In vitro and in silico study of Cassia seeds-derived secondary metabolites on human monoamine oxidase and G-protein coupled receptors

Abstract

Cassia obtusifolia L. seed is one of the most popular traditional Chinese medicine for mutagenicity, genotoxicity, hepatotoxicity, and acute inflammatory diseases. Recently, its neuroprotective activity has been reported mainly via antioxidant mechanisms. There are no reports of neuroprotection via human monoamine oxidase (hMAO) inhibition and G protein-coupled receptors (GPCRs) modulation. The main objective of this study is to delineate and accentuate the neuroprotective effect of Cassia seeds‒derived secondary metabolites via in vitro and in silico hMAO inhibition and GPCRs modulation. The seed extract exhibited good inhibition of hMAO-A isozyme with an IC50 value of 86.89 ± 3.80 μg/mL. Among the solvent‒soluble fractions, EtOAc fraction (IC50: 20.82 ± 5.04 μg/mL) was the most potent inhibitor of hMAO-A isozyme followed by CH2Cl2 (IC50: 40.06 ± 3.58 μg/mL) and n-BuOH fraction (IC50: 93.49 ± 0.27 μg/mL). In isozyme‒B, inhibition by seed extract and solvent‒fraction was mild with an IC50 value range of 56.28 ‒ 246.50 μg/mL. Bioassay-guided isolation led to thirty‒six known secondary metabolites along with two new naphthalenic lactone glycosides, (3R)-cassialactone 9-O-β-D-glucopyranoside (37) and (3S)-9,10-dihydroxy-7-methoxy-3-methyl-1-oxo-3,4-dihydro-1H-benzo[g]isochromene-3-carboxylic acid 9-O-β-D-glucopyranoside (38). Almost all metabolites showed selective inhibition of hMAO-A isozyme. In particular, aglycons obtusin (3), alaternin (8), aloe-emodin (9), emodin (10), questin (12), and rubrofusarin (13) and glycosides cassiasid (15), toralactone gentiobioside (26), and 38 showed promising inhibition of hMAO-A isozyme with IC50 values 0.17 to 11 μM. Compounds 8 and 12 were active against hMAO-B with IC50 values 4.55 ± 0.09 and 10.58 ± 0.28 μM, respectively. The enzyme kinetic study revealed the mode of enzyme inhibition, and an in silico molecular docking simulation predicted that Ile335, Tyr407, Phe208, and Ile180 are the determinant interacting residues for hMAO-A selectivity. Furthermore, the effect of MAO‒active major anthraquinones 8, 9, 10 and 12 on various GPCRs (hD1R, hD3R, hD4R, h5-HT1AR, and hV1AR) modulation was evaluated via cell-based functional assays. Among them, 10 and 8 showed a concentration-dependent agonist effect on hD3R and antagonist effect on hV1AR. The half-maximal effective concentration (EC50) of 10 and 8 for hD3R was 21.85 ± 2.66 and 56.85 ± 4.59 μM, respectively. The reference agonist dopamine had the EC50 value of 4 nM. Compound 12 showed moderate hD3R agonist effect with 34% of control agonist response at 50 uM, and 9 showed mild hD3R antagonist effect. On hV1AR, 10 and 8 showed potent antagonist effect with IC50 values of 10.25 ± 1.97 and 11.51 ± 1.08 μM, respectively. Interestingly, 9 and 12 did not have any observable effect on hV1AR. Only 8 was effective in antagonizing h5-HT1AR (IC50: 84.23 ± 4.12 μM). The results of computational study revealed that ‒ hydroxyl group at C1, C3, and C8 and a methyl group at C6 of anthraquinone structure are essential for hD3R agonist and hV1AR antagonist effect; the H‒bond interaction of 1‒OH group with Ser192 at a proximity of 2.0 Å which 8 lacked role for the higher stability of 10‒hD3R complex and higher potency of 10; and interaction of the 7‒OH group of 8 with Trp358 is a determinant for the observed antagonist effect of 8 on h5‒HT1AR. Overall, this study characterizes anthraquinones, especially alaternin (8) and emodin (10) as principal components in the management of neurodegenerative disorders, particularly anxiety and depression, and supports Cassia seeds as a functional food. However, investigating in vivo effects of these compounds in receptor knock-down, knock-out and/or knock-in mice models deserve particular attention shortly.