Inhibition of glycogen synthase kinase 3β (GSK-3β) is considered to be the central therapeutic approach against Alzheimer’s disease (AD). In the present study, boiled water extracts of the Kangen-karyu (KK) herbal mixture and its constituents were screened for GSK-3β inhibitory activity. KK is used in traditional Kampo and Chinese medicines for improving cognitive function. The GSK-3β inhibition potential was evaluated by using the Kinase-Glo luminescent kinase assay platform. Furthermore, enzyme kinetics and in silico modeling were performed by using AutoDockTools to demonstrate the mechanism of enzyme inhibition. KK extract significantly inhibited GSK-3β in a concentration-dependent manner (IC50: 17.05 ± 1.14 μg/mL) when compared with the reference drug luteolin (IC50: 2.18 ± 0.13 μM). Among the six components of KK, extracts of Cyperi Rhizoma and Salviae Miltiorrhizae Radix significantly inhibited GSK-3β with IC50 values of 20.68 ± 2.50 and 7.77 ± 1.38 μg/mL, respectively. Among the constituents of the roots of S. miltiorrhiza water extract, rosmarinic acid, magnesium lithospermate B, salvianolic acid A, salvianolic acid B, and salvianolic acid C inhibited GSK-3β with IC50 values ranging from 6.97 to 135.5 μM. Salvianolic acid B was found to be an ATP-competitive inhibitor of GSK-3β and showed the lowest IC50 value (6.97 ± 0.96 µM). In silico modeling suggested a mechanism of action by which the hydrophobic, π–cation, and hydrophilic interactions of salvianolic acid B at ATP and substrate sites are critical for the observed GSK-3β inhibition. Therefore, one of the mechanisms of action of KK against AD may be the inhibition of GSK-3β and one of the active components of KK is the root of S. miltiorrhiza and its constituents: rosmarinic acid, magnesium lithospermate B, and salvianolic acids A, B, and C. Our results demonstrate the pharmacological basis for the use of KK against AD.

Symphyocladia latiuscula (Harvey) Yamada is a red alga with a myriad of bromophenols accompanied by a diverse array of biological activities. The main purpose of the present study was to characterize the anti-Alzheimer’s disease activity of bromophenols from S. latiuscula via inhibition of cholinesterases (AChE and BChE), β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and glycogen synthase kinase-3β (GSK-3β). The results of enzyme inhibition assays demonstrated 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol ( 1 ), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether ( 2 ), and bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether ( 3 ) as potent inhibitors of aforementioned enzymes. Among the tested bromophenols, 3 showed multifold higher inhibition of all of the tested enzymes. Enzyme kinetics revealed different modes of inhibition, and in silico molecular docking simulation demonstrated the importance of the 7–OH group and bromine number for H-bond and halogen-bond interactions, respectively. Similarly, 1 – 3 at 20 μM concentration showed more than 50% inhibition of self-induced Aβ 25–35 aggregation. These results suggest that bromophenols from S. latiuscula , especially highly brominated ( 3 ), may represent a novel class of anti-Alzheimer’s disease drugs.

Cholinesterase, β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and glycogen synthase kinase-3β (GSK-3β) are the three main enzymes responsible for the early onset of Alzheimer’s disease (AD). The main aim of the present study was to delineate and accentuate the triple-inhibitory potential of arylbenzofurans from Morus alba against these enzymes. Overall, the enzyme inhibition assays demonstrated the prominence of mulberrofuran D2 as an inhibitor of AChE, BChE, BACE1, and GSK-3β enzymes with IC 50 values of 4.61, 1.51, 0.73, and 6.36 μM, respectively. Enzyme kinetics revealed different modes of inhibition, and in silico modeling suggested that mulberrofuran D2 inhibited these enzymes with low binding energy through hydrophilic, hydrophobic, and π–cation interactions in the active site cavities. Similarly, in Aβ-aggregation assays, mulberrofuran D2 inhibited self-induced and AChE-induced Aβ aggregation in a concentration-dependent manner that was superior to reference drugs. These results suggest that arylbenzofurans from M. alba , especially mulberrofuran D2, are triple inhibitors of cholinesterase, BACE1, and GSK-3β and may represent a novel class of anti-AD drugs.

Kuwanon G (KG) and benzofuran flavonoids such as mulberrofuran G (MG) and albanol B (AB) isolated from Morus sp. are reported to exhibit anti-Alzheimer’s disease, anti-inflammatory, fungicidal, anti-cancer, anti-bacterial, and anti-tyrosinase properties. We investigated the inhibition of mono- and diphenolase activity of mushroom tyrosinase by KG, MG, and AB. KG and MG displayed acceptable inhibition activity compared to kojic acid. AB did not show any activity up to 350 µM. MG displayed six-fold higher inhibition of l-tyrosine oxidation (IC50 = 6.35 ± 0.45 µM) compared to kojic acid (IC50 = 36.0 µM). Kinetic studies revealed that KG and MG inhibited monophenolase activity of tyrosinase in a competitive manner. Docking simulations of KG and MG demonstrated favorable binding energies with amino acid residues of the active sites of tyrosinase. Our investigation of the structure-activity relationship of the fused benzofuran flavonoids (MG vs. AB) implicated the methyl cyclohexene ring moiety in tyrosinase inhibition. The enzyme substrate and relative structural analyses demonstrated that KG and MG from Morus sp. could be useful natural tyrosinase inhibitors in foods or cosmetics.

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