Unveiling the phenology and associated floral regulatory pathways of Humulus lupulus L. in subtropical conditions.

MAIN CONCLUSION: The hop phenological cycle was described in subtropical condition of Brazil showing that flowering can happen at any time of year and this was related to developmental molecular pathways. Hops are traditionally produced in temperate regions, as it was believed that vernalization was necessary for flowering. Nevertheless, recent studies have revealed the potential for hops to flower in tropical and subtropical climates. In this work, we observed that hops in the subtropical climate of Minas Gerais, Brazil grow and flower multiple times throughout the year, independently of the season, contrasting with what happens in temperate regions. This could be due to the photoperiod consistently being inductive, with daylight hours below the described threshold (16.5 h critical). We observed that when the plants reached 7-9 nodes, the leaves began to transition from heart-shaped to trilobed-shaped, which could be indicative of the juvenile to adult transition. This could be related to the fact that the 5th node (in plants with 10 nodes) had the highest expression of miR156, while two miR172s increased in the 20th node (in plants with 25 nodes). Hop flowers appeared later, in the 25th or 28th nodes, and the expression of HlFT3 and HlFT5 was upregulated in plants between 15 and 20 nodes, while the expression of HlTFL3 was upregulated in plants with 20 nodes. These results indicate the role of axillary meristem age in regulating this process and suggest that the florigenic signal should be maintained until the hop plants bloom. In addition, it is possible that the expression of TFL is not sufficient to inhibit flowering in these conditions and promote branching. These findings suggest that the reproductive transition in hop under inductive photoperiodic conditions could occur in plants between 15 and 20 nodes. Our study sheds light on the intricate molecular mechanisms underlying hop floral development, paving the way for potential advancements in hop production on a global scale.

Antioxidant Activity of Humulus lupulus Phenolic Hop Extracts in Creating a New Pâté: An Element Affecting Fat Stability and Microbiological Quality during Storage.

The aim of this study was to evaluate the effect of hop extracts on changes in the primary and secondary fat oxidation products, physicochemical properties, and microbiological quality of pâté-type offal sausages obtained through the partial replacement of animal fat with vegetable fat. This study demonstrated that the extraction efficiency varied among hop cone varieties, with the highest efficiency observed for the Lubelski variety and the lowest for the Magnum variety. The phenolic compound content was higher in the Magnum cones (2.74 ± 0.11 mg/g dry matter) compared to the Lubelska cones (2.27 ± 0.05 mg/g of product). Additionally, the DPPH radical scavenging activity was greater in the extract from the Magnum cones (4.21 ± 0.09 mg TE/g d.w.) than in the extract from the Lublelski cones (3.87 ± 0.05 mg TE/ g d.w.). Similarly, the extracts from the Lubelski cones exhibited a higher antiradical activity against the ABTS radical compared to the extract from Magnum cones. Throughout storage, a significant increase in the pH value was observed in the control sample and in the samples with a 20% replacement of animal fat with rapeseed oil and Magnum hop extract. However, the addition of Lubelski hop extract resulted in a decrease in the pH value during the 15-day storage period. The samples with a 20% replacement of animal fat with rapeseed oil and 0.1% Lubelski hop extract showed the least changes in water activity during storage. The samples with a 20% replacement of animal fat with rapeseed oil and the addition of 0.2% Lubelski hop extract had the lowest peroxide value and TBARS index throughout the storage period. The addition of hop extract inhibited the growth of the total number of microorganisms in the tested sausages. In the samples with a 20% replacement of animal fat with rapeseed oil, the content of aerobic microorganisms, compared to the control sample, was statistically significantly lower.

Protective Effect of Hop Ethyl Acetate Extract on Corticosterone-Induced PC12 and Improvement of Depression-like Behavior in Mice.

