Myrcene-What Are the Potential Health Benefits of This Flavouring and Aroma Agent?

Physical and Chemical Properties of β-Myrcene

Monoterpenes are a class of terpenes; consisting of two isoprene units (5 carbon bases each) (28). Myrcene (C₁₀H₁₆, molecular weight 136.23 g/mol) (29) is classified as an acyclic monoterpene, with properties listed in Table 1.

Myrcene exists in two isomeric forms, namely β-myrcene (7-methyl-3-methylene-1,6-octadiene) and α-myrcene (2-methyl-6-methylene-1,7-octadiene). The most common is the naturally occurring isomer β-myrcene, which contains an isopropylidene group and is often denoted as “myrcene” in the literature. The other is α-myrcene, which exists in the isopropenyl form (2). In β-myrcene there are three carbon-carbon double bonds (two of them being conjugated) and a gem-dimethyl terminal (Figure 1) (36).

Figure 1

β-myrcene structure.

Biosynthesis Production of β-Myrcene

Monoterpenes are produced in plants by the stereospecific condensation of two isomeric five-carbon units: isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) (37). Geranyl diphosphate (GPP) is thus formed via condensation of IPP with DMADP, by GPP synthase (GPPS) (38, 39). Geranyl diphosphate (GPP) then undergoes hydrolysis catalysed by a prenolpyrophosphatase to form geraniol. Consequently, β-myrcene is produced by the dehydration and isomerization of geraniol (37).

Industrial Synthesis of Myrcene

β-Pinene is an important starting material for the synthesis of β- myrcene. Up until the 1950s, β-pinene was extracted from pine or spruce resins by tapping trees Nowadays, β-pinene can be isolated from the waste streams of paper mills, by distillation and desulphurisation from crude sulphate turpentine (CST). The proportion of β-pinene in CST, depends largely on the age, season, geographical location, and variety of the tree (40).

The most common method to produce myrcene industrially is through the pyrolysis of β-pinene (2). In the future, engineered microbial platforms may provide alternative means for sustainable and environmentally friendly large-scale production of myrcene (41–43).

Analysis, Extraction, and Quantification of β-Myrcene

β-Myrcene is considered an important intermediate compound which can be derivatized to produce numerous end products, such as, citronellol, citronellal, geraniol, nerol and linalool (2). Several methods of determining β-myrcene content have been published and developed to achieve more simple, rapid and efficient quantitative analytical methods (Table 2) (55, 58). Naturally, β-myrcene exists as complex mixtures with other monoterpenes. Thus, it would be difficult to isolate β-myrcene in large quantities from these complex mixtures. Therefore, there is a need for an efficient and economical method for separating β-myrcene from other terpenes. Hydro-distillation and solvent extraction were used extensively followed by Gas Chromatography (GC) coupled with Mass Spectrometry (MS) or Flame ionisation detector (FID) for quantification (58, 59). The drawbacks of these techniques were mainly the tendency of losing essential volatile compounds (including β-myrcene) during the process of solvent evaporation leading to decrease the yield and altering its odour characteristics (59, 60). Also, these methods require a lengthy process from 3 to 4 h of distillation allowing possibility of changing the chemical composition of analyte or risk of indefinable loss (51, 61).

In contrast, extraction efficiency and flexibility of supercritical fluid CO₂ extraction are much better than the earlier methods, since there is a greater control in temperature and pressure parameters (50). The yield of extracts obtained by supercritical CO₂ extraction is highly dependent on the temperature and pressure applied during the extraction process. Higher recovery percentage of β-myrcene was obtained at lower temperatures and pressures of supercritical fluid (62). Also, at 40°C and 20.0 MPa, β- myrcene, L-caryophyllene and α-humulene were detected at higher concentrations allowing for their importance in beverage industry as flavouring and aroma additives (63). The demand for using environmentally clean extraction techniques and the need for faster, more powerful, and cheaper analytical procedures is therefore driving the industrial production into methods like supercritical fluid CO₂ extraction.

