Hop Compounds: Extraction Techniques, Chemical Analyses, Antioxidative, Antimicrobial, and Anticarcinogenic Effects

2.1. Isolation of Main Chemical Compounds from Hops

Distillation has been traditionally used for obtaining essential oils from hops. Different approaches have been followed. The essential oil was obtained by passing steam through ground hops and removing the oil from the condensate by extraction with ether. The process took approximately 4 h for distillation of a 100 g sample of coarsely ground hops in 3 L of water. The effect of reducing the time taken for the usual steam distillation has been studied [12]. In 1969, US Patent 3436319 A was issued [13], by Freiherr Von Horst and Kellner, for their “Thin Layer Steam Distillation of Hop Oil Extract”. The process is described as continuous and was proposed for obtaining the essential oil of hop preparations. The main feature of this process was the advantage of completely recovering the oil of hop from the steam distillate and simultaneously producing a residual hop extract containing other components of said extract in substantially unaltered form.

Traditional isolation methods are more and more frequently replaced by the advanced techniques combining sample preparation, separation, detection, and identification. Application of liquid carbon dioxide (LCO₂) in the range of temperatures from 20 up to 25 °C and in the pressure range from 50 to 60 bar, respectively, was for example reported in 1966 for the extraction of hops [11,14]. Stability of the obtained extract was however insufficient and has undergone a chemical change during storage.

An alternative approach was reported in 1977 by Laws and coworkers [15]. Extraction of hops with supercritical CO₂ was performed at temperatures between 40 and 50 °C and pressures between 150 and 400 bar. A high-quality hop extract was also obtained by using LCO₂ at relatively low temperatures. A patent of Muller [16] described the extraction at a temperature below the critical temperature (approximately 31 °C) but at a pressure higher than the critical one (73.25 bar). Next variation included the addition of a small amount of ethanol as a solvent to the liquid carbon dioxide [17]. The choice of an appropriate solvent represents the key factor for isolation of the compound of interest and depends on a number of factors, including dissolving power, selectivity, inflammability, volatility, and cost. Selectivity of LCO₂ is for example used to provide hop extracts free of hard resins and polyphenolic materials which is not the primary focus of this review article that mainly covers extraction and separation methods for isolation of acidic compounds contained in hop flowers and their antitumor, antimicrobial, and antioxidative effects. CO₂ is thus a substitute for organic solvents of a highly nonpolar character, such as hexane and pentane. The range of organic species which dissolve in LCO₂ includes hop resins and aroma components, paraffins, naphthenes, olefins, alcohols, aldehydes, phenols, esters, carboxylic acids, amines and nitrogen heterocycles, aromates, ketones, ethers, amides, and nitrites [11], but not polyphenols.

Ethanol as a polar solvent dissolves a broader range of hop components and represents a suitable solvent for extraction of polyphenols, compounds with structural phenolic features, which can be associated with different organic acids and carbohydrates. Typically 90% ethanol is used and the resulting extract possesses a very similar composition to the original hops. However, the total content of polyphenols is still lower with respect to the other groups of substances [9]. Besides ethanol, scientific literature reports alternative organic solvents—such as methanol, acetone, and ethyl acetate—as effective extractive media for polyphenols as well as, to a lesser extent, propanol, dimethylformamide, and their combinations [18]. Considering industrial applications, the use of organic solvents is limited due to the fact that nontoxic solvents and the maximum residue levels of several solvents in the extracted foodstuff are strictly specified. Consequently, for the manufacture of hop extracts by breweries, carbon dioxide still remains the most applied solvent. High pressure or supercritical fluid extraction is especially suitable for isolation/fractionation of valuable ingredients from natural raw materials with limited solubility in CO₂ at moderate pressures. The extracts contain nearly all essential oils in hops, as well as a sufficiently high ratio of α-acids (humulones) and less bitter lupulones, besides other components such as hard resins and traces of triglycerides, waxes, chlorophylls, and inorganic salts [19]. The main feature distinguishing CO₂ extracts from those prepared with conventional extraction is the lack of traces of undesired organic solvents. Therefore, supercritical CO₂ extraction has become the industrial process of choice for the production of brewery ingredients.

Despite the abundant literature on extraction solvents and techniques for polyphenols from a different plant and herbal sources, there is surprisingly little information available on the effect of extraction conditions on the polyphenol content and antioxidative activities of hops and its products. Namely, the solubility of a solute depends on the solvent density that may vary considerably with changing extraction conditions, especially when operating in the sub- or supercritical region. With a higher temperature, moving towards the critical point, the solvent power increases. Optimum conditions for the extraction of a particular solute has to be therefore established experimentally by determining phase equilibria of this substance in a given solvent.

Numerous studies have shown that organic solvents without the addition of water represent poor solvents for the extraction of polyphenols [20]. Methanol or ethanol can also be mixed with water in different ratios. Alcohol concentration thus plays a key role in polyphenol recovery [20].

