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Humulene

Humulene, also known as α-humulene or α-caryophyllene, is a naturally occurring monocyclic with the molecular formula C₁₅H₂₄ and a molecular weight of 204.35 g/mol. It consists of three units forming an 11-membered ring with three nonconjugated carbon-carbon double bonds, two of which are triply substituted and one doubly substituted. This is widely distributed in the essential oils of various plants, including Humulus lupulus (hops, where it can comprise up to 40% of the oil), Cannabis indica, pine trees, sage, ginseng, and citrus sources such as orange orchards. In hops, humulene plays a key role in contributing to the earthy, woody aroma of and other brewed beverages. Beyond its sensory properties, humulene is biosynthesized from farnesyl diphosphate in plants and has been isolated through methods like , with yields varying significantly across species from negligible to over 60%. Humulene demonstrates diverse pharmacological activities, including , , , , and anticancer effects, partly through interactions with and receptors as well as modulation of pathways like . These properties position it as a promising natural compound for therapeutic applications, such as treating inflammatory disorders, bacterial infections, and certain cancers, though challenges like low and variable natural yields limit its clinical translation. Additionally, it serves as a precursor for synthetic anticancer agents and exhibits repellent and larvicidal activity against mosquitoes, supporting its potential in .

Chemical Properties

Molecular Structure and Isomers

Humulene, also known as α-humulene, has the molecular formula C₁₅H₂₄ and a molecular weight of 204.35 g/mol. It is classified as a monocyclic , consisting of three units arranged in a single 11-membered carbon ring featuring three nonconjugated double bonds. The systematic IUPAC name for humulene is (1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene, which highlights its cyclic structure with methyl substituents at positions 2, 6 ( dimethyl), and 9. Humulene is structurally related to other sesquiterpenes but distinguished by its monocyclic architecture, in contrast to bicyclic forms like , which shares the same molecular formula (C₁₅H₂₄) but incorporates a fused ring system with a cyclobutane and nine-membered ring. This isomerism arises from differences in carbon skeleton closure, with humulene representing a ring-opened variant of the framework. The compound was first isolated from the essential oils of (hops), from which it derives its name, underscoring its historical significance in plant-derived terpenoids. The of humulene is characterized by all-E () configurations at its three double bonds (positions 1, 4, and 8), contributing to its rigid yet flexible . reveals that the 11-membered ring adopts multiple stable conformers due to low , with empirical calculations identifying key low-energy structures that influence its reactivity in biosynthetic and chemical transformations. These conformational features, including chiral enantiomeric forms in certain derivatives, distinguish humulene from more constrained sesquiterpenes and enable its diverse biological roles.

Physical and Chemical Characteristics

Humulene, also known as α-humulene or α-caryophyllene, is a colorless to pale yellowish liquid at with a below 25 °C. Its is approximately 0.889 g/mL at 20 °C, and it has a of 166–168 °C at or 99–100 °C at reduced pressure (3 mm ). The compound exhibits optical activity, with reported specific rotations ranging from -30° to +30° depending on the source and purity. Humulene is insoluble in but readily soluble in solvents such as , , and , reflecting its nonpolar nature. The aroma profile of humulene is characterized by earthy, woody, and spicy notes, often described as contributing to the "hoppy" scent in and . This sensory quality arises from its volatile structure and makes it valuable in and fragrance applications. Chemically, humulene demonstrates stability under neutral conditions but is susceptible to oxidation, particularly in the presence of air or , leading to the formation of humulene I and II as primary products. Its UV absorption spectrum features a peak around 190 nm, attributable to the π→π* transitions of its conjugated double bonds. Key spectroscopic data aid in its identification: the electron impact mass spectrum shows a molecular ion at m/z 204 and a base peak at m/z 93, with additional fragments at m/z 133 and 119 characteristic of the humulene ring system. The IR spectrum includes prominent absorptions for aliphatic C-H stretches (2950–2850 cm⁻¹) and C=C stretches (1640–1660 cm⁻¹). In ¹H NMR (CDCl₃), diagnostic signals appear for vinyl protons at δ 5.0–6.0 (multiplets) and methylene protons at δ 1.5–2.5 , confirming the three double bonds and cyclic structure.
PropertyValueSource
AppearancePale yellowish green liquid
Density
Boiling Point
SolubilityInsoluble in ; soluble in and organic solvents
Optical Rotation-30° to +30°

