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Humulone

Humulone is a prenylated acylphloroglucinol derivative and the predominant alpha acid in the resinous lupulin glands of the hop plant (Humulus lupulus), serving as the primary source of bitterness, flavor, and antimicrobial preservation in beer through its isomerization during the brewing process. With the molecular formula C₂₁H₃₀O₅ and a molar mass of 362.46 g/mol, humulone is an optically active compound, specifically the R-(-)-enantiomer, characterized by a melting point of 64.5°C and a pKa of 5.0. Its solubility is low in neutral water but increases significantly with temperature and pH, reaching approximately 200 mg/L in boiling wort at pH 5.0, which facilitates its extraction and transformation during boiling. In beer production, humulone undergoes thermal to form iso-alpha acids, such as isohumulone, which account for about 70% of the beer's perceived bitterness at concentrations up to 100 mg/L and also contribute to stability and microbial inhibition, being roughly 20 times more effective against like Lactobacillus brevis at low compared to the parent compound. This favors cis-isomers due to , with cis-isohumulone exhibiting approximately 1.82 times the bitterness of its counterpart. Humulone coexists with related alpha acids like cohumulone and adhumulone, and over 32 derivatives have been identified, including oxidized forms like hulupones that can influence beer's off-flavors if humulone autoxidizes during storage. Beyond , humulone demonstrates biological activities, including positive allosteric modulation of GABA_A receptors, contributing to and effects observed in hops-derived products.

Occurrence and Production

Natural Occurrence

Humulone is a derivative and prenylated primarily synthesized in the glandular trichomes, known as lupulin glands, of the female cones of the hop plant (). These specialized structures are located on the inner surface of the cones and serve as the main site for the accumulation of bitter acids, including humulone, which constitutes the majority of alpha acids in . In terms of concentration, humulone typically comprises 20-50% of the total alpha acids, with overall alpha acid levels ranging from 2% to 12% of the dry weight of hop cones, depending on the variety. Bittering varieties, such as Magnum, exhibit higher concentrations, often reaching 11-16% alpha acids by dry weight. These levels can vary due to genetic factors within cultivars, with humulone being the predominant analog in most cases, though proportions differ across varieties (e.g., higher in many European types). Humulone plays a key role in plant defense, functioning as an antimicrobial agent that inhibits the growth of bacterial and fungal pathogens, thereby protecting the plant from infections. The natural occurrence and yield of humulone are influenced by geographical and cultivation factors, with optimal production in temperate climates such as those in and the of the , where long daylight hours and moderate temperatures support robust growth. levels between 6.0 and 7.0 are ideal for maximizing humulone accumulation, as they facilitate nutrient uptake and minimize toxicities like overload. Adequate exposure, typically requiring full sun for 14-16 hours daily during the , enhances cone development and content, including humulone.

Industrial Extraction

Industrial extraction of humulone, the primary in , involves isolating it from hop cones or pellets for use in and other applications. Early methods in the early relied on liquid-liquid techniques, where were treated with organic solvents to dissolve the resins containing humulone, followed by and solvent evaporation to recover the extract. These processes evolved significantly by the mid-20th century, with the adoption of non-polar solvents like in the 1950s, which selectively targeted the soft resins including alpha acids while minimizing of polar compounds. By the late 1970s and 1980s, the development of using marked a shift toward more efficient and methods, with the first commercial supercritical CO2 facility operational in 1980. In solvent-based extraction, hop material is typically ground into pellets or powder and contacted with a solvent such as or in a percolator or , allowing humulone to dissolve into the liquid phase before the solvent is removed via or . , valued for its low polarity, extracts humulone along with other lipophilic components, yielding concentrates with up to 50% alpha acids, though it requires careful handling due to flammability and potential residue concerns. extraction, still used in some facilities, produces broader-spectrum extracts at around 30% yield of total hop material but with higher inclusion of vegetative matter and poorer retention of essential oils due to volatilization during . Supercritical CO2 extraction represents the dominant modern industrial approach, where CO2 is pressurized to 100-300 and heated to 40-60°C to reach a supercritical state, enabling selective dissolution of humulone from pellets in an extractor vessel; the extract is then separated by depressurization, yielding a concentrated without leaving residues. This method optimizes yields at 90-95% recovery of alpha acids, significantly outperforming traditional processes by reducing impurities and environmental impact through CO2 recyclability. A two-step process can further fractionate the extract, first isolating essential oils at lower pressures before targeting the alpha acid-rich bittering fraction. Co-extraction of byproducts is inherent to these methods, including other alpha acids such as cohumulone and adhumulone, beta acids like lupulone, and essential oils that contribute to aroma but may require subsequent purification steps like or additional partitioning to isolate humulone-enriched fractions. In supercritical CO2 processes, minor amounts of waxes and can also appear, particularly at higher temperatures, influencing the extract's color and stability. These byproducts enhance the versatility of hop extracts for but necessitate to meet purity standards for commercial applications.

