1. Introduction
The hop plant (Humulus lupulus L.) belonging to the Cannabaceae family is a fast-climbing perennial that grows well in temperate regions and produces large quantities of yellow–green leaves from spring to autumn.
The plant tends to climb clockwise around any support to a height of 10–18 m. The leaves are opposite or alternate and have three to five lobes with serrated margins; further, they are rough to the touch and have toothed edges.
H. lupulus contains several phytochemicals [
1], and some of its secondary metabolites have potential value whose accumulation depends on various factors such as the genetic make-up, climatic conditions [
2], and cultivation area [
3]. The inflorescences represent the share of biomass exploited mainly because it is widely used to produce beer. In fact, the leaves and stems, as mass accumulated in the post-harvest period, have been little studied as they are basically considered waste material. However, with a view to a circular economy, also considering the high quantity of biomass, the recovery of this material can be useful in the industrial field as an alternative source of functional molecules [
4]. In fact, previous studies have proposed the use of hop leaves as fodder for animals [
5] or to produce ecological pesticides not harmful to bees [
6].
Thanks to its numerous constituents, such as bitter acids, polyphenols, flavonoids, and terpenic compounds, hop cones have been extensively investigated, highlighting a wide range of biological activities including antibacterial, antioxidant, estrogenic [
7,
8] and, lately, also anticancer mainly linked to the presence of xanthohumol [
9,
10]. However, some molecules belonging to these classes of compounds have also been found in hop leaves [
11,
12]; among these, phenolic compounds are present in sufficient concentrations to be responsible for the antiradical capacity attributed to the leaves [
13]. On the contrary, little or nothing has been reported on the volatile composition of dried hop leaves.
The Chinook hop is a cultivar well known in the American craft beer world, and to this day, the majority of the Chinook’s annual supply comes from Washington hop growers. This variety is a cross between a Petham Golding and a USDA-selected male, and it is characterized by a high content of alpha acids and good-keeping properties. It has a strong pine and resin character with spicy notes, and it can also be slightly spicy [
14].
In this study, the complexity of chemical compounds was explored through an exhaustive analysis of the metabolome performed by combining multiple analytical tools (SPME/GC–MS, PTR-ToF-MS, and NMR). For example, the correlation between SPME/GC–MS and PTR-ToF-MS data allows a complete fingerprint of the volatolomic profile of the analyzed matrix, bypassing the limitations of each instrument. On the other hand, NMR is a powerful technique that, when applied to food, allows for the identification and quantification of secondary metabolites in a single experiment. Therefore, a metabolomic approach was applied in order to describe the volatile and non-volatile chemical profile of the dried leaves of Chinook hop grown in southern Italy.
3. Discussion
In the present work, for the first time, a multi-methodological approach was applied to study the metabolome of dried Chinook hop leaves. The obtained results highlighted the presence of a high number of volatile and semi-volatile metabolites.
Overall, up to now, a small number of previous studies have focused on the chemical characterization of hop leaves, with a very small fraction of these involving the volatile content of untreated plant material. In fact, most of the previous articles mainly concerned the determination of α- and
β-acids of hop inflorescences [
20,
21,
22,
23,
24]. Furthermore, to the best of our knowledge, this is the first work reporting the fatty acid content of hop leaves beyond the investigated cultivar.
Our data showed that α-humulene followed by
β-caryophyllene and α-copaene were the most representative terpene compounds. In our previous study [
25], carried out on fresh leaves, we found that
β-caryophyllene, α-humulene, and
δ-cadinene, in descending order, were the most abundant. This suggests that drying, in general, may influence the volatile profile of this matrix. In addition, as reported by Macchioni et al. [
13], the drying method can induce variations in the content of flavones, thiols, phenols, and pigments in hop leaves. Likewise, genetic and environmental factors greatly affect the content of phytochemical compounds [
26].
The detected emission of several terpene compounds from the leaves was in line with what was previously reported by Stanius et al. [
16], where borneol,
γ-cadinene, estragole,
α-copaene,
β-farnesene,
β-caryophyllene,
γ-muurolene, and
α-humulene were emitted by leaf samples of five Lithuanian hop varieties. These compounds were characteristic of the volatile composition of both leaf and cone hops, albeit with different trends [
16].
Thanks to PTR-ToF-MS analyses, in addition to some terpenes, which represent about 2% of the total VOC emission, a range of low-molecular-weight compounds such as aldehydes, ketones, alcohols, and sulfur compounds was also found. Among them, aldehydes such as acetaldehyde (C
2H
5O
+) and propanal (C
3H
7O
+) were the most abundant identified compounds, whereas alcohols, ketones, and sulfur showed the lowest emission. For example, the emission amount of CH
5S
+ (methanethiol) accounted for 0.2% of the total VOC emission (
Table 2).
