Modulation of the replication of positive-sense RNA viruses by the natural plant metabolite xanthohumol and its derivatives.

The COVID-19 pandemic has highlighted the importance of identifying new potent antiviral agents. Nutrients as well as plant-derived substances are promising candidates because they are usually well tolerated by the human body and readily available in nature, and consequently mostly cheap to produce. A variety of antiviral effects have recently been described for the hop chalcone xanthohumol (XN), and to a lesser extent for its derivatives, making these hop compounds particularly attractive for further investigation. Noteworthy, mounting evidence indicated that XN can suppress a wide range of viruses belonging to several virus families, all of which share a common reproductive cycle. As a result, the purpose of this review is to summarize the most recent research on the antiviral properties of XN and its derivatives, with a particular emphasis on the positive-sense RNA viruses human hepatitis C virus (HCV), porcine reproductive and respiratory syndrome virus (PRRSV), and severe acute respiratory syndrome corona virus (SARS-CoV-2).

A Combination of Polymethoxyflavones from Citrus sinensis and Prenylflavonoids from Humulus lupulus Counteracts IL-1ß-Induced Differentiated Caco-2 Cells Dysfunction via a Modulation of NF-κB/Nrf2 Activation.

We here investigated the anti-inflammatory activity of a polymethoxylated flavone-containing fraction (PMFF) from Citrus sinensis and of a prenylflavonoid-containing one (PFF) from Humulus lupulus, either alone or in combination (MIX). To this end, an in vitro model of inflammatory bowel disease (IBD), consisting of differentiated, interleukin (IL)-1ß-stimulated Caco-2 cells, was employed. We demonstrated that non-cytotoxic concentrations of either PMFF or PFF or MIX reduced nitric oxide (NO) production while PFF and MIX, but not PMFF, also inhibited prostaglandin E2 release. Coherently, MIX suppressed both inducible NO synthase and cyclooxygenase-2 over-expression besides NF-κB activation. Moreover, MIX increased nuclear factor erythroid 2-related factor 2 (Nrf2) activation, heme oxygenase-1 expression, restoring GSH and reactive oxygen and nitrogen species (RONs) levels. Remarkably, these effects with MIX were stronger than those produced by PMFF or PFF alone. Noteworthy, nobiletin (NOB) and xanthohumol (XTM), two of the most represented phytochemicals in PMFF and PFF, respectively, synergistically inhibited RONs production. Overall, our results demonstrate that MIX enhances the anti-inflammatory and anti-oxidative effects of the individual fractions in a model of IBD, via a mechanism involving modulation of NF-κB and Nrf2 signalling. Synergistic interactions between NOB and XTM emerge as a relevant aspect underlying this evidence.

Flavonoids from the Roots of Sophora flavescens and Their Potential Anti-Inflammatory and Antiproliferative Activities.

The phytochemical investigation of the roots of the traditional Chinese medicinal plant Sophora flavescens led to the isolation of two novel prenylflavonoids with an unusual cyclohexyl substituent instead of the common aromatic ring B, named 4',4'-dimethoxy-sophvein (17) and sophvein-4'-one (18), and 34 known compounds (1-16, 19-36). The structures of these chemical compounds were determined by spectroscopic techniques, including 1D-, 2D-NMR, and HRESIMS data. Furthermore, evaluations of nitric oxide (NO) production inhibitory activity against lipopolysaccharide (LPS)-treated RAW264.7 cells indicated that some compounds exhibited obvious inhibition effects, with IC50 ranged from 4.6 ± 1.1 to 14.4 ± 0.4 µM. Moreover, additional research demonstrated that some compounds inhibited the growth of HepG2 cells, with an IC50 ranging from 0.46 ± 0.1 to 48.6 ± 0.8 µM. These results suggest that flavonoid derivatives from the roots of S. flavescens can be used as a latent source of antiproliferative or anti-inflammatory agents.

Prenylated flavonoids with significant anti-hepatoma activity from Daphne giraldii and effects on Fibroblast Growth Factor Receptor 1 (FGFR1).

