Formation and accumulation of alpha-acids,beta-acids,desmethylxanthohumol, and xanthohumol during flowering of hops (Humulus lupulus L.)

Important secondary metabolites, present in hops (Humulus lupulus L.), include alpha-acids and beta-acids, which are essential for the brewing of beer, as well as the prenylated chalcones, desmethylxanthohumol, and xanthohumol, which exhibit interesting bioactive properties. Their formation and accumulation in five selected hop varieties, Wye Challenger, Wye Target, Golding, Admiral, and Whitbread Golding Variety, were quantitatively monitored by high-performance liquid chromatography using UV detection. All target compounds were present from the onset of flowering, not only in female hop cones but also in male inflorescences, albeit in low concentrations. During development from female inflorescences to cones, levels of alpha-acids, beta-acids, desmethylxanthohumol, and xanthohumol gradually increased, while each hop variety exhibited individual accumulation rates. Furthermore, these compounds were present in leaves of fully grown hops as well. The study demonstrated that key compounds for flavor and potential beneficial health effects associated with beer not only reside in the glandular lupulin structures but also are distributed over various parts of the hop plant.     

Genic SSRs for European and North American hop (Humulus lupulus L.)

Eight genic SSR loci were evaluated for genetic diversity assessment and genotype identification in Humulus lupulus L. from Europe and North America. Genetic diversity, as measured by three diversity indices, was significantly lower in European cultivars than in North American wild accessions. Neighbor Joining cluster analysis separated the hop genotypes into European and North American groups. These eight SSRs were useful in uniquely identifying each accession with the exception of two sets of European landraces and a pair of Japanese cultivars, ‘Shinshuwase’ and ‘Kirin II’. An accession from Manitoba grouped with the European (EU) cluster reflecting the group’s genetic similarity to older Manitoba germplasm used to develop ‘Brewer's Gold’ and the gene pool arising from this cultivar. Cultivars grouped closely with one of their immediate parents. ‘Perle’ grouped with its parent ‘Northern Brewer and ‘Willamette’ grouped with its parent ‘Fuggle H’. Wild American accessions were divided into two subgroups: a North Central group containing mostly H. lupulus var. lupuloides and a Southwestern group containing H. lupulus var. neomexicanus accessions. These eight SSRs will be valuable for genotype identification in European and wild American germplasm and may potentially prove useful for marker-assisted selection in hop. PCR products from four previously reported primer pairs that amplify the same intronic SSR regions as do the genic SSRs in this study were compared in eight common cultivars. Different primer pairs generated robust markers at the chs2 and chi loci. However, only the HLC-004B and HLC-006 primer pairs amplified successfully at the chs3 and chs4 loci. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of hop (Humulus lupulus L.) flavonoids on aromatase (estrogen synthase) activity

The aim of this work was to study the effect of the prenylflavonoids xanthohumol, isoxanthohumol, and 8-prenylnaringenin on the activity and expression of the enzyme aromatase (estrogen synthase). The effect of different kinds of beer containing these prenylflavonoids was also tested. Aromatase activity was determined by measuring the release of tritiated water during the conversion of [(3)H]androstenedione to estrone. Aromatase expression was determined by RT-PCR. This assay was carried out in choriocarcinoma-derived JAR cells. The tested prenylflavonoids were able to inhibit estrogen formation, and their IC(50) values were determined, although no effect on aromatase expression was found. Lager beer, alcohol-free beer, stout beer, and xanthohumol-rich stout beer (200 microL/mL) significantly decreased aromatase activity. In conclusion, prenylflavonoids are able to modulate aromatase activity, decreasing estrogen synthesis, with relevance for the prevention and treatment of estrogen-dependent disorders such as breast cancer.     

