Journal of Pharmaceutical Sciences xxx (2020) 1-9

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Journal of Pharmaceutical Sciences

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Drug DiscoveryeDevelopment Interface

Blood-Brain Barrier Permeability of Asiaticoside, Madecassoside

and Asiatic Acid in Porcine Brain Endothelial Cell Model
Nur Aziah Hanapi a, *, Ahmad Saifuddin Mohamad Arshad a, Jafri Malin Abdullah b,
Tengku Sifzizul Tengku Muhammad c, Siti R. Yusof a, **
a
Centre for Drug Research, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
b
Brain and Behaviour Cluster, Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia Health Campus, Kubang Kerian,
16150 Kota Bharu, Kelantan, Malaysia
c
Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Neurotherapeutic potentials of Centella asiatica and its reputation to boost memory, prevent cognitive
Received 7 June 2020 deficits and improve brain functions are widely acknowledged. The plant's bioactive compounds, i.e.
Revised 24 August 2020 asiaticoside, madecassoside and asiatic acid were reported to have central nervous system (CNS) actions,
Accepted 9 September 2020
particularly in protecting the brain against neurodegenerative disorders. Hence, it is important for these
compounds to cross the blood-brain barrier (BBB) to be clinically effective therapeutics. This study aimed
to explore the capability of asiaticoside, madecassoside and asiatic acid to cross the BBB using in vitro BBB
Keywords:
Blood-brain barrier model from primary porcine brain endothelial cells (PBECs). Our findings showed that asiaticoside,
Permeability madecassoside and asiatic acid are highly BBB permeable with apparent permeability (Papp) of
In vitro model 70.61 ± 6.60, 53.31 ± 12.55 and 50.94 ± 10.91
106 cm/s respectively. No evidence of cytotoxicity and
Endothelial
tight junction disruption of the PBECs were observed in the presence of these compounds. Asiatic acid
Natural products
Central nervous system showed cytoprotective effect towards the PBECs against oxidative stress. This study reported for the first
time that Centella asiatica compounds demonstrated high capability to cross the BBB, comparable to
central nervous system drugs, and therefore warrant further development as therapeutics for the
treatment of neurodegenerative diseases.
®
© 2020 American Pharmacists Association . Published by Elsevier Inc. All rights reserved.

Introduction

Centella asiatica (L.) Urb. (Apiaceae), widely recognized as gotu

Abbreviations: Ab, amyloid-b; AD, Alzheimer's disease; BBB, blood-brain barrier;
BSA, bovine serum albumin; C. asiatica, Centella asiatica; CNS, central nervous
system; CO2, carbon dioxide; CPT-cAMP, 8-(4-chlorophenylthio) adenosine 30 ,50 -
cyclic monophosphate sodium salt; DMEM, Dulbecco's modified Eagle's medium;
DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GLUT, glucose transporter;
HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; hBMECs, human brain
microvascular endothelial cells; HBSS, Hank's balanced salt solution; H2O2,
hydrogen peroxide; HPLC, high performance liquid chromatography; ISF, interstitial
fluid; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MTT, 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NaF, sodium fluorescein; NO,
nitrogen oxide; O⁻2, singlet oxygen; $OH, hydroxyl radicals; Papp, apparent perme-
ability; PBECs, porcine brain endothelial cells; PBS, phosphate buffered saline; ROS,
reactive oxygen species; SEM, standard error of the mean; SOD, superoxide dis-
mutase; TEER, transendothelial electrical resistance; TPSA, topological polar surface
area; UV, ultraviolet.
* Corresponding author.
** Corresponding author.
E-mail addresses: aziah.hanapi@usm.my (N.A. Hanapi), sryusof@usm.my
(S.R. Yusof).
kola and known as pegaga in Malaysia is native to wetland areas of
tropical countries including Sri Lanka, India, China, Indonesia, and
Malaysia.1 Regarded as a rejuvenating herb, C. asiatica is recognized
for its traditional use to boost memory, prevent cognitive deficits as
well as improve brain functions, especially in Ayurvedic and Chi-
nese traditional medicine.2 In the setting of neurotoxic insults,
neuroprotective and cognitive enhancing effects of C. asiatica were
observed in multiple rodent models of dementia and neuro-
toxicity.3e6 Published data also suggest that C. asiatica significantly
improved memory performance as tested in rats7 and mice.8,9
C. asiatica consists of numerous triterpene compounds including
asiaticoside, madecassoside, asiatic acid, madecassic acid, betulinic
acid, isothankunic acid and thankunic acid.10 Amongst these tri-
terpenes, the most important biologically active compounds pre-
sent in C. asiatica are asiaticoside, madecassoside, and asiatic acid
(Fig. 1).11 Another important triterpene not shown in the figure is

