1Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 349983
Antioxidant and Acetylcholinesterase Inhibitor Potentials of the Stem Extract of Pternandra galeata
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1 Department of Pharmaceutical
Sciences, Faculty of Pharmacy,
Universitas Airlangga, Surabaya 60115,
East Java, Indonesia.
2 Center for Natural Product Medicine
Research and Development, Institute of
Tropical Diseases, Universitas Airlangga,
Surabaya, 60115, East Java, Indonesia.
3 Department of Chemistry, Faculty of
Mathematics and Natural Sciences,
Tanjungpura University, Pontianak
78124, West Kalimantan, Indonesia
*Corresponding
Suciati Suciati
suciati@ff.unair.ac.id
Received: 12 Jun 2022
Accepted: 01 October 2022
Published: 09 October 2022
Antioxidant and Acetylcholinesterase
Inhibitor Potentials of the Stem Extract of
Pternandra galeata
Suciati Suciati1,2* , Dwiki Nur Inayah 2 , Aty Widyawaruyanti 1,2 ,
Rudiyansyah Rudiyansyah3
ABSTRACT
Background: Pternandra galeata belongs to the family Melastomataceae. It is a native
flowering plant in Borneo Island that serve as food for monkey habitat. There has been limited
study on the medicinal and chemical properties of this plant. Objectives: We investigated
the acetylcholinesterase inhibitory activity and evaluated the antioxidant activity of the
ethanolic extract of Pternandra galeata stem. The total phenolic content in the sample was
also determined. Methods: The acetylcholinesterase inhibitory assays were performed using
Ellman’s method. Two different methods were used to evaluate the antioxidant activity of the
extract by 2,2-diphenyl-1-picryl hydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-
6-sulfonic acid) (ABTS) assays. The total phenolic content was determined by the Folin-
Ciocalteu method by employing gallic acid as a reference. Results: The ethanolic extract of
the P. galeata stems inhibited the AChE enzyme with an IC 50 value of 74.62 ± 0.89 μg/mL.
The sample exhibited antioxidant activity in the DPPH assay with an IC 50 value of 20.21 ± 0.08
μg/mL and 7.68 ± 0.09 μg/mL in the ABTS scavenging assay. The total phenolic content was
164.71 ± 3.33 mg GAE/g extract. Conclusion: The ethanolic extract of the P. galeata stem
can be a promising cholinesterase inhibitor and antioxidant for treating Alzheimer’s disease.
Keywords: Pternandra galeata, Alzheimer’s disease, Acetylcholinesterase inhibitor,
Antioxidant, Phenolic compound.
ORIGINAL RESEARCH
Published 09 October 2022
Doi: https://doi.org/10.17533/udea.vitae.v29n3a349983
Antioxidant and Acetylcholinesterase Inhibitor Potentials of the Stem Extract of Pternandra galeata
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1 Department of Pharmaceutical
Sciences, Faculty of Pharmacy,
Universitas Airlangga, Surabaya 60115,
East Java, Indonesia.
2 Center for Natural Product Medicine
Research and Development, Institute of
Tropical Diseases, Universitas Airlangga,
Surabaya, 60115, East Java, Indonesia.
3 Department of Chemistry, Faculty of
Mathematics and Natural Sciences,
Tanjungpura University, Pontianak
78124, West Kalimantan, Indonesia
*Corresponding
Suciati Suciati
suciati@ff.unair.ac.id
Received: 12 Jun 2022
Accepted: 01 October 2022
Published: 09 October 2022
Antioxidant and Acetylcholinesterase
Inhibitor Potentials of the Stem Extract of
Pternandra galeata
Suciati Suciati1,2* , Dwiki Nur Inayah 2 , Aty Widyawaruyanti 1,2 ,
Rudiyansyah Rudiyansyah3
ABSTRACT
Background: Pternandra galeata belongs to the family Melastomataceae. It is a native
flowering plant in Borneo Island that serve as food for monkey habitat. There has been limited
study on the medicinal and chemical properties of this plant. Objectives: We investigated
the acetylcholinesterase inhibitory activity and evaluated the antioxidant activity of the
ethanolic extract of Pternandra galeata stem. The total phenolic content in the sample was
also determined. Methods: The acetylcholinesterase inhibitory assays were performed using
Ellman’s method. Two different methods were used to evaluate the antioxidant activity of the
extract by 2,2-diphenyl-1-picryl hydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-
6-sulfonic acid) (ABTS) assays. The total phenolic content was determined by the Folin-
Ciocalteu method by employing gallic acid as a reference. Results: The ethanolic extract of
the P. galeata stems inhibited the AChE enzyme with an IC 50 value of 74.62 ± 0.89 μg/mL.
The sample exhibited antioxidant activity in the DPPH assay with an IC 50 value of 20.21 ± 0.08
μg/mL and 7.68 ± 0.09 μg/mL in the ABTS scavenging assay. The total phenolic content was
164.71 ± 3.33 mg GAE/g extract. Conclusion: The ethanolic extract of the P. galeata stem
can be a promising cholinesterase inhibitor and antioxidant for treating Alzheimer’s disease.
Keywords: Pternandra galeata, Alzheimer’s disease, Acetylcholinesterase inhibitor,
Antioxidant, Phenolic compound.
ORIGINAL RESEARCH
Published 09 October 2022
Doi: https://doi.org/10.17533/udea.vitae.v29n3a349983
2Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 29 | Number 03 | Article 349983Suciati Suciati, Dwiki Nur Inayah, Rudiyansyah Rudiyansyah
INTRODUCTION
Neurodegenerative disease, such as Alzheimer’s
disease (AD), has become a concern, especially in
developed countries with a high aging population.
This disease has created social and health problems
since it burdens the patient, family, and health
system (1). Most people with Alzheimer’s are 65
and older, but the early stage of AD can be found
at younger ages. AD mainly affects brain function,
which causes memory loss, diminished judgment
capacity, language impairment, and behavioral
changes (2). This disease is progressive and, over the
years, can develop dementia (60-70% of dementia
is caused by AD) (3). Multiple factors are involved
in the pathogenesis of AD, including deficiency of
neurotransmitter acetylcholine (ACh) and oxidative
stress. The regulation of acetylcholine is controlled
by acetylcholinesterase (AChE) (4). This enzyme
catalyzes the breakdown of ACh into choline and
acetate. Therefore, inhibiting this enzyme will help
maintain the level of ACh in the brain. The use of
cholinesterase inhibitors has shown positive effects
in the improvement of cognitive function of AD
patients (5). Studies have revealed that oxidative
stress plays a significant role in the pathogenesis
of neurodegenerative diseases, including AD.
