Scientific Article by: Izair Jefthé Rodrigues, Onildo Campos, Augusto Queiroz Rodrigues, Gabriel Campos, Denis Souza




ABSTRACT

Anthocyanins belong to the flavonoid group and have been described as having important biological activities, such as antioxidant, anti-inflammatory and neuroprotective actions. The benefits of anthocyanins in neurodegenerative diseases such as Alzheimer's disease, dementia, neurotoxicity and Huntington's disease included a glioprotective effect, contributing to the maintenance of astrocyte homeostasis through their antioxidant effect and the modulation of acetylcholinesterase, which may have favored the observed protection regarding the reduction in memory deficit, reinforcing the promising therapeutic potential of anthocyanins.


INTRODUCTION

Neurodegenerative diseases lead to loss of neuronal function and structure; these diseases are associated with an imbalance between the production of free radicals and antioxidants. Cognitive deterioration is a fundamental element in distinguishing between individuals who do or do not suffer from dementia (5). Neurodegenerative diseases are known to give rise to a large proportion of dementias, especially the most prevalent in humans, ¨Alzheimer's and Parkinson's¨(10).

Alzheimer's is considered a primary dementia, one in which brain functions degrade slowly and gradually and are irreversibly lost. Alzheimer's disease is a major problem that has increased due to the rise in life expectancy of the general population (10,1). It was discovered in 1906 by Alois Alzheimer, using criteria of progressive memory loss, disorientation and pathological markers (senile plaques and neurofibrillary tangles) (1).


At first, Alzheimer's disease was assumed to be a rare condition; later it was considered an inevitable consequence of aging. Since the brain is the main oxygen-metabolizing organ and yet has relatively weak antioxidant protection mechanisms (3), it becomes especially vulnerable to oxidative stress. The accumulation of oxidative stress can induce damage to cells and to the DNA repair system in mitochondrial dysfunction, factors considered key and responsible for accelerating the aging process and the development of neurodegenerative diseases(1).


Tian et al. (2021) used anthocyanins in mice and analyzed volatile organic compounds, which are products or intermediates of body metabolism that can reflect the health status of the organism. In mice induced with Alzheimer's, urine and feces tests showed that the anthocyanin-treated group had a lower content of p-cresol, which derives from amino acid fermentation and is considered toxic to the gut(11).

They also showed a lower content of nonanol, which has a pro-inflammatory effect, given that its metabolites can induce low-grade chronic inflammation, and a higher amount of ketones in both feces and urine, indicating that the anthocyanin with a high fatty acid content underwent oxidation in the gut, which is beneficial to the human body(15).

Anthocyanins are natural pigments belonging to the flavonoid group. Their components are responsible for the color of a wide variety of fruits (grape, cherry, strawberry, among others), flowers (red hibiscus), legumes and vegetables(13). Anthocyanins have high antioxidant power, with effective actions against the oxidative stress of human metabolism, which triggers several chronic non-degenerative diseases such as diabetes, cancer, cardiovascular disease, and neurodegenerative diseases such as Alzheimer's disease, neurotoxicity and Huntington's disease; they are therefore used in the prevention and treatment of these diseases(16).

 

METHODOLOGY 

A systematic search was carried out in the electronic databases of the Latin American and Caribbean Health Sciences Literature Hilton(Lilacs), PubMed and Science Direct for articles presenting benefits of anthocyanin consumption in relation to the prevention and treatment of certain diseases. The searches covered articles from 2011 to 2021. The articles included in this review are original, addressing in vivo and in vitro studies. The terms used were “anthocyanin and its derivatives”, “neurodegenerative diseases”, “neuroprotection”.

In the selection of studies, titles were evaluated first, followed by abstracts. Once selected, these studies were analyzed in full, considering the effect of anthocyanin consumption on disease prevention; those meeting the following criteria were eligible: original articles; observational, clinical and experimental (in vitro and in vivo).