Comparison of odor-active compounds in the spicy fraction of hop (Humulus lupulus L.) essential oil from four different varieties

The "spicy" character of hops is considered to be a desirable attribute in beer, associated with "noble hop aroma". However, the compounds responsible have yet to be adequately identified. Odorants in four samples of the spicy fraction of hop essential oil were characterized using gas chromatography-olfactometry (GC-O) and CharmAnalysis. Four hop varieties were compared, namely, Target, Saaz, Hallertauer Hersbrucker, and Cascade. Odor-active compounds were tentatively identified using comprehensive two-dimensional gas chromatography (GCxGC) combined with time-of-flight mass spectrometry (TOFMS). An intense "woody, cedarwood" odor was determined to be the most potent odorant in three of the four spicy fraction samples. This odor coincided with a complex region where between 8 and 13 compounds were coeluting in each of the four spicy fractions. The peak responsible was determined by (i) correlating peak areas with Charm values in eight hop samples and (ii) heart-cut multidimensional gas chromatography-olfactometry (MDGC-O). The compound responsible was tentatively identified as 14-hydroxy-beta-caryophyllene. Other important odorants identified were geraniol, linalool, beta-ionone, and eugenol.     

Direct thermal desorption-gas chromatography and gas chromatography-mass spectrometry profiling of hop (Humulus lupulus L.) essential oils in support of varietal characterization

The use of direct thermal desorption-gas chromatography-mass spectrometry (DTD-GC-MS) and DTD-GC-flame ionization detection (DTD-GC-FID) for characterization of hop essential oils is described. Four hop varieties (Nugget, Galena, Willamette, and Cluster) from the Yakima valley (Yakima, WA) 1998 harvest were analyzed by DTD-GC-MS and DTD-GC-FID methodology. Approximately 1 g of hops was needed for the analysis. Hop samples were prepared for GC-MS and/or GC-FID profiling in approximately 20 min. More than 100 volatile compounds have been identified and quantified for each hop variety. The results were found to be in good agreement with conventional steam distillation-extraction (SDE) data. A calibration curve for determination of essential oil content in hops by DTD-GC-FID has been generated. Quantitation of hop oil content by DTD-GC-FID was shown to be in good agreement with conventional SDE data. The recovery of key oil components valuable for varietal identification was demonstrated to be highly reproducible and characteristic of each variety analyzed when DTD-GC-FID was used for analysis.     

Constitutive expression of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its derivatives in substantial quantities

Resveratrol, a well-known phytoalexin and antioxidant, is produced by the action of stilbene synthase (STS) in some plant species. Hop (Humulus lupulus L.) plants of the Tettnang variety were transformed with a gene encoding for STS from grapevine. Under the control of the constitutive 35S cauliflower mosaic virus promoter, expression of the transgene resulted in accumulation of resveratrol and high levels of its glycosylated derivatives in leaves and inflorescences. Piceid, the predominant derivative, reached a concentration of up to 560 microg/g of fresh weight (f.w.) in hop cones, whereas no stilbenes were detected in nontransformed controls (wild-type). In transgenic plants the amounts of alpha- and beta-acids, naringenin chalcone, and prenylated flavonoids did not change significantly when compared with nontransformed plants. Transgenic plants showed normal morphology and flower development as did the nontransformed controls. The results clearly show that in hop constitutive expression of sts interferes neither with plant development nor with the biosynthesis of secondary metabolites relevant for the brewing industry. Since resveratrol is a well-known phytoalexin and antioxidant, sts transgenic hop plants could display enhanced pathogen resistance against microbial pathogens, exhibit new beneficial properties for health, and open new venues for metabolic engineering.     

Neuroprotective effects of xanthohumol, a prenylated flavonoid from hops (Humulus lupulus), in ischemic stroke of rats

Xanthohumol is the principal prenylated flavonoid in hops (Humulus lupulus L.), an ingredient of beer. Xanthohumol was found to be a potent chemopreventive agent; however, no data are available concerning its neuroprotective effects. In the present study, the neuroprotective activity and mechanisms of xanthohumol in rats with middle cerebral artery occlusion (MCAO)-induced cerebral ischemia were examined. Treatment with xanthohumol (0.2 and 0.4 mg/kg; intraperitoneally) 10 min before MCAO dose-dependently attenuated focal cerebral ischemia and improved neurobehavioral deficits in cerebral ischemic rats. Xanthohumol treatment produced a marked reduction in infarct size compared to that in control rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor (HIF)-1α, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), and active caspase-3 protein expressions in ischemic regions. These expressions were obviously inhibited by treatment with xanthohumol. In addition, xanthohumol (3-70 μM) concentration-dependently inhibited platelet aggregation stimulated by collagen (1 μg/mL) in human platelet-rich plasma. An electron spin resonance (ESR) method was used to examine the scavenging activity of xanthohumol on free radicals which had formed. Xanthohumol (1.5 and 3 μM) markedly reduced the ESR signal intensity of hydroxyl radical (OH?) formation in the H?O?/NaOH/DMSO system. In conclusion, this study demonstrates for the first time that in addition to its originally being considered an agent preventing tumor growth, xanthohumol possesses potent neuroprotective activity. This activity is mediated, at least in part, by inhibition of inflammatory responses (i.e., HIF-1α, iNOS expression, and free radical formation), apoptosis (i.e., TNF-α, active caspase-3), and platelet activation, resulting in a reduction of infarct volume and improvement in neurobehavior in rats with cerebral ischemia. Therefore, this novel role of xanthohumol may represent high therapeutic potential for treatment or prevention of ischemia-reperfusion injury-related disorders.     