Despite several disadvantages, liquid–liquid and solid–liquid extractions are still the most commonly used isolation procedures. For several years, conventional techniques have been widely accepted, mainly because of their ease of use, efficiency, and wide-ranging applicability [21]. Typically, sterilized contaminant free components should be obtained from food and natural tissues in their chemically natural state. Harmful components from nutraceutical products should be removed; heavy metal recovery and enantiomeric resolution are also possible. Currently, solid phase microextraction is successfully applied for the characterization of aromatic properties of hops and other plant raw materials. On the contrary, for the isolation of nonvolatile compounds, solid-phase extraction, and solvent extraction are successfully used [22]. For sample preparation, including purification and/or isolation of polyphenolic compounds, solid-phase extraction in offline columns has also become a popular and effective method. Moreover, accelerated solvent extraction has been recently applied for the extraction of bitter acids from hops and hop products. Finally, low-temperature levels, high yields, and a short process time represent the main advantages of ultrasound-assisted extraction. The procedure usually requires additional cooling, the main part of the applied energy is thereby transferred into heat to protect heat-sensitive substances. Due to cavitation, the cells of the plant material are highly disrupted [23].

Supercritical fluid extraction, routinely used for the production of bitter acid extracts for the beer brewing industry, represents an effective method for the isolation of both volatile and nonvolatile compounds of hops including essential oils. Operating conditions, temperature, and pressure used for the extraction markedly affect the composition of the obtained extract. Relatively high recovery of volatile compounds is obtained at lower temperatures, whilst elevated pressures and temperatures favor high recovery of bitter acids and resinous compounds. Unfortunately, under the conditions used in the extraction with supercritical carbon dioxide for the preparation of hop extracts for the brewing industry, a large group of biologically active prenylflavonoids (prenylchalcones and prenylflavanones) remains in the plant material. Besides CO₂, water is also relatively often used as an extraction medium, mainly due to the ability to become an excellent solvent for organic compounds and a very poor solvent for inorganic salts above its critical point (374 °C, 218 atm). This means that the same solvent can be used to extract the inorganic and the organic components, respectively. Products obtained in this way are solvent-free, without the presence of coproducts and the operational temperature is in the case of CO₂ low [24]. The application of supercritical fluids (SCF) for extraction of natural substances at even higher pressures (over 70 MPa) than in conventional SCF extractions gives rise to new products from known plant materials such as the isolation of less soluble substances. A method for extraction and dissolution of hop acids, including α-acids, iso-α-acids, β-acids, and their derivatives in aqueous media, also comprising of the formation of quaternary ammonium salts of hop acids with quaternary ammonium compounds or mixtures thereof has been recently developed by Mertens and Pascal [25]. Hop acids containing matter is mixed with one or more quaternary ammonium compounds. Quaternary ammonium salts of hop acids are thereby formed. Their specific advantage is a high solubility in (acidic) aqueous medium compared to the corresponding hop acids in the free acid form. This invention further relates to the use of quaternary ammonium salts of hop acids in the beer brewing process and represents an effective concept of improving the utilization of hop acids, including α-acids and bitter taste contributing iso-α-acids, in this process.

2.2. Analytical Methods

High resolution and the ability to provide precise and accurate qualitative and quantitative data put forth gas chromatography coupled with mass spectrometry (GC–MS) analyses as a valuable tool for taxonomic studies of plants. Various phenolic compounds have been found in beer using different detectors, such as coulometric, electrochemical, and photodiode arrays; ultraviolet–visible spectrophotometry; and low-resolution mass spectrometry [26]. More than two hundred compounds such as essential oils, prenylflavonoids, and bitter acids usually classified as α-acids and β-acid, can be detected and further separated. In one single run, their quantities can be estimated by the means of capillary GC analysis.

Main components of hop essential oils such as monoterpenes and sesquiterpenes comprising humulene, bisabolene, caryophyllene, farnesene, and elemene skeletons are commonly determined by the evaluation of the total volatile content by GC–MS [22]. The method requires the determination of retention parameters (t_R, calculated retention index and Kovats retention index) and m/z values of molecular ions for selected compounds from hop essential oils [22]. The main advantage of the method is that essential oils obtained by steam distillation are ready to use for subsolvent GC analysis after appropriate dilution without additional purification.

α-, β-, and iso-α-acids are known for their high potential to oxidize. Development of simple preparative methods (which exclude other hop constituents such as polyphenols, lipids, waxes, and polysaccharides) for the bitter acid oxide fractions is essential due to their effect on beer properties as well as their potential health benefits [27]. A total content of matured hop bitter acids (MHBA), primarily composed of α-acid-derived oxides, is determined by a quantitative analytical method using high-performance liquid chromatography (HPLC) frequently coupled with atmospheric pressure ionization tandem mass spectrometry (APCI-MS-MS) or with negative electrospray ionization mass spectrometry. “General, HPLC analyses have unfortunately displayed a lack of reproducibility, the disadvantage of consumption of organic solvents and only the major compounds, i.e., the humulones and iso-humulones can be examined by this methodology.” Xanthohumol has been determined in hops by HPLC using UV detection. Though, this technique lacks deficient sensitivity and for the quantitative determination of the minor prenylflavonoids. Higher sensitivity is enabled by tandem mass spectrometry (MS-MS). Liquid chromatography (LC) coupled with (tandem) mass spectrometry has been successfully applied to the quantitative analysis of prenylflavonoids [28].