All these techniques had noticeable disadvantages such as excessive use of solvents, longer operation time and/ or large volumes of sample (60, 62). Also, they require the use of highly sophisticated, uneconomical devices with a limited lifetime (53). Furthermore, these techniques are quite complex in operation and are labour intensive, thus, they are not fully efficient when a routine analysis of a large number of samples is needed.

Solid phase microextraction (SPME) has emerged as an alternative to traditional techniques (53). It has been successfully employed to determine the volatile composition of hop and also in different food products (54, 64). This technique has its own characteristics, such as efficient extraction procedures, short analysis of time, economical with low production cost, and high selectivity and sensitivity when coupled with suitable detection modes. Moreover, by using SPME, all the steps of extraction technique can be introduced within a single process without any interruption, resulting in a high sample processing (53).

SPME can also be used with (GC) and (MS) analysis, to provide a full metabolomic profile of the intended plant (53, 65, 66). Hops of the Saaz variety have been studied by Gonçalves et al. using SPME, allowing for profiling the terpenoid metabolomic pattern found in the essential oil. β-Myrcene was dominated by 53.0% of the total volatile fraction at 40°C. The disadvantages of SPME are that the extraction conditions, age of fibre, and matrix could affect the amount of sample absorbed on the fibre. Controlling these factors can produce a highly efficient and reproducible method for extraction and analysis of β-myrcene. Fourier transform Raman Spectroscopy has also been used to determine the percentage of β-myrcene in mastic gum oil based on band intensity measurements (55). The method is extremely rapid, simple and non-destructive for the sample.

There are still some limitations in obtaining a pure extract of β-myrcene allowing for accurate analysis and quantification. Thus, proper selection of extraction techniques and pre- determined sample parameters are necessary for efficient analysis of β-myrcene.

Pharmacokinetics of β-Myrcene

Most previously published data on the absorption, distribution, metabolism and excretion of β-myrcene have been conducted in experimental animals, such as, rabbits and rats (26). In a pharmacokinetic study, blood levels as high as 14.1 ± 3.0 μg/mL β-myrcene (peak value) were detected 60 min after oral administration of 1.0 g/kg bw β-myrcene to female rats (67). β-Myrcene showed a pattern of elimination mostly by urine with an elimination half-life of 285 min, however no studies examined the possibility of biliary excretion (3, 67). β-Myrcene was mainly distributed in adipose tissue and in several major organs, including the liver, brain, kidneys, and gonads. β-Myrcene is bioavailable in human plasma within 30 min after consumption of a single dose. The high degree of bioavailability in plasma is a crucial step towards its useful usage in the food and beverage industry. Furthermore, β-myrcene reaches the plasma unaltered, with a peak concentration between 2 and 4 h (68). This could partially explain its health benefits and applications on human health. More research is needed to characterise the kinetics of β-myrcene in human metabolism.

Urinary excretion of conjugates of two diols (10-hydroxylinalool and 7-methyl-3-methylene-oct-6-ene-1,2-diol), and two hydroxy acids (10-carboxylinalool and 2-hydroxy-7-methyl-3-methylene-oct-6-enoic acid) was observed in male rabbits administered β-myrcene by gavage (69, 70). Diols were formed due to an oxidation reaction occurred at the 3,10-double bond through a 3,10-epoxide intermediate. The metabolites were isolated by using rat-liver microsomal cytochrome P450 enzymes and confirmed by undergoing enzymatic degradation by β-Glucuronidase/Arylsulfatase.

In rats, several metabolites were isolated from the urine after oral administration of β-myrcene, such as, 10-hydroxylinalool, 1-hydroxymethyl-4-isopropenyl cyclohexanol, 7-methyl-3-methylene-oct-6-ene-1,2-diol, 10-carboxylinalool, 2-hydroxy-7-methyl-3-methylene-oct-6-enoic acid (5). Their formation involved a sequence of oxidation reactions of the terminal double bonds by microsomal cytochrome P450 2B and epoxidation of the 1,2- and 3,10-epoxide intermediates, with subsequent hydrolysis to diols. This CYP-catalysed reaction was inhibited by several non-specific inhibitors of cytochrome P450 preventing from conversion of β-myrcene into 10-hydroxylinalool.