The data obtained by the GC–MS and NMR analyses on the derivatized methanolic extract and the hydroalcoholic extract, respectively, were aligned as both techniques highlighted the presence of compounds belonging to other chemical classes, such as sugars which made up the main fraction and the organic acids. Moreover, taken individually, the GC–MS technique has allowed the identification of a series of alcohols, albeit with a low relative percentage; on the other hand, the NMR technique has made it possible to identify a large number of amino acids.
In general, lH-NMR was widely used to characterize bitter acids and xanthohumol in hop cones [
18,
27,
28], but no NMR study is available on hop leaves. On the other hand, only one study reports the polar metabolite content in hop cones by NMR. [
29]. Asparagine, glucose, and fructose have been reported to be the major amino acids and sugars in hop cones [
18]. Instead, in our study, glutamate, asparagine, and sucrose were the main ones in hop leaves. Regarding bitter acids, they were not detected in hop leaves due to their low concentration [
30,
31].
The obtained results from our investigation highlighted that hop leaves can be a natural alternative source of a large number of bioactive metabolites whose biological effects are well known. In fact, the identified different phytochemical classes play a key role in the prevention of various diseases. Among the most representative compounds, the major detected sesquiterpenes
β-caryophyllene and
α-humulene can exert antibacterial, anti-inflammatory, antioxidant, and anticancer actions [
32,
33]. The fatty acids revealed in hop leaves are also known for their significant effects on human health [
34]. For example, linolenic acid, as the major detected fatty acid, has important physiological actions for plants; moreover, following metabolic conversions into bioactive derivatives, it is expected that it can also treat many diseases [
35]. Conversely, palmitic acid has been reported to exhibit significant antibacterial activity against oral microorganisms [
36], in addition to having metabolic implications [
37]. Furthermore, the structures of many well-known antibacterial and antifungal agents exhibit an amino acid backbone that is crucial for carrying out antimicrobial activity [
38]. In plants, sugars are essential as, in addition to providing carbon skeletons as substrates for tissue growth, they also act as signal molecules, thus influencing metabolic processes [
39]. Among the detected alcohols, D-pinitol, a glucose metabolite widely identified in plants of the Leguminosae family, has been reported to possess several pharmacological activities, including antidiabetic, antioxidant, anti-inflammatory, and chemo-preventive effects [
40]. Finally, xanthohumol, the identified polyphenol in hop leaves, exerts various pharmacological effects such as anti-inflammatory, antioxidant, hypoglycemic, and even anticancer activity [
41].
4. Materials and Methods
4.1. Plant Material
Dried hop leaves from the Chinook cultivar were grown in San Nicandro Garganico (Foggia, Italy) at the Vocino family farm in 2021.
The drying of hop leaves took place in a natural way, inside a dark and ventilated room with a constant humidity level (about 50% RH). After about 3 days, the leaves were vacuum-packed and stored in a cool and dry place. Before the analysis, the dried hop leaves were finely chopped with a grinder (MulinexAR 11, Groupe SEB, France) with a particle size of approximately 1.0 mm.
4.2. SPME Sampling
Using the SPME sampling technique, the volatile profile of the untreated matrix was described. About 2 g of the dried leaves were placed inside a 15 mL glass vial with a PTFE-coated silicone septum. A SPME device from Supelco (Bellefonte, PA, USA) with 1 cm fiber coated with 50/30 μm DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) was chosen to extract the components. The operative conditions followed Nezi et al. [
42] with minor modifications. Component desorption was ensured by inserting the fiber directly into the GC injector maintained at 250 °C in splitless mode.
4.3. GC–MS Analysis of the Dried Hop Leaves
To carry out the analyses of the headspace hop leaves, a Clarus 500 model Perkin Elmer (Waltham, MA, USA) gas chromatograph coupled with a mass spectrometer equipped with a FID (flame detector ionization) was used [
25,
43]. A Varian (VF-1 ms) apolar column was used, and the detected and identified components are listed in
Table 1.