A variety of phenolic compounds, such as hexosides, dihexosides, pentosides, and quinic conjugates, such as feruloyl quinic acid, caffeic acid-O-hexoside, coumaric acid-O-hexoside, sinapic acid-O- hexoside, catechin-O-dihexoside, kaempferol-O-hexoside, and apigenin-C-hexosidepentoside, in beer extracts, of which some have been reported for the first time in beer, were efficiently determined by a LTQ-Orbitrap high resolution mass spectrometer [29]. However, techniques employing high-resolution mass spectrometry are still under intense investigation, since data on identification of the phenolic profile by high-resolution mass spectrometry in analysis of hops compounds are still relatively scarce. These techniques, including ion trap quadrupole-Orbitrap-mass spectrometry (LTQ-Orbitrap-MS), which provides single-stage mass analysis that supplies molecular weight information, as well as two-stage mass analysis (MS/MS) and multi-stage mass analysis (MSn) that provide structural information, have been demonstrated as a reliable tool for the structural elucidation of unknown compounds in complex mixtures such as the total hop extracts.

Isoxanthohumol can be metabolized in the human liver to form 8-prenylnaringenin. Prenylnaringenin is an isomerization product of desmethylxanthohumol and until now, known as the most potent phytoestrogen isolated [30]. Specific cytochrome P450 enzymes are responsible for the O-demethylation reaction. The enzymes that convert isoxanthohumol and 8-prenylnaringenin to their most abundant metabolites were identified.

Evaluation of the results obtained by chemometric methods provides data on phytochemical composition, which is crucial for the standardization and quality control of plant raw materials required by food or pharmaceutical industry.

2.3. Methods and Techniques for Determination of Antioxidative Activity

Polyphenols are extremely important for the physical stability of beer during storage [31]. Oxidation and polymerization of endogenous polyphenols and their interaction with proteins represent the main reason for beer turbidity. Catechin and proanthocyanidins (dimers and trimers of catechin, epicatechin, and gallocatechin) have displayed haze-forming activity with peptides in model systems [32].

Total phenolics and antioxidative activities of hop extracts are commonly determined by well-established methods. 2,2-diphenyl-1-picrylhydrazyl radical (DPPH*) scavenging assay is used to evaluate the antioxidative activities of the extracts, whilst the total phenolics are determined by the reduction of phosphotungstic acid and phosphomolybdic acid [33]. Besides, other in vitro antioxidant assays, such as the oxygen radical absorbance capacity (ORAC) assay, the hydrogen peroxide scavenging (HPS) assay, and the linoleic acid (LA) assay, have also been reported for evaluation of oxidative stability. Using the ORAC assay, cinnamic, caffeic, and ferulic acids; gallocatechin, xanthohumol; and myricetin were described as the most active in hydroxyl and peroxyl radical-scavenging [32]. The effect of phenolic compounds was related with vitamin C, using ABTS and DPPH-scavenging assays. Antioxidant activities are expressed as vitamin C equivalent antioxidant capacity (VCEAC). VCEAC results for phenolic compounds were gallic acid > quercetin > epicatechin > catechin > vitamin C > rutin. It was proposed that monomeric and oligomeric flavonols have the ability to limit LDL oxidation. It was demonstrated that among phenolic compounds, flavonoids with a prenyl group are more effective as inhibitors of LDL oxidation [31,33].

Most of the studies were focused on the antioxidant activity of literature-known compound classes in beer. There is a lack of studies on identification of the key antioxidants in beer. For that purpose, the target antioxidants should be isolated, purified, and determined in their chemical structure by the means of LC-MS and 1D/2D NMR [34].

2.4. Methods and Techniques for Determination of Antimicrobial Potential

Hops have a long history of use as a natural preservative in beer due to high concentrations of unique bitter acids that inhibit the growth of Gram positive bacteria already at surprisingly low concentrations. Staphylococcus aureus is one of the most common Gram positive bacteria causing food poisoning. Its source is not the food itself, but the humans who contaminate the food after it has been processed. Despite the well-known high antibacterial, antifungal, and antiviral activities of hops, there is a lack of information about antimicrobial potentials of individual hop compounds. However, lupulone, humulone, isohumulone, and humulinic acid have shown high antimicrobial activity against certain bacteria like Bacillus subtilis 168 [35]. Isolation of such individual chemical compounds and their subsequent analyses are quite tricky and time-consuming, especially in the case of a complex biological matrix such as the hop extract [36]. Recent investigation reports that seven flavonoids, among them two natural (α,β-dihydro xanthohumol and 8-prenylnaringenin) showed significant activity against methicillin sensitive and resistant Staphylococcus aureus and Staphylococcus epidermidis [37]. Aspergillus niger is a clearly visible spoiling agent of bakery goods that forms black-centered spots on the surface of products. A typical opportunist, Candida albicans is the microbe responsible for most clinical yeast infections, e.g., in mouth infections [38].