Natural Occurrence and Sources

In Plants and Essential Oils

Humulene is primarily found in the essential oils of hops (Humulus lupulus), where it serves as a major sesquiterpene component, often comprising up to 40% of the total oil in certain varieties. In noble hop varieties such as Saaz, humulene levels typically range from 15% to 35% of the essential oil, with concentrations higher in female cones compared to other plant parts. The compound also occurs in various other plants, contributing to their terpene profiles. In cannabis (Cannabis sativa), humulene is present in significant amounts in certain strains, such as White Widow and Girl Scout Cookies, where it can represent up to 15% of the total terpene content and pairs with myrcene for earthy aromas. It appears in ginseng (Panax species), often as part of the root essential oils used in traditional medicine. In sage (Salvia officinalis), humulene is a notable sesquiterpene in the leaf essential oil, alongside β-caryophyllene, contributing to concentrations that vary with distillation time. Clove (Syzygium aromaticum) bud oil contains α-humulene at around 7%, supporting its spicy profile. Similarly, pine needles (Pinus species) yield essential oils with α-humulene at approximately 8%, as seen in Pinus halepensis. It is also present in trace amounts in citrus essential oils, such as from sweet orange (Citrus sinensis), typically comprising less than 2% of the oil. These concentrations differ by plant variety, with female hop cones and specific cannabis chemotypes showing elevated levels. In plant essential oils, humulene imparts an earthy, woody, and hoppy aroma that defines the sensory character of and related species. It also plays a defensive role, deterring herbivores like the tobacco () through repellent effects and inhibiting bacterial pathogens to protect plant tissues from infection. Humulene content in exhibits geographical and seasonal variations, with noble varieties like Hallertauer Mittelfrüh and East Goldings often displaying higher levels—up to 30% or more—compared to those from other regions such as . In , cooler climates and traditional cultivation in areas like the and promote elevated humulene, while warmer seasons or subtropical conditions can reduce concentrations by influencing biogenesis.

In Other Natural and Commercial Sources

Beyond its primary botanical origins, α-humulene occurs in trace amounts within certain animal-derived scents, notably as a component of the volatile blend in Asian lady beetles (), where it contributes to mate attraction alongside compounds like β-caryophyllene and α-bulnesene. This presence highlights its role in non-plant natural chemical signaling, though concentrations remain minimal compared to plant sources. Commercially, α-humulene is primarily isolated from essential oils through , a process that extracts the volatile fraction from cones, yielding oils where α-humulene constitutes 15-50% depending on the variety. Certain varieties, such as Hallertauer Mittelfrüh, exhibit particularly high α-humulene levels, up to 30-40% of the total . In commercial products like , α-humulene derives from additions and undergoes partial degradation during , resulting in concentrations typically ranging from 0.1 to 1 , which imparts subtle earthy and notes below its sensory threshold of about 120 ppb in most lagers. Historically, α-humulene was first identified as a major constituent of essential oils in the , with modern analytical detection relying on gas chromatography-mass (GC-MS) for precise quantification in complex matrices. Under the (ECHA), α-humulene is registered as a REACH substance (EC 229-816-7) since 2009, ensuring compliance for industrial use, though specific purity standards for commercial isolates often exceed 98% as supplied by chemical vendors.