The oven programmed temperature was set initially at 55 °C and then increased to 220 °C at 6°/min and finally held for 15 min. Helium was used as carrier gas at a constant rate of 1 mL/min. MS detection was performed with electron ionization (EI) at 70 eV, operating in the full-scan acquisition mode in the m/z range of 35–550 amu. To identify the volatile compounds, the MS-fragmentation pattern obtained was compared with those of pure components stored in the Nist 02 mass spectra library database. The Linear Retention Indices (LRIs) were also calculated using a series of alkane standards (C8–C30 n-alkanes, Supelco, Bellefonte, CA, USA) and then compared with those available in the literature. Semi-quantitative values (peak area percentages) were obtained by peak normalization without using correction factors. The area of each peak in the chromatographic profile was divided by the total sum of all peaks within the chromatogram. The normalized data was then multiplied by 100 and expressed as a percentage. Analyses were carried out in triplicate.
4.4. Extraction and Transesterification Processes
A 3.0 g sample of the dried and chopped hop leaves was weighed in a flask and extracted with 10.0 mL × 2 of methanol (Carlo Erba, Milan, Italy) at room temperature. After separation from solid residue by filtration, the methanolic solutions were combined and dried under reduced pressure at 30 °C to obtain 0.301 g of solid extract.
A 20 mg extract was dissolved in toluene (4 mL) and transferred into a tube to carry out the transesterification [
44]. In detail, a solution (10 mL) of 1% (
v/
v) concentrated sulfuric acid in methanol was added, and the stoppered tube was left overnight at 50 °C. The solution was cooled and transferred in a separatory funnel, and 20.0 mL of water with 5% NaCl was added. A 20.0 mL × 2 of n-hexane (Carlo Erba, Milan) was used to extract the esters, the upper solutions were combined, washed with aqueous potassium bicarbonate (2%) (Merck, Darmstadt), filtered over anhydrous sodium sulfate (Merck, Darmstadt, Germany) and dried under reduced pressure at 30 °C.
4.5. GC–MS Determination of Fatty Acids (FAs) Content
To describe the FAs content, the same apparatus reported above (
Section 4.3) was used. The GC operative conditions to analyze the transesterified extract were as follows: the injector was set to 280 °C, and the oven temperature was programmed from 170 °C at a rate of 3 °C/min to 260 °C for 15 min. 2 µL of the extract was injected into the column in splitless mode. The identification and quantification of the components were performed as previously described. Analyses were carried out in triplicate.
4.6. GC–MS Analysis of Methanolic Extract (after Derivatization)
To describe the chemical content of methanolic extract, approximately 1 mg was added of 300 µL of pyridine and 100 µL of bis-(trimethylsilyl) trifluoroacetamide (BSTFA) with heating at 80 °C for 50 min. 1 μL of the silylated sample was manually injected at 280 °C into the GC injector in splitless mode [
45]. The analysis was performed using the same apparatus, GC-FID/GC–MS. The oven temperature program was as follows: 70 °C then a gradient of 6 °C/min to 170 °C, a gradient of 7 °C/min to 250 °C, and a gradient of 8 °C/min to 300 for 15 min. The compound identification was based on the percentage of similarity plus comparison of mass spectra (MS) using the software NIST02 (Gaithersburg, MD, USA) data library. Relative percentages for quantification of the components were calculated by electronic integration of the GC-FID peak areas with respect to the percent total of ion chromatograms (TIC%). Analyses were carried out in duplicate.
4.7. PTR-ToF-MS Analysis of Dried Leaves
The volatile profile of the dried hop leaves was performed using, for the first time, a PTR TOF 8000 model (Ionicon Analytik GmbH Innsbruck, Innsbruck, Austria). The ion H
3O
+ was used as a reagent ion for the proton transfer reaction. The use of a PTR-TOF-MS allows the simultaneous detection of a wide range of VOC with a high mass power [
46]. The sample preparation and experimental set-up procedure was followed as previously conducted by Masi et al. [
15] on
Mentha spicata dried leaves.
Subsequently, the weighed samples (five grams of chopped dried leaves) were transferred into a glass jar (1/3 L) and incubated for 60 s. The headspace was sampled at a fixed flow rate of 50 sccm for 3 min. Five replicates of dried leaf hops were analyzed, and between the measurements, the PTR inlet line was flushed with clean air to avoid the memory effect. The experiment was led using the following drift tube parameters: voltage 594 V, temperature 110 °C, extraction voltage at the end of the drift tube (Udx) 35 V, and the acquisition was set to 1 mass spectrum per second. Mass spectral data were collected each second over a mass range between 20 and 220
m/
z using a ratio E/N = 140. In order to have high mass accuracy and easily determine the chemical formula of each peak detected, the internal calibration was performed off-line, using together with known low mass ions (NO
+ peak
m/
z = 29.997 and C
3H
7O
+ peak
m/
z = 59.049) a known compound with high mass ions as the carvone (C
10H
15O
+ peak
m/
z= 151.075). Then, once the chemical formula was identified, each signal detected was tentatively assigned to a specific compound based on the results previously obtained by the same tool or by GC–MS analysis and of the available literature on the volatile compounds emitted from hop leaf products. Lastly, raw data acquisition and processing were conducted using TofDaq software (20th Tofwerk AG, Thun, Switzerland) and the methodology used is reported by Taiti et al. [
47].
