Identification of Polyphenolics from Loranthus globosus as Potential Inhibitors of Cholinesterase and Oxidative Stress for Alzheimer's Disease Treatment

Abstract

Mistletoes are considered to be the potential medicinal herbs due to their rich traditional uses. Loranthus globosus is a Bangladeshi mango mistletoe that has been reported as folk medicine for various ailments and diseases. In an attempt to explore its effectiveness in Alzheimer's disease (AD), we investigated the antioxidant and acetylcholinesterase inhibitory activity of L. globosus. We report that the crude methanol extract (CME) of the plant contains a good amount of polyphenolics and possesses antioxidant and cholinesterase inhibitory activity. Fractionation of CME with solvents of varying polarity revealed the highest activity and polyphenolic content in the ethylacetate fraction (EAF). Correlation analysis revealed a significant (P < 0.05) association of polyphenolics with the antioxidant and cholinesterase inhibitory properties. Using column chromatography with diaion resin, the polyphenolics (EAF-PP) were isolated from the EAF that displayed the potent antioxidant and cholinesterase inhibitory activities. Kinetic analysis showed that EAF-PP exhibited a competitive type of inhibition. A total of thirty-six compounds including catechin and its different derivatives were identified in the EAF-PP by LC/MS analysis. Bioactivity-guided separation approach afforded the isolation of the two major active compounds catechin and catechin dimer from the EAF-PP. Hence, EAF-PP represents a potential source of antioxidants and cholinesterase inhibitors, which can be used in the management of AD.

Figure 1

Antioxidant activities of the extract and fractions from L. globosus. (a) DPPH radical scavenging activity. IC₅₀ (μg/ml): CME, 4.156 ± 0.088; PEF, 11.223 ± 0.248; CLF, 24.617 ± 0.421; EAF, 3.130 ± 0.022; AQF, 7.975 ± 0.225; CAT, 3.41 ± 0.004. (b) Hydroxyl radical scavenging activity. IC₅₀ (μg/ml): CME, 15.60 ± 0.356; PEF, 26.617 ± 0.293; CLF, 31.697 ± 0.570; EAF, 12.623 ± 0.268; AQF, 22.687 ± 0.389; CAT, 11.333 ± 0.356. (c) Reducing power. At 100 μg/ml concentration, the absorbances are as follows: CME, 1.874 ± 0.014; PEF, 1.624 ± 0.036; CLF, 1.117 ± 0.116; EAF, 2.457 ± 0.034; AQF, 1.634 ± 0.006; CAT, 2.660 ± 0.032. (d) Total antioxidant capacity. At 100 μg/ml concentration, the absorbances are as follows: CME, 2.039 ± 0.129; PEF, 1.326 ± 0.009; CLF, 0.954 ± 0.025; EAF, 2.688 ± 0.008; AQF, 1.578 ± 0.098; CAT, 2.251 ± 0.151. Results are expressed as mean ± SD (n = 3). Means with different letters (a-f) differ significantly (P < 0.05). CME: crude methanolic extract; PEF: petroleum ether fraction; CLF: chloroform fraction; EAF: ethylacetate fraction; AQF: aqueous fraction; CAT: catechin.

Figure 2

Lipid peroxidation inhibitory activity of the extract and fractions from L. globosus. IC₅₀ (μg/ml): CME, 56.073 ± 1.176; PEF, 66.003 ± 1.754; CLF, 85.863 ± 0.246; EAF, 25.997 ± 0.246; AQF, 38.087 ± 0.417; CAT, 16.510 ± 0.123. Results are expressed as mean ± SD (n = 3). Means with different letters (a-f) differ significantly (P < 0.05). CME: crude methanolic extract; PEF: petroleum ether fraction; CLF: chloroform fraction; EAF: ethylacetate fraction; AQF: aqueous fraction; CAT: catechin.

Figure 3

Cholinesterase inhibitory activities of the extract and fractions from L. globosus. (a) Inhibition of AChE. IC₅₀ (μg/ml): CME, 153.767 ± 2.409; PEF, 123.367 ± 0.306; CLF, 171.533 ± 5.478; EAF, 64.987 ± 0.669; AQF, 87.417 ± 0.610; DON, 8.351 ± 0.076. (b) Inhibition of BChE. IC₅₀ (μg/ml): CME, 155.733 ± 0.907; PEF, 353.633 ± 3.408; CLF, 391.633 ± 4.561; EAF, 85.270 ± 0.982; AQF, 129.267 ± 1.002; GAL, 8.208 ± 0.105. Results are expressed as mean ± SD (n = 3). Means with different letters (a-f) differ significantly (P < 0.05). CME: crude methanolic extract; PEF: petroleum ether fraction; CLF: chloroform fraction; EAF: ethylacetate fraction; AQF: aqueous fraction; AChE: acetylcholinesterase; BChE: butyrylcholinesterase; DON: donepezil; GAL: galantamine.

Figure 4

Quantitative analysis of the EAF-PP and AQF-PP and assessment of their activities. (a) Total phenolic, total flavonoid, and total proanthocyanidin contents. (b) DPPH scavenging activity. (c) Hydroxyl scavenging activity. (d) AChE inhibitory activity. (e) BChE inhibitory activity. Activities were expressed as IC₅₀. Data represent as mean ± SD (n = 3). Means with different letters (a-f) differ significantly (P < 0.05). EAF: ethylacetate fraction; AQF: aqueous fraction; CAT: catechin; TPC: total phenolic content; TFC: total flavonoid content; TPAC: total proanthocyanidin content; GAE: gallic acid equivalent; CE: catechin equivalent; AChE: acetylcholinesterase; BChE: butyrylcholinesterase; DON: donepezil; GAL: galantamine; EAF-PP: polyphenols from EAF; AQF-PP: polyphenols from AQF.

Figure 5

Lineweaver-Burk plot for inhibition of (a) AChE and (b) BChE by different concentrations of EAF-PP. Results represent the average values (n = 3). AChE: acetylcholinesterase; BChE: butyrylcholinesterase.

Figure 6

Characterization of the compounds isolated from the polyphenolic EAF-PP. (a) TLC profile of the compound 1 and authentic catechin. (b) ¹H NMR spectroscopic data of the compound 2. (c) ¹³C NMR spectroscopic of the compound 2. (d) Chemical structures of the compounds 1 and 2.

Figure 7

Antioxidant and cholinesterase inhibitory activities of the compounds 1 and 2. IC₅₀ values of the compounds for DPPH radical scavenging and inhibition of AChE and BChE were shown. Results are expressed as mean ± SD (n = 3). Means with different letters (a-c) differ significantly (P < 0.05). DPPH: 2,2-diphenyl-1-picrylhydrazyl; AChE: acetylcholinesterase; BChE: butyrylcholinesterase.