Hops Provide an Abundant Source of Nutritionally and Pharmaceutically Relevant Compounds
In the search for plant products and phytochemicals that may prove useful in medicine or industry, the food and beverage processing industries are uniquely positioned to explore innovative techniques for extracting and separating valuable compounds. Hops (Humulus lupulus, Cannabaceae) plants contain many compounds with antioxidative, antimicrobial, anticarcinogenic, and other effects that are used in the pharmaceutical and food industries. Hops flower cones have been a principal ingredient of beer for a millennium, since the discovery of its brewing. Many compounds have been identified in hops of different strains, grown under different conditions, extracted by different methods, etc., some of them novel. In accordance with the biorefinery concept, spent hops biomass may be a rich source of high added-value products. The authors review the extraction and fractionation techniques used to isolate some important hops compounds.
To obtain hops extracts with high polyphenol content for use in food preservation, alcohol and fossil fuel solvents are commonly used. The latter must be tightly controlled to avoid residues. Additional purification steps add to processing time and costs. Overall, water and carbon dioxide (CO2) solvents are safer. Use of gases, including noble gases, in sub- or supercritical extraction gives many natural products. Supercritical CO2 is used, e.g., in the decaffeination of coffee (Coffea arabica, Rubiaceae) and for hops extracts. CO2 easily penetrates plant matter with high solvent power. However, it is largely limited to dry raw materials and for obtaining compounds of low polarity and low molecular weight. Steam distillation has traditionally been used to obtain hops essential oils (EOs). Supercritical CO2 is an effective substitute for highly nonpolar organic solvents. Liquid CO2 (LCO2) effectively extracts resins, aromatic compounds, paraffin, naphthenes, olefins, alcohols, aldehydes, phenol esters, carboxylic acids, amines and nitrogen heterocycles, ketones, ethers, amides, and nitrites, but not polyphenols. For polyphenol extraction, 90% ethanol is most used but produces a lower proportion of polyphenols than other hops compounds. Other organic solvents may be useful for polyphenol extraction; again, in foods, residues are strictly limited. In brewing, manufacture of hops extracts rests primarily on CO2. The big difference between extracts obtained with CO2 and those obtained with organic solvents is the lack of residues in the former.
Despite abundant reports on solvents and methods of polyphenol extraction from plant material, there are few on the effects of extraction conditions on polyphenol content or antioxidative activity of hop extracts. Solid phase microextraction is used to characterize hops aromatic properties, but to isolate non-volatile compounds both solid-phase and solvent extraction is used. Accelerated solvent extraction has been used to isolate bitter acids (BAs) from hops and hops products. Ultrasound-assisted extraction offers lower heat levels, high yields, and short processing time. Supercritical fluid extraction, mainly used in breweries to obtain BAs, is effective for isolating both volatile and non-volatile compounds. Operating conditions, temperature, and pressure affect extract composition. Lower temperatures produce more EOs; high temperatures and pressures, more BAs and resins. A novel method of isolating hops acids uses quaternary ammonium compounds to produce soluble quaternary ammonium salts. Over 200 compounds have been reported in beer via various analytical methods. In hops EOs, monoterpenes and sesquiterpenes like humulene, bisabolene, caryophyllene, farnesene, and elemene, usually measured via gas chromatography-mass spectrometry (GC-MS), predominate.
Hops acids, known for their high oxidative potential, are best quantified using high-performance liquid chromatography (HPLC), often coupled with atmospheric pressure ionization tandem mass spectrometry (APCI-MS-MS) or negative electrospray ionization mass spectrometry (NEI-MS). LC with tandem MS (LC-MS-MS) has been used to quantify prenylflavonoids. Several phenolics, including novel ones, were reported in beer using ion trap quadrupole Orbitrap high-resolution MS (LTQ-Orbitrap HR-MS). This method is under investigation with scant data on phenolic profiles of hops compounds obtained with it. Xanthohumol (XNH) was detected in hops by HPLC + ultraviolet detection. Isoxanthohumol (I-XNH) is metabolized by the human liver to form 8-prenylnaringenin (8-PN), an isomerization product of desmethylxanthohumol and potent phytoestrogen. Cytochrome P450 enzymes causing this reaction have been identified.
