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 unvaluable 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 a richer source than roots; these considerations agree with what reported by Choi et al. (2018) who analysed extracts of Korean Humulus japonicus hop leaves, roots and stems, in terms of polyphenol and antioxidant content, indicating, indeed, that leaves had the highest polyphenol concentration if compared with other studied plant materials. On the other hand, it must be underlined that, nowadays, literature is poor of information about the TPC of vitro-derived hop leaves or roots, so it is difficult to have a valid comparison with similar starting material, and for this reason results reported in this study must be compared with those reported for cones and/or leaves and roots collected from in field grown plants. Scouring the literature on this subject, it seems clear that TPC content could be influenced by several factors, among which starting material, extraction method, solvent treatments, and genotype (Abram et al. 2015). For example, 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 in field, as reported by Proestos et al. (2006). TPC recorded in this study seems to be independent of the extraction procedure adopted; 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 (Ceh et al. 2007; Abram et al. 2015), 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, considering the results reported on extracts from open field hop leaves, since, on the best of our knowledge, this is the first study characterizing vitro-derived hop explants, it is possible to point out that the DPPH antioxidant activity reported in this study is higher than previous (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, also the results on antioxidant activity support the thesis of possible use of vitro-derived hop material as antioxidant source, better if 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 on ABTS assay that did not evidence any difference for any factor analysed, results contrast with what 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, indeed, 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.
The statistical analysis did not evidence, for any of the parameters considered, significant influence exerted by the extraction method; even though, for further analysis, the shaker was the method selected, because, observing the standard deviation, this method guarantees a better reproducibility (Table 1).
Regarding the polyphenol profile obtained by UHPLC-MS/MS technique, the identified compounds (Table 3) 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 plant of hop. 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 leave 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).
Despite of the most hop characteristic 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.
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 interested as other commercial hop varieties. The extracts obtained from both genotypes turned out to be rich in important and characteristic compounds, such as α-acids and β-acids, as well as xanthohumol, with antioxidant, anti-inflammatory, estrogenic, sedative and antimicrobic activities. These results confirm the valuable potential of in vitro culture techniques as valid tool for bioactive compound synthesis, 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.