Despite studies demonstrating pathological roles of Aβ in CAA, no effective treatments for CAA have been established. However, accumulating evidence has suggested the emerging effectiveness of taxifolin as a potential agent in the prevention and treatment of Aβ-associated cognitive dysfunction. Historically, oxidative stress has been reportedly closely implicated in the pathogenesis of age-related cognitive dysfunction, because the rate of oxidative metabolism is higher in the central nervous system than that in other tissues, and oxidative damage in the brain progresses with aging [40]. Furthermore, Aβ is also involved in the production of reactive oxygen species and causes neuronal dysfunction [33]. Therefore, dietary intervention with antioxidants has been expected to alleviate oxidative damage in the brain [40], thereby leading to reduced risk of cognitive dysfunction. Based on this possibility, extensive studies extracted a diverse array of compounds from various plants, characterized them, and addressed the potential neuroprotective effects of various antioxidants. During the course of these researches, taxifolin, a bioactive constituent of various plants, including onions, milk thistle, French maritime bark, and Douglas fir bark [41,42], was found and has become a topic of a great interest as a potential novel therapeutic target. Its biochemical and safety profiles have already been established [43,44]. Taxifolin is known to possess multiple pharmacological actions, such as anti-oxidation, advanced glycation end products (AGE) formation suppression, and mitochondrial protection, and has received increasing attention because of its potential efficacy in the treatment of various diseases including malignancies, cardiovascular diseases, chronic hepatitis, hyperlipidemia, and neurocognitive disorders [45].

We have recently addressed the potential therapeutic effects of taxifolin using in vitro and in vivo approaches and provided the first evidence delineating the novel beneficial effects of taxifolin on CAA [45]. The thioflavin T fluorescence assay and transmission electron microscopy imaging performed by us revealed that the addition of taxifolin to an Aβ₄₀ solution significantly inhibited the aggregation of Aβ₄₀ in vitro, indicating a novel suppressive effect of taxifolin on Aβ₄₀ fibril formation [45]. Furthermore, we investigated the inhibitory effects of taxifolin on Aβ₄₀ fibril formation in vivo using a mouse model of CAA, which expresses the human APP gene with Swedish/Dutch/Iowa triple mutations [45,46]. Quantitative analyses using filter trap assay and enzyme-linked immunosorbent assay showed that the cerebral levels of Aβ oligomers were decreased in the taxifolin group mice, which were fed taxifolin-containing chow, compared with the control group mice, which were fed standard chow. Therefore, these findings indicate that orally administered taxifolin has a novel preventive effect on Aβ₄₀ fibril formation in the brains of CAA model mice [45].

Experiments to assess spatial learning and reference memory revealed that taxifolin significantly suppressed cognitive impairment in these mice compared with controls. As expected from taxifolin’s inhibitory effects on Aβ₄₀ fibril formation, immunohistochemical analysis showed that it reduced the cerebrovascular accumulation of Aβ₄₀ in CAA model mice compared with controls. Furthermore, laser speckle flowmetry indicated that taxifolin significantly restored the reduced cerebral blood flow in CAA model mice. Notably, in conjunction with the reduced cerebral Aβ oligomer levels and improved cerebral blood flow, blood Aβ₄₀ levels were elevated in the taxifolin group mice compared with controls, suggesting that taxifolin facilitated the clearance of Aβ₄₀ from the brain into systemic circulation; this would lead to a neuroprotective effect, contributing to cognitive impairment prevention [45].

Regarding Aβ₄₂ fibril formation, which is closely implicated in AD pathogenesis, a previous meticulous study analyzed the effects of taxifolin on Aβ₄₂ aggregation and β-sheet formation using wild-type Aβ₄₂ or mutant Aβ₄₂ carrying substituted amino acids [34]. The results demonstrated a novel mechanism of action of taxifolin in the inhibition of Aβ₄₂ aggregation. The mechanism is related to the chemical structure of taxifolin: a catechol-type flavonoid, which possesses 3′,4′-dihydroxyl groups on the B-ring [34]. The catechol structure of taxifolin first autoxidizes and then forms o-quinone on the B-ring. This oxidized form, in turn, reacts with Aβ₄₂ by targeting Lys16 and/or Lys28 of Aβ₄₂, resulting in the production of Aβ₄₂–taxifolin adducts. Importantly, Lys16 and Lys28 are located in the intermolecular β-sheet region of Aβ₄₂. Therefore, the Aβ₄₂–taxifolin adduct formation contributes to the inhibition as well as to the destabilization of Aβ₄₂ aggregation, suppressing the elongation phase rather than the nucleation phase in the process of Aβ₄₂ fibril formation [34].

