Part Ⅰ Molecular Mechanisms And Therapeutic Potential Of α- And β-Asarone in The Treatment Of Neurological Disorders

Abstract

Neurological disorders are important causes of morbidity and mortality around the world. The increasing prevalence of neurological disorders, associated with an aging population, has intensified the societal burden associated with these diseases, for which no effective treatment strategies currently exist. Therefore, the identification and development of novel therapeutic approaches, able to halt or reverse neuronal loss by targeting the underlying causal factors that lead to neurodegeneration and neuronal cell death, are urgently necessary. Plants and other natural products have been explored as sources of safe, naturally occurring secondary metabolites with potential neuroprotective properties. The secondary metabolites α- and β-asarone can be found in high levels in the rhizomes of the medicinal plant Acorus calamus (L.). α- and β-asarone exhibit multiple pharmacological properties including antioxidant, anti-inflammatory, antiapoptotic, anticancer, and neuroprotective effects. This paper aims to provide an overview of the current research on the therapeutic potential of α- and β-asarone in the treatment of neurological disorders, particularly neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), as well as cerebral ischemic disease, and epilepsy. Current research indicates that α- and β-asarone exert neuroprotective effects by mitigating oxidative stress, abnormal protein accumulation, neuroinflammation, neurotrophic factor deficit, and promoting neuronal cell survival, as well as activating various neuroprotective signaling pathways. Although the beneficial effects exerted by α- and β-asarone have been demonstrated through in vitro and in vivo animal studies, additional research is required to translate laboratory results into safe and effective therapies for patients with AD, PD, and other neurological and neurodegenerative diseases.

Keywords

α-asarone; β-asarone; neuroprotection; neuroinflammation; molecular role; therapeutic; neurological disorders; Cistanche benefits.

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Introduction

The nervous system, a complex network of nerves and specialized cells, is responsible for the control of the body and communication among its parts. According to the World Health Organization (WHO), neurological disorders are defined as diseases of the central and peripheral nervous systems [1]. Neurological disorders can affect the brain, cranial nerves, peripheral nerves, and spinal cord and include neurotraumatic diseases, such as stroke and spinal cord injury; neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD); as well as neuropsychological disorders, such as depression and schizophrenia [2]. In general, neurological disorders are characterized by acute and progressive neuron degeneration, ultimately resulting in brain dysfunction and neuronal cell death [3]. The underlying molecular mechanisms of neurodegeneration include alterations in phospholipid metabolism, accumulation of lipid peroxides, mitochondrial dysfunction, protein misfolding, abnormal protein aggregation, diminished cellular energy levels, disturbed calcium (Ca²⁺) homeostasis, excitotoxicity, oxidative stress, neuroinflammation, dysregulated hormonal signaling, and apoptosis [4–6].

AD, one of the most common neurodegenerative diseases, is characterized by the progressive worsening of learning, memory, and other cognitive functions with age. At the cellular level, AD is associated with the formation of extracellular plaques consisting of amyloid-beta (Aβ) and neurofibrillary tangles that lead to extensive neuronal loss. By reducing cellular energy levels and increasing oxidative stress, inflammation, and apoptosis, these “senile” plaques and aggregates lead to neuronal cell death [7–9].

PD, the second most common neurodegenerative disease, is characterized pathologically by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) [10,11]. This nigral neuronal loss consequently results in dopamine (DA) deficiency in the striatum (ST), which is correlated with motor deficits such as tremors, rigidity, bradykinesia, postural instability, and gait impairment [12]. Dopaminergic cell death in PD is associated with the development of intracellular α-synuclein aggregates known as Lewy bodies [13].

Cerebral ischemic disease, a common form of stroke, is the fifth leading cause of death and disability impacting one million Americans every year [14]. It is caused by the blockage of blood vessels due to a thrombus or embolus [15]. At rest, the brain receives approximately 20% of the body’s total blood supply and is, therefore, highly sensitive to ischaemic events, and even short-lived ischemia can result in significant cerebral damage [15]. During cerebral ischemia, part of the brain is deprived of oxygen and nutrients, which initiates a cascade of cellular and metabolic events that can result in severe brain damage. A considerable amount of evidence suggests that the release of excess glutamate during and after an ischemic insult leads to glutamate receptor hyperactivity, triggering harmful intracellular effects, including calcium overload and the generation of reactive oxygen species (ROS). This disruption of cellular homeostasis eventually leads to neurodegeneration [16,17].