Central Nervous System Effects and Neurobehavioral Activity

Myrcene is well-known for its anxiolytic and sedative effects, which are both desired therapeutic actions, but may also pose some risks. Sedating agents can create drowsiness and impair motor coordination, and this can be assessed using the “rota-rod test” in animal studies measuring the length of time a rodent can balance on a rotating horizontal rod. A relatively high dosage of 200 mg/kg (1,468 μmol/kg) body wt myrcene, led to a 48% decrease in the time of permanence on the bar in the rota-rod test. The same dose of myrcene prolonged barbiturate sleep time 2.6 times. This was more intense in the presence of citral (20). Similarly, a single oral dose of β-myrcene, prolonged potentiated pentobarbital sleeping time, when administered 60 min before the barbiturate, possibly by inhibiting the barbiturates metabolism via cytochrome P450 (CYP) (78).

The main essential oil obtained from Cannabis sativa L. (Cannabaceae; hemp) (myrcene content: 22.9%), demonstrated measurable effects on the autonomic nervous system in healthy human subjects (n = 5). Inhalation of cannabis essential oil for 5 min improved nerve activity and was shown to relive stress and anxiety (Sweet almond oil was used as a control). The subjects generally felt more relaxed, energetic, calm, and an elevated mood, five min post inhalation. The study also used an electroencephalogram (EEG) to measure brain activity and results showed that there was an increase in theta (4–8 Hz) and alpha (8–13 Hz) brain wave activity in the posterior brain region, which is comparable to the EEG waves of individuals undergoing meditation (90).

Myrcene has also been shown to function as an anticonvulsant agent. Myrcene, obtained from Lippia alba (Mill.) N.E.Br. ex Britton and P.Wilson increased the latency of pentylenetetrazol-induced (PTZ) convulsions and increased the percentage of survival in female Swiss mice (91). Similarly, essential oils from Cinnamosma madagascariensis Danguy (8.9% myrcene) was evaluated in vivo for its anticonvulsant effects in Wistar rats that underwent induced seizures using pentylenetetrazole (PTZ). The study demonstrated the antiepileptic potential, by attenuating convulsions with moderate sedative effects. The possible mechanism of action was linked to glutamatergic and GABAergic neurotransmission (92). On the other hand, Da-Silva et al. (93) were unable to demonstrate the protective role of myrcene against PTZ-induced seizures. They were also unable to demonstrate benzodiazepine-like anxiolytic activity and the anti-depressive and antipsychotic effects of myrcene (93).

β-Myrcene may have significant clinical potential in adjuvant therapies, both as a pure compound and as a part of extract preparations. Popular anxiolytic essential oils are rich in other terpenoid alcohols, such as linalool, geraniol and citronellol, which might work synergistically with β-myrcene. Due to limited studies in human participants, small sample sizes, short duration of β-myrcene application, limited diverse administration of β-myrcene, the potential beneficial effect of β-myrcene on neurological disorders need further and more rigorous assessment.

Antioxidant Activity

Antioxidant agents are accountable for the prevention of ageing and degenerative diseases such as atherosclerosis, cardiovascular diseases, cancer, diabetes and neurological illnesses (94). They also have an important role in inhibiting lipid oxidation within food products. In recent decades, there has been growing interest in the use of naturally occurring antioxidants in food preservation (22).

Selected monoterpenes have been studied for their potential antioxidant capacities, which can be attributed by the presence of conjugated double bonds that create chain breaking antioxidant activity. Chemical antioxidant assays like the DPPH assay (22, 95), are excluded here since they are of no pharmacological relevance. There is no evidence for therapeutic benefits on the basis of such chemical assays (96).

