Anticholinesterases and antioxidant alkamides from Piper nigrum fruits

Abstract The anticholinesterase and antioxidant effects of five different extracts of Piper nigrum were evaluated. Twenty-one known alkamides were isolated from active ethyl acetate extract and investigated for their cholinesterase inhibitory and antioxidant effects. Among them, piperine (2), piperettine (5) and piperettyline (20) exhibited dual inhibition against AChE and BChE, and feruperine (18) was the most potent selective inhibitor of BChE. Molecular docking simulation was performed to get insight into the binding interactions of the ligands and enzymes. In addition, N-trans-feruloyltyramine (3) contributed to the strongest DPPH radical-scavenging activity. The self-induced Aβ aggregation inhibition of 2, 5 and 18 was further evaluated. Results indicated that some alkamides could be multifunctional lead candidates for Alzheimer’s disease therapy. Graphical abstract

transmission and promoting the aggregation of β-amyloid peptide fragments (Alvarez et al. 1997) as well as the compensation of BChE, selective cholinesterase inhibitors (ChEIs) or dual-action AChE-BChE inhibitors present a therapeutic strategy to slow down the progression of AD. Furthermore, the amyloid hypothesis declares that the production and accumulation of oligomeric aggregates of Aβ are a central event in the pathogenesis of AD. Therefore, the prevention of Aβ aggregation has currently attracted considerable attention in the treatment of AD (Glabe 2006). In addition, oxidative stress has been defined as the principal pathological cause of neurodegeneration. Thus, antioxidants are proposed as therapeutic options to combat free radical generation and maintenance (Praticò 2008). A multi-targeted therapeutic approach, which includes ChE inhibitors in combination with antioxidants, Aβ 1-42 peptide aggregation inhibitors, may be a more rational approach to dementia treatment (Williams et al. 2011).
The family Piperaceae is composed of four major genera and nearly 4000 species, and they are reputed in various systems of medicine for their numerous medicinal properties, such as the Indian Ayurvedic system of medicine and folklore medicine of Latin America (Bagatela et al. 2013;Lemos et al. 2013;de Queiroz et al. 2014;Sarwar et al. 2015). Black pepper is the dried ripe fruit of Piper nigrum L., previous studies have revealed its chemical compositions (amide alkaloids, terpenoids, multimeric alkaloids and volatile oil) and activities (immunomodulatory, stimulatory, larvicidal, anti-inflammatory, antioxidant, antifertility, antiulcer, antifungal, antibacterial and anticholinesterase activities) (Ingkaninan et al. 2003;Meghwal & Goswami 2013;Santiago et al. 2015). During our search for ChEIs from natural resources, 95% ethanol extract of P. nigrum showed promising dual AChE-BChE inhibition activity ( Figure S1). This intriguing observation somehow corroborates the use of pepper as an antiepileptic, antianxiety, sedative and sleep-inducing herbal preparations in the Asian folk medicine. However, no systematic investigations have attempted to identify the anti-ChE compounds from P. nigrum. Taking this into consideration, this study aimed to isolate and investigate the constituent(s) responsible for the observed ChE inhibition, antioxidant capacity, inhibitory effect on Aβ 1-42 peptide aggregation, and their interactions with the enzymes by molecular docking simulation.
Compared with 2, 8-11 featuring no methylenedioxyphenyl (MDP) ring presented no inhibitory activity against both enzymes which suggested that the MDP ring is an essential feature in ChE inhibitory activity. Weak activities of 15-17 with MDP ring showed that overlong carbon chain may prevent inhibitors from fully entering the active site of ChE, which diminished the inhibition potency by reducing the enzyme affinity. In contrast, 7 has similar chain length with 15 and similar % inhibition for AChE to 2, but no inhibition for BChE, which showed the replacement of (2-methylpropyl) amido group to a piperidine ring would dramatically affect the anti-BChE activity. More active BChE inhibition and higher selectivity (BChE IC 50 = 12.88 μg/mL; AChE IC 50 > 200 μg/mL) of 18 implied that the hydroxyl on benzyl ring was favourable for the BChE inhibition and selectivity. The anti-ChE activities of 5 were 6 times (AChE) and 1.5 times (BChE) higher than that of 2, which indicated that three conjugated double bonds would benefit the inhibition.
The antioxidant potential of extracts and alkamides were determined via the DPPH radical-scavenging assay (Table S1). The best DPPH radical-scavenging potency (EC 50 = 11.82 μg/mL) was provided by 3, which is closer to that of ascorbic acid. Compound 18, a selective BChE inhibitor, exhibited moderate antioxidant activity (42.84% at 200 μg/mL). By contrast, the most active ChE inhibitors (5 and 20) showed low antioxidant activity (24.14 and 29.68% at 200 μg/mL, respectively). Although 2 indicated a low rate of inhibition in this work, its protection against oxidative damage by quenching superoxides (IC 50 = 1.82 mmol/L) and lipid peroxidation inhibition (IC 50 = 1.23 mmol/L) has been previously demonstrated (Mittal & Gupta 2000).
Molecular docking study was performed to evaluate the binding interaction of 5, 18 and 20 at the active site of enzymes ( Figure S3, Table S2). Inhibitor 5 docked deep into the bottom of active gorge of TcAChE, the oxygen atom of the MDP ring displayed hydrogen bonding with Ser 200 and His 440 of catalytic site, respectively. A hydrogen bonding between the oxygen atom of C-1 and Phe 288 of acyl pocket was observed as well. Inhibitor 5 was also deeply buried inside the hBChE active side gorge, two hydrogen bonds were formed between the oxygen atom of C-1 and Ser 198 and His 438 (catalytic triad residues), and a π-π interaction was formed between Tyr 332 and aromatic ring. The binding result of 5 was in good agreement with its dual AChE-BChE inhibition role in vitro. The interaction of 20 at the active site of AChE was similar to 5. Then, 18 displayed a strong interaction with hBChE and two hydrogen bonds were formed with Ser 198 and His 438 at catalytic triad site ( Figure  S3C). Additionally, a hydrogen bonding was formed between hydroxyl group of aromatic ring and Tyr 128 with a distance of 1.75 Å. It might support the experimental result that 18 was acting as a potent BChE inhibitor.

Conclusion
The present study showed the ChE inhibitory activity of 5 crude extracts and 21 alkamides isolated from black pepper. Compounds 5 and 20 exhibited dual inhibition against AChE and BChE. Meanwhile, 18 acted as the most potent selective BChE inhibitor, its antioxidant activity and inhibition on Aβ self-induced aggregation indicated that it could be multifunctional lead candidate for AD therapy. Its C log P value was 2.53, which implied its good capacity to pass through the blood-brain barrier. Our study indicated that P. nigrum could be used in the prevention of dementia as a food supplement or spice.

Disclosure statement
No potential conflict of interest was reported by the authors.