https://doi.org/10.1016/j.xphs.2020.09.015
0022-3549/© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
2 N.A. Hanapi et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-9
Fig. 1. Chemical structures of principal triterpenes in Centella asiatica.
madecassic acid.11 Administration of asiaticoside, madecassoside,
and asiatic acid were efficacious as neuroprotectants in rodent
models of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-
induced parkinsonism, and in rodent models of stroke.12e18 Asiat-
icoside was reported to repair spinal cord injury,19 protected
against neuronal impairment triggered by ischemia/hypoxia20 and
relieved learning and memory damage instigated by Ab in a rat
model of AD.21 Madecassoside was demonstrated to increase
antioxidant enzyme activities in disease models and reduce
oxidative stress,22 in addition to exhibit protective effect against
myocardial ischemia-reperfusion injury after regional ischemia in
rabbit heart.23 Asiatic acid has drawn more attention from scien-
tists because of its therapeutic potential including protective effect
against spinal cord injury via suppression of oxidative stress and
inflammation in rats,24 as well as protective effect against t-BHP-
induced cellular damage and oxidative stress by modulating Nrf2
signaling through activating the signals of Akt and ERK in HepG2
cells.25 Asiatic acid was also reported to exhibit protective effect
against Ab-induced cell death in the neuroblastoma B103 cells and
decreased intracellular free radical concentration thus reducing
H2O2-related cell death.26,27
To be successful CNS therapeutics, a compound has to be able to
cross the blood-brain barrier (BBB). However, to the best of our
knowledge, data on brain permeation of the compounds are still
lacking. BBB is made up of endothelial cells that established the
wall of cerebral microvessels. The cells form tight junction which
seals the paracellular pathway between adjacent cells.28 The BBB
plays an important role to regulate molecular traffic between the
blood and the CNS by a combination of physical, transport and
enzymatic barriers.29 Maintaining the BBB integrity is critical to
control the chemical composition of brain interstitial fluid (ISF),
which is vital for proper neuronal connectivity, synaptic func-
tioning and information processing.30,31 Disruption of the BBB
permits blood-derived molecules which could be harmful to the
neurons entering the brain, and can lead to neurodegenerative
diseases including AD.32 Oxidative stress which can be triggered by
the imbalance between reactive oxygen species (ROS) and antiox-
idants is one of the factors that can disrupt the BBB function.33 For
instance, ROS was demonstrated to increase endothelium perme-
ability to large molecules in ischemia-reperfusion model of super-
oxide dismutase (SOD) deficient mice.34 In in vitro models,
superoxide and other ROS caused leakage of the BBB in a time- and
concentration-dependent manner.35e37 Lee et al. reported ROS-
induced BBB impairment, deduced from decreased trans-
endothelial electrical resistance (TEER) of primary bovine brain
microvessel endothelial cells when challenged with hydrogen
peroxide (H2O2). However, increase in actin and occludin expres-
sions were demonstrated.35
Taking into account the potential of asiaticoside, madecassoside
and asiatic acid as therapeutics for neurodegenerative diseases, this
study aimed to investigate the capability of these C. asiatica com-
pounds to reach the brain through the BBB using primary porcine
brain endothelial cells (PBECs) as model. The model was previously
used as a tool to investigate mechanisms of the BBB permeability of
plant-based compounds,38,39 and validated as a reliable in vitro
model of the human BBB as it retains most of morphological and
functional characteristics of brain endothelial cells.40 Prior to the
in vitro BBB permeability assay, the PBECs viability and tight junc-
tion function in presence of the compounds were determined.
Realizing that ROS can induce BBB impairment in most of neuro-
logical conditions, the role of these compounds in protecting the
PBECs against a known ROS mediator H2O2 was determined.
Materials and Methods
Materials
Dulbecco's modified Eagle's medium (DMEM), penicillin/strep-
tomycin, heparin, L-glutamine, puromycin, hydrocortisone, trypsin-
EDTA, Hank's balanced salt solution (HBSS) without Mg2þ and Ca2þ,
HBSS without Phenol Red, HEPES sodium salt, 8-(4-
chlorophenylthio) adenosine 30 ,50 -cyclic monophosphate sodium
salt (CPT-cAMP), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT), bovine serum albumin (BSA), sodium
fluorescein (NaF), dimethyl sulfoxide (DMSO), H2O2, asiaticoside
(98.5% purity by HPLC), madecassoside (95% purity by HPLC)
and asiatic acid (97% purity) were obtained from Sigma-Aldrich
Chemical Co. (St. Louis, MO, USA). DMEM high glucose without
Phenol Red, phosphate buffered saline (PBS), fibronectin and heat-
inactivated fetal bovine serum (FBS) were obtained from Gibco Life
Technologies (Grand Island, USA). RO-20-1724 was purchased from
Calbiochem (La Jolla, USA). Corning Transwell® translucent poly-
carbonate filter insert (12 mm diameter, 0.4 mm pore size, 1
10⁸
pores/cm2, 1.12 cm2 growth area) were obtained from Corning
(New York, USA). HPLC grade of acetonitrile and methanol were
obtained from Merck (New Jersey, USA). Deionized water for HPLC
analysis was prepared in-house using ELGA PURELAB Option water
purification system (Illinois, USA). Rat tail collagen was prepared
according to Strom and Michalopoulos.41
 N.A. Hanapi et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-9
Culture of Primary Porcine Brain Endothelial Cells
Microvessels were isolated from porcine brain according to the
protocol published by Patabendige et al. with modifications.42,43 To
obtain primary porcine brain endothelial cells (PBECs), two T75
flasks were coated with lab-prepared rat tail collagen for 2 h fol-
lowed by 7.5 mg/mL fibronectin for another 2 h at room temperature.
HBSS without Phenol Red was used to wash the flasks twice after
every coating. A cryovial containing porcine brain microvessel
fragments was removed from liquid nitrogen and thawed quickly in
a water bath at 37 C. Once thawed, the microvessel fragments were
