Coconut (Cocos nucifera) Ethanolic Leaf Extract Reduces Amyloid-β (1-42) Aggregation and Paralysis Prevalence in Transgenic Caenorhabditis elegans Independently of Free Radical Scavenging and Acetylcholinesterase Inhibition

Abstract

Virgin coconut oil (VCO) has been the subject of several studies which have aimed to alleviate Alzheimer's disease (AD) pathology, focusing on in vitro antioxidant and acetylcholinesterase (AChE) inhibitory activities. Here, we studied an underutilized and lesser-valued part of the coconut tree, specifically the leaves, using in vitro and in vivo approaches. Coconut leaf extract (CLE) was screened for antioxidant and AChE inhibitory properties in vitro and therapeutic effects in two strains of transgenic Caenorhabditis elegans expressing amyloid-β_1-42 (Aβ_1-42) in muscle cells. CLE demonstrated free radical scavenging activity with an EC₅₀ that is 79-fold less compared to ascorbic acid, and an AChE inhibitory activity that is 131-fold less compared to Rivastigmine. Surprisingly, in spite of its low antioxidant activity and AChE inhibition, CLE reduced Aβ deposits by 30.31% in CL2006 in a dose-independent manner, and reduced the percentage of paralyzed nematodes at the lowest concentration of CLE (159.38 μg/mL), compared to dH₂O/vehicle (control). Phytochemical analysis detected glycosides, anthocyanins, and hydrolyzable tannins in CLE, some of which are known to be anti-amyloidogenic. Taken together, these findings suggest that CLE metabolites alternatively decrease AB_1-42 aggregation and paralysis prevalence independently of free radical scavenging and AChE inhibition, and this warrants further investigation on the bioactive compounds of CLE.

Keywords: Alzheimer’s disease (AD); Caenorhabditis elegans; Cocos nucifera; coconut leaf extract; sporadic inclusion body myositis (sIBM).

Figure 1

2,2-diphenyl-1-picryl-hydrazyl (DPPH) scavenging and acetylcholinesterase (AChE) inhibition by ethanolic coconut (Cocos nucifera) leaf extract in vitro. The CLE was tested at varying concentrations in vitro for antioxidant activity and the ability to inhibit standard AChE. (A) DPPH scavenging effect of CLE compared with the control (ascorbic acid). At 100 μg/mL, CLE neutralized DPPH radicals by 93.85% (n = 3). *** p < 0.0001 when compared with the control at the same concentration using a two-tailed t-test. p < 0.05 was used to compare treatment groups 4 to 6 (100–1000 μg/mL) using one-way ANOVA; (B) Dose-response curves comparing the AChE inhibitory activity of CLE and positive control (Rivastigmine) in vitro.

Figure 2

CLE significantly reduces amyloid-β plaque deposits in transgenic C. elegans strains CL2006, independently of concentration. Nematodes were transferred to OP50-incubated NGM plates and allowed to mature for three days at 20 °C, after which they were fed with either vehicle or CLE at varying concentrations ad libitum. The amyloid-β deposits in CL2006 were then counted five days post-treatment, and were pooled in triplicate. Dark portions in the cell wall of C. elegans indicate the protein deposits. CL2006 strains were fed with either (A) vehicle or (C) CLE. Detailed images of the body walls of nematodes fed with (B) vehicle or (D) CLE are shown. White circles identify the aggregate deposits; (E) Aβ deposits were counted per treatment group at varying concentrations of CLE (in μg/mL). *** p < 0.0001 when compared with vehicle using two-tailed t-test. p < 0.05 was used to test for significance between treatment groups using one-way ANOVA.

Figure 3

CLE reduces paralysis prevalence in C. elegans strain CL4176. Nematodes were exposed to (A) vehicle or (C) CLE in concentrations similar to the Aβ aggregation assay in vivo. Aβ expression was induced at 25 °C after 36 h post-treatment of CLE or vehicle at 16 °C for 36 h. Detailed images of the body walls of nematodes fed with (B) vehicle or (D) CLE are shown. White circles identify the aggregate deposits; (E) The proportion of paralyzed nematodes were gathered at 2, 12, 24, and 36 h post-induction. To determine the link of aggregation in paralysis, the aggregation assay was done in CL4176 using the paralysis assay conditions.

Figure 4

Model for Aβ aggregation and paralysis reduction by CLE in transgenic C. elegans. CLE acts by inhibiting reactive oxygen species (ROS) and AChE activities in C. elegans, albeit at high concentrations—both of which would otherwise worsen Aβ-induced pathology. However, at high concentrations, a pro-oxidant effect is expected due to antioxidant excess, resulting to CLE-induced ROS production (broken arrow). At concentrations 9 to 350 times higher than the EC₅₀ of CLE, the effect on aggregation was dose-independent, as shown in Figure 2. Therefore, it is possible that CLE is acting directly through compounds that are anti-aggregatory, which masks the pro-oxidant activity of antioxidants in CLE at higher extract concentrations (red “?”).