Hop Constituents Research Articles

How deprotonation of cohumulone and colupulone influence their UV/Vis and CD spectrum

Cohumulone and colupulone are representatives of α- and β-acids, respectively. These compounds are important antimicrobial hop (Humulus lupulus) constituents, where cohumulone is an important source of the bitter taste of beer. In this study, we examined the pH dependence of UV/Vis spectra of both compounds while CD spectra of cohumulone were also measured at various wavelengths. This facilitated the examination of the protolytic equilibrium of both compounds, where the second pKa value of cohumulone was determined for the first time. Additionally, comparing experimental spectra with spectra calculated using time-dependent density functional theory (TD-DFT) enabled the determination of the most likely deprotonation positions and corresponding species most likely present in the aqueous solution at various pH values. Last but not least, comparing calculated and experimental CD spectra of cohumulone facilitated the determination of the absolute stereoconfiguration of cohumulone.

Identification of Potential Antiviral Hops Compounds against Chikungunya Virus.

Chikungunya virus (CHIKV) is an arthropod-borne virus that belongs to the genus Alphavirus (family Togaviridae). CHIKV causes chikungunya fever, which is mostly characterized by fever, arthralgia and, sometimes, a maculopapular rash. The bioactive constituents of hops (Humulus lupulus, Cannabaceae), mainly acylphloroglucinols, known as well as α- and β-acids, exerted distinct activity against CHIKV, without showing cytotoxicity. For fast and efficient isolation and identification of such bioactive constituents, a silica-free countercurrent separation method was applied. The antiviral activity was determined by plaque reduction test and was visually confirmed by a cell-based immunofluorescence assay. All hops compounds demonstrated a promising post-treatment viral inhibition, except the fraction of acylphloroglucinols, in mixture. β-acids fraction of 125 µg/mL expressed the strongest virucidal activity (EC50 = 15.21 µg/mL), in a drug-addition experiment on Vero cells. Hypothesis for mechanism of action were proposed for acylphloroglucinols based on their lipophilicity and chemical structure. Therefore, inhibition of some steps of the protein kinase C (PKC) transduction cascades was also discussed.

The Impact of Whirlpool Hop Addition on the Wort Metal Ion Composition and on the Flavor Stability of American Style Pale Ales Using Citra® Hop Extract and Pellets

The characteristic hoppy flavor of pale ales is known to be unstable and deteriorates during the beer’s shelf life. Metal ions, in particular iron and copper, have a prooxidative effect and therefore accelerate beer flavor deterioration. Recent studies suggest that hop constituents may complex metal ions in beer thereby suppressing their negative effect on beer flavor stability. Using benchtop wort boiling experiments and quantitating metal ions in the cast wort, this study determined that different whirlpool additions of up to 3.0 g/L hop extract or up to 17.4 g/L pellets significantly reduced metal ion concentrations in wort, especially Fe, which was reduced by a factor of up to 6 compared to the control. Based on identified dose- and product-related effects, 2.5-hL brewing trials were performed using a selection of whirlpool hopping levels with either extract or pellets revealing significant differences in resinous and sweaty flavor. The flavor of the beer hopped with 5.8 g/L pellets was perceived as 8 times more resinous and 9 times sweatier than the control revealing clear differences between extract and pellets, especially at the lower hopping levels. The beer flavor stability was predicted using ESR spectroscopy and verified by performing forced aging experiments at 30 °C and monitoring beer flavor changes by sensory evaluation. Results indicate that both extract and pellet whirlpool hopping improve beer flavor stability by reducing the formation of age-related off-flavors and by supporting the retention of flavors related to freshness with only minor variations between the two hop products.