In vivo myrcene (97–100) demonstrate some relevant antioxidant effects. Ciftci et al. treated female Sprague-Dawley rats exposed to the environmental contaminant 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) with myrcene [with a high dose of up to 200 mg/kg bw per day (1,468 μmol/kg) for 30 or 60 days]. These rats had a decrease in hepatic lipid peroxidation via activation of antioxidant and radical scavenger properties (97). Myrcene, again at a high dose of 200 mg/kg (1,468 μmol/kg) played a neuroprotective role in cerebral ischemia/reperfusion injury in C57Bl/J6 mice. In addition, myrcene increased glutathione along with other antioxidant enzymes such as glutathione peroxidase (GPx) and superoxide dismutase, thereby preventing oxidative damage and protecting brain tissue (99).The effects of orally administered β-myrcene (7.5 mg/kg bw; 55 μmol/kg bw) against ethanol-induced gastric ulcers in male Wistar rats is mediated through antioxidant effects via increased levels of GPx, glutathione reductase (GR) and total glutathione in gastric tissue (98). Importantly, future studies investigating antioxidant activity of β-myrcene, need to use appropriate dosage levels that have therapeutic effects in humans. This requires a comprehensive investigation into the recommended dosage of β-myrcene in humans. Additionally, most studies in the literature utilise chemical assays to detect antioxidant activity of β-myrcene. Future studies should use pharmacologically relevant in vivo or cell-based models to measure antioxidant activity.

Anti-ageing Activity

As myrcene is an effective antioxidant compound, it may play a protective role against UVB-induced human skin photo-ageing. UVB exposure is associated with an overproduction of reactive oxygen species (ROS), which is a primary factor in oxidative skin damage (101). Abnormal production of ROS, activates numerous cell surface cytokines, growth factor receptors and mitogen activated protein kinases (MAPKs) (102). Exposure to UV radiation has also been shown to activate matrix metalloproteinases (MMPs), leading to the atrophy of collagen and elastin fibres (103). To date, only one study has investigated the role of β-myrcene and anti-ageing. Myrcene ameliorated skin ageing via decreased production of ROS, MMP-1, MMP-3, interleukin-6 (IL-6) and increased transforming growth factor type 1 (TGF-1) and type I procollagen secretions in UVB-irradiated human dermal fibroblasts. β-Myrcene treatment also downregulated phosphorylation of MAPK-related signalling molecules. Thus, myrcene may have a vital role against age-associated skin oxidative damage in skin care products (104).

Anti-inflammatory Activity

In vitro β-myrcene is a powerful anti-inflammatory agent. Its ability to lessen inflammation occurs via prostaglandin E-2 (PGE-2) (105). In a study by Souza et al. (106) myrcene was shown to be effective in inhibiting the inflammatory response induced by lipopolysaccharide, including cell migration (leucocytes, neutrophils, mononuclear macrophages and eosinophils) and production of nitric oxide in mouse models of pleurisy. A significant inhibition of γ-interferon and interleukin (IL)-4 was also observed (106). Male Wistar rats with isoproterenol induced heart failure showed signs of cardiac function abnormalities. On the other hand, rats who were pre-treated with myrcene were protected from cardiac failure (p < 0.001) and inflammatory signals were abrogated. β-Myrcene was involved in suppressing fibrotic markers such as matrix metalloproteinases (MMP-2 and MMP-9) and regulating the expression of inducible nitric oxide synthase (iNOS), Transforming growth factor beta (TGF-β) and miRNA (profibrotic agents) with potential future benefits in treating cardiac failure (107).

In an in vitro cartilage degradation model of osteoarthritis, myrcene (25–50 μg/mL; 183.5–367 μmol/kg) showed anti-inflammatory and anticatabolic effects on human chondrocytes. Cartilage degradation and osteoarthritis progression was slowed down. Myrcene decreased interleukin IL-1β-induced nuclear factor-κB (NF-κB) and jun terminal kinase (JNK). It further decreased ERK1/2, p38 activation and the expression of inflammatory iNOS. Myrcene decreased catabolic responses (matrix metalloprotease MMP1 and MMP13), whilst increasing the expression of anticatabolic genes (tissue inhibitor of metalloproteases TIMP1 and TIMP3). Additionally, myrcene decreased the expression of non-cartilage specific collagen I induced by IL-1β, thus promoting the maintenance of the differentiated chondrocyte phenotype (23).