 

 OBJECTIVE

To evaluate the neuroprotective and antioxidant effect of treatment with anthocyanin-rich extract in neurodegenerative diseases such as Alzheimer's, Dementia and Parkinson's.

 

RESULTS AND DISCUSSION

Anthocyanins are natural pigments belonging to the group of plant secondary metabolites known as flavonoids; their main functions in plants are attracting pollinators and seed dispersers, as well as protecting various plant tissues during the stages of their life cycle(16). These anthocyanins are found in fruits such as açaí, plum, blackberry, cherry, fig, raspberry, jabuticaba, jambolan, grape and strawberry, and in vegetables such as red cabbage, purple potato, eggplant, black rice and red rice(4).

They are important bioactive compounds that provide functional nutrients with significant antioxidant activity, which has been attracting great interest, mainly due to their high prevalence in diets, being ubiquitous compounds in many plant-based raw materials. Anthocyanins make up the largest group of water-soluble pigments in the Plant Kingdom, with a molecule made up of two to three parts: an aglycone (anthocyanidin), a group of sugars and a group of organic acids(16).

Anthocyanins, a class of water-soluble flavonoids that are natural colored pigments found in various colorful plants such as fruits and vegetables, have received wide attention for their neuroprotective effects on the Central Nervous System (CNS)(12). Several studies in the literature have demonstrated important biological activities of these compounds. As antioxidant actions, they have anti-aging, anti-inflammatory, antibacterial, vision-protective and other physiological functions, and can also reduce the level of oxidative stress and neuroinflammation in the body and improve microglial vitality(6).

The color of anthocyanins generally changes with pH, and as the pH changes, their color shifts from red to different colors such as purple, orange, colorless and blue(4). As a result, they are widely used in the food, pharmaceutical and cosmetic industries. There are more than 20 known anthocyanins in nature, including cyanidin, delphinidin, malvidin, peonidin, petunidin and pelargonidin; these six are the most common anthocyanins(7).

The stability of anthocyanins depends on the presence of the B ring and the hydroxyl or methoxy groups in their structure, with anthocyanin-3-glucoside (C3G) being the most widely distributed in nature(14). Anthocyanins are considered useful functional food ingredients, as they confer several health benefits and are emerging as promising ingredients for the food industry(16).

Anthocyanins are composed of a basic structure called anthocyanidin or aglycone, which is formed by two aromatic rings (A and B) linked to a heterocyclic ring containing oxygen (C), as shown in Figure 1(8).

 

Figure 1. Chemical structures of six common anthocyanidins (flavylium form) and their sources based on current information. ( A ) Basic structure; ( B ), six common anthocyanidins. * indicates that λ max means the wavelength at which a substance has its strongest photon absorption.


Source: Adapted from Kzenovic et al., 2015

 

Some experiments with older adults diagnosed with mild to moderate Alzheimer's-type dementia showed that daily consumption of 200 mL of anthocyanin-rich cherry juice for twelve weeks improved verbal fluency and short- and long-term memory (21). It was also found that consumption of Concord grape juice (6.3 to 7.8 mL/kg/day – 46% anthocyanins) by individuals over 68 years of age with mild cognitive impairment for sixteen weeks increased neuronal activation in cortical areas and improved memory(20).

Notably, some previous studies have already shown that pretreatment with anthocyanins for seven to ten days was able to prevent memory deficit and changes in the activities of the enzymes AChE, Na+ ,K+ - ATPase and Ca2+ -ATPase, and in nitrate and nitrite levels in the cerebral cortex and hippocampus of rats subjected to an STZ-induced model of sporadic Alzheimer's-type dementia (19).

 

NEUROPROTECTION

Neurotoxicity is defined as any adverse effect that disturbs the normal functioning of the central or peripheral nervous system; it can be caused by a biological, physical or chemical agent capable of altering the integrity of an organism(1). Neurotoxic effects occur through direct or indirect action on the nervous system and may lead to mild and transient conditions, as well as to sequelae that can last a lifetime, with symptoms such as loss of reasoning and memory and mental confusion, among others(10).