Principal Component Analysis of Hop-Derived Odorants Identified by Stir Bar Sorptive Extraction Method

Beers hopped with 79 varieties were brewed in small-scale fermenters, and 127 hop-derived compounds were picked up by comparing with the chromatogram of unhopped beer, after the analysis of the stir bar sorptive extraction gas chromatography-mass spectrometry method. The 127 hop-related flavor compounds were used to examine the interrelationship with the hop varieties by principal component analysis (PCA). Different varieties and amounts of hop pellets based on equal amounts of alpha-acids were added to the cooled wort. Addition of higher amounts of hops resulted in lower concentrations of apparent extracts of beer, suggesting the presence of dextrin-hydrolyzing enzymes in the hops. PCA largely established five directions. Direction I including Cascade, Amarillo, Citra, Simcoe, Mosaic, Wai-iti, and Hallertau Blanc varieties was linked to geraniol and citronellol, both known to contribute to citrus characteristics. Direction II including Bobek, Styrian Gold, and Moutueka varieties was associated with linalool and 6-methyl-5-hepten-2-one. Direction III including Hersbrucker and Saphir was linked to sesquiterpene alcohols. Direction IV was associated with hydrophobic monoterpenes, and direction V was linked to hydrophobic sesquiterpenes. Further study of these hop varieties and constituents can help advance our knowledge in using different hop varieties to achieve defined aroma and taste of beer.

Quantitative Analysis of Prenylated Constituents in Commercial Hops Samples Using Ultrahigh-Performance Supercritical Fluid Chromatography.

The importance of hops (the flowers of Humulus lupulus) as food and an herbal remedy is reflected by a large number of analytical methods published. However, supercritical fluid chromatography, a highly efficient, rapid, and "green" separation technique, has not been considered for hops samples so far. This prompted us to establish the first supercritical fluid chromatography-based protocol for the separation, identification, and quantitation of five prenylated constituents of hops. Hulupinic acid ( 1: ), a prominent oxidation product of hop acids, three flavanones, i.e., 8-prenylnaringenin ( 2: ), 6-prenylnaringenin ( 3: ), and isoxanthohumol ( 4: ), as well as the chalcone xanthohumol ( 5: ) could be baseline separated in lessthan 5 minutes using a Viridis BEH 2-EP column (3.0 × 100 mm; 1.7 µm particle size) and a mobile phase consisting of CO2 and isopropanol. Good results regarding selectivity, accuracy (recovery rates: 85.0 - 113.1%), precision (intra-day ≤ 2.1%, inter-day ≤ 3.5%), and linearity (R2 ≥ 0.99) were obtained for both photodiode array and mass detection. The lowest detection limit at 220 nm was at 0.1 µg/mL ( 1, 3: , and 4: ), with mass detection even at 0.001 µg/mL ( 4: ). As an application example of the validated method, the five hops constituents were quantified in three dietary supplements, one herbal medicinal product, and two batches of hop flowers (Lupuli flos). In most samples analyzed, the major component was 5: (0.01 - 1.02%), whereas the major component in Lupuli flos samples was compound 1: (0.12 - 0.21%). This protocol offers a fast and environmentally friendly alternative to liquid chromatography for the quality control of hops.

The Multiple Biological Targets of Hops and Bioactive Compounds.

Botanical dietary supplements for women's health are increasingly popular. Older women tend to take botanical supplements such as hops as natural alternatives to traditional hormone therapy to relieve menopausal symptoms. Especially extracts from spent hops, the plant material remaining after beer brewing, are enriched in bioactive prenylated flavonoids that correlate with the health benefits of the plant. The chalcone xanthohumol (XH) is the major prenylated flavonoid in spent hops. Other less abundant but important bioactive prenylated flavonoids are isoxanthohumol (IX), 8-prenylnaringenin (8-PN), and 6-prenylnaringenin (6-PN). Pharmacokinetic studies revealed that these flavonoids are conjugated rapidly with glucuronic acid. XH also undergoes phase I metabolism in vivo to form IX, 8-PN, and 6-PN. Several hop constituents are responsible for distinct effects linked to multiple biological targets, including hormonal, metabolic, inflammatory, and epigenetic pathways. 8-PN is one of the most potent phytoestrogens and is responsible for hops' estrogenic activities. Hops also inhibit aromatase activity, which is linked to 8-PN. The weak electrophile, XH, can activate the Keap1-Nrf2 pathway and turn on the synthesis of detoxification enzymes such as NAD(P)H-quinone oxidoreductase 1 and glutathione S-transferase. XH also alkylates IKK and NF-κB, resulting in anti-inflammatory activity. Antiobesity activities have been described for XH and XH-rich hop extracts likely through activation of AMP-activated protein kinase signaling pathways. Hop extracts modulate the estrogen chemical carcinogenesis pathway by enhancing P450 1A1 detoxification. The mechanism appears to involve activation of the aryl hydrocarbon receptor (AhR) by the AhR agonist, 6-PN, leading to degradation of the estrogen receptor. Finally, prenylated phenols from hops are known inhibitors of P450 1A1/2; P450 1B1; and P450 2C8, 2C9, and 2C19. Understanding the biological targets of hop dietary supplements and their phytoconstituents will ultimately lead to standardized botanical products with higher efficacy, safety, and chemopreventive properties.

