Anxiolytic effects of essential oils may involve anti-oxidant regulation of the pro-oxidant effects of ascorbate in the brain

Highlights

• Essential oils (EOs) are bioavailable to brain and consistently reported to produce anxiolytic effects

• Pro-oxidant chemistry of ascorbate drives H₂O₂-mediated signaling in the brain and is likely regulated by inhaled EOs

• Pro-versus anti-oxidant effects of EOs reflect local ratios of ascorbate, EOs and neurotransmitters

• The research methods demonstrate an approach to evaluate the pro-versus anti-oxidant effects of EOs in mixture systems that model actual chemical ratios present in the brain

• Anxiolytic effects of lavender are likely due to driving accelerated turn-over and subsequent production of dopamine

Abstract

Essential oils (EOs) absorbed via inhalation are consistently reported to produce anxiolytic effects. The underlying neurochemical mechanisms, however, are not well understood. High concentrations of ascorbate in the human brain (~10 mM in neurons) implicates this compound as a key signaling molecule and regulator of oxidative stress. In this study, we demonstrate the significant in vitro capacity of ascorbate to produce H₂O₂ in the presence of oxygen at physiological pH values, peaking at ~400 μM for ascorbate levels of 1.0 mg/mL (5.6 mM). In comparison, individual EOs and selected neurotransmitters at similar concentrations produced <100 μM H₂O₂. Systematic studies with binary and ternary mixtures containing ascorbate indicated that EOs and neurotransmitters could variably enhance (pro-oxidant, POX) or suppress (anti-oxidant, AOX) the production of H₂O₂ versus the ascorbate control, depending on the concentration ratios of the components in the mixture. Moreover, the AOX/POX chemistry observed with binary mixtures did not necessarily predict effects with ternary mixtures, where the POX ascorbate chemistry tended to dominate. A model is proposed to account for the ability of compounds with electron-donating capacity to catalytically regenerate ascorbate from intermediate oxidized forms of ascorbate, thus driving H₂O₂ production and exerting a net POX effect; whilst compounds that irreversibly reacted with oxidized forms of ascorbate suppressed the production of H₂O₂ and produced an overall AOX effect. Since the anxiolytic effects of different EOs, including extracts of Lavendula angustifolia (lavender) and Salvia rosmarinus (rosemary), were associated with AOX regulation of H₂O₂ production by ascorbate, it can be concluded that these anxiolytic effects are potentially related to the AOX properties of EOs. In contrast, EOs driving POX effects (eg, Junipenus communis (Juniper) berry EO) are proposed to be more useful for their potential anti-microbial or cancer cytotoxic applications.

Introduction

The dominant brain regions responsible for emotional responses are the amygdala in the limbic system and forebrain areas (i.e. medial prefrontal cortex and anterior cingulate cortex) (Etkin, 2010). Anxiety is a negative emotional response to potentially threatening stimuli and reflects imbalanced and dysfunctional neurotransmitter regulation in these brain emotional centers (Nuss, 2015). The inhibitory neurotransmitter, gamma aminobutyric acid (GABA), is important for the regulation of anxiety and is a key therapeutic target for anxiety disorders (Lydiard, 2003). Neural circuits related to anxiety involve inhibitory networks of GABAergic interneurons that are balanced by the excitatory glutamate neurotransmitter. The modulation of anxiety is explained by decreased inhibitory neurotransmission by GABA or increased excitatory neurotransmission by glutamate (Lydiard, 2003; Nuss, 2015). In addition, the state of anxiety is also associated with monoamine (ie, serotonin, dopamine, noradrenaline) neurotransmitter deficiency, and dysregulation of neurotransmitter receptors (Liu et al., 2018).

A neuromodulator acts as a chemical messenger released by neurons that stimulates and influences the diversity of neuronal populations, including the ‘orchestra’ of neuronal receptors expressed in different ratios. This upregulation of neuronal activity underpins synaptic plasticity and learning (Jiang and Salton, 2020; Pedrosa and Clopath, 2017). It has been suggested that synaptic plasticity is modulated in part by the redox balance at the synapse between reactive oxygen species (ROS) and neuroprotective antioxidants (such as ascorbic acid (AA, vitamin C), glutathione, and catecholamines) (Smythies, 2000). Ascorbic acid, present as the ascorbate ion at physiological pH (Witmer et al., 2016), is a water-soluble ketolactone with strong electron donor (i.e., anti-oxidant) capacity associated with two ionizable hydroxyl groups (Du et al., 2012a; Meščić Macan et al., 2019). Ascorbate is obtained from foods and supplements (Harrison et al., 2014) and as vitamin C, has a key role in regulating oxidative stress in biological systems (Chambial et al., 2013a). In the brain, H₂O₂ originates during intra-cellular mitochondrial respiration (Bao et al., 2009). Active transport of ascorbate into the mitochondria may also contribute to H₂O₂ production (Fiorani et al., 2015). Specifically, H₂O₂ generated in striatal medium spiny neurons (MSNs) upon synaptic glutamatergic depolarization, acts as a neuromodulator either via H₂O₂-dependent excitation of GABAergic neurons in the substantia nigra pars reticulata (SNr), or exerts inhibitory effects in dopamine neurons of the substantia nigra pars compacta (SNc) (Avshalumov and Rice, 2003; Patel and Rice, 2012).

