2018 Imcp
2018 Imcp
Page 1 of 5
Investigational
Medicinal Chemistry & Pharmacology
Abstract
Background: The role of nitric oxide in the pathogenesis and progression of neurodegenerative illnesses is
becoming prominent over the years and the inhibition of acetylcholinesterase (AChE) is considered as a promising
strategy in the management of such diseases. This study aims to assess the inhibitory potential of (1) beauvericin,
(2) para-hydroxybenzaldehyde, (3) indole-3-carboxylic acid and (4) quinizarin isolated from endophytic fungi
Epicoccum nigrum on acetylcholinesterase activity and on nitric oxide production in LPS-stimulated macrophages.
Methods: The inhibition of acetylcholinesterase activity was determined using Ellman’s colorimetric method. Nitric
oxide released from macrophages was determined by measuring the nitrite concentration in culture supernatant
using the Griess reagent and the cell viability was determined by MTT assay.
Results: The tested compounds exerted concentration-dependent activity against both AChE and NO production.
At the highest dose assessed (20 μg/mL) compounds 1 and 4 produced the smallest amount of NO (0.25 μM and
0.5 μM respectively) and their cell viability varied from 88.49% to 93.04%. Compound 1exhibited the most potent
anti-AChE activity (IC50 = 13.49 μg/mL) which however was lower compare to the reference compound galantamine
(IC50 = 8.22 μg/mL), while compound 2 and 3 showed moderate activity.
Conclusions: This study revealed that beauvericin is endowed with a potential against AChE activity and NO
production. Taking into account the respective role of NO and AChE in the occurrence and the prevention of
neurodegenerative diseases, our findings indicate thatbeauvericincan be considered as a good drug candidate that
could be further developed for the prevention of neurodegenerative disorders such as Alzheimer's disease.
Keywords: Acetylcholinesterase, Nitric oxide, Natural compounds, Endophytic fungi, Epicoccum nigrum
*Correspondence: Email: [email protected]; Orcid: https://orcid.org/0000-0003-3568-6711 (Prof. Dr. Jean Paul Dzoyem)
1
Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, Yaoundé, Cameroon; 2Department of Chemistry, Higher
Teachers' Training College, University of Yaoundé I, Yaoundé, Cameroon; 3Phytomedicine Programme, Department of Paraclinical Sciences,
Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa; 4Department of Biochemistry, Faculty of Science, University of
Dschang, Dschang, Cameroon
Citation on this article: Melong R, Dzoyem JP, Tsamo AT, Kapche DGWF, Ngadjui BT, McGaw LJ, Eloff JN. Investigational Medicinal Chemistry
and Pharmacology (2018) 1(2):18; Doi: https://dx.doi.org/10.31183/imcp.2018.00018.
Invest. Med. Chem. Pharmacol. (IMCP) ISSN: 2617-0019 (Print)/ 2617-0027 (Online); © The
Author(s). 2018 Open Access This article is available at https://investchempharm.com/
Melong et al. Investigational Medicinal Chemistry and Pharmacology 2018 1(2):18. Page 2 of 5
an endophytic fungus Epicoccum nigrum associated abnormalities in activities of daily life. Excessive NO
with Entada abyssinica. Their isolation procedure and production has been identified as one of the major
structure elucidation were previously described [10]. causative reasons for the pathogenesis of several
neurodegenerative diseases. Moreover, excessive
Nitric oxide inhibitory activity and viability of LPS- NO synthesis under neuroinflammation leads to the
activated RAW 264.7 macrophages formation of reactive nitrogen species and neuronal
cell death [12].As for now, the use of cholinesterase
The RAW 264.7 macrophages cells were seeded in inhibitors to reverse the cholinergic deficit is the
96 well-microtitre plates and were activated by symptomatic treatment. However NO inhibitors
incubation in medium containing 1 µg/mL LPS alone should also be considered in the therapy of such
(control) or lipopolysaccharide with different diseases. Therefore, the search for compounds with
concentrations of the samples dissolved in DMSO. dual activity that could both inhibit excessive NO
Quercetin served as a positive control NO inhibitor for production and AChE activity will be more useful in
the reduction of NO production. The amount of nitric focused neurodegenerative diseases therapies. In
oxide released from macrophages as well as the cell this study, the NO production and the AChE inhibitory
viability were determined as previously described activity of four natural compounds were evaluated.
[11].
