α-bisabolol β-D-fucopyranoside as a potential modulator of β-amyloid peptide induced neurotoxicity: An in vitro & in silico study

Article abstract of DOI:10.1016/j.bioorg.2019.102935

Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder affecting the elderly people. For the AD treatment, there is inefficiency in the existing medication, as these drugs reduce only the symptoms of the disease. Since multiple pathological

Full text of DOI:10.1016/j.bioorg.2019.102935

Accepted Manuscript
α-bisabolol β-D-fucopyranoside as a potential modulator of β-Amyloid peptide
induced neurotoxicity: an in vitro & in silico study
Mahalingam Jeyakumar, Sethuraman Sathya, Soniya Gandhi, Prabhakararao
Tharra, Venkatesan Suryanarayanan, Sanjeev Kumar Singh, Beeraiah Baire,
Kasi Pandima Devi
PII:
S0045-2068(19)30336-0
https://doi.org/10.1016/j.bioorg.2019.102935
102935
DOI:
Article Number:
Reference:
YBIOO 102935
Bioorganic Chemistry
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1 March 2019
8 April 2019
15 April 2019
Please cite this article as: M. Jeyakumar, S. Sathya, S. Gandhi, P. Tharra, V. Suryanarayanan, S. Kumar Singh, B.
Baire, K. Pandima Devi, α-bisabolol β-D-fucopyranoside as a potential modulator of β-Amyloid peptide induced
neurotoxicity: an in vitro & in silico study, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.102935
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α-bisabolol β-D-fucopyranoside as a potential modulator of β-Amyloid peptide induced
neurotoxicity: an in vitro & in silico study
Mahalingam Jeyakumar,a Sethuraman Sathya,a Soniya Gandhi,b Prabhakararao Tharra,b
Venkatesan Suryanarayanan,c Sanjeev Kumar Singh,c Beeraiah Baire b Kasi Pandima Devi a*
Department of Biotechnology, Alagappa University, Karaikudi - 630003, Tamil Nadu, India.
Department of Chemistry, Indian Institute of Technology Madras- 600036, Tamil Nadu, India.
Computer Aided Drug Design and Molecular Modeling Lab, Department of Bioinformatics,
a
b
c
Alagappa University, Karaikudi - 630003, Tamil Nadu, India.
*Corresponding Author: Dr. K. Pandima Devi,
E-mail: devikasi@yahoo.com

Abstract
Alzheimer’s disease (AD) is a multifaceted neurodegenerative disorder affecting the elderly
people. For the AD treatment, there is inefficiency in the existing medication, as these drugs
reduce only the symptoms of the disease. Since multiple pathological proteins are involved in the
development of AD, searching for a single molecule targeting multiple AD proteins will be a
new strategy for the management of AD. In view of this, the present study was designed to
synthesize and evaluate the multifunctional neuroprotective ability of the sesquiterpene glycoside
α-bisabolol β-D-fucopyranoside (ABFP) against multiple targets like acetylcholinesterase,
oxidative stress and -amyloid peptide aggregation induced cytotoxicity. In silico computational
docking and simulation studies of ABFP with acetylcholinesterase (AChE) showed that it can
interact with Asp74 and Thr75 residues of the enzyme. The in vitro studies showed that the
compound possess significant ability to inhibit the AChE enzyme apart from exhibiting
antioxidant, anti-aggregation and disaggregation properties. In addition, molecular dynamics
simulation studies proved that the interacting residue between Aβ peptide and ABFP was found
to be involved in Leu34 and Ile31. Furthermore, the compound was able to protect the Neuro2 a
cells against Aβ25-35 peptide induced toxicity. Overall, the present study evidently proved ABFP
as a neuroprotective agent, which might act as a multi-target compound for the treatment of
Alzheimer’s disease.
Keywords: α-bisabolol β-D-fucopyranoside (ABFP); Cholinesterase (ChE); Oxidative Stress;
amyloid β-protein; Neuroprotective; Alzheimer’s disease (AD).

1. Introduction
Alzheimer’s disease is a progressive neurodegenerative disease associated with memory
loss and cognitive deficits in aged people. Worldwide, it is estimated that 36 million people
presently suffer from dementia and this number is expected to sharply increase to 114 million
by 2050 [1]. AD is characterized by various pathological features like accumulation of
extracellular β-amyloid protein and intracellular neurofibrillary tangles. The major therapeutic
strategy for the treatment of AD focuses on the restoration of cholinergic functionality to
enhance neurotransmission by increasing the acetylcholine availability in the synaptic cleft [2].
In addition, free radicals are inevitably produced by reactive oxygen species (ROS) and
oxidative stress, which leads to the damage of the macromolecules such as DNA, protein, and
lipids which plays a pivotal role in the onset of AD [3].
Currently, no treatment is available to reverse or stop the progression of the AD. The
available AD drug management is based mainly on cholinesterase inhibitors (ChEIs). For example,
tacrine, donepezil, galantamine and rivastigmine, which have been approved for clinical use can
only modulate the symptoms of AD for a short period of time, or aim at a single target [4] Because
of the flexible and complicated etiology, the “one-molecule, one-target” therapy appears not so
efficient in treating multifarious diseases like AD. They offer only symptomatic treatment but
cannot alleviate the clinical manifestation or development of the disease. Furthermore, many
researchers have devoted their efforts for the development of multi-targeted direct ligands
(MTDLs), which are single molecules (for example, 4-hydroxyl aurone derivatives and fused
tricyclic coumarin derivatives) that can exhibit multiple pharmacological effects, as a positive
approach to fight against the multifactorial nature of AD [3,5]. Since the development of effective
MTDLs can act as a promising drug for the treatment of complex diseases such as AD, there is an
urgent need to screen for drugs with multiple targets.

Nowadays, efficient compounds are derived from natural products and their derivatives
hold several biological properties [6,7]. α-bisabolol is one such natural sesquiterpene alcohol,
which is present widely in natural sources such as essential oils of Matricaria chamomilla with
many medicinal properties. Previous work from our lab has shown that the anti-cholinesterase and
anti-aggregation potentials of the marine seaweed Padina gymnospora is attributed to the presence
of the compound α-bisabolol [8]. However, the major drawback of using α-bisabolol is its
insolubility in biological fluids. In order to overcome the short comings of α-bisabolol, the present
study aims for synthesis of the α-bisabolol derivative, α-bisabolol β-D-fucopyranoside (ABFP), a
glycosidation product of α-bisabolol (which is not commercially available). Moreover, it is a
glycosidic sugar compound and it has been reported that the glycosidation of a molecule increases
its hydrophilicity, and thereby influencing its physicochemical and pharmacokinetic properties [9].
According to the in silico ADME predictions, the glycosidic nature of ABFP has been previously
estimated to cross the (blood brain barrier) BBB due to its suitable pharmacokinetics properties
like Polar surface area (PSA), Calculated partition coefficient (clogP), and Hydrogen bond donor
(HBD) [10]. It is quite conflicting with the purpose of the addition of a glycosides be able to
improve BBB porous capacity, whereas Egleton et al., (2001) has shown that the BBB
penetrability of a peptide was enhanced by the addition of a glucose molecule [11]. ABFP
previously identified from the aerial parts of Carthamus lanatus exhibited cytotoxic activity which
was evident by the Artemia salina assay [12]. In addition, this compound has been evaluated for
cytotoxicity against human and rat glioma cancerous cell lines such as (U-87, U-251, and GL-261)
cells [10], while the neuroprotective efficacy of the compound is not yet known. Hence an
approach has been framed to synthesise the derivative α-bisabolol β-D-fucopyranoside and explore
its neuroprotective potential against Alzheimer’s disease through in vitro and in silico systems.