Depression is a common mental disorder. In recent years, more and more attention has been paid to depression and its etiology and pathogenesis. This review aims to explore the neuroprotective and antidepressant effects of hop components. By establishing an in vitro cell damage model using PC12 cells induced by corticosterone (CORT) and an in vivo depression model through the intracranial injection of lipopolysaccharide (LPS) in mice, hop ethyl acetate extract (HEA) was used to study the protective effect and mechanism of HEA on neuronal cells in vitro and the antidepression effect and mechanism in vivo. The results showed that HEA increased the survival and decreased the rate of lactate dehydrogenase (LDH) release, apoptosis, and the ROS and NO content of CORT-induced PC12 cells. HEA alleviated depressive-like behavior, neuroinflammation, reduction of norepinephrine, and dendritic spines induced by intracerebroventricular injection of LPS in mice and increases the expression levels of BDNF, SNAP 25, and TrkB proteins without any significant side effects or toxicity. Hops demonstrated significant comprehensive utilization value, and this work provided an experimental basis for the role of hops in the treatment of depression and provided a basis for the development of HEA for antidepressant drugs or dietary therapy products.

Xanthohumol-A Miracle Molecule with Biological Activities: A Review of Biodegradable Polymeric Carriers and Naturally Derived Compounds for Its Delivery.

Xanthohumol (Xn), a prenylated chalcone found in Hop (Humulus lupulus L.), has been shown to have potent anti-aging, diabetes, inflammation, microbial infection, and cancer properties. Unfortunately, this molecule has undesirable characteristics such as inadequate intake, low aqueous solubility, and a short half-life. To address these drawbacks, researchers have made numerous attempts to improve its absorption, solubility, and bioavailability. Polymeric drug delivery systems (PDDSs) have experienced significant development over the last two decades. Polymeric drug delivery is defined as a formulation or device that allows the introduction of a therapeutic substance into the body. Biodegradable and bioreducible polymers are the ideal choice for a variety of new DDSs. Xn formulations based on biodegradable polymers and naturally derived compounds could solve some of the major drawbacks of Xn-based drug delivery. In this regard, the primary concern of this study is on presenting innovative formulations for Xn delivery, such as nanoparticles (NPs), nanomicelles, nanoliposomes, solid lipid nanoparticles (SLNs), and others, as well as the received in vitro and in vivo data. Furthermore, this work describes the chemistry and broad biological activity of Xn, which is particularly useful in modern drug technology as well as the cosmetics industry. It is also important to point out that the safety of using Xn, and its biotransformation, pharmacokinetics, and clinical applications, have been thoroughly explained in this review.

More than just a beer - Brewers' spent grain, spent hops, and spent yeast as potential functional fillers for polymer composites.

Beer is among the most popular beverages in the world, with the production distributed uniformly between the biggest continents, so the utilization of brewing by-products is essential on a global scale. Among their potential recipients, the plastics industry offers extensive range of potential products. Herein, the presented study investigated the application of currently underutilized solid brewing by-products (brewers' spent grain, spent hops, spent yeast) as fillers for highly-filled poly(ε-caprolactone)-based composites, providing the first direct connection between spent hops or spent yeast and the polymer composites. Comprehensive by-product characterization revealed differences in chemical composition. The elemental C:O ratio, protein content, and Trolox equivalent antioxidant capacity varied from 1.40 to 1.89, 12.9 to 32.4 wt%, and 2.41 to 10.24 mg/g, respectively, which was mirrored in the composites' structure and performance. Morphological analysis pointed to the composition-driven hydrophilicity gap limiting interfacial adhesion for high shares of brewers' spent grain and spent hops, due to high hydrophilicity induced by carbohydrate content. Phytochemicals and other components of applied by-products stimulated composites' oxidative resistance, shifting oxidation onset temperature from 261 °C for matrix over 360 °C for high spent yeast shares. Simultaneously, spent yeast also provided compatibilizing effects for poly(ε-caprolactone)-based composites, reducing complex viscosity compared to other fillers and indicating its highest affinity to poly(ε-caprolactone)due to the lowest hydrophilicity gap. The presented results indicate that the proper selection of brewing by-products and adjustment of their shares creates an exciting possibility of engineering composites' structure and performance, which can be transferred to other polymers differing with hydrophilicity.

