Vitro-derived hop (Humulus lupulus L.) leaves and roots as source of bioactive compounds: antioxidant activity and polyphenolic profile

Bioactive compounds extracted from plant are of great value for those enterprises interested in the use of natural products; plant tissue culture techniques guarantee a reliable and constant biomass production. Hop (Humulus lupulus L.), with its wealth in bioactive compounds, may represent an invaluable resource. The present study focused on the characterization of vitro-derived leaves and roots of two hop plant types, Cascade and Gianni. Extracts obtained from the selected hop explants were investigated, determining their polyphenolic content as their antioxidant capacity, applying DPPH, ABTS and FRAP assays; moreover, extract molecular profile was obtained through UHPLC-MS/MS. Results confirm the wealthy in bioactive compounds and the antioxidant properties of the tested vitro-derived hop explants. The qualitative characterization of vitro-derived hop tissue extracts evidenced the presence of twenty one different compounds, already identified in open field grown hop plants, such as polyphenols, α- and β-acids, as well as xanthohumol and isoxanthohumol. The obtained outcomes lay the groundwork to further investigate the potential of vitro-derived hop plantlets as bioactive compounds source. Characterizing and recovering secondary metabolites from micropropagated hop plantlets.


Introduction
Natural products, derived from an increasing range of different plants, are drawing the attention of an everyday wider portion of enterprises, interested in application of plantbased additives in food, pharmaceutical, and cosmetic sector. In this contest, hop (Humulus lupulus L.), with its wealth in bioactive compounds, is an invaluable candidate to be exploited (Astray et al. 2020;Kramer et al. 2015;Nionelli et al. 2018;Sanz et al. 2019).
Hop is cultivated all around the world, and, lately, also in Italy, for its cones, key ingredient for beer production, but also historically used in traditional and folk medicine against several diseases (Alonso-Esteban et al. 2019;Liu et al. 2015). Hop bioactive properties are due to the presence of numerous secondary metabolites, including terpenoids, phenolic compounds, alkaloids and bitter acids, related to antioxidant, antimicrobial and antiviral capacities (Alonso-Esteban et al. 2019;Hrnčič et al. 2019;Karabín et al. 2016;Krottenthaler 2009;Roehrer et al. 2019). In particular, the main secondary metabolites identified in hop pertain to different chemical classes as polyphenols (catechin, epicatechin, rutin, coumarin, gentisic acid, caffeic acid, ferulic acid, sinapic acid, quercetin, etc.), α-and β-bitter acids (humulone, cohumulone, lupulone, colupulone), and terpenoids (β-myrcene, caryophyllene, humulene, β-farnesene, α-and β-selinene) (Bocquet et al. 2018;Nezi et al. 2022) ( Table 1). All these compounds are mainly secreted by lupulin glands (Stevens et al. 1997), and accumulated at high concentrations in glandular trichomes of cones, but also detected in the vegetative biomass, such as leaves and stems (Gerhäuser 2005;Kavalier et al. 2011). As stated by Čeh et al. (2007), leaves, today considered a waste, may indeed represent a remarkable bioactive compound resource. On the other hand, the types and amount of bioactive compounds recovered by the vegetative hop biomass could be variable depending on the season, because plant secondary metabolism may be influenced by several factors, as environmental conditions (Abram et al. 2015;Green 1997). Furthermore, the accumulation of polyphenols, α-and β-acids, and xanthohumol in the different plant compartments can be cultivar dependent, as described by Čeh et al. in 2007. The application of in vitro culture techniques could bypass the differences in the secondary metabolite production caused by cultivation conditions. The United Nations for Food and Agriculture (FAO) recognised since 1994 in vitro plant tissue culture techniques as an important tool for the large-scale production of bioactive compounds (Anand 2010;Dal Toso and Melandri 2011;Dias et al. 2016;FAO 2002;Rao and Ravishankar 2002). It is, indeed, well acknowledged that vitro-derived plant organs synthetize the same bioactive compounds produced in corresponding in field grown plants (Scarpa et al. 2022), with the added value of a controlled growing environment that guarantees a more reliable and constant production; in addition, in vitro biomass production is continuous and independent of the season, important detail for deciduous species, like hop. Moreover, in vitro plant tissue culture provides the indispensable stress that triggers the synthesis of secondary metabolites, and specifically of polyphenols, in plants (Buchanan and Jones 2000).