gently added to pre-warmed culture medium containing DMEM
supplemented with FBS (10% v/v), 100 U/mL penicillin and 100 mg/
mL streptomycin, 2 mM L-glutamine, 125 mg/mL heparin and 4 mg/
mL puromycin. The culture medium was then added to the coated
culture flasks. The culture was incubated at 37 C in a humidified
atmosphere of 5% CO2. The next day, the culture medium was
changed to eliminate DMSO and debris. The PBECs grown out of the
microvessels and became confluent after three to four days in cul-
ture. The PBECs will then either passage into Transwell® or 96-well
plates depending on assays to be conducted (see sections below).
From the isolation, two types of microvessel fragments were ob-
tained, i.e. the 150's and 60's named according to nylon meshes
used. PBECs grown on Transwell® inserts from the 60's fragments
that passed the quality control, i.e. TEER value > 200 U,cm2 were
used for permeability assay. The PBECs from 150's fragments were
used for cytotoxicity and cytoprotection assay.
Determination of Cell Viability
Prior to the BBB permeability assay, cell viability assay was
conducted according to Mosmann with modifications.44 The PBECs
(150's) were seeded in 96-well plates coated with collagen/fibro-
nectin at a density of 1
105 cells/cm2. Culture medium was con-
sisted of DMEM supplemented with FBS (10% v/v), 100 U/mL
penicillin and 100 mg/mL streptomycin, 2 mM L-glutamine, 125 mg/
mL heparin. Cells were incubated for 2 days to achieve confluency.
Asiaticoside, madecassoside and asiatic acid were solubilized in
methanol, followed by dilution with culture medium. The final
concentration of methanol in the culture medium was <1%. The
compounds were added to the wells with the PBECs at 20 mg/mL,
followed by incubation of 1 h in the incubator (37 C, 5% CO2). Wells
containing cells without added compounds (untreated cells) were
used as negative control, while wells containing medium only
(without cells) were used as blanks. After the incubation, 5 mg/mL
MTT in PBS were added to each well. The MTT is sensitive to light, so
this step was carried out in the dark. The cells were then return to
the incubator for 4 h. Following that, the culture medium containing
MTT was discarded and replaced with 100 mL DMSO as solubilizing
agent. Absorbance of purple formazan crystals was read at 560 nm
and background absorbance at 690 nm using Multiskan Go Micro-
plate Reader (Thermo Fisher Scientific, MA, USA). The percentage of
cell viability was calculated using the following equation:
% of cell viability
Treated cellsðAbsorbance560  Absorbance690 Þ
¼
100
Untreated cellsðAbsorbance560  Absorbance690 Þ
(1)
In Vitro PBEC BBB Model
In vitro PBEC BBB model was set-up on collagen/fibronectin-
coated Transwell® inserts in 12-well format. The apical and the
3
basolateral compartments are considered as the blood and the
brain side respectively. The PBECs (60's) were seeded onto the in-
serts at a density of 1
105 cells/cm2 and cultured in medium as
described in the cell viability assay. When confluent, the culture
medium was change to serum-free medium supplemented with
550 nM hydrocortisone. The cells were treated with 250 mM CPT-
cAMP and 17.5 mM RO-20-1724 to induce differentiation of the
BBB properties, particularly tight junction tightness.
Measurement of Transendothelial Electrical Resistance (TEER)
The effect of asiaticoside, madecassoside and asiatic acid on the
PBEC monolayer tight junction integrity was evaluated by
measuring TEER using STX-100C chopstick electrode pair con-
nected to EVOM meter (World Precision Instruments Inc., Sarasota,
FL, USA). Approximately 24 h after the serum-free medium change
and the treatment with CPT-cAMP and RO-20-1724, the TEER was
recorded for 30 min at 1 min interval. Asiaticoside (20 mg/mL),
madecassoside (20 mg/mL), asiatic acid (20 mg/mL), DMEM (nega-
tive control), 100% DMSO (positive control) and 1% (v/v) methanol
in DMEM (vehicle control) were added to the inserts separately
after 10 min of TEER baseline measurement. After minute 30, the
inserts were returned to the incubator for another 30 min. The last
TEER was recorded at minute 60. The TEER values were corrected
for resistance across collagen/fibronectin-coated blank inserts
(without cells), followed by multiplication with 1.12 cm2, the
growth surface area of the filter insert to give the final unit of
U.cm2.
In Vitro BBB Permeability Assay
The PBECs on filter inserts with TEER more than 200 U,cm2
were used for permeability assay.45 From stock solution, asiat-
icoside, madecassoside and asiatic acid were diluted separately in
transport buffer consisted of DMEM without Phenol Red sup-
plemented with 25 mM HEPES and 0.1% (v/v) BSA with pH
adjusted to 7.4, to give final concentration of 20 mg/mL. Para-
cellular permeability (permeability through tight junctions)
marker, NaF at final concentration of 5 mM was added to the
buffer containing the compounds. For comparison purpose, NaF
was also assayed separately. To start the experiment, the serum-
free medium was aspirated from the apical and the basolateral
compartments. Pre-warmed transport buffer (1500 mL) was added
to the basolateral compartment whereas pre-warmed transport
buffer containing the compounds and NaF (500 mL) was added to
the apical compartment. The assay was carried out on a shaker-
incubator (THERMOstar, BMG Labtech, Germany) for 1 h at 37 C
while stirring at 150 rpm. After 1 h, the transport buffer was
sampled from the apical and the basolateral compartments for
quantification of the compounds and NaF. Quantification of asi-
aticoside, madecassoside and asiatic acid in the samples was
carried out using HPLC. NaF was detected at 485 nm excitation
and 535 nm emission using a fluorescence microplate reader
(CHAMELEON™ V, Hidex, Finland). NaF in the samples was
quantified from relative fluorescence unit (RFU) vs. concentration
plot constructed using a series of NaF standard solutions. Cleared
volume (CV, in mL) was calculated to derive permeability multi-
plied by surface area product (PS, in mL/min) and thence apparent
permeability, Papp.
total amount in basolateral compartment
CV ¼ V
(2)
total amount in apical compartment
4 N.A. Hanapi et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-9

