Alzheimer’s disease (AD) is a neurological disorder characterized by cognitive impairments and synaptic dysfunctions affecting mostly the elderlies

(Bagyinzsky et al. 2017). The progression of AD is correlated to mitochondrial dysfunction, oxidative stress, protein misfolding, abnormal amyloid-beta

(A) deposition, alterations in calcium homeostasis, and neuroinflammation (Taylor et al. 2016). Currently, there are only five drugs approved to

minimize the progression of symptoms related to AD. These are natural product-based compounds which include the acetyl cholinesterase inhibitors

galantamine, revastigmine, donepezil, and tacrine, and the NMDA receptor antagonist memantine (Huang et al. 2019). These drugs only provide

symptomatic relief to the symptoms of AD but do not support much effectiveness in disease progression and prevention. Hence, the challenge of

finding an alternative treatment or therapy from natural products is warranted. The structural diversity of small molecules has contributed to the

researches in finding potential leads in drug discovery including neurodegenerative diseases. AD is a complex illness revolving in an interconnected

genetic, biochemical, and metabolic pathways (Reus et al. 2020). Hence, a molecule which targets more than one pathological hallmarks in AD is

envisioned as a potential possibility in designing and developing effective drugs against AD.

Isomitraphylline and mitraphylline are oxindole alkaloids which we previously isolated from the Philippine endemic Uncaria perrottetii (A. Rich.) Merr.

(Olivar et al. 2018), locally known as “sungay kalabaw”. These two alkaloids were abundant in the genus Uncaria as isomitraphylline was isolated from

18 Uncaria species, and mitraphylline could be isolated from 20 Uncaria species. Previous reports on these oxindole alkaloids demonstrated their

cytotoxic and antiproliferative effects in various cancer cell lines including their potential as anticancer agents (Yu et al. 2015).

Neuroprotective effects of the oxindole alkaloids isomitraphylline and mitraphylline in damaged human neuroblastoma SH-SY5Y cells

Mario A. Tan1,2 and Seong Soo A. An2

1 College of Science and Research Center for the Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines2 Bionano Research Institute, Gachon University, Seongnam-si, Republic of Korea

INTRODUCTION

As part of our research discovery in finding neuroprotective small molecules from nature and their potential mechanisms, we investigated the

cytotoxicity and protective potential in oxidative stress-induced human neuroblastoma SH-SY5Y cells of the isomeric oxindole alkaloids

isomitraphyline and mitraphylline. The neuroprotective effects of isomitraphylline and mitraphylline against amyloid aggregation were not

investigated previously.

OBJECTIVES

METHODOLOGY

Thioflavin T (ThT) Assay. The ThT assay was conducted using an optimized condition using a 384-well plate (Tan et al. 2020).

Cell Viability. SH-SY5Y cells at 2 x 104 cells/well in 96-well plate were incubated for 24 h. Cells were treated with the alkaloids for 24 h. The mediawere removed, cells were washed with PBS, fresh media were added, and incubated for 30 min. CellTiter-Glo luminescent reagent was added andthe luminescence was read on a multi-plate reader. The % cell viability was expressed relative to the control.

Neuroprotective activity assay. Neuroblastoma SH-SY5Y cells were seeded in 96-well plate at 2 x 104 cells/ well and incubated for 24 h. Afterstabilization, cells were pre-treated with the alkaloids for 6 h before incubation with A1-42 (5 M) or H2O2 (100 M). A solvent control (untreatedcontrol cells), A or H2O2 alone, and alkaloids alone treatments were also included. After incubation, the % cell viability were determined in triplicateexperiments.

Measurement of Reactive Oxygen Species (ROS). After 24 h stabilization, SH-SY5Y (2 x 104 cells/wells) cells were pre-treated with the alkaloids for 2 hbefore incubation with 100 M H2O2 for 4 h. After incubation, cells were treated with 25 M H2DCFDA and incubated for 2 h in the dark at 37 oC.Fluorescence intensity (Ex 495 nm, Em 520 nm) was measured in a microplate reader. The ROS level was calculated as percentage of the untreatedcontrol cells (100%) in triplicate trials.

