Daucane esters from laserwort (Laserpitium latifolium L.) inhibit cytokine and chemokine production in human lung epithelial cells.

BACKGROUND: Laserwort, Laserpitium latifolium L. (Apiaceae), is a European medicinal plant. Its roots and rhizomes were traditionally used as a general tonic and to treat inflammatory and infective diseases. PURPOSE: The anti-inflammatory potential of daucane esters, isolated from underground parts extract of L. latifolium and specific structural features that contribute to their activity were investigated. In addition, we studied their interference with the transactivation capacity of the Glucocorticoid Receptor when added together with a classic glucocorticoid (GC), dexamethasone (DEX). This particular property may be relevant in combination strategies, attempting to circumvent diabetogenic side effects of glucocorticoids upon long-term anti-inflammatory treatments. MATERIALS AND METHODS: Nine L. latifolium daucane esters were isolated and elucidated as derivatives of desoxodehydrolaserpitin, laserpitin and a novel 2ß-esterified laserpitinol analogue. Of all compounds effects on NF-κB- and AP-1-driven pro-inflammatory pathways were assessed using TNF- or PMA-induced reporter gene analysis in A549 cells. Daucanes with a strong and concentration-dependent inhibition of both NF-κB and AP-1, were tested for a potential effect on DEX-stimulated GR-driven Glucocorticoid Response Element (GRE) reporter gene activity. In addition, GRE-driven anti-inflammatory mRNA expression was determined (GILZ and DUSP1). Also anti-inflammatory properties were validated by monitoring effects on CCL-2, IL-6, IL-1ß mRNA expression levels (qPCR) and on CCL-2 chemokine production (ELISA). RESULTS: Daucanes featuring an ester moiety and/or a hydroxy group at positions 2ß, 6α and 10α and especially the novel 2ß-esterified laserpitinol derivative that, in comparison to other isolated compounds, features an additional 9α-hydroxy group, demonstrated suppression of both NF-κB- and AP-1-dependent pro-inflammatory pathways. Remarkably, those entities competitively and concentration-dependently repressed GR-driven GRE-dependent reporter gene activities. The most active compounds inhibited CCL-2 protein excretion and compound 4 downregulated genes coding for IL-1ß and IL-6 induced upon TNF treatment in A549. In absence of TNF, compound 4 upregulated the GRE-mediated anti-inflammatory gene GILZ, but not DUSP1. CONCLUSIONS: Daucane esters are novel anti-inflammatory agents that may, in combination with GCs, potentially improve therapeutic benefit. These results contribute to the ongoing search for novel anti-inflammatory agents as safer alternatives to, or with, GCs.

Extracts of three Laserpitium L. species and their principal components laserpitine and sesquiterpene lactones inhibit microbial growth and biofilm formation by oral Candida isolates.

Antimicrobial properties of extracts of underground parts of three Laserpitium L. (Apiaceae) species, namely Laserpitium latifolium L., Laserpitium zernyi Hayek and Laserpitium ochridanum Micevski, were investigated. The investigated species are widely used as functional foods, as spices and for preparations in traditional medicine for treating complaints connected with infection and inflammation. Furthermore, antimicrobial and antibiofilm effects of laserpitine, the most abundant compound in the chloroform extract of L. latifolium, and guaianolide sesquiterpene lactones, such as, isomontanolide, montanolide and tarolide, principal components of the extracts of L. zernyi and L. ochridanum were assessed. The antimicrobial activity was tested using the microdilution method against five pathogenic bacteria and five fungi, as well as in the microplate biofilm assay on two Candida clinical isolates (C. albicans and C. krusei). Among the extracts, L. latifolium showed the most prominent activity. Isolated metabolites exerted higher effects against fungal than against bacterial strains, isomontanolide being the most active. Interestingly, all constituents showed higher potential on inhibition of biofilm formation than fluconazole, a reference compound. Tested metabolites may be good novel agents with high antifungal and antibacterial potential that might find practical applications in food industry as food preservatives in order to retard the growth of food spoiling microbes, but only after detailed safety assessments.