Isomerization and degradation kinetics of hop (Humulus lupulus) acids in a model wort-boiling system

The rate of isomerization of alpha acids to iso-alpha acids (the compounds contributing bitter taste to beer) was determined across a range of temperatures (90-130 degrees C) to characterize the rate at which iso-alpha acids are formed during kettle boiling. Multiple 12 mL stainless steel vessels were utilized to heat samples (alpha acids in a pH 5.2 buffered aqueous solution) at given temperatures, for varying lengths of time. Concentrations of alpha acids and iso-alpha acids were quantified by high-pressure liquid chromatography (HPLC). The isomerization reaction was found to be first order, with reaction rate varying as a function of temperature. Rate constants were experimentally determined to be k1 = (7.9 x 10(11)) e(-11858/T) for the isomerization reaction of alpha acids to iso-alpha acids, and k2 = (4.1 x 10(12)) e(-12994/T) for the subsequent loss of iso-alpha acids to uncharacterized degradation products. Activation energy was experimentally determined to be 98.6 kJ per mole for isomerization, and 108.0 kJ per mole for degradation. Losses of iso-alpha acids to degradation products were pronounced for cases in which boiling was continued beyond two half-lives of alpha-acid concentration.     

Increase in cone biomass and terpenophenolics in hops ( Humulus lupulus L.) by treatment with prohexadione-calcium

Humulus lupulus L. (hop), a specialty crop bred for flavor characteristics of the inflorescence, is an essential ingredient in beer. Hop inflorescences, commonly known as hop cones, contain terpenophenolic compounds, which are important for beer flavoring and of interest in biomedical research. Hop breeders focus their efforts on increasing cone biomass and terpenophenolic content. As an alternative to traditional breeding, hops were treated with prohexadione-calcium (Pro-Ca), a growth inhibitor previously shown to have positive agronomic effects in several crops. Application of Pro-Ca to hop plants during cone maturation induced increases in cone biomass production by 1.5-19.6% and increased terpenophenolic content by 9.1-87.3%; however, some treatments also induced significant decreases in terpenophenolic content. Induced changes in cone biomass production and terpenophenolic accumulation were most dependent on cultivar and the developmental stage at which plants were treated.     

Hop (Humulus lupulus L.) proanthocyanidins characterized by mass spectrometry,acid catalysis,and gel permeation chromatography

Proanthocyanidins extracted from hops (Humulus lupulus L. cv. Willamette) were subjected to Sephadex LH-20 column chromatography using a step gradient of methanol, water, and acetone. The resulting fractions were analyzed by acid catalysis, electrospray ionization and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and gel permeation chromatography (GPC). The proanthocyanidins contained catechin and epicatechin as monomers and as terminal and extension units. Epigallocatechin was found as extension units. The mean degree of polymerization (mDP) of the crude proanthocyanidins was 7.8, but heptamers were the largest oligomers visible in mass spectra of the whole. In the last-eluted fraction (mDP = 22.2), polymers as large as 20-mers were detected by MALDI-TOF-MS, demonstrating the effectiveness of prior separation in improving MS detection. GPC data correlated well with acid catalysis results, confirming the presence of large polymers that were not detected by MS.     