CV
PS ¼ (3)
t

PS
Papp ¼ (4)
S

where V ¼ volume in apical compartment (500 mL), t ¼ time

(60 min), and S ¼ surface area of the filter insert (1.12 cm2). Values
obtained were divided by 60 to express results in cm/s.
Percentage of recovery of the compounds was determined using
the following equation:

Amount At¼60 þ Amount Bt¼60

% of recovery ¼
100% (5)
Amount At¼0

in which Amount At¼60 and Amount Bt ¼ 60 ¼ amount of compound

in the apical and the basolateral compartments at t ¼ 60 min
respectively, and Amount At ¼ 0 ¼ amount of compound in the apical
compartment at t ¼ 0 min.
HPLC Analysis
HPLC detection for asiaticoside, madecassoside and asiatic acid
was performed according to Rafamantanana et al. with modifica-
tions.46 A volume of 50 mL of the standard solutions and samples
from the permeability assay was injected into Jasco PU-2098 HPLC
system with UV-VIS spectrophotometric detector and controlled by
CromNav software. Chromatographic separation was performed
with a Zorbax Eclipse XDB-C18 column (4.6
150 mm, 5 mm par-
ticle size) at 40 C. The mobile phase used consisted of deionized
water (Solvent A) and acetonitrile (Solvent B) with a flow rate of
1 mL/min, detection at 206 nm and a total run time of 55 min. The
gradient elution was set as (i) 0e15 min, 35% B; (ii) 15e30 min, 65%
B; (iii) 30e40 min, 80% B; (iv) 40e55 min, 20% B. Concentration of
compounds in the samples was estimated from the peak areas that
were constructed by the CromNav software.
Fig. 2. Primary porcine brain endothelial cells (PBECs) cultured in 75 cm2 culture flask.
1000 mM for 1 h. H2O2 at a concentration that cause approximately
40e60% cells damaged was selected in the evaluation of cytopro-
tection effect of asiaticoside, madecassoside and asiatic acid.47 Cells
were pre-treated with each test compound separately at concen-
trations of 1, 5, 10 and 20 mg/mL for 1 h followed by exposure to
H2O2 for 1 h to evaluate dose-dependent cytoprotective effect of
the compounds. Subsequently, cell viability was measured using
the MTT assay as described above.
Statistical Analysis
Data are presented as mean ± standard error of the mean (SEM).
Statistical analyses were carried out using GraphPad Prism 5.0
software (La Jolla, CA). Statistical significance was determined using
One-way ANOVA followed by Dunnett's multiple comparison test.
Differences were considered as significant at *P < 0.05, **P < 0.01
and ***P < 0.001.

