Amyloid β-peptide (Aβ) is a small molecular fragment formed by the two-step proteolytic hydrolysis of β-secretase and γ-secretase under the regulation of amyloid precursor protein (APP), which exists in the form of monomers, dimers, polymers, and fiber polymers, such as Aβ40 and Aβ42 (Lane et al., 2018). Increased Aβ production, increased Aβ42/Aβ40 ratio, promotion of Aβ deposition, and reduction of Aβ clearance result in the formation and deposition of SPs, followed by a complex cascade of inflammatory responses, microglial activation, and cytokine release (Selkoe and Hardy, 2016; Panza et al., 2019; Tiwari et al., 2019). These responses lead to progressive neurogenic damage, neuronal defects, and cognitive dysfunctions. Therefore, reducing Aβ production and removing Aβ deposition may be important therapeutic strategies in the treatment of AD.

Scutellarin (5, 6, 4’-trihydroxyflavone-7-glucuronide) has been identified as a major active ingredient of EBHM and has multiple pharmacological activities, including anti-inflammatory, anti-apoptosis, and anti-oxidation (Wang and Ma, 2018; Yang et al., 2018). Scutellarin has been reported to inhibit the aggregation of Aβ in vitro and prevent Aβ-mediated cell death when applied to cultured neuronal PC12 cells (Zhu et al., 2009). In animal models of AD, scutellarin reduced cognitive dysfunction in APP/PS1 mice, inhibited amyloid deposition, and reduced soluble Aβ42 and Aβ40 levels in the cortex of mice. In in vitro experiments, scutellarin prevented cognitive decline by converting Aβ monomers into low-toxicity amyloid fibrils or protofibrils, and by reducing the levels of highly toxic soluble Aβ oligomers (Zhang et al., 2020). Zeng et al. (2018) further confirmed that continuous administration of scutellarin significantly reduced the latency to find a platform and improved swimming in APP/PS1 mice. In addition, scutellarin reduced the levels of soluble and insoluble Aβ in the brain and plasma of mice; decreased the levels of Aβ plaque-associated gliosis, pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-6; alleviated neuroinflammation; and showed anti-amyloidosis effects (Zeng et al., 2018). In a rat model of chronic cerebral hypoperfusion, scutellarin exhibited an anti-Aβ effect. Treatment with 30 mg/kg scutellarin for 4 weeks significantly ameliorated spatial cognitive impairment and memory deficits in rats with permanent bilateral common carotid artery occlusion (pBCAO). Further experiments showed that scutellarin reduced Aβ formation by inhibiting the expression of APP and β-site APP cleaving enzyme 1 (BACE-1) in the hippocampi of pBCAO rats. Additionally, scutellarin (30 mg/kg) significantly suppressed the expression of glial fibrillary acidic protein and Iba1 in the cerebral cortex and hippocampal tissue, thereby inhibiting the activation of glial cells (microglia and astrocytes) in brain tissues (Shin et al., 2018).

Previous studies have suggested that CQA can improve or enhance the learning and memory abilities of various animal models of AD. For instance, 5-O-caffeoylquinic acid (5-CQA) supplementation significantly reduces hippocampal Aβ plaque formation and neuronal loss. This neuroprotective effect is due to 5-CQA upregulation of the gene encoding low-density lipoprotein receptor-related protein 1 (Aβ efflux receptor) and normalization of the perivascular localization of aquaporin 4, thereby promoting Aβ clearance along paravascular pathways (Ishida et al., 2020). In addition, in the SH-SY5Y cell model, CQA exhibited anti-amyloidogenic properties. 4, 5-di-CQA and 3, 4, 5-tri-CQA could suppress β-sheet transformation and cytotoxicity in SH-SY5Y of Aβ42 in a dose-dependent manner. Furthermore, 3, 4, 5-tri-CQA blocked the formation of Aβ42 oligomers, indicating that 3, 4, 5-tri-CQA may be a potential agent for the prevention of AD (Miyamae et al., 2012).

Tau is a multifunctional protein related to microtubule function, and the stability of the microtubule structure in neurons is conducive to the transfer of nutrients or information molecules between synapses (Stancu et al., 2019). However, in patients with AD, tau does not play a role in promoting microtubule assembly and enhancing microtubule stability. Instead, it separates from microtubules, attaches to other Tau molecules, self-assembles into pairs of helices, and reassembles into NFTs (Gao et al., 2018). The main component of NFTs is the hyperphosphorylated tau (p-Tau) protein, which is neurotoxic and destroys the structure of neurons, leading to abnormal communication and signal processing between neurons and neuronal apoptosis (Gibbons et al., 2019). This suggests that using tau protein as an important target and preventing its spread and accumulation may alleviate the progression of AD disease (Jouanne et al., 2017).