Biosynthesis and Synthesis

Biosynthetic Pathways

Humulene is biosynthesized in via the mevalonate (MVA) or methylerythritol () pathways, which converge to produce farnesyl diphosphate (FPP) as the universal precursor for . The key enzymatic step involves , a I (TPS) from the TPS-a subfamily, which catalyzes the metal-dependent cyclization of (2E,6E)-FPP. The mechanism proceeds through the cleavage of the diphosphate group to generate an allylic farnesyl , followed by an initial 1,11-cyclization to form the humulyl intermediate. This intermediate then undergoes 1,3-hydride shifts or to yield α-humulene, a monocyclic E,E,E-configured , often alongside coproducts like β-caryophyllene. In (), the HlSTS1 encodes the primary humulene , producing α-humulene (∼70% of products) and β-caryophyllene (∼25%) from FPP with a of 0.70 μM. This is expressed predominantly in glandular trichomes of developing cones, where terpenoid biosynthesis genes cluster to coordinate production for aroma and defense. HlSTS1 belongs to a mid-sized TPS family of 87 genes in the hop , with alleles showing functional diversity across cultivars; expression peaks 40–60 days after flowering and is regulated by developmental and stress signals within terpenoid biosynthetic clusters. Recent advances include microbial engineering for humulene production, such as optimization of process steps in H16, enabling enhanced yields from sustainable feedstocks like grass clippings hydrolysate as of 2025. Biosynthetic variations occur across species, reflecting differences in TPS specificity. In (Cannabis sativa), CsTPS9FN, a TPS-a phylogenetically related to HlSTS1, produces α-humulene and β-caryophyllene in a ∼1:2.5 ratio, with high trichome-specific expression driven by MEP pathway regulators like DXS2. This contrasts with the humulene-dominant output in , attributed to subtle active-site residues influencing stabilization and sites; TPS genes expanded recently in , yielding 9–33 full-length orthologs with strain-specific product profiles. Evolutionarily, the humulyl acts as a pivotal precursor, enabling rearrangements to diverse sesquiterpenes like or elemene through TPS promiscuity. This versatility has driven terpenoid diversification in , with synthases evolving via and point mutations (e.g., 1–2 changes altering specificity) since the divergence of lineages like ∼12 million years ago. In , shared ancestral TPS clades underscore humulene's role in adapting volatile profiles for ecological functions, such as herbivore deterrence.

Chemical Synthesis Methods

The first total synthesis of humulene was accomplished by E. J. Corey and S. Hamanaka in 1967 through a stereoselective multi-step sequence that constructed the characteristic 11-membered triene ring system from simple acyclic building blocks, marking a milestone in synthesis. This approach highlighted the challenges of medium-ring formation, relying on controlled olefinations and cyclizations to establish the (E,E,E) geometry of the double bonds. In the 1980s, the McMurry coupling became a prominent method for humulene preparation, as demonstrated by McMurry, Matz, and Kees, who employed intramolecular titanium-mediated reductive coupling of a 1,11-dicarbonyl precursor to forge the in a single step with high . The reaction, conducted under low-valent conditions generated from TiCl₃ and zinc-copper couple, proceeded in approximately 60% yield for the cyclization, followed by and isomerization to yield humulene overall in 15-20% from the precursor. This technique contrasted earlier methods by enabling efficient ring closure without high dilution, though it required careful handling of pyrophoric reagents. Palladium-catalyzed cyclizations emerged in the as versatile modern routes, building on earlier work by Miyaura, Suginome, and , who utilized the intramolecular coupling of haloalkenylboranes to assemble the humulene skeleton regiospecifically. Subsequent refinements, such as Malacria and co-workers' four-component assembly followed by Pd-mediated enone formation, achieved the core structure in 50% yield over the cyclization step, often starting from acyclic precursors like modified derivatives. These methods typically deliver overall yields of 20-40%, with key challenges including maintaining trans geometry and minimizing oligomerization during the 11-membered ring formation due to entropic penalties. Asymmetric syntheses targeting enantiopure humulene-derived forms have advanced in the , exemplified by Barik and Nanda's 2024 route to humulane sesquiterpenoids, which incorporates chiral in early and steps to access optically active scaffolds with >95% ee. Recent improvements integrate hybrid strategies, such as recyclable Pd catalysts in solvent-minimized conditions or bio-derived starting materials, boosting step efficiencies to 60-70% while reducing environmental impact. Post-synthesis purification commonly involves with hexane-ethyl acetate gradients, followed by to isolate humulene as a colorless oil, ensuring removal of polar byproducts and isomers.