Chemical Structure and Properties

Molecular Formula and Structure

Humulone, the principal α-acid found in hops, has the molecular formula C₂₁H₃₀O₅ and a molecular weight of 362.46 g/mol. Structurally, humulone is a prenylated phloroglucinol derivative featuring a central 1,3,5-trihydroxybenzene ring acylated at the 2-position with an isovaleryl group (3-methylbutanoyl) and substituted at the 4- and 6-positions with prenyl side chains (3-methylbut-2-en-1-yl groups). This arrangement forms a characteristic β-diketone system, with the acyl ketone and an enolized hydroxyl stabilized by hydrogen bonding. The core structure can be visualized as a cyclohexadienone ring with hydroxyl groups at positions 3, 5, and 6, the acyl chain at position 2, and the prenyl chains at positions 4 and 6, contributing to its lipophilic nature and reactivity. Regarding stereochemistry, humulone possesses a chiral center at the 6-position of the ring and is isolated from natural sources primarily as the (R)-, although synthetic or degraded forms may exist as racemic mixtures. It exhibits a of [α]_D^{20} = -212° (c = 1.0 in 96% ). Humulone serves as the primary α-acid in , with homologs such as cohumulone differing by the substitution of the isovaleryl side chain with a shorter propanoyl group (ethyl-substituted), while adhumulone features a 2-methylbutanoyl chain; these variations account for the diversity in hop bitter acid profiles.

Physical and Chemical Properties

Humulone appears as a pale yellow to yellow crystalline solid. Its is reported as 65–66.5 °C. Humulone exhibits low solubility in , approximately 6 mg/L at 25 °C and neutral , but is highly soluble in and alkaline solutions due to its ability to form salts. This solubility profile arises from its amphiphilic nature, with hydrophobic isoprenyl side chains and polar functional groups. Chemically, humulone is acidic with a of approximately 5.5, attributed to the enolic in its β-diketone moiety, which facilitates and salt formation in basic media. It is unstable toward light and oxygen, readily oxidizing to form products such as hulupones, which impacts its storage and handling. For quantification, humulone shows strong UV at 275 nm, corresponding to its conjugated enone system. Spectroscopically, humulone's () spectrum features characteristic carbonyl stretches in the 1600–1700 cm⁻¹ region, indicative of its conjugated and functionalities. (NMR) data include ¹H NMR signals for methyl protons around 0.99–1.73 and ¹³C NMR resonances for methyl carbons at 17.77–26.00 , confirming its structural features.

Isomerization

Mechanism of Isomerization

The isomerization of humulone to isohumulone is a thermal and mildly acid-catalyzed rearrangement that occurs during the of in , involving a tautomerization that converts the six-membered of humulone into a five-membered structure in isohumulone. This process is facilitated by the β-tricarbonyl moiety in humulone, which enables enolization and subsequent structural reorganization under the conditions of . The detailed mechanism begins with the deprotonation of the β-diketone system in humulone to form a monoanion, which is the rate-limiting step in mildly acidic or alkaline media. This anion undergoes stereospecific ketonization at the enol function, leading to an acyloin-type that facilitates ring contraction. The rearrangement proceeds through a involving the carbonyl at position 5, resulting in the formation of cis- and trans-isohumulone in a typical of 68:32 under mild conditions. The overall reaction can be represented as: \text{humulone} \xrightarrow{\text{monoanion formation}} \text{acyloin intermediate} \xrightarrow{\text{ring contraction}} \text{cis/trans-isohumulone} No net loss of water occurs in this isomerization. The kinetics of the isomerization follow first-order reaction behavior, with the rate strongly dependent on pH and temperature. Optimal conversion occurs at pH 5.0–5.2 and 100°C, conditions typical of wort boiling, achieving approximately 30–50% conversion of humulone to isohumulone within 60 minutes. The activation energy for this process is 98.6 kJ/mol, and the rate constant at 100°C is about 0.0114 min⁻¹. Under prolonged heating, such as extended times beyond 90 minutes at 100°C, minor byproducts form through pathways, including humulinones from oxidation of remaining α-acids and hulupones from β-acids, which contribute less to bitterness but affect overall . The rate constant at 100°C is 0.0026 min⁻¹, with an of 108.0 kJ/mol. Alternative methods for have been explored, including continuous-flow photochemical using UV light, which selectively produces trans-isohumulones as of 2025.