Oxidative stress causes the loss of neurons and
AD progression to dementia (6-8). Oxidative stress
also involves the accumulation of a toxic peptide,
β-amyloid, in the brain of AD patients (8,9).
Pternandra galeata, locally known as Tiju, is a plant
found on Borneo Island in Indonesia and Malaysia.
It is a small to medium size tree with a height of
up to 27 m. The leaves and flowers of P. galeata
are the food source for Borneo’s monkeys (10).
A Literature search showed a limited study of P.
galeata regarding its metabolites and bioactivities.
The methanolic extract of these plant leaves showed
potential antiamoebic activity against Entamoeba
hystolitica (11). There is no report on the antioxidant
and cholinesterase inhibitory activity of this plant.
In our continuing study searching for potential
medicinal plants and marine organisms to contend
with neurodegenerative diseases, we screened
several plants from Borneo for antioxidant and
cholinesterase activity. The current research focuses
on the antioxidant and cholinesterase activity of the
ethanolic extract of the stem of P. galeata.
MATERIAL AND METHODS
Reagents
The chemical used for cholinesterase assays were
acetylcholinesterase from electric eel (AChE type
VI-S), acetylthiocholine iodide (ATCI), horse-serum
butyrylcholinesterase (BChE), butyrylthiocholine
iodide (BTCI), 5,5′-dithiobis[2-nitrobenzoic acid]
(DTNB), bovine serum albumin (BSA), tris buffer, and
galantamine. The chemicals used for antioxidant
assays were 2,2-Diphenyl-1-picrylhydrazyl (DPPH),
2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS), and potassium persulfate. Folin-
Ciocalteu’s phenol reagent, sodium carbonate, and
gallic acid were used to determine total phenolics.
All reagents were purchased from Sigma-Aldrich.
Preparation of plant extract
The stems of Pternandra galeata (Korth.) Ridl.
was obtained from Landau Village, Nanga Taman
District, Sekadanau, West Kalimantan, Indonesia.
The plant was identified by Purwodadi Botanic
Garden, Indonesian Institute of Sciences, with
identification letter number: B-262/IPH.06/KS.02/
VII/2020. The stems of Pternandra galeata were cut
RESUMEN
Antecedentes: Pternandra galeata pertenece a la familia Melastomataceae. Se trata de una planta con flores nativa de la isla de
Borneo que sirve de alimento para el hábitat de los monos. Se han realizado pocos estudios sobre las propiedades medicinales
y químicas de esta planta. Objetivos: Se investigó la actividad inhibidora de la acetilcolinesterasa y se evaluó la actividad
antioxidante del extracto etanólico del tallo de Pternandra galeata. También se determinó el contenido fenólico total de la
muestra. Métodos: Los ensayos de inhibición de la acetilcolinesterasa (AChE) se realizaron mediante el método de Ellman. Se
utilizaron dos métodos diferentes para evaluar la actividad antioxidante de los ensayos de 2,2-difenil-1-picril hidrazilo (DPPH) y
2,2′-azinobis-(ácido 3-etilbenzotiazolina-6-sulfónico) (ABTS). El contenido fenólico total se determinó por el método de Folin-
Ciocalteu empleando el ácido gálico como referencia. Resultados: El extracto etanólico de los tallos de P. galeata inhibió la
enzima AChE con un valor IC 50 de 74.62 ± 0.89 μg/mL. La muestra mostró actividad antioxidante en el ensayo DPPH con un
valor IC 50 de 20.21 ± 0.08 μg/mL y 7.68 ± 0.09 μg/mL en el ensayo de barrido ABTS. El contenido fenólico total fue de 164.71
± 3.33 mg GAE/g de extracto. Conclusión: El extracto etanólico del tallo de P. galeata puede ser un prometedor inhibidor de
la colinesterasa y antioxidante para el tratamiento de la enfermedad de Alzheimer.
Palabras claves: Pternandra galeata, Enfermedad de Alzheimer, Inhibidor de Acetilcolinesterasa, Antioxidante, Compuestos
fenólicos
INTRODUCTION
Neurodegenerative disease, such as Alzheimer’s
disease (AD), has become a concern, especially in
developed countries with a high aging population.
This disease has created social and health problems
since it burdens the patient, family, and health
system (1). Most people with Alzheimer’s are 65
and older, but the early stage of AD can be found
at younger ages. AD mainly affects brain function,
which causes memory loss, diminished judgment
capacity, language impairment, and behavioral
changes (2). This disease is progressive and, over the
years, can develop dementia (60-70% of dementia
is caused by AD) (3). Multiple factors are involved
in the pathogenesis of AD, including deficiency of
neurotransmitter acetylcholine (ACh) and oxidative
stress. The regulation of acetylcholine is controlled
by acetylcholinesterase (AChE) (4). This enzyme
catalyzes the breakdown of ACh into choline and
acetate. Therefore, inhibiting this enzyme will help
maintain the level of ACh in the brain. The use of
cholinesterase inhibitors has shown positive effects
in the improvement of cognitive function of AD
patients (5). Studies have revealed that oxidative
stress plays a significant role in the pathogenesis
of neurodegenerative diseases, including AD.
Oxidative stress causes the loss of neurons and
AD progression to dementia (6-8). Oxidative stress
also involves the accumulation of a toxic peptide,
β-amyloid, in the brain of AD patients (8,9).
Pternandra galeata, locally known as Tiju, is a plant
found on Borneo Island in Indonesia and Malaysia.
It is a small to medium size tree with a height of
up to 27 m. The leaves and flowers of P. galeata
are the food source for Borneo’s monkeys (10).
A Literature search showed a limited study of P.
galeata regarding its metabolites and bioactivities.
The methanolic extract of these plant leaves showed
potential antiamoebic activity against Entamoeba
hystolitica (11). There is no report on the antioxidant
and cholinesterase inhibitory activity of this plant.
In our continuing study searching for potential
medicinal plants and marine organisms to contend
with neurodegenerative diseases, we screened
several plants from Borneo for antioxidant and
cholinesterase activity. The current research focuses
on the antioxidant and cholinesterase activity of the
ethanolic extract of the stem of P. galeata.
MATERIAL AND METHODS
Reagents
The chemical used for cholinesterase assays were
acetylcholinesterase from electric eel (AChE type
VI-S), acetylthiocholine iodide (ATCI), horse-serum
butyrylcholinesterase (BChE), butyrylthiocholine
iodide (BTCI), 5,5′-dithiobis[2-nitrobenzoic acid]
(DTNB), bovine serum albumin (BSA), tris buffer, and
galantamine. The chemicals used for antioxidant
assays were 2,2-Diphenyl-1-picrylhydrazyl (DPPH),
2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS), and potassium persulfate. Folin-
Ciocalteu’s phenol reagent, sodium carbonate, and
gallic acid were used to determine total phenolics.