In vitro inhibition of the transcription factor NF-κB and cyclooxygenase by bamboo extracts.

Several bamboo species have been used in traditional medicine for the treatment of inflammatory conditions. The present study evaluates the in vitro anti-inflammatory properties of the traditionally used bamboo species Phyllostachys nigra (Lodd.) Munro and Sasa veitchii (Carr.) Rehder to explore their future research opportunities and therapeutic potential as anti-inflammatory agents. The extracts were evaluated for their potential inhibitory activity at the level of NF-κB-induced gene expression and suppression of cyclooxygenase (COX)-1 and COX-2 enzyme activities, representative pharmacological targets for the anti-inflammatory action of glucocorticoids and non-steroidal anti-inflammatory drugs, respectively. The activity of P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder was compared with bamboo species without traditional anti-inflammatory indications. High-performance liquid chromatography with diode-array detection and liquid chromatography-tandem mass spectrometry analyses were performed to phytochemically characterize the extracts. P. nigra (Lodd.) Munro leaf extract potently inhibited NF-κB-induced gene expression, while S. veitchii (Carr.) Rehder leaf extract exerted a selective COX-2 inhibition. The crude extracts consistently showed a more potent bioactivity than the solid phase extraction fractions. P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder both exert anti-inflammatory properties, but act via a different molecular mechanism.

Phenolic compounds and anti-oxidant capacity of twelve morphologically heterogeneous bamboo species.

INTRODUCTION: Despite the growing interest in the use of bamboo for both food and health-related applications because it provides a rich source of anti-oxidants, there is still a lack of information on the responsible secondary metabolites of the great variety of bamboo species. OBJECTIVE: To extend the knowledge on secondary metabolites of different bamboo species and to link anti-oxidant capacity with the different classes of phenolic compounds that are present in the leaves. METHODOLOGY: Chromatographic profiles of 12 morphological heterogeneous bamboo species from different genera were recorded using HPLC-DAD (diode array detector) and LC-MS/MS. In addition, the in vitro anti-oxidant capacity was evaluated using a variety of anti-oxidant assays (1,1-diphenyl-2-picrylhydrazyl, Trolox-equivalent anti-oxidant capacity and oxygen radical absorbance capacity). Using partial least square (PLS) analysis as a chemometric method, the anti-oxidant capacity could be linked to specific groups of polyphenols. RESULTS: Flavones and phenolic acids are the two main polyphenolic classes present in the leaf extracts of the 12 selected bamboo species. Luteolin derivatives and phenolic acids were identified as the most potent anti-oxidants. CONCLUSION: The most abundant classes of phenolic compounds present in a selection of bamboo species were flavone glycosides and phenolic acids. Luteolin flavones and phenolic acids are the main anti-oxidant phenolic compounds in bamboo leaf extract. The information obtained in this study provides further support for the development of bamboo-based anti-oxidant food applications and food supplements.

The intestinal microbiome: a separate organ inside the body with the metabolic potential to influence the bioactivity of botanicals.

For many years, it was believed that the main function of the large intestine was the resorption of water and salt and the facilitated disposal of waste materials. However, this task definition was far from complete, as it did not consider the activity of the microbial content of the large intestine. Nowadays it is clear that the complex microbial ecosystem in our intestines should be considered as a separate organ within the body, with a metabolic capacity which exceeds the liver with a factor 100. The intestinal microbiome is therefore closely involved in the first-pass metabolism of dietary compounds. This is especially true for botanical supplements, which are now marketed for various health applications. Being of natural origin, their structural building blocks, such as polyphenols, are often highly recognized by the human and especially the intestinal microbial metabolism machinery. Intensive metabolism results in often low circulating levels of the original products, with the consequence that final health effects of botanicals are often related to specific active metabolites which are produced in the body rather than being related to the product's original composition. Understanding how such metabolic processes contribute to the in situ exposure is therefore crucial for the proper interpretation of biological responses. A multidisciplinary approach, characterizing the food and phytochemical intake as well as the metabolic potency of the gut microbiota, while measuring biomarkers of both exposure and response in target tissues, is therefore of critical importance. With polyphenol metabolism as example, this review describes how the incorporation of microbial metabolism as an important variable in the evaluation of the final bioactivity of botanicals strongly increases the relevance and predictive value of the outcome. Moreover, knowledge about intestinal processes may offer innovative strategies for targeted product development.