In an aluminum chloride plus D-galactose-induced AD mouse model, scutellarin enhanced horizontal and vertical movements in an autonomic activity test and reduced the escape latency time of mice in the Morris water maze (MWM) test. Scutellarin administration significantly reduced the levels of p-Tau, which indicated that the attenuation of the effects associated with AD following treatment with scutellarin may be due to the resultant decreased levels of p-Tau. In addition, the enhanced levels of acetylcholine and SOD in serum and brain lysates may also contribute to the protective effects of scutellarin (Hu et al., 2018).

Acetylcholine (ACh) is primarily involved in arousal, learning, memory, and motor regulation. ACh is widely distributed throughout the cortex, basal ganglia, and basal forebrain, suggesting that cholinergic transmission is essential for brain function (Hampel et al., 2018). The degree of cognitive dysfunction in patients with AD is closely related to a decrease in acetylcholinesterase (AChE) activity and ACh synthesis (Ferreira-Vieira et al., 2016). The cholinergic hypothesis suggests that cholinergic neurons are affected in the early stage of AD and that the degeneration of cholinergic neurons in the basal forebrain, the reduction of cholinergic neurotransmitters in the cerebral cortex and other regions, and the loss of cholinergic agents involved in ACh synthesis are the main causes of cognitive decline in patients with AD (Machado et al., 2020). Therefore, increasing ACh synaptic levels or selective agonists modulating acetylcholine receptors (AChRs) in the postsynaptic membrane can increase the continuous accumulation of ACh and AChR activation in nerve cells, thereby reversing cognitive dysfunction (Gray et al., 2015; Sultzer, 2018).

Scutellarin also showed its ability to improve learning and memory defects in an Aβ-induced rat dementia model, which was attributed to scutellarin treatment, which significantly upregulated the α4 and α7 AChR subunit protein levels by 24% and 30%, respectively. Moreover, maintenance of AChE and butyrylcholinesterase activities in the brain and plasma of Aβ-induced deficits is also an important therapeutic mechanism of scutellarin (Guo et al., 2011b). A scutellarin–rivastigmine hybrid (compound 15c) had a neuroprotective effect against H2O2-induced PC12 cell injury and could cross the blood–brain barrier in vitro. Moreover, compound 15c also had significant neuroprotective effects in scopolamine-induced cognitive impairment in mice and could decrease the vitality of AChE and increase the vitality of acetyltransferase in the hippocampus of mice (Sang et al., 2015).

With the deepening of the study on AD pathogenesis, neuroinflammatory mechanisms are gradually being recognized to be involved in the pathogenesis and development of AD (Leng and Edison, 2021). Microglia (MG) are the most critical mediators of immune response in the brain. MG in the resting state functions in migration and swallowing and plays a main role in nutrition and support. However, it can be activated into M1 (pro-inflammatory activation) and M2 (anti-inflammatory activation) activation states under the stimulation of internal and external environmental factors (Tang and Le, 2016). Studies have shown that M1 type MG in patients with AD oversecretes proinflammatory factors, such as TNF-α, INF-γ, IL-1 β, IL-6, and other inflammatory cytokines involved in neurotoxicity and neuronal death (Morales et al., 2014). Under physiological conditions, astrocytes are evenly distributed and can secrete neurotransmitters and neurotrophic factors to participate in the maintenance of the normal neuronal microenvironment, signal transduction, immune regulation, and other functional activities (Sofroniew and Vinters, 2010). In the AD brain, abnormal accumulation of astrocytes around amyloid plaques has been observed, indicating that astrocytes have a scavenging effect on Aβ deposition (Rodríguez-Arellano et al., 2016). In mouse models of AD, decreased astrocyte activity has been found, which may be related to increased Aβ deposition and significantly increased clearance load (Kraft et al., 2013). Therefore, regulating the inflammatory state of MG and the function and activity of astrocytes may be potential targets in the treatment of AD (Fakhoury, 2018; Du et al., 2021).

