Philippine Journal of Science

151 (2): 545-561, April 2022

ISSN 0031 - 7683
Date Received: 10 Oct 2021

Cholinesterase Inhibitory Activities and In Silico

Docking Studies of Blumeatin Isolated
from Blumea balsamifera L. DC.

Rosemarie Elloisa P. Acero* and Evangeline C. Amor

Terrestrial Natural Products Laboratory, Institute of Chemistry

University of the Philippines Diliman, Quezon City 1100 Philippines

Cholinesterase inhibition provides symptomatic treatment for neurodegenerative diseases

such as Alzheimer’s disease. Commercially available drugs on the market mostly target
acetylcholinesterase. However, evidence suggests that selective inhibition of butyrylcholinesterase
may lead to greater efficacy with fewer side effects and slower disease progression. The aim of
this research was to isolate a butyrylcholinesterase-selective inhibitor from Blumea balsamifera
L. DC. In vitro cholinesterase assays were used to evaluate the inhibitory activity and mechanism
of action of blumeatin. Molecular docking studies were performed to support the BuChE-
selectivity of blumeatin. Blumeatin inhibited BuChE in a concentration-dependent manner, with
an IC50 of 136.3 ± 12.6 μM and a selectivity ratio of 1.395 for BuChE over AChE. Additionally,
the Ki of blumeatin is 10 times lower in BuChE compared to AChE. Molecular docking studies
confirmed the selectivity, as revealed by the tighter hydrogen bonding of blumeatin with the
BuChE’s active site. Blumeatin acts as a competitive inhibitor and a noncompetitive inhibitor
of BuChE and AChE, respectively. With these results, blumeatin could be a potential lead as a
drug treatment for AD because of its butyrylcholinesterase selectivity.

Keywords: Alzheimer’s disease, butyrylcholinesterase- selective, flavanone, molecular docking

INTRODUCTION prevention. The pathological hallmarks associated with

A l z h e i m e r ’s d i s e a s e ( A D ) i s a p r o g r e s s i v e
neurodegenerative disorder characterized by memory loss
and cognitive impairment. From the World Alzheimer’s
Report in 2019, AD afflicts over 50 million people
globally – a number that is expected to triple by 2050
(Alzheimer’s Disease International 2019). Because the
pathogenesis of AD is poorly understood, available
treatment focuses on symptom relief rather than disease
*Corresponding author: rpacero@up.edu.ph
AD include [1] loss of cholinergic neurotransmission in
brain areas attributed to higher cognitive functioning due
to low levels of acetylcholine (ACh), [2] aggregation of
neurotoxic β-amyloid protein into malignant plaques,
and [3] coupling of hyperphosphorylated tau into
neurofibrillary tangles (Davies and Maloney 1976; Hardy
and Allsop 1991; Polinsky 1998). In the AD brain, ACh
levels decline in the neocortex and hippocampus due to
its excessive hydrolysis by cholinesterases – namely,
acetylcholinesterase (AChE) and butyrylcholinesterase

