Background
Lavender (
Lavandula angustifolia) essential oil consists of a mixture of mono- and sesquiterpenoid alcohols, esters, oxides and ketones, containing linalool, linalyl acetate, 1,8-cineole, terpinen-4-ol, β-caryophyllene and camphor as primary components[
1,
2].
Lavender and its essential oil have been used since centuries due to antiseptic, antimicrobial and sedative effects. In today’s folk and complementary medicine, the oil is applied also for the treatment of conditions such as anxiety, restlessness, insomnia and depression. Administration routes include absorption via the respiratory tract (aromatherapy) or oral ingestion[
3]. Although there is evidence-based information on the pharmaceutical efficacy of lavender oil for the treatment of anxiety-related disturbances[
4], its therapeutic significance was little appreciated for a long time, due to the lack of larger clinical trials, but also due to methodological problems in constituent identification and standardization of such complex multicomponent preparations.
Recently, Kasper
et al. demonstrated the therapeutic efficacy of the lavender oil preparation Silexan for the treatment of subsyndromal anxiety disorder in a randomized, double-blind, placebo controlled trial[
5]. Lavender oil treatment was found to alleviate anxiety related symptoms such as restlessness, disturbed sleep as well as somatic complaints, whereby the product demonstrated good tolerability without provoking greater adverse effects[
6].
However, only few reports on the specific neurobiochemical actions of lavender oil are available. Best discussed in the literature are the anxiolytic, analgesic and anti-inflammatory effects of one of the oil’s principal components, linalool and its derivatives like linalyl acetate[
7]. The antinociceptive potential of linalool was studied in several animal studies, e.g. it has been shown to interfere with glutamatergic transmission in mice[
8,
9] and to modify the nicotinic receptor-ion channel kinetics at the neuromuscular junction[
10].
Importantly, immune activation and inflammation are strongly associated with an increase of mood disorders[
11,
12]. Several biochemical links between psychoneuroimmunology and neuropsychopharmacology have been dissected in the past. The catabolism of the essential amino acid tryptophan, known for its essential role in antimicrobial defence, has turned out as an important link between the immunological network and neuroendocrine functions[
12,
13]. The enzyme indoleamine 2,3-dioxygenase (IDO, EC 1.13.11.52) catalyses the rate-limiting step in the conversion of tryptophan to kynurenine and becomes highly activated in macrophages and is induced also in many other cell types upon exposure to pro-inflammatory cytokine interferon-γ (IFN-γ) signaling in the course of the cellular immune response[
12]. Tryptophan depletion creates an anti-proliferative environment against target cells and contributes to the antimicrobial effects of activated macrophages[
14]. However, a reduction in plasma tryptophan leads in consequence to low serotonin (5-HT) synthesis and further, several tryptophan breakdown products are known to exert neuroactive effects[
11].
During the cellular immune response, in parallel to IDO, guanosine triphosphate (GTP)-cyclohydrolase-I (GTP-CH-I, EC 3.5.4.16) is induced by IFN-γ. GTP-CH-I is the key enzyme in the biosynthesis of neopterin, a marker molecule for immune system activation[
15,
16]. As tryptophan metabolism may occur not only via IDO but also via hepatic tryptophan 2,3-dioxygenase (TDO), a concomitant determination of immune activation marker neopterin is suitable to judge the contribution of inflammation in changes of tryptophan levels[
12]. Neopterin levels and kynurenine to tryptophan ratio (Kyn/Trp) have turned out as useful markers for a variety of diseases that are associated with chronic immune activation such as infections, autoimmune syndromes, malignancies or neurodegeneration[
12,
16].
The aim of this study was to evaluate the effects of lavender essential oil and some of its constituents on tryptophan catabolism, by using the well established model system of freshly isolated human peripheral blood mononuclear cells (PBMC), stimulated or not with the mitogen phytohemagglutinin (PHA)[
17]. Determination of Kyn/Trp and neopterin levels in cell culture supernatants is used as sensitive and reliable read-out for the activation status of PBMCs. The terpene alcohol (-)-linalool, a major constituent of lavender oil that is also contained in several edible plant species, as well as two minor lavender oil constituents (+)-α-pinene and (+)-limonene were chosen as reference compounds for analysis, due to their reported anti-inflammatory properties[
18,
19].
Discussion
Anti-inflammatory properties of lavender oil and its constituents have been reported in several
in vitro and
in vivo studies. Lavender essential oil and constituents have been shown to interfere with key immunological pathways, e.g. nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling as well as cytokine secretion[
19,
23]. E.g., (+)-α-pinene, (-)-linalool and (+)-limonene were able to decrease interleukin-2 (IL-2) secretion and to increase the IL-10/IL-2 ratio in mouse primary splenocytes, which indicates their property to repress Th1 immune activation and suggest a potential inclination towards Th2[
19]. Furthermore, (-)-linalool was able to attenuate the production of lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and IL-6 both in RAW 264.7 macrophages and in mice, and has been discussed as potential anti-inflammatory agent for preventing lung injury[
19,
23].