All reagents were purchased from Sigma-Aldrich.
Preparation of plant extract
The stems of Pternandra galeata (Korth.) Ridl.
was obtained from Landau Village, Nanga Taman
District, Sekadanau, West Kalimantan, Indonesia.
The plant was identified by Purwodadi Botanic
Garden, Indonesian Institute of Sciences, with
identification letter number: B-262/IPH.06/KS.02/
VII/2020. The stems of Pternandra galeata were cut
RESUMEN
Antecedentes: Pternandra galeata pertenece a la familia Melastomataceae. Se trata de una planta con flores nativa de la isla de
Borneo que sirve de alimento para el hábitat de los monos. Se han realizado pocos estudios sobre las propiedades medicinales
y químicas de esta planta. Objetivos: Se investigó la actividad inhibidora de la acetilcolinesterasa y se evaluó la actividad
antioxidante del extracto etanólico del tallo de Pternandra galeata. También se determinó el contenido fenólico total de la
muestra. Métodos: Los ensayos de inhibición de la acetilcolinesterasa (AChE) se realizaron mediante el método de Ellman. Se
utilizaron dos métodos diferentes para evaluar la actividad antioxidante de los ensayos de 2,2-difenil-1-picril hidrazilo (DPPH) y
2,2′-azinobis-(ácido 3-etilbenzotiazolina-6-sulfónico) (ABTS). El contenido fenólico total se determinó por el método de Folin-
Ciocalteu empleando el ácido gálico como referencia. Resultados: El extracto etanólico de los tallos de P. galeata inhibió la
enzima AChE con un valor IC 50 de 74.62 ± 0.89 μg/mL. La muestra mostró actividad antioxidante en el ensayo DPPH con un
valor IC 50 de 20.21 ± 0.08 μg/mL y 7.68 ± 0.09 μg/mL en el ensayo de barrido ABTS. El contenido fenólico total fue de 164.71
± 3.33 mg GAE/g de extracto. Conclusión: El extracto etanólico del tallo de P. galeata puede ser un prometedor inhibidor de
la colinesterasa y antioxidante para el tratamiento de la enfermedad de Alzheimer.
Palabras claves: Pternandra galeata, Enfermedad de Alzheimer, Inhibidor de Acetilcolinesterasa, Antioxidante, Compuestos
fenólicos
3Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 349983
Antioxidant and Acetylcholinesterase Inhibitor Potentials of the Stem Extract of Pternandra galeata
into small pieces, then dried in the shade for seven
days, and finally grounded. A hundred grams of the
powdered stem was soaked with 600 mL ethanol
for 24 hours; then, the filtrate was separated under
vacuum. The residue was re-extracted using the
same procedure three times. All collected filtrates
were evaporated under vacuo to obtain 0.55 gram
ethanolic extract.
Acetylcholinesterase inhibitory assay
The assay was performed based on the modified
Ellman’s method (12-14). The extract was dissolved
in 10% methanol at 1-500 μg/mL. Twenty-five
microliters of sample solutions were mixed with
1.5 mM ATCI (25 μL), 3 mM DTNB (125 μL), Tris
buffer (50 μL), and 0.22 U/mL AChE (25 μL) in a 96-
well microplate. The formation of yellow colored
product, 5-thio-2-nitrobenzoate, was monitored in
a microplate reader (Thermo Scientific Multiskan FC)
at 405 nm every 5 s for 2 mins. Experiments were
carried out in triplicates. Galantamine was used as a
standard, and 10% methanol was used as a control/
blank. The percent of inhibition was then calculated
as follows:
( )% 1 00
Mean velocity of control Mean velocity of sample
Inhibition x
Mean velocity of control
−
=
DPPH radical scavenging assay
The assay was conducted in triplicates based on
modified protocols of Herald et al. and Lee et al.
(15,16). The DPPH solution (0.25 mM) was made
in methanol. Different concentrations of extract
(1.25 – 40 μg/mL) in methanol and standard gallic
acid were incubated with DPPH solutions in 96 well
plates and allowed to stand at room temperature in
the dark for 30 minutes. The solutions were shaken
for 30 s, and the absorbance was measured at 517
nm in a microplate reader. Gallic acid was used as
a standard. The following formula calculated the
percent DPPH scavenging effect:
( ) ( )% 100
abs control abs sample
DPPH Radical Scavenging activity x
abs control
−
=
where abs control is the absorbance of DPPH radical
+ methanol and abs sample is absorbance DPPH
radical + extract/standard.
ABTS Radical Scavenging Assay
The bioassay was performed based on the previous
method of Lee et al. with some modifications (16).
ABTS radical was generated by mixing 5 mL ABTS (7
mM) with 88 𝜇L potassium persulfate (140 nM) and
allowed to stand for 16 hours in the dark at room
temperature. Samples at a concentration range
of 1.25 – 25 𝜇g/mL in methanol were treated with
100 𝜇L of ABTS in a 96-well microplate, followed
by incubation for 6 mins in the dark condition at
room temperature. The absorbance was read at
734 nm in a microplate reader. Gallic acid was used
as a standard. Experiments were carried out in
triplicates. The ABTS radical scavenging activity of
the extract was calculated as above.
Quantification of total phenolic content
The total soluble phenolic compounds (TPC) in the
extract were measured according to the method by
Zhang et al. and Herald et al. with slight modification
using gallic acid as a standard (15, 17). Briefly, 25 μL
solution of extract (1000 μg/mL) or gallic acid (25 –
500 μg/mL) was mixed with water (75 μL) and Folin
& Ciocalteu’s phenol reagent (25 μL) in a 96-well
microplate. The mixtures were allowed to stand for
6 minutes at room temperature, followed by adding
100 μL Na2 CO3 solution (75 g/L). The mixtures were
then incubated for 90 minutes in the dark at room
temperature. The absorbance was recorded at
765 nm, and the TPC was calculated based on the
gallic acid standard calibration curve. The TPC of
the extract was expressed as gallic acid equivalent
(GAE) in milligrams per gram of dry extract.
RESULTS
Acetylcholinesterase inhibitory activity
The ethanolic extract of P. galeata was screened
against the AChE enzyme using Ellman’s method.
In this assay, the enzyme AChE will hydrolyze the
substrate acetylthiocholine Iodide (ATCI), resulting
in the product thiocholine, which will react with the
Ellman’s reagent (DTNB) to form a yellow-colored
compound 5-thio-2-nitrobenzoate that can be
monitored at 405 nm. The presence of the AChE
inhibitor will prevent the hydrolysis of ATCI so that
the yellow-colored product will not be formed (12).