Detection and quantification of provitamin D2 and vitamin D2 in hop (Humulus lupulus L.) by liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry

In this work, ergosterol and ergocalciferol were identified for the first time in hop. In addition, in this article, a simple and reliable analytical methodology for analysis of these compounds in different commercial forms of hop is presented. The performance of the method was assessed by the evaluation of parameters such as absolute recovery (higher than 70%), repeatability (lower than 3 %), linearity ( r(2) > 0.9988) and limits of detection (ranging from 0.034 for ergocalciferol to 0.058 mg/L for ergosterol) and quantification (ranging from 0.113 for ergocalciferol to 0.195 mg/L for ergosterol). On the basis of standard additions applied with the optimized procedure and high-performance liquid chromatography with diode array detection, it appears that the Nugget hop plant (crop 2006) contains 1.84 +/- 0.09 microg/g of ergosterol and 1.95 +/- 0.05 microg/g of ergocalciferol. The identity of the compounds was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry in the positive ion mode. The presence of ergosterol here reported should have great potential for the assessment of hop as related to the fungal contamination proportion and hence the quality of this raw material.     

Comparison of the most odor-active compounds in fresh and dried hop cones (Humulus lupulus L. variety spalter select) based on GC-olfactometry and odor dilution techniques

Application of aroma extract dilution analysis on the volatiles obtained from dried cones of Spalter Select hops grown in the German hop-growing area of Hallertau revealed 23 odorants in the flavor dilution (FD) factor range of 16-4096, 20 of which could be identified. On the basis of high FD factors, trans-4, 5-epoxy-(E)-2-decenal, linalool, and myrcene were identified as the most potent odorants, followed by ethyl 2-methylpropanoate, methyl 2-methylbutanoate, (Z)-1,5-octadien-3-one, nonanal, (E,Z)-1,3, 5-undecatriene, 1,3(E),5(Z),9-undecatetraene, propyl 2-methylbutanoate, 4-ethenyl-2-methoxyphenol, and 1-octen-3-one. Ten of the high-impact hop aroma compounds had previously not been identified as hop constituents and, in particular, 1,3(E),5(Z), 9-undecatetraene has not yet been reported as a food odorant. In an extract obtained from fresh hops, in addition to the odorants found in dry hops, (Z)-3-hexenal was characterized as a further key odorant rendering an additional green aroma note to the fresh material.     

Activation of proestrogens from hops (Humulus lupulus L.) by intestinal microbiota; conversion of isoxanthohumol into 8-prenylnaringenin

Hop, an essential ingredient in most beers, contains a number of prenylflavonoids, among which 8-prenylnaringenin (8-PN) would be the most potent phytoestrogen currently known. Although a number of health effects are attributed to these compounds, only a few reports are available about the bioavailability of prenylflavonoids and the transformation potency of the intestinal microbial community. To test these transformations, four fecal samples were incubated with xanthohumol, isoxanthohumol (IX), and 8-PN. Upon incubation with IX, present in strong ales up to 4 mg/L, 36% was converted into 8-PN in one fecal sample and the estrogenic properties of the sample drastically increased. In an experiment with 12 fecal cultures, this conversion was observed in one-third of the samples, indicating the importance of interindividual variability in the intestinal microbial community. Eubacterium limosum was identified to be capable of this conversion (O-demethylation) of IX into 8-PN, and after strain selection, a conversion efficiency of 90% was achieved. Finally, strain supplementation to a nonconverting fecal sample led to rapid and high 8-PN production at only 1% (v/v) addition. Up to now, the concentration of 8-PN in beer was considered too low to affect human health. However, these results show that the activity of the intestinal microbial community could more than 10-fold increase the exposure concentration. Because prenylflavonoids are present in many beers with IX being the major constituent, the results raise the question whether moderate beer consumption might contribute to increased in vivo levels of 8-PN and even influence human health.     

Phytochemical and morphological characterization of hop (Humulus lupulus L.) cones over five developmental stages using high performance liquid chromatography coupled to time-of-flight mass spectrometry, ultrahigh performance liquid chromatography photodiode array detection, and light microscopy techniques

Hop (Humulus lupulus L.) inflorescences, commonly known as "hop cones", are prized for their terpenophenolic contents, used in beer production and, more recently, in biomedical applications. In this study we investigated morphological and phytochemical characteristics of hop cones over five developmental stages, using liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF-MS), and ultrahigh performance liquid chromatography photodiode array detection (UHPLC-PDA) methods to quantitate 21 polyphenolics and seven terpenophenolics. Additionally, we used light microscopy to correlate phytochemical quantities with changes in the morphology of the cones. Significant increases in terpenophenolics, concomitant with glandular trichome development and associated gross morphological changes, were mapped over development to fluctuations in contents of polyphenolic constituents and their metabolic precursor compounds. The methods reported here can be used for targeted metabolic profiling of flavonoids, phenolic acids, and terpenophenolics in hops, and are applicable to quantitation in other crops.     