The impact of the reference substances in attenuating Th1 immune response agrees with results of our study, which showed that non-toxic concentrations of (+)-α-pinene, (-)-linalool and (+)-limonene were able to inhibit mitogen-stimulated IDO activity in a model system of freshly isolated PBMC.
Also, lavender oil treatment was able to dose-dependently inhibit both tryptophan breakdown and kynurenine formation in supernatants of mitogen-stimulated PBMC. This inhibitory effect could already be detected at lavender oil concentrations that affected cell viability only slightly (0.1 to 0.5%). At higher treatment concentrations, effects on tryptophan and kynurenine were even stronger, however also cytotoxic effects of lavender oil increased. Interestingly, it has been shown that kynurenine metabolites are able to induce Th1 cell apoptosis[
24]. Thus, we suggest that at low concentrations, lavender oil might beneficially influence cell viability by counteracting pro-apoptotic signaling, while at higher concentrations toxicity effects become prevalent. In studies with several compounds in the PBMC model[
17,
20], IDO inhibition preceded substance toxicity phenomena, thus probably being a more sensitive indicator of cell death. Of note, the viability assay used in this study is based on the reduction of resazurin to fluorescent resorufin. Increased conversion rates may also indicate enhanced metabolic activity of cells, which does not always correlate with an increase in proliferation[
25,
26].
Importantly, in mitogen-stimulated cells, a suppressive effect of lavender oil treatment on neopterin and IFN-γ concentrations could be observed. In unstimulated cells, lavender oil treatment had no influence on tryptophan and IFN-γ levels, but the formation of kynurenine and neopterin was suppressed to some extent. As PBMC were preincubated with the lavender oil before PHA addition, we suggest that the oil interferes mainly with IDO and GTP-CH-I stimulation. A basal activity of both enzymes is suggested to be present also in unstimulated cells, probably initiated due to the preceding cell isolation procedure. In unstimulated PBMC, tryptophan levels remained unaffected upon lavender oil treatment, e.g. with a 0.5% oil addition, 86.8 ± 3.2% of the initial medium content of tryptophan, corresponding to ~ 32 μmol/L, was still detectable after 48 h, while a significant reduction of kynurenine levels was observed. For 0.5% lavender oil treatment, kynurenine levels were reduced to 50.2 ± 10.1% compared to the untreated control, which corresponds to a reduction from 2.3 ± 0.7 μmol/L to 0.8 ± 0.1 μmol/L.
Of note, changes in immune parameters, such as impaired activities of immunocompetent cells, and involvement of inflammatory mediators and pro-inflammatory cytokines have been reported to be associated with behavioural alterations by several studies, and cell-mediated immune activation is suggested to be an important factor in distinct mental disturbances[
11]. Behavioural changes can be induced by altered cytokine levels, e.g. studies of IFN-α treated patients showed therapy-induced depressive symptoms associated with activation of neuroendocrine pathways and altered serotonin metabolism[
11,
27]. Within the cellular immune response, pro-inflammatory pathways are strongly induced, including neopterin production via GTP-CH-I and tryptophan catabolism via IDO, and the concentrations of these biomarkers have been found to be altered in mental disorders or diseases associated mood disturbances[
12]. Enhanced neopterin concentrations together with low serum levels of tryptophan caused by increased tryptophan breakdown were shown to correlate with neuropsychiatric abnormalities like cognitive decline and depressive symptoms especially in long-lasting and chronic diseases[
28].
Beside the important role of tryptophan catabolism in the regulation of inflammatory responses[
29], tryptophan is a source for the production of 5-hydroxytryptophan, an intermediate in the biosynthesis of neurotransmitter serotonin. In states of persistent immune activation, availability of free serum tryptophan is diminished and as a consequence of reduced serotonin production, serotonergic functions may as well be affected[
12].
About 95% of the body’s serotonin resides in the gut[
30]. Furthermore, the gastrointestinal tract is rich in lymphocytes. Lavender oil treatment concentrations used for this
in vitro study may appear relatively high, however
in vivo, initial effects on IDO are suggested to be initiated already in the gastrointestinal tract, were such concentrations can be readily reached. In the study of Kasper et al., a treatment concentration of 80 mg/day was able to induce clinically meaningful and statistically significant anxiolytic effects[
5].