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Vol. 151 No. 2, April 2022
(BuChE) (Davies and Maloney 1976). To restore normal
levels of ACh, symptomatic treatment relies on inhibition
of these cholinesterases with drugs such as rivastigmine,
donepezil, and galantamine.
AChE and BuChE differ in substrate specificity and
enzyme kinetics despite sharing 65% amino acid
sequence homology (Soreq and Zakut 1993). AChE is
primarily responsible for the breakdown of ACh in the
healthy normal brain, with BuChE playing a minor role.
BuChE only assists in ACh hydrolysis at high substrate
concentrations, where AChE becomes substrate inhibited
(Masson et al. 2009; Radić et al. 1991). Both enzymes
share the same regions in their 20Å hydrophobic gorge
catalytic active site: catalytic triad (CT; Ser, His, and
Glu), choline-binding pocket (Choline BP), acyl-binding
pocket (Acyl BP), peripheral anionic subsite (PAS), and
the oxyanion hole. The Acyl BP of BuChE houses two
smaller amino acids – Leu286 and Val288 (as opposed
to Phe295 and Phe297 in AChE) – which allows it
to accommodate larger substrates (Macdonald et al.
2012). As a consequence, BuChE also participates in the
metabolism of other larger molecules such as neurotoxic
esters, carbamates, and organophosphates (Taylor and
Radić 1994).
As AD progresses, AChE activity remains unchanged
or decreases to 62–67% of normal levels, while
BuChE activity increases (Perry et al. 1978). BuChE
also accumulates within the amyloid plaques and
neurofibrillary tangles, amplifying their neurotoxicity
(Darvesh et al. 2012; Gómez-Ramos et al. 1994). Selective
inhibition of BuChE can, therefore, be the focus of a new
treatment strategy for AD. Isolation of BuChE inhibitors
may [1] pave the way for a better understanding of
BuChE’s contribution to AD and its relationship to other
pathologies involved in AD such as β-amyloid aggregation
and tau-protein hyperphosphorylation; [2] improve
understanding of the etiology of AD, opening a wide
possibility for the development of new and more potent
treatment options; and [3] lead to treatments with lower
side effects than the currently used inhibitors.
Blumea balsamifera L. DC has been commercially
available as a medication for kidney stone dissolution as
“Re-Leaf” in the Philippines (Cortes-Maramba et al. n/d).
There are over 100 volatile or non-volatile constituents
isolated from Blumea balsamifera L. DC. – including
monoterpenes, sesquiterpenes, diterpenes, flavonoids,
organic acids, esters, alcohols, dihydroflavones, and
sterols (Pang et al. 2014). Extracts of Blumea balsamifera
L. DC. leaves have been reported to display various
physiological activities such as anticancer (Norikura
et al. 2008), anti-genotoxicity and anti-mutagenicity
(Sylianco et al. 1987), antimicrobial (Sakee et al. 2011),
antioxidant (Nessa et al. 2004), anti-plasmodial (Noor
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Acero and Amor: Cholinesterase Inhibitory Activities
and In Silico Docking Studies of Blumeatin
Rain et al. 2007), and anti-obesity (Kubota et al. 2009).
Preliminary data showed an active fraction against BuChE
from the ethyl acetate extract of its leaves that can be
pursued. This study aimed to isolate a BuChE inhibitor
from this active fraction of Blumea balsamifera L. DC.
using BuChE inhibition-guided isolation and explain the
BuChE-specificity of the active isolate via molecular
docking studies.
MATERIALS AND METHODS
Materials and Chemicals
All solvents used for extraction and fractionation were
technical grade and single distilled before use. For
inhibition assays, analytical grade solvents were used.
Electrophorus electricus AChE, equine serum BuChE,
5,5-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent,
DTNB), acetylthiocholine iodide, butyrylthiocholine
iodide, and galantamine hydrobromide were purchased
from Sigma-Aldrich. Rivastigmine tartrate was purchased