Epilepsy is a neurological disorder characterized by temporary abnormal electrical activity in nerve cells [18]. In 2015, around 70 million people had been diagnosed with epilepsy worldwide, with 80% of these cases seen in developing countries [19]. Oxidative stress, glutamate excitotoxicity, and mitochondrial dysfunction, among others, have been implicated in the pathogenesis of epilepsy [20].

Several approaches have been proposed for the management of neuronal dysfunction and cell death associated with neurological disorders. However, current approaches primarily serve to reduce or manage symptoms, and no curative therapies have been introduced that can slow, prevent, or reverse disease development [21].

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Medicinal plants, found throughout the natural environment, represent an immense source of bioactive compounds in the form of secondary metabolites and other bioactive constituents [22]. Secondary metabolites derived from medicinal plants have been shown to exert beneficial effects on the chemical balance in the brain by influencing the function of several neurotransmitter receptors, with positive outcomes on cognitive disorders [23]. Recently, our research has identified α- and β-asarone, found in high levels in the rhizomes of the medicinal plant Acorus calamus (L.), as important secondary metabolites with potential therapeutic benefits for the treatment of AD, PD, and other neurological disorders. The mechanisms underlying α- and β-asarone-mediated neuroprotection are multipronged, and include antioxidant, antiapoptotic, and anti-neuroinflammatory effects, as well as the modulation of various cellular and molecular targets; these actions might ultimately contribute to the ability of α- and β-asarone to attenuate the severity of neurological disorders [24–27] (Figure 1). Some of the specific molecular targets involved in α- and β-asarone-mediated neuroprotection have recently started to be unveiled. For instance, α- and β-asarone have been reported to promote disintegration of protein aggregates (tau, Aβ, and α–synuclein) associated with neurodegenerative disorders [28,29], attenuate lipopolysaccharide (LPS)-mediated neuroinflammation, promote neuronal cell survival, improve motor and non-motor functions, and prevent the neurodegeneration of dopaminergic neurons in the brain (24,25. Antidepressant-like effects of a- and B-asarone have also been described (30l, while Pan et al. [31] showed that B-asarone protected cortical neurons and reduced the infarction volume in an experimental model of ischemic stroke.

Here, we review recent research on the mechanisms by which a- and p-asarone exert neuroprotective effects in vitro and in vivo, to clarify their pharmacological properties and critically evaluate their potential as therapeutics for the treatment of neurological diseases.

Occurrence, Bioavailability, and Pharmacokinetics of α- and β-Asarone

The secondary metabolites a- and B-asarone ((E-/(Z)-124-trimethoxy-5-prop-1-enylbenzene) are highly concentrated in the rhizomes of Acorus mlamus Linn, Acorustatarinowii Schott, and Acorus gramineus Solander, which belongs to the Acoraceae plantfamily (commonly known as "sweet flag") (32). a-asarone as an active phytochemical is also present in the bark of the Mexican tree Guatteria gamers Greenman from the Annonaceae family [33]. A. calamus, either alone or in combination with other herbs, has been extensively cultivated in various tropical and subtropical regions worldwide [32,34,35] and widely used as a traditional medicine for centuries [32]. A. calamus contains several phytoconstituents, including alkaloids, volatile oils, tannins, glycosides (xanthone), essential oils, flavonoids, monoterpenes, steroids, lignin, sesquiterpenes, saponins, mucilage, and polyphenolic compounds [36,37]. In the Ayurvedic system, A. calamus is extensively used to treat numerous inflammatory disorders [36,38–40], and in China, traditional practitioners prescribe A. calamus to treat constipation, digestive problems, and other health issues [41]. A. calamus and its primary bioactive constituents have also been found to reduce stress-induced immunosuppression in rats, resulting in improved immune function [42]. Both α- and β-asarone are widely studied bioactive secondary metabolites, featuring a broad range of pharmacological properties, including antioxidant, anti-inflammatory, neuroprotective, antidiabetic, anticancer, antifungal, antimicrobial, anti-ulcer, anti-allergic, wound healing, pesticidal, insecticidal, and radioprotective properties, among others [36,43–47].