Similarly, a variety of analytical techniques are used to assess beer’s stability in storage through hops’ phenolic content and antioxidative effects. Oxidation and polymerization of endogenous polyphenols and their interactions with proteins are mostly responsible for beer turbidity. Total phenolics and antioxidative activities of hops extracts are generally determined using well-established methods, such as the 2,2-diphenyl-1-picrylhydrazyl radical scavenging, oxygen radical absorbance capacity, hydrogen peroxide scavenging, or linoleic acid assays. Most studies have focused on known classes of beer compounds, and there is a lack of studies identifying beer’s key antioxidants. Target antioxidants should be isolated, purified, and their structures elucidated using LC-MS and 1D/2D nuclear magnetic resonance (NMR) spectrometry.
Hops’ unique BAs inhibit Gram-positive bacteria, but data are lacking on antimicrobial properties of individual hops compounds. Lupulone, humulone, isohumulone, and humulinic acid all show high activity against some bacteria. Seven beer flavonoids had significant activity against methicillin-sensitive and resistant Staphylococcus aureus and S. epidermis. There is a need for new antimicrobials that specifically target oral pathogens. Natural preservatives help lengthen shelf-life of processed foods and improve food safety. Residues from hops extracts, a brewing industry waste product, maybe a rich source of compounds with antimicrobial and/or antioxidant activity. Diffusion methods are most used to assess the antibacterial activity of natural substances. Among many that inhibit microbial growth, flavone, quercetin, and naringenin are highly active. Widely found in plants, all occur in significant amounts in hops extracts and biomass. Hops extracts obtained with supercritical CO2 extraction had significant activity against investigated bacterial strains. XNH had the highest activity against all tested strains. Potential hops antimicrobial agents include α- and β-acids, humulones, lupulones, and isomerized forms of humulones. Among organic solvents, methanol and ethanol are effective in isolating compounds with high antimicrobial activity; n-hexane, less so.
Possible toxicity of isolated plant compounds, including genotoxicity, is assessed in many ways. In most cases, tests to determine genotoxicity may be used, with minor modifications, to determine antigenotoxic potential. The Comet assay, for example, is simple, economical, and can be applied to various cells and tissues to determine DNA damage and repair. Improvements to the basic assay are described. Introduction of specific agents allows observation of the removal of oxidized and alkylated bases through base excision repair and of UV-induced cyclobutane pyrimidine dimers through nucleotide excision repair. Investigators of many diseases are united, the authors say, in the belief that the Comet assay has “enormous potential” for daily clinical practice, with several advantages over other methods. Another universally applicable method for testing genotoxicity is the cytokinesis-block micronucleus assay. It is used to assess chromosomal instability, cell death, and cytostasis through direct and indirect measures of cellular and nuclear dysfunction. Since many natural compounds have pharmaceutical potential, in silico analysis is a reasonable way to limit candidates for more costly experimental testing or to find new, stronger candidates. The European Food Safety Authority has proposed adding computational results to toxicity profiles. Different approaches to in silico toxicity modeling are discussed briefly.
While hops plants are one species, there are three significantly different types (North American, Asian, and European) and many cultivars of each type, developed in the never-ending search for better beer flavor and aroma. Flower cones of female plants are used in beer production, with a bitter taste, aroma, and antimicrobial properties. Hops cones contain several functional groups of compounds. Their secondary metabolites are mostly BAs and their derivatives, polyphenols, and EO components. These groups are presented separately, with the main types of compounds found in hops, their skeleton structures, and representative compounds for each. For example, for hard resins/
polyphenols, multifidol glucosides, almost exclusive to hop, are listed with catechins, flavonols, phenolic acids (hydroxybenzoic, hydrocinnamic, and their derivatives), prenylflavonoids, and stilbenes. Under flavonols, kaempferol and quercetin are named; under prenylflavonoids, XNH and I-XNH/8-PN. Each group of compounds is discussed.