Quantification of xanthohumol, isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin in hop extracts and derived capsules using secondary standards.

Hop is a well-known and already frequently used estrogenic phytotherapeutic, containing the interesting prenylflavonoids, xanthohumol (XN), isoxanthohumol (IXN), 8- and 6-prenylnaringenin (8-PN and 6-PN). Since the use of secondary standards can form a solution whenever the determination is required of certain components, not commercially available or too expensive, it was decided to develop an accessible HPLC-DAD method for the determination of these prenylflavonoids. The amounts were determined in hop extract and capsules, using quercetin and naringenin as secondary standards. After optimization of the sample preparation and HPLC conditions, the analysis was validated according to the ICH guidelines. The response function of XN, 8-PN, quercetin and naringenin showed a linear relationship. For the determination of XN, a calibration line of at least three concentrations of quercetin has to be constructed. The correction factors for XN (quercetin) and for 8-PN (naringenin) were validated and determined to be 0.583 for XN, and 1.296 for IXN, 8-PN and 6-PN. The intermediate precision was investigated and it could be concluded that the standard deviation of the method was equal considering time and concentration (RSD of 2.5-5%). By means of a recovery experiment, it was proven that the method is accurate (recoveries of 96.1-100.1%). Additionally, by analysing preparations containing hop extracts on the Belgian market, it was shown that the method is suitable for its use, namely the determination of XN, IXN, 8-PN and 6-PN in hop extract and capsules, using quercetin and naringenin as secondary standards.

Flavonoids from Artemisia annua L. as antioxidants and their potential synergism with artemisinin against malaria and cancer.

Artemisia annua is currently the only commercial source of the sesquiterpene lactone artemisinin.Since artemisinin was discovered as the active component of A. annua in early 1970s, hundreds of papers have focused on the anti-parasitic effects of artemisinin and its semi-synthetic analogs dihydroartemisinin, artemether, arteether, and artesunate. Artemisinin per se has not been used in mainstream clinical practice due to its poor bioavailability when compared to its analogs. In the past decade, the work with artemisinin-based compounds has expanded to their anti-cancer properties. Although artemisinin is a major bioactive component present in the traditional Chinese herbal preparations (tea), leaf flavonoids, also present in the tea, have shown a variety of biological activities and may synergize the effects of artemisinin against malaria and cancer. However, only a few studies have focused on the potential synergistic effects between flavonoids and artemisinin. The resurgent idea that multi-component drug therapy might be better than monotherapy is illustrated by the recent resolution of the World Health Organization to support artemisinin-based combination therapies (ACT), instead of the previously used monotherapy with artemisinins. In this critical review we will discuss the possibility that artemisinin and its semi-synthetic analogs might become more effective to treat parasitic diseases (such as malaria) and cancer if simultaneously delivered with flavonoids. The flavonoids present in A. annua leaves have been linked to suppression of CYP450 enzymes responsible for altering the absorption and metabolism of artemisinin in the body, but also have been linked to a beneficial immunomodulatory activity in subjects afflicted with parasitic and chronic diseases.

Disposition of hop prenylflavonoids in human breast tissue.