The hop constituent xanthohumol exhibits hepatoprotective effects against acute alcohol-induced liver injury

The hop constituent xanthohumol exhibits hepatoprotective effects against acute alcohol-induced liver injury

Strong antimicrobial activity of xanthohumol and other derivatives from hops (Humulus lupulus L.) on gut anaerobic bacteria.

Anaerobic bacteria, such as Bacteroides fragilis or Clostridium perfringens, are part of indigenous human flora. However, Clostridium difficile represents also an important causative agent of nosocomial infectious antibiotic-associated diarrhoea. Treatment of C.difficile infection is problematic, making it imperative to search for new compounds with antimicrobial properties. Hops (Humulus lupulus L.) contain substances with antibacterial properties. We tested antimicrobial activity of purified hop constituents humulone, lupulone and xanthohumol against anaerobic bacteria. The antimicrobial activity was established against B.fragilis, C.perfringens and C.difficile strains according to standard testing protocols (CLSI, EUCAST), and the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were calculated. All C.difficile strains were toxigenic and clinically relevant, as they were isolated from patients with diarrhoea. Strongest antimicrobial effects were observed with xanthohumol showing MIC and MBC values of 15-107μg/mL, which are close to those of conventional antibiotics in the strains of bacteria with increased resistance. Slightly higher MIC and MBC values were obtained with lupulone followed by higher values of humulone. Our study, thus, shows a potential of purified hop compounds, especially xanthohumol, as alternatives for treatment of infections caused by select anaerobic bacteria, namely nosocomial diarrhoea caused by resistant strains.

Hop Constituents Suppress the Formation of 3-Methylbutanal and 2-Furfural in Wort-Like Model Solutions

The ability of hop constituents to suppress the formation of 3-methylbutanal and 2-furfural in wort-like model systems containing maltose, leucine, and Fe2+ was investigated. From all constituents tested (α-acids, β-acids, iso-α-acids, tetrahydro-iso-α-acids, and spent hops), solely α-acids and β-acids were capable of significantly suppressing the formation of 3-methylbutanal as derived from degradation of leucine. 2-Furfural was also found in the model solutions after incubation, and its formation was diminished by all hop constituents but to a varying degree. A central composite rotatable design was employed, and from all factors tested (maltose, leucine, Fe2+, and α-acids), only maltose and leucine were found to be significant model terms (P < 0.05) for the formation of 3-methylbutanal, whereas α-acids also showed effectiveness but at a lower confidence level (α = 0.1063). Maltose and α-acid concentrations were significant model terms for the formation of 2-furfural. Although Fe2+ concentration was an insignificant factor for aldehyde formation, complexing it with ethylenediaminetetraacetic acid still inhibited aldehyde formation. When no maltose was present, 3-methylbutanal was also shown to be derived from leucine oxidative degradation by hydroxyl radical attack. Hop α-acids were demonstrated to significantly abate this particular pathway by Fe2+ complexation and hydroxyl radical scavenging.

Editorial: Liver Myofibroblasts.

Editorial: Liver Myofibroblasts.

The hop constituent xanthohumol exhibits hepatoprotective effects and inhibits the activation of hepatic stellate cells at different levels.