2. Results and Discussion
2.1. Synthesis of α-bisabolol β- D-fucopyranoside (ABFP)
To prepare the target ABFP 1, the glycosyl trichloroacetimidate (TCA) based Schmidt
strategy [13,14] was adopted. We coupled the α-bisabolol and α-D-fucopyranosyl
trichloroacetimidate 2 to achieve the final product 1. To begin with, our aim was to synthesize D-
fucose based TCA derivative 2 (Scheme 1). Accordingly, D-fucose was treated with benzoyl
chloride, pyridine and 10 mol% of 4-dimethylaminopyridine (DMAP), in dichloromethane at RT
for 16 h. The corresponding tetrabenzoyl fucose derivative 3 was obtained in 95% yield after
chromatographic purification. To do a regio-selective benzoyl deprotection at anomeric position
in compound 3, we chose a two step protocol. Initially, a dichloromethane solution of 3 was
treated with HBr (33% in AcOH) at RT for 2.5 h. After washing the reaction mixture with aq.
sodium bicarbonate, the crude material was used for next reaction. Treatment of the crude
reaction mixture with silver carbonate (Ag2CO3) in acetone and water (20:1), at RT for 1.5 h,
provided the hydroxylated compound 4 with 80% yield for two mixture of diastereomer (1:0.5).
Next, using the Schmidt’s procedure, TCA derivative 2 was prepared. Reaction of
monohydroxy-compound 4 with tricholoroacetonitrile (CCl3CN) in presence of cesium carbonate
(Cs2CO3) at 0 °C for 16 h in CH2Cl2 resulted in the formation of fucose-TCA derivative 2 in
70% yield.
Subsequently, with the essential TCA derivative 2 in hand, we focused on the generation
of our target molecule α-bisabolol β- D- fucopyranoside 1 (Scheme 2) [17]. The α-bisabolol was
treated with 10 mol% of TMSOTf in anhydrous CH2Cl2 at -78 °C for 15 minutes. Next, after the
addition of an ice cold solution of TCA derivative 2 in DCM was added dropwise to reaction
mixture and the reaction was allowed to keep at -20 °C for 2.5 h, after quenching this reaction
with triethylamine (Et3N), reaction afforded the product 5 in 80% yield. Further, the solution of

coupled product 5 in H2O/THF/MeOH (1:2:1 ratio) upon subjection to aq. NaOH (0.25 N) for 1.5
h, resulted in the formation of the α-bisabolol β- D-fucopyranoside 1 excellent yield (98%).
2.2 In silico evaluation of ABFP against AChE and β-secretase
2.2.1 Docking analysis of ABFP with AChE & β-secretase
The 3D structure of ABFP were docked with prepared AChE using Glide XP module
implemented in Schrödinger. The docking scores were obtained with -9.488 kcal/mol, glide
emodel of -31.604 kcal/mol and glide energy of -42.255. The interactions obtained during the
docking were evident with the pictorial representation of 2D and 3D interaction of AChE
complexed with ABFP represented in (Fig.1A). It reveals that Asp74 and Thr75 were interacting
with ligand through hydrogen bond interaction. Subsequently, the complex were further
evaluated for the binding free energy calculation which exhibited the ∆Gbind value of -51.810
kcal/mol and found to be supportive for the docking results (Table1).
2.2.2 Molecular Dynamics Simulation studies with AChE
The complex was further analyzed for molecular dynamics simulation for 20000ps time period
of simulation in order to access the binding interaction stability. The conformational changes in
the structure was analyzed through the calculation of root mean square deviation (RMSD) and
root mean square fluctuations (RMSF) over backbone atoms. (Fig. 1B) (A) shows that the
complex have stable deviations around 1.8 Å after 14000 ps time period of simulation. (Fig.1B)
(B) shows the RMSF of residues of AChE where it reveals that residues from 250-270 and 480-
500 were fluctuating to the highest of 4.4 Å because they are in loop region. (Fig. 1B) (C) shows
the radius of gyration of the protein which clearly shows that the protein does not lose its
compactness during the simulation time period. The histogram and the timeline of the protein
ligand contacts were represented in (Fig. 1C) which reveals that Tyr72, Thr75, Trp286 and
Tyr341 were the most interacting residues over the course period of simulation. During Glide XP

docking Asp74 was interacting whereas the same interaction was lost and this is very clearly
evident with the (Fig. 1D). It also shows that there is no much changes in the secondary structure
of protein during the course period of simulation for 20ns. MD simulation analysis shows that
hydrophobic interaction plays a major role in stabilizing the ligand in its active site whereas the
hydrogen bond interaction with Tyr72, Thr75 and Tyr341 plays less important role.
2.3. In vitro evaluation of acetylcholine esterase (AChE) inhibitory activity of ABFP
Acetylcholinesterase is the enzyme present in nervous tissue that helps to break down the
neurotransmitter acetylcholine. According to the cholinergic hypothesis, a decrease in the level of
acetylcholine in the brain is primarily involved in memory destruction during AD. Since the
inhibition of cholinesterase has a significant role for the treatment of AD [15], the present study
involved the evaluation of AChE inhibitory potential of ABFP. The results clearly illustrated that,
the higher concentration of ABFP (50 µg/ml) showed potent ChE inhibitory activity of 95.869 ±
0.121 % when compared with standard drug donepezil whose % of inhibition was 95.856 ± 0.847
(Fig. 2). Several scientific reports illustrate that naturally derived compounds act as a source of new
biofunctional scaffolds targeting cholinesterase and  amyloid aggregation [16]. The results of our
study suggest that ABFP could be used for anti-cholinergic remedy for AD treatment.
2.4 In vitro evaluation of antioxidant property of ABFP
2.4.1. DPPH radical scavenging activity of ABFP
DPPH is a free radical that is mostly used for antioxidant studies. Several studies have reported
that dysfunction of the neuronal junction is combined with oxidative stress that can involve vital
role in AD [17]. The effect of free radical scavenging activity of ABFP was assessed by the
DPPH assay. As seen in (Fig. 3A), ABFP exhibited potent radical scavenging activity of 68.9 ±
0.44% at 50 µg/ml when compared with the standard BHT (80.3 ± 1.5%) at 50 µg/ml. The result

