GC-MS Identification of Cholinesterase Inhibitory and Antioxidant
Molecules from Leaves of Cnidoscolus aconitifolius (Miller) I.M. Johnston
(Euphorbiaceae)
Authors: Joel Onoja Ojogbane , Joachin Ugwueze Nnamdi 
Published: March 2022

Backgound:
Alzheimer’s disease (AD) is a progressive and fatal neurodegenerative disease,
clinically characterized by memory and cognitive dysfunction. AD affects about 35 million people
worldwide today and is estimated to nearly double every 20 years. Cnidoscolus aconitifolius (Miller)
I.M. Johnston has been reported in Nigerian ethnomedicine as a memory enhancer. There is a lack of
scientific evidence to justify the claims. Moreover, there are no effective neurotherapeutic agents available
for the treatment of AD; hence the need arises to search for new and more effective agents.

Objective:
This study aims to evaluate and identify potential molecules with anti-Alzheimer’s and antioxidant
potentials from Cnidoscolus aconitifolius leaves.

Methods:
The air-dried leaves of Cnidoscolus aconitifolius (Miller) I.M. Johnston (PCG/UNN/0267)
were extracted using the successive extraction procedure based on increasing the polarity of the eluent
in the ascending order of n-hexane, ethyl acetate, and methanol. Phytochemical screening was carried
out on the extracts using standard procedures. Acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE) inhibitory activities were done according to Ellman’s method. Eserine was used as standard.
Antioxidant potentials were evaluated using standard in vitro chemical analyses. A GC-MS (QP2010SE,
SHIDMAZU JAPAN) analysis was done to identify bioactive compounds from the most active fraction.
Statistical analyses were performed using one-way ANOVA followed by Dunnett’s Multiple Comparison
test at α0.05.

Results:
Phytochemical analysis revealed the presence of tannins, resins, saponins, flavonoids, phenols,
carbohydrates, alkaloids, and terpenoids. Ethyl acetate fraction demonstrated the highest acetylcholinesterase
and butyrylcholinesterase inhibitory activity at 1 mg/mL with IC50 values of 0.288 ± 0.00 mg/mL
(82.9% inhibition) and 0.440±0.02 mg/mL ((75.4% inhibition), respectively, compared to eserine
(IC50=0.050 ± 0.01 mg/mL) for AChE and (IC50=0.049 ± 0.00 mg/mL) for BuChE. Metal (ferrous ion)
chelating activity was also high in the ethyl acetate fraction with IC50 value of 0.160 ± 0.00 mg/mL
compared to EDTA (IC50 = 0.085 ± 0.00 mg/mL) at 1 mg/mL. Hydroxyl radical scavenging activity was
higher in the ethyl acetate fraction (IC50 = 0.352 ± 0.01 mg/mL) when compared to BHT (IC50 = 0.074 ±
0.00 mg/mL) at 1 mg/mL. The pro-anthocyanidin content was also higher in ethyl acetate (6.94 ± 0.16
mg cyanidin/g of sample) compared to other fractions. GC-MS analysis of the most active fraction
(ethyl acetate) revealed a total of 56 compounds. The major compounds revealed were: n-Hexadecanoic
acid (Area % of 13.45%; Retention time of 14.863), Phytol (Area % of 5.13%; Retention time of
15.864), Octadecanoic acid (Area % of 4.86%; Retention time of 16.211), 9, 12, 15-Octadecatrienoic
acid (Z,Z,Z) (Area % of 26.85%; Retention time of 16.09), Squalene (% Area of 2.65%; Retention time
of 20.94) and alpha-Tocopheryl acetate (% Area of 1.71%; Retention time of 23.40).

Conclusion:
C. aconitifolius has the potential to inhibit cholinesterase enzymes involved in the pathology
of Alzheimer’s disease. The molecules identified could serve as potential drug leads in managing
Alzheimer’s disease.