Xanthohumol is the principal prenylated flavonoid of the female inflorescences of the hop plant. In recent years, various beneficial xanthohumol effects including anti-inflammatory, antioxidant, hypoglycemic activities, and anticancer effects have been revealed. This review summarizes present studies indicating that xanthohumol also inhibits several critical pathophysiological steps during the development and course of chronic liver disease, including the activation and pro-fibrogenic genotype of hepatic stellate cells. Also the various mechanism of action and molecular targets of the beneficial xanthohumol effects will be described. Furthermore, the potential use of xanthohumol or a xanthohumol-enriched hop extract as therapeutic agent to combat the progression of chronic liver disease will be discussed. It is notable that in addition to its hepatoprotective effects, xanthohumol also holds promise as a therapeutic agent for treating obesity, dysregulation of glucose metabolism and other components of the metabolic syndrome including hepatic steatosis. Thus, therapeutic xanthohumol application appears as a promising strategy, particularly in obese patients, to inhibit the development as well as the progression of non-alcoholic fatty liver disease.

Development of a new high-performance liquid chromatography method with diode array and electrospray ionization-mass spectrometry detection for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L.

The study was aimed at developing a new analytical method for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L. (hop), together with a simple extraction procedure. Different extraction techniques, including maceration, heat reflux extraction (HRE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE), were compared in order to obtain a high yield of the target analytes. Dynamic maceration for 30min with MeOH–HCOOH (99:1, v/v) as the extraction solvent provided the best result in terms of recovery of secondary metabolites. The analysis of hop constituents, including prenylflavonoids and prenylphloroglucinols (bitter acids), was carried out by means of HPLC-UV/DAD, HPLC-ESI-MS and MS2, using an ion trap mass analyzer. An Ascentis Express C18 column (150mm×3.0mm I.D., 2.7μm) was used for the HPLC analysis, with a mobile phase composed of 0.25% formic acid in both water and acetonitrile, under gradient elution. The method validation was performed to show compliance with ICH guidelines. The validated technique was successfully applied to the phytochemical analysis of ten commercial cultivars and twenty-three wild Italian hop genotypes, thus demonstrating to be a reliable and useful tool for the comprehensive multi-component analysis of hop secondary metabolites.

Stability of Active Constituents of Hops (Humulus lupulus) Strobiles and their Ethanolic Extracts during Storage

Stability of Active Constituents of Hops (Humulus lupulus) Strobiles and their Ethanolic Extracts during Storage

Inhibition of human cytochrome P450 enzymes by hops (Humulus lupulus) and hop prenylphenols

As hops (Humulus lupulus L.) are used in the brewing of beer and by menopausal women as estrogenic dietary supplements, the potential for hop extracts and hop constituents to cause drug–botanical interactions by inhibiting human cytochrome P450 enzymes was investigated. Inhibition of major human cytochrome P450 enzymes by a standardized hop extract and isolated hop prenylated phenols was evaluated using a fast and efficient assay based on ultrahigh pressure liquid chromatography–tandem mass spectrometry. The hop extract at 5μg/mL inhibited CYP2C8 (93%), CYP2C9 (88%), CYP2C19 (70%), and CYP1A2 (27%) with IC50 values of 0.8, 0.9, 3.3, and 9.4μg/mL, respectively, but time-dependent inactivation was observed only for CYP1A2. Isoxanthohumol from hops was the most potent inhibitor of CYP2C8 with an IC50 of 0.2μM, whereas 8-prenylnaringenin was the most potent inhibitor of CYP1A2, CYP2C9 and CYP2C19 with IC50 values of 1.1μM, 1.1μM and 0.4μM, respectively. Extracts of hops contain prenylated compounds such as the flavanones isoxanthohumol and 8-prenylnaringenin and the chalcone xanthohumol that can inhibit CYP450s, especially the CYP2C family, which may affect the efficacy and safety of some CYP2C substrate drugs when co-administered.

Fate of 2-sulphanylethyl acetate and 3-sulphanylpropyl acetate through beer aging

Sulphanylalkyl alcohols and their corresponding acetates were investigated in 14 commercial Belgian beers. Although it was the major peak at the pulsed-flame photometric detector, the empyreumatic 2-sulphanylethyl acetate was found at concentrations below its individual odour threshold, estimated at 40 µg/L (0–4 µg/L in most fresh beers, 5–12 µg/L in three fresh high-bitter beers). Both the Ehrlich pathway and hop constituents contribute to this content. In 11 of the investigated samples, synthesis of 2SE-A and 3-sulphanylpropyl acetate (roasted, burned) continued during the first three months of storage. Although below their individual thresholds, these compounds might interact by synergy with other aged flavours. As yeast was absent from most of the investigated bottles, chemical degradation of precursors is suspected. Copyright © 2012 The Institute of Brewing & Distilling