HPLC Method Validation Results

The developed method was validated based on European
Medicines Agency Evaluation of Medicines for Human Use Guide-
line on Bioanalytical Method Validation (European Medicines
Agency, 2011; EMEA/CHMP/EWP/192217/2009 Rev. 1). Stock solu-
tions of the compounds were prepared in methanol. A series of
working standard solutions for each compound were prepared in
the transport buffer consisted of DMEM without Phenol Red sup-
plemented with 25 mM HEPES and 0.1% (v/v) BSA with pH adjusted
to 7.4. These standard solutions were used to determine linear
regression, intra-day and inter-day precision and accuracy of the
method. The linear regression analysis was used to evaluate the
linearity of the calibration curves. The limit of detection (LOD) and
limit of quantification (LOQ) were determined by injecting
sequential two-fold dilution of the stock solutions in the transport
buffer. Compounds were analyzed for three consecutive days
(intra-day and inter-day) to determine the precision and accuracy
of the method.
Evaluation of Protective Effect Against H2O2-Induced Oxidative
Stress in PBECs
Asiaticoside, madecassoside and asiatic acid were investigated
for cytoprotective effect against oxidative stress using the PBECs.
Confluent PBECs (150's) cultured in 96-well plates were exposed to
oxidative stress agent, H2O2 at concentrations ranging from 400 to
Cell Growth and Morphology
Porcine brain microvessel fragments were attached to collagen/
fibronectin-coated flasks within a few hours after thawing. The
PBECs started to grow out of the microvessel from day 1 of culture
and reached 70e80% confluency after 3 days (Fig. 2). The PBECs
were then passaged onto Transwell® inserts. In culture, the PBECs
showed a spindle-shaped morphology and contact-inhibited
property. Treatment with puromycin for 3 days to remove
contaminating cells resulting in absence of other cell types
including pericytes and astrocytes in the PBECs culture as observed
under the microscope.
Cell Viability
The PBECs viability was determined in the presence of asiat-
icoside, madecassoside and asiatic acid at concentration of 20 mg/
mL. As displayed in Fig. 3, the compounds did not show toxic effect
towards the PBECs during the 1 h test period.
BBB Tight Junction Integrity
The BBB tight junction integrity is reflected from TEER mea-
surement. The TEER measurement was recorded at 1 min interval
for 30 min, followed by measurement at minute 60 after returning
 N.A. Hanapi et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-9
120
100
% of cell viability
80
60
40
20
0
Untreated 1% A M AA
cells methanol
Compound
Fig. 3. Cytotoxic effect of asiaticoside (A), madecassoside (M) and asiatic acid (AA)
towards primary porcine brain endothelial cells (PBECs) using the MTT assay. Un-
treated cells represent 100% of total viable cells whereas 1% (v/v) methanol in DMEM
served as vehicle control. Data are mean ± SEM (n ¼ 6).
the cells to the incubator for 30 min. Asiaticoside, madecassoside
and asiatic acid were tested at 20 mg/mL. 100% DMSO, 1% (v/v)
methanol in DMEM and DMEM were used as positive, vehicle and
negative control respectively. The TEER values obtained from each
insert were normalized to initial measurement at t ¼ 0 to give
percentage of initial TEER (Fig. 4). The data showed that 1 h
exposure to asiaticoside, madecassoside and asiatic acid at 20 mg/
mL did not disrupt the tight junction integrity of the PBECs. Mean
TEER values of the PBEC monolayer for each tested group before the
assay were: 628.5 ± 0.5 U,cm2 for 100% DMSO, 417.8 ± 70.9 U,cm2
for 1% (v/v) methanol, 414.8 ± 90.9 U,cm2 for DMEM,
288.7 ± 12.8 U,cm2 for asiaticoside, 399.5 ± 15.5 U,cm2 for
madecassoside and 257.3 ± 17.3 U,cm2 for asiatic acid.
HPLC Method Validation of Compounds for BBB Permeability Assay
Table 1 shows linear equation, linear range correlation co-
efficients, LOD and LOQ for each compound. The plot of the peak
area against different concentrations of the compounds were
constructed as calibration curves. The linearity of the calibration
curve was evaluated using the value of correlation coefficient (r).
The correlation coefficient of each calibration curve was higher
than 0.99. Signal to noise ratio (S/N) was used to determine the LOD
and LOQ of the compounds and estimated by measuring S/N equal
to 3 and 10, respectively. LODs and LOQs of the compounds were in
the range of 0.063e0.63 mg/mL and 0.25e2.5 mg/mL, respectively.
Intra- and inter-day precision were found to be less than 2.3% and
3.6%, respectively, while the accuracy of the intra- and inter-day
were between 94.13e108.99% and 95.41e109.39%, respectively,
indicating that the method was reliable and reproducible.
BBB Permeability of Asiaticoside, Madecassoside and Asiatic Acid
The rate of asiaticoside, madecassoside and asiatic acid to cross
the BBB was determined by conducting in vitro BBB permeability
assay. Apical to basolateral (blood to brain side) permeability of the
compounds at 20 mg/mL was determined using PBEC monolayer
with TEER > 200 U,cm2.45 Mean TEER values of the PBEC mono-
layer used for asiaticoside, madecassoside, asiatic acid and NaF
permeability assay were 469.47 ± 72.40, 530.43 ± 77.78,