from drugstores, as it is commercially available as
Exelon® (Novartis).
Dried and powdered mature leaves of Blumea balsamifera
L. DC. were obtained from Leonie Agri Corporation, Sta.
Rosa, Nueva Ecija. The plant was authenticated at the Jose
Vera Santos Memorial Herbarium, Institute of Biology,
University of the Philippines Diliman, Quezon City, and
a voucher specimen (Accession No. 7083) was stored at
the same place.
Instruments
The melting point was obtained with a Fisher-Johns
melting point apparatus. UPLC chromatogram and
HRMS spectrum were acquired in a UPLC system
(Waters Acquity UPLC®) connected to a quadrupole
time-of-flight high-resolution mass spectrometer (Xevo
G2-XS QTof) with an electrospray interface in positive
mode. The UV spectrum was recorded in AR-grade
methanol using Thermo Scientific MultiSkan GO. IR
spectrum was determined by attenuated total reflectance
(ATR) using an IRPrestige-21 Fourier transform infrared
spectrophotometer. The 1D (1H and 13C NMR) and
2D-NMR (DEPT, COSY, HSQC, HMBC) spectra were
recorded at 500 MHz (126 MHz for 13C) Agilent NMR
spectrometer equipped with a 3-mm One NMR probe and
VNMRj ver 2.3 software.
Extraction and Isolation
Dried and powdered mature leaves of Blumea balsamifera
L. DC. (1.197 kg) were extracted five times, at 3-d
intervals, with methanol. The filtrate was collected and
Philippine Journal of Science
Vol. 151 No. 2, April 2022
concentrated in vacuo at 40 °C and 100 rpm. The resulting
methanol extract was partitioned exhaustively between
H2O/hexane (1:6 v/v) followed by ethyl acetate. The
resulting hexane and ethyl acetate layers were collected
and concentrated at 40 °C and 100 rpm. The remaining
aqueous layer was lyophilized.
Sequential fractionation of the ethyl acetate (20.07 g)
extract using vacuum liquid chromatography on silica
gel (type 60, 70–230 mesh) employing gradient elution
yielded 13 fractions (EV1-EV13). A white powder
(151.6 mg) from EV8, precipitated out upon addition of
methanol, was chromatographed over a silica gel column
under gradient conditions – starting with 100-mL of 1.5:1
hexane: ethyl acetate and increasing 10% increments of
ethyl acetate followed by 50% and 100% methanol – to
afford 12 fractions (EV8G1-12). Fractions EV8G3-6 were
rechromatographed over silica gel at gradient conditions
using the same procedure as above but starting with 2.3:1
hexane: ethyl acetate, giving 10 fractions. Blumeatin (20.1
mg) precipitated out from subfractions EV8G3.4–3.6
upon addition of methanol. It has an Rf value of 0.16 in
2.3:1 hexane: ethyl acetate and can be visualized under
UV (254 nm) and I2. The percent yield of blumeatin with
respect to ethyl acetate extract and Blumea balsamifera L.
DC. dried leaves are 0.1001 and 0.0017%, respectively.
5,3’,5’-trihydroxy-7-methoxyflavanone or blumeatin:
white powder; mp 220-222 oC; HRMS (positive mode) at
3.02 kV (% relative abundance) C16H14O6 [M+H]+ found
303.0870 (100), calcd 303.0869, error 0.3 ppm; UV nm
(MeOH, λmax) 337 sh, 286, 228, 206 nm; IR bands (ATR)
3174.83, 2958.80, 1600.92, 1573.91, 1450.47, 1357.89,
1300.02, 1269.16, 1188.15, 1149.57, 825.53, 717.52 cm–1;
1H-NMR (500 MHz, DMSO-d , ppm) δ 2.72 (dd, J = 17.1,
6 graphpad.com). The same was also used for the calculation
3.0 Hz, H-3a), 3.24 (dd, J = 17.1, 12.6 Hz, H-3b), 3.79 (s,
C7-OCH3), 5.42 (dd, J = 12.6, 3.0 Hz, H-2), 6.07 (d, J =
2.3 Hz, H-8), 6.09 (d, J = 2.3 Hz, H-6), 6.75 (s, H-2’ and
6’), 6.89 (s, H-4’), 9.02 (s, C3’-OH), 9.08 (s, C5’-OH),
12.11 (s, C5-OH); 13C-NMR (126 MHz, DMSO-d6, ppm)
δ 42.16 (C-3), 55.90 (C7-OCH3), 78.68 (C-2), 93.80 (C-
8), 94.60 (C-6), 102.6 (C-10), 114.4 (C-2’), 115.3 (C-6’),
118.0 (C-4’), 129.3 (C-1’), 145.2 (C-3’), 145.8 (C-5’),
162.8 (C-9), 163.2 (C-5), 167.4 (C-7), 197.0 (C-4).
Enzyme Assays
Enzyme inhibitory activity against BuChE and AChE was
observed spectrophotometrically at pH 8.0 and 25 °C with