Exploring the composition and potential uses of four hops varieties through different extraction techniques.

Hydrophilic, lipophilic extracts and essential oil of four hops varieties from Slovenia were examined in this study. Lipophilic extracts were obtained by supercritical extraction (SFE), while for hydrophilic extracts ultrasound and microwave extraction were employed. Essential oils were isolated by the hydrodistillation process. The lipophilic composition of essential oils and lipophilic extracts was determined by GC-MS analysis. Monoterpenes and sesquiterpene hydrocarbons were the most abundant class of compounds in oils (62.27-79.65 %), with myrcene being the most abundant constituent. Limonene and trans-caryophyllene were two terpenes determined in all essential oils while only trans-caryophyllene was detected in SFE samples. Antioxidant, antimicrobial, and cytotoxic activity, determined by applying in vitro assays, was more influenced by extraction technique than by varieties. Molecular docking was carried out to gain insight into the potential cancer protein targets BCL-2 and MMP9, whereby humulene epoxide II displayed good binding configuration within the cavities of the two proteins.

α acid fraction from Hop extract exerts an endothelium-derived hyperpolarization vasorelaxant effect through TRPV4 employing the feedforward mechanism of PKCα.

Until now, the beneficial vascular properties of Hop reported in the literature have been mainly attributed to specific compound classes, such as tannins and phenolic acids. However, the potential vascular action of a Hop subfraction containing a high amount of α or ß acids remains completely understood. Therefore, this study aims to investigate the vascular effects of the entire Hop extract and to fraction the Hop extract to identify the main bioactive vascular compounds. A pressure myograph was used to perform vascular reactivity studies on mouse resistance arteries. Phytocomplex fractionation was performed on a semi-prep HPLC system and characterized by UHPLC-PDA-MS/MS coupled to mass spectrometry. Western blot analysis was performed to characterize the phosphorylation site enrolled. The entire Hop extract exerts a direct dose-dependent endothelial vascular action. The B1 subfraction, containing a high concentration of α acids, recapitulates the vascular effect of the crude extract. Its vasorelaxant action is mediated by the opening of Transient Receptor Potential Vanilloid type 4 (TRPV4), potentiated by PKCα, and subsequent involvement of endothelial small-conductance calcium-activated potassium channels (SKCa) and intermediate-conductance calcium-activated potassium channels (IKCa) that drives endothelium-dependent hyperpolarization (EDH) through heterocellular myoendothelial gap junctions (MEGJs). This is the first comprehensive investigation of the vascular function of Hop-derived α acids in resistance arteries. Overall, our data suggest that the B1 subfraction from Hop extracts, containing only α acids, has great potential to be translated into the useful armamentarium of natural bioactive compounds with cardiovascular benefits.

Quantification of Hop-Derived Bitter Compounds in Beer Using Liquid Chromatography Mass Spectrometry.

Hops (Humulus lupulus L.) are essential raw materials for beer brewing, and the major contributors to beer bitterness are isohumulones (iso-α-acids) and humulinones. In recent years, many breweries have focused on the production of hop-forward beer styles by adding hops after or during the cold fermentation stage, which will tend to release humulinones or other hop-derived bitter compounds. In this study, a LC-MS/MS method was developed for quantification of 60 hop-derived bitter compounds in 25 min. Reverse-phase chromatography with an alkaline methanol/acetonitrile (70:30) mobile phase was used for the separation. The quantitative range was 0.053-3912 ng/mL with correlation coefficient r > 0.99, and the LOQ were 0.26 and 0.053 ng/mL for iso-α-acids and humulinones. Precision (RSD < 5.0%) and accuracy (recovery 86.3%-118.1%) were both satisfactory. The abundance of hop-derived bitter compounds in the dry-hopped beer (Double-India Pale Ale) and the nondry-hopped beer (Vienna Lager) were monitored throughout the fermentation and storage stages, and the formation of oxidation and cyclization products showed difference profiles between these two beers. The quantification results reveal how hop-derived bitter compounds change throughout the brewing process, as well as the influence of hops and brewing techniques on beer bitterness.