In addition to the biochemical properties of taxifolin that play a role in suppressing Aβ₄₂ fibril formation, it is of importance to discuss the physiological significance of taxifolin in the prevention and/or treatment of cognitive impairment. Sequential cleavage of APP by secretases generates Aβ; the rate-limiting step in this process is the cleavage by β-site secretase enzyme (BACE1) [47,48,49]. As the expression levels and activity of BACE1 are elevated in the brains of AD patients [50,51], there is a possibility that Aβ production and fibril formation are enhanced in these patients. Several studies have addressed the underlying mechanisms regulating BACE1 gene expression [48,52,53]. Aβ₄₂ induces the activation of JAK2 signaling pathway, which then mediates the activation of STAT3 signaling pathway [48]. The elevated STAT3 signaling, in turn, activates NF-κB signaling, which enhances the promoter activity of BACE1, thereby upregulating BACE1 transcription [48,52,53]. These signaling cascades promote amyloidogenesis, leading to neuronal injury and cognitive dysfunction.

To address the physiological significance of taxifolin in the suppression of cognitive decline, a study examined whether taxifolin is involved in attenuating signaling pathways for BACE1 expression using a mouse neuroblastoma N2a Swe cell line [48]. This cell line carries a human APP Swedish mutation and, when activated, the cells overexpress the gene, producing Aβ. Biochemical and immunocytochemical analyses revealed that the addition of taxifolin to the in vitro culture of these cells upregulated both the expression and the activity levels of SIRT1 [48], a deacetylase involved in the growth, differentiation, and survival of neurons [54]. Furthermore, the taxifolin-stimulated SIRT1 pathway reduced the activation of STAT3 signaling pathway, thereby downregulating BACE1 expression [48]. Together, these studies suggest novel functions of taxifolin besides the prevention of Aβ₄₂ aggregation: taxifolin exhibits suppressive effects on neuronal Aβ production and subsequent Aβ fibril formation through reduction of BACE1 levels by stimulating SIRT1-mediated inhibition of STAT3 signaling pathway. Notably, the authors further demonstrated that cilostazol also exhibits beneficial effects on N2a cells, as observed with taxifolin treatment, by activating the SIRT1 pathway, alleviating the STAT3 pathway, downregulating BACE1 expression, and reducing Aβ production [48]. In particular, their finding that concurrent treatment with taxifolin and cilostazol results in synergistic suppressive effects on Aβ production and on neuronal cell death suggests novel potential therapeutic strategies for CAA as well as AD. In addition, the SIRT1 pathway stimulated by taxifolin and cilostazol might contribute to neurogenesis and cognitive function by potentially upregulating neuroprotective factors such as brain-derived neurotrophic factor [54].

Inflammation in the brain has been highly implicated in the pathogenesis of AD through the acceleration of amyloidosis [55] and neuronal cell death [48,56]. Studies have reported that in neurons, the activation of a proinflammatory mediator cytosolic phospholipase A₂ (cPLA₂) contributes to age-associated cognitive impairment [57] as well as AD pathogenesis [58,59]. Aβ₄₂ can activate cPLA₂ [60,61], which is responsible for the main enzymatic process of metabolizing arachidonic acid; this ultimately results in the production of prostaglandin E₂ (PGE₂), which is a neuroinflammatory molecule [42,61,62]. Both cPLA₂ and PGE₂ have been reported to cause synapse damage [63].

A recent study has investigated the effects of taxifolin on cPLA₂-related inflammatory pathway and Aβ-induced neurotoxicity using the human neuroblastoma SH-SY5Y cell line and mouse primary hippocampal neurons [42]. Biochemical analysis revealed that the in vitro treatment of neurons with Aβ₄₂ resulted in elevated levels of both cPLA₂ and PGE₂. Furthermore, live cell imaging showed that incubation with Aβ₄₂ inhibited the formation of neuronal dendritic filopodia and dendritic spines. In contrast, the addition of taxifolin to these cultures seemed to combat Aβ₄₂-induced neurotoxicity; taxifolin significantly prevented the increase in cPLA₂ and PGE₂ levels as well as the inhibition of dendritic filopodia and dendritic spines formation in neurons incubated with Aβ₄₂. These data suggest that taxifolin exhibits neuroprotective effects besides its suppressive effects on Aβ production through the downregulation of BACE1 expression [48].