rivastigmine and galantamine as the positive controls.
The reaction mixture consists of 180 μL of 100-mM
phosphate buffer at pH 8.0, 10 μL of 1.0 U/mL BuChE
or AChE enzyme solution, 80 μL of buffered Ellman’s
reagent (10.0 mM DTNB and 17.9 mM NaHCO3 at pH
7.0), and 15 μL of the test sample solution in methanol.
Due to solubility limitations in methanol, blumeatin was
Acero and Amor: Cholinesterase Inhibitory Activities
and In Silico Docking Studies of Blumeatin
dissolved in small amounts of DMSO. After incubation for
15 min at 25 °C, the reaction was initiated by the addition
of 15 μL of the substrate (either 7.0 mM butyrylthiocholine
iodide or 94 μM acetylthiocholine iodide). The increase
in absorbance at 410 nm (BuChE) or 420 nm (AChE)
due to the formation of yellow 5-thio-2-nitrobenzoate
anion was monitored at a 20-s interval for 10 min. The
data were analyzed by plotting the absorbance against
time, where the slope of the best-fit line is equivalent to
the reaction rate. Percent inhibition was computed using
Equation 1. Each concentration was assayed in two trials
and two replicates (n = 2, t = 2). Statistical analysis was
performed using SPSS Statistics 17.0 software.
(1)
IC50 value (inhibitor concentration with 50% inhibition)
of blumeatin with BuChE was determined by getting the
rate of reaction of BuChE with butyrylthiocholine iodide
under the same conditions above over a range of blumeatin
concentrations (16.55–297.9 μM) dissolved in 0.01–0.8%
DMSO. For the mode of inhibition, BuChE was reacted
with varying concentrations of substrate and 136.3 μM
of blumeatin. The same procedure was performed with
AChE using 92.6 μM of blumeatin. For both inhibition
studies, two-fold serial dilutions were tested in duplicate.
The curves for dose-response, Michaelis-Menten, and
Lineweaver-Burke plots of blumeatin were generated
using Graphpad Prism version 8.0.0 for Windows
(Graphpad Software, La Jolla California USA, www.
of Ki and IC50 values.
Molecular Docking Studies
The crystals structures of hAChE and hBuChE were
retrieved from Protein Data Bank (PDB) database (www.
pdb.org) with PDB IDs 4EY7 (resolution: 2.35 Å) and
1P0P (resolution: 2.30 Å), respectively (Cheung et al.
2012; Nicolet et al. 2003). PDB 4EY7 (hAChE) was
selected because of its high similarity to Electrophorus
electricus and the presence of donepezil as the ligand in the
active site (Cheung et al. 2012). For hBuChE, PDB 1P0P
was chosen due to its high resolution and the presence of
butyrylcholine as the co-crystallized ligand (Nicolet et
al. 2003). The ligand and receptor were prepared using
LigPrep and Protein Preparation Wizard of Schrodinger
Suites 2020-3, respectively. The ligand was prepared by
generating all stereoisomers and possible states using
Epik at pH 7.0 ± 2.0 and the force field of OPLS_2005.
The receptors were prepared by removing non-bonded
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Vol. 151 No. 2, April 2022
inhibitors and unwanted water molecules, adding
hydrogen atoms, filling in missing side chains and loops
using Prime, generating het states using Epik at pH 7.0 ±
2.0, predicting pKa using PROPKA, and minimizing the
energy using OPLS_2005 and RMSD of 0.30 Å (Olsson
et al. 2011). For both receptors, the grid was generated
by specifying the active site as the centroid of the grid
with a size of 15 Å. Hydroxyl groups of some residues
in the active site were allowed to rotate (Tyr124, Tyr133,
Ser203, Tyr337, and Tyr341 in AChE; Tyr128, Ser198,
and Tyr332 in BuChE). Flexible molecular docking was
carried out using GRID (Grid-based Ligand Docking
with Energetics) in the extra precision mode without
applying any constraints (Friesner et al. 2006). The
graphic representations of the best-docked poses, those
with the most negative docking score, were rendered in
UCSF Chimera 1.14 (Pettersen et al. 2004).
RESULTS AND DISCUSSION