Biological Activities and Research

Anti-inflammatory and Analgesic Effects

Humulene, particularly its α-isomer, exhibits effects primarily through the inhibition of the nuclear factor kappa B () signaling pathway, which reduces the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). This mechanism involves suppressing activation in response to stimuli like (LPS), thereby mitigating downstream inflammatory responses in immune cells. Additionally, α-humulene interacts with type 2 (CB2) receptors, promoting and actions without inducing psychoactivity associated with CB1 receptor activation. Research from 2015 to 2025 has highlighted synergies between humulene and cannabinoids in cannabis extracts, enhancing anti-inflammatory outcomes beyond individual components. For instance, terpenoid-rich cannabis oils, including humulene, demonstrated moderate suppression of acute inflammation in cellular models, with combined effects amplifying cytokine reduction when paired with cannabidiol (CBD). A 2023 study on hops-derived humulene with CBD further confirmed this synergy, showing improved cellular uptake and anti-inflammatory efficacy in inflammatory assays. More recently, a 2025 preclinical investigation in rodent models reported that α-humulene, in combination with geraniol, significantly alleviated post-surgical pain by elevating pain thresholds, suggesting potential for non-opioid pain management. In vitro studies indicate anti-inflammatory potency with IC50 values approximately in the 10-50 μM range for cytokine inhibition, as observed in LPS-stimulated human monocytic cells where α-humulene dose-dependently reduced IL-6 release by up to 60% at concentrations around 100 μM. In vivo, efficacy has been demonstrated in arthritis-like models, such as carrageenan-induced paw edema in rodents, where systemic administration of α-humulene prevented TNF-α and IL-1β production, reducing joint inflammation comparable to standard anti-inflammatory drugs. A 2024 scoping review underscores the clinical translation potential of α-humulene as a topical anti-inflammatory agent, consolidating evidence from pharmacological studies that support its use in formulations for conditions like dermatitis or arthritis, while calling for further human trials to validate safety and efficacy.

Antimicrobial, Anticancer, and Other Effects

Humulene exhibits notable antimicrobial properties, particularly against Gram-positive bacteria such as Staphylococcus aureus, where α-humulene demonstrates a minimum inhibitory concentration (MIC) of approximately 2.6 μg/mL in vitro. It also shows activity against anaerobic pathogens like Bacteroides fragilis, with an MIC of 2 μg/mL and biofilm inhibitory concentration of 2 μg/mL, reducing biofilm formation through downregulation of efflux pump genes. Recent studies from 2020 to 2023 highlight its potential in disrupting bacterial biofilms, offering a mechanism to combat antibiotic-resistant strains by inhibiting adhesion and metabolic activity at concentrations of 8–32 μg/mL. Against fungi, essential oils rich in α-humulene display moderate antifungal effects, with growth inhibition against Candida species and Saccharomyces at concentrations up to 500 μg/mL, though pure humulene's MIC remains higher than for bacteria. In anticancer research, humulene induces in various tumor cell lines, primarily through the generation of (ROS) and depletion of , leading to mitochondrial dysfunction. For instance, in colon cancer cells such as HT-29 and , α-humulene achieves an IC50 of 5.2 × 10⁻⁵ mol/L and 24.4 ± 2.4 μM, respectively, selectively inhibiting proliferation without affecting non-cancerous cells. Similarly, in lines like , it yields a GI50 of 55–73 μM, promoting intrinsic apoptotic pathways via ROS-mediated damage. These effects underscore humulene's potential as an in cancer therapy, enhancing the of agents like in colorectal and pulmonary models. Beyond and anticancer actions, humulene displays neuroprotective potential by scavenging ROS and mitigating in neuronal models. In a 2025 study using cells, α-humulene protected against H₂O₂-induced with an of 209–221 μg/mL, reducing cellular damage in co-cultures with inflamed macrophages. As an , α-humulene deters oviposition in Aedes aegypti mosquitoes, achieving up to 31% reduction at 5 ppm in leaf oil extracts, with behavioral assays confirming its role in avoiding breeding sites. Humulene's profile supports these applications, showing low in preclinical studies. In extracts, it synergizes with THC and to enhance cytotoxic effects against cancer cells, as seen in 2018 analyses of varieties where humulene amplified CBD's antiproliferative activity. This synergy, part of the , may also briefly augment anti-inflammatory outcomes when combined with cannabinoids.