Hop-derived products may contain xanthohumol (XN), isoxanthohumol (IX), and the potent phytoestrogen 8-prenylnaringenin (8-PN). To evaluate the potential health effects of these prenylflavonoids on breast tissue, their concentration, nature of metabolites, and biodistribution were assessed and compared with 17beta-estradiol (E(2)) exposure. In this dietary intervention study, women were randomly allocated to hop (n=11; 2.04 mg XN, 1.20 mg IX, and 0.1 mg 8-PN per supplement) or control (n=10). After a run-in of >or=4 days, three supplements were taken daily for 5 days preceding an aesthetic breast reduction. Blood and breast biopsies were analyzed using HPLC-ESI-MS/MS. Upon hop administration, XN and IX concentrations ranged between 0.72 and 17.65 nmol/L and 3.30 and 31.50 nmol/L, and between 0.26 and 5.14 pmol/g and 1.16 and 83.67 pmol/g in hydrolyzed serum and breast tissue, respectively. 8-PN however, was only detected in samples of moderate and strong 8-PN producers (0.43-7.06 nmol/L and 0.78-4.83 pmol/g). Phase I metabolism appeared to be minor (approximately 10%), whereas extensive glucuronidation was observed (> 90%). Total prenylflavonoids showed a breast adipose/glandular tissue distribution of 38/62 and their derived E(2)-equivalents were negligible compared with E(2) in adipose (384.6+/-118.8 fmol/g, p=0.009) and glandular (241.6+/-93.1 fmol/g, p<0.001) tissue, respectively. Consequently, low doses of prenylflavonoids are unlikely to elicit estrogenic responses in breast tissue.

Development of a high-throughput LC/APCI-MS method for the determination of thirteen phytoestrogens including gut microbial metabolites in human urine and serum.

The investigation into the potential usefulness of phytoestrogens in the treatment of menopausal symptoms requires large-scale clinical trials that involve rapid, validated assays for the characterization and quantification of the phytoestrogenic precursors and their metabolites in biological matrices, as large interindividual differences in metabolism and bioavailability have been reported. Consequently, a new sensitive high-performance liquid chromatography-mass spectrometry method (HPLC-MS) for the quantitative determination of thirteen phytoestrogens including their most important gut microbial metabolites (genistein, daidzein, equol, dihydrodaidzein, O-desmethylangolensin, coumestrol, secoisolariciresinol, matairesinol, enterodiol, enterolactone, isoxanthohumol, xanthohumol and 8-prenylnaringenin) in human urine and serum within one single analytical run was developed. The method uses a simple sample preparation procedure consisting of enzymatic deconjugation followed by liquid-liquid extraction (LLE) or solid-phase extraction (SPE) for urine or serum, respectively. The phytoestrogens and their metabolites are detected with a single quadrupole mass spectrometer using atmospheric pressure chemical ionization (APCI), operating both in the positive and the negative mode. This bioanalytical method has been fully validated and proved to allow an accurate and precise quantification of the targeted phytoestrogens and their metabolites covering the lower parts-per-billion range for the measurement of relevant urine and serum levels following ingestion of phytoestrogen-rich dietary supplements.

Disposition of soy isoflavones in normal human breast tissue.

BACKGROUND: Despite decades of research on the relation between soy and breast cancer, questions regarding the absorption, metabolism, and distribution of isoflavones in breast tissue largely remain unanswered. OBJECTIVE: We evaluated the potential health effects of isoflavone consumption on normal breast tissue; isoflavone concentrations, metabolites, and biodistribution were investigated and compared with 17beta-estradiol exposure. DESIGN: In this dietary intervention study, healthy women were randomly allocated to a soy milk (n = 11; 16.98-mg genistein and 5.40-mg daidzein aglycone equivalents per dose), soy supplement (n = 10; 5.27-mg genistein and 17.56-mg daidzein aglycone equivalents per dose), or control (n = 10) group. After a run-in period > or = 4 d, 3 doses of soy milk or soy supplements were taken daily for 5 d before an esthetic breast reduction. Blood and breast biopsies were collected during surgery and analyzed with liquid chromatography-tandem mass spectrometry. RESULTS: After soy administration, genistein and total daidzein concentrations, which were expressed as aglycone equivalents, ranged from 135.1 to 2831 nmol/L and 105.1 to 1397 nmol/L, respectively, in hydrolyzed serum and from 92.33 to 493.8 pmol/g and 22.15 to 770.8 pmol/g, respectively, in hydrolyzed breast tissue. The major metabolites identified in nonhydrolyzed samples were genistein-7-O-glucuronide and daidzein-7-O-glucuronide, with an overall glucuronidation of 98%. Total isoflavones showed a breast adipose/glandular tissue distribution of 40:60, and their mean (+/-SEM) derived 17beta-estradiol equivalents toward estrogen receptor beta were 21 +/- 4-fold and 40 +/- 10-fold higher than the 17beta-estradiol concentrations in adipose (0.283 +/- 0.089 pmol/g, P < 0.001) and glandular (0.246 +/- 0.091 pmol/g, P = 0.001) fractions, respectively. CONCLUSION: After intake of soy milk and soy supplements, isoflavones reach exposure levels in breast tissue at which potential health effects may occur.