Structure Elucidation
The 13C NMR spectrum and DEPT experiment showed
16 signals consisting of one methyl, one methylene, six
methine, and eight quaternary carbon atoms – similar to
that reported for blumeatin (Nessa et al. 2004). 1H-NMR
shifts at δ 2.72 ppm (1H, dd, J = 17.1, 3.0 Hz) and δ 3.24
ppm (1H, dd, J = 17.1, 12.6 Hz) are due to diastereotopic
protons of C3 and are characteristic chemical shifts
of flavanones. Both protons couple with H-2 (δ 5.42
ppm, 1H, dd, J = 12.6, 3.0 Hz), as confirmed by the
presence of cross-peaks in its COSY spectrum. The
aromatic protons were assigned based on the HSQC and
HMBC correlations. Protons on ring A, δ 6.07 and 6.09
ppm, couple with each other at a frequency of 2.3 Hz.
Overlapped resonance at δ 6.75 ppm (2H, s) corresponds
to protons attached to C-2’ and C-6’, while that at δ 6.89
ppm (1H, s) is for C-4’ of B ring. Resonance at δ 3.79
ppm (3H, s) is assignable to methoxyl protons attached
to C-7. The resonances at δ 9.02 ppm (1H, s) and δ 9.08
ppm (1H, s) correspond to hydroxyl protons of C-3’ and
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Acero and Amor: Cholinesterase Inhibitory Activities
and In Silico Docking Studies of Blumeatin
C-5’, respectively. The shift at δ 12.11 ppm (1H, s) is from
the hydroxyl group of C-5, which is more downfield due
to the β-C=O group.
Its molecular formula C16H14O6 is confirmed by the
molecular ion peak [M+H]+ at m/z = 303.0870 in the
HRMS. In its MS/MS spectrum, the peak observed at
m/z = 167.0359 corresponds to the product of the retro
Diels-Alder cleavage of blumeatin, as shown in Figure 1.
BuChE Assay-guided Isolation of Blumeatin
BuChE inhibitory activities of methanol, hexane, ethyl
acetate, and aqueous extracts together with HV and EV
fractions and EV8 subfractions at 100 ppm are shown in
Figure 2. Moderate inhibitory activities (10–40%) were
observed for the methanol, hexane, ethyl acetate, and
aqueous extracts of Blumea balsamifera L. DC. Fraction
EV8, with inhibitory activity of 66.37 ± 4.76% at 100 ppm,
was pursued. A white powder precipitated out of EV8,
upon the addition of methanol, which was then subjected
to gravity column chromatography (GCC). From the
GCC subfractions (EV8G1-12), five subfractions have
inhibitory activities ranging from 75–100%, and three
fractions have activities of 50–75%. Subfractions EV8G3-
6 were pooled based on inhibitory activity and TLC
profile then subjected to GCC. From the subfractions
(EV8G3.1–EV8G3.10), blumeatin precipitated from the
pooled EV8G3.4-6 upon addition of methanol.
BuChE and AChE Activity of Blumeatin
Blumeatin inhibited BuChE in a concentration-dependent
manner, as shown in Figure 3. Its IC50, the concentration
that exhibited half-maximal inhibition, is 136.3 ± 12.6
uM. At this concentration, blumeatin has AChE inhibitory
activity of 35.85 ± 0.04%. Given this, the selectivity
ratio of BuChE over AChE is 1.395. The IC50­ values of
blumeatin and the positive controls against AChE and
BuChE are listed in Table 1.
Figure 4 displays the Michaelis-Menten and Lineweaver-
Burk plots of the inhibition kinetics of blumeatin with
AChE and BuChE. From the double-reciprocal plot of
AChE, the regression lines intersect at the same x-intercept
(1/KM) denoting a non-competitive type of inhibition
with a Ki of 413.8 ± 46.2 μM for blumeatin. In BuChE,
blumeatin acts as a competitive inhibitor, as observed in
the slightly positive value of the interception point (1/
Vmax) of the regression lines in the double-reciprocal
plot. It has a lower Ki of 37.98 ± 2.79 μM for BuChE,
suggesting more selective inhibition with BuChE.
Docking Studies of Blumeatin
Molecular docking revealed insights on the interactions
of blumeatin to the active site of AChE and BuChE.
Philippine Journal of Science Acero and Amor: Cholinesterase Inhibitory Activities
Vol. 151 No. 2, April 2022 and In Silico Docking Studies of Blumeatin