Isohumulone Formation and Properties

Isohumulones, the primary isomerized products of humulone and related α-acids, exist as six major structural isomers derived from the three principal α-acids in : humulone, cohumulone, and adhumulone. These include the - and - forms of isohumulone, isocohumulone, and isoadhumulone. During thermal in , the forms predominate, typically comprising about 68% of the mixture, while forms account for approximately 32%, due to the greater thermodynamic stability of the configuration. Compared to humulone, isohumulones exhibit significantly enhanced solubility, reaching approximately 600 mg/L at 20°C in water and up to 520–600 mg/L in buffers at 4.5–5.2, which facilitates their incorporation into aqueous media. Their lower value, around 3.5, promotes ionization at typical levels (4.2–4.6), further increasing and contributing to their amphiphilic nature. This tensioactive property also enhances stabilization by forming complexes with foam-active polypeptides like protein Z, with isohumulone and isoadhumulone showing superior performance over isocohumulone in promoting foam height and retention. Isohumulones demonstrate improved resistance to and oxidation relative to humulone, owing to their structural rearrangement into a more stable five-membered ring system. However, they remain susceptible to -induced degradation, particularly under UV exposure, where both - and - forms break down to generate 3-methyl-2-butene-1-thiol (MBT), the compound responsible for the undesirable "lightstruck" skunky off-flavor at concentrations as low as 7 ng/L. In the absence of , -isohumulones are less stable during storage, degrading primarily to and tetracyclic adducts that contribute to bitterness loss. Analytical determination of isohumulones relies on high-performance liquid chromatography (HPLC), which effectively separates the cis- and trans- isomers and quantifies their individual contributions to total iso-α-acid content, typically ranging from 10–100 mg/L in beer. The international bitterness units (IBU) scale, a spectrophotometric measure, is calibrated primarily against isohumulone concentration, as cis-isohumulone imparts approximately 1.82 times greater bitterness intensity than its trans counterpart, providing a standardized assessment of perceived bitterness.

Synthesis

Biosynthesis in Hops

Humulone biosynthesis in hops () occurs via a polyketide pathway that assembles a phloroglucinol-derived core with prenyl side chains, primarily in the lupulin glands of developing cones. The pathway begins with the condensation of three molecules of and one molecule of isovaleryl-CoA, derived from catabolism, to form phlorisovalerophenone as the key intermediate. This initial step is catalyzed by chalcone synthase homologs, specifically the CHS_H1 gene product, which functions as a type III to generate the linear chain that cyclizes into the aromatic phlorisovalerophenone. Subsequent key steps involve sequential prenylation of phlorisovalerophenone with dimethylallyl pyrophosphate (DMAPP), supplied by the methylerythritol phosphate (MEP) pathway in plastids, to form deoxyhumulone. The first prenylation is mediated by the gland-specific prenyltransferase HlPT1, which regioselectively adds the isoprenoid unit at the C-3 position of the phloroglucinol ring to form the mono-prenylated intermediate. The second prenylation at the C-5 position is catalyzed by HlPT2, yielding deoxyhumulone. Deoxyhumulone is then oxidized at the beta-position of the acyl side chain by a cytochrome P450 enzyme (deoxyhumulone hydroxylase or humulone synthase), yielding humulone, the primary α-acid. These enzymatic reactions ensure the structural complexity of humulone, contributing to its bitterness and antimicrobial properties. Genetic regulation of humulone is tightly controlled, with genes for VPS (valerophenone synthase, a CHS homolog), prenyltransferases, and upstream precursor pathways highly upregulated in lupulin glands during the late flowering stage, coinciding with gland maturation and cone development. Transcription factors, particularly R2R3-MYB family members like HlMYB7 and HlMYB3, act as or activators to fine-tune expression; HlMYB7 acts as a , negatively regulating genes by suppressing activator complexes in lupulin glands, while HlMYB3 functions as an activator in the MBW complex to promote expression in glands. This spatiotemporal regulation maximizes humulone accumulation, reaching up to 20% dry weight in cones. Evolutionarily, the humulone pathway derives from ancient plant defense mechanisms, adapting biosynthesis machinery—such as CHS-like synthases—for producing prenylated acylphloroglucinols as agents against herbivores and pathogens. This pathway likely emerged in the family, with variations in enzyme specificity and product profiles across species; for example, H. lupulus emphasizes α-acids like humulone for bitterness, while non-brewing relatives like H. japonicus produce lower levels with altered patterns, reflecting diversification for ecological niches.