Nuclear factor of activated B-cells (NF-κB) is a protein complex that controls DNA transcription and can be found in almost all common animal cell types (Stormberg et al., 2021). It plays an important role in regulating the immune response, and is implicated in cellular responses to stimuli such as cytokines, free radicals and stress (Ju Hwang et al., 2019). NF-κB activation, as an underlying cause of AD, was found predominantly in neurons and glial cells in Aβ plaque surrounding areas (Yang et al., 2021). In addition, there was a strong correlation between increased NF-κB activity and cyclooxygenase-2 (COX-2) transcription in the superior temporal lobe gyrus of AD patients was also demonstrated (Wang et al., 2014). Moreover, the levels of NF-κB activity were increased in cholinergic neurons in the basal forebrains of AD patients (Li et al., 2019), and could affect AD pathology via increased pro-inflammatory cytokines expression and amyloidogenesis (Xie et al., 2020). Therefore, inhibiting NF-κB are predicted to play a protective role in the development of AD (Seo et al., 2018). Scutellarein (5, 6, 7, 4′-tetrahydroxy flavone) is the hydrolyzed product of scutellarin in perennial herbs. A previous study demonstrated that scutellarein is absorbed more easily following oral administration than is scutellarin (Mamadalieva et al., 2011). In Aβ-treated PC 12 cells, pretreatment with scutellarein and scutellarin increased cell viability, significantly attenuated Aβ-induced cell death, and decreased apoptotic ratios. Furthermore, scutellarein and scutellarin increased the expression level of cytoplasmic NF-κB and decreased the expression level of nuclear NF-κB, suggesting that scutellarein and scutellarin inhibit the NF-κB signaling pathway. In an in vivo study, scutellarein and scutellarin significantly improved the Aβ-induced latency to locate the platform during the acquisition period and increased the number of platform crossings in a spatial probe trial. Scutellarein and scutellarin restored the decreased B-cell lymphoma 2 (Bcl-2) and increased Bax and cleaved caspase-3 levels in the hippocampus of Aβ-induced rats, suggesting their anti-apoptotic effects, although scutellarein had more prominent effects than scutellarin. Scutellarein also showed anti-neuroinflammatory activity, significantly increased cytoplasmic NF-κB and decreased nuclear NF-κB compared to the Aβ group. Thus, scutellarein may inhibit the activation of NF-κB signaling in the hippocampus (Huang et al., 2019).

Baicalin is another major active flavonoid extracted from EBHM and has been shown to have anti-inflammatory and anti-tumor effects (Wu et al., 2013). In an Aβ1-42 induced mice model, a 14-day administration of baicalin (100 mg/kg) significantly ameliorated memory impairment in the MWM and probe tests, attenuated glial cell activation, and decreased the expression of inflammatory factors (IL-6 and TNF-α), suggesting that baicalin ameliorated Aβ1-42 protein-related pathology and cognitive dysfunction via its anti-neuroinflammatory activity (Chen et al., 2015). Furthermore, baicalin administration effectively decreased the number of activated MGs and proinflammatory cytokine levels (IL-1β, IL-18, and iNOS), as well as neuroinflammation-mediated neuronal apoptosis via inhibition of the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasomes and toll-like receptor 4 (TLR4)/NF-κB signaling pathway, and attenuated spatial memory dysfunction in APP/PS1 mice (Jin et al., 2019). In another AD transgenic mouse model, baicalin inhibited Aβ-induced microglial activation and reduced Aβ-induced inflammatory cytokine (IL-6, TNF-α, and NO) secretion via the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway, thus providing a new means for the prevention and treatment of AD (Xiong et al., 2014).

Oxidative stress (OS) refers to a pathological state in which free radicals or other products in the body exceed the body’s antioxidant capacity (Chang et al., 2020). Excessive ROS and reactive nitrogen species (RNS) are produced (Smallwood et al., 2018). Under pathological conditions, a variety of cells, such as cortical neurons and astrocytes, can produce excessive NO, which reacts with superoxide anions to produce more active peroxynitry (ONOO-) and·OH, thus causing potential damage to the body. The OS response is characterized by the production of ROS and RNS and an imbalance in antioxidant defense (Chiarini et al., 2016), which are closely involved in the pathogenesis of AD (Ahmad et al., 2017). Antioxidants are generally divided into enzymatic and nonenzymatic systems. The former includes catalase, SOD, and glutathione peroxidase (GSH-PX). The latter includes vitamins, amino acids, and metalloproteins (Margaritelis et al., 2018). By scavenging free radicals and/or decomposing hydrogen peroxide, the oxidation chain is blocked, which delays the occurrence and development of AD (Khan et al., 2019).

In Aβ25-35 induced AD rats, scutellarin shortened the latent escape period and the time required to pass the original site of the platform, and increased the number of crossings. Scutellarin reversed the reduced activity of SOD and elevated levels of monoamine oxidase (MAO), decreased the upregulation of IL-1, IL-6, and TNF-α in the cortex, and reduced the percentage of apoptotic neurons in the rat brain (Guo et al., 2013). In another group of Aβ25-35 AD-induced rats, similar conclusions were obtained; that is, the application of scutellarin significantly increased SOD activity and decreased MDA activity in brain tissues for 30 consecutive days, and scutellarin played an anti-apoptotic role in the brain tissues of rats (Guo et al., 2011a).

