PART 1 Isolation And Identification Of Phenylethanoid Glycosides From Aloysia Polystachya And Its Activity As Inhibitors Of Monoamine Oxidase-A
Mar 10, 2022
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Affiliations
1 Departamento de Biotecnologia em Plantas Medicinais, Universidade de Ribeirão Preto (UNAERP), Ribeirão Preto, SP, Brazil2 Departamento de Química, Faculdade de Filosofifia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (USP), Monte Alegre, Ribeirão Preto, SP, Brazil
Key words
aloysia polystachya, verbenaceae, antidepressant activity, phenylethanoids, monoamine oxidase inhibitors
Abstract
Aloysia polystachya is used as a sedative and antidepressant by the indigenous populations of Argentina and Paraguay, but the compounds associated with these activities have not been determined. We have separated and identified the main constituents of the hydroethanolic extract of A. polystachya by ultra-performance liquid chromatography-mass spectrometry and confirmed the presence of acteoside, isoacteoside, 6'-acetylacteoside, and 4’,4’’’,5,5’’-tetrahydroxy-6,6’’,3’’’-trimethoxy-[C7–O–C7’’]-biflavone by NMR spectroscopy. Inhibitory activities of the hydroethanolic extract and purified phenylethanoid glycosides against monoamine oxidase-A were assessed using a standard fluorometric assay. The hydroethanolic extract inhibited monoamine oxidase-A activity in a dose-dependent manner with an IC50 of 9.2 µg/mL, while the selective monoamine oxidase inhibitor clorgyline exhibited an IC50 of 0.06µg/mL (0.22µM). Acteoside was the strongest inhibitor of monoamine oxidase-A (IC50 value of 5µM), whereas isoacteoside and 6'-acetylacteoside showed IC50 values of around 10 µM. The results showed that phenylethanoids from a hydroethanolic extract of A. polystachyahave been found to have inhibitory activity against monoamine oxidase-A. It is likely that the mode of action of the acteosides is multi-targeted, involving the downregulation of inflammatory molecules and neutralization of oxidation reactions as well as inhibition of monoamine oxidase-A.
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Introduction
According to a recent report from the World Health Organization [1], more than 320 million people (4.4% of the world population) suffer from depression and anxiety disorders, with around 800 thousand suicides per year being committed as a result of these mental issues. Although treatment with synthetic antidepressants is available, many patients suffer from side effects such as dry mouth, constipation, dizziness, blurred vision, increased appetite, weight gain, insomnia, and kidney problems [2]. For this reason, approaches using complementary and alternative medicine, including phytotherapy, have been used in the treatment of several types of mental disorders [3].
Various preclinical and clinical studies have provided evidence in support of the benefits of plant-based medicines in the treatment of general and specific anxiety disorders [4, 5]. Of particular interest are investigations into the anxiolytic properties of Aloysia polystachya (Griseb.) Moldenke (Verbenaceae), an aromatic species found mainly in Argentina and Paraguay. According to ethnopharmacological studies, local populations use the plant, which is commonly known as a burrito, as a digestive, sedative, and antidepressant tonic [6, 7]. Although the anxiolytic and antidepressant properties of hydroethanolic extracts from A. polystachya have been confirmed by preclinical studies [8–10], no phytochemical investigations have been performed with the aim of identifying the compounds associated with these activities. It has previously been shown that the antidepressant activities of some medicinal plants, for example, Hypericum perforatum L. (Hy-pericaceae) and Peganum harmala L. (Nitrariaceae), are associated with the inhibition of monoamine oxidase-A (MAO-A) [11–13]. The MAO family is distributed throughout the central and peripheral nervous systems and overexpression of these enzymes promotes the oxidative deamination of monoamines with reductions in the levels of the neurotransmitters serotonin, norepinephrine, and dopamine, which result in the onset of psychiatric disorders. Such deamination processes also generate substances such as hydrogen peroxide, oxygen-free radicals, and aldehydes that are responsible for the oxidative stress of cells. MAOs exist in two major isoforms that differ with respect to distribution, substrate specificity, and sensitivity to inhibitors. The MAO-A isoform plays an important role in depression and anxiety disorders, while MAO-B is involved in neurodegenerative diseases [13–17].In light of the above, we hypothesized that the anxiolytic and antidepressant properties of A. polystachya derive, at least in part, from the presence of inhibitors of MAO-A. In order to test this hypothesis, we identified the active principles present in the hydroethanolic extract from leaves of A. polystachya and assessed the effects of the crude extract and the main constituents isolated therefrom on MAO-A activity.
