3.1. In Vitro Inhibitory Activity of Baicalein against BACE1 and AChE
The chemical structure of baicalein (5,6,7-trihydroxyflavone) is shown in Figure 1
. To determine the anti-AD potential of baicalein, its inhibitory activity against BACE1 and AChE was evaluated (Table 1
). The compound strongly inhibited BACE1 in a dose-dependent manner (p
< 0.001) with an IC50
value of 23.71 ± 1.91 µM.
In a previous antioxidant study, baicalein exhibited the most powerful antioxidant effect in chemical scavenging, as well as the most potent cellular antioxidant ability among the flavonoid components of S. baicalensis
]. Chrysin is a flavone structurally similar to baicalein, with two hydroxyl groups at C-5 and -7 on the A ring. This flavone showed much weaker antioxidant ability relative to that of baicalein. It can be assumed that the presence of three continuous hydroxyl groups on the A ring improves its hydrogen or electron donating potential, as well as its BACE1 inhibitory activity. In addition, because oxidative stress abnormally increases BACE1 gene expression and leads to Aβ accumulation, the excellent antioxidant activity of baicalein is expected to influence BACE1 inhibition through delaying or reducing oxidative damage [21
]. Apigenin (4′,5,7-trihydroxyflavone) is found in a wide variety of plants and is structurally similar to baicalein, save for the repositioning of one hydroxyl group (C-6 to C-4′). Apigenin was reported to exhibit poor BACE1 inhibitory activity (IC50
> 100 µM), assuming that a hydroxyl group at C-6 of baicalein provides a partial inhibition of BACE1 [22
In addition, baicalein dose-dependently and significantly (p < 0.001) suppressed AChE with an IC50 value of 45.95 ± 3.44 µM. Although baicalein showed somewhat weaker inhibition of AChE compared with galantamine, a well-known positive control with an IC50 value of 1.30 ± 0.01 µM, it is still considered as potential bioactive compound that could prevent both Aβ aggregation and amyloid fibril plaque formation.
3.4. In Silico Docking Simulation of Baicalein and Its Drug-Likeness Prediction
The in silico molecular interaction of BACE1, AChE, and P-gp with baicalein is summarized in Table 3
. According to the results of docking simulation in Figure 3
a–c, the baicalein-BACE1 complex exhibited negative binding energy (−8.60 kcal/mol), suggesting that the compound is a high-affinity BACE1 inhibitor able to bind strongly with the enzyme. Although baicalein is non-competitive BACE1 inhibitor that docks into a non-catalytic region, such as Ser36, Asn37, and Ile26 residues of BACE1, it also binds to the catalytic region Ser35. The catalytic domain of BACE harbors two aspartic protease active site motifs of the sequence DTGS and DSGT that come together to form the active site of the enzyme. Ser35 is a part of the DTGS active site motif of BACE1 and it was found to interact with a water molecule critical for BACE1 hydrolytic activity [2
]. Moreover, six hydrogen bonds were observed between the oxygen groups of Ser35, Ser36, Asn37, and Ile26 and the three hydroxyl groups of baicalein, with bonding distances of 3.15, 3.27, 2.85, and 2.88 and 2.66 and 2.89 Å, respectively. Interestingly, the hydroxyl moiety at C-6 on the A ring of baicalein simultaneously acted as a hydrogen donor to Ile126 and Asn37 residues and an acceptor to Ser36. In contrast, the oxygen atom at C-5 on the A ring accepted the hydrogen from the Asn37 and Ser35 residues, and the oxygen atom at C-7 on the A ring donated a hydrogen to Ile126. The presence of an H-bond donor or acceptor on the A ring may therefore affect the potential inhibition of BACE1 in the biological evaluation results of baicalein.
As shown in Figure 3
d–f, baicalein was revealed to be active against AChE with a lowest energy of −8.7 kcal/mol. The catalytic site of AChE is located at the bottom of a narrow gorge known as the catalytic triad, composed of Ser203, Glu334, and His447. The hydroxyl group at C-5 on the A ring produced close binding with one of the catalytic triads (His447) through a H-bond (bonding distance 2.94 Å). In addition, the hydroxyl groups at C-6 and C-7 on the A ring of baicalein bound to the oxygen atom of Glu202, with a H-bonding distance of 2.88 and 2.94 Å, respectively. A peripheral site comprised of aromatic side chains including Tyr72, Asp74, Tyr124, Trp286, Phe295, Phe297, Tyr337, and Tyr341 contributes to the catalytic efficiency of AChE [25
]. A total of 11 hydrophobic interactions were exhibited by Tyr72, Asp74, Trp86, Asn87, Gly120, Gly121, Tyr124, Ser125, Ser203, Tyr337, and Gly448. Overall, these docking results were in agreement with our in vitro biological experimental results, demonstrating that baicalein is a crucial BACE1 inhibitor connected with BACE1 by stronger H-bonds than with AChE.