Figure 1. Retro Diels-Alder cleavage of blumeatin.

Figure 2. Inhibitory activity of methanol, hexane, ethyl acetate, and aqueous extracts together with HV and EV
fractions and EV8 subfractions with BuChE at 100 ppm (n = 2, t = 2); *Statistically significant from the
negative control at p < 0.05.

Table 2 lists the Gscore values of the best-docked poses
of blumeatin, galantamine, and rivastigmine. Gscore
approximates the ligand binding free energy, while
the docking score accounts for the Epik state penalties
(Friesner et al. 2006). Table 3 and Figure 5 show the
residues involved in the interaction of blumeatin with the
active sites of AChE and BuChE. Blumeatin is embedded
into AChE by encompassing the region of CT and Acyl
BP. Hydrogen bonding interactions were established
between the following ligand atom-AChE residue pairs:
the oxygen atom of ring B (O7) with Ser203 and Gly121
and the oxygen atom of ring B (O5’) with the backbone

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Vol. 151 No. 2, April 2022 and In Silico Docking Studies of Blumeatin

Table 1. AChE and BuChE IC50 ­­values of blumeatin and the positive
controls.
Compound IC50 for AChE, μM IC50 for BuChE, μM
Blumeatin NT 136.3 ± 12.6
Galantamine 0.3616 ± 0.0445 19.07 ± 1.20
Rivastigmine 106.8 ± 18.2 0.4985 ± 0.0089
NT – not tested; values are expressed as mean ± SD for IC50 (n = 2, t = 2).

Table 2. Gscore and docking scores of blumeatin and the positive

controls with AChE and BuChE.
AChE BuChE
Compound Docking Gscore Docking Gscore
Figure 3. Concentration-dependent nonlinear regression of the score score
inhibition of BuChE by blumeatin (n = 2, t = 2, R2 = Blumeatin –8.857 –8.862 –8.982 –8.987
0.9635).
Galantamine –13.374 –13.391 –8.755 –8.771
Rivastigmine –8.831 –8.881 –8.130 –8.179
of Phe295. Ring A of blumeatin interacted with the π
system of His447 and Phe338, whereas ring B is oriented
towards Tyr 341. Blumeatin simultaneously occupied the bonding with Tyr 332, while the hydroxyl group of ring A
CT, Choline BP, and PAS of BuChE via several tighter (OH-5) acted as a donor and acceptor in the bonding with
hydrogen binding and π-π stacking interactions. For Glu197 and Tyr128. Ring B is engaged in π-π stacking
hydrogen bonding, H3’ of ring B acted as a donor in the interactions with Phe329 and Tyr332. Blumeatin binds

Figure 4. Michaelis-Menten and Lineweaver-Burk plots of blumeatin with AChE (a, b) and BuChE (c, d). R2 values
for the different linear and nonlinear regression lines were > 0.9500 (n = 2, t = 2).

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Vol. 151 No. 2, April 2022 and In Silico Docking Studies of Blumeatin

Table 3. Interaction of blumeatin with the active site of AChE and BuChE.
Enzyme Interaction Residues (subdomain)* Bond distance (Å) Interacting ligand moiety
AChE Hydrogen bonding Ser203 (CAS) 2.132 O7

Phe295 (Acyl BP) 2.576 O5’

Gly121 (Oxyanion hole) 2.697 O7
π-π stacking Phe338 (Acyl BP) Ring A
Tyr341 (PAS) Ring B
His447 (CAS) Ring A
BuChE Hydrogen bonding Tyr332 (PAS) 1.838 H3’
Glu197 (CAS) 2.142 H5
Tyr128 (Choline BP) 2.176 O5
π-π stacking Phe329 (Choline BP) Ring B
Tyr332 (PAS) Ring B
*CAS – catalytic active site; BP – binding pocket; PAS – peripheral anionic site

Figure 5. Top binding poses of blumeatin with AChE and BuChE in 3D (a, b) and 2D (c, d), respectively. In 3D, purple
dotted lines indicate hydrogen bonding with respective distances. Purple arrows denote hydrogen bonding, while
green lines correspond to π-π stacking in the 2D diagram.