There is an urgent need for new antimicrobial ingredients specifically targeting oral pathogens such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, and Lactobacillus sp. Despite the availability of robust preservatives food spoilage and poisoning caused by microorganisms remains a problem that has not yet been brought under adequate control. Natural preservatives as an alternative to the artificial ones are therefore needed to achieve sufficiently long shelf-life of foods and a high degree of safety with respect to foodborne pathogenic microorganisms. Several studies have been performed to investigate whether classical hop extracts, supercritical hop extracts or extracts obtained by isomerization of supercritical hop extracts, could be used as antimicrobial agents against foodborne pathogens and also against microorganisms capable of causing food spoilage due to the high content of phenolic phytoalexins, e.g., flavonoids. Screening of plant extracts and their constituents has a well-established history for searching novel anti-infective agents effective against cariogenic and periodontal bacteria [39]. Nowadays, plant antiseptics are incorporated into a whole range of oral products. The substances responsible for this activity are mainly the hop acids, which are classified as α-acids (humulones), β-acids (lupulones), and their oxidation products. These substances consist of a mixture of homologous compounds [20]. Residues attained in the process of hops extraction, which comprises a waste product from the brewing industry, may be used as a cheap source of natural compounds with dual functions (as an antioxidant and antimicrobial agent) of wide application.

Different techniques according to the Standard Test method ASTM E 2149-01 have been reported to assess antimicrobial activity of Immobilized Antimicrobial Agents Under Dynamic Contact conditions (E2149-01, 2002) [40,41,42,43].

The most extensively used techniques to investigate the antibacterial activity of natural substances and plant extracts are diffusion methods which are based on the use of discs or holes as reservoirs containing solutions of substances to be examined according to Brantner and Grein [44], which studied plants of 28 families, selected on the basis of medicinal folklore reports and literature data, in a screening program. Aqueous extracts of plants used externally for treatment of infected skin lesions were tested for their antibacterial potential. The results indicated that ~60% of the plant extracts tested exhibited some level of antibacterial action. Hole-plate diffusion method is used in the case of pure compounds, whilst the cylinder diffusion method is used for the extracts.

However, in the case of solutions with low activity a larger concentration or volume is needed, which may be problematic when determining the antibacterial activity of extracts, including hop extracts. The limited capacity of discs means that holes or cylinders are preferably used. Among several phenolic compounds present in plant extracts, flavone, quercetin, and naringenin have been proved as highly effective in inhibiting the growth of microorganisms due to their interaction with nucleic acid or proteins [45]. Nonetheless, plant extracts generally contain a variety of flavonoids. The broad range of diverse chemical structures may reflect in different biological activities of extracts [46]. Indeed, diverse extraction procedures give extracts with characteristic phenolic profile and different proportion of single phenolic compounds and this certainly influences the antioxidative properties of the extracts.

Hop extracts obtained by supercritical CO₂ extraction, show significant antibacterial potential against investigated bacterial strains. Xanthohumol has been proven to possess the highest activity against all tested strains. According to the literature, α- and β-acids, humulones and lupulones, and the isomerized forms of humulones might be considered as antimicrobial agents in hop extracts as well. Possible mechanisms of antibacterial activity of bitter acids and their derivatives might include the induced leakage of the bacterial membrane due to their highly hydrophobic character, especially of lupulone [47]. Among organic solvents used for Soxhlet extraction, methanol and ethanol have been proven as efficient solvents for isolation of compounds with high antimicrobial activity. On the other hand, when n-hexane is used as a solvent, the resulting extract is less effective than methanol and ethanol extracts against tested bacteria and fungi.

2.5. Methods for Determination of (Anti)Genotoxic Potential

Even though naturally occurring substances as an essential part of edible plants and their products should pose no health risk, we cannot presume that the same applies for isolated compounds in higher doses and for other formulations especially in the form of phytopharmaceutical drugs. Therefore, also in the case of a naturally occurring substance with proven health-promoting effects, testing for potential toxicity and use of limited concentrations is essential for safe use. Several testing methods and protocols exist for the toxicological evaluation of compounds, some even in silico [48].

Among possible toxic effects, genotoxic effects, causing a detrimental impact on the genetic material (DNA) tend to be in the center of scientific attention as they represent the leading cause of several prevailing diseases of our time, especially various cancer types. In order to prevent the formation of cancer and possibly other genetic diseases, one must try to avoid any exposure to genotoxic agents or/and include or increase the intake of chemoprotective substances capable of delaying, preventing, or inhibiting one or more cancer stages [49,50]. Various potential anticarcinogenic substances can be found also in hops and their effects are presented in the following chapter.