4.8. NMR Analysis
For the untargeted NMR analysis, two types of extraction protocols were performed on the dried hop leaves: the Bligh–Dyer extraction and a methanol extraction.
The first one is a useful extraction method for the isolation of most polar metabolites, obtaining a clean extract free from traces of organic compounds. This extraction was performed according to previous literature articles [
48,
49,
50]. In brief, 0.100 g of the dried hop leaves were sequentially added with 0.8 mL of Millipore grade water and 3 mL of methanol/chloroform (2:1%
v/
v). The extraction was performed through a sonication step at room temperature for 10 min. Then, the extract was sequentially added with 1 mL of chloroform and 1 mL of Millipore grade water to obtain the biphasic system methanol/chloroform/water (2:2:1.8%
v/
v/
v). Finally, the upper hydroalcoholic phase was collected, and the residue pellet was extracted again. The obtained extract was dried under N
2 flow and prepared for the NMR analysis. The dried hydroalcoholic phases were solubilized in a solution of 20 mM phosphate buffer/D
2O containing 0.4 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSPA) and EDTA-d16, and 0.7 mL was transferred in a 5 mm NMR tube.
Regarding the methanolic extraction, the used protocol is an already established method for hops bitter acids and xanthohumol extraction [
30]. In detail, 0.100 g of dried leaves were extracted with 1 mL of MeOD with 10 min of sonication at room temperature. The TSPA internal standard was added to achieve a 1 mM concentration in each extract and directly analyzed by NMR.
Both the extraction methods were performed in triplicate and analyzed with a JNM-ECZ 600R (JEOL Ltd., Tokyo, Japan) spectrometer operating at the proton frequency of 600.17 MHz equipped with an autosampler.
The parameters used for the hydroalcoholic fraction analysis were the following: 90° pulse angle of 9.26 μs, 128 transients, 4 pre-scans, 65 K data points, and a presaturation pulse sequence to suppress the water signal. Regarding the methanol extract, a double suppression at 4.83 ppm and 3.28 ppm was performed. The other experimental parameters were a 90° pulse angle of 9.0 μs, 128 transients, 4 pre-scans, and 65 K data points.
2D NMR experiments, namely
1H-
1H TOCSY and
1H-
13C HMBC for the hydroalcoholic extract and
1H-
13C HSQC for the methanolic one were performed using previously reported experimental parameters [
51]. All the NMR spectra were processed using the JEOL Delta v5.3.1. software (JEOL Ltd., Tokyo, Japan). The 1H NMR spectra were Fourier transformed, manually phased, and automatically base corrected. Both types of spectra were referenced to the internal standard TSPA set at 0.00 ppm.
The metabolites identification was based on literature NMR data on other plant matrices and hop parts [
18,
52,
53], 2D experiments, and standard additions. Signals of the identified metabolites and the internal standard in the hydroalcoholic and methanolic extracts were manually integrated for all the samples. In order to realize an absolute quantification, the area of each signal was referred to as the area of the TSP methyl group, the internal standard with a known concentration of 0.4 mM, taking into account the number of protons (
Supplementary Material). The final quantitative results were expressed as mg/100 g of DW ± SD.
4.9. Statistical Analysis
Statistical analyses were performed with GraphPad Prism 6.1 software. The experiments were replicated three times unless otherwise indicated, and the data were expressed as the average ± standard deviation (SD).
5. Conclusions
Until recently, hop leaves, considered waste material, were less scientifically considered than the cones or the female inflorescences. In our study, with the aim of obtaining compositional information on the dried leaves of the Chinook cultivar, a metabolomic approach was carried out. The findings showed a considerable and diversified content of secondary metabolites, many of which are already known for their bioactive potential.
A deeper knowledge of the metabolome, in the context of a circular economy, is useful for understanding how to better exploit this matrix that has so far been underused in various sectors such as industrial, agricultural, or food. Therefore, this information is fundamental to hypothesize the use of the hop leaves as raw material and for contributing to the enhancement of the cultivation of the hop plants thanks to the multiplicity of uses of its products. In conclusion, this study highlights the usefulness of combining different analytical techniques to fully investigate the metabolomic profile of any useful part of the plant.