Of note, deciphering specific bioactivities of isolated essential oil components is challenging because of the great number of constituents with similar physicochemical properties (e.g. lipophilic, high vapor pressure). In general, the major components reflect quite well the features of the essential oils from which they derive[
31]. However minor constituent may contribute to the overall activity profile by modulating these activities and synergisms can play a major role[
32]. (-)-Linalool is the most studied monoterpene regarding analgesic effects, a more detailed elucidation of its impact on the GABAergic system would help to dissect molecular details on its anticonvulsant, analgesic as well as anxiolytic activities[
33]. For both (+)-limonene[
34] and (+)-α-pinene[
35] antinociceptive effects have been reported, however these effects are suggested to be strongly associated with their anti-inflammatory activities[
33].
The here reported effect on tryptophan breakdown is not a unique property of lavender oil or the analysed constituents. In earlier studies using the identical cell-biological assay, similar effects on tryptophan metabolism have been found by investigating
Hypericum perforatum extracts as well as Δ9-tetrahydrocannabinol and cannabidiol, indicating that the suppression of tryptophan breakdown and neopterin production might be an important but a more general aspect in the action of psychoactive compounds[
36,
37]. Thus, although our findings are from
in vitro experiments only, they might be relevant also for the
in vivo situation.
Furthermore, kynurenine derivatives such as kynurenic and quinolinic acid and 3-hydroxykynurenine are known to be neuroactive and their hyper- or hypofunction is associated with neurological disorders and psychiatric diseases such as depression and schizophrenia[
38]. The quinolinic acid to kynurenic acid ratio in the brain is discussed as a potential measure for conditions linked to excitotoxicity. Although both substances must be synthesized locally, because they are not able to cross the blood–brain barrier, other kynurenine pathway components such as tryptophan, kynurenine and 3-hydroxykynurenine can enter the brain, thus establishing a link between peripheral inflammation and brain tryptophan metabolism[
39]. Additionally, also microglial cells and blood-borne cells within the brain can be stimulated to activate the kynurenine pathway in states of peripheral immune activation[
38].
Scientific reports on the impact of IDO activity for different pathological conditions, including neuropsychiatric disturbances, account for IDO as a potential key pharmacological target. Several IDO inhibitors have been identified yet, and much effort will be necessary to evaluate their
in vivo efficacy. The most prominent IDO inhibitor1-methyl tryptophan (1-MT) has been shown to counteract microbial-induced depressive-like symptoms in animal studies[
40]. Beside the synthetic design of IDO antagonists via rational design strategies, also a variety of endogenous and exogenous antioxidants, such as vitamins, food supplements or preservatives, have been shown to suppress tryptophan catabolism in cellular model systems[
17,
20]. Thereby, the modulation of tryptophan metabolism is suggested to be due to the interference of the test substances mostly with immune activation cascades, rather than directly with IDO enzyme, as often other immune-relevant molecules such as neopterin and IFN-γ are affected additionally. Also in this study, lavender oil treatment was able to reduce neopterin and IFN-γ levels in mitogen-treated PBMC.
Moreover, the increased production of neopterin during inflammation could also indirectly affect neurotransmitter concentrations. In human macrophages, neopterin is produced at the expense of tetrahydrobiopterin (BH
4), an essential cofactor for several monooxygenases including tryptophan, phenylalanine and tyrosine hydroxylase[
15]. Thus, beside serotonin synthesis, also catecholamine formation depends on BH
4 availability. Interestingly, in depressed patients with a history of seasonal affective disorder, significantly lower plasma biopterin and tryptophan levels but elevated neopterin levels were found in comparison to healthy controls[
41]. Thus, reduced BH
4 levels in inflammatory conditions might negatively influence neurotransmitter production.
Conclusion
We could show that lavender oil can suppress mitogen-induced tryptophan degradation and IFN-γ production in vitro, and influence on kynurenine and neopterin formation in activated as well as to a lower extent in unstimulated PBMC.
Also, the constituents (-)-linalool, (+)-α-pinene and (+)-limonene showed dose-dependent inhibitory effects on tryptophan breakdown in PHA-stimulated PBMC. Thus, the IDO suppressing activities of lavender oil might at least partially result from the concerted action of the analysed constituents, where also minor components may play an essential role.
The finding that lavender essential oil, a medicinal plant-derived natural multicomponent preparation, may be a source of pharmacological active substances that interfere with key immune activation cascades such as the IDO and GTP-CH-I pathway, is of central relevance for the understanding of its therapeutic efficacy.
However, as the reported effects might not reflect the whole activity spectrum of lavender oil. Further research is necessary to elucidate other neuro-immunological relevant activities and to confirm the in vivo relevance of our findings.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Compounds characterization: MG; blood donations: HS; idea: JMG, FÜ, DF; cell culture work and read outs: JMG, KB, SS; HPLC measurements: SG, SS, DF; manuscript draft: JMG, FÜ, DF. All authors have read and contributed to the final version of the article. All authors read and approved the final manuscript.