The methods and tests for determining the genotoxic potential can, with minor modifications, in most cases be used for the determination of the antigenotoxic potential as well. The most widespread used genotoxicity test remains the Comet assay as it is straightforward, economical, and can be applied in numerous ways on various cells and tissues for investigating DNA damage at physiological conditions and concentrations [51]. As it is utilized for determining DNA damage and its repair, it is popular in human biomonitoring studies along with environmental studies and in assessing the occupational exposure to genotoxic agents associated with various diseases, and the intrinsic factors that affect DNA damage levels in humans. On the other hand, genoprotection by dietary and other factors as well as the exploration of the effect of repair gene polymorphisms can be addressed too [51,52]. The essence of the Comet assay is in that the studied cells are embedded in agarose, lysed, and electrophoresed at high pH, the DNA strands containing breaks thereby move towards the anode, which under fluorescence microscopy looks like a comet tail. The assay has received numerous improvements, especially by including various DNA modifications like oxidized bases and DNA alkylation with specific enzymes in addition to strand breaks that are primarily/easily detected/determined by the Comet assay. Among the most commonly used enzymes one can find endonuclease III, formamidopyrimidine DNA glycosylase (FGP), and 3-methyladenine DNA glycosylase II [51,53]. As already mentioned, the Comet assay additionally enables the determination of DNA repair, not only DNA damage. For determining DNA repair, DNA damage on cells is initially induced via ionizing radiation, UVC radiation, or other genotoxic agents and the chromosomal aberrations or micronucleus frequency is measured as it represents unrepaired DNA [54]. Then, a decrease of the DNA breaks (relative tail fluorescence) is monitored with the incubation time [52]. By introducing lesion-specific endonucleases after the lysis of the cells it is possible to observe the removal of specific base damages like oxidized and alkylated bases removed by the base excision repair and UV-induced cyclobutane pyrimidine dimers removed by the nucleotide excision repair [54]. The latter additionally enables to assess the repair capacity by measuring the increase of repair intermediates instead of the removal of the damage [54]. Therefore, the associations of investigators of the majority of diseases share a common belief that the Comet assay exhibits an enormous potential for the use in everyday clinical practice [52].

The Comet assay possesses several advantages over alternative methods such as using substrates with a low density of lesions and better simulating real-life conditions compared to other in vitro assays [54]. Only the quantitative polymerase-chain-reaction (PCR) method requires equally small amounts of DNA as the Comet assay; however, the limit of detection of the latter is at least an order of magnitude higher and therefore, inappropriate for a biomarker assay [54]. Compared to HPLC methods, the Comet assay is less precise, but better when estimating background levels, and its FPG-based modifications are even more accurate than HPLC [51].

Another universally applicable method for genotoxicity tests is the cytokinesis-block micronucleus (CBMN) assay. This method is used for determining the standard micronucleus index (MNi), representing a small nucleus lacking part(s) of a chromosome or lagging behind in anaphase during nuclear division [55,56]. This method can additionally be used also for measuring the nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis), or nuclear division rate [53]. Therefore, the CBMN method remains widely applicable for determining chromosomal instability, cell death, and cytostasis through various direct and/or indirect measurements of several aspects of cellular and nuclear dysfunctions. An important advantage of the CBMN method lies in specifically restricted scoring to once-divided cells [55].

Numerous natural compounds exhibit phytopharmaceutical potentials. Therefore, to avoid unnecessary laboratory testing and to decrease research costs, it is reasonable to first perform the in silico analysis in order to reduce the number of potential candidates to be experimentally tested or to find novel, stronger candidates for laboratory tests. Even the European Food Safety Authority (EFSA) has proposed a computational approach to supplement the toxicity profiles of compounds [48]. There exist various computational approaches for investigating genotoxicity of specific compounds. One approach is to use the tools that search the existing (toxicological) databases and compare the substances on the basis of their structural elements or other properties with various mathematical/statistical operations [48]. A completely different approach that is also used by our research group is to perform computer simulations of chemical reactions of DNA with potential (anti)genotoxic agents on the basis of quantum mechanics and/or molecular dynamics. More insights are presented in the concluding chapter.

3.1. Soft Resins (Bitter Acids)

Due to the high content of biologically active compounds, the biological effects of hops foremostly refer to the mature female hop cones (flowerings) and their extracts. Bitter acids and xanthohumol were also found in male inflorescences: their concentrations are similar to those found during early female flowerings [67]. The presence of bitter acids and chalcones was also confirmed in the leaves of fully grown hops even if their levels were generally lower than in the hop cones and were strictly related to hop varieties [67]. The hop leaves also contain volatile compounds, but in much lower amounts than the hop cones (< 0.05%) [4]. Substances found in hop plant are commonly called resins. According to their (in)solubility in hexane, the division to soft and hard resins is generally accepted; soft resins being hexane soluble. Soft resins found in yellow powder secreted by lupulin glands are mainly lupulic acids, chemically di- or tri-prenylated phloroglucinol derivatives, and homologs [4,60,68,69]. Owing to their bitter taste, the term ‘bitter acids’ is adopted in the literature. Among soft hop bitter acids a division into two categories is adopted. α-acids (alpha lupulic acids or humulones) and their homologs represent the first category and the larger portion of soft resins, while the minor part represents the β-acids (beta lupulic acids) with homologs named lupulones [70].