indicates that high antioxidant potency of the ABFP might be due to its electron or hydrogen
donating ability.
2. 4.2. Hydrogen peroxide scavenging potential of ABFP
In living cells, the non reactive hydrogen peroxide when converted into the free radical called as
hydroxyl radicals (OH•), reacts with macromolecule and causes tissue damage [18]. Hence
removing the cytotoxic agent H2O2 is very important for protection of human system. The
scavenging ability of ABFP on H2O2 in comparison with standard L-Ascorbic acid is shown in
Fig. 3B. The results exhibited that ABFP showed maximum H2O2 scavenging potential of 68.4 ±
0.44% (at 50 µg/ml) when compared with the standard L-ascorbic acid 66.8 ± 0.17% (50 µg/ml).
A statistically significant correlation was found between ABFP, standard and control (P<0.05).
The data suggests that ABFP possess significant antioxidant activity, which might donate
electrons to H2O2, thus neutralizing it to water.
2.4.3. Hydroxyl radical scavenging activity of ABFP
Hydroxyl radical is an extremely reactive free radical formed in biological systems and has the
capacity to cause DNA strand breakage, which contributes to carcinogenesis, mutagenesis and
cytotoxicity [19]. The effect of ABFP on the inhibition of free radical mediated damage was
assessed by means of the Fe2+ dependent DNA damage assay. The Fenton reaction generates OH•,
which degrades DNA deoxyribose, using Fe2+ salts as catalyst. The hydroxyl radical may attack
DNA either at sugar or base giving rise to toxic product. Fig. 3C shows the effect of ABFP on the
Fe2+ dependent deoxyribose damage. Whereas, the higher concentration of ABFP and BHT (50
μg/ml) exhibited strong hydroxyl radical scavenging activity of 73.8 ± 0.22 % and 71.7 ± 1.5 %
respectively. The result anticipated that ABFP has high antioxidant potency against peroxidation of
lipids on biological systems through scavenging of hydroxyl radical scavenging activity.

2.4.4. Reducing power of ABFP
Reducing power assay which evaluates the conversion of Fe3+ into Fe2+ in the presence of ABFP,
was measured to determine its reducing power ability. The reducing capability of a compound is
a significant marker of its possible antioxidant activity, which generally depends on its ability to
donate a hydrogen atom that can reduce the lipid peroxidation process [20]. Fig. 4A shows that
ABFP exhibited higher reducing power potential when compared to control and standard L-
ascorbic acid. The results of antioxidant assays indicate that ABFP possess greater reducing
potential and it can be able to prevent the oxidative stress to cellular components.
2. 4.5. Assessment of metal chelating potential of ABFP
The biochemical and neuropathological evidences have suggested that dyshomeostasis of
cerebral biometals such as copper, cadmium, zinc, play a major role in Aβ aggregation and
neurotoxoticity, which plays a central role in AD pathogenesis. For example, Αβ interaction with
metal ions like Cu (II) and Zn (II) and their binding can also induce the toxic peptide aggregation
[21]. The results of the present study (Fig. 4B) demonstrated that the formation of ferrozine-Fe2+
complex in metal chelating activity is inhibited by ABFP (50 µg/ml), which showed the highest
metal chelating activity of 6.25± 0.58 when compared to standard BHT 5.15 ± 0.21 (50 µg/ml).
Currently, several lipophilic metal chelators for example, clioquinol and its derivative PBT2
have been studied in clinical trials which showed encouraging results in AD patients [22].
2.4.6. Total antioxidant capacity of ABFP
The Total antioxidant capacity (TAC) of ABFP were evaluated by phosphomolybdenum method
which shows the ability of a compound to reduction of Mo (VI) to Mo (V) at acidic pH [23]. The
results of TAC showed that ABFP exhibited significant (P<0.05) reducing potential with the
higher absorbance of 0.281 ± 0.02 at (50 µg/ml) when compared to control 0.114± 0.001 and

similar to the standard L-ascorbic acid 0.624 ± 0.06 (50 µg/ml) (Fig. 4C) which indicates that it
act as an excellent natural antioxidant.
2.5 Effect of ABFP on in vitro aggregation and disaggregation of Aβ25-35
2.5.1. Thioflavin T assay The antiaggregation and disaggregation property of Aβ peptide was
quantified by Thioflavin T (ThT) assay [24]. ThT is a fluorescent dye that is used for
quantification of Aβ peptide. This assay was performed at different time interval (24 h, 48 h, 96
h and 9d) in two different phases, namely, phase I & phase II (Fig. 5A).
Phase I: Inhibition of formation of aggregates from oligomers: In phase I study, Thioflavin – T
fluorescence intensity was found to increase in Aβ peptide group from 24 h (21.49 ± 2.41 AU) to
48 h (27.61 ± 1.44) which indicated the commencement of Aβ peptide aggregation. However,
upon treatment with ABFP (5 & 10 μg/ml), reduction in the (ThT) fluorescence was observed at
24 h (17.43 ± 2.84 AU, 13.5 ± 0.86 AU respectively) and 48 h (6.65 ± 1.97 AU, 6.79 ± 0.76 AU
respectively) when compared to the standard drug galantamine (50 μg/ml) (11.81± 2.84 AU at 24
h & 6.00 ± 1.97 AU at 48 h). Thus the result of phase I study indicated the capability of ABFP to
inhibit oligomer formation and thereby confirms its anti-aggregation property (Fig. 5B).
Phase II: Disaggregation of the pre-formed mature fibrils: The results of phase II study
showed that an increase in (ThT) fluorescence intensity was observed from 96 h (29.97 ± 1.40
AU) to 9 days (35.18 ± 2.00 AU). However, reduced ThT fluorescence intensity treatment wwas
observed during ABFP (5 & 10 μg/ml) and galantamine in 96 h (14.4 ± 1.02 AU, 3.84 ± 1.2 AU)
& (6.54 ± 1.02 AU) and 9 days (14.46 ± 1.89 AU, 7.30 ± 1.79 AU) & (15.14 ± 1.16 AU)
treatment. The results clearly indicated that ABFP might disaggregate the mature fibrils (Fig.
5B). Therefore, the result highlighted the potential of ABFP to inhibit Aβ aggregation and

