In vitro effect of selected medicinal plants on β-amyloid induced toxicity in neuroblastoma cells

Abstract

Neurodegenerative diseases occur as a result of the breakdown and deterioration of the neurons of the central nervous system (CNS). They are commonly found in elderly people and are a major cause of morbidity and mortality, thereby imposing severe strains on the social welfare systems. Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Cholinergic deficit, senile plaque/amyloid-β peptide deposition and oxidative stress have been identified as three main pathogenic pathways which contribute to the progression of AD. The current therapeutic options cause several side-effects and have problems associated with bioavailability. Therefore, the need arises to search for new compounds from natural products with potential to treat AD. Seventeen plants were selected for this study based on their documented ethno-medicinal use in improving memory, to treat insomnia, calm agitated people, and other neurological disorders. The plants were screened for inhibition of acetylcholinesterase (AChE) using the TLC and microtiter plate method. A dose-dependent inhibition of the enzyme was observed and 4.5% of all the plants showed low (<30% inhibition) AChE inhibition. The ethyl acetate extracts of the roots of Crinum bulbispermum, Xysmalobium undulatum, Lannea schweinfurthii, Scadoxus puniceus and bulbs of Boophane disticha had the best AChE inhibition. Although the IC50 of these plant extracts were higher than that of the positive control, galanthamine (0.00053 mg/ml), they showed good AChE inhibitory activity considering they are still mixtures containing various compounds. The antioxidant activity of the plant extracts was determined by their ability to scavenge ABTS (2,2´-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) and DPPH (1,1-diphenyl-2-picryl- hydrazyl) radicals. The dichloromethane/methanol (1:1) extracts of Chamaecrista mimosoides (root), Buddleja salviifolia (whole plant), Schotia brachypetala (root and bark), water extracts of Chamaecrista mimosoides (root), Buddleja salviifolia (whole plant), Schotia brachypetala (root and bark) and methanol extracts of the roots of Crinum bulbispermum, Piper capense, Terminalia sericea, Lannea schweinfurthii and Ziziphus mucronata all showed good antioxidant activity (>50%), in both assays. B. disticha contained very promising AChE inhibition and was subjected to isolation of active compounds using thin layer chromatography, column chromatography and preparative thin layer chromatography. Two compounds, 6-hydroxycrinamine (a crinine-type alkaloid) and cycloeucalenol (a cycloartane triterpene), were isolated for the first time from the bulbs of this plant. 6-Hydroxycrinamine, and two fractions, EAM 17-21 21,22 and EAE 11 (which could not be purified further due to low yield), were found to inhibit AChE with IC50 values of 0.445 ± 0.030 mM, 0.067 ± 0.005 mg/ml and 0.122 ± 0.013 mg/ml, respectively. Cytotoxicity of the isolated compounds and two active fractions was determined on human neuroblastoma (SH-SY5Y) cells using the MTT and neutral red uptake assays. 6- hydroxycrinamine and fraction EAM 17-21 21,22 were found to be toxic with IC50 values of 54.5 μM and 21.5 μg/ml as determined by the MTT assay. The isolated compounds and fractions did not show any protective effect against cell death induced by Aβ25-35 possibly due to the poor antioxidant activity of B. disticha bulbs. Cytotoxicity was also determined for the methanol extracts of the roots of C. bulbispermum, T. sericea, L. schweinfurthii and Z. mucronata, as they contained promising antioxidant activity. C. bulbispermum was the most toxic, reducing cell viability by <40% at the highest concentration tested. Z. mucronata and L. schweinfurthii were the least toxic with IC50 values exceeding 100 μg/ml, the highest concentration tested. Three concentrations of the plant extracts that were not toxic, or presented low toxicity, were selected to evaluate their possible protective effect against cell death induced by Aβ25-35. Pretreatment with Z. mucronata and T. sericea roots showed a dose dependent inhibition of cell death caused by Aβ25-35. Pre-treatment with L. schweinfurthii roots resulted in an optimum dose for inhibition of Aβ25-35 induced cell death at 25 μg/ml, while still maintaining 80% viability. The roots of C. bulbispermum at non-toxic dose still maintained >50% viability. This study confirms the neuroprotective potential of some of the plants which had AChE inhibitory and antioxidant activity. In addition, four of the plants were shown to prevent cell death caused by Aβ25-35. These plants can serve as potential leads in developing drugs relevant to treatment of AD. Furthermore, two new compounds present in the bulbs of B. disticha were identified. Additional investigations need to be carried out by applying QSAR studies to modify the structure of the alkaloid with the aim of reducing its observed toxicity.