The anti-inflammatory activity of β-myrcene may not only be credited to its antioxidant potential, but also with its interaction with signal pathway cascades involving cytokines and transcription factors. Thus, plant oils rich in β-myrcene could serve as an option help to alleviate anti-inflammatory diseases and their symptoms.

Adverse Skin Reactions

In a European multicentre study, of 1,511 consecutive dermatitis patients, only one patient reacted adversely to 3% β-oxidised myrcene (containing 30% of β -myrcene) (113), this indicates that myrcene is hypoallergenic on the skin and is safe for topical use. Undiluted β-myrcene was moderately irritating to rabbit skin (114); but was neither irritating nor sensitising after being tested at 4% (n = 25) (115). β-Myrcene (5%) was sensitising to two of eleven patients sensitive to tea tree oil (116).

Acute Toxicity

In mice and rats, the acute oral toxicity of β-myrcene was low, with an approximate lethal dose (ALD) of >5.06 g/kg bw (37,143 μmol/kg bw) and 11.39 g/kg bw (83,609 μmol/kg bw), respectively. Administration of β-myrcene via intraperitoneal injection had a lower ALD in mice and rats (2.25 g/kg bw and 5.06 g/kg bw, respectively; 16,516 μmol/kg bw and 37,143 μmol/kg bw, respectively), which is likely due to drug- induced chemical peritonitis (117). The acute oral LD₅₀ in rats and the acute dermal LD₅₀ in rabbits were reported to exceed 5 g/kg body weight (36,703 μmol/kg bw), following oral administration and dermal application (114).

Subacute and Sub-chronic Toxicity

In a 14 week gavage study (Good Laboratory Practise compliant), male and female F344/N rats and B6C3F1 mice (10/group) were given doses of β-myrcene at 0.25, 0.5, 1, 2, or 4 g/kg bw (1,835, 3,670, 7,341, 14,681, 29,362 μmol/kg bw) for 5 days per week (human equivalent daily dose range: 17.5–280 g) (26). All 4 g/kg (29,362 μmol/kg) mice and rats died within 2 weeks, with other deaths observed in groups administered >0.5 g/kg (>3,670 μmol/kg). At the end of the 14 weeks, renal tubule necrosis significantly increased in rats of each dosage groups (not tested in mice). In rats with a dosage >1 g/kg (>7,341 μmol/kg), increased chronic inflammation, inflammation of the forestomach, mesenteric lymph node atrophy and olfactory epithelium degeneration was observed (26).

In a 90 day toxicity study utilising groups of male and female Sprague Dawley rats (10/sex and group), at the request of the EFSA, β-myrcene was administered in a diet containing 0, 700, 2,100, or 4,200 ppm of β-myrcene daily designed to provide targeted doses of 50, 150, or 300 mg/kg bw/day (367, 1,101, 2,202 μmol/kg bw/day) (118). No effects on mortalities, clinical signs of toxicity, haematology and clinical chemistry parameters and organ weights in the presence of β-myrcene within the diet was reported. Furthermore, the histopathological findings observed were not related to ingestion of β-myrcene and were either incidental or spontaneous. The oral NOEL for both sexes of rats, was the highest dose tests: 115 and 136 mg/kg bw/day (844 and 998 μmol/kg bw/day) for males and females (118). It should be noted that this calculated NOEL, is several orders of magnitude greater than human exposures from β-myrcene (119).