Plant material
Explants destined to extraction were isolated from in vitro cultured hop plantlets of two plant types, the variety Cascade and the ecotype Gianni. Cascade is an aromatic variety selected in 1972, from an unknown American variety, Fuggle and Serebriankermthat, that has become one of the most cultivated all over the world, included several Italian regions (Santagostini et al. 2020); actually, this variety is characterized by a high yield and tolerance to several diseases, and used by the brewer industry for the floral and fruity aroma of its cones (Rodolfi et al. 2019;Santagostini et al. 2020). Gianni is a wild hop ecotype recovered in Emilia Romagna (Northern Italy), and grown in a collection field in Marano sul Panaro (Modena, Italy); over the last years, this ecotype has been phytochemically and morphologically characterized revealing its numerous positive technological attitudes, among which its dual purpose (Mongelli et al. 2015). Both plant types were in vitro established and cultured in 500 ml glass jars, containing the culture medium with the following composition: Murashige and Skoog (MS) basal salt mixture (1x) (Murashige and Skoog 1962), MS vitamin mixture (1x) (Murashige and Skoog 1962), 30 g/l of sucrose and 8.0 g/l of Agar. Culture medium, after adjusting the pH to 5.8 with 1N NaOH, was sterilized in autoclave for 20 min at 121 °C. Glass jars with Cascade and Gianni plantlets were then

Phenolic compounds
Chalcones: Xanthohumol, desmethylxanthohumol Flavanones: Isoxanthohumol, naringenin, ( ±)-8-prenylnaringenin sealed, and maintained in a growth chamber at 25 ± 1 °C and light intensity of 20 μmol m −2 s −1 , under 16 h photoperiod, until their use. For each plant type, about 100 plantlets were recovered from five glass jars (20 plantlets per each jar), in order to obtain a sufficient amount of vegetal material (0.5 -1 g form each analysis) to be used for the extraction step. In addition, the collected material can be considered representative of the secondary metabolite content of the plantlets.

Sample extraction
Plantlets were firstly recovered from the culture medium and their root apparatus was gently washed with distilled water; after that, leaves and roots were excised and treated separately. Specifically, vitro-derived leaves and roots recovered from the different jars were weighted, lyophilized by a Freeze dryer Lio-5P (5Pascal, Milan, Italy), pulverized, and extracted with ethanol/water solution (80/20) with a dilution factor of 1:20, resorting to two extraction procedures: the first was based on the use of a shaker (HS 501 digital shaker, IKA-Werke GmbH & Co, Staufen, Germany; 200 strokes/ minute for 2 h at room temperature), while for the second an ultrasound sonication bath (VWR International, Milan, Italy) was utilized, extracting samples for 30 min at 25 °C (Carbone et al. 2020;Martelli et al. 2020). The extracts were centrifuged at 5000 rpm for 10 min at room temperature (Centrifugette 4206 centrifuge, Alc International, Pévy, France) and the supernatants were diluted 1:5 with distilled water, for further analysis. Each extraction procedure was repeated twice. The moisture and the corresponding dry matter percentages of vitro-derived leaves and roots were calculated on the basis of lyophilisation procedure applied. All the results reported are expressed on the dry matter (DM).

Total phenolic content determination
The total phenolic content (TPC) was evaluated resorting to Folin-Ciocalteau's phenol reagent (Martelli et al. 2020), with some modifications. In brief, 250 µl of extract were mixed with 1 ml of an aqueous solution of Folin-Ciocalteau's phenol reagent (Sigma-Aldrich, St. Louis, MO, USA) (1/10 v/v), and 2 ml of an aqueous solution of sodium carbonate (20%, w/v), and kept in the dark for 30 min. The absorbance at 760 nm was measured using a spectrophotometer (JASCO V-530 spectrophotometer, Easton, MD, USA). To evaluate the quantity of polyphenols contained in the considered samples, a calibration curve was constructed, using gallic acid as reference in a concentration range of 10-100 mg/kg (5 points). All the analyses were repeated twice on each sample extract. In addition, in order to achieve more accurate results, the instrumental software was set up to perform three consecutive measurements on each analysed sample. The same approach was then applied for the other assays used to determine the antioxidant capacity of extracts. Concerning total polyphenolic content, results obtained for analysed samples were expressed as mg GAE/g DM.