Various concentrations of P. galeata extract were
prepared to evaluate the dose-response mode
and the fifty percent inhibitory activity (IC 50 ) of the
extract. The result presented in Table 1 showed
that the extract inhibited the AChE enzyme with
Antioxidant and Acetylcholinesterase Inhibitor Potentials of the Stem Extract of Pternandra galeata
into small pieces, then dried in the shade for seven
days, and finally grounded. A hundred grams of the
powdered stem was soaked with 600 mL ethanol
for 24 hours; then, the filtrate was separated under
vacuum. The residue was re-extracted using the
same procedure three times. All collected filtrates
were evaporated under vacuo to obtain 0.55 gram
ethanolic extract.
Acetylcholinesterase inhibitory assay
The assay was performed based on the modified
Ellman’s method (12-14). The extract was dissolved
in 10% methanol at 1-500 μg/mL. Twenty-five
microliters of sample solutions were mixed with
1.5 mM ATCI (25 μL), 3 mM DTNB (125 μL), Tris
buffer (50 μL), and 0.22 U/mL AChE (25 μL) in a 96-
well microplate. The formation of yellow colored
product, 5-thio-2-nitrobenzoate, was monitored in
a microplate reader (Thermo Scientific Multiskan FC)
at 405 nm every 5 s for 2 mins. Experiments were
carried out in triplicates. Galantamine was used as a
standard, and 10% methanol was used as a control/
blank. The percent of inhibition was then calculated
as follows:
( )% 1 00
Mean velocity of control Mean velocity of sample
Inhibition x
Mean velocity of control
−
=
DPPH radical scavenging assay
The assay was conducted in triplicates based on
modified protocols of Herald et al. and Lee et al.
(15,16). The DPPH solution (0.25 mM) was made
in methanol. Different concentrations of extract
(1.25 – 40 μg/mL) in methanol and standard gallic
acid were incubated with DPPH solutions in 96 well
plates and allowed to stand at room temperature in
the dark for 30 minutes. The solutions were shaken
for 30 s, and the absorbance was measured at 517
nm in a microplate reader. Gallic acid was used as
a standard. The following formula calculated the
percent DPPH scavenging effect:
( ) ( )% 100
abs control abs sample
DPPH Radical Scavenging activity x
abs control
−
=
where abs control is the absorbance of DPPH radical
+ methanol and abs sample is absorbance DPPH
radical + extract/standard.
ABTS Radical Scavenging Assay
The bioassay was performed based on the previous
method of Lee et al. with some modifications (16).
ABTS radical was generated by mixing 5 mL ABTS (7
mM) with 88 𝜇L potassium persulfate (140 nM) and
allowed to stand for 16 hours in the dark at room
temperature. Samples at a concentration range
of 1.25 – 25 𝜇g/mL in methanol were treated with
100 𝜇L of ABTS in a 96-well microplate, followed
by incubation for 6 mins in the dark condition at
room temperature. The absorbance was read at
734 nm in a microplate reader. Gallic acid was used
as a standard. Experiments were carried out in
triplicates. The ABTS radical scavenging activity of
the extract was calculated as above.
Quantification of total phenolic content
The total soluble phenolic compounds (TPC) in the
extract were measured according to the method by
Zhang et al. and Herald et al. with slight modification
using gallic acid as a standard (15, 17). Briefly, 25 μL
solution of extract (1000 μg/mL) or gallic acid (25 –
500 μg/mL) was mixed with water (75 μL) and Folin
& Ciocalteu’s phenol reagent (25 μL) in a 96-well
microplate. The mixtures were allowed to stand for
6 minutes at room temperature, followed by adding
100 μL Na2 CO3 solution (75 g/L). The mixtures were
then incubated for 90 minutes in the dark at room
temperature. The absorbance was recorded at
765 nm, and the TPC was calculated based on the
gallic acid standard calibration curve. The TPC of
the extract was expressed as gallic acid equivalent
(GAE) in milligrams per gram of dry extract.
RESULTS
Acetylcholinesterase inhibitory activity
The ethanolic extract of P. galeata was screened
against the AChE enzyme using Ellman’s method.
In this assay, the enzyme AChE will hydrolyze the
substrate acetylthiocholine Iodide (ATCI), resulting
in the product thiocholine, which will react with the
Ellman’s reagent (DTNB) to form a yellow-colored
compound 5-thio-2-nitrobenzoate that can be
monitored at 405 nm. The presence of the AChE
inhibitor will prevent the hydrolysis of ATCI so that
the yellow-colored product will not be formed (12).
Various concentrations of P. galeata extract were
prepared to evaluate the dose-response mode
and the fifty percent inhibitory activity (IC 50 ) of the
extract. The result presented in Table 1 showed
that the extract inhibited the AChE enzyme with
4Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 29 | Number 03 | Article 349983Suciati Suciati, Dwiki Nur Inayah, Rudiyansyah Rudiyansyah
an IC 50 value of 74.62 μg/mL. Figure 1 showed that
the extract inhibited AChE in a dose-dependent
manner.
Figure 1. Concentration-dependent response of P. galeata
extract against AChE
Antioxidant Activity
The antioxidant property of P. galeata was examined
using DPPH and ABTS assays. In the DPPH assay,
the antioxidant provides a hydrogen atom that
reacts with the stable radical DPPH to form a yellow-
colored non-radical diphenylpicrylhydrazine (18,19).
The principle of ABTS assay is similar to that of DPPH
assay, in which the antioxidant acts as a hydrogen
donor to form a non-radical ABTS. The reduction of
a dark-bluish color of ABTS radical can be monitored
by a spectrophotometer (20). The results presented
in Table 1 and Figure 2 suggested that the extract
of P. galeata exhibited concentration-dependent
radical scavenging activities in both DPPH and
ABTS assays with IC 50 values of 20.21 and 7.68 μg/
mL, respectively.
Table 1. Radical scavenging effect and AChE inhibitory activities
of P. galeata extract and standards
Samples
IC 50 (μg/mL)
AChE DPPH radical
scavenging
ABTS radical
scavenging
P. galeata extract 74.62 ± 0.89 20.21 ± 0.08 7.68 ± 0.09
Galantamine 0.63 ± 0.05 - -
Gallic acid - 2.76 ± 0.02 0.97 ± 0.03
aEach value is the average of three analyses ± standard deviation
Figure 2. DPPH and ABTS radical scavenging effects of different
concentrations of P. galeata extract
Total phenolic content
The total phenolic content of P. galeata extract was
ascertained by the method of Folin-Ciocalteu and
calculated as gallic acid equivalent (GAE). The total
phenolic content of P. galeata extract was 164.71 ±
3.33 mg GAE/g of dry extract (Table 2).