Baicalin may also be a potential anti-OS agent. In an in vitro study, baicalin prevented SH-SY5Y cells against damage by directly interacting with copper to inhibit Aβ1-42 aggregation. In addition, it protected SH-SY5Y cells from oxidative injuries induced by Aβ1-42 aggregation by decreasing H2O2 production (Yin et al., 2011). In the N2a/APPswe cell line, pretreatment with baicalin improved cell viability by increasing SOD activity, inhibiting MDA production, reducing nuclear NF-κB protein levels, and promoting nuclear factor erythroid 2-related factor 2 (NRF2) translocation to the nucleus, indicating that baicalin has anti-oxidative stress effects (Cao et al., 2015).

CQA has various pharmacological properties, including antioxidant activity. For example, 5-CQA reduced the rate of apoptosis, increased cell viability, and improved cell morphology in H2O2-treated SH-SY5Y cells. In addition, 5-CQA alleviated the accumulation of autophagic vacuoles and decreased P62 levels and the LC3B II/I ratio, thereby promoting autophagic cellular degradation and increasing autophagic flux (Gao et al., 2021).

Apoptosis refers to programmed cell death that occurs during cell development or under the action of certain factors through the regulation of genes and their products in cells. Neuronal apoptosis plays an important role in neurodegenerative diseases, such as AD, which can lead to the loss of a large number of neurons (Bredesen, 1995; Caccamo et al., 2017). It is influenced by Aβ, Bcl-2-associated X protein (Bax), Bcl2, caspases, TNF-α, ROS, and enzyme perturbation (Dhivya Bharathi et al., 2019). The mechanisms of neuronal apoptosis include the following: 1) the extrinsic pathway, initiated by cell surface death receptors such as Fas and tumor necrosis factor receptor families; 2) the intrinsic pathway or mitochondrial pathway, initiated by stress conditions, chemotherapeutic agents, and drugs; and 3) activation of caspase 12 caused by endoplasmic reticulum stress, leading to apoptosis (Boatright and Salvesen, 2003). Drugs with anti-apoptotic activity and potential applications in targeting apoptosis in AD include flavonoids and antioxidants of plants (Obulesu and Lakshmi, 2014).

Considering the involvement of Aβ-induced OS in the etiology and pathology of AD, antioxidant therapy to scavenge excess ROS by inducing endogenous antioxidant enzymes is a promising approach to the prevention of AD (Park, 2010). Ding et al. (2015) suggested that baicalin has beneficial effects on learning and memory deficits caused by Aβ in rats. The neuroprotective effects of baicalin may be achieved by its antioxidant and anti-apoptotic activities. Baicalin treatment improves antioxidant capacity by restoring the activities of antioxidant enzymes (SOD, catalase, and GSH-PX) and upregulating their gene expression. In addition, baicalin can effectively prevent Aβ-induced reduction in mitochondrial membrane potential, an increase in the Bax/Bcl-2 ratio, activation of Caspase-9/-3, and the release of cytochrome c. Moreover, baicalin treatment significantly inhibits the inhibitory effect of Aβ on Nrf2 and exerts a stronger antioxidant effect (Ding et al., 2015). COX-2 is a key enzyme in neuronal death and is an important marker of the inflammatory response. The protein expression levels of COX-2 and its upstream gene peroxisome proliferator-activated receptor γ (PPAR γ) is increased in patients with AD and is positively correlated with the level of Aβ. COX-2 inhibitors can slow the pathological evolution of AD by inhibiting COX (Pasinetti, 2002). In a study by Li et al. (2011), baicalin inhibited hippocampal COX-2 expression, antagonized Aβ-induced neuronal apoptosis, and diminished cortical and hippocampal neuron necrosis.

EBHM and its active ingredients may play a therapeutic role in AD via other neuroprotective mechanisms. Baicalin plays a significant role in rescuing mitochondrial dysfunction by modulating mitochondrial fragmentation. Yu et al. (2022) reported that baicalin treatment significantly reversed the altered learning and memory behaviors of an Aβ oligomer-induced mouse model due to improved mitochondrial fragmentation, synaptic plasticity, and rescue of dysfunction via inhibition of phosphodiesterase-4, leading to activation of the phosphorylated Ser637 site of mitochondrial dynamin-related protein 1. In senescence-accelerated prone (SAMP) mice, 3, 5-di-O-CQA administration improved spatial learning and memory via upregulation of phosphoglycerate kinase-1 expression and activation of ATP production (Han et al., 2010). 3, 4, 5-tri-CQA exhibited anti-AD effects in the same AD mouse model (SAMP). Treatment with 3, 4, 5-tri-CQA improved spatial learning and memory by increasing neurogenesis in the hippocampal dentate gyrus and by pro-neurogenic effects in human cells, which might have occurred via activation of the bone morphogenetic protein signaling pathway. Moreover, 3, 4, 5-tri-CQA may be a new agent capable of increasing neural progenitor cell proliferation in the dentate gyrus, suggesting that 3, 4, 5-tri-CQA may be a new therapeutic drug for treating AD (Sasaki et al., 2019).