disaggregation in both phase I & II. This data provides a novel hint that ABFP may be used as an
anti-amyloidogenenic lead for AD.
2.5.2. Microfluorescence assay
The results were additionally confirmed by CLSM analysis. The confocal image confirmed that
less peptide aggregation were observed at 24 h & 48 h, whereas the peptide aggregation was
enhanced at 96 h & 9 d in Aβ25-35 treated group. Co-treatment with ABFP (5 & 10 µg/ml)
exhibited anti-aggregation and dis-aggregation property, which was similar as galantamine treated
group (Fig. 5C). Hence the data revealed that ABFP and galantamine effectively caused anti-
aggregation and also disaggregated the pre-formed mature fibrils of Aβ25-35 during the incubation
period of 24 h to 9 days, when compared to control.
2.5.3 Molecular Dynamics Simulation studies with Aβ25-35
The molecular dynamics simulation of the Aβ peptide was performed in order to achieve
better understanding of peptide aggregation and the role of ABFP in inhibition of Aβ aggregation
for a period of 100 ns simulation. The conformational changes and the aggregation of Aβ
peptides were monitored throughout the period of simulation. During simulation, it was observed
that ABFP plays a noteworthy role in inhibition of Aβ peptide aggregation which is evident from
Fig. 6A. It was observed from the figure that formation of β-sheets has not been noted
throughout the simulation time period due to the presence of ABFP which confirmed the anti-
aggregation property. Fig. 6B shows the interacting residues between Aβ peptide and ABFP in
which Leu34 of Peptide 2 and Ile31 of Peptide 5 was found to be involved in interaction.
2.6 In vitro cell survival of ABFP in Neuro2a cells
MTT assay [25] was performed to evaluate the protective effect of ABFP (2.5- 10 μg/ml) in
Neuro2a cells. The results suggest that ABFP increased the viability of cells in a concentration

dependent manner for 24 h. At the concentration of 5 & 10 μg/ml of ABFP, cell viability was
observed to be 94.3 ± 0.05% and 95.2 ± 0.05 % respectively (Fig. 7A). Hence, this concentration
was fixed for further experimental analysis.
2.6.1. Assessment of protective effect of ABFP against Aβ25-35 induced toxicity by MTT assay
The neuroprotective efficacy of ABFP against Aβ25-35 in Neuro2a cells were evaluated by
MTT assay. Treatment of Neuro2a cells with Aβ25-35 peptide reduced the cell viability up to 60%
when compared to control. Conversely, pre-treatment with ABFP (5 & 10 μg/ml) significantly
(P<0.05) increased the viability of cells to 81.2 ± 0.05% and 96.33 ± 0.06% when compared with
control (100%) and standard drug donepezil (50 μg/ml) (78.57± 0.05%) (Fig.7B). In addition,
these results were further confirmed through phase contrast microscopic examination of the
morphology of cells. The phase contrast microscopic image clearly illustrated that loss of cell to
cell contact and reduced cell viability was observed in Aβ25-35 treatment group, whereas, after
treatment with ABFP (5 & 10 μg/ml), neuronal outgrowth between cells and increase in the cell
viability was obtained as similar to that the control group (Fig. 7C).
2.6.2. Effect of ABFP on ROS production in Aβ25-35 treated Neuro2a cells
The production of ROS is considered to be an important pathophysiological event of AD. This
may cause deleterious effects to biomolecules like DNA damage, protein and lipid oxidation
leading to neuronal dysfunction [26]. In the present study (Fig. 8), treatment of Aβ25-35 (50 M)
with Neuro2a cells for 24 h increased the content of ROS production to about (529.75 ± 3.29
A.U) when compared to the control group (319.09 ± 2.47 A.U). However, pretreatment (for 2 h)
with the fixed concentration of ABFP (5 & 10 μg/ml) exhibited significant reduction in the ROS
formation (433.31 ± 2.84 & 411.95 ± 3.77 respectively). The results indicate that treatment of
Neuro2a cells with ABFP inhibited the ROS production induced by the Aβ peptide.

3. Conclusions
The synthesized compound α-bisabolol β-D-fucopyranoside (ABFP) act as a multipotent
agent for the treatment of Alzheimer’s disease by impeding the cholinergic neurotransmission,
oxidative stress, acting as metal chelators and preventing Aβ aggregation in both in vitro and in
silico experiments. In addition, ABFP exhibited no cytotoxicity and also had neuroprotective
effect against Aβ25–35 induced neurotoxicity in Neuro2a cells. Overall, all the experimental
results concluded that ABFP act as a promising multi-target agent for the management of AD.
4 Experimental sections
4.1. Chemicals
Acetylcholinesterase (AChE) from Electric eel and Thioflavin T (ThT) were purchased from Sigma
Aldrich, USA. Acetylthiocholine iodide (ATCI) and 5,5- Dithio-bis(2-nitrobenzoicacid (DTNB)
were obtained from Hi-media Chemicals, Mumbai, India. Aβ25–35 peptide was procured from
GenScript USA, Inc. α -bisabolol was acquired from Alfa Aesar Chemical Manufacturing Company,
USA. All other reagents, solvents and chemicals used for the experiments were of high grade and
purity.
4.2 General procedure for the synthesis of α-bisabolol β-D-fucopyranoside
4.2.1. 2R,3R,4S,5S,6R)-6-Methyltetrahydro-2H-pyran-2,3,4,5-tetrayl tetrabenzoate (3)
An ice cold solution of α-fucose (500 mg, 3.0 mmol) and DMAP (73 mg.
0.6 mmol) in pyridine (5 mL) and dichloromethane (6 mL), was stirred at 0
°C for 15 min. Next the BzCl was added (2 mL, 18.0 mmol) to the reaction
mixture slowly and stirring was carried for 16 h. The reaction mixture was diluted by CH2Cl2 (20
mL) and 1N H2SO4 (15 mL) and washed with NaHCO3 and brine solution. The combined
organic layer was dried over MgSO4. Evaportion of the solvent under reduced pressure and

purification of the crude by flash column chromatography (Hex/EtOAc, 4:1) gave the benzoyl
protected compound 3 as a white foam (1.6 g, 2.7 mmol, 95%).
1H NMR (400 MHz, CDCl3):  = 8.14-8.11 (m, 4 H), 7.87-7.81 (m, 4 H), 7.67-7.62 (m, 2 H),
7.54-7.58 (m, 4 H), 7.47-7.43 (m, 2 H), 7.30-7.25 (m, 4 H), 6.87 (d, 1 H, J = 3.6 Hz), 6.08 (dd, 1
H, J = 10.6 & 3.6 Hz), 5.99 (dd, 1 H, J = 10.6 & 3.6 Hz), 5.91-5.89 (m, 1 H), 4.64 (q, 1 H, J =
7.0 Hz) and 1.32 (d, 3 H, J = 6.5 Hz) ppm.
13C NMR (100 MHz, CDCl3):  = 166.1, 165.9, 167.7, 164.8, 138.9, 133.8, 133.7, 133.5, 133.4,
130.3, 130.1, 130.0, 129.9, 129.4, 129.3, 128.9, 128.8, 128.6, 128.5, 128.4, 90.9, 71.5, 69.1, 68.0,
67.8 and 16.4 ppm.
IR (neat): 2816, 1725, 1593, 1382, 1348, 1264, 1173, 1033 and 716 cm-1.
HR ESI-MS: [C34H28O9Na]+ = [M+Na]+ Require 603.1626; found 603.1638.
TLC: Rf = 0.4 (4:1 Hex/EtOAc).
4.2.2.(3R,4R,4S,5S,6R)-2-Hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl tribenzoate (4)
To a solution of the compound 3 (1.6 g, 2.7 mmol) in CH2Cl2 (7 mL), HBr
was added (1.5 mL, 33% in AcOH) under Ar-atmosphere. The reaction was
stirred at RT for 2.5 h. Reaction mixture was quenched with saturated
NaHCO3 and extracted with CH2Cl2 solvent. The combined organic layer was washed with brine
solution, and dried over MgSO4. Solvent was evaporated under reduced pressure. The residue
was dissolved in acetone (10 mL), H2O (0.5 mL) and CH2Cl2 (1 mL). The Ag2CO3 (1 g, 3 mmol)
was added in portions, to the reaction mixture and allowed the reaction to stirr for 1.5 h at RT.
The reaction mixture was filtered out through celite and MgSO4. The filtered residue was