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1. Bioavailability and Pharmacokinetics of α- and β-Asarone

Due to their lipophilic character, α- and β-asarone have limited oral bioavailability, which can be improved by increasing their stability and solubility [48,49]. The plasma half-lives of α-and β-asarone are relatively short due to the rapid distribution of these agents to vital organs, such as the liver, spleen, heart, kidney, lungs, and brain [25,48,50,51]. Oral administration of the essential oil (single dose of 200 mg/kg) from A. tatarinowii Schott containing 11% α-asarone and 74% β-asarone to rats revealed that the maximum plasma concentrations were 0.5 µg/mL (tmax = 11 min) for α-asarone and 2.5 µg/mL (tmax = 14 min) for β-asarone, with half-lives in plasma of approximately 1 h [52]. Importantly, α- and β-asarone are distributed extensively throughout the brain, indicating their ability to permeate the blood–brain barrier (BBB), which is often a limiting factor when developing treatments for neurological disorders, including neurodegenerative diseases [24,25]. Lu et al. [49] also reported the rapid absorption and permeation of the BBB by α-asarone in rats. In another study, oral administration of α-asarone at 80 mg/kg resulted in 34% bioavailability [53]. A recent pharmacokinetic study demonstrated that the intravenous (i.v.) administration of lipid nanoparticles loaded with α-asarone resulted in significantly increased α-asarone levels detected in murine plasma and brain parenchyma fractions, compared with free α-asarone, confirming the ability to establish and maintain a therapeutic concentration of α-asarone in plasma that can be rapidly transported across the BBB [54]. In another study, the intranasal delivery of α-asarone to the brain using lactoferrin-modified methoxy poly(ethylene glycol)-poly(lactide) copolymer (mPEG-PLA) nanoparticles showed better BBB permeability without poor bioavailability, compared with i.v. administration. Intranasal α-asarone delivery enhanced the brain-targeting efficiency and reduced liver accumulation [55]. Another study demonstrated that the absolute bioavailability, brain-targeting efficiency, and percentage of nasal-mediated brain delivery of nasally administered PLA-α-asarone nanoparticles were 74.2%, 142.24%, and 29.83%, respectively, and nasal administration decreased drug-induced hepatotoxicity [56]. In an in vitro BBB model, borneol and α-asarone, used as co-adjuvant agents, improved the brain delivery of the central nervous system (CNS) drugs puerarin and tetramethylpyrazine. Because this effect could be counteracted by inhibitors of adenosine receptors, the authors concluded that α-asarone may gain entry to the CNS through adenosine receptors (AR), which represent an important pathway for drug delivery. Additionally, the co-administration of borneol and α-asarone decreased the expression of zonula occludens 1 (ZO-1), an important BBB junction protein, but increased A1AR and A2AAR expression. An in vivo pharmacokinetic analysis confirmed that the co-administration of borneol and α-asarone significantly increased the concentration of puerarin and tetramethylpyrazine in the brain, suggesting that a low dose of α-asarone not only improved the oral bioavailability of puerarin and tetramethylpyrazine but also increased BBB permeability, with α-asarone exhibiting superior permeability enhancement than borneol [57].

An absorption, distribution, metabolism, and excretion (ADME) in silico analysis revealed that β-asarone had good oral bioavailability and binding affinity towards dopaminergic receptors [58]. Furthermore, in silico results indicated that β-asarone was likely to interact with various amino acid residues in both the D2 and D3 dopamine receptors through hydrogen bonds [58]. In the same study, the toxicity of β-asarone was predicted using Lazar and ProTox, computational tools used to forecast the toxic properties of molecules. Lazar predicted that β-asarone is carcinogenic in various rodent models. Computational analysis of acute toxicity using ProTox showed that β-asarone had a high LD50 value (418 mg/kg) and the probability of the compound being mutagenic in Salmonella typhimurium was found to be 0.573 [58]. Another study reported that the half-lives of β-asarone in the cerebellum, thalamus, brainstem, cortex, hippocampus, and blood were 8.149, 2.832, 7.142, 1.937, 1.300, and 1.380 h, respectively [59].