Laboratory Synthesis Methods

The classical laboratory synthesis of humulone was first achieved by Riedl in 1951 through a multi-step process starting from . The initial step involves Friedel-Crafts of with isovaleryl chloride in the presence of a Lewis acid catalyst such as aluminum chloride, yielding the key phlorisovalerophenone. This reaction can be represented as: \text{[phloroglucinol](/page/Phloroglucinol)} + (CH_3)_2CHCH_2COCl \rightarrow \text{phlorisovalerophenone [intermediate](/page/Intermediate)} Subsequent steps include selective protection of hydroxyl groups, double prenylation using 3-methyl-2-butenyl bromide to form deoxyhumulone, followed by oxidation (often to the lead salt) and deprotection to afford (±)-humulone, with an overall yield of approximately 5.7%. Modern synthetic approaches have improved efficiency through transition metal catalysis, particularly palladium-catalyzed methods for installing prenyl groups. For instance, a 2014 method employs dearomative conjunctive allylic annulation on acylphloroglucinol precursors using Pd(PPh₃)₄, enabling sequential allylation and cyclization to construct humulone analogs like allyl-desoxyhumulone in 81–87% yield over key steps. These routes leverage decarboxylative allylation to avoid overalkylation issues common in traditional prenylation. Key challenges in humulone synthesis include achieving during side-chain attachment, as the prenyl groups must align properly to mimic the natural (3R,5R)-, and preventing decomposition of the sensitive β-diketone moiety, which is prone to enolization and oxidation under acidic or aerial conditions. Laboratory synthesis of humulone is primarily applied in studies to trace metabolic pathways, where deuterated or ¹³C-enriched variants are prepared via modified or steps for NMR analysis in research.

Applications in Brewing

Contribution to Beer Bitterness

Humulone contributes to beer bitterness indirectly through its isomerization product, isohumulone, formed during the wort boiling process. Isohumulone, the primary iso-alpha acid in beer, activates human bitter taste receptors in the TAS2R family, particularly hTAS2R1, hTAS2R14, and hTAS2R40, which mediate the psychophysical perception of hop-derived bitterness. This binding elicits a sharp, lingering bitter sensation, detectable at low concentrations and characteristic of beers ranging from 10 to 50 International Bitterness Units (IBU), such as lagers and pale ales. The intensity of bitterness is quantified using the scale, defined as the milligrams per liter of isohumulone equivalents in finished , measured via after acidification. During , humulone efficiency typically ranges from 20% to 40%, influenced by factors like boil duration, , and wort density, resulting in only a portion of added humulone contributing to final bitterness levels. High-alpha hop varieties, such as (14-18% alpha acids, predominantly humulone), are favored for their high humulone content, enabling efficient bitterness addition in styles like India Pale Ales (IPAs) where IBU often exceeds 50. In sensory terms, isohumulone-derived bitterness balances the residual sweetness from malt-derived sugars, creating flavor harmony in balanced beer styles. Excessive extraction or high concentrations, however, can shift perception toward astringency, a puckering distinct from pure bitterness, diminishing drinkability.