De novo biosynthesis of the hops bioactive flavonoid xanthohumol in yeast.

The flavonoid xanthohumol is an important flavor substance in the brewing industry that has a wide variety of bioactivities. However, its unstable structure results in its low content in beer. Microbial biosynthesis is considered a sustainable and economically viable alternative. Here, we harness the yeast Saccharomyces cerevisiae for the de novo biosynthesis of xanthohumol from glucose by balancing the three parallel biosynthetic pathways, prenyltransferase engineering, enhancing precursor supply, constructing enzyme fusion, and peroxisomal engineering. These strategies improve the production of the key xanthohumol precursor demethylxanthohumol (DMX) by 83-fold and achieve the de novo biosynthesis of xanthohumol in yeast. We also reveal that prenylation is the key limiting step in DMX biosynthesis and develop tailored metabolic regulation strategies to enhance the DMAPP availability and prenylation efficiency. Our work provides feasible approaches for systematically engineering yeast cell factories for the de novo biosynthesis of complex natural products.

Xanthohumol, a prenylated flavonoid from hops (Humulus lupulus L.) exerts multidirectional pro-healing properties towards damaged zebrafish hair cells by regulating the innate immune response.

Xanthohumol (XN) is a prominent prenylated flavonoid present in the hop plant (Humulus lupulus L.). Despite undoubted pro-healing properties of hop plant, there is still a need for clinical investigations confirming these effects as well as the underlying molecular mechanisms. The present study was designed to (1) establish the role of XN in non-invasive inflammation induced by chemical damage to zebrafish hair cells, (2) clarify if it influences cell injury severity, neutrophil migration, macrophage activation, cell regeneration, and (3) find out whether it modulates the gene expression profile of chosen immune and stress response markers. All experiments were performed on 3 dpf zebrafish larvae. After fertilization the embryos were transferred to appropriate XN solutions (0.1 µM, 0.3 µM and 0.5 µM). The 40 min 10 µM CuSO4 exposure evoked severe damage to posterior lateral line hair cells triggering a robust acute inflammatory response. Four readouts were selected as the indicators of XN role in the process of inflammation: 1) hair cell death, 2) neutrophil migration towards damaged hair cells, 3) macrophage activation and recruitment to damaged hair cells, 4) hair cell regeneration. The assessments involved in vivo confocal microscopy imaging and qPCR based molecular analysis. It was demonstrated that XN (1) influences death pathway of damaged hair cells by redirecting their severe necrotic phenotype into apoptotic one, (2) impacts the immune response via regulating neutrophil migration, macrophage recruitment and activation (3) modulates gene expression of immune system markers and (4) accelerates hair cell regeneration.

Compounds derived from Humulus lupulus inhibit SARS-CoV-2 papain-like protease and virus replication.