Preliminary results from pharmacokinetic studies indicate the rapid and significant brain permeability of α- and β-asarone, expected to satisfactorily induce significant neuroprotective actions necessary to produce a beneficial therapeutic effect. However, further in vivo studies remain necessary to draw definitive conclusions regarding the ADME properties and overall safety of α- and β-asarone.

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2. Toxicology of α- and β-Asarone: Preclinical Studies

Toxicity studies examining the effects of low doses of α- and β-asarone in rodent models have not revealed severe adverse effects. For instance, Chen et al. [60] reported that sub-chronic treatment with α-asarone (50 and 100 mg/kg, per os [p.o.], for 28 days) did not result in overt behavioral changes (walking, rearing, and grooming) in a seizure model generated in Swiss albino mice. However, α-asarone administered at a higher dose (200 mg/kg, p.o., for 28 days) significantly diminished spontaneous locomotor activity, although no mortality was observed. An acute toxicity test revealed that the oral median lethal dose (LD50) for α-asarone in mice was greater than 1000 mg/kg, with no deaths reported in any test groups [60]. In another study, mice were treated with α-asarone (150, 200, 250, 300, and 350 mg/kg) and survival was recorded for 14 days after treatment. The LD50 of α-asarone was calculated to be 245.2 mg/kg, with 95% confidence limits of 209.2–287.4 mg/kg. Deaths occurred mostly within 24 h after injection, and piloerection, ptosis, dyspnea, and ataxia were the most frequent clinical signs observed [61]. An in vivo subacute toxicity study revealed that the oral administration of β-asarone (100 mg/kg, for five consecutive days) reduced body weight and food consumption without causing mortality in pre-weanling rats [62]. Moreover, the weights of the adrenal glands and heart increased, the thymus weight decreased, and increased single-cell degenerative changes were observed in the thymus following β-asarone treatment. However, no significant changes in hematology or enzyme levels indicating hepatotoxicity were detected [62]. In yet another study, a long-term safety evaluation examining the effects of oral administration of β-asarone at 10 and 20 mg/kg p.o. for 90 days in mice did not reveal significant changes in any hematological parameters; however, blood concentrations of total bilirubin (BIL-T) increased following treatment with 20 and 50 mg/kg of β-asarone p.o. for 90 days; K+ concentrations decreased following treatments with 20 mg/kg/day of β-asarone p.o. for 90 days; and Cl− concentrations decreased following treatments with 50 mg/kg of β-asarone p.o. for 90 days [63]. Following the oral administration of β-asarone at 200 µg/kg for 20 weeks in mice, no obvious toxicity was observed. During an LD50 toxicity study, treatment with β-asarone (500, 750, 1000, 1250, 1500, 1750, and 2000 mg/kg, i.v., for 24 h) did not result in marked behavioral changes, and no obvious toxicity was observed [63]. Mice that died first appeared weak and less active, followed by gradual death, and the LD50 of β-asarone was calculated to be 1560 mg/kg [63]. Taken together, based on sub-acute toxicity tests, β-asarone at doses ≤ 100 mg/kg appears to be safe for clinical use, whereas the safety of doses >100 mg/kg remains unclear. For further information on the toxicology of α- and β-asarone, we refer the reader to the following excellent reviews [27,64].

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Rengasamy Balakrishnan 1,2, Duk-Yeon Cho 1 , In-Su Kim 2 , Sang-Ho Seol 3 and Dong-Kug Choi 1,2,

1 Department of Applied Life Science, Graduate School, BK21 Program, Konkuk University, Chungju 27478, Korea; balakonkuk@kku.ac.kr (R.B.); whejrdus10@kku.ac.kr (D.-Y.C.)

2 Department of Biotechnology, Research Institute of Inflammatory Disease (RID), College of Biomedical and Health Science, Konkuk University, Chungju 27478, Korea; kis5497@kku.ac.kr

3 Research and Development, Sinil Pharmaceutical Co., Ltd., Seongnam-si 13207, Korea; seol@sinilpharm.com