Occurrence of Odorant Polyfunctional Thiols in Beers Hopped with Different Cultivars. First Evidence of an S-Cysteine Conjugate in Hop ( Humulus lupulus L.)

Forty-one thiols, mainly β-sulfanylalkyl acetates, β-sulfanylalkyl alcohols, and β-sulfanylalkyl carbonyls, were recently evidenced in hop. In a beer hopped with the Tomahawk cultivar, most of them were found at higher levels than expected. The aim of the present work was to investigate the polyfunctional thiols in beers hopped with different varieties. A few thiols proved not to come only from hop (mainly 2-sulfanylethyl acetate, μg/L levels, and 1-sulfanylpentan-3-one and 1-sulfanylpentan-3-ol, ng/L levels, internal standard (IST) equivalents). The thiol profile of Saaz-hopped beer proved similar to that of the reference beer produced without hop. A high level of 3-sulfanyloctan-1-ol emerged as an indicator of the use of Tomahawk hop (140 ng/L, IST equivalents; FD (flavor dilution) = 65536). In both Cascade- and Tomahawk-hopped beers, 3-sulfanylhexan-1-ol and 3-sulfanylheptan-1-ol were smelled at high flavor dilutions, although only for the latter, significant amounts of the unreduced 3-sulfanylheptanal were found in hop. As already claimed for hop authentication, 3-sulfanyl-4-methylpentan-1-ol remains a good marker of Nelson Sauvin-hopped beers (548 ng/L, IST equivalents; FD = 65536), together with 4-sulfanyl-4-methylpentan-2-one (128 ng/L, FD = 4096). As illustrated by the huge production occurring during fermentation, accurate prediction of hop varietal impact requires quantitating thiol adducts in hop. S-3-(1-Hydroxyhexyl)cysteine was evidenced here for the first time in Cascade hop.     

Inventory methods for finding historically cultivated hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.) in Sweden

The relationship between the conservation of plant genetic resources and their culture history is intimate. Consequently, biodiversity research must also take into account historical and cultural factors. An inventory of plants collected from all over Sweden was made with the aim of establishing a national Swedish gene bank for once cultivated hop. Only female hop, which could be regarded as cultivated hop on the basis of history, were selected. In this study, two different inventory methods were used: one based on plant habitat and the other involving the use of historical documents, primarily large-scale maps from the first half of the seventeenth century, used to locate their cultivars today. The documented history of the hop, combined with its biology, is the basis for the methods used. Hop is the only Swedish crop that according to a 1442 law had to be cultivated. The law lasted for over 400 years. Since the hop is a perennial, dioecious plant and only female individuals are cultivated, over time very few genetic recombination events are expected. Today, it is possible to connect and identify living plants using historical documents. The degree of connection between today’s living plants and the historical evidence for hop cultivation differ between the two methods.     

The influence of European and American wild germplasm in hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.) cultivars

Microsatellite variation at the nuclear and chloroplast genomes was evaluated for wild European and wild American hops, in order to assess the genetic diversity and origin of cultivated hops. Seven nuclear loci and 32 chloroplast loci were used in the analysis of 182 hop accessions including wild European (68), wild American (48), and cultivars (66). A total of 116 alleles were identified using 7 nuclear microsatellites showing different averages of polymorphism and distribution in the wild American and European accessions and cultivars. Two main groups were established as revealed by several statistical analyses; one including European wild accessions and cultivars and a second group consisting of American wild accessions. Three polymorphic chloroplast microsatellite loci were detected, six alleles were scored which defined a total of five haplotypes that were exclusive or presented different distribution between American and European wild accessions. A major influence of the wild European haplotypes was detected among hop cultivars. To the best of our knowledge, this is the first work reporting the use of chloroplast microsatellites in hops.