Nitric oxide inhibitory activity
Acetylcholinesterase inhibition activity
To evaluate the ability of compounds to inhibit NO
Inhibition of acetylcholinesterase activity was production in activated RAW 264.7 macrophage cell
determined using Ellman’s colorimetric method as lines, the amount of nitrite released in the culture
previously described [11].Then, for the samples with supernatant was evaluated 24hours after the
at least 50% inhibition, the IC50 value was calculated treatment of cells with compounds. Results are
by plotting the percentage of inhibition against the shown in Figure 2. The treatment of RAW264.7 cells
concentrations. with LPS resulted in a significant increment of nitrite
concentration in the medium (control). All the four
Statistical analysis compounds showed various extent of inhibition of NO
The results were presented as means of three production activity. In general, a decline in the
experiments. Values were expressed as mean ± amount of nitrite produced was observed when the
standard deviation. Statistical analysis was performed concentration of the tested compounds increased. At
with GraphPadInStat Software and results were the highest dose assessed (20 μg/mL) compound
compared using the Fisher's least significant 1and 4 produced the smallest amount of NO (0.25
difference (LSD) at a 5% significance level. μM and 0.5 μM respectively) and the cell viability
varied from 88.49% to 93.04%. A the lowest
concentration tested (0.5 μg/mL), compound 1
showed the highest NO inhibitory activity with 3.92 of
CHO
NO released. However, unlike the highest
HO
concentration, at the lowest concentration, the activity
O
N
O
2 of compound 1 was found to be more potent than the
O
O OH positive control quercetin which showed 5.01 μM of
O O NO released. The overall cell viability was more than
N COOH 80%, therefore consistent with the fact that the
N
O
observed NO inhibition was not related to the cell
O O
N O OH toxicity. Compound 1 (beauvericin) is a cyclic
1 3 4 hexadepsipeptide, which was previously shown to
possess biological properties including antimicrobial,
Figure 1. Chemical structures of the tested antiviral and cytotoxic activities [13, 10].Plant
compounds. secondary metabolites generally produced for their
1: Beauvericin, 2: para-hydroxybenzaldehyde, 3: defense mechanisms have been implicated in the
indole-3-carboxylic acid, 4: quinizarin. therapeutic properties of most medicinal plants.
Plants, therefore, provide an invaluable useful
resource as leads in the development of therapeutic
compounds [14].Many compounds from medicinal
Results and discussion plants have been demonstrated as inhibitors of NO
production in LPS-activated macrophages. Their
Neurodegenerative disorders especially Alzheimer's structures can be categorized as flavonoids,
disease (AD) are associated with memory loss, alkaloids, sesquiterpene, polyacetylenes, and lignans
cognitive dysfunction, behavioural turbulence and [15].
Melong et al. Investigational Medicinal Chemistry and Pharmacology 2018 1(2):18. Page 4 of 5
8,00
7,00
Table 1: Acetylcholinesterase inhibitory effects of
6,00 compounds 1, 2, 3, 4 and galantamine.
5,00
4,00 Concentration % AChE IC50
3,00 Compounds (µg/mL) inhibition (µg/mL)
2,00
1,00 50 56.33±1.74
a
1 25 30.00±6.56 13.49±1.74
0,00
0,5 2 5 20 12.5 29.33±9.24
A 6.25 25.33±2.52
Concentration (µg/mL)
50 75.00±9.64
120,00 1 2 3 4 Quercetin 2 25 64.33±6.35 17.50±1.74
b
12.5 38.00±8.72
Percentage of cell viability
Conclusions
AChE inhibitory activity
The compounds were tested for AChE inhibitory In this study, natural compounds isolated from
activity by Ellman’s colorimetric method in 96-welled endophytic fungi Epicoccum nigrum inhibited the
microplate. The results are shown in Table 1 activity of AChE and the NO production in LPS-
representing the % inhibition at different stimulated RAW264.7macrophage cells. To our
concentration tested and IC50 value for those who had knowledge, the activities of compounds investigated
more than 50% inhibition. Galantamine was used as herein are reported for the first time. From this study,
the standard AChE inhibitor in this study which new insights on the beneficial therapeutic potential of
showed IC50 of 8.22 µg/mL.The AChE inhibitory beauvericin especially towards neurodegenerative
activity was highest with compound 1 followed diseases are provided.
by compounds 2 and 3 with IC50 values of 13.49
µg/mL, 17.50 µg/mL, and 23.25 µg/mL respectively.
Compound 4 showed weak activity and gave less Abbreviations
than 50% of AChE inhibition. Many plant-derived
products are either in the clinical trial phase or AChE: acetylcholinesterase
currently used in treatment of several ailments AChEI: acetylcholinesterase inhibitor
AD: Alzheimer's disease
including Alzheimer’s disease[16].Previous studies LPS: lipopolysaccharide
reported that cholinesterase inhibitors could act on NO: nitric oxide
multiple therapeutic targets such as prevention of the
antioxidant activity and the formation of β-amyloid Authors’ Contribution
plaques. However, there is still a need for new AChE
inhibitor lead compounds with lower toxicity and JPD carried out this study and designed the experiments. RM and
ATT isolated the compounds and performed the chemical analysis.
higher central nervous system (CNS) penetration. To JPD and RM wrote the manuscript. DGWFK and BTG contributed
this end, many plants have been studied by bioassay- to chemical structural elucidation. LJM and JNE supervised the
guided approaches for the identification of new AChE work. All authors read and approved the final manuscript.
Melong et al. Investigational Medicinal Chemistry and Pharmacology 2018 1(2):18. Page 5 of 5