evaporated under reduced pressure and purified by flash column chromatography (Hex/EtOAc,
7:3) to give α,β isomer, i.e. compound 4 as white foam (1.1 g, 2.0 mmol, 80%).
Diastereomer ratio (1:0.5)
1H NMR (400 MHz, CDCl3):  = 8.12-8.10 (m, 3 H), 7.99-7.95 (m, 3 H), 7.81-7.77 (m, 3 H),
7.64-7.59 (m, 2 H), 7.51-7.47 (m, 4 H), 7.44-7.41 (m, 1 H), 7.39-7.32 (m, 2 H), 7.28-7.21 (m, 3
H), 6.04-6.01 (dd, 1 H, J = 10.5 & 3.3 Hz), 5.78 (d, 1 H, J = 3.3 Hz), 5.72 (d, 1 H, J = 2.6 Hz),
5.61-5.57 (m, 1 H), 5.01 (d, 1 H, J = 7.7 Hz), 4.66 (q, 1 H, J = 6.6 Hz), 4.13 (q, 1 H, J = 6.2 Hz),
1.36 (d, 3 H, J = 6.4 Hz) and 1.27 (d, 3 H, J = 6.4 Hz) ppm.
13C NMR (100 MHz, CDCl3):  = 166.2, 166.2, 165.8, 133.5, 133.2, 130.1, 130.0, 129.9, 129.8,
129.4, 128.7, 128.6, 128.4, 91.1, 72.2, 69.7, 68.6, 65.2 and 16.3 ppm.
IR (neat): 3402, 2922, 1725, 1593, 1453, 1385, 1105, 1068, 832 and 711 cm-1.
HR ESI-MS:[C27H24O8Na]+ = [M+Na]+ Require 499.1363; found 499.1370.
TLC: Rf = 0.4 (7:3 Hex/EtOAc).
4.2.3.(2R,3S,4S,5R,6R)-2-Methyl-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-
triyl tribenzoate (2)
To a solution of the compound 4 (1.1 g, 2.3 mmol) in anhydrous CH2Cl2
(7 mL), CCl3CN (1.2 mL, 11.5 mmol) and Cs2CO3 (110 mg, 0.33 mmol)
were added. The reaction mixture was stirred for 16 h at RT and filtered
out through celite using CH2Cl2 solvent. The solvent was evaporated under reduced pressure and
the crude mixture was purified by flash chromatography (Hex/EtOAc, 7:3) to give the compound
2 (1 g, 1.46 mmol, 70%) as a white foam.

1H NMR (400 MHz, CDCl3):  = 8.66 (s, 1 H), 8.12-8.10 (m, 2 H), 7.96-7.94 (m, 2 H), 7.82-
7.79 (m, 2 H), 7.66-7.61 (m, 1 H), 7.53-7.48 (m, 3 H), 7.46-7.42 (m, 1 H), 7.35 (t, 2 H, J = 7.6
Hz), 7.26 (t, 2 H, J = 7.6 Hz), 6.83 (d, 1 H, J = 3.6 Hz), 6.06-6.02 (m, 1 H), 5.93-5.90 (m, 1 H),
5.89-5.87 (m, 1 H), 4.65 (q, 1 H, J = 6.4 Hz), 1.30 (d, 3 H, J = 6.5 Hz) ppm.
13C NMR (100 MHz, CDCl3):  = 166.0, 165.8, 165.7, 160.9, 133.7, 133.6, 133.4, 130.1, 129.9,
129.8, 129.3, 129.1, 128.9, 128.8, 128.5, 128.4, 94.3, 71.5, 68.8, 68.2, 68.0 and 16.4 ppm.
HR ESI-MS: [C29H24Cl3NO8Na]+ = [M+Na]+ Require 642.0460; found 642.0429.
TLC: Rf = 0.4 (4:1 Hex/EtOAc)
4.2.4. (2R,3S,4S,5R,6S)-2-Methyl-6-{[(2S)-6-methyl-2-(4-methylcyclohex-3-en-1-yl)hept-5-en-
2-yl]oxy}tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (5)
To a solution of α-bisabolol (53 mg, 0.24 mmol) in anhydrous
CH2Cl2 (3 mL) 4 A° molecular sieves and TMSOTf (5.4 mg,
0.024 mmol) were added at –78 °C under inert atmosphere. An
ice cold solution of the compound 2 (300 mg, 0.48 mmol) in anhydrous CH2Cl2 was added to the
reaction mixture drop wise. Reaction was warmed to –20 °C for 2.5 h. After completion of α-
bisabolol, the reaction was quenched by the triethylamine (4 equiv., 100 mg, 0.15 ml, 0.96
mmol). The organic solvent was evaporated under reduced pressure and the crude mixture was
purified by flash column chromatography (Hex:EtOAc, 4:1) to give the compound 5 (130 mg,
0.19 mmol, 80%) as a white foam.
1H NMR (500 MHz, CDCl3):  = 8.20-8.09 (m, 2 H), 8.00-7.88 (m, 2 H), 7.81-7.73 (m, 2 H),
7.65-7.56 (m, 1 H), 7.53-7.45 (m, 3 H), 7.44-7.31 (m, 3 H), 7.27-7.19 (m, 2 H), 5.78 (dd, 1 H, J
= 10.5 & 7.8 Hz), 5.69 (d, 1 H, J = 3.3 Hz), 5.57 (dd, 1 H, J = 10.5 & 6.8 Hz), 5.18 (broad s, 1

