Anxiolytic effects of essential oils may involve anti-oxidant regulation of the pro-oxidant effects of ascorbate in the brain
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.
Section snippets
Chemicals and reagents
Ammonium ferrous sulfate, sorbitol, xylenol orange, hydrogen peroxide (30%, v/v), catalase, ascorbic acid, individual EO compounds (alpha-pinene, beta-pinene, terpinolene), acetylcholine, Levo-dopa (L-dopa), melatonin and dopamine, were purchased from Sigma-Aldrich Pty Ltd (Castle Hill, NSW, Australia). The EO extracts Lavandula angustifolia (lavender), Melaleuca alternifolia (tea tree), Juniperus communis (juniper berry) were provided by Down Under Enterprises (Darlinghurst, NSW, Australia).
Ascorbic acid concentration in human body tissues
As documented in the scientific literature, the concentration of ascorbate varies widely between tissues and cell types in the human body (Table 1). The highest concentrations of ascorbate are present in neurons (10 mM) and the corneal epithelium of the eye (12.5 mM). In contrast, the glial cells which surround the neurons, the cerebral spinal fluid and synaptic fluid have ascorbate concentrations of 1.0, 0.15–0.25 and 0.2–0.4 mM, respectively. The ascorbate concentrations in plasma
Ascorbate, hydrogen peroxide and neurotransmission
Ascorbate is obtained from foods and supplements (Harrison et al., 2014) and is an important vitamin (vitamin C) involved with preventing oxidative stress-mediated damage in biological systems (Chambial et al., 2013b). As the metabolic processes involving AA are highly oxidative (Covarrubias-Pinto et al., 2015), the human brain is dependent on ascorbate for managing oxidative stress and as such, preferentially retains ascorbate over other organs, and at concentrations higher than most other
Conclusion
This research has demonstrated the important role of ascorbate in driving POX redox chemistry via production of H2O2 in the presence of oxygen. Based on the significant levels of ascorbate in the brain, it is proposed that this chemistry is central to the regulation of ascorbate-mediated ROS production by endogenous and exogenous compounds in the brain. A modified form of the FOX assay using catalase was employed to detect and quantify changes in the production of H2O2 for individual compounds
Declaration of competing interest
The authors declare that they have no conflict of interests with any aspect of the work described in this manuscript.
Acknowledgement
Minoli Aponso is supported by a Monash Graduate Scholarship. The provision of selected essential oils by Down Under Enterprises (NSW, Australia) is gratefully acknowledged.
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