We report here the orchestration of molecular ion networking (MoIN) and a set of computational and informatics assisted structural elucidation approaches in the discovery of 23 new prenyl-flavonoids and 13 known molecules from Daphne giraldii Nitsche (Thymelaeaceae), some of which possess significant bioactivity against hepatoma carcinoma. Daphnegiratriprenylone A (DPTP-A) represents the class of polyprenyl-flavonoids possessing a triprenyl substitution, and was identified with the guidance of mass spectrometry and nuclear magnetic resonance combined with computational approaches. This approach illustrates a paradigm shift in the application of computational tools for the direct assignment of new natural product structures and it was demonstrated to be reliable compared to conventional 2D-NMR techniques. Seventeen compounds exhibited potent and selective activity against Hep3B cells (IC50 ranging from 0.42 to 7.08 µM). Tyrosine kinase FGFR1 has emerged as a potential target of polyprenyl-flavonoids by a reverse pharmacophore mapping approach. We validated that the prenyl-flavonoids effectively inhibit FGFR1 using the Mobility Shift Assay, Western blot and molecular dynamics simulations, and the results suggest significant potency of the compounds towards FGFR1. These findings provide a new chemical class with strong links to traditional medicines, possessing reasonable safety for developing potential therapeutic agents for FGFR1-related diseases.

Heterologous biosynthesis of isobavachalcone in tobacco based on in planta screening of prenyltransferases.

Isobavachalcone (IBC) is a prenylated chalcone mainly distributed in some Fabaceae and Moraceae species. IBC exhibits a wide range of pharmacological properties, including anti-bacterial, anti-viral, anti-inflammatory, and anti-cancer activities. In this study, we attempted to construct the heterologous biosynthesis pathway of IBC in tobacco (Nicotiana tabacum). Four previously reported prenyltransferases, including GuILDT from Glycyrrhiza uralensis, HlPT1 from Humulus lupulus, and SfILDT and SfFPT from Sophora flavescens, were subjected to an in planta screening to verify their activities for the biosynthesis of IBC, by using tobacco transient expression with exogenous isoliquiritigenin as the substrate. Only SfFPT and HlPT1 could convert isoliquiritigenin to IBC, and the activity of SfFPT was higher than that of HlPT1. By co-expression of GmCHS8 and GmCHR5 from Glycine max, endogenous isoliquiritigenin was generated in tobacco leaves (21.0 µg/g dry weight). After transformation with a multigene vector carrying GmCHS8, GmCHR5, and SfFPT, de novo biosynthesis of IBC was achieved in transgenic tobacco T0 lines, in which the highest amount of IBC was 0.56 µg/g dry weight. The yield of IBC in transgenic plants was nearly equal to that in SfFPT transient expression experiments, in which substrate supplement was sufficient, indicating that low IBC yield was not attributed to the substrate supplement. Our research provided a prospect to produce valuable prenylflavonoids using plant-based metabolic engineering.

Perspectives on the design of microbial cell factories to produce prenylflavonoids.

Prenylflavonoids are flavonoid-derived compounds characterized by the presence of a lipophilic prenylated side-chain in the flavonoid skeleton. These compounds are present in several food supplements and food products, and have a wide variety of recognized biological activities, namely estrogenic, antioxidant, anti-inflammatory and anticancer. Since these compounds are present in nature in very low amounts, their extraction from plants is not enough to fulfill the current demand, besides being an inefficient and environmentally unfriendly process. For these reasons, the use of microorganisms as microbial cell factories represents an interesting alternative to produce prenylflavonoids in a faster and cheaper way. Saccharomyces cerevisiae has been used as chassis to produce prenylflavonoids. Moreover, Escherichia coli can also emerge as an alternative chassis to produce these compounds. However, there is still a long way before prenylflavonoids can be produced by heterologous organisms with relevant yields and titers. In this review, we highlight the biosynthetic pathways involved in the production of prenylflavonoids. Additionally, we review the advances, challenges and strategies on the heterologous production of these compounds in a competitive way.