H), 5.06 (t, 1 H, J = 7.0 Hz), 4.92 (d, 1 H, J = 7.8 Hz), 4.03 (dd, 1 H, J = 12.9 & 6.5 Hz), 2.32-
2.22 (m, 1 H), 2.07-1.91 (m, 1 H), 1.90-1.84 (m, 1 H), 1.84-1.80 (m, 1 H), 1.78-1.74 (m, 1 H),
1.73-1.70 (m, 1 H), 1.67 (s, 3 H), 1.64-1.63 (m, 1 H), 1.60 (s, 3 H), 1.59-1.56 (m, 1 H), 1.55 (s, 3
H), 1.49-1.42 (m, 1 H), 1.30 (d, 3 H, J = 6.4 Hz), 1.06 (s, 3 H) and 0.98-0.86 (m, 1 H) ppm.
13C NMR (125 MHz, CDCl3):  = 166.3, 165.8, 165.3, 134.3, 133.4, 133.2, 133.1, 131.3, 130.2,
129.8, 129.7, 129.5, 129.1, 128.6, 128.4, 128.3, 124.9, 120.2, 95.7, 82.3, 72.5, 71.4, 70.1, 69.6,
40.5, 38.2, 30.8, 29.8, 26.7, 25.8, 23.4, 22.8, 21.7, 19.8, 17.9 and 16.6 ppm.
IR (neat): 2969, 2923, 2856, 1729, 1594, 1450, 1263, 1172, 1101, 1023 and 708 cm-1.
HR ESI-MS: [C42H48O8Na]+ = [M+Na]+ Require 703.3241; found 703.3210
TLC: Rf = 0.4 (4:1 Hex/EtOAc)
4.2.5. (2R,3R,4S,5R,6S)-2-Methyl-6-{[(2S)-6-methyl-2-(4-methylcyclohex-3-en-1-yl)hept-5-en-
2-yl]oxy}tetrahydro-2H-pyran-3,4,5-triol (1)
To a solution of the compound 5 (130 g, 0.19 mmol) in
MeOH/THF/H2O (1:2:1; 6 mL), aq. NaOH (2 mL, 0.25 N) was
added slowly and continued the stirring at RT for 1.5 h. The
activated Amberlite (with 1 N H2SO4) was added to the reaction
mixture to pH 7 then reaction mixture was filtered and solvent was evaporated under reduced
pressure. Purification of the crude mixture by column chromatography (Hex:EtOAc, 1:1) gave
the compound 1 (69 mg, 0.18 mmol, 98%) as a white solid.
1H NMR (400 MHz, CDCl3):  = 5.35 (broad s, 1 H), 5.04 (broad t, 1 H, J = 6.6 Hz), 4.39(d, 1
H, J = 6.9 Hz), 3.70 (broad s, 1 H), 3.66-3.50 (m, 3 H), 2.20-2.10 (m, 1 H), 2.0-1.90 (m, 5 H),

1.79-1.74 (m, 2 H), 1.67 (s, 3 H), 1.64 (s, 3 H), 1.59 (s, 3 H), 1.57-1.53 (m, 1 H), 1.52-1.44 (m, 1
H), 1.29 (d, 3 H, J = 5.8 Hz), 1.24-1.22 (m, 1 H) and 1.13 (s, 3 H) ppm.
13C NMR (100 MHz, CDCl3):  = 134.5, 131.2, 124.9, 120.6, 97.1, 81.9, 74.3, 71.9, 71.8, 70.4,
41.0, 37.9, 31.0, 29.8, 26.9, 25.8, 23.5, 21.8, 20.2, 17.9 and 16.6 ppm.
HR ESI-MS: [C21H36O5Na]+ = [M+Na]+ Require 391.2455; found 391.2487
TLC: Rf = 0.4 (1:1 Hex/EtOAc)
4.3 Molecular modelling studies
All the in silico investigations were performed on the High performance Graphics
processing Unit installed with the Cent OS version 7. The hardware stipulations include the Intel
core i7 processor of 8 cores and 16 GB RAM speed whereas the software stipulations include the
commercial version of Schrödinger software package and the academic version of Desmond.
4.3.1 Protein and ligand preparation
The three dimensional crystal structure of the receptor protein Acetylcholinestarase (ACHE)
containing the PDB ID: 4M0E has been obtained from Protein Data Bank (PDB). The crystal
structure of the protein was not ready to be used in the theoretical calculations and hence, the
target protein was prepared through the Protein Preparation Wizard implements in Maestro 11.3
[27] in which Prime loop modelling adds the missing residues. Initially, assignment of the bond
orders, addition of hydrogen atoms and the removal of crystallographic water molecules were
performed. Optimization of the hydroxyl group rotation, hydrogen-bonding network and thiol
hydrogen will be performed with the protassign script which also generates the HIS protonation
ann tautomerization states. This script also performs the chi flips in ASN, GLN and HIS
residues. Minimization of the optimized structure of ACHE is performed with the help of OPLS-

2005 force field till the root mean square deviation (RMSD) of the non-hydrogen atoms reaches
0.3 Å. The ligand molecule α-Bisabolol β-D-fucopyranoside was retrieved from PubChem in its
2-Dimensional structure and optimized into the 3-dimensonal structure using the LigPrep [28].
4.3.2 Molecular docking analysis
The Glide module implemented in Schrödinger possess the docking protocol and it was
carried out in the current study [29]. This tool performs the grid-based ligand docking with
searches accounting for the favorable interactions between one or more small molecule with the
bimolecule, especially the protein. In this docking strategy, the ligand will be docked into the
active site with the threshold of 0.50 kcla/mol for avoiding the minimized pose. The internal
generation of conformations passing through the filters helps in the maintenance of the flexible
ligand sampling. The receptor grid was generated with the predicted active site through sitemap.
Ligand docking was utilized to carry out the docking of all ligands with the various charges.
Initially, the ligand molecule was centered and subsequently they were allowed to rotate around
three enagles. Secondly, the grid-based force field evaluation and the refinement of the docking
including the torsional and rigid body movements of the ligands were supported out through the
OPLS-AA force field. The glide score generation was carried out with the following formula.
GScore=a*vdW + b* Coul + Lipo + Hbond + Metal +BuryP + RotB + Site.
(I)
Where, vdW=van der Waal energy, Coul=Coulomb energy, Lipo=lipophilic contact term,
Hbond=hydrogen-bonding term, Metal=metal-binding term, BuryP=penalty for buried polar
groups, RotB= penalty for freezing rotatable bonds, Site=polar interactions at the active site [30].
4.3.3 Binding Free Energy Calculation
The free energy of the binding for the complexes obtained from the docking protocol were
calculated using the Prime MM-GB/SA approach [31]. During this process, minimization of the