Occurrence of polyfunctional thiols in sorghum beer ‘ikigage’ made with Vernonia amygdalina ‘umubirizi’

ABSTRACTSeveral polyfunctional thiols have been previously identified in beers made from barley and hops. These compounds have not been investigated in beers brewed with ‘non‐Western’ raw materials. Here we have performed a thiol‐specific extraction with p‐hydroxymercuribenzoic acid on a traditional ikigage sorghum beer from Rwandese peasants (use of Vernonia amygdalina just for yeast propagation), and on two pilot beers with addition (or not) of V. amygdalina in the boiling kettle, instead of hops. Gas chromatography–olfactometry, gas chromatography–mass spectrometry and gas chromatography with pulsed flame photometric detection analyses of the extracts enabled us to identify 14 polyfunctional thiols. The well‐known hop constituent 3‐methyl‐2‐buten‐1‐thiol emerged as a key flavour in the unhopped beers containing V. amygdalina (flavour dilution &gt; 262 144). The addition of V. amygdalina during boiling also resulted in the presence of 1‐butanethiol, but the production of 2‐sulfanylethanol and 2‐sulfanylethyl acetate was inhibited. Complementary data are required to understand how V. amygdalina leaves are able to impact upon the Ehrlich pathway leading to cysteine and homocysteine‐derived thiols. Copyright © 2012 John Wiley &amp; Sons, Ltd.

Hops (Humulus lupulus) Inhibits Oxidative Estrogen Metabolism and Estrogen-Induced Malignant Transformation in Human Mammary Epithelial cells (MCF-10A)

Long-term exposure to estrogens including those in traditional hormone replacement therapy (HRT) increases the risk of developing hormone-dependent cancers. As a result, women are turning to over-the-counter (OTC) botanical dietary supplements, such as black cohosh (Cimicifuga racemosa) and hops (Humulus lupulus), as natural alternatives to HRT. The two major mechanisms which likely contribute to estrogen and/or HRT cancer risk are: the estrogen receptor-mediated hormonal pathway; and the chemical carcinogenesis pathway involving formation of estrogen quinones that damage DNA and proteins, hence initiating and promoting carcinogenesis. Because, OTC botanical HRT alternatives are in widespread use, they may have the potential for chemopreventive effects on estrogen carcinogenic pathways in vivo. Therefore, the effect of OTC botanicals on estrogen-induced malignant transformation of MCF-10A cells was studied. Cytochrome P450 catalyzed hydroxylation of estradiol at the 4-position leads to an o-quinone believed to act as the proximal carcinogen. Liquid chromatography/tandem mass spectrometry analysis of estradiol metabolites showed that 4-hydroxylation was inhibited by hops, whereas black cohosh was without effect. Estrogen-induced expression of CYP450 1B1 and CYP450 1A1 was attenuated by the hops extract. Two phenolic constituents of hops (xanthohumol, XH; 8-prenylnaringenin, 8-PN) were tested: 8-PN was a potent inhibitor, whereas XH had no effect. Finally, estrogen-induced malignant transformation of MCF-10A cells was observed to be significantly inhibited by hops (5 μg/mL) and 8-PN (50 nmol/L). These data suggest that hops extracts possess cancer chemopreventive activity through attenuation of estrogen metabolism mediated by 8-PN.

The hop chalcone xanthohumol beneficially affects hepatocellular lipid metabolism