The role of key gut microbial metabolites in the development and treatment of cancer.

In recent years, the role of gut microbial metabolites on the inhibition and progression of cancer has gained significant interest in anticancer research. It has been established that the gut microbiome plays a pivotal role in the development, treatment and prognosis of different cancer types which is often mediated through the gut microbial metabolites. For instance, gut microbial metabolites including bacteriocins, short-chain fatty acids and phenylpropanoid-derived metabolites have displayed direct and indirect anticancer activities through different molecular mechanisms. Despite the reported anticancer activity, some gut microbial metabolites including secondary bile acids have exhibited pro-carcinogenic properties. This review draws a critical summary and assessment of the current studies demonstrating the carcinogenic and anticancer activity of gut microbial metabolites and emphasises the need to further investigate the interactions of these metabolites with the immune system as well as the tumour microenvironment in molecular mechanistic and clinical studies.

Prenylated flavonoids in foods and their applications on cancer prevention.

Functional foods play an important role in health care and chronic diseases prevention, particularly cancer. Prenylated flavonoids are presented in many food resources. They are recognized as neutraceuticals due to their diverse health benefits. Up to now, more than 1000 prenylated flavonoids have been identified in plants. Their food resources are reviewed in this paper. Due to the good safety and cancer prevention effect of prenylated flavonoids, this paper reviews the cancer prevention activities and mechanisms reported in last decade. The structure-activity relationship is discussed. Due to the limited availability in nature, the heterologously biosynthetic technique of prenylated flavonoids is discussed in this review. Inclusion of dietary prenylated flavonoids into human diet is highly desirable. This paper combines the up-to-date information and give a clear image regarding prenylated flavonoids as neutraceuticals.

Preparation of Hop Estrogen-Active Material for Production of Food Supplements.

In recent years, the interest in the health-promoting effects of hop prenylflavonoids, especially its estrogenic effects, has grown. Unfortunately, one of the most potent phytoestrogens identified so far, 8-prenylnaringenin, is only a minor component of hops, so its isolation from hop materials for the production of estrogenically active food supplements has proved to be problematic. The aim of this study was to optimize the conditions (e.g., temperature, the length of the process and the amount of the catalyst) to produce 8-prenylnaringenin-rich material by the magnesium oxide-catalyzed thermal isomerization of desmethylxanthohumol. Under these optimized conditions, the yield of 8-prenylnaringenin was 29 mg per 100 gDW of product, corresponding to a >70% increase in its content relative to the starting material. This process may be applied in the production of functional foods or food supplements rich in 8-prenylnaringenin, which may then be utilized in therapeutic agents to help alleviate the symptoms of menopausal disorders.

Moderate Consumption of Beer (with and without Ethanol) and Menopausal Symptoms: Results from a Parallel Clinical Trial in Postmenopausal Women.

The menopausal transition can be a challenging period for women's health and a trigger of uncomfortable symptoms. Beer is the main food source of isoxanthohumol, a precursor of 8-prenylnaringenin, the strongest phytoestrogen identified to date. As phytoestrogens are reported to reduce perimenopausal symptoms, we evaluated if a daily moderate consumption of beer with (AB) and without alcohol (NAB) could improve menopausal symptoms and modify cardiovascular risk factors. A total of 37 postmenopausal women were enrolled in a parallel controlled intervention trial and assigned to three study groups: 16 were administered AB (330 mL/day), 7 NAB (660 mL/day), and 14 were in the control group. After a 6-month follow-up of the 34 participants who finished the trial, both interventions (AB and NAB) significantly reduced the severity of the menopause-related symptoms (p-value AB vs. Control: 0.009; p-value NAB vs. Control: 0.033). Moreover, AB had a beneficial net effect on psychological menopausal discomforts compared to the control group. As the sex hormone profile did not differ significantly between the study groups, the effects of both types of beers (AB and NAB) are attributed to the non-alcoholic fraction of beer. Furthermore, moderate NAB consumption improved the lipid profile and decreased blood pressure in postmenopausal women.