Evaluation of antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay
The antioxidant capacity of leaves and roots extracts was determined applying the DPPH radical scavenging assays (Abram et al. 2015), with slight modifications. 100 µl of sample extract or standard solution were added of 2.9 ml of a DPPH (Sigma-Aldrich, St. Louis, MO, USA) ethanolic solution (0.05 mM), and kept in the dark for 30 min.
To evaluate the antioxidant capacity, 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich, St. Louis, MO, USA) was used as reference, preparing five different standard solutions (0.1-1 mM) that were utilized for the calibration curve construction. In addition, a blank constituted of 100 µl of extraction solution was also analysed under the same experimental conditions applied for samples. Antioxidant capacity was calculated on the basis of radical inhibition percentage (I%), as follows: I% = [(AbsB-AbsS)/AbsB]*100, were AbsB was the absorbance of the blank and AbsS was the absorbance of sample/Trolox standard solution. Results were then expressed as mg TEAC/ml (Trolox Equivalent Antioxidant Capacity). All the analyses were conducted in double, and three consecutive measurements were performed on each sample.

Determination of radical scavenging capacity by 2,2-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS) assay
To evaluate the radical scavenging activity of hop leaf and root extracts, a second test was conducted using as radical the ABTS (Wu et al. 2021). In particular, an aqueous ABTS (Sigma-Aldrich, St. Louis, MO, USA) solution (7 mM) containing K 2 O 8 S 2 (5 mM) was prepared and kept in the dark under stirring for 16 h. Then, the solution was diluted with bi-distilled water in order to obtain an ABTS +· radical working solution with an absorbance of 0.7 ± 0.2 at 734 nm (JASCO V-530 spectrophotometer, Easton, MD, USA). For sample analyses, 1.9 ml of ABTS working solution was added at 100 µl of sample extract or standard solution, and the reaction was conducted in the dark, at room temperature for 30 min. A calibration curve was prepared utilizing Trolox as reference, as described in the case of DPPH test. The absorbance values measured at 734 nm were used to calculate the I%, and to obtain the antioxidant activity expressed as mg TEAC/ml. All the analyses were repeated twice, and three consecutive measurements were performed on each sample.

Determination of ferric reducing power (FRAP)
The FRAP was determined as reported by Keskin et al. (2019). In brief, the working solution was prepared by mixing 25 ml of sodium acetate buffer solution (pH of 3.6 ± 0.1), 2.5 ml of 10 mM 2,4,6-Tris(2-pyridyl)-striazine (TPTZ) (Sigma-Aldrich, St. Louis, MO, USA), dissolved in 40 mM HCL, and 2.5 ml of an aqueous FeCl 3 ·6H 2 O (20 mM), and warming this mixture at 37 °C for 30 min. After that, 150 µl of leaf and root extract or Trolox standard solution were mixed with 1.85 ml of the working solution, and the resulting mixture was kept in the dark at room temperature for 30 min. The absorbance of all the samples was measured at 593 nm (JASCO V-530 spectrophotometer, Easton, MD, USA), and the ferric reducing power was obtained on the basis of a Trolox calibration curve, in the same concentration range considered for the previous tests, and results were expressed as mg TEAC/ ml. All the analyses were repeated twice, and three consecutive measurements were performed on each sample.

Qualitative characterization of extracts by UHPLC-MS/MS
Leaf and root extracts, fivefold diluted with 0.1% aqueous formic acid (Sigma-Aldrich, St. Louis, MO, USA), were used, also, to perform a full characterization. Analyses were performed through a UHPLC-MS/MS apparatus, composed of an UHPLC Ultimate 3000 separative module (Dionex, Sunnyvale, CA, USA), coupled with a TSQ Vantage triple quadrupole fitted with a heated-electrospray ionization (H-ESI-II) probe (Thermo Fisher, San Jose, CA, USA). The separation of compounds was performed using a RP-C 18 SunShell column (2.6 μm, 100 × 2.10 mm; ChromaNik Technologies, Osaka, Japan) maintained at 40 °C. The mobile phase consisted in water (eluent A) and acetonitrile (eluent B), both acidified with 0.2% of formic acid (solvents and reagent from VWR, Milan, Italy). The elution gradient started at 1% of B for 1 min, then B % increased gradually at 80% after 13 min and it was maintained for 7 min for column washing. After that, the initial conditions were restored in 1 min, with a total run time of 22 min. The flow rate was set at 0.35 ml/min, and the injected sample volume was 2 µl. For compound detection, MS experiments were performed in negative ionization mode. In detail, spray voltage was 3000 V, capillary temperature was set at 270 °C, while vaporizer temperature was 300 °C; nitrogen was used as both sheath and auxiliary gas, with a flow of 50 and 5 units, respectively. Sample analyses were initially carried out using a Full Scan method from m/z 100 to 1500, applying a scan time of 0.5 s. Then, target MS/MS analyses were carried out developing Product Ion Scan (PIS) experiments in order to obtain a better compound identification. Collision energy was set at 30 V and pure argon gas was used as collision gas. The identification of compounds listed in Table 4 was performed by comparing the MS/MS ion spectra with the fragmentation data available in several online libraries such as Mass Bank Europe, ReSpect, PubChem, and MoNA -Mass Bank of North America. Additional MS/MS information was obtained from previous works on hop-derived polyphenols, and other secondary metabolites (Česlová et al. 2009;Choi et al. 2018;Cirlini et al. 2016;Fernández-Poyatos et al. 2021;Helmja et al. 2007;McCallum et al. 2019;Prencipe et al. 2014;Xiang et al. 2021).