Cosupplementation of isoflavones, prenylflavonoids, and lignans alters human exposure to phytoestrogen-derived 17beta-estradiol equivalents.

The microbial metabolism of dietary phytoestrogens varies considerably among individuals and influences the final exposure to bioactive compounds. In view of the increasing number of food supplements combining several classes of phytoestrogens, the microbial potential to activate various proestrogens within an individual was evaluated in 3 randomized dietary crossovers. Treatment allocation was based on participants' eligibility (>45% in vitro bioactivation of >or=2 separate proestrogens by fecal cultures; n = 40/100). After a run-in of >or=4 d, participants were given soy-, hop-, and/or flax-based food supplements dosed either separately (SOY: 2.83 mg daidzein aglycone equivalents/supplement, HOP: 1.20 mg isoxanthohumol (IX)/supplement, or FLAX: 2.08 mg secoisolariciresinol (SECO) aglycone equivalents/supplement; reference intervention) or simultaneously (MIX; test intervention) 3 times/d for 5 d, followed by a wash-out period (>or=7 d) and the second intervention. Before and after each (co)supplementation, spot urine and serum were collected. In total, 22 equol, 19 8-prenylnaringenin (8-PN), and 21 enterolactone (ENL) producers completed the SOY+MIX, HOP+MIX, and FLAX+MIX trials, respectively. The microbial bioactivation of daidzein, IX, and SECO, generally decreased upon coincubation in vitro (equol: 4.4%, P = 0.164; 8-PN: 20.5%, P < 0.001; ENL: 44.3%, P < 0.001) and cosupplementation in vivo (equol: 28.3%, P = 0.009; 8-PN: 35.4%, P = 0.107; ENL: 35.9%, P = 0.003). Although the bioavailabilities of total isoflavones, prenylflavonoids, and lignans were not significantly affected upon coadministration, participants were exposed to lower phytoestrogen-derived 17beta-estradiol equivalents. In conclusion, the bioavailability of phytoestrogens, especially when given in mixtures, is subject to high interindividual variation. These findings support the importance of personalized screening when assessing the efficacy of such products and mixtures.

Hop bitter acids efficiently block inflammation independent of GRalpha, PPARalpha, or PPARgamma.

Hop (Humulus lupulus L.) is an essential ingredient of beer, where it provides the typical bitter taste, but is also applied in traditional folk medicine for sedative and antibacterial purposes. In this study, we demonstrate and compare the anti-inflammatory effect of various classes of hop bitter acids (HBA), including alpha-acids (AA), beta-acids (BA), and iso-alpha-acids (IAA), in fibroblasts, which are important players in the inflammatory response. All three studied classes of HBA blocked the tumor necrosis factor alpha (TNF)-induced production of the cytokine IL6, and inhibited the transactivation of the pro-inflammatory transcription factors nuclear factor kappa B (NF-kappaB), activator protein-1 (AP-1), and cAMP-response element-binding protein (CREB). In this respect, the six-membered ring compounds AA and BA showed equal potency, whereas the five-membered ring compounds, IAA, were effective only when used at higher concentrations. Furthermore, with regard to the mechanism of NF-kappaB suppression, we excluded a possible role for glucocorticoid receptor alpha (GRalpha), peroxisome proliferators-activated receptor alpha/gamma (PPARalpha or PPARgamma), nuclear receptors (NRs) that are also known to inhibit inflammation by directly interfering with the activity of pro-inflammatory transcription factors. Interestingly, combining hop acids and selective agonists for GRalpha, PPARalpha, or PPARgamma resulted in additive inhibition of NF-kappaB activity after TNF treatment, which may open up new avenues for combinatorial anti-inflammatory strategies with fewer side effects. Finally, systemic administration of HBA efficiently inhibited acute local inflammation in vivo.