Redox and Anti-Inflammatory Properties from Hop Components in Beer-Related to Neuroprotection.

Beer is a fermented beverage widely consumed worldwide with high nutritional and biological value due to its bioactive components. It has been described that both alcoholic and non-alcoholic beer have several nutrients derived from their ingredients including vitamins, minerals, proteins, carbohydrates, and antioxidants that make beer a potential functional supplement. Some of these compounds possess redox, anti-inflammatory and anticarcinogenic properties making the benefits of moderate beer consumption an attractive way to improve human health. Specifically, the hop cones used for beer brewing provide essential oils, bitter acids and flavonoids that are potent antioxidants and immune response modulators. This review focuses on the redox and anti-inflammatory properties of hop derivatives and summarizes the current knowledge of their neuroprotective effects.

Prenylflavonoids from fruit of Macaranga tanarius promote glucose uptake via AMPK activation in L6 myotubes.

Skeletal muscle is a major tissue of glucose consumption and plays an important role in glucose homeostasis. Prenylflavonoids, a component of Macaranga tanarius fruits, have been reported to have antioxidant, antibacterial, and anticancer effects. However, the effects of these compounds on skeletal muscle glucose metabolism are unclear. Here, we isolated five prenylflavonoids from M. tanarius fruits, and investigated the mechanism of action of these compounds on skeletal muscle cells using L6 myotubes. We found that isonymphaeol B and 3'-geranyl naringenin increased glucose uptake in a dose-dependent manner. Furthermore, both isonymphaeol B and 3'-geranyl naringenin increased AMPK phosphorylation but did not affect PI3K-Akt phosphorylation. Isonymphaeol B and 3'-geranyl naringenin also increased Glut1 mRNA expression and plasma membrane GLUT1 protein levels. These results suggest that isonymphaeol B and 3'-geranyl naringenin have beneficial effects on glucose metabolism through AMPK and GLUT1 pathway. Isonymphaeol B and 3'-geranyl naringenin may be potential lead candidates for antidiabetic drug development.

Synthetic production of prenylated naringenins in yeast using promiscuous microbial prenyltransferases.

Reconstitution of prenylflavonoids using the flavonoid biosynthetic pathway and prenyltransferases (PTs) in microbes can be a promising attractive alternative to plant-based production or chemical synthesis. Here, we demonstrate that promiscuous microbial PTs can be a substitute for regiospecific but mostly unidentified botanical PTs. To test the prenylations of naringenin, we constructed a yeast strain capable of producing naringenin from l-phenylalanine by genomic integration of six exogenous genes encoding components of the naringenin biosynthetic pathway. Using this platform strain, various microbial PTs were tested for prenylnaringenin production. In vitro screening demonstrated that the fungal AnaPT (a member of the tryptophan dimethylallyltransferase family) specifically catalyzed C-3' prenylation of naringenin, whereas SfN8DT-1, a botanical PT, specifically catalyzed C-8 prenylation. In vivo, the naringenin-producing strain expressing the microbial AnaPT exhibited heterologous microbial production of 3'-prenylnaringenin (3'-PN), in contrast to the previously reported in vivo production of 8-prenylnaringenin (8-PN) using the botanical SfN8DT-1. These findings provide strategies towards expanding the production of a variety of prenylated compounds, including well-known prenylnaringenins and novel prenylflavonoids. These results also suggest the opportunity for substituting botanical PTs, both known and unidentified, that display relatively strict regiospecificity of the prenyl group transfer.

Chemosystematic and evolutionary trends of the genistoid clade sensu stricto (Papilionoideae, Fabaceae).