The most important representatives of α-acids (Figure 3) are humulone (35–70% of total α-acids), cohumulone (20–65% of total α-acids), and adhumulone (10–15% of total α-acids). As their chemical structures are very similar, the names and quantities of representative β-acids are analogous; lupulone (30–55% of total β-acids), colupulone, and adlupulone. Other minor representatives of bitter acids include posthumulone/postlupulone, prehumulone/prelupulone, and adprehumulone [4]. The quantities of both types of acids and their homologs, however, may vary greatly as they depend on the hop variety, climate and cultivation conditions [8,71].

The content of α-acids in different hop varieties also determines the content of xanthohumol, the main polyphenol in hops [8]. When exposed to high temperatures (100–130 °C) and pH (8–10) as in the case of hop boiling during the brewing process, alpha-acids isomerize to iso-α-acids that next to oxidized hop acids—humulinones—predominantly to provide the bitter taste of beer [34,72]. As a majority of hops is grown for the brewing industry, its price is proportional to the alpha-acid content [60].

Kurasawa and colleagues [73] established that dried hop extracts, when administered orally, increase the gastric juice volume without affecting its acidity. On the basis of those experiments, Zanoli and Zavatti [4] suggested that the bitterness (the taste) of hops represents a crucial factor in inducing gastric secretion via the cephalic phase. Later, Walker and coworkers [74] confirmed that the bitter acids comprising of α-, β-, and iso-α-acids are potential key components promoting gastric acid secretion and upregulation of the CHRM3 gene expression.

The bitter taste promoting digestion is not the sole property that can be attributed to bitter acids. Sedative effects of hops have been recognized for centuries; however, not long ago, Schiller and coworkers [75] established that the main component of hops providing sedative effects is indeed the α-acids. Nonetheless, they clearly state that contributions of other fractions like β-acids and essential oils are also significant. Consequentially, preparations for fighting insomnia or sleep disorders consisted of hop cones alone or in combination with other sedative herbs, such as Valerian, have been on the market for at least a decade [8].

As beer mainly consists of water, it would not be stable without additives. Hops, especially bitter acids, not only contribute to its taste, but also stabilize the foam and, even more importantly, exhibit strong antimicrobial activity. The strong antimicrobial activity of β-acids mainly stems from their hydrophobic nature facilitating the interaction with microbial cell membranes [69,76].

On the other hand, it is presumed that ionophore properties of iso-α-acids represent the main mechanism for their antimicrobial action [35,72]. Moreover, Schurr and coworkers [72] propose humulinic acids, the hydrolytic product of α-acids and iso-α-acids, as novel tasteless food preservatives, according to their microorganism inhibitory action.

In addition to antimicrobial and sedative effects, a majority of hop compounds exerts a great variety of other health-promoting activities such as antioxidative [66,77], anti-inflammatory [76] and various anticancer effects.

Chen and Lin [78] proposed that a pivotal mechanism for the chemopreventive action of hop bitter acids represents the induction of apoptosis. Many subsequent studies confirmed that bitter acids trigger apoptosis, but the full mechanism of this action still needs to be uncovered. However, it is known that apoptosis is induced via both pathways: intrinsic mitochondrial and extrinsic [61,78,79]. Bitter acids affect the intrinsic pathway by altering the Bcl-2 family of proteins, and the extrinsic by enhancing the expression of p38 that activates p53 and the TRAIL (Fas and FasL) death-receptor pathway [78,80].

In addition to apoptosis-inducing effects, bitter acids are also capable of inhibiting chemically-induced tumor promotion in vivo [81] and angiogenesis; β-acids are even more active than α-acids in inhibiting tumor development [8,61,82], reducing proliferation [81], and even inhibiting the growth of tumorigenic cells [68].

Finally, several in vivo studies [61,83,84,85] demonstrated that humulone, derivatives of α-acids such as iso-α-acids or rho-iso-α-acids and even hexahydro-β-acids inhibit various animal edema, thereby reducing inflammation. This activity probably stems from interactions with other important enzymes like COX, IKK (NF-kB), and Jun N-terminal kinase with mitogen-activated protein kinases (MAPKs), as was explicitly demonstrated in the case of humulone [84].

3.2. Polyphenols

One of the crucial roles in plant organisms: the protection from all kinds of external threads, has been ascribed to a diverse group of substances called polyphenols. Many of hop polyphenols are, therefore, abundantly found in other plants as well, however, the prenylflavonoid class is, to an extent (quantity), only present in the hop plant. Additionally, in hops, one can also find a group of metabolic products of already described bitter acids called multifidol glucosides.