In a study conducted by Shah et al. (2016), the therapeutic efficacy of anthocyanins against glutamate-induced neurotoxicity in the developing rat brain was evaluated. Treatment with anthocyanins led to a significant reduction in glutamate levels in the hippocampus. Neuroinflammation is known to be a consequence of neurotoxicity when induced by glutamate, and supplemented anthocyanin acts directly on glial cell activation and inhibited protein expression(15).

Anthocyanins reduced the extent of neuronal apoptosis in vitro in Huntington's disease, a neurodegenerative brain disease caused by a mutation in the gene for the huntingtin protein(2). Huntington's disease is characterized by arrhythmic involuntary movements, with psychiatric changes, cognitive decline and progressive dementia. Being genetic, it can be inherited; it has no cure, but chemical medications can be used to relieve the symptoms(6) .

In a study developed by Mollersen et al. (2016), anthocyanins were used as a treatment to demonstrate a reduction in the number of oxidative lesions, with the cytosine-adenine-guanine (CAG) DNA repeat, indicating that anthocyanin treatment in R6/1 HD mice showed satisfactory results on the symptoms of cognitive deficits.

Some in vivo studies have demonstrated the ability of anthocyanins to cross the BBB, being detected in brain regions related to learning and memory, such as the cerebral cortex and the hippocampus. Unmetabolized anthocyanins were identified in the brains of rats fed a diet supplemented with blueberry extract (2%) for ten weeks(18). In another study, a diet with anthocyanins from blackberry extract was given to rats for fifteen days, and 0.25 ± 0.05 nmol/g of anthocyanins were found in brain tissue(21).

An in vitro study evaluated the transport of anthocyanins across a cellular BBB model, suggesting that these compounds and their metabolites were able to cross this barrier, influenced by lipophilicity (22).

Anthocyanins have shown great potential and a beneficial effect against cognitive decline and aging-related neurodegeneration; the mechanisms that may be involved in their neuroprotective capacity include antioxidant activity, anti-inflammatory activity, modulation of neuronal signaling and of gene expression related to synaptic plasticity, and increased cerebral blood flow(14).

Their metabolites are known both for promoting and for inhibiting the gut microbiota and can prevent the proliferation of pathogenic bacteria, both gram-positive (Bacillus subtilis, Staphylococcus aureus and Listeria monocytogenes) and gram-negative (Escherichia coli, Salmonella enterica , and Citrobacter freundi) .Anthocyanins can stimulate the growth of beneficial bacteria such as Bifidobaterium spp., Lactobacillus , Enterococcus spp.in vitro and in humans(8).

The neuroprotective effect of anthocyanins has been supported by in vitro, in vivo and human studies. Pretreatment of human neuroblastoma cells with cyanidin was able to inhibit formation at the extracellular and extracellular/intracellular levels, respectively; cyanidin also prevented the induction of apoptotic events. Mouse neuroblastoma cells (N2A) incubated with the peptides Aβ25-35 and Aβ1-40 and then treated with anthocyanins showed decreased ROS formation, preservation of Ca2+ homeostasis and inhibition of β-secretase enzyme activation (23).

Excitatory metabolism of amino acids and neurotransmitters and various causes (such as β-amyloid (Aβ) aggregation, calcium imbalance, etc.) can damage mitochondria, thus producing a large number of reactive oxygen species and causing mitochondrial oxidative stress. Activation of glial cells, especially microglia, leads to local inflammation and the death of many neurons. Inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL-Iβ) released by microglia first activate astrocytes; cytokines released by astrocytes activate microglia, thereby inducing apoptosis of neural cells and producing neuronal inflammation(23,8,9).

Excessive amounts of many neurotransmitters will produce excitotoxicity in the nervous system, such as glutamate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and kainic acid (KA) receptors, and Ca 2+ and K + will interact with some neurotransmitters (such as glutamate), thereby inducing mitochondrial dysfunction(19).