The Papilionoideae, which comprises 503 genera and approximately 14,000 species, is the largest and most diverse subfamily of the Fabaceae family. In this subfamily, the Crotalarieae, Genisteae, Podalyrieae, Thermopsideae, Sophoreae and Euchresteae tribes are closely related by micro and macromolecular features, thus forming the genistoid clade. This group combines well-known genera, whereas other genera lack phytochemical and chemotaxonomic studies. Thus, this work aimed to characterize the special metabolites in these genera in order to define the chemical profile, the micromolecular markers and the chemical diversity, as well as to evaluate the group evolutionary trends. Flavonoids and alkaloids were identified as chemosystematic markers for the studied tribes due to high occurrence number and structural diversity. Among flavonoids, the flavones and isoflavones predominated. Low protection indexes of flavonoid hydroxyls by O-glycosylation or O-methylation were observed, whereas C-prenylation and C-glycosylation were frequent, mainly at C-6 and C-8 positions. The flavone/flavonol ratio shows the predominance of the flavones. Quinolizidine and piperidine alkaloids were present in most genera. Pyrrolizidine alkaloids were found in a few genera from Thermopsideae, Genisteae and Crotalarieae, which suggests a mechanism of adaptive convergence. Cluster analysis allowed separation of genera for each tribe by chemical similarities. The micromolecular trends of protection of flavonoid hydroxyls and alkaloid oxidation indicate the genistoid clade is through evolutionary transition, which is consistent with its phylogenetic position in the Papilionoideae subfamily.

Combined in silico and in vitro studies to identify novel antidiabetic flavonoids targeting glycogen phosphorylase.

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4'-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines.

Flavonoids as Phytoestrogenic Components of Hops and Beer.

The value of hops (Humulus lupulus L.) in beer production has been undisputed for centuries. Hops is rich in humulones and lupulones which gives the characteristic aroma and bitter taste, and preserves this golden drink against growing bacteria and molds. Besides α- and ß-acids, the lupulin glands of hop cones excrete prenylated flavonoids, which exhibit a broad spectrum of biological activities and therefore has therapeutic potential in humans. Recently, interest in hops was raised due to hop prenylated flavanones which show extraordinary estrogen activities. The strongest known phytoestrogen so far is 8-prenylnaringenin (8-PN), which along with 6-prenylanaringenin (6-PN), 6,8-diprenylnaringenin (6,8-DPN) and 8-geranylnaringenin (8-GN) are fundamental for the potent estrogen activity of hops. This review provides insight into the unusual hop phytoestrogens and shows numerous health benefits associated with their wide spectrum of biological activities including estrogenic, anticancer, neuropreventive, antinflamatory, and antimicrobial properties, which were intensively studied, and potential applications of these compounds such as, as an alternative to hormone replacement therapy (HRT).

Evaluation of prenylflavonoids and hop bitter acids in surplus yeast.

This study developed a high performance liquid chromatography with diode array detection (HPLC-DAD) and tandem mass spectrometry (MS-MS) method for determination of prenylflavonoids and hop bitter acids in surplus yeast, a byproduct from beer brewing process. This method enabled the simultaneous separation of 4 prenylflavonoids and 20 hop bitter acids within 30 min by employing a Hypersil-Keystone HyPURITY C18 column and a gradient mobile phase composed of phosphoric acid aqueous solution at pH 1.6 and acetonitrile. For HPLC-DAD analysis, the limits of detection and limits of quantitation ranged from 0.04 to 0.15 µg/mL and from 0.12 to 0.45 µg/mL, respectively, and the recoveries ranged from 82.6 to 99.7%. The intra-day variability and inter-day variability ranged from 1.37 to 8.82% and from 0.68 to 9.74%, respectively. For qualitation by MS-MS, the positive mode was discovered to possess satisfactory collision capacity and high sensitivity for prenylflavonoids, while the negative mode was more suitable for the ionization of hop bitter acids. The content of hop bitter acids in surplus yeast were higher than that of prenylflavonoids, and isomers and oxidation products of hop bitter acids were found. This study has advantages in identifying more components, short separation time, satisfactory resolution, high accuracy and high precision.

Antiproliferative Effects of Hop-derived Prenylflavonoids and Their Influence on the Efficacy of Oxaliplatine, 5-fluorouracil and Irinotecan in Human ColorectalC Cells.