Table 2. Extract yield and total phenolic content of P. galeata
extract
Sample Extract Yield (%) TPC (mg GAE/g extract) a
P. galeata extract 0.55 164.71 ± 3.33
a
Value is the average of three analyses ± standard deviation
DISCUSSION
Alzheimer’s disease is one of the most prevalent
neurodegenerative diseases that mainly occurs
in older adults. Cognitive decline is an apparent
symptom of this disease, caused by the deficit of
neurotransmitter acetylcholine, particularly affecting
cholinergic neurons in the basal forebrain. Another
factor involved in the etiology of AD is oxidative
stress. It is associated with the mechanism of
Aβ-induced cytotoxicity of cholinergic neurons.
Currently, AChE inhibitors, such as galantamine,
rivastigmine, and donepezil, have been proven to be
the most effective therapy to improve AD patients’
memory and cognitive function (21). In addition,
antioxidants have shown benefits in improving
cognitive function and behavioral deficits in AD
animal models (22). Therefore, it is suggested that
natural antioxidants with potent cholinesterase
inhibitory activity can be a better drug for treating
AD (23,24). In the current study, the stem of
Pternandra galeata was extracted with ethanol. The
an IC 50 value of 74.62 μg/mL. Figure 1 showed that
the extract inhibited AChE in a dose-dependent
manner.
Figure 1. Concentration-dependent response of P. galeata
extract against AChE
Antioxidant Activity
The antioxidant property of P. galeata was examined
using DPPH and ABTS assays. In the DPPH assay,
the antioxidant provides a hydrogen atom that
reacts with the stable radical DPPH to form a yellow-
colored non-radical diphenylpicrylhydrazine (18,19).
The principle of ABTS assay is similar to that of DPPH
assay, in which the antioxidant acts as a hydrogen
donor to form a non-radical ABTS. The reduction of
a dark-bluish color of ABTS radical can be monitored
by a spectrophotometer (20). The results presented
in Table 1 and Figure 2 suggested that the extract
of P. galeata exhibited concentration-dependent
radical scavenging activities in both DPPH and
ABTS assays with IC 50 values of 20.21 and 7.68 μg/
mL, respectively.
Table 1. Radical scavenging effect and AChE inhibitory activities
of P. galeata extract and standards
Samples
IC 50 (μg/mL)
AChE DPPH radical
scavenging
ABTS radical
scavenging
P. galeata extract 74.62 ± 0.89 20.21 ± 0.08 7.68 ± 0.09
Galantamine 0.63 ± 0.05 - -
Gallic acid - 2.76 ± 0.02 0.97 ± 0.03
aEach value is the average of three analyses ± standard deviation
Figure 2. DPPH and ABTS radical scavenging effects of different
concentrations of P. galeata extract
Total phenolic content
The total phenolic content of P. galeata extract was
ascertained by the method of Folin-Ciocalteu and
calculated as gallic acid equivalent (GAE). The total
phenolic content of P. galeata extract was 164.71 ±
3.33 mg GAE/g of dry extract (Table 2).
Table 2. Extract yield and total phenolic content of P. galeata
extract
Sample Extract Yield (%) TPC (mg GAE/g extract) a
P. galeata extract 0.55 164.71 ± 3.33
a
Value is the average of three analyses ± standard deviation
DISCUSSION
Alzheimer’s disease is one of the most prevalent
neurodegenerative diseases that mainly occurs
in older adults. Cognitive decline is an apparent
symptom of this disease, caused by the deficit of
neurotransmitter acetylcholine, particularly affecting
cholinergic neurons in the basal forebrain. Another
factor involved in the etiology of AD is oxidative
stress. It is associated with the mechanism of
Aβ-induced cytotoxicity of cholinergic neurons.
Currently, AChE inhibitors, such as galantamine,
rivastigmine, and donepezil, have been proven to be
the most effective therapy to improve AD patients’
memory and cognitive function (21). In addition,
antioxidants have shown benefits in improving
cognitive function and behavioral deficits in AD
animal models (22). Therefore, it is suggested that
natural antioxidants with potent cholinesterase
inhibitory activity can be a better drug for treating
AD (23,24). In the current study, the stem of
Pternandra galeata was extracted with ethanol. The
5Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 349983
Antioxidant and Acetylcholinesterase Inhibitor Potentials of the Stem Extract of Pternandra galeata
extract was then subjected to in vitro antioxidant
and acetylcholinesterase inhibitory assays.
Acetylcholinesterase (AChE) and butyrylcholines-
terase (BuChE) are enzymes that are related to the
level of acetylcholine (ACh) in the brain (25). Howe-
ver, AChE is reported as the major cholinesterase
enzyme in the brain that catalyzes the hydrolysis of
acetylcholine and shows higher specificity toward
acetylcholine. Therefore, AChE enzyme has been an
attractive target for AD therapy (26). In our study, P.
galeata extract showed moderate inhibition against
the AChE enzyme in a dose-dependent manner. Cu-
rrently, we have not found a report from the genus
Pternandra as a cholinesterase inhibitor. However, a
plant from the same family Melastomataceae, Mico-
nia sp., has been reported to have moderate AChE
inhibitory activity (27). A triterpene, sumaresinolic
acid, isolated from Miconia stenostachya showed
AChE inhibitory activity (28,29).
Oxidative stress has been reported to play an
essential role in AD pathogenesis. Therefore,
preventing or reducing oxidative damage by using
antioxidants for AD and another therapy may
provide better results (24). Plant materials, such as
fruits, vegetables, herbs, and spices, have been
known as the source of natural antioxidants (30). The
current study examined the antioxidant property of
P. galeata by DPPH and ABTS methods. The DPPH
and ABTS free radicals are stable free radicals
widely used to estimate the radical scavenging
activity of antioxidants. The results showed that P.
galeata gave strong radical scavenging activities
in both DPPH and ABTS assays. This is due to the
different reactivity of compounds in the P. galeata
extract to DPPH and ABTS radicals. The reaction of
DPPH radicals depends on the steric accessibility
of compounds to them. Small molecules generally
react better with DPPH radicals compared to large
molecules. ABTS radical can be used to measure
the antioxidant capacity of both hydrophilic and
lipophilic compounds (31, 32). The extract showed a
lower IC 50 value in the ABTS assay than in the DPPH
assay. There has been no report of P. galeata as an
antioxidant. However, a plant from the same genus,
P. azurea exhibited radical scavenging activity
against DPPH, ABTS, and nitric oxide (NO) (33).