Since ancient times, hop (Humulus lupulus L.) has been used in folkloric medicine. Xanthohumol (XN) is the main flavonoid found in hop inflorescences, and dietary intake is up to 100mg/day, largely depending on beer consumption. Furthermore, XN-containing dietary supplements are marketed, and we recently demonstrated that XN exhibits anti-fibrogenic effects on hepatic stellate cells and anti-tumorigenic effects on HCC cells, respectively. Little is known about the effects of XN on (hepatic) metabolisms. The aim of this study was to analyze the effects of XN on hepatocellular lipid metabolism. Methods and Results: Incubation with free fatty acids (FFA) led to a dose-dependent increase of lipid accumulation in primary human hepatocytes (PHH) as assessed by analysis of cellular triglyceride (TG) and FFA levels and as visualized by oil-red-O staining. This induced lipid accumulation as well as basal lipid levels were dose-dependently inhibited by pretreatment of PHH with XN (5, 10 and 15µM). At the concentrations used viability and mitochondrial activity were not impaired. The expression of fatty acid synthase, the key enzyme of lipogenesis as well as diglyceride acyltransferase, the enzyme responsible for the last step in TG synthesis, were impaired by XN. Moreover, XN affected palmitoyl-CoA oxidase and carnitine palmitoyltransferase 1 expression, indicative of impaired beta-oxidation. Conclusion: Together these data indicate the potential of XN to beneficially affect pathophysiological key mechanisms of nonalcoholic fatty liver disease (NAFLD). Thus, based on the known safety profile of hop extracts and XN, respectively, this hop constituent may safe as a potential new agent to prevent NAFLD development and progression.

Abstract A43: Hops (humulus lupulus) induce detoxification enzymes in vivo

Abstract A43 Extracts of hop strobiles, the female parts of Humulus lupulus L., are popular botanical dietary supplements as alternative treatment for menopausal symptoms (1). Cancer preventive properties of hops, which are in part mediated through the induction of detoxification enzymes, have frequently been reported (2). This effect has been mainly attributed to one of its major constituents, xanthohumol (XN) (3). However, the chemopreventive effects have so far only been demonstrated in vitro and have not been confirmed in vivo. Therefore, we conducted an in vivo study in adult female rats and analyzed the effect of oral administration of a standardized hop extract (7.5 g extract/kg BW per day, containing 2% XN) or s.c. injection of XN (20 mg/day) on detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase (GST). Analysis of various tissues revealed that while pure XN and oral hops significantly induced NQO1 activity in liver and colon tissue, only oral hops and not pure xanthohumol induced NQO1 activity in the uterus. Interestingly, hops but not xanthohumol significantly induced GST activity in the liver and mammary gland suggesting that other hop constituents and/or an additional mechanism might be responsible for these effects. Transcription of many detoxification enzymes is regulated through the antioxidant response element (ARE) and its transcription factor Nrf2, which is repressed under basal conditions by Keap1 (4). It has been shown that XN alkylates Keap1 and therefore enhances the concentration of free Nrf2 in the nucleus which results in ARE activation (5). Another pathway leading to ARE activation is mediated by protein kinase C (PKC) that phosphorylates Nrf2 leading to accumulation of Nrf2 in the nucleus (6). To investigate whether this pathway is involved in the observations described above, additional experiments with HepG2-ARE-luciferase C8 cells were performed. The cells were treated with hop extract or XN with or without the selective PKC inhbitor, Ro-32-0432, and the ARE-luciferase induction was determined. The results revealed that hops- but not XN-regulated ARE induction is in part mediated by PKC. Possible active compounds are under investigation. These results suggest that hop dietary supplements have the potential as preventive agents against cancer through induction of detoxifying enzymes. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A43.

Positive antibacterial co-action between hop (Humulus lupulus) constituents and selected antibiotics

The research reported here deals with co-action of the hop (Humulus lupulus)-derived anti-bacterial compounds, lupulone and xanthohumol, with several antibiotics. Among the antibiotics investigated for their co-action, polymyxin B sulfate, tobramycin and ciprofloxacin had a positive co-action in inhibiting selected test bacteria. The disc/well-diffusion assay and the minimum inhibitory concentration test (MIC) were employed to determine co-action. Both Gram-positive and Gram-negative bacteria were used in the evaluation. There was some co-action against all Gram-positive bacteria tested. Surprisingly, there was some positive co-action even against certain Gram-negative bacteria but not against others. Particularly, there was no co-action against E.coli. An antibacterial cream with lupulone, neomycin and polymyxin B sulfate was prepared and showed co-action. Ideas for other practical applications of this effect are put forth. The mechanism of the synergistic effect is briefly discussed but no attempt was made to prove it experimentally.