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Vol. 151 No. 2, April 2022
more tightly with BuChE, as evidenced by the shorter
hydrogen bonding interactions with the active site. These
docking results support the experimental selectivity of
blumeatin with BuChE.
CONCLUSION
Blumeatin was isolated from the leaves of Blumea
balsamifera L. DC. by employing BuChE assay-guided
isolation, and its structure was elucidated by 1D and 2D
NMR and verified by UV, FT-IR, and LC-MS. Blumeatin
is a selective inhibitor of BuChE at 136.3 ± 12.6 μM with
a selectivity ratio of 1.395 for BuChE over AChE. This
selectivity is supported by the molecular docking studies
that showed tighter hydrogen bonding of blumeatin with
the catalytic active site of BuChE. It is a competitive
inhibitor of BuChE and a non-competitive inhibitor of
AChE.
ACKNOWLEDGMENTS
The authors would like to thank the Department of Science
and Technology–Philippine Council for Health Research
and Development for the financial support.
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APPENDIX A
BuChE Activity of Blumea balsamifera L. DC. Extracts

Table A1. BuChE activity of methanol, hexane, ethyl acetate, and Table A3. BuChE activity of EV8 white powder GCC subfractions at
aqueous extract at 100 ppm, with rivastigmine as the 100 ppm, with rivastigmine as the positive control.
positive control. Subfraction % inhibition ± SD
Extract % inhibition ± SD
G1 41.67 ± 1.03
Methanol 28.62 ± 2.34
G2 54.12 ± 2.93*
Hexane 30.68 ± 0.73
G3 83.35 ± 0.37*
Ethyl acetate 32.27 ± 1.13*
G4 84.86 ± 1.76*
Aqueous 32.91 ± 4.33*
G5 79.09 ± 0.95*
(+) control 100.3 ± 0.1*
G6 76.53 ± 0.42*
*Statistically different from the negative control at p < 0.05
G7 50.30 ± 0.69
G8 63.38 ± 2.97*
G9 82.54 ± 1.23*
Table A2. BuChE activity of hexane and ethyl acetate VLC fractions
at 100 ppm, with rivastigmine as the positive control. G10 41.36 ± 0.75*

% inhibition ± SD G11 14.73 ± 14.67

Fraction G12 0.49 ± 7.64
Hexane Ethyl acetate
V1 20.11 ± 8.25 16.56 ± 10.61 (+) control 100.1 ± 0.1*
*Statistically different from the negative control at p < 0.05
V2 44.22 ± 27.80 29.52 ± 8.37
V3 37.04 ± 0.59 49.50 ± 2.60
V4 44.02 ± 4.61 60.75 ± 0.24*
V5 59.92 ± 5.81 64.53 ± 4.42*
V6 60.98 ± 2.33 69.74 ± 1.75*
V7 55.80 ± 14.48* 66.79 ± 0.6862*
V8 48.74 ± 2.05 66.37 ± 4.76*
V9 51.04 ± 4.18* 58.91 ± 0.71*
V10 56.52 ± 1.89* 61.28 ± 5.65*
V11 43.57 ± 4.92 47.05 ± 7.27*
V12 34.10 ± 7.11 41.42 ± 0.78*
V13 9.852 ± 14.056 15.07 ± 2.73
(+) control 100.3 ± 0.1* 99.37 ± 0.42*
*Statistically different from the negative control at p < 0.05

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APPENDIX B

Kinetic Data for BuChE and AChE Inhibition of Blumeatin

Table B1. Percent inhibition of different concentrations of blumeatin

with 0.033 U/mL BuChE and 0.35 mM butyrylthiocholine
iodide.
[Blumeatin], µM % inhibition ± SD
297.93 81.2434 ± 0.7743
264.83 79.2815 ± 3.7258
231.72 76.0535 ± 0.6968
198.62 72.2751 ± 0.1988
165.52 69.7337 ± 3.6403
132.41 53.4203 ± 3.1377
99.31 40.9167 ± 2.2636
66.21 40.6627 ± 3.9204
49.65 29.2080 ± 0.3083
33.10 25.3557 ± 0.6037
16.55 26.7088 ± 3.1735
*132.41 34.4822 ± 3.2604