Hop (Humulus lupulus)-derived bitter acids as multipotent bioactive compounds.

Hop acids, a family of bitter compounds derived from the hop plant (Humulus lupulus), have been reported to exert a wide range of effects, both in vitro and in vivo. They exhibit potential anticancer activity by inhibiting cell proliferation and angiogenesis, by inducing apoptosis, and by increasing the expression of cytochrome P450 detoxification enzymes. Furthermore, hop bitter acids are effective against inflammatory and metabolic disorders, which makes them challenging candidates for the treatment of diabetes mellitus, cardiovascular diseases, and metabolic syndrome. This review summarizes the current knowledge on hop bitter acids, including both phytochemical aspects, as well as the biological and pharmacological properties of these compounds.

Eubacterium limosum activates isoxanthohumol from hops (Humulus lupulus L.) into the potent phytoestrogen 8-prenylnaringenin in vitro and in rat intestine.

Recently, it was shown that the exposure to the potent hop phytoestrogen 8-prenylnaringenin (8-PN) depends on intestinal bacterial activation of isoxanthohumol (IX), but this occurs in only one-third of tested individuals. As the butyrate-producing Eubacterium limosum can produce 8-PN from IX, a probiotic strategy was applied to investigate whether 8-PN production could be increased in low 8-PN producers, thus balancing phytoestrogen exposure. Using fecal samples from high (Hop +) and low (Hop -) 8-PN-producing individuals, a Hop + and Hop - dynamic intestinal model was developed. In parallel, Hop + and Hop - human microbiota-associated rats were developed, germ-free (GF) rats acting as negative controls. IX and then IX + E. limosum were administered in the intestinal model and to the rats, and changes in 8-PN production and exposure were assessed. After dosing IX, 80% was converted into 8-PN in the Hop + model and highest 8-PN production, plasma concentrations, and urinary and fecal excretion occurred in the Hop + rats. Administration of the bacterium triggered 8-PN production in the GF rats and increased 8-PN production in the Hop - model and Hop - rats. 8-PN excretion was similar in the feces (294.1 +/- 132.2 nmol/d) and urine (8.5 +/- 1.1 nmol/d ) of all rats (n = 18). In addition, butyrate production increased in all rats. In conclusion, intestinal microbiota determined 8-PN production and exposure after IX intake. Moreover, E. limosum administration increased 8-PN production in low producers, resulting in similar 8-PN production in all rats.

Microbial and dietary factors are associated with the equol producer phenotype in healthy postmenopausal women.

Equol, a microbial metabolite of daidzein, has been hypothesized as a clue to the effectiveness of soy and its isoflavones but is excreted by only 33% of Caucasians. Microbial and dietary factors associated with the ability to harbor equol-producing bacteria were studied in a randomized dietary intervention trial with 100 healthy postmenopausal women. After a 4-d baseline period, subjects delivered first-void urine, fecal, and breath samples. During the 5-d treatment period, 3 portions of either soymilk or soy germ containing 28.51 and 37.99 mg isoflavone aglycone equivalents/portion, respectively, were administered daily, and on the last day, 24-h urine samples were collected. The urinary recoveries of genistein and daidzein from soymilk were significantly higher than those from soy germ tablets. Because the proportion of equol:(daidzein + metabolites) in the urine did not differ between the treatment groups, subjects were pooled and classified into poor, moderate, and strong equol producers based on this criterion. The strong equol producer phenotype correlated negatively [in vivo, r = -0.478 (-0.256 to -0.893), P = 0.021; in vitro, r = -0.576 (-0.350 to -0.949), P = 0.030] with Clostridium coccoides-Eubacterium rectale counts and positively [in vivo, r = 1.158 (0.971-1.380), P = 0.048; in vitro, r = 1.156 (1.007-1.327), P = 0.039] with the abundance of sulfate-reducing bacteria. Furthermore, persons with a higher PUFA [in vivo, r = 2.150 (1.058-4.371), P = 0.034; in vitro, r = 2.131 (1.144-3.967), P = 0.017] and alcohol [in vivo, r = 1.166 (0.721-1.887), P = 0.050; in vitro, r = 1.850 (1.215-2.817), P = 0.004] intake were more likely to be strong equol producers. Finally, we validated the daidzein metabolism by fecal cultures as screening assay to identify equol producers without dietary intervention.