Beer, the most popular beverage containing hops, is also frequently consumed by cancer patients. Moreover, non-alcoholic beer, owing to its nutritional value and high content of biological active compounds, is sometimes recommended to patients by oncologists. However, the potential benefits and negatives have to date not been sufficiently evaluated. The present study was designed to examine the effects of four main hop-derived prenylflavonoids on the viability, reactive oxygen species (ROS) formation, activity of caspases, and efficiency of the chemotherapeutics 5-fluorouracil (5-FU), oxaliplatin (OxPt) and irinotecan (IRI) in colorectal cancer cell lines SW480, SW620 and CaCo-2. All the prenylflavonoids exerted substantial antiproliferative effects in all cell lines, with xanthohumol being the most effective (IC50 ranging from 3.6 to 7.3 µM). Isoxanthohumol increased ROS formation and the activity of caspases-3/7, but 6-prenylnaringenin and 8-prenylnaringenin exerted antioxidant properties. As 6-prenylnaringenin acted synergistically with IRI, its potential in combination therapy deserves further study. However, other prenylflavonoids acted antagonistically with all chemotherapeutics at least in one cell line. Therefore, consumption of beer during chemotherapy with 5-FU, OxPt and IRI should be avoided, as the prenylflavonoids in beer could decrease the efficacy of the treatment.

Dissection of Dynamic Transcriptome Landscape of Leaf, Bract, and Lupulin Gland in Hop (Humulus lupulus L.).

The hop plant (Humulus lupulus L.) produces several valuable secondary metabolites, such as prenylflavonoid, bitter acids, and essential oils. These compounds are biosynthesized in glandular trichomes (lupulin glands) endowed with pharmacological properties and widely implicated in the beer brewing industry. The present study is an attempt to generate exhaustive information of transcriptome dynamics and gene regulatory mechanisms involved in biosynthesis and regulation of these compounds, developmental changes including trichome development at three development stages, namely leaf, bract, and mature lupulin glands. Using high-throughput RNA-Seq technology, a total of 61.13, 50.01, and 20.18 Mb clean reads in the leaf, bract, and lupulin gland libraries, respectively, were obtained and assembled into 43,550 unigenes. The putative functions were assigned to 30,996 transcripts (71.17%) based on basic local alignment search tool similarity searches against public sequence databases, including GO, KEGG, NR, and COG families, which indicated that genes are principally involved in fundamental cellular and molecular functions, and biosynthesis of secondary metabolites. The expression levels of all unigenes were analyzed in leaf, bract, and lupulin glands tissues of hop. The expression profile of transcript encoding enzymes of BCAA metabolism, MEP, and shikimate pathway was most up-regulated in lupulin glands compared with leaves and bracts. Similarly, the expression levels of the transcription factors and structural genes that directly encode enzymes involved in xanthohumol, bitter acids, and terpenoids biosynthesis pathway were found to be significantly enhanced in lupulin glands, suggesting that production of these metabolites increases after the leaf development. In addition, numerous genes involved in primary metabolism, lipid metabolism, photosynthesis, generation of precursor metabolites/energy, protein modification, transporter activity, and cell wall component biogenesis were differentially regulated in three developmental stages, suggesting their involvement in the dynamics of the lupulin gland development. The identification of differentially regulated trichome-related genes provided a new foundation for molecular research on trichome development and differentiation in hop. In conclusion, the reported results provide directions for future functional genomics studies for genetic engineering or molecular breeding for augmentation of secondary metabolite content in hop.

Biocatalytic access to diverse prenylflavonoids by combining a regiospecific C-prenyltransferase and a stereospecific chalcone isomerase.

Prenylflavonoids are valuable natural products that have diverse biological properties, and are usually generated biologically by multiple metabolic enzymes in nature. In this study, structurally diverse prenylflavonoids were conveniently synthesized by enzymatic catalysis by combining GuILDT, a regiospecific chalcone prenyltransferase, and GuCHI, a stereospecific chalcone isomerase that has promiscuous activity for both chalcones and prenylchalcones as substrates. Our findings provided a new approach for the synthesis of natural/unnatural bioactive prenylflavonoids, including prenylchalcones and optical prenylflavanones with chalcone origins.