Applications and Uses

In Food, Beverage, and

Humulene plays a significant role in the brewing industry, where it contributes to both the bitterness and aroma of through chemical transformations during the process. During , humulene undergoes oxidation to form products like humulene , a compound that imparts a characteristic "hoppy" aroma with spicy and woody notes. This forms more prominently from aged , while beers using fresh show lower levels of humulene , with older leading to increased formation of derivatives like humulene I. Levels of humulene and its derivatives are notably higher in India Pale Ales (IPAs), often reaching 25–100 µg/L due to extensive dry-hopping practices, compared to lagers where concentrations remain lower owing to minimal late-hop additions. In food applications, humulene serves as a natural flavoring agent, enhancing earthy and profiles in various spices and herbal teas. It occurs naturally in plants such as , , , and lemongrass, where it contributes subtle woody and spicy undertones to culinary preparations. As a key component of essential oils, humulene-derived flavorings are recognized as (GRAS) by the U.S. for use in food products, including beverages and seasonings, without requiring premarket approval when sourced from approved natural extracts. In , humulene is incorporated into blends from , , and ginger to promote relaxation and subtle mood enhancement through olfactory stimulation. Its earthy, woody aroma, derived from its structure, interacts with the via inhalation, potentially fostering a calming effect akin to mild . Recent research, including a study on receptor interactions, indicates humulene's moderate binding to receptors may support psychophysiological relaxation, though effects remain non-psychoactive and context-dependent. Sensory detection of humulene's aroma occurs at low concentrations, with an odor threshold of approximately 0.12–0.45 in , allowing its subtle presence to perceptual experiences in diluted forms.

In Pharmaceuticals and Cosmetics

Humulene exhibits potential in pharmaceutical applications, particularly for creams and due to its topical and systemic properties, as demonstrated in preclinical models where it reduced paw edema and levels comparable to dexamethasone. of α-humulene at doses around 50 mg/kg has shown marked inhibitory effects on in models, supporting its use in supplements for conditions like . Recent preclinical trials from 2024-2025 have explored humulene's role in cannabinoid-terpene synergies for , with studies in mouse models indicating that α-humulene raises pain thresholds for post-surgical and pain via activation, though it is less potent than like . These effects further bolster its suitability for pain-relief topicals. In cosmetics, humulene, often derived from extracts, serves as an in skincare formulations to combat anti-aging effects by reducing oxidative damage from UV stress and promoting synthesis. It is incorporated into lotions and gels at concentrations of 0.1-0.2% to leverage its anti-inflammatory properties for reducing skin irritation and enhancing , with formulations combining it with and showing superior efficacy . Regulatory assessments confirm humulene's safety in cosmetics when formulated to be non-sensitizing, with hops extracts deemed safe up to 0.2% under good manufacturing practices, though impurities like β-myrcene require monitoring. Emerging patents highlight humulene-enriched derivatives for topical and oral anti-inflammatory products, such as formulations with 3% humulene alongside and β-caryophyllene for enhanced therapeutic delivery. Formulation challenges include humulene's poor solubility and thermal instability in emulsions, necessitating nanoemulsion techniques with to maintain droplet sizes below 50 nm and prevent coalescence during storage.