Microbial and dietary factors associated with the 8-prenylnaringenin producer phenotype: a dietary intervention trial with fifty healthy post-menopausal Caucasian women.

Hop-derived food supplements and beers contain the prenylflavonoids xanthohumol (X), isoxanthohumol (IX) and the very potent phyto-oestrogen (plant-derived oestrogen mimic) 8-prenylnaringenin (8-PN). The weakly oestrogenic IX can be bioactivated via O-demethylation to 8-PN. Since IX usually predominates over 8-PN, human subjects may be exposed to increased doses of 8-PN. A dietary intervention trial with fifty healthy post-menopausal Caucasian women was undertaken. After a 4 d washout period, participants delivered faeces, blank urine and breath samples. Next, they started a 5 d treatment with hop-based supplements that were administered three times per d and on the last day, a 24 h urine sample was collected. A semi-quantitative FFQ was used to estimate fat, fibre, alcohol, caffeine and theobromine intakes. The recoveries of IX, 8-PN and X in the urine were low and considerable inter-individual variations were observed. A five-fold increase in the dosage of IX without change in 8-PN concentration resulted in a significant lower IX recovery and a higher 8-PN recovery. Classification of the subjects into poor (60%), moderate (25%) and strong (15%) 8-PN producers based on either urinary excretion or microbial bioactivation capacity gave comparable results. Recent antibiotic therapy seemed to affect the 8-PN production negatively. A positive trend between methane excretion and 8-PN production was observed. Strong 8-PN producers consumed less alcohol and had a higher theobromine intake. From this study we conclude that in vivo O-demethylation of IX increases the oestrogenic potency of hop-derived products.

Withaferin a strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity.

The transcription factor NFkappaB plays a critical role in normal and pathophysiological immune responses. Therefore, NFkappaB and the signaling pathways that regulate its activation have become a major focus of drug development programs. Withania somnifera (WS) is a medicinal plant that is widely used in Palestine for the treatment of various inflammatory disorders. In this study we show that the leave extract of WS, as well as its major constituent withaferin A (WA), potently inhibits NFkappaB activation by preventing the tumor necrosis factor-induced activation of IkappaB kinase beta via a thioalkylation-sensitive redox mechanism, whereas other WS-derived steroidal lactones, such as withanolide A and 12-deoxywithastramonolide, are far less effective. To our knowledge, this is the first communication of IkappaB kinase beta inhibition by a plant-derived inhibitor, coinciding with MEK1/ERK-dependent Ser-181 hyperphosphorylation. This prevents IkappaB phosphorylation and degradation, which subsequently blocks NFkappaB translocation, NFkappaB/DNA binding, and gene transcription. Taken together, our results indicate that pure WA or WA-enriched WS extracts can be considered as a novel class of NFkappaB inhibitors, which hold promise as novel anti-inflammatory agents for treatment of various inflammatory disorders and/or cancer.

The hop phytoestrogen, 8-prenylnaringenin, reverses the ovariectomy-induced rise in skin temperature in an animal model of menopausal hot flushes.

