane and methanolic extracts by the Soxhlet system

The extracts of the different cultivars were obtained using the ground plant materials (in a bladed processor; Moulinex-DP700, France) using a Soxhlet system. Initially, the extraction of bitter acids was performed with hexane (150 mL—3 × 3 h), followed by extraction of the most polar species, including xanthohumol, with methanol (150 mL—3 × 3 h). The proportion of solvents used was 10 g of plant material to 450 mL of each solvent (hexane and methanol). The solutions were concentrated using a rotary evaporator and dried with a constant nitrogen flow at 25° C [12]. The extracts produced were stored at 5 °C, in a solid state, under cold storage until the experimental procedures were carried out.

Total phenolic content: Folin–Ciocalteu method

The total phenolic content was performed according to the procedure described by Cicco and collaborators with adaptations [28]. 100 µL of extract solution dissolved in methanol (1 mg/mL) was inserted into Eppendorf tubes, followed by the addition of 500 µL of 10% Folin–Ciocalteu reagent in H2O (v/v) and vortexing (Kasvi-K40/1020, South Korea) for 1 min. The reaction was started by adding 400 µL of 7.5% (w/v) aqueous sodium carbonate solution and incubating at 40 °C for 20 min. without shaking.

The same procedure was carried out with the standard gallic acid at different concentrations (2500–19.53125 µg/mL) to obtain the standard calibration curve and determine the total phenolic content in mg of gallic acid equivalents per gram of extract (mg EAG/g). After incubation, 100 µL of each system was transferred to microplates and the absorbances were measured in an ELISA spectrophotometer (Enzyme-Linked Immunosorbent Assay—Biotek, Winooski, USA) at 740 nm. All samples from the different experiments in this research were obtained in triplicate (n = 3).

Chemical quantitation of bitter acids and xanthohumol using QuEChERS extraction and high-performance liquid chromatography (HPLC-DAD)

To verify the amount of the compounds of interest in the evaluated cultivars (bitter acids and xanthohumol), the methodology proposed by Marques and collaborators was used for quantitative analyzes in hop flowers [29].

The compounds of interest were obtained through salt-assisted extraction (QuEChERS), using 1 g of powdered plant material. The supernatant solutions were collected, diluted in acetonitrile (1:10–1:20) and centrifuged (13,000 rpm, 25 °C, 5 min) for chromatographic analyses. The extractions were performed in triplicate and evaluated individually by liquid chromatography (HPLC-DAD).

Chromatographic evaluations were performed using a high-performance liquid chromatography with DAD detection (Agilent 1260 Infinity, Germany), equipped with a C-18 reverse phase column (Agilent eclipse plus − 3.5 µm, 4.6 × 100 mm). The mobile phases were methanol (A) and 2% aqueous acetic acid solution (B), with a chromatographic run time of 35 min, with a flow rate of 1.0 mL/min. The injection volumes used were 10 and 20 μL, with the following gradient flow: 0–5 min (75% A), 5–15 min (100% A) and 15–35 min (75% A).

Bitter acids showed better resolution at a wavelength of 326.4 nm and xanthohumol at 340.4 nm; these being the wavelengths used for quantitation of these compounds [29]. Standard calibration curves, statistical treatment and calculations of compound concentrations (in g/100 g) were performed using Microsoft Excel 365 software.

DPPH· radical scavenging antioxidant assay

Assays were performed in 96-well microplates, adding 20 µL of methanol solutions of extracts and standards (BHT, xanthohumol) at seven different concentrations (15.625–1000 µg/mL), for the standards and extracts of the Herkules and Chinook-1 varieties, and (62.5–4000 µg/mL) for the extracts of the Chinook-2, Saaz and Cascade cultivars, in triplicate. The reaction was started by adding 180 µL of a methanol solution of the DPPH• radical (200 µmol/L) in the absence of light for 30 min [12]. Absorbances were detected in an ELISA spectrophotometer at the maximum absorption wavelength of the DPPH• radical (515 nm) and IC50 calculations using Origin 9.5® software.