Reproductive Toxicology

In a 3 month Gavage Study of β-myrcene, no effects on the weight of reproductive organs, sperm count or oestrous cyclicity was observed in doses of up to 2 g/kg (14,681 μmol/kg) in rats and up to 1 g/kg (7,341 μmol/kg) in mice (26). Administration of high doses of β-myrcene (1,200 mg/kg bw/day; 8,809 μmol/kg bw/day) on days 6–15 of pregnancy, was found to induce embryofoetal toxicity in pregnant Wistar rats. High doses decreased maternal body weight gain, increased the incidence of foetal skeletal malformations, lowered the number of visible implantation sites and the number of live foetuses. Additionally, foetal weights were lower, in comparison to the control group. The no-observable-adverse-effect level (NOAEL) of oral administration of β-myrcene for maternal and offspring toxicity was 500 mg/kg bw/day (3,670 μmol/kg bw/day) (67).

Two addition studies of β-myrcene on reproductive and development toxicity, have also indicated the adverse effects of β-myrcene on birth weight, peri and post-natal mortality, as well as foetal developmental abnormalities in Wistar rats (120, 121). The NOELS for fertility and general reproductive performance has been estimated as 250 mg/kg bw (1,835 μmol/kg bw) (120) and 300 mg/kg bw (2,202 μmol/kg bw) (121). No data on the reproductive or developmental toxicity of β-myrcene in humans is currently available.

Mutagenicity and Genotoxicity

Myrcene inhibited cyclophosphamide induced sister-chromatid exchanges in Chinese hamster V79 cells and cultured hepatic tumour cells (122). Myrcene had no genotoxic potential in mammalian cells in vitro and did not induce chromosome aberrations or sister-chromatid exchange. β-Myrcene reduced CP-induced sister-chromatid exchanges in human lymphocytes in a dose dependent manner. Also, it did not influence the genotoxicity of methane sulfonate and benzo[a]pyrene (123). Additionally, β-myrcene (doses ranging from 100 to 1,000 μg/mL; 734 to 7,341 μmol/kg) reduced the cytotoxic and mutagenic effects of CP in V79 Chinese hamster cells, when tested with rat liver S9. The authors suggested that myrcene had the ability to inhibit cytochrome P-450 isoenzymes which activates compounds with mutagenic and carcinogenic properties (123).

No evidence of chromosomal aberrations in bone marrow cells of rats administered β-myrcene (0.1, 0.5, or 1.0 g/kg bw; 734, 3,670, 7,341 μmol/kg bw) was observed. Although there was no evidence of myrcene-induced clastogenicity, there was a dose-dependent increase in the mitotic index of bone marrow cells at 24 h (124). Additionally, there was no increase in the frequency of micronucleated normochromatic erythrocytes, a biomarker of both acute and cumulative chromosomal damage, in B6C3F1 mice treated with β-myrcene (0.25 to 2 g/kg; 1,835 to 14,681 μmol/kg) by gavage for 3 months (26).

β-Myrcene expressed antimutagenic activities against aflatoxin B₁ (AFB₁) in Salmonella typhimurium (TA100). Doses of 1.5 and 3.0% of β-Myrcene, showed inhibitory actions of 65 and 73%, respectively when tested with 1.0 μg/plate AFB₁ in the presence of exogenous metabolic activation (rat liver S9) using TA100 and the pre-incubation method (125). The NTP (2010) and (126), concluded that β-myrcene was not mutagenetic based on the negative Ames test using Salmonella strains (TA97, TA98, TA100, and TA1535) with and without metabolic activation. It was also negative in the Escherichia coli test system (strain WP2 uvrApKM101) with and without metabolic activation (S9 fraction from Aroclor 1254-induced rat or hamster liver), and in an in vivo micronucleus assay in B6C3F1 mice (26).

β-Myrcene inhibited the activity of pentoxyresorufin-O-depenthylase (PROD), a selective marker for mono-oxygenase CYP2B1, necessary for the activation of genotoxins in rats (127). β-Myrcene also demonstrated protective effects against t-butyl hydroperoxide induced genotoxicity in human B lymphoid NC-NC cells, which was predominantly mediated by their radical scavenging activity (128).