Statistical analysis
Data obtained from the chemical analyses of vitro-derived leaves and roots were subjected to three-way Analysis of Variance, considering the factors "Genotype", "Plant material" and "Extraction method"; mean separation has been carried out resorting to Tukey's test (p ≤ 0.05) (SYSTAT 13).
Moreover, Pearson correlation test was performed to evaluate the relationship between the different antioxidant activity tests and the total polyohenolic content (IBM SPSS Statistics 26.0 software, SPSS Inc., Chicago, IL).

Chemical characterization of vitro-derived root and leaf extracts from two hop genotypes, Cascade and Gianni
TPC and the antioxidant activity of all the different extracts are reported in Table 2. Leaves of both genotypes showed a total polyphenolic content (TPC) and antioxidant capacity higher than roots.
Statistical analysis evidenced that the only factor influencing the TPC parameter is, indeed, the "Plant Material" used to obtain the extract; in leaves a TPC significantly higher than roots was recorded (6.14 ± 2.26-8.00 ± 0.06 mg GAE/g SS vs. 3.92 ± 0.20-5.25 ± 0.44 mg GAE/g respectively), independently of the genotype or the extraction method applied.
Extracts obtained from vitro-derived leaves and roots of Gianni and Cascade, other than for TPC, were characterized in terms of antioxidant activity. To this aim, three different, Differently from what observed for the TPC, the statistical analysis evidenced that "Genotype" is the only factor influencing the antioxidant capacity of the extracts (Table 2). Specifically, extracts recovered from Cascade samples, independently of the plant material and extraction method considered, have an antioxidant activity significantly higher than those obtained for Gianni extracts (9.23 mg TEAC/ml vs. 6.20 mg TEAC/ml) ( Table 2).
Other than with DPPH assay, antioxidant activity of hop vitro-derived explants has been measured through ABTS assay; for this parameter, no significant differences were observed (p > 0.05; Table 2), nor on the basis of the genotype neither comparing the two starting plant materials used. These results probably depend on the composition of the antioxidant fraction, and on the different response that the compounds may have when subjected to reactions with different radicals.
In the case of results obtained for FRAP assay, a significant interaction between the factors "Plant Material" and "Genotype" was registered (Table 2); specifically, there is a significant difference, between Gianni and Cascade, in the antioxidant activity measured in roots. Indeed, antioxidant activity of extracts from Cascade roots is 1.5 fold higher than that of Gianni samples (14.11 mg TEAC/ml vs. 9.49 mg TEAC/ml).
The statistical analysis did not evidence significant influence exerted by the extraction method for any of the considered parameters; even though, for further analysis, the method based on the use of shaker was selected, because, observing the standard deviation, this method guarantees a better reproducibility (Table 2).
Correlation between phenolic concentration and antioxidant capacity was investigated by Pearson correlation test (Table 3) that evidenced, for Gianni, a significant correlation (r = 0,867), between the results of Folin-Ciocalteau test and those of FRAP assay, suggesting that the reducing activity is mainly due to phenolic compounds. Conversely, for Cascade, a significant correlation (r = 0,914) was registered between DPPH and FRAP assays, meaning that the antioxidant activity is exerted by the same compounds. Considering that the small molecules, acceding easier to the radical site of DPPH, are the most responsible of antioxidant activity, it is very likely that the reducing power, measured with FRAP assay, is due to the low molecular weight polyphenols.