 

Figure 2: Microbe-gut-brain axis system

Source: Adapted from Kzenovic et al., 2015

 

Figure 2: Pathogenesis of NDs under the microbe-gut-brain axis system. A. Excitotoxins. Glutamate is released from neurons and binds to receptors (NMDA, AMPA); together with glutamate uptake by excitatory amino acid transporter 2 (EAAT2) in astrocytes and the action of external factors (e.g., KA), this leads to increased glutamate excitotoxicity, thereby promoting Ca 2+ influx, endoplasmic reticulum stress and pro-apoptotic signaling cascades. B. Neuroinflammation. Interactions between glial cells, especially astrocytes and microglia, predispose to neuroinflammation.

 

Anthocyanins extracted from blueberries and currants reduce ROS production; anthocyanins can restrict the production of pro-inflammatory mediators in microglia and prevent inflammatory cell damage caused by Aβ, thereby exerting neuroprotective effects(19). The related research mechanism shows that anthocyanins inhibit nuclear factor-κB (NF-κB) activity, c-Jun N-terminal kinase (JNK) activation and microglial proliferation to suppress neuroinflammatory responses, suggesting that NF-κB and JNK may be potential targets of anthocyanins(4). Anthocyanins could reduce Ca 2+ dysregulation, ROS accumulation and AMP-dependent protein kinases in the KA-treated mouse hippocampal cell line (HT22) and in the activation of primary prenatal rat neurons (AMPK), and the increase in the percentage of apoptotic cells(6).

This induces abnormal protein aggregation and increases intracellular calcium efflux, which triggers a pro-apoptotic signaling cascade of oxidative stress and mitochondrial dysfunction. Oxidative stress causes excessive levels of reactive oxygen and reactive nitrogen in the mitochondria, exacerbating abnormal mitochondrial function(14).

Ultimately, this leads to the release of apoptosis-inducing factors and the pro-apoptotic signaling protein (cyt c), which then activates the caspase cascade or other apoptotic proteins to form apoptotic vesicles, ultimately leading to neuronal apoptosis. HPA: hypothalamic-pituitary-adrenal axis; enteroendocrine cells; EECs: BBB: blood-brain barrier; CNS: central nervous system; ROS/RNS: reactive oxygen species, reactive nitrogen species(6).

Apoptosis is a direct cause of neurological disorders, and Aβ, lipopolysaccharide (LPS) and glutamate as toxins can cause neuronal cascade responses, including endoplasmic reticulum stress-mediated apoptosis and mitochondria-dependent apoptotic pathways(11). On the other hand, Aβ peptides induce the accumulation of unfolded proteins, leading to abnormal protein aggregation and increased intracellular calcium fluxes; a combination of glutamate and lipopolysaccharide then induces neuronal apoptosis(4).

In another pathway, mitochondria release apoptosis-inducing factors and cytochrome c (cyt c), and cysteine-specific proteases (caspases) or other apoptosis-related proteins are activated to form apoptotic vesicles, which interrupt neuronal apoptosis(6).

 

FINAL CONSIDERATIONS

Several mechanisms were used by anthocyanin in the prevention or treatment of Alzheimer's disease. The study suggests observing common points in analyses of the volatile organic compounds of patients with this disease: anthocyanins act by reducing the pro-inflammatory effect of nonanol, inhibit the fermentation that generates p-cresol, carry out oxidation that is beneficial to the gut and provide for the formation of ketones. In neurotoxic disease, the action of anthocyanin is due to its potential mechanism of reducing neurotoxicity by reducing neural apoptosis; in Huntington's disease, the results show that anthocyanins reacted protectively, reducing the number of oxidative DNA lesions.

Anthocyanins did, in fact, have several beneficial effects in the studies analyzed. When consumed, owing to their high antioxidant capacity, they show that this bioactive compound can be used in the prevention and treatment of chronic diseases, acting in a protective, reducing and cell-modulating way, activating pathways, and can act as a means to prevent and treat diseases such as Alzheimer's, neurotoxic disease and Huntington's disease, among many other diseases.

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