Results and Discussion
The hydroethanolic extract from leaves of A. polystachya was submitted to ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), and the chromatogram so obtained is presented in ▶Fig. 1a. The main components of the extract were purified by column chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), and identified as acteoside (syn verbascoside), isoacteoside, 6’-acetylacteoside, and 4’,4',5,5’-tetrahydroxy-6,6’’,3’-trimethoxy-[C7–O–C7’’]-biflavone by comparison of their 1H- and 13C-NMR, HSQC, HMBC (▶Table 1S, 2S, Supporting Information), and MS data (▶Fig. 1b-e) with values reported in the literature [18–20]. The concentration of acteoside in the hydroethanolic extract, as determined by HPLC, was 108.65 ± 1.3 µg/mg of dried extract. This represents the first record of the constituents of extracts of leaves from A. polystachya, although the essential oil of the plant has been analyzed previously and found to contain the monoterpenes carvone and limonene as the major components [21]. The crude hydroethanolic extract from leaves of A. polystachya inhibited MAO-A activity in a dose-dependent manner (▶Fig. 2a) with an IC50 of 9.2 µg/mL, while the selective MAO inhibitor clorgyline exhibited an IC50 of 0.06µg/mL (0.22µM). The purified acteosides also exhibited inhibitory activities against MAO-A (▶Fig. 2b), with acteoside presenting the lowest IC50 value of 5µM (3.1µg/mL) followed by isoacteoside with an IC50 of 10.1 µM (6.3 µg/mL) and 6’-acetylacteoside with an IC50 of 9.5µM (6.3µg/mL). Inhibition of MAO-A leads to the reestablishment of the levels of serotonin, norepinephrine, dopamine, and tyramine, which are key neurotransmitters in the control of anxiety and depression [16]. Thus, the presence of diverse MAO-A inhibitors in leaves of A. polystachya explains, at least in part, the previously reported antidepressant and anxiolytic activity of the species [8–10]. The use of multicomponent plant mixtures can be advantageous in the treatment of diseases of complex etiology such as anxiety, depression, and other neurological conditions. Currently, the use of phytomedicines in the treatment of diseases that affect the nervous system is based on the paradigm of multi-target-directed ligands, i.e., pharmaceuticals that have multi-target activities resulting from the presence of substances such as polyphenolics with anti-inflammatory, antioxidant, and MAO inhibitory properties that are capable of conferring neuroprotection [20,22–25]. It is worth noting, however, that the polar character of polyphenolic substances could hinder interactions with their molecular targets. Nevertheless, clinical studies have demonstrated that the contact between polyphenolics and nonpolar secondary metabolites present in the extracts may modify the permeability of cell membranes and facilitate the uptake of polar compounds [23]. Thus, complex plant extracts containing multi-target agents that interact with their receptors in a pleiotropic fashion generate a pharmacological synergism that affects numerous processes, including the movement of polar metabolites across cell membranes. In this context, Li et al. [26] employed a zebrafish model to demonstrate that acteoside could penetrate the blood-brain barrier, and proposed that the phenylethanoid glycoside may have a potential therapeutic effect in Parkinson’s disease. Monoaminergic neurotransmitters are the main targets of modern antidepressants since their deficiencies are responsible for the debilitating symptoms of depression. A recent in vivo study demonstrated that the ethanolic and aqueous extracts of Lippia citriodora (Verbenaceae) and their main component acteoside exhibited anxiolytic, hypnotic, and muscle relaxant effects, and these properties were attributed, in part, to an interaction with the type A gamma-aminobutyric acid (GABAA) receptor [27]. Clinical studies have revealed that drugs with the capacity to block inflammatory cytokines, such as TNF-α, or other components of the inflammatory signaling pathway, for example, cyclooxygenase-2 (COX-2), are effective in reducing depressive symptoms in patients with rheumatoid arthritis, psoriasis, and cancer, as well as those suffering from major psychiatric disorders [28]. In this context, it has been reported that acteoside can attenuate the production and release of inflammatory molecules, such as nitric oxide (NO), TNF-α, and interleukin 12 (IL-12) in lipopolysaccharide/interferon-gamma (LPS/IFN-γ)-stimulated macrophages [29], as well as histamine and arachidonic acid in RBL-2H3 mast cells [30]. In addition, acteoside is able to reduce the levels of TNF-α, IL-1β, IL-8, IL-6, and NO, and to activate caspase-1, nuclear factor-kappa-B