BACKGROUND: Selected natural compounds exhibit very good antiviral properties. Especially, the medicinal plant Humulus lupulus (hop) contains several secondary plant metabolites some of which have previously shown antiviral activities. Among them, the prenylated chalcone xanthohumol (XN) demonstrated to be a potent inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). HYPOTHESIS/PURPOSE: Following the finding that xanthohumol (XN) is a potent inhibitor of SARS-CoV-2 Mpro, the effect of XN and its major derivatives isoxanthohumol (IXN), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN) from hops on SARS-CoV-2 papain-like protease (PLpro) were investigated. STUDY DESIGN: The modulatory effect of the hop compounds on PLpro were studied first in silico and then in vitro. In addition, the actual effect of hop compounds on the replication of SARS-CoV-2 in host cells was investigated. METHODS: In silico docking analysis was used to predict the binding affinity of hop compounds to the active site of PLpro. A recombinant PLpro was cloned, purified, characterized, and analyzed by small-angle X-ray scattering (SAXS), deISGylation assays, and kinetic analyses. Antiviral activity of hop compounds was assessed using the fluorescently labeled wildtype SARS-CoV-2 (icSARS-CoV-2-mNG) in Caco-2 host cells. RESULTS: Our in silico docking suggests that the purified hop compounds bind to the active site of SARS-CoV-2 PLpro blocking the access of its natural substrates. The hop-derived compounds inhibit SARS-CoV-2 PLpro with half maximal inhibitory concentration (IC50) values in the range of 59-162 µM. Furthermore, we demonstrate that XN and 6-PN, in particular, impede viral replication with IC50 values of 3.3 µM and 7.3 µM, respectively. CONCLUSION: In addition to the already known inhibition of Mpro by XN, our results show, for the first time, that hop-derived compounds target also SARS-CoV-2 PLpro which is a promising therapeutic target as it contributes to both viral replication and modulation of the immune system. These findings support the possibility to develop new hop-derived antiviral drugs targeting human coronaviruses.

Structure and genetic diversity of Canadian Maritimes wild hops.

Studies on the northeastern American native hops (Humulus lupulus ssp. lupuloides) from the Canadian Maritimes are scarce. This study aimed to evaluate the genetic structure and diversity among 25 wild-collected hops from three Canadian Maritime provinces using microsatellite (simple sequence repeat (SSR)) markers. Based on 43 SSR markers, four distinct subgroups were found, with a low molecular variance (19%) between subgroups and a high variance (81%) within subgroups. The Nei's unbiased genetic distance between clusters ranged from 0.01 to 0.08, the genetic distance between clusters 2 and 3 being the farthest and that between clusters 1 and 2 the closest. Cluster 2 captured the highest overall diversity. A total of 18 SSR markers clearly discriminated hop clones by detecting putative subspecies-specific haplotypes, differentiating clones of native-wild H. lupulus ssp. lupuloides from the naturalized old and modern hop cultivars. Seven of the 18 SSR markers also differentiated two clones from the same site from one another. The study is the first, using molecular markers, to identify SSR markers with potential for intellectual property protection in Canadian Maritimes hops. The SSR markers herein used can be prime tools for hop breeders and growers in the region.

Insight into the aroma quality of 'Callista' cultivar of hop (Humulus lupulus L.): Impact of harvest timing, year, and location.

Hops (Humulus lupulus L.) are essential ingredients in brewing, contributing to beer's flavor, aroma, and stability. This study pioneers an in-depth analysis of the 'Callista' cultivar, aiming to unravel how harvest timing, annual variations, and cultivation location synergistically influence its molecular profile, sensory perception, and biochemistry. Leveraging high-performance liquid chromatography and gas chromatography-mass spectrometry-olfactometry, we identified significant year-to-year and location-based fluctuations in bitter acids-the quintessential aroma constituents in hops. Our comprehensive aroma profiling discerned 55 volatile compounds, marking the first-ever sensory detection of 2-butanone in hops, with its presence showing remarkable interannual variability. This study showed significant differences among the three years tested, whereas hops were perceived "fruitier" and more "citrusy" in 2021, even though the bitter acid and aroma analysis showed that 2022 sticks out due to extremely high lupulone values up to 10% dry cone weight and 78% ß-myrcene in the oil fraction compared to 60% and 45% in 2020 and 2021, respectively. Molecular analysis of key enzymes involved in hop aroma biosynthesis revealed no significant associations with location, but a strong diurnal pattern for all genes. The results indicated that especially the hot temperatures of 2022 may have induced significant changes of cone quality, while 2021 was more interesting from the sensory evaluations, which may justify the usage of viticultural terms such as "vintage" for hop marketing. These findings contribute to a better understanding of the factors influencing hop aroma and quality.

