The Multiple Biological Targets of Hops and Bioactive Compounds

Abstract

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.

Figure 1.

Main bioactive compounds in the standardized spent hop extract. The concentrations of the compounds in the hop extract were determined as described in ref 8. P450 1A2 and the human gut bacterium Eubacterium limosum have been described to metabolize IX to 8-PN.^, While P450 mediated O-demethylation of XH to DMX is theoretically possible, it has not been analyzed in detail and might not be clinically relevant.

Figure 2.

Estrogenicity of 8-PN enantiomers compared with estradiol and the standardized spent hop extract. ERα endometrial carcinoma cells (Ishikawa) were grown in 96 well plate (5 × 10⁴ cells/well) in estrogen-free media for 24 h. Subsequently, they were treated with the spent hop extract and compounds at varied concentrations for 96 h at 37 °C, then washed and lysed. Induced estrogen-dependent alkaline phosphatase activity was evaluated spectrophotometrically at 405 nm by the absorbance of the formed p-nitrophenol from the p-nitrophenol phosphate substrate (1 mg/mL). Estrogenic activity of respective treatments was assessed by comparison to the estradiol control (1 nM). The results represent the mean ± SD of at least three independent experiments in triplicate for each sample. The dose response curves were generated using GraphPad Prism.

Figure 3.

Formation of estrone/estradiol catalyzed by aromatase and aldoketoreductase 1C3 (AKR1C3).

Figure 4.

Inhibition of aromatase by hops and hop compounds. Aromatase activity was determined using the CYP19/MFC High- Throughput Inhibitor Screening Kit according to the manufacturer’s protocol (Corning, Woburn, MA). The NADPH-cofactor mix was prepared and added to the 96-well plate. The standardized spent hop extract, the hop compounds, and the positive control, ketoconazole, were dissolved in acetonitrile, added to the 96-well plate, and preincubated for 10 min after serial dilutions were performed. The compounds were then incubated with the enzyme/substrate mix (CYP19/7-methoxy-4-trifluoromethyl coumarin) for 30 min. The stop reagent was added thereafter, and the fluorescent signal was read at an excitation of 409 nm and emission of 530 nm. The data represent the average ± SD of three experiments. The dose response curves were generated using GraphPad Prism.

Figure 5.

6-PN, an AhR agonist leading to the induction of P450 1A1. 6-PN increases estrogen metabolism after AhR-mediated upregulation of estrogen metabolizing enzymes, P450 1A1 and P450 1B1. AhR induction can be regarded as an antiestrogenic mechanism, since P450 1A1 and P450 1B1 induce 2- and 4-hydroxylation of estrogens and thus limit E₂ concentrations and AhR activation mediates ubiquitination and degradation of ERα.

Figure 6.

XH alkylation of cysteine residues in Keap1 leading to activation and translocation of Nrf2 to the nucleus inducing the synthesis of detoxification enzymes such as NQO1, HO-1, and GST.^,

Figure 7.

XH alkylation of IKK and NF-κB cysteine residues. IκBα sequesters NF-κB in the cytoplasm, yet inflammatory stimuli activate IKK phosphorylation of IκBα and subsequent IκBα degradation. This allows NF-κB to translocate to the nucleus and activate inflammatory genes, such as iNOS and COX-2. XH is anti-inflammatory, since it alkylates cysteine residues in IKK, which prevents degradation of IκBα, and cysteine residues in NF-κB, which prevents NF-κB DNA binding.

Figure 8.

Hops and XH inhibition of NF-kB luciferase. MCF-7 cells were grown in six-well plates (25 × 10⁴ cells/well) for 24 h in estrogen-free media at 37 °C. After 24 h, cells were transfected with the NF-κB-luciferase reporter plasmid and treated with TNF-α (10 ng/mL) and (A) the standardized spent hop extract or (B) XH for 6 h before lysis with passive lysis buffer. NF-κB-luciferase activity was measured using the Promega Dual-Luciferase Reporter Assay System (Madison, WI). Results represent the mean ± SD of three independent experiments analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test; *p < 0.001.

Figure 9.

Hops and XH inhibition of iNOS. RAW 264.7 cells were plated at a density of 12 × 10⁴ cells/mL in 96-well plates and incubated at 37 °C for 24 h. Cells were treated with LPS (1 μg/mL) and (A) the standardized spent hop extract or (B) XH and incubated at 37 °C for 24 h. Griess reagent (150 μL of 0.5% sulfanilamide and 0.05% (N-1-naphthyl) ethylenediamine dihydrochloride in 2.5% w/w H₃PO₄) was added to a 96-well plate containing media collected from cells (50 μL). Nitrite levels were detected after the plate was incubated at RT for 30 min. Absorbance was measured at 530 nm, and concentrations were calculated using a NaNO₂ standard curve. Results represent the mean ± SD of three independent experiments and are analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test; *p < 0.001.

Figure 10.

XH induction of apoptosis at high concentrations. XH treatment initiates intrinsic and extrinsic apoptosis pathways by multiple mechanisms, including downregulation of antiapoptotic (BCL-2) and induction of pro-apoptotic (Bax) proteins, increases in ROS, leading to decreased mitochondrial membrane potential and subsequent cytochrome C release, and activation of caspases. As a result, cleavage of PARP and reduced proliferation of tumor cells has been observed after XH treatments.^–

Figure 11.

XH influence on lipid and glucose metabolism. XH exhibits antiobesity and antiglycemic effects by modulating the AMPK pathway. XH increases ROS, which can directly activate AMPK or decrease mitochondrial coupling, resulting in an increased AMP/ATP ratio, and in turn activate the AMPK pathway. AMPK phosphorylates multiple targets, including PPAR-α and SREBP, which results in increased β-oxidation and lipolysis and decreased fatty acid synthesis and adipogenesis.^–

Figure 12.

Designer extracts. Two spent hop extracts were designed with different concentrations of 8-PN. One extract with higher 8-PN concentrations modulates estrogenic effects in postmenopausal women, yet retains chemopreventive effects derived from XH. The second extract designated for use by younger women (premenopausal extract) contains lower concentrations of 8-PN than the postmenopausal extract but similar XH concentrations with potential chemopreventive properties.

Figure 13.

Summary of biological targets of spent hop extracts.