Environmental Role

Atmospheric Chemistry

Humulene, specifically α-humulene, is a (VOC) emitted biogenically from various vegetation sources, including , herbs, and tropical plants, contributing approximately 1-10% to total emissions in certain ecosystems. These emissions are primarily controlled by temperature and light, with higher rates observed during warmer seasons, and can be enhanced by environmental stresses such as herbivory or . In urban and forested areas, α-humulene fluxes from biogenic sources interact with pollutants, influencing local air quality dynamics. In the atmosphere, α-humulene undergoes rapid oxidation, predominantly via , with an estimated lifetime of about 2 minutes under typical tropospheric conditions (e.g., 30 ppb O₃). This reaction proceeds through the formation of primary ozonides and Criegee intermediates, leading to multi-generational products including epoxides, dicarboxylic acids, hydroxy-oxocarboxylic acids, and oxo-carboxylic acids. of α-humulene yields secondary organic s (SOA) with efficiencies of 20-40%, depending on initial concentrations and relative humidity, as demonstrated in chamber simulations. Additionally, daytime oxidation by hydroxyl () radicals follows H-abstraction pathways, producing peroxy radicals that further contribute to low-volatility compounds and aerosol growth, with lifetimes extending to 30-40 minutes at typical OH levels (2 × 10⁶ molecules cm⁻³). Atmospheric modeling of α-humulene oxidation pathways, informed by chamber experiments from 2011 to 2025, highlights its role in urban haze formation through enhanced SOA production in NOx-rich environments. These studies show that sesquiterpenes like α-humulene can account for significant fractions of biogenic SOA in mixed urban-rural settings, amplifying particulate matter concentrations via multi-phase chemistry. Regarding climate impacts, α-humulene exhibits a low global warming potential due to its short atmospheric lifetime, but its high aerosol formation efficiency contributes to indirect radiative forcing through aerosol-cloud interactions and tropospheric cooling effects.

Ecological and Biodegradation Aspects

Humulene, a emitted by various including (), serves as a key component in plant defense signaling within ecosystems. It acts as a that repels pests, thereby protecting host from herbivory and reducing damage to reproductive structures. For instance, α-humulene inhibits mating in Mediterranean fruit flies (), disrupting pest reproduction and providing indirect defense to fruits and flowers. In addition to pest repulsion, humulene contributes to interspecies interactions by deterring oviposition and feeding in vectors like mosquitoes (Aedes aegypti) and aphids (Macrosiphum euphorbiae). Studies show that α-humulene, often in synergy with related terpenes like β-caryophyllene, significantly reduces aphid survivorship and honeydew production, limiting pest proliferation in agricultural and natural settings. While humulene's role in pollinator attraction is less pronounced compared to other floral volatiles, its presence in plant emissions helps balance defense against excessive herbivory without broadly repelling beneficial insects. Biodegradation of humulene occurs primarily through microbial processes in terrestrial and aquatic environments, facilitating its rapid breakdown and minimizing long-term accumulation. In , aerobic degrade α-humulene efficiently, with studies demonstrating over 60% mineralization within 28 days under standard conditions, corresponding to a of approximately days to weeks depending on microbial activity and environmental factors. This process involves enzymatic oxidation by , such as actinomycetes and pseudomonads, which utilize humulene as a carbon source. In aquatic systems, humulene undergoes enzymatic and microbial transformation, often accelerated by hydrolytic enzymes from aquatic and fungi. These pathways lead to of its cyclic into simpler hydrocarbons and alcohols, with rates enhanced in oxygenated waters. The compound's aids its dissipation, further reducing persistence in runoff or . Humulene exhibits moderate environmental persistence due to its physicochemical properties, with a log Kow of approximately 6.6-6.95 indicating potential for partitioning into organic phases but limited in aquatic organisms owing to its rapid . Overall, its low bioaccumulation factor (BCF < 500) stems from metabolic clearance in biota, contrasting with more persistent pollutants. Hop cultivation for humulene-rich varieties impacts local through alteration and use, potentially reducing native and diversity in fields. However, sustainable practices like with wildflowers and reduced enhance habitats and microbial diversity, mitigating negative effects and supporting . Studies from hop regions emphasize that integrating cover crops in fields promotes balanced without compromising yields.

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