In this article we focus on the compounds encountered solely in hops, more substantial and detailed information on other polyphenols can be found in our review paper [24].

Lupulin glands secrete a mixture of prenylated, geranylated, oxidized, and/or cyclized chalcones along with bitter acids and volatile oils [69].

As polyphenols represent secondary metabolites they can be found in all plant parts. However, it was shown that the ethanol extract of hop cones has a 10-times greater content of phenolic substances than the hop leaves [33]. Presumingly, this could be the reason why hop leaves exert a lower antioxidant activity and were devoid of significant antimicrobial activity. Moreover, the authors also described that the phenolic profiles of the two parts of the hop plant were significantly different. It was suggested that a detailed identification of phenolic profiles is essential before exploitation of hop leaves as antioxidants [33].

Similar to the soft resins, the phenolic fraction of hop cones have been extensively studied. Xanthohumol, that represents more than 1% of dried hop cones is especially interesting and remains a topic of research for several applications [8].

For better transparency, polyphenols are presented separately in corresponding subclasses.

3.3. Essential Oils

Essential oils, as is already evident from the name, represent the essence of the plant, meaning its distinctive aroma. Hop aroma has always intrigued the mankind and it represents the significant portion of beer aroma. It is actually the quest for a better beer aroma that evidently brought so many hop varieties [4,57]. Even though this subject has been in the center of scientific and brewer’s attention for centuries, the whole list of substances contributing to the hop aroma and consequentially to beer taste is still not completed. Following the bitter fraction, hop essential oil compounds are also secreted from lupulin glands of female plants [82]. According to the basic molecular structure hop, essential oils consist of three fractions. In the first hydrocarbon fraction, one can find monoterpenes, sesquiterpenes, and aliphatic hydrocarbons. The second fraction contains oxygenated compounds like terpene and sesquiterpene alcohols. In spite of the fact that many believe that the last fraction of sulfur-containing compounds does not contain important biologically-active molecules, this may not be entirely true. Compounds from the sulfur fraction moreover proved to be important contributors to the hop and beer aroma and prevent its spoiling [34,134].

Clustering analysis of essential oils from 25 different hop varieties has shown that the highest amounts, accounting for 47.1 to 89.3% of the oils, represent β-myrcene, α-humulene, β-caryophyllene, caryophyllene oxide, and humulene epoxide II [135]. This is almost completely consistent with the study of Ligor and coworkers [22] study who listed the most important components of hop aroma as myrcene, α-humulene, β-caryophyllene, and β-farnesene. A newer study where comparative aroma extract dilution analysis was performed on the special flavor hop varieties Huell Melon and Polaris determined myrcene, (3R)-linalool, and 2- and 3-methyl butanoic acid as important variety-independent hop odorants and found (1R,4S)-Calamenene—a new odor-active compound in hops [57]. Considering the division into three fractions, the majority of the hydrocarbon fraction is made up of monoterpenes α- and β-pinene, myrcene, and limonene as well as sesquiterpenes α humulene, β-farnesene (not in all hop varieties), β-caryophyllene, α- and β-selinene, and γ-muurolene [8,82]. During the ripening, processing, and storage oxidation processes occur and yield the oxygenated fraction containing linalool, geraniol, caryophyllene oxide, 2-methyl-3-butene-2-ol, and farnesol forms [8,82].

However, the content of a specific substance in essential oils not only depends on the cultivation conditions, storage, and processing, but the extraction and analytical process importantly impact it as well [22,71].

An important (hop) essential oil component monoterpene myrcene has two sides. According to the International Agency for Research on Cancer (IARC), myrcene, contained in certain popular beverages, is regarded as a potential human carcinogen; however, it is clearly stated that this fact still needs to be further evaluated [136]. On the other hand, both myrcene and linalool significantly inhibited the genotoxicity of 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine and only myrcene, even though less efficiently, inhibited the toxicity of 2-amino-3-methylimidazo[4,5-f]-quinoline [137]. β-myrcene has also shown to be a potent dose-dependent TNF-α inhibitor, stronger than α-pinene and d-limonene, through the phosphorylation of the inhibitor of κB kinase and matrix metalloproteinase-9 (MMP-9) gene expression [138]. Moreover, β-myrcene also inhibited invasion of MDA-MB-231 cells (breast cancer cells) induced by TNF-α [138]. More studies argue in favor of positive health effects of myrcene than of its carcinogenicity, therefore, we cannot exclude it from the list of potential anticancer agents.

Myrcene, extracted together with other essential oil compounds from used hops, exhibited the highest repellency towards insect Rhyzopertha dominica (RD50 = 0.27 A mu M cm⁻²), whereas for yet another insect degrading stored foods Sitophilus granarius, limonene was the most effective repellent [139]. Therefore, not only fresh hops but even left-over hops from breweries can be effective at least as an insect repellent, protecting stored foods [139].