Processing in Brewing

In production, humulone is primarily incorporated through hopping methods during the wort boiling stage. Kettle hopping involves adding hop cones or pellets at the beginning of the boil, allowing sufficient time for thermal of humulone and other alpha acids into iso-alpha acids, which contribute to bitterness. This method typically requires 60-90 minutes of to achieve optimal conversion, as the process follows first-order kinetics with an of approximately 98.6 kJ/mol for humulone . In contrast, late hopping adds hops toward the end of the boil or in the post-boil stage, minimizing to preserve volatile aroma compounds while extracting only limited amounts of unmodified humulone. Isomerization efficiency of is influenced by several parameters. duration and temperature directly affect the rate, with yields increasing roughly twofold for every 10°C rise, reaching 50-60% under standard conditions ( 5.0-5.5, 100°C). Higher , such as 5.6 compared to 5.0, enhances conversion due to the acid-base of the reaction, though excessive alkalinity can lead to degradation. Certain like unmalted grains can reduce efficiency by increasing gravity and promoting of alpha acids with proteins and polyphenols, while addition of calcium or magnesium silicates can increase yields; overall utilization is typically 35-40%. Dry hopping, performed post-fermentation, avoids heat exposure altogether, preserving in its native form without significant and focusing on aroma oils. Following the boil, post-processing steps separate humulone-derived compounds from solid materials. Spent are removed via and subsequent , often using or systems, to clarify the and prevent carryover of vegetative matter that could cause haze. Purified forms, such as isomerized hop extract (IHE)—an of iso-alpha acid salts—are commonly added post- for precise bitterness adjustment, bypassing losses during boiling and . Since the 1970s, innovations like continuous-flow reactors for extracts have enhanced processing efficiency, enabling near-complete conversion (up to 70% yield) under controlled conditions with catalysts such as magnesium ions, reducing reliance on traditional batch . These systems, developed alongside advanced products, allow for solvent-free and improved in large-scale .

Biological and Health Research

Antimicrobial Activity

Humulone exhibits antimicrobial activity primarily against by acting as an that chelates and transports divalent cations such as Mg²⁺ and Ca²⁺ across the cytoplasmic membrane, thereby collapsing the proton motive force essential for bacterial energy metabolism and leading to . This mechanism selectively targets organisms like species, with minimum inhibitory concentrations (MICs) typically ranging from 7.5 to 30 μg/mL, depending on the strain and environmental conditions such as and cation availability. are generally resistant due to their outer membrane barrier, which limits humulone's access to the inner membrane. Historically, humulone has served as a natural in , inhibiting spoilage by acid-tolerant bacteria since hops were first incorporated into in medieval , with its properties quantified in early 20th-century studies, including those from the 1910s that isolated and tested hop resins against beer contaminants. These investigations confirmed humulone's role in extending shelf life by suppressing Gram-positive spoilers like Lactobacillus brevis, a key beer-spoiling organism. The isomerized form, isohumulone, demonstrates enhanced antimicrobial potency, often 10- to 20-fold greater than humulone against pathogens such as and species, due to improved permeability and ionophoric efficiency in the isomerized structure. This increased activity arises during processes where humulone isomerizes under alkaline conditions, amplifying its preservative effects. Beyond brewing, humulone has been explored for applications in and as a natural antibacterial agent, with s issued since 2000 for hop acid formulations that inhibit microbial growth in products like oral care items and surface sanitizers without synthetic preservatives. For instance, humulone-based extracts effectively control foodborne pathogens in coatings, offering an to chemical antimicrobials while maintaining product sensory qualities. In , its incorporation into formulations targets skin bacteria like Propionibacterium acnes, supported by studies on -derived compounds' stability and efficacy in topical applications.

Potential Health Effects

Humulone exhibits properties primarily through its hydroxyl groups, which enable it to scavenge free radicals and potentially mitigate in biological systems. In assays, humulone demonstrates activity comparable to . Research indicates that humulone possesses effects by inhibiting (COX-2) enzyme activity, as evidenced in models where oral doses of 10-50 mg/kg reduced inflammatory markers such as production. These findings suggest a mechanistic link to the moderate health benefits observed with consumption, where humulone contributes to dampening without affecting healthy tissue function. Preliminary studies from the 2000s have explored humulone's potential in , showing induction of in human leukemia (HL-60) cell lines through Fas- and caspase-mediated pathways. Additionally, humulone suppresses activation in mouse skin models, which may contribute to anti-inflammatory and anti-cancer effects. Research remains primarily preclinical, with no large-scale human clinical trials reported as of 2025. Humulus lupulus (hops) extracts containing humulone are (GRAS) by the U.S. for use in food products, supported by its long history in and lack of reported in standard assays such as the . However, at high doses exceeding 100 mg/kg in animal models, it may induce gastrointestinal irritation, though no evidence of mutagenicity or long-term toxicity has been observed at typical exposure levels.