Responses of ascorbate to levels of H₂O₂ can influence cell signaling and thereby mediate synaptic plasticity (Rhee, 2006). In particular, ascorbate is proposed as a neuromodulator of glutamatergic and dopaminergic neurotransmission. Dynamic regulation of extracellular ascorbate concentration is mediated by glutamate–ascorbate hetero-exchange (Rice, 2000). This means that ascorbate released from neurons to the extracellular fluid over a concentration gradient, facilitates glutamate uptake and also increases dopamine levels, and therefore regulates dopaminergic and glutamatergic transmission (Morales et al., 2012). Apart from its impact on suppressing oxidative stress, redox signaling is an important factor in catecholamine-mediated neurotransmission (Ballaz and Rebec, 2019).

Ascorbate is therefore a neuromodulator, with both powerful anti-oxidant (AOX) and pro-oxidant (POX) properties (Harrison et al., 2014), and also functions as an electron donor cofactor in redox-coupled reactions involved with neurotransmitter synthesis (i.e. dopamine, norepinephrine) (Ballaz and Rebec, 2019). Redox reactions involving ascorbate and other compounds also drive cellular detoxification functions and metabolic cycles (Halliwell and Gutteridge, 2015). As such, the human brain is highly dependent on ascorbate. Neurons contain high ascorbate concentrations (~10 mM, (Harrison and May 2009; Rice and Russo-Menna, 1998) and also exhibit high rates of oxidative metabolism (Rice, 2000). Ascorbate directly or indirectly regulates the oxidative turnover of catecholamines by the neuromelanin pathway (Smythies, 2000). Ascorbate deficiency alters the levels of monoamines, which are important redox-active neurotransmitters in neuronal communication (Ballaz and Rebec, 2019; Hansen et al., 2018; Ribeiro et al., 2016). For example, an ex vivo study using rat neostriatum tissue demonstrated ascorbate-mediated suppression of cross-linking between oxidized dopamine and protein cysteinyl sulfhydryls (Hastings and Zigmond, 1994). Furthermore, extracellular neurotransmission activity of dopamine in the striatum of rats was preserved in the presence of ascorbate, which prevented dopamine oxidation (Morales et al., 2012).

Essential oils are readily bioavailable to the brain and have been shown to modulate pathways of neurotransmission affecting emotions. For example, oral administration of Citrus limon (lemon) EO to mice increased the dopamine concentration and decreased the dopamine turnover ratios in the striatum and hippocampus (Hao et al., 2013). In addition, Citrus bergamia (bergamot) EO stimulated glutamate release either by transporter reversal and/or exocytosis depending on the dose, in rat hippocampus (Morrone et al., 2007). Likewise, Eugenia uniflora L. (suriname cherry) EO given to mice produced an anti-depressant effect by regulating monoamine neurotransmission (de Sousa et al., 2017).

In spite of reported observations demonstrating regulation of neurotransmission by anti-oxidants, the mechanism by which exogenous redox-active bioactives such as EOs influence brain health or manage dysfunctions is poorly understood (Fraunberger et al., 2016). This circumstance is complicated by the POX and AOX properties of ascorbate that depend on the redox dynamics of the physiological environment (Duarte and Lunec, 2005). It is expected that the high concentration of ascorbate in the brain, maintained in its reduced state, can produce H₂O₂ in the presence of O₂, and this chemistry is central to the chemical and biochemical turnover of the emotion-related neurotransmitters, and to exogenous bioactives such as EOs and dietary factors.

In this study, the hypothesis was investigated, that the in vitro POX and AOX effects of EOs, in combination with ascorbate and neurotransmitters associated with emotion, influence the production and regulation of H₂O₂ in the brain. In order to validate this hypothesis, this research systematically examined the POX and AOX effects of binary and ternary mixtures of EOs, ascorbate and neurotransmitters under relevant physiological conditions, and evaluated the outcomes in the context of mechanistic drivers of putative anxiolytic effects of EOs.