Furthermore, for the effective prevention and/or treatment of AD, docking simulation between P-gp and baicalein was performed to predict the possible effluence system. As shown in Table 3
and Figure 3
g–i, the lowest binding energy between baicalein and P-gp residues was −8.4 kcal/mol. Three H-bonds were observed in the baicalein-P-gp complex, and Ser975 and Ser725 residues participated in this interaction. As with the BACE1 docking study, the oxygen atom of baicalein at C-6 of the A ring simultaneously acted as a hydrogen donor to Ser725 residues and an acceptor to Ser975, with bonding distances of 2.85 and 3.33 Å, respectively. In addition, the hydroxyl group at C-5 on the A ring formed one H-bond with Ser975, with a bond distance of 3.08 Å. Aside from these H-bonds, other residues such as Phe724, Phe728, Val978, Phe332, Phe71, Phe974, and Leu971 were found to have van der Waals interactions.
Lipinski’s criteria, also known as the “rule of five”, is generally considered a drug-likeness test for the evaluation of oral availability for compounds. It states that poor absorption or permeability of a compound occurs when there are more than 5 H-bond donors, more than 10 H-bond acceptors, more than 5 calculated Log P, and the molecular weight is higher than 500 Da [26
]. Additionally, good bioavailability is more likely for compounds with a total polar surface area (TPSA) of ≤140 Å and ≤10 rotatable bonds (nrotb). It is noteworthy that baicalein complied with all requirements of Lipinski’s rule of five for both oral bioavailability and BBB permeability, as listed in Table 4
Several previous studies have demonstrated that baicalein possesses antioxidant activity by reducing Aβ-induced neurotoxicity in PC12 cells, thus protecting rat cortical cells [13
]. The compound prevented H2
-induced apoptosis and restored mitochondrial dysfunction in PC12 cells [28
]. In addition, in vivo studies have demonstrated that baicalein significantly improves the cognitive performance of Tg2576 mice (10 mg/kg) and inhibits LOX and GSK3β activity, consequently preventing tau phosphorylation in APP/PS1 mice (80 mg/kg) [29
Oral acute toxicity studies are needed to confirm a range of doses for compounds and to reveal the safety of clinical signs. Several studies demonstrated that baicalein expresses no signs of toxicity after 2.8 mg of single-dose administration, demonstrating safety for use with humans [31
]. Another clinical trial demonstrated that baicalein is safe and well tolerated after multiple-dose oral administration (200, 400, and 800 mg) in healthy participants [32
]. Even though natural BACE1 inhibitors such as baicalein possess relatively weaker BACE1 inhibitory properties compared with synthetic ones, they may be free of the side effects caused by excessive BACE1 inhibition.
The BBB not only protects the brain from the entry of potentially toxic substances, but also prevents the passage of therapeutic agents required for treating disorders related to neurodegenerative diseases. Therefore, one important issue in successful anti-AD therapy is the ability of the molecules to cross the BBB. Studies have shown that certain substances with lipid-soluble molecules and a low molecular weight of below 400–600 Da are transported across the BBB [33
]. It is of great interest that baicalein has been reported to cross the BBB, and has been found in the rat brain after oral administration of S. baicalensis
To be considered a promising anti-AD substance, both oral bioavailability and the metabolic transformation of compounds are necessary. Baicalein is the main bioactive constituent of S. baicalensis and is metabolized to baicalin, 7-O-glucuronide of baicalein, following intake by various animals and humans [34
]. On the other hand, another study found that when baicalein was administered (30 and 60 mg/kg−1
) through the femoral vein, intact baicalein was observed in rat plasma, the brain, and bile, and that this compound rapidly penetrated the BBB [16
]. In addition, after oral administration of S. baicalensis
extract (30 mg/kg−1
), both baicalein and baicalin were detected in the rat brain, supporting the evidence that these two compounds could penetrate the BBB. Interestingly, the brain concentration of baicalein was greatly higher than that of baicalin. The brain-to-plasma ratio of baicalein was also twenty times higher than that of baicalin [17
]. Although several studies have revealed that baicalein was converted to glucuronide form in vivo, baicalin as well as baicalein exhibited pharmacological efficacy, including antioxidant, anti-inflammatory, and neuroprotective properties. Baicalein fractionally crossed the BBB and reached the brain, indicating that baicalein could act as a natural anti-AD candidate [37
]. Although further and more detailed studies need to be conducted, baicalein appears to be safe and exerts specific inhibitory properties against BACE1 and AChE, as well as P-gp inhibitory potential. It may therefore be considered a novel potential agent for preventing AD.