Characterization of extracts from vitro-derived leaves and root of Cascade and Gianni, through UHPLC-MS/MS
Extracts from vitro-derived leaves and roots of Cascade and Gianni, after TPC and the antioxidant activity measurements, were further investigated on their qualitative profile, through UHPLC-MS/MS technique (Table 4).
The obtained profiles allowed the detection and the identification of 21 compounds in the extracts. Out of 21, only dihydroxybenzoic acid-O-hexoside and catechin were detected in all the analysed samples. Although Cascade extracts resulted richer in terms of number of compounds detected, the molecular profiles of the two analysed genotypes were almost similar. In addition, leaf extracts presented a more complex profile than that obtained from roots, where only few of the 21 identified compounds were present.

Discussion
The use of plant biomass as bioactive compound source is biased by the susceptibility of secondary metabolism to the growing plant conditions. Vitro-derived plantlets, growing in a controlled environment and in standardized conditions, represent an invaluable matrix for a constant and continuous bioactive compound production. In this study, to evaluate the total polyphenolic content and the antioxidant activity of extracts obtained from vitro-derived hop plantlets, and to set up a valid extraction protocol, two hop genotypes, two types of starting material and two extraction procedures were investigated. Results regarding the TPC parameter evidenced that vitro-derived hop leaves are richer in polyphenolic compounds than roots. Due to the lack of literature on chemical characterization of vitro-derived hop plantlets, results reported in this study were compared with those described for cones, leaves, and roots collected from in field grown plants of hop and/or of related species. As an example, Choi et al. (2018) described the highest polyphenol concentration obtained in leaves, rather than in roots or stems of Korean Humulus japonicus. TPC content could be influenced by several factors, among which starting material, extraction method, solvent treatments, and genotype (Abram et al. 2015). Vitro-derived plantlets used in the present study had a lower TPC than cones of Magnum and Marynka (Kovwalczyk et al. 2013), but higher than leaves collected from field, as reported by Proestos et al. (2006). TPC recorded in this study seems to be independent of the adopted extraction procedure; in literature, TPC lower than that of vitro-derived leaves was obtained resorting to the same extraction solvent (70% ethanol) (Muzykiewicz et al. 2019), while, when methanol was used, results comparable with those reported in the current study were obtained (Keskin et al. 2019). Another important factor influencing the TPC content is the genotype of starting material (Abram et al. 2015;Čeh et al. 2007), differently, the two genotypes analysed in this study presented comparable TPC content in their leaves. The information given by the parameter TPC was completed carrying assays aimed at determining the extracts antioxidant activity. Results obtained with the DPPH assay revealed that extracts from Cascade evidenced an antioxidant capacity higher than those from Gianni. Moreover, DPPH antioxidant activity described in this study is higher than values reported on extracts obtained from open field hop leaves (Abram et al. 2015;Muzykiewicz et al. 2019). Finally, DPPH values reported in this study are comparable, sometimes lower (Thiruvengadam et al. 2015), sometimes higher (Amoo et al. 2012) than those reported in other vitro-derived matrices. So, the results on antioxidant activity support the thesis of possible use of vitro-derived hop material as antioxidant source, after a genotype characterization. Different assays may give different responses, even considering the same starting material and extraction procedure; this is what was observed evaluating the results of ABTS assay that did not evidence any difference for any factor analysed, in contrast with information reported by Choi et al. (2018) who evidenced a radical scavenging capacity higher in hop leaves than in roots. These results probably depend on the composition of the polyphenolic fraction, and on the different response that these compounds may have when subjected to reactions with different radicals. Regarding FRAP assay, the two genotypes responded differently. Cascade root extract antioxidant activity was two-fold higher than that of Gianni; similar trend was reported for cultivars Aurora and H. Magnum, for which a differential genotype response was obtained according to the starting material considered, leaves or cones (Abram et al. 2015). Furthermore, in this study, the factor "Starting material" alone did not seem to exert the same impact, while Choi et al. (2018) report that leaves of H. japonicus showed a reducing power, measured by FRAP method, higher than that of roots and stems.
Regarding the molecular profile obtained by UHPLC-MS/ MS technique, the identified compounds (Table 4) belong to several classes, xanthohumol (prenylated chalcone), isoxanthohumol (prenylated flavanone), kaempferol-3-rutinoside and rutin (glycosylated flavonols), catechin (flavan-3-ols) and phenolic acids, derived both from benzoic acid and cinnamic acid. Of particular interest is the presence of phloroglucinol derivatives, a class of compounds that particularly characterize the hop plant. In detail, α-acids, such as humulone/adhumulone and co-humulone, and β-acids, such as lupulone, colupulone and post-lupulone were the most characteristic components, especially in Cascade leaf extracts. These compounds, typically present in hop cones (Česlová et al. 2009;Helmja et al. 2007;Magalhães et al. 2010;Santagostini et al. 2020) gained valuable interest because of their bioactive properties. Lupulones and humulones are known, indeed, for their antibacterial and antifungal activity, being inhibitor of diacylglycerol acetyltransferase, limiting lipid metabolism, and as a consequence, affecting the composition of cell wall and microorganism membrane (Bocquet et al. 2018), while α-acids and β-acids, and xanthohumol resulted active components against Listeria monocytogenes and Staphylococcus aureus (Kramer et al. 2015).
Other than the most characteristic hop compounds, also flavan-3-ol and flavonol glycosides, as catechin, rutin and kaempferol-O-rutinoside, have been identified in hop cones by Kavalier et al. (2011), and rutin have been detected in strobiles by Magalhães et al. (2010).
Among all identified polyphenolics, few molecules have already been characterized in hop tissues different from cones, such as leaves, roots and stems, as caffeolquinic acids, coumaroylquinic acids, and catechin. These compounds have been, previously, reported in the LC-MS/MS profile obtained from hop tissue extracts, prepared with ethyl acetate (Choi et al. 2018). Differences between the results obtained in the present study and those described by literature could depend on the different investigated plant material (in vitro versus in field plants), as from other parameters, as the solvent type used for the extraction. It was demonstrated that using different solvent mixtures, the profile of secondary metabolites changes, both in number of recovered components as in terms of compound concentration. Helmja et al., in 2007, investigated, by HPLC-MS/MS technique, the composition of hop strobilus extracts, obtaining using different solvent mixtures (methanol, water, acetone, etc.), and different extraction techniques, that polyphenolic and bitter acid profile depended on the method used to recover these metabolites. Differences can be pointed out also considering diverse plant materials and varieties. The present study highlighted, indeed, different profiles for leaves and roots, as between the two plant types, showing that Cascade deriving extracts presented a larger pattern of compounds of potential interest (Table 4). The wider range of polyphenols was indeed observed in Cascade leaves extracts: coumaroylquinic acids, 3-caffeoylquinic acid, and kaempferol-O-rutinoside I and II were not detected in Gianni extracts, while profiles obtained for roots resulted, in general, scarcer. Similar observations can be made regarding isoxanthohumol, and β-acids, as lupulone and colupulone, found in Cascade rather than in Gianni leaves, as more in general for all the α-and β-acids identified, contained prevalently in 1 3 leaves. The presence and concentration of xantohumol and α-acids is, actually, related to hop variety, as demonstrated by Mongelli et al., in 2015. The information about secondary metabolite composition could be useful in the choice of the more promising plant types suitable to give extracts abundant in bioactive components.