Safety Assessment of Hops as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of Humulus Lupulus (Hops) Extract (reported functions include antimicrobial agent and hair conditioning agent) and Humulus Lupulus (Hops) Oil (reported function is fragrance). The Panel reviewed the relevant data related to these ingredients. Because final product formulations may contain multiple botanicals, each containing the same constituents of concern, formulators are advised to be aware of these constituents and to avoid reaching levels that may be hazardous to consumers. For these ingredients, the Panel was concerned about the presence of 8-prenylnaringenin, ß-myrcene, and quercetin in cosmetics, which could result in estrogenic effects, dermal irritation, and genotoxicity, respectively. Industry should use current good manufacturing practices to limit impurities and constituents of concern. The Panel concluded that Humulus Lupulus (Hops) Extract and Humulus Lupulus (Hops) Oil are safe in cosmetics in the present practices of use and concentration when formulated to be non-sensitizing.

Spent hops extract (Humulus Lupulus L.) attenuates inflammation and angiogenesis of the retina via the nuclear factor-kappaB and protein kinase B/extracellular signal-regulated kinase pathways.

Spent hops extract (SHE) is a plant extract containing compounds with proven anti-inflammatory and anti-angiogenic activities. However, extract may exert synergic effects compared to its individual polyphenol components. Inflammatory diseases of the retina may lead to visual impairment, a reduction of the comfort of life, and even blindness due to the formation of new pathological blood vessels. More effective therapeutic options are being sought. The goal of the present study was to investigate the anti-inflammatory and anti-angiogenic potentials of SHE on human retinal pigment epithelial cells (ARPE-19) stimulated by lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The SHE (250 µg/mL) was found to downregulate the gene expression of interleukin 6 (IL-6) to 33% in LPS-triggered cells; it also reduced both matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) mRNA expression to 13% and 43% respectively, and their activity to 82% (MMP-2) and 57% (MMP-9), compared to TNF-α-stimulated cells. Also, SHE modulated the TNF-α-induced expression of vascular endothelial growth factor (VEGF) and endothelial growth factor receptor 2 (VEGFR2). It is possible that SHE inhibited retinal inflammation and angiogenesis by suppressing the nuclear factor kappa B (NF-κB), protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways. Our results demonstrate that SHE has anti-inflammatory and anti-angiogenic potential against retinal diseases. This is the first such study to report on the efficacy of SHE on retinal inflammatory diseases.

Interesting Behavior of Geranic Acid during the Beer Brewing Process: Why Could Geranic Acid Remain at a Higher Level Only in the Beer Using Sorachi Ace Hops?

Hops are among the most important ingredients in beer that contribute to beer flavor. Consequently, novel types of hops have been bred and widely used worldwide. For example, the Sorachi Ace hop imparts characteristic varietal aromas, including woody, pine-like, citrus, dill-like, and lemongrass-like aromas, to the finished beer. In our previous study, the unique volatile compound geranic acid was significantly detected only in the test beer brewed with the Sorachi Ace hop; moreover, the coexistence of geranic acid and other hop-derived flavor compounds could result in the characteristic aroma of the Sorachi Ace beers. In this study, selected hop-derived flavor compounds, including geranic acid, were compared among 17 hop varieties. The geranic acid content in the Sorachi Ace hop was the highest among the studied hops. We also investigated the behavior of geranic acid and related flavor compounds throughout the fermentation process. The content of geranic acid was higher than those of the other compounds during fermentation. Next, we compared the concentrations of these compounds in kettle-, late-, and dry-hopped beers using Sorachi Ace hop. The results revealed that geranic acid remained at higher concentrations from the worts to finished beers despite the decrease in the content of other hop-derived flavor compounds as a result of evaporation and/or other factors during brewing. Further, geranic acid could remain at high levels in the test-brewed beers with Sorachi Ace hops because of its behavior as an acid throughout the brewing process, including during wort boiling and fermentation.