docked poses with the local optimization feature and the complex energy were generated with
the help of OPLS-AA (2005) force field and generalized Born/Surface area (GB/SA) continuum
solvent model. The binding free energy of the complex were calculated with the following
equations [32 a-d]
ΔGbind = ΔE + ΔGsolv + ΔGSA
ΔE= Ecomplex– Ereceptor – Eligand
(II)
(III)
where, Ecomplex, Ereceptor, and Eligand are the minimized energies of the receptor-ligand complex,
receptor and ligand, respectively. Prime uses the SGB model which employs the Gaussian
surface instead of the van der Waals surface for the improved depiction of the solvent-accessible
surface area.
ΔGsolv= Gsolv(complex) - Gsolv(receptor) - Gsolv(ligand)
(IV)
where, Gsolv(complex), Gsolv(receptor), and Gsolv(ligand) are the solvation free energies of the
complex, receptor, and ligand, respectively.
ΔGSA = GSA(complex) - GSA(receptor) - GSA(ligand)
(V)
where, GSA(complex), GSA(receptor), and GSA(ligand) are the surface area energies for the
complex, protein, and ligand, respectively. The reasonable criteria for the choice of best
complexes based on scoring and interaction parameters were shown in XP docking with different
charge model of ligands.
4.4. Determination of AChE activity
Acetylcholinesterase is the main pathological hallmark of AD. Briefly, AChE inhibitory activity
was performed by previous reported method with slight modifications [33]. Different
concentrations (10-50 µg/ml) of ABFP were incubated with 10 l of AChE for 45 min at room

temperature. After the incubation period, 125 l of DTNB (3 mM) was added and the total
volume was made up to 300 µl with 50 mM of Tris-HCl buffer (pH 8.0). Consequently, 50 l of
15 mM Acetylthiocholine iodide was added to the reaction mixture to facilitate the initiation of
the enzyme activity. Then, 3 mM 5-thio-2-nitrobenzoate was added to form the yellow color and
inhibition of absorbance was read at the wavelength of 405 nm using UV-Visible
spectrophotometer (U-2800, Hitachi, Japan). The experiments were performed in triplicates.
Donepezil, was used as a standard drug and the percentage of inhibition was calculated according
to the formula,
Where Sc is the Specific activity of control group and St is the Specific activity of treated group
4.5 In vitro antioxidant assays
4.5.1 Free radical scavenging assay
The antioxidant capacity of ABFP was estimated by method of Blois MS, 1958 [34]. 0.1 mM of
DPPH (0.2 ml) mixed with 3 ml of methanol solution was added to various concentrations of
ABFP (10-50 µg/ml). The change in the colour of the solution from purple to yellow was
measured after 30 mins using UV-visible spectrophotometer (U-2800 model, Hitachi, Japan).
Standard drug BHT (1mg/ml) was used as a positive control and the percentage of inhibition was
calculated according to the formula,
Where (
Absc) is the Absorbance of control and (Abst) is the Absorbance of test.

4.5.2. Hydrogen peroxide scavenging assay
The hydrogen peroxide scavenging ability of ABFP was determined according to the
method of Khan et al., 2012 [35]. Hydrogen peroxide solution (40 mM, pH 7.4) was added to
various concentrations (10-50 µg/ml) of ABFP. The mixture was kept at room temperature for 10
min, after which the absorbance of Hydrogen peroxide was measured at 550 nm using phosphate
buffer as blank. L- Ascorbic acid (1mg/ml) was used as a positive control and the percentage of
was calculated according to the formula,
scavenging activity
4.5.3. Hydroxyl radical scavenging activity
The Hydroxyl radical scavenging activity of ABFP was determined by using deoxyribose
method as described in the literature [36]. The reaction mixture (1 ml) containing 1mM ETDA,
10 mM FeCl3, 10 mM deoxyribose, ABFP (10-50 µg/ml), 10 mM H2O2, 1mM ascorbic acid and
0
phosphate buffer (pH7.4) was incubated for 1 hr at 37 C. The reaction mixture was heated at
95 0 C in a water bath for 20 mins, finally added 0.5 ml of 10% TCA and 0.5 ml of 0.4% TBA to
form a pink colour. The absorbance of hydroxyl radical was measured at 532 nm. Butylated
hydroxytoluene was used as the standard and the % of inhibition of hydroxyl radical was
determined by comparison with control and test sample.
4.5.4. Total Reducing power assay
The reducing potential of ABFP was evaluated according to the method of Moein et al.,
2008 [37]. Different concentrations of the ABFP (10-50 µg/ml) was mixed with 0.5 ml of

phosphate buffer solution (0.2 mM, pH 6.6), 1% of potassium ferrocyanide (0.5 ml) and
incubated at 50 °C for 20 min. After the incubation period, 0.5 ml of 10% trichloro acetic acid
was added and the mixture was centrifuged at 3000 rpm for 10 mins. The upper layer was
collected, mixed with 0.5 ml of distilled water and 100 μl of 0.1% FeCl3. The absorbance was
measured at 700 nm. The higher absorbance of the sample indicates higher reducing power
capacity. Ascorbic acid was used as the standard.
4.5.5. Metal chelating power assay
The metal chelating ability was carried out using the published method of Haro-Vicente et al [38].
In brief, the solution of Ferrous Sulphate (0.15 mM) was added to different concentrations of (10–
50 μg/ml) of ABFP. The mixture of solution was reacted by the addition of 0.5 mM of Ferrozine
and shaken robustly and incubated for 10 mins at room temperature. The absorbance of the
solution was read at 562 nm. BHT was used as a positive control. The rate of inhibition of Fe2+-
ferrozoine complex formation was calculated as follows
Where
Abs c is the Absorbance of control and Abs t is the Absorbance of test.
4.5.6. Total antioxidant capacity assay
The total antioxidant potential of ABFP was evaluated by phosphomolybdenum method [23].
Briefly, different concentrations (10-50 µg/ml) of ABFP were mixed with 3 ml of reagent solution
containing (0.6 M) H2SO4, (28 mM) NaH2PO4 and 4 mM (NH4)2MoO4 and the reaction solution was
incubated at 95 °C for 90 min. L-ascorbic acid was used as positive control and the absorbance was
measured at 665 nm.

4.6. Preparation of Aβ25–35 and analysis of Antiaggregation and disaggregation property
Aβ25–35 peptide (0.5 mg powder form) was dissolved in 500 μl of hexafluoro-2-propanol and kept
in room temperature for 24 h to form monomers. Before start the experiments the dried powder
was mixed with 500 μl sterile MilliQ water.
Phase I: Inhibition of formation of aggregates from oligomers: In phase I study, the monomeric
Aβ25–35 (100 μM) was incubated at 37 °C in 50 mM Tris-HCl buffer (pH 7.4) for 24 h to form
oligomers. To this mixture of oligomers, ABFP (5 & 10 μg/ml) and Galantamine (50 µg/ml) was
added and incubated for 24 & 48 h. After that, samples were taken for Thioflavin –T assay and
Confocal microscopy study from the mixture of 24 & 48 h.
Phase II: Disaggregation of the pre-formed mature fibrils: In phase II study, Aβ25–35 (100 μM)
was incubated at 37 °C in 50 mM Tris-HCl buffer (pH 7.4) for 96 h to form mature fibrils. It was
then treated with ABFP (5 & 10 μg/ml) and Galantamine (50 µg/ml) and incubated for 96 h and 9
days separately. For evaluation of the formation of pre -formed mature fibrils, samples from the
mixture of 96 h and 9 days incubation were taken for Thioflavin –T assay and Confocal
microscopy study (Fig. 5A).
4.6.1. Thioflavin T assay
A fibrils were quantified by Thioflavin T assay [39]. Aliquots (5μl) from the mixture of
samples taken from both phases I & II solutions containing Aβ25-35 with/without ABFP (24 h, 48
h, 96 h, 9d) were added to 5 µM Thioflavin-T and final volume was made up to 300 μl of 50 mM
glycine-NaOH buffer (pH 8.5).The fluorescence intensities were measured at wavelength of
excitation 450 and emission 485 nm using spectrofluorimeter (Molecular Device SpectramaxM3,