512.96 ± 43.25 and 588.88 ± 47.04 U,cm2 respectively (Table 2). As
shown in Table 2, asiaticoside demonstrated highest apparent
permeability (Papp) value of 70.61 ± 6.60
106 cm/s, followed by
madecassoside (53.31 ± 12.55
106 cm/s) and asiatic acid
(50.94 ± 10.91
106 cm/s). The percentage of recovery for
5
Fig. 4. Effect of asiaticoside (A), madecassoside (M) and asiatic acid (AA) on the BBB
tight junction function. Transendothelial electrical resistance (TEER) across the PBEC
monolayer was measured at 1 min interval for 30 min and at minute 60 using WPI
STX-100C chopstick electrode pair connected to EVOM meter. A, M and AA were tested
at 20 mg/mL while 100% DMSO, 1% (v/v) methanol and DMEM were used as positive,
vehicle and negative control respectively. The test compounds and controls were
added separately to the inserts after min 10 TEER was recorded (indicated by arrow).
Data are mean ± SEM with n ¼ 5 inserts for 1% (v/v) methanol and DMEM; n ¼ 3
inserts for A and AA; n ¼ 2 inserts for 100% DMSO and M. Statistical differences are
tested using One-way ANOVA followed by Dunnett's multiple comparison test and
presented as ***P < 0.001 compared with 100% DMSO.
asiaticoside, madecassoside and asiatic acid are 82.24 ± 0.83,
86.17 ± 11.74 and 93.61 ± 12.27% respectively, indicating minimal
loss e.g. non-specific binding. Monolayer integrity was ensured in
all assays by measuring permeation of NaF which was added to
insert together with the tested compounds, and also assayed
separately for comparison. NaF exhibited low Papp in the presence
of all compounds with values of 3.66 ± 0.20
106 cm/s (asiat-
icoside), 3.11 ± 0.26
106 cm/s (madecassoside) and
3.92 ± 0.21
106 cm/s (asiatic acid). These Papp values showed no
significant difference compared with NaF tested alone
(5.10 ± 0.91
106 cm/s), reflecting preservation of tight junction
integrity throughout the assay.
Protective Effect Against H2O2-Induced Oxidative Stress in PBECs
PBECs exposed to H2O2 from 500 mM for 1 h showed significant
reduction in cell viability due to oxidative stress. H2O2 at 600 mM
caused approximately 40e60% reduction of cell viability (Fig. 5a).
Therefore, H2O2 at 600 mM was selected in subsequent study to
evaluate the cytoprotective effect of asiaticoside, madecassoside
and asiatic acid towards the PBECs. PBECs pre-treated with asiat-
icoside showed no significant increase in cell viability compared
with H2O2 (600 mM) even at the highest concentration (20 mg/mL)
(Fig. 5b). PBECs pre-treated with madecassoside showed potential
increase in cell viability in a concentration-dependent manner but
this is not significant when compared to H2O2 (Fig. 5c). Interest-
ingly, pre-treatment with asiatic acid at concentration of 20 mg/mL
showed significant increase in cell viability in comparison to H2O2
exposure (Fig. 5d).
Discussion
The neurotherapeutics of bioactive triterpenes from C. asiatica,
i.e. asiaticoside, madecassoside and asiatic acid are widely reported,
but their capability to cross the blood-brain barrier (BBB) has yet to
be established. The BBB, an important barrier at the level of cerebral
capillaries plays a major role in maintaining brain homeostasis.
Dysfunction of the BBB leads to profound and irreversible injuries
to neurons.48 On this note, evidence of asiaticoside, madecassoside
and asiatic acid on BBB permeation without causing any harmful
6 N.A. Hanapi et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-9
Table 1
Analytical Data of Calibration Curves, Linear Range, LOD and LOQ for the Quantitative Analysis of Asiaticoside, Madecassoside and Asiatic Acid.
Compound Calibration Curve r2
Asiaticoside y ¼ 41,639xe74,157 0.999
Madecassoside y ¼ 79,981x þ 73,680 0.999
Asiatic acid y ¼ 106,709xe14,114 0.998
effects towards the barrier is exceedingly desirable. Utilizing in vitro
primary porcine brain endothelial cell (PBEC) BBB model, perme-
ation of the compounds across the BBB is presented for the first
time in this study. Prior to the permeability assay, a non-toxic
concentration profiles of asiaticoside, madecassoside and asiatic
acid were established at 20 mg/mL, and subsequently leaving the
BBB tight junction integrity intact during the permeability assay.
Pre-incubation of the PBECs with 20 mg/mL of asiatic acid for 1 h
significantly protected the cells from oxidative damage induced by
H2O2, in contrast with asiaticoside and madecassoside under
similar condition. Most notably, all compounds showed remarkably
high BBB permeation, of which asiaticoside showed the highest
permeability, followed by madecassoside and asiatic acid.
Safety-related attrition remains a major issue in drug discovery
and development. Given that central nervous system (CNS) toxicity
is a leading cause of toxicity-related clinical trial failures,49,50 the
search for safer neurotherapeutics is extremely important.50 Sci-
entific evidence documented that asiaticoside, madecassoside and
asiatic acid has acceptable safety, particularly towards brain cells.
For instance, asiaticoside up to 100 mM had no significant cell
growth inhibition where cell viability of human brain microvas-
cular endothelial cells (hBMECs) was reported to be over 95% for