BAs and XNH also occur in male hops inflorescences, at levels comparable to immature female flowers. Leaves of some hops varieties contain low amounts of BAs, chalcones, and aromatic compounds. Soft and hard resins are divided by solubility in hexane; only soft resins are hexane soluble. Soft resins in yellow powder secreted by lupulin glands are mainly lupulic acids and their homologs. Their taste gives rise to the term “bitter acids,” with a division in the literature into α-acids (alpha lupulic acids and humulones, their homologs) and β-acids (beta lupulic acids and their homologs, lupulones). Hops’ α-acids include humulone (35-70% of all α-acids), cohumulone (20-65%), and adhumulone (10-15%). β-acids include lupulone, colupulone, and adlupulone. Quantities of both types differ with hops variety, climate, and cultivation conditions. The content of α-acids determines that of XNH, hops’ main polyphenol. In brewing, exposing hops to high heat and alkalinity isomerizes α-acids to iso-α-acids that then oxidize to humulinones, imparting beer’s bitter taste. The price of hops is proportional to its α-acid content. Beer’s bitterness is the main factor in its promotion of gastric acid secretion and thus digestion. Dried hops extracts, taken orally, boost gastric acid secretion without affecting its acidity. The sedative effect of hops, known for centuries, is attributed mainly to its α-acids, with contributions from β-acids and EOs. Sleep aids often include hops and other plants like valerian (Valeriana officinalis, Caprifoliaceae). β-acids’ strong antimicrobial activity is due to their hydrophobic nature, facilitating their interaction with microbial cell membranes. Iso-α-acids’ ionophoric properties are their main antimicrobial mechanisms. Humulinic acids have been proposed as taste-free food preservatives due to their inhibitory effect on microorganisms. Secondary hops metabolites, as with many plants, may cause specific responses depending on the environment. Thus, a compound may be antioxidative in a healthy cell and pro-oxidative in a tumorigenic cell, leading to apoptosis. Hops’ BAs induce apoptosis through both intrinsic mitochondrial and extrinsic pathways, although this remains to be fully elucidated. BAs inhibit chemically induced tumorigenesis and angiogenesis in vivo, with β-acids more active than α-acids in inhibiting tumor development, proliferation, and tumorigenic cell growth. Several in vivo studies report anti-edema/anti-inflammatory effects of humulone and other BA extracts, probably due to interactions with important inflammatory enzymes, as has been shown for humulone.
Hops’ polyphenols and EOs are discussed, with the most attention among the former given to hops’ unique-in-quantity prenylflavonoids, flavonoids with at least one prenyl or geranyl substitution. This increases lipophilicity and improves binding to biological membranes. XNH, in particular, has promise as an anticancer agent. Of hops compounds tested, it is not only most strongly antimicrobial but acts against malaria and the HIV-1 virus and has been found useful in osteoarthritis, diabetes, and endometriosis. Mechanisms of action are being explored. Its isomer, I-XNH, while a less potent anticarcinogen, is more anti-mutagenic and antiangiogenic with some estrogenic activity. Clinical tests confirm XNH’s hepatic metabolite, 8-PN, as a promising treatment for symptoms of menopause and post-menopause, as are phytoestrogens and other plant polyphenols. Hops EOs represent a significant proportion of beer aroma. While studied for centuries, a full list of compounds contributing to beer aroma and taste does not exist. Also secreted by lupulin glands of female hops, EOs contain hydrocarbons (monoterpenes, sesquiterpenes, and aliphatic hydrocarbons), oxygenated compounds (terpene and sesquiterpene alcohols), and sulfur-bearing compounds. In EOs from 25 hops cultivars, β-myrcene, α-humulene, β-caryophyllene, caryophyllene oxide, and humulene epoxide accounted for 47.1-89.3% of total oil; however, many aromatics occur in varying amounts.
Myrcene, regarded by some as a potential human carcinogen, is also reported to inhibit genotoxicity and TNF-α. It repels some food-degrading insects. β-pinene, found in hops and rosemary (Rosmarinus officinalis, Lamiaceae), dill (Anethum graveolens, Apiaceae), rose (Rosa spp., Rosaceae), and other EOs and α-pinene generate substantial synergy with the chemotherapeutic drug paclitaxel. β-caryophyllene may hop EOs’ most important sesquiterpene regarding biological effects due to its antagonism of cannabinoid receptor 2 (CB2). In vitro studies show β-caryophyllene and its oxide’s significant anticancer effects. β-caryophyllene has potential as an analgesic. Both it and β-caryophyllene oxide enhance the efficacy of standard drugs. Among sulfur-bearing compounds, over 41 volatile polyfunctional thiols have been detected in hop cultivars, including novel S-methylthiomethyl thioesters.
A detailed table summarizes hops compounds by class, extraction method(s) used, and reported health effects. Unfortunately, these effects are not described by methodology (in vitro, in vivo, in silico, or human study), departing from this article’s method-oriented emphasis. In sum, the recovery of valuable phytochemicals from food processing wastes not only reduces environmental stress but can add income streams and should be pursued.
The authors report no conflicts of interest.
Hrnčič MK Španinger E, Košir IJ, Knez Z, Bren U. Hop compounds: extraction techniques, chemical analyses, antioxidative, antimicrobial, and anticarcinogenic effects. Nutrients. January 24, 2019;11(2). pii: E257. doi: 10.3390/nu11020257.