Arne Heyerick

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.

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 products 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.

Disposition of soy isoflavones in normal human breast tissue

BACKGROUNDnDespite 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.nnnOBJECTIVEnWe 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.nnnDESIGNnIn 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.nnnRESULTSnAfter 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.nnnCONCLUSIONnAfter intake of soy milk and soy supplements, isoflavones reach exposure levels in breast tissue at which potential health effects may occur.

A randomized, double-blind, placebo-controlled, cross-over pilot study on the use of a standardized hop extract to alleviate menopausal discomforts

OBJECTIVESnTo examine the efficacy of a hop extract (standardized at 100mug 8-prenylnaringenin per day) for relief of menopausal discomforts.nnnMETHODSnA 16-week randomized, double-blind, placebo-controlled, cross-over study was conducted with 36 menopausal women. The participants were randomly allocated to either placebo or active treatment (hop extract) for a period of eight weeks after which treatments were switched for another eight weeks. The Kupperman Index (KI), the Menopause Rating Scale (MRS) and a multifactorial Visual Analogue Scale (VAS) were assessed at baseline, and after eight and sixteen weeks.nnnRESULTSnAfter 8 weeks, both active treatment and placebo significantly improved all outcome measures when compared to baseline with somewhat higher average reductions for placebo than for the active treatment. After 16 weeks only the active treatment after placebo further reduced all outcome measures, whereas placebo after active treatment resulted in an increase for all outcome measures. Although, the overall estimates of treatment efficacy (active treatment-placebo) based on linear mixed models do not show a significant effect, time-specific estimates of treatment efficacy indicate significant reductions for KI (P = 0.02) and VAS (P = 0.03) and a marginally significant reduction (P = 0.06) for MRS after 16 weeks.nnnCONCLUSIONSnWhereas the first treatment period resulted in similar reductions in menopausal discomforts in both treatment groups, results from the second treatment period suggest superiority of the standardized hop extract over placebo. Thus, phytoestrogen preparations containing this standardized hop extract may provide an interesting alternative to women seeking relief of mild vasomotor symptoms.

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.

Antioxidant Flavone Glycosides from the Leaves of Fargesia robusta

The aqueous methanolic leaf extract of Fargesia robusta var. Pingwu was evaluated in vitro for its antioxidant capacity using the TEAC and ORAC assays. C-Glycosyl flavones, farobin A (1) and farobin B (2), together with three known compounds, tricin-5-O-glucopyranoside (3), 2-O-α-rhamnosyl-6-C-(6-deoxy-ribo-hexos-3-ulosyl)luteolin (4), and luteolin-6-C-glucopyranoside (homoorientin) (5), were isolated from the hydroalcoholic extract of the leaves of F. robusta. The structures of the compounds were determined by spectroscopic analyses including UV, 1D and 2D NMR, and MS. Compounds 1, 4, and 5 exhibited potent antioxidant activity in the TEAC assay, while compounds 1, 3, and 5 showed the highest antioxidant capacity in the ORAC assay.

Transport of hop bitter acids across intestinal Caco-2 cell monolayers.

Several health-beneficial properties of hop bitter acids have been reported (inhibition of bone resorption and anticarcinogenic and anti-inflammatory activities); however, scientific data on the bioavailability of these compounds are lacking. As a first approach to study the bioavailability, the epithelial transport of hop alpha- and beta-acids across Caco-2 monolayers was investigated. Hop acids were added either to the apical or to the basolateral chamber and, at various time points, amounts transported to the receiving compartment were determined. The monolayer integrity control was performed by using marker compounds (atenolol and propranolol), transepithelial electrical resistance (TEER) measurement, and determination of the fluorescein efflux. The TEER and fluorescein efflux confirmed the preservation of the monolayer integrity. The membrane permeability of the alpha-acids (apparent permeability coefficients for apical to basolateral transport (P(appAB)) ranged from 14 x 10(-6) to 41 x 10(-6) cm/s) was determined to be substantially higher than that of the beta-acids (P(appAB) values ranging from 0.9 x 10(-6) to 2.1 x 10(-6) cm/s). Notably, the beta-acids exhibited significantly different bidirectional P(app) values with efflux ratios around 10. The involvement of carrier-mediated transport for beta-acids (active efflux pathway by P-gp, BCRP, and/or MRP-2 type efflux pumps) could be confirmed by transport experiments with specific inhibitors (verapamil and indomethacin). It appears that alpha-acids are efficiently absorbed, whereas the permeability of beta-acids is low. Limiting factors in the absorption of beta-acids could involve P-gp and MRP-2 type efflux transporters and phase II metabolism.

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. Copyright

Hop bitter acids efficiently block inflammation independent of GRα, PPARα, or PPARγ

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.