the period of 12 h exposure.51 Madecassoside showed no effect on
cell viability of immortalised murine microglial cell line (BV2) up to
10,000 mM.52 Meanwhile, 24 h treatment of human neuroblastoma
SH-SY5Y cells with asiatic acid for concentrations ranging from 1 to
20 mM showed no growth inhibitory activity with a 10% decrease in
cell viability at 20 mM.53 With regards to the PBECs, the viability of
the cells in the presence of asiaticoside, madecassoside and asiatic
acid at 20 mg/mL (the concentration to be used in the permeability
assay) showed non-toxic profile as these compounds did not
decrease the viability of the cells, thus further confirmed their
safety as previously demonstrated.
While it is important to establish safety of the compounds in
regards to their toxicity, it is also essential to determine the func-
tionality of barrier tight junctions in presence of the compounds.
This is also important to further validate reliability of the measured
apparent permeability (Papp). Transendothelial electrical resistance
(TEER) is a widely accepted quantitative technique to measure the
integrity of tight junction functions in cell-based models of the
BBB.54 In this study, the real-time TEER during 1 h exposure of the
PBEC monolayers to the compounds showed no disruption to the
tight junction integrity. The TEER values recorded are in the range
of values reported to have minimal tight junction leakiness.45
Preservation of the tight junction function support the findings of
Table 2
Papp Values, % of Recovery and TEER Values for Asiaticoside, Madecassoside, Asiatic Acid and Sodium Fluorescein (NaF).
Compound Papp (
106 cm/s) % Recovery
Asiaticoside 70.61 ± 6.60 82.24 ± 0.83
Madecassoside 53.31 ± 12.55 86.17 ± 11.74
Asiatic acid 50.94 ± 10.91 93.61 ± 12.27
NaF 5.10 ± 0.91 78.16 ± 3.63
Data are mean ± SEM, n ¼ 3 inserts for NaF, n ¼ 5 inserts for asiaticoside and asiatic acid, n ¼ 6 inserts for madecassoside.
Linear Range (mg/mL) LOD (mg/mL) LOQ (mg/mL)
2.5e80 0.63 2.5
2.5e80 0.63 2.5
0.25e20 0.063 0.25
non-toxic nature of asiaticoside, madecassoside and asiatic acid
measured at 20 mg/mL in this study.
The rates at which asiaticoside, madecassoside and asiatic acid
cross the PBEC monolayer grown on the Transwell® inserts were
measured from apical to basolateral compartment as apparent
permeability (Papp, cm/s). As shown in Table 2, C. asiatica com-
pounds are highly BBB permeable, of which asiaticoside demon-
strated the highest Papp value, followed by madecassoside and
asiatic acid. Most notably, the compounds showed higher Papp
values than donepezil, a drug for AD55 CE with Papp value of
30.6
106 cm/s, reported in previous study where the PBEC model
was also used as tool to measure BBB permeation.38 Importantly,
monolayer integrity of the PBECs was ensured throughout the
permeability assay by monitoring NaF permeability which
concurrently added to each insert with the tested compounds. The
Papp values of NaF in the presence of all compounds were relatively
low, comparable with NaF tested alone, reflecting preservation of
tight junction integrity throughout the assay.
The structure of the two glycosides, i.e. asiaticoside and made-
cassoside basically does not obey the Lipinski's rule of five as
opposed to asiatic acid. In accordance with Lipinski's rule of five,
the physicochemical data of asiatic acid56 are in the permissible
range with molecular weight of 488.7 Da, hydrogen bond donor
(HBD) of 4 and hydrogen bond acceptor (HBA) of 5, together with
high lipophilicity value (log P of 5.7), indicating good passive
permeability. High Papp value of asiatic acid could also be influenced
by its lowest topological polar surface area (TPSA) value of 98 Å2,
which is comparable to the recommended threshold of 90 Å2,
which also favors possible passive BBB permeation.57 In contrast,
the physicochemical data of asiaticoside and madecassoside lies
outside the prescribed range.58,59 For instance, both glycosides have
molecular weight of more than 500 Da (959.1 Da for asiaticoside
and 975.1 Da for madecassoside). A count of hydrogen bonds in
asiaticoside (HBD of 12 and HBA of 19) and madecassoside (HBD of
13 and HBA of 20) both surpass their recommended range. Asiat-
icoside and madecassoside also showed low lipophilicity judged
from their log P values of 0.1 and 1.2, respectively. Additionally,
TPSA values of asiaticoside (315 Å2) and madecassoside (335 Å2)
exceedingly lies beyond recommended threshold. Given the phys-
icochemical data of the two glycosides, they are very unlikely to
show passive permeability. However, it is important to highlight
that we do not discard the possibility of both glycosides to
permeate the BBB via transcytosis, a well-documented mechanism
of BBB transport of glycosylated enkephalin derivatives.60 Glyco-
sylation of the parent drugs to glycosylated derivatives led to
Sodium Fluorescein, NaF TEER (U.cm2)
6
Papp (
10 cm/s) % Recovery
3.66 ± 0.20 101.84 ± 14.36 469.47 ± 72.40
3.11 ± 0.26 96.32 ± 14.29 530.43 ± 77.78
3.92 ± 0.21 93.66 ± 6.41 512.96 ± 43.25
5.10 ± 0.91 84.34 ± 7.91 588.88 ± 47.04
 N.A. Hanapi et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-9 7