Conclusions
In the recent years, great interest has aroused about the potential use of bioactive compounds of natural origin, both from food and non-food enterprises. Other than from in field grown plants, it has been demonstrated that these compounds can be extracted from vitro-derived plant material, with the enormous advantage of guaranteeing a continuous and standardized biomass production. Hop (Humulus lupulus L.) plant, in all its parts, is a precious source of bioactive compounds, but its in vitro grown plant material has never been investigated; with this aim, the present study has been carried out to characterize leaves and roots of two hop plant types, Cascade and Gianni, cultivated in vitro.
In conclusion, between the two genotypes considered, explants from the variety Cascade seem to be the most promising, both in terms of total phenolic content and polyphenol profile, but the ecotype Gianni could be considered as interesting as other commercial hop varieties. Extracts obtained from both plant types turned out to contain hop characteristic compounds, such as α-acids and β-acids, as well as xanthohumol, that have been related to antioxidant, anti-inflammatory, estrogenic, sedative and antimicrobial activities. These results confirm the valuable potential of vitro-derived plant material as valid source of bioactive compounds, to be exploit in several industrial sectors, in a constant and sustainable manner, independently of the environment conditions and the season. To sum up, this study led new insights for further research, aimed at increasing and diversifying bioactive compound synthesis, as well as, at widening the number of hop genotypes chemically characterised.
Funding Open access funding provided by Università degli Studi di Parma within the CRUI-CARE Agreement. This research was supported by the project: "HOPLURE-Hop Leaves, an Unexplored REsource" (D91B21005370003), funded by University of Parma, through the action Bando di Ateneo 2021 per la ricercar, and cofunded by MUR-Italian Ministry of Universities and Research-D.M. 737/2021-PNR-PNRR -NextGenerationEU.
Data availability All data generated or analysed during this study are included in this published article.

Conflict of interest
The authors declare that they have no conflicts of interest.
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