(NF-κB), NO synthase and activator protein-1 [31] induced by IL-32 and/or LPS in TH-1 cells and macrophages [32]. Some of these inflammatory mediators, such as IFNs, IL-6, IL-8, and IL-1β, have been found at abnormal levels in both peripheral and post-mortem tissue samples from depressed individuals, and have been related to the symptoms of depression [33]. Activation of these molecules by psychosocial stressors may promote significant functional changes in the brain, leading to the development of depressive behavior and other psychiatric disorders. IFNs, IL-1β, and TNF-α, for example, may increase the expression and function of serotonin, noradrenaline, and dopamine receptor pumps, thus reducing the availability of these neurotransmitters in the synaptic cleft [28]. In addition, IFN-γ, IL-6, TNF-α, and oxidative stress can activate indoleamine 2,3 dioxygenase (IDO), an enzyme responsible for the degradation of tryptophan and, thereby, reduce the concentration of the primary precursor of serotonin synthesis [34].Acteoside, isoacteoside, and 6’-acetylacteoside contain hydroxy-phenylethyl and caffeoyl moieties that are known to be associated with antioxidant properties [35, 36], and acteoside itself exhibits considerable antioxidant activity [37]. Studies have shown that acteoside inhibits the aggregation of β-amyloid peptide (Aβ) in a dose-dependent manner, functions as a neuroprotective agent, and enhances memory, and these properties have been attributed to the antioxidant activity of the agent [38,39]. Considering that numerous phenolic-rich species with antioxidant properties are used in the treatment of neurological disorders [15], it has been suggested that the antidepressant and anxiolytic effects of the phenylethanoid glycosides isolated from A. polystachya may also result from their antioxidant activities. This supposition was supported by Xu et al. [40], who presented evidence concerning the relationship between increased oxidative stress and depression/anxiety. Based on the above, we conclude that the antidepressant properties of the hydroethanolic extract from leaves of A. polystachya, and of the purified phenylethanoids isolated therefrom, can be explained by multi-target modes of action involving the inhibition of MAO-A, downregulation of inflammatory molecules, and neutralization of oxidation reactions. Hence, the results presented herein support our original hypothesis that the anxiolytic and antidepressant activities of the hydroethanolic extract of A. polystachya result, at least in part, from the inhibition of MAO-A. However, the structure-activity relationship of the phenylethanoid glycosides identified in this study requires further attention so that novel molecules can be designed for the treatment of specific neurological disorders.
Materials and Methods
Plant material
Leaves of A. polystachya were harvested at the Farmácia da Natureza da Terra de Ismael (Jardinópolis, SP, Brazil) in January 2016. Plant material was identified by Dr. Lúcia Rossi (Instituto Botânico, São Paulo, SP, Brazil), and a voucher specimen was deposited in the Herbarium of Medicinal Plants at UNAERP with the voucher number
HPM-1213. Permission to evaluate the bioactivities of the extracts from Brazilian plants was granted by the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (no. 02001.005074/2011-19).
Preparation of the hydroethanolic extract of Aloysia polystachya
Leaves (1000g) were dried for 72h in a circulating air oven at 45°C, pulverized, and passed through a 40-mesh sieve. The powdered material was steeped in water: ethanol (20:80; v/v) for 7 days and subsequently filtered through Whatman No. 41 filter paper. The filtrate was reduced to dryness on a rotary evaporator and lyophilized to yield 130.2g of dry crude extract, resulting in a drug extract ratio of 7.6:1.
Phenylethanoid Glycosides in cistanche