Table B2. Reaction velocity of different concentrations of butyrylthiocholine iodide with 1.0 units/mL BuChE/AChE in the absence and
presence of 136.3 µM/ 92.7 µM blumeatin.
Reaction velocity ± SD
[Substrate], µM BuChE AChE
No inhibitor 136.3 µM blumeatin No inhibitor 92.7 µM blumeatin
3750.00 0.4578 ± 0.0176 0.3685 ± 0.0478 0.0543 ± 0.0051 0.0692 ± 0.0042
2500.00 0.4633 ± 0.0049 0.3168 ± 0.0371 0.0555 ± 0.0061 0.0659 ± 0.0015
2000.00 0.4401 ± 0.0101 0.2770 ± 0.0260 0.0614 ± 0.0082 0.0590 ± 0.0106
1500.00 0.4272 ± 0.0093 0.2468 ± 0.0185 0.0619 ± 0.0093 0.0586 ± 0.0054
1000.00 0.3903 ± 0.0091 0.2150 ± 0.0266 0.0593 ± 0.0034 0.0395 ± 0.0051
500.00 0.3158 ± 0.0125 0.1372 ± 0.0205 0.0531 ± 0.0032 0.0308 ± 0.0011
250.00 0.1949 ± 0.0079 0.0820 ± 0.0099 0.0327 ± 0.0090 0.0255 ± 0.0068
125.00 0.1380 ± 0.0033 0.0504 ± 0.0020 0.0199 ± 0.0078 0.0175 ± 0.0037
62.50 0.0731 ± 0.0014 0.0247 ± 0.0058 0.0064 ± 0.0018 0.0092 ± 0.0017
31.25 0.0351 ± 0.0018 0.0172 ± 0.0059 0.0039 ± 0.0005 0.0054 ± 0.0017
15.60 0.0190 ± 0.0015 0.0056 ± 0.0006 0.0023 ± 0.0008 0.0030 ± 0.0006

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APPENDIX C

Spectroscopic Spectra of the Isolated Blumeatin

Figure C1. HPLC chromatogram of blumeatin isolated from Blumea balsamifera.

Figure C2. UPLC chromatogram of [a] blank, [b] blumeatin isolated from Blumea balsamifera, [c] selected HRMS source (tR = 3.70 min),
and [d] HRMS spectrum.

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Figure C3. [a] MS spectrum and [b] MS/MS spectrum of blumeatin isolated from Blumea balsamifera at tR = 3.70 min.

Figure C4. UV spectrum of blumeatin isolated from Blumea

Figure C5. IR spectrum of blumeatin isolated from Blumea
balsamifera in methanol.
balsamifera.

Figure C6. 1H NMR spectrum of blumeatin isolated from Blumea balsamifera in DMSO-d6 at 500 MHz.

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Figure C7. 13C NMR spectrum of blumeatin isolated from Blumea balsamifera in DMSO-d6 at 126 MHz.

Figure C8. DEPT spectrum of blumeatin isolated from Blumea balsamifera in DMSO-d6 at 500 MHz.

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Figure C9. HMBC spectrum of blumeatin isolated from Blumea balsamifera in DMSO- d6 at 500 MHz.

Figure C10. HSQC spectrum of blumeatin isolated from Blumea balsamifera in DMSO-d6 at 500 MHz.

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Figure C11. COSY spectrum of blumeatin isolated from Blumea balsamifera in DMSO-d6 at 500 MHz.

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APPENDIX D

Docking Interactions of Galantamine and Rivastigmine with the Active Site of AChE and BuChE

Figure D1. Top binding poses of galantamine (a, b) and rivastigmine (c, d) with AChE and BuChE, respectively. Purple arrows denote
hydrogen bonding, while green lines correspond to π-π stacking in the 2D diagram.

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