Mitochondrial membrane potential (ΔΨm) assay. After 24 h SH-SY5Y (2 x 104 cells/well) acclimatization, cells were pre-treated with the alkaloids for2 h, and combined with 5 M A1-42 for 24 h. After treatment, 1 M TMRE staining solution was added and incubated at 37 oC for 30 min. Thefluorescence (Ex 549 nm, Em 575 nm) was read in a microplate reader. The ΔΨm was calculated as percentage of the untreated control cells (100%) intriplicate trials.

https://www.creative-bioarray.com/support/atp-cell-viability-assay.htm

RESULTS & DISCUSSION

The embedded figure is the structure of amyloid-beta 1-42.

ReferencesBagyinszky E et al. Journal of Neurological Sciences, 2017, 376, 242-254. Tan MA et al. 3 Biotech, 2020, 10, 424. Huang JM et al. Journal of Natural Products, 2019, 82, 1979–1988. Yu B et al. Europena Journal of Medicinal Chemistry, Olivar JE et al. Journal of King Saud University Science, 2018, 30, 283-285. 2015, 97, 673-698.Taylor JP et al. Nature, 2016, 539, 197–206.

% Inhibition of A1-42 aggregationa

Isomitraphylline (50 M) 61.32 ± 2.61*

Isomitraphylline (5 M) 21.63 ± 5.47

Mitraphylline (50 M) 63.27 ± 3.48*

Mitraphylline (5 M) 19.96 ± 3.85

Phenol Red (50 M) 67.31 ± 3.04b

a The values are expressed as mean ± SD of 3-trial experiment; b Used asthe positive control; * No significant difference with the positive controlat p < 0.05.

Both alkaloids showed a moderate inhibitory effect on the Aaggregation.

The abnormal aggregation of A peptides is one of the pathologicalcharacteristics of AD.

A aggregation leads to the formation of A oligomers, protofibrils, andinsoluble plaques which causes dysfunctions in mitochondria resulting inincreased oxidative stress and neuroinflammation.

The capability of the alkaloids to break the A aggregations based on theThT assay prompted the investigations of their neuroprotective effects inoxidative stress-induced neuroblastoma SH-SY5Y cells from A1-42 andH2O2.

The cell viability assay demonstrated the non-cytotoxic concentrations of thealkaloids to be <50M, hence, 20, 10, and 1 M were used for theneuroprotective experiments.

Thioflavin-T Assay

Fig 1. Neuroprotective effects of the alkaloids on A1-42 (Abeta)-

induced cytotoxicity using the ATP luminescence assay.

Both alkaloids showed protective effects at 10 M and 20 M

concentrations.

(*) - p < 0.05 with the A-treated only group.

Fig 2. Neuroprotective effects of the alkaloids on H2O2-induced

cytotoxicity using the ATP luminescence assay.

Both alkaloids showed protective effects at 20 M concentration.

(*) - p < 0.05 with the H2O2-treated only group.

Neuroprotective effects on oxidative stress-induced cytotoxicity, ROS generation and ΔΨm

Fig 3. Effect of the alkaloids on the intracellular Reactive Oxygen

Species (ROS) level.

Both alkaloids decreased the generation of the ROS in damaged

SH-SY5Y cells at 10 M and 20 M concentrations.

(*) - p < 0.05 with the H2O2-treated only group.

Fig 4. Effect of the alkaloids on the mitochondrial membrane

potential (ΔΨm).

Both alkaloids decreased the mitochondrial dysfunction in

damaged SH-SY5Y cells at 20 M concentration.

(*) - p < 0.05 with the A-treated only group.

CONCLUSION

This study has demonstrated the neuroprotective effects of isomitraphylline and mitraphylline for their therapeutic potentials against AD.

The inhibitions of A aggregation by both alkaloids were demonstrated by the ThT assay, and their neuroprotective effects on A-damaged

neuroblastoma SH-SY5Y cells also supported their potentials.

Reduction of oxidative stress was manifested from their protective effects on H2O2-induced SH-SY5Y cells by reducing the generation of the ROS

levels and mitochondrial dysfunctions.

Hence, these alkaloids could be further investigated and developed as potential therapeutic agents for the treatment of AD.

However, in vivo studies are required to fully explore their therapeutic capacity against AD.