a b
120 120
100 100

% of cell viability

% of cell viability
80 ** *** 80
###
### ###
60 60 ### ###
40 *** 40
***
20 *** *** 20
0 0

Concentration of H₂O₂ (μM) Concentration of A (μg/mL)

c d
120 120

100 100

% of cell viability
% of cell viability

80 80 *
# ## ##
60 60 ##
## ###
40 40

20 20

0 0

Concentration of M (μg/mL) Concentration of AA (μg/mL)

Fig. 5. Cytoprotective effect of asiaticoside (A), madecassoside (M) and asiatic acid (AA) towards the PBECs. Decrease in viability was observed when the cells were treated with
H2O2 for 1 h (a). To evaluate cytoprotective effect of A (b), M (c) and AA (d), the cells were pre-treated with different concentrations of the compounds for 1 h prior to oxidative
stress induced by 600 mM H2O2. After 1 h, cell viability was determined and the data are presented as percentage relative to the untreated cells. Data are expressed as mean ± SEM
(n ¼ 6). Statistical differences are denoted as #P < 0.05, ##P < 0.01 and ###P < 0.001 relative to the untreated cells and *P < 0.05 relative to H2O2 exposed-cells, tested using One-way
ANOVA followed by Dunnett's multiple comparison test.

enhanced BBB transport via transcytosis60 and increased CNS up-
take by glucose transporter (GLUT).61 Recent study of glycosylated
nanodisks also demonstrated high BBB permeation via GLUT-
mediated transcytosis.62 Glycosylation of dopamine using several
linkers was also effective in enhancing dopamine BBB permeation
via GLUT.63,64 For future studies, bidirectional permeability assay or
conducting permeability assay in presence of transporter sub-
strates/inhibitors could be carried out to further dissect mecha-
nisms of permeability for these compounds.
Given the involvement of ROS in the pathogenesis of neurode-
generative diseases, research has focused on the potential benefits
of compounds with antioxidant properties from natural sources
due to their ability to inhibit ROS generation and protect neuronal
cells from oxidative damage or death.65 ROS, which refers to
chemically reactive molecules, including H2O2, nitrogen oxide (NO),
singlet oxygen (O⁻2) and hydroxyl radicals ($OH),66 are generated
naturally within the biological system, playing vital roles in medi-
ating a number of cellular activities such as cell survival, inflam-
mation and cancer.67 When ROS production and antioxidant
systems are imbalance,68 high concentrations of ROS might cause
damage to proteins, nucleic acid and lipids which can lead to
oxidative stress or cell death.69 In circumstances where presence of
ROS is in excess or the antioxidant level in the brain is reduced,
impairment of neurological functions caused by oxidative stress
can further lead to neurodegeneration.69 In this regard, endothelial
cells of the BBB are at the front line in protecting brain cells from
insults including oxidative stress, inflammation, excitotoxicity and
vascular reactivity caused by external agents.70 On that note, the
ability of asiaticoside, madecassoside and asiatic acid to protect the
PBECs against H2O2-induced oxidative stress was determined. We
found no evidence of asiaticoside rescued H2O2-induced oxidative
damage in the PBECs. On the other hand, there is a possibility that
madecassoside protected the PBECs against injury induced by
oxidative stress after exogenous H2O2 treatment in a
concentration-dependent manner. This data are consistent with
current body of knowledge, where madecassoside protected BV2
microglial cells from oxygen-glucose deprivation/reperfusion
injury.52 Pre-treatments with asiatic acid showed protective effects
at 20 mg/mL. This finding is in agreement with previous studies
which proposed that asiatic acid exerted neuroprotective proper-
ties on neuronal cultures against glutamate-induced excitotox-
icity,71 rotenone- or H2O2-based oxidative stress,72 as well as C2-
ceramide-induced apoptosis,73 whilst in rodent models, asiatic
acid attenuated glutamate-induced cognitive deficits.74 Reduction
in oxidative stress markers including plasma NO metabolites, O2
production and upregulation of iNOS/eNOS protein expression have
also been reported in high-carbohydrate, high-fat diet-induced
metabolic syndrome rats after treatment with asiatic acid.75
Conclusion
Integrating the evidences, it is apparent that asiaticoside,
madecassoside and asiatic acid are capable to cross the BBB with
high rate without exerting any toxic effects while preserving the
BBB tight junction integrity. Protective effect of madecassoside and
asiatic acid against H2O2-induced oxidative stress in the PBECs
8 N.A. Hanapi et al. / Journal of Pharmaceutical Sciences xxx (2020) 1-9
provides a good addition to the current body of knowledge on the
compounds neurotherapeutics. Previous findings on multi-
pharmacological properties of the compounds particularly to alle-
viate neurodegeneration justified further development as
therapeutics. This study has added valuable data to further support
the development of the compounds as neurotherapeutics, where
impediment of permeation across the BBB is often a concern.
Declaration of Interest
The authors declare that the content of this article has no con-
flict of interest.
Acknowledgements
This study was funded by NRGS NKEA EPP#1 Research Grant
Scheme (grant number 304/CDADAH/650745/K123) from the
Ministry of Agriculture & Agro-Based Industry Malaysia, and partly
supported by Ministry of Higher Education Malaysia under the
HICoE Programme (grant number 311.CDADAH.4401009). We ex-
press gratitude to Mr. Rahim Ali Musa and Mr. Asokan A/L
Muniandy for their technical support in HPLC work. We thank Mr.
Ranjid Singh A/L Raiyappan and Mr. Maarkandan A/L Krishnasamy
for their help in extracting porcine brains at the Department of
Veterinary Services Penang, Malaysia.
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