Climate-induced decline in the quality and quantity of European hops calls for immediate adaptation measures.

A recent rise in the global brewery sector has increased the demand for high-quality, late summer hops. The effects of ongoing and predicted climate change on the yield and aroma of hops, however, remain largely unknown. Here, we combine meteorological measurements and model projections to assess the climate sensitivity of the yield, alpha content and cone development of European hops between 1970 and 2050 CE, when temperature increases by 1.4 °C and precipitation decreases by 24 mm. Accounting for almost 90% of all hop-growing regions, our results from Germany, the Czech Republic and Slovenia show that hop ripening started approximately 20 days earlier, production declined by almost 0.2 t/ha/year, and the alpha content decreased by circa 0.6% when comparing data before and after 1994 CE. A predicted decline in hop yield and alpha content of 4-18% and 20-31% by 2050 CE, respectively, calls for immediate adaptation measures to stabilize an ever-growing global sector.

ECHOPvir: A Mixture of Echinacea and Hop Extracts Endowed with Cytoprotective, Immunomodulatory and Antiviral Properties.

Respiratory viral infections continue to pose significant challenges, particularly for more susceptible and immunocompromised individuals. Nutraceutical strategies have been proposed as promising strategies to mitigate their impact and improve public health. In the present study, we developed a mixture of two hydroalcoholic extracts from the aerial parts of Echinacea purpurea (L.) Moench (ECP) and the cones of Humulus lupulus L. (HOP) that can be harnessed in the prevention and treatment of viral respiratory diseases. The ECP/HOP mixture (named ECHOPvir) was characterized for the antioxidant and cytoprotective properties in airway cells. Moreover, the immunomodulating properties of the mixture in murine macrophages against antioxidant and inflammatory stimuli and its antiviral efficacy against the PR8/H1N1 influenza virus were assayed. The modulation of the Nrf2 was also investigated as a mechanistic hypothesis. The ECP/HOP mixture showed a promising multitarget bioactivity profile, with combined cytoprotective, antioxidant, immunomodulating and antiviral activities, likely due to the peculiar phytocomplexes of both ECP and HOP, and often potentiated the effect of the single extracts. The Nrf2 activation seemed to trigger these cytoprotective properties and suggest a possible usefulness in counteracting the damage caused by different stressors, including viral infection. Further studies may strengthen the interest in this product and underpin its future nutraceutical applications.

Host-plant phenology mediates facultative ant mutualism in the rare butterfly, Celastrina humulus (Lepidoptera: Lycaenidae).

Host-plant phenology can directly and indirectly mediate the abundance of insect herbivores. Our objective was to determine how host-plant flowering phenology shapes a facultative ant-lycaenid mutualism. The focus of our research was the hops azure, Celastrina humulus Scott & D. Wright, a rare species whose larvae feed on the pollen-bearing inflorescences of Humulus lupulus var. neomexicanus. We used several approaches to evaluate the role of host-plant flowering phenology in this system. First, we monitored larvae over three study years (2020-2022) to parse the role of host-plant flowering phenology and other factors in shaping the likelihood of ant tending. Second, we tested larval performance at various phenological stages of the host plant. We also quantified variation in soluble proteins and secondary metabolites among inflorescences at varying phenological stages. Lastly, we treated artificial sugar-protein baits with extracts from different phenological stages of the host plant; this allowed us to assess how chemical variation among stages could impact ant recruitment. Monitoring results revealed that the likelihood of ant tending was lowest for larvae on host plants with early-stage inflorescences. These floral stages had the greatest concentrations of both soluble proteins and α-acids (humulone and cohumulone), and in the feeding trial, early-stage flowers enabled greater weight gain for larvae. However, extracts from early-stage flowers reduced ant recruitment to sugar-protein baits. Altogether, these results suggest that early-stage inflorescences enhance larval growth while also reducing the recruitment of mutualist ants. This shows an indirect mechanism whereby changing host-plant phenology can mediate herbivore populations through interactions with ants.