USA). The background ThT fluorescence was subtracted from the experimental values of all the
samples. Galantamine (50 µM) was used as a positive control.
4.6.2. Fluorescence microscopic analysis
For the fluorescence microscopic study, 2.5 l of the investigational samples (aliquots
from 24 h, 48 h, 96 h, 9d of Phase I and II experiments) were diluted with 5 μM of thioflavin T
and incubated for 10 mins at RT, and then transferred onto a slide. Aβ25–35 aggregation was
visualized and captured by Confocal Laser Scanning Microscope FV300, Olympus, Japan.
4.7 Molecular Dynamics Simulation
The molecular dynamics simulation of the five copies of the Aβ peptides obtained from
the PDB with the ID: 1QXC were randomly placed with the ABFP with the Desmond package
[32d] with the Optimized Potentials for Liquid Simulations (OPLS) 2005 force field [40] for the
minimization of the system. The peptide structure and the ligand were imported with the Monte
Carlo simulated water model of TIP3Pand solvated in an orthorhombic periodic box with the
buffered space of 10 Å from the edges of the peptide so that it will not collide with the solvation
box which is neutralized with the appropriate number of counter ions and 0.15 M salt
concentration [41a-e]. The simulation system was comforted with the constant NPT (number of
atoms N, pressure P and temperature, T) ensemble condition for the generation of the simulation
data for the post simulation analyses. The steepest descent method helps in the energy
minimization of the prepared system with the maximum of 5000 steps till the gradient threshold
(25kcal/mol/Å) is attained. Equilibration of the system has been achieved with the default
protocol available in Desmond. The equilibrated system were carried forward in order to execute
the molecular dynamics simulation of 100ns time period at the constant temperature and pressure
of 300 K and 1 atm respectively with the time step of 2fs.

4.8. Cell culture
The Neuro 2a cells were purchased from National Centre for Cell Sciences (NCCS), India.
The cells were supplied with Dulbecco’s modified Eagle medium (DMEM) supplemented with
5% CO2, 10% horse serum, 15% Fetal bovine serum, 100 mg/ml streptomycin, 100 U/ml
penicillin antibiotic and maintained at 37 °C.
4.9. Assessment of cell viability by MTT assay
The defensive dose was fixed by using the method of colorimetric MTT assay. Initially, the
Neuro2a cells were plated at 2 × 105 cells/ml (per well) in a 96 well plate and incubated for 24h
for allowing the cells to adhere. Subsequently, Neuro2a cells were pre-treated with various
concentrations of ABFP (2.5-10 μg/ml) for 24 h. Then, the cells were resuspended in 100 μl of
MTT (0.5 mg/ml) and incubated for 4 h at 37 °C. Finally, the MTT was removed from the wells
and washed with PBS and 100 μl of dimethylsulfoxide (DMSO) was added to each well. The
absorbance was measured at 570 nm using multi well plate reader [42].
4.9.1. Neuroprotective effect of ABFP against Aβ25-35 induced toxicity in Neuro2a cells
Based on our recent report that treatment of Neuro2a cells with 50 µM Aβ25-35 caused an obvious
decrease in the viability of the cells [25], the dosage of 50 μM of Aβ25-35 was fixed for further
assays. In this assay, the neuro2a cells (2 × 105 cells/ml) were grown in a 96 well plate and
incubated for 24h. After that the cells were pre-treated with the fixed concentration of ABFP (5 &
10 μg/ml) for 2 h, followed by treatment with 50 μM of Aβ25-35 for 24 h. Subsequently, MTT
solution was added to all the wells and incubated at 37 °C for 4 h. After that, the MTT solutions
were removed and washed with PBS. Finally, 300 μl of DMSO was added and the absorbance
was measured at 570 nm. Results were expressed as percentage of viability and compared with

control samples [43]. In addition, the morphological characteristics of cells were observed in the
phase contrast microscope analysis.
4.10. Measurement of intracellular Reactive oxygen species (ROS) level
The measurement of intracellular ROS was assessed using ROS-sensitive 2′,7′-
dichlorodihydrofluorescein diacetate staining method [26]. In this assay, Neuro2a cells (2 × 105
cells/ml) were pre-treated with ABFP (5 & 10 μg/ml) and then exposed to 50 μM of Aβ25-35 for
24 h. Following treatments, the medium was discarded and incubated with 10 µM of DCFH-DA
for 20 min at RT. After that, phosphate buffer solution (pH 7.4) was used to wash the cells and
lysed with lysis buffer (pH 8.0). Finally, the fluorescence intensity was measured at an excitation
wavelength of 495 nm and an emission wavelength of 520 nm by using SpectraMax M3
Microplate Reader. The amount of ROS produced was determined from the fluorescent intensity
observed in the samples.
4.11. Statistical Analysis
All the experiments were done in triplicates and the statistical analysis was performed by one-
way ANOVA using SPSS 17 statistical software followed by Duncan’s test. P<0.05 was
considered as statistically different between control and treated groups.
Acknowledgements
The authors, MJ, SS and KPD wish to acknowledge the Computational and Bioinformatics
Facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by
DBT, GOI; File No. BT/BI/25/012/2012,BIF), DST-FIST (Grant No. SR/FST/LSI-

639/2015(C)), UGC-SAP (Grant No. F.5-1/2018/DRS-II(SAP-II)), DST-PURSE (Grant No.
SR/PURSE Phase 2/38 (G)) and RUSA 2.0 [F. 24-51/2014-U, Policy (TN Multi-Gen), Dept of
Edn, GoI].
Abbreviations Used
AD, Alzheimer’s disease; Aβ, amyloid β-protein; ABFP, α-bisabolol β-D-fucopyranoside;
AChE, acetylcholinesterase; BBB, blood-brain-barrier; DPPH, 1, 1-diphenyl-2-picrylhydrazyl;
ThT, Thioflavin-T; CLSM, confocal laser scanning microscope; TLC, thin layer
chromatography; DMPD, N,N-dimethyl-p-phenylenediamine; MTT, 3-[4,5-dimethyltiazol- 2-
yl]-2,5-diphenyltetrazolium bromide; DCFH-DA, dichloro-dihydro-fluorescein diacetate
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