The major contributor to the hop aroma from the essential oil fraction is presumably the monoterpene β-pinene found also in rosemary, parsley, dill, rose and other essential oils [107]. It was shown that both α-pinene and β-pinene generated a substantial synergistic effect with the Paclitaxel drug for treating non-small cell lung carcinoma [140].

Among sesquiterpenes found in hop essential oils (and other essential oils from clove, piper, and hemp), β caryophyllene seems to be the most important regarding biological effects. Its spiciness, partially coming from the antagonistic action towards the cannabinoid receptor (CB), is actually more representative of piper and clove aroma than hop [107]. Several in vitro studies have shown that β caryophyllene and its oxide possess significant anticancer activities, affecting growth and proliferation of various cancer cells [141]. Especially β-caryophyllene oxide proved to alter several key pathways of cancer development, such as MAPK, PI3K/AKT/mTOR/S6K1 and STAT3 pathways [141]. As β-caryophyllene activates only CB2 and not CB1, it carries a potential as a novel natural analgesic drug. Additionally, both compounds enhance the efficacy of standard drugs by augmenting their concentrations inside tumorigenic cells [141]. All in all, these compounds represent the future of cancer treatment with natural substances. In the oxygenated fraction, one can also find 2-methyl-3-butene-2-ol, whose concentration increases during the storage of hops [8]. This compound is believed to be the most responsible for the calming effect of hop essential oils [8].

Humulene (α-caryophyllene) found in mixtures together with β-caryophyllene represents an important sesquiterpene substance providing the distinctive hoppy aroma to the beer and exhibiting some mild corticosteroid effects. Humulene gets epoxidized during the brewing process, however, it is the hydrolyzed form that provides its taste, not the epoxidized [107].

The minor fraction, representing less than 1% of hop essential oils, are sulfur-containing compounds like thiols, sulfides, polysulphides, thioesters, thiophenes and terpene derivatives, which may also contribute to the beneficial biological effects exerted by hops [142].

Thioesters have been also detected in thermally not processed unkilned hops, suggesting that they are naturally occurring components of hop essential oils, although present in lower concentrations than after heat processing that causes their additional formation [142]. A study of Guadagni and coworkers [143], on the odor contributions of 25 main chemical components of hop essential oils from Bullion hops, has allocated S-methyl hexanethioate on the second place regarding its contribution (4–8%) to the hop aroma. Moreover, S-methylthiomethyl thioesters have been uniquely found in hops [142].

More than forty-one different volatile polyfunctional thiols, detected in various hop cultivars, also represent key contributors to the hop aroma in beer as their content rises during drying and brewing processes [142,144]. Polyfunctional thiols have been appreciated and widely studied in the enology field as the compounds providing desirable odor notes like rhubarb, citrus, passion fruit, or blackcurrant bud carried by 3-mercaptohexan-1-ol (3MH), 3-mercapto hexyl acetate, 4-methyl-4-mercaptopentan-2-one (4MMP), 3-sulfanyl-4-methyl pentane-1-ol, 3-sulfanylhexan-1-ol, and others [134,144]. In addition to releasing pleasant aromas, 3-mercaptohexan-1-ol also inhibits epigallocatechin gallate (EGCG) oxidation, thereby providing the necessary stability for EGCG to retain its beneficial biological activity [145].

Roland and coworkers [134] investigated in which form one can find thiols, especially 3MH and 4MMP in hops. For 4MMP the free-form represents the most important fraction (up to 95–100%) with precursors found in trace amounts only or even completely absent. However, surprisingly, the contrary was found for 3-mercaptohexan-1-ol, where more than 99% were allocated to its precursors: S-cysteinylated and S-glutathionylated conjugates. In a majority of hop varieties, glutathionated 3MH represents more than 80% of the total amount, only in Barbe Rouge hops the proportion is 50:50. Free 3MH represents less than 1% in all hop varieties, although some studies reported up to 7.1% of the free fraction [134]. In plants in general and also in hops, it is more common to encounter cysteine-S-conjugates as they represent the final product of the glutathione detoxification pathway [144]. Since from glutathione-S-conjugates free glutathione can be released, consumed thiol precursors from hops may importantly contribute to the organism’s detoxification processes and therefore prevent the oxidative stress and other diseases like cancer.

To conclude, organosulfur compounds found in hop essential oils can be beneficial through at least three different ways. Some are carriers of the aroma and flavor and can thereby importantly affect our mental and physical state according to the foundations of aromatherapy. Others prevent oxidation/degradation of important biologically active molecules like EGCG. Some may again release glutathione and can, therefore, contribute to the detoxification of organism, especially from numerous tumorigenic substances surrounding us. Last but not least, even compounds not biologically active on their own can still bring about synergistic effects and therefore enhance health-promoting activities of other more active substances contained in hops or even provide new effects that none of these compounds feature on their own.

An overview of hop compounds, applied extraction methods, and described biological effects is provided in Table 1.