ABTS·+ antioxidant assay

To determine the antioxidant capacity against the ABTS·+ radical cation, an adaptation of the method proposed by Torres and collaborators was performed [30]. The radical cation was obtained by the reaction between 5 mL of ABTS aqueous solution (7 mmol/L) with 88 µL of potassium persulfate solution (140 mmol/L) for 16 h in the absence of light. In microplates, 20 µL of methanol solutions of extracts and standards was added in seven different concentrations (0.016–4 mg/mL), followed by the addition of 180 µL of freshly prepared ABTS·+ radical solution and subsequent analysis by spectrophotometry (ELISA) at 734 nm. BHT antioxidant was used as a positive control and IC50 calculations using Origin 9.5® software.

Antioxidant assay hydroxyl radicals and inhibition of the enzyme xanthine oxidase

The simultaneous assay of xanthine oxidase inhibition and hydroxyl radical scavenging was carried out according to the methodology described by literature [12, 31]. Different volumes (500, 250, 100, 50, 25 and 10 μL) of methanol solutions of extracts/ICE-4 (10,000 μg/mL) and xanthohumol (1000 μg/mL) were added to Eppendorf tubes prior to solvent evaporation. One mL of phosphate buffer (pH = 6.6) was added and vortexed for 1 min and placed in an ultrasonic bath for 10 min. Subsequently, 10 µL of xanthine oxidase enzyme (20 U) was added, and the system was incubated in a thermomixer for 3 h (450 rpm at 37 °C), in the absence of light. The reaction was terminated by adding 10 µL of concentrated hydrochloric acid, centrifuged (13,000 rpm, 25 °C, 5 min) for analyzed by HPLC. The operational conditions of the liquid chromatograph (HPLC–DAD) were the same as described above; however, the injection volume was 10 μL and a chromatographic run of 50 min of with the following gradient flow: 0–11 min (0% of A), 11–35 min (20% A), 35–40 min (40% A), 40–45 min (0% A) and 45–50 min (75% A).

The detection and calculations of uric acid levels (evaluation of the ability to inhibit the enzyme xanthine oxidase) were performed at 278.4 nm, while the dihydroxybenzoic acids (2,3 and 2,5-DHBA), related to the antioxidant capacity, were and quantitated at 325.4 nm [12, 31]. The xanthone mangiferin (10,000 μg/mL) was used as a positive control and IC50 calculations using Origin 9.5® software.

α-glucosidase enzyme inhibition assay

For this assay, solutions were prepared at different concentrations of the extracts/ICE-4 (15.625–1000 µg/mL), xanthohumol (3.90625–250 µg/mL) and acarbose (78.125–10,000 µg/mL) in sodium phosphate buffer solution (50 mmol/L, pH = 6.8).

In Eppendorfs, 20 µL of samples and 20 µL of α-glucosidase (1 U/mL) were added. After 5 min, 40 µL of p-NPG reagent (1 mmol/L) was added and the system incubated for 30 min at (37 °C). Following incubation, 100 µL of 10% sodium bicarbonate aqueous solution was added and 100 µL of the systems was collected for quantitation of absorbances in an ELISA spectrophotometer (405 nm). Assays were carried out without the enzyme (blank), for each concentration value of the extracts, to evaluate the possible interference of the p-NPG reagent on the absorbance values obtained [22]. The drug acarbose was used as a positive control at the same concentrations as the extracts.

Acetylcholinesterase enzyme inhibition assay

The determination of the acetylcholinesterase enzyme inhibition activity was based on the methodology described by literature, modified to TLC (thin layer chromatography) [32, 33]. After 10 min of enzyme application, a yellow coloration is observed in the region of samples that do not present inhibitory action and the formation of a white halo in the “spots” of those that present anticholinesterase activity, are calculated from the diameter of the halos (millimeters) [12]. A methanol solution of eserine (1000 μg/mL) was used as a positive standard.