The mechanisms underlying menopausal hot flushes are poorly understood, although it is generally assumed they result from disturbances of thermoregulatory centres in the hypothalamus. 8-Prenylnaringenin (8-PN) has been identified as a potent phytoestrogen in hops (Humulus lupulus) and there are claims that hop-containing preparations can reduce hot flushes. We have investigated the site of action of 8-PN in a rat model of menopausal hot flushes, in which the tail skin temperature (TST) is increased after oestrogen withdrawal induced by ovariectomy. Daily s.c. administration of either 17beta-oestradiol (E2; 4 microg/kg) or 8-PN (400 microg/kg) significantly reduced the elevated TST after 2 days of treatment. Subcutaneous co-administration of either E2 or 8-PN with the oestrogen receptor (ER) antagonist, ICI 182,780 (200 microg/kg), which is thought not to cross the blood-brain barrier, completely blocked the effect of E2 and 8-PN on TST. The ERalpha- and ERbeta-specific agonists, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (100 microg/kg) and 2,3-bis(4-hydroxyphenyl)-propionitrile (60 microg/kg) respectively, both significantly reversed the raised TST in ovariectomised rats. These observations suggest that the regulation of the vasomotor response by oestrogens and phytoestrogens is mediated, at least in part, by peripheral mechanisms involving both ERalpha and ERbeta.

The prenylflavonoid isoxanthohumol from hops (Humulus lupulus L.) is activated into the potent phytoestrogen 8-prenylnaringenin in vitro and in the human intestine.

Hops, an essential beer ingredient, are a source of prenylflavonoids, including 8-prenylnaringenin (8-PN), one of the most potent phytoestrogens. Because 8-PN concentrations in beers are generally low, its health effects after moderate beer consumption were considered negligible. However, human intestinal microbiota may activate up to 4 mg/L isoxanthohumol (IX) in beer into 8-PN. Depending on interindividual differences in the intestinal transformation potential, this conversion could easily increase the 8-PN exposure 10-fold upon beer consumption. Here, we present a further investigation of the process both in vitro and in vivo. In vitro experiments with the dynamic SHIME model showed that hop prenylflavonoids pass unaltered through the stomach and small intestine and that activation of IX into 8-PN (up to 80% conversion) occurs only in the distal colon. In vitro incubations of 51 fecal samples from female volunteers with IX enabled us to separate the fecal microbiota into high (8 of 51), moderate (11 of 51) and slow (32 of 51) 8-PN producers, clearly illustrating an interindividual variability. Three women, selected from the respective groups, received a daily dose of 5.59 mg IX for 4 d. Intestinal IX activation and urinary 8-PN excretion were correlated (R(2) = 0.6417, P < 0.01). These data show that intestinal conversion of IX upon moderate beer consumption can lead to 8-PN exposure values that might fall within the range of human biological activity.

A first prospective, randomized, double-blind, placebo-controlled study on the use of a standardized hop extract to alleviate menopausal discomforts.

OBJECTIVES: To examine the efficacy of a hop extract enriched in 8-prenylnaringenin (8-PN, the phytoestrogen in hops, Humulus lupulus L.) on relief of menopausal discomforts. METHODS: A prospective, randomized, double-blind, placebo-controlled study over 12 weeks with 67 menopausal women, who were administered a hop extract standardized on 8-PN (100 or 250 microg). The responses were determined by means of a modified Kupperman index (KI) and a patients' questionnaire. RESULTS: All groups, including placebo, showed a significant reduction of the KI both after 6 weeks and after 12 weeks. The hop extract at 100 microg 8-PN was significantly superior to placebo after 6 weeks (P=0.023) but not after 12 weeks (P=0.086). No dose-response relationship could be established, as the higher dose (250 microg) was less active than the lower dose both after 6 weeks and after 12 weeks. Still, a trend for a more rapid decrease of KI was noticed for both active groups as compared to placebo. In particular, the decrease in hot flush score (isolated from the KI) was found significant for both treatment groups after 6 weeks (P<0.01) with respect to placebo. Results of the patients' questionnaire were consistent with those of the KI, with the most pronounced effects being observed for the 100-microg treatment. CONCLUSIONS: Daily intake of a hop extract, standardized on 8-PN as a potent phytoestrogen, exerted favorable effects on vasomotor symptoms and other menopausal discomforts. Hop-derived prenylated flavonoids may provide an attractive addition to the alternative treatments available for relief of hot flushes and other menopausal discomforts.