There has been evidence of the correlation
of phenolic compounds with antioxidant and
cholinesterase inhibitory activities. The presence
of hydroxyl groups in the phenolic compounds is
believed to play a significant role in AChE inhibitory
activity. The multiple hydroxyl groups in the phenolic
compounds can enhance the inhibitory action
of AChE due to their stronger binding capacity
(34,35). Several phenolic compounds have been
reported to show inhibition against AChE and
BChE in both in vitro and in vivo studies, such as
quercetin, resveratrol, curcumin, gallocatechin,
as well as cinnamic acid and its derivatives (35).
Phenolic compounds are also well known to be
essential in the antioxidant activities of medicinal
plants. The antioxidant potential of the phenolics is
predominantly due to the redox capability so that it
can absorb and neutralize free radicals, decompose
peroxide, and quench singlet or triplet oxygen
(36). The number and arrangement of the hydroxyl
groups in phenolics are believed to be closely
related to their antioxidant capacity.
CONCLUSION
The stem extract of P. galeata exhibited antioxidant
and acetylcholinesterase inhibitory activities. The
presence of phenolic compounds in the extract
may be responsible for the antioxidant and AChE
activities. Further study is needed to investigate the
chemistry of the phenolic compounds in the extract.
CONFLICT OF INTEREST
The authors have no conflicts of interest regarding
this investigation.
ACKNOWLEDGMENT
Authors acknowledge Facult y of Pharmac y
Universitas Airlangga for research grant PUF 2021.
AUTHOR’S CONTRIBUTIONS
Concept – S.S; Design – S.S.; Supervision – S.S., A.W.,
R.R.; Resources – S.S., K.I., A.W.; Materials – S.S.,
R.R.; Data Collection and/or Processing – S.S., D.N.I;
Analysis and/or Interpretation – S.S., D.N.I.; Writing –
S.S., A.D.A.; Critical Reviews – S.S., D.N.I., A.W., R.R.
REFERENCES
1. Marchesi VT. Alzheimer’s disease 2012: The great amyloid
gamble. Am J Pathol. 2012; 180:1762–1767. ht tps://doi.
org/10.1016/j.ajpath.2012.03.004
2. Alzheimer’s Association. 2022 Alzheimer’s disease facts and
figures. Alzheimers Dement. 2022;18(4):700-789. https://doi.
org/10.1002/alz.12638
3. Alzheimer’s Association. 2020 Alzheimer’s disease facts and
figures. Alzheimers Dement. 2020;16(3):391-460. https://doi.
org/10.1002/alz.12068
Antioxidant and Acetylcholinesterase Inhibitor Potentials of the Stem Extract of Pternandra galeata
extract was then subjected to in vitro antioxidant
and acetylcholinesterase inhibitory assays.
Acetylcholinesterase (AChE) and butyrylcholines-
terase (BuChE) are enzymes that are related to the
level of acetylcholine (ACh) in the brain (25). Howe-
ver, AChE is reported as the major cholinesterase
enzyme in the brain that catalyzes the hydrolysis of
acetylcholine and shows higher specificity toward
acetylcholine. Therefore, AChE enzyme has been an
attractive target for AD therapy (26). In our study, P.
galeata extract showed moderate inhibition against
the AChE enzyme in a dose-dependent manner. Cu-
rrently, we have not found a report from the genus
Pternandra as a cholinesterase inhibitor. However, a
plant from the same family Melastomataceae, Mico-
nia sp., has been reported to have moderate AChE
inhibitory activity (27). A triterpene, sumaresinolic
acid, isolated from Miconia stenostachya showed
AChE inhibitory activity (28,29).
Oxidative stress has been reported to play an
essential role in AD pathogenesis. Therefore,
preventing or reducing oxidative damage by using
antioxidants for AD and another therapy may
provide better results (24). Plant materials, such as
fruits, vegetables, herbs, and spices, have been
known as the source of natural antioxidants (30). The
current study examined the antioxidant property of
P. galeata by DPPH and ABTS methods. The DPPH
and ABTS free radicals are stable free radicals
widely used to estimate the radical scavenging
activity of antioxidants. The results showed that P.
galeata gave strong radical scavenging activities
in both DPPH and ABTS assays. This is due to the
different reactivity of compounds in the P. galeata
extract to DPPH and ABTS radicals. The reaction of
DPPH radicals depends on the steric accessibility
of compounds to them. Small molecules generally
react better with DPPH radicals compared to large
molecules. ABTS radical can be used to measure
the antioxidant capacity of both hydrophilic and
lipophilic compounds (31, 32). The extract showed a
lower IC 50 value in the ABTS assay than in the DPPH
assay. There has been no report of P. galeata as an
antioxidant. However, a plant from the same genus,
P. azurea exhibited radical scavenging activity
against DPPH, ABTS, and nitric oxide (NO) (33).
There has been evidence of the correlation
of phenolic compounds with antioxidant and
cholinesterase inhibitory activities. The presence
of hydroxyl groups in the phenolic compounds is
believed to play a significant role in AChE inhibitory
activity. The multiple hydroxyl groups in the phenolic
compounds can enhance the inhibitory action
of AChE due to their stronger binding capacity
(34,35). Several phenolic compounds have been
reported to show inhibition against AChE and
BChE in both in vitro and in vivo studies, such as
quercetin, resveratrol, curcumin, gallocatechin,
as well as cinnamic acid and its derivatives (35).
Phenolic compounds are also well known to be
essential in the antioxidant activities of medicinal
plants. The antioxidant potential of the phenolics is
predominantly due to the redox capability so that it
can absorb and neutralize free radicals, decompose
peroxide, and quench singlet or triplet oxygen
(36). The number and arrangement of the hydroxyl
groups in phenolics are believed to be closely
related to their antioxidant capacity.
CONCLUSION
The stem extract of P. galeata exhibited antioxidant
and acetylcholinesterase inhibitory activities. The
presence of phenolic compounds in the extract
may be responsible for the antioxidant and AChE
activities. Further study is needed to investigate the
chemistry of the phenolic compounds in the extract.
CONFLICT OF INTEREST
The authors have no conflicts of interest regarding
this investigation.
ACKNOWLEDGMENT
Authors acknowledge Facult y of Pharmac y
Universitas Airlangga for research grant PUF 2021.
AUTHOR’S CONTRIBUTIONS
Concept – S.S; Design – S.S.; Supervision – S.S., A.W.,
R.R.; Resources – S.S., K.I., A.W.; Materials – S.S.,
R.R.; Data Collection and/or Processing – S.S., D.N.I;
Analysis and/or Interpretation – S.S., D.N.I.; Writing –
S.S., A.D.A.; Critical Reviews – S.S., D.N.I., A.W., R.R.