Using a mouse model of AD based on the hippocampal injection of Aβ₄₂, the authors further examined the effects of intraperitoneal administration of taxifolin on the levels of cPLA2 and of the synaptic marker post-synaptic density protein-95 (PSD-95) and on cognitive function [42]. In line with the findings of the in vitro experiments, taxifolin suppressed the increase in cPLA2 and PGE2 levels in the hippocampus in Aβ₄₂-injected mice. Moreover, Aβ₄₂ injection reduced PSD-95 levels in the hippocampus, but taxifolin treatment significantly suppressed these reductions. Furthermore, animal experiments designed to test recognition and spatial memories reported that Aβ₄₂-injected mice exhibited deficits in cognitive function, whereas taxifolin treatment improved this cognitive impairment. These results suggest that taxifolin exhibits suppressive effects on cognitive impairment in the preclinical settings of AD, potentially through pleiotropic functions including inhibition of Aβ₄₂ fibril formation [34], suppression of Aβ₄₂ production [48], and/or alleviation of Aβ₄₂-induced neurotoxicity [42].

Epidemiological studies have reported diabetes to be a high-risk factor for dementia, including AD and vascular dementia [64,65,66]. Potential mechanisms underlying diabetes-related dementia include multifactorial pathways such as Aβ accumulation, neuroinflammation, small vessel infarcts, and neurodegeneration in the brain [64,65,66,67,68]. Accordingly, prevention and treatment of diabetes is critical to reduce the risk of development and progression of dementia.

Detailed findings regarding the effects of taxifolin on diabetes are limited, but a recent study has demonstrated the anti-diabetic effects of taxifolin and its mechanisms of action through in vivo and in silico approaches [69]. The authors used a rat model of diabetes in which pancreatic β-cells were depleted by intraperitoneal injection of alloxan. They found that taxifolin administration via an intragastric route significantly reduced blood glucose levels in the diabetic rats compared with controls (without taxifolin). To elucidate the underlying mechanisms of the hypoglycemic effects of taxifolin, the authors next examined the effects of taxifolin on α-amylase, a carbohydrate-metabolizing enzyme that elevates blood glucose levels; inhibition of α-amylase is effective in the treatment of diabetes [69,70]. Taxifolin treatment significantly reduced serum amylase activity in diabetic rats compared with controls, consistent with its glucose-lowering effects. These studies suggest that taxifolin exhibits hypoglycemic effects through the reduction of α-amylase activity in diabetic rats [69].

The authors further addressed the potential direct action of taxifolin on α-amylase with computational and docking studies, comprising ligand–receptor docking studies, free-energy calculations, and molecular dynamics simulations [69]. In the flexible docking simulations, the authors selected the bioinformatically determined best-docked poses of the taxifolin–α-amylase complex and analyzed the binding modes of taxifolin with α-amylase. The analysis revealed that taxifolin interacts with the residues Trp59, Tyr62, Glu233, and Asp300 present at the active site of α-amylase through a π–π interaction with the benzene rings of Trp59 and Tyr62 and an H-bond interaction with Glu233 and Asp300. In addition, using a molecular mechanics-based scoring method for binding free-energy calculation, the authors showed that van der Waals and nonpolar solvation-free energies also contribute to the binding affinity of taxifolin for α-amylase. Furthermore, the authors examined the dynamic behavior of the taxifolin–α-amylase complex through molecular dynamics simulations, considering the potential effects of solvent, temperature, and pressure on the complex formation, and confirmed the stable conformation of taxifolin at the active site of α-amylase. Accordingly, these in vivo and in silico findings indicate that taxifolin binds to the active site of α-amylase and inhibits its activity, thus leading to improvement of hyperglycemia [69].

Diabetic nephropathy is a serious diabetic complication [71], and chronic kidney diseases (CKDs) are epidemiologically a high-risk factor for dementia [72,73,74]. Reportedly, in a mouse model of CKD, chronic renal dysfunction resulted in elevated oxidative stress levels in the brain, leading to cognitive impairment [75]. Thus, the prevention and improvement of CKDs would contribute to reduce the dementia risk.

Recent studies have reported the novel renal protective effects of taxifolin using a rat model of diabetes, which was developed through pancreatic β-cell depletion with an intraperitoneal streptozotocin injection [71,76]. Taxifolin treatment significantly improved the renal function profiles in diabetic rats compared with controls, in parallel with improved glucose metabolism [71,76]. Consistent with these results, further histological analyses revealed that taxifolin suppressed necrotic cell death in the renal tissue [76] and alleviated renal fibrosis by inhibiting extracellular matrix accumulation and mesangial matrix expansion [71]. Furthermore, biochemical analyses showed that taxifolin reduced the activation of high-glucose-stimulated proinflammatory pathways in rat and human kidney cell lines [71] as well as in renal tissue from diabetic rats [76]. Taxifolin also reduced the levels of reactive oxygen species produced by these kidney cell lines, which were stimulated with high glucose [71]. These findings indicate the potential renal protective effects of taxifolin in diabetic conditions, further supporting its potential beneficial effects on dementia.