REFERENCES
1. Marchesi VT. Alzheimer’s disease 2012: The great amyloid
gamble. Am J Pathol. 2012; 180:1762–1767. ht tps://doi.
org/10.1016/j.ajpath.2012.03.004
2. Alzheimer’s Association. 2022 Alzheimer’s disease facts and
figures. Alzheimers Dement. 2022;18(4):700-789. https://doi.
org/10.1002/alz.12638
3. Alzheimer’s Association. 2020 Alzheimer’s disease facts and
figures. Alzheimers Dement. 2020;16(3):391-460. https://doi.
org/10.1002/alz.12068
6Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 29 | Number 03 | Article 349983Suciati Suciati, Dwiki Nur Inayah, Rudiyansyah Rudiyansyah
4. Ferreira-Vieira TH, Guimaraes IM, Silva FR, Ribeiro FM.
Alzheimer’s disease: Targeting the cholinergic system. Curr
Neuropharmacol. 2016;14(1):101-15. https://doi.org/10.2174/15
70159x13666150716165726
5. Konrath EL, Passos CDS, Klein-Júnior LC, Henriques AT. Alkaloids
as a source of potential anticholinesterase inhibitors for the
treatment of Alzheimer’s disease. J Pharm Pharmacol. 2013;
65(12): 1701-1725. https://doi.org/10.1111/jphp.12090
6. Christen Y. Oxidative stress and Alzheimer disease. Am J Clin Nutr.
2000; 71(2): 621S-629S. https://doi/org/10.1093/ajcn/71.2.621s
7. Halliwell B. Role of free radicals in neurodegenerative diseases:
Therapeutic implications for antioxidant treatment. Drugs
Aging. 2001; 18(9): 685-716. https://doi.org/10.2165/00002512-
200118090-00004
8. Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in
disease and health. Int J Biomed Sci. 2008; 4(2): 89-96.
9. Butterfield DA. Amyloid beta-peptide (1-42)-induced oxidative
stress and neurotoxicity: implications for neurodegeneration in
alzheimer’s disease brain. A review. Free Radic Res. 2002;36(12):1307-
1313. https://doi.org/10.1080/1071576021000049890
10. Mukhlisi, T. Atmoko, Priyono. Flora di Habitat Bekantan Lahan
Basah Suwi, Kalimantan Timur. Susilo A and Iskandar S (eds).
Balikpapan: Forda Press; 2018.118p.
11. Wardana F, Sari D, Adianti M, Permanasari A, Tumewu L, Nozaki T,
Widyawaruyanti A, Hafid AF. Amoebicidal activities of Indonesian
medicinal plants in Balikpapan, East Kalimantan. Proceedings of
BROMO Conference. ScitePress: 2019;77-82.
12. Ellman GL, Courtney KD, Andres V Jr, Featherstone RM. A new
and rapid colorimetric determination of acetylcholinesterase
activity. Biochem Pharmacol. 1961;7(2):88-95. https://doi.
org/10.1016/0006-2952(61)90145-9
13. Suciati, Laili ER, Poerwantoro D, Hapsari AP, Gifanda LZ, Rabgay
K, Ekasari W, Ingkaninan K. Evaluation of cholinesterase inhibitory
activity of six Indonesian Cassia species. J Res Pharm. 2020; 24(4):
472-478. http://dx.doi.org/10.35333/jrp.2020.195
14. Aristyawan AD, Setyaningtyas VF, Wahyuni TS, Widyawaruyanti
A, Ingkaninan K, Suciati S. In vitro acetylcholinesterase inhibitory
activities of fractions and iso-agelasine C isolated from the marine
sponge Agelas nakamurai. J Res Pharm. 2022; 26(2): 279-286.
http://dx.doi.org/10.29228/jrp.126
15. Herald TJ, Gadgil P, Tilley M. High-throughput micro plate assays
for screening flavonoid content and DPPH-scavenging activity in
sorghum bran and flour. J Sci Food Agric. 2012;92(11):2326-2331.
https://doi.org/10.1002/jsfa.5633
16. Lee KJ, Oh YC, Cho WK, Ma JY. Antioxidant and anti-inflammatory
activity determination of one hundred kinds of pure chemical
compounds using offline and online screening HPLC assay. Evid
Based Complement Alternat Med. 2015;2015: 165457. https://
doi.org/10.1155/2015/165457
17. Zhang Q, Zhang J, Shen J, Silva A, Dennis DA, Barrow CJ.
A simple 96-well microplate method for estimation of total
polyphenol content in seaweeds. J Appl Phycol. 2006; 18:
445–450. https://doi.org/10.1007/s10811-006-9048-4
18. Blois MS. Antioxidant determinations by the use of a stable
free r adic al. Nature. 1958; 181:1199-120 0. ht tps://doi.
org/10.1038/1811199a0
19. Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl
(DPPH) for estimating antioxidant activity. Songklanakarin J Sci
Technol. 2004;26(2):211- 219.
20. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans
C. Antioxidant activity applying an improved ABTS radical cation
decolorization assay. Free Radic Biol Med. 1999;26(9-10):1231–
1237. https://doi.org/10.1016/S0891-5849(98)00315-3
21. Mehta M, Adem A, Sabbagh M. New acetylcholinesterase
inhibitors for Alzheimer’s disease. Int J Alzheimers Dis.
2012;2012:728983. https://doi.org/10.1155/2012/728983
22. Danta CC, Piplani P. The discover y and development of
new potential antioxidant agent s for the treatment of
neurodegenerative diseases. Exper t Opin Drug Discov.
2014;9:1205–1222. https://doi.org/10.1517/17460441.2014.942218
23. Zhao Y, Zhao B. Natural antioxidants in prevention management
of Alzheimer’s disease. Front Biosci (Elite Ed). 2012;1:794–808.
https://doi.org/10.2741/e419
24. Suganthy N, Devi KP. In vitro antioxidant and anti-cholinesterase
activities of Rhizophora mucronata. Pharm Biol. 2016;5 4(1): 118-
29. https://doi.org/10.3109/13880209.2015.1017886
25. Giacobini E. Cholinesterases: New roles in brain function and in
Alzheimer’s disease. Neurochem Res, 2003; 28 (3-4):515–522.
https://doi.org/10.1023/a:1022869222652
26. Lopa SS, Al-Amin MY, Hasan MK, Ahammed MS, Islam KM,
Alam AHMK, Tanaka T, Sadik MG. Phytochemical analysis
and cholinesterase inhibitory and antioxidant activities of
Enhydra fluctuans relevant in the management of Alzheimer’s
disease. Int J Food Sci. 2021;2021:8862025. https://doi.
org/10.1155/2021/8862025
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https://doi.org/10.1590/s0074-02762006000700013
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P, Haqiqi MT, Saparwadi. Screening for potential antidiabetes and
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polyphenols: A therapeutic approach for the treatment of
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https://doi.org/10.1111/cns.12971
36. Jothy SL, Aziz A, Chen Y, Sasidharan S. Antioxidant activity and
hepatoprotective potential of Polyalthia longifolia and Cassia
spectabilis leaves against paracetamol-induced liver injury. Evid
Based Complement Alternat Med. 2012;2012:561284. https://doi.
org/10.1155/2012/561284
4. Ferreira-Vieira TH, Guimaraes IM, Silva FR, Ribeiro FM.
Alzheimer’s disease: Targeting the cholinergic system. Curr
Neuropharmacol. 2016;14(1):101-15. https://doi.org/10.2174/15
70159x13666150716165726
5. Konrath EL, Passos CDS, Klein-Júnior LC, Henriques AT. Alkaloids
as a source of potential anticholinesterase inhibitors for the
treatment of Alzheimer’s disease. J Pharm Pharmacol. 2013;
65(12): 1701-1725. https://doi.org/10.1111/jphp.12090
6. Christen Y. Oxidative stress and Alzheimer disease. Am J Clin Nutr.
2000; 71(2): 621S-629S. https://doi/org/10.1093/ajcn/71.2.621s
7. Halliwell B. Role of free radicals in neurodegenerative diseases:
Therapeutic implications for antioxidant treatment. Drugs
Aging. 2001; 18(9): 685-716. https://doi.org/10.2165/00002512-
200118090-00004
8. Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in
disease and health. Int J Biomed Sci. 2008; 4(2): 89-96.
9. Butterfield DA. Amyloid beta-peptide (1-42)-induced oxidative
stress and neurotoxicity: implications for neurodegeneration in
alzheimer’s disease brain. A review. Free Radic Res. 2002;36(12):1307-
1313. https://doi.org/10.1080/1071576021000049890
10. Mukhlisi, T. Atmoko, Priyono. Flora di Habitat Bekantan Lahan
Basah Suwi, Kalimantan Timur. Susilo A and Iskandar S (eds).
Balikpapan: Forda Press; 2018.118p.
11. Wardana F, Sari D, Adianti M, Permanasari A, Tumewu L, Nozaki T,
Widyawaruyanti A, Hafid AF. Amoebicidal activities of Indonesian
medicinal plants in Balikpapan, East Kalimantan. Proceedings of
BROMO Conference. ScitePress: 2019;77-82.
12. Ellman GL, Courtney KD, Andres V Jr, Featherstone RM. A new
and rapid colorimetric determination of acetylcholinesterase
activity. Biochem Pharmacol. 1961;7(2):88-95. https://doi.
org/10.1016/0006-2952(61)90145-9
13. Suciati, Laili ER, Poerwantoro D, Hapsari AP, Gifanda LZ, Rabgay
K, Ekasari W, Ingkaninan K. Evaluation of cholinesterase inhibitory
activity of six Indonesian Cassia species. J Res Pharm. 2020; 24(4):
472-478. http://dx.doi.org/10.35333/jrp.2020.195
14. Aristyawan AD, Setyaningtyas VF, Wahyuni TS, Widyawaruyanti
A, Ingkaninan K, Suciati S. In vitro acetylcholinesterase inhibitory
activities of fractions and iso-agelasine C isolated from the marine
sponge Agelas nakamurai. J Res Pharm. 2022; 26(2): 279-286.
http://dx.doi.org/10.29228/jrp.126
15. Herald TJ, Gadgil P, Tilley M. High-throughput micro plate assays
for screening flavonoid content and DPPH-scavenging activity in
sorghum bran and flour. J Sci Food Agric. 2012;92(11):2326-2331.
https://doi.org/10.1002/jsfa.5633
16. Lee KJ, Oh YC, Cho WK, Ma JY. Antioxidant and anti-inflammatory
activity determination of one hundred kinds of pure chemical
compounds using offline and online screening HPLC assay. Evid
Based Complement Alternat Med. 2015;2015: 165457. https://
doi.org/10.1155/2015/165457
17. Zhang Q, Zhang J, Shen J, Silva A, Dennis DA, Barrow CJ.
A simple 96-well microplate method for estimation of total
polyphenol content in seaweeds. J Appl Phycol. 2006; 18:
445–450. https://doi.org/10.1007/s10811-006-9048-4
18. Blois MS. Antioxidant determinations by the use of a stable
free r adic al. Nature. 1958; 181:1199-120 0. ht tps://doi.
org/10.1038/1811199a0
19. Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl
(DPPH) for estimating antioxidant activity. Songklanakarin J Sci
Technol. 2004;26(2):211- 219.
20. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans
C. Antioxidant activity applying an improved ABTS radical cation
decolorization assay. Free Radic Biol Med. 1999;26(9-10):1231–
1237. https://doi.org/10.1016/S0891-5849(98)00315-3
21. Mehta M, Adem A, Sabbagh M. New acetylcholinesterase
inhibitors for Alzheimer’s disease. Int J Alzheimers Dis.
2012;2012:728983. https://doi.org/10.1155/2012/728983
22. Danta CC, Piplani P. The discover y and development of
new potential antioxidant agent s for the treatment of
neurodegenerative diseases. Exper t Opin Drug Discov.
2014;9:1205–1222. https://doi.org/10.1517/17460441.2014.942218
23. Zhao Y, Zhao B. Natural antioxidants in prevention management
of Alzheimer’s disease. Front Biosci (Elite Ed). 2012;1:794–808.
https://doi.org/10.2741/e419
24. Suganthy N, Devi KP. In vitro antioxidant and anti-cholinesterase
activities of Rhizophora mucronata. Pharm Biol. 2016;5 4(1): 118-
29. https://doi.org/10.3109/13880209.2015.1017886
25. Giacobini E. Cholinesterases: New roles in brain function and in
Alzheimer’s disease. Neurochem Res, 2003; 28 (3-4):515–522.
https://doi.org/10.1023/a:1022869222652
26. Lopa SS, Al-Amin MY, Hasan MK, Ahammed MS, Islam KM,
Alam AHMK, Tanaka T, Sadik MG. Phytochemical analysis
and cholinesterase inhibitory and antioxidant activities of
Enhydra fluctuans relevant in the management of Alzheimer’s
disease. Int J Food Sci. 2021;2021:8862025. https://doi.
org/10.1155/2021/8862025
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